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N Ee rf Mf boy's i . 4 : mipeten: Cane bby es ‘a oe sieges - ere RS Sys aM Betetet bande eas veh wilt aA? ot Nee : Aynee F aia apie pani Pagina neg eta usr Fis Cohn Tees A is ‘4 t Oar Tt ae iohet 7 : i aijacee ts rseeice ) bangs er paris dpe * : + “+ 44e> eae . h tee » : io Feels » i : : * : + “ hase Melgertaulrs :- se tthe? =A} x . ieee ae “ae . ++ iv “ Y pret ~ weeny abi titra s ve hy jsdahale f Monee ' ees Sahps te ht Ae Vahaty oa Digitized by the Internet Archive in 2009 with funding from University of Toronto http://www.archive.org/details/journalofexperim19broo >) ho “ : He WE ie iT ir A THE JOURNAL OF EXPERIMENTAL ZOOLOGY EDITED BY WILLIAM E. CastLe FRANK R. LILLIE Harvard University University of Chicago Epwin G. ConkKLIN JAcQuEs LorB Princeton University Rockefeller Institute CHARLES B. DAVENPORT Tuomas H. MorGan Carnegie Institution Columbia University HerBert 8. JENNINGS GEORGE H. PARKER Johns Hopkins University Harvard University Epmunp B. WILSON, Columbia University and Ross G. HARRISON, Yale University Managing Editor VOLUME 19 1915 yee THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY PHILADELPHIA, PA. COMPOSED AND PRINTED WAVERLY PR By tae Wituiams & W111 CONTENTS NO. 1 JULY Gang bb. Bripnces. A linkase im Drosophila. .-......05.-..0-20e eee cee e eee eee neem Jacgurs Lorn AND Harpoitpa WasTENEYS. The relative efficiency of various parts of the spectrum for the heliotropic reactions of animals and plants.................- Lesuiz B. Anny. The orientation of Amphioxus during locomotion.............-..-.-- Maynie R. Curtis anp Raymonp Peary. Studies on the physiology of reproduction in the domestic fowl. X. Further data on somatic and genetic sterility.............- Epwin Carteton MacDowe tu. Bristle inheritance in Drosophila. I. Extra bristles. SIC ES etc... . : .: HORS tech Soros ce sr apeln era ie wie ede bn eae 8 NO. 2 AUGUST C.M. Jackson. Changes in the relative weights of the various parts, systems and organs of young albino rats held at constant body-weight by underfeeding for various periods. (| Pius ER EECIES Sh SOE Ree RBREIBREISIot A << cc ee ICCC K.S. Lasutey. Inheritance in the asexual reproduction of Hydra. Ten figures........ Rozert H. Hurcurson. The effects of certain salts, and of adaption to high tempera- tures, on the heat resistance of Paramecium caudatum. One figure..............-. Gary N. Cauxrns. Didinium nasutum. I. The life history. Twelve figures (one NO. 3 OCTOBER Morris M. Weuts. The reactions and resistance of fishes in their natural environment emeres PIPER eH OUITE, ft \ocia cisco 2c. owls bait eee ees ais eee ee dees eases Sua aa ened: T. H. Morean. The predetermination of sex in phylloxerans and aphids. Five text SUSTIREE IRs. sa) TELE ROE eh, SI ee een geet 2 ec Cartes Pacxarp. The effects of the beta and gamma rays of radium on protoplasm. sawenty-lve lmmmern(oMnee Wiates))....2.....- 52 .s-ce se ce vee sree eset eee reese nese? TueEopuitus 8. Parnter. The effect of carbon dioxide on the eggs of Ascaris. Fifteen Bem eeetireg: Wie UMEEEMIAUGSSE: -- 2 ccikss. ss saccs cru di ess bes sec ae do emameimee re es ies ii 243 285 323 355 1V CONTENTS NO. 4 NOVEMBER Rutu J. SrockinG. Variation and inheritance in abnormalities occurring after conjuga- tion in Paramecium caudatum. Twenty figures.............. .. ete dt. an 387 Austin Ratru Mipp.Leton. Heritable variations and the results of selections in the fis- sion rate of Stylonychia pustulata. Seventeen figures................-...-....--005 451 CHARLES ZELANY AND C. T. Senay. Variation in head length of spermatozoa in seven additonal species of imsects:, “Hight figuress..-44.-...-.......7 0. daoeee ee 505 CHARLES ZELANY AND W. E. Marroon. The effect of selection upon the ‘bar eye’ mutant of Drosophila... ‘Five figures:...:: -..).Seed2-292..-..--- + - icueipeee + + oe 515 Mary B.Srarx. The occurrence of lethal factors in inbred and wild stocks of Drosophila. Two diagrams... c2 6.5. Sec d xc e eh nee ce he. toss +++ nn ee 531 Jacques Lorn anp Mary MitrcHett CHAMBERLAIN. An attempt at a physico-chemical explanation of certain groups of fluctuating variation............................ 550 A LINKAGE VARIATION IN DROSOPHILA CALVIN B. BRIDGES From the Zoological Laboratory, Columbia University In the breeding work upon Drosophila done in this laboratory, it has been the practice to allow a female to lay eggs only for a period of about ten days. This is the average length of time from the mating of a female to the emergence of her offspring. But this first brood does not by any means exhaust the eggs of a female; if she is transferred to a fresh culture bottle she will lay as many eggs in this as in the first, and will continue to lay for forty or fifty days. Ordinarily, then, we obtain a sample of from 200 to 400 flies from a female, although three times as many might be obtained. It seemed to me that the full output of each female would give a truer index than the one that we were using. Accordingly, in working out the linkage relations of several mutations, I raised from each of the F; females of a few experiments a second brood. In cases involving the second chromosome a remarkable relation came to light when the results of the second broods were compared with those from the first. There had been a change in the linkage so that both in the totals for each experiment and in a great majority of the individual cultures the percentage of crossing-over had fallen significantly. Or, in other language, the ‘coupling strength,’ or ‘gametic ratio’ had risen. This change, while very interesting theoretically, promises further to become an aid in the study of the mechanism of linkage. In the case of the first (sex) chromosome a large amount of data shows no change from first to second broods. In the case of the third chromosome present data show an increase in the percentage of crossing-over, but because of the small number of cases the rise may not be significant. 1 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 19, No. 1 JULY, 1915 2 CALVIN B. BRIDGES The most efficient experiment by which to determine the amount of crossing-over between gens is the back-cross. Here a multiple heterozygote is tested by mating to the corresponding multiple recessive. When this is done, in the next generation produced, there are two contrary classes representing the original or P, combinations, and two other contrary classes of cross- overs. The percentage of crossing-over is given directly by division of the sum of the crossover classes by the sum of all the classes. It was in this way that the percentage of cross- overs was calculated for the following tables. TABLE 1 P, purple vestigial X wild. B. C. F; wild type 2 X purple vestigial SS NON-CROSSOVERS CROSSOVERS PER CENT REF. = = == TOTAL OF CROSS- A aes Wild type Purple Vestigial Eyes A 178 202 16 16 412 7.8 A’ 152 297 13 14 406 66 | =e B 91 100 | 18 13 292 14.0 B’ 69 104 12 8 193 10.2 aise C 165 150 17 19 351 10.3 C’ 191 216 18 17 442 7.9498 D 140 149 20 15 324 10.8 D’ 116 122 9 4 251 5.20) = Sug E 191 214 | 20 19 444 9.0 EB’ 196 2929 11 22 458 7 oe Neate F 202 226 20 22 470 8.9 F’ 7 298 25 20 470 9.6 | +0.7 G 105 158 17 17 297 11.4 eu 188 232 17 14 451 69 | —45 H 123 140 26 30 319 17.6 H’ 129 179 11 20 339 91 | =a Ists 1195 | 1339 | 154 151 2839 10.7 7.8 | —s8 2nds 1238 1539 «116 119 3010 Total..... 2433 2876 270 270 5849 LINKAGE VARIATION IN DROSOPHILA 3 The mutant races used in these experiments will be fully described in a series of papers by Morgan and Bridges. In this paper I am considering only the small fraction of data in which we have records of more than one brood from a single female. THE SECOND CHROMOSOME DATA The first case in which this relation for the second chromosome was clearly shown was that of purple and vestigial given in table 1. Of the eight females whose tests are given in the table, seven showed a decrease in the percentage of crossing- over and one (F) showed an increase, which, however, was smaller in amount than any of the decreases. Likewise, the totals for the second broods when compared with the totals for the first broods showed a decrease of nearly three units in the amount of crossing-over. That differential viability has little or nothing to do with this difference is evident from the regular totals. This is shown even more clearly by the converse case (‘repulsion’), where the difference is entirely negligible. Tn all of these experiments contrary classes are affected similarly and to a like degree, which would not be the case if viability were causing the change. Of the six females tested (table 2) all showed a decided drop, and the totals show a rather greater drop (5 units) than in the ‘coupling’ case. In obtaining data upon any linkage case it is best to have half the data in the form of ‘coupling’ and half as ‘repulsion’ experi- ments. It has been shown by comparative breeding tests that differential viability can be to a great extent eliminated by careful attention to the conditions of breeding—particularly by breeding in pairs in large culture bottles with the right amount of well prepared food. We may offset even this remaining disturbance by balancing the viabilty of a certain class against itself. For example, let us say that in the case of purple vestigial, the class vestigial is poorly viable. If, then, vestigial occurs in an experiment as a crossover class, that class will be too small and a false linkage value will be obtained. The remedy is to 4 CALVIN B. BRIDGES balance against these flies an equal number in which vestigial occurs aS a non-crossover. In this case the error will be the opposite of the previous one, and by combining the two experi- ments the errors should balance and give a better approach to the true value. If each class is thus balanced the error should TABLE 2 P, purple X vestigial. B. C. F; wild type 2 X purple vestigid 3S NON-CROSSOVERS | CROSSOVERS | PER CENT REF. / == = TOTAL OF CROSS- A Purple Vestigial | iter Wild type |p Oe — — = I 157 178 | 26 21 382 | 12.3 i 200 165 12 14 391 6.7 | —5.6 oy al Sigs 176 23 23 420 11.0 J’ 242 195 | 19 26 482 9.3 —1.7 K 252 227 34 38 551 13.1 Ke 198 178 | 26 20 422 10.9 —2.2 M 205 158 | 27 32 422 14.0 M’ 213 246 14 23 496 7.4 —6.6 N 66 54 6 11 137 12.4 N’ 66 64 4 7 141 7.8 —4.6 O 189 172 30 32 423 14.6 O’ 217 225 13 18 473 6.5 —8.1 Ists 1067 965 146 157 2335 13.0 ! 2nds 1136 1073 » 88 108 2405 pied | Potala. -: 2203 2038 234 265 4740 TABLE 3 Linkage of purple and vestigial with balanced viability (1sts) CLsss Pepe CROSSOVERS TOTAL ple Wilt type. eee ee 1339 157 Purple?232 2 eee eee 1067 154 Vestigial:2 2s nota t 965 151 Purple vestigial: 2.7 025 35. . 1195 146 Total: 2330s ee ee 4566 608 5174 11.8 LINKAGE VARIATION IN DROSOPHILA 5 be very small. If an equal amount of data for ‘coupling’ and ‘repulsion’ be combined, each possible class will appear in the required manner both as a non-crossover and as a crossover. Table 3 combines in this manner the results for the first broods of tables 1 and 2. Of the 5174 flies of the first broods 608 or 11.8 per cent were crossovers. " oe) Bes | ae Ce: 19.1 22.9 per ahs 114 | 7 20| 1 — |268 41 -0.8 15.7 —3.4 19.0 3.9 66 | 147 156 2 SL 426] > 85 || 377) as 16.7 18.0 Gi ae) 108 | Ba a8 3 15|-— —,,| 204\oiee 3.1 2g, iba eee | ie ose) ve? | eo |e 22.3 26.6 6’ | 80 86 | 2 5& i8 9] 1 — |20i] 40 =s.0 13.9 —s4 46.9 sume (ea i Se 7 26 1 2 ./Smom 18.7 21.6 700 30. 92. ce i 5| = = (ees 5.7 Slo den | } 722 | 129 153 | 7 10 34 29/ — 4 | 366 5.7 18.3 21.8 Pagel (ae hos ee 16 12] 1 2 | 489) 58 40.1 11.4 —6.9 ip | | | | | @ A103 “a2T. | 9 2 25 93] 1 eee 17. 21.2 74! | A 18° 12'|-— So Soe “F644... ea ee | 76 | 122 102 |12 9 28 20| 1 2 | 296 8.1 17.2 23.3 76" | ee 287 1S By 18114] i, |/207] 6.3: =2.8 13:8 —3e7 16:0) comme 73 | 140 143 7 ‘% 33/ 2 4 | 358) 4.5 17.3 20.1 ge We a eS 1 16, 2 1 |208) 45 =— 1.7 —5.6 14.1 Sam 80 | 188°, 140. 16) 33 2 24| 2 — | 334 3.3 15.6 17.7 S0° || af o 80, | 7 3 19 15) — 1 | 215 5.1 41.8 16.3 40.7 20.2 +8. 86 i Zeon Ek Mk i8|| ote hobs) ean 14.9 18.9 ge’ 98 ges Bo io 4) — 1°) 207] 18 =30 121 —2.8 ieee 88 4) 108 ge IG CB 3: 2819), 1 Sea 16.5 18.4 ss’ | 10090 ota: sd] ])~CO4 CB | 1 83) 4 2 18.4 41.9 Sie Ists | 1476 1577 | 96 74 339 330| 19 23 |3o34| 5.4 18.1 21.3 Qnds | 1028 1187 |55 41 200 166| 9 7 |2693) 4.2 —1.2 14.2 -—3.9 17.2 —4.1 _— — | —_ —— Total... 2504 2764 | 151 115 539 496 | 28 30 (6627 LINKAGE VARIATION IN DROSOPHILA 9 TABLE 8 Cases from table 7 of change in linkage BLACK PURPLE PURPLE CURVED BLACK CURVED | TOTAL WECTCASGL.. 6.0 est oon sen 7 10 10 27 WEF CTEASG f..y. 2 Sneek 4 2 2 8 Percentage of decrease.... P22 25 19.3 differs from the change for purple curved in any female by 3.8 units, which is practically the same amount. From this point, that there is a negligible correlative change for the values for black purple and purple curved, we obtain confirmation of the view that this change is a general one which affects different sections of the chromosome independently, but on the average to about the same extent. From the percentages of occurrence of the three mutants (table 9) it can be seen that differential viability has been al- most entirely eliminated. In the ease of black, 98 flies hatched for every 100 expected—a very close approach to expectation. In the second broods the viability is somewhat poorer, but since the decreases are uniform no changes in linkage are to be expected from that source. TABLE 9 Viability coefficients (data of table 7) BLACK PURPLE CURVED "it SREP | 0.98 | 0.97 0.97 “D7! 0 Oe oho ee 0.95 0.95 0.94 The results in table 7 are not balanced here by an equal amount of data for each of the converse experiments. To balance the data of an experiment involving three loci requires four sets of data, instead of two, as in the case of two loci. Fortunately, in the case of black and curved there are avail- able data for such a balancing as is given for purple and vestigial in table 3. Only flies in first broods can be used for this balane- ing, as I shall make clear in the conclusion. In a paper by Bridges and Sturtevant (Biol. Bull. ’14), there appear records of 7419 such first brood flies in ‘coupling’ and 10 CALVIN B. BRIDGES ‘repulsion’ experiments. The data presented in this paper bring this total up to 11,353 flies and make the amounts for . ‘coupling’ and ‘repulsion’ almost exactly equal, as shown in table 10. TABLE 10 Linkage of black and curved (1sts) with balanced viability gaat d CROSSOVERS TOTAL nasa Walditiypeser cs. ste cee 2210 644 Bis clt: nc. setts oe oe 2292 619 SUEVEGL acc cede ays cee rt tae 2148 630 lack Cunved errs -1ac-c nee ae 2147 663 Totalte'. iteett. 3: een ae 797 2556 11,858: |) 22.5 chromosome is that of streak and morula. Streak is a domin- ant mutation which occupies a chromosome position far from black in the opposite direction (left) from the loci occupied by the other mutants so far treated. Morula occupies a locus likewise very far from black but in the opposite direction (to the right) so that a great section of the chromosome extending beyond the black curved section in both directions is tested by this experiment. Here also the single case so far tested showed a fall (table 11). TABLE Il P, streak 2 X morula ov. B.C. F, streak 9 X morula Dov i | | | 7 | } OVERS CROSSOVERS } NON-CROSS- f ‘a é i | PER CENT OF | es Streak- Wild | | CROSSOVERS | = Streak Morula seals type | ——— ~ — — —— = — [= = — - — = 82 50 47 40 Sr. X68 42.3 82’ 50 31 26 D417 Si a eed —4,9 In table 12, I have summarized by tables the cases for the second chromosome. Of the sixty tests, forty-nine showed a decrease from first to second broods. This decrease appeared uniformly in each experiment. A more satisfactory method of summarizing the data is that of table 13, wherein the data have been collected according to the linked pair of gens tested. The relative number of LINKAGE VARIATION IN DROSOPHILA 1] TABLE 12 Cases of change of linkage in the second chromosome, from tables 1, 2, 5, 6, 7and 11 TABLES TOTAL 1 2 ) 6 11 WMG CREASG a, es emiae - ake 2 See 7 6 4 | 27 1 49 LRT SEM RAM r ey Perse Meena er 5% 1 0 2 0 8 0 1] Potale.s acu. s its ane Ree 8 6 6 4 Serie i W260 TABLE 13 Change for each linked pair of gens PURPLE | BLACK BLACK | PURPLE STREAK TOTAI VESTIGIAL | CURVED PURPLE CURVED MORULA ; WECKCASE SMe nce ct ees 13 18 7 10 1 49 WeReaASCe aes. 2. toss: if 4 4 2 0 11 Percentage of decrease 3203 127 22/2, Pl 10.1 cases of decrease and of increase for each pair is shown (table 13). The percentages of crossing-over calculated from the totals of the first broods and of the second broods for each pair of gens, have shown in every case a fall which is considerable in amount. Table 13 (last line) gives this decrease in amount calculated as a percentage fall from the value given by the first broods as a standard, as was done in tables4and 8. All of these methods show the same real change in the amount of crossing-over between second chromosome gens in the second brood as com- pared with the first brood from the same female. THE SEX CHROMOSOME DATA Although in the case of the first, or sex chromosome, the data are not as great in amount as for the second, the conclusion is quite certain that here there is no change in the amount of crossing-over with second broods (table 14). Between vermilion and fused (‘fused’ is at the extreme right end of the known plotted chromosome) there is a large amount of crossing-over, and the totals show that the value for the second broods is practically identical with that of the firsts. The five females tested showed a fall in three cases, balanced by 12 CALVIN B. BRIDGES a rise in the other two. More data on the same case is furnished by a triple experiment, which involves as the other locus, bar, a dominant mutant deser bed by Tice (Biol. Bull. ’14). These data (table 15) add four cases of rise to the three given by table 14, and a corresponding four cases of fall to the two from the same source. The value for bar fused shows three cases of fall and five of rise. For the pair vermilion bar the cases stand four against four. The totals in every case show practically no change (table 16). The section from cherry to forked includes nearly all of the known sex-chromosome, and for this whole distance there is no change in the totals, and the females are balanced two against two (table 17). In this last case, that of cherry and sable, the slight change is a fall in the amount of crossing-over. TABLE 14 P, wild 22 X vermilion fused Sh. F; wild type 2 X Fi, wild type dc FEMALES MALES see Non-crossovers| Crossovers | co. | Ppa ed nN iat —— | OVERS 75 Wild 7. | Wer- | Wild type epne ee Ga Fuse | 52 96:9) 30. | 25 .| 16;iuaie 82 | 32.9 5 176 | 64 | 59 | 24 | 19 | 166 | -25.9 952 53 GO..| 22) | 20,/| Sanne 57-_.|, aes 53° 7 | 27 | 21 | Ste S40.) = 69) sates) se | 54 88°}. 38. | 35 (aren) 16 103 29.1 54’ 60 | 20 | 22 8 9 59 28.8 | —0.3 | | | | 57 61}. 20 | 22 Zoe 60 30.0 ot 170) |}. 5a) A ee) tS 144 29.8 | —0.2 58 228). SSH Br WP a 116 20.7 58’ 144 | 64 | 38 | 16 | 15 33 | 23.3 ) eee Ists 433 | 165 | 139 | 60 | 54 218") 278 2nds 626 | 229. | 187 | 83°] 72 | S71 | 27.2q)0ee Total. 2 °.ceee 1059 394 | 326 | 143/126 | 989 Py LINKAGE VARIATION IN DROSOPHILA 13 The summary for the data on the sex chromosome is given in table 18, similar to table 13 for the second chromosome. The total of females tested shows 17 cases of decrease and exactly the same number of increase, so that we may safely conclude that there is no change here from first to second broods. The TABLE 15 P, bar 22 X vermilion-fused io. B.C. F; bar 2 X vermilion-fused id | - DOUBLE | NON-CROSS- SINGLE CROSS- | PER CENT OF CROSSOVERS BETWEEN OVERS CROSSOVERS a | OVERS | | = — = REF. | V BrFu V [Br Fu V BrjFu Vv Ful | } y La (eas & co a — 12 re be lbs 2 & gf |e S8/2\fs 4 ge 4 GEE 4 Say mt |>oa ef Sees Bei | Ss me Se 82 165 165| 68 57 8 7 1 — | 466) 25.9 3.4 28.9 Soe 1040 87 | 26 2 - 4 — — | 245190.4 —5.5 1 —1.8 22.1 —6.8 | 8 | 128 164] 51 39 6 4 — — | 392) 23.0 2 25.5 SoueelO0r 940) 28 30 4 4 = = {2a Oree So Sel SAO a 89 85 105] 2 DB 2 — — | 244) 19.3 2.9 22.1 89’ ie Di aa 2 = i || ei) 9.) TL) eilal PR Seiya | 90 SheemtSo | SORMEEOS: <5 = — = | 234| 24.8 Ail 26.9 90’ Pome SoNmoommde. 4 1 = i Vibe eon Bare Gs +32 36.3 +9.4 | | 91 125 107| 41 31 1 — = | 30€| 23.5 0.7 2 Cieea sole 95980 25, o5 1 — 2 | 250)23.2 —0.3 3.2 +2.5 24.8 +0.6 | 92 He) aeiBs|, A eee 2 —- — | 316 20.6 1.9 22.5 O2 a O0m 05m |g 20 1 — 1 | 265) 99.3 +1.7 iil 928) =). 93 (SeGvAao = 202 — 1 — — | 182) 91.4 0.6 22.0 93’ |) RL el 1 ree 22D eb) -ie1e2 0L9) eee =083) 2366) R106 { 94 84 $6} 31 35 8 1 | — — | 255) 25.9 3.5 29.4 94" Gil Ry] SO) Be 4 = = (IER op? se A) Sen ey Sills Ee ogeeoken se 3 | = --. lossy — oy 2 bet. = 96) aa eea8)| 43) a4. 7 2 — es73coorgne ==) et = wag = OZ) | SSte 064) 95.20). 5 3 = Sy supe = 3:5) = 23.0) = , ee 88 LO eeuIoN esos, 38 1 2 = = |ORos e = 26g = ists- | 1273 1883] 488 371 47 28 1 1 (3537| 29.8 BD) 24.9 2nds | 635 677] 208 188 20 18 5 |1751/99.9-+0.1 2.5 +0.3 24.8 —0.1 Total...| 1908 2060] 641 559 67 46 1 6 5288) 29.9 2.3 24.9 | 4 —F —_—_—— 7 14 CALVIN B. BRIDGES TABLE 16 P, cherry 292 X forked oo. F, wild type @ X Fi cherry 3 ¢. FEMALES MALES | SS = Taye | PER CENT REF. Non-crossovers Crossovers A c | OF CROSS- | A Cherry ti Cherry Forked ay eel oe 25 129 145 | 73 70 65 68 | 276 48 .2 25’ 167 148 | 74 82 66 88 | «310 49.7 | 41.5 36 9 88 | 52 52 Bo. al 190 | 45.2 36 57 76) ||, A= a2 2 30 |. i 5a ae 84 76 86 | 40° 34) .38 26 138 | 46.3 $4’ 62 a 2 39 295 .28 | SiG R457 —0.6 85 114° 86) ease r8 41 53 215 43.7 85’ 98 95 | 48 - 63 52 46 | 209 46.8 +3.1 Ists 415 405 | 208 234 179 198 819 46.0 | 2nds 384 390 | 187 216 167 192 | 762 45.8 —0.2 Total........ 799 795 395 450 346 390 | 1581 45.9 TABLE 17 MALES FEMALES 7 PER CENT REP. Wild Non-crossovers Crossovers Boren OF CROSS- A . 1 : OVERS Cherry type Cherry Sable Ch, Ale ; Bs) 131 101 63 52 38 48 201 42.7 55’ 94 96 52 3] 29 30 142 41.6 —1.1 225 197.) 115 83 67 78 343 42.3 TABLE 18 Change for each linked pair of genes (1st chromosome) CHERRY | CHERRY VERMILION VERMILION BAR SABLE WOOLY BAR FUSED FUSED iBone Décrease ap ccccee ees 1 2 4 7 3 17 [NCreRSG cr ee 0 2 4 6 5 17 Total: £eeseeee cae 1 4 8 13 8 34 Percentage change..... —2.6 —0.4 +0.4 +0.8 +13.6. LINKAGE VARIATION IN DROSOPHILA 1 sing’e exceptional rise in the percentage in the case of bar fused is probably not significant, since the same females gave no rise but a fall in the case of other gens, and the distance involved is so small (2.3 units) that a very few flies make a great apparent difference. THE THIRD CHROMOSOME DATA For the third chromosome I am now able to report only the single case of pink and kidney (table 19). Here four of the five second broods showed rises, and only one a fall. The totals show a rise of 2.4 units, or of 17.1 per cent, on the basis of the first broods. This case in itself is too small for any con- clusion to be drawn. It is possible, however, that in the case of the third chromosome a rise from first to second broods may occur. TABLE 19 P, wild X pink kidney. B.C. F, wild type 9 X pink kidney 0 oS NON-CROSSOVERS | CROSSOVERS PER CENT REF. — 7 | es | PP LOraD OF CROSS- A | ‘ae gine. Binlsenkadney: | OVERS : Z| s see ae: Bee a 17 | 109 97 Erez oat EY 1008 17 98 84 Fie Na 2221 AG | 6:8 | | | | 21 - 131 104 toe ake yay al | 1.3 | 21’ 123 117 30) ane 232 | 14.9 | +1.6 23 lll 91 25 T; Cmenainest |i) 1256 23’ 79 60 | OY trae G40) 211522, | .-+3..6 25 Ses ot gee 218i) 118.8 25! 107 90 14 22 238 | 15.4 |.—3.4 27 7 138 33.7) 26 S634) 14.6 27! 121 105 Sin ienleeaeso | 18.4 | a8 Ists 608 522 109 75 1314 14.0 2nds 528 456 122 71 1177 16.4 | +24 Obalsers we asoe 1136 978 231 146 2491 15.3 16 CALVIN B. BRIDGES CONCLUSIONS Only data which have been obtained under like conditions can be used in dealing with any problem such as comparative link- age. If in the construction of a chromosome diagram the value used for A-B was that calculated from first brood data, and the value B—C was based on the total output of a female, the prediction of new values from the diagram would be in- accurate, unless the linkage remained unchanged throughout the life of the female. But if the diagram is constructed wholly from first brood values, predictions will be accurate. The practical point to be derived from this study is the breeding of only one brood from each female, especially in the second chro- mosome work. Any other condition as a standard could not be fulfilled with certainty for any large body of data. Linkage has been explained by Morgan on a chromosome basis, in accordance with the cytological evidence. It is assumed that gens occupy fixed positions, linearly arranged within the chromosome. In diploid groups each such linear series is represented by two homologous chromosomes, A and a, every locus in the one (A) corresponding to the same locus in its homologue (a). Before maturation homologous chromosomes become paired, side by side, and the members of each pair be- come twisted about each other. At some of the points of con- tact the two strands twist in two, as it were; moreover, the end of A fuses to the other end of a as they lie opposed. Any gens that were in strand A but on different sides of a chiasma point will emerge in different strands because of the crossing-over, and hence will be segregated to different gametes. It is obvious that the closer together in the strand any two given gens lhe, the less is the chance that in any given maturation a chiasma will occur between them, the chiasmas being distributed ac- cording to chance. The basis of linkage is that two gens he in the same chromosome so close together that in less than half the maturing germ cells a crossing-over takes place between them. There are two simple ways in which this scheme could be modified to give the change in linkage here described. We LINKAGE VARIATION IN DROSOPHILA ily must suppose that normally the tightness of twist for any chro- mosome varies, within limits, in the different maturing cells. The length of the section between nodes—the internode—will hence vary correspondingly, but around a modal length normal for that chromosome. It is also probable that crossing-over does not take place at every node but only in a certain per- centage, specific for the chromosome. If the average length of the section between nodes, the inter- node, remains unchanged (that is, if twisting becomes neither looser nor tighter) such a decrease in the amount of crossing- over between given gens can be explained as failure to break and re-fuse, i.e., cross over, at as many of the nodal points as normally. If, on the other hand, the average length of the internode becomes greater (that is, if twisting becomes looser) such an effect as described would be produced while the percentage of breaks per node remains constant. A possible means of de- termining which of these views obtains here is offered by a study of the interference effects in the first and second broods. Interference stands in about the same relation to linkage as linkage does to free Mendelian assortment. In linkage there is a hindrance to the independent assortment of two pairs (A, a and B, b) of allelomorphic gens. Such a case in sweet peas is that of round pollen versus long (pair A, a) and red flower versus purple (pair B, b). In the phenomenon of interference there is a hindrance to the independent linkage of the members of two couples of linked genes, A—B and C-D. In any case of free Mendelian assortment one expects as great a percentage of A to be at the same time 0, as of a to be at the same time B; Phat ipa AG: ab > ab (as, e.g., in 9 232321 orl: 1:1: 1). In cases of linkage this relation is altered by a deficiency of the classes arising by crossing-over. Likewise in cases where two couples A—B and C—D in the same linkage group (chromosome) are crossed together, the number of individuals in the double crossover class may be considerably smaller than expectation according to the proportion above. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 19, No. 1 18 CALVIN B. BRIDGES The development of the idea of interference is an illustration of the advantages of the chromosome hypothesis. The existence of this phenomenon was originally deduced by Muller and Sturtevant from a consideration of linkage as a chromosome process. Linkage had been explained as the result of two loci in the same chromosome being so close together that a chiasma occurs between them infrequently. If the chiasma must occur at a node, then, since the internode has considerable length, there must be on either side of the chiasma space which is free from crossing-over. A couple of gens C—D might lie at such a dis- tance from A—B that when a chiasma occurred between A and B, the length of the internode would cause the next chiasma to occur most often to one side of C—D rather than between C and D. In any one maturing egg a chiasma between the members of one such couple would tend to prevent one between the members of the other couple; consequently, few gametes would be formed which would be the result of both occurring simultaneously, that is, double-crossing-over would be interfered with. While the phenomenon of interference is thus a corollary of the chromo- some hypothesis, it is almost unexplainable upon any other view of linkage. Since the accuracy of the calculation of the amount of inter- ference depends upon the accuracy of the smallest class—the double crossovers—in practice we derive the index of inter- ference from a comparison of the observed percentage of double crossovers with the percentage which would be expected if there were no interference. The percentage of double cross- overs expected without interference is the product of the total percentage of crossing-over between A and B by the like value for C and D. That is, if there is 5 per cent of crossing-over between A-—B, and 10 per cent between C—D, then 5 per cent of 10 per cent would give the percentage of cases in which both occur. The case is exactly similar in treatment to the 9:3:3:1 rat_o of two freely assorting pairs of gens, where 25 per cent of all cases are a and 25 per cent are b, and 25 per cent of 25 per cent gives the percentage occurrence of ab. In order to obtain LINKAGE VARIATION IN DROSOPHILA 19 a convenient index, the converse of interference namely coinci- dence, is calculated as the percentage which the observed double crossover class is of the expected value. If, as described, interference is a function of the chromosome twist, then from observing how the change in linkage here re- ported affects interference, we can deduce the method by which the linkage has been altered—whether by a decrease in the percentage of breaks per node or by a decrease in the number of nodes, that is, by a looser twist. If the twist remains normal and the decrease is due to a de- crease from the normal percentage of breaks per node, then each linkage value will be reduced proportionally. Whenever a crossover does occur it occurs in the same position in which it would normally have occurred, so that within any given section of chromosome as great a percentage of crossovers would be doubles as in the normal condition. The effect is to make the new condition a replica of the old, except that every cross- over value is reduced in a common ratio. In this sort of change in the mechanism, the interference would remain wnaltered, for in the ratio of the expected percentage of double crossovers to the observed percentage, both terms decrease proportionally, so that the value is unchanged. If, however, the average looseness of the twist is increased, a totally different result will be produced. Here the nodes become actually further apart, so that whenever crossovers occur they are no longer in the same average position as formerly but are wider spaced. It then requires a longer section of the chromo- some in order that double crossing-over be possible. This means that for any definite section the number of double cross- overs becomes less, that is, interference rises. On this second hypothesis, then, the closer spaced in relation to the length of the internode the members of the pairs A—B and C—D are, the higher is the interference. The new condition should show the same interference as would be shown under the old condition of a shorter internode by gens correspondingly more closely spaced. By considering the amount of apparent displacement (the decrease in the percentage of crossing-over) 4 20 CALVIN B. BRIDGES we might calculate how much interference should rise if that change be due to an equivalent looser twist. Thus an experi- mental method is provided for the analysis of the mode of twist- ing and the distribution of chiasmas, not only under the changed condition, but also under the normal condition as compared to the changed condition. It is, however, no small task to secure data for such a study, and in any other material than Drosophila the problem would be wellnigh hopeless of solution. At present the unfavorable case of black purple curved furnishes only enough data to give a suggestion as to the mode followed. The data for the calculation of interference in the case of black purple curved are given in table 7 and for the first broods may be summarized as follows: B Pr Cv Bi lpers Cv B Pr pee ish ieee rome d ae XC 3,053 170 669 42 Percentage 77.61 4.32 17.4 1.07 Here the total amount of crossing-over between black and purple is 4.32 + 1.07 per cent, and between purple and curved is 17.4 + 1.07 per cent. The expected percentage of double crossing-over is therefore 5.39 per cent of 18.07 per cent, which is 0.97 per cent. The observed percentage (1.07) of double crossovers was somewhat larger than this. Although the difference is so small that it may be due to chance fluctuation, yet it will be instructive to consider its meaning on the assump- tion that it is not due to chance. The actual increase of 0.1 per cent is a relative increase of 11 per cent over the expected 0.97 per cent (percentage of coincidence 111). But inter- ference which increases the percentage of doubles is a reversal of the ordinary type and the explanation of this ‘negative’ interference is as follows: The preceding considerations have applied to the case in which the length of the average internode is such that when one node lies between A and B, the next node will most often lie beyond C—D. This relationship between the relative position of the gens and the length of the internode is such that a chiasma between one couple will tend to prevent one between the other LINKAGE VARIATION IN DROSOPHILA 21 couple, so that positive interference will result. Let us now consider two arbitrary points, M and JN, so chosen that the dis- tance between them is equal to the length of the average inter- node. Whenever a node chances to occur near M, then the next node will most often occur near N. That is, the chances of a crossover at either point are very greatly increased by the occurrence of one at the other point. In this case, instead of getting less than the expected percentage of double crossovers, we would expect, on the internode hypothesis, to get more than the expected percentage. Interference for two such points may be termed ‘negative.’ The second broods of the black purple curved cross give the following data: Bem Cv: Bay | ereCy, Bi Pr Gy, Bieler Cs: : 2.215 96 366 16 Percentage 82.24 3.57 13.6 .594 From this we find that the interference is zero (percentage of coincidence 100). There has been an 11 per cent rise in inter- ference concomitant with the decrease in crossing-over. This would suggest that the decrease in linkage here studied has been due to an increase in the length of the average internode rather than to a decrease in the percentage of chiasmas per node. Unfortunately, the number of double crossovers obtained is not great enough to establish this change in interference as significant rather than due to chance fluctuation—i.e., the vari- ation is within the limits of probable error. \ oo ua” fre sae ' 304 ,t2% 4 “2 4 7. * Ae ' = a +) ~~ Tere a ‘ a at o . e¢ a eT Nas 2 Lh Bate eek > - “t a yi! ve ' a iar ; . . - . oo a THE RELATIVE EFFICIENCY OF VARIOUS PARTS OF THE SPECTRUM FOR THE HELIOTROPIC REACTIONS OF ANIMALS AND PLANTS JACQUES LOEB AND HARDOLPH WASTENEYS From the Rockefeller Institute for Medical Research, New York I. INTRODUCTION While the older authors had treated the motile reactions of animals to light as an indication of their love for or antipathy to this kind of energy, one of us in 1888 pointed out that we are dealing in these cases with phenomena of orientation comparable to the orientation of plants to light.t. In order to indicate the identity of the mechanism in both cases he proposed the same term for both, namely, heliotropism (or phototropism). Loeb stated in his first full pamphlet (1889)? that (if one source of light be given) the animals orient themselves so that their plane of symmetry falls into the direction of the rays of light, ‘‘ whereby the symmetrical points of the surface of the body are struck by the light at the same angle.’’ In 1897 the same writer expressed the idea that the action of light which caused the heliotropic reactions was chemical.* Since it is reasonable to assume that symmetrical elements of the surface of the body are not only morphologically but also chemically alike, we must suppose that if the symmetrical elements of the surface of the animal are struck by the rays of light at the same angle, the velocity of the photochemical reactions in symmetrical elements of the surface (e.g., the eyes or skin) are the same, since the intensity of the illumination of a surface element varies with the cosine 1 Loeb, J. Sitzungsb. d. Wiirzburger physik.-med. Gesellsch., 1888. 2 Der Heliotropismus der Tiere und seine Ubereinstimmung mit dem Helio- tropismus der Pflanzen. Wiirzburg, 1889. 3 Loeb, J. Zur Theorie der physiologischen Licht- und Schwerkraftwirkungen. Pfliiger’s Archiv, Bd. 66, p. 489, 1897. 23 24 JACQUES LOEB AND HARDOLPH WASTENEYS of the angle of incidence. The influence of the light upon the tension and action of symmetrical muscles must in such a case be identical. The light, if it remains constant, will therefore not cause the animal to alter the direction of its motions. If, how- ever, the light strikes symmetrical elements at a different angle the velocity of photochemical reaction is not the same in the two symmetrical elements and the symmetrical muscles on both sides of the animal will receive unequa! impulses (by reflex) from this source. This will lead to an automatic turning of the animal until its plane of symmetry again falls into the direction of the rays of light. If these premises were true, it followed that the heliotropic reactions of animals should obey the law of Bunsen and Roscoe which says that (within certain limits) the photochemical effect of light is equal to the product of the intensity into the duration of illumination; and one of us predicted that this law would probably be found to hold for animal heliotropism.‘ This prediction proved true for the heliotropic curvatures of Euden- drium, as the experiments of Loeb and Ewald’ showed. Ewald could also show that Talbot’s law holds for the orientation of the eye of Daphnia by light,® and Talbot’s law is an expression of the fact that the physiological effect of light is equal to the product of intensity and duration of illumination. The same aw holds for the heliotropic reactions of plants, as Blaauw and Fréschl had shown.’ It also holds for the human eye.’ In all these cases it should be remembered that the law of Bunsen and Roscoe is a threshold law, inasmuch as it holds only within certain limits. With the reduction of both groups of heliotropic reactions, those of animals as well as of plants, to the same law, namely, that of Bunsen and Roscoe, it is idle to consider further the idea that animals are led to the light because they are ‘‘fond”’ 4Loeb, J. The mechanistic conception of life. Chicago, 1912. 5 Zentralbl. f. Physiol., Bd. 27, p. 1165, 1914. 6 Science, N.S., vol. 38, p. 236, 1913. 7 Blaauw, Rec. des Travaux Botaniques Néerlandais, Bd. 5, p. 209, 1909; Fréschl, Sitzungsb. d. Akad. in Wien, 1908. § Charpentier, Arch. d’Ophthalmol., tom. 10, 1890. SPECTRUM, HELIOTROPIC REACTIONS 25 of it, or that they are turned away from it because they hate it; or that the reactions are the result of ‘“‘trial and error.”’ We may, therefore, conclude that the heliotropic reactions of animals and plants are due to photochemical reactions and that the turning of the animal to (or from) the source of light is brought about automatically if the velocity of photochemical reaction is no longer the same in symmetrical areas of the photosensitive surface. This automatic turning results when the mass of photochemical react on products on .symmetrical points of the surface of the anima (eyes or skin) exceeds a certain value; and the variations of this value determine the relative sensitive- ness of different heliotropic animals. Since this has been stated more fully in- former publications of Loeb we may refer the reader to these publications.°® If the basis of heliotropic reactions is a photochemical process, it follows that heliotropic animals must possess a photosensitive substance, and the question ar’ses: Is this substance identical in all heliotropic organisms or do the photochemical substances differ in different heliotropic organisms? Especially does this question become of interest in respect to the question whether there is a specific difference between these substances in animals and plants. The method to decide this question consists in comparing the relative heliotropic eff ciency of different wave lengths in different organisms. If we fnd that the optimal heliotropic effects occur for one form of organisms in one kind of wave lengths, for another in a widely different wave length of the same spectrum, we may conclude that the photochemical substances in the two cases are different, if deduction be made for the possible screen effect of secondary substances contained in the sensitive organ. The older experiments of the botanists were mostly made with colored screens, which yielded the result that behind red screens only weak or no heliotropic reactions of plants occur, while behind blue screens they occur as well as in mixed daylight. Loeb was able to show in his earlier experiments that the same holds °The mechanistic conception of life. Chicago, 1912; article on Tropisms in Winterstein’s Handbuch der vergleichenden Physiologie, Bd. 4, p. 451, 1912. bo (or) JACQUES LOEB AND HARDOLPH WASTENEYS TABLE 1 DURATION OF LOCATION OF ILLUMINATION, | THRESHOLD IN THE INSECONDS _—_|SPECTRUM, IN MICRA 6300 534 1200 510 120 499 15 491 5 487 4 478 3 aos 4 466 6 448 good for the heliotropic reactions of animals. But these experi- ments are not adequate to decide the question of perfect identity of the photochemical substances in all cases. For this purpose experiments with spectral colors are required. The most re- liable experiments made on plants are apparently those of Blaauw on the spore bearers of Phycomyces and the seedlings of Avena. Blaauw proceeded in the following way: He exposed a row of seedlings of Avena to a carbon are spectrum for a certain time. The seedlings were than placed in the dark and after the proper time it was ascertained which part of the spectrum had induced heliotropic curvatures. By varying the duration of time of exposure to the spectrum it was found that with a minimal time of exposure only certain blue rays, namely, those of a wave length of 478 uu, caused heliotropic bending, while with longer exposure longer waves also became efficient. In this way the minimum duration of exposure for various parts of the spec- trum was ascertained. Table 1 gives his result. The red and yellow parts of the spectrum were ineffective for the intensity and time limits used and the optimum of efficiency was in the blue, in the region between 466 and 478 uu. A shorter series of experiments was made on the fruit bearers of Phycomyces, with the following results: 44 to 47 per cent of the Phycomyces showed heliotropic curvatures after 192 seconds of illumination at 615 yu after 192 seconds of illumination at 550 uu after 16 seconds of illumination at 495 py after 32 seconds of illumination at 450 uy after 64 seconds of illumination at 420 uu SPECTRUM, HELIOTROPIC REACTIONS D7 The number of experiments was limited but they indicate an optimum between 495 and 450uu, in this respect agreeing with the results on Avena. We were anxious to know whether for the heliotropic reactions of sessile animals the optimum is situated in the same region of the spectrum. The number of sessile animals which are sensitive to light is rather limited and we had to make use of the hydroid Eudendrium, which also served in the experiments of Loeb and Ewald. II. THE HELIOTROPIC REACTIONS OF EUDENDRIUM The newly formed polyps are positively heliotropic to light and they react by bending towards the light. The bending occurs in the region near the stem; the method of procedure was as follows: Immediately after the colonies were brought into the labora- tory good stems with from 4 to 8 or more polyps were selected. The polyps were cut off and the stems put into glass troughs filled with sea-water, where they were held in position by being fixed in little holes of a layer of paraffin, on the bottom of the trough. The troughs had plain parallel walls. The stems were exposed during the first day to ordinary light—since light is necessary for the regeneration of polyps!°—and were then put into the dark-room. In the dark-room the polyps developed during the next day. These newly formed polyps are very sensitive to light and were used for the experiment. The trough was then exposed to a carbon are spectrum, the visible portion of which was about 20 em. wide. The spectrum was in a dark- room and all precautions were taken to guard against any reflected or other light from reaching the polyps. The stems were in a row and each one was exposed to a different part of the spectrum. The position of each individual poylp was marked in a diagram at the beginning of the experiment and the polyps were exposed to the spectrum for times varying from five min- utes to five hours. Then the polyps were put into the dark again 10 Loeb, J. Einfluss des Lichtes auf die Organbildung bei Tieren. Pfliiger’s Archiv, Bd. 63, p: 273, 1896. 28 JACQUES. LOEB AND HARDOLPH WASTENEYS and after the proper time (two hours or more) the number of the polyps which had bent to the light in various parts of the spectrum was ascertained. As the reader will notice, the bending of the polyps took place after they had been put back into the dark. This corresponds with the method followed by Blaauw in his experiments on plants and of Loeb and Ewald in their experi- ments on the applicability of the law of Bunsen and Roscoe to heliotropic reactions. We have also made experiments in which the stems were ex- posed long enough to the spectrum to enable them to form their polyps while still exposed to the light. The determination of the wave length to which each stem was exposed was rendered possible through the use of the absorption bands of a solution of didymium nitrate. “With the aid of these bands, we could define accurately the position of each stem and polyp in the spectrum. We are indebted to Dr. E. Butterfield for the exact location of these bands in the spectrum. In noting the result the reader must keep the following facts in mind: When a short exposure to light influences the orienta- tion of the polyps of a stem, this will show itself in the fact that the majority of the polyps of that stem will bend straight to the light. If there is no effect of light, the polyps will grow in any direction but it may happen according to the laws of probability that one or the other may bend to the light. In order to be sure that the light influences the direction in which the polyps bend we must require that so great a percentage bend toward the light that chance may be excluded, before we draw the conclusion that we are dealing with a heliotropic effect. We considered it a positive result when 50 per cent or more of the polyps bent to the light. That they should all bend to the light cannot well be expected, especially in cases of short dur- ation of exposure. The new polyps are extremely delicate and they are not all healthy or strong. Moreover, certain polyps will be partly. screened from the light by the stems. Blaauw, as well as Loeb and Ewald, had to use the bending of 50 per cent of the specimens as a criterion of a positive result. The fact that each stem has only a limited number of polyps creates SPECTRUM, HELIOTROPIC REACTIONS 29 an additional difficulty, in this way: that if by chance one polyp is directed to the light and there are only five polyps on the stem it may appear as if 20 per cent of the polyps had reacted positively, while in reality the stem was not influenced by light at all. To avoid misinterpretations of this kind from influencing the interpretation of results we always give the number of polyps in a stem in the following tables. In indicating the wave length it should always be remembered that the region given is usually the center of a small zone which contained the stem; since the latter is not straight and since the polyps are irregular in position it is not possible to indicate the position by one line in the spectrum. We will now enumerate some experiments. In the first verti- eal column is given the wave length, in Angstrém units, to which the stems were exposed (indicating in the next column the color of the region). In the third column we give the fraction of the number of polyps bending to the source of light over the total number of polyps. In the fourth column we give the percentage of the polyps bending to the light. Each experiment was made with different material on different days, unless the contrary is stated (table 2). From this experiment we may deduce, first, that for this dur- ation of exposure (five minutes) the rays to 5700 A.u. (ie., the TABLE 2 Experiment 1: Exposure of Eudendrium polyps for five minutes to the spectrum WAVE LENGTH IN ANGSTROM COLOR OF THE FRACTION OF POLYPS PERCENTAGE OF POLYPS UNITS SPECTRAL REGION BENT TO THE LIGHT BENT TO THE LIGHT About 6500 orange-red 1/29 (4) About 6000 yellow 0/4 0 About 5700 yellow 0/18 0 About 5300-5345 yellowish-green 5/15 33 About 5100 green 3/12 25 About 4900 blue 11/32 35 About 4735 blue 30/49 62 About 4690 blue 4/21 19 4600 blue 5/22 23 4400 indigo 5/52 10 30 JACQUES LOEB AND HARDOLPH WASTENEYS orange and yellow rays) are absolutely ineffective; that the rays for 5300 to 4900 (i.e., yellowish-green, green, and greenish- blue) are only slightly effective; while the rays of the wave length of 4735 A.u., (i.e., blue) constitute the optimal portion; and that in the indigo the efficiency diminishes again. This result is al- most identical with the one obtained by Blaauw with the seed- lings of oats (table 3). TABLE 3 Experiment 2: Exposure, five minutes WAVE LENGTH IN ANGSTROM COLOR OF THE FRACTION OF POLYPS PERCENTAGE OF POLYPS UNITS | SPECTRAL REGION BENT TO THE LIGHT BENT TO THE LIGHT About 5760 yellow | 0/2 0 5711 green 1/2 (50)? 5100 bluish-green 4/6 66 4800 blue 9/22 41 4735 blue 3/3 100 4700 blue 3/11 27 4676 blue 8/10 80 4500 ae | 3/3 100 4400 | indigo 2/2 100 ultraviolet | 0/11 0 This result was less striking than the previous one through the combination of two circumstances: (1) The material was more sensitive than that used in the previous experiment, and TABLE 4 Experiment 3: Duration of exposure, five minutes WAVE LENGTH IN ANGSTROM COLOR OF THE’ FRACTION OF POLYPS PERCENTAGE OF POLYPS UNITS SPECTRAL REGION | BENT TO THE LIGHT BENT TO THE LIGHT 6200 | orange 0/3 | 0 5600 | yellow 0/5 0 5300 yellowish-green 1/10 (10) 5000 bluish-green 1/10 (10) 4850 | blue 0/6 0 4735 _ blue 8/13 62 4700 blue 1/7 | 14 4450 indigo 0/3 0 4432 indigo 0/2 Pax. 0 4000 _ violet 0/6 0 3850 | violet 1/2 ? 3600 ultraviolet 0/1 0 SPECTRUM, HELIOTROPIC REACTIONS 31 (2) the number of polyps was so small that the error was greater and the results not so uniform. Positive results were obtained in the region between A.u. 4400 and 4735, that is, in the blue and indigo and also possibly in the bluish-green; yellow was ineffective, as before (table 4). The most effective region of the spectrum in this experiment is again the region about 4735 in the blue, the same which has proved the most effective in the two previous experiments. In all the experiments red, orange and yellow, and extreme indigo and violet, were ineffective. The next two experiments (4 and 5, tables 5 and 6) were made TABLE 5 Experiment 4: Exposure to light, four minutes WAVE LENGTH IN ANGSTROM COLOR OF THE FRACTION OF POLYPS PERCENTAGE OF POLYPS UNITS SPECTRAL REGION BENT TO THE LIGHT BENT TO THD LIGHT About 5600 yellowish-green 0/5 0 5400 yellowish-green 6/23 23 5000 bluish-green 4/22 18 4800 blue 4/13 31 4735 blue 18/30 60 4700 blue 2/12 17 4670 blue 9/21 43 TABLE 6 Experiment 5: Exposure to light, three minutes WAVE LENGTH IN ANGSTROM COLOR OF THE FRACTION OF POLYPS PERCENTAGE OF POLYPS UNITS SPECTRAL REGION BENT TO THE LIGHT BENT TO THE LIGHT 5760 yellow 3/11 27 5600 - yellow 1/13 8 5200 green 2/16 13 4900 blue 2/4 50 4800 blue 4/10 40 4735 blue 3/3 100 4700 blue 7/20 35 4676 blue 0/1 0 4500 blue 0/1 0 4432 indigo 3/12 25 4100 violet 0/2 0 4000 violet 1/7 14 3800 violet 0/2 0 32 JACQUES LOEB AND HARDOLPH WASTENEYS with shorter exposure of the polyps to the spectrum, namely, four and three minutes. The exposure of three minutes is the minimum from which a result can be obtained; and the results of table 6 are difficult to account for. The time of exposure is so short that slight differences in the sensitiveness of various stems make themselves felt. Both experiments agree in their result with the previous ones, namely, that the region around 4735 A.u. (in the blue) is the most efficient. It was to be expected that in a longer exposure polyps would bend to the light in both indigo-blue and in green but not in yellow and red. Table 7 gives the result of an experiment with an exposure of fifteen minutes. TABLE 7 Experiment 6: Exposure to light, fifteen minutes WAVE LENGTH IN COLOR OF THE SPECTRAL FRACTION OF POLYPS PERCENTAGE OF POLYPS ANGSTROM UNITS | REGION BENT TO THE LIGHT BENT TO THE LIGHT 3700-4100 | extreme violet 14/30 | 45 4100-4900 | violet indigo and blue | 72/95 76 4900-5400 green and bluish-green 14/37 38 = = | 5400-6700 | orange-yellow toyellow-| | ish-green /? 0 Unfortunately, no record of the total number of polyps formed in the yellow and red was preserved; the number, however, was large. The experiment confirms that the blue and indigo are the most efficient rays while the green are markedly less efficient. The yellow and red rays are inefficient. The longer exposure brings out the heliotropic effects in the extreme violet which do not show with shorter exposure. In the following experiments an attempt was made to ascer- tain the influence of longer exposure. The first question was whether by making the duration of exposure considerably longer we should be able to induce heliotropic curvatures in the yellowish-green, yellow and red. Second, we wished to find out whether solarization effects might be observed in the case of too long an exposure. SPECTRUM, HELIOTROPIC REACTIONS 33 The method was as follows: Different stems of Kudendrium with young polyps (prepared in the way described above) were put successively into the same limited part of the spectrum. Each stem was exposed a different length of time. The purpose was to find out how much time was required to cause the maxi- mum number of polyps to bend toward the light (table 8). TABLE 8 Experiment 7: Eudendrium exposed to light of 4700 A.u. (blue) DURATION OF FRACTION OF PERCENTAGE OF EXPOSURE IN POLYPS BENT TO POLYPS BENT TO MINUTES THE LIGHT THE LIGHT 5 4/25 16 10 8/11 73 20 8/29 28 40 1/3 33 80 18/23 78 160 24/24 100 The experiments show that an exposure of more than ten minutes (for the light intensity used in our experiments) did not essentially increase the percentage of polyps bending to the light. TABLE 9 Eudendrium exposed to light five and a half hours WAVE LENGTH IN ANGSTROM COLOR OF THE FRACTION OF POLYPS PERCENTAGE OF POLYPS UNITS SPECTRAL REGION BENT TO THE LIGHT BENT TO THE LIGHT 4800 blue 7/16 44 4950 blue 1/6 16 5300-5500 green 3/13 24 5720 yellow 0/21 0 6000-6550 orange and red 0/32 0 We made with the same material an experiment in which the polyps were exposed for five and a half hours to the spectrum from blue to red with the result shown in table 9. The experiment shows again, first, that even in five and a half hours the rays from yellow to red are without any heliotropic effect. Second, that the efficiency of rays with wave length of THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 19, No. 1 34 JACQUES LOEB AND HARDOLPH WASTENEYS 4800 and above is less than that of wave length 4700. It is possible but not certain that there are solarization effects. In the next series of experiments a region in the neighborhood of 5600 A.u. in the yellow towards the yellowish-green was selected for the stems (table 10). TABLE 10 Experiment 8: Eudendrium ez- posed various lengths of time to the same wave length (about 5600 A.u.). DURATION OF EX- POLYPS BENT TO POSURE IN MINUTES THE LIGHT 10 20 40 80 160 ooco © In order to make sure that this negative result was not the fault of the material experiments with the same lot of material were carried out in the blue-violet part of the spectrum (table 19: TABLE il Eudendrium exposed for one hundred and fifty minutes WAVE LENGTH IN ANGSTROM COLOR OF THE FRACTION OF POLYPS PERCENTAGE OF POLYPS UNITS SPECTRAL REGION BENT TO THE LIGHT BENT TO THE LIGHT 4220-4500 violet and indigo 21/24 87 4500-4660 indigo-blue 9/11 82 4660-4710 blue 7/10 70 4710-4750 blue 15/15 100 4750-4850 blue 14/16 85 4850-5000 blue-green 7/8 87 This experiment again shows strikingly that the blue and vlo- let part of the spectrum is the effective one. The region between fis and 4750 A.u. is again the most efficient. - In a third experiment of this series the stems were exposed fo a wave length of about 4900 A.u. (blue towards the green) for various periods of time with the result shown in table 12. + SPECTRUM, HELIOTROPIC REACTIONS 39 TABLE 12 Experiment 9: Eudendrium exposed to light of 4820 A.u. (blue) PERCENTAGE OF POLYPS BENT TO THE LIGHT DURATION OF EX~-* |FRACTION OF POLYPS POSURE IN MINUTES! BENT TO THE LIGHT 0 2/9 22 10 5/11 45 ao Ce 8/12 75 40 7/11 64 80 11/13 73 160 5/18 28 With the same material an experiment with long exposure (five and a half hours) in the region of shorter wave lengths, 3750 to 4700 A.u. (namely, from-the blue to extreme violet) was carried out. In all, 52 hydroids out of 66 (i.e., 79 per cent) were bent forward. There was not much difference in the vari- ous regions, probably due to the long exposure. III. CONCLUSION AND SUMMARY OF RESULTS These experiments have shown that the most efficient region in the spectrum for the production of heliotropic curvatures in the hydroid Eudendrium is situated in the blue at \ ~ 4735 Avu. This region coincides approximately with the one found by Blaauw for the seedlings of oats namely \ ~ 4780 A.u. The regions in the red, orange and yellow are practically with- out effect in both Eudendrium and Avena. The heliotropism of the sessile animal Eudendrium and that of the sessile plant Avena are therefore identical even as regards the most efficient wave length. We expect to discuss the effects of different wave lengths upon the heliotropic reactions of motile animals and plants in another paper. CONTRIBUTIONS FROM THE ZOOLOGICAL LABORATORY OF THE MUSEUM OF COMPARATIVE ZOOLOGY AT HARVARD COLLEGE. NO. 259. THE ORIENTATION OF AMPHIOXUS DURING LOCOMOTION! LESLIE B. AREY Observers have differed regarding the question as to which end of Amphioxus is in advance during swimming. Rice (’80, p. 8) seems to have been the first to record observations on this subject: These movements were executed sometimes upon the back, sometimes upon the abdomen in the position of ordinary fishes, it seemed to make very little difference which side was uppermost, but I have never seen them move backwards or tail-end foremost. After circumnavigating the vessel once or twice gradually moving slower and slower, they would stop and‘sink down upon the sand at the bottom. . In a more general statement Steiner (’86, p. 497) came to the same conclusion as Rice, ‘‘sie stellen sich so auf, dass ihre Breit- selte in die verticale Ebene fallt und rasch entfliehen sie (from the stimulus) mit grosser Geschwindigkeit, das Kopfende voran, indem der K6rper schlingelnde Bewegungen macht, an denen der Kopf nachweisbar theilnimmt.’’ Two years later (’88, p. 41) he expressed the same opinion in almost identical language. Parker (08, p. 441) took the opposite view: The locomotion of amphioxus is a rapid, curiously irregular wriggle, often accompanied with somersault-like movements which make it impossible to be sure at any moment whether the animal is swimming backward or forward. The results of momentary stimulation, however, show very conclusively that amphioxus can swim both backward and forward, and that the direction of swimming at the beginning of any | course is dependent upon the part of the animal’s body that was stimu- lated. But how long amphioxus keeps to one form of movement I was unable to discover. The fact that it usually buries itself in the sand tail first leads me to believe that, though it can swim forward, as maintained by Rice and by Steiner, it usually swims backward. 1 Contributions from the Bermuda Biological Station for Research. No. 36. 37 38 LESLIE B. AREY Parker and Haswell (710) in their ‘“‘Textbook of Zoology”’ refer to the more or less upright position which Amphioxus as- sumes after burrowing in sand and then add the following astonishing and somewhat ambiguous statement (vol. 2, p. 46): ‘“‘Tt also swims in the vertical position, - - - - - - .’ However this assertion may be interpreted, it certainly is contrary to fact as far as the West Indian Amphioxus is concerned, and from the description of other writers (see also the frontispiece in Willey’s 94 book), the same criticism undoubtedly applies to the closely allied European species as well. Anyone may easily convince himself that the foregoing quotation either presents a gross error or is highly misleading (according to the alternate possi- bilities of interpretation), if he will observe for a short time the locomotor responses, regardless of the antero-posterior orienta- tion, exhibited by Amphioxus under various natural or experi- mental conditions. While working recently at the Bermuda Biological Station opportunity was afforded me for making observations on the swimming habits of the West Indian lancelet, Branchiostoma caribbaeum Sundevall, a species very similar to the common European -Amphioxus. This animal is found in abundance in the coarse coral and shell sand of Flatts Inlet, which connects the water of Harrington Sound with the outside ocean. Ordinary mechanical stimulation, as by a finely drawn glass rod, gave too active a response for observation and accordingly a milder stimulus was sought. This was found in a weak stream of sea-water forced from a rather large canula; if the jet was weak and was directed vertically through 10 cm. of air and 10 em. of water, the entire body of the animal became subjected to a gentle stimulus formed by the wave front. When thus stimu- lated the locomotor response tends to be less energetic and the - disadvantages of local or directional stimulation are obviated, while it has a further advantage over other mild stimuli such as jarring the containing-dish, inasmuch as individual animals may be singled out for experiment and watched from the begin- ° ning of their course. ORIENTATION OF AMPHIOXUS 39 About thirty individuals were placed in three glass jars con- taining a layer of ‘amphioxus sand’ and were stimulated by the method just described. AIl responses in which the orientation was doubtful and those responses which included wild dashes or ex- cessive somersaulting were disregarded; of the responses recorded, some were observed during the whole course and others were judged chiefly by the orientation at the beginning and partic- ularly at the slowed-down finish of the swimming reaction. A tabulation of observations obtained in this manner is as follows: Foal number Of LESPONRES sae cerns eae few bec cee cae alee 50 ATECETIOMeNd 1 ad VAN Ceres eee AE te nce os ietedaneaens 4] HOsheniOrcend iN) Ad VANCere eee eee nis ne Mais tt ae si os wns Cone 9 In a considerable number of animals the first movement was backward, but the direction was quickly reversed bringing the anterior end in advance; this condition will be discussed later in connection with other experiments. When, however, Amphioxus are free to move in unlimited space, the somersaulting and the quick reversals of direction make accurate observations as to what is occurring during the response very difficult. To obviate this difficulty a large por- celain pan was partly filled with water, giving depths which varied from 0.5 cm. at the edge to 1.25 em. at the center. The movements of Amphioxus under these conditions were as follows: When a stimulus (a bristle or finely drawn glass rod) was applied to the anterior end, the animal responded with a backward spring; if locomotion was now continued, this direction was not main- tained for more than a few centimeters, for a quick reversal occurred, exceedingly difficult to follow, but which seemed to include a doubling end for end succeeded by a partial rotation of the long axis of the animal about its middle point; as a result the Amphioxus swam away with its anterior end in advance and usually at an acute angle with the direction toward which it was heading before stimulation occurred. During subsequent swimming these reversals occasionally occurred and as a result the posterior end of the animal might be in advance 40 LESLIE B. AREY for a short time; in this event, however, another reversal soon restored the former orientation and the normal direction of continued movement was plainly with the anterior end in front. When the posterior end was stimulated the animal sprang forward and if it continued to swim, it proceeded head fore- most, although subsequent reversals usually occurred from time to time. One lancelet of this set was especially instructive; after stimu- lation and the usual energetic response, it would continue swim- ming at a rate of about 1 cm. per second for a considerable distance without reversal; in several instances it more than circumnavigated the dish, a distance of over a meter, yet the anterior end was always carried in advance. Several other animals showed the same behavior but in a less degree; in general, after several responses the reaction was as long but less vigorous than that of a rested animal, and hence was easier to observe. A circular trough 30 cm. in diameter, 0.5 em. wide at the bottom and 1.25 em. wide at the top was constructed by placing a porcelain pan, bottom up, inside a slightly larger pan; the purpose of this arrangement was to give the animal free swimming room but to limit its locomotion to one direction. The results were in agreement with those described previously; it was im- possible to make an Amphioxus swim backward for more than a few centimeters before somersaulting and forward locomotion occurred. A final method, which gave more conclusive evidence concern- ing the orientation of the animal during normal locomotion in unlimited space, consisted in treating one end of the lancelet’s body with an intra vitam stain, whereby through direct obser- vation one could be certain of the animal’s orientation even during the wild dashes that often occur. Objections may be raised to the artificial conditions of the experiments described above, which were devised for limiting the animal’s move- ments; thus it is entirely possible (although I do not believe it to be actually the case) that Amphioxus has a more complicated swimming behavior in the open than when locomoting in close quarters where some of its movements, including perhaps back- ORIENTATION OF AMPHIOXUS 41 ward swimming, are omitted. The whole locomotor response is undoubtedly much more rapid during unimpeded progress, hence it might be argued that although the animal in slower and more deliberate swimming carried its anterior end persistently in advance, yet when moving rapidly, if once reversed to back- ward swimming (and I have shown above that this reversal actually occurs from time to time), the physiological inertia of the animal as it travelled at its highest speed would tend to keep it so oriented until the swimming movements grew less energetic and the animal returned to the deliberate swimming that is characteristic at the end of the course. The data given at the beginning of the paper, in which nine out of fifty animals were recorded as oriented with the posterior end in advance while swimming freely in large aquarium jars, would tend to strengthen this suspicion; on the contrary, the actual results obtained from a study of stained animals did not substantiate such a line of reasoning. If an animal, with the exception of one end, is wrapped in a fold of wet absorbent cotton and laid on a glass plate, the ex- posed end can be immersed without difficulty in the stain; in this case a weak solution of neutral red made up in sea-water was used. The anterior end was the one stained in most cases, for the more open structure of the pharyngeal region offers a larger surface for the reception of stain. After the stain had caused coloration to a deep pink or light reddish shade the animals were allowed to recover over night. When stimulated after such treatment, the stained extremity could be followed with comparative ease, and observations made in this way corroborated my earlier conclusions. Amphioxus does not locomote backward for any considerable distance, even when the response is extremely vigorous; but, after a somersault brings it tail-end in advance, either another reversal follows directly or the animal changes its course and returns more or less in the direction from which it came. I believe the observations recorded in the first experiment of nine lancelets, which were supposed to swim backward, can be explained as follows: When the swimming response is nearing 42 LESLIE B. AREY completion, the vigor of the muscular movements rapidly de- creases and ends in complete collapse (Rice ’80, p. 8; Parker ’08, p. 441). After cessation of movement the animal is carried on a short distance by its own momentum and then sinks slowly to the bottom. According to notes taken at the time, five of the nine animals thus observed were judged chiefly by the: finish of their response; if a reversal occurred just previous to the ces- sation of swimming, it is reasonable to expect that the nearly exhausted animal would not reverse again but would continue tail first with the last feeble strokes which precede complete exhaustion. When Amphioxus has been kept in the laboratory for a short time the anterior half of many animals begins to turn pink and in a few days that end may become decidedly colored. This is presumably a manifestation of an approaching moribund con- dition, although the reactions of the lancelets appearto be practically normal. Observations of a number of Amphioxus in this condition led to conclusions similar to those gained by the study of artificially stained animals. Referring to the quotations above, it will be seen that the views of Rice and of Steiner, although agreeing with mine in the main, show some difterences. Rice’s statement that he never saw an Amphioxus move ‘tail-end foremost’ is not only con- trary to the results given in my tabulation, where nine out of fifty observed animals were so oriented during normal swimming, but is also not in accordance with Parker’s (’08, p. 481; pp. 487— 440) experiments, in which resting animals stimulated mechanic- ally or chemically on the anterior end or mid-body, responded with a backward spring. Steiner’s simple statement that Amphioxus locomotes ‘das Kopfende voran’ is certainly too general and omits entirely any mention of the characteristic backward movements just referred to in the criticism against Rice. My own observations agree perfectly with Parker’s, to the effect that Amphioxus burrows in the sand tail first, but his belief, obtained as an inference from this habit, that the animal usually swims backward is directly opposed to the con- clusion reached by me. ORIENTATION OF AMPHIOXUS 43 It is interesting, however, to see how near Parker was to the real solution of the matter, although it must be said that the whole question was not of major importance in his work; thus (08, p. 481) he says: When the anterior end of an amphioxus resting in a shallow dish of sea water was touched even lightly with the bristle, the animal usually sprang backward, though occasionally forward. The back- ward spring was often accompanied by a somersault-like movement, whereby the animal became turned end for end. When the stimulus was applied to the posterior part of the bos the result was almost invariably a forward leap. The somersaulting is only mentioned by him in connection with the backward spring, and this I have shown is characteristic- ally present at the time of reversal to normal swimming, while in a forward leap it is unnecessary and is usually omitted. The animals mentioned above, which swam slowly for a con- siderable distance in a pan of shallow water, afforded an oppor- tunity to observe the movements of the body during locomotion. The head and tail were bent simultaneously toward the same side; the posterior of all the flexures, which is by far the most prominent, occurs approximately at the level of the atriopore; the next prominent flexure is at about the region of the first gonadic pouches but is much less extensive than the former. When swimming slowly no other flexures are evident except a suggestion of one rather close behind the anterior flexure last described. The occurrence of the two largest flexures just anterior and posterior to the gonadic pouches suggests that these pouches materially increase the rigidity of the body throughout the region where they occur and thus actually determine the position of the major flexures. As might be expected, when a forward spring occurs the first flexure is initiated at the anterior end and muscular activity extends posteriorly like a wave; when an animal leaps backward the reverse is true. As regards orientation during locomotion, I thus conclude that while Amphioxus can swim backward for short distances, its normal orientation in continued swimming is with the anterior end in advance. 44 LESLIE B. AREY LITERATURE CITED Rice, H. J. 1880 Observations upon the habits, structure, and development of Amphioxus lanceolatus. Amer. Nat., vol. 14, no. 1, pp. 1-19, pls. 1-2; no. 2, pp. 73-95. STEINER, J. 1886 Ueber das Centralnervensystem des Haifisches und des Amphioxus lanceolatus, und ueber die halbcirkelférmigen Caniile des Haifisches. Sitzungsber. kgl. Preuss. Akad. Wiss., Berlin, Jahrg. 1886, Halbbd. 1, pp. 495-499. 1888 Die Functionen des Centralnervensystems und ihre Phylogenese. Zweite Abtheilung: Die Fische. Braunschweig, 8 vo., x11 + 127 pp. ParkeER, G. H. 1908 The sensory reactions of Amphioxus. Proc. Amer. Acad. Arts and Sci., vol. 48, no. 16. pp. 415-455. ParKER, T. J., AnD Haswett, W. A. 1910 A text-book of zoology. Vol. 2, Macmillan, London, 8 vo., xx + 728 pp., 537 figs. Wittey, A. 1894 Amphioxus and the ancestry of the vertebrates. Macmillan, New York, 8 vo., x1v + 316 pp., 135 figs. STUDIES ON THE PHYSIOLOGY OF REPRODUCTION IN THE DOMESTIC FOWL X. FURTHER DATA ON SOMATIC AND GENETIC STERILITY! MAYNIE R. CURTIS AND RAYMOND PEARL Some time ago one of the authors (Pearl ’12) called attention to the fact that any single record of non-production or low production could not be accepted as evidence for the absence of the genetic factor for high production, since the failure of a bird to lay might be due entirely to somatic (physiological) causes. Later a detailed description of two such cases was published (Pearl and Curtis 714). In both of these instances the ovarian eggs were formed, but did not enter the oviduct. In one case the funnel mouth was too small to admit a full-sized mature yolk, and in the other there was an apparent lack of tone in the muscles of the oviduct and its ligaments. In both cases the yolks were ovulated into the body cavity and resorbed without causing any apparent disturbance in metabolism. The purpose of the present paper is to record some recent observations on other cases of the same general nature. MATERIAL On September 1, 1914, there were in the Maine Station’s flock of first-year birds, eight apparently healthy birds which had laid few or no eggs, and one which had laid well up to the beginning of the breeding season and then stopped laying. All of these birds were hatched between April 7 and May 21, 1913. Several were from high laying strains. In order to determine if possible the cause of the partial or complete sterility, these nine birds were killed and carefully examined. The observations 1 Papers from the Biological Laboratory of the Maine Agricultural Experi- ment Station, No. 73. 45 46 M. R. CURTIS AND RAYMOND PEARL on them confirm and extend our previous conclusions in regard to somatic and genetic sterility, and also in respect to the ability of a bird to absorb rapidly through the peritoneum yolks dis- charged into the body cavity. DATA Data on the nine cases of partial or complete sterility are given in table 1. From the figures in this table it is possible to com- pare the performance record of each individual, both with the anatomical condition of the sex organs at autopsy, and with her genetic expectation, judged by the performance record of her sisters. SOMATIC STERILITY Birds Nos. 141, 81, 364, and 383 belong to high producing families. Not one of -them had a sister which laid less than thirty eggs before March 1 (Pearl 712). These birds themselves would, therefore, be expected to be good layers. Examination of the final columns of the table shows that Nos. 141, 81, and 364 could not lay for anatomical reasons. The oviduct of No. 141 had burst near the upper end of the isthmus. In the body cavity was a yellow liquid composed evidently of egg mixed with serum. In this liquid were many short tubular pieces of egg membrane of approximately the length of the portion of the isthmus anterior to the tear. The opening in the wall was of such size that it was impossible for an egg to pass it. The oviduct on both sides of the tear was in normal laying condition. There was a normal egg (a yolk en- closed in thick albumen) in the posterior end of the albumen secreting region. In the ovary was a series of five normal yolks, the largest apparently mature, and seven discharged follicles. At the posterior end of the body cavity was an empty collapsed egg membrane on which was a thin layer of shell. The perito- neum was slightly thickened, so that it was translucent rather than transparent. 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During the past year we have been particularly unfortunate in this respect. While making every effort to do his best, the person who operated the trap-nests was psychologically poorly adapted to such work, and consequently the records are marred by a number of such easily detected errors as those referred to above in the cases of birds Nos. 81 and 141, and probably by some others not so readily detected. While this is a very regrettable circumstance, it really is not so serious as it might appear at first sight to be, since, after all, the total number of errors in the records is absolutely and relatively small. ite M. R. CURTIS AND RAYMOND PEARL the probability of getting credited with eggs she did not lay was much smaller than in the preceding cases. Since the maxi- mum diameter of the duct is much greater than a normal yolk or the tumor it is possible that a few small yolks passed the tumor and became the yolks of normal laid eggs. Bird No. 383 when killed was in normal laying condition, with an egg in the shell gland and a series of five normal yolks and four folicles in the ovary. There was no apparent ob- struction to egg-laying. The peritoneum was normal and transparent. As explained in the footnote to table 1, it is probable that three eggs derived from the follicles on the ovary had been laid on the floor of the pen. The other was still in the duct. This bird’s production record shows only three scat- tered eggs and seven nesting records. At the time of autopsy she was apparently in perfect health and capable of producing eggs. Why she had failed to lay was not apparent. The idea occurred to us that possibly she had been habitually laying on the floor. Investigation of the floor egg record for the pen in the laying house in which No. 383 had spent the year did not indicate that this was the probable explanation. The total number of eggs laid on the floor in this pen of 125 birds to March 1 was 92. This was not higher than the average number of unrecorded eggs for a pen of that size, and was not as high as many of the individual birds’ trap-nest records. Also, the attendants pick up all birds seen laying on the floor and a habitual floor-layer is bound to be discovered in the long run. We must, therefore, attribute the failure of No. 383 to lay to some obscure physiological condition. That there can be no reasonable doubt that No. 383 was genetically a high producer in constitution is shown by a careful examination of her complete record in this respect. Her sire was & No. 623; all his daughters, which were hatched before June 1, with four exceptions to be noted below, made records before March 1, of over 30 eggs each: SOMATIC AND GENETIC STERILITY 53 Sire o& No. 623 >< 9 different ? 9 (fIy Le . flr Le) Winter production Over 30 Under 30 Zero Observed 28 4 0 Expected 32 0 0 plus No. 383, the abnormal bird under discussion The four birds giving records under 30 laid respectively 26, 26, 23, and 19 eggs before March 1. These are undoubtedly to be regarded as somatic fluctuations from zygotes carrying the two factors’ for high winter production (over 30 eggs). The bird which laid 19 eggs was not hatched till May 20. Bird No. 316 belonged to a small family of which a part were high and a part low producers. By her own winter production of 55 eggs she had shown herself to be genetically a high pro- ducer. After the first of March she began to make many nest- ing and a few egg records; the last egg record was on April 21; the nesting records continued in normal laying rhythms. Autopsy examination showed that there was a cystic tumor attached to the inner wall of the funnel mouth which practi- cally closed the funnel; the oviduct was in laying condition; the ovary had a series of six large yolks and three follicles; there was some free yolk in the peritoneal cavity; the peritoneum was slightly thickened. It was evident that this bird was normally a high producer, which had gradually developed a cystic tumor. This had at first hindered and finally prevented the entrance of yolks into the duct. From that time on the bird had gone through the normal laying cycles, ovulating the yolks into the body cavity and absorbing them. Bird No. 458 belonged to a family which included both high and low producers. At autopsy the ovary was walled off from the rest of the viscera by a large peritoneal pocket which was attached to the dorsal body wall on all sides of the ovary. The pocket was bulging with the contained ovary and a considerable 3 T, and Le of our former notation. 54 M. R. CURTIS AND RAYMOND PEARL amount of free yolk. A little yolk was oozing through a small hole at the midventral angle of this pocket. There were a few small lumps of yolk among the viscera. When the pocket was opened and the free yolk wiped out the ovary was found to be in normal laying condition. It contained a series of four growing yolks, the largest mature, and seven resorbing follicles. The oviduct was in laying condition. At the time of autopsy, then, this bird was ovulating into the ovarian pocket and absorbing the yolks. The rate of absorption was evidently not equal to the rate of yolk formation. In consequence the pocket was stretched to its capacity and had given way at one point, allowing a little yolk to escape into the body cavity. The egg record of this bird shows neither eggs nor nesting records until December 22. From then on until the end of the year the nesting records follow a rhythm similar to a very slow laying rhythm. There is one egg recorded on February 2, a normal clutch of three on March 13, 15 and 17 and a single egg again on May 26. While it is possible that records of all these eggs are mistakes due to causes discussed in reference to the egg record of No. 141, it is probable that at least the record of the normal clutch in March is authentic, as the errors considered would hardly have resulted in this sort of a record. It seems probable that the pocket was neither congenital nor formed complete at once but that during its growth it first hindered and finally prevented the entrance of yolks into the oviduct. In any case, the immediate cause of the partial sterility exhibited is somatic, in the sense that it is not directly con- nected with or related to the genetic constitution of the bird in respect to fecundity. Bird No. 431 represents a case of somatic steri ty of a different sort from those so far considered, in that the difficulty was not primarily with the genital organs. This bird had a crippled back which interfered with the normal use of her legs; she was in poor flesh; she had never been in a trap-nest either to lay or nest. At autopsy the sex organs were in strictly non-laying condition. There was no visible obstruction between the ovary SOMATIC AND GENETIC STERILITY He and oviduct. It is, however, improbable that the physiological tone of the bird had ever been sufficiently good to allow the formation of yolk. Genetically the bird might have been either a good or a poor layer. GENETIC STERILITY Bird No. 349 belongs to a family in which are individuals with, and individuals lacking, both factors for high fecundity. At autopsy this bird was in laying condition, with an egg in the oviduct and a series of five growing yolks and four discharged follicles in the ovary. Two of the three eggs recorded were laid respectively on the day of death and the second preceding day. These two eggs and the one in the duct account for three of the four follicles. The other may have furnished the yolk for a floor egg, explained as in the case of No. 383. There was no yolk in the body cavity; the peritoneum was normal. This bird had made no nesting records. Whether this bird was the extreme of genetically poor layers or whether her sterility was due to somatic causes too subtle for detection by rough autopsy examination is impossible to state absolutely. The probability, however, is extremely great that this bird genetically carries only L, or L,—that is, has only one dose of any of the factors on which production depends. This is evident from the follow- ing considerations: Sire o& No. 627 < 9 different 92 9 (fInL2 *° fll) | Winter production Over 30 Under 30 Zero Observed 10 13 1 (4+No. 349, the bird under discussion Expected 9.6 12.8 8.2 Now the dam of No. 349 was No. 303 J, whose genetic constitu- tion was fL,L2. flils, with a winter record of 23 eggs, and a record for the year of 79 eggs. Absolutely the most likely result of mating such a bird with a Type 4 male, where, as in the present case, there is only one in the family, is a bird which will make a winter record under 30—that is, one which carries but one dose 56 M. R. CURTIS AND RAYMOND PEARL of either L, or L.. The record of 1 egg for No. 349 on January 26 would, on a strictly literal interpretation, put her in the ‘under 30’ class. It seems clear, however, in view of the rest of her record, and of the fact, already repeatedly pointed out, that March 1 does not represent biologically the absolutely invari- able time of beginning of the spring cycle of production, that she is really a zero winter producer. From a mating like that of o& No. 627 x @ No. 303 J one zero producer in every eight offspring is expected. We may next consider the case of bird No. 249. This indi- vidual had no full sisters. The nature of the mating from which she was produced is shown by the following pedigree. Sire o No. 628 x< 11 different 2 2 (fluLs2 e flils) Winter production Over 30 Under 30 Zero Observed 21 25 4 (+No. 249, the bird under discussion) Expected 19.15 24.98 5 .83 In addition to the above, ~ No. 628 had four other daughters, by three different females, which, because of the smallness of the families and for other reasons, cannot be exactly classified genetically. These four birds were all in the ‘over 30’ class. We have classified No. 249 here as a zero winter producer because all of her 9 eggs were laid between February 24 and March 26. Further, all of the seven nesting records occurring during the early spring suggests that she is the extreme of the low producing segregates. It has been elsewhere pointed out that March 1 does not mark biologically a fixed limit of the winter cycle. Some birds, of which No. 249 is undoubtedly an example, begin their spring cycle a short time before that date. Furthermore, the dam of No. 249, which was bird No. 436 J, was of constitution fll..Fll., with a zero winter record, and a. record for the year of 24 eggs. Such a bird, mated with a male like No. 628, should give one in every four daughters a zero producer. SOMATIC AND GENETIC STERILITY 57 The autopsy findings were in accord with this expectation. The sex organs were in a condition intermediate between laying and non-laying condition; no anatomical obstruction to egg laying was observed; there was no evidence that ovulation had taken place into the body cavity. The case of No. 249 is to be regarded as one of nearly complete genetic sterility. DISCUSSION In the present discussion the term ‘somatic sterility’ is used to distinguish obstructions to egg-laying due to accidents or disease affecting the individual, and not (so far as we have any right to infer) inherited from her ancestors. A distinction is made between sterility due to these causes, which may include not only actual obstructive lesions of the genital organs, but also a general lowering of the physiological tonus of the individual to such an extent that it does not form yolk, and sterility due to a lack of the genes for egg-production. The preceding paragraphs show that three of the four birds which belonged to high laying strains and which did not fulfill the expectation, based on a knowledge of their genetic constitu- tion, failed because of the impossibility of a yolk entering the oviduct. Two other birds belonging to segregating families (one of these had proved herself a high producer by her own winter record) showed the same reason for not aying. Another interesting observation is that four of the five birds known to be ovulating into the body cavity because of some obstructions to the mouth of the oviduct nested in rhythms comparable to the laying rhythm of normal birds. One of the authors (Pearl 12) has already published a similar record of another ‘zero’ bird belonging to a high laying line. There are now several similar records on file. He called attention to the fact that it has been experimentally shown at this laboratory that in cases of ligation, transsection or entire removal of the oviduct without injury to the ovary the * * * * bird goes regularly through the entire process of lay- ing save for extrusion of an egg which is physically impossible. The 58 M. R. CURTIS AND RAYMOND PEARL ‘n’ (nesting) record of such a bird is precisely like a normal egg record showing the same phenomena of rhythm and cycles. Each day’s ‘n’ in the record of such a bird represents an egg which she would have laid, had she been physically capable of doing so. We have later shown (Pearl and Curtis ’14) that in all cases of surgical interference with the oviduct the ovary passes through the same rhythm as in unoperated birds. In such eases the formation of the egg proceeds as far as the obstruction to the oviduct. The whole body of evidence is now so convincing that we can- not escape the conclusion that nesting records are, in the great majority of cases at least, associated with ovulation into the body cavity, or the backing into it of a partly or fully formed egg. Patterson (710) stated that ‘‘the stimulus which sets off the mechanism for ovulation is not received until the time of lay- ing (in cases where birds are laying daily) or shortly there- after.”’ He bases this assertion on the fact that before the laying of the egg the oviduct is inactive, but “‘shortly after lay:ng is in a state of high excitability with the infundibulum usually clasping an ovum in the follicle.” In view of the results set forth in No. VIII of these Studies, and in the present paper, it would appear probable that the con- nection, if there is any connection, between the instinct to nest and to lay (i.e., to expel the completed egg) and ovulation is the reverse of that implied by Patterson. Since birds which entirely lack an oviduct (Pearl and Curtis ’14) and therefore cannot by any possibility lay an egg, still ovulate perfectly well and in a normal rhythm, egg-laying cannot very well be the stimulus to ovulation, as implied by Patterson. The explanation which accords best with our present knowledge is that the instinct to nest and to lay is the normal but not absolute (for example, _ note that No. 364 was ovulating into the body cavity and not nesting) resultant of ovulation, even in cases where the yolk does not enter the oviduct. SEE SOMATIC AND GENETIC STERILITY 59 SUMMARY 1. Birds which are hereditarily high layers may fail to make good performance records because for some anatomical reason it is impossible for yolks to enter the oviduct. 2. Birds which ovulate, or return partly-formed eggs, into the body cavity usually show the nesting instinct. 3. The nesting records show a rhythm similar to egg records of normal birds and it seems probable that they are the normal resultant of the ovulation. 4. Data given in this paper also confirm the following state- ments made in a recent paper (Pearl and Curtis 714): a. In case of stoppage of the duct at any level, the duct on both sides of the point of stoppage passes through the same cyclic changes, coordinated with the cyclic changes in the ovary, as a normal unobstructed duct. The duct functions only as far as it receives the stimulus of the advancing egg. b. Absence of pressure from the funnel does not prevent or apparently greatly retard ovulation. Increased internal pres- sure may therefore be the most important factor in normal ovulation. e. Yolks of partly or fully formed eggs may be absorbed rapidly and in large numbers from the peritoneal surface without caus- ing any serious derangement of normal metabolic processes. LITERATURE CITED PartTerson, J. T. 1910 Studies in the early development of the hen’s egg. I. History of early cleavage and accessory cleavage. Jour. Morph., vol. 21, pp. 101-134. PEARL, R. 1911 On the accuracy of trap-nest records. Ann. Rept. Maine Agr. Expt. Stat. for 1911, pp. 186-193. 1912 The mode of inheritance of fecundity in the domestic fowl. Jour. Exp. Zo6l., vol. 18, pp. 153-268. PEARL, R., anD Curtis, M. R. 1914 Studies on the physiology of reproduction in the domestic fowl. VIII. Onsome physiological effects of ligation, section or removal of the oviduct. Jour. Exp. Zo6él., vol. 17, pp. 395- 424. - 2) a BRISTLE INHERITANCE IN DROSOPHILA I. EXTRA BRISTLES EDWIN CARLETON MACDOWELL Station for Experimental Evolution of the Carnegie Institution of Washington From the Osborn Zoélogical Laboratory of Yale University, and the Marine Bio- logical Laboratory at Woods Hole, Mass. SIX FIGURES The four bristles normally found on the dorsal surface of the thorax of Drosophila ampelophila form a rectangle. The extra bristles studied in the following experiments occur in the two longitudinal rows of the normal bristles, or just mediad or laterad to these rows. Figure 1, showing some random patterns of bristles, gives some idea of the different arrangements possible for the most common numbers of extra bristles. These patterns were drawn free hand, since the overlapping of the bristles prevented the use of a camera. The drawings are not exact, but the relative positions in the two rows are sufficiently accurate to indicate that it would be practical’y impossible to make more than a roughly approximate classification of flies according to patterns, even if a very large number of standard patterns were established. There appears to be no position in the two rows that may not be occupied by an extra bristle, thus making it impossible -to develop any satisfactory homologies. This is especially true in the higher grades. For these reasons the best method seems to be recording merely the numbers of extra bristles. As will be seen, the results based on the data of bristle numbers shows very plainly that any attempt to obtain and use data of patterns of extra bristles would be futile with the present cultural methods. 61 62 E. CARLETON MACDOWELL All matings described below were made in pairs. The vir- ginity of the females mated was determined by the pale pig- mentation which deepens when the cuticula hardens a few hours after hatching. Accurate pedigrees and daily records of the sex and bristle number of each fly observed are on file. The results presented in this paper are based on 350 pedigreed matings and bristle counts of over 54,000 flies. Grateful acknowledgment must be made of the assistance rendered by Prof. T. H. Morgan in suggesting that the extra bristles found at times among his flies might form suitable material for the problem the author had in mind, and also in supplying numerous stocks of flies for observing the frequency of the occurrence of the extra bristles. | O aes ae Nae aa a5 ee : Sis 1 a i - 3 =o 208 a) Eas 0 ee 2 ee Gaets: oe a SS) cm ani tes Fig. 1 Random bristle patterns showing some of the arrangements possible for the most common numbers of extra bristles. The numbers indicate the numbers of extra bristles in the patterns following. THE ESTABLISHMENT OF THE RACE WITH EXTRA BRISTLES The flies which gave rise to a race of Drosophila ampelophila having extra bristles on the thorax were found in a stock of wild flies that had been caught at Woods Hole in 1912 and bred in mass cultures fora year in Professor Morgan’s laboratory. This stock had never been mixed by crossing, and in working BRISTLE INHERITANCE IN DROSOPHILA 63 with the flies there has been found no reason to suspect acci- dental contamination. Flies with extra bristles were occasionally found in this stock. Their frequency is shown in table 1. Vari- ous other stocks were examined and extra bristles were found in smaller numbers. TABLE 1 TABLE 2 Extra bristles found in various stocks Percentages of nor- mate mals in successive Bee a? Ht A EE inbred generations See ls ie s selected for in- 7 | a Th rai ile Ze z crease in bristles. | 2 erate Ac Woods Hole wild........... gg1 | 17| 20 4 | 2 | 434.8 ane ae Palmouth wild. ..........¢. 442 | 3 | | 30.6 ae | PS | az Edgewater wild............ AT4 | 2 | 20.4 ay Pe heey. 30.........-<...| 26G Wee 1| 31.1 Ist | 249 | 44.9 Mermilion....2..2........+: 163) 2 (Baile 2nd | 7139 | 10.0 Patt. ee so| | 0 8rd | 2380 3.1 Orange. 183 | 0 4th 1503 | 2.1 [PECL ee 263 | | 0 Sth = 1819 | 2.8 how. i 123 | | | 0 6th | 2780) 0.9 PMEMUNOED occ oats. a wo eras 189 | by 7th | 5833 | 2.6 Wieehead. 4.0: 0... 142 | | |G 8th | 2343 | 0.8 Mew Work 7120 2)... 3892 | 46 3 | | 49i1.2 9th | 1690 1.4 e ey Se ee |S ae 10th | 2479 | 1.9 | 3.4 11th | 2859 | From the Woods Hole 1912 stock a male with one extra bristle was mated to a female with two extra bristles. Of their off- spring 55.1 per cent had extra bristles. Thirty-three matings of first generation flies with extra bristles resulted in an F2 with 90 per cent extra bristles. The subsequent generations pro- duced by inbreeding brothers and sisters in pairs gave higher proportions of extras (table 2). One might suppose that the occurrence of normals in the later generations indicated impur- ity; but in spite of being normal in regard to the group of bristles selected for observation, these flies frequently show extra bristles on other parts of the thorax, and, further, there will be presented evidence indicating that such normal appearing flies may pro- duce all extra children; in other words, that other than genetic causes may prevent the development. of potential extra bristles. 64 E. CARLETON MACDOWELL To test the stability of the extra race further, three mass cul- tures were started from the 4th and 6th inbred generations and have been carried along from bottle to bottle with no further selection. Occasional counts have been made of these cultures and it has been found that the extra bristles have been retained. Table 3 shows the per cent normals in mass counts. It is evi- dent that there are more normals in some cases in these mass cultures than in the inbred lines, but these normals are more than likely potential extras whose greater frequency is due to the fact that in mass cultures there are larger numbers of flies which are smaller on account of the crowding. It will be dem- onstrated later that there is a relation between the size of the fly and the number of extra bristles. (See the discussion on the influence of environment). INHERITANCE 1. THE FACT OF INHERITANCE That a race of flies constantly bearing extra bristles has been established, indicates in itself that the extra bristles are inherited. To show that this is not pseudo-inheritance, due to certain con- stant environmental conditions, a stock of wild flies caught in | New York has been carried along under the same conditions as the mass cultures of the extra stock, and for a few generations this wild stock was bred in separate pairs as were the inbred selected lines. A small percentage of extras was found in this wild -stock (tables 1 and 4) but this did not increase in time, nor did the progeny of separate pairs show any higher pro- portions of extras. Conclusive evidence that extra bristles are inherited and not primarily due to environment is afforded by crosses, which show also the mode of their inheritance. 2. THE KIND OF INHERITANCE The wild stock New York 1912 was found to have fewer extras than any other wild stock examined and so from this stock normal flies were selected for several generations to attempt to reduce the percentage of extras. A preliminary set of four crosses BRISTLE INHERITANCE IN DROSOPHILA 65 TABLE 3 TABLE 4 Occurence of normals in three mass cultures Three generations of iG | | Sen 1 selection by pairs of aay | Ba. | | ae |male MASS MONTH ge BS | vrass | 7 las aS normal New York CULTURE COUNTED ap 26 | CULTURE | ae fees 2S stock to reduce the ee esl Bs I mee aa A numbers of extra 105B | Oct. 67| 0 | 176 | 10.18/1502.6 bristles and test the Nov. | 107!| 0.9 | 12.50/4324.4 influence of mating Dee. |301 | 2.5) 12.12) 952.1 by pairs on the num- Jan. | 251] 5.1] Pi aiineltese 9 (bers of Orisdes: Feb. | 84] 3.5] 1.13/1938.8 , | | Fumes Mar. | 138| 4.3 | 1.22| 902.2 al | 7 ashe bes May 288 2.5| 4.141801.1 Z| | Nos. extra 105B | Total 1304 3.6] 105B, | 12.90/1560.6 z Bgl = 176 Total | 1375 | 3.1 | | 1.12| 567.1 ete... |S : — 2nd |214265) 2 2nd 218584 9 | 2nd 219290 5 2nd |220/262| 9 | 3rd |241)220) 1 | 8rd [242245 7 | 1 8rd 255 11 between extra females from the first inbred generation, and normal males from the unselected New York 1912 stock, showed in general a dominance of the normal, although less than .5 per cent extras were found. These extras were always of very low grade, +1 (one extra bristle), while the corresponding inbred generation of extras showed a range up to +7 (seven extra bristles) and a mode at + 2. If extra bristles were supposed to be due to environmental conditions it would be difficult to account for their general disappearance in this generation. In the following generation, F:, the extras appear in proportions approximating the simple Mendelian ratio, although the normals are somewhat more numerous than expected. The eztras that -appear in F, include higher grades (table 5A). A second set of crosses between extras and normals, after each race had been selected for a number of generations, was made. The extras used were from the 9th and 10th generations of in- bred selection. In every cross the dominance of normal was THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 19, No. 1 66 E. CARLETON MACDOWELL clear, although a few eztra flies appeared in F;. Among 1419 F, flies, 12 had one extra, 3 had two extra bristles. Sixty-five pairs of normal F, flies were mated. The total counts for their children are 8421 normal, 2499 extra, which give a ratio of 3.3:1. (table 5B). As in the previous crosses there is a_ practical incomplete dominance in F,, whatever theoretical explanation may be given, and in F, the numbers of extras are a little too low, yet there can be no question that this is clear evidence of the existence of a Mendelian factor that influences the number of bristles. To test for sex linkage, five of the above crosses were made with extra males and three were made with extra females. In TABLE 5 Giving the results of crosses between extra bristled parents and normals from wild stock (New York ’12) A—Extra bristled parents from the second inbred generation Fi F2 RATIO SISESSE|) posecsat Extra 2 Number of extra bristles NO. Co: hee Bln NO 2 a 3) ormal| ela | pil aie | geen Normal: |» 0:2 173 | 3 2172] 5/10 1 | 107-4 21 0:2 oe 4 463 47| 60| 11) 2 3a 22.) 0:25 °F 2554) 2 466 | 63) 46 {14 | | 3.8:1 IX 0:3 ae 3, 423 | 58) 49) 11) 2) | 3.5% Total 835 4 | 11 1524 / 173165 37 4 4.0:1 B—Extra bristled parents from the eighth inbred generation 3164) 320: | 1563 | | 311 3:0 301 | 2} —| 8} 994] 89) 86! 52! 23/10) 2 3] | 3.5231 314 2:0 40 | —| —| 5| 762| 67| 61| 46 34 27; 3 2) |1/ 3.1:1 312 | 6:0 93 | 2} —| 15) 1383 | 169102, 93 51)18 8 2) | 2.9:1 313 | 6:0 189 | 21] 15 1487 | 138121 90, 38 20 5 4 | 3.5:1 304 0:3 5] eed) 10h Coes ee ON aL 3.9:1 305 0:2 432 | 3/2| 9 1646 | 166145111 45 31 9 4 Ei 307 0:6 203 — —| 11 1954 192166109 64 33 6 5 | 350 Total 1419 12 3 | 64 8421 843695509258141 34 18 | 1 3.3:1 Totals of all | | } crosses 2254 | 16 3} 75 9945 1016860546 262141 34 18 | ij). geaea BRISTLE INHERITANCE IN DROSOPHILA 67 both types of crosses the children showed dominance of the normal, and all grandchildren gave the same general ratios of normal to extra. Moreover, the distributions of these F, extras were similar in the two types of crosses (table 9 and figure 4). This is enough to indicate that there is no sex linkage involved. 3. MODIFICATION OF THE DISTRIBUTION OF EXTRACTED EXTRAS In comparing the distribution of the extra bristles in the F» of the above crosses with that of the extra bristles in the selected race of the corresponding generations, a marked difference is observed. This is shown in figure 2. In these curves the males and females have been put together and the normals occurring in the inbred have been omitted, since corresponding normal appearing flies in the F, of the crosses could not be separated from the genetically normal flies. The data have been plotted on the basis of 500 in each curve, to facilitate comparison. In general the distribution of the extras appearing in the Ff, of a cross is lower than the distribution of the corresponding gener- ation of uncrossed extras. The mode of the extracted extra bristles from the first cross is at + 1, although the frequency of + 2 is nearly as great; the mode of the second selected gener- ation is at +2. In the later crosses, the mode of the extracted extra bristles is at +1, and the mode of the tenth selected generation is at + 3. With these curves in mind it will be well to compare the ex- tracted extras from flies crossed before continued selection, with the extracted extras from flies crossed after selection. It is readily seen that the extracted curve from the later crosses is strikingly higher than that from the first crosses. The range is much higher; but the mode is still at + 1, even more promi- nently so than in the first case. The standard deviation in the first set of extracted eztras is .695 +.017, that of the correspond- ing inbred generation is .863 =.005. In the later crosses standard deviations are as follows: extracted eztra, 1.829 +.018, inbred, 1.402 =.021 (table 6). It appears to be a very definite fact that the modification of the distribution of extra bristles. by 68 E. CARLETON MACDOWELL Fig. 2. Comparisons of the distributions of inbred and extracted extra bristles in corresponding generations. Upper curves, selected inbreds of the second generation (broken line) and extracted extras before selection (solid line); lower curves, selected inbreds of the tenth generation (broken line), and extracted extras of the corresponding generation after selection (solid line). crossing becomes more marked when parents are selected for several generations before being crossed, yet this is not accom- panied by any increase in variability. 4. SUMMARY It has been shown that the normal number of four bristles is dominant to the extra bristled condition; that the simple Mendelian ratio of 3:1 is closely approximated; that no sex linkage is involved. Further, it has been shown that the flies having extra bristles in the F, of a cross have a distribution lower than that of the selected flies of the corresponding gener- ation, and that this difference is more marked when extra bristled flies are crossed that have been selected for nine generations BRISTLE INHERITANCE IN DROSOPHILA 69 TABLE 6 To compare the means and the variability of extra bristles that appear after across with those in corresponding generations of the extra race. . STANDARD MEANS | NUMBERS AES DEVIATION bit racheauH xtras(Gemy 2) recs. - eee =o. 1.665 | 377 | 0.695 +0 .017 inionredelriras, (Genise2) nase einer to ee 921 | 6418 | 0.863+0.005 — bo ow Ww or Extracted Extras (Gen. 10)... 2253...) 5. 2499 | 1.329+0.018 inbnedehxtirass (GensuO) ieee seem « 3.229 981 | 1.402+0.021 ————— == = —— — a = — — — than when flies of the first selected generation are crossed. The variability of the extracted extra bristles is slightly less than that of the corresponding inbred generation. 5. DISCUSSION The interpretation that seems most simple to apply to the above facts is that there is a positive restricting factor present _ in the wild fly that prevents more than four bristles from develop- ing. Although the eztra race has something added somatically to it, genetically it has lost a restricting factor. Now this condition of extra bristles seems not to be phylogenetically new, as studies on the chaetotaxy of related flies suggest. The mechanism that is most concerned must have appeared when Drosophila ampelophila arose, or before, so the extra bristles are not formed by the origin of a new mechanism, but rather by the removal of the restriction of the more recent mechanism. THE SELECTION PROBLEM The main problem to which the preceding experiments have been contributory, has been the attempt to throw some light on the question of selection as a formative process in evolution. The attempt has been made to increase the numbers of thoracic bristles as much as possible by selecting and inbreeding. Ac- cordingly, after the first pair that came from wild stock, high grade brothers have been mated to high grade sisters. In the following account the first eleven generations of this process will be described. 70 E. CARLETON MACDOWELL 1. DO HIGHER GRADE PARENTS PRODUCE HIGHER GRADE CHILDREN ? To show what basis there may be for selection to progress, comparisons have been made of children from high and low parents from the same grandparents. All the matings in F, can be used for this purpose, as all the parents were sibs. Curves showing the 35 F: fraternities were plotted, but they were too numerous for publication. The children from parents of like grades have been grouped together. Table 7 shows the aver- ages of the males and females, the numbers of flies and of families involved in the various types of matings. No increase in the means is found that directly corresponds to the increase in parental values. However, in grouping together children from parents with one or two extra, and comparing them with children from parents with three or four extra, the higher group has, in general, higher means. The low means of the one 4:4 mating involves too few individuals to make a serious exception. In the second generation there seems to be a basis for some effective selection. Similar comparisons can be made in the later generations. Seven families in F, came from the same grandparents. Thirteen of the parents were + 5 and one, +6. Here is a case where the parents are closely alike and one would expect the children in the different families to be closely alike. The curves and the TABLE 7 To show the relationship between the grades of the parents and the means of their children in the second selected generation GRADES NUMBER MEANS NUMBERS OF OF ee Nae EERIE Male Female Male Female heb 4 1.70 2.0) 397 418 122 3 1.45 1.88 367 375 Zin iW 1.42 1.84. 2061 2204 ERG 3 2.05 2.46 171 229 3:4 2 1.94 2.39 207 220 4:4 1 1.61 1 .98 33 41 BRISTLE INHERITANCE IN DROSOPHILA v1 averages (fig. 3 and table 8) show that this is not the case. The averages for females range from +2.83 to +4.05. This’ set of families gives some idea as to how much variation is possible when the grandparents are brother and sister and the parents all sibs of the same grade. In F, are two sets of cousins. In one group are five families (fig. 3B), grandchildren of 115, with parents of various grades. Considering the female averages (table 8B) it is seen that the parental sums of 7, 8a, and 12 give similar filial averages, whereas the parental sums of 8b and 14 give higher averages. Con- sidering the males, parental values of 7, 8b, and 12 give similar TABLE 8 Six groups of cousins. In each group the parents are sibs; grades of parents and means of offspring arranged according to the sums of the parents’ grades. See figure 3 | | | # ‘NOS. OF 5 |NOS. OF Pome esrs (on o"| comones | Soe | nor. ext SUMOF) CuipReN | ee ENTS | NO; |__| gn | | | 9 | of e [ale] 7/9 | @ 2) aire A children in F, | | | D children in F, | | 71 |5|5| 10 | 2.29 | 3.53 | 58 68) 177|/4/4] 8 | 2.61 | 3.90 278216 72 |5/5)| 10 | 2.46 | 2.83 115105) 166/5/6] 11 | 3.19 | 5.16 | 41) 30 7 |5/5/} 10 [3.15 | 4.05 | 60] 55] 178) 5/9]! 14a | 3.00 | 4.98 | 73] 75 87 |5|5}| 10 | 2.81 | 3.37 | 37| 32) 180/6|8| 14b| 2.85 | 5.19 | 27| 31 88 (5/5) 10 | 2.66| 3.91 | 63] 71 | (ae 89 |5/5 10 | 1.98 | 2.74 | 58 58E children in Fyo | 92. /6/5 11 | 2.87|3.13 16 22 289 5/5) 10 (3.28 | 4.40 687 bad | 28416/5}| 11 | 2.75 | 3.88 122120 B children in Fs a 281 | 5|7| 12a | 3.25 | 4.57 | 31) 19 149 |2/5/ 7 | 2.91 | 4.17 | 91} 81) 290|6|6| 12b| 3.40 | 5.46 | 15) 13 135 |4/4 8a| 2.36} 4.15 | 90 89 | | 134 4 4 8b 3.01 | 4.66 75 93 F children in Fi. hh 148 (3/9) 12 | 2.95 | 4.23 | 39) 88) 413|5|5] 10 | 3.41 | 4.61 | 48) 42 132 |6|8| 14 | 2.78 | 4.97 | 50| 35) 411/6/5] 11 | 3.98 | 5.73 | 46 38 | | | 329 | €|6| 12a | 1.76 | 2.72 |102| 80 C children in F, | lav 412|6|6| 12b| 3.20 | 4.54 | 29| 37 141 |3/4| 7 | 3.27 | 4.54 | 77] 90 | | 155 |4/4| 8 | 3.00 | 4.68 | 11) 11) 146 | 2|7| 9 | 2.22 | 3.90 | 66) 52 | 144 |5|6| 11 | 2.67 | 3.71 | 80) 98| | | 142 |6| 8) 14a | 2.60 | 4.73 | 41) 69) hac 153 |5|9|. 14b | 2.97 | 4.76 | 37) 26 fees | | | Nol 46 # Gore S23? ae ew cae Nol 66% aa antes Ne 5298 | Sore lO2 a BAAN 60% | Va | \ | \ No4ll#® AGP 38 FF No 2844 122 a7 / 120 #? A No!78¥ / \ T3 om \ 7522 y No 289 ¥ GBor 772P ' Ca ae) e ° Fig. 3 Six groups of cousins; in each group the parents are sibs. Solid lines are females, broken lines males. The parents are indicated by squares (males) and triangles (females) above the curves of their children. The fractions follow- ing the numbers of the mating indicate the grades of the parents. The numbers of males and females involved in the curves are given at one side. A, grand- children of no. 56, in the 4th generation; B, grandchildren of 115, in the 6th generation; C, grandchildren of 116, in the 6th generation; D, grandchildren of 143, in the 7th generation; E, grandchildren of 246, in the 10th generation; F, grandchildren of 274, in the 11th generation. sI (SX) 74 E. CARLETON MACDOWELL averages, 14 a little lower, and 8a much lower. In the second group of families in F, (fig. 3C and table 8C) female averages from parental sums 7, 8, 14a, and 14b are similar, whereas those from sums 9 and 11 are a whole bristle lower. The male averages for parental sums 7, 8 and 14b are similar while those from 11 and 14a are lower, and from sum 9 a whole bristle lower. In F; is a set of cousins consisting of four families (table 8D and figure 3D). In this group the low parental sum has the low male and female averages, whereas sums 11 and 14a and 14b are much alike. In F,,) there is a group of cousins in four families (table SE and fig. 3E). The female average for parental sum 12b is considerably larger than that for the sum 10, yet the female average for the sum 12a is about equal to that for the sum 10. Parental sum 11 has the lowest male and female aver- ages. In Fi, a group of four families (table 8 F and fig. 3F) shows averages for parental sum 11 that are above averages for 10 and 12b. Parental sum 12a has averages much below any of the other families. This study of averages must be followed by careful observation of the figures referred to above, which show the frequency distributions of each of the families that has been considered. These curves have been plotted so as to enclose in each polygon similar areas, to facilitate com- parisons. The actual numbers of flies are given both in the tables and the figures. Further data bearing on the question of the relation between the grades of parents and children is to be found in the series of curves representing inbred lines through single matings (table 11 and fig. 6). One case must be especially emphasized. The last generation, No. 216, in the series beginning with No. 18 (fig. 6 A) was produced by flies with no extra bristles. This generation which follows seven selections has a higher distribu- tion than the preceding ones which were from high grade parents. There are no normals from these normal parents, and no females with less than two extra bristles. This extreme case is in ac- cord with all that has been found in regard to the independence of the parental and filial grades. This is further borne out by these inbred lines in the comparison of parents and children in successive generations. —— BRISTLE INHERITANCE IN DROSOPHILA VO So the general conclusion seems to stand, that after the first few generations there is no close relationship between the grades of the parents chosen and the grades of the children. This appears to mean that in regard to extra bristles the flies in later generations do not differ genetically from each other; that in the later generations the variability in the number of extra bristles is due mainly, if not entirely, to conditions outside the germplasm. A further test of this would be to cross with normals flies with many extra and flies with few extra bristles and compare the results in the second generations. If the difference between the many and the few extra bristles be due to genetic factors, this would be shown in the F, of the crosses. Data is given that shows the distributions of extracted extra bristled flies from crosses of high and low eatras with normals of a different race. To avoid any complication that might arise from not consider- ing sexual differences, and at the same time to find evidence in regard to sex linkage, four types of crosses with wild flies have been made: high and low males, high and low females. In table 9 the data are arranged to facilitate comparisons between males and females of like grades, but by comparing alternate lines the relations between high and low grades are clear. The ratios and the distributions are practically alike in all cases (fig. 4). In order to make the curve for high males, including over 800 individuals, more easily comparable with the others, these data were plotted on the basis of 600. In conclusion, comparisons of families in the second generation of inbreeding show a tendency for higher parents to produce higher children, but this is not found in the sixth or subsequent generations; in single inbred lines there is found singular inde- pendence of the parental and filial grades, seemingly normal parents being able to produce all extra children; by the analysis of crosses it is found in the ninth and tenth generations that, as should be expected from the foregoing statements, high and low bristle grades are genetically indistinguishable; that the variability found in the late generations is apparently not due to genetic factors. 76 E. CARLETON MACDOWELL TABLE 9 Showing that low and high grades of extra bristles give the same results when crossed with wild normals; also that reciprocal crosses show that no sex linkage is involved Fy F2 RATIO P _. 1 ae ee NORMAL t TO mail 1 | 2 |normal| 1 | 2 | 3] 4)5 | 6}7 | 8) 9 | exrea Low o'o' X Wild 9 9..../341) 2 1756 156147 98 Silo) Dire S342 Low 92 @ X Wild #'o..../487) 3 | 2 | 1841 188159119) 48 33 10 4 2.8:1 High oo X Wild 9 9... .|282 4} 1 | 2870 307/223/183) 89, 38) 13) 4 | Seael High 992 X Wild oa’... .|203 | 1954 192166109 64 33 6 5 | Sock S “Ss. ox L ly, ato aS ers _~ HIGH Okwito oo S HIGH 9 xwiLo oe i Zz fo) 4 5 G TE 8 9 Fig. 4 Extracted extra bristles appearing in F». of reciprocal crosses of high and low grades of extra bristles with normal wild, showing that there is no facto- rial difference between the high and low grade eztras crossed. 2. RESULTS OF SELECTION a. Total generations On the basis of the conclusions just reached, we should expect to find selection effective in the first few generations and later, ineffective. Data will be presented to show the results of selection in the form of curves for total generations and for single lines. Owing to the difficulty of obtaining high grade females that were unquestionably virgin, the most rigid selection was not always possible, so that even in the later generations some matings were made of low parents. In order to avoid the possibility of hiding a real increase in the offspring from high grade parents by including the offspring from low grade parents, BRISTLE INHERITANCE IN DROSOPHILA rye the children having either parent below certain grades were omitted. Along with these were excluded all flies whose grand- parents or more remote ancestors had been excluded. This reduced the numbers in the total generations very considerably, and this is especially so in the latest generations. In these resulting curves the parents of each generation are included in the curve of the preceding generation. In figure 5 the curves have been plotted in such a way as to include similar areas, al- though representing different numbers of individuals. For this reason the numbers of males and females included are put down beside the curves. The solid line represents females; the broken line, males. The small curves between the larger ones in each case represent the parents selected from the gener- ation above, to produce the generation below. Discussion of the curves for total generations. The original parents selected from wild stock were a male with one extra bristle (+1) and a female with two extra bristles (+2). Their children are represented in the first pair of curves. The large number of normal flies in this generation 44.90 per cent is due to the fact that the mother was not virgin. Disregarding these, the male and female modes are at +2. In the following generation the modes remain at +2 but the upper limit of the range goes up from +4 to +7. The flies chosen as parents from this generation, range from +2 to +7. In the third generation the modes are still at +2 but the male curve has moved a little higher and the female curve is markedly above the male. This is clearly seen by comparing the areas of the polygons above the vertical line drawn at +2. The flies chosen from this generation range from +4 to +7. In the fourth generation the female mode is raised to +3 and a clear advance is shown by the whole curve. Although the male mode is still at +2, the proportion of +1 males has decreased and the pro- portions of +3 and +4 have strongly increased. In the fifth generation the male curve has increased its range over the pre- ceding generation but as a whole has fallen back a very little. The female curve has a much increased range, and a slight sag above the mode, otherwise no change. In the sixth generation 78 E. CARLETON MACDOWELL the male mode still holds to +2 but the proportion of +3 flies has grown to nearly equal the mode. The proportions of +4 and +5 flies have also increased although +5 is the limit. The female mode is actually at +5 but the expected mode is easily seen to be at +4. So far the progress and increase has been steady and unquestionable. The following five generations do not show any such advance. In generations seven, eight, nine, and ten the male mode remains at +2 and the proportion of +3 flies is very slightly less. In the eleventh generation the +2 and +3 males are equal, however the proportion of +1 flies has greatly increased in this generation, so this does not mean any general advance. The female curves have modes at +3 in the 7th and 8th generations, with +3 nearly as high. In the last 3 generations the female mode is at 4 and the curves are in general of the same kind. To show what parents were used in the different generations more clearly than can be done by the curves, their distributions are shown in table 10. The averages of the parents are given in two columns at the left and the averages of their children are in two columns towards the right. This table gives a clear summary of the progress of selection. The averages of the children, males and females, show increase up to the sixth gener- ation, when minor fluctuations appear seemingly without any significant change. If the variability in the last five generations be amenable to selection, a gradual decrease in the standard deviation would be expected, even though some hypothetical physiological limit prevented the means and modes from advancing as before. Fig. 5 Eleven generations of selection. Original parents indicated by a square (father) and triangle (mother) above F; broken lines, males; solid lines females. The flies chosen as parents are arranged in small curves above the curves of their children. In generations 8 and 9 the male parents were all grade 5 and are represented by a square the proper distance from the base line. A finely dotted line means males and females together. The curves are plotted in such a way as to include like areas, so the numbers of individuals included are given at the side of each curve. The upper limits are in some cases drawn on the base line to show that such extremes appeared, although in too small numbers to form a whole unit in plotting. ~ Hie "8 / / \ / \ / \ / 4 \ Fe / 934077 ; 1007 °? ©) 1 We G é r y x \ Z \S——- . : ih / / \ / \ / \ / \ ee s Z105c0 NA 20649 2 N / / \ / \ \ / \ / \ Fe / \ 66800 NE NGGS29 — VER / Ni / \ / \ \ e sa eS fo a — ~ \ \ Fio SIT as 3 ATT? FEN = \ \ \ N Fi 80 E. CARLETON MACDOWELL But the standard deviations show a gradual increase while selection is effective according to the curves and the means, and when selection has seemingly become ineffective, the stand- ard deviations fluctuate, but do not show a gradual decrease. b. Single lines The most immediate facts are presented in the form of the his- tory of five inbred lines through single matings, as shown by curves in figure 6, A, B, C, D, E. The grades of the parents selected from each generation are indicated below the curve of their own fraternity and above the curves representing their offspring. Squares are the males; triangles are the females. Four of the lines start from the same pair of first generation flies. The first two generations which are common ancestors of all four lines, are represented in only one set of curves. The parents of families Nos. 87, 71 and 88 are all sibs in family No. 56. The parents of family No. 116 come from family No. 88. The line headed by family No. 17 starts with a different pair of first generation flies. As the history told by these curves is clear it does not seem necessary to discuss each set of curves in detail as has been done for the total generations. In Table 11, A, B, C, D, E, the means for these curves are given. These curves give the clearest picture of the basic facts. Clear progress is shown in the first five or six generations. In each line are differences counter-balanced in the total generations, yet in no case do these differences modify the general conclusion drawn from the study of total generations. 3. SUMMARY In brief, selection appears to have made advances for six generations. When large enough numbers are observed to afford a counter-balancing of the fluctuations of individual families, the advance is steady. That this increase is really genetic is shown by the increase in the distribution of extras extracted from a cross of long selected flies, over the distribution of extras extracted from a cross of unselected flies. In later generations me: $sr6l FES | ° £22E ° $3161 P96! \ §19Z°N Ni / eo + 839 PPLI S£S°N \ SX , | ‘S1OY}O OY} SB SISUq SUIS OY} UO }O]d 0} S|VNPLAIpUI Moz 009 o40M 9104} FIT ‘ou W UPL “OAINO Yovo opIssuo]e UOATS OLB POA]OAUT SOTY JO saoqumnu oy} :svore pwnbo Jo suosAjod UILOF OF poqyoyd ov soAany ‘so dueray ‘soyeuroy { sorenbs ‘sopwur {Aq TUIOV Ray I10q} FO SOAIND OY} MOTO UMOYS O18 UOT}VIOUDS SUTMOTLOF oy sonpord 0} pojoojos syuorvd oy, ‘soury prjos ‘sayeuroy Seu] uoyorg ‘soyeyy “sol ty Jo aed JUSLO Ip V ULOTF ST FY OUT] SE OUT] UT TY UIOAy ov | OUT] UI syuoaed 4say oyy *¥ out] UT 9¢ ‘ou ulor; YO O18 (28 put TY sou) O put g soul] Jo sjuord 4sayg oyy, *SoUuTT poaqut OAL 9 “SIT 8316 PPE #SII°N ! 3 dd801 PPEOI B0II'N gy \ | VOL. 19, No , OURNAL OF EXPERIMENTAL ZOGLOGY JuLy, 1915 to “s19y}0 OY} SB SISEq OUTES 94} TO ‘QAIND YOR OpIssUO]E WATS OIV PIAJOAUT sory JO Stoqunu -sojduviy ‘sopeuiay fsexenbs ‘sojvar {Aqraseyway ITeqy 0} poqyoojas syuered ayy, “SeUT] Pros ‘soywuray ‘g oul] Ut TZ moss av C] OUT] UI Syuosud ysag O47 Lysay oy, “Sout] porqur oad 9 “By god 0} S[BNpIAIpul Moy 004 B49M 910} HIT “OU GY UL ayy {seein ponba yo suoBAjod w1s10y 04 payjojd av Sadan JO SAND OY} AOTIG UMOYS O18 UOIZIIUET DuLAoTpoy OY} aonpoid ‘soul] uoyorq ‘saeyy “saly ty Jo dvd quosayip B WMory St GY OUT] ‘y aul] ul ge ‘ou moss y30q oI (2g PUL TL “SOU) D PUY gf Sout] Jo SyudrEC \ ‘\ 1 4g! i : PPLE Mery ae ree 4 Vg. ‘ i \ / f eA, $801 sasr \ / are / sez Spe ve #02" FESO! I ! ‘ abooy J \- aSZoru Nar S461 PPPG HSIN tezien M Sat PPEE till sau WCS #992°U $828 fF6O0! #21 gaol PPI01 19, xo, 1 vou. 2 = - = 8 s = = 2 z 2 z ” su > Sie iy ¢ ’ oe oi et yuk “ . ° see ; Mh wo ip} ah la ¢ = He ei se e. gr ; ‘ hed « } . Os < ue aihh ah ded ; : a 9 7 \ il e. a ne Play Yoe .e q = & . q TABLE 10 Showing the distributions of the parents selected and the averages and variability of their children in the eleven generations of inbreeding and selection. PARENTS OFFSPRING Distribution accord-| Nos. of | ; 7 = ing to no. of extra | Means | indi- | Modes Means Standard deviations - bristles g |vViduals o | Ee ° -——-~ —-— = | | Aap a ee (2). | 2 S |ilelsiaisie 7\si9i 2/48) 8\8|8|/8)8] Males Females Males Rumates ° hala | || 3 & o|S 5 Silo Sa } | | ala | o S| 6 || & | i es ee. F; [123810 4—|——|— —|1.962.21 F2 34153703) 2) 2| 1.494.010) 1.453+.009 0.912.007) 0.829.006 Fe |—[14! 7] 4! 511 1|-\—3.253.18 Fs 10901185 2| 2/ 1.916.021 | 2.708+.022 | 1.035.014 | 1.201.017 Fs |—|—!—| 214 1 1}——/5.005.11/Fs | 769 776) 2) 2| 2.219.027) 2.9254.031 1.122.019 | 1.284.022 F, |—|—/-| 512| 2—| 1'—4.305.10,Fs | 662 724) 2| 3| 2.300%.030) 3.480.035 | 1.147.021 | 1.415.025 Fs |—|—\—| 9 4) 7—| 3, 1/4.756.16,Fs | 9341007) 2| 5 | 2.703.026 | 4.055.036 A tod 1.729 .026 Fe |—\——| 62710 2) 2 14.965.79 F7 21052064 2 3 2.507.015 | 3.694.023 | 1.025.010 1.530.016 F, |—|—\—|—/11) 4| 2] 11—'5.006.22|Fs | 668| 668) 2| 4| 2.682.031] 3.878.037 | 1.186.023 | 1.407.027 Fs - —|—|—| 6 2\-|—|—5.005.50,Fs | 152 149, 2) 3) 3.065.069) 4.382.083 | 1.276%.049 | 1.504.059 Fp |—|—|-|—'10 4 5} 1—5.306.40 Fio 517) 477| 2| 4 | 2.615%.035 | 3.800.045 | 1.172.025 | 1.469-.032 Fis |—|-|—|—| 613 1|—|_—5.705.80 Fu | 592 526) 2) 4| 2.0504 044| 3.8314 053 1.589.031 | 1.801+.037 CE EE Raia Si (ede ol) Sel Ee ee TABLE 11 Grades of parents and means of children in five inbred lines (see figure 6) . | 1 j | | % Loki ae NOS. OF : reas ; NOS. OF eo ee een ee eee Pe e — — a ——=— lal|a|o] @ 9. [hay fe alavl?| o 9 |@le ae ates. eat [selena el ca A 2) 17) 2 | 2 | 1.53 | 2.03 |170)192) C 4") 71/5 | 5) 2.29 | 3.53 | 58 68 3) 53) 4 | 3 | 1.57 | 2.57 | 67, 69 (5 115 4) 5 2.97 | 3.35 | 52 91 4 67, 4 | 4 | 2.10 | 2.76 101/110 | 6 |135 4| 4 2.36 | 4.15 | 90} 89 5) 98 5 | 4 | 1.87 | 3.14 | 64 80 7 175 3 | 9 2.18 | 3.93 | 38) 44 6114 4 6 2.42 | 3.16 | 33) 13 8 |205 5 | 5) 2.62 | 4.27 |108118, 7124, 4.) 5 | 2.51 | 3.57 119157 9 239) 6| 6 2.84 | 4.25 | 38 70 8151, 4 4 | 3.32 | 4.98 94119 10 276 5 | 6 2.44 | 3.75 105108 | 9207| 5 | 5 | 2.73 | 3.98 | 93104 11 3347 | 5 3.07 | 4.60 | 46 45 | 10232] 3 | 3 | 3.41 | 5.06 | 37| 18 rae, | | 11267 518] 2.49 | 3.41 |196191] D | 4* 87) 5 ~=8 2.81 Lod 37 32 12377 5 6 | 2.95 | 4.26 149194 | & 110) 5 5 | 2.44 3.68 |104108 ae | | ts |e 6 137) 5 | 8 | 2.95 | 4.45 46 55 B 2 18,1) 1 2.23 | 2.59 129117) | 7 |173) 5 | 5 | 2.83 | 4.59 |109) 97 3| 56,7 | 6| 2.50 | 3.44 116125) | 8 206 5 | 6 | 2.91 | 4.26 | 96 87 4 88 5 | 5 | 2.67 | 3.91 | 63) 71) | 9 250 a | TP eOU beset 134127 5 106 5 | 5 | 2.47 | 3.57 (124) 94 10 |287, 6 | 6 | 2.47 | 3.36 (119/131 6131) 4 4 | 2.50 | 3.20 |187203 11 (825) 5 | 5 | 1.65 | 2.28 105 123 7187| 6 | 6 | 2.01 | 2.42 |170172| eee | | 8216 0 0 2.98 4.56 4850 E 51116 4 4 2.57 | 4.00 | 99120 aa | | 6 146 2) 7| 2.22 | 3.90 | 66) 52 cea wat | 7 191, 4| 5 2.25 | 3.86 | 63 57 | | | | 8 226 3 | 13| 8.51 | 4.60 | 37| 52 4 | | | | 9 268 5 5 3.10 | 4.00 | 59) 70 7 Preceding generations are those given under C. 83 BRISTLE INHERITANCE IN DROSOPHILA 85 (after the 6th) as far as the eleventh, no further advance has been detected. This is what the comparisons of different fami- hes from the same grandparents, or successive generations of inbred lines, had led one to expect. In the second generation there is a tendency for parents of higher grades to produce chil- dren of higher grade, while this is not the case in the later gener- ations where high and low variates seem to have the same genetic potentiality, and in so far as this is true, the variability must be explained by extra germinal causes. ° THE ROLE OF THE ENVIRONMENT 1. INFLUENCE OF FOOD In seeking causes for variability in the number of extra bristles, studies have been made on certain factors of the environment, namely, food and temperature. It was early observed that if any normal flies came from a bottle of inbred selected flies they “were apt not to appear among the first flies to hatch in that bottle. Also it became evident that more of the high grades did appear among the first flies hatched, than among the last. To make this clear the data was arranged to show the distribu- tions of the flies counted on successive days from individual bottles. In many cases it was clearly shown that successive days showed lower and lower distributions of extra bristles. In other cases there was no such decline. However, in all cases the highest flies appeared among the first flies drawn off. That the falling off in bristle numbers is not due to any differences in the fertilized eggs is shown by the fact that whenever there is found such a decline in bristle numbers at the end of a bottle, the first flies hatched from the next bottle into which the same parents had been placed, show bristle numbers as high as those found at first in the preceding bottle. This was found to hold good for all cases, even though the parents were moved into three or more successive bottles. When the changes from bottle to bottle were frequent there was less falling off at the end of a bottle. Since the facts are so clear it seems needless to publish a large number of examples. Table 12 gives 3 ex- THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 19, No. 1 86 E. CARLETON MACDOWELL amples from the 50 families so arranged. In mating No. 78, there was no falling off at the end of first bottle, but the second bottle shows very clear falling off. There is found, then, a certain relation between the time of hatching and the number of extra bristles, due to conditions external to the germplasm, namely, that the last flies from a bottle may be of lower grade TABLE 12 Daily counts of progeny from the same parents in successive bottles, showing that when the bristle numbers are lower at the end of a bottle, the high grades are regained at the begin- ning of the nezxt bottle. | | | | | || | | | ¥ | | MAT- | =e | BOT | x10. \pay| 0} 1)2]3] 415 | 6 |7/8|| ine | S05 |molpay 0} 1/2) 3/4/51] 6 |7/8 NO. | } | | | NO | } | Pae| e =, Nees | te 3 Eee | 131 | ist |10/14| | 1] 1] 2) 4] 5] | | |] 145] 18 | 10] 22) | 21° 53) 3 ae ic ete Wm 2 Se | 23) ne) 5 5d ae Ge el 16 SA oad 24 1) 4) 9| Siheyee yee Deca es, 2712) 8114) 8), 3) >a 20| 5| 9/12] 2 28] 2} 3| 7| 4| 2 7 a | | 1] 30 | 10 5 4 2d 22). | 8] 8) 26) 14/15) 7)2)4 31 | > Wiesel Bye SN ew Mie im bs |, ea} 24 | | 30] 2") 2) ae 7 as Pe a ee fa) 31/1 S22 | 5| 4] 3] 2 |}27| 4|16|29| 15} 2 te ee We oa 3| 45) Sie 3d 26l- |., | 3] iol aa Bh 48a 3) 1) 4/14) 9 i 27 2) 9)20|21| 6| 1]1 6) 1) 4] 1) 2) ee | Le Vena ae 2 2 eat ea | oer | Lad | 1-478) | Ist | 20) | eset 3] 26) 15|18| 3] 106 | 1st | 9/23] | 1| 4/10] 2| 2} 1/1) | 4) i) 5) 12) age | 251 1] 3].15] 8 ei 5} | 6) 16) 4] 2m ele Slee 6} | 3] 6] S| alg ee ee 7 2) 19'| 12:02 . | 2d 26) | 27) 6 Shoals 8 | 21 9).2) 2a ee 7| 15) 33) 12)-111 9| |10| 8| 9| 6 ae) Aa 342) oldie | 540) oul / | 110) 4) 2| 4] 17] 7| | | 10 61° Saag | | i a | 8.) a} jet a 4/2] 5| 2 3d | | 4 ae 1 1 | 12) 2 dl Za eee tie | id ier Wee Pa Pe 2 [iad | <9 1 Mepis 21) Cade ee 19 Lads | ' | | | | | than the first ones, and when this is true, the higher grades reappear in the next bottle It is common knowledge that the last flies from a bottle are apt to be smaller than the first ones hatched. This decrease in the sizes of the flies depends probably entirely upon the amount of food the flies eat while larvae; this amount in turn depends BRISTLE INHERITANCE IN DROSOPHILA 87 upon the condition of the food and the environment. The similarity in the occurrence of decreased bristles and decreased size at the last of a bottle led to the idea that there might be some relation between the two phenomena. ‘To see if smaller flies are apt to have fewer bristles, the total body lengths of 1400 random flies have been measured and their bristles counted. The data have been arranged in correlation tables. From table 13 one finds that up to a certain body size there appears a sort of correlation, in that the smaller the fly, the fewer the extra bristles it is apt to show. Above this size (grade 500) there TABLE 13 Males and females. Correlation between size and numbers of extra bristles SIZE NOS. OF EXTRA BRISTLES GRADES 1 7 a eT = 7 => 0 1 2 | 3 Pes 5 | 6 | 7 8 300 1 | | 325 1 | | | 350 2 aI 1 1 | | 375 2 3 7 1 hy | | 400 2 34 Cas 2 1 425 2 10. Naor 24 6 9 1 450 4 35 73 52 20 3 1 475 5 39 94 76 39 14 6 1 500 2 15 76 90 Avek eal ale 219 4 1 525 11 18 = fan hiner tee iy 5 1 550 1 Fae aie a 1G) 15 1 2 575 | i 5 3 6 5 5 1 600 1 Leewayad 3 | 3 appears to be no constant relation between the increase in size and the numbers of extra bristles. However, it will be noted that above this size there are no normals. It has been concluded that the factor controlling extra bristles is not sex linked; how- ever, nearly all the curves of extra bristled flies that have been presented, have shown that the males have fewer bristles than the females. Correlation tables were made for males and females separately (tables 14, 15). These show strikingly enough, that the males have fewer bristles than the females and are smaller, no male being above grade 500. This does not mean 88 E. CARLETON MACDOWELL that a male may never be larger than this, but the comparison with the females is accurate since males and fema es were both used in each group of flies that was measured. In the mass cultures of extra bristled flies the bristle numbers are somewhat lower and the number of normals higher than in the inbred lines, but the flies themselves being raised in larger numbers and so cramped in close together are much smaller TABLE 14 TABLE 15 Females. Correlation between size Males. Correction between size and and number of extra bristles numbers of extra bristles. SIZE NO. EXTRA BRISTLES SIZE | NO. EXTRA BRISTLES GRADE GRADE 1) ib a Reo VS bases] WU Bie da O | Pats 415 Gs 350 | 1 | 325 1 375 350 1 400 | 375 Wed ei) | 425 | Bail 400 1] *2) 450 3| 5 I] 2 A950 0 NS! ah Alla? AGS) | 2] 7 G3 242 450 | 1 | 10| 29 15) 5 2 500 | | 4| 19) 20:165/4/1 475 | 1| 14 29 15] 11) 7 | 1) 1 525 5) 1} 99\4/1 500|2] 3| 7 6 4/1 550 sO ese ane a Fs : ee 575 1 eae. as We ps OL 600 1 Werk vee ia! TABLE 16 To compare children from low grade parents taken from mass cultures and raised in pairs, with children from high grade parents that have come from continuously inbred lines, selected for increase in bristle numbers / GRADE | OFFSPRING—NOS. EXTRA BRISTLES gio ae ke MEANS sgkiy| Pa) 1 2 3 re ee 6 7 8 | 14 Parents from Mass cultures Doe. ware A 15 36 32 25 18 3 3.00 Bail Dea Ss. | 1h doo. to ee. | Oa 2 2.84 561 Leal“ 1 3 5 5 Bee be ih 1 | 1 | 4.00 Parents from 16th generation of inbreeding and selecting up 555.40 825 1 11 | 26. |. 14 4 4 1 3.55 556.1 UDes 4 32) a), SEL 45 24 9 2.45 ooo | 5b 6 33 | 39 42 31 Li 6 1 | 2.80 BRISTLE INHERITANCE IN DROSOPHILA 89 than the inbred flies mated in pairs. It is also found that the first flies that hatch from a new culture bottle are larger and have increased bristle numbers and low grade pairs separated from mass cultures give children of as high grades as are found in the inbred lines raised at the same time (table 16). The mass cul- tures, from which the parents in this table were taken, were started from the 5th and 6th selected generations and had been running eight months. The three inbred families shown from the 16th generation are the only ones mated very nearly at the same time as the three pairs taken from the mass cultures. This table has a deep significance in relation to the accomplishments of selection in the ten generations between the 6th to 16th. 2. DISCUSSION Thus there is evidence to show that flies below a certain size are apt to have fewer extra bristles than larger flies and that the size is largely dependent upon the condition and amount of food, or, more generally, on the environment; further, that males are shorter and have fewer extra bristles than females, and that the differences between mass cultures and the inbred lines disappear when the flies from the mass cultures are bred in pairs. It seems as though the small underfed flies do not have enough material to develop as many extra bristles as the larger flies can. The measurements used in the correlation tables do not make very satisfactory data, as the distention of the abdomen and so, the length of the fly, varies with the amount of food con- tained, but, however little light the tables may give as to the actual amount of correlation and influence of the environment, it is believed that the errors in measurement due to the varying abdominal contents are not great enough to prevent the con- clusion that such an influence of environment does exist. The fact of most importance at present, whatever its explanation may be, is that the numbers of extra bristles are influenced in some direct or indirect way by the conditions in the bottles. 90 E. CARLETON MACDOWELL With these facts in mind it will be well to return to the gener- ally low percentage of extras in the F: of crosses with normals of other races. It has previously been shown that apparent normals may none-the-less be homozygous for extra bristles; a plausible explanation of this fact seems to be the influence of food. 3. TEMPERATURE The regularity with which a new bottle produces an increase in bristle numbers, if it follows a falling off at the end of the pre- ceding bottle, shows that these variations are not mainly due to temperature. This does not say that temperature may not have a real, however slight, influence on bristle development. A series of preliminary experiments were performed to test the influence of temperature. Sister matings were raised in various temperatures, constant to a small fraction of a degree contigrade. In one experiment different sets of eggs from the same parents were raised in various temperatures and compared with controls in room temperatures. Two main questions were to be answered: Can the number of extra bristles be increased by using higher or lower temperatures? Do constant tem- peratures, irrespective of the degrees, influence the variability of bristles and thus indicate an influence of temperature? In the nine matings employed, 1860 flies were observed. The data were all arranged in curves, males and females separated and together, but no clear evidence was found to show that tempera- ture had any influence, either in producing higher or lower num- bers of extra bristles or by changing their variability. It is very clear that temperature influences the bacterial and fungal florae of the fermenting banana, and due to the chemical activity of the food, the environment of the developing flies goes through wide variations, as is witnessed by the multitude of different shades of odors from a single bottle during its productivity. Should any relation between temperature and bristle number be found in later investigations, it would be extremely difficult to show that this supposed relation was not an indirect effect, act- BRISTLE INHERITANCE IN DROSOPHILA 9] ing through the influence on the food. There can be no question that extra bristles are not influenced by temperature in the way that Miss M. Hoge found applied to extra legs. DISCUSSION The question in relation to which these experiments have most interest is whether Mendelian units can be modified by selection, and whether selection can accomplish anything more than a sifting and sorting of hereditary elements whose origin is still unknown. The tendency is either to hold that the hereditary elements can never be modified by simply propagating certain ones, or to hold that there is no integrity of such seeming ele- ments or factors, and that almost anything can be accomplished by sufficiently long and painstaking selection. Some of the observations will admit both interpretations. According to the selectionist’s view, the modification of the extra bristles appearing in a cross with normal, indicates that a factor has been modified. The restricting factor of the normal gamete may have transferred some of its restrictive properties to its allelomorphic mate in the extra germplasm, so that all the chil- dren formed by the union of two so modified extra gametes, would have fewer extra bristles than if the gametes had been unmodified. Selection made steady advances at first, showing that the gametes of flies of higher grades must be somewhat different from those of the lower grades, and therefore, the factor for extra bristles must be a variable thing, however truly a Men- delian unit. That the progress of selection does not continue after the sixth generation may only mean that the conditions have been so irregular or unfavorable that the real phenomena of the germplasm were entirely veiled. On the other hand, these same facts may be used to form the basis of an interpretation involving one or more smaller, or | accessory, restricting factors, which are found in many low bristled flies (some flies may lack all restricting factors, and still be low grade on account of their small size). The more and 92 E. CARLETON MACDOWELL more complete removal of these in successive generations by selection will permit the numbers of bristles to gradually increase. However, when all the flies become homozygous for the absence of the accessory restricting factors, no further increase by selection could be expected. Since there were no greater irregu- larities in the environment apparent after, than before, the sixth generation, and since flies from the fifth, sixth and seventh generations were being raised on the same lot of food and at the same time, the other interpretation of the failure of selection becomes very weak. And when the evidence showing that there is apparently no genetic difference in the later generations be- tween flies of different extra bristle grades, is reviewed, including as it does the weighty finding that seemingly norma’ parents may produce higher grade extra children than preceding high selected ancestors and the conclusion that after nine selections, high and low bristle grades give the same result in crosses, when all these facts are born in mind, it will be realized that the interpretation of the results by accessory factors seems in closer agreement with the facts than does the alternative hypothesis. The hypothesis of accessory factors needs no elaboration or change to explain the phenomena that accompany crosses with a normal race. The main factor for restriction keeps the num- ber of bristles down to four whenever it is present, so the ac- cessory factors can only be detected when the main one is absent. For this reason a simple Mendelian ratio may be found. From the selected flies some if not all the accessory factors have been removed. These would be present in the normal race used in the cross, and, due to the segregation of these independent restrictors, one would find in F, among the flies lacking the main restrictor, all combinations of the accessory factors, forming groups with various restrictive powers from strong to weak. The strongly restrictive groups would make the bristle numbers lower than in the uncrossed flies under the same conditions, while the weakly restrictive groups would make slight or no modification. This would result in an increase in the propor- tions of flies with few extra bristles, yet the high bristle grades would still be found. This is shown to be the case in figure 2, BRISTLE INHERITANCE IN DROSOPHILA 93 where the inbred and extracted distributions are compared. The first set of curves show this fairly well, but in the second set, in which the extra parents had been selected much longer, the phenomenon is very clear. On the other hypothesis, if the factor for extra bristles were modified in F;, one would expect to find the whole curve of the F, extras lowered, and another supposition would have to be made to explain the occurrence of high grades. Such an added supposition would involve the modification of the factor, sometimes in various degrees, and other times not at all—a supposition easy to make (Castle 714) but difficult to explain. That there is a greater modification in the F, extracted extras when the extra parents had been selected for several generations, than when they had not been selected, is the result expected if the selection had accomplished no more than to drop out certain accessory restricting factors, which being present in the wild parent would produce their effect in the second generation at the normal end of the distribution just as strongly as they did in the crosses before selection. To interpret this without using accessory factors one would have to suppose that a factor for extra bristles, that had been made more extra by selection, was more susceptible to contamination, just as squeezing a sponge will make it take up more water; but as has been shown, this kind of a mechanism will not explain the occurrence of the high extremes in this modified distribution. When one considers that the extra bristled condition is due, not to a single factor of which anything is known, but to the nucleus of determiners that carry the main heritage, such a modification hypothesis becomes vague and confused. The occurrence of extras in the F, of the crosses may ew ex- plained by incomplete dominance, in which case the proportion of F, extracted extras should be too high instead of too low. Their occurrence may be explained by a heterozygous condition of the restricting factors in the wild New York 1912 race, which had been escaped being weeded out by selection. The occurrence of extras in the New York 1912 race suggests such a heterozyg- ous condition. It seems probable that the slightly low propor- 94 E. CARLETON MACDOWELL tions of extras in F, is connected with the modification of their distribution. A strongly restrictive group of accessory restrict- ing factors, even in the absence of the main restrictor may pro- duce a normal fly, especially if the fly happens to be small. Much of the recent genetics work supports the principle of accessory or multiple factors. According to this principle more than one independent factor may influence a single character. Besides the cases which prove unquestionably the existence of such multiple factors by’ means of definite ratios, as the ligula in oats and the red grain in wheat (Nilsson-Ehle ’09), yellow endosperm in maize, (East and Hayes, ’11) and the triangular form of capsule in shepherds purse (Shull ’14) there are a large number of investigations in which the presence of multiple factors is strongly indicated. Since the author has already discussed such cases (MacDowell ’14 a and b) it will be necessary only to add references to the recent work of Wichler (713), [keno (14), Hayes (14), Davenport (713), Lotsy (’13), Phillips (14) and Punnett (714), and to eall attention to the critical discussion of Shull (14). Investigations showing transgressing segregation, in which the F, distributions form continuous gradations but with modes dividing the individuals into groups corresponding to 3:1, or 1:2:1, (Balls ’07, Leake ’11, Biffen ’05) have been interpreted by an hypothesis involving one main factor and accessory factors, similar to the one employed above. Still closer resemblance to the work herein described is that of Castle and Phillips (14) with piebald rats. They dealt with a variable character that proved to be influenced by a Mendelian factor. The distribution of the variations of this character was modified by crosses. No hypothesis other than that of accessory factors could be found to explain all the results. However, Castle still holds that the continued success of selection goes to prove the modifiability of a Mendelian factor. The main difference between the investigations on the piebald rats and the extra bristled flies, aside from the fact that no minus race of flies has been established, lies in the fact that in the case of the flies the progress of selection does not seem to continue after the sixth generation, while in the rats it appears to continue as far as the eee aM a BRISTLE INHERITANCE IN DROSOPHILA 95 selections have been made. Another difference that becomes illuminating in the light of these facts is that the flies have been inbred absolutely, while in the rats the inbreeding was of a fairly low degree. Return selection proved equally effective in the piebald rats. If the interpretation of accessory factors be ac- cepted for the results of crossing the rats, it is evident that at least part of the success of selection was due to the sorting out of these accessory factors. Now the fact of a successful return selection can not be sighted in this case to prove the theory that a factor has been modified, since as long as selection was showing steady progress, it should be possible to start a successful return selection. As long as the original selection was progressing there still is evidence of some heterozygosis of the accessory factors. This would afford a basis for the return selection. At this time any positive statement would be premature, but the results given by an attempted return selection of the extra bristles after the upward progress had seemed to stop, appear to indicate that this return selection is ineffective. Finally the more complicated hypothesis of Pearl (’12) supported by his extensive investigations on fecundity in fowls, must be sighted as one based fundamentally on the conception of multiple factors. Taken then on their own merits, the results presented in this paper do not give critical evidence in support of either the hypothesis of modification or of accessory factors. However, the failure of selection after success for six generations and the prob- able genetic equality of the various bristle grades in the later gen- erations, seem to bear the balance strongly towards the hypothesis of accessory factors. Taken in the light of much of the recent genetics investigations, many of which have close theoretical similarities, it is almost impossible to avoid the conclusion that the interpretation of accessory factors is the more probable. Besides, this hypothesis affords a more thinkable mechanism and is more readily understood and tested. For these reasons this interpretation has been adopted at least as a working hy- pothesis upon which to base further investigations. Experiments are already under way to attempt the isolation of accessory factors and, by crossing, to prove unquestionably their existence. 96 E. CARLETON MACDOWELL CONCLUSIONS From a pair of wild flies a race of Drosophila ampelophila has been established which has regularly more than the normal four thoracic bristles. By selecting high grade parents and inbreeding brother to sister, the number of extra bristles was gradually increased for six generations. From the seventh to the eleventh generations fluctuations were found showing no further increase. The maintenance of the high grades of extra bristles does not depend upon selection as, low grade parents from mass cultures started from the fifth and sixth generations that have run eight months, when raised in single pairs, give as high grade offspring as inbred and selected parents mated at the same time. A Mendelian factor is involved in the inheritance of extra bristles, and as normal dominates eztra, this may be regarded as a dominant factor that restricts the number of bristles to four. This factor is not sex linked, although males are apt to have fewer extra bristles than females. The extracted extra bristled flies have a lower distribution than that of the inbred flies of the corresponding generation, although the high extremes of the inbred race are also found among the extracted extras. There is a greater difference between the inbred and extracted distributions when the cross is made after eight selections than when made after only one selection. Environment influences the number of extra bristles, and since small flies are not apt to have as many extra bristles as large ones, it appears that the amount of food eaten is an important factor. From the above statement an explanation may be found for the fact that an apparently normal fly may be genetically homo- zygous for extra bristles. The hypothesis of accessory factors will explain all the facts, and that of modification of a Mendelian factor may be employed to interpret most of them. BRISTLE INHERITANCE IN DROSOPHILA 97 The similarity with much recent work that has given more or less positive evidence of multiple and duplicate factors, per- suades the author that the hypothesis of accessory factors is probably the best one for the facts. The following hypothesis is adopted upon which to base further work: the extra bristles in Drosophila ampelophila are due to the absence of one main restricting factor, and their number is also influenced by accessory restricting factors, which, in the absence of the main one, produce flies with reduced num- bers of extra bristles. October, 1914. LITERATURE CITED Batts, W. L. 1907 Mendelian studies of Egyptian cotton. Jour. Agric. Sci., vol. 2, p. 365. Birren, R. H. 1905 Mendel’s law of inheritance and wheat breeding. Jour. Agric. Sci., vol. 1, p. 4. Castie, W. E. 1914 Size inheritance and the pure line theory. Zt. f. ind. Abs. u. Verb., Bd: 12, s. 2383. CasTLE, W. E., and Puinurrs, J. C. 1914 Piebald rats and selection. An experimental test of the effectiveness of selection and of gametic purity in Mendelian crosses. Pub. Carnegie Inst. of Washington, No. 195. Davenport, C. B. 1913 Heredity of skin-color in negro-white crosses. Pub. Carnegie Inst. of Washington, No. 188. East, E. M., and Hayes, H. K. 1911 Inheritance in maize. Conn. Agric. Exp. Sta. Bull. No. 167. Hayes, H. K. 1914 Theinheritance of certain quantitative characters in tobacco. Zt. f. indukt. Abstammungs- u. Vererbungslehre, Bd. 10, s. 115. Hoar, M. A. 1915 The influence of temperature on the development of a Mendelian character. Jour. Exp. Zodél., vol. 18, no. 2, p. 241. IkENO, S. 1914 Studien uber die Bastarde von Paprika (Capsicum annum). Zt. f. indukt. Abstammungs- u. Vererbungslehre, Bd. 10, s. 99. Leake, H. M. 1911 Studies in Indian cotton. Jour. Genet., vol. 1, p. 205. Lotsy, J. P. 1913 Hybrides entre espéces d’Antirrhinum. Compt. rend. 4° Conf. Internat. de Genetique, p. 416. MacDowe tu, E. C. 1914a Multiple factors in Mendelian inheritance. Jour. Exp. Zodl., vol. 16, no. 2, p. 177. 1914b Size inheritance in rabbits. With a prefatory note and appen- dix by W. E. Castle. Pub. Carnegie Inst. Washington, No. 196. Nitsson-EnLE, H. 1909 Kreuzungsuntersuchungen an Hafer und Weizen. Lunds Universitets Arsskrift, INGER eAdide 2 yisdero: Nir. 2: Peart, R. 1912 The mode of inheritance of fecundity in the domestic fowl. Jour. Exp. Zoél., vol. 13, no. 2, p. 158. 98 E. CARLETON MACDOWELL Puiturrs, J.C. 1914 Further studies of size inheritance in ducks, with obser- vations on the sexratio of hybrid birds. Jour. Exp. Zoél., vol. 16, p. 131. Punnett, R. C., and Baitey, P.G. 1914 On inheritance of weight in poultry. Jour. Genet., vol. 4, no. 1, p. 23. Suuti, G. H. 1914 Duplicate genes for capsule-form in Bursa bursa-pastoris. Zt. f. indukt. Abstammungs- u. Vererbungslehre, Bd. 12, Heft 2. WicuierR, G. 1913 Untersuchungen iiber die Bastard Dianthus ameria xX Dianthus deltoides nebst Bemerkungen iiber einige andere Artkreuz- ungen der Gattung Dianthus. Zt. f. indukt. Abstammungs-u. Verer- bungslehre, Bd. 10, s. 177. CHANGES IN THE RELATIVE WEIGHTS OF THE VA- RIOUS PARTS, SYSTEMS AND ORGANS OF YOUNG ALBINO RATS HELD AT CONSTANT BODY-WEIGHT BY UNDERFEEDING FOR VARIOUS PERIODS C. M. JACKSON Institute of Anatomy, University of Minnesota, Minneapolis FOUR FIGURES CONTENTS Micenitalcanaemethods. «1.0 aoe eee ce Cees bee fe ct tiveness wie Sets 100 enmincombady and ‘tail 1 ene arene fe ee ei, Salod Sadie a eqee toda nee 106 LSICOLS 6 AE Gear en rE RE a SE a ais Sn OER RR Oe ere 112 BEL UETIMM ES TC “br UN Kea." «7c See ee eRe oeiccar vovacs 3,6 sin sustained oe ee, 3 115 MGS PAUIEE CINE 9 ode t vida sc ke Speeds EET oy Shoe ake eevee cei. 117 SUBIC: Soe eee REC SEI: 6 Sisru.coo tan Bnd SARA Sees © Aue ee Cea son WG INLRAE CCC LS AUTRE So ne ee me eae ct NAA) NA Pe Oe ee Se eae 125 WASCE Aba GE GC MTATI CEM? otc Since the completion of the present paper, I have received, through the court- esy of Professor Donaldson, a manuscript copy of reference tables compiled at The Wistar Institute by formulas for various measurements of the albino rat. These include the body-lengths and tail-lengths, by sexes, from newborn to adult. From these data I have calculated the tail-ratios and find the result in general agreement with the curve shown in figure 2. The tail-ratios calculated from the Wistar tables are somewhat lower, corresponding to body-weights from 30 to 100 grams, however. They also increase steadily, so that at body-weights above 200 grams they le slightly above the curve in figure 2._ The tail-ratio according to the Wistar tables is about 4 per cent higher in the female throughout, when the sexes of equal body-length or body-weight are compared. In rats above 300 grams body-weight, the tail-ratio is about 0.86 in the male and 0.89 in the female. WEIGHTS OF ORGANS IN UNDERFED YOUNG RATS 111 The two observations on rats held constant from the age of three weeks to the ages of thirteen and sixteen weeks would seem to indicate a continuation of the process of elongation of the tail, even beyond the normal ratio at corresponding age, but the number of observations is too small for definite conclusion. The number of animals beginning the experiment at later ages, body-weight constant from age of six to age of thirty-two weeks (2) and from ten to thirty-five weeks (3), is also too small to draw any very positive conclusions. However, so far as they go, they indicate (table 3 b; fig. 2) that between the ages of six and thirty-five weeks there is no marked change in the tail-ratio of rats held at constant body-weight. It will be observed that also in the normal rats between six and thirty-five weeks of age there is no apparent change in the tail-ratio, whereas between three and six weeks of age there is normally a decided increase in the tail-ratio. The lengths of the body and tail are of course determined primarily by the growth of the skeleton. I would therefore interpret the results concerning the lengths of tail and body as follows. In young, growing rats held at constant body-weight, the body and tail tend to increase so as to assume the normal ratio at corresponding ages. This is due to the fact that, as will appear later, the skele- ton continues to grow in a normal manner (though at a reduced rate) in animals held at constant body-weight. There is another possible factor in causing the increased tail- ratio in young, growing rats, which may also apply to the similar relative elongation of the tail found in adult rats (ef. Jackson 15¢). Professor Donaldson points out (in a personal com- munication) that during inanition there may be an arching of the spinal column, producing an actual shortening of the body- length. Such an arching actually does occur, and may be noted especially in young rats during chronic inanition. It is well shown in the stunted rats photographed in figure 1. Of course the greater part of this longitudinal curvature of the spinal column is eliminated by the slight tension exerted in order to straighten out the body when it is measured after death. But it is still 112 Cc. M. JACKSON quite possible that this does not entirely eliminate the shorten- ing of the column. In any event, however, this is probably a factor of minor importance in altering the tail-ratio during inanition. Hatai (08) in a series of five ‘stunted’ rats in which growth had been retarded (but not stopped) by a diet of starch-mixtures from: age of 30 days up to from 127 to 215 days (the final body- weight being from 70.9 to 113.7 grams) finds the average tail- ratio 0.75 as compared with about 0.82 in controls. He notes that: The most conspicuous external differences between normal and stunted rats as shown by the stunted rats are in the length of the body and of the tail, both of which were considerably reduced with respect to the body-weight. This peculiar difference, as is seen from the table, holds true in every case. Further, the ratio between the length of the body and that of the tail is considerably less in the stunted rats than in the control rats. . . . Underfeeding therefore produces short tailed individuals. Recently, however, Dr. Hatai (in a personal communication) states that in other inanition experiments he has obtained dif- ferent results, and that ‘‘rats either grown or kept in a state of chronic inanition (starch feeding, lipoid-free ration and wheat embryo feeding) give a longer tail’”’ in agreement with my results. Morgulis (11) in the salamander Diemyctylus found a rela- tively greater shrinkage in the tail than in the body during inanition; while Harms (’09) found the converse to be true in Triton. HEAD The head (table 4; fig. 3) at three weeks normally forms an average of 22.5 per cent of the body, the average net body- weight being 21.2 grams. In the 11 controls at three weeks, the body-weight (24.6 grams) is slightly higher, and the correspond- ing relative head-weight, 20.6 per cent, somewhat lower. In the rats held constant from the age of three weeks to the ages of six and eight weeks, the average percentage of the head (21.6 per cent and 23.9 per cent) is higher than that of the controls. But the average body-weight in these groups is lower, more WEIGHTS OF ORGANS IN UNDERFED YOUNG RATS 113 nearly the normal above cited, so it is doubtful whether there is any actual increase in the head-weight (either relative or absolute) during the experiment. The larger ten weeks group, however, is nearly equal to the controls In average body-weight, and shows an apparent increase in head-weight from an average of 5.01 to 5.384 grams, or from 20.6 per cent to 22.7 per cent (table 4; fig. 3). In any event, however, the increase in the head-weight is sight, and is not apparent in the two rats held constant from three to thirteen and sixteen weeks (average of the two is 20.6 per cent). On the other hand, there appears to be a slight increase in the weight of the head in the rats held constant from six to thirty-two weeks (15.2 to 17.7 per cent), and from ten to thirty-five (12 to 14.0 per cent). On the whole, therefore, the evidence would appear to indicate that in young rats held at constant body-weight for considerable periods of time there is a slight increase in the weight of the head. This is probably due to the increase in skeletal weight, which in the head probably overbalances the decrease in the weight of the TABLE 4 The head; average absolute weight, average percentage of net body-weight and range indicated e Ze ao | SS aay DESCRIPTION OF RATS 8 5 s 5 LSU SSONEA DUS) Vee RRS EN Ga PRK Gaya & 5 iq : (AND RANGE): GRAMS ee SE Bye Z r=) Normal head at 3 weeks (Jackson ’13)............ 24 PAP? 4.60 (3.44- 5.85) 22.5 (16.3-27.3) KGOMUNONSTALONVECKS! beariaere irercieis oy0\cyeicle areya pee aares 11 24.5 5.01 (4.70- 5.90) 20.6 (18.3-23.0) Body-weight constant 3 weeks (age of 3 to 6 weeks) 7 22.4 4.83 (4.50- 5.10) 21.6 (19.9-23.3) Body-weight constant 5 weeks (age of 3 to 8 weeks) 3 20.2 4.73 (4.60- 4.90) 23.9 (19.9-26.0) Body-weight constant 7 weeks (age of 3 to 10 weeks) 22 23.8 5.34 (4.60- 6.50) 22.7 (20.4-26.2) Body-weightconstant10 weeks (age of 3 to 13 weeks) 1 25.5 5.00 19.6 Body-weight constant 13 weeks (age of3 to 16 weeks) 1 26.0 5.50 21.6 Normal at 6 weeks (Jackson ’13)................... 42 50.0 7.40 (6.30-9.40) 15.2 (11.3-18.0) Gonsrols at 6 WeGkS.-c. eisc.cte een eirin w wieres shee 2 42.4 6.40 (6.20- 6.60) 15.0 (14.6-15.5) Body-weight constant 26 weeks (age of 6to 32 weeks) 2 47.1 8.30 (8.20- 8.50) 17.7 (16.8-18.6) Wornprolsat Ol weeks -masccecte sien Miele oie Poseieepne.c he 6 134.0 13.30 (11.10-14.70) 10.1 ( 8.6-11.7) Body-weight constant 25 weeks (age of 10 to 35 weeks (normal head for corresponding body-weight of 85 grams forms about 12.0 per cent.............. 1 85.5 12.10 14.0 Coneralsiatio2' ANG’ soy WeeKSa-s eee ae decile cis aan 6 189.5 20.30 (17.40-26.30) 10.7 ( 9.8-11.4) 114 Cc. M. JACKSON Head Head 10.1 per cent. Head Fore-limbs 22.7 per cent i 6.9 per cent. 20.6 per cent. Fore-limbs Hind-limbs Fore-limbs 9.6 per cent. 15.6 per cent. 8.5 per cent. Hind-limbs Hind-limbs 15.7 per cent. 15.4 per cent. Trunk 67.4 per cent. Trunk Trunk 54 1 per cent. 53 4 per cent. Controls at 3 weeks Constant 3 to 10 weeks Controls at 10 weeks Fig. 3 Diagram representing the average relative (percentage) weights of parts of the body (head, extremities and trunk) in albino rats held at constant body-weight from the age of three to ten weeks, and in controls at three and at ten weeks of age. WEIGHTS OF ORGANS IN UNDERFED YOUNG RATS Lt5 integument; although, as will be shown later, the average loss in the integument of the entire body is relatively slightly greater than the corresponding skeletal increase (fig. 4). In both acute and chronic inanition in adult rats (Jackson 15 a, ’15 c) the head increases very markedly in relative weight, the loss in absolute weight being but very slight in comparison with the loss in weight of the entire body. EXTREMITIES AND TRUNK The extremities (table 5; fig. 3) were separated at the shoulder- joint and hip-joint, respectively. There is apparently a slight decrease in the relattve weight of the fore-limbs in the young rats held at constant body-weight from the age of three weeks to six, eight, ten, thirteen and sixteen weeks of age. In the case of the rats held constant from three to ten weeks, the apparent decrease is from an average of 9.6 per cent to 8.5 per cent. On account of the small number of observations, however, and the difficulty in separating the limbs (especially the integument) in an absolutely uniform manner, the slight apparent decrease is of doubtful significance. Im the case of the hind-limbs, there is likewise an apparent indication of a slight decrease, but even less marked than in the fore-limbs. The apparent average decrease from 15.7 per cent to 15.4 per cent of the body-weight in the largest group (three to ten weeks, is well within the limits of experimental error. On the whole, therefore, it is doubtful whether there is any distinct and significant change in the weights of the extremities in young rats held at constant body-weight for considerable periods. A slight loss, however, might be accounted for by the shghtly greater loss in the integument (as compared with the gain by the skeleton); especially since the integument of the limbs, probably forms a relatively larger part of the limbs than the whole integument does of the whole body. The trunk was not weighed directly, but its weight was cal- culated by subtracting from the net body-weight the weight of the head and extremities. From what has been said con- THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 19, No. 2 TABLE 5 The extremities; average absolute weight, average percentage of net body-weight and range indicated 3 zZ = : ABSOLUTE WEIGHT PASE AE) EE ES DESCRIPTION OF RATS s 3 . Z (AND RANGE): GRAMS aD een aS a = re a. Fore-iimbs Normal at 3 weeks (Jackson and Lowrey).......-. 4 25.5 2.36 9.3 (7.4-10.90) Controls ais Weeks eee vere e es eee sont w co pene 6 23.8 2.29 (2.00-2.51) 9.6 (8.7-10.60) Body-weight constant 3 weeks (ageof3to 6 weeks) 4 PR PE | 1.68 (1.50-1.90) 7.3 (6.7— 8.30) Body-weight constant 5 weeks (ageof3to 8 weeks) 1 24.6 1.70 6.9 Body-weight constant 7 weeks (age of 3 to 10 weeks) 8 22.3 1.84 (1.60-2.20) 8.5 (7.4- 9.50) Body-weight constant 10 weeks(age of 3 to 13 weeks) 1 25.5 1.80 iil Body-weight constant 13 weeks (age of 3 to 16 weeks) 1 26.0 1.80 6.9 Normal at 6 weeks (Jackson and Lowrey)......... 4 79.2 5.30 6.7 (5.9- 8.22) Gontrols/at'G weeks: )*3..° 205.4: 2st ee eee 1 42.3 3.00 (ath Gontrolsiag tl wrecks ei ooo osc ne eee ee eee 2 127.9 8.90 (7.20-10.60) 6.9 (6.3-— 7.60) Normal at 10 weeks (Jackson and Lowrey)........ 3 141.9 7.60 5.3 (5.21-5.50) b. Hind-limbs Normal at 3 weeks (Jackson and Lowrey)......... 4 25.5] 3.80 14.9 (14.3-15.40) Gontrolsitiamvecksi- 2.00352. ere ee eee 6 Daes 3.75 (3.20-4.13) 15.7 (14.3-15.90) Body-weight constant 3 weeks (ageot 3to 6 weeks) 4 23.1 2.97 (2.55-3.60) 12.8 (11.6-15.10) Body-weight constant 5 weeks (age of3to 8 weeks) 1 24.6 3.40 13.8 Body-weight constant 7 weeks (age of 3 to 10 weeks) 8 22.3 3.38 (3.00-3.80) 15.4 (14.3-15.90) Body-weight constant 10 weeks (age of 3 to 13 weeks) 1 DS 3.20 , 12.4 Body-weight constant 13 weeks (age of 3 to 16 weeks) 1 26.0 3.20 12.6 Normal at 6 weeks (Jackson and Lowrey)......... 4 79.2 11.80 14.9 (14.6-15.40) Gontrolsat Grweeks..4io5-.°. 5-6 + terre ke ee 1 42.3 5.60 13.2 Gontrois'st 10sweeks'*5)2 <4 =. 5-eeeeeeeee ee 2 127.9 19.90 15.6 (15.4-15.80) Normal at 10 weeks (Jackson and Lowrey)........ 3 141.9 22.10 15.6 (14.5-16.50) TABLE 6 The integument; average absolute weight, average percentage of net body-weight and range indicated a = @ E z 2 Z ABSOLUTE WEIGHT | SELATLVE WEIGHE DESCRIPTION OF RATS : j (AND RANGE): GRAMS (nae Gm ee Zz r Normal integument at 3 weeks (Jackson and PEG WECY) Nt. pte ch) eRe er ere ER ee eee teem 13 24.8 5.55 22.4 (18.7 -29.2) Controls'sit3 iweeks: oc oe eran on ee se co 10 25.1 5.59 (3.86- 6.36) 21.9 (18.4 -25.2) Body-weight constant 3 weeks (ageof3to 6 weeks) 8 22.2 2.81 (2.21- 3.21) 12.5 ( 9.82-14.1) Body-weight constant 5 weeks (ageot3to 8weeks)| 3 20.2 2.78 (2.27- 3.30) 13.8 (12.6 -15.5) Body-weight constant 7 weeks (ageot3to10weeks)} 22 23.8 3.41 (2.63- 4.51) 14.5 (12.3 -17.5) Body-weight constant 10 weeks (age of 3 to 13 weeks) 1 25.5 3.50 Tov, Body-weight constant 13 weeks (age of 3 to 16 weeks) 1 26.0 3.30 12.7 Normal at 6 weeks (Jackson and Lowrey)......... 14 64.4 13.46 20.9 (16.7 -25.9) GControlsiat Grweeks.: 06. AA aes OS 2 42.4 8.04 (8.00- 8.08) 17.0 (15.1 -19.0) Body-weight constant 26 weeks (age of fto32weeks)| 2 47.1 6.15 (5.70- 6.60) 13.0 (12.9 -13.1) Normal at 10 weeks (Jackson and Lowrey)........ 10 131.0 24.50 18.7 (15.6 —22.3) Controls stilUsweeks:: 2.20502 toc se ae sec eee ae 6 134.0 | 26.70 (19.40-32.10) 20.0 (17.8 -22.0) Bodyweight constant 25 weeks (age ot 10 to 35 weeks) 3 77.8 14.00 (12.30-17.20) 17.9 (16.6 -20.1) Controls at'o2‘and ao weeks 2... 000255... 22eees ee 6 189.5 | 38.40 (28.10-55.20) 20.1 (18.0 —23.2) * WEIGHTS OF ORGANS IN UNDERFED YOUNG RATS iM Eve cerning the head and extremities, it follows that there cannot be much change in the trunk-weight, since the probable slight in- crease in the weight of the head is off-set by a slight decrease in the extremities. In the largest group, however, held at constant body-weight from three to ten weeks of age (fig. 3), the trunk would apparently decrease from an average of 54.1 per cent to 53.4 per cent. This apparent change is so slight as to be (prob- ably) insignificant. The results concerning the parts of the body therefore fail to indicate any decided change of proportional weights in young animals held for considerable periods at constant body-weight. There is apparently a very small increase in the head, counter- balanced by a corresponding decrease in the trunk and extremi- ties, but the change is so slight as to be of doubtful significance. During inanition in adult rats, there is apparently a relative increase in both head and extremities, counterbalanced by a relative decrease in the trunk (Jackson 715 a, ’ 15c). INTEGUMENT In the rats held at constant body-weight from the age of three weeks to six, eight, ten, thirteen and sixteen weeks, there is a very marked loss in the weight of the integument (including hair and nails; table 6; fig. 4). In the case of the largest (three to ten weeks) group, the decrease is from an average of 21.9 per cent to 14.5 per cent of the body-weight. In terms of abso- lute weight, the decrease is from 5.30 grams (5.59 grams, less correction on account of difference in body-weight, which aver- ages 25.1 grams at three weeks and 23.8 grams at ten weeks) to 3.41 grams, a decrease of about 36 per cent. The decrease would appear slightly greater if the difference in relative weight of the integument for different initial body-weights were taken into account. There is apparently even greater loss at the other ages. It would appear that this loss (which is perhaps chiefly a loss of fat) occurs rather early, as at six weeks (body-weight held constant three weeks) the loss is as great as at subsequent and longer periods. 118 Cc. M. JACKSON Integument integument 14,5 per cent. Integument 21.9 per cent. 20.0 per cent. Ligamentous skeleton Ligamentous skeleton Bans 21.2 per cent. 10.7 per cent. 15.7 per cent Musculature Musculature Musculature 32.0 per cent 41.6 per cent. 31.2 per cent. Viscera Viscera 20.5 per cent. 22 2 per cent Viscera 14.3 per cent. ‘Remainder’ ‘Remainder’ ‘Somat , emainder 13.4 per cent. 10.7 per cent. 10.1 per cent. Controls at 3 weeks. Constant 3 to 10 weeks. Controls at 10 weeks. Fig. 4 Diagram representing the average relative (percentage) weights of the various systems (integument, skeleton, musculature, viscera and ‘remainder’ ) in albino rats held at constant body-weight from the age of three to ten weeks, and in controls at three and at ten weeks of age. WEIGHTS OF ORGANS IN UNDERFED YOUNG RATS 119 In the rats experimented upon at later and longer periods (ages of six to thirty-two weeks and ten to thirty-five weeks) there is also a marked loss in the weight of the skin, though apparently not so great as at the earlier periods. This loss in the weight of the integument is in striking contrast with the results of inanition in adult rats (Jackson ’15 a, ’15c). Here the loss is very nearly proportional to that of the whole body, so the integument nearly maintains its relative (per- centage) weight. From his experiments upon young dogs held at constant body- weight, Aron (’11, p. 29) states that: ‘‘The skin shows a slightly higher percentage of the body-weight in those animals kept at a constant weight than in the normal, control dogs. These figures indicate that, while the (body) weight was constant, the skin increased very slightly in weight.’’ The figures cited show the skin in animals held at nearly constant body-weight to form (in four cases) 12.2 to 14.6 per cent of the body-weight, whereas in three corresponding full-fed controls the skin formed 11.2 to 13.0 per cent. Aron, however, overlooks the fact that he is making his comparison with controls at the end of the experi- ment. In order to judge what changes have taken placed during the experiment, the comparison must be with normal control animals killed at the beginning of the experiment. Aron records but one case which can be used for this purpose. His Dog D (table 13, Experiment IV) killed at the age of 40 days, the begin- ning of the experiment, with body-weight of 1985 grams shows a skin-weight of 320 grams, or about 16.1 per cent of the body- weight. From Aron’s own data, therefore, I would reach the opposite conclusion, viz., that in young dogs held at constant body-weight, the skin suffers a marked loss in weight. This would agree with my results on rats. SKELETON The skeleton (table 7; fig. 4) was prepared in three ways. The bones, together with the cartilages, periosteum and liga- ments, constitute the ‘ligamentous skeleton’ (table 7a). The bones and cartilages, after removal of the periosteum and liga- 120 Cc. M. JACKSON ments by immersion for about one hour in 1 per cent aqueous ‘gold dust ’solution at 90°C., constitute the ‘cartilagmous skele- ton’ (table 7b). Finally the cartilaginous skeleton dried in an oven at 95°C. to constant weight constitutes the ‘dry skele- ton’ (table 7 ¢). An examination of the weight of the ligamentous skeleton (table 7 a) reveals the striking fact that while the body-weight TABLE 7 The skeleton; average absolute weight, average percentage of net body-weight and range indicated FS a E ES eC = RELATIVE WEIGHT DESCRIPTION OF RATS S 6 =O (ce nae eee ane SoS o& »& re sae) | Me a PER CENT or 5 Z Zz rs a. Ligamentous skeleton Normal at 3 weeks (Jackson and Lowrey)......... 13 24.8 4.12 16.6 (13.1 —21.10) Gontrol statis weeks 6. 2 eG - 2sjch See eee 12 24.5 3.90 (3.100- 5.46)| 15.7 (13.5 -17.00) Body-weight constant 3 weeks (ageof3to 6weeks)| 8 22.2 4.04 (3.140— 5.50)| 18.0 (14.6 —23.90) Body-weight constant 5weeks (ageof3to 8 weeks) 3 20.2 4.74 (4.290— 5.50)| 23.7 (22.3 —24.90) Body-weight constant 7 weeks (ageof3to 10weeks)| 22 23.8 4.98 (3.700- 7.14)| 21.2 (17.7 —24.60) Body-weight constant 10 weeks (age of 3 to 13 weeks) 1 25.5 4.80 18.8 Body-weight constant 13 weeks (age of 3 to 16 weeks) 1 26.0 5.20 20.0 Normal at 6 weeks (Jackson and Lowrey)........- 14 64.4 9.02 14.0 (10.5 -20.10) Gontrols at tiweeks acdsee hn -h.cs eee ee Fee Ue 2 42.4 5.48 (4.750- 6.20)| 13.0 (11.2 -14.70) Body-weight constant 26 weeks (age of 6 to 32 weeks) 2 47.1 7.20 (6.800- 7.60)} 15.4 (15.1 -15.60) Normal at 10 weeks (Jackson and Lowrey).......- 10 131.0 15.30 11.7 (10.0 -12.90) Cortrols'atil0 weeks ss osc. os tae ee eee 6 134.0 14.00 (11.400-17.60)| 10.7 (8.5 -14.10) Body-weight constant 25 weeks (age of 10 to 35 WEEKS) oP a ene bose wesc ly eee et ~ eee 3 77.8 | 10.30 (9.400-11.60)} 13.3 (12.7 -13.70) Gontrols'atic2 and: opiweekS-......oe. -aet ete a oo 6 189.5 18.60 (15.600-24.30) 9.8 (8.8 -11.20) b. Cartilaginous skeleton (Fresh) Controls atid weeks---- een on see ea ee co eee 6 22.9 2.60 (2.120- 3.00)|} 11.4 (9.0 -12.90) Body-weight constant 3 weeks (ageof3to 6 weeks) 3 22.9 3.56 (2.930- 4.04)| 15.5 (13.3 -17.00) Body-weight constant 45 weeks (ageot3to 8 weeks) 1 24.6 4.50 18.3 Body-weight constant 7 weeks (age of 3 to 10 weeks) 8 22.4 3.16 (2.420- 4.00)| 14.6 (11.9 -17.10) Body-weight constant 10 weeks (age of 3 to 13 weeks) 1 25.5 4.00 ibyey/ Body-weight constant 13 weeks (age of 3 to 16 weeks) 1 26.0 3.96 1502 Controlsat0’ weeks... -2 ence ose ea eee ee 1 115.0 6.40 5.6 > c. Dry skeleton (cartilaginous) Controlsiat 3oweeks*. .-s.... S:.s<.cn cee sees os ae eee Body-weight constant 3 weeks (ageof3to 6 weeks) Body-weight constant 5 weeks (ageof3to 8 weeks) 5 0.804 (0.710-0.964)} 3.43 (3.17- 4.03) 3 1 Body-weight constant 7 weeks (ageof3tol0weeks)| 9 22, 1 1 1 .091 (1.033-1.172)| 4.98 (4.70— 5.13) .285 (1.076-1.485) .84 (4.76- 7.00) bho ~ oneo Body-weight constant 10 weeks (age of 3to 13 weeks) Body-weight constant 13 weeks (age of 3 to 16 weeks) ControliatsiO weeks: ..< o<<=2% .camos scene sens 26.0 115.0 WEIGHTS OF ORGANS IN UNDERFED YOUNG RATS Tt is held constant the skeleton continues to increase in weight to a marked degree. In the rats beginning at three weeks, there is ‘an increase in the relative weight of the ligamentous skeleton from 15.7 per cent of the body to 18.0 per cent at six weeks:and to an apparent maximum of 23.7 per cent at eight weeks. This latter is probably an exceptional figure, as in the largest group, at ten weeks, the average is 21.2 per cent (fig. 4). This corre- sponds to an increase from an absolute weight of 3.90 to 4.98 grams, an increase of about 28 per cent (or slightly more, if correction be made for the difference in body-weight, average 24.5 grams at three weeks and 23.8 grams at ten weeks). The two cases carried to thirteen and sixteen weeks, respectively, show a slightly smaller relative increase. The rats used at later and longer periods (ages of six to thirty-two weeks and ten to thirty-five weeks) also show a considerable increase in the skeleton, though relatively less than those beginning at the earlier period. The data for the cartilaginous skeleton (table 7b) similarly show a marked increase in rats held at constant body-weight for various periods beginning at three weeks of age. The figures for the largest group (three to ten weeks) indicate an increase from 11.4 per cent to 14.6 per cent of the body. In terms of absolute weight, the increase is from an average of 2.60 grams (body-weight 22.9 grams) to 3.16 grams (body-weight 22.4 grams), an increase of about one-fourth. Subtracting the percentage weights of the cartilaginous skeleton from the corresponding ligamentous skeleton, there is (for the three to ten weeks group) an evident increase of the ligaments and periosteum from 4.3 per cent to 6.6 per cent of the net body-weight. This would indicate that the hgamentous component of the skeleton shares in the marked growth during constant body-weight. Professor Donaldson (in a personal communication) has kindly supplied a series of observations showing that the cartilaginous skeleton in the normal rat changes from a relative weight of about 10 per cent of the body at 20 grams to 7.5 per cent at 50 grams, 7 per cent at 100 grams and 6.7 per cent in rats above 122 Cc. M. JACKSON 200 grams. These weights, however, do not include the inter- vertebral dises. The data for the dried cartilaginous skeleton (table 7 c) indi- cate an even greater increase in the dry skeleton of the rats held at constant body-weight. Thus in rats beginning at three weeks the dry substance increases from 3.43 per cent of the body weight to 4.98 per cent at six weeks of age, 5.49 per cent at eight weeks, 5.84 per cent at ten weeks, 6.31 per cent at thirteen weeks and 6.71 per cent at sixteen weeks. Since the increase in the dry skeleton is relatively greater than that for the (moist) cartilaginous skeleton, it necessarily follows that the skeleton must be losmg in percentage of water and gaining in percentage of dry substance. The percentage of dry substance has been calculated for each individual skeleton included in tables 7 a and 7b, and the averages for each group are as follows: controls at three weeks, 31.4 per cent; constant three to six weeks of age, 33.5 per cent; three to eight weeks, 30.0 per cent; three to ten weeks, 41.7 per cent; three to thirteen weeks, 40.2 per cent; three to sixteen weeks, 44.0 per cent; control at ten weeks, 53.4 per cent. Lowrey (713) finds the dry substance of the ligamentous skeleton in the normal albino rat to increase from an average of 33.3 per cent at 20 days of age to 39.2 per cent at six weeks, 45.9 per cent at ten weeks, 50:4 per cent at five months and 52.6 per cent at one year. From the foregoing it is evident that in rats held at constant body-weight beginning at three weeks, the growing cartilaginous skeleton steadily increases its percentage of dry substance. Thus it tends to change the proportions of water and dry sub- stance as during normal growth. .The percentage of dry sub- stance does not increase so rapidly with age as during normal growth, however, but lags behind corresponding to the retard- ation in absolute growth. During inanition in adult rats, on the contrary, there is a relative decrease in the dry substance, and an increase in water-content (Jackson ’15 c). It has already been noted in a previous section (‘‘Lengths of body and tail’) that in the rats held at constant body-weight WEIGHTS OF ORGANS IN UNDERFED YOUNG RATS 123 beginning at the age of three weeks there is an increase in the lengths of both body and tail. The latter increases more rapidly. however, so that it tends to assume the tail-ratio found in normal rats of corresponding age. This indicates that the skeleton not only continues to grow (though at a reduced rate) while the body- weight is held constant, but also tends to grow in a normal manner, so as to produce the normal ratio of tail-length and body-length. The preceding paragraphs have shown that the increased growth of skeleton affects the ligamentous as well as the cartilaginous and bony components, and that the chemical composition (per- centages of water and dry substance) also changes in a manner tending to assume the normal. The question naturally arises as to whether the skeletal growth during constant body-weight is merely a growth in mass, or is associated with the normal process of differentiation. During the present investigation a few observations have been made upon the development of the normal skeleton, indicating some of the more obvious changes during the age-periods of the rats under experiment, especially between the ages of three and ten weeks. While a detailed study of the developmental changes in the skele- ton is reserved for a separate paper, some preliminary con- clusions may be noted here. In skeletons of rats held at constant body-weight from the age of three to the age of ten weeks, the appearance and fusion of certain epiphyses may be noted as in the normal animal during this period, although in most cases the process appears to be retarded somewhat. The following examples may be cited. In the normal skeleton at three weeks of age, the epiphyses at the ends of the vertebral bodies have not appeared; the epiphysis at the lower end of the humerus is well developed, but not fused with the shaft; the maxilla and mandible present each two molars (on each side), with no visible trace of a third. In the normal skeleton at ten weeks, the epiphyses at the ends of the verte- bral bodies have appeared, and most of them have united with the corresponding bones; the epiphysis at the lower end of the humerus is firmly fused with the shaft; well developed third molar teeth have appeared, both in the maxilla and in the mandi- 124 Cc. M. JACKSON ble. In the skeleton of a rat held at constant body-weight from three to ten weeks of age, most of the epiphyses of the vertebral bodies have appeared, and they usually have united at one end of each bone; the lower epiphysis of the humerus is firmly united with the shaft, as normally at ten weeks; well- developed third molars have appeared, as normally at ten weeks. In a rat held at constant body-weight from age of three to six- teen weeks, the skeletal differentiation was more advanced, corresponding at least to the stage reached normally at ten weeks, and in some respects perhaps even beyond it. These observations will suffice to establish the fact that the skeletal growth during constant body-weight is accompanied by normal developmental changes, as well as changes in chemical composition (percentage of water). In other words, we find not only increase in mass but growth and differentiation appar- ently normal in character, though somewhat retarded in rate. These skeletal characters therefore tend to correlation with age, although influenced also by the general body-weight. The remarkable fact that the skeleton continues to grow while the body-weight is held constant was apparently first observed by Waters (08) who found that calves previously well nourished will continue to increase in height and in width of hip for a con- siderable time, even when increase of body-weight is prevented by under-feeding. He remarks (’08 b, p. 9): Apparently the animal organism is capable of drawing upon its reserve for the purposes of sustaining the growth process, for a con- siderable time and to a considerable extent. Our experiments indicate that after the reserve is drawn upon to a certain extent to support growth, the process ceases and there is no further increase in height or in length of bone. From this point on, the animal’s chief business seems to be to sustain life. This law applies to animals on a stationary live weight as well as to those being fed so that the live weight is stead- ily declining, and indeed to those whose ration, while above main- tenance, and causing a gain in live weight, is less than the normal growth rate of the individual. Such an animal will, while gaining in weight, get thinner, because it is drawing upon its reserve to supple- ment the ration in its effort to grow at a normal rate. Aron (’11) experimented with dogs to determine the effect of a restricted amount of food upon young, growing animals. He WEIGHTS OF ORGANS IN UNDERFED YOUNG RATS 125 found that in spite of constant live weight, the animals ,con- tinued to increase in length and height for three to five months. Thereupon the emaciated animals became weaker and died un- less the amount of food was somewhat increased. From a comparative study of nine bones (the entire skeleton was not measured), Aron concludes that the skeleton during constant body-weight increases In mass and also changes in chemical composition (increase in water-content and protein (?); decrease in fat). The results of this very interesting investigation, while sufficient to establish the continued growth of the skeleton, would be more conclusive if the number of observations were larger, with an adequate number of controls at the beginning and at the end of the experiment. In a recent paper, Aron (’14) records a few observations indicating that malnutrition in children retards growth in length less than body-weight; so that the body may continue to increase in length while the body- weight is at a standstill, or even slightly decreasing. Thus the strong growth tendency of the skeleton during bare maintenance of the body-weight is manifest in the human species, as well as in the calves, dogs and rats. MUSCULATURE Although the musculature (table 8; fig. 4) in the normal rat at three weeks averages 26.9 per cent of the body, according to Jackson and Lowrey (12), the controls in the present series gave a somewhat higher amount, the average being 31.2 per cent. As shown in table 8, the musculature in the controls of the present series also averaged slightly higher than the normal according to Jackson and Lowrey at six and ten weeks. In rats held at constant body-weight from the age of three weeks to six, ten and thirteen weeks, the musculature appears relatively very slightly higher than in the controls at three weeks. The apparent increase from three to ten weeks is from 7.40 grams (7.81 grams, less correction corresponding to the smaller body- weight at 10 weeks) to 7.62 grams, or an increase of 3.0 per cent in absolute weight. In the three to sixteen weeks experiment, 126 C. M. JACKSON TABLE 8 The musculature; average absolute weight, average percentage of net body-weight and range indicated 4 am og DESCRIPTION OF RATS S Be < of Zz Normal at 3 weeks (Jackson and Lowrey)......... 13 Conteoleh at's Weeks ooree te eras es See e noe esau 10 Body-weight constant 3weeks (ageof3to 6weeks)| 8 Body-weight constant 5 weeks (ageof3to 8 weeks) 3 Body-weight constant 7 weeks fageof3tol10weeks)| 22 Body-weight constant 10 weeks (age of 3 to 13 weeks) 1 Body-weight constant 13 weeks (age of 3 to 16 weeks) 1 Normal at 6 weeks (Jackson and Lowrey)......... 14 Controiswmiiomeckincc. os oad-. eo so nok toe sae oe te P) Body-weight constant 26 weeks (age of 6 to32 weeks) 2 Normal at 10 weeks (Jackson and Lowrey)........ 10 Gontroisateomweciss 8. 224... c5- okt so oe 6 Body-weight constant 25 weeks (ageof 10to35 weeks)! 3 Controls-at.22)and/35 weeks... ...52 seb sas200 oe 6 BODY-WHIGHT: NET GRAMS 24.8 ime) ‘a uauwmno oer #F HOON We TABLE 9 ABSOLUTE WEIGHT (AND RANGE): GRAMS 6.67 7.81 (6.90-9.82) 7.04 (5.51-8.05) 6.46 (5.68-7.70) 7.62 (4.58-10.87) 7.90 7.50 21.10 14.90 (14.85-15.0) 16.90 (15.30-18.5) 53.10 55.80 (44.00-69.2) 31.20 (30.10-33.9) 81.20 (64.80-119.7) RELATIVE WEIGHT (AND RANGE): PER CENT 26.9 (20.1-30.2) 31.2 (29.5-35.3) 31.8 (25.1-34.7) 32.1 (31.3-33.2) 32.0 (24.8-36.4) 7 (26.1-35.3) .3 (35.0-35.6) 35.7 (34.8-36.6) 1 (37.4-49.1) -6 (39.3-44.5) 40.1 (39.7-40.7) 42.6 (39.0-50.3) Viscera and remainder; average percentage of net body-weight and range indicated ' & An oa DESCRIPTION OF RATS Ze S55 = of e Normal at 3 weeks (Jackson and Lowrey)......... 13 Controls ats weeks: oe oa ce ee ee ee 10 Body-weight constant 3 weeks (age of 3to 6 weeks) 7 Body-weight constant 5 weeks (age of 3to 8 weeks) Body-weight constant 7 weeks (ageof3to10 weeks) | 19 bo Normal at 6 weeks (Jackson and Lowrey)......... 14 Controls'at Gsweekss--2 Sn ee ee eh 1 Body-weight constant 26 weeks (ageof6to32weeks)| 2 Normal at 10 weeks (Jackson and Lowrey)....-...- 10 Gontrols at 10iweeks 20 t2-6 weg 0a. see ch eee 6 Body-weight constant 25 weeks (ageof 10to35weeks)| 3 Coritrols at.32,and 'Shi weeks 37> 6235 5 BODY-WLIGHT: NET GRAMS RELATIVE WEIGHT OF VISCERA (AND RANGE) PER CENT 21.3 (20.1-24.8) 20.5 (17.9-24.8) 25.0 (20.7-29.2) 26.8 (25.0-28.5) 22.2 (19.4-26.0) 20.4 (18.4-22.9) 19.5 (19.1-19.9) 16.0 (14.9-17.2) 14.3 (13.0-15.7) 16.3 (16.1-16.8) 12.9 (11.9-13.5) RELATIVE WEIGHT ‘REMAINDER’ (AND RANGE) PER CENT 12.9 (4.0-19.9) 10.5 (2.6-15.6) 13.6 (7.1-21.6) 2.3 (1.9-2-7) 10.0 (2.5-16.9) 12.0 (6.5-17.1) 19.1 16.4 (16.1-16.7) 12.5 (1.2-18.4) 13.6 (9.7-16.4) 12.4 (9.7-13.9) 17.0 (13.7-19.5) OF WEIGHTS OF ORGANS IN UNDERFED YOUNG RATS L27 the average is slightly lower. In the six to thirty-two weeks experiment, the musculature appears very slightly higher than in the controls at six weeks, while in the ten to thirty-five weeks experiment, the musculature appears slightly lower than in the controls at ten weeks. In the latter case, however, the controls are too heavy for comparison with those under experiment. In general, it seems clear from the foregoing that in young rats held at constant body-weight the musculature also remains nearly constant in weight, with perhaps a very slight tendency to increase in the majority of*cases. In the course of normal growth during this period, the musculature shows a more rapid growth than any other system, increasing from about 27 per cent of the body at three weeks to 41 per cent at ten weeks of age (Jackson and Lowrey). During inanition in adult rats, the musculature loses approximately in proportion to the entire body, slightly less in acute inanition and slightly more in chronic inanition (Jackson ’15 ¢) | Aron (11) did not weigh the muscles in his experiments on dogs, but infers (p. 29) that: ‘‘Only the flesh, muscles and fat of the body remain as the tissues which must have lost during the course of the experiments.’’ From an analysis of samples taken from the leg muscles, he also concludes that ‘‘The muscles contained only one-half of the normal amount of solids,” the protein being greatly decreased and the water-content increased. Again, however, his comparison is with controls at the end rather than the beginning of the experiment, so that no conclusion can be drawn as to the changes taking place during the experiment in the animals held at constant body-weight. VISCERA AND ‘REMAINDER’ With the visceral group (table 9; fig. 4) have been included the brain, spinal cord and eyeballs, as well as the thoracic and abdominal viscera. According to Jackson and Lowrey 712, this group decreases from about 21 per cent of the body at three weeks to about 16 per cent at ten weeks of age. This is in fairly close agreement with the controls in the present series, except at 128 C. M. JACKSON ten weeks. In this case the controls are considerably too heavy for direct comparison with the animals under experiment, which accounts for the discrepancy. In the animals held at constant body-weight from the age of three weeks to the ages of six, eight and ten weeks, the visceral group shows a distinct increase in weight. This is more marked at six and eight than at ten weeks, which perhaps indicates that the viscera may increase in the earlier part of the experiment, and lose weight later. The experiment from six to thirty-two weeks indicates no essential change in the weight of the viscera. From ten to thirty-five weeks there is a slight gain. On the whole, it may be concluded that during constant body- weight in young albino rats the visceral group as a whole under- goes but little change in weight, with a slight tendency to in- crease, especially in the earlier periods. As will be seen later, however, the individual viscera differ greatly in their reactions. Aron (711) concludes that in young dogs held at nearly constant body-weight the organs in general do not lose weight. On account of the small number of observations, however, and the lack of adequate controls, it is difficult to draw any satis- factory conclusion from his observations upon the viscera. The ‘remainder’ is obtained by deducting from the net body- weight the weight of the integument, skeleton, musculature and viscera. It therefore includes loss by evaporation and escape of fluids, as well as a few small unweighed organs and the masses of dissectable fat. The data in table 9 show a considerable variation, as might be expected. On the whole, however, it appears doubtful whether there is any material change in the weight of the ‘remainder’ in young rats held at constant body- weight for considerable periods. There is undoubtedly a loss in the fat, but this is probably counterbalanced by an increased water-content of the interstitial connective tissues. WEIGHTS OF ORGANS IN UNDERFED YOUNG RATS 129 BRAIN The brain (table 10) in eleven controls at three weeks of age averaged 1.282 grams, or 5.31 per cent of the (net) body-weight. This corresponds fairly closely with Donaldson’s (’08) figure for the normal rat of corresponding weight. In the rats held at constant body-weight from the age of three to six and eight weeks, there appears (table 10) to be a relative increase in the brain, especially at eight weeks, where it forms 6.62 per cent of the body. In reality, however, this relative increase is only apparent, and due to the fact that these animals began the ex- periment at a lower body-weight, corresponding to which the brain is relatively heavier. The (net) body-weights of the two rats at eight weeks were respectively 18.1 grams and 17.8 grams. According to Jackson (13, p. 22), the brain normally reaches its maximum relative weight of about 6.7 per cent of the body when the body-weight is about 15 grams. Thus the final brain-weight of the rats held at constant body-weight from the age of three to eight weeks of age is almost exactly that to be expected if the brain-weight has remained constant. TABLE 10 The brain; average absolute weight, average percentage of net body-weight and range indicated 5 E 2 Qa 2% RELATIVE WEIGHT DESCRIPTION OF RATS =) g z 5 pee eae (AND RANGE): og » & = NICE) S CRN PER CENT Ze Be Z Q Normal at 3 weeks (Donaldson ’08, table 1)........ 52 25.0*| 1.285 5.14 GOnGEOISI Abia WEEKS er be oe tees cee e niiteeocis as il 24.5 | 1.282 (1.187-1.364) 5.31 (4.14-6.20) Body-weight constant 3 weeks (age of 3to 6 weeks) 7 22.1 | 1.195 (1.035—-1.297) 5.44 (4.50-6.33) Body-weight constant 5 weeks (ageof3to 8 weeks) 2 18.0 1.183 (1.180-1.186) 6.62 (6.60-6.63) Body-weight constant 7 weeks (age of 3 to 10 weeks) 19 24.0 1.267 (1.136-1.379) 5.30 (4.38-6.20) Normal at 6 weeks (Donaldson ’08)................ 42 45.0*} 1.441 3.20 Wontroliat Giweeksi; 5...;-ecwmretida sek woe asec aoa 1 AQeda\ wale cue 3.23 Body-weight constant 26 weeks (age of 6 to 32 weeks) 2 47.1 1.478 (1.459-1.497) 3.14 (2.96-3.32) Normal at 10 weeks (Donaldson ’08)...............] 34 foeOF|) 1k559 2.08 Controisat LOsweeksii7. csc as des eae wane es 6 134.0 1.579 (1.512-1.636) 1.21 (0.96-1.44) Body-weight constant 25 weeks (age ot 10to 35 weeks) 3 77.8 | 1.646 (1.603-1.723) 2.12 (2.02-2.18) ISONETOIS at Oo AUC Gov WEEKS. cau anoeiala sic /<.etcccs 32s =. 6 189.5 1.777 (1.657-1.890) 0.97 (0.78-1.24) * Gross body-weight. t Body-weight ot controls at 10 weeks too high for comparison. 130 Cc. M. JACKSON The brain weight has also apparently remained nearly constant in the large group held at constant body-weight from three to ten weeks of age. The average absolute weight is slightly less, but almost in correspondence with the body-weight, so that the average relative weight, 5.30 per cent, is nearly identical with that of the controls at three weeks, the beginning of the experi- ment. Interms of absolute weight, there is a very slight apparent decrease from 1.274 grams (1.282 grams, less correction? for difference in body-weight, which averages 24.5 grams in the three weeks controls and 24.0 grams at ten weeks) to 1.267 grams, a decrease of about 0.5 per cent in absolute weight. In the series held at constant body-weight from the age of six to thirty-two weeks, there is an apparent slight decrease in the brain from about 3.23 per cent to 3.14 per cent of the body, and in the ten to thirty-five weeks series a slight increase (from 2.08 to 2.12 per cent). Considering the small number of observa- tions and the normal variation, however, these apparent differ- ences do not appear to be significant. I would, therefore, con- clude from the data above cited that there is probably no appreci- able change in the weight of the brain in young albino rats held at constant body-weight for considerable periods. Hatai (’04) experimented with a series of young rats with initial body-weights corresponding roughly to those of mine at the ages of six to ten weeks. By giving an unfavorable diet (starch and beef-fat) their body-weight was reduced on the average about 30 per cent. The brain in these cases had appar- ently lost in absolute weight, the average loss being about 5 per cent. These results, however, are of course not directly com- parable with those in which the body-weight has remained constant. In a later experiment, Hatai (’08) by underfeeding with un- favorable diet retarded the growth of a series of five rats, begin- ° It should be noted here as in other cases that the correction for organ-weight is not in exact proportion to the difference in body-weight. Allowance must be made for the change in the relative weight of the organ corresponding to the change in body-weight. a > a’ WEIGHTS OF ORGANS IN UNDERFED YOUNG RATS 131 ning at the age of 30 days, so that at 170 days their average weight was only 91.5 grams, while full-fed controls averaged 146.5 grams. By comparison with ‘second controls,’ younger rats of body-weight similar to the final weight of the stunted series, he found that in the stunted rats the brain-weight was practi- cally identical with that of normal rats of the same body-weight. In other words, the growth in brain-weight had been retarded in the same proportion as the body-weight. On this principle, if the body-weight were retarded so as to permit no growth at all, that is held at constant weight, we should expect practically no increase in weight of the brain. This is in agreement with my results, as above stated. More recently Donaldson (711) has experimented with a larger series (twenty-two litters) of rats held at nearly constant weight (34 grams) from the age of thirty to the age of fifty-one days. In the rats held at constant body-weight, the brain weight averaged 7.7 per cent less than in full-fed controls of the same litters. No direct controls were taken at the beginning of the experiment, but from the normal growth formula it is estimated that the initial brain-weight was slightly less than that found in the retarded rats at the end of the experiment. This would indicate an increase of 3.6 per cent in the brain-weight, while the body- weight was held constant. The large number of observations lends weight to this conclusion, although it would be strength- ened if direct controls were available at the beginning of the experiment. It may be noted that if Donaldson’s normal (Wistar reference tables) rather than the direct controls be taken as the basis for estimating the initial brain-weight in my three to ten weeks series, the result would indicate a gain similar to that found by Donaldson in his series. On the whole, therefore, we may safely conclude that there is but very slight if any increase in the brain-weight of young albino rats held at constant body-weight for considerable periods of time. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 19, No. 2 132 Cc. M. JACKSON SPINAL CORD When the relative weights are compared with the controls at the beginning of the experiment (table 11), or with the theoreti- cal normal according to Donaldson (’08) there appears a very decided increase in the spinal cord at all the age-periods during the experiment. Thus while the body-weight has been held constant from the age of three to that of ten weeks, the spinal cord has apparently increased from an average of 0.179 to 0.243 grams, an increase of about 36 per cent (or slightly more if the initial weight be decreased to correct for the difference of body- weight at three weeks, 24.5 grams, and ten weeks, 24.0 grams). This corresponds to an increase from 0.74 per cent to 1.02 per cent of the net body-weight. The increases at the other age- periods are equally striking. Donaldson (’11) in the experiments previously mentioned also found an increase in the weight of the spinal cord in rats held at body-weight of about 34 grams from the age of thirty days to that of fifty-one days. He does not estimate this increase exactly but from the normal weight of the cord at the beginning of the experiment (cf. Donaldson ’08, table 1) the weight must have increased from about 0.223 to 0.2498 grams, an increase of about 10.7 per cent. While this is not so striking as my results (per- haps in part because my experiments covered a longer period of time) it agrees in indicating during constant body-weight a much stronger growth tendency in the spinal cord than in the brain. This is in agreement with the well-known fact that in general the normal post-natal growth of the spinal cord is rela- tively much more rapid than that of the brain. This growth of the spinal cord is apparently correlated with the increase in trunk-length (Donaldson). EYEBALLS An increase even more striking than that of the spinal cord is apparent in the eyeballs (table 12). In rats held at constant body-weight from the age of three weeks, the eyeballs increase from a relative weight of about 0.50 per cent of the body-weight to 0.64 per cent at six weeks, 0.82 per cent at eight weeks, and WEIGHTS OF ORGANS IN UNDERFED YOUNG RATS TABLE 11 133 The spinal cord; average absolute weight, average percentage of net body-weight and range indicated x a Bo Oa DESCRIPTION OF RATS fe) Be on oF Z, Normal at 3 weeks (Donaldson ’08, table 1)........} 47 (CUD ATONE NR: Gal ger In Ge eR AE aceon den 11 Body-weight constant 3 weeks (age of 3to 6 weeks) 6 Body-weight constant 5 weeks (ageof3to 8 weeks) 2 Body-weight constant 7 weeks (age of 3 to 10 weeks) 19 Normal at 6 weeks (Donaldson ’08)................ 42 MIE TOLS AIL GM WEOKB ots osc. c:s.usraw exist oii a eee 1 Body-weight constant 26 weeks (age of 6 to 32 weeks)| 2 Normal at 10 weeks (Donaldson ’08)............... 32 GantrolsiatelO Weeks \ss.v0c.c. oss. cacle ca vech eee ane 6 Body-weight constant 25 weeks (age of 10to35weeks)| 3 Controlsjatia2iand) 35: weeks.............: sess eeere: a * Gross body-weight. BODY-WEIGHT: NET GRAMS t Body-weight of controls at 10 weeks too high for comparison. ABSOLUTE WEIGHT (AND RANGE): GRAMS 0.180 0.179 (0.152—-0.200) 0.207 (0.149-0.235) 0 .192 (0.188-0.195) 0.243 (0.201-0.314) 0.254 0.286 0.399 (0.378-0.420) 0.333 0.422 (0.341-0.464) 0.520 (0.500-0.550) 0.603 (0.491-0.667) RELATIVE WEIGHT (AND RANGE): PER CENT 12 .74 (0.56-0.89) 96 (0.63-1.25) 08 (1.06-1.09) 02 (0.89-1.17) 57 67 .85 (0.83-0.86) 0.44 0.32 (0.27-0.39) 0.67 (0.64-0.69) 0.33 (0.24-0.44) (—=¥ tb — a) (| The eyeballs; average absolute weight, average percentage of net body-weight and range TABLE 12 indicated 2 q a aa m2 | Oa oie] DESCRIPTION OF RATS iS) Eo of | ie a ae oF a Z Q INormal’ates: weeks (Jackson 713))..2c.<<.0 << 0000 006, 24 Pil Moutrols aio wWECKS. «0k cise hone eeiostal bl wie cele a 10 24.6 Body-weight constant 3 weeks (age of 3to 6 weeks) 6 2) Body-weight constant 5 weeks (ageof3to 8 weeks) 2 18.0 Body-weight constant 7 weeks (age of 3t0 10 weeks) | 19 24.0 Normal at 6 weeks (Jackson ’13).... .............. 42 50.0 (ChaynAnay | 7G) (2) 5 ars ie SA Oh ee a 1 42.4 Body-weight constant 26 weeks (age of 6t0 32 weeks)| 2 47.1 Normal at 10 weeks (Jackson 13).................. 75.0 Sontrolsat 10) weeks™...5. oss heen eee none 6 134.0 Body-weight constant 25 weeks (age of 10to35weeks)| 3 77.8 Wontrolsiatio2 and 35 weeks... 2.0 c66+ecacs oes ts: 5 184.4 * Body-weight of controls at 10 weeks too high for comparison. ABSOLUTE WEIGHT (AND RANGE): GRAMS 0.105 (0.073-0.125) 0.120 (0.110-0.133) 0.142 (0.138-0.149) 0.146 (0.145-0.146) 0.179 (0.130-0.196) 0.153 (0.125-0.175) 0.152+ 0.240 (0.230-0.250) 0.173 0.209 (0.195-0.228) 0.275 (0.249-0.323) 0.260 (0.235-0.296) RELATIVE WEIGHT (AND RANGE): PER CENT 0.52 (0.31-0.73) .50 (0.34-0.69) .64 (0.60-0.72) .82 (0.81-0.82) .76 (0.57-1.00) .382 (0.18-0.40) 36 51 (0.50-0.52) 23 .16 (0.13-0.21) .35 (0.34-0.38) 15 (0.12-0.17) coocooo cococooco 134 Cc. M. JACKSON 0.76 per cent at ten weeks. In terms of absolute weight, the eyeballs have apparently increased from an average of 0.120 grams at three weeks to about 0.179 grams (no correction made for the slight difference in body-weight) at ten weeks, an increase of nearly 50 per cent! In the normal, full-fed rat at ten weeks (average body-weight 112 grams) the eyeballs have reached a weight of only about 0.201 grams (Jackson 713). At this rate, the weight of the eyeballs at a normal body-weight of 75 grams (the body-weight indicated in table 12 as ‘normal at 10 weeks.’ to correspond to the body-weight of the animals held at constant body-weight from the age of ten to thirty-five weeks) would be only about 0.173 grams, or slightly less than that actually reached in the series held at constant body-weight of 24 grams from three weeks to ten weeks of age. The growth of the eyeballs in rats held constant from the age of six to thirty-two weeks, and from ten to thirty-five weeks, is equally striking. No data upon the growth of the eyeballs under these conditions have been found in the literature. I have shown elsewhere (Jackson ’15 a, 715 ¢), however, that the eyeballs lose but very little if any during inanition in the adult albino rat. In connection with the astonishing growth capacity of the eyeballs in young animals at constant body-weight, the possi- bility that the growth of the eyeballs is somewhat independent of that in the body as a whole may be considered, which I have already pointed out (Jackson ’13, p. 24). When the large water- content of the eyeballs is considered (85.6 per cent in the rat at twenty days, according to Lowrey ’13), it is, after all, not diffi- cult to comprehend the possibility of its continued growth, largely by water-absorption, when growth in the body as a whole is at a standstill. THYROID GLAND In young albino rats held at constant body-weight from the age of three weeks to six weeks, eight weeks and ten weeks, there is usually a well-marked loss of weight in the thyroid gland (table 13). In the largest group, three to ten weeks, the thyroid has apparently decreased on the average from about 0.033 per cent WEIGHTS OF ORGANS IN UNDERFED YOUNG RATS 135 TABLE 13 The thyroid gland; average absolute weight, average percentage of net body-weight and range indicated 7 aa Ha | 24 Z aS et a a RELATIVE WEIGHT DESCRIPTION OF RATS 26 = 5 hes aR REEVE Os sats (AND RANGE): Be oe er BE PER CENT as ae Ou ° Z Q Normal at 3 weeks (Jackson '13)..........-..-.-+.- 26 18.7 | 0.0060 (0.0034-0.0104| 0.030 (0.018-0.041) WOMULOlsist o! WEEKS te 4.) eek cansiok sine coe eer 11 24.6 | 0.0078 (0.0064-0.0094| 0.033 (0.022-0.042) Body-weight constant 3 weeks (age ot 3to 6 weeks) 3 23.1 | 0.0053 (0.0052-0.0056 | 0.023 (0.022-0.024) Body-weight constant 5 weeks (age of 3 to 8 weeks) 2 18.0 | 0.0058 (0.0947-0.0068 | 0.032 (0.026-0.038) Body-weight constant 7 weeks (age of 3 to 10 weeks) 18 24.1 | 0.0059 (0.0034-0.0087| 0.025 (0.016-0.036) Normal at 10 weeks (Jackson ’13)................+: 24 110.0 | 0.0165 (0.0100-0.030) | 0.015 (0.008-0.022 of the body to 0.025 per cent. Or, in terms of absolute weight, it has decreased from 0.0078 to 0.0059 grams, a decrease of 24 per cent. (A slight correction should be made on account of difference in body-weight.) No observations were made upon the thyroid gland in the experiments beginning at later ages. During acute inanition in adult rats, the thyroid gland appar- ently loses little or no weight; while in chronic inanition with an average loss in body-weight of about 36 per cent, the thyroid gland loses only about 22 per cent in weight (Jackson 715). There is some uncertainty as to the exact figures, however, on account of variability and difficulty in dissecting out the thyroid gland in an accurate manner. The same, of course, holds true for the present series. THYMUS The normal thymus (table 14) at three weeks forms 0.37 per cent of the net body-weight. This decreases, in rats held at constant body-weight, to 0.075 per cent at six weeks of age, to 0.030 (exceptional?) at eight weeks, and to 0.040 per cent at ten weeks. In terms of absolute weight, the thymus has decreased from 0.091 grams at three weeks to 0.017 grams (loss of 81 per cent) at six weeks, and to 0.0094 grams (loss of 90 per cent) at 10 weeks. No correction for the slight difference in body-weight has been made in these estimates. Normally at ten weeks of age the weight of the thymus should have increased to about 0.24 grams (0.30 grams in the controls). 136 Cc. M. JACKSON A maximum absolute weight of about 0.29 grams is reached by the average normal thymus about the age of 85 days (Hatai 14). At one year, it has undergone a complete age-involution, and forms only 0.02 per cent of the body-weight (Jackson 713). That the weight and structure of the thymus are markedly affected by various adverse conditions has long been known, and the process of involution has recently been thoroughly investi- gated by Hammar and his pupils. Jonson (’09) experimented with young rabbits subjected to acute and chronic inanition. In the latter case, the diet was restricted so as to maintain the young rabbits at constant body-weight (similar to the present experiment with rats). Under these conditions, Jonson found the weight-curve of the thymus similar to that of the body-fat, although during acute inanition the fat decreases somewhat more rapidly. In young rabbits held at constant body-weight the thymus in four weeks is reduced to about one-thirtieth of its initial weight. The cortex suffers the greatest loss, being re- duced to one-twelfth of its initial weight within two weeks of chronic inanition at constant body-weight. It would therefore appear that the process of involution is much more rapid and complete in young rabbits than in young rats at the ages included in the present investigation. In both cases, however, the weight of the thymus in hunger involution decreases most rapidly in the earlier weeks of the experiment. HEART The heart (table 15) in the albino rats held at constant body- weight from the age of three weeks appears to have remained practically constant at about 0.70 per cent of the net body-weight up to the age of ten weeks. In absolute weight, the heart would apparently decrease from 0.167 grams (0.170 grams, less cor- rection for difference in body-weight) to 0.166 grams, a decrease of about 0.6 per cent, which is probably within the limits of experimental error. The very slight decrease at six weeks and increase at eight weeks are also probably not significant. Simi- larly in the rats held at constant body-weight from six to thirty- WEIGHTS OF ORGANS IN UNDERFED YOUNG RATS 137 two weeks and from ten to thirty-five weeks the heart has ap- parently retained almost exactly its initial relative weight. The absolute weight therefore apparently remains unchanged (the differences in the table being due to different initial body-weights). During inanition in adult rats (Jackson ’15 a, 715 ¢) the heart likewise maintains its relative weight, losing in absolute weight nearly in proportion to the entire body (slightly more in chronic than in acute inanition). TABLE 14 The thymus; average absolute weight, average percentage weight and range indicated ABSOLUTE WEIGHT (AND RANGE)! GRAMS RELATIVE WEIGTH (AND RANGE): PER CENT 0.071 (0.034 —-0.135) 0.091 (0.042 -0.123) 0.017 (0.011 —-0.022) 0.0054 (0.0037—0.0071) 0.0094 (0.0054—-0.0170) 0.108 (0.052 —-0.284) 0.24 (0.12 -0.44) 0.30 (0.25 -0.34) 0.37 (0.23 -0.55) 0.37 (0.22 -0.51) 0.075 (0.046-0.0101 0.030 (0.021-0.040) 0.040 (0.019-0.062) 0.21 (0.14 -0.36) 0.23 (0.13 -0.35) 0.23 (0.16 -0.29) * The heart; average absolute weight; average percentage of net body-weight and range ABSOLUTE WEIGHT (AND RANGE): GRAMS lace A oe 2 a oa [oe] DESCRIPTION OF RATS io) Bo Bea] Je . < a a oF ela Zz a Normal at 3 weeks (Jackson ’13).................--| 49 18.7 AGierrrtrta lente UGNWEOKS 55 /-)-12)5,<.c/ata10,=/0 0 ole'oce o/s Sa ninloere meee 11 24.5 Body-weight constant 3 weeks (ageof3to 6 weeks) 4 PP Naif Body-weight constant 5 weeks (age of 3to 8 weeks) 2 18.0 Body-weight constant 7 weeks (age of 3 to 10 weeks) 19 24.0 Normal at 6 weeks (Jackson ’13)................... 42 50.0 Normal] at 10 weeks (Jackson ’13).................. 42 107.2 GreriralsiatalOEWECKS Gera dias vic cesses tele ss oases ac 6 134.0 TABLE 15 indicated & I a ag mm a< OG (ome) DESCRIPTION OF RATS ° zo BE ie BS as oP oF a ise] Normal] at 3 weeks (Jackson ’13)..............-+--- 49 18.7 ControlsiAt sAWeCKS) ce ok sete po s sal ciete ote everest Sos 11 24.5 Body-weight constant 3 weeks (age of 3to 6 weeks) 7 PP4ai\ Body-weight constant 5 weeks (age of 3to 8 weeks) 2 18.0 Body-weight constant 7 weeks (age of 3 to 10 weeks) 19 24.0 Normal at 6 weeks (Jackson ’18)...............-..- 42 50.0 OTERO Sia Ca weeks por ae aera ets tac: re aici ieiese = 1 42.4 Body-weight constant 26 weeks (age of 6 to 32 weeks) 47.1 Normal at 10 weeks (Jackson '13)................-- 75.0 Gontrolsat- LO; weGkss.. caadiecm adem eacelidenss alee « 6 134.0 Body-weight constant 25 weeks (age of 10 to 35 weeks) 3 77.8 Controlsiat:32 andi 35) weeks. sci. cs cctlesc sc eselctere - 6 189.5 0.135 (0.082-0.250) 170 (0.130-0.245) .151 (0.123-0.187) 129 (0.128-0.130) -166 (0.124-0.213) .277 (0.183-0.535) 237 271 (0.255-0. 288) 375 .562 (0.489-0.€87) 389 (0.348-0.450) .873 (0.688-1.29) coooocoocococococo & RELATIVE WEIGHT (AND RANGE): PER CENT 0.72 (0.56-0.93) 0.70 (0.55-0.84) 0.68 (0.63-0.85) 0.72 (0.72-0.72) 0.70 (0.55-0.83) 0.55 (0.44-0.68) 0.56 0.57 (0.57-0.58) 0.50 0.44 (0.40-0.48) 0.50 (0.47-0.52) 0.46 (0.37-0.56) * Body-weight of controls at 10 weeks too high for comparison. 138 C. M. JACKSON LUNGS The lungs (table 16) in rats held at constant body-weight from the age of three weeks to the ages of six weeks and ten weeks appear to lose in weight. (The data at eight weeks are abnor- mally high, as noted in the table). Thus between the ages of three weeks and ten weeks, the lungs apparently decrease from 1.04 per cent to 0.91 per cent of the body-weight; or in absolute weight from 0.250 to 0.218 grams, a loss of about 15 per cent. (A slight correction has been made on account of the difference in body-weight). In the few observations upon rats for longer periods beginning at the later ages of six and ten weeks, there appears to be no material change in the weights of the lungs during the experimental periods. The lung infections frequently found in older and adult rats, rarely occur before the age of ten weeks, and thus do not affect the present series. As Hatai has already noted, rats during chronic inanition appear to be unusually free from lung infection. During inanition in adult rats, the lungs lose weight in about the same proportion as the whole body, thus nearly maintaining their relative (percentage) weight. The loss is slightly greater, however, during chronic inanition (Jackson ’15 ¢). * TABLE 16 The lungs; average absolute weight; average percentage of net body-weight and range indicated m2 2s | "E WEIGHT é : a4 Se | spsotute weicut | PELATIVE WEIS! DESCRIPTION OF RATS | s : | a : | (aupainGelGrams| GND Eases a= az | ee be Normal at 3 weeks (Jackson ’13).................. 49 18.7 0.216 (0.151-0.354) 1.17 (0.86-1.46) Controls ato weekss: © sees sees cttt > es eee 11 24.5 0.253 (0.201-0.290)| 1.04 (0.89-1.12) Body-weight constant 3 weeks (ageof 310 6 weeks) 3 20.9 | 0.192 (0.190-0.197)| 0.92 (0.88-0.93) Body-weight constant 5 weeks (ageof3to 8 weeks) 2 18.0 | 0.280* (0.241-0.319) | 1.56* (1.36-1.77) Body-weight constant 7 weeks (age of 3 to 10 weeks) 19 24.0 0.218 (0.170-0.282)| 0.91 (0.78-1.32) Normal at 6 weeks (Jackson ’'13)................... 39 50.0 0.333 (0.244-0.547)| 0.68 (0.58-0.94) Control at Weeks: | .o5 02.2 etch omens soles sic ee ee 1 42.4 | 0.264 0.62 Body-weight constant 26 weeks (age of 6 to 32 weeks) 2 47.1 0.319 (0.309-0.329) | 0.68 (0.65-0.70) Normal at 10 weeks (Jackson ’13).................- 75.0} 0.49 0.65 Conttirals atl Oiweeks: .2.... 55.04 Ace ee ek os Fy Wo 134.0 0.74 (0.610-0.94) 0.56 (0.45-0.74) Body-weight constant 25 weeks (age of 10 t035 weeks)| 3 | 77.8] 0.49 (0.390-0.60) 0.63 (0.52-0.81) * Lungs in one case abnormally heavy on account of post mortem congestion. ee WEIGHTS OF ORGANS IN UNDERFED YOUNG RATS 139 LIVER The liver (table 17) in the albino rats held at constant body- weight from the age of three weeks to six, eight and ten weeks shows an apparent increase. This increase appears greatest in the shortest series, three to six weeks, increasing from 4.95 per cent of the body-weight (in controls) to 5.89 per cent. Be- tween three and ten weeks the corresponding increase is from an absolute weight of 1.16 grams (1.20 grams, less correction for difference in body-weight; or 4.95 per cent of the body-weight) to 1.28 grams (5.25 per cent of the body-weight), or an increase of about 10.3 per cent in absolute weight. In the later and longer periods, however, there appears to be a decided decrease in the weight of the liver (six to thirty-two weeks and ten to thirty-five weeks series). This may be due to the fact that the latter experiments extended over a longer period of time, or it may be because they were begun at a later age. We may therefore conclude that in young albino rats held at constant body-weight there is, beginning at three weeks, an increase in the weight of the liver (apparently greater at six than at ten weeks of age) ; while in rats beginning the experiment later, at six and ten weeks of age (and extending over a longer period) TABLE 17 The liver; average absolute weight, average percentage of net body-weight and range indicated # E a ill eee % D te : : RELATIVE WEIGHT DESCRIPTION OF RATS 3 5 S o CRS REO ais (AND RANGE): oe ne = ANTE IE is PER CENT Bel Bz Zz Q Normal at 3 weeks (Jackson 713) ..................- 49 18.7 0.87 (0.42-2.08) 4.50 (3.21-5.82) Cou tral sr aihemy COKS: 5c. Tore acre eles ah lew es ll 24.5 1.20 (1.06-1.38) 4.95 (4.03-6.00) Body-weight constant 3 weeks (age of 3to 6 weeks) i 22.1 1.30 (0.89-1.63) 5.89 (4.32 -7.39) Body-weight constant 5 weeks (age of3to 8 weeks) P, 18.0 | 0.92 (0.78-1.06) 5.12 (4.36-5.88) Body-weight constant 7 weeks (age of 3 to 10 weeks) 19 24.0 1.28 (0.80-1.82) 5.25 (3.63-7.00) Normal at 6 weeks (Jackson ’13)................... 42 50.0 | 3.19 (2.18-4.76) 6.48 (5.39-8.46) COM UTC eatiO wWeCKSe sc nosso attri ie eed ass 1 42.4] 2.18 5.15 Body-weight constant 26 weeks (age of 6 to 32 weeks) 2 47.1 2.33 (2.21-2.45) 4.44 Normal at 10 weeks (Jackson ’13).................- 75.0 | 4.50 6.00 onurolsrat; lO} Weeks: :. c/sneaceswotosne aeargesds oe elon 6 134.0 6.72 (4.91-8.66) 4.99 (4.28-5.72) Body-weight constant 25 weeks (age of 10 to 35 weeks) 3 77.8 | 3.11 (2.30-3.51) 4.25 (3 .91-4.74) Gontrols at 32 and!35 weeks... f/).00.9..-..50.0040 6 189.5 7.27 (5.84-9.86) 3.84 (3.47-4.14) 140 C. M. JACKSON there is a decided decrease in the weight of the liver. Thus the liver would appear under these conditions to have a tendency to follow the normal growth-impulse, which increases to a maximum at about the age of six weeks, and decreases thereafter (Jackson AGUD or}: During inanition in adult rats, the liver loses in weight rela- tively more than the whole body, and toa greater extent in acute than in chronic inanition (Jackson ’15 ¢). As the liver is nor- mally subject to great variation in weight, however, (Jackson 713) great caution must be observed in drawing final conclusions. Hatai (’13) found the weight of the normal liver distinctly higher than that in my series, and if his data instead of my controls were taken as a basis for the initial weights, the estimated losses in my experiments would be considerably greater, even the younger rats showing a loss instead of a gain in liver-weight. SPLEEN Taking the controls at three weeks as a basis for com- parison, it appears that in rats held at constant body-weight from the age of three weeks to six weeks the weight of the spleen (table 18) remains practically unchanged (the average at six TABLE 18 The spleen; average absolute weight, average percentage of net body-weight and range indicated ' =) ! B Eo zi Zz | - : ABSOLUTE WEIGHT | REVAL ee DESCRIPTION OF RATS se ee CSharp Bee | aida a 2 Z | az | A Normal at 3 weeks (Jackson ’13)................... | 49 18.7 | 0.055 (0.019-0.145) 0.28 (0.15-0.42) Sonenple ato Weeks: 60) tte eon ee Leet eee Ree fies G 24.5 | 0.091 (0.051-0.130) 0.37 (0.27-0.48) Body-weight constant 3 weeks (ageot3to 6 weeks) 7 22:3 0.087 (0.040-0.172) 0.38 (0.18-0.72) Body-weight constant 5 weeks (ageof3to 8 weeks) 2 18.0 | 0.043 (0.036-0.049) 0.24 (0.20-0.27) Body-weight constant 7 weeks (age ot3to10 weeks) | 19 24.0 0 053 (0.033-0.076) 0.22 (0.16-0.33) Normal at 6 weeks (Jackson '13) ................- 42 50.0 | 0.135 (0.086-0.204) 0.28 (0.19-0.47) Gantrolidt Oi weekSs ~. 0545.64. fic eee ee eee oP 42.4) 0.107 0.25 Body-weight constant 26 weeks (age of 6 to 32 weeks)} 2 | 47.1] 0.139 0.29 (0.28-0.30) Normal at 10 weeks (Jackson °13)...............--.| |} 75.0] 0.225 0.30 Console ep lUimGekS:s.. «ees. de” 8 ae re 1 BG | 134.0} 0.350 (0.300-0.420) | 0.27 (0.21-0.36) Body-weight constant 25 weeks(age of 10to35 weeks)) 3 | 77.8 0.230 (0.230—0.240) 0.30 (0.27-0.33) Controls at 32 and 35 weeks.....................--.| 6 189.5 | 0.620 (0.480-0.750) 0.33 (0.29-0.39) | WEIGHTS OF ORGANS IN UNDERFED YOUNG RATS 14] weeks being probably too high, on account of the inclusion of a litter with abnormally large spleens); while in the rats at eight and ten weeks there is a considerable decrease. In the case of the three to ten weeks series, the decrease would be from 0.091 gram (0.37 per cent of the net body-weight) to 0.053 gram (0.22 per cent of the body-weight), or a decrease of nearly 42 per cent in absolute weight. (No correction has been made for the difference in body-weight, which is 0.5 gram lower at ten weeks). If the normal relative weight of the spleen at three weeks (0.28 per cent, Jackson 713) be taken as a basis of esti- mate, however, the loss would appear considerably less. In the rats held at constant body-weight at later and longer periods (six to thirty-two weeks and ten to thirty-five weeks) there appears to be no appreciable change in the average weight of the spleen. It may be concluded therefore that in young rats held at constant body-weight beginning at the age of three weeks there is a marked tendency to a reduction in the weight of the spleen; while at later (and longer) periods the spleen ap- pears to undergo no material change in weight. It must be remembered, however, that the spleen is normally one of the most variable organs in the body (Jackson 713), and final conclusions should be correspondingly guarded. In adult albino rats during chronic inanition the average loss in weight is nearly proportional to that of the entire body, while in acute inanition the loss appears very much greater (Jackson Bias 715 ©): STOMACH AND INTESTINES The stomach and intestines, including mesentery and pan- -ereas, are considered both with contents (table 19 a) and empty (table 19 b). Considering first the empty canal, it. appears in rats held at constant body-weight from the age of three weeks to increase from about 4.8 per cent of the body-weight to 8.0 per cent at six and eight weeks, decreasing to 6.0 per cent at ten weeks. In absolute weight the increase would be from about 1.13 grams (1.20 grams less correction on account of difference in body-weight) at three weeks to 1.45 grams at ten weeks, an 142 C. M. JACKSON increase of about 28 per cent. Between six and thirty-two weeks there is apparently but little change; while from ten to thirty-five weeks there appears to be a decrease in the weight of the alimentary canal. The number of observations, however, is too small for final conclusion. The most remarkable increase occurs between three and six weeks of age, from an absolute weight of 1.20 to 1.78 grams, an apparent increase of about 48 per cent in absolute weight! (The increase would be even greater if allowance were made for differ- ence in body-weight). Thus, as in the case of the liver and TABLE 19 The stomach and intestines; average absolute weight, average percentage of net body- weight and range indicated = a : zZ : : ABSOLUTE WEIGHT | PELATIVE WEIGHT | DESCRIPTION OF RATS . s z : c (AND RANGE): GRAMS eee o> Bz Z Fea) a. Including contents Normal at 3 weeks (Jackson 713)..............- $555) (0 18.7 1.78 (0.74-3.08) 9.3 (5.5-15.5) Cantrole Abioimvecks 223.3- ooo shee opens eee 11 24.5 2.51 (1.96-3.23) 10.4 ( 7.8-15.4) Body-weight constant 3 weeks (ageof3to 6 weeks) 7 22.1 3.10 (1.67-5.07) 13.9 (7.4-21.3) Body-weight constant 5 weeks (ageof3to 8 weeks) 2 18.0 3.64 (2.58-4.71) 20.2 ( 4.5-26.0) Body-weight constant 7 weeks (age ot 3 to 10 weeks) 19 24.0 3.29 (2.26-6.83) 13.5 (10.3-20.2) Normal at 6 weeks (Jackson 713) ...............-.- 42 50.0 8.05 (4.48-13.40) 15.9 (10.6-23.4) Controls ahiGiweoks cpe.2. sermons. in -beei=ee soee sil 42.4 5.13 12.1 Body-weight constant 26 aie (ageot6to32weeks)| 2 47.1 5.98 (4.71-7.24) 12.5 (10.8-14.2) Normal at 10 weeks (Jackson 713) .............. pes 75.0 9.00 12.0 Controls at lO weesseces co. cous cece tee lene ae 6 134.0 | 11.32 (8.82-14.49) 8.3 ( 7.3-11.2) Body-weight constant 25 weeks(age of 10 to 35 weeks) 3 77.8 7.48 (5.S0-8.81) 9.6 ( 8.1-10.4) Controls atis2:and)35 weeks: <2... 02.5.2 ccncee 6 189.5 11.60 (7.51-14.04) 7.4 ( 5.4-12.2) b. Empty Norme] at 3 weeks (Jackson 713)..................- 16 20.0 0.90 (0.37-1.61) 4.5 ( 2.9- 6.1) Gontrolsiat 3 weeks eore ee cee et eae ean see 10 Zoek 1.20 (0.74-1.69) 4.8 ( 3.1- 6.7) Body-weight constant 3 weeks (ageof3to 6 weeks) 7 22.1 1.78 (1.41-2.51) 8.0 ( 6.3-11.0) Body-weight constant 5 weeks (ageof 3to 8 weeks) 2 18.0 1.44 (1.38-1.51) 8.0 ( 7.8- 8.3) Body-weight constant 7 weeks (age of 3 to 10 weeks) 19 24.0 1.45 (0.96-2.29) 6.0 ( 4.5- 8.6) Normal at 6 weeks (Jackson 713)................-.- 50.0 4.00 8.0 Controlstat 6iweels\.- 2 eh.cc cob ewer eeebe oe eeecr 1 42.4 3.00 if Body-weight constant 26 weeks (ape of 6to32 weeks)} 2 47.1 3.22 (2.85-3.59) 6.8 ( 6.5- 7.0) Normal at 10 weeks (Jackson '13).................- 75.0 5.30 7.0 Controls’at10 ‘weeks? 54 2 sence ancien poe 5 127.9 5.26 4.3 ( 3.9- 4.9) Body-weight constant 25 weeks (age of 10 to 25 weeks) 3 77.8 3.97 (3.48-4.35) 5.4 ( 4.7- 5.9) Controls'at.32 and. 35 weeks...300.) 4.5 <<. ..ns.-255% 6 189.5 9.16 (7.57-10.64) 4.9 ( 4.1- 5.3) * Body-weight of controls at 10 weeks too great for comparison with those held constant 10-35 weeks. WEIGHTS OF ORGANS IN UNDERFED YOUNG RATS 143 following the normal growth tendency, there appears to be in these retarded rats an early increase in the weight of the ali- mentary canal, reaching a maximum at about the age of six weeks, after which there is a decline in weight. Jt may be that the early increase in these organs is diminished later on account of the exhaustion of the stored food-supplies available else- where in the body at the beginning of the experiment. Individ- ual organs and tissues differ greatly from each other in their relative susceptibility to attack and absorption at different times during the course of inanition. The behavior of the stomach and intestines weighed with contents (table 19 a) is very similar to that of the empty canal. Contrary to what might be expected, during constant body- weight, with a restricted food-supply (water ad libitum), the canal does not decrease in contents. On the contrary there is an increase in contents (watery or mucous in character) which may even exceed in relative weight the normal contents in full- fed aminals. The maximum occurred in animals held at con- stant body-weight from the age of three to eight weeks, when the canal with contents formed 20.2 per cent’ of the body-weight! In experiments beginning at later ages and extending over longer periods (six to thirty-two weeks and ten to thirty-five weeks), the canal with contents appears to remain more nearly uniform in weight, with some tendency to decrease. During both acute and chronic inanition in adult albino rats, there is a very marked decrease in the weight of the stomach and intestines, both with and without contents (Jackson 715 a, 715 ¢). SUPRARENAL GLANDS The suprarenal glands (table 20) from the age of about six weeks must be considered separately in the sexes, on account of a distinct sexual difference in their weight, as discovered independently by Hatai (713) and myself (Jackson 713). In the earlier ages, however, there is no apparent sexual difference, hence the sexes are combined. 144 Cc. M. JACKSON TABLE 20 The suprarenal glands; average absolute weight, average percentage of net body-weight and range indicated Zn a Z RELATIVE WEIGHT : oZ ag ABSOLUTE WEIGHT = DESCRIPTION OF RATS = 2 Sac) f (AND RANGE): = z a : (AND RANGE): GRAMS Santen o> a2 Z Q Normal at 3 weeks* (Jackson 713).................- 49 18.7 | 0.0072 (0.0040-0.0140)! 0.0400 (0.023-0.074) Controls StS weeks tec. sees ce cea we ces ho clea wes il 24.5 | 0.0088 (0.0060-0.0108)} 0.0370 (0.025-0.055) Body-weight constant 3 weeks* (age of 3 to 6 weeks) f 7 22.1 | 0.0089 (0.0070-0.0110)} 0.0400 (0.034-0.050) 4 18. ; Body-weight constant 5 weeks (ageof3to 8 weeks) | i * : % ae cite Specks cS Lae J 7m 25.2 | 0.0101 (0.0072-0.012€)| 0.0420 (0.033-0.052) Body-weight constant 7 weeks (age of 3 to 10 weeks) |) jo¢ | - 93 3 | 9 0117 (0.0090-0.0147)| 0.0510 (0.040-0.061) Normal at 6 weeks* (Jackson ’13).................. 42 50.0 | 0.0128 (0.0070-0.0190)} 0.0260 (0.015-0.039) Comnirol aii iweeksest de ease sees os os esa einen 1f 42.4 | 0.0090 0.0210 (- 7 5 2 Body- weight constant 26 weeks (age ot 6 to 32 weeks) } a a 3 eas oe f 20m 117.0 | 0.0220 (0.0150-0.0336)} 0.0185 (0.011-0.025) Normal at 40pweeks (Iackson “13)..--.--.-.2----<-. \ a 99.0 | 0.0253 (0.0180-0.0329)| 0.0257 (0.017-0.033) 3m| 154.7 | 0.0295 (0.0270-0.0326)| 0.0190 (0.017-0.023) GorntralstiplO weokss ck cc poet sb. s oe ck eet are eee 3f 114.0 | 0.0318 (0.0290-0.0346)/ 0.0280 (0.0270.031) 2m} 79.6 | 0.0149 (0.0147-0.0150)| 0.0190 (0.017-0.020) if | 74.2 | 0.0145 0.020 Body-weight constant 25 weeks (age ot 10t0 35 weeks) 3m] 218.7 | 0.0270 (0.0120-0.0310)} 0.0100 (0.009-0.013) Controlsatioo and po weeks... 5.2 cecen cet ences l 3f 160.3 | 0.0320 (0.0280-0.0360)| 0/0200 (0.017-0.024) * No sexual difference apparent. ~ TABLE 21 The kidneys; average absolute weight, average percentage of net body-weight and range indicated e | Ge Z = 23 RELATIVE WEIGHT of Ae ABSOLUTE WEIGHT DESCRIPTION OF RATS =) = S AR EE ve (AND RANGE): & - a g (AND RANGE): GRAMS PER CENT se Bei Zz a Normal at 3 weeks (Jackson 713)...............---- 49 18.7 | 0.271 (0.169-0.531) 1.44 (1.19-1.87) Controlsint 3 weeks. 30.2 25 sotseys 3 coves amwesiee neice 11 24.5 | 0.393 (0.314-0.487) 1.62 (1.33-2.16) Body-weight constant 3 weeks (age of 3to 6 weeks) 7 22.1 | 0.396 (0.328-0.487) 1.80 (1.4€-2.05) Body-weight constant 5 weeks (age of 3to 8 weeks) 2 18.0] 0.339 (0.331-0.347) 1.89 (1.86-1.92) Body-weight constant 7 weeks (age of 3 to 10 weeks) 19 24.0} 0.404 (0.325-0.515) 1.69 (1.44-1.97) Normal at 6 weeks (Jackson '13)..................- 42 50.0 0.616 (0.500-0.943) 1.25 (1.00-1.55) Control sat Oiweeks'e=. «a. -kys< henpe eee s ose ken aee 1 42.4 | 0.570 RR Body-weight constant 26 weeks (age of 6to 32 weeks)| 2 47.1 | 0.613 (0.581-0.645) 1.30 (1.28-1.33) Normal at 10 weeks (Jackson '13)..................- 75.0 | 0.750 “1.00 Controls at. 10 -woelkws soe. 2. . ace 5 ihe cease ee eee 6 134.0 | 1.200 (0.953-1.481) 0.91 (0.81-0.99) Body-weight constant 25 weeks (age of 10 to 35 weeks) 3 77.8 | 0.753 (0.728-0.784) 0.97 (0.92-1.02) Controisatecs anda5 Weeks) ....02 sees es teonen 6 189.5 1.539 (1.275-1.909) 0.81 (0.71-0.86) * Controls too heavy for comparison. WEIGHTS OF ORGANS IN UNDERFED YOUNG RATS 145 In the rats held at constant body-weight from the age of three weeks the suprarenal glands appear to increase slightly in weight at six and eight weeks; and especially at ten weeks, where the characteristic sexual difference has distinctly appeared. In absolute weight, the suprarenal glands have increased from 0.0090 gram (0.0088 gram, plus correction for difference in body- weight; or 0.037 per cent of the body-weight) at three weeks to 0.0101 gram (0.042 per cent of the body-weight) in the male at ten weeks, an increase of about 12 per cent in absolute weight. In the female, the corresponding increase is from 0.0084 gram (0.0088 gram, less correction for difference in body-weight) (or 0.037 per cent of the body-weight) to 0.0117 gram (0.051 per cent of the body-weight), an increase of 39 per cent in absolute weight. Normally the suprarenals during this period are de- creasing in relative (percentage) weight. In the experiments at later and longer periods (six to thirty-two weeks and ten to thirty-five weeks), the changes are apparently not great, but a larger number of observations is necessary before definite con- clusions can be reached. In adult rats during inanition there is little or no loss in the absolute weight of the suprarenal glands, which therefore in- crease markedly in relative (percentage) weight (Jackson 715 a, aoe). KIDNEYS The kidneys (table 21) in rats held at constant body-weight from the age of three weeks show a tendency to increase’ which is more marked at six and eight than at ten weeks. Between three and ten weeks of age the increase is from an average of 0.388 gram (0.393 gram, less correction on account of differ- ence in body-weight; or 1.62 per cent of the body-weight) to 0.404 gram (1.69 per cent of the body-weight), an increase of only about 4.1 per cent in absolute weight. At later and longer periods (six to thirty-two weeks and ten to thirty-five weeks) there is apparently but little change in the weight of the kidneys. The slight differences shown in the table are probably not significant. On the whole, it appears that in young rats held at 146 Cc. M. JACKSON constant body-weight there is a slight tendency to increase in the weight of the kidneys, in the earlier weeks, but little or no apparent difference later. In adult rats during acute and chronic inanition, the kidneys lose in weight relatively slightly less than the body as a whole, thereby gaining slightly in relative (percentage) weight (Jackson ’15 ¢). TESTES AND EPIDIDYMI The weights of testes and epididymi unfortunately were not separated in some cases, which (together with their variability and the small number of observations) makes conclusions some- what difficult. For the testis (table 22 a), the clearest case is in rats held constant from three to ten weeks of age, in which TABLE 22 The testes and epididymi; average absolute weight, average percentage of net body-weight and range indicated = = 2 aw = z ELATIVE W DESCRIPTION OF RATS = S 2 : Casas Sead caus | Z (aD Ghee i ° & ~& PER CENT oF a Zz 2 a. Testes Normal at 3 weeks (Hatai 13)... ...........-....- 20.0*} 0.090 0.45 Normal at 3 weeks (incl. epididymi) (Jackson '13)| 24 21.2 | 0.1347 (0.07€-0.224) 0.63* (0.53-0.78) Gontrols ate weeks.ce. acca eee etal. < cs ses eee 6 25.0 | 0.144 (0.114-0.200) 0.57 (0.49-0.62) Body-weight constant 3 weeks (age ot 3to 6 weeks) 2 22.1 | 0.1337 (0.129-0.137) 0.66* (0.65-0.67) Body-weight constant 5 weeks (age of 3to 8 weeks) 1 18.1 | 0.1767 0.98* Body-weight constant 7 weeks (age of 3 to 10 weeks) 7 25.2 | 0.193 (0.106—-0.331) 0.74 (0.49-1.05) Normal at 6 weeks (Hatai ’13)...............-..-.. 50.0*| 0.402 0.80 Normal at 6 weeks (Jackson ’13)............-...--- 20 49.0 | 0.5927 (0.368-0.958) 1.19* (0.88-1.72) | Body-weight constant 26 weeks (age of 6 to 32 weeks) 1 43.7 | 0.3707 0.84* . Normal at 10 weeks (Hatai '13)................---- 70.0*) 0.774 _ tea et Normal at 10 weeks (Jackson ’13).................- 20 117.0 | 1.7477 (0.678-2.62) 1.51* (0.€3-2.41) | Controls stil0 weeksie. cao osm. s = oe teem oe 3 154.7 | 1.850 (1.608-2.10) 1.217 (1.06-1.49) Body-weight constant 25 weeks (age of 10to 35 weeks) 2 79.6 | 1.2947 (1.401-1.188) 1.62* (1.61-1.64) Gontrols atae and obiweekstos. oa 2.4 1-1 3 218.7 | 1.8447 (1.766-1.932) 0.84* (0.74-0.90) b. Epididymi Normal at 3 weeks (Jackson ’13)............. (estim|ated) 20.0 | 0.200 (?) 0.10 (?) Gontrols.at'3 weeks.s.-:y-o- <2 ae smelses ease eee 6 25.0 | 0.0209 (0.0092-0.0246 | 0.084 (0.039-0.123) Body-weight constant 3 weeks (age of 3 to 6 weeks) 1 23.6 | 0.0144 0.061 Body-weight constant 7 weeks (age of 3 to 10 weeks) 7 25.2 | 0.0209 (0.0084-0.0360); 0.080 (0.040-0.115) Gontrols:at710 “weeks 45-1 seco eee ea ceemie een eas 3 154.7 | 0.4700 (0.4480-0.4920)) 0.310 (0.290-0.330) * Gross body-weight. + Including epididymi. WEIGHTS OF ORGANS IN UNDERFED YOUNG RATS 147 there is an increase from 0.144 gram (0.57 per cent of the body- weight) to 0.193 gram (0.74 per cent of the body), an apparent increase of 34 per cent in the absolute weight. (The average net body-weight was about the same in both cases, 25.0 grams in the controls, and 25.2 grams at ten weeks). For the epididymi (table 22 b), the conclusions are even more uncertain; but there appears to be a slight loss in the relative weight of the epididymi in rats held at constant body-weight from the age of three to six and ten weeks. For testis and epididymis combined (table 22 a) there appears to be an increase in rats held at constant body-weight, excepting the period from six to thirty-two weeks. While no final conclusions can be drawn, the evidence indi- cates that in young rats held at constant body-weight there is an increase in the weight of the testis, but not in the epididymis. During inanition in adult rats, the testes and epididymi ap- parently lose weight in about the same proportion as the entire body (Jackson 715 ¢). OVARIES In rats held at constant body-weight from three weeks to ten weeks of age, there would appear to be a decrease in the weight of the ovaries (table 23) from 0.0066 gram (0.0068 gram, less correction for difference in body-weight; or 0.027 per cent of the TABLE 23 The ovaries; average absolute weight, average percentage of net body-weight and range indicated & =e : ma : z RELATIVE WEIGHT DESCRIPTION OF RATS 06 is 8 (Onb RANGRIGaEA iS (AND RANGE): 5 5 2 ; 2 2) GReM PER CENT oe | 64 Z r= Normal at 2 weeks (Jackson ’13)................... 24 16.2 | 0.0036 (0.0015-0.0067)| 0.0220 (0.011-0.039) Montrolstabio WEOKS... joes c-caraae ete sees Sena sen 5 24.5 | 0.0068 (0.0029-0.0104)| 0.0270 (0.012-0.034) Body-weight constant 3 weeks (age of 3to 6 weeks) 3 22.4 | 0.0067 (0.0048-0.0084)) 0.0300 (0.020-0.038) Body-weight constant 7 weeks (age of 3 to 10 weeks) 12 23.3 | 0.0048 (0.0030-0.0064)} 0.0210 (0.012-0.028) Normal at 6 weeks (Jackson ’13)................... 20 54.9 | 0.0106 (0.0050-0.028) | 0.0216 (0.012-0.035) Normal at 10 weeks (Jackson 713).................. 23 98.8 | 0.0350 (0.0100-0.070) 0.0340 (0.013-0.055) Mankioisiat LO weekss......:5..5cs0 s0ess ae odes 3 114.0 | 0.0237 (0.0297-0.038) 0.0290 (0.027-0.033) THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 19, No. 2, 148 C. M. JACKSON body-weight) to 0.0048 gram (0.021 per cent of the body-weight). This would indicate a decrease of about 27 per cent in absolute weight. The ovary, however, is an organ which (like the thyroid gland) is quite variable and somewhat difficult to dissect out with accuracy, so a much larger number of observations would be required for a final conclusion. HYPOPHYSIS In the hypophysis (table 24) there is normally a sexual differ- ence in weight, observable in rats above 50 grams in body-weight (Hatai 713). As in the case of the suprarenal glands, the hypo- physis normally becomes relatively heavier in the female. In the rats held at constant body-weight from the age of three weeks to six weeks, there is what appears to be a sexual differ- ence, the one male with a hypophysis of 0.0016 gram (0.0068 per cent of the body-weight), while the two female hypophyses each weigh 0.0018 gram (0.0079 per cent of the body-weight). These are probably mere accidental variations, however, as in the three to ten weeks series the difference in the relative weight is insignificant (0.0084 per cent of the body-weight in the males, and 0.0086 per cent in the females). In any event, however, TABLE 24 The hypophysis; average absolute weight, average percentage of net range indicated body-weight and ; a 2 os aeZ = RELATIVE WEIGHT Oo B= ABSOLUTE WEIGHT DESCRIPTION OF RATS fe) =o ss (AND RANGE) s am AN ANGE): Al & S ors (AND RANGE)! GRAMS reicened one ze se 52 Z r=) Normsliat so weekss s@hatai713) =. 5...5525ss0seees 25.07/0.00175 0.0070 Controlsisiicnveeks sien ence oat cron eee 25.5 |0.00178 (0.0012-0.0022) 0.0070 (0.0052-0. 0084) Im. | 23.6 |0.0016 0.0068 Body-weight constant 3 weeks (age of 3to06 weeks) i 99.9 10.0018 0.0079 (0.0076-0.0082) rey Oe ier | frm. | 25.2 |0.0024 (0.0016-0.0024) | 0.0084 (0.0057-0.0110) Body, Welgbt constane (weeks (aze ot to1Uiweets) He 23.6 |0.0020 (0.0010-0.0033) | 0.0086 (0.0043-0.0135) Batis: ae m. |130.0 |0.0054 0.0042 Normal at 10 weeks (Hatai ’13)..................-. {? 130.0 10.0084 0.0065 ARCs 3m. {154.7 |0.0062 (0.0059-0.0067) | 0.0040 (0.0038-0.0042) . Sa Dkk bc has ad nauk 2 ace \3f. —/114.0 |0.0054 (0.0046-0.0067) | 0.0048 (0.0040-0.0062) * No sexual difference apparent. + Gross body-weight. » Ure ee WEIGHTS OF ORGANS IN UNDERFED YOUNG RATS 149 there appears to be a distinct tendency to an increase in the weight of the hypophysis in rats held at constant weight from three to ten weeks of age. In absolute weight, this corresponds in the male to an increase from 0.00178 gram to 0.0021 gram, an increase of about 18 per cent. In the female, the corre- sponding increase is from 0.00168 gram (0.00178 gram, less cor- rection for difference in body-weight) to 0.0020 gram, an increase of 19 per cent. As already shown, the suprarenal glands differ from the hy- pophysis in that they undergo a marked sexual differentation in weight during their growth while the body-weight is held con- stant. The impulse to sexual differentiation in these glands therefore appears stronger in the suprarenals than in the hy- pophysis. Final conclusions should be guarded, however, until a larger number of observations is available. In the adult rat during inanition the weight of the hypophysis decreases in nearly the same proportion as the body-weight (Jackson 715 ¢). DISCUSSION With reference to their growth tendency in young rats held at constant body-weight, the organs may be divided into three classes: (1) those in which the growth tendency is so strong that they continue to increase, even when the body-weight is held constant; (2) those which approximately hold their weight con- stant under these conditions; and (3) those which are unable ‘to maintain themselves and lose in weight. According to this scheme, the organs in rats held at constant body-weight from the age of three to that of ten weeks are grouped in table 25. No grouping of this sort can be entirely satisfactory, because in some cases organs are intermediate in position, and especially because (as has been shown) in many cases the weight of an organ will vary according to the age at which the experiment was begun and the length of the period. For example, the liver, which in the three-to-ten weeks experi- ment shows but a slight gain, shows a larger gain at earlier times, and a loss at later and longer periods. 150 Cc. M. JACKSON On the whole, however, the grouping suffices to give a good view of the results. Group I shows the organs with marked growth tendency to be the skeleton, eyeballs, spinal cord, ali- mentary canal, testes, hypophysis and suprarenal glands. Group II, which approximately maintains constant weight, includes the musculature, brain, heart, kidneys and liver. Group III, those which fail to maintain their weight when the body-weight is held constant, includes the integument, lungs, thyroid gland, ovaries, spleen and thymus. In the same table 25 for convenience of comparison are also grouped the various organs of the adult rat according to their relative loss in weight during chronic inanition (Jackson ’15 ¢). TABLE 25 Comparison of growth tendency in young rats held at constant body-weight from age of three to ten weeks with tendency to maintenance in adult rats during chronic inani- tion. The figures indicate the apparent average percentage gain or loss in absolute weight during the period of experiment é GROUP I GROUP II GROUP Ill Marked tendency to growth in Weight nearly con- Marked loss in weight young held at constant body- | stant in young held | in young held at constant weight. at constant body- | body-weight. Strong tendency to maintenance | weight. Relative loss greater in adult chronic inanition Relative loss sim- | than that of entire body ilar to that of entire | during adult chronic in- body during adult | anition chronic inanition | es 2 per cent of per cent per cent change of change of change ‘| *eyeballs +50.0 | liver +10.3) lungs —15.0 *spinal cord +36.0 | *kidneys +4.1 | thyroid gland —24.0 || testes +34.0 | *musculature| +3.0 | ovaries —27.0 || *skeleton +28.0| brain —0.5 | integument —36.0 sh See {| alimentary canal +28.0 | *heart —0.6 | spleen —42.0 fia soa ade | M. +12.0 thymus —90.0 suprarenal glands F. +39.0 M. +18. hypophysis : ie ; f *skeleton +1.8 hypophysis —25.3 | liver —43.0 Adults during |} *spinal cord —4.0 | *kidneys —26.8 | alimentary cana]; —57.0 chronic inanition || *eyeballs —5.8 spleen —29.0 oss of body- 4 brain —6.6 *heart —32.8 weight 36 per || *suprarenal glands —8.9 integument —38.5 cent) thyroid gland —21.8 lungs —40.0 thymus (2?) testes —40.3 *musculature| —40.8 * Indicates correspondence between the groupings in young and adult series. ee fe WEIGHTS OF ORGANS IN UNDERFED YOUNG RATS 151 This enables us to answer the question as to whether the organs in young animals held at constant body-weight (and thus sub- jected to a chronic inanition) behave in a manner similar to adult rats during chronic inanition. In many cases, the group- ing shows an agreement. The skeleton, eyeballs, spinal cord and suprarenal glands have a strong growth tendency in young held at constant body-weight, and also a strong tendency to main- tain their original weight in adults subjected to chronic inani- tion. The musculature, heart and kidneys approximately main- tain their weight in young held constant, and maintain their relative weight in adults during chronic inanition. In the major- ity of cases, however, the behavior of organs in the young differs materially from that in the adult. Thus the alimentary canal has a marked growth in the young at constant weight, yet it loses heavily during adult inanition. The converse is apparently true of the thyroid gland. To a greater or less degree, this inconsistency is seen in the case of most of the organs, as is evident from table 25. This inconsistency is perhaps to be explained in the following manner. In the adult during inanition the various organs lose weight relatively in inverse ratio to the ability of their cells to extract nutrition from the diminishing quantity available in the surrounding medium and to maintain equilibrium under these adverse conditions. In the young animal held at constant body- weight, the conditions differ in that the corresponding cells have the capacity not only to maintain themselves, but to grow. The growth capacity, as is well known, is different from and to some extent independent of the maintenance capacity. The adult has lost the capacity to grow, whereas the young animal has both the powers of growth and maintenance. Hence their organs behave differently during inanition. The alimentary canal, for example, apparently has a strong growth tendency, but a weak power of maintenance. It is further evident, however, that even the growth capacities of the various tissues and organs differ relatively from each other under different planes of nutrition. Thus under normal con- ditions of growth in the rat between three and ten weeks of age £52 C. M. JACKSON the muscular system shows the strongest growth capacity, and increases with greater relative rapidity than any other system (Jackson and Lowrey). The skeleton is of course growing stead- ily, but at a much slower rate, so that it is decreasing in relative (percentage) weight. Under the adverse nutritive conditions in young animals when the body-weight is held constant, how- ever, the musculature is barely able to maintain its weight, while the skeleton is able to absorb more than its share of the available nutrition, and to grow steadily (though at a retarded rate). There are similar differences among many of the indi- vidual organs, though in some cases (e.g., liver, alimentary canal) there is a certain degree of parallelism between the normal growth tendency and the behavior when the body-weight is held con- stant at corresponding periods. That also the power of maintenance may vary in organs according to the nutritional conditions is shown by the char- acteristic differences in the losses of organ-weight in chronic inanition as compared with acute inanition in adults (Jackson ga Sd fee ep Finally, it should be remembered that the results of the present paper, as well as those concerning adult chronic inanition in a previous paper (Jackson 715 ¢), are based upon the use of a diet wholesome and balanced, but insufficient in quantity. It is probable that more or less different results would follow from other forms of inanition, such as ‘partial inanition’ from a chemically defective or highly unbalanced diet. For example, Hatai (’15) finds a pronounced atrophy of the testis and other characteristic changes in albino rats whose growth had been retarded by a ‘lipoid-free’ ration. Bowin (’80), however, in dogs and rabbits fed dry food only (no water) found, with a loss of about 50 per cent in body-weight, the losses in organ- weight similar to those following total hunger. WEIGHTS OF ORGANS IN UNDERFED YOUNG RATS 153 SUMMARY The principal results of the present paper may be summarized briefly as follows: Young albino rats may be held at constant body-weight for considerable periods by underfeeding. The amount of food required for this purpose decreases as the experiment proceeds. As to the body-proportions, the relative weights of the head, trunk and extremities remain practically unchanged during the experiment. There is apparently a slight increase in the head, counterbalanced by a corresponding decrease in the trunk and extremities, but the change is so slight as to seem of doubtful significance. Of the systems—integument, skeleton, musculature, viscera and ‘remainder’—there is but little change in the weights of the musculature, visceral group (as a whole) and ‘remainder.’ There is, however, a marked decrease in the weight of the integument, counterbalanced by a marked increase in the skeleton. Thus on the low plane of nutrition in the young body maintained at con- stant weight, the growth capacity appears weakest in the skin and strongest in the skeletal system. This is in striking contrast with the normal growth process of corresponding ages, during which the musculature increases with relatively great rapidity and the skeleton lags behind relatively. The increase in the skeleton during constant body-weight appears to involve the ligaments as well as the cartilages and bones. The skeletal growth tends to proceed along the lines of normal development, as indicated by decrease in the water- . content, and by formation and union of various epiphyses. Another evidence of the tendency to normal development of the skeleton is seen in the increased relative length of the tail as compared with the body-length. The teeth also continue to develop normally (formation and eruption of the third molars). The individual viscera may be classified in three groups: (1) There is during the maintenance of constant body-weight in young rats a well-marked increase in the weights of the eye- 154 Cc. M. JACKSON balls, spinal cord, alimentary canal (both empty and including contents), testes, hypophysis and suprarenal glands. The suprarenals undergo sexual differentiation in weight (as occurs normally), but the hypophysis apparently does not. (2) There is no marked change in the weights of the brain, heart, kidneys and epididymi. The liver is variable, showing a definite increase in the earlier periods, but a decrease later. The lungs show a slight decrease in the early periods, but not in later! (3) There is a well-marked decrease in the weights of the thymus (‘hunger involution’), spleen, thyroid gland and ovaries. When the organs are similarly grouped according to degree of loss during chronic inanition in the adult (slight loss, loss pro- portional to body, and loss greater than body), many differences are found on comparison with the corresponding groups in the young during constant body-weight. This is explained as due to to the presence of both the growth tendency and the (more or less different) maintenance tendency in the young animals, whereas in the adult there is only the tendency to maintenance. Both the growth tendency and the maintenance tendency, however, show characteristic differences in the various organs according to nutritional conditions (normal nutrition, acute or chronic inanition). EEO WEIGHTS OF ORGANS IN UNDERFED YOUNG RATS 155 LITERATURE CITED Aron, Hans 1911 Nutrition and growth. Philippine Jour. Science, vol. 6, pp. 1-51. 1914 Untersuchungen iiber die Beeinflussung des Wachstums durch die Ernihrung. Berliner klin. Woch., J. 51, 8. 972-977. Bow1n 1880 Beitriige zur Frage tiber die Trockenernihrung. Dissert. St. Petersburg. Cited by Miuhlmann, Russische Literatur tiber die Pathologie des Hungers. Centralbl. f. allg. Path., Bd. 10, 1899. Donatpson, H. H. 1908 A comparison of the albino rat with man in respect to the growth of the brain and of the spinal cord. Jour. Comp. Neur., vol. 18, no. 4. 1911 The effect of underfeeding on the percentage of water, on the ether-alcohol extract, and on the medullation in the central nervous system of the albino rat. Jour. Comp. Neur., vol. 21, no. 2. Harms, W. 1909 Ueber ‘den Einfluss des Hungers auf die Wirbelsdule der Tritonen. Verhandl. d. deutschen Zool. Gesellschaft, 19 Versamml., 8. 307-312. Haratr, 8. 1904 The effect of partial starvation on the brain of the white rat. Am. Jour. Physiol., vol. 12, no. 1. 1908 Preliminary note on the size and condition of the central nervous system in albino rats experimentally stunted. Jour. Comp. Neur., VOlenLs> no: 2. 1913 On the weights of the abdominal and the thoracic viscera, the sex glands, ductless glands and the eyeballs of the albino rat (Mus norvegicus albinus) according to body-weight. Am. Jour. Anat., vol. 15), sa¥oi, ile 1914 On the weight of the thymus gland of the albino rat (Mus nor- vegicus albinus) according to age. Am. Jour. Anat., vol. 16, mo. 2. 1915 The growth of the body and organs in albino rats fed with a lipoid-free ration. Anat. Rec., vol. 9, no. 1. Jackson, C. M. 1909 On the prenatal growth of the human body and the relative growth of the various organs and parts. Am. Jour. Anat., vol. 9. 1913 Postnatal growth and variability of the body and of the various organs in the albino rat. Am. Jour. Anat., vol. 15, no. 1. 1915 a Effect of acute and chronic inanition upon the relative weights of the various organs and systems of adult albino rats. (Abstract). Proc. Amer. Ass’n Anatomists. Anat. Rec., vol. 9, no. 1, p. 90. 1915 b Changes in young albino rats held at constant body weight by underfeeding for various periods. (Abstract). Proc. Amer. Ass’n Anatomists. Anat. Rec., vol. 9, no. 1, p. 91. 1915 ¢ Effect of acute and chronic inanition upon the relative weights of the various organs and systems of adult albino rats. Am. Jour. Anat., vol. 18. Jackson, C. M., and Lowrey, L. G. 1912 On the relative growth of the com- ponent parts (head, trunk and extremities) and systems (skin, skele- ton, musculature and viscera) of the albino rat. Anat. Rec., vol. 6, no. 12. 156 Cc. M. JACKSON Jonson, Arvip 1909 Studien iiber die Thymusinvolution. Die akzidentelle Involution bei Hunger. Archiv f. mikr. Anat., Bd. 73, 8. 390 ff. Lowrey, L.G. 1913 The growth of the dry substance in the albino rat. Anat. Rec., vol. 7, no. 9. Morcvtis, 8. 1911 Studies of inanition inits bearing upon growth. I. Archiv_ f. Entw. d. Org., Bd. 32, H. 2. Waters, H. J. 1908a Capacity of animals to grow under adverse conditions. Proc. Soc. for the Promotion of Agricultural Science. 1908 b The influence of nutrition upon the animal form. Presented at the Thirtieth Meeting of the Society for the Promotion of Agri- cultural Science. INHERITANCE IN THE ASEXUAL REPRODUCTION OF HYDRA K. S. LASHLEY From the Zoélogical Laboratory of the Johns Hopkins University TEN FIGURES CONTENTS NMLTETOCILGTION ». 2. eee oe en wae ees Sa steid oe btaa press bide mee sie eos 157 Eixperiments of Hanel........-.- 2.2022 - 220s eee eee cet eee 159 Criticisms of Hanel’s experiments.:2..--. 05-2. 22- 2-2-2 ese. eee e ep eee 159 Analysis of Hanel’s data....... 0.025.602 tee eee ee eet e ences 163 mse tleduquestions:. «oc. se eee ore miele ote ici - oi B'S 165 Mimeaniattons i: Hydra. sis. oct Aston ns ioe gee eet it 3+ sions Soe ene 166 Variation in the number of tentacles of H. viridis................... 167 Conditions producing like variation in parent and offspring......... 170 lll, Tiiiethernes oinalingeeaeelesig cpa co bococncocaccsseennpuodcooh opignopo seule 174 Hixperimental methods...........-.---. 2102-2 see c cette eens 175 Detailed examination of two clones of H. viridis.................... 176 Differences in number of tentacles and size..............-..+.4--- 176 Wtherlauierences between the clonesseamae ser aera cit re ner 182 Nature of the difference between Clones A and D..:.............. 183 TENG Oi Giilneie Ghayease InAs eabisnis occdc = cnc dcdn en robes DOosIne moe 186 SCITINT Dy Aad ee Be cle ee Brentano oe. CHA] panto quiere te Reece 190 TV. Inheritance of variations within the clone.....................-.------ 190 IvesemblanGenoicloserrelatlvies vc anne cere ee enero ie eieest ienete tes) er 191 Invi ar GEC OTe Ses eee maeaisioaan co pc ldoes Sse amon maar 195 Tin eveeeireniverey (OL Rios a leis Sete OID ocidihis, ao so cota ancidei actress 199 Wh [Diflepormstetiavaticn's cs £5 ees ae eS ie rs BIG ES 8 os aie Iain pict eee 202 WTh., SCH RRR aooe Ones a eS ea enreee Pieler itr oc ts Poor sean eae 204 [Linieira faa OU Sees ds co Sele aes é aonb Se dein pksons Sob ncide Pere en eS. 205 Lo TORI Een: Coe on © SE San IPO ote ac cisco cIRI Cr aes cara 208 I. INTRODUCTION The theoretical bearing of the work of Johannsen upon pure lines was not widely appreciated by students of heredity for the first few years after its publication; the general applicability of Mendel’s law had not been shown and, hence, the genotype conception was without the support since gained from the evi- 157 158 Kk. .S. LASHLEY dence bearing upon the stability of the unit character. The first attempt to test the general application of Johannsen’s conclusions was made by Elise Hanel. In 1908 she published the results of two years of experimental study of inheritance in the asexual reproduction of the fresh-water polyp, Hydra grisea, and drew conclusions which were quite in accord with the results obtained with self-fertilized lines of beans. Her results were soon called in question, however, by the work of Hase (’09) and the criticisms of Pearson (710), who obtained such con- flicting results that a re-investigation of the subject became necessary. The results of such a renewed, independent study of variation and inheritance in Hydra are presented in the present paper.! In order to make clear the questions at issue and the bearing of the results presented here upon these and the general problems of genetics, it will be necessary to review briefly the work of Hanel and the criticisms raised against it, especially as the latter are complicated and not clearly valid. Before this is undertaken, however, some explanation of the termi- nology of the present paper may not be out of place in view of the past confusion in genetic literature. Johannsen (713), following Shull, has employed the word ‘clone’ to describe a family descended from a single individual by asexual reproduction. The name ‘biotype’ applies to any group of organisms which show the same hereditary constitution (‘genotype’). In the statistical terminology the ‘distribution’ is the natural arrangement of individuals showing variation (‘variates’) in classes. The ‘mean’ is the arithmetical average of the variates. The ‘standard deviation’ (c) is an expression of the average extent of variation from the mean shown by the variates. The ‘coefficient of variation’ expresses the average percentage of variation from the mean in terms of the mean as unity. The ‘coefficient of correlation’ (r) represents an arbitrary measurement of the average degree of resemblance be- tween correlated members of two series of variates. The ‘coefficient of regression’ (Rf) expresses the average extent to which the variations of one series follow the variations of the correlated series. The nomenclature of the genus Hydra has been subject to frequent changes. The names which have been used most frequently by ex-’ 1 This was undertaken at the suggestion of Prof. H. S. Jennings, to whom I wish to express my indebtedness for his assistance and kindly criticism during the course of my work. I wish also to thank Prof. B. E. Livingston for the use of the facilities of the Laboratory of Plant Physiology of the Johns Hopkins University during the summer of 1912. INHERITANCE IN ASEXUAL REPRODUCTION 159 perimental zodlogists are those of Linnaeus. Brauer (’08) applied the rule of priority, thus requiring the nomenclature of Pallas. In 1912 Bedét criticised Brauer’s revision and showed that the species, H. viridis L. should be retained. Since the experimental literature has employed the Linnaean nomenclature this has been used here in order to avoid the constant repetition of synonyms. The names employed are given below at the left, with the revision of Bedét at the right. H. viridis L. H. viridis L. H. grisea L. H. vulgaris Pall. H. fusca L. H. oligactis Pall. H. polypus A. Brauer. Experiments of Hanel Hanel began her work in 1906 and during the two years of her experiments bred clones from 26 wild Hydras, obtaining records of nearly 7000 buds, the descendants, by asexual reproduction, of the original 26. Upon comparing these 26 ‘stem parents’ with their immediate progeny she found that those with a large number of tentacles produced buds having, on the average, a ereater number of tentacles than the corresponding buds of par- ents with few tentacles. From the progeny of the ‘stem parents’ taken singly Hanel selected polyps with a high, an intermediate, and a low number of tentacles and continued this selection for from two to seven generations. Averaging the results obtained, she found that within the single clone the mean number of tentacles of the progeny of individuals selected for four genera- tions was slightly less in the group selected for a large number than in the group selected for a small number, whence she con- cluded that variations within the clone are not inherited. Criticisms of Hanel’s results In 1909 Hase, at the suggestion of Plate, studied the relation of the number of tentacles of Hydra to the age of the polyp and to the number of tentacles of the buds. He verified the obser- vations of Parke (’00) and Hanel that the number of tentacles increases with the age of the polyps, finding an average increase of 2.1 tentacles in a number of polyps kept under observation for 90 days. In recording her data Hanel had taken this fact 160 K.-S, DASHLEY into consideration but, believing from observations upon mass cultures that the adult number of tentacles is proportional to the number which the polyps bear when they produce their first buds, she thought herself justified in computing the adult number of tentacles for young poylps which had produced a single bud. Hase concluded that the increase is not regular and that there is no true adult number of tentacles for any polyp. Hase further believed that the number of tentacles which the buds have at the time of separation from their parents is not proportional to the number of tentacles which the parents bear at that time. He gave data to show that while the number of tentacles of the parents is increasing with age, that of the suc- cessive offspring of these parents remains constant and does not follow the increase shown by the parents. He also cut a number of polyps into two or three pieces and found that they did not regenerate the same number of tentacles that they had borne originally. From this he concluded: Die verschiedene Tentakelzahl ist eine reine Somation und besitzt keinerlei Erblichkeitswert. Als eine reine Linie (im Sinne von Johann- sen) kann man daher die direkten ungeschlechtlichen Nachkommen einer Hydra nicht bezeichnen. Es ist daher auch nicht méglich char- akteristische Typen zu isolieren und sie erblich konstant zu erhalten. Aus gleichem Grund kann man auch keine reinen Linien (im vorigen Sinne) aus einer Hydrapopulation durch Selektion sortieren. This conclusion is far from justified, however, by the evidence which Hase presents. Records of two parent Hydras are given, which increased slowly in their number of tentacles from five to nine, together with records of all the progeny of each. If the two groups are averaged, as has been done in table 1, it is evident that the buds produced by these two parents after they had acquired a large number of tentacles have a higher average than those produced when the parents had few tentacles. Yet from these data, Hase concludes, ‘‘Wir sehen, zuerst, ein schein- bares Mitgehen der Tentakelzahl der Knospen mit der Mutter, aber bald tritt ein vélliger Riickschlag ein.” As I shall show later, this is not true for H. viridis, as it is probably not for H. fusca: the regression is only partial. DO EE ae INHERITANCE IN ASEXUAL REPRODUCTION 161 The experiment upon regeneration likewise fails to support the conclusion which Hase draws from it, since it shows only that the number of tentacles is not regenerated at its full value but fails to disprove that the number regenerated is not proportional to the original number. I have reviewed the experiments of Hase in some detail because they have been generally quoted as proving that the number of tentacles of Hydra is not an heredi- tary character, whereas, at best, they but serve to cast some doubt upon Hanel’s conclusions. TABLE 1 Increase in the average number of tentacles of buds produced while their parents bore the numbers increasing from 5 to 9 (modified from Hase) MEAN NUMBER OF TENTACLES OF BUDS PRODUCED WHILE THE PARENTS BORE THE NUMBER OPPOSITE NUMBER OF TENTACLES BORNE BY PARENTS ~“I SS on > Sr or > w Oo On o> or wo bo A much more serious criticism of Hanel’s work, and of ‘pure line’ work in general, was made by Pearson during the follow- ing year. He subjected Hanel’s data to a more thorough analysis than she herself had done and held that they by no means justi- fied the conclusions drawn. Concerning the evidence for the existence of strains, diverse with respect to tentacle number, he Says: Hanel begins with a very careful investigation of the growth and environmental changes in the character selected, the number of tentacles of Hydra grisea. There is a general agreement with Parke’s results that the number of tentacles changes with age, size, food and place of culture. Differences in these factors can produce very considerable differences in individuals and differences in the averages of differentially treated groups which can amount to as much as 0.5 to 0.8 of a tentacle. These are precisely the order of the average hereditary differences. Thus: 162 K. S. LASHLEY PARENT OFFSPRING No. of No. of No. of No. of individuals tentacles individuals tentacles 9 6 364 6.943 9 7 310 7.296 = 8 166 7.344 4 9 125 7 .383 It will be at once recognized that the differences here are rather less than many of the environmental differences and that there is no security that these 26 foundation Hydras are really represented by differentiated hereditary numbers of tentacles. Yet this table, as it stands, embraces Hanel’s proof that the number of tentacles is an hereditary character in the ‘pure line.’ What evidence is there that any one of the numbers of tentacles attached to those 26 parents is really constitutional and not environmental? When we consider that Hanel’s experiments extended over a period of two years, that the different ‘stem parents’ were col- lected and bred at different seasons, and that no data are given by which it is possible to judge which of the races were kept under a uniform environment this criticism greatly reduces the value of her evidence for the existence of diverse genotypes. The second point of Pearson’s criticism deals with the inheritance of the number of tentacles within the clone. The data were subjected to statistical analysis by Miss Elderton and the cor- relation between relatives was determined for different degrees of relationship, within the population composed of the 26 clones. A comparison of the correlations showed that there is a greater resemblance between siblings and between parents and their offspring than there is between ‘uncle and nephew’ or between grandparents and grandchildren. This, as Pearson points out, can not be due merely to the inclusion of diverse races in the tables of correlation but indicates that there is an inheritance of variations within the clone. From this evidence Pearson concludes that Hanel’s records show, not what she believed them to show, but just the reverse; that regression is only partial, both in the population and in the clone; that variatons are inherited equally in both population and clone. INHERITANCE IN ASEXUAL REPRODUCTION 163 Analysis of Hanel’s data Since this method of treating the data does not deal with individual clones it seemed advisable to carry the analysis some- what farther. I have computed the mean number of tentacles of all individuals in each of Hanel’s clones and the correlation between parent and offspring for each.? The mean numbers of.tentacles of the clones tables 2 and 3. These are given in The mean number of tentacles of Hanel’s clones arranged in the order of magnitude CLONE NO. MEAN NO. OF TENTACLES 6 SINTDDADO OS bs file | “J I “I SOROS Me On Gs ss es .428+0 .040 .438 +0 .020 .670 +0 .058 .677 +0 ..049 .805 +0 .029 .925+0 .054 .926+0.041 .010+0.031 .026 +0 .024 .170+0.031 .184+0.032 .230 +0 .0384 .240 +0 .023 .264+0.041 .287 +0 .056 .319 +0 .032 .3825 +0 .029 .361+0 .050 .367 +0 .046 .384+0 .025 .3893 +0 .023 .400 +0 .040 430 +0 .043 .456 +0 .049 .900 +0 .039 .641 +0 .047 .712+0.044 o 0.771 0.475 0.826 0.816 0.734 0.692 0.750 0.786 0.780 0.721 0.658 0.798 0.906 0.902 0.962 0.784 0.786 0.850 1.005 0.914 6.763 0.938 0.934 1.197 0.862 0.835 0.942 NO. OF TENTACLES OF STEM PARENT fom — IH WVSOCIWAOWOANHNMNONDAANAAGHAAGMNOnN nA 2 Hanel records the variations in her clones in percentages; the tables contain many errors, the sum of the percentages included in one array ranging from 85 to 140 per cent. This leads to error in the means and correlations computed from the tables but these probably average out in the numerous constants determined. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 19, No. 2 164 KS. DASHULNY form an almost unbroken series from the smallest to the largest, and, lacking evidence upon the environmental conditions, give little indication of the existence of diverse races. Further analysis indicates that the clones tended to become more alike after they had been kept under cultivation for some time. The average number of tentacles of the offspring of each of the 26 ‘stem parents’ and the average of all their later descendants were computed and are given in table 4. The descendants of parents with extreme variations tend to revert to the mean. The coefficients of correlation between parent and offspring within the clones (table 3) are extremely variable, but the TABLE 3 Correlation between parent and progeny within each of Hanel’s clones CLONE NO. CORRELATION CLONE NO. CORRELATION ee ACR ener he =. teat 0.170+0.054 14 0.057 =0 .050 DOT RO Le 0 .336=+0 .067 15 0.323+0.040 Dye ee ee ere fe dee 0.469+0 .040 16 0 .050+0.066 Sa ee URE eee ee 0.010+=0.029 17 —0.073+0.040 1 REL. pa tla ee pee —0.009=0 .040 18 — 0.060 +0 .037 Fe de cpae eye ee Ps tee 0.2340 .037 19 0.072+0.041 pee Be po 0.1420 .026 20 —0.087+=0.041 7 Sie 8 Oh Sele eee ee 0.0360 .028 21 0.708+0.030 is epee eee ee ee 0 .068=0 .066 22 0.007 =0 .047 At ae a sees ee ee 0 .006=+0 .047 23 0.178+0.059 1O Ree oh er eee 0.009 +0 .042 24 0.0000 .067 PDS et PR ee es 0.040 +0 .037 25 0.1280 .046 1D ie Pee sn SEL 0.031+0.030 | 26 0.1040 .047 1 EES 8 eee 0.0480 .040 Mean of all...... 0.101 TABLE 4 Regression in the first and later generations of the descendants of Hanel’s 26 ‘stem parents’ ‘“STEM PARENTS ”’; FIRST GENERATION; ALL DESCENDANTS; NO. OF TENTACLES MEAN NO. OF TENTACLES MEAN NO. OF TENTACLES 6 6.943 7.086 7 7.296 7.102 8 7.344 7.347 9 7.383 7.347 INHERITANCE IN ASEXUAL REPRODUCTION 165 majority (75 per cent) are positive and the evidence favors the belief in an inheritance of variations within the clone. The great irregularity of the results, however, makes it impossible to draw from them any law of inheritance within the clone or even to be sure that there is any constant tendency toward the inheritance of variations. Unsettled questions Thus the evidence from previous work bearing upon heredity in Hydra must be looked upon as inclusive and the chief problems of heredity and variation as still unsettled. Hanel maintains the existence of diverse races of Hydra which are distinct in their hereditary constitution. This is denied by both Hase and Pearson but upon different grounds. MHanel holds that, within a population, variations are inherited and that selection is effective in isolating the diverse races, within which there is no inheritance of variations. Hase denies that variations in the character studied are inherited at all and asserts that such variations are purely somatic. Pearson attempts to show that the variations are inherited both within the population and the clone. Analysis of Hanel’s data shows that there is usually a correlation between parent and progeny within the clone; that this may be very high in some cases, and in others, negative, varying around an average of 0.101. Experiméntal analysis of the cause of this correlation is lacking. The chief questions demanding further investigation seem to be the following: Will such a variable character as the number of tentacles of Hydra lend itself to genetic study? Are there other characters of Hydra which vary less within the individual? Given a character which is comparable in different individuals, do races, hereditarily diverse with respect to this character, exist? If there are such races, in what characters do they differ and are they numerous or few? Is there inheritance of individual differences within the population, and if so, what part do diverse races play in this inheritance? Is there actually a correlation between parent and progeny within the clone? 166 K. S. LASHLEY If so, how is it produced and what is its significance? Is there an inheritance of individual differences within the clone? What is the origin of the diversities between races? II. VARIATION IN HYDRA The only variable character in Hydra considered in detail by previous workers has been the number of tentacles. At the beginning of the present experiments I examined a number of polyps in the hope of finding other variable characters suitable for genetic study. Size, body-form, color, relative length of the tentacles, reaction to mechanical stimuli, distribution of the nettle cells, diameter of the mature nematocysts, and reaction to light were compared. Of these only size seemed at all suitable for statistical study. Variations in practically all the characters of Hydra appear, not only between different individuals, but in the same individual at different times. Hase has shown that the number of tentacles, alone, varies too much in the individual to form a character suitable for statistical treatment and Plate (713) has stated the necessity for the use of the number of ten- tacles at some definite stage in the development of the polyps in future studies of heredity. The same individual variability appears in the size of the polpys so that the size at some definite stage of development must be used in comparative studies. The time when the bud separates from the parent marks the occurrence of certain definite physiological processes in the bud and, if the number of tentacles of the bud is taken at this time, it serves as an index of the state of development of the bud at the time when these processes are initiated. This number may be compared, in tests for heredity, either with the number borne by the parent at the corresponding age, or with the number which it bears when the given bud is produced. It is more difficult to find a stage where the size of different polyps is comparable. Growth is very rapid for the first few days after the buds are released and it is not always possible to take measurements of the new buds, as such measurements require a great deal of time. After four or five days for H. viridis, when the buds have matured INHERITANCE IN ASEXUAL REPRODUCTION 167 and begun to produce buds in their turn, growth is much slower, although the polyps continue to increase in size until the fifteenth day or later. For comparisons of parent and offspring, I have used measurements of the size at the age of seven days, or as near this as was practicable. The figures so obtained are greatly subject to chance variation but are problaby as dependable as any which can be obtained for so plastic an organism. Variation in the number of tentacles of Hydra viridis The most frequent number of tentacles (the modal number) found in wild populations of H. viridis is usually 6, sometimes 7, and the normal range seems to be from 4 to 9. Table 5 shows, in percentages, the distribution of variations in H. viridis found by previous investigators. Both local and seasonal differences probably play a part in the production of the differences in these populations. The data shown in table 6 have been obtained TABLE 5 Distribution, in percentages, of variations previously recorded for Hydra viridis NO. OF TENTACLES...........- 4 5 6 7 8 9 10 11 12 13 Ruane 99). oo dates = 2.5} 42.0) 40.0} 12.0) 2.0 Hathaway (’99)........ 1125) 50-0) 3520)) 320 zeenicenn( 2 O00) gob ser. ek. 32.3] 48.4) 12.9] 4.8) 1.6 le reect ara 4.8) 30.6] 44.5} 16.4) 2.9) 0.6 UU 22 .8| 46.5] 20.9| 7.4) 1.4) 0.9 Reeseu(09))z. 0s = taars- i % 24.0) 54.0] 15.0) ? ? ? ASO (COD). sepsis: ates ates 20 | 2h0)) FeO) GSA0) 27/0) SHO) 71) wo] Was || Mees TABLE 6 Distribution, in percentages, of variations in a population of Hydra viridis from Baltimore NO. OF TENTACLES MEAN NO. OF NO. OF TENTACLES Ui POLYPS 3 4 5 6 7 8 vo) —y Oo 0 .6|25.0|51.5|18.0} 3.7] 0.6) 6.988+0.044 | 0.8404 | 167 5 5/30 .9/50 9/11 .3) 0.9 6 .696+0.015 | 0.7332 | 1000 0.7/11.4|41 .4/37.9| 7.1] 0.7] 7.393+0.050 | 0.8827 | 140 Oct 1911... 0.6 Oct., 1913. .| 0.1) 0.4 Apr., 1914.. (EZ/ 168 K. S. LASHLEY from a population in a very small stagnant pond in the neighbor- hood of Baltimore. Very extensive seasonal changes are evident from the mean of the population at different times. Numbers of tentacles less than 5 or greater than 9 probably can not be considered normal for H. viridis from this neighborhood. Buds formed with less than four tentacles are usually small and do not extend their tentacles. They either produce more ten- tacles quickly, or die. The only individuals with ten tentacles which I have found were small-bodied, dark in color, and pro- duced buds very slowly. In H. grisea a large number of ten- tacles seems to present a rather unstable condition. Individuals with more than eight tentacles in my cultures have shown a tendency to divide lengthwise and thus reduce the number of tentacles to the mode. I have observed longitudinal division in only one,specimen of H. viridis, which bore six tentacles at the beginning of division and gave rise to two polyps, each with six tentacles. As individual polyps grow older they form additional tentacles, a condition first observed by Marshall (’82) in H. viridis. Hase has demonstrated a like condition in H. fusca and Hanel in H. grisea. Hanel has found that the addition of tentacles occurs even in polyps which are starving, although hunger tends to inhibit the process, as is clear from table 7. Parke found that the number of tentacles might be reduced and Hanel’s table shows that this reduction is favored by starvation. My own TABLE 7 Changes in the number of tentacles of 129 specimens of Hydra grisea during one month’s cultivation; after Hanel WITH FOOD WITHOUT FOOD Number of tentacles: added’: = 9.22. . 2. . :ocsqesecc- + 2 8 Number of polyps showing change................... 2 6 Number of polyps unchanged... .. ......-0.5.25...--- 31 47 Total number-on polyps... 2.02 se eee eee 62 67 Weanneresse per ndividual...-24.>4-ep eee. eee 0.69 0.16 INHERITANCE IN ASEXUAL REPRODUCTION 169 data verify these results for H. viridis. Figure 1 shows the mean number of tentacles of about 30 polyps from two different clones at successive intervals of two days. Besides the specific tendency to change the number of ten- tacles with advancing age, there are environmental factors which may produce variation in the number of tentacles of Hydra. Hanel’s data, table 7, show that starvation partially inhibits 75 tentacles 10 20 30 Days Fig. 1 Increase with advancing age in the average number of tentacles of polyps from two clones, A and D. The averages are based upon about 30 polyps from each clone. Clone A shows an average daily increase of 3.6 per cent; clone D an increase of 2.0 per cent. the addition of tentacles and causes some to be absorbed. Parke has shown that other unfavorable conditions, such as stagnant water, have a like effect. Injury and regeneration of the oral end also leads to a reduction in the number of tentacles (Rand ’99). These factors produce variation in adult Hydras, but further, as will be shown, they have a like effect upon the buds produced by these adults. 170 K. S. LASHLEY Conditions producing like variations in parents and offspring Age. Corresponding to the increase in the number of ten- tacles with the age of the parents, the buds produced by old polyps have, on the average, more tentacles than those pro- duced by young ones, that is, there is an increase in the number of 6.0 S55 0) 5 10 15 20 25 30 Fig. 2 Increase in the average number of tentacles of successive buds pro- duced by the same parents. The ordinates represent the mean number of ten- tacles of the successive buds. The abscissae represent for Clone A the successive buds produced by 70 parents, for Clone D the buds produced on successive days after the 40 parents which furnished the basis of the curve began to produce buds. tentacles of successive buds with the increasing age of the parent. Figure 2 shows this condition clearly. Data from two clones are included in this figure; for clone A the average number of ten- tacles of the first, third, fifth, seventh, etc., buds are given in the graph; for clone D the averages of all buds produced during suc- INHERITANCE IN ASEXUAL REPRODUCTION iw cessive intervals of two days furnish the basis of the graph. The general trend of both lines is upward, although more mark- edly so in A than in D. The same relation for the two clones was found for the increase in the number of tentacles of the parents (fig. 1). The increase in the parental number of ten- tacles with age is due to some internal factor. Its relation to the increase in the number of tentacles of successive buds will be considered later. Besides this internal factor, there are various environmental agents which modify the number of tentacles of parent and bud in the same direction, and which must be taken into account in any study of inheritance. Effects of starvation. One hundred polyps from a mass culture of a single clone (clone A) were divided at random into two groups of 50 each. One group was fed every second day with small crustacea, the other every sixth day. Both were placed in clean dishes of fresh water every second day, and each polyp was kept in a separate dish. The result of this method was that one group received about three times as much food as the other. The first result of the partial starvation of the six-day group was a reduction in the rate of budding. The well fed group produced an average of 0.203 buds per day for each polyp; the starved group produced only 0.074 buds per day, which gives a ratio of almost three to one, corresponding to the amount of food given. There was also a difference in the average number of tentacles of the buds produced by the two groups, the offspring of well fed parents having a slightly higher average than the others. This is shown in table 8. The difference here is small and may easily have been due to chance. The experiment was TABLE 8 Effects of starvation of parents upon the mean number of tentacles of their buds; all buds recorded within 24 hours after their release PARENTS BUDS Mean no. of tentacles No. of polyps aeserke i No. of polyps NGC aera 7.18+0.06 50 6 .32+0.04 153 Starved....... 7.18+0.07 50 6 .24+0.06 61 172 K. S. LASHLEY TABLE 9 Effects of starvation of parents upon the number of tentacles of their buds; second experiment PARENTS BUDS ee Tentacles + M - of | No. of z M . of -| No. ot . Earkeples eyes pene fentaaley ae Dieroree Clone A Ed: 5 5./serte he 6.84=0.05 25 0 6.59=0.03 127 Siarved ec. so. 6.80+0.05 25 0 6.37+=0.09 44 | 0.22+0.09 Clone D Redes eee O60 0.06 PAs 1 5.76=0.03 195 Siarved met acr.c- 5.92+0.06 25 5.20+0.06 44 | 0.56+0.07 repeated with polyps from two clones and results were obtained which made it certain that starvation of the parent leads to a reduction of the number of tentacles of its progeny. The results of this experiment are shown in table 9. The parents recorded in this experiment were old polyps taken from mass cultures. Their mean number of tentacles already exceeded the means of the clones from which they came, so that any considerable increase in their numbers of tentacles during the brief time of the experiment was not to be expected. Hanel’s data, however, prove that starvation tends to inhibit the addition of tentacles in parents and this experiment shows that the buds of starved parents have fewer tentacles than those of normal ones. This condition, occurring among a large num- ber of polyps subjected to unequal and fluctuating environmental conditions would undoubtedly lead to a slight degree of resem- blance between parent and progeny, even if there is no true inheritance of parental variations. Effect of injury. Twenty mature Hydras were cut in two trans- versely and kept until both halves had regenerated. Each half was kept in a separate dish and the buds which it produced after regeneration was complete were recorded. Altogether, 287 buds were obtained from the 40 freshly regenerated polyps. The mean number of tentacles of the clone from which the orig- inal 20 were derived was, at this time, 6.91+0.03. The mean INHERITANCE IN ASEXUAL REPRODUCTION 173 number of tentacles of the original 20, the oral ends, was 7.05 = 0.09. The mean number regenerated by the aboral ends was 6.42+0.09. The regenerated oral ends produced 146 buds, the aboral ends, 132 buds within 28 days after the operations. The mean number of tentacles of the buds from the oral ends was 6.88 + 0.03; that of the buds from the aboral ends was 6.59 + 0.04; the difference here is 0.29+0.004 in favor of the offspring of the polyps which were required to carry out the lesser regenerative processes. Clearly, the same factors which cause a reduction in the number of tentacles in the parents (the call for an expendi- ture of energy in regeneration) cause also a reduction in the number of tentacles of their offspring. In order to test the length of time during which the after- effects of injury to the parents influence the number of tentacles of the buds, the mean number of tentacles of all first, all second, ’ etc., buds produced after regeneration by the oral and aboral ends was computed. This is shown in figure 3. After the ninth bud the graphs are based upon too small numbers to be sig- nificant. It is clear from this figure that immediately after regeneration the buds produced have few tentacles and that the number of tentacles of successive buds increases rapidly until, after five or six have been formed, the normal condition is regained. These experiments show that certain environmental agents, acting upon Hydra, tend to produce variation in a given direc- tion in both parent and offspring, either, as in the first case, by acting simultaneously upon both parent and bud, or, as in the second case, by affecting the bud only indirectly through a reduction in the vitality of the parent. Although only two such agents have been studied experimentally it is probable that many others have a like effect. Parke has shown that the bacterial conditions leading to depression in his cultures brought about a reduction in the number of tentacles of the polyps. I have found that polyps which have just recovered from de- pression tend to produce one or more very minute buds with few tentacles. Polyps kept in water of high salt content become small and dark-colored and produce small dark-colored buds. 174 K. S. LASHLEY ‘ ‘ i 7.0|_ tentacles : ‘ ' ‘ f O 5 F 10 15 Successive buds Fig. 3 The mean number of tentacles of the successive buds produced by polyps after regeneration; ( ) buds produced by regenerated oral ends; (-—-—) buds produced by regenerated aboral ends; all polyps were taken from the same clone. Temperatures much above or below the optimum have an effect like that of high salt content. The results of the action of these agents resemble the cases of temporary parallel induction found in parthenogenetic reproduction (Agar *13) but the mechanism of the action is probably much simpler since we are dealing here only with a congenital reduction in vigor. III. EXISTENCE OF DIVERSE RACES In addition to these variations resulting from environmental action, are there differences between the individuals of a popu- lation due to some internal, hereditary factors? This question has been tested by breeding a number of clones of H. viridis . under similar cultural conditions. Or INHERITANCE IN ASEXUAL REPRODUCTION 17 Experimental methods Hydra is easily cultivated in the laboratory, provided that a con- stant supply of food is available. The polyps used in the experiments were collected from various sources, usually from ponds containing Nitella or Elodea. In the greater part of the work each polyp was kept separately in a small Stender dish with about 5 cc. of water. It was found necessary to wash these dishes in boiling water every second day in order to keep down bacterial growths and maintain the polyps in good health. A small species of Cyclops was used exclusively as food. Attempts to breed them in sufficient numbers in the laboratory failed and all food material was obtained by towing with a plankton net in an artificial pond. The supply so obtained was further con- centrated so that from one to two hundred Cyclops were given to each Hydra every second day. (A healthy specimen of H. viridis will eat ten or more Cyclops daily and the large number supplied allowed each polyp to capture as many as it could eat). At first, a growing stem of Elodea was kept in the culture with each Hydra but as this was found to be unnecessary and a possible source of contamination it was dis- continued after the first experiment. All the cultures were kept at approximately the same level upon a broad table-top where they were exposed to uniform conditions of light and temperature. The cultures of various clones were divided into small groups and the order in which these were fed and arranged upon the table was varied from day to day. While conditions may have varied in the different individual cultures, there seems to be no reason for believing that such differences did not average out in each of the clones, considered as a whole. Besides the individual cultures a number of mass cultures of different clones were kept under similar conditions. Numbers of polyps were placed in 6-inch battery jars filled with water from the food pond. The water in the cultures was renewed weekly, and where two clones were being bred for comparison the water from the culture jars con- taining the two clones was interchanged frequently. The number of tentacles of the polyps was counted under the Zeiss binocular, and in the case of the polyps in individual cultures the number of tentacles of the buds was recorded within 48 hours of the time when they were released from the parents. The numbers of ten- tacles of the parents were recorded also at the time when each bud was produced. Measurements of size were made in the following way: The polyps, contracted, were placed on a grooved slide under a com- pound microscope. When they expanded until the length was about four times the diameter, an outline of the body was made with a camera lucida. The area of this outline was measured with a planimeter and from this, treated as the area of the longitudinal section of an elipsoid, the volume was computed. This method is subject to about 25 per cent error, but is much more accurate than measurements of a single diameter. The constants given in the following pages were computed 176 K. S. LASHLEY by the simple statistical formulae devised by Pearson and others (Davenport ’04; Yule 712). In the computation of the standard deviations for size Sheppard’s correction was not used. All calculations were made with the aid of the ‘Brunsviga’ computing machine, and later repeated de novo. Detailed examination of two clones On June 10, 1912, twenty-five specimens of Hydra viridis, collected from a limited area in a small undrained pond, were isolated in individual culture dishes. They began to multiply rapidly under the favorable conditions of the cultures. The new-formed buds were each placed in a separate dish, labeled, and the number of their tentacles recorded within 48 hours after they separated from the parents. After a few days it was found impossible to care for all the descendants of the stem mothers and it seemed best to discard the majority of the clones. Two of them, A and D, which seemed somewhat diverse were retained. It soon became necessary again to reduce the number of cultures, as not more than 300 animals could be cared for at one time. An equal number of each of the two clones was therefore selected for high, intermediate, and low numbers of tentacles, and the cultures were so arranged as to give not less than ten progeny from as many parents as possible. Since the selections were approximately equal in both directions from the means of the clones, the average number of tentacles of the two may be used for comparison, although the coefficients of variation may be affected and complications are introduced in the analysis of the data for the problem of inheritance within the clone. The breeding was continued until the latter part of August, when the first experiment was brought to a close, and the polyps on hand were placed in mass cultures and left for two weeks. At the end of this time a single polyp was taken from a mass culture of each of the clones and used as the starting point of a subordi- nate clone (A’ and D’) bred in individual cultures as in the first experiment, except that no Elodea was included in the cultures. Differences in number of tentacles and size. The total number of pedigreed descendants of ‘stem mother’ A obtained before INHERITANCE IN ASEXUAL REPRODUCTION Wy September was 1353, of ‘stem mother’ D, 1395. The distri- bution of the variations in the number of tentacles of the two clones is given in table 10. The means and standard deviations computed from these figures are: Mean o loner Ak 5s ix 6.463 +0 .013 0.7371 Tentacles Clonee sto ote. 5.739+0.011 0.6086 Tentacles Difference (A—D) 0.724+0.017 Tentacles The difference between the average numbers of tentacles borne by polyps from the two clones is here many times its probable TABLE 10 The distribution of variations in the number of tentacles of the descendants of ‘stem mothers’ A and D NEB LRCON TENTACLES: so 5 ci ce na0 5's aie oles siecle celeiele 4 5 6 7 8 9 Number of polyps bearing each num- ber of tentacles Clisai® Al. 6 bie ee ee eee Ne ree 9 93 | 590] 589} 69 3 UTD 1D icc Cece REE CEE SPR ROR ae ee = 30 394 | 883 86 2 0 Percentage of all polyps bearing each number DLO) Ae ood pees 0:6 | “6.9 1-436 143.5.) 75:2 |) 0.2 Ciloiine JD peepee Nie eee nes aoe ene a7) \| Ds) NNO Bya3) | Ora ORAL error and proves that the two clones differed either in their hereditary constitution or in the environment to which they were subjected. The conditions of the experiment seem to rule out the latter possibility and further evidence against an environ- mental cause of the difference was given by further experiments with the clones. The second part of the experiment, beginning with the establishment of the two subordinate clones A’ and D’, from single individuals taken out of mass cultures gave 204 pedigreed descendants from stem mother A’ and 153 descendants of stem mother D’. The number of tentacles of these polyps is shown in table 11. The constants for the distributions given are: 178 K. S. LASHLEY Mean number of tentacles o Glone Aye * |: ater eee PRO See ds 6.907 +0 .026 0.557 Clones fk pe es es Se 5.844+0 .029 0.5387 iDifferentes(AG— Dae ee. os cae 1.0630 .039 After two weeks in mass culture the difference between the two clones persisted and was, indeed, increased by 35 per cent. The significance of this increase will be discussed later. TABLE 11 Variations in clones A’ and D’ NOMBER SOR UE NPROUMR cet. 55:2 crecioe.<.o:0'2 sie widlelmpe tones liens 4 5 6 7 8 SS ee ee Number of polyps GONG vee Fee eh. 5 2 oon ete Beas Hee Bee 3 33 148 | 20 (SGC RD) BESO eee SS oi Pace op Ee eee 2 29 114 7 1 Similar results were obtained from populations of the two clones grown in mass culture. The results from five pairs of mass cultures will be considered. Two battery jars were filled with pond water; to each afew Elodea stalks free from foreign Hydras were added; and each was inoculated with 50 polyps, the one from Clone A, the other from Clone D. Cyclops were added every week for three weeks. At the end of a month 100 polyps were taken from each culture and the numbers of their ten- tacles were recorded. The variations were distributed as shown in table 12, I. The constants obtained from this are: Mean number of tentacles o CloneRAR Cece. 3 553 Ce ee 6.560.056 0.8284 VOU EDD) RAN ao = f.1s2, se aD eS eee 5 .86+0 .033 0.4903 Ditrerences(A:— 'D) sae; 2 eee 0.700.060 Five weeks later samples were again taken from these cultures. Their variations are shown in table 12, II. The constants for these groups are: Mean number of tentacles o ClonevAT eae yee 3s 2 tics nee, tee 6.797 +0 .042 0.5038 CONG eee nok Sons Oe ee 5.457 =0.045 0.5539 Ditterences(At—"))e. 2. (Se See 1.340=0.061 INHERITANCE IN ASEXUAL REPRODUCTION 179 TABLE 12 Distribution of variations in samples taken from mass cultures NAMB ER OR TEN TACIAIS. «2 c1ulae nie sislstcdateretsdeisineticiaieisisiecrereiae ale oe) ¢ 4 5 6 7 8 I After three weeks with plentiful food ClonerAet socio ee ee eee reer sine 1 8) 33 47 10 @loneDs fe Ae Gee ty Ae oes Ace cies 20 74 6 II After five weeks more under same con- ditions COME AR et See he he od NRIs ohare ar yee 1 13 48 2, lone Dice. Re: eee ee Be AS. Se 2 34 34 III After one month with little food Clone A 1 2 see eee 1 il7i 28 4 @lonesD)c (x. 2.82 7 es ee or 26 24 IV After three months in mass cultures (CGlonesAbr sa. link. oe eee 1 12 33 4 GlonerDs. = f5. 200) GSE. See 25 24 1 Polyps from mass cultures to which no food had been added for one month showed the distribution of table 12, III. The constants for these groups are: Mean number of tentacles o Cloves ANT as Fie SOs yh he oo 6.700 .06 0.6403 Clone DS-6 cts eo ee ee EE Grey ne 5.48+0.05 0.4995 Witterences (Ar 9D). 8)... We. 5S. oe ee 1.22+0.08 ; The samples recorded in table 12, IV, were taken from mass cultures three months old. The constants for these groups are: Mean numer of tentacles o SCONE WI i 0 a 6.80+0.06 0.6000 Clone) Peete SNS eee da tees 5.52+0.05 0.5380 WrtierenicerCAR=-O)))i pe eee olan ek. 1.28+0.08 The Hydras in one pair of cultures were given very little food for three months and the water in the cultures was changed very rarely. As a result of these unfavorable conditions the polyps in both cultures were very much reduced in size and in number of tentacles. Clone A was most affected but the difference between the clones persisted even here, as the following constants for these populations show: THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 19, No. 2 180 K.' SA.LASHINY Mean number of tentacles o GClonigyAR ea. nto eee Cee Ore ee os 5.519 +0 .035 0.5325 Clone Drea. -- eee Se Oe se oe sok 5 .346+0 .033 0.4970 Difference i(At— epee. fees s ac ese 0.173+0.048 The second character studied in these clones was the size of the polyps when about one week old. The measurements in- clude 184 individuals of Clone A and 182 of Clone D. The TABLE 13 Distribution of variations in the size of clones A and D CUBIC MILLIMETERS 0.00 0.16 0.32 0.48 0.64 0.80 0.96 J.12 1.28 1.44 1.60 1.76 1.92 | | OF | a: distribution of the variations is shown in table 13. The mean sizes of the two clones are: ClonesA=- ss eens Clovew) se feces 24 28 1 265) 134) ce eS ~I ke 0 28 8 a E: Mean size o cu. mm. cu. mm. ClonevAPe, cok ela toe eee Loe 0.869+0.021 0.4288 Oicinay DE AAS Serene ae an a aes - 0 .3822+0.022 0.1504 - Miwterence:(Ad— 4D) 3s cee, See eee 0.547 +0 .023 The mean age at the time of measurement for members of the two clones was: Gione AT 1 Gays eee eee Clone D 7.8 days - The difference in size is even more pronounced than the differ- ence in the number of tentacles: individuals from Clone A were, on the average, more than twice as large as those from Clone D.? The difference in the appearance of the polyps is shown in figure 4. The numbers measured are relatively small but they extend 3 An attempt was made to determine whether the difference in size was due to a difference in the size of the individual cells, in accordance with the theory of the relation between cell and body size advanced by Popoff (’08). Because of the great mobility of Hydra, accurate measurements could be obtained only for the mature nematocysts. In this character the two clones were identical and if it furnishes an index to the size of other cells the difference between the clones was due to a difference in the number and not the size of the body cells. INHERITANCE IN ASEXUAL REPRODUCTION 181 Fig. 4 Diagram of the size relations of Clones A and D. From camera draw- ings of polyps of mean size. over more than half the period of cultivation so that environ- mental differences should have averaged out. The polyps measured were selected because of their relationships, so that the personal factor, an unconscious selection of extremes, can have played no part in producing the difference. All the differences recorded are between groups which had been kept under environmental conditions as nearly uniform as possible. The polyps in individual cultures were fed in irregular order in order to insure a uniform distribution of food. In the mass cultures the parallel series of the two clones were given uniform treatment and an interchange of water in the parallel cultures was made frequently. The evidence seems to prove conclusively that some internal, hereditary factor caused the differences between the clones. This conclusion is still further verified by an analysis of the data from the individual cultures. In order to test the constancy of the difference, the time during which the clones were culti- vated was divided arbitrarily into intervals of five days and the mean number of tentacles of all buds produced by each clone during each five-day period was computed. Table 14 and figure 182 K. S. LASHLEY TABLE 14 Constants for the number of tentacles of the buds produced by clones A and D during successive five-day intervals from June 25 to August 23 CLONE A CLONE D (A — D) NO. OF PERIOD Mean wese! ten- | . emer Se ten- 7 Tonincles Mise 6.530+0.099 | 0.6056 | 5.500+0.168 | 0.5000 1.030+0.194 7) RE ae 6.115+0.118 | 0.8913 5.462+=0.091 0.6919 0.653 +0.149 aR Moet 6.223+0.046 | 0.7280} 5.479+0.036 0.6204 | 0.744=0.053 4.. 6.142+0.055 | 0.8478 | 5.345+0.027 0.5679 | 0.796+0.061 aT age ieee 6.233 +0 .055 0.7754 5.441+0.036 0.6257 0.7920 .065 Os. Saree 6 .552+0.031 0.6529 | 5.796=0.029 0.5532 | 0.756+0.042 eee 6.795+0.030 | 0.6268 6.103=0.031 0.6386 | 0.692+0.0438 Si. 3 6.681 =0 .033 0.6334 | 5.958+0.023 0.4542 0.723+0.040 Gs. 6.4389 =0 .036 0.6499 5.757 =0 .029 0.5078 0.682 +0 .046 TOS 6.236 =0.038 0.7502 | 5.787+0.030 | 0.4980 | 0.449+0.042 Le caer 6.396 +0 .043 0.5958 5.787+0.036 | 0.5376 0.9720 .056 | iat isthe 6.3840 .090 0.6836 5.775+0.090 0.4875 0.609+=0.128 5 show the result of this analysis. Throughout the entire experi- ment the difference between the clones persisted. By July 25 Clone D had increased in the average number of tentacles of its buds until it equaled the earlier average of Clone A (June 30) but this was accompanied by a corresponding increase in the average of Clone A. The figure shows that the difference re- mained fairly constant in spite of wide fluctuations in the absolute values of the means of the clones. Other differences between the clones. There is a marked differ- ence between the clones with respect to the age at which repro- . ductive maturity is reached, the age at which the parents produce their first bud. This is shown in the following tabulation: Mean age of parents when No. of Jirst bud was produced o parents Glone- Ac see a0 4.806+0.103 days 2.422 248 Clone yD) as roe ate 3.748+0.074 days 1 755 253 Difference (A — D) 1.058+0.127 days The rate of reproduction of the clones differed slightly, that of Clone A being somewhat greater than that of Clone D. The difference, based upon 33 parents of each clone which produced more than 10 buds is 0.0542 buds per day. This difference is INHERITANCE IN ASEXUAL REPRODUCTION 183 ig. 20 30 40 50 60 70 BO 90 “100 Fig. 5 The mean number of tentacles of the buds produced by Clones A and D during successive intervals of five days. The mean number of tentacles of Clones A’ and D’ are included at the right as these form a natural continuation of the earlier experiment. not greater than its probable error and is probably not signifi- cant. The equality in reproductive rate is shown by the fact that while no attempt was made to obtain equal numbers in the two clones during the experiment, the numbers bred were, after two months, almost equal (1353 and 1395). Other differences between the clones which can not be ex- pressed quantitatively were apparent in the living animals. Clone D was, as a rule, somewhat darker in color than Clone A and somewhat more resistant to unfavorable conditions. Polyps of Clone A showed a greater tendency to cling to the surface film and were more active than those of Clone D. Nature of the difference between Clones A and D. The fore- going data prove that the two clones studied are distinct through the presence of some internal, hereditary factor. The ‘Reak- tionsnormen’ of the two are different. But whether this differ- ence is genotypic in the sense of being the result of a fixed con- stitution of the germ-plasm or whether it is due to a spurious heredity like that by which the green color is transmitted is not clear. The agents, other than the hereditary constitution, which might produce such clonal diversities are the direct action of the 184 K..S. LASHLEY environment, the persistent effects of former diversities of environ ment, variations in the commensal relations of the polyps and their zoochlorellae, parasitic infection of one clone, and differ- ences in clonal age from the fertilized egg. The first of these is eliminated by the methods of the experiment. It has been shown that the after effects of injury and ‘depression’ are only temporary. Experiments attempting to produce permanent modification by extremes of temperature, chemical agents, and artificially induced ‘depressions’ have been unsuccessful ;- diver- sities are readily produced but they persist for only a few days. Possible variations in the commensal relations of H. viridis are more difficult to control. Microscopic examination of the two clones failed to reveal any difference in the green bodies of the two clones. As a test of the influence of commensalism upon variations I partially removed the zoochlorellae from some individuals of Clone L (see below) by the method devised by Whitney (06-08). During immersion in glycerin the polyps became smaller but when restored to normal conditions they ate regularly and resumed their original size before beginning to bud. The buds, practically white in color, did not seem other- wise different from the normal members of the clone. I never succeeded in getting out all the zoochlorellae from the polyps so that not enough buds for statistical study could be obtained before the polyps resumed their normal color. Five parasites of Hydra have been reported: Amoeba hydrox- ena Entz, Balantidium hydrae Entz, a species of Ophryoglena (Entz 712), Trichodina pediculus Ehr. (Clarke ’65), and Kerona polyporum Ehr. The true parasitic nature of only one of these, A. hydroxena, has been demonstrated. Trichodina did ‘not occur in any of the pedigreed cultures recorded here, although it is common in wild populations. In October, 1913, an epidemic of A. hydroxena broke out in my cultures, destroying all of them. The symptoms of infection by this parasite are easily recogniz- able and there is no possibility that it occurred in the earlier cultures. Kerona occurs rarely in wild populations here but did not appear in my cultures; the other two parasites have INHERITANCE IN ASEXUAL REPRODUCTION 185 never been seen in this locality. The fungus mentioned by Rand (99) is evidently not a parasite of Hydra. Finally, there is no direct evidence bearing upon the question of the effects of the age of the clone upon its variations. It has been impossible to secure clones of known age since in this locality sexual reproduction in H. viridis seems to be almost completely suppressed. Hydras from wild populations have been examined at all seasons during the past three years and in this time not one with ovaries and only seven with spermaries have been found. The treatment by which Whitney (’07) induced sexual repro- duction was tried but without success. These facts seem to justify the conclusion that sexual reproduction plays but little part in the life history of the green polyp. Fig. 6 The number of polyps (ordinates) which died at the ages in days recorded on the abscissae, during Hase’s experiments. (——) H. fusea, (----) H. grisea. - In the development of theories of ‘life cycles’ in micro-organisms the phenomena of ‘depression’ in Hydra have had a prominent place. R. Hertwig, in particular, has sought to emphasize the relations of ‘depression’ to sexual reproduction, but the work of Frischolz (’09) seems to prove that no such relation exists. Practically all studies of depression have been carried out with polyps in mass cultures and there is no evidence that the epidemics of depression reported were not due to environmental rather than to internal factors. The ease with which depression may be induced by unfavorable environment and cured by putting the polyps in clean water makes it practically certain that depression is a pathological condition. The only other work bearing upon the question of age is that of Hase, who studied the age of individual polyps. He found a mean age of 55.2 days for H. fusca and 94.8 days for H. grisea, with the distribution 186 KE. S./ LASHLEY of ages at death shown in figure 6. The irregular form of the curves and the fact that the vast majority of the polyps died at a relatively early age scarcely speaks in favor of an ‘Altersschwache.’ The curves are similar enough in form to suggest that the deaths were due to two periods of unfavorable conditions (a and b) in his cultures. At no time have I observed an epidemic of depression in the individual cultures belonging to the same clone; less than 1 per cent of the polyps have shown depression and always the closest relatives of these remained normal, a condition which is not at all in harmony with theories of clonal senescense. Further, during the four months that the clones were kept under obser- vation, including more than 20 asexual generations, there was no significant change in the character of either clone. The repro- ductive rate, the first character modified by a reduction of the vitality of the polyps, was as high at the end of the experiment as at the beginning. This furnishes some evidence against the belief that the diversities are due to the clonal age but it is not conclusive. The existence of other races A comparison of the standard deviations of the Clones A and D reveals the fact that clone A was always the more variable of the two. This difference suggested that Clone A was not pure and an examination of other evidence quickly confirmed this view. The variations of the clone tended to a dimodality which does not appear in any other race studied and the greater varia- bility and dimodal form of the variation curve did not persist in Clone A’ which was descended from a single polyp taken from Clone A. The records of the clone were reviewed in order to test whether the greater variability was characteristic of the race or was the result of the inclusion of diverse types as is sug- gested by the condition in Clone A’. One subordinate clone represented by the direct line of descent A1A2blala2ala (the 4 Agar (’14) has shown that diverse clones produced by parthenogenetic re- production may be produced by different eggs hatched at the same time. The differences here are clearly not due to the ages of the clones. Lang (’92) found that only one germinal layer of Hydra takes part in the formation of the buds, and this work, while unconfirmed, suggests that the differences between partheno- genesis and budding may not be so great as is generally supposed. INHERITANCE IN ASEXUAL REPRODUCTION 187 successive buds of alternate generations being labled with the series of letters and numerals) showed an average number of tentacles considerably lower than that of the remaining por- tion of the clone. The polyp A/A was recorded as small, with abnormal tentacles, rapidly becoming normal. The mean num- ber of tentacles of the fraternity from which it came was 6.809 +0.062, that of its immediate progeny was 6.200 +0.023, giving a reduction of 0.609+0.067 tentacles in one generation. 60} percent. 1\ Fig. 7 The distribution of variations in the number of tentacles of Clone A ( ) and of the subordinate clones A1A (———) and A — (A1A) (----~-). The mean of all the descendants of AJA (437) was 6.121= 0.020: that of the remainder of the clone was 6.624+0.013 giv- ing a difference of 0.503 +0.024. The distribution of variations in the two subordinate clones, separately and combined, is shown in figure 7. The dimodality of Clone A is clearly the result of the combining of these two groups (neither subordinate clone breaks up upon further analysis nor does any other method of dividing Clone A reduce its dimodality. The appearance of this diversity gives no evidence of a cumulative inheritance of variations. ‘The change was quite sudden, resulting in a difference after one generation as great as the difference in any later gener- 188 K. S. LASHLEY ation. The individual A7A must be looked upon either as a mutant or as, more probably, a polyp of another clone introduced by accident into the culture.® It seems to be hereditarily differ- ent both from Clone D and from the remainder of Clone A. Diverse races which can be recognized at a glance are extremely rare in the populations near Baltimore. Several times fifty Libs Fig. 8 Diagram of the size relations of two clones kept under similar con- ditions, L and F. From camera sketches of individuals of estimated average size. or more clones have been started with polyps from different localities but only twice have distinct races appeared which were recognizable within a few generations. ‘The first of these were Clones A and D. In August, 1913, a small polyp was found which produced very small buds. At first it seemed unhealthy but the buds increased in size and began to multiply rapidly. Un- fortunately the culture could not be continued and the clone was 5 The opportunity for contamination was given by the use of Elodea stalks in these cultures, as in the early part of the experiment no certain method of free- ing them from foreign Hydras had been devised. INHERITANCE IN ASEXUAL REPRODUCTION 189 TABLE 15 A comparison of the mean number of tentacles of clones T, L, and R =A Bl NO. OF HSS OF STEM “as TORE Ore oe ee RM ce 1c ee 6 .553 +0 .058 56 Gres See tih : 6 .282 +0 .049 46 Reo te 8 .650 +0 .081 40 MWe rah, 5 c-cd .513+0 .033 154 | ib cage cee .184+0 .033 152 Difference (7’— L) 0.328+0.046 lost before many individuals had been obtained. However, the difference between this and another clone, LZ, kept under the same conditions was so marked that it seems certain that the. small race represented a distinct genotype. The size relations of the polyps of the two clones is shown in figure 8, taken from camera lucida sketches of estimated average individuals. More frequently the differences are less pronounced. Table 15 gives the constants obtained for three clones bred during July and August, 1913. Clones 7 and Z were continued after R was discarded and the constants for their full numbers are given separately. They were grown in individual cultures under parallel conditions as in the first experiment and seem to be hereditarily distinct. These are the only races which I have cultivated under exactly parallel conditions and no evidence upon the number and variety of such races is available. Varieties of Hydra in the earlier literature. The extensive literature dealing with Hydra gives frequent suggestions of the existence of local varieties of the three generally recognized species, and one or two descriptions seems really to establish their existence. The statements concerning the size of H. viridis made by Baker, Trembley, Rosel, Pallas, and Kastner differ considerably but are not conclusive. Marshall (’82) de- scribed a variety of H. viridis from brackish water which remained distinct from the fresh water forms even after cultivation for 190 ES: LASHLEY many generations in fresh water. The systematic literature and the studies of gonad production by Brauer, Downing Nuss- baum, Weltner, and Koelitz, suggest the existence of monoecious and dioecious varieties. The experiments of Tower show two types of H. viridis differing in their reaction to the ultra-violet rays (?) and in the time required for regeneration. Annandale has described a variety of H. grisea from India which is char- acterized by having a four-tentacled winter form producing gonads. Whitney has been able to establish varieties of H. viridis without green bodies. Summary To sum up the present section of the work: It has been found that within a wild population of H. viridis there are hereditarily diverse races which differ in their number of tentacles at sepa- ration from the parent, in their size at a given age, and less cer- tainly in other characters. The differences between such races are permanent so long as the races are kept under the same environment. The evidence favors the view that the differences are truly genotypic (with the reservation that they are possibly the result of differences in the age of the clones). IV. INHERITANCE OF VARIATIONS WITHIN THE CLONE Inheritance of variations within the clone may be tested in two ways; first, by an analysis of the resemblance between parent and progeny by statistical methods; second, by a con- tinued selection of variates, which should change the type of the clone if the variations are inherited. As has been shown in the discussion of Hanel’s data, selection of variates within the clone seems, on the average, to have no effect, yet there is usually a positive correlation of parent and progeny with respect to the selected character. Pearson has subjected Johannsen’s data for the selection experiment with beans to a similar analysis and 6] have made many attempts to repeat Tower’s experiments, using different types of ares, with and without interposed glass, but have never obtained the sloughing of the ectoderm which he describes. INHERITANCE IN ASEXUAL REPRODUCTION 191 has shown that in these pure lines there is likewise a correlation between parent and progeny. His explanation of this seemingly anomalous condition and the criticism which has been urged against selection experiments in general where Vilmorin’s method is employed is based upon the small numbers selected. Pearson (10), discussing Hanel’s results, says: In selecting a few isolated individuals in each generation, where non-hereditary influences are so influential, we may break the influence of heredity at each step, and since such influences are equally effective with heredity, the chances are that we will do so once in every two selections. Only by taking large numbers of the high and large num- bers of the low would it have been possible to average out the effects of environmental changes. The only ways in which this criticism can be met are by very extensive selections involving large numbers of individuals, or by a study of variation and environment which shall make possible a differentiation between heritable and non-heritable variations, upon some other basis than that of ancestral correlation. Resemblance of close relatives within the clone Number of tentacles. In all breeding experiments the polyps have been so recorded that the number of tentacles of the buds can be compared with that borne by their parents, both at the time when the buds were produced and when the parents themselves were buds. Comparisons of both numbers must be made in order to answer two distinct questions: (1) do the offspring resemble their parents when the latter were in the same stage of development: (2) are the variations of the parents during their development transmitted? To test the first question the correlation between the initial number of tentacles of the parents and that of their buds was computed. Since there is reason to believe that Clone A is impure, it was divided into the two subordinate clones mentioned earlier and the constants were computed for these. If there is a cumulative inheritance of slight variations this division should affect only the probable errors of the correlation constants. The 192 K. §. LASHLEY coefficients of correlation obtained in this way are given in table 16. Only one of these, that of Clone D’ is more than twice its probable error, and this one is negative. The others are all too small to have any significance. The large negative corre- lation of Clone D’ is the result of the inclusion of a single parent (the stem parent of the clone) which produced a large number of progeny with few tentacles and was thus heavily weighted in the computation of the coefficient. The Clones A — (A1A) and D contain such large numbers both of parents and offspring that it is almost certain that the low correlation is not the result of chance, but truly expresses an almost total absence of similar variations in parent and progeny within the clone. In so far as the coefficient of correlation is a reliable test of heredity we may conclude that there is no inherit- ance of variations in the initial number of tentacles of Hydra viridis. For Clone D the fraternal and grandparental correlations were likewise computed; they are given in table 17. Like the parental correlation, the grandparental is too small to have significance. TABLE 16 Correlation of the initial number of tentacles of parents and progeny within the clone r es OF PARENTS |NO. OF PROGENY Clouse ih ees Le. 0.0038+0.018 251 1395 GlonesAe= (AGA) a ce, ase 8 0.0011+0.023 859 lone MeiA- a ae ee | 0.0342 +0 .032 439 pi Fiitsg. 5 wey Ey Se wg ee 0 .2420+0.051 18 153 lorie Rates ss Spelt CLE ee 0.0310 0.047 28 204. Olena Ee ee EE EY a 0.0750 +0 .054 51 154 TABLE 17 Ancestral correlations, clone D ; | ? | xo. oF panexns [8° OF PROGENY Barentals. 25 .0ret eee cee 0.0038+0.018 251 1395 Grandparental: ssc see: eee | —0.0495+0.018 68 1307 iperial..6 eee eee ee 0.07700 .006 10766 INHERITANCE IN ASEXUAL REPRODUCTION 193 The fraternal is only slightly greater and its value is doubtful. It will be considered again in the discussion of the cause of an- cestral correlation within pure lines and clones. The second question, do the parents transmit to their buds the characters which they have when the buds are formed, in- volves one of two concepts. Either the effects of environ- mental action must be transmitted or the organism must con- tinually change in its hereditary potentialities during its develop- ment in order that its transient characters shall be inherited. Something of the latter conception seems to be implied in Pear- son’s doctrine of homotyposis. TABLE 18 Correlation between the original number of tentacles of buds and the number borne by the parents at the time when each bud was produced 7 NO. OF PARENTS |NO. OF PROGENY Po) 1D oe ee 0.096 +0 .016 251 1395 (CIIGTINE Wao Scan creme 0 .240+0 .009 242 1353 Clone A. Grandparental.... 0 .229+0.013 1094 The correlation of the number of tentacles of the buds with that of the number borne by the parents when each bud was produced has been computed for Clones D and A; these are given in table 18. The high correlation of Clone A is obviously the result of the inclusion of the two diverse strains but a compari- son of the parental and grandparental correlations within this clone shows that there is a slight positive parental correlation which is not the result of the mixture of the two types. (The parental correlation would not be higher than the grandparental if the inclusion of diverse types were the only cause of correlation). This correlation is certainly very small, and that of Clone D is also too small to have significance when considered alone, but the fact that these correlations are greater than the ones obtained for the initial number of tentacles indicates that there is here some factor tending to produce a resemblance between the mature parent and its buds. This is also the impression 194 K.' So LASHINY given by the average parental correlation of 0.101 found for Hanel’s clones. What is the significance of such a small coefficient of corre- lation in an organism so subject to environmental influence as is Hydra? Pearson holds that the presence of variations due to environment would tend to obscure the real correlation be- tween parents and offspring and hide any real inheritance which might exist. My data upon the relations of variation to environ- mental changes indicates that the latter may be more effective in producing a likeness between close relatives than in obscuring such a likeness. Some conditions producing like variations in parent and offspring in Hydra have been considered already. Any great diversities in the conditions of the cultures would re- result in a correlation between parent and progeny, even though there were no inheritance of the variations studied. Such con- ditions have been found by Agar in daphnids and plant lice and probably account for the ancestral correlations found by Warren in these forms. The production of a correlation in Hydra by the action of diversities of environment may be illustrated by a consideration of the change in the mean of clones during long periods of culti- vation. Figure 5 shows that the mean number of tentacles of the buds produced by Clones A and D during the first few weeks of cultivation was low; that it increased gradually during the first six weeks, and then decreased again. All the buds produced during each of these five-day intervals were correlated with one another. This gave the following results: . Coefficient of correlation No. of pairs (lone PAR pet A Nate SAS eS 0.0774=0.0015 95141 Clone TD 6 Sanrio e 0.1313=+0.0014 101872 Correlation of all buds produced in each five day period with all produced in the preceding period (a time interval correspond- ing to a full generation) gives for Clone A a correlation of 0.048 + 0.001 which is greater than any of the parental correlations found for the initial number of tentacles. INHERITANCE IN ASEXUAL REPRODUCTION 195 These correlations show a resemblance between the buds produced within a limited time as great as that found between the closest relatives, although these buds were no more closely related to each other than to those produced during other five day intervals. This same effect would be visible in the cor- relation between parent and progeny whenever several gener- ations are included in the same correlation table, as is necessary in the case of Hydra. Such effects of environmental action seem adequate to account for all the coefficients of correlation given by Hanel’s data and for the very slight positive correlations found for the initial number of tentacles in my own clones. The fact that the number of tentacles of successive buds increases with the increase in the number of tentacles of the parent accounts for the slightly larger correlation found between the number of tentacles of the parents and offspring recorded when each bud was produced. Whether the increase in the number of tentacles of the buds is an inherit- ance of the variations taking place in the parent with growth, or is only a temporary effect of the increased vigor of the parent, must be tested by the success or failure of an attempt to modify the character of the race by the continued selection of variates. The effects of selection within the clone The second method of testing inheritance, that of continued selection of variates, offers a good many practical difficulties in Hydra owing to the sensitiveness of the polyps to environmental changes. A specimen of H. viridis, collected in the late summer of 1914, was used to found a large clone. Its progeny were bred in individual cultures until 85 members of the clone were obtained. These gave a mean of 6.141+0.058 with the dis- tribution shown in table 19. From this clonal population 25 polyps with seven tentacles and 25 with six or less were selected. Each was kept until it produced a bud varying from the mean in the same direction as itself. This bud was then selected and kept in the same way until it in turn produced a bud varying in the same direction, and this selection was continued for three THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VoL. 19, No. 2 196 K. S. LASHLEY TABLE 19 Distribution of variates in the clone from which the fifty parents of the selected lines were taken. The polyp from which this clone was derived bore 9 tentacles Number of tentacles.... ........ 3 re 5 6 7 Ninnber of polyps=.-2- 3". 4. | 1 | 2 9 45 2 85 wes. Se ee | | 6.14140.058 months. At the end of this time a record was kept of all the progeny of the last selected generation in each of the 50 lines. The continued freezing of the food pond made it necessary to bring the experiment to a close when an average of 12 buds had been obtained from each of the members of the last selected generation. The selection covered an average of 6.08 gener- ations in the group selected for seven or more tentacles, and of 7.92 generations in the group selected for six or less. The average number of tentacles of all selected generations of the plus selected group was 7.008, that of all selected generations of the minus selected group was 5.560, giving an average of 1.448 tentacles as the amount of difference per generation between the selected ancestors of the two groups. The first effect of the selection was a marked reduction in the vitality of the group selected for a’ small number of tentacles. The rate of budding of the group was reduced and some of the polyps showed symptoms of slight depression. Four of the buds of the last selected generation of this group, after maturing and producing from four to eight buds, went into a depression which lasted for a week or more and were revived only with difficulty. The reduction in the vigor of the minus selected group intro- duces a complication into the study of the effects of selection, for the transmission of reduced vitality, or of characters depen- dent upon reduced vitality, is not a proof of heredity, unless, indeed, the changed condition prove quite permanent. The total number of progeny obtained from the last selected generation was 583, of which 309 were from the parents selected for a large number, and 274 from those selected for a small number of tentacles. The distribution of variations in the two INHERITANCE IN ASEXUAL REPRODUCTION 197 groups is shown in table 20. The offspring of the plus selected group have an average number of tentacles 0.093 + 0.035 higher than that of the minus selected group, which as an effect of selection is scarcely significant. It is about what would be expected for a strength of heredity expressed by the parental correlation of 0.01 (table 21). Whether this effect of selection proves that the progeny inherit one one-hundredth of the variation of their parents, or is merely the effect of a temporary modification in the vigor of the selected groups may be tested by a comparison of the successive offspring of the last selected generation. Such a comparison shows that the entire difference between the two groups appeared in the first six buds produced. The average number of tentacles of successive buds in the two groups increased TABLE 20 Variations in the number of tentacles of the offspring of the last selected generation of polyps selected for a large and a small number of tentacles 4 5 6 7 8 MEAN Selected for a large number. .. 7 89 204 9 6 .695+0 .023 Selected for a small number.. . 2 9 91 165 7 6 .605+0 .026 Difference in the direction of REL CUO Mee ae Nake AS cicavor. 0.095=+0.035 TABLE 21 Theoretical effect of selection of parents differing by 1.45 tentacles continued for Fn generations when the strength of inheritance is that indicated by the coefficients of correlation given COEFFICIENTS OF CORRELATION 0.50 0.10 0.05 0.01 Difference at successive gen- erations. Parental generation......... 0.000 0.000 0.000 0.000 F, 0.725 0.145 0.073 0.015 F, 1.098 0.276 0.141 0.029 F; 1.274 0.393 0.207 0.043 Fy, 1.362 0.498 0.269 0.057 F; 1.406 0.594 0.328 0.071 F; 1.428 0.689 0.384 0.084 198 K. S. LASHLEY TABLE 22 Averages of the first six and of all later buds produced by polyps selected for a large and of those selected for a small number of tentacles AVERAGE OF FIRST AVERAGE OF ALL SIX BUDS LATER BUDS Parents selected for a large number of LENTACIGH oe eee ene. eo sw ae oe ee 6.677 =0 .029 6.712+0.030 Parents selected for a small number of bentacles:tas. Seen. tue JOS CLE B74) GEAGO = 03034 6 .782+0 .037 Difference in the direction of selection...) 0.217+0.044 — 0.0700 .047 as usual but those of the minus selected group increased more rapidly and to a greater extent than that of the plus selected one. This is shown in figure 9. The averages of the first six and of all later buds of the two groups is given in table 22. The differ- ence in favor of the plus selected group in the first six buds is Fig. 9 The average number of tentacles of the successive buds produced by the last selected generation in the selection experiment; ( ) ancestry selected for a large number of tentacles; (-—-—-—) ancestry selected for a small number of tentacles. 0.217 =0.044. There is no significant difference between the later buds of the two groups. Since not all parents produced the same number of buds it seemed possible that this decrease in the difference between the groups might be due to a larger percentage of the progeny of more vigorous polyps among the buds produced after the sixth, but INHERITANCE IN ASEXUAL REPRODUCTION 199 the data given in figure 9 shows that this is not the case. There is no difference between the averages of the seventh and of the eighth buds of the two groups and all parents produced at least eight buds. The likeness of this result to that obtained from regenerating polyps (fig. 3) is very striking and there can be little doubt that it is due to the same cause, a reduction in the vitality of one of the groups compared. Selection of polyps with few tentacles resulted in the selection of those with low vitality and as soon as these were given time to regain their health they produced buds varying around the mean of the race. Selection produced no permanent change in the type of the clone studied. Inheritance of size From the measurements of the size of polyps in Clones A and D the correlations in size between parents and offspring and grandparents and grandchildren within these clones were com- puted. These coefficients are given in table 23. In every case they are positive and the parental correlation of Clone A is very high, more than six times that of Clone D. The grand- parental correlations are negligible but the low grandparental TABLE 23 Ancestral correlations for size COEFFICIENTS OF COEFFICIENTS OF CORRELATION REGRESSION Clone D Parent and Ofspmimemt 1003.50) 02.86. 7M 0.0580 .035 0.056 Grandparent and grandchildren........ 0 .018+0.036 0.020 Clone A arent ana Ons prince aimee ete ee 0 .358+0 .030 0.285 Grandparent and grandchildren........ 0.030+0 .036 0.048 Clone A 1A IRATenb and. OfSDEMcCa een eee es 0.009+0.106 Clone A — (A1A) RArentyan G OLS prines ye see ee 0..255+=0 .063 200 kK. S. LASHLEY correlation of Clone A shows that the high parental correlation is not wholly the result of the mixture of two races in Clone A. Computation of the parental correlation for the two subordinate clones confirms this, giving the following coefficients: Coefficient of correlation ClonexA AR ee be esc. 5 RS eee fede SoS gt ee 0 .009=0.106 Glorie (Aso CANPAN ea Be 8s coi ca.s cei AA eer cere ree 0 .255+0 .063 After this division the correlation is still significant and is four times as great as that found for Clone D. An examination of the data for possible environmental causes of correlation ve) 6 cumm. 10 20 30° 40-5 50° 60. 709 Days Fig. 10 Changes in the average size of the buds produced during successive ten-day intervals by Clones A and D. The ordinates represent the mean sizes in cubic millimeters of all measured buds produced during each ten-day period; the abscissae, the successive ten-day periods. gives the result shown in figure 10. As in the case of the number of tentacles, there was a considerable change in the mean size of the buds produced at different times during the experiment. The correlations between all buds produced in each of the five-day intervals were: Coefficient of currelation Clones so less sce ee See eee SAE « kent Ree 0.0118 Glone vA FCAT AN is ee pecs See ite eter. phe oie a cree teere Cina 0.0952 There is here the same difference in the size of the correlations of the two clones which appeared in the parental correlation but INHERITANCE IN ASEXUAL REPRODUCTION | 201 the correlation due to the changing clonal mean is too small to account for the parental correlation. No adequate data for a further analysis of the correlation in size is at hand, and it may be that variation in size within the clone is actually inherited but, although conclusive evidence against this is lacking, too much trust should not be placed in the parental correlations. The following points, while based only upon general impres- sions gained during the experiments and subject to correction by further experimental test, indicate some of the factors which may have been instrumental in producing a high parental cor- relation for size. Size is a character which is modified much more readily and quickly than the number of tentacles by changes in the environment. Hydras, placed in a 0.1 per cent solution of NaCl, in a very few days become dark in color, small, and produce small, dark-colored buds. Similar changes in size follow extreme changes in temperature. Upon the restoration of opti-. mum conditions the normal size is resumed very quickly. Star- vation is effective in much the same way but to a lesser extent. When a Hydra has been injured slightly, or has passed through a slight depression it grows smaller and produces small buds but eventually both buds and parents resume the normal size of the clone. The size, indeed, seems to be a matter of the immediate state of nutrition of the polyps. Clone A was more readily modified by such agents than was Clone D. This is shown by the following data for the number of tentacles of polyps from mass cultures with and without food for three months: 8-month mass cultures With food Without food Difference TEE: Ran a a 6 .80+0.06 5.52+0.04 1.28+0.07 Sh ag Dae BO De) eat ee eae 5.52+=0.05 5.384+0.03 0.18+0.06 and by the changes represented in figure 10. Corresponding to this fact, the correlations between the relatives in Clone A are higher than in Clone D, which justifies the suspicion that the correlation between parent and offspring for size within the clone is really due only to the action of the environment. 202 K. S. LASHLEY V. DISCUSSION OF RESULTS The experiments reported show that populations of Hydra viridis consist of races which have different hereditary consti- tutions. The diversity between two such strains persisted for so long as they were kept under observation (1438 days) and so long as the different strains were kept under similar and favor- able conditions they showed no tendency to approach each other in character. The distinguishing characters of the different races studied were not, however, fixed in the sense of remaining constant through fluctuations in the environment but under- went changes corresponding to changes in the environment. In general, the diverse clones responded to such changes in the same way and to the same degree, although in the face of very unfavorable conditions the larger strain was most affected and under conditions of almost complete starvation the strains became much more similar. Little evidence has been obtained as to the cause and funda- mental nature of the difference between the clones. The diverse characters noted, number of tentacles, size, color, and age at which asexual reproduction is begun, may all be modified by changing the environmental conditions and the changes thus produced in the first three characters mentioned are correlated in the same way that they are in the diverse races (large size, many tentacles, and light color occur together) so that it is pos- sible that the diversities in these three characters are due to a difference in some single set of physiological processes. Failure to obtain sexual reproduction in H. viridis has made it impossible to determine the relation of the diverse races to gametic processes and to the supposed life cycle of Hydra but there is certainly no relation between the diversity of the clones and the phenomena of ‘depression’ which have been thought to mark periods in the life cycle. The existence of diverse races of Hydra is in accord with the results of Jennings, Woltereck, Shull, Whitney and Agar gained from the study of clones of other invertebrates and, indeed, the volume of evidence from zoological and botanical literature INHERITANCE IN ASEXUAL REPRODUCTION 203 leaves no doubt that the existence of diverse races within the species is a general condition in all phyla. The problem of inheritance of variations within the clone presents much greater difficulties and there is much conflict between the results of different investigators. The work of Whitney has shown that in Hydatina diverse strains may arise in a clone descended from a single fertilized egg and Calkins and Gregory report similar results for Paramecium. In these cases there is, however, no intimation that the inheritance of variations is a general characteristic of asexual reproduction or that the change is the result of the accumulation of slight variations. The four studies in which there is an appearance of inheritance of continuous variations within the pure line or clone are those summarized by Pearson in 1910; the studies of Johannsen on beans, Warren on Daphnia and Hyalopterus, and Hanel on Hydra. In all this work the evidence for inheritance can be drawn. only from the ancestral correlations, while the evidence from the effects of selection seems to point the other way. The question of the relative values of the coefficient of correlation and of selection experiments hence becomes of great importance. In Agar’s recent study of inheritance in parthenogenesis, where great precautions were taken to rule out the influence of environmental agents, there is no significant correlation between parent and progeny within clones of Cladocera and sufficient evidence of environmental causes of correlation is presented to account for Warren’s results with Daphnia. For Aphids he finds, with Warren, a slight ancestral correlation but the evi- dence for an environmental cause of this correlation, while per- haps not absolutely conclusive, is sufficient to make the ancestral! correlation in these forms of very doubtful value as an index of inheritance. Ewing’s selection experiments with Aphis avenae offer further evidence against the inheritance of variations in these forms. The results recorded in the present paper are quite in accord with those of Agar on Cladocera and show that for Hydra also the ancestral correlation is untrustworthy as a measure of inheritance. 204 K. S. LASHLEY There remain only Johannsen’s data on Phaseolus giving evidence of an ancestral correlation within the pure line. The evidence adduced by Pearson indicates only a slight correlation here and the recent work of Harris (’12) upon the transmission of the effects of unfavorable cultural conditions indicates that the real cause of this correlation is likewise environmental. VI. SUMMARY 1. Populations of Hydra consist of hereditarily distinct strains which differ in initial number of tentacles, size of body, color, age at which asexual reproduction is begun, and perhaps in other characters. 2. In the absence of selection these strains remain distinct. 3. Within populations there is a correlation between the characteristics of parents and progeny and of other close relatives. which is largely due to the existence of these diverse strains. 4. Within the clone there is no significant correlation between the variations of close relatives in the initial number of tentacles. 5. Within the clone there is a slight correlation between the number of tentacles of the buds and the number of tentacles which their parents bear when each bud is produced. ‘16. Diversities of environment tend to produce like variations in parents and offspring and this likeness tends to disappear when the environmental cause is removed. The existence of such environmental agents is sufficient to account for the ances- tral correlations found, even though there is no inheritance of variations. 7. Continued selection of variates in tentacle number results in changes in the vigor of the selected groups. This results in an apparent diversity of the differentially selected groups but the diversity persists only during selection and disappears at once when selection is discontinued. Variations in the number of tentacles of Hydra viridis are not inherited. 8. There is a positive correlation between the variations in the size of parent and offspring within the clone. No statis- tical evidence of an external cause of this correlation is presented INHERITANCE IN ASEXUAL REPRODUCTION 205 but from general considerations it seems probable that this, like the correlation of variations in the number of tentacles, is due wholly to the similar action of environmental agents upon parent and offspring. LITERATURE CITED Acar, W. E. 1913 Transmission of environmental effects from parent to off- spring in Simocephalus vetulus. Phil. Trans. Roy. Soc. London, vol. 203 B, pp. 319-351. 1914 Experiments on inheritance in parthenogenesis. Ibid, vol. 205 B, pp. 421-489. ANNANDALE, N. 1906 The common Hydra of Bengal, its systematic position and life history. Mem. As. Soc. Bengal, vol. 1, pp. 339-359. 1907 Notes of the fresh-water fauno of India. No. 10. Hydra orientalis during the rains. Journ. As. Soc. Bengal. vol. 3, pp. 27-28. Brepét, .M 1912 Sur la nomenclature des Hydres. Zool. Anz., Bd. 39, p. 602. Braver, A. 1908 Die Benennung und Unterscheidung der Hydra-Arten. Zool. Anz., Bd. 33, pp. 790-792. Catxins, Gary N., and Gregory, Louise H. 1913 Variation in the progeny of a single ex-conjugant of Paramecium caudatum. Jour. Exp. Zodl., vol. 15, pp. 467-525. Cxiarke, Jas. H. 1865 The anatomy and physiology of the vorticellidan para- site (Trichodena pediculus Ehr.) of Hydra. Mem. Boston Soc. Nat. Hosts; vole, p. 11. Davenport, C. B. 1904 Statistical methods. New York. Entz, Giza 1912 Ueber eine neue Amdbe auf Siisswasser Polypen (H. oligactis Pall.) Arch. f. Protistenkunde., Bd. 27, pp. 19-45. Ewina, H. E. 1914a Pure line inheritance and parthenogenesis. Biol. Bull., vol. 26, pp. 25-35. 1914b Notes on regression in a pure line of plant lice. Ibid, vol. 27, pp. 164-168. Fries, 8S. 1879 Mitteilungen aus dem Gebiet des Dunkelfauna. Zool. Anz., vol. 2, p. 154. Friscuyoiz, E. 1909 Zur Biologie von Hydra. Biol. Centralb., Bd. 29, p. 182. Hanet, Exise 1908 Vererbung bei ungeschlechtlicher Fortpflanzung von Hydra grisea. Jenaische Zeitschr., Bd. 43, pp. 321-372. Harris, J. A. 1912 A first study of the influence of starvation of the ascendants upon the characteristics of the descendants. Amer. Nat., vol. 46, pp. 313-340. 206 K. S. LASHLEY Hast, A. 1909 Ueber die deutschen Siisswasser-polypen Hydra fusca L., Hydra grisea L., und Hydra viridis L. Arch.‘f. Rass. u. Ges. Biol., Bd. 6, pp. 721-753. Jennines, H. S. 1908 Heredity, variation, and evolution in Protozoa. II Heredity and variation in size and form in Paramecium, with studies of growth, environmental action, and selection. Proc. Amer. Phil. Soc., vol. 47, pp. 393-546. 1911 Computing correlation in cases where symmetrical tables are commonly used. Amer. Nat., vol. 45, pp. 123-128. JOHANNSEN, W. 1903 Ueber Erblichkeit in Populationen und in reinen Linien. Jena. 1911 The genotype conception of heredity. Amer. Nat., vol. 45, pp. 129-159. 1913 Elemente der exakten Erblichkeitslehre. Jena. Lanc, ALBERT 1892 Ueber die Knospung bei Hydra und einigen Hydro-poly- pen. Zeit. f. wiss. Zool., Bd. 54, pp. 365-385. MarsHALi, W. 1882 Ueber einige Lebenserscheinungen der Siisswasser-polypen und iiber eine neue Form von Hydra viridis. Zeit. f. wiss. Zool., Bd. 37, pp. 664-701. Parke, H.H. 1900 Variation and regulation of abnormalities in Hydra. Arch. f. Entw’mech., Bd. 10, pp. 692-710. PEARSON, Kari 1896 Mathematical contributions to the theory of evolu- tion. III. Regression, heredity, and panmixia. Phil. Trans. Roy. Soc. London, vol. 187, pp. 253-318. 1898 Mathematical contributions, etc. On the law of ancestral heredity. Proc. Roy. Soc. London, vol. 42, pp. 386-412. .1901 Mathematical contributions, ete. IX. On the principle of homotyposis. Phil. Trans. Roy. Soc. London, vol. 197, pp 443-459. 1909 The theory of ancestral contributions in heredity. Proc. Roy. Soe. London, vol. 81 b. 1910 Darwinism, biometry, and some recent biology. I. Biometrica, vol. 7, pp. 368-385. Puate, L. 1913 Vererbungslehre. Leipzig. Poporr, M. 1908 Experimentelle Zellstudien I. Arch. f. Zellforsch., XX. Bd. 1, pp. 245-379. Ranp, H. W. 1899 Regeneration and regulation in Hydra viridis. Arch. f. Entw’mech., Bd. 8, pp. 1-384. Reese, A.M. 1909 Variation in the tentacles of Hydra viridis. Science, N.S., vol. 29, p. 433. SHoutt, A. F. 1911 Studies in the life cycle of Hydatina senta. II. Jour. Exp. Zoél., vol. 10, pp. 117-166. INHERITANCE IN ASEXUAL REPRODUCTION 207 Suu, G.H. 1912 Genotypes, biotypes, pure lines, and clones. Science, N.S., vol. 35, pp. 27-29. Tower, W. L. 1899 Loss of the ectoderm of Hydra viridis in the light of the projection microscope. Amer. Nat., vol. 33, pp. 505-509. WarREN, E. 1899 An observation on inheritance in parthenogenesis. Proc. Roy. Soc., vol. 65, pp. 154-158. 1901 Variation and inheritance in the parthenogenetic generations of the Aphis, Hyalopterus trichodus (Walker). Biometrica, vol. 1, pp. 129-154. Wuitney, D. D. 1906 Artificial removal of the green bodies of Hydra viridis, Biol. Bull., vol. 13, pp. 291-299. 1907 The influence of external factors in causing the development of sexual organs in Hydra viridis. Arch. f. Entw’mech., Bd. 24, pp. 524-537. 1908 Further studies on the elimination of the green bodies from the ectoderm cells of Hydra viridis. Biol. Bull., vol. 15, pp. 241-246. 1912 Strains in Hydatina senta. Biol. Bull., vol. 22, pp. 205-218. WotteREcK, R. 1909 Weitere experimentelle Untersuchungen iiber Artver- anderung, usw., bei Daphniden. Verh. d. deutsch. Zool. Gesell., pp. 110-171. Yue, G. U. 1912 An introduction to the theory of statistics. London. 208 K. S. LASHLEY APPENDIX The correlation tables from -which the principal constants given in the body of the paper were obtained are appended here. In all cases where the tables include an earlier and a later gener- ation the ascendants are arranged in the rows, the descendants in the columns. In those cases where there is no qualitative difference between the members of pairs, as when siblings are correlated, the pairs are entered only once, according to the method recommended by Jennings (’11). The tables from which the correlations for size were computed are not included because of their great bulk and the lack of certain evidence upon the environmental modification of size. TABLE 24 TABLE 25 Clone D: Correlation between the initial numbers of tentacles of grandparents Clone D: Correlation between the initial numbers of tentacles of parents and offspring and grandchildren | 4 5 6 7 8 | 4 5 6 7 8 = 1 ae mea | 30 4 2 9 27 38 5 23 12672383 372 -5 394 Ole Ga O ee 242— 7 375 6 76 193 542 49 23 883 6) 84 211-505 11 5 816 7 of 41, 2 86 alte ed Sem col AL 76 8 1 1 2 8 1 ff 2 | 106 369 833 59 28 | 1395 113 344 826 19 5 1307 r=0.0038=+0 .018 r=—0.0495=+0.018 TABLE 26 Clone D: Correlation between the numbers of tentacles of members of the same fraternity - oC 6 7 8 t 6 133 273 24 436 5 896 3503 290 12 | 4701 6. 4550 969 31 | 5550 7 WZ 2 79 6 1029 8326 1360 45/10766 r=0.077 =0 .006 TABLE 27 Clone D: Correlation of the initial number of tentacles of theoffspring uith the number of tentacles borne by the parents when each bud was produced. 4 5 6 7 8 4 9 19 2 30 5| 6 % 267 21° 5 394 6] 8 119 623 110 23 883 7 32. 45 9 86 8 1 1 2 14 255 955 1438 28] 1395 r=0.096+0.016 INHERITANCE IN TABLE 28 Clone A-(A1A): Correlation between the initial number of tentacles of parent and offspring. 4 3 3 5 ie iS OT 6 42 6 14... 140 109 3h 2 296 7 | 23 222 133 67 10 455 8 #-- 3b Ad: 1, T 62 9 1 1 42 412 275 117 13 859 r=0.0011+0.023 TABLE 30 Clone A: Correlation of the initial number of tentacles of the offspring with the number of tentacles borne by the parents when each bud was produced 5 6 7 8 9 10 4 B35 98) 1 9 DMme4Oneos ll 3 93 ies 55) 182 101. 23. 5 590 Mimeloet25) 2210159) 63 5 589 So 9) 28) Ble 8: 9 69 9 ear 3 50 482 468 293 100 10 | 1353 r=0.240+0.009 TABLE 32 Clone D’. Correlation between the ini- tial numbers of tentacles of parents and offspring 4 1 1 2 5 Ay 22) pela 12 29 6. 3. ft | MGieas 114 if 3 1 (i 8 il 1 Sey On Somacol: r=—0.242+0.051 ASEXUAL REPRODUCTION 209 TABLE 29 Clone A1A: Correlation between the initial number of tentacles of parent and offspring x 101 276 51 . 6 r=—0.0342+0 .032 TABLE 31 Clone A: Correlation between the initial number of tentacles of grandparents and grandchildren 5 6 tf 8 9 4 % 1 1 2, 5 4b OM aS als 83 61 Ge lesOee so 139) 104 12 433 || 3 RT AI 510 8 8 feels 2S Si 9 2 il 3 89 224 330 389 62 1094 r=0.229+0.019 TABLE 33 Clone A’. Correlation between theini- tial numbers of tentacles of parents and offspring 6 7 8 9 5 1 2 3 6 3 14 5 33 if 24 7 34 «12 148 8 10 u 3 20 28 104 52 20 204 r=0.031+0.047 210 K. .6. ‘HASHLEY TABLE 34 Clone A: Correlation between the numbers of tentacles of all buds produced in each arbitrary five-day period. Each bud is paired in the table with all the others produced in the same period 4 5 6 7 8 9 4 D189. 355. “270s 729 5 574 5572 4300 484 4 10934 6 17277 35967 3859 102 57205 7 20742 4982 254 25978 8 264 30 294 9 1 1 2 656 23204 61279 9609 391 95141 r =0.0774+0.0015 TABLE 35 Clone A: Correlation of the numbers of tentacles of all buds produced in each five-day period with all buds produced in the preceding five-day period. Each bud is paired individually with all buds produced in the preceding period | Clone D: Correlation between the numbers of tentacles of all buds produced in each 4 5 6 7 8 a 4 9 87 374 372 42 1 885 5 66 846 4999 4552 461 5 10929 6 245 5084 32259 33039 3897 81 74605 7 153 3973-32873 «38143 4650 8994 79886 8 16 495 3866 4352 527 9 9265 9 4 55 93 11 163 489 10489 74426 80551 9588 190 175733 r=0.048+0.001 TABLE 36 Ove cont & arbitrary five-day period; arranged as in table 34 4 5 6 iq 91 1845 2936 261 $993 33381 1993 41613 9265 797 91 108388 77930 12271 r=0.1313+0.0014 8 12 72 484 168 6 742 5100 44439 51362 965 6 101872 THE EFFECTS OF CERTAIN SALTS, AND OF ADAPTA- TION TO HIGH TEMPERATURES, ON THE HEAT RESISTANCE OF PARAMECIUM CAUDATUM ROBERT H. HUTCHISON From the Zoological Laboratory of the University of Pennsylvania ONE FIGURE EFFECTS OF SALTS ON HEAT RESISTANCE The effects of certain salt solutions in increasing the resist- ance of various animals to heat are sometimes quite marked, and it seems probable that all animals of aquatic habit would be similarly affected under the proper conditions. Loeb and Was- teneys (Jour. Exp. Zool., vol. 12, p. 543), found that salts exerted a great influence on the ability of Fundulus to withstand sud- den changes of temperature. The maximum temperature into which these fish could, with impunity, be transferred suddenly, varied with the concentration of the sea-water or of a Ringer so- lution, ‘‘being about 25°C. for a concentration of M/128 or M/64; 27°C. for a concentration of M/32; 31°C. for a concen- tration of M/8; and almost 33°C. for a concentration of M/4.”’ Dextrose solutions were found to lack this protective effect against a sudden rise of temperature. In a previous paper (Jour. Exp. Zool., vol. 15, p. 148) the writer mentioned a few experiments in which the heat resistance of Paramecium caudatum was increased when the animals were transferred to solutions of NaNO;,*NaCl, and KNOs, the whole death temperature curve being shifted two degrees higher on the seale. The results of experiments of this kind have never been satis- factorily interpreted, and there is still lacking a complete expla- nation of the protective action of salts against heat, and of the acclimatizing effect of exposure to moderately high temperatures. Loeb and Wasteneys from their experiments with Fundulus in 211 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 19, No. 2 BAZ ROBERT H. HUTCHISON sea-water, in Ringer solutions, and in CaCl, solutions, conclude that the protective action of these salts is not an osmotic effect, nor a case of antagonistic salt action, but ‘‘a specific effect of the salts of the sea water.’’ But we are left in doubt as to just what this specific effect is. Indeed the results of experiments described below seem to indicate that, in the case of Paramecium at least, the salts have no specific action, or if so, such specific action depends upon the nature of the medium in which the ani- mal has previously lived. No attempt is made to offer any complete explanation. It is desired merely to set forth the facts as we found them as a modest contribution to the knowledge of the heat-resisting properties of living cells, with the hope that from an ever-increasing mass of data some general law may eventually be worked out. The following experiments were carried out during the winter of 1912-13 while working at the University of Pennsylvania under the direction of Dr. M. H. Jacobs. Pure lines of Parame- cium caudatum were used throughout, and the method of test- ing their resistance to heat was the same as that previously de- seribed (Jour. Exp. Zool., vol. 15, p. 133-134). In testing the effects of salt solutions a small quantity of the medium contain- ing the animals was centrifuged and two drops of the dense mass of animals were transferred with a pipette to 10 cc. of the solu- tion in question. Five drops of this solution containing the animals were placed in each of the small glass dishes used. The dishes were covered and arranged in order on the floor of the blood-serum oven. The experiments were always conducted so that the rise from room temperature to 45°C. was accomplished in about one hour. Beginning at 37°C., or lower if necessary, the dishes were removed consecutively, one for each rise of one degree Centigrade. After at least one-half hour, for possible re- covery, the number of living and dead in each dish was counted and the percentage calculated. From the percentages the curves for the fatal temperature zones and the mean for each curve were worked out in the manner described in a previous paper. Two pure lines of Paramecium were studied in some detail, the one growing in a medium of alkaline reaction, and HEAT RESISTANCE OF PARAMECIUM PA the other in an acid medium. The results will be described separately and then compared. Paramecium caudatum in alkaline medium. The medium was prepared by boiling 20 grams of hay in 500 cc. of tap-water for five minutes. Two days later, i.e., about the time of maximum acidity, just enough N/20 NaOH was added to render the fluid neutral to litmus. The culture was then seeded with a single individual isolated from a pure line previously grown in the laboratory for some weeks. The animals developed rapidly and the culture remained densely populated for about four months. The medium soon became dark brown in color and decidedly alkaline to litmus, and retained its alkaline reaction throughout its history. The effect of NaCl solutions was tested when the culture was about ten weeks old. Control experiments were carried out in every case at the same time, using five drops of the unchanged culture medium in each dish. The results obtained with M/100 NaCl are summarized in table 1. Experiments with M/50 NaCl were carried out when the cul- ture was about three months old; the results are summarized in table 2. TABLE 1 Effects of M/100 NaCl on the heat resistance of P. caudatum from an alkaline medium TEMPERATURES IN M/100 nacl: SUM OF THREE EXPERIMENTS 38° 39° 40° 41° 42° 43° Total subjected to each temperature... SRB: || B22 || Bre Waa i alas INimMberOfpd eat see es <2 6 se, eetesi- creas areiur 0 0 19 | 149 | 365 Percentage dead at given temperature. . 0 0 7 40 | 100 CONTROLS: UNCHANGED MEDIUM, THREE EXPERIMENTS Totals subjected to each temperature. . 480 | 514 | 463 | 562 Binmber Of, dea thse coaeicth. 2 oc. 4).uth a. 0 6 | 248 | 562 Percentage dead at given temperature. . 0 lose p00 MEANS OF THE ABOVE FATAL JOIN UG OE EO) Eee ear 42 .03° TEMPERATURE ZONES \Wimmeontrolse.. 21 s.00 eS. : 40 .9° \ 214 ROBERT H. HUTCHISON TABLE 2 The effect of M/50 NaCl on the heat resistance of P. caudatum from an alkaline medium : TEMPERATURES IN M/50 nacl: suM OF FOUR Totals subjected to each tem- PeTAGUTC: Froeac eee eee... .-.| 9S «| 266° ar lore” | -se0 rasa ee Number of deathse.o.0- ss. :..-| © 0 0 0 0 | 200 | 430 Percentage dead at given tem- | DELAGUEG hase cee iia. s < s « 0 0 0 0 0 44 | 100 CONTROLS: UNCHANGED MEDIUM FOUR EXPERIMENTS Total subjected to each tem- PGratate ees eree a: >... oc ekl. ol doo, AGL) || A ae ees NumberiGimesius........:...-.- 0 0 24 | 109 | 490 | 555 Percentage dead at given tem- PCEDUUITOM ee ee. ke ee 0 0 el) 125: 84.3} 100 MEANS OF THE ABOVE FATAL fap M/50 NaCl iiss! re oc eee ele TEMPERATURE ZONES (ai CONMPOISHS Sar ccrte ecu ke = se et 40 3° It will be noted from the tables that M/100 NaCl has raised the mean of the fatal temperature zone of this pure line 1.13°C.., and that M/50 NaCl has raised it 1.8°C., the stronger concen- tration producing a slightly greater effect. Concentrations stronger than M/50 had a toxic effect on this race, deaths occur- ring at much lower temperatures than the controls. The effect of KCl was not studied except that it was found in one experi- ment that M/200 KCI had a toxic effect, all the animals being dead at 38°C. In weaker concentrations it would probably have shown some protective action. Solutions of CaCl, were tested on this same race, and weak concentrations had a marked protective action. When trans- ferred to M/300 CaCl, nearly all the Paramecia died within two hours at room temperature, while in M/3000 CaCl, this race remained alive at room temperature for at least two weeks. Table 3 shows the effect of CaCl, alone, and table 4, the effect of a HEAT RESISTANCE OF PARAMECIUM 215 combination of NaCl and CaCl. When the Paramecia were transferred to a medium consisting of 10 ec. of M/100 NaCl plus one drop of M/100 CaCl, the results shown in table 4 were obtained. TABLE 3 The éffect of M/3000 CaCl, on the heat resistance of P. caudatum from an alkaline medium TEMPERATURES IN M/3000 cacle: SUM OF FOUR EXPERIMENTS 39° 40° 41° 42° 43° 44° Totals subjected to each temperature. .| 286 | 340 | 276 | 357 | 319 | 320 Mfumaber.O1-Geathist:!) ... 2a aos anaes 0 0 O | 194 | 310 | 320 Percentage dead at given temperature. . 0 0 0 54.2} 97.1] 100 CONTROLS: UNCHANGED MEDIUM, FOUR EXPERIMENTS Totals subjected to each temperature. .| 444 | 409 | 316 | 554 Brmaen olodeaths. ....... 2.2 2acs wae ea 0 58 | 314 | 554 Percentage dead at given temperature. . 0 11.7} 99.3] 100 MEANS OF THE ABOVE FATAL {Gime WHV/ S000! CaCI 8. 2 Lc 141 -98° TEMPERATURE ZONES tuisineontnolsene sees A 40 .3° TABLE 4 The effect of NaCl plus CaCl, on the heat resistance of P. caudatum from an alkaline / . medium TEMPERATURES IN Nacl + Cacls: SUM OF THREE EXPERIMENTS 39° 40° 41° 49° 43° 44° Totals subjected to each temperature. .| 237 | 228 | 298 | 391 | 249 | 235 ININDET OL GEACMS 6 <2 oo es. a es 0 0 0 31 | 188 |} 235 Percentage dead at given temperature. . 0 0 0 7.9} 75.5] 100 CONTROLS: UNCHANGED MEDIUM, THREE EXPERIMENTS Totals subjected to each temperature. .| 312 | 294 | 316 | 369 Nimmiberioh deauhse esate t= 0 58 | 312 || 369 Percentage dead at given temperature. . 0 19 98 .7| 100 MEANS OF THE ABOVE FATAL iim NaOh-eACa@ lye 5 usc 32... 49056° TEMPERATURE ZONES Mirnicoriira ls en eke Foes ss 2, 40.3° 216 ROBERT H. HUTCHISON The tables show that in this race the solution of M/3000 CaCl, has raised the mean of the fatal temperature zone 1.68°C. This is very nearly the same as the effect of M/50 NaCl, as shown in table 2. The most pronounced effect was produced by the addition of a little CaCl. to the solution of NaCl. In this case the mean was raised 2.26°C. The significance of these increases is all the more important when one notices that the means of the control experiments are almost exactly the same in all the tests, although they were made at very different periods in the history of the culture. The effect of KNO; on this race of Paramecium is summarized in table 5. The mean death temperature of this race was 1.9°C. higher in M /50 KNO; than in the corresponding controls. In three ex- periments with M/100 KNO; the mean was found to be 41.1°, a rise of 0.3°C. above the corresponding controls, which gave a mean of 40.8°C. TABLE 5 The effect of M/50 KNO; on the heat resistance of P. caudatum from an alkaline medium TEMPERATURES In M/50 KNO3: SUM OF FIVE EXPERIMENTS sie 38° 29° 40° 41° 42° 43° Totals subjected to each tem- perature.. BYdee Gon nes as ans ZOD MO O34) 2/625, 3| 730, O04 eRe Number of Benes Ree re Aa 0 0 0 27 | 190 | 210 | 691 Percentage dead at given tem- DELADULGE Ameer oho hc oe oe 0 0 0 4.3) 25.8) 30.2) 100 IN CONTROLS: SUM OF FIVE EXPERIMENTS Totals subjected to each tem- perature... Whe: 5 state «an 8 hace ay ceed | Feo: |) Gia Ol erat eee Number of fee :, SS ene 45 0 0 | 221 | 500 | 723 |-725 Percentage dead at given tem- PeTabirer-we eee sien: ase 0 39.5} 73.8) 100 | 100 MEANS OF THE ABOVE FATAL | in M/50 KNO3...............-.2.+-0: 41.4° TEMPERATURE ZONES | ieontrols, assis Se 39 .5° _ HEAT RESISTANCE OF PARAMECIUM 217 Perhaps the most curious results of all were obtained when this race was subjected to the influence of M/600 Na,CO;, and to distilled water. Table 6 shows these results and those of the corresponding controls, all of which were carried out at the same time. The results in table 6 show that distilled water was just as effective in its protective action for this race as was M/600 NaeCO;, or M/50 KNOs, or as M/3000 CaCl,. The mean death temperature in distilled water was 1.78°C. higher than the corresponding controls. In cane sugar solutions there was no protective action mani- fest. In one experiment two drops of the medium densely popu- lated with Paramecia were transferred to 5 ec. of M/8 cane sugar. When subjected to heat some deaths occurred at 38° and above. ‘This solution was much less effective than distilled water; indeed, it appeared to have a weakening effect. TABLE 6 The effect of M/600 Na.CO;, and of distilled water, on the heat resistance of P. caudatum from an alkaline medium TEMPERATURES In M/600 Nazco3;: SUM OF TWO EXPERIMENTS 39° 40° 41° 42° 43° Totals subjected to each temperature......... ey] az ISP eaizal 151 NtummMperroted Carta Strer cc percvs cays 5 ckachorsilst ons otek eles 0 6 SE LOS aot Percentage dead at given temperature........ 0 4 23.3} 61.4) 100 IN DISTILLED WATER: SUM OF TWO EXPERIMENTS Totals subjected to each temperature.......,..| 147 | 187 | 165 148 | 188 INTIME Oledeatbsne ster cecrs aes teedee. ce ole O 0 4 97 | 188 Percentage dead at given temperature........ 0 0 2.4) 65.5} 100 CONTROLS: UNCHANGED MEDIUM, TWO EXPERIMENTS Totals subjected to each temperature.........| 260 | 216 | 260 | 209 Niumibers offdeabhserrrycaesiie class cerro oes One S24 209 Percentage dead at given temperature........ 0 53.2) 92.7) 100 MEANS OF THE ABOVE FATAL TEMPERATURE EUS OU A eo ose in distilled water....... 41 .82° mae in) COMbroOlSscedse~ ee. 3. 40.045 218 ROBERT H. HUTCHISON The above results agree with those of Loeb and Wasteneys, in one respect at least, in that they show that the protective action of salt solutions is not an osmotic effect. But when we find distilled water just as effective as three of the salt solu- tions used and almost as effective as M/50 NaCl, and the combi- nation of NaCl and CaCl:, it is apparent that in the case of Paramecium at least, the results are not due to the ‘“‘specific action of the salts.” . Some results of tests with another race of Paramecia in an acid medium detract still more from the specific salt action as a satisfactory explanation. An account of some of these results follow. Paramecium caudatum in an acid medium. The culture me- dium was prepared by boiling 20 grams of hay in 500 ce. of tap- water for one-half hour. The following day it was seeded with a single individual isolated from another pure culture which had been growing in the laboratory for some time. This culture medium retained its light straw color throughout. It was never as densely populated as the alkaline medium and died out sooner, i.e., in about three months. Most of the following experiments were performed when the culture was about two months old. It was still light colored and slightly acid to litmus. Tables 7 and 8 summarize the results of tests with M/4000 CaCl., with M/100 NaCl, and with distilled water. It is to be noted that the control experiments show that the normal heat resistance of this race was somewhat higher than that of the race from the alkaline medium, the average of all the con- trols of the acid medium being about one degree higher than the average of all the controls of the alkaline medium. This is just the opposite effect from that produced by acids and alkalis on the coagulation temperature of proteids. Further, it will be noted that the same salts (NaCl and CaCl.) which gave the most pronounced protective action with the Paramecia from the alkaline medium, actually decreased the resistance of those from the acid medium. Considering the action of the salts alone it might be supposed that their effects were conditioned by the reaction of the medium in which the animals had pre- HEAT RESISTANCE OF PARAMECIUM 219 TABLE 7 The effect of M/4000 CaCl, on the heat resistance of P. caudatum from an acid medium TEMPERATURES In M/4000 caclz: sum oF TWO EXPERIMENTS 39° 40° 41° 42° 43° Totals subjected to each temperature......... 243 |‘206 | 202 | 170 Nimperot deaths: 4... ese see ee ee OF 15200) 202") 170 Percentage dead at given temperature........ 0 97 | 100 | 100 CONTROLS: SUM OF TWO EXPERIMENTS Totals subjected to each temperature.........| 288 | 330 | 148 | 300 | 295 Numi en of death Sos .42 « o = oe pera eee: 5 23: | 224 | 210 | 295 Percentage dead at given temperature........ Delle er 15.4; 70 | 100 MEANS OF THE ABOVE FATAL TEMPERATURE J in M/4000 CaCl,........ B9n5ay ZONES gimacaminoles. he esa ok : 41 .55° TABLE 8 The effect of M/100 NaCl, and of distilled water, on the heat resistance of P. cauda- tum from an acid medium TEMPERATURES IN M/100 Nacl: SUM OF THREE EXPERIMENTS 39° 40° 41° 42°" Totals subjected to each temperature............... 368 | 308 | 299 | 327 MiP erLONG ea bh See et ss ows was 4H ale eomiaeneee ne 0 49 | 242 | 327 Percentage dead at given temperature.............. 0 15.8) 81 100 IN DISTILLED WATER: SUM OF THREE EXPERIMENTS Totals subjected to each temperature............... 395 | 390 | 332 | 429 INGrmEGrOteGe aise 0/40 isiepss <5 an se tiek oom Sate mies 0 72, | 326 || 429 Percentage dead at given temperature.............. 0 18.5) 95.1} 100 IN CONTROLS: UNCHANGED MEDIUM THREE EXPERIMENTS Totals subjected to each temperature............... 662 | 685 | 622 | 770 Nitmmbermiot deaths wemee tre 4. yorg sss es hes oe 0 1 || ay | 7) Percentage dead at given temperature.............. 0 2.2) 30.5} 100 {in M/100 NaCl... . 40.5° MEANS OF THE ABOVE FATAL TEMPERATURE ZONES 4 in distilled water.. 40.3° (in controls.(i2....; 41.1° 220 ROBERT H. HUTCHISON viously existed. But distilled water was found to have the same effect, and some influence other than the salts seems to be the important factor. Taken as a whole, the above experiments seem to point to the conclusion that certain properties of the medium are important factors in the heat resistance of P. caudatum, and that such properties will predetermine whether a given salt solution will have a favorable or an unfavorable effect. Whether this is true for other Protozoa remains to be determined, and a satis- factory explanation is yet to be proposed. EFFECTS OF ACCLIMATIZATION TO HIGH TEMPERATURES ON HEAT RESISTANCE A few experiments were carried out during the same season with a view to determining to what extent continued exposure to moderately high temperatures would influence the death temperature. Several attempts along this line failed, some on account of too sudden changes and some for other reasons. Some success was had with two cultures which were studied at fre- quent intervals during an exposure of over two months to tem- peratures ranging from 28° to 36°C. Pure line cultures of Paramecia were used in these experiments. On January 30, 1913, a culture medium was prepared by tak- ing 35 grams of hay in two liters of distilled water. This was heated for a period of ten minutes at a temperature of about 60°C. This was then divided equally between two culture jars so that each contained one liter of the fluid and about 173 grams of hay. After cooling, each of these cultures was seeded with a single individual taken from a pure line which had been growing in the laboratory for 86 days before this date. The two result- ing cultures, which will be referred to as ‘30-a’ and 30-b,’ may therefore be regarded as of the same pure line and growing in the same medium. On February 11 (12 days after the culture was started) 30-a was transferred from room temperature to a water bath at 28°C. The temperature of the water bath was regulated automatically and never varied more than 0.5° either way. The culture jar was so placed that the level of the cul- HEAT RESISTANCE OF PARAMECIUM 22] ture fluid inside was about one inch below the level of the water of the bath outside the jar. The mercury bulb of the thermome- ter showing the temperature of the water bath was also placed about one inch below the surface of the water, so that it gave . very closely the actual temperature to which the Paramecia were exposed. The culture jar itself was of course kept covered with a glass plate at all times. The constant temperature of 28° was maintained for a period of 27 days. On March 10 the tem- perature was raised to 30°C. and on the 12th to 32°C. After 10 days at 32° the temperature was again raised to 34°. After 16 days at 34° the temperature was raised on April 7 to 35°, and on April 15 to 36°, at which point it was maintained to the end of the experiment. On April 26 the culture was in very poor condition and very few animals were present. A little dry fresh hay was added to the medium but it did not have any favor- able effect and the strain had completely died out by April 29. Strain 30-b was used as a check on 30-a, and was kept on a table in a cool room, the temperature of which varied from 12° to 22°C. On April 29, when 30-a had completely died out, 30-b was still in good condition, although not as densely popu- lated as in earlier periods of its growth. During the course of the experiment the heat resistance of both strains was studied at frequent intervals. The mean of the fatal temperature zone for each experiment was worked out in the usual way and these means are plotted in figure 1. Another pure culture, 11—2, was started from an individual isolated on December 11, 1912. This culture was kept at room temperature until February 14, at which time it was transferred to the water bath at 28°C. From that time on until the cul- ture died out (April 25) it was subjected to the same temperature conditions as was 30-a. The mean death temperatures of a series of tests with this strain are also plotted in figure 1. Ex- amination of the figure shows that the resistance of the control was by no means constant. There was considerable variation, the means varying from 40.5 to 42.3°C. The resistance was more irregular and in general slightly higher during the later course of its history than in the earlier experiments. Mean death temperatures of three races of P. caudatum in degrees Centigrade MM TT 0 00 ag TT Uk A Nr ee WP S007 EAA Mt = 0 A A LNA ATU LT AUT A UT TR NN TU TA GS ATR NA 0 UN kT, HM FRITH TART a TL ULV E T Int ULIMIT AA A WAL WEVA et UT HN HR C0 LL EH UC 0 ITIL tl FTL HAUL TLE. 8 TA Ta 0 A TT i 7 AUT AGE 2H EC nan a HL TH | aA i VA AG it IR iT ae eA i es ea AN BL i mu era THR TAG THLE TU HAAR ATTA SEA A A We) HA A A A 18 HA AN NA AAG a 00001 i 000 NN TE RS “MAMTA HT AL S == AB il ESTIMA A a cA TAA ta A i A A A AGT ATATL TTS AA iil = 00 RT TAA EA A HA TG HATTA AT a GT i 80 A Te Reece ne ean HATTA G | | Ai ii! HH | HH LATHE AA i TM AN ee eK eAArocaS NaS RL ADR A a aT A oo TT Ae eA TE i AT z per liter) REACTION OF FISHES IN PER CENT OF TIME SPENT IN HALVES OF TANK. POSITIVE = IN SALT e KIND OF WATER USED HALF; NEGATIVE = IN TAP OR ALT IN GRADIENT DISTILLED WATER HALF Per cent Per cent positive negative Rig alone:,. 7.5 ok eee Slightly acid tap 30 70 NaNO; + trace Ca (NO3;)2......| Slightly acid tap 79 21 NaNO; + trace Ca (NO3)o......| Strongly acid tap 96 | Cs (N@saalotie.... 75255-1. 7 - 2. Slightly acid tap a ie Ca (NO3)2 + trace NaNO3...... Slightly acid tap 76 14 Cai (NOs)e alone: 2.2 aeeoc - oteee Distilled water 19 81 Ca (NO3)2 + traée NaNO;......| Distilled water 7 13 Ca (NO;3)2 + trace Mg(NOs;):. ..| Strongly acid tap 53 47 256 REACTIONS OF FISHES TO SALTS Pri binations of salts in a gradient, and I present a number of experi- ments of this sort here. They show that fishes recognize and react to combinations of salts according to the prediction which might have been made from the results of previous work upon antagonism. It will be remembered that fishes are slightly positive to 0.01N NaNO, in moderately acid water. It was found, however, that they are negative to this salt in water that is but faintly acid and for this reason the following experiments were run in water which contained less than 8 ec. CO, per liter. The experiments were first run as regular salt experiments such as have been described before, and then the antagonizing salt was added ‘to the salt flow. The concentration of the original salt was always 0.01N and that of the antagonizing salt 0.0002N, i.e., a bare trace was added. The results of these experiments are shown in table 2. A considerable number of experiments was run with each combination in the tap water, and then some check experi- ments were run in distilled water. The results in the distilled water gradients are very similar to those in the tap water. Discussion of the experiments with antagonizing salt combinations Table 2 shows clearly that the antagonistic action of the salts is detected and reacted to by the fishes. This is shown, for instance, in the sodium nitrate experiments; here the fishes were 70 per cent negative to this salt in slightly acid water but when a trace of calcium nitrate was added the negative response fell off to 21 per cent and the positive rose from 30 to 79 per cent. Then in strongly acid water the positive response increased to 96 per cent. The reactions of fishes in any gradient are due to their tendency to move about until they reach an environment that neither over- nor under-stimulates them. Thus they will not remain quietly in water that is strongly acid nor will they do so in water that is neutral. A slight degree of acidity (1-6 ec. CO, per liter, Wells ’15 a) furnishes their optimum stimula- tion as far as H ion is concerned. The reversal in reaction of the fishes in gradients to which a trace of an antagonistic salt 258 MORRIS M. WELLS has been added, must then be due to the fact that this trace of salt lessens the stimulation in the salt end of the gradient. There are three principal factors affecting the degree of stimulation of the gradients referred to in table 2, namely, the original salt (e.g., NaNQO;) the antagonising salt (e.g., Ca(NOs;)., and the acid. Before the antagonising salt was added the fishes were negative to the original salt, even though this meant spending most of the time in a degree of acidity which was slightly above their optimum. With the addition of the antagonising salt, however, they reversed their reaction and became positive to the salt end. The antagonising salt must have diminished the original stimulation in the salt end or have increased the stimu- lation in the acid end, or both. The work upon the effect of acids and salts upon permeability suggests that both factors were concerned. Lillie (10) has shown that calcium salts de- crease the permeability of egg membranes while the salts of sodium increase the permeability. Osterhout (’12, a and b) has shown that sodium salts increase the permeability of plant cells while the addition of a trace of calcium salt maintains nor- mal permeability even in the presence of an excess of the sodium _ salt. Osterhout has also shown that there exists a mutual antagonism between certain acids and salts as for instance be- tween HCl and NaCl, but the salts of calctum and magnesium work with rather than against the acid. In the above experiment, therefore, the addition of the cal- cium salt to the end of the gradient which contained a sodium salt in concentration strong enough to cause the fishes to give a negative reaction, resulted in the fishes becoming positive. This reversal in the reaction of the fishes must have been due to the decrease in the stimulating power of the salt end. It has already been shown that increasing the acidity of water will cause fishes to become positive to concentrations of sodium salts to which they are normally negative (table 2) and it was found that the higher the acidity the higher was the concentration of sodium salt selected by the fishes. Table 2 also shows that the antagonistic action between cal- cium and sodium salts is detected and reacted to, when the con- REACTIONS OF FISHES TO SALTS 259 | Bull-head No. 1 | Bull-head No. 2 Bull-head No. 3 ; 341 Ai sali > 70 aol: (Sea linen! one bee! Orel oe a Se | ae ee ee | & ; Tap Bava Tap | 4 | Tap Sta, Tap | 4 Tap At4 | Tap ae = 3 oS 3 Fe a | o | San pareve. lO 4, Cal 5 4, i 1 a 4 1 1 | 1 | 1 1 1 ! 1 U ! i} ! | | 1 ' | | i] 1 ) i | , 1 u | | | | 4] | | | | | ! a 6] | | eri a, = | | 6] - | | ! ! | i on, oe | ; ] 8 | ! | | | ! | ! | Sa , el ! i} ! i} 4) ESL ss Fig. 2 Showing the reaction of bull-heads (Ameiurus melas) to Ca(Nos)s, alone, and in combination with a trace of NaNO;. The concentration of the ealcum salt was 0.01 N throughout and that of the sodium salt 0.0002 N; experi- ments performed in distilled water; numbers at left indicate time in minutes. centrations of the two salts are reversed, 1.e., when the calcium nitrate is present in 0.01N concentration and the sodium as a trace (0.0002N). The data for table 2 were obtained from graph experiments and also from readings. The same fishes were used in the gradient with the different conditions, i.e., they were first graphed in the gradient with the sodium salt (e.g.) alone and then again after the calcium salt had been added to the flow. To illustrate more accurately this method of experi- ment figure 2 is inserted. The graphs shown in this figure are 3 of those made by 4 bull-heads. The experiments were run as follows: The gradient with only Ca(NO;). flowing in at the salt end, was obtained by allowing the flow to continue for 30 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 19, No. 3 260 MORRIS M. WELLS minutes. The fishes were then taken from the large aquarium and placed in pans of water, numbered 1,ete. The fish from pan No. 1 was placed in the gradient and its movements graphed for 15 minutes. It was removed and No. 2 was placed in the gradient and graphed. This was repeated for Nos. 3 and 4. A trace of NaNO; was now added to the inflow at the salt end; after 20 minutes fish No. 1 was again placed in the gradient and its movements graphed for 15 minutes. This was repeated for the three remaining fishes in the same order as before. The graphs show the marked difference in the reactions of the fishes before and after the trace of sodium nitrate was added. C. PHYSIOLOGICAL STATES AND THE REACTIONS OF FISHES In the discussion so far attention has been called to the fact that in most of the series of experiments, there was a small percentage of the fishes (usually 3-5 per cent) which gave re- actions more or less the reverse of those given by the majority. Such exceptions to the general behavior are common in experi- mental work of all sorts and probably indicate physiological differences upon the part of the organisms. That such physio- logical differences, i.e., physiological states, exist and that they influence very markedly the reactions of the animals has been proven beyond doubt (Child ’13, and Allee 712). Allee and Tashiro (’14) have shown that the reactions of isopods are very closely correlated with the metabolic activity and Allee (12) has shown that by changing the rate of metabolism he can alter and even reverse the reaction of isopods to current. REACTIONS IN PERCENT vex wownee (DATE OF | Eapeme | OmGmAL | arms | OF TIME ne Low O2 | Tap water Normal fishes 1913 1913 1 Nov. 20 | Nov. 22 7, 5 5 95 2 20 DD, 9.9 9.5 25 75 3 20 Pie Aye 2229 32 68 4 20 22 70.6 69.0 20 80 5 20 22 126.0 124.0 91 9 Starved fishes 6 Oct. 16 22 Dita 18.6 44 56 7 16 22 66.0 61.7 50 50 8 16 22 90.9 77.0 34 66 REACTIONS OF FISHES TO SALTS 265 normal rock bass are negative to low oxygen (1 cc. per liter at the low end) as has been shown also by Shelford and Allee (13, p. 236). Large rock bass seem to be an exception to this general rule as they are not always negative to low oxygen, and in some cases seem to definitely prefer the low oxygen end of the gradient, spending a majority of the time there. The cause of this re- action has not been determined but it may have to do with the concentration of hydrogen ion which would probably be a little higher in the low oxygen end than in the high oxygen water, the difference being due to the difference in the effect of the two kinds of water upon the elimination of carbon dioxide by the organism. Fishes Nos. 6 to 8 (table 5) are the individuals occurring under the same numbers in table 4. There it was noted that their reac- tion to the CaCl, had become more positive than the normal re- action and in table 5 it will be noted that these fishes are less sen- sitive to the low oxygen also. Fish No. 5 is also the same in tables 4 and 5 and it will be noted that this fish was positive to both low oxygen and 0.01N CaCl. Experiments in low oxygen gra- dients were not performed with these fishes later in their period of starvation but the data given indicate that as they become somewhat starved they at the same time become less negative to low oxygen. This indicates that their metabolic rate is slower than normal. D. ACCLIMATIZATION AND THE REACTION OF FISHES During the course of the experiments, considerable evidence was accumulated concerning the effect of acclimatization upon the reactions of the fishes. A few experiments with fishes in CO, gradients indicated that these fishes after living for two to three weeks in water whose CO, concentration was 8 to 10 ce. per liter, were more sensitive to the CO, than normal fishes. To determine whether or not the presence of an excess of salt would , result in similar reactions to the salt in a gradient, a series of acclimatization experiments with CaCl, was run. A medium-sized (45-gram) rock bass was graphed in a CaCl, gradient; its normal reaction was decidedly negative to 0.01N 266 MORRIS M. WELLS CaCl. It was now placed in a 20-gallon-jar full of a 0.01N solution of this salt. Each succeeding day it was taken from the jar and its reaction in the gradient graphed, when it was re- turned to the jar. This was continued for 6 days; the con- centration of the solution in the jar was then riased to 0.05N. The fish was left in this solution 4 days longer, being graphed each day. It was then returned to the tap water and graphed again after 2 days. In making the graphs each day, the salt solution was run into the end of the gradient tank, opposite that of the day before. A series of the graphs made by this fish are shown in figure 3. They show the different stages in the process of acclimatization. In short they indicate that the Sept. 16 Sept. 19 Sept. 23 Sept. 24 | Sept. 26 | Control ; CaCle| Tap | CaCl | Tap | CaCh | Tap | CaCl | Tap ' CaCh| Tap Tap Tap I | ' { 1 \ \ t } I | | | | | \ I ! ‘ I ! ! 1 | | l I \ Fig. 3 Showing the reversal in reaction to 0.01 N CaCl:, upon the part of a * 45-gram rock bass (Ambloplites rupestris), after being kept in the salt solution for a week, and the return to normal reaction upon being placed in tap water again. In the experiments the salt was made to flow into alternate ends, but in the chart the graphs have been copied so that the reaction will be more easily seen, by keeping the same relation between the tap and CaCl: ends. REACTIONS OF FISHES TO SALTS 267 fish did become acclimated to the CaCl, solution by the end of a week and selected the higher concentration in the gradient. Then after 2 days in the tap water it was negative to the salt again. A like set of experiments was performed with a small bull- head (6 in. long) with similar results; the acclimatization, how- ever, came sooner. Neither of the fishes was fed while in the CaCl, and this would have an effect upon their reaction. The fact that the rock bass became negative again after being returned to the tap water indicates that the starvation did not account for its positive reaction while being kept in the CaCl, solution. Starvation would tend to increase the negativeness of bull- heads to the salt so the positive reaction upon being kept in the CaCl, can be due to nothing but acclimatization. The difference in the effect of starvation upon the reactions of the two species of fishes to salts is probably due to a difference in the metabolism of the fishes and will be discussed in another paper. E. RESISTANCE OF FISHES TO SALTS The toxic effect of certain salts upon organisms has been the subject for considerable investigation upon the part of other workers (Ringer, Loeb, R. Lillie, and others) and therefore con- siderable is known concerning the relative toxicity of the various salt ions. In the present paper are presented data which indi- cate that much of the work upon the toxicity of salts must be reconsidered and correlated with the chemical reaction of the water. The data show that the poisonous properties of a given salt may vary within wide limits depending upon the amounts of hydrogen or hydroxyl ions present in the solution. 1. Resistance to ammonium salts According to Mathews (’07) the pharmacological action of most salts is due to the ions of the salt. The kind of action depends upon the character of the charge of the ion, i.e., whether positive or negative; the degree of action is proportional to the available energy in the ion. Ammonia salts are peculiar, how- 268 MORRIS M. WELLS ever, in that their toxicity is not due to the action of either of the original ions, but to the products which are derived from the breaking down of the original ammonia compound. Ammonia salts in solution dissociate principally into NH, ions and the acid ion with which the ammonia is combined. There is a hy- drolytic dissociation also, so that there is always present in the solution a small amount of the free acid and the ammonium hy- drate. In considering the reactions of fishes to ammonium hy- drate (Wells, ’15a, p. 236) it was pointed out that the ammonium hydrate in solution is in equilibrium with and is but a small per cent of the dissolved ammonia gas. In the case of an ammonia salt the hydrolytic dissociation of the salt produces the hydrate, which in turn dissociates to give water and ammonia gas. The amount to which the salts dissociates into ammonium hydrate and ammonia gas varies with the salt, being least in the sul- phate and larger in the carbonate (Mathews, l.c.). Mathews further states that it is probable that the action of the ammonium salts is due, therefore, to the hydrate which is formed, and in turn the action of the hydrate is dependent upon the action of the dissociated NH;. This gas is probably in a nascent con- dition just at its moment of origin, when the valencies of the nitrogen are still open. The toxie action of the ammonium salts used in the reaction experiments was found to be very marked when they were dis- solved in the tap water, but was much less when the salts were dissolved in distilled water. Solutions (0.01N) of the chloride, nitrate and sulphate were made up in tap and distilled water and small blue-gills (8-gram) were placed in jars of the different solutions. The temperature was kept constant by setting the jars in running tap water. One liter of solution was contained in each; the results are shown in table 6. The marked increase in the longevity of the fishes in the dis- tilled water seemed worthy of further investigation. Death in the distilled water was in part due to increasing acidity of the solution, as titrations showed a concentration of hydrogen ion at the end of the experiments that must soon have killed the fishes even though no other factor were present. This increase REACTIONS OF FISHES, TO SALTS 269 TABLE 6 Showing the resistance of small blue-gills (3-gram) to .01N concentrations of the chlolide, nitrate and sulphate of ammonium dissolved in tap and distilled water DYING TIME IN THE SOLUTIONS KIND OF WATER Chloride Nitrate Sulphate Tap water from aquarium....... 4.8 hours 3.9 hours 3.5 hours Wistillediwatierer.. cna s ek 18 days 16 days 17. days in the acidity of the solutions was marked in the case of all three salts and the titrations showed that the acidity upon the day of death of the fishes, had increased tonearly 0.001N while 0.0001N is enough to kill these fishes in distilled water when no salt is present. The increase in acidity was not due entirely to the CO, given off by the fishes, as boiling did not remove it. It must, therefore, have come from the acid which had formed from the hydrolysis of the salt. The ammonia formed in the same process had passed off into the atmosphere. It seems clear then that the three salts in question do not furnish a large enough quantity of NH; to kill the fishes, if the salts are dissolved in distilled water. It has been pointed out in a previous paper (Wells 715 a) that the tap water at the University of Illinois contains an unusually large amount of the bicarbonates of Ca and, Mg and that as the water is aerated these bicarbonates dissociate to give the normal carbonate. It was thought that the toxicity of the ammonium salts in the tap water may have been due to the forma- tion of ammonium earbonate and the further dissociation of this salt to give NH; in.toxic quantities. To test this possibility three experiments with 0.01N concentrations of (NH4,).CO; in distilled water, were tried. The dry salt gave a strong odor of ammonia but the solution was too dilute to give any odor at all. After thoroughly shaking the solution and allowing it to stand for 10 minutes, a 10-gram sun-fish was placed in a liter of it. A control was run in distilled water. The result of this experiment, together with those obtained from a number of other experiments are given in table 7. The table shows that am- 270 MORRIS M. WELLS monium carbonate is very toxic in distilled water, that standing does not lessen its toxicity greatly nor does it that of the sulphate in tap water, and finally that the ammonium sulphate is no longer toxic in tap water when the carbonates have been converted into sulphates by the addition of enough sulphuric acid to make the water neutral to methyl orange. TABLE 7 Showing the resistance of fishes to .01N concentrations of (NH4)2CO3 in distilled water; the effect of standing upon the toxicity of ammonium salt solutions; and the non toxicity of a solution of (NH4)2SO4%n tap water when the carbonates have been converted into sulphates. DYING TIME OF THE FISH IN THE SOLUTION Experiment Control 0.01N (NH,)2CO; in distilled H2O........ : 1.7 hours normal Same after standing 24 hrs............... 2.2 hours normal 0.01N (NH,)2SO, in tap water............ 1.3 hours normal Similar solution after 24 hrs.............. 2.2 hours normal 0.01N (NH,).SO, in tap water after the carbonates have been changed to sul- PDALCH CA CPU Biers ctetaSsais 1 24 See fish normal at} control in .01N end of amonth sulphate in or- dinary tap; dead in 2.1 hrs. The experiments upon the resistance of fishes to ammonium salts show clearly that ammonia in any form is toxic to fishes in water containing carbonates. Since practically all natural waters contain a greater or lesser amount of the carbonates in solution as such, or as bicarbonates, the introduction of even very small amounts of ammonia into these waters will be very detrimental to the fishes. Table 7 shows, on the other hand, that the carbonates are not necessary to the immediate existence of the fishes, i.e., the water need not be alkaline to methyl orange as Marsh (’07) claimed. It may of course be that the carbonates are necessary to a successful completion of the life history of some fishes, or to the continued existence of certain species. This point has not been worked out so far as I am aware. REACTIONS OF FISHES TO SALTS DIA 2. Resistance to potassium salts Solutions (0.01N) of the chloride, nitrate, and sulphate, were made up in tap water and a small blue gill (3-5 gram) introduced into a liter of each; the results are shown in table 8. The action of the potassium salts in tap water was checked by placing a fish in a 0.01N solution of the most toxic one, i.e., the sulphate, in distilled water. The reactions of this fish were very peculiar. After 3 days in the solution it was noticed that the fish was losing its equilibrium and it was expected that it would die in a few hours. On the next day, however, it was still TABLE 8 Showing the resistance of small blue gills to .01N concentrations of potassium salts in solution in tap water DYING TIME IN Chloride Nitrate Sulphate Normal on 15th day.......... 15 days 4 days alive and for 10 days more it lived spending much of the time lying on its side but righting itself when touched with a glass rod. Its movements were sluggish and stiff, much as though it were dying from fungus disease. In all, the fish lived for 14 days in 0.01N potassium sulphate solution, which is over three times as long as a fish of the same size lived in the same strength solution in tap water. The long-drawn-out death of the fish is not a phenomenon that is peculiar to potassium salts, however, for it was noted that another small blue gill which was in an ammonium nitrate experiment in distilled water at the same time, gave a similar reaction. This latter fish swam about for three days on its side with the body bent into the bow-shape that often distorts fish after death, especially when they dry out. This suggests that the distortion may have been due to osmotic changes in the tissues. bo ~J i) MORRIS M. WELLS 3. Resistance to sodium salts Experiments with the following sodium salts were performed in tap water: bicarbonate, carbonate, chloride, nitrate, and sul- phate. The solutions were 0.01N and the fishes small blue gills (8-5 grams). The results were as follows: Salt used Resistance of fishes Sodium bicarbonate Normal at end of 15 days; discont. Sodium carbonate Dead after 3 days Sodium chloride Normal after 19 days; discont. Sodium nitrate Dead after 31 days; only 50 cc. water left Sodium sulphate Normal after 20 days; discont. From the above results we see that the sodium salts are not toxic to blue gills when 0.01N concentrations are used in tap water. The carbonate is an exception as the fish dies in this solution in 3 days. It has already been shown (Wells 715 a) that these fishes cannot live in water that is even faintly alkaline and thus the action of the carbonate is due to its alkalinity. It will be remembered that the reactions of the fishes in salt gradients were complicated by the antagonism between the salts and the acid in the water. Loeb was the first to demon- strate that there exists an antagonism between salts and acids, as in 1899 he showed that acid antagonises the effect of NaCl on the swelling of muscle. He suggested that the antagonism depends upon the action of the substances upon the proteins of the tissues. Again, Loeb and Wasteneys (711 and 712) demon- strated the antagonism between salts and acids, in their effect upon the marine fish Fundulus and explained the effect as due to a direct action on permeability. Osterhout (’14) made in- vestigations which show that similar though less striking antago- nism between acids and NaCl occurs in plants; he further states that the antagonism is not as great as that between NaCl and CaCl. To determine the relation of the antagonism between salts and acids to the resistance of fresh water fishes, a series of experiments was run with NaCl and HCl. Table 9 summarizes the results of these experiments. From this table it will be noted that fresh water fishes of the same species and size live much longer in toxic REACTIONS OF FISHES TO SALTS 273 TABLE 9 Showing the antagonism of NaCl and HCl in their toxic action upon fishes; experi- ments performed in distilled water (U. of I.) DYING TIME SIZE AND SPECIES OF FISH KIND OF SOLUTION IN HOURS 2o-pram rock bass............... 0.25N NaCl 18 23-gram rock bass............... 0.25N NaCl + 0.00005N HG] 41 12- gram green spotted sun-fish.| 0.25N NaCl 48 8- gram green spotted sun-fish..| 0.25N NaCl + 0.00005N HCl 144 3-gram green spotted sun-fish..| 0.0001N HCl 48 3-gram green spotted sun-fish..| 0.0001N HCl + 0.12N NaCl | normal at end of month 45-gram green spotted sun-fish..| 0.25N NaCl + KOH to make 14 just alk. concentrations of NaCl when a trace of HCl is added. Also that fishes in toxic concentrations of HCl live longer when NaCl is present. Furthermore, NaCl is much more toxic in faintly alkaline solutions than it is in faintly acid solutions. All this agrees with Osterhout’s conclusions as to the effect of alkalies and acids on permeability. 4. Resistance to the salts of Ca and Mg The only resistance experiments which have been carried on with these salts are some that were performed at Chicago. The experiments with Ca were performed in connection with the acclimatization experiments already discussed. In brief, it was found that the sun-fishes lived very well in 0.01N CaCl, while the bull-heads did not live so well. Other experiments showed this same relation for the nitrate and sulphate but the latter salts were decidedly more toxic than the chloride and the sun-fishes did not live well in solutions of them. An interesting fact was noted in connection with the CaC l, experiments. A medium sized (50-gram) rock bass, after a week in 0.01N solution, showed signs of degeneration of the rays of the tail fin. This degenera- tion continued until nothing but the blood-reddened stub of the tail was left. The other fins were not affected ; the tail fin re- generated when the fish was returned to tap water. 274 MORRIS M. WELLS Day (87, p. 203) states that in a certain lake in the British Isles, there is a race of tailless trout which some authors claim can be traced as due to the action of deleterious matter in the water. Day (loc. cit) also quotes J. Harvie-Brown as saying, about 1876, ‘‘that a contraction of the rays of the tail fins of the trout in the river Carron occurred, and was believed to be due to the continuous pollution of the water through the agency of paper mills.”’ Upon looking up the composition of the waste from the paper mills (Griffin and Little "94, and Phelps ’09) I find that among other substances calcium is always present in large quantities, both as the chloride and in other combinations. Therefore the phenomenon reported by Day was likely due to the presence of an excess of calcium in the water. Marsh (’07) has shown that the waste from paper mills is very toxic to fishes. Calcium is not especially important, however, as the toxicity of the waste is probably due to the excess of acidity or alkalinity, and perhaps to other toxic substances. V. GENERAL DISCUSSION The experiments discussed in the preceding pages will be considered very briefly in one or two phases of their general bearing. From an ecological point of view they emphasize once more the ability of fishes to recognize and react to environ- mental factors in very small concentration. It should be pointed out, however, that the reactions of fishes to salts in solution are by no means so delicate as their reactions to acids and alka- lies, i.e., to hydrogen and hydroxyl ions. As a matter of fact the reaction to salts is complicated by the acid factor in many cases, as, for instance, when the salt gives an acid solution, but more especially in the numerous instances where there exists an an- tagonism between the salt and the acid. Thus fishes may react differently to a given salt concentration in water which is strongly acid and water that is but faintly acid. The resistance experi- ments show, also, that fishes can live in the presence of an acid concentration which would ordinarily kill them, if the proper concentration of the right salts is present. The work of Oster- hout (’15) and others, as well as data presented in this paper, REACTIONS OF FISHES TO SALTS DD indicates that the antagonism between the salts of calcium and magnesium is not nearly so marked as it is in the case of the salts of sodium and potassium. Since the former salts are by far the most common and plentiful in natural fresh waters, the importance of salts in nature in antagonising introduced acids is less than it would be were the salts of sodium and potassium plentiful. The problem is one which will furnish material for some very interesting ecological investigation. The importance of small amounts of ammonia in natural waters has been pointed out in the discussion of these salts. The effects of starvation upon fish metabolism and reaction will be further discussed in another paper. There is an inter- esting possibility brought out by the acclimatization and other data, especially those pertaining to the importance of acids, that will be discussed here. This possibility relates to the movements of organisms in general but the present discussion will be limited to the very interesting migrations of the anadro- mous fishes. The stimulus that causes anadromous fishes to spend part - of their life cycle in fresh and part in salt water has long been a matter for speculation. Such stimulus must be related to the rhythmical metabolism of the animal, for it brings the fishes into the sea or fresh water at certain definite stages in the life cycle. The state of the metabolism of these fishes while they are in the fresh water, must differ very decidedly from that during the period of the life cycle which they spend in the ocean, for these two en- vironments differ in two very important particulars, namely, the fresh water has a low specific gravity and is consistently acid in reaction, while the sea water has a relatively high specific éravity and is consistently alkaline. Also the reactions of the fishes are markedly different. In the fresh water they are posi- tive to current, and, in a gradient, select water that is just on the acid side of neutrality and of lower density than that of the sea. Salt water fishes, on the other hand, are probably negative to a fresh water current, select water on the alkaline side of neutrality and reject water of low specific gravity for that of higher (Shel- ford and Powers 715). The reactions of the fishes in fresh water, THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 19, NO. 3 276 MORRIS M. WELLS therefore, are the reverse of those in sea-water with regard to these three factors, and in the normal life cycle of such anadro- mous fishes as the salmon, this reversal in reaction must occur at least twice, once when the fishes leave the fresh water streams for the ocean, and again when they return. With species of salmon that breed more than once, the reversal must occur more often. There are two general complexes of factors to be considered in an attempted explanation of the reactions of the anadromous fishes, namely, the fish and the environment. Both are made up of physico-chemical factors which are measurable, and to a large degree quantitatively. Of the two complexes, that of the living organism is least understood and perhaps, because it is much more variable and changing than the environmental complex, which, especially in the case of the sea-water, varies hardly at all. For the fishes to live normally in the environ- ment there must exist between the two complexes a more or less complete equilibrium. A disturbance of this equilibrium re- sulting from a change in either of the complexes, will, if great enough or long enough continued, result in the death of the fishes unless by their reactions they seek out another environ- ment which allows their physiological processes to proceed nor- mally. It should be emphasized that the only mode of readjust- ment is through the proper reaction, either physiological or motile upon the part of the fishes, since the environment is much the more stable complex, and there is a great deal of evidence to show that of the two possible reactions upon the part of the living organism, the motile reaction is much more likely to occur than the physiological readjustment, i.e., acclimatization. The data presented in this paper and the one preceding (Wells ’15a) as well as that by Shelford and Allee (’13) and Shelford and Powers (15) show that fishes will react to environmental factors in a way that will tend to remove them from detrimental conditions, long before the adjustment becomes a matter of life and death. Thus we find the salmon leaving the fresh water for the ocean, when, as will be pointed out later, it has been shown (Day °87) that remaining in the fresh water for the entire life cycle would not REACTIONS OF FISHES TO SALTS 277 result fatally either to the individual or to the species. The mechanism, therefore, which is working to preserve the life of the organism is so delicate that 1t produces beneficial reactions upon the part of the animal far in advance of life and death com- plications. The working of this mechanism is undoubtedly closely correlated with quantitative and perhaps qualitative changes in metabolism. These changes in metabolism will have a direct relation to the amount of CO, given off by the organism. It has been shown that a slight increase in the carbon dioxide content of an animal’s blood results in a marked increase in the general irritability, and this increase in irritability would alone result in an increase in the range and vigor of the movements made by the organism. Thus no factor other than increased metabolism need be hypothecated to account for the stimulus which starts the breeding migration of so many animals. The directive factors which result in the animal’s coming into special conditions for the breeding activities are another matter. These ean be none other than the factors, physical and chemical, which are present in the environment. In the general metabolism of fishes, the stage of development of the sex organs plays an im- portant role, and it is very probable that the state of metabolism in these organs furnishes the initial stimulus which causes the animals to start upon the breeding migrations at.a given period of the life cycle. Treadwell (’15) points out that the eggs of the Atlantic palola give off an increasing amount of CO, as the swarming season approaches, and concludes that this indicates that there is probably an internal stimulus which is important in producing the swarm. There can be little doubt but that such internal stimulus is acting; the important fact, however, is that it has been shown that such internal changes in the physio- logical state of the animal may result in very marked changes in the animal’s reactions to environmental factors. Allee (712) has shown that, in isopods, a high rate of metabolism is cor- related with a high percent of positive responses to current and that a lowering of the metabolic rate in the animals will diminish and even reverse the rheotactic reaction. 278 MORRIS M. WELLS If we consider the different reactions of the salmon to current, acidity, and density, at different stages in the life cycle, begin- ning with the hatching of the egg we may proceed as follows. It is a well established fact (Loeb ’13) that in the fertilized egg and newly hatched fry, the rate of oxidation is high, and it seems to be clear (Wells 713) that from this time on, up to sexual maturity the rate runs down. That is, the rate varies inversely with the age of the fish. Salmon eggs hatched in fresh water must develop into fry which are able to live in slightly acid water, of relatively low density, and the fishes must also be positive to current or they will be swept from the stream. This we find is true and thus ability to live in fresh water is correlated at this time with a high metabolic rate. As time goes by, however, the rate of reaction becomes gradually lower until we find the fishes either becoming actively negative, or at least indifferent to current, and they are swept or swim into the ocean. They now live for some time in the alkaline water of the ocean, and are able to withstand its much higher density. The equilibrium between the environment and the organism is again disturbed after a time, however, and we find the fishes once more selecting the fresh water at another period of high metabolic rate, i.e., with the ma- turing of the sex glands. From this it would seem entirely possible that fishes which are normally fresh water forms might be temporarily transformed into salt water forms by regulating, that is lowering, the rate of metabolism. With regard to the selection of the water of greater or lesser density, the data presented in this paper offer an interesting possibility. It has been shown that fresh water fishes whose metabolic rate has been lowered by starvation, will select a notably higher concentration of CaCl. in the gradient than will normal fishes. Also older fishes select a higher concentration than do younger ones. Thus a lowering of the metabolism causes the fishes to choose a medium with higher specific gravity than that normally chosen. It will be remembered furthermore that a stay of a little less than a week in 0.01N CaCl: solution caused a fish that was normally negative to this concentration in a gradient, to become positive. Upon being returned to the tap REACTIONS OF FISHES TO SALTS 279 water the reaction was again reversed and the fish became negative once more. Acclimatization of fishes to salts must certainly be concerned with internal adjustments, for Sumner (’07) has shown that the specific gravity of fishes’ blood is altered when they are changed from fresh to sea-water, and vice versa. An alteration in the density of the blood seems then to result in a reversal in the reactions of the organism to density in the environment. Green (’04) has shown that changes in the specific gravity of . the blood of the salmon occur at the time the fishes are entering the fresh water; the blood gradually acquires a density that averages 17.6 per cent less than that of salmon in sea-water (l.e., p. 454). Jones (’87) has proved that age, exercise, sexual maturity, pregnancy, food, etc., have a measurable influence upon the density of the blood of man, and Sumner (’07) states that there are seasonable differences displayed by fishes, in the osmotic phenomena through their gills. It may be that the specific gravity of the blood of anadromous fishes at different stages in the life cycle, can be used as an index to the physiological changes that are going on in the organism. Aiso the effect upon the organism of a higher CO: production within the tissues must vary with the density of the blood and would probably be more marked when the blood is less dense. An investigation of the changes in the density of the blood of the salmon could perhaps best be begun with the fry in the fresh water streams. As the fishes remain for 2 years or even 3 in the fresh water before leaving for the ocean, a thorough study of the relative densities of the blood and the fresh water could be made in this period. That the instinct which causes these fishes finally to reject the fresh water for that of the sea, is backed by some very strong stimulus is indicated by data given by Day (87). Day speaks of an experiment which was carried on by Maitland in 1880. Eggs of salmon were hatched in fresh water, and the young salmon were placed in ponds shut off from the sea. These fishes ate well and grew vigorously until they were about 23 years old. At this stage in the life history, the indi- viduals are known as ‘smolts’ and it is at the smolt stage that they 280 MORRIS M. WELLS leave the fresh water. In October, 1883, one of the fishes jumped out of the pond onto the bank. By the end of November, several had jumped out onto the bank and died there (they usually jumped during the night or early morning). In the following May, 16 of the fishes were found dead on the bank. Then the following October (1884) they commenced constantly jumping out of the pond and meeting with fatal injuries. It was ob- | served that the fishes did not feed at this latter date; this failure to take food is characteristic of salmon entering fresh water to breed. Examination of the fishes which had jumped out of the pond showed that all were approaching maturity and in the later cases, the eggs and sperm were ripe. An attempt was made to fertilize the eggs with the sperm, with good success. Day states that this second generation was normal and vigorous up to 20 months and concluded that it was definitely proved that a so- journ in salt water is not necessarily for the development of the sexual products. If this is true, the migration of the salmon into the salt water, and back again, is all the more curious. There would be advantages and disadvantages to such behavior but the above data prove that the fishes are reacting to the environ- ment in a way that is not immediately essential though the stimulus seems to be a very strong one. A study of the behavior of these fishes in salt, acid and alkali gradients at different stages in their life history, would undoubtedly prove very suggestive and such a study correlated with physiological investigations of the fishes at similar stages will without doubt solve the question of the movements of anadromous fishes. VI. GENERAL CONCLUSIONS 1. Fresh water fishes recognize and react to the presence of salts in solution. The reaction is one which tends to bring them into their optimum salt concentration. 2. Fresh water fishes (and probably marine fishes also, Shel- ford and Powers °15) are not as sensitive to salt ions as they are REACTIONS OF FISHES TO SALTS 281 to hydrogen and hydroxy] ions. The reactions to either the ions of salts or acids are complicated by the presence of the ions of the other. 3. Fresh water fishes react to combinations of antagonistic salts or to an antagonistic salt and acid, in a way that tends to bring them into a region of optimum stimulation. The phe- nomena of antagonism are thus indicated by the behavior as well as the resistance of organisms. 4. Starvation causes certain fishes (e.g., Ambloplites rupestris, rock bass) to select higher concentrations of salt than those normally selected. Other fishes (Ameiurus melas, bull-head) when starved, select lower concentrations than normally. Over- feeding causes bull-heads to select higher concentrations, than those normally chosen. 5. Rock bass and bull-heads which are normally negative to CaCl, 0.01N solution, become positive after being kept in this concentration for about a week. They become negative again when returned to tap water for 24 hours. 6. The migrations of anadromous fishes are probably corre- lated with rhythmic changes in metabolism. These alterations in metabolic activity are largely the result of internal changes such as occur with the ripening of the sexual products. I am indebted to Prof. V. E. Shelford for proposing this prob- lem and for many suggestions during the work. Iam also under obligation to Mr. Karl A. Clark of the Chemistry Department for helpful criticisms and for the loan of apparatus. 282 MORRIS M. WELLS BIBLIOGRAPHY AutuLeE, W. C. 1912 An experimental analysis of the relation between physio- logical states and rheotaxis in Isopoda. Jour. Exp. Zodl., vol. 13, pp. 269-344. °1913 Further studies in physiological states and rheotaxis in Isopoda. Ibid., vol. 15, pp. 257-295. ALLEE, W. C. AND TASHIRO, SHIRO 1914 Some relations between rheotaxis and the rate of carbon dioxide production in Isopoda. Jour. An. Behav., vol. 4, no. 3, pp. 202-214. Cuitp, C. M. 1913 Studies on the dynamics of morphogenesis and inheritance in experimental reproduction. V. The relation between resistance to depressing agents and rate of metabolism in Planaria dorotocephala and its value as a method of investigation. Jour. Exp. Zodél., vol. 14, pp. 153-206. Day, Francis 1887 British and Irish Salmonidae. Williams and Norgate, 14 Henrietta St., Covent Garden, London. GREENE, C. W. 1904 Physiological studies of the Chinook salmon. Bull. U. 8. Bur. Fisheries, vol. 24, pp. 429-456. GrirFin, R. B., anp Lirttr, A. D. 1894 The chemistry of paper making. Howard Lockwood and Co., New York. Jones, E. Luoyp 1887 On the variations in the specific gravity of the blood in health. Jour. Physiol., vol. 8, p. 1. Littiz, R.S. 1910 The physiology of cell division. II. The action of isotonic solutions of neutral salts on unfertilized eggs of Asterias and Arbacia. Am. Jour. Phys., vol. 26. 1911 The physiology of cell division. III. The action of calcium salts in preventing the initiation of cell division in unfertilized eggs, through isotonic solutions of sodium salts. Am. Jour. Phys., vol. 27, p. 289. ; Lores, Jaccuges 1899 Ueber die Aehnlichkeit der Fliissigkeitsresorption in Muskeln und in Seifen. Pfliiger’s Archiv, Bd. 75, p. 303. 1912 Untersuchungen uber Permeabilitit und antagonistiche Elektro- lytwirkung nach einer neuen Methode. Biochemische Zeitschrift, 47, p. 127. Loes, J., AND WASTENEYS, HarpotpH 1911 Die Entgiftung von Séuren durch Salze. Biochemische Zeitschrift, Bd. 33, p. 489. 1912 Weitere Versuche iiber die Entgiftung von Saiuren durch Salze. Biochemische Zeitschrift, Bd. 39, p. 167. MacCatium, J. B. 1905 On the diuretic action of certain haemolytics and the action of calcium and magnesium in suppressing the haemolysis. Univ. of Calif. Publ. 1-2, p. 93. REACTIONS OF FISHES TO SALTS 283 Marsu, M. C. 1907 The effect of some industrial wastes on fishes. House Documents, vol. 64. Water Supply and Irrigation Papers. 1906- 07, p. 387. Matuews, A. P. 1907 The cause of the pharmaologicol action of ammonium salts. Am. Jour. Phys., vol. 18, p. 58. Meutzer, 8. J.. anp Auer, J. 1908 Rigor mortis and the influence of calcium and magnesium salts upon its development. Jour. Exp. Med., vol. 10, p. 45. OsterHouT, W. J. V. 1914 Antagonism between acids and salts. Jour. Biol. Chem., vol. 19, p. 517. 1915 On the nature of antagonism. Science, vol. 41, p. 255. Puetes, HE. B. 1909 The pollution of streams by sulphite pulp waste. A study of possible remedies. U.S. Geol. Surv. Water Supply Paper 226. Rincer, 8. 1886 Further experiments regarding the influence of small quan- tities of lime, potassium and other salts on muscular tissue. Jour. Physiol., vol. 7, p. 291. SHELFORD, V. E., anp ALLEE, W. C. 1914 Rapid modification of behavior of fishes by contact with modified water. Jour. An. Behav., vol. 4, no. 1, pp. 1-30. SHELFORD, V. E., AnD Powrrs, E. B. 1915 An experimental study of the move- ments of herring and other marine fishes. Biol. Bull., vol. 28, pp. 315-334. Sumner, F. B. 1905 Further studies of the physical and chemical relations between fishes and their surrounding medium. Bull. Bur. Fisheries, vol. 25, pp. 55-108. 1907 Further studies of the physical and chemical relations between fishes and their surrounding medium. Am. Jour. Physiol., vol. 19, p. 61. Wetts, M. M. 19138 The resistance of fishes to different concentrations and combinations of carbon dioxide and oxygen. Biol. Bull., vol. 25, no. 6. 1915a Reactions and resistance of fishes in their natural environment, to acidity, alkalinity and neutrality. Biol. Bull., vol. 29, pp. 221-257. THE PREDETERMINATION OF SEX IN PHYLLOXERANS AND APHIDS T. H. MORGAN Department of Zoélogy, Columbia University FIVE TEXT FIGURES AND TWO PLATES CONTENTS IMGLOUUCTION...~ e444 INE A elle Weve Scns w'6ce Oyo ARRON cc ee eo eae 285 fiberstem-mother sexes acre 1a een e ERIM oe) sete. tite a len Saat eee 287 The polar spindle of the male egg of Phylloxera fallax................... . 289 Phe chromosome cycle of Phylloxera fallax...4...0-2 226... s eee eee . 290 The chromosome cycle of Phylloxera caryaecaulis.....................4.. . 291 Semnaniosml Ebyvlloxers fallax: ete Peer Wetec ati) sss acces 295 The spermatogenesis of the bearberry aphid...... «of AMEE ZS CR eee acca » 207 PeTeoraploidicyst im the bearberry: aphidien-emermceieen ess es oon 301 The omission of synapsis in the parthenogenetic eggs of phyloxerans and 213110 See EC AE I03 e pes Sa OT Ree emer . 304 Elishommen lene tnOSPCCb isch > 6 ous So. Fos Ae ROO he coe eae wos 309 The life histories of certain aphids in relation to predetermination of sex... 313 [Shi] bill ONG OU NN Ge ge tet oe ee EM CO. ccre a 6 Ona o ofc ah une OO ae eee 316 INTRODUCTION Two of the critical stages in the life of the phylloxerans of the hickories have already been shown to be intimately connected with changes in the cytological relations of the chromosomes. One of these stages involves the formation of but a single class of spermatozoa, that corresponds to the female-producing class of other insects. This fact explains why the fertilized egg gives rise to females only. The second critical stage involves the elimination of chromosomes from the small eggs that are to be- come males. This fact explains how the male comes to have fewer chromosomes than the female, and brings him into line with other Hemiptera, in which a similar relation holds. There is a third stage that might also be looked upon as a critical stage; namely, the stage at which the polar body is given off from the egg of the stem-mother; because in P. caryaecaulis after that event all of the offspring from one stem-mother are : 285 * 286 T. H. MORGAN known to produee large eggs, and all of the offspring of other stem-mothers to produce small eggs. It follows, for this species, either that there must be two kinds of stem-mothers, or else that some external factor must determine that one kind of stem-mother produces daughters all of which contain large eggs, and another kind of stem-mother produces daughters all of which contain small eggs. If such effects are produced by the environment they must be wrought before the mother is mature, because all the daughters of a single female are alike in regard to the size of the eggs that they carry. On the other hand in P. fallax some of the offspring of one stem-mother contain large eggs while other offspring of the same mother contain small eggs. In this species there must be only one kind of stem-mother, and either external conditions, or some difference that arises when the polar body of the stem-mother’s egg is extruded, must determine whether a given egg becomes a large egg carrier or a small egg carrier. In either case it became necessary to find out what occurs when the polar bodies of the egg laid by the stem-mother are given off before any further advance in the analysis was possible. During the last two years I have studied these stages, and I am now in position to give the complete history of the chromo- somal cycle. And I can now also give certain additional facts connected with the chromosomes at the time of extrusion of the polar body of the male egg of P. fallax. This new evidence makes possible the interpretation of the entire life cycle of the phylloxerans; an interpretation that has a wider interest and application than relates to the life cycle of this group alone. Before taking up the history of the chromosomes, I may re- eall the salient features in the life cycle of the two species that are to be considered. The life cycle of P. caryaecaulis is shown in figure 92 in my book on ‘‘ Heredity and Sex.’’ The stem-mother that hatches in the early spring produces a gall on the leaf of the hickory. As soon as she is mature she begins to deposit her eggs within the gall. From these eggs winged daughters hatch; all those in one gall contain large eggs, all those in another gall contain small eggs. The winged forms leave the galls and deposit their eggs - 4 PREDETERMINATION OF SEX 287 on the under surface of the leaves of the hickory where they hatch within a few days. From the large eggs emerge sexual females, each of which carries a single egg. From the small eggs, males emerge that are sexually mature at birth. Copulation ensues; the sexual eggs are laid on the stem of the tree. From these fertilized eggs the stem-mother hatches in the following spring. The life cycle of P. fallax is as follows: As soon as the stem- mother is mature she begins to deposit. her eggs within the gall. These eggs give rise in this species to wingless daughters (a few winged daughters are also sometimes produced). Whether the daughters in a particular gall are of two kinds, 1.€., some contain- ing only large eggs and others only small eggs, is not known, but both kinds of eggs are found in each gall. The large eggs, laid within the gall, give rise to sexual females. The small eggs, also laid within the gall, produce males. The sexual forms that hatch from the eggs craw] out of the gall (whether before or after mating is not known), and the single sexual egg that each female carries is deposited on the tree. THE STEM-MOTHER’S EGGS As soon as the galls on the young leaves begin to enlarge in the early spring the stem-mother, one in each gall, begins to lay her eggs. If the eggs and embryos contained in the gall are col- lected and preserved, there is a chance that the last laid egg may be forming its polar body and there is the further possibility that one or more of these may be caught in the anaphase. A very large number of eggs had to be cut into sections before the de- sired stages were found. One wonders in fact that any eggs are actually obtained in this phase of division. In all I have records of six anaphases that give the desired information. The initial question is whether there exists a ‘lagging’ chro- mosome at this time that might indicate the elimination from some of the eggs of a whole chromosome. A more certain de- termination would be the count of the chromosomes in the two anaphase plates; but only an extraordinarily favorable case would allow of this being done. Moreover, several such counts 288 T. H. MORGAN would have to be made in order that the results be significant, for only half of the eggs at most might be expected to show such a chromosome reduction. I think, however, that the pres- ence or absence of the lagging chromosome would make the conclusion reasonably certain. I wish to express here my in- debtedness to Miss Edith M. Wallace who has searched through the material and picked out the critical stages. The drawings are also due to her skill. There are shown in plate 1, figures a tof, six anaphase stages of the polar spindle of the egg of P. fallax and in plate 1, figure m, one anaphase stage of the egg of P. caryaecaulis. In none of these stages is there any evidence of a lagging chromosome, and since these stages range from a very early anaphase (a) ‘to the final stages when the daughter nuclei are reconstructing, there is a strong presumption for the view that no such lagging chro- mosome occurs. This conclusion tallies, moreover, with the es- timated chromosome number. For instance, it was known that in P. fallax there are 12 chromosomes in the equatorial plate of the stem-mother’s egg, and this same number is characteristic of the somatic cells of the embryo that arises from the egg. If no mistake on the latter counts have been made (the somatic chromosomes are elongated, and, therefore, more difficult to count) there could have been no loss of chromosomes in the polar body. In the other species, however, where only six of the eight chromosomes are apparent in most stages, it might be imagined that loss of one of the attached chromosomes in the polar body, while not affecting the visible count, might so alter the internal relations as to furnish a new point of departure. It would not be profitable here to take up at length the possi- bilities involved in such a supposition. I have examined them with some care, and have not found that they would furnish any satisfactory solution. On the other hand, the evidence for P. fallax is so clear, and the similarity in the two types in all essen- tial points is so evident, that I think we may accept this evidence from P. fallax as strongly in favor of the view that all of the chro- mosomes divide when the single polar body is given off from the egg of the stem-mother. PREDETERMINATION OF SEX 289 THE POLAR SPINDLE OF THE MALE EGG OF PHYLLOXERA FALLAX In my earlier work I did not obtain any anaphase stages of the polar spindle of the male-producing egg of this species; although a number of equatorial plates were obtained and figured. For certain reasons, that need not now be given, it became evident that this stage in this species should give an answer to certain questions and a long search for anaphase figures was begun. In P. fallax the eggs are laid one after the other by each female. Hence not more than a few eggs in the desired stage could pos- sibly occur in a single gall, which renders the chance of finding such a stage very small indeed. Nevertheless, one excellent ana- phase was found by Miss Wallace, and is drawn here in plate 1, figure g. As shown in the figure, there are two lagging bodies in the middle of the spindle that are conspicuous by their large size. At the inner pole there appear to be ten chromosomes, at the outer pole eight or nine chromosomes. The equatorial plate from which this figure developed must have contained twelve chromosomes, since this number of chro- mosomes has always been found present in such a plate. Of these, eight were autosomes and have divided, so that eight daughter chromosomes go out into the polar body and eight re- main in the egg. The four sex chromosomes, that are pre- sumably paired at this time remain to be accounted for. If the two bodies seen in the middle of the spindle are two whole X chromosomes, then there canbe but eight in the outer plate (which becomes the polar nucleus). There will be ten chromosomes at the inner pole of the polar spindle. But if the two lagging chro- mosomes represent a single X chromosome, procociously split in two, there will be nine chromosomes in the outer plate (which be- comes the nucleus of the polar body). There will be as before ten chromosomes at the inner pole of the spindle. Unfortu- nately it is not possible to determine, with certainty, whether there are eight or nine chromosomes in the outer plate. I am inclined, nevertheless, to adopt the former interpretation as the more probable, because of the evidence from P. caryaecaulis that bears on the same point. It will be observed that the end 290 T. H. MORGAN result is the same whether the lagging bodies represent two X chromosomes, or one X chromosome precociously split, for on either view eight (half) autosomes and two whole chromosomes (X) pass out of the egg, leaving eight half autosomes and two whole (X) chromosomes within the egg. The polar bodies of three other male-producing eggs are also shown in plate 1, figures fh, i, 7. These show one or two dark staining granules outside of the nucleus of the polar body, which I interpret as the remains of the lagging chromosome. In this respect they behave in the same way as do the lagging chromo- somes in P. caryaecaulis which also fail to enter the nucleus of the polar body. A fourth drawing, figure k, represents a late stage in polar body formation in which twelve chromosomes are distinctly counted in the inner nucleus of the polar spindle. The outer nucleus is only partly present in this section, and the rest of it could not be found in the neighboring sections. This egg must have been a female-producing egg in which there is no lagging chromosome, and in which there are twelve chromosomes at the inner pole. Bringing these facts into relation to those already made out, we can construct the chromosome cycle of P. fallax, and with this as a clue make clear the similar cycle of P. caryaecaulis. THE CHROMOSOME CYCLE OF PHYLLOXERA FALLAX The main phases in the cycle are as follows: 1. There is a single kind of stem-mother in this species whose eggs contain twelve chromosomes that divide when the single polar body is given off, so that twelve chromosomes pass out and twelve remain in the egg (plate 1, a tof). 2. These eggs develop into wingless females that produce large and small eggs, but whether the same female produces large and small eggs could not be determined. Both kinds of eggs are found within the same gall, and therefore come from daugh- ters of one original stem-mother. 3. The large egg—the so-called female-producing egg—gives off one polar body, all the chromosomes dividing at this time (plate 1, k). This egg develops into the sexual female. The PREDETERMINATION OF SEX 291 small egg also gives off only one polar body (plate 1, g, h, 4, 7). At this time the four sex chromosomes conjugate, so that two whole chromosomes pass out into the polar body, as lagging chromosomes, and two remain in the egg while all the other chromosomes divide. The male that develops from this egg has ten chromosomes. 4. The sexual female produces but one egg, the reduction in the number of chromosomes taking place at this time (plate 1, 1). Presumably two polar bodies are given off , leaving six chro- mosomes in the egg. 5. In this species the male has ten chromosomes, two of which are X chromosomes. During the first spermatocyte division these two chromosomes pass into the functional cell, so that all the functional sperm come to have six chromosomes. 6. When these sperm fertilize the sexual egg the total number of chromosomes is again brought back to twelve. THE CHROMOSOME CYCLE OF PHYLLOXERA CARYAECAULIS The main relations of the chromosomes in this cycle are illus- trated in diagram 1. There are four ordinary chromosomes or autosomes (colored black) and four sex chromosomes (repre- sented by open circles). Two of the sex chromosomes are as large as the autosomes and two are much smaller. The latter are in most stages loosely united to the larger sex chromosomes, “ one to each. One of the small chromosomes is mar/red by two cross lines in order to distinguish it from the other one.: I have marked it in this way because, as will be shown, an important fact in the life cycle of this species can be accounted for, if one of the two small sex chromosomes is different from its mate. To the left in the diagram the line culminates in the sexual egg. As seen to the right, the line derived from another stem-mother (the one that contains the marked X) leads to two kinds of males, each of which produces its particular class of spermatozoa, one kind containing the open x and the other kind the marked x. If we first follow down the female line to the left, we see that the egg laid by the stem-mother contains eight chromosomes. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 19, no. 3 292 T. H. MORGAN PHYLLOXERA_ CARYECAULTTS @®% = e? nye && Slo Wicthers 94 os CA \ we G=D A soit ~~ £ Sa G Winged Gen Ce” 8 Plax Plate a o é Petar Plate var sn | a - ey ¥ ea ae eee J ee re e °@ Qy @ © | @ @@0 Petar Spindle | | | ||| Me eee Sfumele ge SF Co < at o> ngals ° 44 Sexual temale +a Fw Wale Fit oy emalocyle @>) Se Cora / Sfrermatecife Diagram 1 Illustrates the chromosomal cycle of Phylloxera caryaecaulis; to the left is shown the line that culminates in the sexual female; to the right is shown the line that culminates in the males; this line splits into two subdivisions after the extrusion of the polar body in the male-producing egg. GF ~ Felar bodies and 49 aa “5 Qs @ CS o PREDETERMINATION OF SEX 293 When the polar body is thrown off all of the chromosomes divide. The migrant that develops from this egg comes to produce large eggs with the full complement of chromosomes in them. All of the chromosomes divide when the polar body is produced by this ege (plate 1, fig. n). The egg itself then develops into the sexual female that comes to produce a single egg. When this egg is ripe the chromosomes have united in pairs, of which there are three. In reality one of these pairs must be thought of as made up of the two large sex chromosomes and the two small chromo- somes attached to them. If we next turn to the line represented on the right of the dia- gram we see that the stem-mother contains the same group of chromosomes as in the other case: (one of the small sex chromo- somes is marked). All of the chromosomes divide when the polar body of the egg of the stem-mother is given off. The egg gives rise to the migrant that comes to produce the small eggs. When the polar body, is about to be produced in these small eggs (or before that event) a shifting of the sex chromosomes takes place, so that the two large X’s come together as do the two smali ones, the latter leaving their former loose attachment to the large chromosomes in order to combine. When the polar body is given off the four autosomes divide, while the conjugated pairs of sex chromosomes separate; a member of the larger pair lags on the spindle and is precociously split lengthwise, while the small pair separate and the outer member does not lag on the spindle (plate 1, fig. 0). It will be seen that two kinds of eggs should result according to whether the marked chromosome passes out into the polar body, or remains in the egg. If it passes out, the male that results will give rise, as shown in the figure, to female producing spermatozoa that contain the open x. If we suppose the sexual egg is fertilized by this kind of spermatozoan, the stem-mother that results will give rise to the female line (to the left). If we suppose that the other kind of sperm—the one with the marked x—fertilizes a sexual egg, a stem-mother will be produced that leads to the male line (to the right). The assumption that there are two kinds of males is not entirely hypothetical; for, in my former paper I pointed out 294 T. H. MORGAN that two kinds of males are found that differ from each other in the relation of the small x to the large X chromosome—in one kind of male the two remain in contact, in the other the two keep apart. And I pointed out that all of the spermatocyte cells of a given individual show one or all show the other relation. This is in accord with the assumption of two kinds of males that arise when the polar body is eliminated. In some of the preparations showing anaphase figures in this species, one of which is redrawn here in plate 1, figure o, the two lagging chromosomes appear unequal in size. In other figures, however, (figs. 5, 6, 21, of my 1912 paper) the two lag- ging chromosomes appear to be equal or subequal. For this reason I stated (12) that the two equal chromosomes represent one large sex chromosome divided into halves. On this view the conjugating pair of small x’s have been separated and moved to their respective poles while only the large X lags as it passes to the outer pole. It will be noticed that superficially the number. of chromo- somes in P. fallax is double that in P. caryaecaulis. This sug- gests that the group in P. fallax represents the double number of chromosomes of caryaecaulis (tetraploid), or that caryaecaulis is P. fallax halved. But if the full number of chromosomes in P. caryaecaulis is looked upon as eight, this relation does not hold, unless one supposes that there are four small chromosomes present also in P. fallax (giving sixteen in all) that are attached permanently to other chromosomes. From this point of view the chromosomes of the one species would be double the number of the other and P. fallax would become XX and XY, while P. caryaecaulis would become X and Y. In other words the small chromosomes would no longer be reckoned as factors in the sex scheme. I have preferred, however, the interpretation given in the diagram because the two small chromosomes conjugate in the male egg when the large sex chromosomes conjugate; and, again, because one of them lags in the first spematocyte division along with the lagging X. But the latter relations may be only a con- sequence of the first, and be due to the absence of a mate at this time. Still, since it goes into the female-producing sperm, and PREDETERMINATION OF SEX 295 not indifferently to either pole, this would seem the more natural designation. Without wishing, therefore, to lay too much stress on the nomenclature, it seems to me that the one I have followed represents more naturally the facts as described in the text. SEX RATIOS IN PHYLLOXERA FALLAX Since the galls in this species do not open to release the sexual forms until a considerable number of them have hatched, and since all of the inhabitants (except for rare cases) are the de- scendents of a single female it is possible to get a fairly good sample of the output of each stem-mother. In order to antici- pate a possible objection, viz., that two or more stem-mothers might be included within the same gall, I opened a number of very young galls and found, with the rarest exceptions, that each gall is the result of the activity of a single female. We are safe in concluding, therefore, that the contents of each gall is the pro- duct of a single stem-mother. But whether her daughters, the apterous ‘migrants,’ are individually small-ege-producers or large- egg-producers can not be determined; because, in this species, the apterous ‘migrants’ deposit each egg as.it matures. Since eggs that are not quite mature look like small eggs they can not be distinguished from them. Only by finding a gall in which a single apterous ‘migrant’ was present that had laid both kinds of eggs would it be possible to settle this question. I have al- ready reported that occasionally the migrants are winged, and that when this occurs each individual contains eggs that were all of one sort, namely, small eggs in this instance, but this does not settle the other question, however probable it may appear, that each individual of this generation produces only one sort of egg. In the following table the counts from 26 galls (July 11) are given. Those with the same letter come from the same leaf. The galls were old and ready to open. The old apterous ‘mi- grants’ were still present, but empty, as though they had about finished their productive life. There were present as many eggs unhatched as hatched; only the latter appear in the record, or 296 T. H. MORGAN rather the individuals that hatched from them. In some of the galls the dwarf forms recorded in my former paper were also found. : Except in a few cases the females were in excess of the males. If galls are opened, when the first sexual forms have begun to hatch, males (one, two, or three, ete.) willbe found. This means, TABLE 1. MALES SEXUAL FEMALES OP 28 36 OP 20 31 OP 6 14 OF 15 2 OP 30 15 AbD 4 11 Sieh 18 32 Abb 29 39 PT 5 6 a 21 28 jigs 15 26 ss 24 33 SS 21 23 ss 2 7 SS 9 18 SS 2 2 SS 14 nT, ss 12 28 SS 18 12 4 27 10 21 ww 4 51 ww 17 30 ww 30 32 ww 8 21 ww 36 66 405 628 no doubt, that males hatch first, or possibly that the first eggs produced are males. Since later more females than males are present, it follows that more female-producing than male-pro- ducing eggs are laid. The most striking departure from the ordinary ratios is that in the first count in WW where there were 7 males to 51 females. PREDETERMINATION OF SEX 297 Galls of the same size on the same leaf must have been sub- jected to as nearly uniform conditions as possible, but the counts from the same leaf are not strikingly at least more like each other than they are like other counts from other leaves. It is idle perhaps to speculate as to the factors that predeter- mine whether a given. egg shall be a large or a small one especially as this may depend, as in the other species, not on the conditions that affect the migrant, but on the conditions that determined the nature of the migrant herself. The recent work of Whitney and of Shull shows that very slight differences in the environment turn the scale in sex predetermination. Differences like those described by them might accompany the differences in food con- ditions of the leaf during the day when starch is being made and during the night when starch is being converted into sugar. If, perchance, such environmental changes affect the stem-mother, the differences as to output shown by the galls, might arise. THE SPERMATOGENESIS OF THE BEARBERRY APHID Stevens and von Baehr have described very completely the spematogenesis of several species of aphids. I have also studied at different times a number of species but as they gave nothing new I have not published the results, except for one very brief account (Jour. Exp. Zodél. ’09. pp. 298-305). There is one species, however, which in clearness and simplicity exceeds all others that I have seen. I give here therefore an accountof some of the critical stages in its spermatogenesis and oogenesis. The data furnish, moreover, an occasion to make certain com- parisons between the phylloxerans and the aphids. The species in question, Phyllaphis coweni Cockerell, forms galls on the bearberry. My material was found north of Quissett, Mass., near Woods Hole, where I have collected material for four summers. I have found the galls on the bearberry during June, July and August. Each contains, as a rule, a single stem- mother—rarely two—and her progeny. As ‘the gall gets older the progeny is seen to be made up of larger individuals with wing pads and in addition a series of immature forms, as well as afewmales. The large individuals with wing pads contain sexual 298 T, H. MORGAN eggs. As I have never found winged individuals within the galls the individuals must leave the galls when ready to expand their wings. The origin of the males puzzled me for some time until I discovered that the stem-mother produces them—at first spar- ingly, but later in larger numbers. The same stem-mother that contains the males also contains young sexual females. Shealso produces at times both males and individuals that contain par- thenogenetic eggs and embryos with six chromosomes. Throughout July and August young galls can always be found at the growing ends of some of the branches. These galls con- tain young stem-mothers, and later some of their progeny. ‘These younger stem-mothers may possibly come from the old stem- mothers, or from belated eggs of the previous year, or from sex- ual eggs of the same year in which they appear. A more de- tailed examination will be necessary to settle this point. The sexual eggs begin to pass through their synapsis stages while the young are still present in the stem-mother but even after the young are born and after some of the eggs have left the ovary, eggs at the outlet may show the chromatin contracted at one side. In the male the two reduction divisions may also take place while the young individual is still within the stem- mother, but other individuals do not contain these stages until after the young males have been born. Entire individuals were preserved in Carnoy solution, the ab- domen. cut into sections, and the sections stained in iron hemo- toxylin. The carly spermatogonial cell contains f ve chromosomes (dia- gram 5). Ata later stage I found what seems to be a contraction fgure, plate 2, figures 1 and 2, when the chromatin is shrunken and lies at one side, but as there is nothing specific about this condition it is with some hesitation that I identify it as the synizcsis stage. The early prophases, plate 2, figures 3, 4 and 5, are interest- ing. The three chromosomes are easily distinguished, even be- fore they shorten into rods. Miss Stevens has described a stage that she calls the synapsis stage; but from her figures it seems not improbable that she has PREDETERMINATION OF SEX 299 seen only the early prophases of the first division or possibly the stage between the first and second spermatocytes. The side view of the first spermatocyte division is shown in plate 2, figure 6. An equatorial plate is shown in plate 2, fig- ure 5, which gives the relative sizes of the three chromosomes. Stages in the later first division, some of them duplicates of each other, are shown in figures 7 to 11. The two autosomes di- vide; the X chromosome is drawn out, and is usually dumb-bell- shaped. It generally shows a well marked longitudinal split. The split is so distinct that the halves appear often like two parallel lagging chromosomes. Only in the latest stages of the division is it apparent that the whole X chromosome passes into the larger of the two cells. As in other aphids, the X chromo- somes are, during this division, often constricted near the middle, which in some species is sometimes carried so far that the two enlarged ends are connected by a mere strand. It is this ap- pearance that has led Miss Stevens in her earlier work to infer that the X chromosomes were really divided at this time. In the aphid of the bearberry the constriction appears at first at the middle of the chromosome. It seems later to pass more and more towards one end, until ultimately, as shown in figures 11 and 13, a small piece only is left at one end, which in most cases is later drawn into the thread; although once or twice I have found cases, as in figure 12, in which this terminal piece appears to have broken away from the strand connecting it with the rest. The difference in size of the two cells varies greatly in the earlier phases, as the figures show. But ultimately nearly all of the protoplasm passes into one cell, the one that contains the X chromosome. The small cell is left with two chromosomes and a small amount of cytoplasm. It never divides again, and later degenerates. Stevens was inclined to think that the small cell may sometimes show a division figure, which subsequently fades away, but I have never seen a case of this kind. The two autosomes in the functional cell begin to lose their condensed condition and spread out into loose masses, as shown in figures 16 and 17. The X chromosome is later in passing into this con- * 300 T. H. MORGAN dition, but does so before condensation sets in again, prepara- tory to the next division. In one case, figure 15, the X chro- mosome failed to draw out of the smaller cell, and one end only lies within the nucleus of the larger cell. This end has begun to open out as the other chromosomes have already done. The condition of the other cells in the cyst makes this interpretation probable. During the resting stage, the X chromosome becomescon- densed, as shown in plate 2, figures 14, 15, 16, 17. The chromo- somes in the larger cells next begin to condense, preparatory to the second division. The X chromosome is distinctly larger than the other two; all three divide equally to form the sperma- tids (plate 2, figs. 18, 19, 20). The double nature of the X chromosome in the first spermato- cyte division is so apparent that it invites speculation concern- ing the nature of the division. Presumably the second divison occurs in the plane of the split, but it is impossible to follow this chromosome through its resting stage. Similar cases for the single X chromosome are known, and I can but follow the usual interpretation, viz. that we are dealing with a precocious divi- sion. The tetrad formation that occurs in such forms as Asca- ris is interpreted as a double division, one of which is precocious. The rapidity with which the two reduction divisions take place, often without an intervening resting stage, indicates that each of the chromosomes, even though mated in pairs, has under- gone the preparatory stages of division. Hence two divisions are necessary to separate the four elements. But if this were the whole of the matter it is not apparent why, in a case like this one, the halves of the X chromosome do not separate from each other at the first division. It is perhaps little more than an evasion of the difficulty to suggest that the divisions in the X chromosome are not sufficiently advanced, when overtaken by the first division. Janssens has proposed a view of the necessity of the two re- duction divisions, based on his observations of the chiasma type. His studies of Batracoseps have shown that two of the four threads of the tetrad sometimes break and reunite so that two PREDETERMINATION OF SEX 301 new threads are made up of parts of each of the two original threads. Under such circumstances a single maturation divi- sion would often produce a dyad in which the two threads are genetically unlike. Janssens assumes that this is repugnant to the scheme of reduction, whose purpose is to give rise to a gamete with a single set of units (gens). This solution of the problem rests on the supposed necessity of pure gametes, and this in turn could be more directly accomplished, it would seem, if the conjugating chromosomes separated without interchanging parts, as they do, in fact, in the male of Drosophila. Granting, however, that ‘crossing over’ does occur as a necessity of the physical conditions prevailing at this time, Janssens’ hypothesis might appear to furnish a solution, if at the same time the ne- cessity for pure gametes could be shown essential to development. Obviously, however, the act of conjugation is a capital arrange- ment to give the zygote a heterozygous make-up, and why a quadruple set of gens instead of a double one would be a disad- vantage is not self-evident. A TETRAPLOID CYST IN THE BEARBERRY APHID In a male of the bearberry aphid one cyst was found in which all the cells have the double number of chromosomes including the sex chromosomes. The cyst is in the first spermatocyte stage. Since the other cells in the same testis and in the testis of the other side are normal the tetraploid condition must have arisen from a spermatogonial cell whose chromosomes but not the cytoplasm divided. The other possibility, namely, that the four autosomes failed to conjugate, would not account for the presence of two X chromosomes, nor explain the doubling in all the cells of the cyst, because conjugation occurs long after the cells of a cyst have become separated. The former interpreta- tion is therefore to be preferred. The cells in this cyst, of which a few are shown in plate 2, figures 21-30, are completing, or else have completed, the first spermatocyte division. Taking the figures in order, we see in f gure 21 four autosomes at each pole— only three show in the larger cell—and two X’s extending from 302 T. H. MORGAN one nascent nucleus to the other. Practically the same rela- tions are shown in figure 22, where however, the two X’s appear to be passing into the larger cell. In figure 23 the two X’s stand end to end as is also the case in figures 24 and 25. It is not pos- sible to determine into which cell they might have passed—pre- sumably, however, one to each cell. In figure 26 this result seems to have been attained, since each cell contained four auto- somes, and one X. In figures 27, 28, 29 and 30, the division having been accomplished, only the larger cell is shown. In each case there are two X chromosomes and four autosomes in the cell. There are some questions here of theoretical interest. The autosomes appear to have acted as pairs, if one may judge by the equal distribution to the daughter cells, which seems to have taken place in many cases, although it can not be established for all cases. As there are four autosomes of each kind their copu- lation in pairs does not seem unexpected. The behavior of the X chromosomes is unique. In some cases they have passed to opposite poles and in this sense have acted as a pair, but in most cases they have passed into the larger cell. The first spermato- cyte division in phylloxerans and aphids is of such a kind, that the X chromosome passes into a particular cell, i.e., it does not pass indifferently to either pole. But as a matter of fact we do not know whether the larger cell into which it passes is prede- termined (by some polar relations in the cell) and the X chromo- some follows this preexisting condition, or whether that cell be- comes the larger one, which happens at the time of division to contain more of the X chromosome (owing, let us say, to its ac- cidental excentric position). If one may judge from the appear- ance of the early stages of the first spermatocyte division, the former alternative may seem more plausible. If this be the cor- rect interpretation then the more usual case of the two X’s going to the same pole in the abnormal cyst would be due to their rela- tion to a particular pole of the dividing cell. The exceptional passage into the other cell would be due to one of them getting caught by the constriction, so that it was necessarily detained in the smaller cell. But the more nearly equal sizes of the PREDETERMINATION OF SEX 303 cells in the cases here figured, when an X passes to each pole, may seem rather to favor the other view, namely, that the size of the cell is determined by the chance direction taken by the X chromosome. If cells like these with duplex number of chromosomes should produce functional spermatozoa, and one such spermatozoon should fertilize a normal egg, the number of somatic nuclei would become nine instead of six and the resulting female would have three X chromosomes instead of two. In subsequent genera- tions the number of X’s might be further increased, if, in fact, viable forms could be produced in this way. It does not seem probable, however, that a condition like that described above for the phylloxerans, in which four X’s are present, could have arisen through irregularities of this kind, because the chance that such a rare phenomenon could supplant the normal type seems too small to make such a view possible. Still, the possibility of a tetraploid organism, arising through failure of a spermatogonial or oogonial. cytoplasmic division must be conceded, especially in the light of the sudden appearance of tetraploidy in Primula and Oenothera. If it be assumed that some advantages, such as an increase in size, give the new type an advantage, then it might in time obtain an independent footing. A most striking and interesting relation is shown in this tetra- ploid cyst, namely, the chromosomes are only half as large as are those at the corresponding stage of the normal spermatocyte stage, as seen in other cysts of the same testis. This relation might be interpreted to mean either that the original mother-cell of the cyst, having divided (incompletely) one time more than the other mother-cells of the other cysts, never made good the size loss of the chromosomes. If the growth of the chromosomes be directly related to the size of the cell that contains them, owing to the amount of substance available in such a cell, the smaller size of the chromosomes in the tetraploid cyst might find a rea- sonable explanation. Such a conclusion would indicate that the stage reached by a cell at a particular phase is determined by the cytoplasm, rather than by the size of the chromosomes. 304 T. H. MORGAN THE OMISSION OF SYNAPSIS IN THE PARTHENOGENETIC EGGS OF PHYLLOXERANS AND APHIDS As is well known, the full or diploid number of chromosomes is present in most eggs that develop by means of parthenogenesis. Whether the presence of the full number of chromosomes has in itself anything to do with the phenomenon of parthenogenesis may well be disputed, because in some forms, as in the male bee, the eggs develop without being fertilized with half the number of chromosomes and in artificial parthenogenesis the half number of chromosomes occurs in some forms, at least. In the phyllox- erans and aphids there is a loss of one or two chromosomes from the male-producing egg that develops by parthenogenesis. There is a further question that is important from a descriptive cytological point of view, namely, whether the parthenogenetic eggs omit the synapsis stage and retain in consequence the full number of chromosomes, or whether they pass through such a stage and the chromosomes subsequently separate. In phyllox- erans and aphids the case is quite clear! and I wish to emphasize the ease and certainty with which the problem can be studied in them. In the bearberry aphid, the ovary that is producing sexual eges (diagram 3) can with certainty be distinguished from the ovary that is going to produce parthenogenetic eggs (diagram 2). In the latter there is no contraction phase of the chromosomes. . A prophase of an oogonial division of a parthenogenetic egg is shown in diagram 2,a. Six chromosomes are distinctly seen and the same number is found in the equatorial plate stage shown in diagram 2,b. At the beginning of the growth period, when the chromosomes begin to take the stain again, scattered threads or strands can be made out, as shown in c, which by further con- traction, d, give rise finally to the six rod-like chromosomes. In later stages, when the egg is about ready to leave the ovary and after that time while it is still acquiring yolk, the six chromo- somes can be distinctly seen and easily counted, as shown by most of the eggs in diagram 2,e and f. In the ovary of the sexual in- dividual the chromatin begins to condense into threads at the 1 Morgan, T. H. Proc. Soc. Exp. Biol. and Med., vol. 7, 1910. PREDETERMINATION OF SEX 305 beginning of the growth period, as shown in diagram 3, a, b, c, and the threads later condense at one side of the nucleus, as shown in d. No details of the process of union of the chromo- somes, that must take place at this time, can with certainty be made out. The figures are as accurately drawn as possible, but Diagram 2 a, prophase of an oogonial division; b, metaphase of an oogonial division with six chromosomes; c, young parthenogenetic ovum just prior to the appearance of the chromosomal filaments shown in d, e, f, young ova for the most part in the lower end of the ovary or else just out of the ovary, showing six chromosomes whenever all of the chromosomes are included in the section. beyond the fact that the chromosomes are condensing, the in- terpretation of the details of the drawings is unsafe. When the chromosomes begin to emerge from the condensed stage, as seen in diagram 3, e, f, g, h, three clumps or rods, or sometimes open MORGAN fb 5 ba 306 pod10Wle dAvY 10 “SUID1oWIe OLR ul ss30 pue pus IOLI9JUB UL S][OO OAT yt 2FEw/U.' T7T7TTwTwSwwaS {oe ee a ‘pou1oy st Apoq avpod oy} a1ojoq uMAO Sunod ‘y fo9vys o1IjdvuUAS oY} WOI] Iynd yyia 4 SOMIOSOMTOIYS 9aLY} OY} YOTYA ut BAO BuNO< ‘6 ‘f ‘a {pua Toyo 4e ode4s stsdvuds IVAO OTOYM JO UOT}OOS ‘p {stsdvuds OyuI Suissed BAO Sunod Bul -moys ‘pryde Art9qivoq oY} JO S[RNPTATPUT [VNXS JO SalIBAO ayy JO pue LOTIO}SOd ayy UI sT[eo ‘9‘q*n ¢g UNBIBBIC, PREDETERMINATION OF SEX 307 rings, appear and in this condition they are carried onto the polar spindle. In the bearberry aphid, the stem-mother produces many male embryos as she gets older. Their presence in the mother serves as an index of the condition of her ovary at this time. An ex- amination of her ovarian eggs at this time failed to show in the chromosomes any process suggestive of synapsis, although no doubt the steps preparatory to the elimination of the two sex chromosome must be taking place at this time. As only two chromosomes are involved it is quite likely that even if they went through a contraction phase independently of the rest of the chromosomes (if such were possible) it would be difficult to recognize such a process. The negative evidence has no special value and is mentioned here only to show that the stages were examined for evidence of synapsis. In the phylloxerans the ovary of the stem-mother continues throughout her life to produce a series of eggs that develop by parthenogenesis. All stages in the development of the eggs can be found in almost any female. There is never in any of them the slightest evidence of a contraction phase, and, since the eggs show exactly the same conditions as do the parthenogenetic eggs of the bearberry aphid, it will not be necessary to repeat here what has already been said. The ovary of a young female that will later reproduce by parthenogenesis is represented in dia- gram 4. The migrant generation of the phylloxerans also produces eggs that will develop by parthenogenesis. In P. caryaecaulis all of the eggs develop at nearly the same time, so that the conditions for the study of parthenogenetic stages are not so favorable as in P. fallax, where the wingless ‘migrants’ produce one egg at a time over a considerable period. In neither species have I seen any evidence of contraction, nor have I seen any other evidence of conjugation of the sex chromosomes, with the exception already noticed where two chromosomes of double size were found in two eggs that would give rise to male embryos later. Here, however, the nucleus was ripe and ready to take part in the for- mation of the polar spindle. The observation only shows that THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 19, NO. 3 308 T. H. MORGAN the two pairs of conjugated chromosomes have already united before the spindle is formed. Von Baehr describes for Aphis saliceti a synapsis stage in the spermatogenesis in which the chromosomes are contracted at one Diagram 4 Section of ovary of parthenogenetic female of second generation, with large nutritive cells at one end and small egg cells at the other end on the ovary. Diagram 5 Spermatogonial cells in prophase stages to left and one cell in metaphase to right; all cells show five chromosomes. side of the cell. As no further details that identify this stage were made out, one can not be certain that such figures repre- sent the true conjugation stage. I have studied the spermato- PREDETERMINATION OF SEX 309 genesis of several species of aphids in the hope of getting better figures of this stage, but have found none better than those given by von Baehr. In those species where the formation of the spermatozoa is a continuous process in the testis of the adult male it is possible to obtain in the same testis all stages, from the dividing spermatogonial cells to ripe spermatozoa. ; oe a | 27 28 29 M. L. HEDGE, DEL. THE EFFECTS OF THE BETA AND GAMMA RAYS OF RADIUM ON PROTOPLASM CHARLES PACKARD From the Zoélogical Laboratory, Columbia University TWENTY-FIVE FIGURES (THREE PLATES) The present investigation has been carried on for the purpose of determining the effects of the beta and gamma rays of radium on protoplasm. Much work has been done during the last ten years on the general effects of the radiations, but the results have been conflicting, and the opinions as to their meaning far from unanimous. Conflicting results are found to occur for two reasons; first, because different types of cells react very differently to the same stimulus; and second, because very different methods of applying the stimulus have been employed on the same kinds of cells. The first point calls for further inquiry as to why cells differ from each other in their ability to absorb the rays emitted by radium; the second, for.a more care- ful analysis of the action of the different kinds of rays. This paper deals with the second point. The three types of radiations given off by radium differ markedly from each other in their physical properties. The alpha rays, which are chemically the most active, possess so shght a power of penetration that they do not reach the object of study, being entirely absorbed by the glass tube in which the radium salt is held. They may therefore be left out of this discussion. The beta rays consist of negatively charged particles which can be deflected in a strong magnetic field. The rays are not homogeneous but are made up of partieles whose speed varies from 0.3 to 0.99 of the velocity of light. The slower particles are deviated more sharply in the magnetic field than are the high speed particles and are much more readily absorbed by 323 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 19, NO. 3 324 CHARLES PACKARD matter. The latter are all absorbed by 2 mm. of lead, or by 250 em. of air. Thus it is seen that the distance of the object to be exposed from the radium is a large factor in determining the intensity of the radiation. The slower beta particles are easily absorbed by thin layers of matter much less dense than lead. A thick mica screen is sufficient to stop them, although they pass through a very thin screen of mica without much loss of energy. Chemically, the beta rays are much less active than the alpha rays, since they are not absorbed as readily. It is therefore ap- parent that the slower beta rays are more active than the more rapid ones since they are absorbed in passing through matter. This point is of importance in conducting an experiment for if these more active rays are unable to reach the object of experiment the effects produced may be very different from those obtained when all of the beta rays are available. The gamma rays are similar to the hard X-rays in many respects, but they travel with much greater velocity. For this reason they are not absorbed to any extent and their effects are of a different order from those produced by the beta rays. In a magnetic field they are not deflected. When they pass through lead of considerable thickness they are transformed into secondary beta rays, similar to the beta rays emitted by radium itself. These rays have been shown by Congdon to produce definite effects on living matter. REVIEW OF LITERATURE Up to the present time very few studies have been made on the effectiveness of the different kinds of rays, or on the relative effects of the slow and rapid beta rays. Guilleminot (07) showed that the beta rays are more effective than X-rays when their luminescent effects are approximately the same. A more thorough study is that of Congdon (712) in which he analyzed the effects of the primary and secondary beta rays on seedlings. He states that the gamma rays from 8 mg. of radium bromide produce no appreciable effect. Of the beta rays, the slow electrons are more effective in retarding growth that are the rapid electrons. EFFECTS OF RAYS OF RADIUM ON PROTOPLASM 325 Abbe ('14) exposed wheat grains to mixed beta and gamma rays for varying periods and at varying distances. ‘‘The uni- versal effect was a depression of growth exactly in proportion to both time and distance. The greatest destruction of seed life was at one inch.’’ In no instances was there any evidence of an acceleration in the rate of growth. Carrel (quoted in Abbe’s paper) found that the gamma rays produce no effect on the rate of cell growth in vitro but that the beta rays bring about a retardation of 25 to 50 per cent. There was no morpho- logical change in the cells. The effect of a short radiation per- sisted through twenty cell generations. Experiments in which there has been no attempt to differenti- ate between the action of the different kinds of radiation have been numerous. I will not attempt to review them here for they have been mentioned in a previous paper and by Hertwig (10) and Richards (14). The point which is of interest in con- nection with the present study is that a strong radiation retards development and may produce many abnormalities. A very weak and short exposure brings about an acceleration (Cong- don ’12). Between these extremes it is possible to radiate developing embryos so that no abnormality results although there is a marked retardation. METHODS The beta rays can be separated from the gamma rays in a magnetic field, since the former are deviable and the latter are not. The device for separating the rays is shownintext figure A. The block is made of solid lead. The capsule containing the radium (50 mg. of the pure bromide) rests in the chamber A, the bottom of which consists of a sheet of lead 2 mm. in thickness. This is sufficient to absorb all of the beta rays projected down- wards on the shelf B. Thus only gamma rays can fall on material placed in that position. When the device is placed between the poles of a strong electromagnet the beta rays are deflected in the manner indicated and fall upon material placed on the shelf C. The path of the rays, which under these conditions 326 CHARLES PACKARD is about 50 mm. may be strikingly demonstrated in a dark room by holding a willemite screen where the rays may fall upon it. The gamma rays may be similarly shown falling on the shelf B. The luminescence of the screen is about the same on both shelves. Secondary beta rays are probably produced in the lead and fall upon the shelf B but the effects they produced, as distinguished from those of the gamma rays, was not studied. The material to be radiated was placed in small glass cells, open on top, and provided with mica bottoms. The cells were placed on the shelves B and C. In some experiments the cell Text figure A was placed directly above the radium capsule and at varying distances from it. The material was thus exposed to both beta and gammarays. By varying the thickness of the mica bottoms of the cells it was possible to screen out the slower beta rays or to utilize them all. With a thickness of 0.1 mm. of mica it was found that very few of the slow beta rays were absorbed. A thickness of 0.10 mm. of mica is sufficient to screen out the slower rays, so that only the more rapid ones could affect the material. These experiments were made at Woods Hole during the sum- mer of 1914. For the preparation of radium and for the electro- magnet with the lead device for holding the radium I am deeply indebted to Dr. Robert Abbe of New York City. I take pleasure here in expressing to him my hearty thanks. EFFECTS OF RAYS OF RADIUM ON PROTOPLASM aad MATERIAL The eggs of Nereis limbata and Arbacia punctulata were used almost exclusively in these experiments. Some work was also done on Drosophila ampelophila and on Paramoecium caudatum. OBSERVATIONS ON LIVING MATERIAL a. The gamma rays With the apparatus employed it is difficult to determine what percentage of the radiations falling on the shelf B is in the form of gamma rays and what is in the form of secondary beta rays. But whatever may have been their nature it is evident that they produce a slight acceleration in the rate of cell division. This is most marked in the eggs of the sea urchin, which were exposed either before fertilization or immediately afterward. Text figure 2 indicates the change in division rate. It is seen that the first indication of cell division appears about 15 minutes before a similar change occurs in the control. This difference is con- tinued in the second cleavage. There is no abnormality in the mode of division and the larvae develop with perfect regularity. The acceleration is not cumulative; the eggs do not divide at shorter and shorter intervals. After a few divisions it is impossible to say whether the acceleration persists or not. Nereis eggs do not respond at all to the gamma rays. The eggs and larvae of Drosophila are not affected appreci- ably by the gamma rays. The rate of growth is not changed and the adults are fertile. Paramoecium is not affected even by long exposures. Indeed I have not been able to see any sign of abnormality or change in the rate of division even after prolonged radiation with both beta and gamma rays. This has been the general result reached by other investigators on Paramoecium. In order to test the effect of the gamma rays acting at a dis- tance I placed the glass cells holding the material 5 cm. above the radium. When the electro-magnet was in operation the beta rays were entirely deflected so that only pure gamma rays 328 CHARLES PACKARD reached the objects. All experiments of this sort were negative, showing that the gamma rays in passing through even a short layer of air, lose much of their effectiveness. b. The beta rays I. Experiments on Nereis. The effect of the rapid beta rays, acting at about 50 mm. distance, is seen in a change both in the division rate and in the physical properties of the protoplasm. After the first cleavage the blastomeres divide at a slower rate than normal, although the mode of division is perfectly regular. The swimming trochophores are normal and as active as the controls, but are always behind the controls in their stage of development. The first cleavage occurs before the control eggs divide, but this apparent acceleration is due to the weakening of the peripheral protoplasm of the egg and not to an acceleration of metabolic processes. When unfertilized eggs are placed directly above the radium, at a distance of 4 mm. the effect is far different. If the rapid beta rays are used alone, the peripheral layers of protoplasm are chiefly affected. If both rapid and slow beta rays areused the egg quickly dies, usually before the first cleavage. In the first case there is a profound change in the mode of extrusion of the egg jelly. _ The normal extrusion of jelly, which has been described by Lillie (711), takes place as follows: As soon as'a sperm becomes implanted in the vitelline membrane the jelly, which has been held in delicate alveoli in the cortical layer of the egg, begins to pass through the membrane forming a thick layer outside of the egg. The alveoli, which have thus been emptied of their con- tents, later become filled with water so that they almost dis- appear from view. But before this occurs their walls may be seen. extending radially out to the membrane of the egg. In the radiated eggs no change can be noted before insemi- nation. The oil droplets and the mitochondria, which can be seen by dark field illumination, are normal. After insemination the jelly is given off, but it has not the usual sticky character, — EFFECTS OF RAYS OF RADIUM ON PROTOPLASM 329 for the eggs do not tend to stick together as in the controls. In a few minutes the vitelline membrane is pushed away from the surface of the egg so that the perivitelline space increases in width. Figure 1, which was drawn from the living egg just be- fore the first cleavage, shows the extent to which this increase may occur. The walls of the alveoli are drawn out so that they extend from the egg protoplasm up to the delicate plasma membrane which lies just beneath the vitelline membrane. The following measurements taken from many living eggs just before the first cleavage and 16 hours after show the extent of this increase in width: DIAMETER OF IAM 5 GG D BEBE OPE MEMBRANE NGA Cy | Se eM MIE ee 87 x 100u 87 x 100u Unfertilized eggs radiated for 90 min.; measured just before cleavage........... _ 90 X 96u 116 X 128. Same eggs 18 hours after insemination... . 90 X 96u 154 X 168u _ Through the kindness of Dr. G. L. Kite who dissected a num- ber of eggs from the same lot from which these measurements were taken, I was able to observe that the physical properties of the protoplasm and of the egg membranes are greatly altered. The membranes are still sufficiently tough to hold together when the dissecting needle is pressed against them, but they are softer than normal and can be punctured without difficulty. The protoplasm, instead of being a fairly firm gel is soft, and flows freely through a small tear in the membranes. The perivitel- line space is filled with a semi-gelatinous substance which stains with various protoplasmic dyes. The fact that this is more fluid than the normal jelly may indicate that it is a mixture of jelly and water. Certainly not all of the jelly is given off after insemination. The treatment with the beta rays has so altered the membranes that they are no longer able to allow the jelly to pass through them in a normal fashion. According to Dr. 330 CHARLES PACKARD Kite the protoplasm has increased its water holding power so that the whole egg becomes soft and fluid. A further evidence of the softened character of the protoplasm is shown in figure 2. The peripheral layer is pulled in abnormally under the influence of the aster. Cleavage occurs in a fair proportion of these eggs, but it is abnormal. After several divisions the embryo resembles a heap of shrunken cells. Occasionally the cells put out cilia. Eggs which are treated with both slow and rapid beta rays for varying periods before insemination do not show so marked an increase in the width of the perivitelline space. But evidently they have been greatly injured since in most cases division is entirely inhibited. Those eggs which divide at all do soina very abnormal manner. These facts indicate that the rapid beta rays acting at a dis- tance of 50 mm. are not strong enough to produce any marked abnormality in development, but cause a pronounced retar- dation in the rate of development. Acting at 4 mm. distance they affect chiefly the peripheral layers of protoplasm. When both slow and rapid rays are used cell division is usually in- hibited altogether. 2. Experiments on Arbacia. In the experiments on the sea urchin I used both fertilized and unfertilized eggs, observing the proper precautions to have all the conditions for growth as favorable as possible. The general effect of the rapid beta rays, acting at a distance of 50 mm. is a retardation, the slowing down commencing almost at once in the case of the fertilized eggs. In text figure B the amount of retardation isshown. The curves were made from an average of many observations made on different lots of eggs. In each case the eggs were exposed for 40 minutes immediately after insemination. It will be seen that the control eggs in these experiments begin to divide about 75 minutes after insemination, and that all are in the two cell stage about 10 minutes after they begin to divide. The radiated eges are much retarded both in beginning to divide and in at- taining the complete two cell stage. At the latter time the controls have begun to divide for the second time. The pause ) ats EFFECTS OF RAYS OF RADIUM ON PROTOPLASM 331 Minutes after Insemination [e) sete 0. ie FREER EEE et CCE She) Cee | ce eS Beer Text fig. B. ‘“‘Sea-urchin eggs radiated for forty minutes after insemina- tion.’’ 4cell b Be innina Coo ie EREBC ae between cleavages is evidently increased in length in later development since after several hours the embryos were far behind the controls. These embryos are not abnormal except in the fact that they are behind the controls. The retarding effect of the beta rays, even after so short an exposure as 40 minutes is therefore permanent, even when the embryos are placed in the best possible environment. Unfertilized eggs respond in the same way except that a longer exposure is necessary in order to obtain the same results. When unfertilized eggs are treated with the slow and rapid beta rays for 30 minutes or more and then inseminated with fresh sperm they throw off a very slight fertilization membrane. Cleavage is much delayed and a'most always irregular. The few embryos which survive for a day show many abnormalities. Most of them never pass through the gastrula stage, and those that do, later show abnormalities of the types familiar to all who have observed the development of Arbacia. A careful de- 332 CHARLES PACKARD scription of such typical abnormalities has been given by Tennant (10). It is evident from both series of experiments that pro- toplasmic changes in Arbacia are very small, if indeed they occur at all. 3. Experiments on Drosophila. The larvae and pupae of Drosophila do not ‘show any external change even after an intense radiation for one hour. It is probable that the rate of development is somewhat retarded, although there is not suffi- cient evidence to prove this point conclusively. When the pupae hatch out, the flies are normally active but are completely sterile inter se and with normal wild stock. The sterility is however, only temporary for after about three weeks the flies become fertile again. The offspring appeared to be normal. Apparently only those germ cells which were in advanced stages of devel- opment were destroyed, while the earlier stages were merely retarded. Summary The gamma rays from 50 mg. of radium bromide bring about some acceleration in the rate of development of the sea urchin, but have no effect on Nereis. Rapid beta rays, acting at 50 mm. distance exert a retarding effect, most marked in Arbacia, but noticeable in Nereis and Drosophila. Acting at 4 mm. these rays affect the peripheral protoplasm of Nereis. When both rapid and slow beta rays are used there is a marked protoplasmic change in Nereis and an inhibition of development. Under the same treatment Arbacia shows little or no protoplasmic change, but a very abnormal development. The chief effect on Droso- phila is a destruction of the germ cells in the later stages of gametogenesis. CYTOLOGY OF RADIATED EGGS In a previous paper I described a number of typical abnormali- ties which occur in the radiated eggs of Nereis. The point was made that in these eggs marked protoplasmic changes occur as a result of the treatment with small amounts of radium, and that the nuclear changes, while present, are not always obvious. EFFECTS OF RAYS OF RADIUM ON PROTOPLASM 333 In this respect the results differ from those of Paula Hertwig (’11) who states that in Ascaris the only effect of radiation is on the nucleus. With larger amounts of radium I have repeated the previous experiments, and am able to confirm my former state- ments in regard to Nereis. Experiments on the sea urchin tend to confirm the observation of Miss Hertwig. This situation illustrates the difficulty of making any general statement on the effect of radium on protoplasm. In the following section I will describe the changes brought about in the eggs of Nereis and Arbacia by means of the rapid and slow beta rays. I. Experiments on Nereis The eggs develop in different ways depending on the nature of the rays employed and on the length of exposure. In general there are three fairly distinct types of development. The first type is seen in those eggs which have been exposed to the rapid beta rays‘at 50 mm. distance before insemination. The peri- vitelline space is not increased in width; the sperm enters much earlier than normal, and the maturation divisions, which are normal, take place at a correspondingly earlier time. About 75 per cent of the eggs thus treated show this peculiarity. The second type predominates when the unfertilized eggs are exposed to the slow and rapid beta rays at 4 mm. from the source. The perivitelline space is at first normal but after a short time in- creases notably in width. The sperm enters early, as in the preceding case, but the maturation divisions are abnormal. Almost every egg is thus affected. The third type is seen when the eggs are exposed to the rapid beta rays acting at 4 mm. dis- tance. The perivitelline space, in about 75 per cent of the eggs, is greatly extended; the sperm usually fails to enter, and the maturation divisions are abnormal. The early entrance of the sperm is a very striking and con- stant phenomenon. In table 1, which is a summary of several experiments performed in different ways, the rate of entrance and the subsequent phenomena of maturation and cleavage are shown. The first column shows the normal course of develop- 334 CHARLES PACKARD ment. Variations from this rate are small if the temperature remains constant. It will be seen that the sperm remains ex- ternal to the egg during the prophase and the early formation of the first maturation spindle, and does not begin to enter until the time of the first anaphase. At that time it is drawn through the membrane, a process which occupies less than 5 minutes. It continues its course inward during the first telophase, and is a& 3 = %y = Saeeay RAPID BETA AT 50 MM., papier gee ees RAPID BETA AT 4 MM., 25 7) Gnade 75 MIN. EXPOSURE z EXPOSURE o3 90 MIN. EXPOSURE s ~ 10 | Prophase Prophase Prophase Prophase 20 | Prophase Prophase; peri- | Prophase; peri- | Prophase; _peri- vitelline space vitelline space vitelline space normal normal wide 30 | lst metaphase at | sperm entering; | Ist metaphase lst metaphase - periphery ist anaphase 35 | same;spermstill | Ist polar body | sperm entering; external extruded anaphase 40 | sperm eas ae in ee apn Ssirahee Very abnormal lst anaphase Oo ies 2n s aaa development metaphase perlivl ie ine with suppres- peers oe sion of the Ist 45 | Istanaphaseand | 2nd = anaphase | sperm in center eee pe a telophase and telophase of egg; abnor- ay, = mal polar di- sperm does not oe enter visions 55 | sperm in center | fusion of pro-| a great variety of egg; 2nd nuclei of abnormalities metaphase 65 | fusion of pro- | cleavage cleavage rare cleavage rare and nuclei abnormal 75 | cleavage TABLE 1 Showing the development of Nereis-eggs radiated before insemination EFFECTS OF RAYS OF RADIUM ON PROTOPLASM Sao in the center of the egg when the metaphase of the second spindle has formed. The pronuclei fuse about 65 minutes after insemination. The second column shows the rate at which these phenomena occur in unfertilized eggs which have been exposed to the rapid beta rays acting at a distance of 50 mm. The perivitelline space is not appreciably widened. The sperm enters about thirty minutes after insemination, that is, about 15 minutes ahead of the control. In the meantime the first anaphase has developed. In other words, the development of the egg has kept pace with the early entrance of the sperm. By the time the sperm is in the center of the egg (in 40 min.) the first polar body has been extruded and the second metaphase figure has developed. Here again, the whole development occurs at exactly the same rate as in the controls. If the living eggs only had been observed the conclusion might have been drawn that the treatment with radium stimulated the eggs to divide at a faster rate than normal. But the cytological evidence just presented shows that there has been no stimulation. The weakened egg membranes have permitted the sperm to be drawn in earlier than usual, but that is all. There are no constant abnormalities to be found in these eggs either during the maturation divisions or during cleavage. The subsequent development is normal, but the larvae develop at a slower rate than the control animals. The effect of these rays under these conditions is therefore to weaken the egg membranes and to bring about a retardation in the growth of the embryo. The effect of the slow and rapid beta rays is shown in the third column. The perivitelline space, which at first is of normal width, increases later in a striking manner which has already been described. The protoplasm at the periphery of the egg is much changed in appearance. The finely granular character seen in normal eggs, is lacking, and in its place is a fairly homo- geneous substance in which are held numerous faintly staining spherules (fig. 3). Occasionally it presents a vacuolated appear- ance seen in figure 4. As the perivitelline space increases in width the entrance cone is also pulled out so that it stretches 336 CHARLES PACKARD across the entire space and reaches the membrane just below the sperm (fig. 4). The sperm enters somewhat earlier than in the controls. The cone does not sink back into the egg protoplasm but remains elevated and the sperm penetrates throughout its entire length (fg. 5). In some instances the sperm does not enter at all. The egg develops normally as far as the first metaphase. In some instances development up to cleavage is normal, but such eggs do not develop far, for they die before reaching the trocho- phore stage. Of the abnormally developing eggs about 20 per cent show a curiously small polar spindle. _ The whole figure is crowded to the periphery of the egg. The polar body, however, is normally extruded and the second maturation spindle is apparently normal. In about 40 per cent of the eggs the centrosomes of the first polar spindle divide to form a tripolar or multipolar spindle (figs. 6 and 7). The chromosomes are perfectly normal and the asters are well developed. But in such cases the first polar body is not extruded. The chromosomes remain in the condensed condition for a considerable period, after which, those nearest the periphery are extruded in the second polar body. The reason for believing that this is the second and not the first polar body is that immediately after its extrusion the chromosomes become vesicular, just as they do in normal eggs after the second polar body has been given off. The mechanism involved in the extrusion of those chromosomes is not clear since in no case have I been able to find any spindles. Either the stage during which they are present has been passed through very quickly, or else the fibers do not stain; figures 8 and 9 show this condition. In figure 8 only a few chromosomal vesicles are shown. The polar body is unusually large, and contains little chromatin. Figure 9 shows a later stage. The polar body has formed completely and the remaining chromosomal vesicles, whose position indicates that they were lying in a tripolar spindle, are now distinct. There are about 28 karyomeres in the vesicles. If the sperm fails to enter, development proceeds in a very different way. As before, multipolar spindles form at the first maturation division. The first polar body is suppressed, and EFFECTS OF RAYS OF RADIUM ON PROTOPLASM Send: the chromosomes, following the extrusion of the second polar body, develop into large chromosomal vesicles. Such an egg is shown in figure 10. The sperm is seen still exterior to the egg membrane which is not lifted far off from the egg. There are at least 40 vesicles present in the entire egg and the karyomeres number about 250. Such a number of vesicles is probably due to more than one division of the chromosomes at the first meta- phase. The karyomeres may have fragmented by direct division. Subsequently they have grown, since each one is fully as large as those found in normal eggs. The third type of development is seen in eggs radiated with the rapid beta rays at 4 mm. distance. The increase in the width of the perivitelline space is much more marked than in the preceding type, for it appears earlier and is greater in extent. More than 75 per cent of the eggs show such an abnormality. In the great majority of cases the sperm does not enter at all, and can be found more than an hour after insemination still outside of the egg. This phenomenon may be due to the fact that the vitelline membrane is so rapidly pushed away from the egg that no fertilization cone could extend far enough out from the egg to reach it (fig. 11). The development of these eggs proceeds normally until the time when the sperm should enter, that is, through the first metaphase. The chromosomes are not extruded, and no first polar body forms at all. The inner centrosome of the spindle divides forming well marked tripolar and multipolar spindles (figs. 11 and 12). Figure 13 shows a small protoplasmic pro- tuberance at the point where the polar body should be given off. This is a very common phenomenon. The chromosomes which are very numerous, owing to the multipolar divisions, now become vesicular, just as they do in normal eggs at the end of the second polar division. In the meantime the second polar body has been extruded although the method, as in the preceding case, is obscure. I could not determine how many chromosomes are extruded at this time, but from indirect evidence I believe the number to be 14, that is, the haploid number. The remaining chromosomes, of which there may be 28 or 42 or even more, 338 CHARLES PACKARD depending on the number of divisions of the first polar chromo- somes, now migrate, still distinct, to the center of the egg, or they may grow in size until they finally fill a large portion of the egg. The latter condition is seen in figure 13. In the former case (and such instances are rare) the egg divides abnormally. The chromosomal vesicles become arranged on the cleavage spindle and are unequally divided in the two daughter cells. The larger number stays in the larger blastomere. The asters are very slightly developed, but the spindle fibers and interzonal fibers are very obvious; figure 14 shows this point. The total number of vesicles remaining in the larger blastomere is about 28, and in the smaller, about 14. Whether these numbers are significant or only accidental cannot be said since there are so few cases of this phenomenon. Many of these eggs extrude no polar body at all. In such instances the chromosomal vesicles fuse together and the karyo- meres spin out into a spireme somewhat similar to that seen in normal eggs immediately after the germ nuclei have fused. But in these nuclei there is no nuclear wall (fig. 15) the chro- mosome lying in a vacuole filled with a very faintly staining substance. These observations point to the conclusion that cells may be stimulated or retarded without suffering any marked morpho- logical injuries. The effect has been physiological since only the rate of metabolism has been affected. But with more severe radiation the retardation is not so apparent because the embryos die before developing far. A comparison of the injuries brought about by the slow and rapid beta rays acting together with those induced by the rapid rays alone reveals the curious fact that the more intense radiation occasions less apparent disturbances. This phenomenon superficially resembles that described by Hertwig, who found that when the unfertilized frog egg is radiated intensely, development after insemination is more normal than when it is radiated more moderately. His ex- planation is that the egg nucleus has been entirely inhibited from taking part in cell division, so that only the normal sperm nucleus divides. But in Nereis development after intense radiation is EFFECTS OF RAYS OF RADIUM ON PROTOPLASM 339 never haploid, and the egg nucleus always develops, though abnormally, through the maturation periods. It is impossible to inhibit completely the activity of the egg nucleus without destroying the entire egg. 2. Experiments on Arbacia. A cytological study of Arbacia eggs, radiated both before and after insemination reveals the fact that the treatment produces very slight effects on the cell constituents. Clear cut abnormalities such as were abundant in Nereis are here very rare. Indeed it is only by careful search that they can be found. This does not signify that radium is incapable of effecting marked cytological changes in sea urchin eggs, but merely that the treatment given in these experiments was not severe enough. But inasmuch as abnormal develop- ment always follows prolonged radiation, it is evident that pro- found changes have taken place which cannot be rendered visible by the technical means now at hand. The amount of visible injury cannot be considered an index to the actual condition of the cell constituents. The eggs were exposed for varying times to the gamma rays, the rapid beta rays, and to a mixture of the rapid and slow type. In those eggs treated with the gamma rays there is no sign whatever of injury. As stated before, the only effect of such a treatment is seen in the slight acceleration of cell division. Exposure to the rapid beta rays likew'se produces no visible cytological changes, but only a marked retardation in the rate of development. When both slow and rapid beta rays are utilized some effect on the cell constituents can be seen. Unfertilized eggs were exposed for 50 to 60 minutes to all the available beta rays, after which they were inseminated in finger bowls. In each experiment a parallel series of exposures was made on a very few eggs. Such a control is necessary since overcrowding of the eggs frequently produces the same abnormali- ties as the radium. By having two controls for each experiment the danger of drawing false conclusions was minimized. In the experiments to be cited there was always a sharp distinction between the behavior of the radiated and the control eggs. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 19, NO. 3 340 CHARLES PACKARD The entrance of the sperm is normal in every case, and poly- spermy is as rare as in the controls. As the sperm penetrates the peripheral protoplasm of the egg it revolves, and an aster de- velops in front of it (fig. 16). The further course of the sperm is not marked by any abnormalities (fig. 17). In about twenty minutes the sperm head becomes closely applied to the egg nucleus where it remains as a distinct cap for some time before it completely fuses with the egg nucleus. In the meantime the sperm aster divides and the daughter asters migrate to opposite sides of the cleavage nucleus (fig. 18). Up to this time the egg nucleus is entirely normal. Before insemination it is filled with a tangle of chromatin threads suspended in a delicate linin network, a condition which persists until the sperm nucleus begins to fuse with it. The first sign of abnormal development appears at this time. Some of the chromatin of the egg nucleus condenses into deeply staining spherical bodies which are scattered throughout the nucleus. As a rule they are comparatively small (fig. 19) but may be very large (fig. 20). In this condensed condition they remain throughout subsequent development and may be seen in the anaphase of the first cleavage lagging behind the chromosomes (figs. 22° and 23). The remaining chromatin at first spins out into very delicate threads, during which time the mingling of the parental chro- matin takes place (fig. 19). This stage is followed by a gradual shortening and thickening of the threads (figs. 20 and 21). In the meantime the astral rays (not shown in the figure) grow in and bécome attached to the rod-like chromosomes which have resulted from the shortening of the chromatic threads. This whole process is normal in every respect. The chromosomes can be counted during the anaphase. Many counts show that development is, without exception, diploid. The normal diploid number is 34. The appearance of the mi- totic figure resembles in many ways the figures of Hertwig (712) In his experiments on Sphaerechinus he radiated the sperm only, and found that the sperm chromatin breaks up into numerous masses of irregular shape which in many instances, are involved EFFECTS OF RAYS OF RADIUM ON PROTOPLASM 341 in the spindle in which the dividing egg chromosomes are located. In my experiments, which are the reverse of his, since only the eggs are radiated, it is the egg nucleus that gives rise to the chromatin masses. That they represent abnormal chromosomes is probable, since the number of these bodies, added to the number of normal chromosomes in any figure, gives the usual diploid number. Evidently, therefore, only a portion of the egg chro- matin has been injured severely enough to produce obvious changes in appearance. The achromatic portion of the mitotic figure is normal in every respect. The further stages of division are normal except for the presence of the injured chromosomes which may le any- where in the spindle. Occasionally they go to the poles where they may be seen still condensed at the telophase, when the other chromosomes have already become vesicular. This brief description indicates that the effects of a short radiation are very slight. There is little evidence that the protoplasm has been injured. A longer exposure undoubtedly would produce more marked injuries, but such an exposure is difficult to make in view of the fact that sea urchin eggs are extremely sensitive to overcrowding and to a prolonged stay in small quantities of water, conditions which are necessarily imposed during radiation. DISCUSSION The character of the response of protoplasm to radium radi- ations depends on the nature of the protoplasm itself, and on the intensity of the exposure. In regard to the first point little can be said except that cells differ from each other in their suscepti- bility, wholly apart from the fact that each cell varies in sus- ceptibility during different phases of its own activity. It has been pointed out that an exposure of thirty minutes to the beta rays will bring about in the developing sea urchin changes which are as pronouncd as those produced in Nereis after ninety minutes exposure to rays of the same intensity. Some Protozoa are entirely unaffected at the end of fourteen hours of exposure, while others are killed in a shorter period. There is a similar 342 CHARLES PACKARD variation in the responses of the Bacteria. Obviously, those cells which are injured contain substances which absorb the rays, while those which are uninjured allow the rays to pass through unchanged. The factors which determine the power of absorp- tion of materials are not well known. “The absorption of beta rays is an atomic phenomenon and is not affected by the chemical combination of the atoms. Such a relation appears to hold generally for all types of radiations emitted by radioactive sub- stances”? (Rutherford). Until more is known on this point it will be impossible to predict what effect a given exposure will produce. These facts throw no light on the nature of proto- plasm, but accentuate the point that the protoplasm of one type of cell differs from that of another cell. Disregarding these differences, it may be said that a weak radiation accelerates, and a stronger one retards cell division. Acceleration is not followed by any abnormality. A careful study of this point has been made by Lazarus-Barlow and Beckton (713) who used exceedingly small quantities of radium on Ascaris eggs. Tests made upon many thousands of eggs showed that cell division is accelerated when the exposure is not too prolonged. After an optimum length of exposure the rate of cell division is gradually retarded. I have shown that sea urchin eggs are accelerated by a weak stimulation. The kind of rays seems to make no difference with the result. In- asmuch as the gamma rays are very penetrating, and therefore are not absorbed to any extent, they are the ‘weakest’ and must be allowed to act for a long time before they can produce any effect. The beta rays are more readily absorbed and will pro- duce an acceleration if not allowed to act for too long a time. If the alpha rays are allowed to act in unison with the other types, acceleration will follow after a few seconds’ exposure. These rays are about one hundred times as effective as the beta rays, and the beta rays are more effective than the gamma rays in the same proportion. These figures correspond roughly to the respective powers of jonization of the rays. Retardation follows a moderate radiation of the beta rays. This effect is not peculiar to them, for if they are mixed with EFFECTS OF RAYS OF RADIUM ON PROTOPLASM 343 gamma rays the results are the same provided the intensity of radiation is equal. The effect is cumulative, and_ persists through many cell generations (Carrel 714). This is shown also in the experiments on Nereis and on Arbacia. It has also been found that a radiated cancer, in which cell division has been retarded by exposure to the beta rays, may be transplanted several times and still show the effects of the radiation. There is no appearance of abnormal development, or of any visible changes in the cell constituents. The treatment, in every case, has served merely to decrease the rate of normal metabolism without disturbing the process itself. A very strong radiation with the beta rays (which must neces- sarily be mixed with the gamma rays) or with all three kinds at once, results in profound morphological changes in the cells. The type of changes thus induced varies in different cells. In the sea urchin and in Ascaris the nucleus is most readily affected. In Nereis, on the other hand, it is the protoplasm which first undergoes degenerative changes. Whether this is due directly to an ionization of the chemical compounds of protoplasm is an open question. Inorganic materials are ionized during radiation, but living matter may not be affected in the same way. A Lytic action occurs, as shown in the liquifying of the protoplasm in Nereis, and in the breaking up of the chromatin of Ascaris and other forms. These profound changes in the physical constitution of the cells is accompanied by changes in the behavior of their con- stituents. In this respect cells differ greatly. In Ascaris the achromatic portion of the mitotic figure is uninjured, while the chromatin is broken up into granules. In Nereis, on the con- trary, the chromosomes split with great precision, but the spindles are abnormal and are sometimes entirely absent. But I have never found an egg so injured that it did not make some attempt, however abortive, to go through its usual development. The hypotheses which have been advanced in explanation of the phenomena which follow a severe radiation were discussed in a previous paper (Packard 714). Hertwig’s view, which is based on a study of forms in which only the chromatin is injured, 344 CHARLES PACKARD is that prolonged exposure may so injure the chromatin that it is unable to play a part in cell division. If the sperm alone is radiated, it merely acts as a stimulus to induce in the egg par- thenogenetic development. If the egg is radiated, the sperm nucleus alone divides, the egg nucleus taking no part in sub- sequent development. A less severe radiation of either element serves to generate in the chromatin a poison which brings about abnormalities in growth. I have been unable to find any evidence of parthenogenetic development either in Nereis or in Arbacia. When the eggs are radiated development is either diploid or does not occur at all. The same is true if the sperm is radiated. If the eggs or sperm are not greatly injured development is diploid but abnormal. It is evident that no generalization on the effect of radiations can be based on the behavior of a single form, for it has been shown that there are several types of response among the cells already studied. Nor can we assume that the effect is directly on the nucleus or on the protoplasm. If it were on the former we should expect that exposure of the cells during the resting stage of the nucleus would be followed by greater abnormalities than would obtain when the radiation is made during mitosis, since in the former period the chromatin is more finely divided and presents a larger surface to the rays. But the reverse is the case. Mottram (713) has shown that Ascaris eggs are eight times more susceptible during division than during the resting stage; that is, there are eight times as many deaths following an exposure made during mitosis than during the latter period. It has also been observed that cancer tissue is much more suscepti- ble to the rays when it is growing rapidly than when it is nearly stationary. This indicates that an explanation for these phenom- ena must take into account the differences between the physio- logical state of the cell constituents during these periods. During the resting period the interchange of material between nucleus and protoplasm is small compared with the amount which takes place during cleavage. At the latter time the EFFECTS OF RAYS OF RADIUM ON PROTOPLASM 345 amount of oxygen which is taken in and of carbon dioxide which is given off is greatly increased. The agents concerned in the utilization of oxygen and in the production of carbon dioxide are undoubtedly the intracellular enzymes which during division are more active than during the resting period. Acceleration of the normal metabolic processes must necessarily involve a quickening of the enzyme action. In like manner, retardation of those processes is connected with a slowing down of the activi- ties of the enzymes. According to Gager (’08), ‘‘The broadest, and at the same time the most definite generalization warranted by the work done so far is that the rays of radium act as a stimulus to metabolism. If the stimulus ranges between minimum and optimum points, all metabolic activities, whether constructive or destructive, are accelerated; but if the stimulus increases from the optimum toward the maximum point it becomes an over-stimulus, and all metabolic activities are depressed and finally completely in- hibited.”” The fact that enzymes may be accelerated or retarded has been shown by Richards (’14 b) who states that an exposure of two minutes to X-rays produces an acceleration in their activity, while an exposure of more than five minutes causes a retardation. If we assume that such reactions are duplicated in the living cell we have a logical explanation for the phenomena which have been described. The results of these experiments suggest further lines for re- search. It has been shown that in the sea urchin the chromo- somes are not all affected in a similar manner, for some are evidently injured while others are not visibly changed. Payne (13) has pointed out that when the egg of Ascaris is moderately stimulated, the chromosomes show marked differences in their reaction to the treatment. After the egg has divided, it is found that the chromatin of the sex cells is noticeably different from that in the somatic cells. This indicates that the two kinds of chromosomes are physically different, as Boveri has stated. This point can be tested by studies on the reactions of the chro- mosomes of those bugs in which the X chromosome can ke 346 CHARLES PACKARD distinguished. And should the behavior of the chromosomes in Ascaris find a counterpart in the behavior of the chromosomes of the bugs we would have a simple and elegant method of testing some of the hypotheses concerning the réle of the X chromosome. SUMMARY 1. Very mild radiation by means of the gamma rays from 50 mg. of radium bromide produces an acceleration in the rate of cell division in Arbacia without producing any abnormalities. These rays have no effect on the development of Nereis or Drosophila. 2. Moderate stimulation by means of the beta rays, obtained by separating them from the gamma rays in a strong magnetic field, brings about a retardation of growth in Arbacia and Nereis, which is followed by no abnormalities. 3. More intense radiation in which both beta and gamma rays are used, results in a liquifying of the protoplasm in the Nereis egg, and the development is abnormal. The eggs of Arbacia show no protoplasmic changes, but the chromatin is injured. 4. There is no evidence for parthenogenetic development. 5. Acceleration and retardation may be caused by a change in the rate of enzyme action brought about by the radium treatment. LITERATURE CITED. ABBE, R. 1912 An improved method of using radium. Med. Rec., Feb. 10. 1914 Radium Beta Rays. Med. Rec., Nov. 28. Conapon, E. D. 1912 A comparison of the alteration in the velocity of growth of certain seedlings through the action of the rapid and slow electrons of the beta rays of radium. Arch. f. Entwickl., Bd. 24. GaceR, G.S. 1908 Effects of the rays of radium on plants. Mem. N. Y. Bot. Gardens, vol. 4. GuILLEMINoT, H. 1907 Effets comparés des rayons X et des radium sur las cellule végétal. Compt. Rend. Acad. Sci. Paris, T. 145. Hertwic, G. 1911 Das Schicksal des mit Radium bestrahlten Spermachro- matins im Seeigelei. Arch. f. mikros. Anat., Bd. 77. Hertwic, P. 1911 Durch Radiumbestrahlung hevorgerufene Veranderungen in den Kernteilungsfiguren der Eier von Ascaris megalocephala. Arch. f. mikros. Anat., Bd. 77. EFFECTS OF RAYS OF RADIUM ON PROTOPLASM 347 Lazarus-BarLow and Beckton. 19138 On radium as a stimulus of cell division. Arch. Middlesex Hospital, vol. 30. Litum, F. R. 1911 Studies in fertilization. I, II. Jour. Morph., vol. 22. 1912. Studies in fertilization. III, IV. Jour. Exp. Zodél., vol. 12. Morrram, J. C. 1913 On the action of beta and gamma rays of radium on the cell in different states of nuclear division. Arch. Middlesex Hosp., vol. 30. Packarp, C. 1914 The effect of radium radiations on the fertilization of Nereis. Jour. Exp. Zo6l., vol. 16. Payne, F. 1913 A study of the effect of radium upon the eggs of Ascaris mega- locephala univalens. Arch. f. Entwickl., Bd. 36. Ricuarps, A. 1914a The effect of X-rays on the rate of cell division in the early cleavage of Planorbis. Biol. Bull., vol. 27. 1914b The effect of X-rays on the action of certain enzymes. Am. Journ. Physiol., vol. 35. RuTHERFORD, E. 1913 Radioactive substances and their radiations. Cam- bridge. TENNANT, D.H. 1910. Variations in Echinoid plutei. Jour. Exp. Zool., vol. 9. PLATE 1 EXPLANATION OF FIGURES All the drawings were made with the camera lucida. Unless otherwise stated the magnification is X 875. Figures 1 to 15 inclusive show the development of Nereis under the conditions described; figures 16 to 23 are of Arbacia. 1 Drawn from the living egg. > 475. This egg had been radiated with rapid beta rays at 4mm. for 1 hour; drawn just before cleavage. The very wide perivitelline space is traversed by delicate strands representing the walls of the alveoli. 2 Section of egg treated as above; 35 minutes after insemination; the softened protoplasm at the periphery has been pulled in under the influence of the aster. 3 Egg radiated for 13 hours with both rapid and slow beta rays, 40 minutes after insemination. The protoplasm at the periphery lacks the usual granular appearance. 4 Same treatment, 40 minutes after insemination; the protoplasm is vacuo- lated. 5 Exposure as in fig. 1. 35 minutes after insemination; the sperm has begun to enter the egg. 6 Same, 45 minutes after insemination; the sperm is now in the center of the egg. 7 Egg exposed to the slow and rapid rays for 30 and 50 minutes after. 8 Same, 70 minutes after insemination. 9 Same, 60 minutes after insemination. 348 EFFECTS OF RAYS OF RADIUM ON PROTOPLASM CHARLES PACKARD Ey eh te Qa $429 92 eM.) 4 j Sa i) > e ed OY Bale eed 2 le . Oboe ® e ®~ = 9 (6% ees ’S*e*°@o > | @%e « & ° - 4 a , ¥ = Fa baal — ~ “Sr — re 3 * . x ring ; s s ‘ Ps a Pd a) ~ ce 7 or al a4, oe ’ i aes - “ (oat hae ms : wy” at Sa? “ >. Me, ee ee em oa Le) heal ¥ ay i oe “4 ie! @ ae THE EFFECTS OF CARBON DIOXIDE ON THE EGGS OF ASCARIS! THEOPHILUS S. PAINTER (Instructor in the Sheffield Scientific School) From the Osborn Zoological Laboratory, Yale University FIFTEEN TEXT FIGURES AND THREE PLATES The material upon which the present study is made was placed in carbon dioxide on July 14, and removed on October 9, of the same year (1913). Professor Boveri, in an attempt to keep the eggs of Ascaris for a long time without allowing them to undergo development, had a number of smears? from one female placed in a stoppered glass jar. The air of the jar was then re- placed by passing a current of carbon dioxide through it for an hour and a half and, after sealing it carefully to prevent the escape of the gas, it was placed in a basement room in the Zoé- logical Institute at Wirzburg. At the time the eggs were placed in the gas, no ill effects were anticipated, consequently, no control smears were preserved to show the exact nuclear condition in which the eggs were at that time; and, after they were removed from the gas, they were placed directly on ice where they remained until used. When a few smears were allowed to undergo full development later, it was found that only part of the worms were normal. Professor Boveri called my attention to the fact and placed the material at my disposal with the suggestion that I determine the cause of the abnormal development which part of the worms showed. Although the material for this study was obtained in Wiirz- burg, the greater part of the work has been done since my return ‘A preliminary note on this subject has been published by the author (1914). * The smears were made on ordinary microscope slides according to Boveri’s well known method. 355 9 THE JOURNAL OF EXPERIMENTAL ZOGLOGY, VOL. 19, NO. 3 356 THEOPHILUS S. PAINTER to America. I take this occasion to express my thanks to Professor Boveri for suggesting the problem and for placing the necessary material at my disposal. In preserving the eggs, a mixture of 4 parts 95 per cent alcohol and 1 part glacial acetic-acid was used. They were stained in toto and mounted in glycerine. DESCRIPTIVE Among the embryos which had been allowed to undergo full development, one finds perfectly normal specimens; specimens in which only one end of the body is developed; and lastly, totally disorganized embryos. The problem was _ primarily to determine the causes which produced the abnormalities observed, but as the work went forward a number of other questions of general interest came up which will be touched upon in the following paper.* A drawing of a normal worm, which developed from an egg exposed for three months to carbon dioxide, is given in figure A. A blunt anterior end with the pharynx and the pointed posterior end may be seen, and, in addition, towards the posterior part of the body the large deeply-staining nuclei of the primordial germ cells. Typically there are only two of the latter but occasionally more occur; in one case six were present. The abnormal embryos are of two general types. One of these has the posterior end of the body fully developed while the anterior end is disorganized. The second type is characterized by the absence of organization in its blastomeres. The first type of embryos is shown in figure B. This occurs in roughly 33 per cent of the embryos (in 54 cases out of 165 examined for the point). The pointed posterior end is clearly seen together with the primordial germ cells, but there is much variation in the degree to which the posterior part of the body is developed. We 3 A brief description of the normal development of Ascaris is given on page 367. Any one not familiar with the cleavage, or with the nomenclature, in this worm, will find it helpful to read this over together with a glance at the schematic dia- grams given. The nomenclature of Boveri has been used throughout the present work. EFFECTS OF CARBON DIOXIDE ON EGGS 357 Text figures A to C may, in rare cases, find fully seven-eighths of the worm normally formed, or we may find nothing but a pointed stump; figure B gives a fairly typical case. An example of the second type of embryo is given in figure C. Such individuals are found in about 40 per cent of the cases (66 cases in 165). The greatest variation is noted in the appearance 358 THEOPHILUS S. PAINTER of such embryos. Typically they consist of a mass of cells, among which one may distinguish the primordial germ cells, but no organization exists and quite frequently it is evident that no cleavage cavity was ever formed in the embryo, consequently, that gastrulation had not taken place. Eggs preserved as they were taken from the ice chest, where they had been since their removal from the CO:, showed a slight amount of development. All of them had divided at least once, and something less than half (227 out of 505 eggs counted) had reached the 3-cell stage. Occasionally, in such a preparation, a 4-cell stage is found. An examination of these 3-cell stages (fig. 1) shows that the S, blastomere has divided to form the A and B cells, while the P, blastomere is in a ‘resting stage.’ The nuclear condition of the A and B cells is normal: as may be seen by the figure, waste chromatin occurs in one or both of the cells showing that the diminution process has taken place, but the two cells do not always lie pressed against each other as is normally the case (compare fig. 1 with text fig. F).. In 50 eggs out of 78, examined at random for this point, the A and B blastomeres were separated, in extreme cases the two cells lying on opposite sides of the P; cell. Among the eggs in the 2-cell stage, 101 out of 278 cases showed the 8, cell dividing, with the chromosomes in the equatorial plate phase. The remainder showed resting nuclei in both the S, and P, blastomeres: A close examination of the equatorial plates in the dividing cells shows an abnormal condition of the chromatin (figs. 2a to 2d). Figures 2b, 2c, and 2d are drawn at a higher magnification. In practically every case (96 out of 101 eggs examined for the point) the chromosomes were found fused together. This fusion seems to affect the ends principally (figs. 2a, 2d) leaving the middle portion free, but in extreme cases even the middle parts are involved and all four chromosomes are clumped together into one mass (figs. 2b and 2c). One very constant feature of this fusion is the formation of what appear to be vacuoles in the fused ends. It is also to be noted that when only the ends of the chromosomes are involved the middle EFFECTS OF CARBON DIOXIDE ON EGGS 359 portions have the normal clumped or lumped appearance which precedes diminution (compare fig. 2d with fig. 9). In rare cases, both the S; and the P; cells were dividing at the same time (fig. 3). When this occurred, the chromatin of the §; blastomere showed the fused condition, while the chromosomes of the P, cell were normal. In the 2-cell stages with the nuclei in the resting phase, no . departures from the normal could be distinguished. When the eggs are allowed to develop a short time before they are preserved, the P, blastomere begins to divide. The elon- gated chromosomes, so characteristic of the primordial germ cells, are always found and aside from the axis of division, the cell appears normal. Here and there a tendency for the four chro- mosomes to break up has been noted (text fig. J and L), but this is not to be regarded, I think, as an effect of the CO.. The point will be taken up in detail later under the heading ‘Anomalies.’ If the eggs are allowed to develop further until half are in the 4-cell stage, a variety of conditions are found in the 4-, 3- and 2- cell stages. Needless to say, these various conditions arise out of the 2- and 3-cell stages described above. Among the 4-cell stages we find three different types of em- bryos. One of these (fig. 5) is perfectly normal, both in the position of the blastomeres and in their nuclear conditions. A second type is characterized by the failure of the four blasto- meres to form a rhombus, as they normally should do (fig. 4).4 Most frequently the planes connecting the two pairs of blasto- meres lie at right angles to one another, but this is variable, every imaginable condition being met with in a large number of eggs. The third type is one where, in addition to an abnormal position of the blastomeres, we find an unequal distribution of the chromatin between the A and B blastomeres (fig. 6; this drawing is of a 3-cell stage, selected because it shows particularly well the ‘It is not to be thought that these various positions which the A and B blasto- meres occupy are just phases of the normal shifting which certain cells of the egg undergo about this period. As will be seen, these positions are retained in later cleavage. 360 THEOPHILUS S. PAINTER unequal distribution of the chromatin). Here we fail to find the usual waste chromatin in either the A or B eell. On the other hand, one of the cells in such eggs always contains a very large amount of deeply-staining reticular chromatin (fig. 6) while its mate lacks this. This is a very constant feature of eggs in which there has been an unequal distribution of the chromatin between the A and B blastomeres. Among the 3-cell stages there is always a certain proportion of eggs which are perfectly normal, as far as can be determined. The S, cell is divided, the blastomeres lie pressed up against each other, and the waste chromatin lying in the cyto- plasm of these cells indicates that diminution has taken place. There is a second type of 3-cell stage which is very striking (figs. 7 and 8). In the egg shown in figure 7, the P: and the EMSt blastomeres have separated—are in a stage of division, in fact while the §, cell is undivided and contains a tetraster. Figure 8 shows the same condition of the 8; cell and in addition we note the enucleated protoplasmic ball lying on top of it. Eggs showing tetrasters usually, if not always, possess this pro- toplasmic ball. In figure 7 it is present, but les under the other cells and is not shown in the drawing. The amount of chro- matin between the four centrosomes of the §; cell is very large. It may be undergoing diminution or we may find the elongated chromosomes still present. In the latter case, one may usually count eight chromosomes lying in the spindles. In text figure D, an egg is shown with a history similar to that of the tetraster eggs, but for some reason, the centrosomes have failed to divide. In this respect it is exceptional, but eight chromosomes and the characteristic protoplasmic ball are both clearly seen. The 2-cell stages usually show the 8; and the P, blastomeres undergoing division (fig. 9). Except for the axes of division, which frequently do not occupy normal planes with regard to each other, the division figures are typical of the untreated eggs; and the lumpy condition of the chromatin of the S, cell, which precedes diminution, is seen. A number of smears of eggs were preserved when the embryos were in or just beyond the 4-cell stage, and in these, we find a = EFFECTS OF CARBON DIOXIDE ON EGGS 361 tremendous variation in the relative proportions in which the various abnormalities described above occur. In one preparation over 50 per cent of the eggs showed a tetraster in the S, blasto- mere, or showed the later effects of it. In other slides only a very small proportion of the eggs showed this abnormality. The proportion of the eggs which showed an unequal distribution of the chromatin between the A and B blastomeres, showed the same variation in different slides. On the other hand, the fail- ure of the blastomeres to form the rhombus is an abnormality found in a large per cent of cases on every slide. Text figure D. In the large amount of material which was preserved at fre- quent intervals after the 4-cell period, it has been possible to follow the results of the abnormal conditions described through the later development. Of course, as cleavage progresses, it becomes increasingly difficult to follow the course of the indi- vidual blastomeres, except in the case of the primordial germ cell. The latter is usually conspicuous on account of the large size of the nucleus. An analysis of the later stages has been facilitated by the use of clay models of the eggs and a com- parison of these with the excellent figures given by Boveri (’99). In this way it has been possible to determine very exactly the plane in which a given cell is dividing, and the relation it will have to the rest of the blastomeres. 362 THEOPHILUS S. PAINTER For the sake of clearness, the course of the different types of eggs will be followed separately through later cleavage in the following description. These will be taken up in the following order: (a) The effects of the abnormal positions which the 8, derivatives take in cleavage. (b) The result of the unequal distribution of the chromatin between the A and B blastomeres. (c) The fate of the tetraster eggs. Among the treated eggs there is always a certain per cent which are perfectly normal. The 4-cell stage, such as shown in figure 5, is followed by the division of the A and B blastomeres in a plane approximately at right angles to the plane of the paper upon which the drawing is given (compare with fig. G). Following this, the P; and EMSt cells divide in the median plane of the embryo (compare with fig. 1), and throughout the later cleay- age, the analyses with models show that the normal develop- ment is continued. The development of embryos in which the A and B blastomeres occupy abnormal positions in the 4-cell stage, may be followed with ease up to the time when the §; derivatives number 16 cells. From this point on, such eggs are not be to distinguished from normal embryos. Figure 4 shows a typical case of the positions which the A and B eell take. In figure 10, we see these two cells dividing. The diminution process is taking place normally, but the planes which the dividing cells occupy, instead of being parallel (compare with the normal as shown in fig. G) are at right angles to each other. The result of such a division is shown in figure 11. In the egg shown in figure 12, the ectodermal cells are eight in number and the EMSt cell has divided in the median plane of the embryo. The P» cell is in the equatorial plate phase of division. The elongated chromosomes characteristic of the primordial germ cell are clearly seen. In figure 13, we see a some- what later stage. Both the P, and EMSt blastomeres have divided. It is especially to be noted that the MSt blastomere does not lie in the median plane of the embryo (the EMSt cell divides into an E and an MSt cell; these normally lie in the me- dian plane of the embryo, compare with fig. 1). It is very rare that we find the EMSt cell dividing in any plane but the EFFECTS OF CARBON DIOXIDE ON EGGS 363 median, but in eggs examined after the division, we frequently find the MSt blastomere lying outside of the median plane. In these cases, the A and B derivatives are very asymmetrically distributed over the dorsal portion of the embryo and it seems that these cells push the MSt blastomere out of its normal posi- tion. This is more apparent on models of the eggs, of course, than in figures. The probable significance of this will be taken up later. Many hundreds of eggs similar to those shown in figures 10 to 13 have been analyzed. The abnormal positions of the A and B cells are retained, there is no shifting to form the rhombus, at least it is not usually realized, the ectodermal cells derived from the 8; blastomere take up positions on almost any part of the egg. Thus the bilateral symmetry of the embryo may be completely lost and, what is for our study more significant, the members of the ventral family, especially the MSt blasto- mere, may be moved out of the median plane. The most striking results of the unequal distribution of the chromatin between the A and B blastomeres is shown in figure 14. Aside from the positions which these two cells have, we see that one is dividing early and that it contains only a small number of somatic chromosomes in the spindle. The mate, on the other hand, shows no sign of division, and it will be noted that it contains a very large nucleus (compare fig. 14 with fig. 6). This result always follows the unequal distribution of the chro- matin, apparently, and the early division of the one cell upsets the cleavage rhythm of the 8, derivatives. If the distribution has been very uneven, then one of the cells may divide twice before its mate cleaves. With a more equal distribution the rhythm is not so markedly upset, but in any event the end result is the same: the 8, derivatives become scattered irregularly over the surface of the embryo, the symmetry or balance of the em- bryo is upset, and probably members of the ventral family are pushed out of their normal positions, as was the case with the egg shown in figure 13. The development of the embryos which showed a tetraster in the S; blastomere, is extremely variable, both in the number of cells formed by the division and in the distribution of the 364 THEOPHILUS S. PAINTER chromatin. A typical case is shown in figure 15. Here there are six cells besides the P,; and EMSt blastomeres. One of these is evidently an enucleated protoplasmic ball, so characteristic of the tetraster eggs. The remainder came from the division. It will be noted that one of the small blastomeres contains a large amount of waste chromatin. This is a very common phenomenon exhibited by such eggs after division. In this egg, we also see that the MSt blastomere is not dividing in the same plane as the P,. Such a condition is seldom met with. The later development of these eggs which have had a tetraster in the 8, blastomere, is extremely abnormal. The §, derivatives divide irregularly; they become scattered over the surface of the embryo or lie in one heap; and there is every indication that they very rarely or never form a cleavage cavity and gastrulate. In later cleavage stages, the abnormalities caused by these tetraster eggs is very striking. One of the marks of such eggs is the presence of the small cell with the large amount of chromatin (fig. 15). This disorganization, however, does not extend to the primordial germ cell, for we find it dividing normally in later stages when the embryo is otherwise totally abnormal. At the time of gastrulation, a majority of the embryos appear normal or exhibit minor irregularities, such as a slight asym- metry of shape. The cleavage cavity is present in such eggs, however, and there seems to be no reason why they should not gastrulate normally. Among such embryos one finds a large number which have no cleavage cavity. This sometimes appears to be due to the fact that the ectodermal cells are too scattered or are too few in number to form it. But in every case the primordial germ cell nuclei may be clearly seen. To sum up the foregoing description, we find that the abnormal eggs are of three types: (a) Eggs in which the A and B blasto- meres have abnormal positions in the 4-cell and later stages. (b) Eggs in which there has been an asymmetrical distribution of the chromatin between the A and B cells. (ce) Eggs with a tetraster in the 8; blastomere. We have now to inquire how these three abnormal conditions arose from the eggs just re- moved from the CO.. And, secondly, what relation these ab- EFFECTS OF CARBON DIOXIDE ON EGGS 365 normalities in cleavage bear to the embryos which had been allowed to undergo full development. Taking up the first question, a close examination of the material has shown that the tetraster condition of the S$, blastomere and the irregular distribution of the chromatin in the A and B cells, is due to the same cause, that is, the fusion of the chromatin in the 8, cell (figs. 2a to 2d). A glance at these figures will show that in part of the eggs, only the ends of the chromosomes were involved and that the middle portions were free. In such eggs a division of the 8; blastomeres occurs but, owing to the fused condition of the chromatin, an equal distribution of it can not take place. Diminution of the chromatin occurs and one blastomere receives a number of small ‘diminished’ or somatic chromosomes, while the other cell receives, in addition to the somatic chromosomes, the whole mass of fused chromatin of the equatorial plate. If the fusion involved part of the chromatin which would normally go to form somatic chromosomes, then one cell would receive this together with the waste chromatin. When this fused mass goes to one cell, it does not undergo degenerative changes but (probably because of the presence of some somatic chromatin) it becomes resolved into a reticulum and fuses with the normal nucleus of the cell. In this way, one cell comes to contain more chromatin than its mate, and in later stages, the cell with the least chromatin divides earlier. Vari- ous stages of this process have been observed in my material. When, however, the fusion involved all of the chromatin, as in figures 2b or 2c, then division appears not to take place. Apparently, the fused condition of the chromatin is responsible for this, but whether this prevented the centrosomes from going apart, or whether the fused mass kept the cell wall from cutting through, is not known. Stages that would decide this point have not been seen, but various other steps in the process have been observed. Thus the one cell comes to contain all the chromatin which should be distributed between blastomeres A and B. At the next division cycle, such eggs showed a tet- raster in the §; cell and eight chromosomes are found in the spindles (fig. D). 366 THEOPHILUS S. PAINTER One very constant feature of the eggs showing a tetraster is the occurence of a protoplasmic ball which les on the blasto- mere showing this condition. There seems to be an intimate connection between the formation of the ball and the failure of the egg to divide. This point will be touched upon again. The abnormal positions which the A and B blastomeres take up is to be traced, in part at least, back to the 3-cell stage, when the A and B cells separated (fig. 1). It is probable, however, that this is not the only source of the abnormality, for not infrequently, one finds eggs like those shown in text figure N. As may be seen in the figure, the A and B cell lie pressed against each other, and are dividing. The interesting thing is that the P: cell, is dividing in an abnormal plane. This condition could come about only in one of two ways. Either there has been a rolling or shifting of some of the cells, or, their polarity has beenchanged. Of the two possibilities the former seems the most probable since in the normal egg, a shifting process is involved which brings the B and P, blastomeres together (compare with figs. F and G). As we know nothing definite about the cause of the shifting in the normal egg, it is useless to speculate over the matter at this time. It seems worth while, however, to point out that the separation of the A and B cell might come about by an increase of surface tension. These cells normally le pressed against each other, but were the surface tension of them increased, as through some action of the CO., then they would separate. If the P, cell were also affected, the effect (that is, the separation) might be still more marked. Before we take up the relation between the abnormalities found in cleavage and those exhibited by the fully developed worms, it will be necessary to describe a few of the more striking points in Asearis development. Thanks to the works of Boveri (99) and Zur Strassen (’96), we know the origin and fate of practically every cell in the young worm. A few schematic sketches of the normal development are given in figures E to I. The first division results in two blastomeres, S, and P, respectively, following Boveri's nomenclature. These two cells have different potentialities. In the next division EFFECTS OF CARBON DIOXIDE ON EGGS 367 cycle, these two cells divide in planes at right angles to each other (fig. E), and furthermore, as is well known, the chromatin of the S, cell undergoes a process of “‘diminution”’ while the P, cell retains the elongated chromosomes. The result of the division is four blastomeres which form a T-like figure (fig. F.). Following this, the cell marked P, shifts around until it comes in contact with the B blastomere, forming in this way a rhombus. Up to this time, one can not speak of an anterior and posterior end of the embryo, but after the shifting, these parts are marked out. The Text figures E to I anterior end lies to the right in the figure, that is, at the A and EMSt side, while the posterior end is indicated by the P; blasto- mere. The A and B blastomeres lie dorsally, as in the figure, the P, and EMSt ventrally, the median plane of the embryo being parallel to the paper and passing through all four blastomeres. The A and B cells now divide in a plane approximately at right angles to the median plane (fig. G), while the P. and EMSt cells divide in the median plane (fig. I). The A and B cells give rise to the ectoderm covering the dorsal and anterior end of the body. The EMSt cell will give rise to the entoderm, part of the 368 THEOPHILUS S. PAINTER mesoderm, and the cells of the stomodaeum. The P» cell, after giving off several generations of ectodermal and mesodermal cells, forms the primordial germ cells. The most important points for us to remember are the following: During later develop- ment the A and B cells grow over the dorsal and anterior end of the body of the embryo. The EMSt and P» cells lie ventrally and posteriorly and form the most important organs of the body. Nearly all of the posterior part of the body of the young worm comes from the P; derivatives. We can now turn to the question of the relation of the normal cleavage of the treated eggs and the worms which resulted from them. Since a certain percentage of eggs always developed in a per- fectly normal fashion during cleavage, it is clear that the fully developed normal worms, such as shown in figure A, arose from this source. It is equally clear that the masses of totally disorganized cells which one finds in figure C are due, in part at least, to the forma- tion of the tetraster in the S: blastomere, with the subsequent abnormal development. No doubt other sources contributed to this class of embryo. The embryos in which the posterior end is only partially dif- ferentiated are undoubtedly to be traced to the eggs with the A and B cells lying in abnormal positions, but the details of how this condition affected the later development are uncertain be- cause we have so little knowledge in how far the later shifting of the blastomeres in Ascaris is due to internal organization,and in how far to simple mechanical relations, such as mutual pres- sure, etc. Admitting this uncertainty at the start, we may give a very simple explanation which appears to agree with all of the observations recorded. A glance at figure I will show that the derivatives of the P; cell form a sort of half keel on the ventral and posterior end of the embryo, and the ectodermal cells, (derivatives of the 8:1) by their division form a more or less symmetrical covering for this. The works of Boveri (’09) and Miss Stevens (’09) have shown that this ventral keel may take place when the A and B derivatives EFFECTS OF CARBON DIOXIDE ON EGGS 369 are absent (as when the §, is killed by ultra-violet light). This indicates a high degree of internal organization in these cells. Here it may be remarked that in these experiments there were no ectodermal cells which might move the members of the ventral family out of the median plane. Were, however, the A and B derivatives present but in positions which would destroy the symmetry of the embryo (for example, were they lying only on one side of this keel, and I have observed cases which approached this) to unbalance the system, so to speak, then it seems very probable that some of the ventral cells would be moved out of the median plane. In the later development of the eggs in which the A and B cells occupy abnormal positions, we seem to see this unbalancing tak- ing place. Quite frequently in such eggs, the majority of the A and B derivatives lie on one side of the keel, and in the later stages, as shown in figure 13, one of the blastomeres (the MSt in this case) is moved out of the median plane. Prior to the division, the EMSt blastomere lies in the median plane and even in the metaphase, it holds this position. I have noted only one or two exceptions to this. After division, however, the MSt cell is frequently found lying out of the median plane, and the most probable explanation is that it has been moved out of this plane by the overlying ectodermal cells. It may well be that other causes were operating to produce the same end effect. In a few cases, the EMSt blastomere divided in an abnormal plane, figure N shows such a case, or, disorganization may have come later. As cleavage went forward in these eggs, the A and B deriva- tives formed the cleavage cavity by mutual pressure and gas- trulation took place. It was not until organ formation began that the effects of the shifting of the MSt cell could be observed. Looked at from a theoretical point of view, since the MSt blasto- mere forms the cells of the stomodaeum (after several divisions) we should expect that were this cell pushed out of its normal position, the resulting embryo would lack this part. The em- bryos in which the anterior end is disorganized but in which the posterior end is normal, seem to fulfill these expectations. The 370 THEOPHILUS S. PAINTER posterior end, coming from the other end of the keel and more or less independent of the derivatives of the 8; cell, would be normally formed, since all the necessary elements were present. It is not to be expected that simply the MSt cell would be affected. No doubt the failure of this cell to take up the proper position causes the whole anterior end to be disorganized, and when the A and B derivatives were very irregularly distributed (when they, for example, lay on top of the P2 cell without touching the EMSt, and I have observed such cases) the disorganization probably extended to the posterior end. It seems likely that the degree to which the posterior end was differentiated is to be correlated with the positions which the A and B derivatives took, and thus we have a series of stages from worms which are seven-eighths normal, to worms in which only the stump of the posterior end is differentiated. In this way we are able to explain the production of the half embryos following the treatment of the eggs with CO.. There is, of course, another way of explaining their production, but this has not been advanced because it did not harmonize, as it seemed to me, with the facts which have been discovered by Boveri and his students. We have assumed that the ectodermal cells com- ing from the 8; blastomere, were more or less indifferent in their nature. This is indicated by the normal development, for the cells divide rhythmically and form the general ectodermal cover- ing for the anterior end of the body. In contrast to these, the cells of the ventral family (P; derivatives) divide very irregu- larly and possess a high degree of specificity, that is, one forms the entoderm, another the mesoderm, or primodial germ cells, and so on. Were we to attribute specificity to the ectodermal cells, then the explanation for the half embryos would be that they were formed since the cells destined for the anterior end were scattered and this part of the embryo was undifferentiated. The latter view does not seem tenable since all work points to the indifferent nature of the S; derivatives. In any event, however, the production of the half embryos is to be traced to the abnormal positions which the A and B blastomeres take in the 4-cell stage. EFFECTS OF CARBON DIOXIDE ON EGGS eel While the great majority of the eggs followed the different types of development outlined above, a small per cent could usually be found in any slide which were abnormal for no apparent reason. One condition rather common, is that seen in figure N, where we find the A and B cells undivided even after the P:; and EMSt cell have nearly completed their division. What the fate of such eggs is can not be definitely stated, but since we may find the A and B cells undivided in later stages, it seems probable that such eggs never gastrulate. The percentage of eggs of this type is small, in any event. We now come to the question, why are some of the eggs af- fected by the treatment with the COs, and why do others develop normally under the same conditions? And why do we find different proportions of abnormalities in the smears of the same female? It was for the solution of these questions that two series of experiments were planned and attempted, but since these were unsuccessful, the working hypothesis upon which they were based, will be given. This explanation is only tentative. It is well known that in any mass of Ascaris eggs, some develop more rapidly than others. When the eggs were placed in the CQ, they underwent a certain amount of development before the oxygen available was exhausted. When the supply of oxy- gen lasted until the nuclei were in the resting stage, no ill effects resulted except such as might arise from a shifting of the blasto- meres. If, however, the eggs were in the equatorial plate phase when the supply of oxygen gave out, then they remained in this state until brought into the air again. Whether the fusion of the chromatin took place in the CO, or whether it resulted later, was to have been determined. The variation in the proportion of the abnormal types is, without doubt, due to the following reason. All the eggs for the present study were taken from the fresh uterus of a single female. Such eggs removed from the end of the uterus have given off their polar bodies and the male and female pronuclei lie side by side until oxygen is admitted. Eggs lying farther back in the uterus are not so far advanced as those lying at the tip, con- THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 19, NO. 3 372 THEOPHILUS S. PAINTER sequently, some of the smears are advanced farther than others; a larger proportion of eggs reach the equatorial plate phase in one slide than in another, and we find as a result a larger pro- portion of tetrasters. It seems probable that in the smear with over 50 per cent of the eggs showing tetrasters in the S; cell, the S: blastomere had been able to bring its division only to the equatorial plate phase. However, on all these points more experi- mental evidence is needed and the author hopes to fill in the gaps as soon as suitable material is available. It is of further interest to ask how the eggs were able to live and develop to a slight degree, in an atmosphere of CO:. Es- pecially, when they had been kept at a temperature where they would normally have developed in some three weeks. These questions are taken up in the following discussion. It has long been recognized that intestinal parasites live under anaerobic conditions, but Weinland (’01) was the first to show the mechanism by which they obtained the oxygen necessary for their existence. It had been known previously that Ascaris contained a large amount of glycogen, and Weinland was able to show that when these animals were kept in a medium without food, this glycogen disappeared and he obtained CO, and valerie acid. He suggested that the gyleogen had been broken down by some animal ferment. Glycogen is one of the complex sugars with the empirical formula of (CsH,,O;)*. According to Wein- land’s ideas, this is broken down in essentially the following way: (CsHi005)* = x (C53H1,0.—valeric acid—CO, and O) A number of authors (Brault and Loepers ’04; Buschs 705, 06; Kemnitz ’11; and Brammertz 713) have shown that the eggs of Ascaris contain large amounts of glycogen. Bram- mertz was able to show that during the formation of the polar bodies, the amount of glycogen diminished in the region where these bodies were being formed. The reason for this was that the glycogen was broken down to furnish the oxygen necessary for this process. For further development the oxygen of the air seems to be essential, although the glycogen in the egg is EFFECTS OF CARBON DIOXIDE ON EGGS Bho slowly used up as development goes forward. Brammertz re- gards this glycogen as a sort of reserve to tide the embryo over unfavorable conditions. He cites one experiment in favor of this view, which is quite similar, in its conditions, to the experi- ments recorded above. He found that if eggs were placed in 70 per cent alcohol, part of them developed as far as the two cell stage before they were penetrated by the alcohol and killed. He regards it as improbable that the eggs could have gotten the oxygen necessary from the alcohol, and thinks there is proof here that the glycogen was used for this purpose. The author has not made any experiments with the eggs treated with CO, but the conditions are so similar with the experiment cited that it seems very probable that the eggs used in my exper- iments were able to live over this period of three months be- cause of the presence of the glycogen stored in their protoplasm. ANOMALIES Under this heading I wish to record some observations made on the treated eggs, which have a bearing on the problems of sex determination and the cause of diminution in Ascaris megalo- cephala. Im the dividing primordial germ cells, occasionally eggs have been seen in which there were, besides the four chromosomes, additional elements (in 13 cases out of 123 eggs taken at random). Typically there is only one additional chromosome, as in figure J, but cases with two, three, four, and even eleven fragments have been seen (fig. L). The way in which the single element behaves during division is shown in figures K, M, and N. Cases with more fragments could not be followed through division. The presence of one or more chromosome fragments in the primordial germ cells of Ascaris megalocephala have been de- seribed by a number of authors, and at the present time two views have been advanced to explain them. According to Boring (09) and Boveri (’09), they represent the accessory chromo- somes in this species. This is the view generally accepted. Kautsch (’13), however, has shown that another interpretation o14. THEOPHILUS S. PAINTER is possible. After an extensive study of anomalies in Ascaris, he comes to the conclusion that these fragments are probably bits of chromatin brought into the egg when the polar bodies did not receive their full share of chromatin. Kautsch approached the question of the accessory chromosome in Ascaris in another way. He counted the number of somatic chromosomes in eggs in which there was only one chromosome, rave Ort nn aad Text figures J to N and he found that they fell into two numerical groups; one cen- tering around 27, the other around 36, somatic chromosomes. These, he suggests, may be male and female numbers. The counts made by Kautsch are too few in number to be conclu- sive, but it is interesting to note that his view falls into line with the work done by Edwards (710) on Ascaris lumbricoides. The latter author, as is well known, found that the accessory chromo- some in this species, is represented by a group of five small chromosomes. EFFECTS OF CARBON DIOXIDE ON EGGS one The observations recorded above, have a certain bearing on the question, since it is clear from them that genuine fragmenta- tion may take place in the chromosomes of Ascaris megalocephala, in some cases all four chromosomes being affected. The fragmen- tation of the chromatin in figure L, however, certainly has noth- ing to do with the accessory chromosome, and since a series may be formed in which one, two, or even all four chromosomes may be broken up, it becomes a question whether or not we can in- terpret such cases as shown in figures K, M, or N, as having any relation to the accessory chromosome. Just what relation the fragmentation in the normal eggs has -to do with that ob- served in the eggs treated with COs, is uncertain, but it does not Text figures O and P seem improbable that they are both expressions of a tendency residing in the chromosomes. A second anomaly found is shown in figures O and P. In figure O, the P, and EMSt cells are both dividing, and it will be seen that both are undergoing diminution. It is especially to be noted that the spindles of the two cells are not parallel. In figure P, another egg is shown in which both the P, and EMSt cells are undergoing diminution. Bsa wPESs je in v gepstie » Be o* i 0 6 ae? a“ a yt? va EMSt 8 P, 9 ; & 4 ed | = ~ + FD > ie * 4 = e ‘ Sa rte ee . @ 9 . a ' a 3 Ss m 4 - ‘ Pa 2 Z ef ~ < © ee . per =F os ee e al y getm st . ad »* td s w ou gi Pa Pa? > ec" ra & ~~ ¥ ~ a a0 ~e® oy, we te a ee 2S ae 4 ar ‘, i) to. 10 y fa 1 383 PLATE 3 EXPLANATION OF FIGURES 12 Showing the effect when the A and B cell divide in abnormal planes; the ectodermal cells number 8 in this egg. 13. Aslightly later stage of the same; note that the MSt cell does not lie in the median plane of the embryo. 14 Anegg showing the effect of the unequal division of the chromatin between the A and B blastomeres. 15 An egg showing the effect of the tetraster formation in the §; cell. 16 Showing the Pz and EMSt cell fusing; note particularly the protoplasmic ball projecting from the fusing cells. 17 Showing the later effect of the fusion of the P2 and EMSt cells; note that diminution is taking place in this cell. 384 Ta EFFECTS OF CARBON DIOXIDE ON EGGS PLATE 3 THEOPHILUS S. PAINTER MSt ? » MSt net op) 13 . “ EMS8t . aS 7ike ball 16 Ve 385 « * o A. 4 ¢ . ' * + \ a? - . y a P -~ . - s ~ . VARIATION AND INHERITANCE IN. ABNORMALITIES OCCURRING AFTER CONJUGATION IN PARAMECIUM CAUDATUM RUTH J. STOCKING From the Zoélogical Laboratory of the Johns Hopkins University’ TWENTY FIGURES CONTENTS Terao OU ChLOMe: -y-1- 5 eee ee ee IER a a ye NS Tbs Waevarsietvce «ue 387 MeMvVMiethods.....,...5.24 ee ET ar a, 390 ileetixnerimental Culbures: cae aera semn Is. oe eas he ey dn Sie eb nk ca tees 391 Wrelypes of Abnormal [Ra cess mesere ere noe sf mise cities Scuba sto cere 397 WeaNature of the Abnormalities: .seeemeseenass +o. l6.- Seat ER Ae 407 VI. The Abnormalities as Hereditary Characters: Variation, Imheritance, ardiseleciGnyery cs. +. .2 24. soe esses 412 Experiments in Selection................... Lc BUS Bie eRre 414 Relation to Biparental Imheritamce........................... 440 Miles summary and Discussion of Resuliiseme sets... 15s. $25... 5058- 444 CV LLILTL, GAS 0 eae PRR PE ono) a 8 449 I. INTRODUCTION The fact that there are two kinds of teratological variation, one caused by gametic constitution and therefore heritable, the other occasioned by the action of environmental conditions, and not heritable, has been recognized for some years. There was for a time an endeavor on the part of many investigators to show that all abnormalities were due to conditions of nurture; but the discovery of the strict mendelian inheritance of a large number of malformations has overthrown that theory. In many of the metazoa both kinds of teratological variation, gametic and en- vironmental, have been studied, and their causes and behavior partly determined. In the protozoa the abnormalities caused by the action of external factors have received by far the great- ‘Part of the work here reported was completed while the author held the Alice Freeman Palmer Research Fellowship of Wellesley College. 387 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 19, No. 4 NOVEMBER, 1915 388 RUTH J. STOCKING est amount of attention, heritable abnormalities having been but little studied. This paper presents an investigation of herit- able abnormalities in Paramecium. The principal points examined in this presentation are the following: The origin and nature of the abnormalities; their relation to conjugation; their inheritance and variation in uni- parental reproduction, with relation to the present day prob- lems of ‘pure line’ work; how precisely inheritance occurs; whether there are variations of kind and degree of abnormality; whether such variations are themselves inherited; whether by selection abnormal stocks multiplying asexually can be altered in their hereditary characteristics or differentiated into two or more hereditarily diverse stocks; their relation to biparental inherit- ance; their relation to survival. ; Previous work on the abnormalities found in protozoa has dealt mainly, as before remarked, with the results of environ- mental action. Jollos (13) subjected a race of Paramecium caudatum to changes of temperature and obtained by this method differences in size, as did Hertwig (’08), Popoff (’08, ’09), and Rautman (’09). Jollos found that these changes are transi- tory.and soon disappear as the paramecia become adapted to their new conditions. In a short preliminary note, which does not present the evidence for his conclusions, he states that in one case he obtained a permanent change. He subjected a race of Paramecium to a high temperature and obtained a race which was permanently small at high, normal, and low tempera- tures. Popoff (09) was able to produce by experimental means two abnormal races of Stentor, one giant and the other dwarf. He centrifuged a dividing Stentor, and so caused an unequal distribution of the nuclear material. The daughter cell which received the smaller part was one-fourth the size of the cell which received the larger part. These two multiplied nor- mally for about a week and during that time retained their abnormal sizes. The cultures were then lost. He obtained another giant race of Stentor by suddenly cooling a dividing individual. The division did not take place and the animal reorganized into a single individual which grew and subsequently —_— a INHERITANCE IN ABNORMALITIES 389 divided, forming a giant race which was kept for about forty- five days, and during that time bred true. Lewin (’10) obtained abnormally-nucleate races of Paramecium by cutting an indi- vidual through the macronucleus. The production of form abnor- malities in Paramecium by cutting and other experimental means has been studied by Calkins (’11), Peebles (12), Balbiani (’93), McClendon (’09), and Jennings (08). They all found that such experimentally produced abnormalities are mechanically handed on to one daughter cell at each division for a longer or shorter time. But such abnormal forms are gradually remodelled dur- ing successive generations, or die, and their normal sisters show no tendency to produce abnormal progeny. The teratological variations that arise spontaneously in a culture multiplying vegetatively have been studied extensively by Jennings (08). With one exception he found that all such forms either die very soon or give rise to a race of normals. In one case he did find that the abnormal individual gave rise to a race of abnor- mals, the deformity being such as to prevent the daughter cells from separating after division, thus forming what have been called ‘double monsters.’ In all other cases the abnormality was not a race character but an individual character and was not inherited. During the course of a year’s work at the Johns Hopkins University I followed the history of several abnormal forms which had arisen in the cultures of normals being carried on by other workers in the laboratory. In no case did these forms give rise to a race of abnormals; in one case a race of normals resulted; in all other cases death occurred either before any divisions had taken place or after one or two irregular divi- sions. In rare cases, Jennings (13) found, abnormalities may arise in the members of split pairs; that is, in the members of pairs separated before conjugation has been completed. But these also never gave rise to a race of abnormals, either dying very soon or becoming entirely normal. Teratological variations arising soon after conjugation have been described in a few cases. Simpson (’01) observed four daughter cells of a normal exconjugant Paramecium, three of which were normal while the fourth was posteriorly split. This 390 RUTH J. STOCKING animal divided eight times and its history was similar to that given above for the experimentally produced abnormalities in Paramecium. The abnormal character was handed on to one cell at each division, and the abnormal animal produced by the eighth division died. All of the sister cells and their progeny were normal. The only other observation known to me on the abnormalities arising after conjugation is made by Jennings in his 1913 paper quoted above. While he found only extremely rare and transitory abnormalities among his ‘split pairs’ and ‘free’ individuals, he found a large proportion of malformations among his exconjugants and their progeny. He describes their different types, characteristics, and constant and continued appearance throughout the course of his experiments; and adds, ‘‘A precise study is greatly needed, as to the minute character- istics of these abnormalities, their heritability, their experi- mental cause, and their cytological basis.”’ It was at his sug- gestion that a series of experiments was started for the purpose of studying these problems, and under his direction that these experiments have been carried through. I wish to express here my most sincere thanks for the constant help he has given during the entire course of the work. II. METHODS In all of the cultures on which this work is based the method of handling, cultivating, and recording was identical with that described by Jennings (713). Conjugation was’ first induced; after the pairs had separated the two members were isolated and cultivated separately in ze per cent Horlick’s malted milk (Peebles, ’12). Each pair was numbered, and the two members of a pair designated a and 6; the races which arose from these exconjugants were called after them in the same way, 2a, 2b, 3a, and so on. Beside the slide cultures, mass cultures were also kept for each race. These were kept in bottles and each bottle was labelled correspondingly, 2a, 2b, 3a, and so on. The method of recording described by Jennings was supplemented in my work by drawings of the abnormal forms. In a few cases INHERITANCE IN ABNORMALITIES 391 this was impossible because of the rapid movements of the ani- mal. For about two weeks after conjugation camera lucida drawings were made of the abnormal forms. These were often less active than the normal forms and after being in the same drop of culture fluid for two days all of the individuals -were more or less sluggish. So with a little patience camera lucida drawings could be obtained of almost all the abnormals. When the number to be examined and drawn became very large, how- ever, there was no time for that procedure and free hand draw- ings were made in the majority of cases, camera lucida only in such cases as seemed of more than ordinary interest. The slide cultures were examined every other day during the cool weather, every day when it became very warm, the individuals counted, the abnormals drawn, selection made of those by which the slide cultures should be carried on, and the others put into the bottle mass-culture corresponding to that race. The individuals selected for slide culture were then washed in the milk solution, and put into fresh fluid on clean slides. Ill. EXPERIMENTAL CULTURES The material presented in this paper is derived from three experimental cultures carried from the fall of 1912 to the spring of 1914. The first experiment, conducted from December 2, 1912, to June 11, 1913, was carried out with exconjugant mem- bers of a wild population which doubtless included members of many diverse stocks. The chief aim of this first experiment was to ascertain whether or not abnormalities were ever inherited, and to what extent. From November 25, 1913 to December 22, 1913, a second experiment was conducted on a group of animals all descended from one individual, constituting there- fore a ‘pure line’ or ‘clone.’ It was concerned mainly with deter- mining the effect of conjugation among the members of a single clone, particularly as to the production of abnormalities; this was brought out by a comparison of exconjugants with mem- bers of split pairs belonging to the same clone. In the first experiment some work on the effect of selection was attempted; but the third experiment, carried out on a wild population from a RUTH J. STOCKING January 14, 1914, to April 1, 1914, was concerned wholly with that problem. In it an attempt was made to ascertain the efficacy of selection as a means of breaking a single clone into two or more lines differing hereditarily in amount or kind of abnormality. In all three of the experiments, in two of which very large numbers of exconjugants and their descendants were studied, a large proportion of the resulting races were abnormal, just as was the case in the exconjugants studied by Jennings (13). In Experiment 1, with a wild population, the proportion of abnor- mals was 36 per cent of the entire 262 exconjugants. In Experi- ment 2, with the members of a clone, the proportion of abnor- mals was much higher, being 81 per cent of the whole 200. In Experiment 3 the number of exconjugants studied was small; the abnormal races formed 48 per cent of the total 28. In the 54 members of the 27 split pairs of Experiment 2, twenty of which were cultivated for 19 days, and thirty-four for 7 days, no abnormals at all appeared. Whenever sets of individuals are isolated without conjugation and cultivated in the same way, no such proportion of abnormals are observed; indeed as a rule no abnormals whatever are obtained. We shall return later to the relation of the abnormalities to conjugation (page 412). The exconjugants from large cultures of paramecia may be divided, on the basis of their subsequent history, into a number of diverse classes, which are summarized for our three cultures in table 1. 1) A few pairs, in some cultures, never separate, but die while united. 2) A considerable number, in some cases, die within 24 hours after separation. 3) Others live after separation—often for a long time—but never divide. 4) A fourth group divide, but produce individuals that are in some way and to some degree abnormal. 5) Finally, a certain pro- portion propagate normally after conjugation, giving rise to typical progeny. The proportions of these different classes are shown in table 1. The single pair that did not separate lived united for five days. The exconjugants that never divide after separation form a large proportion of the abnormal individuals, rising to 28 per cent of INHERITANCE IN ABNORMALITIES 393 TABLE 1 Diverse types of exconjugants, their number and proportion in the three experimental cultures 1) NEVER 2) DieD 3) NEVER 4) PROGENY 5) PROGENY SEPARATED FIRST DAY DIVIDED ABNORMAL NORMAL EXPERI- ae MENT No.| Percent |No.| Percent | No.| Fercent |No.| Percent |No.| Per cent 1 0 25 10.0 74 28 .0 21 8.0 |142 54.0 262 2 2 10 3 15 27 14.0 {133 66.0 35 Wieo 200 3 0 0 0 12 43 .0 16 57.0 28 Total .| 2 0.4 28 ee LOI 20.6 |166 33.9 {193 39.4 490 all in Experiment 1. These often live for some time, the length of life varying greatly. The length of life in days for the 101 exconjugants that never divided was as follows: LLanieilas cobed intaih le ER amen ARR eerneeeincecdcarénicanocce se 08} 5 Ti Cope ad O13 eee bots Number of exconjugants: ESMeriIMent. Lic... .f... duu 5 see OMOe 46310) 45 25 638) (LCA isenenimnent: 2. st. cea. ccc eee Deca 2 ol aOe Os Oy OF (27 TOE Gos eee ce eRS ER ely oor. ec AOU Om Om eOon orl LOL All of the animals of Experiment 2 which never divided were dead by the thirteenth day after conjugation; in Experiment 1 nine such individuals lived for some time after this. Those that live for a number of days often change a great deal in size and shape; many of them become immensely large. Twenty- eight of these individuals were measured; for their lengths and diameters on successive days after conjugation see p. 474. The diversities of size and shape among these individuals is illustrated in figure 1, which shows nine of the individuals of Experiment 1 which never divided, all camera lucida drawings to the same scale. The usual size of Paramecium caudatum on the same scale is shown in figure 5. The largest single individual’ Paramecium that I have ever seen was the first individual listed below for Experiment 1; it measured 520 by 150 microns, as against a usual length of 150 to 200 microns. The smallest individual that never divided was likewise in Experiment 1; it measured 128 by 10 microns. It is evident that there was much greater variability in size, shape, 394 RUTH J. STOCKING and length of life, in these individuals of Experiment 1, than in those of Experiment 2. It appears probable that this is con- Experiment 1 MEASUREMENTS ON Seventh day Ninth day Eleventh day Thirteenth day Fifteenth day _ 520 x 150 340 x 130 320 x 100 220 x 55 450 x 120 420 x 150 420 x 92 260 x 80 400 x 140 420 x 120 220 x 90 400 x 135 390 x 160 350 x 110 350 x 80 340 x 110 350 x 130 340 x 100 340 x 70 220 x 105 330 x 100 240 x 150 300 x 120 290 x 140 240 x 105 290 x 100 300 x 170 280 x 140 210 x 115 200 x 20 210 x 20 190 x 160 165 x 20 250 x 50 129x 10 200 x 35 250 x 60 Ave. 315 x 102 288 x 91 314 x 112 227 x 88 220x 90 Third day Experiment 2 MEASUREMENTS ON Fiith day 240 x 75 240 x 55 215 x 35 210 x 65 195 x 55 190 x 45 175 x 35 Ave. 209 x 52... .. 145 x 35 145 x 35 145 x 35 Seventh day 150 x 20 150 x 20 INHERITANCE IN ABNORMALITIES : 395 nected with the fact that the former culture consisted of wild individuals, probably of diverse stocks, while the latter consisted of members of a single clone. This matter will be taken up later. hie ‘Rs Fig. 1 Nine of the individuals of Experiment 1 which never divided, show- ing the diversity of form and size among this class. In this figure and in figures 2, 3, and 4, the animals were all drawn by means of the camera lucida, and are all to the same scale., (XX 100) As a rule there appeared to be a tendency for these large individuals to gradually decrease in size (as shown by the meas- urements above given). Figures 2, 3, and 4 show the changes in form and size in certain cases. The forms were frequently nearly normal; sometimes very abnormal, as the figures show. These individuals that never divided were usually black and granular, and often changed shape when transferred to fresh culture fluid, becoming swollen at the anterior end; they later resumed their original form. ““SUOTPBIOUSH) “SBT Fh :poweg Aep-OT pay, zfi¢lg foc “gouty 4SBJ JO IOATS UT SSOOXxHT €1 | 8 | $T |'°‘Suoreisuern ‘Mmorg ST | OF | Of | °° Suorjeiouer) “4se,7 ‘poled AVp-OT YI e—|0 | e—[occ ete sony 4SBJ JO 1OAB} UI SSOOXGT FL | SI | $1 | ‘Suoryereusry ‘morg IE | &1 | TL 4° °° “Suorpusouor) ‘4sui7 :polleg Avp-gT yyNo 1 [0 | 6—|ttct ts seury 4} SBJ JO LOA] UTSSOOXHT GI | GI | OT |’ Suorersuery ‘Mojg SE Cole yh |= T |@ |e [tcc sonny 4SBy JO IOART UL SsaoxAT 6 or | 8 ““SuOryeIemer) ‘MOTG OT | &I | OF |’ °° Suorgeiousr ‘48R,q7 ‘poled Avp-0T puooag I-|@ |p fore sour] 4SBJ JO IOAV] UL SSaOxAT €1 | OL | 8 |°**‘suorze1euer ‘Mog GI | ZL | Zt | “suoreisuer ‘4su,7 :polleg Ap-0T 4SIt Cun I UAHWON SANIT aunjnd sspu Lajfv UWOLIaJas pasuDjyog ‘auy sad porsad hop-ua} sad suorvpuauab fo saqunu pony ; 6 AIAVL iQrT quawmisaday 482 AUSTIN RALPH MIDDLETON It is 13.74 selections per line for the fast-selected ‘slow’ lines and 12.79 selections for the slow-selected ‘fast’ lines. Now the aver- age difference of fission rate per line per day for the eighty days of balanced selection immediately preceding the present experi- ment was 0.25 generation per line per day and as a result of the fifty days of reversed selection this difference was wiped out and 15 10 30 50 10 B Fig. 1l-a Polygon of the average number of generations per line per ten- day period produced by the ‘fast’ and ‘slow’ sets of lines of Experiment 1-C (balanced selection for 50 days after 80 days of selection, 40 days of balanced selection and 12 days of mass culture). The continuous line shows the averages for the ‘fast’ set, the broken one the averages for the ‘slow’ set. The ordi- nates are the averages per ten-day periods, the abscissae, the ten-day periods. Fig. 11-b Curve of the difference in favor of the fast lines between the aver- age number of generations per line per ten-day period produced by the ‘fast’ and ‘slow’ sets of lines of Experiment 1-C during its five consecutive ten-day periods of balanced selection after mass culture. The ordinates are the differ- ences of the averages, the abscissae the consecutive ten-day periods. FISSION RATE OF STYLONYCHIA PUSTULATA 483 an average difference of 0.27 generation established in the reverse direction. If this difference shows itself to be heritable, it will appear that it is probably due to the large number of selections prac- tised within the relatively short period of fifty days; it will indi- 10 30 50 Fig. 12 Polygon of the average number of generations per line per ten-day period produced by the ‘fast’ and ‘slow’ sets of lines of Experiment 1-D (reversed selection). The continuous line of the curve of the former fast lines now slow-selected. The broken line is the curve of the former slow lines now fast-selected. The ordinates are the averages per ten-day periods, the abscissae the ten-day periods. cate that the alteration of fission rate is proportional to the number of selections made, rather than the length of time over which the selections are distributed. This matter is tested in the next experiment. Experiment 1-E. Balanced selection after reversed selection, June 2 to June 21, 1914. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 19, NO. 4 484 AUSTIN RALPH MIDDLETON TABLE 10 Experiment 1-D: Actual number of generations and selections per 30 lines per ten-= day period during the five ten-day periods of the second reversed selection of the lines of Experiment 1 AVERAGE TEN-DAY PERIODS FIRST | SECOND| THIRD |F S 0. H OURTH| FIFTH TOTAL PER LINE Total number of generations! 263} 309| 300| 292] 473 | 1637 | 54.57 Average number of genera- GIOHS? SW en bos = 8.77 | 10.30) 10.00} 9.73 | 15.77 10.91 Slow Selected Fast Lines: Average number of selec- GIONS ee techn Ne sees Bene) teal Pe Wei a0) || eGR: Total number of generations} 237 | 273 | 217] 225 | 276]! 1228} 40.93 Average number of genera- TIONS See ee ees see F290) 90 723i Fe500 9220 818 Actual excess in favor of the fast selected ‘slow’ lines....| 26 36 83 67 197 |} 409] 13.64 Average excess in favor of the , fast selected ‘slow’ lines....| 0.86 | 1.20} 2.77] 2.23 | 6.56 Dele The lines from the experiment in reversed selection (Experi- ment 1-D) just described were next continued under the bal- anced selection method for two ten-day periods in order to de- termine whether the differences in the reverse direction obtained in that experiment were true heritable differences of fission rate. The animals were transferred to fresh slides daily. Figure 13 shows the polygon of the average fission rates of these two sets of lines and table 11 gives the actual number of generations produced by each set. From these it appears that the average daily difference of fission rate per line between the two sets of lines is 0.38 generation in favor of the fast selected slow set. It is therefore clear that reversed selection had really pro- duced an heritable difference in fission rate in favor of the former slow lines; the relative rates of fission of the two sets was now reversed. As before remarked, this result is to be expected; the earlier experiments show that’ direct selection pro- duces hereditary differences in one direction; reversed selection FISSION RATE OF STYLONYCHIA PUSTULATA 485 in the same way produces hereditary differences in the opposite direction. General results of the first group of experiments. The experi- ments thus far described have all dealt with parts of a single clone of Stylonychia pustulata; selection has been practised on 10 20 Fig. 13 Polygon of the average number of generations per line per ten-day period produced by the fast and slow sets of lines of Experiment 1-E (balanced selection after the reversed selection of Experiment 1-D). The continuous line is the curve of the reversed selected former fast lines now balanced selected. The broken line is the curve of the reversed selected former slow lines now bal- anced selected. The ordinates are the averages per ten-day periods; the abscissae the ten-day periods. the variations in fission rate within the clone. For one hun- dred and thirty days two halves of this clone were subjected to. selection in opposite directions; this produced a marked and steadily increasing difference in the average fission rate of the two halves. Expressing the excess of generations produced by the fast-selected lines as a percentage of the total number of oe 821 OF OTT | SeUT] MOTY Jo oFuBY . IST 0} get |" Sour] ISUAT Jo oBuNY szet leo igo Itz |o9 |t9 Ito |po Ito |zo Ito |ro leo |eo |t9 |89 |g¢ |s¢ |eo joo jog joo jpg |po po [eg jog [zo jeg jeg [To Ju sour 4ysBy out jo IOABY ul SSOOXGT gzoe oct lot lort loer lort lett lotr lort \ett leat joer leat |eet lect lztr |get |gct lear leat jozt lozt [Per [Zt |2aT Jou jSZt jOCT jzst |Get jeer | OTS gocg lest last Itt lost lost ozt lest lost {set lpr |rst lost ost |2st |sst jest fost |ost fost |T8T [cSt |S2T JOST |TST |TST |PST JEST \S8T [est jest jo" ase nvaox | og | 6z | 8z | 4 | 92 | 92 | $2] e2 | ec | te | 0c | or | st | at | or |r] rier | eri} imjorj6 js |4 /9 |e | |e |e |t UGAWON ANTI J juaunrsadag fo spoiiad hop-o] gf ay) burunp nojs pun yspf ‘ouy yova fiq paonposd suoyniauab fo saqunu pio, :y pun g ‘Be ‘7 sping ‘T quoawplodx gy ol WTAViL 08 ¢ Pepe sepe no. (euler ee en eee eo pm TN ee TD eal Peal Ge luo We@h RG sl Biallieres ecient cei (0) ol MSG ieee! cue tee Mune Me aace -08 Sup oyy aS JOLOARJ ULSsooxG, 90°L 0 TINCMAIRSERIKOMTI Geri ge lathe Meelive NBG O iO OTS CG Get Ren Sei: ie Te NG eS) Ao SBe ie) Idee eee cee oe |e eee mene 98° 0 we |6 | 116 | E19 | or | 6 | FE | ot | ot | oc | TL | 12] OF | OL} SE | Th] TL | OL) OL} TE | TE) St] HE | St) at] 8k | Or) Sr) Pr) age eee a PomMod Avp-(T puovg p8'T 12 | \[r4 A tata WEG ICI SS SSI Wi 2 TE eT ag IT ett I Pale Ge | OMNI Fie Ol 00 Sipe BB OnT pers : -o8 ysBy oly JO LOABY UT SSOOXHT 9¢° 01 0 tee We ce te Wage Ngee Wp ae ree Wh ge EN ae ie pe re ee Wh ae ae he Ce CO) Cee Shai Cee ave hye ie dee) ye ai, SOOT OP SI 0 ae Wee ta NH Wt A Se He eH ae ae a ve PS YE A AE PRM Coe] GE TS Pe TSI vie ET eye ys a ASB UT :powod Avp-OT ISAT : NOI Se ~oa1as |rvsoal og | 6z | 82°] 22 | 92 | 92 | #2 | 2 | vz | 12 | Of | GT | St | Zt | OF | GE] PT] et | OEY 1] or; 6 |} 8 | 4 |9 SF |b |e Ys | T UCN ON WNIT ey so Taras woyepas paouvjpog ‘*auy wad porwad fvp-o] “ad suoyniauab fo saqunu JONIIY 2 ff-] Pwaursad xg It WTavViL FISSION RATE OF STYLONYCHIA PUSTULATA 487 generations produced by both sets, the difference was 6.9 per cent in part 1; 12.8 per cent in part 2; 19.3 per cent in part 3, and 21.2 per cent in part 4. Table 12 summarizes the genera- tions produced by each line of each set throughout all parts of the experiment. It shows that for the fast lines the number of generations ranges from 178 to 187, while for the slow lines the range is but from 116 to 128. Thus there is no overlapping in the two sets; the slowest fast selected line has produced 50 more generations than the fastest slow selected line. To determine whether the difference in fission rate thus pro- duced is heritable, parts of the two sets were removed at inter- vals and subjected to culture without selection (‘balanced selec- tion’). In every case it was found that the difference was herit- able. Also, representatives of the two sets after eighty days of selection and forty days of no selection, were subjected to mass culture for twelve days. Further line culture for fifty days without selection showed that the inherited difference in fission rate still persisted. Thus the inherited difference produced by selection had lasted for one hundred and two days without selection. Experiments with reversed selection showed that the inherited difference could be reversed as readily as it is produced; the originally fast set was thus caused to become the slower one, and vice versa. Continuation of these sets without selection showed again that the difference so produced was heritable. Thus in this case the selection of small individual variations in fission rate has split the single clone (derived vegetatively from a single parent) into two hereditarily diverse divisions (diverse clones). 2. The second series of experiments The results of the first series of experiments appeared so important and in ‘some respects unexpected that it was felt necessary to control them by repetition, beginning again with a single individual, and endeavoring anew to procure from it by selection two hereditarily diverse sets. This was first at- 488 AUSTIN RALPH MIDDLETON tempted with a single individual taken from one of the fast lines of the first set of experiments, giving experiment 2, described below. It was later carried out anew with the progeny of a single ‘wild’ individual (‘third series’). Experiment 2-A. Selection among the progeny of a single individual from Experiment 1. The individual selected for repe- tition of the experiment was one of those belonging to a fast line of the previous experiment. The progeny of this individual did not live well, so that in some cases one or both sets died out before any definite result was obtained. In one case, however, selection of fast and slow sets was continued through nine con- secutive ten-day periods (April 5 to July 3, 1914). During every ten-day period except the first set the fast-selected set produced more generations than the slow-selected one. The results are less striking than in Experiment 1, however, in the fact that on twenty-two days out of the ninety, the slow lines produced more generations than the fast ones. The irregularity appears connected with the high mortality in both sets. How- ever, the average difference in fission rate per line per day in favor of the fast selected set was 0.317 for the first thirty days, 0.757 for the second thirty days; and 0.61 for the third thirty days. Experiment 2-B. Now the two sets resulting from the ninety days’ selection in Experiment 2-A were subjected to balanced selection for ten days. The difference in fission rate persisted to the extent of an average difference in favor of the fast lines of 0.28 generation per line per day, though on one day the slow lines were faster by 0.01 generation per line. Before the end of the next period all lines were dead, owing perhaps to the hot weather. Results. The evidence from these two experiments is, so far as it goes, in the same direction as from those of the first set. Selection produced from the progeny of a single individual two sets differing hereditarily in average fission rate. FISSION RATE OF STYLONYCHIA PUSTULATA 489 3. The third series of experiments Experiment 5-A. September 22 to October 21, 1914. To further test the results thus far reached, and to determine whether they are based on conditions generally occurring in the organism studied, a new wild individual was obtained from a new mass culture brought into the laboratory September 15, 1914. From this, two sets of thirty individuals each, all belong- ing to the seventh filial generation, were obtained, and sub- TABLE 13 Experiment 3-A: Actual number of generations and of selections per ten-day period per thirty lines of the 30 fast and 30 slow lines isolated among the progeny of the single ‘wild’ individual subjected to opposite selections for 30 days AVERAGE TEN-DAY PERIODS FIRST SECOND THIRD TOTAL PER LINE Fast Lines: Average number of selections.| 2.53 1.60 1.50 Total number of generations. 973 634 603 2210 73 .66 Average number of genera- TRS. coe Ae eee 32.43 21.13 20.10 Slow Lines: Average number of selections.| 2.90 1.53 0.96 Total number of generations. 935 581 523 2039 67 .96 Average number of genera- URGE eon eee 31.16 19.36 17.48 Actual excess in favor of the faa lintes.. osc... ; ile 45 py 38 53 80 171 5.70 Average excess in favor of the NESiy INVES oo owe Sees dP a7 2.67 1.90 Percent the difference is of the (ROUEN LAIOTE LYON ae oo3.505 4 Oe OIG 1.99%| 4.86%| 7.10% jected, in the manner previously described, one to ‘fast,’ the other to ‘slow’ selection. In this series only one individual, in place of two, was selected from each line at each change, and the selections were made daily. ‘There was no reduplication of the fastest and slowest lines. Fourteen of the fast and twenty-one of the slow persisted intact throughout experiments 3-A and 3-B. Opposite selection was practised for thirty days. The records are given in table 13. In all three ten-day periods the fast- selected lines produce more generations than the slow-selected, 490 AUSTIN RALPH MIDDLETON though in four days of the first period the reverse is true. In all the other twenty-six days the average of the fast lines was above that of the slow. The table shows a gradual increase in the number of generations produced by the fast lines relative to those produced by the slow, the excess in favor of the fast 10 15 30 B Fig. 14-4 Polygon of the average number of generations per line per three day period produced by the fast and slow sets of lines of Experiment 3-A (direct selection-in opposite directions of the progeny of the second wild individual). The continuous line is the curve of the fast set, the broken line, the curve of the slow set. The ordinates are the averages, the abscissae the three-day periods. Fig. 14-b Curve of the difference (in favor of the fast lines) between the average number of generations per line per three-day period produced by the fast and slow sets of lines of Experiment 3-A (direct selection). The ordinates are the differences between the averages; the abscissae, the consecutive three- day periods. Note the progressive increase of the difference under opposite selection. FISSION RATE OF STYLONYCHIA PUSTULATA 491 being for the three successive ten-day periods respectively 1.99 per cent, 4.36 per cent, and 7.10 per cent of the total number of generations produced in the given period. The gradual increase of the difference between the two sets indicates that this difference was heritable. This gradual in- crease is well shown in the curves of figure 14, giving at a, the average number of generations produced per three-day period by each set, at b the curve of the differences in favor of the fast set. Figure 15 gives the curves of variation of the total number of generations produced by the two sets, showing that they overlap very little. The evidence indicates strongly that the effect of selection is cumulative. Experiment 3-B. To test whether the difference produced by selection is actually heritable, balanced selection was now prac- ticed for twenty-one days, October 21 to November 10, 1914. On every day but one (the second) the lines that had been sub- jected to fast-selection averaged higher than others. Table 14 gives the actual numbers of generations per line for the two ten-day periods. Table 14 shows that during both of its periods the fast lnes averaged more generations than the slow ones and further that the per cent that this difference is of the total number of gener- ations produced by both together was practically constant. Figures 14-c and 14-d show the average difference of fission rate between these two sets of lines, averaged for three-day periods. These two figures emphasize the marked uniformity of this average difference. Hence this experiment shows that the oppo- site selection previously practised had produced a nearly uni- form heritable difference of average fission rate between the two halves of this second clone. The results are thus the same as in our first set of experiments. Experiment 3-C. Effects of conjugation on the results of selection. It appeared of interest to determine whether these inherited results of selection would persist through conjugation. It is well known that conjugation is an ordeal having many effects on eT oh | LOE 86 I 90°CL C9TG =| LL 10'S | 26°T 6g I 46° | OFT 68 0 INGO aov uad “UAV g 8 pL £& £ I 69 PE Pp 8 LE VG 0 92 8& 8& 8 iz && IP aS) €L | 82 | 2 LE | OF | SE OT | 8I | 8T (iG ce £& P OL 6 g lg Ig iat 8T j @ | eb |e) er 19 | 8¢ 83 | 9% | 62 | 6 |G TL 9€ g g 0 Fir eeeessgimqog “g WY *“W Jo oouosyICT Heseeeseeseermaog suolpye1oUues ][vyIOT, renee eeeeeeres Mong suol}e1oues [BJO], been eee eee ereengegy Ssuol}eoUNs [BO], ** Q0UdIO IC 7% |SMOT}BIDUOY ‘MOT vo I SUOT}BIOUO) “4SBIT :ponag ABp-j] puooveg SEE CSCI Cat SUOT}BIOUOL) ‘MOTS SUOTPRIOUOL) ‘4SBiT :poilog ABp-OT IIL UTANON SANIT sas 4}0q fig paonpoud 30707 2Y2 fo st aouasaffip siyy abpzuaouad yoym smoys juad sad paysDuUl UUUN}OI 9Y,], ‘sauy papoajas-psvf ay) fo Loanf ur soouasagfip oy) yum ‘y-¥ quawisoday fo woyoojas apsoddo fo shvp hjiryy 94) Burmoppof fjajprpauuy woryoo}os poouv|eq fo shop auo-Aywamy oy? of y-¥ quowisoday fo sau oy, fo aun wad powod fhvp-ua, sad suoyniauab fo soqunu ponoy :q-g jwourlad xg PL ATAVL 492 FISSION RATE OF STYLONYCHIA PUSTULATA 493 vitality and reproductive power; often changing the fission rate. Conjugation was obtained among the selected individuals of this third set of experiments, and its effect tested. Watch glass cultures were made in zy per cent malted milk from each of the thirty fast lines and the thirty slow lines. On 90 20 15 21 Fig. 14-c Polygon of the average number of generations per line per three- day period produced by the fast and slow sets of lines of Experiment 3-B (bal- anced selections after 'the direct selection of Experiment 3-A). The continuous - line is the curve of the fast set, the broken line is the curve of the slow set. The ordinates are the averages, the abscissae the three-day periods. Fig. 14-d Curve of the difference (in favor of the fast lines) between the aver- age number of generations per line per three-day period produced by the fast and slow sets of lines of Experiment 3-B (balanced selection after the, direct selection of Experiment 3-A). The ordinates are the differences between the averages; the abscissae, the consecutive three-day periods. Note the practical uniformity of the difference under balanced selection. December 4 and succeeding days conjugating pairs were ob- tained, some from the fast lines, some from the slow ones. Though many of the ex-conjugants died, eventually representa- tives propagating normally were obtained from six of the fast lines and four of the slow ones of Experiment 3-B. It will be recalled that these animals, before conjugation, had been subjected to opposite selection for thirty days, then to 494 AUSTIN RALPH MIDDLETON twenty-one days of culture without selection and twenty-three days of mass culture. On December 7, sixty ex-conjugants were isolated from the fast lines (ten from each of six diverse lines), and sixty from the slow lines (twenty from each of two slow lines, ten from each of two others). They were cultivated in the way previously described. Balanced selection (i.e., in effect no selection) was practised for fifteen days. Table 15 gives the numbers of generations produced by the fast and slow lines during the five three-day periods of this experiment. No lines were lost by death, and practically no selection of any sort was necessary. The daily record sheets of this experiment show that during its first five days the slow lines averaged more generations per line than did the fast lines. Table 15 shows this for the first TABLE 15 Experiment 3-C: Actual number of generations of the 60 fast lines of ex-conju- gants and of the 60 slow lines of ex-conjugants during balanced selection for 15 days. The two members of each conjugating pair were individuals of the same line AVERAGE THREE-DAY PERIODS FIRST SECOND THIRD |FOURTH). FIFTH TOTAL PER LINE Fast lines: Total number of gen- CLaliGns ce hase: 378 302 188 | 237 192 | 1297 | 21.63 Average number of generations......... 6.30 5I03.Sals passe |a220 Slow lines: Total number of gen- | erations:.: 17.9 W822) 1885 58 84 38 12 ij 6 6 2 510 CHARLES ZELENY AND C. T. SENAY quency distribution gives a major mode at 27.4 » and a minor one at 28.8 ». The minor mode may be accidental though such an irregularity is not common in frequency distributions in a population known to be homogeneous. There are no data available concerning the spermatogenesis of this species but Montgomery (’06) has described two kinds of spermatids for other members of the family. 4. Euschistus variolarius, a hemipteran. Material was ob- tained during April. All the spermatozoa were not fully devel- oped and selection was necessary to insure the exclusion of the unripe ones. Five hundred measurements were made. The re- sulting curve as shown in figure 5 is distinctly bimodal with modes at 15.1 » and 16.5 » and with an inequality favoring the larger spermatozoa. The ratio between the modes is 1.00 : 1.09. E. B. Wilson (06) described an ‘‘X”’ and a ‘‘Y”’ chromosome for this species. The expected ratio as approximated from his figures of the chromosomes is 1.00 :1.04. This does not agree at all well with the ratio between the modes as given above. This result is contrary to that obtained for several species by Zeleny and Faust (715) which showed a striking similarity be- tween the two calculations. 5. Cosmopepla carnifex, a hemipteran. The material was ob- tained in May and the spermatozoa were all active. Five hun- dred measurements were made. The resulting curve as given in figure 6 shows well marked bimodality with approximate equality in the two groups. The modes are at 18.5 » and 19.9 u and give a ratio of 1.00 : 1.075. There can be no doubt in this case of the existence of two fairly equal size groups. Montgomery (’06) describes two kinds of spermatids for this species, one with seven chromosomes plus an ‘‘ X”’ and the other with seven plus a ‘‘Y.” The figures are not suitable for the determination of the chromatin ratio. The ratio in another Pentatomid, Euschistus variolarius, is 1.00 : 1.04. 6. Passalus cornutus, a coleopteran. Material was obtained at the end of March and the spermatozoa were uniformly ripe and active. Five hundred spermatozoa were measured. Neglect- ing the minor projection at the right, the curve as given in figure VARIATION IN HEAD LENGTH OF SPERMATOZOA 511 7 is unimodal with the mode at 11.7 u. This indicates either a single group or two so close together as to give a unimodal result (see Zeleny and Faust ’15 a, p. 193). There are no published spermatogenesis data for this species. Descriptions for different species of beetles show in all cases, except one, two groups as regards chromatin content. Leaving out the possibilities of random sampling and of selective elimina- tion within the two groups the present species seems to be either without distinction among its spermatozoa or else has two groups differing but slightly from each other. 7. Berosus striatus, a coleopteran. The material was obtained in early April and the spermatozoa were all ripe and active. The frequency distribution of the five hundred measurements is given in figure 8. The curve is distinctly bimodal with approxi- mate equality in the two groups. The modes are at 16.1 » and 17.2 » with a ratio of 1.00 : 1.07. There are no cytological data for this species but as was stated in discussing the last form the descriptions for all but one species of beetles give two kinds of chromosome groups among the spermatids. DISCUSSION The present data as a whole substantiate the view that di- morphism in size of spermatozoa is of common occurrence in those groups of animals in which two chromosomal classes of spermatids are of common occurrence. Of the seven species described five are hemipterans and two coleopterans. In both of these orders a great majority of the species so far studied show a quantitative difference in chromosomal content among the spermatids though in each case there are some species which show no difference or only a slight one. Among the five hemipterans three, Corizus lateralus, Euschis- tus variolarius and Cosmopepla carnifex, show two well marked size groups. The other two species are not so clear. One, Leptocoris trivittatus, is probably dimorphic though the curve is irregular. The other, Reduviolus ferus, is doubtful because the upper mode is very low and may be a chance projection in 512 CHARLES ZELENY AND C. T.’ SENAY a unimodal distribution. It should not, however, be forgotten that the two elements of a population will, when combined, give a unimodal curve in case the modes of the elements are close together. Among the coleopterans one, Berosus striatus, has two well marked and equal size groups. The other, Passalus cornutus, has a single size group or two with modes very close together. Taking all cases so far described there are twenty-three spe- cies involved, including the one given by Wodsedalek (’13). This is a sufficient sample to justify us in stating that there can be no doubt of the validity of the hypothesis presented. The chromosomal dimorphism of spermatogenesis is represented in the active functional spermatozoa by size dimorphism. Control of sex then merely awaits our ability to separate the two sizes in the living condition and to use them in artificial insemination. Fig. 6 Cosmopepla earnifex; frequency distribution of head-lengths of 500 spermatozoa from a single testis. Valueunmicroris:... scl. :- 16.5.0 16.8) 17.25 175,199) 18 SIS 25 HSeQUeREN Re = oc 2c aoe 6 14 16 19 26 42 83 19.6 19.9 20.3 20.6 20.9 21.38 21.6 22.0 42 68 37 14 11 af 9 Fig. 7 Passalus cornutus; frequency distribution of head-lengths of 500 sper- matozoa from a single testis. Value in microns..............110:15" 10°30) 10.45 10.60 10.80 11°@Q7=tiae HFCQUENECY AEs Sait: ies Ss os ee 1 5 + 9 14 26 27 11.30) 11:50 11-70" 11:85. 12.00 12.200aeae 46 65 89 65 59 22 29 12550125709 12985) 1300 13220 14 12 7 a 2 Fig. 8 Berosus striatus; frequency distribution of head-lengths of 500 sper- matozoa from a single testis. Value in microns....... ey ey ete 13.7 14.1. 14.4 14:7 I15c17 1525s PPEQIENCy= ess he eee 4 9 9 10 14 32 41 18.5 18.9 19.2 19.6 19.9 20.3 20.6 7 16 14 10 4 2 913 IN HEAD LENGTH OF SPERMATOZOA VARIATION 19.9 18-5 16.1 514 CHARLES ZELENY AND C. T. SENAY BIBLIOGRAPHY Faust, E. C. 1913 Size dimorphism in adult spermatozoa of Anasa tristis. Biol. Bull., vol. 25. Monteomery, T.H. 1906 Chromosomes in the spermatogenesis of the Hemip- tera Heteroptera. Trans. Amer. Phil. Soc., vol. 20. Witson, Epmunp B. 1906 The sexual differences of the chromosome groups in Hemiptera with some considerations of the determination and heredity of sex. Jour. Exp. Zodél., vol. 3. WopsEDALEK, J. 1913 Spermatogenesis of the pig with special reference to the accessory chromosomes. Biol. Bull., vol. 25. ZELENY, C. and Faust, E.C. 1914 Size differences in spermatozoa from single testes. Science, N. S., vol. 39, no. 1003, p. 440. ZELENY, C. and Faust, E.C. 1915 a Size dimorphism in the spermatozoa from single testes. Jour. Exp. Zodl., vol. 18. ZELENY, C. and Faust, E. C. 1915b Dimorphism in size of spermatozoa and its relation to the chromosomes. Proc. Nat. Acad. Sc., vol. 1. THE EFFECT OF SELECTION UPON THE ‘BAR EYE’ MUTANT OF DROSOPHILA! CHARLES ZELENY anv E. W. MATTOON FIVE FIGURES The selection experiment described in the present paper was made with a view to testing the germinal uniformity as regards the distinguishing characteristic in a recent mutant, the ‘bar eye’ race of Drosophila. In this race the ommatidia are reduced in number and the facets are restricted to a vertical band or ‘bar’ as shown in figure 1. The characteristic appeared in a single male during 1913 (Tice, ’14)? and the whole ‘bar eye’ stock is descended from this individual. The race has under- gone no apparent change during the two years of its existence. Our material was obtained in January 1914 through the kind- ness of Prof. T. H. Morgan. There is a pronounced sexual dimorphism in the number of facets, the males averaging 98.03 and the females 65.06. In every case in the present paper the female number is transformed to the male basis by multiplying it by oe Sh Te There is often a slight difference between the number of facets in the right and that in the left eye. For one hundred indi- viduals the average difference was 0.245 per cent in favor of the left eye. This difference is obviously not significant. The right eye only is given in the present records. The number of facets seems not to vary with the length of the period of development. In five broods counts were made of the facets of the earlier emerging individuals and compared 1 Contribution from the Zodlogical Laboratory of the University of Illinois No. 50. 2 Tice, S. A. 1914. A new sex-linked character in Drosophila. Biological Bulletin, vol. 26, pp. 221 to 230. 515 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 19. No. 4 516 CHARLES ZELENY AND E. W. MATTOON with those of the later emerging ones. The averages for the two are approximately equal. = .A sample of five hundred individuals, 250 males and 250 females, taken from the general population of the mutant showed a range of variation in the number of facets from 45 to 182 with +N \ K } A } eA A B Fig. 1 A. Normal full eye of Drosophila. B. ‘Bar’ eye. The dark areas are the faceted regions. Facet namber 16-30 31-45 46-60 61-75 76-90 91-105 106-120 121-135 136-150 151-165 166-180 181-195 196-210 Frequency 0 2 18 56 113° «164 73 30 24 17 3 1 0 Fig.2 Variation of facet number inthe unselected population of the ‘bar’ eye mutant of Drosophila ampelophila. Five hundred individuals are repre- sented, two hundred and fifty males and two hundred and fifty females reduced to the male level. ; a mean of 98.03 + 0.73 and a standard deviation of + 24.30. The variation curve is shown in figure 2. The average for ten individuals of the normal wild race was 701.1 facets. Unselected stock was carried as a control through the period of the experiment. There was practically no change in the THE ‘BAR EYE’ MUTANT OF DROSOPHILA 517 number of facets, the average of 250 individuals at the beginning of the experiment being 98.04 and of the same number at the end of the experiment 98.03. There is thus no change in facet number in the absence of selection. In making the first selection, food containing eggs and larvae - was removed from the culture of the ‘bar eye’ stock and placed in glass vials. Every twelve hours the individuals which had emerged from the pupal cases were slightly etherized and an estimate was made of the number of facets under the low power of the microscope. Males and females with high or low numbers were selected out, high being mated with high and low with low. Each pair was placed in a small bottle with sufficient food to last until all the offspring of the first brood had reached the adult stage. When larvae began to appear in the bottle the parents were killed by etherization and an exact count was made of the facets. As the offspring emerged in the adult form esti- mates of facet number were again made and high selections were made in the ‘high’ lines and low selections in the ‘low’ lines. The final exact facet counts were in all cases made in killed individuals. The experiment has proceeded far enough so that data are complete for three successive selections in each of three ‘high’ lines, called A, B, and C, and in each of three ‘low’ lines, called D, Eand F. Fifty individuals, 25 males and 25 females from a single pair, were measured for each generation in each of the lines, with the exception of the third generation in line B where only forty-six were available. The data for the individual lines are given in tables 1 to 6 and a summary of the six lines is given in table 7. Figures 3, 4 and 5 give in graphic form the course of the selections, each figure combining a ‘high’ with a ‘low’ selection line. The mean values, the extreme variates and the mid-parental values are here represented in diagrammatic form for each of the selections. In all cases there is a significant shifting of the mean as a result of selection. The general population mean of 98.0 is changed in plus line A to 108.7 by the first selection, to 127.5 by the second and to 135.5 by the third. In plus line B the 518 CHARLES ZELENY AND E. W. MATTOON TABLE 1 Plus selection Line A GENERATION 1 | GENERATION 2 | GENERATION 3 GENERAL POPULATION = 33 = 32 = Bs PATenteieest esses Seiten. Me ae eek 127 133 | 179 | 169| 177 | 195 Mid-parental values.......5............ 130 174 186 HES pri 6% i. 57 ace is oii sos 67 69 84 89 92 95 79 71 88 91 93 97 86 74 90 94 98 | 103 91 76 95 97 98 | 109 92 83 95 | 100} 103} 415 92 88 97.| 103 | 107 deotte 94 89 98 | 110} 108] 124 96 91 99 |} 110°) 1091 oiez 99 94 99} 112) 147 | Wag 101 | 100) 102) T13.ie iyi 102 | 101 | 102) 115) 11S) ie 103 106 118 119 126 134 105 | 106 | 124| 122) 1261) ao3q 107 107 125 125 135 137 107 | 110 |° 125] 1383 | 139 eZ 109 | 115] 130| 134} 140] 142 112 116 133 137 141 143 116 | 118) 142) 1389 | 1429 0as 118:| 128 | 147) 142] 154) i538 118 | 133 |. 148] 148] 159 | 157 122 148 176 153 167 160 127 | 149| 177} 156] 180| 163 134 | 149] 180] 163] 187] 171 140 | 151 | 189] 187} 192! 174 179 | 169| 210} 210} 204| 186 Mean of offspring..........98.03 = 0.73| 108.7 + 2.3 |127.5 = 3.1 | 135.5 = 2.8 corresponding figures for the three selections are 110.1, 128.6 and 141.9, and in plus line C, 116.9, 133.5 and 141.0. In minus line D the general population mean of 98.0 is changed to 88.3 by the first selection, to 85.5 by the second and to 81.7 by the third. In minus line E the corresponding figures for the three THE ‘BAR EYE’ MUTANT OF DROSOPHILA 519 TABLE 2 Plus selection Line B GENERATION 1 | GENERATION 2 | GENERATION 3 ¥ a2) R Twn aoe) GENERAL POPULATION 5 oa 3a n =} 2 n eecomlee | 88) | oleae SMa eet |e |) et | ages ERT OTNGS UE s,s hie eotans CH I eee 139 121 184 165 182 222 Midaparental valves... ...-). 0000-805 «<<: 130 174.5 202 LST tt ee an ce 68 57 86 76 96 89 74 76 90 79 99 95 75 80 92 95 | 100] 104 79 85 93 | 101} 102] 116 80 85 95} 103} 118] 118 82 89 Sra LOA 21 123 89 91 99; 110} 123 | 124 90 SE 101i) Tie) 124) 124 95 95} 108} 113} 128 | - 131 98 Son) 11S eS 119) 130°); ASG 98 97 | 122) 127) 1389] 137 100 | 100 | 125) 131] 141 137 102 | 106 | 126) 134] 145] 148 104} 108 | 127} 137 | 147] 154 105} 118 | 132} 137 | 154] 156 108 | 124) 137 | 139| 156) 159 112} 125) 140) 1438; 158] 163 115 |} 128; 144] 145} 160] 165 131 | 131 | 144] 148] 169! 166 133} 1383} 160) 149] 172) 169 ge 137+) 166) 157 | 175.) 175 Lagoon. 1h.) - 163;) 1975) .189 156 | 148 | 172) 175} 207 | 207 167 | 149) 182] 20 184 | 165] 183] 2 Mean of offspring.......... CYB} == (7/33) WO Ib == 2o0/ | IAS oy S309) )) STR ee selections are 93.9, 89.6 and 84.8 and in minus line F, 94.6, 89.8 and 84.7. As a result of the three minus and the three plus selections the mean value of the individuals in the plus lines (139.5) is greater than the highest extreme individual of the three minus lines 520 CHARLES ZELENY AND E. W. MATTOON TABLE 3 Plus selection Line C GENERATION 1 GENERATION 2 | GENERATION 3 Reduced females GENERAL POPULATION 33 2 r= | 3 mn 2|38| 2 = [=i = IPaATenis tA. 7. tice eee blaine eee 2 178 | 157 Mid-parental values..........:...:-..-- 167.5 Ofisprinigee ces. Se ere ee ss cere = 82 63 85 71 86 85 90 89 93 | 101 96 | 103 97 | 106 99 | 106 100 | 107 101 112 003 | 113 112 121 117 121 118 | 124 121 128 122 130 123 131 124 | 133 126 |. 134 126 136 131 142 146 151 147 152 176 159 208 | 163 Mean of offspring.......... 98.03 = 0.73) 116.9 + 2.6 133.5 + 3.3 | 141.0 + 2.8 (137.0) and the mean of the three minus lines (83.7) is lower than the lowest extreme individual of the three plus lines (89.0). The coefficients of variability as given in table 7 show on the average a slight decrease in the selected stock as compared with the general unselected population. This difference however can- THE ‘BAR EYE’ MUTANT OF DROSOPHILA 521 TABLE 4 Minus selection Line D GENERATION 1 | GENERATION 2 | GENERATION 3 GENERAL POPULATION 33 33 33 2 |3e| 2 |3a| 2 | 38 = os = faa = ao PPE CHON REN 9.05 cb Live codes cee ea ee ele 52 60 69 71 63 58 NMiid-=parental values...........:2%.<++<- 56 70 60.5 errprennr 9.0,‘ Ye ae eee ee 44 56 49 56 48 36 74 74 75 75 68 62 95 94 91 91 87 88 96 98 92 91 88 91 OF) 108 92 92 89 95 ie 203. 95 92 92 98 98 | 103 99 94 97 | 104 100 | 103 | 100 97 | 100; 104 102 | 106; 102; 100] 100] 107 103s 109) -104 | 103. | 103), 115 110 | 110; 105; 103] 104] 115 Mon AUG |) 110: ). 104 | 115 | 116 1G els) 121) 6133) lar 127 Mean of offspring.:........ 98.03 + 0.73] 88.3 = 1.8] 85.5 = 1.8 | 81.7 = 2.1 not be considered as significant because of its irregularity and slight amount. . The progression of the mean is much more rapid in the plus than in the minus lines. The averages for the three plus selec- tions give respectively increases of 13.9, 18.0 and 9.6. The 522 CHARLES ZELENY AND E. W. MATTOON TABLE 5 Minus selection Line E GENERATION 1 | GENERATION 2 | GENERATION 3 GENERAL POPULATION 32 38 38 z s 2 = 3 = s | 22 | 2-| 22 |-3 gees PATONG i: ee fo ee oe soe pee 82 80 64 74 60 62 Midzparental values’. -. +: 224.2500. 522 81 69 61 GRsprage 3.602202. 83 25ers 53 50 50 51 46 57 64 68 58 59 58 60 69 72 60 62 59 60 71 74 65 66 61 63 74 74 68 69 62 63 THE 77 73 7 67 68 84 77 76 76 75 71 85 83 7 76 77 72 86 86 82 80 78 72 87 88 83 83 80 77 88 89 86 85 80 *UEl 91 91 87 89 84 80 92 91 91 92 88 82 COS oe 95 97 94 91 99 | 101 97 98 97 92 101 | 104 98 98 98 92 107 | 106 99 |} 101 99 97 108 | 107 {| 100; 104} 101]; 100 109 | 118; 103; 107} 103) 100 117 | 121} 103 | 109; 1065 406 118 | 121] 114] 113] 106) 109 123°} 122) 122 | 116°) tiie 141 | 154] 1384] 124] 123] 136 Mean of offspring.......... 98.03 = 0.73) 93.9 + 2.0 | 89.6 = 1.9 | %.8 = 1.8 corresponding decreases in the minus lines are only 5.7, 4.0 and 4.6. Regression toward the mean of the unselected population decreases with successive selections. (Table 8.) The average regression in the three plus lines is 0.67 for the first selection, THE ‘BAR EYE’ MUTANT OF DROSOPHILA 523 TABLE 6 Minus selection Line F GENERATION 1 | GENERATION 2 | GENERATION 3 Tm —Wm Om GENERAL POPULATION e 33 m 33 S ss Bere e | ce | 2. ites PSUR TUS Meta sis. ve eed Ea I ee 89 78 76 7fil 61 64 Mid-parental values. ......5.....082 506. 83.5 73.5 62.5 OFS 7g AE ee ae SNE Bain cn clagsers tn cic 63 51 4S 59 58 47 96 | 100 95 95 90 89 96 |} 104 95 97 92 91 he} || Oe 97 98 94 91 100 | 109 99 | 100 95 92 102; 115) 102] 103 98 94 103 | 118 | 106 | 104 99 95 104 | 118; 107| 104; 101; 100 107) 121 | 108 | 109} 104) 107 LOR eet 25 ell Oat 2) et On 6 OR ZS 8 a2) | 6 | es | 30 159 | 151 143 | 122] 114) 137 Mean of offspring......... 98.03 = 0.73] 94.6 + 2.2 | 89.6 +1.9| 84.7 + 1.9 0.60 for the second and 0.57 for the third. For the minus lines the corresponding figures are 0.77, 0.64 and 0.60. On the other hand regression toward the mean of the parental generation increases with successive selections. (Table 9.) The average regression in this respect in the plus lines is 0.67 for the first 524 CHARLES ZELENY AND E. W. MATTOON TABLE 7 Summary of tables 1 to 6 PARENTS OFFSPRING 3 Ext = ens ai Mean number | Standard eeesren a Nuap= facets of facets deviation variation high fon individuals General | population..| 98.03 | 98.03.73) 24.3 24.8 182 | 45 500 Line A. Gene =... 130.00 | 108.70+2.3) 24.6 22.6 179 | 69 50 Geneon 174.00 | 127.50+3.1 32.9 25.8 210 | 84 50 Gens oe 186.00 | 1385.50+2.8} 28.9 2les 204 | 92 50 Line B Gern.gl are. 130.00 | 110.10+2.7 28 .8 262 184 | 75 50 Gene 2 ee. 174.50 | 128.60+3.2} 33.4 26.0 PAN “7f8) 50 Genttse oe 202.00 | 141.90+2.9 29.1 20.4 207 | 89 46 Line C ‘ Genes er GT OUN| eG 90-2 Ol eae O 23.1 208 | 63 50 Gens 2s oe 183250) |133250==323| a ot-o DT 198 | 67 50 Gen. 3: =. =. 196.00 | 141.00+2.8) 29.3 20.8 DIS 95 50 Line D Genel peer 56.00 | 88.30+1.8 18.4 20. 127 | 44 50 Genl-2:. 225 70.00 |} 85.50+1.8 18.9 D2 al: 133 | 49 50 Gene ouster 60.50 | 81.70+2.1 Doe Dees (By |) ats 50 Line E Gen alt 4 81.00 |} 93.90+2.0) 20.9 D2se 154 | 50 50 Gens 2. 2-28 69.00 | 89.60+1.9 20.1 Dec 134 | 50 50 Genkto 2554 61.00 | 84.80+1.8 18.9 Doe 136 | 46 50 Line F Gen. 1 83.50 | 94.60+2.2| 22.9 | 24.2 159 | 51 50 Grens'25 5 er 73.50} 89.80=1.9} 20.3 22.6 143 | 48 50 Genesco 62.50 | 84.70+1.9 20.3 24.0 137 7 50 TABLE 8 Regression toward the mean of the unselected population PLUS SELECTIONS | MINUS SELECTIONS Line A | LineB | Line C eos ee D] Line E| Line F <= Selection.) SEA 0.67 | 0.62 | 0.73 | 0.67 | 0.77 | 0.76 | 0.77 | 0.77 Selection: 22. 5...-0 20. sory: 0.62 | 0.60 | 0.58 | 0.60 | 0.55 | 0.71 | 0.66 | 0.64 Selectiom 35.25 sm 0 eeee BO bas, 0.58 | 0.57 | 0.57 | 0.56 | 0.63 | 0.62 | 0.60 THE ‘BAR EYE’ MUTANT OF DROSOPHILA 525 TABLE 9 Regression toward the mean of the parental generation PLUS SELECTIONS MINUS SELECTIONS Line A | Line B| Line C| A¥€F |Line D| Line E Line F | — MEleECulOl 1:5... wots ete 0.67 0.62 | 0.73 | 0.67 | 0.77 | 0.76 Once | Osan Selection 2................] 0.71 | 0.71 | 0.75 | 0.72 | 0.85 | 0.82 0.77 | 0.81 Selection 3................| 0.86 | 0.82 | 0.88 | 0.85 | 0.85 | 0.83 | 0.81 | 0.83 Fig. 3 The effect of selection in Plus Line A, at the right, and Minus Line D, at the left. The horizontal lines represent range of facet number, the lowest numbers at the left and the highest at the right. The lowest horizontal line represents the original unselected population, the others, beginning at the bottom, respectively the populations after the first, second and third selections. The extent of overlapping of facet number in each generation is indicated by the overlapping of the two parallel lines. The heavy lines give the progress of the mean values. The numbers are these mean values. A dotted line in every case runs from a mid-parental value, below, to the mean of the offspring, above. On the sides of each dotted line are the lines running from the mid-parental value to the values of the extreme individuals of the offspring. The full data are given in tables 1, 4 and 7. 526 CHARLES ZELENY AND E. W. MATTOON Fig. 4 The effect of selection in Plus Line B and in Minus Line E. See the description of figure 3 for further details. The full data are given in tables 2, 5 and 7. selection, 0.72 for the second and 0.85 for the third. In the minus lines the corresponding figures are 0.77, 0.81 and 0.83. The decrease with respect to the mean of the general unselected population indicates that there is real progress during the suc- cessive selections. The increase with respect to the parental generation indicates that the effectiveness of the selection de- creases with successive selections and that there is probably a limit to the number of effective selections. It seems probable that continued selection would not be able to change the number of facets in the mutant stock to that of the original stock from which it was derived. The data presented thus show that selection in the ‘bar eye’ race of Drosophila is effective both in increasing and in decreas- ing the number of eye facets. As a result there can be no doubt of the existence of differences in the germinal constitution as THE ‘BAR EYE’ MUTANT OF DROSOPHILA 527 Fig. 5 The effect of selection in Plus Line C and in Minus Line F. See the description of figure 3 for further details. The full data are given in tables 3, 6 and 7. regards this characteristic among the individuals of a generation. This fact is of special interest because the origin of the race by sudden appearance in a single individual is known and is of recent occurrence. Furthermore the behavior in crosses with the normal wild race shows that the mutant differs from the normal wild race in but-a single Mendelian factor. If this were the only germinal factor involved in facet number we would be compelled to conclude that we have a case of variability in a unit factor. As far as the present selection data go such a provisional hypothesis would not be contrary to the facts. It seems more probable however that facet number in the normal wild race is represented in the germinal constitution by more than one factor and that the modification occurring in the pro- duction of the ‘bar eye’ race involved only one of these factors. That this factor is a most important one is of course indicated 528 CHARLES ZELENY AND E. W. MATTOON by reduction from an average of 701.1*facets in the original stock to 98.0 in the ‘bar eye’ stock. Selection, then, may have an effect because of variability or because of lack of homo- geneity in the race as regards these other factors without regard to the ‘barring’ factor itself. Three possibilities are thus open as regards the explanation of the effect of selection in this case. First, the ‘barring’ unit factor may be variable or may have varied since its first appear- ance in 1913. Second, the ‘bar eye’ race and by inference the original normal eyed stock from which it was derived may con- tain additional germinal factors affecting facet number and these additional factors may be variable. Third, the ‘bar eye’ race and by inference the original normal eyed stock from which it was derived may not be homogeneous with regard to these additional factors. Different factorial combinations may be present in different individuals. Selection in this case would segregate the ‘high’ combinations of factors on the one hand and the ‘low’ combinations on the other, yielding finally two homo- geneous races in which further selection would have no effect. The ‘highest’ possible combination of factors as well as the ‘lowest’ possible may not exist in the original sample of the general population, but by Mendelian recombination it would finally appear. . While the data so far obtained do not enable us to decide which one of these three possibilities or which combination of them is to be considered as active in this case there is some evidence to support the view that the third is at least partly responsible. The increase in regression of the mean toward the mean of the parental population with each successive selection indicates an approaching limit to the effectiveness of selection. This is what we would expect in a population that is heterogene- ous as regards factorial composition. If the unit factors them- selves do not vary, selection must soon cease to have further effect. THE ‘BAR EYE’ MUTANT OF DROSOPHILA 529 SUMMARY 1. Three successive selections for high number of facets in the ‘bar eye’ race of Drosophila increased the mean number of facets from 98.0 to 139.5. 2. Three similar selections for low number decreased the mean from 98.0 to 83.7. 3. The lowest individual, 89.0, in the ‘high’ lines after three selections is higher than the mean of the ‘low’ strains, 83.7, and the highest individual of the ‘low’ lines, 137.0, is lower than the mean of the ‘high’ strains, 139.5. 4. Significant progress was noted in each of the three selections in both ‘high’ and ‘low’ lines. 5. There are some differences in variability in the different generations but no significant change is proven. 6. Regression toward the mean of the general unselected popu- lation decreases with successive selections. 7. Regression toward the mean of the parental populations increases with successive selections. This increase makes it im- probable that ‘bar eye’ stock can be raised to the original level by continued selection. 8. It is apparent from these data that individuals in any generation differ as regards germinal constitution. 9. If this difference in germinal constitution is solely in the unit factor concerned in ‘barring,’ variability in this unit factor must be assumed. 10. It is however more probable that there are other factors concerned in facet number. In that case the selection effect may be due either to variability of single unit factors or to presence of original differences in factorial composition. That it is due in part at least to the latter is indicated by the increase in regression toward the mean of the parental generation with successive selections. THE OCCURRENCE OF LETHAL FACTORS IN INBRED AND WILD STOCKS OF DROSOPHILA MARY B. STARK (From the Zoélogical Laboratory, Columbia University) TWO DIAGRAMS Three main points are dealt with in the following account: 1) The relative frequency of sex linked lethal factors in inbred stocks of Drosophila ampelophila in comparison with their occur- rence in wild stocks. 2) The occurrence of new lethals and their linkage relations to other sex linked characters. 3) The demonstration that an extraordinary sex ratio was due to the occurrence of two different lethal factors each carried by one of the sex chromosomes of the female that gave the ratio in question. | THE RELATIVE FREQUENCY OF LETHALS IN INBRED AND WILD STOCKS A hundred virgin females of a stock of Drosophila which was caught at Falmouth, Mass., in the summer of 1912, were mated individually on November 29, 1912, with males of the same stock. The counts of the offspring from each bottle are shown in table 1. A hundred virgin females from a stock that had been caught at Falmouth, Mass., in the summer of 1911, were mated indi- vidually to males of the same stock early in January, 1913. _ The results are shown in table 3. Numbers 138, 36, 38 and 47 of table 3 show a ratio of twice as many females as males while numbers 43, 53 and 67 are doubtful. In order to determine whether the high ratios would reappear in later generations, virgin females from several of these cultures were mated to brothers. The results are shown in table 4. Of the four sets of tests two (viz., 36 and 38) give ambiguous results, while numbers 13 and 47 give respectively 5 high to 9 531 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 19, No. 4 532 MARY B. STARK TABLE 1 FEMALES MALES FEMALES MALES FEMALES MALES FEMALES MALES 97 98 96* 59 85 81 105 105 134 147 121 113 93 93 88 79 77 79 94 99 73 77 58 47 121 117 116 98 105 108 71 75 138 144 82 82 34 41 92 82 132 129 183 166 112 104 47 45 99 111 117 107 72 74 56 62 122 142 161 160 85 92 93 80 127 128 147 140 52 47 89 77 64 61 62 51 87 92 73 87 122 152 126 120 88 79 96 92 95 110 105 112 68 77 69 70 75 82 50 37 133 112 62 74 103 120 109 111 72 93 154 142 112 120 94 97 126 112 203 172 135 137 112 94 117 123 175 147 42 47 138 141 102 102 165 154 166 150 75 74 98 101 120 86 129 127 100 110 91 80 49* 28 125 112 65 62 98 101 83 72 43 50 113 109 128 122 31 21 157 160 117 105 62 59 42 34 147 115 109 106 40 42 70 49 130 if EL 65 7 48 68 57 52 67 56 82 88 86 72 80 70 *Two matings showed rather high sex ratios, and as this is often indica- tive of a lethal factor, I mated individually six of the virgin females from each bottle (twelve in all) to males from the same bottles. Since each bottle of the twelve (table 2) gave a 1:1 ratio it follows that the somewhat lower values noted in these two bottles was probably merely a chance deviation, or else due to the crowded condition of the bottle which prevented all of the males from hatching out. The males are known to continue hatching for some days after the females have ceased to hatch. TABLE 2 26 F2 94 Fe Females Males Females Males 141 121 87 92 128 106 79 74. 162 150 124 105 72 73 177 164 160 159 232 212 130 138 222 204 TABLE 3 jn nn nn En rrnne rnnnennD nn DEEDES NO. OF PAIR FEMALES MALES SEX RATIO |NO. OF PAIR| FEMALES MALES SEX RATIO 1 290 227 28.2 51 154 162 0.95072 2 121 111 A O9 nal 52 139 108 iL here 3 196 200 0.98 :1 53 56 30 1700/5 1 4 176 173 1.02 :1 54 177 182 Orar ot 5 93 102 Ujore)th 2 AE 55 183 145 e201 6 114 134 0.85 :1 56 75 57 bees Eeaea | 7 132 128 Oa aak 57 194 171 nie ogal 8 115 96 E20) 3 iL 58 203 195 1.04 :1 9 96 92 1.04 :1 59 158 129 P27 Sal 10 95 “99 0:96: 1 60 200 176 ails; gal 11 50 63 Ovo ul 61 134 114 theall@ 2 12 124 142 O28 cal 62 45 41 Wald) eo! 13 232 114 2.03 :1 63 196 183 10 Smael 14 262 229 ig oa 64 134 106 Ie Ato) 3 il 15 187 188 1.0071 65 136 122 MUP a 16 212 203 1.04:1 66 143 152 OLga aL 17 214 159 1535.2 2 67 105 63 1.6675 1 18 247 220 pe Wee 68 147 133 IPA Vee | 19 169 145 1G 69 151 157 0.96 :1 20 106 100 1.06 :1 70 175 159 Aoeseh 21 189 149 Tze ql 121 84 1.44 :1 22 155 127 1.22) e4 12 108 156 HS) gi! 23 193 179 108,20 73 80 71 IE nea 24 206 169 122,21 74 106 98 (U8) 21! 25 230 223 1 503720 75 116 103 2 el 26 182 154 DAS se 76 78 59 esas: £ 27 201 168 120.238 77 60 53 1 dlalegat 28 178 142 122-1 78 106 92 alge 29 153 136 Le2 79 $1 69 fi3 734 30 120 79 1.52:1 80 148 118 ja! eat | 31 166 153 1.08; st $1 79 88 0.90 : 1 32 157 143 PG a 82 103 107 0.96 :1 33 212 155 | Bae fae 83 126 120 1.05 :1 34 225 225 00-2 84 116 95 Peivesd 30 156 161 TOS cet 85 95 94 10171 36 188 $1 Zion > i 86 114 140 Ors 4 37 172 183 799s 1 87 75 68 110 x 38 172 83 AOR GA 88 oa 98 0.99 71 39 77 92 0.83 :1 89 119 140 0.92 :1 40 125 83 neta) Ngee ! 90 140 128 OGL 41 159 155 102.21 91 126 124 1.06 : 1 42 102 95 1 fear! 92 133 137 Or Sieh 43 74 49 Palys 93 155 157 0.99 = 4 44 53 36 1.47 :1 94 157 148 jel Vs) ee! 45 113 Ua 149 95 118 115 £0231 46 69 64 1.09 :1 96 111 88 1265: 8 47 200 93 ea ks cea | 97 148 138 ue Wa 48 8& 89 0.938: 1 98 105 103 EADIE) & A! 49 56 60 0598731 99 127 126 eons al 50 76 64 i192 100 124 120 tO st 534 MARY B. STARK TABLE 4 13 36 NO. OF PAIR FEMALES MALES SEX RATIO |NO. OF PAIR} FEMALES MALES SEX RATIO 1 194 95 2.04 :1 1 116 128 O01 2 2 123 50 2.46 21 2 114 124 0.92 :1 3 123 124 0.99 :1 3 89 63 1.41 :1 4 131 113 DEG *: 1 4 101 107 O.94:54 5 164 61 2.70 :1 5 100 74 1.35 :1 6 108 86 121451 6 102 124 0.82 :1 f 143 53 20. 3 4 119 96 1.24:1 8 82 104 O2r9.- 4 8 92 94 0.98 :1 2 193 173 iE a 9 70 49 1.43 :1 10 148 82 1.80 :1 10 100 80 1-25 38 11 159 143 ; eb Uae 11 194 153 1.2728 12 87 93 0.94 :1 12 109 47 2.32 :1 13 53 59 0.96 :1 13 129 105 1.23 :1 14 119 173 1.50::4 14 124 105 LA8 ss 15 165 152 LelOrea 15 127 116 LAG = 38 47 1 112 56 2.00 :1 1 137 141 0.97 21 2 159 139 teed 2 150 157 0.96 :1 3 125 78 161.22 3 140 105 1. 33e4 4 67 73 0298 2 4 4 85 95 0.89 :1 5 58 46 ioe t 5 115 107 1.08 : 1 6 131 90 L245 24 6 127 77 1.65 :1 7 90 79 O04 tf 185 91 2.03 : 1 8 119 129 0.92 :1 8 201 81 2.45 21 3 ve 78 OCS A 9 119 90 1.328 10 153 86 i DB Co Mea 10 149 150 0.994 11 183 153 LAG 4 if! 201 92 2.20 :1 12 164 iNET 140" =a low 1 doubtful and 4 high to 7 low ratios where equal numbers of each kind are expected. Further tests were therefore made. Pairs were mated from 13, 1; 13, 2; 13, 5; 13, 7; 36; 1274759 47, 8; 47, 11. The counts from these matings are shown in table 5. As expected, the test of 13, 1 shows the presence of a lethal since there were 6 high to 6 low ratios. The test of 13, 2 indicates a lethal with 8 high to 11 low ratios. The test of 13, 5 indicates a lethal with 6 high to 13 low ratios. LETHAL FACTORS IN DROSOPHILA 535 TABLE 5 1300 NO. OF PAIR FEMALES MALES SEX RATIO 1 186 103 LSet 2 118 122 Ose st 3 121 128 1.06 :1 4 116 101 1.1561 5 171 81 tte 1 6 138 46 3.00 :1 Z 120 116 Os 8 107 52 2.06 :1 9 115 110 1.05 :1 10 104 128 O: Si a 11 127 68 1.86 :1 12 174 100 fea fe aaa | 13, 2 NO. OF PAIR FEMALES MALES SEX RATIO 1 159 61 PeGleat 2 128 146 0.881 3 105 53 2001 4 212 94 2.3621 5 139 101 Ass7e a 6 84 74 ait Hf 185 122 ft 101 8 240 100 2A A 9 225 160 1.40:1 10 ae 79 0.98 :1 11 91 69 1.38 :1 12 72 87 0.83 :1 13 109 81 123451 14 108 84 lee fees 15 175 91 1292329 16 222 116 oie 1 17 88 40 ae ta | 18 134 104 129 19 145 47 S081 Two high to 9 low ratios appear in 13, 7, which probably in- dicates a lethal as the parent also had a high ratio (viz., 2.7 : 1). These four tests of 13 show beyond doubt that a lethal was present, since each of the four families tested gave some high ratios. 536 MARY B. STARK . TABLE 5—Continued 13, 5 NO. OF PAIR FEMALES MALES SEX RATIO 1 116 139 0.87 :1 2 103 100 1.03 :1 3 222 109 2.03 :1 4 140 140 1.00 :1 5 159 58 2.75 :1 6 150 168 0.89 :1 7 205 210 0.98 :1 8 107 44 2.4331 9 239 140 1.71 :1 10 164 137 1.20 :1 1 125 117 1.07 :1 12 148 123 1.2027 13 172 156 1.10 :1 14 107 50 2.14:1 15 113 115 0.98 :1 16 100 134 0.75 :1 17 211 150 1.41 :1 18 116 133 0.87 :1 19 155 62 2.50 :1 eer NO. OF PAIR FEMALES MALES SEX RATIO 1 267 229 1.17 2 180 161 {1 3 136 123 1.10 :1 4 208 89 2.32:1 5 106 106 1.00 :1 6 162 87 2.00 :1 7 139 131 1.06 :1 8 119 140 0.85:1 9 121 119 1.01 :1 10 109 76 1.43 :1 11 113 83 1.36 :1 Of the 60 females tested, half (30) should have given 2:1 ratios, while in fact, only 22 gave such ratios to 38 giving normal ratios. Provisionally, this deviation may be ascribed to chance. ~ LETHAL FACTORS IN DROSOPHILA 5O7 The following tests were made of the daughters of number 12 of 36, table 4: TABLE 5—Continued 36, 12 NO. OF PAIR FEMALES MALES SEX RATIO 1 121 72 ee sal 2 151 56 2.8:1 3 130 109 [ie Dron t 159 140 heal Bal 5) 97 115 OLSiel 6 177 108 16% ol 7 135 88 Wee) 8 144 55 AG) 3 I In this test there were 5 high to 3 low ratios showing that 36, 12 was a lethal bearing female. The following tests were made of the daughters of number 7 of 47, table 4. TABLE 5—Continued 47, 7 NO. OF PAIR FEMALES MALES SEX RATIO 1 259 121 eal Cae | 2 235 122 ORT 3 188 78 PS | 4 133 152 0:9 24 5 170 90 M43) Sl 6 185 95 LOFT: vf 147 148 10% 1 8 164 150 Leia Here there are 5 high to 3 low ratios indicating a lethal. In 47;8 the 4 high to 4 low ratios indicate a lethal. In 47, 1 there are 3 high to 7 low ratios. Of the three sets of tests of 47 of table 5 there were 12 high to 14 low ratios which establishes the presence of a lethal in this line. The general outcome of these tests leaves no doubt that a lethal was present in the original females that were tested. Since only half of the daughters of a lethal female are hetero- zygous for lethal and since these females are indistinguishable co (Se) QO MARY B. STARK TABLE 5—Continued 47, 8 NO. OF PAIR FEMALES MALES SEX RATIO 1 146 80 1.8:1 2 93 94 1.0:1 3 201 162 1.2:1 4 124 58 2.1:1 5 147 156 0.9:1 6 196 69 2.8:1 7 150 : 119 1. Sick 8 158 57 2.8 Al AT, 11 NO. OF PAIR FEMALES MALES SEX RATIO 1 142 79 1.79 :1 2 168 173 0.98 :1 3 157 171 0.92 :1 4 159 162 0.98 :1 5 167 124 1.34:1 6 247 124 2.00 :1 7 142 127 1 es 8 190 97 1.96 :1 9 122 146 0.90 :1 10 163 156 1.04:1 from their sisters, it is by chance only that one would choose a female with a lethal factor when testing out a stock. On the other hand if a lethal female is mated to a male having a sex TABLE 6 Tey ke NO. OF PAIR FEMALES MALES SEX RATIO 1 183 156 i A He 2 138 118 sO Ws: 3 155 70 2.22 :1 t 158 139 112 ca 5 154 82 1908 6 142 152 0.90 :1 7 163 152 1 Olase oor WH FH OCoOonoawrh whe mprantoaurk wbdr CHO AOANOArkwWN rH _ LETHAL FACTORS IN DROSOPHILA TABLE 6—Continued 539 to Be WR rR AIO WW OOH oR SOLS ee ee ee Co No) bo or oO =) —) co on woo v9 a hwRort Eaowrmc i) | = ee a ee Se ° 13, 1, 12 114 70 156 136 199 108 154 82 195 102 154 103 Serer 123 60 130 52 98 105 82 3 110 95 140 135 170 50 84 62 99 70 13, 2, 8 83 61 150 62 105 96 156 162 84 39 134 134 120 73 109 64 134 111 13, 2, 19 164 129 207 214 225 211 232 172 182 185 341 120 283 152 193 93 366 167 BPNNPEPNHERPE OF ee 540 MARY B. STARK linked factor close to the lethal factor in question a stock may be obtained in which the lethal females may be selected with great probability. For example: If a red eyed female carrying a lethal is mated to a white eyed male half of her daughters will have the factor for red and the factor for lethal in one X chromo- some and the factor for white and the factor that is the normal allelomorph of lethal in the other chromosome. If such a daugh- TABLE 7 13, 2, 8, 2 (four 1 : 1 ratios omitted) FEMALES MALES CROSSING OVER NO. OF PAIR pee SUS Se | ee | A) Red White Red White ae ] 62 73 11 47 19.0 2 87 78 12 48 20.0 3 99 (hi 23 58 28.4 4 77 67 11 rie) 1630 13, 2, 19, 6 (five 1 : 1 ratios omitted) il 177 94 30 88 25 .4 2 117 19 81 19.0 13, 2, 19, 7 (three 1 : 1 ratios omitted) 1 83 107 23 69 25.0 Z 112 118 22 93 19.1 3 98 81 26 68 Peifedl 4 162 85 14 79 ise 1 5 89 94 20 71 22.0 ; 6 98 87 27 72 Zio 1 18 65 210 82 83 | ter is mated to a white eyed male half of the female offspring will be red eyed and half white eyed. The former getting their red bearing chromosome from their mother will be the lethal bearing females since the red bearing chromosome also carried the lethal factor. By selecting the red eyed females, therefore, in each succeeding generation and breeding them to white eyed males the lethal stock can be maintained. Virgin females from numbers 13, 1, 11; 13, 1, 12; 18, 2, 1; 13, 2, 8; 13, 2, 19; of table 5 were mated to white eyed males. The results are shown in table 6. ——a ee LETHAL FACTORS IN DROSOPHILA 541 The tests give 19 high to 22 low ratios which is the expecta- tion for lethals, 1.e., equality is expected and is approximately realized. Daughters from 2 :1 cultures all of which were heterozygous for white and half of which should be heterozygous for lethal also, were again mated to white eyed males with the results in table 7. There were 13 high to 13 low ratios shown by these daughters indicating a lethal factor. On the basis of these data the locus of the lethal is at 23.7. It is interesting to note that the lethal factor occurred in flies that had been inbred a year and that none appeared in the stock having been inbred only two months. Miss Rawls (Biol. Bull. 13) found her lethal in a stock that had been inbred a year. The lethals described by Quackenbush (Se. 710) and Morgan (Se. 712; and Jour. Exp. Zo6l. ’14) appeared in stocks that had been inbred for some time. To test whether, in general, lethals are more frequent in inbred stocks I mated 100 pairs from wild stock caught at Falmouth, Mass., and 70 pairs from wild stock caught at Harris, Minn. The results are shown in table 8. Tables 1 and 8 show that the counts made of offspring from 270 pairs of fresh wild stocks have no unusual ratios. THE SECOND LETHAL FACTOR On February 10 of 1914, sixty pairs from stock collected in the summer of 1910 were mated. The results are shown in table 9. The next to the last pair of the above table seemed to show the presence of a lethal factor. Sixty virgin daughters from this pair were mated to brothers. Nearly one-half of these gave a ratio of twice as many females as males, as shown in table 10. Virgin females from numbers 7, 8, 12, 23 and 59 were mated to white eyed males. About one-third of the counts of the next generation show the presence of a lethal factor (table 11). Some of these lethal females were again mated to white eyed 542 MARY B. STARK TABLE §8 (a) Falmouth Stock FEMALES MALES FEMALES MALES FEMALES MALES FEMALES MALES 98 iG 129 111 161 132 175 182 144 132 207 216 175 176 171 174 74 78 181 154 195 187 191 190 68 86 182 193 101 118 199 223 129 120 196 205 102 85 100 88 120 117 179 187 154 162 99 106 119 138 191 175 195 190 185 180 178 174 105 97 191 198 ” 100 84 176 175 184 181 199 202 174 194 96 103 173 138 150 | 150 87 128 130 109 186 150 166 183 91 90 156 141 174 169 110 123 98 86 103 88 170 197 175 177 . 100 108 125 152 187 222 176 180 100 93 105 95 220 182 192 171 146 136 84 92 186 157 189 172 108 110 140 188 95 110 175 193 100 92 128 151 192 200 159 17 85 102 127 139 * 174 171 171 159 100 101 157 193 113 LE 177 173 172 160 120 145 167 119 166 184 183 189 109 106 123 120 117 81 192 189 102 100 191 210 159 163 176 180 148 125 134 129 110 107 164 170 167 155 106 102 191 199 171 175 males. It was expected that only a few of the red eyed males would appear in the second generation if red and lethal were closely linked. Results (table 11) show, however, that a great number of red males appear. Such a result indicates that a new lethal had appeared which was located some distance from the factor for white, thus giving a chance for a greater amount of crossing over. | In table 11 it will be noted that the percentage of crossovers is 46. This would place the lethal somewhere beyond the factor for sable and not far from the factor for bar eyes upon the sex chromosome. LETHAL FACTORS IN DROSOPHILA 543 TABLE 8 (b) Harris Stock FEMALES MALES FEMALES MALES FEMALES MALES 69 166 137 142 83 95 126 126 17, 96 176 167 158 139 104 104 91 86 124 121 97 105 175 130 159 216 115 125 140 139 163 132 105 98 94 76 180 170 115 79 138 120 199 196 84 87 116 115 154 155 93 81 183 185 153 153 115 89 163 135 157 167 93 75 107 7. 165 135 104 105 138 120 104 117 93 82 164 161 183 179 110 136 218 229 136 142 112 110 215 210 170 156 139 129 0 196 173 161 144 132 240 244 151 133 90 87 195 236 98 98 125 93 162 165 129 99 88 82 102 98 122 116 121 119 236 230 106 95 137 ir 144 154 70. 90 134 135 166 167 96 79 122° 96 172 165 To determine the approximate location of the lethal upon the chromosome virgin females of the fifth generation (table 11) were mated, some to sable males, and some to bar eyed males. The counts of the second generation of crosses with sable and with bar eyed males are shown in table 12. The 1 :1 ratios are omitted. The data in this table show that the distance of. this lethal from bar is 8.3. If the locus of the factor for sable is 43 and that for bar is 57, the data of table 12 show that the locus of the new lethal is 43 + 23.5 or 66.5, since the distance of lethal from the factor for bar is only 8.3. The locus of the lethal must be ‘to the right’ of both factors since 43 + 23.5 or 66.5 is approximately equal 544 MARY B. STARK TABLE 9 FEMALES MALES FEMALES MALES FEMALES MALES 146 151 164 161 164 169 157 168 82 125 148 131 83 79 183 136 266 239 88 99 197 193 201 216 95 104 114 date 167 160 108 -103 114 111 133 129 76 78 96 106 112 140 140 138 88 86 106 86 150 213 93 120 116 97 105 85 114 95 169 173 100 79 155 230 199 185 176 152 106 11 181 205 131 155 98 103 145 154 102 84 142 119 106 96 160 133 91 64 110 96 171 153 138 110 181 197 250 188 143 135 195 178 252 216 152 135 142 162 150 163 180 149 266 108 139 105 148 134 86 55 to 57 + 8.3 or 65.3 or at 65.6. The locus indicated by both experiments when the data are weighted proportionately and a correction is made for double crossing over is 66.2. In the spring of 1914, an interesting lethal turned up in my 1910 stock. Half of the males hatched out as normal males but of the other half, though able to pass through the different stages of metamorphosis, many of them were unable to escape from the pupa case. Those that chanced to do so fell over on one side when trying to walk. I examined all the appendages carefully but noticed no abnormalities. Nevertheless, the legs did not seem strong enough to support the body, nor did they seem to move coérdinately and for that reason would so often become entangled with one another that the fly could not get them separated and would die from exhaustion in a day or so. Not any of these males lived longer than two days. Whether the first lethal of the 1910 and the lethals of the 1911 flies allow any development of the lethal bearing male at all, is still under investigation. ee LETHAL FACTORS IN DROSOPHILA TABLE 10 545 NO. OF PAIR a a RwWNFOCOWANOAKHWN FS — or 16 = ~I WD WN Ww eS Ee RwWNHe OO 25 26 27 28 29 30 FEMALES 211 197 161 123 201 64 277 253 63 112 96 244 155 69 72 119 168 71 38 90 213 70 208 117 64 85 69 88 231 217 116 92 67 73 69 102 50 12 36 141 60 90 74 61 60 39 69 123 119 MALES SEX RATIO |NO. OF PAIR SEE eee ee PEN WEEP ee enw eEPePnNnNON EE NN milla E26: (dae 48: 00: 84: Aly A028 e435 ox/e 00: POR 68: AVES 00: e178 64: AQ: 00: 50: snile AGE Poiles BSE (052 Al: 96: E28 .88: BSD 1 1 1 1 1 1 1 1 if 1 1 1 1 1 if! il 1 1 1 if 1 if 1 1 ] 1 1 ] 1 1 dl 32 30 34 Bi) 36 ov 38 39 40 41 42 59 60 FEMALES 112 56 176 46 143 At 205 49 6¢ 26 73 28 160 124 109 45 50 173 115 44 64 58 164 73 27 103 56 191 193 148 MALES 118 88 SEX RATIO 0 ON ao KDHE OHROPNONKRPH HEP RPHE NN RNP RP NRPHE Yr OFPHrN Oo Ww © oe)! oOo 2 Oo tis) & .00 : .90 : BALE LOOR: 93°: 34 : 90: oils mY lee A: ie) ow 1 1 1 1 1 1 1 1 1 1 1 1 1 1 J 1 1 1 1 1 1 1 1 if 1 1 1 il 1 1 Since no lethal has been found in the hundreds of pairs of fresh wild stock examined and since lethals have occurred many of the inbred stocks, it may appear that inbreeding (with its constraints of confinement and homogeneous feeding) tends to cause the mutation to appear or else the removal of the flies from the competition that takes place under wild conditions makes possible the preservation and continuance of any lethal factors that may appear. in A discussion of these alternatives would involve a fuller knowl- edge than we have at present of the causes of mutations and the 1910 Lethal No. of Pair 40 a o No. of Pair 40 an 7 (277 129) — 8 (253 125) X¥_, F 1 } é @ 86 te «Ss [12 (244 = 116) X¥-> 4 4 agg (266 108) 8 (170 6 (147 98 (208 soy 8 (228 1 (228 [bo (es) gy ee 13 (212 * The 1:1 ratios are omitted. 546 eS S Q 3 RQs 3 a 6 (175 7 (120 9 (109 1 (133 3 (183 4 (161 7 (160 1 (138 1 (113 2 (166 5 (116 10 (147 11 (153 12 (224 15 (120 2 (121 4 (113 6 (124 9 (117 10 (173 11 (130 13 (121 | 4 (113 6 (139 8 (117 9 (124 Total no. of o's = 3053 Ro's 56 115 124 131 96 109 123 111 134 112 113 TABLE 11 < W o's S Rs Ro's W9s Wo's = (G48 56 135 77) 198) Sosa OOH). 107 61) 135) XW AN(iSt 46° 132 85) 96) a: 6 32 = 42 92 68) 30) 7(isdmr54e 21 108) S (15S bl “52 79) ae) ey, 55 37) 90) 4°88) - 26 78 51) aay) 5 (142 54 96 64) 6 (105 45 92 43) 8.65 (20 76 35) 9(119 61 93 61) 95) 66) 46) 3(151 52 135 82) 68) ra@a4e 59 117 85) 87) dae Guise! Avi. 117 73) 80) Se 30 «S82. 126 67) 84) Rhodes 57. 112 55) QrG28> -6 137 68) 10 (101° 42 80 56) 77) 58) 91) 146) 83) 5 (114 47 96 62) 79) 6(108 56 103 63) 86) On ieee sae 124 74) 69) Wied.) =59: 103 56) (12 (139; 60 141 72) 72) 2(109 34 139 81) 63) 62 e232 sds * 38 80 48) Bi) mire (ban. 46 90 57) 5G) ee moe) 67 112-68) 59) 12 (1385 49 78 70) 69) 13 (109 42 77 58) x W 7 (76 17 51-33) —- 63) 1405 1648 65) No. of cross overs = 1405 The ratio of the cross overs to all the o's = 1405 + 3053 = .46 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, voL. 19, No. 4 547 548 MARY B. STARK frequency of their appearance. For the present, therefore, the fact of the occurrence of the lethals in the inbred stocks is the one important result of this examination. THE PRESENCE OF TWO LETHALS Number 4 of 13, 2, of table 6 yielded a ratio of 27.33 :1. It seemed probable in this case that two different lethal factors were present, one in each chromosome. This might seem to prevent all the sons from developing since each son must get one or the other maternal sex chromosome; but the survival of the three males would be possible through the crossing over TABLE 12 (a) Heterozygous Bar Female X Bar Male FEMALES | MALES NO. OF PAIR CROSSOVER Bar | Bar Nacsa PERCENTAGE : 284 | 108 5 4.4 2 214 93 13 123 3 120 47 5 ie 4 187 86 12 1292 5 173 79 ‘ a : ae o § i 7 187 78 > ne 8 247 122 5 ae 9 138 94 i is 10 166 52 A ae is ids be iB 13.0 A Ha 0 12 13.0 = sae 1 10 12.0 14 230 129 10 a 15 121 56 3 Aa a 60 7 10.0 17 241 107 10 $0 18 159 84 9 a a a a 9 10.0 20 139 50 sf ae 1590 144 Total number of os, 1734; Crossovers = 144. Crossover percentage = 144 + 1734 or 8.3. LETHAL FACTORS IN DROSOPHILA 549 TABLE 12 (b) Red eyed 2 heterozygous for sable and lethal X sable i a CROSSOVER NO. OF PAIR SABLE NORMAL SABLE NORMAL PERCENTAGE 1 45 40 35 6 15.0 2 111 101 80 20 20.0 3 167 112 84 30 26.3 4 110 74 69 21 24.6 > 162 104 95 37 28.0 6 77 59 45 9 17.0 7 149 113 92 22 20.0 8 92 104 58 25 29 .0 9 91 105 65 25 27.7 10 85 78 5d 16 22.5 11 134 115 78 29 27.7 12 111 113 79 21 22.1 13 138 138 89 33 27.0 14 39 38 35 5) 12.5 15 27 02 29 8 21.6 16 121 114 63 21 25.3 IZ 54 = gil 48 14 22.6 18 86 91 80 26 24.5 19 73 83 79 19 19.4 1254 387 The total number of males = 1641; Crossovers = 387. The crossover percentage = 387 + 1641 or 23.5. Therefore, the distance of lethal from sable is 23.5. of one of the lethal factors from one chromosome in the other, thus freeing one chromosome from its lethal factor. TABLE 13 1320 tet FEMALES MALES CROSSING OVER EE ray | eee ee | ||| BETWEEN WHITE, || SEX RATIO Red White Red White SDP ey 1 172 174 32 116 21.6 2.34 2 217 228 30 136 18.1 2.12 3 147 174 29 126 18.7 2.07 4 215 216 28 174 13.9 2.13 5 229 194 44 124 26.2 2.52 6 228 209 30 144 17.2 2.91 7 184 201 35 139 20.1 2.21 8 214 212 25 147 14.5 2.47 9 193 155 38 138 21.6 1.97 550 MARY B. STARK Nine of the daughters were mated individually to white eyed males and gave the results in table 13. All nine daughters gave a 2:1 ratio which is expected if their mother had two lethals. Ten other daughters (from the very high ratio mother) were mated to their red eyed brothers! and gave the results in table 14. Six red females of table 13 were tested and gave the following results: Soe AS 7 FEMALES MALES CROSSING OVER NO. OF PAIR BETWEEN WHITE SEX RATIO Red White Red White SESS SE 1 59 51 4 47 7.8 PIAS | 2 88 84 8 59 12.0 22624 3 87 66 i 72 15.4 18 13, 29858 1 90 80 11 60 1525 | 2.5 2 2, le 7s 10 73 12.0 133 0 3 75 66 11 63 ify pA ES p Three of the sisters by brother No. 1 failed to produce any progeny when transferred to separate bottles. Each of the other sisters, however, showed the presence of a lethal factor. Thus all 19 daughters of the female gave a 2:1 ratio. There can be no question but that the high sex ratio of the mother was due to two lethals. Virgin red eyed daughters of some of these females were then mated to white eyed brothers with results as shown in table 15. Since the mothers of the females used in table 15 were all heterozygous for white and for one or the other lethal, they would, when mated to red eyed males, produce two kinds of daughters; one-half heterozygous for a lethal and the other half 1 If the sons that came through were due to crossing over, then the X chromo- some that went into each is free from lethals and consequently they must be normal males. The normality of these three males was also tested by mating them to wild females. The sex ratio was normal. The daughters of the three males were tested individually and gave normal ratios. — —e LETHAL FACTORS IN DROSOPHILA ap TABLE 14 Ten sisters by brother No. 1 CROSSING OVER NO. OF PAIR RED FEMALES RED MALES WHITE MALES SEX RATIO BETWEEN WHITE AND LETHAL 1 105 8 41 2.14 :1 16.7 3 224 23 108 1.80:1 17.6 5 64. 6 18 WAV ea! 25.0 6 90 7 36 2209 1 16.3 uf 35 2; if 3.88 :1 De, 8 38 Pi 6 4.75 :1 25.0 9 36 3 12 BYE 20.0 Waist. <<: 498 61 136 PyPAkSS SAL 30.9 Three sisters by brother No. 2 1 128 18 38 2.0 sl 32.1 2 176 12 78 2.01 13.3 3 197 11 78 eileeal 12.4 Pie pall 18.6 IVES tarts =. 336 25 109 heterozygous for white. The females heterozygous for white when mated to white eyed males would produce equal numbers of red eyed and white eyed males and females (table 15) except where the lethal may have crossed over to the factor for white (as was the case in numbers 5 and 1, starred in table 15).2. The females heterozygous for lethal when mated to the white eyed males produce the 2 : 1 ratio, also indicated in table 15. Matings were, also, made between the three males and daugh- ters of the sisters of the males with results as follows: (13, 2, 1, 4; Female by W male) F; Female by No. 2 male FEMALES MALES : CROSSING OVER NO. OF PAIR SEX RATIO BETWEEN WHITE Red Red White PD eran Mass 236 6 95 Meas Bal 5.9 1 7 16 85 ilag/ eal 15.8 2 116 9 43 Pepe Ab 7/3 3 125 a 40 Dede: 14.9 4 141 5 61 Det Hae * The other class resulting from such crossing over should contain neither the white nor the lethal, and one such female is recorded in table 15. 552 FEMALES NO. OF PAIR Red White Red 1 72 80 69 2 63 48 49 3 18 8 4 50 32 re 43 51 27 (Sister by No 1 145 72 2 141 75 3 53 67 47 4 30 42 32 5 43 34 50 6 161 34 67 (Sister by No. 1 137 72 2 * 103 107 3 72 86 81 4 164 86 5 101 47 (Sister by No. a* 101 85 64 2 29 24 24 3 168 65 1 97 69 ih 2 78 24 3 153 | 72 The four daughters show the presence of a lethal. MARY B - STARK TABLE 15 (Sister by No. 1 male), F2 (Sister by No. 2 male) Mass Fy» MALES White . 1 male) 3 F» eS ot 2 male), Fs 74 3 male), 3 Fe 14 20 70 - Oe & bo lO SEX RATIO eatin o ro orw x1 on NOWwNrF OF >a oe eee — ai) oo NR bo > ond SSs Nowe eo 2 © — =) #88888 fd kkk pet oO CROSSING OVER . BETWEEN WHITE AND LETHAL 18.2 19.0 Summary: The nineteen tested females of 13, 2, 1, 4 (table 6) Other tests showed that the three males gave a 2:1 ratio. behaved like normal males. (2 : 1) is that two lethal factors were present. The explanation of her high ratio LETHAL FACTORS IN DROSOPHILA Dd3 Table 16 gives some of the counts of the descendants of 13, 2, 1, 4 with white eyed males. TABLE 16 saa Ses MELE CROSSING OVER BETWEEN WHITE Red White Red White EES) LESS 162 85 14 79 15.0 228 209 30 144 17.2 100 84 13 68 16.0 91 91 12 68 15.0 127 123 15 85 15.0 122 125 16 108 12.8 127 110 18 117 toe 115 115 23 119 16.2 95 86 12 57 17.4 142 105 15 88 14.5 137 138 20 118 14.4 43 43 8 39 17.0 87 66 12 72 14.3 90 70 iil 60 15.5 108 102 17 79 IL? 0 te 65 als 53 22.0 76 lal 13 48 Biles 120 140 30 - 120 20.0 119 100 29 90 24.3 130 98 18 69 20a 149 133 22 95 18.8 193 155 38 138 21.6 229 194 44 124 Doee 172 174 32 116 21.6 193 155 38 138 216 184 201 35 139 20.1 Numbers 5 and 1 starred of table 15 show a decided decrease in the number of white males. It looked as if the lethal factor ‘had crossed over into the chromosome carrying the factor for white. To examine this, virgin females from number 1 were mated to white eyed miniature males as shown in table 17. A lethal factor connected with the factor for white is evidently present since all the white eyed females gave a 2 : 1 ratio. The red eyed sisters of the white eyed females in table 17a should bear no lethal if, as the last table indicates, crossing over 554 MARY B. STARK TABLE 17 (a) White eyed granddaughters of sister and brother by a white eyed miniature male NO. OF PAIR WHITE FEMALES WHITE MALES SEX RATIO 1 79 * 44 {eS 2 171 fis 2.431 3 176 88 220 iat + 182 90 2aOncal 5 187 88 2altoal 6 176 63 229.54 7 139 48 es ail 8 182 72 yA a | 9 170 104 126 ret 10: 164 68 2.4:1 had taken place, except when crossing over occurred again in the mother. This was tested (table 17b). TABLE 17 (b) Red eyed granddaughters of sister and brother by white eyed miniature male FEMALES MALES NO. OF PAIR a ee ees Se SEX RATIO Red White Red White 1 48 . 59 20 43 1.54:1 2 76 78 74 58 1.1621 3 82 59 50 50 1:41-4 4 69 68 66 62 1.07 :1 5 76 52 67 51 1208.4 6 65 63 80 61 0.90 :1 7 88 53 29 59 1.60 :1 8 33 34 5 27 2.09 :1 9 42 53 6 35 Peo | 10 79 65 1 44 3.20:1 11 64 99 7 71 2.09 :1 Four of the red eyed females gave a 2 : 1 ratio indicating that crossing over did occur in the mother. To determine whether this lethal is the new one or the original one whose locus was shown to be at 23.7 the white eyed females were mated to eosin? to find the locus of the factor in the chromo- 3 Eosin is an allelomorph of white. A female that has the eosin factor in one . _ X and the white factor in the other X can be distinguished from a pure female with eosin in both X’s. LETHAL FACTORS IN DROSOPHILA 70D somes. If it is a crossover it should be found to have the same locus as the factor with the red. The white eyed females mated to eosin males gave white- eosin’ long winged females (with the factor for lethal): white- eosin miniature females; white eyed miniature males; and a few white eyed long winged males as crossovers. The white-eosin long winged females were again mated to eosin miniature males. This mating gave white-eosin long winged females with the lethal factor, eosin miniature females without the lethal factor, eosin long winged females and, white-eosin miniature females, eosin miniature males, white eyed miniature males, eosin long winged males and white long winged males. The single crossovers are the white miniature and the eosin long winged males, while the white long winged males are double crossovers. In table 18 some of the counts are given. The crossover value of white with the lethal involved is 15.6 and that of the lethal with miniature is 19.9. Therefore the locus of this lethal is at 16.7, that is, 1.1 plus 15.6. The locus of the original lethal was shown to be at 23.7, so that this lethal with a locus at 16.7 must be the new lethal whose advent led to the production of the high sex ratio. The mother of the high sex ratio carried in one of her X chromosomes the original lethal at 23.7 and in the other X chromosome, the one derived from the father, the new lethal at 16.7. If this female contained two lethals some of her descendants should have one and some the other; and these two kinds should be expected to give slightly different linkage values with whit¢. If, then, the values obtained from all of her descendants be plotted they should give a bimodal curve. Diagram 1 was made from such data except that the diagram does not include the data in table 18. In diagram 1 there is a strong mode at 15 which corresponds to the new lethal, but the mode which corresponds to the original lethal gives only a weak mode at 22; indeed the curve is not obviously bimodal, small number of determinations not being sufficient to distinguish clearly between the two lethals. The NO FEMALES OF PAIR White-| Eosin Hm Cb | © ont Ha bw bw bo bs bo “IO Ot — bo bo oo eos'n long 28 34 35 39 81 66 31 34 39 48 28 47 17 93 113 minia- ture 32 22 24 30 62 82 11 30 42 48 TABLE 18 MALES LINKAGE LINKAGE OF WHITE | OF LETHAL Eosin | White White. eosin | Eosin minia-| long ture if 10 24 ill 17 18 14 9 69 36 42 25 25 7 26 17 22 10 51 27 24 15 19 16 14 17 21 11 62 31 37 30 63 80 70 78 39 34 51 46 41 38 38 ae, 45 22 | 38 23 54 26 39 29 24 37 49 4 48 33 38 24 37 25 29 16 7 | 30 67 38 67 29 52 30 66] 25 47 | 25 ood <-33 19 16 27 26 51 32 45 25 46 34 1780 | 1201 = = WITH WITH 7 — io : fe LETHAL | MINIATURE WwW M o7 4 10 6 Ttng7s 24.4 32 7 7 2| 18.8 18.8 29 9 6 0} 20.5 14.0 29 4 9 0} 9.5 21.4 87 15} 28 1° ee 19.0 74 i7 \° Se A fie be 20.1 25 4 5 Oi. 4s 14.7 41 2 9 Oj. 38 17.3 42 5 8 0} 9.0 14.4 59 9 18 0| 10.46 20.9 25 5 10 O25 25.0 48 6 15 G.) 287 7 26 6 10 0} 14.3 24.3 35 6 10 O.) “Hy 19.6 63 12) 723 g| 12 23.4 100 10} 30 1:| 7838 22.0 101 a5 | 437 t| nes 23.1 RS" Sis e tes Sel avoe 23.5 49 15 17 =a ee OL 23.8 78 17 18 iii fe ee 16.6 44 9 13 1| 14.9 20.9 47 16 18 a. SAG 24.0 | 30 9 13 1 | 2980 26.5 | 58 11 17 we Se: 20.7 . 76| 26) 19 3| 23.4 17.7 56 13 14 1| 16.6 18.0 . 64" tat | Pages e- 14.1 69 | 20 16 2} 20.5 17.0 | 47 4 11 2| G.4 20.3 55 13 19 i £509 22.6 53 5 13 ie Ms | iss 49 2 9 G1 Ss 15.0 71 14 15 1. |g 15.8 1004. 238 26 3| 16.0 19.4 | 13\ 2 28 0} 16.4 22.0 . 63 16 23 a fen AS 24.0 ; 50 8 9 SNe Daeg 17.0 57 17 17 0 I> 18a¥ 18.7 54| 18 13 leh eee 16.2 39 8 12 0| 13.5 20.3 28 6 4 FV 90-0 15.0 71 54) Be 1| 14.5 21.9 51 8 13 2} 13.5 20.2 57 |. A ea 1 BEG 12.0 2421 | 524] 685| 48] 6633 8656 DROSOPHILA IN LETHAL FACTORS 558 MARY B. STARK determinations of table 18 are all of the crossover value white new lethal, and are expected to give a unimodal curve with the mode at 15. SUMMARY Lethal factors were found in inbred stock only. Fresh wild stock gave no unusual sex ratios. The first lethal (1;,) was found in stock caught in 1911 and has its locus at 23.7. One female of this stock gave an ex- traordinarily high sex ratio; viz., 83 females to 3 males. That this extraordinary sex ratio is due to the presence of two lethal factors, one in each X chromosome, is shown by the fact that all (not half) the daughters gave a 2 to 1 sex ratio. The new lethal (ls,) that appeared in the female with extra- ordinary sex ratio crossed over, in one case examined, to the X chromosome that carried the factor for white. Its locus is at 16.7. Two other letha's were found in the 1910 stock. The first (I) has its locus at 65.2. The other (l,a) differs from all other lethals in that the lethal bearing males emerge from the pupa case, and die almost immediately on becoming adult flies. AN ATTEMPT AT A PHYSICO-CHEMICAL EXPLANA- TION OF CERTAIN GROUPS OF FLUCTUATING VARIATION JACQUES LOEB anp MARY MITCHELL CHAMBERLAIN I There is a general tendency to visualize the factors which determine the hereditary characters as specific chemical com- pounds. If we wish to carry this view (with which we sympa- thize) beyond the limit of a vague statement, we must either try to establish the nature of these compounds by the methods of the organic chemist, or we must use the methods of general or physical chemistry and try to find numerical relations by which we can identify the quantities of the reacting masses or the ratio in which they combine. Attempts in this direction have been made by the suggestion of Loeb! that phenomena of growth belong in the group of auto-catalytic processes, and by T.B. Robertson’s? and Ostwald’s investigations supporting and enlarg- ing this idea; by A. R. Moore’s’ attempt to show that in hybrids the velocity of development of the dominant character is slower than in the pure dominant breed; and by Loeb and Ewald’s! proof that all the embryos of Fundulus have practically the same rate of heart beat at the same temperature. Since our new experiments are a sequence of this last mentioned paper, we may briefly discuss its contents. 1J. Loeb. Ueber den chemischen Character des Befruchtungsvorgangs Roux’s Vortriige und Ausiitze, Leipzig, 1908. Biochem. Ztschr., 2, 34, 1906. 2T. B. Robertson. Roux’s Archiv, 25, 581, 1908; 26, 108, 1908; 37, 497, 1913. Am. Jour. Physiol., 37, 1, 1915; Robertson and Wasteneys, Roux’s Archiv, 37, 485, 1913; Wo. Ostwald. Ueber die zeitiichen Higenschaften der Entwicklungs- vorgiinge, Leipzig, 1908. 3A4.R. Moore. Roux’s Archiv, 34, 168, 1912. 4J. Loeb and W. F. Ewald. Biochem. Ztschr., 58, 177, 1913. 559 560 JACQUES LOEB AND MARY M. CHAMBERLAIN C. G. Rogers’ has shown that the heart beat of the embryo of Fundulus has a temperature coefficient of the order of the magnitude of a chemical reaction, i.e., that it practically doubles for an increase of temperature of 10°C. Loeb and Ewald found that the rate of heart beat is practically the same in each indi- vidual embryo (of a certain age) for a given temperature, vary- ing only in very narrow limits; so that the rate of the heart beat of any of these embryos could be utilized as a thermometer. .The authors explained this fact on the basis of general chemistry as follows: given a sufficient quantity of substrate the velocity of the reaction is in proportion to the mass of enzyme. If we suppose that the rate of the heart beat is determined by the velocity of an enzyme reaction—which supposition agrees with the temperature coefficient—we must conclude that all hearts of Fundulus embryos must have the same mass of enzyme, since they all beat at the same rate when the temperature is the same. If we consider the rate of heart beat of the Fundulus embryo a hereditary character—which is legitimate—we are forced to the conclusion that each embryo of Fundulus inherits practically the same mass of those enzymes which are responsible for the heart beat. The hereditary factor in this case must consist of material which determines the formation of a given mass of these enzymes, since the factors in the chromosomes are too small to carry the whole mass of the enzymes existing in the embryo or adult. II While the rate of heart beat is approximately the same in each egg (at the right age) and for the same temperature, we notice slight variations, the usual fluctuating variation. It occurred to us that this fluctuating variation might offer a chance for further testing the enzyme conception of the factors of certain hereditary characters. We selected, instead of the rate of heart beat, the velocity of cell division. Loeb* had shown in a former paper that the time from insemination to the first cell division °C. G. Rogers. Am. Jour. Physiol., 28, 81, 1911. °J. Loeb. Pfliiger’s Archiv, 124, 411, 1908. GROUPS OF FLUCTUATING VARIATION 561 in the egg of the sea urchin Strongylocentrotus purpuratus can be so sharply measured and is so nearly constant that it can be used for the establishment of a temperature coefficient and this was later confirmed by Loeb and Wasteneys’ for the ege of Arbacia. Since the influence of temperature is again of the high order characteristic of chemical reactions, we may make the assumption that each egg carries a definite mass of one or more enzymes or catalysers which determine the rate of cell division. If we fertilize a mass of eggs of the same female of Arbacia and keep them at the same temperature, we find that they do not all begin to segment at the same time, and that there is an interval between the cell division of the first and last egg of the group. If we assume that the velocity of the cell division is determined by the mass of enzymes and the temperature, the fact that at t° some eggs divide after 100, others after 101, 102, until, e.g., 113 minutes, we must con- clude that this difference in time is the expression of a corre- sponding difference in the mass of enzymes in different eggs, those dividing in 100 minutes having a greater mass of enzymes than those dividing in 102, 103, ete., and 113 minutes; and that the mass of enzymes varies in inverse proportion to the time required for cell division at a given temperature. On this basis we should have to assume that the latitude of variation in the rate of cell division of a group of eggs is the expression of a corresponding variation in the mass of enzyme in the individual eggs. This idea can be put to a test with the aid of the tem- perature coefficient. If we call m the minimum mass of the enzyme responsible for the first cell division in the slowest eggs, then we shall find a certain greater percentage of eggs with the enzyme mass m +a, a still larger percentage with the mass m + as, and a small number with the mass m + dn where m + dn is the greatest mass of enzyme occurring in an egg. If the eggs with the mass m + an divide at the temperature t° after 100 minutes, they will divide in about Qio x 100 minutes at the temperature (t — 10)°, where Qio is the temperature coefficient for 10°C. at this point; the eggs with the smallest mass of enzyme 7 J. Loeb and H. Wasteneys. Biochem. Ztschr., 36, 345, 1911. 562 JACQUES LOEB AND MARY M. CHAMBERLAIN m, which at t° divide after 113’ will divide at (t — 10)° after Q:o X 113 minutes, since the temperature coefficient must be the same for both types of eggs. If we call the difference in the time of segmentation between the slowest and fastest egg the latitude of variation, this latitude of variation should vary in direct proportion to the temperature coefficient for cell divi- sion if our theory is correct. Iil We will first give the temperature coefficient of cell division for the egg of Arbacia for different temperatures; i.e., the results of measurement of the time required from the moment of insemi- nation to the moment when the first egg in the field was seen to divide. The eggs had been kept in a water bath with constant temperature, and a little before the cell division was expected to occur (which time we knew from the former observations of Loeb and Wasteneys) the eggs were put into a watch glass of the temperature of the eggs and the exact time ascertained when the first egg of the lot underwent cell division. Table 1 gives these times according to Loeb and Wasteneys, and according to our own observations. The reader will notice how closely both values agree.’ Our values are the average of a number of determinations, which show only a negligible variation. Beyond 31° no segmentation occurs. We tried no experi- ments on the latitude of variation beyond 25° or below 9°, since outside of these limits the segmentation is no longer entirely normal. From the results of table 1 we compute the temperature co- efficients for the time from insemination to the first appearance of cell division (table 2). In order to determine the latitude of variation of the time of segmentation—i.e., the interval between the time at which the first egg of a set begins to segment and the time when the last egg segments for a certain temperature, we proceeded as 8 The eggs were always used in the first hours after they had been removed from the animal. The time required for the first cell division was.remarkably constant in different experiments. It is worth mentioning that such constancy is only possible when the temperature is kept constant. GROUPS OF FLUCTUATING VARIATION 563 TABLE 1 Time in minutes from insemination to the cell division of the first egg in Arbacia : } 99 TEMPERATURE LOEB AND WASTENEYS Z mea =< degrees | a0 498 .0 8.0 410.0 411.0 9.0 308.0 297.5 10.0 217.0 208.5 11.0 175.0 175.0 12.0 147.0 148.0 13.0 129.0 14.0 116.0 15.0 100.0 100.0 16.0 85.5 17.5 70.5 18.0 68.0 68.0 19.0 65.0 20.0 56.0 56.0 21.0 53.3 22.0 47.0 46.0 23.0 45.5 24.0 42.0 25.0 40.0 39.5 26.0 33.9 27.5 34.0 30.0 33.0 31.0 37.0 follows: The eggs were inseminated in sea water, and kept in a water bath at the desired temperature. The eggs remained in this water bath until about the time when the first segmen- tation was expected to occur. In the meantime, a second water bath was prepared on the stage of the microscope whose tem- perature was slightly below that of the desired temperature. This water bath contained the watch glass in which the segmen- tation of the eggs was to be observed. The watch glass had therefore the temperature at which the eggs were observed. The temperature of this water bath was also kept constant. When the temperature at which the latitude of variation was observed was very low and that of the air of the room was high a slight error crept in, in as much as the temperature of the THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 19, No. 4 564 JACQUES LOEB AND MARY M. CHAMBERLAIN TABLE 2 TEMPERATURE COEFFICIENT FOR 8/18 =— 6.0 / ae 9/19 cal 4.5 / 65 = 4. 9 = 10/20 , 2a 56 11/21 ees 53.5 146 12/2 i ee = 46 : 13/23 = = 2.8 45.5 116 14/24 we ees 100 15/25 et ea 5/25 40 5 water in the watch glass rose slightly during observation. This error made itself felt in that in the case of low temperatures the actual temperature was occasionally a trifle higher than intended. We shall come back to this point later on. When the eggs had been put into the watch glass, a field with no less than 80 and often as many as 150 eggs was selected, and every minute the number of eggs which underwent cell divi- sion was counted until the last egg had divided. Very often a small percentage of the eggs had remained unfertilized and these of course did not divide.’ In table 3 we give a few examples of the actual measurements of the latitude of variation in the time required from the segmentation of the first to that of the last egg in a field. As far as the irregularities in the first two minutes are con- cerned, they must probably be attributed to the fact that the entrance of the spermatozoa into the eggs occurred somewhat irregularly, the moment of insemination differing in various eggs within one or two minutes. ‘Table 4 gives the latitude of varia- ®°When this number was great the material could not be used since in such cases the spermatozoa no longer entered the eggs simultaneously. GROUPS OF FLUCTUATING VARIATION 565 TABLE 3 Latitude of variation in segmentation time TEMPERATURE NUMBER OF 25° | ia | 22° 22° | 12° | 12° EGGS SEG- MENTED AFTER Number of eggs in field 117 127 116 126 116 100 minutes 1 3 1 1 2 4 3 2 12 6 8 24 15 5 3 34 15 21 49 26 8 4 68 34 33 85 40 10 5 107 44 85 95 51 12 6 10 eggs not 62 103 111 60 16 7 fertilized 79 110 lity 67 19 8 90 116 119 id 20 9 95 7 eggs not 80 24 10 100 fertilized 80 28 11 109 88 32 12 18 eggs not 88 36 13 fertilized 88 38 14 90 49 15 92 60 16 95 75 17 84 18 100 85 19 . 101 20 105 85 21 105 95 22 106 96 23 108 24 8 eggs not 25 fertilized 98 2 eggs not fertilized tion, i.e., the difference in time between the segmentation of the last and that of the first egg in‘a field for different temperatures for all observations made. The averages appear in the last line. This series illustrates the source of error to which we have already alluded, namely, that at low temperatures the times 566 JACQUES LOEB AND MARY M. CHAMBERLAIN TABLE 4 Differences in minutes between segmentation of first and last egg in a field at g° 10° 11° 137 13° 14° | $5°) 18°) 192 WP 21°) 22 is oe a so) a0 | eerlae | on: az. aimee | a0] 3 koe eee 49 | 40| 26] 20] 18 | 19 | 19] 11] 13 | 10 TAZ hg las 47 97 | 22 | (13) | 16 | 12] 131 11 | 9 glol7)'5 64 | i9 | 1s} ia} | 12 9|8| 73 60 20 14, | 12 7|\8\|9 46 18 jelNeaal ler 8|7\8 20 12 8 14 8 14 7 8 8 Mean 52.6 [39.5] 26 [22.5] 19.2 |17.5) 13) 12/12.5] 9.6] 8 7.8 8/8] 5 Min. were lable to be too short when the outside temperature was very high. Thus the value 13 minutes for the temperature of 13° is unquestionably too low, and probably the values 46 and 47 for 9°C. are also too low. At the higher temperatures the values differ much less, since the temperatures approximate much more the room temperature. We are now in a position to compare the expected with the observed result. The expected result is the series of tempera- ture coefficients for the time from insemination to the time’ when the first egg of the set begins to divide; the observed result is the series of temperature coefficients for the latitude of variation, i.e., the time which elapses between the segmentation of the first and last egg in a set. These two sets of coefficients should be identical and table 5 shows the degree of agreement. . A comparison shows that the temperature coefficients for the latitude of variation are practically identical with the tempera- ture coefficients for cell division, and that where a noticeable difference exists it is always in the same direction, namely, the coefficients for the latitude of variation are a trifle too small. We can account for this on the basis of the deficiency in the method we have already discussed, namely that when the tem- perature of observation was low and that of the room high, the GROUPS OF FLUCTUATING VARIATION 567 TABLE 5 Temperature coefficients for latitude of variation TEMPERATURES EXPECTED FOUND ld 9 9/19 4.7 52.6 = Als 12.6 : 10/20 3.8 39.5 aay 9 10 S é E 2 11/21 aa 20) 5) Ae 8 12/22 3.1 22.5 eer 78 /s ‘ 9 13/23 2.8 19.2 ei 8 of 75 14/24 28 Le Wis 8 15/25 255 a2 oie temperature in the watch glass may have risen slightly during the observations. Since in the determination of the temperature coefficient the value for the low temperature forms the numer- ator, it is obvious that the observed temperature coefficients are liable to be a little smaller than they would be without this error. We expect to test this idea next season. THEORETICAL REMARKS It was found in a previous investigation that the time which elapses from the moment of insemination to the moment of the beginning of cell division in the egg of Arbacia, is a constant for a given temperature. On the basis of the enzyme theory this was to be explained on the assumption that the mass of ferments contained in the egg of the sea urchin responsible for this process is approximately constant in each individual egg. This would mean that the hereditary factor determining the rate of cell division consists in determiners for definite quantities of ferments. This idea was put to a test by applying it to the fluctuating variability of this process. While for a given tem- perature the eggs of Arbacia will always begin to segment at the same time, not all the eggs segment simultaneously. Assuming 568 JACQUES LOEB AND MARY M. CHAMBERLAIN that those eggs which segment first have a greater mass of fer- ment than the others, fluctuating variability would in this case be due to differences in the mass of ferment in the different eggs of the same female. If this idea were correct, eggs with the maximum and with the minimum amount of ferment should differ in the rate of segmentation by an amount of time which would vary in direct proportion to the temperature coefficient for the process of segmentation. This theory was tested and it was found that the observed values agree very closely with the expected values; the slight variations found being in the direc- tion of the possible source of error of the method of the experi- ments. These experiments support therefore the idea that the hereditary factor responsible for the rate of segmentation is a determiner for a given mass of certain ferments, and that fluctu- ating variability depends in this case upon slight but definite variations in the mass of those ferments in different eggs. SUMMARY OF RESULTS 1. It is shown that the temperature coefficient for the lati- tude of variation of the segmentation of the egg of Arbacia (i.e., the time between the segmentation of the first and last egg of a group fertilized at the same time) is practically identi- cal with the temperature coefficient for segmentation. 2. It is shown that the fact is intelligible on the assumption that the fluctuating variation in this case is due to a variation in the mass of enzyme contained in the different eggs and sup- _ posed to be responsible for the rate of segmentation. AUTHOR AND SUBJECT INDEX BNORMALITIES occurring after conjuga- tion in Paramecium caudatum. Variation and inheritance of 387 Adaptation to high temperatures, on the heat resistance of Paramecium caudatum. The effects of certain salts, and of 211 Albino rats held at constant body-weight by un- derfeeding for various periods. Changes in the relative w eights of the various parts, sys- tems and organs of young’ 99 Amphioxus during locomotion. of Aphids. The predetermination of sex in phyl- loxerans and 285 Arey, Lestte B. The orientation of Amphioxus during locomotion. 37 The orientation Ascaris. The effect of carbon dioxide on the eggs of 355 Asoxal reproduction of Hydra. Inheritance in the 157 ‘ AR EYE’ mutant of Drosophila. The ef- * fect of selection upon the 515 Body-weight by underfeeding for various periods. Changes in the relative weichts of the various parts, systems, and organs of young albino rats held at constant ‘ 99 Bripcess, Carvin B. A linkage variation in Dro- 1 sophila Bristle inheritance in Drosophila. I. Extra bris- tles. 61 CALKINS, Gary N. Didinium nasutum. I. The Life history. 225 Carbon dioxide on the eggs of Ascaris. The ef- fect of 355 Caudatum. The effects of certain salts, and of adaptation to high temperatures, on the heat resistance of Paramecium 211 Caudatum. Variation and inheritance of abnor- malities occurring after conjugation in Para- mecium 387 CHAMBERLAIN, MARY MircHetu,LoEeB, JACQUES, and. An attempt at a physico-chemical ex- planation of certain groups of fluctuating variation 559 Chemical explanation of certain groups of fluc- tuating variation. An attempt at a physico- 559 Curtis, MayniE R. and Prart, RAYMOND. Studies on the physiology of reproduction in the domestic fowl. X. Further data on somatic and genetic sterility. 45 DPD WINIUM nasutum. I. The life history 225 Dioxide on the eggs of Ascaris. The effect of carbon 355 Domestic fowl. X. Further data on somatic and genetic sterility. Studies on the physi- ology of reproduction in the 45 Drosophila. A linkage variation in 1 Drosophila. I. Extra bristles. Bristle inheri*- ance in 61 Drosophila. The effect of selection upon the ‘bar eye’ mutant of 515 Drosophila. The occurrence of lethal factors in inbred and wild stocks of 531 BKe&ss of Ascaris. The effect of carbon diox- ide on the 355 Eye’ mutant of Drosophila. The effect of selec- tion upon the ‘bar 515 ACTORS in inbred and wild stocks of Droso- phila. The occurrence of lethal 531 Fishes in their natural environment to salts. The reactions and resistance of 243 Fission rate of Stylonychia pustulata. Heritable variations and the results of selection in the 451 Fluctuating variation. An attempt at a physico- chemical explanation of certain groups of 559 Fowl. X. Further data on somatic and genetic sterility. Studies on the physiology of re- production in the domestic 45 Eyeey length of spermatozoa in seven addition- al species of insects. Variation in 505 Heat resistance of Paramecium caudatum. The effects of certain salts, and of adaptation to high temperatures, on the 211 Heliotropic reactions of animals and plants. The relative efficiency of various parts of the spectrum for the 23 Hurcuison, Rospert H. The effects of certain salts, and of adaptation to high temperatures, on the heat resistance of Paramecium cau- datum 211 Hives Inheritance in the asexual reproduction ° 15 [ NBRED and wild stocks of Drosophila. The occurrence of lethal factors in 531 Inheritance in Drosophila. I. Extra bristles. Bristle 61 Inheritance in the asexual reproduction of Hydra. 157 Inheritance of abnormalities occurring after con- jugation in Paramecium caudatum. Varia- tionand 387 Insects. Variation in head length of spermatozoa in seven additional species of 505 JACKSON, C. M. Changes in the relative weights of the various parts, systems and or- gans of young albino rats held at constant body-weight by underfeeding for various periods. 99 LASHLEY, K. S._ Inheritance in the asexual reproduction of Hydra. 157 Length of spermatozoa in seven additional species of insects. Variation in head 505 Lethal factors in inbred and wild stocks of Droso- phila. The occurrence of 531 Life history. Didinium nasutum. I. The 225 Linkage variation in Drosophila. A 1 Locomotion. The orientation of Amphioxus during 37 Loers, JACQUES, and CHAMBERLAIN, Mary Mir- CHELL. An attempt at a physico-chemical explanation of certain groups of fluctuating variation. 559 Logs, JaccuEs, and WASTENEYS, HARDOLPH. The relative efficiency of various parts of the spectrum for the heliotropie reactions of ani- mals and plants. 23 569 570 INDEX yi AcDOWELL, Epwin Cariton. Bristle in- heritance in Drosophila. I. Extra bristles. 6 1 Mattoon, W. E., ZELENy, CHARLES and. The effect of selection upon the ‘bar eye’ mutant of Drosophila 515 MiIppLETON, AvuSTIN RawpH. Heritable varia- tions and the results of selection in the fission rate of Stylonychia pustulata. 451 Morcan, T. H. The predetermination of sex in phyloxerans and aphids. 285 Mutant of Drosophila. Tne effect of selection upon the ‘bar eye.’ 515 NASUTUM. I. The life history. Didinium 220 QUCURRENCE of lethal factors in inbred and wild stocks of Drosophila. The 531 Caen oL of Amphioxus during locomotion. The 37 PACKARD, CuarRLes. The effects of the beta and gamma rays of radium on pro- toplasm. 323 PaintTeR, THEoruttus 8. The effect of carbon dioxide on the eggs of Ascaris. 355 Paramecium caudatum. The effects of certain salts, and of adaptation to high temperatures, on the heat resistance of 211 Paramecium caudatum. Variation and inheri- tance of abnormalities occurring after conju- gation in 387 Peart, RAYMOND, CurRTOs, MAayNie R. and. Studies on the physiology of reproduction in the domestic fowl. X. Further data on somatic and genetic sterility. 45 Phylloxerans and aphids. The predetermina- tion of sex in 285 Physico-chemical explanation of certain groups of fluctuating variation. An attempt at a 559 Physiology of reproduction in the domestic fowl. X. Further data on somatic and genetic sterility. Studies on the 45 Predetermination of sex in phylloxerans and aphids. The 285 Protoplasm. The effects of the beta and gamma rays of radium on 323 Pustulata. Heritable variations and the results of selection in the fission rate of Stylonychia 451 elu on protoplasm. The effects of the beta and gamma rays of : 323 Rats held at constant body-weight by underfeed- ing for various periods. Changes in the rela- tive weights of the various parts, systems and organs of young albino 9 Rays of radium on protoplasm. The effects of the beta and gamma. : . 323 Reactions and resistance of fishes in their natural environment to salts. The 243 Reactions of animals and plants. The relative efficiency of various parts of the spectrum for the heliotropic 23 Reproduction in the domestic fowl. X. Fur- ther data on somatic and genetic sterility. Studies on the physiology of 45 Reproduction of Hydra. Inheritance in the asexual, 157 Resistance of fishes in their natural environment to salts. The reactions and 243 Resistance of Paramecium caudatum. The ef- fects of certain salts, and of adaptation to high temperatures, on the heat 211 SALTS, and of adaptation to high tempera- tures, on the heat resistance of Paramecium caudatum. The effects of certain 211 Salts. The reactions and resistance of fishes in their natural environment to 243 Selection in the fission rate of Stylonychia pustu- lata. Heritable variations and the results of 451 Selection upon the ‘bar eye’ mutant of Droso- phila. The effect of 515 Senay, C. T., ZELENy, CHARLES and. Varia- ation in head length of spermatozoa in seven additional species of insects 505 Sex in phylloxerans and aphids. The predeter- mination of 285 Spectrum for the heliotropic reactions of animals and plants. The relative efficiency of vari- ous parts of the 23 Spermatozoa in seven additional species of in- sects. Variation in head length of 505 SrarK, Mary B. The occurrence of lethal fac- tors in inbred and wild stocks of Drosophila. 531 Sterility. Studies on the physiology of repro- duction in the domestic fowl. X. Further data on somatic and genetic 45 Srockinec, RutH J. Variation and inheritance of abnormalities occurring after conjugation in Paramecium caudatum 387 Stylonychia pustulata. Heritable variations and the results of selection in the fission rate of 451 "TEMPERATURES, on the heat resistance of Paramecium caudatum. The effects of cer- tain salts, and of adaptation to high 211 (Oe ae for various periods: Changes in the relative weights of the various parts, systems and organs of young albino rats held at constant body-weight by 99 VARIATION. An attempt at a physico- chemical explanation of certain groups of fluctuating 559 Variation and inheritance of abnormalities oc- curring after conjugation in Paramecium caudatum 387 Variation in Drosophila. A linkage 1 Variations and the results of selection in the fis- sion rate of Stylonychia pustulata. Hertel Variation in head length of spermatozoa in seven additional species of insects 505 \ ASTENEYS, Harporrs, Lors, JAcQuEs and. The relative efficiency of various parts of the spectrum for the heliotropic reactions of animals and plants 23 Weights of the various parts, systems and organs of young albino rats held at constant body- weight by underfeeding for various periods. Changes in the relative 99 Wetts, Morris M. The reactions and resistance of fishes in their natural environment ee Wild stocks of Drosophila. The occurrence of lethal factors in inbred and 531 7 ELENY, CHARLES and Marroon, W.E. The effect of selection upon the ‘bar eye’ mu- tant of Drosophila 515 ZELENY, CHARLES, and Senay, C. T. Varia- tion in head length of spermatozoa in seven additional species of insects 505 ry hy iad ane ai’) oe Li H a a Le | i] ive ey IBINDING 2 7. JUN 6 6 Re | Ve eee The Journal of experimental 2. zoology J66 v.19 cop.2 Biological & Medical Serials PLEASE DO NOT REMOVE CARDS OR SLIPS FROM THIS POCKET Pe ee eee ea UNIVERSITY OF TORONTO LIBRARY I, hia zerain oh im: x rs * Gominedebee ete ce bt meted (O0t Sn peep er my na haeted ae a etsentinn Wend Pyhiet mes 4 ae Tg btckhe SAE? i Sogetieg, RSF apr epee septa ene ane Boe batsee Peeeint! ane sty) at oe ~~ oot ont heen pag + 4 ] Vines Be aeaies Sorered sesce ne . 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