BIOLOGICAL BULLETIN

OF THE

fiDarinc Biological laboratory

WOODS HOLE, MASS.

EMtorial Staff

E. G. CONKLIN Princeton University.

JACQUES LOEB The Rockefeller Institute for Medical Research

T. H. MORGAN Columbia University.

W. M. WHEELER Harvard University.

E. B. WILSON Columbia University.

Managing BMtot

FRANK R. LILLIE The University of Chicago.

VoLl ME XXIV.

\Vou!)S IIOl.K, MASS. DECEMBER 1912, TO MAY 1913

PRESS OF

THE NEW ERA PRINTING COMPANY LANCASTER. PA.

CONTENTS OF VOLUME XXIV.

No. i. DECEMBER, 1912.

SHULL, A. FRANKLIN. The Influence of Inbreeding on

in Ilydatina senta i

GRAVE; B. H. The Otocyst of the PinnidcT 14

RUTHVEN, ALEXANDER G. On the Breeding Habits of Butler's

Gartersnake i v-

ALLYN, HARRIETT M. The Initiation of Development in Chntop-

tems 21

No. 2. JANUARY, 1913.

GOLDFARB, A. J. Studies in the Production of Grafted. Embryos . 73 PEARSE, A. S. On the Habits of the Crustaceans found in Chcc-

topterus Tubes at Woods Hole, Massachusetts 102

RAWLS, ELIZABETH. Sex Ratios in Drosoplii/a ampelophila 115

Xo. 3. FEBRUARY, 1913. BORING, ALICE M. The Odd Chromosome in Cerastipsocus vows us 125 -

BORING, ALICE M. The Chromosomes of the Cercopidcc 133

LILLIE, FRANK R., AND JUST, E. E. Breeding Habits of the

Heteronereis Form of Nereis limbata at Woods Hole, Mass.. . 147 ABBOTT, J. F. The Effect of Distilled Water upon the Fiddler Crab [69

GLASER, OTTO. On the Origin of Double-Yoked Eggs 175

FOOT, KATHARINE, AND STROBELL, E. C. Preliminary Note on the Results of Crossing Two Hemipterons Species with Refer- ence to the Inheritance of an Exclusively Male Character and its Bearing on Modern Chromosome Theories 1X7

\o. 4. MARCH, 1913-

PEARL, RAYMOND, AND PARSHLEY, H. M. Data on Sex Deter- mination in Cattle -<>5

HARTMAN, FRANK A. Variations in the Size of Chromosomes HARTMAN, FRANK A. Giant Genii Cells in the Grasshopper. . . 2311 \\.\u, PHIL AND NELLIE. The. Fertility of Cecropia Eggs in Rela- tion to the Mating Period 245

WILDER, INEZ WHITTLE. The Life History of I)cs>iia»natlnts

f n sea ' -5 i

iii

IV CONTENTS OF YOLUMF XXIV.

No. 5. APRIL, 1913. WILDER, INEZ WHIPPLE. The Life History of Desmognathus

fnsca 293

HEILBRUNN, LEWIS V. Studies in Artificial Parthenogenesis. . . . 343 COLE, LEON J. Experiments on Coiinliiuition and Righting in the

Starfish 362

STOCKING, RUTH J. A Note on the S per mato gene sis of Tencbrio

monitor 370

No. 6. MAY, 1913. KING, HELEN DEAN. Some Anomalies in the Gestation of the

Albino Rat (Mus Norvegicns albinus) 377

WILSON, EDMUND B. A Chromatoid Body Simulating an Accessory

Chromosome in Pentatoma 392

KEPNER, WILLIAM A., AND TALIAFERRO, WILLIAM H. Reactions

of Amceba protcus to Food 411

Fifteenth Report of the Marine Biological Laboratory 429

Vol. XXIV. December, 1912. No. i.

BIOLOGICAL BULLETIN

THE INFLUENCE OF INBREEDING ON VIGOR IN

HYDATINA SENTA.1

A. FRANKLIN SHULL, DEPARTMENT OF ZOOLOGY, UNIVERSITY OF MICHIGAN.

Page

Introduction I

Description of the experiments i

The measure of vigor 2

Results of the experiments 4

Other evidence relating to vigor in inbreeding 6

Mendelian explanations of vigor 7

Loss of vigor not accounted for by the Mendelian explanations 10

A physiological explanation of vigor 10

Bibliography 12

INTRODUCTION.

In some experiments dealing with the inheritance of certain egg characters in the rotifer Hydatina senta, it has been necessary to inbreed the animals a number of times in succession. Evidence relating to the influence of inbreeding on vigor has thus been incidentally obtained. Since this part of the evidence from the experiments has no direct bearing on the inheritance of the egg characters in question, it is published here separately.

DESCRIPTION OF THE EXPERIMENTS.

All the experiments started from a single female, herself an FI from a cross. From her was bred a parthenogenetic line of 12 generations. Some of the females and males of this line were paired, and a considerable number of fertilized eggs obtained. When the fertili/ed eggs hatched, two of the earliest hatchers, one from a family in which a large proportion of the eggs hatched,

1 Contributions from the Zoological Laboratory of the University of Michigan No. 139.

A. FRANKLIN SHULL.

one from a family in which only a few hatched were selected for further breeding. From these two females were bred two parthenogenetic lines, of four and ten generations; respectively. In each line some females were paired with males of the same line, and from one of the fertilized eggs of each lot a new partheno- genetic line was reared. The members of these were again inbred, and so on, six times in succession. There were thus obtained two series of parthenogenetic lines, each one after the first obtained by inbreeding from the line preceding. In one of these series, numbered I. in the table, the numbers of families in the six lines were 12, 4, 5, 17, 5, and 9, respectively; in the other series (II.) the numbers of families in successive lines were 12, 10, 8, II, 21, and 17, respectively.

I shall attempt to show, in what follows, that there is a pro- gressive decrease in the vigor of these six lines, from first to last.

TABLE I.

SHOWING DECREASE OF VIGOR, AS MEASURED BY VARIOUS CHARACTERS, IN Six SUCCESSIVELY INBRED PARTHENOGENETIC LINES OF Hydatina senta.

vt V

*n u Cfl

Character to be Measured.

Number of Parthenogenetic Line.

i

2

3

4

5

6

I.

Size of family of parthenogenetic female . . . Size of family of fertilized sexual female . . . Number of eggs laid per day

48.4 16.7 II. O 2.27

I/II

42.5

12.8

11.4

1.66 i/3

46.8

12.8

10.3 2.25

2/4

42.5

ii. 5

10. 0

1.93

3/i6

31-0

6-3 9-2 2.25

0/4

22.6

7-3

7-5

2.12

5/3

Number of days required to reach maturity Proportion of cases in which first daughter did not become parent

Same in percentages

14.2

25.0

41.6

II.

Size of family of parthenogenetic female . . . Size of family of fertilized sexual female . . . Number of eggs laid per day

48.4 16.7 II. O

2.27

I/II

30.8 13-7 n.6 1-55

4/9

41.0

13.5 7.9

2.57

2/7

37-0 15-2

7-7

2.20

2/IO

33-8

IO.I

9.6 1.90

8/20

24.8 7-6 8.6

2.OO

7/16

Number of days required to reach maturity Proportion of cases in which first daughter did not become parent

Same in percentages. .

2=;.o

2?.cr

41.6

THE MEASURE OF VIGOR.

Six distinct means of measuring the vigor of the several .parthenogenetic lines are available. They are as follows:

i. Size of family of parthenogenetic females. With few excep- tions every daughter of a female used for breeding was isolated and recorded. The average size of family was computed for each parthenogenetic line separately. Families not completely

INFLUENCE OF INBREEDING ON HYDATINA SENTA. 3

recorded, of which there were a few, were not included in this computation.

2. Size of family of fertilized sexual females. It appears that the male does not appreciably affect the number of eggs laid by. the female with which he is mated. The size of family is deter- mined almost wholly by the female herself. In -determining size of family in this case, I have counted the eggs laid, not the number that hatch, as experiments have led me to conclude that the vigor of the parent is responsible for the number of eggs, but not for their viability.

3. Number of eggs laid per day. The young rotifers were isolated and recorded about the same time each day. To find the average number of eggs per day in a given line, the total number of offspring hatched by that line was divided by the number of days on which they were produced, the days for each family being counted separately. Each family was produced usually in three to five days. In a line comprising nine genera- tions, therefore, the number of days as used in this computation would be 27 to 45, regardless of the fact that several families were producing young at the same time, and that the experiment covered only about 20 days. The first day and the last day on which a given female produced daughters were counted as half days, which they must have been on the average, since the offspring recorded as of one such day must have been in some cases the output of a few hours, in other cases of practically a whole day. It is assumed that the eggs hatched in fairly uniform time, as observations have shown that they do.

4. Number of days required to reach maturity. By this is meant the interval between the laying of an egg and the time when the female that hatches from it begins to lay eggs. This time varies considerably in different lines even when reared under identical conditions. It has been found in some cases that in one line that has passed through over ninety generations partheno- genetically, about three days are required to reach maturity, while a young and vigorous line reared at the same time required less than two days. In the present experiments the time required to reach maturity is an average of the parents of all the genera- tions in a given line. It is the interval between the hatching of the first daughter of the first generation and the hatching of

4 A. FRANKLIN SHULL.

the first daughter of the last generation, divided by one less than the number of generations.

5. Proportion of cases in which the first daughter did not be- come the parent of the next generation. In all my breeding experiments, whenever a new family was started, the first two daughters were set aside for further breeding, though only one of them was ordinarily used. If the first daughter was apparently healthy and vigorous, she was invariably used. If the second daughter was distinctly more vigorous than the first, the second became the parent of the next generation. Sometimes it was deemed advisable to discard both and use the third, fourth, or fifth daughter. Thus, in a vigorous line, the first daughter should usually be healthy enough for breeding. As vigor de- creased there should be an increasing proportion of cases in which the first daughter was replaced by a later member of the family. I was unconscious of any selection that would have favored other than the first individual in the later lines of each series, for the idea of measuring vigor by this method did not occur to me until the experiments were all finished and the data were being com- piled.

6. Difficulty of rearing. As the primary purpose of the experi- ments was not to test vigor, but to obtain a large amount of data regarding egg characters, every effort was made to keep the conditions of nutrition, chemical composition of the medium, etc., at the optimum. To this end the food cultures were changed as frequently as seemed advantageous. If the rotifers became less vigorous, they would be more sensitive to changes in the food cultures, and it would be necessary to renew the latter more frequently.

RESULTS OF THE EXPERIMENTS.

The first five of these measures of vigor can be expressed in figures. The sixth, though not thus expressible because records were not preserved, is not less valuable. Table I. gives the data under the first five headings.

The table shows that, notwithstanding fluctuations, there is an evident decrease in the size of family, of both parthenogenetic and sexual females, from the first line of each series to the last line.

INFLUENCE OF INBREEDING ON IIVDATIXA SEXTA. 5

Notwithstanding fluctuations that are usually small, but occasionally large, the number of eggs laid per day is noticeably less in the later lines than in the earlier ones.

The number of days required to reach maturity remained prac- tically unchanged throughout each series, though the temperature was higher in the later experiments than in the earlier. The first line was reared in November, the last line in June. The higher temperature in May and June should have reduced the time required to reach maturity in those months. In June of the preceding year, two lines which were the ancestors of those given in Table I., but had not been inbred, showed an average time of 1.42 days and 1.56 days, respectively, to reach maturity. The fact that the rate of growth remained practically unchanged in the inbred lines, notwithstanding the increase of temperature in the later experiments, favors the conclusion that the later lines were less vigorous.

The proportion of cases in which the first daughter of a family was not vigorous enough to become the parent of the next generation shows so much fluctuation in the six lines separately that I have combined them two by two. Although the irregu- larities are not thereby completely removed, it is plain that the later lines include a larger proportion of families bred from other than the first daughter than do the earlier lines.

In regard to the sixth measure of vigor, the difficulty of rearing, figures are not available because records were not preserved. It was evident at the time of the experiments, however, that the difficulty of keeping suitable food cultures gradually increased as the inbreeding proceeded Whereas each culture was satis- factory for three or four days in the first experiments, they usually lasted less than two days at the end. This was not due to chemical changes hastened by higher temperatures in the later cultures, for cool periods in the last experiments, when the temperature was lower than the room temperature maintained in the first experiments, did not make the cultures last as long as in the earlier lines. Furthermore, that nothing was wrong with the food cultures themselves was shown by rearing rotifers from an entirely different source, presumably not often inbred, and obtaining from them healthy and vigorous families. The

6 A. FRANKLIN SHULL.

rotifers of the latter experiments must have been more sensitive to adverse conditions.

With all these measures pointing in the same direction, the evidence of decrease of vigor with successive inbreeding seems conclusive.

OTHER EVIDENCE RELATING TO VIGOR IN INBREEDING.

In one of my earlier experiments (Shull, 1911), two partheno- genetic lines of Hydatina were crossed and a new line started from one of the FI fertilized eggs. The FI line was more vigorous, as measured by size of family and rate of growth, than was either parent line. This increased vigor in FI, which Whitney (1912) has since shown to be general in Hydatina, is without doubt the same phenomenon as the decrease of vigor with inbreeding. Later experiments of my own (Shull, 1912), which involved inbreeding twice in succession, though affording some evidence of an attendant decrease of vigor, were in part contradictory. In the light of the present evidence, these contradictions appear to be merely the fluctuations, such as are found in Table I., and all that was needed to clear them up was further inbreeding. So far as Hydatina is concerned, there is thus entire agreement in the results of different experiments and different investigators.

In other animals and in plants there has been accumulated a la-rge amount of information leading to the same conclusion, though not without exception, that inbreeding is attended by deterioration. On the animal side it has long been a common- place among practical breeders that inbreeding, at least in many cases, is followed by a weakening of stock. Results of scientific value have been reported in recent years. Castle (1906) found that inbreeding the fruit-fly Drosophila probably reduces produc- tiveness slightly (though this reduction could be prevented by selection). Moenkhaus (1911) inbred the same fly (Drosophila) and found the operation attended by a considerable increase of sterility (failure of the eggs to hatch). He was not inclined, how- ever, to class sterility as a loss of vigor, since other attributes of vigor were not perceptibly diminished. More decidedly favoring the view that inbreeding reduces vigor is the older work of von Guaita (1898) on the mouse and Ritzema Bos (1894) on rats.

INFLUENCE OF INBREEDING ON HYDATINA SENTA. 7

The great stature of plant hybrids was noted by Kolreuter (1763); numerous examples were cited by Gartner (1849); and experiments with many plants were recorded by Darwin (1876). The value of crossing was known to Beal (1876) who made recom- mendations in regard to the rearing of corn and other plants of commercial value. More recently there have been a number of discussions, accompanying new evidence from plants. Maize has shown in the hands of G. H. Shull (1908), East (1908), and Collins (1910) that inbreeding is accompanied by deterioration, and that crossing between distinct lines brings about an increase of vigor in FI. East and Hayes (1912) obtain similar results in some crosses of tobacco, though not in all, and Wellington (1912) finds that the yield of tomatoes is increased by hybridization.

Further citation of such evidence would be superfluous, as rather full bibliographies have been given in recent papers, notably that of East and Hayes (1912).

MENDELIAN EXPLANATIONS OF VIGOR.

To explain the cases in which inbreeding is accompanied by deterioration, several theories have been advanced in recent years. Some have suggested that inbreeding greatly increases the chance of producing pure recessive combinations; it is neces- sary to assume also that these recessive characters are bad. But others have pointed out that there is equal chance that indi- viduals homozygous for good qualities may be produced.

Two other Mendelian explanations have been offered. One was proposed by G. H. Shull (1908) to explain the greater vigor of FI plants of common maize. He found that successive self- fertilization in corn reduced vigor rather rapidly at first and more slowly afterwards, while crossing two unrelated lines resulted in much more vigorous plants in FI. He attributed the vigor of FI to its "hybridity," and the gradual reduction of vigor with self- fertilization to the gradual establishment of the homozygous condition. Whether there is supposed to be a special set of genes for vigor, or whether heterozygosis in ordinary body characters is held responsible for vigor, Shull does not state. Presumably the genes are not all equipotent, so that heterozygosis in one character may contribute more to vigor than heterozygosis in

8 A. FRANKLIN SHULL.

another character. A similar view is advocated by East and Hayes (1912), but these authors specify that heterozygosis is responsible for only part of the vigor of an individual. The remainder they speak of as "inherent natural vigor" and leave it unexplained.

The other Mendelian explanation is that of Bruce1 (1910). According to Bruce's view, there is an indefinite number of genes concerned with each element of vigor, for example, size. Each element of vigor depends on the number of genes present, but dominance is complete, or nearly complete, so that MmNn con- tributes as much, or nearly as much, to vigor as does MMNN. All these genes are held to be equipotent so that MmNn con- tributes twice as much to vigor as MM, and just as much as XxYy. Vigor is therefore proportional, on this view, to the number of different kinds of gene present, whether in homozygous or heterozygous condition. Essentially the same explanation— the bringing together of dominant characters in the zygote, some of which existed in one parent, others in the other parent was later offered by Keeble and Pellew (1910) to explain greater stature in FI hybrids of certain peas. This would, of course, pro- duce heterozygosis in these characters, but it was the presence of the genes, not their heterozygous condition, to which the authors appealed as an explanation.

As between these last two Mendelian views, the evidence does not now decide; but if either one is correct, the other can, with sufficient work, be proven incorrect.

On Shull's view, according to which vigor depends on the number of genes for which the individual is heterozygous, al- though a single inbreeding of a heterozygous line or a single selfing of a heterozygous individual might, in a few cases, produce offspring heterozygous in just as many genes, and therefore just as vigorous, as its parents; yet successive inbreeding or selfing must, by the laws of chance, eventually result in pure homozygous individuals (homozygous for presence or absence it matters not which). Thus in every pure line (which by definition is homo- zygous) the minimum of vigor has been reached, and that mini-

1 This statement of Professor Bruce's view is taken in part from his paper, in part from correspondence with the author.

INFLUENCE OF INBREEDING ON HYDATINA SENTA. 9

mum must be the same1 for every pure line. Inbreeding must, on this view, always eventually reduce vigor if there is random segregation and recombination.

On Bruce's view, according to which vigor depends on the number of different kinds of gene present, there ought to be some cases in which the inbreeding of a heterozygous line or the selfing of a heterozygous individual would result in F], or F2, or F3, etc., having as many present genes as the parent. Thus a parent having the constitution AaBbCcDd might, on selfing for one or more generations, have a few progeny with the formula AABB- CCDD. Such individuals should be as vigorous as the original parent, or, if dominance is not complete, even more vigorous; and their progeny, produced by self-fertilization, should never show decrease of vigor. Any individuals that came to have the constitution AABBCCdd would be a little less vigorous. Those having the formulas AABBccdd and AAbbccdd would be still less vigorous, and the constitution aabbccdd would represent the minimum of vigor. Thus, if four genes were concerned with vigor, it should be possible to isolate four pure lines, each with its own degree of vigor, which would not thereafter decrease. Each line reaches a minimum of vigor, but that minimum is not the same for different lines.

Either of these two views, as stated, fits the evidence so far obtained from cases in which inbreeding reduces vigor, though evidence could be obtained, at the expense of sufficient labor, which would not fit both. East and Hayes's addition to the heterozygosis view, which would probably be assented to by Shull, namely, the postulation of an "inherent vigor' not dependent on heterozygosis, would make it possible to produce pure lines having different degrees of irreducible vigor. But in no case could a pure line, derived by successive inbreeding from an FI that was more vigorous than its parents, be as vigorous, on the view of Shull, or of East and Hayes, as the original FI; this would be possible, as explained above, on the view of Bruce. If many genes were concerned with vigor, testing the correctness of the two hypotheses on the basis of this distinction would probably require a prohibitive amount of labor.

1 Note, however, the effect of East and Hayes's addition to this theory, discussed below.

10 A. FRANKLIN SHULL.

The view that vigor depends upon the heterozygosis of the individual seems to me inherently more probable than that it is due to the presence of certain dominant genes. The former view admits of a plausible foundation in cell physiology, and the essence of it may be extended to cases of decrease of vigor in which there is no change in the genotypic constitution, and which are therefore without the pale of either theory.

Loss OF VIGOR NOT ACCOUNTED FOR BY THE MENDELIAN

EXPLANATIONS.

It has been shown in a former paper (Shull, 1912) that par- thenogenetic lines of Hydatina may, and usually do, become less vigorous as parthenogenesis proceeds. This conclusion has been confirmed by Whitney (1912). The same phenomenon, though disputed by Woltereck (1911), has been reported in Cladocera by Papanicolau (1910). Some workers have found that clones of Parameciiim decrease in vigor with long continued fission, though Woodruff (1911) has shown that this phenomenon is not uni- versal. In none of these cases, so far as known, is there any change in the genotypic constitution throughout the line; hence a change from heterozygosis to homozygosis is not responsible for the decrease of vigor. The loss of vigor usually spoken of as senescence likewise occurs without, so far as known, any change in zygotic constitution.

The physiological explanation which I am about to offer in- cludes the view that heterozygosis determines vigor, and covers also the cases of parthenogenetic lines and clones just mentioned, and perhaps also of senescence.

A PHYSIOLOGICAL EXPLANATION OF VIGOR. Vigor may be thought of as dependent on the rate of meta- bolism. Lillie (1912), in his studies of fertilization, concludes that the increased metabolism that accompanies the development of the egg is due to an accelerated interaction between nucleus and cytoplasm. The introduction of new nuclear elements into the cytoplasm of the egg, which occurs in cross fertilization, may be supposed to disturb the equilibrium, create a greater reaction between nucleus and cytoplasm, thereby increasing metabolism, and hence vigor. On this view, it is not the fact

INFLUENCE OF INBREEDING ON HYDATINA SENTA. II

that the constitution of the F! individuals is Aim that mal.< - them vigorous, but the interaction of a nucleus1 of constitution Mm with a mass of cytoplasm accustomed, so to speak, to a nucleus of constitution AIM or mm. If it were possible to remove from an egg its own nucleus, and substitute for it a nuclru slightly different, but not so different as to be "incompatible," with a diploid set of chromosomes, and have it develop normally, it should, on my view, produce an individual more vigorous than its parent, even if the introduced nucleus were complch ly homozygous. On this view, a line that has become homozygous need not have reached its minimum of vigor, as it must on both of the other views discussed.

In animals that reproduce by parthenogenesis or fission, the long continued interaction between cytoplasm and nuclei that suffer no change of genotypic constitution, may bring about an approach to equilibrium, thereby decreasing metabolism, and hence vigor.

In like manner, continued production of somatic cells without change of genotypic constitution in the nucleus may cause an approach to equilibrium resulting in senescence in the metazoan individual. The cases in which a high standard of vigor is maintained notwithstanding inbreeding, as in wheat and tobacco, or in the absence of genotypic change, as in Woodruff's para- mecia, are not so easily explained. They may be due to any one of several causes. If metabolism be maintained by a reversible reaction between nucleus and cytoplasm, vigor could be sustained indefinitely. Or the interaction may be kept up by changes in the cytoplasm, changes due to variable nutrition or other external agents. These are mere suggestions.

East and Hayes have suggested a physiological foundation for the heterozygosis view. They hold that increased vigor in hybrids is due to more rapid cell division, and that the stimulus to this more rapid division is given by the presence of genes in the heterozygous condition. To me it seems that the stimulus is due, not to any effect that the two parental contributions to the nucleus may have directly upon one another, but to the effect

1 It is assumed without argument that the representatives of body characters reside in the chromosomes.

12 A. FRANKLIN SHULL.

of a changed nucleus and a (relatively) unaltered cytoplasm upon each other.

Perhaps this suggestion must remain in part mere speculation ; but the science of cell physiology is still young, and much may be discovered that will make the proposed view either probable or improbable.

BIBLIOGRAPHY. Beal, W. J.

'76 Horticultural Experiments. Changing Seeds. Rept. Mich. State Board

Agr., 15, pp. 206-207. Bruce, A. B.

'10 The Mendelian Theory of Heredity and the Augmentation of Vigor.

Science, N. S., Vol. 32, Nov. 4, pp. 627-628. Castle, W. E.

'06 Inbreeding, Cross-breeding and Sterility in Drosophila. Science, N. S.f

Vol. 23, Jan. 26, p. 153. Castle, W. E., Carpenter, F. W., and others.

'06 The Effects of Inbreeding, Cross-breeding and Selection upon the Fertility and Variability of Drosophila. Proc. Amer. Acad. Arts and Sci., Vol. 41, pp. 731-786. Collins, G. N.

'10 The Value of First-generation Hybrids in Corn. Bull. 191, Bur. Plant

Industry, U. S. Dept. Agr., 45 pp. Darwin, C.

'76 The Effects of Cross and Self-fertilization in the Vegetable Kingdom.

London, 482 pp. East, E. M.

'08 Inbreeding in Corn. Rept. Conn. Agr. Exp. Sta. for 1907-1908, pp. 419-

428. East, E. M., and Hayes, H. K.

'12 Heterozygosis in Evolution and in Plant Breeding. Bull. 243, Bur. Plant

Industry, U. S. Dept. Agr., 58 pp. Gartner, C. F.

'49 Versuche und Beobachtungen iiber die Bastarderzeugung im Pflanzenreich.

Stuttgart, 790 pp. Guaita, G. von.

'98 Versuche mit Kreuzungen von verschiedenen Rassen der Hausmaus.

Ber. Naturf. Gesell. Freiburg, Vol. 10, pp. 317-332. Keeble, F., and Pellew, C.

'10 The Mode of Inheritance of Stature and of Time of Flowering in Peas

(Pisum sativuni). Journ. Genetics, Vol. i, pp. 47-56. Kolreuter, J. G.

'63 Dritte Fortsetzung der vorlaufigen Nachricht von einigen das Geschlecht der Pflanzen betreffenden Versuchen und Beobachtungen. Leipzig, 156 pp. (Also in Ostwald's Klassiker d. exakt. Wiss., No. 41, Leipzig, 1893.) Lillie, F. R.

'12 Studies of Fertilization in Nereis. III. The Morphology of the Normal Fertilization of Nereis. IV. The Fertilizing Power of Portions of the

INFLUENCE OF INBREEDING ON HYDATINA SENTA. 13

Spermatozoon. Journ. Exp. Zool., Vol. 12, No. 4, May, pp. 413-454, ii pis. Moenkhaus, W. J.

'n The Effects of Inbreeding and Selection on the Fertility, Vigor and Sex

Ratio of Drosophila ampdophila. Journ. Morph., Vol. 22, pp. 123-154. Papanicolau, G.

'10 Experimented Untersuchungen iiber die Fortpflanzungsverhaltnisse der Daphniden (Simocephalus vetuhis und Moina recliroslris var. Lilljeborgii). Biol. Centralb., Bd. 30, No. 21-24. Ritzema Bos, J.

'94 Untersuchungen iiber die Folgen der Zucht in engster Blutverwandtschaft.

Biol. Centralb., Bd. 14, pp. 75-81. Shull, A. F.

'n Studies in the Life Cycle of Hydatina senta. II. The Role of Temperature,

of the Chemical Composition of the Medium, and of Internal Factors upon

the Ratio of Parthenogenetic to Sexual Forms. Journ. Exp. Zool., Vol. 10,

No. 2, Feb., pp. 117-166,

'12 III. Internal Factors Influencing the Proportion of Male-producers.

Ibid., Vol. 12, No. 2, Feb., pp. 283-317. Shull, G. H.

'08 The Composition of a field of Maize. Rept. Amer. Breed. Assn., Vol. 4,

pp. 296-301. Wellington, R.

'12 Influence of Crossing in Increasing the Yield of the Tomato. Bull. 346,

N. Y. Agr. Exp. Sta., Geneva, pp. 57-76. Whitney, D. D.

'12 Reinvigoration Produced by Cross-fertilization in Hydatina senta. Journ.

Exp. Zool., Vol. 12, No. 3, April, pp. 337-362. Woltereck, R.

'n Uber Veranderung der Sexualitat bei Daphniden. Experimentelle Unter- suchungen iiber die Ursachen der Geschlechtsbestimmung. Internat. Rev. d. ges. Hydrobiol. u. Hydrogr., Bd. 4, Heft i and 2, pp. 91-128. Woodruff, L. L.

'n Two Thousand Generations of Paramecium. Arch. f. Protistenkunde, Bd. 21, No. 3.

THE OTOCYST OF THE PINNID^E.

B. H. GRAVE, UNIVERSITY OF WYOMING.

The conclusions reached in this paper were drawn from a study of the otocyst in three species of lamellibranch belonging to the family Pinnidse.

I became interested in the subject some time ago, when I found what appeared to be an abnormal otocyst in Atrina rigida.1 In the paper referred to, it was intimated that the otocyst of this lamellibranch showed signs of degeneration. Further study of this organ in the same and related species has convinced me that the otocyst of the Pinnidae is functionless.

The following description was written after examining some- thing over sixty specimens by the method of serial sections.

Unlike the homologous structure of other lamellibranchs, this sense organ is situated in the tip of the foot at a very considerable distance from the pedal ganglion. In general, it resembles the ordinary lamellibranch otocyst, but differs in being exceedingly large and compound. (See Figs. I, 2 and 3.)

Instead of being a simple capsule, it ordinarily consists of several lobes, each containing an otolith. (See Fig. 3.) The lobes are quite variable in size, but as a rule they are remarkably large. One by measurement is 780 microns in diameter, while the enclosed otolith measures 520 microns. It will be noted that an object of this size can readily be seen with the unaided eye. In fact the otocyst in question stands out quite prominently.

By reference to the figures it will be noted that the larger number of the lobes of these otocysts lie in contact with each other. This together with the fact that the cavities of two ad- joining lobes are frequently found to be in open communication, indicates that they were formed from an original otocyst by budding. It seems, therefore, that budding accounts for the compound nature of these otocysts.

1 B. H. Grave, "Anatomy and Physiology of Atrina rigida," Bulletin of the Bureau of Fisheries, Vol. XXIX., 1909.

THE OTOCYST OF THE PlXXIDjE.

Be this as it may, the lobes are not all joined in a single mass. In fact, all of the cases figured show two isolated groups. The series of sections from which Fig. i was reconstructed shows that this particular otocyst was formed by two separate invagiiiaiions from the ectoderm. The tubes indicating their ectodermal nature still persist, as illustrated in Fig. 4. The same ectodermal

I 2

FIGS, i and 2 are reconstructions of the two otocysts of a single specimen. Note that each is composed of two separate groups of capsules. Note also that the appearance is such as to suggest its origin through a process of budding.

origion of the otocyst of another specimen, not here figured, is demonstrated by similar ectodermal tubes.

It is generally conceded that the otocysts of all mollusks are ectodermal in origin, but it is unusual for their connection with the ectoderm to remain intact in the adult. Such a connection has not. up to this time been observed except in the primitive unspecialized Protobranchia.1- y

1 Drew, G. A., "Life History of Nucula delphinodonla," Quarterly Jour. Micr. Set., Vol. 44, Part 3, new series.

2Lankester, E. Ray, "A Treatise on Zoology," Part V., page 18.

16

B. H. GRAVE.

It appears that most of the individuals of the Pinnidae lack the otocyst altogether. There is not a trace of such an organ to be found in nine tenths of the specimens! In all, I have found only six with otocysts and in these they are highly variable in size and shape.

Of five specimens of Pinna nobilis from the Mediterranean, not one had an otocyst, and an equal number of specimens of the

3 4

FIG. 3. Reconstruction of a particularly compound otocyst. FIG. 4. Drawing of a section of a single capsule of an otocyst, outlined with a

camera lucida. o, otolith; e, ciliated epithelium; t, ectodermal tube which gave

rise to the otocyst.

small red Pinna from Jamaica1 were examined with the same negative result.

On anatomical grounds, therefore, one is almost justified in concluding that the otocyst of the Pinnidae is functionless.

1 I am indebted to Professor E. A. Andrews for specimens of this Jamaican species.

THE OTOCYST OF THE PINXID/E. \~J

PHYSIOLOGY OF THE OTOCYST.

While at Beaufort, North Carolina,' during the summer of 1911, the writer made a study of the function of the otocyst of Atrina rigidia, but the experiments gave only negative results.

A considerable number of specimens were brought into the laboratory and kept under observation to learn their individual behavior. They seemed to surfer no inconvenience after the removal of the tip of the foot, which supposedly contained the otocyst. The normal activities were continued after the opera- tion as before. The following conclusions seem justifiable:

First. A large per cent, of Pinnas have no otocyst. When one is present, it is abnormally large and curiously pathological in appearance.

Second. Anatomical and physiological evidence seem to indi- cate that the otocyst of the Pinnid^p is undergoing degeneration, and is at present of no functional value.

1 I wish to acknowledge my indebtedness to Hon. George M. Bowers for the use of a table at the fisheries laboratory during my stay at Beaufort.

ON THE BREEDING HABITS OF BUTLER'S GARTER- SNAKE.

ALEXANDER G. RUTHVEN, MUSEUM OF NATURAL HISTORY, UNIVERSITY OF MICHIGAN.

Relatively few observations have been made on the breeding habits of snakes, at least few have been published, and there has appeared in print considerable misinformation on the subject, due principally to wrong identification of species. There is especially a deplorable lack of careful studies of the life-histories of the different forms.1 Concerning the North American forms, we know that some are ovo-viviparous and others oviparous, that copulation probably takes place ordinarily in the spring, although in one species it has been said to occur also in the fall,2 that the young appear in the late summer, and there is some evidence that there is a gregarious tendency in the breeding season that may lead to the formation of "piles" of snakes.3 But of the exact time of copulation, the courtship reactions, the significance and commonness of the "snake piles," the length of the gestation period in the different forms, and kindred subjects only the most meager data have been gathered.

During the present year the writer has been able to get a pair of Butler's garter-snake (Thamnophis butleri Cope) to breed in captivity and has carried the female over the period of gestation. On April 9, which was about the first day in spring when the snakes were at all active in this region, a male and female of this species were found together near Ann Arbor. These failed to copulate in captivity, although the male courted the female assiduously for several days. On April 10, seven specimens were collected, and a lot consisting of five males and a large female

1 An excellent summary of the data on the breeding habits of certain North American snakes is given by O. P. Hay, Proc. U. S. Nat. Mus., XV., pp. 385-398.

2 Coues and Yarrow, Bull. Geol. and Geog. Surv. Terr., IV., 278.

3 For a resume of the literature on this subject see Ruthven, A. G., Bull. U. S. Nat. Mus., 61, pp. 13-14.

18

BREEDING HABITS OF BUTLER'S GARTER-SNAKE. 19

placed together in a cage. The female was courted by the males for five days before she appeared to be ready for copulation.

The method of courtship is exactly as described1 for T. sirtalis, The male or males lie on or closely along side of the female, keep up at intervals a spasmodic movement of the abdomen, and endeavor to maintain a loop of the tail over that of the female and to insert the posterior part of the body, ventral side up, under hers.

Early on the morning of April 15 five males were at the same time endeavoring to copulate with the female, showing at once that the sexual impulse was at its height and confirming the con- clusion that the so-called snake piles are due to this impulse. The exact moment of copulation was not observed but it was within a few minutes of 12 noon. When one of the males had succeeded in inserting one of its hemipeni the right in the cloaca of the female the other males at once crawled away.

The pair remained in coitu for two hours and fifteen minutes. During that time the male endeavored to maintain a position along the back or close to the side of the female and when in this position kept up the abdominal movements, but the female moved rather constantly about, dragging the male often at full length behind her. Occasionally she rolled rapidly over and over as many as ten times, turning the body of the male at the same time,2 but this did not break the connection, confirming Cope's3 state- ment that the hemipenis cannot be withdrawn except by invagination.

When the act of copulation was completed the male was removed and the female carefully cared for. She ate freely, was fed as much as she would eat, and was little disturbed. Under this treatment she remained in the best of health and on Sep- tember 6, about 10 A.M., gave birth to thirteen young. This makes the period of gestation almost exactly 144 days.

It should be remarked that either the length of the period of gestation varies, or the breeding season is of some length and depends upon whether the spring is early or late, for the writer

1 Ruthven, A. G., loc. cit., p. 178.

2 More or less of this restlessness of the female may have been due to her being in captivity.

£Cope, E. D., Kept. U. S. Nat. Mus., 1898 (1900), p. 701.

20 ALEXANDER G. RUTHVEN.

has recorded the birth of a brood as early as August 7,1 and on August 25, 1912, a young specimen collected at Ann Arbor and at the most but a few days old was received by the museum. It is very probable that the length of the gestation period is rather exact for the species, that the snakes breed approximately as soon as the weather is warm enough to permit them to become active, and that the breeding season is