~ Lee Soh Sorestie asics x Inara WUAIe he a i} * a \ iis! 4 ‘ mi “ Tea! ih rau a He i Whts MW ne Fas oo, re ety = as Seeteesnas = Son Se = ain i nine! a ah ae Bt ie ve ation tts rite peek ia ity Moe ses AN os Bt ae os Be TATE a Ieee biG in Hic, th me H RR pil par ; af eet? ae \ ae a Hi 5 eager ae iH iP pat Aaa i Sao Se oe Scenes P< <4 eS = ~ = eo er Fasess See FNS earn ae ee SOS = = tae ote. << Ace Sroaseees Septal Sass Bats Digitized by the Internet Archive in 2009 with funding from University of Toronto http://www.archive.org/details/journalofexperim06broo i b ae A + Ph el ae | — = we. tis Valine ta THE JOURNAL OF EXPERIMENTAL 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 GEORGE 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 3 Managing Editor g e) é [° | aoe VOLUME: VI } PUBLISHED EIGHT TIMES A YEAR BY THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY 36th STREET AND WOODLAND AVENUE PHILADELPHIA, PA, CONTENTS No. 1—January, 1909 ADA SPRINGER A Study of Growth in the Salamander, Diemyctyles viridescens ....... EpmuND B. WILsoNn Studies on Chromosomes. IV. The “Accessory”? Chromosome in Syro- mastes and Pyrrochoris with a Comparative Review of the Types of Sexual Differences of the Chromosome Groups. With Two Plates aud Iwo Figures in the Text..........-.. ea Ay Re ae RPE ie A oc N. M. STEvENs Further Studies on the Chromosomes of the Coleoptera. With Four IPS Gres oe see, ae a em OD oo gS Val NU a A tne Be ere WA An Unpaired Heterochromosome in the Aphids. With Two Plates..... Davip Day WHITNEY The Effect of a Centrifugal Force upon the Development and Sex of Parthenogenetic Eggs of Hydatina Senta. With One Plate.......... Observations on the Maturation Stages of the Parthenogenetic and Sexual Eggs of Hydatina Senta. With Five Figures in the Text.......... No. 2—February, 1909 Epmunp B. WILSON Studies on Chromosomes. V. The Chromosomes of Metapodius. A Contribution to the Hypothesis of the Genetic Continuity of Chromo- somes. With One Plate and Thirteen Figures in the Text.......... J. F. McCienpon EILGZOATE CHOICES AVWiEh diwOrblates serie aae ce ae orcs sila ot fe sc 6 os Cuares R. StockarD The Development of Artificially Produced Cyclopean Fish—‘‘ The Mag- 69 IOI 125 139 147 207 265 nesium Embryo.” With One Plate and Sixty-three Text Figures.... 285 No. 3—May, 1909 RAYMOND PEARL Studies on the Physiology of Reproduction in the Domestic Fowl. I. Regulations in the Morphogenetic Activity of the Oviduct.......... 341 O. C. Giaser anp C. M. Sparrow Me: Vhysiolory of Neumiatorysts. 2.0. 5.2... 52u6.8- ee 361 LoutsE Hoyt Grecory Observations on the Life History of Tillina Magna. With Two Plates, Three Figures and Six Diagrams in the Text... 22.2%. 22-2. poe 383 Cuar_Les R. STOCKARD Studies of Tissue Growth. II. Functional Activity, Form of Regula- tion, Level of the Cut, and Degree of Injury as Factors in Determin- ing the Rate of Regeneration. The Reaction of Regenerating Tissue in the Old Body. With One Plate and Eight Figures in Text....... 433 No. 4—July, 1909 C. M. Cup Factors of Form Regulation in Harenactis Attenuata. I. Wound Reac- tion and Restitution in General and the Regional Factors in Oral Restitution. With Twenty-Four Figures..................++-+-+: iyi R. W. HEGNER The Effects of Centrifugal Force upon the Eggs of Some Chrysomelid Beetles.. With Twenty-Four Figures.: .. 0.2.5.2 2. ee eee 507 Witiram REIFF Contributions to Experimental Entomology. I. Junonia Coenia Hiibner 553 II. Two Cases of Anabiosis in Actias Selene Hiibner.............. 565 J. Frank Dantev Adaptation and Immunity of the Lower Organisms to Ethyl Alcohol.... 57° A STUDY OF GROWTH IN THE SALAMANDER, DIEMYCTYLUS VIRIDESCENS BY ADA SPRINGER Part I [iatide@lureulotne .ocdondoucls bodes GUaOa be TOSo abt E Scid oO Unc OO ORC OcemeT OD COO Ob OnIEToO [Noxnrallinatelof opowtlind-lctsinls\ai-)- «(leis «| olerarcysiere olalttorsielslete efela by ai8 = inieie in) eleidicls = olefale cielsie « Ba esratelormstanydtlomaerierm ters cio cieereice ates ere ernicin seek teienie: (ose e'e (7) , with Re ere tlie pySEN ES TIbd Chet retctere tei erel\otete Peed roe eae erie ional ter 27.8 Bermlicaiorrmaalinitach animal sees c/eac.cc sists.cieie aisie s/ossis etcYayole Sree hea, soee eathe aes ans Sere 20.3 After five weeks’ starvation and subsequent feeding 153 mg. of beef a week for four weeks, the percentage increments in Sets F? and H? (Table XV) were as follows: Increment per cent Bis WAL Se PETIT a lit] eS PSs al ofe= steteeietestal the tails were cut six successive times; four weeks after the last cut the percentage increment was 52.3. In Set G* the weight was so far below the average that the results are of no value, and G* became infected. For Set G* there was no set which could be taken as a comparative control; but comparing the thirteen weeks with a corresponding period of any normal intact set the percentage increment was considerably higher. In fact, the percentage in these two cases, except those obtained after a period of starvation, were the highest percentages obtained in any of the experiments. 12 Ada Springer There are, perhaps. not sufficient data upon which to determine definitely whether or not the percentage increment is greater when small pieces of the tail are cut off successively and when it is cut only once at the base; but there are indications from the weekly records and from the end results showing that the successive cuts produce a greater increment than does the single cut at the base. This would seem to indicate that the increased rate of growth is the direct response to the cut without regard to the regenerating mass; but there are other factors yet to be considered that show the results can not be so simply interpreted. vie RATE OF DECREASE DURING STARVATION IN NORMAL ANIMALS AND IN THOSE WITH THE TAIL REMOVED Six sets, consisting of ten individuals each, were taken November 13. In three sets the tails of the animals were cut off at the base, in the other three sets they were left intact. After four weeks’ starvation a comparison shows the following percentage incre- ments: 1 The injured animals 2 The normal controls Sete (MableXdgv)) ira nce «ys eters eaters 6 Set H! (Table XIV): .-0-...)5s0/1 eee eee 6.5 See ba(Table Xia)! o. oar ee 4.6 Set H2 (Lable XIV): ......-. 1s cccse eee 0.5 Sepls ((PableXaiVy) sace fan6 see eee 8.4 Set HB? (Table XIV): ..-.. «- »:-' MSCENGS ce sae er cate ee 1.965 (lors) 6.5 In the injured Sets E' and E®, the initial weight in one case was greater, while in the other it was less than the initial weight of the normal control sets, G! and G*; yet the percenatge increment in both cases was greater in the injured sets. In Sets E* (Table VII), in which the animals were injured by cutting the regenerating stumps, in E'? (Table VII) in which the regenerating stumps were left intact, and in G' (Table VI), the normal control, the average initial weights and percentage incre- ments were as follows: After In.wt. operation Increment In. ut. Increment grams grams per cent grams per cent Sa Sane 1.904 1.835 21 Foal ite Arata aoe te 2.543 5-4 Taking into consideration the difference in initial weight, the large percentage of the injured set does not appear to be so anoma- lous. This was also the case in the following sets (Table VIII) where the experiment was the same. After In. wt. operation Increment In. wt. Increment grams grams per cent grams per cent See DA gaé sass 1.954 1.878 21-3 Seige ce meee oe 2.089 18.2 SHES" sop gctoomesnencs 2.18 11.4 In Sets H? (Table IX), where the animals were injured by cutting off the tails at the base, and H? (Table XI), where the tails were left intact, the average initial weight, and percentage increments were as follows: A Study of Growth 27 After In. wt. operation Increment In. ut. Increment grams grams per cent grams per cent Siete 18 bcs Sees 1.446 1.27 31.6 SGins bis s505 Soe doopoor 1.65 20.3 The difference in the initial weights was not great, therefore the percentage may be taken as showing more nearly a correct relation between the injured and the normal sets. In Sets F* (Table X) in which animals were injured by cutting regenerating stumps, in F!? (Table X) in which the regenerating stumps were left intact, and in H? (Table IX), the normal control set, the initial weights and the percentage increments were as follows: After In. wt. operation Increment In. wt. Increment grams grams per cent grams per cent Sale dee 1.345 1.268 21.8 I cLGAT oe 1.359 Pols This is the only case throughout the experiments, however, where the percentage increment was not greater in the injured set. In Sets I', I?, I? (Table XII) the tails were cut six successive times, and the conditions of food and temperature were identical. The initial weights and the percentage increments of the injured sets together with those of the normal controls, At and A?, were as follows: After In.wt. operation Increment In. wt. Increment grams grams per cent grams per cent Se IES seanmoe WT 1.425 42. Seba a Gach dsone neconc 2.066 221.3 Sa: IE So poooms 2.118 1783) DiLlals SeteAr a iis cischestcrerierrae 1.661 29.8 28? 1S casgaccee 2.489 1.977 23-5 Initial weights here also seem to account for the irregularities. That the percentage in Set [* is greater than either I? and I}, is probably due to the fact that the average initial weight is con- siderably less. This is true also in the control Sets A! and A’. From the comparisons cited above it is found that in some of the cases the initial weight of the injured sets (before the tails Were cut) was greater than that of the intact control sets, while 28 Ada Springer in an equal number of cases the initial weight before the injury was less than in the intact sets. The percentage increment in the majority of cases, however, was greater in the injured sets. This would seem to indicate that the greater percentage increment in the injured sets was not due alone to a smaller initial weight before the tails were cut. It must be remembered, however, that after the tails had been cut off the weight in the injured sets in every case was less than that of the intact sets. It would seem to follow that the greater rate of growth in the injured sets was connected with the decrease in the volume, by removal of the tails. There is another relation to be considered, viz: whether the condition of the tissues themselves play a role or whether the result depends simply on relative volume, that is, whether there is any difference between two animals of the same weight, one having been a large animal when its weight was reduced by cutting off the tail, the other an intact smaller animal. ‘The data are nsufficient upon which to base a conclusion. The following table shows four sets that were starved for a period of five weeks, after which time they were fed 153 mg. of beef per week; also four sets as controls which had been fed for five weeks, after which they were also fed 153 mg. of beef per week. The initial weights and the percentage increments were as follows: After five weeks’ starving After five weeks of feeding In. wt. Increment In. wt. Incremen grams per cent grams per cent Seta (Mabledix) ie oe a 1.65 20.3 | ‘Set Gt (hable\VE )\- es 11.4 Sete tiai (able X0)po aa 1.345 21.8 Set E™ (Table VIL )...: 12904 2d Set FD (Table X)......- 1.359 23.4 Set E!> (Table VII ).... 2.543 5-3 Set Fs (Table XI)....... 1.056 27.8 Set E2> (Table VIII).... 2.089 18.2 The four sets after starvation gained faster than did the sets after a period of feeding. Set B' (Table II) was starved for seventeen weeks, after which it was fed 153 mg. of beef a week for a period of nine weeks. When the percentage or rate of growth is compared with that of normal sets, At and A? (Table I), for an equivalent period, the result is as follows: A Study of Growth 29 In.wt. Increment In. wt. Increment grams per cent grams per cent a 1b OSS RB e a aC Om On aEEe 0.89 ° 54.2 SetpAde cats Nae shoe crertins 2.066 ZFeleG SECEAS ene tel ayes tetarstare ory 1.661 29.8 After starvation the rate of growth was faster than in the nor- mal animals. When Set B? (Table II) is compared with Sets A‘ and A? the result is the same. This was probably due to the fact that starvation had reduced the initial weight. IV. THEORETICAL DISCUSSION It has been shown that when two animals, one larger than the other, are given just enough food to preserve equilibrium in the large one, the smaller animal gains weight. Assuming that digestion and assimilation in both cases are the same, how may the facts be interpreted? It may be safely assumed that it takes a smaller amount of food to preserve equilibrium in case of the small animal, than in the large one, so that the material over and above that used in actual repair of the body waste goes to form new tissue, to increase the size. According to this view, as the small animal approaches the large one in size, which is the maxi- mum weight for the amount of food taken as a basis, the rate of growth should become less. It takes gradually more and more material to replace waste, because of the new tissue that has been continually added; hence there is less to be used in the formation of new tissue. Even if more food were digested by the larger individual the result would be the same. By feeding the large one more than enough to preserve equi- librium, and the small one the same amount, the large one will gain; but the rate will be slower than that of the small animal, for the same reason as given above; the small one uses less for repair and more goes to form new tissue. When animals are injured by cutting off the tails, the increase in rate of growth that follows may be due either to a stimulus produced by the cut, or to an increase owing to the reduction in weight; that is, to a change in the relation between the body material and the amount of food taken. By cutting off the tails 30 Ada Springer at the base about 15 per cent (average) of the body weight is removed, so that it may be supposed the food material that other- wise would have been used to repair the waste in the tail goes to increase the body weight. It has been shown that the smaller the animal the greater its rate of growth; it has also been found that after a period of starvation and consequent reduction in size, the rate of growth is faster than in the animals in a well-fed condition. It is prob- able that when the body material is reduced in animals by cutting off the tails, the increased rate of growth observed may be due to the reduction in size, that is, in proportion to the amount of food taken, rather than to the stimulus of the cut. Whether in addition to this the cut may also act as a stimulus, cannot be affirmed or denied from the experiments so far carried out. ‘The reduction of the body weight by starvation and by cutting off the tails cannot, however, be considered equivalent factors. In the reduction of the initial weight by starvation the condition of the tissue is changed, while when the tails are cut off the remaining tissue still remains in the same condition as before the injury. SUMMARY 1 Increase in weight in adult Diemyctylus is due to an increase in the size of many of the organs of the body, and not to a stor- age of fat. The converse is also true, that decrease in weight is due to a decrease in the size of the organs. 2 Percentage increment is directly proportional to the amount of food consumed by the individual; the more food consumed the faster is the rate. Rate of growth decreases as the maximum weight is approached. This maximum is determined within limits by the amount of food taken; for a certain amount there 1s a definite maximum or point where there is established a state of equilibrium between waste and repair. By increasing the amount of food the weight may be increased and a newcondition of equilibrium be reached. 3 By cutting off the tails the rate of growth is increased. This increase in rate is probably due to a reduction in the size es yi; | Study of Growth 31 of the animal, although from the data it is impossible to determine whether it might not be due to some extent also to the stimulus produced by the cut. Injury in this case reduces the weight of the body without affecting the amount of food digested, therefore it seems reasonable to suppose that less material is needed for repair (there being less material to be repaired), and more goes to increase the weight than is the case in normal control animals. 4. The initial weight is the main factor in determining the percentage increment or rate of growth; the greater the initial weight the less the percentage increment; that is, the larger the animal the more food is used for the actual maintenance of the body material, and the less goes to an increase in weight. 5 Sex may influence the rate of growth, but so far as the data ‘go only indirectly through the initial weight. The average initial weights of the females were less than those of the males. 6 After starvation and subsequent reduction in the initial weight, the rate of growth is higher than in the normal animals. This increase in rate is probably due to the reduction of the initial weight. 7 During starvation the rate of decrease in weight diminishes as the temperature is lowered. 8 If the maximum quantity of food the animals will receive at the lower temperature be takén as a feeding basis, the rate of growth diminishes with the increase of temperature; that is, the rate is highest at the lower temperature and becomes lower as the temperature increases. The quantity of food the animal will take, however, increases with the temperature. It was found that the quantity of food taken by the animals at the intermediate and higher temperatures was three times that taken by those at the lower. ‘Taking as a feeding basis the maximum quantity of food the animals will take at the intermediate temperature (which was also that for those at the higher) the rate of growth diminishes with the increase of temperature. The rates of growth at the intermediate and at the higher temperatures on their own feeding bases were both higher than that at the low temperature on its feeding basis, but the difference in the rates was not in proportion to the difference in quantity of food taken which was three to one. 32 Ada Springer Temperature, therefore, while influencing the amount of food the animals will take, also influences directly the rate of growth. I wish to acknowledge my indebtedness and to express my thanks to Professor Morgan, under whose direction the work was done. Zodlogical Laboratory Columbia University A Study of Growth 33 TABLE I Ser Al Srv A? Showing normal rate of growth. Fed 153 mg. Showing normal growth as in A}, also the (average) of beef a week rate of starvation epee ie a ee |S | moe ran a0 % © oe e 0 a a S | S ® 2 2 = s 2 . Beeal sola *| 2 ee ae \ae Oct. 16; 6 12.396] 2.066 6 9.968 1.661) 23| 6 11.876] 1.979] —0.087| —4.3 ean 9.856 1.643| —o.018] —r. Za) (6 12.54 | 2.09 0.111] 5-4 6 10.326} 1.721) 0.078 4.6 Nov. 6) 6 14.208] 2.368] 0.278 12.4 6 11.967, 1.994] 0.273 14.7 13} 6 14. 2eQaale— O08 5) a Td! 6 11.995, 1.999} 0.005 0.2 20/ 6 14.22) 2.386|| 0.053] 927.2 6 12.222| 2.037; 0.038 1.8 27 \ 926 14.298] 2.383] —0.003 o.1 6 11.917, 1: 980) —O-O5T| 25 ; Dec. 4) 6 14.735| 2.456] 0.073 ais 6 12.775) 2.129} 0.143 6.9 11| 6 15.167] 2.528} 0.072 Pic 6 13.21 | 2.202| 0.073 Be8 18| 6 15.667) 2.611) 0.083 3-2 6 13.46 | 2.243) 0.041 1.8 24| 6 16.655| 2.776] 0.165 Gar 6 13-572) 2).262|, “O-019 0.8 Began to starve Jans -2|) 6 16.275] 2.712] —o0.064| —2.3 6 12.625] 2.104 52-153 Fad SiG 16.347] 2.724| 0.012 0.4 6 11.719} 1.953) —O.151 TA: Tesi. 16 16.86 | 2.81 0.086 Be 6 11.165| 1.861 cnc 4.8 27\ 16 17.492| 2.91 0.100 ate 6 II.135| 1.856) —0.005) 0.2 29] 6 23 6 tle ege8 Reb: si) - 6 18.294] 3.049] 0.139 \ Dee 6 TO.322|| 0.72) || —OnIgb f 3.8 12" 6 19.087) 3.181 sig) 4-2 6 10 1.667} —0.053) ea 19} 6 19.43 | 3.24 0.059 1.8 6 9.705] 1.618} —o.049 2.9 26| 6 19.135] 3-189] —o.061 Set 6 9.425] 1.571] —0.047 2.9 Mar. 5}. 6 TOh2H | 320ml) | 1o.012 0.3 6 9-275] 1.546| —0.025 1.6 12) (6 TS O45 elso|u—O-049) 1-3 6 8.575] 1.429] —O.117 7.8 I9| 6 18.595] 3-099] —o.059} —1.8 6 32237] =a78| O50 3-9 26| 6 18.065] 3.011] —0.088| —2.8 6 Weg, || 2243) || Osea 6.2 Began to feed 459 mg. (average) of beef a week Apr. 2] 6 19.065] 3.177] 0.166) Gee) 6 7.289] 1.215] —0.075 5-9 9) 6 20.142] 3.357| 0.18 ers 6 7-235] 1.206] —0.009 0.7 16| 6 20.685! 3.447| 0.09 2.6 6 6.955| 1.159] —C.047 3.6 Gee Weave ei ae é From From 4 Oct. 16 to Dec. 18 ....] 2.338} 0.545] 23.3 Oct 16 to Dec. 18] 1.952] 0.582! 29.8 . Ortmoto Feb. 19) ...-|62.053) L.174|| 44.2 Oct. 16 to Nov. 20] 1.849] 0.376) 20.3 ; Weears torNeb: 19) -<.-|/ 2.925] 0-629) 271.5 Dec.24 to Apr. 16] 1.710] 1.103) 64.5 iH Mcte16itolNov, 20)... 2.226|+ | 0.320) 141.3 ;. H 34 Ada Springer TABLE II Ser Bi Set B? Showing the result of starvation and of subsequent Showing the result of starvation and of feeding subsequent feeding | E or | = | a a a} = z (ee eee = a) 2 /¢ é Dapecties Basa ce | ¢ 2 | € | g 2 | = ee | 3 ~? = = a e : ey at 2 | % 2 3 Bal | oe "3 }2)/e}/4/ a] 4 2 8 | 2) eames Oct. 16] 6 | 11.561) 1.927 6 11.756) 1.959 23 6 10.761 1.794) —0.133} Ff 6 11.161, 1.86 ©.099 (ost 30) 6 10.278] 1.713 —o.081) 4.6 6 10.376] 1.729 0.131 Ten Nov. 6| 6 | 9.76 | 1.627) —0.086| Gel 6 9.983) 1.664 0.065) 3.8 13) 6 g.292) 1.548) —o °79 4-9 6. | 9.91 : 1.585 0.079 4.8 20} 6 8.784) .1.464) —0.084 5-5 6 9.068) 1.511, 0.074) 4-7 an GG (| 8.2 | 2966 —0.098, 6.9 6 8.787| 1.465| 0.046) alc Dec. 4) 6 8.059, 1-343} —0.023] 1.6 6 8.31 | 1-385) o:c8)] au 11 6 7-629] 1-271] —0.072| Ga 6 7.722) 1.287 0.098 7.3 18} 6 | 7.12 | 1.187 — 0.084 6.8 6 7-465, 1.244] 0.043 ee) | | Began to feed 153 mg. of beef a week | | | increase | % inc . 24) 6 6.842 1.14 —0.047 4- 6 8.926 1.488) 0.244 iy fel Jan. 2) 6 6.597. 1-1 —o.04 | BES 6 8.435, 1.406, —0.082| —5.6 te eam teh ers ee soy —0.084 7.9 6 9.002) 1.5 0.094 6.4 is 6 5-925, 0.988! —o0.028 27) 6 9.54 | 1.59 0.09 Re 22} 6 5-905 0.984) —0.004, 0.4 6 10.26 | 1-71) ) (Onna 7.2 29 | | \ 3-45 6 | \ Bebe) iG|) 6 | 5-505, 0.918} —0.066 3-45 6 | 12-13 | 1.855, es 2) 4: 2} 5 | 4-45 | 0.89 | —0.028 3 6 | nt 1.89 0.035 1.8 Began to feed 153 mg. (average) of beef a week | | increase| % inc. | | 19 4 5.007] 1.251 0.361| 33-7 6 11.982) 1.997, 9.107 Ga5 26) 3 3-94 | 1-313 0.062 4.8 6 11.95 | 1.991, —0.006| —0.3 Mar. 5} 3 4.085) 1.362 0.049 3-5 6 12.125] 2.021] 0.030 1.4 12) 3 4-005} 1-335) 0-027, —2- 6 11.785] 1.964) —0.057, —2.8 19} 3 4.103| 1.368 0.033 2.4 5 9.685] 1.937) —0.027| —1.4 26} 3 4.105} 1.368 0.00) 0.0 4 8.322] 2.08 0.143] [eyed Apr.) 2 3 4.085] 1.362 —0.006| =O) 3 6.497| 2.166, 0.086 4- 9) 3 4.285] 1.428 0.066) AT. 3 6.49 | 2.163] —0.003| —o.1 16] 3 4-655] 1.552) 0.124| 8.3 3 6.4351 2.145! —o.018] —o.8 eel =| = oa Sle] oe FE a2] @ From | From Oct. 16 to Feb. 12..... 1.408] —1.037) —73.6 Oct. 16 toDec. 18 1.601, —0.715, —44.6 Oct. 16to Dec. 18 ....| 1.557| —0.740| —47.5 Dec.18 toApr.16 1.694, 0.901 53-1 Feb. 1zto Apr. 16....] 1.221] 0.662) 54.2 Dec. 18 to Feb.19, 1.620) 0.753) 46.4 Oct. 16 to. Decirr. .- =. 1.599 0.656 = 41. | DecorntoPebst2—..- 1.08 0.381 35-2 oe cere <<, ai es le a SS ae a —— a Pee A Study of Growth 35 TABLE III Ser C} Ser C? Showing normal rate of growth. Fed 102 mg. of beef Showing normal growth asin C1, Fed 102 (average) a week mg. of beef a week “ t Re ey 2 z | Fy "Ee i 2 5 | So ah | g = 7 z ets a | ‘3 z = 3 2 a g = ety wee g, 2 r= 2 (Se ae ales al Ble. be ee aoe Octs16)5 6 11.118) 1.853 | 6 | 12.661) 2.11 23, 6 11.131] 1.855 0.002) o.1 6 | 12.805) 2.134] 0.024 Tek 30| 6 11.277| 1.879 0.024, 1.2 6 | 12.991| 2.165] 0.031 1.4 Nov. 6) 6 11.966) 1.994 0.115 5-9 6 | 13.821] 2.304] 0.139 6.2 Tai 6 11.815] 1.969] —0.025, —1.2 6 | 13.505| 2.251] —0.053] —2.3 20| 6 11.595] 1.933 —0.036, hes 6 | 13.515 2.252) 0.001) 0.04 ZI) 6 11.79 | 1.965 0.0321 1.6 Gy Pxrghro7is2-2 i — 020521028 DEG (4) -)6 12.305] 2.051 0.086 4.2 6 | 13.85 | 2.308 0. 108 4-7 II 6 Teor || Toes | Cora) nats 6 | 13.777| 2-296] —o.012} —0.5 18} 6 TQeA Ti) 2023 0.295} 14.1 6 | 14.64 | 2.44 0.144 6 Began to feed 153 mg. of beef a week | | 24| 6 14.97 | 2.495| 0.265 Tr-2 6 | 14.815) 2.469) 0.029 iieyt jan=, 2) 6 14.777| 2-463) —0.032]| —1.2 6 | 14.845) 2.474] 0.005 0.2 8} 6 14.977| 2.496] 0.033 Teg 6 | 14.897] 2.483] 0.009 0.3 15} 6 15.44 | 2.573| 0.077 3.03 6 | 14.775] 2 463] —o.02 | —0.8 22) 6 17.324) 2.007) O.3%4) TY. 5 6 | 15.305) 2.551} 0.088 355 29} 6 \ Diop) Gaa | Re Reb. 5| 6 18.105) 3.018] 0.131 2 6 | 16.285) 2.714] 0.163 3- 12| 6 18.997) 3-166} 0.148 4-7 6 15.095 2.516} —0.198| —7-5 I9| 6 19.06 | 3.176] 0.01 0.3 6 | 16.037 2.673 | 0.157 6. 26| 6 18.555] 3.093] —0.083] —2.6 6 | 16-055) 2.673| 0.00 fo) Mar. 5) -6 18.92 | 3.153] 0.06 1.9 6 15.965 2.661 0-012) — O-4 12} 6 18 .365| 3.061] —0.092] —2.9 6 | 15.077) 2.513 SCs) = 557/ 19} 6 18.73 | 3.12 0.059 1.9 6 | 15.072| 2.512) —0.001 0.03 26} 6 18.425] 3.071] —0.049] —1.5 6 | 15.125] 2.521] 0.009 0.3 Began to feed 204 mg. of beef a week Apr 2j/ 6 17-955| 2.992] —0.079] —2.6 6 14.865| 2.477| —0.044| —I.7 9) 6 18.295) 3.049] 0.057 1.8 6 | 15.607) 2.601) 0.124) 8 16} 6 18.965| 3.161] 0.112 3.6 6 | 15.875) 2.646) 0.045] bey) S2|\ 2 | Rona lane Se a 8 08 a 8 From From Oct. 16 to Dec. 18....| 2.04! 0.377] 18.4 Oct. 16-Dec. 18] 2.27 | 0.33 14.5 Dec. 18 to Feb. 19....| 2.703} 0.946) 34.9 Dec. 18-Feb. 19] 2.556] 0.233 9.1 Oct. 16-Feb. 19) 2.391, 0.563, Pe kets 36 Ser D} Rate of growth after cutting tails off at base. Fed 153 mg. of beef a week } Ada Springer TABLE IV E = ere . Ce ae aa ¢ 2 & g = at 8: a les ye = = ° > = A 4 | & < = a Oct. 23 + tails | | 13.516) 2.253] 6 |= zails | 11.916 1.986, 30] 6 13.478 2.246] 0.26| 12.2 Nov. 6) 6 13-477| 2.246) 0.00 0.0 13} 6 14.487, 2.414, 0.168) © 7.2 20, 6 | 13-745) 2-291] -—0.123] —5.2 27| 5 ‘| II-537| 2-307, 0-016 0.6 Dec. 4) 5 | 11-955) 2-391) 0.084 225 II} 5° | 12.767| 2.553) 0.162 6.5 18) 4 10.71 | 2.678} 0.125 4-7 24, 3 8.096, 2.698 0.02 0.7 Jan. 2) 3 7-89 | 2.63 | —o.068) —2.5 3| 3 8.085, 2.695 0.065 2.4 15) 3g 8.2 | 2.733) 0.038 1.4 223 7-545 2.555] 0.218) 8.3 29 3 \ 7-4 Bebe s5|) mes 8.765, 2.922, 0.407 f Fok 12} 3 8.84 | 2.943, 0.021 0.7 19} 3 8.92 | 2.973) 0.03 1.01 26 3 9-12 | 3.04 0.067 Bed. Mars. 45) 93 8.965) 2.988 —o.052)} —1. 12 3 3-78) | 2.926] —0-062))) —z2- 19} 3 8.657, 2.886, —0o.04 |] —1.3 26 3 3252 | 2204 |) OL046)) | — 16 Began to feed 408 mg. of beef a week Apr. 2| 3 3-2 | 2-73 | —O-o11| —3-9 9) 3 9-295} 3-098} 0.368) 12.6 =| 26}; 3 3 9-105] 3-035, —0.063 —2 | >| 2 | 4 ° S| A mE From Oct. 23 to Dec. 24 | 2.342| 0.712 | 30.4 Dec. 24 to Feb. 26 | 2.869) 0.342 | 11.9 Oct. 23 to Nov. 27 | 2.2 0.428 19.5 No. of animals | } | | | | } DNDNDNDANTDAATDAAARAAHA NH Began to feed 306 mg. (average) beef a week 6 Ae 6 From Dec. 18— Jan. 15 ” Total weight + tails 11.491 — tails 9.998 11.474 Ser D? Tails cut at base as in D\ Average weight 1.915 1.666 1.912} Increase 0.246 Began to starve 8.892) Fed 153 11.213 | 11.412 11.865 | 13-105 | 14-25 15. 16.535 16.5 16.955 17.565 16.689 16.737 17-17 | 17.225 18.44 | 3-073 | 19.205 mg. of beef a week 1.869 1 .go2 1.978 2.184 2-315) 2.5 2.756 2.75 2.826 2.928 2.782 2.789) 2.862 2.871 average) Mean - , se ( 0.387) 0.033 0.076, 0.106 0.191 _ 0.256 —o0.006 0.076, 0.102) —0.146 0.007 0.073 0.009 0.202 0.128 Increase 0.702 Per cent inc. 13-7 23.1 1.7 4-9 5. 8.3 2.5 a a7 —0.2 2.7 3°5 hee 0.5 2.5 0.3 Gr7 + Per cent increase ~ A Study of Growth 37 TABLE V Ser E? Ser G? Rate of growth after cutting tails off at base. Fed 153 Normal intact control. Fed as in E? mg. of beef a week | # “ = ) ide! = — e “= ° | ~ oO ° ro) = = oO rs) S 8 5 : 5 as Nass 5 3 5 Z A < 4 oy 7 | < 4 ow |+ tails 20.44 | 2.044 Nov. 13} 10 |-— tails fe) |) ihe) Baas ycehe \ Biegle) 17} 10 | 18.047] 1.805] 0.075 4.2 EO} | 212207) 2.021), O-0% 0.4 20/ 10 18.035) 1.804] —o.001| —0.05 10’ | 20.81 || 2.081] —o.04 | —1.9 27| 10 | 17.83 | 1.783] —o.021| —1.1 IO | 20.067} 2.007} —0.074| —3.6 Dec. 4| 10 18.347} 1.835] 0.052 2.8 IO | 20.095] 2.01 0.003 Oo. II} I0 | 19.067| 1.907| 0.072 3.8 IO | 20.445] 2.045) 0.035 Tey, 16 TO) | 19.217] 1.922] 0-015) 0.7 Io | 20.794] 2.079) 0.034 Al Been oa | gee gS oa Be Outer cty «lin of FERC RMR Te | ieee 3) (= a od =| a | 43 Ca Sire From From | Nov. 13 to Dec. 18....| 1.826] 0.192 10.5 -Noy. 13-Dec. 18, 2.095| —0.032] —1.2 Set E} Ser G! Same as Set E? Same as G? + tails | Nov. 13 22.635) 2.264 TOV) | — tails IO } 19.442| 1.944| 18.865 1.887, | 17\\) 10 18.995 1.899] 0.012 0.6 10 | 18.936 1.894) —0.05 | —2.6 20] 10 19.256) 1.926| 0.027 1.4 Io | 19.655) 1.966] 0.072 Aol 27| 10 20.295| 2.03 0.104 Gaz 10 | 19.535| 1.954 —0.012} —0.6 Dee; 4) 10 20.765] 2.077| 0.047 22 fe) 20.189, 2.019} 0.065, lap? II} 10 21.627| 2.163| 0.086 4. I0 | 21.09 | 2.109] 0.09 re) 18} 10 21.882) 2.188] 0.025 ey 10 | 21.795] 2.18 0.071, BE | Pi 2 “o © | #2 2 e o em | Bs SCE es eie ee o tt o a PH —|—___ | From From Nov. 13 to Dec. 18....| 2.037] 0.301] 14.7 Nov. 13-Dec. 18] 2.062] 0.236) 11.4 38 Ada Springer TABLE V—Continued Ser E3 Set G* Same as Set E? Same as G* 4 ae Rie , 2 | 3 eo g Bly sage ll aes 3 Bi oan = = “3 “3 = — s Be Wor 2 = s = 2 gy = = eo a e ~~ eo I ze is) Ta | o =) i) = KS o ro) 6 5 | & 5 5 S Sul ese y 5 Z B < 5 a Z A < 5 oe +tails | | 18.778] 1.878 Nov. 13] 10 |-— tails Io | 19.65 | 1.965 16.14 | 1.614 17| 10 16.004, 1.6 | —o.014, —o.8 10 | 19.785' 1.979, 0.014 0.7 20/ 10 16.156| 1.616 0.015 0.9 10 | 20.19 2.019 0.030 2. | | 27| 10 16.342| 1.634) 0.018 Wige! Io | 19.895) 1.99 | —0.029) —1.4 Dec. 4] 10 17.349] 1.735| 0.101% 5-9 Io | 20.26 | 2.026; 0.036 17, Dt] to 17.469) 1.747). 0.012 0.6 IO | 20.595| 2.06 0.034 1.6 18} 10 18.62 | 1.862) 0.115 6.3 10 | 20.988) 2.099 0.039 1.8 e%| 2 | #2 Feciige fh s. bese we os lo § S 3.8 ten hae 5S = = he 5 5: SS Ed Ay 8 S| 4 aS ical > al From | From Nov. 13 to Dec. Bec 1.738) 0.248 14.2 Nov. 13—Dec. 18) 2.032} 0.134 6.5 a. Rate after cutting tails off at base. of beef a week Set E3 A Study of Growth TABLE VI Fed 153 mg. Ser G1 Normal control. Fed as in E3 39 No. of animals Total weight Average weight = A le E en | = 8 ¢ 2 e S 4S = Bare lp ree, 2 i ‘S ra lh 38 2 S S Sw has J B Z &

¥ g g = z g g | md 50 4 Oo Us & a o S a 5 2 B 2 3 - 2 2 fo} > 3) be 3 = 5 he ae Rie ee a a | de 3 eee x ———— + stumps 9-52 | 1-904 stumps 5 12.705! 2.541 Decr z5l 5 a5 ©.205 | 0.041 — stumps 9-177 1.835 24) 5 | 10.3 2.06 | 0.225 | I1.5 5 13-11 | 2.622] © OSn/igre Jan. 2| 5 | 10.311 | 2.062 0.002 ©.09 5 12.927| 2.585] —0.037| —1-4 8| 5 | 10.837 | 2.167] 0.105 4.9 5 13.14 | 2.628] 0.043] 1.6 T5)) oS |, LP-3a7) 1) 2-267|10.10 4.5 5 13.414] 2.683) 0.055 e®| 3 | #8 a | ¢ | @8 Geass hve se oo) 2 From From Dec. 18 to Jan. 15 ..... 2.051] 0.432 | 21. Dec. 18 toJan. 15] 2.613] 0.142} 5-4 A Study of Growth 41 TABLE VIII Ser E22 Ser E2b Regenerating tails cut off at base. Fed 153 mg. of Control. Regenerating tails intact. Fed beef a week as in E% 2 a 4 e a Shares le : 12 | : a “3 e — Fe Ss - - S z £, g r=] : # es g = S|) a) g 5 = i lee bog 8 3 i) 5 g 3 iS i S$ 3 B aa ee = 2 | 4 a Pa et ever x + stumps 9-77 5 stumps Dee. 13), 5 0.21 5 10.447| 2.089 — stumps 9-39 1.878 24) 5 10.436 | 2.087} 0.209] 10.5 5 11.165} 2.233) 0.144 6.6 Jan. 2] 5 10.535 | 2.107| 0.02 0.9 5 11.167] 2.233] 0.000 Sir55 II.417 | 2.283] 0.176 8. 5 12.342] 2.468} 0.235 | 10.0 ESle 5 11.63 | 2.326] 0.043 1.8 5 12.545) 2.509] 0.041 1.6 = ee We Seaiicee ieee Ss B| § oe s o| § ae: eo) a aa 2 a | eee From | From Dec. 18 to Jan. 15 ....| 2.102] 0.448 | 21.3 Dec. 18 toJan. 15} 2.299] 0.42 18.2 in 42 Ada Springer TABLE Ix Set HS Set H? Tails cut off at base after 5 weeks’ starvation. Fed 153 Normal control after 5 weeks’ starvation. mg. of beef a week Fed as in H? iff ite Weed ta oe ihe || bee he g Boal Peaster g “a 3 Es | AS, oI | = S = a FT eats g re] S 2 ¥ g a & 2 cS o Sate | a Be 3 3) | @ | § oe = = = £ i S oa oO - rol ~= =) Ls ey te (3) 8 | 8 ee | See ee +tails 14.455 | 1-446 | tails | Dec. 18) 10 1.515 | 0.152 10 | 16.495 | 1.65 | — tails | | | 122702 [eh 2 7 | | 24; 10 15.242 | 1.524) 0.254 | 18.1 10 | 19.222 | 1.922] O-27 2) eeiGew, Jan. 2} 10 16.357 | 1.636] 0.112 The 1o | 18.857 | 1.886, —o.036| —1.8 8} 10 16.562 | 1.656, 0.020 iD 10 | 19.632 | 1.963) 0.077 4 15] 10 17-477 | 1.748| 0.092 5-4 IO | 20.229 | 2.023] 0:06 Paes oes leo) 2 BS g EH > 3) bo oS) OSS aes | = | eee From From | | Dec. 18 to Jan. 15 ....| 1.509] 0.478] 31.6 Dec. 18 toJan. 15) 1.836; 0.373] 20.3 | / | ! A Study of Growth TABLE X Ser F1@ Regenerating tails cut off at base after 5 weeks’ starva- tion. Fed 153 mg. of beef a week Ser Fib Regenerating tails intact after 5 weeks’ starvation. Fed as in F\% 43 2 2 = “a ay) eae ee a z g, 9 ¢ 3 e Tem Mecca Td Gr = Soc Gan ala a sepsis lot ia ee, ee ee ee |e |) a c Pal (a= re ee cea (Par |+ stumps 6.715 | 1.345 | stumps 5 6.795 | 1.359 | Dec. 18] 5 0.185 | _—stumps | 6.34 | 1.268 | 24] 5 Tote || Wabi 0.303] 21.3 5 8.4 1.68 0.321, 20.1 Jane 2)| 25 TGs | oben) Ogee tas 5 8.025 | 1.605] —0.075| —4.5 8) 5 TisiZaa\e L544)) —O-C03|) Or 5 8.309 | 1.662) 0.057 3-4 15) 5 7.897 | 1-579] 0.035 Dae 5 8.6 1.72 058 3-4 22) 5 8.89 | 1.778] 0.199] 11.8 5 9-575 | 1-915] ©.195| 10.7 29 | \ 45 5 \ 2g Feb. 5| 5 | 9-745 | 1-949 0.171) f 4-5 5 10.15 | 2.03 0.115) 2.9 12} 5 10.175 | 2.035| 0.086 4-3 5 | 10.825 | 2.165) 0.133] 6.3 19) 5 10.35 | 2.07 0.035 1.7 5 | 11.192 | 2.238 0.073, af 26 5 10.755 | 2.151) 0.081 3.8 5 | 11.225 | 2.245) 0.007) 0.3 Mar. 5] 5 10.805 | 2.161) 0.01 0.4 5 | 11-335 | 2-267, 0.012) 0.5 12]: =k 105577 | 2-115) —0.046| —2.1 5 | 11-033 | 2.207) +0.06 | —2.6 19} 5 10.555 | 2.111] —0.004] —o.1 5 | 10.915 | 2.183] —o.024) —1. 26] 5 DL DE |) 22222 0.111) Beit Gel shige || 262 0.079, hats Began to feed 204 mg. of beef a week Began to feed 408 mg. of beef a week Apr. 2| 5 | 11.11 | 2.222] 0.000 0.0 5 | 11.395} 2.279] 0.019 0.8 9| 5 DOS 721-340) On rae 4.8 5 12.605] 2.521| 0.242] Io. 16} 5 | 12.405) 2.481 0.15 | 6.2 5 13.205| 2.641 0.12 Gets | le © | > 2 2 es) 8 | £4 Se a. bas From From Dec. 18 to Jan. 15..... | 1.424) ©.371| 21.8 Dec. 18 to Jan. 15] 1.539] 0.361] 23.4 Dec. 18 to Mar. 26....) 1.744] 0.954] 54.6 Dec. 18 toMar.26| 1.81 | 0.903} 49.8 | | » att 44 Ada Springer TABLE XI Seri Ser Fb Regenerating tails cut off in middle after 5 weeks’ starva- Regenerating tails intact after 5 weeks tion. Fed 153 mg. of beef a week starvation. Fed as inF™ lS a = 3 | = E Z, 2 r = z = g # ee ek eee Ss | a |) 2 eas hee) a ae 2 | 8 | 2) ae +tails 6.36 | 1.272 Dec. 18] 5 — tails 5 5.28 1.056 6.286 1.257 Pi 241ihas 7-592 | 1.518] 0.261 | 18.8 5 6.567 | 1.313] (O-2a7ieeete Jan. 2) 5 7-595 | 1.519] 0.001 ©.06 5 6.43 | 1.286) —0.027] 2 8) 5 7-869 | 1.574) 0.055 ae5 5 6.72 | 1.344 0.058 1s ps 8.375 | 1.675] 0.101 6.2 5 6.987 | 1.397; 0.053] 3.8 Rees. wlan | 8 B32 ahs o| es "S| Ae From | From | Dec. 18 to Jan 15. ..-.| 1.466, 0.418 | 28.5 Dec. 18 toJan.15| 1.226 0©.341| 27.8 6-z | £Lo°0 “giSz Lor St gl gto'o 6iz-z |S1e-e1 Sfo'o 661% 261° £1 gi oy Gn wan fo-o Sth-z | Lo tt gt jo fgr'z I‘ £1 Loe go’o boiz |Sg6°z1 9 1g L-1— | teovo— | Srb-z |Lgb-+1 *b— | ggo'o tgoz |Lob-z1 gil 6to°o tgoz |SoS-z1 9 lz ‘uel no 6°8 117‘ LSb-z | bl $1 Cor | rz 6gt°z |S10°t1 z'6 yunrey ll ree IP Meee 9 tz gbz'z |SLb- er gS6°1 |SEL-11 Sog'r |dgr-11 $9 hd Oke) sje] — G+ 6g0°0 spiey + 9°6 LL1‘o spiey — 9 gi LvE-z | go'hi 110°z |Sgo°z1 616°1 jbrS +11 syiey + sted + syrey + oz'z |Foz ti CAR Wessun zbl:1 \bSb-or z°9 giro sey — Boks toro sey — “L Szt-o spre — 9 II 6rf-z |L£16-t1 6g6°1 |gf6-11 1zg'rt |Sz6'or s[iey + syrey + syiey + Igt*z |160°£1 Sgg-1 |Lof-11 g6g°1 |LLr-o1 SS ggo"o sie} — £9 Z1'0 spre} — °8 gti spre} — 9 ¥ “J9q' as glee, |) Lor er Sg6*1 j€SL-11 Til | soson 3 spe] + sprey ++ syrey + 3 Iz |1gz" fr gtg't |S1o°11 Sig'1 | 69°6 th 1L‘o g10"o sprey — I‘Oo— | too'o— sprey — L'o Z10°O spit — 9 lz iS) ghee |Lgh tr 6gg:1 |See-11 CSgt |616°6 ay sre} + spley + syva + = wize |S6E°E1 16g°1 |SbE-rr 1vg't | Sg°6 a +°£— | gor:o— spiey — +-+— | ggo'o— | syrey — S:o— 10°0— syiwt — 9 ot gz-z | gg fr zt61 |bSgr 11 £l9°1 |6to-or s spey + syle} + spit + ‘ ggt:z Let: tn fo'z |gli-zt £g9°1 100s!) © syrey — sey — spp. — 9 €1 ‘Aon b6ghz [Sf6-b1 gir'z |SoL-z1 Sezer | yor ; spe + sprea + spiey + aay ee ee rl) rl ele ee g 8 a 8 Bel ae = 8 8 s Fe B, ~ 5 3 % 4 ih 4 B 4 3 4 3 4 : fis ce e a | ¢ : ae] 3 j oO . % ct . = > . 5 oS ov i S — 0 46 Ada Springer TABLE XII—Continued Set I ...|From Noy. 13 to Jan. 15 ee aye Nov. 13 to Jan. 15 Noy. 13 to Jan. 15 Mean (average) 1.976 bd & 8 x os iS me O27 Ts| Ate, 0.486 | 24.5 9253" [2aes A Study of Growth TABLE XIII Ser G2 Tails cut 6 successive times. Fed 153 mg. of beef a week a|/3 || ; & 80 o = 3 = 4 se else alee z a) J s S 5 ° 2 o 5 be Bee | eel 8: | ok + tails| | 9-437, 1.887) tails Dec. 18] 5 0.22 | 0.034! — tails | 9.235 1.847 + tails | 10.209] 2.042) 0.195) I0. Dec. 24 § | tails | | 0.197, 0.029 — tails| 10.064! 2.013 + tails 10.03 | 2.006, —0.007 =O Jan. 2) 5 tails | | 0.17 0.034| — tails | 9-914 1.983 + tails 10.295] 2.059} 0.076) 3.7 8) 5 tails 0.252, 0.054, — tails| | 9.96 | 1.992 + tails 10.8 | 2.16 0.168} 8.2 15} 5 tails 0.335) 0.067] — tails) 10.366, 2.073, + tails | 0.152 Tis BG || 18.125) 2.2261 — tails | 10.961) 2.192 29) 4 9.055] 2.264) 0.072} 3.2 Bebe =5|| 3 7.465) 2.488] 0.224] 9.4 12] 3 7-615) 2.538) 0.05 1.9 19] 3 7-895] 2.631) ©.093|- 3-5 ZO) 3 8.01 2.670 0.039 1.4 47 Ser G% Tails cut 9 successive times. Fed as in G*b 2 r= ‘6 cts} = = SED als & | ¢g g Soe ee hele Secs tye |) ae glee + tails 9-75 | 1-95 | — tails | | Dec. 18 5 9-545) 1-909, + tails 9-97 | 1.994 | 24) 5 | —Zarls | 0.085, 4-3 9-867) 1.974 | + tails) | 9.76 1.952 ; Jan 2| 5 | = zails| —0.022| —1 I | 9.57 | 1.914) | + tails | 10.047) 2.009 | Sianes — tails —0.095 4.8 9-857) 1.971 + tails) 10.429) 2.086 sis) || eG O.115| 5.6 | 10.07 | 2.014 + tails 10.805) 2.161) 22) 5 — tails 0.147 7 10.622] 2.124) | + tails | | 11.055] 2.211] | 29| 5 | —2ails 0.087; 4. 10.76 | 2.152 + tails) Iniaty ||| 22) Feb. 5) 5 | —tarls 0.148 6.6 | riaisntly | Bare + tails | rN) Deion | | 12} 5 = tails, Garza) 5.2 11.425] 2.285 19] 4 | 9-637| 2.409] 0.124] 5.2 26) 4 Q- 155|) 2.288) 0-121) soar Mar. 5| 4 2.492) 0. 9-967| 48 3 Ada Springer TABLE XIIJ—Continued | a ~ = | 5 3 ra] | = g ew: cree ea | AN agen hal coe = a=: Ss ie = a4) ae 2 ‘ | s B 2 g a = a 2 2 S Sri) sa 2 © 8 6. | Fe = 2 8 - 2 o oO res oul oJ o oO a Re wee Veeco Ge. te ete |e ey ——— — | Mar. 5) 3 8.077] 2.692/ 0.022| 0.8 Mar.12] 4 | 9.825 ree 0.036] 1.4 12 3 7-905) 2.635) —0.047/ —1.7 19} 4 | 9-845] 2.461; 0.005) 0.2 19) 3 7-975| 2.658 4 0.8 26] 4 9-79 | 2.447, —0.014| —0.5 =) 3 7-965 2.655 mectge || Woe Apr. 2] 4 9-555| 2-388] —q.o59| —2.4 Apr. alas 7-965) 2.655 0.000) 9| 4 9-795| 2.448) 0.06 2.4 [Paani 16] 4 | 9-935) 2-484) 0.035] 1-4 “> o- Ss o “o o =I ae) = (les ge| s | 8 S 5 5 » & = 3 5 be S| 8 |e a S| tle From _ From Dec. 18 to Feb. 19...| 2.104] 1.10%) 52.3 Dec. 18 to Mar. 19 | 1.997, 0.974| 48.7 A Study of Growth 49 TABLE XIV Ser F! Ser H! Rate after cutting tails off at base. Starved. Normal intact. Starved. ae | : oi A se oaere 4 GE | "S D 8 g ‘> | ‘Ss z 6 = = a g rs eee 3 8 Braet | eg Fy rte act Safeco a Fy +tails | 10 20.679) 2.068 IO | 20.287, 2.029} Nov. 13 —tails | ! 17-552 rane) 20| 10 17.095| 1.71 0.045] 2.5 10 19.429 1.943) 0.086 An 27| 10 16.077, 1.608) 0.102) 6.1 io 17.605 1.761 0.182 9.8 Dec. 4) 10 15-45 | 1-545| 0.063) 3.9 10 16.615 1.662) 0.099 5-7 11} 10 TA a2 7) lA SC O-i2 7s 10 | 15.935 1.594; 0.068 4.1 1g] 10 13.51 | 1.351| 0.082 5.8 10 15.542) 1.554] 0.04 25 | ae: Z eee : eid be EN Wen ioe ee ee We eA SIN From | | From | Nov. 13 to Dec. 18.... 1.553} 0.404 26. Nov. 13-Dec. 18) 1.791] 0.475 | 26.5 Ser F2 ; Ser H? Same as Set F} Same as H} | { Nov. 13} 10 +tails | | | 19.886, 1.989 10 | 22.445) eae | —tails | | 16.872| 1.687 | 20} 10 16.664 1.666} 0.021 1.2 10 21.303) 221g) 482235 Gee 27| 10 15.47 | 1.547| 0.019 I.1 10 19.76 | 1.976 0.154 7.5 Dec. 4] 10 15.092, T.509) 0.038 ane 4 fe) 19 075) 1.908, 0.068 Beis II} 10 14.177| 1.418 0.091 | 6.2 10 Tes 7i5 758, 0.15 8.1 18| 10 13.162) 1.316] 0.102 7-4 10 16.495. 1.65 | 0.108 6.3 | | | ‘Es | = e 2 sets (a el 3) a ose, avAa eee From | From | | Nov. 13 to Dec. 18....| 1-501)" 9.371 24.6 Nov. 13-Dec. 18 1.947| 0.595 30.5 50 Ada Springer TABLE XIV—Continued Ser Fs Ser H? Same as F1 Same as H} = = 4 P| 2 a 5] 2 mz a 5 2 = ty 2 Se S 5 s 2 Ss S = 5 é $ ° = 2 . < ~ ao o — 7) a Bw | ie | ie | 3 a es) eh ee ms Nov. 13) 10 |-+ tails | 17-52 | 1.752, 10 19.607, 1.961 — tails 2) 5) | 0s )))) 20| 10 14.815) 1.482) 0.068 4-4 to =| 18.771! 1.8771 0.084 43 27| 10 13.825] 1.382! o.1 _ 6.9 10 | 17.307] 1-731| 0.146 8. Dec. 4] 10 13.287] 1 329, 0.053 3-9 Io =| 16.379] 1.638) 0.093 5-5 I1| 10 12.397| 1-24 | 0.089 6.9 10 15.332] b-533| 0.105 6.6 18] 10 | 11.64 | 1.164) 0.076 6.3 10 14.455] 1.446) 0.087 6. “S = is r= oO oO i Mo ean pees \38| 2 eee a - = > @ _ — si) ee (= 22) § 18 | From From Nov. 13 to Dec. 18....| 1.357) 0.386 | 24.8 Nov. 13—Dec. 18 | 1.703] 0.515 | 30-2 A Study of Growth TABLE XV Ser k2 Set H? Regenerating tails. Rate of growth after starvation. Normal intact control after starvation. Fed. 153 mg. of beef a week Fed as in F? eoies. be : e/ 213) : ae a = = “3 3 z on 3s 2 2 gy q = E- oF + r= zr a ae S 5 S S By 5 SP (| = 3 o Sea a g 5 A B < 4 = Z B < 4 = Dec. 18| 10 13.162] 1.316 Io 16.495| 1.65 24| 10 16.437| 1-644] 0.328 22et 10 19.222] 1.922} 0.272} 15.2 Jan. 2) 10 16.04 | 1.604] —o.040 Dot fe) 18.857| 1.886) —0.036| —1.8 8} 10 16.507} 1.651} 0.047 2.8 fe) 19.632) 1.963) 0.077! 4. 15} 10 17.269] 1.727] 0.076 4-4 10 20.229] 2.023} 0.06 | g 22} 10 18.37 | 1.837] 0.11 6.1 fe) 21.69 2.169 0.146) 6.9 29 \ 6.5 fe) | i Soe! Hebs. §|) 10 20.935] 2.094] 0.257 f 6.5 10 24.13) || 22413 0.244] f ee I2| 10 21.465] 2.147] 0.053 225 10 24.89 | 2.489] 0.076) gaz 19} 10 23.035] 2.304) 0.157 Fig 10 25.885) 2.589 0.1 3-9 26} I0 23.095] 2.31 0.006 0.2 Io 25.4 | 2.54 | —0.049] —1.9 Mar. 5) 10 23.294] 2.329] 0.019 °.8 7 | 19.035] 2.719] 0.179] 6.8 12} 10 22.925] 2.293] —0.036| —1.5 5 125272507 On2 00 oe 19} 10 23.095] 2.31 0.017 0.7 5 12.502] 2.5 | —0.007]/ —o.2 26| 10 22.812] 2.281] —o0.029] —1.2 5 11.84 | 2.368] —0.132) —5.4 Apr. 2] 10 22.115; 2.212| —0.069, —3 5 11.595 2.319} —0.049; —2 9 5 11.925| 2.385] 0.066 2.8 16 5 12.44 | 2.48 095 3-9 28) § | 58 jee} & | 5 SS a a .& SS = AE From From Dec. 18 to Mar. 5 ....| 1.822] 1.013 55-5 Dec. 18 toMar. 5) 2.184) 1.069 48.9 52 Ada Springer TABLE XVI Ser A Ser D Rate of growth at high temperature 28.2° (average). Fed Same as A. Starved 7 2 n oats = - 3 = mi = a | Saha emo BAe al sey a23| 4 or) 2 tr SI “3 E | ts 0. BE 2 Sp & a ES 2 2. | = 23/ Silos s e 5 oo em | 5 | 5 oO - poy (3) 5 ia ~ (3) a rok evan ele = ee fs = NP i Mat et fe a Feb. 21 6 [11.8 | 1.967) | Mar. 5) 27.5 | 6 |10.057| 1.676] —o.291) —15.9 6 | 10.225] 1.704 12) 27/.69| On MNO elG MN les 2che—-OstkT|| = OlK: 6 8.95 | 1.492 —0.212) IQ¢2 19] 29. 6 | 8.835) 1.473] —0.053] —3.4 6 7-825] 1.304 —0.188) 13-4 26| 27. 5 | 7-425) 1-485) 0.012 0.8 3 3-49 | 1.16 | —o.%44) 419-6 Apr. 2] 30.6 | 5 || 77-04) | 1-408] —0.077| —5.3 3 3-105] 1.035] —O.i25| meme 9} 29. 5 | 7-095) 1.419] 0.011 0.7 3 2.905) 0.968 —0.067 6.6 FO) 26220 5) ele 5 1.43 0.011 0.7 3 2.797} 0.932) —0.036) 327) « & r= ee i Sha es Sg ie § PS coe TRCN Re ia eae Mega: From | From Mar. 5 | Feb. 21 to Apr. 16} 1.698|—.537| —31 to Apr. 16 4 1.318] —0°772|/eu5oe5 SEr=B ; Set E At room temperature 22° (average). Fed Same as B. Starved Feb. 21 6 |13.08 | 2.18 Mar. 5) 21-5 | 6 (12.355 2.059) —0.121 rey 6 |t0:33 | nl72x 12| 22.1 | 6 |11.327| 1.888) —o.171| —8.6 | eis 1.64 0.081} —4.8 19] 22 6 |10.745| 1.791] —0.097| —5.2 5 | 7-215 | 1-443) Osmo 7 | ae 26| 22 6 |10.465) 1.744] —0.047| —2.6 5 || 6.64. | 1-328) —orrns hes Apray 2] 2353 5 | 8.285) 1.657} —o.087] —5.1 5 | 6.04 | 1.208] —o.120} —9.4 9| 22 5 | 8.475| 1.695) 0.038 2.2 5 || 5-745 | 2-149) —O-Ololmaog 16} 22 5 | 8.647) 1.729] 0.034 1.9 5 | 5-66. | 1.33) |. olorgi es 3 8} e la &| r= Melee ie 2 5| 2) ieee pet Slay "=| 3 From | | From Mar. 5 | | Feb. 21 to Apr. 16 | 1.954 — -451|—23.08 toApr.16..) 1.425) —0.591| 41.4 A Study of Growth Ser C At low temperature 11.2° C (average). Fed TABLE XVI—Continued 5 4 a 20/8 | 2.) @ Peete | eh & Beedle | Sear y 2 8 Sea a |) oe 2 Brot 1S = 2 o eo lhe = < a Feb. 21 6 |10.712) 1.785 Marmmsiergen | 6 111.72, || 1.953 168 12} 8.5 | 6 [11.285] 1.881] —0.072 19 11.5 | 6 |11.585| 1.931 05 26| 12 5 | 9.87 | 1.974] 0.043 Apr. 2) 12. 5 | 9.625] 1.925} —0.049 9 10 5 |10.435| 2.087] 0.162) 16 11.6 | 5 | 9.907) 1.981) —o. 106) aS ~_ o H ras From Feb. 21 to Apr. 16 | 1.883] 0.196} 1.04 Set F Same as C. Starved 53 Per cent | w rv Aro —5.2 a a & = a TS o “< = Ps * = & =| ° 3s 5 e S 3 iS) S ra 3 Ze ete eer tS x 6) Pro-325 ier 720 3 ek Olea Teer y2 0.001 0.05 2 3-222) 1.611] —o0.109 6.5 2 3-005) 1.502] —o.109} is 2 294) e0-47 | — 0.032 2.1 2 2.93 | 1.46 | —O.001 0.06 2 2.865! 1.432| —0.028 1.9 “ays + fre Cue tee = ane é eee VY BK — From Mar. 5 Apr. 16). ..5|'h3576|| —O.289|)— 183 54 Ada Springer TABLE XVII SrtA SET Males. Showing normal rate of growth. Fed 315 mg. Females. Normal rate of growth. Fea of beef per week as in Set A Soh ea here 2 cat cg ee z E & g 5 2 & 2 s S a s 3 S S a a $ 7 face ee ee 2.1 8 | 25) Oct. 22] 10 ree 2.124 10 17.214) 1.721 28| 10 21.978) 2.198] 0.074 . 3-4 10 19.052) 1.905 | 0.184 | 10.1 Nov. 4] 10 22.874) 2.287| 0.089 3-9 10 19.148, 1.915 | 0.01 0.5 11} 10 22.934) 2.293] 0.006 0.2 10 19.402) 1.94 0.025 12 18} 10 23.721| 2.372| 0.079 eye 10 20.343] 2.034 | 0.094 4.7 Pal Ue 24.749) 2.475] 0.103 - 4.2 10 21.806 2.18 (0.146 6.9 IDiee, G3|) ike) 25.085| 2.509]. 0.034 as 10 22.708] 2.271 | 0.091 4. 9} 10 26.366 a 0.128 4.9 10 23.575| 2.358 | 0.087 S)07/ 16} 10 27-397 2.740] + 0.103 3.8 Io 24.394) 2.44 0.082 3-4 23] 10 28.378 2.838) 0.098 3-5 10 25.815) 2.582 | 0.142 5.6 31| 10 28.645) 2.865) 0.027 0.9 10 | 26.387) 2.639 | 0.057 Zak Pup aariceye = 2 “~o Gee Coe ee: se] & lps ees 4 oe Ss a |e From | From : Oct. 22 to Dec. 31 ----| 2.494] 0.741 29.7 Oct. 22 toDec. 31) 2.18 0.918 | 42.1 : : A Study of Growth 55 TABLE XVIII Males. Individuals showing normal rate of growth. No. 1 No. 2 fe ar i) ¢ a i) = © 4 I o a=) “al aie cteg erat Z oa is 2 z ie I ea g enme (liese |g z 5 or) a) 5 bh 5 ev) ‘Oo 5 be jl ca =a | a ad pele. jee | ts és Ock 22) - 1 2.627 I 2.787 28) 31 315 2.672} 0.045 1.6 i Hats 2.755] —0.032} —1.1 Nov. 4; 1 | 315| | 2-687 0.015 0.5 I | 315 |4| 2-855} 0.1 a5 II B35 a 2.720} 0.033 Dez Lh ghd Ee 2.795, —0.06 | —2.1 TS goto || 305 2.910| 0.19 6.7 1) 325 (2 2.02)| | 10.1245 4.3 Pests eel scorch o.24ril Tico EM 325. 7 |'35095| Sonny's 5-4 Dec. 2} 1 | 315) | 3-04] —o.215] —6.8 1 | 315} | 3-098] 0.003] 0.09 Slee e310 naismGg|i- o5123 3-9 I | 298 3-227| 0.129 4. Toler 306) || 2446) 0.283 8.5 I | 308 3-468) 0.241 ie 23 x ||| 308 2439] 0.007) —O.2 I | 329 3-365, —0.103) —3. 31 I 325 | 3-483] 0.044 fez 1/5326 | 3-412] o 047 pe => =| Sea str. [uae so) 8 4 & el Siars Shee ete From | From | Oct. 22 to Dec. 31 ....| 3.055] 0.856 | 28 .o1 Oct.22 to Dec.31| 3.099] 0.625) 20.1 Dec. 2 to Dec. 31 ....| 3.261] 0.443 | 13-5 Dec. 2to Dec.31] 3.255) 0.314] 9.6 No. 3 No. 4 Wc 22) 1 Light | I 2.509} Zh] Ga || Reb iy 1.587] 0.01 0.6 uy (gud | 2.472| = 037 —1.4 Wovem4te. 1 | 315) | 1-659 9.072 4-4 I | 315] @| 2-539] Ses) 2.6 II I 315 a 1.744} 0.085) 4-9 I 315\ = 2.537; —0.002) —0.07 mr | 335 2 12799) G.055| 4-1 Tr | 415 = | 2.5921 0.055] 2.1 or) x | 315| | r-997| 0.198] 10.4 FPS asl) ore) | -138 Raa Dec 2) fT | 3T5 | 1.948] —0.049| —2.4 ui |) ghee 2.81 -08 2.8 OP LE |-297 | 2.09 | 0.142 Te tr | 303 | 3-044] 9.234 7-9 Role Ee |) 325 2.197| 0.107} 4.9 I | 324 =| 2.998) —0.046 —1.5 23} 1 | 318 2.335; 0.138} 6. D |igen |) 9.526 128] 4.1 ani x || 329 2.339] 0.004 O.1 M422 3.287, 0.161 Isc =| ? | => 2 7 = 5 8 i Fa = o 5 \ = 4 (eel a | as ys) ae From From | Wer 2271to Decagt....| ©-958| 0.762) 38.9 Oct.22 toDec. 31 2.898) 0.778 26.8 Ween to Dec 3i.... | 2-143) 0.391] 18.2 Dec. 2 to Dec.31 | 3.048] 0.477] 15.6 Ada Springer TABLE XVI1I—Continued No. 5 3 : & ‘s z | a v J ua = ae =] 3S ° "eo 2 8 al cee. | aes lees E Z a S 4 ow Octs 22) 1 1.885 28) 1 | 315 1.927| 0.042 2.2 Nov 4) 1 | 315| o| 1-959] 0.032 1.6 TE 315} 2 2.035] 0.076 3.8 S| ee ean 2 2.052) 0.017 0.8 25|) ere esiGl! walli2-bE2|| 'Os06 2.8 Deen ez rears 2.222] O.11 Re 9} «1 | 286 2.301] 0.079 3-4 TO|Qe Toe eee. 22438| VOnta 7 |e 5571 23|| ea agi 2.47 | 01032] “a.3 QT ciamea 23 2.52 | /O.05 2 a & 2 a 8 Ss as s o a =e & is From Oct. 22 to Dec. 31 | 2.202] 0.635 | 28.8 Dec. 2 to Dec. 31 | ae ypll telseXeh) | Airdate A Study of Growth TABLE XIx af Females Individuals showing normal rate of growth. No. 1 No. 2 3 2 a a Fa He 5 F-) o = 8 5 o = Sele | eeeih | 2-357) —0.075) —3.1 I 315] > 1-689 0.227| 14.4 Hoek.) 35 = 2.284] —0.073| —3.1 I 315 a 1.807, 0.118] 6.7 TSiigeet || -305 = 2 407\) 0-083) 3)-15 I 315 2 I 79H =SeIt || Sols FR eteee STS) en |) 2-4821 0 O-ors| (O26 I BEG ~cleb 982! “CODES 8.2 Ween 2\))) 1 | 315 2.515 133, 5.8 re || giruks 1.987| 0.035] riety Slee |e i7 2.698] 0.183) 7. rou | 2.186, 0.199 9-5 Tole n | {rt 2.637| —0.061) —2.2 I 321 2.217} 0.031 1.4 2a eeets 927 2.696 O59] 2.2 To 982 2.339] 0.122 58 ull 9 eee Gel » Creel Bes I 323 2.418] 0.079 Bea “o> | ra) |) = “w | w | = | & sh Ss 3 gS a a | eee From From | Oct. 22 to Dec. 31 ....| 2.507) 0.571 22127 Oct. 22 toDec. 31) 1.902 1.030 | 5 4:.2 Dec.2 to Dec. 31 ....| 2-654) 0.278 10.4 Dec. 2 toDec. 31 2.202} ©.431/ 19.5 Ada Springer TABLE XIxX—Continued © Dec. 2 to Dec. 31 | 1.872) | u No. 5 3 & S & S 2 }° g a ry (clan ea cea es 5 icodd oe al Zz Pg ie 5 a Oct. 22] 1 1.447) 28). on logis) | 1-542)" ‘cog! 6-4 Nov. 4 I 315) = 1-552 0.01 0.6 Dil) See sis 2 1.507 —0.045) —2.9 18] x | gry = | 1.567, 0.06 | 3.9 Zs | eh =| 1.724) 0.157, 9-5 Decses2) ok Wesns 1.68 | 0.044) —2.5 9} I | 301 1-943, (0.263, 14.5 ae an gs) a 1.999, 0.056 2.8 23, «I | 321 =| 2-051 0 052} 2.5 aa 1 | 320 | 2.065 0.014) 6.8 g ¢ & ar) - From Oct. 22 to Dec. 31 | 1.756) 0.618 (o} wo oo n wb Ww ON Un TABLE XX Ser € Normal growth. Fed 315 mg. of beef per week ah alee = & alec ahs se Stab flings aaa o re i = as Coa a Nov. 11) 7 6.371; 0.910 18) 7 7-021} 1.003) 0.093 9-7 Za a7 7-929| 1.133] 0.13 sipaea DEC ae Z| yey 7-945 1-135 0-002 o.I 9) 7 8.534! 1.219 0.084 71 16, 7 8.821 1.26 | 0.041 3-3 23| 6 8.92 | 1.486 0.226 | 16.4 “oo 2 2 o - From Nov. 11 to Dec. 23) 1.198} 0.576 | 48.08 —- A Study of Growth TABLE XXxI Ser D Growth at low temperature (10° C. average). Fed as much as animals would eat 59 aN Cees Ola , eee Bee ee | ve | 3 ee ee hee Seles E Saal) eee joes 5 2 e ° BS ° > = oS a a Piss < a Ay Oct. 22] 10 D722 ee 22 28) 10 10 20.731| 2.073 0.351) 18.5 Nov. 4] 10 12 | 21.676] 2.168 | 0.095) 4.4 II} 10 Be | 21.766} 2.177 0.009) 0.4 Testo) |||. tra | 21.746] 2.175 | —0.002} —0.09 Zc) 16. || 10 22.531, 2.253 | —0.078) 3.1 Dec. / 2! 10 Io | 21 763| Ae iG hy | 0.077] SF 9) 10 9 | 22.45 | 2.245 0.069 3.1 16} 10 12 22.041| 2.204 | —o.041| —1.8 23 | e1O) | ime "2a 06n/) 22906)! to-102|) | <4)5 Ai 10m |e 12 22.104] 2.21 SO)e/4)) 72 - } a Pee fore iam ete ae From Oct. 22 to Dec. 31 | 1.966) 0.488 | 24.8 No. 1 (Male) No. 2 (Male) | ce Beles : o Co a, =i ai/2e)_]. : cao) ee ee z SO as ic E FON ey een (iE a 8 } wo 5 o S 5 3 80 8 to) 3 4 ise | 4 A Sherer } 4 A Oct. 22 I lx 374, I I aa 28e= SUS nega (OL86. |) 23150 I 315 | 2.202) O1255] 12.2 Nov. 4) 1 315 | 1.874] 0.14 Foy I 215) | 2.192) —o-o1 | —0.4 | 110 | 1.827) —0.047| —2.5 I 415 || 2.302! O.nr 4.8 18] I 105 | 1.747| —0.08 | —4.4 I ELO! 2.457) On URS 6.5 Ze ey © | I.797| 0.05 2.8 I 105 | 2.532; ©.075 gic Dec, 2), x 105 1.67 | —0.127 =F I 105 | 2.406] —o.126) —5.1 gi or Cmte 697 10.027)|/m 11.6 105 105 | 2.436) 0.03 1.2 16} 1 ° 1.605) =0.092| 5.5 I © | 2.284) —o.152| —6.4 aay 110) | 128 0.195] 11.4 I 105 | 2.422| 0.138 5.8 31| 1 10s | 1.688) —o.112| 6.4 I 105 | 2.415| —0.007| —0.2 ee ee) aoe Oh eae ieee . | el eee Ie | “ea |r eee § From From Oct. 22 to Dec. 31 1.531 O23T4) 20.5 Oct. 22 toDec. 31) 2.181 0.468 7a ey | Ada Springer TABLE XXI—Continued No. 3 (Male) FE | el ce =. 2 | 28]. Were ye a ha a Octi§22) | 2n2Q2 28] 1 315 | 2.607 Nov: 4° 4 315 | 2.89 ri] 2 315 | 2.637 18 I 105 | 2.607 25 ° | 2.567 Dec, 2) 1 @) |) Beguy gy) 105 | 2.538 16] 1 oO | 2.493 23) «1 110 | 2.613 eg) a ° | 2.43 From Oct. 22 to Dec. 31 Mean (average 2.331 Increasé Day/s 0.183 —0.253 —0.03 — 0.040 —0.042 0.023 —0.045 0.12 —0.183 Increase fe) -_ Se) oo Per cent inc. increase > A Study of Growth 61 TABLE XXII Ser D! Growth at room temperature (20° C. average). Fed same as Set D | | = Peal eo lee oe i: ie Fotis eh oat 5 - Bia besa |). & Ee es al ee 2 3 3 Se Pe joe < 5 ow Oct) 22|" 10 20 16.924) 1.692 28| 10 20 | 18.534| 1.853 0.161, 9 Noy. 4 10 zo | 18.987/ 1.899 | 0.046, 2.4 11] 10 19 | 18.492) 1.849 | —0.05 | —2.6 18 9 20 | 17.289] 1.921 0.072 3.8 25) 9 20 | 17.429] 1.937 0.016 0.8 Dec. 2} 9 25 17.09 | 1.899 | —0.038] —1.9 9) 9 20 | 16.696) 1.855 | —0.044) —2.3 16, 9 20 | 17.207] 1-912 | 0.057] 3 23| 9 20 | 17.757| 1-973 0.061 g\au ai] = G) 200) 5172268 |05 Ye OLGN) —O-.O54 || 52.7) | > a ~ lecreee lode. are pees RS: = eects ay Ss er From Oct. 22 to Dec.31 | 1.805; 0 226 12.5 No. 2 (Male) No. 3 (Female) 2 2 | ie tae Beh es Leas pao | See ies eo bee : Se eae ec mee St oe | aul é : oem ek bese: mau ts ie 5 Se 6 || ea 3} m fe) ob 2 = 5 =) Ss. |= Se cu Pg eed 5 Ay Oct. 22) ~ x 1.297 I 2.007 28] 1 q1g | 1.592] 0.295) 20.4 I 315 | 2.134| 0.127] 6.1 Nov. 4] 1 Brn el-O79ln | OLOs7 | 5-3 I BTS ee zotoz 028) ifeg) ET ee 315 | 1.697; 0.018) 1. Ti) .3bs5) |) 2.507) —o-o5 5) 225 18] 1 315 | 1.777| 0.08 | 4.6 re 105 | 2.092) —o.oI5| 0.7 i 105 | 1.864! 0.087 4-7 I 105 | 1.982! —o0.110) 5-4 Deco. 2\ x LOG | pt.52) || 0.044) —2,.9 I ° 1.916 —0.066 aes I 105 | 1.947} 0.127) 6.7 I | 1.822] —0.094 Fc HO| eT o | 1.823] —0.124| —6.5 I On |0 0-727" —-O- 08s 4-7 Zale eT | IO | 1.944| 0.121; 6.4 I 10° | 777| O-e4 Finis Ruel TOS ||| a0 89218 —O. 052) —2).7 I o | 1.7 | —0.077 4-4 fetes. || Stv'e zg 2 eS pee aed Sen aia eee From From Weto22 to Dec. 31... - - 1.594. ©.595| 37-3 Oct. 22 toDec. 31) 1.853] 0.307] 16.5 62 Ada Springer TABLE XxTI Ser D? Growth at high temperature (30° C. average). Fed same as Set D. a | o = ad 4, 5 Se iesec ilo ena ne 2 Ee ~S wn = = eigen | x | ¢ z ro) o. oO = i] = ta] Sh RE Ve ee cla TPR 5 2 WE a < 45 a Oct. 22|' 10 | | 15.849| 1.585] 28} 10 35 15.714| 1.571] —o.o14| —0.8 I Nov. 4] 10 29 15-477| 1-548] —0.023] —1.4 II} 10 28 15.537) 1.554) 0.006] 0.3 18| 10 28 15.287| 1.529 —0.025) —1.6 25 || “10 29 15.245) 1.525) —0.004] —0.2 26 | 12.982) 1.442) —0.083| —5.5 1.492] 0.05 3-4 1.453| —0.039| —2.6 1.420| —0.033| —2.2 I 9 | 9 28 13-427 16} 9 29 =| 13.078] 9 9 31 32 — yb ob wo x Ww oo te} -371| —0.049| —3-5 (average) Mean From Oct. 22 to Dec.31 1.478| 0.214) 14.4 No. 1 (Male) No. 2 (Female) Ely cee : yin flee Mae ee a % | oo) = ieee 8 s wo 5 ‘D 5) bs S mS | 1s 3 5 elie heal bec coe 2 |= |F |] a | @ Ort 22) ar 1.037! I | 1.389 28] 1 315 | 1.047] 0.01 0.9 I 315 | 1.484) 0.095 6.6 Nov. 4] I 315 | 1-014] —0.033) —3-2 I 315) || -432|" —0-052|eaaes LL|) ot 110 | 1.044) 0.03 2.9 I 315 | 1.462] ‘oLog 2 18} 1 105 | 1-012) —O.032| — 3°15 I 105 | 1.547] 0.085 5.6 25) a © | 0.967) —0.045) —4.5 I 105 | 1.637; 0.09 5.6 Dec. 2] 1 105 | 0.947| —0.02 | —2. I 105 | 1.548) —0.089} —5.5 g) 1 o | 0.962} 0.015] 1.5 I 105 | 1.589) 0.041 2.6 16; 1 © | 0.88 | —0.082| —8.9 I ° 23 I 110 | 0.918) 0.038) 4.2 I 105 | 1.479] —O.110] —7.1 30) 105 | 0.915] —0.003! —0.3 I 105 |- 1.423) —0;O56|Ne aes | S e = | Si Z 8 4 3 § 3 2 Ss § © bs e | iS cies = 3) 9 eee From From | Oct: 22 to Dec) 31:..2~2 0.976] 0.122] 12.5 Oct. 22 toDec. 13) 1.406} 0.034) 2.4 A Study of Growth TABLE XXIII—Continued No. 3 (Male) E | cS) eee ailtem Vira tla a | Octar22\ 1 1.677| 28) 2 | 355) | 1-637) —o.04, || =2-4 Nov. 4, 1 | 315 | 1.517| —o.12 | —7.6 15 | me 325, | \n2-$37\ 9-02 i) 18} IIO | 1.524] —o0.013] —0.8 Zee ed ° (| 1.442] —0.082] —5.5 Dec 2) Oo | 1.387} —0.055) —3.8 Chl a © | 1.408) 0.021 ish 16] 1 © || 12425)" 0.007) tora 23D IIO | 1.419) —0.006) —0.4 31; =I OG) | 153045 onrr ce Sea “> | 2 as 8 % 2 S 4 PY From Oct. 22 to Dec. 31 1.49 | 23738) 25 A da Springer TABLE XXIV Ser E} Growth at 20° C. (average). Fed as much as animals would eat eet U boise] 20d em F (ea el oe ae z | sos © a + qe) esi ea an ae | : Es s o 2 g So cS) o 2 a | L | G & a < = & Oct. 22) 10 | 17-377) 12798 28} 10 20 22.126} 2.213] 0.475) 24 Nov. 4) 10 20 21.756 2.176 —o 037} —1.6 | In| 16 4 20 22.356) 2.236, 0.06 2.7 18} 10 19 | 23.899] 2.39 | 0.154) 6.6 25) 10 20 25.364 2.536, 0.146, 5.9 Dec. 2] 10 20 25.89 | 2.589, 0.053) 2 9| 10 21 | 26.637] 2.664] 0.075) 2.8 16, Io 20 27-377| 22739|| (G-074) 227 23| 10 20 | 28.04 | 2.804 0.066 2 3 31, 9 20 | 26.05 | 2.89 | 0.086] 3 “~S alae o = mu Fe) aes From Oct. 23 to Dec. 31 | 2.314] 1.152] 49.7 No. 1 (Male) No. 2 (Male) wae | = | | | . s heel | | 2 Biles sz || & i rahe rN met mies Sea ares 2 | oe [oe S| 2 ge fi) Tae 3 128 8) & 3 Pe eo le 1a, es ; oo 8 ‘3 3 eels oie a 2 |a~| & ne Oct. “al I 2.307 | I i Ds527 28 I 630 | 2.407 | O.1 | 4.2 I 630 | 1.854 0.327 Nov. 4| 1 630 | 2.442 | 0.035 | 1-4 I 630 | 2.017 0.163 LHe AE 525 | 2.494 | 0.052 2.1 I 525 | 2.047 0.03 18) I BRA 26278) Ona Sek I | LPN Weary) 0.21 25) S25 al) 2.855 0.228 | 8.3 | 420 | 2.424 0.167 Deca 2) 1 420 | 2.911 | 0.056 1.9 I 420 | 2.476 0.052 gies 525 | 3-003 | 0.092 3.1 I 420 | 2.53 0.054 16) I 2716 ||\) 32039) |) (0-03 0.9 I 110 | 2.649 ©.119 24) 1 315) |) 3-108) ||| 0.075 2.4 I 315 | 2.58 | —0.069 31| 1 315 | 3-157 | 0.049 Het I 315 | 2.628 0.048 oo 2 “oe 2 From | From Oct. 22 to Dec. 31 ..| 2.732 | 0.850 Per cent inc. _ - yw YN OO HH COO DAN HF He HN PW increase ghich Oct. 22-Dec. 31] 2.077 1.101} §3.05 A Study of Growth TABLE XXIV—Continued No. 3 (Male) alae z Bisel x : é SAS: clea aeaner 2 a a == 4 Ay Oct. +22 I 1.507 28) x 630 | 2.242 0.735| 39.2 Noy.) 4a 4 630 | 2.307 0.065; 2.8 Te 525 | 2.192 | —o.115] —5.1 18} 1 525 | 2.297 0.105} 4.6 Za 525 || 2.687 0.39 | 15.6 Decwr2 I 420 | 2.510 | —o.177| —6.8 9} 1 SS ag22 0.012) 0.4 16] 1 105 | 2.635 Slgiuigh| ve) 772) 315 | 2.437 | —o.198] —7.8 Attila (died) Be od 5 § a a 8 From Oct. 22—Dec. 31 1.972 | 0.930 | 47.1 66 Ada Springer TABLE XXV SED E Growth at 30°C. (average). Fed as in E} a ea ne i he | con ee = Bre 8) bea) eed Sel Oo Oo. of = ) a Oo Be 18 tear Sea ai 5 yA A < 4 oe Oct. 22| 10 16.899] 1.69 28) 10 35 D7etGe | Le 751 mOPO2s) waka g Nov. 4) 10 29 17.661] 1.766] 0.051] 2. II} 10 28 18.227] 1.823] 0.057] 3-1 18! 9 28 18.669] 2.074; 0.251; 12.8 25, 9 29 19.967| 2.218} 0.144) 6.7 Dew 2|/ a9 26 20.002] 2.222) 0.004) 0.1 9 8 28 18.864) 2.358) 0.136, 5.9 16] 8 29 19.406] 2.426] 0.068) 2.8 23) 8 31 19.49 | 2.436] 0.01 0.4 31, 8 32 18.120] 2.265] —0.171| 7.2 ; => fi &| 2 a 3 os By = o b is From Oct. 22 to Dec.-21 1.977] 9.575| 29.07 No. 1 (Male) No. 2 (Male) 2) S | | a ae ] = = 5 = ee TBs g a S oie ca (ee g a fg ay tae ge a 3/35) S) 2 6 os a iS) B S) ao 2) 8 mB A | es S 4 < a | es e | 4 a Oct. 22] 1 2.805 I 1.539) 28] «I 420 | 2.627) —0.178) —6.5 I | ‘420 .|°1.582] ~ olo4g ee Nove 4) S40 | p27 7) n= OLOGu |e ag 1 | 840 | 1.582] 0.000; II I §25 | 2.687] o.110) 4.1 tM. 525° || 2.677) sO: 0gn mEpES jited fa 630 | 2.93 0.243} 8.6 | 630 | 1.862} 0.185) 10.4 734 Me Gan 1 We sd oe 5 Re 1 | 315 | 2-887) of0z5| es Dec. 2} 1 | 420 | 3.003] —0.082} —2.6 I 420 | 2.05 0.163} 8.2 9g] x 525 | 3-245] 0.242, 7-7 I 420 | 2.07 0.02 0.9 16] 1 315 3-327) 0.082) 2.4 I 315 | 1.977) —0.093] 4.5 23} 1 | 315 | 3.288] —0.039) —1.1 I 315 | 1.97 | —0.007} 0.3 Bil | You | apis el lsetew ei | Fol iol re) I 315 | 1.937]. —O:03a|mmEIeG “> o | + a ‘os eo + | 2 8 - = 3 - Pe] a | e8 7 =| 3 ee From From Oct. 22 to Dec. 31....| 2.96 0.311] 10.5 Oct: 27—Dec. 31 | 1-738) 0-398) 2228 A Study of Growth TABLE XXV—Continued No. 3 (Female) ei cr leat | ee) 0 | 6 a0 a is) | 2 ay S 5 ow Oct. 22| I 1.172 28) 1 420 1.322] 0.150) 12. Nov. 4 I 840 | 1.412| 0.090/ 6.5 II) 1 §25 | 1.507] 0.095} 6.5 18 I 630 | 1.724| ©.217| 13.4 25 |e ad 315 | 1.987} 0.263) 14.1 Decs 92 I AZONs|e2 2002!) 07/5] nae Oa 205) | 2.210)) | O.148|) 6.9 16} 1 me) |) AoriGel! Choke) Pal 23, I 110 | 2.140] —0.013] —0.6 31; 1 AT 2016) —-O-O2 Aiea te From Oct. 22 to Dec. 31 | 1.644) 0 944) 57-4 68 Ada Springer TABLE XXVI Ser F Growth at 30° C (average). Fed as much as an mals would eat. } @ = | o r) > oo 5) Ee om Pecos ee r= = = 2 Ss S seg ok?) Ss re) ef A ete Ace eh ee 5 See we: || os ae : . Z = = < 5 am Oct. 22] 10 16.606) 1.661 28 10 35 17.449] 1.745) 0.084, 4.9 Nov. 4| 10 29 18.324) 1.832) 0.087) 4.8 In] <9 28 | 17-679] 1.964] 0.132) 6.9 | 8 28 16.564] 2.071 0.107, 5.3 25) 8 29 ~| 17.092] 2.137, 0.066) 3.1 Dec. 2) 8 26 | 17.919] 2.239] 0.102) 4.6 . g 8 28 18.751] 2.344] 0.105) 4.5 16] 7 29 ©| 16.265] 2.323] —o.021] —o.9 : 23) 7 Bt |) 15. 760l 22521) O-O7nl one Bre 7 Q2 Weta 962|)2-487\"—Onnts|y Gee Se |e Noe 8 From Oct. 22 to Dec. 31 1.899 | 0.476 25.06, No.1 No. 2 ZI | oF =| z é ‘oD os 3 2 a S 42 ga SS 2 z a | a 2 = 3 (3 8| & 2 g soles obs Sa faba S S S ee 5) 5 z tS) os 3S 5 Zi Wren. aa ee 5 a Peon | P= eee eg 5 ay Oct. 22] 3 1.107 | I | 1.632 28] =I 420 | 1.417 0.310) 24.5 I 420 | 1.627 | —0.005] —0.3 Nov. 4] I 525 | 1.389 | —0o.028| —1.9 I 525 | 1-747 0.120] 7.1 ed 2G hoki 0.123} 8.4 I 420 | 2.612 0.865 39.6 18] 1 315 | 1.677 | 0.165) 10.3 I 420 | 2.735 0.123) 4.6 25) X (eA SE tea hh) 0.110] 6.3 I 525 | 2.855 0.120] 4.2 Dee: .2\0 17] 420) |r -912 O.125|) = 6.7 I 420 | 2.872 0.017; 0.5 9} 1 525 | 2.002 0.090} 4.5 I 420 | 2.992 0.120) —4. 16] 1 315 | 2.069 0.067} 3.2 I 315 | 2-945 || —0.047) es eA 415 | 2.033; | —0.036)— 127 I 315 | 2-854) —0-ogne aad eal 315 | 1.978 | —o.055| —2.7 I 315 | 2.674 | —o.180| —6.5 | ae tet likes .¢| 2 See is Ss o 3 ey s og | = 8 £ ae s 5] § . 8 pr Se, ced eae &|. 4 Sia From From | Oct.22 to)Dec.31..|. “12542 | (0287x5624 Oct.22-Dec. 31| 2153 1.042} 48.3 STUDIES ON CHROMOSOMES IV THE “ACCESSORY” CHROMOSOME IN SYROMASTES AND PYRROCHORIS WITH A COMPARATIVE REVIEW OF THE TYPES OF SEXUAL DIFFERENCES OF THE CHROMOSOME GROUPS! BY EDMUND B. WILSON Wiru Two Pirates anp Two Ficures IN THE TEXT Since the unpaired idiochromosome (“accessory chromosome’’) was first discovered by Henking (’g1) in Pyrrochoris apterus L. this species has been reéxamined by only one observer, Dr. J. Gross (07), with results that are in substantial agreement with those that pe had reached in an earlier investigation (’04) on the coreid species Syromastes marginatus L. In both cases his conclusions hre in conflict with the view advanced by McClung (’o2), and first 1 Terminology. With the advance of our knowledge of the chromosomes that form the distinctive differential between the chromosome groups of the two sexes, and between the male producing and the female producing spermatozoa, it becomes increasingly difficult to find a common name that will apply equally to their various modifications. Terms such as the ‘‘accessory,” ‘‘odd” or “‘heterotropic” chro- mosome, or ‘‘monosome,” that are based on the condition of these chromosomes in the male only, are misleading or inappropriate; and some of them are in certain cases inapplicable, even in the male— e. g., in Syromastes, where the ‘‘accessory” chromosome is not univalent but bivalent. Such terms as “heterochromosome” or ‘‘allosome” (Montgomery) seem to me unsatisfactory, since they designate the m-chromosomes as well as the differential chromosomes, though these are obviously of quite different nature. Since it has now become evident that a univalent ‘‘accessory” chromosome in the male is exactly equivalent to what I have called the ‘large idiochromosome”’ in other forms, I think these chromosomes should be designated by the same name, and one that will apply equally to both sexes. While there are some objections to the word ‘‘idiochromosome” as a general term for this purpose I am not able to suggest a better one; and since it has already been thus employed by some writers, I shall use it hereafter in a broader sense than that in which I first proposed it, to designate the differential chromosomes in general, whether they are paired or unpaired in the male, and whether one or more pairs are present. A univalent or odd idiochromosome in the male will be called the unpaired idiochro- mosome (or simply the idiochromosome), while the word ‘‘heterotropic’” may perhaps conveniently be used as descriptive of its passage without division to one pole in one of the maturation divisions. In Syromastes, as will appear, the ‘‘accessory” or heterotropic chromosome represents a pair of idiochro- mosomes; while in Galgulus there are several pairs of these chromosomes. Tue JourNAL oF ExPERIMENTAL ZOOLOGY, VOL. VI, NO. I. 70 Edmund B. Wilson shown to be correct in principle by the work of Stevens and my- self, that half the spermatozoa are male producing and half female producing. This view rests on the following facts. When the male somatic chromosome groups contain an odd number, includ- ing an odd or unpaired idiochromosome (as in Anasa, Alydus, or Protenor) the female groups have one more chromosome, being duplicates of the male groups with the addition of another chro- mosome like the unpaired one of the male. When the male groups contain an even number, including a large and a small idiochro- mosome (as in Lygzeus, Coenus or Tenebrio) the female groups contain the same number, but include two large idiochromosomes in place of a large and a small one. In the first type half the spermatozoa receive the odd idiochromosome while half do not, the former accordingly containing one chromosome more than the latter. In the second type all the spermatozoa receive the same number of chromosomes, but half receive the large idiochromosome and half the small. It follows from these rela- tions that eggs fertilized by spermatozoa containing the odd chro- mosome, or its homologue the large idiochromosome, must pro- duce females, those fertilized by the other spermatozoa males. These cytological results, first reached by Stevens (’05) in Tene- brio (which has a pair of unequal idiochromosomes in the male) and myself (’o5b, ’o5c, 06) in Anasa, Protenor, Alydus and Harmostes (which have an unpaired idiochromosome in the male) and in Lygzeus, Coenus, Podisus and Euschistus (which agree essentially with Tenebrio), have since been confirmed in a con- siderable number of species and extended to several other orders of insects.2, They have recently received indirectly a_ striking experimental confirmation in the important work of Correns (’07), which proves that in the dicecious flowering plant, Bryonia dioica, the pollen grains are likewise male determining and female deter- mining in equal numbers. Gross’s conclusion in the case of Syromastes and Pyrrochoris is opposed to all these results in that only one of the two forms of spermatozoa is supposed to be functional (those containing the 2 See the tabular review in the sequel. _~— | Studies on Chromosomes 7% “accessory” chromosome) the others being regarded as in a certain sense comparable to polar bodies (as was also supposed by Wallace (705). This result was based mainly on the numerical relations, and especially on the belief that in both these forms the number of chromosomes is an even one and the same in both sexes—twenty- two in Syromastes, twenty-four in Pyrrochoris. Since the com- plete reduced number (eleven and twelve in the two respective cases) is present only in those spermatozoa that contain the “accessory” chromosome, Gross argues that this class alone can be concerned in fertilization, as follows: Syromastes....Egg 11 + spermatozoén 11 = 22 (d'or 2) Pyrrochoris ...Egg 12 + spermatozoén 12 = 24 (d'or 2) whereas in Anasa or Protenor the relations are: Anasaise. 7). Egg 11 + spermatozoén 10 = 21(c) Egg 11 + spermatozoén 11 = 22( 2) Protenor....Egg 7 + spermatozoén 6 = 13(c) Egg 7+ spermatozoén 7 = 14( 2) In the hope of clearing up this perplexing contradiction | endeavored to procure material for a reéxamination of the two forms in question, and through the great kindness of Professor Boveri, to whom my best thanks are due, was fortunate enough to obtain an abundant supply of both, though unluckily it includes no female material.‘ As far as the relations can be worked out on the male alone they give, I believe, the solution of the puzzle and bring the two species in question into line with the general princi- ple that has been established for other forms. ‘This is evidently true of Pyrrochoris. Syromastes, however, constitutes a new 3 At first thought this seems to be in harmony with the remarkable discovery of Meves (’03, ’07) that in the male honey bee actual polar bodies are formed which produce abortive spermatids. Butobviously the two cases are not parallel, for in the bee the fertilized eggs produce only females; and this finds a natural explanation, in accordance with the general conclusions of McClung, Stevens and myself, in the assumption that it is the male producing class that degenerate as polar bodies. 4 The material, fixed in Flemming’s fluid and in Bouin’s picro-acetic-formol mixture, is of excellent quality and gave preparations of perfect clearness. The Flemming material is on the whole the best. For single stains Zwaardemaker’s safranin and iron hematoxylin were employed (the latter especially for photographs). Various double stains were also used. One of the best, which I can strongly recom- mend to other workers in this field, is the combination of safranin and lichtgriin, which gives prepara- tions of admirable clearness and is also easy to use and certain in its results. 72 Edmund B. Wilson type that is not yet known to be exactly paralleled in other forms; though, as will appear, the genus Galgulus presents a somewhat analogous case. It does not seem to have occurred to Dr. Gross (as it did not to me until I had carefully studied both forms) that Syromastes and Pyrrochoris might be of different type, but such is evidently the case. I shall endeavor to show that Pyrrochoris is of quite orthodox type, having an odd somatic number in the male and a typical unpaired idiochromosome. Since I am compelled to differ with Dr. Gross in regard to this species, | am glad to admit that the doubts I formerly expressed as to his account of the spermatogonial number in Syromastes, were unfounded. In regard to the female number, on the other hand, I believe he was misled by a wrong theoretic expectation (as he evidently was in case of the male Pyrrochoris), though it is possible that his determination of the apparent number was also correct, as indicated beyond. SYROMASTES MARGINATUS L. Gross’s account of this form was as follows: The somatic groups in both sexes are stated to show twenty-two chromosomes. The ‘accessory’ chromosome arises by the synapsis of two spermatogonial chromosomes, and is therefore a bivalent. It divides equationally in the first spermatocyte division but fails to divide in the second, passing bodily to one pole in advance of the other chromosomes without even entering the equatorial plate. All of the spermatid-nuclei thus receive ten chromosomes and half of them in addition the “accessory.”’ ‘These are the essen- tial conclusions; but they are complicated by the following singular view of the relations between the “accessory”? and the micro- chromosomes or “7m-chromosomes.”’ ‘The chromosome nucleolus of the growth period is supposed not to give rise (as it does in Pyrrochoris and other forms) to the heterotropic or “accessory” chromosome of the spermatocyte divisions, but to the m-chromo- some bivalent—the same view as the earlier one of Paulmier (99) which has since been shown to be erroneous (Wilson ’o5c). But, on the other hand it is believed to arise, not from the Studies on Chromosomes vie: m-chromosomes of the spermatogonia, but from two larger chro- mosomes, while the spermatogonial m-chromosomes are supposed to be converted into the “accessory” (!). I will not enter upon the very ingenious, if somewhat fantastic, conclusions that are based on these results, for, as I shall attempt to show, the results them- selves cannot be sustained in some important particulars. But apart from this I am glad to be able to give the most positive con- firmation of Gross’s interesting discovery in regard to the numer- ical relations in the male. Syromastes 1s indeed a case inwhich the spermatogonial number 1s an even one (twenty-two), while there 1s a heterotropic chromosome in the second division. Half the sperma- tozoa seem to receive ten chromosomes and half eleven, as in so many otherspecies of Coreidz. But as Gross also correctly de- scribed, the heterotropic chromosome is here a bivalent which represents two chromosomes united together. The true numbers characteristicof the two classes of spermatozoa are therefore ten and twelve, respectively. For the sake of clearness I will here point out that this becomes at once intelligible under the assump- tion that the female number is not twenty-two, as Gross believed, but twenty-four; and such I believe will be found to be the fact. That Gross was mistaken—doubtless misled by the earlier conclusion of Paulmier (’99), in which he was at first followed by Montgomery (’o1)—in supposing that the chromosome nucleolus of the growth period divides to form the m-chromosomes, is I think thoroughly demonstrated by my preparations. In the case of Anasa and Alydus I showed (’o5c) that the m-chromosomes are not formed in the way Paulmier believed, but arise from two small separate rod-like chromosomes that are in a diffused condition during the growth period and only condense to form compact bodies at the same time that the condensation of the larger chro- mosomes takes place. I have since found this to be true of many other species. It is confirmed in the case of Anasa by the smear preparations of Foot and Strobell (’07), and I have also since fully established the same conclusion by this method, by means of which every chromosome in the nucleus may be demonstrated.° 5 This is opposed to the conclusion of Montgomery (’06). 74 Edmund B. Wilson Although I have no smear preparations of Syromastes it is perfectly clear from the sections that the facts are the same here as in Anasa Alydus, and other forms. In the early prophases of the first divi- sion (at a period corresponding to Gross’s Figs. 31 to 37) when the plasmosome has disappeared or is greatly reduced in size, the nuclei contain both the chromosome-nucleolus and the m-chro- mosomes. ‘This is shown in great numbers of cells with unmis- takable clearness and after various methods of staining, particu- larly after safranin alone or combined with lichtgriin. In the early part of this period the chromosome nucleolus is at once recognizable by its intense color and sharp contour and is not for a moment to be confused with a plasmosome. The ordinary bivalents are still in the form of ragged pale bodies, having the form of longitudinally split rods or double crosses. The m-chro- mosomes have the same texture and staining reaction, but are much smaller and never show the cross form. While it is diff- cult to show the facts to demonstration in photographs of sections they may be fairly well seen in the following. Photo 18 shows the chromosome nucleolus (not quite in focus,) one of the large biva- lents (two others barely appear) and both m-chromosomes. Photo 1g 1s a similar view (the m-chromosomes more condensed), while Photo 20 shows the m-chromosomes and three of the ordi- nary bivalents. The succeeding changes must be rapidly passed through, since the successive steps are often seen in the same cyst, passing from one side to the other. In these stages the large bivalents rapidly condense and regain their staining capacity, finally assuming a bipartite or quadripartite form. The m-chro- mosomes undergo a similar condensation, being finally reduced to ovoidal or spheroidal bodies. The chromosome nucleolus, on the other hand, becomes somewhat looser in texture and assumes an asymmetrical quadripartite shape, in which form it enters the equatorial plate to form the eccentric “‘accessory” chromosome. The period at which the m-chromosomes condense varies consider- ably, and the same is true of their relative position; sometimes they are In contact, sometimes more or less widely separated, even lying on opposite sides of the nucleus. Photo 21 shows two nuclei, one above the other, in each of which appear both m-chromosomes, Rt ie @ Studies on Chromosomes 75 the chromosome nucleolus and a number of the other bivalents. Photo 22 shows the same condition. Photo 23, from the same cyst, is slightly later, showing the two spheroidal m-chromosomes wide apart, the chromosome nucleolus, and several of the other chro- mosomes. (The chromosomes nucleolus, perfectly recognizable in the preparation, is in the photograph hardly distinguishable from the other bivalents seen endwise.) Up to this point, which shortly precedes the dissolution of the nuclear membrane, the chromosome nucleolus is still immediately recognizable by its deeper color (especially after safranin). There follows a brief period in which this distinction disappears, but the chromosome nucleolus is still recognizable by its asymmetrical form. That it gives rise to the eccentric “‘accessory”’ is, | think, beyond doubt. The evidence is demonstrative that it does not divide to form the m-chromosome¢s, and-that the latter arise from separate rods as described. Gross appears to have seen these rods at a much earlier period (cf. his Fig. 10) and correctly identifies them with the spermatogonial m-chromosomes; but he believed them to give rise to the “accessory.” The relation of the chromosome nucleolus to the spermato- gonial chromosomes cannot be determined in Syromastes with the same degree of certainty as in Pyrrochoris (as described beyond), but the size relations leave hardly a doubt that Gross was right in asserting its origin from two of the larger of these chromosomes. The study of these relations is of importance because I believe they justify the conclusion that the chromosome nucleolus, and hence the “accessory,” is nothing other than a pair of slightly unequal idiochromosomes, which can readily be recognized in the spermatogonial groups. Study of the spermatogonial groups in detail shows that twenty of the chromosomes may be equally paired, while the remaining two are slightly but distinctly unequal in size. These can always be recognized as the smallest of the chromosomes except the m-chromosome. Photos 1 and 2 show two groups in which this clearly appears. These photographs are reproduced in Text Figs. Ia, 1b, with two others, c and d, the chromosomes in question being designated as | and 7. 76 Edmund B. Wilson / It is evidently this pair that give rise to the bivalent “accessory”’ (eccentric) chromosome of the first division and hence to the chromosome nucleolus of the growth period. Gross correctly describes this bivalent as a quadripartite body or tetrad, but overlooked the fact that it is composed of two slightly unequal halves, and these correspond in relative size to the unequal pair in the spermatogonia. This appears unmistakably in a great number of polar views of the first division metaphase (though it is not always apparent) and is clearly shown in Photos 3, 4 and 5- It is evident that the bivalent is so placed in the equatorial Cc Fic. 1. Four spermatogonial chromosome-groups of Syromastes marginatus; a and 6 are reproduc- tions of Photos 1 and 2.* * The drawings are not made from the microscope with the camera lucida but directly upon enlarged photographs of the objects. Since I believe this method to be superior in accuracy for the representa- tion of such small objects I will briefly describe it inthe hope that others may find it useful. The original negatives are taken directly from the sections at an enlargement of 1500 diameters (2 mm. oil immersion, compensation ocular 6). From these negatives enlarged bromide prints are made (with a photographic camera) three times the size of the original negatives (1. e., 4500 diameters) upon double weight paper, which givesa good surfacefor pen drawings. The drawing is then made directly on the print with waterproof ink, and when thoroughly dry the remains of the photograph are bleached out in a mixture of sodium hyposulphite and potassium ferricyanide. The enlarged prints of course show the chromosomes with more or less blurred outlines (though they are clearerthan might be supposed); but by working with an ordinary print and the object before one for comparison the drawings may nevertheless be made with great accuracy. They may be tested and if necessary corrected, by the use of a reducing glass. Studies on Chromosomes Th plate as to undergo an equation division, like the idiochromosomes of other Hemiptera heteroptera. In uniting to form a bivalent before the first division these chromosomes differ from those of most other Hemiptera, but in all other respects up to the end of the first division they correspond exactly with them. But even this difference is bridged by a condition occasionally seen in other forms, for instance in Lygeus and Metapodius.* In the last named form the typical and usual condition is that the idiochro- mosomes are in the first division quite separate, lying eccentrically outside the principal ring of chromosomes like the unpaired idio- chromosomes of other coreids (Photos 6 and 7), and in this posi- tion they separately divide. Exceptionally, however, they lie in close contact (Photos 8 and g), forming an asymmetrical bivalent precisely like that of Syromastes. In both cases this bivalent divides equationally, giving two asymmetrical daughter-dyads, thus i. The exactness of the correspondence up to this point seems to leave no doubt of the homology of this pair of chromosomes in the two forms. In the second division, however, the two species show a remarkable contrast. In Metapodius, as in Lygzus or Euschis- tus, the two idiochromosomes are always united to form an unsym- metrical bivalent which enters the equatorial plate and is separated into its two components, half the spermatids receiving the large one and half the small. In Syromastes, on the other hand, the idiochromosomes remain united and do not enter the equatorial plate at all, but pass directly to one pole where they are included in the daughter-nucleus, as Gross has described (Photos 11 to 17). Owing to this behavior of the idiochromosome bivalent, polar views of the second division always show but ten chromo- somes instead of eleven (Photo 10). In this case therefore half the spermatid nuclei receive two more chromosomes than the others, the two classes having respectively ten and twelve chromo- somes. As the idiochromosome bivalent passes to the pole its two components are usually closely united, and often cannot be 6 The latter remarkable genus, which presents the phenomenon of the ‘‘supernumerary chromosomes” (Wilson ’o7c), will form the subject of a forthcoming fifth “Study.” 78 Edmund B. Wilson distinguished; but in some cases they may still be seen, as in Photo 12. As the nuclear vacuole forms the ordinary chromosomes rapidly diminish in staining capacity, while the idiochromosome bivalent retains its compact form and dark color, like a nucleolus, and thus comes conspicuously into view, particularly after safranin. Its double nature is at this time often more clearly apparent than in the preceding stages. It disappears from view some time after the reconstruction, at a much earlier period than in Pyrrochoris. Only exceptionally in my preparations do the chromosomes of the second division show a quadripartite form as Gross figures them. Their usual form is dumb-bell shaped or dyad-like; though as the two halves separate they are often connected by double fibers, as is the case with many other species of Hemiptera. PYRROCHORIS APTERUS L. As already stated, Pyrrochoris is of different type from Syro- mastes and agrees precisely with other forms having an unpaired idiochromosome, such as Anasa or Protenor. Aside from the interest that this species possess as the one in which Henking first discovered the idiochromosome, it is in other respects a peculiarly interesting form for the study of the general spermatogenesis, particularly in respect to the presynaptic and synaptic periods. I shall here, however, confine myself mainly to the numerical relations and the history of the idiochromosome. Henking him- self somewhat doubtfully concluded that the spermatogonial number was twenty-four: “Ich habe in drei Fallen die Zahl 24 erhalten, in einem Falle die Zahl 23. Da die Bilder tiberall die gleichen sind, so habe ich das Zahlgeschaft nicht an einer grosseren Zahl vorgenommen und glaube die theoretisch zu erwartende Zahl 24 als das Normal ansehen diirfen” (op. cit., p. 688, italics mine). It is clear enough from this that Henking, too, was misled by a false theoretic expectation; and a study of his figures (op. cit., Figs. 6, a, b. c, 7) will show that they are very far from decisive. In the case of the female, Henking speaks much more positively (92) and there is hardly a doubt that his count of twenty-four chromosomes was correct, since he found this number “ unverkenn- ——_—— J Studies on Chromosomes 79 bar” in the dividing odgonia, and in the connective tissue cells of the ovary, and also figured (Fig. 39) a double group (exactly such as I described in Anasa, Wilson ’06, Fig. 2, ), showing forty-eight chromosomes. Gross accepts Henking’s account without question, treating the numerical relations in rather summary fashion as follows: “Die Aequatorialplatte der sich teilenden Spermatogonien enthalt 24 Chromosomen. Dieselbe Zahl hat Henking ausser in den Sperma- togonien auch in den Oogonien gefunden. Ebenso konnte ich in den Follikelzellen der Eirohren, also in somatischen Zellen, kon- statieren. 24 ist also die Normalzahl der ppecies’ (07... ps2 77)- In support of this are given two polar views of spermatogonial metaphases (the female groups are not figured) each showing eight small and sixteen large chromosomes (Figs. 9 and 10). His ac- count continues as follows: The idiochromosome appears ageady in the syanaptic period (synizesis) as a double nucleolus-like body, assumed to be a bivalent body that arises by the synapsis of two of the spermatogonial chromosomes, though none of the earlier stages were followed out. At a later period it appears as a single spheroidal body owing to the close apposition of its two halves. This chromosome divides in the first spermatocyte division, but in the second lags behind the others and passes undivided to one pole, as Henking described. All of the spermatid-nuclei thus receive eleven chromosomes, while half of them receive in addi- tion the idiochromosome. Since both sexes were supposed to con- tain twenty-four chromosomes, Gross drew the same conclusion as the one previously reached in the case of Syromastes, namely, that only the twelve-chromosome spermatozoa are functional. In regard to the spermatocyte divisions my own results are perfectly in accord with Henking’s and Gross’s. As to the spermatogonial number, I must say that after having immediately confirmed Gross’s account of Syromastes (which I examined first) I was fully prepared to find a similar relation in Pyrrochoris. It was therefore with astonishment that I found everywhere twenty- three instead of twenty-four spermatogonial chromosomes. This number appears with diagrammatic clearness in a great number of spermatogonia from different individuals (testes from 35 different 80 Edmund B. Wilson individuals have been sectioned) and is shown both in camera drawings and in photographs. Eight of the latter are snown (Photos 24 to 31), and these same groups are also represented in the drawings, Text Figs. 2, a, b, c, d, e, 7, k, 1, together with four others (7, g, h, 1), also from photographs. Inspection of these photographs and drawings will show that the unpaired idiochro- mosome Is at once recognizable by its large size, which renders 7 & & Fic. 1. Spermatogonial groups of Pyrrochoris apterus (drawn on photographic enlargements, as explained under Fig. 1); a, b, c, d, e, j, k and / are reproductions of Photos 24, 25, 26, 27, 28, 29, 30 and 31, respectively. it almost as conspicuous as in Protenor (heretofore described by Montgomery and myself). I find the size relations not quite the same as Gross describes them. There are, as he states, eight chromosomes that are considerably smaller than the others; but two of the others are but slightly larger. The remaining twelve paired chromosomes are much larger, though the contrast is in my material not so great as Gross figures it. The idiochromo- Studies on Chromosomes 8I some is nearly twice as large as any of the others, and is obviously unpaired. I have examined a large number of spermatogonial groups with great care with a view to the possibility that this chro- mosome might in reality be double, but am thoroughly convinced that such is not the case. This is unmistakably evident when this chromosome has the form of a straight or only slightly curved rod (Photos 24 to 28, Text Fig. 2, a to z), and these constitute the great majority of observed cases. I have, however, found a few cases where it has a very marked sigmoid curvature; two or three of these give at first sight the appearance of two chromosomes in contact (Photos 29, 305 313 Wext-HieJa57, k/). ~Evem here clase study shows that it is a single body; but such forms might readily mislead an observer having a preconceived idea of the number to be expected. That this is a single chromosome that is identical with ‘the idio- chromosome of the growth period and the maturation divisions is placed beyond doubt by a study of the presynaptic stages, which were not examined by either Henking or Gross. ‘This period is of such interest in Pyrrochoris as to merit a special study. With only a single exception I know of no other form in which the history of the idiochromosome and the succession of the stages can be so completely and readily followed at this time. Throughout this whole period, beginning with the telophases of the last sperma- togonial division, the idiochromosome can be traced step by step as a single body, and it is evidently identical with the large un- paired spermatogonial chromosome. In the stages that immediately follow the last spermatogonial telophase (Photos 32 and 33) the chromosomes still retain their boundaries, though they show a looser texture, vaguer outlines and diminished staining capacity (by which characters the post- phases are readily distinguishable from the prophases). ‘The large chromosome (idiochromosome) is clearly distinguishable at this time, both by its size and by its deeper color. In the stages that immediately follow a remarkable contrast appears between this chromosome and the others. ‘The latter rapidly lose their visible boundaries and their staining capacity, breaking up into a fine net- like structure in which traces of a spireme-like arrangement may 82 Edmund B. Wilson sometimes be seen. The idiochromosome, on the other hand, retains its identity and deep color and now appears as a conspicu- ous elongated body (“caterpillar stage’). Though its outlines are still somewhat ragged and its color less intense than in the succeeding stages, it already appears in sharp contrast to the pale reticulum (Photos 34 and 35). It sometimes extends straight across the whole diameter of the nucleus; but beside such forms, in the same cysts, are often curved and shorter forms. At this time it is usually surrounded by a distinct clear space or vacuole, as I hope the photographs may show; and there are also in the nucleus from one to three much smaller nucleolus-like bodies which (on account of the staining reactions) I believe to be plas- mosomes, but these soon disappear. Splendid pictures of these and the following stages are given by the safranin-lichtgriin combination, which shows the idiochromosome at every stage bright red, while in properly differentiated preparations the reticu- lum is pure green.? The idiochromosome now takes up a pe- ripheral position and the clear space surrounding it disappears. It acquires a more definite contour, stains still more intensely, and rapidly shortens until it is converted into a condensed ovoidal or spheroidal chromosome nucleolus that may be traced without a break through every stage up to the prophases of the first sperma- tocyte division. As it shortens it may undergo a variety of form changes. In what I regard as the typical process it shows no indication of duality at any period up to the full contraction phase (synizesis) being progressively reduced to a short rod and finally to an ovoidal or spheroidal body (Photos 36 to 42). In the mean- time the nuclear reticulum contracts more and more, usually towards one side of the nucleus, becomes coarser in texture, and increases in staining capacity, until at the climax of the process 7 The effect of this stain depends in some measure, of course, on the relative degree of extraction of the two dyes. My method is to stain in safranin for two to four hours and then to place the slide at once in strong alcoholic solution of lichtgriin for ten to twenty seconds. This is at once followed by rapid washing in 95 per cent and absolute alcohols. The alcohol is then replaced by clove oil and the latter by xylol. In all cases the chromosomes of dividing cells and the chromosome nucleolus of all stages appear brilliant red, the achromatic fibers and general cytoplasm pure green. The relative intensity of red and green depend on the length of immersion in the green solution. The description here given applies to sections rather strongly stained in the green. Studies on Chromosomes 83 it forms a close knot, or rounded mass, staining almost black in haematoxylin, at one side of which is the idiochromosome (now a condensed chromosome nucleolus). ‘These structures lie in a large clear nuclear vacuole, as shown in Photos 39 to 42. ‘The stage thus attained is the characteristic contraction phase or synizesis, which in this species is extremely marked.° In the safranin-lichtgriin preparation at this period the chro- mosome nucleolus is, as always, intensely red. The synaptic knot varies with the relative intensities of the red and green, being in some preparations distinctly red, in others pure green, in still others of mixed appearance. In the succeedingstage the chromatin emerges from the synaptic knot in the form of separate spireme threads which lose their staining capacity for haematoxylin and in the double stain are again pure green (Photos 43 and 44). In the middle and late growth period they are still more or less green but contain red granules. In the prophases of the first divi- sion they at last lose their affinity for the green and finally appear pure red; but this does not occur until just before the dissolution of the nuclear membrane. Since the idiochromosome always retains its intense red color it may thus be followed from stage to stage with great certainty. The study of the whole cycle of changes from the last sper- matogonial division onward gives certain very definite results in regard to synapsis in general, and especially in regard to the idio- 8 Many recent writers have expressed the opinion that the synizesis stage is an artifact produced as a shrinkage product, though Miss Sargant (’96) stated very explicitly that she had seen it in the living cells, and this has recently been confirmed by Overton (’o5). I can fully substantiate this in the case of Anasa tristis. The perfectly fresh testis, gently teased apart in a Ringer’s fluid in which the sperma- tozoa continue their active movements, very clearly shows nearly all the features of the spermatogenesis, including the number, shape and size relations of the chromosomes, their characteristic grouping and behavior in the spermatocyte divisions, the double rods, crosses and other prophase figures, the spindle fibers and asters, and even, I believe, the centrosomes. In this fresh material the synizesis stage appears in essentially the same form as in the sections, the nuclear knot lying in a large clear vacuole. These nuclei only appear in the same region of the testis as in sections, and they show a conspicuous contrast to those of earlier and later stages that lienear bythem. Inthe post synaptic stages the chromo- somes, in the form of spireme threads can be seen again spreading through the nuclear cavity. These observations leave no doubt in my mind that the synizesis is a normal phase of the spermatogenesis in these animals, though it is not improbable that the contraction may be somewhat exaggerated by the reagents. It is evident, however, from such studies as those of the Schreiners (’06) and others that the synizesis does not occur in some forms. 84 Edmund B. Wilson chromosome. Concerning the first point I will here only indicate one principal conclusion. It is quite clear that in Pyrrochoris (and I think the same holds true in other Hemiptera) sy napsis, or the conjugation of chromosomes two by two, does not occur in the closing anaphases of the last spermatogonial division as was described by Montgomery (’oo) in Peripatus and Euschistus (‘‘Pentatoma’’), by Sutton (’o2) in Brachystola, by Stevens (’03) in Sagitta, and by Dublin (’05) in Pedicellina. Although the number of chromosomes in the postphases immediately following this division (Photos 32 and 33) cannot be exactly made out, it is perfectly evident that it is not the reduced number but approxi- mates to the somatic number (twenty-three). “The chromosomes, therefore, have not paired two by two in the spermatogonial ana- phases. It is equally certain that this stage does not pass directly into the synizesis but is separated from it by a long “resting period” (Photos 34 to 38)—as is demonstated by the topographical relations as well as by the progressive stages of the idiochromo- some—in which the ordinary chromosomes lose their sharp boundaries and their affinity for nuclear stains. In this respect Pyrrochoris shows a close similarity to Tomopteris, as described by the Schreiners (’06), whose original preparations, by the kindness of Dr. Schreiner, I have had opportunity to examine. This comparison has convinced me that synapsis occurs at the same period in both—whether by parasynapsis (side to side union) or telosynapsis (end to end union®) or in some other way I am not prepared to say. There can be no manner of doubt that the first division of the bivalents is a transverse one, as described by Paulmier and Montgomery; but it has been rendered evident enough by recent studies on reduction that this in itself gives no trustworthy evidence regarding the mode of synapsis. The direct investigation of the process in the Hemiptera presents great difficulties. The foregoing general conclusion regarding the time of synapsis is of importance for the more specific one in regard to the idio- chromosome. During the entire earlier presynaptic period the ®T have for some years made use of these terms in my lectures on cytology. Studies on Chromosomes 85 elongated idiochromosome is manifestly a single body. As it shortens and condenses to form the chromosome nucleolus, it shows a considerable variety of forms; and the rate of condensation also varies, cells that are already entering the synizesis stage being sometimes seen in which the idiochromosome is still distinctly a rod (Photos 35 and 36). In most cases it is at this time a single ovoidal or spheroidal body; but not infrequently it appears more or less distinctly double (Photos 37 to 39). ‘This condition is however not produced by a previous synapsis of two chromosomes, as Gross believed, but arises, I think, from a tendency of the chromatin to accumulate towards the ends of the rod; and when this is very marked it may assume an appearance of duality, even in the ear- lier stages (Photo 37, below), though this is relatively rare. In the later stages a double appearance is not infrequent, dumb-bell forms being thus produced, which sometimes give in the synizesis stage apparently double bodies. ‘The earlier stages conclusively show that this is a secondary appearance. In the later (postsyn- aptic) stages (Photos 34 and 35), and throughout the growth period, it always appears as a single spheroidal body. In view of these facts I think the conclusion inevitable that the chromosome nucleolus is a univalent chromosome that arises by the condensa- tion of the unpaired large chromosome of the spermatogonia. I have little to add to Henking’s and Gross’s acounts of the maturation divisions. As will be seen from Photos 45, 46, 50, 51, the size relations are correlated with those of the spermatogonial chromosomes. In polar views of the first division appear with great constancy four smallest chromosomes, one slightly larger one, and seven still larger ones, or twelve in all. The idiochromo- some is one of the largest, but cannot be distinguished from the others (as is also the case in Protenor). ‘This is obviously due to the fact that the idiochromosome is still a univalent or single chromosome, while each of the others represents two of the sper- matogonial chromosomes united. Since all have nearly the same dumb-bell shape as seen in side view, the idiochromosome appears from the pole approximately but half as large, relative to the others, as in the spermatogonia. The same size-relations appear in the second division, but all the chromosomes are much smaller, as the photographs clearly show. 86 Edmund B. Wilson I have not succeeded with Pyrrochoris (as I have with several other genera) in obtaining photographs of both anaphase daughter groups showing all the chromosomes; but it is perfectly evident that all divide equally in the first division, and all but the idio- chromosome in the second. This chromosome lags behind the others and then passes undivided to one pole where it is included in the daughter nucleus (Photos 47 to 49) as Henking described. This pole thus receives twelve chromosomes, the other but eleven. As in a number of other species the idiochromosome retains its compact form and deep-staining capacity long after the reconstruc- tion of the nuclei and the breaking up of the other chromosomes. It may thus be distinguished (especially well in safranin prepara- tions) up to a rather late period stage of the spermatids, even after the tails have grown out. It finally disappears from view, and the mature spermatozoa show no visible indication of their dimorphism. GENERAL If my conclusions are correct, Pyrrochoris agrees exactly with other forms in which an unpaired idiochromosome is present. Syromastes however presents a new type in which the “accessory” chromosome is not univalent but bivalent, and in which accord- ingly half the spermatozoa receive two more chromosomes than the other half. If we may apply the same rule to Syromastes as that which holds for other Hemiptera we may expect the sperma- tozoa that receive the “accessory” to be female-producing, the others male-producing. ‘The fertilization formulas for the two species considered in this paper should therefore be as follows: PYRROCHORIS Egg 12 + spermatozoén 11 = zyote 23(c) Egg 12 + spermatozoén 12 = zygote24(Q) SYROMASTES Egg 12 (including J and/)* + spermatozoén 10 = zygote 22 (including J and i)(@) Egg 12 (including Iand/) + spermatozodn 12 (including J andi) = zygote 24 (including i, Tyas) * The formation of a reduced female group of this composition may readily be explained if it be sup- posed that in synapsis the two small idiochromosomes couple with each other to form the bivalent ii, the two large ones to form the bivalent JJ. Studies on Chromosomes 87 The correctness of my deduction may readily be tested by a reéxamination of the female groups. Gross, it is true, states that he has found but twenty-two chromosomes in the female (follicle cells); but I think no one is likely to consider as in any way conclusive the single figure that he gives in support of this (op. cit., Fig. 111). Not less than five of the twenty-two chromo- somes figured are deeply constricted; and any one of these might in reality be two chromosomes in contact. I hope that Dr. Gross himself may be willing to reexamine this point, in view of the possibility here suggested. It is however also possible that the two members of each of the idiochromosome pairs in the female may be united to form a bivalent, in which case the female would apparently show but twenty-two chromosomes; but even if this be so the two members must separate again when transferred to the male. In regard to Pyrrochoris, there 1s little doubt that the determina- tion of the female number by Henking and Gross as twenty-four was correct; and since the idiochromosome in the male is the largest of the chromosomes we may expect the female groups to show two such chromosomes." I should state the expectation less confidently in the case of Syro- mastes if it stood entirely alone; but another case has now been made known in which the male and female groups differ by more than one chromosome. ‘This occurs in the genus Galgulus, which has been worked out in my laboratory by Mr. F. Payne (whose results are now in press)" on material collected by myself. The following facts are very clearly shown in this form. The sperma- togonial number is thirty-five, the female number thirty-eight. In the second division five of the chromosomes are always asso- 1 Henking’s figures (’92) give considerable evidence that such is really the case. His Fig. 83 of the first polar metaphase shows one of the twelve bivalents fully twice the size of the others; and the same is true of Fig. 68, which shows a side view of the second polar spindle, though not all the chromo- somes are shown. With this accords his Fig. 39 of a double group from a connective tissue cell of the female showing forty-eight chromosomes, of which four, of nearly equal size, are nearly twice the size of the others. This agrees precisely with the relation shown in a double group of Anasa figured by me in a former paper (’06, Fig. 2,k) which shows twice the normal number of both the largest and the smallest chromosomes. 11 Since published in Biol. Bull., xiv, 5. 88 Edmund B. Wilson ciated to form a definite pentad element of which four pass to one pole, one to the other, while the remaining fifteen chromosomes divide equally. Half the spermatozoa thus receive sixteen chro- mosomes and half nineteen. From these facts it is clear that the sixteen-chromosome class must be male producing, the nineteen- chromosome class female producing, according to the formula: GALGULUS Egg " + spermatozoin % — 3 = zygote n — 3 (co) Egg ™ + spermatozoén ” = zygoten( 92) This case, together with that of Syromastes (if my inference regard- ing this form be correct) shows that we must considerably enlarge our previous conceptions as to the relations between sex produc- tion and the chromosomes; for we can no longer hold that only a single pair are involved. In Syromastes there are two such pairs, in Galgulus several pairs. A COMPARATIVE REVIEW OF THE TYPES OF SEXUAL DIFFERENCES OF THE CHROMOSOMES It is evident that a greater variety of types exists in regard to the sex differences than was indicated in the brief general review given in the third of my “Studies” (Wilson ’o6.) In that paper I distinguished three types, examples of which are given by Protenor, Lygeeus and Nezara; but the number must now be increased to at least five, and possibly to seven, of which I will now give a brief synopsis. With the exception of Syromastes and Diabrotica this synopsis includes only species of which both sexes have been accurately determined. Forms like the aphids, in which idiochromosomes have not yet been positively identified, have been omitted. Seventeen of the species are here reported for the first time (one or both sexes) from my own results hitherto unpublished. [I am indebted to Dr. Stevens for permission to include her results on the Diptera and on Diabrotica, which are now in press (’o8a, ’o8b). ee a SAP Studies on Chromosomes 89 I Both sexes with the same number of chromosomes; a pair of equal idiochro- somes present in both. No visible difference between the two classes of sperma- tozoa or between the male and female somatic groups. FERTILIZATION FORMULA n Mi z Egg " + spermatozoon ” = zygoten (d'or 2) Described Case Specie Order Family Authority 3’ Somatic No. 2 Somatic No. Nezara hilaris Say Hemiptera heteroptera Pentatomide 14 | 14 | Wilson (’06) To this type belongs also Oncopeltus fasciatus Dall, one of the Lygeide. It is further probable that here belong many forms in which no visible sexual differ- ences are to be seen, and in which idiochromosomes have not been identified. If a particular pair of chromosomes, corresponding to idiochromosomes, are of general occurrence, though not visibly distinguishable from the others, it is probable that this type represents the most frequent condition in animals generally. II Both sexes, and both classes of spermatozoa, with the same number of chromo- somes. The male with a pair of unequal idiochromosomes, half the spermatozoa receiving the large one and halfthe small. In the female a pair of equal idiochromo- somes like the large one of the male. FERTILIZATION FORMULA Egg Egg including J) + spermatozoén ™ (including7) = zygote n (including I’) &° § Pp z sf ys § (including 7) + spermatozoin ” (including J) = zygote n (including IT) 2 wjBwufs go Edmund B. Wilson Described Cases Species Oebalus pugnax Fab. Euschistus fissilis Uhl. ictericus L. servus Say tristigmus Say variolarius P. B. Ccenus delius Say Stiretrus anchorago Fab Podisus maculiventris Say \ (spinosus) f Banasa dimidiata Say calva Say Lygzus turcicus Fab. bicrucis Say Tenebrio molitor Chelymorpha argus Trirhabda virgata canadense Drosophila ampelophila Musca domestica Calliphora vomitoria Sarcophaga sarracinie Scatophaga pallida Tetanocera sparsa Eristalis tenax Order Hemiptera heteroptera Hemiptera heteroptera Hemiptera heteroptera Hemiptera heteroptera Hemiptera heteroptera Hemiptera heteroptera Hemiptera heteroptera Hemiptera heteroptera Hemiptera heteroptera Hemiptera heteroptera Hemiptera heteroptera Hemiptera heteroptera Hemiptera heteroptera Coleoptera Coleoptera Coleoptera Coleoptera Diptera Diptera Diptera Diptera Diptera Diptera Diptera Family | Pentatomide Pentatomide Pentatomide | Pentatomide Pentatomide Pentatomide Pentatomide Pentatomide | Pentatomide Pentatomide | Pentatomide Pentatomide | Pentatomide | | Tenebrionidz Chrysomelide Chrysomelidz Chrysomelide S A | o Somatic No. 2 Somatic No. Authority Wilson | Wilson ’osb, ’o5c, 06 | Wilson ’06 Wilson i & Montgomery ’o1 iN Q Wilson °o6 Wilson ’06 Wilson ’osb, ’o5c, ’06 | Wilson { o Montgomery “or i 2 Wilson ’osb, osc, ’06 Wilson ’07b | Wilson ’07b Wilson ’osb, ’o5c, ’06 | Wilson Stevens ’05 Stevens ’06 Stevens ’06 - Stevens 06 Stevens ’o8a Stevens ’o8a | Stevens ’o8a Stevens ’o8a Stevens ’o8a | Stevens ’o8a Stevens ’o8a * See Type Ila. Ill The female chromosome groups with one more chromosome than the male. Male with an unpaired idiochromosome and an odd spermatogonial number, half the spermatozoa receiving the idiochromosome and half being without it. Female with an equal pair of idiochromosomes like the unpaired one of the male. Egg Egg wsws FERTILIZATION FORMULA (including J) + spermatozoén % — 1 = zygote n — 1 (including/) 3 (including J) + spermatozoén ™ (including J) = zygote n (including II) 9 Studies on Chromosomes gi Described Cases g | 2 | Species Order Family A Bs | Authority Raya ioae | | oma ae | Largus cinctus H.S. Hemiptera heteroptera | Pyrrochoride II 12 | Wilson succinctus L. Hemiptera heteroptera | Pyrrochoride 13 14 | Wilson een Pyrrochoris apterus L. | Hemiptera heteroptera | Pyrrochoride | 23 | 24 | \ cocina gI Alydus pilosulus H. S. | Hemiptera heteroptera | Coreide 13 | 14 | Wilson ’osb, osc, ’06 | ), ae a. i Hemiptera heteroptera | Coreide ey rs al | ‘ aie bids Ol | ’ Protenor belfragei Hag-| Hemiptera heteroptera | Coreide Tay 4s ane ine = 06 Leptocoris trivittatus | Say Hemiptera heteroptera | Coreide 13 | 14 | Wilson Archimerus calcarator Fab. Hemiptera heteroptera | Coreide 15 | 16) Wilson Pachylis gigas Burm. | Hemiptera heteroptera | Coreide 15 | 16 Wilson Anasa tristis DeG. Hemiptera heteroptera | Coreide 21*) 22 | Wilson ’osb, ’o5c, 06, 07a lf go y armigera Say Hemiptera heteroptera | Coreide 25K) ez ‘ ee a sp. | Hemiptera heteroptera | Coreide 21 | 22 | Montgomery ’06 Euthoctha galeator Fab. | Hemiptera heteroptera | Coreide 21 | 22 | Wilson Leptoglossus phyllopus L. Hemiptera heteroptera | Coreide 21 | 22 | Wilson Margus inconspicuus H. S. Hemiptera heteroptera | Coreide 23 | 24 | Wilson Chariesterus antennator Fab. Hemiptera heteroptera | Coreide 25 | 26 | Wilson Corynocoris distinctus Dall. Hemiptera heteroptera | Coreide 25 | 26 | Wilson Aprophora quadrang- | ularis Hemipterahomoptera | Jasside 23 | 24 | Stevens ’06 Peeciloptera septentrionalis Hemipterahomoptera | Fulgoride 27 | 28 | Boring’o7 pruinosa Hemiptera homoptera | Fulgoride 27 | 28 | Boring’o7 Elater, sp. Coleoptera Elateride 19 | 20 | Stevens ’06 ’ Blatta germanica Orthoptera | Blattide 23) 24 f ean Anax junius Odonata | ea 27 | 28 | LeFevre and McGill 08 *This number, disputed by Foot and Strobell (’07a, b), has since been confirmed by my own reéxami- nation (’07a, ’o8) and by that of Lefevre and McGill (’08) and others. Cc g2 Peel B. Wilson IV Female groups (by inference only) with two more chromosomes than the male. In the male a pair of unequal idiochromosomes, half the spermatozoa receiving both these chromosomes, and hence two more than the other half. In the female (by inference only) two such pairs. FERTILIZATION FORMULA Egg ™ (including I, i) + spermatozoin® — 2 = zygote n — 2 (including I,i) S Egg ™ (including J,) + spermatozoén % includingJ,/) = zygote n (including J, I, i, i,) Q (by inference only) ‘ Described Case | | Z| | 2 Specie Order Family = 5 Authority a | $s COM 2Or eoees Hemiptera heteroptera | Coreide 22 CC 2 marginatus L y \ 2 Wilson (inferred) v. . Female groups with three more chromosomes than the male. Half the sperma- tozoa receiving three more chromosomes than the other half. Egg % + spermatozoén” — 3 = zygote n — 3() Egg % + spermatozoén ™ = zygote n( 2) Described Case $|e [pee] cass Specie | Order Family : : Authority | bale een Galgulus oculatus Fab.) Hemiptera heteroptera | Galgulide | 35 | 38 | Payne ’o8 At least two of the foregoing types may be complicated by the presence of certain additional chromosomes, present in some individuals but not in others of the same species, to which I have applied the name of “supernumerary chromosomes. ””” The number of these varies from one to six in different individuals but is constant in the same individual. In some forms (Metapodius, Banasa) these supernumer- Wilson ’o7b, ’o7c. A detailed description is now in preparation. Studies On Chromosomes 93 aries accompany a typical pair of unequal idiochromosomes (as in Type II). In other forms (Diabrotica), the supernumeraries accompany an unpaired idiochromo- some (as in Type III). In these cases definite numerical formulas cannot be given, since the distribution of the supernumeraries is variable and both sexes show a variable number of chromosomes in consequence (directly known only in Meta- podius.) Forthe present these cases may most conveniently be treated as sub-types as follows: . Ila Forms that agree with Type II except that certain individuals may possess, in addition to a pair of idiochromosomes, one or several supernumerary chromosomes. The cases described, with the numbers of chromosomes observed, are as follows: Si 8 Species Order Family Somatic | Somatic Authority No. | No. Banasa calva Hemiptera Pentatomide 26 | 26 Wilson ’o7b heteroptera [26+ 1] Metapodius terminalis Hemiptera Coreidz 21* 22 Wilson ’07b, ’08 heteroptera 22 22+1 22+1 2-+2 22+2 | 2243 22+3 | 22+4 | femoratus Hemiptera Coreide 22 | 22+1 Wilson ’o7b, ’o08 heteroptera 22at2| 2242 2243 | 22+ 4 granulosus Hemiptera Coreide {) = 232 | Brees Wilson ’o7b, ’o8 heteroptera | 2241 | * This number occurs only in Montgomery’s (’o6) material of this species, identification of which though probably correct, is not absolutely certain. This case will be considered in a later publication. Illa Forms that agree with Type III except that certain individuals may possess, in addition to an unpaired idiochromosome, one or several supernumerary chromo- somes. Described cases as follows: 94 Edmund B. Wilson |e 9 | Specie Order Family Somatic | Somatic Authority No. No. Diabrotica 12-punctata__ Coleoptera | Chrysomelide | 19 Stevens ’07, 08 soror | I9+I 19+2 19+3 19+4 Despite the apparent diversity of the types that have been enu- merated all conform to the common principle that the spermatozoa are of two classes, equal in number, that are respectively male producing and female producing. In the case of Type I this is no more than an inference, since the two classes cannot be distin- guished by the eye; but its great probability will be admitted in the fact that the forms with equal idiochromosomes are connected by forms (such as Mineus) in which only a slight inequality exists, with those in which the inequality is very marked (Wilson ’o5a). The facts now show that the difference between the two classes of spermatozoa is not always confined to a single pair of chromosomes, but may affect two pairs (Syromastes) or even a larger number (Galgulus). It is noteworthy that in every case where a quantitative difference of chromatin exists between the sexes it is always in favor of the female, whether it appear in a larger number of chromosomes or in the greater size of one of them. But I must again emphasize the fact that this quantitative differ- ence cannot be considered as the primary factor that differenti- ates the two classes, for in the first class such a difference does not exist, while in Metapodius, even in the same species, it is some- ‘8 T based this type on the facts observed in Nezara, where the idiochromosomes are equal in size in both sexes. This is not in accordance with the later observations of Montgomery (’06) who believes that in the Hemiptera generally the two components (paternal and maternal) of every chromosome pair are at least slightly unequal—though he finds the idiochromosomes of Oncopeltus equal as I have also since observed. A reéxamination of Nezara confirms my original account of this form, though in some individuals the idiochromosomes often appear very slightly unequal. A careful examination of the other chromosomes, particularly the small m-chromosomes (which are most favorable for the purpose) in Alydus, Anasa, Archimerus, Pachylis, and other genera, leads me to a very skeptical view of Montgomery’s general conclusion on this point. It is true that the two members of each pair vary slightly in relative size, and are not always exactly equal; but, in my material at least, it is clear that Studies on Chromosomes 95 times the female, sometimes the male, that has the larger number and quantity. I therefore adhere to the view that if the primary and essential difference between the two classes of spermatozoa inhere in the chromosomes (there 1s of course room for difference of opinion on this point) it must be, or originally have been, qualitative in nature. Since the appearance of my third “Study,” in which some general discussion of the sex chromosomes was offered, there has appeared an important paper by Correns (’07) on the higher plants, the results of which, as he points out, harmonize remarkably with those based on the cytological evidence. The most important of his results is the experimental proof obtained by hybridizing experiments on Bryonia, that in the dicecious species the pollen grains are male producing and female producing in equal num- bers, quite in accordance with the view put forward by McClung (02) in regard to the spermatozoa of insects and proved to be correct in principle by the work of Stevens and myself. ‘That the same result should appear from investigations carried out on such different material and by such different methods certainly gives good ground for the belief that as far as the male is concerned the phenomenon is at least a very general one. Professor Correns points out in some detail the extraordinarily close parallel between his experimental results and the cytological ones of Stevens and myself; but the interpretation that he offers differs materially from both those that I suggested in an analysis of my observa- tions (Wilson ’o6). According to my first interpretation (Castle’s) both sexes are assumed to be sex hybrids or heterozygotes. ‘The conclusion of Correns is that, in respect to the active sexual ten- dencies of the gametes that produce them, only the male is a sex hybrid or heterozygote (3 (2)), while the female is a homozy- gote (2). This interpretation explains the numerical equality this is merely a casual fluctuation, the general rule being equality. This variation appears in dif- ferent cells of the same cyst (as may be seen with especial clearness in the m-chromosomes in side views of the second division where errors due to foreshortening may be eliminated). It would be indeed strange if these relations were subject to no variation whatever. M4 Tt is necessary to an understanding of Correns’s view to bear in mind that the gametes are not considered to be ‘‘pure” in the original Mendelian sense, but to bear both sexual possibilities, one of which is ‘‘active,” the other ‘‘latent.” 96 Edmund B. Wilson of the sexes in accordance with the Mendelian principle without the necessity for assuming selective fertilization. It is so simple, and seems to be so clearly demonstrated in the case of Bryonia, that its application to the interpretation of sex production in general is very tempting. Correns himself believes it “very prob- able” that his conclusion will apply to all the dicecious flowering plants, and possible that it may also hold true of animals (op. cit., pp. 65, 66). It is evident that in their superficial aspects the cyto- logical results seem to bear this out. Wherever the sexes show visible differences in the somatic chromosome groups the female groups consist of two series in duplicate, while the male groups show two series that are not duplicates, only one of them being identical with one of the female series. As far as the chromosomes are concerned, and from a purely morphological point of view, the female is therefore in fact a homozygote, the male a hetero- zygote, in these animals. But when more closely scrutinized from this standpoint the interpretation seems by no means so clear. As I showed in my third “Study” the odd chromosome of the male must be derived from the egg; and if this chromosome bears the sexual tendency, it must under Correns’s hypothesis carry the female tendency—which is a reductio ad absurdum, since it is not accompanied by a male-bearing mate or partner in the male. I think this brings clearly into view the following alter- native. Either the females of these insects must be physiolog- ically heterozygotes (as I assumed), or the so-called “sex chromo- somes”. (idiochromosomes) do not bear the sexual tendencies but only accompany them in a definite way. Which of these possi- bilities is the true one may be left to further research to decide. I will only point out that Professor Correns carefully considers the difficulties that his interpretation encounters in some other direc- tions, and admits that it must be modified in certain cases—for example in the honey bee and in Dinophilus, in which latter case he too is compelled to admit the possibility of selective fertiliza- tion. The parthenogenetic females of such forms as the aphids and phylloxerans, which produce both males and females without fertilization, are still considered by Correns as homozygotes, the production of males being assumed to be determined, if I under- Studies on Chromosomes 97 stand his conception, by the activation of the “latent’’ (not to be confused with the “recessive”) male possibility in the male pro- ducing eggs. ‘This is doubtless an admissible assumption, though it seems to me to put a considerable strain upon the general hypothesis. ‘The more natural view would seem to be the one directly suggested by the facts, 1.e., that the parthenogenetic stem- mother aphid is a heterozygote, the male tendency being in the condition of a Mendelian recessive. But [-will not enter upon a discussion of this question, which is now in a condition where a little observation and experiment will outweigh a large amount of hypothesis. I think, however, that the first of the interpretations that I suggested (following Castle) should not be rejected without further data, and especially not until the question of selective fertilization has been put to the test of direct experiment. ZoOlogical Laboratory Columbia University February 13, 1908 WORKS REFERRED TO Borine, Aice M. ’07—A Study of the Spermatogenesis of twenty-two Species of the Membracidz, Jasside, Cercopide and Fulgoride. Journ. Exp. Zodl., iv, 4. Correns, C.’07—Die Bestimmung und Vererbung des Geschlechtes. Berlin, 1907. Also (in abbreviated form) in Arch. f. Rassen- u. Ges.- Biologie, iv, 6. Dusiin, L. I. ’05—The History of the Germ-cells in Pedicellina Americana. Ann. IN: Y. Acad: Sci... xvi} 1: Foor, K., and Strospett, E. C. ’07a—The “Accessory Chromosome” of Anasa tristis. Biol. Bull., xii. ‘o7b—A Study of Chromosomes in the Spermatogenesis of Anasa tristis. Am. Journ. Anat., vii, 2. Gross, J. ’04—Die Spermatogenese von Syromastes marginatus. Zool. Jahrb., Anat. u. Ontog., xx. ’07—Die Spermatogenese von Pyrrochoris apterus. bid., xxiii. Henxine, H. ’91—Ueber Spermatogenese und deren Beziehung zur Eientwick- lung bei Pyrrochoris apterus. Zeitschr. f. Wiss. Zool., li. *92—Untersuchungen tiber die ersten Entwicklungs-vorginge in den Eiern der Insekten, Ill. zd, liv, 1. 98 Edmund B. Wilson LeFevre, G., and McGI1t, C. ’08—The Chromosomes of Anasa tristis and Anax. junius. Am. Journ. Anat., viii, 4. Meves, F. ’03—Ueber “Richtungskérperbildung” im Hoden von Hymenopteren. Anat. Anz., xxiv. ’*o7—Die Spermatocytenteilungen bei der Honigbiene, etc. Arch. mik. Anat., Ixx, 3. Montcomery, T. H. ’00—The Spermatogenesis of Peripatus, etc. Zool. Jahrb., Anat. u. Ontog., xiv. “o1—A Study of the Chromosomes of the Germ-cells of Metazoa. Trans. Am. Phil. Soc., xx. °04—Some Observations and Considerations upon the Maturation Phe- nomena of the Germ-cells. Biol. Bull., vi, 3. ’06—Chromosomes in the Spermatogenesis of the Hemiptera Heteroptera. Trans. Am. Phil. Soc., xx. McCiunec, C. E. ’o2—The Acessory Chromosome—Sex Determinant? Biol. Bull. iii. Overton, J. B. ’05—Ueber Reduktionsteilung in den Pollenmutterzellen einiger Dikotylen. Jahrb. wiss. Bot., xlii, 1. PautmigR, F. C. ’99—The Spermatogenesis of Anasa tristis. Journ. Morph., Supplement. Payne, F. ’o8—On the Sexual Differences of the Chromosome groups in Galgulus oculatus. Biol. Bull., xiv, 5. SARGANT, ETHEL ’96—The Formation of the Sexual Nuclei in Lilium martagon. I, Odgenesis. Ann. Bot., x. ScHREINER, K. E. and A. ’o6—Neue Studien tiber die Chromatinreifung der Geschlechtszellen (Tomopteris). Arch. Biol., xx. Stevens, N. M. ’03—On the Ovogenesis and Spermatogenesis of Sagitta bipunctata. Zool. Jahrb., Anat. u. Ontog., xviii. ’o5—Studies in Spermatogenesis with Especial Reference to the “Acces- sory Chromosome.” Carnegie Institution, Washington, Pub. no. 36. *o6—Studies in Spermatogenesis. II. A Comparative Study of the Hetero- chromosomes in certain Species of Coleoptera, Hemiptera and Lepidoptera, with Especial Reference to Sex Determination. Ibid., Pub. 36, II. ’o8a—A Study of the Germ-cells of Certain Diptera, etc., Journ. Exp. Zodl., v, 3. *o08b—The Chromosomes in Diabrotica, etc. Ibid., v, 4. Sutton, W. S. ’02—On the Morphology of the Chromosome group in Brachystola magna. Biol. Bull., iv, 1. occade, ae Studies on Chromosomes 99 Wa tt ace, L. B. ’05—The Spermatogenesis of the Spider. Biol. Bull., viii. Wassi1EFF, A. ’07—Die Spermatogenese von Blatta germanica. Arch. mik. Anat., ix Witson, E. B. ’o5a—Studies on Chromosomes. I. The Behavior of the Idiochromosomes in Hemiptera. Journ. Exp. Zodl., 11. °o5b—The Chromosomes in Relation to the Determination of Sex in Insects. Science, xx, p. 500. ’o5c—Studies on Chromosomes. II. The paired Microchromosomes, Idiochromosomes and Heterotropic Chromosomes in the Hemip- tera. Ibid., ii. ’*06—Studies on Chromosomes. III. The Sexual Differences of the Chromosome Groups in Hemiptera, with some Considerations on the Determination and Inheritance of Sex. Jbid., iii. ’07a—The Case of Anasa tristis. Science, xxv, p. II. *o7b—Note on the Chromosome groups of Metapodius and Banasa. Biol. Bull. ’o7c-—The Supernumerary Chromosomes of Metapodius. Read before the May Meeting of the N. Y. Acad. of Sci. Science, xxvi, 077. ’*08—The Accessory Chromosome of Anasa tristis. Read before the Am. Soc. of Zodlogists, December, ’07. Science, xxvii, 690. EXPLANATION OF PLATES All of the figures are reproduced directly from photographs by the author, without retouching. The originals were taken with a Spencer ¥; oil-immersion, Zeiss ocular 6, which gives an enlargement of 1500 diameters. The admirable method of focusing devised by Foot and Strobell was employed. a are reproduced at the same magnification. Prate I (Photos 1 to 5, 10 to 23, Syromastes marginatus;.6 to 10, Metapodius terminalis; 24 and 25, Pyrro- choris apterus). 1and2. Spermatogonial groups of Syromastes; copied in Text-fig. 1, a, b. 3 to 5. Polar views, first maturation metaphase; m-chromosome at the center, idiochromosome- bivalent (“‘accessory’” chromosome) outside the ring at the left. 6 and 7. Corresponding views of Metapodius, typical condition with the two separate idiochromo- somes outside the ring at the left. 8 and g. The same; exceptional condition, with the idiochromosomes (at the left) in contact. 10. Polar metaphase, second division, Syromastes. I1 to 17. Side views of the same division. The duality of the idiochromosome appears in 12, 16 and 17. 18 to 23. Early prophases of first maturation division, Syromastes. Each of these shows the separate m-chromosomes, and in all but No. 20 the chromosome nucleolus (idiochromosome bivalent) also appears. 24 and 25. Spermatogonial metaphases of Pyrrochoris (copied in Text-figs. 2, a, b). TUDIES ON CHROMOSOMES Edmond B. Wilson. PAW Ee The Journal of Experimental Zoology, Vol. VI, No 1. WILSON, PHOTO NN ; Pirate II Pyrrochoris apterus 26 to 31. Spermatogonial groups, each showing twenty-three chromosomes, including the large unpaired idiochromosome; 30, 31 illustrate the rare case in which the latter appears double, owing to marked sigmoid curvature. These photos are copied in Text-figs. 2, c, d, e, j, k and /, respectively. 32 and 33. Post-phases shortly following last spermatogonial division; the chromosomes still distinct, idiochromosome recognizable by its large size and deeper color. 34 and 35. Presynaptic stages following the last, showing “‘caterpillar” stage of idiochromosome and small nucleoli. In the last two the shortening has begun. 36 to 38. Further condensation of the idiochromosome; initial stages of synizesis; apparent duality of the idiochromosome in two of the cells. 39 to 42. Synizesis, showing various forms of the chromosome nucleolus. 43 and 44. Early post-synaptic stages. 45 and 46. Polar metaphases, first spermatocyte division. 47 to 49. Side views of second division. 50 and 51. Polar metaphases, second division. STUDIES ON CHROMOSOMES PLATE 1!1 Edmond BR. Wilson. The Journal of Experimental Zoology, Vol. VI, No 1. WILSON, PHOTO. FURTH -R STUDIES ON THE CHROMOSOMES OF THE COLEOPTERA BY N. M. STEVENS Wirk Four Prates In three previous papers (’05, ’06, ’08) the chromosomes of several species of Coleoptera have been described and figured, and the role of the heterochromosomes in sex determination dis- cussed. The following pages are a further contribution to our knowledge of the character and behavior of the heterochromo- somes, and of the methods of synapsis in this order of insects. The methods used*in handling the material have been the same as in previous work: fixation with Gilson’s mercuro-nitric, Flemming’ s chromo- aceto- -osmic, and Hermann’s platino-aceto- osmic fluids, and stain‘ag with iron hematoxylin or thionin. The aceto-carmine method has been used in testing fresh material, and as a check on the section method. PHOTINUS PENNSYLVANICUS (FAM. LAMPYRID#) PHOTINUS CONSANGUINEUS (FAM. LAMPYRIDZ) In my 1906 paper on the spermatogenesis of Coleoptera and other insects, the spermatogonial plate of one of the fireflies, Ellychnia corrusca, was shown on FI. XIII, Fig. 236. The mate- rial was obtained from adults in September at Woods Hole. Only spermatogonia and growth stages of the spermatocytes were present in the testes. “The spermatogonial plate contains nineteen chromosomes (Fig. 1), two V’s, two long rods and fifteen shorter rods. Constancy in form made this material seem very favorable for study of the individuality of the chromosomes, but I have not been able to get the maturation stages. Two other fireflies THE JouRNAL oF ExPERIMENTAL ZOOLOGY, VOL. VI,NO.1. {4 J 102 N. M. Stevens however have been studied, Photinus pennsylvanicus ad Pho- tinus consanguineus. In Photinus consanguineus, all of the spermatocyte s iges were found in the testis of the adult in summer at Cold Sprin Harbor, and dividing spermatogonia were obtained from the larva in October. In the adults of Photinus pennsylvanicus ‘nly ripe spermatozoa were present; in the larve collected in October and November, only spermatogonia and growth stages of the sperma- tocytes. A large number of larvz were collected and kept during the winter in battery jars with a piece of turf in the bottom. Some of the jars were placed in the greenhouse, others in a cool basement room. The material from both sets of jars was tested from time to time with aceto-carmine, but not until May when the larvee were beginning to pupate, were any maturation mitoses found. From May 8 to May 13 both larve and pupe furnished good material. Asin Tenebrio molitor, the pupz contained favor- able divisions in somatic cells. Fig. 2 is the equatorial plate of an odgonium of Photinus pennsylvanicus, from an ovary sectioned in October. There are twenty chromosomes, two longer and two shorter than the others. The two smallest correspond to the small odd chromo- some (x) of the male, seen in Fig. 3, the spermatogonial plate of nineteen chromosomes. Fig. 4 is an equatorial plate from a male somatic cell, found in mitosis in the digestive tract of a male pupa. In Ellychnia corrusca the synizesis and synapsis stages are similar to those previously described for several of the Coleoptera (Stevens ’06, Pl. IX, Figs. 37 to 42, 61 to 62, and Pl. XII, 153 to 154; Nowlin’o6, Pl. I, Figs. 2to5, and Pl. Il, Figssge to 54)—a dense group of short loops atone end of the nucleus in synizesis, followed by a stage in which the loops straighten and unite in pairs. In Photinus pennsylvanicus these stages are quite different. After the last spermatogonial division, the chromo- somes evidently remain condensed for some time, for we find many cysts on the border line between spermatogonia and spermato- cytes in which the nuclei have the appearance of Fig. 5. A slightly later stage shows the chromosomes more crowded together and at one side of the nuclear space. The next stage, which might rt = Further Studtes on the Chromosomes : 103 be called the synizesis stage, shows the odd chromosome (x) still condensed, and the others forming a rather fine and closely wound spireme (Fig. 7). The fine spireme of Fig. 7 gradually thickens and spreads out (Fig. 8), and later loses much of its staining quality (Figs. g and 10). In these later growth stages the spireme winds in such a way as to appear in tangential sec- tion (Fig. 10) to radiate from the heterochromosome (x). There seems to be no question in this case but that synapsis must occur in the stage shown in Figs. 5 and 6, since the spireme, once formed (Fig. 7), remains unbroken until the prophase of the first maturation division. When the chromosomes come into the first spermatocyte spindle, nine of them are often typical tetrads, and one a dyad (Fig. 11). A comparison of x which is univalent with the bivalents con- vinces one that this is a reducing division for the bivalents and quantitative for the odd chromosome. In early metaphase the chromosomes viewed from one pole of the spindle are circular or oval in outline (Fig. 12), but in metakinesis and anaphase (Fig. 13) nine are dumb-bell shaped and one, the heterochromo- some, is circular. Figs. 14 and 15 show the typical metakinesis and early anaphase, while Fig. 16 is a late anaphase. The second spermatocytes all contain ten chromosomes, of which one, the daughter heterochromosome (x) usually stands out to one side of the equatorial plate (Fig. 17), and nearer one pole of the spindle in metaphase and anaphase (Figs. 18 and 19). In most of the Coleoptera where an odd chromosome has been found, it passes undivided to one pole of the first spindle, and divides in the second division, as in the Orthoptera and many Hem- iptera homoptera; but in Photinus we have a case like that of Anasa and several other Hemiptera heteroptera where the unpaired chromosome undergoes its quantitative division in the first spermatocyte while its bivalent companions are being separated into their univalent elements. In Photinus consanguineus the number of chromosomes is the same as in Photinus pennsylvanicus, twenty in the female and nineteen in the male. The chromosomes of the daughter plates are easily counted after the cell has divided (Fig. 20), 104 N. M. Stevens proving that the heterochromosome is not divided in this mitosis and that the spermatozoa are dimorphic, half of them containing ten, the other half nine chromosomes. The chromosomes of an egg follicle cell are shown in Fig. ars and those of a spermatogonium in Fig. 22. The synizesis and synapsis stages are quite different from those of Photinus penn- sylvanicus, and similar to those of Ellychnia. ‘The synizesis stage has the short crowded loops (Fig. 23). The synapsis stage is less distinct than in some of the cases. previously described. One occa- sionally finds a nucleus with the longer synaptic loops (Fig. 24), but more often synapsis and union of chromosomes occur at the same time, giving a mixture of loops, and spireme with sharp angles of which Fig. 25 is perhaps a fair specimen. ‘The hetero- chromosome may be seen in this stage but is more conspicuous in the later pale spireme stage (Fig. 26). Fig. 27 is the first spermatocyte equatorial plate, Fig. 28 and Fig. 29 the metaphase and anaphase, showing the unpaired chro- mosome dividing late. In fact, it frequently divides so late that the two daughter-heterochromosomes are still quite close together and connected by linin fibers in the metaphase of the pairs of second spermatocytes, as shown in Figs. 30 and 31. A pair of daughter plates are given in Fig. 32, showing that as in P. pennsyl- vanicus, the heterochromosome does not divide in the second spermatocyte. Usually it lags behind the daughter plate to which it belongs, so that the two anaphases (Figs. 29 and 33) are charac- terized by a pair of daughter heterochromosomes, and by a single heterochromosome, respectively. These two species of Lampyridz are the only cases which have been found, where the unpaired heterochromosome divides in the first spermatocyte instead of the second. In one, Photinus con- sanguineus, it divides very late, in a stage which 1s a late anaphase or telophase for the other chromosomes, while in the other species, P. pennsylvanicus, it divides at the same time with the other chromosomes, or only slightly later. It will be interesting to study the spermatogenesis of other Lampyridz for comparison on this point. An abundance of adult material of several other species has been secured and examined, but only spermatozoa Further Studies on the Chromosomes 105 were found. It will therefore be necessary to obtain the larve or pupz before the maturation stages can be studied. LIMONEUS GRISEUS (FAM. ELATERIDZ) In previous work an odd chromosome was found in two species of Elateride (’06). In both, the male number of chromosomes was nineteen, and in one the female number (twenty) was determined (o6, Pl. XIII, Fig. 229). In both species the unpaired chromo- some was the smallest one. In Limoneus griseus there are seventeen chromosomes in the sper- matogonia (Fig. 34), and the heterochromosome (x) is the largest. The synapsis and synizesis stages are similar to those of Photinus pennsylvanicus. ‘The most conspicuous stage in the transition from spermatogonia to spermatocytes 1s one 1n which the condensed chromosomes appear as approximately spherical bodies which nearly fill the small nucleus (Fig. 35). In Fig. 36 the chromosomes are united and somewhat elongated. Elongation continues until all traces of the individual chromosomes, with the exception of the odd chromosome (x), are lost in the fine, closely wound spireme with which the heterochromosome remains connected by linin threads (Fig. 37). As the nucleus enlarges, and the spireme becomes thicker and less stainable, the heterochromosome shows a central vacuole (Fig. 38), and a little later it appears like a spireme wound about in plasmosome material and still connected with the much paler general spireme (Fig. 39). At this point it resembles in its behavior the “accessory” of Orchesticus and Xiphidium (McClung ’o2, Pl. VII, Figs. 4, 5, 12). In the later pale spireme stage (Fig. 40) the heterochromosome is again con- densed. In the first spermatocyte spindle the odd chromosome appears in the equatorial plate in metaphase (Figs. 41 and 42), does not divide, but lags behind the daughter plates (Fig. 43). The chro- mosomes of a pair of daughter plates are shown in Fig. 44. In the telophase (Fig. 45) the heterochromosome holds the hama- toxylin after the other chromatin has been almost entirely de- stained. In the second spermatocytes (Fig. 46) the unpaired 106 N. M. Stevens chromosome (x) frequently divides somewhat later than the others. Polar plates of the two classes of second spermatocyte mitoses are shown in Figs. 47 and 48. So far as investigated the Elateridz have an unpaired heterochromosome which differs from that of the Lampyridz in dividing in the second spermatocytes. NECROPHORUS SAYI (FAM. SILPHIDZ) Necrophorus sayi has an unpaired heterochromosome, while Silpha americana (’06, Pl. XI, Figs. 141-150) has an unequal pair of heterochromosomes. Necrophorus also differs from Sil- pha in having a much smaller number of chromosomes, thirteen in the spermatogonia (Fig. 49), while Silpha has forty. The synizesis stage is of the finely wound spireme type with the odd chromosome usually visible. ‘There is no preliminary stage that can be pointed out as a synapsis stage, but as the chromosomes remain united in a spireme up to the prophase of the first maturation division, when they appear in the reduced number, it seems certain that synapsis must occur at the close of the last spermatogonial division before the synizesis stage. ‘The bivalents of the prophase of the first spermatocyte mitosis (Fig. 50) have the appearance of two spermatogonial chromosomes united end to end, and in the spindle they are merely somewhat shortened (Fig. 52). The first spermatocyte has seven chromosomes with the unival- ent one oftenest at the center of the group (Fig. 51). Fig. 52 shows the seven chromosomes of one spindle drawnat three different levels of the same section. Here the odd chromosome is at the periphery of the plate. ‘The centrosomes in this form are very large. Polar plates of one spindle are shown in Fig. 53, and in Fig. 54 equa- torial plates of the second divisions which proceed as in other sim- ilar cases giving the usual dimorphic spermatids, containing in Necrophorus six and seven chromosomes. CHRYSOMELA SIMILIS (FAM. CHRYSOMELIDZ) Most of the Chrysomelidze have an unequal pair of hetero- chromosomes, but Chrysomela similis, like the Diabroticas, has an odd chromosome. At the close of the synizesis stage this Further Studies on the Chromosomes 107 form often shows synapsis with unusual clearness (Fig. 55): Fig. 56 shows a late growth stage with the spireme still staining more deeply than in most cases, and Fig. 57 the equatorial plate of the first spermatocyte with twelve chromosomes. Metakinesis of several of the bivalents and division of the centrosome are shown in Fig. 58, and an early anaphase in Fig. 59. ‘The second sperma- tocytes contain eleven and twelve chromosomes (Fig. 60), as do also the spermatids and spermatozoa. ‘The sperm heads (Fig. 61) have a large middle piece which stains in iron hematoxylin, but not in thionin. LISTOTROPHUS CINGULATUS (FAM. STAPHYLINID#) STAPHYLINUS VIOLACEUS (FAM. STAPHYLINID#) Three rove-beetles have been examined with a rather small amount of material in each case. All have an unequal pair of heterochromosomes. Listotrophus cingulatus has twenty-six chromosomes in the spermatogonia (Fig. 62), one being very small. The heterochromosome pair is distinguishable in the synizesis stage, which is of the spireme type, and in the later growth stages both members of the pair are clearly separated and associated with a large plasmosome. ‘The chromosomes of the first sperma- tocyte are shown in Figs. 63 and 64, and those of the second divi- sion in Figs. 95 and 66. In the biue rove-beetle, Staphylinus violaceus, the heterochro- mosome air associated with a plasmosome is shown in Fig. 67. The firsc spermatocyte contains twenty-two chromosomes (Fig. 68), ard the unequal pair shows clearly in a section of a spindle (Fig. 99). “The two second spermatocyte equatorial plates appear in F’gs. 70 and 71. Another brown rove-beetle, not identified, has twenty-eight chromosomes in the spermatogonia and fourteen in che spermatocytes. TETRAOPES TETRAOPHTHALMUS (FAM. CERAMBYCID#) CYLENE ROBINIA (FAM. CERAMBYCID#) Tetraopes, the common red milkweed beetle, has twenty chro- mosomes. ‘Iwo spermatogonial plates (Figs. 72 and 73) show the different appearance of the chromosomes in different cysts. 108 N. M. Stevens The two smallest are the heterochromosomes. ‘The synizesis and synapsis stages are of the loop type, though not especially clear. Fig. 74 is the equatorial plate of the first maturation divi- sion; and Figs. 75 to 77, metaphase and anaphase, show the une- equal pair of heterochromosomes dividing either earlier or later than the other chromosomes. Only one specimen of Cylene gave any maturation divisions. The remainder of the testes examined contained only spermatids and spermatozoa. The number of chromosomes is the same asin Tetraopes, twenty. The larger heterochromosome shows the peculiarity of holding the stain longer than the other chromosomes. Figs. 78 and 79 are metaphases of the first division from an iron haematoxylin preparation much destained. EPICAUTA CINEREA (FAM. MELOID#) EPICAUTA PENNSYLVANICA (FAM. MELOIDZ) Two varieties of Epicauta cinerea, one with all gray elytra and the other with a lighter gray border around the elytra, were studied, and the chromosomes in both, as well as in Epicauta pennsylvanica, found to be of the same number and character— nineteen large and one small chromosome in the spermatogonia. The synizesis and synapsis stages are of the loop type and the maturation divisions result in spermatids one half 0. which con- tain the small heterochromosome, and one half the large one. Figs. 80 to 83 show the chromosomes of the spermatog »nia, first and second spermatocytes. The larger heterochromosoine holds the stain as in Cylene. PENTHE OBLIQUATA (FAM. MELANDRYID#) Only one pair of Penthe obliquata has been captured. Uhe ovaries and one testis were fixed in Gilson’s mercuro-nitric fluid, and the other testis in Flemming. The Flemming mater al alone gave any satisfactory results. The presence of an unequal pair of heterochromosomes is shown in Figs. 84 to 86—a sperm 1- togonial plate, a section of a first spermatocyte spindle, and two second spermatocyte equatorial plates. Further Studies on the Chromosomes 109 CICINDELA VULGARIS (FAM. CICINDELIDZ ) In Cicindela primeriana (’06, Pl. XIII, Figs. 198 to 206) the number of chromosomes was twenty, and the heterochromosome pair a large trilobed bivalent. In Cicindela vulgaris the number is twenty-two, three larger than the others (Fig. 87). In the first spermatocyte spindle the conspicuous elements are the trilobed heterochromosome group and a four-lobed or cross-shaped macro- chromosome (Fig. 88). The divisions are like those of Cicindela primeriana. OTHER CHRYSOMELID Among the Chrysomelidz, several other cases of an unequal pair of heterochromosomes will be briefly referred to. Lema trilineata (Figs. 89 to 92) has thirty-two chromosomes, one very small. ‘The synizesis stage is of the loop type followed by synapsis. Doryphora clivicolis is quite similar to Doryphoria 1o-lineata (06, Pl. XII, Figs. 151 to 186), and the character of the hetero- chromosome group is much more easily determined. The reduced number of chromosomes is seventeen, instead of eighteen as in 1o-lineata. “The chromosomes of the first and second maturation divisions are shown in Figs. 93 to 96. Chrysochus auratus has the loop type of synizesis and synapsis and a typical pair of quite unequal heterochromosomes (Figs. 97 and 98). ‘The reduced number is thirteen. Haltica chalybea, the steel-blue flea-beetle, has twenty-two chromosomes in the spermatogonium (Fig. 100). Only occasionally a specimen of this species has been found in the net, and these have been studied with the aid of aceto-carmine. No drawings have been made of synizesis, synapsis or growth stages. Fig. 101 is a prophase showing the heterochromosomes (fh, and h,) and another con- densed pair of chromatin elements which may be m-chromosomes. In the later prophase when the chromosomes are coming into the spindle, the heterochromosomes are often widely separated (Fig. 102), and the same is true of the metaphase (Figs. 103 to 105), so that twelve chromosomes show in the equatorial plate of the first spermatocyte (Fig. 106), but in the late anaphase (Fig. 107), the I1IO N. M. Stevens two heterochromosomes are always found between the two polar masses of chromatin separating like any other unequal pair. In the telophase and youngest spermatids they are usually still sepa- rate from the general mass of chromatin (Fig. 108). An abund- ance of material for further study of this form is much to be desired. The chromosomes of Coptocycla clavata are very similar to those of Coptocycla guttata (Nowlin ’o6). There are eighteen in the spermatogonium, the twosmallest being the unequal pair of hetero- chromosomes. ‘The testes of Lina laponica were examined with the hope that there might be some perceptible difference in chro- mosomes corresponding to the dimorphism described by Miss McCracken (’06) but none was found. The first spermatocytes contain seventeen rather small bivalents, one of which is quite unequal, and the second equatorial plates show clearly the usual dimorphism. MISCELLANEOUS A number of the Coccinellidz have been found to have nineteen large and one small chromosome in the spermatogonia and ten in the spermatocytes as in Adalia bipunctata (’06, Pl. XIII, Figs. 193 to 197). An unequal pair has also been found in one of the Rhynchophora, Phytonomius punctata, and in Obera tripunctata, one of the Lamune. DISCUSSION The character of the heterochromosomes has now been deter- mined for more than fifty species of Coleoptera, belonging to sixteen families. In twelve species an unpaired heterochromo- some has been found, in all of the others an unequal pair, and in Diabrotica soror and Diabrotica 12-punctata (’08) from one to four small supernumerary heterochromosomes may be present in addition to a large unpaired one. In connection with the work on the odd chromosome in the Coleoptera, new material of Stenopelmatus (’05) has been studied, and the spermatogonial number determined as forty-seven instead of forty-six. It was also possible to count one cyst of second BMGs somes sooocmeeGssne 130 Wile eneral(discusslOmia.\.!tors sro i tesip cle se = iis sSNA 4 oS ofS Fee teary erenI ee ee ties tae 133 WL SOUnneIa 7, Soop Rees > RO eT aOR ee aE et iota PE eR mins < Seen a ae 134 \WUIILT - LUSGGievITRROSS Fitaie eran hich Dieser tae Cie te Rei eee eee ee. Ie ae 135 i INDRODUGLION The eggs of Hydatina senta are very favorable for experiments with the centrifugal machine. The adult females which contain eggs in stages up to and including the first maturation spindle may be centrifuged at the rate of twenty thousand revolutions in two to three minutes without any apparent injury. The animal is so transparent that the eggs can be seen immediately after cen- trifuging and their condition recorded. ‘The materials in the egg are separated into three distinct zones, a pink zone, a middle clear zone and a gray zone. This strat hed material only becomes partly redistributed in the egg before cleavage and sometimes scarcely any redistribution Bes place. After the egg is laid the polar body is formed and the first cleavage appears in thirty- five 10 forty-five minutes. The eggs develop within forty-eight to seventy-two hours and produce in most cases normal embryos. Tue JourNAL or ExPERIMENTAL ZOOLOGY, VOL. VI, NO. I. 126 David Day Whitney The following experiments and observations were made at the suggestion oe under the supervision of Prof. T. H. Mor- gan. II RELATION OF THE FIRST CLEAVAGE PLANE TO THE STRATIFI- CATION OF THE MATERIAL OF EGGS CENTRIFUGED WHEN THE GERMINAL VESICLE IS INTACT If several hundred animals are centrifuged at the same time and are examined immediately many can be seen to contain eggs, the contents of which are sharply differentiated into three zones. As the animals are thrown down during the centrifuging process in any position the stratification of the eggs may lie in any relation to the median longitudinal axis of the female. The pink zone may be toward the head or foot of the animal, toward the side next the stomach or on the opposite side, or in any other inter- mediate position. The gray zone is always on the opposite side of the egg from the pink zone and the middle zone between the two. [he germinal vesicle is found at the junction of the clear middle and pink zones and can be easily recognized owing to its large size (Fig. 1). From the various lots of females, centrifuged twenty thousand revolutions, eight were selected containing the pink zone toward the head of the animal and the gray zone toward the foot. The germinal vesicle was plainly visible near the line between the clear middle and pink zones. Each of these eggs was carefully watched while it remained in the oviduct of the female as well as after it was laid. In every egg the first cleavage appeared at that end of the egg which con- tained the pink pigment. ‘The first division was unequal as it is in the normal egg. In some cases all the pink zone was cut off in the smaller cell while in other cases only a part of it was included in this cell. The gray zone was always included in the larger cell. Six other females which had the pink zone of the egg toward the foot of the animal and the gray zone toward the head were isolated and the further history of each egg was carefully followed. Effect of a Centrifugal Force upon Development and Sex 127 The first cleavage in all these eggs appeared in the end which contained the pink material. It is hardly possible that in these fourteen eggs the pink material and the germinal vesicle were driven in each case to the animal pole. If this assumption is correct these two lots of eggs indicate that the position of the first cleavage 1s determined by the induced position of the germinal vesicle at either end of the egg. When, however, the germinal vesicle is carried to the side of the egg with the pink material, that 1s, when it comes to lie midway between the two ends of the egg, it moves later toward one of the ends of the egg since the first cleavage in such cases is at one end. III RELATION OF THE FIRST CLEAVAGE PLANE TO THE STRATIFI- CATION OF THE MATERIAL OF EGGS CENTRIFUGED WHEN THE MATURATION SPINDLE IS PRESENT Some of the centrifuged females were found containing eggs stratified into the three characteristic zones but showing the maturation spindle apparently at the side of the egg, where it normally forms, in the clear middle zone (Fig. 2). In cases where the pink zone was in the end of the egg toward the head of the animal the first cleavage sometimes appeared in that end of the egg and cut off the pink zone in the small cell. At other times it appeared at the opposite end of the egg and included the gray zone in the small cell. In other cases where the gray zone was inthe end of the egg toward the head of the animal the first cleavage sometimes appeared in that end but at other times it appeared in the opposite end of the egg. When the eggs were centrifuged so that the stratification was from side to side, instead of from end to end the first cleavage usually cut off a part of each of the three zones in each cell. Apparently the first cleavage appeared at that end of the egg Nearest to the maturation spindle irrespective of the stratified zones of the egg. 128 David Day Whitney IV CONDITION OF THE YOUNG ANIMALS WHICH DEVELOPED FROM CENTRIFUGED EGGS If the materials of the egg which are differentiated into zones by the centrifugal force are important formative substances their displacement and their different location in the first cleavage stage ought to cause abnormalities in the embryos and in the mature animals which develop from such eggs. The following experiments which are only a few of those carried out will serve to indicate to what extent the embryos form centri- fuged eggs were affected by the displacement of the materials. Experiment I. January 11, 1908, at 8:30 p.m., many females were centrifuged about twenty thousand revolutions and then placed in a watch glass containing tap-water and allowed to lay their eggs. At 10 p.m., thirty-seven eggs were isolated. January 13, at 9:30 a.m., there were in the dish thirty-two apparently normal young females, three winter eggs and two dead parthenogenetic eggs. Experiment V (Control). January 21, at Io p.m., two hun- dred and eighty eggs that had been laid by females which had not been centrifuged were isolated in a watch glass containing tap- water. January 22, at 8 p.m., two hundred and forty normal young animals were removed from the dish. January 23, at I1 a.m., six normal young removed and of the thirty-four unhatched eggs which remained thirty-one were winter eggs and three were dead parthenogenetic eggs. Experiment XI. March 14, 7:45 p.m., several hundred females were taken from the culture jar and placed in a watch glass con- taining tap-water. No eggs had been laid by 8 p.m. These animals furnished material for the following experiments. Lot A. At 8:35 p.m., twenty-five eggs which had been laid since 8 p.m., were isolated and centrifuged twenty thousand revolutions. March 15, at 12:40 p.m., sixteen young normal females were taken out of the dishand nine unhatchedeggs remained. At g p.m. the nine eggs were still unhatched. March 16, at 10 a.m., two of Effect of a Centrifugal Force-upon Development and Sex 129 the eggs had hatched. Of the seven remaining eggs one was a winter egg, three were dead parthenogenetic eggs and the other three contained living embryos which were apparently unable to break through the egg envelope. Lot B. March 14, at 8:40 p.m., twenty eggs-were isolated and allowed to remain undisturbed. At 9:10 p.m., these eggs were centrifuged in the same way and treated as Lot A. March 15, at 1:30 p. m., seventeen young normal females were in the dish and three unhatched eggs which contained living embryos. At 9 p-m., two of these eggs had produced normal females but the other was still unhatched. Lot GC. March 14, at 8:45 p.m., thirty eggs were isolated and allowed to remain undisturbed. At 9:45 p.m., these eggs, in some of which the first cleavage and in others the second and third cleavage had appeared, were centrifuged and treated as Lot A. March 15, at 1:40 p.m., fourteen normal young females were removed from the dish and sixteen unhatched eggs remained At g p.m., seven other normal young females were removed. March 16, at Io a.m, two other normal young females were removed. Seven eggs remained. One was a winter egg, two were dead parthenogenetic eggs and four contained living embryos. Lot D (Control). March 14, at 10:30 p.m., thirty-three eggs were isolated. March 15, at 12:30 p.m., thirteen normal young females and two normal young males were removed from the dish. At g p.m., seventeen other normal young females and one normal young male were remov d. In Experiment I the eggs were centrifuged when they contained the germinal vesicle or else the maturation spindle. In Experi- ment XI, Lot A, they were centrifuged in the stage when the polar body is forming or just after it was formed. In Lot B the eggs we e centrifuged just before the first cleavage and in Lot C dur- ing and after the first cleavage. In some of these experiments the young animals which de- veloped from the centrifuged eggs seem to die sooner if not fed than do the animals developing from normal eggs. Also there 130 David Day Whitney are more cases of the embryos unable to get out of the egg after they are fully formed. ‘They can be seen writhing and twisting inside the egg and sometimes lived there as long as seven days. These cases of abnormalities were due perhaps to the food material in the egg being displaced and therefore unable to nourish certain muscles which are well supplied with food material in the normal embryo. Consequently the young animal was weaker in certain parts of its body. In a few cases the eggs began to develop but apparently soon ceased and never produced embryos which showed any ciliary movement. In normal embryos the ciliary movement around the head can be seen several hours before hatching. Whether the early death of such eggs is due to abnormal cleavage, mis- placed egg substance, or misplaced chromosomes in division 1s not clear. It is nevertheless apparent from these experiments that a very high percentage of normal young animals develop from eggs that have been centrifuged in the various stages of their early development. Vv) THE PROPORTION OF MALE AND FEMALE PRODUCING FEMALES IN THE FIRST, SECOND AND THIRD GENERATIONS AfPTER CENTRIFUGING If the dislocation of the egg substances has any influence on sex it should become evident by following the history of individual eggs in which the zones of stratification are differently arranged in their relation to the first cleavage plane. The following data give the result of experiments carried out to examine the question. Experiment XXXI. March 5, at 2:15 p.m., a female contain- ing a large egg was centrifuged twenty thousand revolutions. At 3:10 p.m., the egg had been laid and was in the first cleavage stage. [he pink zone was entirely included in the sma'ler cell (Fig. 3). On March 6, at 11 a.m., a normal young female was swimming about in the dish. Food was then added. She produced eggs Effect of a Centrifugal Force upon Development and Sex 131 and on March 8, at 10 a.m., four young daughter-females were present in the dish. This female produced twenty-nine eggs which developed into females. “wo of these daughter-females matured and produced males and twenty-seven matured and produced females. Experiment XX XIII. The conditions, size of egg, and the arrangement of egg material in the first cleavage were approxi- mately the same as in Experiment XXXI. A normal young female developed from the egg, matured and produced males. Experiment XX XVII. March 5, at 2:15 p.m., a female con- taining a large egg was centrifuged. At 4:30 p.m., the egg that had been laid was in the first cleavage stage. The small cell included portions of the pink and clear zones, Fig. 4. On March 6, at II a.m., a normal young female was swim- ming about in the dish. Food was added. This female grew to maturity and produced twenty-five eggs, all of which developed into females. One of these daughter-females matured and _ pro- duced males and twenty-four matured and produced females. Experiment XLIV. The conditions, size of egg, and the ar- rangement of the egg material in the first cleavage were approxi- mately the same as in Experiment XXXVII. A normal young female developed from the egg, matured and produced twenty- five eggs, all of which developed into female-laying females. In later generations males appeared. Experiment XXXII. March 5, at 2:15 p.m.,a female containing a large egg was centrifuged. At 3:30 p.m., an egg had been laid and was in the first cleavage stage. ‘The small cell included por- tions of the three zones (Fig. 5). On March 6, at 9 a.m., a normal young female was present in the dish. Food was added. ‘This female matured and produced only three eggs. One of these eggs developed into a male-laying female and the other two developed into female-laying females. This small production of eggs was due to the scanty amount of food given to the female. Experiment L. March io, 1 p.m., a female containing a large egg was centrifuged. At 3 p.m., the egg had been laid and was in the first cleavage stage. [he small cell contained about two- 132 David Day Whitney thirds of the gray zone and a portion of the clear zone (Fig. 6). On March 11, at II a.m., a normal young female was swim- ming about in the dish. Food was added. ‘This female matured and produced fifteen eggs which developed into females. One of these daughter-females produced males and the other fourteen produced females. Experiment LV. The conditions, size of egg and the arrange- ment of the egg material in the first cleavage were approximately the same as in Experiment L. A normal young female developed from the egg, matured and produced fourteen eggs all of which developed into female-laying females. In later generations males appeared. Several small male eggs were centrifuged in the same manner as the largeeggs. Insome of these the first cleavage plane appeared so as to cut off all the pink zone in the small cell and in others it cut off some of each of the three zones. In both cases apparently normal males were produced. None of the females in the above experiments produced the normal number of eggs, which is forty to fifty, because of poor food conditions. In former experiments it has been shown that the percentage of male-laying females in a family of daughter-females may vary from 0 to 50 per cent and also that the percentage of male-laying females in one generation is no indication what it may be in the next generation. Experiments XXXI to LV. In these experiments the appear- ance of male-laying females from the various forms of centri- fuged eggs does not seem to be markedly different from normal cases. In the experiments where the daughter-females of a family were all female-laying females males always appeared in the later generations, thus showing that no pure female-laying female strains were produced. Moreover, large (female) eggs never produced male animals nor did small (male) eggs ever produce female animals. Effect of a Centrifugal Force upon Development and Sex 133 GENERAL DISCUSSION Lyon has found in the eggs of the sea-urchin, Arbacia, that the first cleavage plane is always at right angles to the plane of stratifi- cation of the egg material. Lillie has shown that the stratification, caused by centrifuged force, of the material in the egg of Chztopterus plays no part in determining the position of the polar lobe. When the germinal vesicle is still intact the egg has a well defined polarity, that 1s, one end is the animal and the other end the vegetative pole. If the germinal vesicle is driven to the vegetative pole the polar spindle which develops from it always migrates to the animal pole and there forms the polar bodies. In later work on sea-urchin’s eggs Morgan and Lyon show that, “while the cleavage conforms strictly to the induced stratification, the gastrulation does not conform to the symmetrical arrangement of the materials. The exceptional cases show that there is no necessary relation between stratification of the materials as such and the embryonic axes.” However, in the eggs of Cumingia Morgan has shown that the stratification of the egg material does not influence the position of the first cleavage plane. He says, “This difference is due to the shifting of the nucleus in the egg of the sea-urchin, while the spindle in Cumingia retains its original orientation.” It must be borne in mind that the above results were obtained from eggs centrifuged at different stages in their maturation. The eggs of Chztopterus were centrifuged when in the germinal vesicle and maturation spindle stages, while the eggs of arbacia were centrifuged when in the female pronucleus stage. ‘The eggs of Cumingia were also centrifuged when in the maturation spindle stage and those of Hydatina in the germinal vesicle stage as well as in the maturation spindle stage. When the eggs of these different animals are centrifuged in the maturation spindle stage the spindle is not usually moved from its original position and consequently the first cleavage take place precisely as it does in normal eggs. When the eggs of Chztopterus and Hydatina are centrifuged 134 David Day Whitney in the germinal vesicle stage the later histories of the germinal vesicles differ. In the egg of Chztopterus the maturation spindle which develops from the germinal vesicle, according to Lillie, migrates to the animal pole if it does not happen to be located at that pole, while in Hydatina the maturation spindle never migrates from the end of the egg into which the germinal vesicle is driven by the centrifugal force. In the egg of Arbacia the female pro- nucleus may be so oriented by centrifugal force that the direction of the first cleavage plane is due rather to its location and follows in consequence the stratification of the materials in the egg. None of the previous workers have reared the embryos from centrifuged eggs to maturity because the forms upon which they worked were not suitable for such experiments but Hydatinais an exceptionally favorable form for such work. Eggs were centrifuged in various stages of maturation so that the zones of egg materials were differently arranged in their relation to the first cleavage plane, thus making it possible that in some cases the pink material of the egg would be included in the cells that make up the anterior end of the embryo while the gray material would be included in the cells of the foot region or vice- versa. In other cases the material would be more or less equally distributed in the anterior and posterior regions of the embryo. In no case was the sex of the eggs changed and such eggs pro- duced a very high percentage of normal young males and females. Furthermore the young females grew to adult animals and _pro- duced normal offspring of which the sex ratio was apparently normal. It would, therefore, seem that the effect of centrifugal force upon the eggs of Hydatina senta is not sufficient to cause any noticeable change of structure or of sex in the animals that develop from them. VI. SUMMARY 1 When the unsegmented eggs of Hydatina senta are centri- fuged twenty thousand revolutions the materials in the eggs are stratified into a pink zone, a clear middle zone and a gray zone. Effect of a Centrifugal Force upon Development and Sex 135 2 Ifeggs are centrifuged a short time before maturation when the nucleus is intact the nucleus is carried to the top of the clear zone against the bottom of the pink zone. 3 Very little redistribution of the egg materials takes place before the first cleavage. In consequence the segmented egg retains the distribution of materials impressed on it by the centrifugal force. 4. The first cleavage plane always appears at that end of the egg at which the pink zone and germinal vesicle are located. It forms across one end as in the normal egg separating a smaller and a larger cell. 5 The egg centrifuged after the polar spindle has formed shows that the spindle does not move from its original position. Its location determines the position of the first cleavage plane in so far as this appears at the end of the egg nearest to where the spindle lies. 6 Normal animals, both males and females, develop from centrifuged eggs and these have been reared to sexual maturity. 7 The sex of animals developing from large (female) or small (male) eggs is not affected by the centrifugal force nor is the sex ratio in the descendants of females developing from centrifuged eggs altered. Zoological Laboratory Columbia University April 25, 1908 LITERATURE Hertwic, O. ’97—Ueber einige am befruchteten Froschei durch Centrifugalkraf hervorgerufene Mechanomorphosen. Sitzungsbere preuss. Akad. Wiss. Berlin, math-phys. Klasse. °99—Beitrage zur experimentellen Morphologie und Entwicklungs- geschichte. 4. Ueber einige durch Centrifugalkraft in der Ent- wicklung des Froscheies hervorgerufene Veranderungen. Arch. f. mikr. Anat., liii. °04—Wietere Versuche iiber den Einfluss der Centrifugalkraft auf die Entwicklung tierischer Eier. Arch. f. mikr. Anat., Ixiti. Lituiz, F. *’06—Observations and Experiments Concerning the Elementary Phe- nomena of Embryonic Development in Chztopterus. Journ. Exp. Zodl., vol. iii. 136 David Day Whitney Lyon, E. P. ’o6—Some Results of Centrifugalizing the Eggs of Arbacia. Amer. Journ. Physiol, xv. Morean, T. H. ’06—The Influence of a Strong Centrifugal Force on the Frog’s Egg. Arch. f. Entw. Mech., xxii. °o7—The Effect of Centrifuging the Eggs of the Mollusc, Cumingia. Science, n. s., vol. xxvii, no. 680, pp. 66-67. Morcan AnD Lyon ’07—The Relation of the Substances of the Egg, separated by a Strong Centrifugal Force, to the Location of the Embryo. Arch. f. Entw.-Mech., xxiv. WertzeL, G. ’o4—Centrifugalversuch an unbefruchteten Eiern von Rana fusca. Archiv. f. mikr. Anat., xiii. DESCRIPTION OF PLATE Fig. 1 Section of a centrifuged egg, showing the three zones and the germinal vesicle located near the boundary of the pink andthe clearzones. Gilson mercuro-nitric fixing fluid and Heidenhain’s iron hematoxylin stain. Fig. 2 Section of a centrifuged egg showing the three zones and the maturation spindle in the clear zones. Yolk granules are lodged against the achromatic figure. Bouin’s fixing fluid andHeiden- hain’s iron hematoxylin stain. Figs. 3to06 Free hand semi-diagrammatic drawings of living eggs in the first cleavage stage which were centrifuged when in the oviducts of the females. The fine stippling shows the position of the pink zone, the coarser stippling shows the position of the gray zone, and the clear space indicates the clear zone. Fig. 3 Experiment XXXI—The pink zone included in the small cell and the gray and clear zones are in the larger cell. Fig. 4 Experiment XXXVII—About one-half of the pink zone and a portion of the clear zone included in the small cell and the other part of the pink zone, all the gray zone, and a part of the clear zone are included in the larger cell. Fig. 5 Experiment XXXII—Portions of each of the three zones in each of the two cells. Fig. 6 Experiment L—About two-thirds of the gray zone included in the small cell while the other third of the gray zone together with all of the pink zone and nearly all of the clear zone are included in the larger cell. EFFECT OF A CENTRIFUGAL FORCE UPON DEVELOPMENT AND SEX Davin Day WHITNEY 5 Tue JourRNAL or ExPERIMENTAL ZOOLOGY, VOL. VI, NO. I. OBSERVATIONS ON THE MATURATION STAGES OF THE PARTHENOGENETIC AND SEXUAL EGGS OF HYDATINA SENTA BY DAVID DAY WHITNEY HU TEC CLIN GEL ONL orator aya ate (6 oes ese toyota fete el fajcicle/ hey sielavorsisieqsje's aire) e:e versie sere olsieiets) Gscteve Slate wate eres 137 ie Watertaliand method sreryetar ait eeersarve = eieteraie a sels ae oe wef srove ess Sicreiaiaie Se wetoteelee the 138 EE Bemialeien geminata As IS soso TSE ee Oe OTe o ciskae Halo eas ME Lane eRe ele mots 140 I MES GTe. Sebhott oo bats 5 6 Opal OWb 6 to oo Eco Crem Il ster eae arg ole ear ne Ae ge Re eri 141 Wi ANEAWSTRSEE Ss ero Alec cig Bre ERI PACS ee CSIRO ACER CRE EE Ear eee ond Se 142 Wit Grenaiaalll teres ons he atc ba ene rs Sc CR ON ene pS ee 143 DUST RASS SATYNNEL ALG pare Pety ah eneyepetacay= eotere Vote ravel ae aVeVGs G0 Ghats Gres via Dave @ vial ele «lone, dente’ domargviolo geile ators 144 WIDOL ULSI RNRE SS Sacre SO che Seo EIA pO See AL aC a ene Re MRR REP ot PS RY 145 I INTRODUCTION Despite the experiments that have been carried out by several workers to discover how sex is determined in parthenogenetic eggs the attempts to show that such external factors as tempera- ture or food influence the result do not appear to have been suc- cessful. Attention has turned more recently to the possibility that there are internal factors in the eggs that are all-important in producing males or females. As early as 1845 Dzierzon brought forward very strong evidence to show that the eggs of the honey-bee, Apis mellifica, always develop into males if unfertilized, but if fertilized they develop into females (queens or workers). In other words, internal rather than external agents bring about the result. This theory has been often attacked and strongly defended, and now seems to be generally accepted. In the aphids Balbiani and Stevens find that the same female may produce both male parthenogenetic and fertilized or winter eggs. Lauterborn finds the same phenomenon in the Rotifer, Asplanchna, and Issakowitsch in a Daphnid. Whether the eggs that are fertilized are originally male eggs or develop from a dif- Tue JourNAL or ExPpERIMENTAL ZOOLOGY, VOL. VI, NO. I. 138 David Day Whitney ferent part of the ovary is not certain, but the evidence seems to indicate that they are male eggs. In the two kinds of parthenogenetic eggs of some of the aphids Stevens finds that there is no reduction in the number of the chromosomes during the formation of the polar body. Lenssen, in a study of the parthenogenetic eggs of the Rotifer, Hydatina senta, finds that there is a reduction in the number of the chromo- somes during the formation of the polar body in the male egg but no reduction in the female egg. Weismann found that both kinds of parthenogenetic eggs of Polyphemus (Daphnid), certain Ostracods and Rotifers produced only one polar body while the fertilized eggs produced two polar bodies. Blochmann and Stevens found the same relation to hold ' for certain aphids. In Lisparis dispar, a parthenogenetic Lepidopteran, Platner found that two polar nuclei were formed. In the bee apis, Bloch- mann, Paulcke, Petrunckewitsch and others find that the partheno- genetic eggs which develop into male animals give off two polar bodies. In the Rotifer, Asplanchna, Mrazek, Erlanger and Lauterborn found that the female parthenogenetic egg gave off one polar body and that the male parthenogenetic as well as the fertilized egg gave off two polar bodies. In the parthenogenetic eggs of Hydatina senta Lenssen thought that the male egg gave off one polar body and the female egg gave off none! At the suggestion and under the supervision of Prof. T. H. Morgan the following work upon the eggs of Hydatina senta was done with the view of obtaining more light upon the maturation stages and their relation to the determination of sex. I am also indebted to Prof. E. B. Wilson for many valuable suggestions and criticisms. II MATERIAL AND METHODS The Rotifers were collected and reared in cultures as described in a former paper. The first maturation spindle is formed before the egg is laid and in order to study the early maturation stages animals con- Maturation of Parthenogenetic and Sexual Eggs 139 taining eggs were killed and fixed in masses of thousands and sectioned in toto. Many eggs were found in the desired stages, but as the eggs are filled with yolk granules of various sizes it was exceedingly difficult to find many sections in which the yolk granules were not mingled with the chromosomes. Hot sublimate acetic, Bouin’s fluid, strong Flemming, Gilson, Carnoy, and alcohol acetic, were used as killing and fixing fluids. Some good preparations were obtained by each method, but alcohol acetic gave the best results in obtaining equatorial plates; for it coagulated the cytoplasm of the egg in such a way as to embed the yolk granules in its meshes, thus leaving the spindle and its chromosomes free from yolk granules. ‘Thousands of animals were sectioned and about three hundred good slides were made. Sections were cut 5 in thickness in 51° to 52° C. paraffine. Many parthenogenetic females were also isolated separately and the sex of their offspring determined, for those eggs first laid, before the females were killed and sectioned. ‘The general nature of the maturation stages of such eggs was determined before a more detailed study was made of the eggs in the mixed slides. After the eggs are laid the envelope around them is so thin and at the same time so exceedingly impervious to fixing fluids that the eggs usually collapse in the process of fixation. Sometimes a few do not collapse in alcohol acetic but, however careful one may be, by the time the eggs are embedded they have shrunken. In such eggs the yolk granules are so crowded in among the chromosomes and stain so darkly that no satisfactory results can be obtained. In order to free the spindle from these granules the eggs were first centrifuged. In sections of such eggs the maturation spindle remained in the clear middle zone of the egg and was often entirely free from yolk granules. As only a few sections of these eggs were made no good stages were found in which the chromosomes could be counted but the method gives promise of results that can not be obtained in other ways. Heidenhain’s iron hematoxylin was used chiefly and gave the best results although many other stains were tried. In order to see the polar bodies the eggs, some time after they I 40 David Day Whitney were laid, were put into Schneider’s aceto-carmine for about thirty seconds and then into a water-glycerine solution (1 drop in 5 cc. of water). ‘he blastomeres become separated and the polar bodies can be readily seen. III FEMALE EGG The female egg is easily distinguished from the male egg by its larger size and is never mistaken for the winter egg which may be of equal size, but has a much thicker envelope around it, besides containing the conspicuous sperm nucleus. In the female parthenogenetic egg the number of chromosomes was never definitely determined but many spindles in metaphase were seen in side view, containing numerous chromosomes (20 to @ ee aA is 08°, 3 ut ‘ ~ + A Be Fig. 1 Female parthenogenetic egg. A, equatorial plate of the polar spindle, showing twenty- three to twenty-five chromosomes; B, prophase of polar spindle, showing twenty-two chromosomes. 30). In one polar view of a metaphase twenty-five chromo- somes were seen, Fig. 1, 4. In a prophase twenty-two dumb-bel shaped chromosomes were seen 1n one section (Fig. 1, B) and in the adjoining section there were four other dumb-bell shaped chromosomes together with one that was not constricted. No anaphase or telophase stages were found although hundreds of eggs were examined. Lenssen found the chromosomes somewhat scattered about on the equator of the maturation spindle and con- cluded they were in an early anaphase but since he considered the unreduced number to be ten or twelve chromosomes the twenty or more chromosomes that he saw were probably in an early meta- Nore—The drawings of the chromosomes were made as carefully as possible with a camera under a 1.5 mm. Zeiss apochromatic and compensation ocular 6. They were then enlarged with a drawing camera about three times, corrected by comparison with the objects, and reduced by one-third in repro- duction. Maturation of Parthenogenetic and Sexual Eggs. 141 phase instead of in anearly anaphase. He never saw a telophase stage and decided without any evidence that the chromosomes never separated beyond the early anaphase stage and that later all the chromosomes form the segmentation nucleus. This is probably not the case because one polar body can always be seen near the periphery of the egg after the first cleavage, in total mounts prepared by the method already described. Some- times a constriction can be seen across the middle of the polar body giving it the appearance of being divided into two parts. In the two-cell stage of the egg after the two blastomeres separate the polar body is always found in the space between the two cells (Fig. 2, 4-B). In the four-cell stage it is seen at the point of junc- ture of the four cells (Fig. 2, C). ~ ™~ A sae —— Fig. 2 Female parthenogenetic egg. 4, B, eggs in the two-cell stage, showing one polar body; C, egg in the four-cell stage, showing one polar body at the intersection of the two cleavage planes. TV MALE EGG The male egg is much smaller than the other two kinds of eggs and has a thin envelope around it similar to that of the female parthenogenetic egg. The maturation spindle was seen several times when the chromosomes were in metaphase, anaphase and telophase stages. In two cases of telophase ten and fourteen chromosomes respectively were counted on one end of the spindle (Fig. 3, C-D). Polar views of the metaphase stage showed clearly eleven to thirteen chromosomes (Fig. 3, 4-B). “They were always less in number and larger in size than the chromosomes in the metaphase stage of the female parthenogenetic eggs. 142 David Day Whitney Three polar bodies are to be found near the periphery of the egg close to the line of meeting of the blastomeres. One was usually larger than the other two and often at a little distance away from them (Fig. 4, 4-B), although in one instance the three polar bodies were close together and seemed to be of the same size (Fig. 4, C). 4 1 Oe . SX te fre is pits 7 he ots on “VP : i m y ‘ = D Fig. 3. Male parthenogenetic egg. A, B, equatorial plates of the polar spindle, showing twelve to thirteen chromosomes; C, D, polar spindle in telophase, showing ten to fourteen chromosomes. Lauterborn states that in the male parthenogenetic egg of Asplanchna the first of the two polar bodies which was extended usually divided. Lenssen concluded that only one polar body was formed because he saw the maturation spindle in the telophase stage. He did not follow the history of the chromosomes in the later stages and conse- quently never saw any polar bodies. Fig. 4 Male parthenogenetic egg. A, B, eggs in the four-cell stage, showing three polar bodies, two of which are smaller than the other; C, egg in the two-cell stage, showing three polar bodies of nearly the same size. Vi WINTER. EGG The fertilized or winter egg has a very thick envelope. An oval shaped small body which is probably the sperm nucleus is always found near the egg nucleus. ‘The chromosomes were seen in sections on the maturation spindle (side view) in all stages but in only two anaphase stages (Fig. 5, Maturation of Parthenogenetic and Sexual Eggs 143 C-D), could they be counted because of being too closely crowded together. ‘The polar view of the metaphase in the alcohol acetic fixation gave the best results. Fourteen chromosomes were seen in several sections of different eggs (Fig. 5, 4-B). The chromo- somes were of about the same size as those in the metaphase of the male parthenogenetic egg and were much larger in size and less in number than those in the metaphase of the female partheno- genetic egg. j a e8 es er LD eae e i j ‘ i i § @| jt ile ous He g - e. . €3 RY Cw 8 008 2 ~~ F -w@ e393 e 9 Fore e%e we « : @ e i ) A ee B G we ay fF : \’ D Fig. 5 Winter or fertilized egg. A, B, equatorial plates of the polar spindle, showing fourteen chromosomes; C, D, anaphases of the polar spindle, showing twelve to fourteen chromosomes on each end of the spindle. On account of the thick and opaque egg envelope, the polar bodies were never seen. \l GENERAL DISCUSSION Although Lenssen was mistaken in regard to the number of the chromosomes nevertheless he was firmly convinced that the num- ber in the maturation stages of the male parthenogenetic and the fertilized egg were the same and that the number in the female parthenogenetic egg was greater. By comparing my Figs. 1, 3 and 5, it will be seen that this conclusion is confirmed. ‘The greatest number of chromosomes seen in an equatorial plate of the male egg was possibly thirteen and the number seen in an equatorial plate of a winter egg was fourteen. The chromosomes of both eggs in the same stages were of the same size. In the female parthenogenetic egg the greatest number of chro- mosomes seen was twenty-five (Fig. 1). “The chromosomes were 144 David Day Whitney very much smaller than in the other two kinds of eggs and usually were so crowded together that it was impossible to count them except in a very few cases. These observations show that there is probably a reduction in the number of chromosomes in the male parthenogenetic and winter egg but no reduction in the female parthenogenetic egg. The former case would be similar to what occurs in the honey- bee. In the aphids Stevens found that there is no reduction in the number of chromosomes in either of the male or female par- thenogenetic eggs but only in the fertilized egg. It appears that in different animals parthenogenetic eggs vary in the number of polar bodies that they give off. The male egg of Asplanchna, Hydatina and Apis gives off two polar bodies while the male egg of aphids gives off only one. If it is true that the male egg when fertilized becomes the winter egg which develops into a female it seems evident that the reduction in the number of chromosomes and the formation of the second polar body is not in itself the factor that determines the ultimate sex of the egg. The sperm would seem to introduce a factor that determines the sex of the embryo. ‘This idea is strongly suggested by the evidence that Meves has brought forward in the case of the honey- bee in which he finds that only one kind of functional sperm is produced. Morgan also finds a similar phenomenon for certain Phylloxerans. If the same process occurs inthe sperm of Hydatina the cause of the change in sex of the male egg may be at least surmised. Vil SUMMARY 1 In the female parthenogenetic egg of Hydatina senta there is no reduction in the numberof chromosomes during maturation. One polar body is extruded. 2 Inthe male parthenogenetic egg there is a reduction in the number of chromosomes during maturation. Two polar bodies are formed, one of which subsequently divides. 3 In the winter egg, that becomes fertilized, there is a reduc- tion in the number of chromosomes during maturation, and since Mths sd Maturation of Parthenogenetic and Sexual Eggs 145 a similar process of reduction takes place in the parthenogenetic egg that becomes a male it would seem to follow that the sex of the embryo from this egg is changed by the spermatozoon. Zoblogical Laboratory Columbia University April 25, 1908 LITERATURE CITED. Basianl, E. G. ’69—’72—Memoire sur la génération des aphides. Am. Sc. Nat. Ser, 5. 2oolen tie Tis VeOOsy be t4, 18705) E25, 1872: Biocumann, F. ’88—Ueber die Richtungskérper bei unbefruchtet sich entwick- elnden Insekteneiern. Verh. naturh. med. Ver. Heidelberg, N. F., vol. iv, no. 2. ’8g—Ueber die Zahl. der Richtungskérper bei befruchteten und unbe- fruchteten Bieneneiern. Verh. naturh. med. Ver. Heidelberg, N. F., vol. iv, pp. 239-41; fr. R. Mic. Loc., 1889. Braver, A. ’94—Zur Kenntniss der Reifung des parthenogenetisch sich entwick- elnden Eies von Artemia salina. Arch. mikr. Anat., vol. xi. CastLe, W. E. ’03—The Heredity of Sex. Bull. of the Mus. of Comp. Zool. Harvard College. vol. xl, no. 4. Dzterzon, J. ’45~76—[For a complete list of the writings of Dzierzon, see Biblio- theca Zoologica, ii, p. 2270.] ERLANGER U. LAauTERBORN ’97—Ueber die ersten Entwickelungsvorgange im parthenogenetischen und _ befruchteten Raderthierei. Zool. Anz., vol. xx. Issakowitscu, A. ’05—Geschlechtsbestimmende Ursachen bei den Daphniden. Biol. Centralb., xxv. Laurersorn, R. ’98—Ueber die cyclische Fortpflanzung limnetischer Rotatorien. Biol. Centralbl., xviii. Lenssen ’98—Contribution 4 |’étude du developpement et de la maturation des ceufs chez Hydatina senta. La Cellule, xiv. Maupas, M. ’9g1—Sor le déterminisme de la sexualité chez |’Hydatina senta, Ehr. G@URS Acs Se. Paris, cxin. Meves, F. ’07—Die Spermatocytenteilungen bei der Honigbiene. Arch, mikr. Anat.s: xx. Moraan, T. H. ’08—The Production of Two Kinds of Spermatozoa in Phylloxerans. Functional “Female Producing” and Rudimentary Sperma- tozoa. Proceedings of the Society for Experimental Biology and Medicine, vol. v, no. 3. 146 David Day Whitney Mrazexk, AL. ’97—Zur Embryonalentwickelung der Gattung Asplanchna. Jahresb. béhna Ges., 2, pp. I-II. Nusssaum, M. ’97—Die Entstehung des Geschlechts -bei Hydatina senta. Arch. mikr. Anat., xlix. Pautcke, W. ’99—Zur Frage der Parthenogenetischen der Drohnen. Anat. Anz., vol. xvi. PetruNKEwItscH, A. ’o1—Die Richtungskorper und ihr Schicksal im befruch- teten und unbefruchteten Bienenei. Zool. Jahrb., vol. xiv. Puitiirs, E. F. ’03—A Review of Parthenogenesis. Amer. Phil. Soc., vol. xlii. Pratner, G. ’88—Die erste Entwickelung befruchteter und parthenogenetischer Fier von Lisparis dispar. Biol. Centrabl., vol. viii, no. 17. ’8g—Ueber die Bedeutung der Richtungskérperchen. Biol. Centrabl. vol. vill. Stevens, N. M. ’o04—A study of the Germ Cells of Aphis rosz and Aphis cenotheree Journ. Exp. Zool., vol. ii. WEISMANN AND IscHikawa ’88—Weitere Untersuchungen zum Zahlengesetz der Richtungskorper. Zool. Jahrb., vol. 11. WEIsMANN, A. ’80—Parthenogenesis bei den Ostracoden. Zool. Anz., iii. Wuitney, D. D. ’07—Determination of Sex in Hydatina senta. Journ. Exper. Zool., vol. v. Witson, E. B., ’°05—Studies on Chromosomes. Jour. Exp. Zoél., vols. iiand iii. STUDIES ON CHROMOSOMES V THE CHROMOSOMES OF METAPODIUS. A CONTRI- BUDION TO THE HYPOTHESIS OF THE’ GENETIC CONTINUITY OF CHROMOSOMES’ BY EDMUND B. WILSON Witn One Piate anp THIRTEEN FIGURES IN THE TEXT The genus Metapodius (Acanthocephala), one of the coreid Hemiptera, shows a very exceptional and at first sight puzzling relation of the chromosome-groups which has seemed to me worthy of attentive study by reason of its significance for the hypothesis of the “individuality” or genetic continuity of the chromosomes. The most conspicuous departure from the relations to which we have become accustomed lies in the fact that different individuals of the same species often possess different numbers of chromo- somes, though the number in each individual is constant. An even more surprising fact is that in all of my own material every male individual possesses at least 22 spermatogonial chromosomes, including a pair of unequal idiochromosomes like those of the Pentatomidz, while in Montgomery’s material of M. terminalis every male has but 21 spermatogonial chromosomes, one of which is a typical odd or “accessory” chromosome (unpaired idiochro- mosome).? The present paper presents the results of an investigation of these relations that has now extended over nearly four years, in the course of which serial sections of more than sixty individuals 1 Part of the cost of collecting and preparing the material for this research was defrayed from a grant of $500 from the Carnegie Institution of Washington, made in 1906. I am indebted to Rev. A. H. Manee, of Southern Pines, N. C., for valuable codperation in the collection of material, and to Dr. Uhler, Mr. Heidemann, Mr. Van Duzee, and Mr. Barber for aid in its identification. 2 By Professor Montgomery’s courtesy I have been enabled to study thoroughly his original prep- arations and to satisfy myself of the correctness of his account (Montgomery ’o6). I also owe to him a number of unsectioned testes of the same type. THE JouRNAL or EXPERIMENTAL ZOOLOGY, VOL. VI, NO. 2. 148 Edmund B. Wilson have been carefully studied. ‘These individuals belong to three well marked species—M. terminalis Dall. and M. femoratus Fab. from the Eastern and Southern States, M. granulosus Dall. from the Western—all of which show a similar numerical variation.’ My first material, including sections of two testes of M. terminalis (Nos. 1, 2) from the Paulmier collection, long remained a complete puzzle and led me to the suspicion that the material was patho- logical. ‘This possibility was eliminated by the study of additional material of the same type; but the contradiction with Montgom- ery’s results on the same species suggested that his specimens were not. correctly identified (Wilson ’o7a). Continued study at length convinced me that this supposition too was probably un- founded. If the identification was correct, as I now believe it was, M. terminalis 1s a species that varies not only in respect to the individual chromosome number but also in respect to the sex- chromosomes, certain individuals having an unpaired “accessory” chromosome, while others have an unequal pair of idiochromo- somes. The latter condition alone has thus far been found in M. femoratus and M. granulosus. ‘The essential facts, and the general history of the spermatogenesis, are otherwise closely similar in the three species. The range of variation in the number of chromosomes is in M. terminalis from 21 to 26,in M. femoratus from 22 to 27 or 28, and in M. granulosus from 22 to 27, the particular number (or its equivalent in the reduced groups) being a characteristic feature of the individual in which it occurs. I do not mean to assert that there is absolutely no fluctuation in the individual. In this genus, as in others, apparent deviations from the typical number fre- quently are seen, and real fluctuations now and then appear; but the latter are so rare that they may practically be disregarded. That the number may be regarded as an individual constant (subject to such deviations as are hereafter explained (p. 185) is abundantly demonstrated, not only by the agreement of large numbers of cells from the same individual but perhaps even more 3A complete list of the individuals examined, arranged by localities, is given in the Appendix at p--202, Each individual is there designated by a number by which it is referred to in the text and description of figures. Studies on Chromosomes 149 convincingly by the definite correlation of the spermatocyte- groups with those of the spermatogonia of the same individual. This is shown in the following table, which summarizes the facts thus far observed.‘ SUMMARY Somatic number terminalis femoratus | granulosus First spermatocyte (spermatogonia or division ovarian cells) of 9 rsp i MeN NT or 2 53s yc Re GeO eee ene | Il Gy | fe) ro) fo) ° 23150 Sob Gn COE Oe 12 3 4 3 ° I ce) 230): oc oS eae CO ae 13 fiestas 2 ° 2 2 fe) ME es ece Gi.fa end a's « 14 3 sg lie a (ae 4 ° Bieneces ree sicccesceccece | 15 2 2a ° ° I I 0.0 uA ROO eee 16 I o | 2 | 4 | 2 (GP iich Soe CO Reece 17 Pe prore Whe 2c | fo) co) I ° SRA Nerestests «seein aietie2 | | fo) Salle Foe SOR eee Distribution in the whole group Total somatic number Number of males Number of females | Totals acs | Dil 5 -.3qe tec AAC aoa 9 ro) 9 TEse8c ia AGRO Ee aE 7 4 II PE oac08 Ce Oe ee I 7 | 4 II Ui oote boee Boe ee aeereo ees oe 9 4 13 2G soagediche oaoHe pag eAgep aaoge 3 3 6 2 san hdic uC CCE 7 3 10 (Ga) \s.gttg 68 Be eee ee I | ro) | I okt (C7 cece eo nee Ose eee ) I | I Mt ere ans fea tsas oe Ses 3 19 62 4 The somatic numbers of the males are in each case determined from the dividing spermatogonia. Those of the female are from dividing cells in various parts of the ovary—mainly from the region just above or below the end-chamber—some of them undoubtedly folicle-cells, others probably young nutri- tive cells or odgonia. The chromosome-groups from different regions differ considerably in size, but otherwis show the same general characters. With a very few exceptions the number of chromosomes has been determined by the count of several groups from the same gonad, in many cases by the count of a very large number. In many individuals hundreds of perfectly clear equatorial plates may be seen and the evidence is entirely demonstrative. In seven of the males (owing to lack of mitoses, or to defec- tive fixation) the somatic number has been inferred from that shown in the spermatocyte divisions, or vice versa; but with a single exception both numbers have been directly observed in other individuals of the same type. I am therefore confident that the numbers are substantially correct as given. In case of the female, only the somatic numbers can be given, since the maturation-divisions are not available for study. 150 Edmund B. Wilson The material of terminalis is from New Jersey, Pennsylvania, Ohio, North Carolina, South Carolina and Georgia; that of femor- atus from the three states last named; that of granulosus from Arizona. ‘The variation of number is independent of locality, and individuals of the same species showing different numbers were often taken side by side on the same food plants. It is equally independent of sex, as the table at once shows. I am unable to find any constant correlation between the number of chromosomes and any other visible structural characters of the adult animals. Such an astonishing range of variation in the chromosome num- ber in the same species seems at first sight to present a condition of chaotic confusion. But, as I shall endeavor to show, the first impression thus created disappears upon more critical examination. Detailed study of the facts proves that the variation is not indis- criminate but affects only a particular class of small chromo- somes that are distinguishable from the ordinary ones both by size and by certain very definite peculiarities of behavior. ‘These chromosomes are absent in all of Montgomery’s material; in my own they are sometimes present, sometimes absent, the total num- ber varying accordingly. [he chromosomes in question are the ones which in earlier papers I have called the “ supernumeraries.””S In behavior they show an unmistakable similarity to the idiochro- mosomes; and for reasons given beyond I believe them to be noth- ing other than additional small idiochromosomes, the presence of which has resulted from irregularities of distribution of the idio- chromosomes in preceding generations. The relations seen in Montgomery’s material form the converse case, the small idio- chromosome having disappeared or dropped out. I shall try to show that both cases are probably due to the same initial cause. 5 Wilson ’07a, ’07b. I first discovered this phenomenon in the pentatomid species Banasa calva (’osb) describing the single supernumerary as a “‘heterotropic chromosome.” Later (’o7a) a single supernumerary was found in certain individuals of Metapodius terminalis, and other numerical varia- tions in this species and in femoratus and granulosus were briefly recorded; but at that time I did not yet fully understand the facts. Banasa calva is the only form oustide the genus Metapodius, in a totalof more than seventy species of Hemiptera I have examined, in which supernumerary chromosomes have been found. Miss Stevens (’o8b) has recently found in the coleopteran genus Diabrotica a condition that is in some respects analogous to that seen in Metapodius. Studies on Chromosomes 151 A GENERAL DESCRIPTION Since the phenomena as a whole are somewhat complicated, | have thought it desirable to bring the most essential facts together for ready comparison in a preliminary general account illustrated by a limited number of selected figures (Figs. 1, 2). The funda- mental type of the genus is, I believe, represented by individuals that possess 22 chromosomes in the somatic groups of both sexes, and in which no supernumeraries are present (Fig. 1, d-/). Two of the chromosomes are a pair of very small m-chromosomes, like those of other coreids; two are a pair of idiochromosomes consist- ing in the male of a large and a small member, in the female of two large ones; while the remaining 18 are ordinary chromosomes or “autosomes.”” ‘These chromosomes have in the spermato- genesis the same general history as in other Hemiptera heteroptera. In the first division the idiochromosomes are separate univalents, their position being typically (but not invariably) outside a ring formed by the nine larger bivalents within which les the small m-chromosome bivalent (Fig. 1, d, Photo 2). This division accordingly shows 12 separate chromosomes (one more than the reduced or haploid number.) In the second division, as des- cribed beyond, they are always united to form a dyad or bivalent, composed of two unequal halves, and the number of separate chromosomes is 11. The spermatogonial groups possess 22 chro- mosomes (Fig. 1, ¢) of which the small idiochromosome may often be recognized as the smallest of the chromosomes next to the m-chromosomes; but it does not differ sufficiently in size fromthe other chromosomes to be always certainly distinguishable.* In the growth period the idiochromosomes, as usual, have the form of condensed deeply-staining chromosome-nucleoli, while the other chromosomes are in a vague, faintly staining condition. ‘They are usually in contact but not fused (Eig. 1, 7, Photo 25), thus form- 6 In considering the relative size-relations it is important to bear in mind that the apparent size, as seen in polar view, varies considerably with the degree of polar elongation. Still more important is the fact (which I have emphasized in a preceding paper) that in the first division univalent chromosomes always appear relatively much smaller than they do in the spermatogonia. This is the case with the idiochromosomes and the supernumeraries, which are always readily recognizable in the spermatocyte- divisions, but are often difficult to distinguish in the spermatogonia. 152 Edmund B. Wilson EXPLANATION OF FIGURES Fic. 1 About one-fourth of the figures were drawn upon enlarged photographs by the method described in a preceding paper (Wilson ’o9). The others are from camera lucida drawings. In all cases the form, size, and grouping of the chromosomes are represented as accurately as possible. The form, size, and general appearance of the spindles are shown, but no attempt has been made to represent the exact details of the fibrille. Figs. 1 and 2 are enlarged about 3300 diameters, the others a little less than 3000 diameters. Lettering, in all the Figures I, large idiochromosome or odd chromosome; /, small idiochromosome; m, m-chromosome; f, plas- mosome; s, supernumerary chromosome. In cases where s and : are both present and of equalsizeit is impossible to distinguish between them. In such cases I have as a rule designated as 7 the one lying nearest to J; but this is quite arbitrary. It should be noted also that J cannot always be distinguished from the smaller of the ordinary bivalents. — Studies on Chromosomes Fic. I M. terminalis a-c (No. 3), 21-chromosome form; a, first spermatocyte metaphase; b, spermatogonial metaphase; c, nucleus from the growth period. d-f (No. 19), 22-chromosome form, stages corresponding to above. g-t (No. 20, Photo 4), 23-chromosome form, one large supernumerary. j-l (No. 43), 23-chromosome form, one small supernumerary. 154 Edmund B. Wilson ing a very characteristic bipartite body; but in a good many cases they are separate (Fig. 6, c,d, Photo 26). A large and very dis- tinct plasmosome is also present. Such a group of 22 chromosomes may be regarded as the type of which all the other forms may be regarded as variants, and probably as derivatives. In forms having more than 22 chromo- somes the increase in number is due to the presence of from one to six supernumeraries. [hese vary in number and size in different individuals, but both are constant in a given individual. Their maximal size is equal to that of the small idiochromosome (in which case they are indistinguishable from the latter); such forms will be. called “large supernumaries.” Their minimal size, (“small supernumeraries’’) is about the same as that of the m- chromosomes; but from the latter they are always distinguishable, in the male, by a quite different behavior in the maturation pro- cess. When a single supernumerary is present it may be either large or small, its size being (with slight variation) constant in the individual. When more than one is present all may be of the same size (the most usual condition) or they may be of different sizes, the relation being again an individual constant. Whatever their number or size their behavior is essentially the same as that of the idiochromosomes. In the growth-period they have a con- densed form and are typically united with the idiochromosomes to form a compound chromosome-nucleolus, the components of which are often distinctly recognizable and vary in number with the number of the supernumeraries. In the first division they divide as separate univalents, and this division accordingly shows as many chromosomes above 12 as there are supernumeraries— i.e., if the spermatogonial number be 22 + n, the number in the first division is typically 12 + ». ‘Their typical position in this division is, like that of the idiochromosomes, outside the ring of larger bivalents, though there are many exceptions. In the sec- ond division they are, as a rule, again associated with the idio- chromosomes to form a compound element, though not infre- quently one or more of them may be free from the others. A definite correlation thus appears in each individual between the number and relative sizes of the chromosomes seen in the Studies on Chromosomes 155 maturation-divisions and in those of the spermatogonia; and it also appears in the number and size of the components of the chromosome-nucleoli when these can be distinctly recognized. Figs. 1 and 2 illustrate this correlation and epitomize the most essential facts. “hese figures have been selected from a much larger number to show the clearest and most typical conditions. Some of them are enlarged from the photographs reproduced in Plate I. Many others, with an account of secondary variations, are given beyond. Each horizontal row of figures represents three stages of the same type which, with two exceptions, are all from the same individual. ‘The left hand figure in each row shows the typical arrangement of the chromosomes in the metaphase of the first spermatocyte-division, the middle figure a spermato- gonial group, and the right hand one a nucleus from the growth period, to show the chromosome-nucleolus together with some of the diffused ordinary chromosomes. Fig. 1, a—c (terminalis, No. 3), represent these three stages in an individual of the 21-chromosome type (Montgomery’s material) showing 11 chromosomes in the first division, 21 in the sper- matogonia, and a single chromosome-nucleolus in the growth period. (Additional figures of this individual in Fig. 3.) Fig. 1, d-f (terminalis, No. 19), show the 22-chromosome type, with a small idiochromosome present in addition to the large one. The small idiochromosome (z) 1s distinguishable in Fig. 1, e. (Additional figures in Figs. 4-6.) Fig. 1, g-1 (terminalis, No. 20), show the 23-chromosome type, with one large supernumerary. In the spermatogonial group (h) this chromosome and the small idiochromosome are probably rep- resented by the two designated asi and s. The nucleus from the growth-period (7), shows the plasmosome (p) and a tripartite chromosome-nucleolus formed by the idiochromosomes and the supernumerary attached in a row (cf. Photo 27; additional figures in Figs. 7-8). Fig. 1, ;-/ (terminalis, No. 43), show a 23-chro- mosome group with one small supernumerary. This clearly appears in the spermatogonial group (s); and the small idiochro- mosome (7) is also distinguishable. In the nucleus from the growth-period (/), the supernumerary and small idiochromosome 156 Edmund B. Wilson are united (7, s) the large idiochromosome (/) being separate. (Additional figures in Figs. 7, 8.) Fig. 2, a-c (terminalis, No. 21), show the corresponding stages in an individual of the 24-chromosome type, with two large super- numeraries. ‘Their identification in the spermatogonial group is somewhat doubtful. (Additional figures in Fig. 10.) Fig. 2, d, e (terminalis No. 34), show a 25-chromosome type with three large supernumeraries. [he growth-period (7) is from an individual of granulosus (No. 54) that is possibly of the 26- chromosome type. (Additional figures in Fig. 12.) Fig. 2, g, Ah (femoratus No. 42), and 7 (granulosus, No. 60) show the 26-chromosome type with four large supernumeraries. (See Photo. 28, additional figures in Figs. 9g, 10.) Fig. 2, ;-/ (femoratus, No. 40), are from a very interesting indi- vidual of the 26-chromosome type, with two large and two small supernumeraries (additional figures in Figs. 9, 10). “The sperma- togonia of this individual (&) uniformly show 26 chromosomes, including four very small ones (two m-chromosomes, two small supernumeraries), but the large supernumeraries and the small idiochromosomes are doubtful. No case was found in which all of the six components of the chromosome-nucleolus could be seen; 1 shows five of them, including the two small ones. B ADDITIONAL DESCRIPTIVE DETAILS I will now give a somewhat more detailed and critical account of the facts. “Taken as a whole, the series (including nearly 300 slides of serial sections) presents a profusion of evidence on many cytological questions that could not be adequately described save in a large monograph; but I will here limit the account mainly to ‘tthe numerical and topographical relations of the chromosomes. The clearness of the preparations is such that nearly all the prin- cipal phenomena might have been illustrated by photographs (of which upwards of 200 have been prepared). ‘Thirty of these are reproduced in Plate I, less for the purpose of giving the evidence in detail than of illustrating its character to those not directly familiar with this material. Studies on Chromosomes o. @ é hb : / Frc. 2 a-e, M. terminalis; f, 7, granulosus; g—h, j-/, femoratus. a-c (No. 21), 24-chromosome form, two large supernumeraries. d-e (No. 34), 25-chromosome form, three large supernumeraries. f (No. 54), growth-period, 25- or 26-chromosome form. g-h (No. 42 Photo 8), 26-chromosome form, four large supernumeraries. i (No. 60), 26-chromosome form, growth-period. #-! (No. 40), 26-chromosome form, two large and two small supernumeraries. 158 Edmund B. Wilson 1 Individuals having twenty-one spermatogonial Chromosomes, including an unpaired Idiochromosome. Small Idiochromo- some and Supernumeraries absent To this group belong only the specimens, all males, collected by Montgomery at West Chester, Pa., of which I have examined nine individuals, all of which have essentially the same characters.’ Montgomery (‘or) originally described these forms as having 22 eed chromosomes but subsequently ( ’06) corrected this to 21, describing the phenomena as agreeing in all essential respects with those seen in Anasa and other coreids. A study of the orig- inal preparations has enabled me to confirm this later account in every essential point. After the synizesis or contraction phase of synapsis (as in all individuals of the genus) the ordinary chromo- somes appear in the form of rather delicate spireme-like threads, longitudinally split. In later stages of the growth-period they shorten, become irregular, lose their staining capacity, and assume the vague, pale condition characteristic of so many other forms. In the early prophases of the first division they become more defi- nite, stain more deeply, and appear as coarse longitudinally split rods that often show an indication of a transverse division at the middle point, or in the form of the double crosses as described by Paulmier in Anasa (’99). In the later prophases they condense still further to form nine compact bivalents which finally arrange themselves in a more or less regular ring. The equatorial plate of the first division always shows in polar view 11 chromo- somes (Fig. 3, a,b, Photo 1). In the most typical case the univa- lent idiochromosome lies outside this ring, but it sometimes lies in or inside it. The small m-chromosome bivalent is always near the center of the ring. In side view the larger bivalents are either dumb-bell shaped or more or less distinctly quadripartite, in the 7 These were taken from magnolia trees. In the summer of 1907 I collected in the same locality two males and three females, all from blackberry bushes. To my disappointment, these differ from Montgomery’s specimens, one male having 22 spermatogonial chromosomes, the other 23; while the ovarian cells have in one female 23 and in the other two 24 chromosomes. It is possible that a different species fell into Montgomery’s hands, perhaps an introduced form; but both the structure of the testis and the character of the chromosome-groups agree so exactly with my own material that I now believe that Montgomery’s identification was probably correct. Studies On Chromosomes 159 latter case appearing dumb-bell shaped as seen in polar view. The eccentric idiochromosome is of nearly the same size as the smallest of the large bivalents and is often indistinguishable from the latter except by its position. All these chromosomes divide equally in this division, the m-chromosomes usually leading the way in the march towards the poles, while the idiochromosomes often lag slightly behind the others. The second division likewise shows 11 chromosomes in polar view (3, c, d); but the regular grouping characteristic of the first division 1s now usually lost, the ring formation being often no longer apparent, while either the m-chromosome or the idiochro- mosome may now occupy any position.* In this mitosis all the chromosomes divide except the idiochromosome which lags behind the others and finally passes undivided to one pole (Fig. 3, e-A, Photos 14, 15) as Montgomery described. ‘The nucleus formed at this pole thus receives 11 chromosomes, the sister nucleus but 10, precisely as in Anasa, Narnia, Chelinidea or Leptoglossus. This is proved beyond all doubt by polar views of the anaphases, showing the sister groups lying one above the other in the same section (Fig. 3, 4). In the particular example figured the idio- chromosome lies eccentrically, but this is quite inconstant. The spermatogonia (Fig. 3, 7, 7) always show 21 chromosomes, a largest and a smallest pair being always distinguishable. ‘The unpaired idiochromosome cannot be distinguished from the others. The m-chromosomes are usually equal, but sometimes appear slightly unequal. . In the growth-period the m-chromosomes and the idiochromo- some have the same history as in other coreids. ‘The former are 8 The regrouping of the chromosomes in the second division, first described by Paulmier (’99) in Anasa tristis, is characteristic of the Coreide generally, an eccentric position of the idiochromosome being a nearly constant feature of the first division but not of the second. Failure to recognize this fact in the case of Anasa tristis seems to have been one of the main sources of error in the entirely mis- taken conclusions of Foot and Strobell (’07a, ’07b) regarding this species. (Cf. Lefevre and McGill, ’08.) Demonstrative evidence on this point is given by polar views of rather late anaphases in which every chromosome of each daughter plate may be seen in the same section. Such views, of which I have studied many, both in Anasa and in other genera, show that oneof the chromosomes may indeed occupy an eccentric position, and may there divide; but in such cases the odd chromosome is always found elsewhere in the group, lying either in or near one of the daughter-groups and not in the other. When the odd chromosome is eccentric it is found in one of the daughter groups but not in the other. 160 Edmund B. Wilson typically separate, and at first diffuse (as in Anasa or Alydus). Later they condense to from two spheroidal bodies that conjugate in the late prophase to form the central small bivalent and are almost immediately separated again by the division. ‘The idio- Fy ee ow ® e$ % ® - «© ©’ of e. nd er = XY oa ih ee °er Fic. 3 M. terminalis (Montgomery’s material (Nos. 3-11), 21-chromosome form) a, b, first division, polar view (Photo 1); c-d, second division; e, f, g, side views of second division (Photos 14, 15); 4, sister-groups from the same spindle, in one section, anaphase second division, one showing 10 chromosomes the other II. i-j, spermatogonial groups, 21 chromosomes; k-/, early and late growth-period. chromosome has throughout the early and middle growth-period the form of a single Steele or ovoidal intensely staining chro- moRban eranclewine. which shows in brilliant contrast to ae other chromosomes (Fig. 3, &, /, Photo 24). This body is sometimes slightly constricted in the earlier period. Later it is always con- Studies on Chromosomes IOI stricted, assuming the bipartite form in which it enters the equa- torial plate to form the eccentric chromosome. ‘Throughout the growth-period a large plasmosome is also present, usually separate from the chromosome-nucleolus. In properly stained sections these two bodies differ so markedly in staining reactions that they cannot for a moment be confused. In hematoxylin preparations the chromosome-nucleus is intensely black, the plasmosome pale yellowish, bluish or gray. In Montgomery’s safranin-gentian preparations (though now somewhat faded) the former is bright red, the latter bluish or nearly colorless. There are no females in Montgomery’s material; but in view of the relations known in many other related forms it may safely be concluded that the 11-chromosome spermatozoa are female-pro- ducing, and that the female somatic number in this race Is 22. 2 Individuals with twenty-two Chromosomes in the somatic Groups of both Sexes including a pair of unequal Idiochromosomes in the Male, and a Pair of equal large ones in the Female This condition has been found in seven males and four females, all three species being represented. The three species closely agree in all the phenomena. To the males of this type precisely the same description applies as to the foregoing case except a small idiochromosome 1S present in addition to the “odd” “accessory”” chromosome. The latter is now indistinguishable ae a “large idiochromosome, ” and the identity of these two forms of chromosomes, on which | have laid stress in former papers, is thus fully demonstrated. ‘This appears most clearly in the maturation divisions. In the first division the chromosomes show the same grouping as in the 2I- chromosome forms, but a small idiochromosome accompanies the “accessory,” frequently lying beside it outside the principal ring, though sometimes being in or inside the latter (Fig. 4, a-j, Photos 2, 3). This chromosome is always recognizable as the smallest of all the chromosomes except the 7-chromosomes, and it is in general about half the size of the large idiochromosome or slightly less. All the chromosomes now divide equally (Fig. 4, /, 162 Edmund B. Wilson Photo 11), 12 chromosomes passing to each pole. The second division immediately follows without the intervention of a “resting stage,’’ and the chromosomes undergo the same regrouping as that described for the 21-chromosome forms. As this takes place, the two idiochromosomes conjugate to form an unequal bivalent (precisely as in Lygaeus or Euschistus); so that when the equato- rial plate reforms but 11 (instead of 12) chromosomes appear in polar view (Fig. 5, a-c, Photo 12). The idiochromosome-biva- lent now usually lies near the center of the group (contrasting with the first division), and the m-chromosome is usually not far from it. Such views are almost indistinguishable from those of the 21-chromosome individuals, since the small 1diochromosome is covered by the large one and only appears in side view. In the course of the division the idiochromosome bivalent separates into its two components, which pass to opposite poles, while all the other chromosomes divide equally. The idiochromosomes at first separate more rapidly than the other daughter-chromosomes(Fig. 5,7» 4), as in other genera, but as the division proceeds the reverse condition prevails, so that the two idiochromosomes are seen lag- ging on the spindle between the diverging daughter groups (Fig. 5 il). In the later stages one passes to each pole. ‘There is much variation in this process. Often the two move at the same rate so that in the late anaphases one may be seen entering each pole (Fig. &, /, Photo 17). Not uncommonly, however, one or the other lags behind upon the spindle (usually the large one, though Fig. 5, ;, shows the reverse case) giving a condition that exactly ae that seen in the 21-chromosome forms (Fig 5, m, n), but earlier anaphases in the same cysts at once show the difhovences It is no less conclusively shown by polar views of the late anaphases, in which each daughter-group is seen to consist of 11 chromosomes, ten of which are duplicated in the two while the the eleventh is in one case the large, in the other the small idio- chromosome (Fig. 5, 4, 7, 5, t). The difference between the two types is shown with almost equal clearness by the chromosome-nucleoli of the growth-period. In the 21-chromosome type, as already stated, this body is single. A similar appearance is sometimes given in the 22-chromosome indi- Studies on Chromosomes e f Fic. 4 22-chromosome forms a-l, first division; a,b, term. No. 19, typical (Photo 2);c, term. No. 12 (Photo 3); d,e, fem. No. 29 f. term. No. 12; g, k, term. No. 19, idiochromosomes united; 7, fem. No. 29, same condition; j, gran. No. 47; k, fem. No. 29, first division, side view, idiochromosomes united; /, fem. No. 46 (Photo 11), first divi- sion, anaphase, division of both idiochromosomes. m-q, spermatogonial groups; m, term. No. 19; , 0, fem. No. 46; p, q, fem. No. 29. r-t, ovarian groups; r, term. No. 24:5, term. No. 44;1, term. No. 23, exceptional form and grouping. 164 Edmund B. Wilson viduals, owing to close union of the two idiochromosomes. But in very many cells of this period the chromosome-nucleolus con- sists of two very distinct unequal moieties, in contact (Fig. 6, a, b, Photo 25), or not infrequently widely separated (Fig. 6, c, d. Photo 26). When in contact they form a double body closely similar to the idiochromosome-bivalent of the second division. There can be no question of confusing either of these bodies with the plasmosome, since the latter, showing its characteristic stain- ing reactions, is also present. In the late prophases of the first division the idiochromosomes, if previously united, almost invariably part company to divide as separate univalents, as in other Hemiptera; but they usually remain near together outside the principal ring. Only very excep- tionally do they divide together. The spermatogonial groups (Fig. 4, m-—q) uniformly show 22 chromosomes, and in some cases the small idiochromosome may be recognized by its small size (m, q). This is, however, not nearly so marked as in the first division, since it now appears rela- tively twice as large, owing to the univalent character of the other chromosomes, and often it cannot certainly be distinguished from the smaller of these (1, p). These facts make it clear that if the small idiochromosome be supposed to. disappear, the entire series of phenomena would be- come identical with those shown in the 21-chromosome individuals, the large idiochromosome now appearing as the odd or “acces- sory’ chromosome. The unreduced female groups of this type (ovarian cells) are closely similar to those of the male (Fig. 4, r+) but a small idio- chromosome can never be distinguished. The absence of this chromosome cannot be so convincingly shown in Metapodius as in such forms as Lygaeus or Euschistus, owing to its greater rela- tive size. Nevertheless, after the detailed study of many female groups I am convinced that this chromosome is not present, and that all the chromosomes may be equally paired. Apart from analogy, therefore, I think the conclusion reasonably safe that in Metapodius, as in other forms, the unequal i1diochromosome- pair of the male is represented in the female by a large equal pair, a Ss Studies on Chromosomes 16 Fic. 5 22-chromosome forms a-c, second division, polar view; a, fem. No. 19; b, fem. No. 28; c, gran., 47 (Photo 12). d-p, second division, side view; d-h, fem. No. 29, metaphases, separation of idiochromosomes; 1 J, term. No. 19, anaphases, lagging of one idiochromosome; k-m, gran., No. 47, late anaphases (Photo 17); n, term., No. 19, late anaphase, lagging large idiochromosome; 0, fem., No. 46, exceptional condi- tion, both idiochromosomes passing to one pole (Photo 18); p, term. No. 19, similar form; gq, r, term., No. 19, sister anaphase groups, from the same spindle; s, t, fem., No. 29, the same. 166 Edmund B. Wilson and that, accordingly, the usual rule holds in regard to fertiliza- tion. Exceptional conditions. ‘There are two conditions, rarely seen, that are of interest for comparisons with other species. Now and then the idiochromosomes fail to separate for the first division, but remain in more or less close union to form an asymmetrical bivalent, which in side view is seen to form a tetrad (Figs. 4, /-1, k, Photo 3). This bivalent undergoes an equation division, in this respect agreeing with the conditions uniformly seen in Syro- Fic. 6 M. femoratus (No. 29) 22-chromosome form Four nuclei from growth-period showing diffused ordinary chromosomes, condensed chromosome- nucleoli and plasmosome; in a and b the two idiochromosomes are united to form double chromosome- nucleoli (Photo 25); in c and d they are separate (Photo 26). mastes (Gross ’04, Wilson ’og), and differing from that occurring in the Coleoptera or Diptera (Stevens ’o6, ’08a). A rarer but more interesting deviation from the type is the failure of the idiochro- mosomes to separate in the second division, both passing together to the same pole (Fig. 5, 0, p, Photo 18). Since the other chromo- somes divide equally it may be inferred that in this case one pole receives 12 chromosomes and the other but 10. ‘This has been seen in only three cells and is doubtless an abnormality. It may however, possess a high significance as forming a possible point — eee eS 7) Ree oe Studies on Chromosomes 167 of departure for the origin of the whole series of relations observed in the genus. 3. Individuals possessing twenty-three Chromosomes; one Supernumerary This condition exists in all three species and has been found in seven males and four females. In four of these males the super- numerary is large (of approximately the same size as the small idiochromosome, as in Fig. 1, gz); in three it is no larger than the m-chromosomes (as in Fig. 1, j-/), and is indistinguishable from the latter save in behavior. In each case, as already described, the spermatogonia show 23 chromsomes and the first division 13; and in those showing a small supernumerary in the first division the spermatogonia always show three very small chromosomes. The grouping in the first division, though conforming to the same general type, shows many variations of detail, as may be seen from Fig 7, a-/, Photos 4-6. It is a curious fact that the form of grouping is to some extent characteristic of the individual. For example, the typical arrangement, with both idiochromosomes and supernumerary outside the ring, is very common in Nos. 43 (Fig. 1, ;-/) and 20 (7, a-c), very rare in Nos. 1, 2 (Fig. 7, 2) and 49 (Fig. 7, j-h). In No. 49, very many of the first division meta- phases show both supernumerary and small idiochromosome lying inside the ring (Fig. 7, g-h). I am unable to suggest an explanation of this. In this division all the chromosomes divide equally (Fig. 7, 7-p), so that each secondary spermatocyte receives 13 chromosomes. The usual regrouping now takes place, and the idiochromosomes couple as usual to form an asymmetrical bivalent. ‘The super- numerary sometimes remains free (1. e., not attached to any other), in which case 12 chromosomes appear in polar view (Fig. 8, b,d@). Much more frequently the supernumerary attaches itself to the idiochromosome bivalent to form a triad element, polar views now showing but 11 chromosomes (8, a, c), one of which is compound. The three components of such triads usually lie in a straight line, the supernumerary being attached sometimes to the small idio- 168 Edmund B. Wile? Fic. 7 23-chromosome forms, one supernumerary a-h, first division, polar views, one large supernumerary; a-c, term., No. 20, typical grouping; d-e- gran., No. 48; f, g, 4, gran., No. 49 (Photo 5). i-l, first division, polar views, one small supernumerary; 7, term. No. 1 (Photo 6); --/, term. No. 43 typical grouping in k. m-p, first division, side-views; m and n (term. No. 43) show division of I, 7, m, and small s; 0, term., No. 20, division of I, 7, and large s; p, term., No. 43, division of m, 7, and small s. q-s, spermatogonial groups from individuals with one large supernumerary; q, r, term., No. 20; s, gran., No. 49. t-y, spermatogonial groups from individuals with one small supernumerary; t, u, term., No. 43; v-y, term., No. 2 (Photo 29). Studies on Chromosomes @ « . @ a 0% eS & E oe ee ~ ; re @e* Oy eee es Db 8 ;@ d e@./ @ @ & : gy € » @ 6 xf ee? @e.r & 0 Og @ Ge @o4 £ h e@ e ©@ o @ e = a ¥ r e e < @ eS.” 0e e, pe J RP iw / HUN | gPPon,: wR, rae uti Hel ne ) on | A lal An aa U2 0 é se WW 0 Wp 170 Edmund B. Wilson chromosome, sometimes to the large, or not infrequently lying between the two (Fig. 8, g, h, 0-9). Fic. 8 23-chromosome forms, one supernumerary a-f, polar views, second division; a, gran., No. 49, large supernumerary attached; 5 (same cyst)super- numerary free; cd, similar views of terminalis, No. 43, with small supernumerary; e-f (No. 43), sister groups from same spindle, pclar views. g-m, side-views, second division, from gran., No. 49, with large supernumerary, free in j, attached in the others. n—u, similar views from individual (term., No. 43) with small supernumerary; in u the supernumer- ary is free. ries Studies on Chromosomes 7a In the ensuing division, if the supernumerary lies free it passes without division as a heterotropic chromosome to one pole (8, 1). When connected with the idiochromosome bivalent it passes to one pole attached to one or the other of the idiochromosomes (Fig. 8, k-m, p-t). In either case one pole receives 11 chromosomes and one 12 (Fig. 8, e, 7); but since the supernumerary may accompany either idiochromosome four classes of spermatid nuclei are formed, namely: (1) 1of7=11 (2) 1o+7+5=12 Io +7= II io+I+s= 12 (3) (4) As described in an earlier paper (’07a), there is a tendency for the supernumerary to be associated more often with the small idiochromosome than with the large, and classes 1 and 2 are accord- ingly more numerous than 3 and 4. I was formerly inclined to attribute importance to this as pointing to the more frequent occurrence of the supernumerary in the male than in the female. The larger series of data now available leads me to doubt whether it has much significance; for if (leaving the 21-chromosome forms out of account)-the whole series of forms be taken together, one or more supernumeraries are found in 27 out of 34 males, and in 15 out of 19 females—about 80 per cent in each case. It appears therefore that in the long run the supernumeraries are distributed between the two sexes with approximate equality. Figs. 7, g-s show spermatogonial groups from individuals with one large supernumerary, but in none of them can this chromosome or the small idiochromosome be certainly distinguished. Fig. 7, t-y are from individuals with one small supernumerary, each showing three very small chromosomes. In ¢ and wu the small idiochromosome is doubtful. Fig. 7, v—y, on the other hand, are from an individual (terminalis, No. 2), showing great numbers of very fine spermatogonial groups, in almost all of which the small idiochromosome is at once recognizable. ‘The same is true of a second individual from the same locality. ‘These two individuals, from the Paulmier collection, were the first material | examined and found so puzzling until the examination of another similar individual, No. 43, cleared up the nature of the second division. 172 Edmund B. Wilson 4. Individuals with twenty-six Chromosomes; four Su pernumeraries It will be convenient to consider this type before the 24- and 25- chromosome forms, since the material is more favorable for an account of the remarkable phenomena occurring in the second division. Of these individuals there are seven males and three females, all three species being represented. Unfortunately very few perfectly clear spermatogonial groups are shown; but the spermatocyte-divisions and cells of the growth-period are particu- larly well shown and in large numbers of cells. In all but one of these individuals the four supernumeraries are large and of nearly equal size. In one (femoratus No. 40) two are large and two small. The latter case, already shown in Fig. 2, ;-/, is further illustrated by Fig. 9, A, 7, 7, n, 0. Two of these (h and 7) show but three supernumeraries in the first division, a common appear- ance in this individual (see p. 186). Fig. 9, a-/, show varying arrangements of the 16 chromosomes that appear in the first division, the most typical ones being & and/. Ing, a-c, k,/, both idiochromosomes and the four supernumeraries lie outside the ring. In 9g, g, all but the large idiochromosome are inside the ring. In some of these slides the compound chromosome-nucleoli are shown with great distinctness in many cells of the growth-period. This body usually has the form of a flat plate that lies next the nuclear wall (Fig. 10, g, r) so that a clear view of all the compo- nents can only be had in tangential sections. Thus viewed (Fig. 10, s—u, Photo 28) it may often be seen to consist of six components one of which (the large idiochromosome) is about twice the size of the others and is usually at one side or end of the group. The other five evidently represent the small idiochromosome and the four supernumeraries. In side view (Fig. 10, 9,7) not more than three or four of the components, can as a rule be recognized. In a considerable number of cases these six chromosomes are not aggregated to form a single body but form two or more simpler bodies. The second division in these forms presents an extraordinary a Studies on Chromosomes ) @° - @ M am e oes Yar See *¢ . 8s ee gh @° nd 2 @9@ o. e° e°; & e °8 e -°@ . a cae ess Se. @ i i ee ed = / Fic. 9 26-chromosome forms, four supernumeraries a-g, first polar, supernumeraries large and equal; a-d, fem., No. 42; e, gran., No. 55; f, gran., No. 59; g, gran., No. 60. h-j first polar, from (fem., No. 40, with two large supernumeraries and two small; all of these are shown in j, (cf. Fig. 2, ;), while in h and 7 one is missing (see p. 186). k, first polar, term., No. 36; / from same individual (Photo 9). m-o, spermatogonia groups; m, fem., No. 42, abnormal group with 27 chromosomes; ”, 0, fem., No. 40. showing two small supernumeraries. p-q Ovarian groups, gran. No. 61. W 174 Edmund B. Wilson appearance which I[ at first thought must be due to an artificial clumping together of the chromosomes through defective fixation; but the study of very many of these figures convinced me that such isnot the case. Asin the preceding types, ten of the chromosomes, including the m-chromosomes, have the form of symmetrical dumb-bell shaped bodies which are equally halved in the ensuing division. ‘The remaining chromosomes are usually aggregated to form a compound element (Fig. 10, h-/, Photos 22, 23) in which may be very clearly distinguished the same components as those that appear in the chromosome-nucleoli of the growth-period; and the size-relations make it evident that one of them is the large idiochromosome, one the small, while four are the supernumer- aries. In other words, these six chromosomes, which divide as separate univalents in the first division, have now again conju- gated to form a hexad group. This compound element almost always lies near the center of the group. Polar views of this divi- sion accordingly show typically 11 chromosomes, of which the central one is compound (Figs. 10, a—g, Photo 13). Not infre- quently, however, one or more of the supernumeraries may be sep- arate from the others (Fig. 10, 7, g), the apparent number in polar view varying accordingly. In side views the grouping of the components of the hexad element is seen to vary considerably though the large idiochro- mosome is more frequently at one end of the group. In the ensu- ing division the other ten chromosomes divide equally, while the hexad element breaks apart into two groups that pass to opposite poles (Fig. 10, /-p). The distribution of the various elements is dificult to determine exactly, since they always lag behind the others in the anaphases and are scattered along the spindle in such a Way as often to give confusing pictures. ‘The study of many such anaphases leads me to conclude, however, that at least one of the smaller components always passes to the opposite pole from the larger one, while the other four undergo a variable distribution. In Fig. 10, /, the group is just separating into three toward each pole; in 10 m, it is quite clear that three of the small ones are pass- ing to one pole, while the large one and two small ones are passing to the other, and Fig. 10, 7, is probably a similar case. In these Studies on Chromosomes 175 cases it seems clear that each pole receives 13 chromosomes, as follows: a rofl +2s—13 DOr 2503 Fig. 10, 0, on the other hand, shows a perfectly clear case in which the hexad element has separated into a 2-group and 4-group: Fig. 10, p, shows what is probably a later stage of the same type. In both these cases one pole appears to receive 12 and one 14 as follows : Gye) se dl ae ies 3 b 1o+1+5=12 one pole receiving but one supernumerary, and the other three. The cases in which all of the components may be clearly recog- nized in the anaphases are comparatively rare, and in the greater number of them the distribution of the supernumeraries appears to be symmetrical. Of their unsymmetrical distribution in some cases there can be no doubt (and the same is true of the t4-chromo some form, as described beyond). The few undoubted cases of this all show one to one pole and three to the other (as in Fig. 10, o- p), and I have never found a case in which all four pass to the same pole. It seems, therefore, probable that in the 26-chromosome type there are at least six classes of spermatozoa, as follows: (1) 10+ T+2s=13 (2) 10+ f+25= 13 (3) 1o+ 2+ s=12 (4) 1lotit+ 3s= 14 (5) to +: T+ 35 = 14 ; (6) 1o+%+ s=12 It is possible that the following four additional classes may be produced: (7) 10+ 1+ 45=15 (8) 10+: = ti (9) 10+ 7 = II (10) 10+7+ 45 = 15 Perfectly clear spermatogonial figures of this type were rarely found, though many of them show approximately 26. The nor- mal group of fem., No. 42, is shown in Fig. 2, h. “Two groups from fem. No. 40 (with two small and two large supernumeraries) are shown in Fig. g, n, 0, each having 26 chromosomes including four small ones (cf. Fig. 2, ). Two ovarian groups from gran., No. 61, 176 Edmund B. Wilson Fic. 10 26-chromosome forms a-g, second division, polar, d from fem. No. 40, the others from fem. No. 42; a, (Photo 13) b, c, show a single central hexad; ine and g the components are more loosely united; in d and f one supernumer- ary is free. k-p, side-views, second division, from fem. No. 42 (Photos 22, 23) explanation in text. q-u, growth-period, gran.. No. 60; 7 and r show the compound chromosome-nucleolus in oblique and side-view, s, t, u, en face. Studies on Chromosomes Fic. 10 178 Edmund B. Wilson Fic. 11 24-chromosome forms, two supernumeraries. a-e, term., No. 21, first polar, showing various groupings; g, the same, gran., No. 52 (Photo 7). h, term., No. 21, second polar, tetrad element near center. i-0, somatic groups from individuals with two large supernumeraries; i—/, spermatogonial groups from term. No. 21; m, n, ovarian groups from fem. No. 31; 0, ovarian group, fem., No. 45. p-r, spermatogonial groups from fem., No. 22, with one large supernumerary and one small; Photo 30). s—w, second division, side-view; s, term., No. 21; tw, gran., No. 52 (Photo 21). 79 Studies on Chromosomes e - rT x a ee & : Pa e e2qre a e —— @fe ~®? e S e&® 5) & ‘ @e 2 ee ene @ S | a ey co_8 ce .® 02,08 Ses? e&@ @@ ® = Fic. 11 180 Edmund B. Wilson are shown in Fig.g, p,q. Fig. 9, m, shows a spermatogonial group from fem., No. 42, that is abnormal in showing with perfect clear- ness 27 instead of 26 chromosomes (cf. Fig. 2, /). 5 Individuals with twenty-four Chromosomes, two Supernumerartes The material for these individuals and those of the 25-chromo- some class, is less satisfactory than in the preceding case, but the relations are undoubtedly quite analogous to those just described. The 24-chromosome class is represented by g males and 4 females, and occurs in all three species. In one of the males one of the supernumeraries is large (of the same size as a small idiochromo- some) and one small; in all the others both are large. Additional figures of the first Cran showing variations in the grouping, are given in Fig. 11, a-g; of spermatogonial groups in Figs. 11, 7-7. Of particular i ‘terest is the male, term., No. 22, shown in Photo 30 and in Fig. 11, p-r._ ‘This individual was, unfortunately, immature showing only spermatogonia and cells in the growth-period; but many perfectly clear spermatogonial groups are shown. ‘These groups uniformly show 24 chromosomes, of which three are very small, while in many cases two others are slightly but distinctly smaller than the others. ‘The latter are evidently the small idio- chromosome and the larger supernumerary, while the three small ones represent the m-chromosomes and the small supernumerary. In the second division the two idiochromosomes and the super- numeraries are frequently united to forma tetrad element, various forms of which are shown in Fig. 11, s-w. ‘The distribution cf these four components is not so well shown in this material as in that of the 26-chromosome class, described above. It is, however, clear that this distribution is inconstant. In cases like those shown in Fig. 11, s, ¢, itis probable that the tetrad divides in the middle, so that each idiochromosome is accompanied by a supernumerary, and each pole receives 12 chromosomes. The cases shownlaml Fig. 11, v, w, prove however that this 1s not always the case; for in w the large idiochromosome Is seen passing to one pole while both supernumeraries, attached to the small idiochromosome, Studies on Chromosomes 181 are passing to the other. In this case one pole receives 11 chromo- somes, the other 13. It is evident that in this form there is the possibility of forming six classes of spermatozoa, as follows: (1) 10+ J=11 (2) 1o+ #+25=13 3) 1o+ 72+ s=12 (4) 10o+7+ s=12 (5) 10+ IT+2s= 13 (6) 10+7 =11 In none of these individuals is the material very favorable for the study of the chromosome-nucleoli. “‘Vhey are always evidently compound, but only in a few cases can the components be clearly recognized (as in Fig. 2, c). 6 Individuals with twenty-five Chromosomes, three Supernumerartes No individuals of this type were found in M. femoratus. The other two species are represented by three males and three females but here again the material does not admit of exhaustive study. In one of the females, two of the supernumeraries are large and one small, the ovarian cells showing 25 chromosomes, of which _three are very small (Fig. 12, :-k), a condition seen in every group of which a clear view can be had. ‘The two larger supernumer- aries cannot, however, be certainly identified in any of these. In all the other individuals the supernumeraries are of the larger form. Fig. 12, a, 6, show the first division in one of these cases (term., No. 34); c-g are spermatogonial groups from the same indi- vidual; 4, an ovarian group of the same type. Fig. 12, m—p, are from a doubtful case in which nearly all the first division figures show three supernumeraries (n, 0), but a single case (7m) shows distinctly four. 7 Individuals with twenty-seven Chromosomes; five Supernumeraries. This class is represented by a single very interesting male of granulosus (No. 57), in which only the first division can be satisfac- torily examined. Many polar views of this division show 17 chromosomes (Fig. 13, a-1, Photo 10), of which two are always 182 Edmund B. Wilson smaller than the others. One of these, always central in position, is evidently the m-chromosome bivalent. Of the remaining six, one 1s in most cases decidedly smaller than the others—a relation 25-chromosome forms, three supernumeraries a, b, first polar, term., No. 34; c-g, spermatogonial groups from same individual; 4, term., No. 38. ovarian group; 7k, ovarian groups from term., No. 27, with two large supernumeraries and one small; ], gran., No. 58, ovarian groups, three large supernumeraries. m,n, 0, first division, p, second division from gran., No. 54, with three or four supernumeraries. of which the constancy is attested by the nine figures given of this division. It is evident that in this individual there are four large supernumeraries and one small; and although nospermatogonia Studies on Ch rormosomes I 83 are clearly shown it may be inferred that the somatic number is 27. The chromosome-nucleoli in this individual are evidently com- pound, but in no case can all the components be clearly recognized. The second division shows, as a rule, 11 elements in polar view, the central one being compound (Fig. 13, ;-k), but the distribu- tion of the compound element could not be determined. @7 Ge Ge g o os A . wre 85 fer 2 @%;, @- oss @@e@ °° ee” aie & @ @°:: P) Ba % @ a b G d ‘ @/ o%e. ee ee e"s e ®e . ae?) (f°. ete feof 8 * #2: —? Gee os: ed @> C fe g h Fic. 13 27 and (?) 28-chromosome forms a-i, first division, from gran., No. 57, having four large supernumeraries and one small 7 (polar) and k (side-view), second division, same individual (Photo 10). /, ovarian group from fem., No. 33, having three large and two or three small supernumeraries; in this group appear 28 chromosomes. 8 Individuals with twenty-eight (?) Chromosomes; six Supernumeraries. The last case to be considered is that of a single female of femora- tus (No. 33), in which the number is either 27 or 28. A single perfectly clear ovarian group, shown in Fig. 13, /, shows beyond 184 Edmund B. Wilson doubt 28 chromosomes, including five smallest ones and three or four next smallest. A few other less clear groups were seen in which appear but 27 chromosomes, the missing one being one of the smallest. In these cases one of the small ones may be hidden among the larger ones; but it is also possible that the 28-group is an abnormality. In this individual there are probably three larger supernumeraries and either two or three small ones. C SUMMARY AND CRITIQUE 1 In the genus Metapodius the number of chromosomes is constant in the individual but varies in different individuals from 21 to 27 or 28. The number 21 appears only in the males of M. terminalis (Montgomery's material). 2 The number is independent of sex and locality, and is not correlated with constant differences of size or visible structure in the adults. 3. The variation affects only a particular class of chromosomes. 4. The 22-chromosome forms represent the type from which all the others may readily be derived. ‘These forms possess a pair of unequal idiochromosomes which show the same behavior as in Lygzus or Euschistus, all the spermatozoa receiving 11 chromo- somes, and half containing the large idiochromosome, half thesmall. 5 In the 21-chromosome forms the small idiochromosome has disappeared, leaving the large one as an “odd” or “accessory” chromosome. Half the spermatozoa accordingly receive 11 chro- mosomes and half to. 6 Numbers above 22 are due to the presence of from one to five or six additional small chromosomes which show in every respect the same behavior as the idiochromosomes, and are probably to be regarded as additional small idiochromosomes. In the growth period they have a condensed form and are typically associated with the idiochromosomes to form a compound chromosome- nucleolus. In the first division they divide as separate univalents. In the second, they are typically (though not invariably) again associated with the idiochromosomes to form a compound element. The components of this element undergo a variable distribution Studies on Chromosomes 185 to the spermatid nuclei. All the spermatid nuclei receive the haploid type-group of 11 chromosomes, half including the small idiochromosomes and half the large; but in addition each may receive one or more supernumeraries. The total number of chromosomes in the sperm nuclei is therefore variable in the same individual. 7 Both the number of the supernumeraries and their size, indi- vidually considered, are constant in the individual. The first question that the foregoing report of results will raise is whether the number and size relations of the chromosomes in each individual are really as constant as I have described them. I have for the most part selected for illustration and description the more typical conditions; but, granting the accuracy of the figures, does such a selection really give a fair presentation of the actual conditions? It is almost needless to say that very many cases might have been shown that would seem to give conflicting results. By far the greater number of these discrepancies are, I believe, only apparent. Numerical discrepancies of this kind are very often evidently due to mere accidents of sectioning or to the super- position or close contact of two or more chromosomes. Again, apparent discrepancies in the size relations of the chromosomes, as seen in polar views, very often arise through different degrees of elongation (particularly in the maturation divisions). But apart from such apparent variations, real deviations undoubtedly occur in almost all of the relations described. Now and then, for exam- ple, a spermatogonial or ovarian group is found that clearly shows one chromosome too many (as in Fig. g, ™m),° and the same is true of the first spermatocyte-division, but such cases are very rare. The former case is probably a result of an abnormality in the forma- tion of the chromosomes from the resting nucleus, the latter not improbably to a failure of synapsis. Again, both spermatogonial and spermatocyte-cysts are occasionally found in which the num- ber of chromosomes is doubled or quite irregular. ‘These are * A perfectly clear case of this has been found in the pyrrochorid species Largus cinctus (a particu- larly fine form for study). In this form the normal male number is 11, the female 12; but in one female three cells were found each of which shows with all possible clearness 13 chromosomes, very many other cells showing the normal number. 186 Edmund B. Wilson probably due to an antecedent nuclear division without cell divi- sion, or to multipolar mitoses such as now and then occur in both spermatogonia and spermatocytes. As regards the chromosome-nucleoli of the growth-period, the contrast between those of the 21 and 22-chromosome forms, or between either of these forms and those with higher numbers is usually at once apparent; but in very many cases where more than one supernumerary 1s present the number of components can only here and there be clearly seen. Contrary to what might be expected from their compact form, the compound chromosome nucleoli seem to be rather difficult of proper fixation, their components often clumping together or breaking up more or less when they coagulate. I infer this from the fact that different slides differ materially in the clearness with which these bodies are shown. Two discrepancies, apparent or real, should be especially men- tioned. One is the difficulty of recognizing the larger supernu- meraries in the somatic groups. As already explained, these chro- mosomes, like the idiochromosomes, appear relatively much larger in the somatic groups than in the first maturation division (owing to their univalence in the latter case); but we should expect to recognize them more clearly, at least in the female groups, than is actually the case. ‘This is perhaps due to their undergoing a greater degree of condensation than the others during the growth- period; but I am not sure that this explanation will suffice. A second discrepancy, which may involve an important conclusion, is that in perfectly clear views of the first division, the number of supernumeraries is often less than would be expected from the spermatogonial groups. ‘This is notably the case with femoratus, No. 40 (Fig. 9, 4-7), which has clearly 26 spermatogonial chro- mosomes, but very rarely shows 16 in the first division, the usual number being 15. A similar discrepancy has been noted in other individuals, and in several of the types. Since the typical num- ber in all these cases appears in some or many of the first sperma- tocytes, | long supposed the occasional deficiency to result from an accident of sectioning. I now incline to believe, however, that in some cases one (or possibly more) of the supernumeraries may really disappear (by degeneration’) during the growth-period, Studies on Chromosomes 187 and that this may be one way in which their progressive accumu- lation in number in successive generations is held in check. For the foregoing reasons it cannot be said that any of the rela- tions described appear with absolute uniformity or fixity. The condition typical of each individual must be discovered by the study and comparison of large numbers of cells. [ will only say that prolonged and repeated study has thoroughly convinced me that the relations, as described, may be regarded as being on the whole individual constants. ‘his judgment is based primarily on the exhaustive study of a few of the best series of preparations of individuals of the 21, 22, 23, and 26-chromosome types, in which the facts are quite unmistakable and have given the point of view from which the less favorable material of other cases may fairly be examined. D DISCUSSION OF RESULTS The principal significance of these phenomena seems to me to lie in their bearing on the general hypothesis of the “individual- ity” or genetic continuity of the chromosomes; but they are also of interest for a number of more special problems which [| will first briefly consider. The Relation of the Chromosomes to Sex-production in Metapodius The conditions seen in this genus seem to be irreconcilable with any view that ascribes the sexual differentiation to a general quanti- tative difference of chromatin, whether expressed in the number or the relative size of the chromosomes. In all known cases of constant sexual differences in the chromosomes it is invariably the female that possesses the larger number of chromosomes or the greater quantity of chromatin,’ and this has naturally suggested the view that this difference per se may be the sex-determining factor. As I have pointed out before (’0g), such a view is inapplicable to cases like Nezara or Oncopeltus, where the idiochromosomes are of equal size and no quantitative sexual differences are visible; yet the phenomena in these genera are otherwise so closely similar 10 See review in Wilson ’og. 188 Edmund LER Wilson to those seen in other insects that I cannot doubt their essential similarity also in respect to sex-production. In Metapodius the facts are still more evidently opposed to the quantitative interpretation. The number of chromosomes has here no relation to sex-production; and, as will be seen from the table at p. 149, in the forms with supernumeraries the relative frequency of high numbers and of low is nearly equal in the two sexes. If my general interpretation of the chromosomes in this genus be correct, a like conclusion applies to the total relative mass of chromatin in the two sexes; for all individuals alike possess the type-group of 22 chromosomes (Montgomery’s form excepted) while the supernumeraries represent the excess above this amount. I have endeavored to determine whether this appears in direct measurements, independently of my general interpretation; but have found this impracticable for several reasons. Very consider- able differences in the apparent size of the chromosomes are pro- duced by different degrees of extraction; but this will not account for the considerable differences seen in the same slide when the extraction is uniform. It is evident that the actual size of the chromosomes varies with the size of the cells; for example, both in Metapodius and in many other genera, the chromosomes in the larger spermatogonia near the tip of the testis are larger (in many cases much larger) than those of the smaller spermato- gonia of other regions. How great the differences are may be appreciated by a comparison of the figures. For example, in the spermatogonial groups of No. 2 (23 chromosomes, Fig. 7, vx), the chromatin mass is obviously much greater than in those of No. 21 (24 chromosomes, Fig. 11, 7-/)._ In the 25-chromo- some female groups shown in Fig. 12, :-k (No. 27), the chromatin mass 1s evidently much less than in the 21-chromosome male group shown in Fig. 1, 6, or in the 23-chromosome male groups of Fig. 7, v-x. Conversely, the 22-chromosome female group of No. 44 (Fig. 4, s) shows a much greater chromatin mass than in the corre- sponding male group of No. 46 (Fig. 4, 0), or the male 24-chromo- some group shown in Fig. 11, 7. Evidently, therefore, the relative mass of chromatin can only be determined by means of accurate measurements of both the Studies On Chromosomes 189 chromosomes and the mass of protoplasm, but I have found the errors of measurement of the cell size to be too great to give any trustworthy result regarding the relative chromatin mass. Despite the difficulties in the way of an accurate direct deter- mination, I believe the facts on the whole warrant the conclusion that the relative chromatin mass shows no constant correlation with sex. The most probable conclusion is that the male-produc- ing spermatozoa in Metapodius are distinguished by the same characters as in other forms having unequal idiochromosomes, the former class being those that receive the large idiochromosome, the latter those that receive the small one, irrespective of the super- numeraries that may be present in either class. For reasons that I have elsewhere stated, I believe that if the idiochromosomes be the sex-determinants their difference is probably a qualitative one, and since the small idiochromosome may be lacking it would seem that the large one must in every case play the active role— perhaps as the bearer of a specific substance (enzyme ?) that calls forth a definite reaction on the part of the developing individual. If this be so, we can comprehend the fact that the presence of additional small idiochromosomes (supernumeraries) in either sex does not affect the development of the sexual characters in that SEX. b The possible Origin of the unpaired idiochromarcme (“odd”’ or “accessory Chromosome) and of the Supernumerartes The explanation of the unpaired idiochromosome offered in the second and third of my “Studies on Chromosomes” (’05, ’06) was suggested by the fact that various degrees of inequality exist in the paired idiochromosomes, there being an almost continuous series of forms connecting those in which the idiochromosomes are equal (Nezara, Oncopeltus) with those in which they are so very unequal that the small one appears almost vestigial (Lygzus, Tenebrio). It is evident that by the further reduction and final disappearance of the small member of this pair the large one would be left without a mate, and its history in the maturation process would become identical with that of an “odd” or “accessory” 190 Edmund B. Wilson chromosome. | still believe that this explanation may be applic- able to many cases; but a different one seems more probable in the case of Metapodius and perhaps may be more widely applicable. This was suggested by the observation (p. 166) that in a very few cases, in 22-chromosome individuals both idiochromosomes were seen passing to the same pole in the second division. ‘The rare- ness of this occurrence shows that it is doubtless to be regarded in one sense as abnormal. But even a single such event in an original 22-chromosome male, if the resulting spermatozoa were functional, might give the starting point for the whole series of relations ob- served in the genus, including the establishment of an unpaired idio- chromosome. ‘The result of such a division should be a pair of spermatozoa containing respectively 10 and 12 chromosomes. The former might give rise at once to a race having an unpaired idiochromosome and the somatic number 21 in the male (as in Montgomery’s material). ‘The latter might similarly produce an individual having in the first generation a single supernumerary chromosome and in succeeding generations an additional number. This appears from the following considerations: 1 If a to-chromosome spermatozoon, arising in the manner indicated, should fertilize an egg of the 22-chromosome class (hav- ing 11 chromosomes after reduction) the result should be a male containing 21 chromosomes, the odd one being the large idiochro- mosome derived from the egg. Such an individual would be in «no respect distinguishable from those of Montgomery’s material, and would similarly form male-producing spermatozoa containing 10 chromosomes and female-producing ones containing 11 (includ- ing the unpaired idiochromosome). Visible changes attending a process of desiccation’ . 05... c cece cee cece nec cer ne esseerce 223 NileeWerree or desiccation attamediDy; PAVOdIN ar. wie. 2 oe alale vie sis cieie(ole.o s/a\n'elslo[o.0\= isle) es « © =} « 9 | REM iewrs Ol avisiarl GOtlens, oateieteleleloiejaiciaie oie\cfoielatciavelo « elalelolere eynieielaiateCclofels isloleicie(s 228 2 Evidence for the view that a true desiccation OCCUIS,.........0..eeeeeececeeeeeseeee 231 VII_ Effect of desiccation on the life processes of Philodina................-. ge Sapo awoRasr 236 am loa NIGMS CHECESOUGESIGCAHOUamce mein a itislelats siateiels olelsrals arejeheisratoisicjore tenet ober nistrisiats 236 2 Influence of the previous condition of the animal..................eceeeeeeeeeeeee 237 3 Influence of the conditions attending the desiccation............0-ceeee cece eeeeees 238 FLEE et AO} Glamis te Sonido euOdO Boa dOnad dcop acdc 500s 6papoacugar 238 b Effect of temperature at which drying OCCUTS.........000eeeeeee cece eeeeeeeee 242 GupiitecvOne uration! Of GesiGatlOnarsc —Rapport sur la Question soumise a la Société de Biologie par Pouchet. MM. Pennetier, Tinel, et Doyére au Sujet de la Reviviscence des Animaux Deséchés. Lu a la Société de Biologie le 17 et le 24 Mars 1860 par M. Paul Broca au Nom d’un Commission composee de MM. Balbiani, Berthelot, Brown-Séquard, Dareste, Guillemin, Ch. Robin et Broca, Rapporteur. Mem. de la Soc. Biol. (3), ii, 1-139. Davis, H. ’73—A New Callidina: with the Result of Experiments on the Desicca- tion of Rotifers. Mon. Micr. Journ., ix, 201-209. Doyere, M. P. L. N. ’42—Memoire sur les Tardigrades. Ann. des Soc. Nat. (2), XVill, I-32. EnRENBERQ, C. G. ’38—Die Infusionsthierchen. Leipzig, p. 492, 1838. Facciou!, F. ’91—De la prétendu revivscence des Rotiféres. Archiv. Ital. de Biol., 360-374, 1891. FrEDERICQ, L. ’89—La lutte pour |’existence chez les animaux marins Paris, 1889. FROMENTEL, E. de *77—Recherches sur la revivication des rotiféres, les anguil- lules et les tardigrades. C. R. Assoc. Franc. l’avance. des sci. vi, 641-657. GavarrET, J. ’59—Quelques, Expériences sur les Rotiferes, les Tardigrades, et les Anguillules des Mousses des Toits. Ann. des Sci. Nat. (Zool.), (4), x1, 315-330. Hazen, T. E. ’99—The Life History of Sphzrella lacustris. Mem. of the Torr. Bot. Club, vi, 3, 1899. Hupson, C. T. ’73—Remarks on Mr. Henry Davis’ Paper “On the Desiccation of Rotifers.” Mon. Micr. Journ., ix, 274. The Desiccation of Rotifers. Journ. Roy. Micr. Soc. (2), vi, 79. Hupson, C. T. anp Gosse, O. H.’ 89—The Rotifera or Wheel Animalcules. Lon- don, 1889. Effects of Destccation on the Rotifer 263 Jennincs, H. S. ’04—Contributions to the Study of the Behavior of Lower organ- isms. Carnegie Inst. Publications, 1904. Kocus, W. ’90—Kann die Kontinuitat der Lebensvorginge zeitweilig vollig unter- brochen werden? Biol. Centralb., x, 673-686, 1890. EcxsTEIn, K. (’83)—Die Rotatorien der Umgegend von Giessen. Zeitschr. f. Wiss. Zool., xxxix, 343-344. 1883. LEEUWENHOEK, A. von, 1719 —Continuatio Arcanorum Nature. Epist. 144 ad Henr. Bleysvicium Lugd. Batav., 1719. Leipy, J. ’74—Remarks on the Revivication of Rotifer vulgaris. Proc. Acad. of Nat. Sci., Phila., p. 88, 1874. NEEDHAM, T., 1743—A Letter Concerning Chalky Tubulous Concretions, with some Microscopical Observations on the Farina of the Red Lily, and on Worms Discovered in Smutty Corn. Phil. Transact., xlii, 634-641, 1743. PoucueEt, F. A. ’59—Expériences sur la resistance vitale des animalcules pseudo- ressuscitants. Comp. Rend., xlix, 886. *59—(2) Nouvelles Expériences sur les animaux pseudo-resuscitants. Comp. Rend., xlix, 492. (3) Recherches et expériences sur les animaux resuscitants. Paris: J. B. Balliere et fils, 92 pp., 1859. Preyer, W. ’91—Ueber die Anabiose. Biol. Centralb., xi, 1-5. 1891. Ritzema Bos, J. ’88—Untersuchungen tiber Tylenchus devastatrix. Kihn. Biol. Centralb., vii, 650-659. 1888. Srack, H. J. ’73—The Desiccation of Rotifers. Mon. Micr. Journ., ix, 241, 1873. SPALLANZANI, L., 1787—Oeuvres: Opuscules de physique, animale et végétale, etc. trans. Jean Senebier. Tome, ii, 203-285. Zacuarias, O. ’86—Ko6nnen die Rotatorien und Tardigraden nach vollstandiger Austrocknung wieder aufleben oder nicht? Biol. Centralb., Vi, 230, 1886. PROTOZOAN STUDIES “ BY J. F. McCLENDON With Two Pirates While studying the Protozoa under Dr. H. 8. Jennings in 1904- 1905 I was impressed by the number of the theories relating to the physiology of these minute organisms and began to devise experiments with a view to testing some of the ae With- out attempting to draw very general conclusions from these experi- ments it is hoped that they will at least suggest further problems and make clearer the fact that Protozoa are very complex organ- isms. Not until quantitative studies of several forms are made will the physiology of the Protozoa be understood as clearly as that of the higher vertebrates. We cannot until then be sure of the significance of reactions such as are described in this paper and for this reason they are not fully discussed. I. REACTIONS OF AMCEBA PROTEUS TO MINUTELY LOCALIZED STIMULI! The reactions of Amceba to various stimuli have been described by various writers, but with the aid of the apparatus shown in Figs. 1 and 2 a more precise localization was possible.? As has been the case with other studies on Protozoa, so here, a detailed study reveals complexities comparable with those found in higher organisms. Mechanical Stimulation Amcebe were stimulated with an extremely fine glass needle. The time was counted with a metronome and distances measured 1 These experiments were made at Randolph-Macon College, Ashland, Va., in 1907. 2'To any one wishing to have apparatus like this made I would be glad to furnish descriptions or other data. Tue JouRNAL or EXPERIMENTAL ZOOLOGY, VOL. VI, NO. 2. 266 F. F. McClendon with an ocular micrometer in one eye piece of a Zeiss binocular microscope. The results of a large number of experiments show that the Amoeba does not respond to mechanical stimuli of very small area unless they be repeated at short intervals of time (one to two seconds), and that this interval is in inverse ratio to the area stimulated. Even when a glass needle was thrust through the Ameeba so that the end protruding from the other side was seen, no response was obtained, but the Amceba moved along as usual, the needle cutting a path through the protoplasm until the Ameeba had passed beyond it. When an Amceba was cut in two gradually by an extremely fine glass needle pressed upon it horizontally, the cutting produced no reaction that could be detected either in the piece with, or the piece without a nucleus. Some time elapsed before the non-nucleated piece behaved dif- ferently from the nucleated. Chemical Stimuli It was found impossible to confine fluids poured out of capil- lary tubes to very small areas, so I resorted to the following in- direct method: A fine copper wire was ground to a needle point and further sharpened by erosin in acid. ‘This copper needle was stuck into the ectosarc of the Amoeba. The mechanical effects should be no greater than those of the glass needle (i.e., unnoticeable) but the metallic copper, and colloidal particles flying off from it should chemically affect the adjacent protoplasm. Marked local changes occurred, and if the needle remained in the protoplasm long enough the adjacent area was killed. It appeared to me from a number of observations that this stimulus produced responses very quickly in remote parts of the Ameeba. This was very difficult to test for the following reasons: (1) ‘The Amceba may be considered as constantly receiving stimuli from one or more directions. It is probable that some of these stimuli come from within and are very variable. An additional stimulus must be very strong to produce a reaction that can be distinguished from others. (2) The Amoeba may be considered as a closed bag of ectosarc containing endosarc in the “sol”’ stage, and a con- traction of the ectosare at one place might produce hydrostatically Protozoan Studies 267 an extension at a distant point. Dellinger (’06) supposed that there are strands of denser protoplasm running through the endo- sarc, and gives as evidence the observation that an elongated ingested diatom will move freely along with granules in the endo- sarc when it lies lengthwise to the direction of flow, but will stop when turned sidewise, as though the meshes between the strands were greater than the breadth but less than the length of the diatom. ‘The same effect might be produced, however, by the resistance of the ectosarc when indented by the diatom. The diatom with its silicious shell would probably be heavier than the endosarc and press against the “ventral” wall of ectosarc. If it were lighter than the endosarc it would press against the “dorsal” body wall of the Amceba. In either case the resistance against being swept along by the current of endosarc would be less when it lay lengthwise than when it lay crosswise to the direction of flow. To demonstrate this I measured the force required to pull an ordinary glass slide over the surface of a soft gelatine plate under water. It required 2548 dynes when placed crosswise but only 2078 dynes when placed lengthwise. The same would hold for the diatom if it were heavier than or lighter than the endosarc. The current of endosarc would act on a larger surface when the diatom were placed crosswise but whether this would be sufficient to overcome the increased resistance it is impossible to determine without knowing the viscosity of endosarc and ectosare. But without morphological evidence to the contrary we may safely assume the endosarc to be without a fixed structure. To eliminate the hydrostatic effect I took advantage of the reaction of the Amceba which removes a strongly stimulated area from the source of the stimulus. Ordinarily this is done by local contraction of the ectosarc in the region stimulated. However, if this area is in the middle of a flat side, such a contraction is of no avail and [ have observed none. Furthermore, if the opposite point of the Ameeba is in contact with the substratum, its con- traction would not aid in the removal of the stimulated point. By studying Amoebz both from the side after the method of Del- linger and from above, I learned to distinguish from above those 268 F. F. McClendon portions that were attached to the substratum. I found froma large number of observations that an Ameeba stimulated in the center of a large flat area over an attachment to the substratum, by introducing into the ectosarc a copper needle, showed a tem- porary stoppage of the extension of pseudopodia in the most remote parts. ‘The interval of time was in half the cases less than that calculated for the movement of hydrogen ions in aqueous solution (.03 mm. per second). The reaction tme in Amceba is considerable (though apparently very variable) and allowing for it, it is probable that in all cases the stimulus traveled at a speed greater than .o3 mm. per second. Lest there be an electrostatic action at a distance I “grounded” the copper needle and repeated the experiment many times but with the sameresults. Inorder to facilitate observation I selected large Amcebe moving along without dorsal or lateral pseudopodia. ‘These gave the same results. The Food Taking of the Ameba From the above results, and observations on food taking I pro- pose the following hypothesis to account for the latter process: Chemical and physical influences of the medium cause a har- dening and shrinkage (by loss of water) of the ectosarc (Rhum- blers “Geletinisirungsdruck’’). Chemical processes within prevent this hardening from extending to the endosarc, and dissolve portions of the ectosarc that are displaced inward. ‘The medium affects different portions of the surface to different degrees, causing regional differences in degree of hardening and shrinking, thus producing amoeboid movements. A food body being protoplasmic and therefore similar to the substance of the Amceba might, in lying near an Ameeba, protect it from these outside influences. The protected region would become more fluid, and shrinkage of other regions of the surface would press it out toward the food until it touched it. The food would be pushed along and sometimes rolled over and would rub on the surface of the pseudopod pro- ducing mechanical stimuli of sufficient frequency to cause a local shrinkage of the ectosarc. This stimulus would spread through the protoplasm but being very weak and rapidly growing weaker Protozoan Studies 269 would cause the contraction of only a small area. Beyond the contracted area the protoplasm would continue moving toward the food and surround it from the sides. Probably many other factors enter into and complicate the process and sometimes make it resemble the food taking of higher animals. 2. THE EFFECTS OF CENTRIFUGAL FORCE ON PARAMCECIUM? Methods For short periods of time the hematocrit attachment of a hand centrifuge was used. For longer periods I made an electric cen- trifuge. I made several centrifuges that could be run by a one- fortieth horse power or a one-twentieth horse power hot air motor. The University of Missouri furnished me with a Bausch and Lomb electric centrifuge with a special revolving arm of 158 mm. radius, carrying two one-half drachm vials. I enclosed this in a close fitting chamber which increased the speed by preventing radial air currents. With shunt winding 4000 revolutions per minute were obtained. ‘The speed was regluated with a circular rheostat having 32 stops. In most cases I used gum arabic (or other gums) dialysed through filter paper until it was neutral to litmus, to buoy up the Parameecia in the centrifuge, and I repeated these experiments without gum up to as high a speed as the Parameecia could survive. For a convenient index of the centrifugal force the formula n?r was used —where 7 is the number of revolutions per minute and r the radius in millimeters. In earlier experi- ments the revolutions could not be counted with a speed-counter and had to be calculated from the gear, and the results were prob- ably too high owing tos lipping of bands. The word outward is used to denote direction from the axis of the centrifuge and :nward toward the axis. ‘The recorded experiments are on Paramoecium caudatum but Parameecium aurelia gave simular results. For permanent preparations I found the best method to be fix- ation for one minute in I per cent chromic acid and staining from three to five minutes in Biondi’s methyl green, orange G and acid 3 These experiments were carried on during the session of 1906-1907 at Randolph-Macon College, Ashland, Va., and continued during the winter of 1907-1908 at the University of Missouri. 270 F. F. McClendon fuchsin mixture with a little less fuchsin and of about one-fourth saturated strength. To facilitate changing rapidly from one fluid to another the hematocrit was used to precipitate the Para- meecia. [he chromatin was stained green, plasmosomes orange, cell granules red or orange, trichocysts red and cilia and discharged trichocysts sometimes green. Every part of a whole mount could be studied with the 2 mm. Zeiss apochromatic objective so but few sections were cut. Experiments After centrifuging 15 minutes with n’r = 13,950 X 10% the heavier substances of the food vacuoles and phosphate crystals if present lie in the extreme outer end of the Paramcecium and some may even be forced through the ectoplasm. Next to these come the micronucleus and then the macronucleus. Fig. 3 shows a specimen subjected to this force five minutes. The chromatin has been precipitated so violently as to stretch the nuclear wall, but otherwise the macronucleus has not been displaced. It appear as though the macronucleus were attached in some way, but the appearance might be due to the nuclear sap being less dense than the endoplasm or to the viscosity of the endoplasm preventing the rapid precipitation of the whole macronucleus. ‘The anterior end of the Paramcecium was in this case turned outward. Dr. Lyon (’05) showed that this is usually the initial orientation, but the geotropic reaction may be strong enough to turn the anterior end in the opposite direction, as is shown in Fig. 5. Fig, 4 is drawn from a specimen subjected for half an hour to less force (n’r = 6200 X 10°). The micronucleus is almost in the extreme outer end of the animal. ‘The precipitation of the chromatin has greatly stretched the wall of the macronucleus and the wall has burst at its inner end. In a lot that were centrifuged longer one Paramcecium was found to be without macronucleus or micronucleus or even scattered chromatin material. I have this specimen stained and mounted and have examined it repeatedly without finding a trace of chromatin. Whether the wall of the macronucleus burst as in the preceding case and the nuclei disinte- grated, or whether the nuclei were forced through the wall of the - Protozoan Studies 271 Paramcecium and lost, or whether the Paramcecium is the result of a division in the centrifuge in which one daughter was pre- vented from receiving nuclear material by the centrifugal force, it is impossible to say. I have found what appeared to be division stages in specimens just taken from the centrifuge. However, if the centrifugal force had been great the process of division in these specimens was usually not continued, but the elongate and sometimes partially constricted animal would swim about for days without undergoing much change before death. Fig. 5 is of a specimen centrifuged 24 hours, n’r = 742 x 10°. ‘The poste- rior end was turned outward, and both nuclei have crowded into that end. All the Paramcecia subjected to this experiment were very small at its close, probably due to loss of water under the increased pressure. Judging from many individuals, the time that the macronucleus remained displaced after removal from the centrifuge was about the same as the time it had probably remained displaced in the centrifuge. Many exceptions to this rule, and the fact that some individuals changed their orientation while in the centrifuge, makes an exact statement impossible. E. P. Lyon (05) showed that Parameecia centrifuged for some time are not negatively geotropic. Ifthe geotropic reaction be due to the pressure in one direction of substances in the Paramcecium of specific gravity different from the surrounding protoplasm, (statolith theory) an abnormal displacement of such substances ° might upset the mechanism of geotropism. I found in con- firmation of Lyon’s results that Parameecia recently taken from the centrifuge are not negatively geotropic but apparently swim as often in one direction as in another and gradually reach the bottom by their own weight. Great care must be used in this experiment to rid the Parameecia of gum if they have been put in it, as Ostwald (’06) has shown that altered viscosity of the medium may change the sign of the reaction. Jensen (’93) has shown that Parameecia are positively geotropic on hot days, and the friction of the air may raise the temperature of fluids inthe centrifuge. ‘The effects of a rise of temperature and increase in viscosity due to a little gum in the medium would tend to neutralize 272 ¥. F. McClendon one another. It has been shown above that the pressure in the centrifuge reduces the size and probably increases the density of the Parameecia. This might cause them to go to the bottom even though they preserved a negatively geotropic orientation. I found the time elapsing before the return of the negatively geo- tropic reaction to roughly correspond to the time required for the return of the nuclei to their normal position. ‘This might indicate that the nuclei in normal position acted as statoliths. ‘The fact that the Parameecia are constantly revolving on their long axes does not prevent the application of the statolith theory, because Parameecia moving horizontally do not react to gravity (Jennings ’04); it is only when they start to swim downward that they react. I kept Parameecia in the centrifuge for various periods of time up to one week to test whether distance from the nucleus would effect the structure of the ectoplasm, and obtained only negative results. Probably the circulation of the endoplasm is sufficient to equalize the distribution of substances diffusing out of the nucleus. In case gum solution was used control experiments of Parameecia in gum but not centrifuged were made. The gum was eaten and gave the Parameecia a slightly swollen, vacuolated appearance. Some of the experiments are tabulated below: nr | Tie | RESULT 164 X 10) 7 hrs. | Many nuclei displaced, they had returned in a few hours. 384 X 10 15 min. | Many nuclei displaced, they had returned in 15 minutes. 585 X 10 | 1 day | Many nuclei displaced, they had returned in 1 day. 585 X 10} 14 days | Many nuclei displaced, they had returned in 30 hours. 585 X 10| 3% days | Many nuclei displaced, they had returned in 24 days. 585 X 10| 4 days | Similar results, the displacement was in some cases transmitted to products of | fission. 585 X 10] 5 days | Similar results. 585 X 10! 6 days | Similar results. 764 X 10 6 days | Similar results, many misshapen.* 1318 X 10) 1 day | Similar results, many misshapen.* 1316 X 10} 4 day | Similar results, some dumb-bell shaped. In those marked with * no gum was used. A marked effect on the rate of division was noticed in individ- uals taken from the centrifuge. It has been shown by Calkins Protozoan Studies 272 and his students that the rate of division may be increased by various changes in the chemical composition of the medium. ‘To eliminate error from this source I was careful to have the medium of the centrifuged individuals and the control exactly the same. In the table below, the divisions of three individuals centrifuged 24. hours (n?r = 585 X 10°) are compared with the divisions of three control individuals. Both daughters of the first division of No. 1 were kept, and one of them designated as ta. In all other cases one daughter of each division was thrown away. | Days | | No. | Fea ea eee ie cat Ti ea | in, | Torar I] 2} 3) 4] 5 6 7| sl Q TOLL 1213 1415 16117 18 19 20)21/22 29) et ieee | a | Centrifuged | | hil | Ve Paiteie | hom | Tie oc OO CR NOE OU DD CaReen are J 1 11 toad ogg agg KD 0G 00 dqcC 00 4 Tf 6006010, COS oS Coe Ieee 1} 110odc ogo 0100G0 God da0 ZES SC BO e SoA eS ee | o 1] | 1] of ol do Oo 110 OO DO gd 0d 3 Ree ciate acvaosie'e crear» of 1] 1] 1] oO Of Oc} O 1] of Of Of | x 1 Of 1] Of Of of of x 8 Control | | aia | | igs a | Tsonod Men ooo en Good Serie Oo} oO Ijc Spe Mes tS AS PTS At ee ORS ES Cee 3 Da sd pot bed pat Coe Eee | 99 0 109.000 1100000 odead....)..).... 2 2s gsidgise taGH Sen eee ace 90 1] c] 9 OO q fo) o Sl sl alsa apd eae cals Sel I | } | In the three series from the centrifuge (not counting ta) there were 15 divisions in 23 days whereas in the check there were only 6 divisions in that time and two of the series had run out. In the table below three individuals centrifuged 32 hours at the same rate are compared with three in the check: Days No. A Tora | | | | I 2 3} 4) 5] 6 7 8) g|t0)£1/12)13|14|15|16|17|18|19 20 BiG e ici ic cls alin ci eck Centrifuged | | | | lesa Deere etapa a Sea \es o7a, cies la aos “I] 2} 3] Oo} 1} oldejad)../../..). (alan lod eel ae 7 DESC DO SOUR OEIC CE cer I] 1] 1] | oO s}e area I ol 1} 1| oc 10 2. cnS GOS SUS BOE See poe Deere 2a) 3). 1| Toe) 3] (9) cl ra) a Ee} alerle aes 13 | | Control bon | | REP assisasse s claid weleia a ss ole e aes 1] 1| 2} 1| oldejad) | | | | 5 Dold cat Cone Teneo enee 1| 1] 1] ©] o| oldelad| | | [fel rll B Bp cree ev iele ars sctavelaiac pees = « o} 1| o| o} i\dejad| | | | | 2 J } 274 F. F. McClendon In the above experiment there were in 20 days, 30 divisions in the three series from the centrifuge and only 10 in the control. Dur- ing the five days in which the control lived, there were 16 divisions in the centrifuged series. Many experiments with Parameecia centrifuged from one hour to six days, both with and without gum, gave similar results, only the effect was not so marked in the longer periods. All the above lots were from the same culture. It was stated above that many Parameecia are misshapen in the centrifuge. The end into which the nuclei are precipitated is bulged out by them, and in a few cases the other end is also bulged out (by the accumulation of substances of low specific gravity £) giving the animal the form of a dumb-bell. I fed Parameecia on egg yolk, and globules of a dark brown fatty substance were formed in the endoplasm in such numbers as to make it appear black. These black Paramcecia were subjected to as great a centrifugal force as they could stand, in some cases for two or three days. The fatty substance being of low specific gravity, accumulated in the end opposite the one into which the nuclei were precipitated. The result was a pear shaped body, the large end being black and the small end of the normal Paramcecium color. So great was the difference in the specific gravity of the two ends that the animal could only with difficulty assume a horizontal position or turn the small end uppermost. No marked increase in rate of growth or reproduction that could be ascribed to the stimulating effect of the lecithin in the yolk was observed. . It was noted above that some of the Parameecia when taken from the centrifuge appeared to be undergoing division. More often the body was abnormally elongated, with or without a con- striction in the middle. In these cases the end not containing the nuclei shriveled after a day or so, and the animal died within a week. It may be that the centrifugal force keeping the nucleus in one end prevents its division though the animal is large enough to form two, and the portion around the nucleus attempts to form itself into an individual, thus causing the constriction in the middle. If such a division is actually completed I have never observed it. When Parameecia are centrifuged without gum in the medium, Protozoan Studies 275 the bacteria upon which they feed are all precipitated to the outer end of the receptacle. Although the centrifugal force may be such that the Parameecia can swim against it, the lack of bacteria elsewhere may cause them to remain in the outer end of the recep- tacle in the area made acid by the bacteria. Here some of them are pressed out of shape and may be reduced to thin lamelle. I have seen such forms after being taken from the centrifuge swim about for days in this flattened condition, and sometimes finally regain their normal proportion. More often the Paramcecium turns a number of times while being pressed in the outer end of the receptacle and is reduced to an irregular mass. Such masses may grow to large size and develop several buccal grooves. They may also divide and some of the products of division be irregular, while others form normal Parameecia. A curious case is shown in Fig. 6, though as this specimen was fed on egg its abnormality is probably as much due to the bulging out of one end by the fatty substance as to the pressure of the wall of the receptacle. Its condition when removed from the centrifuge is shown at a. The large end is three-lobed and it appears as though it were about to divide itself longitudinally into three individuals. ‘The next day there are four lobes on the large end and two on the small end (d). On the third day two of the constrictions have disappeared and the animal is almost divided longitudinally into two. In this con- dition it died. One of the irregular individuals taken from the centrifuge and isolated in small watch crystals, divided into two daughters that appeared normal in every respect save that each bore a long horn on the oral side. Dr. H. S. Jennings found a Paramcecium sim- ilar to one of these, in an old culture, and found that for a number of generations (i.e., as long as the series lived) the horn was passed to one of the products of each division and the other was normal. I thought it would be interesting to compare the transmission of these horns produced mechanically, with his observations. The diagram in Fig. 7 presents the results: The irregular individual taken from the centrifuge is represented by a; it divided to form the first two daughters, each with a horn. Some daughters are represented by small sketches, the others by the letters meaning 276 F. F. McClendon normal and /, bearing a horn. One of the first two daughters has the horn nearer the anterior end the other nearer the posterior end. After each division the horn is in a different position, and we can predict the position of the horn in each generation by drawing an imaginary line bisecting the animal in the preceding generation transversely. Many of the “normal” daughters were kept many generations without showing any abnormality in their offspring. Although one series died out in the sixth and the other in the eighth generation there is no reason to believe that the horns would have been lost had the series lived. Sometimes the horn grew and at other times decreased in size but in the later genera- tions it was as large as in the earlier. It is easy to see why such deformities are seldom found in nature, for in case one is pro- duced, even if the deformed individual has an equal chance with normal ones of living and reproducing, after seven generations less than one per cent of its offspring will show the abnormality. The main difference between the results of the reproductive process here and in the Metazoa is the transmission of acquired characters to a small per cent of the progeny in case such charac- ters do not cause the death of their possessors. To speak of a germ plasm in Parameecium without morphological evidence might seem unwarranted. 3. ON ABNORMALITIES PRODUCED BY ENCYSTMENT AND OTHER CAUSES IN PARAMCECIUM AURELIA.* The encystment of Paramececium putridum was described by Lindner (’99) that of P. busaria by Prowazek (’g9) and that of P. caudatum by Simpson (or). I have repeatedly observed Para- moecium aurelia forming a thin membranous cyst in which it might be confined for a week but in which it was killed by drying. For this reason it might be wrong to compare these cysts with those observed by others in Paramcecium. ‘These cysts are most often seen 1n the interior or on the surface of bacterial zodglea and | thought at first that the Paramcecia were simply entangled in the zodglea, but as other Paramcecia were seen at the same * These observations were made at the University of Missouri. ere Fe Protozoan Studies 277 time making their way with ease through the same zodglea at least part of the wall of the cyst must be secreted by the Para- meecium. During the formation of the cyst the animal contin- ually rotates inside of it and the ciliary coat is never lost. ‘The cyst gradually contracts until it is shorter than the occupant, which may have the anterior end folded over the middle of the body or the ectoplasm thrown into folds. After the animal has been in the cyst for some time the folding of the ectoplasm may assume the character of an invagination of the anterior (rarely posterior) end. ‘The invaginated ectoplasm seems to be in large measure absorbed, for after several days the occupant of such a cyst looks like merely the posterior end of a Paramcecium. If the cyst is opened or if one waits until the occupant comes out of its own accord, the latter will swim about and ingest its food as though an anterior end were superfluous. I found numbers of Parameecia without anterior ends, and a few without posterior ends, in the old culture in which the encystment was found. One of these is represented in Fig. 8, a. It was isolated in a watch crystal and remained in the form of the figure two days. On the third day it had changed to the form shown in Fig. 8, b. This might be interpreted as a division in which the reduced vitality of the animal prevented the complete separation of the daughters, followed by a partial division of one of the daughters. The speci- men was lost so that its later history could not be followed. Some abnormalities seem to be the result of the plasticity of the adoral side at the time of conjugation. A pair of conjugants were isolated, and when they separated the adoral regions were drawn out into prominences (Fig. 9). Such prominences are gradually absorbed although they may remain for several days. These prominences do not seem to be of the same nature as the horns shown in Fig. 7 since so far as the investigations go the horns do not and these smaller prominences do disappear. If Parameecia are shaken up with broken glass all those that are not killed or cut in two regain their normal form in a short time (and even fragments if they live regenerate after a longer period) although they may be so torn and mashed out of shape as to bear little resemblance to the type. Probably the form of a Paramcecium 2.78 F. F. McClendon cannot be permanently changed without the formation in it of one or more new chemical substances. In other words we might hypothetically consider the horn alluded to as of the nature of a graft differing chemically from the Paramcecium to which it was attached. 4. ON VARIATIONS IN PARAMCECIUM CAUDATUM AND P. AURELIA® In making the foregoing studies I was interested in inquiring into the identity of the species used. In the experiments at Ashland, Va., only one form was used. It had but one micro- nucleus and agreed in other respects with descriptions of Para- moecium caudatum. At the University of Missouri two forms were found, one similar to that studied in Virginia and another having two micronuclei, and in this respect agreeing with descrip- tions of Paramececium aurelia. Calkins (’06) found a case of P. caudatum acquiring two micronuclei and some of its offspring losing one micronucleus and becoming normal P. caudatum again. He further states that the number of micronuclei is the only invariable character for separating caudatum from aurelia and therefore they are probably the same species. Whether caudatum and aurelia form two species or not cannot be decided from the data at hand but I have evidence to show that these two forms are quite distinct. In none of the numerous cultures which I have kept for months and in one case a year, have | found indi- viduals with different numbers of micronuclei in the same culture. No characters of outward form were found that would serve to separate caudatum and aurelia. ‘The size character is best studied in graphs of the lengths of a large number of individuals of each culture (Fig. 10). In each curve in Fig. 10 the lengths of the indi- viduals measured in fractions of a millimeter are plotted as abscis- sas and the number of individuals in a class represented by arbi- trary units on the ordinates and marked at the top by a cross. The curve itself is merely to aid the eye in comparing measure- ments from one culture with those from another, as are also the 5 These observations were made at the University of Missouri. Protozoan Studies 279 continuations of the .1, .2, and .3 mm. ordinates. The height of one curve is not to be compared with that of another as it depends on the number of individuals measured and the number of classes, but the spread and limits of the curves are to be compared. The specimens were measured after killing in one per cent chromic acid, a mechanical stage was used to prevent any unconscious selection and the measurements for some curves were made with an ocular micrometer, and for other curves with a camera lucida. Fig. 10, a represents the lengths of 218 individuals from a culture of P. caudatum from Hinkson Creek, Columbia, Mo., kept in the laboratory one month. Individuals .2 mm. in length form the class of greatest frequency. ‘The next curve (b) is of 234 indi- viduals from a pond near by. ‘The class of greatest frequency is .1g mm. The third curve (c) is of 219 individuals from Ash- land, Va. They had enough food for health but not enough for reproduction. ‘The class of greatest frequency is .182 mm. After feeding this culture 24 hours on hay infusion, 184 individuals were measured (d), and the class of greatest frequency calculated at .I91 mm., showing that the majority had increased in length. It will be noticed that the curve extends farther to the left. This is due to the fact that many individuals had recently divided and were shorter than before feeding. ‘These and all other cultures of P. caudatum gave a nearly symmetrical curve. Such was not the case with P. aurelia (shown in the last two curves). The curve e is plotted from the lengths of 297 individuals from an old cul- ture found at the University of Missouri. The class of greatest frequency 1 is .133 mm. Hence most of them are shorter than the majority of P. caudatum, but it will be seen that the second mode of the curve is composed of individuals longer than the majority of P.caudatum. At first I thought some individuals of caudatum had gotten into the culture, but an examination of the micronuclei of a large number of individuals both large and small proved that not to be the case. The next curve (d) is of 127 individuals from a culture from a drain ditch at Columbia, Mo. It shows two modes similar in position to those in the preceding, but the highest is of the largest individuals (.26 mm. in length). From these two cultures of P. aurelia I isolated individuals of different lengths 280 F. F. McClendon and started separate cultures from them, which were kept for months. In every case the progeny of one individual showeda curve of as little spread as those given of P. caudatum. Thus it was possible to obtain a culture of minute individuals or one of giants or one of medium size. Subjecting a culture to higher tem- perature or increased salinity decreased the size of the individuals while lower temperatures increased the size of the individuals. P. aurelia of this region must then be dimorphic or else it hap- pened that in starting both the above cultures (e and /) only large and small and no medium sized individuals were procured. Large samples of water containing decaying leaves, etc., from various places developed no cultures of Parameecia, so that more data for aurelia was not obtained.. We may say then that aurelia differs from caudatum in the presence of two micronuclei and that some aurelia are smaller than the smallest caudatum. It has frequently been noted that conjugating individuals are smaller than non-conjugants. [| think that the fact that the mouths of conjugants are closed is sufficient to cause the smaller size. By comparing curve c (of individuals fed less) with curve d (of individuals fed more) you may note that those fed less are smaller. However, no matter how little food is given Parameecia they can still take in water through the mouth and be swelled up with vacuoles, which is not the case with conjugants. Note that the variation of the well fed individuals is greater than that of the poorly fed. Dr. Pearl (’07) found the same to be true of non- conjugants as contrasted with conjugants. In conclusion, a note on the effect of a certain food may be of interest. It is well known that hay infusion has to be often re- newed to keep a culture of Parameecia in good condition. If the culture is left in the same infusion the individuals become smaller, sluggish and finally die. However, if a considerable amount of cane sugar be added to the culture, the Parameecia are very little affected at first but after several weeks become more active and live for months. Whether this be due to alcohol that appears or to bacteria produced in the culture was not determined. Protozoan Studies 281 APPENDIX Dr. Jenning’s paper entitled “Heredity, Variation and Evolu- tion in Protozoa. [” (this journal, vol. v) appeared after this paper had been sent to press. I would emphasize a probable difference between the process of heredity in Protozoa and in Metazoa other than the difference in complexity. In Protozoa the “germ-plasm,”’ whether it be all or part of the individual, is probably equally as accessible to the environment as the “soma.” I use the words “germ plasm” and ‘soma’? for brevity. BIBLIOGRAPHY Of Part 1 BERNSTEIN ’00—Chemotropische Bewegung &c. Arch. f.d. ges. Physiol. 80 Bd. De.uincer, O. P. ’06—Locomotion of Ameeba and Allied Forms. Jour. Exper. Zo6l., vol. iii. Jennincs, H. S. ’04—Contributions to the Study of Lower Organsims. 6. The Movements and Reactions of Amoeba. Publication No. 16. Car- negie Inst. McCtenpon, J. F. ’07—Experiments on the Eggs of Chatopterus, etc. Bio. Bull., vol. xii. Matuews, A. P. ’o7—An Apparent Pharmacological Action-at-a-distance by Metals and Metaloids. Amer. Jour. Physiol., vol. xviii. MaxweLt, S. S. ’07—Is the Conduction of the Nerve Impulse a Chemical or a Physical Process? Jour. of Bio-chemistry, vol. iii. Montcomery, E. ’81—Zur Lehre von Muskelcontraction. Arch. f. d. ges. Physiol., 25 Bd. RuumBLER, L. ’98—Physikalische Analyse von Lebenserscheinungen der Zelle. I. Arch. f. Entwicklungsmech. d. Organismen, vol. vii. ’*o5—Zur Theorie der Oberflachenkrafte der Amoben. Zeit. wiss. Zool., 83 Bd. SUTHERLAND, W. ’06—A Molecular Theory of the Electric Properties of Nerve. Amer. Jour. Physiol., vol. xvii. Of Part 2 Catkins, G. N. anv Lies, C. ’02—Studies on the Life History of Protozoa, II. Arch. f. Protistenk., 1 Bd. 282 F. F. McClendon Carkins, G. N. ’04—Studies on the Life History of Protozoa. IV. Jour. Exper. Zool., vol. i. CzaPEK ’02—Stoftwechselprozesse in der geotropisch gereizten Wurzelspitze, etc. Ber. deutsch. bot. Gesell., 20 Bd. Darwin, F. ’03—On Artificial Production of Rhythm in Plants. Ann. of Bot., 1903. Hatal ’03—Lecithin a Stimulant to Growth. Amer. Jour. Physiol., vol. x. Jensen, P. ’93—Ueber den Geotropismus niederer-Organismen. Arch. f. d. ges. Physiol., 53 Bd. Jennincs, ’04—The Behavior of Parameecium. Jour. Comp. Neur. and Psy., vol. xiv. KuawkINE, M. W. ’88—Le Principe de l’Hérédité et les Lois de la Mécanique en Application a la Morphologie de Cellules Solitaires. Arch. Zoo. Expér. et Gén. (2) Tome 6. Lyon, E. P. ’05—On the Theory of Geotropism in Parameecium. Am. Jour. Physiol., vol. xiv. : ’o7—Results of Centrifugalizing Eggs, 1 and II. Arch. f. Entwicklungs- mech. d. Org. 23 Bd. Mavupas, E., ’88—Recherches Expérimentales sur la Multiplication des Infusiors Ciliés. Arch. Zoo. Exp. et Gen. (2) Tome. 6. MirropHanow, P., ’03—Nouvelles Recherches sur |’Appareil Nucleaire des Para- mécies. Arch. Zoo. Exp. et Gen. (4), Tome i. Moreau, T. H., ’06—Influence of a Strong Centrifugal Force on the Frog’s Egg. Arch. f. Entwicklungsmech. d. Org., 22 Bd. Mortier, D. M. ’oo—The Effect of Centrifugal Force on the Cell. Ann. Bot., vol. xiii. OstwaLp, WoLFcANG, ’03—Zur Theorie der Richtungsbewegungen schwimmen- der niederer Organismen I. Archiv. f. d. ges. Physiologie, 95 Bd. *o6—II. Archiv. f. d. ges. Physiologie, 111 Bd. Wipers, E. ’oo—Inutilité de la Lécithine comme Excitant de la Croissance, etc. La Cellule, T. xvii. Of Part 3 LinpnER, G. ’99—Die Protozoenkeime in Regenwasser. Biol. Cent’bl., xix Bd. ProwazeEk, S.’99—Kleine Protozoenbeobachtungen. Zool. Anz., xxii. Smmpson, J. Y. ‘or—Studies in Protozoa. I. Pro. Scott. Micro. Soc. ili. et we, Protozoan Studies 283 Of Part 4 : GrrassIMow ’04—Physiology of the Cell. -Hertwic, R. ’03—Wechselverhaltniss von Kern und Protoplasma. “Miinchen. = (Lehmann). - Peart, R. ’07—Biometrical Study of Conjugation in Paramecium. Biome- trika., vol. v. Pirate I Fig. 1 “Mechanical hand” for moving a fine pointed instrument in stimulating Amoeba. When in use it is clamped to the stage of a Zeiss binocular microscope (Greenough model) so that the whole appara- tus except the point of the instrument lies to one side of the field of the microscope. When necessary two can be clamped to the same microscope. A crude form of this apparatus, figured and described in an earlier paper (McClendon, ’o7), had the disadvantage of surrounding the field of the microscope on three sides and sometimes colliding with the watchglass or slide on which the objects were placed. By turning the milled head on the left an instrument held in the clamp is moved crosswise, by turning the milled head in the center the instrument is moved back and forth, and by turning the one on the right it is moved vertically, all three are held in the hand and turned by different fingers. Fig. 2 A simpler form of the same apparatus. The milled head moving the instrument vertically is high above the other two. PROTOZOAN STUDIES J. F. McCrenpon ree | I i. a ae Fitiea ieee = Wy aiZ WS SSK SSS SSS SS Tue J OURNAL oF Ex PERIMENTA DB L ZOjLOGY, VOL. VI, NO. 2. Prate II Fig. 3 Paramecium caudatum, (optical section) centrifuged 5 minutes; nr = 13,950 X 10% Fig. 4 Ibid., centrifuged 4 hour; n?r = 6,200 X 10%. Fig. 5 Ibid., same scale, centrifuged 24 hours; n? r = 742 X 108. Fig. 6 a. P. caudatum fed on hens’ egg yolk and centrifuged two days, n?r = 764 X 10%. 5, the same one day later. c, the same one day later. Fig. 7 Genealogical table of descendants of a, a P. caudatum mutilated in the centrifuge (6 days, n*r = 764 X 10°). hk = horned and n = normal individual. Fig.8 a= P.caudatum from old culture. 6 = the same two days later. Fig. 9 A pair of ex-conjugants showing deformities produced by conjugation. Fig. 10 Series of curves showing variation in length in P. caudatum and P. aurelia. a, P. cauda- tum (218 individuals) from Hinkson Creek, Columbia, Mo. 5, P. caudatum (234 individuals) from a pond at Columbia, Mo. c, P. caudatum (219 individuals) from Ashland, Va., on maintenance. d, P. caudatum (184 individuals) from Ashland, Va., well fed 24 hours. e, P .aurelia (297 individuals) from old culture at the University of Missouri. f, P. aurelia (127 individuals) from a drain ditch at Columbia, Mo. | . PROTOZOAN STUDIES PLATE II J. F. McCienpon -mm 2mm 23mm caudatum 10 ** Tue JouRNAL oF EXPERIMENTAL ZOOLOGY, VOL. VI, NO. 2. eid as ner’ mak DEVELOPMENT OF ARTIFICIALLY PRODUCED CYCLOPEAN FISH—“THE MAGNESIUM EMBRYO” BY CHARLES R. STOCKARD Wirth One Pirate AND SIXTY-THREE Text Ficures REELS MR TSN Ta cso ie fate scree aaa aie rasn sins eha) ofa tv ene wre! 'biainin sig ieialelorevelaza's olele a0 Os 285 eae Meera LCC ei errr coi ee rire to aN clic: tyaie's Essig winds Ga viveghaulew ee adie a aes 287 Cyclocephali and Monstra Monophthalmica Asymmetrica Bich eceibh eeeyaia Ss thal arate a epaar agains 292 a_ Living cyclopean monsters from the first indication of the defect to the time of hatching..... 294 Mire MS UEEILELONT PCY CLOT ABS tetefe esis <2) ofeielo) a) isis) era70 = 'c,v's «= v= s\0)sisie’e sia'o syerarsiere@ eis © ole ele 298 aetavae Monstra Monophthalmica Asymmetrica ....1.....20000.+sceen sect evccacreee 304, - Tioturiy BIE eRe EIR gs Aeon ootap leds n Shad Conn Ones aene DOr ae aoc Sr ase sccorc 305 a Earliest indication, exact position and condition of the eye...............eee eee eee renee 305 PEpeEGENp lebe CYClo pias COUDIEIEYESI -2)6.ctefes =1c\5 +1 are 2 -0'e ole o/s ose iors viv eie'cisie.e ole alten ciel ajeinye 310 Beeeedect smpleicyclopeaneye and NOmMal braiM. .. 0.02... s 2 eee ee cc ese ieee aes lenm ers 311 d Extreme cyclopia; from the abnormally small anterior cyclopean eye to entire absence of BUGS . Js nde Baa doe Clo DOGS Te Bop CeO ACO OO BOA CENA Aa eo reet nr micic ryoreot-c - 315 Incomplete diprosopus, with three eyes and one additional lens............--.0--00 eee eeeeeeeee 322 Morphology of Monstra Monophthalmica Asymmetrica ......... 200s eee cece eee eee ences B25 Independent origin and self-differentiation of the crystalline lens....-......-..--0000 0 eee ee eres 328 (Tian ud! sardGnitths ite-es ¢oaccu1€ coe Sheet teen heer. Senne Oo nmeonnn a otorr 330 | THEI). .oceds4adind sda Sade CD DOR Dee Dg OS Oe abn SD de aan pone MD rrecrpcan oa cicrcia errs 334 CBE LStFRG al cock oeS Re Rina ot Op oe Bie eee ene eee nse ante are 336 INTRODUCTION Development is the resultant of the interaction between the inherent tendencies contained within the egg substance itself and the external conditions which surround and act upon this sub- stance. The usual interaction of these factors gives rise to normal animal forms. When, however, either factor is changed an unusual form results; in the one case there arises a germinal variant, and in the other an anomaly occurs as a response to the strange external environment. ‘The product of development, the formed animal, is then to a certain extent a creature of its environ- ment. On the other hand the importance of the internal factors must be recognized although modern experimental work often- THE JourNAaL or ExPERIMENTAL ZOOLOGY, VOL. VI, NO. 2. 286 | Charles R. Stockard times points in a direction which would indicate that these factors may be largely modified in their influences by the external con- ditions. Most monstrosities or abnormalities in development are due to the action of external factors, either mechanical, as pressure, or chemical. Mammals, birds and reptiles, with their complex embryonic-membranes, offer many opportunities for the produc- tion of secondary abnormalities arising from unfavorable mechan- ical or physical conditions. Fcetal amputations and scars, mem- brane fusions distorting facial development, and many other such deformaties are in most cases probably due to secondary influences on development. Besides these there are deformities of a dif- ferent nature, such as the excessive monsters, monstra in excessu, in which certain organs have over developed or produced super- numerary parts; and contrasted with them are defective monsters which fail to complete themselves and are therefore less than a normal individual. It is with this latter class, monstra in defectu, that the present study is concerned. These defective individuals may be grouped into two sub-classes: first those in which certain organs fail to complete themselves, as in cleft palate, hare-lip, arrests in the development of the heart and other parts of the circulatory system. Second, individuals in which certain paired organs occur singly or without mates. “True Cyclocephali or cyclops monsters find their place in this last group. Cyclops monsters have long been known to occur in manand other mammals and are described in many of the earliest medical works. In these beings the one eye is in the middle line of the face and often shows external evidence of a double composition. The nose which normally arises above the eyes and grows down between them as the face develops is here mechanically prevented from descending by the presence of the median eye in its path. The foetus, therefore, has a proboscis-like nose above the eye. The brain and other parts of the body are sometimes deformed though they may be normal. Among the lower vertebrates true cyclops monsters have been recorded by Spemann (’04) as resulting from mechanical injuries to the eggs of the amphibian, Triton tzniatus. These mon- se POe tie oy Artificially Produced Cyclopean Fish 287 sters were double-headed with one or both heads showing the cyclopean defect and were not of the usual single cyclopean type found in man and other mammals. Two years ago (1907) I carried out experiments in which I was able to produce typical single cyclopean fish. ‘This was the first record of the occurrence of cyclopia among fishes. It is also the first case of consistently producing vertebrate monsters such as are known in nature by changing the chemical environment in which the eggs develop. ‘These embryos are in main details similar to the mammalian cyclops, having a single median eye and anteriorly placed double nasal pits. The monsters were produced by allowing the eggs to develop in sea-water in which there was an excess of MgCl,. Cyclopia occurred in a large percentage (at times 50 per cent) of the embryos. The discovery was made so late in the spawning season that it was impossible to investigate the details of the cyclopean defect or rear the embryos to hatching in order to observe their ability to swim ortosee. The method of produc- tion, however, offered such an exceptional opportunity to obtain abundant material for studying all stages of development and degrees of cyclopia that this more extended survey was under- taken. The following account includes a comparative study of cyclopean embryos from the earliest appearance of the optic vesicle to the perfectly formed free-swimming fish with a functional cyclopean eye. The experiments were conducted in the Marine Biological Laboratory at Woods Holl, Mass., during the past summer, while occupying one of the rooms of the Wistar Institute. MATERIAL AND METHOD. As in my previous experiments, the eggs used were those of the teleost fish, Fundulus heteroclitus. The method of producing the defect was much the same as that previously employed although expanded and modified in many ways. During the early part of the season it was difficult to find 288 Charles R. Stockard solutions of the proper strengths and the eggs were either killed or. unaffected. After a few experiments, however, a strength of MgCl, in sea-water was found that gave a large percentage of cyclopia, in many cases again causing 50 per cent of the eggs to form such individuals. ‘This was a 1% m solution prepared as follows: 19 cc. of a molecular solution of MgCl, in distilled water was added to 41 cc. of sea-water. This is not then an actual 18m MgCl, solution but it is 18 parts molecular MgCl,. Mak- te the solution in this way adds to the sea-water, water lacking all of its constituents except the Mg and thus increases in a greater proportion the excess of Mg present. Cyclopia occurred in a series of similarly prepared solutions ranging as follows: 18 M, 17 M, 35M, 35 My Bo My 35 M and 22 M 60 MgCl. A point of importance is that the proportion of oe embryos produced gradually rises in this series up to the 18 M solution and then falls off again. To illustrate concretely, in Experiment VII the 16 m solution caused 12 per cent of the eggs to form cyclopean embryos, the 17 M gave 30 per cent, the 18 M 22 per cent, while te M gave 50 = cent with the cyclopean defect. Sennen, the series, the zo M falls off to 30 per cent and the 21M gives 23 per cent, while in the 22 M no cy clopia occurred and the eggs were all killed. It must be born in mind that these percentages are for the eggs that formed embryos and not for the total number of eggs first put into the solution. The peculiar fact is, that in a series of MgCl, solutions we reach a place where a maximum number of cyclopean embryos occur and in strengths both weaker and stronger than this the number of cyclopean individuals is less. If the defect is due to osmotic pressure, we should not expect a greater pressure to bring about a more normal development. If the action is chemical, we do not usually reach a chemically effective dose and find that a greater dose is less effective. It might be argued that below the point of maximum occurrence of the cyclopean defect, the solutions are insufficient to effect any but the weaker embryos, so that a small number of cyclops appear; above this point the solutions are so strong that all except the hardiest embryos die in early stages and those sur- viving are so resistant that only a few give the cyclopean defect. Artificially Produced Cyclopean Fish 289 At the maximum point the normal or ordinary individuals, which predominate, would be affected, and here the greatest number of cyclopean embryos occur. As I previously mentioned, the MgCl, is found to be rather toxic to these eggs during the earlier stages of development. Many die at this time, but in the medium strength solutions 70 to 80 per cent live and form embryos and in the weaker solutions often more than go per cent live. After the early embryo is formed, however, the high death rate falls and a dead embryo is of rare occurrence in any of the solutions. Many embryos were kept in the solutions thirty days and some hatched in strengths as strong as 1s M. If, on the other hand, the eggs are removed from the solutions when sixty or seventy hours old, when the cyclopean condition is readily distinguishable, and placed in sea-water they grow much better and many hatch normally. Some of the cyclopean fish came out on the twelfth day after fertilization, though usually they were much slower in emerging. The control embryos hatch in from eleven to twenty days, depending chiefly upon the temperature. Solutions of MgCl, in Distilled Water Distilled water solutions of MgCl, of several strengths; 1° M, 11 M, 12 M, 1% M, 14 M and 15 m were not effective. The eggs either died during early stages or developed into embryos with two normal eyes. I had found (1906) that salts of lithium induce the same typical defects in Fundulus eggs in both sea-water and distilled water solutions. Such solutions have opposite conditions of pressure, being in one case hypertonic and in the other hypo- tonic and thus remove all question of osmotic effects as a cause. It was hoped that Mg might also act in the two solutions which would have made it certain that the direct action of the magnesium ion is responsible for the cyclopean condition of these embryos. The problem of cyclopean formation seems, however, to be more complex. It involves the action of magnesium in the presence of certain or all of the sea-water salts. 290 Charles R. Stockard Solutions of MgSO, and Mg(NO,), in Sea-water Sea-water solutions of MgSO, prepared in a similar manner to the MgCl, solutions above were employed. ‘The following SEES M, oo M, $+ My > Ms 0 My so My Go Ms > M Go M, 24 M, 25 M, and 27 M were ineffective, the eggs in these solutions developing ee with very few deaths at any stage. Meg(NO,), solutions in sea-water caused typical cyclopia indis- tinguishable in all respects from that produced in MgCl,. The following strengths were used: 12 M, 14 M, 15 M, 18 M, 17 M, 18 M, 18 M, and 22 Mm. These Mg(NO,), solutions also killed many embryos iene the early stages of development. Cyclopia occurred in from 4 per cent to 40 per cent of the eggs in 22 M, 1s M, 18 M,16M,andi3mMm. These strengths are comparable to those most effective for MgCl,, both as to the amount of magnesium present and as to osmotic pressure. Mixtures of MgCl, + NaCl; MgSO, + NaCl; and Mg (NO,), + NaCl Mixtures of MgCl, and NaCl were added to sea-water as fol- lows: 12 cc. of a molecular solution of MgCl, was added to 12 cc. of NaCl, and 36 cc. of sea-water was then taken to make the entire quantity up to 60 cc. This solution will be spoken of as 1M + 1M, the first term referring to the MgCl, present and the second to the NaCl. On this basis the ployee mixtures were used: 1M +1M,4M +1M,2.M + 1M, 2, M + 1M, in which the MgCl, was wae pad the Weel beet constant, andim +4 M, +M + 4M, .$,M + 4M,4M + 4M, in which the anes NaCl was erated also. Such mixtures caused the se Sioa of cyclopia, the best results were obtained in } M + 1 M, where as times as many as 25 per cent occurred. The 4; M + 1M gave in one case 30 per cent of cyclopia. The {, 1M cape II per cent. It will be seen that the amount of MgCl, present in these mixtures is less than that necessary to cause similar results when used alone. This is a peculiar fact and one for which I know of no explana- tion. Similar results (Stockard ’o7b) were found with mixtures Artificially Produced Cyclopean Fish 291 of salts in distilled water where the final pressure was less than that of sea-water, the normal medium of theeggs. It is also true that if such substances as the sugars be added to a salt solution, a smaller dose of the salt becomes effective in the presence of the sugar. Morgan (’06) first called attention to this peculiar fact in studying the effects of solutions upon developing frogs’ eggs. This would seem to indicate that the effects were due to osmotic pressure conditions and by slightly raising the pressure with another element the effective agent was assisted in its action, but my lithium experiments (1906 and 1907b) are against such a view. A number of mixtures of MgSO, and NaCl were tried, all giv- ing negative results. Mixtures of Mg(NO,), and NaCl as fol- lows were used: }M + 4M, M + 1M and 7, M +1 ™M. The first two caused eggs to develop cyclopia. These are mix- tures closely similar to the effective MgCl, and NaCl solutions. We conclude that cyclopean monsters are produced in Fundulus eggs by the action of sea-water solutions of MgCl,, Mg(NO,), and mixtures of MgCl, and NaCl and Mg(NO,), and NaCl. No other solutions of the many I[ have tried during three summers gave similar effects. Other salt solutions and sugar solutions exerting practically the same osmotic pressure also fail to cause cyclopia. Another argument opposed to the view that osmotic pressure is the cause is the fact that Fundulus embryos are so resistant to changes in pressure. Since two Mg salts give similar results when used in sea-water solutions, it seems probable that the action of Mg, either directly or indirectly, is responsible for the result. Eggs have been subjected to this action before the first cleavage, during the two-cell stage and just before going into four cells, with similar results. No attempt was made to determine at how late a stage the cyclopean condition could still be caused, though it could doubtless be induced after the eggs had passed much beyond the four cell stage. The fact is that cyclopia may be caused in an egg which has started its development normally and which would have given a two-eyed embryo. The idea of a germinal origin of the defect in this case seems excluded. Cyclopia in this instance is the result of unusual external conditions. 292 Charles R. Stockard. CYCLOCEPHALI AND “‘MONSTRA MONOPHTHALMICA ASYMMETRICA ” The magnesium solutions induce the formation of two distinct types of eye monstrosities. ‘The first type is the typical cyclopean monsters, which exhibits a series of individuals showing various degrees of cyclopia. Beginning with a normal individual having eyes in their usual position, we find others in which the eyes are slightly inclined forward and somewhat closer together than usual; or the eyes are still more approximated and occupy an unusually anterior position. (See the diagram, Fig. 1). Next in the series are individuals with their eyes approximated but still distinctly separate, having two optic nerves and two eyeballs with their choroid coats in intimate approximation. We next find the true cyclopean eye which still shows a double nature having two optic nerves; the retina has a paired arrangement and either one or two lenses may occur, depending upon the degree of distinctness of the two components. ‘This eye generally occupies a ventro-median position and looks forward, inclining slightly downward. ‘The eye in others is completely single, showing no indication of a compound structure; it has one optic nerve, a single retinal arrangement, one lens and one pupil. ‘This is the perfection of cyclopia and many embryos possessing such an eye are apparently normal in other respects, except the mouth and nose. They have a ty pically bilateral brain and are perfectly capable of free-swimming movements. Passing beyond this stage of cyclopia, we find embryos which have gone to the extreme and show only a defective antero-median eye. In some indi- viduals the eye is represented merely by a choroid vesicle. ‘The step beyond this is the entire absence of the eye. Diagram Fig. 1 gives a schematic illustration of the various degrees in the cyclo- pean series thus outlined. The histological conditions shown by such a series will be considered beyond. It is important to understand that this series is made up of different individuals showing various degrees of cyclopia and that a cyclopean monster does not pass through these steps in its development. The cyclopean defect is foreshadowed in its final condition when the optic vesicle first separates itself from the brain. Artificially Produced Cyclopean Fish 293 Fig.1 Diagrams of the various conditions of the cyclopean defect as shown by the “Magnesium embryos,” from the normal 4 to complete absence of the eye G. Fig:2 Diagram of the monstrum monophthalmicum asymmetricum series, from one defective eye B to complete absence of one eye E. 204 Charles R. Stockard The second type of optic defect caused by magnesium is a new monstrosity and may be termed Monstrum monophthalmicum asymmetricum, the monster with one asymmetrical eye. It has only one perfect eye which represents one of the normal pair and occupies the usual lateral position. ‘This eye is in all cases perfect while its mate may be indicated by either a small eye, by a mere cellular mass representing an optic cup, or all indications of the second optic cup may be wanting. (See Fig. 2.) This peculiar one-eyed condition exists in many of the embryos in the magnesium solutions. Had such a defect resulted from a mechanical opera- tion, it would probably have been interpreted to mean that one eye anlage was injured and the other not. With the solutions, however, we get a clear case of the gradual dropping out of one eye by comparing different individuals, and here as in cyclopia the defect is present from the earliest appearance of the eye, and is not due to a gradual degeneration, or arrest during develop- ment. A study of sections of these embryos makes the conditions clearer. a The Living Cyclopean Embryos from the First Indication of the Defect to the Time of Hatching The optic vesicles appear in most eggs when about thirty hours old; at this time the blastopore is just closing and the embryo is well mapped out on the embryonic shield. Many attempts were made to select cyclopean individuals at this stage but it could not be done with a great degree of certainty, since some embryos are always slow in giving off the optic vesicles and these at times appear to have only one, but when examined some hours later are found to be normal. A number of eggs were selected, how- ever, at thirty hours old which proved to be cyclopean on later examination. At about forty hours the defect is plainly detectable so that one may arrange the eggs very accurately into two groups, the cyclo- pean individuals and the normal. After such a separation, none of the normal embryos ever exhibited the cyclopean defect in later stages, although kept in Mg solutions. A number of such tests as this in connection with the study of sections convinced me that Par - Artificially Produced Cyclopean Fish 295 the cyclopean condition existed as such from the first appearance of the optic vesicles, and no subsequent fusion of the two optic vesicles or cups took place after that time. A forty-two hour embryo is shown in Fig. 3. It is seen to be well formed and the optic vesicles are clearly outlined on either side of the head. Fig. 4 illustrates a cyclopean individual of the same age. The single optic vesicle occupying a ventro-median position is shown through the transparent embryo. ‘This young individual with its newly formed optic vesicle shows a typical cyclo- pean condition, and no indication is seen of two separate elements that would later fuse. Other embryos at this age have abnormally twisted cephalic regions and show no indication of eyes, although the cyclopean eye might easily be concealed by the bent brain (Fig. 5). Such embryos at later periods are found to be cyclopean and to have narrow tubular brains showing more or less abnormal bendings. When the embryos are about three days old, the brain has expanded and presents a distinctly bilateral appearance; the optic cups are well developed and the lenses are partially formed (Fig. 6). A cyclops monster at this time has a well formed body and the brain is often normal, though in Fig. 7 it is inclined toward the narrow tubular condition and is anteriorly twisted. ‘The ven- tromedian eye is clearly seen through the brain and the outline of its lens is distinct. A somewhat younger, sixty-five hour, embryo is shown in Fig. 8 with a superficially perfect brain and two optic cups intimately approximated. The telencephalon is seen to protrude beyond the eyes, as is the case in the normal individual (Fig. 6). Three four-day embryos are shown by Figs. 9, 10 and 11. The brain and spinal cord at this time are clearly mapped out by a coarse pigmentation, the two hemisphere-like portions (corpora bigemina) of the mid-brain are distinctly formed and the eyes are large with the lens clearly outlined within the cup. A cyclopean monster with a perfectly formed large ventro-median eye is illustrated by Fig. 10. Comparing its brain and other parts with the normal (Fig. 9), one fails to find any important deviations. The abnormal condition of the narrow tubular brained cyclops, 296 ; Charles R. Stockard Camera lucida sketches of living embryos from MgCl2 solutions Fig. 3 A normal embryo of forty-two hours, the two optic vesicles present. , Fig. 4 A typical cyclopean individual of forty-two hours. The singlemedian eye (0. V.) is rep- resented in circular outline. Fig. 5 Anembryo of same age, twisted brain, no optic vesicle shown. Fig. 6 Normal seventy-two hour embryo. ‘ Fig. 7 Cyclops of same age. The eye, op.c, in ventro-median position. Fig. 8 Sixty-five hour embryo, two ventrally approximated optic cups. Fig. 9 Normal four day embryo—bilateral brain outlined. Fig. 10 Four day cyclops, large ventro-median eye and typically bilateral brain, op.c, the eye. Fig. 11 Four day cyclops with narrow tubular central nervous system. Figs. 12 and 13. Five day cyclops, narrow tubular brain with waist-like constrictions dividing them into fore, mid and hind-brain regions. Ventro-median eye. Fig. 14 Five day cyclops with ventro-median eye and dorsally humped brain. 297 Artificrally Produced Cyclopean Fish 298 Charles R. Stockard Fig. 11, is evident. Fig. 12 shows a common type of cyclopia with the three primary brain regions separated by waist-like con- strictions. [wo other variations of the narrow tubular condition are found in Figs. 13 and 14. The embryos are five days old and no changes of importance occur from this time until the hatching period is reached, except the usual progressive development of the eye structures. The normal embryos generally begin hatching when about twelve days old, one cyclopean monster hatched at this time but most such indviduals were much later than the normal in coming out. A twelve day cyclopean fish is seen in dorsal view in Fig. 15 and ventrally in Fig. 16. The large cyclopean eye projects forward and occupies the position usually taken by the mouth at thistime. A slight indention along its mid dorsal line suggests its double nature, although the ventral view (Fig. 16) shows this same eye to possess only one pupil and lens. ‘The brain of this specimen is practically normal. An embryo with the two eyes intimately approximated is shown in front view in Fig. 17. The eyes are joined and each looks forward in a direction slightly towards the side to which it belongs. A common variety of cyclopean fish is one in which the eye is unusually small and occupies an extremely anterior position; Fig. 18 shows such an embryo. ‘This variety is usually unable to hatch, although a few were assisted in breaking through the membrane. They swam rather abnormally, owing to a twisted condition of the body. A dorsal and ventral view of a cyclopean fish is shown in Plate I, Figs. A and B. ‘This indicates the striking appearance pre- sented by these embryos. b Free-Swimming Cyclopean Fish Many embryos, showing the cyclopean defect in various degrees, hatched normally and were capable of swimming in a manner indistinguishable from ordinary two-eyed fish. ‘These monsters gave many indications of ability to see. “They went to the more brilliantly lighted side of the dish with the normal ones. ‘They darted away in a normal fashion when any object was placed in front of the eye, while similar objects put at equal distances from Artificially Produced Cyclopean Fish 2.99 their tails caused no excitement. In two instances they lived for ten days, which is about as long as the two-eyed embryos can survive without food. At this time the entire content of the yolk- sac has been absorbed. The embryos in nature doubtless begin feeding previous to this stage. ‘The cyclopean individuals appear to be as active as the normal and their ability to live would seem to depend only upon the possibility of their obtaining food. A normal fish eight days after hatching is illustrated by Fig. 23. The mouth projects forward beyond the dorsal tip of the head and the two eyes are lateral in position. A cyclopean embryo eight days after hatching is shown in Fig. 24. Here the two eyes are united and occupy the position which the mouth has in Fig. 23. In Fig. 25 a perfectly cyclopean eye is shown in dorsal view: the same individual is seen in lateral and ventral views in Figs. 26 and 27. This fish swam in a normal manner. In the lateral position the mouth is shown projecting ventrally as a pro- boscis-like structure. This condition is due to the fact that the single antero-median eye occupies the position normally assumed by the mouth and thus obstructs the usual forward growth of its structures. [he mouth, therefore, remains ventro-posterior to the eye and grows downward, presenting the proboscis-like appearance. Such a condition recalls in a striking way the nose of the mam- malian cyclops. In mammals the cyclopean defect is accom- panied by a proboscis-like nose situated in the forehead above the median eye. The nose in normal development grows downward to its facial position, but in cyclopia the median eye obstructs its path and forces the formation of the proboscis-like organ in the forehead. The same explanation holds for the fish’s mouth where the eye prevents its forward growth, producing the proboscis- like organ. It is interesting to find that the mouth in cyclopean fish stands in a position so as to fall in the gill series as number one, all the gills and the mouth have the same general direction. I have found that in Bdellostoma the mouth arises in a manner similar to the gills and actually at first arches dorsally and only secondarily arches ventrally. It may have originally been a member of the gill series, as Dohrn (1875) has long thought. It would be 300 Charles R. Stockard Camera sketches of the living embryos in magnesium solutions Fig. 15 Dorsal view of twelve day cyclopean monster, the antero-median eye with furrow indicating its double nature. Fig. 16 Ventral view of the same individual, the eye possesses a single pupil and lens. Fig. 17 A twelve day embryo, ventral view showing two eyes intimately approximated. : Fig. 18 Fourteen day embryo. Small extremely anterior cyclopean eye with protruding lens, extreme cyclopia. Fig. 19 A five day Monstrum monophthalmicum asymmetricum; the left eye has no mate. Fig. 20 A similar twelve day monster lacking its left eye. Fig.21 Anincomplete diprosopus monster seventy-two hours old. Two brains, two normal lateral eyes and one perfect middle eye, the other middle eye indicated by the circular lens L. Fig.22 The same monster when eighteen days old, three perfect eyes. The embryo hatched three hours after this drawing was made and swam abnormally. g 301 Artificially Produced Cyclopean Fish 2 [es sel ie aa 302 Fig. 23 Fig. 24 Fig. 25 Fig. 26 Charles R. Stockard Camera sketches of free-swimming fish Normal individual. JM, its anteriorly placed mouth. Incomplete cyclops, two eyes joined and occupy the position usually taken by the mouth. Dorsal aspect of a perfect cyclops. Antero-median single eye. Lateral view of same. The mouth M is forced by the eye to remain in a ventral position and hangs down as a proboscis-like structure. Fig. 27 Ventral view of same fish, note perfectly single eye, one lens and one pupil, ys., yolk-sac. a Artificially Produced Cyclopean Fish 303 Giga cron Let SO Pae> 2 Pad ee 3 = tae AP ee SS hel wala SS eI AA La >, = 25 304. Charles R. Stockard interesting to know whether the “‘cyclopean mouth” is functional. The mouth does not possess a wide opening as it would normally although a small aperture is sometimes distinguishable near the end of the proboscis. No attempt was made to feed the embryos. c Living Monstra Monophthalmica Asymmetrica These asymmetrical one-eyed monsters may also be identified in early stages of their development. ‘They have a single eye situated on one side of the head. Such an eye appears in some cases as though it were cyclopean and one might easily imagine the cyclopean eye becoming displaced from its usual median position to one side or the other of the head. Studying such eyes in section, however, clearly shows their single unmated origin and condition. An embryo of this kind is shown when five days old in Fig. 19. The brain is slightly abnormal and the pigmentation scarce for such a stage of development. ‘The eye occupies the usual place of the paired eye of that side. A twelve day embryo shortly before hatching is illustrated by Fig. 20. The shape of the body and of the head is comparatively normal. ‘The unpaired eye is slightly forward of its usual position. Many of these embryos hatched. A few of them swam in circles, often whirling around with great rapidity, much as Japan- ese waltzing mice do. Others swam in irregular spirals and only progressed in a straight direction with difficulty. This peculiar one-sided manner of swimming is not due to asymmetrical vision, but results from a defective muscular arrangement, the animal’s body being slightly bent or twisted so that it is unable to straighten perfectly. Some embryos with this eye on one side had normally straight bodies and these were capable of swimming in a direct course with apparently as much ease as a two-eyed fish or the symmetrical cyclopean embryos. These monsters also lived, free-swimming, for some time. In a few cases their mouths were perfect, but in others the mouth parts were distorted or twisted by an asymmetrical condition of the ventral head region. Artificially Produced Cyclopean Fish 305 MORPHOLOGY OF CYCLOCEPHALIA It was mentioned above that the optic outpushings became visible on the sides of the brain at about thirty hours after fertiliza- tion. At this time the brain of Fundulus is a solid mass of cells without a central ventricle. ‘The optic bodies are not hollow at first, but are solid outpushings which later develop central cavities. The cavity generally forms in the optic outpushings while the brain is yet solid. Dareste (’91) has advanced hypothetically the idea that if the anterior vesicles of the brain did not develop, a contact would be maintained between the “parties retiniennes”’ up to a certain time and consequently they would unite to give a median cyclopean eye. If this were in reality the cause of cyclopia we might expect all Teleosts like Fundulus to be normally cyclo- pean since in them the eyes arise while the brain is without a ven- tricle. Spemann (’04) finds in cyclopean Triton embryos that although the tube is hollow, the eye anlagen are defective from the beginning. The matter of a closed brain would then seem to be unimportant in a consideration of the causes of cyclopia. a Earliest Indication, Exact Position and Condition of the Eye When forty-one hours old, the brain as shown in trans-section by Fig. 28, is still a solid mass. The two normal optic out- pushings have developed small cavities but no indication of invagination of the vesicles or ectodermic lens structures are seen. A section through the optic region of an apparently one-eyed monster when forty-one hours old, is shown by Fig. 29. The sections of this series show only one ventro-lateral eye vesicle. The vesicle is large and distinctly optic in nature, while on the opposite side is shown a thick cellular wall from which the brain is becoming separated. Such an individual resembles more a Monstrum monophthalmicum asymmetricum than it does the cyclopean type. Fig. 30 shows a transverse section through the eye of a forty- nine hour embryo which exhibits a perfectly clear case of cyclopia. Here the brain is beginning to form a cavity and the optic vesicle with a well defined central cavity is just invaginating to form the 306 Charlies R. Stockard optic cup. This eye occupies an almost ventro-median position and is united to the brain by a solid cellular stalk. Its contact with ectoderm from which the lens will arise is not established as the head-fold does not yetextend back to this point. Aneye in such a ventral position will oftentimes come in contact with the ecto- derm at a later stage than would a normal lateral eye. Ordinary two-eyed individuals at this age (forty-nine hours) were, like this cyclops, just beginning to form the optic cups and the lateral ectoderm over the incipient cups showed a slight thickening, the earliest indication of a lens. As a rule the cyclopean eye is some- what slower than the normal in its rate of development and may generally be compared with the eyes of slightly younger two- eyed individuals. Several embryos at this age lack eyes entirely and belong to the blind variety presently. to be described. Two-eyed embryos when fifty-four hours old possess well- formed optic cups and lenses still connected with the ectoderm, although projecting into the cavity of the cup. The nasal pits are clearly marked ectodermal invaginations in an anterior and slightly median position relative to the eyes. The brain possesses a well developed central cavity. A cyclopean eye of a distinctly double composition from a fifty-four hour embryo is shown in cross-section by Fig. 31. The optic cup is bilateral and two lens anlagen are indicated by the thickened ventral ectoderm. ‘The section of the brain dorsal to this eye is small and hollow. It is a portion of the diencephalon which is between a larger telen- cephalon and a much larger mid-brain. ‘This eye would finally have produced a large median cyclopean organ of the double type with two retinal areas and two lenses. Its connection with the brain is through two closely approximated stalks and two optic nerves would probably have formed later. Comparing such an eye with that of Fig. 32, of like age we find that here the optic cup is single and one lens is forming. Both sections show the eye in practically similar positions. The embryo from which Fig. 32 was taken possesses a well formed telencephalon and two lateral nasal-pit thickenings of the anterior ectoderm. A horizontal section of a fifty-four hour double-eyed cyclops Artificially Produced Cyclopean Fish 307 Fig. 28 A trans-section through the optic outpushings of a normal forty-one hour embryo. The brain is solid and cavities are just forming in the optic outpushings. Fig. 29 Trans-section through the single optic vesicle (0p.v.) of a forty-one hour embryo from to M MgCl,. The optic process is situated laterally and no indication of a like process exists on the other side. Fig. 30 A slightly oblique section through the cyclopean optic vesicle of a forty-nine hour embryo from 39 M MgCl, op.v. optic vesicle. Fig. 31. Cross section through double cyclopean eye of fifty-four hour embryo from $4 m MgCh, op.c. optic cup; L, lens thickening of ectoderm; Br, normal bilateral brain. Fig. 32 Section of single cyclopean eye in similar embryo. L, lens; op.c. optic cup, small solid diencephalon above; X, guide figure indicating the plane of the sections. 308 Charles R. Stockard is given in Fig. 33. Such a section is most instructive. The condition of the eye is much the same as that shown by the trans- verse section, Fig. 31. ‘The cup is double and two ventral lenses are present. ‘The section passes below (ventral) the diencephalon so that no part of it shows; the telencephalon is indicated in front of the eyes and a thickening of the forward ectoderm shows the nasal plate, posteriorly or behind the eyes the mid-brain 1s cut in horizontal section. A sagittal section of a typical cyclopean embryo is shown by Fig. 34. Here we see the eye and the brain in the third dimension. The telencephalon in front, the diencephalon above the eye, and behind this the large mid-brain with a spacious median cavity. In front of the eye is also shown a median ectodermal thicken- ing, the double nasal pit. The eye is single and exactly ventro- median in its position and connects in a more lateral section with the brain at about the point where the telencephalon and dien- cephalon join. The lens and retina are differentiating into their typical structures. One may obtain a clear mental reconstruc- tion of the cyclops monster at this age by comparing Figs. 31, 32, 33 and 34, the transverse, horizontal and sagittal mid-planes of the cyclopean eye. The early stages just described illustrate the cyclopean defect in its various degrees, and the eye throughout its development retains the original condition of singleness or doubleness. No evidence whatever can be found of subsequent fusions during development. ‘Two clearly approximated eyes arise in that con- dition and remain so without fusing to give a double cyclopean eye, and a double eye never attains to the single condition by a more intimate union of its parts. The statement made in my (1907a) former paper, p. 257, that “‘the fusion of the two components may take place at different periods within a certain limit”? is incorrect, as I (1908) have pointed out in a short note on the subject. This statement was one of interpretation and was based on a comparison of late embryos which showed different degrees of cyclopia. It seemed from such an incomplete study that the eyes were more or less double or compound, depending. upon the stage in development at which they had become approxi- Artificially Produced Cyclopean Fish 309 ia 00% gi. O)1 ‘ 0) 3 ig) ges Fig. 33 Almost horizontal section through a double cyclopean eye of a fifty-four hour embryo in 33M MgCle. See guide figure X for plane of section. N>p., nasal plate; L, lens; op.c., optic cup; Tc., telencephalon; M/b., mid-brain. Fig. 34 Sagittal section (guide figure 7) through typical single cyclopean eye showing its ventral position below the diencephalon Dc. The nasal pit, Np. is median; L, lens; op.c. optic cup; Tc., telencephalon; Mb., mid-brain. 310 Charles R. Stockard mated. ‘The point is one which can only be proven by a number of direct observations on all ages of cyclopean embryos and care- ful study of sections; such a study has convinced me that no fusion of the eyes takes place after they are once clearly given out from the brain. It seems advisable for later stages to consider groups of embryos showing various degrees of the cyclopean defect. b Incomplete Cyclopia; Double Eyes Under the term incomplete cyclopia may be considered indi- viduals with eyes abnormally close together although separate Among Fundulus embryos such individuals exist and a series of stages connect these embryos with those in which the two eyes are intimately connected or joined together. An individual of this kind when sectioned will show the eyes as in Fig. 35. This sec- tion is from a four day embryo, the two eyes are united in the median line of the head and both are perfect eyes with a lens, single retina and one optic nerve. ‘The choroid coat as indicated by the heavy line is just beginning to form. Fig. 36 shows a section of two eyes which are more intimately united. This case is the common “hour-glass”’ eye of cyclopia. The two eyes are independent, except for their waist-like connection and each has its lens, single pupil, retina and distinct opticus. The optic nerve of the right component is seen entering the optic cross at the base of the brain. ‘The brain in this embryo is remarkably perfect, as it is in many cyclopean monsters, and I see no reason whatever for attributing the defect to a “single brain” or any other gross malformation of the cephalic region. Many embryos with deformed brains possessed two normal eyes and the converse is true, many normal brains were accompanied by cyclopean eyes. Leaving the “hour-glass” eye, we find the double-eye shown in Fig. 37, having a common optic chamber each half of which is supplied by one component. ‘[wo lenses and two pupils are present and generally two optic nerves, although they may run so nearly parallel that the two are difficult to distinguish. A single nasal pit is present in the embryo from which Fig. 37 is a section. All of the cyclopean monsters possess two distinct auditory vesicles. Artificially Produced Cyclopean Fish Edy Fig. 38 is a section through a unique double eye; no other such case was found. The two retinal components are connected along their median dorsal line within the brain and extend down facing one another. They are like the two sides of a leguminous pod; between the two a single lens is placed suggesting the seed in the pod. Enclosing the ventral part of the retinal components is a choroid coat shown in heavy black. This choroidal coat does not fully encompass the retinal areas, a part of which extends dorsally far up into the brain. The anterior end of the eye is V-shaped insection. ‘The optic cup anlagen in this case must have been closely united from their first origin in the brain, since por- tions of the retinal region are still contained within the brain itself, yet during development they did not fuse into a single eye. A single nasal pit is present and the mouth. is ventral and pro- boscis-like. An almost single eye is indicated in section, Fig. 39. The choroid coat surrounds the retina, the latter showing slight traces of its compound nature. ‘Two lightly staining regions of nerve tissue are seen and the entire eye is unusually wide laterally. The single lens is normal. ‘The brain here is also normal and the eye occupies a ventro-median position. A further union of the eyes gives the c Perfect Single Cyclopean Eye and Normal Brain The cyclopean eyes are in many cases perfectly single, resem- bling in all respects, except their position, one eye of a normal pair. They are placed immediately ventral and their antero-posterior mid-plane is in the median line of the embryo. ‘The brain in such a cyclops is often normal in all general respects. Figs. 40 and 41 represent horizontal sections through the brain regions of such a cyclopean fish when seventy-seven hours old. Fig. 40, the more dorsal section, passes through the mid-brain and shows the two lateral, hemisphere-like bodies (corpora bigemina) with well formed cavities. Behind these the section cuts the floor of the hind-brain for some distance and finally crosses it where the head bends. Passing ventrally through a number of sections, we find the one shown in Fig. 41. Here only a small ventral 312 Charles R. Stockard Transverse sections of different degrees of double cyclopean eyes Fig. 35 Section of eyes in four day embryo, the two eyes united. Choroid coat beginning. Fig. 36 Section of “‘hour-glass” eyes, the optic nerve of the right component entering the normally bilateral brain. From asixteen day embryo, the retine and lenses differentiated. r.o.n., right optic nerve; Ch., choroid coat. Fig. 37 Section of eye in hatched embryo. Double-eye with two pupils and two lenses. Retina undifferentiated. Fig. 38 Hatched cyclops, section through the peculiar eye with two components facing and lens between them (see text). Fig.39 Section through almost single cyclopean eye, only indication of its compound nature paired retinal arrangement. Brain normal. Guide figure X indicates the plane of all sections and the eye position in the several specimens. 7 313 Artificially Produced Cyclopean Fish = © wv j =e B Qi: YON,» Sel Te at IT NA 314 Charles R. Stockard part of one of the corpora *bigemina is cut and the completely single eye with its lens is found lying ventrally and in a median position. The double olfactory pit is seen in front of the eye and somewhat to one side of the head. ‘The posterior part of the section runs below the hind-brain and finally cuts it as the head bends just in the middle region of the well formed auditory ves- icles. “The section thus presents the three sense organs, the single cyclopean eye, the nasal pits united into a double pit; the paired ear vesicles alone are in their usual positions. A transverse section through the eye of a four day embryo is illustrated in Fig. 42. The retina is unusually wide laterally but no other indication of doubleness is shown. ‘The choroid coat is beginning to form and the eye is connected with the floor of the brain by a single cellular stalk. ‘The retina at this age is only slightly differentiated and there is no arrangement into layers. “This embryo has two distinct nasal plates. Several of the cyclopean fish show the nasal plates separate, although they are usually represented by an anterior double plate near the middle line. A nine day embryo of which Fig. 43 represents a section through the eye has a finely developed brain, well expanded laterally and perfect in general shape and structure. ‘The eye is completely single and the retina is partially formed into layers; the lens 1s almost transparent and the vitreous humor is being formed about it. The eye has all structures closely similar to those in a paired eye of this age and would doubtless have functioned had the embryo hatched. This specimen has a single nasal pit. Another cyclops of perfect structure when studied in sections at thirteen days old showed the mouth posterior to the eye, hang- ing as a ventral proboscis-like mass. . Two nasal plates were present and the eye was single. This eye, Fig. 44, was unusually far forward and although the retina was well differentiated into layers the humor had not perfectly formed behind the lens. The small section of the brain is shown in Fig. 44 to be bilateral and not unusual in appearance. Passing forward through the series of sections to a place where the anterior end of the cyclopean eye- ball stops, a minute lens is found lying in a ventro-median position, Artificially Produced Cyclopean Fish 315 Fig. 45. This lens, although only nine micromillimeters in diameter, has differentiated and shows perfect lens fibers arranged in the usual concentric fashion. It has no connection whatever with the eye, nor with any part of the central nervous system. The small lens doubtless originated and differentiated its tissue in an independent manner. The independent origin and self- differentiation of lenses will be clearly shown in a following sec- tion of this paper. Fig. 45 also illustrates the two lateral nasal plates in section. The cyclopean eye is thus seen to be at times single in nature, showing no trace of a double composition. This may be con- sidered the climax or perfection of cyclopia, if such an expression is permissible. Eyes not completely united, or double-eyes, are the incomplete or imperfect cyclopean condition, while the single condition reduced or distorted may be termed extreme cyclopia. d Extreme Cyclopia: From the Abnormally Small Anterior Cyclopean Eye to Entire Absence of Eyes Many cases are found representing the condition of extreme cyclopia. They may be considered in order, beginning with the least modified. In discussing the living embryo mention was made of those with a small cyclopean eye placed far forward (Fig. 18). Sections of such eyes show them to be of a more or less imperfect nature and sometimes deeply buried in the tissues of the head. Fig. 46 shows a section through the small eye of a hatched embryo eighteen days after fertilization. This eye is placed in the extreme anterior tip of the head and the section shows on the right side pigment spots which lie on the front end of the forehead. The eye is unusually small and the living embryo was abnormal, being unable to swim directly forward. The nasal pits are united in the anterior eye region and a pro- boscis-like mouth is situated ventrally. Two still more abnormal cyclopean eyes are shown in trans- verse section by Figs. 47 and 48, both from thirteen day embryos. In Fig. 47 the eye is close to the single olfactory pit, the retina 1s differentiated into layers, but the lens is larger than the optic cup so that it cannot fit completely into it. The brain of this individ- 316 Charles R. Stockard Sections of perfectly single cyclopean eyes Fig. 40 Horizontal section through mid-brain showing its corpora bigemina, Cb, and floor of hind brain, Hd, in seventy-seven hour cyclops. Fig. 41 A more ventral section of same series, E, the Cyclopean eye; o/.p., olfactory pits united. Hb, hind-brain and Av., auditory vesicle; Cb, floor of one mid-brain lobe. Guide figure X gives plane of each section. Fig. 42 Trans-section of a four day single cyclopean eye in exact ventro-median position. ch, choroid coat; p, pigment spot. Fig. 43 Similar section of nine day eye. Humor cavity behind the lens. Note perfectly bilateral brain. p, pigment spot. Fig. 44 Section of single median eye below perfectly bilateral brain, thirteen days old. Fig. 45 A more anterior section in same series as Fig. 44. The forward tip of the eye ch is seen. A small lens L lies free near the ventral ectoderm; o/.p., olfactory pit; p, pigment spots on anterior end of brain. Guide figure X indicates plane of all sections. Dies 317 Artificially Produced Cyclopean Fish 318 Charles R. Stockard ual is abnormal and the eye is out of the median line. The em- bryo of Fig. 48 was abnormal with the brain distorted so that the cyclopean eye was slightly to one side and far out beyond the head. The retina differentiates into layers but the lens lies out of the central position, and would be unable to function efficiently. A peculiar condition is found in the embryo from which sec- tions shown in Figs. 49, 50 and 51 were taken. ‘This very small eye was again in an extremely anterior position, though almost in the median line. The lens is as large as the optic cup and pro- trudes far out beyond its edge. Fig. 49, the most anterior section of the three, passes through the great circle of the spherical lens and shows it entirely outside the optic cup. On passing back in the series to where the lens is less in size, we reach the anterior edge of the optic cup and choroid coat, Fig. 50. Continuing back in the series of sections, the lens disappears and the optic cup alone is shown in F ig. 51. The lens in this eye is clearly too large fot the accompanying cup as was also the case with the two eyes just described. The size of these lenses is, therefore, independent of the size of the optic cup. Lewis’ (’04) idea that the cup regulates the size of the lens does not apply to these embryos, nor does the tule for the amphibian that the origin of the lens is dependent upon the influence of the cup. A step beyond this condition of a small anterior eye with its ill-fitting lens may be illustrated by an embryo in which the eye is a minute choroidal sphere buried in mesenchyme below the brain and in the median line. In life this specimen seemed entirely eyeless, but sections showed this small eye-like structure (Fig. 52) in the position typically taken by a cyclopean eye. Such cases as this emphasize the necessity of sections in order to cor- rectly interpret the conditions of cyclopia and conclusions based only on superficial studies are necessarily unreliable. The nasal pits were in the normal lateral position. Passing back in the sec- tions to the region usually occupied by the two eyes, it will be seen that on one side a typical lens occurs (Fig. 53). The lens is well differentiated and completely isolated from all connections with either nervous or eye tissue. A band of muscle is seen in the figure to touch the inner edge of the lens. Artificially Produced Cyclopean Fish 319 The occurrence of this lens recalls at once Herbst’s (or) argu- ment regarding the independent origin of the lens. He held that “af the lens really developed uclgpanteete of the optic cup, then in the case of median cyclopia the two lateral lenses should arise in their usual positions; but they do not, and furthermore, the cyclopean cup gets a lens from ectoderm out of the usual lens- forming region.”’ The Fundulus embryos show lenses arising at times in pias usual places and often in other places, independ- ently of the optic cup. We may suppose that in these embryos certain areas of the ectoderm are at times out of their normal posi- tions, and thus explain the promiscuous distribution of independ- ent lenses. Finally, embryos exist in which no indication of the optic cup can be found, these may be said to have passed beyond the ex- treme cyclopean condition. ‘They are not ordinary individuals that are merely blind, since the mouth is usually distorted and sometimes the snout-like structure which accompanies cyclopia is present. ‘his suggests the possibility that the “proboscis- mouth” is not entirely due to its normal position having been usurped by the cyclopean eye. Some of these embryos have free lenses and others no optic parts at all. Figs. 54 and 55 are two transverse sections from the same embryo, the anterior one shows a lens lying against the olfactory pit but free from all connection with the central nervous system. Fig. 55 shows a second lens lying close against the brain tissue. This embryo has no indication whatever of optic cups, and seemed eyeless in life. Other indi- viduals when carefully examined in section had neither an optic cup nor any lens-like structures. We have thus reviewed a series of forms beginning with the usual two-eyed embryos and passing through all degrees of double eyes to single cyclopean eyes, to extremely small cyclopean eyes, to individuals finally with only lenses present and no optic cups and others with neither lens nor cup. 320 Charles R. Stockard The extreme cyclopean condition Fig. 46 Cross-section of hatched embryo, small cyclopean eye located in anterior tip of head. The nose is anterior to this section. , pigment on ‘‘forehead” of embryo. Fig. 47 Section of thirteen day embryo. Small cyclops eye with large lens, differentiated retina and abnormal brain partly surrounding the eye; o/.p., nasal pit. Fig. 48 Section of thirteen day cyclops with eye far forward and out of median line beneath an abnormal mass of the brain. i Figs. 49, 50 and 51 Sections of a small anterior cyclopean eye with large lens projecting out of optic cup. The first section Fig. 49, is most anterior, the great-circle of the spherical lens, Fig. 50, tip of lens in the edge of optic cup, and Fig. 51, center of optic cup behind the lens. Figs. 52 and 53 Sections of thirty day embryo which seemed eyeless in life. Brain abnormal. Fig. 52, the cyclopean eye is represented by a choroid vesicle, E. The more posterior section, Fig. 53, shows a perfect lens L, in the usual lateral position, but no optic cup exists. A band of muscle m is between the lens and brain. Figs. 54 and 55 Sections of two lenses L, one forward by the olfactory pit, o/.p., the other more pos- terior and surrounded by brain tissue. No optic cup present in this nine day embryo. | é : { Artificially Produced Cyclopean Fish 321 Charles R. Stockard OW N N INCOMPLETE DIPROSOPUS WITH THREE EYES AND ONE ADDITIONAL LENS A most valuable object for study was an incomplete diprosopus monster which appeared in my solutions. ‘This individual had two heads separated as far as the lateral eye region. It appeared as indicated by Fig. 21 when seventy-two hours old. The two brains are separate, almost back to the auditory vesicles. “Two normal eyes are shown in outer lateral positions while between the heads one eye, perfect in shape, is mated with the outer eye of the left head and a circular body occupies the usual position of left eye on the right head. The embryo seemed normal in other respects and was in a vigorous condition. The monster when eighteen days old had developed to the usual size and was still hardy. At this time it presented a striking appearance as indicated imperfectly by Fig. 22. Three large eyes normal in form and capable of movement looked out from the double head. All visible evidence of the circular body shown near the middle eye when seventy-two hours old had disappeared. The middle eye was clearly paired with the left eye of the left head component and the right eye of the right head seemed mateless. A single pair of auditory vesicles were present. The young fish respired and twisted vigorously within the membrane. Three hours after this drawing was made, the embryo hatched and swam about in a circular fashion, the body not straightening perfectly. The free living animal was kept for five days and then preserved for sectioning. The sections show the presence of two brains, one spinal cord and one normal mouth leading into a pharynx with its series of gills, while a second short throat is present in the right head. ‘There are two notochords back to the middle of the yolk-sac and one from there on. The rear end of the medulla becomes single and only one pair of ear vesicles are present. ‘There are two olfactory pits anterior and median to the lateral eyes. Three perfectly normal eyes exist. They possess clearly dif- ferentiated retina, irides, humor chambers and lenses. “Two of these eyes are connected in the usual way with the brain of the Artificially Produced Cyclopean Fish 323 left head and one with the brain of the right. Fig. 56 is a section showing the middle eye somewhat back of its center so as to bring the edges of the other eyes into the figure. The middle eye is more anterior in position than the two lateral ones, owing to the slight obliquity of the left head. A distinct lens is showaa in the cup in Fig. 56. On going backwards in the series we reach a sec- tion passing through the middle of the two lateral eyes and the posterior end of the middle eye (Fig. 57). ‘The section shows dorsally the huge double brain and ventrally a central throat and most interesting of all a fourth lens. ‘This lens lies against the outside choroid coat of the middle eye and is in just the position (recognizing a displacement due to development of the middle eye) to be the lens of the left eye of the right head, if such an eye were present. We thus have in this double head three typical eyes and the fourth represented by a free lens. It was impossible to detect the clear lens in the living embryo which emphasizes again the necessity of sections for a definite interpretation of the conditions existing in these monsters. Conclusions drawn from observations on the living eggs without the comparison of sections may be incomplete. ‘The sections further make clear the nature of the circular outline shown against the middle eye of the seventy- two hour embryo (Figs. 21 and 57). Comparing the figures of sections and those of the whole embryos, it will be remembered that the sides of the sections are transposed, since the drawings of the total embryos are made from a simple microscope and the sections from a compound microscope which inverts the image. This incomplete diprosopus monster increases the series of eye monstrosities so that it passes through the cyclopean group to beyond the normal. The diagram (Fig. 58) illustrates in a simple way the various conditions we have considered and emphasizes the continuous nature of the series. Beginning at one end with eyeless individuals, we pass gradually through a series with small buried cyclopean eyes (which may be indicated in the diagram by a palpebral opening, such as similar mammalian cyclops would show), to the perfectly single cyclopean eye, to the double eye with one lens and pupil, to the hour-glass eye with two lenses and two pupils, to two independent but closely approximated eyes, next to Charles R. Stockard 324 FX yes of hatched incomplete dipro- Section through anterior median eye and edges of lateral e Bro, the two brains Br Fig. 57 additional fourth lens L. S5 Fig. 56 sopu posterior part of middle eye, and an More posterior section through middle of lateral eyes Guide figure ¥ makes both sections clear. Artificially Produced Cyclopean Fish 325 the normal condition and finally beyond to the incomplete dipro- sopus with three eyes and a fourth lens.’ The idea of arranging monsters in such a series including the normal is due to Prof. H. H. Wilder. Vv ce magnesium embryos” from entire Fig. 58 Diagram of the various conditions shown by the absence of the cyclopean eye J, to deeply buried eye IJ, perfect single cyclopean eye III, double-eye, IV, two approximated eyes V, eyes unusually close together VJ, normal VI/, three eyes and fourth, lens VIII. The normal is a mean from which different degrees of abnormal- ities are but greater or less deviations. It is possible to arrange almost any type of abnormality in such a series. Supernumerary arms or legs on one side might exist in various individuals in dif- ferent numbers down to the single normal one; other specimens could be found showing degenerate or small arms and finally armless or legless individuals are known. MORPHOLOGY OF MONSTRA MONOPHTHALMICA ASYMMETRICA A brief description of the asymmetrical monophthalmica mon- sters in life has been given above, but their true nature and structural conditions are impossible to detect without sections. It is found that here again a continuous series exists, beginning with the ordinary two-eyed individual through all gradations to the complete disappearance of one eye. The section through the middle of the eyes in a normal embryo of thirteen days old is illustrated in Fig. 59. The eyes, of course, are equal in size and alike differentiated structurally. In the salt solutions, however, many embryos occur with one eye perceptibly 326 Charles R. Stockard smaller than its mate. A section through the eyes of an embryo of this kind when seventy-six hours old is shown in Fig. 60. The left eye is decidedly smaller than the right and possesses a cor- respondingly small lens. From the comparative study of a num- ber of individuals it may be safely stated that this difference in size between the two eyes will not be overcome later, nor on the other hand will the small eye degenerate or disappear. The em- bryo will hatch with its eyes in dissimilar conditions comparable to the state of things shown by this seventy-six hour stage. The brain is normal and two nasal plates are present. An embryo closely similar to the one just described was sec- tioned after hatching. Its large eye appears as in Fig. 61. More anterior sections show a small eye looking forward with a some- what protruding lens in its cup. Behind this small eye is another lens lying free in the ectoderm (shown in Fig. 61). This lens is perfectly differentiated and appears to have arisen independently. A further reduction of the eye is shown by Fig. 62. In this thirteen day embryo the left eye is perfect and the right is rep- resented by a small cellular mass lying close against the brain. The lens of the right side is entirely wanting. In life the head was slightly one-sided, obviously on account of the asymmetrical eye development; no indication of the cellular mass could be detected and the embryo seemed truly one-eyed. A section of another seventy-six hour individual which in life also seemed to be one-eyed is illustrated by Fig. 63. The brain is normal and almost bilaterally symmetrical, an ordinary left eye exists but there is not the trace of an indication of the right optic cup. An ectodermal thickening represents the right lens in process of formation in the position that it would typically occupy. This lens anlage must have arisen independently of a stimulus from an optic cup and is well removed from the brain, so that no direct stimulus from that source can be responsible for its appearance. Other one-eyed individuals showed complete absence of all parts of the second eye, the lens as well as the optic cup failing to arise. The occurrence in the Mg solutions of these one-eyed embryos as well as the cyclopean embryos suggests that the chem- Artificially Produced Cyclopean Fish 227 63 ‘61 Monstra monophthalmica asymmetrica Fig. 59 Section through eyes of normal thirteen day embryo. Fig. 60 Section of seventy-six hour embryo with one normal and one small eye and perfect brain. Fig. 61 Section of the normal eye of a hatched embryo; a small eye with a lens is situated more ante- riorly on the other side and behind this is a third lens, L, shown in the figure on the left side. Fig. 62 Section of normal eye in thirteen day embryo, the other eye is represented by the cellular mass, e, close against the brain. Fig. 63 Section of normal eye in seventy-six hour embryo, the brain is bilateral and perfect, but no indication exists of the right optic cup although the ectoderm of that side has formed a lens thicken- ing L. 328 Charles R. Stockard ical influence exerts a peculiar inhibition of that process of out- pushing or separation by which the optic vesicles arise. Such an idea will be more fully considered in the general discussion given below. The unequal eyes may possibly result from an unequal allotment of eye material to one side or the other. A major portion might go to the right side and a minor part to the left, or the entire eye an- lagen might by chance occur on one side. ‘This in a sense would be lateral cyclopia. Such reasoning is of course purely hypothet- ical. INDEPENDENT ORIGIN AND SELF-DIFFERENTIATION OF THE CRYSTALLINE LENS Spemann (’o1), Lewis (’04), and others have concluded from experiments on amphibian embryos that there is no localization of lens-forming material in any given area of the ectoderm. They further held that the formation of a lens is dependent upon a stim- ulation of the ectoderm through contact with the optic-vesicle or cup. Spemann (’05) in discussing the question of the self-dif- ferentiating power of the lens concluded from a consideration of Schaper’s (’04) experiments on the frog that the lens is not capable of self-differentiation, but that a continued influence or contact of the optic-cup is necessary to cause the lens-plate or lens-bud to develop into a typical lens. LeCron (’07) has recently shown that the lens in Amblystoma is not self-differentiating. I (’07d) found in embryos of the blind Myxinoid, Bdellostoma stout, that a lens-thickening formed in early stages while the optic-vesicles were near the ectoderm. During development the optic cup becomes distantly removed from the ectoderm and the lens-plate disappears as if it were unable to continue development independ- ently of the optic cup contact. On the other hand Mencl (’03) has claimed that the lens in Salmo salar is at times formed independently of the optic cup influence and Spemann (’07) has recently modified his attitude. Spemann finds that in a certain species of frog, Rana esculenta, the lens may arise independently of the optic cup. This lens also Artificially Produced Cyclopean Fish 329 continues to develop and differentiates typical fibers. Most con- clusive evidence favoring the independent origin and self-differ- entiation of the lens is furnished by the Fundulus embryos now under consideration. Attention has been called repeatedly to the occurrence of lenses having no connection with other optical parts. It may be well at this time to summarize these cases which clearly show that in Fundulus the lens may arise independently and continue its devel- opment and differentiation. Fig. 63 illustrates the budding off of the lens from ectoderm on the side of the head which lacks entirely an optic cup. Fig. 61 shows a lens fully differentiated though lying freely in the mesen- chyme of the head. It will be recalled that this is a supernumerary lens; the large and small eyes of the embryo both possess lenses. An optic cup can not be responsible for this third lens. Fig. 57 of the incomplete diprosopus shows the fourth lens of the double head entirely outside the optic cup of the third eye which possesses alens. Figs. 54 and 55 show two lenses in an embryo that pos- sessed no trace of an optic cup. Fig. 53 indicates a lens in its usual position but no optic cup is present. In Fig. 45 a tiny lens is found in front of a cyclopean eye which possesses its own lens. Many other similar illustrations could be given. No one could hold that this indiscriminate collection of lenses, all of which are entirely isolated from any connection with optic cups or other eye parts, as well as in nearly all cases from the brain itself has arisen through direct stimuli derived from the optic cups. It is also evident that the lens after its formation continues to self- differentiate. It seems to me that in Fundulus the case is clearly proven that lens formation does not depend upon a direct stimulus from the optic cup. Such a dependence as advanced by Lewis (’04) for the frog is not, therefore, of universal application, nor is the view tenable that the differentiation of the lens depends upon a con- tinued stimulus from the optic cup. 30 Charles R. Stockard oS) DISCUSSION AND CONCLUSIONS The foregoing facts furnish important information as to the cause and manner of development of cyclopia, and the facts bear directly on previous ideas concerning this subject. By treating the fish eggs with magnesium solutions, it is conclu- sively shown that the experimenter has the power without mechan- ically injuring the egg or embryo to cause what would have been a two-eyed individual to become a cyclopean monster. ‘This undoubtedly is a case of the occurrence of cyclopia through the action of external influences on the developing egg. I conclude, then, that cyclopia does not necessarily result from germinal varia- tions, but I make no claim that it may never arise in such a way. On the contrary, there is no reason why cyclopia should not occur through germinal variations as readily as does any other new fea- ture. The fact that mammalian cyclopean monsters do not sur- vive, or even if it be proven that the free-swimming cyclopean fish are incapable of living or reproducing, does not argue against the possibility that cyclopia may in cases be due to germinal variation. Such a statement is emphasized by a case I (’07c) recently recorded. In a flock of sheep in North Carolina two entirely legless lambs appeared in the spring of 1907. Again in 1908 two other similar lambs have occurred, one being the offspring of a mother which had previously borne a legless individual. These lambs were unable to feed without assistance and in nature would doubtless have died shortly after birth, but their peculiar occurrence in this flock is very probably due to germinal variations, either within the mother or father, or both. Students of inheritance consider sports to be due to germinal variations and the ability of such sports to survive depends merely on their adaptations to the sur- roundings and not in the least on their manner of origin. No reason can be given why a cyclopean individual might not occur as a sport due to sudden germinal variations. From the experi- ments contained in the present paper, however, it may be emphat- ically affrmed that cyclopia 1s not always due to germinal origin. Spemann (04) through an ingenious Be hed of experiment, produced double-headed Triton embryos which exhibited various ~~ Artificially Produced Cyclopean Fish 331 degrees of cyclopia. ‘The eggs of this salamander when constricted about the periphery of the first plane of cleavage with a fiber- like ligature gave monsters with two equal heads. When the lig- ature was oblique with reference to this plane one of the heads was cyclopean to a greater or less degree. Spemann thought the defective head due to the loss of the anlagen of certain parts, con- sequently these parts never began development and organs sit- uated lateral to them developed in contact from the start. In other words parts between the eye anlagen fail to form and thus the anlagen come in contact and so develop from the beginning. ‘This explanation is of course entirely speculative, but it is supported in a manner by experiments which according to Mall (08) Lewis has performed on the fish embryo. Mall states that Lewis found by pricking the extreme anterior end of the embryonic shield in Fundulus eggs that many of the eggs develop into cyclops embryos. It was found in some that the prick had destroyed the “nose” only. ‘This experiment shows conclusively that it is the absence of tissues between the eye arlagen that allows them to come together and unite.” The above explanation no doubt holds for some cases of cyclo- pia produced by cutting or pricking; there it is evident that tissue is destroyed and the destruction of median tissue may cause the regions containing the eye anlagen to unite. It is difficult to apply this explanation to all cases. In the ‘Magnesium embryos,” why should tissue between the eyes fail to form and not other tissues; why are the nasal pits united in some cyclops and separate in others? A close microscopic examination of the brain floors in cyclopean and two-eyed embryos shows no absence of recog- nizable parts in the former. The monstra monophthalmica asym- metrica are also to be explained; here one eye in some cases fails to come off from the brain. Is this due to the absence of its early anlage? ‘The very small cyclopean eye sometimes buried deeply in the head, and the eye shown in Fig. 38 which is partly inclosed within the brain, as well as the entire absence of an eye, suggest another explanation that may apply to all cases in the magnesium solutions. The small eyes close together, cyclopia in various degrees, the 332 Charles R. Stockard imperfect formation or absence of one eye and entire absence of eyes are all conditions common to the magnesium solutions and very rare or never occurring in other solutions, nor in the hun- dreds of eggs observed developing in sea-water. The conditions are, therefore, probably due to a common cause, and I suggest hypothetically that this cause is an inhibitory or anesthetic effect of the magnesium on the process of outpushing and separation of the optic vesicles. Magnesium exerts a decidedly anzsthetic effect upon both vertebrate and invertebrate animals and is an inhibitor of muscular activity. It might possibly inhibit the giv- ing off of the optic vesicles or prevent their separation in the brain, so that both might come off together as in cyclopia, and it might have caused the eye in Fig. 38 to be arrested when only halfway separated from the brain; the absence of one eye and complete absence of eyes would be perfect inhibition. It is necessary to find a definite point in the strength of the solutions in order to obtain the proper amount of inhibition for many weaker eggs are killed during early stages. The strongest argument against such an hypothesis 1s the fact that Mg in distilled water solutions fails to cause cyclopia, whereas its anesthetic or inhibiting powers should be most active in such a solution. Dareste’s (’91) idea that cyclopia is caused by a closed brain or the failure of the anterior vesicle to develop is unsupported, since in Triton with the hollow-brain tube present Spemann finds that the defect occurs. In Fundulus the optic outpush- ings are normally given off while the brain is yet solid, so that according to Dareste all of these fish would be cyclopean in nature. Schwalbe (’o6) in his Morphologie der Missbildungen des Menchen und der Tiere, considers cyclopia to result from unusual pressure exerted during early stages of development which does not cause the lateral parts to grow together but prevents them from developing at all. This position is somewhat in accord with the hypothesis suggested above. If pressure prevents the grow- ing apart laterally of the anlagen which normally require energy to accomplish their separation, then by anesthetizing a part, one accomplishes practically the same thing as by applying pressure. Bats Artificially Produced Cyclopean Fish EKK: =) The part in anesthesia lacks energy to grow out laterally, thus the two eye anlagen remain together in the floor of the brain and come off as one median vesicle either double or single, depending upon the extent of separation possible under the given degree of pres- sure or anesthesia. Mall (08), in his recent memoir on the causes underlying the origin of human monsters, gives an excellent survey and discussion of the evidence furnished by experimental teratology. In the body of the paper is presented a strong case in favor of external influence during development as the chief cause of many mon- strosities. Here we may consider only the discussion of cyclopia. The idea of fusion of the two eye vesicles during their develop- ment is advocated, but the present evidence is against this posi- tion and is in accord with Spemann’s (’04) view of an early defec- tive anlage. Mall also inclines toward the idea of the single brain as being primarily responsible for cyclopia, but it is shown by embryos considered here that cyclopia often accompanies perfectly bilateral and bilobed brains, neither does a retarded growth of the frontal process necessarily follow in cases of cyclopia. Experiments uphold the statement “that every egg has in it the power to develop cyclops monsters.” ‘The germinal theories of cyclopia are shown by the experiments to be unnecessary as ex- planations of its cause. ‘The possibility of its occurrence through germinal variations, though to my mind extremely slight, is not entirely excluded by experiments. ‘The experiments conclusively show the origin of cyclopia through external influences. Much could be said pro and con regarding the significant nature of the cyclopean fish embryos as a specific response to a definite chemical environment. ‘The suggestion is evident, though highly hypothetical, that cyclopia in man and mammals might be due to a similar chemical cause, an excess of Mg salts in either the mother’s blood or the amniotic fluid surrounding the developing embryo. The Magnesium embryo is as typical of these Mg solutions as is the now classic lithium larva of the sea urchin produced by Herbst (92, 93) in his Li solutions, or Morgan’s (’04) lithium frog em- bryos produced in a similar way. They all tend to show that dif- 334 Charles R. Stockard ferent chemical conditions may each induce by their actions a specific type of larva from a given variety of egg. SUMMARY 1 The eggs of the fish, Fundulus heteroclitus, give rise to a large percentage of cyclopean embryos when subjected during their development to solutions of magnesium salts in sea-water. Similar results follow if the eggs are placed in the solutions either before cleavage or when in the two or early four-cell stages, later stages were not tried. ‘his is the first instance of repeatedly causing, by the use of chemical substances, vertebrate monstros- ities such as are known in nature. 2 The peculiar embryos with the median cyclopean eye are able to hatch. Many of them swim about in a perfectly normal manner, darting back and forth to avoid objects placed in their field of vision as readily as do two-eyed individuals. 3. The cyclopean fish is exactly comparable to the monstrous cyclops of man and other mammals. Both have a median eye either double or single in its structure. The nose in the mam- malian cyclops is a single proboscis-like mass above the eye. The nasal pits in the “ Magnesium embryos” are sometimes united and sometimes separate, but the mouth hangs ventrally as a pro- boscis-like organ strikingly suggesting in form the nose in mam- malian cyclopia. The mouth of Fundulus normally occupies an extremely anterior position but in the cyclopean fish the eye has usurped this place, thus preventing the usual forward growth of the mouth elements and forcing them to remain ventrally as the proboscis-like mass. (See Figs. 25,26, 27.) In cyclopean mammals a similar mechanical explanation accounts for the condition of the nose. The median eye obstructs the path of down-growth which passes normally between the eyes, and forces the nose to form above the eye as a proboscis on the fore- head. 4 A study of more than 275 living cyclops monsters and of many of these in section shows all degrees in the defect. Eyes unusually close together, intimately approximated eyes, the double Artificially Produced Cyclopean Fish 335 eye in a median position, the single cyclopean eye, an extremely small anterior eye, a deeply buried ill-formed cyclopean eye, and finally an entire absence of the eye. The embryos exhibit these various degrees of the cyclopean defect from the earliest appear- ance of the optic outpushings, and in no case was cyclopia due to a union or fusion of the two eye components after they had originated distinctly. 5 Asecond type of monster designated as Monstrum monoph- thalmicum asymmetricum, the monster with one asymmetrical eye, was also common in the magnesium solutions. ‘These indi- viduals have one perfect eye of the normal pair but the other is either small, poorly represented or entirely absent. ‘This condi- tion is also present from the first appearance of eye structures and is not due to degeneration or arrest of development. 6 Both types of monsters often form lenses independently of the optic cup stimulus. ‘These self-originating lenses are also capable of perfect self-differentiation, forming lens fibers and appearing as transparent crystalline bodies. Snel facts oppose the idea that the lens during its origin and development is in a dependent relationship with the optic cup, and show this view not to be of universal application. 7 ‘The experiments conclusively prove that eggs may be in- duced to develop into cyclopean monsters by external influences. These influences do not mechanically injure or destroy certain eye regions as does cutting or pricking. It follows, therefore, that cyclopean monsters appearing in nature are not necessarily due to germinal variations, but are far more likely the result of some unusual external influence during development. 8 ‘The occurrence of the various eye monstrosities shown by embryos which develop in magnesium solutions are all probably due to a common cause and I suggest the following hypothetically : Magnesium which possesses a decidedly anzsthetic effect on most animals and is inhibitory in its influences on muscular activity may retard through degrees of anesthesia the optic outpushings in Fundulus embryos and thus account for the total absence of eyes, small eyes, eyes which failed to develop energy necessary for their normal separation and the other unusual conditions which 336 Charles R. Stockard have been considered in detail in the present article. This view, of course, is hypothetical and objections to it are recognized. Cornell University Medical College New York City, October 1, 1908 LITERATURE, CITED DareEstTE, C. ’91—Recherches sur la production artificielle des monstruosités ou essais de tératogénie expérimentale. pp. 366-383, Paris. Dourn, A. ’75—Der Ursprung der Wirbelthiere und das Princip des Functions- wechsels. Leipzig, pp. 1-87. Hersst, C. ’92—Experimentelle Untersuchungen tber den Einfluss der verander- ten chemischen Zusammensetzung des umgebenden Mediums - auf die Entwickelung der Thiere. I. Theil. Zeitsch. f. wis- sensch. Zool. iv, pp. 446-518. ; °93—Experimentelle Untersuchungen. II. Theil. Mittheil. aus der Zool. Station zu Neapel, xi, pp. 136-220. *o1—Formative Reize in der thierischen Ontogenese. Ein Beitrag zum Verstandniss der thierischen Embryonalentwicklung. Leipzig. LeCron, W. L. ’07—Experiments on the Origin and Differentiation of the Lens in Amblystoma. Am. Journ. Anat., vi, pp. 245-258. Lewis, W. H. ’04—Experimental Studies on the Development of the Eye in Am- phibia. I. On the Origin of the Lens. Rana palustris. Am. Journ. Anat., 1, pp. 505-536. Matt, F. P. ’08—A Study of the Causes Underlying the Origin of Human Monsters. Journ. Morph., xix, pp. 1-361. Menc1, E. ’03—Ein Fall von beiderseitiger Augenlinsenausbildung wahrend der Abwesenheit von Augenblasen. Arch. f. Entw’mech., xvi, pp. 328-339. Morean, T. H. ’03—The Relation Between Normal and Abnormal Development of the Embryo of the Frog, as Determined by the Effects of Lithium Chlorid in Solution. Arch. f. Entw’mech., xvi, pp. 691-712. *06— Experiments with Frog’s Eggs. Biol. Bull., xi, pp. 71-92. ScHaPeEr, A. ’04—Ueber einige Falle atypischer Lensenentwickelung unter abnor- men Bedingungen. Anat. Anz., xxiv, pp. 305-326. SCHWALBE, E. ’06—Die morphologie der Missbildungen des Menchen und der Tiere. I. Teil. Allgemeine Missbildungslehre. Jena, pp. 1-230. _-. —- oe ——a - . A a ee a age rie Bes Saws | see eee ——--snissenceaeliedaniAdenmen ea OT Artificially Produced Cyclopean Fish 237 SpemMann, H. ’o1—Ueber Correlationen in der Entwickelung des Auges. Ver- handl. der Anat. Gesellsch., 1gor. °o4—Ueber experimentell erzeugte Bappelbilduneen 3 mit cyclopischem Defekt. Zool. Jahrb. Suppl. vii, pp. 429-470. 7o5—Ueber Linsenbildung nach experimenteller Entfernung der pri- maren Linsenbildungzellen. Ausfiihrlich: Zool. Ang., xxviii. 7o7—Neue Tatsachen zum Linsenproblem. Zool. Anz., xxxi, pp. 379- 386. StocKarD, C. R. ’06—The Development of Fundulus heteroclitus in Solutions of Lithium Chlorid, with Appendix on its Development in Fresh Water. Journ. Exp. Zodl., ili, pp. 99-120. ’o7a—The Artificial Production of a Single Median Cyclopean eye in the Fish Embryo by Means of Sea-water Solutions of Magnesium Chlorid. Arch. f. Entw’mech, xxiii, pp. 249-258. o7b—The Influence of External Factors, Chemical and Physical, on the Development of Fundulus heteroclitus. Journ. Exp. Zodl., iv, pp. 165-201. o7c—A Peculiar Legless Sheep. Biol. Bull., xii, pp. 288-290. °o7d—The Embryonic History of the Lens in Bdellostema Stouti in Relation to Recent Experiments. Am. Journ. Anat., vi, pp. 511-515. *o8—The Question of Cyclopia. Science, n.s., xxviii, pp. 455-456. Wiper, H. H. ’o8—The Morphology of Cosmobia. Am. Journ. Anat., viii, PE 350,54°: EXPLANATION OF PLATE I. Fig. A Dorsal view of a cyclopean embryo in almost natural colors. The large antero-ventral eye shows a slight furrow indicating its double nature. Fig. B The same embryo when the egg is rolled back towards the top of the page. A somewhat ventral view showing the single pupil and lens, the double condition of the eye is only indicated from above. Sle moar ae ARTIFICIALLY PRODUCED CYCLOPEAN FISH Cuarces R. SrocKArpD THE JouRNAL or EXPERIMENTAL ZOOLOGY, VOL. VI, NO. 2 PLATE I RicuHeEoul, del In Figure 32, Plate II, of Miss Stevens’s paper on “ Further 33 Studies on the Chromosomes of the Coleoptera” (vol. vi, no. 1), one chromosome has been omitted; the figure should appear thus: Hy §2... 2 ‘4 SPUDIES ON THE PHYSIOLOGY OF REPRODUCTION IN THE DOMESTIC FOWL I. REGULATION IN THE MORPHOGENETIC ACTIVITY OF THE OVIDUCT’ BY RAYMOND PEARL INTRODUCTION This paper forms the first in a series in course of preparation in this laboratory, all dealing with various phases of one broad, general problem. It is desirable that at the beginning of such a series a statement should be made outlining the problem under investigation and, in a general way, the standpoint from which it is to be attacked. It is the purpose of this introduction to give such a statement. When the work of this laboratory was organized one general line of investigation which suggested itself as of first-class impor-: tance, both from the theoretical and practical standpoint, was the study of egg production in the domestic fowl. A high average yield of large eggs uniform in size and color is a matter of enor- mous importanceto the poultry industry. How can it be obtained ? Can high egg producing capacity be bred into a strain? Can feeding produce it? These are the questions which practical poultrymen in the experiment stations, agricultural colleges, and elsewhere are trying to answer. The zeal for inquiry in these directions is greatly stimulated by the obvious fact that at some time or other during the history of poultry under domestication there has been a very great increase in egg production over what obtains in the wild representatives of the genus Gallus. If the thing can be done once, why not again? 1 Papers from the Biological Laboratory of the Maine Experiment Station, No. 7. Tue JourRNAL or EXPERIMENTAL ZOOLOGY, VOL. VI, NO. 3. 340 Raymond Pearl It takes but brief consideration of these economic points to con- vince one that behind them lies a very broad and complex biolog- ical problem, on which light must be obtained before there can be any hope of solving the practical questions. ‘This is the problem of the physiology of reproduction in the hen. Egg production is a definite, if complex, physiological process. In the production and laying of an egg a long series of events are involved; a num- ber of different organs of the body play a part. Before we can hope to control egg production with any precision or certainty it is necessary to learn in detail what is the normal course of events in the production of an egg; how and in what ways each of these events may be modified or influenced by external circumstances; and to what extent each of them is an inherited matter. The physiology of the organs concerned in egg production must be worked out in detail. In order that a comprehensive idea may be gained of the scope of this problem, let us examine the following skeleton outline of the factors and processes immediately concerned in egg production and the points which must be investigated in attempting to get light on these processes. I Physiology of egg production within the individual. A Processes occurring in or relating to the ovary. 1 The development of the egg and its yolk up to the time of ovulation. Resorption of yolk. 2 Ovulation proper. The rupture of the follicle. 3 Fecundity. B_ Processes occurring in the oviduct. 1 Movement of egg to the outside. 2 Formation of albumen. 3 Formation of the several egg membranes. 4 Formation and determination of the shape and color of the shell of the egg. C Intra-individual variation and correlations in regard to the points enum- erated under A and B. Homotyposis. D Behavior in its relation to egg production and reproduction in general. 1 Mating instincts and habits. 2 Brooding instincts and habits. Physiology of Reproduction in Domestic Fowl 341 Il Phystology of egg production within the race. A Variation in egg production. 1 Intra-racial | in regard to each of the points enumerated under I 2 Inter-racial } above. 3 Mutation. 4 Seasonal distribution of egg production. B_ Inheritance of egg-producing ability. Considered with reference to each of the points enumerated under I above. 1 In pure-bred lines. 2 Under hybridization. C_ Evolution of egg-producing ability. 1 Influence of selection. 2 Egg production in the wild progenitors of domestic poultry. 3. Fixation of egg producing ability as a racial character. Ill The influence of environmental factors (in the broadest sense) on the processes enumerated above. Nutrition. Housing. Meteorological factors. -wW Nn & Drugs. 5 Other environmental agents. IV) The relation of internal factors to, and their influence upon the processes enum- ’ erated under I and II. V Pathological and teratological cases relating to egg production. This outline, while not as extensive or complete as it might be made, gives a fairly comprehensive view of the general scope of the problem which forms the subject of the present investigation. Each topic in the list suggests, of course, a wholeseries of problems, but even to enumerate all these would take far more space than is available here. All that is desired at present is that the broad outlines of the general problem on which we are working shall be clear to the reader. On account of the extent of the subject it is necessary to publish the results of the work upon it in a series of separate papers. ‘The skeleton outline given above will serve as a means of coordinating the separate papers, and making clear the * The general standpoint which regards variation, heredity and other factors of evolution as physio- logical problems has been well set forth by Jensen (’07) and Jennings (’o8). 342 Raymond Pearl relation of each to the general problem. Statements and discus- sions of the subsidiary problems connected with each of the topics in the outline will be given in connection with the detailed treat- ment of those topics. In attacking this problem we are bound to no exclusive method of investigation. The observational, experimental and statistical methods will be used as they appear to be demanded by the exi- gencies of the case. The writer’s standpoint is that the problem is one in general physiology, involving questions having to do both with individual and with racial physiology. On a certain number of the points covered in the outline work has been com- pleted and will be published as soon as possible. On _ other phases of the problem the work is well advanced though not ready for publication. The present paper deals with a definite and circumscribed topic falling under [B4 and IC of the outline. It is well known by poultrymen that the first eggs laid by a pullet often differ from the normal eggs and from eggs laid later by the same bird in regard to both size and shape. This implies a process of regulation in the continued activity of the oviduct in shaping successively laid eggs. The present paper deals with the detailed analysis of a clear-cut and unusually pronounced case of such regulatory ac- tivity of the oviduct. THE MORPHOGENETIC ACTIVITY OF THE OVIDUCT A bird’s egg is an object of very characteristic shape. While the form of the egg varies in different species, and also within the single species, all such variation is comprised between relatively narrow limits. The conformation to type in the case of eggs from birds of a single species is in most cases quite close. It is usually still closer in a series of eggs laid by the same bird, as in the case of any of the domesticated fowls. The production of a series of definitely and characteristically formed bodies all conforming closely to a type by an organism implies that the organs concerned in this production have a morphogenetic function along with others. It is a well established fact that the shape of the egg of any Phystology of Reproduction in Domestic Fowl 343 bird is determined in the oviduct. The yolk as it leaves the ovary is spherical in form, except as it may be deformed through the action of gravity. As it passes down the oviduct it is sur- rounded by albumen, and finally at the lower end by the so-called “shell”? membrane, or membrana testacea. It is probable that the egg is not given anything approaching its characteristic form until after the formation of this membrane. As to exactly where and how the egg is given its form by the oviduct there is some difference of opinion and definite evidence is lacking. Szielasko (05), who has paid particular attention to this problem, after reviewing the older literature of the subject expresses the opin- ion that the egg is given its definite form in the uterus. He says on this point (p. 289): ‘“‘Das Geprage, welches die Eiform der verschiedenen Species aufweist, kann in der Tat, wie Grassner vermutet, nur von dem Uterus verliehen sein; denn solange das Ei im Oviduct verweilt, ist seine Form variabel, da es jeder Um- hullung entbehrt. Die erste Hille, membrana testacea ge- nannt, wird dem Ei erst im untersten Abschnitt des Oviductes un- mittelbar vor der Miindung desselben in den Uterus—im sogen- annten Isthmus—-umgelegt. Auch durch diese Membran wird dem Ei noch keine bestimmte Gestalt gegeben. Diese resultiert erst aus der Umlagerung der harten Kalkschale, welche im Uterus geschieht. Hier ist also das formgebende Organ, hier muss demnach die Untersuchung angreifen.”’ This was also the early view of the matter. Wahlgren writing in 1871 states as a matter of common opinion that: “Hier (i.e., in the uterus) erhalt das Ei seine Schale und seine Form.”’ The most recent worker in the subject, Thompson (08), while making the statement (p. 112), “The egg, just prior to the form- ation of the shell, is, as we have seen, a fluid body, tending to a spherical shape and enclosed with a membrane,” which would cer- tainly seem to imply that he supposed the definite shape to be given to the egg in the uterus, proceeds to develop a theory of the method by which the egg gets its form which seems, as he states it, to in- volve the activity of nearly the whole oviduct in the process. Direct observation shows that after the membrana III 15 DAE Nepales: Ill 17 Plosanneds IiI 19 DBeyetetasinioie Til 21 ANGeao0Gar S522, DF=cangode Til 24 UV GeSdouec DT 25 OM sonoade Ill 27 VhEcoawee Ill 28 asnpoose Ti “30 Rinisiars sin.a0 Ill 31 eeonocee IV 3 Teo apebee DV cst S: Uipasenecee Vie: 6 { Desabooee IV 8 2 Sgoeoee EVES IO 2 cage INV a at) ;hesoones Vers JP@snaodane IV= 15 OS ogeeoor IV 16 De sie cst IV 18 Betrassiciae EV S19 DRC Satan 5 te IV “or mm. 69.3 66.7 63-7 64.5 61.6 65.7 61.1 59-1 63.8 63-9 59-1 60.3 58.6 60.6 mum mm. 33.8 B5ar 35-6 36.0 58) 35-5 36.6 36.4 Spe2 35-9 35-5 36.6 37-3 37-6 egg lost 36.2 37-6 | Maxi- | Length | | | breadth | | | | TABLE 1 Index Data Regarding the Eggs of Ordinal number of egg eee eee! see weee see e wees seen ceee se eeeoes eee eeene eee eeeee eee oees 1908 on N ANN WY BV & [Se ee ee | re OW NN > BW v Ww Hen No, 183 * | wow a oa | is} ao mm, mun Nn 58. Date laid) Length | Maxi- mum breadth mm. Www Ww NNN W Ww nN CO) oS DOAN N AN ON DAN ConA YWYP HP NO DO VRP HW AN SP FW PH DHO NAN VAI DAP AP HH COMO OhWOW PUY O WW WWW WW WH WW WH WW WW WH WwW = be Ti He | 37° oo | 349 350 Raymond Pearl 3 The progressive change in the dimensions of these eggs is in each case gradual, but not absolutely steady. Instead there are fluctuations up and down in each of the characters studied. These fluctuations appear from mere inspection of Table I to be distributed in a random manner. It will be shown in the next section of the paper that this is in fact the case. 4 No egg after the first shows the pyriform shape due to a concavity of the lateral contour of the egg. The eggs laid after the first are simply elongate ovate in form. 5 After the first 12 eggs had been laid the form of the egg was (barring random fluctuations) very close to the normal. The further progressive change towards the normal was exceedingly gradual. With regard to the absolute size of these eggs it may be said that they were all noticeably smaller than the average for the breed. The weight of the first egg laid was 37.9 grams. ‘This was the lightest egg ever laid by hen No. 183 so far as is known. ‘The weights of the first 12 eggs laid are shown in Table II. TABLE II Weight of the Eggs of Hen No. 183 Ordinal number of egg Weight in grams Ordinal number of egg Weight in grams I 37-9 7 47-2 2 45.8 8 44-4 3 46.8 9 44.8 4 47-4 10 46.3 5 45-3 II 42.7 6 46.9 12 47-1 From this table it will be seen that the second and succeeding eggs were distinctly heavier than the first egg laid. After the sec- ond egg was laid there was no definite change in the weight of the eggs. All the changes that occurred in weight after that time were the chance up and down fluctuations about an average point. There was no definite tendency for the eggs to become heavier or lighter as more were laid. Consequently after some 50 had Physiology of Reproduction in Domestic Fowl 351 been laid the weights of the eggs were no longer taken. ‘There seems to be no particular reason for reproducing here more than the weights of the first dozen eggs shown in Table II. In general it will be seen that all of these eggs were below the average for the breed in size. WHAT IS THE CHARACTER OF THE PROGRESSIVE CHANGE TOWARDS THE NORMAL IN THE SHAPE OF THESE EGGS? In order to answer this question recourse must be had to analyt- ical treatment of the data set forth in Table I. Such analysis may best be begun by exhibiting graphically the changes in the different dimensions of the successively laid eggs. The length and breadth may be considered first. In plotting these dimensions only the first 25 eggs are taken. ‘The reasons for stopping these diagrams at this point are the following: (1) To get the whole 87 eggs into a single text figure involves such a reduced scale as practically to destroy the effectiveness of the diagram for analytical discussion. (2) The dimensions of the eggs after the 25th fluctuate up and down about what is practically a straight line. This being the case it is not necessary in each of the diagrams to carry the line out to the end of the data of ‘Table I. The lengths of the first 25 eggs are shown graphically in Fig. 1. In this diagram the abscisse denote the ordinal position of the eggs in the whole series laid. ‘The ordinates denote the length of the eggs in millimeters. From the diagram it appears that: 1 The length of the eggs decreases very rapidly in the first 5 laid. 2 The rate of decrease in length becomes progressively slower with successive eggs. 3 It results in consequence that the line of plotted lengths (disregarding chance fluctuations) is decidedly curved at its begin- ning, but approximates more and more to a straight line as it proceeds. The breadths of the first 25 eggs to be laid by hen No. 183 are 352 Raymond Pearl shown graphically in Fig.2. The plan of this diagram is the same as that of Fig. 1. From this diagram it is apparent that the increase in the breadth of the eggs which occurs with successive laying follows, on the whole, a straight line. The actual observations zigzag up and down, but the underlying steady tendency appears to be for the breadth of the eggs to increase at a slow, but uniform rate, or, in other words, in a straight line. ‘This is in marked contrast to what has been seen for the length. Turning now to the index (100 x breadth + length) which may be taken as measuring shape we have the discussion of a series of values which have been seen to follow a straight line divided by a dimension which follows a decided curve. It would be expected that the quotient (index) so obtained would exhibit a decided curve when plotted. ‘That this is in fact the case is shown in Plate II, where the zigzag line represents the observed indices of the successively laid eggs. Examining this diagram it is seen that to a more pronounced degree the statements made regarding the changé in the length of the eggs apply to the index, if “increase” is in each case substituted for “decrease.’’ The index increases in value very rapidly at first and the rate of increase becomes progressively slower with successive eggs. In the case of the index the whole 87 eggs are included in the plotted curve. Now since the length-breadth index is a better measure of the shape of the egg as a whole than either the length or breadth alone it is desirable to deal with this character in the further analytical study of this case. It was decided to graduate the index curve. Now the shape of the curve, rising sharply at the beginning, curv- ing smoothly and quickly and then running off nearly horizontal, only curving very gradually, suggests at once tothe eye that it is a logarithmic curve. Furthermore, previous experience with similar data suggested a logarithmic as the proper curve. Accord- ingly a curve of the type y=a+bx4+c log x was fitted to the observations, as a first trial. In obtaining the constants of this equation a table of values of S (log x) S (log x), “* ead 353 Physiology of Reproduction in Domestic Fowl fgt ‘on uey Aq prey s3da Sz ysry jo ysueT 33q jo Joquinyy [euIPIG "131 posers «| ‘WU Ul YyysUeT 354 Raymond Pearl and S' (x log x) calculated in this laboratory® was used with great saving of labor. ‘The resulting curve was y = 49.0241 — .ogio x + 11.7669 log x where y denotes the length-breadth index and x the ordinal num- ber of the egg in the whole series laid. Calculating the ordinates of this curve we have the set of values shown graphically by the smooth curve in Plate II. It is clear that this curve is a wonderfully good graduation of the observations. It is so good, in fact, that it is apparent that this logarithmic curve is the analytical expression of the manner in which the change in the shape of the eggs of No. 183 occurred. It is possible now summarily to state the facts regarding the shape of the eggs of hen No. 183 as follows: The first egg laid by this hen was abnormally long and narrow; the eggs subsequently laid ap- proached more and more to the normal in shape. This change in shape was tn accordance with a logarithmic curve of the type y=atbx +clogx wherein y denotes the length-breadth index of the egg, x its ordinal number in the series laid, and a, b and c are constants. It will be noted that the smooth curve shows a tendency for the index as observed to decrease after reaching a maximum along in the region of the 5oth to 60th eggs. The turning downward of the theoretical curve comes about from the fact that the term .ogIo x in the equation is negative. This decrease is not to be interpreted as due to any tendency for the eggs to change from the normal to- wards the abnormal after a number have been laid. On the con- trary there is every reason to believe that it is merely a chance result due to ending the observations at the particular point where they were ended. ‘The observation line fluctuates up and down as the result of chance factors. It happened by chance that towards the end of the series the “down” fluctuations predominated to an extent sufficient to change the sign of the line term (x term) in the equation and turn the fitted curve slightly downward. ‘There is * This table, which is very useful in fitting logarithmic curves to any sort of data by the method of least squares, will shortly be published. ane i ene: behest 9h mn Physiology of Reproduction in Domestic Fowl 355 C2 “tan CAS C5 ieee Ole) 16. U7 Sale (5 Ordinal Number of Egg Fig. 2 Breadth of first 25 eggs laid by hen No. 183 “WU UI WIpeag 356 Raymond Pearl no doubt that had the observations been extended to 100 eggs these ‘““down” fluctuations would have been offset by an “up” set, and the theoretical curve would have shown no downward tendency at its upper end. This is indeed directly indicated in the value of the 87th observation. This egg had the highest index of any ever laid by hen No. 183. If the observations had been stopped at 60 eggs the theoretical curve again would not have shown the slight downward tendency at the upper end. That it does show this is simply an accident resulting from ending the observations at a particular point. DISCUSSION OF RESULTS The facts which have been set forth above are of interest in connection with two questions, viz: (1) the physiology of the deter- mination of the shape of the egg and (2) the more general ques- tion of regulation in morphogenesis. With regard to the first of these questions there are two features of the present case which lend strong support to the view that the shape of the egg is determined by the active contractions of the muscular wall of the uterus. (Cf. p. 6 supra, and Szielasko (’05) p. 289.) These features are: (a) that the eggs laid by this hen were not all alike or even approximately alike. There were great differences in the shape of different eggs. (b) That the shape of the eggs changed in an orderly and progressive fashion (regulation) as they were successively laid. It is hardly conceivable that these two things both could have occurred with the uterus playing sim- ply a passive part and only influencing the shape of the egg through the elasticity of its walls. It might possibly be maintained that the uterus wall became more and more stretched peripherally as more eggs were laid, and that this would account for the thick- ening and rounding up of the egg in a purely passive way. But if this position is taken one Is at a loss to explain the sudden occur- rence of a relatively long narrow egg in the middle of a whole series of approximately normal ones. An example of such occur- rence is given by egg No. 36, and another by egg No. 62. The only reasonable conclusion on this point appears to be that Physiology of Reproduction in Domestic Fowl 357 the muscular activity of the walls of the uterus determines the shape of the egg. ‘The results further show that this morphoge- netic activity of the oviduct may be of a definitely regulatory char- acter. Turning to the consideration of the regulation shown in this case the chief point of interest lies in the precise manner in which the regulatory change follows a logarithmic curve. ‘Though the biological processes involved are quite different in the two cases the type of change is exactly the same in the successive produc- tion of leaf whorls and branches in the normal ontogenetic devel- opment and growth of Ceratophyllum (cf. p. 8 supra) and in the successive production of eggs by this hen No. 183 which has been seen to be a regulatory process in the strict sense. “The approach from a condition of wide deviation from a final type to that type is in both cases along a logarithmic curve. ‘This means that nor- mal ontogenetic development and growth on the one hand, and regulatory development on the other hand have at least one character or principle in common. ‘This principle may be set forth as follows. Whenever a developmental or growth proc- ess follows a logarithmic curve it means that the amount of change which occurs in any given time interval, say between time A and time B, is strictly proportional, either directly or inversely to the total amount of change which has occurred before time A, or, in other words, to the condition in which the organism finds it- self at time A. Furthermore, the rate of change is proportional to the time during which the process has continued. Thus to take a concrete illustration, the amount of growth occurring in a time period A to B in an organism exhibiting a logarithmic growth curve is proportional to the size which the organism has already attained attime A. In growth this relation is inverse: the larger the organ- ism (1.e., the more it has grown) at any given time, the smaller will be the growth change in the next subsequent unit time inter- val. The longer the process continues and the nearer it comes to its final goal, the slower 1s the rate of progression towards that goal. It is of much interest to find both normal ontogenetic and regu- latory changes alike in this respect. In the case of Ceratophyllum (Pearl ’o7) it was found that in 358 Raymond Pearl addition to the logarithmic approach of successively formed structures to a type (“first law of growth”’) there was also a reduc- tion of variability with successive whorl formation (“‘second law of growth”). Of such a reduction of variability with continued formative activity we find no evidence in the case described in this paper. If such a law obtained in this case it would be expected that the zigzag line in plate II would exhibit progressively smaller and smaller fluctuations up and down about the smooth curve the farther out on that curve one went. The diagram shows that this is not the case. The fluctuations are just as frequent and extensive at the end of the curve as at the beginning. The only difference is that they are about a different mid-point in the upper part of the curve from what they are at the start. SUMMARY 1 The plan of a comprehensive investigation of the problem of the physiology of reproduction in the domestic fowl is set forth in outline, and a statement is made of the general standpoint from which the problem is being attacked (pp. 339 —342.) 2 A description is given of a case in which the first egg laid by a certain pullet was very abnormal in shape. ‘There was a pro- gressive change in the successive eggs laid by this pullet. ‘This change was of a regulatory character, the eggs finally coming to be normal in shape. 3. It is shown that this progressive regulatory change follows a logarithmic curve, and the significance of this fact is discussed. 4 The data obtained in this case are held to warrant the con- clusion that the shape of the egg is determined by the muscular activity of the walls of the uterus. . Phystology of Reproduction in Domestic Foul 359 LITERATURE CITED Cusuny, A. R.—On the Glands of the Oviduct in the Fowl. American Journal of Physiology, vol. vi, pp. xviii and xix. Driescu, H. ’01—Die organischen Regulationen. Vorbereitiingen zu einer Theorie des Lebens. Leipzig. (Engelmann), 1go1, pp. xv and 228. Jennincs, H. S. ’08—Heredity, Variation and Evolution in Protozoa. I. The Fate of New Structural Characters in Paramecium, in Connec- tion with the Problem of the Inheritance of Acquired Characters in UnicellularOrganisms. Jour. Exper. Zodl., vol. v, pp. 577- 632, 1908. Jensen, P. ’07—Organische Zweckmassigkeit, Entwicklung und Vererbung vom Standpunkt der Physiologie. Jena, 1907. 251 pp. Peart, R. ’07 (with the assistance of O. M. Pepper and F. J. Hagle)—Variation and Differentiation in Ceratophyllum. Carnegie Institution of Washington. Publ. no. 58, 1907. Sz1etasko, A. ’02—Die Bildungsgesetze der Vogeleier beziiglich ihrer Gestalt. Gera-Untermhaus, 1902. Sz1ELasko, A. ’05—Die Gestalt der Vogeleier. Journ. f. Ornithol. Jahrg. 53, pp. 273-297, 1905. Tompson, D’Arcy W. ’o8—On the Shape of Eggs and the Causes which Deter- mine Them. Nature, vol. Ixxvili, no. 2014, pp. I1I-113, 1908. Waa tcren, FR. ’71-’72—Ein Ei im Ei. Journ. f. Ornithol., Jahrg. 1871-72, pp. 260-265. EXPLANATION OF PLATES Prate I Shows photographs of the first 12, the 18th, 30th, 42d and s4th eggs laid by hen No. 183. The order of arrangement of the eggs on the plate is shown in the following scheme. Top he Deft Pgh, Oi i% 8 piece 18 30 42 (54 Bottom PHYSIOLOGY OF REPRODUCTION IN DOMESTIC FOWL PLATE I RayMonp PEARL Tue JourNAL or ExPERIMENTAL ZOOLOGY, VOL. VI, NOs 3 Priate Il Diagram showing the observed change in the length-breadth index (100 breadth + length) of the first 87 eggs laid by hen No. 183. The zigzag line gives the observations and the smooth curve the graph of a curve of type y= A+ Bx + Clogx fitted to the observations by the method of least squares. eRe as PHYSIOLOGY OF REPRO. Ray} PLATE It Tue JourNnat or ExPERIMENTA | \ | pHYSIOLOGY OF REPRODUCTION IN DOMESTIC FOWL Rayvuonp P: mee ate SEE Eh il mel) HEE SE EEEEEEE, | THE PHYSIOLOGY OF NEMATOCYSTS? BY O. C. GLASER AND C, M. SPARROW MPT EN ERESEAULCEX OED et eteraefatet ator oreotatey oro oeey ale eiotnietereCaleisisiaiel ois #/2\«/oTelo © sie'oi6]e > \0 6 vie we d'v.oie\e sislerevaiscelavelels 361 - heucreilewalman Ges geo see oo gucboundudee 000 DD UCAD DOD OUBDORSOADBODCCOOm HO AUaCoL 362 MDE Ke TATIeT tah Wit ll) VON GAP ae of elclete lala efelerele| “lel «icles ciela)© nicl «| vje\ele\e 0 slelelyie\sis/a\eje «1 e\sieie vies 364 IV Experiments with the tentacles and acontia of Metridium.............0..ceceeeeeeeee 366 eee Xperiments with isolated nemiatOCysts.-)< distilled water many many ACO TAS cpgdboadote soGeoels distilled water many many FEHUACIE SH enicie cic eforeicie sisie'sie saturated sugar solution none | none TEI Dosidoongaededooe one saturated sugar solution none | none PERHEICGrinietcic's:s% «isis. ss s'e's idem followed by H2O dist. many few EDRs od6 SCO SRO OGDRDN idem followed by HO dist. all | none PETEACIES (cic s.ncise Sass se > saturated sodium chlorid many | many ECA ec bier e sie ie cis st saturated sodium chlorid few many PENEACIES oie)eccis 0 vlsvers as 310, 3 Kleinenberg’s picro-sulfuric few | many CODE a ae orci ioversiclsls =»: sat Kleinenberg’s picro-sulfuric none all PENLACIOS fei soiree 3 nxt c's rere sublimate-acetic few many TEES Sooo CDR OO BETTE sublimate-acetic none all FEntaClescinstoleisiejacs ssi se oss saturated mercury bichlorid none none COMM Ac etarciece. c's sis ore diate hsteie saturated mercury bichlorid none none FOUCAGIES H.C o15 fans iieres v5.00 acetic acid many many BACON Arie aero seg sc sero 3 acetic acid : none | all EPREAGIEG EI tet cicie/G, 12) cictel> «.c hydrochloric acid few many REO ds cceclses es ccs s's sess hydrochloric acid none | many ERR UAGIEG Sephari nic coe. s 2 ccs ammonium hydroxid few many BEOUUA Rss exe ais ininis'Sio{oreie 9's ammonium hydroxid none all EEREACI Se tetorelescts sier(s.5 3.0). = 95 per cent alcohol none many PRONG dary aeie se cleisosein is ae = 95 per cent alcohol many many Benitaclesrjias cars oo «ayes 4 chloroform none many OB Aleta cferte cisieie + eVare chloroform none many EPHEACIES eye is otere 0:5: 2,6 6s ether none many MEOW Aa cfeiets cies: oe sth wicis c's ether many many PENGACIES Iso cisieaais's ke ae chloretone none many BACON A sie terspsevistee sf ve ste chloretone many many Beefaclesercrays 2 o(-<<{2)- 22% mechanical pressure many many COTE enters alrie a sio,2) #231 014)s mechanical pressure many many RERGACIES -jos-$ e)sfe\sres ais os «2s heat 100° C. many many ROMEEA MS elcls ete fis1s secretes heat 100° C. many many Fentaclessapmicn is car's seme as heat 0 °C. none none CGA tere clotelefeovcyais) "= 2,3/,6 heat! O07, none none FENEACIES Ws ticle sais e.cte ois a0 « alternating current many many ABORT pe aie SAO ne: alternating current none all 368 O. C. Glaser and C. M. Sparrow It is not necessary to give a detailed analysis of the experiments summarized in Table I. In general they indicate that specific chemical effects are not involved, and further that any theory which attempts to explain the discharge of nematocysts, must take account of the nematocyte. ‘This particular phase of the subject, however, can be more profitably discussed after the experiments on isolated nematocysts have been reported. ‘These also will explain some of the above results which at first sight may appear puzzling. EXPERIMENTS WITH ISOLATED NEMATOCYSTS A nematocyst is a membranous capsule, one portion of which is prolonged into a thread, ending ina point. In its undischarged state, this thread is introverted, and is stored inside the capsule of which it is an organic part. In addition to the visible flament, the capsule contains certain invisible chemical substances. On the basis of this knowledge, we may make certain assump- tions regarding the causes that bring about eversion of the thread, and these assumed causes can then be tested experimentally. We may assume that in order to bring about discharge, it 1s neces- sary to raise the internal pressure of the capsule to a point at which it can overcome the effect due to the uniform external pressure to which the capsule is subject, plus whatever resistance to eversion is offered by the construction of the capsule itself. We may as- sume further, that the capsule is a membrane, semi-permeable to aqueous solutions, and that it contains substances capable of absorbing water. We may assume also that the membrane is specifically permeable to certain ions, although, if the results of the experiments can be explained without this assumption, postu- lation of specific permeability becomes unnecessary. These assumptions were tested experimentally. The results which have been actually obtained appear to be explicable by any one, or any probable combination, of the following factors: increase of internal pressure; decrease of external pressure; reduc- tion in the resistance to eversion due to the construction of the capsule. The Physiology of Nematocysts 369 Mechanical Pressure The effect of mechanical distorting pressure was studied by mixing the nematocysts of Physalia or the tentacles of Metridium with granulated salt and grinding the material between glass plates. Sometimes the salt was omitted, and ground glass plates were used. After treatment in this manner, the nematocysts were examined. In those cases in which salt was used, this was dis- solved before observations on the results of the treatment could be attempted. In this way a considerable mechanical distorting pressure was applied to the individual capsules, and though the nematocysts of Metridium, on account of their minute size and their delicacy, gave inconclusive results, those of Physalia gave very positive ones. Many partial discharges were obtained. Pressure on the cover glass of a preparation of Physalia nettles also causes many partial discharges. Such pressure as was used in these experiments distorts the capsules, and is effective because the internal pressure of the nematocysts is raised by distortion. Uniform External Pressure That the effects of distorting pressure have been correctly inter- preted, is clearly shown by the effect of high uniform external pressure. Such pressure was applied by allowing the nemato- cysts to be drawn up into a capillary tube provided at one end with a reservoir filled with mercury. The open end of the tube was then sealed and the mercury made to expand. The pressure obtained in this manner, calculated from the con- traction of the air bubble inside the tube, and from the bursting strength of the tube, was from 50 to 100 atmospheres. No nemat- ocysts ever discharged when treated in this way. Solutions In Table II are presented in condensed form the results of experiments undertaken to discover the effects on isolated nemat- ocysts of the same solutions which had previously been employed on the living tentacles and acontia of Metridium. ‘The isolated 370 O. C. Glaser and C. M. Sparrow nematocysts of Physalia were not used in this series of experiments for reasons which will become clear later—all the results pre- sented in this section are based on isolated Metridium nemato- cysts secured by the maceration method. TABLE II Saturated sugar solution............ ... eae Idem followed by HeO dist...............--..--. Saturated sodium chlorid) \ 255.0 2.5.5 senna oe “strontium chlorid ..........:......... One Siillate ad soc & oe a ear ee mapnemum sulfate-.2.- ..c2 a2 he3 nen e-s sodium: carbonate »2220 > ©. -50.s eck potassium) carbonate 4-.-2--.8).5- Kieinenberg’s picro-sulfuric..:. 22... -..2+54-5-- Sublmiate acetic. ot 3.7. 5 nes oye! < chs gis Gis cierelnd, ae aieiie chen ete 412 Bee tlertwi or Stee ern plasM ay THEORY foc ores) o/s cposens ote o's) sik etla aye os avs Where ean reed aps tLe 415 LES SUUIRET EIS 75 Seve Baer Nbte sche COE cee RON nC aR 427 I INTRODUCTION ‘Tillina magna was first described in 1879 by Gruber, who found the organism in great numbers in a fresh water culture which had been sent from Vienna to Freiburg, and who regarded it as an intermediate type between Colpoda and Paramecium. Kent, in 1880, placed it in the family Enchelinidz of the Holotrichous ciliates because of its oral cilia, although he admitted that the pharynx was strikingly like that of Conchopthirus. Biitschli, in 1888, mentioned Tillina magna merely as a synonym for Conchop- Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, in the Faculty of Pure Science, Columbia University. Tue JourNat or ExperIMENTAL ZOOLOGY VOL. Vi, NO. 3. 384 Louise Hoyt Gregory thirus magna which he described as the only fresh water form in the genus Conchopthirus, the remainder being parasitic in land and fresh water molluscs. Since 1888 no mention of this form has been made in any classification or investigation. In November, 1906, an infusorian was found in the laboratory of Columbia University, in an infusion of horse manure which had been standing for a month. It was identified by Professor Calkins as Tillina magna, and because of its characteristic tongue of cilia in the oral region, in addition to its general coating of cilia; I have classified it in the order Holotrichida, sub-order Trichostomina, family Chiliferidz, thus taking it from the family Enchelinidz where Kent had placed it, also from the order Hetero- trichida where Biitschli placed it as synonymous with Conchop- thirus, and with Gruber classifying it as a type closely related to Colpoda and Paramecium. Because of its rarity and unusual power of reproduction, result- ing in a rapid increase of numbers, and its apparent ease of culti- vation in artificial surroundings, Professor Calkins suggested that I study its morphological characteristics, its methods of reproduc- tion, its reaction to stimuli, and the process of conjugation, in fact, as much as possible of the processes taking place in the life history, with the view of verifying the work of some previous investigators, and of offering, if possible, further facts for the discussion of such fundamental problems of biology as repro- duction, artificial parthenogenesis, encystment, and the interrela- tions of nucleus and cytoplasm. I wish to express my thanks to Professor Calkins for his helpful suggestions and criticism through- out the course of my work. In November, 1906, two strains were started from the wild material. In January, 1907, I endeavored to find more wild stock in the original culture jar, but was unsuccessful. The entire stock had epee For two years I have made and examined cultures, but in no case have I found the organism. Gruber, in his early article, mentioned the fact that the organism remained in the medium for only a short time, disappearing on the appearance of other protozoan forms. This would accord with Peters’ idea of a gradual succession of forms in a protozoan The Life History of Tillina Magna 385 culture, brought forward in 1904. From his observations on the appearance of Stentor in the culture medium, Peters concluded that, as there is a constant change taking place in the growth of a culture caused by fermentation, there is also a corresponding change in the life of the culture. Paramecium, Euglena and others appear at an early period when the fermentation is active. Stentor, on the other hand, does not appear until the extreme acidity has decreased, at which time the earlier forms begin to die out. “There must be some other reason, however, to explain the nonappearance of Tillina in the new culture jars, which were examined very fre- quently. Possibly the organism is an intestinal parasite of the horse, which may also lead a free living existence for a short time. Its non-appearance in the fresh jars might be explained in this case if the culture material was not infected. II MATERIAL AND METHODS Since Tillina stock was found in a jar containing a culture of horse manure, in order to have the artificial medium as near like the normal as possible, a solution was made of ten grams of man- ure in 60 cc. of water. ‘This was brought to a boil, filtered and allowed to stand. In general it was found better to use the me- dium that was 24 hours old. Fresh medium was made every two or three days and the cultures were examined every day. Attempt was made to find another medium. A hay infusion was prepared according to the method of Calkins (’02). Then the animals were brought gradually into the new medium, starting with a solution of } hay infusion, and } medium, and increas- ing the amount of hay each day. In no case could the animals live in pure hay infusion, death occurring almost immediately. I was able to carry a culture for about a month in a solution of 4 hay and 4 medium, but they were not as healthy as those in the straight medium. An oat infusion was tried, with no better suc- cess, and the so-called “normal”? medium was finally decided _ upon as best for the experiments. The methods used in these experiments are the same as those of Calkins (’02) and Woodruff (’05). In brief, a small chamber 386 Louise Hoyt Gregory was made of a depression slide; two glass supports held the cover glass over the hollow center, covering an area that would hold about twenty drops of liquid. ‘These slides were kept in moist chambers, which were subjected to ordinary room temperature. All possible care was taken to prevent contamination. ‘The slides were washed in boiling water, supports and covers were kept con- stantly in water, when not in use, and the cloths used in drying were for that purpose only. Capillary pipettes were used for the transference of the individuals, and these were kept separate from those used for other purposes. A large pipette was used expressly for the filling of the chambers. Both living and fixed material were studied. The living indi- viduals were isolated by means of a fine pipette and studied in a hanging drop. Material was fixed at frequent intervals, and sec- tions as well as total mounts were made. For embedding methods see Calkins (’07). The best fixative was found to be a saturated solution of corrosive sublimate, to which had been added Io per cent formalin solution in the proportions I0 : 100 cc. saturated solution. The least shrinkage resulted with this method. For staining the total mounts, Heidenhain’s hematoxylin, and hemacalcium, gave the best results. Eosin and picro-carmine were farily good stains. Delafield’s hematoxylin, saffranin, eosin and methylene blue, were tried, but the results were.not good. The sections cut 5 microns in thickness, were stained in general with iron hematoxylin, both with and without the counter stain of eosin. Other stains were tried, but none proved to be satis- factory. III MORPHOLOGY AND PHYSIOLOGY Tillina magna is a large ciliate having the shape of a bean or a kidney. Gruber in his description states that the average length is 200, and that he often found larger forms. In only a few cases, however, have I found it measuring even 200, in length, and never more than this, the size varying from 100-200, in length, and from 70 to 180” in breadth. The average length and breadth of fifty individuals was 160% and 100 respectively. The anterior half of the body is broad and blunt, the posterior half tapering. The The Life History of Tillina Magna 387 posterior region is easily recognized by the presence of a highly characteristic lobe-like process of the dorsal surface, in which lies the contractile vacuole, and which extends out beyond the body proper on all sides, especially at the right posterior edge. A continuation of this edge is extended along a depression on the left side and ventrally into the peristomial region, and appears finally as a tongue-like ridge lying on the floor of the pharynx. This tongue gradually diminishes in width, and disappears near the inner end of the pharynx. While the body in general is com- posed of colorless protoplasm, pigmentation is found only in this posterior lobe, which normally is filled with a mass of black gran- ules. ‘These granules are present when the young individuals break away from the cyst, and cause the lobe to stand out in sharp contrast from the main portion of the body, which at this time is without food vacuoles, and is practically colorless. ‘The lobe is somewhat similar to that described in Colpidium colpoda. It differs in being much more developed, and in being found in the posterior rather than the anterior region (Plate I, Fig. 1). The mouth is situated on the ventral surface in the anterior half of the body, and extends from the region near the middle line out toward the left side, where the peristomial region runs into it. According to Gruber, the peristome is lacking. ‘This is a mistake, I think, as in every individual the mouth is always in its central position, with a definite peristome leading to it. ‘There is no vestibule; the mouth leads directly: into a long tubular, curved pharynx or cesophagus, relatively much longer than that of Col- poda. The pharynx, with its ridge-like tongue, bends anteriorly inward toward the right side of the body, then it turns sharply toward the posterior region, and ends just below the nucleus, its walls widening out like a funnel (Plate I, Fig. 2). The entire surface of the body is covered with many longitu- dinal bands or striz, which indicate the insertion lines of the cilia. The strie are arranged on the surface in a similar manner to that described by Schewiakoff for Colpidium colpoda. On the dorsal surface the lines pass from the anterior to posterior end in straight parallel rows, bending toward the left posteriorly. On the ventral surface, however, they converge about the mouth, which, accord- 388 Louise Hoyt Gregory ing to Biitschli, is the general rule, for when the mouth has shifted from its original, anterior, terminal position to a ventral region, the lines of cilia are moved also. ‘The peristomial and pharyngeal, as well as the membrane or plate region, are covered with long fine cilia, which are easily seen in the pharynx, about the mouth, and along the entire edge of the poster or lobe. ‘These are of uniform size, and three times the length of the cilia covering the body. The ectoplasm is differentiated into two parts, one, the cuticle, or pellicle, which is a thin membrane covering the entire surface of the body; the second, the cortical plasm or alveolar laver (But- schli, 1888), which lies directly beneath the cuticle. ‘The cortical plasm is a definite, well differentiated layer, easily distinguished from the endoplasm (see Text Fig. 1 a). The cuticle and outer Fig. 1 a. Section through the cortical plasm and endoplasm showing the sharp differentiation between the two structures, also the position of the basal bodies in which the cilia take their origin. X 1200. b. Semi-diagramatic surface view showing the raised squares and the insertion of the cilia. X 1200. portion of the cortical layer is raised to form minute papilla, such as have been described in Lembadion (Bitschli ’87), Paramecium (Butschli “81, Maier ’03, Schuberg ’o5), Frontonia (Schuberg ’05), Colpidium colpoda (Schewiakoff ’87), Ophryoglena, Chilo- don, Bursaria (Maier ’03), Opalina, Nyctotherus (Maier ’03, Bessenberger ’03.) A surface view or section shows the body to be divided into small squares or hexagons, the “Feldchen” of Maier (Text Fig. 10). These squares are raised in their centers, forming papille, which are definitely and clearly seen in profile on the edge of the body. At each corner of the squares is a deeply staining body, the basal granule, from which a cilium takes its origin. ‘Thus the cilia lie The Life History of Tillina Magna 389 in the furrows, and the striz truly indicate the lines of cilia inser- tion. ‘The basal granules are not situated deep in the cortical plasm, but lie near the surface, almost directly under the cuticle, leaving below a wide clear space of granular cortical plasm. A comparison of the cortical plasm and ciliary structures of Tillina with those described in other forms, shows that there may be considerable variation even among related types. In Para- mecium caudatum and Frontonia leucas, according to Maier and Schuberg, the surface of the body is divided into hexagonal or thomboidal figures. ‘These, however, are not raised in their centers as in the case of Tillina, but are hollowed out. ‘The sides of the fields are raised to surround the hollow centers, in which lie the basal granules which give rise to the cilia. The papillz at the edge of a section, are the raised boundaries of the hollow areas, the striations on the body indicate the lines of cilia insertion. Another variation is seen in the structure of Colpidium colpoda, and of Lembadion, according to Schewiakoff and Bitschli. These forms agree with Tillina in having raised areas, but differ in the fact that the basal granules lie in the centers of the squares, not at the corners, and the striz represent merely the furrows between the papilla. In the case of Opalina, Bursaria, Ophryoglena and Nyctotherus, according to Maier and Bessenberger, the arrange- ment is the same as has already been described for ‘Villina. Below the basal granules in the cortical plasm, there is a layer of clear granular protoplasm. If trichocysts are present, they should be found here, bus I have no evidence of their presence; Gruber and Kent mention the layer of definite trichocysts in Tillina, but Biitschli (’86) later correctly interpreted this as the alveolar layer. ‘Trichocysts, therefore, are unquestionably absent. The contractile vacuole is of the simplest type. ‘There is one large vacuole situated in the cortical plasm on the dorsal surface of the posterior lobe, and communicating directly with the exte- rior. [here are no definite canals or reservoirs in communication with the vacuole. It has a membrane and is a stable structure, not being constantly reformed. The endoplasm is a fine granular substance containing many food vacuoles. Although there is no definite basement membrane 390 Louise Hoyt Gregory separating the endoplasm from the cortical layer, there is a thick- ening of the protoplasm that marks the limits of each layer. As in the majority of infusoria, the nuclear material is different- iated into two structures, a large macro-nucleus, and one or many small micro-nuclei. “The macro-nucleus lies dorsal to the pharynx in the anterior half of the body, near the middle line, not in the extreme anterior region as is stated in Gruber’s account. Neither is the early statement true, that the nucleus is visible only in young forms in which the protoplasm is less dense. I have been able to see the macro-nucleus at all stages of growth, in the large ma- ture forms as well as in the small young individuals. In the fixed material the nucleus is always visible, staining with different de- grees of intensity, and surrounded by a definite membrane. ‘The question of a nuclear membrane has long been a subject of dis- cussion. Many investigators, among whom are Butschli (’98), and Albrecht (’03), assert that no membrane exists. Butschlidoes not consider the membrane of an infusorian a true one, since it is transitory, and may have the same reactions as the cytoplasm. Albrecht, experimenting with sea urchin eggs, found that if the nuclei were forced from the egg by compression, and brought in contact with another, they would flow together, hence he believes that there is no nuclear membrane. Marcus (’07) repeated Al- brecht’s experiments, using Actinosphzrium, and obtained oppo- site results; that is, the nuclei did not flow together. Albrecht probably broke the membrane when compressing the eggs. I have compressed Tillina, and both the macro- and the micro- nuclei were forced from the body. ‘They always retained their normal shape, and showed the presence of a membrane. This, however, may vary at times in its definiteness, and in two cases seemed to have been broken, allowing the nuclear fluid and cytoplasm to mingle. The shape of the macro-nucleus varies. It is usually an ellip- soidal body, the long axis measuring 504 to 70, the short axis 20/1 to 30u. At other times the shape may be spherical or like the letter U (Text Fig. 2, a,b). The different forms may all be derived from the normal ellipsoidal one, and may represent a certain stage in the preparation for division. ‘Ihe macro-nucleus The Life History of Tillina Magna 391 varies in its staining reactions. At one time it may be filled with a vesicular achromatic ground substance in which are embedded many large deeply stained chromatin masses, which often take the form of threads or loops (Text Fig.3, abc). Another individual may have a nucleus in which the achromatic material 1s faintly stained, and in which there is no indication of the presence of chromatic material having lost its power of taking the nuclear stain The micro-nuclei are small spherical bodies 5 in size, situated close to the macro-nucleus, either embedded in the larger nucleus, or at the edge. The homogeneously staining chromatic mass is surrounded by a clear non-staining area which separates AX Fig.2 Types of nuclei. These may be transition stages between the normal elipsoidal nucleus and the spherical nucleus of the division cyst. XX 400. . Fig. 3 Sections through the normal elipsoidal nuclei showing characteristic appearances of the chromatic substance. it from the membrane always present. ‘They vary from four to ten innumber. Many times [ have found two in close proximity, indicating a late division. Once only I found what seemed to be a spindle formation. The bodies are so small that it is impos- sible to distinguish the internal structure. Intracellular digestion in Protozoa has long been a disputed question. Engelmann (’79) and Le Dantec (’92) concluded from their experiments that the digestion was due to the presence of an acid medium. Mouton and Mesnil (’g0) came to the opposite conclusion. Greenwood (’94) made extensive experiments on the digestion of the gastric vacuoles in Carchesium, and came to the conclusion that the original vacuole is not the digestive vacu- 392 Louise Hoyt Gregory ole, but that the food particles are forced in the protoplasm, and later are gathered into the true digestive vacuole where digestion takes place in an acid medium. Metalnikoff (03) has offered the last suggestion in which the two opposing results are combined. In his experiments on feeding paramecia with alizarin, he finds the beginning of digestion taking place either in an acid medium indicated by the yellow color of alizarin, or in an alkaline medium indicated by the red color of alizarin, the main and final digestive processes, however, always take place in an alkaline medium. Thus the digestive processes of the Protozoa closely resemble those of the Metazoa in which is found the pepsin ferment fol- lowed by the pancreatin action. Attempts were made to duplicate these experiments in case of Tillina, but in all cases the coloring matter was not taken in. The endoplasm is well filled with large food vacuoles which are in different stages of digestion. Some are crowded with bacteria, and stain deeply with hematoxylin, indicating that digestion has not proceeded far; some show lighter areas at the ends of the vacu- oles, an indication of digestion in those regions; in others the proc- ess has gone on to a greater extent, and only a slight amount of undigested material remains, which 1s stained a pale gray in comparison with the black stain of the fresh food particles; finally vacuoles are found with no trace of the presence of food; these can hardly be distinguished from the contractile vacuole, position only being the means of identification. IV REPRODUCTION AND ENCYSTMENT I-ncystment is an expression of certain physiological conditions in the cell during which different functions may be performed. The cyst may be temporary only, and for the purpose of repro- duction or digestion, or it may be permanent, affording the organ- ism a condition of rest as well as a protection from an unfavorable environment. ‘The reproductive cysts are of two kinds, those in which simple division takes place, and those in which so-called sporulation occurs. Cohn, in 1853, described s mple division within a cyst, in the case of Prorodon teres, one division only taking place. Carter, The Life History of Tillina Magna 393 in 1856, descr bed a similar process in Otostoma, and Stein in 1859 the same for Colpoda cucullus. Butschli, in his work of 1888, has brought together the results of previous ‘investigations, and has class fed those forming division cysts into three groups as follows: Group 1. “Those which always form division cysts before sim- ple division takes place: Colpoda cucullus, Holophrya multi- cilia, Amphileptus, Trichorhynchus, Lachrymaria. Group 2. Those in which it is doubtful if cysts are always formed before division: Prorodon, Actinobolus, Holophrya gula, Enchylys tarda, Ophryoglena, and here Biitschli places Tillina, wh ch, I believe, rightly belongs in the first group. Group 3. Those which may or may not encyst before division: Leucophrys patula, Glaucoma scintillans. Today the most complete, and in fact, the only detailed descrip- tion of division cyst formation is that published by Rhumbler in 1888, in his paper entitled “A Study of Cyst Formation and the Developmental History of Colpoda cucullus.” The division cysts may be either oval bodies in which the protoplasm divides but once, forming two individuals, or spherical bodies in which one or two divisions may take place, resulting in the formation of two or four individuals. He describes a permanent opening in the surrounding membrane of these division cysts through which the contractile vacuole discharges its contents during the early history of the formation of the cyst. Later the contractile vacuole rotates, and its contents are discharged within the mem- brane outside of the body. The newly formed individuals make their escape through the opening in the membrane. Reproduction in the case of Tillina takes place exclusively by the formation of spherical cysts in which the protoplasm may divide once or twice, to form two or four individuals. “The forma- tion of these cysts varies in frequency depending a great deal upon the amount of food present. Under favorable conditions, when the division energy is normal, and the food has been given at regu- lar intervals, each individual will encyst on the average of once a day, and by two divisions will give rise to four young individuals, the entire process covering a period of about twelve hours. The 394 Louise Hoyt Gregory record shows that this rate of division may rise to 2.5 divisions per day (25 divisions in ten days), and that it may sink as low as .3 of a division per day (3 divisions in ten days). (These figures represent the average of the division rates of four individuals for ten day periods.) On comparing the frequency of division with that of Paramecium aurelia, Calkins (’02, ’o4), and with that of Oxytricha fallax, Woodruff (’05), the records showthat the division rate of Paramecium varies from a maximum of 1.7 divisions per day, to .07 divisions per day (in other words, from seventeen to seven-tenths division in ten days); while that of Oxytricha varies from 3.5 to .2 divisions per day (or from thirty-five to two divi- sions in ten days). ‘These records show that the division rate of Tillina never reaches the height of that of Oxytricha, neither is it able to remain at such a low rate without dying out, as that of either Oxytricha or Paramecium. ‘The protoplasm of Tillina seems to lack the responsive as well as the endurance power of these two other forms. When the young individual breaks from the cyst it is about one- fourth the normal size, but perfectly formed, containing few if any food vacuoles. It swims about, taking in food and growing rapidly until in about six hours it has reached its normal size, and is well filled with food vacuoles, which gives it a dark color. As Biutschli states, it seems probable that these cysts are usually formed after the body has reached its maximum size. ‘This can- not be the only condition, as he cites the case of Amphileptus, which encysts in order to digest. I have found cysts varying in size, showing that the capsules may be formed when the maxi- mum size of the individuals has not been reached. The first indication of a preparation for division is the notice- able change in movement. ‘This gradually becomes slower and slower until finally, the individual comes to rest on the bottom of the depression slide, or near the glass supports. ‘The cilia tem- porarily disappear. A thin membrane is then secreted covering the entire surface of the body, and rotation begins within the newly formed membrane, indicating the reappearance of the cilia. Ex- cretory particles are discharged from the posterior region. As the rotation proceeds, the normal elongated bean shape is grad- The Life History of Tillina Magna 395 ually lost, the peristomial region disappears, and the posterior lobe is absorbed and the large elongated macro-nucleus is shifted in position. If, in the changing of position, one end of the nucleus is moved, the U-shaped form, already mentioned, will result. The spherical nucleus may also be explained as a result of the rotation. In these cases, however, the nuclear changes have anticipated division, as the membrane is not yet formed. ‘The mouth, as well as the nucleus, is changed in its position, Finally, when the spherical form has been reached, nothing is visible save the nucleus, contractile vacuole, food vacuoles and the cortical layer, which is still prominent just below the membrane. Bitschli states that the presence of this layer within the cyst is the exception rather than the rule. There is some question in regard to the history of the mouth during the formation of divi- sion cysts. [he mouth is not visible in the living cysts, possibly being concealed by the presence of many food vacuoles; neither is it always found in sections of the division cysts. It seems most probable that the old mouth disappears at an early stage, and that a new one is formed before the appearance of the first plane of division. ‘This would explain the fact that in sectioned material a mouth is found in some sections and not in others. ‘This division cyst differs from that of Colpoda cucullus in being always spherical in form, and in having no definite opening in the cyst wall. The first indication of division is in the elongation of the nucleus with a slight constriction in the center (Plate II, Fig. 1). Almost at the same time a constriction appears in the membrane, extend- ing toward the center in such a way that the division plane will pass through the nucleus and the mouth. ‘The contractile vacuole lies at one side, and passes to one of the daughter halves, a new one being formed in the other half. After the first division, or more often, before the first division has been completed, there is a shifting of the daughter nuclei and mouths, and the second plane of division appears at right angles to the first (Plate II, Figs. 2, 3, 4). During this process the macro-nucleus divides by simple division. In some cases the chromatin stands out in sharp contrast to the light faintly staining achromatic ground substance. ‘The chro- matin may be arranged in lopes or masses, in the central portion of 396 Louise Hoyt Gregory the nucleus, or at the extreme edge. In a few cases it seemed to have disappeared at one region and the nuclear substance mingled with the cytoplasmic material. While the micronuclei divide by mitosis, this division is inde- pendent of cell division. During the latter process, four micro- nuclei may pass directly into one daughter cell, and five to the other, or six into one and seven into the other. All of the bodies are of the same character as those described in the normal forms, having a homogeneous central mass, surrounded by a clear area, the whole body being bounded by a thin but definite membrane. After the planes have cut through, and the four young cells are separated, rotation takes place individually within the membrane, until the normal form is assumed. Usually the young individuals overlap, as the space is not large enough for all four to be on the same plane. As they increase in size, the membrane is broken, and they escape from the cyst fully formed, with the posterior lobe, mouth, contractile vacuole—all organs in position, but of one- quarter the normal size. The formation of sporulation cysts is not common among the Infusoria. Reproduction takes place, in general, by the simple transverse division of the body, either within or without a cyst membrane. One of the few examples of the formation of sporu- lation cysts has been described by Rhumbler in the history of Colpoda cucullus. The food vacuoles are first eliminated, and then a thin membrane or velum is secreted slowly on the outside of the cell membrane. A space is left between the two membranes into which the contractile vacuole discharges its contents. “There is no opening in the membrane, and the cell floats in its own cavity. The body gradually loses its normal form, shrinks to one-half the original size, and assumes a spherical shape. ‘The cilia are lost, the nucleus is invisible, all assimilation particles are eliminated, and finally there is nothing left save a homogeneous mass of pro- toplasm which ulitmately divides to form eight, ten, or twenty spores. Evarts, in 1873, described a somewhat similar process in Vorticella. Under certain conditions, such as a changed environment, un- usual heat or cold, or because of some internal stimulus, perma- The Life History of Tillina Magna 397 nent cysts are formed. If fresh medium is not given, and if many individuals are allowed to remain together in a small space, as is often the case with the oldest reserve stock, then the individuals round up and secrete a definite thick gelatinous membrane. ‘The cysts are usually smaller than the division cysts, both because the former contain few if any food vacuoles, also because the very young individuals may encyst as well as the mature individuals, if the conditions are favorable for such a result. ‘The protoplasm within the cyst is dense, the nucleus is the only part remaining of the normal body, the mouth, pharynx, contractile vacuole, all having disappeared. As the cysts become older, the membrane becomes thicker and harder; a second membrane, however, is not secreted. Individuals may be induced artificially to form cysts at any period. If they are placed in tap water, or in a sugar solution, encystment will occur within a short time. Changes in tempera- ture will bring about the same result. Cysts will be formed within twenty-four hours if the individuals are subjected to a tempera- ature of 38° C. The same conditions that cause encystment will often result in the disappearance of the membrane, and the renewal of active life. If encysted forms are subjected to the same tem- perature (38°) that caused the encystment of the free individuals, the majority will lose their membrane, and again lead a free life. Similar results are obtained if the cysts are put in tap water, or in fresh medium rich in food. ‘The age of the cyst is important for this point, for the older the cyst, the more difficult it is to bring the organism out. [Twenty-four cysts that were ten days old, were put in fresh medium and twelve were kept at room temperature, the other twelve were put in the water bath at a temperature of 38°. Not one came out of the cysts. April 6 12 cysts (8 days old) were put into a water bath at a temperature of 38° C. 8 nonecameout. They were transferred to normal environment. Io all came out. II 12 cysts were kept in room temperature. IZ II came out of cysts. April 11 12 cysts were put in water bath 38° C 12 8 came out. 398 Louise Hoyt Gregory June 13 9 cysts were put in rain water at room temperature. 14 5 came out of cysts. From these experiments it will be seen that there is no definite rule in regard to the numbers that can be forced from the cyst. There is a great deal of individual difference, and the same proc- esses that will be effective in one case will have no results in another case, even though the age and the environment have been the same. Generally, however, starved forms will always encyst, and a change in environment, either of food or of temperature, will cause the encysted form to assume its free living existence. Minchin has suggested the cause of this may be the influence of salts in the solution upon the membrane, an external cause, or the stimulus may come from the interior of the cyst acting on the membrane, the original stimulation coming from the new environ- ment. It either case the cause would be primarily an external one. In general, the reproductive and protective cysts differ but little from one another save in the thickness of membrane and presence of food vacuoles. Rhumbler has found a more important differ- ence in the presence of a definite opening in the reproductive cysts of Colpoda cucullus. Since Rhumbler’s observation is the only evidence, we may consider this an exception. In Colpidium also a reproductive cyst may become a permanent protective cyst, and in Tillina I have seen individuals that had encysted for division secrete a thicker membrane, until finally a permanent cyst was formed. ‘This may happen either before or after division; in the latter case each individual secretes a thick membrane. There is also a close relation between the reproduction within the simple division cysts and the sporulation cysts. If the divi- sions are simultaneous, the process might be regarded as sporula- tion, if, however,they are successive, the process is division. The conditions in Tillina must be considered an intermediate stage, for in reproduction, resulting in the formation of four individuals, the divisions are not simultaneous, but nevertheless, they follow each other so rapidly, that the second plane appears before the first division has been completed, and the four individuals mature at The Life History of Tillina Magna 399 approximately the same time. In the history of Colpoda cucullus, division within the cysts may take place either by forming two, four or eight individuals, by successive divisions, in a similar manner to that observed in Tillina, or by forming as many as twenty or more individuals simultaneously, a process of true sporulation. An interesting question in regard to the reproductive cysts, is the significance of single and double divisions forming two and four individuals respectively. ‘This was found to take place also in Colpoda, but not in similarlyshaped cysts as in Tillina. Rhum- bler describes the single divisions as taking place only in the oval cysts, and even then, only very seldom. I have tried to find if there is any relation between the number of single divisions and the vitality of the protoplasm. Diagram I shows the number of single and double divisions in the main & culture during a period of eight months. At the beginning, when the vitality was high, and the divisions frequent (e.g., periods 1-26) there are more double divisions than single, while in some five-day periods there were no single divisions at all (e.g., period 2, 6, 10, 21). As the vitality decreased, the number of single divisions grew more frequent (e.g., periods 28, 29, 31, 35, 36), and at the end of the history, there were more single than double divisions (e.g., periods 42, 44, 46, 48-50). Thus the appearance of single divisions might be considered an indication of low protoplasmic vitality. V CONJUGATION Although the life history of Tillina magna was carefully watched throughout thirteen months, or through 546 generations, no con- jugations or indications of conjugations were observed. Calkins (04) was able to bring about conjugation at almost any period by putting in small watch glasses, masses of paramecia that had col- lected about the edge of the culture jar. Similar attempts were made with Jillina, but with no results. Neither individuals of the same nor individuals of different ancestry, starved or well fed, gave any evidence of conjugation. ‘The experiments of Joukowsky (’98), and of Woodruff (’05), show that many hy potrich- ous ciliates do pass through many generations without conjuga- tion. Joukowsky carried Pleurotricha lanceolata through 460 “SUOISIAIP 9[3UIS OY} ‘OUTT UOxOIG OY, “SUOISIAIP aQnop 24} SayeoIpur au] snonuUOD ayT, “(YIUOUT 9Y3 0} spowed xis) spoted {ep aay 10j apr sroSesaae ayy, “gf PANINI [oWWO ayy Jo A1o}sTY aIWUa ay BuLINp suOIstAIp a[SuIs Jo JoquINU a4} 0} WONPIEI SI} PUL SUOISIATP a]qnop Jo Joquinu 94} SMoYs wriseip oy 7, I NvuovIG, Taal Ws 4 ' t—. The Life History of Tillina Magna 401 generations, and Woodruff watched Oxytricha fallax through 860 generations. On the other hand, conjugation has been studied by Maupas in the case of Colpoda, which in form is closely related to illina. ‘There are two possible explanations of the non-appearance of conjugation stages: the first, that the condi- tions under which the experiments were carried out lacked the proper stimulation for conjugation, the protoplasm never reaching the miscible condition which Calkins described as characteristic of conjugating paramecia; the second possibility is that Tillina may be an intestinal parasite, and that the conjugation processes are carried on under very different surroundings from that in which the normal simple division takes place. ‘The fact that the main stock was lost or disappeared from the culture jar, in which they were originally abundant, is the reason that I was not able to experiment with large numbers of wild material. VI OBSERVATIONS ON THE LIFE HISTORY The A culture, consisting of four individual lines of the same ancestry (A1, A2, A3, A4) was started on Noyember 7, 1906, and died on March 1, after having passed through 210 generations. Diagram II was made according to the methods of Calkins. ‘The curve represents the average number of divisions per day, of each individual for ten-day periods, thus representing as a whole, the division energy or general vitality of the protoplasm of the A culture. The curve is similar to those already made for Paramecium and Oxytricha. There are the same periodic rises and falls in the division rate which Woodruff has termed the “rhythms.” ‘The curve is a normal one with the exception of the unusual decrease in the division rate at the fifth period. This, however, is explained by the fact that an unexpected fall in the temperature of the lab- oratory took place during the Christmas holidays, and during this period many encysted permanently, and all of the lines suf- fered a marked decrease in vitality. Aside from this exception, there is a gradual downward tendency in the curve, indicating a general decrease in the vitality, and marking the approach of ‘spouad Avp-uay Jo ssaquinu ay} Juasaidar wssiosqe ayy, *S[eNPIAIPUT INOJ Jo UOISIAIP JO ayer ATiep advsVAv ay} Juasaidar sajyeutp1o sy, “spolsed Aep -u2} IO} pasessAe UOISIAIP Jo ey ‘wOIeIaUed YIOIZ ay) UI (Lobr ‘1 ‘1eP) YstUY 0} “(gor ‘L *AON) JvIS Wosy Y aiNyNo eUseU eUTTTT, Jo Aro\sty a3a;duI0D II Wvuovig, The Life History of Tillina Magna 403 the end. The sudden drop at the last period, and the following death of the culture was unexpected, and cannot be explained. In the ninth period the division rate averaged 1.8 per day, then suddenly many began to encyst, and the division rate fell to 1 per day. Beef extract, alcohol, K,HPO,, and KCl were used as stimulants, but with no effect, and the cultures died out March I, when A2, the last one to divide, had been encysted for twelve days. ‘The stock material was low, otherwise I think the culture might have been saved. ‘The same sudden drop in the division rate, near the end of the life history, has been noted by Maupas in his culture of Stylonychia, and by Woodruff in his culture of Gastrostyla steinii. In each case a period of comparatively high division rate preceded the sudden death. The B culture was started at the same time as the A culture, but from different stock. ‘This culture as a whole was carried through 405 days, a period of thirteen months, during which it passed through 548 generations. The main or control culture, consisting of four lines, B1, B2, B3, B4, lived through 403 generations during 250days. ‘These lines thus designated were not stimulated at any time in order that the effects of stimulation in the other lines might be more apparent upon comparison. Diagram III shows the life history of this culture. The curve is a normal one, falling naturally into the rhythmical periods already mentioned. ‘There is a gradual de- crease in vitality from beginning to end. At the 24th period the rate of division increased to some extent, chang ng from an aver- age of .87 to 1.37 divisions per day. All the lines increased rap- idly in numbers during this period; this was due undoubtedly to the unusual hot weather. At the end of this period the lines were weak, and all save two stimulated lines, died, in spite of extreme efforts to save them. At the beginning of the 12th period, at the time of the death of the A culture, although the main lines of the B series were divid- ing on the average of two divisions a day, it was thought best to try the effect of stimulants on the division rate as previous inyesti- gators had found that certain stimulants would increase the vital- ity of the protoplasm, and enable it to renew its life process in "Jooq WIA poze aanypnd = — - "OHM YA pajeas} aingynd = — porod yrgt ‘(poued taht Surnp parp) ew yyy *OdHeM YA pojvesy jou aanqynd = ~ - 9 YY "OdHeM YA pe}ver) oingpno *9INj[Nd [O1;U09 IU} Puodas "OG H*¥ Yl pajvesdy jou oinqyno — porod ys1£ ‘tT puodas FODH*Y YIM pajvas} ainyno = — pouad pfz ‘(poued yySz Surnp parp) fou0s = ~ _ VOdH®M YA pojvoy oinyjno = —— pouod yjoz “ojo “syes YAM JuoUALIT) —= X “UOISIAIP JO ayer Alup odvs9Av ayi juasoidos sayeuIpsO oy, “(yuoUT yIva 0 sported aery}) sporrad Avp-u9} 1OJ padvsoar UoIsIAIp Jo AVY ‘uoHLioued yaghS aya ut (Lobr “gt *9aq) Yysruy 0} (9061 ‘2 *AON) JavYs Woy g anno eUseUN eu Jo Aroysty ajapdurog TIL Wvuovicg OV 9 -te ier St 8G 0¢ a _ — e TO co ut { Gt t vz | za eal GZ | | | | | LOGT | ager Dwi i wiOlare {I We AM. heseesy Dav ATAL | ¥NAT 1 ava | wav vaew | goes NV S IH AON The Life History of Tillina Magna 405 times of depression. Accordingly, six sets of experiments were started. Experiment 1. Bs5, BO, were treated with sugar. Experiment 2. B7 and B8 were treated with tap water. Experiment 3. Bg, Bio, B11, B12, were treated with K,HPO,,. Experiment 4. B13, B14, B15, B16, were treated with pan- creatin. Experiment 5. B17, B18, Big, B20, were treated with beef extract. Experiment 6. B25, B26, B27, B28, were treated with alcohol. The records for the sugar experiments are as follows: February 26, Bs and B6, from stock of Br and B2, were put into a solution of 5, sugar plus tap water; February 27, individuals were pale, but normal; fresh solution was given; February 28, both individ- uals encysted as if for division; March 2, Bs divided unequally, and Bé6 encysted permanently; March 5, both lost. Thinking there might be a sufficient amount of salts in ordi- nary tap water to sustain life, and disregarding the difference in density also, since many well-fed individuals that had become sluggish, had resumed their normal activity when put in tap water, a water culture of two lines, B7 and B8, was started from the stock of B3 and By. ‘These are the records: February 26. 3Bz7 and B8 transferred to tap water. February 27. B7 and B8 divided once; individuals pale, and were put in fresh water. February 28. B7 encysted for division; B8 divided twice. Marchi. 87 divided three times, B8 divided once. March 2. No division. March 3. No division; both permanently encysted. In the case of the experiments with potassium phosphate, four lines were started from the stock of B1, B2, B3, B4. Each was _ put in a solution containing ten drops of medium and one drop of j, , sol. of H,PO, Bg was left in the solution one day, Bio two days, Bri three, and Br12 four days. Bg and Bio were lost, and the lines were filled in from B12. At the end of the 17th period, a second treatment was given, but this had no effect, and the lines died out at the end of the 2oth period. 406 Louise Hoyt Gregory B13 and Br4 were placed in a solution containing ten drops of medium, and one drop of medium plus pancreatin. B16 and Br5 were put in a solution containing 4 plain medium and 4 medium plus pancreatin. ‘his last solution proved to be too strong, and both individuals died after four days. ‘These lines were filled in from B13 and B14, both of which were doing well. ‘The variations in this series are abrupt, and the entire set died at the end of the 17th period. Individuals treated with beef extract seemed to have their vitality increased to a greater extent than those treated with any of the other stimulants. At the 12th period four lines were started from the main culture. ‘These were put in a solution of beef extract (fresh pieces of beef were put in cold water and brought to a boil, then allowed to cool). B17 was treated two days, B18 for three days, and Brg and B20 for four days, the medium being changed on the third day to fresh extract. On April 20, at the end of the 16th period, the vitality seemed to be decreasing, and Big and B20 were stimulated, B17 and B18 being left untouched. The result is evident, B17 and B18 died at the end of the 2oth period, while Brg and B20 lived fifty days longer, dying out on the 25th period, at a time when there was great mortality among all the lines. This period followed one of high rate of division caused by an unusual rise in temperature. The experiments with alcohol were started on June 23, for which four lines, B25, 26, 27 and 28, were used. One drop of a solution of 50cc. H,O +1 cc. 100 per cent alcohol was added to the medium in which B25 and B26 were living, and two drops of the above solution were added to the medium in which B27 and B28 were living. ‘This was changed each day. B25 died in four days, the line was filled in from the stock B26. B28 died in three days, and the line was filled from the stock of B27, and all four lines were then treated with two drops of the alcohol solution. ‘This treat- ment had no stimulating influence, and on July 8, the entire cul- ture died, having lived 16 days. A second culture was started October 1, and died on October 17, this also having lived 16 days. The results of the first series of experiments may be briefly stated at this point: The Life History of Tillina Magna 407 The effect of the sugar solution was to lower the vitality, to produce abnormalities, and finally to cause death. The tap water, likewise, was found not to be a successful me- dium, though the detrimental effects were not as quickly noticed as those due to the sugar solution. “The comparison of the records for B3 and B4, the non-stimulated lines, with those of B7 and B8 show the effects of the water medium at a glance. B3 B4 B7 B8 IVS RNEINT Dyfoo oda dab doe aD bawaOCUGOOOUCEGD ON SdSD 2 ° I I INEST REEES AN Oop ib od Sb BAAD Ota oc ADDON ETO rarenCc 2 2 co) 2 wilcas | SIS SeGé ne Banguasacuoe dae soon op aoc. 2 2 3 I WUZTEL, "SBA BOO ROBO CO cIerIOD DOR COUOnnOOOOo Er 2 2 ° ° MII MIDE SASEE Sc) oaths! ero: o.sieisis scvicts eesie 205 S1s hele steno esis I 2 ° ° The effects of the pancreas, potassium phosphate and beef ex- tract are seen best in Diagram IV, where the histories of each treat- ment together with the non-stimulated cultures, are shown. Of the three curves, the one indicating the pancreatic treatment shows the most abrupt changes or greatest fluctuations. All three curves show a decrease in vitality during the 12th period, The pancreas series recovered most quickly from the depression, changing from a rate of 1.1 divisions per day in the 12th period, to 1.6 divisions in the 13th period. During the following period, it fell again to .g only to rise again to 1.2 in the 15th period, and then to die out quickly in the 17th period. The potassium phosphate series is much more gradual in its rises and falls. ‘The curve is very much like that of the main culture. It maintains a slightly higher average, however, through- out its history. The history of the beef series shows the greatest actual effect. The division rate is higher than normal throughout the life of the culture. ‘That one treatment was not sufficient to carry the series along, is shown by the fact that the lines re-stimulated on the 17th period lived while the lines which received but the one initial stimulus died during the 2oth period. ‘Though the main line lived as long as the re-stimulated series, yet its average rate of division was lower, and the general condition of the individuals was much poorer and weaker. ‘The beef extract undoubtedly had a strength- ening effect. CONTR rT. ee On NO ON — Dow eT one Ns) OMFS DOSN + Dmo 6 5 Hm mej, Pomme, JN) DOA O00 SLO Dooce ~ POT ASSIU M LOSS ae 20 18 16 14 12 10 i 6 4 9 | | | MAR | APR | MAY | JUNE | JULY tplro. 13 14.) ge 16 17118 19 20 121 22 23124 25 | - | | | | rf | | ! eos | Diacram IV Histories of treatment with beef, pancreas and potassium phosphate during a period of four and one- half months, together with the history of the control culture during the same period. Rate of division averaged for ten-day periods. In the beef history the broken line indicates the vitality of the individuals that were not treated again at the beginning of the 16th period. | The Life History of Tillina Magna 409 Calkins and Lieb (’03) found that alcohol prevented to some extent the fall into periods of depression, and prevented the extinc- tion of the lines. Woodruff (’08) finds that alcohol may have oppo- site effects, causing an increase in the division rate at one time, and a decrease at another time. He also states that when an increase in the division rate takes place, this effect is not lasting, but is soon followed by a period of low vitality, even below normal. All experiments treating Tillina with alcohol solutions of different strengths proved fruitless. In every case the division rate was lowered, the vitality weakened, and the lines thus treated died out in a short time. In the 20th period, a second series of K, HPO, experiments was ’ started, the individuals being taken from the main culture, as was the first set, and treated inthe same way. ‘This culture proved to be the most successful of all, and was the one to live through the very serious depression period of July, and also furnished the last individual to die on December 15, 1907. As Diagram III shows, at the beginning of the 23d period, this culture was divided, Bg and Bro being left untouched, while B11 and B12 were treated with a second stimulation. As a result of this treatment, the re-stimulated lines lived, while the Bg and Bro lines died. At the beginning of the 25th period, on July 5, there was a marked decrease in the division rate of all the lines, and the control culture died on July 14. The beef culture was treated again, but succumbed atthesametime. An extract of calf’s brain was given, but this had no effect. Alcohol also, was found to have no influ- ence. Finally the numbers of stock material and individuals in the K,HPO, culture were reduced to nine, all of which survived this period of extreme exhaustion. ‘This culture, four lines of which became the main culture, was stimulated again on July II, and again on August 5. At the beginning of the 31st period, the main culture was left untouched, and a new culture of four lines from the stock of B12 was re-stimulated. ‘The diagram shows that the main culture died at the end of the 34th period, while the new culture, which was again stimulated in the 32d period, increased in vitality, the division rate averaged 1.26 divi- sions per day, the highest point reached during the life history, 410 Louise Hoyt Gregory with the exception of the 26th period. Finally, in the 35th period, there was a second period during which the vitality suffered, when all save the two lines were lost. Fortunately the stock material was in better condition than in July, and the cultures were renewed. At this time, a third beef culture was started and carried along with the newly stimulated K,HPO, culture. The entire set was again treated on October 21, 31, and November 14. Both cultures seemed to respond to the first stimulus only, and from the 36th period showed a gradual weakening in the vital- ity. Abnormalities appeared, the division plane not always pass- ing entirely through, or sometimes unevenly through the encysted organsim. Calcium and potassium nitrate were used as stimu- lants (one drop of a 3; solution being added to 10 drops of medium), but nothing seemed effective, and gradually the lines died out, sometimes by the formation of abnormalities, more often by the formation of permanent cysts. Attempts were made with dilute HCL to dissolve this cyst membrane. ‘This was unsuccessful, and the last individual formed its permanent cyst on December 16, the culture having passed through 546 generations in 13 months. No attempt was made to keep permanent cysts alive after they had been formed for more than ten days. Possibly if some indi- viduals had been kept for longer periods, they might have event- ually resumed their normal condition. During the ten-day periods 26, 27, a few experiments were made to compare the effects of an initial, daily and weekly treatment with one drop of 315 X; solution K,HPO,. From the few experi- ments, the results seem to indicate that a repeated treatment increases the vitality to a greater extent than an initial treatment. If, however, the treatment is too frequent, the accelerating effect is lessened, and is finally lost. Woodruff found that in comparison with the control culture, an initial treatment (30 minutes) of a solution of =x, K,HPO, causes a slowing of the division rate, while a daily causes a marked inhibition. The results are practically the same in both series of experiments, though the response of Tillina to the treatment was very slight in comparison with that of Oxytricha. 7 The Life History of Tillina Magna 411 VII REGENERATION AND CENTRIFUGING The experiments of Nussbaum (’88) on Oxytricha and Gastro- styla, of Gruber on Stentor, of Balbiani (88) on Trachelius and Prorodon, of Verworn (’95) on Thallassicolla, all prove that non- nucleated fragments of protozoa will not develop while nucleated fragments regenerate easily. A few experiments were performed with Tillina. A single individual was placed in as small a drop of water as possible. Then by the aid of a simple microscope, transverse, longitudinal or oblique cuts were made with a sharp scalpel. ‘The individuals being minute and constantly moving, made the operation somewhat difficult, and often but one-half would live, the other being crushed by the knife. Eight success- ful experiments were performed in which a longitudinal cut was made. Of these, six left halves regenerated in 24 hours, and two right halves. ‘Ten transverse cuts were made, and in two cases both halves regenerated. Of the other eight, four posterior and four anterior halves regenerated, showing that there is no differ- ence in the regenerative power of the anterior and posterior regions. In two cases regeneration, growth to normal size, and a single division took place within 24 hours. Only a few oblique cuts were made, and these were unsatisfactory, as one section was too small to regenerate. On the whole, Tillina has a remarkably high degree of regenerative power. Regeneration, however, will not take place if the halves are put into tap water. They seem to require the full degree of density in order to recover their normal conditions. Experiments were made centrifuging four groups of ten indi- viduals each 50, 100, 300, and 500 times respectively. The results were not definite, lack of material preventing extensive experiments. In all the centrifuged individuals, the nucleus tended to be shifted forward. In those centrifuged 100 times practically all of the nuclei were sent to the anterior end of the body. ‘Those centrifuged 300 times showed a scattering of the pigment throughout the body, as well as a shifting of the nucleus. In one case among those centrifuged 500 times, the pigment was sent in a mass to the anterior end, together with the nucleus. In 412 Louise Hoyt Gregory all cases, the body is shown to be plastic and unstable. Attempts were made to try the power of regeneration in the centrifuged individuals, but this was not successful as the organisms were in too weak a condition, and went to pieces on being cut. VIII GENERAL CONSIDERATIONS Artificial Rejuvenesence Like the division rate of Paramecium and of Oxytricha, that of Tillina shows the same rhythmic variability, representing peri- odic variations in the vitality of the protoplasm. Unlike Parame- cium and Oxytricha, the division rate of Tillina does not indicate as definite a response to treatment with salts. Such substances, apparently successful in other forms, seem to have been effective only in raising the vitality slightly above the normal, and increas- ing it sufficiently to carry the protoplasm through periods of weak- ness, and the question arises, has the protoplasm been rejuve- nated? According to the definition of Woodruff: “a cycle is a periodic rise and fall in the fission rate, extending over a varied number of rhythms, and ending in the extinction of the race unless it is “reyuvenated’ by conjugation or a changed environment.” Following the definition, the first impression would be that the 25th period of the Tillina curve marks the end of the first cycle, the stimulation of the K,HPO, of two periods previous, affording the changed environment the influence of which carried one cul- ture through the period of lowered vitality. If this is true, a second cycle ends at the 35th period, and the third at the goth period. In all, then, there would be three cycles, the first lasting eight months, the second and third three months each. A careful study of the history of Tillina has convinced me that the curve of vitality represents one cycle only. As has already been men- tioned, there was no markedly high period of activity resulting from a stimulation as in the case of Paramecium and Oxytricha. The slight impetus that was given was only temporary in its effect, and after a comparatively short time a re-stimulation was neces- sary to carry the individual along at even a normal rate. From The Life History of Tillina Magna 413 the beginning to the end of the history there has been a slow decline in the division rate. ‘This was not noticed during the first eight months, yet the diagram shows its presence. At the end of eight months, a stimulus seemed necessary and was given in K,HPO,. This caused a slightly higher rate of division, but it could hardly be spoken of as having caused “rejuvenation,”’ for almost immediately the vitality diminished, and a second, and soon a third stimulus was needed, this exhaustion appearing more and more frequently as the end drew near, and the stimulants having less and less effect on the protoplasm, finally failing absolutely in their potency. From these observations [| am convinced that an artificial rejuvenation of the protoplasm, in the sense of Calkins and Wood- ruff, has not taken place at any time in the history of ‘Tillina magna, and that the effects of the stimulants have been to pro- long rather than to renew life. But after all, is it not a question as to the meaning of “ Artificial Rejuvenation?” According to Calkins and Woodruff, this term has been applied to protoplasmic changes induced by chemical or mechanical means, which result in a reorganization of the body indicated by a renewal of metabolic processes and a high division rate. A marked change in the protoplasm of Tillina has not taken place after treatment with salts. Nevertheless, some action occurred which enabled the stimulated culture to hold its own, while the non-stimulated cultures died. Is this not practically the same, only to a lesser degree, as that which Calkins and Wood- ruff found in their so-called artificial rejuvenation of Paramecium and Oxytricha? During certain periods in the life history, the vegetative activ- ities of the organisms become exhausted and ‘ ‘physiological death” (Hertwig’s term) follows, unless some stimulus is given to renew the vitality of the protoplasm. In such a condition, Tillina shows but a slight degree of sensitiveness in its response to the treatment with beef and potassium phosphate. Death is averted and the organism is enabled to hold its own during the period of low vital- ity. Oxytricha, in a similar period of vegetative exhaustion, responds to a greater degree to stimulation, not only is death pre- 414 Loutse Hoyt Gregory vented but the vegetative elements again become active, and a com- paratively high division rate follows, though not for a continued period. Paramecium aurelia shows a still higher degree of sen- sitiveness of the protoplasm. As a result of treatment with salts, the protoplasm renewed its activities, a high division rate followed, and this condition continued through a period of six months. Such a marked response to stimulation brings us close tothe facts of artificial parthenogenesis. The unfertilized egg may be con- sidered to be in a state of physiological depression, its vegetative activities are undeveloped and unless some stimulus is given, it will die. Experiments have shown that through treatment with salts, the egg renews its activities, divides, and development follows as if normal fertilization had taken place. Thus, if an unfertilized egg is stimulated artificially to develop, the term artificial partheno- genesis is applied. If a protozoan is artificially stimulated to renew its weakened activities, the term artificial rejuvenation is used. Both terms apply to different degrees of the same pro- toplasmic reaction, and are relative only. Artificial rejuvenation must be applied to the condition found in Tillina as well as to those of Oxytricha and Paramecium. The term cycle, likewise, is relative only. If we can speak of but one in the life history so far known in JTillina, why should we speak of more than one in the history of Paramecium or Oxytricha where the difference in the vitality of the protoplasm is one of degree only? Enough consideration has not been taken of the fact that not only does each individual vary in its degree of sensitiveness at different periods in the life history, suggested by Towle, and shown by the rhythms of Woodruff, but each individual of the same species as well as of different species has its own peculiar protoplasmic reactions. Woodruff himself, has failed to consider this fact in his last paper on the effects of a varied environment on Paramecium. He has carried a culture of Paramecium for a year on a medium that has been constantly changed, and so far he finds no indication of any marked periods of weakness such as Calkins found appearing at fairly regular intervals in those forms kept on a constant hay infu- sion diet. From this Woodruff concludes that the unchanged diet was abnormal, and caused the periods of low vitality which The Life History of Tillina Magna 415 have been prevented in some way from appearing in the proto- plasm of individuals kept onavaried medium. He can not logically compare his results with those of Calkins for he is not dealing with the same protoplasm, and unfortunately he carried no control series on a hay infusion diet. As a result, although his own con- clusions are not thoroughly established, he has given added proof of the individuality of the protoplasmic reactions of Paramecium. Thus we have all gradations in the response of protoplasm in a state of vegetative exhaustion to an artificial stimulus. The facts of the weak response of the slightly sensitive protoplasm of Tillina stand at one extreme, and the facts of artificial partheno- genesis involving an extremely sensitive protoplasmic condition, at the other extreme. he terms that have been applied to one set of facts must be applied alike to all and as a result they can have no definite meaning. Above all, the facts of the varying de- grees of sensitiveness of the protoplasm of individuals of the Same species as well as of different species must be kept in mind in interpreting changes that take place during the life history of an organism. T he “ Kernplasma”’ Relationship T heory. In 1903, Hertwig stated his theory of the “Kernplasma’’ rela- tion, which, briefly, is as follows: In a normal condition there is an established balance between nuclear and cytoplasmic mass, brought about by a continual interchange of nuclear and cyto- plasmic material. ‘This balance is unstable, and under certain conditions, such as starvation, overfeeding, or change in tem- perature, it is lost, and there arises an excess of nuclear or of cyto- plasmic material as the case may be. As a result of this abnor- mality, the cell is unable to carry on its ordinary metabolic activi- ties. Finally it falls into a state of depression, which will ulti- mately result in death unless certain regulatory processes take place, which will restore the normal size relations. This normal condition may be brought about by a self- -regulatory process of the cell itself, i in which the enlarged nucleus gives up some of its excess material to the cytoplasm, or vice versa; or the normal relations may be restored by the introduction of a foreign element 416 Louise Hoyt Gregory through conjugation. This is the only means of recovery from ~ deep depressions. ‘Thus conjugation is a regulatory process to — bring about the normal relations between the nucleus and cyto- — plasm. Upon this theory, Hertwig has founded his theory of the ~ origin of sex cells and the determination of sex. Thus Hertwig believes that an excess of nuclear material is the cause of the ; periods of depression; that conjugation is the means of relieving this depression, and that the conjugating cell is equivalent to a — depression cell. i In a recent paper, Popoff (’07), a student of Hertwig, reaches | the following conclusions: A culture of Stylonychia, kept from April 1 to July 16, showed periods of high vitality, which alternated with periods of low vital- — ity, or depression periods. ‘These latter periods were accompa- — nied by a cessation of the ordinary life processes, also by morpho- — logical changes. These morphological changes included a great reduction of the ~ body size, from 360-320 to go—200y and also a correspondingly ~ large increase in the size of the macro-nucleus. ‘This change in the size relations he considers the cause of the depression periods. As the depression periods became more and more serious, fewer individuals were able to rally by a self-regulatory process, which took place by a fragmentation of the nucleus, or by a direct expul- sion of nuclear material into the cytoplasm. The tendency to conjugate is found only in deepest depression — periods, and is the means of restoration to normal conditions. Popoff has made his curve of the general vitality from daily records of the division of ten individuals, and finds that in the — life history of three months and a half, five periods of depression 5 appear, the first and second a month apart and the last three two weeks apart. Changes in food or temperature often cause fluctu-— ations in the daily records and a curve made from such records : is hardly as reliable as one made from the records averaged for as longer period. If the curve of Stylonychia 1 is plotted from average — 2 records of five or ten day periods, it will be found to correspond to the curves of Paramecium, Oxytricha and Tillina, each showing — the rhythmic periods of high and low vitality. (See Diagram V.) a a gel a be: Lia ‘yodog jo spotiod ,, uorssardap daap,, ayy are gi ‘fr ‘or ‘b spouag *spoued Aep-2ay 10J posessavr uoIstAIp Jo ajey “yodog jo spiode1 oy} wory poyord st aAmnd ayy, ‘*eryDAuOTAIG Jo Ar10jsty aa7duI0D A Wvuoviq 418 Louise Hoyt Gregory The first four of the depression periods of Popoff (e.g., periods 4, 10, 13, 16) are merely the periodic falls in the division rate that occur in the normal rhythms, and from which recovery is autono- mous. Such periods are not to be confounded with the serious periods of low vitality, which end in the death of all, unless some external influence, such as artificial stimulation or conjugation takes place. Calkins found in his study of Paramecium that conjugation took place during the period of maturity, after a number of divi- sions had been passed, and before a decrease in vitality had begun. The cell at this time of maturity is recognized by a certain “mis- cible”’ quality of the protoplasm which is characteristic of many conjugating cells. This cell, however, is not a degenerate cell, but a mature cell in a physiological condition such that unless it is stimulated by conjugation or artificially by chemical or mechan- ical means, it may degenerate and die. Popoff states that there is a great decrease in size during the periods of low vitality. He states that the size varies from 360 to gov, but he gives no definite data for definite periods, and no data regarding the great increase in the size of the macro-nucleus, or the change in the ratio between the size of the nucleus and that of the cell body. With the view of investigating the relation of nucleus and cyto- plasm to the general vitality, | have made measurements of all the material which I have preserved, obtaining the length and breadth of both nucleus and cell in every case. Unfortunately the accurate dates of the early material were not kept, and could not be used. Material was found, however, which was taken from 20 ten-day periods. ‘The ratio for each individual was computed and the average made from the ratios of all the individuals of that period. An example may make the method more clear. One of the individuals of the 26th period measured 120» in length (=L) and 7oy in breadth (=W), and the nucleus measured 45 in length (=1) and 20 in breadth (=w). The ratio was written as follows: L:W:: 1: w= +" x = =.701 the 2 7O The Life History of Tillina Magna 419 coefhcient or resultant. Eighteen individuals belonged to this period, and the eighteen results were averaged and found to be 825. Acurve was then made of the resultants of the twenty periods obtained in a similar manner. If the nucleus (1 : w) increases in size, the resultant is correspondingly raised; if the nucleus becomes smaller the resultant is decreased, therefore in periods of lowered vitality, when, according to Hertwig and Popoff the nucleus is supposed to have increased greatly at the expense of the cytoplasm, we should expect to find the resultant increased and the curve varying in the opposite direction from that of the general vitality. The two curves in Diagram VI show that the duceasien of the resultants vary sometimes in the same direction, and sometimes in the opposite direction from those of the general vitality. Dur- ing the periods 25-31 inclusive, the curve of the resultants is seen to follow that of the division rate, and the actual measurements show that when the body enlarges, the nucleus increases also in size. Usually if the length of the body is longer than the average the length of the nucleus has also increased, and the relationship is the same. During the 26th period, one of fairly high activity, in which the number of divisions per day was 1.6 (16 in ten days), the average length and breadth of eighteen individuals was 1324 and 79, of the nucleus, 54 and 25. During a period of slightly less activity, the 36th, when the division averaged 1.3 per day, the average length of six individuals was 126y, the breadth 77», the length of the nucleus 44, the breadth 29. In this case the size of both the cell and nucleus was diminished. Again in the 37th period, when the number of the Mises had been reduced almost one-half, the average length of the cell was 1481, the breadth g6y, the length of the nucleus 40, the breadth 364. In this case we see that both the nuclear and the cell size increased, in spite of the fact that the vitality of the protoplasm was diminishing. Finally, in the 38th period, during which the records show almost no division, the beef lines averaging .3 divi- sions in ten days, the cell body and nucleus enlarged, the length and breadth of the cell body being 183 and 82», and that of the nucleus 564 and 45. ‘The largest individual found belonged to his period, measuring 200% by rgom, and the nucleus measuring i] ‘spotsad yaS pure pzt ay 10J a[quyiear 49M Sp10I01 ON ‘spolsad Kep-uay Suipuodsais09 ay) JO sTENpPIArpur wos pouteiqo “M2 ] = ALi T ONT oyy Jo SiuP ~4pnsor podeIoAr ayy Wor; opeur 9AIND 9Y} ST VUTT ua 01g eU1L ‘sporod Avp-ua} Ud9}XIS suunp oangyns q oy) jo AyyeyA [eroued 94} Se}eoTpul oul snonuyuos oy 7, IA Wvuovid % The Life History of Tillina Magna 421 100” by 50u. ‘These facts seem to prove that there is no relation between the amount of nuclear material in the cell, and the general vitality of the protoplasm. In other words, the periods of weak- ness are not caused by an excess of nuclear material. “The nucleus. may or may not increase in size during periods of low activity; if an increase does take place, it is generally found that the cyto- plasmic material has increased also, and the ratio between the two is the same as in the periods of high activity. Formerly it was thought that as age advances the cells diminish in size. Wood- ruff, however, found that there is no diminution in size until just before death, the shrinkage then being normal, since the metabolic functions had practically ceased. I have found a similar condi- tion in the protoplasm of Tillina. ‘The size averages of nucleus and cytoplasm are practically the same throughout the life history. Finally, in order to make my points clear, I will summarize the chief differences between the results of Popoff and my own. If the curve of Stylonychia made from Popoff’s data is plotted in the same manner as that of Jillina, it will be found that four out of the five so-called depression periods resolve themselves into normal rhythmic fluctuations, from which recovery takes place without external influence, and Hertwig and Popoff are quite wrong in considering them “depression periods” in Calkins’ sense. he fifth period which marks the end of the cycle may or may not have been a true period of depression, as all died, not being stimulated in any way. Actual measurements of Tillina, show that during normal fluc- tuating periods, the so-called “depression periods” of Popoff, as well as in the actual periods of weakness, there is but little and no regular change in the size relations of nucleus and cytoplasm. The curve plotted from the resultants obtained by averaging the ratios of cell area to nuclear area, shows no definite relation to the curve of vitality as would be expected on the theory of Hert- wig and Popoff. The size of Tillina has been found to be practically the same at the end of the life history as at the beginning. The work of Maupas and Calkins has shown that conjugation does not take place during the depression periods, but prior to 422 Louise Hoyt Gregory this and at a “‘period of maturity,” a fact indicating that the con- jugating cell must not be considered a cell in a state of depression. Enriques, in a recent paper, has criticised the methods and re- sults of Calkins and Popoff. He is strongly opposed to any theory of physiological and germinal death, and of senile degeneration. He considers all periods of low vitality to be caused by changes in temperature, action of bacteria, or irregularity in giving fresh food medium. In other words, the results of Maupas, Hertwig, Calkins, Popoff and others, he regards as due to poor culture methods. For the most part, Enriques’ criticism has been made with an incomplete understanding of the methods used in these experi- ments. Popoff, it is true, has given but a meager account of his methods. One Stylonychia mytilus was isolated in a watch glass. Colpidium was used as food. His methods of preparing and oe ing the food are as follows: “Diese Beobachtungen machte ich gelegentlich meiner experi- mentellen Untersuchungen uber das Verhaltnis zwischen Kern- und Plasmagrésse bei der Teilung von Stylonychia mytilus bei verschiedenen Temperaturen. Genaueres uber die in dieser Richtung gewonnenen Resultate werde ich demnachst mitteilen. Dieses holotriche Infusor ist leicht immer in grossen Mengen zu haben, indem man Blatter von Kopfsalat in ein grosseres Glas mit Wasser bringt. Dieselben miissen gut gewaschen sein, um die anhaftenden Cysten moglichst zu entfernen. 2 oder 3 Tage spater, nachdem eine schwache Faulnis in dem Glase sich entwickelt hat, bringt man einige Colpidien in die Kultur hinein. Dies geniigt, dass nach weiteren 3-4 Tagen die Kultur von Col- pidien wimmelt. Man muss immer darauf achten, dass die Sty- lonychien eine solche Nahrung nicht vertragen. Man giesst am besten jede 2 Tage die Halfte von dem Wasser der Futterkultur ab, fullt frisches Brunnenwasser nach und bringt wieder dazu einige frische Salatblatter. Die den Stylonychien zugefuhrte Nahrung muss in kleinen Portionen sorgfaltig mit enier starken Lupe durchmustert werden, damit man versichert ist, dass keine anderen Infusorien sich darin befinden. Wird zufallig die Fut- terkultur durch Oxytrichen oder andere Raubinfusorien verun- The Life History of Tillina Magna 423 reinigt, so ist sie nicht mehr brauchbar. Das wasser und die Nahrung der Stylonychienkukur muss unbedingt jeden Tag griindlich gewechselt werden.”’ Enriques makes the following criticism of these methods: “Die Flissigkeit ist jeden Tag substituiert, mit Kopfsalatinfus, wo viele Colpidium leben; es scheint aber, dass er die kleinen Kulturglaser nicht wechselte; das ist eine sehr wichtige Vorsicht, da die Fliissigkeit die der Glasoberflache anhangt, oft zu reich an Bakterien ist, so dass es nicht genugt, die Flussigkeit zu wechseln. Ein Kopfsalatinfus ist kein konstantes Nahrungs- mittel, auch wenn es immer eine bestimmte Zeit vor dem Gebrauch prapariert wird; sonst ist auch die Quantitat der Nahrungsflussig- keit nicht konstant, die den Infusorien gegeben wird. Die Tem- peratur war natirlich nicht konstant. Is folgt von diesen ‘Tat- sachen, dass die Stylonichien sich mit einer unregelmassigen Frequenz teilen missen; das ware nur verhindert, wenn die In- fusorien den oben citierten Einflissen gegeniiber nicht so em- pfindlich waren, wie es zu bekannt ist, um es noch zu betonen. Wir koénnen nicht genau die Zahl der Generationen seiner Versuche berechnen, weil wenn einen ‘Tag 10 Stylonichien vor- handen, und spater z. B. 15 gefunden, und diese auf 10 wieder reduziert sind, man nicht wissen kann, ob die bleibenden die- selben sind, wie friher, oder TVochterindividuen.”’ Popoff does not state whether the watch glass was changed at the same time as the medium. If this were not done there would be the possibility of bacterial growth. It is to be supposed, however, that this precaution wastaken. Experiments have shown that slight changes in temperature have no effect, in the long run, on the growth of the culture. Popoff states that the temperature varied but 2° during the entire period. Popoff’s method of reduc- ing the number of individuals to ten each day is not accurate for daily records. The average for longer periods, however, would be the same whether the number was reduced to ten or one. Enr:ques says in regard to Calkins method: “Bei Calkins Versuchen sind die Paramacien, ohne experi- mentelle Griinde den Bakterien gegenuber als unempfindlich betrachtet; Calk'ns meint, dass man die Kulturen nur von Zeit 424 Louise Hoyt Gregory zur Zeit durchsehen brauche, was von seinen Tabellen klar ge- macht ist; es ist aber auch klar gemacht, dass genau diejenigen Male, da die Kulturen fiir mehrere Tage sich tberlassen sind. Depressionen erscheinen.”’ This criticism is based upon a complete misunderstanding of the facts and methods. Calkins says in his studies on the Life History of Protozoa I: “‘In my experiments one individual is isolated every day or every two days . . . . Fresh culture medium is used at every isolation, and a single specimen is trans- ferred to it with as little of the old mediumas possible It does no great harm to leave the culture for a longer period ae twenty-four or forty-eight hours. The bacterial growth is not detrimental to the Paramecium. ‘The rate of division is, however, slightly reduced on the third day, and very much reduced on the fourth, while the turbidity becomes less and less. If no fresh infusion is added to the slide, division stops altogether, and symp- toms of starvation become evident in the Infusoria.”’ Thus it is not the presence but the absence of bacteria that causes a slower division rate. Periods of low vitality occurred at regular intervals regardless of whether the culture had been examined and changed every day, or every two or three days. Great pre- caution was taken in all of the experiments to prevent contamina- tion of any kind. “The more apparatus used the greater the danger of injuring the cultures by deleterious foreign matter such as alcohol, acids, other Protozoa, etc. To avoid untold accidents, I am accustomed to wipe dry the slides, cover glasses and cover glass supports, using a clean cloth which is used for no other pur- pose. [he same care is taken with the pipettes . . I take particular care of the one used for transferring the indpadeed Paramecium from one slide to another.”’ Enriques may have had some ground for criticising Popoff’s methods, and is right in saying “Dass die Versuche von Popoff keine neue Basis ia die Wdeicranesaieone gebracht haben.” The criticism of Calkins’ work, however, is based on a false inter- — pretation of the facts, and as a result, is of little value. The true nature of the relationship between the nucleus and the cytoplasm is still an open question. ‘The results of Gruber, Nuss- The Life History of Tillina Magna 425 baum, Boveri and others have established long ago, the fact that growth and differentiation cannot take place without the presence of both materials. ‘There has been some evidence that the nucleus actually gives up some of its chromatic material to Protozoa, also in the maturation processes of the egg. Lillie (02) finds somewhat similar evidence in the developing egg of Chztopterus. In the preparation for the division into two cells, there is a definite flowing of nuclear material into the cytoplasm, to become the granules of the endoplasm. Also in eggs differentiating without cleavage, he finds a definite relation between the microsomes of the cyto- plasm and the chromatin of the nucleus, the one originating from the other. ‘These are eggs in an abnormal condition, yet it is of value to find the same processes taking place under forced condi- tions as in the natural development. Evenin Tillina, two instances were found where there seemed to be a breaking down of the nuclear membrane at one point, allowing the nuclear material to mingle with the cytoplasm. ‘The individuals were not in an abnor- mal condition, on the contrary, they were taken at a time of rela- tively high activity, a condition not to be explained by the theory that the nucleus had become too large in relation to the size of the cell, and was regulating itself by this means. Boveri (05) offers what might be considered evidence for Hert- wig’s theory, namely, that in his experimental studies on larve, he finds the cell volume to be proportional to the number of chro- mosomes, and the number of cells proportional to the chromosomal mass, thus the size of the nucleus would seem to determine that of the cell. He adds, however, that a certain quality as well as quantity of nuclear material is needed to bring about the most favorable results. Minot in his recent book “Age, Growth and Death” has ad- vanced a theory somewhat similar to that of Hertwig. In brief, he believes that the segmentation of the ovum is a process of rejuv- enation, that is, the ovum must be considered an old cell with an excess of protoplasmic material. In order to regain the proper balance between the nuclear and cell size, segmentation takes place, young cells are produced and the nuclear material is in- creased at the expense of the protoplasm. When this process 426 Louise Hoyt Gregory ceases there is an excess of nuclear material, and the protoplasm then begins to grow and to become differentiated. This is sen- escence, which, according to Minot, begins in the two-cell stage. In other words, rejuvenation implies the increase of nuclear mate- rial, senescence the increase of protoplasmic material, both proc- esses being due to changes in the size relations of nucleus and cytoplasm. ‘These processes take place especially in the growth and development of the Metazoa. He has little faith in the view that these same processes take place in the Protozoa, and does not accept as final the results of Maupas and Calkins on the degener- ation of the protoplasm and the appearance of old age in the life cycle of a protozoan individual. Begging the question, he de- mands proof of an excess of protoplasmic material in the cells which are in a condition of lowered vitality before he will accept the view of senescence in Protozoa. Hertwig, on the other hand, would have an excess of nuclear material in the cells in a weakened condition. Both investigators are concerned with size relations only, and have failed to recognize the importance of the constant changes taking place in the quality of the nuclear and protoplas- mic material. ‘There is certainly a possibility, if not a probability, that they both have confused effect with cause. In the ordinary metabolic processes of digestion, assimilation, etc., physiological changes are constantly taking place which affect the nature of the nucleus and cytoplasm. If these processes are disturbed in any way, there is, as a result, a detrimental effect upon the character of the protoplasm, and certain morphological changes set in. Calkins has shown that at certain periods in the history of Paramecium, the activities connected with the ordinary digestive functions of the cell were affected, and as a result, the macro-nucleus became more dense, and the endoplasm crowded with undigested food particles. This condition would end in physiological death unless salts were given which would stimulate the processes of digestion and enable the cell to resume the normal conditions of growth and division. Again, at a later period, a more serious depression occurred, in which the ordinary digestive functions were not affected, the endoplasm and macro-nucleus being normal, on the other hand the cortical plasm and the micro- The Life History of Tillina Magna 427 nucleus were abnormal and germinal death followed. In neither case of depression was the size of the nucleus abnormal in its rela- tion to the size of the cell body. Because of these facts, and of those resulting from actual measurements of Tillina, it seems more probable that if the nucleus is greatly enlarged as in the case of Stylonychia, this must be explained as -the result, rather than the cause of the depression, and the definite increase in proto- plasmic material which Minot found, is the result of senescence not the cause. In other words, the evidence at the present time seems to indicate that the morphological changes taking place in the cell are due to physiological changes in the metabolic action of the cell. IX SUMMARY 1 Tillina magna is a ciliated infusorian, belonging to the family Chiliferidz, suborder Trichostomina, order Heterotrichida. Wild material was found but once only in an infusion of horse manure. All attempts to find more material were unsuccessful and the possibility arises that the organism is an intestinal para- site of the horse. 2 The size varies from 100-200» in length, and from 70-1804 in breadth. 3 The organism is recognized easily by the presence of the characteristic dorsal posterior lobe, a portion of which extends as a tongue into the peristomial region, finally disappearing in the floor of the cesophagus. ‘The entire structure is covered with long fine cilia. 4. The surface of the body is covered with striations which indicate the insertion of the cilia. The cilia have their origin in the basal bodies which lie in the cortical plasm at the corners of the raised fields into which the surface of the body is divided. 5 The nuclear structure consists of a large macro-nucleus and a varying number of very small micro-nuclei which lie at the edge of or embedded in the macro-nucleus. 6 Reproduction in Tillina takes place by the formation of division cysts, within which the protoplasm divides to form two 428 Louise Hoyt Gregory or four individuals. ‘This process must be considered an inter- mediate stage between ordinary division and true sporulation. The formation of two individuals only within the cyst has been found to be an indication of low vitality of the protoplasm. 7 Permanent cysts are formed under certain conditions, such as unusual heat or cold, or lack of food. Experiment shows there is no general rule for the recovery of the normal condition after the organism has formed a permanent cyst. Often the same abrupt changes that caused encystment will bring about the free living condition. 8 Experiments on regeneration also show that Tillina possesses a high order of regenerative power. In twenty-four hours an anterior or a posterior half of the body will regenerate its lost half, and divide to form two daughter individuals. The few centrifuging experiments that were made demonstrate the lability of the protoplasm. Inevery case the nucleus was shifted forward, and the pigment which ordinarily was present only in the posterior lobe, was scattered throughout the protoplasm, or sent in a mass to the anterior end. g Conjugation was not observed, although all possible means to bring about favorable conditions were employed. 10 Two cultures of Tillina magna have been carried on. Cul- ture A, consisting of 210 generations, extended from November 1, 1906, to February 18, 1907; culture B extending from November 1, 1906, to December 15, 1907, having passed through 548 genera- tions. The life history of each culture is represented by a curve plotted from the averages of the four lines in each culture, and again for ten day periods. 11 The curve which represents the general vitality of the proto- plasm shows the normal rhythmic fluctuations observed by Wood- ruff. 12 The protoplasm of Tillina is not as sensitive to changes in environment as that of other forms. Treatments with K,HPO,, beef, pancreatin, calf’s brain, caused only a slight increase in the ~ division rate. Rejuvenation took place, but only to a slight degree. Since the term rejuvenation must be a relative one only, the term cycle loses its value, and we may consider the life histories of Til- lina, Paramecium and Oxytricha as composed of one cycle. The Life History of Tillina Magna 429 13. Each protozoan individual has its own characteristic degree of sensitiveness that differs from all others of the same family, as well as from all others of different species. 14 Actual measurements of nuclear and protoplasmic size, at different stages in the life history, show that there is no relation between an excess of nuclear material and a period of low vital- ity. Such periods of weakness may or may not be accompanied by changes in the size relations of the nuclear and cytoplasmic material, in no way are they caused by such variations. The true cause must be sought in the physiological not morphological changes taking place within the cell. Zoélogical Laboratory. Columbia University, January, 1909. LITERATURE BESSENBERGER, E. ’03—Ueber Infusorien aus asiatischen Anuren. Arch. f. Pro- tistenk. Boveri, TH. ’05—Zellenstudien, v. Butscuu, O. ’76—Studien iiber die ersten Entwickelungsvorgange der Eizelle der Zelltheilung, und der Conjugation der Infusorien. Abh. d. Senckenb. nat. Gesellsch. Frankfurt a.M., x. *83—Protozoa. Bronn’s Klassen und Ordunngen des Thierreichs. *94—Protoplasm and Microscopic Foams. Carkins, G. N., ’01—The Protozoa. Columbia University Press. ’o2—Studies on the Life History of Protozoa. I. The Life Cycle of Parameecium caudatum. Arch. f. Entwickelungsmech., xv. CALKINs AND LieEB ’02—Studies on the Life History of Protozoa. II. The Effect of Stimuli on the Life Cycle of Paramcecium caudatum. Arch. f. Protistenk, i. Carkins, G. N. ’02—Studies on the Life History of Protozoa. III. The Six- hundred and Twentieth Generation of Paramcecium caudatum. Biol. Bull., ii. ’04—Studies on the Life History of Protozoa. IV. The Death of the “A” Series of Paramcecium caudatum. Conclusions. Journ. of xp: Zool i °06—The Protozoan Life Cycle. Biol. Bull., ii. 430 Louise Hoyt Gregory Conn, F. ’53—Beitrage zur Entwickelungsgeschichte der Infusorien. Zeit. f. wiss. Zool., iv. Enrigues, P. ’08—Die Conjugation und sexuelle Differenzierung der Infusorien. Archiv. fiir Protistenkunde, xii. Evarts, E. ’73—Untersuchungen an Vorticella nebulifera. Zeit. f. wiss. Zool. xxiii. GreENWwooD, M. ’94—On the Constitution and Mode of Formation of ‘Food Vacuoles”’ in Infusoria, as Illustrated by the History and Proc- esses of Digestion in Carchesium. Phil. Trans., 1894. GruBeEr, A. ’79—Neue Infusorien. Zeit. f. wiss. Zool., xxxiii. Hertwic, R. ’03—Ueber Korrelation von Zell-und Kerngrésse und ihre Bedeutung fiir die geschlectliche Differenzierung und die Teilung der Zelle. Biol. Cent., Bd., xxiii. Jouxowsky, D. ’98—Beitrage zur Frage nach den Bedingungen der Vermehrung und des Eintrittes der Konjugation bei den Ciliaten. Vehr. 5 Nat. Med. Ver. Heidelburg, xxvi. Kent ’80—A Manual of Infusoria, ii. Linu, F. R. ’92—Differentiation without Cleavage in the Egg of the Annelid Chztopterus pergamentaccus. Arch. f. Entwick. mech., xiv. *o6—Observations and Experiments concerning the elementary Phe- nomena of Embryonic Development of Chztopterus. Journ. of Exp. Zodl., iil. Marcus ’07—Ueber den Aggregatzustand der Kernmembran. Gesellschaft Morph. und Phys. Minchen. I. Mater, H. N. ’03—Uber den Feinen Bau der Wimperapparate der Infusorien. Arch. f. Protistenk, 111. Maupas, E. ’89—Le rajeunissement karyogamique chez les Cilliés, Arch. d. zodl. expér. et gén. 2me SEr., Vil. *88—Recherches expérimentales sur ]a multiplication des Infusories ciliés. Arch. d. Zool. expér. et gén. 2me sér., vi. METALNIKOFF, S. ’03—Uber die intracellulare Verdauung. Sebastopol, vi. Mincuin, E. A. ’03—The Sporozoa. ‘Treatise on Zodlogy. Lankester, i. Minot, ’o8—Age, Growth and Death. Putnam, N. Y. Peters, A. W. ’04—Metabolism and Division in the Protozoa. Proc. Amer. Acad. Arts and Sci., xxxix. Poporr, M. ’07—Depression der Protozcenzelle und der Geschlechts-zellen der Metazoen. Arch. f. Protistenk, sup., 1. Ruums-er, L. ’88—Die verschiedenen Cystenbildungen und die Entwickelungs- geschichte der holotrichen Infusoriengattung Colpoda. Zeit. f. wis. Zool., xlvi. ScHEwiakorF, '89—Beitrage zur Kenntniss der holotrichen Ciliaten. Bibliotheca Zool. Heft. 5 The Life History of Tillina Magna 431 ScHuBERG, ’o5—Ueber Cilia und Trichocysten einiger Infusorien. Arch. f. Protistenk, vi. Srein, F. *83—Der Organismus der Infusionsthiere. Leipzig. Tow te, E. W. ’o4—A Study of the Effects of Certain Stimuli, Single and Com- bined, upon Paramecium. Amer. Jour. of Physiol., xii. Wooprturr, L. L. ’o5—An Experimental Study on the Life History of Hypotrich- ous Infusoria. Journ. Exp. Zodl., i. °o8—Effects of Alcohol on the Life Cycle of Infusoria. Biol. Bull., xv. *08—The Life Cycle of Paramecium when subjected to a varied environ- ment. Am. Naturalist vol. xlii. Prate I Fig:1 Dorsal view of Tillina magna showing general external characteristics as well as the character of cortical plasm, endoplasm, and micro-nuclei lying near, or embedded in the large macro-nucleus. X 560. P Fig.2 Ventral view of Tillina magna showing especially the region of the mouth peristome and cesophagus, with the tongue-like continuation of the posterior lobe on the floor of the peristome and continued through the mouth, disappearing finally in the floor of the esophagus. X 560. THE LIFE HISTORY OF TILLINA MAGNA PLATE I Louise Hoyt Grecory THE JourNAL or ExPERIMENTAL ZOOLOGY, VOL. VI, NO. 3 Pirate II Photographs showing different stages in the process of encystment for division. Figs. 1, 2, 3 were taken with the same magnification. Fig. 4 was taken with a higher magnification. ; Fig. 1 A section through an individual encysted for division. The macro-nucleus has elongated and has become constricted. The first division plane is shown appearing at right angles to the long axis of the nucleus. Food vacuoles in different stages of digestion are shown. X 450. Fig.2 A section showing the beginning of the second division plane. X 450. Fig. 3 Section through an encysted form which has divided twice to form four individuals. Portions of the four individuals are seen. The nucleus shows in but two. X 450. Fig. 4 Section through an encysted form which has divided twice. Three of the four individuals show. The beginnings of the ciliated mouth regions and the nuclei are visible. X 530. THE LIFE HISTORY OF TILLINA MAGNA PLATE II Louise Hoyt Grecory ) f ‘ WrGeag _ Tue Journat or ExPeriMENTAL ZOOLOGY, VOL. VI, NO. 3 ' SLUDIES OF TISSUE GROWTH II FUNCTIONAL ACTIVITY, FORM REGULATION, LEVEL OF THE CUT, AND DEGREE OF INJURY AS FACTORS IN DETER- MINING THE RATE OF REGENERATION. THE REACTION OF REGENERATING TISSUE ON THE OLD BODY BY CHARLES R. STOCKARD Pathological Laboratory, Cornell University Medical College, New York City Wirn One Pirate anv Ercut Ficures 1n THE Text PMR ea HERAT CULO Uy tater aorar ete ayevetetars oleae soy stcro) ecieicteveTaTar el foie) fejcisveie/ois tale) ii skoieis/e ore/e)isyelererevelsrare*otatel ste 433 Meee ttcerra lean MethOcSes < cvs.ccicists sleveisieis o's eis o.cis disis s/s alelerelere o)sVieierelale Soiaibociersiee 010% 434 II The influence of activity and rest on the rate of regeneration ..............eseeeeeeeees 435 IV Form regulation and arrest of regenerative activity in pieces of the medusa disk .......... 38 iV) Relation of the levellof the cut tothe rate of regeneration...........0...0scecceseceeese 442 VI Relation of the degree of injury to the rate of regeneration...............-2eeeeeeeeeee 446 VII The relation between the rate and amount of regeneration and the physical condition at? (ae euiiiell lyaih re éaas otemtioone sae reOaceni eo TOoc Soe nen SUAS a ano er oernr 462 SoU SEIEENTEI ATS RATIC) (COT.CLUSTOUS |= wveicy oy eje/eus.eie. = eissei (912, 0y010,41 «/~) 2:6 4°%.0/e spe invs a a oreiersre o.eisivieie sieissee 466 PME ALIICSICICE yercrer- afetsfasat cre ieesva ee’ 'eie) svetascaieinye)aine arevete ) scare sysisue: cae ei eceierar a atacavareuels slot @ 468 I INTRODUCTION The present contribution is the second of a series of studies aiming towards an analysis of the factors controlling the rates and limits of tissue growth. The problem is complex, yet the investigator has full access to experimental methods in consider- ing the processes of regenerative growth. Factors regulating regeneration are probably identical with those determining pri- Mary or generative growth and if the former could be identified and controlled it might become possible on the one hand to main- tain some forms of growth indefinitely or on the other to suppress certain excessive growths of a pathological nature. Tue JouRNAL or ZooLoGy, VoL. vi, No. 3: 434 Charles R. Stockard My (08) previous study on regeneration in the Scyphomedusa, Cassiopea xamachana, considered the influences of certain in- ternal factors on the rate of regenerative growth. The present account is also concerned with the rate of regeneration in Cas- siopea and in addition includes a study of two species of brittle stars, Ophiocoma riisei and Ophiocoma echinata. The level at which the cut is made determines the rate of the ensuing regeneration in the three animals employed. The nearer to the center of the body the tissue is removed the more rapidly will the regenerating tissue grow. An arm regenerates at a faster rate when amputated near its base than when amputated at a more distal point. The growth of the new tissue gradually be- comes slower, however, as the original size is approached. An analogy is found in embryonic growth, the smaller embryo in- creases proportionately much more rapidly in size than does the larger and older embryo. ‘The rate of increase becomes contin- ually slower as the adult size is approached and finally growth ceases at this limit. The relation between the extent of injury and the rate of regen- eration will be considered in each of the three species. Finally, the evidence furnished by animals regenerating different amounts of tissue will be reviewed in order to ascertain the nature of the influence exerted by the new growing tissue over the old body substances. The experimental part of the investigation was conducted in the Biological Station of the Carnegie Institution at Tortugas, Fla. I wish to express my thanks to the Director, Dr. Alfred G. Mayer, for the facilities supplied for this work and for his kindness in preserving the ophiurans after I had gone from the laboratory. The remainder of the work has been done in the Pathological Laboratories of Cornell University Medical College for the Hunt- ington Fund for Cancer Research. II MATERIAL AND METHODS ‘The medusz are abundant in the somewhat stagnant water of the moat surrounding the old fort at Dry Tortugas Islands. This by the originally equal diameters. I have calculated the specific — amounts on the basis of final diameters merely to show the erro- — neous impression obtained by using such a method. —— a es Studies of T1ssue Growth 459 The upper half of Table [IX summarizes the data from O. riisei and presents the following points of interest. First, individuals of Ophiocoma ritset when kept under identical conditions increase in body size the slower the more arms the individual 1s regenerating. This fact is not likely due to the incapacity of the more injured specimens to secure food since the compartments in which all were confined are small and the individuals seemed equally able to traverse this limited feeding ground. A probable explanation is that the new regenerating tissue possesses an excessive capacity for the assimilation of nourishment and consequently those speci- mens regenerating more new arms were less able to increase in body size than those regenerating fewer. Table IX shows secondly, that the rate of regeneration of each arm bears no relation to the number of regenerating arms, or in other words, the extent of injury. Column five gives the specific amounts of regeneration for each arm when calculated by dividing the average arm lengths by the final disk diameters. ‘The last two figures in the column indi- cate the error of such calculation. Calculating the specific amounts of regeneration on the basis of the original average diam- eters which were practically equal in all the groups we obtain a series of numbers bearing the same relations to one another as are shown by the numbers in column four. Ophiocoma echinata, a spiny, grayish, mottled brittle-star, was experimented upon in exactly the same fashion as Ophiocoma rusei. Its response to different degrees of injury was much more pronounced than that of the species riisei. Again five groups of individuals of the same average size were selected and operated upon so as to remove different numbers of arms I cm. from their bases at the disk. Referring again to Table IX the lower half represents a tabulated summary of the data from Ophiocoma echinata. ‘Two facts of importance are here also to be recognized. First, the fourth column giving the average arm lengths for each group shows that the rate of regeneration decreases as the extent of injury increases. Each new arm grows fastest from those individuals regenerating a single arm and successively slower in the groups growing two, 460 Charles R. Stockard three, four and five new arms. These differences in rates are significant when compared with their probable errors. Secondly, it is to be noted that the average of final disk diameters in Ophiocoma echinata is practically equal in all of the groups. This fact might be reconciled with the smaller increase in disk diameters shown in the more injured groups of Ophiocoma riisei, TABLE Ix Tabulated summary showing the rates of regeneration in brittle-stars when regenerating different numbers of arms O phiocoma riisei | AVERAGE | No. OF [FINAL DISK) No. OF | LENGTH OF nce ieee = DIAMETER — RE>) AEM-BUDE|| 2s aunt MENS IN MM. | MOVED. | AFTER 49 | DAYS, IN MM. fe aes 2 SR Ae 42.9 2.340.104 ais = «ielsictsicielalaiele\aela\elolere 539 IX Review of the effects of centrifugal force upon developing eggs..........2+.0eeeeeeeeeeeee 540 Mee WNEICIStTa EO Of Eel P PICONLEMESE (oteisteor-\e0e\e1-:)ciciri-i-/s s{eiele's)sis/e/etolejsi= s/ofelaisle/eie) leiel-ielere 540 2 The restitution of the egg substances after centrifuging...............0ceeeeeeeeeeee 546 ppebnedeeortneere whenlcentritin ped sjerey=icels -ela'21 te -\eiev=(ele lale’s (ols ajstelolelalels »\clelelsl=lavsrerata 547 PMP UTE TALC Ot CEVelO PUACLIEs a ciciei- + o\c:syeicinsassis sis spoleieictaswtere\s wis e)eldle Ziureje ereleivie lore leis/eletaga oveys 547 Reb eesicentritu pedi DelOre de pOSItlOs o)ac.\e)-ve o/o\s)s « \sieheisie)» «\ je) #1 s¥+[e)=)0)e/61#/ eleiojaisiejaieleiaisi=ia\=\oie 548 Ul Ginn: 204.05 00000 SoC bo Ca poe Abe SS SHO O Go abO er anveeaetipeba ne copease bodidund dD 548 BPM PRIECE AUK Ger ealate 1 valores ovcta.clsieig.t1s(e)s)2i0;8le;s\a/ere 6: 4.5%0\e = e/cie"s,alove duals dice esa stee. a ajelstayeia'e sisiiaye'gs eve 550 I INTRODUCTION It has been found that insect eggs are definitely oriented within the ovaries and the exact position of the future embryo seems to be determined -already at this early period. This fact has led many embryologists to believe that the eggs of insects are very highly organized. If this is true a redistribution of the contents of the egg would have a profound effect upon the development of the embryo. In order to obtain a rearrangement of material a centrifugal machine was used successfully, as is shown’ by the experiments described in Part VII of this paper. 1 Contributions from the Zodlogical Laboratory of the University of Michigan. No. 125. Tue Journat or ExperimeNTAL ZoOLOGY, VOL. VI, NO» 4. 508 R. W. Hegner During the course of the study of the germ-cells in some chryso- melid beetles? a disc-shaped mass of granules (Fig. 9, g. c. d) was discovered in the freshly laid eggs suspended in the peripheral layer of cytoplasm at the posterior end. I have called this struc- ture the “‘pole-disc.” ‘These granules are taken up by the germ- cells in the course of their migration and apparently determine the character of these cells; on this account I have called them “germ- cell determinants” (Hegner ’o8 )). It was hoped by means of centrifugal force to scatter the granules of the pole-disc and obtain an embryo either without germ-cells or with germ-cells in various parts of the body. It was also thought possible that the pole-dise might move as a whole and, becoming massed in some other region of the egg, might influence at this point cells which would ordinarily become body-cells. As will be seen later some data were secured but not enough to warrant any definite conclusions. So far as I have been able to learn from the literature no experi- ments with centrifugal force upon the eggs of insects have ever been performed successfully and in only one case has any arthropod egg been tested in a centrifugal machine (Lyon ’o7). Lyon merely says: “The ovarian eggs of the common garden spider could be separated by one minute’s centrifugalizing into two layers”’ (p. 169). The experiments described below were begun at the University of Wisconsin in the spring of 1908 and were continued at the Marine Biological Laboratory at Woods Holl, Mass., where I occupied a room subscribed for by the Wistar Institute of Anatomy and Biology. ‘The material was further studied at the Zoological Laboratory of the University of Michigan. II MATERIAL AND METHODS During the course of this work eggs of the following beetles were used for experiments: Calligrapha multipunctata, C. bigsbyana, C. lunata, Leptinotarsa decemlineata and Lema trilineata. ‘The posterior ends of these eggs are fastened to the leaf on which they 2 The Origin and Early History of the Germ-Cells in Some Chrysomelid Beetles. Accepted for publication by the Journal of Morphology. Centrifugal Force upon Beetles’ Eggs 509 are laid. In the case of Calligrapha multipunctata and C. bigs- byana the eggs can be definitely oriented as is explained in the next part of this paper. A number of beetles were kept in the laboratory and the eggs were marked on the anterior-ventral sur- face with a small spot of waterproof india ink. ‘The exact time of deposition was recorded in all cases. The eggs are not always in the same stage of development at the time of laying, but all those in one batch are approximately in the same condition. When the eggs had developed to the desired point they were placed in small indentations in a block of parafhn. ‘The entire block containing the eggs was then lowered to the bottom of a glass tube of an ordinary water-power centrifugal machine. ‘The eggs were then 15 cm. from the axis of rotation. “The number of revolutions per minute was not accurately determined, but was probably between 1500 and 2000, although in some cases (those described in experi- ments C. M. 1 and L. D. 1 and 2) a slower rate of speed was used (360 revolutions per minute). ‘The eggs when taken from the centrifugal machine were left in the cavities in the paraffin block with the heavy end down until they were fixed. In previous work I found a modification of Petrunkewitsch’s fluid the best for killing and fixing the eggs. ‘This was used entirely for the centrifuged material, although control eggs were fixed in a number of the common mixtures. Eggs were stained in toto with Mayer’s hzmalum acidulated with 2 per cent of glacial acetic acid, or with alum cochineal. Sections were stained on the slide principally with hemalum followed by Bordeaux red. One difficulty in doing experimental work with the eggs of Calli- grapha is that only a few are laid at one time (eight is the average number) and, as the conditions of the experiments frequently are responsible for the destruction of some of these, no series contains very many successive stages. There are also causes for trouble in making preparations. In some instances the eggs stuck fast to the chorion at the outer end, where the contents had been strongly driven against it; the chorion could not be removed from these without injury to that part of the egg. After eggs have been centrifuged they are more difhcult to section than before because the large deutoplasmic spheres collect 510 R. W. Hegner at the end away from the axis of rotation and a breaking out is frequent in this region. III THE ORIENTATION OF THE EGGS OF CALLIGRAPHA BIGSBYANA It has been known for more than twenty years that the eggs of insects are definitely oriented within the ovaries of the adults. Hallez in 1886, finding this to be true of the ova of Hydrophilus and Locusta, expressed the fact in his “‘ Loi de l’orientation de l’embryon chez les Insectes” as follows. ‘‘La cellule-oeuf posséde la méme orientation que l’organisme maternel qui l’a produit: elle a un pole céphalique et un pdle caudal, un coté droit et un cdté gauche, Fic.1 A diagrammatic drawing of C. bigsbyana clinging to the under side of a willow leaf and showing _ the orientation of the egg in the ovarian tubule and after deposition. : Fic. 2 Four eggs of C. bigsbyana laid intworows. a. = anterior. d. = dorsal. /. = left. p. = j posterior. r. = right. x. = anterior ventral surface where the spot of India ink was placed as a guide — for orienting the eggs during the experiments. , une face dorsale et une face ventrale; et ces différentes faces de la — cellule-oeuf coincident aux faces correspondantes de l’embryon.” No difficulty is experienced in distinguishing the anterior from the posterior end of the eggs of Chrysomelid beetles as it is always — the posterior end which first emerges from the vagina. This end is fastened to the leaf on which the egg is laid and subsequently — becomes the posterior end of the embryo, regardless of the posi- _ tion of the leaf. In only two species (Calligrapha multipunctata _ and C. bigsbyana) of the many Chrysomelid beetles examined could the right and left sides of the egg be accurately determined. The egg eine of these insects is as follows: “‘ The beetle selects Centrifugal Force upon Beetles’ Eggs Rit a leaf and clings to its under surface. ‘The tip of the abdomen moves rhythmically up and down about fifteen times at intervals of a little less than one second. ‘This results in the exudation of a drop of viscid, colorless fluid about one-third the transverse diameter of the egg. ‘The egg is forced out a moment later and carries with it this drop of fluid by means of which it is fastened to the leaf. When the egg reaches the leaf it is pushed back away from the beetle (Fig. 1), which then moves to one side and again begins the rhythmical movements which precede the laying of another egg. In this way eggs are laid in a double row as shown in the accompanying figure (Fig. 2), but frequently three or more may be laid in one row. ‘The intervals between the layings of the individual eggs average one minute and twenty seconds” (Hegner 08 a). ‘Two to nineteen eggs are laid at one time, the average number being eight. Fig. 1 “Hele ee the orientation of the egg of C. bigsbyana lying in the ovary and also the final position after it has been laid. IV THE EFFECTS OF GRAVITY UPON THE DEVELOPMENT OF THE EGGS OF INSECTS That the position of the insect egg after laying has no influence upon the development of the embryo was proved by Wheeler (1889) in the case of Blatta. This author kept capsules from fourteen to twenty days in the following positions: “tr Resting with the lateral faces perpendicular and crista uppermost. “2 Resting on the crista with the lateral faces perpendicular. “3 Resting on the left lateral face. “4 Resting perpendicularly on the anterior end. “‘s Resting perpendicularly on the posterior end. “In all these cases the eggs developed normally, without the slightest indication of displacement in position or alteration of shape in the embryo; whether they were forced to develop with their heads pointing up or down.” The conclusion reached was that “the force of gravitation has no perceptible effect on the development of the eggs of Blatta 512 R. W. Hegner Wheeler also proved that the antero-posterior differentiation of the embryo of Leptinotarsa decemlineata is not affected by changes in the position of the egg after laying, but is predetermined in the ovary. During the course of my work with Calligrapha, eggs were taken as soon as laid and placed inevery possible position. “The embryos were found to be in no way affected by the orientation of the egg with respect to gravity. The only exception to the rule that gravity has no influence upon the development of insects’ eggs seems to be that of Hydrophilus aterrimus reported by Megusar (’06). The eggs of this water beetle are laid in a boat-like cocoon which is kept in an upright position in the water by means of a peculiar mast. Megusar found that if these cocoons were inverted, thus also inverting the eggs within, the development of the eggs was retarded and a deformity in the embryos resulted. ‘The small number of larve that hatched lived for only a short time. V yo ofrquiy uy i *yuatses 0} Suruursoeq ysnf{ euRAgssiq “> Jo oAIquia UY *2A00I8 [PIJUIA dy) puke pug UIE oY] SUTMOYs PO sINOY Om}-AJIIG PULAGSSIq ‘> JO 38a Ue JO advJINS [eAJUDA aYJ JO MITA pue rorzajsod ayy ye dnosd e wrr0} syjao-w1e3 yerprownad ayy, ‘wortsodap sajye sanoy amoy-Aytaay vueAqssrq *> Jo 33a ur Jo Mata aovyANG ‘9 ¢ 9 ‘OM S “Og bony £917 514 R. W. Hegner in the peripheral layer, but collect about them a number of granules (germ-cell determinants, Fig. 17, g c d) which they encounter in this region and continue their migration until they are entirely separated from the blastoderm. ‘These are the primordial germ- cells (Fig.3, p.gc). The first change noticed in the blastodermisa crowding together of the cells on the ventral surface of the egg. This results in the formation of a broad longitudinal band of closely aggregated cells, the ventral plate. The edges of this plate are e a } J 7 8 , if . 4 - Fic. 7 Surface view of embryo described in Series C.B. 2, c. The embryo has begun to broadea and shorten. . Fic. 8 An embryo of C. bigsbyana in which the tail-fold is coincident with the posterior end of , the egg. c.ap. = cephalic appendage. p.= posterior. t.ap.= thoracic appendage. 1.f. = tail-fold. — thrown up into two folds; these spread out in the posterior region — extending to the end of the egg where they pass around the pri- mordial germ-cells and meet on the dorsal surface. The ventral plate now decreases both in length and in breadth and a longi- tudinal concavity, the ventral groove, appears. The germ-band can now be recognized; it covers the entire ventral surface of the egg except a wedge-shaped area anterior to the groove (Fig. 4, gb). Centrifugal Force upon Beetles’ Eggs 515 The germ-band becomes narrower as development advances; its posterior end pushes around that end of the egg and up on the dorsal surface. The lateral folds gradually cover over the ventral groove and the amnioserosal fold grows forward from the posterior Fic. 9 Longitudinal section through an egg of C. bigsbyana four hours after deposition. = ged. = germ-cell determinants. gn.= germ-nuclei copulating. Ahb/. = “‘Keimhautblastem.” p. = pos- terior. v.m. = vitelline membrane. y. = yolk. end to meet the anterior fold (Fig. 5). The segmentation of the germ-band and the lengthening of the entire embryo now pro- gresses rapidly. The cephalic extremity extends almost to the 516 R. W. Hegner anterior end of the egg and the tail-fold extends a little more than half-way up on the Morea surface (Fig. 6). The tail-fold now begins to recede as the embryo shortens and broadens (Fig. 7) and in a short time coincides with the posterior end of the egg. The embryo now grows laterally around the yolk (Fig. 8), its various parts being situated approximately in the positions they occupy at the end of about six days when it hatches as a larva. VI THE STRUCTURE OF THE EGG OF CALLIGRAPHA BIGSBYANA AT THE TIME OF DEPOSITION At the time of laying the eggs of Calligrapha bigsbyana are not always in the same stage of development, although usually polar body formation is taking place. The egg figured (Fig. 9) was — fixed four hours after deposition. ‘The polar bodies have already been produced in this egg and the male and female nuclei are in the act of conjugation. ‘The egg consists of a large central mass of yolk and a comparativ ely thin peripheral layer of cytoplasm, the “Keimhautblastem” of Weismann. The interdeutoplasmic spaces are filled with cytoplasm which is connected with the “Keimhautblastem” by delicate strands of the same material. The enormous amount of yolk contained in the eggs of these insects makes the identification of other substances extremely dificult. The yolk-globules range in size from large deutoplasmic spheres to small granules, and, as the dissolution of some of them — is continually taking place, one is unable to determine where yolk _ ends and cytoplasm begins. The only accumulations of cyto- plasm large enough for eee are those surrounding the ~ nuclei ae the aie mass, and the peripheral layer, the “Keim- — hautblastem.” No differences in composition or staining qualities were observed between the cytoplasm of these two regions. ‘The ‘““Keimhautblastem” consists of a fluid ground substance in which are suspended very fine granules. It is a homogeneous layer of cytoplasm everywhere except at the posterior end of the egg. At this point there is a disc-shaped mass of larger granules imbedded within the inner portion of it. These granules stain deeply with hematoxylin. They are easily seen not only in sections but also — PS ras oe Centrifugal Force upon Beetles’ Eggs KEG in eggs that have been properly stained in toto. Because of their ultimate fate, as explained in the introduction, I have called these _ granules the germ-cell determinants (Fig. 9, gc. d). Vil THE EFFECTS OF CENTRIFUGAL FORCE UPON EGGS CENTRI- FUGED AFTER DEPOSITION _ Table I presents in concise form the main points in the series of experiments which have been selected for detailed description. Besides the thirteen series noted here there are also two tables (XI and XII) which give the results of a number of other series of experiments which were not considered of sufficient importance to describe at length. TABLE I List of the experiments described in detail | Age when centri- Length of time ie) 3] oO i=] co iS) f. S) 5 Name | Number of series fuged | centrifuged C. bigsbyana C.B.4. | ° 15 min. to 4 hrs.) post. end in MG CBA ° 4 hours | ant. end in 7 s C.B. 10 ° 6 hours | side in 5 : C.B.9 ° _ 6hours | post. end in 4 ©.Bs2 14hours | 1 hour = 7 & CLG 21 hours | 2 hours ‘ C. multipunctata C.M. 1 fo) | 16 hours ie 4 C. lunata C.L. a 1 hour 12 hours So Cia ghours | 12 hours ant. end in ¢ L. decemlineata IDB a 2 hours | § min. to 24 hrs| post. end in “ fi pean 7} ° | 5 days le hls” . % L.D. 2 fo) | 7 days 7 q Lema trilineata | Ie c 2 hours | co — Series C.B. 4—Table II mr: eggs of this series were centrifuged as soon as laid. They . ere held in place with their posterior ends towards the axis of ‘rotation. “Two eggs were removed at the intervals indicated in Table II; one of these two was fixed immediately, the other was allowed to develop. If the latter did not hatch within a period ‘several days longer than the normal hatching time it was fixed. 518 R. W. Hegner TABLE II Calligrapha bigsbyana—Series C.B. 4 Interval between Number of Age when cen-Length of timeend of experi- experiment | _ trifuged centrifuged ment and fixa- BO SS tion C.B.4,a | Control C3400. | c 15 minutes fo) C.B. 4,¢ fo) 30 minutes ° C.B. 4,d ° 1 hour ° C.B. 4,e ° 2 hours ° Posterior end CBi 4. ° 4 hours ° toward axis of C.B. 4, g ° 15 minutes 6 days rotation Normal larva C.B.4,h ° 3° minutes 6 days Normal larva C.B. 4,1 ° 1 hour 10 days Did not hatch C.B.457 ° 2 hours 10 days Did not hatch C.B.4,k ° 4 hours 10 days Did not hatch The progressive effect of a centrifugal force upon the distribution of the contents of the egg is shown by these experiments. They also furnish data concerning the amount of disturbance neces- sary to prevent the hatching of a normal larva. C.B. 4, a. Sections of the fresh control egg of this series show a condition similar to that illustrated in Fig. g. C.B. 4, b. An egg centrifuged for fifteen minutes is very slightly affected. The “Keimhautblastem” has apparently not been changed at all. The yolk shows a partial redistribution; the larger, heavier globules have begun to move toward the outer end of the egg, z.e., the end away from the axis of rotation, and the inner portion of the yolk mass is almost entirely composed of the smaller globules. ‘The pole-disc occupies its normal position at the posterior end of the egg; all about it are small, irregular vesic- ular spaces which are no doubt caused by the accumulation of the lighter fats in this region. No polar bodies could be discovered in the sections of this egg, but no significance can be attached to this fact as they cannot always be found in normal eggs. C.B 4, c. The effects of centrifugal force applied to this egg for thirty minutes are similar to those just recorded for C. B. 4, 6.5 the changes however are more pronounced. We findthat there are Centrifugal Force upon Beetles’ Eggs 519 more of the large yolk-globules near the outer end and less of them at the other pole. ‘There is also a slight thickening of the ‘‘ Keim- hautblastem” at the sides of the egg near the innerend. The pole-disc is present in its usual position, but it is surrounded by a greater number and larger, irregular vesicles than in C.B. a,b. C.B. 4, d. An egg taken from the centrifugal machine at the end of an hour is definitely stratified, two distinct layers being visible. ‘There is a small cap of orange-colored material situated at the extreme inner end, while the rest of the egg representing the other layer has changed in color because of the redistribution of the yolk. ‘The intense yellow color of the outer end is due to the invasion of a vast number of large deutoplasmic spheres into that region. No definite layers can be distinguished in this large por- tion, since the change in color from bright yellow at the outer end to pale yellow at the inner end is gradual. A longitudinal section through this egg is shown in Fig. 10. Most of the large yolk- globules lie in the outer region; the interdeutoplasmic spaces are entirely free from the cytoplasm which usually fills them. ‘The “Keimhautblastem” has been forced almost entirely away from the outer end and from the periphery of the outer third of the egg and has added its mass to that of the inner region. At the extreme inner end one large, bud-like protrusion (one-third the short diameter of the egg) and several smaller ones have formed. ‘They _are covered externally by a thin layer of “ Keimhautblastem”’ and are composed of a great number of vesicles. A similar vesicular portion was noted in the two eggs described above (C.B. 4, 5 and c), but it has in this egg reached such proportions that we shall hereafter call it the vesicular zone. ‘This is the material which appeared bright orange in color in the living centrifuged egg. Only one nucleus could be discovered in the entire egg. ‘This is situated at one side near the inner end, as shown in Fig. 10, 7. The pole-disc has moved from its position at the end and has traveled en masse away from the axis of rotation. It has carried that portion of the ‘‘Keimhautblastem” in which it is suspended along with it, producing a distinct depression at one side of the inner end of the egg. ‘The sections containing the pole-disc fell outside 520 R. W. Hegner of the vesicular zone so that a figure has been introduced to show the change in position of this structure (Fig. 11, gc. d). C.B. 4, e. A third layer makes its appearance if a fresh egg is centrifuged for two hours. Before fixation this appears as a =<-----V.Z. 1—-—-— --- ------V. 14. Fic. 10 Longitudinal section through egg C.B. 4, d. Fic. 11. Longitudinal section through the posterior end of egg C.D. 4, d, showing the effects of a centrifugal force applied for one hour upon the position of the pole-disc (ge.d.). Fic. 12 Longitudinal section through the posterior end of egg C.B. 4, e, showing the effects of a centrifugal force applied for two hours upon the position of the pole-disc. Fic. 13 Longitudinal section through the posterior end of egg C.B. 4, f, showing the effects of a centrifugal applied for four hours upon the position of the pole-disc. gc.d. = germ-cell determinants (pole-disc). hb/. = ‘‘Keimhautblastem.” n.= nucleus. p. = posterior. pt. = pathway made by the outward movement of the pole-disc. v. = ventral. v.z. = vesicular zone. y. = yolk. Fic.14 Transverse section through egg C.B. 10, c. Centrifugal Force upon Beetles’ Eggs 521 colorless cap at the outer end. ‘The sections show it to consist of a small mass of gray material which is heavier than the large yolk-globules. I shall call this the gray cap. It is composed of very small granules, does not stain like yolk nor as intensely as the cytoplasm of the “Keimhautblastem.” The pole-disc has moved forward during the second hour the egg was centrifuged, and now lies anterior to its original position about one-fourth of the total length of the egg. In its progress it has pushed its way forcibly through the yolk mass, leaving a long, narrow, open path- way behind it (Fig. 12, pt). No nuclei were found in the sec- tions. C.B. 4, 7. This egg was centrifuged for four hours and then fixed. A surface view of the egg stained in toto revealed a large central, colorless bud at the posterior (inner) end surrounded by a number (at least seven) of smaller buds. These are produced by wrinkles or folds in the surface of this region, due either to poor fixation or to a decrease in turgidity at the inner end. ‘The entire egg seems to have been shortened slightly antero-posteriorly by the continued application of centrifugal force. The longitudinal sec- tions made of this egg are not perfect, certain portions of the outer end being lost because of the accumulation of large yolk-globules (yolk which is not imbedded in cytoplasm is liable to break on the knife in cutting). I cannot be positive, therefore, of the presence of a gray cap in this egg. ‘There is little doubt, however, that this structure was not absent in this instance, since the other eggs similarly treated possess a gray cap. ‘The vesicular zone has increased in size during the last two hours this egg was centrifuged, and has been folded into larger bud-like prominences than were noted in the last egg described (C.B. 4, ¢). The pole-disc has made further progress in its journey away from the inner end. It has now reached a point about one-third of the total length of the egg anterior to its original position (Fig. 13, gc. d). The open pathway which was observed behind it in C.B. 4, e, has become closed and the “ Keimhautblastem”’ that was pulled in with it has passed back and taken part in the vesicular layer. C.B. 4, g- A normal larva hatched from an egg centrifuged for fifteen minutes with the posterior end towards the axis of rota- 522 R. W. Hegner tion. ‘The hatching period of approximately six days is the nor- mal one for eggs of this species. C.B. 4, h. This egg, which was centrifuged for thirty minutes, also developed normally, the larva hatching in six days. C.B. 4,1, 1 and k. None of these eggs continued its develop- ment farther than the early cleavage stages. TABLE III Calligrapha bigsbyana—Series C.B. 3 Interval Numberof Age when cen-Length of timebetween end of : ; experiment trifuged centrifuged experiment and Baealreon aca fixation C.B.3,.4 Control C.B. 3,5 ° 4 hours ° Anterior end C2B=3;16 ° 4 hours 37 hours toward axis of | Did not develop C.B.3,d ° 4 hours 61 hours rotation ‘ CB.35e ° 4 hours 96 hours 2 CB. 3, ° 4 hours 6% days * Series C.B. 3—Table III Series C.B. 3 will serve to show the effects of a centrifugal force applied for four hours to fresh eggs with their anterior ends toward the axis of rotation. C.B. 3, a. The control egg was normal and in a stage slightly younger than that shown in Fig. 9. C.B. 3, 6. When taken from the centrifugal machine at the end of four hours this egg appeared stratified ina manner exactly like that of C.B. 4, e. Longitudinal sections show a gray cap at the heavy outer end, a middle zone of yolk and an inner light vesicular zone. ‘The distribution of the “‘Keimhautblastem” is also similar to that in an egg centrifuged with the opposite (pos- terior) end toward the axis of rotation, 7.2., it has moved toward the lighter end of the egg. The inner pole is creased and folded as in C.B. 4, e. One nucleus is present near the vesicular zone. The pole-disc remained at the posterior end of the egg near one side; Centrifugal Force upon Beetles’ Eggs 523 it fell outside of those sections containing parts of the gray cap so that I was unable to determine whether it is of greater or less specific gravity than the later substance. C.B. 3,¢, d, e, and j. These eggs did not develop very far, although the youngest (3, c) contained a number of nuclei in the course of disintegration. Sections of the other eggs (3, d, e and 7) show a further dissolution of the nuclei, the vacuolation of the ““ Keimhautlbastem”’ and other evidences of catabolism. TABLE IV Calligrapha bigsbyana—Series C.B. 10 Interval Numberof Age when cen-Length of timebetween end of) ; I experiment | trifuged centrifuged experiment and cree tf BLES | fixation | C.B. 10, a | Control C.B. 10, b | fo) 4 hours ° Right side in* CB -.10;\¢ ° 4 hours ° Ventral side in C.B. 10, d ° 4 hours 36 hours Right side in C.B. 10, e ° 4 hours 48 hours Right side in C.B. 10, f ° 4 hours 60 hours Ventral side in C.B. 10, g fo) 4 hours 9 days Right side in C.B. 10, h ° 4 hours 9 days Ventral side in * This means that the right side of the egg was placed toward the axis of rotation. Series C.B. 10—Table IV ‘These experiments were undertaken to detemine if the position of the embryo upon the egg can be changed by altering the distri- bution of the cytoplasm. Four of the eggs were oriented in the centrifugal machine so that their right sides were toward the axis of rotation, the other three with their ventral surfaces toward the center. C.B. 10, a. The control egg was in an early cleavage stage. C.B. 10, b and c. No differences could be discovered between an egg centrifuged with its right side turned inward and one with its ventral surface in the same direction, either before or after 524 R. W. Hegner fixation. ‘The sections also show a similar arrangement of mate- rials. Fig. 14 represents a transverse section through C.B. 10, c. There is a light vesicular zone at the side which was turned toward the axis of rotation; this is folded into bud-like prominences just as we found to be the case in C.B. 4, e, and others. The yolk- globules are distributed in the usual manner, the large ones being on the heavy side. The “Keimhautblastem” has moved away from the side of greater specific gravity and toward the lighter side. No gray cap could be found. It is probable that the material which produces this zone has all been thrown to the outer side, but the area is too great to allow of any perceptible accumula- tion. C.B. 10, e, f, g and h. No one of these eggs developed beyond an early cleavage stage. ‘The nuclei then disintegrated and the amoeboid masses of cytoplasm in which they lay became vacuo- lated as did also the “ Keimhautblastem.”’ TABLE V Calligrapha bigsbyana—Series C.B. 9 Interval Number of Age when cen-Length of time between end of : : experiment trifuged centrifuged experiment and Sse Rema fixation C.B. 9,4 Control C.B. 9, b ° 6 hours ° C.B. 9, ¢ ° 6 hours 41 hours Posterior end C.B. 9, d ° 6 hours 65 hours toward axis of C.B. 9, e fe) 6 hours 89 hours rotation C.B. 9, f fo) 6 hours 10 days Series C.B. g—Table V Although these eggs were taken as soon as laid, sections through C.B. 9, a show that they were in a rather advanced cleavage stage when the experiments were begun. They represent a condition intermediate between those of series C.B. 2 (Fig. 17) and C.B. 4 (Fig. 9), resembling in structure an egg ten hours old. »- Centrifugal Force upon Beetles’ Eggs 525 C.B. 9, 6. Three zones are recognizable in this egg corre- sponding to those already described in egg C.B. 4, e and, although centrifuged for six hours, no noticeable difference is discernible in the distribution of material in this egg and one of nearly the same age which was centrifuged for only one hour (C.B. 2, b). The nuclei of many of the vitellophags are distorted or disintegrating. The granules of the pole-disc have, as in normal eggs, become imbedded in the cytoplasm of the primordial germ cells; the latter occupy their usual position at this stage between the vitelline mem- brane and the blastoderm at the posterior pole. C.B. 9,c. ‘Two eggs were fixed forty-one hours after they were taken from the centrifugal machine. One of these did not develop, its nuclei disintegrating and the “Keimhautblastem” becoming vacuolated; the other carried an embryo with a distinct ventral groove (Fig. 15). Superficially this embryo resembles that of a normally developed egg of this age except that it does not reach as far anteriorly on the ventral surface, but extends farther around the posterior end and up on the dorsal surface (compare Figs. 4 and 15). It is evident that under the influence of centrifugal force the nuclei and “ Keimhautblastem” have become massed in the posterior half of the egg, where development has continued. This egg if it had been allowed to develop would no doubt have produced an embryo resembling that described under C.M. 1, 5 (Fig. 21). Sections of this egg show a rearrangement of the yolk- globules, a condition being reached similar to that illustrated in Fig. 9. ‘The gray cap and vesicular zone are still present. C.B 9, d. One of two eggs preserved sixty-five hours after being taken from the centrifugal machine did not develop; the other produced a shapeless mass of tissue, no definite organs being distinguishable. Fig. 16 is a diagram of a sagittal section through this egg. The gray cap and vesicular zone are still present, the former at one side of the outer end of the egg, the latter just dorsal to the embryonic tissue. R. W. Hegner 526 Fe EN Se Ay ee ce RT Sat / ‘sanuvis yok =F ‘yok = *€ “yt SuNuouo ur poinfur sem $89 oroym oovyans [eUAA JOIAIUe = ‘OX ‘aORyINS -10p UO puLq-uwIa8 Jo pus IoLa\sod = + *9UOZ AL[NIISVA = *z'a *sBeydoTpaA = sa *aAoorS [eIQUIA = “da ‘saxayds ypOA T[eUIS = *sK°s — *s]]a9-UUIAT [eIp -rowud = 93d = *taponu oruapoyseyqaid = *w'yqd = sa01saysod = *¢ = ‘soxoyds yod adaey Ssh pwayseyquneywoy,, = "/qyy “syURUTUIIOJap ]]99-W49d = ‘pod ‘deo Avid = 9d = *purq-wi0s = qs = ‘anssyy atUOAIquIo = “7:9 “WIApoyseTq = */q ‘IOMOjUe = ‘y ‘LI “BIg jo advjs oy] ur 33a ue Jo syuaqUOD ay} JO uoNNqiysip ayy uodn moy ouo s0y porydde sor105 [eSnyzuyUe ev Jo saya oy Burmoys “q ‘z *g'D 3d9 YSNoIY} uONoes [euIpnyduOT gi ‘ong ‘uontsodap 104ye simoy use}INOJ Istp-ajod pur rapnu ,,{WroJseTqINVYUOY,,, oY} Jo VONNqrysIp [eurr0U oYy SurMoys ‘vy ‘z “gD do YSNoIy] UONIes [euIpnyuoT Lr “ony *pua r010jsod ay} ye onssty Jo sseur ssopodeys ve Surmoys ‘p “6 *g*D 3da ysnory) UONIS [euIpNysuOT gr ‘ONT “by -Sry ym oredmog +9 6 *g'a B30 Jo aovjinspemuoA Sr ‘ong ‘Ol ‘Gl BA - see ae = ‘q ale oe Dee Centrifugal Force upon Beetles’ Eogs 2 fo) ee 5 TABLE VI Calligrapha bigsbyana—Series C.B, 2 _ | Interval eo on Age es cen- Length & time}, tween end of . Pepericnent | trifuged centrifuged bevedinea’ aad Orientation Remarks fixation B24 | control Posterior end C.B. 2,5 14 hours 1 hour ° toward axis of Mees 256 14 hours 1 hour 48 hours | rotation normal embryo CBs 2,.a2 14 hours 1 hour | 6 days normal larva Series C.B. 2—Table VI The eggs used in these experiments were laid at 7 p.m. on July 19. One egg was fixed at the end of fourteen hours; the others were at the same time placed in the centrifugal machine with their posterior ends toward the axis of rotation and subjected to the usual number of revolutions for one hour. C.B. 2, a. Fig. 17 shows the “ Keimhautblastem,” the pole- disc and the distribution of the nuclei in the control egg, aged four- teen hours. ‘he yolk is not included in this figure, as its distribu- tion is similar to that of the freshly laid egg (Fig. 9). The two groups of nuclei, those which form a more or less regular layer near the periphery and will fuse with the ‘‘ Keimhautblas- tem” in a few hours producing the blastoderm (pd/. 1), and the vitellophags (vt) scattered about in the yolk, are quite clearly marked at this stage. When taken from the centrifugal machine a colorless layer of material was observed at the outer end of the egg; this is the gray cap occupying a position similar to that noted under C.B. 4, e. The color of the egg was deep yellow posterior to the gray cap and gradually faded out toward the inner end until near that pole it was almost colorless. A bright-yellow cap, the vesicular layer, occupied the extreme inner end. A sagittal sec- tion of this egg is shown in Fig. 18. At the anterior end is the heaviest substance in the egg, the gray cap. Just posterior to this we find the largest deutoplasmic spheres which gave to the living egg its bright-yellow color. The spaces among these are 528 R. W. Hegner free from cytoplasm. The yolk-globules become smaller and smaller posteriorly until they cease altogether in the middle region, where smaller and lighter yolk granules take their place. At the posterior end there are many irregular vacuoles caused by the accumulation of fat in this region. During the hour the egg was under the influence of centrifugal force the preblastodermic nuclei (Fig. 17 pbl. n) migrated outward until they fused with the “Keimhautblastem” forming the blastoderm. ‘The “ Keimhaut- blastem” in the mean time flowed away from the anterior end of the egg, adding this portion to that posterior to it and producing a blastoderm in the latter region decidedly thicker than usual. The nuclei in the blastoderm seem to have been influenced by the centrifugal force; those near the central region have apparently been drawn out of their normal spherical shape and are now oval. As the inner pole is approached the nuclei become less and less oval until at theextreme end they are sphericalas normally. The vitellophags have migrated toward the axis of rotation and the outer end is free from them altogether, while a greater number than usual are present near the posterior end. ‘The centrifugal force used has apparently had no effect upon the position of the nuclei of the vitellophags in relation to the mass of cytoplasm which surrounds them, as in every case the nucleus is in or near the center. The direction of division of these vitellophags, how- ever, seems to have been influenced for we find in almost every instance that the daughter cells produced by a recent division lie one posterior to the other, 7.¢., in the direction of the centrifugal force. The germ-cell determinants have found their way as usual into the primordial germ-cells at the extreme posterior end of the egg (Fig. 18, p. gc). C.B. 2, c. A normal embryo (Fig. 7) was produced by this egg, which was fixed forty-eight hours after being centrifuged. Not the slightest difference could be discovered between an in toto preparation of this egg and a normally developed egg of the same age (63 hours). Sagittal sections show that the yolk has under- gone segmentation and that the yolk-spheres and yolk-granules are equally distributed throughout the entire yolk mass. ‘The germ- cells have migrated from the posterior amniotic cavity through the Centrifugal Force upon Beetles’ Eggs 529 pole-cell canal and into the embryo and lie near the end of the tail- fold. ‘The gray cap has not entirely disappeared, but what I take to be a remnant of it is situated at the dorsal anterior surface. C.B. 2,d. A normal larva hatched from this egg in the average length of time required for eggs of this beetle when developed under natural conditions. TABLE VII Calligrapha bigsbyana —Series C.B. 5 Interval Numberof Age when cen-\Length of timebetween end of , : experiment _trifuged centrifuged experiment and essa es | fixation | GiB: 5, 4 | control Posterior end | C.B.5,b5 | 21 hours 2 hours ) toward axis of | CB. s,.c 21 hours 2 hours | 27 hours rotation | normal embryo C.B.5,d 21 hours 2 hours 6 days normal larva Series C.B. 5—Table VII This series of experiments was performed in order to discover if centrifugal force would have any appreciable effect on an egg in which the blastoderm has already been formed, and if so whether or not the egg would at this late stage continue to develop and eventually produce a larva. C.B. 5, a. The eggs of C. bigsbyana at the age of twenty-one hours have usually reached a stage in which a blastoderm of a single layer of cells completely covers the central yolk mass. Scattered about irregularly among the yolk-globules are numerous vitellophags. At the posterior pole are a number of cells lying in a closely packed group between the vitelline membrane and the ege (Fig. 3, pgc.); these are the primordial germ-cells (pole-cells) which a few hours earlier migrated through that part of the pos- terior end occupied by the pole-disc, taking the granules of which this is composed along with them. C.B. 5, 6. The application of centrifugal force for two hours has very little effect upon an egg twenty-one hours old as seen in 530 R. W. Hegner surface view. ‘he surface at the inner end is creased and folded Just as was found to be the case with younger eggs (C.B. 4, e). Longitudinal sections through this egg present a distribution of material similar to that with which we are already familiar. A gray cap is present at the outer end (Fig. 19, g. c); the largest deutoplasmic spheres are adjacent to the gray cap, and there is a gradual decrease in the size of the yolk-globules until near the inner end where these are lacking altogether giving way to the vesicular zone. Most of the vittellophags have passed into the Fic. 19 Longitudinal section through egg C.B. 5, b., showing the effects of a centrifugal force applied for two hours upon an egg covered by a blastoderm. Explanation of letters same as Figs. 15-18. vt,a, = vitellophags which have fused with the blastoderm. inner half of the egg; a number of them seem to have fused with the ‘“‘Keimhautblastem” in the equatorial region. As was the case with the “‘ Keimhautblastem”’ in the younger eggs (C.B. 4, e) — the superficial layer (blastoderm) has become thinner at the outer _ heavy end until it is barely visible at certain points; its mass has been added to that toward the inner end. ‘The primordial germ- cells occupy their normal position at the posterior end of the egg. C.B. 5, c. An egg in the condition just described was allowed . a Centrifugal Force upon Beetles’ Eggs 531 to develop for twenty-seven hours and then preserved. Exter- nally the embryo it carried appeared to be normal in every respect. It was in a slightly younger stage than that of C.B. 2, c, shownin Fig. 7. Upon sectioning it was found that the vesicular zone had disappeared entirely, that the yolk had segmented and both this and the vitellophags had regained their normal distribution, but that there still remained a small amount of the heavy gray cap. The embryonic tissue seems to have sustained no ill effects from the centrifugal force. C'.B. 5, d. A normal larva hatched from the remaining egg of this series in the average period, six days. Series C.M. 1. Two freshly laid eggs of Calligrapha multipunctata placed with their posterior ends inward were centrifuged for sixteen hours at a rate much slower than that applied to the eggs in most of the other experiments. At the end of this period three perfectly distinct zones could be recognized by their colors. ‘The nearly uniform pale-orange color of the normal egg had given way at the inner end to bright orange; at the opposite pole was a whitish cap, while the comparatively large central zone faded gradually from bright yellow at its outer end to pale yellow where it joined the inner orange stratum. C.M. 1, a. An in toto preparation of one of these eggs which was fixed immediately after being taken from the centrifugal machine shows that the three zones do not differ in color only, but are composed of three different substances. Sections of this egg show a stratification similar to that already described for C. bigs- byana (C.B. 4, e). The stage of development, however, is unlike that of any egg so far examined. Fig. 20 shows the nuclei aggre- gated in the inner portion of the egg. The “ Keimhautblastem”’ | at the sides of the egg and surrounding the folded vesicular zone contains many nuclei producing a kind of blastoderm. ‘The vitellophags have accumulated in the inner portion of the | central zone. Many of these are either dividing by amitosis or seem to have recently completed such a division. ee . “ypok = ‘auoz avpnoisaA = ‘za = “sdvydoyayA = “sa *[eIVUDA = ‘a ‘winapouroys = ys *umapoyso0ad =i ‘oksquia yo pua rouaysod = *a'd = -x0uaysod = *d ‘deo fvxd = ‘9d = sanssy oyUoAIquIa = y'9 "[USIOP = “P “WIIPOIse[] = “74 ‘adepuedde = dv * ‘ofaquia Jo pua roWajUe = ‘ay *y[04 yA Jo opisino padojaaap aavy YIIYM soArquia SUrMoYs “['Q"T Saag WI] 8399 JO SMITA DIvJANS apig Ez pure TZ “SOIT “pua so11ajsod ay} ye HOA ay3 Jo aptsyno OAIquio Javp v Jo yuourdofaAaap oy) SU;MOYSs “4 “TWO 83d JO MATA VIVJANS [RINeT 17 OL] ‘v ‘TWD 332 yBnosyy UoNdas [eUIpNysUO'T O% “OL R. W. Hegner 532 Centrifugal Force upon Beetles’ Eggs 533 C.M. 1,6. Fig. 21 represents a surface view of the right side of an egg like that just described which was allowed to develop for nine days. ‘The embryo has continued to develop at the inner (posterior) end. Its orientation is normal except that the entire embryo has shifted its position posteriorly so that the posterior end instead of being coincident with the posterior end of the egg is now part way up on the dorsal surface. A small mass of embry- onic tissue is imbedded in the large mass of yolk. Normally this yolk would be surrounded by the embryo and become included within the mid-intestine; in this case a dwarf embryo has developed without growing around the nutritive material. TABLE VIII Calligrapha lunata—Series C.L., a = | | Interval | | Number of [Age when cen-Length of time between end of 5 é ‘ 5 : : Orientation | Remarks experiment trifuged centrifuged experiment and fixation C.b.a,1 1 hour 12 hours ° Posterior end CL.a,2 | “ - 55 hours toward axis of C.L. a, 3 s s 79 hours ‘rotation | ‘ if 24 days hatched in 6 days + i 18 day larva series CL. a—Table VIII The effects of centrifugal force upon the eggs of C. lunata are shown by this series of experiments. ‘The results, as may be seen from a comparision of the above table and the following descrip- tions, differ only in minor details from those recorded for eggs of C. bigsbyana similarly treated. C.L. a, 1. This egg was stratified by the centrifugal force into three layers, a gray cap at the outer heavy end, a middle yolk zone with large deutoplasmic spheres at the outer end gradually decreasing in size toward the inner pole and a light vesicular layer at the extreme inner end. Longitudinal sections resemble those of C. M. 1 shown in Fig. 20. There are a number of nuclei present scattered about among the yolk-granules near the inner end of the middle zone. Each nucleus is approximately in the 534 R. W. Hegner center of the amoeboid mass of cytoplasm in which it lies embedded; the whole apparently has moved en masse toward the lighter end of the egg. ‘The pole-disc is situated between the vesicular layer and the middle zone; it is probable that its change of position is due, not to any movement of the granules, but to the accumulation of lighter fats posterior to it. C.L. a, 2. The only redistribution of material that has taken place since this egg was taken from the centrifugal machine is a movement of the “Keimhautblastem,”’ resulting in several large accumulations at the periphery in the middle region. The nuclei have disintegrated and the “Keimhautblastem” has the vacuolated appearance indicative of its early dissolution. No larva could possibly have developed from this egg. C.L. a, 3. Sections of this egg show a continuation of the cata- bolic processes mentioned in C. L. a, 2. C.L. a, 4. The only egg which was not fixed before the end of the hatching period seems to have developed normally, as it produced a normal larva. I can account for this only on the assumption that the eggs of this series were differently affected by the centrifugal force and that C.L. a, 2 and C.L. a, 3 were too severely injured to continue their development while C.L. a, 4 was able to readjust itself to the new conditions imposed by the change in the position of the egg contents. A perfect series of sagittal sections was made through this larva; they showed no irregulariues in the size, position or structure of the internal organs. The reproductive organs (female) are in their proper positions. TABLE IX Calligrapha lunata—Series C.L.I. | Interval Number of | Age when cen-; HELE of time Ponies end of Gaeaehae Raise experiment trifuged | centrifuged experiment and fixation | C.L.I. a control | Anterior end C.L.I. b. ghours | 12hours ° toward axis of Gile 5 x 24 hours rotation Chia 4 - 48 hours Cre ss = 4 days hatched ; a q s Centrifugal Force upon Beetles’ Eggs 535 Series C.L. 1—Table IX The effects of centrifugal force upon the eggs of C. lunata when oriented with their anterior ends toward the center are shown by these experiments. C.L.1, a. The control egg of this series proved to be in a stage similar to that already described for C. B. 9, a. C.L. 1, 6. After being centrifuged for twelve hours this egg showed the customary three strata. Longitudinal sections resem- ble those of C. M. 1 (Fig. 20); they differ from them only in the absence of a well-defined blastoderm in the inner region. C.L. 1, c. During the twenty-four hours since this egg was taken from the centrifugal machine the yolk has had time to redistribute itself to some extent and many of the larger globules are present at the lighter end. Development has proceeded and the inner half of the egg is one large syncytium in the center of which is the vesicular zone containing a few nuclei. C.L. 1, d. Sections of this egg may be compared with that of C.B. 9, d,shown in Fig. 16. There is a mass of tissue at the inner end which is thrown up into folds, but no definite structures are distinguishable in it. C.L. 1, e. The only egg that was allowed to develop through- out the entire hatching period produced a larva at the end of six days. ‘This larva is apparently normal. It was preserved when three days old. TABLE X Leptinotarsa decemlineata—Series L. D. 1 | Interval aoe of | Age ves cen-|Length of time | between end of Ghtamten | Raatke experiment trifuged centrifuged | experiment | and fixation | | ED: 1,1 control £.D.1;2 2 hours 5minutes | | Posteriorend hatched in 6 days £.D.1,3 oa 1ominutes | /toward axis of i L.D.1, 4 i 20 minutes | rotation ¥ ED. 1,5 . 45 minutes — L.D.1, 6 ss 13 hours S L.D.1,7 “ 23 hours a 536 R. W. Hegner Series L.D. ].—Table X The above table (Table X) shows the results of a graded series of experiments upon eggs of Leptinotarsa decemlineata centri- fuged from five minutes to two hours and one halt. These eggs, including the control (L. D./, 1) all hatched at the same time, showing that the amount of centrifugal force has no perceptible influence upon the rate of development. seres 1b.D: a L.D.1. A number of fresh eggs of the potato beetle, Leptino- tarsa decemlineata, were centrifuged at a low rate of speed (360 revolutions per minute) for five days. They were oriented with their posterior ends toward the axis of rotation. ‘The resulting embryos (Figs. 22 and 23), which of course would not have hatched, are very similar in appearance to that described under C. M. 1, b. The heavy substances in these eggs are apparently non-essential for the development of the embryo, being made up principally of nutritive yolk. When deprived of this material a dwarf embryo is produced at the inner end of the egg. series LD. 2 Another batch of potato beetles’ eggs were centrifuged at the same rate of speed for seven days. Dwarf embryos developed at the inner light end in every case. No sections were made of these embryos. Series Le T. 1 A number of eggs of Lema trilineata were centrifuged with their posterior ends turned inward. In all cases the stratification induced resembles that of the eggs of C. bigsbyana similarly treated. Table XI presents the data obtained from a number of experi- ments which have been selected from fifteen series of the eggs of Calligrapha lunata. Eight of these centrifuged eggs produced larvz in the normal hatching period; of these, four were centrifuged with their posterior poles toward the axis of rotation, three with their anterior ends toward the center and one with its side turned Centrifugal Force upon Beetles’ Eggs 537 TABLE XI Calligrapha lunata Interval Number of | Age when cen-| Length of time between end of, Gielen Weniasten experiment trifuged centrifuged experiment and fixation Cl.id,3 ° 23 hours post. pole in | hatched in 6 days Cu, 3 ° 1 hour 8days “s not hatch, high speed C.L. e,2 4 hour 3 hours | 7 days bs | did not hatch ila, 4. 1 hour 12 hours | a | hatched in 6 days rly, 2 1 hour | 13 hours 7 days | : | did not hatch Coboy Bae 8 hours 13 hours | 11 days ‘ | ts C.L. h,3 24 hours 2hours | < | hatched in 6 days Cer. x, 5 47 hours 6 hours 7 days | | did not hatch CE. s;2 so hours 2 hours o | batched ia 6 days C.L. q,2 ° 2 hours 7 days _ ant. pole in ' did not hatch CL. n> 2 4 hour % hour | ss _ hatched in 6 days C.L. 4, e g hours 12 hours | s 4 C.L.i,2 | r4hours 2 hours qdays | ¥ | did not hatch C.L. k, 2 14 hours 2 hours | ts hatched in 6 days . | : hatched in 6 days C.L.b,2 1 hour 20 minutes | onone side {arate if high speed TABLE XII Leptinotarsa decemlineata | Tnterval Number of | Age when cen- Length of time between end of Gasaeee | Rants experiment trifuged | centrifuged experiment and fixation | + hour 2hours 7 days | post. polein | did not hatch . e 10 days 5 * 4 hour 4 hours 6 days | = £ “* 8 days f: | “ 1 hour | rdhours hatched in 6 days 23 hours | 1 hour vs ‘: 24hours | 2 hours i ; 24 hours 3+ hour © ad ° | 3 hours 7 days | ant. pole in _ did not hatch ° | © | S | hatched in 6 days 24 hours 4 hour ¥ > 538 R. W. Hegner inward. ‘The age when the eggs were centrifuged ranges from freshly laid to fifty hours. ‘The length of time centrifuged ranges from twenty minutes to twelve hours. It is obvious that there is no definite total amount of centrifugal force which will prevent the hatching of the egg. ‘The orientation of the egg is apparently of no importance. The data given in Table XII have been selected from eleven series of experiments upon the eggs of Leptinotarsa decemlineata. There are too few items in this list to warrant any general con- clusions, but the experiments tend to show that an older egg has greater chances of producing a larva after being centrifuged than does one experimented upon a short time after deposition. Both eggs oriented with the posterior end toward the axis of rotation and those with the anterior end toward the center gave rise to normal larve. VIII THE EFFECTS OF CENTRIFUGAL FORCE UPON EGGS LAID BY CENTRIFUGED BEETLES. I Experiments with C. bigsbyana Series C.B. 12 A female C. bigsbyana was centrifuged at the usual rate of speed for two hours and fifteen minutes with her posterior end toward the axis of rotation. When taken from the machine she seemed to suffer no ill effects but proceeded to walk about and feed as usual. ‘Three days later, July 24, five eggs were laid; two of these were fixed at once and the other three allowed to develop. The former showed no outward signs of any disturbances due to cen- trifugal force. Sections also failed to disclose any rearrangement of materials. ‘The eggs that were left to develop were fixed at the end of eight days. A superficial view of one of these is shown in Fig. 24; a shapeless mass of tissue lies imbedded within the disinte- grated yolk mass. penes CB-13 The same beetle as that of Series C. B. 12 laid a second batch of five eggs two hours after the first five were deposited. Two of 2 Ma ] Centrifugal Force upon Beetles’ Eggs 539 these which were fixed immediately showed no effect of centrifugal force; the other three hatched in six days. 2 Experiments with Leptinotarsa decemlineata peries L.D. } At 3:30 p.m. on July 17 a female L. decemlineata was centri- fuged for one hour with her posterior end toward the center. Fic. 24 Surface view of egg C.B. 12 laid by acentrifuged beetle. ap. = appendage. e.t.= em- bryonic tissue. p. = posterior. s. = space between two yolk masses. y. = yolk. One egg was laid an hour after being centrifuged and others were laid at irregular intervals until g:30 the next morning, when the total number reached seventy. ‘The first egg laid, as well as all of the others in the series, showed a stratification produced by centri- fugal force. Only two layers, however, resulted, no gray cap being discovered in the sections. ‘The vesicular zone is not as large as in older eggs centrifuged outside of the body of the mother for a 540 Ro i. Hegner similar length of time, but the yolk-globules have a distribution almost exactly like that induced in the latter. Many of the eggs were allowed to develop; all of these hatched in six days. Series L.D. m The same beetle as that of Series L.D. 7 laid a batch of eggs at 7 p.m. July 19, 1.¢., fifty-one hours after she was centrifuged or thirty-three and one-half hours after the first lot were deposited. No effects of centrifugal force could be discovered in sections of these eggs. Normal larve hatched from those eggs which were not preserved. Series L.D. o A third batch of eggs were laid by the beetle of L.D. 7 at 1 p.m. July 20. The preserved eggs showed no effects of centrifugal force; the others hatched in six days. Series L. D. ¢ A fourth lot of eggs were laid by the same beetle as in L.D. 7 on July 22. These agreed in every respect with those described in Series L.D. m. IX REVIEW OF THE EFFECTS OF CENTRIFUGAL FORCE UPON DEVELOPING EGGS rt Distribution of the Egg Contents The most noticeable result obtained by centrifugal force is the redistribution of the materials contained in the egg because of the differences in their specific gravities. A number of cases have been reported of eggs whose contents are normally visibly different and localized in particular regions. For example, Boveri (’01, a, p. 145, Fig. 1, and ’o1,b, Taf. 48 and 49, Figs. 6-22) found three hori- zontal zones present in both unfertilized and fertilized eggs of Strongylocentrotus lividus. These zones could still be recognized in young blastula. Wilson (’o4, p. 68) states that, “The Denta- lium egg shows from the beginning three horizontal zones, an equatorial pigment-zone and two white polar areas. Each of the Centrifugal Force upon Beetles’ Eggs 541 polar areas includes a specially modified protoplasmic area proba- bly comparable to a polar ring.” Conklin (’05, a, p. 211) says of the Ascidian egg (Cynthia): “All the principal organs of the larva in their definite positions and proportions are here marked out in the 2-cell stage by distinct kinds of protoplasm,” and again on p. 216 this author states that “the substances of the ectoderm, mesoderm and endoderm are recognizable in the unsegmented ege.”’ In another place (Conklin ’o5, b, p. 220) we find the state- ment that, “Three of these substances are clearly distinguishable in the ovarian egg and I do not doubt that even at this stage they are differentiated for particular ends.”’ Many other eggs that do not exhibit a normal stratification and are apparently homogeneous throughout take on a zone-like appearance under the influence of a strong centrifugal force. Morgan (’06) found that when the unsegmented eggs of Rana sylvatica are revolved 1600 times per minute for seven minutes the pigment and yolk are driven to the top of the egg, leaving a clear polar field. Similar results were obtained in toads’ eggs in three minutes. Lyon (’06, ’07) was able to induce four layers in the egg of the sea-urchin, Arbacia. “Two layers were obtained in eggs of the starfish, Asterias (Lyon, ’07). The annelid, Che- topterus, exhibited three layers. The same author also centri- fuged the eggs of the Ascidian, Cynthia, the Gephyrean, Phascolo- soma, and the common garden spider; the eggs of Cynthia and Phascolosoma were stratified into three layers, those of the spider into two. Lillie (06) found that not only in the unsegmented eggs of Chetopterus but also in the two, four and eight celled stages three zones appeared in each cell when placed under the influence of centrifugal force. The contents of the egg of the mollusk, Cumingia, may be separated into three zones (Morgan, 08). The eggs of the rotifer Hydatina senta were centrifuged by Whitney (’og) while still within the mother. Three distinct layers resulted: a pink zone, a clear middle zone and a gray zone. The eggs of Calligrapha bigsbyana are when laid of a nearly uniform pale-yellow color. When subjected to a strong centri- fugal force for a sufficient length of time three zones are distin- 542 R. W. Hegner guishable: (1) a bright-orange light zone at the inner end (the vesicular zone, Fig. 20, v. z), (2) a comparatively large central mass composed of yolk globules which are largest at the outer heavy end, gradually becoming smaller until indistinguishable from cytoplasm at the inner end, and (3) a colorless layer (the gray cap, Fig. 20, g. c) at the extreme heavy end. ‘These three zones are produced when the eggs are oriented either with their posterior (C.B. 4, e) or their anterior (C.B. 3, 5) ends toward the axis of rotation. When placed with their sides toward the center only two layers are induced, the vesicular zone and the yolk zone. Three layers may be obtained in fresh eggs (C.B. 4, ¢) in eggs which have reached a late cleavage stage (C.B. 2, b, Fig. 18) and in eggs which are covered by a blastoderm (C.B. 5, a, Fig. 19). The gray cap. The material of the gray cap is the heaviest of the egg contents. It is composed of very fine granules whose positions before being driven to the heavy end of the eggs could not be determined A fresh egg when centrifuged for one hour does not exhibit this layer (C.B. 4, d, Fig. 10). At the end of two hours, however, a distinct gray cap is present (C.B. 4, e). Eggs in late cleavage stages require a lesser amount of centrifugal force in order to produce this structure (C.B. 2, db, Fig. 18). We conclude from this that either the gray cap material is liberated during develop- ment and the egg fourteen hours old (C.B. 2, 5) contains a greater quantity of it, or else some condition of the yolk mass at this age allows it to pass more rapidly toward the heavier end. Longitu- dinal sections through egg C.B. 2, c (Fig. 7) show that although the embryo has developed normally the material of the gray cap is still at the heavy end where it was driven by the centrifugal force. A like condition also exists in a slightly younger egg (C.B. 5, c). It is evident that the gray cap substance is not necessary for the normal development of the embryo. The vestcular zone. The light fats which probably produce the vesicular zone at the inner end of the egg collect very quickly under the influence of centrifugal force. An egg centrifuged for only fifteen minutes (C.B. 4, 5) has a small number of vesicular spaces near the pole-disc. Continued application of centrifugal force results in a greater number of these vesicles until at the end of Centrifugal Force upon Beetles’ Eggs. 543 one hour a very distinct zone may be recognized (C.B. 4, d, Fig. 10). The surface of the egg in this region is in every case wrinkled and folded as though the volume had decreased at this end and the firm layer of ‘“Keimhautblastem” had become pulled in (C.B. meee, 10; C.B. 4, 7, Fig..13; C.B. 10, .c, Fig. 14; C.B. 5, a, Fig. 19). This may, however, be due to poor fixation, as these folds are not visible in the eggs before they are killed. The vesicular zone is present as such for some time after the eggs are taken from the centrifugal machine. It is not visible in sections through eggs C.B. 2, c, and C.B. 5, c, which carry normal embryos, but is present in C.B. 9, d (Fig. 16), which has produced a shapeless mass of tissue at the inner (anterior) end. ‘This would indicate that the material of which this region is composed is required for normal development. However, I do not believe that this is established by the few cases observed. The yolk zone. A very slight amount of centrifugal force is necessary to cause a noticeable disturbance in the large central yolk mass. The largest yolk spheres, as shown in C.B. 4, }, are thrown to the outer heavy end within fifteen minutes after the egg is centrifuged. A more marked distribution of yolk globules results from a longer application of centrifugal force. A redis- tribution takes place very quickly after the eggs are removed from the machine; this is shown distinctly in sections through eggs C.B. 2, c, and C.B. 5, c. No redistribution took place in eggs revolved at a slow rate of speed for along period; the yolk remained at the heavy end in these cases and the embryos, failing to grow around it, became dwarfed as shown in Figs. 21, 22 and 23. The yolk has been shown to be the densest substance in the eggs of other animals; for example, in the frog’s egg the white yolk is the heaviest material, as Born (’85) proved by sectioning those that had been rotated. The cytoplasm. The peripheral layer of cytoplasm is lighter than the gray cap material or the yolk; continued application of centrifugal force causes it to rise to the inner end of the egg (C.B. 4, d, Fig. 10), where it becomes part of the vesicular zone. ‘The cytoplasm filling the interdeutoplasmic spaces also accumulates in this region. 544 R. W. Hegner The cytoplasm of the beetle’s egg is not made incapable of development by centrifugal force, since an embryo may be pro- duced after a profound change in its arrangement. Gurwitsch (704, 05) concludes, from his experiments upon the eggs of am- phibians and echinoderms, that no vital structure of the cytoplasm is destroyed by the forcible passage of yolk granules through it. Morgan’s conclusions from his experiments with the eggs of Rana palustris are, on the contrary (’02, p. 306), that “The most impor- tant effect, however, of a strong centrifugal force is the direct injury to the protoplasm of the lower hemisphere of the egg.” The nuclet. In C.B. 4, d (Fig. 10) one nucleus was found near the inner end of the egg. The nuclei of eggs which are in late cleavage stages rise toward the lighter pole (compare Figs. 17 and 18). In later stages the nuclei of the blastoderm are not affected, but the vitellophags move through the yolk toward the inner end. In every case the nucleus with its amoeboid accumulation of cytoplasm moves as a whole, the nucleus remaining approxi- mately in the center of the cytoplasmic mass. Lyon (’06) found that the nucleus in the egg of the sea-urchin, Arbacia, is less dense than most of the other constituents. In centrifuged eggs of As- terias and Phascolosoma the nucleus is next to the lightest sub- stance (Lyon ’o7); this is also the case in Hydatina senta (Whitney ’0g, p- 135). In Paramecium caudatum the nucleus is heavier — than the endosarc and is driven to the outer end by the centri- fugal force (McClendon ’o8). Similarly Andrews (’03) has found that in seeds the nucleus is always of higher specific gravity than the cytoplasm, cell sap and oil drops. When the membrane dissolves the nuclear sap escapes, leaving the heavier chromatin behind. Thus we find that the spindle does not rise toward the lighter end of the egg. Lillie (’06, p. 179) found in Chetopterus that the maturation figure is not moved by centrifugal force, but is usually fixed at the periphery. Some- times, however, it was torn loose (p. 184), when it moves as a whole, the chromosomes and spindle never being separated by the — centrifugal force. ‘The same is true of Hydatina senta (Whitney — 09, p- 155). Morgan (’08, p. 446) makes the following state-— ment after a study of the effects of centrifugal force upon the eggs — { Centrifugal Force upon Beetles’ Eggs 545 of the mollusk, Cumingia: “In general, a resting nucleus may be forced to the lighter pole of the cell owing to the presence in the nucleus of nuclear sap, but the chromosomes and the spindle are more difficult to move, since they have nearly the same specific gravity as cytoplasm. When they move they do so as a whole, which shows that the spindle figure when present is a definite structure.” It is seldom that mitotic figures are found in sections of beetles’ eggs and none was present in any of the many centrifuged eggs that I have examined. ‘The nucleoli of the centrifuged eggs of Chry- somelid beetles seemed not to be affected, but were found in all parts of the nuclei irrespective of the direction of the centrifugal force. The nucleolus is heavier than the nuclear sap in the ova of the lobster. Its eccentric position was noted by Bumpus (’91, p. 225); later Herrick (’95, p. 155) proved that it falls to the lower side of the nucleus “like a shot within a tennis ball.” Lyon (07, p. 168) reports that the germinal vesicle is forced to the light end when unmatured eggs of Asterias are centrifuged, but that the nucleolus is heavier. The germ-cell determinants. Figs. 11, 12 and 13 are from longitudinal sections through the posterior ends of eggs which had been centrifuged one hour, two hours and four hours respect- ively. ‘They show that the pole-disc moves en masse toward the heavy end of the egg and that it carries with it the “ Keimhaut- blastem” in which it is suspended. In Fig. 11 there is a slight indentation in the surface at the posterior end; in Fig. 12 the pole- disc has penetrated farther into the yolk, leaving an open pathway (pt) behind it. This pathway is really unbroken, but appears cut across in the figure. A third stage is shown in Fig. 13, where the pathway has become closed and the group of germ-cell deter- minants is on its way toward the anterior pole. ‘The eggs (Series C.B. 4) were oriented with their posterier ends toward the axis of rotation. No definite conclusion could be reached concerning the comparative specific gravity of the pole-disc, but a section through an egg centrifuged with its anterior end toward the center (C.B. 3, 5) leaves little doubt that it lies between that of the gray cap andthe yolk. The fact that the pole-disc moves asa whole, 546 R. W. Hegner likewise the vitellophags, seems to show that, contrary to what Lillie (’0g) finds to be the case in annelid eggs, there is here good evidence of mass movements of protoplasmic areas. 2 The Restitution of the Egg Substances After Centrifuging The results obtained by several investigators from experiments with centrifugal force upon the eggs of a number of species of ani- mals seem to prove that, as Conklin has recently stated (’08, p. 94), “when different substances of the egg are displaced by strong centrifuging they tend to come back to their normal positions unless prevented by partition walls which have formed in the mean time.” Morgan (’o06) found that the pigment of the toad’s egg does not return to its original position after the removal of the egg from the centrifugal machine. In Arbacia if the centrifuged eggs are left unfertilized readjustment begins and the eggs appear nearly normal after several hours (Lyon ’07, p. 163). In fertilized eggs of Arbacia, Morgan and Lyon (’07, p. 157) claim that the materials displaced by centrifugal force do not become rearranged to any extent before cleavage begins. In Cumingia the induced distri- bution of the egg contents is to a large extent retained (Morgan ’08). Very little redistribution of the egg materials takes place before the first cleavage in Hydatina senta (Whitney ’09, p. 135). The nuclei of Paramecium caudatum, as reported by McClendon (08), slowly regain their normal positions after removal from the centrifugal machine; in some cases this took several generations. Andrews (’03) states that the contents of centrifuged seeds gradu- ally return to their original arrangement, but if kept dry this process may take several months. There are no cell walls in the eggs of beetles when in the process of cleavage to hinder the rearrangement of materials that have been driven out of their normal positions by centrifugal force. Nevertheless readjustment takes place very slowly if at all. The yolk globules which are the first to become displaced are also the first to redistribute themselves, and we find them occupying their usual positions twenty-seven hours after the end of the experiment (C.B. 5, c). The substance of the gray cap does not become rearranged. ‘The vesicular zone in some cases disappears in a Centrifugal Force upon Beetles’ Eggs 547 short time (C.B. 5, c, and C.B. 2, c); in other cases it is still present after sixty-five hours (C.B. 9, d, Fig. 16). The cytoplasm under- goes a partial restitution, but in those cases where most of it has accumulated in the inner region the embryo is formed at this place Giigs. 16, 21, 22 and 23). 3 The Age of the Egg when Centrifuged The general statement may be made that the older the egg the more chances there are of its normal development after peaentae: ing. Morgan (02, p.265) states that the eggs of frogs “which have divided once or twice will withstand a greater rate of revolution than those that have not divided. Moreover, eggs that have seg- mented a number of times, so that the content is divided by cell walls, will develop normally at rates of revolution that kill or pro- duce abnormalities 1 in unsegmented eggs, or eggs just beginning to segment.” The age of the egg also determines to a certain extent the amount of stratification. In the eggs of Chztopterus (Lillie ’06, p. 184) the stratification is not so pronounced before the break- ing down of the germinal vesicle and there is no gray cap formed. Lyon (’07, p. 168) could not distinguish any layers in unmatured eggs of Asterias. The beetle’s egg becomes stratified more quickly if centrifuged when in a late cleavage stage than when fresh (compare C.B. 4, d, Fig. 10, and C.B. 2, b, Fig. 18). Eggs that have reached the blastoderm stage are more difficult to influence (C.B. 5, a, Fig. 19). he experiments described in this paper show that eggs in the blastoderm stage or older almost always produce Horie emiryes and some times larve (C.B. 2, c; C.B. 2, d; CB. 5, c; See ce Lh, 3; CL. s, 2; C.L.k.'2; L.Dk. 5; L.D. an, 6; Pt). n, 7). 4. The Rate of Development The effect of agitation upon the rate of development is not cer- tain because in several of the experiments reported the tempera- ture was not carefully regulated. Meltzer (’03, p. 250) siates that the eggs of the sea-urchin, Arbacia, develop into an advanced cleavage stage more quickly than normally if they are violently 548 R. W. Hegner shaken. Some experiments by Morgan (’04, p. 96) upon the toad’s egg seem to show that agitation hastens the development. Whitney (’06, p. 47) finds that “‘mechanical shocks and vibrations are not effective in accelerating the early segmentation of the fertilized eggs of Arbacia, Asterias, Fundulus and Ctenolabrus.” The development of the eggs of Hydrophilus aterrimus is retarded if their position is reversed with respect to gravity (Megusar ’o7). The sea-urchin egg develops more slowly than is normal after being centrifuged; this is probably due to the resistance to cleavage offered by the cap and pigment (Lyon’o7, p. 166). McClendon (08) found the rate of division of centrifuged Paramecia to be greater than that of normal animals. When seeds are centrifuged and restitution is slow the growth is retarded (Andrews ’o03). Centrifugal force seems to have no influence upon the rate of development of those beetles’ eggs that produced normal embryos and larve. A large number of observations give the average period for the development of the eggs of C. multipunctata, C. bigysbyana and C. lunata as five and two-thirds days (Hegner ’08, a). Ina great many cases normal eggs do not hatch under six and one-half days. Practically all of the centrifuged beetles’ eggs hatched in six days. 5 Eggs Centrifuged Before Deposition In the majority of cases the eggs laid by centrifuged beetles show no rearrangement of material and the production of an embryo or larva is not impeded. ‘The exceptions to this are the eggs described as Series C.B. 12; here two abnormal embryos were produced by eggs which had been centrifuged within the mother before the germinal vesicles had broken down. No definite cause can be given for this irregularity. X SUMMARY 1 Eggs of Chrysomelid beetles when oriented in a centrifugal machine with either their posterior or ante1ior ends toward the axis of rotation, and subjected to 1500-2000 revolutions per minute for from one to twelve hours, become stratified into three layers: (1) a light vesicular zone at the inner end, (2) a heavy granular Centrifugal Force upon Beetles’ Eggs 549 gray cap at the outer end, and (3) a comparatively large intermedi- ate mass of yolk, the larger globules lying at the outer end of this layer. 2 The gray cap is induced by a lesser amount of centrifugal force in an egg containing many cleavage nuclei than in a fresh egg. Either the gray cap material is liberated during development or else some condition of the yolk mass in the older egg allows it to pass more rapidly toward the heavier end. ‘The gray cap material is not necessary for the normal development of the embryo. 3 The vesicular zone becomes visible after fifteen minutes of centrifuging. It is composed of fat imbedded in cytoplasm. This zone disappears during development. 4 The yolk globules are distributed throughout the inter- mediate region of the egg; the largest spheres are at the outer heavy end. It takes very little centrifugal force to cause this rearrangement. Restitution to the normal condition takes place soon after the egg is removed from the centrifugal machine. 5 The cytoplasm is lighter than the gray cap material or the yolk, but heavier than the fat of the vesicular zone. ‘The passage of the cytoplasm to the light end of the egg does not incapacitate it for the production of an embryo. 6 The nuclei are apparently equal in specific gravity to the cytoplasm. Cleavage nuclei and vitellophags rise to the inner end of the egg; the nuclei of the blastoderm of older eggs are not visibly influenced by centrifugal force. 7 The germ-cell determinants move en masse from their usual position at the posterior end toward the anterior end when the former is placed inward. ‘The further history of these granules has not been traced. 8 Restitution takes place very slowly. Those substances easily displaced are also the first to redistribute themselves. The cytoplasm seldom regains its normal position, but produces a | dwarf embryo outside of the yolk at the light end of the egg. g The age of the egg determines the susceptibility to centri- fugal force and the future growth of the embryo. In general an egg in a late cleavage stage becomes stratified sooner than a 550 R. W. Hegner fresh egg. Eggs centrifuged when in the blastoderm stage or older almost always produce normal embryos and sometimes larve. 10 Centrifugal force has no influence upon the rate of develop- ment of eggs which produce normal embryos or larve. 11 The orientation of the embryos produced by centrifuged eggs is not affected by centrifugal force. Dwarf embryos, how- ever, are frequently formed at the posterior ends of the eggs; these never produce larve. ! 12 Inthe majority of cases the eggs laid by centrifuged beetles — produce normal larve. 13 The eggs of insects, although supposed by many embryol-— ogists to be the most highly organized of any animal eggs, may — have their contents profoundly disturbed without preventing the i : aos. See: production of a normal embryo. The cytoplasm and nuclei of — centrifuged eggs are forced out of their usual positions, but often normal development takes place. This would indicate that a high degree of organization does not prevent the egg from adapt-_ ing itself to changed conditions. The University of Michigan. February 19, 1909. LITERATURE Fa Anprews, F, M. ’03—Die Wirkung der Centrifugalkraft auf Pflanzen. Jahrb. wiss. Bot., Bd. 38. Born, E. ’85—Biologische Untersuchungen. I. Ueber den Einfluss der Schwa auf das Froschei. Arch. f. mikr. Anat., Bd. 24. Boveri, Tu. ’o1, a—Ueber die Polaritat des Seeigeleies. Werh, der Phys. Med. — Ges. zu. Wiirzburg, N. F., Bd. 34. S ’o1, b—Die Polaritat von Ovocyte, Ei und Larve des Strongylocentrotus — lividus. Zool. Jahrb. Abth. f. Anat. u. Ontog., Bd. 14, Heft 4. Bumpus, H. C. ’91—The Embryology of the American Lobster. Journ. of Morph., vol. 5. ; Conx.tn, E. G. ’05 a—Organ-forming Substances in the Eggs of Ascidians. Biol. Bull., vol. 8. | ‘05 b—Mosaic Development in Ascidian Eggs. Journ. Exp. Zodl., vol. 2. *o8—The Mechanism of Heredity. Science, N. S., vol. 27. a" Centrifugal Force upon Beetles’ Eggs 551 Gurwitscu, A., ’04—Zerst6rbarkeit und Restitutionsfahigkeit des Protoplasmas des Amphibieneies. Anat. Anz. Erganzungsheft z., Bd. 25. *o5—Ueber die Zerstérbarkeit des Protoplasmas im Echinodermenei. Anat. Anz., Bd. 27. Ha.iez, P. ’86—Loi de l’orientation de l’embryon chez les insects. Compt.- rend., tome 103. Hecner, R. W. ’08, a—Observations on the Breeding Habits of Three Chrysomelid Beetles, Calligrapha bigsbyana, C. multipunctata and C. lunata. Psyche, vol. 15. "08, b—The Effects of Removing the Germ-Cell Determinants from the Eggs of Some Chrysomelid Beetles. Biol. Bull., vol. 16. Herrick, F. H. ’95—The American Lobster: a Study of its Habits and Develop- ment. Bull. U.S. Fish. Com. Lituiz, F. R. ’06—Observations and Experiments Concerning the Elementary Phenomena of Embryonic Development in Chetopterus. Journ. Exp. Zo6l., vol. 3. *og—Polarity and Bilaterality of the Annelid Egg. Experiments with Centrifugal Force. Biol. Bull., vol. 16. Lyon, E. P. ’o6—Some Results of Centrifugalizing the Eggs of Arbacia. Am. Journ. Physiol., vol. 15. ’o7—Results of Centrifugalizing Eggs. I. The Specific Gravity of Eggs and the Changes in Specific Gravity Occurring During Development. II. Effects of Centrifugalizing Unfertilized Eggs on their Develop- ment. Arch. Entw’m., Bd. 23. McCtenpon, J. F. ’o8—The Effects of Prolonged Centrifugal Force on Parame- cium. Am. Journ. Physiol., vol. 21. Mecusar, F. ’06—Einfluss abnormaler Gravitationswirkung auf die Embryonalent- wicklung bei Hydrophilus aterrimus Eschscholtz. Arch. Entw’m., Bd. 22. Metmzer, S. J. °03—Some Observations on the Effects of Agitation upon Arbacia Eggs. Am. Journ. Physiol., vol. 9. Morean, T. H.—’o2 The Relation between Normal and Abnormal Development of the Embryo of the Frog as Determined by Injury to the Yolk Por- tion of the Egg. Arch. Entw’m., Bd. 15. ’o4—The Dispensability of the Constant Action of Gravity and of a Centri- fugal Force in the Development of the Toad’s Egg. Anat. Anz., Ba. 25. ’°06—The Influence of a Strong Centrifugal Force on the Frog’s Egg. Arch. Entw’m., Bd. 22. ’°o8—The Effects of Centrifuging the Eggs of the Mollusc Cumingia. Science, N.S., vol. 27, pp. 66-67 and 446. 552 R. W. Hegner Wuee er, W. M. ’8g9—The Embryology of Blatta germanica and Doryphora decemlineata. Journ. of Morph., vol. 3. Wuirney, D. D. ’06—An Examination of the Effects of Mechanical Shocks and Vibrations upon the Rate of Development of Fertilized Eggs. Journ. Exp. Zo6l., vol. 3. ’og—The Effects of a Centrifugal Force upon the Development and Sex of Parthenogenetic Eggs of Hydatina senta. Journ. Exp. Zodl., vol. 6. Wison, E. B. ’04—Experimental Studies in Germinal Localization. I. The Germ-Regions in the Egg of Dentalium. Journ. Exp. Zool., vol. I. CONTRIBUTIONS TO EXPERIMENTAL ENTOMOLOGY’! I. JUNONIA COENIA HUBNER BY WILLIAM REIFF Towards the end of September I| received, through the kindness of Mr. Jacob Doll, of Brooklyn, N. Y., a considerable number of caterpillars of Junonia coenia Hubner which had been found near Brooklyn in a portion of Long Island that is rather damp and over- grown with bushes and low plants. Here the butterflies had been rather abundant one or two months previously. Mr. Doll assures me that the frequency of J. ccenia in this region is not at all the rule. Although the butterflies are never completely absent in any year, there are usually only a few caterpillars to be found. J. coenia in the northern states belongs, therefore, to the cate- gory of butterflies that appear in great numbers only during cer- tain years. It is, moreover, probable that the abundance of this form is due to migration from the south, and that this migration to the north would be the stronger the dryer and hotter the season. The summer of 1908 would seem to justify this supposition. The caterpillars, which, as I have said, were present in num- bers, fed on Gerardia purpurea L., which ts cited as their favorite food-plant by Scudder in his work on “The Butterflies of New England.” As I could obtain no material of Gerardia, I gave them Linaria linaria Karst, Plantago media L., and Plantago major L. Plantago media was not eaten at all; Linaria linaria only unwillingly, but Plantago major with evident relish. A considerable portion of the material unfortunately died of flacherie (flaccidenza). As soon as I recognized the disease I, of Contributions from the Entomological Laboratory of the Bussey Institution, Harvard University. No. r. THE JourNAL or ExPERIMENTAL ZOOLOGY, VOL. VI, NO. 4. 554. William Reiff course, isolated the apparently healthy animals, but too many of them had already contracted the disease. The first symptom of flacherie appeared as a complete apathy, both in feeding and in movement. Later the animals crept slowly up the walls of the breeding cage and remained motionless under the lid, where they died in the course of a couple of days. As a rule, one pair of pro- legs was fastened to the gauze of the wall, while the anterior and posterior portion of the caterpillar hung down on the right and left side. The dead caterpillar at once decomposed, and often dropped into several pieces at the least touch. The caterpillars were reared at out-of-door temperature. Pupa- tion began the sixth of October. The time of pupation, that is, the time which elapses between the attachment of the caterpillar and the completed pupa, differs according to the prevailing tem- perature. At 23°C. pupation took place in from 10 to 12 hours; 18° C. caused the caterpillars to pupate in 17 hours; 9° C. in about 48 hours; 6° C. in 60 hours, and below + 4° C. pupation did not take place at all. The caterpillars, which I subjected for a long time (up to 8 days) uninterruptedly to a lower temperature, vary- ing between 0° and +3° C., died, no matter in what stage they happened to be. I used for this purpose caterpillars of all ages: animals that had not yet completed their second moult, up to those which had all attached themselves for pupation. In the original locality Mr. Doll had found caterpillars of all sizes as late as the end of October. Indeed, there were even some freshly hatched butterflies, together with flown specimens of the preceding generation. In his letter to me he suggested the question as to whether the caterpillars might not hibernate, since it was obviously impossible for the majority of them to pupate during this same year, as the food-plant was mostly frozen and the temperature was becoming lower from day to day. Iam now of the opinion, after the above experiments, that J. ccenia never passes the winter in the northern states as a caterpillar, but that all caterpillars die as soon as the temperature sinks to +3° C. for several days. The pupa is not quite so sensitive to continued cold as the caterpillar, but still to a considerable degree. All the pupz which I exposed to a constant low temperature (—5° C. or — ny Contributions to Experimental Entomology 555 below), for more than forty-eight hours, died.? Still, very pro- nounced cold, for example —15° C., never injured the pupz if they were exposed to it only about an hour, even when the experi- ment was repeated as often as three times daily for several succes- sive days. Hence, J]. ccenia could not hibernate in the northern states even as a pupa since a constant temperature of —5° C. or below for several days is by no means rare in winter. Through the influence of cold the pupa takes on a very dark ground color, while the white punctate markings increase in dis- tinctness and also in size. If one exposes pupz to higher tem- peratures ( + 38° C. and above) all the colors pass over into a light red in which one sees small black dots. In both cases the newly acquired color persists till the butterfly hatches. If caterpillars attacked by flacherie still have the strength to pupate, the progress of the disease becomes apparent in the color of the pupa, which is a uniform blue-black. “The decomposition process is like that of the caterpillars. Just as caterpillars of all ages may die of flacherie, the period at which it occurs in the pupa also differs with different individuals. I had, for example, a pupa which died about six hours before it should have hatched as a butterfly. ‘The head, thorax and legs were completely developed, the marking of the wings was almost perfect, but the body was decomposed. In this case, therefore, only the body of the animal was affected with the disease, and this, for some peculiar reason, had not spread to all parts of the pupa. But ‘that the disease al- ways first makes its appearance in the abdomen was shown by the reddish pupz obtained from experiments with high tempera- tures. When these had flacherie the progress of the disease from the tip of the abdomen to the head could be accurately followed in the increasing change of color. All the pupz used in the experiments and the control were kept in the room at a temperature of about 22° C. and care was taken to provide the necessary amount of moisture in the receptacles con- taining them. A// the butterflies hatched after being kept in the room from ten to thirteen days, both the control pupz and the In all experiments I used pupz that were 10 to 12 hours old. Perfectly fresh pupe, with their chitinous investment still soft, died within 12 hours at—2° C. 550 William Reif pupz that had been treated experimentally with cold or heat. I must call attention to the fact, however, that only experiments with intermittent temperatures were undertaken, and that subjection to the excessive temperatures never extended beyond three days. No experiments were made with high constant temperatures, while the experiments with lower temperatures gave no butter- flies when I subjected them to a cold of —3° C. for seven days. The hatching of the butterflies usually took place about noon between 11 and 2 o'clock, and, moreover, only when the weather outside was fine. Although the pup were kept in a warm room, not a single butterfly hatched when the barometer was low. I come now to an account of the color peculiarities of the but- terflies hatching from pupz exposed to different temperatures. EXPERIMENTS WITH HIGH TEMPERATURES An incubator was used for this purpose and accurate regulation of the heat was accomplished by means of a thermostat. Care was taken to keep the air in the apparatus moist. All the pupz which I exposed to a temperature of 45° C. for more than two hours, or such as were exposed to 44° C. for more than five hours, died. Of all the pupz which remained in the apparatus five hours at 43° C., only one later produced a butter- fly, but this was a complete cripple. All the pupz endured very well for several hours a temperature of from 40° to 42°C. My method was to expose the pupz on the first day to a temperature of 40° C. for four hours, the second day to 41° C. for five hours, and on the third day to 42° C. for four hours. All the butterflies which I obtained from these heat experiments differ from the normal form by their sharper and more vivid color- ation. ‘The otherwise chocolate-brown ground color of the wings is more blackish, so that both the white and red markings stand out more strongly. In some individuals the white cross-band on the anterior wings is somewhat broadened. ‘The orange-colored band between the margin and the eye-spots of the hind wing 1s more luminous, broader and crosses not only the hind wing, but also sometimes shows itself in the form of an uninterrupted pro- Contributions to Experimental Entomology 557 longation to the tip of the anterior wing. ‘The eye-spot markings of the hind-wing remain rather constant, while the eye-spots of the anterior-wing vary more or less. With the enlargement of the white apical spot there is a correlated great accentuation of the small eye-spot lying immediately behind it. ‘The ring of the sec- ond and larger eye-spot further back was in some cases suffused with black scales. It often happens that the elements of a new pattern are added; thus, for example, one cannot overlook the tendency to add to the row of eye-spots already existing other markings of the same kind. Moreover, black dots, which are often surrounded by a feeble ring, make their appearance. The lower surface of the hind-wings 1s, without exception, deep red- dish-brown and has a och, recognizable pattern. This color- ation is not, however, the result of higher temperatures, for it is present in all of the individuals taken in late autumn in the north- ern states, as | have been able to ascertain by an examination of a large amount of material which Mr. Doll kindly permitted me to study. Specimens from the more southerly range of the species have a much lighter lower surface. EXPERIMENTS WITH LOWER TEMPERATURES For producing lower temperatures [ used a mixture of pounded ice and cooking salt, and the experiments with intermittent tem- peratures consisted in subjecting the pupz three times daily for periods of two hours to a low temperature which, in most cases, was for the first day —7° C., for the second day —8° C., and for the third day —9° C. In the intervals the pupz were left at +6° C., and after the last exposure were taken into the room tempera- ture. All the healthy pupz produced butterflies which in the color- ation and marking are a complete contrast to those which I have described in the foregoing paragraphs. In these specimens, too, the ground color of the wings is dark, but this deepening of color extends over all the elements of the pattern. The white cross band of the anterior wings is evanescent so that it is represented only by a distinct, light-colored cloud. |The orange-colored band, 558 William Reiff which runs parallel with the outer margin, is almost completely supplanted by the dark ground color of the posterior wings, and is no longer to be detected in the anterior wings. The small white apical spot is very variable. Although it has all disappeared in rather light-colored specimens, it continues to remain more dis- tinctly visible in dark specimens. All the eye-spots are diminished in size through the effects of cold. Instead of there being new adventitious markings, the markings already existing have begun to disappear. The most posterior eye-spot of the hind wings often consists only of a tiny dot, while the remaining eye-spots towards the outer margin lose their rings, and the ground color, therefore, goes over into the inner marking. ‘The two red spots towards the anterior border of the fore-wing are least influenced by these changes. ‘The inferior surface of the anterior wings corresponds to that of the upper surface. ‘That of the hind wings is uniform silver gray, with a pale brownish tint and a scarcely perceptible pattern. WHAT CONCLUSIONS MAY BE DRAWN FROM THESE EXPERIMENTS? We may assume as established beyond question that the genus Junonia had its origin in the tropical zone, where even today most of the species of the genus are to be found, and whence the species migrated and spread in the direction of the two poles till a limit was set to their expansion by the coming of the glacial period. The more the northern regions were covered with ice, the more Junonia had to retreat to the south. ‘The conditions in the subtropical zone, in which the genus was able to spread in all directions without meeting with any obstacle, were different. After the glacial period, and hence not so very long ago, a gradual northward migration again began. There are species of Junonia in the tropics both of the old world and the new world, but they are to be found in the north temperate zone only in North America, where the north is not separate from the south by mountain ranges, although it has been shown that North America passed through a longer glacial period than Europe (J. Hann, Handbuch der Klimatologie, Stuttgart, Contributions to Experimental Entomology 559 1883). [he species of Junonia differ, therefore, from those of the very closely related, ubiquitous genus Pyrameis Hb., which, according to Standfuss (Handbuch der palaarkt. Gross-schmet- tetlinge, Jena, 1896), also had their origin in the tropics. Gen- erally speaking, however, the species of Junonia are less adaptable than those of Pyrameis. The only northern representative of the genus Junonia isia member of the ccenia group, and I believe that this species had already developed as an independent form before the glacial period, while it was advancing towards the north, and had at least become sufficiently stable to remain as a species after it had been pushed back to the south. When later the road to the north was again open, it was all the easier for this species to follow the path of its ancestors and take possession of the region which the preceding generations had to leave on the approach of the ice age. If we now glance at the species of Junonia which are exclusively peculiar to the tropical fauna of America, we are surprised to find that no single species of the group is at all as brilliantly colored as coenia. All of them have a rather dull coloration and fewer or at any rate smaller eye-spots. ‘The most nearly allied form from which ccenia could be supposed to have arisen, is J. genoveva Cramer. Jamaica seems to be the true home of this species, but it has already spread so far over South America that the northern- most limit of its range coincides in part with the southernmost limit of that of J. coenia. Its pattern, which everywhere remains constant, except for the sporadic appearance of slight differences in shade of color, proves that genoveva is phylogenetically a very old form. ‘That genoveva and ccenia are to be regarded as two decidedly distinct species and are not to be united in one species, as, for instance, Dyar has done in his “List of North American Butterflies”’ (Washington, 1902), is proved by the facts, first that there are no true transitions between the two species; second, that from none of the experiments with high temperatures did there result a butterfly which even approximated to the genoveva type. Nevertheless, at least a partial reversion to the primitive form should have taken place through high temperatures if coenia were not a species that had been fixed for a long time, for, since the 560 William Reiff tropics are the original home of its ancestors, atavism could be produced only through the influence of heat. I could obtain no reversional forms by this means, but, on the contrary, all the but- terflies departed still further from the series of forms which we have been considering; hence, the supposition lies near at hand that in the north temperate zone ccenia has become so far detached from its original home that the species has already lost the ability, or has already become too old to produce atavistic forms. ‘This would agree with the view which | have previously stated, that coenia had already become a stable species in the nearctic fauna before the glacial period, and that it continued to exist further south as coenia during this period, and then again took possession of the north after the expiration of the ice age without change in its coloration or markings. Another fact that lends support to this view is that the nearly related Pyrameis atalanta L., and P. cardui L., react much more decidedly to the influence of tempera- ture, so that the. phylogenetic age of these species‘must be much less than that of J. coenia. Nor does the distribution of these species contradict this view, for atalanta, and especially cardui, belong to the best flyers among the butterflies. If, therefore, the forms which one obtains through heat, and which are more or less decidedly modified in one direction, show no reversions, they must necessarily be regarded as progressive forms. It could be objected, perhaps, that heretofore in the experi- ments of European investigators, progressive forms, so far as heat- ing the pupz is concerned, have been produced by constant and only moderately high temperatures, but we must bear in mind that in the middle zone of the United States, where J]. ccoenia is most abundant, the temperature that prevails during by far the greater portion of the year is the one which was artificially produced in the European experiments. Hence I was, of course, obliged to expose the pupz to a relatively higher temperature. The experiments of European investigators have shown, however, that in one and the same species, pup exposed for a certain time to moderate abnormal temperatures, may produce in a correspond- ingly shorter time the same varieties as are produced by employ- ing high temperatures. If the heat forms that have been obtained Contributions to Experimental Entomology 561 of J. coenia are really to be regarded as progressive, no case of atavism should appear among them, but, on the contrary, new markings should make their appearance, such as have not been found heretofore in other Junonia species of this group. The absence of all reversions was considered above, and I have also, while describing my experiments, called attention to the appear- ance of new characters in the pattern. ‘These differences in the markings consist mainly, to repeat once again, of the decidedly broader and more vividly colored, reddish orange band on the hind wings, in the appearance of the band in the anterior wings and the origin of new ocellate markings in the characteristic row of eye-spots. Through the kindness of Mr. Doll, of Brooklyn, I have been able to examine a large series of Junonia species, which were partially caught and partially bred under natural conditions. Now, among the many individuals of the ccenia group in this collection, there are a few which show very clearly the characters just mentioned. One specimen, in fact, has on its hind wings a completely normal, fully-developed, third eye-spot. I regard it, therefore, as highly probable that J. ccenia is even now in the process of changing in the direction indicated. ‘This is suggested also by the rather frequent occurrence in nature of the difference in the breadth of the orange-yellow band of the hind wings, for if the progressive form were to be conceived as still to be initiated in the indefinite future, the characters described should not be found in nature and ccenia should be much more constant in its pattern in the southern portion of its range than it is. But how are we to regard the cold forms that have been obtained experimentally ? These could not be cases of atavism, since a re- version through the influence of cold would be impossible in a species whose original home is in the south. No doubt J. ccenia is in the act of adapting itself to cold. If, through my experiments, diametrically aberrant forms had arisen, there would be great difficulty in reaching a conclusion; but as only one direction of variation is observable, we can place all the specimens in a single common group, which has just the opposite coloration from that of the warm forms. The principal characters may be again reviewed; the general darkening of the upper surface of the wings, 562 William Reiff the white and red scale formations are replaced more or less by black, eye-spots all smaller and tending to disappear, and the disappearance of the halo surrounding the eye-spot so that the center begins to shade over into the ground color. It is to be noted also that the two red spots on the anterior border of the anterior wing are scarcely changed in the slightest degree, either in these or in the experiments with warmth, and this must point to a great phylogenetic age for this portion of the markings which is SO Se of the species of Junonia. If, however, as I have said, there is no atavism in these cold forms, we must regard the changed specimens as being without doubt progressive forms. It seems peculiar, and certainly it may be advanced as an objection, that one species of butterfly can have two prospective forms, which nevertheless, are in strongest contrast to each other. Let us examine this question more closely. As IJ have stated before, J. coenia will probably take on, in the not very distant future, a somewhat different coloration and pat- tern in the portions of North America which it has continuously inhabited. In these regions and because of the mild winters, there has been no obstacle to the survival of the species, but the conditions have been quite different in the northernmost portions of its range, for here J. ccenia can never become indigenous on account of the severe cold of winter unless the species acquires the habit of hibernating. As I have said in the beginning of this paper, caterpillars and pupz of ccenia die at what would be a very slight fall of temperature for the northern winter. Hiber- nation of the eggs can hardly come into the question, for the last generation of our Pyrameis species, which, as I have several times remarked, are closely related to Junonia, does not pair till spring. Moreover, it is also known that butterflies pair, as a rule, only after having flown for a considerable period. For the generation of ccenia which hatches in its northern range from the end of October on, there would not be sufficient time to attain sexual maturity, but this insect does not, as a rule, hibernate in the north even as an imago. No doubt the last generation attempts this, but only a few specimens survive the winter. How could we other- wise understand that in spring the butterfly is a great rarity in Contributions to Ex perimental Entomology 563 places where it was found in great numbers during the previous fall? A good parallel to this case is that of the Pyrameis atalanta L., and P. cardui L., of which Standfuss says verbally in his “Handbuch der palzarktischen Gross-schmetterlinge,’’ Jena, 1896: “Although individuals of atalanta and cardui may be observed during high summer and fall as a rule in quite as great numbers and sometimes even greater numbers than the butter- flies of antiopa, polychloros, c-album, io, and urticz, these two species are in general very much more rarely seen in the spring than the series just mentioned. Only after very mild winters are atalanta and cardui found to occur frequently in spring. Our severe winters evidently kill off most of the individuals of these two species, which have not yet sufficiently acclimated themselves to such low temperatures, and are also much more rarely seen hibernating than the numerous species we have sup- posed to originate in northern latitudes. Although the second generation of atalanta and cardui is not exactly rare during most years, this is due to the extraordinary flying powers of these species, which continually push forward from milder into more inclement regions.” (p. 302.) We may, therefore, agree with the view which Scudder advances in his “ Butterflies of New England,” that hibernation is extremely rare in J. coenia throughout its northern range. It would seem that, notwithstanding the much greater phylogenetic age of coenia than of atalanta and cardui, it is much more difficult for the former species to accommodate itself to severe cold. But if ccenia is to take permanent possession of the northern portions of its range, and not be continually recruited in this range by specimens from the south, it must acquire the ability to hibernate. It seems prob- able that the imaginal insect is destined to do this, although it 1s not impossible that the pupa may acquire the ability, for, as | have stated at the beginning of my paper in connection with the data received from Mr. Doll, caterpillars of all sizes are found in the field till late in the fall, and this, of course, means also the pres- ence of pup. If such pup should hibernate the butterflies aris- ing from them would certainly not have the same color pattern as those whose whole development occurs during the warm sea- 564. William Reiff son. We should expect, therefore, to see the two generations of J. ccenia taking on a seasonal dimorphism in their northern range, and the causes of this would be just the opposite of those which have operated in the production of the seasonal dimorphism of the European Araschnia levana L. Concerning the importance and origin of the seasonal dimorphism in this latter species, A. Weismann has given an extended and clear account in his “Stu- dien zur Descendenz-Theorie,”’ Leipzig, 1875. I am, therefore, of the opinion that the coenia which I obtained from experiments with cold, show the direction which the colora- tion and markings of the winter form will take in future time, if the pupa should acquire the ability to hibernate. That this time is still very far distant, is shown, for example, by Doll’s collection of ccenia, which among its great riches, shows only one specimen that approximates to the cold forms. If we now bring together what has been shown by the experi- ments with warmth and cold, we come to what is certainly the correct conclusion, that J]. coenia Hb. is about to produce a local form in its southern range, and that in its northern range it will bring forth a seasonal dimorphic species after a considerable period of time if the pupa, instead of the imago, should acquire the ability to hibernate. A few words may be said about the melanistic form of Junonia coenia, the ab. negra Felden. Through the kindness of Mr. Doll, of Brooklyn, and Mr. H. H. Newcomb, of Boston, I have been able to exarhine a fine series, together with transitions, of this aberration, which is known only from the southern and south- western range of the species. Its origin in nature is probably to be traced to extremely dry heat acting on the pupa, but this can only be decided by further experimentation. It is certain, how- ever, that negra is not to be placed in the same group with the darker cold forms, since even in the most strongly melanistic speci- mens the eye-spots are still distinct and of normal size, whereas in the cold forms, even in light-colored specimens, there is always a diminution or incipient dissolution of these spots. Whether it would be right to regard the ab. negra as a partial reversion to J. genoveva, I will not undertake to decide. Apart from the — Fie ne i ee ee " Sl hd Contributions to Experimental Entomology 565 small differences in the form of the wing and in the pattern of the upper surface, the lower surfaces of both series of individuals present such a strong contrast to the ab. negra that it would seem impossible to draw any conclusion without more extensive inves- tigation. In conclusion I would call attention to some aberrative speci- mens of J. ccenia which I raised from normally treated pupz, which, however, had an abnormally strong depression in the wing cases. ‘The eye-spots are all greatly enlarged, in part elongated and so large on the posterior wing that they come in contact with each other. Mr. Doll has in his collection several specimens that exhibit the same peculiarity, and were probably produced by the Same causes. Another pupa, left under normal conditions, also, probably in consequence of some pressure to which it had been subjected during pupation, exhibited a slight but sharp depression in the right anterior wing case. The butterfly which hatched from this pupa showed an absence of scales in the corresponding region of the wing. Experiments undertaken for the purpose of ascertaining the effect of sulphuric ether vapor on the developing coloration of the butterfly led to no result, as the pupz died. In conclusion I wish to express my gratitude to Mr. Jacob Doll, Mr. H. H. Newcomb and especially to Prof. W. M. Wheeler for their kind aid in my investigations. II. TWO CASES OF ANABIOSIS IN ACTIAS SELENE HUBNER In his second volume of Experimentelle entomologische Stu- dien, Sophia, 1907, Prof. P. Bachmetjew discusses on pp. 684-686 the anabiotic condition in insects. By means of calorimetrical measurements on Lepidopterous pupz he established the fact that the juices of the insect body do not completely congeal till they have been reduced to —4.5° C., but that at this temperature the Insect does not yet die. Permanent cold rigor, or death, sets in, on the contrary, under very different conditions, as the author has shown in the first volume of the work to which I have referred. If, now, the juices are congealed, Bachmetjew further states, 566 William Reiff the insect is in a condition in which no metabolism can take place, for the circulation of the blood has then become impossible. An insect without metabolism cannot be regarded as being still alive, but nevertheless it is not dead, since it has not reached the point of permanent cold rigor. It is, therefore, in a transitional con- dition, a lifeless or anabiotic stage. ‘To aid in the understanding of the conception of anabiosis Bachmetjew’s explanation may be repeated in his own words: “The anabiotic condition is not one of lethargy, for in lethargy metabolism still takes place, although very slowly, till the insect finally dies of inanition. The condition under discussion can be better compared with that of a clock in which the pendulum has been intentionally stopped. Of course, the clock has not been injured, but it does not go. On pushing the pendulum the clock shows that it is still intact; and just as the clock with a motionless pendulum can remain uninjured for an indefinitely long period of time, so, presumably, an insect could remain for an indefinitely long time in the anabiotic condition without dying.”’ (Vol. 11, p. 685.) From some pupz of Actias selene imported in November, 1908, for hybridization experiments during the coming summer, there hatched as early as the twentieth of November a female moth, which I left out-of-doors in a wire cage till November 22d inclusive. At this time the temperature for several days had been unusually high, and was undoubtedly responsible for the premature hatching of the insect. From the twenty-third of November on I subjected — the moth to a continuously low temperature, varying from —3° C. to —6°C. Daily observation showed that the moth remained unchanged and motionless, with spread wings, on one of the walls of the wire cage, and that it responded to no external stimuli. It should be noted, however, that the stimuli were never sufficiently — severe to injure the specimen. In order to ascertain whether it was still alive, I carried it over on the third of January, 1909, to the outside temperature, which was on that day about +5° Cm Although the moth retained the same attitude as before, it never- ! theless moved its antennz and legs when it was stimulated, but the slight rise in temperature was not sufficient to cause it to move spontaneously, nor did January 4, with an average temperature Contributions to Experimental Entomology 567 of about +7° C., produce any change. Only when the unusually warm fifth of January arrived, which had an average temperature of about 17° C., did the moth, of its own accord, move its anten- nz, legs and wings and wander about languidly in its cage in the manner so often observed in the females of the allied Saturniids. On the evening of the fifth of January, I placed the insect in a moderately heated room in order that it might not be subjected to a sudden change of temperature that has been predicted by the Weather Pee On the following morning, however, it was dead. Contrary to my expectations, this female selene did not lay a single unfertilized egg during her whole life period. An examination of the very much swollen and unchanged abdomen, showed, however, that the ovaries were perfectly normal. On the third of January, 1909, which was a mild day, there escaped from the Actias selene pupz that had been left out-of- doors another specimen, which proved to be a male. This speci- men was left till the fifth of January, inclusive, in a cage of its own out-of-doors, and was then treated from the sixth of January to the fourth of February inclusive, in the same manner as the formerspecimen. On the fifth of February, which had an average temperature of +6°C., I took the moth out of doors again, where it remained till the afternoon of the seventh of February, when it died. The temperature of the sixth of February averaged about + 13° C., while the average for the seventh of February was about +4°C. With the single exception that in this second experiment the insect reacted more readily and in a more pronounced manner to the rise in temperature—the males of the allied Saturniids are always very lively—and even made attempts to fly about its cage on the warmest day (the sixth of February) the conditions coincided with those of the individual on which I first experimented. Summarizing the results from the time of hatching of both moths we have the following table: 568 William Reiff | i | o.U°8 : a oh zZEe z AGAIN IN a a a | EX. LARVA | IN TEMPERATURE IN ANABIOSIS DEATH Oo. Bee a | TEMPERATURE 20 6 & real ON AReyE OC.) | —i2co)— 621. = OCCURRED | 3 Z = & 2 18 | ABOVE o° C. = 28 < | } 32 deals with the adjustment of Paramecia to distilled water. Part II, the present study, has to do with the adjustment and immunity of Stentor and Spirostomum to ethyl alcohol. At this point I should like to express to Prof. H. S. Jennings, under whose direction the following researches were made, my highest appreciation for the sincere sympathy he has shown at all periods of my work upon this subject. IMMUNITY OF STENTOR AND SPIROSTOMUM TO ETHYL ALCOHOL I INTRODUCTION The effect of alcohol upon living matter has long been a subject for observation and experimentation. Its marked influence upon man makes it a problem of the greatest practical concern to the race. Aside from this, the nature of its action, especially in the case of its use in moderation, is of the highest scientific interest. Within the past decade notable advancement has been made in this latter direction. Probably the work of no single investigator has been farther reaching in its results than has that of Atwater.*® From his extensive and thorough-going investigations we have ® Published 1908, Amer. Jour. Physiol., xxiii, pp. 49-63. ® Atwater, Physiological Aspects of the Liquor Problem, vol. ii, 1903. 574 F. Frank Daniel learned much of the real nature of the relation that alcohol bears to the process of metabolism. ‘The former notion that alcohol facilitated the storing up of fat by retarding metabolism has largely given place to the view that alcohol is itself oxidized in the body, thus preventing or retarding the oxidation of other materials. Further than the fact of its oxidation but little is known. In the following investigations I have undertaken a study of the general effects of alcohol upon single-celled organisms. For work of this sort much depends upon the type of cell selected. Probably no single requisite is more important than that the organ- ism_be_of sufficient si ermit ready determination of the moment_of death The blue Stentor (S. coeruleus) meets this requirement admirably. In addition, its habit of attachment gives it a marked advantage over more active infusoria, and its characteristic reaction to light makes it easily obtainable in larger numbers relatively free from _deébris. In determining the fatal dose of alcohol for Stentor it will be. well briefly to survey the effects of weak percentages in general. II PRELIMINARY EXPERIMENTS A Effects of Minimal Amounts of Alcohol Alcohol even in very low percentages is generally regarded as a ‘protoplasmic poison.” Its deleterious effects in small .doses depend much, however, upon the kind of organism studied. Hodge’ in experimenting upon developing yeast found a de- crease in division, in solutions contajping as low as zy of I per cent of alcohol, the average number of cells in this strength being only 992 per cubic centimeter as compared with 2061 under nor- mal conditions. On the other hand Maltaux and Massart® oi ee and a for “‘one ie Penee of the life cycle” increases the division rate. n io. Parambciumn cig, fureher important to nots aan it 1s cae important to note that the increase was lost do Taramecnr 11s further portant fo note that the merease time. 7 Hodge, 1897, Pop. Sci. Monthly, vol. 1, pp. 594-603. 8 Maltaux, Marie et Massart, Jean. 1906, Recueil de l’Institut Botanique, vi, pp. 269-421. 9 Woodruff, L. L., 1908, Biol. Bull., xv, pp. 85-104. Immunity of Lower Organisms to Ethyl Alcohol 575 Richardson” in determining the death-point for a fresh-water medusa found that from 3'5 per cent to ;'5 per cent of alcohol proved lethal to this form within a short period of time. Reid Hunt" in his fascinating Studies in Experimental Alcohol- ism has obtained in higher animals the first positive evidence, so far as | am aware, of direct injury due to minimal doses of alcohol. These experiments upon white mice made it evident that amounts of ethyl alcohol far too small to produce indications of intoxication are capable of rendering the animal more susceptible to a definite poison,—acetonitrile. sIn my experiments I have found protozoa comparativel ist- _ weeks at a time. Solutions of alcohol as high as 4 per cent in strength were in all cases early destructive of the protoplasm of Stentor and percentages of 2 and 3 per cent produced death after a period of six and two hours, respectively. B- Reaction to Stronger Percentages 1 Description In the following study two very different strains of Stentor— which we may designate as E and F—were employed. These showed marked differences in their reactions to ethyl alcohol. Type E, composed of cells of immense size, deeply pigmented, and actively free-swimming, came from a black infusion of de- cayed vegetable material. “Type F on the other hand—composed of cells of medium size, and usually attached, grew in a clear medium of tap water and Chara. These latter cells maintained a sturdy condition with slow rate of division for long periods of time. The two types in a sublethal medium (e.g., 1 per cent alcohol) showed the following characteristic differences in behavior. The : . a = large cells E upon subjection to the alcohol were stimulated to 10 Richardson, 1888 (July), Asclepiad. 4 Hunt, Reid, 1907, Bull. No. 33, Hyg. Lab. U. S. Pub. Health and Mar. Hosp. Serv., Washington, BC. 576 F. Frank Daniel marked activity. After having remained in the medium for a day an acceleration in division was observed which resulted in cells of smaller size. ‘Type F, on the contrary, in this weak solution of alcohol showed slight increased activity upon subjection, and practically no increase in the rate of division upon remaining in the medium for a short period of time. When these two types had been kept in a weak solution for some time, and were then placed in stronger alcohol, they showed still more important differences. Type F gained a marked immunity as a result of remaining in the weak solution, while type E showed very little effect of this sort. ‘This difference will be brought out in full in the following investigations. 2 Method of Studying Immunity Tests for the immunity resulting from a weak solution of alcohol may be made in one of two general ways. Either (1) the com- parative resistance period of alcoholized and control (normal) animals to a known fatal percentage may be determined, or (2) the ability of alcoholized animals to live in a solution which was previously destructive may be tested. The two may be stated more comprehensively in the following questions: 1 What is the effect produced upon the general resistance of an organism by rearing it in a weak chemical, for example alcohol, and testing it to a strong solution of the same substance. 2 Can animals by living in a weak concentration of a substance be made to survive a stronger percentage of the same? For example, can an organism which is normally killed within a few hours by a 2 per cent solution of alcohol be made to live in the same strength ? 3. Experimental As has been mentioned, the answer to these questions depends upon the animals used. Type E, kept for different periods of time in a weak medium and then tested to a stronger percentage, did not give marked evidence for immunity on either of the tests mentioned above. ‘Type F, on the other hand, reared similarly, Immunity of Lower Organisms to Ethyl Alcohol 577 gave clear evidence of immunity when tested by either of the meth- ods. ‘This will be seen from the two experiments which follow. The first of these, in which F was tested for an increased resist- ance to a fatal dose, was made in the following way: Into each of two dishes, designated respectively as A and C, Was put 5 cc. of a natural culture medium, containing twenty normal Stentors. ‘lo A was then slowly added 1 cc. of 6 per cent alcohol, forming a I per cent acclimatizing medium. After a few days the animals from A (acclimatized to 1 per cent alcohol) and from C (control) were tested to a 6 per cent concentration— each experiment consisting of a drop (either of A or of C, contain- ing a single Stentor) added to 1 cc. of 6 per cent alcohol. The - death-point was taken as the instant at which ciliary movement ceased.” ‘The period of resistance—the number of seconds that the organism survived—was marked in seconds. The experiment shows the following comparative results: Experiment I. RESISTANCE OF STENTOR OF TYPE F TO 6 PER CENT ALCOHOL AFTER LIVING 4 DAYS IN I PER CENT ALCOHOL A Four Days in 1 Per Cent Alcohol C Control Animals Seconds Seconds PER PABURETTAISEO Diss 5, -[s.0. 5 2)5,c18. 8 0/010 ele Siei0 0's Keo. VExp dy GiliaystOpasacastsieee meen: = 100 PRGUNAESEOP e's < lac isis ees Sies.aisis “516 « 130 DUC AYStO Panweneisiren asics 145 2) Bile GD Dies aaa etc eee 300 q ciliaystop: ssf. st oa nmstetes ale 120 BMCTMABSEOPs 2oyci'sis c. s/s ose 76 ss storia 720 a. ciltaistopresee settee ara 120 FRCRIGESEO Piano cls. <0 cIe's xha.a\sd Brie sd 220 GeCilia-Sto pect weer 160 Scilla Sst Pew sei) e rere tal 75 GRCUIATSOD se). vi Fe sie ce oer eaine mec 330 QiciliaStopys scite seis se straitetner= 245 MOR CUIAtSEOP siere/eic'nie « atelorcteiele ts ere 420 TOT CUayStOP jade arate niiegye ss 255 Average resistance = 257.5 Average resistance = 258 From the above it is reasonably certain that the low increase in resistance shown in Experiment III was not due to an injury attributable to the 1 per cent acclimatizing fluid, for in the same strength, to which the animals were subjected so gradually as to be without injury, no acclimatization whatsoever resulted. Since no injury and very doubtful acclimatization is evident in a medium of I per cent strength, the question naturally follows : May not the low degree of immunity be due to the fact that the medium is too weak to produce an immunizing effect ? To test this, Stentors were brought through a graduated series of transfers to the highest percentage in which they could live without apparent injury—a 1.5 per cent solution. A test to 8 per cent alcohol of animals from this medium resulted as follows: 584 F. Frank Daniel Experiment VII RESISTANCE OF STENTORS OF TYPE E TO § PER CENT ALCOHOL, AFTER LIVING 4 DAYS IN 1.5 PER CENT ALCOHOL A Four Days in 1.5 Per Cent. C Control Seconds Seconds Exp. Treiliatstop a. waccer tere eee 40 Exped Cilia stops... cieieniw onic eer eee 50 Drcilia StOP weses titties aes go 2) Cilia ‘SCOP 5s ss seieiere aisie)si-e'e oe eee 35 AECUE SN) he sccan daa ao Unoeoe. 45 2 Gilialistop.s--9.< ose see ee 75 MICHA SLOP aserece ate aa eciciere 75 AICI ATSEOP ce steele oe oie eee 55 RELICS 0) Dp naco 5 aaubebmosoucd: 30 Pptet 80) Hoeedomanapaaonues omc 30 Gretlta sto peeae sweeie steiner 35 Giciliarstaps..2.ceeeens sete 105 7 CUlaStOP) see cites aes ore = 45 7 Cilia, SLOP’ " See ne —— Immunity of Lower Organisms to Ethyl Alcohol 587 individual differences in their susceptibility to alcohol—some quickly adjusting themselves to it, others doing this with greater difficulty. Similar variability in results may be due to the fact that the acclimatizing medium is too strong or too weak. In either case there may be entire lack of immunity, or immunity may be deferred. Lack of immunity may be due either to a medium too weak to produce any effect, or one so strong as to produce lasting injury. Deferred immunity may be due to the fact that the medium 1s so weak that a long period is necessary for producing an acclimatiz- ing effect, or it may come in a stronger solution when the first effect is injurious, but is later replaced by the acquirement of immunity. In several cases I have noticed that animals tested soon after subjection to the acclimatizing medium showed a decrease in resistance. ‘This in some cases lasted several hours; in others it was of short duration and was followed by evident adjustment. In an acclimatizing fluid of medium strength, an early evidence of immunity may be expected from average animals by the end of the fourth or fifth hours. The degree of possible tolerance reached evidently depends upon factors similar to those which we have just set forth. Among these may be especially mentioned—the effects of differ- ent percentages of the acclimatizing media, the period of time during which the animals are subjected to such media, and the condition of the organisms at the time of subjection. In my work upon type F, a medium of 1 per cent alcohol has been found most satisfactory. It has, therefore, been adopted as the standard acclimatizing fluid in which all of the animals of A, with but few exceptions, have been reared. If the medium in which the animals are kept be of a strength lower than 1 per cent the immunity produced by it may be expected to be propor- tionately less in degree at a given time than that from a I per cent medium—provided, of course, the strain suffers no perma- nent injury from this latter strength. Using then a 1 per cent solution of alcohol as a unit we may pro- 588 Ff. Frank Daniel ceed to experiments which determine the relation that slightly differing strengths bear to different degrees of immunity. In a solution of 0.5 per cent the evidence for adjustment to alcohol though sometimes slight is usually clear after a short time. In this strength evidence may be expected which by the end of the fourth day is definite. By this time the degree of immunity pro- duced by two acclimatizing media—for example one of 0.5 per cent and the other of 1 per cent strength—should be sufficient to be compared, and their differences noted. An experiment follows in which animals from 0.5 per cent and I per cent media were tested to 6 per cent killing fluid. Both were further controlled by the resistance of normal animals. Experiment IX RESISTANCE OF STENTORS OF TYPE F TO 6 PER CENT ALCOHOL AFTER LIVING 4 DAYS IN SOLUTIONS OF DIFFERENT STRENGTHS A Four Days in 1 Per Cent Alcohol Seconds EXP eytaCiliay SLOP ye see ctslate slolte ayet ators eiels 190 BECHIal SLOP a cislelaie ste ietetaiae clot is telote 330 Gucilialstopecen-ciestearst- ciyroe cil 140 AV ENINEY Sapo onogoosqcoseooLcondS 180 3 GUE Sie oosondoboepocdoedoNd 350 Giciliakstoperietseemeee tee ste 160 FACUIAIELOP Semester eclselemine testers 195 C Control ONE Aoi Nesodohcasccanpanconsec 180 Seconds Oicilia stoprcacicteis sate darelacieeisercle 240 Exp: 1. cilia’ stop.;..5.0. 0 esse eee 130 HOMCIMARSEGP see otarefol otter eiciertel ae = 330 Zz cilia’ stop... : 80 156.5 80.5 From Experiment XII it is seen that the increase is much like that given in type F of Stentor. Although in Spirostomum there is a greater range of variability yet the striking feature in the many cases observed was that although the normal animals varied in their resistance from time to time the ratio between acclimatized and unacclimatized (control) animals remained practically the same. Immunity of Lower Organisms to Ethyl Alcohol 597 From experiments in an 8 per cent solution, another point of extreme interest was observed. Organisms often showed the first signs of injury within the usual time—one to two minutes—and disintegration followed in the usual way. Upon reaching mid-body, however, this was suddenly stopped. At the point of injury a round plug of proto- plasm formed, filling up the wound. ‘Thereupon the cilia resumed a backward stroke and the body moved forward in a normal fashion. This phenomenon was observed again and again as the method by which the organisms often prolonged life for considerable periods of time. In the same way that type E of Stentor was tested on the second, fifth and seventh days, we may test Spirostomum to see the general indications of resistance. These results, shown in a condensed way in the following table, are different from those obtained in type E of Stentor. Experiment XIII RESISTANCE OF SPIROSTOMA TO 8 PER CENT ALCOHOL AFTER LIVING IN I PER CENT ALCOHOL. A = LIVING IN I PER CENT ALCOHOL. C = CONTROL Second Day Fifth Day Seventh Day A Cc A c A Cc Seconds Seconds Seconds Seconds Seconds Seconds 45 go 3h) ge 2K) 45 205 150 240 220 260 55 110 45 75 72 195 Wee) 55 190 Jo go 330 40 go 65 215 110 55 50 200 150 255 50 go 50 120 105 7o 70 190 85 95 65 255 60 60 50 225 go 205 7o 190 60 95 92 79 45 rO% eae, Average resistance ....124 104 165 83.5 168.5 65.5 In the foregoing series only a slight increase for the acclimatized animals is shown on the second day. It will be noted, however, that the normal resistance (104 seconds) is high. In other cases 598 F. Frank Daniel a better increase was shown at the end of the first day than is here given on the second. Probably the most typical results for the series are those on the fifth day. On the seventh a slightly higher average obtains for A, but C is less resistant. Had it been typical, however, there would still be a ratio slightly above two to one. Another point may be noted in passing. ‘The control animals, unlike those of Stentor, instead of increasing in resistance, showed a gradual decrease. A further study was made in Spirostomum of the immunity shown at considerably later periods of time. The greatest difficulty encountered was in keeping the acclim- atizing medium A at its usual strength. In order to do this two plans were tried. In one the culture was changed every few days; in the other it was kept in ground glass vessels which were sealed and set at constant temperature until the time of experiment. The latter method was adopted as giving better results. An experiment under these conditions follows in which the organisms were tested after eleven days in the acclimatizing and control media. Experiment XIV RESISTANCE OF SPIROSTOMUM TO 8 PER CENT ALCOHOL, AFTER LIVING II DAYS IN I PER CENT ALCOHOL A 11 Days in 1 Per Cent Alcohol C Control Seconds Seconds Expr giiCiliaestO Pease ee nest erin 190 Exp: 1 ciliaystops..<. 4,440. 30 AMEE Sioa acoadecoogaseunasse 50 2 cilia StOP: «<5 i. + 2211s Sa Z| Gch ei) sboudenadoucstacdds: 85 2 cilia stop. 22. ae eee 25 ATE NEU REO Msscoohecna kebenausoc= 95 4 cilia stops... 2:22 eee GaGlren Sia yascanocobdobedabhouss 95 § cilia: Stop. 2... «ej >to eee ee Giciltaistop-ni.ss ste se see eats 390 G6 icilia) stop). <2 2 ses oe 30 FGUENA an oaotos Sania Soon c 45 7 Cilia’ StOps .'s.00 40+ <2) 65 SieilG@rstop. che. sege eee oe 285 8: cilia stop... 53.2:.5/.. 0.2 Ouciliagsto pases seee sore aie: 150 9. cilia stops... o..0% 7: eee ROVGIASSEO Dire eer ern ereiereleraraterey=ts 7o 10 \cilia Stop. ois. ss Adee eee 30 145-5 64 We see from this that although the resistance of the control was low, the acclimatized animals have retained their immunity for this period of time. Immunity of Lower Organisms to Ethyl Alcohol 599 A later series at the end of the second week gave the following similar results: Experiment XV RESISTANCE OF SPIROSTOMA TO 8 PER CENT ALCOHOL AFTER 14 DAYS IN I PER CENT ALCOHOL A 14 Days in 1 Per Cent Alcohol C Control Seconds Seconds ISPEMTECUISEStOP es ceiceie ps aioe vines ale eis 215 13s (ET POTEN ENG 10) Noor con nGUn OBO A OOse 25 SPRUE AUSEG Pa fays'scc10)=35 miele» e/eie ereraoie 60 ZiCilia) StOP ere «alelsiciere stele eho otal ote 125 RICINARSLOD ninie(sictci0/«'o'15' «1010141 s\vie.s ovajeie's 250 [5 CRINEMAZC| Da Linde ¢ SGGc IG on OC re 55 GTEHIARSEOPaais'0:5 <8 5)0.< + xels oe ots eer 230 G6 ciliate stops iiecnie ao steve ots ete lela wci 5° BMEIFRCTL A SLOP inis\.c 00 cin.s «iso, n\0/0 woe oe 65 PECULARSEO Dujereiets fale sfaiereateeloeareTate 95 BBOCMIAESLO DE. 20's eisie% 2. «cee a eicte 55 8 ciliaistOpancoet is -ainste leiAe pees 30 3) GUE Si0) Sec. guocamenigess JenGure 95 Qiciliatstopeaeer re saci et 120 EEMCTIANSEO P)sia'oroitin) ele aie) = oie. 0(e/+ «3/011 50 TO\ciliaistop: «sesso cera 30 E2735 61 From these two experiments it appears probable that immunity remains as long as the killing fluid is not markedly weakened. A study of Spirostomum shows a marked degree of similarity between its reaction and that of type F of Stentor. In both there was a well marked immunity which by the end of the fourth day had reached a degree of constancy. The experiments just described show further that the immunity which was constant on the fourth to the seventh day was still present when tested at a much later date. With this we may conclude the present study and pass to a con- sideration of another phase of the nature of immunity. IV SPECIFICITY OF IMMUNITY Ehrlich*® found that white mice which were immunized to ricin were still susceptible to another poison—that of abrin. ‘The immunity conferred by the one substance in this case did not carry with it protection against a different substance. In other words its action was specific. 16 Ehrlich, P., 1891, Deutsche med. Wochenschrift no. 12 (ricin), no. 14 (abrin). 600 F. Frank Daniel Whether the same specificity holds in the case of single cells where a lower degree of immunity obtains, is a question to which considerable interest attaches. ‘The problem may be stated thus: Will acclimatized unicellular organisms, which show a stronger resistance to alcohol, also demonstrate an increased resistance when tested to a fatal dose of another and different substance? To determine the point in question organisms which I have found could be rendered immune to ethyl] alcohol have been tested: (1) to substances radically different from alcohol and (2) to sub- stances in a way related to alcohol. In the first case normal and acclimatized animals of both Stentor’? and Spirostomum were tested to hydrochloric acid and to sodium hydroxide. In the sec- ond, they were subjected to other alcohols—methyl alcohol and glycerin. In these studies attention has been directed to two things: First, to the physiological effects—that is, to what the organism did; and secondly, to the chemical effects, or to what changes the chemical produced. The first point was to see whether normal and acclimatized animals acted in the same way or differently in the same killing medium. A second was to determine whether there was any dif- ference in chemical effect upon the two sets—the acclimatized and control animals. If such differences were noted either in behavior on the part of the organism or in action on the part of the chemical, a further proposition would be to see if any relation exists between the two. A The Action of Ethyl Alcohol (C,H,0H) As a standard for subsequent observation and experiment we may repeat earlier experiments in order to see what takes place when control and acclimatized animals are subjected to a fatal dose of ethyl alcohol. Normal unacclimatized Stentors (type F) when thus subjected to a 6 per cent killing fluid showed slight or no bodily movement. At from 15 to 35 seconds there was a strong antero-lateral (aboral) 17 In this study when Stentor is mentioned type F is meant unless otherwise stated. Immunity of Lower Organisms to Ethyl Alcohol 601 bulging and extrusion of the protoplasm, followed by a rapid loss of color. The body cilia ceased moving early, but the membranellz continued with strong stroke until nearly the time of death. Acclimatized Stentors, on the other hand, upon subjection to the 6 per cent alcohol usually remained motionless. Early distor- tion, then uncertain beat of cilia up to 45 seconds, was followed by an increase in ciliary activity. ‘This finally became so vigorous as to shake the whole body mass of protoplasm. In this, as in our previous study, an extreme tenacity of life was manifest. Thus both in behavior and in chemical action differences were produced. The relation the one bears to the other, however, is difficult to see. Notwithstanding the fact that a stronger external effect was produced upon the acclimatized animals than upon the controls, the former survived a greater period of time in a lethal percentage of alcohol than did those of the control animals. We may now examine the phenomena in acclimatized and con- trol animals when hydrochloric acid is used as the killing fluid. B The Action of Hydrochloric Acid (HCI) 1 General Effects Unacclimatized Stentors tested to an ,“, solution. In a concen- tration of this strength an early rotation was noted which ceased soon after injury began (15 to 30 seconds); the membranellz stopped early. The body upon losing its color became brown. A little later contortions of the protoplasm were followed by a splitting away of the body mass from the cell wall and a forming of this mass into a coagulum. After this action no ciliary motion was seen excepting in the buccal cavity. A cclimatized Stentors. With the exception that a slightly greater activity was shown in these than in normal Stentors no difference either physiological or chemical could be detected. The most striking phenomenon of the above study, observed both in normal and acclimatized animals, was the peculiar way in which ciliary activity was stopped. Just before death, pro- 602 F. Frank Daniel nounced contortions of the protoplasm were the forerunners of a wave-like action which passed posteriorly, splitting away the endo- plasm from the pellicula and forming of the endoplasm a typical coagulum. As a result of this action, all ciliary movement ceases, excepting that of the buccal pouch. (At this location, pellicula and endoplasm were not early separated.) Destruction came about more rapidly in the case of acclimatized animals than of the controls; this may be seen from the following experiment upon resistance (Stentor). But the reason for a much earlier splitting and coagulum formation in the one case than in the other is by no means clear. > 2 Effects Upon Resistance In the following experiments upon both Stentor (type F) and Spirostomum, a notable difference was seen between normal and acclimatized animals when tested as to their endurance in a lethal dose of hydrochloric acid. In both cases the acclimatized animals were reared in a I per cent medium of alcohol. ‘These at the end of four days showed an increased resistance to 6 per cent alcohol. ‘They were then tested to =“, and ;™, concentrations of HCl, respectively. Experiment XVI RESISTANCE OF STENTOR AND SPIROSTOMUM TO Hcl AFTER ACCLIMATIZATION IN A I PER CENT SOLUTION OF ALCOHOL a_ Stentor b Spirostomum Tested in ,™. Sol. HCl Tested in ;M~ Sol. HCl A & A € Acclimatized Control Acclimatized Control to Alcohol to Alcohol Seconds Seconds Seconds Seconds EXpon 1 Giltayetaprec sc. ssf--0 ce 60 T7 ON ee EXpo eT CitiayStOp... cane oat. 150 150 2 CINANSEOD 2). «-e01- ie « otc * 210 210 DGita sStO Poise 40 285 Gicttatstop. aos er ee 155 150 PuGHIA SEO Peiate's oral sia Care 40 270 FiGUIAIELOP se 'sis.< rie eras 140 160 S jeiial sto pe esa ns se 50 195 Sicilia Sto pir semieniaiopn crs 200 180 Qiciiavstope ass. cess 7O 390 Oy cilia tap. efrere 145 260 TO CUA StOPs wees cniere 45 420 TOICUAaNStOP. we ele reinieters 160 170 Average resistance.... = 78.5 249 Average resistance.... = 159 178.5 Immunity of Lower Organisms to Ethyl Alcohol 603 Both for Stentor and Spirostomum not only was there no increase in resistance of acclimated animals when tested to hydrochloric acid but there was—especially in Stentor—a clear and unmistak- able weakening. Animals which normally resisted ,%, HCl for 249 seconds, if first accustomed to alcohol, showed an average endurance of only 78.5 seconds. Spirostomum, while showing less injury, in no case showed advantage by first being acclimatized to alcohol. For Spirostomum it will be noticed that a much lower suscepti- bility to HCl was shown. Although its controls were tested in a medium eight times as concentrated as that used for Stentor, they gave a resistance period almost as long as did normal animals of Stentor; while its acclimatized animals were much more resist- ant to this solution than were similar acclimatized Stentors in the much weaker solution. This lack of susceptibility to acid on the part of Spirostomum has in large part its explanation in the contour of the cell. In Stentor it was seen that in the formation of the coagulum the large rounded mass of endoplasm was drawn away from the pellicula, and that this separation is what causes the cessation of ciliary movement. In Spirostomum, however, the mass of endoplasm is long and columnar and forms into a coagulum more slowly and with a much less destructive effect. The coagulum in passing along the body becomes shorter and thicker. This rounding together with slight constrictions in the cell wall impedes its course, thus allowing the posterior cilia to continue beating often for long periods of time. Thus we see that while the formation of a coagulum is destruc- tive to Stentor, the same process gives advantages to Spirostomum when tested to a fatal percentage of hydrochloric acid. C The Action of Sodium Hydroxide (NaOH) 1 General Effects Normal unacclimatized Stentors in a solution of =, NaOH gave the following characteristic reactions: Slight movement for a brief time (15 seconds) followed by a loss of the membranellz 604 Ff. Frank Daniel and a prominent protrusion of the membranellar plates. Color— a beautiful sea green—extruded. Body usually burst and rapid destruction followed—the cilia remaining active as long as a par- ticle of form remained. Acclimatized Stentors. Stentors which had been acclimatized in I per cent alcohol, upon subjection to ;%, NaOH turned rapidly around and around for a few seconds. ‘The body wall then gave way and destruction followed with extreme rapidity. In a comparison of acclimatized and unacclimatized Stentors two differences are noticed. Acclimatized animals show a greater activity upon subjection tosodium hydroxide and this chemical acts with greater rapidity upon the acclimatized forms. The most notable thing observed in the study of sodium hydrox- ide was in relation to the pellicular membrane. If the pellicula remained intact, life was often prolonged for a remarkable period of time; if on the other hand the pellicular wall gave way, the cell was dissolved with extreme rapidity—leaving only traces of pro- toplasmic granules in the surrounding medium. Rapidity of death to those animals which have remained for a brief period of time in alcohol has a possible explanation in the fact, previously noted, that acclimatized Stentors, even in alcohol, show an early distortion. A rupture of the pellicula in NaOH means immediate death. If the process of acclimatization to alcohol makes a rupture of this sort more probable, it would natur- ally follow that if the organisms are first subject to a weak per- centage of alcohol and then tested to a fatal dose of sodium hydrox- ide they would meet an earlier death. ‘This is in agreement ‘with what is seen in Experiment XVII. 2 Effects upon Resistance In the following experiment acclimatized and unacclimatized animals of both Stentor and Spirostomum were tested to see the period of time that they could withstand an ;%; solution of so- dium hydroxide: Immunity of Lower Organisms to Ethyl Alcohol 605 Experiment XVII RESISTANCE OF STENTOR AND SPIROSTOMUM TO,“ NaOH AFTER ACCLIMATIZATION IN I PER CENT ALCOHOL a Stentor b Spirostomum A Cc A (e: Acclimatizedto Control Acclimetized to Control I Per Cent I Per Cent Alcohol Alcohol Seconds Seconds Seconds Seconds Exp. 1 cilia stop......... go 220 Exp.) 2 ellia stops sseatewiiaes 35 30 PRCULAVStO Pos. << «s/s 180 225 DeCiliaestOpectjees aes 40 30 @ieilia stop. .-...... 135 110 9 ciliatstopeesdtaestita 20. 35 Areiia stop. ..... 0s 215 420 ASCIMANSEO Ps steele slais 5 20 20 5 cilia stop......... 60 270 BG. Ciltapsta pe somalsie sioiae 25 30 6 cilia stop......... go 45 6 ciliastapicenee earls 30 35 Peciia stop-........ 195 255 Pe Cita stopajeeeiete quo» 25 50 8 cilia stop....... son 45 220 Sicilia stopritsiaecie/st)- 16 20 60 g cilia stop......... 130 330 O\cilia ‘sto pees san)adone 35 40 FOVCtlia StOp. . <2. . 0's 60 225 TO) Cilia stOp. ssiseeienie ns 40 60 Average resistance. = 126 232 Average resistance. = 29 39 It will be seen that Stentors are much less susceptible to sodium hydroxide than they were to hydrochloric acid. In a solution of NaOH eight times as concentrated (;™;) practically the same control resistance was shown as in (,%,) HCl (232 seconds, 249 seconds respectively). But that this is in no sense general for single cells may be seen from Spirostomum, which was more resist- ant to the acid than to an equal concentration of the base. This similarity of susceptibility to equal concentrations of acid and base was not in Spirostomum due alone to the retarding action of the coagulum in acids, but partly also to the fact that in bases the body burst early and disintegrated with great rapidity. In both of the foregoing studies acclimatized animals which gave an increased resistance to alcohol, when tested either to hydrochloric acid or to sodium hydroxide showed no such increase. On the contrary they invariably demonstrated a lower degree of endurance. ‘Thus the immunizing action of the alcohol was in these cases clearly specific. We shall now turn to a consideration of the second group of substances. 606 F. Frank Daniel Whether animals acclimatized to weak concentrations of alco- hol have gained a benefit which will help them when tested to a fatal dose of a substance kindred to alcohol is a question of closer interest. We shall first study this in one of the lower alcohols. D_ The Action of Glycerin C,H, (OH), 1 General Effects Normal unacclimatized Stentors. Subjected to a molecular concentration of glycerin (*) the animals remained motionless for an instant, then suddenly began backing and contracting rapidly. Membranellea became non-functional, usually within 45 seconds. ‘The peristome (membranella) was soon lost. Signs of plasmolysis—especially in the posterior part of the body—fol- lowed. The color was retained to a marked degree. Long after the membranellz had stopped beating the body cilia continued in activity (the opposite effect from that of ethyl alcohol). ¢ Stentors acclimatized in 1 per cent alcohol. In the same con- centration of glycerine these gave reactions which could not be distinguished from those described above. A single peculiarity in acclimatized animals may be mentioned. In a number of cases these assumed a characteristic pipe-shaped appearance, similar to that seen when normal Stentors were kept in water of very great purity. The most characteristic phenomenon observed from the action of glycerin was the loss of the peristome or membranelle. This was noted alike in acclimatized and unacclimatized animals and in solutions varying in concentration from a molecular solution to a concentration of one-fourth molecular strength. The first signs of injury to the organism came as a stoppage of these peristomal cilia. The peristome then became detached in a ribbon-like fashion and either hung from a point of attachment or, as Was more usual, was entirely lost. A phenomenon of great interest was the regeneration of the peristome after its Joss in the manner just described. In a sub- lethal concentration those animals which gave off the peristome in the afternoon had by the following morning regenerated a new one. Immunity of Lower Organisms to Ethyl Alcohol 607 Johnson*’ has shown that the process of regeneration in Stentor is similar to what occurs in the intricate process of division. In the cases of loss of the peristome which I have just described, however, no active condensing and separating of the nodes of the meganucleus took place. A part of the body simply constricted off and the meganuclear nodes were seemingly undisturbed. 2 Effects upon Resistance The animals in the following experiment were reared in I per cent alcohol and then tested in comparison with control specimens to a molecular solution of glycerin: Experiment XVIII RESISTANCE OF STENTOR AND SPIROSTOMUM TO m GLYCERIN, AFTER ACCLIMATIZATION IN I PER CENT ALCOHOL a_ Stentor b Spirostomum A Cc A Cc Acclimatized to Control Acclimatized to Control I per cent I per cent Alcohol Alcohol Seconds Seconds Seconds Seconds Exp. 1 cilia stop......+... 180 240 Exp. 1 cilia\stopraces-- siete 120 240 PACA StOp....--p0-- 240 450 2 Gilia Stops wisiatele «101-1 240 180 Qicilia stop........-. 150 255 Queiliay tO peer eeetiele ats 150 240 gacilia stOp.....2...- 120 330 As Cilia) SEO esis etoleiaieier= 180 300 cilia) stop... ..-. 100 285 RiciiastOpaeeer eee 180 200 6 cilia stop.......... 135 300 Gy ciliaistop.yeaseeei ere 210 240 PACU ADSEOP sco 10> « 205 250 7ecilide SLO Psi mille 180 240 8 cilia stop.......... 190 340 Sicilia stopseccadtee aes 200 180 Oicilia stop.......... 150 225 Oo ciliaystopre eee eee 180 250 10 cilia stop.......... 165 300 HO) Cilia) SEOPs asim -eleleteare 210 240 Average resistance. = 163.5 297-5 Average resistance. = 185 231 While, as we have before seen, no difference either in behavior or in chemical action could be detected as between unacclimatized and acclimatized animals, yet in this study on comparative resist- ance a difference was noticed. Ina solution of the above strength both cases gave clear evidence of the specificity of the immunity 18 Johnson, Herbert P., 1893, Jour. of Morphol., viii, pp. 467-562. 608 F. Frank Daniel due to acclimatization in 1 per cent alcohol. ‘The resistance to ¥ glycerin was decreased by remaining in alcohol. In a weaker solution, however, while Spirostomum showed a similar specificity, Stentor was far less regular. In an ¥ solution in one case it showed a slight advantage in its acclimatized ani- mals (A = 516”, C = 462”). Study in a weaker solution (*) gave results still more difficult to interpret. Animals reared in I per cent alcohol gave a resistance to % glycerin of 1293 seconds; those in 0.5 per cent, 1696 seconds, and those reared in normal culture medium, 1600 seconds. Even in these cases a certain degree of specificity of immunity is evident, for in no place is there a degree of increase in the resist- ance to glycerin approximating that given to ethyl alcohol. E The Action of Methyl Alcohol (CH,OH} 1 General Effects é In methyl alcohol, a lower member of the alcohol group, the following condition was found: Normal unacclimatized Stentors subjected to an 8 per cent solu- tion: Movement slight or animal altogether quiet; body bulging antero-laterally much as in the case of ethyl alcohol. Loss of body color; strongly marked distortion in 80 to 105 seconds; membranellz around groove persistent; frontal field retaining color and its cilia active after the posterior part of the body was colorless. Acclimatized Stentors. Stentors acclimatized in I per cent ethyl alcohol when subjected to 8 per cent methyl alcohol showed the following effects: Early movement; membranelle stopped somewhat as in glycerin; an occasional animal with membranellz lost, but the peristome was not cast off in a ribbon-like band as occurred in glycerin. . Slight, if any, difference could be seen in the behavior of the acclimatized and the control animals in the above comparison. In both, the membranellz had a resistance mid-way between that shown in ethy] alcohol and in glycerin. Immunity of Lower Organisms to Ethyl Alcohol 609 A considerable difference was seen between the action of methy! alcohol and that of glycerin upon the color and cilia of the frontal field. Both color and ciliary activity of this region were long re- tained in methyl] alcohol while in the glycerin both were lost at a comparatively early time. 2 Effects upon Resistance In Stentor and Spirostomum, the comparative resistance for both normal and acclimatized animals is shown in Experiment XIX which follows. In this Stentor was tested to an 8 percent concentration and Spirostomum to a Io per cent solution of methyl alcohol. Experiment XIX. RESISTANCE OF STENTOR AND SPIROSTOMUM TO METHYL ALCOHOL, AFTER LIVING IN I PER CENT ETHYL ALCOHOL a_ Stentor b Spirostomum Tested to 8 Per Cent CH,OH Tested to 10 Per Cent CH,OH A c A € Acclimatized to Control Acclimatized to Control Iper cent Ethyl Iper cent Ethyl Alcohol Alcohol Seconds Seconds Seconds Seconds ExpemeteeitaystOp.... 2.02.26 5: 480 240 Expt} ciliaystop scence ens 25 25. PRCA SEO o:e(0 25012 2 sec 240 360 2 iGiliastopaeemelolcercls 50 35 AeCHianstOP...<-...+.-. 180 240 Aciliavsto paces 20 30 HEGUTAUSEODS:. v:c eis 2's cis 270 105 Ay Clan SUO Pe sterertatetlcicns 45 30 GRCIUARSEOP. 010/141 lela, 120 270 FUEL Si) ooecbaoemoe 7O 80 BECHIARSEOPass:0- : pe eras ase cS STO 7F gs Soar ssistae HA ob aig wre atetine weg Wiad pTehpeg by! vt ty eros Alttotetastite a + Neate are) Aly ath yi at ee “ego 4 pgs Stra ree he oye # io ie rt i i pe hate owas ti Sree ts eee qt yao) eg tie" ne