if Seah relate et / 3 ag y ot okey F { thea ey t fy bpp eee et Seerte tere > t Nght Lar toy tert ty $70 Mes ee Ht A pteter ate. a O52 8 (Xe: ot At Ie ea nal ions MEE EC NLS if ene hehiat ta chen 3 He ¥ a’. wataineasys beta hy City ¢ Teter uete Satan ‘ cart, hfe’ trtee scare seein ached fy Pot Takes te sehr: Peete RUE ; Tne MMe Re aes Bete! alts f qy i maha Shou! Bs os Quik rt, ‘g 53 Df ga taky wate Tis asia Batlle Aen EMSC A SEE LE! tarsal ete be ite THE JOURNAL OF EXPERIMENTAL ZOOLOGY EDITED BY WiuuiaM FE. CastLEe Frank R. LILuiE Harvard University University of Chicago Epwin G. CoNKLIN Jacques LorB Princeton University Rockefeller Institute CuHarLes B. DAVENPORT Tomas H. Moraan Carnegie Institution Columbia University HERBERT S. JENNINGS GEORGE H. PARKER Johns Hopkins University Harvard University Epmunp B. WILSON, Columbia University and Ross G. HARRISON, Yale University Managing Editor VOLUME 17 1914 THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY PHILADELPHIA, PA. (COMPOSED AND PRINTED AT THE -WAVERLY PRESS es (Br THE WiLL1aMs & Wirxins Ci : BALTIMORE, gis S.A. we ly = Mes unant CONTENTS NOE JULY EK. J. Lunp. The relations of Bursaria to food. II. Digestion and resorption in the food vacuole, and further analysis of the process of extrusion. Eight figures and two ee eels @ se 46,6 6b ws 6)N 8 Ol uses Ss wie le els © #1. eJel= eee sls sie @) @ 0.6) 6) ep) 0)(6)|.0 8) vie 6 6) ua sive (6 \6)a | s = 0s & Ole 01018 ’CuHartes W. Metz. Chromosome studies in the Diptera. I. A preliminary survey of five different types of chromosome groups in the genus Drosophila. Diagram and twenty-six figures (plate) oe ss. a 6) nee os a ein 00 © (6) 8) 0 © @ 0 em, 0\6 8 a) 8)10.¢) @ ae eels» © vim so je) wie = = = elale ee C.M. Cuitp. Studies on the dynamics of morphogenesis and inheritance in experi- mental reproduction. VIII. Dynamic factors in head-determination in Planaria. Two figures WO \@ 0 whe a 6 6. 4))0\ 6) ee 16. 0 8 © 0 © 0,00 6 @)[6\ a,'6\0 0. e086, 00 = 00.0) 6 6 8 8)\0.u 6 o 6 «pe se 5 0 6 86 0 8 « = Seis 018 sie T. H. Morean. Two sex-linked lethal factors in Drosophila and their influence on the sex-ratio. Seven figures CHO CeCe mC Cacarer? ChUI Care r mC nny CML MCat het tery CO CU) COCuty Cs Cy Cty Ceci tI Jacques Lors. Cluster formation of spermatozoa caused by specific substances from Roscor R. Hype. Fertility and sterility in Drosophila ampelophila. I. Sterility in Drosophila with especial reference to a defect in the female and its behavior in heredity Pie \e\/e) ele s)/0) «040, «© 0) © (6) .0 (o)[e! v.08) |v. u 0's .0\ (ee) ©) 0) 0) 010) 2) 6) w 08 ©, 8.0 6 0, 6, « 6) 016! 0 wiv, 0.0 410) 0/s) 0) 9,,0 etellele ele ele) sie) = NO. 2 AUGUST Roscor R. Hype. Fertility and sterility in Drosophila ampelophila. II. Fertility in Drosophila and its behavior in heredity. Nine diagrams................-...seeee Brapiey M. Parren. A quantitative determination of the orienting reaction of the blowfly larva (Calliphora erythrocephala Meigen). ‘Twenty-four figures.......... a S. J. Hotmes. The behavior of epidermis of amphibians when cultivated outside the DOM Sem SCVMM HELIER Soh cier oie ete revere cre telve Foye ae eteceie) cists ajo-e « stcuelerenerecraicts elcid oeateiatel ake el tetarete Watpvo SHumway. The effect of thyroid on the division rate of Paramaecium. Three GINO EAC es 6 AR a REDE HIG Oe rE aOR eR TOPO crn CMC ram es aie cord o's dpie 45 61 81 1238 141 173 213 lV CONTENTS NO. 3 OCTOBER "Ty. H. Morean. A third sex-linked lethal factor in Drosophila. Three figures........ 315 HERMANN J. Mutter. A gene for the fourth chromosome of Drosophila Etuet Nicuoitson Browne. The effects of centrifuging the spermatocyte cells of Notonecta, with special reference to the mitochondria. Six figures............... 337 Roscoe R. Hype. Fertility and sterility in Drosophila ampelophila. III. Effects of crossing on fertility in Drosophila. IV. Effects on fertility of crossing within and without an inconstant stock of Drosophila. Twelve diagrams..................... 343 Eveanor L. Cuark anp Exrot R. Ciarx. On the early pulsations of the posterior lymph hearts in chick embryos: their relation to the body movements. Two charts. 373 RAYMOND PEARL AND Maynik R. Curtis. Studies on the physiology of reproduction in the domestic fowl. VIII. On some physiological effects of ligation, section, or MERON A OF, GE OVECUICE 20.5.5 am, cocteia 5 5 ees bose chee te tole UATE Oe & ORO tae eee ».. 395 NO. 4 NOVEMBER ~ x LoranpDE Loss WoopruFF AND RHODA ERDMANN. A normal periodic reorganization process without cell fusion in Paramaecium. Sixty-six figures (four double colored PLATES he pec atc talus cc ch: Sete hac ane tote cette aa eta ns a fey sees oc RA ed 425 Ross G. Harrison. The reaction of embryonic cells to solid structures. Fourteen HOUMES esc 5 Ce Rees sc SG Serie OR erie ERs cy eee SINGLET 2 os oe aE ein 521 Davin Day Wuitney. The influence of food in controlling sex in Hydatina senta..... 545 THE RELATIONS OF BURSARIA TO FOOD II. DIGESTION AND RESORPTION IN THE FOOD VACUOLE, AND FURTHER ANALYSIS OF THE PROCESS OF EXTRUSION EK. J. LUND Zoélogical Laboratory of the Johns Hopkins University EIGHT FIGURES AND TWO PLATES CONTENTS TLa Alea" EEPOLE Vo 1 Rage eerie Se RO NR Abe eh aA ee Re OR Rane Ema a i NMeatentalrandame lhodsaanuat ti scnee ecient inci Cae bits castors Nee arae 3 To what extent can yolk be considered a food for Bursaria?.................. 5 TETRA TORUS (0; Ueno een sca Moi inte Set mere a ees Oe Pett Tt na OAM 5 Ce aCe 8 1. The food vacuole; formation and physical changes..............-.--- 8 2, Chemical changes imtheimood yacuole...25-). 2.6 Sevdcn ese. +s sare ea 12 3. Effect of quantity of vitellin eaten, upon the average velocity of Co Ir Zersh 00) | Ne Re an Mee CR AA iy ae TRA ae 9 OLR ON gS ACR 15 4. Effect of congo red upon the average velocity of digestion, extrusion, SCRE OCR RE RAS Pe EMME aR aS yo Th Aig ARS Fst OmkeNe 22 Birestion ang resorphoniol fai, cage sacs os cles Sos oe eiiela tyres + 4 gees Rae 26 1. A demonstration of fat digestion and resorption in the food vocuole ORAEUTSEM PNA hoe cet eames eae Oca a ee a Rape sta ay ene Ica 26 2. Role of fat in growth and energy requirement....................5005- 28 3. Is fat formed from vitellin or starch in Bursaria?............,....++-- 29 The nature of some of the factors which bring about extrusion................ 30 i Hxperiments with paramin oil and: oltve! oil... sec casas aaa 30 2. Effect of mass or volume upon the extrusion reaction..............-. 34 pSiTTSTE E:T At PRE AAICD Ae ey, I Rei DOR aE RE ay ts ce PPAR ATS OE A Bat 38 ANT ETREY ATURE, CONSE ie ah, obi Gis Siediln o Pe He Ome ice hoeorcees Sie manera okey co .om een Gee fy INTRODUCTION To obtain exact information on the processes of metabolism in single-celled organisms, and particularly the Protozoa, experi- mental procedure must be based upon simple, well defined and reproducible conditions, so that the work may be repeated and verified. Attempts to fulfill this requirement are subject to the 1 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 17, No. 1 gJuLy, 1914 2 E. J. LUND danger that the conditions may become so artificial as not to war- rant conclusions concerning what occurs under usual (‘normal’) conditions of the organism. Nevertheless, a certain degree of simplicity may often be obtained in the conditions of experimenta- tion and yet coincide with the usual conditions of a varied en- vironment to a sufficient degree so that the results give a definite answer to questions which are directed toward finding out what the principles are which underlie the processes. The following experiments have been carried out with an BAIS to fulfill the above requirements. In a previous paper (Lund 714) some of the most important external relations of Bursaria to food have been presented, in- cluding a demonstration of a selective extrusion among the food vacuoles. The following paper deals with the processes which take place in the food vacuole, and with the conditions under which it exists as an active system in this unicellular animal. Proteins, fats, and carbohydrates in the form of starches are present in the material eaten by Bursaria, as may easily be de- tected by direct observation or by: the aid of microchemical tests. Protein in the form of other living ciliates, flagellates, etc., is taken in and digested. Many of these Protozoa which serve as food contain fat globules and starch or amylum grains, and hence all three food substances may at times be found present in the cytoplasm of Bursaria from flourishing cultures. Since there is disagreement between results of different investi- gators, working with the same Infusoria; but more particularly because there are indications that similar forms among these animals differ in their food metabolism, the statements here made refer only to Bursaria and may or may not be valid for others, except where specifically stated. RELATIONS OF BURSARIA TO FOOD ° 3 MATERIAL AND METHODS Simple types of the three classes of food were found in lipoid- free egg yolk (vitellin), pure olein, and starch grains of various kinds. Potato starch was the most serviceable because grains of a very uniform size can be obtained by repeated decantation of — a suspension, and hence results may be expressed quantitatively in terms of unit volume. Fresh hard boiled yolk—a combina- tion, chiefly, of lipoids and protein—was also used. Olive oil of the best grade obtainable gave the same results as prepared olein. ~** From the yolk of the hen’s egg was prepared a protein (vitellin) that was perfectly lipoid-free, so far as could be determined; this was done as follows: An egg was boiled to hardness (15-20 minutes), and the granular portion of the yolk was kneaded gently to a moist, fine, floury pulp. This was placed loosely ina Soxhlet apparatus and extracted with an alcohol-ether mixture, for eight to ten hours; in this way the fat and lecithin were re- moved. The vitellin was then washed several times with fresh alcohol-ether mixture, removed from the Soxhlet and while still moist, gently kneaded to a fine white powder. This was left to dry at 25°C. for twenty-four hours. After drying was completed the residue was gently rubbed between sheets of filter-paper. When the steps mentioned above were carefully carried out, the grains of vitellin were found to be separated. This white dry powder was kept in a dry bottle in a cool place, and used as a food stock. When the animals were to be fed, a suspension of the powder was made in tap or distilled water and rubbed up; then left to settle, and the supernatant liquid with the smaller particles was poured off. This process of separating the smaller grains from the larger ones was repeated until a perfectly clear suspension ‘ of the protein replicas of the uniform hard-boiled grains was obtained. It is important to note that some eggs yield much more uni- form yolk grains after boiling, than others. Only those eggs were used in this work which were satisfactory in this respect. 4 E. J. LUND It seems highly probable from the results, that all traces of lecithin and fat had been removed by this method. Cold ether or cold alcohol extraction alone is not sufficient to remove all the lipoid content from hard boiled yolk. This will appear from results (p. 29) of experiments in which such vitellin grains were used. By this method of preparation, lipoid-free vitellin may be ob- tained in very uniform grains, and this permits one to fulfill a condition most important for precise experimentation. One can feed definite unit quantities to a single cell} and this makes it possible to attack a number of questions regarding metabolism that would otherwise be inaccessible, and to express the results in quantitative terms. Fresh hard-boiled yolk served as a protein-lipoid diet, which could be fed in the same way, for the vitellin grains’ are simply the protein matrix of the fresh hard boiled yolk grains in which the lipoids and any other alcohol-ether soluble substances are imbedded. There is, of course, no means of knowing whether the vitellin of the unextracted yolk grain does not undergo change during ether-alcohol extraction; but the fact (demonstrated by the experiments) that in the food vacuole digestion of the protein in these two forms occurs in the same way and with about equal readiness, shows that the process of fat extraction does not alter the chemical or physical nature in such a way as to interfere with the digestion and resorption of the vitellin. In all the experiments where necessary, the animals were placed in 500 ce. of tap water twenty-four hours previous to feeding. The result of this was that the food and débris con- tained in the cell had been discharged during the twenty-four hours of starvation, and hence a cell with perfectly clear cyto- plasm was obtained for the experiments. By this means a parti- cularly desirable uniformity in physiological condition of the organisms was obtained. The material was from the same wild stock cultures as that used in the experiments reported in my earlier paper. RELATIONS OF BURSARIA TO FOOD 5 In most of the experiments the organisms were fed singly, and large numbers were used, so that any individual variations play a minor part, if any, in the final results. Where temperature regulation was necessary the experiments were carried on in an oven kept constant to within 1.5°C. TO WHAT EXTENT CAN YOLK BE CONSIDERED A FOOD FOR BURSARIA As yet it has not become possible to propagate pure races of ‘Bursaria from single individuals, or in culture, by feeding it an artificial diet of vitellin or fresh yolk; so that it must be re- membered in conjunction with the facts presented at this time, that the full requirements for the maintenance of life and re- production have not been fully satisfied, and to this extent the ideal conditions have not been met. But to prove that vitellin and fresh yolk are drawn upon for the energy requirements and growth of the cell Experiment I is given: Experiment 1. Three sets of 74 individuals each were starved in tap water for 24 hours. Set A was fed with fresh hard boiled yolk in suspen- sion prepared as described above. Set B was fed vitellin; while Set C was not fed. Each individual in the three sets was left to eat as much as it would in 20 minutes. They were then picked out and washed once in tap water which had been boiled, from this they were removed to watch glasses each containing 2 cc. of boiled tap water. The water was boiled in order to kill any bacteria, or other organisms sometimes present in small numbers in the tap water. Two individuals were placed in each watch glass and these were set m moist chambers which were kept side by side. There was therefore no difference in the temperature at which the three sets remained during the experiment, although a daily fluctuation in temperature of 4 or 5°C. occurred. Such variation, however, does not affect the results of the experiment for the particular end in view. It was found that after feeding twenty minutes nearly all of those in the fresh yolk suspension (A) had eaten large quantities, while those in the vitellin suspension (B) had on the average not eaten so large a number of ‘grains as those in the fresh yolk suspension, although in both Set A and Set B, each individual had eaten more than one grain. All the indi- viduals in the three sets were treated identically except in feeding. All were normal and active at the beginning of the experiment. At the end of every 24 hours detailed records were taken for each individual, as to whether it had a normal form or had undergone dedifferentiation— 6 E. J. LUND which is under many conditions a typical reaction—and if the latter were the case, to what extent it bad proceeded. Death was taken to have occurred when the cell had ceased to move and began to disinte- grate. In this way a record of the effects of the food on the maintenance of form, degree of activity and length of life, was obtained, so that from these the means can be taken and compared. To save space, instead of giving the detailed results in the form of tables they are given by curves and averages. Curves A, B and C of figure 1 represent for the three sets respectively, the longevity or death rate. Points on the abscissa indicate the time in hours, while points on the ordinate represent the number of individuals which were still alive at the time the record was taken. The curves A and B of Sets A and B respectively, show clearly (1) that the yolk and vitellin had a definite effect in prolonging the life of the cell. The relation of curves A and B to each other will become clearer when we have considered later experiments (p. 28) which show, other things being equal, that we should expect longer life from individuals fed both lipoids and protein. By comparing the average length of life in the three sets we find that Set A lived 4.98, Set B, 5.39 and Set C 3.20 days. Sets A and B, therefore, lived on the average about two days longer than the unfed Set C. (2) The vitellin grains underwent total di- gestion and resorption in most cases, while the fresh yolk grains were generally only partially digested, this was especially the case where more than 2 or 3 grains were eaten. (3) The animals which had been fed were on the average more vigorous than those of Set C. (4) The animals of Set A grew to be larger in most cases than those of either Set B, or Set C. Many of Set A became twice as large as those of Set C, while those fed vitellin were on the average larger than any of Set C. The maximum size was reached about thirty-six hours after feeding. There can therefore be no question but that both the fresh yolk and the vitellin entered into the chain of metabolic proc- esses and were in part at least, drawn upon by the cell for its energy requirement. But very few of the organisms divided. Whether yolk or vitellin is a sufficient diet for cell division as RELATIONS OF BURSARIA TO FOOD *pey you ‘StL (Q PAIND) O 409 OTTYM “UTTTOITA poj SBM (q 9AIND) { 499 ‘yjoX YSo1J pay SBA (Y PAIND) Y OG “Yovo spenprarpur pL JO S}OS 9014} UI OjI] JO YASUI] oy} Surmoys soamny) T ‘SI "SYH 88z y9z ov 12 z6l gl vvl ozt 96 ZL ay vz 8 B.S. LOND well as for growth can not be answered at present. But for the questions dealt with in the present paper the important fact to establish is that the yolk or vitellin can be drawn upon for the energy requirements and growth of Bursaria. PROTEIN. DIGESTION 1. The food vacuole; formation and physical changes The passage of the grain into the body is brought about par- tially by ciliary action at the base of the buccal pouch; but large grains or masses of food are pushed through the base of the gullet and into the endoplosm, by what seem to be contractions of the wall of the base of the gullet, and also apparently by activity of the endoplasm about the gullet behind the food. During this process of swallowing, a liquid comes to be included about the food so that when the vacuole separates from the base of the gullet liquid surrounds the yolk grain. Where does the liquid enclosed with the grain come from? Part of it is derived from the external medium, as is readily determined by direct observation of the process of swallowing. But the liquid in the vacuole, when the latter is separated from the base of the gullet, is likewise partially made up of an acid secretion from the cytoplasm of the lower portion of the gullet, as will be demonstrated below. When the vacuole has formed it is usually carried toward the middle of the cell and may remain practically stationary, especially if it is large. If movement takes place, which is nearly always the case when the vacuole is small, then the vacuole may traverse any part of the cyto- plasm, and in any direction. In Bursaria there is no such regu- larity in the course of the food vacuole as has been described for Paramecium by Nirenstein (’05); and for Carchesium by Green- wood (’94). Often digestion and resorption begin and are completed in one and the same place, without any circulation of the vacuole. Residues are extruded from a small area on the dorsal side of the body. The first visible change which takes place is the absorption of the liquid which has been enclosed with the grain during the RELATIONS OF BURSARIA TO FOOD 9 formation of the vacuole. This is always definite and can generally be readily observed and followed throughout the process. This is exemplified by the following experiment: Experiment IT. Single normal individuals which had been placed in tap water 24 hours previously, were each fed a single grain of fresh yolk and the time interval noted between the separation of the vacuole from the base of the gullet and the point of complete disappearance of the liquid about the yolk grain. Table 1 shows records from ten individuals each fed one grain. From the table it will be seen that the duration of the process is relatively uniform. The point when all the liquid about the grain has been absorbed was determined to within about thirty seconds. TABLE 1 Experiment II INDIVIDUAL NUMBER 1 | 2) 3 4 | 5 5} Gi [3 | 9 0 10° AVERAGE L Number of minutes for resorption of AG al liquid about yolk grain............ 4 | 33 3 43) 7 f+] 33 5 | 4.45 When more than one grain is enclosed in the vacuole the same process of absorption takes place, following the separation of the vacuole from the base of the gullet; the vacuole membrane be- coming closely applied to the surface of the grains as is shown in figure 2. Different vacuoles in the same cell are quite independent as to the absorption of fluid. To illustrate, figure 3 is given. Here the new vacuole contained a comparatively large amount of liquid about the grain (in most cases the quantity of liquid is less), and came to be located close to an older vacuole in which the process of digestion of a Paramecium had practically been com- pleted, and which contained a considerable quantity of liquid. The vacuole containing the digested Paramecium was unaffected in size by the absorption of the liquid about the yolk grain in the newly formed vacuole. An explanation on the basis of osmotic relations within the cytoplasm and in the vacuoles, assuming the vacuole membrane 10 E. J. LUND to remain in a uniform condition, might perhaps account for the visible difference in the vacuoles in this and similar cases. But a conception of this process may more properly be obtained if we Ke, ) mer ays eel eS e, POO fy? Fig. 2 Shows the resorption of the liquid included with the yolk grains during formation of the vacuole. A, food vacuole just separated from base of gullet; B, the same, five minutes after separation. Fig. 3 Showing the independence of vacuoles with respect to resorption of liquid contents. The larger vacuole containing a partially digested Paramecium is not affected by the resorption of the fluid in the vacuole containing the fresh yolk grain. B drawn 53 minutes after A. think of it as a process of imbibition of the liquid by the colloidal cytoplasm, accompanied by changes in the permeability of the vacuole membrane. The problem here must be similar to that RELATIONS OF BURSARIA TO FOOD 11 which will confront us later regarding the absorption of the liquefied products of digestion. Within one or two hours after the grain comes to lie in the vacuole, the beginning of solution becomes apparent as a lique- faction and consequent rounding off of the corners and_ edges. This proceeds in exactly the same manner as when cubes of coagulated egg-albumen are digested by the action of pepsin- hydrochloric acid. At the end of digestion no solid remains of the vitellin grain can be seen; all that is left is more or less liquid in the vacuole. Another important visible change in the vacuole consists in the usual second appearance of liquid about the vitellin grain. It seems most in conformity with the observed facts to consider this as a consequence of the formation of soluble products of digestion, and as being due to the fact that the rate of resorption of these liquid products is less than the rate at which the lique- faction of the vitellin grain takes place. The reason for this conclusion is partly based upon the fact that in some cases the whole process of digestion of the vitellin may go on and be completed without the appearance of any visible liquid between the grain undergoing solution, and the vacuole membrane. In this case it is obvious that the rate or power of resorption is equal to or greater than the rate of solu- tion of the grain. Furthermore, it is to be expected that as digestion of the protein continues, the affective osmotic con- centration of the cleavage products increases and this obviously - may bring about an increase in the liquid contents, provided that the permeability of the vacuole membrane (resorption) does not undergo a simultaneous, proportional increase. In other words, the absence or presence of liquid during digestion of vitellin is a resultant of two sets of conditions (a) the rate of digestion and (b) the rate or power of resorption, one factor of which is the degree of permeability of the vacuole membrane. This further agrees with the fact that digestion of a vitellin grain is not always at a uniform rate. A rapid solution of the grain sometimes begins shortly after eating, changing later to a slower one. Even 12 E. J. LUND food vacules formed at the same time, of the same material, and in the same individual, may behave diversely as to the resorption of liquid, and also in rate at which digestion takes place. But this individuality of behavior in different vacuoles does not prevent the conception of equilibrium from being applied to the phenomena of resorption. Essentially the same observable physical changes take place in vacuoles containing fresh yolk as has been described for vitellin. Further evidence for this view will be given on page 25. 2. Chemical changes in the food vacuole Some of the chemical conditions in the food vacuole of Bur- saria during digestion of vitellin can be shown by the use of sensitive indicators adsorbed by the grains. Vitellin or fresh yolk grains stained with neutral red remain bright red during the whole process of digestion. Grains stained in an alkaline alcoholic solution of alizarin, quickly lose the blue color and remain colorless throughout the digestive process. Similarly, grains stained in an aqueous blue litmus solution change to red and remain red until nothing remains of the grains. These three indicators agree therefore in showing that the whole process of digestion of vitellin takes place in an acid medium, and that during no part of the process does alkalinity appear, as it does in food vacuoles of Parmecium and some other ciliates. Grains of vitellin or yolk stained with congo red become dark brown after one, two or more hours, and continue to remain | dark brown throughout digestion. In some grains no change in color from red to brown takes place, but what differences in conditions account for this has not been determined. ‘Two other indicators were used, Tropaeolin 00 and diethylaminoazo- benzene. Vitellin or fresh yolk stained with Tropaeolin 00 showed little or no change in color. Diethylaminoazobenzene likewise showed no change. This result is due to the fact that these two indicators are not sensitive to very weak acids or strong acids in high dilution. RELATIONS OF BURSARIA TO FOOD 13 The points of origin of acid as well as the rate of acidification of the vacuole contents was determined. Table 2 gives typical records of the total time for completely acidifying grains in fifteen out of fifty individuals which were fed with fresh yolk stained in blue litmus. No noticeable difference existed between the acidification of fresh yolk and that of vitellin. The time in seconds which it took to change completely the blue litmus- stained grain to red, is given by the numbers underneath the number of the individual. The average of the observations in table 2 is 38 = seconds. TABLE 2 Total time in seconds for acidifying fresh yolk grains stained in blue litmus. The table shows typical records of fifteen out of fifty individuals in which the time for acidification of each grain was taken. The grains which are represented by the numbers opposite to the brackets were included in the same vacuole. The other numbers, not opposite brackets, represent grains, each of which were in a separate vacuole, i.e., swallowed separately INDIVIDUAL NUMBER 1 2 | 3 65 6 7| 8 9| 10) 11. 12 13 14 45 | | | | | Ist grain......... 15 20 ‘ 25) 20, 65 45 60 80 4515 ‘ | 45 (35 | 55 Od grain..........| 35 30 | 20 15 40 40 10 30 20 30 {30| )60 }35 | 30 3d grain..........| 50) | 55 | | 15 80 | 50 20, 30 40| \50 |35 | 25 4th grain......... ali’ <40 | 15 | | 25) 60 45| |40| [35 Sth grain......... | | so] | 20 hole Weal | 60} {30 | Gil grain’. ...>.:.: | 2p | 30 | Fy et | 45 | 7th grain......... | | | 20 Wa 75 SED Pram 4.2% 4,54 | | 180, he. 30 It was evident that the time of acidification depended mainly if not solely upon three conditions, as follows: (a) the size of the grain; the larger the grain, other things being equal, the longer it took for the last trace of blue color to disappear. For example; grain number 8 of individual number 5, table 2, was a very large grain; while grain 2 of individual number 8 was very small, hence the difference in time. (b) The physical consistency of the grain must be assumed to determine its permeability and hence its time of acidification. (c) The concentration of the acid secreted into the vacuole. @. 14 E. J. LUND It has so far not been possible to determine the relation between the number of grains eaten and the time of acidification of each, because litmus is slightly toxic to Bursaria and hence stained grains of even fresh yolk are not eaten very readily. The arrow at a in figure 4 shows the point at which the acid first begins to be secreted into the forming vacuole. This is indicated by the sudden appearance of a faint pink color about the edge of the erain which gradually increases. Figure A in plate 1 shows the degree of acidity reached before the grain leaves the gullet. Fig. 4 Outline drawing of Bursaria showing steps in the formation of food vacuoles and resorption of the liquid enclosed; a, place at which acid secretion begins. Grains of relative size shown will not as a rule be rejected after they have passed the point indicated by b. As the grain passes through the mouth surrounded by the mixture of the acid and the external medium, the change toward red progresses rapidly until the grain’s interior has been reached by the acid (plate 1, figs. A, B and C). When the acid had reached the center of the grain the times noted in table 2 were taken. There is no way of telling whether acid is continuously poured into the vacuole after it has left the end of the gullet. No evidence was obtained as to the nature of the acid secreted, nor as to whether it is in combination or in the free condition. * RELATIONS OF BURSARIA TO FOOD 15 The physical and chemical changes taking place in the vacuole containing vitellin or fresh yolk grains, which have been de- scribed above, are briefly summarized in plate 1. Here the series of figures from A to L, inclusive (series 1 and 2), show the usual course of the process of digestion and resorption of fresh yolk when the latter is retained throughout the process. Series 1 and 3 show the usual course of digestion and resorption of vitel- lin, when the rate of resorption is less than or equal to the rate of digestion. Series 1 and 4 show the same when the rate or power of resorption is equal to or greater than the rate of digestion of the vitellin grain. The successive figures do not represent the condition at equal intervals of time, but are typical stages in the process. Frequently there occurs an alternate presence and absence of liquid or smaller variations in the quantity of liquid around the grain during the stages from H to L, inclusive, and from H’ or H” to L’ or L”, inclusive. Thus the figures from H, H’ and H” to L, L’ and L” respectively, represent this process only as it proceeds typically in the majority of cases. 3. Effect of quantity of vitellin eaten upon the average velocity of digestion Since each cell could be fed just the desired number of grains of practically uniform size, the effect of the quantity of protein upon the average rate of digestion, and upon certain other processes could be determined. Experiments arranged to find out what the relation is between rate of digestion and the quantity eaten, were tried with both fresh yolk and vitellin. Fresh yolk was found unsatisfactory for this purpose, since the grains were generally not retained but extruded before digestion was completed. This was especially true when a large quantity of fresh yolk had been eaten (cf. Experiment VIII, p. 34). Under usual conditions vitellin fed to -the animals was not extruded, and this was especially true if care was taken not to let the animals eat too many grains. The average limit in the number of grains that could be eaten and retained -was determined from a large number of observations 16 E.. J. LUND during the work. In these it was found that when normal, active animals were fed vitellin under what seemed to be the best experimental conditions, five to seven or even nine grains were retained, and digestion of the whole mass continued to com- pletion, leaving no residues. In the experiments, to determine the average rate of digestion of vitellin the maximum number of grains fed was six, and it was found that with this number as the maximum extrusion very rarely took place. Rise in temperature accelerates the digestive process, but the variations due to temperature were eliminated, since all the sets of individuals were side by side in an oven kept at 25 to 26°C., and the fluctuations in temperature due to taking the moist chambers from the oven while the records were taken, were the same for each set of individuals. The organisms used in each experiment were from the same healthy culture. They were starved in tap water twenty-four hours previous to the beginning of the experiments, so that a clear cell was obtained. When the animals were to be fed, a large number were removed from the 500 ce. dish of tap water in which they had been starved, to an 8 cc. dish containing tap water. Some of the vitellin suspension prepared as described above (p. 3), was added to the tap water in the dish containing the animals. The quantity of vitellin suspension added varied according to whether it was de- sired to feed a small or large number of grains to each individual. If a large number of individuals were to be fed only one grain, a very weak suspension was added; the result was that the rate of feeding was slow, and hence gave sufficient time to remove the individuals as soon as one grain had been swallowed. As feed- ing went on, the individuals that had eaten the desired number of grains were picked out with a pipette and placed in separate dishes containing tap water. In this way, under favorable con- ditions, it was possible to obtain several sets of individuals - simultaneously, each individual of each set having eaten a defi- nite number of protein grains. After feeding, the animals were RELATIONS OF BURSARIA TO FOOD 17 removed from the dishes with tap water, and two individuals were placed in each watch glass in order to lessen the labor in taking the records. Each watch glass contained 2 ec. of tap water. The watch glasses were placed in moist chambers. In order to avoid the effect of individual variations in the rate of digestion of equal numbers of grains, forty-eight individuals were used in each set. Effects upon the result due to variation in size of the grains were therefore also practically avoided. Digestion was taken to be complete when all the solid contents of the vacuole had become luquefied. The results of two experiments are given. Experiment III, February 12, 1913: Table 3. Each individual of Set A, Set B and Set C was fed 1, 3 and 6 grains respectively. Exami- nation of each individual was made and records taken, 33, 5, 64, 8, 93, 11, 123, 14 and 22 hours from the time of feeding. The rate of digestion and resorption when a large number of grains have been fed is in many cases slow toward the end of the process; so that it is sometimes difficult to tell at just what time (within 1 or 14 hours) the last remains of solid protein disappear. For this reason eighteen hours was taken as the average time of complete digestion in those individuals of Set C which had small protein residues at the end of fourteen hours and none at twenty- two hours. All of Set C showed complete digestion at the end of twenty-two hours, In each of the three sets (table 3) individuals numbered 2 show a longer time for digestion than their partners numbered 1. This has no significance, because it is due to arbitrarily calling the first individual that had completed digestion, number 1, and the last one, number 2; when the record was taken. The same is true for table 4, Experiment IV. It will be seen from table 3 that the average number of hours which it takes to complete digestion in the three sets A, B and C, is not directly proportional to the number of grains eaten, i.e., as 1:3:6. Before further consideration of these results the next experiment will be described. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 17, No.1 LUND J. 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J. LUND Experiment IV: Table 4. In this the procedure was the same as in the previous experiment, but the animals were taken from a different culture, and instead of 3 sets of 48 individuals each, 4 sets of 48 individ- uals each were used. Each individual of Sets A, B, C and D were fed 1, 2, 3 and 6 grains, respectively. Four individuals (marked X in the table) of Set C extruded part of the six grains and are therefore not counted in the results. The temperature was 25 to 26°C. Examinations were made 33, 5, 63, 8, 93, 11, 123, 14, 153, 17, 183, 20 and 223 hours after feeding. In this experiment the possible slight error in the average time for complete digestion in Set C, Experiment III, due to the approximation of the time of complete digestion in some of these individuals, is eliminated, because observations were made regularly throughout the whole 223 hours. And the conditions were on the whole better than in Experiment III. The results of both experiments agree in showing that the aver- age total amount of vitellin digested per unit of time is greater in individuals fed a larger quantity than in those fed a smaller one, i.e., this quantity is greatest in Set D> Set C> Set B> Set A. The average time, found by experiment, for the complete digestion of one grain or its equivalent in volume or mass, in the sets of individuals of the preceding two experiments is given in column 2 of table 5. Now if we establish a limiting case, by supposing that the average quantity of vitellin digested per unit of time, is a func- tion only of the amount of surface of the substrate exposed to the action of the digestive agent, then we should expect to find (provided further that the total surface of each grain in the individuals fed more than one grain was exposed to the action of the digestive agent), that individuals fed 6, 3 and 2 grains each, would complete digestion of all in the same length of time as it takes to digest one grain. Hence, if it takes 8.56 hours (Experi- ment IV) to digest one grain, then in Sets B, C and D we should expect to find that the quantity of vitellin equal to the volume of 1 grain, would undergo digestion in the times shown in table 5, column 3. RELATIONS OF BURSARIA TO FOOD PA The results are uniformly less than those found by experiment (column 2). Now since we have constructed a limiting case for the maxi- mum average rate of digestion on the assumption that the rate is in direct proportion to the surface of substrate exposed, we may on the same assumption establish a limiting case for the mini- mum average rate of digestion, by assuming that the surfaces exposed to digestive action in the four sets, are to each other as the surfaces of spheres of vitellin having volumes 1, 2, 3 and 6. Calculating this on the basis of the rate of digestion found by experiment for one grain (Set A), we obtain the values given in column 4, table 5. It will be noted that these values, based on the above assumption are all greater than the values found by experiment. The results of both experiments agree. However, the intermediate position of the obtained results (column 2, table 5) does not exclude the possibility that the digestive process TABLE 5 Summary of and calculations for the average velocity of digestion of vitellin, based on results of Experiments III and IV, tables 3 and 4 COLUMN 1 2 3 4 5 6 ses [gaze [ggees jeez | 2 miele Sea 8 BEa7s B33 EXPERIMENT Ser so) g.2° ents |phosa | BE a3 233 aUn aS ae gae nae = (= ao 500) Sok ROO QHRZAGD| S Bo | g88 [5o253\sge2 aise. =| 22 es | go | gEGSElgegeeeseset) =! go | gaa. [atest esos ziags, | © e3 BGes 2. O88 BLoORS a Rims 3 33 286s | Goss Sloas | Sore 4 Zi < 6) 0 |< > | hours hours | hours hours Set A...) 1 7.06 | | 7.06 ts ae Set Bu: 3 5:05), |, “2850 4280, || 4506), aes) i pa Set C...| 6 DBD Me Tek |). Sc88 a: | 124846: | RGnee | = == ee | (| Set A... 1 8.56 | 8.56 IV. Febru-}| Set B...| 2 5.62 499) | 6.76 6.04 7.96 ary 18,1913 )| Set C...| 3 4.25 2.85 | 5.93 4.94 | 7.38 Seti D:.2)/ 116 3.04 W742, | 4S70N Wa oe4Ou, W744 pi, E. J. LUND really conforms to the law of a heterogeneous chemical reaction (between a solid and a liquid). The values in column 5, table 5, were obtained by substitution in Arrhenius’ formula t=kV M for the rate of digestion in the dog as found by London.? The values obtained by means of this formula agree well, within the limits of experimental error, with those found by experiment in the case of Bursaria. In column 6, table 5, are given the values of & in the formula t=kV M. The agreement is not so good in Experiment III as in Experiment IV for reasons given above, although the variation in the constant k of both experiments, and especially that of Experiment IV, is well within the limits of experimental error. If it were possible, it would be interesting to determine what the value of k would be for other food substances. The experiments show that in spite of individual variations among the organisms and the variation in the process in different vacuoles in the same individual which were mentioned above, the sum total gives a definite result, and shows that the average rate of digestion of vitellin under rigid experimental conditions follows a definite law. 4. Effect of congo red upon the average velocity of digestion, extrusion, etc. | In order to discover some of the changes which take place in the reactions of the cell when protein (vitellin grains) was changed by letting it adsorb a substance from solution, the following experiment was carried out. Congo red was chosen since its toxicity to Bursaria is low when compared to that of many dyes, 1 The intermediate position of the values found suggests agreement with the results of the experiments of Bayliss (’04), who found that ‘‘with concentrations of casein up to about 4 per cent, the velocity of digestion is proportional to the concentration of the substrate . . . . . whilst with more than 8 per cent, inverse proportionality sets in’’ (cited from Euler: General chemistry of the Enzymes, p. 186). Furthermore the results also suggest the possibility that the quantity of active agent produced in the cell or vacuole is not directly propor- tional to the quantity of vitellin eaten. 2 This is, =kVM; where t is the time for complete digestion, M the amount by weight eaten, and k a constant depending upon the nature of the food. RELATIONS OF BURSARIA TO FOOD 23 and since vitellin grains readily adsorb it in considerable quantity, depending upon the length of time during which the grains are left in the aqueous solution of the dye. _The results of the experiment clearly show: (a) that the ad- sorbed congo red very markedly interferes with or prevents di- gestion of the parts of the vitellin grain toewhich the congo red has been adsorbed, and (b) that it brings about a condition which leads sooner or later to an extrusion of the contents of the vacuole; (ec) that it may exert a greater or less toxic effect from within the vacuole, upon the cell, which may lead to an earlier death than if the unstained vitellin had been eaten. In short: the chem- ical nature of the substance taken into the vacuole of Bursaria may determine in various ways what many of the conditions and reactions of these organisms will be, and especially, in this connection, the action of the digestive agent and the process of extrusion.? The following are the results in brief. Experiment VI. Grains of vitellin of uniform size, prepared as de- scribed above, were stained twenty minutes in a deep red aqueous solu- tion of congo red. They were then washed several times until no more stain could be washed out. An unstained portion of the same vitellin sample was washed the same number of times in tap water; this was fed to the control individuals. Forty normal individuals previously starved in tap water for 24 hours were each fed one grain of the stained vitellin. Similarly a control set of 40 individuals were fed, each one grain of the unstained vitellin. All the conditions and material were the same in the two sets, except that the individuals of one set was fed stained grains, those of the other set unstained ones. Both sets of individuals were kept in watch glasses, each containing 4 cc. of spring water. Two individuals were placed in each watch glass. Records of each individual were taken 2, 4, 6, 7, 9, 11, 13 and 22 hours after feeding. Table 6 gives the results. Table 6 (a) shows that the partially digested or undigested congo red stained grains were to a large extent extruded, while in the control set, all retained the unstained 3 [.have here used the term ‘chemical nature,’ and in my previous paper (Lund 14) such térms as ‘toxicity’ (p. 29), ‘specific chemical properties’ (p. 41), with a general meaning. The finer distinction between the effects due to chemical and physical properties of a substance, as for example its solubility in the plasma membranes, state of colloidal aggregation, chemical reaction with the protoplasm, etc., remains an open question. 24 E. J. LUND protein grains. This, therefore, shows that the chemical nature of the substance in the vacuole may, and does in this case, deter- mine whether it shall be extruded or not. Table 6 (b) shows the effect of the adsorbed dye upon the process of digestion of vitellin. The stain retards and sometimes even prevents digestive action. ‘This was seen from the fact that the corners and edges of the grains where little or no unstained yolk exists, persist more markedly than those of the unstained grains. The stained grains, nevertheless, often became smaller by shrinkage, caused apparently by solution of the protein from the deeper parts of the vitellin grain, which may have been stained less deeply. That the adsorbed dye interferes, to some extent, with the maintenance of the normal form and outline of the animal is shown by the results of table 6 (c). The fact that, for example, the total number of normal individuals of those fed a stained grain has increased rather than decreased at the eleven-hour record, since the nine-hour record was taken, is due to regulation of form, either by closing and opening the oral pouch, or by undergoing TABLE 6 Showing the results of feeding one grain of vitellin stained in congo red, to each of a set of forty individuals. For comparison (control) another set of forty individuals were each fed 1 grain of unstained vitellin TIME IN HOURS AFTER FEEDING 2{ 4] 6| 7| 9/11] 13| 2 (a) Total number of individ- Stained: (8.0) ah gh eol ale). gi aghoetes uals that had extruded } need 000000 0 0 grains before death J ae oar (Dy Bete member ian nas aiceac dae oo 0000000 uals that had completed’ | Ty, stained | 0 1 4! 10 22| 28| 37 digestion of grains DLs aiaaie tment Cc) iota) mune 08 avi cried eee 38) 35) 27 26) 15| 24! 23] 14 uals that were Hormal ini) 77 ieined|. 0. 37| 38| 37, 38 34 331 35! 29 Stamedeuoaeeneeee o.0 0 0 0 3 11 Unstained........ 000 0 0 OF O 2 form J (d) Total number of individ- uals dead (e) Total number of individ-| uals with a perceptible | | Stamed.).-..0\:... 700 0 3 2 0 0 amount of liquid 2 Unstained........| O| 4 vacuole ~J = ies) — Ww — ler) w bo «- RELATIONS OF BURSARIA TO FOOD 25 more fundamental morphological changes. Set B> Set A. This becomes more apparent if we calculate the percentage of the total number of grains fed which was present in the individuals at the times when the records were taken. In order to bring out these relations more clearly the curves in figure 8 were notes from the percentages given in table 7. Now, from what has thus far been said, and from the records, we have no proof that the loss in the number of grains which did RELATIONS OF BURSARIA TO FOOD 30 occur was not, in part at least, due to complete digestion and resorption of the missing grains. But this difficulty ‘is almost wholly avoided in this experiment, because the process of diges- tion and complete resorption of fresh yolk is much slower than that of vitellin alone. This is mainly due to the fact that the fat of the fresh yolk is digested and resorbed at a slower rate than the vitellin of the yolk grain (cf. plate 1, figs. H—L, and figs. H’-L’). This fact is further shown in the records of the total numbers of grains present in Set A, table 7, for twenty individuals out of forty-eight, still contained remains of the one grain fed tharty hours previously, this should be compared with the average time for digestion of one grain of vitellin in Experiments III and IV, table 5, column 2. Now, from these facts regarding the difference in rates of digestion of one grain of vitellin and fresh yolk, it is not to be expected that the individuals of Sets B and C would show as rapid a rate of digestion as if they had been fed an equal number of grains of vitellin; and therefore it becomes clear that if the comparatively small total number of grains pres- TABLE 7 Summary of results of Experiment VIII (January 24, 1913) showing the effect of the quantity of fresh yolk eaten, upon the course of the extrusion reaction in Bursarva. Three sets of forty-eight individuals each were used. Each individual in the Sets A, Band C, was fed 1, 3 and 6 grains, respectively | HOURS AFTER FEEDING 24 30 Ohad lace Zena | te tae tow * < — = Sa $ 7; 5 al | > | {| Total number of grains) | | | ee 2: || present........... .. | 48 | 47| 46 | 34 | a2 | a2 | 32 | 30 | 28 | 20 ss 4 Per cent present of to- | | viduals fed | Palla Penh aaa | | | | 1 grain each | Ce SPAS | | UI isd somite age? « (100 97.9+ 95.8+ 70.8+66.6-+66.6+60.6+62.5+ 56.3 41.6+ | ‘ i ae wad ies == l Set B. Forty-{ | Total number of grains | | | | eight indi-]| present.............. 144 | 137) 97 | 44 | 44 | 43 | 31 | 29 | 27 19 viduals sal Per cent present of total | 3grainseach|| numberof grainsfed. 100 95.1+ 67.34-(80.5-+)30.5-+ 29.8 21.5 10.14 18.7+ Ae | 49 | 30 26 17 10 Set C. Forty- {| Total number of grains | eight indi-|| present.............. | 271/219 | 165 | 98 | viduals fed {| Per cent present of total | | | many grains number present at | | | | i each a three hours...2.5...- 100 81.14 00.8+ 30.1++|18.0 | 11.0} 9.5 6:2=F) (30 36 E. J. LUND HRS. Fig. 8 Curves plotted from percentages in table 7, Experiment VIII, showing the effect of mass or volume of fresh yolk eaten, upon the extrusion reaction. Curves A, B and C are plotted from Sets A, B and C, respectively. The points on the ordinates represent the percentage of total number of grains fed, which still remained in the vacuoles at the time indicated by the same points on the abscissa. RELATIONS OF BURSARIA TO FOOD 30 ent, towards the end of the experiment, in the individuals of Sets B and C, was caused by rapid and complete digestion, then the effect of mass on the rate of digestion for fresh yolk would have to be relatively vastly more pronounced with fresh yolk grains than is true for vitellin; and this is not the case. The first parts of the curves (fig. 8) bring out the pointsclearly, for here the error, that would be caused by the loss of grains due to complete digestion and not to extrusion, is practically avoided, since the rapid fall in the number of grains takes place during the first part of the experiment, before the digestive process could have been effective in causing total disappearance of the yolk grains; especially is this true for Set C, Curve C. On the other hand, Curve A which we should expect to be markedly affected by digestion, does in fact show only a slight fall, compared to that of Curves B and C. It will be noted that the amounts of fall of the curves show the same sequence as the number of grains fed. The, results therefore actually demonstrate beyond question, that the quantity of fresh yolk eaten is a determining factor in the process of extrusion. The greater the mass or volume the more effective is the stimulus from the contents in the vacuole. A quantitative or intensity factor as well as a qualitative factor therefore enters and determines whether or not extrusion of fresh yolk shall take place. Now from the results of Experiment VIII we have as yet no evidence showing in what way the mass or volume affects the process; 1.e., whether it is an intensity effect due to chemical or mechanical stimulus or both. Another important fact in connection with the extrusion re- action is that the stimulus from the contents of the vacuole is most effective in bringing about extrusion during a rather limited period (4 to 6 hours with fresh yolk grains) after the formation of the vacuole. This will be seen from the sudden drop in the curves in figure 8, with subsequent tendency to retention of the remains of the yolk. There is a process of functional adjust- ment (loss of irritability?) with the continued action of the stimu- lus from the contents of the vacuole. Roughly it may be ex- 38 E. J.. LUND pressed by saying that a reaction by digestion is substituted for one by extrusion. “These facts appear in.a clearer way when the course of extrusion is studied in single individuals. A further analysis of these responses must be kept for a future time, since the data from other experiments in which were used other substances are not sufficiently complete. SUMMARY 1. A quantitative method (p. 3) was worked out, by means of which it was possible to study the processes in the food vacuole of Bursaria. 2. Yolk and vitellin may be drawn upon as food for the energy requirements and growth of Bursaria. 3. The liquid of the newly formed food vacuole is partly made up of the external medium, and partly of an acid secreted by the base of the buccal pouch. After a few minutes this liquid is resorbed and the vacuole membrane becomes applied to the yolk grain. The vacuole contents remain acid in reaction throughout the process of digestion of vitellin and yolk grains. 4. Sooner or later after the initial resorption of liquid about the grain, digestion begins. Digestion may or may not result in the second appearance of liquid in the vacuole, according to the principle that whenever the rate of solution—this perhaps in part depending upon the concentration of the cleavage agent— is greater than the rate of resorption, then the liquid products of digestion accumulate more or less about the grain, while if the rate of solution of the grain is slower than the rate of resorp- tion, then the products of digestion are removed as fast as they are formed. Equilibrium between these processes in the vacuole may be established during digestion of vitellin with much, little, or no liquid present in the vacuole. 5. The average time for complete digestion of vitellen in Bur- saria was found to be directly proportional to the square root of the quantity of vitellin eaten, i.e., the relation expressed by Arrhenius’ formula t=kV y was found to hold to within the limits of experimental error. ‘ RELATIONS OF BURSARIA TO FOOD 39 6. Congo red adsorbed by vitellin grains and fed to Bursaria interferes with or prevents digestion of the parts of the vitellin grain to which the dye has been adsorbed and causes an early extrusion. 7. Olein is digested and resorbed by Bursaria while paraffin oil is not affected. Lipoids and fats play an important rdéle in promoting growth in Bursaria. No evidence was obtained for the formation of stainable lipoid from pure vitellin. Starch or amylum grains are not digested. 8. The time of extrusion is determined by the quality (chemical) and the quantity or intensity (chemical, physical or both) of the stimulus from within the vacuole by the substance eaten. 9. The maximum tendency.to respond by extrusion to the stimulus from the vacuole contents, exists within a limited time (4 to 6 hours with fresh yolk) after feeding. LITERATURE CITED ARRHENIUS, 8. 1909 Die Gesetze der Verdauung und Resorption. Zeitschr. f. Physiol. Chem., Bd. 63, pp. 323-377. GREENWOOD, M. 1894 On the constitution and mode of formation of ‘food vacuoles’ in Infusoria as illustrated by the history of the processes of digestion in Carchesium polypinum. Phil. Trans. Roy. Soc., London, vol. 185-B, pp. 355. Lunn, E. J. 1914 The relations of Bursaria to food. I. Selection in feeding and in extrusion. Jour. Exp. Zoél., vol. 16, pp. 1-52. NIRENSTEIN, E 1915 Beitrige zur Ernahrungs-physiologie der Protisten. Zeits. f. allgem. Physiol., Bd. 5, pp. 485-510. 1910 . Uber Fettverdauung und Fettspeicherung bei Infusorien. Zeits. f. allgem. Physiol., Bd. 10, pp. 137-149. Stanrewicz, M. W. 1910 Etudes expérimentales sur la digestion de la graisse dans les Infusoires ciliés. Bull. Intern. Acad. des Sc. de Cracovie, Série B, pp. 199-215. PLATE 1 EXPLANATION OF FIGURES Typical stages in the process of digestion and resorption in a food vacuole of Bursaria containing a single grain of fresh yolk or vitellin. A Degree of acidity attained by the vacuole as shown by litmus at time of separation from the end of the gullet. The pink edges indicate that acid has been secreted from wall of gullet; figures B and C, further stages in same process. A to E, inclusive, show course of resorption of liquid enclosed during formation of vacuole. G to L, inclusive, typical stages of digestion and resorption of fresh yolk (protein and lipoid). G and H’ to L’, inclusive, typical stages in digestion and resorption of vitellin- liquid present in the vacuole. G and H” to L’’, the same as H’ to L’ except that products of digestion are resorbed as soon as they are formed. Change indicated by figures under z lasted on the average 38 seconds; change shown by figures under y about 43 to 6 minutes. 40 RELATIONS OF BURSARIA TO FOOD PLATE 1 BE. J. LUND oO ae) y Y ite ro =) A a “ ES w x ¥ Nae COO SOSEO 4] PLATE 2 EXPLANATION OF FIGURES A and B_ Low power, camera lucida drawings of two sister cells stained to show fat 42 hours after separation. Cell A was not fed; cell B was fed olive oil 18 hours after separation from sister cell. The large drop of oil in B represents that re- maining in the vacuole. a and b, high power, camera lucida drawings in one focal plane of area indicated by squares in A and B, respectively. 42 RELATIONS OF BURSARIA TO FQOD PLATE 2 E. J. LUND 43 4 Loe aaa 9 ve oe a x ms i . i ¥. ‘ . WE, ‘ rr. - . ’ ’ é i* ‘ 8 veiw 5 oe ~ , 7 * . . 3 ne . . i . ? . ‘ . . + ‘ an) Lo a « q bh 8: 2 . a ry "* ¥ y ow bg. CHROMOSOME STUDIES IN THE DIPTERA J. A PRELIMINARY SURVEY OF FIVE DIFFERENT TYPES OF CHROMO- SOME GROUPS IN THE GENUS DROSOPHILA CHARLES W. METZ *+ From the Department of Zoédlogy, Columbia University DIAGRAM AND TWENTY-SIX FIGURES (PLATE) To the student of chromosomes and chromosome behavior a cytological study of the Diptera presents many features of inter- est. Some of these have been described or mentioned by Miss N. M. Stevens in connection with her work on the sex chromo- somes,! but aside from this I know of no serious attempts at such a study. When compared with the detailed and critical works on the chromosomes of the Hemiptera, Orthoptera, and Coleoptera, this lack of knowledge of the Diptera is surprising, and can be explained only by the fact that the latter are in many ways un- satisfactory objects for cytological study. The difficulties in such a study are admittedly numerous, but notwithstanding, much can be accomplished, and the interest of the questions in- volved seems to me to more than justify the extra effort expended in their investigation. For this reason a series of such studies has been undertaken, the extent of which will depend upon the time and facilities available. Some are under way at present and others will be taken up subse- quently. The results I hope to present in a series of papers, to which the present is introductory. 1So far as known to the writer, the papers of Dr. Stevens are the only ones dealing with the chromosomes of the Diptera. They are four in number: The chromosomes of Drosophila ampelophila. 1907. Proc. VII Internat. Zodl. Cong. eee 131 45 34.0 Lethalamimia tures: eee eee 814 323 40.0 White mimiatures.<7 cc eee ene ee 994 397 40.0 SEX-LINKED LETHALS IN DROSOPHILA 97 THE SECOND LETHAL FACTOR In an experiment with certain stock which had been inbred for three years, a pair produced: 73 females and 16 males (= 5:1) These numbers represent the total output of this pair, or at least all the flies that were produced from one bottle. (A) Twenty-two of the seventy-eight females were mated to white miniature (of which two pairs produced nothing). (B) Twenty- nine of the seventy-eight females were mated to eosin vermilion males (of which six pairs produced nothing). (C) Twenty- four females of the seventy-eight were mated to eosin miniature males (of which four pairs produced nothing). The experi- ments with white and with eosin should give similar results, since white (w) and eosin (w®) are allelomorphs. The sixteen males were mated to eosin miniature, white miniature, and eosin vermilion females, but every one of these males proved sterile. The output of the three lots of females mated to the respective males mentioned above is shown in A, B and C of table 10. Under each column, A, B, C, the progeny is provisionally classi- fied, first, into those that give apEren Ea a 1:1 ratio, and then those that gave approximately a 2:1 ratio (or higher). A horizontal line separates these two classes. The classification into these two groups may appear arbitrary, since the ratios in the two classes come very near to each other in some cases. For the present it will suffice to explain that the 2:1 ratio is expected if one lethal is present, the 1:1 ratio if no lethal is present. From table 10 one can get no clue as to the location of the lethal factor that is assumed to be upsetting the normal sex- ratio, but by breeding the females to a male that carries the same sex-linked factors as their fathers, the location of the lethal factor is revealed. 98 T. H. MORGAN Crosses with white miniature males A number of daughters were taken from No. 12 (indicated by a star in table 10) and bred in pairs to white miniature males. Since No. 12 was carried further, I have selected it first for illustration. The data are arranged in table 11, under two headings, viz., those pairs that gave a sex-ratio of 1:1, and those that gave 2:1. The data from pairs with a 1:1 ratio show nothing except the association of white and miniature. The data from the 2:1 ratio, in which the second lethal is present, show no males in the TABLE 10 A B Cc : g ey) ig (ies ¢ 2 | alow Bi heen a ee a 20) Jaa aa uf 11 | 60% 44a a ee One 8 Soy CGnimeot | 1.3 VIL | 53891) 44) 1.25 a Te selec Bilin BOr ql made cl) 128 10a 8 9) ee |) AS 9 aoe ate G64 ab tet eS as ‘San a 54 | 53) | ate Say Vian ee . | ales TAH 63 il, G40 || wal m |. 24 | 20 hates 9 | 69 | 49 | 1.4 TE | 358, 0H) 5G aad Dp | 67. or aie IS 55 Gl Sd. 1) 084i" ey a ey levee ih oe ieee eke | Baert Veda 1G I: gilts sli el 0 tes GV) BAS alee 8, 65 |.-47' | aaa DZ) GA ede | 8 XV Ova aaa ole de \t1 calc 1S 8) 1 068.0|" Oa eV I Ost 83. eT este 5S. ah” 8a" |) seed | DE 670 56) | 12a a e794) Seoimn eae PAGO T i) 48 ES XK | 4d 88 hy eS be®s 6 | 46, Aisa ATMO 35, eae SOD BY | BRIS Aa 2 ky Meo) ea eames Cee AN OA NSS ONERATINT |) ROI | 40h ele ee NGS one an ne TOR S938: ily 155 saaieail| eaenaealll imine 53 ci + 19) hoe 11 Geez, eae 119 T | 79 Ny 8% eral hes ale G2: eso maren ig |p EP Ne SRS} LOOM ee Pee) te Oye 80 | 31 2.8 APG da | 268i aeeeo TV) 64:71 926-9) 204) ni? 58 20 ees NS Sa avec he Mes: a i BA Vil TA ST 288. oo: Mae) | 2Senn amare! 19 Sb asehi eee Vii 16 5S SAE) Gg a | Bly Cie 20) || «BS el WAS GCA VOY Dba GSei eta Sen aaondiely bat 10 3p oe8 RIT |< 531) eee | VAL | YOGI 30a one DERI) (20) A ae SEX-LINKED LETHALS IN DROSOPHILA 99 red-long class, which is the lethal class and, as will appear, also the double cross-over class. One of the single cross-over classes is red miniature; the other, white long. Both are smaller than the non-cross-over class, white miniature. TABLE 11 (From No. 12 of table 10) | ¢ ee cptetres 52 8 ae pare fe a Ro: eee ese eM ag. OL SRS a Ge INO Bte les Gen RENCE nate eee Wes Re Ae eae 1 i aay oe NP SSS aha 28 © ead 87 he 20 led 3 eeeO itl 12 chew 5 Sees Ally 10y [wk cleats 7 7 DCE PO Tig i Gagne 0h) ay i oS ee: 8 osmogl se Ne TS an) ied 28. The30". | Or Gees 9 Soll | 2Gua Id) ie Se ie 22n lee27>!, | ISNA) 2S oie 13 Set See elt Nt ale Ae OO lk 2 > Ate ME TeRi a I) wal A 15 25 Paget sient) UontGalk 1S) 0 280! Tse aG a ieee 18 27 | 26 | 14 | 16 | 38 | 33 |, 13 | 20 8 19 DA S260 OI, 10 sh BF) Ae 21 SO i G2ep i oelh Se FID. eebrohy 10...) 6: ales 22 aa 251 PSP Ge 2a 08 | 9. | 13 i 26 | 26.92 250 ota 22 1 O20) 22 S| IES 29 ee ea ey ee er ae | Eee Total | 392 | 344 | 162 | 163 | 245 | 332 | 159 | 132 2 BO lags 155 tO! SOKO ee 3, ) 14 Wieoen 4* OS ae” 16 late 0 | 19 3 Til 239 5* AG 5 tly AA N20 lo? AO) Vek 6 | 25 6 Dita FO 12 Oo 9 ase aS aee 11 kee 16s 2 100.) 1a (Pa ee oa mee 2) Fase Ps 12 ier He2e en alse | a2) Or pat 5 8.0 (2M 14 Pe NOs bee. OC AO OG" ae aerye O°.) alte 16* ae te 8. | Ole On det. a.) CO -es 17 ae lag). 98 | ia | 0 | or 3 1) 61). ees 20 Depa ade i leh sO) St 2 | -0:, | aaa 23 38 | 34 | 18 | 19 0/535 3 lent: || 24 Bf) 24 Te io 0) |, 3h 5 7. ete 25 2th | 31, oh ES tO 0", 19 2) |. - 2) lees 27 De 27 cea UW eel Os STZ ae Sy A eed 28 I es a Va 2 i= Us ee | 0 30 Bole, |e 20) 7 0 | 33 5) 6 eee 31 Si eon re tay | ade Ot aer k aade eG) a / aes Se Btn = = | | | 0 | 436 | 54 | 104 | . 100 T. H. MORGAN : In each generation the females that gave the highest sex- ratio were selected to breed from. Whatever disadvantage this method of selection may have, it at least makes more probable the retention of a 2:1 ratio. TABLE 12 (From No. 16 of table 11) Pieanomteoeslok. . No. ae |} Be | ce) BE) 08 | Bede cee ee ) 2° EF a ES ge BA a HS ae a 146 495 | to 494 | 0 i 30) 7 1) aoc) iaee b* 532-32 1). Ber 19 0 | 36 9 1 |) oe c* 17 a7 "49, 908) ag 0 | 26 6 4 |e d a7 Say a ety ee ati @ i. 29 3 5) eg e Ban ee 9 ee Seay a) 0 | 29 2 4 | S38 f Loew! 3004 20 Ge 15 0 | 20 2 9. |. asae g 55-| 49 | 20 | 28 0 | 66 9 |) 14) 9) ae h 7 ae; 3 0 | 40 6 1 || Some i 20 | 20 Geile ao Onn a 4 A 117586 Total 367 | 327 | 164 | 159 0 |290 | 48 | 45 | | | TABLE 13 (From No. b of table 12) Ges fe | | % a) (ee ear eee en es No. Wee Cod BEE PY x Bas treeilN) 4.06 a gene eam nets < Cleese ee lc se | 1 Be 98 eG. eats 0 | 19 6 | 3 ee 2 35 Wh 530,. 4 alo ets 0 | 36 9 | at) joe 3 39) |. 30 sod RIG al) LOro aon AM 2 aa 4 Wetec es Pass ol eee a ee | 6. |. 2 ae 5 |} 36 | 44 | 21 Gy 1, 20" | 228 4 Oo | ag 6 be36 nse | c16 Sl, Oe 1 988 5 1 igo 7 AA 9G «| Sener a. O71. 35 3 | Sores 8 46°) 49 | 25°] 25 NO) 44 Ce peo 9 | 43 | 34 9 6 0 | $35 eet 1 es: 10 Bigot a5. 41" Sa 6 Oi. || Kok) Hee ol) 3a 11 35h. 3801) 15 laws 0 |) 45 | 4 1 {oan 12 908 1 87 | 27 G90 0 | 2 | 10 | 10 | 25 Total | 467 | 410 | 230 | 162 | 0 | 304 | 51 | 47 | 2.6 | | | SEX-LINKED LETHALS IN DROSOPHILA 101 From lot 16 of table 11 several virgin long-red females were mated in pairs to white miniature males. Their offspring are shown in table 12. Each pair shows in its offspring that the second lethal was present in the long winged red mother. A further generation was reared in pairs from red females of lots b and ¢ of table 12. The offspring recorded in tables 13 and 14 show that the 2:1 sex-ratio continues. Returning to table 11 there are records of two lots taken respectively from No. 4 and No. 5. The offspring from these pairs are here shown in tables 15a and 15b. Both show that lethal II is present as indicated by the absence (or rarity) of the red long males. Before examining the preceding tables 10 to 15 in detail it may be helpful to have in mind certain theoretical relations. The location of factors for white (w) and for miniature (m) have been already determined. If we are dealing here with a lethal factor, it is essential to determine its position in relation to white and to miniature. For reasons that will appear later it is here assumed that this lethal lies between white and minia- ture (fig. C). TABLE 14 (From No. c of table 12) o Oo | Bl ee D D % | D Oi sean é ae Srl eee | eee g a Sr eGo AiGeli mete: [al6. wit cet enely ee Ge llaae el PI SA bee lee hes REO ee me ee 1 27 uy 27 \a A | 26 0 | 39 Bean was | «28 2 26 | 35 6 |215 Oh e238 Wo Sb. |. 2a 3 bao ( -59-" | 730, (7 932 0 | 56 7 2 | oe 4 61 | 49 | 39 | 13 0 | 44 7 31 toe 5 40-9981 16> | V16\='|.~ 0) 1-30 D 5 || aaa 6 29 | 35 7 eats 0 | 26 3 7. |e 7 praG, 4 le 20) «|g 0°| 50 6 9. |Wg3 8 SA LU EM ls O2 |F 32 1) £0 7 | ears 9 BAG Sie ec ORC net 0 | 34 6 4 oli aae 11 | 59 | 55 | 27 | 25 On) 67 8 7) Wee 12 Pete 10s), anette <0.) 28 3 1° ieies 13 Peale hots en | te oO, | Se 5 3/725 Total 479 | 450 | 227 | 229 O,>|.455° | 69. | 56. |e oe THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 17, NO. 1 102 T. H. MORGAN : We are concerned with the heterozygous females only. Of the two sex chromosomes of these females, the one of maternal origin is indicated by the heavier line in the diagram (fig. C), the other, of paternal origin, is indicated by the lighter line in the figure. In the present case there entered from the maternal side the three factors: normal (red) eye (W), the second lethal (1,.), and normal wings (M); there entered from the paternal side the three factors: white eye (w), the normal allelomorph of lethal (L.), and miniature wing (m). In the case of this lethal the possibilities of interchange of factors between these chromosomes are shown in figure C, at b, c, d. If the crossing-over takes place between white and lethal, the case is indicated in (b). The two gametes that re- sult from this single cross-over are WL.m and wl,M; the latter never becomes realized in the male because he carries the lethal (1,). Similarly, if the crossing over takes place between lethal and miniature the two gametes that are produced are W |. m and w L: M, of which the former carries the lethal factor. There is another possibility, viz., the double crossing-over (d). TABLE lia (From No. 4 of table 2) oa RED WHITE | RED | WHITE RED WHITE | RED | WHITE | LONG 9 MIN. 9* | MIN. 2 | LONG 2 LONG MIN. o& | MIN. Go’ LONG o ms ae ry: 4 5 é a 26 22 14 | 12 0 23 3 | 6 b 23 19>) tt] 13 1 12 3 8 c 34 39 21 18 0 28 10 8 d 30 24 12 16 2 15 5 9 Total 113 104 | 58 59 3 78 | 21 3l TABLE 15b (From No. 5 of table 2) RED WHITE | rep | ware | rep «| ware | Rep | ware NO. | LONG °) MIN. 9 | MIN. 9 LONG Q | LONG MIN. | MIN. o LONG b | Se | ila | Ga Sy ile 0 te JY oe if CAPA 34 307+} 8 So Ol ae els 6 Total | 50 AGE ae id 1° tok Og ae 6 wie SEX-LINKED LETHALS IN DROSOPHILA 103 w 1, se W1.M \s WLiem w Ee m Wi I, M wl.M 7 Ti aa W Ls m Ww lL, M WE TS a