si . Re A” ORIN Ye yee NTT SP aR oer ” “ pa - hersedhumthcnoeee cate Bec Pa see erie ee Ta as cee Mt wy a: ay MS i) thy i Uy My f war ih toa Aes ve i Ag sii Kd a y, \ if i Digitized by the Internet Archive in 2009 with funding from University of Toronto http://www.archive.org/details/journalofexperim26broo Ss THE ‘JOURNAL EXPERIMENTAL ZOOLOGY EDITED BY Jacques Lors The Rockefeller Institute Wivuiam B. Caste Harvard University Epmunp B. WILson Epwin G. CoNKLIN Columbia University Princeton University THomas H. MorGan CuarLes B. DAVENPORT cies Columbia University Carnegie Institution Grorce H. PARKER HERBERT S. JENNINGS Hass University Johns Hopkins University RAYMOND PEARL FRANK R. LILLIE Maine Agricultural University of Chicago Experiment Station and Ross G. HARRISON, Yale University Managing Editor Jy yO £7 dw VOLUME 26 1918 : Jofi fig THE WISTAR INSTITUTE OF ANA'TOMY AND BIOLOG*’ PHILADELPHIA, PA. mh ‘ ‘ ue A Cn tou f 4 Til eae ny it in ‘ain CONTENTS No.1. MAY Heten Dean Kina. Studies on inbreeding. I. The effects in inbreeding on the growth and variability in the body weight of the albino rat. Four- tpn SETA TTC) ok UL A te 2 32 EN re eR cc 1 P. W. WHITING AND HELEN nee Kine. Ruby-eyed dilute gray, a third allelomorph in the albino series of the rat.....................-...+..-- 55 M. F. Guyer anp E. A. SmitH. Studies on cytolysins. I. Some prenatal PHEETALOt lene ETT LLD GUIS seen tee fc =): cs a ee ie oo A 65 Witiram A. Kepner AND ARNOLD Ricu. Reactions of the proboscis of Planaria albissima Vejdovsky. ‘Ten figures...................-.-...... 83 S.Hatar. Onseveral effects of feeding small quantities of Sudan III to young bing) Taber ~Lhree: chanigme ys. .8 a doe ser. 5 ope ostns bese 101 CHARLES R. SrocKarRD AND GEORGE N. PApaNnicoLtaovu. Further studies on the modification of the germ-cells in mammals: The effect of alcohol on treated guinea-pigs and their descendants. Nine tablesandnine figures... 119 No.2. .JwEY Oscar Rippie. A demonstration of the origin of two pairs of female identi- cal twins from two ova of high storage metabolism. Thirteentables...... 227 B. W. Kunxeu. The effects of the ductless glands on the development of the TSN OS bee Sond Sa aan OS eA: Comoe Coe e ae eenEe << eae 255 Mario Garcra-Banus. Is the theory of axial gradient in the regeneration of MOM A Onhe Os yA taeigie eek eS... GRR oe re ok 265 ALFRED C. Reprretp. The physiology of the melanophores of the horned toad Phrynosoma. Eight text figures and five plates............. 275 Heten Dean Kina. Studies on inbreeding. II. The effects of Sqectgihne on the fertility and on the constitutional vigor of the albino rat. Two LETS fe Ce Si: Bo Be Ont 1 COE Oe eC ne Se 2.0): Sn rere, ee 335 W. J. Crozier. The amount of pation material ingested by holothurians (Sue HODUSi ees bwOUCHAn ES ee aera ts. oo PRE Sno owes ee 379 No. 3. AUGUST H. H. Newman. Hybrids between Fundulus and mackerel. A study of paternal heredity in heterogenic hybrids. Four figures................ 391 W.C. Auer ann E. R. Stern, Jr. Light reactions and metabolism in May- fly nymphs. I. Reversals of phototaxis and the resistance to potassium cyanide. II. Reversals of phototaxis and carbon dioxide production. ICDC: Sg Sega G loa fe ii auees.e + seve id conn bware 423 IV CONTENTS AtvaLyn E. Woopwarp. Studies on the physiological significance of cer- tain precipitates from the egg secretions of Arbacia and Asterias. Two Chantsrandethmeeni Gunes... .1 ace rice ieee Deoceis bance Soe eh S.O.Masr. Effects of chemicals on reversion in orientation to light in the colonial form, Spondylomorum quaternarium...................2..+0-- A. FRANKLIN SHULL. Relative effectiveness of food, oxygen, and other sub- stances in causing or preventing male-production in Hydatina......... Suinicut Matsumoto. Contribution to the study of epithelium movement. The corneal epithelium of the frog in tissue culture. Nine figures........ AUTHOR'S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE MARCH 2 STUDIES ON INBREEDING I. THE EFFECTS IN INBREEDING ON THE GROWTH AND VARIA- BILITY IN THE BODY WEIGHT OF THE ALBINO RAT HELEN DEAN KING The Wistar Institute of Anatomy and Biology The rapid development of the new science of genetics has - opened up many fertile fields of investigation and it has also re- vived interest in the problem of inbreeding which has been dor- mant for many years. Charles Darwin (’75, ’78) considered the subject of inbreeding so important that not only did he collect all available data regarding it, but he himself carried on a series of inbreeding experiments that extended over a period of eleven years. Darwin’s experiments on plants were followed by those of Crampe (83), of Huth (87), and of Retzima-Bos (’93; ’94) on various species of mammals. The conclusions reached by each of these investigators can well be stated in the words of Darwin (78): “The consequences of close interbreeding carried on for too long a time, are, as is generally believed, loss of size, con- stitutional vigor and fertility, sometimes accompanied by a tendency to malformation.”’ Darwin adds, furthermore: ‘That any evil directly follows from the closest interbreeding has been denied by many persons, but rarely by any practical breeder and never, as far as I know, by one who has largely bred ani- mals which propagate their kind quickly.” On account of the almost universal prejudice against inbreed- ing, or because the results of former work seemed conclusive, the problem of inbreeding was practically ignored by scientists after the publication of Retzima-Bos’ results in 1893, and only within the past decade has it again received any serious consid- eration. The recent experiments of Gentry (’05) on swine, of Castle et al. (06) and of Moenkhaus (11) on Drosophila, and THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 26, NO. 1 MAY, 1918 2 HELEN DEAN KING of various stockbreeders on horses and cattle (Chapeaurouge, 709; Anderson, ’11) have shown conclusively that there is no general physiological law forbidding inbreeding and that the re- sults obtained depend very largely on the character of the stock that is inbred. As Chapeaurouge has stated: Die Erfolge und Miserfolge naichster Inzucht hingen klar und sicher nicht nur von der Gesundheit und Konstitution der Stammel- tern und den dusseren Verhéltnissen ab, in welechem die Tiere gehalten werden; sondern auch vor allen Dingen von der richtigen Auswahl der Tiere zur Weiterzucht, welche nie die allgemeinen Bedingungen des zuchterischen Zweckes gegeniiber den speziellen aus dem Auge lassen darf. Je mehr die Haltung den natiirlichen Verhiltnissen oder den Bediirfnissen der Zuchttiere entspricht um so weniger sind tible Folgen zu befiirchten. There are a number of questions of economic as well as of scientific import which recent work on inbreeding does not answer: Does close inbreeding, if carried on for a long period of time, ever lead to degeneration if only the best animals, from sound stock, are used for breeding? If degeneration does fol- low from this kind of mating, does it affect merely the body size, vigor, and fertility, or does it also influence body form and modify the structure and action of the central nervous system? If no evil results appear, can inbreeding be used to improve a race by combining the best of the dominant characters with any de- sirable recessive ones that may appear? Finally, does inbreeding change the normal sex ratio, as Diising (’84) and others have maintained? Taking advantage of the opportunity which the animal colony of The Wistar Institute of Anatomy and Biology afforded for carrying on an investigation that might answer some of the above questions, a series of inbreeding experi- ments on the albino rat was started in the spring of 1909. As the work, although still in progress, has already extended over a period of eight years, it seems advisable that some of the results obtained should be published, since they lead to rather definite conclusions. The present paper, the first of a series, gives a detailed account of the investigation, with an analysis of the effects of close inbreeding on the growth and variability EFFECTS OF INBREEDING ON BODY WEIGHT 3 in the body weight of rats belonging to fifteen inbred genera- tions; subsequent papers will deal with the effects of inbreeding on fertility on constitutional vigor and on the sex ratio. To avoid needless repetition, a complete bibliography will be given in the final paper. I wish: to express my great obligations to the Director of The Wistar Institute, Dr. M. J. Greenman, for placing at my dis- posal ample facilities for carrying on the investigation and for his unfailing interest in the problem. I am much indebted, also, to Dr. H. H. Donaldson for advice and criticism that have been most helpful when difficult situations have arisen at various stages of the work. 1. MATERIAL AND OUTLINE OF THE EXPERIMENT The albino rat (Mus norvegicus albinus) is a domesticated variety that is well adapted to confinement and is easily cared for. It breeds throughout the year, producing litters of rela- tively large size, and under proper environmental conditions from three to four generations can be obtained in a single year. This combination of characteristics makes the rat very useful as a laboratory mammal, and exceptionally favorable material for a study of the problem of inbreeding. The experiments were started with a litter containing four rats, two males and two females, which was born on the 10th of May, 1909. This litter was selected from a number of new-born litters in the general stock colony merely because it happened to contain the number of individuals required for starting the in- vestigation, and not because of the ancestry, the size, or the vigor of the individuals. One of the females in the litter was called ‘A,’ and her descendants form the ‘A series’ of inbreds; the other female was designated as ‘B,’ and her descendants form the ‘B series’ of inbreds. The plan of the experiment required that females A and B, as well as all of the females subsequently used for breeding, should be mated twice with a brother from the same litter and then twice with an unrelated stock male. Since the mating of 4 HELEN DEAN KING brother and sister from the same litter is the closest form of inbreeding possible in mammals, no other form of mating has ever been used to obtain inbred litters. In every generation all females used for breeding have belonged to inbred litters; none of them have ever been taken from ‘half-inbred’ litters obtained by the mating of inbred females with stock males. In the early part of the experiment the number of breeding animals was, of necessity, small. In every generation after the sixth about twenty females from each series were used for breeding, so that approximately 1000 young were obtained in each generation. All four of the rats used in starting the experiment were killed when they were no longer wanted for breeding purposes. Each rat was weighed, measured, and carefully dissected. When the various records were compared with the norms for the albino * rat (Donaldson, ’15) it was found that all of the rats were under the average body weight for their age, but that they were nor- mal in all other respects as far as could be determined by the usual methods of laboratory procedure. The faet that these individuals were sound, healthy animals and normal in all essential respects is a point on which I wish to place special emphasis in order to forestall the possible criticism that the re- sults obtained in this work were due to the use of an exceptional strain of rats. In the earlier generations the inbred rats exhibited all of the defects which are popularly supposed to appear in any closely inbred stock. Many females in both series were sterile, and those that did breed usually produced only one or two litters which were generally of small size. A considerable proportion of the rats were dwarfed, or stunted in their growth, and many of them developed malformations, particularly deformed teeth. The animals showed, also, a steady decline in vitality in suc- ceeding generations and usually died at a relatively early age. If the experiments had been discontinued at this point the re- sults would have been a confirmation of the conclusion reached by Darwin and by several other investigators, that inbreeding invariably leads to sterility and to physical degeneration. EFFECTS OF INBREEDING ON BODY WEIGHT 5 Fortunately for this work, many rats in the general stock colony, in which there was no inbreeding, exhibited the same characteristics as the rats belonging to the inbred strain. It was evident, therefore, that the unfavorable condition of the animals could not justly be attributed to inbreeding alone. On investigation it was found that all of the rats were suffering from malnutrition due to the character of the food that they received. At the time that these experiments were begun the rat was used as a laboratory mammal in only a few of the larger research institutions in the country, and little was known of the environmental and nutritive conditions best suited to its needs. Following the plan of feeding in general use in other animal colonies, the rats were fed chiefly on bread soaked in milk and on corn; meat and vegetables being given only once each week. Such a diet does not furnish the proportion of food elements that the rat requires if it is to be kept in good physical condition: there is too much starch, too little protein. In the spring of 1911 a radical change was made in the rats’ food. Milk and fresh bread were eliminated from the diet and ‘scrap’ food, consisting of carefully sorted table refuse, was fed once each day; cobeorn being kept in the cages as an extra ration. Such a diet has proved to be a most satisfactory one, and it has been used continually up to the present time, except that dog biscuit has been substituted for cobeorn as extra food supply. This change was made last year following the loss of a consid- erable number of animals through intestinal disturbance caused by the eating of fermented corn. A very marked improvement in the general condition of all of the rats in the colony was noted very soon after the diet was changed. The animals gained in size and in weight, sterility almost disappeared, and the average number of young in the litters was increased. From this time on malformations were no longer common, and not a single instance of deformed teeth has been discovered in the thousands of animals that have been bred in the colony during the past five years. Simply by a change in the food characteristics said to typify the ‘dire effects of inbreeding’ were eliminated, and up to the present time they 6 HELEN DEAN KING have not reappeared, although the rats have been carried through twenty-eight generations of brother and sister matings. In the inbred colony, up to the sixth generation, very little selection of breeding animals was possible; any females that would breed at all were used to continue the series. The change in food was made at the time that the animals of the fourth inbred generation were reaching maturity. Inthe course of the two following generations the effects of malnutrition gradually disappeared, and in the sixth generation most of the rats were of normal size and relatively large litters were being produced. After this time large and vigorous animals were available for breeding purposes, and it became possible to make a careful selection of the breeding stock. From the seventh generation on the selection of the individ- uals which were to serve as progenitors of the succeeding gener- ation was always made among the newborn young, as the sexes can readily be distinguished at this time (Jackson, 712). In the A series of inbreds, which is called the ‘male line,’ all litters containing an excess of female young were always discarded; in the B series, the ‘female line,’ litters with an excess of male young were never reared. Unless the individuals in the litter were of normal size and vigorous at birth they were killed at once. The young which were retained remained with their mother until they were one month old, when they were again carefully examined, and if they did not come up to the norms for stock animals of like age they were discarded. If the young rats ful- filled all requirements as to body weight and vigor they were returned to the cage to be reared as possible breeding stock. This rigid selection left in each generation, as a rule, at least three times the number of animals that were required for breed- ing. When the rats become sexually mature, at about three months of age, they were again inspected, and any that were be- low normal in any way were rejected. Generally only one female of a litter, the first to breed, was taken to continue the line. If, however, the individuals were unusually large and vigorous, two, very rarely three, breeding females were taken from the same litter. EFFECTS OF INBREEDING ON BODY WEIGHT 7 Since the change in the food in 1911 and the removal of the colony to new quarters in 1913, the environmental conditions under which the rats were reared have been as uniform as it was possible to make them. All inbred rats, and also the stock animals used for controls, have been subjected to the same con- ditions of light, of temperature, and of nutrition, and they have been cared for in a similar way. Any differences between the two inbred series, or between inbred and stock animals must, therefore, be ascribed to causes inherent in the individuals; they cannot be attributed to the varying action of environment or nutrition. 2. THE GROWTH IN BODY WEIGHT OF INBRED RATS In view of the results that earlier investigators (Crampe, Ritzema-Bos) obtained in their inbreeding experiments with rats, little attention was paid to the fact that the body weights of the animals in the, earlier inbred generations were consider- ably less than the norms for stock albino rats of like age. When the individuals of the sixth inbred generation became mature, it was noted that many of them were much larger than stock animals of the same age. This fact was so at variance with the generally accepted belief regarding the effects of close inbreed- ing on body size that it seemed desirable to make a study of the weight increase with age of individuals in the later generations of the two inbred series. From the seventh generation on from three to five litters of each inbred series were weighed, first when the animals were thirteen days old, again when they were weaned at thirty days of age, and thereafter at intervals of one month until they were fifteen months old. At the thirteen- and _ thirty-day periods animals of the same sex were weighed together and the average body weight for the group recorded, as at these ages individual differences in body weight are, as a rule, too small to make separate weighings necessary; at all other ages individual records were taken. The litters that were used for a study of growth in body weight were all selected at birth on the same basis as the litters 8 HELEN DEAN KING that were to be reared for breeding purposes. There was no culling of the less desirable individuals, however, and all mem- bers of every litter were reared and weighed at the ages noted. When the animals became mature the largest and most vigorous pair in each litter was usually used for breeding. As the growth of very young rats depends largely on the amount of nourishment that they receive from their mother, litters of medium size, containing from five to eight young, were, as a rule, those selected for weighing. Such litters, more- over, represent the general run of individuals in a colony more fairly than do very large or very small litters in which animals with extreme body weights are often found. It was not always possible to weigh adult animals at exactly the ages designated in the various tables, but the weighing of a litter was omitted if it could not be taken within one week of the time specified. In weighing experiments of this kind there is always an un- avoidable error due to the presence of a greater or a less amount of undigested food in the alimentary tract. To obviate this source of error as far as possible the rats were weighed in the morning before they had received their daily food ration. Animals that were obviously ill and females known to be preg- nant were never weighed, while the weight of suckling mothers was not recorded if it was below the previous record. In the rat pregnancy cannot be detected with certainty until about the thirteenth day, so undoubtedly many gravid females were weighed unknowingly during the course of this investigation. The increase in the body weight of a female as the result of pregnancy cannot be very great up to the thirteenth day, how- ever, since Stotsenburg (’15) has shown that the weight of a fetus at this time is only 0.04 grams. Hrrors in the records due to the inclusion of the weights of pregnant females were doubtless balanced by the weights for animals that were in early stages of pneumonia when there was no external evidence of the disease. The present paper gives data showing the weight increase with age in 333 males and in 306 females belonging to the first fifteen generations of the inbred group. Altogether these gen- erations comprised a total of 1601 litters, containing 11,657 EFFECTS OF INBREEDING ON BODY WEIGHT ) individuals, of which the majority were the progeny of brother and sister matings, the others were the offspring of inbred fe- males and stock males. The number of animals for which weight records were taken is, it is hoped, large enough to be fairly representative of the inbred colony as a whole and to give results that have some statistical value. No animals belonging to the first six inbred generations were weighed at regular intervals, but fortunately a series of weight records is available that will give some idea of the size of these rats after they became adult. In order to ascertain the effects of close inbreeding on the weight of the central nervous system, one of my colleagues, Dr. 8. Hatai, made careful autopsies of the four rats used in starting the experiment and he also exam- ined a large number of their descendants. From the record cards for these animals I was able to obtain the body weights of a considerable number of rats belonging to the first six gen- erations of the two inbred series. The body weight data. for only the best-of the animals reared during this period were used in the present case; records for all individuals that were noted as having deformed teeth or other malformations were excluded, as well as the records for those animals that were ob- viously runts. Table 1 shows the body weight records for 92 males and for 73 females belonging in the first six generations of the A series of inbreds: table 2 gives similar data for 85 males and for 64 females of the B series. Only one record for each individual was obtained, i.e., the body weight at the time that the animal was killed. The ‘mean age in days,’ as given in the first column of table 1 and of table 2, shows the median points in thirty periods that correspond to the ages at which the rats of the later inbred generations were weighed. For example, under the mean age ‘151 days’ are grouped the body weights of all of the animals that were killed when they were from 136 to 165 days of age. As no records were taken of animals that were less than 105 days old the first age group given is that for 120 days. TABLE 1 Showing the increase in the weight of the body with age for rats belonging in the first six generations of the A series of inbreds MALES FEMALES a GT Body weight in grams Namnbes Body weight in grams ie prea S (oT of indi- of indi- Average | Highest | Lowest | VidU@I8 | Average | Highest | Lowest viduals days 120 200.1 252 134 11 142.8 193 106 13 151 188.2 267 133 24 127.5 165 103 15 182 218.2 317 135 17 132.2 199 105 5 212 183.6 227 138 5 123.7 153 102 8 243 239.8 | 293 iSound C71 eoe\) TSN 77 2 273 225.0 292 155 11 146.4 218 112 5 304 246.2 273 203 5 137.6 143 128 3 334 274.0 By 225 3 189.0 225 162 5 365 310 ie 164.8 215 143 5 395 273.0 294 269 3 UAT Ah 191 151 6 425 166 Es 455 164 8 180 152 5 92 73 * Record not used in constructing graph. TABLE 2 Showing the increase in the weight of the body with age for rats belonging in the first six generations of the B series of inbreds MEAN AGE days 120 151 182 212 243 273 304 334 365 395 425 455 MALES FEMALES Body weight in grams Body weight in grams ING ber of indi- Average | Highest Lowest viduals Average 205.8 266 142 24 149.7 210.6 257 141 16 149.4 241.6 345 146 15 136.0 239.7 315 157 12 119.2 270.5 304 238 4 169.7 215.6 241 185 6 161.0 233.07 287 174 + GHZ 249 i 191.6 223 .0 257 202 3 163.6 167.8 V2.2 85 * Record not used in constructing graph. 10 170 211 185 173 189 193 189 213 185 193 213 Highest | Lowest 136 113 109 100 149 131 138 161 144 139 145 Number of indi- viduals = ANOonrhE ROE DE nNS lor) rs EFFECTS OF INBREEDING ON BODY WEIGHT ia A comparison of the corresponding data for the individuals of the two series of inbreds is best made through the graphs in fig- ure 1 constructed from the data for the average body weights at different ages as given in table 1 and in table 2. In figure 1 the graphs for the body growth of the males are considerably higher than those for the females, since the male rat, after reaching maturity, is normally a much heavier animal than the female of like age. Considering that only one record . EEE Growth in body weight. Albino Rat. (1 320 Hee eeeEee tt ECE HH Seauea sueeeeee! EE 300} See CdEnoEeR Hee ri T To cH 280f11 Body weight in grams PERE EC EEE EEE Ere i ro TEEEEEEEe er Se ee 260 FLEE EEE EE EEE EEE EEE EERE ert oH EEE a aes Be HH PA 240 + t cor p | — t cot eau cooe ~~ t 's' t UY t t rook EEE Her ey aan t sort waa_|Series BOT { rr EEE Senneaees Eeeeet rH | tH HH +H 4 t iH 200|=+- tH FON } Females i 180) a 4 - Series B 160 a 7 t } Series A 140 120 eo t 100) 80) t 60) 4 2! Age in days 0 20 40 60 80 100 120 140 160 1 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 Fig. 1 Graphs showing the increase in the weight of the body with age for males and females belonging in the first six generations of the two series of inbred rats (data in table 1 and in table 2). was taken for each animal the corresponding graphs for the two series, especially those for the females, run remarkably close together. It is evident, therefore, that there was no significant difference between the two series as regards the growth of the individuals during the first six generations of inbreeding. Table 3 gives the body weight data for all of the individuals of the first six inbred eenerations for which records were taken (a combination of the data in table 1 and in table 2). 1 HELEN DEAN KING TABLE 3 Showing the increase in the weight of the body with age for rats belonging in the first six generations of the inbred series. A combination of the data in table 1 and in table 2 MALES FEMALES MEAN AGE Body weight in grams INGIIGOR Body weight in grams Arcee or = of indi- of indi- Average |} Highest Lowest viduals Average | Highest | Lowest viduals days = 120 204.0 266 134 35 144.4 193 106 17 151 197.2 267 133 40 136.6 211 103 26 182 229.1 a 135 32 139.1 199 105 10 212 218.3 138 18 122.2 173 100 12 243 248.0 304 182 15 Zed 189 149 6 273 2217 292 155 1E7/ 152.8 218 112 9 304 240.6 287 174 9 156.9 189 128 15 334 267.7 312 225 4 190.4 225 161 11 365 244.7 310 202 4 164.2 215 143 10 395 PHN) 294 269 3 172.9 193 139 11 425 166 ilies 455 168.1 2138 145 9 aT 137 * Record not used in constructing graph. A comparison of the data in table 3 with body weight data for a series of stock albino rats reared as controls for the inbred series after the change to ‘scrap’ diet had been made (table 13) will show to what extent malnutrition decreased the body size of the individuals in the first six inbred generations. Graphi- cally this result is shown in figure 11 and in figure 12 (compare graph D with graph A and graph C). Body weight data were taken, at the intervals stated, for 99 males and for 76 females belonging in the seventh to the fifteenth generation of the A series of inbreds. The average body weights of the males at different age periods are shown, by generations, in table 4: corresponding data for the females are given in table 5. Data for 57 males and for 93 females belonging in the seventh to the fifteenth generations of the B series of inbreds are shown according to generations in table 6 and in table 7. TABLE 4 Showing, by generations, the average body weight at different ages of 99 males belong- ing in the seventh to the fifteenth generations of the A series of inbred rats GENERATIONS AGE 7 8 9 10 11 12 13 14 15 days grams | grams | grams | grams | grams | grams | grams | grams | grams 13 18 18 18 18 16 19 19 18 18 30 43 45 46 4] 44 44 49 47 42 60 150 WE 134 129 102 127 134 123 123 90 201 151 199 214 177 184 186 193 189 120 242 196 249 264 227 229 244 233 204 151 274 252 287 289 257 249 Die 259 235 182 30621 6286 | S0Sslsta.| 279.) 273: (291 | 27s: | 206 212 STA Sls | ose 2090)| 288) 1 2934) Sst 1) 22837206 243 329 341 342 318 302 295 317 293 286 273 355 | 352] 364] 305| 321) 312| 327| 304) 300 304 357 345 334 310 314 319 333 302 303 334 358 | 368i aokoae 305 |s 327.) 316 1 339))\ 307 | S307 365 S74) 377 399 |, 300) 336) 324.) 353%) 37> 3l5 395 375 | 370} 386] 307| 360| 318] 3438| 318) 312 425 412 | 384] 404| 307) 354] 334] 355 | 330) 321 455 424 | 383] 403 | 324]! 343 | 339 | 344] 322); 320 Number of rats welshedeeee an .- 2= 6 6 9 14 12 10 15 15 12 TABLE 5 Showing, by generations, the average body weight at different ages of 76 females belonging in the seventh to the fifteenth generations of the A series of inbred rats GENERATIONS AGE if 8 9 10 ily 12 13 14 15 days grams | grams | grams | grams | grams | grams | grams | grams | grams 13 16 15 16 15 15 18 17 17 18 30 40 a7, 42 aa AL 41 45 45 40 60 126 73 AG A107 90 | 102] 110| 108] 105 90 160 | 117| 156| 184| 139| 145] 157] 162] 144 120 172) | ast Stl k4s| AST | SiGe 727) tah |, 163 151 190) 163°) 205 | 189) 182 187 | 192) 485 | 188 182 190 | 182| 217| 208| 189] 195 | 196.| 204] 203 212 17 IOSA Oke) 193 | a844|4)218,4 215°) 209)! 211 243 189 | 200| 223| 202) 200| 203| 216] 214] 218 273 187 | 219) 229) 223) 192) 226), 218) 217 | 222 304 921 | 221 | 236 209] 239] 221 | 222| 222 334 B17 || O20 242) (222 | 244) 218 | 223 || 227 365 952 | 256| 256} 223| 238| 220| 226| 226 395 232 253 | 225| 253 | 227| 228| 224 425 252 244 | 260*| 268*| 230] 226] 219 455 241 | 257 | 257*| 243 | 224| 219] 217 Number of rats : weighed.......... 4 9 7 8 10 8 9 10 11 * One record only. 13 TABLE 6 Showing, by generations, the average body weight at different ages of 57 males be- longing in the seventh to the fifteenth generations of the B series of inbred rats AGE days 1 3 30 60 90 120 15 1 182 212 24 3 273 304 39 4 365 39 42 45 Number weighed 5 5) = tS) of rats * One record only. GENERATIONS if 8 9 10 11 12 13 14 15 grams | grams | grams | grams | grams | grams | grams | grams grams 21 21 20 18 19 19 19 20 20 49 49 53 49 40 47 50 52 49 170 | 158| 140] 172] 149| 140] 144| 132| 145 944.| 197 | 211 | 217 | 220) 212} 193) 210) 201 277 | 280| 253] 265] 262] 240| 225) 255] 233 322 | 293 | 288} 291] 300| 264] 274; 279| 267 344 | 328| 340] 334] 330] .278| 283] 300] 288 376 | 331 | 360] 340] 353} 290] 297] 305) 303 372 | 348| 367 | 336] 368] 286 | 303| 302] 308 454*| 356 | 371*| 316] 367| 306) 309) 311) 311 477*| 361 | 380*| 335] 408| 313 | 313) 322] 311 BGA || ated 381 | 337) 303*) 330) 321 364 | 345*| 333] 373 | 328 | 326%] 362 | 336 369 365*| 365 | 336%) 334*| 351 | 339* 365* 353 357*| 339* 347* 343*| 330* 3 Uf 3 5 9 8 6 a 9 TABLE 7 Showing, by generations, the average body weight at different ages of 93 females belonging in the seventh to the fifteenth generations of the B series of inbred rats —= = | GENERATIONS \ AGE if 8 9 10 11 12 13 14 15 days grams | grams | grams | grams | grams | grams | grams | grams | grams 13 18 18 it74 17 18 |, “is 18 18 18 30 45 40 50 4g} 39] 39 46 48 45 60 135 | 119] 122] 125] 113] 120| 104] 104] 107 90 187 | 168) | 168 | 170 | 5465. | 1495|) 9474) a6 abt 120 187 | “48 | 180 | 174.) °182.| §167)) 169° | 183) 172 151 204 | 200] 200} 199| 191 | 186] 195} 196| 191 182 213 | 196| 205 | 208| 200] 195| 196| 214] -202 212 216 | 213] 199| 217| 202| 204] 204] 210) 206 243 908 | 216 | Site), 217 | $225 1 212s) 2144) 216 ||, 204 273 243 | 196*| 218 | 212*) 236 | 208] 220] 219| 214 304 246 | 201*| 217| 239| 243| 207| 225| 223] 217 334 935 | 204*| 239 Daly |) aie 243 | e237 || ©2024 365 249 | 210*| 242] 262%] 249] 215] 238| 241 | 229 395 301* 941 | 275%] 241 | 212.) 8B26'| -239.| 229 425 323* 261*| 238 | 207 239 | 234 455 317* 280*| 244 231 | 235 Number of rats 5 weighed.......... 7 ai 5 11 12 10 9 12 16 * One record only. 14 EFFECTS OF INBREEDING ON BODY WEIGHT . py Tables 4 to 7 are inserted chiefly for reference, although they bring out two important facts more clearly, perhaps, than do any of the other tables. The data, as given in.these tables, show that rats belonging to the earlier generations of the inbred series did not live as long, as a rule, as did the individuals in the later gen- erations. This was particularly noticeable in the individuals of the Bseries. Up to the twelfth generation only three rats in the B series (two females and one male) lived to the age of 455 days; in subsequent generations many individuals lived for the entire weighing period of fifteen months, and some of them were kept until they were nearly two years old. One who be- lieves with Crampe and Ritzema-Bos that continued inbreeding necessarily lessens vitality and so shortens the life of the indi- vidual meets here with the seemingly paradoxical fact that the animals that belonged to the later inbred generations outlived those that belonged to the earlier generations. In this experiment the use of only the most vigorous animals for breeding purposes has seemingly overcome any tendency that inbreeding might have to shorten the life of the individuals. The second point of interest brought out by tables 4 to 7 is that in each generation of the two inbred series the average body weight of the males exceeded that of the females at every age for which records were taken. At birth the male albino rat is slightly heavier than the female, whether the animals belong to a stock or to an inbred strain (King, ’15b). Data for the growth in body weight of the albino rat, as recorded by Donaldson (06), show that as early as the seventh day after birth the growth of the female is more vigorous than that of the male, and that the female is, as a rule, a relatively heavier animal than the male up to about fifty-five days of age. Ferry’s (13) growth data for the albino rat (Donaldson, 715; table 65) confirm Donaldson’s findings. In Jackson’s (’13) data for the albino rat, ‘‘the excess of average weight was invariably in favor of the male at birth, and also in the majority of cases at all succeeding ages; while the records obtained by Hoskins (16) show that the albino female is a heavier animal than the male only at the age of about six weeks. In a series of stock albino 16 HELEN DEAN KING rats reared as controls for the inbred series (King, ’15 a) the ma- jority of the males exceeded the females in body weight at each weighing period. As no records were taken when the animals were just six weeks old, the relative size of the two sexes at this age was not determined. At thirteen days of age, as tables 4 to 7 show, the average body’ weight of the inbred males was only one gram more than that of the females. Such a slight difference as this would be negligible, considering the possible error in the weighings due to the varying amount of food in the alimentary tract, save for the fact that it is found in every generation group. In some litters the females, were, on the average, heavier than the males at thirteen and also at thirty days of age, and in a few instances the weight of the largest female surpassed that of the smallest male even when the animals were sixty days old. Although the albino female, whether stock or inbred, has a relatively smaller birth weight than the male, she soon comes to have a body weight that is very nearly equal to that of the males in all cases, and often exceeds it. Even though the absolute body weights are less at any given age, therefore, the female grows more vigorously than the male during the first few weeks of postnatal life. An early acceleration in the growth of the fe- male also occurs in the guinea-pig (Minot, ’91; Castle, ’16), and it finds a parallel in man, as Donaldson (’12) has pointed out, since during a certain phase of development, when children are from thirteen to sixteen years old, the average weight of girls is greater than that of boys; while at all other ages boys, as a rule, are the heavier. é The relative growth in body weight of males and of females in corresponding generations of the two inbred series, or in suc- ceeding generations of the same series, can be determined by referring to the data in tables 4to 7. Fora comparative study of the body growth of the individuals in the two series it seemed advisable to combine the data for three succeeding generations. Data for the A series of inbreds are given in table 8. The growth graphs shown in figure 2 were constructed from the data for the males of the A series, as given in table 1 and EFFECTS OF INBREEDING ON BODY WEIGHT 1 TABLE 8 Showing the average body weights at different ages of inbred rats of the A series separated into three groups according to the generation to which the individuals belonged MALES FEMALES ee Generations | Generations | Generations Generations | Generations | Generations 7-9 10-12 13-15 7-9 10-12 13-15 days grams grams grams grams grams grams 13 iksjoal 17.5 18.4 15.5 16.0 17.5 30 49.4 43.4 43.6 42.5 40.0 43.2 60 122.3 118.3 127.0 98.9 98.5 107.6 90 186.0 193.2 188.9 137.5 157.9 153.6 120 230.6 242.5 _ 228.1 160.5 178.5 168.3 151 269.5 267 .9 256.9 181.5 191.0 186.2 182 304.3 290.4 276.7 192.2 195.3 201.9 212 320.2 293.2 287 .1 189.8 196.2 211.2 243 337.1 305.9 298.3 205.5 201.7 216.0 273 357.4 312.5 307 .7 NAR 215-1 219.4 304 349.5 314.5 3133.0 Dea 219.4 DOA 334 369.0 315.4 318.4 Doe 234.9 223.9 365 379.2 318.1 327-1 252.2 235.2 224.7 395 375.6 323.6 323.6 232.0 239.5 226.4 425 399.4 327.0 332.0 252.5 254.0 223.8 455 403.5 336.0 330.2 241 .0* 251.6 219,.0 * One record only. in table 8. In this, as in some of the other figures, the space between the graphs was slightly widened, where properly the lines would run very close together or overlap, in order that the course of each graph might be clearly followed. | Figure 2 shows that the body growth of the males in the three generation groups of the A series of inbreds progressed at about the same average rate until the animals were 150 days of age, as is indicated by the position of graphs A to C. At this point there was a marked acceleration in the growth of the males belonging to the group comprising the seventh to the ninth generation (graph A) which continued until the end of the weigh- ing period. Graph D, representing the growth of the males of the A series during the first six generations, begins at the 120- day period, as no younger rats belonging to these generations were weighed. It seems almost incredible that this graph can . THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 26, No. 1 18 HELEN DEAN KING represent the adult body size of the progenitors of the animals whose growth is indicated by graph A, since rarely do we find a group of mammals having in the adult state a body size so much ereater than that of its immediate ancestors. Growth in body weight. Albino Ratt Ht EAH SEeeeaeeaee! 400) SeriesA Males + r+ : t ECEEEEEE An 380} + + as Ltt | SHEE Eee Teri ti 360} Hat 340 Body weight in grams + 320 f ai x Cc 300) : au 280 i aa Cott D 260 BaD” 2 t 240 hae ; + i 220 (freae Poor H 200 90m t 180) i 160FE sau: 140 ri (Aas i 120) .- ia t t f T aoee Cf rt rt 5 | t =e t eee + 100 t ; = + t Ht HH iereertestaet i 80) a EEE EEE ee Crt ro t f { BRE eee eee eee ee eee a i | T 1g 60 Pret a ooh Torr Ht gy tt seeneteeeiet FECeEEEE SHEE ey i HEE EE eeeenccee E i URES ooo HEH BEER EEEEEEEEEEEEEEEEEEECEEEE EEE EH EEE ah — | am ro i } a re Hoe “TETt Age in days | t Lt +4 | 1H CeCe T ae t I it Corer BI tH Hy tt tee 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280:3C0 320 340 360 380 400 420 440 460 480 Fig. 2. Graphs showing the increase in the weight of the body with age for males belonging to various generation groups of the A series of inbreds (data in table 1 and in table 8). A, graph for males of the seventh to the ninth genera- tions inclusive; B, graph for the males of the tenth to the twelfth generation in- elusive; C, graph for the males of the thirteenth to the fifteenth generations inclusive; D, graph for the males of the first six inbred generations. Graphs showing the growth in body weight of females belong- ing to the three generation groups of the A series are shown in figure 3 (data in table 1 and in table 8). The females of the first six generations of the A series were very much smaller than the females of the later generations at every age for which records were taken, as the position of graph D in figure 3 clearly shows. There was practically no difference in the rate or in the extent of the body growth in the groups EFFECTS OF INBREEDING ON BODY WEIGHT 19 comprising the seventh to the fifteenth generations, as graphs A, B and C cross and recross each other at various points and run as close together as would any set of graphs constructed from the data for different series of individuals. Data for the growth in body weight of the individuals of the B series, arranged in groups of three generations each, are given in table 9. 320—eH Fr{Growth in. body weight Albino Rat.--Pr rrr r tt T aI t Ht Soneoou0 soorat jy 280 Het | 1 J ret sence PEE sane | DoT it Trt cesatt ry i Tet 260|-| Body weight in grams paraiaisiciale { . Cer ' a8 - - 40H- f sy) 3 EECEEEEE Ee ag SEEEDEEEE 201; i i t ef Age in days ‘ ~ + tte 1 ste 1 jeapeesess' tase 0 20 40 60 80' 100 120 140 160 180 200 220 240 260 280:300 320 340 360 380 400 420 440 460 480 Fig. 3 Graphs showing the increase in the weight of the body with age for females belonging to various generation groups of the A series of inbreds (data in table 1 and in table 8: lettering as in figure 2). From the average body weights at different ages of the males of the B series of inbreds, as given in table 2 and in table 9, the graphs in figure 4 have been constructed. In the B series, from the first weighing until the last, there was a marked difference in the body weights of the males in the four generation groups, since all of the graphs in figure 4 are distinctly separated except at one point (865-day period). Graph A runs higher than any of the other graphs from the be- ginning until the end of its course, thus indicating that in this series of inbreds also the males of the seventh to the ninth gener- ation were heavier animals than the males in the later genera- tion groups. Males in the first six generations of the B series TABLE 9 Showing the average body weights at different ages of inbred. rats of the B series sepa- rated into three groups according to the generation to which the individuals belonged MALES FEMALES rie Generations | Generations | Generations | Generations | Generations | Generations 7-9 10-12 13-15 7-9 10-12 13-15 days grams grams grams grams grams grams 13 21.0 19.1 19.8 17.9 ilee 18.2 30 50.0 47.2 50.0 43.8 44.8 46.2 60 156.9 151.4 140.7 120.3 119.6 105.4 90 218.9 213.4 201.6 W258 163.3 153.6 120 273.6 259.7 237.6 182.8 175.9 174.6 151 299.0 284.3 271.6 201.3 190.4 193.5 182 335.3 309.6 289.7 205.3 200.6 205.4 212 347.5 323.4 301.7 210.4 209.5 206.7 243 358.3 ovale: 304.9 211.6 PAW c O 210.5 273 Ola ee 330.4 310.2 222.2 PAUP «1 PALE all 304 380.5 353.2 316.5 225.6 222.4 2A S 334 363.1 359.0 323.1 230.6 228.6 230.8 365 360.4 342.4 344.2 238.4 229.3 234.3 395 369.0 BID of 342.2 261.3 236.4 232.6 425 365 .0* 353 .0 348 .0 323,05 230.4 236.4 455 347 .0* 331.5 Slizf (Oe 256.3 233.2 * One record only. TABLE 10 Showing the average body weights at different ages of inbred rats of the combined series (A, B) separated into three groups according to the generation to which the individuals belonged MALES FEMALES a Generations Generations Generations | Generations Generetions Genemtions days grams grams grams grams grams grams 13 19.2 18.1 18.9 16.8 16.9 17.8 30 49.6 44.9 47.6 43.0 42.8 45.1 60 135:5 131.8 DS PAS 7 10) % 106.4 90 195.6 200.9 193.1 154.1 160.7 153.6 120 248 .6 250.5 231.4 172.1 NG 6% 171.8 151 281.1 274.3 262.2 191.2 190.7 190.2 182 S56 298 .0 279.5 199.2 198.5 203.5 212 328.7 305.7 291.6 203.4 202.2 208.3 243 343.9 311.4 300.0 208 .6 209.4 213.3 273 361.6 316.1 308.5 217.4 215.8 218.2 304 359.8 322.2 314.2 222.8 220.8 2215 334 367.3 324.1 319.3 228.7 232.8 226.6 365 374.0 325.5 329.2 243.7 232.4 228 .6 395 374.4 331.4 325.3 246.6 238.3 229.0 425 396.2 330.5 333.4 276.0 240.8 229.6 455 403.5 336.7 333.0 279.0 253Ra 224.6 EFFECTS OF INBREEDING ON BODY WEIGHT 21 were quite as inferior to their descendants in body size as were the males in the corresponding generations of the A series (graph D). The growth in body weight of various groups of females be- longing in the B series is shown graphically in figure 5 (data in table 2 and in table 9). Graphs A to C in figure 5 were drawn so that the lines are distinct. These graphs should lie very close together and -Growth in body weight. Albino’ Rat. cay Coe = Series B Males QUIT tT Eeet - : T TT aestetil rE neaaaee! 380 SEE EEE EEE TEECE EE EEE EEE CE tia 1 Ht oe gag beenGGGbeaaiesendge- \.jaun Agags rt EEE gp" .dieeeess ‘SGSSSSEREEE ne 7 360) + iH + i! Jz Eo EEE Sea ae gaa : oe waseasr ett. At Bot 340 ia 2 aI rite a oss fara Body weight in grams Or Ot Poe + HAC ia cance ) seuaaar ousdeaa anes caudcensssssscssssaeeeeeesese 4 ia pee eat 300 eH : 280 1 4 ru 20h Peet Age in days © 20.40 60 60 100 120 140 160 180.200 220 240 260 280 300 320 340 360 380 400 420 440 460 460 Fig. 4 Graphs showing the increase in the weight of the body with age for males belonging to various generation groups of the B series of inbreds (data in table 2 and in table 9: lettering as in figure 2). overlap in many places, since the data in table 9 shows that the actual differences between the corresponding body weights of the various groups are very small. The position of graph D indicates that adult females of the first six generations were very much smaller animals than their descendants of like age, as was the case in the A series also. The data given in table 8 and in table 9 have been combined in table 10. py HELEN DEAN KING Figure 6 shows graphs for the weight increase with age in all of the inbred males for which weight records were taken (data 1 in table 3 and in table 10). As the position of the graphs in figure 6 show, males of the seventh to the ninth inbred generations (graph A) were animals of unusually large size and they were considerably heavier, after reaching maturity, than their own progeny of like age. At the 365 day period the space between graph A and graph B in- dicates a difference of about 50 grams in the average body +t} + +t t + t+ 320 | Coe Growth in body. weight. Albino Rat. Sad SSS00000000 88 EEE ey x8 Hiiine T Ht H |Series B _Females(} aI zeeeeet Pere seecse sanent EEEEEEEEEEEHt : A EEE EERE EEE EERE EEEEEEE 280|11 Body weight in grams {1 ce ECE EEE EEE 260 ame | | a 1 | ZA Peet at rT aa eoaee Py SaESSReRee JaBBERS Hi ye <5? c 220-4 HEHE eae sana SEgeaeSeneuag HEE 4-4 +t 2: T t ofsetteede EEE 180 igeereere! D: 160 Eee SG5BE WOR ay Ht + : | 120) tH i s i t f t 100 eet on cd Sooseerd J jane T 7 , sal t a’ >F ++ dese 4 / A ia t = y /) famaweboos 40-7} I hit { rrr BHaae! it a 20 fe } aeeeet : coo rH ORR eRaeas ! EOE i Age in days | T i cetet a EERE EEE EEE BEEEEEEEEEEEEEEEEEEEE Soe e0eeoueeseseoaceoeae! Soeeeeeoes t 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 Fig. 5 Graphs showing the increase in the weight of the body with age for females belonging to various generation groups of the B series of inbreds (data in table 2 and in table 9: lettering as in figure 2). weights of the two groups of animals. From the tenth genera- tion on the course of growth in body weight was practically the same in all inbred males, as is shown by the fact that graph B and graph C run very close together throughout their entire length. Figure 7 shows graphs for the body weight increase in the series of inbred females (data in table 3 and in table 10). The relative position of the graphs in figure 7 show that, with the exception of the animals in the first six inbred genera- PTA Growth in body weight. Albino Rat | sestseses SSSGE509' 4855>~ ag 2 SSS88585 ee ae SUSERS" AEF SE0~ \_ S08 S885 SSeS ee eee Sees eee eee HA gee 7 208" wan eaeEB! TI SSRESRRSSEeE8 SESREESERSEREEEEERED 22084 SERRE RSEREESSREREED SEREREESHEDSEEEEESEy SRSSSEESESESEEEESY Z | Y, ’ ~ Snnpeen! _sSSUSESSSEECEEEETSEESESEEESESTESSESES Dor 40 60 80 100 120 140 160 180 200 22 Fig. 6 Graphs showing the increase in the weight of the body with age for males belonging to various generation groups of the two series combined (A, B). Data in table 3 and in table 10: lettering as in figure 2. SS EGrowth in body weight Albino Rat - seefsfateaestati Beuusee0ecnSSGEECeeSsscancess i ¢ UE SESUESSUR SSSR SSS GR GREE SSG ESSEeSes 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 4 Fig. 7 Graphs showing the increase in the weight of the body with age for females belonging to various generation groups of the two series combined (A, B). Data in table 3 and in table 10: lettering as in figure 2. 23 24 HELEN DEAN KING tions (graph D), the body increase with age was about the same in all the various generation groups of inbred females up to the time that the animals were one year old. After this age the females in the group comprising the seventh to the ninth genera- tions were somewhat heavier than the other females, but their excess In body weight was very much less than that in the cor- responding groups of males (fig. 6). On referring to the data in table 4 to table 7 it is found that in both series of inbreds the average body weights of the animals in the seventh, eighth and ninth generations were greater than those of animals in the tenth and subsequent generations. In the seventh generation, particularly, females as well as males were exceptionally heavy animals at all ages for which records _ were taken. The largest males yet obtained belonged in the seventh generation of the A series of inbreds; these rats weighed at their maximum 460, 482, and 511 grams respectively. The largest female in the series was a member of the seventh genera- tion of the B series of inbreds, and she weighed 323 grams when she was 425 days old. The probable cause for the unusual weight of the animals in the seventh to the ninth inbred genera- tions will be considered later. Data showing the range in variation and the averages for the body weights at different ages of the weighed individuals in the A series of inbreds (seventh to fifteenth generations only) are given in table 11. Similar data for the rats of the B series are shown in table 12. The graphs in figure 8 were drawn to show the relative growth in body weight of the males belonging to the two inbred series (data in table 11 and in table 12). The males of the B series of inbreds had a heavier average body weight than the males of the A series up to the last weigh- ing period (455 days), as the position of the graphs in figure 8 show. The crossing of the graphs at the end has no significance, since the largest males of the B series died before the final weighing. Graphs showing the growth in body weight of the females in the two inbred series are shown in figure 9: the data for these graphs are given in table 11 and in table 12. TABLE 11 Showing the increase in the weight of the body with age for 99 male and for 76 female rats belonging in the seventh to the fifteenth generations of the A series of inbreds MALES ; . FEMALES AGE Body weight in grams Number Body weight in grams Number individ- individ- Average | Highest | Lowest uals Average | Highest Lowest uals days . 13 17.9 21 15 99 16.1 22 14 76 30 45.6 82 35 99 41.8 67 33 76 60 123.0 176 72 95 102.3 159’. 71 74 90 189.8 248 120 99 150.3 193 93 63 120 233.9 305 174 94 ‘170.0 212 133 63 151 263.4. 360 204 94 187.6 224 157 65 182 287 .4 367 216 91 197.5 236 167 62 212 296.8 420 238 85 202.8 248 158 59 243 309.5 419 234 84 209.9 245 171 56 273 322.6 435 265 74 217.0 251 167 53 304 BRYA lee 415 267 70 221.0 257 181 42 334 328.9 415 268 63 227.2 268 196 37 365 336.2 428 289 59 229.8 282 198 35 395 335.9 433 283 51 230.3 274 206 31 425 345.9 448 281 44 2321.2 268 206 PA 455 361.3 473 306 25 228.3 257 182 20 TABLE 12 Showing the increase in the weight of the body with age for 57 male and for 93 female rats belonging in the seventh to the fifteenth generations of the B series of inbreds MALES FEMALES AGE Body weight in grams Number Body weight in grams Number peiscial eees ess). individ- Average | Highest Lowest uals Average Highest Lowest uals days 13 19.8 27 16 57 18.0 25 14 93 30 48.6 75 35 57 45.1 72 30 93 60 148.5 190 110 57 115.2 147 80 93 90 210.1 266 149 ay/ 161.1 197 117 68 120 254.1 331 218 55 176.8 198 134 73 151 282.9 365 232 55 193.4 229 156 67 182 | 307.5 407 253 52 21320 241 168 70 212 319.4 410 254 44 208 .2 247 174 60 243 D200 408 259 39 212.5 261 179 54 273 30087 454 275 32 213.3 278 177 47 304 344.3 477 290 28 221 1 281 179 40 334 344.4 385 299 18 230.3 290 179 26 355 348 .2 376 298 if 233.7 276 194 29 395 355.1 3874 333 11 237 .2 301 206 23 425 353.2 365 339 5 239.4 323 210 19 455 336.6 |° 347 320 3 245.0 317 212 13 Growth in body weight. Albino Rat Body weight in grams Males T ape Series A oH T Series B a Age in days ro cr relat 20.40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 460 Fig. 8 Graphs showing the increase in the weight of the body with age for males belonging in the seventh to the fifteeth generations of the two series of inbreds (data in table 11 and in table 12). $205 See ea “Growth in body weight. Albino Rat. sat aneareg, 300 tEE== rita } Females FH f : ry f Z PC EE EEE eee t coo i 280) ‘ody weight in grams sousssesss PERE oH I t i +t oes Pett PeCHeeH f Ht u i Fay ett 260 a tt 4 ra PEPE pee Eee 1 ! es elaine { mae | ert pte Series B 240 +4 i | t t EEE muadauhodie, ro EEE “3 - Series A | 220 EERE EER eee seaa 200} | I on, ai } i SHH HH : ao oanae | | ime : 180 -- : . i t t wae — : t GHEE ort a Tt 1 Diet LLLET 1 Foret T EEC EEE 160) SESSSERReeRy 8 - Ht } Pot Eee ay | i I { i a im nSeo! sf Tt it T T 140) : enw 4 — - +--+ “ i ISRRREEEee Ct t | { ce roo Poo rag mt | SEE SGaggaaes rf t 120) rH anena Sea { t ct 100F-4 Sonuse aes 1 80 aeeael St Eo SSSEuar / a Zoos SoSSnSRemnen T | I ar T 40) | Lig t 20K met tH | | ; T Age in days + t ap! : 7 oa EEE EEE EEE EEE EH aeeeeae O 20 40 60 80 100 120 140 160 180 0 220 240 260 280 300 320 340'360 380 400 420 440 460 480 Fig. 9 Graphs showing the increase in the weight of the body with age for females belonging in the seventh to the fifteenth generations of the two series of inbreds (data in table 11 and in table 12). 26 EFFECTS OF INBREEDING ON BODY WEIGHT 20 The graphs in figure 9 run close together throughout their entire course, but the graph for the B series is higher at all points than that for the A series. It appears, therefore, that growth in body weight was somewhat more vigorous in the fe- males of the B series than in those of the A series. It seems a rather significant fact that in both figure 8 and figure 9 the graphs run parallel from the beginning until the end of their course; they do not cross and recross at various points, as one might expect would be the case with graphs for two series of rats from the same ancestral stock that were reared simultaneously under the same environmental conditions. Fe- male B and her mate, the ancestors of the B series of inbreds, were heavier animals at the time that they were killed than were the progenitors of the A series of inbreds, female A and her mate. Body weight in the rat is so dependent on physical condition, however, that a single weighing of the animals when they were at an advanced age would not necessarily give a true idea of the relative size of the animals at an earlier age period. The difference in the size of the two pairs of rats with which the experiments were started, together with the fact that after the sixth generation the descendants of female B were relatively heavier animals than the descendants of female A, point to the conclusion that the difference in the size of the animals in the two series was not due to chance or to environment, but that it was dependent in some way upon the inheritance of genetic factors for growth. | Table 13 gives the body weight data for the total of 156 males and 169 females in the seventh to the fifteenth inbred generations for which weight records were taken (a combination of the data in table 11 and in table 12). Table 18 brings out one fact of interest: the average body weight of the male inbred rats increased with age up to the end of the weighing period when it was 358.7 grams; the average body weight of the females was at its maximum at the 425 day period, and then fell off slightly at the final weighing. Indi- vidual rats show a pronounced difference as regards the time that they attain their maximum body weight and, as a rule, the 28 HELEN DEAN KING TABLE 13 Showing the increase in the weight of the body with age for 156 male and 169 female rats belonging in the seventh to the fifteenth generations of the two series (A, B). A combination of the data in table 11 and in table 12 MALES " FEMALES AGE Body weight in grams Number Body weight in grams Number fhaivide Magid Average’ | Highest Lowest uals Average | Highest Lowest uals days 13 18.6 27 15 156 17.0 25 14 169 30 46.5 82 35 156 43.6 72 30 169 60 132.6 190 72 152 109.4 159 71 167 90 197.3 266 120 155 155.9 197 93 i3l 120 241.4 331 174 149 WADo0/ 212 133 136 151 270.6 365 204 149 190.5 229 156 132 182 294.7 407 216 143 200.8 241 167 132 212 304.5 420 238 129 205.8 248 158 119 243 313.8 419 234 123 PMN 261 171 110 273 325 .0 454 254 106 Dilla 278 167 100 304 BVA oll 477 267 98 221.0 281 179 82 334 332.3 415 268 81 228.4 290 179 63 365 338.8 428 289 76 231.5 282 194 64 395 339.3 433 283 62 2338.2 301 206 54 425 346.6 448 281 49 235.6 323 206 40 455 358.7 473 306 28 234.9 317 182 33 females reach this point at an earlier age than do the males. The records for these inbred rats show that in many cases the maximum body weight in both sexes came when the animals were seven or eight months of age, at which time they were at the height of their reproductive activity (King, 716), and then gradually decreased; other rats increased steadily in body weight until they were sixteen or even eighteen months old. Autopsies made on a considerable number of unusually large rats indicate that the later weight increase is chiefly an accu- mulation of adipose tissue and is not, therefore, to be consid- ered as true growth. Under the very favorable climatic conditions of California, Slonaker (12 a) found that the albino rat reaches its maximum body weight, as a rule, by the age of fifteen months. In one EFFECTS OF INBREEDING ON BODY WEIGHT 29 series of animals whose weight records were taken at frequent intervals from the time of weaning until natural death, Slonaker found that the ten males attained their average maximum body weight of 247.5 grams when they were 434 days of age, and that the six females reached their maximum weight of 151.8 grams somewhat earlier than did the males i.e. at 375 days of age. After the maximum was reached there was a slow but steady f porerareretsiet Hee frit tit aGrowth in body weight. Albino Rat /- 444+ AH rte Grae a FETE aaa sqsuugua! HEE { snansaoem eee H+I geaaeaa ttt puseueues EERE EE EEE EEE EH Sheet at Fig. 10 Graphs showing the increase in the weight of the body with age for males and for females belonging in various generations of the two series combined (A, B). Data in table 3 and in table 13. A and B, graphs for individuals in the seventh to the fifteenth generations inclusive; C and D, graphs for animals in the first six inbred generations. decline in body weight, although the animals appeared to be in good physical condition, and some of them lived to be nearly four years old. The graphs in figure 10 show the weight increase with age for the total number of males and females in the two inbred series for which weight records were obtained (data in table 3 and in table 13). 30 HELEN DEAN KING The graphs in figure 10 show in a very striking manner the great difference between the size of the rats in the first six in- bred generations and those in the later generations. At the- 300 day period the space between graph A and graph C repre- sents a difference of 87 grams in the average body weights of the two groups of males. Females of the earlier generations (graph | D) were likewise far inferior in body size to the females of sub- sequent generations (graph B), and at 300 days of age the space between graph B and graph D indicates a difference of 64 grams in favor of the females in the later generation group. The earliest data on the growth in-body weight of the albino rat are those of Donaldson (’06) who studied the growth changes in a series of animals reared at The University of Chicago. Other investigators, Jackson, Slonaker, Ferry and Hoskins have pub- lished records for the growth in body weight of various series of albino rats reared under different environmental conditions. All of the latter data agree, in the main, with those of Donald- son, although as the rat is very responsive to external conditions some series of records show more rapid and vigorous growth than others. Donaldson’s growth graphs for albino rats may be taken as representing the average run of stock animals. His graph for the males is reproduced as graph A in figure 11, and that for the females is shown as graph A in figure 12. As controls for the present series of inbred albino rats thir- teen litters of stock albinos, comprising fifty males and fifty females, were reared in The Wistar Institute animal colony under the same environmental and nutritive conditions as the later generations of the inbred series. In selecting litters for controls care was taken to pick out only those in which the young were large and vigorous at birth. The animals chosen, there- fore, represent the best, not the average, stock in The Wistar colony. The average body weights at various ages of the males and females in this selected group of stock albinos, reproduced from table 3 of a previous publication (King, 715 a), are given in table 14. EFFECTS OF INBREEDING ON BODY WEIGHT 31 Figures 11 and 12 show graphs for weight increase with age in two series of stock albinos (graphs A and C), together with eraphs representing the growth of animals in the inbred series (graphs B and D). The graphs for the various groups of males are given in figure 11. Ht 7Growth in body weight Al Se eeeeeepece: EH Males EET Samascenuuen! “F&, ++ i. GE Gy Pq + souseeees 5 cceeeSeoEs at Ht aaa { i Ht 0 aoe Pope aes PA EEE t 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280, 300 320 340 360 380 400 420 440 460 480 Fig. 11 Graphs showing the increase in the weight of the body with age for different series of male albino rats. A, graph constructed from Donaldson’s data for stock albinos; B, graph for males belonging in the seventh to the fif- teenth generations of the two series of inbreds combined; C, graph constructed from data for a selected series of stock albinos used as controls for the inbred strain; D, graph for males belonging in the first six generations of the two series combined. In figure 11 graph B, representing the growth of the males in the inbred series, runs higher than Donaldson’s graph for stock albinos (A) from the beginning until the end of.its course. At the 243 day period the space between these graphs represents a difference of about 18 per cent in the average body weights of the two series of animals. At all points, except the thirty day period, graph B is higher than graph C which shows the body 32 HELEN DEAN KING growth of the selected stock males reared as controls for the inbred group. Data in table 13 and in table 14 show that at 13 days of age the inbred males weighed, on the average, 1.4 grams more than did the stock males of the same age, but that at thirty days of age the average body weight of the stock group was two grams more than that of the inbreds; after this age inbred males increased in body weight much more rapidly than did the stock animals. When the rats were at their prime, at eight months of age, inbred males were about 12 per cent heavier than the males of the control series. The above analysis of data shows that not only were inbred males of the seventh to the fifteenth generation much heavier than the general run of stock animals at any given age, but that they were also larger, except at the thirty day period, than the selected stock controls reared under similar environmental conditions. Graphs showing the weight increase with age for various groups of stock and inbred females are given in figure 12. In figure 12 graph A, which indicates the body growth of the females of Donaldson’s series of stock albinos, is not strictly comparable to the other graphs, since it was constructed from the actual weight data for unmated females only up to the ninety day period, beyond this point the data used were those of un- mated females corrected to accord with the weights of breeding females as caleulated by Watson’s (’05) formula: all other graphs were based on the actual weight data of breeding females. Graph A runs lower than either the graph for the inbred females (B) or that for the stock controls (C) during the period in which the actual weight records were used in constructing the graph, but later it is considerably higher than the other graphs. It would seem as if the corrective factor introduced in Watson’s formula was much too high, since no series of actual weight records for the albino rat yet reported comes up to the standard required by Donaldson’s graph. In figure 12 the graph for the growth of inbred females (B) and that for the females in the control series (C) run very close together throughout their entire length. From the thirty to EFFECTS OF INBREEDING ON BODY WEIGHT 33 the sixty day period graph C is a little above graph B, but at all other points graph B is the higher of the two. At the 243 day period the space between the graphs represents a difference of only 0.76 per cent in the average body weights of the two groups of females, although the inbred females were 3.7 per cent heavier than the stock females when the animals reached one year of age. ; These results indicate that the rate and the extent of the growth in inbred females was about the same as that of the fe- males in the selected stock controls during the adolescent period, oe $ “Jaanauaeoes! > -auBeUgO "4 _-acaudedeccsscesescstses gat can gare fs fale aaa) ry 1 280} =| Sody weight in grams, wt rt Eee Seer | seteei: seseettaiie secesacerseesessaasead saieeectrecatesattacaiil a ci H CH Age in days 4 LEEPER EEE EEE EEE Fig. 13 Graphs showing the increase in the weight of the body with age for males belonging in the fifteenth generation of the two series of inbreds com- bined (A, B), and for males in the series of stock controls. SSSSREER_/ generation is further corroboration of the linkage of the two loci. The ruby-eyed dilutes have an eye-color distinctly lighter than that of the red-eyed yellows. The ruby-white compound, C,C., which we have called fawn from the coat color, has an eye-color still lighter. A comparison of eye-colors in the rat has shown a gradation from black to pink as follows: black, PP. RR. CC.; red, PP. rr. CC.; ruby, PP. RR. C,C,; light ruby, PP. RR. C,C..; pink, pp. RR. CC.; light pink, PP. RR. C.Ca. Other combinations may show intermediate grades. The coat-color of the ruby-eyed dilutes is a very light sepia showing more or less yellowish white. The yellow tinge is probably due to excretions from the skin, since there are appar- ently no yellow granules. Albinos and hooded rats also show this yellow tinge in the hair, especially as they grow older. Wright (15) has shown quadruple allelomorphs in the albino series of guinea-pigs. He has further discussed the matter (17 a) in a general scheme of color inheritance for mammals. A comparison of conditions in the rat with conditions in the guinea- pig will be of interest. In the wild Norway rat the hairs are white at the base. Very eradually black pigment granules appear toward the tip and in all of the larger hairs these increase without interruption until the hair becomes intense black. In the hairs of intermediate and small size, however, the black pigment usually gives place to yellow granules which extend almost the entire length. The ex- treme tip is black. Some of the smaller hairs have black pig- ment in a single uninterrupted row of granules and others are white. A few of the smaller yellow-banded hairs have the band rather narrow and close to the tip. In almost all cases, however, when the yellow band occurs, it is very wide. In the black rat most of the hairs are white at the base, becoming black or dark sepia toward the tip, while a few of the smaller hairs are white throughout their entire length. 62 P. W. WHITING AND HELEN DEAN KING In fully colored ticked guinea-pigs the hairs of the back are sepia at the base and black toward the tip except that a narrow yellow band occurs on almost every hair at the short distance from the tip. In blacks the hairs are similar except that the band is lacking. Thus the intense guinea-pig is darker than the intense rat, for the hairs of the latter are white at the base. As described by Wright, there is in the guinea-pig a gradual decrease in the amount of yellow pigment from the intense, CC, through the various dilute combinations, CaCa, CaC-, CaCa, until the red- eyed dilute, C-C;, shows no yellow at all. In this animal the hairs are dark sepia banded near their tips with white. The dilute carrying albinism, CzCo, has light sepia hairs banded with light cream. Wright explains the correlation of the lighter sepia with the presence of yellow by assuming that in the animal CaCa there is competition in the oxidation of chromogen between the compound black producer, enzyme I-—II, and the yellow pro- ducer, enzyme I. In the animal C;C, the yellow producer has been removed and enzyme I-—II is free to act on chromogen with- out competition, All of these grades of pigmentation in, the guinea-pig are darker than the ruby-eyed dilute rats, as is also the most dilute combination obtained in the guinea-pig, the red-eyed carrying albinism, C;C.. In this animal the hairs are light sepia to the base and are ticked with white near the tip. The ruby-eyed dilute gray rat, A.C;C,, has no yellow pig- ment, but the yellowish tinge, as seen in the white rat, is notice- able in the lighter parts of the hairs; the black pigment is re- duced to a very light sepia and is confined to the tips of the hairs. In the ruby-eyed light sepias, a.C;-C,, the pigment is likewise very dilute, but extends from the tips well down the hairs. ‘The base, however, is without pigment, as in the black rat. Thus the agoutiand non-agouti dilutes can be distinguished, not by the presence or absence of bands, as in the guinea-pig, but by the greater extent of the pigment in the non-agoutis. The coat-color of the fawns, C-Ca, is extremely dilute, midway between that of the homozygous ruby-eyes and that of the Albinos. The hairs of agouti red-eyed yellow rats, A.rr, are yellow and RUBY-EYED DILUTE GRAY RAT 63 white. The larger hairs are white, while those of intermediate and small size are white at the base and tip only. For the rest of their length they have large yellow pigment granules. In the non-agouti red-eyes, aa.rr, the hairs are almost entirely white except for the yellow stain similar to that which occurs in white rats. There may be a very slight amount of extremely dilute sepia pigment in some individuals. The double recessive, ruby-red, C;C,.rr, expected in the F, generation from the cross of ruby-eyed dilute gray by red-eyed yellow should be practically white even in the presence of the agouti factor, for the Albino type of dilution found in the dilute gray eliminates yellow, while red-eyed yellow dilution eliminates sepia. It is also to be expected that the double recessive, ruby- pink, C,C,.pp, will have white hair. y SUMMARY A new Mendelian variety of the Norway rat known as ruby- eyed dilute gray has been found near the Zoological Laboratory of the University of Pennsylvania. The hair is light sepia at the tip and grades to white at the base. The eye color is ruby. The new variation is recessive to intense pigmentation. When the dilutes were crossed to black-hooded rats all the F, indi- viduals were intense and the F, generation showed thirty-three intense and fourteen dilutes. Ruby-eyed dilution is allelomorphic with albinism. The F, individuals, called fawns, are intermediate both in hair and in eye color. Fawns when bred together produced eighty ruby- eyed dilutes, one hundred and fifty-six fawns, and eighty Albinos. Ruby-eyed dilutes crossed with red-eyed yellow rats produce rats of the wild type. The second generation shows evidence of linkage of the two factors, since double recessives did not appear. No linkage is apparent with hooding or with non-agouti. In the agouti dilutes the sepia pigment is restricted to the tips of the hairs. The non-agouti are more heavily pigmented. 64 P. W. WHITING AND HELEN DEAN KING LITERATURE CITED CasTLe, W. E. 1916 Gametic coupling in yellow rats. Carn. Inst. Wash. Pub. . No. 241, part III: 175-180. Caste, W. E., AND Wrigut, SewaLt 1915 Two color mutations of rats which show partial coupling. Science, N. 8., XLII, August 6. Wuitine, P.W. 1916 A new color variety of the Norway rat. Science, N.S., XLIII, June 2. Wriaunt, SEwAatt 1915 The Albino series of allelomorphs in guinea-pigs. The Amer. Naturalist, XLIX, March. 1917 a Color inheritance in mammals. Journ. of Hered., vol. 8, May. 1917b II The mouse. Ibid., August. 1917c III The rat. Ibid., September. 1917d IV The rabbit. Ibid., October. 1917e V The guinea-pig. Ibid., October. e AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE MARCH 2 STUDIES ON CYTOLYSINS I. SOME PRENATAL EFFECTS OF LENS ANTIBODIES M. F. GUYER AND E. A. SMITH From the Zoological Laboratory, University of Wisconsin 1. INTRODUCTION Ever since the pioneer work of Buchner in 1889 and of Beh- ring and Kitasato in 1890, there has been a lively interest in the subject of so-called immune sera. The last few years have brought forth a flood of literature on antitoxins, agglutinins, precipitins, bacteriolysins, hemolysins, cytotoxins or cytoly- sins, and opsonins—all the fruition of these earlier investigations. Inasmuch as the first discoveries were concerned with certain bactericidal properties artificially produced in the sera of ani- mals through the injection of various species of bacteria, and because of the tremendous importance of these facts in furthering our knowledge of infection and immunity, the field became at once the province of the bacteriologist and the pathologist. Following closely upon the heels of the earlier discoveries came a brilliant series of practical applications in diagnosis, prophy- laxis, and therapeutics, until it is not to be wondered at that investigators became absorbed in the medical phases of the subject. It is clear, however, that the phenomena in question all have their broader biological aspects, and it seems time 0 see if the knowledge already gained in this field may not be utilized in a new attack upon certain fundamental biological problems. | The work already done on precipitins, in fact, shows how these methods may be applied. When rabbits are injected with several doses of serum prepared from horse-blood, for example, their blood develops a substance not present in the blood of untreated 65 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 26, NO. 1 66 M. F. GUYER AND E. A. SMITH rabbits. If the serum containing this substance is mixed with horse-serum in vitro, a cloudiness results, due to the precipita- tion of part of the protein in the horse-serum. The newly en- gendered ingredient of the sensitized rabbit serum which pre- cipitates the horse-protein is termed a precipitin. The material injected, in this case horse-serum, is spoken of as the antigen. Not only can precipitins be formed against the blood-serum of an alien species, but against a wide range of substances, such as bacterial products, milk, peptone, globulins, and various albumins. It is this specificity against a distinctly alien albumin that renders the precipitin test one of ready application in the medico- legal differentiation of human blood and various human albu- minous substances from those of other animals. Not only is the precipitin test useful in discriminating be- tween non-related species, but it may prove to be important in establishing the taxonomic position of new forms and in confirm- ing or changing the classification of groups already known, since among closely related species the specificity of the reaction is not absolute. For instance, Nuttall, who made observation on some 500 different animals found that rabbit-serum highly sen- sitized with human blood-serum reacts, though in varying degree, with the blood of all mammalia; a less strong serum, besides reacting on human blood, also causes a precipitate in the blood of anthropoid apes (chimpanzee, orang-outang, gorilla) and in a less degree in the blood of other monkeys; whereas a weak serum reacts with human blood and produces only a slight cloud- iness in the blood of the anthropoid apes. He found that the same quantitative differences exist in antisera specific for each of the large vertebrate classes, birds, reptiles, and amphibia. Thus by the precipitin test a differential scale of actual relation- ships can be established. The degree of activity of sensitized sera is judged by the dilution in which they will react. A properly sensitized serum will give a distinct reaction in blood diluted 1000 times. There are records of reactions with blood diluted 20,000 times or even 50,000 times, while Ascoli, indeed, reports obtaining a specific STUDIES ON CYTOLYSINS 67 reaction with a serum sensitized to egg albumin upon mixing it with egg albumin diluted 1,000,000 times. The delicacy of such tests can be appreciated when one knows that ordinary chemi- cal tests cease to give reactions in blood diluted 1000 times. As long ago as 1895 Bordet found that the blood of guinea- pigs which had been repeatedly injected with the red corpuscles of the rabbit acquired peculiar properties. Serum prepared from these sensitized guinea-pigs, when placed in a test-tube with rabbits’ blood, not only caused the agglutination of the red blood corpuscles, but even rapidly dissolved them. The serum of untreated guinea-pigs was incapable of doing this or did it only feebly. Bordet showed further that this enhanced solvent action of the serum of animals treated with rabbit cor- puscles existed only for the red cells of the rabbit, not for those of other species of animals. Exceptions to this rule have since been found, though in the main the action is a specific one. The action is known as hemolysis, and the substance in the serum which brings about solution of the red cells is termed a hemolysin. Hemolysins are now known to be special members of a gen- eral class of substances termed ecytotoxins or cytolysins. For just as alien red blood cells lead to the production of hemolysins, so various other materials, as leucocytes, nervous tissue, sper- matozoa, and crystalline lens, when injected into the blood of an unrelated species, will form lytic substances more or less specific for the antigen used in the sensitizing process. All the cyto- lytic sera so far studied have been found to be more or less hemolytic, and it is probable that none acts exclusively upon its own antigen. The important fact, for our purposes, is that although a particular cytolytic serum may affect some other tissues, it vigorously attacks the special tissue used as antigen. The exact nature of antibodies, such as precipitins, eytoly- sins and others, or the manner in which they are engendered is not known. The blood is probably in the main the carrier rather than the producer of such bodies. While the leucocytes may be one source of certain constituents, it is probable that various tissues of the body are responsible. There is evidence to show 68 M. F. GUYER AND E. A. SMITH that for some bacteria, at least, the bone-marrow, spleen, and lymph nodules produce the antibodies. Although presumably distinct from one another, the various classes of antibodies seem to have many points of similarity, as, for instance, their methed of origin, their reaction to heat, and their mode of operation. Chemically their natures are still unknown. Many of themseem tofunction through the combined action of at least two separate constituents. In some instances perhaps more constituents are involved. One of the compo- nents developed in response to injected foreign substances, such as bacteria, cells, dissolved proteins, various ferments, toxins, and venoms, though variously named by different workers, is commonly called the immune body or amboceptor. The other, an ingredient of normal serum, is termed the complement. Whether a single complement acts alone or a series of comple- ments operate in conjunction with the amboceptor is a matter of dispute. Complement, in general, decomposes readily upon warming much above blood heat or spontaneously upon stand- ing. It seems much less stable in every respect than the ambo- ceptor, though the latter is the specific constituent developed through the introduction of an antigen. The union of such foreign substances as amboceptors with a living cell is possible, according to Ehrlich’s conception, be- cause of the existence of a series of secondary chemical con- stituents or groups attached to the main living molecules of that cell. To these he has given the name of receptors. Since this term is In common usage, it is perhaps well to retain it until a better one for the existing condition is forthcoming, even though all workers would not subscribe to the implica- tions it suggests. While receptors are supposed by Ehrlich to have a great variety of functions; he regards that of assimila- tion as of particular importance. ‘To combine with these re- ceptors any given substance (e.g., amboceptors) must possess, supposedly, very definite constitutional or configurational rela- tions to the receptors in question. ; One view looks upon the complement present in all serum as a sort of a ferment possessing digestive properties. It is power- STUDIES ON CYTOLYSINS 69 less until cells of the kind used as antigen have been rendered vulnerable by the action of the specifically generated immune body. Even the complement in an animal’s own serum will suffice to dissolve one of its own tissues if the proper amboceptor is introduced. Views as to just how these various elements combine and react are nearly as numerous as the investigators who have studied them. The explanation vouchsafed by the Ehrlich school for the lack of absolute specificity, is that certain of the different tissues of the body have receptors of the same kind, so that anyone of such a group of tissues might serve as an antigen for all the others. In other words, according to them, specificity is not a matter of cells, but of receptors. There is urgent need for further investigation of cytolysins not only as regards this question of tissue specificity, but also as to the degree of specificity or lack of specificity for the ho- mologous tissues on groups of related organisms. Furthermore, cytolysins have been developed so far for only a very limited number of tissue elements. The reports of pathologists who are seeking to develop specific cytolytic sera for cells of pathological origin, particularly malignant tumors, are increasingly discourag- ing, since apparently the distribution of common receptors is too widespread to permit the formation of antisera with sufficient specificity for medical purposes. But this difficulty need not block the biologist in his efforts to secure certain other results. If it is possible to originate in living organisms antibodies which will destroy particular tissue elements, is it not possible to secure similar selective action on Certain parts of the de- veloping embryo? Moreover, if we are ever to break through the apparent impass which has enveloped our long-standing problem of the inheritance of somatic modifications, or that of provoking specific modifications in the germ through direct operation of external agencies, is not the employment of cyto- lysins possibly a line of attack which may yield fruitful re- turns? ,If a special serum will single out and destroy a certain element of the adult, is it not possible that there is sufficient constitutional identity between the mature substance of that 70 M. F. GUYER AND E. A. SMITH element and at least some of its material antecedents in the germ, that they too may be influenced specifically by the serum in question? In an attempt to find answers to these and kindred questions, the authors of this paper have undertaken a series of experi- ments to produce specific antenatal effects in fetuses by means of cytolysins; to determine how early in an embryo cytolytic effects may be secured; to obtain cytolysins which will be op- erative in the developmental stages of certain periodically re- newed structures, such as feathers; and particularly, to test the possibility of securing specific effects through the germ itself. The present paper concerns itself mainly with the antenatal effects secured in rabbits and mice by means of fowl serum sensitized with lens. Experiments more or less similar to those here recorded are still in hand and others are soon to be under- taken on a more extensive scale. 2. EFFECTS OF LENS CYTOLYSINS ON FETAL RABBITS The lenses of rabbits were used as antigen and chickens were employed as the source of the antibodies. The lenses, immedi- ately upon removal from the dead animal, were pulped thor- oughly in a mortar and diluted sufficiently with normal saline solution to permit of injection into the peritoneal cavity of the fowl by means of a hypodermic syringe. In order to prevent injury to internal organs, the fowl was held back downward that the viscera might settle away from the ventral abdominal wall, and the puncture was made just under the tip of the breast bone. At this point the needle enters easily, no internal resistance to the discharge of the liquid from the syringe is encountered and the fowl apparently suffers no pain. When the fowl was ready for removal of the serum, killing proved to be more practicable than drawing off blood from the living bird. To secure the blood in sterile condition, the fowl was anaesthetized with ether until insensible, the feathers were hastily plucked from the neck and the latter washed in‘ alcohol, the skin was cut entirely around the neck near the head and STUDIES ON CYTOLYSINS Fal peeled back, the oesophagus and trachea were transected and turned back so as not to discharge into the receptacle for catch- ing blood, the head was then quickly severed near its base with heavy scissors and the neck thrust into a wide-mouthed sterile test-tube or flask. Blood removed in this way was chilled in the dark for one or more hours and then centrifuged for some twenty minutes to separate the serum from the clot. The serum so obtained was, after warming to blood heat, injected into rabbits through the TABLE 1 pxponimnxt | DATE Se eee | acre |iltreicere cc. cc November 1 le ah, &! 2 8 15 I November 8 | l, 2, 3, 4 2 6 15 ri ’ || November 15 2 2 8 We) || November 22} 1, 2,3, 4 2 8 UES January 17 5-11 6 20 2-5 10) 8) te ero caer January 19 5-11 6 20 2.5 January 22 5-11 6 20 2.5 f March 8 12-16 4 20 2.5 ve March 15 12-16 4 15 25 jo aes March 17 12-16 4 15 2.5 || March 20 12-16 4 15 2.5 * All rabbits half grown. marginal vein of the ear. All operations including those per- formed on rabbits were done with sterilized instruments and vessels and under as aseptic conditions as possible. Experiment 1 In this experiment four fowls eight months old were used for sensi- tizing with lens material, and two pregnant rabbits, each of which had previously born normal litters, were the recipients of the sensitized fowl serum. On November 1, 8, 15, and 22, respectively, the four fowls were in- jected intraperitoneally, each time with the lenses from an adult rab- bit (table 1). The lenses were pulped and diluted with 6 to 8 ce. of im M. F. GUYER AND E. A. SMITH normal saline solution. On each date each fowl received 1.5 cc. of this mixture, containing approximately one-half of a lens. On December 6 one of the fowls was killed and bled. On December 7 the blood which in the meantime had been kept in the dark at a tem- perature of about 5°C. was centrifuged and the decanted serum used for injection into the two pregnant rabbits, each of which, between 10 and 11 a.m., was given 6 ce. of the undiluted serum (table 2). The in- jection was made directly into the blood stream through the marginal vein of the ear. By 2.30 p.m. each rabbit showed evidence of illness, particularly individual A, which was passing what appeared to be bloody urine at frequent intervals. Both had apparently recovered by the next day. December 9. Both rabbits were given a second injection of serum from another of the sensitized fowls which had been killed on De- cember 8. This time the dose for each rabbit was changed to 5 ec. of serum diluted with 3 ec. of normal saline solution. The animals showed no ill effects after this treatment. December 11. Treatment similar to that of December 9 was given, the serum being from the same source. December 14. Each rabbit was injected ‘with 6 ce. of the undiluted serum of a third one of the sensitized fowls, and this dose was repeated on December 15, using serum from the same fowl. December 18. Each rabbit was given 6 ec. of undiluted serum from the fourth fowl. . December 30. Rabbit B, an Albino, gave birth to seven young. When the young finally opened their eyes, one had a slight opacity of about one-half of the lens of the left eye. This cleared up after three days. A second one had the entire left eye noticeably smaller, with the lens opaque. This opacity has persisted as has also the smaller size of the eye. At the present writing (October 15) this individual is about fully grown. While its right eye seems to be perfectly normal, the left is but little larger than that of a newly born rabbit. A third individual had a cloudy rim around the edge of the lens which slowly cleared after several days. The four other young, in so far as one could judge from external appearance, had normal lenses. But since young of another mother subjected to similar treatment had watery or liquefied lenses (see ex- periment 5), a condition which was not ‘detectable until the young were killed and the eyes dissected, it is possible that cytolytic effects existed in the lenses of some of these four apparently normal individuals. One, in fact, when killed four months later (experiment 5) was found to have a watery and diffuse lens. By that time, unfortunately, two of the other three had been disposed of so that it was impossible to determine the consistency of their lenses. The last one is being kept for further breeding experiments. She is at present mated with the dwarfed-eyed one. Rabbit A failed to bear young. Since she is the one recorded as passing what appeared to be bloody urine after the first injection, it is EXPERI- MENT DATE OF INJECTION TEES. VS Fe December 7 December 9 December 11 December 14 December 15 December 18 January 31° February 3 ~I February February 3 February 7 February 13 Mareh 31 April 3 April 5 STUDIES ON CYTOLYSINS TABLE 2 SE- eae Betton DOSE OF SERUM REMARKS RABBIT FOWL A and B} 1 | 6 ce. serum A and B; 2 | 5 ce. serum + 3 | December 30, B had 7 ce. normal sa- young line Visible externally in A and B} 2 | 5 ce. serum + 3 three individuals: 1 ec. normal sa- partly opaque lens line in left eye (cleared A and B) 3 | 6 ce. serum up); 1 opaque lens A and B) 3] 6 cc. serum in left eye (per- A and B) 4 | 6 cc. serum sisted); 1 cloudy rim (cleared up) May 19, 1 killed; watery lenses Cand D) 5 | 7 ce. serum + 3 | On February 15; rab- normal saline bit C bore 5 young (Camb) 6 | 10 ec. of a mix- (all apparently nor- ture of 8 ce. mal, though 1 de- serum + 3 cc. veloped abnormal normal saline incisor teeth) ©, D' 7 | 10 cc. of a mix- | On February 16, D ' ture of 8 ce. bore 4 young (all serum + 3 ce. apparently normal) normal saline B 6 | 7 cc. serum + 3 | March 6, B bore 6 cc. normal sa- young. Great dis- line _ crepancy ims size B 7 | 8 cc. serum + 2 between individual ec. normal sa- members of litter line B 8 | 8 cc. serum + 2 | ec. normal sa- line E 12 | 10 ce. of mixture | April 24, 8 young of 10 parts se- born. Eyes weak rum + 2 parts and watery normal saline E 13 | 10 ce. of mixture | May 19, 1 killed and of 10 parts se- | lenses found to be rum + 2 parts | liquefied normal saline E 14 | 11 cc. of above mixture 74 M. F. GUYER AND E. A. SMITH not impossible that she aborted at this time. She was remated suc- cessfully later and is the subject of experiment 2. Experiment 2 Rabbit A of the former experiment was mated on January 10. She was not further injected with sensitized serum, as it was desired to find if the effects of the earlier injections might hold over and affect the young in utero. On February 12 she gave birth to eight young. None showed any perceptible effect of the lens cytolysin administered to the mother during the earlier experiment. Experiment 3 In this experiment seven fowls were used for sensitization, and two pregnant rabbits, C and D. January 17, six lenses of adult rabbits were ground up in a mortar and diluted with 20 cc. of normal saline solution. Each of the seven fowls was given a 2.5 ec. dose of this suspension intraperitoneally. This treatment was repeated January 19 and again January 22. Two of the fowls were bled January 30 in the usual way, and on January 31 the two rabbits, C and D, were each injected through the ear vein with 10 cc. of a mixture of serum 7 parts and normal saline solution 3 parts. On February 3 serum was taken from another of the sensitized fowls, and the two rabbits were each given a 10 ce. injection of a mixture of the serum 8 parts with 3 parts of normal saline solution. This was repeated on February 7 with se- rum from yet another treated fowl, using the same proportion of serum and normal saline solution as on February 38. On February 15 rabbit C bore five young. All were apparently normal at birth, though one rapidly developed abnormally large incisor teeth and had to be killed April 24 to prevent its slow starvation. The eyes of all were appar- ently normal. Rabbit D bore four young February 16, all with nor- mal eye structures.as far as could be judged from external appear- ances. The young of both of these experiments had been disposed of before the importance of determining the consistency of their lenses was realized. Experiment 4 Rabbit B of experiment 1 was mated again January 24 to January 30. She was injected at intervals with serum from the sensitized fowls used in experiment 3, namely, on February 3 with a mixture of 7 cc. of serum and 3 of normal saline solution; on February 7 with a mixture of 8 ce. serum and 2 ee. of normal saline solution, and on February 13 with a similar dose. On March 6 she gave birth to six young which ranged in size from an extremely small one to one considerably larger than the others. No eye defects were visible. Again, because the eyes were of normal size and transparency, the young were disposed of, unfortunately, without determining the texture of the lenses. ~ Or STUDIES ON CYTOLYSINS Experiment 5 Five fowls were injected March 8, 15, 17, and 20, respectively, with lenses from young (about half-grown) rabbits, four lenses being used each time, so diluted with normal saline solution that each fowl re- ceived 2.5 ec. of the mixture. Rabbit E was injected in the usual way with serum from these fowls, the doses being: March 31, 10 cc. of a mixture of 10 parts of serum and 2 parts of normal saline solution. April 3, 10 cc. of a mixture of 10 parts of serum and 2 parts of normal saline solution. April 5, 11 ce. of a mixture of 10 parts of serum with 2 parts of normal saline solution. On April 24 a litter of eight young was born. All were tardy in getting their eyes open, one being particularly delayed in this respect. The eyes of all when opened seemed weak and watery. By May 17 all eyes seemed normal and most of the young were given away as pets in order to reduce the number of individuals which must be car- ried over the summer by a caretaker. On May 19 one remaining young one of this litter was killed. An examination of its eyes re- vealed the fact that its lenses were liquid, though still transparent. The lenses of three normal rabbits of the same age were examined and found to be fibrous and fairly firm. This suggested that the young in the litters of the other females which had been treated with serum might also have had lenses similarly affected though not visible from the exterior. Unfortunately, as already recorded, these had been disposed of before the discovery was made. One male of the litter produced in experiment 1 was still available. It was killed May 19, about four months after birth, and its lenses were found to be very watery and diffuse when compared with those of normal rabbits of the same age. F Since this same liquefaction of lens was found in the lenses of young mice in similar experiments which were being carried on by the senior author in California in the meantime (cf. section III), there seems no reason for doubting that it is an effect pro- duced by a foreign serum sensitized to crystalline lens. A new series of experiments is being undertaken for further enlight- enment on this point. None of the adult females used in the foregoing experiments showed in the lenses of their own eyes any effects of the treat- ment. The tabulated data of the experiment are shown in tables 1 and 2. 76 M. F. GUYER AND E. A. SMITH 3. EFFECTS OF LENS CYTOLYSINS ON FETAL MICE The experiments on mice were performed by the senior author at the Scripps Institution for Biological Research at La Jolla, California. His thanks are due this institution for many courte- sies. He is particularly indebted to Dr. F. B. Sumner and Mr. H. H. Collins for their generosity in adding to his stock of mice, for identification of species, and for information about breeding and rearing Peromyscus. Chickens were used as the source of antibodies and lenses of Peromyscus maniculatus gambeli were employed as antigens. The fowls, averaging three and eight-tenths pounds in weight, were gradually sensitized, as specified in table 3, by repeatedly injecting emulsified lens intraperitoneally. To prepare this emulsion a number of lenses were ground up in a mortar in a little normal salt solution. When thoroughly pulped the mix- ture was thinned with more of. the salt solution so that it could be readily injected by means of a hypodermic syringe. The pro- portions of lens and of saline solution in the various injections are specified in table 3. The individual fowls injected, ten in number, are arbitrarily designated by the letters A, B, C, ete., tO: The purpose of the present experiments was to build up a lens eytolysin which when injected into pregnant mice (Peromyscus maniculatus gambeli) would have a solvent effect on the lenses of the young in utero. Judging from the results of earlier ex- periments on the rabbit, no effect on the lenses of the mothers was anticipated. Precipitin tests Inasmuch as there is no visible way to tell when serum is adequately sensitized for use as a cytolysin beyond trying it out directly, and since both time and the supply of pregnant females were limited, a series of lens precipitin tests were made with various of the fowls after the fifth and sixth injections, respec- tively in order to be sure that they were responding to the lens proteins. While there is possibly no necessary connection be- STUDIES ON CYTOLYSINS Fi tween the precipitin and the cytolysin reactions of the blood, it was felt that if the lens had so sensitized the fowls that precipi- tins were formed, one might infer that cytolysins had also been generated. When ready to make these precipitin tests, several other species of Peromyscus were available from the experimenter’s own and from Dr. Sumner’s collections, hence it was possible to make a series of comparative tests. These are set down in full in table 4 because of the rather interesting relationships indi- cated. Since the tests were merely incidental to the other work, they are to be looked upon as suggestive rather than as TABLE 3 bate FOWLS INJECTED MOUSE TENSES NOEMAL SAUt'| DOSAGE PRR cc. cc. March 24...| 5 (A, B, C, D, E) 26 15 3 Mearchrolis sO CAn Bea vand Eto: 42 27 3 E died) April 7.....| 9 (as above) 42 27 3 Apri[ 10....| 9 (as above) 42 Bf 3 April 13.....| 9 (as above). 96 45 5 April 24..... Su(An BD Ca) ehenGene Tr. 50 28 BAG: J died) finished experiments. Judging from the results of these few tests, which are in harmony with the well-known work done several years ago by Nuttall,! it might be well worth some one’s time to choose a genus such as Peromyscus and make extensive and ac- curate precipitin tests of various kinds on the different species with the view of finding physiological relationships and deter- mining how these correspond to present taxonomic grouping and to geographical distribution. Interesting disclosures regarding conditions in hybrids should also come to light. For such work one should have narrow, very carefully graduated centrifugal tubes by which the amounts of precipitation could be accurately measured. 1 Blood immunity and blood relationship, Cambridge University Press. 78 M. F. GUYER AND E. A. SMITH In making the precipitin tests the lenses were taken from four individuals of each species of mice to be tested, ground up ina mortar and diluted with 3 cc. of normal salt solution. _The mix- ture was then filtered and to the resulting fluid 15 drops of fowl serum, sensitized as in table 3 with lens of Peromyscus mani- culatus gambeli, were added. In the case of Peromyscus cali- fornicus insignis, which is much larger than other members of TABLE 4 SPECIES OF MOUSE TESTED PRECIPITATION AFTER EIGHTEEN HOURS Serum of Fowl A. April 30 1. Peromyscus maniculatus gambeli _ Abundant 2. Peromyscus californicus insignis About one-third that of 1 3. Peromyscus eremicus fraterculus About one-third that of 1 4. Reithrodontomys megalotis longicauda | Very slight clouding 5. Perognathus fallax fallax None Serum of Fowl B. May 4 1. Peromyscus maniculatus gambeli Abundant 2. Peromyscus californicus insignis Nearly one-half that of 1 3. Peromyscus eremicus fraterculus Very slightly more than that of 2 4. Perognathus fallax fallax None Serum of Fowl C. May 9 1. Peromyscus maniculatus gambeli Abundant 2. Peromyscus eremicus fraterculus About one-half that of 1 3. Peromyscus maniculatus sonoriensis Almost as abundant as 1 4. Peromyscus maniculatus rubidus Very slightly less than 3 5. Perognathus fallax fallax None the genus, four young individuals of about the size of adult P. gambeli were selected, because it was desired to have the amount of lens material used in each species as nearly the same as possible. After eighteen hours any precipitate that had ap- peared was concentrated in a centrifuge in order to get a more accurate estimate of the relative quantities formed. It will be observed from table 4 that all species of the genus Peromyscus gave positive precipitin tests to serum sensitized STUDIES ON CYTOLYSINS 79 with lens of Peromyscus maniculatus gambeli, though the two subspecies of maniculatus (sonoriensis and rubidus) stood much closer to gambeli than did any of the others. This bears out the relationships as established by taxonomists. Reithrodontomys megalotis longicauda, the long-tailed harvest-mouse, which be- longs to the same family as Peromyscus, gave a slight reaction, while Perognathus fallax fallax, the short-eared pocket-mouse, of an entirely different family, gave only negative results. Effects of antibodies on the fetus In attempting to get cytolytic effects on the young in utero, sixteen mice which had previously been mated for the purpose or which were obviously pregnant were used. Of these five proved TABLE 5 MOUSE NUMBER eek eee eee, REMARKS 1 to 9 inclusive | May 1 A Nos. 1 and 2 died. No. 3 had 3 young May 2. No. 4had 3 young May 4 5 to 9 May 4 B No. 5 had 2 young May 6 6 to 9 May 9 re 6 to 9 May 18 C Nos. 6, 7, 8, dead, May 19. No. 9 died ' May 20 10, 11 (controls) | May1,4| Normal | No. 10 died. No. 11 had 4 young May 6 not to be pregnant, leaving eleven for the test. Two of these, used as controls, were injected with serum from a normal, non- sensitized fowl, so that nine were available for injection with the cytolytic serum. It will be seen from table 5 that the mortality was heavy and that only females which were well advanced in pregnancy and which, therefore, got but one or two doses of serum, brought forth their young. At each treatment every female was injected with 1 cc. of the sensitized serum. Species of Peromyscus maniculatus gambeli only were used. The period of gestation in this form is twenty-one days and the young do not open their eyes until about sixteen days after birth. It will be seen from table 5 that only three (Nos. 3, 4, and 5) of the females injected with the sensitized serum survived and bore young. Of these, No. 3 lost one of her young a few days 80 M. F. GUYER AND E. A. SMITH after birth, No. 4 ate one of hers, and No. 5 lost one of hers at the end of the first week. Thus, only five young of the females treated with sensitized serum survived long enough to get their eyes open. Viewed externally, the eyes of these five young appeared normal. All showed the usual heavy pigmentation and no de- fects were apparent in the lenses. On May 23 all were decapi- tated, the eyes were removed immediately and the lenses care- fully dissected out. In this way abnormalities which were invisi- ble in the living animals came to light. One of the young of No. 3 had both lenses normal in consist- ency and transparency, the other one had the left lens normal, the right markedly clouded. One of the young of No. 4 had both lenses translucent, the other one had an opaque zone around the periphery of the left lens and a relatively large, scar-like white area in the right. From table 5 it will be noted that parents 3 and 4 had received only one dose of the sensitized serum, and that late in pregnancy. The remaining young one, from a mouse (No. 5) which had been given two doses of the serum had both lenses affected, the left being opaque and abnormally small, the right transparent but of a liquid instead of the nor- mal fibrous consistency. The entire eyeball in fact, which con- tained the dwarfed lens was upon removal readily seen to be smaller than the other eyeball, or than that of a normal mouse of the same age. The four young of the surviving injected control, No. 11, were killed the same day and were found to have lenses of normal size, texture, and transparency. It would appear, therefore, that unsensitized fowl serum has no perceptible effect on the lenses of the unborn young. As a further control, six normal young, two sixteen days old with eyes just opened, and four fifteen days old with eyes not yet open, were also killed and examined. Their lenses were all transparent and of fairly, firm texture. The lenses of the treated mothers were also carefully exam- ined, but all had remained of normal consistency and trans- parency. STUDIES ON CYTOLYSINS ral 4. CONCLUSIONS It seems legitimate to infer from the foregoing experiments on rabbits and mice that lens tissue of such forms when injected into fowls excites the production of specific antibodies which may attack in utero the lenses of the young of the species used as antigen. This reaction is not invariable, however, since, so far as one can determine by direct observation, a majority or even all of the individuals of a litter may not be acted upon, or a given individual may be affected in only one eye. The reason for such uneven effects is not apparent. It occurs to one, at first thought, that possibly the placenta is impervious to such antibodies except as occasional rupture of placental blood-vessels might permit of direct mingling of fetal and ma- ternal blood. But such an hypothesis does not account for the fact that only one eye of an individual may be affected. More- over, it seems improbable that mere accident to the placenta would account for such closely similar results as were found in both mice and rabbits. The liquefactions described would indicate a true cytolytic effect. Of the several proteins composing the lens, one is fibrous, and it is upon this that the sensitized serum seems to have operated. Whether or not it had also affected the other ingredients could not be determined. Whether the clouding and opaquing which occurred in others of the lenses should be regarded as the result of a cytolysin or of a precipitin is problematical. Further experiments are necessary to clear up this point. The important fact is that such opacity can be induced by specific sera and that it may be- come permanent. The most striking case among the rabbits would indicate that if the opacity is attributable to a precipitin reaction, a cytolytic effect accompanied it. For in this instance the affected lens has remained so reduced in size that the whole eye has been markedly dwarfed. In so far as the literature on cytolysins records positive re- sults, it leads one to expect specific effects in the immediate animal injected. But as already noted, no such effects were THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 26, NO 1 82 M. F. GUYER AND E. A. SMITH observable in any of the injected females. A possible explana- tion of the lack of effect on the mothers may be that, because of meager circulation of blood in the lenses of adults, the quan- tity of cytolytic serum which reaches a lens is insufficient to affect it. In the developing eye of the young, the circulation is probably much fuller. The lenses in such forms, moreover, are in the process of formation and are not the fibrous masses which exist in older animals. For these reasons the lenses of immature animals are probably more susceptible to cytolytic and kindred agents. The fact of chief interest is that visible specific structural modifications can be engendered in the young in utero by means of specifically sensitized serum. The present paper is to be regarded as a report of progress in a more extensive series of experiments. AUTHOR'S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE MARCH 2 REACTIONS OF THE PROBOSCIS OF PLANARIA ALBISSIMA VEJDOVSKY WILLIAM A. KEPNER AND ARNOLD RICH From the University of Virginia TEN FIGURES CONTENTS Materraleandamethods:---¢nies-- > aoe sere SAG Inia Mace Reh SI Oe Mere OO INGRVOUSISW SLANE Meietie ne Nel vase cme eer etn Dare te err aiare a eater erat as arate ce oles 602 Anatom ycoti bewproboseis) ts) ,ceves | ers Std eis sted ee ite ae 602 Norma lefunetionin gots hespRObOScIs=-aeaaeer coe eee eicr eae 603 HUERELIONS, OFEECE OLD LODOSCIS ere 4cs7.5 cage ee lowe Fens aire ei Asie cuekaln © suche, eee Seer 604 Factors determining inhibition of the proboscis.........................-.. 607 (3) hithtsmotachielconditions 4) ee-n eben ease lane eee ieee 609 (b) en euralim hibition yess ees eerie ae toe See 617 IRE NOUTIO NA 1H Hovore|-Coht AU REYaNG| FaIRO OL OR pen a oogumcopesoconsasaeaadaddinonge s006¢ 610 DNL CHTSIGH I PR oy ee ee Hee Lr eR ee Ne RS crete sincera tae Pa 617 DeraiarerCilede. a5sae pets cise ieee Pareto ee oes lot ne eats Go's SIR aaa 618 MATERIAL AND METHODS The planaria upon which we worked is found in the brooks and spring runs of the mountains about the University of Vir- ginia. Collections were made most conveniently by gathering Vaucheria masses, leaves and stones from the bed of these mountain streams. When such material has been kept in an aquarium jar of tap-water, the worms rise to the surface. We have had no difficulty in keeping these animals for a week or more in tap-water that is free from infusion masses. The animals so collected were fixed in chrom-aceto-formalin and aceto-sublimate solutions. Golgi preparations werealso made, but these yielded us no information concerning the nery- ous system. Iron haematoxylin and Mallory’s connective-tissue stain gave us our best results in studying the nervous system of the flatworm. * 84 WILLIAM A. KEPNER AND ARNOLD RICH NERVOUS SYSTEM The central nervous system of this triclad consists of a pair of dorsal ganglia, connected by a transverse commissure and of two ventral nerve-trunks which give off at more or less regular intervals lateral and mesial branches. We have been able to trace a pair of the mesial branches of the ventral nerve-trunks towards the base of the proboscis, so that there is good reason for believing that the proboscis has a definite connection with the central nervous system, such as Gamble (01) describes for triclads in general. As early as 1897 R. Monti (97) wrote of the ventral nervous system of Dendrocoeles, “J’interprete done les cordons longitudinaux comme une chaine ganglion- naire non encore différenciée.”’ Our own histological studies of Planaria albissima show that the ventral nerves are not mere bundles of nerve-fibers, but present a series of ganglionic masses along their entire extent. Steiner (98) found that these ganglia exercised local control, for isolated posterior portions of Pla- naria neapolitana moved under control of ganglia in these parts of the body. The mesial branch of each of the ventral nerve- trunks leaves the ganglion that lies near the base of the proboscis to enter the proboscis. ANATOMY OF THE PROBOSCIS The proboscis, when freed, is a slightly tapering, almost cylin- drical tube, its posterior end being the wider. Near the ante- rior end—the fixed end under normal conditions—there is a muscular ring, which acts as a sphincter. Both the inner and the outer surfaces of this tubular organ are densely covered with cilia. The wall is highly muscular. A definite nerve- plexus has been describe and figured (Gamble, ’01) for the proboscis of planarians. From what we have seen of the his- tology of this organ, the proboscis does not greatly differ from the description of the anatomy of the proboscis of other Pla- naria. We have left the detailed histological study of this organ for a later piece of work. It is important, however, to mention here the presence of numerous glandular ducts which REACTIONS OF PROBOSCIS OF PLANARIA 85 extend throughout the entire length of the proboscis and open by means of pores along the margin of the mouth. The cell- bodies of these unicellular glands lie within the mesenchyme ven- tral and both posterior and anterior to the base of the proboscis. It is to be noted, therefore, that when a proboscis has freed it- self from the body proper, all of its reactions are carried on without the aid of the cell-bodies of its peculiar unicellular glands. NORMAL FUNCTIONING OF THE PROBOSCIS Normally, as is well known, the proboscis functions as a pre- hensile organ. When the normal proboscis is ingesting food it is extended and its oral end lies projecting well out beyond the mouth of the proboscis-sheath. The oral end, as it ap- proaches food, opens and closes, operating as a grasping, funnel- shaped structure. In addition to this muscular play of the cir- cular lip of the proboscis, there is a movement of the cilia which line its lumen. This ciliary activity aids in carrying food into the opening and closing mouth. A peristaltic wave arises be- hind the food thus carried into the mouth, and this wave next travels anteriorly, driving the food ahead of it against the closed sphincter of the proboscis. After a mass of food has been col- lected near the sphincter, the latter opens and delivers the food to the enteron. This reaction of the fixed proboscis to food is not to be observed frequently under laboratory conditions. We have seen such reactions only twice, while none of the ninety members of the class in general zoology saw it. Food is not, therefore, readily accepted by the uninjured animals when they are being observed in the small amount of water present as the specimens are studied under the compound microscope. We have, however, been able to feed them with ease when they are retained in watch-glasses containing tap-water, thus making it improbable that the tap-water was highly injurious to the speci- mens, as Walter (08) found to be the case for the worms and tap-water with which he worked. 86 WILLIAM A. KEPNER AND ARNOLD RICH REACTIONS OF FREED PROBOSCIS Our attention was first directed to the conduct of the pro- boscis of this planarian while a class in general zoology was studying the worm. It was observed, in many cases, by the members of one section of this class, that, while they were study- ing the specimens under supported cover-glasses, the animals had cast off their proboscides which were swimming .about, oral ends first, by means of their external cilia and ingesting various solid objects. In one instance Mr. Geiger called the attention to the fact that the freed proboscis of his specimen had turned upon its own body and had ‘eaten a hole right through it.’ This observation suggested those made by Leidy (’47) on a planarian which had more than one proboscis. He said, If one of these animals be punctured or cut, one or more of the proboscides will be immediately protruded as if they existed under pressure, and will move about in all directions, appearing as if entirely without the control of the animal; or if one of the animals be crushed between two slips of glass so that the proboscides will be torn from their attachment, they move about involuntarily, always in a line forwards or towards the mouth, which they do by contracting the stomachal extremity towards the oral, the latter remaining fixed. In this progressive course they constantly contract and dilate; the mouth opens and any matter in the vicinity rushes in, when it is closed and the matter passes onwards, and by the alternate contraction and dilata- tion of different parts of the same tube, it is thrown backwards and for- wards several times, and finally violently expelled at the torn extremity. : In fact, these curious independent movements caused me at first to mistake the organs for viviparous young. Darwin (43) also recorded that the proboscis of a land pla- narian of Brazil lives long after the body has been destroyed by salt. Bardeen (’01 a) described briefly the normal food reactions of Pla- naria, and shows that a decapitated specimen will not find food ma- terial in a dish, although such a specimen could “‘be made to eat if it were placed on its back on a slide in a small drop of water. Under the conditions mentioned, the pharynx is usually protruded, and will ‘Mr. W. H. Taliafero called our attention to this reference and that which we have made from Walter (’08). REACTIONS OF PROBOSCIS OF PLANARIA 87 engulf bits of food placed in the mouth.’’ An experiment was performed in which the part of the head in front of the eyes was cut off. Such specimens, from which merely the tip of the head had been removed, reacted normally to food. It is also shown that specimens from which the part of the body posterior to the pharynx has been removed feed like normal worms.” We have observed that the reaction of the incomplete pro- boscis of Planaria albissima is variable. When it lacks more than the cell bodies of its basal glands, it shows but little de- parture from normal conduct—the three codrdinated move- ments of food ingestion being carried out. eS There is greater variability in the reaction of the proboscis when the sphincter at its base is removed, as the following obser- vations indicate. Fig. 1 A, transverse cut removing sphincter of proboscis; proboscis inactive except for ciliary movement. A specimen was cut transversely at A, figure 1, so as to free a proboscis which lacked the sphincter. The reaction of the cilia of the external surface of this proboscis carried it from the proboscis sheath. The proboscis then lay for ten minutes by the side of a piece of planarian body and then, without showing any reaction to the food, slowly moved away. There were no opening and closing movements of the mouth on the part of this specimen and there were no peristaltic movements in the wall of the proboscis. In another experiment, after two parts had been removed from the body of the specimen by transverse cutting (fig. 2, B and C), a third cut was made so as to pass through the base of the proboscis (D). Here again the pro- boscis, less a sphincter, swam from the sheath by ciliary activity and then lay quite inactive. Next the proboscis was severed ? Quoted from Pearl (03), p. 523. 88 WILLIAM A. KEPNER AND ARNOLD RICH as indicated at (Ff). Immediately the oral end opened and closed in an indifferent manner, so that food, which lay near the mouth, was not ingested. No peristaltic waves arose in the aboral part of the organ. Finally, in a third specimen (fig. 3), we had a case in which the freed proboscis was quite active when complete (G); but there were evident no longer any of the movements which play a réle in the ingestion of food when the oral fourth of the proboscis was removed (H). A marked variation from the reaction of the above incomplete proboscis is A —r Fig. 2. A, entire animal; B, dorsal ganglia and anterior portion of body re- moved; C, poster portion of body cut away; D, proboscis severed through base, sphincter removed, proboscis then swam from sheath and remained inactive; E, free proboscis; F', proboscis severed transversely, oral end opened and closed in an indifferent manner. oe oer | that of the specimen shown or represented in our figure 4. Here the sphincter (D) was cut away, and though the movements of the remainder of the proboscis were not normal (more spas- modiec), still the proboscis mouth explored for food and ate. The larger part of this proboscis swam about, and in coming by its own detached sphincter accepted the latter by greedily ingesting it. Unless, therefore, the proboscis be complete enough to per- mit of all of the three coédrdinated movements being effected, no two and usually no one of this set of movements will be well carried out. REACTIONS OF PROBOSCIS OF PLANARIA 89 FACTORS DETERMINING INHIBITION OF THE PROBOSCIS We have been interested most in an effort to determine what the inhibitory influences are which control the fixed proboscis in such manner that it rarely ingests objects under conditions D a iGo tical Fig. 3 B, dorsal ganglia and posterior extremity of body cut away; C, more of anterior portion of body removed; D, posterior half of proboscis-sheath cut away; H, entire proboscis-sheath removed, proboscis becoming active (F); G, proboscis active after autoamputation; H, proboscis inactive after oral fourth was cut away. favorable to microscopic study, thus making the fixed proboscis stand in sharp contrast to the freed proboscis, which latter under laboratory conditions may readily be demonstrated in- gesting food and other solid objects. It has occurred to us that one of these inhibitory factors may be external and the other internal. 90 WILLIAM A. KEPNER AND ARNOLD RICH The observations of the men of the class in general zoology suggested that perhaps a disturbance of thigmotactic conditions resulting from shallow water or pressure of the cover-glass were responsible for the autoamputation of the proboscis and that its reaction was not due to disorganization of the body or parts of the body. This suggestion is further supported by two ob- servations made later by ourselves. On two occasions we had ee De ee ae Fig. 4 B, two-thirds of proboscis-sheath and posterior portion of body cut away, proboscis active and ingesting body proper; C, proboscis active after auto- amputation; D, sphincter of proboscis removed. drawn away the water from specimens, so that the worms had but a mere film of water over them. In these specimens, thus stranded in quiet shallow water, it was noticed that the proboscis had broken from the body in each case and was swimming or wriggling about within the sheath. ‘To one specimen water was added as soon as the proboscis had freed itself. After the ad- dition of this water the proboscis left the sheath and swam about in the water quite as actively as did the body proper ' from which it had been separated. In the other specimen the body proper, while in the shallow water, became so intimately REACTIONS OF PROBOSCIS OF PLANARIA 91 fixed to the slide that when water was added it appeared as a distorted mass which the freed proboscis was ingesting. More- over, the proboscis when inactive lies within a sheath, the walls of which lie more or less in contact with the quiet proboscis. It has been noted that, when the proboscis is extended to pro- ject from the mouth of the sheath, the part that lies free is the portion of the proboscis that is actively contracting and mov- ing. The part within the sheath shows little or no activity until food passes through it. Hence we tentatively inferred that ab- sence of contact with the walls of the proboscis-sheath excites the proboscis into activity. The following experiments were made to determine whether the absence of thigmotactic stimuli excites the proboscis beyond the inhibitory control of the central nervous system. 1. A specimen had its anterior and posterior ends cut away (fig. 3, B). Next an additional part of the anterior end was am- putated. There was up to this step no reaction on the part of the proboscis (fig. 3, C). The posterior half of the proboscis- sheath was next cut away and as yet the proboscis remained quiet (fig. 3, D). When, however, all of the sheath was removed, al- though the proboscis remained joined to the fragment of body at its base and hence in connection with a part of the central nervous system, it became active at once, turning upon the frag- ment of body and ingesting part of it (fig. 3, F). F inally, the proboscis underwent autoamputation and swam about attempt- ing to ingest objects. This reaction and autoamputation of the proboscis may have been due, in this case, to a mechanical injury of the central nervous system. 2. In this second experiment we had the proboscis incited to activity under other conditions. Here, after the anterio and posterior ends had been removed (fig. 5, B), the proboscis left the sheath when an effort was made to pull away the sides of the proboscis-sheath with two needle points. While the sheath was thus temporarily spread the proboscis swam out. Here again there was a temporary disturbance of the thigmotactie condi- tions existing between the proboscis and its sheath: but the ques- tion of disturbing the central nervous system, during the opera- tions, also presents itself. 92 WILLIAM A. KEPNER AND ARNOLD RICH 3. Finally, we have in our third experiment a case in which a posterior part of the body was amputated so as to leave at least two-thirds of the proboscis projecting free (fig. 4, A, B, C). Here the possibility of mechanical injury of the con- trolling portion of the central nervous system during the opera- tion is much less, and yet the proboscis immediately became ac- tive and turned anteriorily in an effort to ingest part of the body to which it was attached. The proboscis in this specimen soon underwent autoamputation. This last is a strikingly exceptional case and is the only example in which we have it suggested that thigmotactic stimuli, inde- pendent of nervous control, are factors in the inhibitory control A B fae eS) Fig. 5 3B, anterior and posterior extremities removed. On attempting to open sheath with needle points, proboscis underwent autoamputation. of the proboscis. Even here there may have resulted an injury of the ganglia or nerves of the proboscis, so that the breakdown of the inhibition was due to nervous injury and not to thigmo- tactic disturbances. We have, therefore, no clear case that shows that thigmotactic disturbances will alone excite the proboscis into abnormal activity. Our experiments, however, indicate that the central nervous system acts as an inhibitor to the proboscis. Not all of the central nervous system seems to be directly concerned with this inhibitory control. 1. The dorsal ganglia of a specimen were amputated (fig. 6, A), and no marked disturbance of the proboscis followed. Ten minutes later a second cut was made midway between the base REACTIONS OF PROBOSCIS OF PLANARIA 93 of the proboscis and the first cut (B) ; three waves passed over the proboscis during five minutes, otherwise it was quiet. Then a cut was made quite near the base of the proboscis (C) and the latter writhed in the sheath for two minutes, then became in- active. The next cut was made at the base of the proboscis, and at once the proboscis became active and wriggled out of a tear in the sheath that had been made accidentally. After ninety seconds the proboscis suffered autoamputation (D, EF). A (aaa Fig.6 Dorsal ganglia amputated; B, more anterior portion removed; C, addi- tional removal of anterior part of body; D, cut made near proboscis base, pro- boscis in sheath; EZ, proboscis underwent autoamputation and wriggled from rent made accidentally in sheath. 94 WILLIAM A. KEPNER AND ARNOLD RICH 2. A specimen was impaled upon a needle point near the right-hand margin of the proboscis. The animal, in freeing it- self, made a tear in its body that passed obliquely from near base of proboscis-sheath posteriorily to right margin of body (fig. 7, A). The proboscis swung out from this rent and pro- jected as a passive, quiet object. Next the sheath and poste- rior part of the body were torn away leaving the proboscis trail- Fig. 7 A, rent made at right side of proboscis near its base, proboscis swung out, inactive; B, posterior portion of body removed, proboscis inactive; C, dorsal ganglia removed, proboscis inactive; not accepting food (F’); D, further portion of anterior part of body removed, proboscis inactive, not accepting food (F); E, cut made near base of proboscis, proboscis active. ing behind the swimming animal (8). In this condition the proboscis remained perfectly quiet. The dorsal ganglia were amputated and a piece of food pushed after the swimming ani- mal near the mouth of proboscis, but as yet there was no re- action on the part of the proboscis (C). One-half or more of the remaining anterior portion of the body was cut off. After REACTIONS OF PROBOSCIS OF PLANARIA 95 this the specimen swam no longer and a piece of food was placed in contact with the oral end of the proboscis; still the proboscis remained quiet (D). Finally, when the portion of the body proper was reduced to a piece relatively as small as indicated in figure 7, EH, the proboscis became active at once, swung to and A B c 2 F c Fig. 8 A, rent made by animal tearing from needle point upon which it was impaled; B, similar rent made on other side, completely exposing proboscis, proboscis inactive, not accepting food (F); C, portion of one side of body cut away at proboscis base, proboscis yet inactive, not accepting food (F); D, dorsal ganglia amputated, proboscis inactive, not accepting food (F'); HZ, cut near base of proboscis, proboscis active. fro, and in a moment broke away and swam about ingesting fragments of its own body for more than seven minutes. 3. Figure 8 shows a rent made in a specimen by impaling it on the right side of the body near base of the proboscis and holding it on the needle until the animal tore from the needle. In a like manner a similar rent was made at the left side of the 96 WILLIAM A. KEPNER AND ARNOLD RICH body. When this second rent was made, the posterior part of the body was torn away (B). In neither case when the rents were being made did the proboscis show any reactions, though food (Ff) was pushed after the projecting proboscis of B. The projecting part of the body to right of the proboscis was ampu- tated, and as yet no reaction of the proboscis followed (C). About half of the remaining anterior portion of the body was next removed. The specimen no longer swam and when food (F) was placed near the oral end of the quiet proboscis, the latter showed no response (D). Finally practically all of the body A. Fig. 9 A, cut made removing left lateral nerve-trunk from region of pro- boscis, proboscis inactive; 6, cut made removing dorsal ganglia and right lateral nerve-trunk from region of proboscis, proboscis active. proper was removed, as indicated by EH, and immediately the proboscis set up exploratory movements and a swinging to and fro. Eventually the proboscis broke away and, as it swam through the water, pushed a piece of its own body ahead of its mouth. Further evidence of the inhibitory influence of the central nervous system is to be seen in experiments like the following, in which the specimens were cut more or less longitudinally instead of transversely. 4. In specimen shown in figure 9 a lateral cut was made with a razor, which severed the connection between the proboscis REACTIONS OF PROBOSCIS OF PLANARIA 97 and the ventral nerve of that side of the body. There was no evidence of reaction of the proboscis. Then the other margin of the body with its ventral nerve was cut away, and, though there was no damage done to the sheath or to the base as such, the proboscis at once suffered autoamputation and swam about ingesting food (B). A SSS F g.10 A, cut made on right side of body too shallow to include lateral nerve- trunk, proboscis inactive; B, cut on left side removing lateral nerve-trunk, pro- boscis projected, but remained inactive; C, second cut made on right side remov- ing other lateral nerve-trunk, proboscis active. 5. Figure 10 shows an experiment that yields evidence of this nervous inhibition. First a very narrow edge of the body was cut away—too narrow to include the nerve-trunk. There was no reaction of the proboscis. Then, from the other side a strip was cut deep enough to take away the nerve-trunk of that side. THE JOURNAL OF EXPDRIMENTAL ZOOLOGY, VOL. 26, NO. 1 98 WILLIAM A. KEPNER AND ARNOLD RICH The proboscis projected itself from the sheath, but hung there perfectly quiet, showing no signs of activity. After several minutes a second strip was cut more deeply from the side from which the first narrow slice had been taken. This second cut was deep enough to remove the nerve-trunk of that side, and the proboscis at once underwent autoamputation and became active. It is thus indicated that the severing of connection between the proboscis and both lateral nerve-trunks is necessary, when the sheath is intact, for the removal of inhibition sufficiently to cause the proboscis to become hyperactive. The manner in which the basal ganglia have been removed in our experimenting is rather crude. Needles, scalpels, and razors were used in this work. It is quite possible that in these opera- tions not only were the nerve connections destroyed, but pres- sure in a varying degree must have been brought upon the proboscis-sheath. This pressure as it varied might have dis- turbed the thigmotactic conditions within the proboscis-sheath. This latter contingency may account for certain variations in the reaction of the proboscis as observed by us. For example, two of three animals that had been severed but once, and that near the bases of their proboscides, underwent autoamputation of their proboscides and had their prehensile organs leave their sheaths. The proboscis of the third specimen wriggled actively within its sheath, but only swam out of the sheath when a hole was made in the sheath with a needle. In addition to this variation of reaction on the part of the proboscis, each of us has had cases in which, after the ganglia adjacent to the base of the proboscis had been removed, no autoamputation resulted. More experimentation is needed to determine the cause of this variation. At present we can only suggest that in cases like these last ones we had caused no disturbance of thigmotactic conditions in the sheath when we were removing the ganglia, hence the proboscis was not excited and showed no response, though the inhibition of the nervous system had been destroyed. It is indicated by the above experiments that the removal of the ganglia posterior to the base of the proboscis does not ma- terially disturb the control of the organ. The amputation of REACTIONS OF PROBOSCIS OF PLANARIA 99 the more anterior portions of the central nervous system may result in slight disturbance of the proboscis, while the ganglia adjacent and anterior to the base of the proboscis ordinarily act as inhibitors to the ingesting reflexes of the proboscis. REACTION TO FOOD OF FREED PROBOSCIS The ability of the proboscis that had undergone autoampu- tation to distinguish between food and non-food was tested in the following manner: After a proboscis had separated from the body it was washed in several changes of fresh tap-water to free it from any solutions or particles of its own body, than might ‘serve as a stimulating agency to the proboscis. Frag- ments of washed cover-glass were placed into clean water that contained such a washed proboscis. In some instances these fragments of glass were accepted by the proboscis and were passed by the sphincter and thrown from the organ as food is handled by free proboscides. The ingesting of objects by a freed pro- boscis displays no choice, hence it is a reflex. CONCLUSIONS 1. All proboscides of Planaria albissima that have been sev- ered from their adjacent ganglia show some reaction by dis- turbed movements within the proboscis-sheath. Most of the proboscides thus separated from the central nervous system underwent autoamputation while lying within the sheath. Ina relatively few instances we have found that, unless the pro- boscides in addition to being cut off from the central nervous system have been excited by a disturbing of the thigmotactic conditions within the sheaths, they do not undergo autoamputa- tion. In all cases, however, the disturbance of the thigmotactic conditions of the sheath so excites the proboscis that, without the in- hibitory control of the adjacent ganglia of the central nervous sys- tem, the proboscis suffers autoamputation and acts as an independent reflex organism. 2. The freed proboscis is able to carry out the three co- ordinated muscular movements involved in the mechanics of 100 WILLIAM A. KEPNER AND ARNOLD RICH food ingestion, but this only when the entire musculature of the proboscis is intact. The freed proboscis had not the ability to distinguish between food and non-food. The exercise of choice is made possible only through the functioning of the central nervous system. LITERATURE CITED Darwin, CHaruLes 1843 Journal of researches during the voyage round the world in H. M.S. Beagle, 2nd ed. New York. GamBiE, F. W. 1901 The Cambridge natural history, vol. 2, London. Lerpy, JosepH 1847 Description and anatomy of a new and curious subgenus of planaria. Proc. Acad. Nat. Sci. Philadelphia, vol. III. Monti, R. 1897 Sur le systéme nerveux des Dendrocoeles d’eau douce. Arch. Italien. de Biol. T. 27. PEARL, Raymond 1903 Movements and reactions of fresh-water planarians: a study in animal behaviour. Quart. Journal Micr. Science, vol. 16. SreINER, J. 1898 Die Functionen des Centralnervensystems und ihre Phylo- genese: Dritte Abth. Die wirblellosen Thiere. Braunschweig. Water, Herpert E. 1908 The reactions of planarians to light. Jour. Exp. Zool , vol. 5. AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE MARCH 30 ON SEVERAL EFFECTS OF FEEDING SMALL QUANTI- TIES OF SUDAN III TO YOUNG ALBINO RATS! S. HATAI From The Wistar Institute of Anatomy and Biology THREE CHARTS Daddi’s (’96) observations on rabbits, guinea-pigs, and fowls which showed that Sudan III mixed in oil is readily absorbed from the alimentary tract, and is ultimately deposited in the adipose tissue, induced numerous investigators to study the problem of the metabolism of fat by the use of this dyestuff. Recently Ehrlich and his pupils demonstrated that certain dyestuffs can stain some micro-organisms in vivo, and indeed in some instances such chemical substances thus introduced into the animal body produce a curative effect. This new line of investigation, now called ‘chemo-therapy,’ induced numerous investigators to ex- amine the possible affinities between organisms and various dye- stuffs, including Sudan ITI, and consequently the literature on the feeding of Sudan III is quite large. I shall therefore review only a few studies which are intimately concerned with my own experiments. Riddle (08) fed Sudan III to hens during the laying period, and demonstrated that this dyestuff is easily absorbed into the egg and the yolk fat intensely stained. In another paper, Riddle (’10) gives further observations on feeding Sudan III to domestic fowls, and not only extends his 1 This research was closed and set aside in January, 1916, because it had not given the information originally sought, and the further study of the atrophy of the thymus and other viscera did not fall within our program. The problem of the changes in the thymus has now been taken up by Dr. Ivan Wallin, and in view of the fact that the work is to be carried on, it has seemed proper to pub- lish the results already in hand.—s. H. 101 102 S. HATAI earlier observations on the deposition of Sudan III in ova as well as in the soma, but reports also his incidental observations on the growth of the chick fed with Sudan III, as well as those on the question of fat metabolism. In brief, he finds that Sudan ITI tends to retard the growth of the chick. He also notices the production of defective feathers among these test chickens. Gage (’08) repeated Riddle’s experiment and found that while Sudan III is deposited in the hen’s egg, it does not appear in new-born albino rats whose mother has been fed with this dye- stuff for some days previously. Gage thinks that the placenta prevents the entrance of Sudan III into the fetal circulation. In 1912 Mendel and Daniels published their extensive observa- tions on fat metabolism, using not only Sudan III, but several other fat-soluble dyestuffs. These investigators touch on nu- merous interesting problems regarding the fate of the dyes after these are taken into the animal body, and I shall later on refer to this important paper. Another study was made by Corper (712), who fed guinea-pigs with several dyestuffs which are soluble in fat, including Sudan III, with a view to studying the biochemistry and chemo- therapy of tuberculosis. Corper fed the animal with a rather large dose (0.025 grams per diem) of Sudan III for long periods (over 200 days) without apparently producing any ill effects. While I was feeding Sudan III to young albino rats to deter- mine whether it appeared in the newly formed myelin sheaths, I noted that the rats which were receiving a small amount (about 8 to 9 milligrams per diem) of Sudan III mixed in olive oil, not only failed to grow, but appeared in many respects strikingly abnormal. Preliminary examination of the organs revealed the fact that most of these organs were distinctly altered in appearance. The most noticeable alteration was the complete atrophy of the thymus in every one of these seven test rats. Inasmuch as these alterations were not recorded by previous investigators, and par- ticularly because of the behavior of the thymus towards Sudan III, the present investigation was undertaken. FEEDING SUDAN IIl TO YOUNG ALBINO RATS 103 METHOD OF INVESTIGATION For this investigation albino rats alone were used. The rats were from 27 to 33 days of age and were still running with mother. The litter was divided into two groups; one group, the controls, was fed with Austin’s dog biscuit, and the other, the tésts, received approximately 0.008 to 0.020 milligrams of Sudan III (Griibler’s) in the form of a solution in olive oil well mixed with about 5 grams of the powdered dog or rat biscuit. The amount of oil? used was about 1 to 2 cc. per rat per day. When 20 milligrams of Sudan III were given the dose was found to be too strong for the young rats to withstand for more than one week, and indeed at the end of one week the test rats were so emaciated and sick that it was necessary to return the animals to the normal-diet for recovery. Altogether 135 rats belonging to 19 litters were used, and from the study of them I can present a few examples of the growth of the body and organs of rats fed with Sudan III mixed in olive oil. In each series the controls and the test animals were always from the same litter. GROWTH OF BODY IN WEIGHT Table 1 gives the growth record for five of the series. These are typical. The corresponding graphs for the growth of the body in weight as the result of Sudan III feeding are shown in chart 1 for series I, II and III and chart 2, for series VIII. Both charts show clearly that the rats fed with Sudan III do not grow as well as their litter mates of the control group. The amount of Sudan III administered was small (8 milligrams), nevertheless it is sufficient either to inhibit the growth entirely or retard it to _a considerable extent. In general the younger rats appear more sensitive to Sudan III than the older. In fact it has been found that the rats with body weights of more than 50 grams show a high resisting power to this dyestuff, and it requires a consider- ably longer period to produce marked results. 2 The oil used in all of these tests carried the trade-mark: Extra fine—James Wagner (Philadelphia)—Made in France. It was not tested chemically. 104 S. HATAI TABLE 1 Showing the growth in body weight of albino rats fed with Sudan IIT dissolved in _ olive oil, contrasted with the control rats fed with the normal laboratory diet. In series X the control rats were fed with the laboratory diet plus olive oil alone. SERIES I, II, AND III SERIES VII. SERIES X Sudeep Sudan SSO regen Control peer anya Control aaa aii See 10-15 8-9 mgms. mgms. i te lara fai fh | tir Pettepetieie+aciie tae Days after Sudan IIT feeding Initial 30.0) 32.1 AAA 302 5)\| SORON| AIO 28 eal 29 a7. | 32240) 32a if AGES | 41 9r | o85)'384-6) | S6e2n AZe a tobe ONO SoSal Esse 10 A) 3 | 29.2'| 41.3 | 34.8 14 68.4) 58.9 F150) 38.9 | (39208) 502084629) 30h 48h Or 3549 17 | Ole PSOr4a O2konlsons 21 86,5] 24087a) $900) 40.4.) AOK4 bbe oN OO Gill 5 Os es7a4: 25 OBI I Bee! | Cesiy Za Gy | SYM ert WW Sioeeb ly (RileAe |) S¥Rea 28 97.8) 80.9 | 103.4) 42.3 | 42.0 | 55.0 | 68.4 | 29.6 | 69.1 | 36.4 31 106.4] 87.8 | 111.0) 42.8 | 41.5 | 51.5 35 THORS | S90 S2 FAS 54250 Tale be 00 Series I 28 days old at the beginning of the experiment. Series II 29 days old at the beginning of the experiment. Series III 33 days old at the beginning of the experiment. Series VIII 27 days old at the beginning of the experiment. Series X 28 days old at the beginning of the experiment. Riddle found that chicks do not grow well on a Sudan III diet, and furthermore the Sudan chick produces defective feathers. Unfortunately, Riddle does not give data concerning the body growth, and thus the extent of the injurious action of Sudan ITI in his experiments cannot be judged. As has been stated al- ready, the majority of investigators fed Sudan III to the adult . animals. This may account for the fact that they did not notice any toxic action of the Sudan. It is also possible, however, that rats are more susceptible to Sudan III than either rabbits or guinea-pigs. This point needs to be further investigated. Since the test rats in the earlier series received Sudan III mixed with olive oil, while the control rats did not get any oil FEEDING SUDAN III TO YOUNG ALBINO RATS 105 at all in their ration, it is conceivable. that the olive oil might be the cause of this phenomenon, and not the Sudan dye itself. In fact, it has been noted by Adler (711) that olive oil is highly toxic to rabbits and when given to the young, the growth is BODY WEIGHT SE ren 110, cite BSge ade a HEHEHE aie BeBe 100 a= B Baa 90 PEEEEEEE EEE A [s] Enea scat cast fovaetasite Bas 80 710 BS Ba |_| 60 BEBE zo Rae SEES ewe: sedesiaceterecstestesttaratastt ef fa cam a | ML | | Phe EREDAR eawsaa 44 50 EREDAR BREARRGEBD TT = al (al (2) a nese r | | ao | AL a | — 40 a paneer I EEE Raragene a0 Re REO Ia a aan eee e le eae so4ne-an REE EEE EEE BEE - je cae Swale EEE EEE EEE PEE EEE EEE CCE EEE EEE Beni Ee eae slate Be iat de Pe bei le imisiehensioeteh: [ets isles er nt: Pie ich BE rE tae aan SEeBagaa aeSRSas 0 10 20 30 40 DAYS Chart 1. Showing the effect of feeding a small quantity of Sudan III on the growth of the young albino rats. Series I, I and III. o----o Control e——e__ Experimented materially retarded. He states that 6 ee. per kilo killed in sIx days when fed daily; 5 cc. per kilo weight did not kill, but pro- duced secondary anemia with blood crises presenting the picture of pernicious anemia. 106 S. HATAI I have therefore carried two series of experiments in the fol- lowing manner. One half of the litter received biscuit and olive oil, while the other half received Sudan III in addition. The amount of oil given was the same in both series. The results show, table 1, that the olive oil alone does not modify the growth of the body and, furthermore, the organs in the controls were not at all modified (table 4). The growth of the rats fed with So Peas" SOUS SSEEESEEETEUCEEGEEE eeaeteyas a ae aacenooe can Be HEEB SESSERSEeS BREREEEROReCEEe BRE BEBREEDoEaeEe 20 ERREEERREEEaaBs SRERBSERSEee 0 10 20 30 DAYS. Chart 2. Showing the effect of feeding a small quantity of Sudan III on the growth of the young albino rats. Series VIII. O---- 0 Control ) @ Experimented biscuit plus oil and the rats fed with biscuit plus oil and Sudan III is shown in chart 8. I am therefore confident that the olive oil alone, as here used, is not toxie to the growing rat when given to the amount of | to 2 cc. per rat per day. It must be stated, however, that I have not yet tested the rat with higher doses of olive oil. FEEDING SUDAN III TO YOUNG ALBINO RATS 107 GENERAL APPEARANCE OF THE RATS FED WITH SUDAN Ill The rats fed with Sudan III appear like those severely under- fed. The hair becomes rough and erected, the spine is curved, thus producing an appearance of elongated limbs. Neverthe- less, the rats are very lively and hop around and play with each other as actively as the controls. The urine becomes deep pink usually within two or three days after the Sudan II feeding is BODY WEIGHT GMS. Sx Tf ae a a a 0 10 20 30 DAYS Chart III. Showing the growth of the albino rats which received a small quantity of the olive oil in the normal ration contrasted with the rats which received in addition a small quantity of Sudan III. O---- 0 Control (oil fed) ee Experimented begun. The feces are hard and black in color and ether extrac- tion reveals the presence of the dye in a considerable amount in them. ‘The rats are eager for the experimental ration for the first two or three days. but afterwards they consume but very small quantities. The skin around the urethral opening is usu- ally wet and stained pink, owing possibly to lipuria. I have not determined the maximum length of time during which the rat can survive on this Sudan III ration. 108 S. HATAI TABLE 2 Showing the growth of the organs of the albino rat as affected by feeding with Sudan III plus oil. Dose 10 to 15 mgms. Younger series INITIAL MEASURE- SUDAN III AND OLIVE OIL FEEDING CONTROLS LENGTH = MM. MENT WEIGHT = GRAMS fee ae oteelsatrolsgtie|iat29 Bodylencth. <1) Meee. 8.) 94.8 94.7 94.5 96.3 (yas Matslencinee sameness a iho 78.5 | 79.3 PS) ie) 96.0 Body weight... er... 0. || 22.9 21.5 OBA 24.3 44.2 Braiiicss) a5 See eee cee | 369° It ete SB Oa emIg Ree aL eso 1.463 KG dn ys: tux o>. eee ee eee || O2319) | 505292 0.323 0.354 0.566 DA VET ga os ie Sem eee de! | 1.260 9) Save mee OS 7a e304 3.102 Spleens... 4) eee eete.| 10:066" | MOROSIe me ONOGSE a MONOGS 0.218 IR AM CCAS Aes eRe ee ee 0.166 | 0.200 0.226 0. 248 0.449 AB yAnUs, 3.) Ree en: | 0.043 | 0.012 | 0.012 | 0.007 0.127 Suprarenals7. 4 See ate ore. ¢ 0.009 | 0.009 0.010 0.010 0.014 Thyroid. .c.0. 6) ae retry st | 0.005 | 0.005 | 0.005 | 0.006 0.008 Ely pophysisseceen sere eee | 0.0014 | 0.0012} 0.0013 | 0.0014 0.0020 PEStGS 2:00 yA ae. 0.130: 4) 402098=)| “01087 11405087 0.157 Ovaries: s--co eee eae | 0: 0086300051 0.0038 0.0084 Age: (days); saeneenees|, 28 33 35 38 38 GROWTH OF ORGANS AS AFFECTED BY THE FEEDING OF SUDAN III In order to determine the effect of Sudan III on the growth of the organs, young rats just weaned were treated in the following manner. One or two rats were measured and the weights of the organs determined at the beginning of the experiment. The re- maining rats were examined at different intervals in order to compare the organ weights at later ages with those at the begin- ning of the experiment. At the end of the experiment the rats which had been kept as controls and which were receiving the normal ration were examined together with the last test rats. The object of this final procedure was to show the amount of growth the organs should have made had the rats not been sub- jected to the Sudan III diet. The last test group was also util- ized to determine the percentage of water and the lipoids in the organs. The results of these examinations are given in table 2 for the younger, and table 3 for the older series. FEEDING SUDAN III TO YOUNG ALBINO RATS 109 TABLE 3 Showing the growth of the organs of the albino rat as affected by the feeding of Sudan III. Dose 8 to9 mgms. Older series INITIAL MEASURE- | SUDAN III AND OLIVE OIL FEEDING CONTROL LENGTH = MM. MENT WEIGHT = GRAMS 37462 ngeye eee - 2 See 27439 Bodyalengthesrrren ss er 108.1 TMG 116.3 119.5 15de2 Roa penton heee abe oe eit cit 2 86.2 94.8 99.0 109.0 139.4 Body weight........ 33.9 fl ell 40.1 44.7 98 .4 ISTaND ils Seika at eos comets ier oe 1.379 1.412 1.412 1.438 1.598 Ket ney 8 shes ae sa ee 2 0.461 0.473 0.482 0.446 0.794 TGivierss oe) ee ee eras ce 2.063 PD ASE 2.887 22615 3.861 Spleen). ° Aa yeepes see see. ss) 0.142 0.151 0.222 0.273 0.550 Ban CREaS! eee a oe ee ses oe 0.280 0.302 0.341 0.421 0.703 AUREUS: + Styrene 0.107 0.073 0.067 0.031 0.273 Supranenalse reac ct du. a. 0-2 0.014 0.013 0.014 0.013 0.023 GN nivawey (0 WAR chore: Glocieane c.g eee a 0.008 0.014 0.015 0.018 0.020 Eby popiysiste+ 52h ap ssc! 0.0016} 0.0018; 0.0019| 0.0019) 0.0040 ESTES Hee Cyeay Ao 8 SETI Lert s es 0.202 0.199 0.0229} 1.262 Oivaries# ches ey: 7 eee es eae 0.0107; 0.0084 0.0065} 0.0152 INGENIGAYS Rae eee is2 Shi. lags 29 36 43 64 64 From table 2 we note that despite the fact of almost no growth of the entire body of the test animals, such organs as the liver and pancreas made a striking increase in weight throughout the entire period of the Sudan III feeding. On the other hand, the thymus gland was greatly atrophied, and the sex glands also showed a marked diminution inweight. Noneof the other organs showed noticeable loss when compared with those of the rats examined at the beginning of the experiment. These alterations just noted appear also in the second series of the experiments (table 3), where a much smaller dose of Sudan III (8-9 milligrams) has been given. I may add here that al- though on the average the kidney weights of the Sudan-fed rats do not differ much from their initial weights, nevertheless the individual records show that in the majority of cases the kidneys of the test rats have suffered a slight atrophy. I am thus inclined to believe that the kidneys also undergo a slight atrophy as the 110 S. HATAI TABLE 4 The weights of the organs determined in both control and Sudan III fed rats at the end of the experiment SERIES IV SERIES VII AND VIII gPRIES IX AND X Dose Dose Dose ABSCESS Le 8-9 mgms. 10-15mgms.| Control | 8-9 mgms. VeRO! SN Control |Sudanand| Control |Sudanand| oiland | Sudan and 1o¢+19 | oil fed for 29 oil fed for biscuit | oil fed for 21 days 28 days 20 28 days PMCof) tl f9; 29 2c Body length......... 129.0 102.7 146.5 110.0 142.5 109.5 Marlilenethe.....s02 7 113.0 83.7 137.0 105.0 130.0 101.0 Body weight......... 52.0 26.1 | | 7958 36.8 71.9 33.9 BYES Wh pa ee Se ane er ge 1.490 1.338 1.516 1.4383 1.604 1.478 Eeantaetan eee. 0.372 0.190 0.371 0.191 GANGS et eee 0.473 0.27 0.721 0.394 0.641 0.423 GIVER ys Cette 2.219 1.992 3.073 2.046 2.588 2.174 ID(Tan ye ene cosas manana 0.516 0.344 0.488 0.277 Sleehieg rarer 0.255 0.123 0.167 0.079 0.148 0.074 IPSNCEeas. ase. asc aoe 0.425 0.268 0.686 0.3865 0.591 0.348 AMONAITER fon anekeob occ 0.146 0.015 0.256 0.030 0.206 0.012 Suprarenals.......... 0.015 0.009 0.025 0.010 0.019 0.011 AM tKaRONG AS snadaoweeoe 0.009 0.010 0.020 0.010 0.0138 0.008 Hypophysis.......... | 0.0023 0.0013 0.0029 0.0018 0.0029 0.0019 MP OStes nie ect ere: 0.341 0.071 0.818 0.201 Ovaries! eee. 0.0097 0.0070 0.0149 0 .0052) Ager(days)ie set 48 48 50 58 57 57 result of feeding Sudan III. Some of the organs, however, in- crease in weight. ‘The increase of the liver in weight seems to be due to an accumulation of fat, as this ean be shown histologi- cally, as well as by the amount of lipoids extracted (page 113). The increased weight shown by the pancreas may be due to the hyperactivity of this organ in connection with the metabolism of fat. The atrophy of the thymus gland as the result of feeding Sudan IIT is most noticeable. The rate of thymus atrophy seems. to depend on the amount of Sudan III administered. A larger dose appears to reduce the thymus much more quickly than a smaller dose. Since the thymus is highly sensitive to almost any abnormal physiological condition, such as an exposure to Roentgen rays, inanition, etc., we may assume that the present FEEDING SUDAN III TO YOUNG ALBINO RATS 111 TABLE 5 Showing the percentage of water in the various organs of Sudan III fed rats con- trasted with those of control rats ce pene taller le are Sudan | , | Sudan | a | Sudan Control | Sudan eee eee) eet | ee or 2P+19 * | 2A+19 ear 2g Body length...... 129.0 | 102.7 | 153.0 | 120.2 | 146.5 | 110.0 | 142.5 | 109.5 Body weight...... 52.0} 26.7 | 99.0) 46.4) 79.8 | 36.8] 71.9 | 33.9 AGEs CRYS i aecen 2= 48 48 63 63 58 58 57 57 BGO: o,f 2 2ae ea 81.6 | 83.0] 80.7) 83.0) 80.9] 80.8| 81.6] 82.1 Brain.. MOs4\ 29.0 |) 792 One 7826.) (79-0 We78:6Ht 7928 | 7866 Heart. tae. * Tisae gaeGn) 07.0 WW 6eS\\ 77148 | 27-2 HKadneys!) se. oon. 1078) 1) ©2620 | Fone areal Ocoee coed | 2OLO) Get HOVCR hae) eae 73.1 67.1 70.9 68.2 (AO POLe are Wie (2d lene. Skee TEIN LEN El cone ON eae el>(eudbe or | dasa\ |) eek Fes eee ae 78.2 | 80.2] 79.2) 80.0| 79.4} 79.8 Panereas. 4.955... 2:40 16.9) C7 Say ionke 86929" |) 7426 (b6922) || “76:0 Phy muse 3. 52200 13. G7\ SO° 94 97927 | 80258) 792% | 78-1 instance of atrophy may be due either to the abnormal nutri- tional condition or possibly to a direct toxic action of Sudan III on the thymus. I have given in table 4 (see also the last two columns in tables 2 and 3) the weights of the organs determined in both control and test rats at the end of the several experiments. The organs given in table 4 were used for the determination of water and lipoid content, as well as for the demonstration of the presence or absence of Sudan III in various organs, by both microscopic and extraction methods. As we should expect, the: organs of the test rats are smaller than those of the controls. It is interesting to note also that even the liver and pancreas, although they have made a continuous growth during the ex- perimental period, are yet far behind those of the control rats. I wish to call special attention to the case of the control rats which received the same amount of olive oil as the test rats (series [X and X). In this case we notice that the control and test rats show the same degree of difference in the organ weights as in 112 S. HATAI TABLE 6 The percentage of water in the brain of Sudan III fed rats contrasted with that in the brain of the control rats | BRAIN WEIGHT, BRAIN WEIGHT, | GRAMS. | GRAMS | PERCENTAGE PERCENTAGE AGE | NO. SEX | OF WATER AGE NO. SEX OF WATER | Control Sue Contro] Sudan days ek Pinel 35 C=10+22 | 1.407] 1.381} 57 C=1¢ | 1.492) 1.422 S*=27 +12 |80.070|79.820 — lot '79.160,78.700 35 C=1¢7+4+19 1.414] 1.324) 57 C— 1a 1.712] 1.535 S=17+192 |80.560)79.960 S=1¢ 7900078. 500 36 C=20 +192 | 1.440) 1.377| 60 CS 18 | 1.598] 1.470 S=27+192 |80.24080.070) | S=19 78.730 )78 .330 a7 | C=19 | 1.483] 1.415] 61 | CG=19 | 1.561] 1.528 S=1¢ (79 .920|79.720 iS) Sailey 78.910, 78.620 | | 48 C=17+4+19 1.490, 1.338) 61 C=H16 1.750) 1.534 S=27+4+19 ree S=19 79 060,78 930 550 Clare 1.434| 1.397; 68 | C=1¢ | 1.596) 1.455 3 = 12 179.170/78.780| S=17+192 |79.000/78.370 *S. Sudan III fed. the other cases where their controls did not receive any oil at all. This shows that the olive oil alone, in the amounts here given, does not produce any noticeable effects on the organ weights. The percentages of water in the various organs are given in table 5. From table 5 we see a considerable difference in the water content between the control and test rats. The organs which give a diminished percentage of water are the liver, spleen, kidneys, and brain, while those which give an increased percentage are the blood, pancreas, and lungs. The greatest difference between controls and test rats is shown in the blood, pancreas, and liver. Although the brain gave a very small difference in the water content, nevertheless this difference is highly constant. With a view to illustrating the constancy of difference in the percentage of water in the brain, I have compiled table 6. FEEDING SUDAN III TO YOUNG ALBINO RATS 183 In this table the means from all the twelve series of experi- ments are given. We notice that in every instance the per- centage of water in the Sudan III brain is less, and thus the dif- ference in the water content between the control and the test rats cannot be doubted. - The full meaning of these alterations in the percentage of water in the several organs I am unable to explain; nevertheless, it seems evident that in the case of the liver the diminution of water is mainly due to the accumulation of fat. From two determinations I have obtained the following results for the amount of fat in the liver: CONTROL SUDAN III per cent per cent CES We conccorsdocopcanensa| Gl 35.52 | Cold ether only used. Case 2. st Ieee hee arene 20.94 36.95 | Ether and alcohol extraction. The histological examination shows clearly that the greater part of the liver was infiltrated by fat. In the case of the brain I found a significant increase in the amount of lipoid in the test rat, as will be seen from the following: CONTROL SUDAN III per cent per cent Alcohol-ethermextractess sas oen soa aeeee 42.15 43 .94 This increase in lipoid may account for a slight diminution of water content in the test rat, but this requires further study. A slight diminution of the water content in the case of the kidneys and the spleen may also be due to a slight increase of 1 pin content as in the case of the brain, but I have no data to prove this. The increased water content noted in the lungs and pancreas I am also unable to explain at this moment. It seems, however, highly probable that in the case of the pancreas, hyperfunction in connection with the metabolism of fat may account for this. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, Vou. 26, NO. 1 114 S. HATAI As to the high water content of the blood of the rats fed with Sudan III we may attribute it to the condition of profound anaemia. The blood examination kindly made by Dr. Rivas (University of Pennsylvania) shows a loss of 12 to 40 per cent in the number of erythrocytes. I have made an alcohol-ether extraction of the dry blood and obtained the following results: CONTROL SUDAN III * ‘ per cent percent Allcohol-ethertextract nese oes eee 1.56 3.66 This higher content of fat in the blood may indicate that the elimination of fat from the blood has been much disturbed. GENERAL REMARKS Various alterations produced as the result of feeding Sdan III to the growing albino rat have been presented. We found that the alteration is not limited to a mere retardation of the growth of the body and organs, but that the composition of these organs is also modified. It is strange that most previous investigators who fed Sudan III to rats, as well as to several other animals, failed to notice any toxic effect of this dyestuff. This failure was perhaps due to the fact that most investigators used adult animals for their investigation, without very careful testing, while Sudan III is strongly toxic only to the growing animals. Riddle, who fed Sudan III to young chicks, noticed a retarda- tion of the growth of the body, as well as defective feathers. This investigation by Riddle, on one hand, and my own, on the other, shows clearly that Sudan III is injurious to young animals at least. I have already mentioned the high resistance of the rats whose bodies weigh more than 50 grams, but I have not tested the reaction of the fully grown adult rats to Sudan III. I have, how- ever, tried feeding Sudan III to female rats which were nursing young. In these cases a toxic action of Sudan III was found without exception. The lactating glands react promptly to the dye, and failure of the secretion of milk is evident within a day FEEDING SUDAN III TO YOUNG ALBINO RATS 115 or two. Thus in many instances I was obliged to give the mother food free from Sudan III in order to keep alive the young rats which were depending on her. So far as these nursing fe- male rats were concerned, the effect of Sudan III is similar to that in young rats which have just been weaned; that is, it arrests erowth and causes emaciation, curvature of the spine, rough hair, etc. No examination of the organs in the suckling young has yet been made. It seems clear from this that Sudan III is toxic to nursing females, as well as to their suckling young. It is of interest to note that extraction with ether shows a small trace of Sudan III in the following organs: liver, pancreas, lungs, and kidneys, but fails to show even a trace of Sudan III in the brain, spleen, or heart. Corper (’12) demonstrated the pres- ence of Sudan III in the liver, and often in the lungs, of guinea- pigs, but always failed to find it in the brain, spleen, heart, testes, or adrenals. Mendel and Daniels (’12, 713) found Sudan III in the liver (four out of five cases), but never in the kidneys of the rat. This discrepancy as to the presence of Sudan III in the kidneys might be due to the fact that the rats used by the pres- ent writer were young and were also heavily dosed. Highly interesting are the questions how Sudan III produces such profound alterations in the growth of the body and organs and how we are to explain the chemical alterations found in the organs. Riddle, who noticed a retardation of growth and the formation of defective feathers in the chicks fed with Sudan III, considers that the stained fat becomes less easily available to the organism and thus Sudan III produces effects similar to simple starvation. This question of the availability of stained fat was taken up by Mendel and Daniels, who, however, combat Riddle’s conclusions and think that the stained fat is just as readily available as un- stained. Although I have not made a special study on this point, nevertheless it seems clear that the alterations found in the test rats cannot be explained, as merely the result of simple inanition, as proposed by Riddle. A high degree of anaemia, fatty infil- tration of the liver, as well as the nephritic condition, point to pathological alterations rather than to the phenomenon of simple inanition. 116 S. HATAI It appears to me, on the other hand, that in the test rats the normal metabolism of fat must have been much disturbed, as can be seen from the presence of a large quantity of fat both in the liver and blood, and thus we may consider that the stained fat also is less readily utilized. Another interesting question is the origin of the lipoids con- tained in the organs. We found that Sudan III is practically absent, even after five weeks of continuous feeding, in nearly all the organs so far examined. Particular interest attaches to its absence from the brain in which nearly 50 per cent of the solids are represented by lipoids. Moreover, the amount of Sudan III to be recovered from the liver, pancreas, lungs, and kidneys is almost negligible. Microscopical examination fails completely to reveal any trace of Sudan III or stained fat in the organs even when the extraction mass yields a trace of it. Since Sudan III is so intimately combined in fat, and wherever fat appears Sudan IIT is always found, and furthermore since Sudan III stains not only fat or oils, but fatty acids also, it seems prob- able that if the cell elements should absorb the fat or fatty acids for building up the organ lipoids, we might expect Sudan III to be absorbed aiso. There is no a priori reason to suppose that Sudan III alone is rejected, while the fat or fatty acids containing it are capable of being absorbed. This result suggests that the lipoids found in the organs may not be formed by the fat or fatty acids furnished from without, but are elaborated by the organ itself; in other words, the organ lipoids are endogenous and not exogenous in origin. CONCLUSIONS 1. Albino rats 27 to 33 days old were fed with Sudan ITI mixed in olive oil. The amount of Sudan III given daily was about 8 or 9 milligrams in one series and 10 to 15 milligrams in the other. A dose of 20 milligrams was found to be too large. 2. In all cases Sudan III retards to a considerable extent the normal rate of the body growth. Olive oil alone as given by me does not produce this effect. FEEDING SUDAN III TO YOUNG ALBINO RATS 7 3. Most organs maintain nearly their original weights; some change in weight. The liver and pancreas show a steady in- crease, while the thymus, testes, and ovaries show a striking diminution. The rapidity of the thymus atrophy seems to be proportional to the amount of Sudan III administered. 4. The rats fed with Sudan III show a high degree of anaemia, and the reduction of erythrocytes may be as high as 40 per cent. 5. The composition of the organs is more or less altered. We find an increase of water content in the blood, lungs, and pan- creas, while a reduction occurs in the liver, spleen, kidneys, brain, and heart. 6. The reduction in the water content of the brain belonging to the Sudan III fed rats is small, but it is highly uniform and occurs in all of the series without a single exception. It was noted also that the brain belonging to the Sudan III fed rats gives an alcohol-ether extract nearly 1.5 per cent greater than in the control brain. 7. The aleohol-ether extract of the dry total blood is signifi- cantly greater (2.1 per cent) in the Sudan III fed rats than in the controls. 8. Extraction with ether shows a small trace of Sudan III in the following organs: liver, pancreas, lungs, and kidneys, but failed to show even a trace in the brain, spleen, or heart. LITERATURE CITED Apter, H. M. 1911 Experimental pernicious anemia. Proc. Soc. Exper. Biol. and Med., vol. 9, no. 1. Corper, H. J. 1912 Intra-vitam staining of tuberculous guinea-pigs with fat- soluble dyes. J. of Infectious Diseases, vol. 2, no. 3. Dappt, L. 1896 Nouvelle méthode pour colorer la graisse dans les tissus. Archiv. Ital. de Biol., vol. 26. Gags, S. H. ann Gace, 8. P. 1908 The cause of the production of down and other down-like structures in the plumages of birds. Biol. Bull., vol. 28, no.’ 3. 1910 Studies with Sudan III in metabolism and inheritance. J. Exp. Zool., vol. 8, no. 2. : MenpeEL, L. B. anp Dantes, AMy L. 1912-1913 The behavior of fat-soluble dyes and stained fat in the animal organism. J. Biol. Chem., vol. 13. Pa or" 1 AUTHORS’ ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, MARCH 2 FURTHER STUDIES ON THE MODIFICATION OF THE GERM-CELLS IN MAMMALS: THE EFFECT OF ALCOHOL ON TREATED GUINEA-PIGS AND THEIR DESCENDANTS CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU From the Department of Anatomy, Cornell Medical School, New York City NINE FIGURES CONTENTS 2. Quality of the experimented and control animals...................... w ie3) cal M2} co) x B o B + a B o + lex ° Q ra) =] Q co |=! os) ° © A fa) ° = ro) E Be na a. Contrast between the immediate effects of alcohol taken by inha- lationvandby stomach, <7. 48 i: eck eI on ee b. The vigorous condition of the animal after daily inhalation of alco- hol: for, Tonm periods. 1. S72 eae ete ys eee eas id lew Agee Seles 5. A general comparison of the progeny from alcoholic lines with those frony normal lines usenet ae wee oles oa ebiee oe ue, eee . Absorption of embryos in utero and abortions of parts of litters: methods of detecting ‘these processessse sce iO ein ee ole 7. A comparison of the qualities in the different generations of the alco- holic lines as they become further removed from the generation di- TEGCY -LECALEG 4. c2(y $3 EROS lane sina LIS Uae ned LAER MEE rhc 8. A comparison of animals from directly treated fathers and fathers of alcoholic stock with animals from directly treated mothers and mothers of alcoholic stock and with others from both parents of alenolie- SloGk.\.. 2 i. 75a see ae fsa gabe ge sober k ice. Lites 9. A comparison of lines from only male ancestors alcoholic with lines from only female ancestors alcoholic and with those from both male abeefenisie ANCEStOTS ALCONGMC. 6... 520. v is eects Geese vncsctcacces 10. Treating males with alcohol for one and two generations compared with treating females for one and two generations....................-. {t. The sex-ratio in relation to paternal and maternal alcoholism and to the treatment of male and female ancestors with alcohol........... 119 lor) 142 156 159 120 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU 12. The birth weights and rate of growth in the normal and the alcoholic SOTLES 2 oes 1S ee ee oie eis eae te sice fotapnds a eso v's. 9c Oe 199 18. The records of normal males and females paired successively with nor- mal and alcoholic mates; the crucial demonstration of the effects of alcokolismyongtberois primey sy niet: «suo css, nels ae eee 205 14. The contrasted qualities in the control and the alcoholic series........ 208 15. Generaltconsideravlonsrerr certo merit Ser tite. oats 5 3 ch.3, se oon eee 212 Literahunretcrted ites esac ine tae a tras MIU obs chee Sas Seine 225 1. INTRODUCTION The present contribution presents the results obtained during the sixth and seventh years of an experiment on the modification of mammalian germ cells by the treatment of parental generations with alcohol. A number of new facts are added to our previous findings, and the data now permit a more thorough analysis. Treating the results obtained during these two years separately may be looked upon as taking a cross-section of the entire experi- ment. And when this isolated portion of the investigation is compared with the previous studies, it supplies a further most important control for the experiment as a whole. The earlier reports of this investigation (Stockard, ’12, 713, and 714; Stockard and Papanicolaou, 716) were made after the first two years, three years, and five years of its progress. These reports showed, in what seems to us a definite way, that the germ cells in either the male or female mammal may be changed or affected by a chemical treatment administered to the body of the individual. The progeny derived from such chemically treated animals showed more or less marked deviations from the normal in many definitely measurable qualities, such as their mortality records, structural appearance, nervous reactions, and ability to reproduce. ‘The treatment also affected in the male, the crucial germ-cell test for mammals, their ability to beget CHERERE when mated with normal females. In general it may be stated that the offspring Bataaeed when treated males were paired with normal females were inferior in several respects as compared with other offspring from the same normal mothers bred to control males of exactly the same origmal stock. Further, when the male offspring from treated fathers MODIFICATION OF THE GERM-CELLS IN MAMMALS 17t were mated with normal females, the individuals resulting from such matings were as a group decidedly inferior to the young produced by normal females when mated with control males. This group inferiority was not only present in the grandchildren, or F, generation, but also in the F; generation descended from aleoholized great-grandparents. Fortunately, since these experiments were first reported, sev- eral similar studies by other investigators using the methods here employed have been conducted on other mammals and birds. Our results have been corroborated, though the response to the treatment has in some instances been thought to differ from that shown by the guinea-pigs. Useful and suggestive interpretations of the results have been advanced, yet certain points of view are presented with which we are not always able to completely agree. The bearing of these studies on the present results will be dis- cussed in a section beyond. In particular we are indebted to Pearl (17) for his recent characteristically clear and exact analysis of the influence of alcohol fumes on domestic fowls and their progeny. This study has suggested to us the importance of a considerable amount of data contained in the card-catalogue records of our animals which had not been fully valued in the previous discussions of the ex- periments. In the present report we have followed several of Pearl’s ideas in more completely separating the qualities to be contrasted between the alcoholic and control lines. As might be expected, various objections have been advanced from time to time regarding the cause and explanations of the results which we have reported on the effects of alcohol in the guinea-pig. In all cases, however, the objections have been raised either by persons entirely unfamiliar with these animals and their breeding qualities or by others who have not been sufficiently interested or careful to read the descriptions of the animals and breeding methods used. It has been suggested on certain occasions that the defects and degenerate conditions which have been reported in our alcoholic lines were probably present in the original stock on which the experiment was con- ducted. Such a remark in the face of the experimental control 122 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU which has been fully described scarcely warrants discussion, yet we should like to state in the beginning for the benefit .of the casual critic who may not wander through the following pages the real nature of the original control. A group of forty animals, eleven males and twenty-nine fe- males, was obtained from a reliable breeder in the early fal of 1910. These animals were all under one year old and strong and vigorous in appearance; most of the females were pregnant. All the females were kept until they had produced a normal litter of young. Their production was what would ordinarily be obtained from healthy guinea-pigs; all of the young were nor- mal in appearance and about 80 per cent of them survived under the by no means perfect system of care then employed. Three males and six females, after the test matings, were then taken for alcohol treatment. The choice was entirely random, there being no evident marks of superiority or inferiority in any of them as compared with the other animals retained as normal control. One of the three males selected for treatment lived to be more than seven years old, and the others were all healthy, strong animals that lived long and bred vigorously. These treated males were mated with alcoholic females and with nor- mal females. The same normal females were mated at different times with normal males and such offspring were considered control. From the beginning of the experiments it may be said that the same normal female often serves as part of the experi- ment, being mated to alcoholic males and again as the control. The same is true of normal males; they are frequently mated successively with alcoholic females and normal females. From this original stock the normal animals, both males and females, have invariably given rise to average normal offspring when paired with normal mates, while, on the other hand, the treated animals being part of the same breed, have in the quality of their offspring shown a decidedly inferior condition even when paired with normal mates. After the experiment had been in progress for eighteen months, in March, 1912, a new stock of animals of an entirely different source from the first lot was introduced. Again, after testing MODIFICATION OF THE GERM-CELLS IN MAMMALS Zs their breeding ability by one normal mating, certain of this lot were taken for alcohol treatment, and these animals were bred both separately and with the original lot. Yet the records of the alcoholic and normal individuals were again different. Finally, in October, 1915, when the experiment was five years old, we obtained four new stocks of guinea-pigs from different dealers and introduced them into the experiments in various ways along with our now pedigreed lines from the old stock. The records of these new animals as well as our old lines known for three or more generations regarding inbreeding and other conditions are to be considered in the present paper. These experiments bring out additional facts in the study, and we believe they supply an unquestionable control on the previous results. In other words, this may be taken as a new study con- sidering the conditions of 1,170 guinea-pigs born from various alcoholic lines as well as from normal control animals. About 600 of the animals are born of alcoholic lines with no inbreed- ing in any case back through their great-grandparents. About 300 of them are from alcoholic lines and at the same time some- what inbred; these are for all considerations treated separately from the straight alcoholic lines. The control animals with -which the alcoholics are compared are of the same blood lines as the alcoholics and are also not inbred. 2. QUALITY OF THE EXPERIMENTED AND CONTROL ANIMALS a. Selection of animals As briefly mentioned above, the control and the first treated individuals are derived from exactly the same original stock. Luring the progress of the experiment other animals have been subjected to the treatment, and these in many cases are of known pedigree for several generations in our colony. In all cases only vigorous animals are used for the treatment and they are invariably tested by being mated at least once before the treatment is commenced. This precaution is undoubtedly of much importance, equally as important as knowing the blood lines, in selecting normal breeders. These test matings are 124 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU further strengthened by the fact that the same normal males are mated with alcoholic females and with normal females, and normal females are mated with alcoholic males and again with normal males as a control, ete. In this way the experimental and control animals are actually in some cases the same individuals and in all cases they are constantly being bred together. There is no question that the animals treated with alcohol and the control are equally general or random samples of the popula- tion. Yet there is a marked contrast between the records of their offspring and descendants. b. Inbreeding The alcoholic lines which we shall analyze in detail in the following considerations are practically devoid of inbreeding. Almost all of these animals are known in our colony for three or more parental generations, and we mean in stating that they are not inbred that a given individual in their ancestry never ap- pears more than once back through the great-grandparent gen- eration. In the first table to be considered the straight alcoholic lines may be compared with other lines that are not only alco- holic, but also inbred, usually to a slight degree, and it is seen that inbreeding in either the alcoholic or the control to a lim- ited degree gives no indication of any significantly injurious effects. In our former report (16) there were shown to be more in- jurious effects in the alcoholic inbred lines than in the non- inbred. This difference has now disappeared on account of the fact that the animals in the former table were more closely in- bred and were earlier generations than the bulk of those in the present consideration. The degree of inbreeding in the inbred lines is now much reduced as compared with the earlier table, and the records have improved. This difference between the earlier and the present results indicates that inbreeding in these alcoholic lines may be easily carried to a degree which will make the injurious effects more marked. We have avoided even the slightest approach to such a degree of inbreeding in the straight MODIFICATION OF THE GERM-CELLS IN MAMMALS 125 alcoholic lines. The pedigrees of a great majority of these alcoholic animals could readily be given to cover several genera- tions, but it does not seem advisable to enter into this detail, since there is no possible chance that any differences which might exist between the normal and alcoholic lines are due to different degrees of inbreeding among the individuals of the two groups. And further, in all the groups it is entirely out of the question that any difference between the records of the control and the records of the alcoholic may be due to the control having been by chance originally good breeders and the alcoholics originally bad. The control animals are in almost all cases either sisters, brothers, parents or other blood relations of the treated animals. c. The number of animals alcoholized Recognizing the great variability in the breeding results from the different individuals in a group of higher animals, such as mammals, it has been deemed entirely essential to make our experiment on a considerable number of males and females. The mating records of two normal male guinea-pigs are fre- quently quite different even though paired with the same fe- males. It is also highly probable that different individuals will differ in their susceptibilities and responses to the treatment, so that the records of two or three males might easily prove con- fusing even though all might exhibit some effects of the experi- mental treatment. Thus the following twenty-eight males have been treated with alcohol, and a number of matings from each of these and their descendants have supplied the breeding records. The first three are from the original 1910 stock Nos. 4 3, 50%, and 6, and the remaining twenty-five are animals bred and reared in the colony or from the newly introduced stocks: Nos. 437, 45%, 700, 72, 800, 812, 678 0, 8870, AN 9132, ae 1299, 1577, 1687, 1830, 3020, 3536, 3650, AN ~ AA U / im Wc — J 5747 493 2, iN a 771¢,A ‘8899, 1091¢, 11342, A 1153.4, 13826¢ and 13272. 126 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU In the case of the females an attempt has also been made to lessen the error caused by indiv'dual differences in breeding ca- pacity and in responses to the treatment by using a number of animals. Thirty-four individuals have been treated in all. Many of these females were bred for a number of times as con- trol before being subjected to the fume treatment, after wh ch they are placed of course among the alcoholics. Their earlier breeding records are therefore part of the control data and their subsequent records part of the data included for the alcoho ic lines. The same thing is true of a number of the males men- tioned above. In none of these cases can it be objected that the animals had become too old for normal vigorous breeding while being used in the alcoholic lines. We have constantly guar ed against breeding the alcoholic animals after there is any question as to age affecting their breeding capacities when compared w th the normal breeding cycle of these guinea-pigs. The treat- ment of the large majority of the animals is begun when they are less than one year old, and they have a vigorous breeding span of at least four years. The individua' females wh ch have been subjected to the aleohol fumes are the following: The first six are from the original 1910 stock, Nos.8?,9¢?,10¢9,119,129, and 34 92; the following twenty-eight are animals reared in the colony or from the newly introduced stocks: Nos. 559, 57°, OO (G0N), OS” 62°90 649, 659 (66 9, 88510"2 90 os aiiivaes NA 1589, 1619, 6549, 8479, 8659, 9469, 7— 1229, 2009 ES Ne 228.0, 397-9, 11399, »A 7969; 1002.9 1105, N= A as A 1468 2 and 1469 ¢. There are no contrasts between the histories and capacities of the experimented and control animals that can be fairly ac- counted for as due to differences in either their origins, blood lines, or relationships. As far as experiment and control with biological material may be practically useful, any differences which may exist between the records of the alcoholic guinea- MODIFICATION OF THE GERM-CELLS IN MAMMALS MP7; pigs and the normal control lines are due to the treatment ad- ministered to the alcoholic lines. We further believe that if the differences which do exist between the alcoholics and control are so slight that the crudest mathematical calculations are -n- sufficient to indicate their presence, the experiment has then produced no data of biological interest or importance since conducted on animal material of such complexity as a group of mammals. This statement is made with no intention or pre- sumption to question the real importance and value of modern biometrical methods, but is only what we believe should apply to this particular experiment. 3. EXPERIMENTAL METHOD AND THE CARE OF ANIMALS Throughout these experiments alcohol has been administered to the guinea-pigs by a method of inhalation which was devised in the beginning. The animals to be treated are placed in fume tanks fully described and illustrated in an earlier communica- tion (Stockard, ’12) and absorbent cotton soaked with com- mercial 95 per cent ethyl alcohol is placed on the floor of the tank beneath a wire screen on which the animals stand. The fumes of evaporating alcohol very soon saturate the atmosphere of the tanks and the guinea-pigs introduced into this saturated at- mosphere are allowed to remain until they show distinct signs of intoxication. During the earlier years of the experiment they remained for one hour each day in such tanks, but during the past twelve months we have increased the treatment to two hours per day for the males and three hours for the females. This longer treatment is much better in that the animal, of course, gets a larger dose and its tissues may become more quickly influenced by the treatment. The animals may remain until they are completely intoxicated, in which case they are unable to walk, and therefore lie in a typical drunken stupor, or they may be affected to such an extent that they attempt to walk and in so doing stagger and fall in a manner characteristic of the drunken state. The amount of treatment here employed, however, does not produce complete intoxication. It would be perfectly possible with an elaborate system of measurements to determine exactly the quantity of aleohol iumes 128 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU each individual receives per day, per month, or per year, but, as we have pointed out before, such knowledge would be of no advantage either to us or to others in estimating the results of these experiments. No two individuals would be affected to exactly the same degree by the same dose, and as is the case with man the later influences of the treatment no-doubt differ in different individuals. There is also no particular interest here in the amount of alcohol used, since our primary problem is whether or not an active chemical substance may be given in sufficient amounts to the parent mammal to produce effects upon its offspring or descendants by modifying its germ-cells, or in the case of the pregnant female by acting through the mother on the developing embryo. We have thus employed, as stated in our previous reports, a simple physiological index of the amount of treatment, giving enough each day to perceptibly influence or intoxicate the ani- mals, but not enough to produce a complete drunken stupor. Animals may remain for very long times in these treatment tanks when alcohol fumes are not present without in any way suffering for want of breathing space. ‘This method has many advantages so far as the general health of the individual animal is concerned over drinking alcohol into the stomach, as will be discussed in the following section. The only object in choosing alcohol as the treating agent is on account of the fact that considerable knowledge exists as to its physiological actions on certain animal tissues and it is known to be an active organic substance that might produce effects. It had further been used by one of us (Stockard, ’10) in producing various developmental abnormalities in fish embryos which could be treated directly with diluted alcohol, and the general nature of the effects on these embryos had been studied. A final advan- tage in using alcohol in such experiments is the ease with which it may be administered to the animals by the inhalation method, which we have described. Caging and care of animals. All of the guinea-pigs, both the experimental and the control animals, are kept in the same type wooden cages. These are group cages, each containing twenty MODIFICATION OF THE GERM-CELLS IN MAMMALS 129 compartments one foot high by one foot wide by two feet long. Each compartment is sufficient to fully accommodate one “emale with her litter of young or three adult animals. In all of the cages some of the compartments are occupied by the alcoholic animals and others by the control so that the cage accommoda- tions for the two classes are identical. The cages are thoroughly cleaned, the floor sprinkled with sawdust and fresh hay put in daily. In addition to the hay, which is eaten with relish, the animals are fed every day with fresh carrots and several times per week oats are given with occasional cabbage or kale. It is also important for their perfect health, though not necessary for their existence, that guinea-pigs be given fresh water every day during the warmer months and several times per week during the winter. This is frequently neglected in keeping these ani- mals since it is commonly thought that they get a sufficient amount of water from the green foods. At the present stage of this experiment, along with several other problems now being studied, a stock of over 500 animals is constantly kept on hand. One reliable keeper devotes his entire time to cleaning the cages and feeding. He in no case discriminates in his treatment of different animals and from the cage numbers is unable to know all of the alcoholic line animals or the controls. From the beginning of this experiment, in making the matings a male is placed in a compartment with one female during her heat period (Stockard and Papanicolaou, 17); in this way there is no opportunity for preferential or choice matings. A male might discriminate in his behavior between an alcoholic and a normal female if in a compartment with the two, as Pearl be- lieves his roosters have done when placed in a pen with both - normal and alcoholic hens. After the male has remained in the pen for one month, the female is carefully examined and at this time with some practice the investigator may feel the small embryos in the horns of the uterus. The male is removed and the female remains alone in the compartment. A list of all preg- nant animals, both alcoholic and control, is kept and their com- partments are examined both morning and late afternoon of each day in order to detect an abortion should it occur, since the THE JOURNAL OF EXPERIMENTAL ZOOLOGY. VOL. 26, NO, 1 130 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU female may devour the early aborted young. In addition to this, each pregnant female is reéxamined once or twice and the number of fetuses in the right and left horns of the uterus re- corded each time on her catalogue record. By this method it has been found that a number of females may often absorb their embryos, either one or all, and so give birth to a smaller litter than originally began development or to none at all. The absorption of individual embryos seems so far as we have detected not to interfere with the development of the remaining ones. These examinations of pregnant females have been repeatedly controlled by opening the animals and ob- serving the contents of the uterus, and the examinations in all cases have been very accurate. This thorough watch over the females has furnished us much more exact data as to prenatal deaths, early absorptions, etc., than were contained in our former reports. The entire care of the animals has been much improved during the past two years. Our records for monsters and other weak- ened conditions are, therefore, somewhat reduced; yet the same marked contrast between alcoholic and control is present even though the weakened alcoholic lines have no doubt profited more by the improved methods of care and feeding than have the healthier controls. The defects are also the same in type as those formerly observed, though not so marked in degree. 4. THE INFLUENCE OF ALCOHOL INHALATION ON THE INDIVIDUAL The immediate effects on guinea-pigs of inhaling alcohol are somewhat similar to those observed after drinking it. As stated above, the animals after some time become unable to walk with- out staggering as a result of loss of muscular coérdination and finally reach, with a long treatment, a state of complete alco- hohe stupor. The presence of alcohol in the blood of the guinea-pig after the inhalation treatment is readily detected by even simple chemical tests, as we have frequently pointed out. Other in- vestigators also find that alcohol is easily introduced into the MODIFICATION OF THE GERM-CELLS IN MAMMALS 131 general system of birds and several mammals by this method. Pearl (’16 b) definitely recognizes the fact that alcohol is readily taken into the system by the inhalation method, but makes the following statement regarding the effect: “It is true that it is prac- tically impossible to induce by the inhalation method in animals habituated to alcohol that state of muscular incodrdination which is usually, but by no means always, the most striking objective symptom of the condition of being drunk.” Our observations on guinea-pigs show them to respond very differently in this re- spect from the fowls used by Pearl. In the case of guinea-pigs habituated to alcohol, it is very easy by the inhalation method to induce a state of muscular incoérdination due to the drunken state and finally a complete anaesthesia, the muscles being en- tirely relaxed and the animal unable to move. It may be that fowls are peculiar in their reaction to aleohol and 1: may also be extremely difficult to administer to them a highly effective dose without fatal results. Such an idea is suggested by the fact that Pearl does not get the gross symptoms of intoxication by leav- ing his fowls in the tanks for one hour, yet they ‘ accumulate a fatally toxic dose of alcohol by staying in the same tank under the same conditions for from twenty minutes to half an hour longer.’’ Guinea-pigs do not at all react in this manner after an hour or two in the tank they may show signs of intoxication by becoming groggy, with their muscles generally relaxed so that when lifted their bodies are almost entirely limp. Yet they have not consumed anything near the fatal dose, since they may remain in the same tank under the same conditions for even two or three hours longer before becoming completely intoxicated so as to be unable to move; and in order to inhale a fatal dose they must remain still longer, at least six or seven hours We have treated only one fowl, a white leghorn cock, in our tanks. This bird responded much as the guinea-pigs do show- ing decided muscular incoérdination, staggering and frequently almost falling as it walked. He was also able to withstand a long treatment and never, though treated several times, did he show any tendency to suddenly accumulate a fatally toxie dose as Pearl found his fowls to do. 132 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU a. Contrast between the immediate effects of alcohol taken by inhalation and by stomach An important point to keep in mind when considering these animals intoxicated by the inhalation method is that on being removed from the tanks they use up the alcohol in their systems very rapidly and also begin to throw off alcohol by respiration. The intoxication is, therefore, of short duration so that the animal may be fairly well recovered within half an hour or perhaps only a few minutes, depending upon the amount of the treatment. In other words, this is an acute short, intoxication closely com- parable to an ether anaesthesia from which the animal readily recovers when the fumes are no longer inhaled, but which during the inhalation may give a complete intoxication. On the other hand, a drunken condition resulting from taking alcohol into the stomach is of much greater duration since the gradual absorption of the alcohol continues for a longer time before the system begins to burn it up or throw it off to such a degree that the amount present begins to be continuously reduced, permitting the animal to slowly recover from the drunken state. A guinea- pig receiving a dose of about 25 ec. of 15 per cent alcohol into its stomach will be decidedly intoxicated within fifteen or twenty minutes, and the extent of intoxication will increase until the animal becomes unable to walk or stand and lies in a drunken stupor. Such a condition may persist for six or seven hours or longer, and the body temperature may be lowered from one to even four degrees Fahrenheit. It seems to us, therefore, that the chief difference between inhaling aleohol and drinking it into the stomach is that in the first case the action of the substance on the animal system is of shorter duration, lasting but little longer than the length of the sojourn in the fume tanks—a short acute effect—while alcohol in the stomach is gradually and continuousiy absorbed for a considerable length of time so that the animal’s tissues are acted upon for hours after receiving the dose. Another very serious phase of the stomach alcohol, aside from the typical intoxication effects, is its tendency to derange the animal’s powers of diges- MODIFICATION OF THE GERM-CELLS IN MAMMALS 133 tion and thus to cause very injurious results. The inhalation method is accompanied by no such complications. We have now considerable data bearing on this problem and are conducting an experiment to determine the quality of the effects on the animal body and the progeny produced when dilute alcohol is taken into the stomach of guinea-pigs for long periods of time. The results of this study are to be compared with the data from the fume-treated animals. b. The vigorous condition of the animal after daily inhalation of alcohol for long periods A number of the guinea-pigs have now been treated with alco- hol fumes almost to a state of intoxication six days per week for from five to six years. Few guinea-pigs in captivity live so long atime. There were two males treated for over six years, one of which lived to be more than seven years old. So far as we know, this is the longest life reported for a guinea-pig. The treat- ment was continued with these very old animals but they were not used for breeding. In no case when the treatment was be- gun on an animal over three months old could any injurious effects on its general welfare or length of life be discovered. We have called attention to these facts in our previous publications. There are certain direct injuries resulting from the inhalation of ethyl-alcohol fumes during the early stages of the treatment. The mucosa of the respiratory tract is considerably irritated during the first few months and secretes freely while the ani- mals are in the tanks, causing a watery flow from the nostrils and mouth. The membranes become more resistant as the treatment goes on and later little effect can be noticed. This irritation has never given rise to any noticeable inconvenience to the animals. The surface of the eye is also greatly irritated during the first few months, causing an abundant secretion from the lachrymal glands while in the fume tanks, and finally re- sulting in many instances in an opacity of the cornea. In some cases this opacity disappears after a few weeks and the animal is again able to see, yet some of the animals treated for several years have remained entirely blind. 134 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU A number of the treated animals have died and many others have been killed at various times during the progress of the ex- periment. Their organs and tissues have been carefully exam- ined at autopsy and later studied microscopically. All tissues have appeared practically normal and none of the various well- recognized pathological conditions occurring in human alcoholism have been discovered. Tissues from animals treated as long as three years have been carefully studied, and the heart, stomach, liver, lungs, kidney, and other organs present no noticeable con- ditions that might not be found in normal individuals. Alco- holized animals are usually fat, but no fatty accumulation has been noted in the parenchyma of any organ. Several males and females have been semicastrated during the experiment, and the ovaries and testes have been found to be in a generally healthy condition. It has seemed, however, that the ovaries of treated animals as well as all animals of the alco- holic lines show an unusual tendency to become cystic as com- pared with the ovaries of normal individuals. We have not, however, made sufficient comparisons to give the foregoing statement any greater weight than a mere supposition. The general condition of all animals under the fume treatment is particularly good, and, as stated above, they continue to grow if the treatment is begun on individuals before they have attained full size, and all become fat and vigorous, taking plenty of food, living long, and behaving in a typically normal way. The accompanying illustrations of five treated animals photo- graphed along with control individuals show their perfectly normal appearance. In figure 1 is seen two male guinea-pigs and from the photograph as well as in life it would be impossible for any one to detect signs of physical inferiority on the part of one or the other. Yet the animal on the right, No. 8007, was four days less than 5 years old when the photograph was made and had inhaled alcohol over one hour per day, a sufficient dose to give signs of intoxication, for six days per week, during four years, two months and five days. During the last seven months he had inhaled alcohol fumes two hours per day. He is per+ fectly well and alert, as the photograph clearly shows. His MODIFICATION OF THE GERM-CELLS IN MAMMALS 135 companion on the left is a normal animal, No. 150, being 4 years and 3 months old when photographed. The sober exist- ence of this male has not given him any advantage in appear- ance over the old alcoholic; both are very good males, each weighing almost 900 grams when photographed. This is well above the weight of the ordinary adult guinea-pig. Figures 2 and 3 show again on the left the same normal animal, No. 1507, in order that the reader may obtain a more definite impression of the uniformly good condition of the three alco- holic males. The alcoholic male No. 72 on the right in figure 2 was 5 years, 1 month and 10 days old when photographed, eee Fig. 1 The animal on the left is a normal male, No. 150, over four years old. The one on the right, No. 80 o, is almost five years old and had been treated with the fumes of alcohol six times per week for four years and two months, yet is seen to be in a vigorous condition. weighing over 900 grams. He had been treated with alcohol fumes one hour per day until the last seven months, when he was treated two hours per day for six days per week. The entire duration of his treatment when photographed was four years, two months and five days. Figure 3 shows on the right alcoholic male No. 70. This animal was 5 years, 1 month and 11 days old when photo- graphed, and weighed 885 grams when 5 years old. He had re- ceived the same amount of alcohol treatment as the other two. The three were bred and reared in our colony and are above the average male guinea-pig in size and vigor. They have been good breeders as young normal specimens, as well as during 136 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU their aleoholic careers, but there has been a decided difference in the quality of their offspring during the two periods. Figures 4 and 5 show two female alcoholics photographed along with the same black male No. 116. In figure 4 the alcoholic is an albino, No. 659. She was introduced into the experi- ment with the second stock from a new source in March, 1912. This female had been treated with alcohol fumes for two years, seven months and seventeen days when photographed. During the first two years of the treatment she inhaled one hour per day for six days per week and during the remaining seven months was treated for three hours per day, until fairly well intoxi- cated each time. The normal male No. 116 was 4 years, 83 Fig. 2. The animal on the left is the same control individual, No. 150 <7. The one on the right is an alcoholic male, No. 72, which was more than five years old and had been treated with alcohol fumes for four years and two months. months old when photographed. The female No. 65 gave : or- mal young before her treatment began, but now produces off- spring with very poor records. The female No. 158 is shown on the left in figure 5. This animal was produced in our colony from normal parentage and was 4 years and 3 months old when photographed. She had been treated for fourteen months one hour per day and for three hours per day during the last seven months. She is a large vigorous female. These photographs illustrate to some extent the fact that the treated animals themselves are little changed or injured so far as their normal appearance goes, and should there be inferior qualities in their offspring these cannot MODIFICATION OF THE GERM-CELLS IN MAMMALS 137 be attributed to a condition of general depression in the parents, but more clearly to a peculiar action of the strange chemica ma- terial in the blood upon the glands of reproduction or the germ cells of the males and females. In his study of the influence of aleohol inhalation on the do- mestic fowl, Pearl has found the treated individuals to respond in a way closely similar to our treated guinea-pigs. He has fortunately reported his results in much more thorough detail than we, yet the facts contained are practically the same for the two groups of animals. The mortality records of treated fowls show an advantage over similar records from untreated Fig. 3 Two male guinea-pigs. One on the left the normal animal, No. 150, more than four years old. On the right, No. 70 o’, more than five years old and had been treated with alcohol fumes for four years and two months. control. Our ecard catalogue contains the record of every death that has occurred among the guinea-pigs since the beginning of the experiments, and we may state in a general way that the mortality statistics for the treated animals is certainly as good and perhaps slightly better than those of the control. Pearl has very naturally considered these findings in connection with the ‘‘widespread popular opinion that life-insurance statis- tics have ‘proved’ that even the most moderate use of alcohol definitely and measurably shortens human life.’ On careful investigation of the statistics Pearl finds them to be entirely un- convineing and to be based on biological evidence insufficient to prove anything. This is exactly in line with our own experi- 138 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU ence in studying the literature of any phase of human alcohol- ism. We have studied very thoroughly the literature relating to the influence of alcoholism in men and women on their prog- eny and, including the study of Elderton and Pearson, find it to suffer from the defects which Pearl points out in the longevity studies. Some of these contributions we shall discuss beyond, but none give any exact statement as to the amount of alcohol consumed or the length of time during which it had been con- sumed or any definite information as to other conditions or the general behavior of the individuals considered. The data are usually collected by persons entirely untrained and incapable of Fig. 4 On the left normal male No. 116, almost five years old, and on the right an alcoholic female, No. 65, more than five years old that had been treated with alcohol fumes for about two and one-half years. accumulating biological evidence. These extremely inexact records are often subjected to very careful and exact mathe- matical analysis which tends to give a scientific aspect to the consideration, but in no way improves the quality of the incor- rect data used. Unfortunately, this renders it difficult to make comparisons between the responses of human alcoholics and those of selected animals used in well-regulated experiments. Yet aside from the above, even should the data relating to the influence of alcohol on human longevity justify a comparison with experimental results, we feel that such a comparison could not properly be made with either Pearl’s observations on the effect of aleohol on the mortality record of fowls or ours on the life record of alcoholized guinea-pigs, since in both experiments MODIFICATION OF THE GERM-CELLS IN MAMMALS 139 the animals have been treated by inhalation of alcohol fumes, while human alcoholics have taken the substance into the stomach. The difference between the effects on the treated individual of the two methods of administering alcohol cannot be too strongly urged. By the inhalation method the individual ex- periences only the stimulating or with further dosage the intoxi- cating and anaesthetizing effects of aleohol. As far as we have detected there are no injurious secondary effects on the indi- vidual’s welfare resulting from habitual inhalation of ethyl- alcohol fumes. The results are very different, however, when the guinea-pigs drink daily doses of 15 per cent ethyl alcohol. Fig.5 Thesame male, No. 116, is shown on the right and an alcoholic female, No. 158, is on the left. She was more than four years old and had been treated with alcohol for over two and one-half years, yet she is in no way injured in appearance. Only a few animals and a short time are sufficient to demon- strate the fact. A number of animals were given alcohol into the stomach at the beginning of these experiments and their diges- tion and metabolism were so deranged by the treatment that we were forced to devise and adopt the inhalation method as a more likely means of conducting an experiment of long duration. It has been shown by us for guinea-pigs, and Pearl has dem- onstrated with fowls, the prosperous manner in which animals withstand the inhalation of alcohol vapor. We may now give briefly the effects on three guinea-pigs of drinking daily doses of alcohol for only three weeks. The ani- mals, Nos. 1739, 10987, and 1184.7, at the beginning of the 140 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU experiment weighed respectively, 822, 635, and 527 grams, the female being old and the two males young growing specimens. They were each given about 20 ec. of 15 per cent ethyl alco- hol in tap water daily, except that once each week they were given almost 30 ce., which was a completely intoxicating dose. The 20-ce. dose causes all of them to be groggy for a few hours after drinking it; the effect increases for an hour or so and then gradually wears off. There is only slight if any change in rectal temperature. The animals seem fully recovered on the follow- ing day and have a normal appetite, but do not eat so ravenously as do untreated individuals. When 30 ce. of 15 per cent alcohol is given in three 10-ce. doses at fifteen-minute intervals the animal is badly intoxicated and unable to walk within fifteen or twenty minutes after the last dose. The hind legs are particularly uncertain, the animal often tumbling over almost on its back, kicking frantically and having great difficulty in righting itself. Should its mouth come in contact with food the guinea-pig will chew in a peculiar man- ner, seeming in all reactions to be typically drunk. After one and a half or two hours the animal lies on its side with its trunk muscles often undergoing spasmodic contractions several times per minute, if taken up or made to move it struggles and falls panting in the drunken condition. By this time the body tem- perature may have fallen as much as 2 degrees below the pre- treatment record. After three hours it is still unable to stand or walk and is breathing heavily with a temperature as much as 25 degrees Fahrenheit below normal. After four hours the con- dition is about the same and so for several hours longer until it gradually begins to recover and by the following morning it is fully recovered, but shows in its appearance the effects of the experience of the previous day. When animals are given five partial and one complete intoxi- cation by stomach alcohol per week they begin after a few days to regurgitate some of the stomach contents on receiving the first swallow or so of alcohol, but after this they take the dose without further disturbance, though they resist taking it more and more each time. Their desire for food is somewhat reduced as the treatment is continued. MODIFICATION OF THE GERM-CELLS IN MAMMALS 141 After the first week No. 173, the old female that should have weighed the same or gained in weight under normal conditions, had lost 50 grams, or 6 per cent of her total weight. The two young males should have gained, No. 1098 gained 17 grams, only 2.6 per cent of his weight, while No. 1184 lost 4 grams or prac- tically stood still. Their weight records for the indicated inter- vals are as follows: 1732 109871 11847) grams grams grams Wea ets. rete rds cess 822 635 527 1.4 ya a 2 oe eet es Peer rea 772 652 523 ily Pa) Se nico cere rene Oe 740 656 477 RVIsEV RO SMe tartan Sr yee ccc cke ars ele te 759 659 469 AfbtaSe Die aan MO Spcke an 8 eacette cate aie, eee 735 637 483 Arne Wns aes s coe tad ail omee 775 621 475 The alcohol was taken from May 7 to 28, and during that time the first guinea-pig lost 63 grams, or 7.6 per cent of its original weight. Of the two young males one gained 24 grams, or 3.7 per cent of his weight, while the other lost 58 grams, or 11 per cent of his original weight. During the next two weeks after the treatment stopped the male that had gained 24 grams lost 38 grams, so that at this time each animal weighed less than when it began to take alcohol. This may have been a rather strong dose, but allowing for that, it was readily recognized that these animals were suffering from the treatment, while other guinea-pigs inhaling alcohol for three hours per day until groggy showed no injured appearance. Ani- mals taking alcohol into the stomach suffer mainly on account of the injurious effects on their digestion. Alcohol acts on the gastric mucosa in such a way that the individual is placed at a disadvantage in handling its food and the ill effects observed are more largely due to this derangement of digestion than to the toxic action of alcohol on the animal system. Alcohol in the stomach makes the case complex, while we believe that inhaling alcohol gives effects simply due to the chemical action of alcohol itself on the tissues. For these reasons we do not believe that comparisons are easily made between the conditions of animals 142 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU that have inhaled aleoho! fumes and the conditions of other ani- mals that have taken alcohol into the stomach, since the latter individuals may be reacting more to a deranged digestion than to alcoholic intoxication. Therefore, there is objection to mak- ing comparisons between the mortality records of animals treated with alcohol by the inhalation method and the reports on the effect of alcoholism in man. Yet, on the other hand, it may be possible that the influence of aleohol on the germ cells of an anima’ is the same whether the alcohol reaches the reproductive glands by being inhaled into the lungs or swallowed into the stomach. Such a position is not inconsistent with the discussion above if we take into ac- count the possible, though unknown, effects of the deranged metabolism of the parent on the germ cells. 5. A GENERAL COMPARISON OF THE PROGENY FROM ALCOHOLIC LINES WITH THOSE FROM NORMAL LINES The consideration above has brought out the fact that the in- halation of aleohol fumes sufficient to produce partial intoxica- tion six times per week for long periods does not cause any easily recognized disadvantages in the general bodily condition or powers of existence of guinea-pigs. Pearl’s experiments demon- strate the same fact in connection with the domestic fowl. This is, o course, leaving out of account the irritating effects of the fumes.on the surface of the eye which may result in bl ndness, although even this is no handicap to either feeding or epro- duction under cage conditions. I’, then the genera’ body tissues are not sufficiently injured to cause an easily noticeable change in their powers of function, why should the germ cells be particularly susceptible to the treatment? The germ cells within the body of a mammal are undifferentiated genera'ized cells with no known function except to exist and await their time to develop. The soma or body, in respect to the germ cells, is simply a culture medium in which they live. The nour- ishment necessary for their existence is delivered to them by the body fluids. Any strange chemical substance which may find its way into the body fluids will reach the germ cells, and should this substance be sufficiently active and injurious in its effects MODIFICATION OF THE GERM-CELLS IN MAMMALS 143 the germ cells may be so modified as to render them incapable of normal development. This might easily occur without differentiated somatic tissues being sufficiently damaged to greatly impair their usual functions. In other cases, and prob- ably as a rule, the somatic tissues are also injured by any offen- sive substance present in sufficient quantities to modify the germ cells, and there are many reasons for believing this to be the result in several chronic human infections. One must not infer from these statements that the germ cells are readily injured by poisons taken into the system; indeed, they seem on the contrary to be protected to a remarkable degree against such effects, and for this reason it is difficult to obtain a substance which may be used in experimental studies on the modification of the germ cells. Should the germ cells be modified through the action of any substance, the point of particular importance is that all cells arising from such a modified germ will be similarly modified, since they are merely products of its division, and thus the soma and germ cells of the resulting individual will deviate from the normal in proportion to the degree of the primary modi- fication of the cells from which it arose. Provided the change is one of such a nature that the cell or its parts are unable to recuperate, for example, if their specific chemical or physical make up be altered, then not only will the generation resulting from the originally modified germ cells be affected, but all future generations arising from this modified germ plasm will likewise be affected. It seems also highly probable that should such results occur, the modifications to be observed in the somatic generations will be of a generalized nature affecting the organism in various ways so as to render its development less vigorous, its chance of sur- vival less certain, and its ability to behave in a normal fashion more or less hampered. In certain cases the animal might really show no evident signs of its altered character. It seems to us, on the other hand, that only through the very rarest chance, one in possibly thousands, would any of the small number of definite characters under observation happen to be modified by their response to the treatment. The inheritance of coat-color, 144 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU for instance, may not be affected, although the germ plasm might be so seriously altered as to give rise to the most extremely ab- normal individuals. The same would apply to the very few other characters in mammals the inheritance of which have been studied from the Mendelian standpoint. Finally, then, the fact that the soma seems little injured by the alcoholic inhalations is in no way an index of what may be expected from the development of the germ cells of guinea-pigs which have been under habitual treatment. Arlitt and Wells have very recently reported that the admin- istration of alcohol in the food of male white rats for two or more months results almost constantly in the appearance of marked degenerative alterations in the testicles although other organs were apparently uninjured. They find that these changes affect the steps of spermatogenesis in inverse order to their occurrence, so that for some time before sterility and complete aspermia result, the animal is producing spermatozoa with all possible degrees of abnormality. The probable relation of such phenomena to the production of defective offspring is obvious. A general survey of the progeny from the normal and alcoholic lines as a whole will first be undertaken and is based on the data presented in table 1. In this table the animals are arranged in four groups, the first column containing the records of those produced by normal control matings without inbreeding, the third column records of normal animals somewhat inbred, while the second column gives similar records for animals produced in the alcoholic lines without inbreeding, and the fourth-column animals are not only alcoholic, but also somewhat inbred. The table contains in all records of 1170 animals, from our catalogue numbers 613 to 1909 except 126 animals that could not properly be included such as 39 new stock adults, 22 killed for different purposes during early embryonic life, 31 derived from mothers with only one ovary, and others too heterogeneous in origin, as those from ancestors treated during pregnancy, ete., to be cer- tainly placed. They represent, as stated above, the animals produced during the sixth and seventh years of the experi- ment and none from the earlier years. The figures of the first horizontal space may be used to indicate 145 MODIFICATION OF THE GERM-CELLS IN MAMMALS “+9'S) 408 xsl “%8E'| “sla wel (os v (> \ Ds (2 (%+9 2) “807% “Sle 68S el if: (ay C S (9) ae c i (%1ZS) LST LSSS Use I & S vy ‘S iG yor %ilO %&t"l ALT OlT = %6S-8I N (%Lo-GS) etl kslo xpele %sise 81 (lg. ira lore @) S + S G | (%+L+E) al) - Gl iw tc Si (%STS9 ) Hj Sil. lke (5 S SU) gO YRS YOKE %SORI X%ee-99 %ool I; El 96 Sis Si cS WT | tOS 22 (titi) qi \vS sow ODT \BHLLPBesoay” %tSIT %6L0S —“ —__ Gt OF +t oS Se sty © Redees haltns OONG 86= %Iete N@%SG 17) MESES YOST %IIIIZOCIL %O0I OS gol 8 Seat G (%5-tiyt “of -sew THT ABW Porvny %9C bo “YS a Aa Cyn mel oa me © ion ad (sS Ga L POAGUt jeEWAONY [| Ti S ASST WBr ~ECOO %ZL-|8 “Z1LS18}] Y%os %7 ACESS xBL148 %ool c Ss %ES El SSUlf NOYO| 681 = %6L-31 N(%7tg-9¢) “IIIB GtS ~90-6E “%ls-Bl ~%cvsl %OL MSIE %III Bir'si (%+E'}) (4c+'0) (plo sGuows Uaym 4p OOS UCULSsa}) po7Zts.lapupy (%98'C) (%8S'S) (pjO SUJLOWS LeU %Lol “bed %G68:L UESG ~%9801 %F |'BOOGULUL BIOW) S 1) Sj S POZISIOAOC YtIT 40T %0E+ %I9'O © Gl | v os K6 aalpafeg OO! = (%|g'z7 ) Peep [eyo iG, Golluoce ci i eeciiee a)! ta. © G Ll S 4 ¢ @ (%58 62 ) (%R08+-) Si OS it 8 vz Se ORS ULUHAA paley POSS OANPOWOSld ‘paqiosqy SULUOW & !QAO POAT (%+l OL ) (%TE'IS ) +2 6L GI 2) eet y 7 Ve, SRR Sigel leecell tl! fi si Gs ©! az = cS I ee t Ee (ev0°S1) 9E ivf 4ow LU 42aH!| OBvisayy (+s-¥) & -ofyvw LUT 4241, BBs soy SAVES WRESLE %SO VT —— eS oe OLE TI 8S SUT Cy OG or ol Sr ecce a Ca oo SOUL] PRUHION) SSM DIIOHOOIY GNY IWWHONW WOUd ANSESOYd SGHL JO STHOODSY ALNVWNO GNY ALNVLOW Bl Mehyae 1 26, NO. OF EXPERIMENTAL ZOOLOGY, VOL. THE JOURNAL 146 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU the productivity of the different lines. The numbers 1 to 5 in- dicate the number of young, one, two, three, four, or five produced by a female in a single litter. Litters of five indi- viduals are the largest that have occurred from this strain of guinea-pigs. The average number of young in a litter from the normal lines is 2.77, and of the 233 animals included in this column 24.03 per cent of them were born in litters of one or two young. About 39 per cent were born in litters of three, while 37.33 per cent of the animals were members of large litters of four and five individuals. There were only a few normal in- bred animals, as shown in the third column, but their general occurrence in the different-size litters was about as in the straight normal lines, half of the animals were born in litters of three, and almost 30 per cent in larger litters, and only about 20 per cent in litters smaller than three. The average litter happens to be in the small number of inbred animals a little higher than in non-inbred stock. The arrangement of the young in large and small litters in the alcoholic and alcoholic inbred lines is almost exactly the re- verse of what we have just seen for the normal. Again, a little less than half of the animals occur in litters of three. But over 30 per cent of the individuals are from litters of only one or two, while about 20 per cent are born in litters of four or five. Stated in other words, in the normal lines one and one-half times as many individuals are born in litters of four or five as in litters of one or two, while in the alcoholic lines one and one-half times as many are born in litters of one or two as in litters of four or five. The explanation of this, we believe, is as follows: About half ° of the pregnancies in this stock of guinea-pigs should result in litters of three, as is found to be the case in all of the lines of table 1. All litters of less than three young are due in the first place to a low productivity on the part of the female as is prob- ably indicated by the production of more than one-fifth of the normal young m such litters. In the second place, small lit- ters are frequently due, particularly in the alcoholic lines, to the death and absorption in utero or early abortion of one or more members of an originally large litter. The absorption in utero of such embryos, often of rather large size, may occur in a nor- MODIFICATION OF THE GERM-CELLS IN MAMMALS 147 mal guinea-pig, yet such a phenomenon is not very common, although in the alcoholic lines it is frequently observed. We shall consider this process below, the only point of interest here being its effect on the size of the litter. The exactly reversed percentages of individuals born in large and small litters in the normal and alcoholic lines, as shown by the table, may indicate that one-third of the animals in alcoholic lines that are born in litters of one or two were originally in litters of three, four or five. For example, the normal lines have in all well over 12 per cent more animals born in litters of four or five than in litters of one or two, and the alcoholic lines have over 12 per cent more in litters of one or two than in litters of four or five, and this 12 per cent probably has been thrown from the larger into the smaller litters on account of early abortions and absorptions which occur in the former. The too frequent oc- currence of small litters is undoubtedly indicative of not alone an actually low productivity, but a very early prenatal mortality. Another occurrence also partly due to an early prenatal mor- tality is the failure of a mating to produce a result. No doubt in rare cases fertile guinea-pigs may be mated during the heat period of the female, as these have been, without a following conception. In the normal lines four out of eighty-eight matings, or 4.54 per cent, failed, giving negative results, while in the alcoholic lines three times as many matings failed, and very probably this excess represents those cases in which not only a part of the litter is lost through an early prenatal mortality, but the entire litter is destroyed. Of course, some cases of actually infertile matings are also represented. By this ‘early prenatal mortality’ is meant the absorption or loss of an embryo before it is of sufficient size to be detected on carefully feeling the uterus through the body wall of the mother. With experience an embryo eight or ten days old may be de- tected by an external examination of the uterus. Through our routine examination of the females after being with the males for one month, any embryo lost after this time will have been discovered and is definitely recorded in the third horizontal space of the table. If absorption or early abortion of one or more embryos in a litter may actually be observed to occur after as 148 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU much as ten days of development, there must certainly be a pre- natal mortality of some extent previous to this time. Experi- ments with the eggs of lower forms which develop outside of the mother, permitting direct observation, speak for the great pre- ponderance of an early embryonic mortality, many such eggs dying during the cleavage and gastrular stages when subjected to even slightly unfavorable conditions. We have some direct evidence on ‘early prenatal mortality’ in female guinea-pigs which have been examined by operation after repeated ‘mating failures.’ The ovaries of some such animals contain corpora lutea of pregnancy indicating that an embryo had been present shortly before the examination. Pearl records that the eggs from alecoholized fowls are to a high degree infertile. This he believes is due to many of the germ cells as such having been killed by the treatment. By infertile, Pearl means, of course, that no fertilization or zygote formation took place, yet it is extremely difficult in all cases to detect whether the early stages of development may not have occurred and been followed by death and degeneration. The death may have occurred during the cleavage or gastrular stages while the egg was yet in the uterus of the hen and many of the ‘infertile eggs’ might really be classed among the early pre- natal mortalities. We make these suggestions merely as pos- sibilities which to us are somewhat tempting, since if there was actually an early prenatal mortality in some of these ‘infertile eggs’ it would bring the effects of the alcohol treatment on the fowls and mammals still closer together. It is only through our recent analysis of the size of litters and mating failures, along with careful examination of the pregnant females, that we have become aware of the sometimes frequent very early embryonic death. The second horizontal space shows the number of young from the several lines that reached maturity, or lived over three months. Here again the size of the litter is an important factor. It may be stated generally that the power of survival of a guinea- pig varies inversely with the size of the litter in which it is born. We shall see beyond that this is also true of their birth weight, growth rate, and certain other qualities so that in mak- MODIFICATION OF THE GERM-CELLS IN MAMMALS 149 ing comparisons between young guinea-pigs it is important to know whether the individuals concerned occurred in litters of equal size. In the normal lines all individuals born singly survived, and, as the seventh space shows, 30 per cent of them were unusually large or over size when three months old. Normal animals born in litters of two or three survive in about 84 per cent of the cases and are often of large size. The members of litters of four survive in only 62.5 per cent of the cases and are not gener- ally vigorous animals. The records show that 80 per cent of the young in litters of five survived, but this is very unusual and is due probably to the small number involved, and possibly to a slight extent to the extreme care with which the pregnant females with the larger number of young were handled. This extreme care, however, only saved 13.33 per cent from the same number of aleoholic-line young born five in a litter. The second column indicates that over 81 per cent of alcoholic animals born in litters of one or two are capable of survival. Such a record is almost as good as the control, showing how very strong the members of small litters are and indicates again that an early individual selection may have played some part, since no doubt there has been a prenatal mortality among the weaker individuals which originally existed in some of these litters. This is emphasized further by the fact that the members of litters of three survive in only 60.93 per cent of the cases. Here the prenatal mortality has not played so severe ardle and many weaker individuals are born. The power of survival of animals born three in a litter from the control is about 23 per cent better than from the alcoholic lines. Only 48 per cent of the aleoholic- line individuals from litters of four were able to live three months. Recognizing the small numbers involved, only 13.33 per cent of the aleoholic guinea-pigs born in litters of five were viable. It thus appears that when the alcoholic animals produce large litters the quality of the young is very poor, whereas their small litters contain animals with good survival records. ‘There is little doubt that this apparent difference in quality is in part due to a prenatal selection which, in the case of the small litters, has eliminated most of the weaker individuals and left only the 150 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU stronger to be born. In addition to this, it must also be recog- nized that the ability of the female to properly nourish the mem- bers of the large litters is somewhat overtaxed. Three or less than three embryos are very well nourished by normal mothers. It must be recognized here that the inferior records of the alco- holic lines are not alone produced by alcoholic mothers, but come also from alcoholic fathers as following tables will show. The survival records of the normal inbred lines are about the same as those from the straight control, and are almost equally superior to the alcoholic lines. The alcoholic inbred animals have a survival record closely similar to the straight alcoholic lines, and again decidedly inferior to either the normal or normal inbred lines. The fifth horizontal space contains the mortality records which are the reverse of the survival records just considered. However, we have given here not only the actual mortality in litters of different sizes, but have corrected the total mortality record on the basis of the occurrence of large and small litters and their mortality in the different lines as compared with the control. We have also expressed the mortality in numerical proportion in the several lines, taking the control as 100. The total mortality in the normal lines is 22.31 per cent. This is a very good record, since it not only includes the postnatal mor- tality, but all exact prenatal mortality as well. We mean by exact prenatal mortality those cases of absorption in utero and premature abortion which were actually observed, and not those calculated on the basis of size of litter, mating failures, ete., as was discussed in connection with the productivity of the different lines.., The total mortality of the normal inbred is 21.95 per cent, or almost the same actually as well as when corrected for litter sizes as the straight normal lines. The total mortality of the alcoholic lines without ibeeaiee was 35.52 per cent, or almost 1.6 times greater than the mor- tality of the control. But this does not fully represent the real difference between the two lines unless it be corrected on the basis of the mortality record for the different-size litters in the alcoholic and the normal. The mortality is much higher among MODIFICATION OF THE GERM-CELLS IN MAMMALS Eyl the members of the large-size litters than among those in the small litters, and the large litters are 1.7 times more frequent in the control than in the alcoholic lines. The mortality is corrected on the basis of the normal records as follows: The rate for the normal animals born one in a litter is zero; two in a litter, 15.21 per cent; three in a litter, 16.66 per cent; four in litter, 37.5 per cent, and five in litter, 20 per cent. _ On this basis what should be the number of alcoholic animals dying in the several different-size litters?) The numbers should be zero instead of 7 for one in litter animals; 24.64 instead of 30 for two in litter animals; 46.48 instead of 109 for three in litter animals; 37.5 instead of 52 for individuals born four in litter, and 3 instead of 13 for five in litter. These numbers give a total of 111.62, which divided by the number of alcoholic animals, 594, shows a mortality percentage of 18.79. On the basis of the control mortality for the different-size litters, this is what the mortality should have been in the alcoholic lines, yet instead of 18.79 per cent it was actually 35.52 per cent, or almost double the normal rate. Again to express the corrected mortality in the alcoholic lines in terms of the control as 100, we find that for every 100 of the control animals that die 189 from the alcoholic lines die. The last column shows the 302 aleoholic inbred animals to present a still worse record. The actual mortality here is 39.07 per cent, or one and three-fourths times higher than in the con- trol. Here again correcting as in the preceding cases, the mor- tality on the basis of the control record in the different-size litters, it should normally be 18.59 per cent, but instead the mortality is 2.1 times greater than this among these alcoholic inbred animals. In other words, for every 100 control animals that die 210 alcoholic inbred individuals succumb. While the normal inbred animals, although their numbers are small, pre- sent a slightly better record than the straight control, 98 of these dying to 100 of the control. In the third and fourth horizontal spaces of the table the total mortality is divided into the prenatal and postnatal deaths. The proportion of prenatal to postnatal death in the different lines presents peculiar arrangements that will be seen to exist, not only 152 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU in this, but in several of the tables to follow. The prenatal records include embryos that die and are absorbed in utero, never passing to the outside, other embryos and fetuses which die and are passed out or born prematurely, and finally full- term young which die shortly before birth and are, therefore, still born or born dead. The postnatal deaths include all ani- mals dying before reaching three months of age, at which time guinea-pigs are about mature. In the control lines 51.92 per cent of the total mortality oc- curred before birth or was prenatal, while 48.08 per cent of the deaths occurred after birth. Considering the numbers in- volved, it therefore may be said that the pre- and postnatal mortalities are about equal in the straight control lines. ‘There is no evidence here of a particular tendency on the part of the young animals to succumb at any given or critical stage in their development. The numbers contained in the normal inbred column are cer- tainly too small to be considered. In both the alcoholic and the aleoholic inbred lines where the numbers involved are considerable (the records showing 329 deaths among 896 animals), the prenatal mortalities are double the postnatal deaths. The alcoholic column shows 70.14 per cent of the total mortality to occur before birth, while only 29.85 per cent of the individuals that died were lost after birth. The last column gives for the alcoholic inbred animals 65.25 per cent of the total mortality as prenatal and only 34.74 per cent as postnatal. This consistent arrangement in the two columns indicates a tendency on the part of the weak and subnormal individuals of the alcoholic lines to suecumb during early stages of their development. Such an interpretation is exactly in ac- cord with and is substantiated by the high early prenatal mor- tality which exists in these lines as indicated by the size of their litters and frequent mating failures when compared with the control. A mortality arrangement of this kind accords with what is known of almost all weak or diseased stocks—there is a very high loss during the early stages of development, as well as during MODIFICATION OF THE GERM-CELLS IN MAMMALS 153 later embryonic or uterine life. Furthermore, many individuals - die very soon after birth, while those that happen to survive the periods shortly following birth are often capable of an almost or quite normal existence. The mortality in the control is low, but half of this, or a high proportion, occurs after birth. The mortality in the alcoholic lines is high, but only a low proportion, about one-third of this, occurs after birth. It may be added further that the young alcoholics which die after birth in the majority of cases die within a few days, while the control young that die after birth are more likely to be scattered along over a number of days or weeks. It is thus seen that in both the alcoholic and alcoholic inbred lines there is a decided tendency for the developing embryos and young to succumb during the early periods of their development. This would suggest that these affected individuals were often incapable of passing through the early critical stages of uterine life. But if they were sufficiently fit to survive these periods, their chance for existence was good, so that their postnatal mortality, although actually higher than the control, was pro- portionally much lower. Thus we have a somewhat rigid in- dividual selection taking place during the stages of uterine life, so that the sum total of the individuals at a given stage is of a better average quality than during any previous stage and vice versa. Therefore, as is clearly shown beyond, those ani- mals of the alcoholic lines which live to become mature and prove to be fertile are a strictly selected few and in each gen- eration the proportion of strong to weak individuals through this selection constantly tends to increase. The sixth horizontal space shows a complete absence of de- fective individuals in either the normal or normal inbred groups. It may be stated here that during the entire seven years of this experiment not one grossly defective or deformed individual has appeared in the nonalcoholic or control lines. This is a rather remarkable record for any group of animals, and it speaks strongly for the perfection of the original stocks from which both the control and the alcoholic lines have been derived. 154 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU In the alcoholic lines about 25 per cent of the individuals were grossly defective. By defective is meant those specimens which show deformities, such as one abnormally small eye, cataract or opaque lenses, deformed limbs, paralysis of the limbs, gross tremors which make the animal incapable of locomotion or proper feeding, ete. There are slightly more defectives in the alcoholic inbred groups, 3.31 per cent in all. The next line records the over-size or unusually large animals, those weighing more than 500 grams when three months old. Among the control 80 per cent of the individuals born singly or one in a litter grew to be unusually large specimens. More than 10 per cent of those in litters of two were also unusually large, and 5.53 per cent of the three in litter animals are included in this class. None of those born in litters of four or five were able to attain such a size. Of the total control animals over five and one-half per cent were of this large size, while only about half as many from the alcoholic and alcoholic inbred lines at- tained such a distinction, yet in both treated groups there were over two and one-half per cent of large specimens. The last line of the table shows the occurrence of unusually small animals, those weighing less than 300 grams when three months old. Among 233 control animals only one such individual appears, 0.42 per cent. The alcoholic lines contain more than three times as many of these as the control, but still very few, only 1.34 per cent. ‘The numbers in the normal inbred column are too small for consideration. Among the 302 alcoholic in- bred animals there were eleven under-size specimens, or 3.64 per cent. This is almost three times as high a percentage as occurred in the alcoholic lines and over eight times as high as is recorded for the control animals. Comparing the present results with those of our earlier papers, particularly with the similar table 2 (16), it will be noticed that the numbers involved are almost twice as great and the records of the animals considered are decidedly better than were for- merly shown. This improvment in the quality of all lines is due to several factors. In the first place, the breeding methods have been decidedly improved since studying the oestrous cycle of MODIFICATION OF THE GERM-CELLS IN MAMMALS 155 the females and determining the exact time of the ‘heat periods’ (Stockard and Papanicolaou, 17). This has enabled us to pair the animals at the most favorable periods and thus to obtain far better and more exact mating records than was possible on the basis of the previous conceptions of the guinea-pig’s sexual be- havior. Secondly, the housing, care, and feeding of the animals are decidedly better during the last three years than during previous times, and on this account the mortality in all lines has been reduced, but as might be expected, the weaker alcoholic lines have profited more by this improved condition than have the control animals. For example, the mortality record of the control has been lowered only a little more than 3 per cent, while in the alcoholic lines it has been lowered a much as 18 per cent. This improvement in the alcoholic lines is also partly due to the existence of more late-generation animals with many normal ancestors. Thus, although the lowered mortality record of the alcoholic may not be entirely due to the better living condi- tions, yet it serves as a striking illustration of the difference in response to the change on the part of the control animals and the alcoholics. The previous somewhat unfavorable state did not greatly impair the powers of existence of the control ani- mals, but it did evidently eliminate some of the weaker alco- holic individuals that might have survived under more ideal arrangements. It must be recognized, in the third place, that for the alcoholic inbred animals the degree of inbreeding among the later gen- erations here included is less intense than was the case with earlier generations in the former reports. And for this reason the previous rather decided differences which were shown be- tween the straight alcoholic group and the alcoholic inbred ani- mals have almost, though not entirely disappeared. Lastly, the fourth point of difference to be borne in mind in comparing the earlier and present records is that there are now more late-generation alcoholic descendants with less affected material in their total germ-cell complex than was true of the ani- mals in the former tables, which as a group were composed of generations closer to the direct alcohol treatment. For ex- 156 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU ample, an animal derived from a directly aleoholized father and a normal mother could be said to contain half affected and half normal germ plasm, whereas another in whose pedigree the only aleoholic individual was an aleoholized grandfather, would undoubtedly contain a smaller amount of affected stuff. Finally, then, in the ight of the facts involved, the general table presents an impression closely similar to that derived from the previous records of these experiments, but it adds data of much importance for a clearer understanding of the problems concerned. The improvement in the present records over the former cnes might suggest that should the methods of breeding and caring for the animals reach perfection, the differences between the alcoholic lines and the control might be entirely erased. This would be possible if the improvement was due alone to method, but such a suggestion ignores the fact that the improvement is more largely due to the presence of late-generation animals with only a small amount of alcoholic germ plasm in their ancestry and a large number of normal progenitors. The analysis of the following table 2, in which the several generations are treated separately, will fully substantiate the validity of the foregoing statement. : Before considering this table, however, we may discuss briefly the phenomenon of absorption of embryos in utero and our methods of examining pregnant females in order to fully record the fate of all embryos that begin to develop. A knowledge of this prenatal mortality is involved not only in the table just studied, but in several of those that follow. 6. ABSORPTION OF EMBRYOS IN UTERO AND ABORTIONS OF PARTS OF LETTERS: METHODS OF DETECTING THESE PROCESSES After having observed the course of pregnancy and the size of the litters produced in a large number of cases, we became con- vinced that many of the small litters delivered at full term were only partial litters. Particularly in the alcoholic lines it became evident that abortions of one or two members of a litter might MODIFICATION OF THE GERM-CELLS IN MAMMALS 157 occur without hindering the further development to term of the remaining members. It was also recognized as is known even for the human female that embryos might be absorbed in utero. In the guinea-pig we have found that the absorption of one or more embryos in utero, as is true of partial abortion, may not interfere with the further normal development and birth of the remaining members of such litters. When it was realized that these absorptions and abortions of parts of litters were taking place, the necessity arose of definitely detecting each case in order to make the prenatal mortality records approach correctness. A systematic examination was, therefore, begun of every female after being with a male for one month up to within a week or ten days of delivery. The female to be examined is allowed to stand on a flat sur- face and the investigator with both hands presses the ventral abdominal wall so as to feel with the fingers the horns of the uterus against the dorsal abdominal wall. With considerable practice the small embryos and placentae may be definitely counted within one or both horns of the uterus. The num- ber of embryos and their position in the two horns of the uterus are noted on the record card of the female. After this initial examination she is reexamined once or twice during the preg- nancy and each time the number and position of the embryos with the date of examination are recorded. The number of young finally born helps to show how nearly correct the exam- inations have been. The records now contain several hundred such examinations and show that absorption of embryos may take place not only during early stages, but after the fetuses have attained consider- able size. The difference between absorption and partial abor- tion may usually be recognized by the fact that the embryo being absorbed may exist for some time as a small lump in the uterus, while the aborted embryo disappears from the uterus and leaves no palpable remains. There are exceptional cases in which the uterus is unusually swollen or congested after the abortion and these on being felt would still seem to contain a partial embryo. The cages of the pregnant females are exam- 158 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU ined every morning and afternoon and aborted embryos or pla- centae are generally located, yet instances do occur of early abortion possibly during the night in which no trace of the aborted material is found, since the female very quickly attempts to eat the aborted products. Fig. 6 On the right a normal 19-mm. embryo taken from the right horn of the uterus of an alcoholic female. The left horn of the uterus contained the degenerating mass shown on the left which was attached to a small placenta and represents an embryo in the process of being absorbed in utero. The mother had an aleoholized father. During the two years which supply the data for the present study the females have been very carefully and consistently examined throughout their pregnancies, and the records of ab- sorbed and premature or aborted young are very accurate for all MODIFICATION OF THE GERM-CELLS iN MAMMALS 159 periods after the embryos are of sufficient size to be detected by this method of external examination. ‘To convey some idea of how accurately one may detect a structure by palpation through the abdominal wall of the guinea-pig, it may be stated that a slightly cystic ovary has frequently been diagnosed by such an examination. A normally developed embryo 19 mm. crown rump length is shown in figure 6 and near it is seen an amorphous embryonic mass 2 mm. in longest diameter which represents the other member of the litter. The two were in different horns of the uterus. The placenta of the normal embryo was of the usual size, while the one associated with the arrested specimen was only about one-half as large. The entire mass of the smaller ovum in the uterus was about that of a ten-day specimen, while the normal individual was a typical twenty-day specimen. This case was detected by external examination and was merely opened in order to use the embryos for illustrating the phenomenon. In the explanation of the figure the ancestry of the embryos is given. The intrauterine absorption of embryos, as stated above and indicated in table 1, may occur in normal guinea-pigs. A. W. Meyer (’1t7) has very recently described the histological con- ditions found in partially absorbed embryos which he had ob- tained during a study of the prenatal growth of the guinea-pig. There is considerable data from our study to indicate that this absorption of embryos is somewhat more frequent in the alcoholic than in the normal lines. 7. A COMPARISON OF THE QUALITIES IN THE DIFFERENT GEN- ERATIONS OF THE ALCOHOLIC LINES AS THEY BECOME FURTHER REMOVED FROM THE GENERATION DIRECTLY TREATED It has been mentioned in discussing the improvement of the records in table 1, as compared with our previous reports, that this advantage is partly due to the larger number of late-gen- eration animals at present included. We may now analyze the alcoholic lines for a comparison of the qualities of the early and 160 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU late generations, F; to Fs, on the basis of their productivity and mortality records. Such an analysis is of particular importance to test in the first place whether the effects of the alcohol treat- ment on the germ cells are permanent, altering their qualities in inheritance, and in the second place whether an increasing amount of normal germ plasm acquired with each generation may tend to offset the original alcoholic effect by dilution. Table 2 contains the data from the non-inbred alcoholic lines divided into different generations. The first. vertical column gives the records for 233 control young as a standard of com- parison. ‘These are the same records shown in the normal column of table 1, except that in the present table we have included in the first horizontal line under each group the average birth weight of the litters produced. This is termed the average litter weight and is recorded in grams. For the normal stock this average productivity is 197.12 grams; that is, the average weight of all the litters at birth was this amount. The average litter weight is In a way associated with the average litter size, since a litter containing several young though each individual may not be so large, will probably weight more than a litter of fewer or of one young. Thus a group having a higher average litter than an- other group will also probably have a higher average litter weight, though this is not necessarily the case, as will be seen on com- paring the several columns of the table. The second column contains the alcoholic line animals. This again is the same 594 alcoholic animals shown in the second column of table 1 and is given here for comparison with the four following groups, each of which is a certain portion of this total column. The average productivity for the alcoholic animals is 170 grams, or 87 grams less than the control, and when cor- rected on the basis of the average litter size, it is 5.6 grams less than it should be according to the normal standard. The third column gives the records of 186 animals with one or both parents treated with alcohol, the F; generation. Thirty- three of these animals also had a slight alcoholic history in their ancestry, and thus the entire group are not pure F; alcoholics. The proportion of large and small litters in this column is about 161 MAMMALS THE GERM-CELLS IN OF MODIFICATION Ver, MN aay o/tat : : : : / $= %St0t N(%blLI)| Grl=%c0 IoN (%i9-0¢) TLI=%688) N(%OSTS)| OEv=%s98i NC%io'cr)| G81=%6LRIN (%4z5-S¢) O0f =(%I2° tt) %09 Uslseltlezqloe %0l | fog Wav Whe elgeel Lewic | Loo) xsvvedargrxsal A | 9998 es Goce visa %evsi | %or le Gya1%ie'ss Sy Se agile as | ) IOS GD Oo os) Sl wupieaiae Olea al €l tS 60) O€ L Seo Sie ib Te is eS GF Ge oh eee Rae (%99°99) (%80'0S) (400) (%S8 67) (2f80°8+) Ome. al Braces 6 Ov we) C ae 43 tA =i) Wan eee (%LL'LL) (%t6°69) (fa'0l) (%+0L ) (%t6'1S) iG ole -Sle 2 lc le +1 + alfa 2) (0) +2 6L 6) (oy Gy Wh ae GA Ee Zt : 1 ha tot ghar oul eS a? a eye %S8't8 %8E69- XEG9T blee9 689 +L dL. fool xor-9l MSL va Yor M59 Url el Lev) OG eS 4049 2EI'8 is'Sh aibsl Leses veg e882 | eeccel Web akow9 ~arI8 Misi8 | Yoa ar aeeeexel4g L001 GC SG sri cglie iG i vy Etl 9B tt G 8b OL! Zel IS @l St sb 6¢ O} i 4 Gre aS! Boas Cs eae ci a7) is +os Cet 2 (ee (esede “rf yow CHS tN wfyww] (%rocnaIe-wefyow | “(o%tg4)e wef yew ¢ APRLOLI [AM rayil eSV-eay [asia Zbl yAA Vay! 4S [SGI YM Layyl] DBOIPY |.So-oL| Yara} PBwosryy | aBTULG) yr sayi| eSvaony TIT NAMI IPVAIAY L+’'@ -VO}pL!] ABs ayy IS-@ ADH] DHOAII oY LeU -\OH!) OPvaaiy LL°@ Vy aSonew %\9 OF %Le'yt %Go'0% BB TS % +L LI LIE Lee 6l ¥lo'eS YEE'LE %SO' ve i ——— a —— i —— pat im cA — —— G* Ov 99 9t Ol Ol tL %61.901 sz G 3 06 +5 6 SI OO! 6LT %9) BF S| @L O6 9¥ Ol Srey re J) Gi er Oma Serta et Coe Si iae Gee (ig eae | 4 S $4UD.10d |s fud.00d puDig- poars sJUdied PWS - OOS | PUB. — {vd1S-fVIIS]-poo.iS puro stud.i0d -f0a1$ oppor cae Ean S$ ea, puis Bee Gear Senuoiiod HIOFS JOWAOKZ reer PUDIS POLOOlE YPM] S(OUMUO DjOYOD Wy] SPOUNUI DNOYOd YY SJPLUUIUWE DjOYO| 7] SPOULIUE DIOYODS | MOOS. OiNOHOONy SHib JO" SVOREVMENSSO Waele PoP jOJOL SysUOW & UI FIM POld U10G \\Ifg OANLOWO-1d ‘Paqiosgy SYsUOW S a AQAO poary Joqunu JeyOL AHL NI ONIYYNODO SIVWINY JO SGYOOAY SHL 4O WOSMVaWOD Y ING Seale etal THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 26, NO. 1 162 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU the same as in the total alcoholic column, 34.6 per cent of the animals were born in litters of less than three and only 17.74 per cent in litters of more than three. The normal record as pointed out before is just about the reverse of this. The average- size litter in which the F; animals occur is 2.51, which is slightly larger than for the total alcoholic column, but the average weight of these litters is less than for the entire alcoholic lines, being 165 against 170 grams. As compared with the control the average productivity of this column is 32 grams low, and when corrected on the basis of the average-litter size, the litters are then more than 13 grams less than the control standard. The mating failures are about the average alcoholic result, 12.94 per cent. The mortality record of the F,; animals is not so good as for the entire alcoholic group, only 56.98 per cent of them living longer than three months as against 64.47 per cent. The total mortality is 43.01 per cent, and when this is corrected on the basis of the normal mortality for the various-size litters in which the individuals occurred, we find that the F; mortality is almost 2.3 times the control record, or 230 against 100. The corrected mortality here as compared with the entire aleoholic group is 230 against 189, or 41 points higher. The proportion of prenatal to postnatal mortality corre- sponds closely to that of the entire alcoholic group and contrasts with the control in the same way as discussed in considering table 1. Finally, then, the F; group of animals from either one or both treated parents, are inferior to the alcoholic group as a whole in having a higher mortality record and in occurring in litters of a lower average weight although of equal average size. The fourth column contains the records of animals more than one generation distant from the aleohol treatment; that is, those having treated grandparents, great-grandparents, or great-great- grandparents, or combinations of these, F., F;, and Fy generations. All of the alcoholic animals from column 2 are included in this column, except the third column of F, animals; there are thus 408 individuals. MODIFICATION OF THE GERM-CELLS IN MAMMALS 163 The distribution of the animals in large and small litters is closely the same as in the two preceding columns, over 30 per cent being in litters of less than three and 20.09 per cent in litters larger than three. ‘The average-size litter and the average litter weight are just about what is found for the total alcoholic group and somewhat better than for the F; group. The percentage of surviving animals is a little better than the total alcoholic group and considerably better than the F, group. The prenatal and postnatal mortality proportions follow the typical arrange- ment for the alcoholic lines, the prenatal being about two and one-third times higher than the postnatal. The total mortality among these animals is about 10 per cent lower than for the F; group and slightly below the record of the total alcoholic lines. When the mortality is corrected in terms of the normal mortality for the different-size litters and stated on the basis of 100 for the control stock, it becomes 172 as against 230 for the F, column and 189 for the all generations alcoholic column. The fifth column records 147 animals still further removed from the treated generation; these had treated great-grandpar- ents or great-great-grandparents or both, ”the F; and Fs genera- tions. Some of these animals may have had only one or two alcoholic ancestors out of eight or sixteen; therefore, the pro- portion of modified to normal germ plasm is often very small. The arrangement in large and small litters differs from the other alcoholic groups and approaches that shown by the nor- mal lines very closely, there being a higher percentage born in large litters than in small. The average-size litter is larger than in the three preceding columns, although still well below the control. The average litter weight is low when compared with the normal lines and only about the same as in the three preceding columns when taken in connection with the average size of the litters. When corrected for the average size, the weight of the litter falls more than 10 grams below the control record. The mating failures still show the high percentage of the alcoholic lines, being over three times as many as in the control. 164 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU A greater percentage of individuals survived than in any of the preceding groups except the control. The proportion of prenatal to postnatal mortality shows the arrangement characteristic of the aleoholic groups. As a matter of fact, the prenatal mortality is really unusually high, and this is probably due to the high percentage of large litters, as among these the prenatal mortality is most frequent. It is as though the animals of this group had produced almost as high a pro- portion of large litters as the control animals and still they were not sufficiently good quality as compared with the control to keep down the prenatal mortality in these high litters. The total mortality when corrected on the normal rate for the litter sizes and expressed on the basis of 100 for the control becomes 145. This is a decided improvement over the other alcoholic groups, although poor in the light of the control. From a survey of this column it may be concluded that ani- mals as far as three generations removed from the direct alcohol treatment are still differentiated as a group from the control in regard to the weight of the litters in which they are born, the tendency of the matings to result in failure, the high proportion of prenatal mortality over postnatal, and the total mortality which is one and one-half times higher than the normal. All of these differences exist in spite of the fact that more and more normal germ plasm has been introduced during each generation until some of these animals may have had as many as six or seven normal great-grandparents against one or two treated or alcoholic great-grandparents, though the average of course had somewhat more treated ancestry than this. One of the F; individuals, descended from treated great- grandparents, is shown in figure 7. The animal on the left was a non-inbred female, No. 803, with six of its eight great-grand- parents treated with alcohol and only two, on the paternal side, were normal. Its great-grandparents may be written thus: A indicating alcoholic and N normal, the @ on the left, in the formulae: [(AxA) (AxA)] [(NxA) (AxN)]. The animal on the right is an ordinary normal guinea-pig born on the same day as the small degenerate specimen which weighed only one-third MODIFICATION OF THE GERM-CELLS IN MAMMALS 165 Fig. 7 On the left a non-inbred female, No. 803, with six of its eight great- grandparents treated with alcohol and only two on the paternal side not treated. She was small and degenerate and lived only one day. On the right is shown a normal animal born on the same day, the two being photographed on one plate. Fig. 8 Two F; guinea-pigs born in the same litter from a normal father and a mother derived from four alcoholized grandparents. The albino female, No. 955, on the left weighed at birth 90 grams, the small defective male on the right weighed only 38 grams and died within two days; the sister is still alive. 166 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU as much and lived only one day. Although some young from control parents do die shortly after birth, they are not so un- usually small nor degenerate in appearance as the defective young of the alcoholic lines. Another even more striking example of the small defective animals appearing in the F; generation is shown by the photo- graph, figure 8. The two individuals in this picture were born in the same litter. Their mother was a black and red animal from four aleoholized grandparents and their father was a nor- mal albino male, [(AxA) (AxA)] [N]. The F; animal on the left, No. 955, is an albino female weighing at birth 90 grams. She is thus an unusually large animal to be a member of a litter of three and is of the type of the normal albino father. Her small degenerate brother on the right weighed only 38 grams at birth, had a severe tremor which rendered him incapable of nor- mal progressive movements, and he lived only two days. His degeneracy and black and red color are both qualities for which he was indebted to his aleoholic mother. A marked discrepancy in either size or condition between two members of the same litter at birth is entirely lacking among our control lines. It is rarely so decided as this case illustrates, yet very frequent in the alcoholic lines and particularly in the F, and F; generations. A number of illustrations of this type could be continued to show that the quality of the later generations from alcoholized ancestors is decidedly subnormal. Such conditions as the above occur not only in spite of the introduction of normal germ plasm which tends to overshadow the alcohol effect, but also in spite of a rather harsh individual selection which is at work tending to improve the stock with each generation. Almost all of the badly defective individuals in the alcoholic lines are lost early in their career, as is shown by the high prenatal mortality; other less defective ones die soon after birth, such as those pictured above, and only the best live to become fertile adults. It is thus found that even this selected group mated with many normal individuals still pos- sesses enough of the mcdified germ plasm which resulted from the early alcohol treatment to cause their offspring to be inferior to MODIFICATION OF THE GERM-CELLS IN MAMMALS 167 the control animals in a number of important qualities that ren- der them less capable of survival. These two factors, the constant introduction of more normal germ plasm and the elimination of all the weaker alcoholic indi- viduals so that only the stronger reproduce, may finally in late generations so purify the aleoholic lines as to cause them to attain a condition equally as good as the normal. The sixth and last column of table 2 may illustrate such a condition, though it contains the records from only a few ani- mals. These animals are descended from one or more treated ereat-great-grandparents, the F, generation. They are four generations removed from the alcoholic treatment. The average-size litter is almost as large as in the control, and on the basis of its size it is actually heavier than the control average. It may be said from the evidence shown that the pro- ductivity here is equally as high as in the control. A higher percentage of individuals survived than among the control, and even though the mortality figures are small there was certainly no tendency toward a high prenatal mortality. On the contrary, there was scarcely any prenatal mortality, so that the record in no way resembles that of the alcoholic lines. On the basis of 100 for normal stock mortality, the mortality here cor- rected for litter size is only 84, or 16 per cent better than the normal. It is actually in the table 5 per cent lower than the control. After having considered the last column, the F, animals with their very good record, it should be recognized that these same animals are included with the F, and Fs; individuals in the fourth and fifth columns. Their presence in these columns, particularly in the fifth, has tended to incline the records toward the normal. One must realize, therefore, that the F, and F; animals if considered alone would present even stronger aleoholic records than are indicated in the fourth and fifth columns. The table shows that the nearer to the direct alcohol treatment an animal is produced, the more inferior in quality it will be as a result of the high amount of modified germ plasm contained in the germ-cell complex from which it arises. Therefore, the records 168 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU of F, individuals are worse than the records of the sum of all alcoholic generations, as is seen on comparing the third column with the second. The later generations being further and further removed from the treatment and having less and less modified germ plasm on account of the constant introduction of normal stock are progressively improved until finally the Fy gen- eration has its modified germ plasm diluted to such a degree that its record is on par with the control. The ancestors of these late-generation animals were also suc- cessively selected from the least affected of the alcoholic stock, being those animals capable of survival and reproduction, while the most highly affected died or were sterile and incapable of reproduction. We assume the probability that the more nearly normal animals with stronger bodies also carry germ cells that are less affected than those in the more degenerate individuals. Most of the grossly defective individuals which reach maturity are sterile as evidence in this direction. Thus individual selection being in this case a selection of germ plasm as well as soma, helps materially to improve the quality of the later generations. 8. A COMPARISON OF ANIMALS FROM DIRECTLY TREATED FATHERS AND FATHERS OF ALCOHOLIC STOCK WITH ANIMALS FROM DIRECTLY TREATED MOTHERS AND MOTHERS OF ALCOHOLIC STOCK AND WITH OTHERS FROM BOTH PARENTS OF ALCOHOLIC STOCK Are the general conditions induced by directly treating the father with alcohol the same as those resulting from treating the mother, and are they equal in extent? Do fathers of alcoholic ancestry beget offspring of better or worse quality than off- spring produced by mothers of similar alcoholic ancestry? Or are the effects of the alcohol treatment on the germ cells, which is expressed through several generations, carried with equal degree by both the alcoholic father and the alcoholic mother? We shall attempt in this and the following section to supply data which may serve to partially, at least, satisfy these queries as well as furnish an analysis of several other more detailed propositions. 169 MODIFICATION OF THE GERM-CELLS IN MAMMALS Dil 90tT= [8t= ghi= %oS\Bl N (%8O1S) |%99 BIN (AEETE) 1989 WIS AUG Bb tA % IK) YooltscAsse}. S LET 4+] E be L9TI I pease (We Ge erat aL Peep |PjoL ©) (Gs Ee El G S Ol ba eee | yo (Hb le) (%ET ST) (Ie oF) (%tL TC) sysuowW ¢ CPS rd SP Ny 2S al ce) oO ord (@) pe) | @) ) (2) (0) (0) U(ULiM ( Sete cilia emt cuiceal § bet Geet moe (UFIM poiy : UNO} [IPs (%e6-t9) (%9L +l % ¥0°69 glo ahd f a3 {eI ; \ - $i Zl dey fj uy \ ) PANnpeuMsid AS (GS tok G ac os i Sy) (oa (e) ts oO St GG oO ae 6 tile fe oe Mig tee 1 letee et GFEt | ‘peq sosqy 8 89) (+b°09) C48T 59) (%9%°69) (%%9-L9) (% IL-9) (Lib) (%99-99) ssl uwqs¢srB Ul [dor yoy vklsaleal yo18) euecauleccaveces| asl eles ai46 %o8 |b tadrosigznel tils8] 105 4ypIb xsl, 29999 WCE TEL ULL Yoo | Mees ae oL ees “L001 SU;UOU © O ve BS HI] Ze J Bhat L | oO Ml BL Of OF] O Ie bE +e TI] ZT I GL oc 9 | Oo Ol tw + @y i Ri Sit 5) (BAO POA] a sae | fs cae SC | Ss ra ote Co | able | ace | ok © veer Gs {a all 3 661 +Lt al] (%S4¥-9)9 jioh-pond (yoke El aiwfww (4eslO+l \ywJuvw THEI yonposd aay | bis ynpoAd way |60-1LI4yonpord aay tal 78) 6S (669-2) 9 phew kyg-annel awfaew | eerory9 “of ww TRE donpord-sray [SL-9L! yanposd ara [GGoL)4yonposd aSuroay BUT Aay(>Bwarny | E9g°c Ad [eBvsoray | ppt aay aborray | ie-o AAW Bway 1F9°T raysPBvroy | st-c sat |>Bvrray -l od wnu ees atte eel = MSEET ulsoe “LITLE leal-a = uirow Iuesy = acler |vicey * eecle ‘bell = —asyou | %ot nov Mestre) fs sg 8¢ BLol Bl) G orsalos 8 |g te 9E 9€ tI | O be tL eH SI | FS be gol ge L| Gace zu dng Oo 9) LS gio i | oO tl4cgig9 Gaa4 ts G6 WN is we Gee Se ss eee Weal cr a ae sep te lee tie eG Heat 6 UE | ee ee TT Doyoo© | Diyoyoo)e | D'oyooje SL U9.10d Yjog |.194,OLY AJ UO} soupefA|UO +6S peposi, | popeoip sjua.ied Yo] 1ayjoul AuO} -roype f Aju |sjuated Yrog|ouZ,OUL AUC) soupe ft Kua sor If Jdogox (pepnyoul 4s-lifsuope.1aUcyyie)| ; Howerstee Pei Vip sieae apis SOR C) ©) Risse JOU ng UBISeP D'OYOo|e SYoYoO De fo sJeWUlUe [IY | fo spusied YM sjeUUe ZIOUOdIV OVIOHODIY SLNAYYd HLOd YO SITOHOOIY YAHLOW “OIMOHOOTY SI YSHLWa AINO NaEHM ANASOUd SHI VO SLOBSda3 GHL 4O SISAIVNY NW WES Bel, (UolPe.idUas 4s41f) sLUded Pepedll YIM S;EUIUL DILOUODI|VY 170 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU Table 3 is an arrangement of the records of animals on the basis of paternal and maternal alcoholism. ‘The first group of animals are those from parents treated directly and having no other alcoholic history. Thus the total 153 differs from the total 186 animals with treated parents in the third column of table 2, since in thirty-three cases the former group had not only treated parents, but also treated ancestors. The second group contains all the animals with parents of alcoholic descent, but not di- rectly treated, the total number is 408; these are the same ani- mals that compose the fourth column of table 2. The third or last group contains all of the 594 non-inbred animals of the alcoholic lines. The individuals in each of the three groups are separated into three classes. The classes of the first group are those with only father treated, those with only mother treated, and those with both parents treated. In the second group the young are classi- fied as those from only father aleoholic, which means the father was descended from treated ancestors which may have been either treated males or females. In other words, this is not the record of a pure alcoholic male line, but merely the alcoholic effects, if any, that reach the recorded individual through an alcoholic father regardless of the origin of his aleoholism. The second column of this group shows the records of animals from alcoholic mothers. Here again the mother’s alcoholism may be due to treatment of any of her ancestors, male or female. It is not a purely female alcoholic line, but a maternal alcoholic line. The third column of the second group shows records of animals from parents both of which were alcoholic. In the entire second group the alcoholism of the parents is ancestral, not being due to direct treatment, while in the third group the alcoholism is either direct, ancestral, or both. The third group is, therefore, an arrangement of all the animals from alcoholic lines for a comparison of the influences of maternal and paternal alcoholism. In the first column of table 3 it is seen that when the father only is treated the results contrast decidedly with the control. MODIFICATION OF THE GERM-CELLS IN MAMMALS 7a! There is a high percentage of small litters and a low percentage of large litters, thus giving next to the lowest average litter con- tained in all the records, only 2.30 against 2.77 for the normal. The average litter weight is very low on account of the small average litter size. When this is corrected for the proportion of weight to number of individuals in the control litters these small litters from the treated fathers weigh more for their size than do the control, being over 6 grams heavier. This is not an actual advantage since the majority of young born in small litters of one and two are larger than those born in high litters of four or five. The percentage of mating failures is unusually high, 23.52 per cent against only 4.54 per cent in the control. All of these facts would seem to indicate that the treatment of the fathers had evidently lowered their productivity or fertility, causing them to fail to sire offspring in almost one-quarter of the matings and to beget unusually small litters in the other three-quarters of the cases. There must have also been a high ‘early prenatal mortality’ in view of the remarkably great per- centage of small litters and high percentage of mating failures. We must necessarily divide the mortality into prenatal and postnatal, and the prenatal again into ‘early prenatal,’ as in- dicated by the small average size litter and high number of mating failures, and ‘late prenatal’ based on the exact observa- tions of absorptions, abortions, and still births. Two-thirds of the offspring from treated fathers survived against over three-fourths from the control. The prenatal mor- tality is a larger proportion of the total than in the normal. The total mortality when corrected to the normal rate for the differ- ent-size litters in which the animals were born is 178 in terms of the control as 100. This is only slightly below the mortality rate of 189 for the entire non-inbred alcoholic group. When the mother alone was treated the records of the off- spring differ considerably from the above. The percentage of small litters is only slightly higher than the percentage of large litters, and the average-size litter, 2.78, is as large as the nor- mal. There are very few mating failures, in this regard again 172 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU almost a normal record. The productivity of these treated mothers is high and the size of the litters would indicate a very low ‘early prenatal mortality.’ Here, however, their good records stop. Although the litters contained as many individuals as the con- trol litters, their average weight was 26 grams below the nor- mal. The large litters from treated mothers actually weighed only as much as the very small litters from treated fathers; there- fore, the individual members of the litters from treated mothers were unusually small animals. The ‘late prenatal mortality’ was proportionately very high—three times the postnatal. Thus many of the young died in utero or were still-born, and those that were born alive were small specimens. The total mor- tality was 51.08 per cent, corrected for the litter sizes, and ex- pressed in terms of the control as 100 it becomes 281—the highest mortality on record. We see from the table that treating the mother with alcohol does not appreciably affect her productivity, but greatly depreci- ates the quality of offspring to which she gives rise. While in the case of the alcoholic father the productivity is greatly re- duced, and although the quality of offspring which he begets does not compare favorably with the control, it is considerably superior to that from the treated mother. In the treated mother the alcohol may act not alone on the ova or germ cells, but on the developing embryo as well, while in the father it acts, of eourse, on the germ. cells alone. Does the difference between the qualities of the offspring from these two cases represent the action of the treatment on the developing young in utero? Further, does the reduced productivity on the part of the treated male indicate that the spermatozo6n or male germ cells are more sensitive to the treatment than the egg? The remain- ing columns of this and the following table may throw some light on these questions. During the period of the experiments now under consideration practically no matings between treated males and females have been made, as the third column of this group shows. MODIFICATION OF THE GERM-CELLS IN MAMMALS Wi The’next group in table 3 are animals derived from parents of alcoholic descent which had not themselves been treated. These are the same 408 animals recorded in the fourth column of table 2. The first class in this group are animals obtained from fathers of alcoholic ancestry and normal mothers; the second class are from mothers of alcoholic ancestry and normal fathers, and the third class are animals produced by two alcoholic parents. Ags mentioned above, the alcoholic father or mother may owe their condition to either male or female or to both male and female ancestors. These are not purely male or female alcoholic lines such as will be found in the next table. A comparison of these three columns with the normal records shows clearly the alcohol effects, though not so strongly ex- pressed as when the father or mother is directly treated. The father and mother columns of this group differ very little from one another, which is in marked contrast to the striking differ- ences when the fathers and mothers are directly treated, as seen in columns 1 and 2. In the present columns all of the modified conditions are due to an injury of the germ cells in the treated ancestral generations. This is equally as true of the alcoholic- mother column as of the alcoholic father. For example, the mortality records in the alcoholic father and mother columns are about the same, while there is a remarkable discrepancy between the mortality records of young from treated fathers and treated mothers in the first two columns. The extremely high mortality, largely late prenatal, among the offspring of directly treated fe- males is to some extent due to the direct action of the alcohol upon the early developing embryo in utero. If this action could be eliminated the treated father and mother columns of the first group might become as nearly similar as the alcoholic father and mother columns of the second group. The individuals in the latter two columns are on an average about the same dis- tance removed from the ancestral alcohol treatment, and, there- fore, the records would be little affected by a correction on the basis of the generations treated. When both parents are from alcoholic ancestry the produc- tivity is considerably lowered as shown in the third column by 174 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU the high percentage of small litters and low percentage df large litters and consequently the very low average litter of 2.31. This is likely due to the male partner in the combination, as the preceding columns would suggest. The average litter weight, however, is high, so that the individual members of the litter are as heavy as the normal; this, again, may be due to the male influence as expressed in the high early prenatal mortality. The mortality records, though markedly inferior to the nor- mal, show an advantage over the two previous columns. ‘There is probably a high ‘early prenatal mortality’ as indicated by the low average litter, but the ‘late prenatal mortality’ is lower than in any of the foregoing columns except that of the treated fathers, where again the litter was very small and the prob- able ‘early prenatal mortality’ high. This close association be- tween the small litters and the low late prenatal mortality makes it seem all the more probable that the litter size is associated with an ‘early prenatal mortality’ that occurs so near the begin- ning of development that it cannot be directly observed. On the other hand, this result could be interpreted as due to a lowered fertility. If this were brought about through an elimi- nation of the weaker germ cells we might except also the asso- ciated low late prenatal and postnatal mortality, and would have a condition in exact accord with Pearl’s interpretation of the results on fowls. We should be glad to accept such an ex- planation, but for the considerable amount of evidence in our records which points towards a high ‘early prenatal mortality’ rather more than infertility as the underlying cause of the small litters and low late mortality. It must also be remembered that the infertility among the fowls was found in the females as well as the males, while here it would be confined to the males only. The shght advantages which appear in favor of the records from both parents alcoholic as compared with records from alco- holic mothers or fathers are due largely to the distance from the treatment of the generations concerned. In the majority of cases the generations are more remote in the both-parent column than in.either the father or mother column, and on the basis of MODIFICATION OF THE GERM-CELLS IN MAMMALS 175 the evidence shown in table 2 this may readily explain the apparent advantages. The last three columns show the results of the first two groups combined and in addition contain a few records from mixed cases that could not be properly included in any of the previous classes; for example, animals with one parent of aleoholic ancestry and the other parent directly treated, ete. Here again there is considerable contrast between the alcoholic- father and the alcoholic-mother columns, these differences being due to the influence on the totals of the F, records from the treated-father and treated-mother columns of the first group. The productivity when only the father is alcoholic is low, the litters being small and over 21 per cent of the matings result in failure. It may be inferred that there was a rather high ‘early prenatal mortality.’ The average litter weight, however, was about as good as normal. The late prenatal and postnatal mortality records are better than those from the alcoholic mothers. The average-size litters from the alcoholic mothers was rather large and the mating failures were much less frequent than from the alcoholic fathers, indicating a lower probable ‘early prenatal mortality.’ The average litter weight was lower than from alco- holic fathers, taking into account the size of the litters in the two classes. The total mortality from alcoholic mothers was high and the proportion of late prenatal to postnatal was ex- cessive. It is thus seen that a high prenatal mortality is fol- lowed by a low postnatal death rate, and this is in accord with our assumption that a high ‘early prenatal mortality’ will be followed by not only a low postnatal, but also a low late pre- natal mortality. In other words, the more thorough the elimi- nation of defective embryos and fetuses the greater the prob- ability of survival for the selected few that remains to be born. The last column with both parents alcoholic has a mortality record as good as the alcoholic-father column and better than the alcoholic-mother, but this is only apparent and not real. The column contains only one individual from directly treated parents, and consequently the alcoholic treatment was applied 176. CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU on the average to more remote generations than was the case in the two single alecoholic-parent columns. In spite of the generations concerned, there is a higher per cent of small litters and a lower per cent of large litters here than in any other class in the entire table. Consequently there is also the lowest average litter. In so extreme a case there was no doubt a high early prenatal mortality. The average litter weight is actually low, but allowing for the small-size litter the average birth weight of the individuals is about as much as the control, again indicating that an individual selection has occurred through an elimination of the weaker embryos during the early develop- mental stages. The extremely small-size litter and the high ‘early prenatal mortality’ may also in addition to the generations concerned ex- plain to some extent the relatively low total mortality and es- pecially the lower rate of late prenatal mortality as compared with postnatal. The questions involved in the present section may be still further analyzed by rearranging the data on the basis of only male ancestors treated or only female ancestors treated instead of only father alcoholic and only mother alcoholic. ‘Table 4 pre- senting this arrangement will be reviewed in the following sec- tion, after which several points of interest may be better discussed. 9. A COMPARISON OF LINES FROM ONLY MALE ANCESTORS ALCO- HOLIC WITH LINES FROM ONLY FEMALE ANCESTORS ALCOHOLIC AND WITH THOSE FROM BOTH MALE AND FEMALE ANCESTORS ALCOHOLIC The records tabulated on the basis of male or female ancestors treated supplement the arrangements in table 3, where the groups are classed for only father or mother alcoholic. In table 3 the alcoholic father may owe his alcoholism to the treatment of any of his ancestors, either male or female or both. The alcoholic effects, if any, are there due to the paternal ancestry. The same applies to the groups with only mother alcoholic. MODIFICATION OF THE GERM-CELLS IN MAMMALS 177 In table 4, on the other hand, the groups with only male ancestors treated owe their modified conditions, if such exist, entirely to the effects of the treatment on male animals, though the individual being considered may have inherited this alco- holic effect through its mother. Thus animals in the columns with only male ancestors treated were not necessarily derived from alcoholic fathers, but may have been produced by alco- holic mothers which, however, owe their alcohohc condition to one or more treated male ancestors. The table permits a comparison of the action of the treatment on the male germ cells and the transmission of the effects with the action of the alcohol treatment on the female germ cells and the effects transmitted to the different generations. While the last table permitted a comparison of the animals derived from males of alcoholic stock with others derived from females of alcoholic stock. The two tables serve to analyze very completely the problem of the parts played by the sexes in the acquisition and transmission of the effects of the alcohol treatments. The three columns in the first group of table 4 are the same as those of the first group of table 3, being the records of F; animals derived from treated fathers, which have only one male ancestor treated according to the table 4 arrangement, and F; animals derived from treated mothers or from only the one female ancestor treated. This group was discussed in review- ing the third table. The points of chief interest in the present connection are the decidedly inferior conditions of the off- spring from the treated females ‘as compared with those from the treated males, in so far as their measured mortality records and birth weights per litter are concerned. On the other hand, the records from treated males suffer as regards the ‘early prenatal mortality’ indicated by the small average-size litter and the high percentage of mating failures, while the records of the treated.females in regard to these conditions are equally as good as those of the control animals. The next three columns of table 4 are highly important, since they contain the results of matings when, first, only male an- cestors are treated; second, when only female ancestors are THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL, 26, NO. 1 178 CHARLES R. STOCKARD AND GEORGE N. PAPANICOLAOU WwSi\= ebIl= qe tS\= g6l= \gt= 8lis 4MEBELN (%LebZ) bo bl MW (~EC-OH)|VeTIN CLL-9S)]| Wb MLE f)| IT -Bl W(%ba°BC) {WSL -BIN (%ETLE) ATV N(%8I 1G), 98 N (Uce'es) alloca aissi4979] ails tare dle vor |ysqrteetel zine zegsl 28617 : ata Lal asbl| Wnibewwlezecrl 49c| 208457 Ale Beh 81 fol ‘slays | nbn a | POOP |BEOL oof pb €i97e Bec II Gg Oercige (G Wolo aac te ts ollie G7 || OO Or! leo? [lll aah ap Ol QV al qe) Siete et Seta Ag. (Na ae so OIG a ea ce ee DU Ss ete) | EP tae all YAL SE #10 GE) (LE ot) K ) ¢ g (2) SUPUOW SE og +i i og +i | (o+ Iz \ Sie c Tae iG Sita Cee ghee) (56649) (%b+°89) (%29°GL) (%8t+9) (tp be) (ae +L) ; UNOQIIUES 3 OANPCWO.14 Cpe tte y Vo) eee oy INA xe Gall @ Saas ¥3 (Exe Gill OP 0 gstt 9 ¢ mlz ages. 46m Lt We |) Sp ete ee Gii-CNe ee Cleese Se toe Ih Pogjiosgy (%tg°aL) (%99°bS) (%tt-€9) (%99°%]) (C4Sb IL) (ASU) (ib 8) (%99°99) Ack ACEL All Yoo] — oes Lebol. 04898 24001 SYUFUOW S| os sv bl | @) I Ue Sil S) JOAO Poa eat Aa ase ler Ae es . TG 09 (% It syernns ws (%es ee) 8 iy) yaw Lg Li yenposd ay | 82 °0L) pnpordasvamy Lees LIL YLLLL sl ol S€+l € GS 7% 5 0 \ Sree] ASSSSVISUCEEL | Agvet yecer ASL Yo8 feccel zULLcreegey eM tne O 92 Sb bb +i] o ti EPLE + | zt al t9 +5 Cl Sy Rats GW Ol) ty ae ee Sie ora a o6l 1h €tl 181 (Ae (Zoey Welw (meee )E\whrew Boe bored pow [(%bL-ciy il yofsow | (40°81 iv) Uw Uisol) *pardsaray |] Le ili por “sou | 18-8491 prnpad oy | cla ynpad-sey | isa Ll yonposd sony Joe AloLsts ls 18 %09 tT bev ie 7 Chg Ee MI rel 19555 18 Gath Oo 9% Sh ib El pea tara Lh stl (Yel) rl sof yew $C CLI ypnpoad 1907 Lee sayy ray] 99t sayiseay | ceo sal aray lero AB Aray | 9g Aa Amayy | OST Aap ara’ BLT Ap Aemy | OT Layt|>Svr>aV7 oqunu %rb El SBS IL GI = Brae |eogce %LLIE | %EE'bI EGE | Ger %rege [VISEC YES Yell %sJor | ~or 40% reel SEBS NES ye [e9be pees (ee eee eee we Ze ma || ies and je 4,o| O JE 18 8F GI} O 8 gol oe G | sl 9e ob v7 SI Qo 9& 18 OG +I Oo tl g&& 8 + | ol +e 9 Be ON OC DIMES Si (oye Ao 24 Fall wh) Be al Oa KB Ne ol) Tease Te pects Ste fate all 4 tS Ge ee hs tee) sya GG Cec ae ge PIPCIA} SAOL| POLLS} Sof] PAPLI-4S.10|PoLLd-If S.10f |PA;Ce1y $10} |po-eo.l S902 68 9|saoue § z ro) fac VA o days 4/24/15—9/24/15 Meo: He 5 8.372 1 | Continuous activity Average days between (16)| 7.7; 3:8 6 | 8.360 clutches = 7.7 days (67 152°) 11/19/15—3/15/16 2 | Continuous activity 11.4, 5:4 1. | S285 Average days between (10)! clutches = 9.9 days} 8.4) 5: 6? 0 | 8.370 (107 :10¢)? 4/28/16—7/14/16 6.8) 3:4 1 | 7.655 3 | Continuous activity WEIGHT OF FIRST AND SEC- OND EGGS OF CLUTCH, AND NUMBER o': 2 FROM EACH Ratio =| 4 (2) P oO — Q os Ss s | = 5 S 3 5 eB Ele 91H 2nd |8.79/3c' : 39} (0) Ist |8.16/1¢% :49) (3) 2nd |8.60\20 :32)| (3) Sing. |8.03) — :12}(0) Ist |8.03/4% :09)|(0) 2nd |8.44)1¢ :39| (0) Sing. |8.55] — :19/(1) ——$—— | —— | —— Ist |8.22/80 :29/() 2nd_ |8.52/3c7: 427 0) Ist |7.55/1% : 32] (0) — | | —— 2nd {7.75/20 :19) (1) Average days between (8) | 7.4| 1:7 2 | 7.502| Ist |7.39)107 :39) (1) clutches = 7.1 days (47 ——<—. | ————————__| -—— 2nd {7.61/00 :49/(1) 2119) 7/15/16—3/25/17 Ist |7.78|/7ch :39| (2) 4| Interrupted activity 14.5/12:10} 3 | 7.820) 2nd (7.78150 : 792} (1) Average days between (13) clutches = 14.5 days Sing. |8.23] — :19/() (127 : 109) Total from Ist of clutch = 17 skpixse cos ome oe 278 Se AGapulVe COlGr Changes. eee arts Sete Salen ae ta: eras 279 4. Color changes due to nervous excitement....................... 279 III. External stimuli involved in melanophore reactions.................. 280 i. Hilumination and: temperaguress, sas 04 oes, one's - od bey ecia tit ine . 280 2 BEE COLOR. Of LHeCS MUSCLES ie nant a te Men een ae eile erase etesdrcy os 282 or viechantealstinmraly }$4 5) ae ieee sae OR NLL ae SERA SRL 283 AS ersdre shinies yd... coe bees: Jeigkcdtee lars . act. 283 De aN OS HOM sy SUTONELE BOE, 265 Nac et Se eR ak ak a os 8 i Ss 8 283 SAUBOUN ACEH a meee erat Selene eb hel SDE. Salina tate wel ney Da eras 284 IV. Receptors involved in melanophore reactions.......................- 284 1. Direct action of stimuli upon melanophores....................- 284 21 Receptors of noxious stimuli; 44.32. ws hatyeur. .8 whe). toed. Gap 289 3. Receptors involved in adaptive reactions....................... 290 SELES ME Urner e oka Cece, Ca rae a areca ee rner Lee te Aa 291 V. Coérdinating mechanisms of melanophore reactions.................. 291 Ls Coérdinative, action. of the} circulationer.s: 5. sell!) . .. ene beseee 292 a. Influence of the respiratory function of the circulation upon TNC LAMO ON OUEM ys Wan ls Hoketadl tet Ages ste ane dapr de area octet acres 292 b. The coérdination of melanophores by hormones............ 292 ce. The nature of the hormone involved in melanophore reac- LOTS PO MECMES CSE, a a aie OO OS ee aa OV, ea eee 295 2. Coérdinative action of the nervous system....................-- 306 a. The nervous control of melanophores....................-. 306 b. The nervous paths of melanophore reflexes................. 310 TERS) ae er id tel chee Beach nN oes cd bo rcniacte th sf SHON 4 ~ reget 312 ! Contributions from the Zoélogical Laboratory of the Museum of Compara- tive Zoélogy at Harvard College. No. 309. 275 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 26, NO. 2 276 ALFRED C. REDFIELD VI. (Discussion ie en aos tee eas 2 oie ssh oie ee sone ee enn 312 1. The nature of the contraction of melanophore pigment.......... 312 2. Résumé of the reactions of the melanophores of the horned toad. 314 3. Comparative physiology of the melanophores of vertebrates..... 316 a. The influence of hormones upon melanophores............. 316 b. The codrdination of melanophores and smooth muscle...... 317 ce. The physiological basis of the emotions.................... 319 VillanSummiany: Seen’ see Ae eae) be eee eRe. soveecr eae 319 Ville siniternvure-civedteenen..0. Seen net)... s ekoa ey bie Lathe ecco 5 + Syme eee TEC AGE TO CBee note, tee ee eT OE eee nt, Rane PN ee) NAPE BP Loy ch oh 323 I. INTRODUCTION The results of an investigation of the reactions, codrdination, and function of the melanophores of the horned toad Phyrnosoma cornutum Harlan are described in the following paper. The facts which are presented possess, in addition to their intrinsic interest, a bearing upon several aspects of comparative physiology. Earlier studies upon the melanophores of lizards indicated that in the chameleon (Briicke, 52; Keller, 95) the color changes are produced under the influence of nerves which cause the melano- phore pigment to contract, while in Anolis (Carlton, ’03) the nerve impulse appears to cause the opposite effect, an expansion of the melanophore pigment. The present investigation was initiated in the hope of gaining more light upon the relation of the nervous system to the melanophores. It very soon became evident that the melanophores are not only under the control of nerves, but that certain of their reactions result from the direct effect of stimuli upon them, while other reactions indicate clearly that some coérdinating mechanism other than the nervous system is involved. The discovery that these reactions are due to a hormone (Redfield, ’16) and the identification of the hormone as adrenin form the most novel result of the investigation. A few observations have already been made upon the color changes of this lizard by de Grijs (99) and upon the closely related species Phrynosoma orbiculare, by Wiedersheim (Hoffmann, 90, p. 13853), and Phrynosoma modestum, by Weese (’17). Parker (’06) has made a more intensive study of the reactions of the melanophores of Phrynosoma blainvillei to illumination MELANOPHORES OF THE HORNED TOAD Dit and temperature. The great mass of data upon the chromato- phores of other lizards has been reviewed recently by von Ryn- berck (06) and by Fuchs (14). . Horned toads have been obtained from collectors in various parts of Texas and Oklahoma. ‘They may be had in large num- bers between April and September. In the laboratory they were kept in a large sunlit cage, where they thrived on a diet of meal worms. In summer the animals were kept in cages out of doors and fed on various insects in season. Horned toads do not feed if kept in the laboratory through the winter. They become greatly emaciated and usually die before spring If these lizards are placed in a dark, cool cellar, they go into hiberna- tion and remain in very good condition all winter. Experiments have been carried on in the Zoological laboratory of Harvard College at Cambridge, Massachusetts, in the physi- ological laboratory of the Harvard Medical School in Boston, and in the laboratory of the United States Bureau of Fisheries at Woods Hole. I wish to acknowledge my indebtedness to Dr. G. H. Parker for his kindness in directing this investigation and to Dr. Walter B. Cannon for valuable advice on certain phases of the work. Il. THE MELANOPHORE REACTIONS OF THE HORNED TOAD 1. Description of color changes Under certain conditions the ground color of the upper surface of the horned toad is fuscous.?. Across the back run three irregu- lar bands of fuscous-black, bordered posteriorly with a bright amber-yellow line, which stands out prominently in contrast to the dark ground-color. Similar fuscous-black bands, not bor- dered with yellow, extend across the legs. The flattened scales which extend in a row along the sides of the body are black at the base, white at the tip. Under other conditions, the ground- color becomes drab, drab-gray, or cinnamon-buff, frequently flecked with black. The amber-yellow lines across the back no * The color nomenclature of Ridgway (12) is used throughout this description. 278 ALFRED C. REDFIELD longer stand out in contrast with the ground-color, but the fus- cous-black bands now become very prominent both on the back and legs. The lateral scales become white throughout, with the exception of two on each side which are located at the ends of the fuscous-black cross bands of the back. These scales rarely be- come entirely free from pigment. Plate 1 illustrates the extremes of these color changes.’ The color changes of lizards have been shown by Briicke (’52), Keller (’95), and Carlton (’03) to be due to the migration of pig- ment contained in cells known as chromatophores, which are situated in the dermal layer of the skin. An examination of sections of the skin of the horned toad reveals the presence of chromatophores, filled with black pigment, such as Keller (’95) has called melanophores. When the melanophore pigment expands the ground-color becomes fuscous; when it contracts this portion of the skin becomes drab. The fuscous-black bars across the back and legs contain such an abundance of non- motile pigment that their color does not change. In addition to the melanophores there exist in the skin pigment cells containing a yellow, pigment. Whether this pigment is motile has not been determined, certainly it causes no prominent color changes. 2. Daily rhythm of color changes At night the melanophore pigment of the horned toad is con- tracted, giving the lizards a pale appearance. In the early morning this pigment expands and the skin becomes uniformly dark. During the heat of midday the melanophore pigment contracts again, but as evening approaches there is a second ex- pansion, followed finally by a contraction once more as night sets in. Thus the pigment is contracted and the animals pale at night and at midday; the pigment is expanded and the animals dark in the morning and afternoon. 3 Photographs fail to bring out fully the contrast between the dark and the pale condition of the skin. This is because the skin is tinged with yellow, a non-actinic color. Orthochromatic plates and a color screen have been used, but without complete success, in an attempt to overcome this difficulty. MELANOPHORES OF THE HORNED TOAD °* 279 3. Adaptive color changes Horned toads show striking color changes which depend upon the color of the environment in which they live. If they are placed in a pen which is lined with black cinders, the ground- color becomes so dark that the transverse bands are almost indis- tinguishable. When such animals are transferred to a pen lined with white sand, the ground-color becomes very pale in the course of a few days. The contrast between these pale animals and horned toads which have been left in the cinder-lined cage as controls is illustrated in plate 1. This change in color becomes noticeable within one day after the lizard has been placed in its new environment, and reaches a maximum within one week. Upon the adaptive state of the chromatophores is superimposed the daily rhythm of color change. Thus horned toads which are adapted to a dark background become paler at night and at mid- day, but never attain the extreme paleness of specimens adapted to a lght background. Similarly, horned toads which are adapted to a light background become darker in the morning and afternoon, especially on cool days, but never attain the dark color of specimens which are adapted to a dark environment. 4. Color changes due to nervous excitement Whenever horned toads are thrown into a state of nervous excitement the melanophore pigment throughout the entire surface of the body is contracted and the animal assumes the maximal pale condition. This reaction is observed whenever one of these lizards is subjected to any treatment which causes it to struggle in an effort to free itself. Simply holding a horned toad upon its back, attempting to pry open its mouth, or pinching its tail may suffice to cause this reaction. A male horned toad, attempting to copulate with a female, has been observed to become very pale at a time when the pigment of the other ani- mals in the pen was expanded fully. The contraction of the pigment which accompanies nervous excitement occurs more rapidly than the two other types of reac- tion which have been described. The complete change in color 280 ‘ ALFRED C. REDFIELD may occur in as short a time as three minutes and never requires longer than ten minutes. This reaction is also a dominant one, occurring irrespective of the condition which the cycle of day and night and the color of the environment has imposed upon the melanophores. III. EXTERNAL STIMULI INVOLVED IN MELANOPHORE REACTIONS 1. Illumination and temperature The series of color changes which accompanies the cycle of day and night is dependent upon the accompanying changes in light and temperature. The rhythm of the color changes may be broken by a disturbance of the normal rhythm of the changes in light and temperature. Thus on cool days the midday con- traction of the melanophore pigment does not occur; on cloudy days or when the animals are buried in the sand the skin may remain in the pale condition throughout the entire day. If a horned toad is exposed to bright daylight or to sunlight which is not too warm, after a short time an expansion of the melanophore pigment occurs. If the animal is placed in the dark, the melanophore pigment is contracted. These changes may be followed most clearly by observing the appearance and disappearance of the dark areas upon the bases of the lateral scales. The rapidity with which these changes occur in either direction varies from ten minutes to one-half hour, depending upon the intensity of the illumination, the temperature, and the idiosynerasy of the indiv:dual. In order to determine whether the effect of light upon the melanophores is due to the heat developed by its action, the following experiment was performed. An apparatus was devised in which the temperature could be kept quite constant and to which light could be admitted at will. This apparatus consisted of a glass aquarium 30 cm. in diameter and of equal height. Into this was inserted a second glass vessel 15 cm. in diameter. Between the walls of these two Jars ice or water of any desired temperature could be introduced. By this means the air in the MELANOPHORES OF THE HORNED TOAD 281 inner jar could be kept within a degree or two of a given tem- perature. In the inner jarwas placed a layer of sand, which served to keep it floating upright and gave a natural support for the animal. This jar was kept covered to maintain the temperature and humidity as constant as possible. When desired light was excluded from this apparatus by inverting over it a large paste- board box. November 26, 1913. Two horned toads, the melanophore pigment of which was expanded, were placed in the apparatus at 10.45 A.M. They were illuminated by the light from the overcast sky. Warm water was placed in the outer jar. After one hour (11.45 A.M.) the temperature of the air in the inner jar had risen to 40°C. and the melanophore pigment of the animals had contracted. One-half hour later (12.15 P.M.), the temperature of the air in the inner jar had fallen to 26°C. and the melanophore pigment had ezx- panded slightly. Ice was added to the water. Nearly two hours later (2.00 P.M.), the temperature was found to be 16°C. and the pigment was fully expanded. The apparatus was then covered with a box to exclude the light. After one-half hour (2.30 P.M.) the melanophore pigment was still expanded. ‘Temperature 17°C. The ice-water was replaced with warmer water. One hour later (3.40 P.M.) the temperature had risen to 25°C. and the pigment had contracted again. It remained in this condition as the temperature was raised to 36°C. at 4.20 P.M. Repeated experiments confirm the conclusions which may be drawn from these data. It makes no difference in which order the changes of temperature and illumination are arranged. The following summary indicates the condition of the melano- phore pigment under various conditions of temperature and illumination: 1. High temperatures produce a contraction of the melano- phore pigment, irrespective of illumination. 2. Low temperatures produce an expansion of the melanophore pigment, irrespective of illumination. 3. At intermediate temperatures (30° to 20°C.) the state of the melanophores is conditioned by the illumination, light causing an expansion and darkness a contraction of the pigment. 282 ALFRED C. REDFIELD Light and heat thus act in opposite ways and their effects need not be confused. The expansion of the melanophore pigment in the hight cannot be due to heat, as this stimulus causes a con- traction of the pigment. In certain individuals light dominates over temperature in determining the state of the melanophores through a larger range of temperatures than that indicated above. Some horned toads maintain a contracted condition of the pigment in the dark at temperatures as low as 5°C. The pigment of others may expand upon illumination when confined at temperatures as high as 37. These conclusions are in very good agreement with those which Parker (06) drew from experiments upon Phrynosoma blainvillei. By their means may be explained the rhythmic changes in the color of horned toads which are correlated with the cycle of day and night. The expansion of the pigment in the morning is due to the stimulation of light. The heat of midday causes a con- traction in spite of the light, but as the air cools in the afternoon the light effect again dominates and the pigment expands. When the light fails, at night, the pigment becomes contracted as a result. 2. The color of the substratum It has been pointed out on a preceding page that the color of the environment has a marked effect upon the condition of the melanophores. Seg & 0°6 | FF BG I'L | 16h y) z9 | 29 OI OF IeTeT SI 6°2 | SOT IZ 08 | G49 | 8 08 | 9ST At Go el ouy 1Z €8 | S6r $Z 62 | St9 Ig GL | 9&T SI [Sse lace We 6:3 SSsi 1Z 92 | e99 | Sg Qucale2GT 02 pL | 9ST like Lis: Ost ree Phe) Pela (Saw MACY ZS | OFT LI 08 | 2ST 61 LL | PST 02 QZ, e219 108 Gees scar SI 0°8 | F9I 02 LS |2021 IZ OS ale SSe =a oe Ones -2ET LT 0:8) sero 0 SL | 9ST 02 S20 ieGapes co $9 | 92 ral CG eaetGr 91 Cole seal LI Coa mdcree|a2e OL | 99 8 9°9 | OOT CT €'8 | Cri py I'Z | 00F | 99 LuOna ang 6 ZL | 601 GT 08 | 621 91 29 | SPE | gg TG 49 IT 19 | 98 ial 89 | ZOI Gl 89 | 9IF 19 8'¢ | 8g OL i lhe [EET GT Le vOen SI 0.0" |} 86z er .68 92 | OF 9 ¢’9 | zg 8 PAOr lee ral oS | 60T re GaP. Gl G ye (eye ¢ Gib 9G G L908 rca Oe eer g ©0861 g G°9) |ELz i G56 i 06 |6 I 09 |9 I Oe leh I sue Seve | asp ae | wa nS re |" Pe Sia | asuioay | [tI0I, [®IO.L oSBIOAW roquUINN |1oquUIn jy odUIOAW Iaquinyy }40q On Ny aBVIOAY JoquInNy |1oquin AUVWWOS peIqut-j[By) $19}41] YIANOLT 6°9 | sce"s | SBF 9°9 | SZT 92 SrZ ai rs GZ pL | 98T G2 GL | 802 LG pL | 661 1X6 Wie paso 1K6 £9 | ZI £@ SeZe 189T £% Lik \ OLS LZ 89 | SI 1G VL TZT &% 9°9 | 6&1 12 G4 | OST GG GL | StI 0G 8:9 | HI IZ gg. T&T 0G 69 | SOT LT LZ | Gen LI cad IOT 9T o9 | ¥6 GT CO mea ean SI ake W584 tea og | 8 L L9 | 02 1€ OFLealee I anos Jo ToS Cane roquinyy |zequinyy (porqur-speq) $10991] PITY L (porqut) 810}41] puooseg saluas UALLIT syot pasqur fo satuas py ay? fo suoyvsouab aay-hzyuany suf ay) fo yova ur paonpoid sayy ayy fo az1s abpseav ay, pun Laquinu ay} Burmoys T ATaV.L (perqut) 810441] J8It 7 Ge-T a BNO HINO r BO NOILY -HONGS 339 EFFECTS OF INBREEDING ON FERTILITY AND VIGOR e'2 | oge‘zt| gco'tT | 22 | Se2'% | 20e | 82 | 9240'S | GIF | 82 | 9008 | 29H | 69 | BIZE | BOF | 2-1 Lode COO sek Rega soc 02 jE GSI GZ je fay 9% O'L | P8T 92 GZ O'L | $89 | 26 TROVE SH LI 2 Oe ee dT 9% 0°L | 681 1B 9°2 | 202 Js bz T'S) =| S27 Omen 62 ¥'8 | 921 CT (a aati | 0% 8'°8 | S61 rad 2°8—|-181 ree 82 SL | PSL | S6 S681 61 8°8 «| TIS ¥G 0'8 | 01% 9% LO Wht 9% 2% gL | S19 | 2 OZ | ZIT 91 Daa lree 8T Reyes Ged ¥ oa roun ¥2 IZ 1 2ae|e S6Gnonlt be 0°8 | 88 I Paden Per 1Z 8°9 | 6LT 9% Oia | PLT 9% 02 ’'8 | 869 | &8 yL |:96 SI 8'°8 | S6T 2% Z°6 | 18 ¥% LL | 981 ¥Z 61 JL @B09 a1, 08 ae | ial! 0z ea ely 02 8°2 | OST &% €'L | 891 £2 81 yL | P19 | 2 9°9 | II LT GeSh eee 1Z Hepa al {04 rh TL | 8ST rad LI pL | 799 | 68 6 On -\saer 61 9°8 | 061 eG 9°L | P8T ¥Z ¢'9 | 8ST ¥ 91 Gee |e Ceae Ly G8) | OFT LI Qi: St SAT = 8 208 6°9 | S&T 02 ¢'9 | T8I 0% GI TZ | og | 08 GPL? CBT ST 08 | TOT 0% 9 | SST 13 L°9 | OPI IZ #1 Hele | Ouse | p2 08 | ¥OT €1 62 | OST 61 0°8 | 89T IZ €L | PST 12 g1 GoF 1 P OGGe aan 9'°¢ | 89 ral T'S | PST 61 St | S27 02 6°9 | 68T 02 ZI PLE (Leen Ee Z'8 | 6FI 81 OL) | Ser ST L°9 | Sa 61 6°S | ZIT 61 II OL | 6h | ¥9 9°9 | &6 ial 9°9 | LOT 9T G8 | SFT LT T'9 | FOL LI Ol Gree lt Sok. \E6 6b | 6F Or PL VLE LI 0°6 | €8T 02 29" | 981 0% 6 Ore: ICCge? =| 9F Orz sete € ipa a3 gI 9°8 | 621 CT Ore =|: SOT cI 8 9°9 | 998 | gg SL: | 82 Or 6°9 | POT CT 0°L | SOT cI 8g | 62 cI L Lk | (Oy | 2g 26 | b, 8 GP AOE iat See. |) SEE cI O°L | 90T CT 9 gL | 96¢ | TZ e'L | 96 eI €°8 | OST ST I'L | 89 0z cg | 881 02 ¢ SL | 60C-." | 6c 0°9 | 8I g 08 | 9¢ y) 9°8 | 69 8 09 | 99 II 17 (OT 2 ial cr |6 z Ze | 9% ¢ I'¢ | 98 L g TS) |: 2g L O76) 56 I oe | 21 z ¢'6 | 6I j oo | a z z o¢ | ¢ I og |g I I Zn | eto | ae ena po) eu, | rt nol Shs | Maat RUE | aro | aa ate ae |e esvioay | [BIO 1810L oBB10Ay JoquInN |equInN oBBIOAY JaquIn NY |toquin osuioay Joquinyy |1equin yy ogvIOAY ieaquiny |JequinNy a ee ee) NOLLY (porquryyeq) 819941] YWNOT | perqurypey) 810991] PAL (perqut) 810441] puosag (parqur) 810391] 3811 cae AUVAWOS SHINAS UALLIT SDL palqui fo saras g ay) fo suorpnsauab aay-fAjzuan) ysaf ay) fo yooa ur paonpoud s1ajqy ay] fo az18 abpsaan oy] PUD sequin ay} Burnoysy & ATAVL cING HELEN DEAN 340 cy | zop‘ezl sore | gz | poze | eco | 8:2 | 6999 | zss | 62 | SSH'L | GE | BO | PLEO |, E96 | S2-T GL see | 281 6°9 | OFG GE GL | SPE SP 92 | 968 Gg 8°9 | 298 GS GZ G2 9st | O6T L9 | PSC SE G24 | GSé SP GL | 688. Gg GL | 168 GG VG LL 61E'T | OLT 6'L | 896 VE &L | 908 GP 08 | 82Zé LV SL | L9€ LY &% OL GOST | O6T O02 | 69 9€ 68 | 62PF 8¥ 68 | Vr ig O'L | 218 €¢ GG SL | IPT | PST 62 | 662 8& 68 | 198 bP 0°8 | 90F 1g 2 | S28 1g 1Z Gk | VOGT | SLT LL | 812 8G OL | 908 bY 92 | 807 &¢ 69 | 298 &¢ 0% OL ele | 99T 92... | Vie 8G €°8 | 49 ww G8 | 668 LV TL | && LV 61 82 208 T | Sot VL | 69¢ Gg G8 | g&é 6€ G8 | LL OF €L | OEE 9F 8I 62 6Sr'T | P81 GL | 16d OF y8 | 98& OF ts Ns 6P GL | 89€ 6P LT GL GCI T | 6ST 09 | POT 66 9°Z | TTE OF LL | OVE SY ZL9 | LOE SY 9T 82 GAG: Daleeor T8 | 926 ins v8 | OVE Iv LL | Vee cP OL | c0E &P cil! GL Sid I | TOL GL | 892 9¢ V8 | &h iy L’L | && GP LQ | 182 GP iat LL 666 1 | 6ST 62 | 19¢ && 92 | 908 OF 08 | 8h ev € ZL | vTé &¥ &T 9°L LOTT | LPT GL | 802 66 18 | 908 8& G8 | 62E OV TL | 82 OF at V2 OFI'T | PST 64 | ¥82 9& O'S | G08 8& VL | 862 OF ¥9 | 982 OF II a 4 PEOT | IFT VL | O&@ Te VL | 89¢ 9€ T'8 | 108 LE €9 | S&& LE Ol 02 O16 66T Job = Werk GG GL | 8% && €'8 | 90& LE G9! | TES LE 6 Syd GLL sO OL | 22 Il 6°9 | POT 83 G8 | 122d G& GL | 0&3 of 8 (ay) 992 IIT €L | 681 61 el) ee O€ GL | VES Ié 8g | O8T I€ L 69 | SF LOT GL | Let 61 4°9 | 881 83 EL | 066 0€ 9°9 | 002 0€ 9 GL GS6 eal 9°90) 268 &@ G72 4) ¥8¢ tte 9°L | 882 8é 9'9 | 0&2 8& “| G9 GhP 89 TZ | v9 6 GL | 800 il! €L | 9 02 Vee lei acelt ixé V O8S O8T 9& GY 1G g eS ave L GS! 469 Or Te aie bey vl € 8°9 LET 0G LG | & v GL | 98 5 92 | OP 9 HOF Face | G 8°9 PE | OrG 4/56 I OOF a9 i OMe ane I 0-9 oF G I OVP a enp 9} 41] 10 a}! od 10341] tod 19}}1] 10d re et i a a ea a Paleo eed aga ater laude osuiaay | [BIOL TOL | ogeroay [PAU 9N TOqWINN | sggioay TOUUNN |FOQUNN | ogg roay TOQUNN |OQUNN | oggioay Jequin Ny |zoquanyy eae. (perqur-jyey) 810941] YQANoy | (poarqur-jyey) 810741] pry (poarqut) 810341, puodveg (porqur) 810341] 48417 qaeee AUVWWOS SATUAS WALLIT g 91qQD) WL pu J 91QD) U2 DIDp ay} fo UOYMULquios D :(q‘V) soles paiqur on} oyz fo suoynsoudsb aaif-hjywon) ys.uf 947 fo yooo wr poonpoud $1991) 9y7 fo azis abp.9av pup saqunu oy) Bumoyy € WIAvV.L 341 EFFECTS OF INBREEDING ON FERTILITY AND VIGOR S&F 62 | 6P8'S | ESF Ce ODL eb Coe deal G2, leOcusc alt= Ore De IRSSe-e Ln Ole tt PLC OL | 988 GG G'L | OS 2 2£ | 906‘¢ | Fle LL | SPS IZ O'S | 292 Sant amiSPO cay One GL | 98F cg O'S | 129 Lie \@e09 oe eee ay Peek} 15) 62 O'S | $99 GL | n0Gs Tak Zoe Toy lade OF file \akor Gave Neale ba POL Ge etic 9¢ G9 | 108 aod| sje y, h d d CHA iG RERT Ree eer OR yee Sear ee sone wa eee doquinu |oOquINN |roquinyy | zequinu goquinyy |zoqunyy toquinu |.» ODBIOAY | [RIOT [BJO], . | osB10OAW OSBIOAW AUVAWOS porqur-j[By) 810941, YANO, L9 Zh | O89 92 G6 o8 | &98 vOr €8 G8 | 662 ¥6 18 G8 | G69 ¥8 99 82 | Ogg OL OF L9 | 698 g¢ 104}, 10d sunod jo sequinu $1091] jo gjenpin | 19941] stpULego jo Joquiny |1oquinyy aseroay |OUUnN roquin yy (peaquy-jeq) $10}}1] PALL (pezqut) 810}}1] puosag SQIUaS UALLIT 69 | gees CSP 14 | &FS 9L 14 CPL POT €°L | G69 $6 OL | 88S $8 ¢’9 | 6SP OL 8) Geel ss, LS seed go} Sempra | 830997] -Iputjo | yo TOquInt lroquinn |1equinN ddBIOAY (perqur) 810941] 481 So-1 GC-8% 6o-61 8I-t1 vI-IT OI-2 9-1 SNOLLV -HaNaD S]DL poique fo sarias y oy) ur poonpoud 8.19771) 9y7 fo 9218 obn.oan pup woqunu 947 sdnoib uoynwauab snorua sof Burnoyy b WTAV.L 342 HELEN DEAN KING An examination of table 4 shows that all of the litters produced in the first generation group of the A series were smaller, on the average, than corresponding litters in the later generation groups. The relatively low fertility of the animals in the early generations was not due to inbreeding, but to the fact that these individuals suffered from malnutrition. As soon as the nutritive conditions were improved there was at once an increase in the number and in the size of the litters produced, as the data for the fifth and for the sixth inbred generations show (table 1). As indicated in the last column of table 4, the groups compris- ing the tenth to the twenty-fifth generations of the A series . Showed, as a whole, comparatively little variation in the average size of the litters. The maximum average size (7.8) came in the group including the fifteenth to the eighteenth generations. This maximum was, however, only 0.1 greater than the average litter size for the preceding and for the following group, and therefore it can have little, if any, significance. Litter data for various generation groups in the B series of in- breds are shown in table 5. As the average size of the litters produced in the first gener- ation group was greater than that in the second group (table 5), it might appear that the fertility of the breeding females in the B series was not lessened by malnutrition. In the beginning of these experiments many more females of the B series than of the A series were completely sterile, but the females of the B series that did breed were the more productive. Malnutrition, in this instance, was a selective agent that helped to eliminate the tend- ency to sterility in the B series by preventing the breeding of any except the most fertile females. In the B series the maximum average size of the litters was found in the group comprising the nineteenth to the twenty- second generations, but, as was the case in the A series, this max- imum was not great enough to be considered significant. Litter data given in table 4 and in table 5 have been combined in table 6. The data for each of the two inbred series, as well as that given in table 6, shows that in all generation groups the first litter cast 343 EFFECTS OF INBREEDING ON FERTILITY AND VIGOR VL 988‘ZI| 999'T Femail) Wisterd LOE & 2 ola 61h 82 | 909'€ | Z29F 6°9 | 612‘E GZ-1 VL $20'S | €L16 GL | 948 GG v8 | 667 IZ LL | £Lg GL 9°L | @2g SC-8G 92 CFOS | FFE Sie \eS&F 69 G8 | 102 G8 08 | 662 OOT EZ | OT 6C-61 GZ 8zS°Z | PEE G2 | ves €Z CaS 202 €8 Ged \ 629 68 G95 12879 SIT VL 602% | 662 y2Z | SSP 19 62 | €09 92 y 2, .} 909'- 18 £9 | LYS FI-IT TZ ZE9‘T | BES 9 | [ve L§ OL | C&P 19 V8 | c9g L9 69 | LIP OI-L Siz 8146'T | FLT $2 | 961 GZ LL .| VEE €P SL | 068 0g GOP) Sh Oissr SuRge Fol tpur jo |SONTIO sundae go] SIUEPLA | Sioa fsenas yo) SEMPL | SMT Funds 0] TAPIA | SOIT [sos yo] STERPLA qequinu | zoquine | “OAM | aquinu | se jo equinu | Pur jo yo zequinu | PUT yo jo zequimu | ~tPUr jo azuoay| mor | (OL | sauoay POUHAN [OqUINN | Sseroay POUWEN [QUINN | os eroay [OUMN |OUTUNN | oserony [AUN [requ SNOILY -HGNAD perquy-jrey S10}}1] BANE, | (perqur-spey) $1099] PITT, (perqur) 810341] puosag (perqur) 810391] SIL AUVANOS SGIuas YUALLIT spt pauqua fo sares g ay] Ur poanpoud 3.19971) 947 fo azis abpsaav pup waqunu ay? sdnoib uoynsauab snorina 1of Buimoygy $ ATAVL | 1 | 75] 131] 205] 361} 388) 551] 528) 466] 333] 209| 94 | 49 | 11] 5 | O _ ee eee S EFFECTS OF INBREEDING ON FERTILITY AND VIGOR 351 In the A series of inbreds the range in litter size was from one to seventeen. The litter of one was cast by a female of the nine- teenth generation that was suffering from pneumonia and had to be killed three days after parturition. This is undoubtedly a case where the physical condition of the female prevented the normal development of all of the embryos except one; the other embryos probably became atrophic and were absorbed. The litter containing seventeen members occurred in the fifteenth generation. All of the individuals were born alive, but they were all very small, weighing not more than three grams each: the average weight of the albino rat at birth is about four grams (King, ’15b). In the B series, as table 8 shows, the range of variation in litter size was not as great as that in the A series: no litters smaller than two or larger than fifteen were obtained. In both series litters containing seven young were the most frequent, while those with eight young were only slightly less in number. Figure 2 shows graphs for litter frequencies in the two series of inbreds that were constructed from the data given in table 8. In figure 2 each graph rises quickly to the modal point at seven, falls slowly at first and then rapidly. The drop in graph A at the point of 6 has apparently no significance, since there is no similar drop in the B graph. Each graph is a simple frequency curve with one modal point, and is exactly the sort of graph that one would expect to obtain from the data for litter frequencies in a large series of animals belonging to a pure race. C. Puberty Under normal conditions puberty tends to appear at approxi- mately the same age in the different individuals of a given race, but the time of its appearance is seemingly more dependent on the growth changes incident to age than it is on age itself. In the albino rat both males and females attain sexual maturity when they are about two months of age (Donaldson, ’15), but the environmental and nutritive factors that hasten or retard growth have considerable influence on the reproductive activity of the THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 26, NO. 2 352 HELEN DEAN KING 5 Number of Litters a EEE CE Gee SS (GREER We ee (SRREEE fi oS 2 at eee ares oe 2 anicteit LH srasTEseraeeee FEH-HEEETH Size of Litter 44 EEGGSRSao! Saee0i~ “Vee Sa Suny Ra ee [a SSSSSeeen. _ _ UP Ce oO Wt 12 138 14 16 (6 {7 b a Fig. 2 Graphs showing the frequencies of litter size in the two series of inbred ~ rats (data in table 8). EFFECTS OF INBREEDING ON FERTILITY AND VIGOR 3093 individuals. If young rats are fed exclusively on a meat diet, puberty is considerably delayed (Watson, ’06) ; the same effect is produced by underfeeding (Osborne, Mendel and Ferry, ’17). According to my observations, the time of year in which the ani- mals are born affects their subsequent growth and also the time of their maturing. Rats born in the winter and early spring grow rapidly, and usually breed at about three months of age; those born in the summer and autumn grow more slowly and compara- tively few of the females cast litters before they are four months old, many not breeding until spring, which is the season of the most pronounced sexual activity for the rat. Convincing evi- dence that age alone does not determine the beginning or the end of the reproductive life of the rat is given by Osborne and Mendel (15, ’17), who found that Albino females, stunted at an early age by underfeeding, were completely sterile until they were properly nourished, when they grew rapidly, attained a normal size, and were able to breed long after the age at which the meno- pause usually appears. According to Darwin (’75) and others, favorable environment tends to delay sexual maturity, though not necessarily to decrease fertility. Since these inbred rats were reared, for the most part, under environmental conditions that seemed well adapted to their needs, and since they lacked the stimulus to reproductive vigor which is said to come from outcrossing, it might be expected that they would tend to mature much later than stock Albinos which were not inbred. Table 9 shows the approximate age at which the breeding females belonging to various generation groups of the two inbred series cast their first litter. The records for the first generation group, given in table 9, confirm Osborne and Mendel’s findings that underfeeding tends to retard sexual maturity, since they show that about one-half of the breeding females in this group did not cast their first litter until they were four months old. In subsequent generations, when the animals were adequately nourished, they began breed- ing at a much earlier age. Under the conditions of this experi- ment, inbreeding seemingly hastened the onset of puberty, for 354 HELEN DEAN KING in both series, as the inbreeding advanced, there was a marked tendency for relatively more of the females to breed at the earliest possible age. About 30 per cent of the breeding females in the eighteenth to the twenty-fourth generation group of each series threw their first litter at or before the age of ninety days; only a small proportion of them failed to breed before reaching the age of four months. As a whole, the females of the A series of inbreds tended to mature slightly earlier than the females of the B series, but the TABLE 9 Showing the approximate age at which breeding females in various generation groups of the two inbred series (A and B) cast their first litters ———— os BE SERIES A SERIES B SUMMARY (A,B) mn Pea| 3, |82 [8g |88 | >, |88 [gs | 88 | 3, |88 (88s | 88 a OP GRE WIEN eS f | Sos sees | a 2\so /e2e | eC mee | go) oo Blas iG) ef.) || Geo ealesewe ll: tek ey || (tale noo ales a2 Bn | ae eeelee | See | ae (SSS eae | S¥s | Be eee eee | ee sHal @|Seclass | 83°] 3 | 832 issey | 835] 3 | 88° |sey | see ‘Bho | es BROS So o>| ao Si 9 So S32 | a3 a>| 320 o> Zz Pee~ pH Ble Ho oO] , HS .& ne S |p Ho bo] , aS SH HES |, hott] , HS HES | 02 | 520/505 a| S00 | 62 | 5.00 |5028a| 507 | 68 | GO (508 a] G20 o ‘=! a a Ay B a ie a al Ay Ay a 1— 5} 58 | 12.0) 836.2 | 51.8} 56 | 14.3 | 41.1 | 44.6 | 114 | 13.1 | 38.7 | 48.2 6- 9} 70 | 21.4] 68.6 | 10.0 | 67 TATA V VCD Sr a6 | eon tors LON) 7849) 2520|) A 396) Sl ASEGr 74 te) 1236S) ONS) 7280) ano 14-17) 94 °).28.7) 62:8.)/8.5 | 89 | 25.8.1,62.9.) 11.3 | 183) |.27.3)| 62.9.) 928 18-21} 104 | 36.5) 62.6 | 0.9 | 100 | 31.0 | 64.0 | 5.0! 204 | 33.8 | 68.3 | 2.9 22-24} 76° | 31.5| 60.6 | 7.9 | 75 | 20.0 | 69.4 | 10.6 | 151 | 25.8 | 65.0 | “9.2 1-24) 486 | 27.1) 61.6 | 11.3 | 468 | 19.8 | 65.3 | 14.9 | 954 | 23.5 | 63.4 | 13.1 difference between the two series was not great, and correspond- ing records were in nearly as close agreement as were those for litter size. The youngest breeding female in the inbred strain was a member of the A series of inbreds, and she was eighty days old when she cast her first litter of five young. As the gestation period in the albino rat is about twenty-two days (Donaldson, 15), this female must have conceived when she was two months old. Kirkham and Burr (715) state that one of their Albino females gave birth to a litter when she was only seventy-seven EFFECTS OF INBREEDING ON FERTILITY AND VIGOR 399 days old; while Lantz (710) reports a case in which an albino rat was said to have produced a litter at the age of fifty-six days. This last case is certainly a remarkable one, and its parallel has not been found among the 50,000 rats bred in our colony. The last section of table 9 shows that, after the tenth genera- tion, there was no marked change in the proportion of females that bred at three and at four months of age, respectively. Nearly 24 per cent of the total number of females used for breed- ing cast their first litter by the time they were three months old; over 60 per cent of them bred for the first time when they were between ninety and one hundred and twenty days of age; while about 13 per cent did not breed until after they were four months old. The latter group was made up, for the most part, of females that were born in the summer or autumn. Although Diising (’84) states that inbred animals tend to mature very early, I do not think that inbreeding alone was responsible for the fact that relatively more of the females in the later than in the earlier generations of these inbred rats bred at three months of age. In these experiments, when two or more females of a litter were reared as possible breeding stock, the first female that became pregnant was the one taken to continue the line, provided she fulfilled all requirements as to size and vigor. Thus the manner in which breeding females were selected pre- served those individuals that tended to breed at an early age, and this tendency to early maturity, if heritable, must have been retained in the stock and intensified to some extent through continued brother and sister matings. Inbreeding, aided by selection, would thus seem to be the factor involved in producing a strain of rats in which the females attained sexual maturity at a relatively early age. D. Sterility Sterility occurs normally in the Albino, as in other strains of rats, and therefore it might be expected to appear at times in any strain, regardless of whether the animals were inbred or outbred. Crampe (’84) states that of 221 Albino females which he selected for breeding forty-six, or 20.8 per cent, were sterile. 356 HELEN DEAN KING Out of 124 stock Albino females reared in our own colony during the past three years and intended for breeding purposes thirty- two, or 28.8 per cent, were completely sterile, while about 10 per cent of those that did breed cast only one or two litters. Un- fortunately, no records have been kept that give information regarding the exact proportion of sterile females in the first six generations of the inbred series. ‘The number was relatively very large, and must have included at least one-half of the total number of females that lived to be six months old. Sterility in these females was, for the most part, the result of poor nutrition, and it disappeared as soon as the nutritive conditions were im- proved. Loeb (717) has shown that in the guinea-pig ‘‘under- feeding prevents maturation of the follicles and thus causes sterility which lasts as long as the effect of the underfeeding is present in the ovary.’”’ In the guinea-pig, as in the rat, adequate nutrition reéstablishes normal conditions in the ovary and sterility almost entirely disappears. In Drosophila, according to Castle et al. (06), low productive- ness (sterility) is directly transmitted by inheritance and is amenable in selection. In the rat, sterility seems to depend not entirely on genetic factors, but to a marked extent upon condi- tions, such as malnutrition and disease, that act unfavorably upon reproduction. In the present experiments, by selecting for breed- ing only the most vigorous individuals (which it seems were also the most fertile), sterility in as far as it may depend on genetic factors would seem to have been practically eliminated from the strain, and it has not reappeared even after twenty-eight genera- tions of brother and sister matings. Of the 954 inbred females that were used for breeding during the course of these experiments, 653, or 68.5 per cent, cast four litters each, and many of them, kept for body-weight records, produced several other litters which were not recorded. Of the females that did not cast the required four litters, the great majority died from pneumonia, or were killed because they showed unmis- takable evidence of illness. A few of the females stopped breed- ing after producing one or two litters, although they were appar- ently in good physical condition and were paired for several EFFECTS OF INBREEDING ON FERTILITY AND VIGOR 357 months with males that were known to be fertile. A postmortem examination of the reproductive organs from several of these semi-sterile females showed, in every instance, an inflamed con- dition of the ovaries or of the uterus which would render repro- duction impossible. Barrenness in these cases was doubtless due to disease and not to any inherent tendency to sterility. A similar diseased condition of the reproductive organs has been found to be responsible for the partial sterility of stock Albinos. 2. THE CONSTITUTIONAL VIGOR OF INBRED RATS The best criterion by which to gauge the so-called ‘constitu- tional vigor’ of any animal is undoubtedly its power of repro- duction, since that is of the utmost importance for the continua- tion of the race. There are, however, other important tests for vigor that can be applied, such as the rate and extent of growth, agility, mental alertness, resistance to disease, and ability to live to an advanced age. According to Darwin (’78), “‘the effects of close interbreeding in animals, judging from plants, would be deterioration in general vigor, including fertility, with no neces- — sary loss of excellence of form.’”’ This would seem to indicate that, whatever tests were applied, closely inbred animals and plants would show marked inferiority when compared with individuals of the same species that were not inbred. That such a sweeping generalization is not justified is shown by the results of anumber of recent inbreeding experiments: the work of Shamel (05) on tobacco, of Stout (’16) on chicory, and of Hayes and Jones (717) on tomatoes give no indication that self-fertilization in these plants causes a loss either of vegetative or of reproductive vigor; Gentry’s (05) experiments on swine, and those of Castle et al. (06) and of Moenkhaus (’11) on Drosophila show that any loss of vigor that might come from inbreeding can be entirely overcome by the proper selection of breeding stock. The present series of experiments on the rat are the first recorded for any mammal in which brother and sister matings were made continuously for twenty-five successive generations. In the inbreeding experiments with rodents made by Crampe, by Ritzema-Bos, and by von Guaita, matings were made between 358 HELEN DEAN KING animals related in various degrees, and they were made as often between parent and offspring as between sibs. Ritzema-Bos states: ‘‘Bemerkenswert ist namentlich das Result, dass die Paarung zwischen Geschwistern viel schlechtere Erfolge leferte als die Paarung zwischen Mutter und Sohn, resp. Vater und Tochter.”’ Presumably, therefore, my inbred strain, in which all breeding females came from litters produced by the matings of sibs only, would show an even greater evidence of deterioration in vigor than did the rats inbred by Crampe and by Ritzema-Bos. Data already given show that these inbred rats were much more fertile than stock rats reared under the same environmental conditions, so it is evident that their reproductive vigor was not impaired. In their ability to withstand disease inbred rats com- pared favorably with stock rats. The rat scourge, pneumonia, was quite as prevalent among stock animals as among the inbreds and took its toll of lives as frequently and as quickly in one strain as in the other. Parasitic infection was as common in the stock colony as in the inbred, and severe changes in temperature were followed by just as many deaths among stock animals as occurred in the inbred strain. The rat’s power of resistance to disease and to unfavorable environmental conditions did not appear to be lessened by inbreeding under the conditions of these experiments. Records for the growth in body weight of a considerable num- ber of rats belonging in various generations of the two inbred series show the approximate age at which death occurred in all individuals that did not live to the end of the weighing period, which came when the rats were fifteen months old. As similar records were recently obtained for a series of stock animals, it is possible to compare the relative length of life in the two strains and thus to determine whether inbreeding tends to shorten the life of the individuals, as it might be expected to do if it impaired the general vigor of the animals to any extent. Table 10 shows the mortality at different ages in such of the A series of inbreds as were used for the determination of the effects of inbreeding on the growth in body weight, given in the first paper of this series. For convenience the data were arranged in generation groups: the last group includes the findings through EFFECTS OF INBREEDING ON FERTILITY AND VIGOR 309 the twenty-third generation only, as the weight records for ani- mals belonging in the twenty-fourth and in the twenty-fifth gen- erations are not yet completed. As all of the animals reached the age of three months, the first mortality record given is that for animals at six months of age. On examining the mortality data for the males, as given in table 10, it is found that comparatively few of the animals in any generation group died before the age of six months, and that over 50 per cent of them lived to be more than one year old. A comparison of the corresponding records for the various genera- tion groups shows unmistakably that the animals belonging to TABLE 10 Showing the mortality at different ages in a group of 236 males and 179 females belonging in the seventh to the twenty-third generations of the A series of inbred rats PER CENT MALES LIVING AT PER CENT FEMALES LIVING AT GENER-| NUM- VARIOUS AGES NIGMB EL VARIOUS AGES ATION |BER OF OF GROUPS | MALES | | FEMALES) | 6 mos. | 9 mos. | 12 mos. | 15 mos. | 6 mos. 9 mos. 12 mos.| 15 mos. 7-10) 35 91.4 | 71.4 54.3 45.7 28 92.8 67.8 25-0°| 1027 11-14) 52 ODES A 71k Siete 38.4 sy 97.3 83.8 56.7 Soe 15-18} 60 /|100.0 | 75.0 Saeo 26.6 47 97.8 2H 59.5 29.8 19-23} 89 | 98.8 | 88.7 Tou 46.0 67 98.5 82.1 | 67.1 |} 46.2 7-23) 236 96.2 | 78.8 61.9 39.4 179 97.2 77.6 | 56.4 34.1 the later generations tended to be longer lived than did those in the earlier generations. The mortality data for the females of the A series are much like those for the males, the most noticeable difference being found in the records for the first generation group where only 10 per cent of the females lived to be fifteen months of age. Taking the animals of the A series as a whole, about 4 per cent of them died before they reached the age of six months; 20 per cent did not live to the age of nine months; 50 per cent were dead at the end of one year, and only about 35 per cent lived to be fifteen months old. Mortality data for individuals belonging to various generation groups of the B series are shown in table 11. 360 HELEN DEAN KING In the earlier generations of the B series the mortality in both males and females was considerably greater than that in the animals belonging to the A series: only 5 per cent of the males lived to be fifteen months old, while not a single female reached this age. For the later generation groups the data for the B series were very similar to those for the A series. As a whole, however, the animals in the A series lived longer than did those in the B series. The data given in table 10 and in table 11 have been com- bined in table 12. This table shows also mortality data for 377 stock albino rats reared in The Wistar Institute animal colony during the past three years. Included in the latter series are the TABLE 11 Showing the mortality at different ages in a group of 151 males and 231 females be- longing in the seventh to the twenty-third generations of the B series of inbred rats PER CENT MALES LIVING AT PER CENT FEMALES LIVING AT GENER-| NUM- VARIOUS AGES NUMBER VARIOUS AGES ATION |BER OF OF GROUPS | MALES REMADES (o> >> SS ne 6 mos. | 9 mos. | 12 mos. | 15 mos. 6 mos. 9 mos. | 12 mos. | 15 mos. 7-10} 18 | 94.4 | 50.0] 38.8 11-14; 30 | 86.6 | 70.0 | 26.6 15-18} 43 {100.0 | 69.8 | 51.1 19-23) 60 [100.0 | 93.3 | 76.6 5.5 34 19.4.) 23255) 1766 6.6 43 90.47 |. GSLs) 3459") 216.2 27.9 64 96.9} 76.5] 54.6] 26.5 6.6 90 | 100.0 | 86.6 | 77.7 | 53.3 32.4; 231 94.3 | 70.6] 54.51 31.1 7-23 | 151 | 97.2 | 76.8 | 54.9 records, elsewhere published (King ,’15), for fifty males and for fifty females of selected stock that were reared as controls for the inbred strain. The mortality ‘data for the inbred rats, given in table 12, show that close inbreeding did not tend to shorten, but to lengthen the span of life in both males and females: 50 per cent of the animals belonging to the last group lived to be fifteen months of age, while in none of the other groups did even 30 per cent of the individuals attain this age. It is probable that the relatively high death rate in the animals of the earlier generations was due to the fact that the rats had not regained the vigor that was so greatly impaired in their ancestors by malnutrition. EFFECTS OF INBREEDING ON FERTILITY AND VIGOR 361 Donaldson (’06) has assumed that the span of life in man is thirty times that of the rat, and therefore that a rat of three years corresponds to a man of ninety years. Considering the relatively small proportion of men that live to be nonagenarians, one would not expect to find many rats in any colony living to three years of age, yet under the equitable climate of California, Slonaker (12) succeeded in keeping two of a series of sixteen albino rats beyond this age, and one of them lived for forty-five months, or the equivalent of one hundred and twelve years of human life. At various times during the past five years a number TABLE 12 Showing the mortality at different ages in a group of 387 males and 410 females belonging in the seventh to the twenty-third generations of the two inbred series (a combination of the data in table 10 and in table 11). Data are also shown for the mortality in a serves of stock albino rats comprising 199 males and 178 females AGES PER CENT MALES LIVING AT PER CENT FEMALES LIVING AT GENERATION VARIOUS AGES VARIOUS AGES BER OF andre BER OF oO GROUPS 5 FE- 6 mos. | 9 mos. | 12 mos.| 15 mos.} saALES | 6 mos. | 9 mos. |12 mos. ! 15 mos. 7-10 53 | 92.4 | 64.1 | 49.0 | 32.0 62 | 85.4 | 43.5] 20.9] 4.8 11-14 82 | 89.0 | 70.7 | 47.1 | 26.8 80 | 93.7 | 73.7 | 45.0 | 25.0 15-18 103 {100.0 | 72.8 | 52.4 | 27.1 97.3 | 74.8 | 56.7 | 27.9 19-23 149 | 99.3 | 90.6 | 74.5 | 50.3 | 157 | 99.3 | 84.9 | 73.3 | 50.3 7-23 387 | 96.3 | 78.0 | 59.2 | 36.7 | 410 | 95.6 | 73.6 | 55.3 | 32.0 Stockseries..| 199 | 98.9 | 85.9 | 63.8 | 28.1 178 | 95.5 | 87.6 | 70.2 | 37.6 of inbred and of stock Albinos were kept in our colony in good physical condition until they were about two years old. We have never attempted to keep any rats beyond this age. Osborne, Mendel and Ferry (’17) state that out of ninety-one albino rats kept under ordinary laboratory conditions during their entire lifetime, “17 (19 per cent) died under one year of age; 48 (53 per cent) died between one and two years of age; and 26 (29 per cent) lived more than two years, the oldest one reaching an age of nearly 34 months. From these figures it is evident that less than a third of the rats in our colony may be expected to live to be more than two years old.” In another paper these 362 HELEN DEAN KING authors (15) state: ‘‘ Fully half of our stock rats have died before the age of 600 days.’ Unfortunately, the mortality data given by Osborne, et al. are not in a form which makes it possible to compare them directly with the records for these inbred rats. It would seem, however, from the results as given, that their — animals tended to live longer than the rats in my inbred strain. The rhortality data for the series of stock Albinos, given in table 12, ean be directly compared with those for the inbred rats given in the same table, since both series of animals were reared under similar environmental conditions and the records were taken at the same age intervals. Relatively more of the stock than of the inbred males were living at six, nine, and twelve months of age, but only 28 per cent of the stock males lived to be fifteen months old, while 37 per cent of the inbred males attained this age. The records for the female groups show that relatively as many inbred as stock females lived to the age of six months, but that more of the stock than of the inbreds were living at nine, twelve, and fifteen months of age. Taken as a whole, therefore, lon- gevity in the inbred strain seemed to be somewhat less than that in the stock controls. Some of the inbreeding data for animals which Darwin (’75) collected were so at variance with his own results on plants that he was foreed to admit that; ‘‘manifest evil does not usually follow from pairing the nearest relations for two, three, or even four generations.’”’ In a long-continued series of inbreeding experiments, therefore, the deleterious effects of inbreeding would supposedly be more accentuated in the later than in the earlier generations. A comparison between the mortality rec- ords for stock animals and those for the inbred group comprising the animals in the nineteenth to the twenty-third generation should show the effects of inbreeding on longevity much better than the comparison between the groups as previously made. Such a procedure is the more justifiable, perhaps, because these two groups of animals were reared in the colony simultaneously. While in the two male groups only about 1 per cent of the animals failed to reach the age of six months, relatively more of the inbred than of the stock maies were living at all other age periods noted: EFFECTS OF INBREEDING ON FERTILITY AND VIGOR 363 the final records for the two groups show a difference of 22.2 per cent in favor of the inbred animals. In the female groups the span of life in the inbreds also tended to be longer than that in the controls, but the difference was not quite as marked as in the case of the males: the final records show a difference of only 12.7 per cent. It appears, from the above comparison of data for stock and inbred rats, that continued inbreeding, under favorable environ- mental conditions and with the aid of selection, cannot only lessen the tendency to early death caused by malnutrition, but that it can extend the average span of life in the rat considerably beyond that found in the stock controls. Constitutional vigor, as judged by the longevity of the individuals, is therefore not invariably lessened by continued inbreeding. In table 10 and in table 11 it will be noted that the mortality data for the first generation group indicate that the span of life in the females, particularly in the B series, was much shorter than that in the males. The reason for this ‘selective mortality’ is not clear, although it may be that the females were not able to throw off the effects of malnutrition quite as readily as were the males. In both inbred series, after the tenth generation, the mortality in the females at any age period was practically the same as that in the corresponding group of males. Data given in table 12 show that stock females tended to live longer than stock males: a reversed relation seemed to hold for the inbred rats. Taking the inbred colony as a whole, I am inclined to the opinion that the females, as a rule, tend to live longer than do the males. More males than females usually die as the result of a sudden, sharp change in temperature, and the impression one gets from working daily with the animals is that the males are far more sus- ceptible to pneumonia than are the females, and that they are sooner attacked by various parasitic pests, such as lice and ear- mites. White (’14) states that in India the bubonic plague is a more fatal disease to male than to female rats, thus indicating that the female is stronger, constitutionally, than the male. These results are in accord with the findings for the human race: census reports and various statistical tables that have been com- 364 HELEN DEAN KING - piled show, as does the investigation of Pearson et al. (’03), that the duration of life in women is longer than it is in men and that women are the less susceptible to disease at all ages. The various physical defects, so prevalent among Crampe’s (83) inbred rats, were all found among my inbred rats at the beginning of these experiments, but they were due to malnutri- tion, not to inbreeding, since they entirely disappeared when the animals received proper food. Among the thousands of inbred animals that were reared during the past five years some few, not to exceed a dozen in all, lacked one or both eyeballs. This defect has also appeared, at times, in stock animals. On the average, one in every 10,000 rats born in the stock colony is tailless. This abnormality, as Conrow (’15, ’17) has shown, involves the skeletal structure in the entire pelvic region. The inbred colony has con- tained only one tailless individual as yet. Unfortunately, this rat was destroyed by the mother soon after birth, so it was not carefully examined. Neither of these defects appears to be heritable, and neither can be due to inbreeding, since each has appeared also in a stock that is outbred. No other abnormalities of any kind have appeared in the animals of the inbred strain up to the present time when the individuals of the twenty-eighth generation are approaching maturity. The findings in this series of experiments, therefore, do not give support to Ritzema-Bos’ contention that inbreeding tends to cause ‘‘eine gréssere Pradis- position fir Krankheiten und das Entstehen von Missbildungen.”’ When a considerable number of animals belonging to any series exhibits various kinds of malformations, it is safe to assume that either environmental and nutritive conditions are unfavorable to normal development, as in the early part of the present series of experiments, or that there is an inherent weakness in the stock used that is brought out and accentuated by random inbreeding, as seemed to be the case with Crampe’s rats. No data are available for a direct comparison between stock and inbred rats as regards their relative activity at different ages, but several series of experiments have been made in different psychological laboratories in which the behavior of rats from this inbred strain was compared with that of stock controls. EFFECTS OF INBREEDING ON FERTILITY AND VIGOR 365 In the inbred rats of the earlier generations the brain and spinal cord were decidedly below the normal weight of these organs in stock animals of like age and body weight. ‘‘ From the fourth to the tenth generation the relative brain weight remained, on the average, constant at six and one-half per cent less than that of the normal control rats” (Basset, 14). The habit formation in a number of rats that belonged in the sixth and in the seventh in- bred generations was tested at Johns Hopkins University by Basset (’14), who found that these animals were inferior to stock rats in their ability to form habits, and that they show less reten- tion of a habit, and were longer in relearning it, than were the controls. Inbred rats belonging in the twelfth and in the fourteenth gen- erations were sent to Harvard University where Mrs. Yerkes (16) studied their behavior and compared it with that of stock albino rats obtained from The Wistar Institute colony and from a different source of supply. The general conclusion reached by Mrs. Yerkes was that ‘‘inbred rats learned a trifle more slowly than the stock rats, both in the maze and in the discrimination experiments, but that they carried discrimination of lightness and darkness further, and showed the most pronounced difference only in their greater timidity and instability of behavior.” Temperamental differences between stock Albinos and inbreds of the fourteenth and the fifteenth generations were investigated at Harvard by Utsurikawa (’17). The results obtained showed that inbred rats were less active and more savage than the out- bred rats, and that they responded more quickly and in greater amount to momentary auditory stimulation than did outbred rats. The two strains were found to differ also in ‘‘restlessness or continuity of response.’’ Inbred rats showed the greatest restlessness”’ in case of momentary and repeated auditory stimu- lation and less in case of continued stimulation, whereas for the outbred animals the reverse is true.’”’ These temperamental differences between inbred and stock rats would seem to indicate that inbred rats are more ‘high strung’ nervously than are out- bred rats. Nervousness is a trait manifested by many thorough- bred animals, and it is particularly characteristic of the racehorse. 366 HELEN DEAN KING The nervousness of the horse is undoubtedly the result of continued selection, since breeders consider that an animal must have this trait highly developed if it is to be a success on the track. If nervousness is a trait that is transmitted by inheritance and amenable to selection it is probably also a trait that would tend to be intensified by close inbreeding, and therefore it might be expected that rats closely inbred for many generations would be somewhat more nervous than outbred stock controls, as Utsur- ikawa found to be the case. When the last two series of investigations were completed the animals used were sent to The Wistar Institute where they were killed and carefully examined by Dr. Hatai. It was found, as Mrs. Yerkes states, that the inbred rats had a somewhat greater body length and body weight than the stock rats, and that they showed a brain weight in relation to body length and body weight that was only from 0.002 per cent to 0.006 per cent less than that of stock rats. Since the inbred rats of the sixth and of the sev- enth generations had a brain weight about six and one-half per cent less than the normal (Basset, ’14), it would appear, from Mrs. Yerkes’ findings, that somewhere between the seventh and the twelfth generations the animals entirely recovered from the effects of malnutrition and became normal again with respect to the relative weight of the central nervous system. They have remained normal in this regard up to the present time, as autopsies made at various periods on animals of the later generations have shown. With the return of the central nervous system to its normal weight relations, the inbred rats must have regained much of their lost mental vigor, since in behavior tests animals of the fourteenth generation were found to be inferior to stock animals only in that they were slower and less active. The lesser activity of the inbred rats Mrs. Yerkes ascribes to ‘‘a greater timidity and a greater susceptibility to environmental conditions.” Savage- ness, wildness, and timidity are heritable behavior complexes, according to R. Yerkes (’13), and since no attempt was made in the course of these experiments to eliminate these traits by selec- tion, it is not surprising that they were manifested in a somewhat intensified form after many generations of close inbreeding. EFFECTS OF INBREEDING ON FERTILITY AND VIGOR 367 3. DISCUSSION Wherever inbreeding has been practiced it has usually been accused of producing anything and everything undesirable that has appeared in the offspring. The following quotation from Mitchell (’65) is quite typical of the belief that prevailed among zoologists, as well as among the laity, until the past decade, regarding the effects of consanguineous marriages: Consanguinity in parentage tends to injure the offspring. This injury assumes various forms. It may show itself in diminished via- bility at birth; in feeble constitutions, exposing them to increased risks from the invasion of strumous disease in after life; in bodily defects and malformations; in deprivation or impairment of the senses, especially those of hearing and sight; and, more frequently than in any other way, in errors and disturbances of the nervous system, as in epilepsy, chorea, paralysis, imbecility, idiocy, and moral and intellectual insanity. Sterility or impaired reproductiveness is another result of consanguinity in marriage, but not one of such frequent oecurrance as has been thought. Stock breeders, also, have been imbued with the idea that in- breeding is always inimical to constitutional vigor and that it leads to sterility. For these reasons most of them have opposed the mating of animals related even in a remote degree. During the past few years it has been shown by a number of carefully controlled experiments that inbreeding does not necessarily produce the evil effects that have been attributed to it, and that the results obtained in any inbreeding experiment depend, pri- marily, on the soundness of the stock that is inbred; secondarily, on the selection of animals for breeding purposes, and, finally, on the environmental conditions under which the animals live. Haphazard inbreeding of inferior stock under unfavorable en- vironmental conditions has produced many of the failures for which inbreeding alone has been held responsible. Since the experiments of Crampe (’83), of Ritzema-Bos (’93, 94), and of von Guiata (’98, ’00) have furnished the classic ex- amples of the dire effects of inbreeding on rodents, it may be well to examine these experiments in some detail to see whether the unfavorable results obtained cannot be traced to some cause other than inbreeding per se. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 26, No. 2 368 HELEN DEAN KING Crampe’s inbreeding experiments were begun, in 1873, with an Albino female and a white and gray male. From the mating of these rats he obtained the litter of five young which formed the basis of his breeding stock. These animals were inbred, in various degree of relationship, for seventeen successive generations. Crampe states that many of the animals were sterile and that others lost their reproductive instincts at the end of the first year. Various kinds of malformations appeared; the animals were seemingly too weak to resist disease of any kind, and they died at a relatively early age. The weakness of these rats and their ~ susceptibility to disease, as well as the high degree of sterility among them, all point to the probability, as Ritzema-Bos sug- gests, that Crampe started his experiments with animals taken from a defective stock. Since results similar to Crampe’s were obtained in the early part of my own experiments, I am inclined to the opinion that inadequate nourishment was a factor that was responsible, in great part, for his failure to maintain the stock in . good physical condition. Ritzema-Bos started his investigation in 1886 with a litter of twelve rats that was obtained from the mating of an Albino female and a wild Norway male. ‘These rats were inbred, in various ways, for six years, during which .time, Ritzema-Bos states, “about thirty generations were obtained.”’ There is evidently some inaccuracy in this latter statement. The female albino rat does not cast her first litter until she is about three months old; wild rats do not breed, as a rule, before they are four or five months old. Assuming that all of the females used in Ritzema- Bos’ experiments bred at the earliest possible age, 1.e., three months, only four generations could possibly be produced in a year: this would give a; maximum of twenty-four generations at the end of six years. In my own experiments an average of about three and one-half generations a year were obtained. Ritzema-Bos gives data showing the average size of the litters and the number of infertile matings during the various years in which the work was in progress. These data have been repro- duced in table 13. EFFECTS OF INBREEDING ON FERTILITY AND VIGOR 369 During the first three years, as table 13 shows, there was little diminution in the average size of the litters produced. In the three following years, however, litter size decreased considerably, and at the end of the investigation the litters averaged less than one-half the size of those obtained in the beginning. ‘These results certainly justify Ritzema-Bos’ conclusion that: “Die fortgesetze Zucht in engster Verwandtschaft vermindert das Fortpflanzungsvermégen, kann sogar schliesslich vollkommene Unfruchtbarkeit verursachen.”’ Lloyd (12) has suggested that the deterioration in Ritzema-Bos’ stock might have been due to overcrowding, since many varieties of rats will not breed in close confinement. TABLE 13 Showing Ritzema-Bos’ data for the average size of the litters and for infertile matings in a series of inbred rats YEAR PRE ne oe Spee, YOUNG | pBpR CENT INFERTILE MATINGS 1887 7.5 0.00 1888 oul 2.63 1889 de 5.55 1890 6.5 17.39 1891 4.2 | 50.00 1892 3.2 41.18 Von Guaita obtained a number of white mice from a strain that had been inbred by August Weismann for twenty-nine gener- ations. How these mice were inbred I do not know, since I have not been able to find any account of the details of this experiment. Von Guaita crossed these white mice with Japanese waltzing mice, and then inbred their descendants for five generations. The data regarding the average size of the litters obtained in these two sets of investigations are shown in table 14. Weismann’s data, given in table 14, show that the average size of the litters decreased directly as the inbreeding advanced, and so appear to indicate that inbreeding lessened the fertility of the mice. In this experiment there seems to have been a very great difference in the number of litters that were produced in the vari- ous generations. In the first two generations there was an aver- 370 HELEN DEAN KING age of about twenty-two litters to the generation: in the last nine generations the average was only about three litters to a genera- tion. Such a small number of litters as that produced in the later generations of this series does not afford an opportunity for a careful selection of breeding stock, neither does it furnish sufficient data to make the results of statistical value. In the successive generations of mice bred by von Guaita there was, to quote Davenport (’00): ‘‘a reduction in fertility of about 30 per cent, and this is probably due to close inbreeding.” In order to make this deduction from von Guaita’s data, however, it TABLE 14 Showing the number and average size of the litters in twenty-nine generations of white mice inbred by August Weismann, and in seven generations of hybrid mice inbred by von Guaita AVERAGE commnamioxs | “OMDER OF | UMEER Or LITTER | 1-10 219 6.1 Weismann’s data for white mice....... 4 11-20 62 520 ( 21-29 29 4.2 ( 1 7 4.4 | 2 15 3.0 Von Guaita’s data for hybrid mice..... ) ; = a | 5 30 Behe 6 11 PA is necessary to combine the records for three generations, as Davenport did. If the records for the various generations are considered separately, or grouped by twos, there is not the steady decrease in fertility with advancing inbreeding that Davenport’s grouping of the data implies. Taking the data for the first two generations together, the average size of the litters was 3.7; for the next two generations there was an average of 4.0 young per litter; in the final group the litters averaged 2.7 young. Since the crossing of varieties is supposed to increase vigor and fecundity it seems strange that the F; and the F, litters in this series should contain a smaller average number of young than is found in the EFFECTS OF INBREEDING ON FERTILITY AND VIGOR orl normal litter of either of the varieties that were crossed (five to six young per litter). Since crossing did not restore the normal fertility of the individuals, it would seem as if there must have been a strong tendency to sterility in each of the strains crossed. If such were the case, continued inbreeding, apparently without selection, would bring out this latent character and intensify it. It seems rather remarkable that, of the many writers who have cited the results of the above series of experiments as proof that close inbreeding lessens fertility, not one, to my knowledge, has emphasized the fact that all of these experiments were made with hybrids and not with a pure strain. Hybridization in itself, as many investigators have noted, often produces a most marked effect on fertility. Some hybrids are equal, or even superior to the parent stock in fertility others are completely sterile; and among the hybrid offspring from various crosses all grades of productiveness from normal to complete sterility have been found. When hybridization increases fertility, its most marked effect is usually found in the animals of the F,; and F, generations, and in later generations productiveness, as a rule, tends to decrease. In connection with another problem, I have for several years been breeding the F; hybrids between the wild Norway and the albino rat, and I have also inbred various strains of ‘extracted’ rats, brother and sister, for several generations. Careful records have been kept of the litter production in all of these strains. While the great majority of the F, hybrid females are fertile, at least 25 per cent of the F, females are completely sterile, and about 10 per cent of those that do breed have only one or two litters. None of the ‘extracted’ strains that I have studied have even been as fertile as the inbred Albinos. The increase of sterility and the diminution in litter size with continued in- breeding has been very marked in some of these strains, but this lessened productiveness has been due, I believe, to hybridization, and it has not been influenced by inbreeding save in as far as in- breeding has intensified the tendencies which acted unfavorably upon productiveness. By rigid selection of only the most fertile individuals for breeding, from a large potential breeding stock, it might be possible to eliminate from the ‘extracted’ strains of rats are HELEN DEAN KING the tendency to sterility that is seemingly caused by hybridiza- tion. Such a selection was not attempted, apparently, in any of the series of experiments cited above, nor was it done in my own wofk with hybrid stock. The experiments of Crampe, of Ritzema-Bos, and of von Guaita show unquestionably that fertility in hybrid rats is diminished by random inbreeding, but they cannot legitimately be used to give evidence regarding the effects of inbreeding on the fertility of a pure race. Other series of inbreeding experiments made on pure strains of rodents show that inbreeding does not necessarily lead to a marked decrease in fertility. Neither Schultze (03) nor Cope- man and Parson (’09) found inbred mice less productive than the outbred strain; Castle (16) did not find any great decrease in fertility in various races of rats inbred for seventeen generations. In the inbreeding experiment with guinea-pigs that has been carried on for several years at the Bureau of Animal Industry in Washington, there is, to quote Popenoe (717): ‘‘no general deterioration. While a few strains have run out, others are nearly as vigorous as are the control families.”’ Results comparable to the above have been obtained with other animals. It is well known that inbreeding has been used extensively, and with very favorable results, in the building up of various strains of thoroughbred horses and cattle (Wriedt, ’16), and the productiveness of these strains has not been greatly lessened. In the extensive series of inbreeding experiments with Drosophila, made by Castle et al. (’06), it was found that ‘‘in- breeding probably reduces very slightly the productiveness of Drosophila, but the productiveness may be fully maintained under constant inbreeding (brother and sister) if selection is made from the more productive families. . . . . Selection has a much greater influence on fertility than inbreeding, so that selection from the most productive pairs is able to more than offset the effects of inbreeding.’’ The effectiveness of selection in increasing the fertility within an inbred strain is shown with great clearness in Moenkhaus’ (’11) experiments with Drosophila. Moenkhaus was able to establish two distinct strains, one of high and one of low fecundity, by selecting, from among the variable EFFECTS OF INBREEDING ON FERTILITY AND VIGOR Be offspring of the fourteenth generation of a closely inbred race, pairs of individuals showing very different degrees of productive- ness and then inbreeding their descendants. Moenkhaus con- tinued some of his lines for seventy-five generations and found that close inbreeding (brother and sister) was not deleterious either to fertility or to vigor. Hyde (’14) has found also that in certain strains of Drosophila sterility is an inherited character that is not influenced by inbreeding, and that ‘‘selection is an effective agent in controlling it.” In the present series of inbreeding experiments on the rat, the productiveness of the strain was decreased by malnutrition during the early generations, but normal fertility was restored as soon as the animals were adequately nourished. In later generations the fertility in the inbred animals was greater than that in the series of stock controls reared under similar environmental conditions. Thus even after a high degree of sterility had been introduced into the strain it was not retained in spite of the fact that close inbreeding was continued. In the later generations any tend- ency to sterility that appeared was evidently suppressed by selection. In the rat, as in Drosophila, selection seems a more potent factor for good than inbreeding is for evil. During the past few years it has been shown, by a series of brilhant experiments, that characters tend to be inherited in groups and that this grouping depends upon the fact that the genetic factors involved are not segregated independently in gametogenesis, but tend to be linked together (Morgan et al., 715). In these experiments with the rat it has been found that animals that are large and vigorous when young tend to mature early, to be very productive, and to live to an advanced age. While all of these characters are influenced to a considerable degree by environmental conditions, it is evident that they must all depend to some extent upon heritable genes, since they are transmitted from generation to generation. A selection of breeding animals on the basis of size and early maturity has meant also selection for high fecundity and for characters that represent superior vigor of constitution, it would seem as if the genetic factors involved must tend to be inherited together, although they are probably not linked as are many of the genes in Drosophila. 374 HELEN DEAN KING Wentworth’s (’13) experiments with Drosophila indicate that the supposed weaknesses from inbreeding are due to “the mere segregation of factors for lower vigor.”” Assuming that a similar segregation of these factors occurred in the inbred rats during the early generations, individuals containing the factors for ‘lower vigor’ were evidently eliminated by the selective action of mal- nutrition, and only those animals containing dominant genes for ‘high vigor’ were able to survive and to perpetuate their kind. Neither inbreeding nor selection is creative in its action. Selec- tion can act on fertility only by preserving those individuals that contain genes for characters favorable to reproduction; inbreed- ing conserves these characters, and, to a certain extent, intensifies them. The action of both selection and inbreeding can be nullified by unfavorable conditions of environment or of nutrition which may produce a rapid deterioration in the fertility of any stock, regardless of the way in which the animals are bred. It was shown by the work of Darwin (’78), as well as by a~ number of more recent experiments (Shull, 10; East and Hayes, 12; Hayes and Jones, 17), that crosses between different varie- ties of plants often produce hybrids that possess greater reproduc- tive vigor than either parent stock. This result is due, according to East and Hayes (’12), to “the stimulation of vigor through heterozygosis.”’ Inbreeding, these authors state, “tends to isolate homozygous strains which lack the physiological vigor due to heterozygosity. Decrease in vigor due to inbreeding lessens with decrease in heterozygosity and vanishes with the isolation of a completely homozygous strain.” If the latter is a good strain, because of its gametic constitutional and natural inherent vigor, it is ‘‘ready to stand up forever under constant inbreeding.’ The results obtained in these inbreeding experiments with the rat accord with the theory of East and Hayes to some extent. The effects of inbreeding on the fertility and on the vigor of the rats were obscured in the early generations by the action of mal- nutrition, but it would appear that the animals lost very little of their constitutional vigor during this time, since adequate nutri- tion soon restored the normal productiveness of the strain and its eeneral vigor as well. Apparently at about the tenth generation, EFFECTS OF INBREEDING ON FERTILITY AND VIGOR 375 the inbred rats became sufficiently homozygous for vigor to become fairly constant. Beyond the point, as the data show, there was little variation in the fertility or in the longevity of the animals up to the twenty-fifth generation. Selection and favor- able environment kept the strain at a point of high productive- ness, but, under the conditions of the experiment, they did not increase vigor beyond the stage which was reached at the tenth generation. As already stated, no attempt was made in the course of these experiments to influence fertility by selecting breeding animals from large or from small litters. Whether selection can act in the rat, as it does in Drosophila, and produce strains of high and of low productiveness within a line that has been inbred for many generations is a problem for the future. As the strain has been very fertile for many generations it seems very improbable that any sudden loss in fertility will occur in the future, unless sterility appears as a mutation which cannot be eliminated by selection. While corresponding records for the two inbred series (A,B) are in close agreement; there are, nevertheless, differences be- tween the series that have persisted from the very beginning. Female A, one of the two females with which the experiment were started, showed a relatively high degree of fertility since she gave birth to five litters, containing thirty-five young, before she was killed at the age of one year: female B, a sister of female A, cast only one litter of five young, although she was paired con- tinuously for several months and appeared to be in good physical condition. The two litter brothers with which these females were paired showed no marked differences in size or in vigor. The rats of the A series (which were descended from female A) were, as the records show, somewhat more fertile than the rats of the B series (the descendants of female B), and they also tended to mature earlier and to live longer. The differences found were not very marked in any case, and they might well be ignored were it not for the fact that in all of the characters noted the animals of the A series were superior to those of B series. Environment cannot be held accountable for these differences, since the two series of inbreds were kept constantly under similar conditions of 376 HELEN DEAN KING light, of temperature, and of nutrition. Although the two series were descended from the same ancestral stock, apparently there was an inherent difference in the gametic constitution of the two pairs of rats with which the experiment was started, which persisted from generation to generation and produced the effects noted. While the inbred strain of rats that has been developed in the course of these experiments is seemingly superior to the average run of stock Albinos in body size, in fertility, and in longevity, I do not claim that this superiority is due solely to the fact that the animals were inbred, neither do I wish to assert that, in gen- eral, inbreeding is better than outbreeding for building up and for maintaining the general vigor of arace. The two forms of breed- ing are not mutually exclusive: each has its merits, and the one should supplement the other to bring out the best in any stock. The favorable results that have been obtained in these experi- ments have been achieved through the constant selection of only the best animals from a larger number available for breeding purposes and by keeping the environmental conditions as uniform and as favorable as it was possible to make them. These experi- ments have fully demonstrated, I think, that even in mammals the closest form of inbreeding possible, i.e., the mating of brother and sister from the same litter, is not necessarily injurious either to the fertility or to the constitutional vigor of a race even when continued for many generations. Success or failure in inbreeding experiments depends chiefly, it would seem, on the character of the stock that is inbred, on the manner in which the breeding animals are selected, and on the environmental conditions under which the animals are reared. ‘There is no warrant, therefore, either in theory or in fact, for the dogmatic assertion of Kraemer (13) that: ‘‘continued inbreeding must always result in weakened constitution, through its own influence.”’ 4. SUMMARY 1. The present paper gives data showing the fertility, the time of puberty, and the longevity in two series of albino rats (A,B) that were inbred, litter brother and sister, for twenty-five gener- ations. EFFECTS OF INBREEDING ON FERTILITY AND VIGOR 377 2. Data given for the A series of inbreds comprise 1752 litters containing 13,116 individuals, or an average of 7.5 young per litter (table 1); records for the B series of inbreds include 1656 litters having a total of 12,336 members, or an average of 7.4 young per litter (table 2). The two series combined comprise a total of 3408 litters which contained 25,452 individuals. For the entire strain the average size of the litter was 7.5 young. 3. In any litter series of albino rats, whether the animals are inbred or outbred, the first litter cast is the smallest of the series, as a rule; the second litter is the largest; while the third and fourth litters are about the same size and a little smaller than the second litter. 4. The size of any litter cast depends chiefly on the age and physical condition of the female, and is not affected by the related- ness or the unrelatedness of the parents. 5. A comparison of the data for the inbred strain with data for litter size obtained from a series of stock Albinos reared under the same environmental conditions as the inbred strain shows that each litter of the stock series was relatively smaller than the corre- sponding litter in the inbred group. For the entire series of 424 stock litters the average size was 6.7 young per litter. This average is 0.8 less than the average for litter size in the inbred strain (table 7). 6. In the A series of inbreds the range in litter size was from one to seventeen; in the B series it was from two to fifteen. In both series the most frequent litter size was seven (table 8). 7. In the early generations of these inbred rats malnutrition greatly delayed the time of puberty in the animals. In the later generations, under favorable nutritive conditions, the animals bred at a relatively early age. 8. While the records give no definite information regarding the number of sterile animals in the inbred strain, they show clearly that inbreeding did not decrease the productiveness of the animals. Of the 954 females that were used for breeding, 653, or 68.5 per cent cast the required number of four litters. Where partial sterility occurred in apparently healthy females it was found to be due to a diseased condition of the reproductive organs. 378 HELEN DEAN KING 9. The constitutional vigor of these rats was apparently not im- paired to any extent by inbreeding. Only two kinds of malfor- mations were found in the animals of the inbred strain after food conditions were improved: one individual was born tailless and about a dozen individuals lacked one or both eyeballs. Both of these defects occur in outbred stock Albinos and neither appears to be heritable. 10. Under the conditions of these experiments the span of life in both the males and females in each of the inbred series was in- creased. The records show that inbred males tended to live longer than did inbred females: a reversed relation was found in the animals of the control series. In the inbred colony as a whole, the females seemed to be longer lived than the males and they were less susceptible to disease at all ages. 11. According to the behavior tests that were made, inbred Albinos are slower, less active, more timid and nervous, and some- what more savage than stock Albinos that are outbred. 12. High fecundity, early sexual maturity, and vigorous growth are characters that seemed to be inherited as a group in the inbred strain of rats. It seems probable that the genetic factors on which these characters depend do not segregate independently, but tend to combine in gametogenesis. 13. The animals of the A series were slightly more fertile than the animals of the B series, they attained sexual maturity earlier, as a rule, and they lived longer. These differences probably depended in some way on a dissimilarity in the gametic consti- tution of the two pairs of individuals with which the experiments were started. 14. The results obtained in these experiments do not accord with the general view regarding the effects of inbreeding, since they indicate that inbreeding per se is not necessarily inimical either to fertility or to vigor. Success or failure in any series of inbreeding experiments would seem to depend on the character of the stock that is inbred, on the manner in which breeding animals are selected, and on the environmental conditions under which the animals are reared. — AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, JUNE 1 THE AMOUNT OF BOTTOM MATERIAL INGESTED BY HOLOTHURIANS (STICHOPUS)! W. J. CROZIER Bermuda Biological Station for Research TWO CHARTS I. It has repeatedly been suggested that animals which obtain their food by the ingestion of sandy or muddy bottom materials harboring minute organisms may exert an important influence upon the local topography of the sea-floor. No attempts seem, however, to have been directed toward estimating the amounts of material which may actually be ‘worked over’ in this way. Large holothurians, such as Stichopus, Actinopyga, and to a lesser degree species of Holothuria, are found in great profusion upon shallow littoral bottoms among ‘coral’ reef islands in the warmer seas, and Gardiner, Verrill, Mayer, and other observers agree in attributing to them a particular significance for the disintegra- tion and scattering of caleareous bottom deposits. These rela- tively huge holothurians are very plentiful, they usually contain a considerable amount of sand, and the bottom is in places thickly strewn with their castings; it is very natural to suppose that the feeding activities of these animals may have an effect upon the sea-bottom not unlike that, so carefully described by Darwin, which earthworms produce in the soil. Thus it might be considered that the production of many fine sand particles would be facilitated by mutual grinding in the intestinal tract, and that the intestinal juices might also aid in the solution of calcareous fragments. From this point of view, as well as with the object of securing data for use in a study of digestion in these holothurians, I have endeavored to estimate the amount of bottom material which may be passed through the digestive 1 Contributions from the Bermuda Biological Station for Research. No. 88. 379 380 W. J. CROZIER tract of the large Stichopus moebii Semper, which is so abundant on grass-free bottoms of sandy mud about the Bermuda Islands. An adequate estimate of this nature requires information upon the following points: 1) the maximum amount of material usually contained in the gut of Stichopus of different sizes; 2) the fre- queney with which holothurians of different sizes feed; 3) the frequency-distribution of sizes in the Stichopus population; 4) the actual numerical abundance of Stichopus in regions of known area. It is necessary to explain, in connection with 1) and 2), that Stichopus is very convenient for a study of this kind, since as a rule the intestine is filled, completely, before defecation is begun; and that an appreciable interval elapses between defecation and subsequent feeding, so that the gut is for a time entirely, or almost entirely, empty of solid contents. Feeding may begin before the intestine has been emptied completely, but each filling of the gut seems to be handled as a unit; characteristically, the whole con- tents of the intestine are voided rather rapidly and in a con- tinuous mass. Hence the long castings which are to be seen upon the bottoms where Stichopus lives. These observations were confined to shallow-water situations, and the ‘quantitative’ data refer to a season (September—Novem- ber, 1917) when the temperature of the water was between 17° and 24°C., usually below 20°C. Stichopus occurs down to depths of 8 fathoms, and possibly more. It is almost certainly more abundant, however, in shallow water along the shore. II. 1. The amount of sand held in the intestine of Stichopus just before evacuation is begun appears to be somewhat variable, in animals of about the same size, even when they are collected in the same locality. Large numbers of them were opened, at various hours of the day and at different stages of the tide, and from the examination of the alimentary tracts of these individuals thirty-nine were selected which seemed to be filled to the maximal extent. The gut contents, in each of these cases, were removed to a finger bowl, well washed with rain water, the sand filtered off, dried in the sun, and weighed. All of these animals were obtained upon one kind of bottom, near Agar’s Island. Figure 1, BOTTOM MATERIAL INGESTED—-HOLOTHURIANS 381 A, shows the relation obtained between the length of a Stichopus and what may be called the maximal, or ‘full,’ intestinal capacity. The errors involved in weighing the contained mud are probably no greater than those incidental to the measurement of the length of a Stichopus. The intestine in the instances considered ‘full’ was tightly packed with sand, as was also the oesophagus and buccal chamber. In the region of the stomach proper, at least in its anterior half, the material was less closely compacted. cms. cms. : +130 10 o °o Length of alimentay tract Length of Stichopus “10 904/111) BO) 1) 2401) 1: GOs: 60 70 gms. Weight of Intestinal Contents Fig. 1 A (scale to the left)—relation between length of Stichopus and the average weight of the washed, air dried, contents of the ‘full’ alimentary tract. B (scale to the right )—Same for the length of the intestine (from figure 2). The several regions of the alimentary tract of Stichopus, it may be mentioned incidentally, are more sharply delimited than text- book descriptions of holothurian anatomy would lead one to sup- pose. The short white buccal chamber is followed by a very thin-walled, highly contractile, oesophagus, which is more or less heavily pigmented by a substance belonging to the ‘echinochrome’ or ‘antedonin’ group. This cesophagus appears to exert a suctorial function; but the activities of the different segments of the alimentary tract may be better considered in a subsequent 382 W. J. CROZIER article. In the pigmented oesophagus lies the region at which the anterior connections of the gut are ruptured in the process of visceral autotomy. The oesophagus leads into the stomach, which occupies the remainder of the first (‘dorsal’) loop of the gut, passing posteriorly into the reflexed intestine. The intestine itself is white, and of uniform diameter throughout its length down to the sphincter constriction at the point of entrance into the cloaca. The stomach, two or three times the diameter of the intestine, contains yellowish or orange digestive fluid, and by peristaltic movements mixes this fluid with the ingested mud. The material which is passed on into the intestine is there com- pacted and for several hours undergoes a process of segmentation before being voided in a more or less continuous mass. The total length of the gut increases rapidly with the size of the Stichopus (figure 2), and the maximal capacity is less than proportional to the cube of this length (figure 1, B). The length of the alimentary tract was estimated in a number of ‘full’ indi- viduals, as the intestine is very quickly shortened by contraction when cut from ‘empty’ specimens. The direct proportionality between gut-length and length of individual (figure 2) seems to show that the judgment based on the ‘full’ condition (employed in the construction of figure 1, A,) was sufficiently accurate. The fact that the stomach is never entirely filled with sand, but al- ways contains a fair volume of digestive juices, probably accounts for the deviation from expectation regarding the relation between length and gut-capacity (figure 1, B,); as it is, the ratio—(dry weight of maximal contents): (cube of the length of gut)—de- creases with increasing length of animal. These determinations show that when filled to its maximum the gut of an average Stichopus of ordinary length (27 cms.) contains about 46 gms. (dry weight) of bottom material; this is mainly composed of calcareous fragments of various kinds. [If it ‘ate’ but once in the twenty-four hours, such an individual would pass through its intestine, and deposit in the form of castings, some- thing like 1.4 kilos (dry weight) of bottom material in one month, at this time of year (November). This estimate is, as a matter of fact, much too low. BOTTOM MATERIAL INGESTED—HOLOTHURIANS 383 2. It is rather difficult to determine exactly the number of times per day that these holothurians fill the intestine. Labora- tory feeding experiments are quite useless, as the animals usually will not eat when confined in vessels having vertical walls up which they may creep. The castings do not retain, in the field, a recognizable form for more than several hours, so that little help can be had from that quarter. Moreover, these animals move about to an extent which one would scarcely predict. When viewed from the shore, they seem ponderously sluggish,— cms. 30 N a Length of Stichopus i) {2} 60 60 70 80 90 100 110 120 130 cms. Length of alimentary tract. Fig. 2 Relation between length of animal and length of gut, in Stichopus moebii. a part of the bottom,—yet they do in fact exhibit quite decided migratory movements, particularly in a vertical direction, in appropriate places. Several attempts to get Stichopus to feed upon a substratum prepared by mixing lamp-black with the natural mud, in order that the mud might be recognizable if swallowed, were unsuccessful. The examination of normal intestinal contents plainly showed that the periods of emptying were not characteristically coincident with tidal events. At- tempts to discover the existence of any other form of regularity THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 26, No. 2 384 W. J. CROZIER in the feeding activity of Stichopus led to the discovery of a somewhat surprising condition. Three fairly representative localities were chosen for detailed observation, the places in question being sufficiently near to- gether so that they could be inspected in rapid succession. The first was a shallow, well protected, bight in Crow Lane, immedi- ately to the north of Saltus Island; the second, along the east shore of Agar’s Island; the third, along the north side of a narrow ridge of rock projecting westward from an island situated at the mouth of Fairyland Creek. Stichopus was abundant at each of these stations. When the contents of the alimentary tract of a dozen or more specimens from one of these places were examined, it was found that as a rule the great majority of the individuals were in precisely the same condition as regards the presence of ingested mud. At certain times of the day all of the individuals in any one locality were filled to an identical degree. The Stichopus at the different stations were, however, in very differ- ent conditions as regards contained mud. Thus, on several oceasions each of 12 Stichopus taken at 8:30 a.m. near Saltus Island was found with the gut completely empty; while those at Agar’s Island and at the mouth of Fairyland Creek were uni- formly ‘one half full,’ the posterior portion of the intestine being empty, although they were collected only a few minutes after the first lot had been obtained. Repeated collections of this sort, at different hours of the day, convinced me that all the holothur- ians in any one locality were usually to be found with the intestine filled to about the same extent. The reason for this curious uni- formity I am not yet in a position to discuss, but it is a condition very useful for the purposes of the problem treated in the present paper. The next step consisted in determining the rapidity with which the intestine of Stichopus is filled and emptied. Observations were made in the three localities previously mentioned; the work was of necessity restricted to periods of fine weather. During storms the holothurians do not remain near the surface, but in exposed places, retreat to a depth of several fathoms. They BOTTOM MATERIAL INGESTED—HOLOTHURIANS 385 also move into deeper water at nightfall, and gradually creep up to near the surface during the morning hours. I quote from a record of observations made during a single day; these records are typical of my findings upon other occasions. December 12, 1917 Agar’s Island 7:05 a.m. visible 7:30-8:00. Castings by 10 of the 12 inspected animals. 9:30. 10 full, 1 filling, 1 emptying, 1 empty (13 opened). 11:00. Of 8 animals, 4 had begun castings. 11:30. 8 dissected; 5 empty, 2 well filled, 1 with oesophagus and stomach full. 4:30 p.m. No castings No castings Rocky spit near Fairy- land Creek 8 :00 A.M. visible 10:30-11:00. Of 63 ani- mals, 60 in the pro- cess of defecating. 4:30 p.m. Of 48 animals, all had castings near them or were de- fecating. No castings Bay north of Saltus Island 8:30 a.m. No castings apparent; 5 animals opened had the in- testine well filled. 10:00. Castings plenti- ful; of 6 opened, only 1 animal had sand in the gut. 2:00 p.m. No castings; 11 opened; all were full or at least (2 eases) 3 full. 4:45. Of 28 animals, all exhibited castings. visible; 4 examined had the _ intestine well filled. It follows that these holothurians were probably engaged in filling the intestine at least three times in the twenty-four hours. During daylight, the process of filling and emptying appears to occupy, at a temperature of about 20°C., a period of about 5-6 hours. In utilizing these results for further calculations, I have considered that in any given littoral situation frequented by Stichopus each animal fills the gut twice each day. It seems reasonable to make this restriction owing, 1) to the fact that at night the holothurians retreat into deeper water, and 2) to the fact that in stormy weather they remain well below the water furface, except in protected places; this restriction is probably of a character to diminish, rather than to enhance, the calculated tilling of the sea-bottom by Stichopus. The observations on feeding showed that animals of all sizes between 10 and 30 cm. length fill the intestine with about the same frequency. 386 W. J. CROZIER TABLE 1 Estimates of the quantities of bottom material swallowed by Stichopus moebii at certain localities in Bermuda. The calculations based on the number of Stichopus present in the areas measured, their average size, and the assumption that they eat twice each day = ~ _ - 2 GRAMS PER LOCALITY AREA pete Sy eT Diag ert iar Jeet ATars sds and ee ee eee el OOO X35 97 eo Dien Spi ead. . Se: ener 100 X 25 37 6.5 28.0 Marshall Islanders eee. | 100. 50 675 13.5 29.0 Harrington Sound’. ?-.02. 2). 75 X 50 67 2.9 8.3 Fairyland Creek............ 100 X 15 63 5.0 25.0 * The number seems fairly constant in any one locality; for example, in the small area studied on the south side of Marshall Island, between 600 and 700 young Stichopus (between 10 and 18 ems. length) were constantly present from August to November. There is a very decided tendency for all those in one local- ity to be of about the same general size. III. Employing the reasonable, and I believe exceedingly moderate, assumption that, on the average, Stichopus fills the in- testine two times each day throughout the year, and taking into account the average size of the individuals locally concerned (with the aid of fig. 1, A), the calculations exhibited in table 1 were made in order to obtain some idea of the magnitude of the effects which may properly be ascribed to the feeding activities of Stichopus. It is further assumed that the specific weight of the bottom-material is the same in the different localities mentioned, an assumption which is well within the limit of error of other parts of the ealculation. I have no desire to convey the im- pression that these figures (table 1) possess any notable precision, but Iam unable to see that they could be made more exact with- out the expenditure of an unprofitable amount of energy. It will be noted that any inadequacy attaching to these esti- mates is in all probability such as to make the results too small rather than too large. Yet the average figures appear to be of considerable magnitude. It seems that for each square meter of bottom, in the localities studied, between 2.7 and 29 gms. of cal- careous deposits are passed through the gut of Stichopus each day. This leads to the conclusion that on the average, in these BOTTOM MATERIAL INGESTED—HOLOTHURIANS 387 places, something like 6.8 kilos (dry weight) per square meter per year is eaten by Stichopus. The amount really concerned cannot very well be much less; it may be one and one half to two times as great. If we attempt to figure the weight of bottom material eaten by Stichopus in one of the partially enclosed sounds at Bermuda, such as Harrington Sound, which has a superficial area of about 1.7 square miles, we find that probably not less than 500,000 kilos, or say between 500 and 1000 tons, of bottom substance passes through the intestine of this holothurian each year. This cal- culation, necessarily of a rough order, is based on the assumption that Stichopus is about as frequent in Harrington Sound as in the average of places listed in table 1, and that the holothurians eat but twice each day; these assumptions are admittedly rough, but their respective inadequacies probably tend to counteract each other. IV. The geological importance of the feeding activities of Stichopus is determined by the magnitude of the changes which may be produced by any or all of the following influences: 1) in moving about, the‘holothurians may carry from place to place some portion of the bottom deposits; as subsequently liberated in castings, the ingested material may thus be carried near to the water surface, exposed to wave action, and redistributed over a new section of the bottom; 2) the mutual attrition of caleareous fragments, especially when the ingested mass is under- going segmentation in the intestine, may produce in a mechanical way particles of a fine degree of subdivision; 3) the intestinal fluids may dissolve part of the caleareous material; if subsequently precipitated in floeculent form, upon being expelled into the sea, this material would assist in the accumulation of ooze. The first of these three factors is undoubtedly of some conse- quence. The castings are readily broken up by currents, even at a depth of several fathoms. The mucus which surrounds and impregnates the ejected mass may have an action, as a protective colloid, in assisting the dispersal of the finer particles by the water, but this action can only be of a brief and temporary char- acter, since the slime is soluble in sea water. 388 W. J. CROZIER The second influence has, I think, been in the past over rated. Microscopic examination shows that delicate bryozoan skeletons, sometimes in pieces 5 mm. x 2 mm., pass through the intestine apparently unscathed, as do also bits of echinoid spines 5—6 mm. in length. There is no detectable increase in the amount of finely ground material in the last centimeter of the intestine as compared with that in the oesophagus. I made a number of tests in which the contents of the oesophagus, or of the buccal chamber, were compared with those of the last portion of the intestine, by suspending the mud in tall cylinders of water; these cylinders were shaken well, and the contents allowed to sediment. The proportion of fine material to that of coarser grade was in all cases the same. When opening the intestine of a Stichopus, one gets the impression that the contents are more finely divided than are those of the oesophagus, because most of the smaller par- ticles are on the outside of the densely compacted mass. Never- theless, some grinding may take place, but this factor seems to be relatively unimportant. The partial solution of calcareous fragments may be of greater significance. The yellow fluid contained in the stomach of an ‘empty’ Stichopus gives with indicators an apparent acidity of P, = 5.0-6.5. Fluid obtained by centrifuging the stomach contents of animals engaged in feeding showed acidities varying from 4.8 to 5.5, the latter being most common. ‘There seems to be an active secretion of acid at the time of feeding. This acidity is adequate to dissolve some calcium carbonate, and is in fact greater than that of the rain water which forms stalactites in the limestone caves at Bermuda; the freshly fallen rain water is at about Px = 6.0, while that caught directly as it dripped from the tip of stalactites in several caves which I investigated was at about Px = 7.9-8.0. This seems to be the most important influence which Stichopus exerts, geologically; namely, the solu- tion of a small amount of calcium carbonate, which is probably soon precipitated again when the intestinal contents are ejected into an alkaline sea water (Pax = 8.1-8.2). In this way these holothurians may have played a not inconsiderable part in the excavation of lagoons, and in the formation of muddy deposits,— BOTTOM MATERIAL INGESTED—-HOLOTHURIANS 389 even though they do not devour corals as Darwin at first believed from the reports made to him and from the supposed masticatory function of the stone-ring. SUMMARY 1. A preliminary estimation has been made of the amount of bottom material (mainly calcareous) deposited in littoral situ- ations about the Bermuda Islands which may be passed through the intestine of the large holothurian, Stichopus moebii Semper. 2. This estimate is based upon the fact that it is possible to obtain a fairly accurate idea of the rate of feeding in Stichopus, and of the maximal contents of the gut in individuals of different S1Zzes. 3. In certain typical areas frequented by this species the amount of bottom material passing through the intestine of Stichopus is roughly 6 to 7 kilos (dry weight) per square meter per year. 4. It is estimated that in the enclosed sink Harrington Sound the amount of bottom deposit annually eaten by Stichopus is perhaps 500 to 1000 tons. 5. The fluid stomach contents of Stichopus are sufficiently acid to dissolve some calcium carbonate. The mutual attritionof particles in the intestine is probably of small significance for the formation of finely divided particles.? Pembroke, Bermuda, January 5, 1918. 2 Some of the observations recorded in this paper were made with the aid of apparatus purchased by means of a grant to the Director of the Bermuda Bio- logical Station from the C. M. Warren Fund of the American Academy of Arts and Sciences, to aid in certain investigations of seawater and the chemical com- position of body fluids of marine animals. te wat fata ah tal ae iets eo vt} 4 0 Pa 0 ee care oh * ae pee 5 fi pre Be ta Bee A Wy ape kaceaiieey Wl Paves ond awene oben itey tnd is tude ible Ly denne hic Wy AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE HYBRIDS BETWEEN FUNDULUS AND MACKEREL A STUDY OF PATERNAL HEREDITY IN HETEROGENIC HYBRIDS H. H. NEWMAN Hull Zoological Laboratory, University of Chicago FOUR FIGURES I. INTRODUCTION The present status of heterogenie hybridology in animals Experiments in hererogenic hybridization of animals have been performed largely upon two groups, Echinoids and Teleosts. A much larger amount of attention has been paid to the former than to the latter group, although it now seems certain that the Teleosts in a number of important respects are more favorable than the Echinoids for this kind of work. The list of investi- gators who have contributed to the literature of hybridization among the Echinoids is imposing, including as it does such names as Boveri, Seeliger, Morgan, Vernon, Driesch, Delage, Stein- bruck, Loeb, Doncaster, Peter, Fischel, Herbst, Godlewski, Kupelwieser, Hogedoorn, King, Moore, Tennant, McBride, Fuchs, Debaisieux, Shearer, and DeMorgan. Only a few, largely Americans, have studied hybridization in Teleosts and the work has been comparatively superficial. Only the following seven or eight investigators have contributed to this field: Appelléf, Bancroft, Hertwig, G. and P. Loeb, Moenkhaus, Mor- ris and Newman. It is my conviction that had the same amount of attention been applied to Teleost hybridization as has been given to that of Echinoids, we would have a vastly better under- standing of the truth about heterogenic hybridization phenomena that we now have. 391 THE JOURNAL OF PXPERIMENTAL ZOOLOGY, VOL, 26, No 3 Auaust, 1918 392 H. H. NEWMAN The hybrid situation in Echinords A close comparison of the results of hybridization in Echinoids with that of Teleosts has served to place the whole matter in a new light and to emphasize the need of further work on Teleosts. It seems necessary in making such a comparison to show graph- ically the interrelations of the species that have been crossed. The following table gives in italics the genera of Echinoids used in all of the various experiments, together with an outline of their classification (table 1). TABLE 1 GENUS FAMILY SUBORDER ORDER SUBCLASS Arbacia Arbaciidae Arbacina Echinus Hipponoe Triplechinidae et Diade- i Toxopneustes Mohining f lade Regularia moida Strongylocentrotus \ ; Sanneneeicis Strongylocentroidae Echinocardvum Spatangidae Sternata | Atelosto- | Irregu- mata laria A goodly number of investigators have crossed Strongylo- centrotus and Sphaerechinus, using a number of species of each genus. Such crosses are of merely intergeneric width and are hardly to be classed as heterogenic. Interfamily crosses have been carried out between Echinus of the family Triplechinidae and Sphaerechinus of the family of Strongylocentrotidae, and there is little difference of opinion as to the evidences of paternal heredity. Inter-sub-order crosses between Arbacia of the suborder Arbacina and members of the suborder Echinina have given less definite results as to the hereditary effect of the foreign sperm, Driesch stating that the larvae were pure maternal and Fischel that the paternal influence is shown in form, size, pigment, and skeleton. Neither author was able to rear the larvae far enough to study very definite characters. HYBRIDS BETWEEN FUNDULUS AND MACKEREL 393 Only one cross of greater than suborder width has been made within the bounds of the class Echinoidea and that is of sub- class width. McBride and Fuchs have independently crossed a representative of the subclass Regularia with one of the sub- class Irregularia, using Echinus and Echinocardium. The percentage of fertilizations is so low and the larvae are so un- healthy that no definite conclusions were reached by either author. By means of chemical aids to insemination, ‘crosses’ of inter- class width have been made between Echinoidea eggs and the sperm of Astereridea, Holothuroidea, Ophiuroidea and Crinoidea. Though extremely few larvae resulted—and these were decidedly abnormal—they were described as pure material. By the same chemical means Echinoid eggs have been inseminated with the sperm of Mollusca and Annelida, and occasional stunted larvae have resulted that also are said to be pure materinal. These interphylum ‘crosses’ mark the extreme limit of heterogeneity in hybridization, if indeed they are hybrid phenomena at all. One step further and we would have crosses of subkingdom value (Protozoa x Metazoa) or even of kingdom value (plants x animals), which would amount to a reductio ad absurdum. It must be borne in mind that, in Echinoids, ‘crosses’ of greater than suborder width are impossible in nature and can be made only by the use of chemical agents that serve to break down the normal incompatibility of the heterogenous germinal materials, and may well play a réle analogous to that of parthenogenetic agents. ‘Crosses’ of greater than suborder width are described as being purely maternal, although the criteria for this judgment are ex- tremely doubtful since the embryo and larvae reared are highly pathological. No paternal heredity is to be expected in view of the fact that the foreign chromatin forced into the egg unnaturally, utterly fails to coéperate in cleavage or in more general cell metabolism, but remains an inert mass till gradually eliminated by absorption. An exception appears to exist in the case of the cross between Echinus and Antedon, in which Godlewski claims a normal behavior of the paternal chromosomes, except that 394 H. H. NEWMAN the latter come to be indistinguishable from Echinus chromo- somes; yet the larvae are pure maternal in so far as they are any- thing definite. In these wide ‘crosses’ it appears reasonable to conclude that the foreign sperm at least assists in the initiation of development, duplicating in a sense the rdle of partheno- genetic agents. According to Loeb: ‘‘The egg behaves exact!y as we should expect from the fact that the spermatozoén removes only certain obstacles for the development. of the egg, but does not cause its development by carrying any activating enzyme.” It may be concluded, then, that in Echinoidea crosses of generic and family width exhibit paternal heredity, that when Echinoids are ‘crossed’ with other classes or other phyla that heredity is not concerned, but merely initiation of development. The really interesting and critical phases of Echinoid hybridiza- tion lie between these extremes: in crosses of subordinal, ordinal, and subclass width, and there are, unfortunately, very few data on this point. A eross of subordinal width was studied by Driesch and by Herbst. Their results and conclusions are diametrically op- posed; the former concluding that the hybrid larvae were purely maternal and the latter that the paternal influence was seen in several characters. More work is needed upon crosses of this width. A cross of subclass width was made by McBride and repeated by Fuchs with results quite different in the two cases, and it seems certain from what these authors say that the larvae are so few and so abnormal that little conclusive evidence as to heredity is available. In conclusion it may be said that the results of hybridization ex- periments of greater than interfamily width among the Echinoids have been entirely inconclusive, and it would appear necessary to transfer our attention to Teleost material if we wish for any approach to a definite answer as to the question whether heterogenic hybrids show paternal heredity or are merely parthenogenetic vn character. HYBRIDS BETWEEN FUNDULUS AND MACKEREL 395 The hybrid situation in Teleosts as compared with that in Echinoids Taxonomically the group Teleostei is of lower value than the group Echinoidea, the former being merely an order of the sub- class Teleostomi of the class Pisces, while the latter is a class on a par with the class Pisces. A group of Echinoidea correlative with the order Teleostei is the order Diademoida of the sub- class Regularia. A strict comparison of hybrid phenomena would therefore be limited within the confines of these two. orders, Diademoida and Teleostei. As a matter of fact, only one Echinoid cross has been made that exceeds the limits of the Diademoida. No one has as yet succeeded in crossing a Teleost with any of the Ganoid orders of Teleostomi nor with any other subclass of Pisces, much less with members of other classes of vertebrates or with invertebrate phyla. By the use of appropriate chemicals it might conceivably be possible to inseminate Teleost eggs with other than Teleost sperm, but my impression is that the micro- pyle would be too much of an obstacle for a type of sperm very different from that of a Teleost. Even if a foreign spermatozoon could be forced past the micropyle barrier, it would doubtless be unable to initiate development for the same reasons that the several sorts of parthenogenetic agents, that have been extensively tried upon Teleost eggs, have failed to bring about any develop- mental result; for Teleost eggs, unlike Echinoid eggs, are extreme- ly refractory to parthenogenetic agents and would therefore probably fail to respond to foreign sperm. Hybridization ex- periments among Teleosts are, therefore, confined entirely within the order and at most will be of subordinal width. A very large number of crosses of subordinal, of family, and of generic width have been made. A companion of the following table (table 2), which represents the classification of the genera of Teleosts, with table 1 will be of interest. A survey of this table shows that nearly half of the genera used are members of the large suborder Acanthopterygii, just as most of the Echinoid genera used in crosses belong to the suborder Echinina. The four suborders in the table have been crossed in a great many ways and with varying degrees of success. At 396 H. H. NEWMAN least as wide a cross as any possible within the bounds of the genera given in this table is that between the first and the last genera, Fundulus and Scomber. In general it may be said that any Teleost will cross with any other with the exception of vivi- parous species, and those with peculiar breeding or brooding habits, such as the pipe-fishes, where secondary obstacles to cross insemination present themselves. It is also worthy of note that no artificial aid to insemination is necessary In any cross. In the case of crosses between closely allied species, as, for instance the various species of Fundulus (F. heteroclitus, F. ma- jalis, F. diaphanus), in which paternal heredity is as obvious in many ways as is maternal, in that paternal characters are often TABLE 2 Genera of Teleoster used in hybrid experiments GENUS FAMILY SUBORDER Fundulus : : : P ; ‘ids Hapl Conatodon } Cyprinodontidae aplomi Gasterosteus | : - 3 Gasterosteidae Cateasteomi Apeltes Menidia Atherinidae | pi AMS Pronotus Stromatidae / Morone Serraninae | Stenotomus Sparidae | preeosoanel Tatidne Acanthopterygii Tautoga Scomber Scombridae | dominant over maternal. The same is clearly true for crosses between two genera of the same family as when the genus Gas- terosteus is crossed with Apeltes, both genera belonging to the family Gasterosteidae. In this case a large percentage of healthy hybrid larvae hatch and they are by no means pure maternal. Crosses of interfamily width such as that between representatives of the different families of Acanthopterygii give similar results, m so far as success in development and strength of paternal heredity are concerned, as do crosses of suborder width and need not be considered at length. As a very typical case of heterogenic hybridization of suborder width among the Teleosts I have chosen that between Fundulus heteroclitus of the suborder HYBRIDS BETWEEN FUNDULUS AND MACKEREL 397 Haplomi, and Scomber scombrus of the suborder Acanthop- terygii. This cross between mud-minnow and mackerel is an excellent one for our purposes because it shows in the clearest way what many other crosses show, but show in a less obvious way. One reason for making an intensive study of one particular heterogenic cross was because I have felt that certain incorrect conclusions as to the nature of heterogenic Teleost hybrids had been published by well-known writers. Moenkhaus holds that the development of these hybrids is pure maternal and that the cleavage rate of the hybrid egg is unaltered by the foreign sperm even if the cleavage rate of the paternal species is much slower or much more rapid than that of the maternal species. I have shown that this is incorrect by the use of more exact meth- ods of comparison. There is an acceleration of cleavage and development, accompanied by a heightened vigor when two very closely allied species are crossed. In any crosses except those of very closely allied species the rate of cleavage and subsequent development is retarded and more or less abnormal (subnormal)} larvae result. This does not depend directly on the distance of the cross, for sometimes crosses of suborder width show less retardation and a higher viability than crosses of generic width. Loeb, on the basis of certain rather unfortunately chosen crosses, supports Moenkhaus in his statement that cleavage rate and that of subsequent development is pure maternal and goes farther in claiming that heterogenic hybrids are pure maternal in their hereditary characters. He thinks the hybrid larvae, which in all of his experiments appeared to be quite unhealthy, are merely pure maternal or parthenogenetic larvae that are poisoned by the presence of materials brought in by the sperm. The argument in favor of the pure maternal character of devel- opment, which implies parthenogenesis, is that ‘if the develop- ment of the egg were caused by an enzyme carried into the egg by a spermatozoén, developing eggs should be accelerated by a spermatozoon of a species developing at a faster rate.’’ This conclusion does not appear to me to follow at all, for we must not forget the high specificity of enzymes. An enzyme that is ¢a- pable of setting up a high rate of activity in one species of proto- 398 H. H. NEWMAN plasm would not be expected to activate so readily another species of protoplasm. It might conceivably retard develop- ment beyond the normal rate even in a slowly developing egg and still play the typical réle of sperm materials in heredity. This is, I believe, exactly what happens in all heterogenic hybrids, for they all exhibit more or less pronounced evidences of early and long-continued retardation. That the sperm actually do cooperate in development even in suborder crosses is shown by the unmistakable cases of paternal heredity. It is inconceivable that a foreign sperm could function in heredity without effective functioning in development, and any hybrid in which paternal heredity is demonstrated is neither a parthenogenetic individual nor pure maternal. It is just exactly this pomt that the present experiments demonstrate, to my mind at least, beyond controversy. I have selected the Fundulus x Mackerel cross out of nearly one hun- dred crosses that I have personally made among the Teleosts because it is especially favorable. In no sense, however, is it exceptional or peculiar in character, merely a little clearer and more diagrammatic than others that might have been chosen. Any one of half a dozen other crosses would have done nearly as well. Il. EXPERIMENTAL Differences between adults of Fundulus heteroclitus and Scomber scombrus Fundulus and the mackerel are sharply contrasted in all of their adult characters as might be expected in representatives of differ- ent suborders. They differ radically in habitat, ecological relations, breeding habits, eggs, and larvae. Fundulus is a minnow with shore-feeding habits, is found in both salt and brack- ish water, is tolerant of foulness in water and to low oxygen and high CO, concentration. The large eggs (25 mm. in diameter) are laid during a clasping act on the part of the male which insures fertilization of the eggs with a minimum expenditure of milt. The eggs sink to the bottom and adhere to stones and seaweeds at or near the bottom by means of the sticky egg HYBRIDS BETWEEN FUNDULUS AND MACKEREL 399 envelope, which probably also protects the eggs from injury of various kinds. In correlation with the fact that eggs are ferti- lized immediately on their emission from the oviduct, it is note- worthy that the spermatozoa live only a few minutes, at most five, in seawater. Consequently, any females that have been isolated for any appreciable length of time could not possibly earry sperm upon their bodies. Therefore any criticism of these results, based on the assumption that occasional heterogenic hybrids that go through successfully to hatching might be due to chance fertilizations by sperm of the same species, have no foundation in fact. All that one has to do to obviate the possi- bility of any such contingency is to isolate the Fundulus females and handle no Fundulus males during the hybrid experiments. This was done in every case. The mackerel, in contrast with Fundulus, is a rather large fish, frequenting the off-shore waters except when they come in closer to breed. They are intolerant of water impurities, die soon in aquaria and show themselves extremely sensitive to lack of oxygen or high CO, concentration in the seawater. The eggs are about 1 mm. in diameter, typically pelagic in character, but differ from most pelagic eggs in having a distinct pinkish cast. The milt is extremely abundant and is evidently shed in clouds in order to ensure fertilization of the scattered eggs. The spermatozoa live in aquarium seawater for about twenty minutes and probably live even longer in natural seawater. Differences between embryos and larvae of the Fundulus and mackerel The egg of the Fundulus develops much more slowly than does that of the mackerel, taking about two weeks to hatch as compared with about two and a half days for the latter. Only in late larval stages are there significant differences of bod- ily structure and proportions. These do not need description here as none of the conclusions here brought out have to do with such general characters. Our attention must be focused upon one type of character, the peculiarities of the yolk and body chromatophores. 400 H. H. NEWMAN Fundulus has two well-defined types of chromatophore: a type of melanophore (black chromatophore) characterized by large squarish body and few short branches, showing also a tendency to fuse into syncytia, and a red or red-brown chromatophore which in its definitive or fully expanded condition is very intri- cately branched. The black type, if unhealthy, may remain small, and give out.a few slender branches, but is never a very intricately branched cell in pure Fundulus embryos. The red chromatophores also may be relatively simple or unbranched if the embryo is pathological or retarded. The mackerel larvae also have two types of chromatophore equally characteristic of the species and quite distinct from those of Fundulus. One type is a melanophore which is characterized by a small core or body and very slender anastomosing branches. They never exhibit the tendency to fuse into syncytia. The second type is an olive-green chromatophore that occurs in the larvae both on body and yolk sac. Usually there are two large green cells just back of the eyes, two more a short distance behind the otic vesicles, and two or three adjoining the Kupfer’s vesicle on the yolk sac. There are no red cells in mackerel nor any green cells in Fundulus. So in these two opposed char- acters there is a sharp contrast, and the finding of a red chromat- ophore in a mackerel egg-hybrid or a green chromatophore in a Fundulus egg-hybrid could not be interpreted as within the range of variability of the maternal species or as a pathological occurrence. avn) sletiis « sn nivea& tegen a ee es eyes t, OLD Pbteen Onl ponnvernnyimipiss. Wy Lk eo APS S628 3. Negative vs. positive nymphs; both treated with HCl............... 531 HXpPeriMentia wiihapOpasseuMMerAnldes ye od lstaes ks EMwiealda oo bck utinw ee. SOD Experiments with, aleohol and. strychnine i... 200). onan win wea ew ena ils 537 Diseuasloniint mile art fede wee aise ickeee dsb b-Allt Ainge las) eraattli 540 Candlusions-ais |. 2. .0F ak Basie aiolss Gas orescca sid f. UES MG oello cab 544 LiteraturescttOdid rast eetpranere tie el aii roe Oat ee e (bekethaiseen 1 dia 545 423 THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 26, No. 3 424 W. C. ALLEE AND FE. RB. STEIN,, JR. I. REVERSALS OF PHOTOTAXIS AND THE RESISTANCE TO POTASSIUM CYANIDE! HISTORICAL The idea that there is a relation between the fundamental metabolic processes and the signs of the reaction of animals to stimuli is not new. It has also occurred to some investigators that there may be a relation between the rate of these metabolic processes or part of them and the phototactic reaction. Holmes (05) found that Ranatra is made more positive by conditions that cause an increase in activities and made more negative by opposite conditions. Carpenter (’05), studying the light reac- tions of Drosophila, concluded that the more stimulated the animels were, the more positive they became. Jackson (’10) came to the conclusion that the changes in re- sponses to light, which he obtained with the amphipod Hyalella, are not due to chemical changes of the eye or skin, as Loeb (10) had suggested, but are rather due to a sudden stimulation or shock to the nervous system. Mast (11, p. 283) states that in Arenicola, ‘‘Any condition which serves as a depressant tends to cause the young larvae to become negative,’ and he concludes (p. 287) that ‘“‘The facts 1) that the light reaction may be affected in a given organism by so many contrasting conditions; 2) that the same change in external conditions may ceuse opposite reactions indifferent organisms, and 3) that the sense of the reaction may be changed without any immediate external change—indicate that these responses are due not to a direct and specific effect of the environment on some definite chemical compound within the organism, but rather to the effect on the organism as a whole.”’ Bohn (’12) separately reached a somewhat similar conclusion when he decided that there are two kinds of sensibility, one to light and one to shade, and that these correspond to antagonistic 1 The experiments upon which part 1 of this paper is based were performed at Williams College in 1913-14. Certain tests were repeated by the junior author in the spring of 1915. The nymphs were identified by Mr. W. A. Clemens, of the Cornell Limnological Laboratory, to whom we express our thanks. LIGHT REACTIONS—METABOLISM—MAY-FLY NYMPHS 425 chemical reactions. Causes that accelerate oxidations tend to make animals positive to light and causes that inhibit oxidations produce reactions to shade. Bohn’s conclusions are based on his own and Drzewina’s (’11) observations. Phipps (15) found that when amphipods, negative to light, are treated with potassium cyanide, chloretone, or were subjected to decreased oxygen tension or to starvation that many of the animals reversed their reactions both to light intensity and to the direction of rays. None of these workers measured the effect of the substances upon the metabolism of the animals under investigation. Indeed, this has been done only by MacCurdy (13), who found that the negative starfish Asterias forbesi gives off less carbon dioxide in sunlight than in shade. He made no effort to control the reac- tion to light. Many investigators, beginning with Loeb (’04), have recorded reversals of sign of the phototactic response which could be pro- duced at will; the problem which we set for ourselves was to find whether or not there is any correlation between the sign of the reaction to light and the rate of metabolism of the animal as measured by resistance to potassium cyanide or by carbon dioxide production. For the first part of this inquiry the May- fly nymphs Leptophlebia sp? and Epeorus humeralis (Morgan) were chosen because of their abundance near the laboratory and because Wodsedalek (711) had found the phototactic reactions of the May-fly nymph Heptagenia interpunctata is readily reversed by chemicals. ECOLOGICAL NOTES The Leptophlebia and Epeorus nymphs studied lived in moun- tain streams with stony beds and rapid currents such as are quite common in the Berkshire Hills. Their distribution was most carefully studied in Tunnel Brook (near Hoosac Tunnel). In the early autumn when few leaves had fallen the nymphs lived on the under sides of stone and were almost invariably facing up- stream. Their clinging ability enabled them to maintain them- 426 W. C. ALLEE AND E. R. STEIN, JR. selves in the swift current. In April and May large numbers of Leptophlebia were found among submerged leaves in the quieter parts of the brook. At the same time Epeorus was most abun- dant under stones in more rapidly moving water. Neither were found on the upper surface of stones until late in May when the adults were emerging in large numbers. At this time hundreds of Epeorus nymphs could be seen on the upper side of any large stone in the brook, all headed against the current. METHODS The phototactic tests were made in a large, light-tight wooden box to which light was admitted through adjustable slits. The opening was near the bottom of the box so that the light entered the end of the experimental dishes. During the experiments described in the first part of this report a north window fur- nished the light source. The experimental box was painted dead black inside. The rear was curtained with a heavy black, cloth. A semicircular opening in the floor of the box opposite the light inlet permitted the observer to sit with head and upper body in- side the box without introducing an appreciable amount of light. The light-reaction tests were carried on in oblong glass dishes measuring 12 x 5.2 x 2.2 em., which were placed with the long axes parallel with the light rays. When it was necessary to cool the dishes to keep the temperature at or below tap temperature, the experimental dishes were placed in shallow glass trays and packed in ice on all sides except that toward the window. Control dishes were kept under identical conditions save for the factor under experimentation. The nymphs were kept in the laboratory in aluminum tea balls which hung in running tap water which was similar in salt and gas content to the water of their native streams. Most of the experiments were performed before the animals had been in the laboratory five days. LIGHT REACTIONS—-METABOLISM—MAY-FLY NYMPHS 427 REVERSALS OF PHOTOTAXIS In experiments upon phototaxis the nymphs were selected from a number which had stood a short time in tap water exposed to the light conditions under which the test was to be made. Then, if the experiments were to be upon negative animals, only decid- edly negative nymphs were selected. During the course of the experiments nymphs were subjected to various strengths of sul- phuric, hydrochloric, and acetic acids; potassium and sodium hydroxide; potassium, sodium, calcium and magnesium chlorides; ethyl alcohol, chloretone, caffein, strychnine, and to temperature changes. The two species with which we worked reacted oppositely to light. Epeorus was normally positive while Leptophlebia was normally negative. The former is the more definite in its reac- tion, as shown by the ratios of the average negative (N) and positive (P) responses of the controls. Thus Epeorus with P : 90 seventeen sets of control readings gave a N ratio of il while ea : 5 Leptophlebia with thirty such controls gave 12 Quantitative reversals were obtained with Epeorus with ethyl alcohol, calcium chloride, and with a decrease of tem- perature, Alcohol was the most efficient reversing agent used with this species. At tap temperature (about 12°C.) the best results were obtained with a 2 per cent solution. When the tem- perature was lowered 5° or more, better results were obtained with a 1 per cent solution. Under both conditions quantitative reversals were obtained with positive nymphs and with the control strongly positive throughout. With Leptophlebia more of the chemicals tried gave quantita- tive reversals. Hydrochloric and sulphuric acids, potassium and sodium hydroxides, potassium cyanide, and potassium chloride gave 100 per cent reversals of negative nymphs while the controls were predominantly negative throughout. Sodium and calcium chlorides gave 80 per cent reversals with the controls over 80 per cent negative. Magnesium chloride, chloretone, and caffein had little effect. Alcohol, as with the other species, gave a high percentage of reversals, although quantitative results were rare. . C. ALLEE AND E. R. STEIN, JR. 428 ‘aAOUI ATOATIOV OF poze[NuTys AT[voTuRYoour 910M LADY} YY} SoyROIpUT 9 “poyB[NUIT}S AT[VOTUBYOOUL JOU oIOM STRUITUB OY} YVY} SoyVoIpuT x * x | 001 Ore 625% Ic-Z4T | & 8 |§ 8-0 8i7 GG F 996) ¥ T-0 teh tol altiesdateherSiy-ymenl}o als SrA 0,0) (0) x Pre isewit ate Neale TT 89 |S °8-0'9)S8 SI F 699) ¥ ax 9°24 0 IT-0'9 |6F IN &200°0 *k « |96& OT + 69T| ¥ V 89S | 6 F (99-0 EST L ¥ 1&3) ¥ asv |89/;9809 ($8 IN &200°0 * Pe ee 2 9°0 + 6 | ® Wee Se SeLn\c) Sa0ee a 9 +F 6L | * asv |&8|$ 8-08 fF IN &200°0 etqo[ydoydaT * + | 96 SUP 6 FL L6 | ¥ V 8°98 | 9'F 0 S-€ S/0l SUS > + O'8h¥ | ASV | 98 0 TT-0'9 (61 |N 100000'0 * PE |p teh 6 OT + 8hT| ¥ &T 9° |S°8-0'7F/8 02 * LPS ¥ L 6¢|02-G°€ |8 |N 10000°0 * ae |i Pea One VW Ses 5ie259)0 S057 61 oT + §9T| ¥ ad ®*vV |09)084 8 j|Z41 IN 100000 * eben |W) te 9 + TTT] * V 8°99 | FP 98-0 )0¢ 8+ eoT/*¥ | “ASV |69|060'9 (2 IN 10000°0 sajgnurue ‘OQ ‘2ap “UU “UU saynuru ‘2 ‘bap “UU “UU snio00dy € WOIstarcT | V uoIstatq D n Pl QD D 4 > a) z D mn re > n % D Bid [ei On | SG eo 5 ad SI > 3 g | as SI > 5 S Gz Bat ee el ata S S ci E g lee = cE ed Q z z 2g eis) 2 bie is 25 ie cS) = 5 is zu > rs | S | SF le salece de] 4 e | #@ 1G 4 igs] 4 a F 3 ge | © (839 E ee ts en ak 4 : Sols ee re 3 3 us ogo aU f0 ayD4 4an0) 9Y}) fiyypIYa.L00Y} BaDY PyNoYs JDY) S]DULUD WO S7]NSAL S}IqQLyxa 97QD} BY) fo P UoIMmig ‘vigazydojzdaT fo wsr0 -qojau fo avs ay} sainspaut fij}0041p “N GOO'O (2412YN 07 SD OS]P "72 Saunspaw fiz}IaLvpUr *N [00000 0 PUD sydwfiu snsoady f0 ajD4 DYOQnjaUW ay} sainspaw fijjoa1yp apruvlia wnissvjod fo sUuoYNpOS JDUWLOU TOOOO'O 42Y}2YN 07 SDV 8]89} fo synsau ay) Burnoys T @IaVL LIGHT REACTIONS—METABOLISM—MAY-FLY NYMPHS 429 DOES RESISTANCE TO POTASSIUM CYANIDE MEASURE THE RATE OF METABOLISM IN MAY-FLY NYMPHS? In this work the cyanide resistance method of Child (’13) was used without modification except in the strength of the cyanide solutions employed. In this method with a relatively high concentration the animals with a higher rate of metabolism (ef. Geppert, 99, Hyman, 716) die before those with a lower rate. In much weaker solutions acclimatization occurs and the effect is reversed. The resistance of the nymphs was tested in 500-cce. Erlenmeyer flasks. Control experiments showed that the more sensitive Epeorus nymphs could live from five to fifteen days in this amount of unchanged tap water, provided the temperature was approximately constant. Some difficulties were encountered in determining the death point. The nymphs were usually observed every thirty minutes in later experiments every twenty minutes), and those that were apparently motionless were removed with a large pipette and care- fully inspected under a lens. If no motion was apparent they were stimulated with a needle. If they failed to show any mo- tion under these conditions they were considered dead. It is obvious that this treatment would stimulate live nymphs and more frequent inspection would increase rather than decrease the experimental error. Epeorus proved to be much less resistant to the cyanide than Leptophlebia. With the former (table 1) a solution 0.00001 normal gave a measurement of metabolism by the ‘direct method;’ with the latter the same measurement was obtained by a strength of 0.0025 normal. With Epeorus 0.000001 normal was found to measure metabolism by the acclimatization method. With this dilution so great care was necessary in order to maintain the solution at even its approximate strength that no serious tests were run. A new solution of 0.1 normal potassium cyanide was made up weekly, and from this dilutions were made to the desired strength. With extreme dilutions and with certain preliminary experiments which ran several days the solutions were made up fresh twice daily. 430 W. C. ALLEE AND E. R. STEIN, JR. The inquiry as to whether resistance to potassium cyanide in May-fly nymphs is affected by the rate of metabolism of the ani- mal was prosecuted along the following lines: 1) What is the relation between the resistance of large (old) and small (young) nymphs? 2) What is the effect of stimulation upon the resistance to the cyanide? 3) What is the effect of differences in temperature? The results of these experiments are summarized in table 1. Nymphs of Epeorus were less resistant to 0.00001 normal potas- sium cyanide when they were small, or stimulated, or at increased temperature. All of these conditions cause a higher rate of metabolism (Child, 713; Allee, 714). On the other hand, in a 0.000001 normal solution the smaller (younger) nymphs were more resistant than the larger ones, which is what the theory demandsaf a solution of this strength to measure indirectly the rate of metabolic processes. Leptophlebia in 0.0025 normal solution was less resistant when young, or stimulated, or when the temperature was increased so that this strength of cyanide directly measures the metabolic rate of these nymphs. A solution 0.00025 did not indirectly measure the rate of metabolic processes of these nymphs and the experiments were not continued long enough to find a solu- tion strength that would do so. In all the above instances in which the evidence is that cyanide resistance does measure the rate of metabolism the differences in the survival times exceeds twice the probable error and we may safely hold them to be statistically significant. RELATION BETWEEN THE SIGN OF LIGHT REACTION AND RESIST- ANCE TO THE CYANIDE 1. Epeorus The average survival time of fifty-one positive Epeorus nymphs (table 2) which had been taken directly from tap water was 108 = 5 minutes. These nymphs averaged 5.4 mm. long. A reversal of the phototactic reaction of forty-seven other posi- LIGHT REACTIONS—METABOLISM—MAY-FLY NYMPHS 431 tive Epeorus nymphs was caused by treatment with alcohol (1 or 2 per cent) and decreased temperature (2° to 8°C.). These reversed nymphs gave a survival time of 131 + 6 minutes in the same strength of cyanide. Their size average was 6.5 mm. The difference in survival time is only 2.1 times the probable error, and since the difference may have been affected by the difference in size, too much emphasis cannot be laid on these results. The survival time of eighteen nymphs of the same species that failed to reverse in the alcohol-reduced temperature treat- ment was 88 + 3 minutes. Their average length was 6.1 mm. The nymphs that were reversed by this treatment lived forty- three minutes longer in the cyanide than those that remained TABLE 2 Showing the relation between the sign of the phototactic reaction of Epeorus nymphs and their resistance to potassium cyanide AVERAGE AVERAGE NUMBER | AVERAGE > NUMBER | AVERAGE aes AEaOn tenor. | SURVIVAL | TREATMENT BEFORE KILLING | poopy LencTH | SURVIVAL TIME TIME | ! Positive nymphs | Negative nymphs mm. minutes mm. minutes 51 5.4 108 + 5 Tap water 18 Gel 88 + 3 Alcohol 47 6.5 j|131+ 6 Lowered temperature 11 6.1 |160+16 positive. Since this is 4.8 times the probable error, it must be significant. The majority of the Epeorus nymphs treated with alcohol and decreased temperature reversed their reaction to light and had a slightly (perhaps questionably) lower rate of metabolism than the control animals and a decidedly lower rate than those not re- versed by the treatment. The nymphs that remained positive, although given the aleohol-low temperature treatment, appar- ently had been stimulated by the alcohol, for they showed a higher rate of metabolism than the control animals. This can be explained by assuming that the alcohol acted as usual, first stimulating and later depressing. If this be true, the fact that alcohol plus reduced temperature was more effective than the 432 W. C. ALLEE AND E. R. STEIN, JR. latter alone becomes significant if the metabolic differences prove to be causal rather than incidental or resultant, for the difference between a stimulated period followed by depression may well be greater than mere depression. Sometimes quantitative reversals were obtained by reducing the temperature. Eleven nymphs that had been thus reversed (average length 6.1 mm.) were killed in cyanide and gave an average resistance of 160 = 16 minutes. This is fifty-two min- utes longer than that given by the control nymphs which is 2.5 times the probable error and is probably significant. Taken altogether, the evidence here presented indicates that reversed Epeorus nymphs have a lower rate of metabolic activity than do positive animals. One other observation confirms this idea. Epeorus nymphs collected in October were kept in a large aquarium that also contained some fresh-water mussels. In December and January the nymphs were found to be dying in large numbers. When tested all were negative to light, although when first collected they had given almost quantitatively posi- tive reactions. Obviously the metabolic process of the nymphs was strongly retarded and this is correlated with their reversal to light. 2. Leptophlebia Leptophlebia nymphs were usually negative in their reaction to light giving a ratio of twelve negative to five positive animals. The average survival time of sixty-one untreated nymphs (average length 7.9 mm.) that gave the usual negative light reaction was 131 minutes. Forty-two positive nymphs (aver- age length 7.8 mm.) under conditions similar in every way re- sisted the same strength of cyanide 130 minutes. Thus there was no difference in the metabolic condition of these two groups that could be measured by the cyanide resistance method. Hydrochloric acid was very effective in causing reversals in Leptophlebia. The survival time of thirty-six nymphs _ so reversed (average length 7.7 mm.) was 127 minutes. Twenty nymphs similarly treated that remained negative (average length 7.8 mm.) gave a mean survival time of 120 minutes. This LIGHT REACTIONS—METABOLISM—MAY-FLY NYMPHS 433 difference is of course negligible as is also the difference between these acid-treated animals and the control. Aleohol was also effective in making these negative nymphs positive. Fourteen nymphs that were made positive gave a resistance of 91 + 7 minutes to the cyanide as compared with 130 + 4 minutes for the sixty-one control animals. This is 3.5 times the probable error. The eleven nymphs that were treated with alcohol and remained negative showed some stimulation TABLE 3 Showing the sign of phototactic reaction of Leptophlebia nymphs and their resistance to cyanide pirate Silchar sunvivan TREATMENT BEFORE KILLING pelclty pbs g SURVIVAL Positive nymphs | Negative nymphs mm. minutes mm. minutes 36 ott 127 HCl 20 7.8 120 14 7.8 Of == 7 Alcohol 2 per cent 11 7.7 |109+9 9 7.8 119 H2SO.4 8 7.8 115 Acetic acid 2, 7.0 136 7 8.5 127 NaOH 8 7.8 146 8 6.9 132 KOH 2 7.0 130 9 6.8 1183) KCN 2s Uc4 103 3 8.3 115 KCl 2 8.0 103 6 8.7 100 NaCl 4 8.7 105 MgCl Ye 8.0 129 2 7.8 129 CaCl, 3 (lees 123 6 7.8 161 Chloretone 8 8.1 165 108 7.8 123 Totals and averages 64 7.8 129 42 7.9 131 + 4 Tap water control 61 7.8 |130+4 when tested with cyanide, but not so much as the positive nymphs. Considering the numbers tested and the small deviation from the resistance shown by the control animals, none of the results obtained with other reagents and listed in table 3 are significant with the exception of those with chloretone. This drug was not very efficient in causing reversals, but did cause decided de- pression, as measured by the cyanide method, and caused re- versals in about 30 per cent of the nymphs treated. 434 W. C. ALLEE AND E. R. STEIN, JR. The average effect of all these reagents upon the resistance to cyanide, if such is worth anything, shows no marked difference between positive and negative treated animals nor between the treated animals and the control. From these experiments with Leptophlebia we have the inter- esting results that these nymphs were reversed without affecting their resistance to potassium cyanide (HCI and averaged results) ; with accompanying stimulation (alcohol) and with accompanying ’ depression (chloretone). The quantitative reversal of positive Epeorus nymphs and negative Leptophlebia by 1 per cent alcohol in the same ex- perimental dish at the same time was repeatedly demonstrated. Thus conditions absolutely identical caused opposite reversals in the two species. At first sight this would appear to mean that both positive and negative animals were reversed for the same reason. This is not necessarily true. In the tests to find the strength of potassium cyanide that would directly measure the metabolic rate of the two species it was found that Leptophlebia was only one-fourth as sensitive as Epeorus. Since the Lepto- phlebia are much more resistant, a strength of alcohol that only stimulated them may have clearly depressed Epeorus. That such is the true explanation is indicated by the effect of alcohol on the resistance of the two species of nymphs to cyanide. The Epeorus that had been made negative were found to be depressed, while the Leptophlebia that were made positive were clearly stimulated. II. REVERSALS OF PHOTOTAXIS AND CARBON DIOXIDE PRODUCTION?" METHODS The experiments upon which the second part of this report is based were carried on at Lake Forest College upon a May-fly 2 This section is based on experiments by the senior author, now being continued, which were started in the spring of 1916. They were made possible by money grants from the Elizabeth Thompson Fund and from the Bache Fund of the National Academy. 3 The nymph whose reactions are described in the sccond part of this paper is Heptagenia pulchella Walsh. We are indebted to Professor J. G. Needham for this identification. LIGHT REACTIONS—-METABOLISM—-MAY-FLY NYMPHS 435 nymph belonging to the Heptageninae. These nymphs are quite common in Pettibone Creek (Shelford, *13, maps) where they are usually found in the stones between riffles. Pettibone Creek is a brook about the same size as the Berkshire streams mentioned in the preceding part, but with much less rapid current. The nymphs were kept in the laboratory for long intervals during the winter in well aerated running-water aquaria. Ani- mals to be experimented on were transferred to room-tempera- ture aquaria aerated by means of an air-pressure pump operated by water pressure. The experiments were conducted as at Williamstown save that a daylight, concentrated filament, 100-watt Mazda (C2 of the General Electrical Company) was used as a source light. The experimental box was placed in a darkened room so that only light from the source lamp could enter during the experiment. In most of this work an assistant plotted the reactions of individual nymphs to light and manipulated their change to experimental solutions. Careful controls were run. When the nymphs were to be changed to a new experimental solution, the controls were changed in the same manner to fresh tap water. The assistant also prepared the pairs of nymphs for their carbon dioxide test in such a way that the experimenter had no idea which was the experimental and which the control nymph. The carbon dioxide production was determined in Tashiro’s biometer (Tashiro, 713, 717) as follows: The assistant placed two nymphs, whose rate of carbon dioxide production was to be com- pared, momentarily on filter paper and then transferred each to a shallow glass cell. The nymphs used were of the same size and were selected so that the experimental factor was the only known cause for varia- tion in their rate of carbon dioxide production. The containers were marked for future identification. These were handed to the experimenter and immediately placed in the apparatus. Within five minutes from their removal from the experimental dishes one could get an indication of their relative rate of carbon dioxide production. Under optimum conditions the entire process of determination could be repeated at the rate of three per hour. ‘This was much in excess of the usual rate. 436 W. C. ALLEE AND E. R. STEIN, JR. TABLE 4 9/1/16. 1:20 p.m. Put two lots of eight May-fly nymphs each in aquarium water in dark box. Light: 60-watt Mazda, 50 cm. distant. The nymphs had been in the laboratory two days. Temperature aquarium 21.5; of room 22. Lor 1 LOT 2 a= =| ar = 1230 sae 2 5 Be alle.32 1 1 Giln2 1 5 1: 48 1 Gh \ 2 6 5S 2 1 5 8 2:01 3 “Gy, || Al 1 6 2: 06 1 2 5 1 2 5 2:07 iL 1 1 5 1 1 6 2: 08 1 1 1 5 1 7 2:09 1 1 6 1 a 2:10 1 1 6 1 a PAS Ui 2 1 5 2 6 2:12 if 1 6 2 6 PAB M83 1 2 5 2 6 2:14 1 1 6 1 7 PAB U5) 2 6 1 7 2:18 * UF 1 i bs * Positive one has been positive throughout put in biometer 2:20 with a negative one that has been negative all the time. Washed with CO: free air 5 minutes. Positive nymph in left chamber, negative in right. Bubble 2: 25. First ppt. Rt. 2: 28. Much more left 2: 38. Took out biometer, 2: 40. Both active; put back in dark box in separate dishes. :00 Both reacting as before testing in biometer. 20:8 Do: :45 Do. lOR Do: :15 Replaced in biometer as before. 25 Bubble. :27 First left, positive. :40 More on left as before. :45 Out of apparatus. Reintroduced at negative end of separate dishes. :00 Nymph that had been positive throughout, still positive; other nega- tive as before. 6:00 Both negative. 7:40 Nymph negative throughout now positive, other negative. Light on all night. 8:00 a.m. Both negative. arth Pp PP Rw 1 WwW LIGHT REACTIONS—METABOLISM—MAY-FLY NYMPHS 437 Some idea of the nature of experimentation together with its effect on the nymphs may be gained from table 4. This table is a slightly expanded copy of a portion of the laboratory record for the day. It shows that during almost four hours of observation, in which time three biometer tests were made, the nymphs maintained their original light reaction and that each test showed the positive nymph had the higher rate of carbon dioxide production. : Whatever the faults of the method used, it at least has the merit of being always comparative. As was to be expected, large nymphs gave off carbon dioxide more rapidly than small ones and active nymphs more rapidly than inactive ones. These nymphs are strongly thigmotactic, and this usually caused them to remain quietly in their container. Occasionally one would move. Such determinations were of course thrown out. , PHOTOTAXIS AND CARBON DIOXIDE PRODUCTION IN UNTREATED NYMPHS These nymphs, like Leptophlebia, are usually negative to light. This normal reaction to light is graphically shown in chart 1 together with the preliminary and control records shown in charts 2, 3 and 4. About 20 per cent of the untreated nymphs were positive to light. When these were tested they were found to have a higher rate of carbon dioxide production than the negative nymphs. ices a Their N ratio, based on 332 non-selected control readings was 9.5 This is indicated by the graphs in columns 1 and 2 of chart 1 and the details are given in table 5. Exposure to light for considerable time sometimes caused negative nymphs to reverse their light reaction and become positive. Such animals were found to be more stimulated, as determined by the rate of carbon dioxide production, than simi- lar nymphs that had not been exposed to light (table 6). This is just the opposite to the result obtained by MacCurdy with nega- tive starfish which he found gave off less carbon dioxide when exposed to strong light. Chart 1 1 + 5/22/17 2 3 + 5/22/17 — + 5/19/17 4 5 6 ro w nw w UP & ca peyypwo supa - - - - oe a o on 2 o ° ~ ~ ~ Foidart A . TRUN N 6 ~ on o oe ” © © © rc) s 50' omitted = rs) 6 ry ir) Fe UT aU UCR LAL LL = = a a nn a rotebecrorbrercrboreboo bbb beer a i | a S a } 3 S > = = s 5 3S 3 8 S OT A EE EE EAA EE A AT (eT | Unt 8 TOO CC CN OCC I OC PC CC ey S TODO UO IO OIC OU RL LC is} 8 rey 8B B& & 3 8 8 More C05 ~ +) © n nm n 8 8 & 8 SO CC OCCT CL PCC UC CC WL UCC CC PO PL LL LL PW COp test before exposure 8 8 TO OY | 8 g & 8 8 8 g Heeb eee beet beoeebooebeecn mlitimelolotiitbebtharteidtibteet teeter beeen roel eee ether artreietererr tines ara 438 LIGHT REACTIONS—METABOLISM—MAY-FLY NYMPHS 439 TABLE 5 Showing the comparative rate of carbon dioxide production of positive and negative nymphs under control conditions (p. 525) SIZE DATE MORE COz + = mm. mm. 9/ 5/16 r-8.0 8.0 | Positive 9/ 1/16 6.0 6.5 | Positive 6.0 6.5 | Positive 9/ 2/16 525 6.0 Positive 5.5 5.5 | Positive 10/26/16 7.0 7.0 Positive 10/27/16 (a5 8.0 | Positive 10/28/17 tal 7.0 | Positive 7.0 7.0 | Negative. More active before test 8.0 8.0 | Negative. More active before test 11/ 2/16 6.5 7.0 | Positive RyAW BWA 11.5 12.5 | Positive utes 12.5 | Positive 3/13/17 IL) THEO Positive 0 11.0 | Positive 11.0 11.0 | Positive 11.0 11.0 Positive 11.0 11.0 | Positive. Not marked. Demonstrated 3/15/17 10.0 10.0 | Positive 3/16/17 10.0 10.0 Positive 3/20/17 11.0 11.0 | Positive 3/21/17 11.0 11.0 Positive 3/22/07 10.0 10.0 Positive Number tested 23 pairs. Positive more, 21. Negative more, 2. Chart 1 Showing graphically the reaction of six untreated nymphs to light from a 100-watt daylight Mazda (C2) placed 50 cm. from the experimental dishes. The charting was done by an assistant from whose records these graphs were copied. The scales show time in minutes. The left-hand side represents the positive end of the dish, i.e., the end toward the light. Where the line is approx- imately straight vertically the nymph was resting quietly. Curves and kinks show movement which did not markedly change the position in the dish in respect to light. Column 1 gives the reactions of a nymph that was predominantly positive to light, which after twenty-three minutes’ exposure gave more carbon dioxide in the biometer than the negative nymph whose reactions are recorded in column 2. Column 3 shows a nymph made positive by long exposure to light. In this case the reversal came suddenly. Column 4 gives the same result ob- tained by a different method. Perhaps column 3 represents a ‘tropic’ and column 4 a ‘trial’ reaction. Columns 5 and 6 show the reactions of two nymphs that were tested in the biometer before exposure to light. The animal with the higher rate of carbon dioxide production became positive. THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 26, NO. 3 440 W. C. ALLEE AND E. R. STEIN, JR. TABLE 6 Showing the effect of long exposure to light upon carbon dioxide production SIZE TEMPERATURE MINUTES MORE CARBON EXPOSED DIOXIDE Exposed Unexposed Exposed Unexposed mm. mm. deg C. degC. 13 13 21 19 120 Exposed 13 14 21 19 150 Exposed 10 10 23 21 204 Exposed 10 10 23 21 234 Exposed 10 10 15 13 52 Exposed EXPERIMENTS WITH HYDROCHLORIC ACID 1. Effect on negative nymphs As with the other species studied, hydrochloric acid was one of the most effective reagents in causing reversals. In one set of carefully controlled, fully plotted experiments there were a total of 220 control or preliminary readings lasting from fifteen to ninety-three minutes. Of the nymphs thus tested, thirty-seven were or became positive without other treatment than exposure to light. This is 17 per cent of the number tested. In this same series of experiments 125 nymphs that had been consistently negative through a preliminary testing period of at least fifteen minutes, were treated with N/25 hydrochloric acid. Under this treatment, seventy-five nymphs, 60 per cent, became positive. Of the fifty nymphs that did not reverse, twenty-five were kept under observation for less than twenty-five minutes and only two were kept until they died. If it had been the pur- pose of the experiments to ascertain how many nymphs could be reversed by the treatment, doubtless about 90 per cent would have become positive before death resulted. The typical effect of the acid upon the light reactions of these nymphs is shown in chart 2. The graphs show that the nymphs may reverse soon after being placed in the acid or the.reversal may come only after long exposure. Forty-eight per cent of the reversals came within fifteen minutes, but reversals occurred after seventy minutes’ treatment. Death frequently followed close upon the later type of reversals. LIGHT REACTIONS—METABOLISM—MAY-FLY NYMPHS 441 The effect of this treatment upon the carbon dioxide produc- tion is shown in table 7, which lists all the determinations, and in table 8, which partially analyzes the results listed in the pre- ceding table. The biometer tests show that the nymphs were stimulated when first put into the acid and that this period of stimulation lasted approximately fifteen minutes. The time limits varied with different individuals. After this period of stimulation the nymphs were depressed. This gives two periods in the carbon dioxide production corresponding to the two periods in the re- versals by this strength of the acid. The tables show that all nymphs were not tested immediately after reversal, but of those whose carbon dioxide production was found within two minutes after reversal and which had been stimulated by the acid the average time of treatment was 13.2 minutes. Thirteen of the twenty-five nymphs so tested had reversed within ten minutes after being placed in the hydrochloric acid. On the other hand, the average time of treatment of the animals similarly reversed and measured, but giving more car- bon dioxide in the control than in the acid, was twenty-seven minutes, and seven of the seventeen nymphs reversed after twenty-seven to thirty-seven minutes’ treatment. From these experiments it appears that either a marked increase or a decrease may accompany phototactic revérsals of these nymphs when treated with hydrochloric acid. 2. Effect on positive nymphs About 20 per cent of the Heptageninae tested were positive to light when first tested or became positive under the influence of exposure to light for a short time. This positive reaction is much less stable than the usual negative reaction. Of fifteen carefully plotted tests lasting from 15 to 114 minutes, seven, or 47 per cent, showed a change to the usual negative reaction without treatment. In nineteen tests lasting from 1 to 68 minutes seventeen of the nymphs, 89 per cent, were made negative by N/25 hydrochloric acid. Most of these became negative within the first five min- utes of treatment and, as was to be expected from the preceding 1 + Water Water o o - « re ~ ~~ - S 6s a ms > TN DR PULLS POLLY LOCOCO POC Changed to Changed u/25 HCl to water B Ss hontintimobiibenbtt ttt cite | POROUS VOL LOL BB fete Bee 3/25 HCl hee fas 8 5/26/17 Vvvrrelovssebertcetomtthicelere tition bedeeedoane B 8 8 ° 2 8 OU Water rrslrecetlictorobertotiiteteeebeebire Pein 2 6/15/17 6/15/17 — rteelinrber toda 3 4 + 5/18/17 4-5 /16/27 (3 4 3 o : 5 & = 3 O or s a mn % Ps aa os TOTO OC UCC CPO CN S 8 8 OJ CTU POCA POCO PCP Chart 2 5 6 + 5/ee/i7 —|+ 5/28/17 -— Water Water biilaie ~ - a) ~ rege & IANA ——$f\>-—_—_—_—— poecrdomevletttdieliebe eed! pitetlitereliseieborteebitnelioiecbaareedes o : S UO CCOT OCC CO TOU COO OCC RCTODCCR CACC C RI cUUDT CUOUON OCC CLT UCC CCUCTCCCUL jCCICCy MCC CIOL pCRCICICy POU CCRC WUCICIC PUR cI. CICLO PURICIRG TUGUICIGS NUON UT IMU UR CO CID i] 9 1/2 minutes omitted & S = a = Ss - i] [as 3S 8 S & Changed Changed to to water |¥/25 HCl Havant TION No 000000 0000) 0) OOo 0G On oo On oo Oo 8 2 Ss 8 aa £ 19‘omitted Less COp More C02 prrtiloliebiebeeleledbectd babble ebb ben bcd boeicbei prrtrdapeeeborseelorire teeters Less C02 ] wore C02 442 LIGHT REACTIONS—METABOLISM—MAY-FLY NYMPHS 443 experiments, they were found to be stimulated by the treatment (table 9). Two instances of the reversal of positive animals by acid treat- ment are given in the first two columns of chart 4, p. 450. In both instances shown the reversals are typical in that they are very marked and occur almost directly upon the start of acid treatment. In the second instance shown the experimental dish was twice turned end for end and each time the nymph moved directly negative. Here we have the same reagent making negative animals posi- tive by either stimulating or depressing them and making positive animals of the same species negative by stimulating them. 3. Negative vs. positive nymphs; both treated with HCl The treatment with hydrochloric acid did not cause all the nymphs to reverse their light reactions. When animals that had been made positive were compared with those still negative, it was found (table 10) that the former had a higher rate of carbon dioxide production when the time of exposure had been approxi- mately the same for both nymphs compared. Almost all the nymphs tested came from the preliminary period of stimulation. Theoretically, nymphs long exposed to acid and positive would give less carbon dioxide than those more recently placed in the acid and still negative. This possibility was not tested, since the results can easily be interpolated from the tests given in pre- vious tables. Chart 2 Showing graphically the light reactions of four nymphs treated with N/25 HCl and their carbon dioxide production as compared with that of their control nymphs. Column 1 shows two reversals with the acid treatment, the first of which occurred within three minutes. The reversals shown in column 1 and that in column 4 were accompanied by stimulation. On the other hand, the reversal shown in column 5, which came after twenty-three minutes’ exposure to the acid and which was allowed to remain positive for thirty-five minutes before testing, showed depression. The preliminary test in columns 5 and 6 is not charted, but was essentially like the first eight minutes shown. It will be noted that in this test the control became positive due to exposure to light and was therefore more stimulated than the ordinary negative control. Other tests show depression by treatment with acid for this length of time when com- pared with negative nymphs. 44-4 W. C. ALLEE AND E. R. STEIN, JR. TABLE 7 Showing the effect of N/25 hydrochloric acid upon carbon dioxide production. All the nymphs here listed were decidedly negative in their light reaction before being placed in the acid SIZE SIGN OF LIGHT REACTION f _ | eee) MORE GO: NACL IN pesclebi ce oa a HCl Control AND TEST 13 16 a = Acid 2 1 13 13 + + Acid 2 1 11 11 “+ — Acid 2 1 13 13 oe — Acid 3 1 13 13 + = Acid 5 2 13 13 + “= Acid 5 2 12 12 + _ Water 5 2 11 11 — — Water 6 1 10 10 + = Acid 6 1 10 10 + = Acid 6 1 1 11 + — Acid 6 1 12 12 a — Acid a 1 10 10 +- — Acid a iL 11 11 -- Acid 8 1 10 10 a ~ Acid 10 1 10 10 + - Water 10 7 11 Hal a - Acid 12 1 11 11 2 — Acid 12 3 Wy 2, + + Acid 12 1 13 13 + _ Water 1 1 13 13 a. -- Acid 13 2 13 13 — + Acid 14 2. 11 11 + _ Water 14 4 15 15 + = Water as Us 15 13 13 _ + Acid 16 1 14 14 a a Water 17 1 13 13 — -- Acid Ue 1 13 13 _ Water 17 1 13 13 - Water 18 1 13 13 + _ Acid 18 1 12 12 _ — Water 19 1 11 11 oe _ Water 20 12, 11 11 a _ Acid 21 0 12 2 + + Water 2D, 1 14 14 a5 — Acid 23 1 11 11 - = Acid 23 0 14 14 aa _ Water 28 1 13 13 + — Water 28 5 10 10 + + Water 28 a LIGHT REACTIONS—METABOLISM—MAY-FLY NYMPHS 445 TABLE 7—Concluded SIZE SIGN OF LIGHT REACTION MINUTES POSS esata ees ere + — HCl Control AND TEST 10 10 - — Water 29 0 11 11 + = Acid 30 5 ili 10) + - Water 31 6 13 13 + = Water 33 1 10 10 -- — Water 33 0 12 12 oe _ Water 33 1 12 12 + _ Water 35 1 12 12 a. — Water 37 1 15 13 bE — j Water 37 1 13 13 os _ Water 38 38 18} 13 + ~ Water 41 1 13 13 + _ Water 41 14 11 11 a _ Water 42 33 13 13 “bE _ Water 44 1 11 11 _ _ Acid 44 0 10 10 — = Acid 48 0 13 13 a _ Water 51 14 13 13 -- _ Water 58 3 13 13 + — Water 59 7 ial il ob _ Water 67 0 13 13 + + Water 70 30 51 10 + - Water 78 10 TABLE 8 Showing relative carbon dioxide production of experimental and control nymphs analyzed on the basis of length of exposure to hydrochloric acid. This table is based on table 7 MORE CARBON DIOXIDE MINUTES IN HCl HCl Water Part I. Showing all tests listed in table 7 1— 5 6-10 11-15 16-20 21-25 26-30 31-40 41-50 51-60 61-70 71-80 SOON OCOKF WN NO & Rew WEN eR OrW be TABLE 8—Concluded MORE CARBON DIOXIDE MINUTES IN HCl HCl | Water - Part Il. Showing results of the biometer tests made within two minutes after reversal 1— 5 6 1 6-10 7 1 11-15 5 i 16-20 2 3 21-25 3 1 26-30 1 2 31-40 0 5 41-50 1 2 67 0 1 TABLE 9 Showing the effect on carbon dioxide production of treating positive nymphs with N/25 hydrochloric acid until they became negative SIZE SIGN OF LIGHT REACTION MINUTES So ee soe | eee ee HCl Control HCl Control AND TEST 13 13 = _ Water 2 2 12 12 — -- Acid 3) 3 13 1133 = = Acid 3 2, 13 13 — — Acid 3 3 10 10 = =e Acid 3 2 12 12 _ _ Acid 4 1 13 13 — — Acid 4 4 14 14 _ + Acid 9 1 TABLE 10 Showing the relative carbon dioxide production of negative nymphs made posilive by treatment with N/25 hydrochloric acid, compared with those similarly treated but not reversed SIZE MINUTES IN HCl MINUTES | Hoc eee eee a ae fe = TEST 13 13 + 2 2 i 14 14 + 4 4 2 12 12 + 3 3 1 14 14 + 10 10 1 12 12 oa 13 13 13 14 13 — 14 14 3} 12 12 + 15 15 3 12 174 + 15 15 6 ati ial + 25 25 2 1 ili Both same 32 29 3 11 11 + 9 21 2 13 13 + 15 15 2 446 LIGHT REACTIONS—METABOLISM—MAY-FLY NYMPHS 447 EXPERIMENTS WITH POTASSIUM CYANIDE In the experiments with the negative Leptophlebia potassium cyanide had proved an effective reagent in causing reversals. With these Heptageninae at a concentration of N/500 it was almost as effective in causing reversals as hydrochloric acid. Of thirty tests run, 57 per cent were reversed by the cyanide while the controls showed no reversals at all. Biometer tests proved that the cyanide clearly depressed the nymphs. The details are listed in table 11 and specimen results are shown in chart 3. In all cases the nymphs were washed in water after treating with KCN in order to keep the potassium from in- terfering with the CO, determination. - In addition to the carbon dioxide tests, one nymph died while moving positive, another just after reaching the positive end. In all ten nymphs died in the experimental dishes as a result of the cyanide treatment, of these six had reversed their reaction to light shortly before dying. TABLE 11 Showing the effect of N/500 potassium cyanide upon nymphs negative to light and upon their rate of carbon dioxide production SIZE SIGN OF LIGHT REACTION i EWS SSE Sa a Pew co | eee | Seren KCN Water KCN Water AND TEST 13 13 op — Water 3) 2 12 12 a ~ Water 4.5 1 14 14 “= oa Water 6 3 13 13 + _ Water 7 2 12 12 - Water 8 2 13 13 — — Water 6 1 13 13 “fh — Water 9 3 13 13 + _ Water 14 4 13 13 —- oa Water 16 5 12 12 + _ Water 19 2 12 12 + — Water 41 1 pooloeeeeeeee heehee 7 no © o ~ o o > rey n ~ Ss cs) & a a B&B 8 8 S B bs = COUT PLLC) POCO PLLC LOCO LOL LL MIU PL VLU PLC LLL ULL LLG LBL LLU CLL CRUE CCL UCL UC 8 : & N 8 8 aL 2 +6/10/17 +6/10/17 — Water Changed to KCN Deal 15' later Water Changed to KCN Dead 8 8 s re S 8 & 8 8 3 & be] Es a = & S i} Ss © o =) a ao - we - DT CL PP DO OB UL VLU PL PLL LL 8 reboot ceecoebccrbcebeb be lererbecerebebodarebocerbirrebieeatinne Chart 3 3 + 6/10/17— Water Less CO> 448 4 5 6 6/10/17 — + 6/10/17 6/12/17 — KCN KCK ~ cy ry n o - - o oe o o pethidine ieboebeere bea Less COo 6/10/17 Less COp KCR 6/11/17 KCN @ o o © ) 5 im} i} - & - & 6/11/17 KCN a a = MAMMA AT TC A Te PLA VOUT LLL ITY CC CCC RCC COC PC CC CC POL & cI TPL LL I DO 3 13 a UTR CL Oe 2 SL eA LAL OL SUL OU CCE Ca Lc Died withi 5 minutes 6/12/17 6/10/17 KCN 8 KCN 8 3 2 ns n 8 & 8 8 Peevetlieberetisreeti beter teebceebrtrrrebeeedeecbceetiriefaccebireerlceleriebriebireelicrecel 8 8 mobo beibeb bbe Pa beelirebirgdiacatas More COp Hid LIGHT REACTIONS—METABOLISM—MAY-FLY NYMPHS 449 EXPERIMENTS WITH ALCOHOL AND STRYCHNINE Ethyl alcohol had less marked effect in causing reversals among these Heptageninae than with the species used in the first part of the work. In twenty-five well-studied cases, only five nymphs were by treatment with 2 per cent solution reversed. Of the fifty-two accompanying preliminary or control readings, one nymph became positive. When alcoholic nymphs were tested in the biometer (table 14), it was found that alcohol acts here according to its usual effect as a narcotic, first stimulating and later depressing. Some typical results obtained with the alcohol treatment are given in chart 4. . This shows two tracings of the reactions of nymphs that were not reversed by the treatment and of one that was, together with a type of control reaction which occurred frequently. Some preliminary experiments with strychnine showed a marked increase in the activity of the nymphs without a reversal of their light response. The animals usually remained at the negative end of the dish in almost continual motion. In the biometer, they usually showed a markedly increased carbon dioxide production. I am not able to state why these nymphs did not reverse their light reactions, but offer these results with alcohol and strychnine as evidence that all stimulation and depression do not cause reversals in phototaxis. Chart 3 Showing the light reactions of ten nymphs treated with potassium cyanide together with three preliminary test periods and one complete control. In the cases where the preliminary and-control graphs are not given it is under- stood that they are in no essential respect different from those shown. In order to economize space the time spent in changing from water to cyanide is not fully shown as it was in chart 2. Where such a change is indicated it must be understood that two or three minutes elapsed. In all cases given in columns 5 and 6 the control, with which the carbon dioxide production of the treated nymphs was compared, is not given in the chart. For text reference see p. 535. lorlopdiebrb eet te prelimbic beber i a o a - cay POCA POCO) PICO OL PL %& Ss = & " 8 UU PC Le PUPECP PETE dp Eee ep Peep eet Le Pa Chart 4 1 2 S S ; 6/1 / P70 ea ney T/T 4/28/17 4/27/17 Water Water Water Water 2) 2 ry os e ~ - a o o “4 oe © o i) s Ui) PAOLO PLAC. OW WC LO OL BEC HE Hacirbobi loeb beet I=) i-3 PLD eS UATE APES UARATYARTOTRSETE APU USAT RIE TAP 6B - ic) Ca e & Faoloebobd bebe baeebcdo died = = a a Changsd to Changea N/25 HCL to water i & o 3 & » & Changed More COg Less CO2 to Alc .2% wy oS rm Ss Water Water B'omitted 18‘omitted 6/12/17 6/12/17 8 Hlavrtlercerbiveielosioebiria bien Cha ngea to Alc .2% 8 & A ICC AOL Hlttt UCT PO) PL PLL PO WA 8 g & B £ 8 8 F UIUTUTICN PULL PGC RL) POLCOLOC CELA. COL OC PO LO HW 8 8 HC] N/25 Reversed Reversed Less polo beeeeebee ie =More COp More CO5 = than controlj|/ than control- seecrlitrotetrretimtebel tebe beree deeb lary 15' 5 4/20/17 Alcohol omitted Less C02 More C02 aie 4/20/17 Water 15" omitted 8 ree 8 ry 8 B 8 8 s 3 8 5 & = & 8 i=} 5 ° o a o a . s 1 a BUUUUL TRU UU UL OL PC CI COL OC OC CC CO CP BC scietdoreettopeibisstiedietititicrbtdabiebebrcticcbobec ebb LIGHT REACTIONS—-METABOLISM—MAY-FLY NYMPHS 4051 TABLE 12 Showing the effect of 2 per cent ethyl alcohol upon the phototactic reaction of nymphs negative to light and upon their carbon dioxide production SIZE SIGN OF LIGHT REACTION MORE CO2 ope ey Alcohol Water Alcohol Water 10.5 10.5 ss aE Alcohol 7 8.5 8.5 =F ae Alcohol 8 12.0 12.0 — _ Alcohol 9 9.0 9.0 - = Alcohol 9 11.0 11.0 — — Water gl 9.0 9.0 _ _ Water gl 8.0 8.0 ~ = Alcohol 10 Les ilo ok aa Alcohol 10 10.0 10.0 a + Water 10! 10.5 10.5 — _ Water 10 Ay 11.0 11.0 f+ a Alcohol 11 9.0 9.0 — — Alcohol 12 11.0 11.0 — Alcohol Ue 11.0 11.0 — — Alcohol 13 9.0 9.0 _ _ Alcohol 16 10.5 10.5 + — Alcohol 28 10.0 10.0 — Water 44 TL 1S — = Water 50 8.5 8.5 _ + Water 50 10.5 10.5 - — Alcohol 52 11.0 11.0 = Water 55 10.0 10.0 _ _ Water 83 9.0 9.0 _ - Water 180 1 These control nymphs were more active than the experimental ones before the biometer test was made. Chart 4 Showing the reactions of two positive nymphs to light after treat- ment with N/25 hydrochloric acid together with their controls and the result of treating three nymphs with 2 per cent ethyl alcohol with one control tracing. The control shown in column 6 remained quietly at the negative end during almost all of its exposure and yet gave more carbon dioxide than the more active alcohol treated nymph whose response is shown in column 5. The nymph shown at the bottom of column 1 became negative immediately upon treatment with the acid. The experimental dish was then twice turned end for end each time the nymph moved directly away from the light. 452 W. C. ALLEE AND E. R. STEIN, JR. DISCUSSION Since this is an inquiry into the problem of a possible relation- ship between the sign of the phototactic reaction and the general rate of metabolism of May-fly nymphs, it is pertinent to question the effectiveness of our means of measuring the rate of the meta- bolic processes. The cyanide-resistance method is best checked by comparing results obtained with it with those given by Ta- shiro’s method of determining carbon dioxide production. This has been done for the isopod Asellus communis (Allee and Tashiro, 14). There it was found that the two methods gave the same results in direct tests and that two isopods subjected to daily variations of oxygen tension for ten successive days with daily quantitative estimations of carbon dioxide production by Dr. Tashiro behaved according to expectation based upon previous experience with the cyanide method. Regarding the work with Tashiro’s biometer in measuring the carbon dioxide production, I consider this the best, though by no means the fastest, method of making such comparative car- bon dioxide tests as those recorded in this paper. It is less complicated than the electrolytic determination of the hydrogen ion concentration and more accurate than the colorimetric meth- ods. The instrument is somewhat complicated in appearance, but it is in reality as simple in manipulation as a modern micro- scope equipped with oil-immersion lens, mechanical stage, and camera lucida. I have repeatedly demonstrated end points to coworkers at Woods Hole and even to college freshmen. My only change from Tashiro’s technique (’17, p. 109) consists in the use of a low-power binocular in reading end points. The sources of error in the method as applied to May-fly nymphs are: 1, May-fly nymphs are water-dwelling animals and were tested in as nearly a dry atmosphere as possible. 2. Five minutes or more intervened between the time the nymphs were taken from the water and the first indication of the relative rate of carbon dioxide production. During this interval the nymphs must be picked up, partially dried, and placed in glass containers. LIGHT REACTIONS—METABOLISM—MAY-FLY NYMPHS 453 Regarding these points it must be borne in mind that the readings here given are all comparative ones in which the control and experimental nymphs were treated exactly alike. Since one must needs be taken from the water before the other, this was varied in the different experiments. The nymphs often lived twenty-four hours in the apparatus and at times they lived as long as forty-eight hours in the slight amount of moisture present. 3 Difference in carbon dioxide production may be due to difference in movement. This source of error was eliminated by the simple method of throwing out all tests where movement occurred. 4. Unconscious personal preference for one of the nymphs producing more carbon dioxide. This was eliminated by the experimenter’s ignorance of which was experiment and which was control. The relationship between the general metabolic processes of animals and their reaction to light may conceivably be one of the following: 1. Conditions that depress positive animals may make them negative (Mast, Bohn, ‘Drzewina) and conditions that stimulate negative animals may make them positive (Holmes, Carpenter, Bohn, Jackson). 2. The above relationship may be reversed (Phipps in part). 3. Conditions that stimulate animals may cause reversals of their normal reaction or vice versa. 4. Conditions that markedly change the metabolic rate may cause reversal of either positive or negative animals. 5. Changes of metabolism accompanying changes in light reac- tions may be incidental or resultant rather than causal. 6. The relation between metabolic processes and the reaction to light may vary in different species so that no general law can be worked out. 7. There may be no relationship between the rate of metabol- ism and the phototactie reaction. With the positive Epeorus nymphs only depressing agents were used and these caused reversals. With both the negative 454 W. C. ALLEE AND E. R. STEIN, JR. species both stimulation and depression resulted in reversal and in positive members of the Lake Forest Heptageninae stim- ulation of positive nymphs caused reversal. In Leptophlebia thirty-six tests with hydrochloric acid shewed no effect on the average resistance to potassium cyanide. The biometer tests with the Heptageninae give the explanation. When first sub- jected to the acid the nymphs are stimulated; later they are de- pressed. If taken during the first period the resistance to cya- nide would offset that of the later period and so yield an average about the same as the control. An examination of the carbon dioxide production records shows that about as many nymphs were stimulated as were depressed by the treatment, so that a general average not considering the time factor would show re- versals with no relation to the rate of carbon dioxide production. In the May-fly nymphs studied the results obtained demon- strate that there is a relationship between the rate of metabolic processes and the sign of the phototactic reaction. It is also clear that reversals are accompanied by either a marked stimula- tion or marked depression. The experiments indicated but do not prove that the stimulation or depression is causal. From one point of view it makes little difference whether the metabolic changes are causal or only symptomatic; the fact that they are correlated at all is important. If the change in metabolic conditions is causal, the fact is evi- dent that all changes do not cause reversals. This was shown particularly by the action of the ethyl alcohol upon the Like Forest nymphs. This stimulated and later epressed the nymphs with or without an accompanying reversal. On the assumption that metabolic change is causal the non-action of alcohol in 80 per cent of the cases might be explained by supposing that it did not cause a change quantitatively large enough. This suggestion is supported by the observation with the other species that alcohol accompanied by decrease in tem- perature ws more effective in producing reversals than alcohol alone and by the fact that in general these species were more susceptible. - LIGHT REACTIONS—-METABOLISM—MAY-FLY NYMPHS 455 The idea that a certain quantitative change must occur before reversal in light reaction takes place is also supported by carbon dioxide determinations made before the nymphs were exposed to light. In some of these, as, for example, the results shown in columns 5 and 6 of chart 1, the nymph with the higher speed of carbon dioxide production became positive upon exposure to light while the other was decidedly negative. It more frequently happened that both nymphs so treated were negative in spite of the fact that one had a higher rate of metabolism than the other. Evidently the biometer is more sensitive to changes in carbon dioxide production than is the :ight reaction. Strychnine, again, did not cause a high degree of reversals, but it did strongly stimulate the nymphs. ‘There is no question but that this stimulation is as strong as that caused by hydro- chloric acid which caused a high percentage of reversals. This difference in result can only be explained by the assumption that while light reversals are accompanied by changes in metabolism that are probably causal, all such changes do not cause reversals in reaction to light. The untreated positive nymphs were found to give off more carbon diox'de than untreated negative ones. These negative animals often moved back and forth in the dishes, spending the major part of the time at the negative end. Some nymphs do this more than others. This brings them to the positive end more frequently and gives them more opportunities to come to rest there. A reversal apparently on this plan is shown in column 4, chart 1. It appears that this is one mechanism for reversal to light and here the relation between metabolic rate and the reversal is obvious. The higher the metabolic rate, the greater the tendency to move back and forth; the more random move- ments, the greater the chance of becoming acclimated to the positive end and of reversing the normal reaction. All eversals even under the influence of light alone were not of this type, witness column 3, chart 1. This reversal, like most of those experimentally obtained by the use of chemicals, was not preceded by random movements, but took place as though the anima! had suddenly discovered the attractiveness of the positive end and must needs go there even though it died in the attempt THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 26, No. 3 456 W. C. ALLEE AND E. R. STEIN, JR. (as in cyanide reversals). This excursion was often the first one the nymph had made to the positive end and frequently the nymphs remained there until they died. The reason for ‘he rela- tion of metabolic rate with this type of reversal is not evident, especially when the reversal followed a very strong depression. An explanation of reversals sometimes advanced (Ewald, ’13) is that animals change in their sensitivity to light and hence reverse their reactions. On this basis the change in sensitivity is causal; the light reversal and change in carbon dioxide produc- tion, resultants. In May fly nymphs these supposed resultants are correlated in such a way that either stimulation or depression may accompany reversals from negative to positive light reac- tions. ‘This means that either increasing or decreasing sensi- tivity to light will make negative May-fly nymphs positive, or that an increase (or decrease) in sensitivity wil. cause now de- pression and now stimulation. Either of these necessary assump- tions expand the sensitivity hypothesis beyond the limits justified by known facts. CONCLUSIONS The light reactions of the positive May-fly nymph, Epeorus, was reversed by treatment with alcohol, lowered temperature, calcium chloride, and other reagents. Nymphs so reversed had a lower rate of metabolism, as measured by resistance to potas- sium cyanide, than normal untreated nymphs. The negative nymph, Leptophlebia, was similarly reversed with accompanying stimulation or depression as measured by resistance to the cyanide. A negative nymph belonging to the Heptageninae was reversed in its light reactions with accompanying increase or decrease in carbon dioxide production as measured by Tashiro’s biometer. The experiments conclusively demonstrate that the photo- tactic reaction of these nymphs is correlated with the metabolic condition and indicate, but do not prove, that certain changes in metabolism cause the reversals in reaction to light. All nymphs that reversed their light reactions we e ether stimulated or depressed, but stimulation or depression did not necessarily involve phototactic reversal. LIGHT REACTIONS—-METABOLISM—MAY-FLY NYMPHS 457 LITERATURE CITED Auten, W. C. 1914 Certain relations between rheotaxis and resistance to po- tassium cyanide in Isopoda. Jour. Exp. Zodl., 16, pp. 397-412. Auer, W. C., anp Tasurro, Surro 1914 Some relations between rheotaxis and the rate of carbon dioxide production in Isopods. Jour. An. Beh., 4, pp. 202-214. Boun, Greorces 1912 Les variations de la sensibilité en relation avec les variations de l’Etat Chimique Interne. C. R. Acad. Sci. Paris, 154, . pp. 388-391. Carpenter, F. W. 1905 Reactions of the pomace fly Drosophila ampelophila Loew to light gravity and mechanical stimulation. Am. Nat., 39, pp. 157-171. Cuitp,C.M. 1913 Studies on the cynamics 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. Zodl., 14, pp. 153-206. 1913. Certain dynamic factors in experimental reproduction and their significance for problems of reproduction and development. Arch. Entw. Mech., 35, 8. 598-641. Drzewina, ANNA 1911 Sur la résistance des crustacés au cyanure et les effets sensibilisateurs de cette substance. C.R. Soc. Biol., 70, pp. 855-857. Dr7ewina, ANNA, ET Bonn, GeorGEs 1912 Modifications des réactions des animaux sous l’influence du cyanure de potassium. Ibid., 70, pp. 855-857. Ewautp, Wotraana F. On artificial modification of light reactions and the influence of electrolytes on phototaxis. Jour. Exp. Zodl., 13, pp. 591- 612. Gerrert, J. 1899 Uber das Wesen der Blausiure-vergiftung. Zeitschr. klin. Med., 15, 8. 208-307. Homes, S. H. 1905 The reactions of Ranatra to light. Jour. Comp. Neur., 15, pp. 98-112. Hyman, L.H. 1916 On the action of certain substances on oxygen consumption. ‘I. The action of potassium cyanide. Am. Jour. Physiol., 40, pp. 238- 248. Jackson, H. H. T. 1910 Control of phototactic reactions in Hyalella by chem- icals. Jour. Comp. Neur. and Psych., 20, pp. 259-263. Lorn, J. 1904 The control of the heliotropie reactions in fresh-water crus- taceans by chemicals. Univ. of Calif. Publ. in Physiol., 2, pp. 1-3. Logs, J. 1912 Comparative physiology of the brain and comparative psy- chology. MacCurpy, Hansrorp 1913 Some effects of sunlight on the starfish. Sci., N. S., 38, pp. 98-100. Mast, 8. O. 1911 Light and the behavior of organisms. Wiley, 410 pp. Purpps, C. F. 1915 An experimental study of the behavior of Amphipods with respect to light intensity, direction of rays and metabolism. Biol. Bull., 28, pp. 210-222. 458 W. C. ALLEE AND E. R. STEIN, JR. SuHetrorp, V. E. 1913 Animal communities in temperate America. Chicago, 362 pp. TasHiro, SHtro 1913 A new method and apparatus for the estimation of exceedingly minute quantities of carbon dioxide. Amer. Jour. Physiol., 32, pp. 137-145. 1917 A chemical sign of life. Univ. of Chicago Press, 142 pp. WopsEDALEK, J. E. 1911 Phototactic reactions and their reversal in May- fly nymphs. Biol. Bull., 21, pp. 265-271. AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, JUNE 24 STUDIES ON THE PHYSIOLOGICAL SIGNIFICANCE OF CERTAIN PRECIPITATES FROM THE EGG SECRETIONS OF ARBACIA AND ASTERIAS ALVALYN E. WOODWARD Simmons College, Boston, Massachusetts TWO CHARTS AND THREE FIGURES CONTENTS LOS IGRI Tore TCL KO) spe eae Paice Jee Pon Bane Wine Aly 7A 7 a ee 459 II. Concerning the factual basis of the fertilizin theory................... 461 A. Concerning egg secretions in general........................0000- 462 B. Concerning the secretions of Asterias and Arbacia in particular.. 464 iy ihysiologicaleanal ysis ie yt cpt ee, | peu cee at na thos ocr: 464 2 General chemicals properticsacwess: vse fo. eso caine sank: 471 3. Precipitation of specific elements from the egg secretions andathei properties: ee ek ote Oss SOD ln IO 475 C. Concerning inhibitors............. BEES ES METS es eS DRM AS ROTA Meas 479 HLCInOV aL Ob ferguuiziher 26 eee wee Wie st WL tre gle la Bie 479 2. Combination with ‘sperm receptors’.....................--- 479 3. Combination with the ‘spermophile group’ (agglutinin) of FOUL UZ ya Leas APE, SNPS RS REY ORLA ST RGERSe s Eatee yh begs 480 4. Combination with the ‘ovophile group’ (lipolysin) of fertilizin 480 5: Combination withi ‘epg Treceptorss.... 0. 2s: bee ec se eos 482 G:-Unclassitied’ inhibitors:,..- ea ee. ee ee. eee 484 III. Concerning activators—particularly lipolysin—and theories of activa- UO ee ae eit, cls. MAS 5 coopster aie 484 YUM CLAIM ACELY ATION: 05h)... sie ape hate | tae thes oy eee 485 BaP Accivaiioneby SDGrM, ... 0/46) 0} 50 1 Estimated. photographs). The others were unaffected. When, late in the season, Asterias eggs were again resistant, this experiment was repeated. Out of ten batches of eggs treated, every one attracted sperm decidedly better than the control. 490 ALVALYN E. WOODWARD Since the inhibitor present in the egg is a compound of an unsaturated fatty acid, iodine should saturate the acid and enable the egg to develop. The validity of this reasoning is shown by experiments in which Miss Hague and the writer (’17) used iodine as a parthenogenetic agent with Arbacia eggs. The curves in fig. 2 and the data in table 16 summarize the results by TABLE 15 The effect of salt solutions on resistant Asterias eggs. The amount of reagent indi- cated in the first column was added to 100 cc. sea-water. The eggs were treated with these solutions for twenty minutes, washed, and inseminated PERCENTAGE OF CLEAVAGES A B Cc D Contre! Insemi- Control Insemi- | Insemi- | Insemi- nated nated nated nated Untreated controls........ 0 4 1 11 5 25 Calcium chloride 4 ec. 2.5 molecular..... 20 24 22 2 ce. 2.5 molecular..... 10 38 15 1 ce. 2.5 molecular..... il i 18 5 ec. 2.5 molecular..... 1 7 9 Lithium chloride fl (Os IOI Ee Maas oes 6 36 16 ATcemnmormals aac. ee 3 53 19 D Ges woman sea aao ee 1 9 31 I (on TeVereONeS eos ee 1 8 25 Potassium iodide AZCEMMOTIM AMEE: seer e 3 48 26 WO (os INOMITEN a Rae ee Boe iL 47 23 il Ges WON oe soosoeue it 10 25 ODiecenormalene. ae 1 10 15 Note.—The addition of very dilute KCNS (about 1 ce. of al per cent solution to 100 cc. of sea-water) likewise produced a great increase in the number of eggs fertilized. giving the average number of cleavages obtained by each method of treatment. The fact that the length of time during which the eggs are subjected to iodine does not affect the number of cleav- ages, indicates that the reagent not only enters the egg imme- diately, but also affects it immediately to its fullest extent. This strengthens the idea that iodine accomplishes its result by combining with the unsaturated fatty inhibitor. The membranes 491 EGG SECRETIONS OF ARBACIA AND ASTERIAS “OUIPOT 0} posodxo o10M S830 oY} YOryA Sutanp soynurur jo zoqunu oY} O}BOIPUT S9AIND 9y4 uo SioquInU oY, “Juese o1oUesoUNy AIvd vfse sUIPO! Sursn Aq pourv4qo sosuvyo jo osvyuoo10d oy} SUIMOYS SOAIND Z ‘BIyq LOY IYOS AUPOl JO YfOllb A g 7 YY ty wy S q ODNDY 2 ) 0 Ot I40-4 N oe 492 ALVALYN E. WOODWARD formed on these eggs were remarkable in resembling the mem- branes on eggs fertilized with sperm more closely than those on the usual parthenogenetic eggs—another reason for the belief that iodine reacts with the normal inhibitor and so permits the normal activator to function. | - Arbacia eggs, like those of Asterias, become resistant to ferti- lization toward the close of the breeding season. Their fertility may likewise be increased by allowing them to stand in dilute iodine solution ten minutes before insemination. It should be noted that iodine treatment, like any other form of ‘doctoring,’ TABLE 16 The parthenogenetic effect of iodine ciorremen| 12% | ASCE | APR oe Sean eee a SOT RTON ene IODINE IODINE IODINE IODINE IODINE . SOLUTION | SOLUTION | SOLUTION | SOLUTION | SOLUTION |SOLUTION ae} £ s & s iS 2 £ 5 B B 5 5 5 5 B Q jor o a Q Q Q An. > a a> > > > > > eo) ee a0) iso) q q q jen) =F a= ae =e aR ee ar ae Mea minutes 225 2 | SGN, PAE |p AL We BO e aS) 4s} 9 | il Sills aa hs 6 5.0 1 Hh |\ 118) G) PRP A Bere a Wakes: 6 8 3 5 7.5 7 4 | 18 | 19 | 29 | 27 | 26 | 28 (ay | 5) 2 3 10.0 3 3 Ge 2A 25 a ZO ne Om le en, 4 | 13 3 6 PAN) |) 7 53 Oy EE whey NEO) | malay |) PAS) Mos). a3 3 4 15.0 Py Notas WMC We Pass |i sy ||, 228s |) TKN) 1 2 5 3 5 20.0 21 | 24 9 TON TRIM AE |) HEN Gs) 2 § 4 3 25.0 | 15 | 20 8 | 16 7 9 does not improve eggs that are already fairly normal, like those in the last column of table 17. In this connection it is interesting to recall the experiment of E. P. Lyon and L. F. Shackell (10). They found that iodine, as indicated by the starch reaction, disappeared more rapidly from sea-water in the presence of unfertilized eggs than of eggs which had been fertilized. From this they concluded that the unfertilized eggs are more permeable to iodine than the fertilized. In the light of my own experiments, it seems more probable that the unfertilized eggs contained more unsaturated fathy acid with which the iodine might combine. EGG SECRETIONS OF ARBACIA AND ASTERIAS 493 On the other hand, if the eggs be allowed to settle in test-tubes and only the supernatant fluid be tested, it will be found that the liquid above the unfertilized eggs combines less readily with io- dine than does that above the fertilized eggs. The first inference from this was that the fertilized eggs had excreted unsaturated fatty acid into the sea-water. If, however, we add to the water poured off from unfertilized eggs an amount of sperm suspension equal to that present in the other tube, we find that iodine is ab- sorbed in equal amounts from each. ‘Therefore, the results were due to the presence of sperm in the water above fertilized eggs, rather than to unsaturated acid excreted at the time of fertilization. TABLE 17 The effect of iodine on resistant or overripe Arbacia eggs PER CENT (eee lOkce wer es. sperm sCOntnO lent t) are ee ote oot ae ee 24 29 81 2. 10 cc. eggs +1 drop saturated iodine 10 minutes + SPE e ere ees eee nee ahi ats Sle tap olals peas RS oe 28 39 40 3. 10 cc. eggs + 2 drops } saturated iodine 10 minutes + SP GSTUD SE o/s sicoy de easyer cwanaa nee ache SERN CO EEN Fs een Shs 0 36 44 62 4. 10 cc. eggs + 1 drop } saturated iodine 10 minutes + | SETS S Atak Pe Oe ed EN Ue genet Apet he 50 48 | 61 5. 10 ce. eggs + 1 drop } saturated iodine 10 minutes + S] Oo) tO Caen Bacercicht es EY IOC Ree OM oo KES Oe Soe 31 Neither Asterias nor Arbacia lipolysin seems to combine with iodine. The chemical parthenogenetic agents, however, react with the fatty inhibitor, and, as we saw, the egg secretion, one of the three physiological activators, also combines with it to produce a compound soluble in water. According to this view, then, the resting egg contains enzymes which control metabolism, unsaturated fatty acid which inhibits enzyme action, and lipolysin, which reacts with the unsaturated fatty acid to make it innocuous. We can conceive that the rela- tive amounts of these substances may change from time to time in the same egg and may differ in different eggs. If there is more than sufficient fatty acid present to combine with all the lipolysin, the surplus will inhibit the action of the enzymes and the egg 494 ALVALYN E. WOODWARD will lie dormant. As the egg ‘ripens,’ however, it secretes more and more lipolysin. If this accumulates within the egg to such an extent that the inhibitor is relatively greatly reduced, the metabolic enzymes become extremely active and development, or cytolysis results. Perhaps natural parthenogenesis, such as occurs in Daphnia and aphids is the result of such conditions. The membrane of starfish and sea-urchin eggs is easily perme- able to lipolysin, so that it does not normally accumulate within the egg. If, however, the eggs are crowded in a small amount of sea-water or, which amounts to the same thing, are placed in a bath of lipolysin, the loss of this substance from the egg is pre- vented. It therefore reacts w th the inhibitor, and the egg de- velops—a case of autoparthenogenesis. Any other method which disturbs the ratio of activating enzymes to fatty acid so that the proportion or effectiveness of the enzymes is increased must lead to development or cytolysis. As already stated, many chemicals have this effect, and produce parthenogenesis. B. Activation by sperm Can the action of the spermatozo6n be explained along similar lines? Its effect probably varies with the group of animals. Ostwald (’07) measured the amount of peroxidase and catalase in 1) extracts of Amphibian eggs; 2) extracts of sperm; 3) a mixture of the two extracts. He found that the mixture contained con- siderably more enzyme than the sum of (1) and (2). Hence we may conclude that in Amphibia the sperm carries into the egg something which changes pro-enzymes into enzymes. It in- creases the activator. On the other hand, Amberg and Winter- nitz (11) were unable to find any more oxidase or catalase in echinoderm eggs laked after fertilization than in those laked be- fore. Moreover, Warburg (’08) and Loeb and Wasteneys (’13) found the same rate of oxidation in parthenogenetic as in fertilized sea-urchin eggs. S nee, in this group, fertilization does not in- crease the amount of oxidase in the egg, the spermatozoon may act by reducing the amount or effect of the inhibitor. EGG SECRETIONS OF ARBACIA AND ASTERIAS 495 In conclusion, I wish to express my gratitude to Dr. O. C. Glaser, who suggested this problem and directed the work; to Dr. F. R. Lillie for the privileges of the Marine Biological Lab- oratory, Woods Hole, and to the several colleagues who kindly allowed me to demonstrate to them various reactions. IV. SUMMARY In confirmation of the work of Lillie and Glaser, it was found that Asterias and Arbacia eggs secrete into the supernatant sea- water a substance which causes the sperm of the same species to be activated, aggregated, reversibly agglutinated, and paralyzed. The secretion is also a parthenogenetic agent. Further study of its physiological properties brought out: 1. That its presence is necessary for the fertilization of the egg, since, a. Immature eggs of Asterias, which cannot be fertilized, pro- duce a secretion with less than one-sixtieth the agglutinating power of that produced by the same eggs when mature. b. Eggs from which the secretion has been washed do not de- velop when inseminated. If, however, secretion be added before insemination, they develop. c. Arbacia eggs which are ‘resistant’ to fertilization late in the season, also produce little secretion. They fertilize normally if secretion is added. 2. That the secretion has a dual nature, as shown by the fol- lowing facts: a. It reacts with both the sperm and the egg. b. Boiling destroys its value as a parthenogenetic agent, but not as an agglutinin. ; c. Perivisceral fluid of the same species inhibits autopartheno- genesis, but not agglutination. A qualitative study of the chemical properties of the secre- tion confirmed Glaser’s observations, and indicated: 1. That it does not dialyse through a collodion sac, and so is probably colloidal. 2. That it contains carbon and nitrogen, but gives no clear response to protein tests. It gives a faint yellow color, in the 496 ALVALYN E. WOODWARD xanthoproteic test, however, which indicates the presence of tyrosine, phenylalanine, or tryptophane. 3. That two substances can be precipitated from the same secretion : a. By saturation with (NH;).S8O,, a sperm agglutinin is thrown down. b. By a method of Robertson and others, using BaCl. and acetone, a parthenogenetic agent is obtained. In an attempt to learn whether or not enzymes are present, it was found that the agglutinin resembles an enzyme in the effect upon it of x-radiation. It also follows the law of Schiitz and Borissow, i.e., rate of action = K+/C ferment. The secre- tion does not give a positive reaction to tests for oxidase, cata- lase, or a proteolytic enzyme. Since the parthenogenetic agent dissolves a fat obtained from the eggs, it may contain a lipase. Hence it was called, provisionally, a lipolysin. A study of methods of inhibiting the initiation of cleavage brought out that both autoparthenogenesis and sperm fertiliza- tion are inhibited by: 1. Asterias and Arbacia serum, which may, like mammalian sera contain antiferments specifically protecting the cells of the organism itself. 2. ‘Purple x’ and ‘Salmon x,’ substances obtained by boiling the testicular or ovarian tissues of Arbacia and Asterias. 3. Antifertilizin, obtained from eggs which have been freed from fertilizin. This inhibitor may be identical with 4. An unsaturated fatty acid obtained by extracting the eggs with ether. Parthenogenetic agents fall’ into three classes: 1) The fat solvents, including lipolysin, ether, chloroform, butyric acid, ete. 2) The halogen compounds—iodine and various salts. 3) Physi- cal methods, which may change the physical or quantitative relations of substances within the egg. Not only do these agents produce development in normal, uninseminated eggs, but they change resistant Asterias and Arbacia eggs so that they may be fertilized. EGG SECRETIONS OF ARBACIA AND ASTERIAS 497 A consideration.of the above facts makes it seem probable that the factors tending to produce development in the resting egg are of the nature of enzymes. The action of these, Jobling found, may be inhibited by unsaturated fatty acids. The egg remains in the resting stage so long as the action of these enzymes is inhibited by the unsaturated fatty acid. The egg itself, when mature and in a suitable medium, produces a lipolysin which binds this inhibitor. The efficiency of the inhibitor may also be lowered by physical and chemical means. In some groups, the spermatozoon appears to bind the inhibitor, in others, to increase the activity of the enzymes. V. LITERATURE CITED AmBpERG, S., AND M.C. Wrnternitz 1911 The catalase of sea-urchin eggs before and after fertilization, with especial reference to the relation of cata- lase to oxidation in general. Jour. Biol. Chem., vol. 10, pp. 295-302. Euter, H. 1912 General chemistry of the enzymes. J. Wiley & Sons, New York. Fucus, H. M. 1914 Studies in the physiology of fertilization. II. The action of egg secretions on the fertilizing power of sperm. Archiv f. Entw’- mech. d. Organismen, Bd. 40, pp. 205-252. Guaser, O. C. 1913 On inducing development in the sea-urchin (Arbacia punctulata), together with considerations on the initiatory effect of fertilization. Science, N.S., vol. 38, pp. 446-450. 1914a The change in volume of Arbacia and Asterias eggs at fertiliza- tion. Biol. Bull., vol. 26, pp. 84-91. 1914b A qualitative analysis of the egg-secretions and extracts of Arbacia and Asterias. Biol. Bull., vol. 26, pp. 367-386. 1914c On auto-parthenogenesis in Arbacia and Asterias. Biol. Bull., vol. 26, pp. 387-409. Gop.Lewskl1, E. 1911 Studien iiber die Entwicklungserregung. II. Antagon- ismus der Einwirkung des Spermas von verscheidenen Tierklassen. Archiv f. Entw’mech. der Organizmen, Bd. 33, pp. 233-254. JoBLinG, J. W., AnD W. PererseN. 1914a Soaps as ferment-inhibiting agents. Jour. Exp. Med., vol. 19, pp. 239-250. 1914b Nature of serum anti-trypsin. Jour. Exp. Med., vol. 19, pp. 459-478. Just, E. E. 1915 a Initiation of development in Nereis. Biol. Bull., vol. 28, pp. 1-17. 1915 b An experimental analysis of fertilization in Platynereis mega- lops. Biol. Bull., vol. 28, pp. 93-114. 498 ALVALYN E. WOODWARD Lituin, F. R. 1912 The production of sperm iso-agglutinins by ova. Science, N.S., vol. 36, pp. 527-530. 1913 a Studies of fertilization. V. The behavior of the spermatozoa of Nereis and Arbacia with special reference to egg-extractives. Jour. Exp. Zool., vol. 14, pp. 515-574. 1913 b The mechanism of fertilization. Science, vol. 38, pp. 524-528. 1914 Studies of fertilization. VI. The mechanism of fertilization in Arbacia. Jour. Exp. Zool., vol. 16, pp. 523-590. 1915 Sperm agglutination and fertilization. Biol. Bull., vol. 28, pp. 18-33. Lituie, R. 8S. 1911 The physiology of cell division. Am. Jour. Physiol., vol. 27, pp. 289-357. 1912 Certain means by which starfish eggs naturally resistant to fertilization may be rendered normal, and the physiological conditions of thisaction. Biol. Bull., vol. 22, pp. 328-346. Logs, J. 1910 The rdéle of alkali in the development of the sea-urchin. Proc. Soc. Exper. Biol. and Med., vol. 7, pp. 119-120. 1912 The mechanistic conception of life. Univ. of Chicago Press. 1913 Artificial parthenogenesis and fertilization. Univ. of Chicago Press. 1914 Cluster formation of spermatozoa caused by specific substances from eggs. Jour. Exp. Zool., vol. 17, pp. 123-140. 1915 On the nature of the conditions which determine or prevent the entrance of the spermatozoon into the egg. Am. Nat., vol. 49, pp. 257— 285. 1916 The organism as a whole. G. Putnam’s Sons. New York. Logs, J.. anp M. M. CHAMBERLAIN 1915 An attempt at a physico-chemical explanation of certain groups of fluctuating variation. Jour. Exp. Zool., vol. 19, pp. 559-568. Lors, J., anp H. WasteNrEys 1913 The influence of hypertonic solution upon the rate of oxidations in fertilized and unfertilized eggs. Jour. Biol. Chem., vol. 14, pp. 469-480. Lorvennart, A. S. 1905 Further observations of the catalytic decomposition of hydrogen peroxide. Am. Jour. Physiol. vol. 13, pp. 171-185. Lyon, E. P., anp L. F. SHacketn, 1910 On the increased permeability of fer- tilized sea-urchin eggs, following fertilization. Science, vol. 32, pp. 249-251. MacMunn, C. A. 1885 On the chromatology of the blood of some inverte- brates. Quart. Jour. Mic. Sci., vol. 25, pp. 469-490. McCienpon, J. F. 1910 Further proofs of the increase in permeability of the sea-urchin’s egg to electrolytes at the beginning of development. Science, vol. 32, pp. 317-318. Martuews, A. P. 1913 An important chemical difference between the eggs of the sea-urchin and those of the starfish. Jour. Biol. Chem., vol. 14, pp. 465-467. Ostwatp, W. 1907 Uber das Verkommen von oxydative Fermente in den reifen Geschlechtszellen von Amphibien. Biochem. Zeitschr., Bd. 6, pp. 409-472. EGG SECRETIONS OF ARBACIA AND ASTERIAS 499 Ricuarps, A. 1914 The effects of x-rays on the action of certain enzymes. Am. Jour. Physiol., vol. 35, pp. 224-228. RicHarps, A., AND A. E. Woopwarp 1915 Note on the effect of x-radiation on fertilizin. Biol. Bull., vol. 28, pp. 140-148. Rosertson, T. B. 1912 On the isolation of odcytase, the fertilizing and cyto- lysing substance in mammalian blood sera. Jour. Biol. Chem., vol. 11, pp. 339-346. Warsura, O. 1908 Beobachtungen iiber die Oxydationsprozesse im Seeigelei. Zeitschr. Physiol. Chemie, Bd. 57, pp. 1-16. Witson, E. B. 1901 Experimental studies in cytology. II. A cytological study of artificial parthenogenesis in sea-urchin eggs. Archiv f. Entw’mech. d. Organismen, Bd. 12, pp. 529-596. Woopwarp, A. E. 1915 Note on the nature and source of ‘purple x.’ Biol. Bull., vol. 29, pp. 135-137. : Woopwarp, A. E., anp F. 8. Hague 1917 Iodine asa parthenogenetic agent. Biol. Bull., vol. 33, pp. 355-360. PLATE 1 EXPLANATION OF FIGURES Photomicrographs of inseminated Asterias eggs: 1 and 2 Controls. Note that the spermatozoa are scattered. This shows especially well in the right hand part of 1. 3. Eggs from the same female treated for one hour with 0.3 per cent ether in sea-water, washed, and then inseminated, with the same sperm suspension as above. Note the spermatozoa in halos around both mature and immature eggs. 500 EGG SECRETIONS OF ARBACIA AND ASTERIAS ALVALYN E. WOODWARD PLATE 1 AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY THE BIBLIOGRAPHIC SERVICE, JULY 19 EFFECTS OF CHEMICALS ON REVERSION IN ORJENTA- TION TO LIGHT IN THE COLONIAL FORM, SPON- DYLOMORUM QUATERNARIUM S. O. MAST Zoological Laboratory of the Johns Hopkins University CONTENTS IMtrOguCtONer swan ee ee Wess olan eo) tape) elect eit Bi eh Pe Rea ete 503 WT ATOTV APIO PITIe TN OUS #9 ty: eases tes oes ee ee Ras De ape eget tN a obs acs oa ticgatels 506 Effects of chemicals—General statement:.............5... 0-0 e cece eee e eee 507 Eifiee tof acids: sh fio Sea eb Feo . 2) Sas ORs Fe 508 he CusO fell calli shee eee Asc ss eet eee eee ot tes en deed ae peer eee 512 iecwotechemicalsconcentranlone see riee ee ae ok cee ee ee se ee ae 514 Effect of time-rate of change in chemical concentration................... 515 Bfffect)of phy sioldgical istatéS Citi! .. lee ei, We 2. PE ODID. 516 Effect of anesthetics.!............ OS PONIES Teer] A ey Leste Mere etree oi 516 Effect of temperature, and illumination. .............0--2--+44eseehee seers 517 IDSC USSTOTIE RE er eee er, Sele resets Sete Re ed ns IR EE SPR tie et 5 halo ehors 518 Serene ey ss c : HENS Num- | Num- | Num- | Num- | Num-| Num- | Num- | Num- CENT | ber.of | ber of | ber of | ber of | ber of | ber of | ber of | ber of FO 29 | 2 heats 99 F9 29 | | May 16:..2......\- 0.01 oO | 28 o | 23 | O 26 One 26 Mis MG ot ccs 5. 0.02 07 | s21 0}. 22 OF 7" |) OR e Niece 2068) 0.01 4 | 14 o>} 38 0 | 2 | 0 | 21 Wears as 24) 0501 4) iO. ih ats Geil BA W632 | 0 | 35 Wisi 124.22 eS OCO2@ I ease ae 0 | 13 1 | 21 | 0 | 19 Mary 280 22 ee: 0102) iva) 2 0 | 19 Ores eal 2 Mayo0es | 0.03 OF4) “10 3 | 43 hee Tce RRC ke: &? May Sif-n58 5) | 0.025 9 | 30 0 | 40-|44 | 55 3) «63 Funes 4 wes Nie 10202 alee 4s a ci Se 2 Se 4e. | 10miP Gd Matalspen- 2.8... seeeeoes* | 167 12 | 214 13° eoOT 24 | 319 Percentage of o'Q ......| 23 (an 53} 4.2 | 6.9 Manure scum was added as food, and the dish was set under a bell jar in an atmosphere of which 40 per cent was oxygen. 3) A third dish was filled with the dilute creatin solution, manure scum was added as food, and the dish set in air. 4) The fourth dish, as general control, was filled with untreated spring water, manure scum was added as food, and the dish was set in air. After from twenty-four to forty hours the rotifers were re- moved from the dishes. The offspring hatching from eggs laid in that period were reared to maturity in spring water, being fed with manure scum. The rotifers used in this experiment were received from Prof. D. D. Whitney, who collected them at Lincoln, Nebraska. Experiment 6. This is a duplication of experiment 5 in every respect, except that the rotifers used were part of a line received from Prof. A. L. Treadwell, who in turn obtained them from Prof. D. D. Whitney, then at Wesleyan University. They are presumably from the same New Jersey stock as was used in Dr. Whitney’s former work. All the preceding experiments in this MALE-PRODUCTION IN HYDATINA Bad paper, with the exception of experiment 5, were performed with these New Jersey rotifers. Table 8 shows the results of experiment 6. While the last three divisions of table 7 show differences of the kind expected, these differences are small. Comparisons based on the first three divisions of that table would indicate that Euglena is about 18 times as potent in increasing male- production as is oxygen. Or, deducting from the total effect of Euglena the 1.1 per cent which a comparison of the second and third divisions of the table would indicate was due to oxygen, the effect of the Euglena as food would be nearly 17 times as great as that of oxygen. A different result, however, is found in table 8. The first three divisions of the table show that oxygenation increases the proportion of male-producers 5.1 per cent, Euglena 20.2 per cent. Deducting from the total effect of Euglena the fraction charge- able to oxygen alone (20.2 — 5.1 = 15.1), Euglena as food is nearly three times as effective as oxygen in increasing male- production. TABLE 8 Showing the effects of oxygen, Euglena, and creatin upon the proportion of male- producers in a New Jersey line of the rotifer Hydatina senta | | as : | —— AN s | Ad 703 EUGLENA AND MANURE, SCUM MANURE SCUM MANURE SCUM STRENGTH = AND OXYGEN- |AND UNTREATED), OF sGaaneee | ATED CREATIN CREATIN | eoees = CREATIN : - SOLUTION | SOLUTION z ‘ BEY | SOLUTION | Bs Bens Num- | Num-| Num-| Num-| Num-| Num-| Num- | Num- CENT | ber of | ber of | ber of | ber of | ber of | ber of | ber of | ber of | #9 | 99 | #9 ae NRO fete) Te | | | UNC MAOS oa 4 0.0 19 11 2 29 2 19 0 28 Bri ay ie ne eee | 0.02 3 2 5 | 44 bea soar ||” Soe US Le oe 0.02 3 19 0 21 0 6 0 11 JUNC EL oo eee 0.02 2 22 Oilete: 1 29 0 12 June 4 ee 0.02 4 | 28 Ghul. 26 1 29 4 23 Totals... a hen oe Sh 82 19 | 135 } 9 | 116 12 | 101 Percentage of 0 @ ...... 27 .4 12.3 1.2 10.6 538 A. FRANKLIN SHULL Response to oxygen in the Nebraska line The failure of the Nebraska line in experiment 5 (table 7) to show any marked effect of oxygen is the only inharmonious result obtained in this study. It seemed possible that this line was not as sensitive to oxygen as the New Jersey line used in the other experiments. If this were the case, any other response to oxygen might prove less marked in the Nebraska line than in the New Jersey line. Such a response to oxygen in this species was found in earlier experiments. In a former paper (Shull, *15a) it was shown that races of rotifers might differ very markedly in the place of egg-laying. One race laid its eggs very largely on the bottom or sides of the dishes; another race chiefly attached to the surface film of the water. In connection with later oxygen experiments (Shull, ’18), it was shown that placing the dishes in an atmosphere containing an excess of oxygen caused the eggs to be laid more largely on the bottom than when the dishes were kept in air. If the Nebraska line lacked responsiveness to oxy- gen, this lack might be evidenced by failure of oxygen to alter the place of egg-laying. This possibility gave rise to the following experiment. Experiment 7. On each of the days named in table 9 several rotifers of the Nebraska line were placed in each of two dishes of water and fed manure scum. One dish was set under a bell jar in an atmosphere of which 60 per cent was oxygen, the other placed under a bell jar in ordinary air. After a period of from sixteen to eighteen hours, the eggs laid on the bottom and at the surface film in each of the dishes were counted. Asa check upon the results, a similar test was simultaneously made upon the New Jersey line. Table 9 gives the results. While the Nebraska line is here shown to be responsive to oxy- gen, the controls show that it normally laid more of its eggs at the bottom than did the New Jersey line. In view of this normal difference between the two lines, it is difficult to judge of the rela- tive effect of oxygen upon them, and the discrepant results in experiments 5 and 6 probably remain unexplained. MALE-PRODUCTION IN HYDATINA 539 TABLE 9 Showing the number of eggs laid at the surface film of water and at the bottom of the dish by two lines of rotifers, one from Nebraska, the other from New Jersey, when kept in air and in a 60 per cent oxygen atmosphere. The excess of oxygen causes more eggs to be laid at the bottom, but it 1s questionable which line is most affected this way, since their behavior is different under the same conditions NEBRASKA LINE NEW JERSEY LINE Air Oxygen Air Oxygen DATE Eggs | Eggs | Eggs | Eggs | Eggs | Eogs | Eggs | Eggs laid at | laid at | laid at | laid at | laid at | laid at | laid at | laid at surface |bottom | surface |bottom | surface |bottom | surface |bottom December /Sia. Silk. Fe". 5 25 0 22 16 12 2, 29 Decemberwileis.e so e=. |) 28 23 1 44 28 16 3 Wie Decemberulan. yissnso ae 4 24 0 28 1 5 1 3 Mecembern20ss..5. 4-28. 5. 6 24 0 13 32 26 0 25 ARO GALS Sp eeeeys det tee se 43 96 1 107 Tif 59 6 | 79 Percentage at surface... 30.9 0.9 56.6 7.0 | DISCUSSION While this paper was in preparation an article by Whitney (17) on a similar subject, the relative effectiveness of oxygen and food as sex determiners, was published. These two papers, however, in no sense duplicate, since they approach the problem from different angles and with different criteria of judgment. A considerable part of Whitney’s paper is devoted to experi- ments to show that green organisms as food increase the pro- duction of males in other rotifers than Hydatina. It is to be regretted that many of these experiments were performed with mass cultures without controls. If these rotifers are like Hyda- tina in producing males in ‘epidemics,’ the lack of controls is most unfortunate. It is well known to every student of Hyda- tina that, especially in lines that produce only a moderate pro- portion of males, these males often appear in well-defined ‘waves,’ and it has been shown that these epidemics not infrequently have a rather regular periodicity (Shull, 715b). If, in an experi- ment without control, a line which regularly produces numerous males once a month, the experimenter, ignorant of the interval 540 A. FRANKLIN SHULL between periods of many males, should attempt to alter male- production by introducing something into the culture five or six days before the beginning of the period of males was due, he could use any one of a dozen agents and obtain the same result, namely, an increase of male-production a few days later. Notwithstanding the lack of controls, Whitney’s conclusion that the use of green organisms for food in these several species of rotifer increases male-production is probably correct; it is un- doubtedly correct in the case of Hydatina, as Whitney (’14) sat- isfactorily showed in an earlier paper and as I have confirmed in my experiments. The chief question is how much of this effect of green organisms is due to nutritive differences, how much to other agents associated with the green organisms. One of Whitney’s experiments bears upon this point, but the results are not easily understood. That experiment is repre- sented by his table 5 (Whitney, 717). Information on which one could base a judgment of the significance of this experiment is not given. The cultures were apparently of some size, all of them containing green organisms. These cultures had been in existence from five to thirty days, presumably in the sunlight (either direct or diffuse), before the beginning of the experiment. Some of them were then excluded from the sunlight, but for how long a time is not stated. If the duration of the darkness was not prolonged, it is to be expected that the average oxygen content would be appreciably greater than in a culture which had never been kept in the light. The experiments described in table 4 of this paper, test No. 5, indicate that oxygen accumulated in photosynthesis is only very gradually lost. It is not clear, then, that the oxygen content of the cultures in the darkness was as much lower than that of the sunlight cultures as Whitney sup- posed. It is almost certain that, unless they were excluded from the sunlight for a considerable period, these dark cultures contained appreciably more oxygen than a culture never kept in the sunlight would contain. E It is a pertinent question, therefore, whether the smaller amount of oxygen in the cultures in darkness was as effective as the larger quantity in the sunlight. Some of Whitney’s cultures MALE-PRODUCTION IN HYDATINA 541 in the darkness were aerated, others not, and he appears to have suspected that if oxygen were a male-producing agent, the more oxygen there was present the more males there should be. In the experiments described in this paper there is little difference, in its effect on male-production, between water saturated with a 40 per cent oxygen atmosphere and water saturated with a 60 per cent oxygen atmosphere, although it was shown that 60 per cent oxygen atmosphere left the oxygen content of the water very plainly greater. What difference there was indicated that the lower concentration of oxygen was the more effective. It can- not be accepted without question, therefore, that aeration of the water, in addition to the use of green food with its incidental oxygen, should further increase the male-production. Disregarding the above objections to the experiments on which table 5 of Whitney’s paper is based, we may examine the results of those experiments. Instead of comparing only selected parts of his table 5, the whole table may be included. Since in some of the cultures more mothers were used than in others, merely adding together the offspring of parents that were treated in the same manner would give to those cultures having many mothers undue weight. A more just method of combining like cultures is to average the percentage of male-producers in all cultures; each culture is then as weighty as the others, regardless of the number of parents used. By this method of combination, Whitney’s table 5 is converted into the following: Per cent male-producets Sunlveht mwitheaeration. .2502 i . ee arises sae eionce.. ee ath. ete 54.5 Darkness hvaeraion +: 31 5+.¢.2 is pp ee pane es sels = os OR aides 19.0 DaGknessy awit OOWteteratlOny e Oy) 6a es +e) aa {eo ofan 6 "Woe or = - a = = So ee ~ = f \ -- ; : — - Pa | - c tf / Ku < - 3 4/ : - - i J a ae — Ss ¢ Yess s SIS a> 4 —_— : - = - a _—_—-- -_~ > <7 “= oe = = E i ———— Se : Fig. 2. Vertical section of corneal tissue cultivated in plasma. Experiment XXXVIIL, 5, showing the epithelial movement (£) along the endothelial surface (ed). Age of the culture, four days. Drawn from one of the serial sections. t, cut ends; ep, epithelial, ed, endothelial surface; c, part of connective tissue of cornea. X 98. b. Movement on tissue. The next important type of the epi- thelial movement is that on the corneal tissue. By virtue of this, the growing epithelium spreads over the edges of the frag- ment and along the endothelial surface. Figure 2 shows an ex- ample of this very clearly. A movement of this type occurred in some cases on all edges of a fragment and in others only on a part of it, combined with other types. It was also observed that part of the cut end of the epithelium might remain almost inac- tive, while in the other part marked activity occurred. c. Other types of movement. Whenever the growing epithelial cells came into contact with the cover-glass, they moved actively over it. The character of cell movement, however, was essen- 500 SHINICHI MATSUMOTO tially the same as on the endothelial surface. The advancing edge of the growth was composed of a very delicate sheet of protoplasm, showing amoeboid movement, with numerous branching hyaline pseudopodia. When the delicate membrane spread out to an extreme degree, it resulted in the breaking up of the sheet into isolated masses. Ina small percentage of the cultures the spreading of cells over the outer epithelial surface of the cornea (fig. 1, #’) as well as along the lower surface of the culture medium (fig. 1, S) was noted. The elements of the connective tissue showed neither active growth nor movement. The study of the lymphocytes found in the corneal tissue needs special investigation. 2. Movement of the epithelium cultivated in serum Generally, the cornea cultivated in serum showed some details of cell movement, different from those noted in plasma cultures. No amoeboid migration of the cells into the medium took place. During the first and second hours, the cells of the edges became clear and round, sometimes even swollen in appearance; they soon started to move. The most striking and constant phenomenon noted in the use of serum was the spreading out of epithelium over the tissue itself, especially on the endothelial surface. The epithelial rim extended usually parallel with the cut edges, showing amoeboid movement on the advancing border. The rapidity of spreading out of cells was sometimes quite remarkable. In a small per- centage of the cultures, movement of cells was also observed on the original outer epithelial surface of the cornea. If single cells or a part of the rim were in contact with the cover- glass, they clung to it and spread over the surface of the glass with marked activity. The boundaries of individual cells were often hard to distinguish under the microscope. When such prepara- tions are properly impregnated with silver nitrate (Lewis and Lewis, ’12) the intercellular spaces can be clearly demonstrated (fig. 3). When the delicate membrane had extended to the EPITHELIAL MOVEMENT ah utmost degree, the cells along the periphery became loose and isolated. The appended tables give some examples of the frequency of various types of epithelial movement in plasma (table 1) and in serum (table 2), respectively. Fig.3 Experiment XLIX, 2. Silver impregnation, demonstrating the bound- aries of individual cells. Drawn from a part of actively moving border closely attached to the lower surface of cover-glass. Cultivated in serum; three days’ growth. G, advancing border. X 450. 3. Velocity of epithelial movement The velocity of cell movement in vitro exhibits a considerable variation under different circumstances, and, as stated before, THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 26, NO. 3 002 SHINICHI MATSUMOTO TABLE 1 Cultures in plasma CELL MOVEMENT NUMBER SERIES NO. | OF PREPA- | On On On eats RATIONS Into | endothelial | epithelial On __| surface : medium antice aerncate || COVED glass | film 50 a 18 14 14 1 1 50 b 14 5 13 1 46 10 3 10 4 45 6 5 5 2 W 1 1 43 20 13 Lil 2 ih (G2) 52 6 5 4 1 3 E7/ 10 9 3 2 4 61 10 8 6 2, 2, 1 62 2 2, 2 2 2 63 34 24. 33 102 33 18 oll 106 15 15 14 104 15 iL 15 107 6 iat 110 s 6 8 111 9 4 9 212 18 11 lyf totale 239 149 206 10 20 3 2 combinations of several types of movement are often to be seen in a single preparation. During the period of activity, a change in the type or direction of movement was often observed, and such a change in one part was apt to modify the movement of neighboring cells. Single cells or small groups of cells, as a rule, moved more freely than those in the spreading membrane. The consistency of the plas- ma, the liquefaction and the contraction of the fibrin influence both the rapidity and the type of movement into the plasma. The temperature has both direct and indirect influences on the movement. There is also individual variation which cannot be attributed to any of the above factors. For these reasons, the velocity of the epithelial movement varies from hour to hour, and it is very difficult to analyze the factors influencing it. Usually, the most vigorous activity occurred during the first twenty-four hours. For instance, the epithelium on a piece of EPITHELIAL MOVEMENT 553 TABLE 2 Cultures in serum | CELL MOVEMENT SERIES NO. Gees | - | - |. cee Ogee | Abeer: glee Sraiciheval epithelial ae | vactace oS ae | surface surface = film 56 28 28 94 28 28 5 1 58 7 6 1 60 8 a 1 139 ln | 7 138 rea 6 1 140 5 5 135 Sah. S | | 165 14 14 166 12 12 167 15 15 208 23 23 1 219 1S 18 221 | 20 20. | 232 | 7 if | 233 | 9 9 | 234 9 9 Motales.-e le, 220 212 1 5 1 3 cornea cut radially into eight parts, spread out into the plasma within twenty-four hours in an extension which was even larger than their original area, though not in the same degree of rapidity at all cut ends. Similarly, in successful preparations, it was seen that the whole endothelial surface was covered within twenty-four hours by the epithelium spreading over the entire circumference of the piece. When the moving borders met each other, move- ment ceased. Some measurements are given in table 3. TABLE 3 MOVEMENT ON MOVEMENT INTO PREPA-| ENDOTHELIAL SURFACE PLASMA RATION oe ae 24 hrs. | 48 hrs.| 5hrs. | 24 hrs. | 48 hrs. Experiment 210, plasma culture, i ROIS | OVA O25) 1 0200s OFS an ORS at 21°C. Dl OA Ose bee Mois Oss | iste Bo WO Ws. Wes Os el | iL 4 | 0.14 | 0.65 | 0.7 2 LO! a n089 Om OSI g ie OR IO cORO Ss OS Sam mleO 6 0.1 0.3 0.9 Cs Ot e067) a029 SoMa Osa Mes Average movement in milli- MMEGELS Hy. bil eee testes epee 0.12 | 0.68 | 0.838 | 0.087) 0.7 |1.16 20 hrs.| 40 hrs.| 60 hrs. Experiment 211A, plasma cul- 1 Oi ee 0) ture, at 20°C. Dy 0.8 | 4.0 | 5.4 3 Tea | 2 bie S Average movement in milli- MLE GETS cess = a arsine eons O77 | 228 |-3-4 20 hrs. | 40 hrs. 20 hrs. J Experiment 211B, plasma cul- TWO e ae) the | ture, at 20°C. Zr ORG Okt ay lee. elo) ANT KOLOn EOE, De Ona ORS 0.2 65086 29} 205.6 Average movement in milli- MC TCTSE Saves tee ao vei wceciek, s 0.65 | 0.78 20 hrs Experiment 208, serum culture, 1 0:3 at 20°C.* A Ne! 3 | 0.8 4 10.8 5 | 0.6 6) KOR Average movement in milli- ENE GETS heen 5) eae EE Te 0.6 *Pieces which measured: 1.2 x 0.6 mm.; 1.8 x 0.5 mm., 2.0 x 0.6 mm., 2.4 x 1.1 mm., and 0.8 x 0.8 mm., were entirely covered up with moving epithelium in seventeen hours. 554 EPITHELIAL MOVEMENT DOO II. ON THE REACTION OF EPITHELIAL CELLS TO CERTAIN SOLID SUPPORTS 1. Movement on flat surfaces a. On glass and celloidin. We shall first consider movement on the glass cover-slip.' There is difficulty by means of usual culture method in bringing the epithelial rim into contact with the cover-glass from the beginning. Attempts to do this by reducing the plasma failed, as the epithelium did not show activ- ity unless the medium was used in sufficient quantities. In the later experiments, the piece of cornea (cut off tangentially to eyeball) was placed on the cover-slip, mainly with the inner surface down, and subjected to slight pressure by means of thin silver wires or glasses and kept in that position for a certain period after mounting with serum, so that the cut ends came into contact with the glass. When the silver wires were properly placed and controlled under the microscope, it was possible in almost every instance to bring certain parts of the moving epi- thelium into contact with the cover. Of course, where even a minimal space existed between the tissue and cover-glass surface, the epithelium crept on the underlying tissue. Cover-slips coated with celloidin were also used. Thus the movement of the epithelium on celloidin and glass surfaces, respectively, could be compared. A comparison could also be made in the same preparation, by employing a cover-slip, only one half of which was coated with celloidin. Such preparations must be handled carefully, otherwise a change of cell form readily occurs. No characteristic difference whatever in the mode of movement on the two different surfaces could be noticed. If the epithelium erew out vigorously, closely attached to the cover-slip, each cell became flattened into an exceedingly thin layer. In some instances it was seen that the advancing border became very | Tt should be stated here that the cover-glasses used in these experiments were throughly cleaned and washed in the vapor of distilled water, as is usually done to remove any trace of alkali, in order to exclude any chemical influence from that source. (Ostwald-Ruther, Physiko-chemische Untersuchungen. ) 556 SHINICHI MATSUMOTO irregular. This was most marked when active cells on the border, showing unusual protoplasmic activities, tore themselves loose from their connection. Similar conditions, however, were often to be noticed in the actively mobile epithelial layer growing out into firmly clotted plasma. In the epithelial movement on the endothelial surface, such a condition but rarely occurred; as a rule, the cells moved more uniformly, showing a smooth advane- ing border. In other instances small pieces of cover-glass or celloidin mem- brane were placed on the endothelial surface and pressed against it, so that a certain portion or the whole of this surface, which is a favorable support for moving cells, was covered. In these cases the epithelium was also seen to move over the celloidin or glass, if they were suitably mounted and the cells were not injured. Where the cell movement on such artificial supports failed, examination proved that the placing of the membrane was not suitable; thus, by control of pressure, it was possible to direct the moving cells to these artificial surfaces, though this was not always an easy matter. At any rate, it is an established fact that the epithelium is able to creep on objects of such nature that chemical influences are excluded. b. On dead cornea. If in a part of an explanted piece of cornea an epithelial defect existed, it was seen that the epithelial cells were able to cover the spot, spreading out from all edges, prac- tically with the same rapidity as on the endothelial surface. The same was true of a spot which had been killed by touching it with a heated needle. The cells injured by the latter procedure became round and detached, and the growing epithelium crept beneath them along the surface of the killed tissue. Preparations were made of large strips of corneal tissue (about 2x 4mm.), across the center of which a sharply defined epithelial defect was produced, after which one half of the remaining corneal tissue was killed by means of a heated needle, making the tissue surface look opaque and wrinkled. In such preparations it was seen that the epithelial movement over the wound occurred uni- formly, covering both the live and the dead tissue with the same rapidity. EPITHELIAL MOVEMENT 507 Analogous experiments were made with pieces of cornea in which an epithelial defect existed at one extreme end and in which the tissue at the other end had been killed; similarly, the cell movement on a burnt wound was compared with a control prep- aration, in which the underlying tissue was left intact with simply an epithelial defect. The use of tissue, vitally stained by neutral red? or nile blue, facilitated the observation of epithelial movement on such wound surfaces. Analogous observations were made by using the surface of the cartilaginous layer of the sclerotic coat of the frog’s eye. This tissue could readily be isolated from the other layers after boiling, and it was then thoroughly washed before using. It was trans- lucent, showing characteristic convexity, which admirably fitted on the inner surface of the similarly curved cornea; even without any pressing, the two tissues would le so closely together in the culture medium (serum) that the epithelium from the cut ends of cornea crept over the cartilage. It proved helpful, how- ever, if the pieces were slightly pressed together. Out of twenty-six experiments, movement of the epithelium over the cartilaginous plate took place in twenty-one; in three cases no movement was observed owing to injury of cells. An example of this is illustrated in figures. 4 and 5. 2. Movement of fiber-like supports Next, the response of the epithelium to the various fiber-like supports were tested, such as spider web, silk fiber, glass wool, and asbestos. a. Spider web as support. Experiments were made similar to those of Harrison (’14), using spider webs. In each preparation a sufficient quantity of serum was used as culture medium. Figure 6 represents one of the preparations of the series; many cultures were found where the cells clung to the fibers. Out of thirty cultures twenty-four were positive, four doubtful, two infected. 2S. Matsumoto. Demonstration of epithelial movement by the use of vital staining. This paper will appear in the next number of this journal. DOS SHINICHI MATSUMOTO b. Silk fiber as support. Other experiments were made, using silk fibers. In these a number of fibers of raw silk were stretched on the glass ring and cover-glass, imitating the experiment with the spider webs. Out of twenty-nine cases there were fourteen that gave positive results, one being infected. Chas: : t Sg oo‘ eo Siar aie Sf. Za i “i : ye Mee. 25 \4 NS a : Fig. 4 Experiment 233, 4, showing epithelial movement (#) on the boiled cartilaginous plate (ch) of sclerotic coat closely placed on the endothelial surface of cornea. Cultivated in serum; forty hours’ growth. ¢, cut ends of the piece of cornea are clearly visible through the transparent cartilagineous plate. X 98. Similar experiments were made by Carrel and Burrows (’11a) with embryonic chick tissue. c. Glass wool as support. Glass wool (Merck) was thoroughly cleaned and sterilized, then placed in the culture, so that the epithelium might attach itself to the fibers. Out of twenty-eight cultures sixteen gave positive results. An example is shown in figure 7. EPITHELIAL MOVEMENT 599 d. Asbestos fiber as support. Asbestos fibers were ignited and treated with pure conc. HCl; the fine cloudy suspension was col- lected and washed in distilled water to remove completely all traces of HCl, and then sterilized before use. In the serum culture of the cornea the fibers were mixed densely so that, they appeared like a nest, in which the tissue lay. AS the fibers were very fine, they did not hinder microscopical exam- ination, if not too densely placed. en a> “a =e > “Ss =, Fig. 5 Experiment 233; 4. Same preparation as shown in fig. 4. Portion of vertical section, drawn from one of the serial sections. ep, epithelial surface of cornea; c, connective tissue; ch, cartilaginous plate, placed on the endothelial surface of cornea; f, cut end of cornea. Note the epithelial movement (E) on the sclerotic cartilage (ch). Forty hours’ growth. X 98. Thirty cultures were made in this group, with positive results in twenty-two. Figure 8 shows an example of this series. e. Movement on pith, etc. Further studies were made with pith. Thin pieces were used, which were kept in distilled water which was changed every three days for a period of over three months. Experiments showed that the epithelium was able to cling to the cell walls of the thin piece of pith. Figure 9 shows clearly 560 SHINICHI MATSUMOTO the growing out of epithelial cells on the support. Outof twenty- seven experiments nineteen gave positive results. On the shell membrane of hen’s egg, used as support, after thoroughly washing and boiling, a similar condition was observed. \ a y gw ee fe X ) a ee) 3 “5 \ > 2 & WS Ge \ Ae , Sa ENE é E all 33 - z - : E + es = (Oe = Z 9) F \ j € tf \. } 6 7 Fig. 6 Experiment 238, 11. Epithelial movement in serum on spider web- In the neighborhood of the explanted tissue (c) a good many isolated cells, round in shape, were noticed, which are omitted in this figure; they showed no active movement. ¢, edge of the piece; above, some cells move out in sheets on the cover-glass. Compare Jour. Exp. Zoél., 17, 521, figs. 4 to 7,12. x 98. Fig. 7 Experiment 49, 7. Epithelial movement on glass wool (gw); culti- vated four days in serum. ¢, edge of tissue. > 98. III. RESUME This paper deals with the movement of corneal epithelium of the adult frog in vitro. Frog cornea is very suitable for this purpose, as it is so transparent and thin that it permits of direct observation of the cell movements. The corneal epithelium cultivated in plasma shows various types of movement, according to the nature of the substratum EPITHELIAL MOVEMENT 561 (fig. 1). The movement is of an amoeboid character. As a rule the cells have a strong tendency to cling to their own kind and thus extend in sheets, although under certain conditions active movement of isolated cells is also to be seen. In the majority of cultures movement into the medium or along the endothelial surface (or both) takes place according to the consistency of the culture medium. The fact, that the epi- Cc Fig. 8 Experiment 238, 1. Epithelial movement on fibers of asbestos: eulti- vated forty hours in serum. At the extreme left cells are to be seen moving along cover-slip. Note the adaptation of single cells to asbestos fibers. X 450. thelium moves along the epithelial or endothelial surface is very important from various points of view; such a movement may easily be overlooked in the culture of non-transparent tissue, such as skin. In the preparations in which serum is used, no migration of the epithelium into the medium takes place. There is mainly a movement of cells over the tissue, especially on the endothelial surface. 562 SHINICHI MATSUMOTO Naturally, the question arises, whether the epithelial movement on the tissue, which is so frequently to be seen in the fluid me- dium, is chemotactic or thigmotactic in nature, It might even be that the type of movement is due both to mechanical and to chemical influences acting simultaneously. However, the epi- thelium is able to move with practically the same velocity both on a substratum where the covering epithelium has been simply Fig. 9. Experiment 239. Corneal piece cultivated in serum with pith; three days after explanation. Note the epithelium clinging to the piece of pith (p) moving out of the cut end (¢) of cornea (c). X 98. seraped off and on one where the underlying tissue has been killed by heating. The movement in the latter cases cannot, therefore, be dependent upon chemotactic influences from the liv- ing tissue. The same is true of the movement taking place on the surface of the cartilaginous plate of the sclerotic coat, pre- viously killed by boiling. Furthermore, the epithelium is able to move on the surface of glass, on a celloidin film, and also on such fiber-like supports EPITHELIAL MOVEMENT 563 as spider web, glass wool, asbestos, ete., when it is brought into contact with them. The cell movement on the glass and celloidin film is very vigorous, sometimes more rapid than on the endothelial surface. In the movement on such supports chemotactic influences are to be considered as excluded. It has been repeatedly observed by various writers that a suit- able support for the growing cells is an important requisite; Harrison (14) demonstrated lately the importance of such fac- tors for the movement of embryonic cells very clearly. The facts above described confirm this view, that stereotropism plays an important role in cell movement. The behavior of corneal epithelium in vitro serves to throw some light on the mechanism of epithelial growth in vivo. The experiments show clearly that the epithelium is able to extend from the cut end quite rapidly in sheets into the medium (plasma), or on the tissue (plasma and serum), and can cover a large area, whereas mitotic cell divisions are not necessary at all. That Oppel (12) as well as Osowski (14) did not observe amoeboid activity of the epithelium was perhaps due to the diffi- culty of direct observation. Special, careful observation on the movement of epithelium of warm-blooded animals, however, is necessary. In conclusion, I wish to express my indebtedness to Prof. R. G. Harrison for his direction and valuable advice. 564 SHINICHI MATSUMOTO LITERATURE CITED CaRREL AND Burrows 1911 Cultivation of tissues in vitro and its technique. J. Exp. Med., 18, 387. 191la An addition to the technique of the cultivation of tissues. J. Exp. Med., 14, 244. Cuampy 1914 Note de biologie cytologique. Quelques résultats de la méthode des cultures des tissus. Arch Zool. Exp., 54, 307. Harpe 1916 Some observations on the virus of vaccinia. Ann. d’l inst. Past., 30, 299. Harrison 1910 The outgrowth of the nerve fiber as a mode of protoplasmic movement. Jour. Exp. Zodél., 9, 787. 1914. The reaction of embryonic cells to solid structures. Jour. Exp. Goole ii, o2e Houtmes 1914 Behavior of ectoderm of amphibians when cultivated outside the body. Jour. Exp. Zoél., 17, 281. LAMBERT AND Hanes 1913 Beobachtungen an Gewebskulturen in vitro. Virch. Arch., 211, 89. Lewis AND Lewis 1912 Membrane formations from tissues transplanted into artificial media. Anat. Rec., 6, 195. Lors 1898 Uber Regeneration des Epithels. Arch. Entw.-Mech., 6, 297. 1902 Uber das Wachstum des Epithels. Arch. Entw.-Mech. 13. 1907. Uber einige Probleme der experimentellen Tumorforschung. PeAewstanoe 1912 Growth of tissue in culture media and its significance for the analysis of growth phenomena. Anat. Rec., 6, 109. LOWENSTEIN 1913 Experimentelle Untersuchung iiber Regenerationsvorginge in der Kaninchencornea. Arch. Ophth., 85. OppEL 1912 Causal-morphologische Zellenstudien. V. Mitteilung. Die active Epithelbewegung. Arch. Entw.-Mech., 35, 371. 1913 Demonstration von Epithelbewegung von Froschlarven. Anat. Amz. Ane ilidia: Osowsxkr 1914 Uber active Zellenbewegung in Explantat von Wirbeltierem- bryonen. Arch. Entw.-Mech., 38, 547. Peters 1885 Uber die Regeneration des Epithels der Cornea. Inaug.-Diss. Bonn. Ruta 1911 Cicatrization of wounds in vitro. J. Exp. Med., 13, 422. Sauzer 1911 Uber die Regeneration der Kaninchen-Hornhaut. Arch. Augenh., 69, 303, ete. UnLeNHUTH 1914 Cultivation of the skin epithelium of the adult frog. J. Exp. Med., 20, 614. INDEX LBINO rat. Studies on inbreeding. I. A The effects of inbreeding on the growth and variability inthe body weight of the Albino rat. Studies on inbreedirg. II. The effects of inbreeding on the fertility and 1 on the constitutional vigor of the......... 335 Albino rats. On several effects of feeding small quantities of SudanllIto young.... Albino series of the rat. Ruby-eyed dilute gray, a third allelomorph in the.......... Albissima Vejdovsky. Reactions of the pro- DOscisiopelananiayna pees eee eee ee Alcohci on treated guinea-pigs and their de- scendants. Further studies on the modi- fication of the germ-cells in mammals: Hee eC tyoh ee creysiccniairs Slevin enero ALLEE, W. C., AND STEIN, E. R., Jr. Light reactions and metabolism in May-fly nymphs. 1. Reversals of phototaxis and the resistance to potassium cyanide. II. Reversals of phototaxis and carbon di- Oxide;pradiuctionss = eee ee eee eenne Allelomorph in the Albino series of the rat. Ruby-eyed dilute gray,athird............ Antibodies. Studies on cytolysins. I. Some prenatal efiectsiotlenshs.. ser eee Arbacia and Asterias. Studies on the physio- logical significance of certain precipitates from the egg secretions of................. Asterias. Studies on the physiological signifi- cance of certain precipitates from the egg secretions of Arbacia and................. 4 Axial gradient in the regeneration of Tubularia supported by facts? Isthetheory of ..... ANUS, Marto Garcra. Is the thecry of axial gradient in the regeneration of Tubularia supported by facts?.......... Body weight of the albino rat. Studies on ink reeding. ile The effects of nibread int iepentea by holothiciane ThSamMounvOu seas eee Bottom raaterial (Stichopus). ARBON dioxide produetion. Light re- actions and metabolism in May-fly nymphs. I. Reversals of phototaxis and resistance to potassium cyanide. IT. Reversals of phoatotaxis and........ Chemieals on reversion in orientation to light in the colonial form, Spondylomorum quaternarium. Effects of.....:......... Corneal epithelium of the frog in tissue cul- ture. Contribution to the study of epi- thelislimovements het 205). a: Crozier, W. J. The amount of bottom ma- terial ingested by holothurians (Sti- chopus) . Culture. Contribution to the study of epi- thelial movement. The corneal epithe- lium of the frog in tissue................. Cyanide. Il. Reversals of phototaxis and ear- bon dioxide production. Light reactions and metabolism in May-fly nymphs. I. Reversals of phototaxis and the resistance COMPOUHSRVUM ds sien ce Sheek eres Cytolysins. I. Some prenatal effects of lens antibodies. ‘Studiesion....A.canas «ee eee 101 265 - 265 423 503 pee production. Light reactions and metabolism in May-fly nymphs. I.Re- versals of phototaxis and the resist to potassium cyanide. 11. Reversals of phototaxis and carbon.. ............ 423 Ductless glands on the dev gppaent of the flesh flies. The effects of the.. cere Ap) FFECTIVENESS of food, oxygen, and other substances in causing or prevent- ing male-production in Hydatina. Rel- BEIVE. Ao se.S Seo Reet Egg secretions of Arbaciaand ‘Asteri las. Studies on the physiological significance of certain precipitates fromthe Meas Epithelial movement. The corneal epithe- lium of the frog in tissue culture. Contri- bution to the study (Cline hee oie SO f Epithelium of the frog in tissue culture. Con- tribution to the study of epithelial move- ments. vhecornealee eee eee eee 545 EEDING small quantities of Sudan III to young albinorats. Onseveral effects (ARE Rc SOR OM Gn MEAT cae ee eee 101 Fertility and on the constitutional vigor of the albino rat. Studies on inbreeding. 11. The effects of inbreeding on the.......... 335 Flesh flies. The effects of the ductless glands on the development of the.. 255 Flies. The effects of the ductless glands « on the development of the flesh... 255 Fly nymphs. I. Reversals of phototaxis and the resistance to potassium cyanide. II. Reversals of phototaxis and carbon dioxide production. Light reactionsand metab- olismiinwMay=5-0) sss eee eee 5p 2 BR} Food, oxygen, and other substances in caus- ing or preventing male-production in Hydatina. Relative effectiveness cf. 521 Form, Spondylomorum quaternarium. Ef- fects of chemicals on reversion in orienta- tion to light in the colonial.........../... E Frog in tissue culture. Contribution to the study of epithelial movement. The cor- nesllepithelim ohthe ener eee sae eee eee Fi Fundulus and mackerel. A study of paternal heredity in heterogenic hybrids. Hybrids between ee Aare serene arte Coe ae OO ERM-CELLS in mammals: The effect of aleohol on treated cuinea-pigs and their descendants. Furtherstudies on the mG OCU CAHIONTO UNG seer ae an eee ne 119 Glands on the development of the flesh flies. The effects of the ductless............... 255 Gradient in the regeneration of Tubularia sup- ported by facts? Is the theory of axial...... 265 Growth and variability in the body weight of the albino rat. Studies on inbreeding. I. The effects of inbreedingon the..... 1 Guinea-pigs and their descendants. Further studies on the modification of the zorm- cells in mammals: The effect of alcohol Onitresteds, sass eee : Guyer, M. F., anp Situ, BE. A. Studies on cytolysins. I. Some prenatal effects of lens antibodies........... 565 566 INDEX ATAI, S. On several effects of feeding small quantities of Sudan II] to young SL bInNIO Tats ote oot ee eee ae eee oe 101 Heredity in heterogenic hybrids. Hybrids between Fundulus and mackerel. A stidyronpatermallecn eters peat eee eae 391 Heterogenic hybrids. Hybrids between Fun- dulus and mackerel. A study of paternal heredity isi. noo tee ie ete ee 391 Holothurians (Stichopus). The amount of bottom materialingested by............... 379 Horned toad Phrynosoma. The physiology of the melanophores of the............... 275 Hybrids between Fundulus and mackerel. A study of paternal heredity in heterogenic hy DEAStae eee cone ere ee oes 391 Hydatina. Relative effectiveness of food, oxy- gen, and other substances in causing or preventing male-production in........... 521 NBREEDING. I. The effects of in- breeding on the growth and _ variability in the body weight of the albino rat. StUGITESTON =a leeee em Ss set e Oeeiee Rae sie 1 Inbreeding. II. The effects of inbreeding on the fertility and.on the constitutional vigor ofthealbinorat. Studieson.............. 335 Ingested by holothurians (Stichopus). The amount of bottom material .............. 379 7 EPNER, Wim11am A., anp RicH, AR- NoLD. Reactions of the proboscis of Plsnaria albissima Vejdovsky........ .... 83 Kina. HeLren Dean. Studies on inbreeding I. The effects of inbreeding on the growth and variability in the body weight of the AIDIMO WA noc eee te ee Sees eee Kine, Heren Dean. Studies on inbreeding. IL. The effects of inbreeding on the fertil- ity and on the constitutional vigor of the PALL OMENS case eee es eee ol eo OD Ikinc, Heten Dan, anno Wauirtrne, P. W., Ruby-eyed dilute gray, a third ailelo- morph in the Albinoseries of therat..-...- 55 Kunxket, B. W. The effects of the ductless glanas on the development of the flesh FECES SOREL Sa petite SEES Fe hi eh geraichon cya 255 i | RRS antikodies. Studies on cytolysins. I. Some prenatal effects of..............:. 65 Light in the colonial form, Spondylomorum quaternarium. Efiects of chemicals on re- version in orientation 10.............. S52 TAB Ligkt reactions and metabolism in May-fly nymphs. I. Reversals of phototaxis and the resistance to potassium cyanide. IT. Reversals.of phototaxis and carbon dioxide PEGAUCHON™ cheese oie eee cee esos ere 423 Ma cee A study of paternal hered- ity in heterogenic hybrids. Hybrids between Pundulusjand= 2.2222 2. dae. 391 Male-production in Hydatina. Relative effec- tiveness of food, oxygen, and other sub- stances in causing or preventing.......... 521 Mammals: The effect of aleohol on treated guinea-pigs and their descendants. Fur- ther studies on the modification of the Term=Cellsi New. wes oetaeee eras 119 Mast, S.O. Effects of chemicals on reversion in orientation to light in the cslonial form, Spondylomorum quaternaritm. .. 503 Materialingested by holcthurians (Stichopus). ‘The amount of boptoOml-ss-- 440 6e ee eee Matsumoto, SHinicu1. Contribution to the study of epithelial movement. The cor- neal epithelium of the frog in tissue Culture osc ase es eee eee 545 May-fly nymphs. I.-Reversals of phototaxis and the resistance to potassium cyanide. Tl. Reversals of phototaxis and carbon dioxide production. Light reactions and MmetaVONSMAn.-.< 0. eee ee tee 423 Melanophores of the horned toad Phryno- soma. Thephysiology of the............ 275 Metabolism. A demonstration of the origin of two pairs of female identical twins from two ova of high storage..-......5......... 227 Metabolism in May-fly nymphs. I. Rever- sals of phototaxis and the resistance to po- tassium cyanide. II. Reversals of photo- taxis and carbon dioxide production. Trahtireachionsiand... .9.