A te ae Ney eg be i Shted tp bate CELA wna gage tie brits ae a ee ata gt CRN Pent a Sesh * nents 4 tee “A sae A porsh to ' Le ‘africa *e on Setters ole ay te : a se Ban Scie Li tgs 2 “uv, an aint tba beee ke tombe? Saar tes bs ew areas ae ee fe cei Raya wantin eae eines, pe se ae Wan Re w ue inte As) “ Stee vue we' ‘ - Vide ry tut AV EERE e Cae et HP aetanarta \ oy | va \ es AS aul hy abi i . (ave bts sonia w he ' TSP 8 Ain Chain te tet 4 ae AG be Se thee joys seed ats fir utatast page teh a Chad My Vee ey vA i. Be ve FeO ons Venn wt ota a ha Ce ee pares e othe ’ ev eet Righ Aer Aa 4 4 ON a ead fa] tataee ey 4 : bn Sean ‘ ot on ’ ven ) ya she 4 . a sence Boek yeyeAEs ier y eee al teeerb ret . be h pyle’ 8 Si ene : ‘ is. 4 SSorishisieae thats - 4 A ‘ : aes ‘ Sa eee Ai 4 # than) eee ae ee wa ain hv ie gt see trinintatancch ents oie ( Patra Satna Nae e Mg . ; + . ue, . 48 Bus ee if Bhd Seok : : ae a \ ot ibs, PR ‘ bs 3 . P iy . a Ve wh rata rere ane ee 1 may i tie take Uae sees era whe erat 7] Vins com * Tew Tee mere YU ts TONE tte Woy: aft ‘ys Mi deemnsetryg te ERE TEN ee ~~ Pa Re a Atemeaet t re Kee) WO ASTM SG Ae arte $0 Sante wees ee a> 2 Beanie . Aw oT int rn kee ae R Wath WW eM Ae papas i ERRATA wr eed Aerie! Oat, Wes LAY ‘Reaces ae tas co sieves a wig Sree Eee tear. be ee Pe ge 5 WMA way ra SES SE: ae we eres earn ites ie Cees sist od cP et AFA pea S - Sahat = saereries raped ae Retras te ree hae ae wma Pat ner yee ween Hey a ” + P4uas St ates? ores KPIS TES, & hee mieiee Se ca Okage age pepe tebe age oa a oe Bap eat Digitized by the Internet Archive in 2009 with funding from University of Toronto http ://www.archive.org/details/journalofexperimO8broo Yao i i yy %; By, fi eve an yay > as &, b+ es t a ; 7 é > e i eee em 3S OU RNA L OF IXPERIMENTAL ZOOLOGY EDITED BY WILLIAM E. CASTLE FRANK R. LILLIE Harvard University University of Chicago EDWIN G. CONKLIN JACQUES LOEB Princeton University University of California CHARLES B. DAVENPORT THOMAS H. MORGAN Carnegie Institution Columbia University HORACE JAYNE sEORGE H. PARKER The Wistar Institute Harvard University HERBERT S. JENNINGS CHARLES O. WHITMAN Johns Hopkins University University of Chicago EDMUND B. WILSON, Columbia University and ROSS G. HARRISON, Yale University Managing Editor ca VOLUME VIII ees ee Py i ' cs THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY PHILADELPHIA, PA. 1910 — = —_ CONTENTS No. 1—January, 1g1o G. H. Parker The reactions of sponges, with a consideration of the origin of the Memwalsmpsteni. VWithothree ioibes... 2: SA aeweee bak iw be. I H. G. Kriss The reactions of zolosoma (Ehrenberg) to chemical stimuli. With [NOG LY SRN BSG 6 < SE A gana oe 2) ce 43 Asa ARTHUR SCHAEFFER Selection of food in stentor ceruleus (Ehr.) With two figures.......... 75 No. 2—March, rgto A. J. GotpFars Light as a factor in the regeneration of hydroids. as 0 2 ee ee ee ie H. H. NEwMan Further studies on the process of heredity in fundulus hybrids. 1. The influence of spermatozodn on the rate and character of early Brera ae NVtIt SEMEN MOUIEERM PII gi) i Sony pel? eas, dian agen a's 143 Oscar RIDDLE / Studies with Sudan III in metabolism and inheritance................ 163 W. E. Caste: The effect of selection upon Mendelian characters manifested in one SESEO cil Pe Ee pe cio r , ea 185 No. 2—Continued. W. A. MatTHENY effects of alcohol on the life cycle of paramecium. With one figure... 193 N. M. STEVENS ‘The chromosomes in the germ cells of culex. With fifty-two figures...... 207 N. M. STEVENS An unequal pair of heterochromosomes in forfcula. With forty- Ciphe Heures) eso f en ce ke ate cine ae a rr 227 No. 3—May, Igo A. M. Banta A comparison of the reactions of a species of surface isopod with those of a subterranean species. Part 1. Experiments with light. With SIX HSUTES. ao estos. Sea eines Melee eee ee eee Aa 243 AARON FRANKLIN SHULL Studies in the life cycle of hydatina senta. 1. Artificial control of the transition from the parthenogenetic to the sexual method of reproduc- tion. With one figure. ..c3< 2.2... o2 pee aoe 311 No. 4—June, Ig10 E. Newton Harvey The mechanism of membrane formation and other early changes in developing sea-urchins’ eggs, as bearing on the problem of artificial parthenogenesis. With two figures...........:. . 72255: Wittiam Morton WHEELER The effects of parasitic and other kinds of castration in insects. With eight figures... bs. vere os ie Sed ene ee 377 A. M. Banta A comparison of the reactions of a species of surface isopod with those of a subterranean species. Part It. Experiments with mechanical stimulation. 2.26... eo eas va cls on tgs 439 A. H. EstaBrook Effect of chemicals on growth in paramecium. With one figure..... yee ton) G. H. PARKER Olfactonymreactionsum hshesw.. ee <7 ae eee 4. Oa nee 533 CONTRIBUTIONS FROM THE ZOOLOGICAL LABORATORY OF THE MUSEUM OF COMPARATIVE ZOOLOGY AT HARVARD COLLEGE. E.L.MARK, Director. No 204. THE REACTIONS OF SPONGES, WITH A CONSIDERA- TION OF THE ORIGIN OF THE NERVOUS SYSTEM BY G. H. PARKER Professor of Zodlogy in Harvard University Wiru Turee Ficures MURERN EE COCA ULC C1 CSR SP lacey cre fe, cts, help es eas ehh = s:/41 sab aecahag eRe oar geaeale iS) «sito ose: sclsisln Seiclaleitte/ starwars aveveilts ate 2 Pe emGture Ot Stylotellati scr. .1- . b aaseeiyeas es =. sae Abi: AGU Oo aonb on Sete h ra semanas 5 MMIC ACHOHS Qi SUMO tel aay tfaj otter ihn tele nie 40 IT IN SRODUCTIONZ Previous attempts at the discovery of the nervous system of sponges have been made almost exclusively from an anatomical standpoint and with such negative results that Vosmzer and Pekel- Tue JourNat or EXPERIMENTAL ZOOLOGY, VOL. VIII, NO. I. i) G. H. Parker haring (’98, p. 18) believed themselves justified in declaring that the cells of sponges “are not connected in a way so as to enable them to conduct stimuli from one cell to another”’ and that these animals are therefore ““destitue of the principle, the significance of which culminates in nervous tissue.”” It was the chief purpose of the investigations recorded in the present paper to ascertain whether there was any physiological ground for the assumption that sponges possess a nervous system, or whether from the stand- point of their activities, as well as of their structure, they showed no evidence of nervous organs. The general inertness of sponges has doubtless long deterred investigators from attempting a study of their reactions, and it must be confessed that even on close examination they show only a few form of inconspicuous response These few types of movement, however, are of considerable interest, for, as the following account will show, they throw con- siderable light not only on the question of the nervous system, in sponges but also on the still more fundamental problem of the origin of the nervous system in general. The species on W hich my work was done was Stylotella helio- phila Wilson, a monaxonid demosponge belonging to the order Helichondrina. ‘This species will be describes in a monograph on the sponges of Beaufort, N. C., soon to be published by Dr. H. V. Wilson, and I am indebted to Dr. Wilson for having called my attention to this sponge, which in all respects was extremely satisfactory for the work I had planned. My investigations were carried out in June and July at the Beaufort Laboratory of the United States Bureau of Fisheries, and I am under obligations to Commissioner G. M. Bowers for the privilege of okie at this laboratory and toits director, Mr. H. D. Aller, for generous pro- vision during my stay there. 2. STYLOTELLA UNDER NATURAL CONDITIONS Stylotella heliophila is found in great abundance in the shallow water near the Beaufort Laboratory. It grows in masses about as large as a double fist and is attached to stones, oyster shells, and Fike materials. It is dirty orange-yellow or ereenish yellow Reactions of Sponges W in color and, though sometimes simply massive in habit, it gener- ally rises in finger-like processes from an incrusting base (Fig. 1). It is found near low-water level, and some colonies are so situated that at the spring tides they may be continuously expose d to the air for as long as four hours. Asa rule the massive form is charac- teristic of those colonies which from time to time are exposed to the air; the long-fingered type 1s limited almost exclusively to such as are never uncov eral by the sea even at the lowest tides. A Stylotella grows on the upper surfaces of stones, etc., in shallow Fig. 1. Side view of a colony of Stylotella heliophila Wilson, about natural size. From a photo- graph taken by Dr. H. V. Wilson. water, it is often in strong sunlight for the greater part of the day and in fact when uncov med by the tide it may also be exposed to the extreme heat of the sun for hours ata time. That it not only survives under such conditions but seems’even to court sunshine has doubtless given occasion to its specific name heliophila. The water in which it thrives is often deeply laden wuth sediment and this at times may shield it partly from the sun’s rays, but when the water is clear or the sponge is exposed to the air, it receives 4 G. H. Parker the full force of these rays yet without any apparently disastrous effects. If a colony of Stylotella in natural position in quiet, clear sea- water 1S closely examined, its numerous oscula, which occupy either the tips of the fingers or slignt elevations on the surface of its body, will be found as a rule to be widely open, so that an observer can look far down into the interior of the animal and see much of the branched gastral cavity and the excurrent canals leading into it. Alenough the fingers of the sponge are generally not much over seven to eight millimeters in diameter, the oscula may measure as much as four and a half millimeters in width when fully expanded thus giving a considerable view of the internal cavities. If such a colony is suddenly lifted out of the seawater into the air, the water rapidly drains from it and the airrushes through its oscula into its internal chambers. On returning such a sponge to the sea, the air thus introduced is with difficulty dis- lodged and may eventually as large bubbles distort and deform the sponge. Sponges that are exposed to the air on the beachby the natural fall of the tide show no such inclusions of air,and an examination of them: in seawater brings to light the fact that their oscula are all firmly closed thus preventing the entrance of air. The steps of this closure can be easily followed by watching a sponge that is gradually becoming exposed to air when, ina quiet sea, the tide is falling. Under such circumstance the oscula remain open till they come into direct contact with the air when, with about three minutes, they close. If now the sponge colony is moved into deeper water, theoscula will reopen in from seven to ten minutes. If oscula at different levels on the same sponge are watched, those that come in contact with the air first, close first and those that are situated ata deeper level do not close until they in turn have been exposed to the air. These conditions are easily reproduced 1 in the laboratory. ‘hus if a colony of fresh sponge is carried into the laboratory and eee in a glass vessel in which a current of seawater in kept running, on exposing the tip of any finger to air its osculum will close in a few minutes to reopen after it has been reimmersed in seawater for about ten minutes. On quickly removing a sponge from the sea the chim- Reactions of Sponges 5 ney-like membranes around the oscula very generally collapse showing that they are delicate structures. That the normal closure of the osculum is not a purely mechanical collapse of this kind is seen from the fact that when a sponge in which the closure has taken place by eradual exposure to air 1s returned to sea- water, its oscula do not flap open but can be seen to expand only gradually as by the relaxation of a sphincter. It is quite clear that the closure of the osculum is a definite response, which, among other things, prevents the entrance of air into the cavities of the sponge when, by a fall of the tide, the sponge becomes exposed to the air. Another response which can be observed in Stylotella in its natural state is seen on comparing specimens that have been exposed for some time to the air on the beach with specimens still in the water. The latter as a rule have a plump appearance and a relatively smooth surface, whereas those that have been in the air look somewhat shriveled, and their surfaces are roughened as though their flesh had shrunken down on a rather resistant skele- ton. At first sight it would seem that the sponge had shriveled simply because under action of gravity the water had been drained from it, but that this shriveling is probably not thus produced, but is dependent upon a positive contraction of the flesh of the sponge, is seen from the fact that the same shriveled, rugose appearance can be assumed by a sponge im seawater under conditions to be described later. “These two reactions, the clos- ing and opening of the oscula, and the shriveling and filling out of the common flesh of the body, are the most obvious natural responses exhibited by Stylotella. 3- STRUCTURE OF STYLOTELLA Stylotella is an encrusting sponge that usually throws up longer or shorter fingers (Fig. 1.) hese fingers, which represent the individuals in «he colony, may attain a length of four centimeters and each one carries near its distal end usually one, sometimes two or more oscula. When fully expanded the oscula are roundish openings in a dome-shaped elevation or at the end of a more 6 G. H. Parker chimney-like projection. When contracted they are completely closed and the delicate tissue about them is puckered i into a slight spine-like elevation, the point of which represents the real position of the osculum. The largest oscula when fully open measure, as already stated, about four and a half millimeters in diameter. From each osculum a branched gastral cavity extends through the substance of the sponge either down the length of a finger or into the massive body, depending upon the position of the oscu- lum. In the fingered forms the gastral cavities le either near the axis of the fingers, and are thus buried in the substance of the sponge, or on the surface of the fnger, im which case they can be Fig. 2. Radial portion of a transverse section of Stylotella; the flesh of the sponge is tinted, the cavities are untinted; on the extreme left is the dermal membrane pierced by two ostia that lead into a large subdermal cavity, from which incurrent canals lead to the flagellated chambers, which in turn open by an excurrent canal into the gastral cavity at the extreme right. 25. traced on the outside as translucent-walled canals well down the length of the finger. [Excepting the regions where the gastral cavities show from the outside, the whole external surface of the sponge is faintly rugose and of a dirty- yellow color. Th einternal structure of the sponge 1s well seen ina transverse section of a finger. On the outside of such a section (Fig. 2) 1s a well defined membrane pierced in many places by dermal pores or ostia. ‘These openings are roundish or oval in outline and, as seen in living bits of membrane torn from the outer surface of the sponge, they measure from ten to twenty micra in diameter. The ostia lead into relatively large sub-dermal cavities, which Reactions of Sponges 7 often form a definite layer around the whole finger diectly under the dermal membrane From these sub-dermal cavities pass off the incurrent canals, which lead centrally into the flagella ted cham- bers. These chambers are usually spherical in ean measur- ing about twenty to forty micra in diameter and forming a more or less compact la yer surrounding the gastral cavity; they con- nect with this cavity by short, irregularly branched, excurrent canals. Where the castral cavity 1s ease to the external surface of the sponge, there are apparently few or no ostia, sub-dermal cavities, or flagellated chambers, but the dermal membrane and the lining of the gastral cavity coalesce to form the translucent wall already Paationedt In the living condition of the sponge the layer of flagellated chambers is orange in color while the other parts of the eaeee are mostly dirty- ee in tint. I have not studied with any fulness the histology of stylotella but in good osmic-acid preparations cut into sections ten micra thick and stained in picrocarmine much of the cellular structure of this animal can be made out. The outer surface of Stylotella is covered with a dermal epithelium composed of polygonal cells that are usually extremely thin, though at places they show a con- dition approximating that of a cuboidal epithelium. ‘The sub- dermal cavities, incurrent and excurrent canals, and gastral cavity are lined with a very flat epithelium, whose presence is difficult to demonstrate unless it is well preserved and cut ata favorable angle. The flagellated chambers are of course lined with a layer of rela- tively large choanocytes. In some places the dermal membrane seems to be made up of nothing but the dermal epithelium and the lining epithelium of the sub-dermal cavity and is therefore extremely thin. In most regions, however, it contains several other kinds of cells. Of these one set is represented by elongated, spindle-shaped cells, the so-called myocy tes, and these are arranged like irregular sphincters around the ostia. They also surround abundantly the sub-dermal cavities, gastral cavity, osculum, etc. Structurally they have the appearance of a primitive kind of smooth muscle-fiber. In some places in my preparations they seem to lie directly on the exposed surfaces of the canals and cavities that they bound as though they were merely elongated 8 G. H. Parker epithelial cells, as in fact they are believed to be in many other sponges ( (Minchin, ’oo, p. 46; Schneider, ’ 02, p.260). Even admit- ting, however, that in Stylotella they are in all cases covered by an epithelium, the epithelium is certainly in most instances so extremely thin that these cells are almost in contact with the sea- water passing through the cavities that they surround. In the region of the osculum the myocytes are especially numer- ous and form a conspicuous sphincter on the inner face of the oscular collar and internal to the mass of longitudinally arranged spicules which surround this opening. Asa result the contraction of the osculum is accomplished without much folding of the surface Fig. 3. Transverse section of the base of an oscular collar of Stylotella. The central cavity is the osculum, which, as is shown on the right is directly surrounded by a sphincter of myocytes, external to which is the tissue containing the spicules. X 35. of the sponge next the oscular cavity, for this surface 1s immedi- ately in contact with the contractile material, if in fact it is not contractile itself. On the other hand the outer substance of the oscular membrane and the palisade of spicules are thrown into many folds in contraction as though they passively followed the constricting ring of myocytes to the outside of which they are attached: (Pip: 2): This palisade- like arrangement of the rigid siliceous spicules is the only one that erent allow an easy con- traction and expansion of the osculum and it is in strong contrast with that of the spicules in the rest of the sponge, in which these bodies show no such grouping. Reactions of Sponges 9 Although the larger apertures, including the osculum, possess well defined sphincters by which they are closed, I have never been able to find in Stylotella systems of radiating fibers by which they might be opened. Now and then I have seen what seemed to be slight radiating systems, but they were always associated with closed or partly closed openings and might perfectly well have owed their origin to the mechanical stretching of the elastic tissue in the neighborhood of a sphincter. I am inclined to believe therefore that the myocytic sphincters in Stylotella work against the general elasticity of the body tissue, which may have a slight radial arrangement in their neighborhood, rather than that they oppose a well defined system of radial myocytes. The absence of radial fibers in many sponges in which sphincters occur has been noted by Minchin (’00, p. 46). 4. REACTIONS OF STYLOTELLA A. Movements of the Oscula The opening and closing of the oscula in Stylotella, as already mentioned is the most obvious of the responses of this sponge. lf a colony under ordinary conditions is examined, some of the oscula will almost cettainly be found closed, though the majority will be widely open. Ifa small colony 1s closely inspected under a low power of the microscope, the open oscula will be seen to emit a large number of minute particles indicating that a current is setting out through these openings. In what seem to be closed oscula a minute but otherwise similar current can often be detected showing that they are really not closed. Some oscula, however, show absolutely no current, though I have invariably found that when in such cases the oscular tip was cut off, the current almost instantly could be seen, and I believe, theref ‘re, that the oscula do close completely and thus check absolutely the current that ordinarily passes through them. In order to get some idea of the natural movements of the oscula, a vigorous colony of Stylotella was isolated and three of its oscula were kept under approximately hourly observation for three days. The results of these observations are summarized in Table I. 1O G. hae Parker TABLE I Times in hours and minutes during which in the course of three days oscula 1,2, and 3 were open or closed TIME IN HOURS AND MINUTES OF EACH SUCCESSIVE TOTAL TIMES NUMBER OF PERIOD OF OPEN OR CLOSED STATE IN 72 HOURS THE OSCULUM 7 Open Closed | Open Closed} Open | Closed| Open | Closed, Open | Closed Osculumt.........) 0.45 | 2.00] 19.05 | 3.20] 20.15 | 7.50] 2.35 | 16.10] 42.40 | 29-20 @sculuin'25..--6 MUe5O) 3220n\, 241.20) | 120) || 2.00 67.10 | 4.50 Osculum)3y.-4 ee. O.45 | (0.25 | 20.407) 2.315) 23/.50)| (OL 15) || 22010 68.45 | etl Since the three oscula whose conditions are recorded in Table 1 were on the same colony and near together and were exposed to almost identical surroundings, the fact that osculum 1 was closed on the average one hour in every two and a half, while oscula 2 and 3 were Bee only one hour in every eighteen, must be attrib- ve to the difference in constitution of osculum I as contrasted with that of the other two. The condition of general openness as exemplified by oscula 2 and 3 is doubtless typical for these organs. At least in any vigorous cake under normal conditions, the majority of the Sa willbe found open much of the ttme. When an osculum opens or closes, it does so in response to some stimulus. To ascertain what the effective stimuli are in this form of response, I have studied the oscular reaction in relation to mechanical and chemical stimulation and to heat and light. a. Mechanical Stimulation When at low tide a specimen of Stylotella was transferred from the shallow water of the outside to the laboratory tank, an opera- tion that required about ten minutes, it was found that the anima that in the outside water had most of its oscula open usually had the majority of them closed when it had arrived in the laboratory, notwithstanding the fact that it had not once been exposed to the air during this transfer. At firstit was suspected that this closure of the oscula was due to the disturbance caused in loosening the sponge from the bottom, etc., but it was found that this was not Reactions of Sponges II so, for, if a sponge after it has been dislodged, is left in the out- side seawater, its oscula remain open. Change in illumination was also suspected of being the cause of the contraction. But if a sponge in its natural situation in full sunlight is suddenly shaded to an extent not unlike the diffuse daylight of the laboratory, the oscula still remain open. The reduction in the intensity of the light, then, is not the cause of the contraction. After this the effects of currents was tried, for, so far as could be judged, the chief difference between the condition of the sponge in its usual habitat and in the laboratory, aside from recent disturbance and illumina- tion, was that in the first situation it was in moving seawater and in the second it was in the same water standing still. An aqua- rium with a free circulation of water was set up and sponges were placed in this in such situations that they caught the full effects of the current. The results were very uniform. Sponges from the exterior often arrived in the laboratory with many of their oscula closed. On putting these specimens into the aquarium under a strong current of seawater they almost invariably opened their oscula within ten minutes. The following record from my laboratory note-book will give a good idea of the character of these changes. 12:40 p.m. A sponge brought directly from the outside was placed in the aquarium with a strong circulation of seawater. Many of the oscula were closed. 12:45. Oscula began opening. 1:10. All oscula have been widely open for some time. Sea- Water current is now cut off. 1:12. Many oscula are closing. 1:14. Most oscula are closed. Seawater current is now turned on again. 1:18. Oscula have begun opening. 1:25. Most oscula are open. 1:39. Oscula remain open. This and many other similar experiments pointed to the impor- tance of currents in keeping the oscula open, but this form of experiment did not show what particular aspect of the current caused the osculum to open or to remain open. Did thesponge r2 G. A. Parker give out excretions which in quiet water gathered to suchan extent in its immediate neighborhood as to cause its oscula to close and only on the removal of these by a current of water would the oscula open, or did the current carry oxygen to the sponge or actin a purely mechanical way to induce the opening of the oscula? To test these matters the following simple apparatus was con- structed. ‘Three cylindrical glass aquaria of considerable size were placed at three levels so tee the water from the uppermost aquarium could be siphoned freely into the intermediate one from which the water overflowed into the third. Having filled the apparatus with seawater, it was possible to keep it running continuously with the same seawater by returning that which collected in the third or lowest aquarium: to the uppermost one. If now the current of seawater carried away excretions from the sponge or brought oxygen to it and these operations had anything to do with the opening of the oscula, the use of the same water over and over again ought soon to bring on a condition that would no longer cause the oscula to open. But sponges placed in the current of the middle aquarium remained with their oscula open for hours in seawater that had been used many times over. More- over the oscula closed quickly when the current was cut off and reopened soon after it was started again. I therefore believe that the mechanical stimulation of a current of water is an effective means of opening or keeping open the oscula Stylotella. ‘These first experiments were made on whole colonies of Stylo- tella and only the general condition of their oscula was recorded. I next turned to the individual sponges, the so-called fingers, to ascertain what parts of the finger must be exposed to the current to induce an opening of the osculum or the reverse. To test this question | placed a colony of Stylotella in a strong current of sea- water and, when the oscula were well opened, I bevered a glass tube over a vertical finger so that the tube protected the whole length of the finger from the laterally impinging current but was at no place in contact with the finger. “The water in this tube on examination was found to be for the most part quiet;1ts condition, however, did not interfere with the slight currents produced by the sponge itself. Although the osculum of the finger under examination was fully Reactions of Sponges 13 open when the tube was lowered over the finger, it closed in seven minutes after the tube was in position and remained so for a quar- ter of an hour. I now inserted a small tube into the upper end of the large tube and ran a gentle current of seawater down into the end of the large tube where the finger was situated. hus the sponge was again in a current of seawater and in fourteen minutes its osculum was fully open. On cutting off this current, the oscu- lum closed in six minutes. From these experiments itis quite evi- dent that when no current of seawater impinges on a finger, its osculum closes and when such currents do strike the finger the osculum opens. It was noteworthy that during the time of these experiments the oscula in the immediate neighborhood of the one tested showed no changes in reference to those observed in the individual within the tube, but they remained for the most part persistently open in the general current of seawater. The next question that naturally suggested itself was how much of a finger must be exposed to a current of seawater to induce the opening of itsosculum. To test this, I placed the glass tube over the distal half of a finger leaving the proximal half exposed to the generalcurrent. I found, however, that the current eddied up into the tube and thus impinged on a part of the sponge supposed to be protected from it. To check this I inserted a small ring of cotton- wool between the free end of the tube and the sponge. Under these conditions the osculum closed in eight minutes even though the lower half of its finger was in a strong current of seawater. This form of experiment was repeated with only the distal fourth of the finger protected from the current, and again the osculum closed in seven minutes. Thus it is only necessary to have quiet water around the outermost fourth of a finger to cause its osculum to close, and a strong current on the proximal three- fourths of the finger will not induce the osculum to open. I next reversed these experiments and attempted to ascertain how much of the distal tip of a finger must be exposed toa current to induce the opening of its osculum. In making these trials, a piece of light-weight brass-tubing was cut to such a length that when it was slipped down over a vertical finger of the sponge, it [4 G.H. Parker covered the finger all but the tip. ‘The space between the oscular tip and the tube was filled with cotton-wool and the whole allowed to stand in quiet seawater. After the osculum had been closed for about 4 quarter of an hour, a gentle current was started across the end of the tube so that it impinged on only the oscular membrane. In three minutes the osculum showed signs of opening and in eight minutes 1t was fully open. This form of experiment was many times repeated with essentially similar results. It is therefore necessary for the current to impinge on only the oscular tip of the finger in order that the osculum shall open. The closing of the osculum in quiet water and its opening in a current of water are then both very local reactions and cannot be induced from points on the finger a quarter of its length (about half a centimeter) from the osculum. If the oscula Stylotella close simply because the water in their immediate vicinity ceases to move and not in consequence of the acculumation of waste products or lack of oxygen, they probably close in the air on a falling tide because of the same mechanical conditions. If in the laboratory an inverted test-tube full of air 1s lowered over a finger whose osculum is open till the oscular mem- brane just comes in contact with the air, the osculum closes in about three minutes. [he same result can be obtained when the test- tube contains washed hydrogen in place ot air. Hence this reaction is not due to the oxygen of the air, but is very probably induced by a purely mechanical condition of quiescence into which the tip of the sponge finger passes in going from the water inte the gas. If an osculum opens to the mechanical stimulation of a current and closes in its absence, it is reasonable to suppose that it might respond to the stimulation produced by touching it with a bristle or stroking it with a fine brush, but my attempts in these directions were not eaciee Touching or stroking an oscular membrane inside or outside when the osc lean was open and ina current of seawater never resulted, as might have been expected, in a con- traction of the osculum. Similar attempts on the outside of a closed osculum in quiet seawater occasionally resulted in a partial opening of the aperture, but these occurrences were so irregular -Reactions of Sponges 15 and at such lengthy periods after the application of the stimulus that no reliance could be placed on them. So far as my observations on mechanical stimulation go and they are full only in reference to currents, itis quite clear that an osculum closes quickly (in from about three to eight minutes) in quiet seawater or air, and opens more slowly (in Sou about seven . to fourteen minutes) in a current of seawater. The fact that the oscular closure is quick and its opening relatively slow supports the view that I have already advocated from the standpoint of the structure of these parts, namely, that the sphincters are myocytic and work against the general elasticity of the surrounding tissues. Hence closure might be expected to be rapid and expansion rela- tively slow. b. Injury In making preparations of Stylotella for physiological tests it became quite apparent thattheclosing of the osculum was a common accompaniment of cutting the sponge. If a finger of Stylotella is cut off about a centimeter from the osculum, that aperture even in a current of seawater is likely to close within a short time and to remain closed for an hour or more. The oceurrence of an oscular closure is much less likely, if the finger is cut off at two centimeters from the osculum than at one centimeter. If the cutis made at half a centimeter from the osculum that opening closes very quickly and may remain so for as much as a day. If a finger instead of being cut off, 1s only cut into one on side, there is less likelihood of contraction than in the preceding cases. A finger cut into on one side at one centimeter from the osculum retained an open osculum, and the same was true when the cut was half a centimeter from the osculum. But when a cut was made three millimeters from the osculum, this aperture closed in nine minutes and remained so over a quarter of an hour. If a pin is stuck into a finger of Stylotella, its influence on the osculum depends on the distance it is from that aperture. Atone and a half centimeters no certain response was observed, but at half a centimeter the osculum closed in about ‘ten minutes and remained so several hours, though it eventually opened. This 16 G. H. Parker observation 1s in accord with what Merejkowsky 7a. Seay found to be true of Rinalda; if the oscular edge of the sponge is struck several times with a needle, the osculum quickly contracts and remains so several minutes, after wes it more slowly opens. The same is said by Merejkowsky (p. 14) to occur in Suberites. As might be inferred from the statements already made, the injuries done to one finger of Stylotella have no influence on the condition of the oscula of neighboring fingers, nor do injuries inflicted on the common flesh of the colony between fingers influ- ence the oscula of these fingers. The nature of the stimulus produced by cutting the flesh of a sponge seems to be rather mechanical than otherwise. Such an injury besides mechanically disrupting tissues does little more than liberate juices from the substance of the sponge. These juices, however, when collected and discharged artificially and with great freedom in a normal sponge with open oscula, do not cause the oscula to close. [am therefore led to believe that the closing of the osculum on injury to an adjacent part of the sponge is due to the mechanical disrupting of tissues rather than to the effects of the juices that are liberated. If, however, the stimulus from the injury is chiefly mechanical, it results in a very different form of response from that due to currents, for the latter cause an opening of the osculuin while the former induce its closure. c. Chemical Stimulation Since the oscular sphincter of Stylotella is made up of tissue that has a striking resemblance to smooth muscle, I tried the effects of a number of drugs on this sphincter to ascertain whether or not they influenced this organ as they did the smooth muscle of the higher animals. ‘The drugs used were ether, chloroform, strychnine, cocaine, and a tropin dissolved in seawater. This water was then used in the circulating apparatus already described (p. 12), so that sponges could be exposed toit in currents. I also tested in the same apparatus the effects of diluted seawater, of freshwater, and of seawater deprived of its oxygen by boiling. When a sponge whose oscula were open ina current of pure sea- water suddenly had this changed for a current of seawater con- Reactions of Sponges 17 taining a half per cent of ether, the oscula closed in from three to three and a half minutes. When in place of pure seawater, sea- water containing a half per cent chloroform was used the oscula closed in a minute and a half to two minutes. The sponges treated with ether-water reopened their oscula in about two hours; those that had been subjected to chloroform-water did not reopen in less than four hours and some never recovered. Since both ether and chloroform-water induce a closure of the oscula, even when they are applied to the sponge in the form of a current, I regard these drugs as vigorous stimulants and of the two, chloro- form is the more effective and, as in so many other cases, the more harmful. When a current of seawater containing one part of strychnine to fifteen thousand parts of seawater was substituted for a current of pure seawater, the sponges closed their oscula in from eight to twelve minutes. Thus strychnine must be regarded as a stimu- lant to contraction. Sponges whose oscula had remained open for some time in a current of seawater, were subjected to a current containing one part in a thousand of cocaine, whereupon their oscula closed in from seven to ten minutes. All such sponges reopened their oscula after having been in a current of pure seawater for about half an hour. Since the sponges closed even in a current, cocaine at the strength used must be regarded as a vigorousstimulant to closure. In a solution of one part of cocaine in ten thousand parts of sea- water, the oscula remained open as in pure seawater, but, as the following observations show, this drug was not without its effect. A particular osculum was found on several trials in pure seawater to close in from four to five minutes after the current had ceased. On subjecting this osculum for some fifteen minutes to a current of cocaine in seawater, one to ten thousand, it was found that on the cessation of the current the osculum closed in from eight to nine minutes. After an hour in a current of pure seawater the rate of four to five minutes was reéstablished. A weak solution: of cocaine, then, inhibits slightly the closure of the osculum. A cocaine solution of one part in fifty thousand of seawater 18 G.°H. Parker could not be distinguished in its action on the osculum from pure seawater. A solution of one part of atropin to one thousand parts of sea- water checked the rapidity of oscular contraction much as the stronger of the two effective solutions of cocaine did, and a solu- tion of one part of atropin in ten thousand parts of seawater could not be distinguished from pure seawater. Although the observations on the actions of these various drugs as given in the preceding paragraphs are insufhcient to admit of any detailed analysis, the results are in agreement with what is known of the action of these materials on smooth muscle. To this type of muscular tissue chloroform 1s more destructive than ether, strychnine renders it especiaily contractile, and cocaine and atropin inhibit this property somewhat (Grotzner, ’04, p. 65). This evidence, therefore, supports the view that the sphincter myocytes of sponges are in the nature of primitive smooth muscle fibers. The effects of dilute seawater and of freshwater itself on the oscular mechanism were tried in the circulating apparatus. Ifa sponge whose oscula have been open in a current of seawater for over av hour is flooded with a current of water composed of one- fourth fresh water and three-fourths sea-water, the majority of the oscula contract somewhat in twenty minutes, after which they remain partly open. Ina mixture of half freshwater and half sea- water the oscula contract but do not close completely. In three- fourths freshwater and one-fourth seawater, the oscula contract in about seven minutes but do not close. In pure freshwater, they remain expanded as though dead, but even after having been twenty-four minutes in fresh water, such sponges will revive in running seawater, though their oscular collars are seriously damaged and are regenerated only after severaldays. AsStylotella inhabits the shallow waters near the shore, it must often be sub- jected after heavy rains to the effects of diluted seawater, but, as the observations recorded above indicate, it would not be seriously damaged by these changes and would probably protect itself against them by oscular constriction. To prepare seawater free from oxygen a large volume was Reactions of Sponges 19 boiled vigorously for some time to discharge the contained gas and after this had been accomplished the water was set aside in a tightly stoppered vessel to cool. Sponges in a current of normal seawater and with open oscula were suddenly subjected to a cur- rent of seawater thus deoxygenated drawn with as little exposure to the atmosphere as possible from the storage vessel. Their oscula closed in from ten to twelve minutes. On returning them to a current of ordinary seawater, they reopened their oscula in from fifteen to twenty-five minutes. Lack of oxygen will there- fore cause the oscula to close. d. Heat and Cold The seawater in which Stylotella was found living in the neigh- borhood of the laboratory in June and July had a temperature of about 25° to28°C. Inacurrentof this water the oscula of Stylo- tella will often remain open many hours together. If the tem- perature of the current was changed to about 35° C., the oscula often constricted slightly, and the same was true at 40° C. At 45°C. the oscula in five or six minutes went into a state of flabby contraction, and if this temperature was maintained for a con- siderable time much of the sponge died. At temperatures lower than the normal, from 25° to 9° C., the oscula remained open and outward currents could be demonstrated. Thus low tempera- tures were apparently without effect on the oscula and high tem- peratures called forth a partial contraction. j e: Light Sudden changes from the most intense sunlight to the most com- plete darkness were not followed by any Shear able movement of the oscula in Stylotella, which in this respect follows the general statement made by Minchin (’00, p. 89), that adult sponges are not sensitive to light. B. Movements of the Dermal Pores or Ostia The movements of the dermal pores or ostia in Stylotella were not so easily demonstrated as those of the oscula were. “The small 20 G. H. Parker size of the ostia makes a direct determination of their condition almost impossible and consequently the presence or absence of a current of water through them was taken as an indication of their state. [he demonstration of this current has been accomplished from the earliest times (Carter, ’56, Lieberktthn, ’56, Bowerbank, 58) by the addition to the water of some such substances as car- mine, starch, or indigo, whose particles could then be followed as they were carried in the moving water. Latterly this method has been severely criticised by von Lendenfeld (89, p. 592), who claims that even these small suspended particles mechanically stimulate the sponge and cause it to close its ostia. Von J.enden- feld has used milk as an indicator and has found no objection to it. With Stylotella it is easy to demonstrate the ostial currents with carmine, ete., and so far as I could discern this material could be used without causing partial closure of these apertures. In fact I must agree with Bidder (’96, p. 32) that the carmine particles seemed to have no effect whatever on the ostia, but were swept into the interior of the sponge with great freedom for hours at a time. It must, however, be confessed that not only carmine but even milk is an unnatural substance for a sponge and as Stylotella lives in water that ordinarily contains much fine suspended material, I found it necessary only to watch this substance to gain all the information that was needed as to the direction of ostial cur- rents, their strength, ete. In testing the ostia | usually pinned a finger of sponge under the microscope in a small glass aquarium so arranged that a continu- ous current of seawater could be kept running through it, and by watching the suspended particles along the sides of sucha prepara- tion under a magnification of about ninety diameters, 1t was com- paratively easy to ascertain whether the ostial currents were runningornot. Asarule the objective of the microscope was used as an immersion lens and plunged under the surface of the sea- water. In making these observations it was, however, necessary for the time being to stop the current of seawater that was running through the small reservoir, otherwise the movement of the sus- pended particles over the surface of the sponge was so rapid that it was impossible to tell whether they entered an ostium or glided Reactions of Sponges 21 by it. When this current was shut off the osculum often closed and under such circumstances, as might have been expected, the ostial currents ceased. “To be certain that the cessation of these currents was due to the closure of the outlet, | cut off a closed oscu- lum and found that the ostial current almost immediately began again. Moreover when I ligated the cut oscular end ofa finger on which ostial currents could be easily seen these currents ceased at once and on the removal of the ligature the currents recommenced. From these observations it is quite clear that the osculum con- trols in a purely mechanical way the current within the sponge. When the osculum is open this current may run; when it is closed the current ceases even though the ostia are open and the choano- cytes continue tobeat. In view of these facts I regularly removed the oscular ends from fingers of Stylotella on which I wished to test the ostia. Although the presence of an ostial current is conclusive evi- dence of the openness of the ostia, its absence 1s not proof that the ostia are closed even supposing that the oscular end 1s cut off, forit is conceivable that the choanocytes may cease to beat, in which case the cessation of the current would be misleading as to the con- dition of the ostia. For some time | was puzzled as to a means of meeting this difficulty, but a simple method finally suggested itself and was adopted. If the oscular end of a finger of Stylotella 1s cut off atsome distance from the osculum, the cut face includes not only the gastral cavity and some of the flagellated chambers, but also the sub-dermal cavities. An examination of the currents from such a cut end will show a large, slow, central current emerg- ing from the gastral cavity and a considerable number of smaller more rapid currents entering the surrounding sub-dermal cavities. These cavities form a set of intercommunicating spaces over the whole surface of the sponge, and the currents that set into them at the cut end depend purely upon the action of the choanocytes. If, now, no inward currents can be detected at the ostia but cur- rents can still be seen to enter the sub-dermal cavities at their cut ends, it is clear that the absence of lateral currents is due to the closure of the ostia and not to the cessation of the choanacytes. In this way, then, [ used the presence of ostial currents to indicat 22 G. H. Parker that the ostia were open and their absence, when coupled with the presence of sub-dermal currents, to indicate that these apertures were closed. Mostof the tests that were carried out on the oscula were repeated on the ostia and the results will be stated briefly in the following paragraphs. a. Mechanical Stimulation A finger of Stylotella from which the oscular end has been cut off may be kepta long time in running seawater in apparently normal condition. Wed the current of seawater is temporarily shut off, small suspended particles can be seen to drift slowly up to the sur- face of the finger and disappear by suddenly darting into the ostia. In this way she ostia can be demonstrated tobe open. Many par- ticles are too large to enter these apertures and they will accumu- late on the surface of the animal in quiet water. When, however, the general current is set going again, it sweeps these larger par- ticles away and leaves the surface of the sponge relatively clean. f after the ostia have been demonstrated to remain open for some time in running seawater, this current is permanen tly shut off so as to leave the sponge in quiet water, the ostia may con tinue open for many hours during which the surface of the sponge often becomes deeply buried under an accumulation of particles most of which are too large to enter the ostia._ Inno case has a cessation of the seawater current been followed by a closure of the ostia as with the oscula. ‘The ostia, then, differ from the oscula in that they remain open in both quiet and circulating seawater. Prepared fingers of Stylotella in which strong sub-dermal and oscular currents could be seen but no ostial currents were persent, were kept in some instances in running seawater, in others in quiet seawater without, however, Pelnines any eqidenee to show that either state of the seawater caused the opening of the ostia. Flow- ing seawater and quiet seawater both seem to have no effect on the opening or closing of the ostia in Stylotella. Even when Se loci is covered with a deep layer of silt, its ostia can often be demonstrated to be open. Under ordinary cir- cumstances, however, it 1s not usual to find this sponge thus Reactions of Sponges 23 covered. Its natural habitat is in a current of seawater and though this water may often be heavily loaded with suspended matter, its current seems to be sufficient, as already noted, to remove many particles which, from their size, accumulate on the surface of the sponge. But this is not the only means for the removal of these larger particles. A close inspection of the outer surface of Stylotella will show that it is regularly inhabited by several animals; chief among these are young ophiurans, caprellas, and a species of copepod. All these a nals: and especially the copepods, keep up an incessant movement over the surface and loosen and dislodge much of the accumulated drift. The cope- pods and probably the other forms find much to feed on in this omnium-gatherum, and their relation to the Stylotella seems to be of a symbiotic character. “These organisms together with sea cur- rents are responsible for the generally clear character of the sur- face of the sponge. But even when this sediment is abundantly present on Stylotella its ostia remain open. I have also failed to find any evidence in favor of the view that when the ostia are closed the accumulation of silt on the surface of the sponge will cause them to open. I’xposure to air likewise seems to have no effect on the ostia. A finger of Stylotella on which the ostia were freely open was exposed to air for about a quarter of an hour. Upon reimmersing it In seawater the ostial current could be seen at once. I[t was again put in the air, this time for three-quarters of an hour, where- upon it was reimmersed and the ostia again gave evidence of being freely open though the finger as a whole had the shriveled appear- ance characteristic of sponges that have been exposed sometime to the air. Stroking the surface of Stylotella seemsneither to bring about a closure nor an opening of the ostia. In this respect they are as irresponsive as the oscula. So far as mechanical stimulation is concerned, the ostia are very unlike the oscula. . The oscula are responsive to water currents and their absence and the mechanical effects of exposure to the air; the ostia are uninfluenced by any of these changes and are apparently also undisturbed by sediment. 24 G.H. Parker b. Injury The great majority of fingers cut from Stylotella, if put directly under the microscope, show no ostial currents. As a rule these currents begin to appear in from ten to fifteen minutes after the finger has been cut off. When a finger that has established its ostial currents is cut in two, these currents often cease in the two parts though sub-dermal and oscular currents can be easily demon- strated. Aftera quarter of an hour the ostial currents can usually be seen again in such pieces. Not all specimens show these con- ditions, but they are of common enough occurrence to justify the conclusion that a considerable incision in Stylotella produces in its neighborhood a temporary closure of the ostia. In this respect the ostia resemble the oscula. c. Chemical Stimulation To seawater containing ether (4 per cent) or chloroform (4 per cent) the ostia closed even more quickly than the oscula did, and on fingers whose ostia were closed, the presence of ether or chloro- form in the surrounding water did not induce these apertures to open. Strychnine, one part in fifteen thousand of seawater, was followed by a gradual closing of the ostia, a condition already observed by von Lendenfeld (’89, p. 608) in other sponges. To one part of cocaine in a thousand parts of seawater open ostia closed in about ten minutes and closed ostia remained closed. ‘To one part of cocaine in ten-thousand parts of seawater the ostia remained open or if closed in the beginning, they oper in about eight minutes. Of this drug von Lendenfeld (809, p. 640) states that the stronger solutions cause a contraction of the ostia, which is true, and that the weaker solutions leave these apertures un- changed, which is probably not wholly correct, for they inhibit to some extent the contractibility of the sphincters. To atropin, one part ina thousand of seawater, the open ostia remained open and the closed ones opened in about nine minutes. ‘Thus atropin probably also inhibits the action of the sphincter. As the actions of these various drugs on the ostial sphincters is very similar to their action on smooth muscle, it 1s probable that the ostial myo- Reactions of Sponges 25 cytes, like those of the osculum, are cells not inappropriately described as primitive smooth muscle-fibers. In water composed of three-quarters seawater to one-quarter freshwater, the ostia remained open, and a strong ostial current could be seen at the end of twenty minutes. When the ostia were closed the effect of this mixture was to induce an opening of the ostia in about a quarter of an hour, after which a strong ostial current continued to flow. ‘To mixtures of half seawater and half freshwater oscular, and sub-dermal currents as well as ostial currents ceased in about ten to twelve minutes, showing that though the ostia probably remained open, the currents ceased esas of the collapse of the choanocytes. Closed ostia in this mixture opened slightly and then all currents ceased in from nine to ten minutes. In fresh water all currents ceased immediately. “These mixtures of seawater and freshwater so far as their effects can be seen, influenced the ostia much as they did the oscula, in that they induce a partial but imperfect contrac- tion. In seawater rendered free from oxygen by boiling and subse- quently cooled to 28° C., the ostia remained open or, if closed, hey opened in from seven to ten minutes. This reaction was precisely the reverse of that of the osculum and to make close com- parisons of the two I prepared several fingers of Stylotella by cutting them off rather short at the proximal ends thus perman- ently opening the gastral cavity and by leaving the oscular end intact. ‘These preparations were placed one after another under the microscope in pure running seawater and after the oscula were freely open the current of pure seawater was changed for one of deoxygenated seawater. Under these conditions ail the oscula closed in about ten minutes, if not completely at least nearly so, and the ostia remained open, their currents now dis- charging chiefly through the cut end of the gastral cavity. Deoxy- genated water, then, is a means of closing the oscula and opening or leaving open the ostia. To seawater containing juice expressed from an oyster, either fresh or foul, the open ostia remained open and their currents seemed at times to increase. I was never able to demonstrate with certainty that to these materials the closed ostia would 260 G. H. Parker open, though occasionally they seemed to. Like the oscula, the ostia were indifferent to seawater containing juice from the body of Stylotella itself. d. Heat and Cold P reps ared fingers of Stylotella in which the ostial currents were running vigorously continued to exhibit these currents after the ae ture of the seawater had been changed from 28° C. to 6° C., and fingers in which the ostia were dnsed at 28° C. opened ies after thet sponge had been a few minutes in water at 35° C. At 40° C. all currents, sub-dermal and oscular as well as ostial, became rapidly feeble and then stopped, and at 45° C. these cur- rents ceased abruptly as though the heat had caused the choano- cytes to stop beating. ‘This view is supported by the fact that few fingers of Selorella ever recovered after having been sub jected to seawater at 45° C. for any length of time. Cold water at 9.5° C. caused all currents to run more slowly, but did not bring about a closure of the ostia. In fingers in which the ostia were closed these organs did not open after having been a quarter of an hour in seawater at 9° C. In these specimens the sub- dermal and oscular currents became sluggish on reducing the temperature of the water, hence the effect of the low tempera- ture was probably chiefly on the choanocytes. e Light As in the case of the osculum, I have observed no effect from intense sunlight or shadow on the opening or closing of the ostia. C. Movememts of the Body as a Whole Aristotle in the fourteenth chapter of his fifth book on the his- tory of animals makes the interesting statement that the sponge is supposed to possess sensation because it contracts if it per- celves any movement to tear it up and it does the same when the winds and waves are so violent that they might loosen it from its attachment. He further adds in his charaeterisae way that the natives of Torona dispute this. The idea that the common Reactions of Sponges 2 | flesh of the sponge is contractile is not without modern support. Merejkowsky (78, p. 14) states that if Suberites is so placed that it is partly out of water, it will curve the body until it is under water as much as possible, and if the body is then covered with water, it will return to its former position. It must be evident, from what has already been stated, that Beh ebithacommon flesh of Stylotella is con tractile. As already noted, specimens our of water quickly assume a shriveled and rugose appearance as though the flesh had contracted on a resist- ant skeleton, a condition which it also quickly assumes in quiet seawater. Moreover, if a sponge is placed partly in running seawater and partly in the air, the portion in the seawater remains smooth and that in the air becomes rugose. Specimens made rugose either in the air or in quiet water soon recover their smooth appearance on being placed in running water. Air or quiet water may then cause a contraction of the common flesh of Stylotella, a condition counteracted by running water. The contraction of the common flesh can also be seen well around some of the larger cavities, such as the gastral cavity. If a long finger of Stylotella whose two ends have been cut off and whose gastral cavity extends along one of its sides is placed in quiet seawater, the gastral cavity is soon indicated by an external groove due to the apparent collapse of its wall. “This groove, how- ever, 1s caused not by collapse, but by the contraction of the com- mon flesh which as partial partitions or even travecula is abundant about the sides of the gastral cavity. On returning the finger to running water the flesh relaxes and the groove mostly disappears. Although the common flesh of Stylotella is unquestionably con- tractile, I have never observed that the body of this sponge as a whole moves in consequence of this contractility. Thus in no instance have I seen a partly immersed finger of Stylotella bend farther into the water, though I have let fingers stand in a posi- tion favorable for this for over a day. Nor have I ever observed fingers to turn in conformity to the direction of the current. Thus some fingers of Stylotella are not directed straight upward, but have their tips turned to one side or the other, so that the oscula open laterally. A number of these were set, some with oscula fac- 28 G. H. Parker ing the current, some with these openings away from the current, and others sidewise to the current. After three days none of these had materially changed their directions, thus giving no evi- dence of a general movement of the body. I also attempted to get evidence of the general movement of the body through geotropic responses. Stylotella ordinarily grows with its fingers and oscula directed upwards, as though it was negatively geotropic. A large colony was, therefore, kept in- verted in an aquarium of circulating seawater for about a week on the assumption that the fingers might turn from this unusual position, but at the end of this period there was no apparent change of position. This observation, however, does not prove that Stylotella i is not geotropic. Slight evidence of geotropism is to be found in its mentad of regenerating oscula. Whena moderately long finger of Stylotella is cut off and the whole of its oscular end, removed, the cylindrical body thus resulting will under favorable conditions form a new osculum. Whether tnis regeneration will take place at the end nearer or farther from the former osculum seems to depend chiefly on the position of the piece of sponge in reference to gravity. If the end that was nearer the former osculum is uppermost, it alwaysregenerates the new osculum; if it is down, the opposite end very generally regenerates the new organ. ‘Thus in the regeneration of the osculum Stylotella shows some slight geotropic activity, and while it must be admitted that the common flesh of this sponge 1s con- tractile, this contractility does not seem to result in movements of the body as a whole such as might be looked for in geotropic and other like responses. It is possible that in this sponge the skeleton, which is well developed, is too resistant to allow the body as a whole to be bent, and that, therefore, the contractility of the common flesh can make itself manifest only in the local ways already mentioned. D. Currents The currents of sponges, which were supposed by many older naturalists to reverse in their direction from time to time and to depend upona systole and diastole of the body of the sponge, have Reactions of S ponges 29 been generally acknowledged since the time of Grant (’25, 120. ’27) to be uniform in their direction and to depend upon the action of cilia-like organs. Some years ago Miklucho-Maclay (’68) and Haeckel (72) maintained that a reversal of the current could occur, Dut more recent observers have not confirmed this state- ment. In the thousands of living individuals of Stylotella that | have examined I have never seen an exception to the rule that water enters the ostia and sub-dermal cavities, when open, and makes its exit through the osculum. Moreover I have never found a living specimen of Stylotella in which currents could not be demonstrated. Even in those in which the ostia and oscula were closed and no external evidence of currents could be seen, the cutting off of the oscular end and the consequent exposure of the gastral and sub-dermal cavities always was followed by the appearance of characteristic currents. It seems to me probable that under nurmal conditions the choanocytes beat incessantly in Stylotella. The currents produced by them would then be controlled by the opening and closing of the ostia and the oscula. It must be borne in mind, hee cus that a continuous current does not necessarily mean that all flagellated chambers are con- tinuously at work. Some may cease from time to time without causing the general current to cease. All that the presence of a continuous current really proves is that all flagella ted chambers are not inactive atonce. It would not be surprising to me, how- ever, to find, if evidence could be obtained, that the action ofthe choanocytes is uninterrupted. The fact that a current could always be demonstrated in all fingers of Stylotella by cutting off the oscular ends leads to the conclusion that, aside from the ostia and the oscula, there is no other complete check on the current such as prosopylic or apopylic sphincters, etc. If the ostial and oscular sphincters are the organs of control for the currents in a sponge, they must be strong enough to resist the pressure produced by these currents, and when these apertures are closed the tissues of the sponge must also withstand a certain strain produced by the working of the choanocytes. Doubt has been expressed by some writers as to the ability of the body of a sponge to meet these mechanical requirements, but as no one, so 30 G. H. Parker far as I am aware, has ever attempted to measure the pressures involved, it seems useless to urge such objections till actual meas- urement has been accomplished. It is comparatively easy to determine the pressure produced by the activity of the choan- ocytes of such a sponge as Stylotella. “[o make this measure- ment the following simple device was employed. Currents cease Opens and remains open.) Currents strong, then cease | No reaction. , ' Currents become slow (Normal aeeee ea eeenee- |) Normal | Remains open or if closed) OPE engeneneddostocdl NG ? Currents cease ? Currents cease » INO'reactionS. 95.02.2540: No reaction 34 G) Hs Parker This organic independence in Stylotella also appears in the almost complete absence of transmission from part to part. “The opening and closing of an osculum on one finger in accordance with the condition of the surrounding water has no influence on adjacent oscula even though they be only a centimeter or so dis- tant. Extensive wounds, which can be made with much local precision, influence oscula or ostia only within a very close range. Transmission at best cannot be over a much greater distance than a centimeter or so. Nor is the nature of this transmission at all nerve-like. A cut made about 3 mm. from an osculum was fol- lowed by the closure of the osculum only after eleven minutes, though this osculum had previously closed in quiet water in from four to five minutes. “The form of reaction resembles that seen in the vertebrate iris, in which in response to a point of light the iris contracts locally, the contraction gradually spreading through the whole organ ( Hertel, 07). In the sponge, as in the iris, we are probably dealing with the direct stimulation of smooth muscle, which when locally contracted stimulates by its contraction the adjoining resting muscle and thus a slow form of transmission is accomplished through the muscle substance itself. These studies of the reactions of Stylotella support the conclu- sion arrived at from earlier anatomical investigations to the effect that sponges possess nothing that may with propriety be called nervous tissue. Their reactions, which have the general charac- ter of great simplicity and independence, are, I believe, entirely due to the direct stimulation of choanocytes or myocytes which are either on exposed surfaces or close to them and which, at least in the case of myocytes, exhibit a form of progressive stimulation that resembles sluggish transmission. Sponges are metazoans possessing muscular but not nervous tissue. §. ORIGIN OF THE NERVOUS SYSTEM In seeking evidence on the origin of the nervous system, investi- gators have naturally turned to primitive metazoans, and the coelenterates have afforded the principal material for speculation on this subject. ‘These speculations took their origin in the dis- Reactions of Sponges 35 covery by Kleinenberg (’72) of the so-called neuromuscular cells in hydra. These cells, which have since been found in great abundance in many other ccelenterates, were believed by Kleinen- berg to contain the germ of the nervous and muscular systems of the higher metazoans. According to him the elongated basal process of the neuromuscular cell was the contractile or muscular element, and the cell-body that reached from the exterior to the muscular part was the receptive and transmitting or nervous part. In his opinion this cell became divided into two, one cell to become purely muscular, the other purely nervous, and these two cells, thus derived directly from the primitive neuromuscular cell, were supposed to be the forerunners of the muscular and nervous systems of the higher animals. Kleinenberg thus conceived mus- cular and nervous organs to have had a common origin and to have undergone a simultaneous differentiation. The neuro- enlace cell theory was favored by Van Beneden (’74), who claimed that in Hydractinia the intermediate condition between a neuromuscular cell and its two derivatives was to be seen, but Bergh (78) showed this claim to be based on inaccurate obser- vation. . The study of the nervous system and sense organs of marine coelenterates led Oscar and Richard Hertwig (’ 78) to the con- clusion that the so-called neuromuscular cells were not nervous but merely muscular, and they proposed for these elements the name epithelial muscle-cells. They also pointed out that the nervous system of the ceelenterates consisted of sense-cells and ganglion-cells and they believed that these two kinds of cells to- gether with the epithelial muscle-cells were simultaneously differ- entiated from among the elements of the corlenterate epithelium. Thus they did not trace the origin of nervous and muscular tis- sue to a single cell but to a layer of cells from which the three types just named were supposed to arise by simultaneous differ- entiation. his view, though slightly modified by such workers as Havet (’o1), who declared that what the Hertwigs called gang- lion-cells were more strictly speaking motor-cells, has been more or less tacitly accepted by most modern students of the neuromus- cular mechanism of celenterates (Schaeppi, ’04; Wolff, ’o4; Hadzi, ’0g; Groselj, ’o9g). 36 G. H. Parker The views of Kleinenberg and the Hertwigs, as this brief survey shows both contain the common element of simultaneous and inter-related differentiation of nervous and muscular elements. As contrasted with this aspect of the question Claus (’78) and Chun (’80) claimed an independent origin for these two types of tissue and that their connection was secondary. ‘The ground for this opinion, at least as maintained by Chun, ts chiefly the condi- tion found in vertebrates where in ontogeny it 1s very probable that nerve and muscle are independently differentiated and secondarily united. ‘Uhus this opinion gets 1ts support from highly specialized rather than from primitive metazoans. The view as to the origin of the nervous system, or better of the neuromuscular mechanism, to which the study of the activities of sponges has led me, is in strong contrast with the opinions that have already been expressed. The fact that sponges have an organized musculature, though they show no evidence of nervous organs, leads me to the conclusion that nerve and muscle have not differentiated simultaneously, but that muscular tissue has pre- ceded nervous tissue in order of evolution. The condition in sponges is absolutely contrary to the statement of Kleinenberg (72, p. 23) that there are no animals with muscles and without nerves, nor is 1t consistent with the view of the Hertwigs (’78, p. 165) and their followers that these two kinds of tissue differentiate simultaneously. Muscular tissue unassociated with anything that can reasonably be called nervous tissue certainly occurs in these primitive metazoans, and muscle of this kind directly stimu- lated, 1. e., without the necessary intervention of other cells, is in my opinion the initial stage in the growth of the neuromuscular mechanism. The next step in ie process is, I believe, that realized in most coelenterates, 1. e., a muscular mechanism to which has been added certain receptive cells, sense cells, that serve as delicate organs for bringing the muscles into action. This step is the first step in the differentiation of true nervous tissue, though it is the second in the growth of the neuromuscular mech- anism as a whole. At this point my view is in strong contrast with that of Claus and Chun who, as already stated, have main- tained that nerve and muscle arose independently. This I do Reactions of Sponges 37 not believe possible, for | agree entirely with Samassa (’92) when he declares that a nervous mechanism without muscles or other effectors is inconceivable. In my opinion nervous tissue has differentiated not independently of muscle, as claimed by Claus and by Chun, but in most intimate relations with it and as a more effective means of bringing it into action than direct stim- ulation is. Primitive muscles, then, as independent effectors, were centers around which the beginnings of nervous differentia- tion probably occurred, in that certain peripheral cells came to be specialized as receptors for stimuli and excitors of muscular activ- ity, a condition now realized in ccelenterates. From this standpoint it must be clear that the histogenesis of primitive nervous tissue involves cells that are in contact with the exterior on the one hand and with muscular tissue on the other. The conditions realize almost perfectly the requirements of the well-known theory of neurogenesis advocated by Hensen (’64), and I, therefore, believe that this theory is a truthful portrayal of primitive neurogenesis. I do not admit, however, that it pre- sents a correct picture of the histogenesis of vertebrate nerves. In this problem the evidence seems to me to be strongly in favor of the initial separateness of nerve and muscle amd their secondary union, an operation which in my opinion is a coenogenetic modi- fication of the primitive process. But whether the axis-cylinders of vertebrate nerve-fibers are outgrowth of neuroblasts or not, 1s a question that has no direct bearing on the one herein discussed, the differentiation of the primitive nervous system. Such a primi- tive nervous system, essentially receptive in character, 1s, how- ever, merely the beginning of that structure which in the higher metazoans is designated as nervous. This primitive nervous system is not in any appropriate sense to be called centralized. Its diffuse character, from an anatomical as well as from a physio- logical standpoint, is well known, and only after the nervous structures have become concentrated either in the periphera lepithelium, as in some worms, or on separation from this epithe- lium, as inthe higher metazoans, is a condition arrived at which necessitates the formation of true nerves, and allows the establish- ment of common paths (Sherrington, ’06), a condition which 38 G. H. Parker may be appropriately called centralized. When this concentra- tion takes place it usually occurs near the chief group of sense organs and gives rise to what is conventionally called the brain. In nervous differentiation, then, the chief central organ or brain follows, in its early evolution, the lines of sensory differentiation. The differentiation of the complete neuromuscular mechanism as possessed by the higher animals, has occurred, I believe, in three successive steps; first, the formation of independent effectors, tor as seen in the muscles of sponges; secondly, the addition of receptors to such effectors, as seen in what I have elsewhere called the receptor-effector systems of the coelenterates; and finally, the differentiation near the receptors of adjusters or central organs con- cerned primarily with easy transmission from receptors to effectors (Parker ’o0g). 6. SUMMARY 1 Stylotella under natural conditions closes its oscula and contracts its flesh when at low tide it is exposed to the air. 2 Its outer surface is perforated by many ostia which lead _ to large subdermal cavities, these in turn connect through incurrent canals with the flagellated chambers from which excurrent canals pass to the gastral cavity and the osculum. 3. The flesh of Stylotella contains many myocytes, which are arranged as sphincters around the ostia, internal cavities, and osculum. ‘These sphincters apparently work against the general elasticity of the flesh and not against radiating systems of myo- cytes. 4 The oscula close in quiet seawater, on exposure to air, on injury to neighboring parts, in solutions of ether (0.5 per cent), chloroform (0.5 per cent), strychnine (zst0s), cocaine (qy00), and in deoxygenated seawater. “They contract but do not close in diluted seawater and at temperatures higher than normal (35° to 45° C.). They remain open in currents of seawater, and their closure is ey by solutions of cocaine (yot00) and of atropine (x00), and in fresh water. They are apparently uninfluenced by low EMperaCuncs; by weak solutions of cocaine (s9h00) and of atropine (yo)00) and by light. Reactions of Sponges 39 5 The ostia close on injury to neighboring parts, in solutions of ether (0.5 per cent). chloroform (0.5 per cent), strychnine (xs407), and cocaine (yess). They open in solutions of cocaine (zoho). and of atropine (zoos), in dilute seawater, deoxygenated seawater, and warm seawater (35° C3: They are apparently unaffected by mechanical stimulation, except injury, by low tem- perature, and by light. 6 The choanocyte currents cease in solutions of ether (0.5 per cent), and of chloroform (0.5 per cent), in diluted seawater and at high temperatures (40°—45° C.). They become slow at low temperatures (9° — 10° C.), and fast in solutions of strychnine (¢sbov). In deoxygenated water they first become fast and then cease. 7 The flesh of Stylotella is capable of contraction, but such contractions give the sponge only a shrivelled appearance with- out changing its general form. 8 The currents in Stylotella are constant in direction and give no evidence of reversal. They are controlled by the ostial and oscular sphincters. They produce a pressure equivalent to 3.5 to 4 millimeters of water. The pressure necessary to break through the closed ostia is 10 to 15 millimeters of water and through the closed oscula somewhat more. g The reactive organs of Stylotella, the ostia, the oscula, the flesh, and the choanocytes, are all more or less independent of one another and their action is changed by direct stimulation. In the ostia, oscula, and flesh contraction 1s accomplished by spindle-shaped cells, the myocytes, which resemble primitive, smooth muscle-fbers. 10 The body of Sty lotella is almost without transmission and such transmission as Is present is so. sluggish 1 in character and so silght in range as to resemble transmission in muscles and not in nerves. It is probable that Stylotella possesses no organs that can reasonably be called nervous. 11 The nervous and muscular systems of metazoans were not differentiated simultaneously (Kleinenberg, O. and R. Hertwig) nor independently (Claus, Chun), but muscles, independent effec- tors, as represented by the anne of sponges, were the first of the neuromuscular organs to appear and these formed centers 40 G. lake Parker around which the first truly nervous organs, receptors, in the form of sense-cells developed giving rise to a condition such as 1s seen in the coelenterates today. ‘To this receptor-effector sys- tem as seen in modern ccelenterates was added in the higher meta- zoans the adjuster or central organ, thus completing the essen- tial parts of the neuromuscular mechanism as seen in the higher metazoans. 7. BIBLIOGRAPHY. Bercu, R. S. °78—Nogle Bidrag til de athecate Hydroiders Histologi. Videns- kabelige Meddelelser fra den naturhistoriske Forening in Kjgben- havn, 1877-78, 29 pp., 1 Taf. Bipper, G. ’96—The Collar-cells of Heterocoela. Quart. Jour. Micr. Sci., new ser., vol. 38, pp. 9-43., pl. 2. BowErBANK, J. S. °58—Further Report on the Vitality of the Spongiade. Rep. 27 Meet. Brit. Assoc. Adv. Sci., pp. 121-125, pl. 1. Carter, H. J. ’56—Notes on the Freshwater Infusoria of the Island of Bombay. No. 1. Organization. Ann. Mag. Nat. Hist., ser, 2, vol. 18, pp. 115-132, 221-242. Cuun, C. ’80—Die Ctenophoren des Golfes von Neapel. Fauna und Flora des Golfes von Neapel, Monogr. 1, xvitt + 313 pp., 18 Taf. Craus, C. ’78—Studien tber Polypen und Quallen der Adria. Denkschr. Akad. Wissensch., Wien, Bd. 38, pp. 1-64, Taf. 1-11. Grant, R. E. ’25—Observations and Experiments on the Structure and Func- tions of the Sponge. Edinburgh Philos. Jour., vol. 13, pp. 94-107, 333-346. °26—Observations and Experiments on the Structure and Functions of the Sponge. Edinburgh Philos. Jour., vol. 14, pp. 113-124, 336-341. °27—Observations on the Structure and Functions of the Sponge. Edin- burgh New Philos. Jour., vol. 2, pp. 121-141, pl. 2. GroseELj, P. ’og—Untersuchungen tiber das Nervensystem der Aktinien. Arbeit. zool. Inst. Wien, Tom. 17, pp. 269-308, Taf. 1. Grirzner. P, ’04—Die glatten Miiskeln. Ergebnisse der Physiol., Jahrg. 3, Abt. 2, pp. 12-88. Hanzi, J. ’og—Ueber das Nervensystem von Hydra. Arbeit. zool. Inst. Wien, Mom. 17, pp:225-226, Late1—2: HarckEL, FE. ’72—Die Kalkschwamme. Bd. 1. Berlin, 8vo, xvi + 484 pp. Havet, J. ’o1—Contribution a létude du Systéme nerveux des Actinies. La Cellule, tome 18, pp. 385-419, pl. 1-6. Hensen, V. ’64—Zur Entwickelung des Nervensystem. Arch. pathol. Anat., Physiol., klin. Med., Bd. 30, pp. 176-186, Taf. 8. Reactions of Sponges 41 Herte, E. ’07—Experimenteller Beitrag zur Kenntnis der Pupillenverengerung auf Lichtreize. Graefe’s Arch. Ophthalmol., Bd. 65, pp. 107-134. Hertwic, O., unp R. Hertwic. ’78—Das Nervensystem, und die Sinnesorgane der Medusen. Leipzig, 4to, x + 186 pp., 10 Taf. KLEINENBERG, N. ’72—Nydra. Eine anatomisch-entwicklungsgeschichtliche Untersuchung. Leipzig, 4to, vi + 90 pp., 4 Taf. LENDENFELD, R. y. ’89—Experimentelle Untersuchungen tiber die Physiologie der Spongien. Zeitschr. f. wiss. Zool., Bd. 48, pp. 406-700, Taf. 26-40. LieperkUmn, N. ’56—Zusatze zur Entwickelungsgeschichte der Spongillen. Arch. Taf. 18, Fig. 8-0. MeErREJkKowsky, C. 7 tudes sur les Eponpes de la Mer Blanche. Mém. Acad. Imp. Sci., St. Pérersbourg sér. 7, tome 26, no. 7, 51 pp., 3 pl. Mrxiucuo—Mac ay, N. ’68—Beitrage zur Kenntniss der Spongien, I. Jena. Zeitschr. Ba. 45 pp: 221-240, Vat. 4-5: Mincuin, E. A. ’oo—Sponges. A Treatise on Zoology, edited by E. Ray Lan- kester, part 2, 178 pp. Parker, G. H. ’og—The Origin of the Nervous System and its Appropriation of Effectors. Popular Sci. Monthly, vol. 75, pp. 56-64 137-146, 253- 263, 338-345. Samassa, P. ’92—Zur Histologie der Ctenophoren. Arch. f. mikr. Anat., Bd. 40. Ppa £57-2435 Lat. 8-12. Scuaeppl, T ’o4—Ueber den Zusammenhang von Muskel und Nerv bei den Siphonophoren. Mitth. Naturwiss. Ges. Winterthur, Jahrg, 1903-04, pp- 140-167. SCHNEIDER, K. C. ’02—Lehrbuch der vergleichenden Histologie der Tiere. Jena, 8 vo, xiv + 988 pp. SHERRINGTON, C. S. ’06—The Integrative Action of the Nervous System. New York, 8 vo, cvi + 411 pp. Van BENEDEN, E. ’74—De la distinction originelle du testicule et de l’ovaire; charactere sexual des deux feuillets primordiaux de |’embryon; hermaphrodisme morphologique de toute individualite animal; essai d’une théorie de laf€condation. Bull. Acad. Roy. Sci. Lett., Beaux- Arts, Belgique, sir. 2, tome 37, pp. 530-595, pl. 1-2. VosmaER, G.C. J. anp C. A. PEKELHARING ’98—Observations on Sponges. Verhandl. Kon. Akad. Wetensch. Amsterdam, sect. 2, deel. 6, no. 3, 51 pp-» 4 pl. Wotrr, M. ’o4—Das Nervensystem der polyoiden Hydrozoa und Scyphozoa. Zeitschr. allg. Physiol., Bd. 3, pp. 191-281, Taf. 5-9 . a . i ‘ i i i j | ve 1 fi i i " i Hy Sine eect t-te oP -pey esses eteys sia ees xeiceel. Q Fig. 2. Llustrating the ‘‘push ball” method of getting rid of objectionable particles. The arrows show the direction of beat of the discal cilia. c, mass of carmine grains; f, funnel: m, membranelle; mo., mouth; p, pouch. masses which are, after some time, either by a special reaction or by accident—I didn’t determine which—dropped over the edge of the disk, opposite the pouch. ‘The carmine masses move slowly and it can be readily seen that the motion is altogether due to the action of the discal cilia. This method of preventing the ingestion Selection of Food in Stentor Ceruleus (Ehr.) 87 of indigestible particles is very effective. Very few particlesever get into the pouch so long as this mode of ciliary action takes place. This modification of behavior appears gradually and also disap- pears gradually; and during the transitions the action is imperfect, some particles passing into the pouch while others are carried to the right before dropping into the pouch. This phenomenon is interesting in that the discal cilia, which act usually ina certain definite codrdinated way, are able to change their behavior so as to act in an entirely different but still coordinated way. What is more remarkable still perhaps is that some of the cilia beat in the same direction in both cases, while some others beat 1n an exactly opposite direction, the rest of them beating in every conceivable direction between their usual direction of beat and its direct oppo- site. [his is a very good example of the extreme plasticity of behavior of such an organism as Stentor. Under the same conditions in which the foregoing change of behavior was observed there was found another method by means of which the Stentor made its ingesting apparatus ineffective. This was done by contracting and staying contracted. Contrac- tions for more than several minutes have not heretofore been recorded, but in one set of Stentors I observed continuous contrac- tion for more than two and three-quarter hours. ‘There was not a single relaxation during all this time and it is possible that this state of continuous contraction lasted longer than two and three- quarter hours, for the observation was notcontinued until relaxa- tion occurred. The body cilia remained constantly reversed in this case, but the membranelle frequently alternated between the usual and the reversed beat. Both groups of these cilia beat less vigorously than when relaxed or free swimming. The pouch and funnel were closed and no particles whatever were ingested. Stull other Stentors under similar circumstances differed i behavior from all the above. Under stimulation of dense sina: of carmine some Stentors swam with the foot ahead continuously for over three hours. ‘The membranella were sometimes beat in the ordinary and sometimes in the reversed way, but always with a less vigorous beat than when the Stentor is fully extended attached, and in water with few particles present. There was no 88 Asa Arthur Schaeffer spiral turning or revolving on the long axis. ‘The Stentors swam ina circle as one would expect from a consideration of the cres- centic shape of the partly extended Stentor. ‘This method of behavior also resulted in the ingestion of but very few particles. These are probably the eines constituents of what may be called the normal behavior of Stentor. We shall next consider the ex- periments which were designed to answer the question: Can Stentor discriminate between food and indigestible particles? EXPERIMENTS WITH SPECIFIC PARTICLES We have just seen that the normal behavior of Stentor is very complex for an organism of such simple anatomy, especially as far as the movements and action of the cilia are concerned. We saw that there are at least four distinct groups of cilia—five, if the pouch and funnel cilia are considered as two groups—each of which has a more or less definite thing to do under ordinary circum- stances; but the moment that conditions obtain which are unusual, the behavior of one or more of these systems of cilia changes. We saw that any one of these four or five groups of cilia can change its behavior while the rest of the ciliary apparatus beat in the usual manner; or all the cilia of the entire Stentor can change direc- tion and force of beat upon occasion so that entirely changed behavior results. In fact with these four or five groups of cilia which may beat independently of each other and vary their be- havior in different ways, 1t seems hardly possible that a situa- tion could confront a Stentor which could not satisfactorily be met by having recourse to the many possibilities of the varying behavior of these ciliary systems. The next problem then was to devise an experiment that should test the efficiency of this highly adaptive ciliary apparatus in discriminating between food and indigestible particles. Experiment 1. Discrimination between Phacus and Sulphur For this purpose roll sulphur was ground up into a fine powder and then thoroughly stirred with a large quantity of water. After Selection of Food in Stentor Ceruleus (Ehr.) 89 the coarser particles had settled down the finer particles were siphoned off to be used in the experiment. The water in which the sulphur was stirred up was filtered water from the Stentor cul- ture, so that the results of the experiment cannot be attributed to any peculiar qualities of the water. Some of the particles of sul- phur were sucked up into a pipette together with some living Phacus triqueter, as previously described. ‘This mixture of Pha- cus and sulphur was then fed on to the disk of a normally behav- ing Stentor in the fully extended condition, and the pathand fate of each particle recorded. ‘The results follow: The particles are numbered in the order in which they reached the disk of the Stentor. The sulphur particles are denominated ‘‘s,” the Phacus “‘p.” Thus, 7s in the ‘‘rejected” column signifies that the seventh particle was sulphur, and that it was rejected. Where several numbers are bracketed, it signifies that these particles were fed simultaneously. In the column headed ‘‘loops” is shown the number of loops made by the particle. (See p. 11). The column headed ‘‘size” gives the size of the sulphur particles in units of the size of Phacus. Thus I, means same size as Phacus, .5 means one-half that size. TABLE I Experiment 1. Discrimination between Phacus and Sulphur | PARTICLES) PARTICLES | || PARTICLES PARTICLES LOOPS SIZE } LOOPS SIZE EATEN | REJECTED | | EATEN | REJECTED = =| oa | Ly (tsp | Ip | | | 16p 2p | | | eps | 3P | | 18p 2 4P | 19P) | 2 5P | 20p | \* [6s | I | 21p | \ys I | 22p 8s 2 I 23p gs z “ 24P 10s 3 I | 25s I IIs 2 I | 26s 3 5 12s I | | 27S 3 5 13s | I I |= 28s 3 5 [14s I | | 29s 3 AS SUMMARY Eaten, 12 Phacus and 1 grain of sulphur. Rejected, 3 Phacus and 13 grains of sulphur. go Asa Arthur Schaeffer In the above experiment there were eaten 12 Phacus and 1 grain of sulphur, while 13 grains of sulphur and 3 Phacus were rejected. It is evident therefore that in this case there 1s some sort of dis- crimination between Phacus and sulphur. Experiment 2. Discrimination between Starch Grains and Phacus In another experiment designed for the same purpose but in which iodine-stained potato starch grains and Phacus triqueter were used, even more sharply defined results were obtained. ‘The technique was similar to that of the preceding experiment, except that two pipettes were used, one for starch and the other for Phacus. The starch was stained with iodine to facilitate obser- vation. Previous to feeding, the starch was washed very thor- oughly to remove all the superfluous todine. The results are as shown in Table II. This experiment shows conclusively that Stentor can and does discriminate between two kinds of particles differing as much as Phacus and starch grains do from each other. The possibility of coincidence is entirely ruled out of court in that the stream of particles was changed at least seven times from starch to Phacus and from Phacus to starch. In the whole experiment there are only four “mistakes” at the most, including the rejection of a swarmspore twice and of a Coscinodiscus. But later experiments will show that the rejection of the swarmspore and Coscinodiscus were probably not mistakes, so that there occurred in this test only one mistake, that of ingesting particle numbered 6g—a starch grain. The size of the starch grains was variable, being from one-eighth to four times the size of a Phacus specimen. This shows that in this experiment, as in the preceding, size was not the determining factor in the selection. Another point worth noticing is that there are no loops in the paths of the particles Seton and that there are very few particles rejected without loops. The 45 loops of par ticle 32 are probably due to the fact that the Stentor was at that time lying on its side with pouch upper- most, thus making the removal of the particle more difficult than usual. When the animal turned over the particle was gotten rid of. The usual number of loops does not exceed 10 in any one Case. Selection of Food in Stentor Ceruleus (Ehr.) gl s = starch grains; p = Phacus; c = Coscinodiscus; sp = swarmspore. Where (—) occurs it signifies that the group in which it is found was broken; that is, some of the members of the group were eaten while the others were rejected. Thus, in group 36p, 37p, 38p, (—), which consisted of four particles fed simultaneously, the first three were eaten while the last one was rejected. Size of starch is given in terms of Phacus. TABLE II Experiment 2. Discrimination between Starch Grains and Phacus EATEN | REJECTED LOOPS SIZE EATEN | REJECTED LOOPS | SIZE Ip [36p 2p }37P 3P |38P 4p \(—) 39¢ | 5P 40p | 6p 4tp AAP | | ges = 4 8p | 43s 3 Uh) 9P | ek Sa ee: a 1op ! ee? ice: 5 IIp f46p | | 12p | i 47P 13p | | 48p 14s | I | 49P | 15s I | if Sop | 16s | I | 5p | 17s 3 | I 5p | | 18s 6 | I | Sap 19s 3 I | J sap 2o0p | yy aE | J s6p | 22p | 57P | 23P | \.geseeere. | 24p | | S9P | J 2sp | {Gop ) 26p 6ip | 27P | 62p 28s 8 1 \63p | 29s 8 | I | 64p | 30s 3 Se Ui ae | f3is 2 | AGS { 66p dee, , Seah Fe | 338P 2 68s 3 | 4 34P | 69s | ni 35P 7Os thon I 30 minute intermission 71s Beye: I | || SSS eS EEE SUMMARY Eaten, 50 Phacus, and 1 starch grain. Rejected: 18 starch grains, 1 Coscinodiscus, and 1 swarmspore. 92 Asa Arthur Schaeffer To determine what would be the effect of feeding in a mixed stream three kinds of particles, two that were not food and one that was food, natural starch grains, powdered glass, and Phacus triqueter were sucked up into different capillary pipettes and fed to a normal Stentor with the following results. Experiment 3. Discrimination between Starch Glass, and Phacus p = Phacus;s = starch grains; g = particles of glass. The particles of starch and glass were selected of a size about equal to that of Phacus. TABLE III Experiment 3. Discrimination between Starch, Glass and Phacus EATEN REJECTED LOOPS EATEN REJECTED LOOPS EATEN REJECTED LOOPS us {18s 38 35P_| il 2s | 4 19s a3) | 36p II J 3s [20s | 38 37P \ 4s 21p | (38p 5s (22p 39P 6s \23P 40p 7s (24p 41p 8p 25p (42g 9p 26p 3 ee 1p nip 8 442 IIp \ 28p | 2 (45g 12p | 29p 4 46g J 3p | 30p 3 tae \ r4p 31p 3 48g [15s 32P 498 4} 16s 33P 4 50g 17s 34P 2 51g ; 5260) SUMMARY Eaten, 21 Phacus and 1 starch grain. Rejected, 12 starch grains, 11 particles of glass, and 7 Phacus. At this point the Stentor contracted and swam away. ‘The dis- crimination in this experiment is of about the same degree of accuracy as in the two preceding experiments. The specimens of Phacus which were rejected may not represent a mistake in dis- crimination at all, but may be due toa condition of partial satiety, Selection of Food tn Stentor Caeruleus (Ehr.) 93 which will be taken up a little later. If this is the case the Stentor discriminated very well indeed what was Phacus and what was glass or starch. An interesting feature of this experiment is the number of loops that are recorded for the Phacus which were rejected and the absence of loops in the paths of the particles of glass. So far, these experiments show that Stentor can select Phacus from a stream of mixed particles in which there are one or two kinds of indigestible particles mixed with Phacus. There are two possibilities as to the way this selection is accomplished. First it may be that Stentor ingests from a mixed stream only one kind of food particles (such as Phacus triqueter), and rejects all other kinds of food and indigestible substances. ‘The other pos- sibility is that Stentor ingests all sorts of food particles and re- jects all sorts of particles that are not food. “To determine which of these alternatives is the one which actually obtains, the follow- ing experiment was performed in which two kinds of food par- ticles, Phacus triqueter and Euglena viridis, were fed in a mixed stream with two kinds of indigestible substances, powdered sul- phur and powdered glass. The following are the results: Experiment 4. Selection of Phacus and Euglena from Sulphur and Glass The Stentor upon which this experiment was tried was the same one which had submitted to the second experiment, on the pre- vious day (p. go). It still contained considerable amounts of partly digested food which probably represented the Phacus that were eaten the day previous. ‘The starch grain which was eaten in Experiment 2 was not to be seen, and it is probable that it was voided some hours after it was ingested. That it was digested is made highly improbable in view of the work of Meissner (’88) who found that nearly all of the potato starch which the Stentors ate was not digested, and that some of the starch which remained in Stentors for over 48 hours was practically in the same con- dition as when fed. ‘There is therefore no good ground for sup- posing that any part of the behavior of the Stentor in this experi- ment was due to the fact that a starch grain was ingested on the 94 Asa Arthur Schaeffer previous day. As later experiments will demonstrate, however, the ingestion on the day before of the 50 Phacus has probably in- fineneed the behavior of Stentor, and it is pretty certain that the condition of partial satiety which is exhibited in the beginning p = Phacus;e = Euglena; s = sulphur; g = giass; t = encysted Trachelomonas volvocina. TABLE IV Experiment 4. Selection of Phacus and Euglene from Sulphur and Glass | EATEN REJECTED LOOPS SIZE EATEN REJECTED LOOPS SIZE Ip 5 21g 3 I 2p 3 222 3 2 3P 2 238 Zo 5 4p 248 5p 252 I 6p 26s 25 7p 27s 2 25 8p 28s 2 1 gp 29s 3 I 1op f 30s 5 % IIp | | \ 31s 5 35 I2p 32e {13P 33e (-) 14p 34e (S) 15p 35t (©) | 16p 36e Gis 17p 37P 18p | 38p a 39P | 20g 3 I 40p | SUMMARY Eaten, 15 Phacus and 4 Euglene. Rejected, 8 Phacus, 6 particles of sulphur, 6 particles of glass, and 1 Trachelomonas. of I:xperiment 4 was due to this cause. | think it willbe pretty clearly shown that the rejection of the first three Phacus in Experi- ment 4 was not a “mistake” on the Stentor’s part but probably represents a transition from a condition of partial satiety to one of hunger, brought about by the stimuli of the Phacus upon the pouch and funnel. The Phacus numbered 14, 15, 16, 17—-the last four of a group of nine--were rejected probably because of the Selection of Food in Stentor Ceruleus (Ehr.) 95 inconvenience or impossibility of swallowing as many as nine Phacus at once. In very hungry Stentors six are sometimes swallowed at one gulp, and once a Stentor was observed to swal- low seven, but in groups of more than seven some were always rejected. So then in this experiment there are probably no mistakes in discrimination whatever unless the rejection of a Trachelomonas volvocina is considered a mistake. But the Trachelomonas was in the resting stage and inactive, as were also the swarmspore and the Coscinodiscus in Experiment 2. There is a possibility therefore that the inactive particles are rejected and that only moving, active particles (organisms), are ingested. To deter- mine whether selection is made upon this basis, some Euglena viridis were taken from a culture and killed in various ways, by heat, alcohol, acetic acid, etc. “They were then thoroughly washed and sucked up into capillary pipettes, those killed by heat in one pipette, those killed by alcohol in another, and so on. Another pipette was then filled with normal living Euglena. Normal Sten- tors were then isolated and fed with these Euglenz in various con- ditions in a mixed stream, precisely as was dene with the various substances in the preceding experiments. There was no discrim- ination observed between any of the differently prepared Euglenz. The dead Euglenz were eaten with the same readiness as were the living. Some Stentors rejected about equal numbers of each, while others rejected all of both kinds. Similar experiments were tried using Phacus and Trachelomonas as food, with the same results. There was no selection between the living and the dead organisms. Another possible way of explaining how the rejection of the Trachelomonas, Coscinodiscus, and the swarmspore was made, is that Stentor may be able to distinguish the different kinds of food particles from each other, and that certain kinds of food may be eaten with more readiness than others. This kind of selection may, perhaps, take place under all conditions, or only under cer- tain conditions. It may depend on the relative number of the different kinds of particles, or the order in which they come. To determine this matter a number of experiments were designed. 96 Asa Arthur Schaeffer ‘The first experiment was designed to show whether selection was exhibited when Phacus triqueter and Phacus longicaudus were fed in mixed order, Phacus triqueter being much more numerous than Phacus longicaudus. Experiment 5. Discrimination between Phacus triqueter and P. longicaudus (See Table V) Out of the five Phacus longicaudus which were fed with the 1760 Phacus triqueter, four were rejected and one was ingested. The four rejected ones were the last members of the respective groups in which they were fed, and the one which was ingested cane first in the group of two—-166/, 167. It was not positively ascertained in any of my experiments whether discrimination 1s nicer among the last members of a group than among the first, but the evidence seems to point that way. But even if this should turn out not to be true, it seems clear that actual discrimination between Phacus triqueter and P. longicaudus took place in this experiment. Experiment 6. Discrimination between Different Species of Organisms In another experiment designed to further show selection of one or more kinds of food particles from as many as six different spe- cies of organisms, the following results were obtained. There were fed in mixed order, E sola viridis, Euglena deses, Phacus triqueter, [elhatems loresnee elie: ‘Trachelomonas hispida, and Trache- lomonas Palvociian All these organisms were fed from a single pipette on to the disk of a Stentor as in the preceding ex- periment. For results see Table VI, p. gg. Experiment 7. Discrimination between Different Species of Organisms Immediately following the above experiment | fed another Stentor with the same sorts of flagellates (but omitting the Eug- lena deses which is difficult to handle in a capillary pipette owing to its habit of sticking to the walls) with the results shown in Table VI} p. 100. Loops are not recorded. Selection of Food in Stentor Ceruleus (Ehr.) oF The numbers followed by/ are Phacus longicaudus; all the other numbers represent Phacus triqueter. TABLE V Experiment 5. Discrimination between Phacus triqueter and Phacus longicaudus EATEN REJECTED LOOPS EATEN fa ee \2 | 46 3 | (-) 0. | ©) Be. | 49 6 | Vi 50 bin | ; 51 ( 8 | 52 | \ 83 10 | 54 (| | J 55 oul | LO ig. 57 14 ji 58 15 We) 16 17 61 {18 | | 62 19 63 ee | 64 i) 65 22 (66 (23 67 . 68 25 | (69 {26 70 \27 | l 71 28 | ee 29 ©) 3° 31 | 32 76 33 77 f34 iets 78 (35 ore f36 | 80 \37 | fas | | {82 \39 the [40 | iO) 41 4 42 . 44 3 REJECTED LOOPS | EATEN REJECTED LOOPS 56 59 60 81 83 84 85 86 { 87 | 88 21 RPwWwW - wewnnr -& Ww 129 130 131 96 5 98 4 BF. | 13 100 3 101 | | | 1131 | 2 114 | 6 fxs 6 \116 5 117 4+ {118 8 \z19! 3 126 4 128 11 132 I mS 98 Asa Arthur Schaeffer TABLE V—continued EATEN REJECTED LOOPS EATEN REJECTED LOOPS EATEN REJECTED LOOPS 133 | 149 » (1661 (-) 134 3 | {(-) 150 6 167 135 I | te) 151 4 168 (136 | 152 \C) 169 ) 137 | {153 170 (138 | 154 4 171 /139 355 OE fo SE 4 \ 140 156 | (-) 173 4 141 | 157 | Neiiles SiGe: I (142 | | 158 1 175 2 143 | 159 8 176 2 144 | | 160 1177 179] I 145 | | 161 178 146 | 162 ©) 38 r70re I 147 | 163 180 | 6 148 | | {164 | (©) 1651 1 At first sight neither Experiment 6 or 7 seems to show that Stentor eats some kinds of food with more readiness than other kinds, for some individuals of each of the various kinds of flagel- lates were eaten while some of every kind were rejected. But upon closer examination it is found that the latter parts of the experiments are different from the earlier in several respects. A larger proportion are rejected at the end of each of the experi- ments than at the beginning. Of the organisms eaten the variety is much more extensive at the beginning than in the latter part of the experiments. All the organisms that were fed in the begin- ning of both experiments were ingested, but at the close only Eu- glena viridis and Trachelomonas volvocina were ingested, all the other kinds being rejected. Only a single ‘Trachelomonas his- pida was ingested. It is clear that there occurred a change in the physiologic state of Stentor as each of the experiments pro- gressed. In the sixth experiment, of the six different kinds of flagellates that were fed, Euglena viridis and Trachelomenas vol- vocina were eaten with the greatest readiness. After the twenty- second organism had been fed, ten Euglenz viridis were ingested and five were rejected. ‘The fact that not all the Euglenz were Selection of Food in Stentor Caruleus (Ehr.) 99 ev = Euglena viridis; ed = Euglena deses; pt = Phacus triquetur; pl = Phacus longicaudus; th = Trachelomonas hispida; tv = Trachelomonas volvocina. TABLE VI Experiment 6. Discrimination between Different Species of Organisms iy FATEN | REJECTED, LOOPS 2 | REJECTED, LOOPS EATEN | REJECTED LOOPS ipt f3cev | {sopl 2ed \©) | 31ev \ 60ed (3ev (32pl 61ed 62pl 4 4eV 4 33ev | (sev (sant | 63pl bev 35pl | 64ch* 5 7ev f36pl 6stv 2 8pl \37pl 66tv Zs gev | f38pl | 67pl loev \39pl 68pl 4 ipl | 4opl | 5 Fy 12pl_ | 4lev "Three mipute intermission. Pi- 13pl | 42tv pette was refilled with food or- r4pl | | f4ath organisms. 1spl | (4gev | 16ev 45th | ogev 17pl 46th | 7oev 18pl | 47ev 4 7lev lyev | 48pl 72ed 2opt | 49tv 73eV 21pl | soev (74ev 22pl | 51ev 475€V [23ev | 52pl | \ 76ev 24ev 53th | 77ev 25ev 54ev 78ev \C-) 26ev 55ev 79eV (27pl 56th 8oev | bee 57ev | Siev | 29pl 58th *Food particle 64 was a Coleps hirtus. ingested does not prove that there was no selection going on in this part of the experiment, for the Phacus triqueter and the T'ra- chelomonas hispida are without exception rejected. The explan- ation of this apparently capricious selection of some Euglenz and the rejection of the rest is probably to be sought in the changed 100 Asa Arthur Schaeffer condition of the Stentor brought about by the particles which were just previously ingested. That is, the Stentor was probabl, in a condition of partial satiety, where less and less food is taken even if it should meet all the requirements of a perfect food under con- ditions of hunger. ‘The actual proofs of sucn conditions of par- tial and complete satiety will be taken up later on, but it may be pointed out here that in the sixth experiment the Stentor appar- ently grew less and less hungry as the number of ingested particles increased. Particles designated as in Experiment 6 TABLE VII Experiment 7. Discrimination between Different Species of Organisms EATEN | REJECTED | EATEN REJECTED EATEN | REJECTED EATEN REJECLED Ip] | | (1spl 30eV 45tv 2pl | 16pl 31pl 46tv 3pl |) 17pl 32pl 47th 4pt | ((-) 18pl 33eV | 48tv 5pl | r9pl 34pl 49pl 6pl 2opl 35ev Sotv 7pl | 21ev 36pl | Sipt 8th | 22pl | 37eV | 52pl gev f23ev | 38ev | 53tv | Iopt | LO) 24pl | 39tv 54eV 11pl 25eV | 40th 55th 12pl 26eV | 41pl 56th 13pl | 27eV | 42pl 57tv 14pl 28pt 43pl s8ev 29pl 44tv 5gev The proportion of Euglena viridis and Trachelomonas volvo- cina was not the same in the two experiments, so no conclusions may be drawn with regard to which of these two kinds of organ- isms is eaten with the greater readiness. But it is of course clear that Euglena viridis and Trachelomonas volvocina are “ preferred”’ by the Stentor to Phacus triqueter, P. longicaudus, and Tra- chelomonas hispida; which is only another way of saying that Stentor expresses a choice in the food which it eats. Selection of Food in Stentor Ceruleus (Ehr.) 101 Experiment 8. Discrimination between Organisms of Similar Size and Shape As a final test in food discrimination in which the path and fate of each particle was recorded the following experiment was de- signed and performed. ‘Two kinds of organisms were used, one being Trachelomonas volvocina and the other a species of Euglena which upon very slignt disturbance contracted into a spherical mass of almost exactly the same size as that of a Trachelomonas. The organisms were fed from a capillary pipette in a mixed stream, the as of Euglenz and Trachelomonas being as nearly equal as possible. The loops were not recorded. ‘The results follow: = Euglena; t = Trachelomonas; th = Trachelomonas hispida TABLE VIII Experiment 8. Discrimination between Organisms of Similar Size and Shape | | | EATEN | REJECTED EATEN | REJECTED | EATEN REJECTED EATEN REJECTED Ie Werée 32e fave 2e ree | 33t | | \48e 3e 18t 34t 4ge 4e 1gt f 35¢e got 5t 20t \ 36e ji sit 6t [s'|amey Me™ aye \ s2t fen | f 22e | 38t 53t 8e NEG) | ezze 39t | | ' 54e gc 24t 40e 55t | roth 25t f ait 56t Ile | 26e \ 42e || 57e 1942 ||| 27e 7 43t 58e ryt | 28e | 44e 59t 40 || 29e | 45t | 6oe 1st, || 30€ | | 46t 61t 3Ie | 62e Of the 62 organisms 32 were Euglenz, 29 Tracheolmonas vol- vocina, and 1 I. hispida. Seventeen Euglenz were eaten and 15 rejected; and of the Trachelomonas 3 were eaten and 27 rejected. That the Euglenz were eaten with more readiness than the Tra- chelomonas is evident from an inspectoin of these figures, which 102 Asa Arthur Schaeffer show that 56 per cent of the Luglena were eaten andonlyto per cent of the Trachelomonas. But a better and truer idea of the accuracy of selection is obtained by a study of the results as set forth in the table. As in the sixth and the seventh experiments, the beginning of this experiment differs from the latter part in that the variety of ingested particles is greater in the beginning, and also in that the proportion of food ingested as compared with the amount fed, is greater in the beginning. ‘There seems also to be a marked change in the basis upon which discrimination is effected; it evidently became more restrictive towards the end. What is the cause of the change in the degree of restrictiveness in selection in these experiments? Is the change due to differences in the food or to an alteration in the Stentor itself? Evidently different food particles possess different strengths or powers of giving the stimulus that causes Stentor to ingest them. The stimulus from Phacus triqueter 1s stronger than that from Trachelomonas hispida; and that from Euglena viridis 1s still stronger than that from Phacus triqueter. “Uhis is shown by the fact that at certain times (when nearly replete), Stentor takes only Euglenz (or Phacus if Kuglenz are not present), from a mixed stream of organisms. ‘here probably are slight differ- ences between the strengths of the stimuli from different individ- uals of the same species, but these differences are evidently less than those between different species, for when nearly satiated, Stentor takes only the particles of one species. Yet we find in the experiments described above that specimens of the same organism (as Euglena) are at first eaten without any exception, while later less than half of them are eaten. The change in the proportions eaten as the experiment progresses are well seen in the following tabulation of the results of the eighth experiment. The experiment is divided into successive groups of about ten particles to each group. Selection of Food in Stentor Ceruleus (Ehr.) 103 TABLE VIlla Tabulation of Results of E. sparaen 8 | TRACHELOMONAS TRACHELOMONAS EUGLENAE EATEN || EUGLENAE REJECTED | : | afer EATEN REJECTED GROUPS : | a _——— | Number | Per cent | Number) Per cent | Number | Percent | Number Per cent 1 | | } } | | 2 1 I-10 6 | 86 | I | 14 2 | 66% I _ 333 11-21 ° ° 2 100 I II | 8 89 22-30 4 57 3 43 fo) fe) 2 100 31-40 | 3 50 3 50 ° ° 4 100 41-50 | 2 40 a 60 fc) ° 5 100 51-62 | 2 | 40 3 60 ° fo) 7 100 As this table shows, the percentage of Euglena eaten decreases steadily throughout the experiment, from 86 per cent to 40 per cent; while that for Trachelomonas decreases from 662 per cent too. The percentage of Euglena rejected increases Font 14 per cent to 60 per cent; of Trachelomonas from 33 per cent to 100 per cent. Similar changes were found in all the experiments of like character with Stentor, including a number which I do not publish in detail. It is evident that this regular change cannot be due to chance variations in the food, but must be due to an alteration in the physiologic state of Stentor itself. In some way the Sten- tor changes as it takes food, so that stronger and stronger stimuli are required to set off the ingesting mechanism. Such a change is of course parallel with what we observe in higher organisms. We find therefore that we have in this unicellular organism physiologic conditions corresponding to what we call hunger and satiety in higher forms. Differences in behavior due to hunger and satiety have not heretofore been demonstrated for those pro- tozoa that secure their food by means of an alimentary vortex. The experiments we have just described, primarily designed to test the power of selection, indicate strongly the existence of such differences. Let us now turn to experiments that were planned to test this matter. We wish to determine what differences exist between hungry and satiated Stentors, and whether there are inter- mediate conditions, manifesting themselves in differences in be- havior. 104 Asa Arthur Schaeffer Experiment 9. Effect of Hunger on Behavior in Feeding Eight Stentors with some of their own culture solution were placed into a small preparation dish. After they had attached themselves | selected a large individual and fed it with Phacus triqueter with the result shown in Table LX. Experiment 10. Effect of Satiety on Behavior in Feeding At the close of the foregoing experiment the dish of Stentors was set aside until next day when it was found that the Stentor which was fed in this experiment contained a considerable num- ber of small brownish bodies. These upon close examination were found to be Phacus triqueter in a stage of partial digestion. The protoplasm was extracted from the Phacus and the chloro- phyll was changed in color. Here was then a good opportunity to see whether the ingestion of Phacus on the previous day had any effect upon the ingestion of food particles today. A mix- ture of Euglena viridis and Phacus triqueter was fed from a cap- illary pipette on to the disk of Stentor with the results shown in Table X. Perhaps the most notable difference between these two experi- ments is in the number of food particles ingested. In the ninth experiment 73 out of 118 particles were eaten, the majority being ingested in the first half of the experiment, while in the tenth only 10 were eaten out of 75, and these ten were distributed compara- tively evenly. A large number of experiments performed upon well-fed Stentors showed substantially the same results as Ex- periment 10. In no case where the membranellze and pouch cilia beat normally, 1 e., where their movement was not reversed, did it happen that every single particle was rejected. Some few were always ingested if the experiment was sufhciently extensive. “The lowest ratio of particles eaten to those fed was 1 to 12. When the particles were fed very rapidly the ratio was very much in- creased. But if the particles are fed at the rate of about 100 an hour, results like those of Experiment 10 will be obtained. Sten- tors from artificial cultures in which food 1s very plentiful, and in which the Stentors thrive and reproduce very rapidly, show sub- stantially the same results as are shown in Experiment 10. Selection of Food in Stentor Ceruleus (Ehr.) 105 TABLE IX Experiment 9. Effect of Hunger on Behavior in Feeding R —__— aa FATEN REJECTED LOOPS | EATEN | REJECTED LOOPS EATEN | REJECTED LOOPS fi | | 47 I | J 87 a 2 | | 48 2) | \ 88 4 3 | | 49 3 89 | ra ; Bre: = } 7 |Stentor contracted; hit with the as : | > | ; pipette. x 9 3 es | | [so | 2 | aes J8 | 5 2 4 I Ke | sz | 2 95 | 3 10 | 3 | 3 | I } 96 I Il | i I | 97 I 12 | 55 I 98 | 13 56 5 99 14 57 100 (-) 66 2 | | 109 UI f | f | { 24 | } SS eee : 25 68 |At this point a Stylonychia was f | | 26 69 | fed twice to the Stentor but was (-) 27 7 7° | rejected each time. On being 28 | Fis | fed the 3d time the Stentor held 29 | 72 | the S. in the pouch for an instant 30 } (73 | and then contracted, setting the (-) 31 ie 74 | S. free. The S. was fed again (-) 32 5 f I | to the Stentor and was eaten. 33 | G) 76 4 | The Stentor then contracted for 34 (77 a few minutes. 35 78 36 F [110 I 37 & 80 2 | | 111 I 38 81 (| | 4 112 I =) 39 She || 82 | 113 I 40 | 83 2 | 114 I (-) a I | ba f 11s | es (Contracted; hit with the pipette. } (-) 116 2 re maak Oneps a +4 a4 4 me) 118 I Wie). | 745 | 85 : \ 46 | C)) 86 2 106 Asa Arthur Schaeffer e = Euglena viridis; p = Phacus triqueter TABLE X Experiment 10. Effect of Satiety on Behavior in Feeding EATEN REJECTED LOOPS EATEN | REJECTED LOOPS | le I f 44e I 2p i \ 45e I 3P I J 46e I { 4e 1 \ a7e [es se 1 48e | 2 be I 48 fed again (49e) | 3 7e I soe f 8e I : Sip I \ ge I 52p 1 1oe I 53P I 11p 4 1 54P I 12e 2 55P I f 13e | 56p \ 14e _57P I 15e I 58p | I J 16e | 59e 3 \ (-) 17e. I | _ Bit of Debris I 18e. 1 | Fed again (Bit of Debris) I 18 fed again (age) r || | { 60p I 18 fed again (20e) I \ 61p I 18 fed again (21e) Ge | 62p I 18 fed again (22e) I 63p 1 18 fed again (23e) I Bit of Debris 1 18 fed again (24e) I f 64p | I 18 fed again (25e) I \ 65p ee: 18 fed again (26e) 3 66p | I 18 fed again (27e) 2. 67p 2 18 fed again (28e) I | 68p 3 18 fed again (29e) I = 30€ I At this point the Stentor contracted. When again if 31p expanded, Stentor reversed the cilia when the ; (-) 32e 1 || stream from the pipette reached the disk. After 33e about a minute the cilia beat normally. 1 “4 ,, ———__ f35e I 69e 2 : \ 36e yl 70e I 37€ I (71e | (38e I 72e | 39 I (-) 73¢ 3 | {4oe 2) ) 74e 1 4le 1 75€ I Selection of Food in Stentor Caruleus (Ehr.) 107 Perhaps the most significant difference of all between the ninth and the tenth experiment is with regard to the occurrence of loops in the paths of the organisms which were fed. These two experi- ments are typical in this respect of nearly all the experiments which form the basis for this paper. We find, first, that nearly all the loops occur in the paths of the rejected particles. As it happens i in these two experiments, no particle is rejected without at least one loop in its path. Second, when loops occur in the paths of ingested particles they are gener- ally found only in those experiments where a comparatively large proportion of the particles are ingested, or in other words, when the Stentor is hungry or only in the first stages of satiety. Third, in extensive experiments like the two preceding, more loops occur in the first half of the experiments than in the last half, both in the ingested and in the rejected particles. Fourth, very few loops occur in feeding hungry Stentors. Fifth, very few loops occur in a satiated Stentor. Sixth, the maximum number of loops is found when the Stentor is in the first stages of satiety. Seventh, in a stream of mixed particles including food and bits of glass or sand, the glass or sand is generally rejected with fewer loops than the food particles. A conspicuous case of this is seen in the third experiment. What is the real significance of these loops? Are loops the result of fatigue of the ingesting or of the rejecting mechanism, or are they correlated with a certain physiologic state of Stentor in such a way as to be a factor in the selection of food? It appears clear that the loops are not due to fatigue of the ingesting or re- jecting mechanisms, nor of the apparatus for receiving the stimuli. Fewer loops are made when the Stentor is satiated than when only partial satiety sets in; this would not be the case if the occurrence of loops were due to fatigue, as will presently appear. Further, when excessively small particles are fed, such as the Euglenz in the eighth experiment, as many as 11,000 may be eaten and several times that number rejected, all within two or three hours, so that fatigue could hardly occur from handling a few hundred as in Experiments g and 10. Again we have seen that discrimination is more precise as the Stentor becomes satiated. This shows that 108 Asa Arthur Sch ae ffer the apparatus for receiving stimuli has not become effectively fatigued, Thus it appears clear that the occurrence of loops is not due to fatigue; we must look for the explanation in some other change in the physiologic state of the animal. As noted above, the loops appear as the animal approaches satiety, so that there can be little doubt but that the change in the degree of hunger is what brings on the loops. The loops, as we have seen, almost always occur in the paths of particles that are finally rejected. It appears that the rejecting mechanism is not set in operation by the first slight stimulus from an objectionable particle, but the stimulus seems to be summated with every successive loop that is made, until it is finally strong enough to cause rejection. In the first stages when the animal is hungry the ingesting mechanism 1s more readily set off than that for rejection; near repletion, the rejection appa- ratus is more readily set off; and as repletion advances the reject- ing apparatus 1s continually more and more readily set off, re- quiring therefore fewer loops. ‘The apparatus for receiving stim- uli seems therefore to be in a state of continual change, so that stimuli which readily set off the ingesting reaction when the ani- mal is hungry have but a slight effect when the Stentor is nearly replete, and finally have no effect at all, or set off only the rejecting reaction. A difference is seen in the fact that when a particle is accepted, this is done (as a rule) at once, without loops, while rejection is done only after some delay, with the occurrence of loops. Only when the Stentor is fully satiated does rejection occur instan- taneously. In the state of incomplete satiety, rejection is a slow and uncertain process, as if the stimulus for rejection had first to gather strength before rejection could occur. ‘Thus the number of loops which occur before rejection depends on the degree of hunger. ‘There is doubtless a similar though reverse effect of hunger on the ingesting apparatus; it seems cer- tain that a stronger stimulus is required to set off the ingesting reaction when the Stentor is partially satiated than when very hungry. But the evidence is not so clear as for the rejecting apparatus owing to the fact that loops rarely occur in the path of Selection of Food in Stentor Ceruleus (Ehr.) 109 a particle that is to be ingested. ‘The nature of the assumed re- jecting and ingesting mechanism and the way selection is brought about, will be discussed later on. We have thus far dealt mainly with what may be called stages of moderate hunger and moderate satiety. ‘There still remain the states of utter hunger and of surfeit to be described. The condition of extreme hunger may be produced by putting a number of Stentors with as little of their culture solution as pos- sible into clear tap-water and keeping the water as free from bac- teria as possible. ‘There is no evident difference in behavior between Stentors in the condition of extreme hunger and those moderately hungry. The condition of utter satiety is more interesting. “UVhe behav- ior of a Stentor in such a state is very different from the normal behavior. The following experiment fully illustrates this. Sev- eral hundred small paramecia were placed with a few Stentors in a watch glass for two days, so that the Stentors could feed upon them. ‘The largest of the several Stentors was then selected for feeding with Trachelomonas hispida. The Stentor was filled with a number of globular masses of slightly brownish transparent material which probably represented as many broken down para- mecia. The Trachelomonas were fed from a capillary pipette as usual. Following are the results. Experiment 11. The Effect of Utter Satiety on Stentor In this experiment the figures, 1 to 24, signify as many Trach- elomonas hispida which were fed to Stentor. As in preceding experiments, they are numbered in the order in which they were fed. Stage “pipette presented,’”’ means that the end of the cap- illary pipette which contained Trachelomonas hispida was brought within about half a millimeter from the animal’s disk, and that a very slow stream of water was then caused to flow against the disk. All the other stages of behavior are self-explanatory. - Asa Arthur Schaeffer Pipette presented 1 Prachelomonas hispida rejected with 1 loop Contraction All cilia normally active Pipette presented Slowing of cilia Pipette removed Contraction for 4 minutes Cilia normally active Pipette presented Contraction Pipette removed Cilia normally active Pipette presented Slowing of cilia Pipette removed Cilia normally active Pipette presented Slowing of cilia Pipette removed Cilia normally active Pipette presented Slowing of cilia Pippette removed Cilia normally active Pipette presented Slowing of cilia Pipette removed Contraction Cilia normally active Pipette presented Slowing of cilia Pipette removed Cilia normally active Pipette presented Slowing of cilia Pipette removed Contraction Cilia normally active Pipette presented Slowing of cilia Pipette removed Cilia normally active Pipette presented Slowing of cilia Pipette removed Contraction Cilia normally active Pipette presented Slowing of cilia (Pipette continued) Bending away Pipette removed Cilia normally active Pipette presented Slowing of cilia Bending away Pipette removed Cilia normally active Pipette presented Slowing of cilia Bending away Pipette removed Cilia normally active Put a Trachelomonas on the disk near the pouch with a very thin glass rod. 2 rejected, with 5 loops. Pipette presented Slowing of cilia Pipette removed Cilia normally active Trachelomonas presented with glass rod. 3 rejected with 7 loops Trachelomonas presented with glass rod 4 rejected with 4 loops Pipette presented Selection of Food in Stentor Caruleus (Ehr.) III Slowing of cilia 10 rejected with 4 loops Pipette removed II eaten Cilia normally active 12 (Coscinodiscus) eaten Trachelomonas presented with glass 13 rejected with 8 loops rod 14 réjected with 4 loops 5 rejected with 4 loops 15 rejected with 8 loops Pipette presented 16 rejected with 5 loops Slowing of cilia 17 rejected with 3 loops Pipette removed 18 rejected with 3 loops Cilia normally active 1g rejected with g loops Pipette presented Stream veryslow 20 rejected with 3 loops 6 rejected with 3 loops 21 rejected with 4 loops 7 eaten 22 rejected with 5 loops 8 rejected with 4 loops 23 rejected with 7 loops g rejected with 8 loops 24 rejected with 2 loops This Stentor was at no time as fully extended as the average Stentor is when not containing much food, nor was the ciliary action quite so strong and vigorous as when normally hungry. This experiment is a good example of a remarkable change in the physiologic state of Stentor in that the Stentor was slowly brought from a condition of surfeit to one of only partial satiety. The change was a gradual one inasmuch as five Trachelomonas im- pinged upon the disk before the change was complete. A single Trachelomonas caused apparently no visible change in behavior. There seems to have been required the summated stimuli from five Trachelomonas before the state of surfeit could be changed to one of only partial satiety. There was also a gradual decrease in irritability arising from the stream of water from the pipette. At first this caused contrac- tion. A little later only a slowing of cilia occurred—the initial stage of contraction. Still later the Stentor bent away from the source of the stimulus, and finally the stream from the pipette no longer caused any visible reaction. This decrease in irritability is parallel with the decrease in satiety but is probably not due to the same cause. ‘The cause of the decrease was probably the frequent repetition of the stimulus, the faint stream of water from the pipette, for a marked decrease in irritability resulted before the glass rod was used. But the 112 Asa Arthur Schae ffer change from a condition of surfeit to one of only partial satiety was caused by the impinging of five Trachelomonas on the Sten- tor’s disk. This decrease in the condition of satiety does not represent a similar rate of decrease of food in the Stentor’s body. We see therefore that a particular state of hunger in a Stentor does not directly nor necessarily accurately represent the amount of food in the Stentor at the given moment. What thestate of hunger actually represents is the condition of the organ for receiving stim- uli from external food particles. ‘This is influenced: (1) by the past history of the amount and kind of stimulation from external particles; (2) by the amount of food in the Stentor’s body. Other peculiarities of behavior attendant upon the condition of satiety are the following: 1 Extension is always sub-maximal. Instead of being ex- tended as fully as possible with the disk spread so as to present the greatest area to the base of the vortex set up by the membran- nee the Stentor is only partially extended and the disk is smaller. The animal does not extend perpendicularly upward or horizon- tally from its base of attachment, but generally hangs downward, or frequently lies upon some debris, etc., if possible. 2 The aboral side is more strongly convex when replete than when hungry. This posture may be related in some way to the voiding of excrementa, though no evidence could be obtained to show that this Is true. 3 There is a marked decrease in the activity of the membran- ella. ‘his may have much to do with the degree of extension in Stentor. Strong action of the membranellz tends to pull the disk away from the foot, and therefore full extension may be partly due to the strong beat of the membranellz. If the mem- branellz beat only in a weak manner there is no such pull upon the Stentor, and as a result it lies prone or hangs downward from its point of attachment. ‘This is made still more probable by the fact that hungry, free-swimming Stentors are seldom as fully extended as attached ones. 4 Satiated Stentors are very irritable to stimuli affecting the swallowing or rejecting mechanisms. In hungry Stentors one Selection of Food in Stentor Caruleus (Ehr.) 113 can poke around the pouch a good deal before a Stentor contracts, but in a satiated specimen the faintest touch generally causes con- traction. But mechanical stimuli on the sides of the body do not seem to cause contractions more readily in replete than in hungry Stentors. 5 If the stimuli are not too strong, contraction is often re- solved into stages, and only the first of these may be passed through, instead of all of them as is the case when the Stentor is hungry.. Thus contraction may be resolved into the following separate stages. (a) Cessation ofaction of the membranella. (b) Closure of the pouch. (c) Gradual rounding up of the anterior and posterior portions of the Stentor into an oblong mass. If the stimulus still continues, complete contraction follows; but this act changes the form and size of the already partially con- tracted Stentor very little. No such slow contraction takes place in a hungry Stentor where, if the mechanical stimulus is strong enough to cause cessation of action of the membranellz, the entire process of contraction occurs instantaneously. EXPERIMENTS WITH MIXTURES OF PARTICLES In this part will be considered experiments in which the food particles were not fed and observed individually, but in which the Stentors were surrounded by mixtures of particles of various kinds. The purpose was to determine whether the animals can make a selection from among the different particles of such a mixture. After remaining for various periods of time in such mixtures, the Stentors were placed on a slide and compressed with a cover glass. It was then possible to estimate with some accuracy the relative amounts of the various kinds of particles that had been ingested. For such experiments we can use only particles that remain long in suspension, so as to maintain their relative distribution. Heavy particles cannot be employed. It is obvious that this method gives less precise results than employed in the foregoing experiments. But by its use cer- tain additional problems can be attacked. We have seen that Stentor discriminates between different sorts of particles fed in T14 Asa Arthur Schaeffer succession; can it also make a selection from among particles that are intimately mixed? And why does it at times take particles that are not good for food, such as carmine? In the first series of experiments the following procedure was adopted. In each of several small preparation dishes there was placed 10 to 20 cc. of filtered fluid from the Stentor culture, to- gether with about 30 Stentors that contained no solid food. Into some of the dishes was introduced a mixture consisting half of carmine particles, half of an admixture of Chlamydomonas with some very small Euglena. In others, serving as control carmine alone was introduced, in amount equivalent to the total quan- tity of particles in the other dishes. The contents of the dishes were thoroughly stirred, and they were then placed in a dark box to prevent the Chlamydomonas from collecting at the lighted side of the dish. After half an hour the Stentors were examined under the microscope. “The average content for each Stentor was about 1500 Chlamydomonas, about 85 Euglenz, and carmine of the bulk of about 10 Euglena. Sev- eral Stentors had ingested about the same amount a food, but no carmine whatever. About half the Stentors were left surrounded with these sub- stances for 24 hours. ‘These then contained a much greater amount of Euglena and Chlamydomonas than before, but in about the same proportions. But the carmine content was prac- tically nil. This is probably explained by the fact, brought out in the first series of experiments, that discrimination becomes more perfect as hunger becomes less. Having become in the later hours nearly satisfied, the Stentors discriminated more accurately against the carmine, and meanwhile that which they had ingested in the first hours of the experiment had been egested in the natural course of events. The result was not due to the settling of the larger particles of carmine to the bottom, since when I added fresh carmine, none of it was ingested. It is also improbable that the Stentors had become “educated” to the fact that carmine 1s not food, as will be shown later. In the control dishes where only carmine was present, the Sten- tors had ingested an amount of carmine equal to about two-thirds Selection of Food in Stentor Ceruleus (Ehr.) 115 of the quantity of substance taken in by the Stentors in the dishes containing both food and carmine. ‘There was variation among different individuals in the amount taken, but none were entirely devoid of carmine. A similar series of experiments in which india ink was substi- tuted for carmine gave the same results, except that only about half as much india ink was ingested as carmine. These experiments show that in an intimate mixture Stentor can discriminate and select with a high degree of accuracy such minute food particles as Tilman Sone among indigestible particles like carmine or india ink. In some cases the accuracy of selection is almost perfect. Further, it is seen that the larger amounts of carmine are eaten when no food is present, while little or none is ingested when food is present also. In another experiment the Stentors came from a culture where food was more abundant. ‘They were fed with mixtures contain- ing one or more of the following: yeast, carmine, india ink, Eu- glene, Trachelomonas. Nine different combinations of these materials were made in separate dishes. Equal quantities of the different substances were employed in each experiment. At the end of an hour the Stentors of each dish were examined as to the materials ingested. The different combinations employed and the results obtained are given in the following series. 1 Mixture of yeast and carmine. Result: Stentors at the end of an hour contained about twice as much yeast as carmine. The total bulk ingested was equal to that of about 600 Euglene. 2 Yeastandindiaink. Result: Less yeast inpeated than in I. Very little ink was eaten—about the bulk of three Euglene. 3 Euglenz, Trachelomonas, and carmine. Result: Many Eu- glene and Trachelomonas ingested. Carmine to the bulk of about 15 Euglenz. 4 Euglene, Trachelomonas, and ink. Result: Same quantity of Euglenze and Trachelomonas a in 3. Ink to the bulk of 1 Euglena. 5 Yeast, Euglene, Trachelomonas, and carmine. Result: More Euglene and Trachelomonas then yeast. Carmine to the bulk of 9 Euglene. 116 Asa Arthur Schaeffer 6 Yeast, Kuglenae, Trachelomonas, and ink. Result: More Kuglenz and Trachelomonas than yeast. No ink. 7 Ink. Result: Ink to the bulk of 3 Euglene. 8 Carmine. Result: Carmine to the bulk of 20 Euglene. 10 ©Yeast, Euglena, and Trachelomonas. Result: More Eu- glena and ‘Trachelomonas then yeast. Only about half the Stentors of each dish were examined at the end of an hour. ‘The others were allowed to remain in the dishes until next day. Examination then showed somewhat the same results as are described above. In dish 4 however, no ink was found in any of the Stentors, and in all the dishes in which ink and carmine were placed the amount of these substances ingested by the Stentors was less. [The amount of yeast in the Stentors in dishes 6 and 5 was also considerably less than on the previous day. his decrease may have been due, in part at least, to the torulas sinking to the bottom, notwithstanding the frequent stir- rings to wu the dishes were subjected. These experiments show that Stentor can discriminate between the torulas of yeast and the grains of ink or carmine, and that selec- tion is more perfect for example, between yeast and ink than between yeast and carmine ‘They show also that less ink and car- mine are eaten when food is present than when not. But in the case of dish 1 the Stentors are found to contain more carmine when the yeast is also present than when.only carmine is present asin dish 8. I am unable to explain why this is so in this particu- lar case. These two sets of experiments are probably sufhcient to show that Stentor can discriminate as well when enormous numbers of particles touch the disk and pouch continuously for 24 hours, as when a small number are swept into the pouch in rather slow suc- cession, and that very minute particles of different sorts can be sorted as accurately and rapidly as large particles. But all these experiments were performed when ay different particles were mixed in about the same proportion. ‘The question next came up: What will happen when the proportions are changed? Will Stentor select food particles from particles that are not food as accurately when the latter are greatly in excess? or in a mixture of different Selection of Food in Stentor Ceruleus (Ehr.) 117 kinds of food particles, will the ratio of ingesta vary directly with the proportion of the amounts of the different particles as they occur in the mixture? or does a variation in the different kinds of particles, in the matter of proportion, have no effect upon the amount of each kind ingested? Trachelomonas volvocina, T. hispida, Phacus longicaudus, P. triqueter, Euglena viridis, and E. deses were placed in a small preparation dish with about to cc. of filtered Stentor culture solu- tion. About 100 times as much carmine as food organisms was added and into this mixture several Stentors were placed, and the dish set in the dark for about an hour. Examination at the end of that time showed that about ten times as much food was eaten ascarmine. ‘This is practically the same result that was obtained in dish 3, described above, where the proportions of Euglena, Trachelomonas, and carmine were equal. Another series of experiments bearing upon this point was per- formed with mixtures of a species of Euglena and a colorless flagel- late, probably a species of Chilomonas. ‘The Euglenz were about 304 long and about 12 wide; Chilomonas, 18 long and 8y wide. These two organisms were mixed in various proportions. Stentors were then introduced and the dishes set in the dark for two hours. The contents of the dishes, together wi h the results obtained, follow: 1 Mixture of Euglena and Chilomonas in proportion of 4 : I. Result: Ten times as many Euglenz ingested as Chilomonas. 2 Chilomonas and Euglenz in proportion of 4: 1. Result: Four times as many Evuglene ingested as Chilomonas. One typical Stentor contained 200 Euglenz and 54 Chilomonas in 80 vacuoles. 3 Chilomonas and Euglenz in same numbers. Result: About six times as many Euglenz ingested as Chilomonas. 4 Many Chilomonas. Result: Stentors contained as many Chilomonas as Stentors in dish 6 contained Euglenz. 5 FewChilomonas. Result: Comparatively few Chilomonas eaten. 6 Many Euglene. Result: Many Euglenze eaten,—from 5,000 to 11,000. 118 Asa Arthur Schaeffer 7 Few Euglene. Result: Comparatively few Kuglene eaten. Some peculiarities of behavior of the Stentors in the above series of experiments may be worth mentioning. About 60 per cent of the Stentors were swimming freely. In dishes 4 and 6 where the Chilomonas were dense, the Stentors swam continuously with the foot ahead. About one-third of all the free-swimming Stentors in dishes 2 and 4 (where the Chilomonas were dense) were in a state of almost maximum contraction. ‘These Stentors conta‘ned very few Chilomonas. Nearly all the free-swimming Stentors in the other dishes were extended. Some of the attached Stentors in the dishes 2 and 4 were maximally contracted. These contained the smallest number of food organisms. Some of the other attached Stentors rolled masses of Chilomonas on the disk in the “push ball”’ fashion as described on page 86. The other attached Stentors in these dishes continually reversed their cilia. The attached Stentors in the Euglena dishes, 1, 3, 6 and 7, kept all the body cilia beating forward, whether all the Euglena were rejected or all eaten. We have seen that Stentor takes less waste matter (carmine, etc.) when food is present, more when it is absent. ‘This brings up another interesting question. In order that the animal shall reject substances not good for food, must it first have taken a certain amount of food? If so, how much food must be taken before discrimination begins? And in order to induce discrimina- tion, is it necessary that food should be actually ingested, or 1s mere contact with food all that is required? As to the quantity of food necessary to induce discrimination the experiments just described show that Stentor discriminates against carmine (or Chilomonas), about as completely when the proportion of Euglenz present is very small (about 1 : 100) as when more Euglenz are present. [tis impracticable to work with smaller proportions of Euglena, since in such cases one cannot keep the various kinds of particles uniformly distributed. To determine whether actual ingestion of food, or only contact with it, is necessary to induce discrimination, the following exper- ment was tried. The waste material, carmine, was mixed with rather large paramecia. ‘The latter may serve as food for Stentor, , Selection of Food in Stentor Ceruleus (Ehr.) 119 but they are rarely captured, though they frequently come in con- tact with the Stentors. Five dishes contained equal amounts of tap water and carmine particles; into some of these there was introduced also certain quantities of food particles, in others none. Fifty Stentors were placed in each of these dishes, and then were examined after 20 minutes to determine the relative amounts of carmine taken. ‘The conditions and results are as follows. 1 Carmine alone. At the end of 20 minutes the Stentors con- tained carmine to the bulk of about 400 Euglene. 2 Several thousand paramecia plus carmine. 3 Several hundred paramecia plus carmine. In dishes 2 and 3 none of the Stentors contained any paramecia. Their carmine content was about half that of the Stentors of dish 1, a bulk equivalent to that of about 200 Euglene. 4 Trachelomonas hispida, T. volvocina, and carmine. ‘The Stentors contained a very few specimens of Trachelomonas. Car- mine to the bulk of about 80 Euglene. 5 Euglena viridis and carmine. The Stentors had in 20 minutes taken each about 275 Euglenz, and carmine equivalent in bulk to about 4 Euglenz. These results seem to indicate that the presence of the paramecia in dishes 2 and 3 decreased the amount of carmine taken up by the Stentors. The paramecia frequently touched the disk, and were often swept into the Stentors’ pouches but always escaped, and it is probable that this touching of the discal or pouch cilia by the paramecia altered the physiologic condition of the Stentor in much the same way as if several paramecia had been eaten. It might be thought that the bacteria carried over with the paramecia produced the result described above, but this is probably not the case, since In many experiments of this nature carmine was put into a portion of filtered paramecia or Stentor culture solution which was full of bacteria, and yet the maximum quantity of car- mine was taken up. | The evidence thus indicates that it is only necessary for food particles to touch the Stentor’s disk or pouch in order that Stentor should then to some extent reject the waste particles. A question related to the one we have just considered is the fol- 120 Asa Arthur Schaeffer lowing. Does not Stentor react or behave differently to such indigestible particles as carmine or india ink after being in contact with them for a few minutes or halfan hour? In other words, may not the Stentor under these conditions be “educated” into reject- ing carmine after having for some time ingested this substance? ‘The paper by Metalnikow, referred to in the introduction, bears upon this very point. This author worked upon paramecium and found that if these organisms are fed with carmine for 15 days or so, they cease to take up this substance. “Fittert man Paramecien sehr lange Zeit hindurch nur mit einem Farbstoff, z. B. mit Karmin, so kann man haufig nachstehende interessante Er- scheinung beobachten. Anfangs, wahrend der ersten Tage des Futterns, bilden sich in einem jeden Infusor 30-40 kleine, gaf arbte, Karmin enthaltende Vacuolen. Hierauf nimmt die Zahl der Vacuolen immer mehr und mehr ab. Bereits nach wenigen Tagen enthalt ein jedes Infusor nicht mehr wie 15-20 Vacuolen, wahrend nach 10-15 Tagen nicht wenige Infusorien angetroffen werden, welche keine einzige gefarbte Vacuole mehr enthalten. Nach einem noch grosseren Zeitraum héren sammtliche Infusorien auf Karmin zu verschlucken” (/. z., p. 183, 184). I attempted to verify Metalnikow’s results upon Stentor and expected at the outset to find perhaps a greater capacity in Stentor to acquire such a “ganz neue Fahigkeit” than in Paramecium, because of the generally accepted notion that Stentor is more highly developed as far at least as its “action system”’ is concerned. It would be in line with the results obtained by my other experi- ments on Stentor. But I was disappointed, as the following experi- ments show. In 5 large covered dishes were placed 60 watch glasses, each one being filled with about 5 cc. of fresh, weak, filtered hay infusion. In 6 of these glasses were placed Stentors; in 6 more were placed Stentors and paramecia; ratio, I :10; 6 others contained Stentors and Euglenz, 1 :100; 6 others contained Stentors, paramecia, and india ink; 6 others, Stentors, paramecia, and carmine; 3 others Stentors and india ink; 3 others, Stentors and carmine; 6 others, paramecia; 3 others, paramecia and india ink; 3 others, paramecia and carmine; 6 others, paramecia and Euglena; 3 others, para- Selection of Food in Stentor Ceruleus (Ehr.) 121 mecia, Euglena, and carmine; 3 others, paramecia, Euglenz, and india ink. One hour after these experiments were set up all the glasses were examined. All the Stentors which were placed in ink or carmine mixtures, whether paramecia were present or not, contained ink or carmine but the amounts were variable. More carmine was found in the Stentors than ink. Likewise all the paramecia placed in ink or carmine mixtures contained vacuoles of these substances. The highest number of vacuoles was from 40 to 50. The lowest number observed was 4. More ink than carmine was found n paramecia. All the glasses were examined once a day after they were set up. Three days after the experiment was begun, some paramecia were found with very few vacuoles of carmine. The smallest number was 4. These paramecia were then placed in fresh car- mine, and in half an hour they contained 50 or more vacuoles each. ‘The carmine and ink content of the Stentors had so far remained constant. Seven days after the experiment was set up, no decrease in the number of vacuoles of ink or carmine in either paramecia or Stentors was found. But one paramecium was found with no carmine vacuoles. This paramecium was then placed with fresh carmine and for over an hour no carmine was taken. It was then placed in fresh ink and in 30 minutes 6 vac- uoles of ink were found to have been ingested. This result is probably due to the fact that the grains of india ink are smaller than the grains of carmine. Ten days after the experiment was set up many paramecia in those glasses where the least ink and carmine happened to be, contained very few vacuoles of these substances, but upon adding some fresh carmine or ink these paramecia at once filled up on these substances as they had done in the beginning of the experi- ment. [he paramecia in the glasses where the carmine or ink was dense contained about the same quantity of these substances as at the start. About the same may be said for the Stentors. In the glasses in which the ink or carmine was dense the Stentors were filled with these substances. In other glasses the Stentors con- tained very little ink or carmine; but upon refilling, these sub- stances were again taken up as at the beginning of the experiment. 122 Asa Arthur Schaeffer Fifteen days after the experiment was begun, Raphidium developed in the glasses and the Stentors died. About six of the dishes which contained paramecia and ink or carmine were how- ever in good condition. ‘These paramecia showed no decrease in the number of vacuoles of these substances. After two or three days these paramecia also died. These results are quite at variance with those described by Metalnikow. I therefore contrived another experiment which eliminates certain possibilities of error to which the above experi- ment and those of Metalnikow were open. A clinostat was set up with a cylindrical jar revolving on the horizontal axis once in every 15 minutes. Seven small vials were then filled with Stentors and paramecia in 5 cc. of their own culture solution. In 3 of these vials was placed carmine; in the other 3 india ink; and the other was left for a control. The vials were corked and placed loosely in the jar revolved by the clinostat. As the clinostat revolved the j jar, the vials rolled around, and as they did so their contents were shaken up thoroughly about three times every 15 minutes. In this way the contents of the vials were distributed uniformly for the thirty-three days throughout which the experi- ment was carried on. The Stentors, paramecia, and culture solu- tion were placed in the vials in the same proportion in which they existed in nature. A little fresh carmine or ink was added every few days. The contents of the vials were examined every day, whenever possible, in this manner. [he contents were poured out into a small preparation dish and placed under a binocular micro- scope. The condition of the Stentors and paramecia was then easily observable. In this wav the water in the vials was also thoroughly aerated. About 30 Stentors and 500 paramecia were placed in each vial. The following are the tabulated results for the paramecia. [he table shows the number of paramecia which con- tained no ink or carmine on the dates shown. Selection of Food in Stentor Ceruleus (Ehr.) 12 OW OCTOBER NOVEMEER — | : ea DAMES fe mcGisical oic'e ® 22 | 23 24 26 27 28 29 | 30 a4 5156 7| 9 |10/11 12|13|16|18| 20) 23 a _ laa be ies oc ae ———}— al4|2|6/r1/1/1/ololo]ojolo}oljojoj/o}ojojojolojo Carmine vials 4b) 2 | 3 2|tl/olololole}1}olo 2) 2.6 o}o|o|o|o 0° Ken Sts | 1}o|oljolo ©05|"0) 10) 16) |o)| © * 010 10 0! 01) 00 (2j2{rj1/1rlolo/rlojol/ojojojojojojo/rjo Ink vials..... ENEO: || T |ed |KO) {0} || ©) |/O7} 0; ko o}ol|o o}ol|o Pit 2/o0l/olololololololjol/olololo o|olo}ololo | | Many paramecia in the act of dividing were observed to have from 5 to 15 vacuoles in them. Only one paramecium was found to contain no ink or carmine in dividing. The paramecia of November 4 and 12 which had no carmine or ink in them were of abnormal, boomerang shape. The number of vacuoles in the paramecia in these vials was almost constant as long as the experiment was carried on—over 33 days. Attempts were made at counting vacuoles and the average high number was about 70. About 5 per cent of the paramecia were found to con- tan only from 5 to 15 vacuoles. In a number of cases such indi- viduals were isolated and fresh carmine or ink added, when they would invariably fill up again. Ink was generally eaten more readily than carmine. The paramecia acted normally as long as the experiment was continued, and there was reason to believe ee the experiment could have been continued further if it had been deemed necessary. As for the Stentors, throughout the entire run of the experiment, none contained neither ink nor carmine. It was impossible to count the vacuoles because of their irregular sizes and shapes. The ink and carmine content of the Stentors was fairly constant. No decrease in the amounts of these substances in the Stentors was found throughout the entire experiment. It is clear from these experiments that if either Stentor or paramecium can be “educated”’ in such a way as to refuse to eat carmine, etc., after feeding carmine to these organisms fora month, it can only be done under very special conditions. Metal- nikow does not describe his experiments in detail, but from what 124 Asa Arthur Schaeffer I was able to gather from his paper with regard to the method of work our results disagree utterly. What is the reason for the disagreement? Metalnikow says that after his paramecia had ceased to take up carmine he stirred up the solution and yet the paramecia refused to ingest any more carmine. In my work [ also found this to be true, but l attributed this to the fact that the carmine is no longer mixed in the same way as 1t was originally, for the mucus excreted by the paramecia, and other cellos matter in the water, cause the carmine particles to stick together, so that the paramecia no longer take this kind of carmine with readiness. Further Metalnikow states that if one takes some of these paramecia which have refused to eat carmine and places them in fresh carmine, none is taken up. I have also tried this many times, but in almost every case I found that the paramecia again took up fresh carmine. In some few cases | found paramecia which did not eat carmine under any circum- stances, even if they were placed in fresh solutions, but close exam1- nation nearly always showed that these rare individuals either were deformed or had just previously divided, and so were unable structurally to ingest the grains of fresh carmine. It appears possible that Metalnikow unfortunately got hold of some of these individuals. But there is another statement in his paper which is hard to reconcile with the one quoted above. “Halt man die Infusorien bei massig hoher ‘Vemperatur (d. h. bei 10-15° C.; die Versuche wurden wahrend der kalten Jahres- zeit angestellt), so tritt wahrend mehrere Tage keine Uheilung ein. “Jeden Tag untersuchte ich ein solches ungeteiltes Infusor auf seine Fahigkeit hin Karmin zu verschlucken. Im Verlauf von zwei, dre (in seltenen Fallen auch 4) Tagen, wahrend welcher Zeit es mir gelungen war die Infusorien von einer Theilung abzuhal- ten, konnte ich nicht bemerken, dass ihr Verhalten zum Karmin irgend welche Abanderungen erfahren hatte. Jeden Tag weiger- ten sich die Infusorien hartnackig Karmin zu fressen. Kaum aber hatte sich ein Infusor geteilt, so anderte sich sein Verhalten plotzlich und es begann von neuem Karmin zu fressen. In den- jenigen Fallen wo ich das Infusor in den Thermostat oder an Selection of Food in Stentor Ceruleus (Ehr.) 125 einen warmen Ort (von iiber 20° C.) vebrachte, trat eine [heilung meistens sehr bald ein und die Tochterinfusorien begannen sofort Karmin kornchen zu verschlucken.”’ Metalnikow thus finds that although paramecia learn in some way to refuse carmine after being fed with it for some time, yet as soon as they divide, the daughter paramecia at once take up car- mine as before. But he also says that after paramecia had been left in carmine for 15 days or longer, most of them contained no carmine whatever. Whether there were only very few paramecia dividing in this latter case, or whether the paramecia after several generations inherited this acquired character of refusing to ingest carmine, we are not told. These important details as well as others to which attention is called above, are not given in Metal- nikow’s paper, which is indeed only a preliminary paper. But until such details are made known, and until Metalnikow’s experiments are described in detail, so that they may be verified, a discussion of our results which disagree in every essential par- ticular, is futile. THE BASIS OF SELECTION A series of experiments will now be taken up which were designed for the purpose of ascertaining if possible upon what basis selection in Stentor is exercised. We have seen from the experiments up to this point that Euglena are eaten with more readiness than Phacus, and that carmine is more readily eaten than india ink, and that between living and dead organisms no selection seems to be exercised. Whatcan be the basis of discrim- ination that gives such results? So far as we can tell there are only two possible methods by which Stentor can discriminate between two different substances; one is by a chemical sense (“tasting” and “smelling’’), and the other by a tactual sense (“touching’’). Does Stentor select a particle by “ touching”’ it, or by “tasting it,” or by both methods? (The psychological terminology is used here merely for convenience. By tasting and touching are meant reactions to chemicals and to contact and form, respectively.) 1260 Asa Arthur Schaeffer In the first experiments, organisms which are readily eaten by Stentor were altered as far as their chemical nature was concerned. Some were cooked, some soaked for a considerable time in alcohol, others in 10dine, osmic acid, mercuric chloride, tannic acid, etc. Kuglenz, ‘Trachelomonas, and very small paramecia were the organisms employed for these purposes. After they had been treated with the chemicals mentioned, they were thoroughly washed and sucked up into pipettes. Hungry Stentors were iso- lated; then normal living organisms, and also those treated with the chemicals, were fed to the Stentors in mixed order, as was done in the first series of experiments. In no case were the chem- ically treated organisms rejected while the living were eaten. Some Stentors ipecced all the organisms, and others rejected some of the living smal some of the chemically treated, while the rest, including organisms of both kinds, were rejected. It was found in previous experiments that Stentor could dis- criminate between Phacus triqueter and P. longicaudus, and between Trachelomonas hispida and T. volvocina, etc. In the experiment just preceding Stentor did not discriminate between living organisms and organisms killed with osmic acid, iodine, etc. Now it seems aviansies that Stentor selects upon a chem1- cal basis, since it is hard to understand how there could be a greater difference in “ taste’? between living Phacus triqueter and living P. longicaudus, where there was selection, than between living Phacus triqueter and specimens of P. triqueter killed with acids, iodine, etc., where there was no selection. But to test this result further another set of experiments was performed in which the form and surface texture of food organisms was changed to a greater or less extent, but the chemical nature was left, as nearly as possible, in the same state as in the normal organism. Paramecia and Euglenz were used for these purposes. By means of very small platinum knives these organisms were cut into halves or quarters, or even smaller pieces, and then fed in a stream with whole living organisms of the same species. ‘There was no discrimination. ‘The pieces were eaten as readily as the whole specimens. Many of these organisms were then mashed and minced into very fine fragments so that the original form and Selection of Food in Stentor Ceruleus (Ehr.) 127 surface texture was quite destroyed. This “jelly”? was then sucked up and fed to Stentors together with normal living organ- isms of the same species. [he whole specimens were eaten but the “jelly” was rejected. The Stentors bent away or reversed their cilia the moment the mashed paramecia or Euglenz touched the disk or pouch, exactly as they do when a cloud of carmine ink, etc., is similarly caused to come in contact with the Stentor. Small starch grains were then mixed with this jelly and diluted and fed to the Stentors. No starch grains were eaten. The Stentors still reversed their cilia when any of this mixture touched the disk. Starch grains (potato and corn) were then fed in solutions of various strengths of sugar, beef juice, pork juice, pepsin, Liebig’s Extract of Beef. The starch was invariably rejected. This same thing was tried with carmine and india ink, but the same results were got here as were derived from the controls; no more carmine or ink was ingested if pork or beef juice, or sugar, was present, than when these were absent. Stull other experiments were carried on in which the food organisms were soaked in various chemicals such as iodine, anilin dyes, alcoholic solutions of quinine, etc., and then only part of the superfluous chemical was washed away, so that when fed, a little of the iodine, quinine, etc., would be in solution and thus give the Euglenz or paramecia quite a different “taste” from that which these organisms normally possess. These experiments are very difheult to carry out, owing to the fact that when iodine or quinine is just a little too strong, the Stentors contract the moment these substances touch the disk, and yet if one washes these chemically treated organisms a little longer one cannot be sure that any iodine or quinine remains to affect the Stentor before the organisms are swallowed. ‘The experiments are also somewhat uncertain, for in the case where an organism is eaten, we cannot be sure whether the quinine, dye, or iodine, etc., had any effect on the Stentor at all. But the following are the results. When the food organisms were not too vigorously washed, the Stentors that were fed with them bent away or contracted the moment the stream from the pipette touched the disk. When the washing was carried a little further the organisms were ingested as were the living organisms. 128 Asa Arthur Sch ae jfer Now if Stentor selects its food upon a chemical basis we should expect something like the following to occur. 1. Living organ- isms ought to call forth a different reaction from those cooked or chemically treated, since 1t seems evident that the “taste”’ of these two classes of food is very different. 2. When living organisms are fed we should expect the behavior to differ from what occurs when they are fed in sugar solution, etc. 3. We should not expect carmine or ink to be eaten. 4. Starch grains would probably be eaten if soaked in, and fed with, paramecium juice; and the minute fragments of paramecia would also probably be eaten 1f Stentor selected its food upon a chemical basis. But as a matter of fact the reverse is true in each case. But let us see what Stentor would probably do if it selected its food upon a tactual basis. Under tactual stimuli all those of size, weight, form, and surface texture, and discrimination might be made upon any one or all of these factors. It is clear from the outset that size plays little or no part in the selection, for all sizes of organisms from bacteria to paramecia are ingested. So we have only to consider weight, form, and sur- face texture. Selection upon the basis of weight alone would result in rejecting all substances which differed in weight (specific gravity) from living organisms. ‘This would explain the rejection of glass, sand, starch, etc., and the ingest on of carmine and ink. But it prob- ably would not explain selection obtaining between Phacus and Euglena or between Euglena and Chilomonas, or between a rapidly moving organism like Halteria and any other particle. Selection on the basis of form alone would also fail to explain all the results obtained in the experiments. For a Phacus differs more from a Euglena in form than from a starch grain, ete. Nor would the factor of surface texture completely explain all the results obtained in the experiments. It seems clear therefore thatas far as my experiments go, no single quality such as weight, or form, etc., is decisive for setting off the Stentor’s ingesting mechanism in all cases where discrimina- tion occurs. [tis probable that more than one factor serves as a basis for discrimination. Selection of Food tn Stentor Ceruleus (Ehr.) 129 But whatever the basis is, itis constantly varying. ‘The degree of hunger, as we have seen, has a marked influence upon selection. Hunger perhaps does not form part of the real basis of selection inasmuch as it only influences the degree of accuracy in discrimi- nation. The same statement may be made as regards mere con- tact with food. As was shown in experiments described on page 119, the mere contact of paramecia with Stentor seems, like hun- ger, to have increased the degree of accuracy in discrimination. The experiments then in this part show that Stentor probably selects its food upon a tactual and not upon a chemical basis. Further the experiments seem to indicate that more than one of the factors, weight, form, or surface texture, serve as a basis for discrimination. THE SELECTIVE MECHANISM We have seen that there are two mechansims by means of which discrimination is directly brought about. The ingesting mechanism is set into action by certain qualities in particles, when the Stentor is hungry. The absence of these qualities, or the presence of qualities which are objectionable to Stentor, call into action the rejecting mechanism. ‘These two mechanisms, taken each by itself, are constantly varying with regard to the strength of stimulus required to set either into action. As the Stentor passes from a hungry toa satiated stage the ingesting apparatus is continually less and less easily set off. But with the rejecting apparatus the reverse 1s true. As the Stentor grows more and more satiated, constantly weaker stimuli set off the rejecting mechanism until finally all particles stimulate it into action. These changes are due to the constantly varying physiological state, brought about by the constantly accumulating food in the body of Stentor. That is, the amount of food ingested regulates solely the ease with which the ingesting or the rejecting mechan- isms are set off. But in some cases there is evidently a direct effect produced in the stimulus-receiving organ by the stimulus. Such cases are those experiments in which carmine and food par- ticles were fed, sometimes mixed, and sometimes each substance 130 Asa Arthur Schaeffer by itself. Some Stentors from the beginning of the experiment rejected carmine apparently only because food was also present. The rejecting and the ingesting mechansism may therefore be acted upon and their mode of action in consequence changed, both by the physiological state as determined by the amount of food in the animal, and by the condition of the stimulus-receiving apparatus as influenced by the amount of previous stimulation. Whenever a particle possesses qualities that stimulate to a greater or less extent both the ingesting and the rejecting mechan- ism, the action of the cilia in the pouch and funnel are not thor- oughly coordinated either for ingesting or for rejecting, and as a consequence loops are formed in the path of the particle. It seems from the experiments that the stimuli from the objectionable features of a particle are summated more rapidly than the stimuli from the desirable features; for 80 per cent of all the particles that | had loops in their paths were finally reyected. We also find that a much larger number of loops occur when the Stentor is beginning to be replete than when hungry or when almost satiated. At the stage when the animal is just beginning to be replete, stimuli of medium intensity are required to set off either the ingesting or the rejecting apparatus. ‘he stimuli are more nearly of equal inten- sity, or in other words, the mechanisms are set off more nearly with equal readiness at this stage than at any other. This 1s probably the reason for the larger number of loops at this stage. Selection between food and indigestible particles in Stentor is an almost perfect adaptation. With very few exceptions only particles (organisms) of food value are ingested. Indigestible particles of many sorts which have for thousands of generations not come in contact with Stentor are nevertheless rejected with accuracy. SUMMARY 1 Stentor ceruleus exercises a selection among the particles that are brought to its food pouch by the ciliary current. The selection is brought about by changes in the beat of the cilia of the pouch and funnel. Certain particles are rejected by a localized Selection of Food in Stentor Ceruleus (Ehr.) 131 reversal of the cilia; others are carried to the mouth and ingested. Of two particles within the funnel at the same time one may be thus rejected while the other is ingested. Selection thus occurs not only from among particles reaching the pouch successively, but from a large number reaching the pouch at once. 2 Stentor discriminates very accurately between organisms (Phacus, Euglena, etc.) and indigestible particles (carmine, glass, sulphur, starch, etc.), ingesting the former and rejecting the latter. 3 Stentor discriminates between different kinds of organisms, eating some (Euglena, Phacus triqueter) with great readiness, while others (Trachelomonas hispida, Phacus longicaudus) are rarely ingested. 4 States of hunger and satiety, and intermediate conditions, are shown to exist in Stentor by differences in the behavior toward food. The animal discriminates more perfectly (1. e., more restrictively), when almost satiated than when very hungry. When very hungry it may ingest many indigestible particles (car- mine, india ink, etc.) 5 The amount of a given substance ingested. depends upon what other substances are present. Stentor in water containing indigestible particles, such as carmine, may ingest much of the latter; if the water contains in addition many organisms fit for food, very little of the indigestible matter is ingested. 6 It was not found that Stentor and paramecium become lastingly “educated” to reject certain sorts of food that they have formerly taken. Such changes as occur in selection seem to be mainly matters of hunger and satiety. 7 Stentor selects its food upon a tactual basis and apparently not upon a chemical one. That is, Stentor reacts in selecting food, to physical properties only or chiefly, and not to seenical properties. 8 Stentor in a condition of satiety differs in many respects from Stentor in a condition of hunger. In satiety we find the following conditions: a. Extension is always submaximal. 6. The aboral side is more strongly convex than the oral side. c. There is a marked decrease in the activity of the membranellz. d. Stentors respond much more readily to mechanical stimulation 132 Asa Arthur Schaeffer at the disk. e. If the stimuli are submaximal, contraction is often only partly accomplished. g ‘The amount of food in the body of the Stentor at any given moment is not an accurate register of the degree of satiety; the latter depends upon what recent stimuli have been received, as well as on the amount of food in the body. REFERENCES Burscnit1, O. ’87—89—Protozoa. Vol. i of Bronn’s Thierreich. Leipzig. — EHRENBERG, C. G., °38—Die Infusionsthiere als vollkommene Organismen. Leipzig. Entz, Gera ’88—Studien iiber Protisten. Auftrage der kénigl. Ung. Naturw. Ges. Budapest. Hoper, C. F., anp Aixins, H. A. ’93—The Daily Life of a Protozoan. Amer. Journ. of Psychology, vol. vi. Jennincs, H. S. ’02—Studies on Reactions to Stimuli in Unicellular Organisms. vii. Amer. Journ. of Psychology, vol. vi. Meissner, M. ’88—Beitrage zur Ernahrungsphysiologie der Protozoen. Zeitschr. f. wissensch. Zool. Bd. 46. MetTALNikow, S. ’07—Uber die Ernahrung der Infusorien und deren Fahigikeit ihre Nahrung zu wahlen. ‘Travaux de la Societe Imp. des. Naturalistes de St. Petersbourg. Bd. 38, Lief. 1, No. 4. STEIN, F. v. ’67—Der Organismus der Infusionsthiere. Bd. I. Leipzig. Verworn, M. ’89—Psychophysiologische Protistenstudien. Jena. EIGHT As: A FACTOR IN THE. REGENERATION. OF HYDROIDS SECOND STUDY A. J. GOLDFARB Zoélogical Laboratory, Columbia University, New York Three different kinds of hydroids are found abundantly in the harbor of Woods Hole, Mass. Of these Tubularia (Parypha) crocea reaches its most luxuriant growth about the end of June and then declines in numbers and vitality toward the end of July or the first part of August, at which time they disappear completely. In the meantime Eudendrium ramosum begins to grow about the first part of July, reaches its maximum growth about the end of July and then gradually disappears. [Pennaria tiarella comes last, toward the end of July, and persists until the beginning of September. All three kinds may be found on the same piles at the same time. In a previous publication' the curious effect of light upon the regeneration of Ek. ramosum was described. From those studies it was evident that the idea of the simple and direct effect described by Loeb,? needed radical revision. In brief the facts for E. ramosum are as follows. Under ordinary conditions, hydranths are replaced in about 48 hours. As the hydranths live only a few days, they are replaced again and again by new ones. If the number produced on successive days be examined, it is found that the largest number are regenerated within a few days after their removal, and that the number steadily decreases on succeeding 1 Goldfarb, A. J. Experimental Study of Light as a Factor in the Regeneration of Hydroids. Journ. Exp. Zodl., vol. 3, 1905. *Loeb, J. The Influence of Light on the Development of Organsin Animals. Pfliiger’s Archiv., Bd. 63, 1895. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 8, NO. 2. 134. A. “f. Goldfarb days, until about the 13th day, after which no more hydranths are produced. When these colonies were removed to a dark chamber, dark enough not to effect photographic plates, the number of hydranths produced in this chamber, and the rate of their development, was not materially altered. Regeneration took place in the dark as well, or almost as well, as in the light. But this statement holds true only during the first cycle’. Ia after this period a pro- found change occurred, as a result of which these hydroids did not regenerate a single hydranth so long as they were kept in the dark. ‘This treatment rendered them remarkably responsive to light, so that exposure always induced the regeneration of hy- dranths. But the surprising feature was the intense sensitive- ness which hydroids so treated seemed to possess. Exposure to shaded light of 15 seconds sufhced to stimulate the regeneration of a Seale series of hydranths. No explanation of this peculiar relation to light was given. It cannot be said that E. ramosum is dependent upon light by virtue of substances produced by symbiotically pecociaed organisms. Nor is it dependent directly upon light for its sustenance in the manner of plants. The following experiments were undertaken with the idea of finding what influence light has upon the regeneration of the other two closely associated hydroids, to ascertain whether this extreme sensitiveness to light is common to the other forms, and _ possibly to determine what significance this fact has in the life history of these plant-like animals‘. The procedure was practically the same in all the experiments. The hydranths were removed from the selected stalks and the same number were placed in wide shallow dishes containing the same quantity of sea-water. To prevent the rapid accumulation of bacteria, the water was changed at first daily, and in the later experiments, on alternate days. The controls were kept in the shaded light of the laboratory, the others were prepared, in the * Vide Journ. Exp. Zool. Vol. 3, 1905. * The experiments were made at the Marine Biological Laboratory, Woods Hole, Mass. For the priv- ilege of working therein I am indebted to the Director, Prof. F. R. Lillie. Light in the Regeneration of H ydroids 135 manner just described, and kept in a dark chamber. The manipu- lation, change of water and counting of hydranths of the latter stalks were all done in the dark room, into which the only light was that which came through a double thickness of ruby glass. The temperature of the controls varied slightly from the others, the difference never exceeding 2 to 24°C. As each new hydranth was fully developed it was removed. Thus the figures in the tables represent the actual number of regenerated hydranths. TUBULARIA (PARYPHA) CROCEA This is a colonial hydroid usually made up of unbranched stalks, though not infrequently small branches are developed. Stalks were cut off quite close to their bases, their hydranths were then removed at approximately the same level for all the stalks of a series, 1. e., either at the base of the hydranth or the middle of the stalk, etc. The results obtained among the controls and among those maintained in the dark chamber are given in the accompanying table. In Experiment 1, the hydranths were removed about five- eighths inch from the apical ends. No branching stalks were used. ‘The 24 controls were prepared in the light, 40 others were prepared and kept in the dark. In Experiment 2, there were 20 controls, and 42 inthe dark. In Experiments 3 and 4, all hydranths were removed in the light. In Experiment 3, there were 20 con- trols, 60 in the dark. In Experiment 4, the hydranths only were removed, leaving one part of a colony of about 50 stalks, con- nected at their bases, to serve as a control, and another part of the colony was placed in the dark. In all there were 114 controls and 192 1n the dark. As in E. ramosum, the hydranths are regenerated ordinarily within two days after their removal. In the dark, their first appearance was somewhat delayed, though all subsequent regen- eration took place at the normal rate. This initial retardation is seen in Experiment 1, where 24 controls regenerated 21 hy- dranths on the second day and 1 on the third day. The 40 kept in the dark regenerated 5 on the second day, while 23 appeared on 130 A. “f. Goldfarb TABLE I EXPERIMENT I EXPERIMENT 2 EXPERIMENT 3 EXPERIMENT 4 Inthe Inthe Inthe Inthe Inthe Inthe | Inthe Inthe Dark Light | Dark Light | Dark Light | Dark Light No. of stalks 40 24 42 20 60 20 50 50 Regenerated Regenerated Regenerated | Regenerated 2d day 5 21 4 14 I 3 ° fo) 3d 23 I 10 8 8 3 ° ° 4th 6 5 3 2 4 5} ° ° sth 3 4 | fo) fo) I 3 ° I 6th 3 ° | 3 ° | 3 I I 2 7th 4 I | I 2 | 4 Z I 10 8th I Ov l| I I | 3 ° 6 5 gth ° ° ° ° | 2 I 7 4 1oth 2 fe) | ° ° ° I 7 2 rth ° ° | fo) ° ° ° 9 2 12th ° ° ° I 2 ° ° 4 13th ° ° | ° I I ° 3 ° Total Reg. 47 32 22 29 | 29 21 34 30 Per cent Reg. 117 133 52 145 | 45 105 68 60 | ig | Exposed 15 minutes 14th ) ° ° | I 15th ° I fo) ° 4 fo) Exposed | Permanently | 16th ° ° 4 ° ° 3 ° 17th ° ° ° ° ° ° ° 18th 2 ° fo) ° I fe) 2 ° 1gth I ° I fe) ° ° ° ° 20th fo) fe) ° ° 21st I fe) fo) ° 22d ° ° ° 24th | ° ° 25th fe fc) | | TOTAL NUMBER NUMBER PER CENT | OF STALKS REGENERATED | REGENERATED Injthetdarke sina cincletsto ore to etocietsiatsrsei newer 192 132 68 An ‘thelipht-t enteric sb ce cr teen IN | 114 112 99 Light in the Regeneration of H ydroids 137 the thirdday. In Experiment 2, 20 controls regenerated 14 and 8 hydranthson the second and third days respectively, while 42, in the dark, regenerated 4 on the second and 10 on the third day. The same relation obtains in Experiment 3. In Experiment 4, 50 controls regenerated 1, 2, 10, 5, 4, and 2 hydranths on the 5th, 6th, 7th, 8th, gth and toth days respectively, while the 50 in the dark regenerated 0, 1, 1, 6, 7, and 7 hydranths in the same time. The curve representing the number of hydranths regenerated on successive days, are analogous and that given for Eudendrium. The maximum number produced at any one time appeared within a few days after the beginning of the experiment, and the num- ber steadily decreased on the following days until no more were produced. In Experiment 3, no more hydranths appeared after the roth day, in Experiment 2, after the 13th day, in Experiment 4, after the 14th day, and in Experiment 1, after the 15th day. Though the observations were continued for over 25 days the con- trols no longer produced any hydranths. In this respect Tubu- laria crocea behaves exactly like Eudendrium. While the controls no longer regenerated after 7, 10, 13 and 14 days respectively, those kept in the dark produced new hydranths during 10, 13, 8 and 17 days for the corresponding series. ‘This difference in time, namely, 3 days, was much greater than the initial retardation. ‘This may be interpreted to mean that the prolonged darkness continued to retard the development of the hydranths. Beside this retardation, the removal of light also inhibits develop- ment to a considerable degree. Even during the first cycle, while the number of hydranths that were regenerated was large, the maximum (maximum per cent rather than the absolute num- ber is here referred to), for any one day was always less than among the corresponding controls. Also the total per cent regen- erated during a definite period, 13 days for example, was about 31 per cent less in the dark, than in the light. Darkness then exerts certain definite and measureable effects even during the first period or cycle, and in these respects the behavior of this hydroid is not unlike that of Eudendrium. But it is only after this first cycle, that the stalks kept in the dark 138 A. 7. Gold farb come to differ so radically from the controls. ‘The latter by this time were quite spent, i. e., they no longer produced hydranths in the light. ‘Those in the dark, likewise produced no more hydranths but upon exposure to light not a single but a series of hydranths were regenerated. In Experiment 1, and 2,4 and 5 hydranths, respectively, were regenerated in this manner. An exposure of but 15 minutes sufhced to stimulate the production of several hydranths. In this respect also Tubularia closely resembles Eudendrium, though the latter was far more sensitive to light, as shown by the eee number of hydranths regenerated after exposures, by the eee: regenerating period, and by the briefer light stimulus that ee to bring these about. There are two minor points that may be mentioned at this place. (1) Individual records made it quite certain that stalks kept in the dark could regenerate a second and third time. (2) Colonies whose stalks had been separated behaved in exactly the same manner as those that were not so separated from the colony. The experiments were drawn to a close by the lateness of the season, when good healthy stalks were no more to be procured. It would have been interesting to have ascertained with far more exactness the minimal exposure that would have stimulated a regenerative cycle, to have ascertained whether a second or third eae could have been induced by such brief or briefer exposures. But the facts, so far as they go, clearly indicate that Tubularia crocea behaves essentially like Eudendrium. During the first cycle, regeneration takes place in the dark almost as well as in the light; that after this period regeneration occurs only after the stalks are exposed to the light. ‘The two hydroids differ in that longer exposures are required to stimulate Tubularia, and that fewer hydranths result from such stimulation; in other words Tubularia is less sensitive to light than Eudendrium. The uni- formity of the results in the four experiments bespeaks the correct- ness of these conclusions. Light in the Regeneration of H ydroids BS? PENNARIA TIARELLA It has been already pointed out that this hydroid lives with T. crocea and E. ramosum on the same piles and at the same sea level. Like Eudendrium, it is very much branched, and experi- ments showed that the regeneration of similar pieces from these two hydroids gave practically the same results. New hydranths were regenerated in about 48 hours after their removal, and in this regard resembled the other two hydroids. These newly formed nycuauthe were removed daily, so thatthe figures actually represent the number of different hydranths regen- erated. There is one disturbing factor that has to be reckoned with, namely, the tendency to produce “roots” in place of hydranths particularly after thigmotactic stimuli, such as contact with the side of the dish, or with other stems. In as much as the size of the stalks, of the dishes, the number of stalks in each dish and other conditions were quite the same among the controls and among those in the dark, this disturbing factor may be fairly assumed to be constant in both sets of stalks. In some colonies this ten- dency to root formation is so strong that nearly all cut ends pro- duced roots instead of hydranths. The regeneration of Pennaria differs from the other two hydroids in several respects. In the first place, the curve represented by the number of new hydranths on successive days, was not so definite as in Tubularia or Eudendrium, due in all probability to the tendency to heteromorphic “root”? formation. Yet the curve of Pennaria approximated very closely that of the other two hydroids. This 1s shown as follows. The maximum number of hydranths produced on any one day, appeared during the early part of the experiment, and if continued fora long period the number towards the close of the experiment was always very small; and these hydroids, it might be added, were decidedly smaller, 1. e., one-half to one-third the normal size. In Experi- ment 5, the number produced on the 2d to the 6th days inclusive, was 12,15, 16, 13,11 hydranths. During succeeding 5 day in- tervals, the greatest number that appeared on any one day was 140 A. “f. Goldfarb 13, 8,4 and 2. Greater irregularities took place in Experiments 6 and 7. In Experiment 6, 8 was the largest number of hydranths present at one time during the first 5 days and during succeed- ing § day intervals the largest number was 14, 17, 9, 10, 5, 13, and 5 hydranths. In Experiment 7, the figures for the same in- tervals were 10,10, and 7 hydranths. In the pecond place, new hydranths were regenerated during a longer cycle than either of the other two By deoides 23 days in Beceaede 7, 26 days in Experiment 5, and 35 days in Experi- ment 6, and would in all probability have continued to regenerate for a longer period. After so protracted an interval the hydranths were not only fewer as mentioned above but were decidedly smaller or malformed. The most decided difference was observed in the behavior of those stalks that were placed in the dark. From the first not a single hydranth was regenerated from these stalks. Although a little over one fpaueaad branchlets and pedicels from various colonies were placed in the dark, in no instance was a hydranth produced. Pennaria unlike Tubularia and Eudendrium requires no preliminary treatment in order to bring about a total cessation of the regenerative processes. As might have been anticipated, the stalks in the dark required particularly long exposures in order to stimulate the formation of new hydranths. Exposures of 2 minutes and 5 minutes (Experi- ment 5) proved totally inefhcient. Exposures of 3, 5, 8, 10, 15, 20, 25, 30, and even 60 minutes (Experiment 6) were equally inadequate, even though the exposures were made in the direct rays of the sun. Exposures of 2 hours, 3 hours and 4 hours were sometimes ineffective and sometimes produced hydranths. In I-xperiment 6, for example, 200 pedicels exposed for 2 hours regenerated during the next five days only 4 hydranths, while the controls regenerated 70 hydranths in the same time. Exposures of 3 and 4 hours gave no better results than the 2 hour exposures. The hydranths so produced were frequently dwarfed. The mini- mal time required to stimulate regeneration could be still further reduced, in some colonies, by the simple expedient of exposing the hydroids daily. Instead of two or more hour exposures, regenera- Light in the Regeneration of H ydroids 141] tion could be induced with far more certainty by exposures of one hour or one-half hour for several successive days. And although very few hydranths were produced at any one time they appeared on as many as 5 separate days. ‘Thus, daily half hour exposures resulted in 0, 0, 3, 6, 0, hydranths in one set of stalks and 0, 0, I, I, I, O in another set. One hour exposures daily gave rise to Bona, 1) tO: ande,/0, '0,'45.0; 0, and 0} 0,2, 1, 2, 2*hydranths in 3 different sets of stalks. If left in the light for a whole day, hydranths were almost certain to appear in every experiment and these were large and numer- ous. Though the absence of light was inimical to development yet no permanent injury resulted from prolonged retention in the dark. For on returning the stalks to the light many normal sized hydranths were immediately regenerated. After 10 days in the dark the stalks in Exper:ment 5, were brought into the light and produced G, 1, 4,8, 0, 5, 55 1,0, 3, 0; 2, ©, 4, 1,°3 hydranths during the next 16 days. In Experiment 6, after 16 days in the dark 0, 14, 31, 26, 68, 17, etc., hydranths were regenerated on successive days, There is a variety of Pennaria that is found attached to eel grass not far from shore. ‘This is said to belong to the same species as that found on the much more shaded piles of the wharves, though it differs from it in several respects. Some experiments were made upon this variety, to determine whether the difference in habitat was reflected in a difference in the amount of light required to stimulate regeneration. ‘The stalks of this variety, it was found, required as a rule longer periods of exposure to stimulate the development of new hydranths. In other respects their behavior was quite the same. Loeb? in his account of the regeneration of Eudendrium ramo- sum stated that this hydroid never regenerated in the absence of light. He undoubtedly confused Eudendrium with Pennaria tiar- ella. In conversation with the writer Loeb expressed the opinion that this was probably the case. These comparative studies make it perfectly clear that so re- markable a sensitivenes as that displayed by Eudendrium (after the first cycle) finds no parallel among the other two closely 142 A. y: Goldfarb associated hydroids. On the other hand the absence of light is not so directly preventive of hydranth formation among Tubularia and Eudendrium as in Pennaria. ‘hese hydroids living practi- cally in the same environment agree in that after they have ceased to odes hydranths they may be stimulated to regener- ate them by light and vice versa its absence retards and ulti- mately alte development. But the conditions and the de- gree to which light is effective varies with each hydroid. In P arypha and Badendaum the absence of light inhibits regenera- tion only after a prolonged preliminary period. In Pennaria it is a coditio sine qua non from the very beginning. SUMMARY Light is a well defined factor in the regeneration of these hydroids, but the degree of effectiveness and the duration of the preliminary period required to render the hydroids susceptible to light stimuli varies. Eudendrium ramosum has a long pre- liminary cycle during which regeneration takes place in the dark ‘almost -as well as: in tee light. After this period, no regeneration occurs so long as stalks are maintained in the dark. A very brief stimulus, that 1s, an exposure to the light of 15 seconds, sufhced to call forth a series or cycle of hydranths. New series of hydranths could be produced again and again by repeated exposures. Like Eudendrium, Tubularia crocea also has a preliminary period of about 13 days during which hydranths are developed almost as well as in the light. re the expiration of this cycle, regeneration may be stimulated by exposure to the light of about 15 or more minutes. Pennaria tiarella diiters from the other two hydroids in that there is no preliminary cycle." sirom the beginning hydranths are never regenerated in the dark. They may be stimulated to develop only by long exposures of 2 hours or more, or by exposures of one-half to one hour daily. * Roe He k ol UDIES OF THE PROCESS OF HEREDITY IN FUNDULUS HYBRIDS! H. H. NEWMAN Wirn Seven Text-Ficures I. THE INFLUENCE OF THE SPERMATOZOON ON THE RATE AND CHARACTER OF EARLY CLEAVAGE In a previous paper on Fundulus hybrids’ it was stated that the developmental rhythm of the young embryo was distinctly influenced by the foreign spermatozoon as early as fourteen hours after fertilization. It was also conjectured that the influ- ence of the male cell was operative at a much earlier period, although it did not manifest itself in a measurable degree. Attempts were made to test the influence of the foreign sperm upon the early cleavage rhythm, but the results were largely negative owing to the crudeness of the methods employed. As the writer was convinced that these results needed reéxam- ination, the work was resumed during the summer of 1909. This time the methods proved to be sufficiently refined to suit the case and positive results were obtained. The treatment was statisti- cal in the sense that very large numbers of eggs were examined, and this involved an amount of tedious labor and eye-strain probably out of proportion to the value of the results obtained. During the earlier attempts much difficulty was experienced in obtaining satisfactory data concerning the rate of cleavage. It seems a simple enough matter, to one who has not made the at- tempt, to enumerate the 2- and 4-cell stages in a batch of a thou- sand eggs or more. Inreality, however, the difficulties are numer- 1 Contributions from. the Zodlogical Laboratories, University of Texas, No. 100. 2 Journal of Experimental Zodlogy, vol. v, no. 4. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 8, NO. 2. 144 fea Jaaks Newman ous and not entirely surmountable for reasons that will soon become clear. A large percentage of the eggs show intermediate stages between the 2- and the 4-cell condition, and in many cases the first two blastomeres undergo the second cleavage at different rates, pro- ducing 3-cell conditions. In earlier studies all eggs were counted as 4- ccell stages if the second cleavage had begun, thus grouping into one Gees all stages from the end an the 2-cell to the beginning of the 8-cell rite In practically all of the earlier experi- ments the additional error was made of allowing the development to proceed a little too far, so that nearly all ae the eggs had at least begun the second cleavage. In later stages (8-, ie and 32- cell conditions) it became a matter of great difficulty to assign the various individuals to one of two classes, for the reason that cleavage is far from regular either in time rate or the arrange- ment of the cells. In all experiments described in the previous paper, where the eggs of Fundulus majalis were involved, the results show a slightly more rapid rate of cleavage in the hybrid than in the pure-bred eggs. The eggs of F. majalis were found to be much more suitable for a statistical treatment of the rate of cleavage than those of Fundulus heteroclitus because of their larger size, the greater contrast between the color of the yolk and of the blastomeres, and also, a matter of extreme importance, the fact that the eggs if fertilized in a small amount of water, remain entirely separate from one another and can be kept evenly distributed in a single layer on the bottom of large flat dishes. All eggs can thus be kept under identical conditions, so far as illumination, tempera- ture, and oxygen supply are concerned. The eggs, moreover, are not sticky and can readily be handled with needles or pipette. Those of F. heteroclitus, on the other hand, invariably cohere in clumps so that the innermost eggs are more or less cut off from light and oxygen. It is possible, of course, to separate these clumps, but the operation is a slow and tedious one and before it can be completed many eggs will have been hindered in their development. Even after these eggs have been separated they are difficult to handle as they remain somewhat sticky for a long Heredity in Fundulus Hybrids 145 time and tend to adhere in a very unpleasant way to the instru- ments used for handling them. For these reasons, the eggs of F. heteroclitus are deemed unfit for experiments of the present sort and attention has been given solely to the eggs of F. majalis. The species F. majalis develops only about two-thirds as rapidly as the species F. heretoclitus, the former reaching the hatching period in about three weeks; the latter in about two weeks. ‘The same general time ratio prevails throughout the entire develop- mental process. In F. majalis the second cleavage occurs in about four hours after fertilization, while in F. heteroclitus it occurs in about three hours. Hybrid eggs (F. majalis 9 F. heteroclitus *) show a slight acceleration of early cleavage as compared with pure-bred F. mayalis eggs. Expecting the influence of the foreign spermatozoon to be very slight at so early a period as the second cleavage, a very large number of eggs were used and as many experiments were per- formed as the time would permit. In all experiments it was found necessary to distinguish several stages between the 2- and the 4-cell conditions. As a rule the following stages could be distinguished: a. Complete 4-cell, shamrock-shaped, with each blastomere separated from its neighbors by a deep notch (Fig. 7). b. Incomplete 4-cell, with the notches of the second cleavage less deep than those of the first (Fig. 6). c. 3-cell, in which the second cleavage had occurred in one of the blastomeres and not in the other. d. 2-cell with second cleavage furrow just beginning (Fig. 5). e. Complete 2-cell, with the blastomeres separated by deep notches (Fig. 4). 7. Incomplete 2-cell, with the first cleavage just beginning or incomplete (Figs. 1, 2 and 3). For the sake of brevity these stages are designated as follows: 4-cell, 4-minus, 3-cell, 2-plus, 2-cell, 2-minus; the 4-minus being valued at 34, 2-plus at 24, the 2-minus at 14, and the rest at face value. Eggs that show no signs of cleavage are designated as “‘un- cleaved.”” The majority of the latter have either failed by chance 146 H. H. Newman to be fertilized or are incapable of development. A few might develop if allowed more time. No attempt, however, has been made to distinguish between unfertilized and retarded eggs, as cytological methods would be required. In all experiments every precaution was taken to treat the lots of eggs used for pure and hybrid strains exactly alike. In some of the experiments a mixed lot of egos from several females and the - mixed milt of several males of Beri species yielded good results, while in other cases the eggs of one selected female were divided and fertilized by the milt of two or more selected males of each species. In everyinstance the eggs after stripping were thoroughly mixed by stirring and shaking and then divided into two approxi- mately equal lots, which were fertilized at the same instant by abundant milt obtained by macerating the ripe testes of selected males. When development had proceeded to the desired point the two lots, pure and hybrid, were killed at the same instant in equal amounts of picro-sulphuric acid. It was found advanta- geous to examine and count the eggs while in this killing solution because the clear definition of the blastomeres 1s lost if the eggs are transferred to alcohol. ‘To obtain the best results the examina- tion and enumeration of eggs should be made within a few days after killing. After the counts have been made there are obviously several methods of dealing with the data thus obtained. ‘The best method involves a more or less arbitrary valuation of the various stages in terms of blastomeres, the uncleaved and unripe eggs being omitted from consideration. ‘The total number of blastomeres divided by the number of eggs will give the average condition of the lot and will furnish a antennal comparison between the stages of advancement of the pure and hybrid strains. A simpler method consists of a mere comparison of the relative percentage, in the two lots, of more and less advanced conditions, the most significant comparison being that between all 4-cell stages (includ- ing 4-minus) and all 2-cell stages (including 2-plus and 2-minus), 3-cell condition being ignored because not strictly assignable to either class. Another simple method involves a comparison of 4-cell, 2-cell and intermediate stages in the two strains. A full Heredity in Fundulus Hybrids 147 account of a fourth and considerably more searching method of comparison, which was used in the last two experiments, precedes Experiment 6. The three methods of comparison just described were used in the first four experiments and were in each case mutually con- firmatory. For the sake of brevity and uniformity they will, in each experiment, be designated as follows: Method I. Comparison of average number of blastomeres. Method II. Comparison of all 4’s and all 2’s. Method III. Comparison of strict 4’s, strict 2’s and interme- diates. EXPERIMENTAL DATA Ex periment I Eggs of several females and milt of two males of each species used. Killed three hours and forty-five minutes after fertilization. TABLE I | PURE-BRED HYBRID STAGE VALUE Tice = ——— = wae a = ‘ No. of 3 No. of Bios ob ses Blastomeres Newel Eee: Blastomeres COM ery es ia o.5 01% 4 74 296 167 668 (STC k ans ere 32 62 217 65 227% “02 Eee an 33 99 23 69 220) NORE SESORHGeoe 2¢ | 56 140 28 7o =o) | et ee 2 101 202 64 128 BEMTUS: & 5) o:2\e « 14 3 4% 6 9 SRotale ec. yavnsees 329 958% 353 11714 There were 235 uncleaved eggs in the pure-bred lot and 218 in the hybrid. Method I. Comparison of average rane of blastomeres. The average in the pure-bred lot is 2.91 +; that in the hybrid lot 3-31 +. Method II. Comparison of all 4’s and all 2’s. In the pure-bred Jot there are 41.33 + per cent of 4’s and 48.63 + per cent of 2’s; in the hybrid lot 65.72 + per cent of 4’s and 27.76 + per cent of 2’s. 145 H. H. Newman Method III. Comparison of strict 4’s, strict 2’s and interme- diates. In the pure-bred lot there are 22.49 + per cent strict 4’s, 30.69 + per cent strict 2’s and 45.89 + per cent intermediates; in the hybrid lot 47.30 + per cent of strict 4’s, 18.13 + per cent of strict 2’s and 32.86-+ per cent of intermediates. The per cent of 2-minus conditions is too small in both strains to furnish a basis of comparison. Ex periment 2 Eggs of several females and milt of several males of each spe- cies used. Killed three hours and fifty-five minutes after fertili- zation. TABLE II PURE-BRED HYBRID STAGE VALUE : | ee No. of Eggs EO! | No.of Eggs ane Blastomeres Blastomeres 4oGelly iromperettesret 4 86 344 | 336 1344 A=IMINUS 0/2 oye oes 33 85 297% 125 437% B=cell a nie wares 3 19 57 30 go 2-plus 24 40 100 71 106} DaCO lc tacstensyseexe: esc 2 44 88 76 152 Dass S peter keres ¢ 13 18 27 23 35% Alto tall evae celeste eater toot 292 9134 661 216¢4 Method I. Comparison of average number of blastomeres, the average in the pure bred-lot is 3.13 +; that in the hybrid lot 3.27 +. Method II. Comparison of all 4’s and all 2’s. In the pure-bred lot there are 58.56+ per cent of 4’s and 34.93 + per cent of 2’s; in the hybrid lot 69.74 + per cent of 4’s and 25.71 + per cent ofa" s: Method III. Comparison of strict 4’s, strict 2’s and interme- diates. In the pure-bred lot there are 29.45 + per cent of ‘strict 4%s5 15.06 + per centof strict 2’s and 49.31 + per cent of intermediates; Heredity in Fundulus Hybrids 149 in the hybrid lot, 50.83 + per cent of strict 4’s, 11.49 + per cent of strict 2’s and 34.19 + per cent of intermediates. The percentage of 3-cell and of 2-minus conditions is somewhat smaller in the hybrid than in the pure-bred strain. There were 442 uncleaved eggs in the pure-bred lot and 222 in the hybrid lot. It often happens that there is a larger percentage of heterogenic than of homogenic fertilizations. At present the factors governing this condition are not sufficiently understood to warrant a discussion. Experiment 3 . Eggs of a considerable number of females and milt of two choice males of each species used. Killed four hours and forty minutes after fertilization. TABLE II PURE-BRED HYBRID STAGE VALUE | ; iu ae! ¥ at if No. of Eggs Hoes No. of Eggs ee : Blastomeres Blastomeres ACO aE ohats, Heredity in Fundulus Al ybrids 151 Method II. Comparison of all 4’s and all 2’s._ In the pure-bred lot 20.44 + per cent of 4’s and 70.69 + per cent of 2’s; in the hybrid lot 26.50 + per cent of 4’s and 62.89 + per cent of 2’s. There is only a slight difference in the percentage of 3-cell stages in the two strains. Method III. Comparison of strict 4’s, strict 2’s and interme- diates. In the pure-bred lot there are 5.45 + per cent of strict 4’s 53-15 + per cent of strict 2’s and 36.62 + per cent of intermediates; in the hybrid lot, 7.24 + per cent of strict 4’s, 50.17 + per cent of Strict 2’s and 40.45 + per cent of intermediates. The percentage of 2-minus stages is more than twice as large in the pure-bred as in the hybrid strain. Ex periment a The eggs of several females divided and fertilized with the milt of two males of each species. Killed four hours after fertilization. In this case a surprisingly small number of the pure-bred eggs developed, while a large number of the hybrid eggs cleaved nor- mally. On account of the very small numbers of developing pure- bred eggs only the first method of comparison will be used. The average number of blastomeres in the pure-bred lot is 2.67 +; that of the hybrid lot 3.61 +; showing a marked difference in the same direction as in more successful experiments. Ex periment 6 In order to make the comparison between the pure-bred and the hybrid strains more searching, the eggs of one large female were divided into two approximately equal lots and fertilized with the mixed, expressed milt of several males of each species. Careful drawings had previously been made of seven stages between the t-cell and the 4-cell conditions. These were made at approxi- mately equal time intervals and were numbered from 1 to 7, be- ginning with the earliest stage. All eggs, so far as was possible, were examined and grouped according to their resemblance to the figures. In case an egg falls between two figured stages it is assigned to an intermediate group, e. g., an egg that falls between 152 Jeghe Fa, Newman stages 4 and 5 1s classed as 43 3. The average condition can then be readily obtained by assigning to each egg a value corresponding to the hgured group to which it belongs and dividing the total of these values by the total number of eggs. ‘The result should give a very accurate numerical statement of the relative rate of cleavage in the two strains. ‘he accompanying figures and table give in abbreviated form the results of the experiment. el: ee pac) -(e) kK) The average stage of the pure-bred lot is 5.11 +; that of the hybrid lot, 5.57 +, a difference of nearly half a stage. Another method of dealing with these data 1s to compare the percentage of eggs assigned to the lower half and the upper half of the table, allowing the dividing line to fall below stage 5. This shows a predominance of the more advanced stages to be more marked in the hybrid than in the pure-bred strain. The mode in both cases is stage 6, but there 1s greater skewness of curve toward the 7 end of the curve in the hype than in the pure-bred array. Another point worthy of note is that there 1s a much larger per- centage of irregular cleav ages in the hybrid lot than in the pure- bred. This in Foie might. be used as evidence of the very early formative influence of ae sperma tozoon. The proportion of 3-cell stages is very nearly equal in the two Heredity in Fundulus Hybrids 153 strains and has not been considered in the calculations because this condition is attained by only a comparatively few eggs and these could not be assigned to any of the figured classes. TABLE V PURE-BRED HYBRID FIGURED = = Aiea STAGES Ten tien Nf i s Me che ees Value in Terms Magsot Bees Value in Terms of Stages of Stages I 2 2 3 3 2 2 4 2 4 3 3 y) ; 3 32 2 Gi fo) fo} 4 3 12 I 4, + 12 54 5 22 5 13 65 ut 55 53 17 | 932 14 77 6 Dy) | 162 38 228 64 I 6s 10 65 7 2 | 14 9 63 | Ao tale ca: 84 | 429 94 5244 _ — | -—— — — — TABLE VI PURE-BRED HYBRID | P t P t | No. of Eggs | peo No. of Eggs | ere of Eggs of Eggs | | | BELOW) orate ac.s 2 - | 25 28.57 + 12 12.76 + sand above........ 60 qLeA2 | 82 87-2411 Ex periment 7 As it became difficult to obtain any more good material it seemed advisable to reéxamine the eggs.of one or two earlier experiments, using the more refined methods described in the last experiment. One hundred eggs of each strain were drawn out at random by a disinterested person from the material used in Experiments 2 and 4. Each egg was assigned .to its class and the average condition determined as before. 154 H.H.Newman The material from Experiment 2 showed the average condition of the pure-bred eggs to be 5: 26 +; that of the hybrid, 5.39 +. ‘The material from /xperiment 4 showed the average condition of the pure-bred eggs to be 4.72 + that of the hybrid 4.76+. The difference here is very slight, put in the same direction as 'n the other cases. No doubt another random selection of eggs from the material used in E xperiment 4 would have shown a more marked difference than that just recorded. Summary of Experimental Data and Conclusions The tabular summary, Table VII, of the first four experiments will enable the reader to see at a glance that, no matter what method of comparison is used, there is a developmental balance in favor of the hybrid strain. The last two experiments, somewhat more searching in char- acter, show that the hybrid strains develop more rapidly than the pure bred. In all cases the accelleration in developmental rhythm must be due the introduction of the spermatozoon of the more rapidly developing species. In Experiment 6 it is clear, in addition, that the form of cleavage was affected by the foreign sperma tozoon, in that there was a strong tendency toward fiicrgtllac ay | in cleavage even in the early stages described. We conclude that the male germ cell begins to exercise its hered- itary function at a far earlier period than is commonly supposed. II THE ROLE OF THE SPERMATOZOON IN EARLY DEVELOPMENT The question of the precise réle of the male cell in early develop- ment has received much attentionof late. It has come to be recog- nized that the spermatozoon has two separate functions, that of initiating development and that of imparting to the offspring the characters of the male parent. There seems to be a strong tend- ency today to regard the hereditary function as one that operates only aftera Aa of abeyance, during which the hereditary char- acters of the young embryo are Aenea solely by the structure Heredity in Fundulus Hybrids 155 TABLE VII eal eon | EXPERIMENT I | EXPERIMENT 2 | EXPERIMENT 3 | EXPERIMENT 4 a Zz SUBJECT OF a - COMPARISON = aaah ae i = 8 Pure |Hybrid| Pure Hybrid) Pure | Hybrid Pure | Hybrid 2 — = =s el | — | | I Number of developing | CIT Sea ECO ete eal 329 ey || ey 661 | 1283 | 1277 587 566 | | | [Average number of | | | | | | blastomeres........ 2.91-+| 3.31-+] 3-13-+| 3-27+) 3-19+) 3-44-+| 2-464) 2.58+ Excess in average num- | | ber of blastomeres | in favor of hybrid SEGA, Bas stelstes reseed .40 -14 ine || | -12 Per cent of all 4-cell | | StAPES eosin esta 41 -33-+ (65-72-1158. 56-1 (69-741 65.08-+78.77-++ |20.44-+ 26. 50+ Per cent of all 2-cell) | | Stagesta ts sha .. 48.634 |27.76-+ 34.93-+ 25-71+/29-15+ 15.11-+ |70.69-++ 62 .89+- Excess in per cent of | all 4-cell stages in favor of hybrid strain 24.39 11-18 | [r3.69 | 6.06 Per cent of strict 4- | Gellstapes st. <0). 22.49-+47.30+ 29.45-+ 50-83 /21.42+134.53-+) 5-454) 7-247 Per cent of strict 2- | cell stages ..........30.69-+ 18.13 15.06-++ 11-49+)18 40 9-16-+|53-15-+ 5o.17+ Per cent of intermedi- Ate SEAPES! danas 45.89+ 32.86-+ 49.31-+134-19+ 60.17+156.14-+ |36.62-+ 40.45 Excess in per cent of | | | strict 4-cell stages in favor of hybrid strain 24.81 | 21.38 IZ3-11 | 1.79 | 156 H.H.Newman of the egg protoplasm. This point of view has been clearly ex- pressed by one of its leading exponents* in the following words: ‘Fin: illy as evidence that taneratee may take place through the cy toplasm of the egg, reference must be made to the everenele important work of teas and Godlewski. By concentration of hydroxyl-ions Loeb found that it was possible to cause the sperma- tozoa of starfishes and ophiurans to fertilize the eggs of sea- urchins. ‘The embryos and larve resulting from such crosses showed only the characteristics of the mother. Later Godlewski, using the same methods, was able to fertilize the eggs of a sea- urchin with the sperm of a crinoid, and although such hybrids were raised to the larval stage, they showed only maternal character- istics. Still more, enucleated urchins eggs fertilized by crinoid sperm produced gastrule of purely rchan type. These results demonstrate, as Boveri admits, that the chromosomes of the sperm do not in this case influence or modify the cytoplasm of the ege cell; while the experiments on the enucleated egg show that the characteristics of the organism, at least as late as the gastrula stage, are derived entirely from the egg cytoplasm. “ Boveri long since showed that the ore stages of development, perhaps as Ate as the blastula or gastrula, are uninfluenced by the spermatozoon and are purely i penal in type; in the case of God- lewski’s hybrid larvae, he supposes that the sperm chromosomes remain permanently inactive. But however this result is to be explained, it may be considered as definitely settled that the early development of animals is of purely maternal type, and that it 1s only in stages later than the gastrula, and consequently after the broad ics of development and the general type of differen- tiation have been established, that ay influence of the sper- matozoon begins to make itself felt; and itis equally certain that this type of differentiation 1s predetermined in the cytoplasm of the mature egg cell, rather than in the egg nucleus. “On the other hand, there is no AGaie that the differentia- tions of the egg cytoplasm have arisen, in the main, during the ovarian history of the egg, and as a result of the interaction of 4 Conklin, E.G. Science, N.S., vol. 27, no. 168, p. 98. Heredity in Fundulus Hybrids I Wh nucleus and cytoplasm; but the fact remains that at the time of fertilization the hereditary potencies of the two germ cells are not equal, all the early development, including the polarity, symmetry, type of cleavage, and the relative positions and proportions of the future organs being predetermined in the cytoplasm of egg cell, while only the differentiations of later development are influenced by the Sperm. In short, the egg cytoplasm fixes the type of development and the sperm and egg nuclei supply only the details.” My own observations on the early stages of the process of heredity and an examination of the experimental evidence, that lies at the foundation of the above view, together with a number of more recent contributions along the same line, force me to take a position on certain questions decidedly opposed to that of Conk- lin. Is the specific sSymmerty, polarity, Gor ex pressed solely in the egg and not in the spermatozoon or in the various types of somatic cells? It is scarcely necessary to point out that the sperm cell at all stages af development shows just as pronounced a polarity as the ege—more so in later stages. This polarity is largely the expres- sion of a definite relationship between nucleus and cytoplasm and is doubtless specific and hence characteristic of all cells of a given organism. No doubt this polarity expresses itself in a somewhat different fashion in different kinds of cells, but these special mani- festations are, I believe, of secondary importance. When the spermatozoon for example, undergoes an exaggeration of its specific polarity and symmetry during the end stages of its devel- opment, when most of its cytoplasm 1s converted into a locomo- tor mechanism, it becomes a specialized cell with a definite func- tion, and departs from the specific cell type. Is not the same true of the egg, a cell in which the primitive specific polarity and symmetry have been distorted by the large accumulations of inert nutritive material? It is entirely probable therefore, as Lillie has shown, that the real polarity and symmetry are characters of the ground substance common to all of the cells of the organism. Since then a fertilized egg is a product of the more or less com- plete fusion of two cells with the same inherent specific polarity and symmetry, 1t appears somewhat extreme to state that “all I 58 lin ee as fe Newman of the early development, including the polarity, symmetry, type of cleavage, and the relative positions and proportions of the future organs are predetermined in the cytoplasm of the egg.” sit See aey settled that the early development of animals 1s of purely maternal type, and that it 1s only in stages later than the gas- trula, and consequently after the broad outlines of development and the general type of differentiation have been established, that the in- fluence of the spermatozoon begins to make itself felt”? The experiments detailed in an earlier part of this paper show that the spermatozoon exercises an hereditary influence upon the rate of development at the earliest possible period when it could be noticed or measured, and would seem to indicate that the hereditary function of the male germ cell begins to operate imme- diately, not after a period of abeyance. Godlewski has also shown in his hybrids that there is a well marked retardation in the cleavage as early as the 4-cell stage. There is evidence also that the form of cleavage is subject to the influence of the spermatozo6n, as was indicated in Experi- ment 6, where in the hybrid strain there was a preponderance of irregular cleavages. This phenomenon is seen to much greater advantage in orice cross, produced by fertilizing the eggs of Cy Titans variegatus with the sperm of Funduluct heteroclitus. In this case the whole cleavage is decidedly irregular after the 4-cell stage. Fischel' has shown that in a number of E:chinoderm hybrids the male influence 1s expressed structurally in the early blastula stages, not only in the general size of the embryos, but in the actual size and shape of a cells. ‘he sperm also seems to be responsible for the production of a number of early monstrosities in which the “broad outlines of development” have been decidedly distorted. Such typical monstrosities are very common among Fundulus hybrids and there is no doubt that similar conditions are found in all hybrid experiments. : Another phenomenon that has caught the attention of many observers is the wide range of variability among hybrids. This * Archiv. f, Entw. Mech., vol. 22, pp. 498-525. Heredity in Fundulus Hybrids 159 increased variability frequently manifests itself from the first and must be considered as one of the early effects of the foreign sperm. Can the data derived from remote hete TOGENIC CTOSSES, ae as those described by Loeb and Godlewski be safely used as criteria for positing a theory of normal biparental inheritance ? An examination of the work of the two experimenters mentioned and of several other contributions of more recent date, reveals the fact that the spermatozoon in no case functions completely or normally. In fact, as Loeb himself has suggested, 1t seems highly prob- able in crosses between different orders, such as echinoids and crinoids, that the spermatozoon performs only one of its functions, that of initiating development, and that the process of develop- ment is thenceforth parthenogenetic. In Godlewski’s experiments it can scarcely be doubted that the sperm nucleus enters the egg and fuses with the egg nucleus. The figures show, however, that the chromatin Tagen remains inactive and that the male pronucleus, instead of increasing in size until it equals that of the female pronucleus, remains in its concentrated condition until it fuses with the latter. “This fusion has every appearance of a mechanical absorption of a foreign particle. In no place does Godlewski indicate that the chromatin of the sperm nucleus takes part in the mitotic divisions of cleavage. It operates only to the extent of slightly hindering the rate of early cleavage and probably is soon entirely absorbed by the egg protoplasm. A still clearer case is that described by Kupelwieser,’ who fer- tilized the eggs of an echinoid with the sperm of a mollusc. In this case both description and figures show clearly that the sperm nucleus never breaks up into chromosomes, but remains inactive in the form of a mere lump of inert substance, apparently com- pletely incompatible with the materials of the egg nucleus. In this form it is carried along through several cleavages and is sub- sequently absorbed. It should not be surprising then to find such hybrids, if hybrids they may be called, showing pure maternal * Arch. f. Entw. Mech., vol. 27, pp. 434-462. 100 H.H.Newman characters. The very fact that there is no sign of a paternal influence should only serve to emphasize the importance of the nucleus as a factor in determining the character of early develop- ment. Bataillon,® fertilizing the eggs of several species of Anura with the sperm of the Urodele Triton, obtained results very closely in accord with those of Kupelwieser. There was no real nuclear amphimixis, but the sperm nucleus remains in a mass and soon degenerates. When individuals belonging to two genera of the same order are crossed there is ede less Se gan ve sTivay: as\ awnule- Herbst,’ for example, has made an extremely careful study of the behavior of the paternal chromatin in the hybrids produced by fertilizing the eggs of Sphzrechinus with the sperm of Strongy- locentrotus, in which he found that the male chromatin in some cases divides more or less completely into chromosomes and takes part in the mitosis of early cleavage, in others it seems refractory and shows a tendency to go undivided to one motitic pole, and in still others it becomes scorer into a small separate nucleus in the 2-cell stage. Evidently at no period does the male chroma- tin function normally. When two species belonging to the same genus are crossed there is sometimes an approach toward complete compatability of the nuclear materials of the two parents. In the two species of Fundulus used in the above experiments we have a case in point. Here there is no visible difference between the chromosomes of the two species and the male chromosomes seem to behave quite normally in cleavage from the first division onward. One would aaa be justified, therefore, in drawing conclu- sions concerning the normal process of heredity from Bee such as have been described where there 1s every evidence that the pater- nal contribution 1s either eliminated at a very early stage of develop- ment or functions in a decidedly abnormal manner. Is there cytological justification for the statement that “ the char- 6 Archiv. f. Entw. Mech., vol. 28, pp. 43-48. Archiv. f. Entw. Mech., vol. 27, pp. 266-308. Heredity in Fundulus Hybrids 161 acteristics of the organism, at least as late as the gastrula stage, are derived entirely from the egg cytoplasm’? The above statement is based largely upon certain experiments of Godlewski, in which enucleated sea-urchin eggs fertilized with crinoid sperm produced gastrulae of purely urchin type. Exam- ination of Godlewski’s records shows that in all these experi- ments only four eggs developed at all and these did not produce typical larvae. ‘That this very meager piece of evidence is inade- quate and unsatisfactory seems to be the opinion of subsequent workers on echinoderm hybrids. It 1s certainly not sufficiently well established to form the basis for any important conclusion. There is undoubtedly a close correlation between the degree of normal functionality of the male nucleus in early development and the degree of hereditary influence exerted by the latter. This was shown in clear fashion by Baltzer,* who crossed four species of sea-urchins in all possible ways and noted thatwhen there was a complete elimination of paternal chromatin a pure maternal type of larva resulted, and when the male chromosomes continue to function more or less normally the larvae showed an admixture of maternal and paternal characters. The results here discussed seem to point to the conclusion that the nuclear material is the chief factor in determining the char- acter of early development. As Fischel has ably pointed out, the réle of the spermatozoon 1s from the beginning formative in char- acter 1n that it 1s able to place the stamp of its own specific char- acters upon the early developmental stages of the organism, while the egg cytoplasm furnishes only the material for the formative operation of the combined nuclear material of the two parents. § Zool. Anz. Bd. 35. STUDIES WITH SUDAN III IN METABOLISM AND INHERITANCE OSCAR RIDDLE From the Laboratories of Experimental Therapeutics and Zoology, University of Chicago li, lisyaaoreliveGoiny SR BAS Acie 5 islae Pesce S COR CRO OICREME ts Be meghions cor ici aie (OP oN te, Ss eee 163 Tf, TES ers Pee in ee erty pee OEE 6 OCC en ote Crate OS cP ta aE Me tic it ae eer r 165 1 Membranes through which Sudan is known to pass ...............2seeeeeeeees 165 2 The mechanism of the transfer and deposition of Sudan within the body.......... 166 3 The uses to which Sudan has been applied in experimental biology and medicine... . 167 AeSudanslile anda therpiementsrins 16 Heritan Conver oes) ai tte tertile tetra) ole)2 <2 ele 168 UIE BxcperumentalemethOdsrs..ty 0. airs liste © «oe sleet etal tags hace) ve Aelele.e Fis nee! oboe 169 TAY. RYARDILISONTGE ores etna Sioa Geen ote Uae ate ticm.c chen bs corny au Gone aa betaon scammer 170 Dosen OL Suse Oh OVE cohncoodead enor socuwn aoe dded secosanceeooesar 170 Disjatersilararny Ole Sue kan IMO ha Waele, Ane gk ote oooh kook doo eens oc ge 171 The rate and conditions of absorption and deposition of Sudan III.............. 172 V. An unsuspected action of Sudan. (An apparent tendency of Sudan to lessen the “‘avail- abilitygy Obmatsmine the) Orpanism)) |r. ac acetate elicit 174 Byolloysreniemieene ces, RD Fp Rico dcineo.au.c oe eo bue deur doe Ubaeclve eOanEe onaue 174 @hemiicalievid ence eros 5 pets cove cs cteveia ts £25 eacshoued oeudns Satayays coy eenerete ty olay a fauealieeas crates, fase, tins shal 176 Wile DI SCUSSLON) amd iCribl Ques, 55.25 <2e1e ststcis «1m ajeuectelaval Ses srerslsiava)s sl aiageilase Sat, 2 agains sapys 4-121 177 Vin QUGnne AL Ost s8e adas coe een aA ee ace por OnneGRat: Dh. sc Roane a aae eee ar car 182 VAIL, (Dili ityeril yeas Shee omAc eee p aor bnc Oaonnon Casa ouneanuaddnun ppenbuogoe vabodeuane 183 INTRODUCTION In a paper presented before the American Society of Zoologists, December, 1907, the writer (07) described and demonstrated, among other things, the deposit of Sudan III in the egg of the domestic fowl. A very brief abstract of this paper was published in Science, June, 1908. All of the data concerning the method of introducing the color into the eggs, and the significance of this in metabolism and inheritance, as well as the data concerning an interpretation of the white and yellow yolk of vertebrate ova, were then written up together. It now seems advisable however, to treat the first two subjects apart from the last since the phenomena 104 Oscar Riddle of inheritance involved in the transmission of this aniline dye have attracted an amount of attention which make a fuller treatment necessary.! It is important that any statement concerning the :mheritance of this or another pigment be accompanied by a statement of the facts concerning the behavior of this substance in metabolism. ‘That is to say, if we make statements concerning the passage of a substance through follicular cells and of its deposit, distribution, behavior or development within germ cells and in their derivatives, it is obviously important to keep in view what we know regarding the way this substance passes through other membranes; how it exists, 1s distributed, behaves or develops in somatic cells. It is of value to realize how these things happen in any membrane or cell of the body, because it is this same how, or in other words, the mechanism of such successive transforma- tions in the germ, that is the very se/7 of inheritance. Again, if we state our results on inheritance in terms of metabolism we are, in this case at least, less liable to exaggerate the importance of our ' Puring the two years since this paper was prepared and read other workers have under- taken and reported work along similar or related lines; and quite recently, within four months, three papers have appeared which make it seem advisable to divide this paper into two parts, and to publish in full without further delay the part most intimately connected with the present title. One is the more readily persuaded to this division and immediate publication because of a communication which appeared in Science, September, 1909. | Ur. Ludwig Sitowski there directs attention to the fact that he had secured the deposit of Sudan III in the eggs of moths, and had published an account of his experiments as early as 1905, but that his paper has been over- looked until now by the writer and apparently by others. With pleasure the writer hastens to make acknowledgment of his excellent work. Only the timely appearance of his communication together with the fortunate circumstance of delay in the publication of this article have made it possible to give his work the credit it deserves. It is hoped that Er. Sitowski wil! realize that the place and title of his publication were such as to make it not difficult for American workers to overlook. Sitowski evidently has not seen even the abstract of the writer’s paper but only a short notice or review of it which was written by H. A. in the Zeitschrift f. d. Ausbau der Entwick- lungslehre, Bd. III, Heft 2, 1909. The writer’s report of this work has received notice in several quarters, and in almost every instance he has been credited with the works of others, or to others has been attributed work done by him. In these shiftings he usually has fared better than he deserves; but it scems that the results thus far obtained in this field should be brought together in such a way that it may be made plain just what has been done and incidentally who did the work. It is partly for these reasons that the entire literature on Sudan, so far as the writer has been able to find it, is here brought together and an outline of the results given. Sudan III in Metabolism and Inheritance 165 results; while at the same time we get a close view of the sort of mechanics which is back of this type of “inheritance.”” We may state at once our belief that we are here dealing with a noteworthy, though very simple, form of inheritance, but one which seems by no means sufhcient to illuminate hereditary processes in general. By the use of Sudan the writer has been able to demonstrate some hitherto unrecognized features of fat metabolism, particu- larly as it occurs in birds. It seems both convenient and helpful to report these results in connection with the present survey of Sudan in metabolism and inheritance. HISTORICAL The historical background of the present studies is furnished by a view of the state and source of our knowledge of the four following topics: (1) The living membranes through which Sudan will pass, and the points at which it 1s deposited in the body. (2) The mechanism of the transfer and deposition of Sudan within the body. (3) The uses to which Sudan has been put in experimental biology and medicine. (4) Sudan III, and other pigments, in “inheritance.” In summarizing the data on these topics it has been considered to the advantage ofthe reader, to include the results of the writer as well as those of other investigators whose findings have been published since the preliminary report of his own work. The summaries, therefore, are believed to be complete to date. (1) Membranes Through which Sudan is Known to Pass Intestinal mucosa: birds and mammals, (Daddi, ’96). Intestinal mucosa: human, (Franz and Stejskal, ’o2). Intestinal mucosa: moths, (Sitowski, ’05). Embryonic intestine: yolk-sac of chick, (S. H. & S. P. Gage,’o8). Renal epithelium: human, (Franz and Stejskal, ’02). Epithelium of egg chamber: moths, (Sitowski, 05). THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 8, NO. 2. 100 Oscar Riddle Follicular epithelium: birds, reptiles, mammals, (Riddle,’07 b). Epithelium of mammary glands: rat, (Gage, Stotsenberg, ’08 b). Thecee of Corpus luteum: rabbits, (Riddle, this paper). Peritoneum: birds, (Riddle, this paper) (2) The Mechanism of the Transfer and Deposit ion of Sudan within the Body Practically all we know concerning the mechanism of the transfer and deposition of Sudan we owe to the feeding experi- ments and observations of Daddi, to the studies on the solubility of the stain by Pfltiger and Nerking, and to the chemical researches of Michaelis. ° Daddi (’96) found that when fed to rabbits, guinea-pigs, pigeons and fowls, Sudan passes through the intestinal epithelium, enters the circulation and is deposited in the adipose tissue of the body generally; that it is a specific fat strain, and that in its introduc- tion and deposition in the body it 1s always associated with fat. Biedermann, ('98) was the next to use Sudan experimentally. He fed the stain to Tenebrio molitor and found that although the intestinal contents became colored, the body fat was not colored.’ Hofbauer (’00) made the mistake of supposing that only natural fat and not soap.and fatty acids, was able to carry the dye through the intestinal epithelium, (he used Alkanna which re- scinliles Sudan in its solubilities, and he also refers to Sudan). He thought, therefore, that by use of the stain he could determine in Phieh form the fats are absorbed from the intestine. The paper containing this error, two other papers which repeat it, together with the three or four papers devoted to exposing the error, furnish nearly one-half of the literature dealing with Sudan III. Pfliiger (00) and Friedenthal (00) simultaneously pointed out Hofbauer’s mistake, showing that under the conditions furn- ished by the intestine Sudan 1s solablemon only in fat but in bile, in sodium soap and in glycerine. Friedenthal, however, declared that soaps have no power to dissolve the stain in the absence of free fatty acids. Nerking (’00) immediately showed that entirely ? Prof. T. H. Morgan informed the writer that in a fly, Drosophila, he has recently obtained a simi- Jar result. Sudan III tn Metabolism and Inheritance 167 neutral soaps do have the power to dissolve Alkanna, Lackroth and Sudan. It may be pointed out that Sitowski (’05) repeated Hofbauer’s error although he based no conclusions upon it. White- head (’09) without knowing of Hofbauer’s work attempted to solve the same problem ina like manner. Mendel (09) has shown that Whitehead was further mistaken in his observation that after a dog has been fed with Sudan, the lymphatics of his mesentery remain unstained. It may be stated here that there is no difficulty what- ever in finding the stain in the lymphatics of fowls, if these be examined two or more hours after being fed the dye. Michaelis (‘o1)made very careful chemical studies of Sudan and related compounds. He considers the staining of fat a physical and not a chemical process, and draws the conclusion from his work that the phy sical properties of a body depend upon its chem- ical character since the dye molecules, to be soluble in fat, must have a very definite constitution. He concludes, “fat will be stained by those azo-dyes which are ‘indifferent’ in the sense of possessing no salt-forming groups.’’ Mann (’02) has given some. consideration to the nature of the union of Sudan with fatty com- pounds, and on the basis of Michaelis’ studies, states (p. 310) that “the union between Sudan III and oleic acid is a chemical one depending on the oxidation of the unsaturated fatty com- pound. Therefore, the action of Sudan III and similar dyes 1s analogous to that of osmium tetroxide, the only difference being that azo-dyes form additive compounds with the fat without loss of color, while osmium tetroxide, after having formed addi- tive compounds, is readily decomposed owing to the high valency of the osmium.” From either view—point, or indeed from any possible viewpoint, it seems certain that the dye is bound to the fatty constituents, cannot loosen from them, and is dragged with them mechanically, so to speak, wherever they may go. (3) The Uses to which Sudan has been applied in Experimental Biology and Medicine As has been noted, Daddi introduced the stain into _histo- logical work, used it as an intra-vitam stain, and by its means stud- ied the foci and extent of various fatty degenerations of the liver I 68 Osca if Riddle and muscles. The futile attempts of Hofbauer, Sitowski and Whitehead to determine the form in which fats are absorbed from the alimentary tract have also been mentioned. Franz and von Stejskal (02) made extended studies on the tat metabolism of a chy- luricby meansof the stain. When stained fat was fed to the patient the colored fat appeared in the urine in four hours and continued always less than twenty-four hours. When, however, stained fat was injected subcutaneously into the shoulder it did not appear in the urine. Sitowski with this stain undertook the solution of certain prob- lems of digestion in the caterpillar of certainmoths. Hemade little progress with these problems, but in the course of his investiga- tions discovered a deposit of the dye in the eggs (primary oocytes) of these forms. The writer has used Sudan in a number of studies and for several purposes. He will mention at this point merely its use in determining the time and rate of growth of the eggs (primary odcytes) of fowls and turtles; in the study of some spec- ial features of fat metabolism; and to gain some information as to the inter-relation of the soma and germ cells, including transmis- sion and inheritance behavior. Gage has followed the first report of our results with further studies on the behavior and distribu- tion of this dye in the developing fowl, 1ts appearance in the milk, and with negative results, its passage through the placenta. (4) Sudan III and Other Pigments in Inheritance The only recorded cases known to the writer, of the de- posit and persistance of foreign or maternal pigments within germ cells, are the following: Schmidt (’91) found that Alkanna- colored fat was taken up by apparently all plant cells; the ovules of these plants are not specifically mentioned as obtaining part of this coloring matter.? Pizon (or) states (p. 170) that “the first pigment of the larva (Botryllides) proceeds from the maternal organism by migration.”’ His observations are not conclusive. Sitowski (’05) fed Sudan to caterpillars and obtained the stain within their eggs. He now reports (’0g) the presence of the stain 3 Fat colored with Alcannin had been used by Pfeffer (Osmotische Untersuchungen, 1877) to color the fat being injested by Myxomycete plasmodia. Sudan III tn Metabolism and Inheritance 169 in the somatic structures of larvae hatched from such eggs. The writer (07) caused birds and turtles to deposit quantities of Sudan within their eggs. S. H. and S. P. Gage (08) have hatched Sudan-containing eggs of the fowl and noted the re-dis- tribution of the stain in the somatic tissues. In addition to these cases, however, it should be noted that it is practically certain that the natural coloring matter of the eggs of the salmon pro- ceeds from the muscles of the fish. It is quite certain that the jat which is in these muscles, and in which much if not all of the coloring matter resides, is transferred to the ovary and to the growing eggs. These lipochromes of the muscle fat doubtless remain fixed to the constituent fatty acids, when this fat is broken up in the muscles and is thrown into the circulation; from the blood or lymph, we believe, the two enter the ovum together, precisely as in the case of Sudan-stained fat. EXPERIMENTAL METHODS Laying hens were fed Sudan III in three ways, viz.: in gelatine capsules, dissolved in egg-yolk, butter or animal fat, or by enclos- ing small lumps of the stain in pieces of bread (no fat). The results were very similar in all cases. With birds, the method of feeding seems quite immaterial since the stain apparently always meets with enough fats within the alimentary canal to carry con- siderable quantities of it through the intestinal wall. The dose varies from one-half gram to three milligrams. For most studies, particularly those dealing with problems of metabolism, large doses are to be avoided; from three to twenty milligrams have been found most useful, Many birds were fed the stain at inter- vals of thirty-six, forty-eight and seventy-two hours; series of eggs from birds thus fed were obtained, were hardboiled, sectioned under water with a sharp razor and then examined, these latter operations being done chiefly to learn the rate of growth of the ova. In other cases the birds were killed at such time after feeding as was demanded by the points under investigation. The stain was introduced into the bodies of chicks and rabbits also by injection of its solution in a mixture of oleic acid and alcohol. A widely variable quantity of the solution was injected 170 Oscar Riddle into the peritoneal cavities of these animals (also into brachial veins of the chick, and ear veins of the rabbit). The method of feeding the stain to turtles will be described with the results of that work. RESULTS OF THE EXPERIMENTS Deposition of Sudan III in Ova The eggs of hens fed as described above almost | invari- ably showed marked quantities of the pigment deposited in the yolks. Anovum which had undergone its final and rapid growth in a bird which was being fed at regular intervals of thirty-six, forty-eight or seventy-two hours would show in section a series of evenly-spaced, concentric circles of orange-red, these alternat- ing with other circles of light yellow, the natural ground color of the yolk. The width of any Sudan-colored circle could have been regulated at will at the time of feeding; much stain giving the wide rings of red, and little stain resulting in narrower rings of less intense color. If the birds be killed a few hours after feeding all of the larger ova are found to be deep red on the outside; if, however, the bird be not killed until one or two days have elapsed since the feeding, these ova will have a perfectly normal external appearance, and only an examination of the in- terior of the eggs will reveal the presence of the stain. This is, of course, a consequence of the rapid growth of these ova. One successful attempt was made to stain the ova with Sudan injected into the peritoneal cavity. Four injections of a mixture of alcohol and oleic acid forty per cent each, to which traces of sodium carbonate were added, were given within a period of forty-eight hours. Four eggs were subsequently laid by this bird and were found to contain the dye. In the case of the turtles the records are as follows: Three very large females of Emydoidea blandingii were heavily fed with Sudan for three weeks during July and August. The stain was put with butter into capsules of large size and these were pushed with long-slender forceps into the stomach, while the neck was stretched and the mouth held open with other forceps. All were killed five days after the last feeding. All of the larger ova Sudan ITI in Metabolism and Inheritance 171 showed the characteristic color of Sudan at their peripheries. The thin, follicular membranes were slightly tinged with red. In January four similar turtles were fed and killed in the same manner. In none of these cases could Sudan be found within the eggs, although some of the follicular membranes seemed. very faintly stained. This different result during the two seasons is of consider- able interest from the standpoint of determining the season in which the eggs of turtles grow. If the eggs had been growing (depositing yolk) in January, they should have taken up the stain. The fact that they failed to do so is evidence that they are not growing in January. The definiteness of this finding is somewhat vitiated however, by the writer’s observation (og) that the di- gestive capacity of these forms is very low in midwinter; and by the further observation that the forms which were fed the Sudan in winter sometimes regurgitated parts of it. It cannot be stated as certain, therefore, that as much Sudan was put into the blood of the turtles in winter as in summer. (The turtles were kept from summer until January in aquaria containing water at outside temperature. At the beginning of the feeding experiment they were brought into water at summer temperature, about 20°). The ovaries from rabbits injected (with the same solution as for the birds) once or twice daily for a week, were examined. Only two of these animals survived the injections long enough to be con- sidered seriously. One of them showed no certain traces of the stain anywhere in the ovaries, the other, only in the corpora lutea. This work on the rabbits was shared by Prof. S. A. Matthews. Deposition of Sudan III in the Soma Fowls heavily fed on Sudan, for even a day or two, usually show upon examination a reddish color in all their adipose tissues, most prominently in subcutaneous and peritoneal fat. This butconfirms Daddi. In addition to his findings, however, it was determined that if newly hatched chicks be fed the stain during the growth of the juvenile plumage the feathers also take up the stain and become distinctly red in color; (the Sudan-containing offal was often and completely removed from the brooders and pens to prevent its be- 172 Oscar Riddle ing mechanically scattered over the outside of the plumage). The claws and bills of the birds likewise. become highly colored, but one cannot be perfectly certain that this color is not of external origin. Injection of the stain gave very similar results; in these cases, how- ever, a more diffuse color was obtained, no attempt was made to color the feathers and no staining of the intestinal wall was noted. These birds laid down colored fat after having been given the stain by injection into the peritoneal cavity. After feeding the stain to turtles one finds but traces of Sudan deposited in somatic tissues. [his is undoubtedly due to the fact that they store fat extremely slowly, and that their bodies actually contain but little fat. “The further fact of the difficulty or slowness of digestion which the writer (09) has found especially to charac- terize the turtles, may also be important in this connection. The subcutaneous fat and the intestinal mucosa were the only parts other than the ova and follicular membranes in which the writer found the stain deposited in turtles . No injection of the dye was attempted in these animals. Rabbits apparently ingest Sudan much more slowly than do fowls. Nevertheless, upon continuous feeding red-colored fat becomes visible in all parts of their bodies, subcutaneous fat every- where, peritoneal and kidney fat, the intestinal wall and corpora lutea. Daddi noted a similar distribution (except in the corpora lutea) in rabbits and guinea-pigs. A similar distribution of color results from the injection of the stain (except for the mucosa). The Rate and Conditions of Absorption and Deposition of Sudan ITI Almost no attention has been given by previous writers to the rate at which Sudan is absorbed and desposited. Since this really represents the rate at which fat is absorbed and de- posited, 1t becomes a matter of considerable interest. Similarly, the conditions of its deposition and non-deposition when it is brought within the blood-stream, have nowhere received consid- eration, except, of course, that it has been generally noted that it is deposited in fats. The writer is able to report approximately correct data on these points as they were obtained in the study of the fowl only. Sudan III tn Metabolism and Inheritance 173 Many birds were killed soon after feeding with Sudan; the time intervening between feeding and killing ranging from one-half hour to days and months. From the examination of these birds it was learned: (1) That the stain may appear in the mesenteric lymphatics within a period of seventy minutes after feeding; (2) at end of two to three hours after heavy feeding Al perceptible ; amount of stain is laid down in the rapidly growing ova; (3) the body fat becomes colored much more slowly than the yolk fat; (4) the sev- eral regions of body fat are not all colored simultaneously, even the subcutaneous fat of some regions remaining colorless at a time when subcutaneous fat elsewhere is quite red. ‘This last fact seems to indicate that there are differences between these several “‘storehouses”’ of fat; that some are centersof a mostactive commerce, there being in these a continuous loading and unloading of wares; whereas there are other storehouses of fat whose portals during normal conditions at least, are quite tightly closed. In regard to the conditions under which Sudan is, or can be, deposited, we have determined the following facts: (1) Sudan can be deposited only in growing ova. Indeed, fora perceptible amount of the stain to be taken up the ova must grow more rapidly than do those ova of the fowl which are less than 5 mm. in diameter. (2) Sudan can be deposited with difficulty, and only in small amounts, in a fowl that is not being fed and is thus made to use its store of fat instead of being allowed to grow new fat. ‘These results have been verified on so many birds that there is no doubt of their being €. tirely reliable. It cannot be said that the starved animals did not get the fat into their circulation because of failure to absorb the stain under the starving conditions, for some of these birds were given the stain by injection, and they too showed just as decidedly the results stated above. One cannot but see in these two results the very strongest evidence that while in the body, Sudan III clings at all times to the fats or their constituent fatty acids, and so goes quite mechanically wherever these particles go, it 1s indeed, attached to them. (3) There is moreover in lightly-colored fat a marked tendency of the stain to remain in this fat in the living animal and not to leave it for other contiguous fat. This wes shown by the sharpness of the inner edges of the bands of stain in the ova, as well as by one’s ability to circulate stain through the 174. Oscar Riddle body of animals not depositing fat, without coloring certain regions of fat. (4) It was found that within the ovum the Sudan is de- posited in the germinal disc and in the latebra in smaller amounts than elsewhere. ‘This is undoubtedly to be associated with the lower fat content of these regions of the egg. AN UNSUSFECTED ACTION OF SUDAN An Apparent Tendency of Sudan to Lessen the Availability of Fats in the Organism. Since 1904, when the writer first fed Sudan to chicks, several things have come under his observation which indicate that Sudan-stained fatis not as available,—does not splitup and yield its ener gy to the organism as readil y—as does the unstained jat. If this could be positively established it would be a very im- portant fact, possibly giving some clue as to what “availability” of foods rests upon. We might, perhaps, then proceed so to treat certain foods or constitutents of the tissues as to increase or decrease at will their utilization or destruction within the body. Some special effort has been made to get positive data on this hitherto unsuspected action of Sudan. It must be admitted that conclusive data have not been obtained; in their absence the writer can only submit the following record of efforts,—a few observations and experiments which seem to contain some bits of evidence: Biological Evidence (1) Young chicks which were given Sudan with their food ate much more than those not fed the stain; they seemed always hungry and did not grow as well as the other birds of the same age and breed. This, of course, may easily have another explanation than the one suggested. (2) Ina certain “starving” experiment it happened that birds three months old were used, five of which had been given three heavy feedings of Sudan during the two days immedia tely preced- ing the starving period. T ene five Sudan-fed birds were all dead eters any of pas four non-fed ones showed very great signs of weakness. Three of the five dead birds were carefully cme They showed Sudan in patches of subcutaneous fat, in other patches along theneck, bebind the occiput and even distinct traces Sudan III tn Metabolism and Inheritance EG in peritoneal fat. On the other hand, the muscles showed extreme waste. Two of the birds from the other pen were now sacrificed for comparison. ‘They showed hardly a trace of fat. Dipping them into an eighty per cent alcoholic solution of Sudan failed to reveal more than traces. The muscles, however, were obvioulsy larger and much better preserved than in any of the Sudan- fed birds. Sudan was found to be a non-toxicant as many birds were fed several months and one adult hen was fed the stain almost continously for ten months without visible injurious effect. This fact; together with those mentioned above, lead one to suspect that the: presence of the stain in the fat made this fat in these birds less available than if unstained, and that under the new conditions the energy of the proteins (of the muscle, etc.) became more avail- able than that of the fats. (3) If birds be fed considerable quantities of Sudan while growing a plumage it will be found that the “fault-bars’”’‘ of the feathers become more pronounced in extent. It has been shown conclusively that any decrease in the nutrition of the feather germs produces these effects. Attention has elsewhere (Riddleso7"p- 172) been specifically called to this power of Sudan to produce fault-bars or defective areas in feathers and to the fact that this seems to be due to a starving effect produced by the Sudan. (4) It has also been pointed out by the writer (08, p- 174) that if young chicks in their downy plumage be “starved”’ for a time, or fed Sudan in quantities, there is a common result in the two cases, namely, an inhibition of the growth of most of the definitive feathers and a long retention of the downy plumage. This is evidence of the sort we are just now examining, since these Sudan effects so closely parallel “starving” effects. The following case is perhaps less valuable evidence Ne the same kind. (5) It has been observed that many laying hens cease to lay eges after having been fed considerable quantities of the stain. The effect here is again the same as that resulting from a with- drawal of food; it may, however, have other causes as well. (6) The above and similar observations led to the following experiments. The one here recorded was made after nos. 7 and § 4 See Oscar Riddle on the Genesis of Fault-bars in Feathers and the Cause of Alternation of Light and Dark Fundamental Bars. Biol. Bull., vol. 14, pp. 328-370, 1908. 170 Oscar Riddle had failed to satisfy. Six plymouth rock hens in good condition, not laying, were isolated for the experiment. Quite at random three of these hens were taken and given food + Sudan capsules for two days; the other three were given food but no Sudan. The birds were then all removed to a pen where they could get no trace of food; only water was given them. ‘The weight of each bird was taken at the beginning and on each third day of the ex- periment during its fifteen days of duration; the object of all this being to learn which group of birds would lose weight faster. It was thought that those using most fat would lose least weight and vice versa; viz., that energy must be supplied to the birds during life, that if they secure this energy from the fats of their bodies instead of from their protein, they will need to use fewer grams to obtain any desired amount of energy.since the energy content of fats is to that of proteins as about 9.3 to 4.1. An important part of the records of the weighings unfortunately has been mis- placed and the writer cannot give the exact figures; but the net result showed that each of the Sudan-fed birds had lost, at the end of the period, a higher percentage of its initial weight than had either of the non-fed birds. Chemical Evidence (7) It was thought that if the stained fat were less available to the organism, as seems to be the case, this might be connected with a decreased power of the fat-splitting ferment to split such fat. The following attempt was made to determine this point; lipase was prepared from the castor bean and equal quantities of this was put into flasks, one set containing oil +Sudan, the other pure oil only. Flasks of the two sets, left on the shaking machine and given time for hydrolysis, were then titrated with nj10 Na OH, and compared. It was found, however, that the strong and per- sistent color of the Sudan so obscured the expression of the indi- cators that it was quite impossible to determine the neutral point in the Sudan-containing flasks. The titration method of estimat- ing the rate of hydrolysis of the fat, therefore, had to be given up. An attempt was next made to determine the rate or amount of digestion in the two sets of flasks by measurements of their elec- Sudan III in Metabolism and Inheritance 177 trical conductivities. This proved impracticable because of the extremely low conductivity of the oils. ‘The writer is therefore not prepared to state whether the presence of the stain in the mole- cule of fat has any effect upon the power of lipase to hydrolize that molecule. . (8) Itseemed advisable next to learn the effect of the presence of the stain on the rate of the spontaneous oxidation of the fats. By a method described elsewhere® Prof. A. P. Mathews and the writer, in connection with other work, made a few experiments on the rate of oxidation of linseed o1l with and without the stain. It was determined that the oxidation proceeds more slowly in the oil + Sudan than in pure oil. Light has, however, such a profound effect upon the rate of oxidation that it is perhaps possible to attribute much or all of the retardation measured in our experi- ments to the absorption of light rays by the Sudan. The ques- tion that has been raised of the lessened availability of Sudan- stained fats must then be left without conclusive answer, but with such evidence as the preceding statements afford. DISCUSSION AND CRITIQUE The main facts at hand have already been given in a rather long historical statement and in the preceding account of the writer’s own results. The specific statements on the several topics of fat metabolism need not be again referred to. The general question of the basis or source of usefulness of Sudan III in such studies as the present may, however, be touched upon here. We can now consider too another most interesting aspect of our sub- ject, namely, the significance in inheritance of the observed trans- mission of this aniline dye from soma to germ cell, and its redis- tribution among the daughter cells of the germ. We treat the former topic first. In the study of the problems of fat metabolism, what is it that gives value and significance to the use of Sudan III? The answer must be that it is because Sudan sticks to fat or fatty con- stituents as long as they remain such in the body. Where the * See article by A. P. Mathews, O. Riddle and S. Walker, The Spontaneous Oxidation of Some Cell! Constituents, Abstract in Journ. Biol. Chem. vol., 4, p. xx, June, 1908., 178 Oscar Riddle / original’ stained fat goes, we believe from our experience, the stain will go also; the tell-tale color of the Sudan betraying at once both the presence and the source of the fatty materials in transforma- tion. We are thus enabled to study such aspects of fat metabo- lism as involve transfer, and re-deposition of fat, etc., which have been open to almost no other means of attack. Indeed, few organic constituents of the body other than the fats, are open to such methods of study even now. ‘The sum of our present information shows quite clearly that the Sudan holds to the constituent fatty acids even when the i integrity of the fat molecule is lost; this, dur- ing its transfer within the body fluids, through practically all of fhecmenibramestofthe body, and during re-synthesis, in whatever part of the body this may occur. In Alt these states and relations the pigment maintains the union; apparently only during the OXI- dation and final destruction of the fat is the alliance beoben! How far the oxidation must proceed before the disunion occurs, the writer is unable to say. The facts already brought forward concerning the behavior of Sudan in several aspects of fat metabolism furnish some solid ground upon which to base a discussion of the transmission and “inheritance”? phenomena involved in the passage of Sudan into the egg and the embryo. We can get a clear vision of this field of fact if we now focus on two points: What are the processes con- cerned in the entrance of the dye into the egg, and in its re-distribu- tion in the newly arising cells of the embryo? How do these pro- cesses compare and contrast with processes known to be involved in inheritance and developmental phenomena? ‘The answer to these questions should bring into relief a safe estimate of the sig- nificance of the transmission phenomena in question. The facts absolutely support the view that the passage of the stain through the follicular membrane, which has here been shown, is in no way unlike its passage through the intestinal epithelium or any other membrane. The Sudan, playing here an entirely passive role, is taken mechanically to whatever point the fat goes and remains with the fatty acids wherever they again bacon anchored through resy nthesis into fat. The processes involved "This holds true apparently when the fat is /ightly stained. Statements made elsewhere furnish the necessary qualifications, and the evidence. Sudan III tn Metabolism and Inheritance 179 in the re-distribution of the stain and fat in the cells which arise by division of the egg, are not different. Here we must believe that each cell of a dividing pair will carry stain in very close propor- tion as it carries fat. This is the testimony obtained from all so- matic tissues and the writer has shown that the general conditions of this statement are fulfilled in the oocyte and egg itself, since the germinal disc and the latebra of these stages take least stain (intra vitam) and are known to contain least fat. When in the course of development there arises a variety of body regions, some of which are less favorable for oxidations and therefore more favorable forthe storage of fats, the stain-containing fats may become transferred to these regions of the embryo, precisely as occurs in the somatic tissues of the adult. Localized areas of stained fat thus arise during embryonic life. If now one compares and contrasts these processes with those known to accompany inheritance, 1. e., developmental processes, some interesting features appear. There is, to be sure, transmis- sion of the dye from soma to germ, there is a persistence of that which is transmitted to such an extent as to cause this soma obviously to display the “new character.”’ If in the chick the body fat were used up in egg production,’as was elsewhere noted to occur in the salmon, some of the dye would of necessity again be deposited in the several eggs next formed; these eggs would in turn supply the somatic tissues developing from them. But this must inevitably come to an end in a few generations, the stain, sooner or later, having become diluted’ to the vanishing point. Again, there 1s absolutely no new growth of the material forming this “character,’’ nor is there any ‘eel change either in early or in late phases of the life cycle. NMeeehological change does however, accompany each change in the disposition of fat eiehaehe organ- ism, the color-picture thus being a moving one, different in aaa succeeding stage of development. These striking contrasts with what we recognize as the basic things in dees denen ical phenomena may well cause many to inquire: Why do we stop at all to consider the phenomena under 71 have observed a hen to lay four eggs after the beginning of a “starvation” experiment; the last of those eggs was laid on the twelvth day of starvation and much of the fat of its yolk was undoubtedly derived from the body fat of the bird. 180 Oscar Riddle observation, as inheritance phenomena! ‘The reason is that in- quiry and reflection seem to attest that this behavior of aniline dye is not an isolated thing in nature but that certain behaviors are known which are universally treated as “‘hereditary,” and which rest upon essentially the same base. ‘There is then, a group of cases which exhibit the simplest known inheritance phenomena, and which may be considered in the light of, and be largely ex- plained by our experience with Sudan. At the outset we call attention to the s'mplest analogy: the fact that the entire fat content of the egg yolk, which in the egg of a fowl aggregates several grams, is without doubt transmitted from the soma to the egg in the same way that Sudan is _ trans- mitted. That is to say, the fatty acids, which are re-s ynthesized into fat within the yolk pass from the soma (1. e., from within the body fluids) through the follicular membrane as these same fatty acids; the fatty ed constitutents of the egg-yolk by no means originating within the egg. ‘This conclusion follows as a logical necessity from our knowledge of fat metabolism elsewhere in the body, as well as from the special findings of Henriques and Hansen (03), who report the recovery of the specific and foreign fats of the food from the egg-yolk of the fowl. Moreover, from the stand- point of our general knowledge of metabolism it is not to be ex- pected that these constitutents of the egg-yolk should reach the latter in any other form. The protein of the egg-yolk, must also be conceived as having entered the egg, or at least to have ap- proached it,in a simpler form than protein, namely, as amino acids, etc.; the reconstruction of these doubtless occur chiefly within the egg itself and in this way give rise to the complex and special proteins of the egg. These things which occur in the formation of every egg, these ‘transmissions’ of amino and fatty acids from soma to germ, are cited because some biologists have considered the passage of a molecule of dye, ( iGo pebenvene “azo”? @ naphthol) from soma to germ, a thing not at all to be expected. Perhaps this state of thought is but an echo of the thoroughness of our long instruction on the wide gulf supposed to separate germplasm and somato- plasm; on an implied immunity proceeding from follicular walls, and an inviolate incorruptibility thought to preside over Sudan III tn Metabolism and Inheritance ISI all that lies within a vitelline membrane. Nevertheless the anal- ogy holds: the mass of fat and protein in the egg of the fowl is transmitted from parent to germ. This fat, moreover, neither immediately disappears nor undergoes equal distribution in the embryo, but like the Sudan it persists and becomes specifically localized. As an interesting example of such localized persistence the writer cites the toad’s egg in which Miss King (08) has pointed out that masses of yolk from the developing egg persist in the new germ cells, and that the yolk masses serve to mark off these germ cells as such. Here occurs a passing over of certain con- stituents from one germ cell to the next generation of germ cells. Of course it cannot be asserted positively in this case that the identical fat molecules of the first egg were contained in those of the succeeding generation. Miss King merely asserts the continuity and persistence of the morphological picture fur- nished by aggregates of such molecules. The transmitted fats however, exhibit one more advanced stage of complexity of be- havior than does Sudan, due to the new combinations they can enter into and the readiness with which their molecules can be both built up and torn down in the organism. A second analogy of the transmission and temporary persistence of Sudan we find in the cases of hereditary immunity, observed hitherto chiefly in mammals. In these cases, as 1s becoming well known, the immunity secured by the foetus through the placenta or germ may be of longer or shorter duration, often covering only a fraction of the span of a single generation. [tis from the stand- point of the type of transmission displayed by Sudan that these inherited immunities are to be interpreted. Sitowski has given the analogy of the passage of parasites (spirochzete and other protozoa) from the soma into the repro- ductive cells; the analogy is not complete, as he has pointed out, since in the case of the parasites we deal with living, active forms which seek out the germ cells. Other differences might well be noted. Bacteria also are known to reach germ cells in a similar way. Of much more interest and weight is the analogy between the behavior of Sudan and that of the glow substances of the glow- THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 8, NO. 2. 182 Oscar Riddle worm. In Lampyris and Pyrophorus these substances are to be found in the eggs and in every inter vening stage up to the adult. [tis true that in this case the substances, in contrast to the Sudan although not contrasting with the fat, do increase in amount at given points in the cycle. But the general features of the two cases agree so closely that actual kinship of behavior seems certain. In conclusion, we may emphasize the fact that the transmis- sion behavior of Sudan is the simplest of a simple class, and one of which we can render mechanical explanation at practically every point. Itshows to us the simplest form of inheritance, if the above analogies be granted. If the entrance of fat into the egg and its persistence there is as stated; if the hereditary 1mmuni- ties are of similar origin; or if the “glow” susbtances mentioned owe any considerable amount of their persistance to the type of mechanics with which we have been dealing in the case of Sudan, then this latter substance has proved of value in giving a view detailed and clearcut, of the mechanism of some phenomena which have been generally considered inheritance phenomena. The writer does not forget, however, the striking contrasts which these cases present to the great bulk of developmental phenomena, and which seem to present quite a different magnitude of complexity. He wishes to acknowledge his inability, for the present at least, to state how the simpler cases here considered are to enter very deeply into a solution of the more complex ones. It need hardly be pointed out, after our detailed account of the action of Sudan, that these studies furnish no basis whatever for the inheritance of acquired somatic characters. SUMMARY 1 Sudan III fed to fowls and turtles is deposited in their grow- ing ova. 2 Ova and soma of birds and mammals take up this stain after injection into the circulation or peritonial cavity. 3. The dye molecule is closely united with the constituents of the fat molecules and does not usually separate from them in the body. 4 Evidenceis obtained indicating that the fat of certain regions Sudan III in Metabolism and Inheritance 183 (in fowls) may increase actively in amount whilst other regions of fat take up no new molecules of fat whatever. 5 The stained fat may appear in the mesenteric lymphatics as soon as seventy minutes after feeding. Perceptible amounts may be deposited on the periphery of growing ova one or two hours later. 6 The stain is taken up very slowly, or hardly at all by birds which are being starved and thus made to decrease their store of fat. 7 Fat stained with:Sudan is apparently less available to the organism than is unstained fat. 8 The stain which is passed through the follicular epithelium into the egg, 1. e., into the newly arising organism—shows there a selective distribution; least stain being found in those parts of the egg which contain least fat, namely, the germinal disc and latebra. g The significance in inheritance of our experience with Sudan lies: (1) in the fact that here we get—through relatively accurate knowledge of the properties and physiological behavior of this aniline dye—a clear picture of how particles of the food or soma become a part of the germ or new generation; (2) in the emphasis which it lays upon the fact that the normal constituents of the egg@ have a comparable history; (3) in the seemingly perfect par- allel which it offers in explanation of the inheritance of immunity, etc.; (4) and the possible light which this extremely simple form of inheritance may throw upon the mass of developmental and inheritance phenomena which seem to be of a much higher order of complexity. BIBLIOGRAPHY (Sudan) Daovp1, L. ’96—Nouvelle méthode pour colorer la graisse dans les tissus. Archives Ital. de Biol., vol. 26. BIEDERMANN, W. ‘98 Beitripe zur vergleichenden Physiologie der Verdauung, I. Pfluger’s Archiv, vol. 72. Horsauer, L. ’oo—Kann Fett unverseift resorbirt werden? Pfliiger’s Archiv, vol. 81. Prucer, E. ’00—Ueber die Resorption kiinstlich gefarbtes Fett. Pfliiger’s Archiv, vol. 81. FRIEDENTHAL, H.’oo—Ueber die Permeabilitat der Darmwandung f tir Substanzen von hohem Molekulargewicht. Centralblatt fiir Physiolgie, vol. 14. 184 Oscar Riddle NeRKING, ]. ‘oo—Ueber das Lésungsvermégen von Seifen fiir fertlésliche Farb- stoffe. Pfliiger’s Archiv, vol. 82. Micnae is, L. ’o1r—Ueber Fettfarbstoffe. Virchows Archiv, vol. 164. Mann, G. ’02—Physiological Histology, Oxford. Franz K. anpd von SteysKat, K. R. ’02—Ueber das Wesen der Fuvopaischen Chylurie. Zeitschr. fiir Heilkunde. (Intern. Med.) vol 23, p. 441. SitowskI, L. ’05—Spostozenia biologiczne nad molowcami( Biologische Beobach- tungen uber Motten). (Contribution a la biologie des teignes). Bull. intern. de l’Academie des Sciences de Cracovie. Rippie, O. ’o7a—A study of fundamental bars in feathers. Biol. Bull. vol. 12. ’o7b—The rate of growth of the egg-yolk of the chick, and the significance of white and yellow yolk in the ova of vertebrates. Paper before the Amer. Soc. of Zodl., Chicago, Dec., 1907. Abstract. Science, n.s., vol. 27, p. 945, 1908. 08—The cause of the production of down and other down-like structures in the plumages of birds. Biol. Bull., vol. 14, no. 3, 163. Gace, S. H. ann S. P. ’o8a—Sudan III deposited in the egg and transmitted to the chick. Science, n. s., vol. 28, p. 494. ’o8b—Coloration of the milk in lactating animals and staining of the grow- ing adipose tissue in the suckling young. Paper before the Amer. Soc. of Anat., Balto., Dec., ’08. Abstract. Anat. Record, vol. 3, no. 4, p. 203, 1909. WuiteHEaD, R.H. ’og—A note on the absorption of fat. Amer. Jour. of Physiol., vol. 24, no. 2. Menpe1,L. B. ’o9—The absorption of fats stained with Sudan III. Amer. Jour. of Physiol., vol. 4, no. 5. Sitowsk1, L.’og—On the inheritance of aniline dye. Science, n. s. vel. 50, Sept. 3, p- 308. ( General ) ScumipT, R. H.’g1—Ueber die Aufnahme und Verbreitung von Oelen durch Pflan- zen. Flora, vol. 1, 74, New Ser. 40. Pizon, A. ’o1—Sur la pigmentation des Tuniciers et la mobilité de leurs granules pigmentaires. Comptes Rendus, Ac. Sc. vol 132, p. 170. Henriques, V. anp Hansen, C. ’03—Uber den Uebergang des Nahrungsfettes in das Hithnerei und ueber die Fettsaure des Lecithins. Skandinav. Archiv. Physiologie, vol. 14, no. 6. Kino, H. D. ’o8—The oogenesis of Bufo lentiginosus. Jour. of Morphology, vol. 19, no. 2. Ripp.e, O. ’og—The rate of digestionsin cold-blooded vertebrates: the influence of season and temperature. Amer. Jour. Physiol., vol. 24, no. 5. THE EFFECT OF SELECTION UPON MENDELIAN CHARACTERS MANIFESTED IN ONE SEX ONLY! W.._E. CAST EE In the Journal of Experimental Zoology, vol 7, No. 4, Miss McCracken reports extensive and important observations upon the inheritance of the alternative race-characters of silk-moths, univoltinism (one generation a year) and bivoltinism (two gene- rations a year). ‘The inheritance she characterizes as non-Men- delian on the ground (1) that when a cross is made involving the contrasted race-characters neither condition appears to be uni- formly dominant and (2) that in subsequent generations neither condition can be wholly freed from the other, that 1s neither be- haves as an extracted Mendelian recessive, and (3) that the proportions of univoltins to bivoltins following a cross does not approximate an ordinary Mendelian ratio, but changes from generation to generation according as selection is made for one condition or the other. It seems to me, however, that these reasons are not sufficient to establish the non-Mendelian character of the inheritance, but on the contrary are entirely consistent with a Mendelian interpre- tation. In the first place i it is to be observed that the inheritance is strictly alternative. All broods are either bivoltin or univoltin in character. This is prima facie evidence in favor of a Mende- lian interpretation. ‘The two essential features of Mendelian in- heritance, dominance and segregation, are both strongly in evi- dence throughout the entire experiment. ‘The only obscure points from a Mendelian standpoint are these: (1) Is dominance re- versed within the series, and (2) are the ratios obtained Mendelian ratios. Confusion in the interpretation arises fron the fact that the univoltin or bivoltin condition is manifested only in the female line, though transmitted through both sexes. The female silk- moth hatched in the spring of the year lays a batch of eggs and ! Contributions from the Laboratory of Genetics, Bussey Institution, Harvard University, No. 5. 186 WE. Castle then dies. ‘These eggs either hatch and produce a second genera- tion of moths the same season (bivoltinism) or else hold over to the next spring before hatching (univoltinism). All the eggs laid by the same moth behave in the same way, regardless of the charac- ter of the male that fertilized the eggs, as well as of the character of the moths which are to develop from the eggs. “Thus a univol- tin mother may produce both univoltin and bivoltin daughters, but neither sort will hatch from the egg before the following spring. And the eggs of a bivoltin (spring generation) mother will all hatch the same summer, regardless of whether her female descen- dants are to function as bivoltin or univoltin egg-layers. It therefore becomes somewhat difficult to trace the decent of the contrasted race characters. And to free either condition from the other, when they have once been crossed,is doubly difficult because the germinal constitution of the individual can be de- tected neither in the adult males, nor in the second (summer) generation of females. Plant-breeders have encountered puzzling conditions of a some- what similar nature, but happily have found a complete and simple explanation of them. ‘This explanation, I believe, will apply with modifications to the case under discussion. In maize, red color of the seed-coat (or pericarp) is a Men- delian dominant to its absence (white seed-coat), but the red color of the seed-coat is of purely maternal origin, and has no re- lation to the transmission of red or its opposite by the embryo lying within the seed-coat. If the plant of red-seeded maize is pollinated with pollen from a white-seeded variety, red seed 1s produced though the contained embryos are heterozygous, red (white). Now if this seed is planted, again only red seed will be produced, the heterozygous mother plants showing only the domi- nant character in the ears which they bear. But the embryos contained within the second crop of seed will be of three sorts in accordance with Mendel’s law, viz: 1RR, 2RW, and 1WW. Plants raised from embryos of the first two sorts will bear red ears, but a plant raised from a WW embryo will bear white ears. even though the seed-coat which covered that embryo was red, And if such WW plants are self-pollinated or pollinated interse, no red ears will be obtained thereafter, as shown by Locke (06). ao —_— — — Selection upon Mendelian Character 187 The behavior of red pericarp color is throughout this experiment consistently that of a Mendelian dominant, the only peculiarity of the case being that the dominant character is manifested only in maternal structures, not in paternal ones or in those of the embryo itself. Again, if white-seeded maize 1s pollinated with pollen from a red-seeded variety, the color of the seed is not affected, though the contained embryos are heterozygous, R(W). The seed ac- cordingly is white, but plants raised from it bear red ears. And if another generation of plants is raised from such red seed, these prove to be of three sorts, as in the reciprocal cross, viz: IRR, 2RW, and rWW. The RR and RW plants bear red ears, the WW plants bear white ears, though all the plants alike were raised from red seed. It follows that a red seed-coat may cover an embryo of any one of these sorts, RR, RW, or WW, but a white seed-coat may cover only two of these three sorts of embryos, viz: RW or WW. For the white seed must have received a maternal contribution of white, though the paternal contribution may have been either R or W. If accordingly one selects seed by color alone fein a mixed race of red and white, neither the red seed nor the white seed will breed true, either at the outset or after repeated selections. ‘This fact, however, is not inconsistent with a strict Mendelian behavior of pericarp color in heredity. li in the supposed case selection is carried out on a considerable scale, we can predict with consider- able accuracy what the proportion of red to white ears will be following each selection. Reciprocal crosses between red seeded and white seeded vari- ties yield the same results so far as seed-color in the hybrid plants and in their offspring is concerned. The F, hybrid plants bear only red ears. Accordingly it is impossible in this generation to make any selection for seed-color. Some of the F, plants bear red ears, some white ears. Here then selection may begin. If one saves only white ears for seed in this and subsequent genera- tions, the proportion of white to red ears in each successive crop should be approximately as shown in Table 1; if on the other hand one saves only red ears for seed, the proportions should be approximately as shown in Table 2. 188 W.E. Castle A glance at these tables shows with what persistency a Mendelian character manifested as a maternal character only may be ex- pected to crop out in the progeny of a mixed race, even when re- peatedly excluded by selection. The dominant character, red, TABLE 1 Expected results of selecting white ears only from a mixed race produced by a crass between a pure red and a pure white variety of maize GENERATION WHITE EARS RED EARS PER CENT WHITE SELECTIONS MADE lot poodcem mae fo) all fo) Mic scodhpatognes I 3 25 fo) l iowonehcacsocons I I 50 I MASpHacmqesoCtober A I 75 Z lta sidnaburaedamee 7 I 87.5 3 lWieaanmAo cheHoone 15 I | 93-7 4 DE icone S a Onee 31 I 96.8 5 1 ema oc oceranen 63 I | 98 .4 6 Le ono Ree 127 I | 99.2 7 1 A ac do Rea iia Bere 255 I | 99.6 8 I cease decor Sir I | 99.8 9 lode a oer 1023 I 99.9 10 TABLE 2. Expected results of selecting red ears only from a mixed race produced by a cross between a pure red and a pure white vartety of maize GENERATION RED WHITE PER CENT RED | SELECTIONS MADE | es | De aert ood Ear en ae all ° 100 ° | Oe Roan Oo Bes Gee 3 I 75 ° : edete le foaisiiviisisy ott coke 5 I 83.3 I a Oa ORSON 7 I 87.5 2 spa schelele sashes sy° |] 9.1 I 90.4 3 1D OSTA Oe | 1223 I 92.5 4 lee Oped Bond | 15.5 I 94 5 Laan doo aac CNN 19.0 I 95 6 Baia lerans coke stone hes 23.4 I 95-9 7 has the lead at the outset, since all F,, plants bear red ears, and this ascendency it holds through three successive selections, but beyond that point selection for the recessive character, white, takes the lead and this lead it increases at each successive selec- tion. Nevertheless after ten selections have been made the white Selection upon Mendelian Character 189 series still produces one red ear in a thousand, and the red series produces a considerably larger proportion of white ears.? Let us now compare with these series the results obtained by Miss McCracken in selecting for the conditions bivoltinism or univoltinism in a crossed race of silk-moths. The original cross (1g04) was made between a univoltin female and a male of bivoltin race. Ten of the F, female moths were tested, and six of these proved to be bivoltin, four univoltin in character. This looks like a Mendelian 1:1 ratio and suggests that one or the other parent in the original cross was heterozy- gous. But the results obtained by Toyama (’06) indicate that univoltinism is dominant over bivoltinism, and there is nothing in the results of Miss McCracken at variance with this idea. If so, the original female was a heterozygote, U (B), and when mated with a bivoltin male produced offspring half heterozygous, U (B. half pure bivoltin, BB. Matings of the F,, offspring with pure bivoltins would have settled this point, but no such matings were made. We are left therefore with only such information as is afforded by five matings of the F, individuals cnter se and by twenty-four matings of the F, males with univoltin females said to be of pure race. These twenty-four females at any rate, proved to be all uni- voltin, but there is reason to think that not all of them were ho- mozygous in that character. For of thirty tested females ob- tained from this cross, two proved to be bivoltin. Either, there- fore, univoltinism is not always dominant over bivoltinism, or else one or more of these univoltin females of pure race was in reality heterozygous, U (B). There is nothing in this assumption at variance with the statement that bivoltinism did not occur in the race from which these twenty-four univoltin females came. For the results of Doncaster (’08) on Abraxas, of Miss Durham (08) on canary-birds, and of De Vries (’08), on Oenothera show that in a wide variety of organisms one sex may be regularly heter- ozygous in gametic composition without resulting i in the produc- tion of a single recessive individual, except in picial out-crosses. 2 These tables show us what a serious task it would have been for our Puritan ancestors to eliminate from their harvests the occasional red ear which caused such joyous confusion at the New England husking-bees, even had their austere consciences demanded the undertaking. Igo W. E. Castle If all the univoltin females employed in these twenty-four matings had been heterozygous univoltins, while their mates were half of them pure biv oltin, half heterozygous univoltin, then we should have expected the offspring to be as four bivoltin: five univoltin, or 1:14, instead of the observed 1: 14. It seems probable, there- fore, that the twenty-four univoltin females were not all heterozy- gous, or else that the F, males mated with them were univoltin in character to a greater extent than their sisters, the tested F, fe- males. The data given are insufhcient for testing either hypothe- sis adequately. In either case we should expect the subsequent generation to contain a mixture of univoltin and of bivoltin fe- males, as actually observed, but in what proportions they occur- red would depend upon a number of contingencies. Concerning these we are largely without information, so that no Mendelian ex- pectations of much value can be calculated. Nevertheless I have calculated one such set of expectations which is contained in Table 3. It is based on the following contingencies: (1) That univoltinism is uniformly dominant over bivoltinism, and that, therefore, all bivoltin females transmit that condition only. But since the character of the male mate is in every case uncertain, it is assumed (2) that the males are in every generation of the same sorts as the females, and occur in the same proportions. 3) The actual mates of each group of females are assumed to have been such as one would obtain by random selection, that is they are univoltin and bivoltin in the same proportion as the popu- lation from which they are taken. If, for example, a group of individuals contains thirty univoltins and twenty bivoltins, and from this group five females are fee it is assumed that 3 of them are univoltin and two bivoltin. (4) The F, offspring pro- duced by the original cross are es to have been half pure bivoltins, half heterozygous univoltins. (5) The “pure” U fe- males, mothers of series A, are assumed to have been heterozy- gous in one out of seven cases. It will be seen from Table 3 that selection for B is, in nearly every case, attended by a reduction in the percentage of univol- tins (increase in the percentage of bivoltins) as we should expect, though this reduction is less rapid than we should expect. Con- trary to Miss McCracken’s view, the bivoltins are not in excess, } ~ - > Selection upon Mendelian Character 191 but are deficient. On the other hand when selection is made for U, there is observed no increase in the percentage of univoltins. Such increase we should expect in a series of successive selections for U, but not of necessity as a result of a single selection. “Thus in series EF following a single selection of U females, we expect a smaller percentage of U females than in the parental series, A’. TABLE 3. Percentages of univoltin females observcd in the several series and thetr relations to the expected Mendelian percentages. NUMBER OF PER CENT PER cENT | SERIES RESULT OF MOTHS TESTED UNIVOLTIN EXPECTED [phe rea ect oe Oe 30 93-3 94.6 airs se eyarsps 316 89.9 84.5* | 1 selection of U Kiameeas ees 3 | 743 88.7 | 2 selections of U a Le ch SS enn eee 209 85.2 51.4 | 1 selection of U,1of,B 15 {2 (ise) eae (?) 74 17.4 | 1selectionof U,2 of B raya cto arals 6 | 4c 50 DER s 55 chs 5A) 30 86.6 23.4 1 selection of B Gee eet ii win 23 69.6 12.1 2 selections of B Neerepece eters = 12 | 50 6.1 3 selections of B Jee(@g09); 2... or. (?) 33 ger 4 selections of B Weer. hacer tt 21 | 80.9 59.2 _ rselection of B, 1 of U 1b. 0 ORS Coe II | aT 70.2 | 1 selection of B, 2 of U Gh, See 31 71 30.2 2 selections of B,1 of U FGigoo) 2:42. =. (?) 43 14.6 3 selectionsof B,1 of U *Miss McCracken’s ‘‘Table of descent” is not in agreement with her text as regards the derivation of series E. I have assumed the correctness of the text, that series E is derived from the univolts (not the bivolts as shown) of series A’. But we do not expect a second selection for U to be attended by a further decrease in the percentage of U females, as is observed in series K, and series L. ‘This, as Miss McCracken observes, is a matter deserving explanation. It is unfortunate that we do not know from what particular broods the males were taken in each series. Without such knowledge a complete Mendelian analysis is impossible. If the parents of K and L happen to have been chosen from broods in which bivoltin individuals predominated, the high percentages of bivoltins in those series are fully explained. On this point it is sufficient to quote a paragraph from Miss 1Q2 W. E. Castle McCracken’s paper, p. 756. “In 1907, and again in 1908, pre- caution was taken to make a number of matings within each brood. It was found that all the tested females furnished by a few broods, particularly in 1908, were bivoltin-producing. Many of the broods furnished univoltin-producing females only and others furnished females part of whom were bivoltin-producing and part of whom were univoltin-producing. In each case these females were mated with males of similar ancestry.” ‘This 1s very clear evidence of Mendelian behavior of the characters univoltinism and bivoltinism. Had the author traced the descent through individual broods throughout her experiments instead of lumping them into series, 1am confident she would never have characterized the inheritance as non-Mendelian. Even without this, had selec- tion been made continuously for univoltinism within the mixed race, as was done for bivoltinism in one case, it can scarcely be doubted that the percentage of univoltins would have increased steadily, though probably less rapidly than bivoltinism in the reverse sort of selection. This at any rate is what we should expect if univoltinism is dominant. Compare Tables 1 and 2. On the whole, notwithstanding the incompleteness of the data, we are, I believe, justified in concluding that univoltinism is a Mendelian dominant to bivoltinism. For when from a mixed race produced by crossing, selection is made for either condition, bivoltinism increases faster than univoltinism. ‘The fact that bivoltin mothers may produce univoltin daughters when mated with males of unknown character is entirely in harmony with a Mendelian interpretation. It is unnecessary to assume a mysteri- ous “pull of ancestry,” a delayed “conjugation,” or the “masking of an anlage” for a series of years followed by its reappearance, so long as a simpler explanation in line with established principles of inheritance fully accounts for the phenomena observed. It may not be out of place to repeat that if one is to test fairly in a particular case the Mendelian or non-Mendelian character of inheritance, the line of descent must be known through individ- uals, not through masses of individuals. The futility of the mass- method of dealing with inheritance phenomena has been sufhciently illustrated in the results of the biometric school in England. PERE CTS OF ALCOHOL, ON THE LIFE CYCLE OF PARAMECIUM W. A. MATHENY From the Biological Laboratory of Clark University With One Ficure Calkins and Lieb (1) made some experiments with alcohol on paramecia and found that “. . . alcohol has no effect when taken in too weak doses, and too powerful an effect when taken in over ee doses.” “ when a mec dose is given | ie alcohol és 4 parts of Mu ) the effect is a eaaenued stimulus he sustains the high rate of division even during periods of depression of the control series.’ “ There is no doubt that for a time at least, alcohol will prevent death during periods of depression.”” “ there is evidence that . . . the general vitality would decrease under the constant stimulus asit does under treatment with hay infusion alone, although much more slowly.”” “ Notwith- standing the more rapid living, the ‘general vitality does not seem to be affected badly by the alcohol.” Woodruff (2) found that: 1. ‘Minute doses of alcohol will decrease the rate of division at one period of the life cycle and increase it at another period of the life cycle.” 2. “When alcohol increases the division rate, the effect is not continuous, but gradually diminishes and finally the rate of divi- sion falls below that of the control, followed by fluctuations above and below the rate of the control.” Since the acute and chronic effects of alcohol on the Paramecium have not been clearly shown, these experiments were started with that end in view. 194 W. A. Matheny The single-celled animals are especially well adapted for the investigation of physiological activity. “Che ease with which they lend themselves to experimen tal methods and the simplicity of their structure make it possible to arrive at an accurate analysis of the effects of the stimulus in question. ‘This problem was suggested by Prof. C. F. Hodge under whose direction a series of Salar experiments are now being carried on. METHODS Hay infusion was used at first as a culture-medium, but later :t was found that alfalfa, on account of its uniformity, gave much better and more reliable results. A comparison of these culture- media is given in [able 1. The infusion was made as follows: Fifteen grams of hay or alfalfa were boiled for one minute 1n 1500 cc. of tap water. ‘This was prepared each day late in the afternoon and was used the next morning. A“wild”’ paramecium was taken from a hay infusion in labora- tory and placed in a watch glass in 8 ce. of fresh solution. At the end of twenty-four hours the hay solution was renewed. On the second day there were paramecia enough present to start the experi- ment. At first, six cultures, each comprising four lines, were carried. Afterwards others were added. Using a pipet drawn out to a fine point, four individuals were transferred to four depression slides, each having a capacity of about five drops. Pure infusion was added and this started the control culture. In a similar manner cultures were started in the qollongaes percentages of alcohol, per cent, fo per cent, a5 per cent, ¢, per cent, aon pemceur A few days later cultures were started in 2 percent, 3 per cent, 4 per cent, 5 per cent, and 6 per cent solutions. In order to determine the resistance of paramecia to alcohol in greater quantities, the following experiment was performed. An individual was taken from the control and isolated in pure infu- sion. When it had made three divisions, a culture of four lines was started in pure solution for control. Another culture of four ae | E ffects of Alcohol on Paramecium 195 lines was started in a4 per cent alcohol infusion. ‘This alcohol medium was increased by § of 1 per cent each day. ‘The culture died when transferred into g per cent solution. Separate pipets were kept for each culture and great care was taken to transfer as little infusion as possible along with each indi- vidual. A lens having a magnification of ten diameters was used entirely in transferring specimens with the pipet from one slide to another. ‘This was easier than using a compound microscope. The slides were kept in moist chambers to prevent evaporation of the infusion. ‘These were stender dishes nine inches in diameter and three inches deep. In the bottom there was moist sand one inch deep on which rested four glass rods and on these rods were placed the depression slides. ‘Then over all was placed the tight fitting cover. Cover glasses were not used on the depression slides. itach day the rate of division was recorded for each of the cul- cures. When the count was made, an individual from each line was isolated on a clean depression slide in four drops of the culture medium. ‘The four lines in each culture were averaged to get the daily rate of division, and this result was again averaged for eight day pericds. In this way, fluctuations in division rate are elim- inated and we have a more reliable and comprehensive result than would be obtained by carrying a single line. The rate of division was taken as the index of the physiological condition of the organisms. All previous investigators in this held have considered this to be the most accurate indication which is available. Believing that more reliable data could be had if the alcohol was mixed directly with the culture medium in the desired proportion, this plan was followed entirely. This method eliminates a pos- sible source of error found in the “dropping”’ process which has been used extensively in similar experiments. A COMPARISON OF HAY INFUSION AND ALFALFA INFUSION AS CULTURE—MEDIA On January 6 the six cultures were started in ordinary hay infu- sion. From the first, the rate of division was low and it was plain 196 W. A. Matheny that something must be done to revive them. On January 19 the paramecia were transferred to an infusion made of alfalfa. They at once showed a marked increase in the rate of division. The division rate has since been uniformly high and devoid of depres- sion periods, except one or two of very brief duration, due to fall of temperature in the laboratory. TABLE 1 Table showing comparison of Hay Infusion and Alfalfa Infusion as Culture Media. The Alco- hol was mixed directly with the media as indicated below. In column 1 are given the average gen- erations of paramecia in hay infusion for thirteen days. Column 2 shows the average generations of the paramecia in alfalfa infusion during the succeeding thirteen days. 1 | : CONTROI I PER CENT 10 28 | 50 100 PER CENT PER CENT PER CENT | PERCENT DAYS = — i 7 : —— = = (1) (2) I 2 I 2 I 2 I 2 I 2 I I 25 | Tae Geilee 2G. | ales2 Sok bed || Uo ALY Nf Shean or 2S 2) I a5 2 2 BACON FG 22.750 | 225 2eAQu Lea aso. Be Te7 Siete 3 3: | 4.25 | 3.25 | 4.25] 2.50] 3.61 | 2.50 50 | 3. 4.25 | 3 4-5 4 AD Sa O25) Hea. (SOS hel Fg OES TS Bq a\rae mews ll eeey/G) Osi 5 ce Bie Serauliasc Sasi legis 6.86) |'4°75 | (862i) 5 7-§9.|| 4esaluaqasn 6 6.25 | I0 12 | 55 TO} 125 |"4\ Qtr (6.62) | T0468" |r7- 9.71 5-75| 9.87 7 7.87 | 11,12 | 7.37 | 11.73 | 6. 10.86 | 7.87 | 11.68 | 8.25 | 10.83 | 7 25] 10.12 8 862) ) 12.87) | 8.12 | 13523 | (625 | 12336 | 9.12 || 13-68) 9.251) r2c45esaznl ero Qe Waig62, 114262) )88.87 5) rae734| 75 13.86 9.87 | 16.36 |10. 14.57 | Qe2simaans 10 10.1 | TORQ 799371 773) leo 16.36 | 9.87 | 19.36 10.2 17.57 | LOstalea7eay II 10.8 17.80 10.1 19.48 | 8.25 | 17.36 | 9.87 20.36 |12. 19.57 | 10.7 | 18.96 12 12.1, | T9F80)|1O-6 P2173) (8825 17.86 |10.6. | 22.29 |I2.2 || 21.197) 1216" |l2onaG Nay || aeeyeet | 20:80 12.4. | 22.98 |'8-75°| 19.08 In2~-3 || 23666) 113%5)) | 22820 ea oe E ffects of Alcohol on Paramecium 197 GENERAL EFFECTS OF ALCOHOL ON THE DIVISION RATE TABLE 2 Chronological table showing average generations of Paramecia in alcoholic alfalfa infusions. First experiment DATE CONTROL ; 10 | 25 30 100 : PER CENT PERCENT | PER CENT PERCENT | PER CENT Jan. 7 1.25 2.25 it. 1.25 1.25 | Ie 8 De B75 71-945 Lipa De | 1.75 9 Be 3-25 2105 25 ae | ae 10 4-25 4. 3: 3-75 4. | 3-75 we 5. 425 325 4-75 5° 4-5 12 6.25 5-5 4. | 6.62 7: Gas 13 | 7.87 Tea) 6. 7.87 8.25 7.25 14 | 8.62 8.12 6.5 8.98 9.25 7-25 15 | 9-62 8.87 Tok 9.87 10. 8.25 16 10.12 9-37 8. 9.87 10.25 9.25 17 10.87 10.12 8.25 9.87 12 Io. 18 10.87 10.62 8.5 9-87 12.25 10.75 19 12.12 12.49 8.75 10.62 eet 12.5 20 KQE37 13.74 10.12 12.37 14.5 14. 21 | 15.12 15.24 11.24 Weiee yy, 15-75 1 els 22 | 17.12 17.24 12.99 15.24 17.37 17.12 23 18.99 19.24 14.49 17-49 19.12 18.87 24 20.99 Pa ie dt 16.74 19.55 21.24 20.87 25 21.99 22873 17.49 20.55 22.36 pop B 12 26 23-74 24.23 18.99 22.55 24.36 DTA: 27 25.49 26.73 20.49 25.23 26.48 26.17 28 28.24 29.73 22.99 28.23 29.48 28.85 29 e/a 31-48 24.49 on! soe 55235 3° 30-24. 33°23 “Io 3) Beas} 33-23 SS 31 33-24 35.48 25-99 33-16 34-85 33.60 Feb. 1 34-74 36.73 26.24 34-53 36.6 35-35 2 35-74 37-48 27.49 35-53 37-6 36.35 3 37-24 39-48 28.49 37-53 39-1 37-85 + 38.74 40.98 29.74 39-28 40.1 39-35 5 40-49 A223 30°99 40-2 41-35 PHAWANAWO NYA WHY NKHHKHN HN AY mn “© RoOnMNnNn BN + & HOO Se we em me om x 3 PERCENT | PERCENT Ded ii 4.8 ae 7.8 ye 9.2 G2 10.9 8.7 123.09) 9-9 14.4 10.1 15.4 10.6 7a 10.8 19.3 11.8 20.6 13-3 220 13.8 24.2 15.8 25.5 17.1 26.9 18.6 28. 20.1 29.7 Dit aie 23et 32 8 23.8 34.8 25.6 35-8 273 37-9 28.3 3959 Boas 40.7 32.6 42.9 34.8 45-3 35-8 47-5 37-3 48.8 | 39-3 50.8 40.6 52.8 42.6 55- 44.8 57- 46.4 58.5 47-1 60.5 48.1 61. 49.6 61.8 50.6 64.3 52.1 65.7 53-6 67.4 55. 2 69.2 56.4 79-5 57.6 7p rob) 293 4 PER CENT or An FWY WK = yey SES C\dS Merits Sy] SS) GA os v wn RPNN NNW COW Ww 40. > nan BONA AA HK = YOR A 5 PER CENT IAW www 6 PER CENT nn Como WOON DANN & VY ew me mM AAW WY © ONAN A bv Died 202 W. A. Matheny TABLE 3—Continued | 2 3 | 4 en ae a PER CENT PER CENT PER CENT | Mar. 9 72 | 73-3 | 61.3 55-3 10 7By.G Hex 62.8 55.8 11 | 75.2 76.3 65.3 56.3 12 Tae. A a5 66.3 | 56.6 13 79-7 79- 67.3. | 56.6 14 81.5 0.5 68 .6 56.9 15 | 82.5 81.9 | 70. | Seog) 16 84.5 83.4 72. | Died 17 | 86.5 85. | 73% | 18 | 88.3 | 86.5 75: 19 go | 88.5 Gah 20 92.2 | go.5 78. 21 | 94-4 92.5 80. 22 95-7 | 93-9 82.2 23 Opa | thoy 83-4 24 99.4 | 98.5 | 84.2 25. aicot6. )) 99:7. _—|| 86. 26 101.9 101. 87. 27 103.8 102.” 87.8 28 105.6 104.2 | 89. 29 106.6 105.4 | 90.5 30 107.8 TO7e3 ps g1.8 | 31 110 109.4 | 93-3 Apr. I 1 tay I11.3 94-7 || 113.9 113.4 96.2 3 57 | 115.3 97-9 | 4 117.5 | iy fase) | 98.9 5 T1829" || 118.9 IOI .2 6 119.7 119.7 103.2 Te wreTG es!) ilaaaa 105.4 8 | 124. 123.2 106.2 9 | 125.5 125.5 107.2 10 | 126.5 126.5 108 II | 127 .3 128.7 109.3 12 | 128 .8 128.7 110.8 | 13 | 130.6 130.8 112.8 14 | 132.8 iReYe Ye 114.8 15 | 135.2 134.8 116.3 16 | 136.9 136.8 117.8 1 | 138.4 138.5 | 119.3 18 | 140.1 140.6 121 19 | 141.8 141.9 123 20 | TAGES 143-6 123.7 21 145.8 146.1 124.0 DATE Apr. May - ike) Effects of Alcohol on Paramecium TABLE 3—Continued 203 2 3 CONE ROS PER CENT* | PER CENT 146.8 147-3 126.6 en 148.8 128.6 149.6 150.6 129.6 150.8 152.6 130-3 151.8 153 -6 tds 152.8 154.8 132.8 | 154.1 156.1 133.8 155.8 157-3 135.8 157.4 158.3 137-1 158.4 160.2 138.4 159.8 161.4 140-7 161.1 162.9 e+? 163.2 165.4 345;7 164.7 167.1 146. 166.3 168.6 147-5 168.8 170.9 149-5 170.6 172.1 LESS) 172.6 174-3 ES2e7 173.6 175.8 154-7 175.6 177-8 158 177-8 | 179.8 160 180. | 182.3 162. 182.5 | 184.6 ee 184.5 186.1 166. 186.5 | 187.9 Le 188.5 189.7 he 190.5 192.2 172.5 192.3 194. 1335.) a) 193.8 195-5 “fen 194.8 196.5 176. 195.8 | 197.5 R/T | 196.8 | 198.5 al 198 .8 | 2007-5 ae | 200.8 202.5 182. 202.8 204.3 ag | 204.3 205.8 85. 206.3 207.8 186.5 207.3 209. 187.5 209.3 210 189. 211.3 De IgE 213.3 215.5 193% 215.8 217-5 Pee 218. 219-5 195-5 220 221.5 5 204 W. A. Matheny EXPERIMENTS WITH ALCOHOL ‘The first series of experiments was started January 6 and carried on until June 4. The percentage of alcohol given, varied from 1 per cent to zoo per cent. The different cultures, with one exception, showed remarkable uniformity in division rate. The exception was the culture in |, per cent infusion. From the very beginning and uniformly throughout the experiment these individuals showed a slower rate of division. But since there were three cultures in weaker alcohol solutions and all of them equaled the control in division rate, we conclude that this exception was caused by sources other than the alcohol. When the experiment was discontinued the control had reached 243 generations, the 1 per cent culture 245, the 75 per cent cul- ture 211, and the 5 per cent culture 248. The #5 per cent culture was discontinued in the 227th generation at which time the control was in the 224th. ‘The 1—100 per cent culture was discon- tinued in the 184th generation while the control was in the 178th. The second series of experimentsjwas started January 26. The percentage of alcohol varied from 2 per cent to6 percent. At the end of the 15th day, the 6 per cent culture was dead. During this time it had reached only the 8th generation, while the controlwas in the 22nd. ‘The 5 per cent culture lived 25 days and reached the 25th generation while the control was in the 39th. The 4 per cent culture died during the 5oth day in the 57th generation at which time the control was in the 82nd generation. When the 6 per cent culture died it was 14 generations behind the control, the 5 per cent culture was also 14, and the 4 per cent culture was 25. The experiments were discontinued on June 4. The 3 per cent culture had reached 196 generations and was 24 generations behind the control. It might be noted here that this culture seems to mark the beginning of the effects of alcohol. The 2 per cent cul- ture was in the 22Ist generation, and showed no effects whatever from the stimulus. ‘The control was in the 220th generation. Depression periods were never in evidence. When the tempera- Effects of Alcohol on Paramecium 205 ture of the laboratory was allowed to run low on holidays and Sun- days there was a marked lowering of the division rate which affected all the cultures alike. ‘This temporary check always dis- appeared when the temperature came back to the normal state. CONCLUSIONS Our experiments show that: 1. There is no evidence that alcohol acts as a periodic or con- tinued stimulus. 2. There is no evidence that the general vitality would decrease under the constant stimulus of minute doses. 3. Alcohol in minute doses, 2 per cent or less, has no effect whatever. ; 4. When a medium dose ts given, for example 3 per cent, the general vitality 1s weakened. 5. If alcohol is given in greater strength than 3 per cent the rate of division is lowered and the organisms finally die. LITERATURE (1) Gary N. Carkins and C. C. Lies ’o2—Studies on the Life-History of Protozoa. 2. The Effects of Stimuli on the Life-Cycle of Paramecium caudatum, Archiv fuir Protistenkunde. (2) Loranpe Loss Wooprurr ’o8—Effects of Alcohol on the Life Cycle of Infusoria, Biological Bulletin, vol. 15, no. 2, July. 200 —— CONTROL W. A. Matheny eleva Sa le7e4 Ss 1-507 ee tlw The ordinates represent the 0000 2/ average daily rate of division. ee vi . . soseere o The figures below indicate the f number of eight-day periods. Scabans 5% 2 3 4 5 6 / 8 9 10 11 12 13 14 15 16 17 18 19 T T Ale T T T Tiree aT T “Un T cece ae) T Chart showing division rate of cultures averaged for eight day periods. THE CHROMOSOMES IN THE GERM-CELLS OF CULEX N. M. STEVENS Bryn Mawr College Wirn Firry-rwo Ficures In the summer of 1905, Miss Boring and I collected material for the study of the spermatogenesis of the mosquito, but the germ- glands proved not to be sufficiently well fixed. In 1907 | spent several days studying aceto-carmine preparations from the larve and pupz of some California mosquitoes. Naturally I expected to find one or more heterochromosomes, but nothing of the kind could be detected either in the growth stages of the spermatocytes or in the maturation divisions. “The number of chromosomes was small, only three in one species and four in the other, but it was not an easy matter to determine whether or not the pairs of univa- lents were exactly equal. In October of this year (1909) I accidentally discovered an abundance of larve and pupz of Culex (sp.'not determined, prob- ably C. pungens), in a small pond where I was able to collect the material up to November 22. ‘This time I determined the location of the testes and ovaries, in the third segment from the end of the tail—removed the anterior segments, and secured good fixation in Flemming’s fluid and fairly good in Gilson’s mercuro- nitric. The larger part of the material was dissected and the germ- cells studied in aceto-carmine preparations. With careful sealing it has been found that these slides can be kept in usable condition ‘April 7, 1910. It is now quite certain that pupe of two species were used in this work. All of the material that can be obtained from the same pool this season will be examined with a view to determining the conditions in the germ cells of each species breeding there. A species of Anopheles with 6 chromosomes in the spermatocytes has already been found. 208 N. M. Stevens for several weeks, if the material has not been too deeply stained in the beginning. ‘he fixed material was stained either with tron- haem: itoxylin or with thionin, both giving good results especially with the Flemming fixation. Prana: of cells and chromosomes drawn from sections are only about two-thirds as large as those taken from aceto-carmine preparations. ‘The difference is mainly due to shrinkage in fixing fluids and alcohols, though the acetic acid probably swells the structures slightly. “he majority of the figures were taken from aceto-carmine preparations and_ those taken from sections will be designated as such. A few odgonia were found in mitosis in various young ovaries. In all cases the chromosomes were paired in prophases and meta- phases before metakinesis (Figs. 1 and 2), as previously described by the author in several species of Muscidz (08). Two longer pairs are present with one pair considerably shorter. “The homol- ogous chromosomes composing the pairs are apparently equal in length. As in the Muscide, each of the six chromosomes divides longitudinally, and pairing of the daughter chromosomes prob- ably occurs in the telophase, for very early prophases show the chromosomes paired and twisted together forming three spireme threads which gradually shorten and separate for mitosis. Fig. 3 shows the chromosomes of an oocyte in an early growth stage ne the paired chromosomes still distinct, and Fig. 4 the nucleus of a somewhat later stage showing three separate spireme threads of different lengths. Whether these separate spiremes later unite to form a single thread | have been unable to determine, but that parasynapsis occurs immediately after the last oogonial mitosis is certain, and it is equally certain that the chromosomes are sim- ilarly paired 1 in earlier generations of the oogonia. Fig. 5 1s an outline camera drawing of a testis stained in aceto- carmine and considerably flattened under the cover- glass. In the first cyst at the tip (a) were resting spermatogonia, in the sec- ond (b) anaphases and telophases Be spermatogonial mitoses. Then followed cysts (c,d, e) containing synizesis stages, and growth stages of the first spermatocytes, one cyst (f) in a stage inammedie ately following the first maturation division, several cysts of sperma- tids (g, h, 7, ;) and masses of spermatozoa (k) pressed out through in Chromosomes in Germ-Cells of Culex 209 the broken wall of the testis. The two testes are situated one on each side of the digestive tract in the third segment from the end of the tail of the pupa. Maturation occurs mainly, if not wholly, during the pupa stage. Fig. 6 is a good specimen of an early prophase of spermatogonial mitosis, showing three long granular chromatin threads, one of which already shows its double character. Fig. 7 1s a section of a nucleus showing the twisted chromosomes of a later prophase stage. Fig. 8 is a spermatogonium in metaphase, showing the pairs separated and apparently all equal; Fig. 9 the chromosomes from a similar stage, in outline, so as to show the full length of each chromosome; and Fig. 10 a late prophase from a Flemming- iron-hematoxylin section. In no one of these figures would one suspect that one pair of chromosomes might be unequal, but in the testes of two individuals I found seven plates of a different character, one of which is shown in Fig. 11, with the shorter pair of chromosomes apparently composite, each consisting of a longer and a shorter portion, the longer components equal, and the shorter unequal and suggesting a case of unequal heterochromo- somes such as occur in the Muscidz (708). Had I not found these cases, Six in one testis and one in another, I should have said that there was no evidence in the mosquitoes of any such hetero- chromosomes as occur in so many other insects, and are clearly present in the nearly related Muscide. A very distinct synizesis stage occurs in which the granular and beaded chromatin threads are wound about a large nucleolus, which in Flemming material stained with thionin, is yellowish in early stages and gradually acquires a staining quality nearly equal to that of the chromosomes. Figs. 12, 13 and 14 were taken from the same section to show an early synizesis stage with a pale plasmosome, a later stage with blue-staining plasmosome, and a pale spireme stage with the plasmosome stained a deep blue (Fig. 14). This series suggests an extrusion of chromatin sub- stance from the spireme during the synizesis stage and an absorb- tion of the extruded material by the plasmosome. In Culexsit is quite certain that parasynapsis occurs in each cell generation 210 N. M. Stevens of the germ cells in the telophase. Other cases’ where synapsis is known to occur either before or after synizesis have indicated that the two phenomena have no necessary connection. In a recent paper Miss King (08) describes a separation of chromatin substances and rejection of masses of deeply staining material from the spireme during synizesis in the odcytes of Bufo lentin- ginosus. Something Gaile was observed by Miss Boring (07) In connection with the synizesis stage of the spermatocytes of three species of Ceresa ( Pl. II, Figs. 62-67, 82 and 93). In the former case the rejected Shmomeeane substances were observed in the form of nucleoli and also as a deposit on the nuclear mem- brane; in Ceresa they formed a dense plate at the base of the bouquet of short chromatin loops in the synizesis stage, and later became divided up into several dense masses distributed over the inside of the nuclear membrane and gradually disappearing in the later growth stages and prophases of the first maturation division. Buchner also describes a “Chromidial-austritt” during the syni— zesis stages of the odcytes of Gryllus campestris where granules of material staining like chromatin are extruded from the nucleus in the region where the ends of the chromatin loops touch the nuclear membrane (’og, Pl. 21, Figs. trg—121). The change in staining quality of the plasmosome in Culex may therefore be regarded as further evidence that the synizesis stage of both oocytes and spermatocytes is probably a period during which some modification of the chromatin occurs preliminary to matur- ation. Whether the rejected material visible in some cases, 1s waste material or substances which have some function connected with the growth stages of the germ cells, we can only surmise. All fheouel the synizesis aa growth stages of the spermato- cytes of Culex, there is absolutely no sign of any condensed hete- rochromosomes, only a plasmosome and a spireme, or perhaps three separate spireme threads. When the spireme begins to shorten and thicken one can occasionally be sure that it 1s not * As examples where synapsis occurs before synizesis, I might cite from my own work, Photinus penn- sylvanicus and Limoneus griseus (’o9, Pl. I, Fig. 5-8; Pl. II, Figs. 31-38), while in many other species among the Coleoptera synapsis occurs at the close of the synizesis stage as in Chelymorpha argus (’06, Pl. LX, Figs. 37-43) and Photinus consanguineus (’o09, Pl. I, Figs. 23 and 24). Chromosomes in Germ-Cells of Culex PLAN continuous. In Fig. 15 one double end was seen above the plas- mosome, and a segment with both ends free at a lower focus. From this stage on, the three chromatin threads shorten, thicken, and each separates into its two parallel components. Fig. 16 is an early prophase showing the three long twisted pairs, Fig. 17 a later stage with shorter, thicker twists, and Fig. 18 a slightly later stage from a section, showing a very characteristic appear- ance of the three pairs about the time that the spindle 1s formed. Fig. 19 is also from a section, and shows the plasmosome still present but pale again. In both testes from one individual, examined in aceto-carmine preparations, there was one cyst of prophase stages, two of which are shown in Figs. 20 and 21, where one pair of chromosomes was condensed while the others were still pale and granular. The material was collected on November 22, after freezing weather, and a very unusual number of cells were in the prophase and metaphase of the first maturation mito- sis. [he chromosomes and cells were both smaller than usual, and I thought that maturation must have been hastened by high temperature following cold, and that the stages shown in Figs. 20 and 21 were abnormal. Later, however, I found a trace of the same phenomenon in perfectly normal material well fixed with Flemming and stained with thionin;1. e., one pair of chromosomes becoming condensed in advance of the other two. The con- densed pair in Fig. 21 resembles closely the shorter pair in Fig. 18 and other similar stages; and, together with the inequality ob- served in seven spermatogonial equatorial plates (Fig. 11), indi- cates that the smaller pair of chromosomes in Culex may have some of the characteristics of the heterochromosomes of other insects. Fig. 22 is a typical first spermatocyte in metaphase or meta- kinesis, the spindle being formed within the elongated nucleus. In Fig. 23 the same Pees me are shown separately. The middle one (4) is the shorter pair of the spermatogonia and pro- phase stages and the ring is the figure 8 of Fig. 18, 6. In rare cases all three pairs may come into the spindle in the form of rings, but usually only one pair takes this form. Fig. 24 1s a very frequent prophase appearance, showing one ring with over- 212 N.M. Stevens lapping ends, and the other pairs, one of them simply crossed, the other changing from the parasynapsis arrangement of the earlier prophase to the telosynapsis method of union of the chromosomes in metaphase. Fig. 25, a and 6, shows a diflerent method of union of the ends. ‘They most often overlap, but may unite and split giving the cross-form so familiar in insect spermato- genesis. Fig. 26 shows an unusually well marked cross; it is the same ring-shaped chromosome with the outer ends separated, the other pairs being already in metakinesis. Fig. 27 is a later stage showing the ring chromosome about to separate later than the other pairs. Figs. 28 and 29 show again both methods of union of the chromosomes in telosynapsis. Fig. 30 is an extreme case of overlapping. Figs. 31 and 32 show two other groups, in each case the group of three being from the same spindle. Fig. 33 1s a rare case in which all three pairs appeared as rings in the spindle. Figs. 34 and 35 are prophase and metaphase stages from sections. The anaphase in Fig. 36 shows that the over- lapping ends of a pair of chromosomes may remain attached side by side until quite a late anaphase instead of pulling out into the end to end position seen in Figs. 23 and 31. It is interesting to find in Culex a clear case of parasynapsis in oogonia, oocytes, spermatogonia and spermatocyte prophases, and then to see these same chromosome pairs appearing in the first maturation meta- kinesis as though united end to end (telosynapsis), and not only this, but to be able to trace all the changes from parasynapsis to telosynapsis in some of the preparations. In the Muscidie the chromosomes pair side to side (parasynapsis), and separatein the first maturation mitosis in a manner closely resembling many cases of longitudinal division, going to the poles in the nin of V’s while in Culex the pairs become more or less perfectly united end to end (telosynapsis) and then separate as V’s. In many cases one can only infer from the position of a pair of chromosomes in the spindle what the method of synapsis has been. In Culex, if one saw only the metaphases and anaphases one would certainly say that it was an undoubted case of telosynapsis, but the fact is that we have here a case of intimate and prolonged parasynapsis somewhat similar to that observed by Strasburger and his school Chromosomes in Germ-Cells of Culex 213 in both somatic and germ cells of various plants, the clearest cases being described by Overton (og) in Thalictrum purpurascens and Calycanthus floridus. In a number of spindles, most of them from one testis, the left hand chromosome pair (a) of Fig. 23 was found irregularly frag- mented (Figs. 37, 38, 39), and even unequally divided as in Figs. 4o and 41. ‘These cases of fragmentation were very rare and were found among a larger number of cells containing normal groups of chromosomes, but constrictions such as appear in Fig. 23, a, were frequent. Fig. 42 shows a still deeper constriction in the same chromosome. ‘These cases of fragmentation and constric- tion suggest that this particular chromosome pair may be com- posite, and I should therefore not be surprised to find other species of Culex with a larger number of pairs of smaller chromosomes, or even to find more than the expected number in somatic cells of this species. In the telophase of the first maturation division the chromosome fuse and then large vacuoles appear as in Figs. 43 and 44. Later one finds a distinct nuclear membrane and chromosomes lying on this membrane as in Fig. 45, where cell a is drawn to show an optical section through the nucleus, and cell } a tangential section. In the prophase of the second division (Fig. 46) the chromosomes are already divided longitudinally, and they always come into the spindle divided and much tangled (Fig. 47). Asin the first divi- sion the spindle forms in the elongated nucleus. In the aiiaphase the V-shaped chromosomes move out of the tangled metaphase and form regular polar groups (Figs. 48 and 49). A telophase is shown in Fig. 50. As a final effort to decide whether the smaller pair of chromo- somes is equal or unequal, | went through my sections again and made camera drawings of the plainest cases of prophase group- ing and of the various metaphase forms (Figs. 51 and 52). The components of the pair in prophase are always more or less twisted and foreshortened, so that a slight difference like that in- dicated in Fig. 11 might be difficult to detect. In Fig. 51, a tod are from prophasesof the first spermatocyte, e froma late sperma- togonial prophase. Similar cases may be seen in the metaphases THE JOURNAL OF EXPERIMENTAL ZOOLOGY VOL. 8, NO 2. 214 N. M. Stevens of Figs, 8 and g. In all of these cases | should suppose that the pairs were equal if the question had not been raised by the seven spermatogonial plates represented by Fig. 11, and also by the occurrence of an unequal pair of heterochromosomes in each of the nine species of Muscide previously described (08). In Fig. 52, a to j, the same pair of chromosomes is shown in metaphase and metakinesis. It is possible in each case that one chromosome is slightly larger than its mate, but the difference is certainly not conspicuous, saa if present, 1s obscured by the fact that the outer, free ends of the elements usually turn in different directions. DISCUSSION Heterochromosomes If we define a heterochromosome as one that remains condensed through the growth stages of odcytes or spermatocytes, then we must say that such are not present in Culex. We have seen, however, that one pair of chromosomes may be condensed in advance of the other two pairs, in an early prophase of the first maturation mitosis, and in the spermatogonia of two individuals we have found evidence that an unequal pair of small chromosomes is combined with a larger equal pair, which, we would suggest, may control the behavior of the smaller unequal heterochromo- some pair, preventing it from remaining condensed during the growth stage. On the other hand the tendency of the hetero- chromosomes to remain condensed may account for this pair of chromosomes sometimes appearing in condensed form earlier than the other two pairs in the prophase of the first maturation division. It is certain that in most cases the heterochromosomes, if present, are so intimately fused with another pair of chromo- somes that it 1s rarely possible to detect their presence, the slight difference in length of a pair of long, twisted chromosomes being difficult to determine. The case of Culex is an interesting one in connection with that of several species of Lepidoptera (Stevens ’06, Dederer ’07), Nezara (Wilson ’05) and Forficula* (Zweiger ’06) and Anisolabis *In the case of Forficula auricularia, the author finds an unequal pair of heterochromosomes entirely distinct from the lagging pair described by Zweiger as present in some individuals and absent in others. , es ~ y.) Chromosomes in Germ-Cells of Culex 215 (Randolph ’o8) in which an equal pair of chromosomes has been described as resembling the odd heterochromosome and the un- equal heterochromosome-pair of other insects, in that it remains condensed during the growth stage of the spermatocytes. ‘The conditions in Culex would indicate that in other cases where no heterochromosomes have been found, they may nevertheless be present, combined with other chromosomes in such a way that a slight inequality in size may easily escape detection, and that their characteristic behavior during the growth stage of the sperma- tocytes may be changed by the influence of the pair with which they are combined. As to the relation of the heterochromosomes to sex determination further discussion seems to be of little value until we get more evidence in connection with experimental breeding. Synizests The synizesis stages of Culex apparently have no relation what- ever to the phenomena of synapsis, but are interesting in that they afford further evidence that synizesis is a period of recon- struction in which certain elements present in the spermatogonial chromosomes are either rejected as waste material or are isolated in order that they may perform some function in connection with the growth stages of the germ-cells. Synapsts Perhaps the most interesting point in the history of the germ- cells of Culex is the fact that, as in the Muscidz, pairing, or syn- apsis, occurs in connection with each spermatogonial and odgon- ial mitosis as well as in anticipation of maturation. I have not been able to study somatic mitoses in Culex, but in the Muscidz a similar pairing was found in follicle cells of the ovaries, and it may therefore be true that pairing of homologous chromosomes occurs In connection with each mitosis throughout the life history of these insects, as Overton thinks probable in the case of sev- eral of the higher flowering plants whose cytology he has studied. Both in the Muscidz and in Culex, all the evidence indicates 216 N. M. Stevens that parasynapsis of homologous chromosomes occurs in the tel- ophase of each mitosis of the germ-cells, and that this intimate relation of the paternal and maternal members of the pairs per- sists from one mitosis to the next, when in the odgonia and sperma- togonia, each chromosome divides longitudinally, but in matura- tion the two members of a pair separate and go todifferentcells. It is of especial interest to see in Culex a perfectly clear case of para- synapsis change in some cases to an equally clear case of telosyn- apsis before metakinesis, while intermediate ring-stages and cases of overlapping ends also occur. SUMMARY 1 The number of chromosomes in Culex sp. 1s six in oogonia and spermatogonia and three in the spermatocytes. 2 There is some evidence that a small unequal pair of hetero- chromosomes is combined with a larger equal pair of chromosomes. 3 During the synizesis stage, the staining quality of the plasmo- some changes in such a way as to indicate that it has received chromatin material from the chromatin threads wound about it. 4 Parasynapsis occurs in each cell-generation of the germ- cells, the homologous maternal and paternal chromosomes being paired in telophase and remaining so until the metaphase of the next mitosis. 5 Parasynapsis of homologous chromosomes often changes to telosynapsts in the metaphase of the first spermatocyte. Bryn Mawr College December 20, 1909! BIBLIOGRAPHY Borinc, A. M. ’07—A Study of the Spermatogenesis of ‘Twenty-two Species of Membracide, Jessidae, Cercopide and Fulgoride. Jour. Exp. Zodl., vol. 4. Bucuner, P. ’og—Das accessorische Chromosom in Spermatogenese und Ovo- genese der Orthopteren, zugleich ein Beitrag zur Kenntnis der Reduktion. Arch. f. Zellforschung., vol. 3. Deperer, P. H. ’07—Spermatogenesis im Philosamia cynthia. Biol. Bull., vol. 12. 'Received for publication Jan. 15, 1910. Chromosomes in Germ-Cells of Culex 217 Kino, H. D ’o8—The Odgenesis of Bufo lentiginosus. Jour. Morph., vol. 19. Overton, J. B. ’og—On the Organization of the Nuclei in the Pollen Mother Cells of Certain Plants, with Especial Reference to the Permanence of the Chromosomes. Ann. of Botany, vol. 23. Ranpotpu, H. ’o8—On the Spermatogenesis of the Earwig, Anisolabis maritima. Biol. Bull., vol. 15. Stevens, N. M. ’o6—Studies in Spermatogenesis. II. A Comparative Study of the Heterochromosomes in Certain Species of Coleoptera, Hem- iptera and Lepidoptera with Especial Reference to Sex Determina- tion. Carnegie Inst., Washington, Pub. 36, No. 2. o8—A Study of theGerm Cells of Certain Diptera, with Reference to the Heterochromosomes and the Phenomena of Synapsis. Jour. Exp. Zool., vol. 5. ‘og—Further Studies on the Chromosomes of the Coleoptera. Jour. Exp. Zodl., vol. 6. Witson, E. B. ’og—Studies on Chromosomes. I. The Behavior of the Idio- chromosomes in Hemiptera. Jour. Exp. Zodl., vol. 2. ZwericerR, H ’o6—Die Spermatogenese von Forficula auricularia. Zool. Anz., 30. Also Jena. Zeitschr. f. Nat., vol. 42. 218 N. M. Stevens DescrirTION oF FIGURES The figures were all drawn with camera lucida. Fig. 5 was drawn with Zeiss 16 mm, compens. 4; all others figures with Zeiss 1.5 mm, compens. oc. 12. The plates were reduced one-fourth. Fig. 1 Odgonium showing 3 pairs of chromosomes in equatorial plate. Ac-c-prep. Fig. wv Three pairs of chromosomes from another o6gonium. Ac-c. Fig. 3 Chromosomes and plasmosome (p)froma youngoécyte. Ac-c. Fig. 4 Nucleus of an older odcyte showing 3 separate spireme threads. Ac-c. Fig. 5 Outline camera drawing of a testis mounted in aceto-carmine and flattened by pressure on the cover-glass. a= cyst of resting spermatogonia; b = spermatogonia in anaphases and telophases; c, d,e = synizesis stages; f = second spermatocytes in stage seen in Fig. 44; g,h,1,j = spermatids; k = masses of spermatozoa. Ac-c. Fig. 6 Spermatogonium from tip of testis, early prophase showing one chromatin thread sepa- rating into its component chromosomes Ac-c. Fig. 7 Section of a nucleus of a spermatogonium showing twisted pairs of chromosomes in prophase of mitosis. Fig. 8 Spermatogonium showing three pairs of chromosomes in metaphase Ac-c. Fig.g Chromosomes from another spermatogonium in outline. Ac-c. Fig. 10 Section of a spermatogonium showing all three pairs of chromosomes in late prophase. Fig. 11 Chromosomes from one of 7 equatorial plates found in spermatogonial cysts in two indi- viduals. The smaller pair of chromosomes appears to be composed of an equal and an unequal pair combined. Ac-c. Fig. 12. Early synizeses stage from material fixed in Flemming and stained in thionin Plasmosome (p) pale yellowish. Sec. Fig. 13 Later synizesis stage from same section, plasmosome (p) blue Fig. 14 Spireme growth stage from same section, plasmosome deep blue. Fig.15 Late growth stage of first spermatocyte, focusing from the surface of the nucleus down to the plasmosome (fp), and showing one end of a spireme double. Ac-c. Fig. 16 Prophase of a first spermatocyte mitosis showing the three twisted pairs of chromosomes. Ac-c: Cells of Culex Chromosomes in Germ- 14 220 N. M. Stevens DeEscripTIoN oF FiGuRES Fig. 17 Later prophase. Ac-c. Fig. 18 Still later prophase from a section; showing the shorter pair (a), a figure 8 (b) which later appears as a ring, and a second long pair (c). Fig. 1g Section of a prophase showing the plasmosome (p) still present. Figs. 20 and 21 Exceptional prophases in which one pair, apparently the shorter one, was completely condensed earlier than the other two pairs. Ac-c. Fig.22 Typical first spermatocyte metaphase. Ac-c. Fig. 23 Chromosome bivalents from the same cell drawn separately. Ac-c. Fig. 24 Chromosomes from a slightly earlier stage, a spindle prophase. Ac-c. Fig.25 a and 6 The ring chromosome. Ac-c. Fig. 26 Three chromosome pairs from one spindle showing the ring-chromosome forming a cross where the ends are united. Ac-c. Figs. 27, 28,29 Other groups of chromosomes, each group from one spindle. Ac-c. Fig. 30 Pair of chromosomes showing extreme case of overlapping, from a metaphase. Ac-c. Fig.31 Another metaphase group. Ac-c. 222 N. M. Stevens DescripTIoN oF Ficures Fig. 32 Metaphase group in outline. Ac-c. Fig. 33. Exceptional group of three rings. Ac-c. Figs. 34 and 35 Late prophase or early metaphase, from sections showing a single chromosome pair. Fig. 36 Anaphase, showing the ends of one pair still overlapping and attached. Ac-c. Figs 37-41 Exceptional cases of fragmentation and unequal division of the chromosome pair a of Fig. 23.. Acc: Fig. 42 Another case of irregular constriction of the same chromosome. Ac-c. Fig. 43. Pair of second spermatocytes. Ac-c. ——. Chromosomes in Germ-( Cells of ‘6 ‘ulex nN N WW 224 N. M. Steven: DescrRIPTION oF FiGuRES Fig. 44 Second spermatocyte; condition of the nucleus when the sister cells separate. Ac-c. Fig. 45 Second spermatocyte; sister cells in later stage; a optical section through center of nucleus, b surface section of nucleus, chromosomes distributed over the nuclear membrane. Ac-c. Fig. 46 Prophase of second maturation mitosis showing chromosomes already divided longitudinally Ac-c. Fig. 47 Metaphase of second division,—chromosomes always tangled. Ac-c. Figs. 48 and 49. Anaphases, Fig. 49 much flattened. Ac-c. Fig. 50 Telophase. Fig. 51 The smaller pair of chromosomes in prophase, a — d from sections of first spermatocytes, e from a spermatogonium. Fig. 52 The smaller pair of chromosomes in metaphase and metakinesis of the first maturation mito- sis, from sections. Chromosomes in Germ-Cells of Culex 225 ) AN UNEQUAL PAIR OF HETEROCHROMOSOMES IN FORFICULA N. M. STEVENS Bryn Mawr College With Forty-E1cur Ficures In 1906, there appeared in the Zool. Anzeiger, vol. 30, no. 7, a preliminary paper entitled, “Die Spermatogenese von For- ficula auricularia” by Herbert Zweiger, and the same year this author published in the Jena Zeitschrift, vol. 42, a more elabroate paper under the same title. Zweiger found a variable number of chromosomes, 24 or 26 in the spermatogonia and 12 to 14 in the spermatocytes. He de- scribed a chromatin nucleolus in the growth stages of the sperma- tocytes; and in some of the first spermatocyte anaphases, a lag- ging pair of chromosomes which he calls “ das accessorische Chromo- som.” He also states that in some cysts two such are found, making 14 in all. The numerical conditions were constant for each cyst, but not for all cysts of the same testis. In the second spermatocytes he found 12, 13 or 14 chromosomes. Carnoy (85) described the same species as having 10 to 14 chromosomes in the spermatocytes. La Valette St. George (87) found 12 in first and 12 to 14 in second spermatocytes. Sinéty (’o1) gives the numbers as 24 in spermatogonia and 12 in spermatocytes. [hese authors did not deal with the question whether or not heterochromosomes are present in Forficula. Last summer while I was collecting material at the Marine Station in Helgoland during the third Sage. in July, I chanced to find an abundance of Forficula and identified the insects with the aid of the laboratory collection, as Forficula auricularia. Having previously gone over the question as to heterochromosomes in 228 N. M Stevens. Anisolabis maritima, a related form, with Miss Randolph (08) I was interested to see some preparations of Forficula, so I put up a number of testes in Gilson’s mercuro-nitric fluid. In September and early October | found what appeared to be the same species in E isenach, Germany, and preserved more material. In November some of each set of material was embed- ded, sectioned and stained with thionin, at the Zoologisches In- stitut, Wurzburg. On examining the preparations with the micro- scope, | was considerably surprised to find a perfectly clear case of an unequal pair of heterochromosomes in the first spermato- cytes, and this is my excuse for adding another paper to the litera- ture on the spermatogenesis of Forficula auricularia. In the resting spermatogonia the chromosomes remain con- densed, as figured by Zweiger, and are for the most part in con- tact with the nuclear membrane. Fig. 1 was drawn by focusing from the surface of the nuclear membrane down to about the cen- ter of the nucleus. In every case where favorable equatorial plates were found the number of chromosomes was 24. Figs. 2 and 3 were taken from different sections of the same testis, and Fig. 4 from another individual. Occasionally one finds a sug- gestion of a synizesis stage, but this stage 1s certainly very incon- spicuous and probably very brief. Fig. 5 shows one such nucleus with one isolated chromosome which corresponds fairly well in size to the larger heterochromosome. ‘There is no evidence as to when synapsis occurs. At the beginning of the growth stage of the spermatocytes one finds the chromosomes passing from the concentrated condition of the spermatogonia (Fig. 1) through a transition stage (Figs. 6 and 7) into a spireme stage (Fig. 8), in which the chromatin thread is slender and pale in both thionin and iron-hzmatoxylin prepara- tions (Fig. 8). “The heterochromosome pair (x) 1s clearly distin- guishable in these stages, and one, two or more plasmosomes are present (p). The spireme soon shortens and thickens (Figs. g-11) and fre- quently the heterochromosome pair may be seen to be composed of a larger and a smaller chromosome (Figs. 7 and 11). Fig. 12 shows other forms which the heterochromosome pair may assume —e— Pair of Heterochromosomes in Forficula 229 during these stages. Sometimes it contains a vacuole as in Fig. 10, especially in late growth stages. In the preparations stained with thionin the heterochromosomes can be distinguished from the plasmosomes with comparative ease, on account of their dif- ferent staining qualities. The spireme segments and splits longitudinally at about the same time (Fig. 13). Sometimes the daughter segments spread apart and twist as in Fig. 14, but they soon fuse again (Fig. 15) and most of the segments assume the form of loops, U’s and twists (Figs. 15, 16, 17). Fig. 18 shows a variety of prophase forms. ‘The twists and figure 8’s are formed from such loops as are seen in Fig. 15, and further condensation gives the U and ring forms. The U- form is the most common (Fig 19), but rings and twists, and figure 8’s with the ends twisted together are frequently seen. Figs. 20, 21, and 22’show transition stages from the rings and U’s to the dumb-bell form in which the chromosomes come into the spindle. Occasionally one sees an incipient cross (Fig 21, a). Fig 22 shows a variety of intermediate and transition stages. Fig 23 1s a tan- gential section of a nucleus in a late prophase, showing the hetero- chromosome pair (x) and three ordinary bivalents. Figs. 24 and 25 also show late prophase stages. In Fig.25 the tetrad charac- ter of the bivalents is evident. In a spindle-prophase and meta- phase, the tetrads are sometimes as clear as in Fig 26, both in thio- nin preparations and also in preparations where the differentiation of iron-hematoxylin has been carried to just the nght point. The first spermatocyte metaphase usually looks like Fig. 27 from the Eisenach material, or Fig. 28 from the Helgoland collection, the heterochromosome pair being at one side of the plate and often remaining out of the plate longer than the other pairs (Fig. 28). Figs. 29 and 30 are earlier and later metaphase plates with the heterochromosome (x) distinguishable at one side of each group. In thionin preparations and in the paler iron-hematoxylin slides one finds some spindles in which the tetrad nature of some of the bivalents 1s shown both by longitudinal furrows and by the attach- ment of the spindle fibers to the split ends of the dumb-bells (Fig. 31). A similar stage seen from the pole of the spindle is shown in Fig. 32, some of the chromosomes showing the split. Fig. 33 gives 230 N. M. Stevens several views of the unequal pair in different stages of longitudinal splitting. [t will be seen from the figures and description given, that I find no evidence that the two halves of the bivalent chromosomes remain folded together parallel with each other, as described by Zweiger; but the rings and U’s and V’s all gradually straighten out so that the univalent elements of the dumb-bell-shaped chromo- somes stand end to end in the spindle, and the bivalents often appear as typical tetrads. Figs. 34 and 35 are early anaphases showing separation of the univalent components of both the unequal heterochromosome pair and the equal pairs. In my material the anaphase stages (Figs. 34, 35, 36) usually show nothing like Zweiger’s “‘accessorisches Chro- mosom”’ (Zweiger, ’06, Zool. Anz., Fig. 13; Jena Zeit., Fig. 25), but occasionally one finds it (Figs. 37 and 38). Fig. 37 is from the Eisenach material and Fig. 38 from a Helgoland preparation. The former preparation consisted of a single pair of testes in which it was possible to count the chromosomes in a number of spermato- gonial plates: all had 24 (Figs. 2 and 3). There were also a large number of first spermatocyte plates, containing 12 chromosomes without exception. ‘There was only one small cyst of second sper- matocytes in metaphase; 12 chromosomes were counted in 24 cases, 13 in 2 and if inone. It is therefore evident that in this individ- ual the lagging pair could not be an additional pair of accessory chromosomes making 26 for the spermatogonial number (Zweiger’s explanation). Many pairs of daughter plates of the first sper- matocytes were counted and 12 daughter chromosomes found in every case (Figs. 39 and 40). In these two pairs of daughter plates the heterochromosomes x,, and x, were distinguishable. In the second spermatocytes the usual number of chromosomes is 12 (Fig. 41 6), but in all testes in which this stage was at all abun- dant, there were occasional 11’s (Fig. 41, a) and 13’s (Fig. 41, cg) in the same cysts with the 12’s. Multipolar first spermatocyte spindles are sometimes seen, but they are not frequent enough to account for the irregular numbers in the second spermatocytes. Then, too, the numbers are always 11, 12 and 13 and there is no reason why multipolar mitoses should not give numbers both Pair of Heterochromosomes in Forficula 231 larger and smaller. Therefore, although I have succeeded in find- ing very few cases of a lagging chromosome like that described by Zweiger and shown in Figs. 37 and 38, I am inclined to connect the irregular numbers in second spermatocytes with this chromosome, which, judging from its size, and from the behavior of the super- numerary heterochromosomes 1n Diabrotica soror and Diabrotica 12-punctata (Stevens ’o8), | think must be a precocious division of the smaller heterochromosome, x,. In Fig. 41, e, two of the 13 chromosomes are unusually small, lie side by side, and were paler blue than the other chromosomes, a difference which 1s often notice- able between the larger (x,) and smaller (x«,) heterochromosomes in metaphase of the first maturation mitosis in preparations stained with thionin. In this case the number, 13, may be due to precocious division of x, in the daughter cell to which it passed in the first mitosis. In such cases as are figured in Figs. 37 and 38, if one daughter element of the lagging chromosome goes to each second spermatocyte the numbers should be 12 and 13 with one smaller chromosome in each cell. In Figs. 41, d, 7, and g, one of the 13 chromosomes is unusually small (s).. The number 11 1s more difficult to account for, unless the two heterochromosomes some- times go to the same daughter cell; of this | have seen no evidence. In two cases I found that one chromosome which was out of the equatorial plane had been removed in another section, leaving I1, and in a few spindles seen from the side one chromosome has been considerably out of the equatorial plane. ‘This may account for all of the 11’s, All of the figures of 13’s (Figs. 41, e—g) were cases where metakinesis had not begun, and it was perfectly certain that 13 distinct chromosomes were present. “The chromosomes usually divide regularly as in Fig. 42 and give daughter plates con- taining 12 chromosomes as in Fig. 43, but occasionally one sees a lageing chromosome (Fig. 44) which in some cases is pulled out somewhat irregularly between the two groups of fused chromo- somes as though dividing (Fig. 45), but 1s more commonly plainly included in one of the spermatids without being divided (Fig. 46). In tact, I have found no case where this chromosome was clearly divided as is the case with the lagging chromosome of the first division (Fig. 37,x,). “This lagging chromosome of the second 232 N. M. Stevens maturation mitosis 1s always paler than the polar mass of fused chromosomes, and is apparen tly about equal in bulk to one of the two lagging elements seen in Fig. 37. I should therefore think that the most Probables interpretation of the irregular number of chro- mosomes in the second spermatocytes (13) and of the two lagging chromosomes is that the first (Figs. 37 and 38) is a precocious divis- ion of the smaller heterochromosome (x,), and that the second (Figs. 44-46) is one of the products of such a division, already uni- valent and therefore not to be expected to divide again. In my material the irregularities in division and in numbers are compara- tively infrequent. As the number 24 has always been found in the spermatogonia and 12 in the first spermatocytes, it would seem that only the spermatids which are the result of two regular divi- sions and therefore contain 12 chromosomes can become func- tional. The occasional cases of a lagging chromosome in the first spermatocyte mitosis suggest that the smaller heterochromosome in Forficula is in somewhat the same uncertain condition as to its behavior in maturation as is the case with the supernumerary heterochromosomes in the Diabroticas, which divide sometimes late in the first spermatocyte division and sometimes in the second division, thus giving rise to irregular numbers. The heterochromosomes of two sizes can be distinguished in the spermatids as shown in Figs. 47, a and b and Figs. 48, a and 6, each pair from one section of the same cyst. DISCUSSION In respect to the unequal heterochromosome pair, which judg- ing from analogy with other insects, is probably to be associated with the determination of sex, my results differ from those of all others who have worked on the spermatogenesis of Forficula auric- ularia. Either the inequality in this pair of chromosomes has been overlooked, or the species is variable as to the character of its heterochromosomes in different localities. I thought at first that the difference in size of the heterochromosomes (x,, and x,) in the Helgoland material was somewhat more conspicuous than in that celles ted.in Eisenach, but on further examination the dif- ae a. ee Pair of Heterochromosomes in Forficula 233 ference between the two lots of material proved to be slight, if indeed there be any difference. The small heterochromosome is often flattened so that it looks smaller in side than in face view (Figs. 28 (H), 35 (H), 24 (E), 27 (E), 3 (E), 33 (E))._ Ineither material the inequality in size of the components of this pair is conspicuous enough so that it is dificult to understand how any- one who was looking for heterochromosomes could overlook it. As to the method of formation of tetrads [ should disagree with Zweiger and agree with Sinéty in finding them formed in a manner typical for many insects, by transverse division and longitudinal splitting of the telosynaptic pairs of bivalents. The indications are either that Forficula auricularia must be variable as to number of chromosomes in different localities, or that it is a composite species made up of several small species dif- fering in number and behavior of their chromosomes. According to the latter supposition which seems to me the more probable, Sinéty’s material with 24 and 12 chromosomes in spermatogonia and spermatocytes respectively, 1s a different small species from mine with usually 24 and 12 but sometimes I1 or 13 1n the second spermatocytes, and both are different from Zweiger’s which has 24, 26 or 28 in spermatogonia and 12, 13 or 14 1n the spermatocytes. The peculiarity about Zweiger’s numbers that I am unable to understand, is his finding the number of chromosomes different in first spermatocyte cysts of the same testis. I have always found the number in the first spermatocytes of insects constant for the individual. In cases like Diabrotica soror and D. 12- punctata, I get variable numbers in the spermatogonia and first spermatocytes of different individuals, but for each individual the spermatogonial and first spermatocyte numbers are constant while the second spermatocyte number is variable, as [ find it in Forficula. SUMMARY 1 In my material of Forficula auricularia, collected in Helgo- land and Etsenach, Germany, I find 24 chromosomes in the sper- matogonia, I2 in first spermatocytes, and usually 12 in second 234 N. M. Stevens spermatocytes and spermatids, though 11’s and 13’s are occasion- ally found in each individual. 2 An unequal pair of heterochromosomes 1s present in the first spermatocytes. “he components of the unequal pair separate producing dimorphic second spermatocytes, spermatids and sper- matozoa. 3 “Das accessorische Chromosom”’ of Zweiger appears to me to be a precocious division of the smaller heterochromosome, giv- ing rise to irregular numbers of chromosomes in the second sper- matocy tes. Bryn Mawr College January 10, 1910. — or i - — Pair of Heterochromosomes in Forficula 235 BIBLIOGRAPHY Carnoy, J. B. °85—La Cytodiérése chez les Arthropodes. La Cellule, T. 1. Ranpotpu, H. ’o8—On the Spermatogenesis of the Earwig, Anisolabis maritima. Biol. Bull., vol. 15. StnEty, R. de ’o1—Recherches sur la Biologie et |’Anatomie des Phasmes. La Cellule, vol. 19. Stevens, N. M. ’o8—The Chromosomes in Diabrotica viltata, Diabrotica soror and Diabrotica 12-punctata: A Contribution to the Literature on Heterochromosomes and Sex Determination. Journ.Exp. Zodl., vol. 5. ZweIGER, H. ’o6—Die Spermatogenese von Forficula auricularia. Zool. Anz., vol. 30. Also Jena Zeitschrift, vol. 42. v. La VaLetre Sr. Georce °87—Zellteilung und Samenbildung bei Forficula auricularia. Festschrift fur Kolliker. 236 N. M. Stevens DescRIPTION oF FiGuRES The figures were all drawn with camera lucida, Zeiss 1.5, 0c. 12. Lettering on figures p = plasmosome. xe = smaller heterochromosome. x = heterochromosome pair. S = division product of x2. x, = larger heterochromosome. Fig. 1 Nucleus of resting spermatogonium. (H,) Figs. 2 and 3 Spermatogonial equatorial plates, 24 chromosomes. (E1*) Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. 4 5 6 7 8 9 10 15 16 Spermatogonial equatorial plate, 24 chromosomes. (Hi*) Synizesis stage. (H,) Transition stage from synizesis stage to spireme stage. (H1) Later transition stage (E2) Early growth stage, showing heterochromosome (x) and two plasmosomes. (H1) Later growth stage. (H:) Similar growth stage, showing vacuolated heterochromosome (x). (Ez) Growth stage showing the bivalent heterochromosome (x). (He) Various forms of the heterochromosome from same cysts as Fig. 7 and Fig. 11. (He and Ee) Early prophase showing split segments. (Ez) Similar stage showing the daughter segments separated and twisted. (E1) Later stage showing loop-form of segments. (E1) Section of a nucleus containing three different prophase stages, split segments, V-shaped chromosomes, and twists. (E2) Fig. Fig. Fig. Fig. (H;) 17 18 1g 20 Slightly later stage, showing U’s and twists. (Ez) Various prophased forms, stage of Fig. 17. (Ez) Later stage, showing U-shaped chromosomes and the heterochromosome x. (H,) Later stage, showing U’s and dumb-bells, x in outline above the U-shaped chromosome. * H,; and Hz = Helgoland material; E; and Ez = Eisenach material. & I: 8 ; 2 5 z 3 2 3 4 SSN ale & 1:58 12 2 I I I 5 2.64 3 | 1.86 % 3205 13 4 4 ° ° 1 1.77) e 3:08 \yorpaia 6 2 co) ) I 1.68) P rs) 3:10 16 3 zi oe he NS aa 1.54) or 3:15 rare 2 I fo) ie I jy he4a | | | —2.45 | THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 8, NO. 3 260 A.M. Banta TABLE II[—Continued. Lamp changed to section-1 end of tank TIME OF cr SECTIONS OF TANK =a Fane oe “Sa Sal ; a son Cia. | AVERAGE RECORDS NS Vag eZ 3 4 5 6 soaatee 3rd 14 3 I 2 I I 1.91 § 3:184 | i S ne cs : ; ; ak 2.34 z 3:20 9 6 2 fo) 2 3 2g g ger ‘ga 5 ° ° 5 2.82 3 3:22 3 II I 2 I 4 2.95 3 8:23 5 8 2 2 I 4 2.91/ 3.16 Z Bens 3 9 3 ° I 6 a023 3:25 2 8 ° ° 6 7 3-91 & 3:26 2 6 5 Oo 3 6 3-54 & 3:27 Ont eG 5 4 I is 3-71 c 3:28 ° | 9 | 4 4 I | 4 3 45 3-45 $ 3:29T Bat 25s cam ees 4 3 Pes 4.24 z 3230 Se) 5 ain bes 3 3 3 3-32 E : 3- 3 3:35 I 5 4 2 2 | 8 4.05 a. *3 8 : 2 oe ; . : | 43 eat = 32404 2 4 2 3 2 9 4.18f é os, wa ees Teste ce Oe : 4:15 ° I I 2 3 15 | 5.36 : 4:19 ° 2 I I 4 | 14 | 528 eS 4:27 | ° I I ° 3 leg Race % 4:31 fo) I I ° 3 | 17 Wen yec= 5 4:45 ° I I ° 3 | 17 5-55 oO as etre : I 0 Sn nes 5-45 | | 3.254 * General movement at sections 1 and 2. 7A recoil from section-6 end, as at this time more individuals were moving from than toward the section-6 end of tank. $Some individuals still wandering about. seemed fairly well settled at 3:15 p.m., an hour and thirty-two minutes after the experiment was begun. All but one were in the negative half of the tank and 18 were in Section 1, while the mean average position was 1.41; a change in a negative direction of 2.45 since the beginning of the experiment. At this juncture the light was changed to the opposite end of the tank and a record of positions made as soon as possible after the change. The animals began to respond to the light stimulus Reactions of Isopods to Light 261 quickly and a very general and consistent migration toward the negative end continued for about seven minutes, when the photokinetic effect became apparent in the turning back of many individuals upon reaching the negative end of the tank. At 3:29 p-m., more seemed to be moving toward the positive than toward the negative end. These movements were clearly due to photokinesis, for the animals had by this time been in the tank long enough to have become thoroughly adjusted to it. At 3:40, 22 minutes after the position of the light was changed, consider- able wandering about in the tank was noticeable, but 51 minutes after changing the light, at 4:09, the animals seemed pretty well confined to the negative end, though wandering about there to some extent. After 4:27 the movements were slight. During the 14 hours following the reversal of the direction of the light, the mean average position changed from 1.91 to 5.45, representing an average movement of 3. 54 in a negative direction. The above experiment is typical for the reactions of Asellus. The phototactic influence is often slower in asserting itself than in the experiment here recorded, but the other influences appear in this experiment in a characteristic way. A number ofexperi- ments with the same intensity yielded similar results, bearing out the conclusion that Asellus is decidedly negative to such an in- tensity after previously being in diffuse daylight. The results of another experiment with Asellus following ex- posure to diffuse daylight are given in Table III, the intensity of the light in this case being only 3 C.M. (19 c.p. incandescent at 2.75 m. from middle of tank.) As these animals had been in the tank in diffuse daylight within the glass ring for 55 minutes before the experiment began, they seemed fairly well settled and were near the center of the tank. While they were not very active at the time the ring was removed, yet, with freedom to move in any direction, they responded very promptly to the directive light. The r response was so prompt and the movements so general that at first accurate counts could not be made. A part of this movement was due to photokinesis, rather than phototaxis, for the number and position of those in the positive end varied considerably. Whereas two or three min- 262 A.M. Banta TABLE III ASELLUS ComMMUNIS (36 individuals) February 23, 1906 Previous exposure: diffuse daylight Illumination: horizontal, 3 C.M.; lamp at Section-6 end In tank within glass ring 55 minutes before experiment began TIME OF = SECTIONS OF TANK -{- MEAN MAKING Le a ee ae S| AVERAGE ) RECORDS 1 2. 3 4 5 | 6 | POSITION 5 | | | rz) | | | & 10:50 ro) ro) 18 | 18h, sco" Ble Cee 3.50 a 10:51 10 12 ae | 2 |) cue al 4 2.67 Bs 10:52 12 P| Bh coal 7 | 3 DG 2 e 10:53 14 i oval use tera Ti DeA2 | Pextanne | | aos 10:54 18 8 3 2? || 4 I 2.14 Oe | | | aay C8) 10:56 14 8 7 | I 2 2.39 es | a, oO 10:58 rs, 4 5 6 a 4 3 2.58 ues bo 11:00 14 9 4 2 6 5 2.89 Shuto | o 11:05 16 6 5 2 I 6 2.56 3 cal I1:41 Il 7 dea 3 | 8 3 2.94 a | o 11:44 | Io 6 2 | 8 3 7 2216 E 12:23 | 16 6 Beal wag fe) 6 2.53 & o 22: 17 4 I “il 5 5 2/275 i 2235 II of 3 te Hl 3 II 3-31 5 3°45 15 7 3 5 2 4 P20 0)5) 5:05 20 6 2 | 3 I 4 | 2.19 —1.31 utes after the light was turned on only 8 or 10 individuals were in the positive end of the tank, at 11:44, nearly an hour after the experiment began, 18 were in the positive half and their positions were very different from what they had been three minutes be- fore (11:41), when 14 were in the positive half of the tank. This general activity was very pronounced at 11:05, 15 minutes after the experiment began. At 11:41 I made the following note in my record book “quite active and apparently a general move- ment toward + is beginning.” ‘This apparent general movement was due largely to the photokinetic activity, which at this par- ticular time happened to produce a general movement in a posi- tive direction. At 12:23 the activity was still very marked, but at 2:25 1t was much less pronounced, and at 3:45 and 5:05 there were only slight movements. Reactions of Isopods to Light 263 The experiments described above serve to illustrate the series in which Asellus after exposure to diffuse daylight was subjected to illumination with light of various intensities from 2.5 C.M. to 2855 C.M. Many of the reactions were less pronounced than those described above, and sometimes the results were not very definite. Generally such cases were readily explicable as due to the apathy of the animals after they had once become thoroughly settled in the tank; for such indefinite results usually came from testing animals which had been in the tank 24 hours or longer. From the experiments with Asellus communis when subjected to light of various intensities after previous exposure to diffuse daylight, the following conclusions are drawn: 1 Asellus does not respond to light below about 2.5 C.M. in- tensity, but responds to light from 2.5 C.M. to 2855 C.M. inten- sity. 2 This response consists of two factors, a non-directive pho- tokinetic effect and a directive negative phototactic effect. The former is often the prevailing influence at the start, but the pho- totactic influence becomes the effective one as the photokinetic effect decreases and eventually disappears. B. After being in Darkness In conducting experiments on Asellus which had been kept in darkness before the beginning of observations, the following method was pursued. The animals were first placed in the tank and left in the darkened room. In addition to darkening the room, the outer tank was covered with light-proof screens. After the animals had been thus kept in the dark for the desired length of time, the screens were carefully removed and the lamp, which had previously been placed in position for the experiment, was suddenly made light. Mention has already been made of the fact that Asellus does not respond to light intensities of 1 C.M. or less. The statement holds true whether the Asellus has been previously exposed to light or has been in the dark. Several intensities between 0.001 C.M. and 1 C.M. were tried, but definite responses were not 264 A.M. Banta obtained. ‘This lack of response to light of 1 C.M. or less (being found in animals after their retention in darkness when their response is normally positive, as well as after their exposure to light when their response is normally negative) may be taken as evidence that Asellus is not at all sensitive to light of such in- tensities. The range of intensities to which Asellus failed to respond was, as far as the evidence went, the same for animals previously in darkness and those previously in light. After retention in darkness for several hours, Asellus gives a positive response to light of intensities between 2.5 C.M. and 80 C.M. (19 ¢.p. incandescent at 0.49 m. from middle of tank). TABLE IV AseLtus communis (16 individuals) January 13, 1906 Previous exposure: darkness (in tank) for 40 hours Illumination: horizontal, 2.5 C. M.; lamp at Section-1 end TIME OF | of SECTIONS OF TANK = | MEAN | | | MAKING =—s sd AVERAGE c | io) RECORDS | 1 2 3 4 5 ns} POSITIONS = | | | = re | | 2 8:19 4 | Eee | ° 2 | 4 3.38 a 8:20 4 2 I I | 4 3.62 2 a 8.2 4 3 ° 2 Sep 3-4 £ 8:22 ol I 2 | g 2 aul 2/103 Bs 8:23 6 | 2 I 4 or || Z 27R 5 | _ 8:24 8 I 4 eo | I 2 2.44 & 8:25 7 1 3 Ce i 2 2 2.47 | 2 8:26 7 I 4 ch | I 2 253 | 5 8:27 8 I 4 oF I 2 2.44 2 8:28 8 I 4 o | I 2p | 2.44 2 8:29 8 I 4 ° te 2 | 2.44 & 8:30 8 I 4 ° | I 2 2.44 re 8:31 8 I 2 Tomes I 2 2.47 _ * | : & 8:32 7 ol I 4 Cong I 2 2.53 s 8:33 7 2 3 i, ° a 2.62 2 8:34 8 I 4 Onna fo) a || ols I 8:35 Son 3 2 fe) I 2an| 2a g 8:36 | 9 2 I I I 2 | 221 a3 8:37 | 9 2 Tae I Taal 2 2.31 2 :0 2 || I °o (| 2 2.1 7a 9:05 9 | 9 5 9:32 | 9 3 I I oF 2 Dag Reactions of Isopods to Light 265 TABLE V AsELLuS comMuNIs (18 individuals) December 12, 1906 Previous exposure: diffuse daylight Illumination: horizontal, 80 C. M.; lamp at Section-6 end Experiment began as soon as animals were in tank TIME OF ~ ge SECTIONS OF TANK a MEAN & MAKING — — _ — —$ $$$ AVERAGE — RECORDS 1 2 3 4 5 6 POSITIONS Re vo 2240 ° ° 9 9 fo) ° as oe c 2:41 Za 5 5 4 fe) fo) 255 g 2:42 II 3 2 I fe) I 1.83 é ei 2:43 8 4 3 fo) 2 I 2.28 aE, 2:44 Oa 4 2 I I 4 2.94 3 5 2:45 |} 10 heed ° fe) I 5 272 ag 2:46 DIG 2 I ° I 3 2.28 pie 2:47 6 2 4 3 ° 3 2.89 5 2:48 5 3 3 I I 5 3-28 S 2:49 BF ha? 3 + 2 4 3-67 3 50 i 2 4 4 2 3 3-5 e 2261 9 2 2 I 2 2 pls a BiaGe Tigh 5 I I 2 2 2.56 as = 2:53 12 ° ° ° 3 3 2.5 v —1.0 Satisfactory positive reactions were obtained from Asellus after it had been kept in darkness for three or four hours or longer. Often when the intensity was only 2.5 C.M. the positive response was not very evident. The most marked positive response fol- lowed exposure to intensities between about 11 C.M. (19 cp. incandescent at 1.32 m. from middleof tank) and 80 C.M. Asellus exposed to an intensity of 2855 C.M. often appeared positive at first, but remained so for only a very short time, then becoming negative. Table IV shows the results of an experiment with 16 Asellus when subjected to light of 2.5 C.M. intensity after retention in darkness for 40 hours. This Table shows that the animals began to respond to the directive light within three or four minutes and that within sixteen minutes virtually the maximum reaction had taken place. Few random movements occurred in this experi- ye hs 266 M. Banta ment, hence little photokinetic effect was to be inferred. The reaction was clearly positive and continued positive until the close of the experiment, although in another experiment the same individuals following their exposure to diffuse daylight were nega- tive to the same intensity of light. ‘Two additional experiments are here given to show.by contrast the difference in the nature of the responses when the animals had 1) 5 min. 10 min. 15 min. Fic. 4. Asellus communis (18 individuals). Graphic representation of results of experi- ments shown in Tables V and VI. — Broken lines based on Table VI. previously been exposed to light and when they had been in dark- ness. December 12, 1905s 16 Asellus after exposure to diffuse daylight were placed in the tank and at once subjected to light of 80 C.M. intensity. Table V and the continuous line in Fig. 4 show the results of this experi- ment. The experiment was contin- ued for only thirteen minutes; the response was negative and very marked. Many of the in- dividuals started up rather quickly, and all which responded at once moved away from the light. A little later two started toward the light. By that time, however, some had reached the negative end and were turning back. The record made at this time—-two minutes after the start—shows the maximum re- sponse, as judged by the change in mean average position. It will be seen that the photokinetic effect in this experiment was very marked, but that, never- theless, the positive phototaxis Reactions of Isopods to Light 267 was evident almost immediately. After the lapse of thirteen minutes, when the experiment was discontinued, the animals were closely covered and left in darkness until the following day. The same animals were then experimented with wilder exactly the same conditions as on the previous day, except that in the meantime they had been in darkness for 16 hours, whereas on the day before they had previously been exposed for a long time to diffuse day light. There was one other difference in the conditions of the two experiments, but 1t was non-essential; in the first case the animals were in the tank for only a short time before the experi- ment began, whereas in the second case they had been in the tank about 24 hours. Other experiments showed that this would, in any case, influence only the time at which the reaction occurred and that it would not affect the nature of the reaction. Table VI and the broken line of Fig. 4 show the results of this experiment. TABLE VI AsELLus communis (18 individuals) December 13, 1906 Previous exposure: darkness (in tank) for 16 hours Illumination: horizontal, 80 C. M.; lamp at Section-6 end | | TIME OF - SECTIONS OF TANK. —- MEAN = MAKING AVERAGE gS) RECORDS N = i) 1 2 | 3 4 5 6 POSITIONS “ a | TSS 6 I | | 1 2 8 3-890 c= | =} 11:16 6 I fo) 3 2 6 3.67 g Ss 11:17 4 3 I 2 4 4 3.61 2 §& 11:18 4 I oy 2 4 7 4.22 = 8 > oe c - 11:19 4 I aaa 3 ° 9 4-17 ‘3g a oO 11:20 3 I oy 2 2 9 4.44 LS 11:21 3 I oI 3 I 10 4.56 os . a | 11:22 3 I I 3 I 9 4.39 2 11:2 B I Oi ° is 4.67 s 11:24 I 2 tel 2 2 if) 4.78 | } r 11:25 ° 2 cant 3 2 fe) 4-94 a= 11:26 ° ° 3 2 ° 13 5.28 ie 11:2 o | ° I 2 I 1 - 56 S 7 4 Simo) Pe 11:28 i 7 ° rt fi I ) 15 5-44 eS t 268 A.M. Banta The response was somewhat less prompt in the second experi- ment, but it was as definitely positive as the response in the first experiment was negative. Numerous experiments were also made with other intensities (3 C.M., 11 C.M., and 25 C.M.). The results support the con- clusion that Asellus after being kept in the dark 1s positive to all intensities of light between 2.5 C.M. and 80 C.M. Some of these experiments were continued for periods varying from 3 to 5 hours and the positive response persisted throughout the whole time. The maximum response, however, usually occurred between 30 and go minutes after the experiments began. When Asellus was subjected to a light intensity of asmuchas 2855 C.M. the animals very soon sane the negative end of the tank, although for a short time he response was often positive. These experiments indicate that after retention in darkness Asellus is positive even to light of fairly high intensities, but that with intensities as high as 2855 C.M. it very soon becomes negative. From the foregoing experiments with Asellus when subjected to horizontal illumination of various intensities, after being in darkness, these conclusions have been reached: 1 Asellus does not respond to intensities of 1 C.M. or less. 2 It does respond to intensities from 2.5 C.M. to 2855 C.M. 3 This response is largely a direct phototactic effect, since the animals before exposure to specific intensities of light had been retained in darkness in the tank and of course had become thor- oughly settled there; but the photokinetic element is also recog- nizable. 4 The immediate response to intensities from 2.5 C.M. to 80 C.M. is positive and continues to be so for at least 3 to 5 hours. 5 With an intensity of 2855 C.M. the response is often posi- tive at first, but soon becomes negative. By way of a general summary of the effect of horizontal illu- mination upon feaing it may be said, thatafter exposure to diffuse daylight it is negative to all intensities greater than about 2 CME It does not respond to lower intensities. Light has both a pho- tokinetic and phototactic effect upon Asellus. The photokinetic effect is often sufficient to mask for a time the phototactic Reactions of Isopods to Light 269 influence, but this influence, even though in some cases it may not be asserted at once, ultimately appears, the photokinetic effect meanwhile becoming less pronounced. After retention in darkness for a few hours, Asellus is positive to intensities between 2.5 C.M. and 80 C.M.; but to intensities of 1 C.M. or less it does not respond. To an intensity of 2855 . M. Asellus is generally negative, though sometimes it is positive at first, but in that case it very quickly becomes negative. 3. Cecidotea The experiments upon Cacidotea were made under precisely the same conditions as in the case of Asellus. Czacidotea was found to be still less responsive to light than Asellus. Even after the conditions of experimentation and observation had been so refined that Asellus was seen to be definitely responsive to hori- zontal illumination, Cacidotea long seemed quite unresponsive, exceptina purely photokinetic way. Finally, however, numerous careful experiments showed that Cecidotea was feebly responsive ina directive way to horizontal illumination. This species, like Asellus, after being transferred to the tank, kept up random move- ments for some time, doubtless as the result of thigmotactic or some other non-directive stimulus, and these random move— ments were even more marked and longer continued—on an average, nearly twice as long—than with Asellus. The glass ring was often used to confine the Cxcidotea until they should become thoroughly settled in the middle of the tank. Because of the general reluctance of the animals to move or to respond to light vances after they had once become settled, many of the experiments were begun as soon as the animals were placed in the tank. In such cases directive results from light stimulation ordinarily appeared only after the lapse of some time; for the movements due to thigmotaxis or other non-directive stimuli predominated at first. With Cecidotea, as with Asellus, the effects of stimulation by light appeared to be both photokinetic and phototactic. In many cases when animals almost or quite settled in the tank were sub- 270 A.M. Banta TABLE VII Caciporea stycia (26 individuals) November 7, 1906 Previous exposure: diffuse daylight Illumination: horizontal, 2855 C. M.; lamp at Section -6 end Records begun 5 minutes after animals were transferred to tank TIME OF _ SECTIONS OF TANK + MEAN MAKING - = | AVERAGE RECORDS 1 2 3 4 5 6 __ POSITIONS 9:20 I ey | 6 16 3 fo) BT: 9:25 OF a 39 9 1 3 | 3-46 9:30 3 9 10 I 2 3.50 9:35 2 2 6 6 4 6 4.0 9:40 4 2 6 9 2 3 3-46 9:45 6 ats 8 3 4 3-54 9:50 5 a. | 6 4 fe) 7 3.42 9355 5 2 6 6 2 5 3-50 10:00 6 eT 5 5 I | 3-54 10:05 qi Fl Bee 3 2 6 | 3-27 10:10 7 ae 1 | 2 3 4 || 3.58 10:15 ai 5 2 & I 6. | 28 10:20 5 5 3 3 3 fo 3-58 10:25 6 3 6 3 4 4 | gag 10:30 | 9 3 3 3 2 6 | eloutls 10:35 | 9 2 6 4 ° 5 2.96 10:40 7 4 5 4 ° 6 BEI 10245 ip 6 4 3 I 5 3-0 10:50 7 5 3 4 I 6 3-19 10:55 6 5 4 4 2 5 3-23 11:00 6 7 4 3 fe) 6 2.08 11:05 5 6 7 ° 2 6 aE II:10 5 6 4 2 I Seo 3-46 11:15 a] 4 I 5 6 | 3-42 11:20 9 5 2 ° 9 Bion 11:25 6 3 5 fo) 4 8 3-65 11:30 5 ° 6 2 4 9 4.04 11:35 5 2 4 ° 8 7 4.96 11:50 8 6 I 2 5} 6 Ba1'5 11:55 7 9 2 I 2 4 2.85 12:00 7 4 5 2 I 7 Bua 12:50 9 3 | 9 I 1 B 2265 12:55 8 6 f I fo) 4 2.65 1:00 | 8 6 7 I ° 4 2.65 1:05 | ar 6 5 2 ° 2 2.23 1:10 12 3 3 5 fe) 3 2.50 Change in mean average position between the first and the last observation f Reactions of Isopods to Light 271 TABLE VII Continued. 1 TIME OF — SECTION QF TANK + MEAN ey: MAKING - - — ~ AVERAGE a so l os RECORDS 1 2 3 4 5 6 POSITIONS 3 Ie — = a 2 a 10 2:30 J og ° 9 ° I 3 2.42 0 a, | i} 2335 12 I 9 ° I 3 2.46 o = : 6 2.62 oh SS, 3330 II 3 I I 4 .62 .& 3335 II 4 6 ° ° 5 2.58 5 2 3340 II 3 7 fo) I 4 2.58 Bie a 4:20 14 2 5 fe) ° 4 Oe) 4 “2 | 218 a fo) 4:2 14 3 4 ° fe) 5 2a ao 5:35 17 4.7) ¥ ° ° 2 1.77 oO E jected to stimulation by directive light the photokinetic effects were very marked. The apparent phototactic effects occurred only when these photokinetic effects had become less evident. Further, the photokinetic effect with Czcidotea is stronger in comparison with the apparent phototactic effect than it is in the case of Asellus, where the phototactic effect is fairly well marked. Czcidotea was subjected to various intensities of horizontal illumination from 5 C.M. (8 c.p. incandescent at 1.3 m. from middle of tank) to 2855 C.M. (772 c.p. 6-glower Nernst lamp at 0.52 m. from middle of tank). No clearly directive responses were obtained to intensities lower than 80 C.M. (19 ¢.p. at 0.49 m. from middle of tank). Czecidotea usually shows a negative response to intensities of 80 C.M. or greater. “Table VII shows the results of an experiment with twenty-six Czcidotea, which had previously been exposed to daylight, when they were subjected to horizontal illumination of an intensity of 2855 C.M. The records were begun five minutes after the animals were transferred to the tank. This 1s a fairly typical experiment. The animals having been in the tank only five minutes when the records were begun, the thigmotactic and other influences due to transference to the tank were shown to good advan tage. More than an hour elapsed after the beginning of the experiment before any marked indication of a directive “esas to light appeared, and that response was prob- 2.72 A. M. Banta ably a chance result since from about 10:30 a.m. (when the first nega tive response seemed indicated) to 12:00 the mean average position shifted back and forth, at one time apparen tly indicating a negative response and at another a positive one. | From 12:50 to the close of the experiment at 5:35 the apparent negative photo- taxis predominated and the other influences became less and less effective. The mean average position during the course of the experiment shifted from 3.77 to 1.77, a movement of 2 In a nega- tive direction. Fig. 5 represents the results of the experiment recorded in Table VII. The loci of the curve show the mean average posi- tions of the animals during the course of the experiment at inter- vals which were at first fifteen minutes apart, but later not so close together. Table VIII shows the results of an experiment similar to the one last discussed. But in this case the animals had been in the tank 9:20 10:20 11:20 12:26 1:20 2:20 3:20 4:20 5:20 Fic. 5. Cecidotea stygia (26 individuals); Nov. 7, 1906; previous exposure, diffuse daylight; illumination horizontal, 2855 C. M. Reactions of Isopods: to Light 539 TABLE VIII ate Ca#ciporea styoia (21 individuals) November 14, 1906 Previous exposure: diffuse daylight Illumination: horizontal, 2855 C. M.; lamp at Section-1 end Animals in tank 14 hours before experiment began | TIME OF + SECTIONS OF TANK. — | MEAN MAKING | AVERAGE RECORDS 1 2 3 4 5 6 POSITIONS | | 755) 5 z zs 7 3 3 Sob 8:00 4 ° I 8 4 4 3-95 8:05 4 ° I 7 5 Aan | 4.0 8:10 2 I I 7 4 Sen 4.09 8:15 3 I 3 6 2 6 4.0 | & 8:20 3 2 2 4 5 5 4.0 S 8:25 2 2 6 6 zi 2 25571 g 8:30 2 u 5 at 4 5 47-05 pe 8:35 3 ie | 2 | 8 6 I 3-76 ey 8:40 I 2 | At 6 4 4 4.05 a 8:45 I 2 4 7 2 F 4.05 e 8:50 Z 3 3 3 4 6 05 re 8:55 2 2 3 i 2 5 4.05 os 9:03 fc) 2 5 @ 2 9 4-52 es: 9:07 fe) I Sa 3 2 10 4-71 g gilt 2 3 2 | 4 | 2 8 4.19 z g:15 ° Gi oll. age? Cayl| «22 7 4.38 ie 9:20 I 3 PA ose? 2 8 | 4.24 Ps 9:25 I I ae Roa 2 9 4.62 3 9330 I Ea ie 63 5 3 8 4-52 & 9:35 I I | I 6 2; fe) 4.76 5 9:40 I Bo | I 6 2 9 4-57 = 9:45 | ° 2 I 6 | 2 fe) 4.81 5 Boome): 1 3 s lame | 9 ro a ee 9:55 ey | I 2 | 27 gee 4.81 ¥, 10:00 ° BV ee an 5 3 11 5.05 5 10:05 ° rey | 2 4 I 13 5.09 Y 10:10* Ts | Tet 2 5 I 10 4-7 | 10:15 2 2 | 2 3 ° II 4.5 | 10:20 I eae 3 a I ia | 5.05 10:25 I 3 2 2 “0 | 12 4.65 10:30 Ds || I 2 I 2 12 4.8 10:35 I 3 ° 2 Dey” G3 4.85 10:40 fo) I fo) 4 fo) 15 5-4 10:45 Cy] I I 2 I | I5 5-4 | | | —1.88 *One defective individual removed. 274 A.M. Banta / TABLE VIII Continued Direction of light reversed TIME OF \y ae SECTIONS OF TANK. e MEAN MAKING : ye fie : AVERAGE RECORDS 1 2 3 4 5 6 | POSITIONS 10:50 1 2 I ) I 13 | 495 10255 2 2 1 2 Faeoi|| Bod) 4.55 s 11:00 2 2 I 2 Zale 4.65 as} q 11:05 2 2 ro) 3 yi | 9 | 4.6 % 11:10 3 I I 2; I II 4.58 & I1:15 4 I 3 2 I 9 4.1 | oD é | 3 11:30 3 3 3 2 | ° 6 3.65 | 11:40 5 4 3 1) | ° 7 Aho | 3 11245 5 4 2 1 ° 8 ass 6 12:50 6 3 4 I 2 4 ayn ae g Soa 1:05 5 4 3 2 | 2 4 a) | Sees TIO 5 4 3 Zee I 5 3-25 8 3 | Oo, 2 1:15 5 4 4 aa I 5 an eae 1:20 5 3 4 ay Ws a 5 3-3 | = | o 1:25 5 3 5 Te) | I 5 3\.2:5 2 1:30 5) 3 5 | I 5 3-25 I 1:40 5 3 4 2 ° 6 | 3-35 8 2235 5 5 4 I ° 5 3-95 aS 3:30 4 5 4 2 I 4 3-15 Fe ae a i) 3245 3 6 5 I 2 3 3.1 q 4:30 4 6 4 2 I 3 2.95 o Reis 5 4 6 I 2 2 2.85 | —2.10 14 hours before the experiment with light began, whereas in the former case they had been in the tank only five minutes. More- over, in the case recorded in Table VIII the light was transferred from one end of the tank to the other during the course of the experiment in order further to test the efficiency of the light as a directive influence. Since the animals, before this experiment began, had beenin the tank over night, the thigmotactic or other disturbing influences due to their transference to the tank was eliminated. The pho- tokinetic effect was therefore easily recognizable. It appeared within a few minutes after the exposure to light began and con- Reactions of Tsopods to Light 275 tinued to be rather conspicuous for about 24 hours. Therecords set down in Table VIII] were not made at sufficiently frequent intervals to show well the random movements due to this influence. As these movements became less general and less vigorous, the animals were found to be more and more in the negative parts of the tank. By 10:45, 2 hours and 50 minutes after the experiment started, most of the animals were fairly settled and 18 out of 20 were in the negative half of the tank. This represented a change in mean average position of 1.88 in a negative direction. Possibly further change in this direction would have occurred if the experiment had been continued without modification, but at this time the light was changed to the other end of the tank. Having already been exposed to a high intensity for about 3 hours, the response to this change was not very prompt, and the animals did not shift as near to the new negative end as might have been expected. How- ever, the change in mean average position was very decided in the 63 hours during which the experiment was continued, being 2.10 in a negative direction. From these experiments, which are typical of the series, it is evident that Cacidotea responds negatively to intensities from 80 to 2855 C.M. In general the maximum negative response was reached in approximately 2 to 3 hours, but there was consider- able variation in the length of this period depending somewhat upon the intensity of illumination and also upon the length of time the animals had been in the tank before the experiment began. After once responding to horizontal illumination, and becoming fairly well settled in the negative end of the tank, the animals did not move toward the positive end again to any marked extent, 1.e., they did not become less negative. Experiments were made with intensities from less than 1 C.M., (I c.p. incandescent at 1.3 m. from middle of tank) to 80 C.M. It was shown conclusively that Cacidotea is never positive in its response to horizontal illumination; but whether or not it is more responsive following retention in darkness or whether it then THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 8, NO. 3 276 A.M. Banta responds to lower intensities than when previously in light, was not directly determined. There is, however, convincing evidence that after exposure to strong light for some time Cecidotea is less responsive to light. ‘This was shown in the experiment de- tailed in Vable VIII, in which at the beginning of the experiment a definite negative response occurred in 2 hours and 50 minutes, three-fourths of the animals under observation having in that time collected and become settled in the extreme negative end of the tank. But when the light was then changed to the other end of the tank, there resulted a less definite response even after a much longer time (6? hours) of exposure. “The mean average position was changed a great deal, to be sure, but part of that change would have occurred as the result of the normal non-directive movements of the animals. No very pronounced tendency to collect in the extreme negative end appeared in the course of the entire 6? hours. Hence it seems evident that these animals after exposure to 2855 C.M. for 2 hours and 50 minutes were less respon- sive to the influence of directive light. It seems very probable that with Cecidotea there are not two factors (i.e., phototactic and photokinetic) in the response to horizontal illumination, as there clearly are with Asellus, for with Czecidotea the whole reaction seemed clearly attributable to photokinesis. Conviction that such was the case led to a par- tial test of the matter, as the result of which it may be said that no direct phototactic reactions were observed with Cecidotea. The responses were clearly and purely photokinetic, the animals starting up in any direction in which they happened to be turned. Def- nite orientation or direct movements in a negative direction did not occur at all. It was different with ANclinee in which definite orientation and direct responses on the part of individuals very generally occurred. In order to determine if the difference in luminosity between the two ends of the tank was sufficient to account for the collecting of Czcidotea in the end farthest from the light under the influ- ence of photokinesis alone, tests were made to determine the actual luminosity in various parts of the tank. The following method was employed. A six-glower Nernst lamp of 772 c.p. Reactions of Isopods to Light 277 intensity.was used. It was placed at the distance of one meter from the nearer end of the outer tank. ‘The photometer was placed close to the opposite end of the outer tank, so that the light from the lamp had to traverse the water in both tanks. The luminosity at the photometer (1.72 meters from the lamp) was measured by using as a standard of comparison a single-glower Nernst lamp whose candle power had been previously determined. In like manner was determined theluminosity at the photometer at the same distance after removal of the tanks. On the basis of these determinations the water used in the tanks in these experi- ments was found to cut down the light passing through it at the average rate of 4 per cent for eachcentimeter. By calculation, the luminosity - each part of the tank was then determined. The luminosity at the middle of imaginary section number 1, the section nearest the lamp, was found to be 620 C.M., that at Sec- tion 6, 251 C.M., ora little more than two-fifths that at Section I. When, however, the lamp was placed at a distance of only 20 cm. from the outer tank, the distance at which it often was used in the experiments, the difference in luminosity at the two ends of the tank was still more. A computation based on the above determination showed that when the six-glower Nernst lamp was used at 20 cm. from the near end of the outer tank the luminosityat the middle of Section I was 7863 C.M., whereas at section 6 it was only 1065 C.M. or less than one-seventh as great as at Section I. ‘These Aveaitianews while they can be only approximate, show that the difference in luminosity between the two ends of the tank (and the difference would of course be greater the nearer the lamp was to the tank) was very considerable. In order to obtain high intensities of illumination the lamp was, in most of the experiments, used very near to the tank; hence the difference in luminosity between Sections 1 and 6 was usually very great. Since the difference in illumination between the two ends of the tank was so great, there is sufficient basis in that fact for explain- ing the collection of Cecidotea in the negative end of the tank as the result of photokinesis alone. Photokinetic movements were very marked with Cecidotea 278 A.M. Banta when subjected to an 80 C.M. or a greater intensity, and occur- red almost without exception in all such experiments. The re- sponse was noticeably more prompt and more vigorous in animals at the end nearer the lamp than in those farther from the source of light. ‘The activity, as stated before, took the form of random movements. ‘There was no evidence of a selection by the individ- ual in the direction of these movements with resulting orienta- tion such as occurs in the random movements discussed by Holmes ‘Kols: The ultimate positions in which the animals settled were the result of a certain degree of attunement to a definite intensity of light, which rendered them less subject to the photokinetic influence, so that when their random movements carried them into the negative end they were less and less affected by the inten- ‘sity of lien prevailing there and finally became so attuned that the intensity at the negative end of the tank did not affect them in a photokinetic way. Hence the settling at the negative end was the result of photokinesis. Ceecidotea, although for some time very active, sooner or later became acclimated and came to rest under any intensity of illumination. In the experiment recorded in Table VIII the animals after appearing to respond in a negative phototactic way, became in the course of 2 hours and 50 minutes so acclimated to the existing conditions of illumination that when the light was transferred to the opposite end of the tank, they responded much less definitely than at the beginning of the experi- ment. Before the experiment began they had been subjected to diffuse daylight; when settled in the negative end of the tank they were acclimated to two-fifths the intensity to which they were subjected after the light was transferred to the opposite end of the tank. These and other similar experiments afford good evidence of acclimatization to light and seem to support the statement that the responses of Czcidotea to horizontal illumina- tion are due to photokinesis and not to phototaxis. From the foregoing experiments with Czcidotea when sub- jected to horizontal illumination the following conclusions are drawn: Reactions of Isopods to Light 279 1 Czcidotea is not very responsive to horizontal illumination. 2 The thigmotactic and other influences due to the transfer- ence of the animals to the tank are more potent with Cecidotea than with Asellus, and the effect continues longer. 3 Cecidotea does not respond to intensities of illumination less than about 80 C.M. 4 Cecidotea responds negatively to intensities from 80 C.M. to 2855 C.M. 5 This response is not direct, i. €., itis nota direct phototactic response. 6 Light produces with Caecidotea a very decided photokinetic effect, which continues for some time. As this effect decreases, the animals gradually settle at or near the negative end of the fans, 1. ¢., they eradually become acclimated to the lower inten- sity of alain and when acclimated to the conditions of illumination at the negative end of the tank they accumulate there. 7 With horizontal illumination both Asellus and Czcidotea respond in a negativ e€ way to moderate intensities as well as to all stronger intensities; but below certain ranges of intensity both are indifferent. Czecidotea is indifferent to light intensities below 80 C.M., Asellus to intensities below 2.5 C.M. Hence Czcidotea is indifferent to a considerable range of intensities (2.5 C.M. to near 80 C.M.) to which Asellus is more or less responsive. 8 However, after having been in the dark for several hours, Asellus is positive to intensities of light between 2.5 C.M. and about 80 C. M.; and sometimes it is positive for a short time to an intensity of 2855 C.M., but in that case it very quickly becomes negative. hes foes or. exposure to any intensity to which it responds at all, Asellus is positive. 9 Although Asellus is not very responsive to horizontal illumination, it is decidedly more responsive than Czcidotea to those intensities to which it responds at all. This difference appears in the directness and the relative promptness of the re- sponse given by Asellus as compared with the irregular and tardy response of Czcidotea. 10 This difference in the reactions of Asellus and Czcidotea 280 A. M. Banta tolightis sufficient to account both for the occurrence of Cacidotea in caves and subterranean waters in general and for the virtual non-occurrence of Asellus in such situations; for the negative res- ponse of Czecidotea to light would aid in directing it into caves and keeping it there; but Asellus after being in darkness becomes positive, and therefore would move toward the light, 1.e., out of a cave, in case it had by chance made its way into one. II. VERTICAL ILLUMINATION Tt. Methods and Apparatus The apparatus used in the experiments with vertical illumina- tion 1s shown in vertical section in Fig. 6 (p. 281), a dark and a light field were secured by using the same two tanks as in horizon- tal illumination and causing one-half of the inner tank to be illum- inated by vertical rays while the other half was kept as dark as possible. A rectangular lamp-container (LC), similar to the one used in the experiments with horizontal illumination, was sus- pended in a vertical position by means of two stout cords passed through pulleys so that it could be raised or lowered as desired. A broad V-shaped piece of blackened sheet iron (C) was placed as a roof over the top of the lamp container in such a way as to pre- vent the light from escaping above into the room and at the same time to Agar a ready means for the escape of the heated air from around the lamp. Within the lamp-container at one side was fitted an adjustable partition (P) made of two thicknesses of paste- board. This partition was lengthened at the lower end by means of a heavy card-board fastened between the two thicknesses of pasteboard in such a way that it could be moved freely in the plane of the partition. The lower edge of this partition was made to bisect the inner tank, fitting into it closely and extending down to the surface of the water when the tank was filled to a depth of 3cm. The partition was practically light proof; but in order the more effectually to shut out all light from the dark end of the tank, that end was carefully covered over with a black cloth supported by a large piece of blackened pasteboard (S'). Black cloth was Reactions of Isopods to Light 281 tightly fastened around the lower end of the lamp-containez and closely drawn around the illuminated end of the tank as well as the edges of the partition, so as to form a light-tight hood between Fig. 6 Diagram showing in vertical section the apparatus used in experiments with vertical illumina- tion. C, sheet-iron roof of lamp container; CS, cloth screen; IT,inner tank; LC, lamp container; OT, outer tank; P, partition; S, horizontal screen; SL, vertical slate; T, table; X, place of observa- tion. 282 A.M. Banta the lamp-container and the light end of the tank. ‘The only light which could reach the dark region came through the water from the illuminated half of the tank. A 6-glower Nernst lamp sus- pended within the lamp-container was the one most used. The partition was set at such an angle within the lamp-container that the middle of its edge was immediately below the Nernst lamp This secured rays of light at right angles to the long axis of the tank at the plane of diveon Koes the dark and the light re- gions. In these experiments a sharp plane of separation between a strongly illuminated and an entirely dark region was desired. To the eye this plane seemed very distinct, shoueh the dark region was somewhat illuminated, perhaps to one- fiftieth the intensity of the light region, due to the diffusion of light through the water and Ale to reflection from the ground bie bottom of the inner tank. ‘The ‘sides and end of the ‘Teed half of the tank were non- reflecting, since they were lined with sheets of slate painted dead black. Usually the observations were made from a position (X) near the dark end of the tanks, the eye being placed slightly below the level of the water in the tank. By looking through the glass ends of the two tanks and the water within, one was able to observe the whole surface of the inner tank and the animals upon it with- out changing his position or disturbing the light hood. High intensities of illumination were generally used ad little dificulty was experienced in counting the Ee eal in either the light or dark regions. If the ‘lamination was not sufhcient to enable one to see readily the animals in the darkened region, their outlines could be observed by bringing the eye into ane a position that the ani- mals would appear eile against the relatively intensely illuminated space beyond. This faethodl of observation wasseldom necessary, however. The 6-glower Nernst lamp was used at a distance of about 30 cm. from the floor of the tank. It produced considerable heat at the surface of the water, but the heat thus produced was not apparent at the bottom of the tank, for it was not enough to affect a thermometer bulb placed there. According to Melloni ( cf. Mast Reactions of Isopods to Light 283 ’06, p. 387, footnote) a layer of distilled water 9.21 mm. thick transmits only 11 per cent of the total incident radiation. ‘Tap water transmits only a very little more. Hence the amount of heat reaching the bottom of the above mentioned tank would be less than 0.2 per cent of the total incident radiation.’ To guard against a rise in temperature of the water in the experimental (inner) tank, the water of the outer tank was con- stantly renewed by siphons, one drawing off the water at one end while the other replenished the tank at the other end. This arrangement proved effective. The intensity of illumination at the bottom of the tank, allow- ing 4 per cent reduction in actual luminosity for each centi- meter of water through which the light passed (see page 277), was 6983 C.M. (772 c.p., 6-glower Nernst lamp at 30 cm. from surface of water, which was 3 cm. deep). The luminosity in the dark region of the tank, while not determined, was apparently near, though below, the threshold of stimulation for directive response in Asellus, and certainly much below that in Cecidotea. Sometimes the experiment was started by placing the animals in the illuminated part of the tank and observing their reactions atonce. At other times the animals were placed in the tank and allowed sufficient time to become thoroughly settled before thes were exposed to the light. Separate treatment of the experi- ments on the basis of this difference is unnecessary, for the dis- advantage of the random movements of the animals in the former case was balanced by the disadvantage of the apathy of the animals in the latter case. In both the ultimate responses were the same. Numbers of individuals were experimented with at the same time, as in the experiments with horizontal illumination. 2 Tf the first 9.21 mm. of water transmits 11% of the incident radiation, the second 9.21 mm. would transmit II per cent of its incident radiation, 1.e., 11 per cent of 11 per cent or 1.21 per cent of the incident radiation at the surface of the water. The third 9.21 mm. of the water would transmit 11 per cent of its incident radiation, or 0.133 per cent of the incident radiation at the surface of water. The remaining 2.37 mm. of water would still further reduce the amount of heat reaching the bottom of the tank. 284 A.M. Banta 2. Asellus As in all other cases, Asellus and Cecidotea were experimented with in succession and under the same conditions. ‘The reactions of Asellus will be described first. With vertical illumination of 6983 C.M.* intensity the photokin- etic effect upon Asellus was very marked. If the animals had only recently been placed in the tank, the random movements brought about by thigmotaxis or other influences due to transference to the tank were likewise very marked, but because of photokinesis the animals in the illuminated region were more active than those in the dark region. When the influence of the transference to the tank ceased, the difference in activity between the animals in the illuminated region and those in the dark region was increased. When given time to become acclimated to the tank before the experiment was begun, many of the animals began to move about within a half minute or less. With Asellus the characteristic in ter- mittent movements continued in most cases as long as the ani- mals were in the light region, but the activity was always less when they were in the dark region, so that there was a decided photokinetic effect. Concerning the usual movements of Asellus, 1t may be said that normally the animal moves by short stretches or ‘‘runs,’’ between which it pauses for a time. When more active, its runs are longer and its pauses shorter. It normally comes to a stop by a gradual slowing of its movements, as 1t might appear to do if the stop were due to a loss of momentum. When stimulated in any manner just after the beginning of one of these runs, or near the end of it, the animal often stops almost instantly. Sometimes, however, the movement is immediately accelerated by the stimulus, for the run is more rapid and longer than it would otherwise have been. If the stimulus is applied during the middle of a run, it seems less likely to be effective. When an Asellus has been stop- 3 This intensity was produced by the use of a 772-c. p., 6-glower Nernst lamp at 30 cm. from the sur- face of the water, which was 3 cm. deep. In calculating the intensity of the light at the bottom of the dish, where the animals were, 4 per cent was deducted for each cm. of water through which the light passed. Reactions of Isopods to Light 285 ped by some stimulus, 1t often remains perfectly quiet for a time. But if the stimulation be kept up, the animal usually waves its antennz about in a characteristic manner, lifts its head slightly, turns the anterior end of the body to one side or the other a little, and perhaps turns the entire body more or less and begins to move again. All of these movements may occur in succession, or any one may occur without the others, or only the first part of the series may be gone through with; but the series occurs often enough to be characteristic, particularly with stimuli that are non- directive. This strongly suggests a motor reaction in the sense in which Jennings uses the term. It is not that stereotyped type of reaction, however which characteriszes the typical motor reac- tion. With these preliminary statements regarding the actions of Asellus, we are in position to consider the actions of the species at the plane of division between the dark and the light regions. If headed toward the illuminated region, the animals sometimes stop abruptly when partly across the eee or immediately after crossing it. This stopping occurred often enough to indicate that it was due to the sudden action of the light on a animal. If the animal in one of its runs, reached the plane before it was well under Way, or when it seemed near the end of such an excursion, this abrupt stop was more likely to occur than if the animal crossed the plane while well under way, since in the latter case any stimulus, as has been stated, is likely to be less effective than when the ani- mal is moving more slowly. Sometimes stopping near the line was apparently due to causes other than that of suddenly coming into the light, e. g., the animal may have reached the end of its run. But in other cases the stopping was so abrupt and the reac- tion so characteristic of Asellus when stimulated, that without question the reaction was due to the influence of the light. If the animal stopped at the plane, or so little bey ed it that the characteristic movements following stimulation brought its head back partly or entirely over the plane, it almost invariably turned into the dark region at once or followed along the plane a short distance and fae entered the dark. Animals which on entering the light region met the plane at a very oblique angle 286 A.M. Banta seldom failed to turn back into the dark region, unless they crossed the plane in the middle ofa run. It must not be thought, however, from the above statement that an immediate turning back into the dark region was the rule. For, in the first place, the majority of the ae crossed the plane without stopping at all, and secondly, not more than one-fourth of those which appeared to be stopped by the sudden illumination, turned directly back. The sequence and character of the reactions at the plane were by no means inv rariable. “he animals,even when apparently made to stop by the influence of the light, sometimes merely waved the antennze somewhat, or lifted the head a little and moved on in the same direction as before. Animals which the sudden effect of the light did not cause to stop when going into the illuminated area sometimes showed an immediate acceleration in movement. If such acceleration in movement did not occur at once it appeared very soon. The same statements may be made with reference to those individuals which stopped at the plane but did not find their way back into the dark region at once; after the first pause they showed quickened movements either at once or very soon thereafter. Hence, in any case, though the animal might pause on first entering the illuminated region, phocolmene effects very soon appeared. Of more general occurence than the stopping or turning back of the animals upon entering the illuminated region was the stop- ping of the animal just within the dark region when entering it from the light. It frequently happened that soon after starting an experiment in which 25 or 30 Asellus were used, from four to eight individuals at a time would be seen just within the dark region, where they had stopped as soon as their impetus allowed after passing beyond the reach of the light stimulus. No attempt was made to determine the reaction time at the plane, but the distances beyond the plane of the positions at which the animals stopped were apparently determined by the individ— uals’s reaction time and its momentum when it reached the plane. Occasionally the animals, after starting across the plane in to the dark region, would turn back into the “lumped region, but this was exceptional. 2 ea eee Se i es Reactions of Isopods to Light 287 A few of the records of observations on the actions of individual Asellus at the plane of division between the light and the dark areas are here transcribed to illustrate the details upon which are based the general statements which precede. April 28, 1906. rz:ro._ One Asellus climbed up side of tank at the plane. Came from dark and returned to dark. 11:15. An individual came from dark headed into light and immediately turned about. 11:25. A large & moved back and forth across the plane several times and for eight minutes did not get far from the plane. It finally went into dark. 2:15. One individual came to the plane from light, paused and then crawled up side just on the plane remaining on plane for approximately a minute, then went to dark. 2:15. At start it was noted that after the Asellus crossed the plane into dark they often paused much longer than usual. At one time five were noted just within the dark, all having heads toward dark. 2:32. One individual headed into light but soon turned back, then moved along plane for sometime, moving partly into light, and after about 14 minutes turned away into dark. 2:32. Another went into light and immediately hurried off and scarcely stopped until it had made a circuit of tank and gotten into dark, where it stopped for some time and then moved away very deliberately. 2:37. One started into the light end but stopped as soon as it reached the plane, and turned back in two or three seconds. The above were notes made hastily the first time Asellus was observed under these conditions, and are given here, first, because they show characteristic reactions of the animals; and, secondly, because they were made before I had formed any very definite conception of the movements and actions of the animal under these conditions of experimentation, and when I was entirely unprejudiced toward any explanation of the animal’s actions at the bounding plane. The eyes of Asellus, as might be expected, played a conspicuous part in the reactions at the bounding plane. Whether the animal was headed from the illuminated region toward the dark region, or vice versa, the effects appeared as soon as the eyes were across 288 A.M. Banta the plane. If the animal, upon turning back after it had crossed over into the illuminated region, got into such a position that one eye was in the dark region, its immediate return to the dark space was almost certain. Meeting the plane ata sharp angle brought one eye into the light, while the other was still in the dark; hence the turning back into the dark that almost invariably occurred i in such cases. When an Asellus followed along the bounding plane for a short distance before turning into either area, the head was the part of the body which “found” the plane and directed the animal in following it. Whatever the direction of the animal’s movements, they were never medified until the eye reached the bounding plane. This was taken to indicate that the animal when approaching the illuminated region was not capable of distinguishing that region until the eyes were very near the plane or were aeaily ‘lenoted by the strong light; likewise that when moving in the light toward the dark region 1t was equally incapable de detecting that region until the eyes were quite near the plane or actully eiicd bey aa the reach of the light. ‘The ac- tions of the animal in following a along the plane atk in finding its way back into the dark region are Biel due to the fect of unsymmetrical stimulation of the two eyes. The general movements of the animals as a whole and the ulti- mate positions taken by Asellus under such conditions will next be considered. In Table IX are given the details of an experiment with vertica! illumination by means of the apparatus previously (p. 280) described. In this case 24 individuals (Asellus) were transferred to the tank and left for one hour in diffuse daylight. The room was then darkened and the artificial light (6983 C.M.) turned on. The observations given above (p. 287) were made during this experiment. In the first column, at the left, are given the epochs at which observations were made; in the two other columns are given the numbers of individuals found in the illuminated region and in the dark region respectively at these epochs. At the beginning of this experiment the number of individuals in the flltinainn eed aah wee very nearly equal to that in the dark end. But the photokinetic effect appeared very promptly, and TABLE IX TABLE {[X Continued AseLLus communis (24 individuals) WOhEeER Gn : TIME OF April 28, 1906 ee ILLUMINATED | NUMBER IN MAKING Previous exposure: diffuse daylight RECORD | REGION | DARK REGION Animals in tank for I hour. (6983 se m.) NUMBER IN 11:00 6 18 penis ILLUMINATED | wuMRER IN 11:01 6 18 ae CEOS DARK REGION wide 4 20 a eb (6983 c. a.) | 11:03 2 | 22 - 11:04 I 23 10:20 II | 13 11:05 1 23 10:21 10 14 11:06 4 20 10:22 | 8 16 11:07 5 19 10:23 4 20 11:08 4 20 10:24 6 18 11:09 3 21 10:25 9 15 11:10 I 23 10:26 Il 13 11:11 3 21 10:27 15 9 11:12 4 20 10:28 12 12 11:13 4 20 10:29 12 12 11:14 3 21 10:30 12 12 11:15 4 20 10:31 | 7 17 11:16 a 21 10:32 5 19 1:17 4 20 10:33 2 22 11:19 5 19 10:34 3 21 11:20 9 15 10:35 4 20 11:21 8 16 10:36 | 4 20 11:22 9 15 10:37 6 18 11:23 9 15 10:38 9 15 11:24 9 15 10:39 10 14 Teac i 17 10:40 II 13 11:26 6 18 10:41 9 15 11:27 5 19 10:42 7) 17 11:28 qi 21 10:44 6 18 11:29 2 22 10:45 6 18 11:30 3 21 10:46 6 18 11:31 3 21 10:47 9 15 11:33 2 22 10:48 7 17 11:34 I 23 10:49 9 15 11-35 I 23 10:50 9 15 11:36 I 23 10:51 10 14 ULF 2 22 10:52 12 12 11:40 2 22 10:53 9 15 12:10 I 23 10:54 | 8 16 B2535 2 22 70255 7 a Average for the Fou +t aS a whole time. 6- 18+ 10:57 9 15 a 10:59 eee 5 19 Average per cent for whole time. 24.9% 75 -1% 290 A.M. Banta the number in the illuminated end rapidly decreased. However, the photokinetic effect usually did not cease at once when the animals entered the dark region, hence many of them on reaching the end of the tank, turned back and often wandered into the illumi- nated region again. Since in this experiment the animals had been in the tank but an hour, the thigmotactic or other influence due to the transference of the animals to the tank was still effective. These influences caused the animals to move about so vigorously that they kept entering the illuminated region rather freely for over an hour. However, since the activity was less in the dark end, that factor alone served to keep the number in the dark region in excess of that in the illuminated region. When the thigmotac- tic and photokinetic influence became less strong the number in the illuminated region became quite small and remained so. The observations of Table [X will serve to illustrate the nature of the experiments of this series, which are summarized in Table X. In all these the Asellus were confined in the tank, one half of which was exposed to vertical illumination of 6983 C.M., while the remaining half was very faintly illuminated. . This series of experiments shows Asellus to be extremely respon- sive to vertical illumination of 6983 C.M. intensity. In the end virtually all of the animals, remained in the dark region entirely out of range of the strong light. By way of general summary of this series of experiments with Asellus the following conclusions are drawn: 1 The photokinetic effect in the illuminated end of the tank is very marked. 2 Photokinesis causes some of the animals to start up sud- denly soon after they are exposed to the light, and a generally increased activity soon results. 3 Whenheaded toward the illuminated space the sudden illum- ination of the animal upon crossing the plane between the dark and the illuminated region sometimes causes the animal to stop abruptly. 4. When the animal is made to stop by this sudden illumina- tion, it often reacts in a characteristic way. “eJep pazoesjsqe asay} wos ATeIeUI you uatiiadxa as1jU2 VY} Jo eIVP 9YI WOIy UAL] o1e UWINOD Ys] oY} UI UAAIS sjUddIed aBeIdAe OY Ly —€-$z +b bio iho om SER, OO rio OS OOOOS Ganzi oO. wo. 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Banta 5 his characteristic reaction sometimes brings the animal’s eyes back to the plane, in which case the animal seldom fails to return into the dark region. 6 Animals which meet the plane at a sharp angle usually enter the dark regions whatever may be the direction in which they are headed. 7 Individuals which, in going into the illuminated region, pass the plane without being stopped by the sudden influence of the light, sometimes show increased activity almost instantly. At other times the increase comes less quickly. 8 Individuals entering the dark region from the illuminated one very generally stop on crossing the plane separating the two, but in cases where the individual does not stop a decrease in activity occurs. g The eyes are the effective light receptive organs of this ani- mal, as is shown by the actions of the animal at the plane between the dark and light regions. 10 Unsymmetrical stimulation of the two eyes governs the animal’s movements at the plane. 11 The photokinetic effect keeps the animals moving for some time. It causes them to recoil from the dark end of the tank and then to continue to go back into the illuminated area. 12 More than half of the animals collect within the dark region on the average within 4.3 minutes after the experiment starts. Virtually all the animals ultimately settle down and remain in the dark region of the tank, this occuring on the average 44 min- utes after the experiment starts. 3. Cecidotea The experiments upon Czcidotea with vertical illumination were made under exactly the same conditions as were those upon Asellus. However, two preliminary experiments were made upon Czcidotea with intensities lower than 6983 C.M. In one of these experiments (the bottom of) one end of the tank was illuminated with an intensity of g7 C. M. ( 60 c.p. incandescent at 75 cm. from surface of water); in the otherthe intensity was 172 C.M. (49c.p. Reactions of Isopods to Light 293 Nernst at 50 cm. from surface of water). In the first case 12 ani- mals were used and the experiment was continued for 3 hours. In the second 24 animals were used for 24 hours. Little or no photo- kinetic effect appeared in either case, and the animals did not exhibit a tendency to remain in the dark region rather than in the light one. When two days later, the same animals were exposed to 6983 C.M. intensity, very definite reactions were obtained. It is therefore. safe to conclude that Cecidotea is little responsive to vertical illumination of an intensity of 172 C.M. or less. As with Asellus, vertical illumination of 6983 C.M. intensity produced a decided photokinetic effect upon Cecidotea. Random movements due to recent transference to the tank like- wise appeared in the experiments with Cecidotea. But such movements are of only passing interest here, for they merely resulted in a delay in the ultimate settling of the animals. During the period of these random movements the activity was greater in the illuminated than in the dark area. This difference in activ- ity in the two areas became more evident as time passed, the in- fluence due to recent transference to the tank presumably becom- ing less strong. The photokinetic effect was less marked and less prompt in its appearance with Cecidotea than with Asellus, but there was no mistaking its existence. However, in the case of Czcidotea, when it had once become settled 1n the tank, the response to this influ- ence was quick compared with the usual tardy response to light. With many individuals the response came in the course of a min- ute or two,and the majority of the individuals were stimulated to begin moving within ten minutes. From general preliminary observations it seemed that Czcidotea sometimes reacted in a direct and definite manner to the change in illumination at the plane of division between the illuminated and the dark regions. In a general way these reactions were like those of Asellus under like conditions, but they occurred much less often, so that 1t was hard to decide whether there really were definite reactions, and whether the observed actions at the bound- ing plane were not due to other causes than the change in illumina- tion. To test these supposed reactions of Czcidotea an arbitrary 294 TABLE XI A.M. Banta Caciporea styGia (18 individuals) October 5, 1906 Previous exposure: diffuse daylight TIME OF MAKING RECORDS 10:36 10:37 10:38 10:39 10:40 10:41 10:42 10:43 10:44 10:45 10:46 10:47 10:48 10:49 10:50 10:51 10:52 10:53 10:54 10:55 10:56 10:57 10:58 10:59 11:00 11:01 11:02 11:03 11:04 11:05 11:10 11:11 rus 02 11:13 T1:14 11:15 NUMBER IN ILLUMINATED REGION in tank, for 1% hours. NUMBER IN DARK REGION | 16 17 16 13 13 13 13 II II 10 10 II 10 II II i] ~ coemm ON NIN On CWO OO LG NOSES) OO) I OOOO SN Tin Om za Cn tS _ ot (ofe “T= ae) ‘Oo Cc CO TABLE XI Continued TIME OF NUMBER IN MAKING ILLUMINATED | NUMBER IN RECORDS eS CrOn DARK REGION 11:16 7 na 11317 7 i 11:18 7 ff 11:19 6 vs 11:20 8 6 11:21 9 9 11:22 8 es 11:23 7 Set 11:24 8 is 11:25 7 oy 11:26 7 Aa 11:27 5 13 11:28 5 3 11:29 5 13 11:30 5 13 te fl 11 11:32 7 a Te33 8 10 Dig 4, 8 10 a3 9) 8 rte) 11:36 8 11°37 6 12 11:38 5 3 | 11:40 7 a 11:41 7 =e 11342 6 a 11343 5 13 11:44 4 14 11245 5 3 11:46 4 14 11:47 5 13 11:48 5 13 11:49 5 13 11:50 6 a 11:51 6 *) 11252 6 nd 11:53 uf Il Seeks 7 11 mE55 7 II 12:00 6 a Reactions of Isopods to Light 295 TABLE XI Continued TABLE XI Continued £. 2 e383 2 Eases pa eis See SES ETE: TIME OF | wumper IN | TIME OF NUMBER IN | NUMBER IN NUMBER IN MAKING | ILLUMINATED | MAKING ILLUMINATED | DARK REGION | DARK REGION RECORDS | REGION RECORDS REGION | * 12:05 5 13 1:20 3 15 1:09 3 15 | 1:21 3 15 1:10 3 15 1:36 3 15 Mes 3 15 | = Tet 2 16 ‘Average for 1:13 3 15 whole time.. .|7.3—,0r 40.5% 10.7-++, or §9.5% 1:14 3 15 SSS SSS 1:15 3 15 | Average after one 1:16 3 15 OUTS mertenics: = | 4.6—, or 25.5% |13-4, 0174.5 % Waly 3 15 | | 1:18 3 15 |Average after 24 1:19 3 15 | hours..-...-. 2.9g—,or 16.3% 15.1-+,0r 83.7% plane was established in the middle of the illuminated region for comparison with the boundary between the light and dark regions. The hood around the illuminated part of the tank was then opened somewhat to permit observations from above, and the actions of the animals when they passed through the arbitrary plane and like- wise when they passed the boundary between the dark and illumi- nated regions were carefully watched and compared. It was found, after observing Cecidotea passing through these planes repeatedly, that the reactions at the bounding plane between light and dark were not very marked. However, out of a total of 2126 observations at the two planes, 50 cases were noted in which the animal after starting across the bounding plane into the illum- inated region turned about and remained within the dark re- gion whereas only 30 cases of similar sharp turning about of ani- mals moving away from the dark half of thé tank occurred at the arbitrary plane. Conversely, only 16 cases were noted in which the animals upon entering the dark region from the illuminated one turned back sharply and remained within the illuminated region, whereas 23 cases were noted in which a similar turning about of animals moving toward the dark region occurred at the arbitrary plane. There were 208 cases in which the animal on entering the dark from the illuminated regions stopped when crossing the 296 A.M. Banta boundary plane; only 172 such pauses occurred at the arbi- trary plane. These results, while not very decided, still indicate that Caecidotea is sometimes affected by the abrupt change in the intensity of illumination at the plane of separation between the light and dark regions. : The general movements of Cacidotea, when experimented on in numbers, under the conditions already described (one half of the tank being dark, the other half being under vertical illumina- tion of 6983 C.M.) were fairly definite and no lengthy series of experiments was necessary to demonstrate them. In addition to the general photokinetic effect, which incited the animals to move- ment and kept those in the illuminated region more active than those in the dark one, there existed a very pronounced tendency for the animals to congregate in the dark region. Table XI gives the results of a characteristic experiment of the series. In this experiment 18 Czcidotea were placed in the tank already described and under the same conditions of vertical illum- ination (6983 C.M. intensity) as were employed with Asellus (p. 288). These animals had been in the tank in diffuse daylight for 14 hours before the records began, but just before the light was turned on they were all driven into the region that was about to be illuminated. Naturally, if all the conditions were the same in both halves of the tank, one would expect the animals to distri- bute themselves equally in the two. But when, the other condi- tions remaining the same, the light conditions are different in the two halves, any marked difference in distribution is clearly attrib- utable to reaction to light. In this experiment, starting with practically all the animals in the illuminated region, an equality of distribution had become established in the course of 16 minutes. But this equality did not persist; after a few fluctuations on either side of equality dur- ing the next period of abowt thirty minutes, the number in the dark region became permanently greater than that in the illumi- nated part, and although there were some fluctuations, the tend- ency was toward a constant increase of numbers in the dark re- gion, which finally reached a maximum in about two and a half hours. Although this result was accomplished rather slowly, it 297 f Isopods to Light 7ons O React Bjep paiorrsqe asoy} wosy Apatow jOU “JuouTTIedxe oITJUa BY} JO IVP IY} WI] USye] VIL UUIN]OD 4se] VY} UI sadeIBAR YT, x | g zt z°61 ul gt y I LET. ||| wire storm sufi jorersy siiese sae Hae by econo) 4.16 ope laxe. ele \aheleliove ehh svahsrelelale.ssanerenonelss evan pe isrerers Cov eerces 5 squoutsedxa jo Jas aitjua 10J saseIIAYy ~- == iV E SS = a os =e oe or} + Fa z 2 || See ae Wee I tsi | ae | SIS eae I) AS | ° tz BWP PD IG Gefolnn Hei otria/o) £°+t tt cm ot tz | ee ew PL. 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Banta was sufhciently obvious and definite, as it also was in all other similiar experiments. For the whole time of the experiment the average number of animals in the illuminated area was 40.5 per cent of the entire number of individuals in the tank, but for the last half hour only 16 per cent stayed in the illuminated portion. Table XII gives in condensed form the results of the experi- ments of this series. From this table it may be seen that the average length of time from the beginning of the experiment until the majority of the animals began to collect in the dark region of the tank was 17 minutes, and the average time until the maximum response had been virtually reached was 1 hour and 18 minutes. The average per cent remaining in the illuminated region for the whole time of the experiments was 33 —, and the average per cent remaining in the illuminated region at the close of the experiment was 19.2. Hence it seems clear enough that under these conditions of illumination the Czcidotea tend to collect in the dark end of the tank and that once collected and settled, they remain there. If it be granted that Cacidotea sometimes reacts to the abrupt change in illumination at the plane of division between the dark and the illuminated regions, then the reactions are due to the effects of unequal stimulation upon the different parts of the ani- mal’s body, and to a tendency in the animal to react in a charac- teristic manner when strongly stimulated. ‘This characteristic reaction in Czcidotea consists in a more or less abrupt turning about and starting off in a new direction. This reaction may aid the animal in getting partly within the dark region when once entirely across the plane and in the illuminated region. This occurs so rarely, however, as to be practically negligible. Those general reactions to the light which were manifest in an increased activity in the illuminated region were of course photo- kinetic, and the collecting of the animals within the dark region was likewise due to photokinesis. The animals were from the first more active in the illuminated than in the dark region. ‘This alone would soon cause individuals to collect in the dark region, where the activity was less. When the photokenetic effect ceased to be sufficient to cause the animals to recoil from the end of Reactions of Isopods to Light 299 the tank in the dark region so as to reenter the illuminated region, then the animals remained in the dark region. The foregoing experiments indicate: 1 That under the conditions described, Czecidotea was little responsive, either in increased activity or in collecting within the dark area, when exposed to intensities of g7 C.M. and 72 C.M.; but 2 That with an intensity of 6983 C.M., a very decided photo- kinetic effect was produced; 3. That this photokinetic influence caused most of the ani- mals to become active in from 30 seconds to about ten minutes after they were exposed to the light; 4 That thus the activity was greater in the illuminated region; less pronounced in the dark region; 5 That sometimes Cecidotea, like Asellus, apparently reacted at once to the sudden transition in illumination between the dark and the illuminated regions; 6 That this reaction sometimes appeared as a sudden turning back into the dark region after the stopping at the plane between the dark and the illuminated regions, when headed towards the illuminated part. 7 The influence of the change in illumination was also shown by the fact that Cecidotea when headed toward the dark region, did not often turn back into the illuminated region after stopping at the bounding plane; whereas, when headed in the opposite di- rection such turnings into the dark region were not rare. 8 In addition to the general photokinetic effects upon Cecidotea the vertical illumination of 6983 C.M. intensity caused the animals ultimately to collect and remain within the dark region. g* The majority of the animals began to appear in the ae portion of the tank within 17 minutes after the beginning of the experiment. 10 Practcally the maximum number was collected in the dark region within about i+ t hours, and when once settled there they Pe ned, in that region. Ii Certain reactions an Cecidotea at the plane of division be- tween the dark and the illuminated regions are apparently due to 300 A.M. Banta the effects of unequal illumination upon different parts of the animal’s body. 12 Cxecidotea sometimes reacts in a characteristic manner to the sudden influence of the light, and this occasionally assists in directing the individual into the dark region. 13 The general reactions, consisting in increased activity within the light region and an ultimate assembling in the dark region, were due to photokinesis. “The assembling and remaining within the dark region occurred when photokinesis was not strong enough to cause the animals to recoil from the dark end of the tank so as to reenter the illuminated region. Nagel (’94) has called the reactions of animals to sudden illum- ination “photoskioptic” and to sudden shading “skioptic.” Applying Nagel’s terms here, it may be said that both Asellus and Oxtdeies are both photoskioptic and skioptic, since both species (or certainly Asellus) sometimes immediately respond to the sud- den change in illumination encountered in passing from a dark region to an illuminated one, and also on passing from an illumi- nated to a dark region. Yerkes (’¢3, p. 306) states that with the jelly fish Gonionemus murbachii, an “increase in light intensity uniformly causes a motor reaction in quiescent individuals, and the inhibition of movement in active individuals. Decrease in light intensity usually causes the inhibition of movement in active in- dividuals, but rarely does it act as a stimulus to activity in case of resting animals.”’ With rAselligs and Czcidotea the photoskioptic and skioptic responses are more variable than in Gonionemus. Both species of crustacea when active sometimes respond to a sudden increase in the intensity of illumination by an inhibition of movement, at other times by an abrupt acceleration of movement; when quies- cent, Asellus often responds to increased intensity of illumination by moving at once. If in motion, Asellus very often responds to a sudden decrease in the illumination by coming to an abrupt stop. ‘These experiments with vertical illumination have shown that both species respond 1 in a photokinetic way and ultimately collect in a dark region. Asellus responds more quickly and more gener- Reactions of Isopods to Light 301 ally than Czecidotea, smaller percents of Asellus than of Cecidotea entering and remaining within the light region. Asellus very generally reacts at once to the sudden change in illumination at the plane of separation between the illuminated and dark regions, whereas Czecidotea only occasionally reacts at that plane. Mi. ILLUMINATION BY DIRECT SUNLIGHT WITH THE RAYS AT RIGHT ANGLES TO THE LONG AXIS OF THE TANK As in the experiments with vertical illumination, the tank was so arranged in this series as to have a dark and an illuminated region with a sharp plane of separation between the two. This series of experiments was made at a south window in the basement of the Museum. ‘The available space directly within this window was large enough toenable one to make use of the direct sunlight for only a limited period (about two hours daily), but fortunately this period came at about mid-day. Just what the sun’s luminosity ordin- arily is at mid-day in Cambridge was not determined. Naturally so many factors. affecting the lemme are concerned that any determination or estimate of it at one time would have little value for any other time. To my eye the brightness at the tank under direct mid-day sunlight ordinarily ean approximately twice as great as that produced by the 6983 C.M. artificial light so often used. In these experiments the inner glass tank filled with water to a depth of 3 cm., was used without the outer tank. Under one half of the tank was placed black cloth of several thicknesses. This extended exactly to the middle of the tank, and was fitted to the end and sides and over the top of that half. To further ex- clude the light from this part of the tank, a black card-board par- tition extended down from the edge of the cloth at the median plane. This partition just cleared the surface of the water, so that the only light which entered the dark end came through the water below the partition. Of course the so-called dark end was consid- erably illuminated owing to the diffusion of light through the water but the plane between the illuminated and dark regions was never- theless very sharp, so that the contrast between the two wasstrong. The tank was placed upon a light but rigid box so that it could 302 . A.M. Banta be shifted occasionally in order to keep the sun’s rays approxi- mately atrightangles to the long axisof the tank. This mechanical disturbance, w ie exceedingly undesirable, was non-directive in its effects. - It did, however, tend to increase the activity of the animals and thus indirectly increased the average percentage in the illuminated region, rather than that in the dark region. Fur- thermore, since ine shifting did notoccur oftener than every 15 min- utes, and since within fifteen minutes after the experiment started practically the maximum response had occurred, the results were already virtually attained before any shifting was necessary. In these experiments the temperature of the water rose rapidly, but since the water used was 3 cm. in depth and the sun’s rays en- tered it somewhat obliquely, they passed through a thickness of more than 3 cm. of water. Hence it is safe to say that heat was not the effective stmulus in these experiments. i ie Asellus The following table (XIII) shows the results of one of these experiments, in which 43 Asellus communis were used. After obervations at intervals of sixty seconds for about half an hour, at 11:01 the illuminated end of the tank was quickly darkened and the one previously dark was suddenly illuminated. At 11:30 a return to the initial illumination was made. This table shows that Asellus very promptly avoids direct sun- light under the conditions of these experiments. The animals in the illuminated region showed great activity. ‘This appeared very promptly with most individuals, nearly all beginning to move within one minute after the sunlight was allowed to reach them. This soon resulted in bringing all the individuals into the dark area. In no case did it require more than about five minutes for nearly all the animals to find their way into the dark region; once there, very few came back at all. In most cases those which did come back remained in the sunlight for only a minute or even less. After each disturbance es by shifting the tank to compensate for the earth’s motion, several usually came out into the illuminated region; but they remained in the light only a Reactions of Isopods to Light 303 TABLE XIII ASELLUS COMMUNIS (43 individuals). October 12, 1906 Dark and illuminated regions reversed TIME OF MAKING RECORDS 10:34 10:35 10:36 10:37 10:38 10:39 10:40 10:41 10:43 10:44 10:45 10:46 10:47 10:48 10:49 10:50 10:51 10:52 10:53 10:54 10:55 10:56 10:57 10:58 10:59 11:00 11:01 Averages for whole time Dark and illuminated regions normal | | | NUMBER IN | NUMBER IN |} ILLUMINATED DARK REGION | REGION 26 17 be NH w “oO - + is) 7 Averages for 1.9—, Or 4.4% |41.1+,0r 95.6%|| whole time TIME OF MAKING RECORDS 11:03 11:04 11:05 11:06 11:07 11:08 11:09 NUMBER IN NUMBER IN ILLUMINATED | DARK REGION REGION 2 43 15 28 25 18 36 7 41 | 2 41 2 41 2 40 3 42 I 42 I 42 I 43 < 43 2 42 I aie 3 41 z; 41 2 43 © 43 iS 43 S 43 c 42 I 43 < 43 < 42 I 43 2 43 2 43 © 39-2—, Or 91%] 3.8 +, or 9% | 204 A.M. Banta TABLE XIIM—( Continued) Dark and illuminated regions returned to normal || TIME OF NUMBER IN NUMBER IN | TIME OF NUMBER IN | NUMBER IN MAKING ILLUMINATED DARK | MAKING | ILLUMINATED | DARK RECORDS REGION | REGION | RECORDS REGION | REGION | | | | | 11:33 39 4 | 11:53 ° 43 11334 10 a3 11:54 fo) 43 Dai 6 37 || 11:55 ° 43 11:36 I | 42 || 11:56 ° 43 L337 I 42 11:57 ° 43 11:38 I | 42 | 11:58 ro) 43 11:39 2 41 | 11:59 fe) 43 11:40 2 | 41 | 12:00 fe) 43 11:41 I | 42 12:01 ° 43 11342 I | 42 12:02 ° 43 11343 | 2 41 12:03 ° 43 11:44 fe) 43 12:04 ° 3 11:45 fe) 3 12:05 ° 43 11:46 I 42 12:06 fo) 43 11:47 ° 43 12:07 fo) 43 11:48 ° | 43 12:08 ° 43 11°49 ie | 43 11:50 fo) | 43 | Averages for | | 11:51 fe) | 43 | whole time. 1.8 +, or 4.3 %|41.2—, OF 95.7% 11352 fo) | 43 | very short time. No careful study of the animal’s actions at the bounding plane between the illuminated and dark regions was attempted, but numerous instances were noted in which animals started into the illuminated area and at once turned back. Several other experiments upon Asellus under the influence of direct sunlight were made and the results were fully as striking as in the observations tabulated above. On the average for the whole time of the experiments of this series only 9.6 per cent of the ani- mals were in the illuminated area. By way of summary it may be said that Asellus was extremely sensitive to direct sunlight, which it avoided if opportunity was afforded, and that immediately upon entering the illuminated area it often turned back. Reactions of Isopods to Light 305 2. Ceacidotea Under the same conditions of illumination several experiments were made with Czecidotea. The results of one of these, in which 21 individuals were employed, are shown in detail in Table XIV. This table shows that under the conditions of illumination de- scribed, Czecidotea, like Asellus, tended to collect in the dark region. The photokinetic effect appeared quite promptly with the ani- mals in the illuminated region, though it was less prompt than with Asellus. However, most of the animals began moving within from three to five minutes, and within eight minutes this activity had led a majority of them into the dark region.. After entering the dark region they returned to the illuminated one much more than Asellus did; but even with Cacidotea the num- ber which kept returning was comparatively small. The number which started into the illuminated region and turned back within a few seconds was such as to suggest that the influence of the change in illumination at the plane of division between the two regions was in many cases immediate. A more careful study of this point seemed desirable, but an unobscured sun at mid-day came so seldom at that time, that the necessary observations were not made. The other experiments made with Czcidotea under the same conditions gave results in entire accord with those of the experi- ment discussed above. The average per cent of Czcidotea in the illuminated area for the whole time of all the experiments of this series was 22.4. Ceecidotea, then, was quickly affected in a photokinetic way by direct sunlight, though not so quickly as Asellus. This activity incidentally led the animal into the dark region, from which it very generally did not return. Czecidotea just entering the illum- inated region from the dark one sometimes turned back into the dark region at the plane of division between the two regions. Comparing Asellus with Czcidotea when subjected to illumina- tion by direct sunlight with the rays at right angles to the long axis of the tank, Asellus proved decidedly the more respon- sive. It was affected more quickly by the sunlight, sooner 306 CA#cIDOTEA STYGIA (21 individuals ) A.M. Banta TABLE XIV October 3, 1906 TIME OF NUMBER IN NUMBER IN | TIME OF NUMBER IN MAKING ILLUMINATED DARKENED | MAKING ILLUMINATED RECORDS REGION REGION RECORDS REGION 10:56 11 10 11:45 I 10:57 10 II | 11:46 2 10:58 7 14 | 11:47 2 10:59 af 14 | 11:48 I 11:00 8 13 11:49 2 11:01 5 16 11:50 2 11:02 6 15 || 11:51 2 11:03 6 15 11:52 2 11:04 4 17 Tne G3 2 11:05, 3 18 11:54 I 11:06 7 14 | 11:55 I 11:08 10 II 11:56 fo) 11:09 10 II 11:57 I 11:10 Gl 14 11:58 2 11:1 7 14 11:59 I 11:12 4 17 12:00 I 11:13 3 18 12:01 2 11:14 6 15 12:02 B 1T:15 8 13 12:03 4 11:16 4 17 1 12:04 5 11:17 5 16 | 12:05 6 11:18 5 16 || 12:06 3 11:19 5 16 | 12:07 3 11:20 2 19 | 12:08 3 TU21 2 19 12:09 3 TUs23 4 17 | 12:10 3 11:24 5 16 | 12:13 ° 11:25 6 15 | 12:14 ° 11:26 5 16 12:15 2 11:27 3 18 12:16 2 11:28 4 17 12:17 2 - 11:29 4 17 11:30 2 19 ||Averages for 11:31 2 19 || whole time. {3.8+, or 18.+% 11232 3 18 NUMBER IN DARKENED | REGION 17.2—, or 82.—% Reactions of Isopods to Light 307 entered the dark region, and less often returned to the illu- minated one. A general discussion of these results is reserved for the second part of this paper. IV. SUMMARY OF REACTIONS TO LIGHT Tt. With horizontal illumination 1 Asellus communis exposed to horizontal illumination is not responsive to intensities of light of 1 C.M. or less. 2 It is very decidedly affected in a photokinetic way by those light intensities to which it responds. 3. Itis also affected in a phototactic way by horizontal illum- ination. Following exposure to such light, it is negative to an in- tensity of 2.5 C.M.or more. [tis neutral to an intensity of 1 C.M. or less. After retention in darkness for a few hours, it is positive to such intensities as call forth any response (2.5 C.M. or more); but to an intensity of 2855 C.M. the positive response is only momentary. 4 Its response appears to be direct, being produced by the effects of unsymmetrical stimulation of the two eyes of the animal. 5 Cecidotea stygia is not responsive to light intensities below about 80 C.M. Itis negative to such intensities as it responds to at all—8o0 C.M. or more. 6 ‘This response of Czcidotea is photokinetic in its nature, the random movements causing the animals ultimately to settle in the negative end of the tank, where the intensity of illumination 1s least; to this intensity the animals soonest become acclimated. 7 After considerable exposure to strong light both Czecidotea and Asellus become less reactive to it. Conversely, following retention to darkness they are both apparently somewhat more responsive to light. 11. With Vertical Illumination With one half of the tank“ illuminated by light of 6983 C.M. intensity falling vertically upon it, and with the other half as THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 8, NO. 3. 308 A.M. Banta dark as it could be made under the conditions, there being a sharp plane of demarkation between the halves: 1 Asellus is more active in the illuminated region than in the dark one. 2 The photokinetic effect upon Asellus is such that for a time it very generally recoils from the end of the darkened half of the tank and reénters the illuminated region. 3. Asellus showsimmediate responsiveness to the sudden change in the intensity of illumination, these responses manifesting them- selves in inhibition of the animal’s movements. Sometimes it stops on entering the light region—whereupon it occasionally turns back, but more often this happens when it first enters the dark region. Sometimes Asellus shows immediately accel- erated movements when it enters the illuminated region, but when this 1s not the case increased activity usually appears very soon. 4 ‘The animals (Asellus ) ultimately collect and remain within the dark region. 5 The reactions of Cacidotea resemble those of Asellus, but Czecidotea reacts at the bounding plane much less often and less decidedly than Asellus. Czecidotea collect within the dark region more slowly than Asellus and do not remain there as exclusively as do the latter animals. 111. With Illumination by Direct Sunlight To the sun’s rays at right angles to the long axis of the tank both species are more responsive than to an illumination by arti- ficial (Nernst lamp) light of 6983 C.M. intensity, although they respond in much the same manner. As under the other methods of illumination, Asellus is decidedly more responsive than Czec'dotea. Reactions of Isopods to Light 309 V. BIBLIOGRAPHY Banta, A. M. ’07—The Fauna of Mayfield’s Cave. Carnegie Institution of Wash- ington, Publication No. 67, 114 pp., 2 pls., 13 fig. Cuitton, C. ’94—The Subterranean Crustacea of New Zealand: With Some Gen- eral Remarks on the Fauna of Caves and Wells. Trans. Linn. Soc., 2 ser., Zodlogy, vol. 6, 1893, part 2, pp. 163-284, pl. 16-23. Dusots, R. ’89—Sur la perception des radiations lumineuses par la peau, chez les Protées aveugles des grottes de la Carinole. Compt. Rend. Acad. Sci., Paris, tom. 110, pp. 358-361. E1GENMANN, C. H. ’90—The Point Loma Blind Fish and its Relatives. Zoe, vol. I, no. 3, pp. 65-72, pl. 2, 3. ’oo—The Structure of Blind Fishes. Pop. Sci. Mo., vol. 57, no. 1, pp. 48- 58, 8 fig. ’°00*—The Blind fishes. Biological Lectures; Marine Biological Laboratory at Woods Hole, 1899, pp. 113-136. ForseEs, S. A. ’76—List of Illinois Crustacea. Bull. Ill. Mus. Nat. Hist., vol. 1, no. I, pp. 3-25. Garman, H. ’92—The Origin of the Cave Fauna of Kentucky, with a Description of a New Blind Beetle. Science; vol. 20, no. 508, pp. 240-241. Hamann, O.’96—Europaische Hohlenfauna. Eine Darstellung der in den Hohlen Europas lebenden Thierwelt mit besonderer Beriicksichtigung der Hohlen Krains. Jena, 8°., 296 pp. 6 Taf., 150 Fig. Hoimes, S. J. ’05—The selection of Random Movements as a factor in Phototaxis. Jour. Comp. Neurol. and Psychol., vol. 15, no. 2, pp. 98-112. LankeEsteER, E. R. ’93—Blind Animals in Caves. Nature, vol. 47, no. 1217, p. 389. Leypic, F. ’83—Untersuchungen zur Anatomie und Histologie der Thiere. Bonn., 8°.,174 pp. 8 Taf. Mast, S. O. ’06—Light Reactions of Lower Organisms. I. Stentor coeruleus. Jour. Exp. Zool., vol. 3, no. 3, pp. 359-399, fig. 1-6. NaceL, W. A. ’94—Der Lichtsinn augenloser Thiere. Biol. Centralbl., Bd. 14, pp. 364-393. Packarp, A. S. ’88—The Cave Fauna of North America, with Remarks on the Anatomy of the Brain and Origin of the Blind Species. Mem. National Acad. Sci., vol. 4, 1886, 156 pp., 21 text fig., 27 pl. Parker, G. H. ’o5—The Stimulation of the Integumentary Nerves of Fishes by Light. Amer. Jour. Physiol., vol. 14, no. 5, pp. 413-420. Payne, F. ’07—The Reactions of the Blind Fish, Amblyopsis spelaeus, to Light. Biol. Bull., vol. 13, no. 6, pp. 317-323. 310 A.M. Banta Ricnarpson, Harkiet ’05—A Monograph of the Isopods of North America. Bull. U.S. Nat. Mus., no. 54, litt + 727 pp., 740 fig. RoucGemont, P. de ’76—Die Fauna der dunkeln Orte. Munchen, 40 pp., 5 Taf. Semper, K. ’81—Animal Life as Affected by the Natural Conditions of Existence. New York, 8°, xiv + 472 pp., 2 maps, 106 fig. Veypovsky, F. ’o5—Ueber einige Stisswasser-Amphipoden. III. Die Augenre- duktion bei einem neuen Gammariden aus Island und tiber Ni- phargus caspary Pratz aus den Brunnen von Miinchen. Sitzb. bohm. Gesell. Wiss., Prag, Jahrg. 1905, no. 28, 40 pp., 2 Taf. Vir£, A. ’97—Remarques sur les organes des sens du Sphoeromides Raymondi, n.s., Stenasellus Viréi,n.s., et de quelques asellides. Compt. Rend. Acad. Sci. Paris, tom. 125, pp. 131-132. ’99—Essai sur la faune obscuricole de France. (Thése.) Paris, 8°, 147 pp., 4 pl. Yerkes, R. M. ’03—A Study of the Reactions and Reaction Time of the Medusa Gonionema murbachii to Photic Stimuli. Amer. Jour. Physiol., vol. g, no. 5, pp. 279-307. STUDIES IN THE LIFE CYCLE OF HYDATINA SENTA I. ARTIFICIAL CONTROL OF THE TRANSITION FROM THE PARTHENOGENETIC TO THE SEXUAL METHOD OF REPRODUCTION. AARON FRANKLIN SHULL Department of Zodlogy, Columbia University With One Ficure UL RRGINEUTO Nes oo. denbloep be lube Donadgae U-Cu0de abeocguuETooe SOT OL OR OE Gone tare Op Ran r ge 311 ‘Eqgillgm anal dartetaiQore Bape mince e cece iad toc d..Ac,Cnnce SRC SERSice Seas rine Oak cre Oc oeror 316 LEYPUAMMTEMGS oF gtd DOAASO DOO EN GOO GSMS icp OCHO Oe tia Cea a ie IAT = Cire eerie eee ere tee one 319 INGENTA REOUTSe Obs PaLtHeno PeMetl Ef SELLE fai tetetera erotela as ov.12)-Var=tat=rolte, o]aso:syelatYerate, s/o) «/sietsiole late = 319 Variability of the percentage of male-producers in related strains under like conditions........ 320 Influence of quantity of food culture on percentage of male-producers.................----- 320 Influence of age of food culture on percentage of male producers...................... 328 Influence of substances in water on percentage of male-producers.............2.00000 ee eeee 330 Influence of breeding from different parts of the family on percentage of male-producers...... 337 ; Influence of size of family on percentage of male-producers ........ a aveenhet oof te retsiat ans 340 identinyorsexualiepos, andimalele ges. co\c a cuatelo en's = Ser 1O ayo satan tatals elejakete: Qoreyerese «alec e 342 PAPE EIRATS VOMEESU LEG relete tated eee tep Ranson scare giels «ais enie. Sie cio is SOs chicos wie mia ecaye Slsicyu's aise saree le Stasyevete 344 LD ISGEESIO1) 6 6 ge ROR AO ES ONO CBE EOe COO CRD OS EE eee A pee ner noire nie nee ge err eee 345 SMH petra ply tere taysrct w sie ake storey Nake iat J axe Sale harsher ej eveteiah tte oimteretaen nchaieotoass Sige e Heed Meesld ke SOCe 354 . INTRODUCTION In the attempt to solve the problem of sex determination, 1m- portant experimental evidence supposed to bear on the question has been derived from the rotifer Hydatina senta. It seems prob- able, however, that the phenomena referred to in Hydatina do not bear on sex-determination. It is practically certain that the mother of the males is the sexualfemale. What at first seemed, therefore, a question of sex-determination relates instead to the transition from the parthenogenetic to the sexual phase of the life cycle, so that one could substitute the term sexual female for the term male-producer in describing the phenomena. The qe Aaron Franklin Shull important problem in Hydatina, therefore, 1s to determine what condit ons, whether external or internal, bring about the change from the parthenogenetic to the sexual mode of reproduction, and so affect the proportion of male-producers. With a view to testing anew the influence of the various agents which have been supposed to alter this proportion, the experiments described in this paper were performed. ‘The results of the experiments do not support the view that any of the proposed agents have a direct influence on the proportion of male-producers; but they do give evidence of another factor, not hitherto suggested, a factor which not only accounts for the results of the present experi- ments, but affords a simple probable explanation of the results upon which the previous contradictory conclusions were based. The results of the experiments indicate that the presence of cer- tain substances dissolved in the water in which the rotifers are reared may exert a potent influence on the proportion of male- producers; and they make it probable that the agents formerl , thought to exert such an influence either have no influence at all, or exert it only indirectly by first affect ng the dissolved sub- stances. [he previous contradictory Ponclisions may thus be brought under a common point of view. The above conclusion regarding dissolved substances was reached only after a number of tests had been made of the factors previously bel eved to influence the proportion of male-producers; in the account given in th’s paper, the experiments are described approximately in the order in which they were performed. The | fe cycle of Hydatina senta is as follows: From the fert- lized or resting egg there hatches invariably a female. This fe- male may,under favorable circumstances, produce parthenogenet- ically 40 to 50 offspring, all of the same sex, which so far as I have observed is always the female. These females may in turn pro- duce 40 to 50 young, all the offspring of one parent being of the same sex; but while some of the females produce females, others may produce males. Thus the females may be spoken of as male- producers or female-producers. “The number of male-producers ina family varies within wide limits; it may be zero, or it may be 100 per cent. When males have appeared in a colony, resting Life Cycle of Hydatina Senta 313 eggs may also appear provided the young females copulate within a few hours after hatching. The winter eggs are recognizable by their large size, and thick shell which is covered with many processes like the nap or pile of a fabric. The parthenogenetic eggs have thin shells; those yielding females are usually larger than those hatching into males, but eggs of intermediate size may be of either sex. Most of the above facts were reported by Maupas (’g0a). In later investigations, the same author (Maupas (’gob) found that when young females were given every chance to copulate, no males were produced and about the same percentage of females produced winter eggs as produced males when peels was pre- vented. From this he concluded that the winter or resting eggs are fertilized male eggs, and that young females destined to pro- duce females can not be fertilized. Maupas (’g1) also performed experiment from wh ch he concluded that the proportion of male- producers depends on temperature. ‘The offspring of five fe- males kept at 26° to 28° C. included 97 per cent of male-producers, while five sister females at 14° to 15° C. yielded only 5 per cent of male-producers. Five other females which were kept in the cold while they laid the first half of their output of eggs, and at 26° to 28° C. while laying the last half, yielded 24 per cent of male- producers in the former lot, and 81 per cent in the latter. Six other females were alternated between high and low tempera- ture, and the highest percentage of male-producers came from eggs laid at the high temperature. The possible influence of temperature was afterwards examined by Nussbaum (’97), who got only negative results with tem- perature differences. Nie been sexperiments showed, he thought, that starvation increased the proportion of male-producers. He tried to reconcile Maupas’s findings with his own as follows: Mau- pas probably did not isolate the young rotifers as they hatched, so that his aquaria soon came to contain many individuals. At the h'gher temperature, the animals multiply so much more rapid- ly and each one eats so much more, that the quantity of food put into the dishes at the outset soon became exhausted. The en- suing starvation, Nussbaum supposed, effected the increase in 314 Aaron Franklin Shull the number of male-producers. ‘Temperature, according to his explanation, was only indirectly responsible for the change. He further suggested that Maupas may have determined the sex of the offspring by the size of the egg, which, as Nussbaum himself first pointed out, is not a safe criterion. Nussbaum believed that these two explanations, together with “chance,” sufficiently ac- counted for Maupas’s remarkable results. It will be profitable to state briefly the evidence which led Nussbaum to the above conclusion regarding starvation. His experiments are open to doubt on the ground that they were not usually controlled. Possibly some experiments were controls of others where it is not apparent from the text. Nussbaum rarely mentions controls, hence it 1s probable that in most cases he did not intentionally institute them. His general method seems to have been to raise the rotifers under such circumstances as he could provide, watch the course of the experiments, and record the changes, such as scarcity of food, gradual change of tempera- ture, etc., that occurred. In certain temperature experiments, however, the controls were definitely maintained. When an aquarium showed signs of scarcity of food and males afterwards appeared in it, the experiment was taken as evidence of the influence of starvation. Evidence of scarcity of food was of four kinds: (a) The presence of many females in the same aquar- ium, in which case there must have been less food for each one, even if scarcity was not otherwise apparent; (4) a low rate of egg production; (c) partial emptiness of the gut of the animals; (d) direct observation of the food. ‘There were about eighty experi- ments in all. It is often difficult to decide whether starvation occurred, for there were all degrees from starvation to good feed- ing. I have tried to examine the published data impartially with the following results. 1. In 13 experiments many females were left in one aquarium (from which it might be supposed that the food supply for each was deficient), and males appeared; and in four experiments where distinctly few females occupy the same aquarium, no males appeared. Butin atleast one other experiment (54), many females lived together without producing males. Life Cycle of Hydatina Senta 315 2. In three experiments a rather low rate of egg production (from which partial starvation was inferred) was followed by the appearance of males. 3. In 12 experiments, where abundant food was supplied and the experiment continued for several days, only females were produced. 4. In seven experiments, where hunger was evidenced by the small quantity of free food present or the state of fullness of the gut, males appeared later; but in seven other experiments hunger was not followed by the production of males, and in five others males appear without preceding hunger. It appears that Nussbaum draws his chief support from the cases of inferred starvation in (1) and (2) above, since those in (3) and (4) are contradictory. As the rate of egg-production varies considerably even with abundance of food, the conclusion that starvation increases the proportion of male-producers rests largely upon the cases where the food of a single aquarium was divided among many individuals. The conclusions of Maupas and Nussbaum were tested by Punnett (’06) in several experiments carried out with great care. He isolated each young female and followed its history individ- ually, which neither of the preceding investigators seems to have done. He was unable to secure in three generations an increase in the proportion of male-producers by starving the young females for some hours after hatching. Variations of temperature from 8° to 23° C. yielded no results, though the animalswere kept four to eight days near each extreme. Punnett thought he found evidence, however, of strains, each yielding a rather definite pro- portion of male-producers. He recognized three types of parthe- nogenetic female; one yielding many male-producers (ca. 40 per cent), one few male producers (ca. 2 per cent), and one no male- producers. His general conclusion was that external conditions had no influence on the sex of the offspring, but that this was de- termined by an internal factor, the zygotic constitution. The character of the male and female elements uniting in the winter egg would, according to his interpretation, determine the ratio of male-producers in the parthenogenetic generations that fol- 3106 Aaron Franklin Shull lowed, and that ratio would be fairly constant regardless of ex- ternal conditions, until the parthenogenetic series was terminated. Whitney (’07) made more extensive experiments, including several thousand individual records, which sustained Punnett’s conclusion that neither temperature nor food influenced the per- centage of male-producers. He found no evidence, however, of constant strains, for he was able to derive lines yielding many male-producers from lines yielding few, and vice versa. He attempted to explain Maupas’s results in two ways. First, he found that at the high temperature used by Maupas (26° to 28°C.), male-producers laid from two to four times as many eggs as did female-producers. Maupas probably assumed that the number of eggs was approximately the same for each, and in this way, inane concludes, introduced an error. Second, Whitney found that the male- producers appeared chiefly in the early part of the family, and since the high temperature used by Mau- pas reduced the size of the families, the proportion of male-pro- ducers was accordingly raised. ‘That the first explanation 1s invalid will be shown later. On the second point, some light is thrown by data given in this paper. In view of these conflicting results, it seemed highly desirable that the whole question be reexamined. I undertook the work at the suggestion of Prof. T. H. Morgan, and I am indebted to him for suggestions and encouragement throughout. PROBLEM AND METHODS The problem in Hydatina, briefly stated, was to discover the factom OF factors, either external or internal, which determine the proportion of male-producers. In the experiments to be described, each female was isolated in a Syracuse watch-glass, and kept in about 2 cc. of Great Bear Spring water. The food used was chiefly a colorless flagellate, Polytoma uvella. Ihave had much better success with this than with Euglena or its allies. It was originally secured from a small stream containing kitchen drainage on the Palisades inGrantwood, N. J., in June, 1909, and has been readily propagated from one Life Cycle of Hydatina Senta 317 culture to the next since that time. Cultures were made by immersing fresh horse-manure, tied up in cheese-cloth, in about three times its volume of water. Manure giving a rich brown solu- tion gave much better results than paler solutions. After the manure had beenextracted for one to several days, Polytoma uvella from an older culture was introduced. In from one to five days it was abundant enough to use. The quantity of water in a food culture varied from one pint to several quarts; the smaller cul- tures were ready to use earlier, and also passed their optimum in shorter time. Cultures were made up fresh at intervals of one to several davs; no culture was found tolast satisfactorily longer than four or five days, usually less. Pipettes used in handling the young females were heated in a gas or alcohol flame before using a second time. Pipettes used for food cultures were never used in transferring rotifers; at no time in all my work were any rotifers of this species found in the food cultures. In all experiments performed at Columbia University, the Syracuse watch-glasses were heated in an oven to a tempera- ture of about 200° C. before being used again. In work done at Cold Spring Harbor, L.I.,the dishes containing females from which I was breeding had been placed in boiling water for several min- utes after previous use. Dishes for aoa ale to be kept only until the sex of their offspring was determined, and then discarded, were washed carefully and allowed to dry thoroughly, but not heated. In the course of the summer, 300 of these dishes were tested by placing in them water and food, but no rotifers. Not a single rotifer ever appeared in any of these dishes. Further- more, had there been any adhering rotifers or eggs, these would have appeared later when the records were made. Had not the presence of a foreign rotifer been evident from the nearly equal size of two of them, it must frequently have occurred in a series of families producing many males, that the two rotifers yielded offspring of different sexes. Outof over nine thousand records from unheated dishes, there was not one case of this kind. I con- clude, therefore, that no error has been introduced by failure to heat dishes before a second using. 318 Aaron Franklin Shull TABLE I Showing number of male- and female-producers in a series of 81 generations of H ydatina senta bred under the most favorable circumstances attainable. Male-producers are designated 3\Q , female-producers 29. | seer cane | NO. OF NO. OF | pee see xo.ok | NO.OF | PER CENT ATION | YOUNG | we As | ATION YOUNG we =? org’? Gece ee cae = z = 1 June 29 19 gt |} 34" Auge 25 7 45 2) 3°) ed 25 35 17 3 44 3 \July 2 ° 18 I 36 19 fo) 41 4 3 | 39 13 iy 37) 20 ° 44 5 | 5 | 26 9 | 38 22 ° 7 6 | 6 23 16 | 22 fo) 35 7 | 8 41 | 9 |) 39 23 fe) 25 8 9 38 16 40 25 6 32 9 | II 31 | 12 ain 26 31 8 10 12 | 26 20 az, 27 3 28 11 14 24 14 | 43 29 20 30 12 | 15 | 5 48 +4 30 5 40 13 | 16 Do jo 4Ou Ul Bas Sept. 1 29 19 14 17 4 | 46 46 3 28 fe) 15 18 38 | 13 47 5 13 18 16 | 20 9 39 48 8 ° 37 17 | 22 41 12 49 10 41 18 23 41 12 10 13 33 19 25 20m || 8 50 12 ° 44 20 | 26/33 hae Le Ly. 14 9 43 21 27 | 23 lit 2.6 52 16 g* 3 22 29 8 | 28 | 53 18 o* 1 23 30 I | 30 ho 20 o* 1* 24 31 10 17 | 385 22 21 33 25 |Aug. 1 ° 8 | 56 24 6 42 I | 4 48 57 26 17 14 26 aul 2 19 58 28 28 13 3 | fe) 52 59 Oct I 25 26 27 4 | fo) 35 60 3 I 50 28 6 ° 40 61 5 | 28 15 29 8 ° 42 62 8 37 2 30 9 | : 36 63 10 4 34 31 10 7 37 64 12 13 32 32 12 ° 39 65 14 12 33 13 ° 50 66 15 ° 31 * Remainder of family not recorded. Life Cycle of Hydatina Senta 319 TABLE I Continued | NO. OF | DATE OF NO. OF DATE OF ee eee NO. OF NO. OF oi a NO. OF NO. OF | PER CENT » A- _~A a sENERA- Zn ( | m0) TION YOUNG SF fae ae TION YOUNG SP oe | he |) ee aE =| aare = | 67 \\Oct.. 17 ° 35 76 Octay 31 ° 14 | 68 18 ° 48 Nov I 8 20 69 | 20 10 4 77 | 2 I 23 70 | 21 | fo) 17 78 | 3 fo) 43 jh 23 4 27 79 5 4 33 7 a 25 | 9 34 80 8 12 14 73 27 | 2 17 81 10 2 5 74 28 31 13 12 2 9 75 30 6 32 LCUAIL 5 iGo Gets Bn ER OTe Ie ne Ge RIA ROR Roi ae cnet? 992 2262 30.4 EXPERIMENTS Normal Course of Parthenogenetic Series Experiment I. A stock of rotifers had been brought into the laboratory about the middle of May and there multiplied rapidly. After being kept in a dish for about four days, the rotifers were nearly all dead, but many winter eggs had been laid. The culture was then placed in a refrigerator at a temperature, of 10° to 14° C. until June 26, excepting three days from June 16 to 19. On June 26 it was brought to room temperature, and on June 27 a young female was isolated from it. From this female was bred a series of 81 generations under what seemed to be the best obtainable conditions. ‘The first member of each family became, when possible, the parent of the next generation. Usually only one family was reared in each generation. The purpose of this experiment was to ascertain the nature of the fluctuations in percentage of male-producers, which might occur without intentional alteration of the conditions by the experimenter. The results are shown in Table I. 220 Aaron Franklin Shull The data show that, besides the considerable fluctuation in the percentage of male-producers which appears between one genera- tion and the next, there may also be long-continued periods in which few male-producers appear, followed by equally long periods in which they are abundant. The generations between one winter ege and the next do not behave as a strain having a fairly con- stant proportion of male-producers Variability of the Percentage of Male-producers in Related Strains under Like Conditions Experiment II. ‘To determine what difference in the percent- age of male-producers must be obtained to give positive indica- tions of the influence of a given agent, two series of generations were reared under like conditions. Both series were reared in the same water, at the same temperature, and were fed approxi- mately equal amounts of the same food cultures. The experiment was performed twice, A and B, Table II. In A, the parents of the two series were first cousins once removed; in B, fourth cousins. The difference in the first experiment is about six per cent, in the second less than 2 per cent. In the former case, where the difference between the two percentages of male-producers 1s greatest, the ratioof the higher to the lower percentage is about 1.1 to 1.0. In experiments designed to test the influence of a given agent, unless the ratio of the higher to the lower percentage of male-producers is greater than I.1 to 1.0, 1t is not safe, therefore, to infer from a single experiment that the agent in question has any influence; and the greater this ratio, divs stronger 1s the evi- dence of such A tts In case of an agent Rene but shght effect, this effect should be shown by numerous experiments giving small differences of practically uniform sign. Influence of Quantity of Food Culture on Percentage of Male- producers. Experiment III. On July 22 two sister individuals, respec- tively the first and second of their family, from the 17th generation Se Life Cycle of Hydatina Senta 321 of Experiment I., were isolated. One with its progeny was abun- dantly fed (five to ten drops of the culture) on what was considered the best food. The other with its progeny was fed only so much as was judged would be necessary to maintain life and enable them to produce a moderate family. The shorter families indi- cate that partial starvation actually occured, but this starvation TABLE II Showing the number of male- and female-producers in two series of generations from related parents, both sertes being reared under like conditions. Series I | Series IT No. oF ExPrEri- paced = MENT | DATE OF DATE OF ATION First |NO- OF | NO.OF |PERCENT) NO. OF NO. OF PERCENT YOUNG S29 one) CF SD TOUNG J'2 QQ or 52 [a b . | July -2 31 a July 2 re 18 ; Se ee 15 aa. ae 13 3 5 23 15 5 26 9 4: 6 25 26 6 23 16 5 8 34 16 8 41 9 6 9 36 12 9 38 16 7 uu 24 22 II 31 12 8 13 23 26 12 26 20 aes aaa, | 53. | gona a5 || 013, | 65.5 Beets sass. I | July 22 xf 27 | July 22 41 os 2 ant 15 34 23 41 12 3) | 26 28 16 25 30 18 4 21) 13 31 26 33 17 5 28 18 14 27 24 26 6 29 10 22 29 8 28 3 5 7 7 ge 25 15 30 I 30 31 ° 3 | vat Aug. « 3 33 ar 10 17 | 9 3 7 29 «i! Aug. I ° 8 | I 4 48 10 4 o 39 3 e 19 3 ° 52 II 4 a 35 SEO EA iia esas os sd cae Bel5O 270 | 935.7 193 322 37-4 322 Aaron Franklin Shull TABLE Ii Showing number of male- and female-producers in a series of 55 generations of Hydatina senta which were well fed, and a series of 54 generations which weve starved. WELL-FED STARVED. = , ie NO. OF | DATE OF NO: OF | NO. OF PER NO. OF DATE OF No. oF NO.OF | PER GENERA- FIRST yA CENT OF GENERA- | FIRST nA | CENT OF TION YOUNG e . | * ot Q TION YOUNG a : | : ° | (of 9 Tis studs July 23 41 12 | 1 |July 23 27 23 Dae 25 30 18 i 2 25 | 20 5 Bde tys 26 295) | |eeel7 O35) 25 102) ae ||, CELE Bitte | 27 22 e260 3 27 ene 7 | | [unee | Zgi!| . 48 28 | 4 28 20 ot Nel Gees | 30 I 30 | 5 29| 14 6 Se 31 10 17 14.9 6 31| 3 11 5326 Sines: ‘Aug. fo) 8 7 Aug. 1 | ° 8 | | 4 48 | 5] x “oun meg Ohiese 3 2 19 | 3 oy I. hie) | Beier se 9 4| 3 || oclilieae TOniasee | 4/| © | 35 Xe Lon | 6; o 30 Te aaste | 6 o | 40 J II 8 15 14 Wj co | 8 fo) | 42 12 | Q I al 26.5 Lets tin | 9 2 36 7.2 13 | II I 25 j Taieenyeh | 10 7 37 Ta ull 12 6 | 2k Miao t | 12 ° 39 15 14 10 Il 25.0 Of ae 13 ° 5° 4.9 16 16 ° 12 7 15 7 45 17 18 14 19 USisv eae 17 B 44 18 19 9 26 30.2 LQmaevees| 19 fo) 41 2.2) 19 21 ° 8 20m aca 20 ° 44. 20 22 fo) 7 21 ah ° 7 21 24 9 24 37.8 22 ° 35 22 26 16 10 | 2 Here 23 ° 25 25.6 23 27 | kG 12 9) ena 25 6 32 24 29 | 5 23 37.6 DAs oleate 26 31 8 25 30 9 13 J sa erro 27 3 | 28 Aye Wee 2 6 16 26 . 29 20 | 30 pho) ay 4 4 | 6 36.8 QT oyna | 30 S| om) 28 6 11 14 | DO rarroie Sept I 29 | 19 29 8 2 14 ) | ZO | 3 28 Loe) PSOne 30 10 10 23 16.0 2On ec 5 13 18 j | 31 12 ° 26 Bilieressyys 8 fo) 37 32 13 I | A Dieta: 10 4 41 33 15| 10 6 agra 10/13 33 oe 34 17 |) ~O*S es aaC ere 12 fo) 44 *Remainder of family not recorded. Life Cycle of Hydatina Senta 323 TABLE Il[I—continued WELL-FED | STARVED eae || PATE: oF NO. OF | NO. OF PER cent] NO. OF | PATE OF | wo. oF | No. OF PERCENT GENERA- FIRST go | 99 or M9 | GENTEAN I} FIRST | V6 9.0 lor gig TION YOUNG | | | TION YOUNG | | —_—s RA dse: Sept. - 14 9 43 | 35 19 o* 1* Borman - 16 g* 3* 27.6 36 6|Sept. 21 oF 1* 36 18 of iS} 37 23 ° 6 nace sien 20 o* 1* ) 38 24| 14 | 12 Boies. 22 21 33 7 | 26.2 39 26) 12) || 267 | A Clomnen 24 6 42, ||| 40 28 | 9 17 f a8 AOS? 26 17 14 x 41 |Oct Pe 22) i GS) AMMecet st 28 a WY sae) 42 4) I 27 a53 Te pee Oct. I 2 26 | | 43 cal 2ON Nile 9 ete soe | 3 1 coe || 67-2 44 8 13. =} -20-| 7 a ae 5| 28 15 | | te ater 10 7 18 >| 39.7 Ale eis: 8 37 2 46 II we | LG | ASE ee 10 II 34 47-2. | 47 13 12 j 22 } hy ee 12 13 32 j 48 16 ° 18 | : Ageia | 14 2 1) 49 17 38 ie AQ Se: | 15 | © 31 | 50 18 | 19 13 eacec 17 | ° 35 pS 51 20 5 24 Gens ¢ 18 fo) 48 52 aa he 7 25 pone rete 20 10 4 53 23 16 } 19 || es Ce lerone 21 fo) 17 | 54 25s \ Lou ore A 21.9 Gare 23 | 27 | 55----- 25 Dy tee | | UGhell s Gahan aaa tel 553 1652 25.0 | 466 810 36.5 *Remainder of family not recorded. continued throughout life, and was not concentrated in the first few hours after hatching. The results are shown in Table III. There is a decidedly greater proportion of male-producers among the starved generations than among the well-fed. Since there is considerable fluctuation in the well-fed line, covering long periods, it is instructive to divide the experiment into parts, as shown in Table IV. Had the experiment ended with the family started on July 27, or had it included only the period from August 26 to September 5, or from September 21 to October 12, there would have been THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 8, No. 3. ‘ 324. Aaron Franklin Shull no very clear evidence of any influence of food on the proportion of male- producers; what evidence there is in these parts of the experiment would have gone to show that starvation reduced the percentage of male-producers. But in those parts of the experiment from July 28 to August 25, and from October 13 to October 25 the evidence is very decisive on the other side; and the period from September 6 to September 20, while not so marked, also points strongly to the conclusion that starvation 1s accom- panied by an increase in the proportion of male-producers. TABLE IV A summary of the data given in Table IIT, dividing it into periods according to the percentage of male-pro- ducers in the well-fed series. | WELL-FED STARVED Limitinc Dates | a 7 Ino. OF G'Q|wo. or 9Q| 7FR CENT lino. oF GQ No. or 9 Q| 7P™ CENT | or SP ¥ or J'2 Juliy23" to Juliy:277sstecc052 Se cuces | 127 73 63.5 72 47 60.5 July 28itoyAup-2ce saosin ai 50 754 0.6 125 265 32.0 Aug. 2OitOISEDE: Giri eiacis seme 129 153 45-7 55 80 40.7 SEP ft OitO SePts 20s mjehas -e-te cher 35 203 TAs 7) 34 100 25.3 Septs 2IMtOO ct wiz esos cic ctous ote 187 261 41.7 93 157 lige OctsiatojOcta2 geri vteesce 25 208 | ToOs7 87 161 35-0 These apparently conflicting results do not show that the food culture nas no influence on the proportion of male-producers. It is to be noted that the proportion of male-producers in the starved line is much more constant than in the well-fed line. The change in the proportion of male-producers from one group to the next is‘always of the same sign in the starved series as in the well-fed, but is lessin amount. This greater constancy of the starved fami- lies is well shown in the figure (see opposite page), in which the percentage of male-producers in each series is represented by a curve. [lo eliminate the minor fluctuations from one generation to the next, the experiment has been blocked off in five-day peri- ods in July and August, and six-day periods after September 1. The last period includes seven days. The aim was to include 375 “ull PpeAre ys 941 jo yey SI 9AIND peop 20L “SoT[IUUe J Pe%Are ys jo S9IIVs PB ul pue “Saq[Iuiey PoeF-[[os jO SeTIos B ur si9onpoid-syeur jo uonsodoid 94} Jo vsAIny yi “OMY 4 Gl 6 SION ala te € des 62 ves vl 6 b'Sny OF Gl O€ Sv Life Cycle of Hydatina Senta 09 gz Aine é 9 40 932] US0104 320 Aaron Franklin Shull TABLE V. Showing number of male- and female-producers in three series of gencrations of Hydatina senta which were abundantly fed; and three control series, derived from siste’s, that wee sta-ved, WELL-FED STARVED | No. or | - : . — _ eRe Garp DATE OF PER DATE OF | | PER MENT ae rinst | | | cenror|| rirst aes Oe NO leenem YOUNG Je e | 9 YOUNG JS | em Je | | | 1; SoSRpeone 1 Aug. 18 | 2 47 Aug. 18 14 19 2 | 20 2 46 | 19. 9 26 3 21 | fo) 49 2s ° 8 4 23 fo) 30 22 fo) 7 5 25 | 2 28 24 | 9 24 6 26 | 34 11 26 LO eee 7 | 27 I 41 27 15 12 8 29 | 8 40 29 5 23 9 30 | 7 260m | 30 9) ae Io _(|Sept. 1 | 9 29 | Sept. 2 6 | 16 ia | 3 11 20 4 4 6 12 5 17 27 an) 6 11 14 ng Talla ole 28 | 8 2 14 | 14 9 | 12 24 10 10 23 | 15 10, 4 34 12 ° 26 16 12 | I 39 otal eects tence, aait peu ae aaieas | 125 519 19.4 110 241 pause) B I ‘Aug 25 | 2 28 Aug. 25 fo) 21 2 2} 34 II 26 15 12 | 3 25) I 41 | 4 8 | 20 | 4 29 | 8 40 2 12 | 16 | ce 30 7 26 30 20) ele 6 Sept. 1 9 x || Sept. 1 14° We etsy 7 | 3 11 20 aalee Oe 10) | 8] Belle 07, 27 5 | 8 a | 9 | 7| 14 28 7 eae 17 10 Gp eeet2 24 9) Ag 10 | II 10 | 4 24 II 3 26 12 12 | I 39 3] 17 17 | LE Gtall eect. Anca easeme ere Leo GR Nt, Psa) | 129 180 41.7 Life Cycle of Hydatina Senta 3 727 TABLE V—continued W BLL-FED | STARVED —— = = b | a ee eee ae a E OF | | pER | DATHOF | E MENT |GENBRA- ua | NO. OF | NO. OF aa | ae | NO. OF | NO. OF cit TION ; - : s YOUNG | oS? a |0OFG'9 || yYoune | w¢ | pe or o' 2 } | | | Benn | 1 |Aug. 24 3 35 Aug. 24 | fe) | 42 2 25 | Oo) 40 | 26 8 3 3 28 o (| 44 28 | fo) 2 4 29 iy il eh | | 29 | 7 12 |. § ra (eee ey oe 31 9 | 16 | Gi |Sept. a 2 10 40 | Sept. 2 a) eS | 7 3| 17 20 | | #\ 29 13 8 ye 4 | 5 | I 7 —— a | | | | SROtal er ness ke Sorts cee mere el) 76 256 | 22.8 | 58 | 113 33-9 AGrranlGntOtalecs ees -. 9 109 185 37.0 4 22 | 86 2053 OEWMZOF- Fi. sa: 5 25 109 18.6 eee) 6H) il 59 9.2 Siero le ds oie acs gir 70805 ® 64 349 15.4 TABLE XIV Showing number of male- and female-producers in a series of familtes of H ydatina senta bred from the first- born, and another series from the fifteenth-born of each successive generation, all being the progeny of a single individual. A and B are separate experiments. First-Born FirrEENTH-BORN EXpERI- No. OF DATE OF | | DATE OF | MENT GENERA- FIRST |No. OF | No. OF PERCENT) FIRST NO. OF | NO. OF PERCENT TION YOUNG | oe QQ | or DQ) Younc JP | GD jor’? | a SE EE ee eee EES ee [RAR BR OEe I July 20 On Neat | July 21 29 18 2 22 ar || 12 | 23 8 38 3 ee Ca zy 4 45 4 Zr oo 18 27 | o | 36 5 26 33 17 29 3 40 6 27 23 26 31 15 9 7 29 8 28 8 30 I 30 9 31 10 | 17 | NGG Ri AS ope ee eas 196 | x91 50.6 59 186 24.0 lA ae | Tees | ae Oo, | 8 | Aug. 2 5 44 | I 4 48 | | 2 2 2 19 | 5 ° pee | 2 ° 52 | 3, | 4 c 35 7 2 35 fel Nahe a ie ie a hc? 8 | aOR Oat 5 8 ° 42 | 6 9 ae 36 SPs Mites te) Nie GH beet aed 8 280 ve) | 5 165 2.9 340 Aaron Franklin Shull tain necessary exceptions. The experiment was performed twice, A and B, Table XIV. In A, it was necessary to use one eighth member, and in B one twelfth and one eighteenth member instead of a fifteenth. In B, one second-born was used instead of a first- born. From these two experiments it appears that breeding from the later parts of the family results in fewer male-producers unless the percentage of male-producers yielded by the first-born is very low. Influence of Size of Family on Percentage of Male-producers Whitney (07, p. 13) endeavored to explain Maupas’s high percentage of male-producers in his temperature experiments as due in part to the shortening of the families. He plotted the position of the male-producers in 23 families, and found the great majority of them to appear in the first two-thirds of the family. If the conditions of the experiment curtailed the family by simply omitting the last third of each one, which consisted almost wholly of female-producers, the proportion of male-producers would be greatly increased. As Whitney’s conclusion was based on a small number of fami- lies, | have collected data from 349 families, varying in size from II to 56, comprising about 12000 individuals, bred during the summer and early autumn of 1909. Table XV groups this data according to the size of the family. An examination of any one of these groups of families plainly shows that there is no accumulation of male-producers near either end of the family. The small numbers in the last four places in each group are in part due to the fact that most of the families did not reach the maximum length of their respective groups. It is conceivable that the difference in the proportion of male- producers caused by starvation is referable to the shortened fami- lies. In general, partial starvation was found to increase the pro- portion of male-producers. It might be supposed that the num- ber and position of the male- een Nae in the family was prede- termined; if the middle third of the family were destined to be Life Cycle of Hydatina Senta 341 largely nale-producers, the mother would be able notwithstand- ing the small quantity of food to prolong her family to include all the male-producers. But if the middle third of the family were destined to be chiefly female-producers, then partial starvation would prevent this middle third from being produced at all. If this be the true explanation of the higher percentage of male- producersin thestarved families, then those families which reached more than the average length should show an accumulation of male-producers in their latter portions. ‘There were 76 starved families in my experiments, including 1876 members, or an average of 24.6 per family. Of the 41 families that contained more than TABLE XV Showing the number of male-producers occupying the various places in their respective families, compiled from 349 families. | NUMBER OF MALE-PRODUCERS OCCUPYING EACH PLACE IN FAMILY No. OF SIZE OF FAMILY a ItIEs | eal SI ae ae I] 2] 3! 4) 5) 6) 7] 8 OTOL I|2\13/14) 5)16/17| 18) r9}20)21)22123)24)25)26.27:28 Gimli ncr 25 cee 32 | 3) 4 6121013 11141012 8 8 8| 7 9 7| 7 9 101212 1211013 10111313 ADCO seit sis.31.¢,- 61 6 g 12,13 18 21118115 15 15|12|17 16,20)17 21]23)25 22/22 16 22/21 22.2025 25 26 41-45.........| 36 3} 2) 6 8! 4) 8 8) 8irx 6 7] 8) 6 9 gtort Q 111213 141414 14.13.15 12 BOSAO Ns te: 35 | 2! 41 7| 4 6 5) 6 6 8| 8 a 4! 6 Sixx) gfr4\r4lx1/13| 913) 9 8] 9 g1013 Sans aie 46 | 3] 7] 31 8 913 1215 14 12 1613161717 19115 21 2012017 18/14 171014 13 9 Bes Osech Aa .| 45 7| 81 7 9 1014 1013 14 18 16.14 1517.21 221920 181817 161617 11 78 4 Bi ooaPOeee 830 5) 7| 9) G1oj12\12 7 1110 13,12)12)14/10 11/12 11 6 8) 6 3 2| 2| O MO 20a coy = 34 4\ 5| 9| 8| 8) 6 7 8 7I10 2| 8 810110 7] 4 4, | © | BES ote ctl as 29 4| 6 5) 71x01 81 7 7] 6 9) 5] 3] 2} 3] 2 | | | | 29 3031 32333435 3637 38 394041 42.43 44 4546.47 48.49 50,51 52.53 54.55 56 ae 51-56. 32 Paleatslechestial chadtntag ester leah 8! 811 9} 8 9 i 7 5| 4) 3) 2) 1 AOREOD sa cteatls 3 61 26302825 22.24|24|19 2123 20\20)20|22|15|16|14 11 9| 8 4 2) 41-45. 4 36 1215 121114, 11)12\14 13,10 11/11) 811 6 5 1 | RGEC. ai... - 35 12|12\t0} 12/10] 8 5) 5) 5] 3] 1] oO | | Bie Gest se +. 46 710) 5] 3 43 I | | | | 26-20) 2.. 62.5: 45 3) 1 | 1 | | bri | | 7) oe eee ea 31 | | | | 16-20.. 34 Sid emer 29 | | 342 Aaron Franklin Shull 25 members, 34 lay between 26 and 35 inclusive. ‘The position of the male- -producers i in these is given in Table XVI. It is evident, I think, that there 1sno accumulation of male-pro- ducers at either end of these families,and hence that the shorten- ing of the families by starvation 1s not responsible for the increased percentage of male- producers i in partially starved families. TABLE XVI Showing number of male-producers occupying the various positions in their respective families, compiled from 34 partially starved families of more than average length. Position IN FAMILY NUMBER OF MALE-PRODUCERS OCCUPYING EACH PLACE IN FAMILY 2021 Lass 24252627 27 28 29) x03! sas 34135 S1zE OF FAMILY No. oF FAMILIES ] | | | I 2) 345 d 7, 8) B gtcburarsansiins SUGEISY Lshe alae 4 4 ee es ae | | | i a dda oa 9101011 9 10) eae 8) 8) Sa 3) O} 1} ae a7 7| 7|10\12| 910 9 guyz n1 001) | Ho} 718 5 313210 | | | Identity of Sexual Eggs and Male Eges Maupas (gob) inferred from certain numerical relations that the resting eggs of Hydatina senta are fertilized male eggs, and that upon females which are destined to produce females impregna- tion has no effect. Subsequent investigations have only tended to make the identity of male eggs and sexual eggs more probable, but direct observations to establish the point have been wanting. Previous workers have all found that each female laid only one kind of egg; (1) parthenogenetic male eggs, or (2) partheno- genetic female eggs, or (3) fertilized resting eggs. If the resting eggsare fertilized male eggs,it would seem poseibles by limiting the number of spermatozoa fhe enter a female during copulation, to secure from her some resting eggs and some male eggs. Since one female can rarely lay over ca Of 17 resting eggs, andl usually lays only Io or 12, even under favorable circumstances, the num- ber of spermatozoa must be less than ten to afford any consider- able probability of getting both kinds of eggs from the same female; or at least less than ten must be successful in entering 2 O O eggs. Life Cycle of Hydatina Senta 343 Experiment XIV. Over a hundred fertilization experiments were made to test this point. Young females within the first six hours after hatching were placed in several drops of water with a few vigorous males about a day old. ‘They were watched until copulation occurred, then separated by taking them up in a pipette and squirting them vigorously out again. The time of copulation varied from 2 to 25 seconds. But from every one of these females that did not produce females parthenogenetically, I obtained only resting eggs or only male eggs. Some of the fer- tilized females laid small eggs toward the last of their output, often not any larger than the majority of male eggs; but these eggs had the thick shell and characteristic markings of winter eggs, and they did not hatch even when kept for days, so they were classed as resting eggs. Later, a number of fertilized females were reared for cytological study. The method of securing them was as follows: A female was isolated and allowed to produce at least one daughter to show that she was a female-producer. ‘There was then placed in the dish with her a male-producer that had been producing young for some hours, together with the 12 to 15 males which had already hatched from her eggs. As the young females hatched in this dish, they nearly all copulated, and were isolated at intervals as as in the other experiments. It thus happened that the male- producer was always about a day older than the female-producer in the same dish, so that the males wereold, or sometimes all dead, when the last females of the family hatched. These late females were sometimes not fertilized, and produced males. On September g, 1909, a young female, the last member of a family of 46, hatched under the conditions described above, and was isolated. On September to she had laid two large eggs, of the shape of resting eggs; but though their shells were thicker than those of parthenogenetic eggs, they were considerably thinner than those of most resting eggs. Without examining themcare- fully with a microscope, to determine the markings of the shell, I set the dish aside to see whether the eggs would hatch in a few hours, as they probably would if they were parthenogenetic eggs. 344 Aaron Franklin Shull By September 11, the same female had laid several small eggs, also with comparatively thin shells. On September 12 there were 23 small eggs in the dish; no more were laid. On September 13, a numberof males were swimming in the dish, and some of the small eggs were then only empty Stele Fifteen males in all appeared in the dish, the last two on September 16; the remaining eight small eggs did not hatch. On SHINS 21 a young female was foundin the dish, and one of the large egg shells was broken and empty. ‘The other large egg had not hatched Dec. 17, when it was discarded. Thus, winter eggs and male eggs were secured from the same parent. Since so far as known all the parthenogenetic eggs of one individual are of the same sex, there seems to be little room to doubt that winter eggs are male eggs that have been fertilized. The bearing of this is pointed out elsewhere. SUMMARY OF RESULTS The proportion of male-producers in Hydatina senta may be re- duced, even to zero, by rearing the rotifers in the water of old food- cultures, from which the protozoa have been removed. This effect is due to substances dissolved in the water. If, instead of being reared in the water of old food cultures, the rotifers are bred in spring water but fed from old cultures and not fresh ones the proportion of male-producers may be likewise reduced, but in less degree. cere ation may be accompanied by a higher proportion of male- producers; but this is probably due to the reduced amount of dis- solved substances incidentally introduced with the food. No evidence of so-called “‘sex-strains”’ has been found; more or less constant differences attributed to “strains” may have been due to the use of food cultures containing different quantities of dissolved stuffs. ‘ Families bred from the last daughter of a family include on the average fewer male-producers ana families bred from the first daughter. This may be due to the accumulation of substances in the water in which the parent was reared. Life Cycle of Hydatina Senta 345 Male-producers are not more abundant at one end of the family than at the other, regardless of whether the family be large or small. One female may lay both fertilized eggs and male eggs. DISCUSSION The first stage in the solution of the problem undertaken in these studies seems to have been reached in Experiments [X and X. The results of these experiments indicate that in Hydatina senta the proportion of male-producers is reduced by certain dis- solved substancesin the water in which the rotifers are reared. The full force of this discovery is not at first apparent, and the explanation of the two experiments in question 1s not the measure of its importance. Not only may nearly all the results of experi- ments dealing with the proportion of male-producers, which are described in this paper, be accounted for by this new factor; but practically all the work of previous investigators, which led to contradictory conclusions, may be simply explained by the same means. It is thus possible, without wholly rejecttng the conclu- sions of earlier workers, to bring their apparently discordant re- sults under a common point of view. In starvation experiments, it is not practicable to use a smaller quantity of protozoan food, without at the same time introducing a smaller quantity of the substances dissolved in the food culture. The results attributed to starvation may in reality be dependent on the reduced quantity of such substances. ‘The difference in the proportion of male-producers apparently resulting from starvation is of the same sign as should result (according to Ex- periments [X and X) from the influence of these dissolved sub- stances; and as the differences obtained in the starvation experi- ments (III and IV) are not greater than may easily be explained by this factor alone, 1t may be doubted whether starvation per se has any effect whatever. Nussbaum’s conclusion that starved rotifers yielded more male-producers than well-fed ones, seems at first sight to be justified; but the effects which he noted were probably due, not to the scarcity of protozoan food, but to less concentration of certain substances in the water. 340 Aaron Franklin Shull A similar explanation is at hand for the ‘‘sex strains”’ described by Punnett. The various series of generations in which this investigator found constant differences in the proportion of male- producers were probably reared on different food. Though the dates of the experiments are not given, it seems almost certain, from the author’s account of them, that they were performed at different times, hence different food must have been used. It is thus possible that the constant differences which Punnett noticed were due to constant differences in the nature and quantity of the substances contained in the food cultures. It is conceiy- able that “strains” may occur in the sense that families derived from rotifers having very different histories may behave differently with respect to the proportion of male-producers. Work is needed to decide this point. But Punnett’s explanation does not apply to series of families derived from sister individuals, and re- course to it 1s not necessary in other cases if the food cultures are different. The influence of the character of the food cultures is again shown in the experiments (V and VI) with old and new food. The roti- fers fed from the old cultures include fewer male-producers than do those given fresh food. As the protozoa in the two cultures were apparently equal in all respects, the cause of the difference in the proportion of male-producers must be sought in the liquid portion of the culture. If it be supposed that the substances in the cultures, which tend to reduce the number of male-producers, accumulate with increasing age of the culture, the smaller pro- portion of male-producers in families fed from old food is placed in harmony with the other experiments. Such an accumulation of substances in old food cultures may account for other phenomena. The experiments (XI and XII) in breeding from the first and last members of the family may, on this assumption, be brought into harmony with the general conclusion. At the time when the first daughter in a family was hatched, the food culture from which her mother was fed was three to five days old; when the last daughter was hatched, the same food culture was eight to eleven days old. ‘The food culture had grown older in the dishes with the rotifers, just as it had Life Cycle of Hydatina Senta 34.7 in the culture jars, and had probably, notwithstanding its diluted state, gone on accumulating the same dissolved sub- stances. This aging of the food culture is probably not sufficient to account for the very large differences obtained in some of the experiments; but in addition to this factor, there 1s the possibility that the products of metabolism of the rotifers themselves have the same effect as the substances derived from the food cultures. These two factors may account for the result of the four experi- ments in which there is a markedly higher proportion of male- producers among the first-born. If, in the experiments, it be- came necessary to change the water in one of the dishes shortly before the end of the amily and hence to add new food, the last daughter would be hatched under approximately the same condi- tions as the first. In such a case the result might be like that of the second part of Experiment XII, a nearly equal proportion of male-producers in both first- and last-born. Unfortunately, when these experiments were performed, I did not greatly sus- pect the influence of dissolved substances in the water. I have no notes, therefore, to show whether or not the water and food were changed as I have suggested. I only know that such changes were occasionally made, but do not know where. The conclusion that substances in the water cause the varia- tion in the percentage of male-producers is quite in harmony with the nearly uniform results of the experiments (VII and VIII) with drainage water; and since food cultures must be frequently changed, fluctuation in a long series of generations, like thatin Experiment I, 1s probably due to the samecause. ‘Thus practi- cally the whole range of phenomena so far noted, which relate to the variable proportion of male-producers may be dependent on this one factor. Some of the explanations I have offered must be provisional only. Iam prepared to find that several factors are at workinstead of one; but the simplicity of the explantion in every case has led me to extend it tentatively to several phenomena where its validity can be established only by further work. So far in this discussion, nothing has been said regarding tem- perature, the factor to which Maupas attributed the most extra- ordinary differences in the proportion of male-producers. Some 348 Aaron Franklin Shull experiments of my own which, because they are too few to be con- clusive and because other experiments along the same lines are still in progress, are reserved for a future paper, seem to indicate that temperature hassomeinfluence. But asitis probable that the details of Maupas’s conclusion can not stand, | have been led to seek for an explanation of his results. Whitney, it will be remem- bered, has already offered two possible explanations. First, he dis- covered that at high temperatures a male-producer laid two to four times as many eggs as did female-producers at the same temperature. Maupas peobanly supposed that the out-put of eggs was the same for each, hence his 97 per cent was in part accounted for, Whitney believes, by larger families. This explana- tion, however, would only account for an excess of males, whereas Maupas obtained an excess of male-producers. It made no differ- ence in Maupas’s experiments whether a male-producer laid 15 eggs or 50, she counted only one toward the g7 per cent in the ent To sustain Whitney’s point it would be necessary to show that a female whose offspring are largely male-producers lays more eggs at a high temperature than does a female whose off- spring are largely cone -producers. Such evidence is not, I be- lieve, forthcoming. The second soln offered by Whitney to account for Maupas’s results was that at a high temperature shorter families were produced than at a low temperature. He found from an examination of 23 families that the male-producers occurred chiefly in the first two-thirds of their respective families. If these families were shortened by cutting off the last members, which were nearly all female- producers, te percentage of male- -producers would be increased. It appears, however, from the examina- tion of a very much larger number of families (Table XV) that the male- -producers are not accumulated at either end of the fami- ly. It also appears that a short family is not a long family minus its last portion. Families containing 46 to 50 members have their maximum number of male producers among the 25th to 30th members. If a family of 31 to 35 were the same as a family of 46 to 50 with its last 15 members omitted, the maximum number Life Cycle of Hydatina Senta 349 of male-producers should here also appear among the 25th to 30th members. But it does not; the maximum is among the 15th to 20th members. The same fact emerges from a comparison of any other two groups in Table XV. A short family is not a cur- tailed long family;itis built on a plan of its own, which 1s approxi- mately the same, relative to its length, as that of a large family. Either the family is completely worked over, or elimination occurs all along the line, from beginning to end of the family, and not at the end alone. Since in the light of new data these two proposed explanations are inadequate, I have been led to seek for others. Firstly, Mau- pas may have used different food for the two parts of his experi- ments, but his account is too brief to enable us to Judge on this point. Another possible, and I believe more plausible, explana- tion is found in the effect of breeding from different parts of the family. It appears that breeding from the first member of suc- cessive families may yield many more male-producers than does breeding from the last member. When the conditions are such as to produce many male-producers among the first-born, the difference may be very great,—57 per cent and 14 per cent respectively in one case. Whether this phenomenonis due to aging of the food culture in the dish with the parent, or to accumulated metabolic products of the rotifers themselves, or to any other factor, does not concern us here. The fact remains that the first- born may yield more male-producers than the last-born. If Maupas reared the first five members of the family at a tempera- ture of 26° to 28° C., and only decided to institute a control experi- ment when it became apparent that the first families would be largely male-producers, then the sister individuals used for the control and placed at a temperature of 14° to 15° C., must have been late members of the family. Maupas does not tell us that these experiments were performed simultaneously, and it would have been very natural to have tested the high temperature to see whether it offered any probable results Bee beginning any formal experiments. I offer this explanation only as a sugges- tion, but it seems to me a probable one. 350 Aaron Franklin Shull Since all the positive results upon which earlier conclusions were based may readily be explained as due to substances in the water, let us see whether the negative results offer any obstacles. ‘There is but one point of any considerable importance on which my results are seemingly at variance with those of previous workers. ‘This relates to the question of starvation. The experi- ments of Punnett and Whitney went to show that quantity of food, or any concomitant factor, had no influence upon the pro- portion of male-producers. ‘Though I have arrived at an opposite conclusion, my results are not, 1t seems to me, opposed to their results. Starvation in the experiments of Punnett and Whitney was limited to a period of hours after hatching, whereas in mine it continued throughout life. It was supposed that the female would be more susceptible early in life than afterwards. If the sex of the immediate offspring of a female were to be affected, probably only those factors which operated early in life would be of any avail. If the effect of (apparent) starvation is not notice- able until the second generation, there may be late stages in the development of oogonia and eggs which are more susceptible to the influence of starvation (really the small amount of certain substances in the water) than are the very early stages shortly after hatching. In Nussbaum’s experiments and in mine, this influence occurred in late stages as well as early, and it is impossi- ble to state whether the critical period, if such exist, occurs at one stage or at another. Whether my starvation experiments differ essentially, therefore, from those of Punnett and Whitney, seems to depend on whether the influence of the substances dissolved in the water is feltin the first generation or the second. Both Maupas and Nussbaum discussed this point with regard to certain external conditions, but disagreed in their conclusions. Some light is thrown on this question by the experiments with the fil- trate from old food cultures. Among the females transferred from spring water to the filtrate, some were male-producers; but of the females of the next generation, none were male- producers. ‘This shows that the full effect of the filtrate is not apparent until the second generation. Whether the proportion of male-producers among the females transferred to the filtrate Ww ~ Life Cycle of Hydatina Senta 3 was altered by the change of medium, the numbers used were too small to decide; but the influence felt in the first generation 1s at most only a fraction (probably a small one) of that apparent in the second generation. Since this is the case, it is readily seen how a reduction in the quantity of substance in the water at a comparatively late stage of the rotifer’s life, when the oogonia or eggs are well developed, might result in more male-producers in the next generation, whereas a similar reduction just after hatching might have no effect. This view harmonizes with the result that, while starvation throughout life is followed by an increase in the proportion of male-producers, starvation for only a short period after hatching has no effect. There remains the further possibility that starvation just after hatching may defeat its own purpose. Doubtless some unassimi- lated food comes over from the egg to the young female. How long this lasts has not been determined. Since a young female eats very soon after hatching, to deprive her of food must dis- turb her normal processes; but 1t seems to me doubtful whether as great a degree of starvation has thereby been obtained as has been supposed. Not less important than the relation of my experiments to those of previous workers, is the examination of the experiments for defective points. The assumption that male-producers appear chiefly in a given part of the family, and that reducing the size of the family may be accomplished by omitting, unaltered, some specific portion of the family, have been shown to be with- out foundation. As a corollary of this, the similarity of large and small families with respect to the relative position of the aie producers in them shows that in general the value of experi- ments with Hydatina is not diminished because the families are small, provided the requisite aggregate number of individuals is obtained. Death losses sometimes invalidate experiments, and must always be taken into account. If any safe conclusion is to be reached, the differences-caused by the conditions of the experi- ment must be so great as to make the death losses insignificant, or it must be shown that these losses are not selective. In the experiments with the filtrate from old food cultures, it has been Aaron Franklin Shull ~~ Al N shown that the death losses are probably not selective; and even if selective they are, in certain experiments, entirely insignif- cant. Nothing has been said on this point regarding the stary- ation experiments. If it could be shown that the shortening of the families in these experiments were due to death of many of the female-producers, a considerable increase in the propor- tion of male-producers would be accounted for, and the experi- ments might only show that when the rotifers were starved many female-producers died. “That the starved families were smaller was due chiefly to the fact that fewer eggs were laid, and only in small part to failure of the eggs to hatch or to death of the young rotifers. But no amount of elimination of female-producers could result in an increase in the absolute number of male-pro- ducers. Such an increase in the number (as well as proportion) of male-producers is found in the second and sixth parts of Table IV, an increase too great to be insignificant. I conclude, there- fore, that death losses do not vitiate the results of the starvation experiments. Previous workers with Hydatina have spoken of the problem which its varying proportion of male-producers presents, as one of sex-determination; but it is open, as I have indicated in the introduction, to interpretation as a change from the partheno- genetic to the sexual phase of the life cycle. This view was adopted by Morgan (’07, p. 346). The assumption necessary to support this view was that the male- -producers are the sexual females, which assumption was based on the numerical relations found by Maupas (’gob), the cytological evidence of Whitney (09), and the analogy afforded BF Asplanchna ( (Lauterborn, 98, p- 178). Since among many thousands of females laying only eges that develop parthenogenetically, not one has ever been found to produce offspring of both sexes, my observation that a male-producer may also lay resting eggs, though not a complete demonstration, leaves little doubt that male eggs and sexual eggs are identical. My observation does not exclude the possi- bility that female eggs may also be fertilized, but Maupas’s experiment menmoneel in the introduction and the chromosome counts made by Whitney make this improbable. ae Life Cycle of Hydatina Senta 353 If resting eggs are fertilized male eggs, and never fertilized female eggs, male-producers are the sexual females. The appear- ance of male-producers then becomes merely a transition from the parthenogenetic to the sexual phase of the life cycle, and is not different from that in certain aphids. In aphids it has been shown (Slingerland, ’93, and others) that external conditions may influence the occurence of the sexual generation, and Issak6witsch (707) and Woltereck (’og) find the same true of daphnians. There is a much larger body of earlier literature upon the subject, but any discussion of this, or any attempt to relate the phenomena in the rotifers to those in other groups, is purposely deferred until my data are more complete. In cases where the sexual female is distinguishable from the parthenogenetic female, there has been no confusion of the phenomena of the transition from one phase to the other with sex-determination. Where, as in Hydatina, the sexual is not externally distinguishable from the partheno- genetic female, the inauguration of the sexual phase appears to be merely the addition of males, hence the application of the term “sex-determination” to the phenomena. The external .imilar- ity of the two kinds of females does not alter the essential nature of the case. Under this view, the interesting features of the life cycle of Hydatina senta are that the sexual eggs may de- velop without fertilization, in which case they sedis males, and that the two sexes of the sexual phase do not first appear simultaneously. The sexual female always appears one genera- tion earlier than the male, for she is, if unfertilized, the gence of the males. Accepted by The Wistar Institute of Anatomy and Biology, March 6, 1910. Printed June 22, rgto. Aaron Franklin Shull oS) wn oN BIBLIOGRAPHY IssakowirscH, A. ’07—Geschlechtsbestimmende Ursachen bei den Daphniden. Archiv f. Mikr. Anat. u. Entw., Bd. 69, pp. 223-244. LAuTERBORN, R. ’98—Ueber die zyklische Fortpflanzung limnetischer Rotatorien. Biol. Centralb. xvi, p. 173-183. Maupas, M. ’goa—Sur la multiplication et la fécondation de |’Hydatina senta Ehr. Comp. Rend. Acad. Sci. Paris. T. 111, pp. 310-312. ‘gob. Sur la fécondation de |’Hydatina senta Ehr. Comp. Rend. Acad. Sci. Paris. T. 111, pp. 505-507. ‘91—Sur la déterminisme de la sexualité chez |’Hydatina senta. Comp. Rend. Acad. Sci. Paris. T. 113, pp. 388-390. Moraean, T. H.’07—Experimental Zodlogy. | Macmillan Co.,pp. xii +454. Nusssaum, M. ’97—Die Entstehung des Geschlechts bei Hydatina senta. Archiv f. Mikr. Anat. u. Entw., Bd. 49, pp. 227-308. Punnett, R. C. ’06—Sex-determination in Hydatina, with some Remarks on Parthenogenesis. Proc. Roy. Soc., B, Vol. 78, pp. 223-231, with 1 pl. SLINGERLAND, M. V. ’93—Some Observations on Plant-Lice. Science, xxi, Jan. 27, p. 48. Wuitney, D. D. ’07—Determination of Sex in Hydatina senta. Journ. Exp. ZoOl., v, no. 1, Nov., pp- 1-26. ‘og. Observations on the Maturation Stages of the Parthenogenetic and Sexual Eggs of Hydatina senta. Journ. Exp. ZoOl., vi, no. 1, Jan., pp. 137-146. Wo ttereck, R., ’og— Weitere experimentelle Untersuchungen iiber Artveran- derung, speziell tber das Wesen quantitativer Artunterschiede bei Daphniden. Verh. Deutsch. Zod]. Gesell., pp. 109-172. => THE MECHANISM OF MEMBRANE FORMATION AND OTHER EARLY CHANGES IN DEVELOPING SEA- URCHINS’ EGGS AS BEARING ON THE PROBLEM OF ARTIFICIAL PARTHENOGENESIS E. NEWTON HARVEY Columbia University Witu Two Ficures The first visible change occurring in many eggs after the entrance of a spermatozoon is the appearance, at the periphery of the egg, of a fertilization membrane. Although observed and discussed by many authors, very little experimental work has been done on the mechanism of its formation. Yet such an investigation may give a clue to the nature of the change initiating development. This summer (1909) a study of membrane formation in Echino- derm eggs was undertaken. ‘The experimental work was _per- formed in part at the Biological Laboratory of the Carnegie Institution at Tortugas, iene and in part at the Marine Bio- logical Laboratory at Woods Hole, Massachusetts. I wish to express my thanks to Dr. Ralph Lille for the use of some reagents and to the Wistar Institute of Anatomy for a table at the latter station. Iam also indebted to Dr. 1. H. Morgan for very kindly criticising this paper. The forms experimented on at Tortugas were Toxopneustes variegatus and toa less extent Hipponoé esculenta. At Woods Hole Arbacia punctulata was used. I shall discuss the early changes taking place in developing eggs under seven heads, viz: 1 The efficiency of acetic acid in forming membranes at different temperatures. 2 The mechanism of membrane! formation. 1 Unless otherwise stated, by membrane, the fertilization or vitelline membrane is meant. The sur- face film or plasma membrane of the egg is spoken of as the egg membrane. The two have very different properties both chemical and physical. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 8, NO. 4. 356 E. Newton Harvey The chemical nature of the membrane. The migration of the pigment granules of Arbacia eggs. Loss of pigment in Arbacia eggs. Surface tension changes in fertilized and unfertilized eggs. “IAQ & YW The action of development-starting substances in general. I. THE EFFICIENCY OF ACETIC ACID IN FORMING MEMBRANES AT DIFFERENT TEMPERATURES A well known method of determining whether a given process occurring in organisms is chemical or physical in nature, is to compare its temperature coefhcient with the temperature coefh- cients of various known physical or chemical phenomena. In this way it has been shown that the rates of increase of the heart beat, conduction of the nerve impulse and many other organic processes are due to chemical processes since they are accelerated to the same degree by a rise of temperature as is the velocity of chemical reaction. ‘The latter are distinguished from the great majority of physical processes, in that they are affected enor- mously by a rise in temperature. Chemical reactions proceed two to three times more rapidly with every 10° rise in tempera- ture. [he same method may be used to see whether the action of a given substance 1s chemical or physical in nature. My object in studying the effectiveness of acetic acid at dif- ferent temperatures was primarily to test Loeb’s hypothesis, that the reason the fatty acids are the most efhcient acids in calling forth membrane formation is because of their property of dis- solving lecithin and other lipoids. Solution is a physical process. The solubilities of most substances are not greatly affected by temperature. Unfortunately the exceptions to the above rule are mostly exhibited by fatty substances. As nothing is known of the solubility of lecithin in acetic acid at different temperatures, a definite answer as to the action of acetic acid on the eggs cannot be given. Certain other possible actions, however, are excluded and certain others included by my results. These will be dis- cussed after giving the results. Membrane Formation 357 The experiments were performed on the eggs of Toxopneutes variegatus. Perfectly normal membranes may be produced on returning to sea-water after the acid treatment. With Hipponoé the membranes formed after treatment with acetic acid are very close to the egg and almost invisible with the low power, except on slightly high focus when they appear separated from the egg surface by a very fine clear ring. ‘This is not apparent in the untreated eggs, and in those eggs which have not responded to the treatment. In all experiments the usual precautions against contamination with spermwere taken. About 2 cc. of sea-wa ter, densely crowded with eggs, was pipetted to 100 cc. of the acid sea-water at the proper temperature. The eggs were then removed from the solutions at intervals to sea-water at 29°C. (the normal summer temperature of the water at Tortugas) and examined for mem- brane formation. ‘The temperatures given were readings taken at the beginning and end of the experiment. Controls were always kept. EXPERIMENT I Fune 30, 1909. Eggs taken 4.45 p.m. 24 ce. a acetic acid to 50 cc. sea-water. TIME IN MINUTES AFTER 5.05 P.M. TEMPERATURE = 7 2 t | 4 I | 2 3 a Se | ect Poanene ee ae ————— | { MOeaNO 4 Crates ercie sists none | none | very few 50% 50% 75% | 100%, 100% LPS Cae anncenae none none | occasional | very few none 65% | 10% none 27 SEa(0s (Carneqodn or occasional | 50% 50% 10% Gceasional) none | none none | EXPERIMENT II July 1,1909. Eggs taken 7.30 a.m. % 3 Cc. ao CH3COOH to 50 cc. sea-water. TIME IN MINUTES AFTER 8.15 A. M. TEMPERATURE | y | | | t | + | I 14 2 | 3 Avni Guess CO | — | | HicetG Caen none | none | none occasional | occasional | 50% | a few) a few | . EQp-20— Catioe s ar | none none | occasional 30% | 70% 30% | a few| a few 297-305 Caso. | 50% | 70% 90% 100% 90% 40% | none | none | none E. Newton Harvey EXPERIMENT III Temperature 23°-24°C. ce. ‘. ACETIC TO §0 CC. S. W. TIME IN MINUTES AFTER 1.20 P. M. 1 cc It 2 3 4 6 THAIN As eA ee oe none none 25% 45% 50% 80% QSEAITIN See eer ee ee Re occasional occasional 70% * 90% 60% ORM. 4. Sone eG ae occasional 10% 90% go% go% none Tem perature 34°-33°C. | ; i ] Fe a. LE HLsOUb eee o's or Bek Ooo oA none 10% 100% 80% none none ; 5 | ies | ni nt) LNG Ace « Oi ae Rens rie 5 ee occasional | 100% 9o% none | none none Giminis.-4 ok eee eee 20% 100% none none | none none *Missed. The above tables may be simplified as follows: EXPERIMENT I OU ptimum time of exposure Tem perature Minutes Ser ret OEE Ee CEM Ie oat Grn Cine o ond conocer oratana of 7 DO rapersite cera rey clenssesch aes, sell Ske! revere, tel suc) Sactiagn coutsen sie stetepensbarersiey okege kerr 4 BIC Se decusuckogensier ie wy spat, otesta y/o ee tndo tal y tor ole ehstate of Renata eccretertere ea teken tore i EXPERIMENT II Optimum time of exposure Tem perature Minutes LV Ee Ue ee Tene hon sane or tar tons Oboe bean eases eos one 4 HO Sis cs cus yeystols te s20 1c te lanchar ec reselists fe. starsva vegche Les Stee siebeber=y Te, ele oy Ravan ecey eek tats 3 oa MET ADO ROA HOE OA She ODE L AOU caSbodeonoddaggoooaasnoUneS 14 There is about a halving of the time of exposure required for a rise of 10° C. When the optimum concentrations instead of the optimum times are compared as in Experiment III the following result 1s obtained: Membrane Formation 359 TEMPERA- . TIME OF EXPOSURE OPTIMUM CONC. OF ACID ture ee Sat = Poe 6 cc. X acid to 50 cc. s. w. a GS. se. svi alan ale Talia! aval aenieiai canal nin ’ 3 areas SaaS 2-3 cc. acid to 50 cc. s. w 33 2-3 To 5 Ss . exe 3-4 cc. N acid to 50 ce. s. w. 3 MINUTES... 62-6611 eee eee eres 5 % > , N “4 ‘ (33 14-2¢c. N, acid to 50 cc. s. w. 6 ont ’ faze 3 | ce) acid te 5o'cexs. 9, GRTUTE SE Ae eee eee eee ote ae 39 ee ees ; K i ) 33° 14 cc. N acid to 50 cc. s. w. Both the optimum-time and optimum-concentration figures show a large increase in the efficiency of acetic acid with a rise of temperature of 10° C. Expressed in terms of a temperature- coefiicient (Q),, the increase amounts to a doubling, thus: Kt Qu-y =? t + 10 in which Q,, is the ratio of a constant at a temperature f degrees, to a constant at t + 10° C. The very marked efficiency at 36° may be an additive effect as high temperatures are known to cause membrane formation in both sea-urchins and starfish eggs. I did not succeed in producing membranes on Toxopneustes eggs by exposure to sea-water at those temperatures and for the times used with the acid treatment. What changes might the dilute acetic acid bring about in the egg of a sea-urchin which would result in the formation of a mem- brane? The chief possibilities are three. It might: 1 Dissolve out the lipoids at the periphery. 2 Change the surface tension of the egg directly, in that the composition of the medium about the egg is altered. Instead of egg protoplasm—sea-water, we have egg protoplasm—acid sea- water. 3. Combine with some of the egg proteids. The first possibility has already been discussed. The surface tension between two phases is only very slightly influenced by temperature. For the same reason diffusion rate and degree of dissociation of CH,COOH would be negligible factors. 360 E. Newton Harvey There remains only the most probable action, an actual com- bination of the acid with some of the egg proteids, the rate of formation of this compound varying with the temperature as do other reactions (07, = 2\—3)), Greeley’s’ results with HCl also bear out this conclusion Assuming that the acetic acid actually takes part in some reaction’ in the egg ,which is it? Is the membrane—for there is now ample evidence (to be discussed below) to show that the membrane is not present before fertilization—a resultof the union of CH,COOH with some egg substance? It can bedefinitely said that this is not the case. The many substances which will produce membranes are so diverse, chemically, that it is incon- ceivable they should all combine to form the same substance (membrane) or even by their presence bring about its formation. It is obvious that heat and mechanical agitation could not act in this way. ‘The membrane 1s all ready to be formed yet 1s pre- vented from so doing by something. It forms only another example of the so-called “stimulus reactions,” which have been compared to the setting off of a charge of gunpowder by a spark. The change which “sets off”? the membrane formation as well as the reactions into which I believe the acetic acid enters will be discussed in the second division of this paper. II. MECHANISM OF MEMBRANE FORMATION I shall first propose an explanation, of how a membrane may be conceived to form about a system of interacting substances, (as an egg), and then discuss somewhat more fully various facts connected with its actual production. As observed in the living egg, almost immediately (14 to 3 minutes) after the addition of sperm the membrane substance becomes separated from the egg surface by spaces. ‘These spaces fill with a fluid, unite and enlarge, thus pushing out the membrane 2 Greeley: A. W. Biol. Bull., iv, 1902-3, p. 124. Greeley did not interpret his results with refer- ence to chemical action. 3 Reaction is used throughout this paper in the same sense as in chemistry. Membrane Formation 301 some little distance. ‘Two separate events take place, the forma- tion of the membrane, and its separation from the egg. Mechanism In order to simplify conditions as much as possible let us con- sider what would occur under certain conditions in an egg cell (Fig. 2) in which everything has been removed except those sub- stances directly connected with the membrane reaction. Its 1) MEMBRANOGEN 1] CO, + MEMBRANE 2 SUBSTANCE: Fics 1 and 2. inorganic analogue would be represented by an hypothetical cell (Fig. 1) containing cane sugar and appropriate enzymes. The ractions indicated above will proceed until all the sub- stances are present in definite proportions. Equilibrium is then attained. ‘The egg (cell) membrane is impermeable to the con- tained substances and also to the salts of sea-water. ‘This repre- sents the condition,in the mature sea urchin egg. Suppose now a momentary change of permeability occurs so that CO, + mem- brane substance (CO, + C,H,OH) may pass out of the system. This upsets the equilibrium and the reactions proceed in the direc- 362 E. Newton Harvey tion of the arrows until checked by a second accumulation of reaction products and equilibrium is again attained. The membrane substance, in contact with sea-water, hardens, (presumably an oxidation and comparable to the hardening of silk in the air) thus forming a film. Some proteid substance formed just behind the fertilization membrane (possibly a small amount of the membranogen diffuses out during increased per- meability) would absorb sea-water and push the membrane out. A second increase in permeability would result in a repetition of the process with the formation of a second membrane. The principle of Gibbs as apphed by Metcalf may help in understanding how this reaction can proceed so readily at the cell boundary. If, in a solution, a reaction occurs, one of the products of which lowers the surface tension of the mixture, the commencing of the reaction will be favored at the surface and the products will collect at the surface. The role of the acetic acid in membrane formation would be the increasing of the permeability of the egg membrane. ‘This 1s pre- sumably iene about by a combination of CH,COOH with some of the Site proteids, a change with which increased per- meability 1s assumed to be connec cel At the end of this paper | shall give some further general evidence for the permeability theory.’ The actual process of membrane formation as observed under the microscope reveals nothing contrary to the above theory. Herbst® in 1893 cut sections of eggs, fixed at intervals from immed- lately after fertilization tll the pushing out of the membrane. He describes the clear “Protoplasmasaum” becoming plainly thicker just before a portion of 1t becomes lifted off and he inter- preted this to indicate a secretion. It is not very resistant first but later becomes quite firm. ‘The pushing out from the ‘Metcalf: Zeit. Physic. Chem. 52 p., 1905; also Héber, Physikalische Chemie d. Zelle und Ge- webe, 2 ed. Leipzig, 1906, p. 209. 5See my preliminary report (Year-book, Carnegie Inst., Washington, no. 8, pp. 119, 1909), and Science, n. s., Xxx, p. 776, 1909, also similar evidence by Lillie, R. S. (Biol. Bull., xvii, p. 202, 1909). and McClendon (Science, n. s., xxx, p. 454, 1909). 6 Biol. Centralb., 13, 1893, p. 14. Membrane Formation 363 egg is due to “eine gallertartige Substanz, welche durch von aussen aufgenommenes Wasser aufquillt.”” Loeb? has recently expressed the opinion that an “Eiweisskorper”’ or lipoid is the substance concerned in the absorption of sea-water and resultant separation of the membrane. I had come to similar conclusion this summer before hav- ing read Loeb’s or Herbst’s papers. The facts are as follows The fluid between the egg and fertilization membrane has too great a volume to have come from the egg without a corresponding di- minution in size. It must be chiefly sea—water. It at least contains considerable chlorides (as shown by precipitation with AgNOs). The fertilization membrane is very freely permeable to the salts of sea-water, relatively impermeable to sugar and proteids. A small concentration of a sugar or proteid (even though its osmotic pressure were far less than that of sea-water) would be capab!e of absorbing sea-water through a membrane perfectly permeably to sea-water. Double Membranes I have already mentioned the result we should expect in a *hypothetical sugar cell if the permeability of its membrane should be a second time momentarily increased, namely, a second escape of the reaction product. In the egg a second membrane should be formed by substances causing a second increase of permeability. ‘This actually occurs. Tennent’ has recorded a second mem- brane formed by sperm on starfish eggs which had previously been treated with CO, and formed membranes. I was unable to get the above result in sea-urchin (Toxopneustes) eggs treated with CH*COOH followed by the addition of sperm. Only those eggs which had been subjected to the acid treatment too short a tme to form membranes could be fertilized. “They segmented normally. The spermatozoa were apparently unable to pass membranes formed:by acid. 7 Loeb, J.: Arch. Entwm., xxvi, 1908, p. 82. ‘Tennent: Journ. Exp. Zodl., 31, 1906, p. 538. 364 E. Newton Harvey Chloroform saturated sea-water causes membrane formation in a large per cent of Toxopneustes eggs. If the eggs have been fertilized first, beautiful examples of double membranes can be obtained by after treatment with CHCl, saturated sea-water." The second membrane (due to CHCI;) is just as distinct as the true fertilization membrane and lies half way between it and the ege@ surface. “hese eggs are also more normal looking than unfer- tilized eggs subjected to CHCl, sea-water treatment as the cytoly- tic changes caused by the CHCl; do not take place so rapidly on eggs with membranes already formed, probably because the mem- brane offers some resistance to its ready entrance. ‘This second membrane can be formed on eggs which are in the two cell stage and also up to early blastule. Inthe two cell stage it is as distinct asin undivided eggs and sur- round each of the blastomeres. The greater the number of blastomeres the less distinct does it become and the more closely does it surround each cell. The space between it and the cell surface also becomes less transparent as if filled with some other constituents of the blastomeres which have diffused out. The fact that membranes can be produced so late in segmentation stages indicates a considerable similarity in the constitution of the egg during early cleavage stages." An extremely fine and delicate second membrane is formed on fertilized Arbacia eggs placed in four times concentrated sea- water as also on the evaporation of the sea-water on a slide. This is accompanied by loss of pigment and other constituents of the eggs. Attempts to produce secondary membranes in fertilized ‘Toxop- neustes eggs with CH,;COOH failed, even though they were treated long enough to bring about the characteristic effects of over treatment. ° Herbst had already performed this experiment. Loc. cit. 10On examining the literature I find that Loeb (Arch. Entwm. 23, 1907, p. 479) has already recorded membranes formed on individual blastomeres. In one case 2-4 cell stages were obtained by CaCl, treatment (50 cc. § m CaCl, + 1.6 cc. NX. NaOH). In another case 2-16 cells were produced by hyper- tonic treatment. In both cases when sperm was added the individual blastomeres became entirely surrounded by a membrane and dwarf gastrulae resulted from their further development. Membrane Formation 365 Different Types of Membranes Loeb” has cited several instances of development without mem- brane formation in sea urchins and R. Lillie.” has mentioned such a case in starfish. ‘The best known case is presented by develop- ment after treatment with hypertonic sea-water. I have repeated this experiment of Loeb’s (using 100 cc. sea-water + I5 cc. 24 m KCI for 1 hour 20 minutes) and find that there are membranes formed on these eggs exactly like those formed on Hipponoe eggs treated with CH;COOH already mentioned. They are very close fitting and might easily escape notice. Membranes which push out only very slightly from the egg surface may be produced by sperm fertilization at high and at low temperatures (15° to 20° C. with Toxopneustes at Tortugas, and 32° C. with Arbacia at Woods Hole). ‘The eggs are mixed with the sperm for about one-half minute and then placed in the sea-water at the proper temperature. Cases of development without membrane forma- tion seem to be rather cases of development without pushing out of the membrane. Another type of membrane is obtained by allowing eggs to stand at room temperature for 28 hours. When sperm is added practically all the eggs become surrounded by a thick membrane adhering to the egg surface closely. When the egg divides this surrounds each of the blastomeres, which become quite spherical, Similar membranes are formed by sperm fertilized eggs in Ca- free sea-water. That the fertilization membrane is :,ot present as a surface film in unfertilized eggs which is later pushed out is shown by the fact that unfertilized eggs dissolve completely in concen- trated H,SO, while fertilized eggs dissolve all but the membranes. Loeb. J. (1) On fertilizing with sperm after 48 hours standing in sterilized sea-water. Pfliigers Archiv. 93, p. 59, 1903. (2) By hypertonic sea-water. Univ. Calif. Pub. Phys., ii. p. 83, 1905. (3) After treatment with pig serum some eggs form no membranes. These may segment and develop into larvae. Arch. f.d. ges. Physiol. 124, p. 250, 1908. (4) Some eggs of Asterina form no mem- branes after acid treatment, yet develop into small blastula, Univ. Calif. Pub. Phys. ii. p. 153, 1905. ZR, Lillie. After 20 hours in, KCN eggs are warmed. No membranes form yet segmentation 2000 takes place. Journ. Exp. Zodl. v, p. 386, 1908. 300 jibe Newton Harvey | EIB) Ue CHEMICAL NATURE OF THE MEMBRANE I.xperiments were also undertaken to determine the composi- tion of the fertilization membrane. Arbacia eggs were used. Their membranes are insoluble in mKOH and NaOH even on short boiling, although the eges et entirely colorless only a few granules being Sehle: On wrolone- a boiling and evaporation when the strength of the alkali must approach 24 m, the mem- branes dissolve or at least become so broken up as to be invisible. In cold concentrated H,SO,, the membrane 1s insoluble while the egg substance first chars reddish brown, later becoming Shey invisible so that only the spherical fertilization membrane is apparent. Unfertilized eggs without membranes dissolve entirely in concentrated H,SO,. In concentrated HCl there is no solution of the membrane. The egg contents become a clear shrunken granular mass. Eggs in the two cell stage show the division between the blastomeres as a clear line. Concentrated HNO,, NH,OH and glacial acetic acid act like HCl. 1 was unable to demonstrate any proteid in the membrane by the xanthoproteic test although this may be due to its thinness. At any rate the membrane apeoned colorless while the egg con- tents were turned a bright yellow. Lilhet® has recently expressed the opinion that the fertilization membrane ts “‘a paptogen membrane consisting mainly of protein material.” Such a membrane would be much more delicate and easily ruptured than the fertilization membrane is. Besides there appears to be little if any protein in it as shown by its insolu- bility in pepsin HCl, caustic alkalies and concentrated H, SOe It may be compared to the cellulose layers formed about plant cells after division, or to the chitinous skeleton formed in insects by the hypodermal cells. In composition it is probably one of the albuminoids. Lille, R.: Biol. Bull., xvii, pp. 202, 1909. Membrane Formation 367 SUMMARY The essential points brought out in the preceding pages may be summarized as follows: The action of acids in producing membranes on unfertilized sea-urchin eggs is due to their combination with some substance in the egg but the membrane is not the product of this combina- tion. In composition the membrane is probably an albuminoid. It is not present as such before fertilization. The essential condition for its formation is an increased per- meability of the egg surface for a membrane substance which passes out and hardens to the membrane in contact with sea- water (a secretion). Double membranes may be explained on the above theory. Several types of membrane may be produced under different conditions and it is probable that the secretion of the membrane substance always takes place although it may remain close to the egg surface. IV. MIGRATION OF THE PIGMENT GRANULES OF ARBACIA EGGS The second visible change which takes place after fertilization in Arbacia eggs is the m gration of the red pigment granules to the surface. In the mature unfertilized and the immature eggs they le distributed throughout the cytoplasm. This change takes place within ten minutes after fertilization and invariably whenever membrane formation takes place, no matter by what means brought about, whether by acid, by osmotic treatment or by sperm. It is thus associated with membrane formation and may be explained as follows: Most small particles suspended in fluid media become negatively charged and there is additonal evidence that these pigment containing bodies are so charged. Lillie has brought together evidence that the centrosomes are MR. Lillie: Am. Journ. Physiol., xv, p. 46, 1905. 368 E. Newton Harvey negative regions. When the micromeres are formed the pigment is prevented from entering them by the large and prominent asters, then present. Even when cut off from the pigmented area of centrifuged eggs these cells are relatively free of pigment. The granules are thus repelled by the centrosomes. If an increase of permeability is the change initiating the development of an unfertilized egg the same potential differences (between exterior and interior, and different regions of the cell) might be expected that takes place in muscles during stimulation. These potential differences are quite general in the functioning of various tissues (nerves, glands, sensitive plants, etc). “Their origin is most easily accounted for by variations in differential permeability of the cell to anions and cations.*® Lillie*” has discussed this theoretically in a recent paper. Without going into details it may be said that “with the appearance of an increased permea- bility the peripheral regions of the protoplasm must become, for a time at least until the potentials are equalized, positive relative to the interior.” Such pigment granules if negatively charged would be drawn by the electrostatic attraction of the now positive egg surface, to the surface, providing of course, the potential difference were high enough. A calculation (by Lillie) of this based on the observed changes in muscle cells has given a value of 14 volts percm. ‘This would be ample to account for the migra- tion actually observed in Arbacia eggs. The orientation of small particles with relation to the asters occurs in other eggs. Fischel'® in staining sea-urchin eggs with intra-vitam dyes noted a migration of particles stained with neutral red, toward the nucleus and aster, the formation of ‘an ellipse about the spindle-fhgure and a ring about each daughter nucleus where the cell divides, and finally, during the resting stage, even redistribution throughout the cytoplasm. ‘This pro- cess is repeated during each cell division. ‘8 Lyon, E. P.: Arch. Entwm., 23, p. 67. 1907. 16 See Bernstein, Arch. f. d. ges. Physiol. 1902, xcii, and Brunings, id. xcviil, and c. 1903. 7 Lilhe, R.g: Biol. Bull. xvi, p. 207-208, 1909. ‘8 Fischel, A.: Anat. Hefte, 37, p. 863, 1899, also Arch. Entwm., xxii, p. 526, 1906. Membrane Formation 369 ? V. LOSS OF PIGMENT IN ARBACIA EGGS Since the pigment of Arbacia eggs is soluble in water it follows that the membrane of the chromatophore granules must be imper- meable to the contained pigment, otherwise it would diffuse through the cytoplasm and out of the eggs (providing the egg membrane were also permeable to it). If the eggs are heated diffusion of the pigment into the sea-water takes place, showing an increase in permeability in both the chromatophore and plasma membranes. I have never noticed a permeability (to pig- ment) of the former independent of the latter. ‘There is also evidence that the impermeability of the granule membrane is dependent on the plasma membrane. If unfertilized eggs are crushed slightly under a cover glass, part of their contents will flow out into the sea-water and round up. ‘The colored granules which have been pressed out immediately lose their pigment while those within the original fraction still retain it. I do not think this can be due to crushing because the bodies are so small. There is also a difference in the optical properties of the surface newly formed about the extruded protoplasm as compared with the old. The same phenomenon is observed when eggs are placed in hypotonic sea-water (sea-water one third, distilled water two- thirds. ‘The eggs swell and their surface layer becomes indis- tinct. When this occurs the pigment immediately disappears from granules and diffuses throughout the cytoplasm. At the same time in many eggs a thin irregular membrane separates. A loss of the pigment, which becomes a yellow-red color, occurs in four times concentrated (by evaporation) sea-water'!®. The same solution occurs in CH,COOH in concentrations greater than those required to produce membranes. This index of determin- ing permeability changes has been used by Lillie?® working on muscle cells. Pure isotonic solutions of electrolytes which cause '9 See Loeb’s description of this in Strongylocentrotus U. Calif. Pub. Physiol. ii, pp. 73-81, 1905. °° Lillie, R.: Am. Journ. Physiol. xxiv, p. 14, 1909, and id. p. 459. 370 k. Newton Harvey contraction in the muscles, bring about a loss of pigment in the cells of the same organism, Arenicola larva. McClendon?! mén- tions that the parthenogenetic agents which he has used bring about a loss of pigment in sufficient concentration, and Loeb” had already emphasized the cytolytic nature of membrane form- ing substances although Interpreting it in a different way. VI. SURFACE TENSION CHANGES IN FERTILIZED AND UNFERTI- LIZED EGGS In the same paper® which was cited in discussing the cause of the movement of the pigment granules of Abaca eggs to the surface, Lillie has pointed out the relation which should exist between changes of permeability (accompanied by ionic inter- change) and the surface tension of the membrane in question. An increase of permeability should be accompanied b y an increase in surface tension (in as much as the surface tension of a film is greatest when the potential difference between its two sides 1s least). ‘This actually does take place in Echinoderm eggs. Egos from the same females are often somewhat irregular in shape, frequently being elongated, twice as long as wide. Sometimes 40 per cent of ences eggs are in this condieee and | have seen practically all the mature eggs of Toxopneustes irregular just after shedding. It is hard to realize that such small bodies can exhibit the shapes they do especially if they are compared with other fluid systems of the same size, as oil droplets, which main- tain their spherical form through their high surface tension. If such irregular eggs are fertilized with sperm or treated with CE. ,COOH ene is an immediate change. They all become spherical, indicating an increase of surface tension. ‘The round- ing of eggs on engi tion takes place quite generally. Whether this is actually due to a change in potential difference resulting from increased permeability is not so certain but it is significant that the change occurs just after the entrance of a spermatozoon or after treatment with acid sea—water. “1 Science, n. s. XXX, Pp. 454, 1909. * Loeb, J.: Arch. f. d. ges. Physiol. 122, p. 196, 1908. @ Tillie, R.: Loc. cit.,p. 204. Membrane Formation eu Sea urchin eggs (Arbacia and Toxopneustes) also round up on standing for some time in sea-water thus indicating an increase in surface tension. They also become fertilizable by foreign sperm on standing™ (ca. 6 hrs.) or by treatment with an acid or alkali (in concentrations too weak to cause membrane formation. ) It appears as if the change undergone by the eggs on standing were in the direction of increased permeability and that the egg must start toward development in order to be fertilized by foreign sperm. Some eggs do undergo division on standing but it is probable that accessory ee are responsible for this. In order to determine further the nature of the change taking place in mature unfertilized sea-urchin eggs on standing in sea- water, and especially if this change were in the direction of increased permeability, I tried if any less acid were required to cause mem- brane formation six hours after shedding. At this time the eggs of Toxopneustes become fertilizable by foreign sperm. ‘The fol- lowing table is a typical result: Fuly 14, 1909. Eggs taken 12.30 p.m. Temp. 33° to 34°. CONCENTRATION—CC. ACID TO 5O CC. SEA-WATER TGC 5 z 3 4 6 ee after taking MGEPINTIIECS< eres so ain c dare none 10% | 100% 80% | mone none Dernier ohik s Sete ais heels very few 100% 90% none | none none ee = 6 hours after taking WP MIMITHEES xc since A none very few 100% 80% | none none Bi ricit 0 BR OLR CET very few 30% 90% | very few none none Another experiment like the above gave a similar result, the optimum treatment, both as regards time and concentration, about coinciding three-fourths and 5? hours after taking the eggs. If any, a very slightly longer treatment appears more favorable. Certainly the eggs require no less acid to causé membranes to form after standing for six hours. In the above experiment the per cent of eggs which could be caused to form membranes was *4 See Tennent, Biol. Bull. xv, p. 127, 1908. JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 8, No. 4. 272 E. Newton Harvey less in the lot which had stood. ‘Vhis 1s also the case when eggs are fertilized with their own sperm. I tried this experiment with Hipponoe with the same result. Apparently the increase in surface tension on standing is not connected with an increase of permeability as | believe the increase after fertilization to be. ‘The increase in surface tension may account for the entering of starfish sperm which could not enter immediately after shedding of the eggs, for Loeb! has expressed the opinion that the surface tension of the egg and sperm are the determining factors in the entrance of the spermatozoon. As the sea-urchin egg stands a decreasing per cent of its own sperm’ becomes capable of entrance while an increasing per cent of foreign sperm may enter. VII. ACTION OF DEVELOPMENT-STARTING SUBSTANCES IN GENERAL Throughout this paper a momentary increase in permeability of the egg membranes has been mentioned as the fundamental change underlying membrane formation and _ the initiation of development. Eggs in which no membranes are formed are excited to development by the same means as eggs which do secrete a membrane, so that this increase of permeability is probably a change occurring after the entrance of a spermatozoon in all eggs. A consideration of the various par- thenogenetic agents bears this out. A classification of the present known means of causing eggs to develop is as follows: 1 Hypertonic solutions (with an OH ion concentration 10-8). Raising the os- motic pressure of the medium by electrolytes or non-electrolytes or evaporated sea-water. The most universal method for Echinoderms, Annelids, Molluscs and Vertebrates. 2 Hypotonic solutions and distilled water (Asterias and Arbacia), Shucking. 3 Mechanical agitation (Annelids and Star-fish). 4 Temperature changes (Echinoderms). *5 Loeb.: Dynamics of Living Matter, p. 163. Membrane Formation 373 1 Short exposures to high temperatures. 2 Long exposures to low temperatures. 5 Electrical shocks. 1 Charging eggs (Strongylocentrotus) Delage. 2 Induced shocks (Arbacia) McClendon. 3 Constant current (Asterias) (7) Shucking. 6 Chemical reagents. 1 Specific actions—K, Mg; Mn Niand Co. (7) (Delage) 2 Alkaloidsand glucosides (saponin, solanin, pilocarpin, strychnin, quinin, hyocyamin, nicotin.) 3 Tannin and re'ated substances. 4 Fat solvents (ether, chloroform, benzol, alcohol). 5 Bile salts (Na taurocholate and glycocholate). 6 Blood sera (of rabbit, pig, ox, and certain worms). 7. Acids and alkalies. 7 Absence of oxygen (weak CNK and O-free sea-water). 76 A glance at the above classification will show the general similarity in the means of stimulating muscles and sensitive plans and of exciting unfertilized eggs to develop. They are both stimulus responses, and may be expected to show a common underlying cause conditioning the response. I have discussed this in a preliminary note in Science?? and quote from it: “ A con- siderable mass of evidence now exists, especially emphasized in recent papers of Ralph Lillie,2’ that stimulation of muscles is effected by a momentary increase in the permeability of the muscle membrane to CO, allowing its more ready escape during contraction. CO, is the chief end product of the energy-yield- ing reaction on which contraction depends and its removal from the cell allows the reaction to proceed (during contraction) to a new equilibrium (of rest) when checked by a second accumulation * Absence of oxygen and low temperature as well as hypertonic solutions (in part) seem to act as correcting agents, setting the oxidations in the egg on the right path to proper development, (Loeb), and as such do not come for discussion within the scope of this paper. 27 Science, n. s., ¥Xx, p. 694, 1909. 28 Lillie, R. S.: Am. Journ. Physiol., xxii, p. 75, 1908}; xxiv, p. 14, 1909; and xxiv, p. 459, 1909. 374 E. Newton Harvey of CO,. ‘The increase of permeability on stimulation removes the condition which 1s preventing the contraction. “The movements of sensitive plants can best be explained as due to an increase in permeability of the cell membranes relative to the turgor main- taining substances. he important point is that processes in general brought about by stimulation are connected with changes in permeability. Morgan expressed the situation clearly when he compared the means of causing development to a stimulus. The best method of denne permeability changes is by the use of pigment containing cells, such as red blood corpuscles. The escape of haemoglobin serves as an indicator of increased permeability. This process of haemolysis occurs frequently in cases of organic poisoning and 1s manifested in living animals by haemoglobinuria. In the laboratory loss of haemoglobin (from erythrocytes) can be brought about in various ways, by strongly hypertonic as well as by hypotonic solutions. Brah- machari** regards the laking in hypotonic media to be due to some other cause than the actual rupture of the corpuscle by absorption of water. High (heat laking at 60° C.) temperatures, condenser discharges, and a great variety of chemical substances also allow the haemoglobin to escape. Of the latter may be men- tioned acids (especially fatty acids) and alkalies, glucosides and alkaloids (saponin, solanin, pilocarpin), tannin, and related sub- stances, fat solvents (chloroform, ether, alcohol, benzol), the bile salts (Na glycocholate and taurocholate), soaps, haemolysins of foreign blood sera and of animal (cobra, spider, crotalus venom), plant (Amanita and Helvella) and bacterial poisons.*° The list given above coincides almost exactly with the list of chemical substances starting development. As yet there have been no experiments on the poisons mentioned but it is highly probable that reptile, fungus, and bacterial poisons will be found as efficient in causing development as Loeb has shown the bile 28 Brahmachari, U. N.: Biochem. Journ. iv, p. 280, 1909. 5° For a discussion of means and substances causing haemolysis, see Stewart, G. N. Journ. of Phar- macology and Exp. Therapeutics, i, 1909, p. 49. Heinz, R..: Handbuch der experimentellen Pa- thologie u. Pharmakologie, Bd. i, p. 392, Jena, 19¢4. Membrane Formation ( 375 salts, solanin and saponin to be. The specific ions may well increase the permeability of egg cells, for their action on other tissues is of this nature. In the alkali and alkaline earth metals this has been very clearly brought out by Lillie’s work on Areni- cola larvae, already mentioned. It seems probable that the induced increase of permeability brings about the development of an egg for the same reason that the increase on stimulation brings about contraction in muscle cells, namely, by permitting, at the proper time, the escape of some reaction product which is preventing, by its accumulation, the further proceeding of reactions in the egg. This is one very important way in which chemical equilibria may be upset in fluid mixtures surrounded by membranes whose permeability may vary. The reaction product which escapes may be simply CO,, (as appears to be the case in muscles) or some more complex sub- stance, (as the membrane substance) or both. A discussion of this with practically no experimental data would be useless, however. The facts which indicate a momentary increase in permea- bility of the surface membrane, as the first change taking place in the development of an egg, may be summarized as follows :3! 1 The general similarity in the means of stimulating eggs to divide, and the means of stimulating muscles and sensitive plants. These may be broadly classified as chemical, mechanical, elec- trical, thermal, and osmotic. 2 The fact that the chemical substances which start partheno- genesis cause in other cells an increase in permeability (haemoly- sis of red blood corpuscles and loss of pigment in pigment bearing cells). 3 Evidence that stronger concentrations of development starting substances cause loss of pigment in pigmented eggs. 4. That a secretion is the first visible change occurring in many eggs. : 5 That a migration, of pigment-containing granules to the cell surface in Arbacia eggs is caused by a region of positive change 31 These are quoted unchanged from my article in Science. 376 E. Newton Harvey at the surface resulting from ionic interchange accompanying increased permeability after membrane formation. 6 ‘That an increase of surface tension, which must accom- pany a change of potential at the surface, is quite general in naked eggs after fertilization, as indicated by their rounding up when previously they had been irregular in outline. Pot seth TS OF PARASITIC AND OTHER KINDS OF CASTRATION IN INSECTS: WILLIAM MORTON WHEELER With Ericur Ficures I. THE EFFECTS OF STYLOPIZATION IN WASPS AND BEES The perusal several years ago of a very interesting paper by Pérez (86) on bees of the genus Andrena infested with Stylops led me to undertake a similar study of our North American wasps of the genus Polistes parasitized by Xenos. I began to collect stylopized P. variatus during the autumns of 1898 and 1899, while I was living in Chicago, but the wasps proved to be too scarce to serve my purpose. During the summer of 1900, however, while I was spending my vacation at Colebrook, in the Litchfield Hills, Connecticut, I noticed many specimens of Polistes metricus Say infested with Xenos (Acroschismus) wheeleri Pierce and I at once began to collect them.? In ten days during the latter part of August | gathered one thousand specimens of the Polistes from flowers of the golden 1 Contributions from the Entomological Laboratory of the Bussey Institution, Harvard University No. 20. 2 There may be some doubt about the specific names of the host and parasite here mentioned. I have called the wasp P. metricus as this is the name under which it is commonly known and because our extremely variable species of Polistes are in a state of great taxonomic confusion. Miss Enteman, who has studied them very extensively (’04), would probably refer my specimens to P. pallipes Le- peletier, while others would be inclined to regard them as belonging to P. fuscatus Fabricius. Brues (70g) and I had identified the parasite as Xenos peckii Kirby, but Pierce (’08), regards it not only as speci- fically, but also as generically distinct. He has given it the name wheeleri and placed it in a new genus (Acroschismus) because it has the cedeagus ‘‘ considerably dilated at the base, arising between two claws,” whereas Kirby’s species is placed in another new genus, Schistosiphon, because it has the cedeagus “cleft at the apex.’’? The old genus Xenos of Rossi he restricts to the European species (vesparum Rossi and jurinei Saunders). Although these generic distinctions may prove to be valid, I shall use the old name Xenos in the present paper.) THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 8, No. 4. 378 William Morton Wheeler rod (Solidago canadensis) within an area of less than a square mile and noted the sex of each individual and the number, sex and posi- tion of the Xenos parasites which had protruded their heads between the gastric sclerites of the wasps. A further study of the form and coloration of the hosts was undertaken in the hope of detecting modifications, like those seen by Pérez in stylo- pized Andrenz. My observations, however, gave much less in- teresting results than those obtained by the French naturalist, and | therefore refrained from publishing them and awaited an opportunity to continue them on additional material. This opportunity, however, has not presented itself, so that I have de- cided to give my observations for what they are worth, in the hope that they may be amplified by some other more fortunate ob- server. My preserved Xenos material was turned over partly to Miss Enteman, who published a short paper on the genital ducts of the females (’99), and partly to Mr. C. T. Brues who published a brief account of the embryology of the parasite (’03). The table on the page opposite contains the results of counting the sexes of both host and parasite on the different dates of collecting. From this table the following conclusions, valid only, of course, for the particular summer and locality in which the insects were collected, may be drawn: 1. Of the total number (1000) of Polistes metricus, 251 or fully 25 per cent were stylopized. ‘This is a high percentage, though as will be shown, it has been exceeded in the statistics of other observers. It may be regarded as too great, first because the parasitized individuals, being more sluggish, would be more easily caught, and second, because my interest in such specimens would lead me to exercise greater care in capturing them. I[ would say, however, in answer to such objections, that | attempted to collect the wasps at random without noticing whether they bore parasites or not, that a long handled net was sede In captur- ing them, and that the table contains only specimens in which Xenos hed already protruded their heads between the gastric segments of the wasps. A number of apparently unifested wasps were dissected and were found to contain larval parasites, so that the actual percentage of parasitism was even greater than that indi- cated in the table. Effects of Castration in Insects 379 6 cS a o s s z 2 3 rad re oS a o “4 S eI 8 Fe S 7s ‘6 Ss 3 a as moa ce eee tel a ee | Bl eau gee los 6. | ye 3 os 3 aw om © Se 2 es) = n es 5 my 5 a 2 tea ee hee res | eo ees | Beene si | Bs 20 rede cl oS is it ts I 7 Ss i = i) A Se | oe Aad at as an 3 | 3 5 - i) ee a eH Z August I 14 60 4 56 3 ° 33 85 Gi 14 2 16 72 3 69 3 ° 3 67 58 9 3 19 31 5 26 14 z 12 55 49 6 4 20 108 5 103 43 3 40 89 73 16 5 21 73 6 67 18 ° 18 36 24 12 6 22 143 6 137 12 z 9 19 10 9 7 23 66 15 51 20 2 18 50 36 14 8 24 137 36 101 21 5 16 40 B2 8 9 27 167 50 | 117 29 8 21 55 34 21 10 29 143 7 136 30 2 28 66 56 10 | = = pe | Totals 1000 | 137 863 251 | 2235 226 562 443 11g Aver. | and per 100 13-7 86.3 | PRI Ppl 22.6 56.2 44.3 11.9 cent | | { 2. [The number of male Polistes increased very suddenly Aug- ust 23 to 27 and then fell off still more abruptly. Apparently these collections were made at the time of the emergence of the male brood for the particular locality. ee dlne aes: difference in the ratio of male to female Polis- tes (1 : 6.3) is to be accounted for partly by this temporary appearance of the males and partly, perhaps, by the fact that this sex is much more wary and therefore more difficult to capture than the females. 4. While the total number of females examined was somewhat more than six times as great as that of the males, the number of females stylopized was fully nine times as great as that of the stylopized males. As the male brood of the Wasp appears late in the season this may be due to a partial immunity of this sex from the attacks of the parasites, since Brues (’05) ) has shown that the triungulin Xenos must enter the wasp larve in the spring or early summer (de infra, p. 393.) 380 William Morton Wheeler 5. The table shows that the sexual ratio of the Xenos (3. 7 males to 1 female) was almost the reverse of that of the sexual ratio of the Polistes. ‘That the male parasites should be nearly four times as numerous as the females is easily explained, however, from the fact that the males are so much smaller than the females that more of them can develop to maturity in a single host. In addition to these more general conclusion, a number of more Fig. 1. Specimens of Polistes metrica heavily parasitized by Xenos (Acroschismus) wheeleri. special deductions may be mentioned, based on the daily tables which are too long and complicated to be inserted here: 1. The number of Xenos in a single Polistes varied from I to it. The latter number was taken from only three wasps and these were all females. Ten Xenos were taken from a single individual, also a female, and as a rule the higher numbers, 1.¢., 5 to 9 were all taken from wasps of this sex, but one male contained 8 of the parasites. In the great majority of infested specimens only one Effects of Castration in Insects 381 or two Xenos were present. The table shows that the average number in ali the infested wasps was about 2.4. These numbers probably represent the few survivers of an originally much greater number which had lived as larvze in the individual larval w asps. Brues (’03) took as many as 31 larve of X. pallidus of both sexes from a single larva of the Texan P. annularis! 2. Both sexes of the Xenos may occur in the same Polistes, but when the number exceeds 4, the Xenos are all males. In only one case did | find as many as 3 female Xenos in the same host; in all other cases there were only one or two. In 45 of the 251 infested Polistes, or in nearly 18 per cent, Xenos of both sexes occurred. Hence while there is undoubtedly a tendency, as Brues has observ- ed (03), for the sexes to be the same in the same host, this 1s so far from being a general rule, that the sex of the parasite cannot be supposed to be determined by its host. 3. When more than one female Xenos is present in the same Polistes, they are of the same size but each is smaller than the females occurring singly in a wasp. 4. When both sexes inhabit the same Polistes the heads of the females protrude between the more posterior segments, whereas the cephalic ends of the male puparia may protrude between any of the segments behind the first. The heads of the females there- fore usually appear from under the posterior edges of the fourth or fifth abdominal segments. ‘This is obviously an adaptation to the greater length of the female parasite, which has to lie stretched out in the abdomen of its host and could not protrude its head between the more anterior segments without bending its body. Sometimes both sexes protrude their heads side by side ein under the tergite or sternite of the same segment. Sometimes one sex is on the dorsal, the other on the ventral side of the same wasp, but protruding from the same segment. 5. When the female Xenos protrudes its head between two ter- gites, it les with its ventral surface uppermost, 7.¢., its dorso-ven- tral orientation 1s the reverse of that of its host: when it protrudes its head between two sternites, it lies with its ventral surface down- ward, 7.e., with the same dorso-ventral orientation as the wasp. This is obviously an adaptation to copulation with the winged 382 William Morton Wheeler male, for the latter must have to insert its penis along the ventral surface of the head of the female and immediately under the over- lapping sternite or tergite of the host. That several of the conclusions drawn from the table on page 379 cannot have general validity is shown by comparing them with the statistics of other observers. Horne (’72) says that the speci- mens of Polistes hebraus which he observed in India were ‘“‘ex- tremely troubled with Stylops (Xenos), every fifth or sixth one taken having a female of one under one of the segments of the abdo- men.” Theobald (’92) found that among 180 Andrena lapponica taken in England during 1887, 105 or 58 percent contained Stylops; of 60 bees of the same species, taken in 1888, 54 or go per cent were badly stylopized. He believes that the female Andrenz are more afflicted with the parasites than the males, and he records the num- ber of Stylops found in the 54 bees taken during 1888 as comprising 33 females and 21 males; 2 females each contained 2, 3 males con- tained 2, 25 females and 18 males 1 each. The corresponding numbers for 40 stylopized specimens of Andrena nigroznea were 3 females each with 3 Stylops, 1 male with 3, 3 females with 2, 5 males with 2, 16 females with 1 and 12 males with 1, making 22 females and 18 males. On the basis of these figures Theobald differs from Perkins (’92), who found the males of various Andrenz and Halicti more frequently stylopized than the females. This author says that he has seen hundreds of stylopized male Halictus tumulorum, but has never seen a female in this condition. Although Theobald’s conclusions agree with my own, his data do not furnish very strong support in favor of his contention, since in A. lapponica the ratio of parasitized males to females is 1: 1.5 and in A. nigrozenea only 1:1.2. Skinner (’03) counted 34 stylopized individuals among 140 Polistes texanus, which he found at Pecos, Texas. He says that “most of the Xenos appeared to be females and only 4 males were secured.” The percentage in this case is very similar to that which I found in P. metricus. Brues (’05) has published some statistics on-two colonies of the Texan P. annularis infested with Xenos nigrescens Brues and X. pallidus Brues. In these cases the amount of para- Effects of Castration in Insects 383 sitization was very great. In one nest there were 86 wasps, 44 or 51 percent of w hich contained X. nigrescens. There were from one to seven in each wasp (anaverage of 2.6 per host), and of the total number of Xenos (94), 91 were males and only 3 females. In the other nest there were 42 wasps, and 36 or more than 85 per cent were stylopized. The total number of the parasites—in this case X. pallidus —was 125 (81 males and 44 females); the highest num- ber in a single wasp being 10, the average per host 3.6. Fuller consideration must be given to the effects of the stylopids on their hosts. This may properly begin with a résumé of the excellent work of Pérez (1886) who examined stylopized speci- mens of 47 species of Andrena. The effects produced by Stylops in these bees is so considerable as to render their specific deter- mination difficult. This is not surprising perhaps, when we con- sider the vast number of closely related species in the genus. All the known specimens of certain “species”? (F. Smith’s Andrena insolita, separata and victima) have been found to be stylopized, which gives force to Pérez’s opinion that these are not true species but merely parasitized individuals of forms that are already known under other specific names. Pérez describes minutely the fol- lowing modifications as characteristic of stylopized ‘Andrenz: (1) The abdomen is shortened and swollen and therefore more globular, the shortening being due to an attenuation of the termi- nal segments. (2) The head is usually smaller than that of nor- mal specimens. (3) The villosity of the abdomen is more abundant, longer and more silky, especially on the terminal seg- ments, and its color is often greatly altered, becoming lighter and more reddish or fulvous. The villosity of the thorax may undergo similar but less pronounced changes. (4) The puncta- tion of the body becomes finer, denser and more superficial in correlation with the pilosity, which arises from the punctures. These changes are common to both sexes and therefore affect specific characters. They give the specimens a peculiar pseudo- specific facies. Pérez therefore rightly warns against basing new species of Andrena on stylopized individuals. The following changes affect the secondary sexual characters: (1) The normal males of the genus Andrena, as in many other 384 William Morton Wheeler genera of bees, have a greater amount of yellow or white on the face or clypeus or on both than the cospecific females. Stylopiza- tion tends to diminish this hl zht color yery perceptibly and hence to make the face of the male resemble that of the female. In the female the parasites produce the reverse effect, making the face resemble that of the male. “It is dificult to find a stylopized male of A. labialis, ¢.g., whose face 1s normally colored and, on the other hand, it 1s quite as rare to find a stylopized female of this species having the face entirely black.” (2) The normal fe- male Andrena differs from the normal male in the structure of its hind legs, the tibiae of which are modified for collecting pollen. They are always robust and incrassated and have a brush of long, curved hairs, especially on their internal surfaces. Similar hairs are found also on the femora, coxee and metapleura. “The metatar- sal joint of the hind legs is also kilated or enlarged and is furnished with rows of stiff hairs on its lower surface. In the male the hind tibize and metatarsi are slender and bear only short, sparse, straight hairs and this 1s true also of the coxze and metapleure. The pres- ence of Stylops in che abdomen of the female diminishes the de- velopment of the pollen-collecting apparatus to such a degree that the hind legs become like those of the male. The reverse occurs 1n eelupiced males, the organs under consideration be- coming more enlarged and approxima ting to the female type in their lect aL Ka modifications in this sex, however, are rarer than in the female and in ae sexes they vary greatly in different stylopized individuals. (3) The frontal furrow near the internal orbit of the eyes, which s ae with velvety pubescence, 1 is well- developed in the normal female, but feeble or absent in the normal male. In stylopized Andrenz this furrow may undergo diminu- tion of development in the female and becomes accentuated in the male. (4) Although the female Andrena has 12-jointed, the male 13-jointed antennz, there is no modificationof the numberof joint in parasitized individuals. “The antennz of the normal sexes may differ in the length of the second funicular joint. In one species, A. Trimmeriana, the second funicular of the normal female is as long as the two succeeding joints taken together, whereas in. the normal male this joint is at most half as long as the succeeding Effects of Castration in Insects 385 joint. In the stylopized male of this species Pérez found the sec- ond funicular attaining to two-thirds the length of the third joint and to this extent approximating to the conditions in the female. (5) The normal female Andrena bears a fringe o long hairs, the ana fimbria, on the edge 0 the fifth adbominal sternite, but tn s fring: is lacking in th normal male. Stylopization tends to sup- press the development of the fimbria or causes it to disappear com- a in iS female and more rarely has the reverse effect on the male. ) The sting, which is peculiar to the female, is reduced in size In ee eed individuals, the copulatory organ of the male is also reduced in length and becomes narrower and less curved, while the paramera tend to become atrophied. Pérez concludes from these observations that, so far as the secondary sexual characters of Andrena are concerned, the modif- cations induced by the Stylops are not merely attenuations, but actual inversions of development. “The stylopized Andrena, male or female, is not merely a diminished male or female; it 1s a female which takes on male attributes; a male that takes on the characters of the female.” The intimate correlation which exists between the structure and instincts of all organisms, leads one to look for instinct peculiarities corresponding with the morphological inversions described above. Pérez found only one stylopized female Andrena which had its hind legs charged with pollen, and he therefore concludes that the stylopized bees rarely or never forage or build nests like the normal females. Normal and peace bees of both sexes, however, visit flowers as this is not a unisexual instinct, and hence the triungulins produced by the Stylops have an opportunity to move off onto the plants, climb onto normal foraging bees and thus get transferred to the brood in incipient nests. In this way the perpetuation of the parasites is insured through a line of bees capable of nourishing them. The internal changes due to stylopization have been studied by Newport (48), Ben and Perkins (g2). All of these authors find that the testes and ovaries are not destroyed by the parasite but are more or less reduced in size, in the male sometimes only on the side of the body bearing the Stylops. Inthe female the oécytes 356 William Morton Wheeler or ova degenerate in their follicles and are evidently quite incap- able of Ae dlejansins in the male there may be ripe spermatozoa in at least one of the testes. Perkins found motile spermatozoa in all the stylopized males which he dissected, and Pérez mentions a male of Andrena decipiens taken 1m copula, so that this sex may retain, at least occasionally, not only the normal mating instincts, but the ability to fecundate normal females. The parasites before maturity live on the fat-body and blood-tissue of ae hosts and do not attack the other organs directly. These undergo partial atrophy through lack of nutrition. Observations similar to those of Pérez fave been published by Saunders (’82) and Schmiede- knecht (83). Turning now to Polistes, we find that in this genus the secondary sexual characters are in certain respects quite as clearly developed as in the andrenine bees, but as wasps do not collect pollen, the hind legs show no special modifications in the female. The fol- lowing are the main external sexual differences observable in Polistes metricus: “The male has a slender thorax and long, nar- row abdomen. The antenne are 13-jointed, with a long, slender funiculus, not enlarging towards its tip; the second funicular joint is little if any longer than the two succeeding joints taken together. ‘The face is long and narrow, with a pair of longitudinal grooves running from the antennal insertions to the clypeus and separated by a prominent longitudinal welt or elevation. The clypeus is flat or even slightly concave and its surface is impunc- tate. The whole face and clypeus, the anterior surface of the anten- nz to within a few joints of the tip of the funiculus, the anterior surface of the coxa, femora and tibia, a series of transverse bands or spots on the abdominal sternites behind as well as in- cluding the first segment, are sulphur yellow. The two large ferru- ginous spots on the first abdominal segment are usually well- developed. In the female the thorax is proportionally stouter and the ? Though the publications of these authors antedate the article above reviewed, we are not to infer that this implies priority of discovery. Pérez says that he originally called the attention of these in- vestigators to the facts and had himself published a preliminary account of his researches as early as 1880 in the Revue Internationale des Sciences, Tome I. E ffects of Castration in Insects 387 abdomen is decidedly shorter. The antennz are 12-jointed, with a shorter funiculus slightly enlarging towards its tip; the second funicular joint is nearly as long as the three succeeding joints taken together. ‘The face is decidedly shorter than that of the male, the grooves and welt much less pronounced and the clypeus is convex and coarsely punctate. The face is black, with the internal orbit and sometimes portions of the clypeus, the anterior surface ~ of the scape and of the two first funicular joints, the anterior surfaces of the tibia and apical portions of the femora, ferruginous. The sulphur yellow is restricted to the tarsi and the posterior border of the first abdominal tergite, and the ferruginous spots on the first abdominal segment are obscure or wanting. The wings are often somewhat more deeply infuscated than in the male. In stylopized Polistes metricus of either sex I fail to find any modifications of a morphological character which could be definitely attributed to the presence of the parasites. A few of the more heavily stylopized females were abnormally small, but with these exceptions, all the wasps were of normal stature. No modifications of the antennz nor of the structure and proportions of the face could be detected. A study of the coloration, however, yielded more positive results, but even here, owing to the great range of color variation to which P. metricus likew all our other species of the genus, is subject, the results are not capable of very precise formulation. In the coloration of the face stylopized males show no tendency to approach the female. In 14 out of 25 heavily stylopized females I find the clypeus of the usual black or dark brown color; in the remaining I1 it is more or less ferru- ginous or yellow. Some specimens have the free border of this eee sulphur yellow or its whole surface ferruginous, or only its posterior border or sides of this color. One specimen has the clypeus ferruginous with a small black spot in the center. It would be possible to regard these cases as approximations to the male type of coloration due to parasitism, were it not that per- fectly normal, unstylopized females not infrequently exhibit the same erythrism of the clypeus. I have not seen a sufficient num- ber of P. metricus from different localities to be able to determine JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 8, NO. 4 388 William Morton Wheeler whether the percentage of this modification is so much greater among stylopized than among unstylopized individuals as to show that it must be attributable to the influence of the parasites. | am inclined to believe, however, that it is part of a more general erythrism which affects also the abdomen of many parasitized i in- dividuals. This region, to a varying degree in such specimens, but undoubtedly to a greater degree i in those that are most heavily sty lopized, takes on in both sexes alike a distinct ferruginous tinge which is usually most pronounced towards the posterior borders of the tergites and sternites. Sometimes it may be very strongly developed as in one rather small female taken August 29, and bearing three male Xenos. In this case the second gastric segment is entirely ferruginous, with the exception of a black anteromedian triangle, and the posterior half of each of the remaining segments and the whole clypeus, except its anterolateral corners, are rich ferruginous. [| have failed to notice in the legs, wings and antenne of either sex in stylopized specimens any color modifications that could not be regarded as falling within the wide limits of normal specific its. The color modification here described is not confined to styl- opized specimens of P. metricus. It has also been observed by Brues (’03) in two of the Texan species, Ps, Tubiginosus and annu- laris. “The stylopized Polistes,” he says, “can he recognized even before the heads of the pupa cases begin to appear between the sclerites of the abdomen, by their males color. They seem never to become as darkly colored as normal specimens. [his lighter color of parasitized specimens seems to apply only to the ongi- nally dark species, in P. rubiginosus there seems to be but slightly if any lighter coloration. In the specimens of P. annularis from which I raised Xenos, all of them females, the faded appearance is especially noticeable upon the dorsum of the abdomen. The first abdominal which is normally piceous with a narrow apical yellow band 1s in this case almost entirely bright ferruginous, or is ferruginous with the border yellow. The rene of the abdo- men is rormally piceous, but the posterior margins of the seg- ments, especially the second and third tend to become more or less broadly dull ferruginous in stylopized specimens.’ Effects of Castration in Insects 389 There is also a modification of behavior in stylopized Polistes. Several observers have noticed that such individuals are more sluggish, that they fly about less actively, and Brues (’03) has found that they are less inclined to use their sting, probably because the voluminous parasites interfere with the exsertion of this organ. A similar inability is observed in queen honey-bees with ripe ovaries and in worker honey-bees with their crops full of honey. The peculiarities of behavior in stylopized wasps are such as would be expected in parasitized organisms for these al- most invariably exhibit a general reduction of vitality due to malnutrition. Fig. 2, Abnormal abdomens of Polistes metrica; d and B, dorsal; C, ventral; D, lateral view. Among the unstylopized female Polistes taken at Colebrook there were three specimens with abnormal abdomens. Sketches of these are shown in Fig. 2. The segments in some cases were partially divided on either the right or left side, and in one case there were several supernumerary sclerites. It might be inferred that these abnormalities were the result of stylopization, for although no Xenos were found in the specimens, these parasites may have been present in the larve from which the anomalous individuals developed. I doubt this, however. At any rate, the anomaly in question is not peculiar to wasps that are subject to 390 William Morton Wheeler stylopization or indeed to insects. Janet (’03) describes and figures a very similar abnormality in Vespa rufa, an insect that is not afflicted with Stylops or Xenos, and Cori and Morgan (’92) show that similar abnormalities are not uncommon in earthworms and cestodes. In the case of Polistes the abnormality must be produced either in the early embryonic stages while the metameres are forming or at the time of the formation of the abdominal sclerites in the pupa. We may conclude, therefore, that Xenos produces no modih- cations of the secondary sexual characters of its Polistes host com- parable to those produced by Stylops in the bees of the genus Andrena, but merely a tendency to a reddish coloration of the abdomen and face, a tendency which, so far as the abdomen 1s concerned, 15 manifested equally by both sexes. This general lightening of color in stylopized Polistes and its reddish tinge remind one at once of the similar changes observed by Pérez in Andrenz, although in the latter insects it seems to be confinea to the pilosity. Pierce (’0g, p. 32), cites the following observations, which show that a similar change of color was long ago observed by Saunders in stylopized bees of the genera Pros- opis and Hyleus: ‘Prosopis gibba occasionally exhibits irregular rufous patches on the abdomens of affected individuals (Saunders, *50). Prosopis rubicola exhibits color changes regularly. The nymphs of those Hylai which are likely to produce the pale-colored specimiens (H. versicolor), which prove, as anticipated, to be only avariety of the H. rubicola consequent upon parasitic absorption, may usually be identified within one or two days of their final metamorphosis by assuming a yellow tinge, and may be set apart as certain to produce male parasites. (Saunders ’52.)” It is not easy to account for this modification. Brues 1s inclined to believe that “‘ tke reason that the reddish Polistes are not affected, is that red is a more primitive color than piceous and that the color simply becomes arrested at this stage and does not tend to become so before the red stage.”” The question of the developmentof varia- tions of color in the species of Polistes 1s a very complicated one, as Miss Enteman (’04) has shown, and a number of possible explanations of the erythrism of stylopized individuals might be ~ Effects of Castration in Insects 391 suggested. The ontogenetic explanation suggested by Brues is one of these, implying that a red stage precedes the brown or black of the mature form of dark species like P. metricus. This is borne out by the development of the color pattern in such species. On this view stylopization inhibits color development in an ontogenetically and presumably therefore in what corresponds to a phylogenetically earlier stage. A second explanation is, how- ever, suggested by Miss Enteman’s studies. ‘These tend to show that the dark-colored races or species of Polistes are due to cold and moisture, the lighter yellow and red forms to heat and aridity. This seems to be clearly indicated in the distribution of the spe- cies, e.g., in such extreme forms as the yellow P. texanus and the black canadensis. It is possible, therefore, that the erythrism of stvlopized P. metricus, which in normal coloration 1s closely related to P. canadensis, is due to withdrawal of water from the tissues by the developing parasites. ‘his does not contradict the ontogenetic and phylogenetic explanations but supplements them, if we suppose that the primitive yellow or red color cannot pass on to the piceou’ or black stage unless the tissues contain a sufh- cient amount of water. Miss Enteman has shown that the piceous or black color is in the form of pigment granules in the chitinous cuticle of the wasp’s integument, whereas the yellow is deposited in the hypodermis. Erythrism is probably due, therefore, to a diminution in the cuticular pigment which permits the yellow hy- podermal pigment to shine through. As both kinds of pigment are the result of metabolism in the pupa, we can see how a disturb- ance of metabolism either through withdrawal of water by the para- sites or through other causes might lead to the deposition of a smaller amount of the black pigment and hence to erythrism. It is more difficult to account for the absence of all modifications of the secondary sexual characters in stylopized Polistes, when such modifications are so evident in Andrena. We may, perhaps, account for this difference on one of the following hy potheses: . As will be shown in the sequel, complete extirpation of the ee in young larval insects, has produced in the few species on which it has been performed, no appreciable effects on the de- velopment of the secondary sexual characters. This indicates that 392 William Morton Wheeler these characters may be so fixed and so nearly independent of the gonads, except, perhaps, in the very earliest larval or late em- bryonic stages, as to remain quite unaffected in their development after the gonads have been completely removed. ‘The degree of this independence may be supposed to differ in different insects and even in different individuals of the same species. It may be slight or almost absent in Andrena and very well marked in Polis- tes and this may account for the differences between the stylo- pized specimens in the two genera. The difference in the manifestation of changes in the second- ary sexual characters may, however, be due to ethological differ- ences between the two genera. Andrena has only male and female forms and both under normal conditions are adequately fed in their larval stages. In Polistes the larvz of the earlier broods in the annual series, as Marchal has shown (’96, ’97) are poorly fed and as a result become sterile females, or workers. As imagines they maintain themselves in a sterile condition by appropriating very little of the food they collect to their own use, since they at or.ce lavish it in feeding the succeeding broods. Hence the females of these earlier Beeade become Celok in the first place through alimentary castration of the larva from which they develop, ae in the second place, maintain themselves in this condition as adults through the nursing or nutricial function (nutricial castra- tion). “These peculiar phenomena will be more fully discussed in the second part of this paper. Owing to these two formsof physio- logical castration inhibition of the development of the reproduc- tive organs 1s a common and normal occurrence in Polistes females, and he parasitic castration induced by Xenos would not be ex- pected to produce somatic changes of such magnitude or of such a nature as Pérez has observed in Andrena, all the females of which are normally fertile mothers. In other words, the effects of the Xenos on their hosts is of the same nature as the alimentary castration to which all the earlier broods during the seasonal development of the Polistes colony are normally subjected, and this probably accounts for the absence of any specific effects on stature and structure and the evident ease with which the volu- minous parasites are borne and tolerated. Effects of Castration in Insects 393 In the case of the male Polistes the matter is not so readily explained, since this sex 1s not subjected to the two forms of nor- mal physiological castration just mentioned. But it should be noted that the effects of stylopization on the secondary sexual charactersof the male even inAndrena are rarer than in the female (wide, p. 384), owing to the fact that castration 1s much less complete in this sex, as both Perez and Perkins have shown. This is, no doubt, also the case in Polistes, for the development of the testes requires much less food than does that of the ovaries, and the presence of the Xenos probably, therefore, has much less effect on this sex. It has long been known that male puparia and adult female Xenos are found only in the late summer or fall brood of Polistes in the brood, namely, which consists of fertile females and males that are to mate and provide, after hibernation of individuals of the former sex, for the formation of new colonies during the en- suing spring. Brues (’05) captured on May 22 a large over-win tered female of P. rubiginosus containing a female Xenos ni grescens that gave birth to a lot of triungulin larve. Evidently, ie lore, the larvae of the wasp must be infested with triun- gulins in the spring, soon after the colony is founded. How come it then, we are led to ask, that the adult Xenos appear only ir wasps belonging to the last or autumn broods? If these wasp: really belong to so late a brood they could not become infested unless we suppose that the triungulins hang about the wasps nest for a long period before entering the larvae. As this assump- tion is very improbable, we seem to be forced to the conclusion that the wasps that bear the Xenos in the late summer really belong to early broods which have been greatly retarded in their larval and pupal development. Dodd (’06) and Howard (’o8) have published some interesting ne ated which show that the larvze of other insects (Lepidoptera, Formicide) parasitized by chaleidids are greatly retarded in their growth and development. If this occurs also in Polistes larvz infested with Xenos,as seems probable, we may be able to account for the facts and understand how the single generation of Xenos manages to survive till the following spring to insure the perpetuation of the race in healthy, 304 William Morton Wheeler Incipient colonies of the wasps. ‘The triungulins are, in all probability, carried to these colonies by hestihe wasps from the flowers onto which they crawl from their mothers after hibernat- ing in their hosts. Since the foregoing paragraphs were written Pierce’s fine monograph of the Strepsiptera has appeared (’og). This work contains such a full summary of all that has been published on this remarkable group of insects, together with so much new matter, that | should have thought it unnecessary to publish the preceding pages, but for the on that they were written for the purpose of elucidating a problem which Pierce treats only incidentally. Of the many interesting facts contained in his paper I shall cite only a few which have an immediate bearing on the matters considered above. The fullest statistics given by Pierce relate to two large colonies of Polistes annularis infested with Xenos pallidus. pes colonies, which were collected at Rosser, Texas, September 23, together contained 1553 wasps, 1311 males and 242 females. Of these 266, or 17.1 per cent were stylopized, 259 being males and only 7 females. The highest number of Xenos observed in a single wasp was 15, and this occurred in a male specimen! Pierce also cites some statistics published by Austin (1882) on 50 Polistes metricus collected at Readville, near Boston, Mass., August 20, 1879. Of these wasps, 14 of which were males and 36 females, g or 18 per cent were stylopized (2 males and 7 females). Pierce figures the abdomen of a male wasp (Leionotus (Odynerus) annulatus Say) which has the sclerites much distorted as in the P. metricus shown in Fig. 2. Concerning his specimen, which contained a female Leionotoxenus hookeri Pierce, he says: “It seems that in pushing itself out between the segments the parasite completely split the dorsal tergites of segments three, four and five and split segment two half way to the base. The parasite was located behind segment three.’ He cites the observations of Pérez on the effects of stylopization in Andrena and adds the following modifications observed by Crawford in specimens of Andrena crawfordi infested with Stylops crawfordi: Puncturation of abdomen less strong, punctures finer and sparser;,especially noted on second segment. Effects of Castration in Insects 395 “2. In females with male parasites the basal joint of the hind tarsi is narrower, approaching the shape of the corresponding joint of the male tarsi; this joint not noticeably narrowed in female with female parasites. “3. Scopa of parasitized female thinner, plumosity shorter, not so silky. “4. Out of six males with male parasites two show the second transverse cubital gone in both wings; one has stubs at each end, however, in right wing; one has the transverse cubital slightly interrupted in both wings. Out of about 110 nonparasitized males none show any variation. “5. Out of 38 females with male parasites one has the left wing with three submarginals, the right wing with two submar- ginals; one has two submarginals in both wings but right wing with a stub of the nervure; one has first transverse cubital of the left wing one-half gone; forty-five nonparasitized females show no variation. “None of the other salient alterations found by Pérez could be expected in this species because of the close resemblance of the two sexes. Andrena crawfordi is a very generalized bee.” Pierce also calls attention to a single ecieed specimen of A. advarians in his collection, with a spurious nervure in the third discoidal cell, and believes that parasitism may affect the trachea- tion of the wings, a modification not observed byeletes. II GENERAL CONSIDERATIONS. By employing the word “castration” in a broad sense to mean any process that interferes with or inhibits the production of ripe ova or ripe spermatozoa in the gonads of an organism, and not merely in the concise original meaning as the sudden and complete extirpation of the gonads, we are ee to bring to- gether a number of interesting but hitherto rather scattered facts which have a bearing on ne correlation of the primary and secondary sexual characters. An adequate consideration of these facts would go a long way, I believe, towards preparing us for a profitable study of ie recondite problem of sex determination. 396 William Morton Wheeler Owing to the limits of this paper and to the fact that the depend- ence of the secondary on the primary sexual characters in verte- brates has been recently analyzed by several authors, notably by Herbst (o1) and Cunningham (08), I shall confine oy remarks very largely to the arthropods. ‘Taking the word “castration” in ae broad sense suggested above, we may distinguish: . Surgical, or true castration, 1. e., the sudden and complete eee ioe ro eae or female gonads, so that the organism is deprived of its primary sexual characters, if we do not include in this term also the gonad-ducts and copulatory organs. ‘This operation is of the greatest experimental significance, since, when performed at the proper ontogenetic stage, it has been shown to lead in many animals to interesting modifications of the secondary characters of each sex. 2. Physiological castration. Under this head may be included at least three different forms of inhibition in the development of the gonads, leading to a failure of the individual to develop its primary sexual eet. or, in other words, to an inability to function as a male or a female. This inhibition is brought about by an insufhcient supply of nutriment and appears as the result of a well-known law, according to which the organism provides in the first instance for the growth and differentiation of its soma and develops its gonads on the nourishment in excess of that required for normal growth in stature and the complete differ- entiation of the various tissues. The following three forms of physiological castration may be distinguished: A. ite atar, castration. This term was originally given by Emery (’96) to the suppression of gonadic deqelapmcat through insufficient feeding of the organism ones ¢ its larval life. B. Nutricial castration. This term was first used by Marchal (97) to designate the maintainance of the gonadsin an undeveloped condition in the adult, owing to the latter’s devoting itself to nursing the brood of other fertile individuals instead of itself taking on the reproductive function. C. Phasic castration. I use this term, for lack of a better, to include all the cases in which the gonads are inhibited in their development by seasonal or ontogenetic (growth) conditions. This Effects of Castration in Insects 397 form of castration is not sharply marked off from the two preceding but may be made to include them, since both alimentary and nutricial castration can be suspended during the life time of the individual and normal reproduction supervene. 3. Parasitic castration. ‘This term was first introduced by Giard (87, etc.) in a series of studies on crustacea. It refers to the suppression or destruction of the gonads by parasites. By, enlarging the scope of Giard’s decnaen we can distinguish two Pie: we parasitic castration: A. Individual parasitic castration, which 1s induced in certain organisms when they contain parasites, and B. Social parasitic castration, which occurs in ants when one colony in becoming parasitic on a colony of a different species eliminates the sexual individuals of its host. A number of illustrations will bring out the fundamental resemblances between these different methods of suppressing the reproductive function and the resulting modifications of the somatic characters of the individual or of their equivalents in animal societies. I. Surgical castration. The pronounced modifications of the secondary sexual charac- ters observed in vertebrates, especially in birds and mammals, from which the gonads have been removed during early life, or in which these organs have become diseased, have led some inves- tigators to look for corresponding modifications in the secondary sexual characters of insects subjected toa similar operation. One observer, Hegner (’08), has succeeded in castrating the embryos of a chrysomelid aca (Calligrapha multipunctata) by removing the very young sex-cells as soon as they are segregated in the protoplasmic accumulation at the posterior pole of the egg during the formation of the blastoderm. Although Hegner’s experiment, which consisted in pricking the chorion at die pos- terior pole and allowing the sex-cells to flow out, was successful to the extent of demonstrating that the embryo may continue its development after the operation, nothing but a few young larvee 398 William Morton Wheeler were obtained. he experiment therefore, throws no light on the question with which we are here concerned. Much more important are the results of experiments per- formed by Oudemans, Kellogg, Meisenheimer and Regen in cas- trating larve. Oudemans (’99) was the first to attempt surgical castration in insects. He removed one or both gonads from male and female caterpillars of the gypsy moth (Ocneria dispar) before the last and second last moults. About one-third of the caterpillars (30 out of 86) survived the operation and produced moths. From a study of these, the Dutch investigator concluded that castration has no influence, either on.the external appearance, 7.¢., on the secondary sexual characters, or on the behavior of the moths, since the castrated males copulated, though they had no sperma- tozoa, and the females, though they had no eggs, nevertheless stripped from their abdomens the mass of long hairs in which they normally oviposit. Females castrated only on one side laid eggs, and three normal females that copulated with castrated males, laid eggs which developed parthenogenetically. Kellogg (04) succeeded in castrating silk-worm caterpillars (Bombyx mori) after the second, third and fourth moults by burn- ing out the gonads with a hot needle. This method was very inferior to that emploved byOudemans. Not only was the mortal- ity of the caterpillars greater, to judge from Kellogg’s remarks, but the complete destruction of the gonads was obviously much less certain. Like Oudemans, he failed to detect any modifica- tions of the secondary characters of either sex in cases in which dissection of the adult moths proved that the gonads had been completely destroyed. More recently Meisenheimer (07) has carried out much more elaborate experiments than either of his predecessors, on about 600 Ocneria dispar caterpillars, of which 186 yielded imagines. The smallest caterpillars castrated were between the second and third moults, and about 3? cm. long, but he also used those be- tween the third and fourth and between the fourth and fifth moults. He was able to remove the gonads even before the second moult but the larva were too delicate to survive the operation. Effects of Castration in Insects 399 Three series of operations were performed: first, the removal of both gonads; second, the removal of the gonaals together with the gonad-ducts; and third, the transplantation. of testes into female and of ovaries into male caterpillars. “The transplantation of ovaries was more easily performed than that of the testes. In these cases the transplanted organs not only developed to their normal size, but the ovaries in some cases even United with the male vasa deferentia. In one case a single transplanted ovary united with one of the vasa deferentia, and as the testes of the opposite side developed, an artificial hermaphrodite Was produced. Meisenheimer describes the results of his operations as follows “Oudemans’ and Kellogg’s experiments established the fact that the removal of the gonads exerts no influence on the secondary sexual characters. My results agree with these to the extent that in my experiments the originally male caterpillar always produced a male moth, the female caterpillar a female moth. The general habitus of the respective sex was always perfectly preserved, both in the form of the body, the structure of the antenne and the color- ation of the wings, and this was true of the operations, both in the case of the castrated mothsand of the artificial hermajyhrodites. But on examining, in a comparative way, the material Gbtained, a certain effect of the operations seems, nevertheless, to be notice- able. The moths subjected to the two kinds of operation may be arranged in series, which in the males vary from dark, to light forms and pass over in the females from a whitish to a darker color.” But, as Meisenheimer observed, there is cor: siderable color variation in both sexes of normal gypsy moths, and this was true also of his control series, though he believed the variations to be greater in those developed from operated caterpiilars. The specimens with transplanted organs, however, showed no greater modification than those of the castrated series. it is especially noteworthy that in the cases of transplantation there was no change in the copulatory or other organs, though these had not yet developed at the time of operating. Herice, although Meisen- heimer made artificial hermaphrodites, he did not succeed in producing artificial gynandromorphs.4 | ’ } 4 Unfortunately I was unable to secure a copy of the first part of Meisenheimer’s final mono- graph (’o9) till after the manuscript of my paper had gone to ress. The review here given of his experiments is, ther:fore, inadequate. 400 It will kL formed onl As such ex] yield differe 10) has rece ; tris >)» In tion experin the insects tion brings the operat were remo) last larval; in the last held and occupying were Mai William Morton Wheeler ed that the preceding experiments were per- lometabolic insects of the order Lepidoptera. ts on ametabolic insects might be expected to Its, it is interesting to record that Regen (og, ‘cceeded in castrating crickets ( (Grylluscampes- paper he gives us Pee more than an orienta- rformed for the sake of determining whether irvive the operation, but his second contribu- nd more satisfactory data. In order to perform ircotized the crickets with CO,. The testes 40 males (20 in the second last. and 20 in the nd the ovaries were removed from 10 females ‘hese 50 individuals were released in the open ned to the burrow which it 1s in the habit of ut its larval life. The operated individuals itting off portions of their wings, and near their bur s were placed with records of the necessary data. 9- ckets had reached maturity Regen recovered g males t! ncastrated in the second last, 13 of those cas- trated arval instar, and 6 females. “The insects were left in s. Ten days later he found that the crickets had ¢) ws and there was a tendency for them to as- socia +h consisting of a male and female occupying a hol i. Several individuals had migrated to other parts ow in which Regen experimented, but he suc- ceede: gand placing in a terrarium 10 males (4 castrated in the and 6 in the last larval instar) and one female On tl ens he made the following observations: ool ginal males, part of which had been castrated during the chirped thic shrill a manp had been castr. rare intervals. “9. The behay and part during the second last larval instar, ut the remainder of their lives in as lively and normal males. Only one of the males, which i the last larval instar, chirped feebly:and at the castrated males towards the females was the same as that ©f normal individuals. They enticed the females with theirs) >! idulation and whena female approached, E fects of Castration in Insects 401 emitted a soft, whirring sound, and tried to afhx their sperma- tophores to her, for “3. The glands which secrete the spermatophore envelopes produced these structures up to within a short time of the death of the crickets and therefore performed their function independ- ently of the testes. In external appearance the spermatophore envelopes of castrated males were in all respects like those of normal males (In some cases they were somewhat smaller), and contained a white secretion, which was less abundant than in normal sperma- tophores. “5. The markings of the anterior wings, or tegmina and the development of the stridulatory organ showed no modifications. “6. The females were unable to distinguish between normal and castrated males. They followed the Sait of the latter, mounted their backs and permitted them, as if they were normal males, to arbe their spermatophore envelopes near the genital orifice. . The castrated female behaved like one that had not’been et She thrust her ovipositor into the earth and made motions like a normal female, so that she had every appearance of desiring to oviposit. As time went on this “ oviposition ‘ became abnormal, as the female kept on thrusting her ovipositor into the earth but only to a slight depth.” Regen assured himself of the completeness of castration in these crickets by dissection and by examination of the spermato- phores, which were found to contain no spermatozoa. He also kept a series of castrated individuals in captivity from the time of operation, and when these reached maturity they were found to behave exactly like the individuals that had been permitted to mature in the field. His experiments, therefore, gave results in complete harmony with those of Oudemans, Kellogg and Meisen- heimer. It must be admitted that his insects were all castrated in rather late stages. He informs us, however, that during the sum- mer of 1909 he successfully castrated a number of much younger larvae, measuring only 5 to 8 mm., and that these had grown to a length of 20 mm., by December 1909 when he wrote his second paper. At that time the females were readily distinguishable 402 William Morton Wheeler from the males by their ovipositors. He intends to remove the spermatophore g elands from someof the malesof this series and also from some uncastrated males and to report on the results in a further publication. 2» Alimentary Castration The best examples of this form of castration are to be found among the social Hymenoptera, 7.e., among the social wasps, bees and ants. In these insects the majority of the female larve in each colony become what are called workers, because they are fed on a limited diet, grow very slowly, pupate more or less prematurely and hence as adults, or imagines are smaller in stat- ure than the normal females of their respective species. These workers are also distinguished by other morphological and etho- logical peculiarities. Owing to their inadequate nourishment as larvee, their ovaries are, as a rule, in a very rudimentary condition. Very striking examples of this alimentary castration are seen in the incipient colonies of ants, while the mother queen is bringing up her first diminutive brood of workers, in the species of Carebara, the queens of which are more than 1000 times as large as their sterile offspring, and in Pheidologeton, in which there is nearly as great a difference beween the stature of the queen and that of the smallest workers. In bumblebees, honey-bees, social wasps and most ants this difference is less pronounced, but it is never- theless perceptible and clearly traceable to larval starvation. Opinions differ as to whether the other characters peculiar to the worker forms of these insects are the result of underfeeding, but itis evident that none of these can be regarded as an approach to the male type of structure. In other words, notwithstanding the very decided inhibitory effect of larval starvation on the develop- ment of the ovaries in the adult workers of the social Hymenop- tera, the soma does not tend to become like that of the male, but merely departs to a greater or less degree from that of the female type. This departure 1 is usually in the direction of greater simpli- fication and is most pronounced in the ants, the GEER of which are wingless, have a smaller and much simpler thorax and smaller eyes and ocelli. Effects of Castration in Insects 403 The social Hymenoptera, however, are not the only insects which practice alimentary castration. A very interesting case is also seen in certain aphids of the genus ee e.g., in the Ph. carye-fallax recently studied ay Morgan (’o9). The stem- mother, or fundatrix of this insect makes and inhabits a hollow gall on hickory leaves. She lays numerous eggs which may give rise to two kinds of offspring. The eggs first deposited produce individuals that grow up to form the wingless sexupare, (Fig. 34), Se, a J > 7 NN fe ‘a Fig. 3. Large wingless female of Phylloxera carye-fallax; B and C, dwarf females of same, drawn to same scale as A. (After T. H. Morgan.) while the eggs laid later give rise abruptly to very small females, (Figs. 3B and C), which Morgan calls ‘ “supernumerary or dwarf females.” These he describes as follows: “In the larger galls as many as 46 eggs may produce the large individuals, ad then the smaller series abruptly begins; while in the smallest galls only one to three or four or more large individuals are produced when the smallseriesbegins. ‘There seems to be here not a prede- termined number of large and dwarf females, but the conditions JOURNAL. OF EXPERIMENTAL ZOOLOGY, VOL. 8, NO. 4. 404 William Morton Wheeler of life determine when the one kind ceases to be produced and the other begins. ‘The two types of individuals must, however, be Vaetsanel by alternative possibilities possessed by each egg. The supernumerary or dwarf females differ from their large wingless sister-forms, and from the young of the latter in a num- ber of points. The shape of the body is entirely different and resembles that of the sexual male; but it differs from the male in two important respects; first, the dwarf individuals have a very long proboscis which in this species is absent in the male; second, there are no testes within the abdomen as in the males, where they form a relatively enormous mass. Otherwise the dwarfs are so similar in external form to the sexual males that their true nature was uncertain until they were studied in serial sections. These showed the absence of the testes and the presence of rudimentary ovaries and ducts resembling those of immature parthenogenetic females. There was nothing to indicate that the dwarfs could become sexual females. In fact the latter con- tain each an enormous egg when they hatch.”” Morgan believes that the dwarf females “‘are destined to a brief existence, and die without progeny," and he gives good reasons for supposing that they owe their origin to eadeant He feeding of their parents. In other words, we bane here a case of alimentary castration differing from that of the social Hymenoptera only to the extent that the mother insect provides her egg with an inadequate amount of yolk instead of feeding the larva from day to day on an insufh- cient amount of food. The resemblance of the dwarf females of Phylloxera carye-fallax to the workers of ants and other social insects is very striking, although it seems not to have been noticed by Morgan. Perhaps the Selle known “high” and “low”’ types of male in many insects, notably of the Seamed and Lucanide are to be regarded as the results of larval feeding. If this is the case, the low males may present examples of alimentary castration. This peculiar male dimorphism certainly bears more than a superficial resemblance to the female dimorphism of the social Hymenoptera. These may, indeed, be said to have high and low females, which, like the corresponding forms of the opposite sex in Scarabzidz, Effects of Castration in Insects 405 are sometimes connected by intermediates In ants the soldiers and desmergates represent the intermediate forms.* But no one, to my knowledge, has studied the testes of beetles with dimorphic males, with a view to ascertaining whether these organs are more imperfectly developed in the low than in the high individuals. ‘he low males undoubtedly approach the female in form, and might, therefore, be said to assume the secondary char- acters of this sex, were it not for the consideration that in a large number of scarabzeid and lucanid species and genera both sexes have thesamesimpleform. Thisindicates that the low male simply fails to develop its secondary sexual characters and hence returns to the ancestral type of the species in which these characters were either very feebly manifested or were altogether absent. G. Smith (05a) has shown that in the Scnmale dee and Lucanide, as well as in certain crustacea (Tanaidz), “the differentiation into high and low males within the limits of a species has widely in- fluenced the progressive differentiation among the different closely related species of many groups.” This is somewhat more clearly expressed by saying that there are also high and low species in certain groups, the lanes species of certain genera having a more pronounced male dimorphism than the smaller closely allied species. This is also true of the sexual dimorphism of female ants, as is seen in such generaas Solenopsis and Camponotus and among the genera of the subfamilies Dolichoderinz, Camponotinz and Mymicinz. It will be shown in the sequel that there is alsoanother way of accounting for the “high” and “low” forms of some insects. In this connection, I may briefly consider two cases which, if correctly reported, would appear to represent a complete loss of the reproductive organs by alimentary castration carried back into the early larval or embryonic period. Adlerz (’86) and Miss Bickford (95) failed to find any traces of ovarian tubules in workers of the common pavement ant, Tetramorium cespitum. If this negative observation be correct, the workers of this ant must be regarded as utterly sexless. In my opinion, however, renewed investigation ° For a fuller account of the conditions in these insects the reader is referred to my paper on poly- morphism (’o07). 406 William Morton Wheeler is required to establish the truth of this statement. ‘he other case 1s even more doubtful. Silvestri (06) recently described Copidosoma truncatellum, a chalcidid which is polyembryonic and infests the eggs and caterpillars of moths belonging to the genus Plusia, as possessing two very different larval forms. One of these he designates as “asexual” and states that it lacks every trace of the reproductive organs. It is very unlike the ordinary sexual larva in having a vee head, well-dev eloped mandibles and a very slender Baad cedesliis body, and never lives beyond the larval stage. Silvestri believes that it has been developed for the purpose of breaking down the tissues of the host caterpillar and of thus rendering them more easily assimilable by the sexual larvz which alone develop into imagines. The following considerations seem to me to cast considerable doubt on this Interpretation: First, the asexual larvee hgured and described by this investigator are suspiciously like certain very young ichneumonid larve, and as their development is not satisfactorily traced to the same cell- masses from which the sexual Copidosoma larve arise, it 1s not improbable that the two larval forms really belong to two very different parasites. In other words, Silvestri’s Plusia caterpillars were probably infested with ichneumonid in addition to Copido- soma larvae. Second, I have been unable to find any larve of the asexual type in a number of American Plusia gamma caterpillars which were heavily infested with Copidosoma truncatellum. Third, as in many species of Chalcididz larve of Silvestri’s sexual type are able by their own endeavors to break down and assimi- late the tissues of their host, it seems improbable that a single spe- cies should have developed a peculiar sexless and moribund larva for this particular purpose. He Nutricial castration The abortive or rudimental condition of the sexual organs seen in the cases of alimentary castration may be normally pro- longed and maintained throughout the adult life of the workers among the social Hymenoptera, when these insects are compelled to live on the slender remnant of food that remains to them after Effects of Castration in Insects 407 they have satiated their queens and the young broods which are continually hatching from her eggs. Marchal (’97) has called attention to this ise in the wasps, and it has long been known to obtain in ants and the social bees, though the causal connection between the protracted immaturity of the ovaries in adult workers and their primary function as nurses had not been sufficiently emphasized. The form of castration which is thus produced is, however, not necessarily permanent. If the trophic status of a colony becomes highly favorable, or if the queen dies, the ovaries of one or of a number of the workers may undergo active growth and produce eggs capable of normal development. In such cases the workers may be said to usurp or to supplement the function of the queen, but owing to the fact that the adult insect cannot modify its external characters, there is no visible differ- ence between the sterile and fertile workers, except in the size of the abdomen, and even this may be so slight as to escape observation. The primary cause of nutricial castration is to be sought in the instincts of the individual itself, whereas alimentary castration would seem to be attributable to the instincts of the individual’s living enviroment, 7.e., to its nurses. This distinc- tion, however, is probably more apparent than real, since as | have suggested in a former paper (’07), it is possible that the worker larva is from the beginning an organism predisposed _ to assimilate only a portion of the nourishment with which it is provided by its nurses. The growth and development of the larva obviously does not depend on the amount of food admin- istered to it but on the character and rate of operation of its assimilating mechanism. A larva may be very voracious, but its tissues may be constitutionally unable to appropriate more than a limited portion of the food which enters its alimentary tract. Ihe administration of highly assimilable food, as in the case of the “royal jelly” which is fed to the larval queen bee, may be, as I have maintained (’07), primarily for the purpose of accelerating the development of her ovaries, and the secondary characters of this insect, which are mostly of an abortive charac- ter (smaller sting, shorter wings, smaller hind legs) may be the result of this development. 408 William Morton Wheeler Nutricial castration is not confined to the social insects but occurs also in mammals during the periods of gestation and lac- tation and in birds during incubation, as the result of a very simi- lar inability of the organism to expend in reproduc tion the ener- gies demanded by Fe exigencies of the nursing function. 4. Phasic Castration. The forms of sterility which I include under this term, though temporary, cannot be sharply distinguished from the cases of alimentary and nutricial castration, since both of these may ‘be abolished during ontogenetic development and yield to a fer- tile phase, as, e.g., when worker ants become gynecoid and nym- phal termites become supplemen tal males and females. We may, indeed, say that the great majority of animals exhibit alimentary castration during their embryonic, larval and juvenile stages, but that this is not universally true is shown by the many examples of neotenia and pedogenesis scattered through the animal king- dom. There are, however, several cases of temporary castra- tion which, though intimately dependent on the trophic condition of the individual nevertheless do not properly fall in the categories previously considered. ‘The following may serve as examples: A. Many hermaphroditic animals are protandric, 1. e., develop only their male reproductive organs at a very early stage and do’ not mature their female reproductive organs till after the testes are partly or wholly exhausted. Some of the most extreme cases of this phenomenon are seen in the epicarid crustacea and in the singular parasitic worms of the genus Myzostoma. In the crustacean Danalia the individual becomes a functional male while it is still a minute and active larva. Later this form at- taches itself to the abdomen of a crab, loses its limbs, and develops a long proboscis which penetrates the tissues of its host. The abundant nutriment thus acquired enables the parasite to grow rapidly. Its ovaries then begin to enlarge, while the remains of its testes degenerate and are devoured oF phagocytes, and the creature teenies atemales Av very. similar condition occurs, as I showed several years ago (’96) in certain species of Myzo- Effects of Castration in Insects 409 stoma (e.g., in M. pulvinar von Graff). In these striking exam- ples the animal is only potentially hermaphroditic, since func tion- ally it exhibits seasonal gonochorism through phasic castration of the ovaries during its youth and of the testes during its adult stages. B. Geoffrey Smith (’o5 a, ’0g) has called attention to a very striking form of phasic castration in decapod crustacea: ‘ Dur- ing the breeding season the males of Inachus mauritanicus fall Fig. 4. Males of Inachus mauritanicus. A small breeding male with swollen chele; B non- breeding male, with slender chele; C, large breeding male with swollen chela. (After Geoffrey Smith. ) into three chief categories: Small males with swollen chelz (Fig. 44), middle sized males with flattened chelew (B), and large males with enormously swollen chele (C). On dissecting specimens of the first and third categories 1t is found that the testes occupy a large part of the thoracic cavity and are full of spermatozoa, while in the middle-sized males with female-like chelz the tes- tes appear shrivelled and contain few spermatozoa. These no..- breeding crabs are, in fact, undergoing a period of active growth and sexual suppression before attaining the final stage of devel- 410 Willtam Morton Wheeler opment exhibited by the large breeding crabs.” This same con- dition was previously observed by Faxon (’85) in male crayhsh belonging to the American genus Cambarus. Of course, the three stages ened by Craik are separated by moults. Ob- mously we have here a condition like that observed in many male fishes, amphibians and birds, which lose their secondary sexual characters during the seasons when they are not breeding. Smith regards the phenomenon as “ obviously parallel to the ‘high and low dimorphism,’ so common in lamellicorn beetles,” but this is a mistake, as ‘Cunningham (’08) has shown, for we are here confronted with a case of seasonal sexual dimorphism. Nothing comparable to the condition described above is seen in insects, for the reason that these animals either do not mature their gonads till after they have attained their fixed and final imaginal instar, or if they become sexually mature as larvz or pup (neo- tenic and peedogenetic aphids, cecidomyids, chironomids, etc.) they do not develop beyond this stage. It is not improbable, howev er, that insects which live several years in the adult stage and have seasons of sexual activity alternating with seasons of infertility, may exhibit great periodical changes in the size and development of the reproductive organs. I have been unable to find any observations on this subject in the entomological litera- ture. 5. Indwtdual Parasitic Castration. The first zoologist fully to appreciate the importance of para- sites in suppressing the reproductive function and in incidentally affecting the somatic characters of their hosts was Giard. He publi shed some twenty papers (’69—02) on a great variety of cases which he observed not only among animals bat also among plants. The cases to which he devereds most attention were the decapod crustacea, especially species of Stenorhynchus, Portunus, Carcinus, Cancer, Platyonychus, Eupagurus, P alaemon, Gebia and Hippolyte, which are infested with extraordinary cirriped and bopyrid parasites of the genera Sacculina, Portunion, Bopyrus, Probopyrus, etc. Within more recent years these studies have Effects of Castration in Insects 4II been continued and deepened by Geoffrey Smith (’06, ’og) on the spider crab Inachus mauritanicus infested with the cirriped Sac- culina neglecta and by Potts (’06, ’0g) on hermit crabs (Kupa- gurus meticulosus) infested with the cirriped Peltogaster curva- tus. A summary of the work of these two authors will not be out of place here, since they have reached rather definite con- Fig. 5. Specimens of Inachus mauritanicus to show effects of parasitic Sacculina neglecta. A normal male; B, normal female; C, male infested with Sacculina (final stage ); D, abdomen of infested female; FE, infested male in an early stage of its modification. (After Geoffrey Smith.) clusions not without a bearing on the various cases of parasitic castration in insects and other organisms to which I shall have occasion to refer. According to Geoffrey Smith (’og) the abdomen of the normal male of Inachus mauritanicus “is small and bears a pair of copu- latory styles, while the chelipedes are long and swollen (Fig. 54). In the female (Fig. 56) the abdomen is much larger and trough- 412 William Morton Wheeler . shaped, and carries four pairs of ovigerous appendages; the che- la are small and narrow. ‘Now it is found that in about 70 per cent of males infected with Sacculina the body takes on to varying degrees the female characters, the abdomen becoming broad as in the female, with a tendency to develop the ovigerous appendages, while the che- la become reduced ( Fig. 5C’). ‘This assumption of the female characteristics by = ani under the influence of the parasite may be so perfect that the abdomen and chela become typically female in dimensions, while the abdomen develops not only the copulatory styles typical of the male, but also the four pairs of ovigerous appendages typical of the female. The parasitized females, on the other hand, though they may show a degenerate condition of the ovigerous appendages ( Fig. 5D), never develop a single positively male characteristic. On dissecting crabs of these varying categories it is found that the generative organs are in varying conditions of degeneration and disintegration. “The most remarkable fact in this history is the subsequent behavior of males which have assumed perfect female external characters, if the Sacculina drops off and the crabs recover from the disease. It is found that under these circumstances these males may regenerate from the remains of their gonad a perfect hermaphrodite gland, capable of producing mature ova and sper- matozoa. The females appear quite incapable, on the other hand, of producing the male primary elements of sex on recovery any more than they can produce the secondary.” The following account is quoted from Pott’s summary (’0Q) of his own edie on the modifications induced in Eupagurus by Peltogaster and of Smith’s observations: “The difference between the sexes of Eupagurus is shown only in a couple of external characters, the position of the generative apertures (asin all Decapods) and the character of the abdominal appendages. The abdomen of the hermit crab is furnished on one side only with a few appendages, insignificant, but with definite functions. It is in the female that we see the full development of the appendage as a swimmeret with two equal branches, the inner one provided with long hairs affording a secure anchorage for Effects of Castration in Insects 413 countless eggs while the outer one is of equal size 1n both sexes, and in both by its paddle-movement maintains respiration cur- rents in the shell. No use has been found for the outer branch in the male and so has become quite rudimentary, but the effect of the parasite Peltogaster 1s to stimulate the growth of this rudi- ment. There isof course great variability of response to this stimu- lus but those individuals which experience the maximum amount of change possess swimmerets exactly similar to those of a mature’ female, even in the assumption of the curious branched or barbed hairs which in this case can never bear eggs. As in the spider crabs so here, the female appeared incapable of the reverse change, and the large number of hermit crabs with typical female append- ages and sealed genital apertures are undoubtedly to be regarded in part as modified males. “A protest will conceivably be uttered against the attribution of a special sexual significance to the development of typical swimmerets in the male in both spider crabs and hermit crabs. It is of course well known that in the larval stages of these Crustacea biramous abdominal appendages are found in both sexes to be subsequently reduced or lost in the male. Lest this, then, be deemed a happy opportunity for applying the term “reversion”’ to this phenomenon I[ hasten once more to point out that when the male develops biramous abdominal swimmerets they are of the type associated with female maturity, and that the specialized nature of their nursing-hairs cannot well be associated with ances- tral conditions. “Both Sacculina and Peltogaster inflict sterility upon their host and apparently entire abortion of the gonad generally is the final consequence. On the external appearance of the parasite the eggs of the female shrink through absorption of their yolk and the formation of spermatozoa is after a time suspended in the male. The testis of the spider crab dwindles and disappears without undergoing any particular histological change; but in the hermit crab it is curious to note the presence of large cells with large nucleus and abundant protoplasm in sections of the testis. These instantly suggest ova in their appearance and call to mind the instances of the occurrence of such cytological elements as a nor- 414 William Morton Wheeler mal experience in the testes of many animals. In sand-hoppers (Orchestia) to quote a well-known case (and there are many others in the Crustacea) spermatozoa are produced in the anterior part of the young testis while posteriorly the whole space 1s occupied by two or three large ova (vide Boulenger ’08). ‘The particular interest of the phenomenon in this case is its association with a definite cause, that is, parasitism. We are also able to come to some conclusion as to the degree in which such a condition can be called true hermaphroditism. Some striking evidence is offered by spider crabs which were once infected by Sacculina but which have outlived their parasite and recovered from its influence. Such crabs occur in nature in fair frequency and the only reminder of their former condition is the chitinous ring on the abdomen which surrounded the peduncle of the para- site. Afterthe death of the external part of the Sacculina the root system may continue to exist in the host and it 1s only when this has disintegrated and been absorbed that regeneration of the gonads hecanes rapid, for the still living roots repress the devel- opment of the sexual organs as effectually as the living parasite. A few crabs however were found in which the gonads had again attained full size and maturity. One was a female with a well- developed ovary and four were males only shghtly modified ex- ternally, with elands producing large quantities of spermatozoa. The remaining four cases were em EAble for the crabs showed with a complete external hermaphroditism the corresponding gonads. In all four animals the reproductive gland consisted of a male part with ripe spermatozoa, and a female division with large pigmented ova. The ducts were usually absent, but one individ- ual possesssed both vasa deferentia and oviducts. The sequel to these observations is given by the experimental evidence which Smith then obtained. It was attempted to destroy the parasite by removing the external part and the crabs so freed were kept ander comfortable conditions for several months and the few survivors then killed. Regeneration had obviously occurred to a considerable extent, but the gonads were nearly always unisexual. In one individual alone, which was externally a hermaphrodite there was a gonad similar to those just described. In spite of the Effects of Castration in Insects 415 comparatively small number of cases with fully formed herma- phrodite glands we are not going too far in definitely asserting a connection between their occurrence and parasitic influence, for bisexual gonads have to my knowledge never been met with in Decapod Crustacea under normal] conditions.’ Butit thus ap- pears that the curious condition in the hermit crab ts an incipient stage corresponding to the perfect hermaphroditism of the “re- covered”’ spider crabs, and if the action of the parasite in absorb- ing surplus nutrition were withdrawn the young ova in the testis of the hermit crab would become large and pigmented like those in the spider crab. “These two cases have been described at some length as ex- amples of extreme modification. In other Decapod Crustacea which are infected by the same parasite an effect is observable which is similar in kind but not in degree. The common shore crab of England (Carcinus) is commonly afflicted (if affliction it be) by Sacculina. Here again the male undergoes modification while the reverse change never occurs in the female. The narrow abdomen of the male is often exchanged at the moult after infec- tion for one much broader but never attaining the full female width. One may look in vain, however, for any reduction of the copulatory styles or for the appearance of the smallest rudiments of swimmerets. The closure of the genital apertures nearly al- ways follows parasitic attack in spider crab and hermit crab; but they never become blocked up in shore crabs with Sacculina. Yet the external change is apparently greater than that produced in the reproductive glands. Dissection in every parasitized male showed vasa deferentia of the characteristic milky white color due to countless masses of spermatophores all packed with spermatozoa. The testes though reduced, then, always remain in reproductive activity. The parasites which infect spider crab and shore crab are practically identical and presumably exert a very similar stim- ulus yet the results are markedly different. It 1s obviously the host which offers a different reaction in the two cases. In another ® Tn a footnote Potts states that “Calman in the recently appeared volume Crustacea of Ray Lan- kester’s Treatise on Zoology refers to the unpublished observations of Wolleback on normal hermaphrod- it'sm in certain deep-water Decapoda.” 6 410 William Morton Wheeler crab (Kriphia) examined by Smith there was infection both by S: ae and by a parasitic [sopod crustacean. Here the nature of the parasite governs the result, and crabs with Sacculina alone never showed the least trace of modification, while changes closel y similar to those described above occurred in those which har- boured the Isopod.”’ Geoftrey Smith (’05 b) has also described parasitic castration in Inachus dorsettensis by a sporozoon (Aggregata inachi) which - lives in the intestine of the crab and induces modifications not unlike those induced by Sacculina. Smith says that of fifty males of 1. dorsettensis examined, “‘seven specimens were clearly dis- tinguished by having the flat chelz characteristic of the females, w hile the abdomen was much broader than is the case in normal males of a corresponding size, thus converging on the female con- dition. In one specimen there was present on the under side of the abdomen a pair of swimmerets which are characteristic of the female, these appendages being altogether absent in the normal males.” Dissection of these crabs showed the intestine “to be covered with cysts of Aggregata inachi, the body cavity was also full of liberated sporozoites, the haemolymph having a milky ap- pearance due to the crowded presence of these Bodice The testes were in all cases disintegrated, only the vesiculze seminales remain- ing. Iwo modified males were also found to contain the cysts of Aggregata inachi, but in none of these males were there larger quantities of sporozoites in the haemolymph, so that it appears that the hermaphrodite external characters are assumed by the in- fected male at the moult which follows the liberation of a large quantity of sporozoites.” Smith made no observations on the infected female Inachus, as this sex is much rarer than the male. The foregoing examples of parasitic castration in crustacea have been reviewed at some length, because they show the phenomenon in its most striking manifestation. Guard as early as 1888 (b) published a long list of other animals and plants known to be cas- trated by what he calls “gonotomic’ ” parasites. ‘The most inter- " esting examples, apart from Andrena and the crustacea just con- sidered, are thecastrationof the nemertean Lineus obscurus by the orthonectid Intoshia linea, of the planarian Leptoplana tremellaris Effects of Castration in Insects 417 by Intoshia kefersteini, of the brittle, star Amphiura squamata by theorthonectid Rhopalura giardi and by a copepod (Fewkes 88), of the snailsof the genera Paludina, Lymnza and Planorbis by dis- tome sporocysts (Distomum militare, retusum, etc.), of the crus- tacean Cyclops tenuirostris by larval distomes (Herrick 783), of the bumble bees (Bombus) by the extraordinary nematode Spherularia bombi, and of the males of various North American squirrels and chipmunks (‘Tamias lysteri,Sciurus hudsonius and leucotis) by the bot-fly Cuterebra emasculator as described by Fitch (’59), Riley and Howard (’89) and Osborn (’96). Among plants Giard cites the castration of the fig by Blastophaga grossorum, of Melandryum album (Lychnis dioica) by Ustilago antherarum and various grasses by smuts, ergots, rusts, etc. The case of Melandryum and Ustilago which was repeatedly studied by Giard(’69, ’87a, 88d, ’8ga) bears a curious resemblance to that of the male crab infested with Sacculina. The Melan- dryum is “normally dicecious. The young flower is Fermaphro- dite but in certain individuals the ovaries abort, in others the stamens remain rudimentary. When the parasitic fungus develops on a male plant, it fructifies in the stamens, but when it falls on a female plant, it seems at first as though it could not fructify and that the infested plant must profit accordingly. But this 1s not the case, for the plant develops its rudimentary stamens completely in order to permit the fructification of the parasite, just as the male Stenorhynchus enlarges its abdomen in order to protect the Sacculina fraissei.”’ Castration frequently occurs in plants through petalody, or petalomania, 7. e. the conversion of stamens or carpels into petals, preducing the well-known ‘double’ flowers. Molliard (o1) has produced petalody experimentally in Scabiosa colum- baria by artificially infecting the plant with the nematode Heterodera_ radicicola. And this investigator, Meehan (’00), Giard (02) and Cramer (’07) cite a number of observations which indicate that petalody is often the result of infection of a plant with root-fungi. Veuillemin (’07) has observed in Lonicera infested with aed a suppression of the carpels and a distinct androgeny of a certain number of the flowers. 418 William Morton Wheeler Instead of da: to review the various examples of parasitic castration cited by ¢ niard in his paper of 1888, and in many of his later publications, it will be preferable to describe as briefly as possible a number of selected examples, especially some that have come to light more recently among insects. ‘The stylopized Polistes and eee having been adeaeeely described in the first part of this paper, will bee omitted. Grassi and Sandias (’93) describe a remarkable case of parasitic castration in termites. They find that worker and soldier ter- mites have the intestinal cacum, which occupies much of the ab- dominal cavity, distended with enormous numbers of parasitic Protozoa belonging both to the Ciliata (Dinonympha, Pyrsonym- pha, Ticheny pha) ) and to the Gregarinida. The Ciliata have been studied by several authors, notably by Leidy (77, ’81), Grassi (’85), Kent (’85), Porter (’97), and Dodd ( 06). In ter- mites Bees with these parasites the reproductive organs, both male and female, remain small and undeveloped, apparently as the result of the pressure exerted on them by the distension of the cecum. ‘The parasites are absent in the very young termites and in the sexual forms, which are fed onsaliva. Grassiand Sand- ias infer that the Protozoa must either be killed off or, at any rate, prevented from living and growing in the alimentary tract of saliva-fed individuals. These investigators are inclined, there- fore, with some reservations, to regard the development of the two sterile castes in termites as the result of infection with pro- tozoan parasites. his infection is, of course, readily brought about as the workers and soldiers are not fed on saliva like the sexual forms but on dead wood and on the feces of individuals belonging to the same castes. The researches of Grassi and Sandias have received a certain amount of confirmation from Brunelli (’05), who finds that queens of Calotermes flavicollis and Termes lucifugus sometimes become infested with the parasitic Protozoa, and that when this happens the young oocytes in their ovaries degenerate. Calotermes queens are more susceptible to this form of castration then the queens of Termes. Brunelli explains the winged soldier observed by Grassi and Silvestri’s (703) 48 workers of Microcerotermes struncki with Effects of Castration in Insects 419 well-developed reproductive organs (40 females and 8 males), as being instances of fertility brought about by a disappearance of the Protozoa through some unknown cause. Such fertile soldiers and workers would be comparable to the “recovered” spider crabs above described, except that there is no tendency towards hermaphroditism. It is not altogether improbable that the high and low males among the Scarabaeidae, Lucanida and Forficulide are produced AR is iY f § ; Fig 6. 4, normal worker of Pheidole commutata; B and C mermithergate of same in dorsal and | teral view. in some such manner as the workers and soldiers of termites. It is certainly suggestive that all three of these families of insects live on decomposing vegetable substances and in situations where they become very readily infected with gregarines. Giard (94a) has given good reasons for supposing that the high and low males of Forficula, which were made the basis of a statistical study by Bateson (g2), are produced by differences in the number of gregarines they harbor in their alimentary tract. The French THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 8, No. 4. 420 William Morton Wheeler observer says: “‘It is, indeed, possible to predict from the length of its forceps whether or not a male Forficula possesses gregarines and whether these are present in greater or lesser numbers. Since these parasites produce a diminution of a secondary sexual char- acter, that is, the length of the forceps, without bringing about absolute sterility (complete castration being ereennonalie it not infrequently happens—and this is the case both on the beaches of Wimereux and on the Farne Islands —that the individuals with short forceps, namely, those containing parasites, are more nu- merous than the individuals with long forceps.” Giard is inclined to believe that similar conditions may obtain in such beetles as Xylotrypes gideon, Oryctes nasicornis and other Scarabzide with high and low males. The low males of these beetles, however, are not to be regarded as having acquired female characters, but as having lost the male characters, so that, as Giard remarks, the “infested individuals are generally padomorphic as compared with the normal form.” In two of my former papers (’o1, ’07) I described a peculiar case of parasitism in a Texan ant, Pheidole commutata. The larve of this insect are occasionally infected with nematodes of the genus Mermis and develop into peculiar forms, which I have called mermithergates (Figs. 6B and 6C). These are much larger than the normal workers ( Fig. 64), which they nevertheless resem- ble in the structure and small size of the head, although they possess small ocelli and in this respect resemble the queens. In thoracic structure they approach the soldier form while the gaster is enor- mously distended with Mermis and retains scarcely any vestiges ‘of the fat- body, reproductive organs and other viscera. The behavior of these parasitized aol: is also peculiar, since they never excavate the soil, nor care for the brood like the nor- mal workers, but run about in a state of chronic hunger, begging food from their uninfested nest-mates. Emery (’go, ’04) has re- corded the occurrence of mermithergates in quite a series of neo- tropical ants, including Pheidole absurda and several Ponerinz of the genera Odontomachus, Neoponera, Ectatomma, Pachy- condyla and Paraponera. In the cases described by Emery and myself only the worker Effects of Castration in Insects 421 forms were infested and modified by the Mermis, but Mrazek (08) has recently shown that the virgin queens of the European Lasius alienus may become infested mach this worm and that when this occurs the insects develop abnormally small wings (Fig. 78). These individuals, or mermithogynes, as Mrazek calls them, have been seen by other investigators and described as brachypterous to distinguish them from the normal macropterous individuals of the species. After seeing Mrazek’s paper I examined a small collection of seven brachypterous and as many macropterous females of La- sius neoniger (a form closely related to alienus) which I had taken from a single colony near Manitou, Colorado, August 9, 1903. Three of the short winged individuals were dissected and each was found to contain a large coiled Mermis, 53 to 55 mm. long, which filled out the whole abdomen, so that in the living indi- viduals there could have been little left of the reproductive organs and other viscera. here is nothing unusual in these females except the small size of their wings, which measure only 6 to 6.5 mm. in length, whereas those of normal L. neoniger females measure 10 to 11 mm. ‘These observations show that the queens of our American Las may be affected by Mermis in exactly the same manner as the queens of the related European species. The species of Mermis are not, however, the only known gono- tomic nematodes. A much more extraordinary form is Sphezru- laris bombi, which has been known ever since the days of Réaumur (1742) to produce sterility in the hibernating queens of bumble- bees. According to Leuckart (’87), who has written the best and apparently also the most recent account of Spherularia, infested bees are sometimes found, “which have not a single ma- ture egg in their ovaries. Structurally these organs are perfectly developed and have ova in the blind ends of their ovarioles, but ripe eggs are lacking. In other specimens one may find in addi- tion to the young, also some ova of perfectly normal dimensions.” He says that he has “never seen an infested queen which had the ovarioles as uniformly and richly provided with eggs as are the ovaries of healthy bumble-bees at the same season. As a rule, one finds only a few eggs, sometimes only a single egg.” These 422 William Morton Wheeler bees are therefore unable to found colonies, according to Schnei- der and Leuckart. They keep flying about till late in June and then die, whereas uninfested queens have started their colonies and no longer fly at large after the beginning or middle of May. Spherularia occurs only in the queens, and has never been found in those that have become mothers of colonies. It would be in- teresting to know whether the colony-founding instincts of Fig. 7. 4, normal female of Lasius alienus; B, mermithogyne of same species (After Mrazek. ) infested queens show the same tendency to atrophy as the ovaries. As the bees become infected in their imaginal instar, apparently while seeking their winter quarters, the parasites can produce no modifications in the external characters. The Lasius mermithogynes described above recall some ob- servations of Kinckel d’Herculais (’94.) on Algerian grass-hoppers (Stauronotus maroccanus and other species) infested with flies of the genus Sarcophaga. The maggots of the flies are entopara- sitic, devouring the fat-body, and, according to Kiinckel d’Her- culais, also absorbing the oxygen dissolved in the blood-plasma of E ffects of Castration in Insects 42,3 their hosts. ‘The results are an atrophy of the reproductive organs (parasitic castration) and a weakening of the wing-muscles, so that the grasshoppers have a disinclination to fly. For this latter condition, which is described as a “‘kind of rhachitis,’? Kunckel d’Herculais suggests the name “‘aptenia.” Like the brachyptery of the Lasius mermithogynes, it points to an intimate correlation between the development of the reproductive organs and_ the wings, a correlation which 1s also clearly demonstrated in most insects by the coincident maturation of the former and full devel- opment of the latter organs at the beginning of the imaginal instar. The extensive literature on entoparasitic Diptera and Hymen- optera, if carefully searched, would probably yield a number of accounts of parasitic castration. Pantel (’og), in an important paper, distinguishes both direct and indirect parasitic castration as the result of the infestation of lepidopteran larva with the larvz of tachinid flies. In the former case the fly larve live in the testes of the lepidopteron and destroy the gonadic elements directly. In the latter the gonads suffer atrophy through the action of the parasites on the other viscera. ‘The only cases | have found in which the host shows a modification of its external sexual characters as the result ofsuch castration, are the homoptera Typhlocyba hippocastani and douglasi, which are described by Giard (’894, ’8gd) as being infested with a dryinid hymenopteron, Aphelopus melaleucus and a pipunculid dipteron, Chalarus (Ateloneura)spuria. The females of both species of Typhlocyba, when castrated by Aphelopus, have the ovipositor much reduced ; the Chalarus alone seems to have less effect on this organ. The penis of the male T. douglasi islittle modified by either EE the parasites, but in T. hippocastani infested with Chalarus, this organ shows a decided reduction in size and simplification of structure so that the specific characters become profoundly modified. None of these modifications, however, indicates any tendency to take on the characters of the opposite sex. _6. Social Parasitic Castration This category 1s not sharply marked off from the preceding, for if we define it as including those cases among social insects 424 William Morton Wheeler in which the individuals that represent the reproductive organs (1.e., the males and queens) of the colony considered as an organ- ism of a higher order, are castrated by parasites, we should perhaps include also the Lasius colonies containing mermithogynes and the queens of Bombus infested with Sphzrularia described in the foregoing paragraphs. But in these cases itis merely prospective colonies, so to speak, which are castrated, since neither the mer- mithogynes nor the parasitized Bombus queens have as yet be- come mothers of colonies. For this reason I have treated them as cases of individual parasitic castration. Here belongs also the production of pseudogynes in Formica colonies infested with the peculiar myrmecophilous beetles of the staphylinid tribe Lomechusini (Lomechusa and Xenodusa) which I have considered at length in a former paper (’07). These beetles tend to sup- press the development of the annual brood of virgin queens since the worker ants of parasitized colonies either neglect the queen larve or endeavor to convert them into workers, after the period during which this change can be successfully accomplished has eat The results of this behavior is the production of the non- viable pseudogynes and the gradual degeneration of the colony. In this case also the colony is not castrated, but the mothers of prospective colonies may be said to suffer from misapplied alimen- tary castration. Leaving all these cases out of account we have left only those in which a parasitic colony of insects prevents the development of or destroys the fertile sexual individuals of the host colony in which it lives. As parasites of this type I may mention the vari- ous slave-making ants (Formica sanguinea and Polyergus rufescens and their various varieties and subspecies), the temporary social parasites (Formica rufa, exsecta, exsectoides, etc.) and the perma- nent social parasites of the genera Anergates, Wheeleriella, Epi- pheidole, Sympheidole and Epcecus. There are other social para- sites that do not destroy the reproductive individuals of the host colony, for example, the bees of the genus Psithyrus, which live in the nests of bumble-bees, and among ants such species as Lep- tothorax emersoni, Formicoxenus nitidulus and Harpagoxenusg sublevis. Stillotherants, such as the species of Strongylognathug, Effects of Castration in Insects 425 do not destroy the queen of their host colony ([etramorium ces- pitum), but since the workers of this colony prefer to rear the small sexual forms of the parasites instead of their own bulky males and females, the development of future colonies of the host species is rendered impo ssible and we have here again a case of prospective social castration. The conclusion which we reach after marshaling this long series of illustrations of the various forms of castration 1s that among insects the only case in which destruction or inhibition of the reproductive function clearly results in any modifications of _ the secondary sexual characters comparable to the modifications observed in vertebrates under like conditions, is that of the sty- lopized andrenine bees as described by Pérez. In all the other cases extirpation of or injury to the gonads may indeed result in modifications of the somatic or secondary sexual characters, but the latter do not take on the peculiarities of the opposite sex. The most striking illustrations of the truth of this statement are the insects that have been surgically castrated. These show that the secondary sexual characters must be so independently and so immovably predetermined and at so early a period in the onto- geny that complete extirpation of the gonads during prepupal life fails to produce the slightest curtailment or modification either in the secondary sexual characters or in the sexual instincts of the adult insect. This conclusion renders it imperative to rein- vestigate the cases of stylopization in the andrenine bees for the purpose of ascertaining whether Pérez’s interpretation is the only one which they will yield, especially since it has been shown in the first part of this paper that the study of stylopization in Polistes leads to a very different view and one in complete harmony with the other cases of castration in insects. It is interesting to note that castrated crustacea, to judge from the observations of Giard, Geoffrey Smith, and Potts, show modi- fications like those of castrated vertebrates and not like those of the insects. This isin all probability due to the fact that the devel- opment of the primary and secondary sexual characters is grad- ual and continuous in the Crustacea and vertebrates, whereas both these characters in insects are arrested in their develop- 420 William Morton Wheeler ment and remain unaffected by the surrounding processes of growth and differentiation till the imaginal stage is attained. In holometabolic insects the secondary sexual characters are, of course, segregated in the imaginal discs, or histoblasts, and even in hemimetabolic and ametabolic insects there must be a similar isolation of the cell-materials which will produce the somatic sexual peculiarities of the adult. The opinion here advocated, namely, that in insects the pri- mary and secondary characters are very loosely correlated dur- ing ontogenetic development or in a very different manner from what they are in vertebrates or even in the crustacea, receives indirect support from two interesting classes of facts. One of these classes comprises the anomalies known as gynandromorphs,which, though always rare, are nevertheless much more frequently found among insects than among any other animals. These anomalies consist in combinations of male and female somatic characters in the same individual, usually in such a manner that the two lat- eral halves or the anterior and posterior portions of the body are of different sexes. In the former combination the reproductive organs may be hermaphroditic and correspond with the sex of the Heleee of the body in which they lie, but this 1s not always the case, and in anteroposterior, or frontal, or in mosaic,or decus- sating gynandromorphs, which exhibit an irregular mingling of the the sexual characters, the gonads may nevertheless be unisexual. Herbst (or) and Driesch (’07) have emphasized the obvious inference that these various arrangements of the male and female characters cannot owe their ongin to internal secretions, or hormones, and indeed all those who have speculated on the ori- gin of these anomalies are unanimous in holding that they must arise either from peculiarities in the structure of the egg or from irregularities in its fertilitation or early cleavage stages at the very latest. Among recent speculations on the origin of gynan- dromorphism those of Boveri (02) and Morgan (05, ’0g) may be mentioned. Boveri believes that the gynandromorph arises from an egg which has segmented prematurely, so that the male pro- nucleus unites with one of the cleavage nuclei. Morgan is of the opinion “that the results may be due to two (or more) sperma to- Effects of Castration in Insects 42 7 zoa entering the same egg, one only fusing with the egg nucleus and the dies not uniting, but developing without combining with any parts of the egg nucleus.’’ These hypotheses have no very cogent facts to support them and I fail to see how they have any advantage over the hypothesis which was advanced by Donhof as long ago as 1860, to the effect that the gynandromorph arises from the fusion of two eggs, only one of which, in the case of the honey bee, is fertilized. In its original form Donhof’s hypothe- sis is incomplete, but I believe that its plausibility is increased by addition of the following considerations. We may assume with Beard (’02), von Lenhossék (’03), Reuter (’07), Morgan (’og) and others that the gonochoristic Metazoa produce two kinds of eges, male and fei file: which may or may not differ in size but differ in sex even as oocytes. Now we know from zur Strassen’s researches on Ascaris (’98) that two eggs may fuse and neverthe- less give rise to a single embryo of perfectly normal structure though of twice the normal size. In Ascaris the fusion occurs after the oocytes have reached their full growth, but -a fusion of younger oocytes would be, in all Hee not only more readily accomplished but lead to the formation of a single embryo of the normal size. The structure of the ovarioles of insects indicates that it would be a very easy matter for two young oocytes to be- come enclosed in the same follicle, too easy, indeed, to accord, at first glance, with the fact that gy nandromorphs are such rare anomalies. But if two female or two male oocytes fused no gy- nan dromorph would result, and the chances of either of these fu- sions of like oocytes occurring would be quite as great as that of two oocytes of opposite sex. If this be the way in which gynandro- morphs arise, we should have to explain the occurrence of the lateral type of the anomaly by supposing that the plane of fusion of the two eggs be omes the median sagittal plane of the future insect, whereas in the frontal type this plane would be transverse to the longitudinal axis. Finally, ig the mixed and decussating types we should have to suppose that the male and female egg-mate- rials are mixed or interpenetrate one another toa variable degree. The hypothesis here sketched has the advantage of permitting of some slight cytological verification, for microscopic examination 428 William Morton Wheeler of the ovarioles of a large number of Lepidoptera, which seem to present the anomaly in question more frequently than other insects, might reveal an occasional inclusion of two oocytes in the same follicle or even various stages in their fusion. Or if hives are ever again found like the famous :ugster hive, in which so many gynandromorphous bees were produced, the cytologist will have an opportunity to test the hypothesis here advocated by a careful examination of the ovarioles of the queen. But no matter what view we hold in regard to the origin of gynandromorphs, we are compelled to admit that they demon- strate the very early and rigid determination of the secondary sexual characters, the possibility of their complete development even when the gonads of the corresponding sex are lacking and their independence of internal secretions. ‘To this extent they con- firm the results obtained by Oudemans, Kellogg, Meisenheimer and Regen in their castration experiments. Indirectly they indi- cate that the insect egg not only has its primary sexual characters determined long before fertilization and independently of the later nuclear or chromosomal phenomena, but that even the secondary sexual characters are in some manner also prede- termined at this early stage. Where great differences of stature are secondary sexual characters, as in phylloxerans, some aphids and rotifers, we find corresponding differences in the size of the male and female odcytes. ‘This is, of course, quite in harmony with tre remarkable predetermination of the embryonic regions of the insect egg. Long ago Hallez (86) and I (89, ’93) showed that in many insect eggs the regions corresponding to the ventral and dorsal, right and left, and cephalic and caudal portions of the embryo are clearly established long before the maturation divisions. The second class of cases, which indicate that the primary and secondary sexual characters of insects may develop indepen- dently of one another, are found among certain species of ants, the males of which, though developing g compl and external geni- talia of the usual type, hare nevertheless become decidedly femi- nine in their secondary sexual characters. That this condition 1s an expression of degeneration seems to be indicated by the fact Effects of Castration in Insects 429 that it occurs only in parasitic species of the genera Anergates, Formicoxenus and Symmyrmica or in species like those of the gen- era Cardiocondyla, fechnomyrmex and Ponera, which form small, scattered colonies, often with a tendency to lead a secluded or subterranean life. In the three parasitic genera the males are always wingless and resemble the females and workers in the struc- ture of their bodies. The resemblance to the worker is very great Fig. 8. A, winged male of Ponera coarctata in profile; B, winged male of P. eduardi; C, subergatomorphic male of the same species; D, ergatomorphic male of P. punctatissima ( After Emery.) in the case of Formicoxenus. In Cardiocondyla and Ponera we have a number of species whose males show a similar approxima- tion to the worker and female type, and in one species of the latter genus, P. punctatissima, shown in the accompanying figure (Fig. 8D) the male is indistinguishable from the worker except in the structure of the genitalia. We have here, therefore, a true inver- sion of the male, so far as its secondary sexual characters are con- 430 William Morton Wheeler cerned, apparently as an adaptation to ethological requirements, although the primary sexual characters have remained unaffected. If it be true that the rudiments of the secondary sexual char- acters are set aside so early in the development of insects and re- main uninfluenced by the internal secretions, we can understand why these characters exhibit no modification in cases of surgical castration and why the modifications induced by alimentary, nutricial and parasitic castration bear the aspect of inhibitions or retardations of growth. Normal imaginal development in insects, as is well known, depends on the amount of food accumu- lated during larval life and stored up in the fat-body. In insects surgically castrated during their younger stages there is nothing to hinder the accumulation of this reserve material, and all the imaginal characters, including the secondary sexual characters, are thereby enabled to Hegel normally and completely. But in insects that have been underfed or are infested with parasites the reserve materials are either prevented from accumulating or are consumed, so that the imago may have great difficulty in de- veloping its imaginal characters. It 1s not surprising that under such conditions the secondary characters are more or less reduced or aborted, as we see in the forceps of parasitized Forficula males, the thoracic and cephalic horns of male Scarabzidz, the mandi- bles of male Lucanidz, the wings of female Lasii, and many of the other cases cited above. There is simply not enough nutri- ment to permit of the full growth of the characters under consid- eration. Their Todiien tone therefore, 1s readily explained in insects as due to malnutrition and we are not compelled to invoke the internal secretions, or hormones, which play such an impor- tant and interesting réle in the sexual physiology of vertebrates. 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Apténie et castration parasi- taire. C.R. Acad. Sci. exviii, 1894, pp. 1106-1108. (Transl. in Ann. Mag. Nat Hist. (6) vol. xiv, pp. 74-7 Effects of Castration in Insects 435 La Baume, W. ’10—Ueber den Zusammenhang primarer u. sekundarer Ge- schlechstmerkmale bei den Schmetterlingen und den tibrigen Glieder- tieren. Biol, Centralbl. xxv, 1910, pp. 72-81. Ley, J. ’77—On Intestinal parasites of Termes flavipes. Proc. Acad. Nat. Sci. Phila., 1877, pp. 146-1409. ’81—-The parasites of the Termites. Journ. Acad. Nat. Sci. Phila. (2), vill, 4, (1881) pp. 425-447. von LenuossEk, M. ’03—Das Problem der geschlechtsbestimmenden Ursachen. Jena, 1903. LeuckarT, Rud. ’87—Neue Beitrage zur Kenntniss des Baus und der Lebens- geschichte der Nematoden. Abhandl. Math. Phys. Cl. d. Kgl. Sachs. Gesell. Wiss. xiii, no. 8, 1887, pp. 567-704, Taf. i-iil. Marcual, Paur. ’96—La reproduction et l’évolution des guépes sociales. Arch. Zool. 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Journ.’ Morph. vu, 2, 1892, PP- 245-251, 3 figs. ’°o5—An alternative interpretation of the origin of gynandromorphous Insects. Sci. xxi, 538, 1905, pp. 632-634, 1 fig. ’og—A biological and cytological study of sex determination in Phyl- loxerans and Aphids. Journ. Exper. Zool. vii, 2, 1909, pp- 239-352, I pl., 23 text figs. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 8, No. 4. 436 William Morton Wheeler Mrazek, At. ’o8—Myrmekologické Poznamky. II]. Acta Soc. Ent. Bohemia, v, 4, 1908, pp. 139-146, 4 figs. Newport. '48—The natural history, anatomy and development of Meloé. Second Memoir. Trans. Linn. Soc. London, xx, 2, 1848, p. 335. Osporn, Herperr. ’96—Insects Affecting Domestic Animals. Bull. no.5, n. ser. U.S. Dept. Agri. Div. Entom. Washington 1896, pp. 302, 170 figs. Oupemans, J. Th. ’99—Falter aus castrirten Raupen, wie sie aussehen und wie sie sich benehmen. Zool. Jahrb., Abth. f. Syst. xi, pp. 71-88, pls. i-v. 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Zur Strassen, O. ’98—Ueber die Riesenbildung bei Ascaris-Eiern. Arch. Ent- wick. Mech. vu, 1898, pp. 642-676, 2 pls. g figs. CONTRIBUTIONS FROM THE ZOOLOGICAL LABORATORY OF THE MUSEUM OF COMPARATIVE ZOOLOGY AT HARVARD COLLEGE, E. L. MARK, Director, No. 210 A COMPARISON OF THE REACTIONS OF A SPECIES OF SURFACE ISOPOD WITH THOSE OF A SUBTER- RANEAN SPECIES PARTE TI A. M. BANTA Ieee Kperimedts withime chanical stimulation, serie terers'- emrerateeelctsyatrn (eae . ..: ».. 9/8) 4} 1 7 ASO eh Mi J r/ 1] 2 as pe 3 DAS a CAg se | I 2 SULOPOdS s,s =: - 7|8| 6 5} 3 gy a} I 3 59 |53 [36 j20 | 9 \10 jg 2g} 3) 6) 4) 8) 14} 218/318) 9/9 j25 (= a) SS eee 168 66 21 14 51 THE JOURNAL OF EXPERIMENAL ZOOLOGY, VO. 8, NO. 4. 452 A.M. Banta 4) 20 extreme responses were given by Cecidotea and only 25 of the tests; 10 of which were upon the flagellum of the antenna, failed to produce any reaction at all. With Asellus the same tests produced no extreme responses, while there were 63 failures to respond out of a total of 80 tests. Hence it appears that Czecidotea is quite responsive to the weakest current used while Asellus is only slightly so. The same relative lack of sensitiveness noted in the flagella of the antenna, when stimulated by the bristles, was again noted with the currents. ‘To stimulation by localized currents the fla- gella in both species are rather insensitive. ‘This is especially true with Czcidotea, whose flagella seemed less sensitive to this sort of stimulation than those of Asellus. To stimulation by bristles the reverse was true for in those experiments the flagella of the antennez were more sensitive in Cecidotea than in Asellus. A possible explanation of this discrepancy is that bristles tend to produce vibrations more than currents do and that the flagella are probably quite sensitive to frequent vibrations. This will be taken up again when the discussion of the reactions to sound is reached. ‘The portions of the body of Asellus most sensitive to stimulation by currents are the head and the bases of the anten- na. ‘They were almost the only parts responsive to the weakest currents. In Czcidotea the maximum sensitiveness 1s less con- fined to the head and flagella of the antennz than in Aselius, since it is shared somewhat by the uropods and legs. This point was well brought out by using current 5 with a number of pairs of individuals. Czecidotea was found to be somewhat sensitive to this current upon the head, base of the antenna, uropods and legs, but Asellus was seldom sensitive to it at all and never except occasionally upon the head and bases of the antenne. From these experiments upon the effects of mechanical stimu- lation by localized currents of water the following conclusions are drawn: 1. Czecidotea 1s much more sensitive to water currents than Asellus. 2. The sensitiveness of Czecidotea is only a little less marked Reactions of I sopods 453 with the weakest than with the strongest of the four currents used, while the sensitiveness of Asellus almost disappears within the same range of stimulation. 3. The threshold of stimulation for Cacidotea is considerably below that for Asellus. 4. The flagella of the antennz in both species are only slightly sensitive; contrary to what is usually true, to water c rrents this organ is more sensitive in Asellus than in Ceecidotea. 5. The most sensitive parts of the body in each species are the head and bases of the antennz, but the uropods and legs of Czcidotea are only slightly less sensitive than such other parts. 3. With the Concussion Produced by a Falling Solid Body Striking upon a Surface of Wood A third method of testing the relative sensitiveness of Asellus and Czcidotea to mechanical stimulation was by trying the effect of a mechanical vibration produced by the concussion of a falling solid body upon a surface of wood. A steel ball and a lead shot were used. The steel ball weighed 3.505 grams; the shot 0.542 grams. At first only the steel ball was used and it was dropped through a distance of from 50 cm. to2cm. Since the threshold of stimulation was not reached for either species by the concussion produced by the steel ball, the lead shot was used in an endeavor to get a weaker stimulus. The momenta of the two falling bodies were calculated,® and the falling distances chosen so that the concussions produced by the two bodies would form a graded series. The metal balls were dropped upon the flat, smooth surface of a pine board 30 cm. long, 1g cm. broad and 1.2 cm. thick. The balls were dropped from between the thumb and forefinger at various levels, as determined by a meter stick, and made to fall ata particular place on the board. The isopod to be experimented upon was put into a Waxed-bottomed stender dish 6 cm. in diame- 5 The effect of the friction of the air and the difference in the hardness and density of the two bodies were not taken into account. 454 A.M. Banta ter. [he dish was placed near the center of the board and 8 cm. from the place where the ball struck the board. In order to ob- serve slight movements of Czcidotea during the tests, these ani- mals, which are white, were placed in a dish the bottom of which was black. For a similar reason the rather dark Asellus was ex- perimented upon in a dish having a whitish bottom. In every case the animals were allowed to remain from 15 minutes to an hour in the dish to become accustomed to the new surroundings before the experiments were begun and no animal was experi- mented with until it had apparently begun to act and move nor- mally after its transference. In these tests the following grades of stimuli were used. The steel ball was dropped from 50 cm., 20 cm., 10 cm., 5 cm., and 2 cm., and had the following momenta at the instant of striking the board:: 1007, 004,401, 347,.and:219 ©) G- S-sumies.¢ In using the lead shot for the lower range of stimuli, it was deemed advisable as a check to test the reactions of the animal to vibrations produced by both the steel ball and the lead shot when they collided with the board with the same momentum. Hence the shot was dropped 83.6 cm. and the ball 2 cm., so that in each case the bodies struck the board with a momentum of 219 C. D.S. units. The shot was used also from 50, 30, 20, 10 and § cm., producing momenta of 169, 131, 107, 76 and 54 C.G.S. units. The whole series of momenta used then was (1) 1097, (2) 694, (3) Agr, (4) 347, (§) 219 (steel ball), (6) 219 (shot), (7) 16073) eieae (9g) 107, (10) 76 and (11) 54 C.G.S. units. These will bexetemed to in future as momenta of grades I to 11 respectively. In test- ing an animal the greatest momentum was first used for 20 trials. After each individual trial the result was recorded and the animal allowed to come to rest again, if it had responded to the stimulus. If the animal seemed erratic in any way, subsequent sets of 20 tests were made till constant results were obtained. After com- "momentum = mv V=YV 2S¢ .'.momentum = m] 2sg m= mass, s= distance through which the body falls, v = velocity, and g = gravity. Reactions of Isopods 455 pleting the tests with one momentum for an Asellus, the dish con- taining the animal was gently removed from the table so as to be beyond the influence of the vibrations, and the experiments were repeated on a Cecidotea. This method of procedure was fol- lowed for each pair of animals through all the eleven momenta used. The kinds of reactions were designed by numbers as follows: O, no reaction. 1, sight movements of antenne or other appendages. 2, more extended movements of antennz or other appendages. 3, movements of appendages and bending anterior end of body. 4, same as “2” or “3” followed by the animal’s crawling. 5, crawling at once. For each test the number corresponding to the reaction was recorded. ‘Table V shows one of these records complete. Table V indicates that the responsiveness of this Asellus was most pronounced to the vibrations produced by the falling bodies striking the wood with 1097 and 694 C.G.S. units of momentum, and that there was a rapid decrease as the lower momenta were reached. With Cecidotea the reactions were most pronounced to the higher momenta used and a rapid decrease in responsiveness occurred with momenta below grade 4, but the responsiveness is not entirely lost even with momenta of grade 11, only 54 C.G.S. units. Czcidotea responded not only more often but with a greater vigor of reactions to all the grades of stimuli. These two individuals are typical of the reaction of the two species as Table VI will show. From Table VI Asellus is shown to be less reactive than Czci- dotea to this sort of stmulation. This difference appears not only in the number of the responses, but in the vigor of the re- sponses, and in the range of responsiveness, for Asellus is not sensitive at all to such stimulation when the momentum 1s less than 169 units, while Cacidotea appears somewhat responsive to as low a momentum as 54 units. Asellus gave the following aver- 4.56 A.M. Banta TABLE V ASELLUS COMMUNIS NO. I, OF g MM. LONG RODY USED STEEL BALL | LEAD SHOT — | == Distancevofpballl ami crn’ is, a aetelepsthavele ncececssey saci FO) 120), Tol 5s 2 183.6 §0| 30) 20) Io 5 Momenta in C.G.S. units.................+.../1097| 694) 491) 347) 219], 219] 169) 131) 107, 76) 54 Gradesiof momenta-ciasee citar nee ee VoL eri Sel eee | 5. | 6] 7 | 8 |oumMoyan - SaaS Ou! 401/20; ||MO) Calm Ouleeo Tessa O30 500 Va: oe beenal Cosl) sonlligtonte | ZEN) $49 On)! 20x Os elon TO, ° 2h. Os|/ On ne Onll MOG ERO || Os}, On}, 204) 205] 504] Mos) soa | | | 3 OOD O | of o | o| WeZoye|| atoth |p Uefophl fees) lloras | ° | | ° | OM ta| cou|n-Ohllecor neon RE AaCtlOns\istersieices e078 tacit otek ens ananee s eyefouscoee + {iat Som) OE | o | ° | o} of | 2 Ip e2alleOnltgoultata | o} 0| (agral at Os 09) stOs| annie 44) | con Pasiine, | o | fo) | e251 |= ON] MO: | Ta) PO OM |anO 2| 1 Ti |) 10) |) 2Oyl|eonl 0 ll a Weel “for akon | o| o hee | 4 | 1) 0))| On| Mono | Ba Bu) On| Od, OullOn| nou arll wey cos! aug ton eon ued | 0 4s Poul aula oulltno | o| | | | | is Numberiof'responsesin2ovtrials.:....0.\.00c5-] Tal 13) 4 45) orale I | Sumisoliresponsesiiscr epee oot epee owaletete tetera el ote | 33 Sf Zt ss IP ee Naas Averageivigor of respomses®. . -cis -is inieiclorei 2 24|) 28) a | od) 28 eee ih Reactions of Isopods TABLE V C#CIDOTEA STYGIA, NO. I, rote. 10 MM. LONG LEAD SHOT 5 54 | | 10 10 11 | 20 107, 76 9) | 8 3° || 7 STEEL BALL BODY USED - 1097) 694| 491) 347 219] 219, 169 131 aS MOmMer tame GG) Se UNIS «se 5 aersisicialcvencea os Gradesyol momenta) s.. 0. -s eee cece ate] Dis fan cesOlmialliincmicssrie ns see esa aser ct IRGHREMONS Sse clecwis carts ce cs Fe (o} fo} (e) ee eet oO Ow t+ - (0) 4-7) “io) ene is Tah O Doh nd + | = a fo), ao} “i(0) me: a ie 2 ¥ || Cxcsumel Oo} (OF om —_—_—————— (ne w BO st eS a ~ Oo +o + Dr Sites isa) By WOOL aw t+ isa) nN ~ OM - O — m= OW isa) nwo coves) Bene —— = Number of responses in 20 trials.............. AXVela Pe wi POLORTESPONSES).e. da cinis sins sors = eee SUEUGHTESHONSES occ cucioe, «oh ciet- aay eases ale Se A.M. Banta TABLE VI Summary of the reactions of six Asellus communis and six Cacidotea stygia to mechanical vibrations produced by the concussions of bodies falling with various momenta. The body used in producing the con- cussion, the distance of its fall, its momentum at the time of producing the concussion, and the grade of tts momentum, are indicated in successive horizontal columns at the top of the table. For each individual tested are given in italics the total number of reactions obtained out of 20 trials for each momentum used, and the sum of these reactions on the basis of their vigor. Following the summaries for the six pairs of individuals are given in the last four horizontal columns the total number of responses, the average number of reactions out of 20 trials, the average sum of the reactions for each 20 trials, and the average vigor of the reactions for each momentum used. For example, Asellus No. I gave 14,13, 4, 4,1, 1, 1, 0, 0,0, and 0 reactions to the differ- ent momenta used, and the sums of the vigor of these reactions were 33, 36, 4,8, 2,1, 2,0, 0, 0, and o ASELLUS BODY USED | STEEL BALL | SHOT -. es 2 [Ces il | ees = » l | l | Distamceottalldn vem: vce a tocatisy, oft ow Sel §o) 20, Io) 5] 2 |83.6 50, 30, 30) Io & MomentaximiCiGeSsunltsirccciystic te eraser LOOT) 694 471 347| 219] 219 169 131) 107] 76) 54 Grades ofmomenta.n. y.4as es ee see | 1) 213) 4) 5] ©) 7 | 8.) on mone No.1, 2,9 INO-ofrespomses) onto tjo tue oor 14 13 AN 4n\ 0 | HEN) is mm. long i Vigoriofwesponses.<.1q8 ef-s] (33) 230l) Ao Sal waa aa | No. 2, 2, 8.5 \ Nozofresponses.:.....-0 sec: 16, 16) | 4| Ir} 2\| oO | fam.long | Vigorofresponses............-| 27] 21 34. Fal) Wille 24 eo | No. 3; 2,9-4| No.of responses...-.........-.| 9] 5 6. 31) -oull oll wom | | mm. long Vigor ofresponses.,. 3... -2..- LO} 2) a7h|, Sail On| OnE RO, | No.4, 2,9 INGiohinesponses®.cne1 & n =< | TABLE VII, Parr 2 406 CHCIDOTEA STYGIA A.M. Banta Lu an a me “-) ~ Sie 3 ee C < . ~ on ye ~ 3 ~ + & ae. 3 Of Lae OM OF ica (0) iene 10) FO) et ae m7 0 wm O = a cs 2 Na) el ts ae . Oo & CS UNIO On IA Ae OO 6 ini 2 fad ion n | | 4 oral eal Hee! gy yo |HR8lBs! 2 Zz Zz Zz jez Zz Zz Zz Zz Zz Z v2 Zz Z RIiZe| =e | & |peipalo}]a}] 6 |e eM Mace || Cialecre| eealagcie| Sell Solace Sale: eco S vi Ux Q Q |Snlao! s = < cS se | < < < ) & S 15u/09! & & 5 4 o] & 5 5 5 = 4 4 z rs = 5 S| wR 2h = = > c ow w wo ioe] ne wo ioe] les] fee] ive] w ive] & fee} oo o w | me Et & & ee aS & & & & ye & & ist & & i] & ic] & ical + 1S es SP iS) iS iS) iS) Be)» Was i) to i) 4 H H H H fora) gals e ; rn Sater steal eSee cw Hazen |S Stlmce alse eae lll seg ON eae |e are | ONES | ovat he aLva ela) t= 1 ee ae i i ee Oh gr ba F - Fah = : 4 4 BS \io8|] © So | S|\mam HY 4 4 He |e Zw) we | wo 4 4 4 4 Holo 4 el H Ke) 7 Z 2! 06 OF SAS se: |e. | Z oS | eo |le|o | 6 oS oS eM Aeeco eeee eco | Oe lo VOR Tort Bail Oita. Ino ieoel scl Gel 0 | Oe | oulic cz 26} =a ap) lott BB | a 5 a a |o 4 Oy | oy DN 2 7 Di a 7 fon an ea | a lee \SS\e6 ei | taal t=O hak ba etc Per= ret ple Ss et |45|44 S ss a1 45 SAN eee S zAiGS 36/9 z Saisie >Sl>o S R/)Ss [isteas|[is <4 2 B ; S sal eed ‘ | | Soe : : 61 | SI | ZT | OT | ST | PI | €T CLS Ol al 26.1 Si eZee On eS ay ee eee Cea) INANINAIXY JO YAEWAN | | \ “juauitsag.xa ay1 Butanp spotiad snotive ayy qo ydno. ays fo fo pua saddn ays ut Siaquinu ay) Uaais a. Saul] Durpaarons ays uy *saury aor ystf ayj ut uaasd auv sjuautadxa ays fo Susuussaq ays w ysnony ayi fo pua waddn ay} ut sppnpiarpus fo saquinu ay) puv aynuim icg Siajeustjuad ut juadiny ay fo yiBua.qs ay) “pasn S]DUIUD fo 4aquinu aul ‘ap aud ‘squautiad xa aud fo 4aqQuinu au ‘vajom. fo SJUIAANI yum SN1]9S F uo squautsad xa 6y] fo S]iDJap auios uaais auD 2]/qV1 Siyd uy TA WIA L 473 Reactions of Isopods ‘unos Sururolpe ayy Ul papsOdeI SUONBAJasgO IoYIINy puke yur) oy Jo pud JaMOT OY} 0} UDATIP a19 STRUTIUR IY g ‘uunjoo Juiutofpe ayy ur usATs st yorya Jo prodar ayy ‘Ap SuIMO]OJ ey pue yysu seao panuyuos sem quoursadxy ¢ ‘ay £ pasvoaddop asuodsad jun oui edesaAy ‘ry br asuodsar WNUNxeW [YUN au advIOAY qi | pb | ob gh | 4 zig 8 jar, |B |e [e rey inl Boe |e ames etiepeet aia paseaisap _ | | | asucdsad [13UN 11e}8 10}Je sNoYy UT out], 4% | $1 | ¥1 131} k | & | ¥¥ | $e | I ATE 1% |% | eh ¢ PgR 20 ge | bas a "+++" "* pgzimo90 asuodsas | : | “XUU [IQUN j4e]s Ja\je sInoy Ur oUt], — = ee es = Searls aa | © C\\4E 5 \h9 |° | GQ) |e I | |g 6 | Th | ieee sor anaes caer" “SuiULOUT SUIMOTIOJ OY J, | ad et | | | | | Miavewe Aptech ORE ee Oe UENTIIS BH eee er | 2 | Z | | i Np ro eR aC TRS cece eT | —|9 6 | | 2 | Vie CMP fete i 1 9 le cuccta ers hrm sancti Gec, BeOS yt ¢ ee | | = aes EP AEN pea Cho nee etic el “ry GZ Ty = — Ol g 6 | nee ewe) eho foe era prey aa tos Gere | FTot Seige maa | 9 9 ai | — | = esol) 8 Fy Ura west duel diay ss Aisles Pe TE Ta Lo) ue | § = = |= 9 | fa fe | + eee “3149 Sale| E |+ |6 Bec: |= | \z | lg | Ss isiea nent 23 =e isa a | € 6 9 | |= Z | le | S YH DW on ccnre-teranc Ooo 5 sees say § — af > [eten | peat he Ieeme6 =| |= |= | gel Gg) a” ede vet ee alicees RRR et = py | 9 = ere | € f—aie g | tee Hea he | Tie E EA betes ctrcercregucit er PORN ec ay = |e = |e L 6 ee |e | |= | Neer y hee: a | ¢ bE FS carne Si eae Ee 13 ae = hy Aen ee eye oe | ¢ Iisa lee = ¢ g DN A Laksies a eine an ac reese eer = |Z S | ey es es ae ee iS 9 sl al — | lz L Bde raha Nert oe a eliarat ooireen ele aS FES “ry Ee | de Yb) a er ia | — re iil sell A £ lz lou Pe) A ait : Dahan . “ay Ez OL! ig 9 = —/9 — he | 4 6 |* Ne fie | - | [2 if Repeal ne pases an bese deeses ery ag alt eae | | + + 6 gs |-|- == eae lz L Gault ; seer ks E ‘yz cl 5 | 2 18 th os 19 9 9 |Z 9 SA ee ee | ee [i gies > yu Uy i Rae Ia SEEM ap —}|—|g |Z emt ae Ria 16 raat) | ao ig Bldee here) a Seth tes Pi eS esea eee ea 9 as =} | 2 Z 3 g 8 \ Ba. Sei = y ete iene, BSW) [y/o provorer coinireto torr rae Oy ano c =< yo 4 4 TABLE IX This table gives tne details of the experiments on Cecidotea with currents of water. The plan of this table is like that of Table VIII already described. A.M. Banta AVG ONIGGOdad WOU GAQONILNOO INAWINAdXa AVd ONIGAOANd WOU GHONIONOOD INAWIYaAdxXa a goor ‘oz YaaNaOAG 2S oa t+ AVG ONIGAOAAd WONT GAANIL -NOO INAWIYAdXA ‘AVG GUIHL AVG ONIGAOaad WONT GAONIL -NOD INANIAAdXa ‘AVG GNOOAS (o} wm 00 go6r ‘61 xaawaoaa si ae wm sS go6r ‘gi uaawaoga | © Se ees wD ‘ vill coal o goOr ‘gz AAMWAAON Ss a ° 1 goOr ‘gt AMHWAAON | O SS a Ht goOr ‘61 AAHNWAAON ie) ae : ° = goOr ‘£1 AdAHWAAON ° RSE w a goor ‘6 uaawaAon | BB ih go6r ‘g ATH#WAAON © ee | + = 2 Se a: & ew ° z a 45 z Bene ge & me be a a ~ : * - f n . eS ese iaeroe be a] COs as ° a " x G 4 aes a a TAS) Me a Zz i = Gta ae 8 =) ke . Z oom 5 Sela Bx a O c Eieia | a Saal VE Sta Zan S Number in upperend atstart....... Aitericiminiyy. menicwet. omits ear NOMM SN ee whee Sey. ea er: iGO aad tntomacrmmcc dens 475 Reactions of Isopods +hLz rs *17—6 ‘dxa ynoyqitAy *sadeIOAy ‘uunjoo dururofpe ayy ur uaatd sr yorya JO prodaz oy ‘Aep Surmoyjoj ays durunp pur yystu s9AO ponuyuos sem quowredxy . % | | | | gS | | 2 te ul . ul asearo asvaso -op ou -ap ou ue CE eee aR ee eta i | | | korg mee 0 Br Sia) 8| opener + | }t I | — ee ~~ lke == | I or 8 — = $ ¢ =* is BA ate i aie = — —_ = — 6 — — — — | 9 = iS = I = == — = L L 6 — — = as = = = Ol — = HE, + — == Z 8 — L _ _— 9 8 | I 8 | — — — — — _- L I — 9 9 t = 6 | ¢ g = | — | — -- —};—};—- Ld 6 8 z ate = = = L = heat 6 9 Zz 9 a. — — a L — 6 6 + = —|}— $ = — + 6 = t = —- | — = — = 9 6 -— I 1x4 ul OSPO1D | -op C00 (a ca Ppeseolaap asuodsai yuu yes Jaye sinoy ur suty A ae ig te * petIns3s0 asuodsa “xeu youn J1R4\S 4ayje sanoy Ul duITT, a< 8 pores esses ss -GUTUTOUL SUTMOTTOF OUT, 470 A.M. Banta remained in the upper end for about three-quarters of an hour, after which time the animals that entered the light seldom re- mained long. Czaecidotea was not often swept back. It wandered about in the light upper end for a time and entered the dark end again apparently by accident. Often, too, 1t turned back into the dark as soon as it came to the light area. Under the last conditions of experimentation Asellus seems more rheotactic at first than Cecidotea, but this reaction persists for a shorter time than in Ceecidotea. Probably a better way of describing this state in Asellus is that at first 1ts rheotaxis is stronger, as compared with its negative phototaxis, than Czci- dotea’s, but that its rheotaxis soon gives place to negative photo- taxis; while Cacidotea does not so soon cease to enter the light as Asellus, it never enters and collects in the light in large num- bers. Its negative phototaxis is from the first stronger than its rheotaxis. In regions of caves the cave streams, as I know them, ordinarily originate underground and flow out of the caves, although there are many exceptions to this rule. In Mayfield’s Cave, near Blooming ton, Indiana, which is the source of some of my material, there is no inflow into the cave from above ground except through sink holes in times of heavy rains or thaws. ‘The stream is merely an outlet for underground drainage. The experiments in which the upper end of the trough was darkened while the lower remained light, thus resembled in min- 1ature the conditions met with in this cave. Judging from the results of these experiments Asellus and Cecidotae ought both to enter such a cave, but Asellus being less persistently rheotactic would be less inclined to follow up the stream and remain within the cave. Czecidotae, while less strongly rheotactic at the begin- ning, is persistently so, and hence would tend to creep persistently into the cave. The added fact that Asellus, after having been in darkness for some hours, is positively phototactic would induce the animals to leave a cave after once having become indifferent to the current, provided that they were carried by accident to within a glimmer of light. The experiments in which the lower end of the trough was Reactions of [sopods 477 darkened and the upper end illuminated afforded conditions somewhat resembling conditions in caves which are entered by streams from the outside. Under such conditions, judging from the results of the experiments, Asellus, when carried to the inlet, would tend to leave the cave because of its relatively stronger rheotaxis. In addition to this factor, however, the positive pho- totaxis of Asellus after having been in darkness would also aid. Czecidotea, on the contrary, 1s always negatively phototactic, and while it shows a tendency under these circumstances to enter the light because of its rheotaxis, it 1s probable that this form of reactions is always subordinate to its negative phototaxis. oD Ys ORS GEE OOD Attempts were made to ascertain if a difference in the food of Asellus and Cecidotea was possibly an important factor in deter- mining the habitats of the two species. To this end specimens of Cecidotea were taken direct from the caves in Indiana and pre- served in formaline before they had an opportunity to feed upon other substances than those they might get in their normal sur- roundings and the same was done with Asellus communis ob- tained near Cambridge, Mass." The digestive tracts of each were then carefully examined to determine their contents. Afterward several individuals of each species were kept in clean tap water without food for several days and then placed in separate jars with bits of decayed leaves and living Ceratophy- lum and subsequently examined to determine what was taken for food when living and dead plant tissue were equally present. With Cecidotea the evidence pointed to the conclusion that the animal in the cave collects for food whatever decaying or- ganic matter it happens upon. Naturally dead plant tissue forms lM ancasellus tenax dilata, the common and extremely abundant Asellid from southern Indiana, was similiarly treated and examined. It was found that the food of this species from the region about the cave in Indiana is practically the same as that Of Asellus communis near Cambridge, Mass. 478 A.M. Banta the main part of its food. ‘The digestive tracts contained rela- tively small amounts of mineral matter. ‘Those placed with liv- ing and decaying plant tissue ate relatively small amounts of green alow, probably iat that taken in with the decayed plant tissue. From the evidence obtained from the series of examinations of Asellus it seemed probable that it takes its food either from sur- faces of dead plant tissue or from living plants. In cases where material apparently quite recently taken as food was found in the digestive tract there was always more or less fresh plant tissue and those individuals which were deprived of food for a time and then placed with both the decaying leaves and the living Cera- tophyllum seemed to have collected nearly all their food from the Ceratophyllum. There was, however, no evidence to prove that green plant tissue was a necessary portion of the food of Asellus. It probably could live upon decaying organic matter exclusively, as Czcidotae does, but it partakes of bee kinds of food and will use living plant tissue largely when both living and dead are present. In every case the digestive tracts of those Asellus examined con- tained a large percentage of mineral matter, in most cases as much as 85 per cent of the contents being inorganic. It will be remem- bered that the digestive tracts of even those Cacidotea which were taken directly from the caves contained very little mineral matter. The difference between the amounts of mineral matter taken with the food in the two species is remarkable in caves where Czcidotea obtains its food, organic matter is extremely scarce, while, where Asellus lives, the proportion of organic to mineral matter is very many times as great. This difference sug- gests that Cxcidotea possesses a superior discriminative power in selecting its food. Such ability would be of immense advantage to an animal in a cave. The lack of discrimination in selecting food clearly indicates, I believe, that Asellus is incapable of meet- ing the food conditions within caves. Reactions of Isopods 479 IV. GENERAL DISCUSSION The significance of the various separate results obtained in the different series of experiments has in several cases been pointed out in summarizing these results. Cecidotea stygia and Asellus communis have quite similar habits, except for the fact that the former lives almost exclusively in subterranean waters, whereas the latter, though occasionally found in cave water, is extremely rare there. Its occurrence in caves has been mentioned only a few times. When collecting in caves | have never seen either Asellus communis or Mancasellus tenax dilata, although both species occurred near caves in which I did much collecting, and the latter species was present 1n immense numbers immediately outside the caves. Czecidotea, when found above ground, has occurred only in water near and immediately connected with underground waters. These two animals, though differing in habitat, are much alike in many of their reactions to stimuli. Both are negatively photo- tactic to such intensities of light as they respond to at all; both respond in like manner to various tactile and mechanical stimuli, and the regions of the body most highly sensitive to these stimuli, are nearly the same in the two species; both are rheotactic, re- sponding to currents of water in like manner. When subjected to the influence of a current of water, in a trough one end of which was strongly illuminated, both species, though for a time re- sponding to the rheotactic stimulus alone, soon, in most cases, reacted to the light alone. Their food is nearly the same, both species feeding largely upon decaying plant tissue. The following differences in the reactions of the two species were noted: 1. Asellus is decidedly more responsive to light stimuli and responds to much lower light intensities (2.5 C. M.) than Czcido- tea (80 C. M.); after retention in darkness Asellus is for a time positive to a considerable range of intensities (2.5 C. M. to 80 or more C. M.). Czxcidotea is never positive. 2. Cecidotea, on the other hand, is decidedly the more respon- 480 A.M. Banta sive to mechanical stimulation, this difference appearing in the number and vigor of its responses and in its having a decidedly lower threshold of stimulation than Asellus. 3. Cecidotea, though at first less rhetotactic than Asellus, is persistently rheotactic, whereas rheotaxis with Asellus is only temporary. When the stimulus of strong illumination in the lower part of the trough was added to the stimulus of a current, the Cecidotea more persistently remained within the upper dark end. Although there are considerable differences between the re- sponses of the two species in minor details, these are, after all, not so much differences of kind as of degree. Physiologically considered, the two species are very much alike. The relatively slight sensitiveness to light shown by Cecidotea, as compared with Asellus, is what one might expect from the responses of eyeless animals 1h general, as Sonera with those which possess eyes, For example, Dubois (’89, pp: 358-359) found that Proteus, the blind salamander of the European caves, responded to light from a projec tion lantern. Semper (’89, p. 79) states that Proteus is sensitive even to daylight. higenmann (700, pp. 113-116) found the blind fish, Amblyopsis, sensitive to strong light etidenly thrown upon it as well as to diffuse daylight, for see animals, when kept in a pool in the open air, where they remained concealed among rocks during the day, swam about freely in twilight and at night. It has been shown by Payne, (07) that Amblyopsis reacts to light from a “100 c.p.” acetylene lamp at 32 inches from the end of the aquarium; but with this intensity of illumination an average of only 54 per cent of the individuals were in the end farther from as against 460 per cent in the end nearer to the light. With eoneide ein greater intensi- ties he found the reactions were much more decided. Hence it seems that the lower intensity used by Payne (’07, p. 318) 1s near the threshold of stimulation for Amblyopsis. There is no reason to suppose from any of these observations that such animals respond to low intensities of illumination. Reactions of lsopods 481 The nicety with which even slight sensitiveness to light may regulate an animal’s movements is well illustrated by the follow- ing case. Parker (’05, p. 418) found sensitiveness to strong light in the skin of the tail of Ammoccetes, and pointed out the sig- nificance of this in relation to the burrowing habit of the animal. Here again, the sensitiveness was to intense light only. The eyes of ee animals probably subserve all light-recep tive functions necessary for swimming about, but in burrowing the integumen- tary organs of the tail serve to distinguish intensities sufficiently to direct the animal to continue burrowing until completely cov- ered. Here, asin the other cases cited, the organs are not highly discriminating light-receptive organs, but they sufhce for the regulation of the animal’s movements. Among the higher animals those that possess degenerate, or poorly developed eyes are such as in general live in dark situa- tions. Their light-perceiving organs are of advantage to them only in aiding them to remain within a suitable environment. Highly developed light-receptive organs are of no advantage in their dark habitat, and organs capable of perceiving only consider- able changes in intensity of illumination are sufficient to serve as a check to keep them in their proper surroundings. Czcidotea nearly always lives in absolute darkness and ordi- narily has little occasion to discriminate between intensities of illumination. If, however, in approaching the mouth of a cave it becomes subjected to increased illumination, its light-receptive organs ordinarily are discriminative enough to prevent its leaving its subterranean abode. Whether its light-perceptive organs were adapted to its cave habitat or whether its cave habitat was adopted because its organs suited it for such surroundings, does not here concern us. At any rate, Czxcidotea’s light-receptive organs are sufficiently discriminating to serve their part in regu- lating the movements of the organisms within caves. We have no reason to suppose that a species lives where there is extremely little light because it is extremely responsive to light. On the contrary, we have every reason to suppose the opposite— that when an animal lives where there is little light and where it has little opportunity or occasion to be Pdacnecd by light, it will 452 A.M. Banta be little responsive to this influence. It is a part of the adaptive economy of nature, a frugality long noted with reference to eye structures, but apparently less remarked with regard to light per- ception in general. Such animals as the cockroach and Oniscus (cf. Cole, ’07, pp. 373-380) remain in relatively closely circumscribed and restricted dark situations by day. While their habits are such that highly discriminative eyes would be of little use to them, slightly acute organs for discriminating differences in intensity serve them very well, aiding them in reaching and remaining concealed within their diurnal haunts. Asellus often lives in aay exposed situa- tions, but, like Oniscus, it tends to seek the darker of the avail- able regions. It possesses better powers of discrimination than Cecidotea, and this serves it well in aiding it to find quite small and restricted shaded areas, which Czecidotea can not do. The experiments discussed in this paper clearly show that the subterranean species, Cacidotea stygia, has greater sensitiveness to mechanical stimulation (whether purely tactile or vibrations) than its near relative, Asellus communis, which lives above ground. Czcidotea proved decidedly the more sensitive, both in having a lower threshold of stimulation and in responding more generally and more vigorously to such stimuli. Hence these isopods furnish an undoubted case in support of the common belief that cave animals have acquired greater sensitiveness to mechanical stimulation than their near cele living in other situations. Since Czecidotea was shown to be much less sensitive to hght than Asellus, its greater sensitiveness to mechanical stimulation is a good illustration of the principle of increase 1n sensitiveness to one sort of stimulation in compensation for the partial loss of sensitiveness to another, 7.¢., the organism is so adapted to its environment that when one influence ceases to regulate 1ts move- ments another acquires increased importance and in a measure replaces it. There still remains one point to be considered —the bearing of these experiments on a possible explanation of the occurrence of the cave species within caves rather than outside of them and the Reactions of Isopods 483 occurrence of non-caverniculous species outside of rather than within caves. One needs to experiment with the two species for only a short time to be struck with the similarity of their responses to various influences. The minor differences in their reactions, however, are very significant in relation to the habitats of the animals. While Czecidotea is responsive to only fairly high intensities of light (80 C.M. or greater),it is always negative to any intensity to which it responds at all. Hence, if outside a cave, its light reactions alone would tend to lead it into a cave if there were one near, while if it were in a cave and wandered into the light near an outlet, its negativity to light would prevent its leaving the cave and passing into waters above ground. If Asellus were near a cave, its response to light would at first tend to direct it into the cave. But, after having been in darkness within the cave for a time, it would again become positively phototactic, so that if it came in reach of light from the outside it would escape. Again, Czcidotea, while less strongly rheotactic than Asellus, when first subjected to a current, is more persistently rheotactic. In experiments combining the effects of a current of water with light stimulation under such conditions that when the animals moved against the current they passed into a darkened region, it was shown that Czcidotea remained in the darkened region more persistently than Asellus did. This, as already stated, repeats in miniature the usual conditions found in cave streams, since they generally flow out of caves rather than into them. In such cases, even if Czcidotea were not rheotactic, its greater tenacity in holding to the substratum would better enable it to make its way into caves and remain there; whereas, if the stream were swift, Asellus could not hold its own against the current. In the experiments with light combined with a current of water under such conditions that when the animals moving against the current passed out of a darkened region into a strongly illuminated one, the other cave stream condition (found in streams which enter caves from above ground) was in a way duplicated. In those experiments Asellus was more vigorously rheotactic at first, but Czcidotea more persistently so, although under these condi- THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 8, NO. 4. 484 A.M. Banta tions neither persisted in entering the light for any great length of time. Asellus, however, after having been in a cave in darkness for a time, would become positively phototactic and would then have both its rheotactic and its positively phototactic impulses to direct it out of the cave. The suggestion that cave conditions involve fewer mechanical disturbances in underground waters than are found in out-door streams and pools, and that the relative immunity from mechan- ical disturbance within caves 1s a factor in determining the dis- tribution of the one species within caves and the ine outside, seems hardly worth following up. The difference in the food Piten by the two species seems to be a factor in determining their habitats. Czcidotea eats nearly exclusively dead plant tissue, even when provided with living and dead tissue in equal abundance. While Asellus takes much fresh plant tissue in addition to the dead plant tissue which it also feeds upon, it is not shown that living plant tissue forms a neccessary part of its food, so that apparently it might feed upon dead plant tissue exclusively. There is an extremely small amount of organic matter and a great prevalence of sand, etc., in caves as canna with the amounts of organic matter and inorganic matter in the situations in which Asellus lives. In view of this fact the relatively very small amount of dirt and small particles of mineral matter within the digestive tract of Cacidotea as compared with that of Asellus suggests that Cacidotea exercises more discrimination than Asellus in taking its food. The importance to an animal living in a cave of a superior ability in selecting its food could not be overestimated, since the organic matter there is so scanty. If Czcidotea possesses such an advan- tage over Asellus that alone may be a very important factor in determining the suitability of Cecidotea to a cave habitat and the Micali) of Asellus for the same locality. To recapitulate, we find some apparent importance in the factors of the animals’ food and their different rheotactic responses in determining the habitats of the two species, but the one factor which seems of most importance, and which alone affords an explanation of Cacidotea’s living in caves and Asellus not living Reactions of Isopods 485 in caves, is the different responses to light, —Czcidotea always responding negatively to such intensities as it responds to at all, and Asellus, after having been in darkness, being positively phototactic for a time. Since Asellus is so closely related to Caecidotea morphologically and physiologically, it would seem that if under stress of circum- stances any epigeal animal could suddenly become a cave inhab- itant, Asellus might be expected to be capable of undergiong such change in environment. Asellus is in many ways apparently nearly suited for cave habitation, and in time it may become fur- ther modified so that it will be capable of living in caves, but it is not fitted for cave life. Its physiological reactions are such that it is prevented from taking up cave life, and further its apparent lack of discriminative ability in selecting food renders most improbable the continued existence of a straggler of this species within a cave. This case fails to support Lankester’s (’93, p 389) theory of the sudden and accidental origin of cave animals, but rather lends support to the theory that cave animals must become closely adapted for cave life before they are capable of taking up such existence. V. SUMMARY Mechanical Stimulation 1. Asellus and Cecidotea respond in much the same manner, and corresponding parts in the two species are the regions of greatest sensitiveness. 2. Czcidotea is decidedly more responsive than Asellus, react- ing more often, more vigorously, and to weaker stimuli. 3. The threshold for Czcidotea is below that for Asellus. Rheotaxis 1. Asellus and Czcidotea are rheotactic and respond to currents in like manner. 486 A.M. Banta 2. Cecidotea, though at first less rheotactic than Asellus, 1s persistently rheotactic, whereas the rheotactic response with Asellus is only temporary. Photic and Rheotactic Stimulation 1. When subjected to the influence of a current of water in a trough the upper end of which is strongly illuminated, both spe- cies, though for a time apparently responding to the rheotactic stimulus alone, soon react principally to the light. 2. When the upper end of the trough is darkened, Czcidotea more persistently remains within the darkened upper end. Food 1. Czcidotea and Asellus take about the same food, but Asellus eats much live plant tissue with the decaying plant tissue. 2. Asellus takes in vastly more debris and particles of mineral matter with its food than Cecidotea. General Habits 1. Czcidotea stygia and Asellus communis are not only struc- turally similar, but their habits and reactions to various stimult are very much alike although there are several minor differences in their reactions. 2. The cave species is decidedly less sensitive to light than its above-ground relative. 3. Cave animals do not have need for highly discriminating light-receptive organs. Their movements are well regulated by light-receptive organs which are capable of disnneuicnnne only considerable intensities of illumination, so that when approaching the outlet of subterranean waters their negative response to light restrains them from passing beyond the limits of caves. 4. The subterranean Cecidotea is clearly very much more sensitive to tactile stimulation than its epigeal relative. Reactions of Isopods 437 5. Cecidotea, being less sensitive to light stimulation than Asellus and more sensitive to mechanical stimulation, affords an illustration of compensative sensitiveness to one influence for a partial loss in sensitiveness to another influence. Habitat There are several possible factors which determine the habitat of one of these species to be within and the other outside of caves. 1. There is a remote possibility that the relative freedom from mechanical disturbance within cave waters 1s a factor in deter- mining the existence of Cacidotea in caves. | 2. The difference in the reactions of Asellus and Czcidotea to light affords an explanation of the occurrence of Czcidotea in caves, and subterranean waters in general, and the virtual non- occurrence of Asellus in such situations; for the negative response of Cecidotea to light would aid in directing it into caves and keeping it there; but Asellus after being in darkness becomes positive, and therefore would move toward the light, 7.e., out of a Cave in case it had by chance made its way into one. 3. Cecidotea, being more persistently rheotactic than Asellus, and being able in creeping to hold its own against a current better than Asellus, would not readily be swept away by a stream flowing out of a cave. Asellus, on entering a cave under such conditions, would soon lose its rheotactic response and further would become positively phototactic in a few hours. Hence, if it by chance came near the mouth, it would react to the light and escape. 4. The reactions to the influence of a current of water com- bined with the influence of light afford an additional factor in determining the distribution of the two animals. 5. An apparently superior discrimination on the part of Czcidotea in selecting its food may make it possible for Czecido- tea, rather than Asellus to live in caves where organic matter is extremely scanty. ~ 488 A.M. Banta BIBLIOGRAPHY Banta, A. M. *10o—A comparison of the reactions of a species of surface iso- pod with those of a subterranean species. Part I. Journ. Exp. Zool., vol. 8, no. 3, pp. 243-310. Core L. ].’07—An experimental study of the image-forming powers of various types of eyes. Proc. Amer. Acad. Arts and Sciences, vol. 42, no. 16, pp. 335-417. Dusots, R. ’89—Sur la preception des radiations lumineuses par la peau, chez les Protées aveugles des grottes de la Carniole. Compt. Rend. Acad. Sci. Paris, Tom. 110, pp. 358-361. E1GENMANN, C. H. ’oo—The blind fishes. Biological Lectures, Marine Biologi- cal Laboratory at Woods Hole, 1899, pp. 113-136. Homes, S. J. °05—The selection of random movements as a factor in phototaxis. Jour. Neurol. and Psychol., vol. 15, no. 2, pp. 98-112. LankesteErR, E. R. ’93—Blind animals in caves. Nature, vol. 47, no. 1217, p. 389. NaceEt, W. A. ’94—Der Lichtsinn augenloser Thiere. Biol. Centralbl., 14, pp. 264-393. Parker, G. H. ’05—The stimulation of the integumentary nerves of fishes by light. Amer. Journ. Physiol., vol. 14, no. 5, pp. 413-420. Payne, F. ’01—The reactions of the blind fish, Amblyopsis spelzus, to light. Biol. Bull., vol. 13, no. 6, pp. 317-323. SEMPER, K. ’81—Animal life as affected by the natural conditions of existence. New York, 8vo, xvi + 472, pp. 2 maps, 106 figs. YeRKES, R. M. ’03—A study of the reactions and reaction time of the medusa Gonionemus murbachii to photic stimuli. Amer. Jour. Physiol., vol. 9, no. 5, pp. 279-307. ea, a, EFFECT OF CHEMICALS ON GROWTH IN PARAMECIUM A. H. ESTABROOK From the Laboratery of Experimental Zaology Fohns Hopkins University With ONE Ficure CONTENTS TESTO GEL OH opsteye a aches << atsns Yas ake ea cctools seas atatn,c/ a0 seaunye¥e Bie v's e eteraid an lacsaterete aia fer< al are, sla ee ose. 490 Weerieriabancy Wri Hodges. cost Sar ce Ah Nie hed TON AS) Sea Tg deh 491 Growing Gistiledkwa tent. aea sets yee ces = oss) - yiches cielRtbe as SiS nae. 6:2) a ea Sas eens iets Atego 492 Effect ofapure distilledtwaters:,..<\etutetoyterar tela) vials ciccnns « eee A deere seade 492 Efectiot addingyNa€)ita the distilled’ water: ...02..- << -l-scloee ne ees te) oleae 494 Growth nisodiumchlondes.c.mc/se cats ia orate steset stale mse seis @ Sins oie aoe ofalelovetersvaiere evslete wre oie sia ayes 497 Preliminary experiments on the effect of NaCl on the form and dimensions of ACER LES Sate reer tae (aves shal at sveinve ctapors/ ste Gr ouetenet darcy apace ibeust otaasc ahs Seles a ake etearakorer etn 498 Directmeasurementiok growth alter fissloWesc- .- 42 ef tasteless aaa 501 ROle of osmotic pressure in effects of sodium choloride....................200-- 504 Comparative resistance of adult and young Paramecia to NaCl.................. 508 Are Paramecia in stages preparatory to division like adult or like the young in their FesistanceyeOrebeSaltes ncn. 98.2 Rae ens te es OA sie Sa scinre SEA A eae rar ah 508 Acchinatizationy tor Nal) os. cutee eis Wee ete oe ciate a eis atone t ravine eee nates 509 EfectsiOh nico une OD) prO wth. ts Seana clare a oe Sale seem tn oes ova Nortel fase 2 Se ne acne oe ee Nabdare 511 Efecto mcotine upomadul/Parameciaeee asa. --)4:2,c0))sceess aoe tee tae 511 Effectiotmico tine oniprowin ey Parame clas wantstsle ates << 2 eicats easjesje'e siege cio eee 513 Different resistance of young and adults to certain concentrations............... 516 BhectsronstnychMine ONT ero wth cc teint eters aca nis tects otters oe alae anes a a 516 Effects offstuychnine uponsadulbebaramecia./. «0.22 ciaede nae « -le nrledss > seeeles 517 Growth of Paramecium in strychnine after fission.............---2-- eee eeeeeuee 518 Comparative resistance of adult and young Paramecia to strychnine ...... .... 521 Efectn ot dleohol onl prow thsi epee eines et ania © a olniel rons mis ae a oiajes. wd Aslarn af flemane Sa 523 Effect: of alcohol ‘onssrawiip, Ear aletldy jo.c 2s seh ds one nes eae veda eee 523 Resistance of adulérandsyoungaine alcohol: 05 .j.c-.2% 00 - da eee n+ esas eee 528 Riecthontood, on: prowing: ParaMeclay cafe so teieisie score 2's are aint Sects sin Ao c vin oles seminar 529 Summary and discussion... ...-. 2. .s0e. cece eee cece cece eect eee eect e eee eee n ee enes 531 SSPE rete ae crc typ A ahh ois a ENA Oe a SIE eS ois ha ate cated ni « Y's, oka: sxntevaies ctr Sout 534 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 8, NO. 4. 490 A.H. Estabrook INTRODUCTION Most studies of growth have been made upon organisms com- posed of a multitude of cells, so that the growth observed is a complex resultant of the growth of the component cells. It is evident that in producing such a resultant, the method of growth of each single cell might be very different from that of the organ- ism as.a whole. The present paper examines the growth of a single cell, as affected by various agents, particularly chemicals. There have been but few studies of the growth of single cells. Jennings! (’o8) studied the normal growth of the infusorian Paramecium, showing that it follows a curve that is similar, 1n a general way, to that of the growth of a multicellular animal having determinate growth. Popoff (°09) investigated the relative growth of snaene and cytoplasm in a ee of infusoria. Balls (08)) examined the growth of the Sore-shin- fungus, particularly the effect of temperature upon it. In this organism the growth of the cell is indeterminate, as in many of the hgher organisms. The common infusorian Paramecium has served as object of the present study. The normal growth of Paramecium 1s thus summarized by Jennings: ? In the following investigation, I have endeavored to find out some of the factors determining growth in Paramecium, and to discover whether size may be Cormeen modified by external conditions. The effect of the following chemicals upon the growth of Paramecium has been studied: sodium chloride, the alkaloids nicotine and strychnine nitrate, and alcohol. ‘Vhe growth in pure water, containing no food or salts, has likewise been studied. I wish here to acknowledge my indebtedness to Prof. H. Jennings, under whose direction the work was done, for valuable suggestions and criticism. I also wish to thank Prof. B. FE. Living- ston for his interest in the experiments on osmotic pressure. 1 Loc. cit., page 441. 2 Loc. cit., page 448. Effect of Chemicals on Growth 491 MATERIAL AND METHODS In all the experiments one pure line of Paramecium has been used, that designated by Jennings* as 1,. This was the largest race he isolated, measuring from 200 to 230microns, orabout one-fifth of a millimeter. All the specimens used are descended from a single pair of conjugants which he selected from a wild culture. Thus, by using animals all derived from a single pair, any possible differences due to diverse ancestry are excluded and only variation within a a pure line has to be dealt with. The Paramecia were kept in stock in large flat dishes holding about 2 to 3 liters. The hay infusion in ie they were kept was made fresh about once a week, by pouring off some old infusion and adding new hay and fresh water. The Paramecia were thus kept in good condition and afforded an abundance of material at all times. Paramecia undergoing division were picked out from the stock culture with the capillary pipette, and put on a slide with a con- cave depression. At the moment when the two halves separated, one of the daughter cells was placed in a few drops of hay infusion in one concavity on a ground glass slide, the other daughter cell was placed in a similar concavity on the same slide, this latter depression containing the solution whose effect was to be studied. The time at which the halves were isolated was noted, and after a certain interval they were taken out, killed and measured separately. This gives a direct method of measuring growth, as the specimens, can be killed at any age desired. The control in hay infusion gives a normal curve of growth and acts as a check, while the other shows directly the treet of the solution upon the normal growth. The fact that the two halves come from the same mother cell gives an almost ideal condition of experimenta- tion, since the twoorganisms studied are of the same age, their pre- vious history is the same, and they are in the same’ physiological condition at the moment of separation. The Paramecia were taken out by means of the capillary pipette, gathered into one or two drops of liquid and then killed by sud- * Loc. cit., page 494. 4.Q2 A.H. Estabrook denly adding a large amount of Worcester’s solution (a saturated solution of HgCl, in 10 per cent formalin). When Paramecia are properly killed by this method no distortion takes place. They were then measured by projecting them with the Edinger projection apparatus, and measuring the image with a millimeter scale. Lenses of such combinations were used that three microns in the object were equal to one millimeter in the projected image. The measurements were then converted into microns by multiply- ing by three. GROWTH IN DISTILLED WATER Before studying the effects of chemicals as upon growth it is well to examine growth in a fluid containing as small an amount of dis- solved chemicals as possible; that is, distilled water. Any growth taking place in distilled water will evidently not be due to food materials present in the medium nor is it likely to be due to any specific salts, since these are almost lacking. After the nature of growth in distilled water has been determined, the specific effects of substances dissolved in the water can be studied. Daniel (08) showed that if Paramecia are introduced directly from hay infusion (which has a high salt content) into pure dis- tilled water, it is the sudden change that injures them. He also showed that they could be made to live in pure distilled water if they were introduced into it gradually. The distilled water used in the experiments was made by Dr. G. F. White of the Johns Hopkins Chemical Laboratory. Ordi- nary laboratory distilled water was redistilled from sulphuric acid and potassium chromate, then distilled from barium hydrate and condensed in block tin. This water gives a conductivity of about >< TOR’. Effect of Pure Distilled Water In the experiments, one half of a dividing specimen was placed in hay infusion, such as was known to be favorable to growth: the other was placed in pure distilled water. Before transferring Effect of Chemicals on Growth 493 to the distilled water, the specimen was washed once in a con- siderable quantity of the same distilled water, in order to remove any trace of hay infusion remaining on it. At the end of five minutes specimens in the hay infusion had increased in length from 139.5 to 153.3 microns. Those in the distilled water had grown precisely the same amount. Now, however, the injurious action of the distilled water began to show by the fact that the specimens in it began to shorten and thicken, while those in the hay infusion continued to increase in length and decrease in thick- ness. At the end of fifteen minutes the control specimens measured 159.6 x 42.7 microns, those in the distilled water 149.7 X 53.1 microns. The latter are thus actually shorter than they were ten minutes earlier. At the end of thirty minutes they were all dead, while those in the control were strong and active. The compara- tive measurements are given in [able I. TABLE I Comparative measurement in microns of Paramecium in pure distilled water and in hay infusion CONTROL HAY INFUSION PURE DISTILLED WATER AGE | Toe Length Width Ne a: Length Width a as Specimens Specimens Min. ° 139-5 42.6 9 5 Toes 44.0 12 153-3 47.0 12 15 | 159.6 42.7 | 13 149.7 53-1 13 30 | Normal | 15 All dead Swollen 15 | It is notable that a certain amount of growth did occur in the distilled water in the first five minutes, in spite of the fact that the latter is quickly injurious and soon fatal. It seems clear that the injurious result was here due, as in Daniel’s experiments, to the sudden change from the fluid with the high salt content to one with a low salt content. 404 A.H. Estabrook Effect of Adding NaCl to the Distilled Water The effect of adding a small amount of some salt to the dis- tilled water was next studied. For this purpose sodium chloride was used. When enough sodium chloride was added to the dis- tilled water to give a ,N, solution, the results are those given in able T+ TABLE II Com parative measurements in microns of Paramecta on i a NaCl dissolved in pure distilled water, and 1n hay infuston CONTROI ine acl AGE ‘f | Length Width NG a Length | width | Ne: @ Specimens | | Specimens | : = | Min 9 [> 839.5 42.6 9 | 30 167.2 38-7 8 164.3 39.3 9 go petra 27200 a 14 169.5 375 al 14 Hours 5 185.7 41.6 | 15 165.0 | 37.2 | 15 24 174.9 54-9 | (10to)'17 184.8(5) | 48.6 (5) | 10 | 5 alive 5 dead | | From this table it is evident that the addition of the small amount of the NaCl to the distilled water has a very good effect on the vitality and growth of the animals. Most of them are alive at the end of twenty-four hours. The growth is only slightly retarded in the salt solution as compared with that in the hay infusion. At the end of twenty-four hours, however, half the animals in the salt solution were dead or dying. The living specimensin the salt measured 18.48 48.6 microns, and were thus larger than the controls, which were 174.9 X 54.9 microns. But it must be noted that seven of the ten specimens that were in the In this and the following tables, whenever division has taken place, the figure enclosed in paren- theses is the number of specimens at the beginning of the experiment, the other number indicates the number of specimens at the end of the experiment. In this case there were 10 specimens at the begin- ningof theexperiment. These increased by division to 17 specimens at theend, allof which were measured. Y Effect of Chemicals on Growth 495 control at the beginning had divided, giving seventeen specimens, and the difference in sizes is due to the fact that some of these seventeen specimens are young and not fully grown. I should say however, that the five in the salt were not quite so large as normal. Evidently growth that is almost normal in amount and rate can take place i in a fluid that will not sustain life for any great length of time. The effects in this experiment are explained by the one next performed. The animals were placed in pure distilled water plus a sufficient amount of sodium chloride to make a % solution. The results are shown in Table III. TABLE III Com parative measurements in microns of Paramecia in = NaCl dissolved in pure distilled water, and in hay infusion CONTROL | N, NaCl a Sa | a BU ea AGE ieee Length Width | No.of | Length Width | No. .of Specimens | | Specimens | | Min | | ° 148.5 50.7 8 | go 183.7 45.0 | I2 188.7 47-1 | II Hours | | 5 200.1 58.2 14 | 177-3 45.9 12 24 199.2 63.9 (21 to) 27 2p ies 60.9 21 As the table shows, growth in 70 NaCl took place almost as well as in the hay infusion. At go minutes, those in the salt were as large as those in the control. At 5 hours, they had fallen somewhat behind, but at 24 hours they were of about the usual adult size. The lack of food had prevented them from dividing, while 6 of the 21 control specimens had divided, giving 27 alto- gether. The larger average size of the specimens in the salt solution was doubtless due to this division in the hay infusion; some of the specimens in the latter were young and not full grown. Thus a % solution of NaCl in distilled water has the necessary amount of salts for Paramecium to live in it with normal growth. The amount of salts in the ,, solution was evidently not sufficient 100 to keep the animals alive for a long time. 496 A.H. Estabrook faa NaCl solution in pure distilled water also gave interesting results, as shown in Table IV. TABLE IV Comparative measurements in microns of Paramecia in BE NaCl dissolved in pure distilled water, and in hay infusion CONTROL | acl | ae Length | Width AGE et Length | Width Ne a Specimens | | Specimens Min. | | | ° 139.5 42.6 9 | | 30 | 171.6 37.8 | 10 | 164.1 | 40.5 | 10 go 182.0 37.8 15 166.6 «| B7)a | 15 Hours | | | 5 | 191.4 | Ae 15 | 161.7 36.6 | 14 24 183.0 | 58.8 (1oto)13 | 185.1 | 46.8 10 At the end of 30 minutes, the Paramecia in the 3, NaCl solu- tien were shorter than the controls. Growth was still retarded at gO minutes and at 5 hours. At the end of 24 hours the speci- mens in the salt were long but not so thick as the controls. The controls, however, had increased from 10 to 13 specimens, so that those in the salt were only slightly retarded in growth. Those in the salt showed evidences of injury. PABLE NW: N NaCl dissolved in pure distilled water, and in hay infusion Comparative measurements in microns of Paramecta 1n CONTROL a NaCl | sid Length Width | No. of Length Width No. of ; Specimens | : | | Specimens | | | 7 Min. | ° 146.4 46.2 | 10 | | mee acti 475 9 153-9 44-4 i 15 159.9 Ga e. || 13 Tine | 44.7 10 30 168 .3 43-5 10 164.4 | 47-4 | 9 60 189.0 42.0 13 166.5 (2) | 43-5 (2) 13 | 11 dead | 2 alive | Effect of Chemicals on Growth 497 A X NaCl solution in pure distilled water had no effect on growing Paramecia at 5 or 15 minutes of age (see Table V). At 30 minutes, those in the salt were slightly smaller than the con- trols; while at 60 minutes, of 13 specimens in the salt, 11 were dead and 2 were smaller than the controls. To sum up the results with distilled water, we find that pure distilled water killed Paramecia that have been living in hay infusion by the great change from the high to the low ale content. When the Paramecia are put into distilled water with enough NaCl present to make it a ,, solution, the injurious effect is not so marked and becomes very noticeable only at 5 to 24 hours. A X NaCl solution in distilled water seems to have the necessary cale content to keep the Paramecia in normal condition. The 5, and NaCl solutions in distilled water have a much higher salt content than is beneficial, and so cause a certain amount of inhibition of growth. More light upon this matter is given by the further work upon NaCl which is to be reported next. GROWTH IN SODIUM CHLORIDE Perhaps the chief purpose of the present paper was to study the effects on cellular growth of certain well-known poisons, particularly nicctine, Asati) and strychnine. In order that there should be no danger of mistaking for specific effects of these poisons symptoms ie are likewise produced by other and un- related chemicals, I first made a careful study of the effects of ordi- nary sodium chloride when added to the infusion in which the animals live. This substance, though not commonly accounted a poison, has of course most deleterious effects when present in too great amounts. ‘These effects, particularly on growth, we shall now examine. r A X, NaCl sclution was made by dissolving 0.146 grams of soldium choride in 25 cc. of hay infusion. he being ae most concentrated solution of the salt that was used, any strength desired was made by dilution of this with hay infusion. The same hay infusion without the sodium chloride was used for the control experiments. 408 A.H. Estabrook Preliminary Experiments on the Effect of NaCl on the Form and Dimensions of Adults Adult Paramecia put into a NaCl solution died after some time. They first became thin, then shorter and much thinner, and slowly died. The appearance of Paramecia as they are dying is very characteristic; they become much swollen, sometimes twice as thick as normal, sometimes longer, or they may be shorter; the protoplasm contains many vacuoles; the animals swim about slowly. The exact moment of death is dificult to determine in this condition, as vitality seems to remain a long while, and even after they have become motionless, they may sometimes be made to move again by stimulating them with a capillary rod. To determine the effect on dimensions, a large number of speci- mens were put into a N, NaCl solution (see table VI). TABLED Vi Dimensions of adult Paramecia in a NaCl. | NO. OF SPECIMENS TIME | LENGTH | BREADTH MEASURED Min. Normal at beginning | 204.5 47.0 50 3 191.2 41.2 | 50 5 | 188.6 | 40.8 ix) 8 186.0 39.7 50 10 | 183.8 39-3 50 15 180.1 | 38.7 50 25 183.8 43-4 50 40 181.2 AIST 5° At intervals of 3 minutes, about 50 were taken out, killed and measured. Most of the animals died about 10 to 20 minutes after being put into the salt, but some, more resistant than the others, remained alive 60 minutes. The curve (Fig. 1), plotted from these figures, is thus based on different numbers of individuals E ffect of Chemicals on Growth 499 LENGTH 190 MICRONS 5 ei eS \ 40 O 10 20 30 40 TIME IN MINUTES THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 8 NO. 4. 500 A.H. Estabrook in different parts; it 1s nevertheless instructive. The changes in the form and size of Paramecium shown by the curve, are the same as seen under the microscope. ‘The first effect is an imme- diate decrease in the width, and at the same time, a shortening of the organism. Further decrease in length takes place, and then, as death approaches, the Paramecia become thicker and slightly longer. I have designated this form of curve as the death curve, as later work shows that its appearance 1s always followed by death. Adult Paramecia put into a X, NaCl solution died in one-half to 3 hours, none living in this strength longer than 3 hours. ‘The same general effects were observed as in the §, NaCl solution, except that, extending over a longer period, they were not so marked. Adult Paramecia put into a X, NaCl solution became thinner for about 20 minutes, then gradually regained their normal size and continued to live normally in that solution. In order to see if X, NaCl and the more dilute solutions of NaCl had any effect upon size in adult Paramecia which were kept in them for longer periods of time, the following experiment was tried: One hundred Paramecia taken from the stock culture were put into each of the following solutions, X, %,, X, and X NaCl, and a control composed of hay infusion (the same as that used for the solvent in the NaCl solutions). The Paramecia remained in the solutions 48 hours, were then taken out, counted, killed, and measured. This was done several times but as the results were in every case the same, only one series is reported here (see table VII). TABLE VII Dimensions of Paramecia that have remained 48 hours in various strengths of sodium chloride, compared with controls. Measurements are in microns and each is the average taken from 50 specimens. 100 specimens in each watch glass at beginning. g A g NO. OF SPECIMENS | LENGTH WIDTH AFTER 48 HOURS Control 194 190.2 48.5 3, NaCl 220 173.05 49.0 A. NaCl 180 181.1 49.8 fy NaCl 210 188.4 49.1 N. NaCl 234 182.4 48.4 E ffect of Chemicals on Growth 501 The table shows that these concentrations of NaCl have had no character stic effect either upon the rate of division or upon the SIZe. Only in the case of X NaCl was there a slight indication of the modification of the rate of division, this concentration standing on the border line between the injurious and non-injurious solu- tions. In table VII, roo Paramecia in X; NaCl had increased to 220, while an equal number in the control had increased to but 194. In another case, 50 specimens left in X, NaCl for 48 hours had increased to but 64, while in the corresponding control 109 had been produced. Direct Measurement of Growth after Fission Growth in solutions of NaCl was next measured directly by placing in the solutions to be examined specimens that had just divided, as described on page 491. X- NaCl in hay infusion. The measurements in hay infusion to which NaCl had been added, and in the same infusion without the salt, are given in table VIII. TABLE VIII Comparative measurements in microns of growing Paramecia in N. NaCl dissolved in hay infusion, and in hay infusion. | CONTROL WN, NaCl Fn : a. AGE | | Length Width Soe tencth | Width ene ee | Specimens Specimens | | = Min | 5 | 152.9 50.4 18 | 153-2 | 37-2 16 Io | 162.5 52.2 22 | 161.7 7-2 II 20 | 166.9 Rte, 14 162.1 39-1 15 40 186.9 49.6 19 141.5 2.0 II 65 182.4 6 Ati4 II 50.1 18 142. For the first 10 minutes after separation, the halves in the salt solution increased in length at the same rate as the controls, then the length decreased rapidly for 40 minutes, when the Paramecia in the salt had become shorter than they were at the time of sepa- 502 ve ka a ke Estabrook ration. All those in the salt were dead at 60 minutes; many had died earlier; the latter, of course, were not measured. “The width of those in the salt decreased very rapidly in the first 5 minutes, then remained the same up to 60 minutes, when the animals died. N NaCl tn hay infusion. able [X gives the measurements 20 for growth when N, NaCl was added to hay infusion. TABLE IX Measurements of growth in = NaCl tn hay infusion. | CONTROL N. NaCl | | | | | eta | Length | Width NS a | Length | Width Ne Gs | Specimens | Specimens Min. | | fo) 144.3 ie 7 | 5 156.3 49-5 13 | 171.6 | 43-8 12 15 162.9 50.1 10 | 163.8 40.8 | 10 30 | 171.0 46.5 8 | 168.0 45-6 | 10 60 | 189.3 45-3 | 12 | 174.9 43-5 10 go | 187.8 Agsbl- | 14 i rs6e9 ARE ORs | 14 | | | | | 6 alive | | 8 dead Hours 6 | Allalive | 50 44 alive | 50 | | 6 dead 25 198.6 =| 58.5 (50 to) 56 | 181.8 | 5211 | Allalive | | | | 23alive | | | | | 16dead | 11 Meas. 40 All alive | | (56 to) 65 | 39dead_ | 50 | | For the first 20 minutes in this solution, the growth in length was normal. The width, however, showed a marked decrease, similar to that shown in the X, NaCl. At the end of 30 minutes, however, the Paramecia were normal in both length and width. At 60 and go minutes those specimens in the salt were shorter than the controls. After go minutes the Paramecia in the salt showed great variations in growth. Shortly after the age of go minutes specimens began to die, although some were alive at 30 hours. At the age of 40 hours none were alive in the salt, while E ffect of Chemicals on Growth 503 the controls had increased from 50 specimens to 65. At 25 hours, some of each were killed and measured. ‘The controls measured 200.7 X 59.4 microns; those in the salt, 181.2 53.4 microns. This experiment shows that .X. NaCl has a definite effect upon growth, inhibiting it after 60 minutes and finally causing death. It also shows that there is a great variation in the resistance of the animals to this strength of the chemical, some dying in 6 hours while other live 30 hours. Growthin NaCl. In X, NaCl we have a strength of the chem- ical which may or may not affect growing Paramecia, depending on the conditions. In one of my experiments, there was appar- ently no effect of the salt upon the growing Paramecia at any time. Those in the salt grew to the same size as the controls (see table X). TABLE X Measurements of growth in a NaCl. | | CONTROL a, NaCl | 4 AGE | Length Width Nee Length | Width gr: | Specimens Specimens Min fo) 144.3 Gpeee? 8 go 186.3 44.7 10 186.3 44.7 10 Hours 24 192.3 66 | 10 186.6 60.9 10 | | In another experiment, under what seemed exactly the same conditions as before, the X, NaCl had a slight inhibiting effect upon the growth. The measurements are given in table XI. At the ages of go minutes, 5 hours, and 24 hours, those in the salt were shorter than the controls; sufficient numbers have been taken to give the figures some weight. I have worked out the probable errors for the averges, and find that, taking three times the probable error of each and substracting from the greater and adding to the less, in each case we still have significant differences, greater at gO minutes and 5 hours than at 24 hours. Thus it seems from the analysis of the figures that there is in this case an 504 A.H. Estabrook actual, though slight, inhibition of growth due to the X. NaCl. Thus = isiontthe border line between a strength that will and one that will not affect erowth i in Paramecia. Nand N.NaClinhay infusion. Neither of these concentra- tions has any characteristic effect on the growth. See tables XII and XIII. Role of Osmotic Pressure in Effects of Sodium Chloride We may now ask whether these effects of NaCl are due to the osmotic pressure of the sodium chloride in solution acting on the Paramecia, or to the direct chemical effects of the substance, or to both. By means of freezing point determinations,’ we find that the osmotic pressure of ordinary hay infusion is .44 atmospheres. In table XIV is given the osmotic pressure of the different solu- tions of sodium chloride which we have used. By examination of the table we find that an increase in the osmotic pressure from .44 atmospheres (that of hay infusion), to 1.841 atmoshperes (that of X NaCl in hay infusion), has only a slight effect on the growth. Growth that was almost normal took place in X, NaCl in hay infusion. But when we increase to an osmotic pressure of 2.528 atmospheres, that of X NaCl in hay infusion, there is a marked effect on the growth, which 1s even more marked at the pressure of 4.546 Gea Ones > NaCl.) This would seem to indicate then that an increase ae osmotic pressure had an injurious effect on growing Paramecia. To give 5 The osmotic pressure of the hay infusion has been found by the lowering of the freezing point method. When the lowering of the freezing point has been determined by the Beckman apparatus, the osmotic pressure is computed from the following forumula: (22.3 iv 86° On Pa= AX Where A is the lowering of the freezing point, 22.3 is the osmotic pressure of a weight norma! solution, and 1.86° is the lowering of the freezing point caused by a weight normal solution. The freezing point of a sample of hay infusion such as was known to be favorable to the growth of Paramecium was found to be .036 below that of pure distilled water, and by applying the above forumla the osmotic pressure of the hay infusion was .44 atmospheres. Effect of Chemicals on Growth 505 TABLE Xl Measurements of growth in N. NaCl. y CONTROL 4 fy NaCl Ace Length Wad Length | width | No of Specimens | Specimens Min. | ro) 145.0 50 10 go 198.3 = .987 42.1 20 | 176.55==1.230 45-1 20 Hours | | 5 119.59=1.476 41.6 34 | 190.80 .897 43-8 | 35 24 | 206.17 1.758 Gish 18 | 192.34==2.136 57-2 18 —— —__— — | — TABLE XII Measurements in growth of => NaCl CONTROL SALT AGE Penh Width eee Teepe Width a Specimens | Specimens Min ° 145.8 | 53-4 9 | 30 174.0 | 49-5 9 174.0 46.8 | 10 go 201.9 | 45-9 17 201.0 45-3 17 Hours | | 5 200.7 44.4 20 196.2 | iets | 21 24 219.4 63.6 14 224.8 64.8 15 TABLE XIII Measurements of growth in 50 NaCl CONTROL SALT AGE Length Width hee a Length Width Br Specimens Specimens Min. ° 131.1 49.8 ii 5 145.2 47-7 7 | 145-2 48.9 7 15 159-3 47-4 9 160.5 46.8 10 30 168.9 45.0 II | 1971.3 3% * | II go 194-4 45-6 19 189.3 46.8 19 Hours 5 194-7, 42.3 20 193.8 43.8 20 24 198.4 60.0 22 202.9 58.2 20 506 A.H. Estabrook further light upon the part played by osmotic pressure, a .07g N solution of cane sugar was made by dissolving 2.681 grams of sugar, which had been rendered free from all impurities, especially sodium chloride, in 100 grams of pure water. This solution has an osmotic pressure of 2.087 atmospheres and is isotonic with a solution of NaCl in distilled water. Halves of Paramecia just divided were used, one-half put in the sugar, the other half in the NaCl solution, both being carefully washed free of any hay infusion. Now if the injurious action upon Paramecia which we have observed is due to osmotic pressure alone, we would expect the two solutions to have the same effect upon the animals in this experiment. The results are shown in Table XV. Up to the age of go minutes, the specimens in the sugar did not grow as large as normal, but did grow faster than those in the salt. At 5 hours, those in the sugar were slightly larger than at go minutes, but still smaller than normal, while those in the NaCl were dead. At 24 hours those in the sugar were not as large as at 5 hours, but it must be noted that there was no food in the sugar solution. The animals in the sugar were alive, but small, however, at 48 hours. This experiment then shows that osmotic pres- sure has an effect upon growing Paramecia, but that the injury caused by the sodium chloride is not due entirely to the osmotic pressure of the solution, but is also due to some other factor. The effects caused by the action of the sodium chloride then, are due to the osmotic pressure exerted by the sodium chloride plus the direct action of the salt upon the protoplasm of the Paramecium. So far as is known the action of the sugar is due entirely to its osmotic pressure. To see the effect of even higher osmotic pressures upon Para- mecium, growth in a XN NaCl in hay infusion was compared with the growth in an isotonic solution of sugar, 7.e., a .16 N solution in hay infusion. Growing Paramecia were used, and 1t was found that the Paramecia died in both of these solutions at about the same time, 10 to 15 minutes. The inability of the Paramecia ® The measurements for the osmotic pressure of sugar solutions are taken from some unpublished work of Prof. H. N. Morse of the Johns Hopkins Chemical Laboratory. Effect of Chemicals on Growth TABLE XIV. 57 The osmotic pressures in atmospheres of different strengths of NaCl at 20° C. dissolved in distilled water and tn hay infusion. The osmotic pressure of hay infusion is assumed to be .44I atmospheres. | STRENGTH oF NaCl o. Pp. oF NaCl pissorvep 1N DISTILLED WATER } o. P. oF NaCl pissoL_vep IN HAY INFUSION ro | 4.105 sd 2.087 $0 Uetine) an -870 fo | 658 100 432 TABLE XV. Comparative measurements of in microns Paramecia in 20 in distilled water. N, NaCl in distilled water, and in .079 N sugar | GRowTH IN ,N. NaCl 1n GROWTH IN .079 N SUGAR | DISTILLED WATER IN DISTILLED WATER at's : No. of ; No. of | Length | Width i Length Width 3 | Specimens | Specimens Min fe) | 140.7 50.4 10 140.7 Reich | 10 fe) | 142.5 42.0 10 158.4 45.0 9 30 | 164.4 | 42.7 12 165.0 43-2 | 12 gor” =| 163.5%" 45-3 | 3 173-1 47.0 | 15 Hours | | & | All dead 10 175.5 50.1 10 24 | Alldead 154.2 48.60 | on 48 | Alive 10 TABLE XVI. Table showing different effects of N. NaCl upon adult and young Paramecia. | AFTER HOURS ADULT, 32 SPECIMENS YOUNG, 32 SPECIMENS | 6 Alive Dead Alive Dead 12 32 19 13 40 32 28 28 4 3 29 7 The data for osmotic pressure given here have been compiled from the formule given on pages 41-44 in ‘‘The Role of Diffusion and Osmotic Pressure in Plants,” by B. E. Livingston, Chicago, 1903 508 2. H. Estabrook to grow in these solutions, which have a large amount of the chem- cal present in them, is probably due as much to the extraction of water from the cells as to the direct action of the chemical upon the life activities. Comparative Resistance of Adult and Young Paramecia to X, NaCl eae in the work it was noticed that adult Paramecia put into aes NaCl solution continued to live, the solution having appar- ently no effect upon them, while young Paramecia put into the solution just after division died, before the end of 30 hours (as set forth above). A careful test of the varying resistance in the two cases was made as follows: Into each of the two depressions of a single slide was put a few drops of X, NaCl solution. Into one was put an adult Paramecium, into ake other a young one from a fission just completed; both the adult and the young coming from the same stock culture, so that the past history would be nearly the same. Thirty-two pairs such were arranged and their further fate observed. The results are shown in Table OVAL This experiment and other data given above (page 500), shows that adult Paramecia may live normally and grow in a solution that sooner or later kills Paramecia if they are put into the solution just as the two halves separate at division. Are Paramecia in Stages Preparatory to Division Like Adult or Like the Young in their Resistance to the Salt? To test this the following experiment was tried: Paramecia which were preparing for division were selected. “They were then grouped in pairs, those being put together which were in the same stage of preparation for fisson. (This is very easy to do with much accuracy, since one accustomed to working with Paramecia can tell within a few minutes when a dividing Para- E fect of Chemicals on Growth 509 mecium will separate). One of a pair was put into hay infusion, the other one of the pair into a §, NaCl solution in hay infusion. It was found that all the pairs separated within 15 minutes of each other, so that there is little room for error. They were allowed to grow for 24 hours, and then killed and measured separately. Of 20 sets of pairs the results are as follows: Control; hay infusion; 17 dividing Para- N, NaClin hay infusion; 17 dividing Para- mecia gave 37.specimens measuring at the age mecia, mates of those in control experiment, of 24 hours: 213.264 += 1.566 X 71.19. gave 34 specimens measuring at the age of 3 dividing Paramecia gave 6 normal speci- 24 hours: 205.059 = 2.466 X 71.64. Mens not measured. 3 dividing Paramecia gave 6, all died. Taking into consideration the probable errors of the measure- ments, there is evidently no significant difference in size of the two sets. However, three of ches dividing Paramecia in the sodium chloride, after separating to form 6 specimens, died, while the corresponding controls remained normal. Why six out of forty in the NaCl should die, while the rest throve as well as the con- trols, is difhcult to explain. Possibly there is a critical early period in which injury may occur; if the animals survive this, they flourish. The experiment taken as a whole shows, however, that the chemical is not so injurious to the young if the parents have been subjected to it a short time before fission. Acclimatization to NaCl We have seen above that certain concentrations of NaCl de- crease growth. It was therefore determined to cultivate Para- mecia in one of these solutions, to see 1f a smaller race could be obtained from L,, or if possible, this race might changeinto one of the other races described by Jennings.’ It was very easy to acclimatize Paramecium to a * NaCl solution. If introduced directly into such a solution, Paramecia, as we have seen, die in a few minutes. The method of procedure for producing acclima- tization was as follows. Adult specimens were put into a SLoc. cit., page 494. 510 A.H. Estabrook N. NaCl solution for 24 hours, then for a like period into ¥,, then Omi) into §, where they will now live and multiply. Fifty Paramecia were put into hay infusion and 50 into ;4y NaCl in the same hay infusion. At the end of 24 hours, 50 of the con- trol were put into fresh hay infusion, and 50 of those in the 3) NaCl were put into X, NaCl. At the end of the 24 hours, there were, as the result of some division, 69 1n the control culture, 64 in the salt. Forty-four of the control were put into — hay infusion, and the 64 in the salt were transferred to the +, NaCl. At the end of 48 hours, the control numbered 54; those in 4; NaCl, 75. Fifty of each were killed and measured. The average meas- urements of the 50 controls were 188.7 x 64.8 microns, of the 50 in 3, NaCl, 170.7 x 70.02 microns. The Paramecia in the salt, as well as those in the control, were large and active. There was plenty of bacterial food present in both, ate only se cae difference being the smaller size of those in the NaCl. After four days, the specimens in the salt solution stopped dividing, while some had divided in an abnormal way. In many the division was in- complete, so that two specimens remained connected. Nothing of the sort was seen in the controls, the latter continued to divide normally. After the Paramecia had been in the ;) NaCl 6 days, many began to die. An attempt was made to save these by put- ting hein into fresh hay infusion containing ;‘y NaCl, but when a few were introduced into this from the old foals they all died in one to two hours, while those remaining in the old 4 NaCl solution continued to live for nearly 20 hours more. The controls remained normal and alive. Thus it is easy to acclimatize Paramecia to ‘, NaCl for a short period, but itis more difficult to get them into “perfect relation to their changed environment. ae animals in the salt at the latter end of the experiment were evidently in a very unstable equilib- rium with their environment. Possibly the effect was caused by the accumulation of the sodium chloride in the cells. Summarizing the work with sodium chloride, we find that the addition to the culture medium of sufficient NaCl to make a 3 or 3» solution permits the young specimens to grow for a few min- utes after fission, then inhibits growth, finally causing death. E ffect of Chemicals on Growth F 511 3) NaCl is on the border line; it sometimes decreases growth, sometimes not, dependinig on the conditions. More dilute solu- tions do not effect growth. Young specimens are injured by 3% NaCl, while old ones are not. If the parents are subjected to this concentration for a short while before fission, the injury to the young is less. Paramecia can be temporarily acclimatized to a solution that would at first kill them, but after some days 1n such a solution fission becomes abnormal and ceases and the animals die. The injurious effects of sodium chloride are due partly to increase of osmotic pressure, partly to specific chemical action. EFFECTS OF NICOTINE ON GROWTH Having studied the effects of the comparatively innocuous sub- stance, NaCl, I now undertook to examine the effects of nicotine (C,,H,,N.). This alkaloid is so poisonous that only minute quantities are required to produce an effect; so minute that the osmotic pressure produced by them is clearly of no importance in the results. The effect of nicotine on single cells is of interest in view of its effect on man (see Lee, Langley, and others). Effect of Nicotine upon Adult Paramecia One hundred adult specimens of Paramecia taken at random from the stock culture were put into watch glasses containing known amounts of nicotine dissolved in hay infusion. These were allowed to remain for 50 hours. The results are given in table XVII. From this table it is clear that strengths of nicotine from I—1,000 up to 1~-20,000 kill adult Paramecia in different times varying from 2 hours to 50 hours. Those in the 1-40,0000 solution and the more dilute solutions continued to live. In order to determine whether there was any effect upon the size a number of specimens were taken from these solutions, killed, and measured (see table XVIII.) 512 A.H. Estabrook TABLE XVII Effect of different strengths of nicotine upon Paramecta. 100 specimens each in watch glass at beginning of ex periment. PARTSIORENTCOTENT Tl STARTED FER. I, 1909, 12:30 P. M. IN PARTS OF HAY — = — RUSION. | Feb. 1, | Reb=pr; | Feb. 1, | Feb. 3, Feb. 3, 2B ME) 5PM ik soe M: | 10 A. M. 3 P.M. E=TOOO iieicte. Sele Allideadii a ieneanaeercr WV eiebece tase: | \e:eale-s, oferta ae ee EIAs a adtao AS | Alldead | All dead | ese eadon: \\or Getcbeey RES | age ae ee but 2 | | I USnosomouaade Alldead | Few alive ANCES I Seeesueces I cvsndtsco- but 60 | | 12,000 ns ioenio ee Allidead = yi Allidead) WI) aa. snce ee | Sere os Gone oc but 30 | | I=25SOO siete ee sodead | More dyingse ie eAllidead sui yas asec || c-roioue cere TGR SOOO ei essicpta sete: sodead | More dying 10 alive | All dead | otecehepieterae iGKeHoeohGandawncue Allalive | Allalive Many dying | 6 alive All dead T=20;000h ee cine ye All alive All alive Some dying | 5 alive — 3 alive I—AO;COOM eee | All alive | All alive Allalive | Allalive | Allalive T=SO;0COmaae eaten: | All alive Allalive | Allalive | Allalive All alive T—1OO!COO sea eee: | Allbalive | Allalive All alive All alive All alive Control hay infu- SIODN at Riots stoke | All alive All alive Allalive | —— All alive All alive TABLE XVIII Dimensions of Paramecia that have remained 50 hours in various strengths of nicotine compared with con- trols; measurements are in micorns, and each is the average taken from 50 specimens. | LENGTH WIDTH At beginning.ot expernment-aseaae eee ee eee ee ae oer ee | 21Q)40 ||| 46.3 Controlvatiend’ tak face cat eee oe Oia SA aT EER Pee ake | 260.7 | 67.5 IEA O,OCO Mor setets tala venrets sta s) varie ER eye GCE et ee CE ETL Ce | 213-Q all 63.4 T=RO\GOO WA abs SIRs Se gee CE ne LR cae Teo ea SE ees 210.2 64.0 (0.0 0,c.o Pee eins PEM OAD Gb an wo CaO RS oO od Hin Bane oo oatcg saan den les 214.5 63.4 It is seen that the Paramecia in the nicotine were about the same size at the end of the experiment as they were at the begin- ning. The controls, however, had increased in size from 219.4 X 460.3 microns to 260.7 x 67.5 microns. This would seem to indi- cate that those in the nicotine did not grow at all durng the period of the experiment. Effect of Chemicals on Growth Effect of Nicotine on Growing Paramecia 513 Growth in 1-2,500 nicotine. Growth in nicotine was measured directly in the way previously described (page 491). One of the two products of fission was placed in pure hay infusion, the other in hay infusion containing nicotine. In a solution of 1 part of nicotine to 2,500 of hay infusion, the results are as shown in table XIX. TABLE XIX Measurements of growth in I-2,500 nicotine. | CONTROL NICOTINE id jh) eneek Width No: Of oy |) tength Wideh |. Mout Specimens Specimens Min | ° | 145.0 53-0 fe) 5 159-7 Fol 12 155-4 59.2 12 we 164.1 58.2 II | 157-8 62.4 | fe) 20 172.1 56.2 II | 156.5 69.8 II ae 169.0 57.6 9 163.5 65.1 | ie) 60 | 191.7 52.6 9 160.2 68.7 9 Hours | | 2 All alive | II 4 alive 7dead_ | II 5 | Allalive | II | 11 dead | Il | ( { TABLE XX Measurements of growth tn I-5,000 nicotine. CONTROL NICOTINE AGE | Length | Width ADC! ene Width — | | Specimens | Specimens Min | | ° 132.6 Cy’ 10 | 5 ri ees 55-0 9 149.7 52.2 | 10 15 156.3 Galal 10 152.1 54-3 10 30 167.7 51.6 II 162.3 54-7 9 60 185.4 50.1 Il 168.3 Exelon 9 Hours 9 197.7 52.5 II 175.2 49.2 7 24 212.1 62.1 25 174.9 48.4 25 8 dead 10 alive 7 meas- : | ured. 48 All alive | 18 18 dead 18 514 A.H. Estabrook At the end of five minutes, though there was some growth, those in the nicotine were not quite so large as the controls. At 30 minutes, they were still growing, but continued to be smaller than the controls. At 60 minutes, they began to decrease in size. At the end of 2 hours, of 11 specimens in the nicotine, 4 were alive and 7 dead. It is to be noticed that some growth took place in a solution of nicotine which later killed the organism. Growth in I-5,000 nicotine. In one part of nicotine to 5,000 of hay infusion, growth took place in the same general way as in the I-2,5000 solution (see table XX). The Paramecia grew in the nicotine, but were not at any time so large as the controls. At the age of g hours, those in the nico- tine measured 175.2 x 49.2 microns, while the controls were 197.7 52.5. Atthe age of 24 hours, those in the nicotine were begin- ning to die, and at 48 hours, all those in the nicotine were dead. Growth in I-10,000 nicotine. In a 1—10,000 solution of nico- tine, the Paramecia grew at about the same rate as those in the I-5,000 (see table X XI), remaining somewhat behind the con- trols. The difference is not great, but the results indicate a slight in- hibition of growth caused by this amount of nicotine. All those in the nicotine were dead at 45 hours. Growth in 1-20,000 nicotine. The results with 1—20,000 nico- tine were variable (see table X XII). Up to 5 hours, the growth was not evidently affected. At later periods the specimens in the nicotine were sometimes smaller, sometimes larger, than the controls. At 24, and 48 hours, they were distinctly smaller. Thus the effect of such a quantity of nicotine shows only after a considerable period. Growth in I-30,000 nicotine. In 130,000 nicotine, growth took place as in the control (see table XXIII). At the end of 24 hours, the measurements in the nicotine were 193-2 X 63.9 microns, while the controls measured 187.5 x 68.7 microns. ‘The difference is without significance; it 1s partly due to the fact that one of the controls had divided, the two small specimens so produced reducing the average length. E ffect of Chemicals on Growth 515 TABLE XxI Measurements of growth in I-10,000 nicotine. | CONTROL NICOTINE AGE | | v | Length = Width Pewee Length Width Bok | | Specimens Specimens | Min. | ° 145.8 53-7 10 5 | 153-6 §1-3 9 152.1 55.8 10 15 166.8 52.5 10 163.2 54-3 9 30 175.2 Giles 10 171.6 48.9 9 60 192.9 Gaby 12 183 .3 ey 10 go 189.9 go.1 9 185.1 51.0 12 Hours | 5 180.9 45.6 9 170.7 52e5 8 24 186.0 55.8 5 177.6 49.8 5 45 Allalive 17 17 dead 17 TABLE XXII Measurements of growth in I-20,000 nicotine CONTROL NICOTINE AGE ui Length Width [+ No-ot Length Width New ek Specimens Specimens Min. ° 141.9 48.0 9 go 187.8 43-5 8 185.1 45-9 II Hours | Gi) 180.0 45.6 Co) 188.4 48.6 10 24 187.2 56.4 10 173-7 54-9 9 48 | 195.2 62.4 (roto12 185.2 eee 8 TABLE XXIII Measurements of growth in I1-30,000 nicotine CONTROL NICOTINE AGE Length Width By oF Length Width ae Specimen Specimens Min. | ° 139.8 49.5 10 go 167.4 — 46.2 10 168.9 45-3 10 Hours a ee 44-40 5 180.6 44-4 0 | : 24 187.5 68.7 (12 to)13 193.2 63-9 | 12 THE JOURNAL OF EXPERIMENTAL ZOOLOGY VOL, 8, No, 4. 510 A.H. Estabrook Different Resistonce of Young and Adults to Certain Concentrations Interesting results were reached in the effect of nicotine upon Paramecia of different ages. Nine adult specimens were put into a I~5,000 solution of nicotine. Nine specimens just divided were putinto the same solution. At the end of 2 hours, 5 of the adult specimens were dead, while only 2 of the young were dead. At 20 hours, all g adults were dead, while all but 2 young were alive. At the end of 30 hours, all the young were dead. Some adult Paramecia were put into I-20,000 nicotine. These were all dead at the end of two days. Some specimens just di- vided, put into the same strength of nicotine, grew to normal size and lived for 5 days, but did not divide. Thus in these grades of nicotine the young Paramecia are more resistant to the chemical than adult specimens. In NaCl, on the other hand, as has been shown, the adult specimens are more resistant than the young specimens. Summing up the results with nicotine, we find that a certain amount of growth takes place in the stronger solutions of nicotine, though these same solutions later kill*the organism. This is true for solutions up to and including 1-10,000. In I—20,000 nicotine solution there was a very slight retardation of growth at late stages. In the 1—30,000 solution the nicotine had no effect on the growth. EFFECTS OF STRYCHNINE ON GROWTH Strychinne has long been known to be a powerful poison for protoplasm. ‘The work of Schulze, Binz, and others has shown its action on Protozoa, but there are no studies as to its effect upon growth in smaller quantities than lethal doses. Calkins and Lieb have shown that, in small quantities, strychnine in- creases the rate of division in Paramecium, but they did not study its effect upon growth and size. It will be interesting to deter- mine whether there is increased size at the time when the animals show the increased rate of division, described by the authors mentioned. The general effects of this powerful poison upon cel- lular growth are likewise of interest. E ffect of Chemicals on Growth 517 Effects of Strychnine upon Adult Paramecia A ,;) solution of strychnine nitrate was made by dissolvin .0387 grams of that salt in 1,000 cc. of distilled water. This solu tion then contained about one part of strychnine nitrate in 2,50 of water. As this was the strongest solution used, other strength desired were made by further dilution of this with hay infusion The 1-2,500 solution of strychnine killed Paramecia in a very few minutes. A I~12,500 solution caused them to swell up and become vacoulated almost immediately, although they remained alive and moving for several hours. With more dilute solutions the effect is not so immediate or marked. ‘To determine the effect of the more dilute solutions, watch glass preparations (as with NaCl and nicotine) were made. Strengths of strychnine up to I-37,500 killed a great many of the Paramecia. In the I-50,000 there were as many at the end of 48 hours as at the be- ginning. The normal rate of division was maintained in the I-125,000 and more dilute solutions. There was no effect at any time upon the size (see table XXIV). TABLE XXIV Table showing rate of division of Paramecia in different strengths of strychnine and in hay infusion. 100 specimens in each watch glass at beginning. STRENGTH OF NUMBER OF SOLUTIONS USED SPECIMENS AT END Control I | 179 Control II 217 Control If] 200 I-12,500 3 1-25,000 23 1~37,500 83 I-50,000 99 1-7 5,000 182 I-125,000 162 I-150,000 198 1-200,000 | 207 3 I-250,000 193 5 1s AS H. Estabrook GROWTH OF PARAMECIUM IN STRYCHNINE AFTER FISSION Growth in strychnine was measured directly in the way pre- viously described (page 491). Growth 1n 1-12,500 strychnine. For the first five minutes after separation, those in the alkaloid grew to the same length as the controls. At 15 minutes, those in the strychnine decreased in length until they were as short as they were at the time of fission, and remained so for 60 minutes, when all were dead. The width of those in the alkaloid increased very rapidly in the first 30 minutes (see table X XY). TABLE XXXV Measurements of growth in I-12,500 strychnine. CONTROL STRYCHNINE pa | : | No. of | No. of Length Width Seaver Length Width Seecieeae Min | | ° | 133.8 54.0 | 6 5 | 154.8 54.0 8 153.0 59.2 8 15 | 165.0 53-4 | 5 134.4 69.0 5 30 178.5 52.2 | 7 134.7 82.2 9 60 All alive | 10 All dead 10 TABLE XXVI Measuremente of Growth 1n I-50,000 strychnine CONTROL STRYCHNINE AGE | ; 5 | GNonor. em ae No. of : Length | Width Specimens Length Width Specimens — M | Min. | fc) 134.1 51.9 | 7 5 143-4 50.7 | 10 145.8 51.0 10 15 151.1 49.2 | II 150.9 49.8 II 30 160.5 47.1 12 | 155.2 Si dirg/ 12 60 182.7 AZA5 | 11 1507 53-2 12 go 189.0 42.9 II 164.1 48.1 II Hours | 2 All alive | 10 Swollen and 10 | dying E fect of Chemicals on Growth 519 TABLE XXVII Measurements of growth in I-75,000 strychnine CONTROL STRYCHNINE AGE Benaeh Width 2 of Tewath Width No. of Specimens ; Specimens Min | ° 137.4 49.2 10 5 149.4 48.0 9 150.3 49.2 9 15 159.3 48.6 10 157.8 | 49.2 10 30 165.6 43.8 10 161.4 47-7 10 go 191.4 40.5 10 171.0 46.8 10 Hours 5 190.8 40.8 12 163.8 49.8 II 24 All alive | 13 3 barely 13 alive; 10 dead | Growth in 1-50,000 strychnine. Normal growth took place in I~50,000 solution of strychnine for the first 15 minutes (see cable MXVI): Further growth then took place, but was not so rapid as in the control. At the end of 2 hours those in the strychnine were dying. Growth in I- 75,000 solution of strychnine. For the first 15 min- utes after separation, the Roles in the strychnine increased at the same rate as the control (see table X XVII). Further growth took place, but was not so rapid as in the con- trol. At 24 hours, of 13 specimens in the strychnine 10 were dead and 3 dying. Growth in 1~-100,000 strychnine. Normal growth took place in I-100,000 strychnine up to the age of 30 minutes, the specimens in the alkaloid growing to the same size as in the control (see table XXVIII). Growth then continued, but not so great in amount as in the control. At 5 hours, those in the strychnine were much shorter than the controls, but at 24 hours they had almost reached nor- mal size again- At the age of 48 hours, the Paramecia in the strychnine had not grown as much as the controls; 14 specimens 520 A.H. Estabrook in the strychnine had not divided and measured 181.2 * 44.1 microns, While 14 specimens in the control had increased by divi- sion to 33 and these measured 197.4 51.6 microns. Thus we find that in the 1-100,000 strychnine normal growth takes place up to the age of 30 minutes, then further growth ensues, but is not so great in amount and the specimens do not become as large at 48 hours as in the control. Division is also stopped in this strength of strychnine. Growth in 1-125,000 strychnine. ‘Vhis concentration has no ehect on the growth (see table X XIX.) > TABLE XXVIII Measurements of growth in I-100,000 strychnine CONTROL | STRYCHNINE ee ee | = : a AGE Tenens Width Mook vo) Waahenses Width NO. eh | Specimens | Specimens Min | ° 142.1 52.8 8 5 145.8 50.7 10 146.4 | 50.1 10 15 149.7 51.9 | 9 156.6 | 525 10 30 162.3 | 46.2 Il 161.1 48.3 12 | go 191.4 | 45.6 9 179-4 | 51.9 9 Hours | 5 | 183.5 Anat II 153-3 47-4 12 24 199.3 7O.5 20 196.3 49.5 22 48 197.4 51.6 (14 to) 33 | 181.2 44.1 14 TABLE XXIX Measurements of growth in I-125,000 strychnine CONTROL | STRYCHNINE AGE | No. of | Tenet Pal widths || ae | Tength | Width New | pecimens | _ Specimens | . | | | Min. | fo) 139.2 49.8 8 30 166.2 | 45.6 9 | 169.8 | 46.8 6 go 188 .1 | 43-2 10 | 188.1 45.6 10 Hours | 5 191.5 46.4 13 194.8 48.0 | 15 24 206.3 52.6 17 206.1 59-7 18 48 220.5 55.5 (5 to) 10 MOny | 54.6 (5 to) 8 E ffect of Chemicals on Growth 521 Growth in 1I-200,000 and I--250,000 strychnine. It was noted above that certain investigators had found that minute quantities of strychnine increased the division rate in Paramecium; in order to determine whether increased size accompanied this ‘ncreased rate of division, more dilute strengths were used. In both 1-200,000 and 1-250,000, growth was exactly the same as in the controls (see tables XXX and XXXI). There was no evidence at any time of either increased size or increased rate of division. Comparative Resistance of Adult and Young Paramecia to Strychnine. Twenty adult Paramecia and 20 young Paramecia from the same stock culture were put into I-25,000 strychnine solution. RESULT | ADULT; 20 SPECIMENS | YOUNG} 20 SPECIMENS After | Alive Dead Hours 2 18 2 Some swollen 5 18 2 All dead 12 9 9 All dead 24 18 All dead The same experiment was again tried using I~50,000 strych- nine in place of the 1-25,000. The results follow. ADULT; 20 SPECIMENS YOUNG} 20 SPECIMENS After Hours 6 All alive Few swollen 6 Allalive ~ Dying II All alive All dead 24 13 dying, 7 dead All dead It is evident from these experiments that adult Paramecia have a much greater resistance than young specimens to the stronger solutions of strychnine. Summarizing the effects of strychnine, we find that when speci- mens just after fission are introduced into hay infusion contain- ing strychnine at concentrations of from I-12,500 to I—75,000 a A.H. Estabrook §22 TABLE XXX Measurements of growth in 1-200,000 strychnine CONTROL STRYCHNINE AGE f Length Width aoa Length Wide ene Specimens | Specimens = 2 = —-— -—__—-|-—--_— Hours fo) 145.0 50.0 10 5 195.9 39-9 i fe) 199.2 45.0 9 24 196.1 55-1 20 193-3 59-5 20 48 201.3 48 .3 (29 to) 39 205 Bal | (29 to) 30 TABLE XXXII Measurements of growth in 1-250,000 strychnine CONTROL STRYCHNINE | AGE J | Length Width Peace Length Width“) vem Specimens Specimens : an mn ; Fe ; | Hours fe) 145.0 50.0 10 24 193.8 50.1 10 191.1 55-5. 10 48 196.4 44.3 (20 to) 32 203.8 48.7 | (20to) 27 TABLE XXXII Measurements of growth in 5 per cent alcohol CONTROL ALCOHOL AGE . N ; L Length Width 0. of Length Width ae Specimens k Specimens Min. \e 135;°3 44-4 | 10 5 146.7 45-9 | 10 137.7 44-7 9 30 163.2 39-3 | 8 146.4 45-3 | 10 go 165.8 39-5 | II 153.0 44.4 9 Hours 3 176.1 39.0 II 156.9 Gy2}07/ ie) 5 Allalive 15 Dead 15 sp. | 1S certain amount of growth takes place in the first few minutes, then the animals die. almost normal in amount. No evidence appeared of increased size or increased rate of fission due to strychnine in any strength. In strychnine at I-100,000, growth 1s Effect of Chemicals on Growth 523 Adult Paramecia are more resistant than young specimens to the stronger solutions. E ffects of Alcohol on Growth Introductory. So much has been written in the last few years, and such diverse results reached on the effect of alcohol upon higher animals that it will be interesting to study the effects of alcohol upon growth in single cells. Calnis and Lieb,® in 1902, showed that “alcohol had no effect upon Paramecium taken in too weak doses and too powerful an effect when taken in over strong doses,” also “when a medium dose was given, /.e., one part of alcohol in 2 500 of hay infusion, the effect 1s a continued stim- ulus which sustains the high rate of division even during periods of depression of the eareeal series. Woodruff,!® in 1908, found that alcohol increased the rate of division at certain periods in the life cycle of Paramecium and decreased it at others. He also found that the increased rate of division was not lasting, but that doubling the amount of alcohol again caused a rapid cell division for a Ganied period. It will be of value, then, to see what effect different amounts of alcohol will have upon growing Paramecia, and whether minute quantities will cause increased size or increased rate of division. Ejfect of Alcohol on Growing Paramecia Growth in 5 per cent solution of alcohol in hay infusion. Young Paramecia just after separation were used, in the way described on page 4g1. Some growth took place in a 5 per cent solution of alcohol in hay infusion, but the animals did not grow so fast at any time as the controls.’ All those in the dleakol were dead at the end of 5 hours (see table XXXII). Growth in 3 per cent alcohol. A certain amount of growth took place in 3 SE cent alcohol, but the specimens in the alcohol did * Loc. cit., page 364. 10 Loc. cit., page 85. U The slides containing the alcohol cultures of Paramecia were kept in a separate chamber from the the controls. In the moist chamber containing the alcohol cultures. the bottom was covered by a 5% solution of alcohol and inthis way loss of alcohol by evaporation from the culture drops was prevented. The control cultures were of course kept in water vapor alone. In the other strengths of alcohol studied, similar precautions were taken. 524 A.H. Estabrook TABLE XXXIII Measurements of growth in 3 per cent alcohol CONTROL ALCOHOL AGE < Length Width NO of Length Width Sieh of | SPecimens | Specimens Min | | ° 139.2 45.6 10 30 162.0 39.3 10 153-9 39.6 10 go 187.0 38.2 18 173.1 39-3 16 Hours | 5 191.7 | B72 | II 185.7 48.0 10 é | | a 12 | All alive | | 20 All dead | 20 not at any time grow as fast in the alcohol as in the control ( see table XX XIII). At 12 hours, all those in the alcohol were dead. Growth in 2 per cent alcohol. At the age of go minutes, Para- mecia had grown to normal length, but were not so thick as the controls. At 5 hours, they were slightly shorter in length and much thinner than the controls (see table XX XIV). At 24 hours, those in the alcohol measured 196.1 x 56.5 microns, and had not divided at all, while the controls had increased from 22 to 42 specimens. Thus in 2 per cent alcohol animals had grown to about normal size, but did not divide. The effect of the alcohol is shown mainly on the inhibition of division. Growth in 1 per cent alcohol. At the end of go minutes, Para- mecia growing in I per cent alcohol were slightly larger than the controls. At 5 hours and at 24 hours those in the alcohol had grown to the same size as the controls. Thus 1 per cent alcohol has no effect upon the growth (see table XX XY). Growth in 1-500 alcohol. As was mentioned above, it has been found by certain investigators that minute quantities of alcohol cause increased rate of division. In order to see if increased size accompanied this greater division rate, the effects of more dilute solutions of alcohol were tried upon growing Paramecia. Paramecia grew to exactly the same size in the 1-500 alcohol as in the control (see table XX XVI). Growth in 1-2,500 alcohol. In a solution of 1 part alcohol in 2,500 of hay infusion, Paramecia grew to the age of 5 hours, at the Effect of Chemicals on Growth 525 TABLE XXXIV. Measurements of growth in 2 per cent alcohol. | | CONTROL | ALCOHOL AGE . | No. of | No. of — | Length Width | Specimens Length Width Specimens } ieee oe [is Min. | ° 135.0 45.0 10 | go 191.0 = 52hea | 9 | 192.0 43-2 9 Hours | | 190.2 45-7 9 186.3 36.6 9 24 22 specimens inc\reased to 42\(22 to) 42 196.1 56.5 DD, TABEL XXXV. Measurements of growth in I per cent alcohol. CONTROL. ALCOHOL. au hee a rues le | No. of | Length | Width Specimens Length Width Specimens Min | fo) 135.0 45.0 10 | go 189.0 42.6 9 197-7 4373, | 9 Hours | 5 | 202.2 44-7 13 205.6 40.8 | 15 24 | 210.6 67.7 34 213.6 69.6 | 33 TABLE XXXVI. Measurements of growth in 1-500 alcohol CONTROL | ALCOHOL AGE. | l) Novos) | No. of | Length Width | Specimens Length Width Specimens | = | {ine Min. | | ; Gaile Sige.o bs 48.0 | Ic | go 185.2 38.4 | 18 186.6 | 37-9 | 16 Hours Ip 190.5 38 4 | 21 187.4 41.1 21 24 204.7 O4eS a 29 | 202.0 63.1 30 same rate as in the control (see table XX XVII). At 24 hours, however, those in the alcohol were 6.2 microns longer than the controls. If the probable errors of the length are inspected, this <7A0 le a A.H. Estabrook TABLE XXXVII Veasurements of growth on 1-2,500 alcohol. CONTROL ALCOHOL AGE NO. OF NO. OF Length Width SPECIMENS Length | Width SPECIMENS | 2 Min | fo) 133.8 40.5 10 30 161.0 40.3 9 156.6 | 28.1 10 Te) 171.0 Rice 10 168.3 37.8 10 Hours | 5 179.7 40.5 10 178.6 38.7 9 24 199.7 63.6 21 205.9 | 65.5 20 1.479 2.094 difference becomes insignificant. So the result would indicate that the alcohol had not increased the growth above the normal amount. Growth 1n hay infusion of Paramecia treated for 30 minutes after fission with 74 per cent alcohol. ‘Yo see if treatment with a strong solution of alcohol for a short period would have any lasting effect upon the animals when transferred to normal conditions, or whether the animals would be inhibited in growth for the actual time in which they were in the alcohol, the following experiment was tried: Paramecia just after fission were put into 74 per cent alcohol and allowed to remain. At the age of 5 minutes, and 30 minutes, they were shorter than they were at the time of separa- tion. hey were also swollen and vacuolated and motionless. Water evidently had been extracted from the Paramecia by the strong alcohol (see table XX XVIII). At the end of 30 minutes the Paramecia in the alcohol were taken out and put into hay infusion. Fifteen minutes after being taken out of the alcohol or at the age of 45 minutes, they had increased from 121.8 microns (the size at which they were transferred from the 74 per cent alcohol to the hay infusion), to 147.9 microns, being then sightly larger than at the time of fis- sion. No further growth took place in the Paramecia that had been treated for 30 minutes with the alcohol until shortly after 5 hours. At that time the Parmecia began to increase in size, and E ffect of Chemicals on Growth TABLE XXXVIII 527 Measurements of growth of Paramecta treated with 7s per cent alcohol for 30 minutes after separation and then placed in hay infusion CONTROL Paramecia in Hay EXPERIMENT | Infusion AGE - es | | | Length | Width | No-o | |Length | Width | No.of | Specimens | | Specimens Min. ° 142.2 44-4 | to ~— | in 7 3 per cent | | alcohol | 5 144.6 45.0 | 9 in 74 per cent 123.3 48.9 10 | alcohol | | 30 150.3 | 42.0 10 in 7% per cent 121.8 54-0 8 | alcohol 45 Nigd tll An. | | 9 30 minutes in | 147-9 41.7 10 alcohol, 15 | minutes in hay | infusion 60 WS7eOU) Aze7 ea 8 30 minutes in 144.7 46.6 9 alcohol, 30 | | minutes in hay | infusion Ars. 2 184.5 38.6 16 | 30 minutes in | 148.4 39-4 13 | | alcohol, go | minutes in hay | infusion | 5 | EFS 42.3 II 30 minutes in | 137-4 42.8 11 alcohol, 44 | hours in hay | infusion 24 Gea WN via 16 30 minutes in _ 160.6 51.7 13 | alcohol, 234 | hours in hay | | infusion. 48 \e a18752) 52.7 (6 to )g 3°. minutes in | 184.9 | 59-4 (6 to) 11 alcohol, 474 | | | ; hours in hay | infusion | 528 vite H. Estabrook at 24 hours they measured 160.6 % 51.7 microns. ‘They were at that time shorter than the controls, which had not been treated at the beginning with the alcohol. At the age of 48 hours, the control had increased from 6 to g specimens and measured 187.3 53-7 microns, while those that had been treated with the alco- hol for 30 minutes after fission, and then placed in hay infusion, had increased from 6 to 11 specimens, and measured 184.9 X 59.4. At 48 hours the Paramecia had entirely recovered from the effects of the initial treatment with alcoholand had regained theirnormal size. Thus we find that 75 per cent alcohol inhibits growth completely, and that the inhibitory effect lasts for some hours after the ani- mals have been removed fromit. But finally the inherent tend- ency to grow overcomes the effects of the alcohol, and the speci mens regain their normal size and rate of growth. Resistance of Adult and Young to Alcohol There is no such marked difference in the resistance of a young and adult Paramecia to strong solutions of alcohol as we find with the other chemicals. In a 5 per cent solution young and adults die in about the same time (2 to 3 hours). Ina 3 per cent solution the adults are slightly more resistant than the young, living in the solution about 12 hours, while the young die in about 5 hours. The relation of alcohol to erowth casts some light in the role of osmotic pressure in aireabueie the effects of chemicals on growth. We find that nearly normal growth takes place in 2 per cent solution of alcohol; this has an osmotic pressure of 8.24 atmos- pheres, while Paramecia die quickly in aX, solution of NaCl, with an osmotic pressure of but 4.55 atmospheres. ‘This indicates that changes in osmotic pressure have only a small part in the effects of these chemicals upon growth. Thus the effects of alcohol on growth in Paramecium are in a general way similar to the effects of the other chemicals studied. The stronger solutions, 5 per cent and 3 per cent, inhibit growth somewhat, and the animals finally die. Normal growth takes place in 1 per cent alcohol. No evidence was fame that minute quantities of alcohol increase growth. ‘The adult Paramecia are slightly more resistant to alcohol than the young. E ffect of Chemicals on Growth 529 Effect of Food on Growing Paramecta At almost every step in these experiments, the question arose as to what part the presence or absence of food plays in the growth of Paramecium. We have seen that during the first go minutes, growth took place very fast. To what is this rapid growth due? Is it due to ingested matter or to imbibition of water? Growing Paramecia were put into hay infusion containing India ink, and it was found that no particles of the India ink were ingested into the body of the animal until about 30 minutes after fission. ‘Thus the marked increase in size which takes place in the first 30 min- utes in a growing Paramecium cannot be due to ingested matter of any sort. The only other explanation for the increase in size is the imbibition of water. To determine directly the effect of food upon growth in the early stages, the growth of Paramecium in tap water containing rather little bacterial food was compared with the growth in feck hay infusion. Halves of divided specimens were put into hay infusion, the other halves of the same specimens into tap water which had been boiled and then aerated. At the end of one hour, 35 specimens in the hay infusion measured 173.1 40.9 microns, 35 in the tap water 171.4 x 40.6 microns. We find that the same amount of growth has taken place in both. The growth in tap water as prepared above was then compared with that hay infusion in which there was dense bacterial growth. Specimens Just after fission were used and kept in the different culture fluids one hour. At the end of that time, the Paramecia in the hay infusion with plenty of bacterial food present measured 169.8 42.0 microns (10 specimens), while those in the tap water with practically no bacterial food grew to about the same size, 171.3 X 42.3. From this experiment and the experiment before, growth to the age of 60 minutes would seem to be independent of the amount of food present, as the same amount of growth takes place in the tap water with no food present as in the hay infusion with the large amount of bacteria. To see what effect food will have upon growing Paramecia for longer periods of time the following experiment was performed: 530 A. lig Estabrook Some hay infusion was allowed to stand in a warm room until it became turbid from the growth of bacteria. Part of this was then filtered through a new Pasteur-Chamberlain bougie. “The filtrate was thus rendered free of all bacteria and was also exactly the same, chemically and otherwise, as the hay infusion with the bacteria in it before filtering, except for the absence of the bac- teria. Halves of dividing Paramecia were put into these two solutions, one with bacteria, the other without. At the end of go minutes, there was, as was to be expected, no difference in size. At the age of 5 hours, the Paramecia in the hay infusion with the bacterial food were shorter and thicker and measured 200.1 60.5 microns (14 specimens), while those growing in the media with- out bacteria were longer and thinner;11 of these measured 210.9 45.0 microns. At the age of 24 hours the presence of food has had a marked effect. Twenty-eight specimensin the hay infusion with the bacteria had increased by division to 49 specimens, and these measured 193.5 62.2 microns, while the 28 specimens in the medium without food had not divided at all and measured 205.2 X 53.4 microns. It must be said here, however, that some growth of bacteria had taken place in the solution which was sterile in the beginning, as it 1s impossible to wash Paramecia absolutely free from adhering bacteria. Yet at 24 hours, there was evidently an abundance of bacteria in the one medium, and almost none in the other. Thus in the early stages of growth up to the age of about go minutes the presence or absence of food material in the medium has no effect on the size of the growing animals. As we have seen earlier, the greatest amount of growth takes place in the first go minutes and our experiments indicate that this increase in size is due mainly to imbibition of water. From then on, the presence of food in the culture fluid has an effect on the size to which the Paramecia will attain. With plenty of food present the animals grow shorter and thicker, and divide sooner, than those kept in a medium with less food. It is to be noted that in the experimental work in the general effects of the different chemicals studied, the question of bacte- rial food does not enter in the interpretation of the results. It was E fect of Chemicals on Growth 53! found that in no case did the presence of any of the chemicals in the hay infusion stop the normal growth of the bacteria, the culture medium with the chemical in it becoming turbid with bacteria at about the same time as the control hay infusion. For this reason there will be about the same amount of food in one culture medium as the other, and the question of food affecting the general results will be negligible. SUMMARY AND DISCUSSION 1. The investigations do not show that any of the substances studied have what could be called a specific or characteristic effect on growth. When very weak they have no effect whatever. In greater concentrations, all retard the later stages of growth, at the same time manifestly interfering with the other vital processes of the organism. When still stronger even the early stages of growth are impeded or prevented; in such cases the organ- ism is quickly killed by the chemical. In no case is the growth affected without other injury to the organism. On the whole it appears clear that the effects on growth are secondary; they are consequences of the interference of the chemical with the other vital processes of the animal, and do not appear unless such inter- ference exists. Essentially the same effects on growth appear whether the interference with other vital processes is due merely to the absence of any salts in the medium, to the presence of undue quantites of such a common substance as sodium chloride, or of minute quantites of such poisons as nicotine, alcohol or strych- nine. None of the substances studied, whatever the amount present, has a tendency to increase the normal rate or amount of growth (although the presence of a small amount of sodium chlo- ride permits this normal growth to occur, when it otherwise would do not so). 2. The early stages of growth show in certain respects a remark- able independence of the surrounding medium. In many cases, as we have seen, a certain amount of growth takes place under conditions which later destroy the organism. At fission the organ- ism seems to have a certain potential of growth, due largely to THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 8, NO. 4. 532 A.H. Estabrook internal conditions; it has a strong tendency to grow in a perfectly definite way, at a definite rate, the rate giving a curve of a definite form. It grows for a time in this way in spite of the almost com- plete absence of the salts that are necessary for its continued existence, and in spite of the presence of actively injurious chemi- cals, which in a short time kill the organism. Whatever growth occurs tends to follow the normal growth curve. The substances within the organism, plus water, seem all that is necessary for this process, and it persists for a time in spite of positively injuri- ous external conditions. No evidence was found that a race of a given typical size can be transformed by any of the chemicals studied into a larger or smaller race. Their effects on growth seem due to intent with the vital processes, resulting i in pathological conditions in other respects as well as in growth. Continued action of the chemi- cals that interfere with ‘growth usually sooner or later cause death. The causes of fies observed temporary changes in size in a given race under differing cultural conditions are probably to be eolehe for in variations in the nutritive and other conditions of the normal environment, particularly in conditions that affect the rate of fission. 3. On the precise nature of the deleterious action on growth in the case of the different chemicals the investigations gave little light. In the case of sodium chloride it appears possible, as we have seen, that a part of the injurious action is due to osmot.c pressure, while a part is not. But in the case of nicotine and strychnine the minute quantites employed show that the osmotic pressure plays no part, yet these chemicals produce essentially the same effects on the erowth as do undue amounts of sodium chloride. In the case of alcohol the growth occurs in solutions having a much higher osmotic pressure than the deleterious solu- tions of sodium chloride. Thus all the facts taken together seem to indicate that disturbance in osmotic relations plays little part in producing the effects on growth, even in the case of such substances as sodium chloride. 4. As to the processes in growth itself, it appears clear that the increase in size in early stages, up to 60 to go minutes (at Effect of Chemicals on Growth 533 which time half the growth in length has occurred), is due al- most solely to the imbibition of water. Up to this time growth occurs in much the same w ay whether there are food substances present in the water or not; as we have seen, it takes place when all the solid substances are removed from the fluid by filtering, or even 1n pure distilled water containing a little sodium eae. After about ninety minutes the presence or absence of bacterial food in the medium has a noticeable effect on the growth. If plenty of bacterial food is present the animals are thicker, but do not grow so long as with little food; this 1s owing to the fact that where food 1s abundant, fission takes place more frequently. The animals may, however, reach the normal length in a medium containing almost no food; we have seen that this occurred in distilled water containing a little NaCl. Butin such cases fission does not occur; this growth by mere imbibition of fluid can mani- festly not continue for more than one generation. 5. In investigating the different resistance of young and adult Paramecia toward the stronger solutions of the chemicals, it was found that adult Paramecia were much more resistant than very young animals toward sodium chloride, strychnine, and alcohol, while the reverse was true with nicotine, the young being more resistant than the adult. It would be naturally eeneeed from the work done on higher animals that the adult would be more resistant. The feversal of the effects found in nicotine are probably due to some specific effects of the nicotine which are not present in the other chemicals. 6. Acclimatization of Paramecium to strengths of sodium chloride, which would kill the animals if introduced directly, was found to be easy, but this acclimatization was not permanent and the animals finally died. In acclimatizing Paramecia to a § NaCl solution the ratios of the different salts within the cell are “probably changed to such a degree that it takes a long time for the cell to regain its eamilibeiaa so as to continue its Se metabolic processes. Acclimatization of Paramecia to the other chemicals was not attempted. 534 A.H. Estabrook BIBLIOGRAPHY Batis, W. L. ’°o8—LTemperature and growth. Annals of Botany, vol. 22, pp. 557-591. Binz, C. '67—Ueber de Einwirkung des Ch'nin auf Protoplasma-Bewegungen. Arch. fir Mikr Anatomie, vol. 3, pp. 383-389. CALKINS AND Lis, ’02—Effect of stimuli on the life cycle of paramecium caudatum. Arch. fur Protisten kunde, Bd. 1, pp. 355-371. DanigL, J. F. ‘08 —The adjustment of Paramecium to disti led water and its bearing on the problem of the necessary inorganic salt content. Am. Jour- nal of Physiology, vol. 23, pp. 48-63. ENNINGS, H.S. ’o08—Heredity, variation and evolution in Protozoa, II. Proceedings | ys , £ of the American Philosophical Society, vol. 47, no. 190, pp. 393-540. Lanctey, J. N. ’05—On the reaction of cells and of nerve endings to certain poisons, chiefly as regards the reaction of striated muscle to nicotine and to curare. Journal of Physiology, vol. 33, pp. 374-413. Ler, W. E. ’o08—Quart. Jour. Exp. Physiology, vol. 1, pp. 335-358. Poporr, M. ’og—Experimentelle Zellstudien, i. Ueber die Zellgrosse, ihre Fixierung und Vererbung. Arch. fiir Zellforschung, Bd. 3, pp. 124-180. ScHuLzE, M. ’63—Das Protoplasma der Rhizopoden und der Pflanzenzellen. Pp. 68. Leipzig Eng’emann. Wooprurr, L. L. ’o8—Effects of alcohol on the life cycle of infusoria. Biological Bulletin, vol. 15, pp. 85-104. Accepted by the Wistar Institute of Anatomy and Biology, May 5, ro1o. Printed August 18, roto. CONTRIBUTIONS FROM THE ZOOLOGICAL LABORATORY OF THE MUSEUM OF COMPAR- ATIVE ZOOLOGY AT HARVARD COLLEGE. E. L. MARK, Director. No. 209. OLFACTORY REACTIONS IN FISHES G. H. PARKER The fact that the olfactory apparatus, both peripheral and central, is very well developed in most fishes has led many mor- phologists to ascribe to these animals a keen sense of smell; but this opinion has been unsupported by physiological evidence, for up to the present time investigators of the subject have not been able to demonstrate any form of stimulation or reaction charac- teristic of this apparatus in water-inhabiting vertebrates. The observations of Aronsohn (’84, p. 164), that a goldfish, which ordinarily will eat ant pupz with avidity, will not take these pupe after they have been smeared with a little oil of cloves, are not conclusive evidence that the fish scents the oil, for it is entirely possible that this oil merely irritates the skin of the fish’s snout and does not stimulate the olfactory apparatus at all. Nor is the discovery madeby Steiner (788, p. 47), that the spontaneous appropriation of food by Scyllium ceases on the removal of the cerebral lobes or simply on cutting the connections between these lobes and the olfactory bulbs, satisfactory evidence that the olfac- tory apparatus in these fishes is an organ of smell rather than a receptor for taste or some closely allied sense. Nagel (94, p. 184) noted that the front portion of tne head of Barbus was as sensi- tive to sapid substances after the olfactory tracts had been cut as before that operation, and Sheldon (’o9, p. 291), who has stud- ied the dogfish with great fulness, demonstrated that the decided sensitiveness of the nostrils of this fish to weak solutions of oil of cloves, pennyroyal, thyme, etc., was not influenced by severing the olfactory crura, but disappeared on cutting the combined maxillary and mandibular branches of the trigeminal nerve. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 8, NO. 4. 536 G. H. Parker Evidently the nostrils of fishes, like those of the higher verte- brates, are innervated by fibers from the trigeminal nerve, and it is this nervous mechanism rather than the olfactory apparatus that 1s stimulated by the substances that have ordinarily been applied by experimenters. In fact, so far as the olfactory appar- atus of the fishes and seaplane is concerned, we must agree with Nagel (’94, p. 61) that no one thus far has discovered any- thing positive concerning its function. It is, therefore, a matter of interest to record what seem to be unquestionable reactions dependent upon the olfactory apparatus of our,common fresh- water catfish, Amiurus nebulosus. Amiurus nebulosus is a bottom-feeding fish possessing fair powers of sight and unusual gustatory organs located not only in the mouth and on the general outer surface of the body, but especially on the eight Harbiee about the mouth (Herrick, ’03). It is a hardy fish, living well in confinementand undergoing opera- tions with sucess. It possesses near its anterior end a pair of nasal chambers each of which is provided with two apertures, one anterior, the other posterior. ‘The anterior aperture is nearly circular in outline and is located on a slight conical elevation somewhat anterior to the root of the dorsal barblet. The pos- terior aperture is slit-like in form and lies immediately posterior to the same barblet. “The anterior aperture is apparently always open; the posterior one seems capable of slight closure, but is usually freely open. By keeping catfhishes a few days without food, they can be made most eager for it, and if into an assemblage of such individuals, a few fragments of fresh earthworms are dropped, the excitement that ensues will last some time after the final piece of worm has been swallowed. During this period the fishes swim about excit- edly in the lower part of the aquarium, now in this direction, now in that, and frequently sweep the bottom with their barblets. As can be noticed when the feeding actually occurs, the fishes seldom seize a fragment of worm till their barblets have come in contact with it. Y et before they have thus touched any food, they show a marked degree of excitement and it is this initial nervous state that would lead an observer to suspect that they scented their food. Olfactory Reactions in Fishes 537 I, therefore, took this phase of their activity as the one to be tested in connection with their olfactory apparatus. A number of fishes that had been without food for several days were isolated in small vessels of water and, after an hour or more, when they had come to rest, they were tested with a solution filtered from a mixture of freshly chopped earthworms and tap- water. By means of a very fine glass pipette a small amount of this solution was discharged directly over the anterior olfactor aperture of a given fish and the fish was then closely watched. Notwithstanding the most careful manipulation, more or less of this solution ane be seen at times to be swept into the mouth of the fish by the respiratory current and may well have stimu- lated the gustatory organs in that cavity. The subsequent move- ments of the fish were extremely irregular, and, though I was reasonably sure that as a result of the application of the solution the fishes respired more deeply and fully than before, I could not be certain that this reaction was not due to oral stimulation. Though I could see that some of the solution applied to the nasal aperture was sucked into the mouth, I was unable to make out whether any of it really entered the nasal chamber itself. As it is essential for the stimulation of the olfactory surfaces that the exciting material shall make its way to them, I turned next to the accessibility of these surfaces from the exterior. The nasal apertures of the catfish are apparently always open and when a fish is swimming with some vigor through the water its motion doubtless drives a current of water through each nasal chamber. I tried to demonstrate this current indirectly by mak- ing a fish swim for five minutes through water containing a small amount of starch in suspension and then comparing the con- tents of its nasal chambers with that from the chambers of a fish that had been held motionless for a like period of time in the same water. So far as the comparison was concerned, the results were inconclusive, but the microscopic examination of the freshly opened nasal chambers led to the discovery that they were lined with cilia which were beating vigorously and persistently. To ascertain whether ee cilia produced a current of water through the nasal chambers, a freshly prepared fish-head was £38 G.H. Parker immersed in water and the nasal apertures were tested with a mixture of water and carmine. By this means it was easy to demonstrate that a current of water entered the anterior nasal aperture and emerge from the ae one, as has already been shown in Amia by Brookover (’10, p. 77), and that the water passed through the olfac tory ee x the catfish in from eight to ten seconds. It is therefore certain that even in the resting fish a continuous current of water is coursing through the nasal chambers from anterior to posterior, and, fine from the posi- tion of the nasal apertures on the body of the fish, this current is probably accentuated by forward locomotion. If the nasal chambers of a resting fish are continuously provided with a flow of water from the exterior by which odorous material may be carried to the olfactory surfaces, the failure of the animal to respond to such material must have some deeper seat than the receptive organ. Since what appeared in the normal catfish to be a scenting reaction was observed only when the animal was in locomotion, it seemed to me that this condition might be the only one under which olfactory responses would be esiubiaees and that the resting fish represented a state wholly unfavorable for such reactions. In other words, it seemed possible that only when the central nervous organs were discharging impulses to locomotion were they in a condition to transmit in :pulses eminating from the olfactory receptors. With this idea in mind, I set about devising a new line of experimentation to be carried out on the actively swimming fish. As a preliminary to a revised method of procedure, five normal fishes were placed in a large aquarium over night that they might become accustomed to their surroundings. In this aquarium were then hung two wads of cheese-cloth, in one of which was con- cealed some minced earthworm. The fishes, which were swim- ming about near these wads, were then watched for an hour and their reactions in reference to the wads were recorded. The wad without worms was passed by the fishes many times and did not excite any noticeable reaction. The wad containing the worms was seized and tugged at eleven times in the course of the hour notwithstanding the fact that from time to time this Olfactory Reactions in Fishes 539 and the other wad were interchanged in position. Not only did the fishes thus openly seize this wad, but, when in its neigh- borhood, they would often turn sharply as though seeking some- thing but without success, a form of reaction seldom sheduved near the wad which contained no worms. ‘!wo other sets, of five normal fishes, each, were tested in this manner and with similar results. It was perfectly clear to anyone watching these reac- tions that the fishes sensed the difference between the wad of cloth with worms and that without worms. To ascertain what receptive organs were concerned in ve re- actions just described, I took from among the fifteen normal fis ses already tested two sets of five each and prepared each set aiffer. ently by subjecting its members to a special operation. One set was etherized, and, through a small incision between the eyes, their olfactory tracts were cut thus rendering their peripheral ol- factory apparatus functionless. From fishes of the other set all the barblets were removed whereby their external gustatory or- gans were partly, though not wholly, eliminated. After these operations both sets of Robes were liberated in the large aquarium where they remained for over two days. At the expiration of this time, thev were carefully inspected and tested. They swam about in an essentially normal way and members of both sets snapped bits of worm from the end of a hooked wire much as a normal fish does. I[ therefore judged them to be in a satisfactory condition for experimentation. The tests were begun by introducing into the large aquarium containing the ten ies a wad of pene cloth ui which were hidden some minced earthworns and recording the kind of fish that visited it and the nature of their reactions. During the first hour the wad was seized 34 times by fishes without barblets but with normal olfactory organs and, though often passed by fishes with cut olfactory tracts, 1t was “nosed”’ only once by one of these. Inext substituted a wad of cheese-cloth without worms for that with worms and recorded the reactions of the fishes for a second hour. ‘Though members of both sets frequently swam by this wad, none at any time during the hour seized it or even nosed it. These tests w ere repeated on the same fishes for two suc- 540 G.H. Parker ceeding days and with essentially similar results. On the second day the w ad with worms was seized 16 times during the test hour by fishes with normal olfactory organs and on the third day 54 times. On both these days the fishes with their olfac tory tracts cut made no attempts on the wad with worms nor did any fish at any time nose the wormless wad. The movements of the two sets of fishes when in the neighborhood of the wad containing minced worms were characteristically different. “Che fishes with their olfactory tracts cut swam by the wads without noticeable chang; those without barblets, but with their olfactory appa- ra‘ us intact almost always made several sharp turns when near the ‘vad as though seeking something, and then either moved slowly away or swam more or nes directly to the wad and began to nose and nibble it. These reactions were so clear and so character- istic chat when taken in connection with the conditions of the fishes, they lead inevitably to the conclusion that the olfactory apparatus ‘of the catfish is serviceable in: sensing food at a distance much beyond that at which the organs of taste are capable of acting; in other words, catfishes truly scent their food. Whether such olfactory reactions as those that have just been described are really due to smell or not is regarded by some authors as an open question. Nagel (94, p. 56), who has discussed this matter at some length, concluded on rather theoretic grounds that fishes could not possibly possess a sense of smell and that their so-called olfactory organs act more as organs of taste than of smell. Possibly the whole matter is merely one of definition. With human beings smell differs from taste chiefly i in the concen- tration of the stimulating solution and not, as was formerly sup- posed, on the state of the stimulating material, for, though we usually say that we smell gaseous or vaporous materials and taste liquids and solids, all these substances are in reality dis- solved on the moist surfaces of wnich ever sense organ they stim- ulate. The most striking difference between taste and smell with us is that we smell extremely dilute solutions and taste only very much more concentrated ones. Asa result we recognize the presence of many distant bodies by smell and not by taste, for the very minute amount of material that reaches us from the dis- Olfactory Reactions in Fishes 541 tant body will form a solution on our moist surfaces that will be stimulating for our organs of smell but not for our organs of taste. Hence our olfactory organs as compared with our organs of taste are what Sherrington (’06) has called distance receptors, a desig- nation justly emphasized by Herrick (08). Although this dts- tinction between taste and smell is one of degree rather than of kind, it seems to me reasonably sound and it ae eel holds in the case of the catfish much as it does with us, for this fish responds through its olfactory organs to solutions too dilute to affect its gustatory organs, and the nature of the response to olfactory stim- ulation (seeking food, etc.) is such that the olfactory organ in this fish can be called appropriately a distance receptor. I therefore believe that the cathsh, though a water-inhabiting animal, pos- sesses an olfactory organ that is as much an organ of smell as is the olfactory organ of the air-inhabiting vertebrates. 542 G. H. Parker BIBLIOGRAPHY Aronsoun, bk. '84—Beitrage zur Physiologie des Geruchs. Arch. fiir Anat. u. Physiol., physiol. Abt., Jahrg. 1884, pp. 163-167. Brookover, C. ‘to—The olfactory nerve, the nervus terminalis and the pre-optic sympathetic system in Amia calva L. Journ.Comp. Neurol. Psychol., vol. 20, pp. 49-118, pl. 1. Herrick, C. J. ’03—The organs and sense of taste in fishes. Bull. U.S. Fish Comm., for 1902, pp. 237-272. Herrick, C. J. 08—On the phylogenetic differentiation of the organs of smell and taste. Journ. Comp. Neurol. Psychol., vol. 18, pp. 157-166. Nace, W. A. ’94—Vergleichend-physiologische und anatomische Untersuchungen uber den Geruchs- und Geschmackssinn und ihre Organe. Bibl. Zool., Heft 18, viii + 207 pp., 7 Laf. | SHELDON, R. I.’0g—The reactions of the dogfish to chemical stimuli. Journ. Comp Neurol. Psychol., vol. 19, pp. 273—311. SHERRINGTON, C. S. ’06—The integrative action of the nervous system. New York, 8vo, xvi + 411 ‘pp. STEINER, J. ‘88—Die Functionen des Centralnervensystems und ihre Phylogenese. Braunschweig, 8vo, xu + 127 pp. Accepted by The Wistar Institute of Anatomy and Biology, June 0, toro. Printed August 18, 1910. _ TP ws) i ‘ae in me 1 AM bash | : ; 4 f a it ’ 2 ‘tee S. H Le W F - 7 i | 1 j ; * Ge Y 5 of —% j : J i) = ~¢ ' mi F r 7 - : . >» - 5 9 - ap : ; F 4 ' a » _ a) 4 Py ; i ae ae ine may Ar e re =) BINDING SECT ~ Tor em eee “ep 1080 L The Journal of experimental 1 Zoology TAR Veo —~Y COD. 2 BioMed per. PLEASE DO NOT REMOVE CARDS OR SLIPS FROM THIS POCKET UNIVERSITY OF TORONTO LIBRARY ie Wa 3 if 4 y lee trate fs ‘'y Me eles itt teteyl ehaan aa ag : a ee hoa hue “ Pita ” F) a ah rad We oive ry Weed cate tte " P Vsovyibri stil by bony 4 fois Vi4e% Hag the ge v Voy ny erp wae POD le etviee ey dey ety bolts ? 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