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_ BIOLOGICAL BULLETIN 


Marine Biological Laboratory 


WOODS HOLE, MASS. a 


ceanian Instiige 
SX i. \ 


A ~ JUN 30 1915 


Editorial Statt Na as 


E. G. Conxiin—Princeton University. 
Jacques Lors— The Rockefeller Institute for Medical Research, 
Gerorce T. Moore— Zhe Missouri Botanical Garden. 
, : T. H. Morcan— Columbia University. 
W. M. WxHeEeELer—Aarvard University. 
E. B. Wirtson— Columbia Universzty. 


Managing Loitor 
Frank R. Liririe— Zhe University of Chicago. 


VoLUME XXVIII. 


WOODS HOLE, MASS. 
‘JANUARY TO JUNE 1015 


TT 


P 
THE NEW ERA 
‘LAN 


CONTENTS OF VOLUME XXVIII. 


No. 1.. JANUARY, IQI5. 
(usta hs) nitvaronio; Development in Nereis. 0)... . 
LILLIE, FRANK R. Sperm Agglutination and Fertilization...:.. 
Wireman, H. L. Observations on the Spermatogenesis of the Gall- 
LUM, [DSO ARO GPU REGO (MAGI) <3 ea a A a a cle 
SweEzy, OLive. Egg Albumen as a Culture Medium for Chick 
MRISSUUCIS Sooke: SOU e ee CBee .a Gn 8 AS eet a i gia ga came a 
WERBER, E. I. The Influence of Products of Pathologic Meta- 
bolismion the Developine NeleostOuwny ee 


No. 2. FEBRUARY, I9Q15. 


Lores, Leo. An Early Stage of an Experimentally Produced 
Intrauterine Pregnancy and the Spontaneous Parthenogenesis 
Oihe Hoos mmuike Quary oj the Guinea Pigs) sean. 

GARREY, WALTER E. Some Cryoscopic and Osmotic Data...... 

LoEB, JACQUES. Concerning Brachet’s Ideas of the Réle of Mem- 
ORAM eR OLMGAMOM AU NHCHEN ZOOM seme ey tae a ee 

Just, E.E. An Experimental Analysis of Fertilization in Platy- 
ORCUS GGL OLDS je oe Sie pee a Nea rN OMEE LIne Oa eke tire Sk Maks Nel 

LINDON ODWiNe ES POKoGysts aman Ajnelid ses. 4.0 alee 

No. 3. Marcu, Io15. 

SUTHERLAND, G. F. Nuclear Changes in the Regenerating Spinal 
Cord of the Tadpole of Rana clamitans................... 

Ricuarps, A., AND Woopwarp, A. E. Note on the Effect of X- 
IROCVOOD: Ol, TG RIMTAUES oe hs ols) PAB ols os ee Bl 

GLASER, OrTo. Can a Single Spermatozoén Initiate Development 
hip. LAUTAN OE RON se Se, AY eh LA REE PE Shs OR an LD 

Curtis, MAyNiE R. Studies on the Physiology of Reproduction 
UO HUE IDO GORE IPODS” DLA G ENE ian ou a We eee 


No. 4. APRIL, IQ15. 
Wootsey, CARRIE I. Linkage of Chromosomes Correlated with 
Reduction in Numbers Among the Species of a Genus, also 
Wahine nS pectesroj. the Locustid@s: 28s. el: 


111 


140 


149 


154 


iy 


1V CONTENTS. 


- SHULL, A. FRANKLIN. Periodicity in the Production of Males in 


LSC HOR S.”’ Cre D es GIS bo oo Go oc aoc 
Linton, Epwin. Note on Trematode Sporocysts and Cercarieé in 
Marine Mollusks of the Woods Hole Region............... 
Purers, C.F. An Experimental Study of the Behavior of Amph- 
pods with Respect to Light Intensity Direction of Rays and 
Metabolism..... At eR One Ma TA ye ny Ui aba She dscns: 
Ewine, H.E.. A ‘Case of Persistent Melonisi ae ae ae eee 
LILLIE, PRANK IR: Siudves of Fertilization ee 


No. 5. May, 1915. 
Moore, ARTHUR R. On the Rhythmical Susceptibility of Develop- 
ang Sea Urchin Eggs to Hypertonic Sea Water..........-.% 
LILLIE, RALPH S. On the Conditions of Activation of Unfertilized 
Starfish Eggs under the Influence of High Temperatures and 
Fatty Actd (SOuUutows oc. a See oe 
BupincTon, R. A., AND HARVEY, H. F. Duivision Rate in Ciliate 
Protozoa as I eens by Thyroid Constituents . 


SHELFORD, V. E., AND Powers, E. B. An Experimental Study of 


the Movements of Herring and Other Marine Fishes......... 


No. 6. JUNE, 1915. 
Seventeenth Annual Report of the Marine Biological Laboratory. . 
Haraitt, Cuas. W. Regenerative Potencies of Dissociated Cells 
Of FIV CPOMCEUSC. 1 oxy. Wie Lhe se ee eee 


SPURGEON, CHARLES H. The Eyes of Cambarus setosus and Cam- 


barus DEUUWCIdUs oooh hos Ck Ss CE eee 
Morcutis, S., Hower, PAaut E., anD Hawk, P. B. Studies on 
Liussues of Fasting Animals... .s55)) hee ee ee 
McInpoo, N. E. The Olfactory Sense of Coleoptera... ....-.%-: 


187 


Shah 


- Aarine sBioloatcal aboratory : ee 


woons HOLE, MASS. 


‘Laue, Frank R. - Sperm Aaglatination and ee 18 ae 


‘Observations. Om the Spermatogenesis of the 


| Gall Py Dr ee erinacet i Mayr he 


: Swezy, oe ee Albumen as: a Culterre Medium efor 


; a i Cndee Tissue. 7 NM Ee See 4 an 


Wenner, B i The Influence ae Pee of Pathologic. 


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Vol. XXVIII. January, Tors. No. 1 


SIOLOGICAL BULLETIN 


INITIATION OF DEVELOPMENT IN NEREIS. 


D, 13, US. 


(From the Marine Biological Laboratory, Woods Hole, Mass., and the Physiological 
Laboratory, Howard University School of Medicine, Washington, D. (E>) 


If any apology be needed for merely adding to the long list of 
eggs susceptible to agents of artificial parthenogenesis it may be 
suggested that initiation of development in annelids possesses 
some interest since annelid ova respond only with difficulty 
to agents that induce development.1 We need but recall the 
case of Chetopterus (Loeb, ’orb, Lillie ’o2, Allyn), of Amphitrite 
(Loeb, ’o1a, Scott), of Podarke (Treadwell), of the Pacific Nereis 
(Loeb, ’13), of Polynoe (Loeb, ’07, ’08) and of Nereis limbata 
(Fischer, Lillie, 11, Loeb, 120, ’13)? to justify this statement. 
In all these eggs differentiation without cleavage is far easier to 
obtain than development closely simulating the normal. Among 
diverse agents few only will give cleavage in Chetopterus (Allyn, 
Loeb and Wasteneys). The case of Thalassema stands almost 
alone among annelids in giving development which is, according 
to Lefevre, to a surprising degree like the normal. Another 
instance among annelids of differentiation with cleavage arti- 
ficially induced is worthy of note. 

This report, however, on the initiation of development in 
Nereis by heat has, I think, special significance. The results 
here offered require an interpretation which concerns the funda- 
mental theory of parthenogenesis and fertilization. 


EXPERIMENTAL. 


Certain preliminary experiments date from 1913. The ex- 
periments given here were performed during June, July, and 


1 Bullot claims to have produced normal development in Ophelia with artificial 
means. 
* Loeb’s experiments with Nereis (’12) were apparently incomplete. 
I 


2 BAe. JUST: 


August 1914, at the Marine Biological Laboratory, Woods Hole. 
The majority of the experiments deal with the effect of heat on 
the Nereis egg. Under A these experiments are described. 
Under B are described experiments with KCl. 


A. THe EFFECT OF WARMING ON THE INITIATION OF DEVELOP- 
MENT IN NEREIS. 

Methods.—At first all sea-water used was heated, usually not 
beyond 75° C., to destroy any spermatozoa possibly present, 
cooled, and vigorously shaken before the experiment. But this 
is unnecessary, as my observations showed. I have kept Nereis 
eggs in sea-water during the cool days of June for thirty-six 
hours without even jelly secretion. During several seasons I 
have never found eggs spontaneously developing in sea-water, 
although eggs occasionally extrude part of their jelly. More- 
over, in not a single uninseminated control in ordinary sea-water 


was a developing egg ever found. In many experiments in - 


addition to the uninseminated control a batch of eggs from the 
same animal as those warmed was inseminated. It was thus 
clearly proved that the eggs subjected to warming are in no wise 
abnormal. For fear of contamination, the needless inseminated 
control was discarded in the later work. 

For a given experiment the following procedure was adopted: 

A small flask or a large test tube with a measured quantity of 
sea-water was placed in a large beaker of sea-water. This was 
warmed over an alcohol flame and the temperature kept constant 


by the use of thermometers in the flask and in the beaker. The 


eggs were generally from one female; if from several small ones, 
they were mixed so that the inseminated or uninseminated con- 
trols and the warmed eggs were always the same. The eggs in 
the initial experiments (see below) were either from females cut 
in the warm sea-water or they were put in the warm sea-water 
dry; 7. e., from a thoroughly dried female which was pricked to 
cause the escape of eggs. Eggs were also subjected to heat after 
washing by changing the sea-water several times during various 
intervals of time. By means of a capillary pipette measured 
quantities of eggs were transferred after exposure at varying 
intervals to five or to one hundred c.c. of ordinary sea-water. The 


Pic. on 


INITIATION OF DEVELOPMENT IN NEREIS. 3 


experiments: were performed during the morning and afternoon 
following the evening that the worms were captured. A few 
experiments were performed during the evening of capture. 

The point to be emphasized is that washing in sea-water so 
modifies the eggs that they do not respond readily, or at all, to 
parthenogenetic treatment. 


THE EXPERIMENTS. 

The experiments with heat may be divided into four groups as 
follows: 
I. The initial experiments in which the eggs were cut from the 

animals while in the warm sea-water. 

2. The experiments with dry eggs. 
3. The experiments with eggs in warm “serum.” 
4. The experiments with washed eggs. 


1. THE INITIAL EXPERIMENTS. 


In the initial experiments, worms were cut in 5, I0, 25, 50, 
anc nOOnc cy Of sea-waten at BO, 32-4138 43447 35) 307_C., the: 
worms removed and the eggs exposed for from five to fifteen 
minutes. 

The following experiments selected from a number give the 
details: 

(a) July 22,1:45 P.M. A female put in 100 c.c. of sea-water 
at 31° C. swims actively without discharging eggs. At 1:50, 
the temperature is 35° C. Eggs are cut out, the worm removed. 
Ten samples of eggs are removed to 5 c.c. of ordinary sea-water 
AsmiOllows: "2.08902 NOr 2 259 2):35)) 2:45, 2255 3:05, 4:15, 3:25. 
Bea aes 

At times the temperature rose to 36° C. and once to 36.5° C. 
Many eggs at the time of removal from the warm sea-water 
exhibited membranes standing off at an unusual distance, others 
were darker than normal, and a few had disintegrated. Later 
experiments showed that these changes are due to exposure at 
too high temperature. Even five minutes exposure at 37° C. 
will bring them about. The jelly is formed in the warm water, 
and often at 35° C. or above it is dissolved and disappears. This 
may be shown by examining eggs in India ink ground up in 


4 B. EU ust: 


sea-water. Many developing eggs are devoid of jelly hull, but 
the cortical changes are complete. 

One hour and ten minutes after exposure, some eggs are in 
“blister” cleavages; that is, the protoplasm is irregularly budded. 
One hour and thirty minutes to two hours after the change to 
ordinary sea-water, among all gradations of cleavage-like patterns 
are some normal two and four-cell stages. The next day, Nos. 3 
to 10 showed some real cleavages and a small per cent. of ap- 
parently normal swimming forms. Many are beaded or blis- 


7 


tered, some are unsegmented “‘swimmers,’’ and some two and 
four-cell swimming forms. Some eggs remain in the germinal 
vesicle stage. 

(b) June 23, 11:00 A.M. A female placed in 50 c.c. of sea- 
water at 35.5° C. is rendered immobile but does not shed. Eggs 
are cut out at 11:02, the worm removed. Six samples of eggs 
are taken as follows: 11:18, 11:25, 11:34, 11:40, 11:45, 11:50. 

Many eggs on removal from the warm sea-water show the 
jelly formed. The membranes after jelly formation are still a 
little farther from the eggs than in normal fertilization. Many 
eggs remain in the germinal vesicle stage with the cortex intact. 

1:45 P.M. Fairly normal cleavages in Nos. I to 4. 

June 24, 8:30 A.M. Swimming forms are found in the dishes. 
By far the best are those in Nos. 2 and 3. 

The optimum time of exposure, therefore, lies between twenty- 
three and thirty-two minutes. Later experiments showed that 
the optimum exposure at 35° C. is at or near twenty-five minutes. 

(c) June 24, several experiments were run at various tem- 
peratures. Those at 35° C. confirmed the findings of the previous 
ones. Temperatures ranging from 30° C. to 31° C. give no results; 
regardless of the length of exposure the eggs remain in the ger- 
minal vesicle stage. 

The following experiment of June 24, at 33° C. is typical of a 
number of repetitions at this temperature: 

(d) June 24, 11:15 A.M. A female placed in 25 c.c. of sea- 
water at 33° C. swims actively without discharging eggs. Eggs 
are cut out at 11:15 and samples taken at five-minute intervals 
up to 11:50. The temperature is practically constant. The 
samples taken are masses of eggs with the cortex wholly or (in 


INITIATION OF DEVELOPMENT IN NEREIS. 5 


earlier ones) partially broken down. The cytoplasm is normal 
in color and the membranes normal. 

1:35 P.M., many eggs are in cleavage. 

4:00 P.M., many eggs are in late cleavage. 

June 25, 9:00 A.M. The dishes show a good per cent. of very 
fine ‘swimmers.’ The cleavage seems almost normal. 

Thirty-five minutes’ exposure gives by far the highest per- 
centage of swimming forms. As in all the experiments of this 
group, some eggs remain in the germinal vesicle stage with 
cortex intact. 

If eggs be warmed in “‘egg-water’’ (sea-water charged by eggs 
that have remained in it for several hours) the results are no 
different. 

To sum up, we find that eggs of Nereis cut out in warm sea- 
water and exposed to temperatures ranging from 33° to 35° C. 
develop with cleavage which is closely similar to the normal. 
Some eggs remain in the germinal vesicle stage. For the best 
percentage of swimming forms the optimum exposure at 35° is 
twenty-five minutes; at 33°, is thirty-five minutes. 


2. EXPERIMENTS WITH Dry EGGS. 


Many of the experiments with dry eggs were run with the 
washed egg series. In the majority of cases eggs from one female 
thoroughly dried on clean filter paper were received in a dry 
watch glass.1 These eggs were divided into two lots; one lot 
warmed in sea-water at the given temperature and the other 
washed by changing the sea-water several times, allowed to 
settle, and after draining placed in the warm sea-water. 

A large number of experiments was made with dry eggs, in the 
attempt to determine the quantitative relations early found to 
control the number of eggs developing. Thus, with smaller 
quantities of warm sea-water every single egg quickly forms jelly 
and at least ninety-eight per cent. cleave, but with larger quan- 
tities of warm sea-water the percentages are lower. 

As Miss Allyn found for Chetopterus cleavage appears to in- 

1 With dry eggs one must be careful for the mere drying will initiate changes as 


Ihavefound. Eggs left on filter paper for from five to twenty minutes form jelly, 
a small per cent. cleave and a few swim. 


6 De 135 AUIS. 


terfere with the further development. I have never been able to 
get more than twenty per cent. of these eggs to reach the swim- 
ming stage. If one could determine definitely the quantitative 
relations this percentage might be increased. From the observa- 
tions it appears that the optimum amount of warm water used 
varies; it depends upon the bulk of the eggs. While best results 
are got with small quantities of water, it is possible to use too 
little—three c.c. for instance, for the eggs for a large female. 
Jelly formation and cleavage are induced but swimming forms 
are less numerous than in the case of ten c.c. for about the same 
bulk of eggs. 

The following are typical experiments of this group: 

(a) July 16, 10:30 A.M. Eggs from a dry female in a dry 
. watch glass are divided into two lots; one lot washed, the other 
transferred to 5 cc. of sea-water at 34° C. Samples out as follows: 
at 10:20, 10:50, and at five-minute intervals thereafter to 11:20. 

2:00 P.M. Uninseminated control, no change. Every single 
warmed egg had formed jelly: all have formed polar bodies. At 
least half of these are in cleavage stages. 

July 17. All dry eggs in some stage of cleavage, many of 
which are normal; some swimming forms in many of the dishes 
even after forty minutes’ exposure. 

(b) July 17, 9:58 A.M. Eggs from a dry female divided into 
two lots. Lot A in 5 c.c. of sea-water; Lot B in 20c.c. of sea-water. 
Both exposed to 33° C. 10:01 jelly formation. Eight samples 
taken as follows: 10:05, 10:11, 10:16, 10:22, 10:27, 10:33, 10:38, 
and 10:43. 

Lot A gave at least 95 per cent. of cleavage and a percentage 
of swimming forms in all dishes beginning with No. 3 (the 18- 
minute exposure). Lot B gave 75 per cent. of cleavage and best 
swimmers for 24, 29, and 35-minute exposures. 

(c) Other experiments showed that the highest per cent. 
(100 per cent.) of jelly formation and of cleavage (98 to 99 per 
cent.) is in the smaller quantities of sea-water—5, 6, and 10 c.c. 
—whatever the temperature;a fewswim. With larger quantities 
of sea-water at the various temperatures more eggs remain in 
the germinal vesicle stage. The lower exposures give most 
normal-looking swimming forms—trochophores scarcely to be 


INITIATION OF DEVELOPMENT IN NEREIS. 7 


distinguished from the normal either while living or in sectioned 
material. The higher exposures give more abnormal swimming 
forms. 

For comparisons I have selected the following tables from my 
notes to show the percentages of cleavage and of “‘swimmers”’ 
obtained with eggs from worms cut in warm sea-water and with 
dry eggs. Itis apparent at once that while there is no appreciable 
difference in the percentages of swimming forms after warming 
either the ‘‘cut out” or the dry eggs, there is a marked difference 
in the percentages of cleaving eggs. This is the case in all the 
experiments. 

_ July 12. Two females cut up at 9:55 A.M. in separate flasks 
of sea-water at 35° C. gave the following results: 


Female No. fr. 


Sample Taken Cleavage. Swimming Forms. 


IN@: 2 10:06 65% Po % 
sigice vo 10:13 50 4 
rare 10:15 55 5 
Wael 10:20 67 5 
Semtsiy 10:25 60 9 
Be KG) 10:30 72 3 
re Pati 10:35 | 47 | 7 
eS 10:40 \ 68 4—abnormal 
re IO:45 | 62 very abnormal 
LO T0:50 33 | very abnormal 
Female No. 2. 
Sample Taken Cleavage. | Swimming Forms. 
No. 1 10:07 66% | 3% 
ey es 52 IO:14 42 8 
Dy eis I0:16 33 I2 
ata vd: TO:21 AT | I4 
Stns 10:26 44° 3 
ect) I0:31 74 no) 
iene 10:36 24 2—abnormal 
oer) IO:41 81 | 5s— “* 
crs OFda| 10:46 32 very abnormal 
oF 6G) IO:51 17 very abnormal 


July 20. Dry eggs in 30 cc. of warm water at 35° C. gave the 
following results: 


Exposure, Cleavage. Swimming Forms. 
I8 minutes 90% 1% 
23 se oe I5 % 
30 ce ce Io % 


35 ce GG 1% 


8 1B Be USI. 


With dry eggs one may obtain 100 per cent. cleavage; with the 
eggs cut from worms in warm water one never gets more than 81 
per cent. the average being very much lower as the figures given 
above show. With both kinds of eggs 20 per cent. swimming 
forms is the maximum, the optimum exposure for the various 
temperatures used being the same. 

Experiments show that the use of warm “‘egg-water’’ does not 
improve the results. 


3. THE EXPERIMENTS WITH SERUM EGGs. 


The effect of warming Nereis eggs in the body fluids was studied 
with difficulty mainly because of the scarcity of body fluid in 
Nereis. As Lillie has pointed out this worm is little more than a 
bag of eggs. The amount of blood present is negligible and 
unavailable for. warming experiments. I therefore adopted the 
method used by Lillie—that of cutting up spent females. In his 
study this juice gave results comparable to the perivisceral fluids 
in Arbacia.. For an experiment I minced as many spent females 
as I could get, using a small quantity of sea-water; the juice thus 
obtained is designated as ‘‘serum.” While I think that my 
experiments with this serum are conclusive I wish to point out 
that Nereis is not the most favorable form with which to establish 
the fact of serum inhibition—certainly this is true for the method 
I used. It may be stated at the outset that as Lillie found for 
both Nereis and Arbacia I have found repeatedly that the 
“serum” of Nereis quite definitely inhibits fertilization. Further- 
more, just as definitely does the serum inhibit initiation of de- 
velopment with warming. I cite experiments to give the details: . 

(a) July 15, 10:55. Eggs from one fine large female previously 
dried are divided into four lots. Eleven spent females are finely 
minced to procure twenty drops of ‘‘serum.’’ Ten drops of the 
“serum”’ is added to each of two dishes containing 3 c.c. of sea- 
water; eggs added to both. One lot is warmed at 34.5° C.— 
Lot A; Lot Binseminated. Samples of A are taken at five-minute 
intervals up to 11:35. 2:00 P.M., I per cent. of cleavage in . 
both lots. Next day no swimming forms in either. Eggs from 
the same female, Lot C, warmed in sea-water and Lot D, in- 
seminated, develop. 


INITIATION OF DEVELOPMENT IN NEREIS. 9 


During the afternoon of July 15 this experiment was repeated 
with the’same results. 

(b) July 22. Eggs warmed at 34° C. in serum plus sea-water 
(serum from the bodies of seven spent females cut up in two c.c. 
of sea-water): 10 drops plus three c.c. sea-water, 10 drops plus 
five c.c. sea-water and Io drops plus ten c.c. sea-water. 

Eggs exposed for twenty-five minutes. Less than one per 
cent. developed in any dish. 

During August these results with serum eggs were verified. 
The highest per cent. of swimming forms obtained was one per 
cent.; this was with a very dilute serum. Not only do the eggs 
fail fo cleave but fail in the great number of cases even to form 
jelly. In some cases the development of eggs inseminated in 
serum was farther advanced than the serum warmed eggs. 
Since in the case of the initial experiments the worms were cut 
up in sea-water, it may be that failure of a percentage of eggs to 
cleave is due to the inhibition of the escaping blood and tissue 
juice. With the dry eggs cut quickly on the dry watch glass this 
escaping juice cannot so easily contaminate the eggs. 


4. THE EXPERIMENTS WITH WASHED EGGs. 


In Platynereis sea-water definitely destroys the fertilizing power 
of the egg. Even minute quantities of sea-water will render the 
egg incapable of cleavage although the spermatozoa may pene- 
trate. Moreover, if the eggs of one female remain in a small 
quantity of sea-water, 5 c.c., for instance, for thirty seconds their 
fertilizing power is lost. And yet in nature, inseminated eggs 
begin to be laid in many cases five or six seconds after copulation 
(see Just,’14). In Nereis, therefore, it was thought that washings 
in sea-water by frequent changes through several hours might 
act as the sea-water does in such a surprisingly short time on 
Platynereis eggs. 

During the June Nereis run, then, as many experiments as 
possible were conducted to determine the ‘‘fertilizable”’ period by 
inseminating at intervals eggs that had remained in sea-water 
with and without frequent washings. Lillie has shown for 
Arbacia eggs that the capacity for being fertilized decreased with 
the decreased secretion of fertilizin. He finds for Nereis also 
very much the same relationship. 


Io Be Ey JUS. 


Without going into details, it may be said at the outset that 
the egg of Nereis gradually loses its power of being fertilized and 
eventually reaches the condition of the Platynereis egg where in- 
semination induces maturation only. I cite a single experiment. 

June 28, 9:10 P.M. Dishes of eggs Nos. 1, 2, and 3 were set 
aside. The next day at 2:10 P.M. each dish of eggs was drained 
and divided into two lots—A and B. Lot A in each case was 
inseminated in the water-which had stood over the eggs for seven- 
teen hours. Lot B of each dish was inseminated in fresh sea- 
water. No eggs in either lot of No. 1 developed beyond matur- 
tion. In Lots A and B of Nos. 2 and 3, .1 per cent. or less went 
as far as the two-cell stage. Some eggs in all the dishes were in 
the germinal vesicle stage. No trochophores were found. 

Eggs were frequently tied in bags of filter paper and placed 
in a beaker under running water for twelve hours. In other 
cases they were washed by changing the water at odd times during 
the day. It was found that eggs differ greatly with respect to 
the time that they must remain in sea-water before they lose 
their fertilizing power, but it may be clearly proved that washing 
or staling of Nereis eggs renders them incapable of being fertilized. 
This stage may be reached after three hours in seawater (cf. 
Just, ’12): 

This varying susceptibility proved very disappointing because 
I had suspected, not, of course, the degree of susceptibility present 
in Platynereis, but perhaps such as could be expressed more 
definitely. 

Because of these results with washed and stale egg insemina- 
tion, when the warm sea-water experiments were continued during 
the July Nereis ‘“‘run”’ I was certainly unprepared for the results 
obtained. The following experiments are typical of a large 
number performed almost daily during the July and August 
“TcEb have fe 

(a) July 11, 9:50 A.M. Eggs from one female divided into 
two lots; one lot put in sea-water. This lot transferred from the 
sea-water to warmed sea-water (35° C.). The eggs form jelly in 
the warm sea-water and make a mass which has to be shaken to 
obtain samples. 10:55, many have formed jelly and maturated, 
but most retain jelly with germinal vesicle intact. Some of 


INITIATION OF DEVELOPMENT IN NEREIS. iti 


these eggs again subjected to heat; no results. July 12. Very 
few, I in 1000, swimming. 

(b) July 15, 3:20 P.M. Eggs cut out and washed, put in 6 
 c.c. of sea-water warmed at 35°C. Samples taken at five-minute 
intervals for forty minutes. Next day: Majority are in germinal 
vesicle stage, at least seventy-five per cent. Less than one per 

cent. swimming. 
~ (c) July 16, 9:35 A.M. Eggs washed ten times evening before 
and five times during this morning. Two series: A inseminated, 
B in warmed sea-water at 3:50 samples taken (ten in all) at five- 
minute intervals. Uninseminated control. 

July 17, 1:30 P.M. No development in uninseminated control 
(few have cytolyzed). Inseminated eggs show that few have 
formed jelly (ten to fifteen per cent.). One per cent. have cleaved 
and some of these swim. Of the warmed eggs at least ninety-five 
per cent. are in the germinal vesicle stage with cortex intact. 
Less than one per cent. have formed polar bodies. ° : 

(d) July 16, 10:30 A.M. Eggs from a dried female divided in 
two lots; one lot washed in 100 c.c. of sea-water by changing the 
water four times. 10:40 A.M. In warmed sea-water, 34° C. 
Samples out at five-minute intervals for sixty minutes. 

2:00 P.M. At least ninety per cent. in the germinal vesicle 
stage, small per cent. form jelly and divide. Next day, none 
swim. 

I was tempted to discredit my June experiments after the first 
of these findings. I could only convince myself after running 
series after series of washed and dry eggs along with eggs cut out 
directly into warmed sea-water. Most workers in inseminating 
eggs obtain the sexual products in separate dishes, and add sperm. 
Such procedure succeeds admirably with Nereis giving one 
hundred per cent. of cleavage. But if eggs be cut out of Nereis 
in sea-water, divided in two lots, and washed once or twice, one 
lot being inseminated and the other warmed we get the surprising 
result that while every single inseminated egg develops, few of the 
warmed go beyond maturation. If the water over the eggs be 
changed a few times in ten minutes, ninety per cent. warmed in 
sea-water fail even to maturate. 

This must mean that the egg of Nereis is so susceptible to 


2 E. E. JUST. 


sea-water that warming fails after washing although fertilization 
is still possible. If fertilization be impossible (as in stale eggs) 
warming also produces no effect. 

Washed or stale eggs warmed in sea-water charged by eggs that 
have remained in it for some time do not fare any better than 
those subjected to warmed sea-water; as in the first and second 
series of experiments this ‘‘egg water’’ makes no difference in the 
results. 

I think that these facts are incontrovertible. Washing or 
even residence in sea-water for a short time interferes seriously 
with the effect of heat in initiating development. 

Study of insemination of dry and washed eggs was made. 
Apparently there is a difference here of response to the spermato- 
zoon. The dry egg is more irritable, jelly formation being ex- 
tremely rapid. This is true of dry eggs inseminated in small 
quantities of sea-water. This behavior recalls that of Platy- 
nereis. 

These results, moreover, might suggest that our methods are 
much too crude in the study of these extremely sensitive cells— 
the egg and the spermatozoon. 

Summing up we may say concerning the effects of warming on 
the eggs of Nereis: (1) That while eggs cut out of worms in the warm 
sea-water form jelly and divide in large numbers, a small per cent. 
swimming, some remain in the germinal vesicle stage. (2) That 
at least ninety-eight per cent. of the dry eggs form jelly almost all 
of which cleave: twenty per cent. become trochophores closely re- 
sembling the normal. (3) That eggs in “‘serum’”’ fail to develop 
except in very small numbers. (4) That washed eggs even after but 
two or three washings develop if at all in small numbers. 


B. Errect or KCl In THE INITIATION OF DEVELOPMENT. 


According to Fischer the eggs of Nereis after treatment with 
KCI will go through cleavage and produce trochophores. Lillie 
(11), however, could not get the eggs after KCl treatment to go 
beyond maturation. During three seasons this had been my 
experience. This summer I studied the effect of KCl on washed 
and unwashed eggs. 

If the eggs be washed two or three times before exposing to 


INITIATION OF DEVELOPMENT IN NEREIS. 13 


the action of KCl every egg maturates but never more than one 
in a thousand swims. If the eggs be allowed to remain in sea- 
water from two to twelve hours with frequent changes of sea 
- water the results are about the same. Dry eggs subjected to 
KCl treatment maturate, cleave once or twice, and produce, in 
one experiment at least, seven per cent. of swimming forms made 
up of unsegmented two and four-cell ‘swimmers.’ 


THE EXPERIMENTS. 


5, 10, 15, 20 and 25 per cent. 2.5M KCl were used. It was 
found that 15 per cent. 2.5M KCl in sea-water gave the best 
results. Typical experiments follow: 

(a) August 12, 10:54 A.M. Lot A: Eggs from two females cut 
in 80 c.c. of 20 per cent. 2.5M KCl at 10:54. Lot B: Eggs from 
one dry female put in 3 c.c. of 20 per cent.2.5M KCl. At 11:00 
jelly formation in both. Samples of eggs taken from A and B 
as follows: 


IN[O SSRs, 43: Anes to) SR cece aeecl aio aco (ont aie II:00 
INOS 2 eae See cece 2 cavics vse Gin G) oe een ceaT me II:10 
INTO ssi SaaS etOho a ERP nae As coe aay onde ir giey 
INO NG spel ol orane tous epee eip oly craps THSAS 
INTO SOS a ares reateaic cu eh ee in ES oe eue eta Wat 6332) 
INOSRO RG ce ereetet ctor neice: nie untshav enous enone tes II:40 
IN OMB Preece eee a ease ye Niaseuatey aweoeuteercnres II:50 
INO SIS ei eles erstea rope e ines atenauet svageen ayeneme 12:00 


August 13, 12:00 M. Dry eggs of August 12 (Lots A and B). 
All maturated; some cleavage-like processes and some swimming 
forms after twenty minutes’ exposure or more. Highest per- 
centage (five) of swimmers after fifty minutes’ exposure. These 
are unsegmented, two and four-cell swimmers. 

(b) August 12, 12:05 P.M. Water changed three times on 
eggs during three hours and then placed in 20 c.c. of 20 per cent. 
2.5 M KClinsea-water. Samples taken at five-minute intervals 
up to 1:00 P.M. 

August 13, 1:00 P.M. Washed eggs of August 12, all matur- 
ated; I in I,000 swim. 

Experiments during August 13 and 14 with fifteen per cent. 
2.5 M KCl gave about the same results. 

(c) August 15, 9:30 A.M. Two females quickly cut in I0 c.c. 


14 Ee Ee. jUSI: 


of 15 per cent. 2.5 M KCl and removed. Samples taken as 
follows: 


INHER: 3 guaien is oe Soe eon Dee aaie T0335 
Be A EG Nae tan RR tI tom aN eG 8 10:50 
OME Bish daalasoh tna etaeee ye Pues at a arene ea II:05 
BS Te hs Be Can The Se ds ae UA ee I1:20 
BOAR RUSS. Wes Pap Btls Riots Nant ol RP Petes I1I:40 
Sabet OVER ROM PSEA cee SEN ST ne BON CS ETOCS Can 12:00 
FSD) EE ss ie “altar vah ouch ue rare ake Re ete Pek Meneame 1:25 


INOS Wc lacce Myke etts ole soem 4 per cent 
bere Retet ae MEN SIO ENan a a tron es Tae Mie 
pares ee Hecmerihhd Ne ak ae ahs. Kiger DPa aah ae 
ype BAB ah detae atin ter eee he ee 
CNT) tee tea a Me RMR Se oe EU a re CN Dee Star ne 
BREE M6 hse ap near haere He rch Ge Deltas ts 
Hh fewer tee ee EER ect cass Bee: es 


(d) On August 17, eggs were washed by changing the water six 
times in five hours then subjected to 15 per cent. 2.5 M KCl in 
sea-water, samples being taken at sixty minutes and thereafter 
at five-minute intervals. One tenth per cent. (.1%) was the 
best result after sixty minutes in the KCl sea-water. 

(e) Combination of KCI with Heat..—An experiment of last 
summer was repeated except that dry eggs were used and the 
minimum exposure, five minutes, of the series the only one tried. 
The protocol follows: 

August 17, 1:25 P.M. Dry eggs are put in 10 c.c. of 15 per 
cent. 2.5 M KCl in sea-water for five minutes; jelly formation 
almost at once. Eggs are then placed in 50 c.c. of sea-water at 
35°C. Four lots removed to 100 c.c. of sea-water as follows: 


INTO Hane We Bee ee ae ot weds aie rae a I:55 
SP IDE Ay oh Ath ce TN Aon a Renee tee 2:00 
eet AR ach ARR trie ebee Sane tia Can 2:05 
th) WANA shone Se ee Cet tee ee AO LO 


The next day, at 10:30 A.M., the percentages of swimming 
forms, largely unsegmented found were as follows: 


INOS ase (25 minutes’ exposure to heat)...... 3 per cent. 
pune See (30 a “ ier ahah) eet itis ep ae, 
re Bue ae (35 i ie Bie Rahs heres eyes DR ra. (aay 
tne tae (40 eS ss Daie lint) Muniuaweas DIGG nit it 


1 Tt will be recalled that Allyn used a combination treatment of KCl and heat on 
the egg of Cheloplerus with rather different results from those mentioned here with 
Nereis. Her method however, was different. 


———— 


INITIATION OF DEVELOPMENT IN NEREIS. 15 


It appears, therefore, that with KCl, and KCl and heat, 
washed and unwashed eggs alike will maturate, but that the dry 
eggs alone respond with cleavage or the production of swimmers. 


DISCUSSION. 


In the egg of Nereis Lillie discovered a substance, fertilizin, 
which has the property of agglutinating Nereis sperm. This sub- 
stance may be detected in the water in which the eggs have re- 
mained for a short time. If, however, the eggs be washed by 
changing the water two or three times the fertilizin is no longer 
secreted in detectable quantities, 7. e., there is not enough to 
agglutinate the sperm. Such eggs are none the less fertilizable 
by sperm, giving off at the time of insemination more fertilizin, 
all of which is then utilized or completely thrown off during the 
cortical changes. It therefore follows that at the time of shed- 
ding the egg is laden with free fertilizin ready for secretion. This 
conclusion is supported by additional facts. In the first place 
I have pointed out above that the dry egg or egg in small quan- 
tities of sea-water is hyper-irritable—that is, if jelly formation 
may be taken as index. If one inseminates the eggs of Nerevs 
dry or in small quantities of sea-water the jelly formation is 
extremely rapid. Jelly formation is correspondingly slow in 
washed and stale eggs. The breeding behavior noted night after 
night for several seasons is significant: freshly shed eggs at the 
surface of the sea excite numbers of males to shed their sperm 
around the shedding or recently spent female. Lillie’s experi- 
~ ments (Lillie and Just) on this sperm shedding reflex, moreover, 
prove that the egg loses fertilizin once in the sea-water. The 
“dry’’ and ‘“‘washed”’ eggs of my experiments, then, are physio- 
logically different: the dry egg has all its available fertilizin 
content, the washed egg has secreted part of this substance. 

Lillie has shown that the eggs of Nereis will not fertilize in 
the tissue juices of the animal; my experiments show also that 
the body juice of spent females inhibits fertilization. Unlike the 
washed egg, the ‘‘serum” eggs possess fertilizin but its action is 
inhibited. 

But it is on the basis of experiments on Arbacia that Lillie 
has developed the fertilizin theory as an explanation of the me- 


16 Dy By juSi. 


chanism of fertilization. Without going into details it may be 
said that in Arbacia it is found that the egg secretes a substance, 
fertilizin, whose presence is capable of quantitative determination 
and which is necessary for fertilization since first, eggs washed 
free of it are no longer capable for fertilization; second, fertilized 
eggs no longer secrete it; and third, eggs after membrane forma- 
tion with butyric acid are not capable of fertilization and do not 
give off the substance. The perivisceral fluid of Arbacia, 
moreover, produces an inhibiting effect on fertilization preventing 
the action of fertilizin on the egg. 

My results with warming Nereis eggs parallel to a striking 
degree these facts brought out in the studies of fertilization in 
Nereis and Arbacia (Lillie, *12, ’13a, ’130, 14). Eggs washed 
free of the bulk of fertilizin will not develop however long the 
warming treatment lasts; serum inhibits the artificial initiation of 
- developmental processes; only the dry eggs with their full content 
of fertilizin when suddenly shocked with elevation of temperature 
respond with jelly formation and cleavage. It would seem, there- 
fore, as I have suggested for Platynereis, that fertilizin is just as 
essential for artificial initiation as for normal fertilization. The 
difference seems to be that for artificial initiation more fertilizin 
is required. Further attempts at Woods Hole this summer to 
induce artificial parthenogenesis in Platynerets strengthen this 
belief; a percentage of Platynereis eggs will fertilize in small 
quantities of sea-water; the same bulk of eggs in the same 
amount of water fail to respond when subjected to warming. 

If, therefore, as Loeb (’12a) says, ‘‘fertilization is primarily 
and essentially artificial parthenogenesis’’; or if ‘‘a theory of 
fertilization must also be a theory of parthenogenesis at least 
for the phenomena common to both”’; and if “similarly a theory 

of fertilization must be consistent with the facts of parthenogen- 
~ esis” as Lillie ('14) suggests; these experiments, we are forced to 
conclude, make another link in the chain of evidence which 
supports the theory that fertilization is essentially a process of 
the egg. The spermatozoon initiates the development of the 
egg, as does warming, through the activation and the binding of 
the fertilizin. 


a PO ee gee ee ——— 


ee ee a a 


INITIATION OF DEVELOPMENT IN NEREIS. 17 


LITERATURE CITED. 
Allyn, H. M. 
"12 The Initiation of Development in Chetopterus. BurioL. BULL., 24. 
Bullot, G. 
704 Artificial Parthenogenesis and regular Segmentation in an Annelid (Ophe- 
liad. Arch. Entw.-Mech., 18. 
Fischer, M. H. 
’03-~«Artificial Parthenogenesis in Nereis. Am. Jour. Physiol., 9. 
Just, E. E. 
712 ~The Relation of the first Cleavage-plane to the Entrance-point of the Sperm. 
BIOL. BULL., 22. 
"14 Breeding Habits of the Heteronereis Form of Platynereis megalops at 
Wood’s Hole, Mass. Bror. BULL., 27. 
Lefevre, G. 
’o7 ~©=Artificial Parthenogenesis in Thalassema mellita. Jour. Exp. Zool., 4. 
Lillie, F. R. 
’o2 ~©Differentiation without Cleavage in the Egg of the Annelid, Chetopterus 
pergamentaceus. Arch. Ent.-Mech., 14. 
*tr Studies of Fertilization in Nereis, t and 2. Jour. Morph., 22. 
"12 Studies of Fertilization in Nereis, 3 and 4. Jour. Exp. Zool., 12. 
713 Studies of Fertilization, 5: Jour. Exp. Zool., 14. 
’13b The Mechanism of Fertilization. Science, N. S., 38. 
’14 Studies of Fertilization, 6. Jour. Exp. Zool., 16. 
Lillie, F. R. and Just, E. E. 
13 Breeding Habits of the Heteronereis Form of Nereis limbata at Wood’s Hole, 
Mass. BIOL. BULL., 24. 
Loeb, J., Fischer, M., and Neilson, H. 
‘ora Arch. f. d. Ges. Physiol., 87. 
Loeb, J. 
’o1b Experiments on Artificial Parthenogenesis in Annelids (Chzetopterus) and 
the Nature of the Process of Fertilization. Am. Jour. Physiol., 4. 
’07 Ueber die Allgemeinen Methoden der kunstlichen Parthenogenese. Pflti- 
ger’s Archiv, 118. 
708 Ueber die Entwicklungserregung unbefruchteter Annelideneier (Polynoe) 
mittels Saponin und Solanin. Pfliiger’s Archiv, 122. 
*12a Heredity in Heterogeneous Hybrids. Jour. Morph., 23. 
’12b The Comparative Efficiency of Weak and Strong Bases in Artificial Par- 
thenogenesis. Jour. Exp. Zool., 13. 
13. «Artificial Parthenogenesis and Fertilization. The University of Chicago 
Press. 
Loeb, J. and Wasteneys, H. 
12 Fertilization of the Eggs of Various Invertebrates by Ox-serum. : Science, 36, 
No. 921. 
Scott, J. W. 
*06 The Morphology of Parthenogenetic Development of Amphitrite. Jour. 
Exp. Zool., 3. 
Treadwell, A. L. 
702 Notes on the Nature of Artificial Parthenogenesis in the Egg of Podarke 
obscura. Bio. BULL., 3. 


SPERM AGGLUTINATION AND FERTILIZATION. 
FRANK R. LILLIE. 


In a recent paper on “Cluster Formation of Spermatozoa 
Caused by Specific Substances From Eggs’”’ Loeb (’14) has pre- 
sented a criticism of my theory of fertilization (Lillie 136 and 
14), based on observation of the California sea-urchin Séron- 
gylocentrotus purpuratus. My own observations were made on 
Arbacia punctulata of Massachusetts, and it would appear that 
part at least of Loeb’s criticism was due to certain differences in 
the two forms, for he has now stated (Loeb, 1914), p. 318, foot- 
note) that the ‘‘cluster formation” of the spermatozoa may find 
its explanation ‘“‘on the assumption of an agglutination at least 
in the case of Arbacia,”’ as I maintained; it is therefore not a 
““tropistic reaction”’ as he thought probable from his observa- 
tions in California. This was one of the chief differences of 
opinion. A second one was in regard to the source and sig- 
nificance of the substance in the fluid of egg suspensions that 
caused such agglutination; Loeb maintained that it was merely 
the dissolved chorion (1. e., jelly layer) of the egg, and that after 
this was removed the eggs no longer produced the agzglutinating 
substance, and yet were capable of fertilization; whereas my 
contention was that the agglutinating substance was a secretion 
of the egg soaked up by the jelly, as by a sponge; that the eggs 
produced it for a certain length of time after the removal of the 
jelly, and lost their power of fertilization after they ceased to 
produce it. 

These criticisms cut at the foundation of my theory. Inas- 
much as the correction of the tropistic interpretation of agglutin- 
ation is given only in a footnote to another paper, and no cor- 
rection of the source of the agglutinating substance has yet 
appeared, it is incumbent on me to consider the criticisms 
carefully; at the same time I wish to take the opportunity to 
explain certain points that appear to be open to misinterpretation, 
and to record some new observations. 

18 


SPERM AGGLUTINATION AND FERTILIZATION. 19 


I. ‘‘CLUSTER-FORMATION’’ VERSUS AGGLUTINATION. 


The phenomena exhibited by sperm suspensions of Arbacia 
with which we have to deal are of four distinct types, which it is 
essential to distinguish sharply: (1) activation; (2) aggregation; 
(3) agglutination, (4) mass-coagulation.! (1) That the activity 
of spermatozoa is affected by substances in the sea-water requires 
noargument. The subject is discussed in study V (Lille, 1913a, 
pp. 519-532). (2) Aggregation of spermatozoa may be brought 
about by tropistic reactions. In my paper on the “Behavior 
of Spermatozoa,’ I devoted a great deal of attention to such 
ageregation phenomena and the distinction from phenomena of 
agglutination (1913, pp. 532-548 and pp. 551-552). Among 
other things I pointed out that aggregation as a tropistic phe- 
nomenon implies a gradient,? and that the spermatozoa never 
adhere, however crowded they may be; there is no observable 
physical change of the spermatozoa and the slightest agitation 
suffices to disperse them again. Such tropistic phenomena may 
be exhibited in response to CO, and other acids (Nereis), or 
certain constituents of egg secretions, to mention only chemotaxis. 

(3) Agglutination of spermatozoa on the other hand requires 
no gradient, and the spermatozoa adhere physically to such an 
extent that the agglutinated masses may be preserved intact in 
killing fluids; its degree is a function of the concentration of the 
agglutinating medium, and is also different in different species. 
Agglutination is non-toxic, not limiting the life of the sper- 
matozoa; it is reversible, its duration depending on the concen- 
tration of the agglutinating medium; it cannot be repeated if the 
reaction is complete, at least within the time limits of my ex- 
periments, even though the spermatozoa remain motile;’ finally 
motility of the spermatozoa is a prerequisite to a decided reac- 

1 The reaction here referred to is a lethal phenomenon. It possibly involves 
cytolysis with subsequent adhesion of the spermatozoa. 

2Tt is important to notice that the spermatozoa of suspensions may produce 
gradients through their own activities. Thus I pointed out that autogenous aggre- 
gation reactions in sperm suspensions of Nereis arise from the positive chemotaxis 
of the spermatozoa to their excreted COs:, giving rise to very striking phenomena 
(Lillie, 1913a, pp. 519-521 and pp. 538-540). It is conceivable that such a tro- 
pistic phenomena is involved as a part factor in the agglutination phenomena 


under discussion. 
3 Glaser (1914) also comes to this conclusion. 


20 FRANK R. LILLIE. 


tion; evidently because the physical change on which the reaction 
depends is not sufficient to cause adhesion except when the 
spermatozoa positively collide These six criteria definitely 
define the phenomenon. 

Agglutination is positively distinct from aggregation. It is 
an entirely different biological phenomenon. The two may, 
however, be exhibited simultaneously, as when a drop of egg 
secretion of Arbacia is injected into a sperm suspension of the 
species. In such a case the spermatozoa exhibit positive chemo- 
taxis to one constituent of the egg secretion, and are agglutinated 
by another (the fertilizin). The separateness of these two sub- 
stances was maintained in my first publication on the subject and 
demonstrated by repeated experiments (see Lillie, 1913a, p. 549, 
and 1914, pp. 545-546). 

(4) The phenomenon of mass-coagulation is, on the other hand, 
a lethal irreversible phenomenon. It may be exhibited in response 
to various agents, such as KOH, NaOH, salts of lanthanum and 
cerium,” etc., and in some cases the secretions of the eggs of other 
species or their blood. Hitherto I have not adequately defined 
this phenomenon as distinct from the agglutination phenomena, 
though in my last study (1914), I noted the distinction (p. 541). 
The phenomenon is essentially lethal, but not all destructive 
agents exhibit it; thus acids, so far as I have observed, destroy 
the spermatozoa without causing mass coagulation. The 
phenomenon is irreversible, and this suffices to distinguish it 
from true agglutination, even if no other criterion were available. 
However, it exhibits quite a different aspect from agglutination; 
in the latter the sperm masses tend to take on a spherical form; 
if originally elongated they contract into balls or break up into 
smaller masses which become spherical, thus offering considerable 
resemblance to a phenomenon of surface tension, as Loeb notes. 
The peripheral spermatozoa are in violent movement until the 
time of reversal. In the mass-coagulation reaction, on the other 
hand, there is no such surface tension effect, strands anastomose 

1 Loeb argues that the necessity of movement on the part of the spermatozoa 
for the appearance of this phenomenon removes it from the category of true agglu- 
tination; but this seems to me to be a purely arbitrary criterion. 


2 My attention was called to the action of the salts of these metals by a letter 
from James Gray of Cambridge University. 


SPERM AGGLUTINATION AND FERTILIZATION. 21 


wit4 other strands and form a net-work and the movements of 
the spermatozoa soon cease. 

The substances of egg secretions, which I have hitherto called 
hetero-agglutinins, belong to this category, in some cases at 
any rate. Though I will not assert that there is no such sub- 
stance as a hetero-agglutinin in the real sense of agglutination, 
yet the substance in Arbacia blood, or egg secretions, the effects 
of which on Nereis sperm I have previously studied, should be 
regarded as a toxic rather than an agglutinating substance, 
having the mass coagulant action. As I stated in my last paper, 
p- 541,.1t produces a permanent coagulum in Nereis sperm sus- 
pensions; “‘in this respect the action differs from the iso-agglu- 
tination, which is without toxic effects.” | 

We must keep firmly in mind the distinctions between aggre- 
gation (tropisms) agglutination, and mass-coagulation. Agglu- 
tination, with which we are particularly concerned, is distin-: 
guished from aggregation by the facts that it occurs in the 
absence of a gradient, it involves physical adhesion, and cannot. 
be repeated if the reaction is once complete; it also is characterized 
by a high degree of specificity! From mass coagulation it is. 
distinguished by the facts (1) that it is non-toxic, (2) reversible,. 
(3) dependent on motility of the spermatozoa. Agglutination 
occurs so far as I have observed with certainty only in response 
to egg-secretions of the same species. 

For description of the phenomena of agglutination of sperm 
by egg-extractives of the same species, I must refer to my previous 
paper (Lillie, 1913a@); the phenomenon in Arbacia is a true agglu- 
tination in the sense defined, not a tropistic reaction, nor yet a 
mass coagulation. Loeb has admitted this for Arbacia, and I 
would therefore venture to suggest the probability that the phe- 
nomenon which Loeb has described in Strongylocentrotus and 
termed “ 
reserve as a possible tropistic reaction, is also true agglutination, 
which differs only quantitatively from Arbacia and Nereis. The 


cluster formation,’ which he interprets with some 


1 Loeb admits that the “cluster formation”’ exhibits a high degree of specific- 
ity. It is therefore inconsistent to interpret the reaction, as he also does, as a 
“possible tropistic phenomenon”’ because such phenomena so far as we know do 
not exhibit specificities of this kind. Agglutination phenomena, on the other 
hand, as is well known, commonly exhibit equal specificity of a similar kind. 


Ze, FRANK R. LILLIE. 


conditions under which it occurs, in response to egg secretions 
of the same species, its character, reversibility, and the specificity 
of the reaction are identical with Arbacia. It is apparently, 
however, less pronounced, and therefore not so readily recog- 
nizable of itself as an agglutination phenomenon. Even the 
‘‘apparent surface tension phenomena’”’ which Loeb describes 
for the clusters—‘‘Short streaks or cylinders contract into 
spherical masses, the above described clusters; and long cylinders 
break up into a series of small clusters’ —are the same as I 
previously described for Arbacia (1913a, pp. 550-551). 

Loeb’s interpretation of the “‘cluster-formation”’ as a possible 
tropistic reaction confuses the two sets of phenomena—viz., 
aggregation (a true tropistic phenomenon) and agglutination— 
which sperm suspensions may exhibit to the egg-sea-water of its 
own species. But the aggregation (tropism) can take place 
only when there is a gradient from the secretion to the sperma- 
tozoa. This is realized under the conditions of my experiment 
of injecting a drop of egg-sea-water into a fresh sperm suspension 
beneath a raised cover slip; in such a case the two phenomena 
take place simultaneously viz.; aggregation in the form of a 
ring around or in the introduced drop (depending on concentra- 
tion), and agglutination. These two phenomena are produced 
by two constituents of the egg-sea-water, as I have already 
maintained. 

For the study of the aggregation phenomena therefore it is 
desirable to employ an agent which has no agglutinative action. 
This I did in an extensive series of experiments by the method 
just referred to (1913, p. 533 ff.). To illustrate:—a drop of a 4/100 
dilution of a saturated solution of CO» in sea-water injected into 
a sperm suspension of Nereis in sea-water mounted beneath a 
raised cover-slip is marked within a few seconds by the formation 
of a ring of active spermatozoa within the margin of the intro- 
duced drop, and separated from the general sperm suspension by 
a clear zone nearly free of spermatozoa 1.5 to 2 mm. in diameter. 
I interpreted the ring formation as a positive reaction to the 
attractive substance (CO, and acids generally) ; the spermatozoa 
follow the gradient from the suspension into the drop containing 
CO» a certain distance, 7. e., up to a certain concentration, and 


SPERM AGGLUTINATION AND FERTILIZATION. 23 


are there arrested. The proof of this interpretation is found in 
the fact that, if increasing concentrations of CO, are used, the 
ring forms outside the drop and becomes progressively wider, 
1. €., the migration ceases at a distance from the center which 
increases with COs or acid concentration (see 1913a, pp. 536-538). 
Loeb suggests that the ring formation with a clear external zone 
around it is “‘an indication that the spermatozoa are negatively 
chemotropic to the strong egg-sea-water, and possibly positively 
chemotropic to the more diluted egg-sea-water, or to the dense 
collection of spermatozoa in the ring.’ The latter suggestion is 
of course untenable as a primary cause, for the ring-formation is 


precisely the phenomenon to be explained. It is also unnecessary 


to assume any negative tropism; the ring formation is due to a 
limitation of the positive movement by concentration. This is 
fully discussed in the paper referred to above, but Loeb does not 
allude to the discussion. 


2. THE SOURCE OF THE AGGLUTINATING SUBSTANCE. 


Professor Loeb has also taken issue with me on the question 
of the origin of the agglutinating substance. He regards his 
experiments as proving that the substance which causes the 
‘cluster formation” is not formed in the egg but in the chorion; 
1. é., in the layer of jelly which surrounds the egg. On the other 
hand I regarded it (and still hold to the opinion) as a secretion 
of the egg; with which the jelly of course becomes saturated. 
Loeb’s observations again were on Strongylocentrotus and mine 
on Arbacia. ‘The issue is a real one even though the chorion is 
itself a secretion of the egg in earlier stages. 

Loeb’s conclusion was based on his observation that if the 
chorion be dissolved off in dilute hydrochloric acid in sea-water, 
the naked eggs transferred to sea-water produce no detectable 
amounts of the agglutinating substance any more, whereas the 
acid sea-water contains it in large quantities. My conclusions 
were based on the observation that when eggs of Arbacia are 
deprived of jelly (chorion) by shaking, or a prolonged series of 


1 Glaser (1914) also agrees substantially with me: ‘‘the agglutinating substance 
is located in greatest abundance in the jelly and the eggs also contain this material,”’ 
p- 371. 


24 FRANK R. LILLIE. 


washings, they still continue to produce the agglutinating sub- 
stance in sea-water, though in much diminished quantity; in my 
full paper, which Loeb had not the opportunity of consulting, 
I gave series of measurements on this point (1914, pp. 532-538); 
I also pointed out that in immature ovaries containing many 
primary ovocytes, but some mature eggs, the quantity of agglu- 
tinating substance produced was relatively very smell (1914, 
p- 530), and I therefore suggested that the substance was secreted 
by the eggs at the time of maturation and was soaked up by the 
jelly as by a sponge. The eggs, however, continue to produce 
it after maturation, as I shall show. The immature eggs have as 
thick a chorion as the mature eggs; therefore the agglutinating 
substance cannot be merely dissolved chorion. I recognized the 
possibilicy of the view expressed by Loeb, investigated it as fully 
as possible at the time, and rejected it. 

Since Loeb’s paper has appeared, I have repeated his experi- 
ments and found my former observations and conclusions con- 
firmed in all respects: 

Experrments.—The optimum concentration of HCl for removal 
of jelly without injury to eggs was found to be 50 c.c. sea-water 
- 1.4 ¢.c. N/to HEI. 1.2'c.c. N/1o H@lin Soic:c. sea=water didmon 
fully remove the jelly, and 1.6 c.c. caused too much injury to 
the eggs evidenced by heavy agglutination and later cytolysis. 
In an experiment of July 17, 1914, the three above concentrations 
were used. The complete removal of the chorion in the inter- 
mediate concentration was demonstrated by observation of the 
eggs in a thick suspension of India ink in sea-water; even the 
minutest traces of adherent jelly can readily be detected by this 
method, but it was all gone. The eggs were then washed as 
follows: 10.11 A.M. 42/6 c.c.; 10.40 51/5 c.c.; 10.58 50/4 c.c. 
The supernatant fluid was then tested and found to be free from 
sperm agglutinating substance; thus furnishing proof that all 
originally contained in the jelly had been washed out. At 11.20 
the supernatant fluid was poured off leaving only 5 c.c. in the 
tube. The eggs were allowed to settle, and at 11.25 the super- 
natant fluid was tested and gave a 9-10-second agglutination 
reaction with fresh sperm suspension. Thus these eggs entirely 
deprived of jelly by HCl are producing agglutinating substance. 


SPERM AGGLUTINATION AND FERTILIZATION. 25 


At 4.25 P.M. the eggs were washed again 5/0.7 c.c. and the new 
fluid gave a I4-second reaction. The next morning the same 
eggs were washed again 5.5/1 c.c. The new fluid gave a 6-7- 
second reaction. 

These results may be expressed in a different way: thus in an 
experiment of July 20, a series of eight successive washings of eggs 
deprived of jelly by acid sea-water represented a dilution of the 
agglutinating substance contained in the acid sea-water remaining 
with the eggs of 12,700,800 times. But the acid solvent itself 
was negative at 1/800 dilution: it was of 400 agglutinating 
power. In other words, after the removal of the jelly the eggs 
themselves had produced a sufficient quantity of the agglutin- 
ating substance to account for the tremendous difference; and 
they were still producing it. 

These eggs without jelly are fertilizable, as Loeb states, but 
only 37 per cent. segmented in a heavy insemination of the first 
day in the experiment of July 17, and only a small part of these 
developed to the ciliated stage, none of which were normal, most 
being stereoblastulae and incapable of farther development. 
The result is entirely similar to that described in my last paper 
(study VI, ’14) for the fertilization of eggs deprived of jelly by 
shaking and subsequent washing. 

The same experiment was repeated on July 18, 20 and 21, with 
identical results: the eggs from which jelly i@entirely removed 
by HCI continue to produce the sperm-agglutinating substance 
(fertilizin) so long as they live, but their capacity for development 
after fertilization is much reduced. 

In all experiments at least three concentrations of acid were 
used, and in each experiment it was observed that when the con- 
centration was sufficient to dissolve the jelly there was a good 
deal of agglutination of the eggs, and in the later washings a 
great many eggs broke down liberating their pigment. As I 
have previously shown, broken-down eggs liberate a substance 
(anti-fertilizin) which neutralizes the sperm agglutinating action 
of the fertilizin. Therefore, when a sufficient percentage of the 
eggs are breaking down, the production of sperm-agglutinating 
substance (fertilizin) by intact eggs may be entirely masked. 

I have no intention of disputing Professor Loeb’s observations 


26 FRANK R. LILLIE. 


for Strongylocentrotus. But they merely prove either that 
Strongylocentrotus sperm is not so delicate an indicator as Arbacia 
sperm, or that the method employed by Loeb was inadequate to 
detect small quantities of fertilizin. In Avrbacia the eggs con- 
tinue to charge the sea-water with sperm-agglutinating substance 
after complete removal of the jelly, whether by shaking and 
repeated washings, or by HCl; and the substance continues to 
be formed as long as the eggs remain fertilizable and living, no 
matter how often the eggs are washed. The eggs of Arbacia 
secrete the substance as I previously maintained. It is not merely 
the ‘dissolved chorion.” 

It might possibly be objected to this conclusion that the con- 
tinued appearance of the agglutinating substance in egg suspen- 
sions in sea-water after removal of the chorion indicated merely 
previous adsorption of the substance of the chorion. But the 
indefinite continuance of its production is inconsistent with the 
idea of a mere secondary removal of an adsorbed substance. 
The idea is also inconsistent with the fact that Nereis eggs have 
no jelly at the time laying, but produce a similar sperm agglu- 
tinating substance. In this form the jelly also is secreted by the 
egg after insemination. 

Finally if it can be shown that the jelly of immature eggs is 
entirely devoid of the sperm agglutinating substance, my position 
that this substance is a later secretion of the egg is rigorously 
proved. As noted above I maintained the probability of this 
view in my previous paper (Study VI). This summer my first 
experiments were undertaken to investigate this point anew. 

Fortunately the season was late, and not a single Arbacia was 
ripe when I began work (June 8). This applied to males as well 
as females: so it was impossible at first to secure ripe sperm as 
indicator. I therefore made extracts of immature ovaries to be 
kept for subsequent testing from three females (1, 2, and 3, 
June 8). June 11 extracts of ovaries in sea-water were made 
from females 4, 5, and 6: numbers 4 and 6 contained only ovo- 
cytes; No. 5 had a large number of ripe ova in addition. On 
June 16 extracts I-6 were tested with Arbacia sperm suspension: 
I, 2, 3, 4, and 6 were absolutely negative; no agglutination. No. 
5 gave a strong agglutination reaction lasting about one minute. 


SPERM AGGLUTINATION AND FERTILIZATION. 27 


It is highly improbable that the agglutinating substance had been 
destroyed in five of the six, and retained in the only one (No. 5) 
of the extracts which was made from ovaries containing some 
‘ripe ova. So far as these observations go, the jelly of immature 
ovocytes is free of agglutinating substance. 

Again on June 15 I made extracts from ovaries of three females 
in two of which ripe ova were practically absent, the third bad 
a few. Tested the same day the two former extracts had no 
sperm agglutinating properties; the third gave slight agglu- 
tination. 

The females appeared to mature slightly earlier than the males, 
so that for these experiments I was forced to use rather thin 
sperm suspensions (mixed more or less with immature spermato- 
zoa), which were probably not as delicate indicators as one could 
wish. However the difference between the ovaries containing 
ripe ova and those without was perfectly distinct. Later when 
fully ripe males could be had all ovaries contained ripe ova. 

The following observation also tends in the same direction: 
June 27, 1914—Three females were selected, of which number 1 
was the ripest attainable, the eggs flowing freely out of detached 
ovaries, and very few ovocytes occur; numbers 2 and 3 were the 
least mature attainable; number 2 had very few detachable ova, 
mostly late ovocytes with a sprinkling of ripe eggs; number 3 
had quite a few detachable ova with a large proportion of ripe 
eggs. The ovaries of all three were cut up equally, and sea-water 
added to each to make 10 c.c. When the ova and ovaries had 
settled they stood at 1.5 c.c. in I, at 1.3 c.c. in 2, and 1.5 c.c. in 3. 
After five hours, tests of the agglutinating strength of the super- 
natant fluids were made with clear fresh sperm. 

No. I gave a 10-second reaction at 1/800 dilution. 

No. 2 gave a 6-second reaction at 1/10 dilution. 

No. 3 gave a 7-second reaction at 1/40 dilution. 

Thus No. 1 is 80 times the strength of 2 and 20 times the strength 
of 3. In general the fertilizin production is proportional to the 
ripeness of the ovaries. 

There is not the slightest doubt in my mind about the demon- 
strative character of these observations. The appearance of 
agglutinating substance in the jelly of Arbacia eggs is secondary, 


28 FRANK R. LILLIE. 


and takes place probably at the time of breaking down of the 
germinal vesicle. 

Loeb’s contention that the agglutinating substance is merely 
dissolved chorion therefore does not hold for Arbacia. With 
this his argument against my fertilizin theory also falls: ‘‘More- 
over if it should turn out that the substance which is responsible 
for the cluster formation is identical with the substance which 
Lillie calls ‘‘fertilizin,’’ which is very likely the case, Lillie’s 
theory becomes untenable, since this substance does not, in all 
probability, originate from the egg, but from the chorion and 
since there is, as we have seen, no connection between the presence 
of this substance and the power of the eggs of being fertilized” 
(pp. 136-137—Loeb, -'14). 

In this statement Loeb sums up the essentials of his criticism; 
since I have shown that “‘cluster formation” is true agglutination 
(which Loeb now admits), and that the agglutinating substance 
(my fertilizin) is not dissolved chorion but a true secretion of 
the eggs which continues to be produced after the chorion is 
removed, the entire stated criticism becomes ineffective. There 
7s a connection between the presence of this substance and the 
power of the eggs of being fertilized: the substance can first be 
demonstrated at the time that the power of being fertilized first 
arises, viz., after breakdown of the germinal vesicle; it can be 
demonstrated as long as eggs retain the power of being fertilized, 
whether the chorion be removed or not, and it disappears ab- 
solutely after fertilization, as I showed in my previous paper 


(study VI, p. 553, 1914). 


3. OTHER CRITICISMS. 


Another objection raised by Loeb is that ‘‘the supernatant 
sea-water of the eggs of Strongylocentrotus franciscanus will not 
induce cluster formation of the sperm of Strongylocentrotus 
purpuratus: yet the latter sperm fertilizes the eggs of francis- 
canus,”’ from which he argues that the fertilizin of Strongylo- 
centrotus franciscanus can not be necessary for the fertilization of 
itseggs. Anerror in logic is involved here; agglutination of sperm 
is merely an indicator of the presence of a certain substance, 
which is none the less present in franciscanus even if purpuratus 


SPERM AGGLUTINATION AND FERTILIZATION. 29 


sperm does not reveal it; it may nevertheless be activated by 
purpuratus sperm and this is the essential point in the theory.! 
Agglutination of sperm is of no significance except as indicator. 
As I pointed out in my previous paper, binding of the fertilizin 
by sperm receptors, 7. ¢., the chemical reaction, is a thing entirely 
distinct from agglutination; if such binding causes a certain kind 
of physical surface change of the spermatozoa of suspensions of a 
certain minimum concentration, they agglutinate; otherwise not. 
Agglutination is a valuable indicator that enables us to make 
certain analyses, and that is all. The same principle of fer- 
tilization may hold in the entire absence of sperm agglutination. 

Another objection in which Loeb supports the possibility of 
superposing fertilization on parthenogenesis will be dealt with in 
a separate paper. My contention in this case is that the possi- 
bility of such superposition always rests upon incompleteness of 
the parthenogenetic reaction; if the fertilization reaction be 
complete, whether by parthenogenesis or insemination, it cannot 
be repeated. Everybody admits that eggs fertilized by sperm 
cannot be refertilized; it is a logical impossibility that eggs ‘‘fer- 
tilized” by parthenogenetic reagents should be refertilized. The 
problem of the apparent contradiction involved in Loeb’s and 
Herbst’s contention of superposition works out in the manner 
indicated. A study of this problem by one of my students will 
appear soon. 

Loeb cites as a farther difficulty of my fertilizin theory, which 
he says I have not considered, ‘‘that in addition to the membrane 
forming substance still another, namely a correcting agency, is 
necessary for causation of the development of the egg.”” Though 

1 Loeb states (1914, p. 135): ‘‘If the phenomenon of cluster formation were in- 
separably associated with the power of the eggs of being fertilized, we should expect 
that sperm should only be able to fertilize the eggs of a species if the egg-sea-water 
of the same species caused the cluster formation of the sperm.’”’ I have never 
maintained that agglutination (‘‘cluster formation’’) is inseparably associated with 
the power of the eggs of being fertilized, but merely that a certain substance pro- 
duced by the egg is a necessary factor in fertilization. In some cases this substance 
(fertilizin) produces agglutination of the sperm of its own species, and this reaction 
furnishes an indicator of its amount, when present, or of its absence. In other 
cases such an indicator is lacking: I do not find that supernatant sea-water of the 
eggs of the starfish (Asteria forbesii), for instance, agglutinates its own sperm; but 


I have evidence, to be published elsewhere, that the mechanism of fertilization 
may be explained in the same way as in Arbacia. 


30 FRANK R. LILLIE. 


I cannot accept this statement of the problem, I have nevertheless 
taken into consideration the fundamental fact, to which Loeb 
alludes, in the full account of my experiments, which appeared 
after Loeb’s paper was in press. The fundamental fact is simply — 
that the fertilization process in some cases can be divided in 
two sharply marked stages. This is perhaps most simply and 
convincingly shown by my own experiment (Lillie, 1911) of 
removing the spermatozoon from the egg of Nereis after it had 
already induced the cortical changes, with the result that the 
developmental phenomena came to a standstill before the first 
cleavage. I cannot agree with Loeb that the second stage in- 
volves a factor corrective of an excess action of the factor of the 
first stage. I think it is probable that we have a progressive 
process readily capable of resolution into two stages. 

In my complete paper (Lillie, 1914) I considered the pea 
phase of fertilization with reference to the new theory, and may 
refer the reader to the discussion there given (study VI, pp. 
582-584). Here it is only necessary to point out that the 
‘fertilizin’’ theory is at least as well adapted to account for the 
two stages as the “‘lysin”’ theory. 


4. CONCLUSION. 


I may be allowed to emphasize the essential features of my 
theory with some added light thrown by the work of this summer. 
The fundamental conception is that all agencies initiating 
development of the egg do so by the same means, viz., activation 
of an ovogenous substance, which I have termed fertilizin. This 
conception brings fertilization and parthenogenesis under one 
conception. I further assumed that such activation in the case 
of fertilization was caused by union of a constituent substance 
of the spermatozoon (the sperm receptors) with the fertilizin, 
and that the activation expressed itself by consequent union of 
the fertilizin with certain egg substances (the egg receptors). 
The reaction was thus conceived in terms of the Ehrlich side- 
chain theory, and was represented diagrammatically accordingly. 

That certain chemical combinations form an essential feature 
of the fertilization reaction cannot be open to doubt. I have 
not previously taken into account the consideration that the 


SPERM AGGLUTINATION AND FERTILIZATION. 31 
4 


occurence of such reactions, taking place, as they must, across 
the egg membrane, is dependent on physical conditions of the 
membrane, especially its permeability to the substances con- 
‘cerned. In speaking, as I did, of five blocks to the fertilization 
reaction, I was concerned only with the chemical reactions in- 
volved. There may be other blocks of a physical nature. 
Indeed these were much in evidence in the fertilization of Asterias, 
which I studied in the first part of the summer, and shall report 
on elsewhere. Another important consideration is that the 
reaction must also be dependent on environmental conditions 
such as temperature, ionic constitution of the medium (see Loeb, - 
’14b), etc. Blocking of fertilization may also arise from such 
causes. 

Continuing the exposition of the theory; I identified the fertil- 
izin of Arbacia with the substance found in the fluid of egg sus- 
pensions which causes agglutination of sperm suspensions of the 
same species. This phenomenon cannot possibly be lacking in 
significance, for it furnishes direct evidence of a combination of 
egg and sperm derivatives; the phenomenon itself is not con- 
cerned in fertilization, for a single spermatozoon may fertilize 
an egg. Neither does the absence of such agglutination in other 
species affect in the least the conclusion that may be drawn from 
Arbacia: because we may have a combination of egg and sperm 
derivatives without any sperm agglutination. The agglutination 
is incidental, the combination is the essential thing. 

The fertilizin theory in its essential aspects is not dependent 
on the identification of fertilizin and sperm agglutinating sub- 
stance. I believe in their identity; but if it were proved, as 
Loeb has sought unsuccessfully to do, that the agglutinating 
substance is not essential for fertilization, the fertilizin theory 
would still not be attacked in its essence. The conception that 
initiation of development is essentially a phenomenon of activa- _ 
tion would still stand in opposition to theories of external agents 
acting directly by corrosion (cytolysis), or coagulation, or what 
not. The egg could still be regarded as a self-contained system 
with no more than the usual environmental relations. It is only 
from this point of view that the complex phenomena of parthen- 
ogenesis and fertilization can be united in a logical whole. 


32 FRANK R. LILLIE. 


The theory of the identity of fertilizin and sperm agglutinating 
substance rests upon a considerable body of ascertained facts (see 
study VD), and it gives us at once a point of attack and a working 
hypothesis of considerable value. I have been able to show for 
instance that the origin of the capacity of the egg for being fer- 
tilized can be understood on this basis; that the cessation of 
fertilization capacity can also be so understood; and that the 
physiological sterility (prevention of polyspermy) of fertilized 
eggs is readily explained by the neutralization of the fertilizin 
by a substance (anti-fertilizin) demonstrably present in the egg. 

On the other hand the theory does not postulate that the 
fertilizin of all forms should agglutinate sperm of its own species. 
There may be many forms in which the union of the sperm re- 
ceptors with fertilizin does not produce such physical changes of 
the spermatozoa as to lead to agglutination. In those cases in 
which agglutination does occur we have a reaction very useful in 
analysis; but it cannot be too strongly emphasized that the 
agglutination itself is to be regarded merely as an indicator of 
the essential reaction. 


SPERM AGGLUTINATION AND FERTILIZATION. 33 


LITERATURE. 
Glaser, Otto. 


’14 A Qualitative Analysis of the Egg-Secretions and Extracts of Arbacia and 
; Asterias. Biot. BULL., Vol. 26, pp. 367-386. 
Lillie, Frank R. 

’r112 Studies of Fertilization in Nereis—I. Cortical Changes in the Egg. 
II. Partial Fertilization. Journ. Morph., Vol. 22, pp. 361-391. III. and 
IV: Jour. Exp. Zool., Vol. 12, pp. 413-474. 

’12 The Production of Sperm Iso-agglutinins by Ova. Science, N. S., Vol. 
XXXVI, pp. 527-530. 

’13a Studies of Fertilization. V: The Behavior of the Spermatozoa of Nereis 
and Arbacia with Special Reference to Egg-Extractives. Journ. Exp. Zool., 
Vol. 14, pp. 515-574. 

’13b «=The Mechanism of Fertilization. Science, N. S., Vol. XXXVIII., pp. 
524-528. 

’14 Studies of Fertilization—VI. The Mechanism of Fertilization in Arbacia, 
Journ. Exp. Zool., Vol. 16, PP. 523-590. 

Loeb, Jacques. 

"14a Cluster Formation of Spermatozoa Caused by Specific Substances from 
Eggs. Journ. Exp. Zool., Vol. 17, pp. 123-140. 

’14b On Some Non-specific Factors for the Entrance of the Spermatozoon into 
the Egg. Science, N. S., Vol. XL., pp. 316-318. 


\ 


OBSERVATIONS ON THE SPERMATOGENESIS OF THE 
GALL-FLY, DRYOPHANTA ERINACEI (MAYR). 


H. L. WIEMAN, 


ZOOLOGICAL LABORATORY, UNIVERSITY OF CINCINNATI. 


INTRODUCTION. 


Dryophanta erinacet is one of the gall-producing Hymenoptera 
having two generations in the year: males and females in the 
spring, and females alone in the fall. The fertilized, eggs of the 
bisexual generation produce females exclusively, while the un- 
fertilized eggs of the female generation produce both males and 
females. 

The material for this study was obtained on April 13, 1914, 
from galls occurring on white oak trees (Quercus alba) in the 
neighborhood of Cincinnati. The galls are smooth spherical 
protuberances on the bud-scales, several millimeters in diameter. 
Usually one, but occasionally two, and sometimes three galls 
very close together are found at the end of a single twig. Twigs 
bearing galls were cut off, brought into the laboratory, and the 
cut ends inserted in sand moistened with water. Ten days later 
males and females emerged from the galls, and continued emerg- 
ing for two weeks. Copulation took place immediately after 
emergence. 

These galls supplied all stages of developing males and females 
from the late larva to the imago. For fixation Petrunkewitsch’s 
fluid was used; penetration being facilitated by making a longi- 
tudinal incision through the body wall. In some cases ovaries 
and testes were dissected out, but better results were obtained 
when the organs were left in situ and the entire animal sectioned. 

Embedding was done in rubber-paraffin, and serial sections cut 
10 w in thickness. Two methods of staining were used: safranin 
and light-green, and iron-haematoxylin with or without counter- 
stain. After dehydration the stained sections were cleared and 


‘For assistance in collecting and preserving material I wish to acknowledge my 
indebtedness to Dr. Annette F. Braun. 


34 


SPERMATOGENESIS OF THE GALL-FLY. 35 


mounted in euparal. Euparal offers several advantages over 
balsam as a mounting medium. Thus its use obviates running 
stained sections through absolute alcohol; since sections may be 
transferred directly from 95 per cent. alcohol to euparal. Next 
the index of refraction of euparal is low 1.483. And lastly euparal 
dries quickly, so that sections may be studied at the end of twelve 
to twenty-four hours after mounting, without danger of injury. 


OBSERVATIONS. 


The testes of the late larva and early pupa show primary 
spermatocytes at the end of the growth period as large polygonal 
cells having a reticulated nucleus containing a poorly defined 
nucleolus, often of a bipartite character (Fig. 1). The nucleolus 
does not take the safranin stain as deeply as the chromosomes, 
and thus differs markedly from the chromosome nucleolus of the 
primary spermatocytes of many Hemiptera. 

A true primary spermatocyte division does not occur. Instead, 
a small mass of cytoplasm free of chromatin is constricted off, 
forming the so-called polar body. Preparation for this sup- 
pressed or abortive division begins with a change in the outline 
of the cell, the spermatocyte assuming a pear shape (Figs. 2, 3 
and 4). From the narrow end of the cell and forming the stem of 
the pear, extends a short filar process. At the base of this process, 
which at first glance suggests the tail of a spermatozoon, is often. 
found a light basic-staining spherule which may or may not be a 
centrosome. While these changes are taking place in the cyto- 
plasm the nucleus undergoes a slight contraction and the chro- 
matin passes through a series of transformations terminating in 
the formation of chromosomes (Fig. 4). 

The next step in the process is somewhat uncertain and there 
may be some question as to seriation. It seems that after the 
chromosomes are completely formed, they become massed in 
clumps at one side of the nucleus, and from these masses distinct 
loops extend toward the opposite side of the nucleus (Figs. 5 
and 6). The cell shortens, the filar process becomes less distinct 
(Fig. 6), and a portion of the cytoplasm is constricted off (Figs. 7 
and 8). As this is taking place the nuclear membrane appears 
very irregular in outline but seems to remain intact. Inside the 


36 . H. L. WIEMAN. 


nuclear area the chromosomes are in the form of single rods whose 
free ends extend toward the polar body. There is every appear- 
ance to indicate a resistance of the part of the chromosomes 
against a tension pulling toward the polar body. Distinct spindle 
fibers are not to be seen, but the cytoplasm contains a reticular 
structure which may represent a poorly developed spindle. The 
polar body is quickly cut off from the cell to which, however, it 
may remain attached for a considerable length of time (Fig. 15). 
The free polar body of Fig. 8 belongs to a cell in an adjacent sec- 
tion. Polar bodies cut in various planes are frequently seen in 
the spaces between spermatocytes at this time (Figs. 8 and 16) 
and throughout the second spermatocyte division. The complete 
absence of polar bodies in cysts containing cells with the chro- 
mosomes in the looped condition of Figs. 5 and 6 makes it almost 
certain that the looped stage precedes that of Figs. 7 and 8, in 
which the chromosomes show free ends. 
Preparations for the second spermatocyte division follow very 
rapidly. After the formation of the polar body, the second sper- 
matocyte rounds up; the knot of chromosomes separates into 
distinct, short, thick, curved rods, 12 in number (Fig. 9). 
In the cell figured here, a late prophase, the nuclear mem- 
brane is fairly distinct. Details of spindle formation were 
not observed. Figs. 10 and 11 show characteristic side-views of 
spindles at metaphase. The chromosomes seldom lie in one 
plane so that counting even in polar views is a difficult matter. 
In such views, as in Figs. 12 and 13, 12 chromosomes can be 
counted with considerable accuracy in the majority of cases. 
A characteristic late telophase is shown in Fig. 14 which re- 
sembles to a striking degree a somatic mitosis, and strongly 
suggests that the chromosomes have been divided longitudinally. 
In later stages of this division (Fig. 15) the chromosomes become 
packed into dense compact masses, so that it is impossible to 
determine the number of constituent chromosomes in the 
daughter groups. When reconstitution of the nuclei occurs 
(Fig. 16), these masses break up'into slightly bent rods of ragged 
outline. In cross section these rods appear as dots of which 12 
can often be counted. Counts of the daughter groups of chro- 
mosomes made in this way are not very satisfactory, since one.is 


SPERMATOGENESIS OF THE GALL-FLY,. 37 


never sure that a cross-section includes all of the rods or that a 
single rod has not been cut more than once. 

The spermatids formed by this division seem therefore to be 
equal in size and chromatin content, and all of them develop 
into spermatozoa. There is no evidence of a heterochromosome or 
chromatoid body passing undivided into one of the spermatids. 

By the end of the second spermatocyte division all of the polar 
bodies are detached and show signs of disintegration, fragments 
being frequently seen in the intercellular spaces giving the ap- 
pearance shown in Fig. 16. 

The relatively distinct outline of the chromosomes seen in this 
last figure persists for but a short time and is completely lost in 
the young spermatids. Figs. 17 and 18 are early stages in the 
transformation of the spermatids into spermatozoa. 

Such in brief is an outline of the main features of development 
of the germ cells in the male of Dryophanta from the growth 
period to the spermatids. There is but one true maturation 
division—that of the second spermatocyte. The first spermato- 
cyte division is indicated by the pinching off of a small quantity 
of chromatin-free cytoplasm which forms the so-called polar body. 


DISCUSSION. 


Doncaster in his studies of the gametogenesis of the gall-fly, 
Neuroterus lenticularts, arrived at certain conclusions which may 
be considered at this point. This species of Hymenoptera has a 
similar life-history to that of Dryophanta. Thus according to 
Doncaster the female generation emerges in April from galls 
formed during the preceding summer and immediately lays eggs 
in oak buds (species?). Early in summer the galls appear from 
which males and females emerge. After copulation the female 
lays eggs in the tissues of young leaves at the side of a small vein. 
From the galls resulting, females emerge in the following spring. 

As in Dryophanta, therefore, the fertilized eggs of the bisexual 
generation develop into females; while the unfertilized eggs of 
the female generation produce both males and females. 

Doncaster found that the first spermatocyte division is abor- 
tive—a small portion of the cytoplasm being constricted off as the 
polar body. This is followed by a resting stage which resembles 


38 H. L. WIEMAN. 


the metaphase of a true division, but is distinguished from it by 
the persisting nuclear membrane and the position of the chromo- 
somes at one end of the nucleus near the broad end of the cell. 
No nuclear division takes place but the nucleus becomes oval in 
shape and the chromosomes generally contract to form a compact 
mass lying across its center. Insome cells at least this chromatin 
mass seems to divide—one half passing to each side of the oval 
nucleus. The chromatin may finally disperse and give rise toa 
condition resembling the first spermatocyte in which the chro- 
matin has begun to appear. ‘“‘Possibly the division of the chro- 
matin inside the nucleus, which occasionally seems to occur, is 
the persistent remnant of a true nuclear division, or it may be 
compared with the ‘intranuclear karyokinesis’ described by 
Kostanecki in the parthenogenetic eggs of Mactra”’ (p. 93). 
Toward the end of the rest stage the chromatin becomes grouped 
in the form of large elongate granules or small bands having a 
more or less meridional arrangement under the membrane. 

The second spermatocyte division in Neuroterus is a true mi- 
totic division in which the haploid number of chromosomes, 10, 
appears on the spindle to be equally divided between the daughter 
cells. There is also a small stained body lying outside of the 
spindle which passes undivided to one of the spermatids. 

In the spermatogonia and in mitotic figures of nerve cells in 
the developing nervous system Doncaster finds the halpoid 
number of chromosomes, 10, but in mitoses of immigrant meso- 
derm cells the diploid number, 20. 

The eggs layed by the females of the bisexual generation 
undergo two maturation divisions; leaving 10 chromosomes for 
the female pronucleus. The spermatozoon brings into the egg 
10 chromosomes, and 20 chromosomes appear on the cleavage 
spindles. The parthenogenetic eggs of the female generation 
may be divided into two groups: Those which undergo matura- 
tion and develop into males; those which omit the maturation 
divisions and develop into females. In the first group 10 chro- 
mosomes are found in the cleavage divisions; in the second group 
20. Since any female produces only one kind of egg, there are 
male-producing females and female-producing females. 

Mitoses in the nervous system of all females show the diploid 
number of chromosomes. 


SPERMATOGENESIS OF THE GALL-FLY. 39 


Returning now to Dryophanta I should like first to consider 
the stage represented in Figs. 5 and 6, which I believe corresponds 
to the second spermatocyte resting stage mentioned by Don- 
caster in Neuroterus. The figures at first glance suggest the 
synapsis stage of other insects, but in view of other facts it is 
difficult to interpret the condition as a fusion of chromosomes. 
Earlier stages such as the prophase shown in Fig. 4 display the 
same number of chromosomes as appears in the-second spermato- 
cyte division, 12, which is assumed to be the haploid number 
approximately. Since there is no evidence in Dryophania of 
an intra-nuclear division of these 12 chromosomes into two groups, 
a true synapsis at this time would be equivalent to a second 
‘“reduction.’”’ A more probable interpretation of this “looped 
stage’’ and one that is warranted by a close study of the sections 
is that the limbs of a loop are the halves of a chromosome that has 
undergone a temporary and incomplete splitting. With the 
next step in the process, the formation of the polar body, the 
split disappears and the chromosomes have every appearance 
of being single, solid rods (Figs. 7 and 8). The latter condition 
might of course be brought about by breaking of the loops at the 
middle, but in that event one would expect to find twice as many 
single chromosomes as loops. Such is not the case, for the 
number of unsplit chromosomes is the same as the number of 
loops so far as could be determined. Reversing the seriation at 
this point would of course change the interpretation offered here; 
but the main reason for placing the looped stage before the other, 
as has been mentioned above, is that there is no evidence of 
polar body formation at this time. And to this may be added 
the fact that the outline of the cell at the looped stage as shown 
in Fig. 6 represents an intermediate condition between that of 
Fig. 4 in which there can be no question about polar bodies being 
absent, and Figs. 7 and 8, in which the polar bodies certainly are 
present. 

An actual resting stage, if one occurs at this time, must be of 
very short duration. The second spermatocyte division follows 
very quickly after the formation of the polar body. Fig. 9 
represents a prophase of this division in which the chromosomes 
are surrounded by an intact nuclear membrane. The spindle 


40 H. L. WIEMAN. 


area of the second spermatocyte is rather distinctly marked off 
from the rest of the cytoplasm (Fig. 10) and suggests that the . 
nuclear membrane disappears very slowly. 

Polar views of the metaphase display, as nearly as could be 
determined, 12 chromosomes, presumably the haploid number. 
It would seem that each chromosome is divided quantitatively 
by a longitudinal splitting; although it must be remembered that 
attempts at verifying this conclusion by studying the constit- 
uents of the daughter groups are not satisfactory owing to the 
tangled condition of the chromosomes. ; 

I find nothing resembling the small stained body which in 
Neuroterus according to Doncaster passes undivided to one of 
the spermatids. As Wilson has observed this body is of the same 
nature as the chromatoid body seen in the growth-period and 
spermatocyte-division of Pentatoma. The chromatoid body is of 
rounded form, dense and homogeneous consistency, and after 
double staining with haematoxylin or safranin and light green 
is at every stage colored intensely blue-black or brilliant red, 
precisely like the chromosomes of the division period or the chro- 
mosome-nucleoli of the growth period. Nevertheless Wilson 
finds that the body is neither a chromosome nor any kind of a 
chromosome and takes no visible part in the formation of the 
spermatozoa. In the transformation of the spermatids it wanders 
far into the sperm-tail and is at last cast off altogether. 

I have not yet had opportunity to study the maturation phe- 
nomena of the egg in either generation of Dryophanta, but obser- 
vations confined to individuals of the bisexual generation point 
to general conclusions which differ somewhat from Doncaster’s 
views regarding the chromosomal relations in the alternate 
generations. In the material at my disposal spermatogonial 
divisions are not abundant enough to determine the number of 
chromsomes. While mitoses abound in the somatic cells of male 
larvae and pupae, it is difficult to find good clear metaphases; but 
wherever counts were possible, the number found was 12 (Fig. 
19). In the follicle cells of the ovary I have found it less difficult 
to count the chromosomes. Figs. 20, 21 and 22 are drawings of 
metaphase plates of such cells in which the numbers are re- 
spectively 13, 14 and 13. 


SPERMATOGENESIS OF THE GALL-FLY. AT 


In the somatic cells of both males and females one occasionally 
finds mitotic figures concaining a much larger number of chro- 
mosomes, but such cases are in the nature of exceptions and no 
- one would contend that they represent an average condition. 
If there is such a thing as constancy in the number of chromo- 
somes in the majority of somatic cells, the constant is in the 
neighborhood of 12 in both males and females of the bisexual 
generation. Because this is the number of chromosomes found 
in the second spermatocyte division, I2 is assumed to be the ap- 
proximate haploid number. Now in any case where an accurate 
count is difficult or impossible in the somatic cells, it is always 
possible to determine with certainty that the number is very 
much less than the expected diploid number 24. In view of the 
fact, that in the honey-bee it is said that the somatic mitoses 
show a very much higher number of chromosomes than occurs 
in the gonial cells, somatic mitoses should not be used as a safe 
and reliable method of determining the diploid number. There 
may however.be some significance in the fact that a large number 
of somatic cells of both males and females of Dryophanta contain 
a number of chromosomes that approximates the number found 
in the dividing spermatocyte rather than a multiple of this 
number. 

Any definite statement regarding the origin and significance 
of this condition must await examination of the maturation and 
cleavage spindles of the egg. However, the facts at hand do 
suggest that the males and females of the bisexual generation of 
Dryophanta develop from eggs whose chromosomes have under- 
gone reduction in maturation. The slightly large number of 
chromosomes found in the females somatic tissues may or may 
not be of significance, but if sex determination has its basis in 
the chromosomes, a difference in the method of distribution of the 
chromosomes in maturation may explain why some of these 
eggs develop parthenogenetically into females and others into 
males. 

In a recent paper Nachsheim has summed up in a general 
statement the results of investigations dealing with sex-de- 
termination in Hymenoptera as follows: “Die Mannchen der 
Hymenopteren entstehen aus unbefruchtete Eiern, die zwei 


42 H. L. WIEMAN. 


Richtungskérper abgeschniirt und eine Reduktion ihrer Chromo- 
somenzahl erfahren haben. Sie besitzen also nur ein Chromo- 
somensortiment, das miitterliche, und infolgedessen muss in der 
Spermatogenese die Reduktionsteilung unterbleiben. Die Weib- 
chen der Hymenopteren besitzen beide Chromosomensortimente, 
also die diploide Chromosomenzahl in ihren somatischen Zellen, 
da sie aus befruchteten Eiern ihre Entstehung nehmen oder—bei 
den Blatt- und Gallwespen—zwar ebenfalls aus unbefruchteten 
Eiern, aber aus solchen, die den Reifungsteilungen ihre Chromo- 
somenzahl nicht reduciert haben; entweder findet in diesen Eiern 
iiberhaupt nur eine Reifungsteilung statt, oder beide Reifungs- 
teilungen sind Aquationsteilungen. Der zweite Richtungskorper 
kann also. . . . an Stelle der Spermatozoons treten, d.h. der 
zweite Richtungskérper bringt in Verbindung mit der Eikern 
dasselbe Geschlecht hervor wie der Eikern in Verbindung mit 
einem Spermakern”’ (pp. 220-221). 

My findings in the somatic chromosomes of Dryophanta raises ~ 
the question as to whether females of the bisexual generation are 
produced parthenogenetically from eggs that do not undergo 
reduction in maturation. An examination of maturation stages 
in the egg is necessary to decide this point and material for this 
purpose is being collected at the present time. 


LITERATURE CITED. 
Doncaster, L. : 
’r011 Gametogenesis of the Gall-fly, Neuroterus lenticularis (Spathegaster 
baccarum). Parts I. and II. Proc. Roy. Soc., B., Vols. 82 and 83. 
Nachsheim, H. 
’r3 Cytologische Studien tiber die Geschlechtsbestimmung bei der Honigbiene 
(Apis mellifica). Arch. f. Zellfschg. Bd. 11. 
Wilson, E. B. 
713, A Chromatoid Body Simulating an Accessory Chromosome in Pentatoma. 
Biot. BULL., Vol. 24, 1913. 


44 H. L. WIEMAN. 


EXPLANATION OF PLATES. 


The figures are camera drawings made at table level with Zeiss apochromatic 
objective, 1.5 mm. and compensating ocular, 12. There has been some reduction in 
reproduction. , 

PLATE I. 


Fic. 1. Primary spermatocyte at the end of the growth period. Male pupa. 

FIGS. 2, 3 AND 4. Primary spermatocytes undergoing changes in outline pre- 
liminary to the formation cf the polar body. 

Fics. 5 AND 6. Primary spermatocytes having chromosomes in the form of 
loops or split rods. 

Fics. 7 AND 8. Stages in the cutting off of the polar body. Fig. 8 contains a 
second polar body belonging to a cell in a neighboring section. 

Fic. 9. Prophase of the second spermatocyté division showing 12 chromosomes. 

Fic. 10. Side view of the second spermatocyte spindle at metaphase. 


BIOLOGICAL BULLETIN, VOL. XXVIII. PLATE |. 


H. L. WIEMAN. 


omy 


46 H. L. WIEMAN. 


PLATE II. 


Fic. 11. Side view of the second spermatocyte spindle at metaphase. 

Fics. 12 AND 13. Polar views of the second spermatocyte spindle at metaphase 
showing 12 chromosomes. 

Fic. 14. Second spermatocyte spindle at late anaphase showing a free polar 
body near the upper end of the cell. 

Fic. 15. Second spermatocyte at telophase with a polar body attached to the 
upper daughter cell. 

Fic. 16. Early spermatid, reconstruction of the nuclei. Polar body fragments 
near the upper cell. 

FIGS. 17 AND 18. Stage in the transformation of spermatids into spermatozoa. 

Fic. 19. Metaphase chromosome group in the mitosis of a developing wing, 
showing 2 chromosomes. Young male pupa. 

FIGS. 20, 21 AND 22. Metaphase chromosome groups of ovarian follicle cells, 
showing 13, 14 and 13 chromosomes respectively. Late female larva. 


PLATE Il. 


BIOLOGICAL BULLETIN VOL. XxVII. 


H. L. WIEMAN. 


EGG ALBUMEN AS A CULTURE MEDIUM FOR CHICK 
IUSSUWIE,, 


OLIVE SWEZY. 


Egg albumen as a culture medium for chick tissue in vitro 
has received but scant attention from experimentalists, in spite 
of the fact that it forms the natural medium, in part at least, of 
the embryo chick. Ina recent series of experiments, however, 
results have been obtained which show that all the usual mani- 
festations of cell activity, noted by various observers in other 
culture media, were to be met with in cultures made from egg 
albumen, and have, I believe, demonstrated satisfactorily its: 
entire adaptability to that use. These experiments were carried 
on in the laboratory of Prof. S. J. Holmes, to whom my thanks 
are due for his kindness in giving advice and assistance through- 
out the course of the work. 

The technique followed has been that outlined by Burrows and 
Carrel, modified to suit the different conditions under which the 
work had to be carried on, using embryos varying in age from 
twenty-four hours to fourteen days. Of these it was found that 
the most successful results were obtained from embryos of from 
ten to fourteen days growth, though all showed considerable 
activity. Fragments of all the organs of the body, including the 
brain and spinal cord, were used, but the most active growths were 
obtained from the heart. Several series of preparations were 
made by cutting up the entire embryo into minute particles in 
a small amount of Ringer’s solution and egg albumen, stirring 
and shaking these rapidly for a few minutes and then placing a 
small drop of the mixture on the slide and sealing in the usual 
way. By this process cultures could be made containing but a 
few or even single cells. The medium used has been egg albumen 
alone or mixed with- varying proportions of egg yolk, -Ringer’s 
solution and extract of muscle tissue. Egg yolk proved entirely 
unsatisfactory because of the impossibility of seeing what was 
taking place within it. The best results were obtained from egg 

47 


48 OLIVE SWEZY. 


albumen alone and with mixtures of albumen and muscle tissue 
extract, the latter being prepared from embryo chick tissue and 
added to the albumen either before or after making the culture. 
Egg albumen coagulates to a more or less firm consistency and thus 
gives one of the conditions apparently requisite for the growth and 
activity of the tissue cells. 

Owing to the viscosity of the albumen, considerable care is 
necessary in handling the specimens when it becomes needful 
to transfer the culture to a fresh medium, the usual method of 
procedure being to cut away the old albumen with a sharp knife. 
When, as is frequently the case, the outgrowth seemed to be 
mainly on the surface of the glass, and thus could not be trans- 
ferred in the usual way without the loss of the greater part of 
the growth, another method was used. Inverting the cover glass 
the albumen was removed with forceps and pipette, several 
changes of Ringer’s solution successively placed over the culture 
and, after removal of this, a fresh drop of albumen was added to 
the culture and it was again sealed up. 

The latent period, before the beginning of activity of the 
culture, lasted from half an hour to several days. Usually, in 
good preparation, active amceboid movements began within half 
an hour after being put on the slide. At that time along the 
border of the tissue could be seen the elongated, outpushing cells 
forming a fringe along what was before a clear cut outline, with 
a few scattered cells lying at some little distance from the main 
mass. These cells displayed very active amoeboid movements 
that are less common in the older cultures though still present to 
some extent. When these cells are chilled or disturbed they 
contract and become rounded. Ona number of cultures groups 
of cells showed long clear processes extending outward, some- 
times branched, with the ends breaking up into short filaments. 
These were in all cases cultures which included portions of the 
brain or spinal cord from a four-day chick. An attempt was 
made to photograph one of these cultures but the length of time 
necessary was sufficient to chill the slide and, on examination, 
it was found that the processes had all been retracted. Subse- 
quent incubation had no effect on the culture, though disinte- 
gration did not take place for several days. In all the cultures 


EGG ALBUMEN AS CULTURE MEDIUM FOR CHICK TISSUE. 49 


these processes disappeared, were retracted apparently, in the 
course of fifty to seventy hours and no further evidences of them 
were seen. In the preparations made by shaking up the finely 
cut embryo with Ringer’s solution, a greater or less number of 
single cells were found. In the course of a few days these were 
greatly increased in number with a distinct massing together. of 
the cells, usually along the outer border of the drop of albumen. 
Owing to accidents of various kinds these were not carried along 
far enough to show the tissue formation noted by Carrel. 

The most marked instance of tissue formation was that appar- 
ent in a culture made from the heart of a fourteen day chick, 
which, at the end of twenty days was encircled by a new forma- 
tion five times the diameter of the original piece of tissue. This 
new formation was several cells in thickness and composed of 
fusiform and polygonal cells, sometimes massed together, forming 
a network, or in other places showing distinct cell boundaries. 
Among these cells many showed division figures at various stages. 
Around the outer margin of the mass of cells and extending nearly 
three-fourths of the entire distance around it, the cells had taken 
on a different character. Here they had become flat, thin and- 
elongated in a direction parallel with the margin of the circle. 
This formation was several cells in thickness with the cells closely 
matted together and forming a distinct boundary that was 
conspicuous without the aid of alens. The remaining one-fourth 
of the margin was occupied by cells actively pushing outward. 

To test the effects of cold on the growth of the tissues, the 
embryo was sealed up in a stender dish containing Ringer’s 
solution and placed in the ice box of the refrigerator with the 
temperature but a few degrees above zero, Centigrade. The 
first of these was used the second day and behaved like normal 
tissue. Most of those kept in the refrigerator for a number of 
days became infected with bacteria. The longest period of cold 
storage which gave successful cultures was four days, from Jan- 
uary 31 to February 4. One half hour after making the cultures 
from this embryo the cells were moving out in an active condition 
in four out of the sixteen cultures made. The subsequent history 
of these cultures was the same as that of unrefrigerated tissue. 

The longest period during which tissues have been kept alive 


50 - OLIVE SWEZY. 


without any evidences of necrobiosis has been ninety-three days, 
and in the majority of these cases death has been caused by in- 
fection with bacteria or molds or other accidents, and, not, 
apparently, by any lack of vigor in the tissues themselves. This, 
in general, seems to be true of most of the cultures which appear 
to be in a thriving condition after the second day or third day, 
and especially where renewals of the culture medium have been 
frequent, and precautions have been taken to avoid tearing or 
otherwise injuring the tissues. However disintegration fre- 
quently takes place from no apparent cause. 

Egg albumen presents some difficulties when a stained prep- 
aration from the culture is desired, on account of its avidity for 
stains. In the first stained preparations made it was impossible 
to distinguish the outlines of the cells, and the study of the 
specimen seemed a hopeless task. This difficulty was later over- 
come by the following methods: the cover glass was inverted and 
placed on the mouth of a vial containing a quantity of osmic acid. 
The mouth of the vial was small enough to be completely covered 
by the cover glass and yet not touch the preparation. After 
fixing in this manner for ten minutes the cover glass was placed 
in a stender dish containing distilled water and left for a number 
of hours. Frequent agitation and changes of the water removes 
the greater part of the albumen, leaving the tissue adhering to 
the glass, which may then be put through the alcohols and stained 
in the usual way. With this method very clear preparations may 
be obtained. 


ZOOLOGICAL LABORATORY, 
UNIVERSITY OF CALIFORNIA, 
BERKELEY, CAL., October 13, 1914. 


THE INFLUENCE OF PRODUCTS OF PATHOLOGIC 
METABOLISM ON THE DEVELOPING TELEOST 
OVUM. 


KE. I. WERBER, 


DEPARTMENT OF BIOLOGY, PRINCETON UNIVERSITY. 


In his recent work on pathological human ova, after careful 
sifting of anatomical evidence, Mall! arrives at the conclusion 
that the failure of large numbers of ova to develop normally is 
to be traced to diseases of the uterus. According to his view, 
which is supported by obstetrical and gynecological data, diseases 
of the uterus are the primary cause of the faulty implantation of 
the ovum. This in turn makes proper nutrition of the developing 
embryo impossible thus leading to various degrees of malforma- 
tions by arresting development. The deformed embryo is 
eventually aborted after it has exhausted its inadequate means of 
subsistence in the uterus. Full-term monsters would be born 
from such deformed embryos if they were not hindered in their 
further development by starvation. According to this theory, - 
therefore, an apparently healthy ovum discharged into a diseased 
uterus fails to develop normally owing to its defective implan- 
tation. 

Mall studied largely pathological ova of the first two months 
and the interpretation of the numerous cases described by him 
seems justified. Practically all pathological ova of the early 
months studied by him as well as by other investigators, exhibited 
the condition of faulty implantation, so that it is not unwarranted 
to regard this condition as the direct cause of monstrous develop- 
ment. 

A consideration of some instances of arrested, defective or 
even monstrous development found after full-term birth would 
suggest, however, that there must be also some other factors 

1 Mall, F. P., ‘‘A Study of the Causes Underlying the Origin of Human Mon- 


sters.” Journ. of Morphology, Vol. X1X., 1908; “The Pathology of the Human 
Ovum” in Keibel-Mall ‘‘Handbook of Human Embryology,” 1910. 


5st 


52 E. I. WERBER. 


which primarily interfere with normal development. Such 
defects as rudimentary development of one or both eyes, con- 
genital absence of both arms, hydrocephalus, possibly also cases 
of congenital deafness, to mention only a few that are well 
known to occur, can, in the writer’s opinion, hardly be traced 
to defective implantation. The results of investigations in 
experimental teratology by Panum,! Dareste? and more recently 
by Stockard’ and Bardeen* would seem to suggest that some 
physico-chemical factors may be at work in a great number of 
cases of pathological development. These factors may in some 
instances be the primary cause of terata, while in other cases 
they may be only secondary contributing causes. 

The experimental teratologists subjected developing ova in 
very early stages to changes in the physico-chemical nature of 
the environment and found that various monstrosities could be 
produced under these conditions. It was impossible, however, 
for them to control the results of experimentation, as they could 
not predict the type of monster which would result from the 
employment of the same factors. The experiments of Stockard, 
where a more or less definite monstrosity—cyclopia or monoph- 
thalmia—appeared with considerable certainty in a large per- 
centage of embryos developing in magnesium chloride or alcohol 
solutions, mark a distinct progress in this field of inquiry, because 
they paved the way towards experimental control of monstrosities 
occurring in nature. 

To the writer Stockard’s work suggested the possibility that 
the monstrosities met with in higher animals and man may to a 
certain extent be due to the influence of injurious substances 
found in the circulation under pathological conditions. While 
this hypothesis could not be applied to bacterial toxins on account 
of insufficient knowledge, it seemed that some substances thrown 
into the circulation in various metabolic diseases may be re- 

1Panum, ‘‘Entstehung der Missbildungen,”’ 1860. 

2 Dareste, ‘‘Recherches sur la production de monstrosites,’’ Paris, 1891. 

3 Stockard, C. R., ‘‘ The Artificial Production of a Single Median Cyclopean Eye 
in the Fish Embryo by Means of Seawater Solutions of Magnesium Chlorid,” 
Arch. f. Entwmech., Vol. XXII., 1907; ‘‘The Influence of Alcohol and Other Anzs- 


thetics on Embryonic Development,”’ Am. Jour. of Anat., Vol. X., I910. 
4 Bardeen, C. R., Jour. of Experimental Zool., 1907; Am. Jour. of Anat., Vol. XI. 


TELEOST OVUM. 53 


sponsible for pathological development. Thus the etiology of 
defective or monstrous development would be traced to the 
pathological metabolism of the mother or possibly even of the 
father. For, as Bardeen! has shown, a normal, healthy ovum of 
the toad, if fertilized with sperm which had been injured by 
exposure to the action of X-rays, will give rise to a deformed 
embryo. 

With this idea in mind the writer conducted during the summer 
of 1914 experiments on eggs of Fundulus heteroclitus. The eggs 
of this fish are easily obtained at Woods Hole and are excellent 
material for experimentation. The investigations on the fish 
eggs are of a preliminary character, and were undertaken to 
ascertain the influence of some toxic substances occurring in 
pathological metabolism on the developing egg. 

The number of these substances being rather large while the 
spawning season is limited to a few weeks, it was impossible to 
try more than a few of the chemicals. Urea, butyric acid, lactic 
acid, sodium glycocholate, acetone and ammonium hydroxide 
were tried as to their effect on the development of fertilized eggs. 
Definite results were so far obtained only with butyric acid and 
acetone. 

Ten c.c. of a 1/12—1/14 molecular solution in 50 c.c. of sea water 
was found to give the greatest number of monsters when butyric 
acid was used. The eggs were submitted to the action of this 
solution for 20 hours after they had reached the eight-cell or 
sixteen-cell stage, 7. e., 3 to 3% hours after fertilization. While 
under this procedure numerous monstrosities were at first ob- 
tained, the method failed almost completely in later experiments. 
I therefore employed developing eggs in the first stages of division 
(2- and 4-cell stages) when many monstrosities were produced 
even after a sojourn of thirty hours in the butyric acid solution. 
But it seems to me that the reason why the method failed with 
the eggs in more advanced cleavage stages was that the time of 
exposure was too long, as very many eggs were dead by the 
end of that treatment, and that with an exposure of 10 or 15 hours 
better results would have been obtained. 

There is, however, as important difference in the effect which 


1 Bardeen, C. R., Jour. of Experimental Zool., 1907; Am. Jour. of Anat., Vol. XI. 


54 E. I. WERBER. 


this toxic substance has upon developing eggs in the first and 
second or in the third and fourth divisions. In the former case 
anterior hemiembryos, dwarf embryos with deformities of the 
eyes or of the otic vesicle, and malformations of the most extreme 
kind were predominant, while in the latter deformities of the 
eye such as cyclopia and monophthalmia, etc., were mostly 
observed. In either case, however, there were very few embryos 
in which only the nervous system was affected. In most of the 
deformed embryos all organ systems were more or less involved 
in the malformation. 


Similar results were obtained with acetone in sea water, varying 


in concentration from 20-50 c.c. of a molecular solution in 50 c.c. 
of sea water. In this mixture the eggs remained from 24-72 
hours from the eight-cell or sixteen-cell stage. In every case 
great numbers! of monsters similar to those already mentioned 
were produced. 

The monstrosities in both series of experiments with butyric 
acid and acetone being essentially alike it will not be necessary 
to describe separately the deformities produced by each. 

Cyclopia and asymmetric monophthalmia were found to occur 
rather abundantly. There were also some cases of asymmetric 
monopththalmia in which an open orbit was found on the side 
lacking the eye. It is of some interest to note in this connection 
that the eyeless orbit in such cases is usually closed on the outside 
by periorbital tissues. The anatomy of the head of such embryos 
may probably reveal some interesting conditions. Other cases 
of asymmetric monophthalmia were found in which an apparently 
free eye had developed on the yolk-sac at a considerable distance 
from the embryo. Probably the most striking of the results 
obtained in this investigation were some eggs in which nothing 
could be observed but an eye. In only one case this eye seemed 
to be perfectly developed, while the other solitary eyes had 
‘“‘coloboma’’-defects, the fissure of the chorioid still being 
patent. Only a few (five or six) of these malformations are 
recorded, but in spite of their rare occurrence they are very sig- 
nificant from the standpoint of experimental embryology. At the 


1 No attempt was made to ascertain the percentage of the deformities found in 
these experiments, this part of the work being deferred to later investigation. 


TELEOST OVUM. 55 


present time it is, obviously, impossible to account for the occur- 
rence of these remarkable cases. However, it is hoped that an 
anatomical investigation of early stages in the development 
of eggs subjected to the influence of the environmental modi- 
fications used in these experiments, may give at least a clue as to 
what may have happened in the development of these eggs. 
Practically all other known deformities of the eye such as total 
blindness, or presence of lenses only, or presence of supernumerary 
lenses were frequently found. 

To the student of the physiology of development the occurrence 
in these experiments of large numbers of anterior hemiembryos 
which seem to be closely analogous to those obtained by mechan- 
ical means by Roux,! Endres,? Morgan? and K. Ziegler‘ will be 
of special interest. As will be pointed out soon the formation 
of the hemiembryos in these experiments may also possibly be 
due to similar factors. 

A great number of embryos were hydrocephalic and so far as 
could be determined it is reasonable to expect that an anatomical 
investigation may reveal in some deformed embryos oedematous 
conditions, also herniae and other mechanical obstructions which 
played a part in their formation. 

Striking abnormalities of the heart and blood-vascular system 
were found in all malformed embryos with the exception of those 
which showed only median cyclopia. Some were entirely devoid 
of the heart, while other possessed an exceedingly delicate tube 
in its place which was practically straight and of about the size 
of the intestinal blood vessels in a normal embryo of a corre- 
sponding stage. The rate of the heart beat varies with the 
degree of the abnormality of the organ, and is, as a rule, very slow 
in all monstrous embryos. The range of variation in the develop- 
ment of the blood vessels is very wide. There may be merely 
blood islands scattered on the yolk-sac, rudimentary, imperfectly 
connected, or in some instances more or less normal vessels. 

1 Roux, W., ““Gesammelte Abhandlungen zur Entwicklungsmechanik der Or- 
ganismen,”’ II., 1895. 

2 Endres, H., ““Anstichversuche an Froscheiern,’”’ Sitzber. d. zool.-bot. Sektion 
d. schlesischen Ges. f. vaterlandische Kultur, 1894. 


3’ Morgan, T. H., “The Formation of the Embryo of the Frog,’’ Anat. Anz., 1894. 
4 Ziegler, K., “Zur Postgenerationsfrage,’’ Anatomische Hefte, Vol. LXVI., 1902. 


56 E. I. WERBER. 


Twins were found only in a few cases and only once were true 
‘‘Siamese’’ twins observed. They were much deformed, had 
one common heart and only vestigial eyes. Several eggs were 
recorded, in which an anterior duplicity had developed. In one 
of these latter cases the components of the duplicity were totally 
blind, hydrocephalic, their hearts were very delicate, the blood 
vessels rudimentary and the yolk-sac was covered with dense 
networks of richly pigmented blood islands. 

These monstrous embryos hatch only very rarely, most of 
them dying after the development has reached the stage in which 
the remant of the yolk-sac is in the normal embryo converted into 
the anterior body wall. As far as could be determined from the 
embryos in toto it is the enormously large (oedematous?) peri- 
cardia that mechanically obstruct the formation of the ventral 
body wall. The correctness of this interpretation will be tested 
by microscopic sections of these embryos. 

The mechanism of the formation of the described monsters can 
at this time not even be definitely suggested. The observation 
was made that the yolk-sac in all extremely malformed embryos 
shows a marked decrease in size as compared with that of normal 
eggs of the corresponding stage of development. The greater 
the degree of injury inflicted on the embryo the smaller the yolk- 
sac. It is not impossible that the chemicals used in these experi- 
ments indirectly bring about this decrease in the size of the 
yolk-sac. For it was noticed that the chemicals used in these 
experiments softened the egg-membrane considerably, a fact 
which suggests an increase in the permeability of the egg. 
Owing to both increased permeability of the germ-disc cells and 
to internal osmotic pressure of the yolk-sac, an escape of sub- 
- stance from the yolk-sac might have been caused, which, being 
forced out at different points of the yolk-sac, might have frag- 
mented the germ-disc. Many eggs were observed in which 
this fragmentation of the germ-disc was very evident. Some 
parts of this ruptured germ-disc may be so badly damaged as 
not to beable to develop further, while the remaining fragments, 
even if they are very small, may still give rise to various monsters, 
hemiembryos, dwarfs or even toa solitary eye. Or possibly the 
decrease in size of the yolk-sac of malformed ova may point to 


TELEOST OVUM. 57 


elimination of both yolk-sac and germ substance as an effect of 
the solvent action of the chemicals to which the eggs were ex- 
posed. Whatever the mechanism involved in the production of 
-the recorded pathological ova may be, at the present time, it can 
hardly be more than conjectured. It will be the object of future 
investigations to find a satisfactory answer to this open question. 

There seems to be a close similarity between these cases where 
parts of the germ-disc are apparently lost through elimination of 
some kind and the production of hemiembryos by mechanically 
injuring one of the blastomeres of the developing frog’s egg, as 
described by Roux, Morgan and other investigators. 

The writer intends to continue this work on the teleost eggs 
as well as on the amphibian and hen’s eggs. He also hopes that 
he may in the near future secure adequate facilities for carrying 
on experiments on the influence of the toxic substances of patho- 
logical metabolism on the development of the mammalian em- 
bryo. The plan of this work would be to mate animals in which 
metabolic disturbances had been produced experimentally. 

A complete description and analysis of the results obtained in 
the investigation reported here will be published at an early date. 

The writer takes pleasure in acknowledging his indebtedness to 
Professor C. R. Stockard of Cornell University Medical College 
with whom he on several occasions had discussed some phases of 
the work, and from whom he has received valuable suggestions 
regarding preservation of material. 


MARINE BIOLOGICAL LABORATORY, 
Woops Hote, Mass., 
September 3, 1914. 


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Volenee Vie February, 1915. No. 2. 


PolOLOGICAL BULLETIN 


ee ee 


AN EARLY STAGE OF AN EXPERIMENTALLY PRO- 
DUCED EXTRAUTERINE PREGNANCY AND 
THE SPONTANEOUS PARTHENOGENESIS 
OF THE EGGS IN THE OVARY OF 
THE GUINEA PIG:! 


LEO LOBB. 


The observation on which I wish to report is of great interest 
from several points of view. It explains the negative result of 
our former attempts which aimed at producing experimentally 
an extrauterine pregnancy in the guinea pig. It contributes to 
the understanding of the mechanism of the sexual cycle and it 
makes certain my previous conclusions, which formerly had only 
been probable, concerning the fargoing parthenogenetic develop- 
‘ment of ova in the ovary of the guinea pig, conclusions which our 
previous studies had made very probable. Ina great number of 
previous experiments we made incisions in various parts of the 
uterus of the guinea pig and at different times after copulation.? 
Under these circumstances it certainly must often have happened 
that fertilized eggs left the uterine cavity. But_extrauterine 
pregnancy did in no case take place under such circumstances. 
Even after ligation of the fallopian tubes we were not able to 
observe the occurrence of an extrauterine pregnancy. This 
latter observation is in accordance with some experiments of 
Mandl and Schmidt.’ It was of interest to determine what was 
the fate of the ova which left the lumen of the uterus and passed 
into the peritoneal cavity after fertilization. An observation 

1 From the pathological laboratory of the Barnard Free Skin and Cancer Hos- 
pital, St. Louis. 

2Leo Loeb and John W. Hunter, University of Pennsylvania Medical Bulletin, 


Dec., 1908. 
3 Archiv f. Gynaecol., 56, 1898. 


59 


u“ f DS 


60 LEO LOEB. 


which we made in the course of our continued experiments serves 
to clear up this point. 

Two days, sixteen hours after copulation, incisions were made 
into the uterus of a guinea pig. The weight of the animal at the 
time of the operation was 550 grams. ‘The incisions were longi- 
tudinal and extended through both horns of the uterus up to 
near the point of juncture with the tubes. Besides the longi- 
tudinal incisions a number of transverse incisions into the uterine 
wall were made. Eighteen days after copulation uterus as well 
as one of the ovaries was taken out for examination. The ovary 
was cut into serial sections. Small follicles in the early stages 
of development, as well as other follicles in early stages of con- 
nective tissue atresia were found. In addition there were many 
follicles in the last stages of follicular atresia. There were also 
present several young corpora lutea, the center of which was 
partly filled out by connective tissue, while the center of the 
cavity had not yet been organized by connective tissue. There 
were furthermore present corpora lutea in an early stage of retro- 
gression, as well as yellow bodies, completely atretic corpora 
lutea. These findings correspond to an ovary about three days 
after ovulation. 

Microscopic examination of those parts of the uterus which 
had not been incised during the operation showed cylindrical 
surface and glandular epithelium with numerous mitoses in the 
glandular ducts. The fundi of the glands are somewhat smaller. 
In the lumen of the uterus there are some polynuclear leucocytes, 
a greater number of which are found in the ducts of the glands. 
In the connective tissue of the mucosa as well as in the surface 
epithelium the presence of several small round cells is noted. 
There are very few mitoses in the connective tissue of the mucosa 
which is rich in nuclei. These findings correspond to a condition 
of the uterus about 3-3% days after copulation. 

Near the tubal end of one of the uterine horns, not far from the 
usual situation of the ovary there was a small nodule. This 
nodule was cut in serial sections, and its structure is best ex- 
plained by referring to the illustrations. 

Fig. 1 shows the position of the embryo. 

(a) It lies in the neighborhood of the fallopian tube. 


EXPERIMENTALLY PRODUCED EXTRAUTERINE PREGNANCY. 61 


\ 
(b) Some distance from the embryo we see the cut wall of the 


uterus. 
-(c) In the direction towards the tube we see the musculature 
of the uterus, in the opposite direction the epithelium with the 


Fic. 1. Low power. a, embryo; 0, Fallopian tubes; c, everted walls of the 
uterus. 

A more detailed explanation of the figures is found in the text. 

FIGS. I, 2, 3, 4 and 6 are from microphotographs. Fig. 5 from a drawing. 


glands and the connective tissue is visible. In the detached part 
of c the glands have the character of mucous glands. At this 
place the mucosa of the uterus is everted as a result of the incision. 
If we follow on further sections the position of the placenta which 
surrounds the embryo proper, we find that at some distance from 
the embryo proper it dips into the peritoneal side of the uterus 
at a place above the beginning of the incisions, where therefore 
the uterine lumen is still intact, and it even penetrates into a 
fissure of the musculature of the uterus. Further downwards 
the embryonal placenta extends to the peritoneal tissues of the 
upper part of the incised uterus. Fig. 1 of course represents only 
one section while the description which we just gave is based on a 
study of a number of serial sections. The egg embedded itself 


62 LEO LOEB. 


evidently in the connective tissue between the upper end of the 
uterus and the lower end of the tube and its derivatives pene- 
trated still deeper between the musculature of the uterus in the 
direction from the peritoneal side. 

The character of the embryonal structures and their relation 
to the surrounding tissue are more clearly shown on Fig. 2. 6 is 


Fic. 2. Thedeveloping embryo; somewhat higher magnification. a, embryonal 
structure (neural canal?); 6, Fallopian tube; c, giant cells of the embryonal placenta; 
d, cuboidal cells of the embryonal placenta surrounding cavities; h, hemorrhages in 
the surrounding connective tissue. 


the fallopian tube. a is the embryo, which is surrounded by 
placental structures c and d and other similar not especially 
designated structures. In the periphery of these structures are — 
found extensive hemorrhages into the connective tissues and these 
are in turn surrounded by strands of connective tissue and by 
blood vessels. The entire region between the tubes and the 
outer hemorrhagic zone is filled out by embryonal placenta. 
The embryo proper corresponds to a developing guinea pig at 
a stage directly following the formation of the germ layers. 


EXPERIMENTALLY PRODUCED EXTRAUTERINE PREGNANCY. 63 


A points to a central structure, which probably corresponds to 
the Anlage of the neural tube. Under the abnormal conditions 
under which the embryo must develop, the various embryonic 
structures are evidently somewhat distorted. Fig. 3 shows the 


Cc a d 
i 
i 


Fic. 3. The embryo proper, higher magnification. a, mitosis; c, surrounding 
giant cells; d, a structure which perhaps corresponds to the placental cavities lined 
with cuboidal cells. f 


central part of the embryo at a higher magnification. A points 
to the same cavity as a in Fig. 2. The cell designated by a is 
seen in the process of mitotic division. Other embryonal cells 
also divide mitotically at various places. Surrounding the central 
parts of the embryo we find epithelial structures arranged in 
layers adjoining as is shown on Fig. 2. Giant cells c surround the 
embryo at various places on Fig. 3 in a similar way as seen on 
Fig. 2. A larger number of giant cells are also found at a some- 
what greater distance from the embryo. These giant cells are 
arranged typically around cavities, which are lined by smaller 
cuboidal cells. Don Fig. 2 points to such a cavity lined with such 
cuboidal cells. Perhaps also the canal d on Fig. 3 corresponds to 
such a cavity. The small cuboidal cells often proliferate and 


64 LEO LOEB. 


their proliferation leads to the formation of papillary excrescences 
into the cavities. These excrescences fill sometimes a great part 
of these cavities. Mitoses often appear in these cuboidal cells. 
Fig. 4 shows such a placental structure at a higher magnification. 


a d 
| 


Fic. 4. A placental cavity lined with cuboidal cells.: c, giant cells; d, cuboidal 
cells lining a cavity and forming papillary excrescences; c’, a giant cell penetrating 
into the surrounding connective tissue; v, blood vessels; , hemorrhages in the con- 
nective tissue; w, experimentally misplaced uterine epithelium. 


d points to a cavity lined with cuboidal cells. The cuboidal cells 
form papillary proliferations into the lumen. The cavity bulges 
into the surrounding tissue at d1. The cavity is surrounded on 
several sides by giant cells c and these giant cells protrude into 
the cavity and divide it into two parts. These giant cells have 
the power to penetrate farther into the surrounding tissue inde- 
pendently. Ci represents such a giant cell, which penetrates 
into the surrounding’ fibrous tissue. Surrounding this placental 
structure we find connective tissue in which there are many hemor- 
thages h. U represents a cavity lined with uterine epithelium. 
v represents a blood vessel. Fig. 5 represents a drawing of a 
similar placental structure. D represents the cavities lined with 
cuboidal cells, and partly filled with the proliferated cuboidal 


EXPERIMENTALLY PRODUCED EXTRAUTERINE PREGNANCY. 65 


cells. C are the giant cells surrounding the cuboidal cells. F is 
fibrillary connective tissue in which there are many hemorrhages 
h. There is nowhere a formation of adecidua. visa blood vessel. 


Fic. 5. A typical placental structure. The various letters have the same sig- 
nificance as in Fig. 4. 


As we have already seen on Fig. 4, the giant cells penetrate 
deeper into the tissue, independently of the small cuboidal cells. 
They prefer especially the neighborhood of blood vessels, pene- 
trate the walls of the latter and replace the endothelial cells. 
Blood vessels thus changed are of course thereby weakened, and 
they are no longer as well able to resist to the full extent the blood 
pressure, and thus hemorrhages into the tissue, as so frequently 
seen, result. Fig. 6 shows two vessels v. Giant cells c have 
advanced up to the lumen of these vessels and substitute the 
endothelial cells. In ci also there lies a giant cell in the tissue. 
At many places there are hemorrhages h in the connective tissue. 
In the periphery of the upper half of the section, connective 


66 LEO LOEB. 


tissue surrounds the structure. d points to a cavity filled with 
small cuboidal cells. 

These findings will have to be interpreted in the following 
way. At the time when the incisions were made into the uterus, 
namely two days and sixteen hours after copulation, the ova had 
probably already left the tube and had reached the upper part 
of the uterine cavity. At this time one or more of the ova left 
the uterine cavity through the incision into the uterine wall 


Fic. 6. Placental embryonal giant cells penetrate into the vessel wall. v, blood- 
vessels; c, giant cells substitute vascular endothelial cells; c’, a giant cell lying in 
the host tissue; 4, hemorrhages in the host connective tissue; d, cuboidal placental 
cells of embryonal origin. 


and one of the ova passed around the outer side of the upper end 
of the left uterine horn, and embedded itself in the connective 
tissue between the tube and the upper end of the left uterine 
‘horn. A part of the embryonal placenta in the course of de- 
velopment penetrated farther into the musculature of the uterine 
horn. ‘The fertilization of this ovum had in accordance with the 


EXPERIMENTALLY PRODUCED EXTRAUTERINE PREGNANCY. 67 


general view concerning the time of fertilization of the guinea- 
pig ovum already taken place at the time of the operation. We 
excised the nodule fifteen days eight hours after the incisions had 
‘been made. 

Our description of the embryo clearly shows that under the 
existing abnormal conditions the development of the ovum was 
greatly retarded. The embryo is still alive and even growing, as 
the mitoses, which were found at various places, indicate, but the 
embryo is found to be at a very much earlier stage of development 
than one would expect eighteen days after copulation. The 
embryonal placenta also is only very incompletely developed. 
While the normal placenta of the guinea pig shows a complicated 
structure at this period of development, in our case the embryonal 
placenta consist solely of layers of small cuboidal cells, which 
usually line cavities, and produce papillary excrescences pro- 
jecting into the cavities. On the outer side of these cavities 
_ there are giant cells. The giant cells penetrate also independently 
into the surrounding connective tissue and substitute walls of 
blood vessels, and thus contribute to the hemorrhages which we 
find so frequently. Cuboidal cells as well as giant cells are 
growing actively by mitosis—the latter however to a lesser 
degree. The surrounding host tissue remains passive. The 
embryonal tissue is surrounded by fibrillar connective tissue 
containing the ordinary connective tissue cells. There 1s no- 
where an attempt at the formation of a decidua on the part of the host 
tissue. 

These observations are in entire accord with our former ex- 
perimental findings from which we concluded that in the guinea 
pig solely the connective tissue of the uterine mucosa is able to 
produce decidua in response ‘to artificial stimuli, as cuts and 
foreign bodies while the fallopian tube, peritoneal and other con- 
nective tissue are unable to do so.! 

These additional observations again prove the similarity 1m the 
mode of action of the artificial stimuli leading to the formation of a 
decidua on the one hand and of the ovum on the other hand. Ina 
similar manner as the artificial stumula were not able to call forth a 


1Leo Loeb, Zentralblatt fiir Physiol., Bd. XXIII., No. 3; Journal Am. Med. 
Association, Vol. LIII., p. 1471, 1909. 


68 . LEO LOEB. 


formation of decidua in the peritoneal connective tissue, the ovum 
is likewise unable to do so. 

These observations furthermore clear up the fate of the ovum 
in cases in which it is not able to develop normally in the uterine 
wall. Frequently a fixation of the ovum does not take place in 
such abnormal cases, especially on the smooth peritoneal epi- 
thelium. In other cases however the ovum fixes itself and begins 
to develop in the connective: tissue without however finding the 
necessary decidual reaction on the part of the surrounding con- 
nective tissue. In such cases the development of the embryo 
proper as well as of the embryonal placenta is very much re- 
tarded as compared to the normal development; furthermore the 
embryonal differentiation also remains incomplete and we may 
assume, that after some time the growth ceases and the em- 
bryonal structure is substituted by host connective tissue in a 
similar manner as in the ovary of the guinea pig. We find there- 
fore in the guinea pig no or only a very much retarded and in- . 
complete development of the ovum outside of the uterus. This 
is in all probability due to the fact that the host tissue is not 
suited to receive the ovum and to supply it with the necessary 
food stuffs. In this case the host tissue behaves passively in 
contradistinction to the uterine mucosa. This conclusion agrees — 
with the fact that we find a general parallelism in the ability 
of the uterine mucosa to produce decidua or deciduomata and 
to permit a normal development of the ovum. As I have previ- 
ously shown, various experimental interferences, as for instance 
extirpation of the corpora lutea or of the ovaries, have approxi- 
mately to the same extent an inhibiting influence on the develop- 
ment of deciduomata and of pregnancy. We may thus conclude 
that the ability of the host connective tissue to produce a decidua 
in a normal manner is of significance for the normal development 
of pregnancy. 

We see therefore that in the guinea pig the ovum does either 
not develop at all outside of the uterus or in case an extrauterine 
fixation of the ovum should take place, the development is much 
retarded and soon comes to a standstill. As our present and 


1 As we shall later especially emphasize, the same holds good in the case of the 
parthenogenetic development of the egg in the ovary of the guinea pig. 


EXPERIMENTALLY PRODUCED EXTRAUTERINE PREGNANCY. 69 


especially our previous observations concerning the partheno- 
genetic pregnancy in the ovary of the guinea pig demonstrate,} 
the development of the embryonal placenta preponderates re- 
latively very much over that of the embryo proper, probably 
because as I have already suggested, in contact with the host 
tissue the derivatives of the ovum produce mainly the placental 
structures. This is very marked in the case of the partheno- 
genetic development in the ovary of the guinea pig, where in 
typical cases under those conditions placental structures are 
found exclusively and only exceptionally the embryo proper 
begins to develop. 

These observations explain apparently very well the fact, that 
while in the guinea pig a further going development of the ovum 
is possible after extrauterine fixation, in man a complete extra- 
uterine development is not an infrequent occurrence. Our 
findings suggest as one of the causes for this difference in occur- 
rence of extrauterine pregnancy in man and guinea pig, the fact 
that in the case of man the host tissue offers a more suitable 
soil than in the case of guinea pig; while as we saw in the latter 
the development of the decidua in response to various kinds of 
stimuli takes only place in the connective tissue of the uterine 
mucosa, in the case of man the connective tissue of various pelvic 
organs and even the appendix is able to produce decidua as many 
observations show. In accordance with this interpretation a 
number of observers actually reported the development of a 
decidua in the fallopian tube in cases of tubal pregnancy. It is 
very probable that in tases of tubal pregnancy in which a decidua 
was not found in the tube, we had to deal with stages in which 
the chorionic wandercells had already penetrated deep into the 
host tissue and thus gradually destroyed the decidua; in a similar 
manner in the case of the guinea pig it can readily be seen that 
the wandercells of the embryonal placenta destroy a greater 
part of the decidua. It is very probable that from a certain 
stage of embryonal development on, the decidua is no longer in- 
dispensable as far as the continued existence and further develop- 
ment of pregnancy are concerned. 


1 Roux’s Archiv, Bd. XXXII., p. 662, 1911; Zeitschrift f. Krebsforschung, 11. Bd.» 
2. Heft, 1912. 


70 LEO LOEB. 


Our observations are also of interest from another point of 
view. We know that under ordinary circumstances the corpus 
luteum remains longer preserved in pregnancy than in the non 
pregnant animal. Pregnancy prolongs the sexual cycle. We 
may now inquire into the cause of the prolongation of the life 
of the corpus luteum during pregnancy. Several years ago I 
pointed out, that the growth of the embryo might perhaps directly 
or indirectly prolong the life of the corpus luteum during preg-. 
nancy.! | 

Now we find in our case a small embryo as well as an embryonal 
placenta developing outside the uterus. Notwithstanding this 
fact a new ovulation had taken place about three days pre- 
viously and accordingly the corpora lutea of the preceding 
sexual cycle which had been terminated at the time of the last 
ovulation were degenerated. 

This observation proves that a developing embryo including 
embryonal placenta is in itself not sufficient to protect the corpus 
luteum from degeneration, and to prevent a new ovulation. It 
-is possible that the maternal placenta is concerned in the pro- 
longation of the life of the corpus luteum either alone or in con- 
nection with the embryo, which latter as our further experiments 
have shown, prolongs noticeably the life of the experimental 
placentomata (deciduomata). Indeed experiments which I 
carried out some time ago have shown that the development of 
deciduomata without the development of an embryo is able to 
prolong the sexual period; while normally the sexual cycle in the 
guinea pig has a duration of from 15 to 18 days, it lasts from 20-30 
days after production of deciduomata.2. Whether as a result of 
these experimental interferences also the life of the corpus luteum 
is prolonged will have to be still further investigated. 

Our observations are furthermore of significance for the inter- 
pretation of certain structures, which I found in about 5 per cent. 
of the ovaries of young guinea pigs.’ 

In as much as these structures become absorbed after a certain 
time and are substituted by connective tissue, these structures 


1 Zentralblatt f. Physiol., Bd. XXIV., Nr. 6; Medical Record, June 25, 1910. 

2 Leo Loeb, BIoLoGicAL BULLETIN, Vol. XXVII., July, 1914. 

3 Arch. f. mikrosk. Anatomie, Bd. 65, 1905; Roux’s Archiv, Bd. XXXII., p. 662, 
1911; Zeitschrift f. Krebsforschung, 11. Band; 2. Heft, 1912. 


EXPERIMENTALLY PRODUCED EXTRAUTERINE PREGNANCY. 71 


must in fact occur more frequently than the direct findings 
suggest. We have to deal with formations which resemble closely 
structures of the embryonal placenta, and they originate in 
ovarian follicles. They are either well preserved or are found in 
the process of retrogression and in the end are substituted by 
connective tissue. In two cases I was able to find besides em- 
bryonal structures proper, for instance the Anlage of the nervous 
system. It had been known previously and I myself had de- 
scribed processes which had to be interpreted as the first seg- 
mentations of eggs in atretic follicles which in consequence of’ 
the abnormal conditions under which they took place followed as 
might have been expected an abnormal course.!. The interpre- 
tation that we have to deal merely with the disintegration of the 
ova can be excluded with certainty. Such an interpretation 
would be contradicted by the regularity of the divisions. Fur- 
thermore we may find in these various segments either nuclei or 
the remnants of nuclear spindles and I was able to observe the 
simultaneous presence of a mitosis in each one of the two such 
segments. These segmentations also are found chiefly in the 
ovaries of the young guinea pigs. A somewhat furthergoing 
formation of the first segments in ovarian eggs has recently been 
described in armadillo by Newman.’ ' 

In all these cases we have merely to deal with the first parthe- 
nogenetic segmentations of the ovum, while our observations in 
the ovary of the guinea pig prove a much furthergoing develop- 
ment leading to the formation of embryonal placenta and of 
embryos in the stage of the germ layers within the ovary. It is 
of course natural, as I emphasized on a former occasion, that 
under these abnormal conditions the processes of development 
cannot follow an altogether normal course, and it was therefore 

tLeo Loeb, “On Progressive Changes in the Ova in Mammalian Ovaries, 
Journal of Medical Research, Vol. VI., 1901. Arch. f. mikrosk. Anat., Bd. 65, 1905. 

2H. H. Newman, BIOLOGICAL BULLETIN, XXV., p. 52, 1913. It may be espe- 
cially emphasized that our interpretation of the placental and embryonal structures 
found by us in the ovaries of guinea pigs does in no way depend on the interpreta- 
tion of those changes in the ova within the ovaries of the guinea pig which in common 
with previous authors we held to be early abnormal segmentations of ova, while 
a number of other investigators interpreted them as of a degenerative character. 


There can be no doubt about the presence of further developed embryonal 
structures in the ovaries of guinea pigs. 


72. LEO LOEB. 


desirable that a confirmation of our interpretation of these ovarian 
structures should be obtained. The findings which we have just 
communicated offer the desired confirmation. In our new ob- 
servations we have also to deal with embryonal structures found 
in the peritoneal connective tissue and developing in an abnormal 
situation without being aided by the host tissue through the 
formation of a decidua. We have of course to consider the fact 
that in the ovary the limitation of space is still more marked than 
in the connective tissue on the outer side of the fallopian tube and 
of the uterus. In both cases the placental structures preponderate 
over the embryonal ones proper; in both a retardation in the de- 
velopment is found and a preponderance of certain placental struc- 
tures. Such favored structures are the layers of cuboidal cells, 
lining cavities, forming papillary excrescences into these cavities 
and surrounded at the periphery by giant cells which latter pene- 
trate in both cases into the surrounding tissue, especially around 
the blood vessels, the wallsof which they may perforate, thus giving 
rise to hemorrhages. The identity of both formations, namely of 
the experimentally produced extrauterine pregnancy which we 
have just described and of the embryonal structures developing 
parthenogenetically in the ovary becomes quite evident, when 
one compares the microscopic sections of both of these formations. 
The microphotographs and the drawings also show the similarity. 

The similarity of the embryonal structures proper becomes 
clear through a comparison of Figs. 2, 3, and 6 in the former 
communication (Zeitschrift fiir Krebsforschung),) and of Figs. 
2 and 3 in the present communication. The similarity of 
the placental structures is made evident through a comparison 
of drawings 1,2 and 4 inthe Archiv f. mikrosk. Anatomie,? of the 
Figs. 10, 12, 14 and 15 in the Zeitschrift fiir Krebsforschung with 
Figs. 4 and 5 of the present article. On several of these former 
figures there were also represented the relations of the wandering 
giant cells to the blood vessels and the hemorrhages resulting 
therefrom. 

Our new observations render 1t therefore certain that a fargoing 
parthenogenetic development of ova takes place in the ovaries of a 


1 Loc. cit. 
2 Loc. cit. 


EXPERIMENTALLY PRODUCED EXTRAUTERINE PREGNANCY. 73 


relatively large number of guinea pigs, leading in the first place to 
the formation of placental structures, in some cases however also to 
the formation of embryos in the stage of the germ layers. We have 
discussed the possible causes for this parthenogenetic develop- 
ment on another occasion.1 We have perhaps to deal with a 
development which is caused by changes in the circulation and 
in the exchange of gases at the time and in consequence of the 
rupture of follicles. 

Such an explanation would be in accordance with the fact that 
the first segmentations of the ovum in the ovary of the guinea 
pig are found especially in atretic follicles, that the segmentations 
set in with beginning atresia and then gradually progress. Now 
we know that the atresia of follicles is more marked, than at any 
other time, at the time of ovulation.2 In this connection it is 
especially worthy of notice that the first segmentations of the 
ova in the ovary as well as the furthergoing parthenogenetic 
development, which leads to the formation of embryonal and 
placental structures, is preferably found in the ovaries of young 
animals. The latter, however, occurs occasionally also in some- 
what older guinea pigs. In such cases we may perhaps have to 
deal with structures which originally developed in younger an- 
imals, which then however had remained stationary for a longer 
period of time. 

We have still to discuss the significance of these structures for 
the interpretation of certain pathological formations, namely the 
embryomata and the chorion epitheliomata of the female germ 
gland. The large majority of pathologists assume in agreement 
with the suggestion of Bonnet and Marchand that these patho- 
logical structures take their origin from misplaced blastomeres 
and not from the parthenogenetically developing ovum. As I 
formerly emphasized? our observations make it very probable 
that such pathological formations originate from parthenogenetic- 
ally developing ova. They are therefore the ‘descendants”’ and 
not the “‘brothers”’ of the organism in which they originate. We 
may assume that in certain cases the parthenogenetic develop- 

1 Leo Loeb, Proceedings Am. Philosophical Society, Vol. L., p. 228, 191. 


2 Leo Loeb, Journal of Morphology. 
3 Zeitschrift f. Krebsforschung, loc. cit. 


74 LEO LOEB. 


ment of ova leading to these pathological structures begins only 
after birth. In a similar manner as we saw that embryonal 
placenta as well as the embryo proper can develop from the 
parthenogenetically segmenting ovum and that the embryonal 
placenta can be formed without the simultaneous development of 
the embryo proper, thus chorion epitheliomata may originate in 
the ovary without any accompanying embryonal structures 
proper. In other cases however there develop mainly the em- 
bryonal structures proper or certain of their parts. 

This conception of these structures explains the fact that they 
are mainly found in the germ glands. On the other hand, there 
exists no reason, why we should expect that aberrant blastomeres 
should mainly be found and develop at this place. Furthermore 
I have never been able in the many hundreds of ovaries of guinea 
pigs which I have examined microscopically to find a structure 
resembling a misplaced blastomere. ; 

We still have to explain why these structures are occasionally 
also found in the male germ glands and especially, why teratomata 
occur also, although less frequently, at other parts of the body, 
outside of the germ glands. As far as their occurrence in male 
germ glands is concerned, it might be explained by the fact that 
in a certain number of cases cells of both sexes may be found in 
the same individual, that therefore true hermaphroditism occurs. 
That this is not so rare an occurrence as has been assumed has 
recently been shown by L. Pick.1 We have perhaps also to 
consider the possibility that at a certain stage of development 
also the male germ glands are capable of developing in a similar 
manner as the ova. However at the present time there exist no 
facts supporting such an hypothesis. 

We know furthermore that in the course of embryonal de- 
velopment the germ cells migrate. It is therefore conceivable 
that occasionally one of their number may follow a wrong path 
and thus give origin to the formation of the teratomata outside of 
germ glands. While we are thus able to explain the origin of 
these structures on the basis of a parthenogenetic development 
of ova we do not intend to deny the possibility that under certain 
conditions irregularities in the embryonal development may lead 


1 Cited from a review in the Miinch. med. Wochenschrift, 1913. 


EXPERIMENTALLY PRODUCED EXTRAUTERINE PREGNANCY. 75 


to the transformation of blastomeres or of remnants of not fully 
differentiated embryonal tissues into teratomata, an hypothesis 
which would be in accordance with the finding of misplaced 
blastomeres by W. Roux in the course of the embryonal develop- 
ment of amphibian eggs. 


SUMMARY. 


1. It is possible in the case of guinea pigs to produce experi- 
mentally the first stages of an extrauterine pregnancy. 

2. In a similar manner, as in the case of guinea pigs experi- 
mental interferences of various kinds are not able to call forth 
the production of deciduomata in the connective tissue outside 
of the uterine mucosa after the discharge into the circulation of 
the sensitizing substance which is secreted by the corpus luteum, 
the developing ovum is unable to call forth a decidual reaction. 

3. Under the conditions produced by us experimentally the 
development of the embryo is very much retarded and will in all 
probability come to a standstill after some time. Neither does 
the embryonal placenta develop in an entirely normal manner, 
although quantitatively the embryonal placental structures 
preponderate considerably over the embryonal proper. It is very 
probable that the lack of the decidual and of the typical blood 
vessel reaction on the part of the host connective tissue is the 
cause of this abnormal development. In man an extrauterine 
decidua can develop and accordingly here a fully developed 
extrauterine pregnancy is not rare. As we have shown pre- 
viously the effect of the extirpation of the corpora lutea on the 
formation of the decidua and on the development of pregnancy 
is approximately parallel. This is an additional fact which 
renders probable the significance of the decidual reaction for the 
complete development of the extrauterine pregnancy. The 
decidual reaction is at least one of the conditions which has to 
be considered in this connection. 

4. Notwithstanding the presence of a young, developing em- 
bryo in the extrauterine connective tissue a degeneration of the 
corpora lutea and a new ovulation took place in the ovary. This 
proves that the persistence of the corpora lutea during pregnancy 
does not depend upon a substance secreted by the embryo; it is 


76 LEO LOEB. 


probable that the growth of the decidua perhaps in combination 
with the growth of the embryo prolongs directly or indirectly 
the life of the corpora lutea during pregnancy. Thus far ex- 
periments, which we have carried out in order to decide this 
question, have shown that the presence of living and growing 
deciduomata prolongs the sexual period; furthermore that preg- 
nancy prolongs the life of the deciduomata. Further investiga- 
tion will decide whether or not these effects are exerted indirectly 
by means of the corpus luteum. 

5. Our experiments render it certain that the structures which 
we found in a considerable number of guinea pigs and which we 
formerly interpreted as early stages of parthenogenetically 
developed pregnancies in the ovaries of guinea pigs really represent 
a relatively far going parthenogenetic development of ova which 
may lead to the formation of embryos in the germ layer stage 
which however usually leads merely to the formation of an em- 
bryonal placenta probably as a response of the developing ovum 
to the influence exerted by the contact with the surrounding 
host tissue. We show furthermore the significance these findings 
have for the interpretation of the teratomata and chorionepi- 
theliomata of the germinal glands. 

6. The embryonal wander cells destroy outside as well as within 
the wall of the uterus bloodvessels of the surrounding host tissue 
in the ovary as well as in the peritoneal connective tissue and 
they thus cause hemorrhages in the surrounding host tissue. 


SOME CRYOSCOPIC AND OSMOTIC DATA! 
WALTER E. GARREY. 


Subsequent to the publication in 1905 of data on ‘‘The Osmotic 
Pressure of Sea Water and the Blood of Marine Animals, etc.,” (1) 
‘the author has had occasion in the course of his other investiga- 
tions, to make numerous determinations of the freezing point of 
various sea waters, solutions and bloods; this method having 
been used to check up other methods of obtaining solutions of 
known osmotic pressures. Someof the data thus acquired have 
been correlated, and although somewhat fragmentary, they are 
published in hopes that they may facilitate the work of other 
biologists. 

The determinations have been made with the Beckmann 
apparatus and a differential thermometer, which could be read 
accurately to 0.005° C. When it is remembered that the de- 
pression of the freezing point (A) of a gram-molecular solution of a 
non-electrolyte is (theoretically at least) 1.85° C. below zero, 
that this depression corresponds with an osmotic pressure of 22.4 
atmospheres (at 0° C.), and that the osmotic pressures vary di- 
rectly with the depression of the freezing point, it is seen that the 
osmotic pressure of any solution may be calculated from the 
simple formula: osmotic pressure = 22.4 a. A/I.85. 


SEA WATERS. 


Sea waters are not solutions of absolutely fixed chemical com- 
position, nor have they a constant concentration. While the 
ratios of certain salts are quite constant, there are other variations 
such as the content of absorbed oxygen and carbon dioxide and 
even of the fixed carbonates. J. Loeb (2) has called attention to 
the fact that the free alkalinity, 7. e., the number of HO ions, is 
distinctly higher in the sea water at Woods Hole than at Pacific 
Grove. 


1 From the Physiological Laboratory of Washington University, St. Louis. 
77 


78 WALTER E. GARREY. 


The figures for the depression of the freezing point (A) given 
in Table I. indicate the wide range in concentrations in sea waters 
of different localities. 


AREER IE 
Sea Water from: A—°C. Observer. Reference. 
INaplessar een sascee =2.20 Bottazzi |Arch. ital. de biol., 1897, XXVIII., 61. 
Arcachon a eiaeion eo. —1.89 Rodier /|Trav. des Lab. d’Archachon, 1899. 
Pacific Grove, Cal.....| —1.925 | Greene /|Bull. U. S. Bureau Fisheries, 1904. 
XXIV., 429. 
Pacific Grove, Cal.....| —I.90 Garrey |BroLt. BULL., 1905, VIII., 257. 
Woods Hole: ......... —1.81 . Bio. BULL., 1905, VIII., 257. 
Beaufort, N. C........ —2.04 ae IQII. 
Helsolandmernee eerie —1.90 Dakin Bio-Chem. Jour., 1908, 269. 
In the Kattegat....... —1.66 a 5 ; 
Open BalticSea....... —1.30 af 
Kiel harbor.......... —1.093 kg 


In the following sections further details obtained by the author, 
by means of the cryoscopic method, are given for sea waters of 
some American localities. 

(a) Woods Hole.—Determinations made during the summer of 
1904 have been previously reported (loc. cit., pp. 258-259) show- 
ing the freezing point to be slightly variable between — 1.805 and 
—1.84° C. The average of determinations made the latter part 
of July of six different years gave an average A = — 1.81° C,. 
with which, as will be seen from succeeding data, the following 
solutions are isosmotic: Sodium chloride, 0.52 m; Magnesium 
chloride, 0.29 m., cane sugar, 0.73 m. ‘‘Van’t Hoff’s solution,” 
made from m/2 stock solutions, had a freezing point of — 1.84° C., 
this is so slightly in excess of the concentration of Woods Hole 
sea water that it may be considered isosmotic with it. This 
“Van’t Hoff’s solution’? was made up from half molecular solu- 
tions according to the formula given by J. Loeb, (3) viz: 100 mole- 
cules NaCl, 2.2 molecules KCl, 1.5 molecules CaCl, 7.8 molecules 
MgCl, and 3.8 molecules MgSO,. The traces of bicarbonate 
and phosphate were omitted from the solution, but when added 
in optimum amounts (e. g., I c.c. N/20 NaHCOs3 per 100 c.c. 
solution, as in the procedure of Loeb, p. 35), the solution becomes 
exactly isosmotic with Woods Hole sea water. 

(b) Pacific Grove-—Green in 1904 made freezing point deter- 


CRYOSCOPIC AND OSMOTIC DATA. 79 


minations of the Pacific Grove sea water and found that A= 
— 1.924° C. Garrey in 1905 made determinations, obtaining a 
slightly lower value for A viz., —1.905° C. On the basis of either 
of these figures, it is seen that the sea water in this locality is 
about 5 per cent. more concentrated than at Woods Hole and 
that a correction for this amount must be made if the osmotically 
equivalent solutions are to be calculated from the figures given in 
the previous section (a). | 

(c) Beaufort, N. C.—Working in the laboratories of the U. S. 
Bureau of Fisheries during the summer of 1911, the author made 
the following observations of the freezing point of sea water 
obtained at different localities in that vicinity and under different 
conditions as described in Table II. 


TABLE II. 

IQII. Beaufort, N. C. [X= 'AC, Remarks. 
June 8 |Open sea, outside “Sea Buoy’’ | —2.043 

ee ~ IWharivof U.S:F.C.... 5.2.2. —1I.987 |I1:40 a.m.—tide low, N.E 

wind, previous showers. 

; Whart-of ULS:F.C.. 2. 65... —2.015 |4:00 p.m. tide high 
July 19 |Wharf of U.S.F.C........... — 2.038 

“Open sea, at ‘‘Sea Buoy’’.....| —2.03 

0S BO Neri, 1S I9Coo 650 ¢oeb suo —2.07 |9:15 a.m., tide low 

He ES INAne rate (OAS) ) aha Cag. eeeaa ions era eres —2.06 |3:00 p.m., tide high 

Weare SO SATEY SOUT Gey acy eet eeusne ia eke ool —2.073 

HO ee Soe SIN lovee t AGES she Craniiy Geena retrey ere ara —2.079 |Tide low, strong south wind 
Soe, © WS IBM ob oo oooocdodocuDE —2.05 

- S INWinevar WESHIKCss 6 Gc obeacuCs —2.052 |Tide low 

““"\Newport River, at ‘‘Cross 


FROCK SA erates here oyeteet: —1.707 


From these figures it is seen that the open sea water off Beau- 
fort hasa A = — 2.04° C., and is 12 per cent. more concentrated 
than at Woods Hole. 

(d) Diluted Sea. Water.—The constant necessity for the use, in 
biological investigations on marine forms, of diluted sea water 
and corresponding concentrations of pure salts, has led the 
author to make the determinations found in Table III. Various 
dilutions of Woods Hole sea water were made and the freezing 
points determined. In most cases the densities at these dilutions 
have also been determined by the pycnometer method. The 
figures given in the table are all from actual determinations made 


80 WALTER E. GARREY. 


by the author; when not given they may be approximated by 
interpolation.! 


TABLE III. 
Dilution : Densities of Sea ; 

Woods Hole Distilled NOG: Water Dilutions NaCl eae 
Sea Water \ + { Water at 21.59 C. (Ref. ae Seiko sown. 

c, cm. c. cm, H,O at 21.5° C.) 
Undiluted fo) —1.8I 1.02426 3.04 

Sy Ces nS Gx —1.54 2.6 

Tae Ae Fae —1.35 2.275 

662.55 285 > —1.20 2.00-+ 

60 “ Owned —1.09 I.81 

So) Olan —0.915 I.0123 1.58 

AS is ie es —0.82 I.4 

Ae) Oo) ™ —0.73 I.0096 I.21 

Ri 0 @g 0% —0.64 I.07 

33% “ 662% “ —0.61 1.008 T.02 

SIA wiles GS an —0.505 I.00 

3 OMe x HO. 3 —0.547 1.0073 0.91 

Paes Gemma ts —0.460 T.0062 0.76 

gx0) 90 80 “* —0.37 1.0046 0.60 

TOES: 90 “* —0.187 1.0023 0.30 


CANE SUGAR. 


Attention should be directed to a fact to which Jones (4), 
Morse and Fraser and Berkeley and Hartley have called atten- 
tion, viz., that cane sugar solutions show osmotic pressures 
considerably in excess of what theory would lead one to expect. 
Loeb has shown the importance of this fact for biological work (5). 
From purely theoretical considerations one would expect a molar 
(gram-molecular) solution to show an osmotic pressure only 
slightly in excess of that of Woods Hole sea water. Loeb found 
that it caused a shrinkage of the eggs of the echinoderms even of 
the Pacific, and his experiments caused him to select 6/8 m. cane 
sugar as the proper concentration for the development of Stron- 
gylocentrotus purpuratus. The osmotic pressure of Woods Hole 
sea water by calculation from the freezing point is 21.9 a. (at 
o° C.), a figure which is almost identical with that obtained by 

1 The determinations of Gerlach for NaCl and KCl (Chemiker-Kalender, 1914, I., 
p. 261) and of Schiff for MgCl and CaCl (zbid., p. 265) show, that, for concentra- 
tions of solutions of the magnitudes with which we are dealing and in which these 
salts are present in sea water, the densities are a linear function of the concentration. 


A plat of our determinations shows the same to be true for both densities and 
freezing points of dilutions of sea water. 


CRYOSCOPIC AND OSMOTIC DATA. 81 


calculation for 0.75 gram molecular solutions of cane sugar, using 
the measurements of Berkeley and Hartley. Some of our de- 
terminations of the freezing point of solutions of cane sugar 
illustrate their peculiar osmotic behavior.? 

For a gram molecular solution of cane sugar (342.2 grams per 


liter of solution) we found A = — 2.775° C.; for 34 mol. (256.6 
grams per liter) A = — 1.855°, — 1.86° C.; and for % mol. 
(171.1 grams per liter) A = — 1.15°, — 1.155° C. For these 


three solutions the theoretical depression of the freezing point 
would be to — 1.85°, — 1.387° and — 0.925° C., respectively. 
Comparison of these figures shows how much in excess of the 
theoretical osmotic pressure, that of these solutions really is. 
Morse and Fraser have pointed out that the correspondence with 
the theoretical expectations is greater, if ‘‘weight normal”’ 
solutions are used, 7. e., if the substance is present in a liter of 
the solvent, instead of this volume of the solution. This does 
not account, however, for the full amount of the discrepancy 
found. To illustrate this: It was found in our experiments that 
in making a gram-molecular solution by dissolving 17.11 grams 
of cane sugar in 50 c.c. of the solution (15° C.), it was necessary 
to add only 39.4 c.c. of distilled water; A was — 2.775° C. Had 
50 c.c. of solvent been used to make the corresponding “weight 
normal”’ solution, A would have been — 2.187° C.3 This figure 
exceeds the theoretical A (— 1.85° C.) by 0.337° C., which is 
probably to be accounted for by hydration of the sucrose molecule 
(Callendar (6)). 

In the figures given above it is to be noted that the A of .75 
mol. solution of sucrose (— 1.855° C.) is that which theory expects 
of agram molecular solution, and its osmotic pressure lies between 
that of the sea water at Woods Hole and Pacific Grove. By 

1 The measurements of Berkeley and Hartley were made using other concen- 
trations. The original figures of these workers as also those of Morse and Fraser 
et al. are given in the “‘ Physikalisch-chemische Tabellen,’’ Landolt, Bornstein and 
Roth, 4th ed., Table 179, p. 787. Their original papers are referred to, zbid., p. 790. 

2 The sugar used in these experiments was free of all reducing sugars and had 
been twice recrystalized from glass-distilled water with subsequent drying in 
vacuo. 

3 Calculation of the freezing point of a molecular ‘‘weight normal”’ solution, 


based upon Morse’s figure for the observed osmotic pressure (24.8 a. —o° C.) 
gave a slightly lower figure, viz.: A = — 2.048° C. 


82 


WALTER E. GARREY. 


extrapolation we obtain the following figures for the concentra- 
isosmotic with sea water of: 


tion of sucrose; 


I. Woods Hole 
2. Pacific Grove 


3. Beaufort 


0.73 m. (A = — 1.81° C.) 


= 0.765 m. (A = — 1.90° C.) 
= 0.81 m. (+) (A = — 2.01° C.) 


SALT SOLUTIONS. 


(a) Sodium Chloride—In addition to the freezing points 
of solutions given in Table III., the following have been deter- 


TABLE IV. 
Nee een Made by /\ = OC, Remarks. 

0.65 L. —2.255 

0.65 Gupracic: —2.35 

0.65 (EG, 229 Co —2.50 Madein a flask standardized to 
I5° C. 

0.60 pls —2.11 

0.58 G. —2.03 Isosmotic with Beaufort Sea 
water 

0.54 G. —1.90 Isosmotic with Pacific Grove Sea 
water 

0.54 L. —1.895 

0.52 G. —1.81 Isosmotic with Woods Hole sea 
water. 

0.50 L. —1.735 

0.50 L. —1.74— 

0.50 G. —1.745 

0.50 F. =1.75 

0.50 L. —1.765 

0.50 G. —1.745 

0.444 G. —1.54 


mined by the author on solutions made up by different com- 
The concentrations chosen were somewhat 


petent workers. 


TABLE V. 


MgCl, Concentra- 


tion.1 


0.50 molecular 
0.36 of 
0.35 oy 
0.31 


0.30 
0.29 
0.10 


[NC (C- 


—2.845 
—2.03 

—1.985 
—1.895 


—1.85 
—1.815 
—0.495 


Remarks. 


Isosmotic with sea water at Beaufort. 


Isosmotic with sea water at Pacific Grove (or 0.32 


m. according to Greene’s determination). 


solution). 


Isosmotic with sea water at Woods Hole. 
Dissolved in 100 c.c. of distilled water (not of 


1 Concentration referred to volume of solution, not of solvent. 


CRYOSCOPIC AND OSMOTIC DATA. 83 


to either side of those isosmotic with the sea waters of our coast 
laboratories. 

(b) Magnesium Chloride.—In solutions of this salt some of 
the molecules are dissociated into three ions, which accounts 
for the fact that the osmotic pressure is greater and consequently 
the depression of the freezing point is lower, than that of equi- 
molecular solutions of sodium chloride. The following commonly 
employed solutions have been tested (cf. Table V.). 


ANIMALS. 


In addition to data previously published by the author (loc. 
cit., p. 263), several determinations have been made on the blood 
of animals of the waters of the American coast,'and inland rivers. 

(a) Limulus polyphemus.—In the work referred to it was 
shown that under experimental conditions the blood of this 
animal, like that of other marine invertebrates varies to conform 
in concentration to that of the external medium. We have 
since found that this is true in the natural habitat of these ani- 
mals, thus at Woods Hole (1904) the blood of Limuli depressed 
the freezing point like sea water, to — 1.82° C. At Beaufort, 
N. C., July 20, 1911, the water of the Fisheries “pound” froze 
at — 2.03° C. The blood of four Limuli taken from this water, 
in which they had been kept for several weeks, showed the fol- 
lowing freezing points, respectively, — 2.025°, — 2.03°, — 2.04° 
and — 2.35° C. In the case of another Limulus captured at 
“Cross Rocks” in the Newport River, near Beaufort, September 
8, I91I, the blood A = — 1.71° C. while the water at that 
place depressed the freezing point to — 1.707° C. Such readings 
taken from animals under natural conditions established the 
absolute identity of osmotic pressure of the external and internal 
media despite the differences in their composition. 

(b) Elasmobranchs.—A shark seven feet in length (not identi- 
fied) was captured in the Fish Commission nets at Beaufort, 
July 27, 1911; the sea water froze at — 2.02° C.; cryoscopic 
readings of the blood from the heart and portal vein, withdrawn 
immediately after death, were identical, within 0.01° C., and 
showed A to be — 2.182° C., which again is practically identical 
with that of the Beaufort sea water taken in the neighborhood 
of the fish trap on that date. 


84 : WALTER E. GARREY. 


The A for this elasmobranch is larger than for those tested at 
Woods Hole by the author and later by Scott (7). The figures 
indicate an adjustment to the greater concentration of the sea 
water at Beaufort, a fact which is also borne out by determina- 
tions made on the blood of ‘sting rays,’”’ the blood of four of which 
at Beaufort gave A = — 1.98°, — 2.04°, — 2.03°, — 2.07° C., 
respectively. These depressions are not greater than that of the 
sea water from which the animals were taken although both the 
author and Scott found a slightly greater depression for the blood 
of the dog fish (Mustelus canis) of Woods Hole than for the sea 
water of the laboratories; this water is, however, somewhat less 
concentrated than the water outside the heads. 

(c) Marine turtles—The defibrinated blood of three species 
of marine turtles! caught at Beaufort in 1911 was frozen and the 
A thus determined for each individual is as follows: 


Chelonia mydas A = —0.675° C. 
Cclpochelys kempi A = — 0.687°, — 0.70°, — 0.70° C. 
Caretta caretta A = — 0.69°, — 0.69°, — 0.685° C. 


In the cases of two carettas obtained at Woods Hole in 1913, the 
A found was identical with that given above. These depressions 
(A) are, in all cases, greater than those obtained by Bottazzi 
for ‘‘Thalassochelys caretta’” (A = — 0.61°) although it is 
worthy of mention that the waters from which our animals were 
taken were, if one can judge from Bottazzi’s writings, less con- 
centrated than that from which his specimens were obtained; 
if any adjustment to aqueous media were to take place it would 
be in the direction opposite to that indicated by the above figure. 
It is certainly true, however, that the blood of fresh water and 
land turtles shows a depression of the freezing point which is 
distinctly less than that of marine turtles; Bottazzi found A 
for Emys europa = — 0.463° to — 0.485° C., while for Pseudemys 
elegans of the Mississippi Valley we obtained a depression in 
which A = — 0.48° C. These figures are so much below those 
obtained with the blood of marine turtles that they would seem 
to indicate the possibility of some degree of adjustment to the 
concentration of the external medium; on the other hand it is a 


1 The author is indebted to Mr. Hay for the identification of these animals. 


CRYOSCOPIC AND OSMOTIC DATA. 85 


fact that the land turtles do not show a more concentrated blood 
than do those which live mainly in fresh water. 

This point was put to the experimental test upon the marine 
turtles, Colpochelys kempi and Caretta caretta; the last figure in 
the previous data given above for each of these species was ob- 
tained, with the blood of a specimen which had been kept for 
two months in a tank containing fresh water. There was ab- 
solutely no change in the concentration of the blood of these 
individuals, and we feel justified in concluding that adjustments 
of the nature of those under consideration do not take place in 
these forms at least not within the duration of our experiments. 

(d) Fresh Water Fish.—Preliminary to a study of the effects 
of osmotic and saline media upon fresh water fish (soon forth- 
coming) it was desirable to know the osmotic pressure of the 
blood of forms taken from the Mississippi river. The following 
list contains some forms peculiar to this region on which no data 
have hitherto been given. 


1. Polyodon spathula = — 0.492°, — 0.486°, — 0.50° C. 
2. Scaphirhynchus platyrhynchus = — 0.505°, — 0.507°, — 0.503° C. 
3. Lepidosteus osseous (L.). (‘Gar’) = — 0.487° to — 0.52° C. 

4. Amzia calva (L.) (land locked) = — 0.508° C. 

5. Catostomus teres = — 0.51° to — 0.52° C. 

6. Perca fluviatilis = — 0.498° to — 0.51° C. 


Fresh water ganoids are seen to have blood which is identical 
in concentration with that of fresh water teleosts. All have blood 
less concentrated than that of any of the marine fishes and it is 
conceivable that in the case of these animals some adjustment to 
environment has taken place; such adjustments are known for 
marine fish as has been shown by the author (1) and others (Fréd- 
ericq, Bottazzi, Dekhuysen (8), Dakin, Joc. cit.). 


REFERENCES. 
I. Garrey, Walter E. 
705 Brov. BULL., VIII., p. 257. 
2. Loeb, J. 
’r3 ‘‘Artificial Parthenogenesis’’ Chicago, p. 34 
3. Loeb, J. 
P. 35, loc. cit. 
4. Jones, H. C. 
’93 ~Zeitschr. f. Physikalische Chemie.—XII, pp. 110 and 529; XII., p. 623. 


86 WALTER E. GARREY. | 


5. Loeb, J. 
P. 130, loc. cit. 

6. Callendar 

208 Proc. Roy. Soc., A, 1908, 80, 466. 
7. Scott, G. G. 

13 Annals N. Y. Acad. Sciences, 1913, XXIII., p. 1 et seq. 
8. Dekhuyzen, M. C. 

Arch. Neerland, Sc. Exact. et Nat., Ser. 2, 10, 121. 


Note.—For other literature consult the papers by Garrey! and by Scott;’ also 
Bottazzi, Ergebnisse der Physiologie, 1908, VII., p. 162; cf. also Table I. of this 


paper. 


CONCERNING BRACHET’S IDEAS OF THE ROLE OF 
MEMBRANE FORMATION IN FERTILIZATION.! 


JACQUES LOEB. 


1. A recent publication by Brachet? seems to make it necessary 
to discuss once more the relation between membrane formation 
and development. The writer had shown in 1895 that if oxygen 
is completely withdrawn from the fertilized sea urchin egg no 
development is possible, while the moment oxygen is admitted the 
development can begin again. As he had suggested in 1906 and 
_as has since been proved by O. Warburg, and H. Wasteneys and 
the writer, the entrance of the spermatozo6n into the egg of the 
sea urchin increases the rate of oxidations in the latter (by 400 or 
600 per cent). The entrance of the spermatozoén causes also a 
membrane formation which is very marked in the fresh egg and 
is generally less marked or may appear to be absent if the egg 
has been lying in sea water for a day or more. It has been 
shown, moreover, that the artificial production of a membrane in 
the unfertilized egg by butyric acid has the same influence upon 
the increase of the rate of oxidations as the entrance of the 
spermatozo6n. These and other facts seemed to support the 
view of the writer that an alteration of the surface of the egg, 
which usually but not necessarily results in a membrane forma- 
tion, is an essential feature of the development of the egg. 

More recent experiments by Warburg? have made it very 
probable that the process of oxidations in the sea urchin egg is 
a case of catalysis by iron, which is confined mainly if not ex- 
-clusively to the surface; and this fact, in connection with the data 
mentioned above, seems to indicate that the process which under- 
lies membrane formation in the unfertilized egg may consist in 
bringing about or rendering possible the iron catalysis which is 

1 From the Rockefeller Institute for Medical Research, New York. 


2 Compt. rend. l’ Acad. d. sc., CLIX., 642, 1914. 
3 Warburg, Zeztschr. f. physiol. Chem., XCII., 231, 1914. 


87 


88 JACQUES LOEB. 


responsible for the sudden increase in the rate of oxidations after 
artificial or natural membrane formation. Since fertilization by 
sperm is accompanied by a membrane formation and followed by 
the same increase in the rate of oxidations as is artificial membrane 
formation by butyric acid, it is probable that the alteration of the 
surface (underlying membrane formation) is also the cause for 
the increase in the rate of oxidations in the process of natural 
fertilization. 

2. The writer has time and again stated that the formation or 
non-formation of the fertilization membrane is of only secondary 
importance; what matters are the physicochemical changes 
which underlie the membrane formation and which are responsible 
for the sudden rise in the rate of oxidations of the sea urchin egg 
after artificial or natural membrane formation; and which may 
even occur when for some reason the fertilization membrane is 
modified or when its formation is entirely suppressed. There is 
no doubt that in the writer’s first experiments with the purely 
osmotic method, the fertilization membrane was often very 
indistinct or in some cases even completely lacking, while 
nevertheless the enormous increase in the rate of oxidations and 
development to the pluteus stage ensued.1 . 

It is possible to modify the surface of the unfertilized egg in 
such a way that if it is later fertilized by sperm the abnormal 
character of the membrane formed, or the abnormal conditions 
of the surface, may lead to the death of the egg. The writer 
described such a case in 1909.2, When the unfertilized eggs of 
Strongylocentrotus were treated for five minutes with a hyper- 
alkaline solution of NaCl (50 c.c. m/2 NaCl + 1.0 c.c. N/1o 
NaOH) and then transferred to normal sea water to which sperm 
was added, the eggs were all fertilized but apparently without 
membrane formation, though in reality probably with a tightly 
fitting membrane. They all segmented but perished in the 
blastula or gastrula stage. When, however, the eggs were not 
fertilized immediately after the treatment with alkali but after 

1 The literature of the subject can be found in the writer’s recent book on 
“Artificial Parthenogenesis and Fertilization,’’ Chicago, 1913. 


? Loeb, “‘ Die chemische Entwicklungserregung des tierischen Hies,’’ Berlin, 1909, 
jh Tey 


MEMBRANE FORMATION IN FERTILIZATION. 89 


they had been in the sea water for one hour or more, a more 
normal membrane was formed and the eggs developed into plutei. 
Why did the eggs only live to the blastula or gastrula stage when 
they were fertilized immediately after the alkali treatment? 
Should this have had something to do with the abnormal char- 
acter of the membrane which was formed when the egg was fer- 
tilized immediately after the alkali treatment? Were the cells 
pressed by the membrane which was too tight, and did this 
pressure kill them if prolonged? If this were the case, a tearing 
of the membrane should save the life of the egg. It would be of 
interest to try this experiment. 

3. Ina recent number of the Comptes rendus de Il’ Académie des 
Sciences, Brachet has published an observation which may or 
may not be similar to the one just mentioned. He found a year 
ago that if the eggs of the sea urchin, Paracentrotus lividus, at 
Roscoff, are put for two hours in contact with sperm of Sabdellaria 
alveolata (which cannot fertilize the eggs) and if they are after- 
wards fertilized with sperm of their own species, they develop 
without apparently forming a fertilization membrane. From 
this Brachet concludes that the formation of a fertilization 
membrane is not necessary for development, a conclusion which 
will surprise nobody who is familiar with my first experiments on 
artificial parthenogenesis, or who has ever fertilized eggs which 
have been lying in sea water for several days. Moreover, Brachet 
observed that the sea urchin eggs which are fertilized with sperm 
of their own species, after two hours’ treatment with the sperm of 
Sabellaria, die at the time of gastrulation. The prolonged treat- 
ment of the eggs of Paracentrotus with the sperm of Sabellaria 
seems therefore to have a similar effect as the short treatment of 
the egg of Strongylocentrotus with the alkaline NaCl solution in 
my experiments. 

4. The deductions which Brachet draws seem, however, difficult 
to reconcile with each other. We stated already that he assumes 
that the eggs of Paracentrotus after two hours’ treatment with 
the sperm of Sabellaria form no fertilization membrane after 
fertilization with their own sperm. Yet, he states further that 
these eggs die in the gastrula stage for the reason that they cannot 
hatch; for if he shakes the eggs and thereby destroys ‘‘la couche 


90 JACQUES LOEB. 


corticale’”’ the larve can hatch’and are now able to develop into 
plutei. The only membrane, however, which can prevent the 
eggs from hatching is the fertilization membrane, and it is im- 
possible to harmonize the two statements of Brachet, first, that 
these eggs have no fertilization membrane and, second, that the 
gastrulae cannot hatch unless the membrane of the egg is pierced. 
Professor Goldschmidt, to whom I showed Brachet’s paper sug- 
gested that Brachet probably means by “‘couche corticale”’ the 
hyaline membrane (Herbst’s “‘Verbindungsmembran’’) which 
surrounds the blastomeres and that he assumes erroneously that 
this hyaline membrane forms a continuous layer around the 
blastula in the same way as the fertilization membrane does. 
This is, however, not the case since the hyaline membrane par- 
ticipates in the process of segmentation and forms a distinct layer 
around each individual blastomere, but not a continuous envelope 
around the whole blastula. 

Brachet’s observation is intelligible on the assumption that the 
egg after it has been treated with the sperm of Sabellaria forms 
a very tightly fitting membrane when it is fertilized with its own 
sperm and that this membrane must be torn by shaking the 
egg in order to allow the blastula to hatch (or to escape from 
being killed by the mechanical pressure of the tightly fitting 
membrane?). Brachet found also that it is possible to sub- 
stitute for the shaking of the egg a treatment with butyric acid, 
which as he assumes also tends to remove the obstacle to the 
hatching. This may be correct, but unfortunately he draws 
the further conclusion that the butyric acid treatment must have 
the same effect upon the unfertilized egg as upon the fertilized 
egg which has previously been treated with the sperm of Sabel- 
laria. Leaving aside the fact that the unfertilized egg has no 
membrane, it has been shown that the butyric acid treatment 
raises the rate of oxidations of the unfertilized egg about 400 or 
600 per cent., while acid does not increase, but, on the contrary, - 
lowers the rate of oxidations in the fertilized egg. Moreover, the 
writer has shown that if a fertilized egg is treated with butyric 
acid, in the same way as is required for inducing artificial parthe- 
nogenesis, the fertilized egg is not injured, while the inducing of a 
membrane formation by butyric acid in the unfertilized egg leads 


MEMBRANE FORMATION IN FERTILIZATION. OI 


to the rapid death of the latter, if it is kept at room temperature 
and if it does not receive a second treatment either with a hyper- 
tonic solution or lack of oxygen. This case was fully discussed 
by the writer in a recent paper.! It is therefore not justifiable 
to conclude that the action of butyric acid on the unfertilized 
egg must be identical with the action of the same substance on a 
fertilized egg, treated beforehand with the sperm of Sabellaria. 

Should it be possible that Brachet’s ‘‘couche corticale”’ is the 
chorion or the “jelly”? which surrounds the unfertilized egg? 
But this jelly is normally dissolved when the egg is fertilized. It 
might be conceivable that the sperm of Sabellaria causes a harden- 
ing and a contraction of this jelly which protects it against being 
dissolved by the sperm of the sea urchin and that subsequent 
shaking or a subsequent treatment with acid destroys this jelly. 
But granted this were the case, it would be erroneous to use 
experiments on an artificially altered chorion to draw conclusions 
upon the réle of membrane formation in fertilization or artificial 
parthenogenesis. 

The writer wonders how Brachet (or Herlant) are going to 
harmonize the following well-established facts with their views. 
If the eggs of Strongylocentrotus purpuratus are treated with 
hypertonic sea water for about two hours, they form in most 
cases no membrane and nothing happens to them except that a 
certain percentage of them begin to divide very regularly into 
2, 4, 8, possibly 12 or 16 cells and then stop. Such eggs are to 
all appearances in the resting stage and live as long as the other 
unfertilized eggs if nothing is done to them. If they are fertil- 
ized by sperm each blastomere forms a special fertilization mem- 
brane and now each blastomere develops into a blastula or into 
a pluteus, according to the size of the blastomere. They also 
develop into plutei if an artificial membrane formation is called 
forth with the aid of butyric acid. The writer is inclined to 
explain this phenomenon by assuming that the treatment with 
the hypertonic solution called forth two effects, one of which 
was a peripheral change resulting in an increase in the rate of 
oxidations. This effect is, as the writer has shown, reversible 


1 Loeb, “Weitere Beitrage zur Theorie der kiinstlichen Parthenogenese,’’ Arch. 
f. Entwckingsmech., XXXVIII., 409, 1914. 


92 JACQUES LOEB. 


and was possibly reversed while the eggs were in an early stage 
of development. It seems to the writer impossible to reconcile 
these observations with the purely morphological views of 
Brachet or Herlant. | 
Brachet (like Herlant) tries to explain the phenomena of 
artificial parthenogenesis and fertilization without any consider- 
ation of the striking chemical processes that accompany fertiliz- 
ation and artificial membrane formation. He reverts to that 
standpoint of the pure morphologist which Sachs, in his papers on 
‘“‘Matter and Form in Plants”’ characterized as ‘ 
ism.” This standpoint disregards the sources of energy in life 
phenomena and treats morphological changes as if they required 
no source of energy. It seems to the writer that the fact of the 
necessity of oxygen for development, the fact that mere mem- 
brane formation (both by butyric acid or by a spermatozoén) 
raises the rate of oxidations 400 or 600 per cent, and the fact that 
the amount of rise is identical in both cases, are so striking, that 


‘empty formal- 


these facts cannot be ignored in a theory of the rdle of membrane 
formation in the development of the sea urchin egg. The writer 
has always considered the changes underlying the membrane 
formation as the essential factor in the initiation of development, 
while he considered the formation of a fertilization membrane 
only as a welcome but not essential indicator of the chemical 
changes in the surface of the egg; afact which Brachet, on account 
of his disregard for the chemical processes, has entirely over- 
looked. Brachet, from his purely morphological standpoint, 
erroneously assumes or makes it appear as if I considered the 
formation of a visible membrane as the only and essential act 
in the initiation of development. 


AN EXPERIMENTAL ANALYSIS OF FERTILIZATION 
IN PLATYNEREIS MEGALOPS. 


BE. BE. JUS#. 


Study of the breeding habits of Platynereis megalops revealed 
the fact, as has been pointed out (Just, 14), that insemination 
takes place in the body cavity of the female and that although 
egg laying begins often but five seconds after copulation, the eggs 
will not fertilize when artificially inseminated after exposure to 
the action of sea-water. It is this failure of sea-water insemina- 
tion that forms the basis of the present contribution to the 
analysis of fertilization in Platynereis. In order clearly to inter- 
pret the phenomena of sea-water insemination a study of the 
morphology of the normal fertilization was made (see Just, ’15a). 

The experiments undertaken for the analysis of fertilization 
in Platynerets come under three heads: 

A. Conditions of successful insemination. 

B. Cross fertilization with Nereis. 

C. Artificial parthenogenesis with various agents. 


B and C are taken up mainly because they supplement results 
under A. 


A. CONDITIONS OF SUCCESSFUL INSEMINATION. 


During the summer of 1911, I was studying the maturation and 
fertilization of the Platynereis egg for comparison with those 
processes in Nereis. The methods of insemination used with 
Nereis, cutting out the eggs and sperm in sea-water, gave no 
cleavage. Various trials with the utmost care, using diverse 
methods never gave cleavage. Not until August 24, 1911, did 
I chance to find that normally insemination takes place in the 
body cavity of the female (cf. Just, ’14). 


I. Observations on Eggs Inseminated in Sea-water. 


If eggs and sperm be cut out of Platynereis and mixed in sea- 
water, the phenomena of maturation, sperm attachment, and 
93 


04 E. E. JUST. 


copulation of the germ nuclei may be readily followed; but such 
eggs do not segment nor do they ever develop into swimming 
forms. 


The Living Egg. 

If insemination be made in a suspension of India ink ground 
up in sea-water, the jelly formation may be easily followed: it 
differs but little from the cortical outflow observed in eggs nor- 
mally laid. All eggs, however, do not secrete this jelly; of these, 
some remain in the germinal vesicle stage and others go through 
maturation with all or part of the cortex intact. 

As in the normally inseminated egg (see Just, 15a) no cone is 
present. More often than in the normally laid egg a broad 
plateau of cytoplasm marks the point of sperm attachment. 
The sperm, from one to six, are attached to the membrane above 
this raised cytoplasm or near it. 

Maturation proceeds about as in the normal egg. At matura- 
tion stages slightly later than in the normal egg, the sperm may 
be found in the egg. It moves forward with aster formation. 
The pronuclei meet, remain apposed for a short time, separate, 
and fade from view. This is not true of all eggs; for apparently, 
those in the germinal vesicle stage or in maturation stages with 
cortex intact never engulf the sperm. Moreover, in many eggs 
that are in maturation with the cortical layer gone, one cannot 
find sperm. 

These eggs never divide. At first, 1911, I thought that this 
behavior of the egg was due to injury of the worms. Its sig- 
nificance became clear only after the discovery of the normal 
method of egg-laying. 


The Sectioned Egg. 

During four seasons eggs have been preserved at three and five 
minute intervals upward to two hours after insemination in 
sea-water. Study of the sectioned eggs confirms the findings of 
the study of living eggs. Many eggs remain ovocytes with sperm 
attached or not. Those that go through maturation do so with 
or without jelly formation. Eggs that form jelly are likewise of 
two classes: those in which sperm are found to have penetrated 
and those in which no sperm are found. 


FERTILIZATION IN PLATYNEREIS MEGALOPS. 95 


I have not been able so far to determine any structural differ- 
ences in the ovocytes with and without sperm attached. In the 
case of the eggs that maturate with the cortex wholly or partially 
intact, the spindle may be abnormal. In most cases if it reach 
the periphery of the egg it does so at a point practically devoid 
of cortical cytoplasm. Or again, it may lie parallel to a tangent 
of the egg membrane. 

Those sections which reveal the sperm within the egg are in 
the minority. It appears from experiments several times 
repeated during the four seasons of study that the penetration of 
the sperm depends upon the amount of sea-water used. If the 
eggs be inseminated in a large quantity of sea-water or washed 
(by changing the water several times) very few eggs form jelly. 
With less water more form jelly. Eggs inseminated quickly in 
small quantities of sea-water are capable of engulfing sperm. 

The history of the penetration as known may be briefly given. 
One finds sperm external to the egg at different stages. How it 
gets into the egg I cannot yet state with certainty although this 
point has received most careful study for three years. Material 
has been prepared in every way possible to demonstrate the early 
penetration. So far I have not found the sperm entering the egg 
as a slender thread like that in the normal egg. It can be easily 
demonstrated in the endoplasm. On one slide of the 1911 series, 
for instance, I counted twenty sperm heads with their asters 
lying near the centre of the egg. The sperm head remains for a 
longer time than in the normal egg a black knot with a long 
drawn out thread extending to the single aster. A second aster 
has never been found. The germ nuclei copulate but the eggs 
never cleave. Various stages are found from sixty to one hundred 
twenty minutes after insemination—sixty minutes after cleavage 
in the normal egg. The pronuclei after apposition gradually 
separate and degenerate as discrete nuclear masses. Many eggs 
show only one chromatin mass in process of degeneration; 
doubtless, these are eggs which sperm do not enter. The sections 
of such eggs closely resemble those of Nerezs eggs from which the 
sperm have been removed (see Lillie, ’12). I have repeatedly 
made observations on living eggs inseminated in sea-water and on 
sections. I have yet to find a single cleaving egg. 


96 [Dra 195, {USite 


Two hours after insemination the eggs exhibit cytoplasmic 
stratification; the oil drops later fuse to form one at the vegetative 
pole. Twelve hours after insemination the conditions are the 
same; there is never a swimming form among these eggs. 


2. Nature of the Inhibition to Development. 


It may be very clearly shown that sea-water is responsible for 
the lack of cleavage by the method of “dry insemination.” If 
males and females dried on filter paper be cut up separately and 
the drops of eggs and sperm thus obtained be mixed with sub- 
sequent addition of sea-water, a percentage of the eggs always 
cleave and develop into normal trochophores. I have kept larvae 
from such dry inseminations until they were seven mm. long 
with thirty or more segments, few differing from normally laid 
eggs. There is doubtless an optimum time after mixing for the 
addition of sea-water, but any time upward to two minutes 
gives results. The following is an example: 

August 3, 1912. To determine the time interval after mixing 
dry eggs and sperm before adding sea-water. 


Per Cent. 
Water Added. of Cleavage. 
Res AENOMCE Sie ts SPiyh ese ey nea ela tector Peak ee one uen unica cae Renee 60 
2. Nive Seconds ahterncic cos -pone eh eiuera are toeeces ci onben ee Rrereene 50 
3. ensecondsrahtereame. telecine 90 
4: Twenty: secondsiaiters sac yee e nee ee eee ei 45 


Practically, as soon as eggs and sperm are mixed, sea-water 
may be added. I have not been able to add sea-water quickly 
enough after mixing to prohibit cleavage. If the eggs are allowed 
to stand two minutes the majority are plasmolyzed by the addi- 
tion of sea-water. | 

The amount of sea-water that will permit fertilization has been 
repeatedly determined: 

July 28, 1912, 9:45 P.M. Experiment to determine the maxi- 
mum amount of sea-water that permits fertilization. 

Males and females are thoroughly dried on clean filter paper. 
A male and a female placed in each of the eight perfectly dried 
clean watch glasses. Sea-water added as follows: 


FERTILIZATION IN PLATYNEREIS MEGALOPS. 97 


NG ues Bikte How o onde la Ope IgewLe gle OmiaIod oS DOE LD I drop 

OG py te Side bralete 6/60 Bide 0 Slocproloiphowolp ae 0 CHES cen 2 drops 

OG i Reh eB ERAN O TS AEA EGS He Rime om Celen S ° Aine aha ete 

BCS Sl Sad eR er ee Goo RHO © > Tete easy ART: 

CO ee ea emi oer bo Gia cee Pho Ral el a Ga fat eee neh co BCU RC RRR hie 

BO yale AME VAP) Mi coat en cr alot, Qonra'l iva ace parka, Sirk oh. TN ay (Si. 4h 

OL is a No Ana eon tt coho tN: 0 Pig Din Cate eae ad IO c.c. 

Gs Ta Le oie Baril TL all een ibe, Segemioha n:6 b-GiiG.0\c- Ob CAC SiR no sea-water. 


The worms were then cut up and flooded with sea-water, later 
transferred to fresh sea-water in finger bowls. 

Nos. 1, 2, 3 and 8 gave cleavage; a per cent. of normal trocho- 
phores was found the next morning. In dishes 4, 5, 6 and 7 not 
an egg divided, no swimming forms developed. 

No single observation in the whole work was made as often as 
this; the results are wonderfully precise. As I shall show later 
the experiment quoted was conducted under the optimum con- 
ditions, and yet it shows the inhibiting effect of such a surprisingly 
small quantity of sea-water. All other observations show two 
drops of sea-water for each worm to be the maximum that will 
permit normal fertilization. Inno case have I got cleavage where 
two and one-half drops of sea-water for each worm (2. eé., five 
drops to two worms) were used. While the same pipette was used 
to secure equal drops, the worms, females particularly, vary in 
size. I have usually taken the average females for these experi- 
ments. Such an animal, as found by actual count in three cases, 
has about 11,000 eggs. There is enough variation, however, in 
the size and weight of the worms to make impossible any law 
concerning the lethal amount of sea-water. I believe, never- 
theless, that there is an optimum time for the addition of sea- 
water—equal to the time the sperm are in the female in normal 
insemination; and an optimum amount of sea-water—about as 
much as the worms will take up after thorough drying. 

The results of these inseminations over a period of four seasons 
prove clearly that sea-water except in minute quantity is fatal 
to fertilization. 


Does Sea-water Injure Egg, Sperm, or Both? 


Three explanations of the failure of Platynereis eggs to cleave 
after insemination in sea-water are possible: 


98 By 1d, just 


(a2) Both eggs and spermatozoa are injured by the sea-water. 

(b) The sperm alone are injured by the sea-water. 

(c) The eggs alone are injured by the sea-water. 

The failure of the eggs to go beyond maturation may be due 
to the injurious action of the sea-water on both eggs and sperm 
alike. It would seem reasonable to assume that for internal 
insemination both cells need the perivisceral fluids. It might be 
difficult to conceive how this adaptation in Platynereis could have 
taken place acting on one only of the sex elements. As both eggs 
and spermatozoa are protected by body fluids in normal insem- 
ination, so both are exposed to the lethal action of sea-water. 
Embryologists are all careful when inseminating eggs of forms in 
which insemination normally taken place in the sea not to con- 
taminate the dishes containing ova with the animal’s tissues or 
fluids. Lillie (130, ’14) has shown why this is essential. I have, 
however, repeatedly with success fertilized Nereis eggs dry (see 
Just, 150) doubtless because the body fluid of Nereis is practically 
negligible. And the case of Platynereis is similar to that of 
Nereis; in this smaller worm there is no more fluid; the female 
is a mere locomotor ovary, although the male does have a small 
amount of fluid and a great number of corpuscles. 

The second possibility is that the sperm alone are injured by 
the sea-water. Injury to the sperm through transference from 
the male’s body fluid to sea-water, however, cannot be due to 
difference in osmotic pressure. For as Frédericq has shown, and 
Garrey since for the Woods Hole region, the osmotic pressure of 
invertebrate body fluids is about the same as that of sea-water. 
Moreover, Platynereis sperm in sea-water as far as I could de- 
termine exhibit none of the effects experimentally produced by 
Koltzoff on various sperm cells including those of Nereis (dumer- 
iii?) through treatment by various salt solutions or those con- 
ditions described by de Meyer with hypotonic and hypertonic 
solutions. Insome other way, then, the sperm must be assumed to 
be weakened but still capable of partially fertilizing the egg as the 
Hertwigs, Gemmil, Budington, Dungay, etc.,haveshown. And 
indeed my Platynereis slides of sea-water inseminated eggs show 
similarities to the figures by Lillie of the penetration of injured 
sperm in Nereis; in Platynereis, however, the germ nuclei develop 


FERTILIZATION IN PLATYNEREIS MEGALOPS. 99 


a little farther. Steinach long ago, later Walker (’99, 11) and 
Hirowaki have shown that in mammals the prostate secretion is 
necessary for fertilization. Sea-water, then, might injure the 
" sperm and hinder fertilization by destroying a supporting medium 
necessary for fertilization. (On this point, cf. Gemmil’s ex- 
periments.) 

Finally, a third explanation is possible: the egg alone is injured 
through sea-water treatment. The egg, in this case, may be 
dependent on a substance in the female’s body or on some se- 
cretion of its own necessary for fertilization. Both egg and sperm 
may need body fluids but sperm may be hardier, egg less resistant. 

The seasons of 1912 and 1913 were largely given over to ex- 
periments to determine which possible explanation is valid for 
Platynereis. In 1914, many of these experiments were repeated. 
And I may say at once that the explanation must come under 
the third head as shown by the following experiments. 


The Experiments. 

The plan of the experiments is briefly as following: 

Males and females were cut up separately in dishes of clean 
sea-water. The bits of tissue were carefully removed, the dish 
of eggs being handled with utmost care to prevent unnecessary 
agitation. The eggs and sperm suspensions were filtered after 
having remained in sea-water for varying lengths of time. 
Sexual products treated thus are designated “washed eggs’’ and 
“washed sperm.’” 

Males and females were thoroughly dried on filter paper or 
clean sheer linen. The males were cut up in dried clean watch 
glasses; the females were cut up in the same way or pricked when 

1 That the resistance of eggs and sperm of both Nereis and Platynereis is unequal 
would seem probable from the following: If to a Nereis sperm suspension janus 
green be added the fertilizing power of the sperm is in no wise impaired; or if the 
dye be added to sea-water the living males absorb it readily without any injurious 
effect on the sperm. The same quantities of the dye in sea-water is toxic to the 
egg before or at insemination. Eggs taken from a female Platynereis that has been 
swimming in a janus green-sea-water solution that is not toxic to the males or their 
sperm will not fertilize. Cf. also action of nicotine on Strongylocentrotus sperm 
and eggs as observed by the Hertwigs. 

2 Several methods were used for ‘‘washing”’ sperm and freeing them of sea- 


water, among others that of centrifuging at high speed for six minutes. These 
were all abandoned for the method here described. 


100 E. E. JUST. 


most of the eggs that escaped were collected in dry watch 
crystals. Bits of tissue were always removed. Such eggs and 
sperm are ‘‘dry eggs’’ and “‘dry sperm.” 

For a given experiment eggs and sperm were mixed and after 
an interval of time varying from five to sixty seconds flooded 
with sea-water. Four kinds of inseminations were made: 


Washed eggs X washed sperm. 
Washed eggs X dry sperm. 
Dry eggs xX dry sperm. 
Dry eggs xX washed sperm. 


The experiments fall into two groups: “A.M. inseminations” 
—made the morning after the worms were captured; and “P.M. 
inseminations’’—made during the evening of capture. 

The following table gives a summary of results: 


TABLE I. 
Eggs. Sperm. 5 Group. Development. 
Wrashedieee ree WSN. scan adde ASIN BHoGl IPSIMos Go p5400 None. 
WRISINEG!s bo 500006 Dry A.M. and P.M.......... None. 
DD iny dere mert: DD) reyanrarein eta ieee A.M.and P.M.......... Cleavage and larve. 
ID vere trsasretattcnsisy Washed......... AIM oy oe Sones eoeanee None. 
IDSA auch mabe aOR Wiashediae ssn oe [PAINT Ie kee iraen ene Cleavage and larve. 


Washed eggs, inseminated with dry or washed sperm, never 
reach cleavage stages nor do they ever produce swimming forms. 

I have commented above on the dry egg X dry sperm series. 
These eggs cleave and later produce normal larvae. 

Washed sperm X dry eggs of the A.M. group (1912) did not 
yield cleavage or swimming forms. The worms do not thrive 
well in the laboratory. The practise, therefore, of conducting 
experiments the morning after capture has been since I912 prac- 
tically abandoned. The only test for the vitality of the worms is 
copulation—a test the very nature of which precludes experiment. 
Doubtless, therefore, this set of experiments gave no results 
because the animals were not fit. Study of sections of eggs 
normally inseminated and laid as early as 5 A.M. shows a large 
percentage in the germinal vesicle stage. I have made counts in 
dishes of living eggs to show at the later cleavage stages the pro- 
portion of eggs still inthe germinal vesicle stage. For example, 


FERTILIZATION IN PLATYNEREIS MEGALOPS. IOI 


August 8, 1912, 2 P.M., six hours after laying of 10,851 eggs 
(from one female) six per cent. were still in the germinal vesicle 
stage. Other counts of living eggs and of sections show higher 
percentages. Every egg laid the night of capture cleaves. Dry 
inseminations, day or night, at best never give more than ninety 
per cent. of cleavages. The poor quality of the animals after 
several hours in the laboratory may account for the failure of the 
dry eggs X washed sperm A.M. group to cleave. But since the 
dry eggs X dry sperm A.M. series gives cleavage, I am rather 
inclined to believe that the method used was poor: for instance, 
the filter paper then used was too soft allowing the loss of most of 
the spermatozoa or too much water was left when the dry eggs 
were added. 

The results with dry eggs X washed sperm, P.M. group are 
wonderfully uniform and show conclusively that the sea-water, 
at least for the exposures used, has no harmful effect on the sperm. 
The method used is simple. As soon as possible after capture one 
to three males are cut up in from 8 drops to 20 c.c. of sea-water 
and allowed to stand upward to twenty minutes. (The sperm 
are active after having been in sea-water for twelve hours.) The 
sperm suspension is then filtered. I used a very hard filter 
paper. This paper was then tilted and thoroughly drained 
until under the lamplight the glistening water was thoroughly 
absorbed. A dried female was cut up on the filter paper or 
pricked and the eggs thus procured rolled over the paper to reach 
the sperm left behind or caught in the pores of the filter. The 
whole was then put in a dish of clean sea-water. It would be 
tedious to cite the individual experiments. They show conclus- 
ively that dry eggs inseminated with washed sperm develop in 
normal fashion. 

Now since, as has been shown above, there is a minimal 
amount of sea-water that will permit fertilization, dry eggs 
_ ought to fertilize if put on the filter paper before all the water 
has been absorbed. Such indeed is the case. Moreover, dry 
eggs put in two drops of thin sperm suspension develop. From 
a suspension made by cutting up one or more males in sea-water 
two drops are taken. Dry eggs put in this cleave and next 
morning swim. 


102 E. E. JUST. 


This observation led to a series of experiments (during 1913 and 
1914) designed to ascertain whether or not the density of the 
sperm suspension is a factor in the fertilization of Platynereis. 

These experiments prove in general that the number of dry 
eggs added to sperm suspensions that develop depends upon the 
density of the suspension. The denser the suspension the larger 
the number of trochophores. Moreover, for dense suspensions 
the minimum amount of sea-water permitting fertilization appears 
to be slightly higher than for thin suspensions. Cleavage is 
directly a function of the chances of the spermatozoa reaching 
the egg before the fertilizing substance is lost. 

The time of flooding with sea-water after insemination is also 
important for the highest percentage of cleavage. But these 
factors cannot be expressed with mathematical exactness. Some 
points, particularly with reference to inseminations with dense 
suspension need further experiments to determine their signi- 
ficance. 

That the egg when exposed to the action of sea-water quickly 
loses something necessary for fertilization must be the conclusion 
drawn from these experiments with washed or unwashed eggs. 
Even thirty seconds residence in sea-water, as repeatedly proved, 
is sufficient to inhibit cleavage in every single egg. If dry eggs 
from a single female be put in five cubic centimeters of sea-water 
and thoroughly drained as soon as they settle they will not de- 
velop after insemination although this procedure may take but a 
half minute. The egg alone is affected by sea-water; the fer- 
tilizing power of the sperm is not affected by exposure to sea- 
water. 


3. The Nature of the Fertilizing Substance. 


The fertilizing substance once lost cannot be restored. If 
washed eggs be mixed with an extract obtained by crushing dry 
eggs in one or two drops of sea-water and dry sperm added, 
cleavage does not result. I lay no stress on this, however, for 
it seems to me that such an extract might yield anything. 

The presence of various substances in the sea-water or the 
lowering of the temperature of the sea-water does not prevent or 
restore the loss of this substance. 


FERTILIZATION IN PLATYNEREIS MEGALOPS. 103 


KOH.—Eggs were teased out of the female directly into sea- 
water plus KOH in various proportions. Or, eggs from dried 
females were placed in the solution. After remaining from thirty 
seconds to two minutes in the alkaline sea-water the eggs were 
inseminated dry and flooded with sea-water. In other cases in- 
seminations were made in the solutions. Washed eggs were 
similarly treated. Whatever the method alkaline sea-water 
never gave cleavage. (Cf. sections on cross fertilization and 
artificial parthenogenesis. ) 

Hypertonic and Hypotonic Sea-water.—Egegs, both washed and 
dry, were treated with 244 M KCI + sea-water as follows: 


I. 1 drop 23-M KCl + to drops of sea-water. 
2. 2 drops “ Bt verte Th Se enum on 
SEES aie gies pire Diese Sg 
eee ca iL 'S 7) Oa age 
Gi Sie Gh ein peat pens er ce S 
OxiOiie Ay ape nes hatte Bs 


Dry sperm were added at once and the dishes flooded with 
sea-water after five minutes. Or, after treatment for varying 
number of minutes the eggs were inseminated dry. The eggs 
developed no farther than with KCl treatment alone (see 
beyond); they form jelly and maturate. 

Hypotonic solutions used similarly gave no cleavage. 

Ether.—The following table is a summary of the experiments 
with ether: 


Eggs. Solutions Used. Exposure. Inseminations. 
Washed, .3 to .6 per cent. I to 5 minutes dry; in the solution. 
Dry, oe oe oe be oe oe 
Teased, sé ee ee 6eé “é ce 


“Teased”’ eggs are those got by cutting up the female in the 
ether-sea-water. , 

A few eggs form jelly and maturate after the ether treatment. 
Compared with sea-water inseminations, ether cuts down the 
per cent. of maturations. According to R. S. Lillie (12) star- 
fish eggs resistant to fertilization may be rendered normal by 
ether in low concentration. In Platynereis the condition is 
different. The egg is not rendered resistant to fertilization by 
the action of sea-water; it is weakened through loss of something 


104 ES Ee aust 


by the sea-water since it combines but feebly with the sperm 
The ether as in Aséerias renders the Platynereis egg irritable since 
as shown by the low percentage of maturation more fertilizing 
substance must be secreted. 

KCN.—Inseminations made with washed or dry eggs during 
or after treatment with KCN (1 per cent. KCN and sea-water 
made in various proportions) gave only maturation. But the 
eggs will maturate in KCN alone while in the solutions. (Cf. 
Allyn on Chetopterus.) 

CaClz.—Newman found that CaCl. inhibits fertilization in 
Fundulus through a precipitation effect. I thought that in 
somewhat the same way calcium chloride might through action 
on the cortex inhibit the loss of the fertilizing substance in Platy- 
nereis. M/2 CaCl, added to sea-water in different quantities 
does not inhibit the loss of the substance since after the calcium 
chloride treatment the egg does not fertilize. 

Cooled Sea-water.—Sea-water was cooled to 10.5° C. and dry 
eggs after 30, 60 and 90 seconds’ treatment in 5 c.c. were insemi- 
nated at this temperature or after the cooled water was pipetted 
off. In some experiments the female was kept at the low tem- 
perature for several minutes before the eggs were cut out. 5 C.c. 
of sea-water were used in each experiment. The eggs never 
cleave, but more form jelly and maturate than controls insemi- 
nated in ordinary sea-water. This would seem to indicate a 
slowing down of the secretion. The effect of cold is just the op- 
posite of the effect of ether. Unfortunately, only few of these 
experiments were made. Perhaps they should be repeated at 
lower temperatures. 

Concerning the nature of this substance, some of my earliest 
notes are of interest. After insemination in sea-water I found 
some time later (forty minutes in one case) ‘‘sperm dancing above 
the eggs.’’ In 1914, I found the sperm of sea-water insemination 
active after twelve hours. One does not find this after dry in- 
semination, even with excess of sperm. Sperm in the dishes of 
successfully inseminated eggs are profoundly changed. Study 
of the movements of Platynereis sperm reveals the circular swim- 
ming of echinid spermatozoa, as shown by Buller, Gemmil, 
Winslow, and others (see also Dewitz, Ballowitz, etc.). They 


FERTILIZATION IN PLATYNEREIS MEGALOPS. 105 


finally become quiescent through lack of oxygen! in various 
positions without orientation. After dry inseminations they 
come to rest, as can be seen after flooding the dishes, definitely 
oriented and not in haphazard arrangement. Clustered among 
the jelly hulls, their heads point toward the eggs. On occasions, 
I believed that I demonstrated the agglutination of the sperm by 
sea-water in which the eggs had been lying. The evidence is not 
clear-cut and more recent attempts have failed. The egg 
charged sea-water, however, does activate the sperm. 

I wish to point out the serious difficulties experienced in the 
series of sperm agglutination experiments. In the first place, 
twenty ‘‘large’”’ dried males (two and one half centimeters long) 
do not yield enough sperm and body fluid to make up a drop as 
large as a drop of dry sperm from a very small Nereis. Then 
again the thickest suspension got is largely made up of blood cor- 
puscles. I have never succeeded in procuring a ‘milky sus- 
pension’’—the admixture of corpuscles and body fluid giving 
always a pinkish mixture. And finally, one cannot always get 
twenty or more males necessary to make up even this thin sperm 
suspension. Repeated efforts, therefore, extending through two 
seasons have not been marked with very positive results. 

With Nereis sperm, the case is indisputable. If water in which 
Platynereis have laid eggs be taken it is found to have an agelu- 
tinating effect on Nereis sperm. Thus: 

August 18,1914. At10:15 P.M., ten females laid eggs in six c.c. 
of sea-water each. After five minutes some of this water was 
drawn off—z2o c.c. in all. Nereis sperm suspensions were made 
up fresh at 10:20, 10:30, 11:00 and 11:05. A drop of the sperm 
suspension was mounted on a slide under a raised cover slip. A 
drop of the water taken from the dishes of eggs was injected 
beneath the cover slip. Under the microscope, the quiescent 
sperm appeared at first intensely active, then rushed together 
and formed agglutinated masses among others still free-swimming. 


1 This fact was brought out in 1913 when I was repeating some old observations 
on echinoderm spermatozoa. While experimenting with the sperm of Thyone in 
janus green solutions, I noted after some time had elapsed that cover-slip prepara- 
tions showed that bacteria present previously bluish in color had changed to a 
decided red. Later observations proved that as the dye was reduced in bits of 
tissue under the cover slip the sperm quieted down in various positions. 


106 1B, 185 USI. 


The same experiment succeeds if one uses the water from dishes 
in which uninseminated eggs have remained for a few minutes. 
Washed eggs do not cause agglutination of Nereis sperm; water 
charged «by normally inseminated eggs or uninseminated eggs 
retains its power of agglutinating Nereis sperm after twelve 
hours at least, the reaction coming on more slowly. The freshly 
charged water acting on fresh sperm suspension gives a clear-cut 
and beautiful reaction. 

It may seem far-fetched to argue that the fertilizing substance 
lost by Platynereis eggs when exposed to sea-water is agglutinin 
or fertilizin as discovered by Lillie in Nereis and Arbacia because 
the washed egg, no longer fertilizable by its own sperm, can not 
sufficiently charge the sea-water- to agglutinate Nereis sperm. 
Yet I believe this is the case precisely. The agglutination of 
Nereis sperm by Platynereis egg-water is correlated with jelly 
formation in Platynereis by Nereis sperm. In sea-water in- 
seminations, Nereis spermatozoa are almost as effective as those 
of Platynereis. Added to this is the difference in behavior of 
Platynereis sperm in egg charged sea-water, in sea-water insemin- 
ations, and in dry inseminations. 

The evidence may be scant, but it seems to me sufficient to 
indicate that the substance lost which is necessary for fertilization 
is identical in nature with the fertilizin of Lillie. 


B. Cross FERTILIZATION WITH NEREIS. 


I have mentioned (Just, 14) the fact that it is generally taken 
for granted that reciprocal crossing of Nereis and Platynereis is 
the rule. This led me to attempt cross fertilization. Cross 
fertilization never produces segmentation or development though 
it may induce the maturation process. 

Of the methods used in echinoderm hybridization—those of 
Loeb, Tennent,! etc.: (1) high temperature; (2) treatment with 
fresh water; (3) treatment with alkalis; (4) allowing the eggs to 
stand; and (5) polyspermy—all were tried except the first. 
Since the eggs of Platynereis are normally inseminated in the 
body cavity and therefore with little sea-water, I tried “‘dry 


1Dr. Tennent in 1912 very kindly communicated to me at length his latest 
methods in echinoderm hybridization. 


FERTILIZATION IN PLATYNEREIS MEGALOPS. 107 


inseminations’: 2. e., Nereis males were cut up dry and a drop of 
the sperm without the addition of sea-water added to eggs of 
Platynereis cut up dry. Inseminations were made in a variety 
of ways as the following table of method shows: 


TABLE II. 
SUMMARY OF INSEMINATIONS MADE IN IQII, I912, I913, AND I9I4 
Platynereis sperm on Nereis egg. 
I. Few sperm in sea-water. Fresh eggs in sea-water. 


2. Dense sperm suspension. 


3. Few sperm in sea-water. Stale eggs in sea-water. 
4. Dense sperm suspension. ; 


5. Few sperm, dry, Fresh eggs dry. 
6. Heavy insemination dry. 


. Few sperm, dry. Stale eggs washed. 


~w 


8. Heavy insemination, dry. 


Reciprocal crosses of Platynereis eggs and Nereis sperm were 
made. 

“Stale eggs”’ are eggs that have stood in sea-water for several 
hours. ‘‘Stale eggs, washed”’ are stale eggs on which the water 
has been changed several times. 

These experiments were made repeatedly during four seasons. 
The sperm of Platynerets has practically no effect on the egg of 
Nereis whether fresh or stale, dry or in sea-water. In one ex- 
periment (1911) I got jelly formation in a few eggs. This ex- 
periment later repeated (1913) gave no result. If Nereis eggs 
be inseminated with Platynereis sperm during the evening of 
capture they show no change the next morning. Inseminated 
with Nereis sperm twelve hours after insemination with Platy- 
nereis sperm, the eggs develop normally if anything in greater 
numbers than such stale eggs in ordinary sea-water do. 

Nereis sperm will cause Platynereis eggs to form jelly, the per 
cent. of eggs thus responding depending upon the amount of 
sea-water used and the density of the sperm suspension. But in 
general many of the eggs fail to form jelly.or go through matura- 
tion. Many that maturate do so with the cortex partially or 
wholly intact. Sections of these eggs preserved at three minute 


108 EOE. just 


intervals after insemination have been studied. The sperm does 
not enter; or, if it enters must disintegrate early for I have never 
found sperm nuclei in these preparations. — ! 

Clearly, then, one may not use the eggs of these worms in- 
discriminately. 


C. ARTIFICIAL PARTHENOGENESIS. 


The following agents have been used in an attempt to bring 
about artificial parthenogenesis in the egg of Platynereis megalops: 


Centrifuging, 
KCI, 

NaOH, 
KOH, 

HNOs, 

lath, 


7. Warm sea-water. 


ONES Ae Cie 


The eggs were cut out of the worms in sea-water centrifuged; 
subjected to varying quantities of salt, alkalis, or acids for dif- 
ferent lengths of time; or warmed in sea-water for from five to 
thirty minutes at 35° C. . These methods gave polar body forma- 
tion, cytoplasmic changes, fusion of the oil drops, and finally 
chromatin disintegration in the animal hemisphere. The eggs 
never cleaved. 

Study of the literature reveals the fact that the clearest cases 
of artificial parthenogenesis closely simulating the normal in 
cleavage and in larval development are of those eggs that have 
formed one or both polar bodies when shed: the echinids, for 
example, and the asteroids. Other eggs shed in the germinal 
vesicle stage like those of Polynoe (Loeb ’08), Amphitrite (Loeb 
"oI; Scott.) Nereis (Lillie ’11), etc., give only differentiation 
without cleavage or incomplete cleavage. Loeb and Wasteneys’ 
work on Chaetopterus with ox serum as well as Miss Allyn’s 
on the same egg with heat are exceptions. The great exception 
to the general statement made above is Thalasema (Lefevre) 
where it appears with single substances, acids mostly, normal 
development is closely simulated. On the whole, however, 
ovocytes yield less readily to parthenogenetic agents than 
mature ova. 


FERTILIZATION IN PLATYNEREIS MEGALOPS. 109 


Mathews’ experiments (’o1) on Asterias may in this connection 
be cited. He found that when the eggs of this starfish were got 
while still in the germinal vesicle stage shaking would produce 
development only after the eggs had remained in sea-water until 


maturation was gone through with. Sea-water acts as a first 
stimulus and mechanical shock induces further development. 


So R. S. Lillie (08) on the same egg finds that its responsiveness 
to momentary elevation of temperature as a means of producing 
artificial parthenogenesis ‘‘ varies greatly at different periods in the 
life of the egg.” ‘“‘The most favorable period is some little time 
(10 to 20 minutes) before the separation of the first polar body.” 

Reasoning thus, I thought that I might carry Platynereis eggs 
through maturation with one agent and then through cleavage 
with another. Eggs were, therefore, treated with KCl, KOH, and 
NaOH in sea-water for various lengths of time and then subjected 
to heat, shaking, and centrifugal force. In no case did I procure 
cleavage although the first agent in each case caused maturation. 
With Nereis, on the other hand, KCl and subsequent warming in 
sea-water induces development (see Just 7150). 

It is interesting to note that eggs subjected to heat in the 
minute quantities of sea-water that permit fertilization do not 
develop beyond maturation. Apparently, the conditions for 
successful artificial initiation of development are more exacting 
than those for successful insemination. 

We may conclude, then, that the results of attempted cross 
fertilization and artificial parthenogenesis are harmonious with 
those of sea-water insemination, so far as cleavage is concerned, 
in their negative results. The fundamental questions are: (1) the 
significance of the sea-water insemination and (2) the extent to 
which the results with Nereis sperm and with parthenogenetic 
agents are capable of like interpretation. 


DISCUSSION. 


Any analysis of fertilization must deal with the phenomena 
from the point of view of heredity or of initiation of development. 
Considered as the process of initiating development, fertilization 
may be divided into the stages of insemination, sperm pene- 
tration, and germ nuclei copulation. As Lillie has repeatedly 


110 E. E. JUST. 


pointed out! experimental evidence must be amassed testing the 
meaning of each of these stages. lets 

I. Concerning insemination, as Lillie has shown, the egg plays 
an important part through the production of agglutinins.? For 
both Arbacia and Nereis it has also been shown that chemotaxis 
plays a part in insemination. (Lillie, ’12, ’13a, ’13b, and ’14). 

I believe that Platynereis belongs to this class. I may, how- 
ever, be permitted again to point out the great difficulty attending 
the use of Platynereis eggs on this phase. All the phenomena are 
extremely rapid, the reactions must be very nice. The material 
is unfavorable for any intensive study of agglutination and che- 
motaxis. When one stops to think of the extremely precise 
reactions of the eggs, one gets a hint of the task. The carrying 
over of the smallest drop of sea-water above the maximum to eggs 
from vigorous females within the shortest time after capture will 
prohibit cleavage in every egg. . 

To answer the general question whether or not eggs secrete 
substances that activate the spermatozoa, I believe forms whose 
eggs are inseminated normally in sea-water should be used. So 
far as Platynereis is concerned, agglutination or not, chemotaxis 
or not, the egg must lose a substance or substances when in 
sea-water whose presence is necessary for fertilization. 

2. Study of the normal fertilization of Platyneretis indicates that 
as in Nerets the egg plays the active réle in the penetration of the 
spermatozoon for it actually draws in the passive spermatozo6n. 
After sea-water treatment I have not, as mentioned above, found 
the early stages of penetration in eggs fixed at three minute 
intervals after insemination. Either the sperm penetration is 
unlike that after normal insemination or penetration takes place 
with extreme rapidity. In the later stages of penetration it is 

1 Lectures to classes in embryology, Woods Hole, Mass. 

2 Apparently Buller did not realize that he obtained iso-agglutination of sea- 
urchin sperm, although he speaks of the sperm forming “‘balls’’ and although the 
phenomena of agglutination were well known at that time. Landsteiner the year 
before had secured sperm agglutinating sera. Nougouchi’s work on Nereis sperm ~ 
is of interest: he demonstrated agglutination with snake venom. The experiments 
of Schiicking, von Dungern, de Meyer, and others are well known. An observation 
of Walker’s (’10) is likewise worthy of mention—the agglutination of the sperm of 


the rat when mixed with the seminal vesicle secretion of the same animal. 
Chemotaxis of sperm has been demonstrated for mammals—see for instance, Low. 


FERTILIZATION IN PLATYNEREIS MEGALOPS. III 


clear that the spermatozoa behave in abnormal fashion even 
granting that I may have overlooked the amphiaster. The 
evidence seems to indicate that after sea-water treatment the 
egg lacks the power to engulf the sperm. However, whatever 
the method of penetration one point is beyond contradiction: 
these washed eggs never cleave. 

The observations agree with those of Lillie (14) who notes 
* that some unpublished observations in the case of Nereis show 
that “‘if the cortical changes be induced by artificial means there 
is a brief period in which insemination of the eggs may be followed 
by penetration of the spermatozo6n, but without causing cleavage 
of the egg.’’ Miss Allyn found that after KCl treatment of the 
egg of Chetopterus, the spermatozo6n may enter but its behavior 
is not normal. Kite (quoted from Lillie ’14) finds that sper- 
matozoa injected into star-fish eggs never give cleavage. ji 

In these cases, the interpretation must be that the ‘‘fertiliz- 
able”’ condition of the egg has been destroyed through loss of 
fertilizin before insemination. In the same way sperm may pene- 
trate unripe eggs as Hempelmann has shown for Saccocirrus (so 
too, von Hofsten for Otomesostoma and Shearer for Dinophilus gyro- 
ciliatus). Two yearsago I found that eggs from Nereis limbata just 
before transformation into the heteronereis phase would not fer- 
tilize with active sperm either from the nereis or heceroneris form. 
Moreover, eggs from metamorphosing worms kept for several 
weeks in the laboratory although apparently ripe would not 
fertilize on insemination during the dark of the moon. At full 
moon, sometimes but a few days later, eggs from the same animal 
would fertilize and develop into larve which were kept for weeks. 
We may assume in these cases that the fertilizin is either absent 
or is unavailable. Penetration, therefore, may take place before 
the fertilizable period is reached as well as after it has been 
passed, but the egg is not capable of fertilization. 

3. Apposition of the germ nuclei of Platynereis after sea-water 
insemination may ensue, but never cleavage. After the loss of 
the fertilizing substance, then, the normal fertilization process 
may be closely simulated even to the point of the copulation of 
the pronuclei but development never goes beyond this point. 
In short, the normal fertilization process demands at the very 


Tpn2 E. E. JUST. 


outset the fixation by the spermatozo6n of the escaping fertilizin. 
This takes place in Platynereis almost instantaneously (see page 
93) but brief though this phase may be it cannot be omitted. 

The experiments with Nereis sperm and agents of artificial 
parthenogenesis demand explanation. Eggs such as those of 
echinids used in cross fertilization (Loeb, Tennent, Baltzer, 
Herbst, etc.) or in artificial parthenogenesis when subjected to 
treatment are so subjected with their substances intact. They 
are normally shed in sea-water for insemination and the sea-water 
does not for some time destroy their fertilizing power. Platy- 
nereis eggs when subjected in sea-water to foreign sperm or to 
various agents have lost something through the action of sea- 
water. This very ‘‘something’’ is necessary for artificial par- 
thenogenesis and, moreover, as shown above (for Nereis also) 
must be present in greater quantity than necessary for fertiliza- 
tion. I am emboldened further to suggest that eggs normally 
inseminated in the ovocyte stage yield to parthenogenetic agents 
only with difficulty because they lose fertilizin at the impact of 
the first stimulus—chemical treatment, shock, etc. Sperm 
alone, in most cases, are strong enough by fixation of the fertilizin 
to carry such eggs through their dual phase—maturation and 
fertilization. Whether by sperm, then, or by artificial agents, 
the initiation of development is fundamentally the same.1 The 
egg plays the leading rdéle; it needs but to have its fertilizin ac- 
tivated in order to develop. 

The observations on Platynereis were rendered less difficult 
because of the study of the maturation and fertilization in Nereis. 
For this study I was fortunate to be able to supplement my own 
slides with two series lent me by Professor F. R. Lillie. It isa 
genuine pleasure here to acknowledge my further indebtedness to 
him for his many suggestions and for his stimulating interest in the 
Platynerets studies begun at his suggestion and under his direction. 


MARINE BIOLOGICAL LABORATORY, 
Woop’s Hote, Mass. 


1] think that Martin Jacoby’s experiments support this view. He found (Bizo- 
chem. Zeit., 20, 333-335) that serum from rabbits into which eggs had been injected 
showed an increased power to stimulate parthenogenetic development of the eggs. 
He also found (ibid., pp. 336-343) that an enzyme which may be extracted from 
sperm and from eggs after sperm penetration may be got from parthenogenetic 
eggs. 


FERTILIZATION IN PLATYNEREIS MEGALOPS. 113 


LITERATURE CITED. 


Allyn, Harriett M. 
I2 The Initiation of Development in Chaetopterus. BioL. BULL., 24. 
Budington, R. A. 
932 The Influence of Magnesium Chloride on the fertilizing Potential of Sperma- 
tozoa. Science, N. S., 35. 
Buller, A. H. R. 
200 «6h fertilizing Process in the Echinoidea. Report, British As. Ad. of Sci. 
202 Is Chemotaxis a Factor in the Fertilization of the Eggs of Animals? OQ. J. 
M.S., 46. 
Dungay, N. S. 
’13, A Study of the Effects. of Inquiry upon the fertilizing Power of Sperm. 
BIOL. BULL., 25. 
Frédericq, L. 
-%04 Sur la concentration moleculaire du sang: et des tissus chez les animaux 
aquatiques. Arch. de Biol., 20. 
Gemmil, Jas. F. 
700 ©On the Vitality of the Ova and Sperm of certain Animals. Jour. Anat. and 
Phys., 34. 
Garrey, W. E. 
204 Osmotic Pressure of Sea-water and of the Blood of marine Animals. BIOL. 
BULL., 7. 
Hempelmann, F. 
’12 Die Geschlechtsorgane und -zellen von Saccocirrus. Zoologica, Heft 69. 
Hirokawa, Waichi 
09 Ueber den Einfluss des Prostatasekrete und der Samenfliissigkeit auf die 
Vitalitat der Spermatozoen. Biochem. Ztschr., 19. 
Jacoby, M. 
’r0 Ueber das Verhalten der Sperma- und Eienzyme bei der Befruchtung und 
ersten Entwicklung. Biochem. Ztschr., 26, 336-343. 


Just, E. E. 
’14 Breeding Habits of the heteronereis form of Platynereis megalops. BtOL, 
BULL., 27. 


’r5a The Morphology of the normal Fertilization in Platynereis megalops. 
Jour. Morph., in press. 
*15b = Initiation of Development in Nereis. Biov. BULL., 28. 
Koltzoff, N. K. 
09 ~©60Studien iiber die Gestalt der Zelle, ii. Arch. f. Zellforsch,, 2. 
Landsteiner, K. 
’99 «©6Zuur Kennteris der spezifisch auf Blut kérperchen Wirkenden Sera. Cent. f. 
Bak., 25. 
Lefevre, G. 
202 ~=6Artificial Parthenogenesis in Thalassema mellita. Jour. Ex. Zool., 4. 
Lillie, F. R. 
Ir Studies of Fertilization in Nereis. I. The Cortical Changes in the Egg. 
Il. Partial Fertilization. Jour. Morph., 22. 
’72 III. The Morphology of the normal Fertilization. IV. The Fertilizing 
Power of Portions of the Spermatozoon. Jour. Ex. Zool., 12. 
’13a V. The Behavior of the Spermatozoa of Nereis and Arbacia with special 
Reference to Egg-extractives. Jour. Ex. Zool., 14. 


114 E: Esujust: 


°13b The Mechanism of Fertilization. Science, N. S., 38. 

’r4 Studies of Fertilization, VI. The Mechanism of Fertilization in Arbacza. 
Jour. Ex. Zool., 16. 

Lillie, R. S. 

08 Momentary Elevation of Temperature as a Means of producing artificial 
Parthenogenesis in Star-fish Eggs and the Conditions of its Action. Jour. 
Ex. Zool., 5. 

’72 Certain Means by which Star-fish Eggs naturally Resistant to Fertilization 
may be rendered normal and the physiological Conditions of this Action. 
BIOL. BULL., 22. 

Loeb, J., Fischer, M., and Neilson, H. 

70x Arch. f. d. Ges. Physiol., 87. 

Loeb, J. 
708 Ueber die Entwicklungserregung unbefruchteter Annelideneier (Polynoe) 
mittels Saponin und Solanin. Pfliiger’s Arch., 122. 
Loeb, J., and Wasteneys, H. 
’12 ©Fertilization of the Eggs of various Invertebrates by Ox Serum. Science, 
36. 
Liéw, Otto 

202—’03 + Die Chemotaxis der Spermatozoen in weiblichen Genitaltract. Zitz. 

der Kaiserlichen Math-Naturwissen. Classe. 111-112. 
Mathews, A. P. 

701 Artificial Parthenogenesis produced by mechanical Agitation. Am. Jour. 

Phys., 6. 
de Meyer, J. 

"11 Observations et Experiénces relatives a l’action exercée par des extraits 

d’oeufs et d’autres substances sur les spermatozoides. Arch. de Biol., 26. 
Schiicking, A. 

’03 Zur Physiologie der Befruchtung, Parthenogenese, und Entwicklung. Arch. 

f. d. Ges. Physiol., 98. 
Steinach, E. 

’94 Untersuchungen zur ver gleichenden Physiologie der mannlichen Geschlechts- 
organe insbesondere der accessorichen Geschlechtsdriisen. Arch. f. d. 
Ges. Phys., 56. 

Walker, Geo. 

’99:«€Cf Beitrag zur Kenntnis der Anatomie und Physiologie der Prostata nebst 
Bemerkungen iiber der Vorgang der Ejaculation. Arch. f. Anat. und 
Physiol. 

’10 The Nature of the Secretion of the Vesiculi Seminalis and of an adjacent 
glandular Structure in the Rat and Guinea Pig, with special Reference to 
the Occurrence of Histone in the Former. Johns Hopkins Hosp. Bull., 21. 

"11 The Effect on Breeding of the Removal of the Prostate Gland or of the 
Vesiculi Seminalis or of Both, together with Observations on the Condition 
of the Testes after such Operations on the White Rats. Johns Hopkins 
Hosp. Rep., 16. 


‘ 


SPOROCYSTS IN AN ANNELID.1 


EDWIN LINTON, 


WASHINGTON AND JEFFERSON COLLEGE, WASHINGTON, PA. 


In the summer of 1910, while at work at the United States 
Fisheries Biological Station, Woods Hole, Mass., I was told by 
-Dr. Gilman A. Drew that what were supposed to be cercarize 
had been noticed at different times associated with the annelid 
Hydroides dianthus Verrill among material being used for study 
at the Marine Biological Laboratory. 

Acting upon this suggestion I examined a large number of these 
serpulids on several dates in August of that year. Although 
much of the material was examined very minutely, the worms 
having been removed from the tubes, teased, and everything that 
even remotely resembled a sporocyst further examined, neither 
sporocysts nor cercariz were found. 

In the following summer I secured two lots of these sporocysts 
from this annelid. For the first lot, July 15, I am indebted to 
Dr. Drew, and for the second, July 21, to Miss Margaret Morris. 

In each case the single annelid was lying in a dish of sea water, 
and in the bottom of the dish there were a large number of sporo- 
cysts. These sporocysts were found to contain cercarize in 
various stages of development but no rediz. As they lay free 
in the sea water the sporocysts were for the most part white, or 
bluish translucent white. In some of them there were varying 
amounts of orange pigment of similar appearance to the abundant 
pigment in the annelid. They were short and thick, bluntly 
rounded at the ends, and more or less arcuate. In some cases 
they were curved until the ends almost touched each other. 
Many of the second lot were orange yellow, also many of them 
were actively contractile. A frequent change of shape was that 
from the characteristic short, blunt-pointed sub-cylindrical 
form to a fusiform shape with elongated and slender-pointed 

1 Published by permission of Commissioner of Fisheries. 


115 


116 EDWIN LINTON. 


ends. In this condition they were sometimes straight and some- 
times arcuate (Fig. 1). In each sporocyst there were tailed cer- 


Fic. 1. Contraction shapes of sporocysts, life. 


carie along with various stages of developing cercariz, from 
globular balls of cells, 0.04 millimeter in diameter, to cercarie, 
0.5 millimeter or more in length (Fig. 2). The anterior portion 


Fic. 2. Sporocyst showing cercarie in different stages of development. Cam- 
era lucida sketch of stained and mounted specimen. Actual length 1.04 millimeter. 


of these cercariz is sub-cylindrical and slightly tapering at the 
anterior end. It is marked off from the elongated tail portion 
by a constriction, which, in the mature cercariz, is at about the 
anterior fourth of the entire length. In other words, the tail, 
which is forked at the end, is about three times the length of the 
body. Along the dorsal aspect of the body in a few instances a 
longitudinal row of exceedingly slender spines was noted. As 
this cercaria resembled very closely a cercaria which I have 
found in the scallop (Pecten trradians), I recorded in my notes 
that it was likely that these spines, as in the cercaria from the 
scallop, are remnants of a fin-like membrane, and that the type 
represented by this cercaria is evidently near that of Cercaria 
cri tata L> Val. 


SPOROCYSTS IN AN ANNELID. 117 


On July 19, 1914, through the kindness of Dr. E. J. Lund, I 
had the opportunity of examining another lot of these cercariz 
from this same annelid. Some of these were observed to be 
covered with an exceedingly thin hyaline membrane which be- 
comes constricted at frequent intervals, the constrictions ulti- 


mately being the only part of the 
membrane that is visible. The cer- 
cariz from Hydroides, as was the case 
with those from the scallop, exhibit 
great activity, but the nature of their 
movement is different. Instead of a 
characteristic pecking motion of the 
anterior end, the cercarie from the 
annelid, occasionally, after lying mo- 
tionless for a time, perform exceedingly 
rapid wriggling movements. The an- 
terior end of the body is provided 
with a short, retractile boring appa- 
ratus, shown protruded in Fig. 3. 

In addition to the various stages of 
developing cercariz, other structures 
were observed in these sporocysts. 
With transmitted light these appeared 
to be granular, but with reflected 
light, or, with high magnification, they 
appear to consist of minute oil drop- 
lets, at least in part. In some cases 
they were distributed rather uniformly 
near the surface, in others they were 
massed in the central region. 

The number of these sporocysts is 
very great. In the first lot it was 
estimated that there were between 900 
and 1,000 sporocysts in the dish with 
the annelid. After the worm had 
been lying for a few minutes in a dish 
of clean sea water to which it had been 


Fic. 3. Cercaria with fin-like 
crest. The thin investing mem- 
brane is indicated at the poste- 
rior end. Length of body 0.17 
millimeter, length of tail, 0.52. 


Fic. 4. Anterior end of a cer- 
caria with a crest of slender, 
cilia-like spines. 


transferred, a number, 25 or more, of sporocysts made their 
appearance on the bottom of the dish. The worm was then _ 


118 EDWIN LINTON. 


placed in corrosive-acetic and afterwards sectioned. The sections 
show an immense number of sporocysts (Fig. 5). For the most 


Fic. 5. Transverse section of Hydroides dianthus showing sporocysts embedded 
in the body wall. a, intestine. 


part they are distributed ventrally in the inner portion of the 
body wall, although a few lay among the muscles near the 
exterior, and a few in the epidermis. If I interpret the sections 
correctly, the sporocysts escape from the ventral side of the 
serpulid, where the body wall is comparatively thin, and where 
the sporocysts are in greatest numbers. 

There is considerable variation in the size of the sporocysts. 
The largest noted was 0.70 millimeter in length and 0.28 milli- 
meter in diameter; the smallest 0.17 in length and 0.10 in di- 
ameter. In like manner the cercarie varied in length, but the 
length of 0.12 millimeter for the anterior portion, and 0.36 for 
the tail, or 0.48 millimeter for the whole length, is not far from 
the usual length of a mature cercaria. One cercaria, living, had 
the following dimensions: Length of body 0.17 millimeter, breadth 
0.04; length of tail 0.52, breadth 0.02. 

What were interpreted to be striated muscle fibers were 
noticed in the tails of living cercarie (Fig. 3). These fibers 
extend diagonally backward and inward from the exterior to 
the median line. They were about 0.0017 millimeter in diameter, 
and what appeared to be cross striations were plainly visible 
with a Zeiss D objective. Under an oil immersion lens their 
resemblance to striated muscle was evident. é 


OF THE 


_ Marine ‘Biological ‘Laboratory / 


_ Woops HOLE, MASP. 


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See eO RS earls 


eC Oo  UNEAREH, 1915 


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Sutnertann, G. Woo. | Nuclear hinge) TH ibe amen =: 
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4 Ricans, Ao AND a Note on the Effect - ne Radiation on : 
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“Can a Siele “Spermatozoon Initiate 
Development on. Arbacta? a SESS ea 


USiidies on the Physiology 


154 ea 


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"MARINE BIOLOGICAL. LABORATORY 


Py sae 


i a cy PRINTED AND isSUED BY 
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LANCASTER, PA. 


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Entered Oetober 10, 1902, ‘at Lancaster, Pa., as second- clase mutter, 
under Act of Congress, of J Aly, 16, 1894. 


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Vol. XXVIII. March, 1915. No. 3 


PPIOLOGICAL BULLETIN 


NUCLEAR CHANGES IN THE REGENERATING SPINAL” aati 


CORD OF THE TADPOLE OF RANA CLAMITANS."’ 


GEORGE FRED SUTHERLAND. 


CONTENTS. 

I. Statement of the Problem...........-----2-..-2-eeee eects eet eeeee IQ 
Il. Material and Methods....>......... 1 se OM TERN ES, 3c SOAP ana 121 
Tit, OPSAAVAOMS. obo nGcecu ss ode oc od Go dn GS bacco ocsopogHoeawan op UO ObS 122 
I. Degenerative Changes after an Operation..........-..---+-+-+--:- 123 

2. Enlargement of Nuclei.............-.0 eee ee tee ete 129 

3. Temporary Partial Closing of the Spinal Cord............------- I29 

A, (Call IDIOM 65 oconneccounboogeooccouan ace scos boon GHD e DoE DG I31 

IW) IDYSGURSH elite Gio 6 ables voocs Ob baw Udo Be mine obiG ou 5.b dln o'a cs lolgum pl ciceo mrcro- 134 
1. Amitosis and Fragmentation.............-.-. eee eee eee eee 134 

2. Appearance of Leucocytes......-- 1... 222+. eee eee ett 135 

3. Temporary Closing of the Spinal Cord.............-...--.++-05. 136 

4. Rate of Division. Amitosis vs. Mitosis.......-....+++++-++-++--- 137 

Wi, QWMINIAT) Goon oc coos oancn oon geo uGaddh ode ooguogn sD oooooSECoadGaS 138 
Wil, Ihilbiliqereeyplinys 500 aco cocade anon be godaguadandsanoudoocospevKd Dour 139 


I. STATEMENT OF THE PROBLEM. 


The present paper gives the results of an histological study of 
the early stages of regeneration in the spinal cord of the frog 
tadpole, Rana clamitans. It deals especially with the degen- 
erative nuclear changes immediately following the operation, 
and the phenomena of nuclear division in the formation of the 
new organ. 

Fraisse (1885) studied these stages in several vertebrates in 
order to discover the origin of the regenerated tissues, and pre- 
sented the following conclusions which may be used as a basis 
for a further detailed study. 

“‘t. Sowohl bei Amphibien wie bei Reptilien sind verletzte 
Gewebe nur im Stande, wiederum gleichartig Gewebe zu erzeugen. 
Die Leukocyten iibernehmen bei der Gewebsbildung nur die 

1Contribution from the Zoological Laboratory of the University of Illinois, 
No. 37. 

119 


I20 GEORGE FRED SUTHERLAND. 


Function der Ernahrung; ausserdem nehmen sie zerfallende 
Gewebsproducte auf und assimiliren dieselben, um sie an anderen 
Orten wieder zu deponiren. Niemals werden sie selbst zu 
fixen Gewebszellen, weder in der Bindesubstanz noch sonst wo. 

‘“‘2, Sammtliche der in Frage kommenden Gewebe der Am- 
phibien und Reptilien sind im Stande, sich zu regeneriren; 
entweder direct aus ihren Elementen, oder aus einer Matrix, so 
lange diese Matrix unverletzt ist. Als Matrix fiir die Epidermis 
ist das Rete Malpighii, fiir das centrale Nervensystem das 
Epithel des Centralcanales, fiir die Muskulatur die Muskel- 
kérperchen zu betrachten. 

‘““2, Zuerst regeneriren sich Epithel und Bindegewebe; beides 
scharf getrennt, urspriinglich aus gleichartigen Zellen bestehend, 
die sich spater differenziren.”’ 

There remains the further problem of the stages in the process 
by which the old organs at the cut surface replace their lost 
parts. Two distinct kinds of changes take place in this process, 
(1) degenerative and (2) regenerative. First the injured cells 
at the cut edge degenerate. Then follows regeneration proper, 
or the formation of the new organ from the remaining elements 
of the old. 

There are three ways in which regeneration proper might 
take place. (1) The cells at the cut edge of each organ by 
dividing might extend outward, and in time form the completed 
organ: (2) the cells in front of the cut edge might wander back- 
ward; and (3) the cells in front of the cut edge might divide in 
situ and push backward the more distal cells. These possible 
methods of regeneration will be made clearer by a diagram of 
that part of the hollow neural tube extending forward from the 
cut (Fig. 1). If (1) (division of cells at the cut edge) were the 
method of regeneration, we should find after the operation that 


ANTERIOR 1 Cur 
1] 
4 


Fic. 1. Diagram, explained in the text. 


the cells at the cut surface A, or from A to C, are dividing rapidly 
while from C to B about the normal number of cells is dividing. 


NUCLEAR CHANGES IN RANA CLAMITANS. I21 


If (2) (migration of cells) were the method, we might find no 
dividing cells at all, but should expect to find that the cells from 
B to A or possibly only from D to A are turned with their long 
axes parallel to the longitudinal axis of the spinal cord as if moving 
toward the cut end. If (3) (division of more anterior cells in situ) 
were the method, we should expect to find dividing cells all the way 
from B to C or possibly concentrated in a growing zone ED. 

The present paper aims to give an account of the nuclear 
changes, both degenerative and regenerative, involved in the 
formation of the regenerated spinal cord. 


II. MATERIAL AND METHODS. 


Serial sections were made of tadpole tails killed after various 
regeneration periods. This enables one to follow the process 
from stage to stage. But to get uniform results from this method 
and eliminate individual variations, one must take tadpoles as 
nearly alike as possible at the start, operate on all at the same 
time, keep them under uniform laboratory conditions and make 
sections of several individuals at each stage. 

On October 12, 1913, seventy tadpoles of Rana clamitans, 
varying in length from 30 to 60 mm., were brought into the lab- 
oratory. Two days later they were put into individual finger 
bowls, and forty-four medium sized individuals (32-40 mm. in 
length), chosen to constitute the main series, were grouped by 
twos or threes. Those of each group were as nearly alike as 
possible and each group was treated as a unit in the time of 
operation, killing, etc. The finger bowls were placed side by 
side on a table some distance from the windows so that uniform 
conditions of temperature, light, etc., were insured. None of 
the tadpoles was fed during the course of the experiment, and 
none died from the effects of laboratory conditions. 

On October 15, the first operations were performed. Each 
tadpole was transferred from the finger bowl to a paraffin block 
and approximately one fourth of the tail was removed, with 
a sharp scalpel, at right angles to the plane of the tail. The 
animal was returned to the finger bowl and the removed part put 
into Gilson’s killing fluid. At the end of the period of regener- 
ation, the animals were again taken out onto the block and the 


122 GEORGE FRED SUTHERLAND. 


regenerated tail plus a second fourth of the normal tail was 
removed and put immediately into Gilson’s killing fluid. The 
times of killing were as follows: normal, immediately after the 
operation, I, 3, 54%, 9%, and 14 hours, and 1, 2, 3, 4, 6, 8, 9, Io, 
12, 14 and 16 days after the operation. Usual methods of 
technique were followed. Delafield’s hematoxylin and acid 
fuchsin stain the nuclei blue and the cytoplasm pink, but do not 
distinctly bring out cell boundaries. For the most part sections 
were made in the sagittal plane. 


III. OBSERVATIONS. 


The study was confined to the histology of regeneration in the 
spinal cord, since a preliminary examination showed that this 
organ of all those in the tail was best adapted for a study of the 
present problem. Fig. 2 shows by a sagittal section the spinal 


Fic. 2. Sagittal section through a part of the normal! tail, showing the spinal 
cord and its relation to the surrounding tissues. mc, spinal cord; cc, central canal; 
ntc, notocord; ct, connective tissue; pc, pigment cell. (330 diameters.) 


cord, and its relation to the surrounding tissues. Fig. 3 shows a 
transverse section of the spinal cord alone. It is a hollow tube 
which distally is formed of a single layer of cells. The nuclei are 
very near the inner border of the cells so that there is a wide 
outer zone of cytoplasm but practically no inner cytoplasmic 


NUCLEAR CHANGES IN RANA CLAMITANS. 123 


zone. At this stage in the development of the tadpole, the cells 
near the distal end of the spinal cord show little differentiation. 


Fic. 3. Transverse section through the normal spinal cord, showing the nuclei 
and the outer cytoplasmic zone. cc, central canal. (890 diameters.) 


1. Degenerative Changes after an Operation. 


When a tadpole’s tail is removed the old notocord extends out 
beyond the other tissues, and the connective tissue between the 
notocord and spinal cord:’is usually broken so that the spinal 
cord bends dorsally as in Figs. 7 and 8. A transverse cut through 
the tail leaves the various organs at the cut surface in contact 
with the surrounding medium, the water in which the tadpole 
lives. Sections of tadpoles killed immediately after the operation, 
show the direct effect of the cutting (Figs. 4 and 5). Many. 


Fic. 4. Transverse section through the end of the spinal cord immediately after 
the operation, showing deeply-staining nuclei. cc, central canal; 2m, normal nuclei; 
dn, deeply-staining nuclei. (920 diameters.) 


nuclei and cells are broken and irregular in appearance and may 
be loosened or torn apart from each other. The injured nuclei 
at the cut edge and extending forward with decreasing frequency, 
are homogeneous in appearance and take a deep haematoxylin 
stain. Undoubtedly some of the nuclei are cut, and this accounts 
for the irregularity in shape of a good many. But a good many 
others, also staining deeply, are rounded and smaller than normal 
nuclei. These may be either normal nuclei which under the 


24 GEORGE FRED SUTHERLAND. 


stimulus of the operation are contracted or compressed, or cut 
nuclei which have rounded off. These deeply-staining nuclei, 
whether rounded or irregular in shape, are smaller than normal 


Fic. 5. Sagittal section through the side of the spinal cord immediately after 
the operation, showing the deeply-staining nuclei at the cut end. dn, deeply- 
staining nuclei. (920 diameters.) 


nuclei, so it may be that the chromatin, which stains deeply, is 
condensed on account of the loss of achromatic material. 

The same assumption is borne out by the somewhat different 
appearance of nuclei in the tadpoles killed one hour after the 
operation (Fig. 6). Some are rounded as before; others are 
angular or slightly hour-glass shaped, with rather dense cyto- 
plasm extending out from the corners. If parts of the nuclear 


Fic. 6. Sagittal section through the spinal cord one hour after the operation. 
This shows the “‘contracting’’ nuclei. cc, central canal; dn, deeply-staining nuclei; 
nn, normal nuclei. (920 diameters.) 


membrane were held by the cytoplasm while the nucleus as a 
whole decreases in volume either by contraction or loss of achro- 
matin, the nuclei might present such an appearance. Moreover 
there are gradations from hour-glass-shaped to normal nuclei 


NUCLEAR CHANGES IN RANA CLAMITANS. 125 


and corresponding gradations in size and depth of stain. In 
cases of this sort there are often vacuoles or cytoplasm between 
the nuclei as if the latter had shrunken, whereas in the normal 
cord, the nuclei are so close together that no cytoplasm can be 
seen between them. These facts indicate that normal nuclei 
become deeply staining nuclei by contraction or by loss of achro- 
matic material. 

This “contraction” of nuclei seems to be caused by contact 
with the water or killing fluid, or the succession of the two, as 
well as by direct injury from the scalpel, for other nuclei which 
are in contact with the exterior only through the central canal 
show this phenomenon. In some cases, the end of a nucleus 
nearest the central canal is deeply stained and contracted while 
the other part is normal (Fig. 4). The question immediately 
arises, why does not the water or other external factor enter the 
open neural tube and cause the contraction of the inner parts of 
practically all nuclei in the spinal cord? It is probably because 
of the presence in the tube of some substance which prevents the 
ready admission of external fluids, though capillarity would have 
a similar effect. Since the sections show very little structure 
within the central canal, this content must be liquid or semi- 
liquid. However, in a number of sections there is a rather long 
narrow band of cytoplasmic material which may be the more solid 
part of a semi-liquid substance coagulated by the killing reagent. 
There are other evidences of the presence of such a liquid. The 
sections from two of the tadpoles killed one hour after the oper- 
ation show a coagulation of the outer surface of the blood plasma 
covering the wound, but over the spinal cord this coagulating 
process is delayed. The most plausible explanation seems to be 
that some cerebro-spinal fluid (compared by Barfurth to the 
cerebrospinal fluid of mammals) exerts an outward pressure 
which breaks through any slight hardening of the plasma at this 
point. Perhaps transference of the animal to a medium of 
different density, the killing fluid, aids the outburst. Sections 
of another tadpole killed at one hour show the presence of this 
coagulated plasma over the end of the spinal cord as well as over 
other parts of the tail. 

The outward pressure of a fluid would tend to push out into 


126 GEORGE FRED SUTHERLAND. 


the blood plasma any free elements such as the injured and de- 
generating nuclei with very little cytoplasm and hence little 
connection with other cells; and when this fluid breaks through, 
some of these nuclei may break off and float away. At one hour 
after the operation, broken and small rounded nuclei are seen in 


betes en 


Fic. 7. Sagittal section through the spinal cord and the surrounding region one 
hour after the operation, showing irregularly shaped, deeply-staining nuclei in 
the end of the spinal cord and in the coagulated plasma layer. dn, deeply-staining 
nuclei; pl, plasma layer; cf, connective tissue; utc, notocord. (1,100 diameters.) 


the end of the spinal cord and extending out into the hardened 
layer of the plasma, giving evidence of some force acting outward 
at this time (Fig. 7). Other evidences will be mentioned in 
describing the stages at which they appear. 


NUCLEAR CHANGES IN RANA CLAMITANS. 127 


Three hours after the operation there are fewer of the angular 
nuclei than at one hour and more of the round deeply-staining 
nuclei. The latter vary from the size of similar ones in the 
earlier stages down to fragments. Moreover some of the larger 
of these seem to be in the process of fragmentation, that is, ap- 
pearances indicating stages in direct division are seen. The 
gradation in size and depth of stain at one hour from normal 
nuclei nearly to rounded ones, and the gradation down to frag- 
ments at three hours, as well as the appearances of fragmentation, 
make it fairly clear that normal nuclei just in front of the cut 
edge may contract, become rounded, and fragment. This must 
be a degenerative process. Even finer intermediate steps are 
seen in preparations of later stages. 

Sections of one individual at this period appear very much 
like those immediately after the operation. The deeply-stained 
nuclei are similar, and the spinal cord is not covered either by 
epidermis or plasma, so that a recent outbreak of the cerebro- 
spinal fluid must have taken place. In this case a second contact 

- with the exterior has again started the degenerative process. 

At five and a half hours the spinal cord is entirely covered by 
the thickened plasma layer, in which is a group of fragmenting 
globular nuclei. In one preparation at this time, the epidermis 
has closed-in over the entire wound, and there is a series of stages 
in the degeneration of nuclei. Some are only slightly smaller and 
darker than normal nuclei; others have the angular appearance 
characteristic of nuclei one hour after the operation, while still 
others are round and fragmenting. At this stage there is another 
evidence of the presence of a cerebrospinal fluid. The plasma 
covering the end of the spinal cord is pushed outward, making a 
knob-like extension of the central canal similar to that shown in 
Fig. 8. This did not appear in earlier stages either because not 
enough cerebrospinal fluid was present, or because the plasma 
layer had not coagulated sufficiently to resist the outward 
pressure of this fluid. 

Of the two preparations of tadpoles killed after a nine and a 
half hour interval, one shows the epidermis and plasma covering 
all the wound except the neural tube; the other shows this part 
also covered. In the former, the sides of the neural tube are 


128 GEORGE FRED SUTHERLAND. 


separated as if by a recent outburst of cerebrospinal fluid, and 
deeply-staining rounded and fragmenting nuclei are seen. In 
the second preparation, the deeply-staining nuclei are all small 


Fic. 8. Sagittal section through the end ot the spinal cord fourteen hours after 
the operation. This shows the epidermal layer, the plasma layer, and the knob- 
like extension of the central canal, caused by the outward pressure of the cerebro- 
spinal fluid. ep, epidermis; cc, central canal; pl, plasma layer; ntéc, notocord. -(330 
diameters.) 


and fragmentary. In other words no more nuclei seem to be 
starting to degenerate. 

At fourteen hours, plasma and Soins cover the spinal cord 
though the plasma is pushed outward by the cerebrospinal fluid 
(Fig. 8). There are nuclear fragments in the cord and de- 
generating nuclei in the plasma. Another preparation of the 
same period shows the nerve cord still open to the exterior, as 
well as the nuclear appearance of an earlier stage. 

At twenty-four hours, only a few of the nuclei are slightly 
smaller and darker than the normal. At this time there appear 
near the end of the spinal cord, granular leucocytes containing 
pigment granules and fragments which closely resemble the 
fragments of degenerating nuclei. It may be that the leucocytes 
appear at this time and dispose of nuclear fragments. After one 
day, the degenerating nuclei are too rare to be significant. 

The degenerative process which the foregoing facts seem to 
show, may be indicated diagrammatically as follows: 

Cells directly cut —> broken nuclei — rounded nuclei —> fragments —> disposed 
of by outbreak of cerebro-spinal fluid, or by leucocytes. 


Cells just in front of those cut —> angular nuclei —> rounded nuclei—> fragments 
— > disposed of by leucocytes. 


NUCLEAR CHANGES IN RANA CLAMITANS. 129 


2. Enlargement of Nuclet. 


A few preparations of the spinal cord soon after the operation 
show plainly that the nuclei near the end, but just in front of the 
deeply-staining nuclei, are larger than those of the normal cord. 
The long axes of nuclei close to the edge were measured and com- 
pared to nuclei of the same preparation which are some distance 
forward in the old tissue (Table I.). Immediately after the 


TABLE I. 
- a Nuclear Length Nuclear Length Difference in 
Time of Regeneration. Close to Edge. in Front of Edge. Length. 
INonmall es feriensisra wae cies 7.9 7.5 4 
Immiediatelyyo. 5-2. 22 fase 10.5 7.8 Def 
TMEV Ulca ss siteises ey ave ni eoetioletecavayemere II.3 9.1 2.2 
QPMOULS EEA ome 8.6 Toe 1.3 
SG ROOULS Oia cas iota shaveroucidi suckers I2.8 10.5 Does 
QaSvWOuUnsiecs jas aries eesve wee ances 8.1 8.0 .I 
PAV OUTS tarties ks ear eee 7.6 8.3 —.7 
GEA OR Ge SOO Bones 8.2 6.6 r.6 
DECAY Sheurerie ee spice header caus a) cues Sccbe 8.1 8.0 .I 
UGLADY Sie eta estate ers el seaatnrs 8.2 8.9 —.7 
AWAY Shaver sie tetka ei siaeeed wb eine 8.4 8.2 B 
(Sy GENYES i, Sattler loners mien Re 10.7 10.4 3 


Explanation.—Each measurement recorded here is the average of the measure- 
ments of 9 or 10 nuclei. These were recorded in terms of the spaces of the ocular 
micrometer, but since one space was equal to approximately one micron (.955), 
the measurements were not transposed. 


operation and in the very early regeneration stages, the nuclei 
near the end are larger, but the difference decreases until after 
nine and a half hours it is hardly significant. This enlargement 
might be preparatory to normal division or it might be a swelling 
which is a degenerative change preliminary to fragmentation 
Since this size difference is greatest at the very beginning and 
decreases during the first day until it is no longer significant, and 
since mitotic divisions are not seen in numbers until the third 
day, the enlargement is probably an early stage in nuclear 
degeneration. 


3. Temporary Partial Closing of the Spinal Cord. 


After the degenerative process is complete and the deeply- 
staining nuclei have disappeared, the end of the nerve cord starts 
to close over. By the first day, the nuclei in the end of the cord 


130 GEORGE FRED SUTHERLAND. 


have begun to pull apart, stretching out the connecting cyto- 
plasm (Fig. 11). In general they extend toward the opposite 


Fic. 9. Sagittal section close to the edge of the central canal, showing a row of 
cells, not quite at the end, extending across the central canal. Other sections of 
the series show that the end of the cord is still open. cc, central canal; rbc, red blood 
corpuscles; Jc, leucocyte. (920 diameters.) 


wall of the central canal, thus narrowing the opening at the end. 


Some sections show pseudopod-like cytoplasmic extensions of 
the cells into the central canal as if closing were to be produced 


Fic. 10. Sagittal section through the new spinal cord six days after the opera- 
tion. bv, blood vessel; mit, mitotic figures; tc, notocord; ct, connective tissue. 
(330 diameters. ) 


<i iin eal 


NUCLEAR CHANGES IN RANA CLAMITANS. 131 


by amoeboid movement of the cells. Figure 9 shows a section 
through one side of the cord, in which one layer of cells, not 
quite at the end, is extending down into the central canal. Up 
to about six days phenomena such as these may be seen, but 
sections from six to sixteen days show that the closing is not 
completed within that period. By sixteen days the new tail is 


Fic. 11. Sagittal section through the spinal cord one day after the operation 
showing the granular leucocytes at the end of the cord, and the pulling apart of 
nuclei in the lower part of the cord. cc, central canal; Jc, leucocytes. (920 di- 
ameters. ) 


almost as long as it will become (Durbin, 1909), and the spinal 
cord reaches back close to the epidermis at the posterior end. 
Still these later preparations show the sides of the neural tube 
gaping open, and red blood corpuscles extending forward into 
the central canal of the new cord, as if the pressure of the cerebro- 
spinal fluid is not sufficient to keep them out. 


4. Cell Division. 


In an organ such as the spinal cord in which the nuclei lie 
close together, it is difficult to determine an amitotic division. 
In order to be sure that amitotic divisions do occur, one must find 
continuous stages in nuclear and cellular constriction without 
the formation of chromosomes. Because of the massing of 
nuclei, this cannot readily be determined in the normal spinal 
cord, though the slides were examined with this point in mind. 
The present study gives no evidence that normal nuclei divide 
amitotically, but stages in direct division can be seen in the 


132 GEORGE FRED SUTHERLAND. 


deeply-staining nuclei at the cut edge. Is this amitosis or 
fragmentation? Do the daughter nuclei form normal nuclei, or 
do they divide several times and degenerate? There is no de- 
finite evidence that nuclei which divide directly ever become 
normal again. But at successive stages the deeply-staining 
nuclei become smaller and smaller down to fragments, so that 
the direct division is probably a fragmentation as a part of the 
degeneration of injured nuclei. 

Mitotic divisions can easily be distinguished by the formation 
of chromosomes. All the preparations were examined and the 
distance of each mitotic division from the cut edge was measured. 
The results are shown in Table II. In the sections of the normal 
tail the number of divisions is the smallest, but since up to three 
days the mitoses are scattered and the number of individuals 
small, there is no reason for considering these mitoses anything 
but normal. During the period of degenerating nuclei, there are 
almost no mitotic divisions close to the edge. On the third day, 
the nuclei just in front of the cut edge are proliferating rapidly; 
at four days there are a few divisions past the cut; at six days 
there are almost as many divisions in the new spinal cord as in 
the old; at eight and nine days most of the divisions are in the 
new cord; at fourteen days there are scattered mitoses only, both 
in the old and new cord, and at sixteen days most of the dividing 
cells are in the very end of the new cord. If later divisions follow 
this general trend, it seems likely that the rest of the spinal cord 
will be formed by a growing zone at the tip, and until the new 
cord is complete the number of mitoses near the tip would prob- 
ably decrease gradually. | 

Fig. 12 gives the average number of mitoses in the spinal cord 
at each stage, and therefore represents the rate of growth at these 
times. On the second day there is a considerable mass of tissue 
over the whole wound, though only degenerative changes have 
been taking place in the nerve cord. Beginning about this time, 
the nuclei in the end of the cord loosen and draw apart some- 
what, stretching out the cytoplasm between them (Fig. If). 
This is apparently the first extension in length of the spinal cord. 
At three days active proliferation of cells has begun but the 
pulling apart or stretching toward the cut edge continues. Fig. 


133 


NUCLEAR CHANGES IN RANA CLAMITANS. 


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134 GEORGE FRED SUTHERLAND. 


10 (six days’ regeneration) shows the cells in one part of the cord 
stretched out to such an extent that vacuoles are left between the 


50 


40 
30 
20 


10 


12345678 91011 1213 1415 16 


Fic. 12. Curve giving the number of mitotic divisions in that part of the spinal 
cord within 3 mm. of the edge. Beyond 3 mm. the mitoses are scattered. An 
abscissa represents the period of regeneration and the corresponding ordinate gives 
the average number of mitoses found in the individuals killed at the end of that 
period. 


cells. It is during the period from four to sixteen days that most 
of the increase in length takes place, by active proliferation and 
migration of cells. 


IV. DISCUSSION. 


1. Amitosts and Fragmentation. 


Fraisse in describing the regulative process at about two days 
after the operation, says; ‘‘Bereits friiher machte ich darauf 
aufmerksam, dass am Wundrande eine starke Auswanderung von 
Leukocyten stattfindet, und dass diese es sind, welchen vor allen 
Dingen die Bildung des homogenen, lymphartigen Saumes, 
welcher zuerst die wunde bedeckt, zuzuschreiben ist. Das 
Riickenmark geht nun an meinen Schnitten bis dichte an diesen 
homogenen Saum heran, und die Elemente, welche es zusammen- 
setzen, lassen sich immerhin noch nach 24 Stunden auch an 


NUCLEAR CHANGES IN RANA CLAMITANS. 135 


diesem Saum von einander trennen, dann aber tritt eine bedeu- 
tende Wucherung von Kernen auf, und zwar scheint dieselbe 
auszugehen von den sogenannten Kérnern,! deren Inhalt véllig 
homogen und stark lichtbrechend erscheint. Durch Picrocarmin 
werden diese Elemente ebenfalls stark tingirt, und nun sieht 
man an diesen nahezu gleich grossen Kérnern Kerntheilungen, 
ohne dass jemals eine Spur von karyokinetischen Figuren con- 
statirt werden konnte, in der Weise auftreten, das der Kern oder 
die Korner sich in der bekannten Weise schuhsohlenformig 
einschntiren, und dass dann aus beiden Halften Elemente gleicher 
Art hervorgehen. Nicht nur eine einmalige Einschniirung glaube 
ich beobachten zu kénnen, sondern auch eine mehrfache, so dass 
der Kern sich bei diesem Process nicht nur in zwei, sondern auch 
in mehrere Stiicke theilen kann.” 

Fraisse discusses further the evidence that the nuclei from the 
end of the spinal cord, which are found in the lymph-like border, 
divide amitotically. This agrees with the present observations. 
But he is satisfied to show that direct division does take place. 
So far as my preparations show, there are few evidences that the 
nuclei which divide amitotically afterward become normal nuclei. 
In some of the preparations of stages at which the deeply-staining 
nuclei have almost disappeared, there are a few nuclei which stain 
only slightly darker than the normal ones, and at this time there 
are no stages between these and the fragments. These few 
slightly darkened nuclei may, then, be forming normal nuclei 
again. All other evidence points towards the conclusion that 
at successive stages, these deeply-staining nuclei become smaller 
and smaller as if fragmentation or repeated direct division, is 
taking place. The conclusion from these facts is that nuclei 
which have only started to degenerate may perhaps return to 
the normal condition, but that nuclei that have gone so far as to 
divide amitotically are destined to fragment. 


2. The Appearance of Leucocytes. 
Barfurth (1891), working on the regenerating spinal cord of 
the frog larva at forty-six hours and at three days, makes the 


1 K6rnern-nuclei of the gray substance, which are not present in the distal region 
of the spinal cord. 


I 26 GEORGE FRED SUTHERLAND. 


following statement: ‘‘ Die unterste Theil des regenerirten Medul- 
larrohres beherbergt in seinem Innern und zwischen seinen 
Epithelzellen zahlreiche fettig degenerirende Leukocyten; viele 
kleine und grosse Fetttropfen, die man hier iiberall findet, fiihre 
ich ihrem Ursprunge nach auf solche zerfallene Wanderzellen 
zurtick. Ausserdem finden sich hier auch viele Pigmentkérnchen» 
die wohl bei der regressiven Metamorphose der zerfallenden 
Leukocyten entstehen (Pigmententartung).” 

Barfurth figures the spinal cord of a larva of Triton cristatus 
after the sixth day of regeneration, in which these leucocytes and 
fat drops are shown. His figure is very similar to Fig. 11, which 
shows a section of a tadpole killed twenty-four hours after the 
operation. Both Fraisse and Barfurth mention particularly the 
presence of leucocytes in the early regeneration stages, but in 
the present study, leucocytes were not found in large numbers. 
Up to the end of the first day, none at all were seen close to the 
spinal cord. The earliest stage mentioned by Barfurth is that 
after a forty-six hour regeneration period, and this probably 
accounts for the different interpretation he gives of the origin 
of the “Fetttropfen’”’ or fragments. If these fragments are 
followed back into earlier stages in my sections, they become 
larger and larger and are seen to be identical with the degenerating 
nuclei. To be sure, the leucocytes when they first appear in the 
spinal cord region contain what might be called fat drops, but 
is it not more reasonable to suppose that the leucocytes which are 
present at this time dispose of the fragments of injured spinal 
cord nuclei? 


3. Temporary Closing of the Spinal Cord. 


Barfurth describes the closing of the spinal cord at three days 
by means of cytoplasmic extensions of the cells, such as were 
seen in the preparations used in the present study. ‘Der sich 
wieder ansammelnde Liquor cerebrospinalis driickt nun auf die 
neugebildeten, noch wenig resistenten untern und seitlichen Theile 
des Rohres, und treibt sie kolbenartig auseinander. Die Zellen 
passen sich einstweilen durch ihre Lagerung diesem Druck an 
und behalten spater diese Lage noch eine Zeit lang bei.’’ Bar- 
furth mentions this as a temporary closure of the spinal cord, so 


NUCLEAR CHANGES IN RANA CLAMITANS. 137 


his later preparations evidently show the cord again open. The 
regenerated spinal cord at sixteen days has almost reached its 
maximum length, but it is not yet closed. Whether or not the 
completely regenerated spinal cord is open at the end or closed as 
in the normal tail cannot be answered by the present study. 


4. Rate of Division. Amitosis versus Mitosis. 


Durbin (1909), in analyzing the rate of increase in length 
throughout the regenerative process in the tail of Rana clamitans, 
distinguishes four periods. ‘The operation was followed by an 
interval of low rate, succeeded by one of rapidly increasing rate, 
then by one of rapidly decreasing rate and finally an interval in 
which the rate gradually approaches zero. The first low period 
is explained by a combination of two factors, (a) the shock of the 
injury, and (6) the formation of a cap of embryonic cells which is 
to serve as a basis for the more active regeneration. The second 
or period of rapidly increasing growth is the one in which prac- 
tically all the cells in the new part are undifferentiated and 
rapidly dividing. The third and fourth periods are explained by 
the appearance of differentiation, which lessens the number of 
dividing cells.” 

Fig. 12, based on the number of mitotic divisions in the spinal 
cord, shows these same periods. The initial period of low rate 
covers the first two days; that of rapidly increasing rate includes ~ 
the third to ninth days; the period of rapidly decreasing rate 
extends from the tenth to sixteenth days, and the period of 
gradually decreasing rate, though not covered in the present 
work, would undoubtedly extend on from about sixteen days. 
In the light of this histological study, a somewhat different inter- 
pretation might be given to the initial period. It is during these 
first two days that degeneration of the injured cells is taking 
place. Though at this time a cap of undifferentiated cells is 
being formed over the wound, the spinal cord does not participate 
in the formation of this cap, nor is any such cap formed at the 
end of the spinal cord. Since the spinal cord cells in this part of 
the tail are so slightly differentiated, the new cord is formed from 
the old without the separation of a group of special embryonic 
cells. 


138 GEORGE FRED SUTHERLAND. 


The similarity of the rate curves based on a counting of the 
mitotic divisions with that based on the amount of tissue formed 
at each period, seems to be significant. It shows that the rate of 
tissue formation is closely correlated with the number of mitotic 
divisions. Considering amitosis, this may be interpreted in 
one of two ways—(1) either the number of amitotic divisions is 
similarly correlated with the rate of growth so that the total 
number of divisions both mitotic and amitotic, gives the same 
form of curve as the mitotic divisions alone, or else (2) amitotic 
divisions are not numerous enough to be significant. The former 
explanation is improbable. The nuclear conditions producing 
mitotic division are probably different from those producing 
amitotic division. Different cells in the same region may divide 
by different methods, but it is very improbable that the conditions 
producing one form of division would increase and decrease in 
influence at the same rate and the same times as those producing 
the other form. Moreover, in the present study, no examples 
of direct division,were seen exceptin the degenerating, fragmenting 
nuclei. This similarity of the rate curve of mitotic divisions to 
the rate curve of growth is evidence, other than the negative 
observational evidence, supporting the view that amitotic division 
is not important in the formation of this organ by regeneration. 


V: SUMMARY. 


1. The regenerating spinal cord of the frog tadpole has been 
studied histologically in order to learn the mechanism, or the 
stages in the process, by which the new cord is formed from the 
old. 

2. During the first day after the operation, injured nuclei in 
the end of the spinal cord degenerate. ‘There is first a decrease 
in size, by contraction or loss of achromatin, and then a frag- 
mentation of these degenerating nuclei. The fragments may be 
carried away either by the outbreaking of a cerebrospinal fluid 
or by leucocytes which appear at this time. These fragments 
are parts of disintegrated spinal cord nuclei and not of leucocytes. 

3. From the second to the sixth days there is a temporary 
partial closing of the neural tube, probably by migration of the 
cells near the end. 


ee 


NUCLEAR CHANGES IN RANA CLAMITANS. 139 


4. The new cord is formed by the cells of the old cord near the 
cut edge, by mitotic division and migration. 

5. The number of mitotic divisions at different periods is 
proportional to the rate of regeneration at those periods as de- 
termined by Durbin. Amitotic division, if it occurs, is not 
important in the formation of the regenerated organ. 

6. There is no observational evidence from this study that 
amitotic division does occur in normal regenerating spinal cord 
cells. 

This work was carried on under the direction of Dr. Charles 
Zeleny. His suggestion of the problem, and constant interest 
in its progress are sincerely appreciated. 


VI. BIBLIOGRAPHY. 
Barfurth, D. 
’91 Zur Regeneration der Gewebe. Archiv fiir mikroskopische Anatomie, Bd. 
37, Pp. 406-491. 
’03 Die Erscheinungen der Regeneration bei Wirbeltierembryonen. Handbuch 
der vergleichenden und experimentellen Entwickelungslehre der Wirbel- 
tiere. Bd. 3, Teil 3. 
Boring, A. M. 
705 Regeneration in Polychoerus caudatus. Part II. Histology. Jour. 
Exper. Zoél. Vol. 2, No. 3. 
Child, C. M. 
706 Contributions toward a Theory of Regulation. I. The Significance of the 
Different Methods of Regulation in Turbellaria. Arch. f. Entwicklungsm. 
der Org., Bd. 20, p. 380. 
Durbin, M. L. 
2709 ©=An Analysis of the Rate of Regeneration Throughout the Regenerative 
Process. Jour. Exper. Zoél., Vol. 7, No. 3. 
Fraisse, P. 
785 Die Regeneration von Geweben und Organen bei den Wirbelthieren, 
besonders bei Amphibien und Reptilien. Kassel und Berlin, 1885. 
Morgan, T. H. 
700 Regenerationin Planarians. Arch. f. Entwicklungsm. der Org., Bd. 10, p. 58. 
’or Regeneration. New York. 
Morgulis, Sergius : 
’10 Is Regeneration a Repetition of the Ontogenetic and Phylogenetic Proc- 
esses? Amer. Nat., Vol. 44, p. 92. 
Stevens, N. M. 
?o0r Notes on Regeneration in Planaria lugubris. Arch. f. Entwicklungsm. der 
Org., Bd. 13, p. 396. 
701 Regeneration in Tubularia mesembryanthemum. Arch. f. Entwicklungsm. 
der Org., Bd. 13, p. 410. 
’07 Histological Study of Regeneration in Planaria simplicissima, Planaria 
maculata, and Planaria morgani. Arch. f. Entwicklungsm. der Org., Bd. 
24, DP. 350. 


NOTE ON THE EFFECT OF X-RADIATION ON 
FERTILIZIN.! 


A. RICHARDS AND A. E. WOODWARD. 


The observations of one of the writers that x-rays would 
produce changes in the activity of certain enzymes suggested 
that these rays might perhaps be effective in bringing about 
changes in the action of cell extractives, particularly of fertilizin, 
the descriptions of which by Lillie and Glaser appeared at the 
beginning of the summer. The opportunity was presented to 
test this suggestion during the past summer at Woods Hole, 
since the one of us carried on studies on the effect of x-rays on 
some marine eggs and the other continued the work on fertilizin 
of Arbacia begun by Glaser. This note gives a summary of the 
results. It is realized by the writers that the study is by no 
means a complete one, but it is believed by them that publication 
is justified in view of the facts that the experiments give clear 
evidence on the main point under investigation and that there 
is at present no prospect of opportunity for their further work on 
the problem. } 

In taking up these experiments the writers felt that if it should 
be shown that x-radiation influences the activity of the cell 
extractive called fertilizin, that fact would be of interest from 
several view points: (1) without regard to the nature of fertilizin 
or its role in the fertilization of the egg, it is a substance derived 
from the eggs which has the property of being definitely modified 
by those external agents of which experimental use may be made; 
(2) in cell extractives, of which fertilizin is an example, there is a 
basis for the action of x-rays upon living cells, and doubtless the 
marked effects of the rays upon tissues is partially due to such 
action; (3) the modifiability of its activity by radiation is an 
interesting property of fertilizin; (4) this property may serve as a 
point in determining the relation of fertilizin to enzymes. 

1 Contribution from the Marine Biological Laboratory at Woods Hole, and from 


the Zoology departments of the University of Texas (No. 123) and of the University 


of Michigan. 
140 


EFFECT OF X-RADIATION ON FERTILIZIN. I41I 


The methods used in these experiments were largely those of 
Lillie and Glaser. The solution used as a standard was prepared 
according to the method of Lillie by “adding to a certain number 
of ‘dry’ eggs, double their volume of sea-water, and with occa- 
sional slight agitation allowing ten minutes to elapse. At the 
end of this time the ova were precipitated by 100 revolutions of 
the centrifuge and the supernatant fluid, a clear, golden liquid 
in the case of Arbacia,’ was decanted, (Glaser, 14a). The 
agglutination of fresh sperm in suspension by fertilizin in both 
control and radiated solutions, was tested by the unit concentra- 
tion method of Lillie, of which he says, (130) ‘The agglutination 
reaction of the sperm in the presence of this substance (Arbacia 
fertilizin) is, as noted in previous studies, reversible, and the 
intensity and duration of the reaction is a factor of the concen- 
tration of the substance. The entire reaction is so characteristic 
that it was possible to arrive at a unit by noting the dilution at 
which the least unmistakable reaction was given. This was fixed 
at about a five or six-second reaction, which is counted from 
the time that agglutination becomes visible under a magnification 
of about 40 diameters until its complete reversal. The unit is 
so chosen that a half dilution gives no agglutination of a fresh 1 
per cent sperm suspension.” Further details are given in his 
recent paper (’14, pp. 526-528). One can best observe the details 
of the reaction with the low power of the microscope. The 
sperm suspension is mounted under a cover glass and the drop 
of fertilizin added at the edge of the suspension by means of a 
pipette. The entire process is observed through the microscope, 
and the time elapsing before the complete reversal of the reaction 
is carefully noted by means of a stop-watch. Thus it was 
possible to determine the degree of activity of a given sample of 
fertilizin, and by comparing radiated and non-radiated solutions, 
to measure the effect of the radiation by x-rays. 

Another possible method of studying the effect is suggested 
by the fact that fertilizin can be used to bring about the parthen- 
ogenetic development of Arbacia eggs, the so-called auto-parthen- 
ogenesis. The efficacy of fertilizin before and after radiation 
in bringing about auto-parthenogenesis is a measure of the action 
of the radiation on it. 


142 A. RICHARDS AND A. E. WOODWARD. 


In all the experiments with sperm it has been our policy to use 
only data from clear cut reactions in which the beginning and the 
end of the agglutination were definitely marked. Precautions 
were taken to see that the sperm suspension was fresh and clean. 
Lillie has shown that both of these factors are important, for an 
old suspension becomes inactive and the presence of impurities 
such as blood acts as an inhibitor of the reaction. 

Previous experience (Richards, 14) has shown the radiations 
to be of three kinds in relation to their effect on enzymes depending 
on duration, intensity and distance of the object from the x-ray 
tube; namely, accelerative, non-effective, and inhibitive. Under 
the conditions which usually prevailed in these experiments, a 
short exposure, of about 2 minutes, is accelerative; an exposure 
of about five minutes is non-effective; and one of longer duration 
becomes inhibitive. In view of these facts, similar exposures of 
fertilizin were made and the resulting activity tested as already 
explained. 

In a preliminary experiment on July 8 the following figures were 
obtained as the average of a number of readings of the time 
elapsing before the complete reversal of the agglutination reaction 
after short and long radiation of fertilizin. The fertilizin solution 
used was about 2 per cent. standard strength (in this early ex- 
periment the strength was not accurately determined, but it is 
not strictly necessary under the conditions of this test that it 
should be known exactly). For the control, non-radiated solu- 
tion the average reaction time was 32 seconds; for the 2-minute 
radiation the average time was 33 seconds; and for the 15-minute 
minute radiation it was 23% seconds. This solution was then 
diluted to one-half and these figures obtained : Control, 19 seconds; 
2-min. radiation, 20 seconds; 15-min. radiation, 16 seconds. 
This experiment is incomplete and the differences lie nearly within 
the limits of variation, but they suggest definitely that the short 
radiation rendered the fertilizin slightly more active (that is, 
enabled it to hold the sperm in agglutination longer), and the 
long radiation caused it to be less active than the control. More 
decisive data would have been given had the dilutions been 
continued to unit concentration, a fact which led to the adoption 
of that method in subsequent experiments. 


EFFECT OF X-RADIATION ON FERTILIZIN. 143 


In another experiment (July 14) a 1/50 dilution (2 per cent. 
standard) of Arbacia fertilizin was used. It was separated into 
four parts, of which one (Sc) was kept as a control solution, one 
(S2) was radiated 2 minutes, one (S5) five minutes, and the last 
(S7) seven and a half minutes. The results of these solutions 
when tested for their agglutination time at successive dilutions 
to unit concentration are given in the following table. %Sc 
means control solution diluted to one-half; 14 Sc, diluted to one- 
fourth, etc. The difference between two successive reaction times 
is marked d. Unit concentration is indicated by the asterisk(*). 


TABLE I. 
Succes-| Reac- Succes-| Reac- Succes-| Reac- Succes-| Reac- 
sive Di-| tion | Valuelcive Di-| tion |Value|sive Di-| tion |Valuelcive Di-| tion | Value 
lutions.| Time, | f 2: |lutions.| Time. | Of 2 |lutions.| Time. | Of 2: | lutions.| Time. | of 2: 
Se. |34 sec. S2 |37 sec. S5 /34 sec. S7 |29 sec. 
2 Sc. |22 sec.| 12 4 S2 |23 sec.| 14 | 4S5 |27 sec.| 7 3% S7 |22 sec.) 7 
¢ Se. |17 sec.| 5 %S2 |15 sec.| 8 | +Ss5 |19 sec.| 8 487 |17 sec.| 5 
4 Se. |r0 sec.| 7 $ S211 sec.| 4 | $S5 |12 sec.| 7 %S7 |12 sec.| 5 
Te Sc. |4-5 
sec.*] 5 |a6S2/ 7 sec.| 4 | asS5 |5 sec.*| 7 | isS7/8 sec*| 4 
gx S2 |4 sec.*!| 3 sz $7 |0 sec. 


Inspection of this table shows that the activity of S2 was 
increased by the short radiation, for five dilutions were required 
to reduce it to unit concentration, whereas that state was reached 
in four dilutions in the other three solutions; also the full strength 
of this solution held the sperm in agglutination longer than did 
that of the control, 37 against 34 seconds. In other words, Sc 
was 800 units agglutinating strength, S2 was 1,600 units, S5 and 
57 were each a little over 800 units, and much below 1,600 units 
strength. (Lillie, ’14, p. 527.) 

The number of dilutions required in S5 was the same as in Sc 
and the sperm were agglutinated the same time by both solutions. 
This is in line with the previous experience that a radiation of 
about five minutes’ duration under the conditions of these experi- 
ments is non-effective. However, these figures give an additional 
fact of possible significance which has not been entirely confirmed 
by other experiments either on fertilizin or on enzymes such as 
pepsin. If d represents the differences between the number of 
seconds required for the reversal of the reaction by successive 
dilutions, its value in S5 is practically a constant, 7; but in Sc and 


144 A. RICHARDS AND A. E. WOODWARD. 


S2 it begins as a large number and decreases rapidly: in Sc its 
successive values are 12, 5, 7 and 5, while for S2 they are 14, 8, 4 
and 4. In S7 the values of d are smaller and decrease more 
slowly, being 7,5,5 and4. Thissuggests that the laws governing 
the agglutination reactions by the various solutions are of dif- 
ferent character. But in as much as this interesting result has 
not been generally obtained it is not possible to attach special 
importance to it at this time. It is given merely as suggestive. 
_ The data in the case of S7 indicate that the activity of the 
fertilizin was decreased although the number of dilutions was the 
same as in the control, because the number of seconds required 
for the reversal of the reaction at unit concentration was much 
larger than is usual; yet at a further dilution no reaction was 
obtained. Also the undiluted solution did not hold the sperm in 
agglutination as long as in the control. Furthermore, it may be 
significant that the value of d for S7, as indicated above, are 
smaller than in the case of the other solutions. 

Subsequent experiments along the same line gave similar 
results. They show clearly that radiation by x-rays is capable 
of changing the activity of fertilizin, and in general agree with 
previous work that weak radiation is accelerative and strong 
inhibitive. Some of our experiments were performed during the 
latter part of the summer at the end of the breeding season and 
there were irregularities in the results, but it is believed that these 
irregularities may be attributed to the unsatisfactory condition 
of both sperm and eggs at this season of the year and that the 
statement above gives the true effect of radiation on fertilizin. 

Also during the latter part of the summer the writers tested 
the effect of x-radiation on fertilizin with regard to its power of 
inducing auto-parthenogenesis. Due to the near end of the 
breeding season these results are not entirely trustworthy, but 
they agree fully on one point, namely, that the radiation effects 
changes in the capacity of fertilizin to induce parthenogenesis. 

On August 10 a sperm agglutination experiment was performed 
which possibly throws some light on the irregularity of the auto- 
parthenogenesis and at the same time makes the auto-partheno- 
genesis test doubtfully applicable for the radiation problem. 
This experiment gave data showing that the radiation effects 


EFFECT OF X-RADIATION ON FERTILIZIN. 145 


wore off when the fertilizin had stood for some time. If this is 
true in general it must follow that, since the fertilizin must stand 
in the parthenogenesis experiments, there would be irregularity 
in the results. 

The only tests of the effect of x-radiation on Asterias fertilizin 
were made on July 28, when the fertilizin was divided into four 
portions, as usual. One was kept for a control, one radiated two 
minutes, one five minutes, and the fourth fifteen minutes. The 
fertilizin was then put on mature Asterias eggs, which were 
allowed to stand two hours in the solution. They were then 
rinsed with sea-water and treated with hypertonic sea-water 
(50 c.c. sea water + 8 c.c. 2.5 M. NaCl) for thirty minutes, 
washed again with sea water, and allowed to stand for 12 hours. 
All four lots of eggs showed parthenogenetic development, and 
those treated with fertilizin which had been radiated 2 minutes 
had a much larger percentage of cleavages than either the 
control or the others. 

Several times Arbacia fertilizin was similarly subjected to 
x-rays and then tested for its auto-parthenogenetic effect on 
fresh Arbacia eggs. The experiments are not satisfactory, 
because in most cases eggs from the same females gave abnormal 
results when tested in other ways. The following summarizes 
the more interesting experiments. Percentages were obtained 
by counting about 200 eggs. 


TABLE II. 
Experi- Experi- Experi- Experi- Experi- 
ment I. ment II. ment III. | ment IV. ment V. 
(25) ee(B8l eal b8| oe losl ae |Se 
22/43/28 /43|e8|/42/28|45|28)43 
OG | s2t Vea | sul og| sun, oe!] saul or) sa 
Os gS O> aS Os a Os 5s Os a 
BS |O5 | SS OF | BE /OR | SOR | BE | OF 
Ol]xs ©) || es QO} +128 oO} xs © |] +2 
28 os ae ge A 
[eae POEL ebay eae Leet See eee 
Sperm control 23.8 46.6 
Fertilizin control (unradiated) |30.2| 0 | 9.5] 0 | 5.2] I |20.2] .5 115.5 
Fertilizin 2 min. radiation.....]24.3 |few ]1I4.1| I |10.8| 3 |13.5] 0 |28.5| 2 
Fertilizin 5 min. radiation....j21.6/ “‘ |17.9| 0 | 8.4] o {10.5} oO {87.2 
Fertilizin 15 min. radiation...]17.5| 0 |15.6| .5 | 9.5] 0 |13.6] o {52.1 


Since the effect of x-radiation on fertilizin seems to be similar 
to its effect on enzymes, it is of interest to note the fact that the 


146 A. RICHARDS AND A. E. WOODWARD. 


efficiency of the agglutinin contained in fertilizin, like pepsin 
(Euler, p. 132) varies with the square root of the concentration. 
If the efficiency is measured by the number of seconds the sperm 
remain agglutinated, and the concentration is measured by units 
of strength, the curves in Figs. 1, 2, and 3 are obtained for the 
readings of July 14, August 10, and August II, respectively. 
The average is shown in the dotted line of Fig. 4. If an equation 
is worked out for this curve, we obtain y? = 11x where y repre- 
sents the efficiency and x the concentration. ‘This equation is 
plotted as a solid line on Fig. 4. In the higher dilutions, of which 

a greater number of values were averaged, and where readings 
could be made more accurately, the curves coincide very closely. 
In the less dilute portion the coincidence is not so marked, but is 
still within the limits of experimental error. 

The writers are not now able to offer an opinion as to whether 
or not fertilizin has the character of an enzyme. ‘The coin- 
cidence, however, in the behavior of this substance, when 
treated by x-rays, to that of true enzymes, is indeed striking. 

While the nature and composition of fertilizin are as yet 
unknown, it is a cell-extractive which is capable of undergoing 
changes under the action of experimental agents such as radia- 
tion by x-rays. Possibly it, or its forerunner, exists in the 
egg in combination. Among the other constituents of Arbacia 
eggs, this substance stands as one which, at least in solution 
in sea water, is able to bring about certain reactions on 
the part of sperm, and these reactions are subject to experi- 
mental modification. This justifies the inference that this sub- 
stance or perhaps some similar one within the egg may be 
capable of undergoing modification in its relations to the various 
intra-cellular activities. 

In this modification we may look for the seat of part of the 
changes which are brought about in living tissues and especially 
egg cells by radiation. The Hertwigs, Packard and others have 
shown that the chromatin of such cells is affected, and there is 
good evidence that the cytoplasm as well is influenced. Changes 
in their activity have also been demonstrated in the case of 
enzymes. These experiments add still another to the list of 
substances which are affected by the action of x-rays. It is 


= Oe ee hee eee 


EFFECT OF X-RADIATION ON FERTILIZIN. 147 


probable that fertilizin is simply one example of a group of sub- 
stances which may be the object of such action (but an example 
which may be studied). It is to be noted that these experiments 
render untenable the conclusion of the Hertwigs, that chromatin 
is the chief and perhaps exclusive seat of the effects of radiation 
upon eggs. Fertilizin is a substance doubtless without mor- 
phological representation in the structure of the egg; yet it may 
suffer considerable modification from x-ray treatment. 


REFERENCES. 
Euler, H. 


’r2 General Chemistry of the Enzymes. New York. Wiley. 
Glaser, Otto 
*13, On Inducing Development in the Sea Urchin (Arbacia punctulata) 
together with Considerations on the Initiatory Effect of Fertilization. 
Science, N. S., 38, p. 446. : 
’14a A Qualitative Analysis of the Egg-Secretions and Extracts of Arbacia and 
Asterias. Biot. BuULL., XXVI. 
’14b On Auto-Parthenogenesis in Arbacia and Asterias. Biot. BULL., XXVI. 
Lillie, F. R. 
’73a Studies of Fertilization. V. The Behavior of Spermatozoa of Nereis and 
Arbacia with Special Reference to Egg Extractives. Jour. Exp. Zool., 14. 
’13b The Mechanism of Fertilization. Science, N. S., 38, p. 524. 
‘t4 Studies of Fertilization. VI. The Mechanism of Fertilization in Arbacia. 
Jour. Exp. Zool., 16. 
Richards, A. 
14 The Effect of X-Rays on Certain Enzymes. Amer. Jour. Physiol., 35. 


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CAN A SINGLE SPERMATOZOON INITIATE DEVELOP- 
MENT IN ARBACIA?! 


OTTO GLASER. 


During the summer of 1913 while making the camera lucida 
tracings on which I have based my comparisons between the 
volumes of the unfertilized and fertilized ova of Arbacia,? it 
became necessary, in order to prevent rotation on the part of the 
eggs, and the consequent necessity of readjusting the focus, to 
employ very attenuated suspensions of sperm. The result of 
the highest dilutions used in these experiments, however, gave an 
unforeseen result since the appearance of the fertilization mem- 
branes was either very much delayed, or failed entirely to take 
place. This observation suggested the idea of a mass effect of 
the spermatozoa, and the possibility that this might play a rdéle 
in normal fertilization. 

At that time I had already made observations which had con- 
vinced me that the fertilization membrane in this egg is not formed 
de novo, but is preformed in the unfertilized egg, and simply 
rendered visible by changes occurring at the time of impregnation.’ 

The mechanism through which the fertilization membrane 
becomes visible will be dealt with in detail at another time; for 
the present it is sufficient to say that the absorption of water 
plays an important réle. It occurred to me therefore that the 
prevention of this absorption and perhaps the prevention of 
fertilization itself might be possible even with the employment of 
more concentrated suspensions of sperm, if the eggs were first 
treated with Ca. As a matter of fact, it was either difficult or 
impossible to fertilize eggs so treated. The spermatozoa were 
active enough, but failed to enter, and fertilization membranes did 
not appear ‘The following protocol is typical: In a small watch 


1from the Marine Biological Laboratory at Woods Hole, and the Zodlogical 
Laboratory of the University of Michigan. 
2“*The Change in Volume of Arbacia and Asterzas Eggs at Fertilization,’ Bio- 
LOGICAL BULLETIN, Vol. XXVI, pp. 84-01. 
3“*On Inducing Development in the Sea-Urchin (Arbacia punctulata), together 
with Considerations on the Initiatory Effect of Fertilization,’ Science, Vol. 
XXXVIII., pp. 446-450. 
149 


I50 OTTO GLASER. 


crystal, 4 volumes of fairly dense egg-suspension in sea-water-+2 
volumes n CaCls. After two minutes washed in sea-water. 


12.26 insemination moderate. 
12.28 o fertilization membrane. 
UDO) a 


12.24. i 


bb bc 


Control normal. All eggs with fertilization membranes in 3-5 
minutes after insemination. 100 per cent. cleavage. Hundreds 
of eggs examined in both control and experiment. In Ca-eggs 
very few divisions. 

In connection with these experiments I noticed that insemi- 
nation with great excesses of sperm frequently led to results at 
variance with the above, for fertilization membranes appeared 
about the majority of the eggs despite the use of Ca, and these 
eggs developed. This experience strengthened my belief, not 


only in the validity of the Ca-experiments, but also in the cor- 


rectness of the original idea, namely that the number of spermato- 
zoa that come into contact with the egg may make a difference. 

Encouraged by this result, I diluted a sperm-suspension until 
only the faintest trace of opalescence remained. Several drops 
of this attenuated fluid were then drawn up into a medicine 
dropper of medium size and expelled quantitatively. If the 
dropper, which of course remained infected with sperm, was then 
used to agitate eggs in a small quantity of sea-water by carefully 
drawing the water in and expelling it several times, it was found 
that very soon a few spermatozoa had attached themselves to 
every egg. In an optical diameter, 4 to 5 sperm could easily 
be distinguished, but I awaited further changes in vain, despite 
the fact that the spermatozoa seemed to have reached the eggs, 
exhibited the usual amount of activity, and were potent in 100 
per cent. of the cases when applied in larger quantities to eggs 
of the same lot. The following experiment is illustrative: 


12.17 insemination with infected pipette. 
12.18 o fertilization membranes. 

12.19 O ty i 

12.20 
Toei 
222 
2 


-3C 


WW NN 


DEVELOPMENT IN ARBACIA. I51 


Control normal; all eggs with fertilization membranes in 3 to 5 
minutes. Experimental eggs examined at irregular intervals 
throughout the day, but no increase in the number of membranes. 

Whether the appearance of a fertilization membrane, and im- 
pregnation itself will fail to take place in other eggs under similar 
conditions cannot be predicted, and is perhaps even improbable. 
With the eggs of Arbacia punctulata however I repeated these 
tests so often that I cannot doubt the correctness of my obser- 
vations, and I therefore fail to understand Kite’s! claim that he 
succeeded in calling forth a fertilizatiot membrane in this egg 
by means of a single spermatozoén. I imagine that his method 
involved factors whose importance was unsuspected, since he 
says: ‘The real difficulty with this type of experiment is not the 
size of the spermatozo6n, but the fact that when four or five are 
injected into the egg-jelly, they usually swim out and away from 
the egg. This necessitates the making of many injections in 
order to get a single spermatozo6n to attach itself to the vitelline 
membrane and start the reaction.’ The ‘‘making of many 
injections”’ very likely involves touching the vitelline membrane 


’ 


an equal number of times, which recalls an experiment men- 
tioned in my earlier paper? in which fertilization membranes were 
induced by surrounding the eggs with large numbers of minute 
infusoria. Observation indicated a continuous bombardment of 
the ova. 

A quantitative relation between the rate of appearance of the 
-membrane and the agencies, spermatozoa, normally calling it 
forth is really no more surprising than the efficacy of Ca as an 
inhibitor. Since now sea-water of sufficient hypotonicity will of 
itself call forth membranes’ one may expect the exact reverse of 
the Ca-experiments if one immerses the eggs briefly in hypotonic 
solutions. Such ova, if not submerged too long so that the 


1G. L. Kite, ““The Nature of the Fertilization Membrane of the Egg of the Sea 
Urchin (Arbacia punctulaia),’’ Science, Vol. XXXVI., pp. 562-564. 

2 Science, loc. cit. 

3 In my preliminary communication (Science, loc. ctt.) I considered the method of 
“inducing”’ a fertilization membrane in Arbacia by means of hypotonic sea-water 
new. Schiicking however described this procedure in the year 1903. (Arch. f. 
d. ges. Physiol., Vol. 97, p. 85.) The same method was used on Arbacia eggs by 
McClendon in 1910. (American Journ. Physiol., p. 246.) 


152 OTTO GLASER. 


appearance of the membrane would have to be attributed to the 
hypotonic treatment itself, should be capable of fertilization by 
means of the sperm-infected medicine dropper. Actually under 
these circumstances fertilization with only 4 to 5 spermatozoa 
visible in the optical equator is possible in a considerable number 
of eggs. 

PROTOCOL. 

In a watch crystal 3 volumes of sea-water+3 volumes of dis- 
tilled. Added 1 volume of an egg-suspension in normal sea- 
water. At the instant’ when the first indications of membrane 
‘initiation’? were noticeable added 3 volumes of “double sea- 
water,’’ 7. e., sea-water whose volume had been reduced one-half 
by boiling. By means of a sperm-infected pipette every egg was 
provided with 4 to 5 spermatozoa. In a series of microscopic 
fields the number of undivided eggs was later compared with the 
number that had divided. The results were: 


Experiment I. Experiment II. 

Undivided. Divided. Undivided Divided. 

7 2 4. I 

4 3 3 2 

4 4 12 2, 

7 2 18 (0) 

I 2 6 5 

6 (0) 6 to) 

2 4 7 4 

5 I 8 (6) 

5 4 8 5 

5 2 14 4 

3 2 T4 3 

§ 3 12 3 

5 3 13 Ir 

6 5 8 2 

2 6 6 3 

4 3 7 I 

13 2 

6 I 

6 3 

ako) I 

| 8 iD 

Motalen 72 46 189 45 

Per cent.61 39 81 I9 


Controls: Normal eggs-++usual amount of sperm = 100% Fertiliz- 
ation. Eggs treated as above-++usual amount of sperm=100% 
Fertilization. This is in sharp contrast with the earlier experi- 
ments in which the operations were carried out at the same 


DEVELOPMENT IN ARBACIA. 153 


dilutions but without the brief fore-treatment with hypotonic 
sea-water. Results which harmonize with these but prove less 
satisfactory on account of injuries to the eggs can be gotten by 
the use of heat. In this case one might think of a parthenogenetic 
effect, but in Arbacia at least, it is not easy to confuse the usual 
parthenogenetic cleavage with normal two or four-cell stages. 

It is very easy to misunderstand these experiments and to 
draw wrong conclusions» There is no more doubt in Arbacia 
punctulata than in any other form that a single spermatozoon is 
sufficient to carry out the biparental effect. Furthermore the 
experiments with dilute sperm do not in anyway enable us to 
prejudge what would happen in another egg under similar con- 
ditions nor do they warrant the inference that the initiation of 
development by a single sperm is impossible in Arbacia ova 
deprived of their superficial coverings. I feel very sure of this 
however: In Arbacia the appearance of the fertilization membrane 
after insemination is a sign that the egg investments have allowed 
the sperm to pass through. This passage has been possible 
because the coverings have changed. The change depends on a 
synchronous softening and absorption of water, the latter having 
consequences as the result of which the membrane becomes vis- 
ible. Inasmuch as the becoming visible of the membrane is a 
reliable index of fertilization, and one of the consequences of 
fertilization is the division of the ovum, we may say that the 
initiation of development by a single spermatozo6n in this case 
is impossible because a single sperm cannot effect those changes in 
the egg-coverings which will permit it to reach the protoplasmic 
surface film that lies beneath. The situation is exactly as though 
the entrance to a room were blocked by a barrier which a single 
man could not break down, although a group of ten might. 
Once broken down, any one of the men could cross the threshold, 
but for the opportunity of doing this, the services of the others 
would be needed. With this analogy in mind, the statement 
that a single spermatozoon cannot except possibly under special 
conditions, fertilize the normally invested egg of Arbacia punctu- 
lata, would appear to agree with the facts. 


ZOOLOGICAL LABORATORY, 
UNIVERSITY OF MICHIGAN, 
November 12, 1914. 


STUDIES ON THE PHYSIOLOGY OF REPRODUCTION 
IN THE DOMESTIC FOWL. XII. 


On AN ABNORMALITY OF THE OVIDUCT AND ITs EFFECT UPON 
REPRODUCTION.! 


MAYNIE R. CURTIS. 


In a recent paper Pearl and Curtis (1914) have shown that when 
the passage of an egg through the oviduct is prevented by surgical 
interference with the duct the sex organs pass through their 
normal reproductive cycles. The oviduct functions to the level 
where the passage is interrupted and the egg is then.returned into 
the body cavity. The eggs thus set free may be absorbed without . 
causing any serious disturbance in metabolism. In a paper still 
in press (Curtis and Pearl) it has also been shown that congenital 
or acquired obstructions to the oviduct may occur without arti- 
ficial interference and that the results in such cases are the same 
as in the former cases. 

The following case was recently brought to our attention by 
Mr. J. C. Hawkes, Poultryman at the Maine Agricultural 
College poultry plant. 

A year and a half old Rhode Island Red bird was killed for 
meat. She was well grown, in good flesh and in every respect- 
was perfectly normal in appearance. When an incision was made 
to remove the viscera a full sized membrane shelled egg slipped 
into the opening. Mr. Hawkes then kindly turned the bird over 
to us for examination. 

The eggs and egg membranes shown in Fig. I were all removed 
from the body cavity of this bird. These represented every 
possible stage of absorption of the egg from a normal membrane 
shelled fresh egg to the collapsed empty membranes shown in 
the fourth line of the figure. Some of the eggs and some of the 
empty membranes were free in the body cavity. Some were 


1 Papers from the Biological Laboratory of the Maine Agricultural Experiment 
Station No. 76. 


154 


PHYSIOLOGY OF REPRODUCTION IN DOMESTIC FOWL. 155 


partly or entirely enclosed by peritoneum. In several instances 
two eggs or an egg and a bunch of membranes were walled off 
together. The last line of the figure shows collections of empty 
membranes enclosed in peritoneum. These peritoneal covered 
masses were attached by suspending stringslor folds of peritoneum. 
The large mass at the right end of this line contains a very large 
number of these empty membranes. A larger view of it is 
shown in Fig. 2. The second line from: the bottom of Fig. 1 
shows collapsed empty egg membranes of which some are single 
and some two or three tightly packed together. The three top 
lines of the figure show eggs in various stages of resorption. One 
was a normal fresh egg in a single egg membrane. Ten had 
evidently been normal eggs but at the time of autopsy they con- 
tained a homogeneous mixture of yolk and albumen which had 
lost the gelatinous character of fresh egg albumen. Each of 
these eggs was enclosed in a single egg membrane. The other 
four eggs were double eggs. These eggs were much like the double 
eggs (ovum in ovo) described by Parker (1906), Patterson (1911) 
and by many other writers. (The appended bibliography is 
‘supplementary to the one given by Parker 1906.) 

The eggs of this sort described in the literature had all been 
laid. Most of them have had shell on one or both of the con- 
centric components. The double eggs found in the body cavity 
of this Rhode Island Red hen had no shell on either the enclosed 
or enclosing egg. The nature of the contents of the double eggs 
differed in each of the four cases. In one both enclosed and 
-enclosing egg contained yolk. The yolk and albumen of the 
enclosing egg were somewhat mixed, although they did not yet 
constitute a homogeneous fluid. In fact the currents or streams 
of yolk could be seen in the clear albumen through the semitrans- 
parent egg membrane. The yolk and albumen of the enclosed 
egg were still more distinct although the-yolk membrane had 
already ruptured. The enclosed egg was about the size of the 
normal egg and the enclosing egg (the third egg in the top line) 
was the largest egg found in the body cavity. A second double 
egg was composed of a normal sized enclosed egg which had 
apparently contained the normal egg parts. The contents had, 
however, been reduced to a homogeneous brownish-yellow liquid 


156 MAYNIE R. CURTIS. 


much thinner than fresh egg albumen. The enclosing egg was 
only slightly larger than the egg it enclosed and it seemed 
probable that a second egg membrane had been received directly 
around the first on its passage back up the duct. A third of the 
double eggs had two closely applied egg membranes as in the 
preceding case but the enclosed egg was itself a double egg. The 
inner egg in this series was a small “‘witch”’ or “‘cock”’ egg con- 
taining a little yolk not enclosed in yolk membrane and a small 
amount of normal fresh albumen. The outer egg contained only 
normal fresh albumen. The other double egg was even more 
remarkable in character as it consisted of a concentric series of 
four enclosed eggs. The inner one, like the inner egg just 
described, contained a little free yolk enclosed in normal albumen. 
Each of the successive enclosing eggs contained only normal 
albumen. This whole egg was not larger than a normal hen’s 
CBSE 

These peculiar double formations indicate that an egg did 
not always pass up the duct in time to get out of the way of a 
succeeding egg. In case an egg met another yolk it might 
become enclosed in a double egg or it might change the direction 
of the incoming yolk If the yolk was ruptured and a part 
remained in the duct it might furnish the nucleus for a “cock” 
egg which might then become enclosed in a succeeding egg. 
Apparently the direction of peristaltic movements became at 
times much disturbed, as the last double egg described must have 
passed up and down the duct several times before it was finally 
extruded into the body cavity. : 

The visceral organs of the bird were in normal condition. 
There was a little slightly oily yellowish serous fluid bathing the 
viscera. The peritoneum was very slightly thickened but other- 
wise normal. The ovary was normal with a normal series of 
enlarging yolks and resorbing follicles. It was apparent that the 
bird was in the midst of a normal reproductive period and was 
backing membrane shelled eggs into the body cavity and re- 
sorbing them with great rapidity. 

The oviduct (Fig. 3) was perfectly normal from the funnel 
mouth to the posterior end of the isthmus. Here the tube 
abruptly ended blindly at D. There was no shell gland or vagina. 


ee 


PHYSIOLOGY OF REPRODUCTION IN DOMESTIC FOWL. 157 


The oviduct ligaments were continuous to the posterior end of 
the body cavity. That is the tube ended in the fold of enclosing 
peritoneum while the fold continued to the posterior end of the 
body cavity. The heavy bands of smooth muscle in the ventral 
ligament (see E, Fig. 3) continued to the end of the body cavity 
—several centimeters beyond the end of the tube. The tube 
rounded off smoothly at the posterior end and the ligament 
behind did not present the slightest indication that it had ever 
contained any oviduct tissue. It seems probable that the duct 
had never extended any farther than at present. From the 
embryonic history of the oviduct it is evident that if the actively 
growing point of a duct should cease at an unusually long distance 
anterior to the cloaca a blind oviduct of this form might result. 

The development of the oviduct according to the account given 
by Lillie (1908) begins on the fourth day of incubation as a groove- 
like invagination of a strip of thickened peritoneum on the surface 
of the Wolffian body or embryonic kidney. The lips of this 
groove fuse on the fifth day so as to form a short tube open an- 
teriorly to the body cavity and ending blindly posteriorly. The 
open end of this tube becomes the ostium tube abdominale or 
funnel‘mouth of the oviduct. The posterior end grows backward 
between the strip of thickened peritoneum and the Wolffian 
body. It normally reaches the cloaca on the seventh day. The 
growing point is always a short solid wedge of cells. The duct 
receives its lumen a short distance anterior to this. On the 
twelfth day of incubation the primordium of the shell gland is 
distinctly visible as an expansion of the lower end of this tube. 

The most probable explanation of the abnormality of the 
oviduct found in the case described is that in early embryonic 
development (probably on the sixth or seventh day of incubation) 
the backward growth of the primordial oviduct stopped per- 
manently while the differentiation of the part already formed 
continued in the normal manner. 

As in other cases where the passage of the egg is prevented 
the sex organs passed through their normal reproductive cycles; 
the oviduct functioned as far as the point where the passage was 
interrupted; the eggs were then returned to the body cavity and 
resorbed. The number of eggs and empty egg membranes found 


158 MAYNIE R. CURTIS. 


in this fowl which was apparently in a perfectly normal physical 
condition show that a bird possesses very great power of resorp- 
tion of its own proteins from the peritoneal cavity. Such 
resorption does not necessarily cause metabolic disturbances. 


BIBLIOGRAPHY. 


Brown, J. T. 
Encyclopaedia of Poultry, Vol. 1, p. 137. 
Brown, M. C. 
’10 Freak Eggs. Poultry Digest, Vol. 1V,.No. 11, p. 5. 
Curtis, M. R., and Pearl, R. 
Studies on the Physiology of Reproduction in the Domestic Fowl. X., 
Further Data on Somatic and Genetic Sterility. In press. 
Hargitt, Chas. W. 
’99 ©SSome Interesting Egg Monstrosities. Zool. Bull., Vol. II., pp. —. 
’12 Double Eggs. Amer. Nat., Vol. XLVI., pp. 556-560. 
Henneguy, L. F. 
*rr Ocuf complet de poule inclus dans un autre oeuf complet. Compt. rend. 
Soc. de biol. Par., Vol. LXX., pp. 779. 
Lillie, F. R. 
708 The Development of the Chick. New York. 
Parker, G. H. 
706 Double Hen’s Eggs. Amer. Nat., Vol. XL., pp. 13-25. 
Patterson, J. T. 
*11 A Double Hen’s Egg. Amer. Nat., Vol. XLV., pp. 54-50. 
Pearl, R., and Curtis, M. R. 
Studies on the Physiology of Reproduction in the Domestic Fowl. VIII., 
On Some Physiological Effects of the Ligation, Section or Removal of the 
Oviduct. Jour. Exp. Zool., Vol. 17, pp. 395-424. 
Pick, E. W. 
’11 Egg Abnormalities. Poultry World, Vol. 7, pp. 495. 


fs phate 


MAYNIE R. CURTIS. 


* ? 


EXPLANATION oF PLATE 
Fic. 1. Eggs and egg membranes removed from the 
Island Red fowl. ie 


BIOLOGICAL BULLETIN, VOL. XXVIII. PLATE |. 


MAYNIE R. CURTIS. IKE, Is 


162 MAYNIE R. CURTIS. 


EXPLANATION OF PLATE II. 


Fic. 2. Natural size photograph of the large peritoneal covered mass of egg 
membranes shown at the lower right hand corner of Fig. 1. This is cut across and 
opened back to show its composition. 

Fic. 3. Photograph (greatly reduced) showing the oviduct of the bird from 
which the eggs in Fig. 1 were taken. A =funnel; B=albumen secreting region; 
X =isthmus ring; C =isthmus; D=blind end of the oviduct; H=mass of smooth ; 
muscle in ventral ligament posterior to the end of the oviduct. 


PLATE Il. 


BIOLOGICAL BULLETIN, VOL. XXVIII. 


IRHEGs Do 


3- 


Eines 


MAYNIE Re CURTIS. 


woons HOLE, ‘MASS. 


eS 


e 
eyo 


Vol. XX VIII. April, 1915. No. 4. 


SAIOLOCICAL BULLETIN 


— 4 
yea yan 
ee ® \Wea 
<o 


LINKAGE OF CHROMOSOMES CORRELATED WITH MAY 5 1915 
REDUCTION IN NUMBERS AMONG THE SPECIE 
OF A GENUS, ALSO WITHIN A SPECIES OF 


NV ati i a y ye 7 
‘onal N\use 
iE EOCUSI Ln as ar a 


CARRIE I. WOOLSEY. 


In the following paper it is my purpose to give the result of my 
study of the chromosome numbers within three species of one 
genus of the Jamaican Locustide. 

The material used was collected in Jamaica during the summer 
of 1912 by Professor W. R. B. Robertson, of Kansas University. — 
The testes of all but one individual, No. 416, were removed while 
in the field and fixed in either Bouin’s or Flemming’s fluid. The 
slides were made in the fall of 1912. The clearest and best divi- 
sion figures as well as those in which the chromosomes were most 
crowded together and unsatisfactory were made from Flemming- 
fixed specimens. The sections were cut at about twelve micra 
and iron-hematoxylin proved most satisfactory for staining. 
All drawings have been made at the level of the base of the mic- 
roscope with the aid of a camera lucida. A 2 mm. Spencer oil 
immersion lens and No. 18 Zeiss compensating ocular were used. 
All the figures were magnified to 3,900 diameters and have been 
reduced 1/5, giving a final magnification of 3,120 diameters. 

The specimens studied were identified and classified by Mr. 
A. N. Caudell of the United States National Museum, Washing- 
ton, D. C., as follows: 

588 and 580, adult males of Jamaicana flava, N. Sp., Caudell. 

430, a nymph and 560, an adult male of Jamaicana unicolor, 
Brunner. 

416, 585, 586, 587, four adult males of Jamaicana subguttata 


Walker. 
163 


164 CARRIE I. WOOLSEY. 


438, a nymph and 503, a small male of Jamaicana subguttata. 

In determining the number of chromosomes in the various 
individuals, I have, in all cases, made drawings from not only 
different cysts, but from different follicles ao well. In a few 
instances where the figures are particularly clear and distinct, I 
have taken more than one cell from the same cyst (Nos. 30, 31, 
33) but where this was done I made additional drawings from 
other parts of the testes. There can be no doubt as to the 
character or appearance of these figures. 


OBSERVATIONS. 
Jamaicana flava n. sp. Caudell. 


All chromosomes are of the rod type. One individual shows 
two pairs of peculiarly associated chromosomes in the spermato- 
gonial division figures. 

In my comparison of species, the description of J. flava should 
logically come first since the chromosomes here are all of the rod 
shaped, simple type, and are not sufficiently associated to form 
multiples. They also vary the least in their behavior from what 
we might consider the original or primitive condition of the 
species of this genus. To distinguish individuals of the same 
species, I shall refer to each by number. Nos. 589 and 588 
belong to J. flava. 

In individual 589, I found the number of chromosomes to be 
thirty-five. They are all rod-shaped, varying in size from the 
large unpaired accessory chromosome which I have numbered 
18, through a graded series of pairs indicated by number, 
according to their size from 17 to I. 

In a polar view of a spermatogonial metaphase figure, the 
seventeen or eighteen largest chromosomes are found, as a rule, 
on the periphery in no fixed position or order, with the smallest 
pairs clustered about in the center. This is quite well shown in 
Figs. 3 and 4 while Figs. 1 and 2 show some of the larger chromo- 
somes in the center of the figure where they have probably 
displaced the smaller ones through some accident in previous 
mitoses. There seems to be nothing unusual or irregular in the 
number or relation of chromosomes here either in spermatogonial 
or spermatocyte divisions. 


LINKAGE OF CHROMOSOMES. 165 


In the first spermatocyte stages the chromosome complex 
consists of seventeen undivided autosomes plus the accessory or 
sex-chromosome (Figs. 10 and 11). As division proceeds, the 
unpaired sex-chromosome goes entire to one pole accompanied 
or followed by 17 autosomes. The mates of these seventeen 
autosomes pass to the opposite pole, thus making the number at 
the two poles eighteen and seventeen respectively. Before this 
division takes place, the paired autosomes show the same grada- 
tion in size as was noted in spermatogonial figures (Fig. 11). 
There are several extremely large chromosomes in this individual 
but the change in the series is so gradual that it is difficult to 
say where the dividing line falls. However, I am reasonably 
sure I can pick out at least three pairs in the spermatogonial or 
three undivided chromosomes in the first spermatocyte cells, 
that are larger than the others. 

In 588, the second individual of J. flava studied, a slight but 
- distinct difference can be noted in the spermatogonial figures. 
The number of chromosomes is the same, thirty-five, and all are 
of the rod type varying in size as was noted in No. 589. How- 
ever, in pairing off the chromosomes, I find there are in each cell, 
two pairs of closely related ones. Each member of the pair No. 
16 is always found in close association with a member of the 
pair No. 14 (A, Figs. 5, 6,7, 8). The members of the other pairs 
are distributed throughout the figure very much the same as in 
corresponding figures of No. 589. The largest chromosomes 
always appear on the periphery and the smallest ones in the 
center. These two sets of parallel rods, as may be noted, in the 
figures, have no regular position in respect to each other or to 
other chromosomes in the cell. Sometimes they are near each 
other and as often they are to be found on opposite sides of the 
figure. However, they are never far from the accessory chromo- 
some. * 

Jamaicana subguttata Walker. 

Five individuals out of six belonging to J. subguttata show 
only the rod type of chromosomes. In the sixth individual, a 
distinct variation is found in the appearance of a V-chromosome. 

Of the ten individuals I studied, numbers 416, 585, 586, 587, 
438 and 503 belong to Jamaicana subguttata. The sections of 


166 CARRIE I. WOOLSEY. 


No. 416 broke up: badly so that it was difficult to find a perfect 
division figure. However, a sufficient number were found to 
determine the number and character of the chromosomes (Figs. 
13-14). 

I found no distinctive feature in the number, arrangement, 
or behavior of the chromosomes in the first five individuals of 
this species (Figs. 13 to 29). There are thirty-five chromosomes 
in each and their appearance is very similar to that noted in 
individuals, No. 588 and 589. The accessory chromosome is 
very prominent and in the majority of spermatogonial metaphase 
figures it is found with seventeen of the largest autosomes on 
the periphery, the remaining seventeen small ones being in the 
center of the figure (Figs. 13, 15, 25-29). 

A most interesting feature was found however in the individual 
No. 503. Instead of the thirty-five autosomes of the simple rod 
type that were common to the other members of the genus as 
well as species, I found here thirty-three rods and a large V- 
multiple whose arms are of unequal length. When I attempted 

to arrange the autosomes in pairs, I found two of the large ones, 
-numbers 16 and 14, without mates among the rod type but 
corresponding in size to the arms of the V (Figs. 30 to 33). In 
one cyst I found several perfect cells in spermatogonial metaphase 
showing this size relation between the rods and the arms of the 
V (Figs. 30-31-33). 

After satisfying myself that this multiple chromosome appears 
in all spermatogonial metaphase figures, I examined cells in other 
stages of growth. In the various phases of the first and second 
spermatocyte cells I found the V still present. In the first 
spermatocyte figures the rod mates were often still attached to 
the arms of the V but the break could always be distinguished 
more or less distinctly (Figs. 37 through 50). In first spermato- 
cyte metaphase (Fig. 42), I found the V still attached to or 
united with its mates as were several of the rod chromosomes 
also. In this stage I found fifteen rods without the two in the 
V and the accessory, making in all eighteen chromosomes in the 
first spermatocyte. 

In the second spermatocyte division the accessory chromosome 
divides longitudinally so that each daughter cell receives half. 


LINKAGE OF CHROMOSOMES. 167 


The same kind of division evidently takes place in the V-multiple 
here. In Fig. 52, we can see the one V going over to one pole a 
little after the other chromosomes have passed. What appears 
to be the other half of the V has already reached the opposite 
pole. I did not find another cell showing this conclusively 
but the division of the other parts of the chromatin matter would 
seem to indicate this interpretation. The V at the pole is 
again the size of the multiple in the spermatogonial stage. 


Jamaicana unicolor Brunner. 


The two individuals of J. unicolor differ. One is of the simple 
rod type, while the other shows the two V-chromosomes. 

The spermatogonial metaphase figures of individual 430.1 
show thirty-five chromosomes of the rod type (Figs. 54-57). 
There is nothing in their appearance or behavior to distinguish 
them from individual 589 of J. flava or from individuals 416, 
585, 586, 587 and 438 of J. subguttata and the same holds true 
in later stages of growth. 

In No. 560.1, the second individual of this species, I found 
two multiple chromosomes (Figs. 58-67). In addition to the 
thirty rod autosomes, the accessory and two large V’s are to be 
seen on the periphery of the spermatogonial metaphase figures. 
The chromatin material in this series of slides is so badly massed 
that perfect cells are difficult to find. For this reason I can not 
be certain of the size relations among all the chromosomes but 
I have gained the essential facts for my purpose. The largest 
chromosomes are quite clear and distinct so that I can number 
the mates of the five largest pairs besides the accessory. Figs. 
58-60 show the number and arrangement of the chromosomes. 
Many spermatogonial cells can be found in which the two V’s 
and the accessory are readily distinguishable although the other 
chromosomes may be too badly massed for further study (Figs. 
61-64). 

In the first spermatocyte, the multiples are found attached 
to each other end to end, thus forming an elongated ring. A 
slight constriction appears where the ends of the V’s meet 
(Figs. 65-66). 

The cells in the earlier growth stages were small and the 
chromatin so massed together that I gained nothing from them. 


168 CARRIE I. WOOLSEY. 


DISCUSSION. 


McClung and others who have worked on the chromosomes of 
the Orthoptera believe there is a fixed or definite number for 
each group of related insects, and presumably for all kinds of 
life. Other writers disagree. Miss Browne (’13) has sum- 
marized the work done and results obtained by many investiga- 
tors on varied kinds of life so thoroughly that I shall not go into 
this in detail. I shall, however, review the methods as set 
forth by her whereby the changes in the chromosome numbers 
have been accounted for by the various authors. 

One method is by the fusion or separation of particular chromo- 
somes. Miss Browne in her work on Notonecta (13) and Wilson 
(11) on Negara use this explanation. A change in number by 
a process of fusion was used by McClung (’05) and Robertson 
(15) in the appearance of a multiple chromosome, the former in 
Hesperotettix and Mermiria, the latter in Chorthippus (Steno- 
bothrus) curtipennis. A change by a process of splitting has 
been advocated by several observers, Payne (09) and Wilson 
(Ir) among them. These account for slight or gradual changes 
but wide variations are accounted for by Wilson by a new 
segregation of the nuclear material causing a change in number 
and size relations of the chromosomes, but not in their essential 
quality. Another method whereby a change might take place 
is by an abnormality occurring in mitosis. Wilson (’o9a) has 
described an unequal distribution of the chromosomes to the 
daughter cells in Metapodius. An arrest of cell division after a 
division of the chromosomes has taken place was found by 
Boveri (’05) in sea-urchin eggs. 

My material resembles McClung’s (’05) in that the change 
in number is accounted for by the fusion of chromosomes, these 
giving rise to a multiple (V). However, the composition and 
behavior of this multiple differs from the one described by him 
in so far as its relation to the sex chromosome is concerned. 

These multiple or V-chromosomes resemble very much more 
those being described by Robertson (’15) in Chorthippus (Steno) 
bothrus). Robertson has found that Chorthippus (Stenobothrus- 
curtipennis has seventeen chromosomes and of these, six are V’s 
(three pairs of V’s). Counting each limb of the V as a chromo- 


LINKAGE OF CHROMOSOMES. : 169 


some, he believes there results twenty-three, the number normal 
for Truxaline a subfamily of the Acridide. It was at his sug- 
gestion that I undertook to determine if Jamaicana which I 
found to have these V-shaped chromosomes showed the same 
phenomena. 

In my material the multiple is likewise apparently formed by 
the union of two of the autosomes. As has been done by others, 
I numbered and paired the chromosomes according to size, 
calling the unpaired accessory which is the largest chromosome 
in the complex, No. 18, and grading the others down from that. 
I at once found two autosomes of unequal length, No. 16 and 14, 
without mates as well as the very noticeable V. Since the arms 
of the V are unequal, and correspond in size as well as length to 
the unmated rods, I believe No. 16 is the mate of the long arm 
of the V, while No. 14 mates with the short arm of the multiple 
(Figs. 30-36). The sex chromosome is very prominent and is 
often seen in close proximity to the multiple throughout the 
transitional stages, but it never unites with the components of 
the multiple as McClung found in Hesperotettix and Mermiria. 

In the spermatogonial metaphase cells the V first appears. 
It is always on the periphery of the ring with the apex pointing 
inward, while its rod mates may be anywhere on the periphery, 
sometimes near it, sometimes opposite it. 

From the spermatogonia through the spermatocytes this 
multiple can be traced. It divides longitudinally in spermato- 
gonia. In the first spermatocyte the tetrads divide transversely 
separating the V fromitsrod mates. Half of the second spermato- 
cyte cells receive the V and half receive the rod mates of the V 
(Figs. 37-47). In thesecond spermatocyte we have every reason 
to believe that the V divides longitudinally, each of the two 
spermatids resulting thus receiving a V while from the other 
second spermatocyte two spermatids result which will contain 
the rod mates of the V. 

This is what takes place in the one V-type of J. subguttata. 
The associated rods in No. 588 J. flava (Figs. 5-8) are easily 
distinguishable in the polar view of the spermatogonial metaphase | 
and their size 16 and 14 in the series corresponds to that of the 
arms of the V in No. 503. Their behavior can not be traced as 


170 CARRIE I. WOOLSEY. 


readily as the V, however, since their union is evidently not 
permanent and the rods separate in the later growth stages. 
The fact that they are associated in the early stage forming two 
sets of related rods on the periphery strengthens my belief in 
the manner of the V-formation in the other forms. In the place 
of these two sets of rods, or of the one V and one set of rods in 
No. 503, I found two V’s in No. 560 a representative of J. 
unicolor. Here we find the same thing taking place as in the 
one V type. In the place of a long V formed by the short V 
and the rod mates in the spermatocyte figures, we here find a 
ring formed by an end-to-end union of the arms of the V’s 
(Figs. 65-67). 

It will be seen that throughout the three species of this genus 
of Jamaican Locustide the number of chromosomes, 35 remains 
constant, although their behavior varies not only within the 
genus but even within the species. In seven of the ten indi- 
viduals studied representing three species, there are 35 chromo- 
somes of the simple rod type. As a whole, the division figures 
are clear and distinct and the growth stages can be followed 
with moderate certainty. In the first spermatocyte, the acces- 
sory passes undivided to the one pole making the number in the 
two daughter cells 18 and 17 respectively. When these divide 
in the second spermatocyte, division takes place longitudinally 
and the spermatids number 18, 18, 17 and 17 respectively, the 
last two being without the accessory chromosome. These then 
will produce males when fused with eggs, and the oosperm will 
contain thirty-four autosomes plus the one sex chromosome 
brought in by the egg. Those spermatids with eighteen, will 
produce females, since here there will be two sex chromosomes, 
one from the sperm and one from the egg in addition to the 
thirty-four autosomes, making the number 36 in the females. 

I find, however, an exception from this general rule of 35 
separate rod type chromosomes in each species studied. J. flava 
shows in one individual the presence of two pairs of associated 
rods in all spermatogonial figures. The rods that make up each 
pair or set are unequal and hence can not be mates. In number- 
ing the members of the complex these rank as 16 and 14 in the 
graded series, 18 being the unpaired accessory and the largest 


Pe ee ee 


LINKAGE OF CHROMOSOMES. 171 


in the group (Figs. 5-8). I find by numbering the members of 
the pairs as separate individuals, we have thirty-five chromo- 
somes here as in the other seven forms. In the later stages of 
growth, nothing appears to distinguish this form from its fellow 
No. 589 so we must conclude the rods of the related pairs are 
not held permanently together but are lost after the spermato- 
gonial division among the other rods of the complex. 

In J. subguttata a multiple chromosome appears resembling 
a V which I believe has been formed by the union of two of the 
autosomes. These, by continued association, have finally united 
end to end and we find them forming a V. The members or 
arms of the V are unequal and, as was noted in connection with 
the associated rods, can not be mates, since mates are identical. 
Hence the mates to the components of the multiple must be rods. 
I numbered and paired the chromosomes in the spermatogonia 
according to size and found two rods 16 and 14 without rod mates. 
These correspond with the arms of the V in size and appearance, 
and as many cells show the same relation, it seems to me they 
may be so taken. Figs. 30-36. 

The inequality of the parts of the multiple is especially well 
shown in the first spermatocyte figures. The long arm of the 
V is linked up with a long rod of approximately the same size, 
while a somewhat shorter or smaller rod is united with the smaller 
arm of the V. A slight constriction is to be seen where the rods 
join the arms of the multiple. 

In determining the count for this species, those individuals 
having the simple rod type chromosomes, have of course thirty- 
five in their complex. In No. 503, the odd one in this group, 
there are thirty-three chromosomes of the rod type plus the 
multiple which I believe is composed of two of these autosome 
‘rods united. This then makes the number for the entire species 
thirty-five, the same as was found for J. flava. 

The exception in J. wnicolor contains two multiples or V's 
similar to the one found in J. subguttata. They are apparently 
formed in the same way and probably of the same two chromo- 
somes, Nos. 16 and 14, as the difference in the lengths of the 
‘arms of the V’s corresponds with that in No. 503. In the first 
maturation division, the V’s which compose the elongated ring, 


WZ CARRIE I. WOOLSEY. 


break apart at the constriction and each cell receives one V. 
This splits longitudinally in the next division so that each 
spermatid carries one V multiple. 

J. unicolor contains thirty-one rods and the two multiples. 
If we accept what has apparently taken place in the previous 
cases, we can now say the V multiples are composed of four rods 
which will make the number for the individual thirty-five. 

It seems evident then, that the ten individuals, representatives 
of three distinct species, contain a uniform number of chromo- 
somes regardless of the fact that their behavior differs by the 
appearance of multiples in two specimens and a transitional 
form containing associated rods, not yet forming a V, in one 
individual. 

TABULATED RESULTS. 


, r Total Num 
sae Accession Num-_ Number of | Groups of Rods| NY | “ot Rod Ele. 
PECIess waar eee Cheencieee Associated Vs. ments (Chromo- 
: by 2’s. i somes). 
i} 

Ho, UGE od Soon ae 588 31 2 none 35 
589 35 none none 35 
J. subguttata..... 416 35 none none 35 
438 35 none none 35 
503 33) none T 35 
585 35 none none 35 
586 35 none none 35 
587 35 none none 35 
J.unicolor...... 430 35 none none 35 
560 Bhi none 2 35 


Although I made my drawings by the aid of a camera lucida 
I determined the series of size relations from the slide as well as 
from the drawing, comparing and judging as accurately as I 
could the graded pairs. Asa result of this study, I believe it is 
the same 14-16 pair that we find associated in the individuals 
described, Nos. 588, 503, and 560. Just what significance this 
may have upon the life processes of these grasshoppers I can not 
say. As to outer body characters, there is nothing to indicate a 
variation from those of the simple rod type. 

There is still another interesting association in this material 
that I feel should be noted here. In the cases where a multiple 
is present, it is more or less closely attended by the extremely 
large accessory chromosome. In the No. 503 material, this 


LINKAGE OF CHROMOSOMES. 173 


\ 
really occurs more often than my drawings would indicate. 


Whenever the multiple is seen, whether in an entire cell or only 
in a section, it is quite rare that the sex chromosome does not 
accompany it. In the majority of cases, although my drawings 
do not bear me out in this, spermatogonial figures show these 
two elements on the same half of the periphery. The No. 560 
material shows this particularly well. They are not connected 
as McClung found in Mermiria (’05), and Robertson in Chort- 
hippus ('15) but seem to be influenced or attracted by each other 
so that they are generally found in close proximity. 

It seems to me the individuality or genetic continuity of the 
chromosomes which Wilson (09) speaks of, is pretty well estab- 
lished here in at least the multiple types. In cysts that show 
different stages of division, the V may often be distinguished 
still intact, so that it can be traced from spermatogonia through 
the second spermatocyte still in the V form. It is quite likely 
only one half the spermatids of the one-V type receive a V while 
each spermatid of the two-V type receive one. 

I wish to express my thanks and indebtedness to Prof. W. R. B. 
Robertson for his helpful suggestions and encouragement during 
the progress of this work. 


ZOOLOGICAL LABORATORY, 
KANSAS UNIVERSITY, 
September, I9r4. 


BIBLIOGRAPHY. 
Boveri, Th. 


’05 Zellenstudien V. Uber die Abhangigheit der Kerngrésse und Zellenzahl der 
Seeigellarven von der Chronosomenzahl der Ausgangszellen Jen. Zeit- 
schrift, Bd. 32. 

Browne, E. N. 

*r0 Study of the Male Germ Cells in Notonecta. Journal of Experimental 
Zoology, Vol. 14. No. 1, January, I913. 

’10. The Relation between chromosome-number and species in Notonecta. 
BIOL. BULL., Vol. 20. 

McClung, C. E. 

705 The Chromosome Complex of Orthopteran Spermatocytes. Brot. BULL., 

Vol. 9. 
Payne, F. 

709 Some New Types of Chromosome Distribution and Their Relation to Sex. 

Biou. BULL., Vol. 16. 
Robertson, W. R. B. 
’08 The Chromosome Complex of Syrbula admirabilis. Kan. Univ. Sci. Bull. 


174 CARRIE I. WOOLSEY. 


Robertson, W. R. B. 

"15 Taxonomic relationships shown in the chromosomes of the Tettigide and 
other subfamilies of the Acridide: V-shaped chromosomes and chromosome 
numbers in Acrididee, Locustide, and Gryllide. 

Stevens, N. M. 

*13a Supernumerary chromesomes and synapsis in Ceuthophilus. Brion. BULL., 

Vol. 22. 
Wilson, E. B. 

’0ga_ Studies on Chromosomes, V. The Chromosomes of Metapodius, etc. 
Jour. Exp. Zoology, Vol. 6. 

Ir Studies on Chromosomes VII. A Review of the Chromosomes of Nezara, 
with Some More General Considerations. Jour. Morph., Vol. 22. 


176 CARRIE I. WOOLSEY. 


PLATE I. 
Jamaicana flava n. sp. Caudell. 


In polar views of spermatogonial metaphase figures, the number of chiomosomes 
in both individuals is thirty-five. Chromosomes are paired and numbered ac- 
cording to size, 18 being the single accessory. 

Fics. 1-4. Polar view of spermatogonial metaphase of individual No. 589. 

Fics. 5-8. Polar view of spermatogonial metaphase of individual No. 588, 
showing, A, the oddly related pairs of chromosomes (14’s and 16’s) in each figure. 

Fic. 9. Lateral view of spermatogonial anaphase. No. 588. 

Fic. 10. Polar view, first spermatocyte of individual 580. 

Fic. 11. Lateral view of metaphase of first spermatocyte chromosomes 
numbered according to size. Sex-chromosome going undivided to one pole. 
Individual 589. 

Fic. 12. Prophase of second maturation division or interkinesis. 


PLATE |. 


BIOLOGICAL BULLETIN, VOL. XXVIII» 


Cc. |. WOOLSEY. 


Ail Eyraat 


hpi 


178 CARRIE I. WOOLSEY. 


PLATE II. 
Tamaicana subguttata Walker. 


Spermatogonial figures from four individuals (Fig. 19 exception). The number 
in each polar view is thirty-five. Chromosomes are paired and numbered according 
to size, 18 being the accessory. 

Fics. 13-14. Spermatogonial figures from individual 416. Polar view, meta- 
phase. ! 

Fics. 15-18. Polar view of spermatogonial metaphase figures from individual 
No. 585. 

Fic. tg. Lateral view of first spermatocyte. The tull number of chromosomes 
is not shown. 

Fics. 20-22. Polar view of spermatogonial metaphase figures from individual 
No. 586. 

Fics. 23-26. Polar view of spermatogonial metaphase figures from individual 
No. 587. ; 


PLATE Il. 


BIOLOGICAL BULLETIN, VOL. XXVIII. 


C. |. WOOLSEY. 


A as 


Sarah 


os 


180 CARRIE I. WOOLSEY. 


PuateE III. 
Jamaicana subguttata Walker. 


Spermatogonial figures from two other individuals of this species. The number 
of chromosomes in 438, is thirty-five. In 503 there ate thirty-three simple chro- 
mosomes plus a V-shaped multiple. 

Fics. 27-29. Polar view, metaphase spermatogonial figures of individual No. 
438. 

Fics. 30-36. Polar view, metaphase spermatogonial figures of No. 503. The 
V-multiple chromosome is conspicuous on the periphery. 

Fic. 37. First spermatocyte, late prophase showing the long V bi-tetrad of 
the one-V type. 


PLATE Ill. 


“BIOLOGICAL BULLETIN, VOL. XXVIlle 


a 


182 CARRIE I. WOOLSEY. 


PLate IV. 
Jamaicana subguttata Walker. 


First spermatocyte figures of No. 503. 

Fic. 38. Polar view prophase of first spermatocyte showing fifteen simple 
autosomes, the V-multiple, and the “‘accessory’’ chromosome. The latter is en- 
closed in a receptacle apart from the other chromosomes. 

Fics. 39-41. Lateral view metaphase of first spermatocyte showing the 
multiple chromosome. The constrictions in the arms of the V show where the rod 
mates are about to pass to the opposite pole, breaking away from the multiple at 
the constricted places. The full number is not present but the accessory is promi- 
nent in 39 and 40. 

Fic. 42. A spermatocyte figure showing the full number, 15, of tetrad chromo- 
somes plus the double tetrad, plus the sex chromosome. 

Fic. 43. First spermatocyte showing double tetrad and the accessory chromo- 
some. 


BIOLOGICAL BUL‘ETIN, VOL- XXVIII. PLATE IV. 


Be tee a8 


Lqstetere 


43 


Cc. |. WOOLSEY. 


184 CARRIE I. WOOLSEY. 


PLATE V. 
Jamaicana subguttata Walker. 


Spermatocyte figures of No. 503. 

Fics. 44-46. First spermatocyte anaphase. The multiple chromosome is to 
be seen in each figure. It has lost its rod mates, which have probably gone to the 
opposite pole, and is now the size of the spermatogonial V. By comparing it with 
the multiple before division as seen in Figs. 39-43, what has taken place, is more 
readily seen. 

Fics. 47. First spermatocyte telophase showing the V at one pole and its rod 
mates and the sex chromosome at the other. The latter is split longitudinally. 

Fic. 48. Telophase of a first spermatocyte division. 

Fic. 49. Telophase of first spermatocyte division containing the sex-chremo- 
some and the multiple. 

Fics. 50-51. Telophase of the first spermatocyte or resting period of the second 
spermatocyte. The sex-chromosome is split preparatory to the second spermato- 
cyte division. 

Fic. 52. Second spermatocyte. Sex-chromosome divided. The V-chromo- 
some is here divided and the arms are of the original size seen in the spermatogonial 
figures. 

Fic. 53. Second spermatocyte. Sex-chromosome divided. 


PLATE V. 


Cc. . WOOLSEY. 


186 CARRIE I. WOOLSEY. 


Pate VI. 
Jamaicana unicolor n. sp. Caudell. 


Spermatogonial figures of two members of this species. Thirty-five rods are 
found in the one; thirty-one rods and two V’s are found in the other. Figures 65-67 
are first spermatocytes. 

Fics. 54-57. Spermatogonial figures of individual 430. There are thirty-five 
chromosomes of the rod type here, paired and numbered according to size. No. 18 
is the unpaired sex chromosome. 

Fics. 58-60. Spermatogonial figures of individual 560 showing thirty-one rods 
and two V’s in each. No. 18 is the unpaired sex-chromosome. Just the largest 
chromosomes are paired and numbered. 

Fics. 61-64. Spermatogonial metaphases. Although the chromatin material 
is much massed, the sex and the two V-chromosomes are very prominent and 
distinct. 

Fic. 65. First spermatocyte of individual 560 showing the multiple or bi-tetrad 
formed by the two V’s. 

Fic. 66. First spermatocyte prophase showing the complete number of chro- 
mosomes in individual 560,—fifteen rod tetrads, the bi-tetrad, and the sex-chromo- 
some. 

Fic. 67. The ring-shaped bi-tetrad more condensed in a latter prophase. 


PLATE VI 


BIOLOGICAL BULLETIN, VOL. XxXvIIl. 


Cc. |. WOOLSEY 


PERIODICITY IN THE PRODUCTION OF MALES IN 
HYDATINA SENTA.! 


A. FRANKLIN SHULL. 


INTRODUCTION. 


There is often a well-marked rhythm in the production of males 
in the rotifer Hydatina senta. Generation after generation may 
pass with few or no male-producing females; while later, in a 
few successive generations, male-producers may be abundant, 
only to be succeeded by a period in which male-producers are 
uncommon or wanting. Although this rhythm has not been 
mentioned by all students of the life cycle of Hydatina, and has 
been emphasized by few of them, it can hardly have escaped 
notice by any one who has bred this species for several months. 
In another genus, Asplanchna, Mitchell (1913) has laid stress 
upon this rhythmical appearance of males, as a basis for certain 
theoretical conclusions, and has called attention, by way of 
generalization, to the same rhythm in Hydatina. 

Regarding the cause of this periodicity, there is not general 
agreement. Mitchell, writing of Asplanchna but extending his 
conclusions to rotifers in general, appears at times to regard the 
rhythm as the effect of an internal factor, and again as due to 
environmental conditions. He says ‘“‘this rhythm is not the 
result of external conditions”’ but “‘is not absolutely independent 
of them.’? Later he adds that ‘male production ... is a 
matter of physiological potential and under the more or less 
direct control of nutrition’’ (p. 229). At other places he states 


that ‘‘male production . .. is a phenomenon all but wholly 
under nutritive control...’ (p. 246); “these male and non- 
male producing strains ... exist ...and... these strains 


are also produced by nutritive changes”’ (p. 247); and “qualita- 
tive and quantitative changes in nutrition will be found the 
universal sex-controlling factors in this group’”’ (the rotifers) 

1 Contribution from the Zodlogical Laboratory of the University of Michigan. 


2 Op. cit., p. 228. . 
187 


188 A. FRANKLIN SHULL. 


(p. 253). Whitney (1914), on the other hand, has no hesitancy 
in ascribing the periodicity of male production to external 
factors; thus, in mentioning earlier work of his own, ‘‘he was of 
the opinion that whatever the potent factor was that sometimes 
caused only females to be produced, and at other times caused 
nearly all males to be produced, it must be an external factor.” 
On the basis of his recent work he attributes this periodicity to 
alternation of the active and quiescent states of the protozoan 
food of the rotifers. 

In my own work on Hydatina during several years, certain 
lines have been bred so long, and so many families completely 
reared, that further light may be thrown upon the rhythm of 
male production. In the following pages evidence is first pre- 
sented, bearing on the regularity of the periodicity of male 
production, and the probable independence of this periodicity of 
the environment. Later the supposed evidence that male pro- 
duction is correlated with nutrition is discussed. 


REGULARITY OF THE PERIODICITY OF MALE PRODUCTION. 


Conditions Necessary to the Demonstration of Periodicity. 


In parthenogenetic lines that produce many males, rhythm is 
not as easily demonstrated as in lines producing few males. If 
the proportion of males in a line producing many males be repre- 
sented by a curve, there are so many irregularities in it, so many 
minor humps even in the periods of few males, that the larger 
humps are less striking. If the lines produce few males, on the 
other hand, the whole curve may be so lowered that the periods 
of depression are below the base line; that is, there are no males 
at all in these periods. In such cases the rhythm may be quite 
striking. 

Further difficulty in detecting rhythm is introduced by rearing 
only one family in each generation. ‘There are great individual 
differences between families taken at the same period, so that 
the family chosen may or may not be an average of all families 
that might be reared at the same time. Furthermore, the method 
of publishing the results, merely giving the total for the one 
family in each generation, often makes the rhythm appear less 
definite; because it often happens that in three or four successive 


PERIODICITY IN THE PRODUCTION OF MALES. 189 


generations, in a period of many males, the males are produced 
mostly by the daughters at the end of the family in the first of 
these generations, by the daughters in the middle of the family 
in the second generation, and by the daughters in the first part 
of the family in the third generation. In the manner in which 
these results have been published, the males of the first and third 
generation (appearing under date of the beginning of their 
respective families) seem to be four or five days apart, whereas 
they may be only one day apart, or even hatched on the same day. 
It seemed advisable to obviate as many of these difficulties as 
possible, and to obtain evidence of periodicity less open to 
objection. The method adopted was as follows: First, a line 
was selected which was producing only a moderate number of 
males, with the expectation that the periods of male production 
would be completely separated by periods in which there were 
no males at all. Second, instead of isolating all the daughters of 
a single family in each genertion, parts of a number of families 
were isolated. This second precaution was taken to smooth out 
irregularities and to reduce the apparent length (and hence the 
overlapping) of the periods, both of many and of few males. 


Lines Exhibiting Periodicity. 

The line selected to meet the above conditions was one of the 
F, lines from a cross between an English and a Nebraska line, 
described in an earlier paper (Shull, ’15). Several females of 
this line, all of approximately the same age, were placed together 
in a dish. When they reached maturity, the daughters of the 
early part of their families were rejected; but the daughters 
produced after about 24 hours of egg laying were preserved. 
From these daughters the “‘sex ratio’’ (ratio of male-producing 
to female-producing females) was determined, and from them 
also the next generation was reared. The first daughters were 
in each case rejected because, as I have shown before (Shull, ’10), 
the first daughters are less commonly male-producers than are 
daughters in the middle of the family. 

The daughters selected for rearing the next generation were in 
like manner kept together in one dish until about 24 hours after 
their first daughters appeared. At this time a second lot, all of 


190 A. FRANKLIN SHULL. 


about the same age, was isolated to determine the sex ratio of 
the second generation, and so on. 

From 30 to 100 daughters, all of nearly the same age, and from 
the same parts of their respective families (end of first day of 
egg-laying) were recorded for each generation. A new genera- 
tion was, by this method, secured every three days, as a rule, 
instead of every two days, as is possible when the first daughters 
of a.family are used as parents of the next generation. 

The number of male-producing and female-producing females 
in each generation thus recorded is given in Table I. Male pro- 


TABLE I. 


SHOWING THE NUMBER OF MALE-PRODUCING (o'@2) AND FEMALE-PRODUCING 
(2 9) FEMALES IN A LINE oF Hydatina senta. 
The male-producers occur periodically. 


Date. Number of o& 9.|Number of @ 9. Date. ieee of o' 9.|Number of 9 9. 

Jan 16 2 24 Mar. 14 (0) 44 
19 Co) 24 I7 (0) 38 

22 I 35 20 (0) 42 

25 3 33 23 ie) 23 

28 19 46 26 8 40 

30 14 46 29 36 48 
Feb. 2 (0) 56 Apr. 1 7 q1 
5 (0) 40 4 I 51 

9 2 38 7 ©) Sil 

I2 (0) A3 iO) to) 40 

I5 to) A7 13} I 36 

18 (0) 48 16 fo) 35 

2I (0) 13 19 Co) 43 

24 ) 35 22 3 85 

27 17 aI 26 68 51 
Mar. 2 13 27 20 32 23 
5 6 29 May 2 18 34 

8 2 36 5 3 50 

II O 42 8 fo) 44 


duction in this line, when bred as described above, showed a 
well-marked periodicity, the interval being about one month. 
Conditions were purposely kept fairly uniform; but even if it 
were not possible to prevent changes of the medium, it is scarcely 
probable that any external condition favoring male production 
should have recurred with such regularity. Moreover, were this 
periodicity due to external factors, the intervals between periods 
of male production should be the same in all lines bred at the 
same time and subjected to the same conditions. That this 
was not the case will now be shown. 


PERIODICITY IN THE PRODUCTION OF MALES. IQI 


In another line, bred for a much longer time than was the line 
described above, there was equally clear evidence of regular 
periodicity, though the extent of the waves of male production 
was not determined. A line of rotifers obtained from England 
in the fall of 1912, described in another paper (Shull, ’15), has 
been reared up to the present time. No considerable numbers 
of individuals of this line have as a rule been isolated, hence the 


sex ratio can not be stated; but during the time when the 
periodicity of other lines was being examined, several dozen 


TABLE II. 


SHOWING DATES BETWEEN WHICH MALES WERE PRODUCED IN AN ENGLISH LINE 
_OF Hydatina senta. 


No males appeared between one period of male production and the next. 


Number of Period of Dates Between Which Males 
Male Production. Were Found. 
RED LTS Cee he Sie Sess arora role SN aee SURES cakes February 13 to 25, 1913 
SeConle ts seeker a earsuttetencuole cheumenaaievays April 17 to 26, 1913 
AN cbhgele Aia-crceaectencia oe oeeees Cia CCID orca June 19 to 24, 1913 
TOUTE Ayo evatetvetneeel cisiiseceteds icuecetensiioss August 24 to 30, 1913 
EG RU lavreeeeper seay cees sisnele ola lisusuamenesenavcecvene ve: = October 31 to November 15, 1913 


individuals of most generations were reared in two or three 
dishes. As the number of males was always small in this line, 
males were found in these ‘‘mass’’ cultures only occasionally. 
They nearly always appeared in the dishes containing several 
successive generations, and then were wanting for a considerably 
longer period. There was thus a rhythm of male production, 
which, as shown in Table II., proved to be fairly regular and 
definite, though the number of male-producers in each period 
was not known. 

The interval between periods of male production was, in this 
line, a trifle over two months. Inasmuch as this line was bred 
in part simultaneously with the one recorded in Table I., the 
difference in the interval of male production in the two ites 
(one month in the first, over two months in the second) effec- 
tually disposes of any suspicion that this rhythm was induced 
by external conditions. 

A third line which showed evidence of periodicity in the produc- 
tion of males was obtained from Nebraska in 1912, and has 
been reared ever since. This line is also described in a recent 


192 A. FRANKLIN SHULL. 


paper (Shull, ’15). Complete families were isolated from early 
in November to December 15, 1912, but no males appeared. 
During the time when periodicity of male production was being 
studied, representative mass cultures were reared. These cul- 
tures showed males in small numbers only, and at times separated 
by wide intervals. The times of male production, as far as 
known, are shown in Table III., though the proportion of males 
was not recorded. 

The periods of male production in the Nebraska line are 
separated by intervals of three to five months, the interval 


TABLE III. 


SHOWING TIMES AT WHICH MALES WERE PRODUCED IN A NEBRASKA LINE OF 
Hydatina senta. 


Number of Period of Male Production. Dates Between Which Males were Found, 
PTS beep deas cleic erasteeyt ue ieee eS January 15 to February I, 1913 
SCO Gear aE ota cMaaRE I os acidic April 22, 23, 1913 
SDNaia: Gl gohan 2 25 Sit aoe ace n aes eee August Io to 16, 1913 
JSTONSDE(C] Ne sri ane eo le Mee mec tL capa. 8 December 7 to II, 1913 
Observation wanting! 

Sib 0) UN Ga) ae ie ENE eR Be coer A Gd November 1, 1914 


increasing with the age of the line. Though no complete families 
were reared, during any of these ‘“‘waves”’ of male production, 
it was evident that in the later periods there were fewer males 
than in the earlier ones.® 

In each of the three lines described, there was a well-marked 
rhythm in the production of males. The great regularity of 
this periodicity, especially in the first two of these lines, and the 
fact that the lines differed considerably from one another in 
regard to the interval between periods of male production, forbid 
the assumption that the waves of male production were brought 
on by specific external conditions. 


1 Observations were wanting from March 8 to June 10, 1914. Probably not 
more than one period of male production fell between these dates, hence the next 
males recorded are to be regarded as belonging to the sixth period. 

2 A single male-producing female appeared in this ‘“‘wave”’ of male production. 

3 In the English line described in Table II., while the intervals between periods 
of male production did not increase, with the age of the line, the number of males 
in successive periods plainly decreased. This is a confirmation of a conclusion 
which I formerly drew from lines bred through shorter periods, namely, that the 
proportion of male-producing females gradually decreases with the age of the line. 
Whether, as Mitchell suspects, this decrease is due to uniformity of conditions, is 
a question not answered by the evidence. 


PERIODICITY IN THE PRODUCTION OF MALES. 193 


No statement here made is to be construed, however, as a 
contradiction of my former claim that external conditions may 
alter the extent of male production.! Few biological facts are 
more firmly established than that external factors modify the 
life cycle of Hydatina. The results described above merely 
show that, under fairly uniform conditions, there is nevertheless 
a periodicity in the production of males which must be due to 
internal factors. . 

PERIODICITY AND NUTRITION. 

Mitchell (13) has pointed out that in Asplanchna periods of 
male production are also often, perhaps usually, periods of 
vigorous growth and rapid reproduction; and he concludes there- 
from that male production is a result of high nutrition. This 
conclusion may be correct, but it is scarcely logical, since co- 
incident events are not always related to one another as cause 
and effect. But assuming as Mitchell does that size of family 
is a guide to nutrition, let us examine all the sources of informa- 
tion that are extensive enough to be of value, to determine, if 
possible in this way, the relation of nutrition to male production. 
In my own work in the past few years, there have been two lines 
in which hundreds of families have been reared. By collecting 
all of the families of the same size in a single line, and recording 
the proportion of male-producers, it should be possible to discover 
to what extent size of family and male production are correlated. 
Obviously one must not collect in the same group families belong- 
ing to two or more unrelated lines, for one of these lines may 
have larger families, and at the same time (but from other causes) 
either many or few male-producers, so that the groups of families 
of large size would have on the average a correspondingly high 
or low proportion of male-producers. Such an apparent correla- 
tion would have no significance. Within a single line, however, 
no such error could affect the results. The two tables herewith 
presented (IV. and V.) are each compiled from families belonging 
to a single line. ; 


1 My discovery several years ago of internal differences between parthenogenetic 
lines of Hydatina senta, the result of which is a different proportion of male pro- 
ducers in each line, is characterized by Mitchell as a “return to the position of 
Punnett.” Since Punnett never found an effect of external conditions, and since 
I never repudiated my experiments proving the effect of external conditions, there 
can have been no “return.” 


194 A. FRANKLIN SHULL. 


Notwithstanding the great fluctuation of the proportion of the 
male-producing females in families of different sizes, it might be 
possible to see in the percentages given in the last columns of 
these two tables (or perhaps only Table IV.), a slight increase 
from top to bottom, and hence a correlation between size of 
family and male production; though the degree of correlation 


TABLE IV. 


SHOWING SIZE OF FAMILY AND PROPORTION OF MALE-PRODUCING (o'Q@) AND 
FEMALE-PRODUCING (2 9 ) FEMALES IN A SINGLE PARTHENOGENETIC LINE 
oF Hydatina senta. 


6 : Number of Number of Number of Percentage of 
Size of Family. nemulices Jo. 29, 39. 
Ito 5 7 3 13 18.7 
6 to 10 I5 I5 96 13.5 
II to 15 16 47 158 22.9 
16 to 20 20 28 322 8.0 
2I to 25 20 73 388 15.8 
26 to 30 21 I52 437 25.8 
31 to 35 33 174 909 16.1 
36 to 4o 27 DUB 816 20.7 
AI to 45 29 281 977 22.3 
46 to 50 26 324 931 25.8 
51 to 55 I9 285 410 28.6 


certainly can not be high. However, even if such correlation 
exist, it does not follow that the percentage of male-producing 
females is dependent upon the degree of nutrition, which deter- 
mines size of family. The one way to test the effect of nutrition 
is to alter it artificially, and note the results. That has been 
done in my starvation experiments (Shull, ’10, pp. 320 ff.), with 
results that were positive but of such a character that they could 
be explained as due to changes in the chemical composition of the 
medium, rather than changes of nutrition. Mitchell (13) ob- 
jects that in drawing conclusions from these starved families, 
I have regarded only the totals; had I observed individual 
families, he believes, I would have reached a different result. 
Mitchell states that the male-producing females in my starvation 
experiments did not appear in the smallest families, which were 
presumably the offspring of the most starved parents, but in the 
larger families, produced by the better nourished females; and 
from this supposed fact concludes that abundant male production 
is due in Hydatina, as he believes it to be due in Asplanchna, to 


PERIODICITY IN THE PRODUCTION OF MALES. 195 


high nutrition (coupled with irregularities of nutrition). How 
far his statement that the male-producing females are in the 
larger families is correct, may be seen from Table VI., in which 


TABLE V. 


SHOWING SIZE OF FAMILY AND PROPORTION OF MALE-PRODUCING (o'@) AND 
FEMALE-PRODUCING (9 Q ) FEMALES IN A SINGLE LINE OF Hydatina senta, 
DISTINCT FROM THAT IN TABLE IV. 


3 . Number of Number of Number of Percentage of 
Size of Family. Families! Ge. , | : 
Ito 5 12 2 38 5.0 
6 to 10 17, 13 121 0.7 
Ir to 15 19 67 183 26.8 
16 to 20 29 105 AI5 20.1 
21 to 25 B2e 152 587 20.5 
26 to 30 i7 48 418 10.3 
31 to 35 22 149 577 20.5 
36 to 40 15 113 463 19.6 
41 to 45 18 76 704 9-7 
46 to 50 IQ 192 716 21.1 
51 to 55 | 8 106 { 314 25.2 


the families of the starved line in the experiment referred to 
(Shull, 1910, Table III.) are tabulated. The group of families 
containing I to 5 daughters, and that of families numbering 41 
to 45, are omitted because there is but one family in each group. 


IAs Wl 


SHOWING SIZE OF FAMILY AND. PROPORTION OF MALE-PRODUCING (o'@) AND 
FEMALE-PRODUCING (92 @ ) FEMALES IN A STARVED LINE OF Hydatina senta 
DESCRIBED IN A FORMER PAPER. 


The greatest proportion of male-producers is in families of medium size. 


f Number of Number of Number of Percentage of 
Size of Family. Families. FQ. 29. FQ. 
6 to 10 7 a 49 12.5 
Ir to 15 3 6 34 15.0 
I6 to 20 6 39 66 iyo 
21 to 25 8 88 IOL 46.5 
26 to 30 16 172 273 38.6 
31 to 35 8 96 169 Bor 
36 to 40 2 14 64 17.0 


In this table it appears that the greatest number of male- 
producing females is found, not in the largest families, but in 
those of medium size. It may also be recalled that the distribu- 
tion of the male producers with regard to size of family, in these 
starved families where nutrition was known to have been variable, 


196 A. FRANKLIN SHULL. 


_is not the same as the distribution in the well-fed families of 
Tables IV. and V., about whose nutritive conditions we know 
only that which size of family tells us. The argument that the 
numerous male-producing females of the starved line were 
produced as a result of high nutrition of their parents, loses much 
of its weight when it is shown that these male-producers were 
not chiefly in the largest families. 

It is not to be asserted that nutrition has no effect upon male 
production. Indeed, Whitney (’14) has presented new evidence 
that qualitative differences of nutrition do affect male production. 
It is not clear what relation Whitney’s results have to the ques- 
tion of periodicity of male production, whether changes of 
nutrition can be made to destroy the rhythm, or wholly to 
alter the interval, or merely to modify the extent of male produc- 
tion. My own starvation experiments, referred to above, left 
the intervals between the periods of male production unaltered, 
but the waves of male production and the intervening periods 
of female production were rendered less striking. I attributed 
the effects shown in these experiments to the chemical nature 
of the medium, and not to nutrition. Until experimental 
evidence indicates the contrary to be true, it is safest to assume 
that nutrition also, when it affects male production at all, does 
not alter the interval between periods of male production, but 
merely the extent of male production. 

To summarize: Three lines of Hydatina, bred through many 
months, showed fairly regular periodicity in the production of 
males. One line exhibited relatively abundant male production 
every month; another every two months; while in the third the 
interval varied from three to five months during the period of 
observation. The fact that the interval between the periods of 
many males is quite regular in some lines, and is not the same. 
in all lines reared simultaneously, indicates that this periodicity 
is due to an internal factor. Hundreds of families were examined 
to determine whether the largest families, which were presumably 
offspring of the best nourished parents, contained the greater 
number of male producing females, as Mitchell assumes they do. 
In well nourished lines there is some doubt whether there was 
any correlation between size of family and number of male 


PERIODICITY IN THE PRODUCTION OF MALES. 197 


producers; in starved families, on the other hand, the greatest 
numbers of male producers were not in the largest families, but 
in those of medium size. On the statistical evidence as a whole, 
the influence of quantity of nutrition upon male production is 
held _to be “not proven.” When qualitative differences in 
nutrition affect male production, the interval between periods of 
many males probably remains unchanged. 


BIBLIOGRAPHY. 
Mitchell, C. W. 
’13. Sex Determination in Asplanchna amphora. Jour. Exp. Zodél., Vol. 15, 
No. 2, August, pp. 225-255. 
Shull, A. F. ‘ 
‘Io Studies in the Life Cycle of Hydatina senta. I. Artificial Control of the 
Transition from the Parthenogenetic to the Sexual Mode of Reproduction. 
Jour. Exp. Zodél., Vol. 8, No. 3, May, pp. 311-354. 
‘15 Inheritance in Hydatina senta. II. Characters of the Parthenogenetic 
Females and Their eggs. Jour. Exp. Zodl., Vol. 18, No. I, pp. 145-186. 
Whitney, D. D. 
’r4 The Influence of Food in Controlling Sex in Hydatina senta. Jour. Exp. 
Zool., Vol. 17, No. 4, November, pp. 545-558. 


NOTE ON TREMATODE SPOROCYSTS AND CERCARIA# 
IN MARINE MOLLUSKS OF THE WOODS HOLE 
REGION. 


EDWIN LINTON. 


In the summers of 1909 and 1910, while engaged in the study 
of the parasites of fishes at the laboratory of the Bureau of 
Fisheries, Woods Hole, Mass., I examined a number of inverte- 
brates for larval stages of trematodes. The results of these 
examinations, although rather meagre with respect to the 
number of species found, are not without interest. 

The following species of mollusks were examined: Crepidula 
fornicata, C. plana, Ilyanassa obsoleta, Littorina littoria, L. rudis, 
Modiolus plicatulus, Mya arenaria, Mytilus edulis, Neverita 
duplicata, Pecten trradians, Purpura lapillus, Urosalpinx cineria, 
Venus mercenaria. Besides these several species of crustaceans 
were examined, also one annelid, Hydroides dianthus. 

Larval trematodes were found in only two species of mollusks, 
viz. Ilyanassa obsoleta and Pecten irradians. No trematode 
parasites were found in any of the crustaceans. A sporocyst 
found in the annelid, Hydroides dianthus, has already been 
reported.’ 


I. SPOROCYSTS AND CERCARLE FROM ILYANASSA OBSOLETA. 
FIGURES I-6. 


Snails of this species were examined on six occasions. In all 
but one of these examinations sporocysts were found. On each 
occasion a considerable number of the snails were examined with 
much care, the several organs being teased under a lens. None 
of the sporocysts, however, were seen in place, in all cases having 
been found lying at the bottom of the dish in which the snails 
had been dissected. 

The following extracts from my notes made at the time of 


1 Published by permission of Commissioner of Fisheries. 
2? BIOLOGICAL BULLETIN, 


198 


TREMATODE SPOROCYSTS AND CERCARIA. 199 


collecting will give details of frequency of occurrence with such 
other observations as seem to be appropriate. 

1909. July 3, 34 snails examined, no parasites found. 

July 19, 110 snails examined, 21 sporocysts found. The 
sporocysts were inactive and contained tailless cercariz which 
were very active. So far as examined the sporocysts in this lot 


Fic. t. Sporocyst containing cercariz, from Ilyanassa obsoleta; in sea water 
flattened under cover glass. Length 0.86 millimeter. 


contained relatively few cercariz, 9 being the greatest number 
seen, and as few as 2 noted in one sporocyst. 
Three sporocysts lying free in sea water had the following 
dimensions in millimeters: 
NE CTIO CM eer earys eterno aeons eat aren a ave 0.62 0.75 0.88 
Breadthsiy. satis eracaci cise seekswal ore a 0.30 0.26 0.30 
Two cercariz, killed under cover-glass over flame, had the 
following dimensions: 


ILGMRWNs ob oacec 5b Shon NO HONGO Oke Oe OPI aa ear 0.31 0.42 
Breadth antehnOteann ce rrrerctuse sis mie ss sacs s eon ares 0.05 0.07 
mic dene geyeieia te, Sere eacielscensthe eines 0.13 0.14 
POStEHIOns Scuraieeiye ces cess 0.06 0.06 
PTET OLE SU CEL peer cyeyeivareis clcnsy nist susciielone. @ cusioviene 0.04 0.05 
Wenitrallsi eke ryieevene javenevcye el arousl cusvay ors (a dedus Gnatayece 0.07 0.06 


July 20, 120 snails from North Falmouth. A dozen or more 
of these were dissected under a lens in the endeavor to find ex- 
actly where the sporocysts occur but without finding any in 
place. All the snails were then picked to pieces, washed, and the 


200 EDWIN LINTON. 


water decanted. About 100 sporocysts were obtained. These 
lay motionless on the bottom of the dish. They were easily seen 
on a black background, being whitish translucent. They were 
short oblong with rounded ends, often slightly arcuate. 


Fic. 2. Sporocyst from Ilyanassa obsoleta, flattened under cover glass, fixed 
over flame, stained and mounted in balsam. a, cercarie. 6, germinal cell masses 
and young stages of cercarie. Length of sporocyst 0.80 millimeter. 


July 22, about 300 snails from Tarpaulin Cove were broken 
open and about 80 of them removed from their shells. A few of 
these were looked over carefully, much of the material being 


TREMATODE SPOROCYSTS AND CERCARIA. 201 


teased and examined with the aid of the compound microscope. 
No sporocysts were seen in place. Sporocysts were found on the 
bottom of the dish in which the snails that had been removed 
from their shells were lying, also in the dish which held the snails 
still in the broken shells. It would appear that the sporocysts 


Fic. 3. Cercaria in sea water, ventral view, showing excretory vessels in front 
of ventral sucker, rudiments of testes, etc. Length 0.25 millimeter. From 
Tlyanassa obsoleta. 


are rather loosely lodged in the mantle cavity, since they make 
their appearance when the broken shells and partly exposed ani- 
mals are shaken about in the water. Some 600 sporocysts were 
obtained from this lot in a short time in this way. When the 
snails were picked to pieces, washed, and the water decanted, an 
increased number of sporocysts were obtained. The number of 
cercarie in these sporocysts was greater than that recorded in the 


202 EDWIN LINTON. 


lot collected on July 19. One sporocyst when opened liberated 
Ao cercariz. Some of these were immature. An anterior spine 
was noted for the first time on these cercaria. It is embedded 
in the tissues of the head and may be seen protruding its sharp 
tip at the extreme anterior end in certain stages of extension 
while the cercaria is actively contracting. What were taken to 
be excretory vessels were seen extending from near the lateral 
margins of the oral sucker. They appear to unite in front of the 
oral sucker and again behind the ventral sucker. There was a 
large and conspicuous excretory space near the posterior end 
which communicated with the terminal pore by a slender canal. 


Hig hod OO P0000: 1 


Fic. 4. Free-hand sketches of anterior end, life; showing oral spine, minute 
spines on sutface of body, and anterior excretory vessels. a, dorsal view. Di- 
ameter of oral sucker 0.04 millimeter. 6, Ventral view of another specimen. 
Diameter of oral sucker 0.06 millimeter; length of oral spine 0.017 millimeter. 
From Illyanassa obsoleta. 


A cercaria, flattened slightly and fixed over the flame, had the 
following dimensions in millimeters: Length o. 24, breadth o. 10, 
oral sucker 0.041, ventral sucker 0.057, length of anterior spine 
0.020. The posterior end was truncated as if slightly retracted. 

July 23, 50 snails from a small salt water pond were removed 
from their shells and carefully dissected; about 350 others were 
broken open and stirred about vigorously. After a careful 
search 14 sporocysts were found. The pond from which these 
snails came, while salt, did not have free communication with 
the sea. 

In 1910 a lot of snails that had been kept in a dish of sea water 
for several days were opened on different dates with the following 
results: 


TREMATODE SPOROCYSTS AND CERCARI:. 203 


August 24, 24 snails were removed from their shells, picked to 
pieces with forceps, washed, the water decanted, and about 10 
sporocysts found. These were linear oblong, frequently arcuate 
and slipper shaped, thickish. Dimensions in millimeters: 


Wetle that te ys) stances cre Seo 0.66 0.56 0.60 0.56 0.70 


0.55 
BREACHES con accodoooo5. 0.25 0.26 0.25 0.25 


0.33 0.25 


Fic. 5. Cercaria from [lyanassa obsoleta, stained and mounted in balsam, dorsal 


view, showing oral spine, rudiments of prepharynx, pharynx, testes, etc. Length 
0.30 millimeter. 


August 26, 24 snails were examined that had been opened and 
removed from their shells the day before. No sporocysts were 
found. Another lot of 24 were opened and examined on this 
date. No sporocysts were found. 


204 EDWIN LINTON. 


August 29, 36 snails examined in the usual way. No sporo- 
cysts were found. 


The following notes were made on material that had been 
stained and mounted in balsam. 


The shape of the cercariz varies greatly but seems to be due to 


exvU 


Fic. 6. Cercaria from Jlyanassa obsoleta, in balsam. 


Length 0.30 milli- 
meter. 


exv, excretory vessel; m, oral sucker; oe, esophagus; ~, prepharynx; ph, 
pharynx; f, testes; v, ventral sucker. 


different methods of fixing, or, at least to different degrees of con- 
traction. When fixed under pressure they are more or less 


TREMATODE SPOROCYSTS AND CERCARIA. 205 


elongated. When much flattened they are usually long ovate, 
the greatest width being at the ventral sucker, which is near the 
middle of the length, thence they taper towards each extremity 
but more towards the posterior than the anterior end. When 
less compressed they may be long fusiform, or subcylindrical. 
The body is covered throughout with exceedingly minute spines. 
When the cercarie are fixed without pressure they may be ovate, 
short fusiform, always thickish, frequently arcuate, the neck 
especially having a tendency to be bent ventrad. The suckers 
are nearly equal but there appears to be some variation. In 


Fic. 7. Sporocyst with cercarie, from Pecten irradians, life, under slight 


pressure; two cercariz escaping from one end of sporocyst. Length 1.78 milli- 
meter. 


most cases the ventral sucker is slightly larger than the oral. 
The apertures of the suckers are variable depending on the state 
of contraction when fixed. Frequently the aperture of the ven- 
tral sucker is transverse. In one case it was elongated axially. 
The aperture of the oral sucker was in most cases nearly circular. 
The pharynx is subglobular and lies near the anterior border of 
the ventral sucker. The intestinal rami were not distinctly 
shown. The anterior spine is not easily seen in the mounted 
specimens. The stained and mounted material does not usually 
show more of the excretory system than the posterior vessel 
which is very conspicuous in the living worms. In some of the 
mounted specimens this posterior vessel was evident; in others 
it could not be distinguished. In the flattened, oval individuals 
it was not seen (fig. 5). In the cylindrical forms it was usually 
visible (fig. 6). Rudiments of reproductive organs appear in all 
the older cercariz. The most conspicuous of these are two lat- 


206 EDWIN LINTON. 


erally placed subglobular bodies which are situated a short dis- 
tance behind the ventral sucker. These I take to represent the 


Fic. 8. Sporocyst with cercarie, from Pecten irradians, stained and mounted 
in balsam; slightly crushed under the cover glass. a, wall of sporocyst; b, cercariz; 
c, germinal cell masses and young stages of cercariz. Length 0.60 millimeter. 


testes. In front of the left testis may be seen, in some, a granu- 
lar mass opposite the left posterior margin of the ventral sucker 


TREMATODE SPOROCYSTS AND CERCARIA. 207 


(figs. 3 and 5). This is probably the ovary. Behind the testes 
on the median line is a dense granular mass which is doubtless 
the beginning of the uterus. Granular masses which fill the 
body, but are most dense along the lateral margins may represent 
the beginnings of diffuse vitellaria. 

The cercariz of this species resemble Cercaria linearis Lespes, 
but the sporocysts are different. 


2. SPOROCYSTS AND CERCARI4Z2 FROM PECTEN  I[RRADIANS. 
FIGURES 7-—I0. 


In the summer or 1909 I examined 361 scallops on nine dates 
from July 3 to August 27 but found no sporocysts. In August, 
1910, I examined 6 large scallops from Quisset Harbor. They 
had been kept in a vessel of sea water in the laboratory for two 
days before they were examined. After removing one valve the 
animals were shaken vigorously in sea water. A few- small 
sporocysts were found in the bottom of the dish in which the 
scallops had been shaken. The scallops themselves were then 
examined carefully for sporocysts but no more were found. The 
sporocysts were elongate and slowly contractile with a tendency 
to become arcuate. The larger examples at rest in sea water 
measured 0.70 millimeter in length and 0.42 in breadth; length 
of one of the smaller specimens 0.30, breadth 0.15. A specimen 
compressed under a cover glass was 1.78 in length and 0.36 in 
breadth. These sporocysts contained numerous slender, tailed 
cercariz. One of the latter in alcohol was 0.40 in length and 
0.024 in breadth; another, length 0.20, breadth 0.027, length of 
body 0.085, length of tail 0.115. The first sporocyst examined 
had what appeared to be an actively contractile papilla at each 
extremity. These apparent papilla proved to be cercariz par- 
tially liberated from the sporocyst, but evidently held by the 
wall of the sporocyst contracting around them. All the cer- 
cariz, both in the living and preserved specimens, are long and 
slender, the tail, in all cases, except immature specimens, being 
considerably longer than the body. In fully extended examples 
the tail may be two or three times as long as the body. When 
they are liberated from an active sporocyst they exhibit a pecu- 
liar jerking movement of the tail and posterior half of the body, 


208 EDWIN LINTON. 


the anterior end meanwhile being bent ventrad and performing 
a kind of pecking movement. This characteristic behavior of 
the anterior end is plainly in part due to the jerking movements 
of the posterior portions, and in part to the alternate protrusion 
and retraction of a short, proboscis-like organ at the anterior end. 
These movements suggest adaptations to enable the cercariz 
to penetrate the soft membranes of the secondary host. In 
some freshly liberated individuals a thin, hyaline, membranous, 
fin-like border was distinguished. On one of these cercariz this 
membrane was observed to be broken up into slender rod-like 
processes which resembled long cilia. The posterior extremity is 
divided into two slender branches. This forked extremity was 
also seen to be surrounded by a thin membrane in some fresh 
specimens. The structure of both body and tail is coarsely 
granular. Rudiments of what probably represent the oral 
sucker, and the pharynx were distinguished. When a sporocyst 
is crushed, there are seen, in addition to the cercariz, of which 
there may be many stages of development, some granular 
material and balls of cells. 

While the prevailing shape of these sporocysts is long and 
slender considerable variation exists. Both sporocysts and cer- 
cariz are much like those found in the.annelid Hydroides dian- 
thus. The cercaria resembles Cercaria cristata La Valette. 


25 JPRS 


While the examination of numerous specimens of the edible 
mussel (Mytilus edulis) resulted negatively, so far as trematode 
larvee were concerned, a few cysts were noted, some of which may 
have been caused by trematodes. At my request Dr. Irving A. 
Field, who was opening large numbers of mussels in connection 
with his study of the development, and experimental work on the 
food value of this mollusk, handed to me those that in any way 
appeared to be abnormal. The number of such was small. 

On July 24, 1909, 2 mussels were brought to me by Dr. Field, 
which he thought to be in poor condition. They had been 
cooked, so that there was no opportunity to examine them alive. 
One of them had about 15 cysts from 0.5 to 2 millimeters in 
diameter, a’ong the edges of the mantle, and 4 on the foot, 1.5 to 


TREMATODE SPOROCYSTS AND CERCARIA. 209 


2.5 millimeters in diameter. One of these cysts when crushed 
proved to be filled with small granular cells irregular in outline. 
Besides these, 3 small pearls were found in the mantle. There 
were a number of small white cysts embedded in the mantle of 
the other mussel. These contained pearls, 58 small pearls hav- 
ing been obtained from them. Some of them were multiple. 
They measured from 0.3 to 1.12 millimeters in diameter. 

On August rr some small cyst-like yellowish masses of similar 
appearance to those collected on the 24th, were found on the foot 
and mantle of a mussel. Their contents resembled leucocytes. 
A smear preparation revealed round cells of different sizes, the 
prevailing size being about 0.01 millimeter in diameter, with very 
strongly staining nuclei. A very careful examination of over 100 
mussels made on different dates failed to yield any parasites. 

It is perhaps worthy of note that the redia stage is omitted 
from the larval stages of trematode development which I have 
found in the invertebrates of the Woods Hole region. 

Reference may here be made also to another abbreviated 
trematode life history in the case of the distome, Parorchis avitus, 
from the Herring Gull,! where miracidia, still within the ova in 
the later folds of the uterus, contained each a single well-de- 
veloped redia. 

1 Proceedings of the U. S. National Museum, 46: 551-555. 


AN EXPERIMENTAL STUDY OF THE BEHAVIOR 
OF AMPHIPODS WITH RESPECT TO LIGHT IN- 
TENSIEY, DIREGRION TOF RAYS VAN DE 


METABOLISM. 
Cele PTR Ss: 
I. Hm troduction. css eins sd sic see eps Rees eacueuate tenes casiacsaace dl er ieag els eee 210 
VA INTeth ods ic s3 cickadce signer cord eee atnie elms ue ic emcee make a Se 210° 

II. Responses of Untreated Amphipods to Light Intensity and to Direction of 
FRAY Sisija said aioe: 2c tee te Syeee gal Saas te day see ede SoS oiee eel or aera neue ae eee 213 

III. Responses of Treated Amphipods to Light Intensity and to Direction of 
TRAY Sic. 3) aes) gr eta eros Anuar ep chen e|\elaaa ze, acoSs one: sacar eee ee 216 
A‘. Potassium! Cyanide are eae 6 aoe Sas Oot eae ae a eee 216 
BB; Chloretone ys aii. Ook Abeta tear ena ele ahe ictaicle She lreteine (Senne eRe een 216 
( Grples) 21 e172 110 (0) a ee ee nary Co eI ey otai yi a a er emer os renee CEE ANS "Gvo-0''6,"6 e. 217 
De wow Oxygen Contentsi aciacece ser ererates cece suena suo a Sener 217 

IV. Sides of the Experimental Tank, in Relation to the Lamp, occupied by the 
Amphipodssts = ats teins stots Hered Maielete teehee eee eee 210 
View Conclusionsiands Discussions renee aee ence ee eco ee 220 
Al, SiiuTMMATA,, 665 ose ASO APA R a tere gl OU re ite Oe e's, Sg 220 
B. Metabolism, Physiological States and Reactions................ 221 
Bibliography es iedsc a e0 ae eee ee ee US ee 222 


I. INTRODUCTION. 


The object of this study was to determine the effect of reagents 
and conditions affecting metabolism upon the reactions of am- 
phipods to intensity and direction of light rays. The reagents 
and treatments used were potassium cyanide, chloretone, star- 
vation and lowered oxygen content. All experiments were per- 
formed in the laboratory either upon amphipods just brought in, 
or upon those kept in captivity from one to ten weeks. . 

Three species of amphipods found in the vicinity of Chicago 
were used in the experiments, namely, a swift stream species, 
Gammarus faciatus (Say); a sluggish river and lake species, 
Hyalella knickerbockert (Bate); and a pond species, Eucran- 
gonyx gracilis (Smith). 

(A) Methods.—In all the experiments a dark room was used. 
The special apparatus was a light grader, designed and first used 
by Yerkes (’02) and described with diagrams by Mast (’I1, p. 61) 


210 


BEHAVIOR OF AMPHIPODS WITH RESPECT TO LIGHT. 211 


and Shelford (14). In the light grader the animals are kept 
during experiments in a small rectangular tank having glass 
sides. A false bottom in this tank allows running water to pass 
through and thus keep the water the animals are in at constant 
temperature. Midway between this tank and the nernst lamp 
in the grader there is a partition having a triangular aperture and 
this aperture is covered by a lens. By this means an intense 
field of light is made to fall upon one end of the small tank when 
the latter is placed at the focal point of the lens. This field of 
intensity shades off to darkness in the opposite end of the tank 
because of the triangular opening, thus making an intensity 
gradient. The light which passes through the glass sides of the 
small tank is reflected by mirrors to a dead black wall in another 
part of the grader. 

When ready for the experiment the animals were placed in the 
small tank, usually three at a time, and allowed to remain in 
darkness for a short time to recover from the shock of handling. 
Then the light was flashed upon the tank and immediately the 
animals were released from the glass tube with which they had 
been confined. Every thirty seconds the relative positions of 
the animals in the tank were recorded. In part of the experi- 
ments 40 readings each were taken, the first 10 of which were 
discarded because of the excitement of the animals due to hand- 
ling and to the flashing of the intense light upon them. In the 
remaining experiments 25 readings each, with the first five dis- 
carded, were found to be enough to give typical results. 

In each set of experiments the tank occupied by the animals 
was placed in three different positions: position 1, at right angles 
to the direction of the light rays; position 2, at an angle of 45° 
to the direction of rays with the dark end nearer to the lamp; 
position 3, at an angle of 45° to the direction of rays with the 
light end nearer to the lamp. The animals were first released 
in the field of intense light and a series of readings taken; then 
the same animals were released in the dark end, and oftentimes 
also in the place of medium light, the readings being repeated 
in each case. 

About 50 of the 257 experiments performed were eliminated 
because of avoidable errors in preliminary work. The animals 


212 C. F. PHIPPS. 


were selected at random from the pan when experimented upon 
and the reactions under different conditions were compared. 

When treating the animals with KCN and the other reagents 
they were kept in shallow glass dishes with a glass cover plate 
sealed on with vaseline to prevent evaporation. In the ex- 
periments with these animals the same solutions which they had 
been kept in were used in order to avoid the possibility of any 
stimulation which might occur by changing to tap water. The 
untreated. amphipods were kept, and experimented on, in tap 
water. Filtered tap water was used in working with starved 
animals. 

It was necessary, first, to establish a standard for the normal 
reactions of untreated amphipods, and then to compare with 
this any different reactions of treated amphipods. The series 
of experiments on untreated amphipods, by which the standard 
for normal reactions was established, form a good control series 
with which to compare the reactions of treated amphipods. 
Untreated amphipods were considered normal if they were 
negative to intense light when the tank was in position I or 
position 3. They were also considered normal if they were 
positive to intense light when the tank was in position 2. The 
reaction of the animals in position 2 of the tank shows that the 
direction of rays has a stronger influence than light intensity, 
for, though negative to intense light, in no case in any of the 
experiments with untreated animals with the tank in this posi- 
tion did the majority remain in the dark area. We may con- 
clude from this that amphipods are negative to direction of 
rays. With treated amphipods these normal reactions were 
reversed in many experiments as will be shown later. 

It was impossible to separate the influence of light intensity 
from that of direction of rays in cases where the tank was in 
position 3. Normally with the tank in this position both light 
intensity and direction of rays force the animals to the dark end. 
The percentage of those seeking the dark end when the tank 
was in this position was much greater than when the tank was 
at right angles to the rays, showing again the negative reaction 
to the direction of rays. The ray direction does not function 
when the tank is at right angles to it, except possibly to force 


BEHAVIOR OF AMPHIPODS WITH RESPECT TO LIGHT. 213 


the animals to the side of the tank farthest away from the lamp. 
Comparative figures which will be given later indicate at least 
that ray direction may force the animals to the side farthest 
from the lamp. Possibly the data as regards light intensity 
vs. ray direction has been over-emphasized. 

We can base our conclusions definitely on the effect of the 
direction of rays only upon reactions with the tank set in position 
2. The standard for this experimental work is based therefore 
both upon light intensity and direction of rays. 


Il. RESPONSES OF UNTREATED AMPHIPODS TO LIGHT INTENSITY 
AND TO DIRECTION OF RAYs. 


Table I. shows the reactions of untreated amphipods when the 
tank was at right angles to the direction of light rays (position 1). 
Not only are the three species compared but also the stock kept 
in the laboratory several weeks is compared with that freshly 
brought in. 

TABLE I. 
UNTREATED AMPHIPODS. 


Experimental Tank at Right Angles to Direction of Rays (Position 1). 


Normal Reactions. Reversed Reactions. 
Speci In Cap- Read. 
pecies. 5 5 3 
tivity. A Read- Read- 
ity ings. ieee %+.| ga. |%—. ae b+.| ou. |o—. 

Gammarus..... 4 weeks I50 I50 37 | 21 | 42 fo) fo) (0) 

2 days 120 60 27 | 19 | 54 60 54 | 26 | 20 

Hyalella....... 4 weeks I30 130 16 GR Way o}.0 to) 

I day I50 I50 13 || TO) | 9/7/ (0) (0) fe) 

Eucrangonyx...| 6 weeks I50 I50 7 | ux | FO (0) Co) (0) 

1 day 6 | Go | 20] 7 igo ll q| @| © 


+ indicates a positive reaction, « an indifferent reaction, — a negative reaction. 


The strongest negative reaction to intensity is shown by the 
Hyalella and Eucrangonyx species, 73 to 77 per cent. This 
means that in the 150 half minute readings, e. g., Eucrangonyx 
6 weeks stock, only 13 per cent. of the animals were found in the 
area of intense light, 11 per cent. in the medium or dim light 
and 76 per cent. in the dark area. Although 13 per cent. were 
in the field of intense light at the time the readings were taken, 
yet, in most instances, the stay in that area was but momentary. 
If the animals in their ‘‘random movements”’ or ‘‘busy explora- 


214 (Co Ip IPIBGIE IES), 


b 


entered the field of intense light they were plainly stimu- 
lated and usually darted back quickly to the dark area. 

The typical reaction for the species, negative to intense light, 
was that given by a majority of the untreated amphipods in the 
series of readings taken, and the group was considered to give 


tions’ 


reversed reactions only when a majority, in a series of readings, 
was found in the region of greatest light intensity. Such a 
reversal was found with one group of Gammarus, fresh stock, 
where in 60 readings the majority, 54 per cent., were found to 
be positive to intense light, while but 20 per cent. were negative, 
at the time of the readings. Another group of Gammarus fresh 
stock, however, was just as strongly negative to intensity. In 
this case of reversed reaction the animals remained in the inten- 
sity field much more constantly than did those of other groups 
which showed strong negative reactions. These few reversals 
with Gammarus may be due to the intense light, or to some factor 
not recognized. Mast (11) has reported cases where long con- 
tinued or increased light intensity has reversed the phototactic 
reactions of certain animals. The terrestrial form of amphipods, 
Orchestia agilis, is negatively phototactic when first exposed to 
light, but becomes positively phototactic with bright light, the 
stronger the light the quicker the reaction. 

When the tank was placed in an oblique position so that it was 
at an angle of 45° to the direction of light rays with the dark 
end nearer the lamp (position 2), the majority of reactions in all 
the experiments performed were normal. As stated above, this 
normal reaction is based upon both light intensity and direction 
of rays, and as the direction of rays exerts a stronger influence 
than does intensity, the animals were forced to the light end of 
the tank, when the tank was in this position, and so appear 
positive to intense light. The percentages of normal reactions 
(positive readings), with the tank in position 2, are not so high 
as in position I (negative readings). This undoubtedly is due 
to the fact that the influences of intensity and direction of rays 
were working against each other when the tank was in position 2. 

The position of the tank was again changed so that it was at 
an angle of 45° to the direction of rays with the light end nearer 
the lamp (position 3). In all except one series of readings, 


BEHAVIOR OF AMPHIPODS WITH RESPECT TO LIGHT. 215 


namely with Gammarus fresh stock, the large majority of re- 
actions were normal, that is, negative to light intensity and to 
direction of rays. In this one case of reversed reaction with 
Gammarus the same animals were used as in Table I., where a 
reversed reaction is shown. With the tank in position 3 the 
percentages of normal readings were much higher than is true 
of the other positions of the tank. This is due to the combined 
action of intensity and ray direction in position 3, both together 
forcing more animals to the dark or negative end of the tank 
than does intensity alone when the tank is in position 1. The 
average percentage is 7814 negative reaction for position 3 of 
the tank and 661% for position I. 

In the above three positions of the tank a larger percentage of 
animals freshly obtained was negative both to light intensity 
and to direction of rays, in all cases except one series of readings, 
than were the animals kept in the laboratory for several weeks. 
Some factor or factors associated with long captivity apparently 
had an effect in lessening the negative responses of the amphipods 
Possibly the metabolic 
rate was depressed by laboratory conditions causing a tendency 
to a reversal. 


to intensity and to direction of rays. 


Table II. shows the percentage of experiments giving normal 


TABLE II. 
UNTREATED AMPHIPODS. 


Experimental Tank at Right Angles and at Oblique Angles to Direction of Rays 
(Positions I, 2 and 3). 


No. of Experi- Normal Reac- | Reversed Reac- 
ments Per- No. of Read- | tions, Per Cent.| tions, Per Cent. 
formed. mats of Experiments. | of Experiments. 
Laboratory stock....... 33 1,174 100 (0) 
Rico SHOE Koc oceanddaae 27 990 85.1 I4.9 
MR OEAL SH rha. snpst Senora 60 2,164 93.3 6.7 


and reversed reactions with all untreated amphipods, both with 
the tank at right angles and at angles of 45° to the direction of 
rays. 


216 C. F. PHIPPS. 


III. RESPONSES OF TREATED AMPHIPODS TO LIGHT INTENSITY 
AND TO DIRECTION OF RAYS. 


(A) Potassium Cyanide.——Only stock kept in the laboratory 
for some weeks was used in these experiments. Three different — 
strengths of cyanide were tried, N/100,000, N/125,000, and 
N/150,000. Both reversed and normal responses occurred with 
all three. Probably the N/100,000 is not too strong for work 
with these animals and quicker results may be obtained with 
this strength. The animals were kept in the different solutions 
for varying lengths of time, one to nine days. 

In Table III. the reactions of the three species are shown with 
the tank in position 2. In this case the Hyalella gave no majority 
of reversals in any series of readings, though the other two 
species showed strong reversals. Where the animals were ex- 
posed a longer time, as with Eucrangonyx, all the experiments 
gave a majority of reversed reactions. When the tank was in 
position I or 3 the results were very similar to those in position 2. 


TABLE III. 


AMPHIPODS TREATED WITH POTASSIUM CYANIDE. 
Experimental Tank at an Angle of 45° to Direction of Rays with the Dark End Nearer 
the Lamp (Position 2). 


Normal Reactions. Reversed Reactions. 
Species ie (aids Bed ame 
SMES tivity. 3 “lRead- Read Exposed. 
ree, ings h+.|%x. | S—. ings. +. | bo |%—. 
Gammarus...|6 weeks | 320 | 270) 61 | 27 | 12 | 50 0 | 43 | 57 | 1-3 days 
Hyalella..... 2 weeks | 180 | 180] 73 & || Be (0) (0) 0 |2-3 “ 
Eucrangonyx.|9% weeks | 90 Ol ol O Io) ad | ae |) Se loo “ 


(B) Chloretone-——For these experiments a solution of 0.0025 
per cent. was used, which was strong enough to give a very 
perceptible odor of chloretone. The animals were kept in this 
solution from 8 to 12 days before experimenting. This length 
of time exposed undoubtedly was a factor in causing many 
more reversed reactions than with other treatments. 

When the tank was in position 1 the Gammarus showed a 
much larger per cent. of reversed than normal reactions, but 
the other two species were all normal in the majority of readings. 
In the other two positions of the tank all three species had strong 


BEHAVIOR OF AMPHIPODS WITH RESPECT TO LIGHT. 


PN 


reversals, and the Hyalella and Eucrangonyx gave no majority 


of normal reactions in any series of readings. 


TABLE IV. 


AMPHIPODS TREATED WITH CHLORETONE. 
Experimental Tank at an Angle of 45° to Direction of Rays with the Light End Nearer 
the Lamp (Position 3). 


See Table IV. 


; Normal Reactions. Reversed Reactions. 
Guetie In Cap- ao Time Ex- 
S- tivity. = L. osed 
y ings. or G4. | to. +s —. Ae Lt .| ha. |%—. P 
Gammarus...|8—9 weeks} 150 | 60 | 35 | 10 | 55 | 90 | 56 | 17 | 27 |8-12 days 
Hyalella..... Abty oes 60 (0) fo) 0 | 60 | 95 2 3 Oy 
Eucrangonyx.| 1% “ 60 0) o| o| 60 | 57 4 | 36 ove 


(C) Starvation—Amphipods from each habitat were kept in 
filtered tap water and starved from 4 to 14 days before experi- 
menting. Reversed reactions occurred with all three species, 
some after short treatment, others only after long treatment. 
Table V. shows reversed reactions with Hyalella and Eucran- 
gonyx when the tank was in position 2. Gammarus gave reversed 
reactions with the tank in positions 1 and 3, but the majority 
were normal with the tank in position 2. 


TABLE V. 
STARVATION TREATMENT. 
Experimental Tank at an Angle of 45° to Direction of Rays with the Dark End Nearer 
the Lamp (Position 2). 


| Tel Normal Reactions. Reversed Reactions. 
F In Cap- i Time Ex- 
Species. tivity. eae. Read- a+.| da.) %— Read- £4.| Ga.|%— posed 
ngs i ings. 5 
rae a a) 
Gammarus... 6% weeks} 60 | 60 | 56 | 19 | 25 0} oO] o|4 days 
Hyalella..... 7 a 120 | 60 | 71 8 | 24 | © | 383 | au | ar iGo % 
Eucrangonyx.\13-3 “‘ AG) || OO) || AG || Bie |) 26) |) CO |) B® || wH |) Sie gaia 


(D) Low Oxygen Content—The oxygen content of tap water 
was reduced by using a machine, devised by Shelford and Allee 
(13), for deaérating water by a process of heating and then 
cooling to the required temperature. The oxygen content was 
reduced in some experiments to as low as 0.79 c.c. per liter. 
The dish containing the amphipods was filled with this low 
oxygen water and the cover sealed down with vaseline. Amphi- 


pods began to die after an exposure of about one day to low 


218 C. F. PHIPPS. 


oxygen content of from 0.79 c.c. to 1.51 c.c. per liter. Only 
Gammarus gave a majority of reversed reactions in any series 
of readings (see Table VI.), and this took place with the tank 
in positions 1 and 3. The other two species gave no majority 


of reversed reactions in any position of the tank. 


TABLE VI. 


Low OxyYGEN CONTENT. 
Experimental Tank at Right Angles to Direction of Rays (Position 1). 


Normal Reactions. Reversed Reactions. ‘ 
we Total ee me of 
tock. : x- 2 per 
Readings. Her ae | cree |e ee d+.| be. |6— posed Liter. 
Gammarus... I20 60 (0) 5 OF 60 | 67 | Ir | 22 |r0 hrs.| 0.79 
Hyalella..... 60 60 | 34 | 18 | 48 (0) fo) O}|s7aeaen I.51 
Eucrangonyx. 60 60 | 23 | 18 | 50 0 0) o |22% “ | 0.99 
In Table VII. the percentage of experiments is given showing 


normal and reversed reactions with all treated amphipods, both 
with the tank at right angles and at angles of 45° to the direction 
of rays. 

TABLE VII. 


TREATED AMPHIPODS. 


All Three Species. Tank in Positions I, 2 and 3. 


Normal Reversed 
No. of No. of Reactions, Reactions, 
Treatment. Experiments Readings. Per Cent. of Per Cent. of 
Performed. Experiments. | Experiments. 
Potassium cyanide..... 54 I,520 BB 27.7 
Chloretoneanenseeeeer 27 810 44.5 55-5 
Stanvavloneeare eee 20 900 69.0 31.0 
Low oxygen.......... 22 720 68.2 31.8 


Table VIII. shows that the percentage of reversals in the three 
species is quite different under the same treatment. Also, in 
the same species, the percentage of reversals varies for each 


reagent used. 
TasLeE VIII. 


TREATED AMPHIPODS. 
Reversed Reactions only. 


Gammarus, Hyalella,4 Eucrangonyx, 
Per Cent. of Per Cent. of Per Cent. of 
Experiments. Experiments. Experiments. 
Reversed reaction with KCN......... 39.3 Gols Gigi) 
ss - “Chloretone.... 60.0 66.6 Baoe 
s “ ““ Starvation.... 66.6 18.2 25.0 
ee +4 “Low Oxygen... 36.3 50.0 0.0 


BEHAVIOR OF AMPHIPODS WITH RESPECT TO LIGHT. 219 


IV. SIDES OF THE EXPERIMENTAL TANK, IN RELATION TO THE 
Lamp, OCCUPIED BY THE AMPHIPODS. 


At each reading, when the position of the animals in relation to 
intensity was taken, their position in relation to the sides of the 
tank was also taken. The object was to determine, if possible, 
whether the direction of rays influenced the animals to seek the 
side of the tank farthest from the lamp. 

The results are shown in Table IX. The readings have no 
reference to the dark end of the tank. In positions 1 and 2 the 
largest per cent. of the animals was found on the side of the tank 
farthest from the lamp, while in position 3 the majority were 
found on the side nearest the lamp. This is not conclusive, 
however, and these lateral positions in the tank may be due 
only to ‘‘random excursions’’ or ‘“‘busy explorations’”’ that 
Holmes (’o1) speaks of as characteristic of active animals. 
Possibly the animals were reacting towards their own shadow, 
in positions I and 2, rather than to ray direction. It is interesting 
to note that there is very little difference in the results between 
treated and untreated amphipods. 

In none of the experiments was there evidence of orientation 
either to light intensity or to direction of rays. 


TABLE IX. 


TANK AT RIGHT ANGLES TO DIRECTION OF RAYS (POSITION 1). 


Side Ueaee from Mean: Dacition! en ae to 
Untreated Amphipods...... 48.5% TUM 40.2% 
Treated Rey Resa ete 49.2 14.9 35-9 


TANK AT ANGLE OF 45° TO DIRECTION OF RAYS WITH DARK END NEARER THE 
LAMP (POSITION 2). 


Untreated Amphipods...... | 52.0% 7.7% | 39.4% 
Treated RMON Ee ae 38.2 


TANK AT ANGLE OF 45° TO DIRECTION OF RAYS WITH LIGHT END NEARER THE 
Lamp (POSITION 3). 


Untreated Amphipods...... | 35.7% | II.5% | 52.8% 
Treated Gober Wie 50.4% 


220 @) ha PHLPPS:: 


V. CONCLUSIONS AND DISCUSSION. 


(A) Summary.—t. In an experimental tank set at right angles 
to the direction of light rays and graded from intense light to 
darkness, pond, stream and river amphipods, as a group, seek the 
dark area, therefore are negative to light intensity. 

2. When the same tank is set obliquely, at an angle of 45°, to 
the direction of rays with the dark end nearer the lamp, the 
amphipods are forced to the light area, even though they are 
negative to intense light. The stimulus of the direction of rays, 
to which the amphipods react negatively, has a stronger effect 
than the stimulus of light intensity. 

3. If the experimental tank is set at an angle of 45° to the 
direction of rays so that the light end is nearer the lamp, normal 
amphipods, as a group, seek the dark end. In this case the 
direction of rays exerts the same stimulating effect as light 
intensity in forcing the animals to the dark area. This again 
shows a negative reaction to intensity and to direction of rays. 
A larger percentage show negative reaction, with the tank in this 
‘position, than when the tank is at right angles to the light rays. 

4. When treated with certain depressing agents many of these 
amphiphods become reversed in their reactions to light intensity 
and to direction of rays. : 

5. Freshly obtained amphipods give a larger percentage of 
negative reactions, both to intensity and direction of rays, than 
do amphipods which have been kept in the laboratory for some 
time. 

6. In these experiments there is no evidence of orientation of 
amphipods either to light intensity or to direction of rays. 

7. Changes in the metabolic processes (physiological states) 
of the amphipods were undoubtedly the cause of reversed 
reactions in this series of experiments. 

Some of the above results have been obtained also by other 
investigators. Holmes (oI) says that all aquatic amphipods 
studied by him were negatively phototactic, although three 
species of land amphipods studied were positively phototactic. 
Loeb (’04), writing about experiments on Gammarus pulex and 
other animals, says that ‘‘whatever increases the activity tends 
to increase the positive reaction to light, while anything which 


BEHAVIOR OF AMPHIPODS WITH RESPECT TO LIGHT. 221 


tends to quiet the animals tends to make them negative.”’ He 
adds also that the Gammarus pulex, which is negative to light, 
can be made positive by adding to the water a little carbon 
dioxid, hydrochloric, oxalic or acetic acid, ether, chloroform, 
paraldehyde, alcohol, esters and all ammonium salts. Boracic 
acid, according to Loeb, does not reverse these amphipods, but 
Jackson (’10), in repeating Loeb’s experiments, but using Hyalella 
knickerbockert, found that a saturated solution of boracic acid 
does cause a reversal, the same as the other reagents. Jackson 
also found that some other acids and some alkalies produce the 
same effect. These reversals took place, however, only when 
he dropped the animals into the solution, for when he put the 
animals into distilled water and gradually added the chemicals 
no reversals took place. 

McCurdy (’13) says that ‘‘sunlight modifies the normal physio- 
logical changes taking place in protoplasm, checking some of the 
processes and probably accelerating the others. A starfish in 
the light moves to the shade because of disturbance by light of his 
metabolism.” A part of this disturbance was due to there 
being ‘‘less COs given off by the starfish when it was put in the 
sunlight.” 

(B) Metabolism, Physiological States and Reactions.—Before 
answers can be given to many. questions that arise, much more 
work along these lines must be done. Other methods and treat- 
ments must be used, such as high oxygen content, caffein, acids, 
alkalies, carbon -dioxid content, etc. | 

From the data obtained from this series of experiments it is 
evident that the responses of aquatic amphipods, like those of 
many other animals experimented upon, are related directly 
to the physiological state or condition of the animal. Anything 
which disturbs the rate of metabolism of the animal alters the 
response to stimuli. Allee (’12) with isopods, Child (10) with 
planaria, Wodsedalek (11) with may-fly nymphs, and other 
investigators have found this to be true. 

In these experiments on amphipods then, the reversed re- 
actions are caused by some change in the metabolic processes 
of the animals. Potassium cyanide depresses the metabolic 
processes by decreasing oxidation. Oxidation is decreased by 


222 C. F. PHIPPS. 


decreasing the ability of the tissues to take up oxygen. Chlore- 
tone is a soporific and has a depressing or inhibiting effect upon 
certain metabolic processes. Starvation decreases metabolism 
by removing the material to be oxidized. Such reagents and 
treatments are known to have specific effects on metabolism 
and they also cause reversals in phototaxis, therefore the re- 
sponses are related to the metabolic rate of the animal. 

Jennings (’o4) says that physiological states are the most 
important determining factors in reaction and behavior. By 
physiological states he means the varying physiological condi- 
tions as distinguished from permanent anatomical conditions. 
Can we be sure that such physiological states do exist? If we 
subject animals to the same external conditions and give the 
same stimulus, and the animals react differently, then the 
difference must be due to variations in internal conditions; else 
the reactions would always be the same. A stimulus changes 
the physiological state of the animal as a whole, and this change 
in physiological state induces a certain type of reaction. 

My thanks are due to Dr. V. E. Shelford under whom this 
work was done, and to Dr. W. C. Allee and Mr. M. M. Wells 
for valuable suggestions. 


BIBLIOGRAPHY. 
Allee, W. C. 


’32. An experimental Analysis of the Relation between Physiological States and 
Rheotaxis in Isopoda. Jour. Expt. Zool., Vol. 13, No. 2. 
Banta, A. M. 
’13. Experiments on the light and tactile reactions of a cave variety and an 
open water variety of an amphipod species. Proc. Soc. Exp. Biol. and 
Med., Vol. 10, May 21, 1973. 
Cushny, A. R. 
*t0 Pharmacology and Therapeutics. t1oth ed. 744 pp. Phil. 
Holmes, S. J. 
701 Phototaxis in Amphipods. Am. Jour. Physiol., Vol. 5. 
203. Sex Recognition among Amphipods. BiIoL. BULL., Vol. 5. 
?05 The Selection of Random Movements as a Factor in Phototaxis. Jour. 
Comp. Neur., Vol. 15, pp. 98-112. 
Jackson, H. H. 
’r0 Control of Light Reactions in Hyalella. Jour. Comp. Neur. and Psy., - 
Vol. 20. 
Jennings, H. S. 
704 Physiological States as Determining Factors in the Behavior of Lower 
Organisms. Carnegie Inst. of Wash. Publ. 16, p. 100. 
’06 Behavior of Lower Organisms. Columbia Univ. Press. 


BEHAVIOR OF AMPHIPODS WITH RBSPECT TO LIGHT 223 


Loeb, J. 


204. The Control of Heliotropic Reactions in Fresh Water Crustaceans by 
Chemicals. Univ. Calif. Publ. Physiol., Vol. 11, No. tf. 
Mast, S. O. 
‘tz Light and the Behavior of Organisms. J. Wiley & Sons, N. Y. 
Saunders, W. J. 
’11 A study of the Behavior of Amphipods with Notes on their Life Histories. 
Unpub. Master’s Thesis, Lib. Univ. of Chicago. 
Shelford, V. E. 
’13) Animal Communities in Temperate America. Univ. Chicago Press. 
*14 An Experimental Study of the Behavior Agreement among the Animals of 
an Animal Community. BIoL. BULL., Vol. 26, No. 5. 
Shelford and Allee. 
’13 The Reactions of Fishes to Gradients of Dissolved Atmospheric Gases. 
Jour. of Exp. Zool., Vol. 14, No. 2. . 
Weckel, A. L. 
207 The Fresh Water Amphipods of North America. Proceed. of U. S. Nat. 
Mus., Vol. 32. 
Wodsedalek, J. E. 
’11 Phototactic Reactions and their Reversal in May-fly Nymphs. BIOL. 
BULL., Vol. 21. 5 
Yerkes, R. M. 


202 Reaction of Daphnia pulex to Light and Heat. Mark Anniversary Volume. 


A CASE OF PERSISTENT MELANISM. 
H. E. EWING. 


The occurrence of melanism is a phenomenon of wide dis- 
tribution in nature, being recorded among animals belonging to 
a great many classes and orders. Our records show that while 
the occurrences are frequent and are found in species belonging 
to many of the larger zodlogical groups, yet the actual numbers 
of melanic individuals found among the individuals of any one 
species, in any one region is usually extremely small in com- 
parison with the total number of normally colored individuals 
of the same species found in the same region. Because of this 
rarity of these black-colored individuals the appearance of 
melanic forms has been very generally regarded as being due to 
sporadic though at times oft-repeated, sporting. Such melanic 
forms do not usually persist racially. It was Darwin who 
years ago noted that sports of almost all kinds were ruthlessly 
eliminated in the struggle for existence; and sporting in the 
form of melanism apparently has offered no exception to this 
general rule. 

Perhaps one of the best examples of the racial persistence of 
melanism is that of the melanic form of the moth, Amphidasys 
betularia, which existed as a rarity in England some years ago, 
but which now has replaced the typical form about some of the 
manufacturing districts. This persistence of melanism has been 
explained on account of the environment in these districts being 
changed by the smoke from factories which darkens the vegeta- 
tion in general, by the killing of lichens and by the depositing 
of black soot, and in this manner gives an advantage to the 
melanic forms by making them less conspicuous than the normally 
colored individuals. 


THE APPEARANCE OF MELANIC ROSE CURCULIOS. 


During the summer of 1913, while in the Willamette Valley in 


western Oregon, I came across instances of melanism among 
224 


A CASE OF PERSISTENT MELANISM. 225 


individuals of one of our common beetles that were quite striking. 
We have in the Willamette Valley, as in almost all other sections 
of the country, the well-known rose curculio, Rhynchites bicolor 
Fab., which feeds chiefly upon the buds and flowers of wild and 
of cultivated roses. It will feed, however, upon a few other 
plants, especially the buds of wild blackberries, which grow so 
abundantly along the streams in western Oregon. 

This weevil is about one fourth of an inch long, and in all the 
sections of the country where it has been observed outside of the 
Willamette Valley is of a red color above; while the underparts 
of the body, and sometimes the head and beak, are black. The 
red in some instances extends forward so as to include the head 
and beak. When viewed feeding on the roses, the dark under 
surface of the body is largely concealed so that the weevil 
appears almost entirely red. 

The red color of the dorsal surface is possibly protective to 
the species when it is feeding on the petals of wild or cultivated 
roses, as the color harmonizes with the red of many roses, and 
for this reason might make the individuals much less conspicuous 
‘objects to hungry birds and other enemies. 

I found feeding along with the red individuals of Rhynchites 
bicolor Fab. individuals which were totally black. At first I 
suspected that these black individuals were of a different species. 
Upon looking the matter up I found that they had in a few 
instances been collected, and were called Rhynchites eneus Fab., 
a black species, which is common in the eastern part of the 
country. 

During the summer of 1913 I demonstrated that the two forms 
would breed together in captivity. However, of the several 
larve that I obtained from the eggs deposited none reached 
maturity. Following these experiments I made a critical study 
of the characters of our two forms found in Oregon, and failed to 
observe any differences in structure whatever, hence considered 
the black ones only as melanic individuals of the common rose 
curculio. In order to get the opinion of a specialist in Coleoptera, 
I showed specimens of the two forms to Dr. Van Dyke, of the 
University of California. He stated that the two were the same, 
and that the black form found in Oregon was not the Rhynchites 


226 ! H. E. EWING. 


@neus Boh. of the eastern states. I may here add that the real 
Rhynchites @neus Boh. is a very hirsute species; it has well- 
developed punctate striations on the elytra, and prominent 
marginal elytral grooves. Our black Oregon form has none of 
these characters pronounced,—the hairs are very small, the 
punctations in the elytral strie are almost obsolete, and the 
marginal grooves of the elytra can hardly be noticed. 


MATING AND OTHER HABITS. 


During the spring and summer of 1914 I made many field 
notes on these two forms of Rhynchites. I found that the two 
were constantly associated. They fed together in the same way. 
on the same rose bushes and even on the same buds. They 
were found to feed together on wild blackberries; they emerged 
from winter quarters at the same time, and, finally, they were 
repeatedly found to be interbreeding in nature. 


ATTEMPTS AT REARING HYBRIDS. 


Four of the individuals of the black form which were found 
mating in nature with red individuals were confined with their 
red mates in separate breeding cages. Here they continued to 
breed for over a month, after which they began to die. In the 
meantime the females had laid large numbers of eggs; but out 
of this large number, including several score, I am very sorry to 
state that I did not succeed in rearing a single individual to 
maturity. The whole trouble this year was that the buds of 
the roses soon died after they were punctured by the female, 
and fell to the ground, taking with them the developing larve. 
In these dead, shriveled rose-buds the larve invariably died. 
Even their transference to fresh rose-buds was of no use, for 
they would not stay in these buds when placed there artificially. 
None of these larve pupated. 

It may be well to state that in all the literature which I have 
examined I have failed to find a single record of this weevil 
being reared from the egg to its adult state. Its life history 
has not been worked out by complete breeding experiments, 
although repeated field observations, together with fragmentary 
notes on the life history, have shown that it produces but a 
single brood a year and hibernates in the adult stage. 


A CASE OF PERSISTENT MELANISM. De7 


MATING STATISTICS. 


Having been foiled in my attempts at breeding these beetles, 
I decided to attack the problem from a different standpoint, 
namely, to observe the mating habits in nature, to see if the 
black individuals mated as freely with the red ones as with 
those of their own.color. During the months of May and June 
I made fourteen different observational trips to some patches of 
wild roses at the west end of the town of Corvallis, Oregon. I 
collected every pair that was observed mating, as well as those 
that were not observed mating; these were collected so as to 
obtain population statistics. The results obtained were as 
follows: o 


Motalenitimpecmotmacines ObSsenvedeinee aA 0 ste eine ei erie creer eae 53 
Normal Rhynchites bicolor X normal Rhynchites bicolor .............++++4+05. 4A 
IMI@laNe Torin S< TAS eNC HOM. gssbous0c0doeugouunuEuoUoSOGOC ig CeEAS ence te) 
INjomangll JR, Opeakor Gt S< iaSlasone tori Og, Goboncbancengoouuctuaosnouosgacucs 3 
iMiglaiG oH XX imorminall IR, WAeolor? QD ocinaarsdcasucdosusosoee AOR Se kik 5 eee 9 


Thus we find that out of the total number of matings observed 
in nature, 83 per cent. were between normally colored individuals, 
while 16 per cent. were between melanic individuals and normal 
ones; no matings being observed between two melanic forms, 

Although we can not prove anything in regard to the exact 
status of these two forms of Rhynchites from these observations 
alone, yet we may speculate, somewhat, in regard to a few points. 
They tend to indicate: 

1. That the normal intergeneration of these two forms is 
followed by the segregation of the characters following the 
dominance in the first cross. 

2. That the black form is recessive to the red. 

3. That racial characters have become fixed without natural 
barriers, without isolation of any kind or without change of 
habit but purely through the segregation of the characters in 
the germ plasm. 

POPULATION STATISTICS. 


The population statistics obtained for the same patches of 
wild roses that the mating statistics were obtained from, and 
at the same time are as follows: 


228 H. E. EWING. 


IRS GH ILESHOLCOLOTALIA De, p11 © E012) ltemen eerie ae ea ee eee eta eee 588 
i BY Fe Qed ap a) cin 0 AMES ee een eS, al HL seni cena) Each an LU OAHU RIAD Koala tol A'S! gh.a\Gco.10 0d 68 
Total for both: fOpmMS eee el ieee ieee eee Goh. Serena Mien eate en esse ek eee 656 


Thus out of the total of 656 individuals counted, 68, or I1 
per cent. were melanic. This percentage is so large that I 
think that no one would suspect the occurrence of melanism in 
this species to be due to the sudden sporting of a great many 
individuals, but rather to the persistence of melanism through 
segregate inheritance of the melanic types from a few or even 
a single sport progenitor. In other words either melanism or 
other characteristics associated with it in this instance have 
been advantageous to the black forms under the particular 
conditions of the Willamette Valley to such an extent that a 
black or melanic race has been evolved in direct competition 
with the normal type of the species from which it sprang, and 
has not been ruthlessly eliminated as a race as is usually the 
case with melanic forms. 

The future of this black race of rose curculios will be interesting 
to watch. I hope to be able soon to breed these forms success- 
fully, and also hope to be able to ascertain what the conditions 
are in the Willamette Valley which apparently make melanism 
advantageous to this curculio. A description of this melanic 
form is here given. Even in the Willamette Valley its distribu- 
tion differs from that of the normal R. bicolor. aH 


Rhynchites pullatus new variety, or species. 


All the individuals as yet observed are black throughout. 
Body and appendages clothed with very fine short hairs which 
are not noticeable with the naked eye. Snout subequal to the 
tibia of the first pair of legs in length, bent almost straight down- 
ward in the male, but extending forward so as to be plainly 
visible from above in the case of the female. The antennz 
arise from a position a little in front of the middle of the snout. 
in the male and a little behind the middle of the snout in the 
female. Antennz well clothed with hairs; three distal segments 
much broader than the others and forming a club. Dorsal sur- 
face of thorax punctate. Around the margins of each elytron 
is a distinct groove. The longitudinal punctate lines are rather 
indistinct, and may be wanting. Femora of anterior legs some- 
what swollen. Length of body excluding snout, 5.5-7.0 mm. 


STUDIES OF FERTILIZATION. 


VII. ANALYSIS OF VARIATIONS IN THE FERTILIZING POWER OF 
SPERM SUSPENSIONS OF ARBACIA. 


FRANK R. LILLIE. 


Il, INSABROWWCTUCINs Go 6b os0GsGo0b 0s a EIA ROR i 0 2 Sth yO eR NE oR 2290 
Il. TESSETDIBISUADT ESTE SN SAU oh Saeko gc Rete a. CU Os (OA a aaa 231 
Teme VIC LOOSE. eres none e seats tecstrchiae torte etchrs cd salto olga fommineseatel tage teres ebouaiiews, auohc heise 231 
D, Wine Onions (CLI Of IDARITOTIS soo be6sorc cbse cose ocoacooosobOCDD 233 
3. Curves of Successive Half-Dilutions...............-+1+--++-222- +e 235 

4. Time as a Factor in the Fertilizing Power of Sperm Suspensions of 
Dutienent CONCENITALIONS radii ene ee tore enrol ent oa 239 
5. Other Factors in the Fertilizing Power of Sperm Suspensions........... 244 
ALND TSCUSSEO Niswarsyenatern seme neye raises woes se lel or cnleirephaager alan els lejensr Sete etred ae ee taafogey eo cere cegalca)telts fe 246 


J. INTRODUCTION. 


In his epoch-making ‘‘Expériences pour servir a l’histoire de 
la génération des animaux et des plantes’”’ published in 1785 the 
Abbé Spallanzani describes among his numerous experiments on 
fertilization and artificial parthenogenesis some determinations 
concerning the minimal quantity of sperm necessary to fertilize 
the eggs of the frog. He found that he could get perfect fertili- 
zation with seminal fluid diluted 2,720 times with water. At 
greater dilutions the percentage of fertilized eggs began to fall 
off, but some eggs fertilized up to a dilution of about 20,000 
times. He calculated that the weight of the ‘spermatic par- 
ticles’ necessary to fertilize an egg was 1/2,994,687,500 of a grain, 
and that the volume of the egg in proportion to the volume of 
spermatic particles necessary to fertilize it is as 1,064,777,777: I. 
In 1824 Prevost et Dumas confirmed these calculations. 

So far as I know such experiments have not been repeated. 
The reason for this would appear to be that when it was once 
established that only a single spermatozoén unites with each 
ovum in fertilization all such quantitative studies of the fertilizing 
power of sperm dilutions appeared to have lost their point. So 
long as it was assumed (as was generally the case) that the fertiliz- 


ing power of the spermatozoén is a function of its motility 
; 229 


230 FRANK R. LILLIE. 


alone, that is, of its capacity to ‘‘penetrate the ovum,” there 
could be no object in quantitative studies. But as it came to 
be recognized that the fertilizing power of the spermatozoGn is 
associated with some definite substance that it bears, possibly 
either a lysin (Loeb) or an activator (self), the problem assumes 
a different aspect; for it is obvious that if the sperm should 
lose such a substance in any way, its fertilizing power would be 
lost even though its motility should be preserved unimpaired. 
In such a case the relative fertilizing power of sperm suspensions 
could not be measured either in terms of concentration or of 
activity of the spermatozoa. Variations in the fertilizing power 
of suspensions of known concentrations might, therefore, be a 
measure of the loss of the postulated fertilizing substance. On 
reflection it is obvious that the spermatic substance in question 
must be loosely bound to the sperm, because it exerts its first 
effect, that of inducing cortical changes in the egg, before pene- 
tration, as I have shown for Nereis, and Loeb for certain hybrid 


_combinations; at this time, therefore, the spermatozo6n must set 


free its receptors! (activators). 

Recently Glaser (1913 and 1915) has maintained that in 
Arbacia more than one spermatozo6n is needed for fertilization 
of the egg, even though only one actually penetrates. The 
observations on which this conclusion rests are no doubt correct, 
under the given conditions, and I have made similar observations, 
as will appear in the course of the present paper. But it by no 
means follows from the observations that a single spermatozo6n 
may not be adequate under other conditions (and this can be 
demonstrated). We cannot, however, deny a priori the possi- 
bility that for the initial phases of fertilization a number of 
spermatozoa may be of assistance though only one enters and 
is concerned in later phases. If the phenomena of fertilization 
are to receive a physiological, and ultimately a chemical, inter- 


1In study VI. (1914), I propounded a theory of fe1tilization according to which 
the initiation of development of the egg is due to activation of an ovogenous 
substance, which I named fertilizin, contained in the cortex of the egg. In fer- 
tilization such activation is caused by a certain constituent of the sperm, which I 
called the sperm receptors; and the action of the fertilizin thus aroused must be on 
certain substances of the egg which I named in general egg-receptors. From a 
chemical point of view therefore we must have an interaction of three substances 
(or groups of substances), viz., sperm receptors, fertilizin, and egg receptors. 


STUDIES OF FERTILIZATION. 231 


pretation, quantitative questions may be of serious significance. 

It would seem to be a perfectly simple matter to determine 
the greatest dilution of sperm at which any fertilization takes 
place, and to express in the form of a curve, from percentages of 
eggs fertilized, the rate of loss of fertilizing power due to dilution. 
This was the very simple problem with which the present investi- 
gation began. However the results were in the highest degree 
contradictory; the same lot of sperm might vary in a period of 
half an hour from 1/1,024 to 1/9,000,000 (or less) of 1 per cent. 
dilution in its power to fertilize the same percentage of a single 
lot of eggs. The investigation, therefore, turned to the problem 
of such variations and their cause. 


Il. EXPERIMENTS. 
1. Methods. 


Quantitative methods cannot possibly be as rigorous in a 
problem of this kind as in a purely chemical problem. In the 
first place we have to deal with variable reagents in the ova and 
sperm of Arbacia; and in the second place the initial measure- 
ments must be made rather hurriedly, so as to ensure freshness of 
the reagents, and under conditions that do not injure their 
vitality; the available quantities of material also limit the 
methods of measurement. 

Sperm.—The standard for measurements of sperm dilutions 
is the “dry sperm”’; 2. e., the thick creamy mass that exudes 
_ from ripe testes of Arbacia. If a ripe male be opened and in- 
verted in a dry Syracuse watch crystal a certain amount entirely 
free from foreign admixture usually flows from the genital pores 
and collects in amass in the crystal. While this may in certain 
cases be as much as 2 c.c., usually it is a much smaller quantity. 
It is quite impracticable to measure this by graduated pipettes; 
I have therefore used a drop of this dry sperm from bulb pipettes 
of fairly uniform openings as a standard, and, reckoning 30 such 
drops to the cubic centimeter, have made “I per cent. sperm 
suspensions’’ by the addition of such a drop to 3.3 c.c. of sea 
water. This is the standard suspension from which most of 
the experiments proceed, and all sperm suspensions are expressed 
in fractions of such a I per cent. suspension. Given perfectly 


232 FRANK R. LILLIE. 


dry sperm, the initial variation due to the method cannot be 
very great in relation to the tremendous range of variation in 
fertilizing capacity of sperm due to other causes. Indeed it is a 
vanishing quantity. 

Eggs Egg concentration is a factor of relatively slight 
significance within the limits of the experiments, as will appear 
from the facts to be presented. Within a very wide range it 
does not affect the result measured in percentage of fertilized 
eggs. It is measured roughly by allowing washed eggs to settle 
for half an hour in a 100-c.c. graduated cylinder, and expressing 
the quantity there settled as a percentage of the entire fluid. 
There is of course for every concentration of sperm an egg con- 
centration that is above the optimum for percentage of fertiliza- 
tion. But, as will be seen from the tables, such egg concentration 
lies beyond the concentration used in most experiments. 

In most cases segmentation of the eggs was used as the criterion 
of fertilization, but membrane formation was also used in some 
cases, especially in high concentrations of sperm where many 
eggs failed to segment owing to polyspermy. 

Formulation of Results —The fertilizing power of sperm suspen- 
sions is expressed in curves whose ordinates are percentages of 
fertilization, and the abscisse a geometrical series of dilutions 
of I per cent. sperm in powers of 2. This method was adopted 
for the abscissee because of the method of successive half dilu- 
tions used in many experiments, and because the enormous range 
of fertilizing power made it impossible to compare results on 
one scale with an arithmetical progression. When it is realized 
that the fertilizing power may cease at 1/156 of I per cent., or 
extend to 1/90,000,000 the necessity of the geometrical series in 
the abscissz will become apparent. 


2. The Optemum Curve of Dilutions. 


We may begin with the optimum curve of dilutions (Curve 1), 
because this answers most completely, and probably fully, to the 
current expectation that a single spermatozo6n suffices for the 
fertilization of an egg. This curve is prepared from data of ex- 
periments calculated to bring eggs and sperm together in the 
freshest possible condition of the sperm. In general measured 


STUDIES OF FERTILIZATION. 233 


quantities of washed eggs were put in measured amounts of sea- 
water, and measured quantities of definitely calibrated sperm 
suspensions added and stirred in as uniformly as_ possible. 


BY 23 BAL Bis BO) Diy 


I9 20 21 


: i 
ieeeee 
Cee 


LIN 


I5 16 17 


14 


T3 


© i@) i 
: : 


12 
CURVE I. 


8 


: 
: 
L. 


100 
9 
8 
70 
60 


A control of unfertilized eggs in sea-water was always kept to 
guard against chance fertilizations. To illustrate: the last four 
determinations of the curve were made as follows: In four 


234 FRANK R. LILLIE. 


crystallization dishes were placed 1,000 c.c. sea-water (A), 
3,000 c.c. sea-water (B), I,000 c.c. sea-water (C), 3,000 c.c. sea- 
water (D). To each was added 2 c.c. of a washed egg-suspension 
(about 3 per cent. to 5 per cent.). The sperm was then pre- 
pared as follows: (1) one drop dry sperm to 3.3 c.c. sea-water at 
9.43 A.M. =1 per cent:; @) I cc. of sperm 1 to 90) Ge. )sea- 
water 9.43.30 A.M. = 1/100 per cent.; (3) I c.c. sperm I to 999 c.c. 
sea-water 9.45.30 = 1/1000 per cent. To A was added I drop 
sperm 2(1/100 per cent.) at 9.43.45; to B one drop sperm 2(1/100 
per cent.) 9.44; to C one drop sperm 3(1/1000 per cent.) 9.45.45; 
to D one drop sperm 3(1/1000 per cent.) 9.45.45. An assistant 
stirred in the sperm thoroughly as added. The sperm concentra- 
tion in A was therefore 1/100 X 1/30 X 1/1000 = 1/3,000,000 
per cent.; in B it was 1/9,000,000 per cent.; in C 1/30,000,000 
per cent.; in D 1/90,000,000 per cent. 1/3,000,000 per cent. 
falls between 21 and 22 on the scale, and the others as shown. 
The exact times of mixing the sperm are given because, as will 
appear beyond, time is an extremely important factor with 
reference to fertilizing power. 

To appreciate the extent of this dilution it may be said that 
beyond a dilution of 1/10,000 per cent. (between 13 and 14 on 
the scale) one can rarely find a single spermatozo6n in the jelly 
of the fertilized eggs. At about 1/2000 per cent. (1I on the 
scale) the sperm suspension does not even appear opalescent. 
We may therefore feel reasonably sure that beyond about 14 or 
15 on the scale a single spermatozo6n certainly suffices to com- 
pletely fertilize an egg. 

In further elucidation of the curve I may say that the critical 
(steep) part was covered by several determinations for each 
point. Thus there are five determinations averaged for the 
positions between 13 and 15. Seven between 15 and 18, five 
between 18 and 20, and six between 20 and 21. The determina- 
tions beyond 21 are single determinations. For the first part 
of the curve up to 13, there are numerous determinations. There 
are great variations in the single determinations compared with 
one another; these averages must therefore be regarded only as 
approximate values. With a sufficiently large number of deter- 
minations the irregularities between 15 and 17 and between 19 


STUDIES OF FERTILIZATION. 235 


and 22 would no doubt disappear. But it is improbable that 
the general form of the curve would undergo any essential 
change even with a much more extensive series of determinations. 


3. Curves of Successwe Half-dilutions. 


In contrast to these results, and for the purpose of defining 
the character of the main problem sharply, we may next con- 
sider the fertilizing power of a series of half dilutions of a I per 
cent. sperm suspension. The curves from these experiments 
furnish an almost incredible contrast to the one already given; 
as an example we may examine the following strikingly regular 
curve, Fig. 2. The first member of this series was a 1/8 per cent. 


OME One ee ANE SY Olin’) Seer One LONae TL 


sperm suspension freshly prepared, thus falling in position 3 on 
iierscales 6 cc. or this was taken (Nos 1); to 4 ¢.c,of 1, 4 cc: 
of sea-water was added (No. 2) = 1/16 per cent., (1/2*); to 4 c.c. 
of 2, 4 c.c. of sea-water was added (No. 3) = 1/32 per cent., 
(1/2°); this was continued eight places to 1/2. . Four drops 
of a 10 per cent. egg-suspension was then added to each, and the 
percentage of segmented eggs was counted three hours later. 
Plotted they give the above curve. In this case it will be seen 
that the fertilizing power almost ceases at 1/2!° = 1/1024 per 
cent. sperm suspension. The eggs and sperm were not at fault 


236 FRANK R. LILLIE. 


because a parallel control series, in which the same quantities 
of the same lot of eggs were first placed in the same quantities 
of sea-water and sufficient of the original I per cent. sperm 
suspension added to make similar sperm dilutions, showed over 
95 per cent. cleavage in each case, and actually 99 per cent. in 
No. 8 of the control where the sperm dilution was 1/1200 per cent. 
As a further control it may be added that eggs which fail to 
fertilize in such relatively concentrated sperm suspensions may 
all be fertilized by the subsequent addition of a trace of perfectly 
fresh sperm. 

The type of experiment just cited was the first undertaken, 
and for a time it seemed to offer an almost insoluble problem, 
though the real explanation turned out to be extremely simple. 
I have twenty curves from similar experiments, fourteen of which 
run out absolutely from the third to the twelfth place on the 
scale (4. e., from 1/8 per cent. to 1/4096 per cent.); in the remain- 
ing 6 (as in the curve just given) the dilutions were not carried 
far enough to reach the zero point, but they agree in principle 
with the others. 

A number of control experiments demonstrated the relative 
lack of significance of the actual sperm concentrations. As one 
of these I may mention experiment C of August 3. In this case 
a series of sperm dilutions in powers of 4 was made from 1 per 
cent. The proportion of eggs fertilized ran off to I per cent. at 
1/45 (1/2) and to o at 1/46 = 1/4096 per cent. But one drop 
of a O.I per cent. suspension of the original I per cent. sperm 
added to eggs in 200 c.c. sea-water fertilized 94 per cent. of them 
(control for sperm). Thus the control fertilized almost perfectly 
at 1/60,000 per cent. dilution, whereas the fifth member of the 
series of dilutions 1/2! (1/1024 per cent. sperm) fertilized only 
I percent. The actual concentration of the sperm is thus not the 
most significant thing. 

This is also brought out strikingly in the following experiment 
(August 14). A series of half sperm dilutions was made as usual 
(Series A) 2 c.c. in each dish; to a second series (series B) of 
dishes was added 2 c.c: sea-water each and 4 drops of an egg- 
suspension. The numbers of series B were then inseminated by 
one drop each of sperm from the corresponding number of A, thus 


STUDIES OF FERTILIZATION. 237 


diluting the sperm about 1/60. To each of the A series (except 
1) four drops of the same egg-suspension was then added. The 
resulting percentages of fertilization are given in Table I. 


TAB IE aL: 
A. B. 

Ro (2%) = I. (1/60%) — 99% 
2, (u/296) = Co5% 2. (1/120%) —99.5% 
So CYL) = OSV 3. (1/240%) — 98% 
4. (2/8%) — 99% 4. (2/480%) — 60.5% 
5 (/O%) = 5% 5 (H/CSO%) = 52% 
Ge (/B2%)) = GO5%6 6. (1/1920%) — 8.5% 
7. (1/64%) — 21% 7. (1/3840%) — 1.5% 
8. (2/128%) — 6% 8. (1/7680%) — o 

Go § (CYBIS%) = Bas Ve 9. (1/15360%) — o 


If we compare A and B in this table it will be seen that while 
it is true that B runs out earlier than A, nevertheless the fertiliza- 
tions in the two series are not proportional to concentrations of 
sperm; for instance A 9 at 1/256 per cent. fertilizes 3.5 per cent. 
of the eggs, whereas B 3 at 1/240 per cent. fertilizes 98 per cent., 
B 4 at 1/480 per cent. fertilized 60.5 per cent., B 5 at 1/960 per 
cent. fertilizes 51 per cent. It is obvious that it is not concentra- 
tion but condition of the sperm that is significant, which comes 
out with extreme emphasis in a control of this series. In this 
control, 1 drop of A (1 per cent. sperm) was added to 8 drops of 
the same egg suspension in 1,000 c.c. of sea-water 2 minutes 
after the other inseminations, thus making a 1/30,000 per cent. 
(1 X 1/1000 X 1/30) sperm suspension; every egg fertilized; the 
percentage of cleavage was 100 per cent. 

The question then arises, what is this condition of the sperm 
which causes such loss of fertilizing power? We may note the 
following points: (1) To bring out the lack of significance of 
the absolute concentration of the sperm, in several of the experi- 
ments with successive half dilutions, counts were made of the 
numbers of spermatozoa seen in the egg-jelly of members of the 
series with no fertilizations: Thus on July 16a series of half dilu- 
tions ran out to o in the seventh crystal (1/128 per cent. sperm) : 
in ten eggs selected at random from this crystal, an average of 9 
spermatozoa was counted in the jelly and in contact with the 
membrane of these eggs; but, as the upper and lower surfaces 


238 FRANK R. LILLIE. 


could not be examined, the whole number must have been at 
least double; in No. 8 of the series, an average of five spermatozoa 
was counted with each egg; in No. 9 an average of 1.2; No. 10, 
1.4; No. 11, 0.9. Similar counts were made in other cases. 
But in fertilizations under optimum conditions all of the eggs 
may fertilize in dilutions of sperm so great that it is almost 
impossible to find spermatozoa in the jelly of the eggs. (2) 
The spermatozoa are active and the eggs readily fertilizable in 
such a series as the above. Repeated observations were made 
on this point; which would be tedious to relate in detail. 

It may be noted that in the fertilization under optimum condi- 
tions the eggs were first placed in sea-water, and given quantities 
of sperm then added; whereas in the experiments with successive 
dilutions eggs were added to sperm suspensions already made up. 
This suggested that the order of adding eggs and sperm might 
be of significance in some way. However, this does not appear 
to be the case. 

The possibility remained that the repeated handling of the 
sperm in successive dilutions decreased their motility. Micro- 
scopical examination did not confirm this idea; and subsequent 
experiments disproved it, as the fundamental factor at least. 

Thus it would appear that the only real difference between the 
optimum and minimum conditions of the fertilizing power of 
sperm dilutions is a time factor; under what I have called the 
optimum conditions the final dilution is made from a relatively 
concentrated sperm suspension in the presence of eggs; but under 
the conditions of successive dilutions time elaspses before the 
eggs are added. 

Thus in Curve 2 the preparation of the series of sperm dilutions 
from the original 1 per cent. suspension occupied 22 minutes 
before the eggs were added. In Curve 1, on the other hand, 
less than a minute elasped from the time of preparing the 1/100 
per cent. and 1/1000 per cent. sperm suspensions used in the 
last four determinations to the time of their use in inseminating 
(see p. 234); and the final dilution was made in the presence of 
the eggs. 

The time factor is the real explanation as will be shown im- 
mediately. But at first sight it did not seem a very probable 


- 


STUDIES OF FERTILIZATION. 239 


explanation for two reasons: in the first place the time from 
preparation of the original 1 per cent. sperm suspension to that 
of addition of eggs is usually less than twenty-five minutes, which 
is-usually considered too short a time for injury to sperm; and 
in the second place, after the addition of eggs to the sperm- 
dilution series, in several control experiments the original 1 
per cent. sperm suspension was shown to be capable of fertilizing 
at I1/30,000 of 1 per cent. (1/2 ca.) by addition to eggs in sea- 
water. If the sperm suspensions lose their fertilizing power with 
time, it must be that the significance of time im this respect varies 
inversely to concentration. As soon as such a proposition is 
formulated it is easily tested experimentally, and this was done 
in a thorough fashion. Ps 


4. Time as a Factor in the Fertilizing Power of Sperm Suspensions 
of Different Concentrations. 


The experiments under this head were performed in three ways: 

A. A considerable quantity of the sperm suspension to be 
tested was made up, and divided in several equal parts in a series 
of bowls; measured equal quantites of the same egg-suspension 
were then added to members of the series at definite time inter- 
vals. This method was followed for sperm dilutions from 1/300 
per cent. (between 8 and 9 in the scale) down. B. Measured 
amounts of the more concentrated sperm suspensions were added 
at time intervals to measured quantities of eggs in equal amounts 
of sea-water. C. Finally, to control the data in section 3, a 
series of sperm suspensions, made by successive half dilutions 
as in section 3, was divided in two equal series, and eggs were 
added at once to the one series, and after a time interval to the 
second. 

A. The following table gives the data under method A. The 
figures at the head of each vertical column give the sperm dilu- 
tion in fractions of the I per cent. sperm suspension; below is 
given the place of such a sperm suspension in the scale of powers 
of 2. The figures in the columns give the percentages of fertiliza- 
tions for inseminations made at the time (age of the suspensions 
in minutes) indicated at the left. To illustrate the method of 
experimentation for one column which will serve for all the rest, 


240 FRANK R. LILLIE. 


Wai 1. 


SHOWING RATE OF LOSs OF FERTILIZING POWER OF SPERM SUSPENSIONS. 


fe) ° fo) 5 ° ie) Q 9 9 9 8 8 
0/96 |100 | 99.5|/95 |95-5 |97 |9I |97 97-5 |88 |91.5| 58 | 40 
I bee OWS || AH s oo oc (7 ee hee PR a hear gee leanne 42 I 
2/99.5/98.5]..... sol 75 50.0 bil GO) ODS ||OS) | COL | SO0So o's ollocces 
3 100 OMS I ha eee: Gola St deur acaodl Buca roll hau etichenl keeaaenetc cae 4 16 
All SJ9)o5}|| @5j055}|5 3 6 30 -++-(76 |..../34.5 | 93.66] 85.5) 49.5 | 13 | 15.5 cof: 
OMAR ees S etl OBASMINS 2 al-toe.ste SOsSierehseal| arersyens tem EAA ee O5 || 
8] 98.5] 65.5|....- BEN ey | PONT A 15 || Giles || FWB3o5\| 7 7 | 17S | eee 
16] 85.5] 2 | 90.5|45 | 26 KOS SIg aldo Be T4 |36 4.5| I (0) (0) 
32| 96.5] 67.5} 30.5] 1.5] oO 4.5) 9 it (0) 4 Ce) 0.5 (0) (0) 
64| 2.5] 19 fo) 0.5| 0.5 | 3.5| 2 (o) fo) 4 fo) (0) (o) Co) 
E20) Bs B lloacoc Pail Kraeersretnl WA eeact cis eho leech i Reena cere olan a lore o:4 
8—9 |Q-10] ITO-++ |II +|11I—12|12 —|12—-13 13-14|t4+ HATE, 105) 5° 16 —|16—17|18 — 


we shall give the experimental data for 1/30,000 per cent.: 
August 18, 1914. The eggs of two females were taken at 9.50 
A.M. and washed at 10.04, 10.06 and 10.23 (150 c.c. of sea- 
water being used in each washing). A series of 7 Syracuse 
crystals was then laid out with 10 c.c. of sea-water in each. 
To 1 was then added 5 drops of the egg-suspension. A single 
drop of fresh dry sperm was then added to 333.3 c.c. sea-water 
at 10.37 making a 1/100 per cent. sperm suspension, and 1 drop 
of this was added to crystal 1 at 10.37.30 and stirred in by an 
assistant making a 1/30,000 per cent. (1/100 X 1/10 X 1/30) 
sperm suspension in presence of eggs. One drop of the 1/100 
per cent. sperm was also added to crystals 2-7, which contained 
no eggs, at 10.38, making 1/30,000 per cent. sperm suspension 
in each. To No. 2, 5 drops of the same egg-suspension was 
added at 10.40, to No. 3 at 10.42, to No. 4 at 10.46, to No. 5 at 
10.54, to No. 6 at 1iaro; to No. 7 at 11.42) “At 2:207EeNiaimay: 
assistant, Mr. Cohn, then estimated the percentages of segmented 
eggs in each crystal, by first thoroughly mixing the eggs, then 
assembling them, taking a sample, and making two counts of 
100 each, which were averaged. 

The table shows (1) that the effect of time up to 64 minutes 
is to diminish the fertilizing power of the suspensions at every 
dilution represented. (2) That the rate of loss of fertilizing 


ee ee eee 


at ae 


STUDIES OF FERTILIZATION. . 241 


Power increases with dilution, 7. e., the effect of time varies in- 
versely to concentration of sperm. This is brought out very 
clearly by the following curves (Fig. 3) of loss of fertilizing 


© § 4 US 2) 25 FO SG AO ag GO 55 CoO OF 7 


CURVE 3. 


power of sperm suspensions at different concentrations. The 
abscissee represent age of sperm suspensions in minutes; the 
ordinates represent fertilizing power as expressed in percentages 
of segmenting eggs. Each curve stands for a given sperm 
dilution. Curve 1 represents loss of fertilizing power of a 
1/300 per cent. sperm suspension, curve 2 of a 1/3000 per cent., 
curve 3 of a 1/30,000 per cent. and curve 4 of a 1/120,000 per cent. 
sperm suspension. 

B. On August 6, I prepared a series of seven sperm dilutions 
in powers of 4 from I per cent. to 1/4096 per cent. Each of these 
was then used to fertilize a measured quantity of egg-suspension 
at the intervals given in Table III. 

For the fertilizations 10 c.c. sea-water was measured out in 
advance in Syracuse crystals and 5 drops of a 5 per cent. egg 
suspension added to each. For each fertilization 1 drop of sperm 
was added and stirred in. It will be observed that I per cent. 
sperm lost none of its fertilizing power so far as this test went; 
1/4 per cent. fell off from 96.5 per cent. to 16.3 per cent.; 1/16 
per cent. from 46.5 per cent. to 0; 1/64 per cent. from 0.5 per 
cent. to o in the second place; whereas the greatest dilutions did 


242 FRANK R. LILLIE. 

TABLE III. 

Fertilizations. 
Sperm Dilution. Made at A. B, G JD). £. 
2.50 P.M. | 3.01 P.M. | 3.26 P.M. | 3.35 P.M 4.03 P.M. 

Tee yO 5 tea 2.20 P.M. 909 % 100% 100 % 00.5% 09.5% 
Bo WG s > oo 2.28 P.M. 96.5% 68% 61.5% 18.5% 16.3% 
235 U/TO% » 2.30 P.M. 46.5% 18% 8.5% a Ye, 
A. 1/64%.. 2).22) PN. 0.5% o% 0% © % 
5. 1/256%. 2.34 P.M. fo) o% 0% 
6. 1/1024% 2.36 P.M. to) 0% 
7. 1/40906%..| 2.39 P.M. 


not fertilize at all. 


It should of course be noted that the sperm 


suspensions used were diluted 300 times in the actual insemination 
(10 c.c. = 300 drops of sea-water, and one drop sperm added). 

Thus time is an important factor in the fertilizing power of 
sperm dilutions from 1/4 per cent. down. The matter cannot 
be stated with great accuracy, but we can say in general (referring 
to Table II.) that sperm suspensions from 1/300 per cent. to 
1/1200 per cent. lose their fertilizing power nearly completely in 64 
minutes; from 1/2400 to 1/24,000 in 32 minutes; from 1/30,000 
to 1/60,000 in 16 minutes; from 1I/120,000 to 1/240,000 in 6 
Table III. shows loss of fertilizing power of higher 
concentrations by a different method. 

C. We are now in a position to understand the principal reason 
why the curves of successive half or quarter dilutions of a I 
per cent. sperm suspension run off so rapidly. The reason is 
that the preparation of the series requires time, 10 to 20 or more 
minutes. In the early experiments of this kind the significance 
of brief periods of time was not recognized, and so no time records 
were kept; but I have 13 curves with accurate time records. 
Of these I reproduce only two (Fig. 4). For this experiment 
(Sept. 5), (1) 6.6 c.c. of I per cent. sperm was prepared, 9.30 
A.M. (2) 4 c.c. of I was transferred to a Syracuse watch crystal 
and 4 c.c. of sea-water added (= 1% per cent.). (3) 4 c.c. of 2 
was transferred to crystal 3 and 4 c.c. sea-water added (= 4 
per cent.) and this was continued to 12 numbers (finished at 
9.39.30 A.M.). The suspensions 2-12 was then divided in two 
equal amounts of 2 c.c. each, making series A and B.. To each 
crystal of series A 2 drops of a I per cent. egg-suspension were 


minutes. 


STUDIES OF FERTILIZATION. 243 


added at 9.47, that is, 17 minutes after the 1 per cent. sperm was 
prepared. Twenty-eight minutes later (10.15 A.M.) two drops 
of the same egg suspension were added to series B. The only 
difference between series A and B is the time factor. The per- 
centages of cleavages were counted for both series, and the plotted 
results given the curves. Considerable loss of fertilizing power 


On Tiy O 2h Use ye Aly Shiny Ok, Zor nO ON nd Oly a Te 


Too 


has occurred in series B as compared with series A. Now, if we 
compare these time intervals with those given in Table II. we 
see that, in the curve of series B the last number, which is a 
1/2" or 1/2048 per cent. sperm suspension, loses its fertilizing 
power completely in 36 minutes (7. e., from 9.39.30 to 10.15.30) 
which corresponds very well with the rate of loss of fertilizing 
power in a 1/2400 per cent. sperm suspension. This agreement 
is rather closer than usual; in some cases the series of 14 dilutions 
ran out at higher concentrations in about the same time; but 
in no case, I think, did they require more time. This suggests 
some possible stimulating effect of the successive changes which 
causes the spermatozoa to lose their fertilizing power more 
rapidly than under the time factor alone. 

Gemmill (1900) observed that the duration of vitality of 
spermatozoa of sea-urchins and limpets. tested by their move- 
ments or by the fertilizing capacity, varies greatly “ according to 
the amount of sperm used in proportion to the volume of sea- 


244 FRANK R. LILLIE. 


water in which it was shed.”’ ‘‘Whenasmall quantity of sperm 
was mixed with a large quantity of sea-water, the duration of 
vitality of the spermatozoa is short, but when the converse pro- 
portions are used, it is greatly lengthened.” ‘‘ By taking sperm 
from a sea-urchin and mixing it in different vessels with different 
quantities of sea-water, one obtains sets of spermatozoa, which will 
retain their vitality for a rising series of terms, e. g., 8, 12, 16, 24, 
48 and 72hours. For the longest term, the proportion of sperm- 
atic fluid to sea-water should be not less than 1 to 10.” 

Gemmill was thus dealing with the same phenomenon with 
which we are concerned. He gives, however, no exact quantti- 
tative data and relatively few experiments were performed. He 
attributes the results to (1) greater activity of the spermatozoa, 
and consequent earlier exhaustion in the more dilute-suspensions 
and (2) to dilution of the “spermatic fluid”’ by which he sup- 
poses the spermatozoa to be nourished. 


5. Other Factors in the Fertilizing Power of Sperm Suspensions. 


In the large number of experiments carried out to test the 
fertilizing power of sperm suspensions the general form of the 
curves is remarkably constant. Some, however, are quite ir- 
regular, and it was never possible to get exactly the same curve 
in the repetition of any experiment. A few of the irregularities 
may conceivably be due to error, as for instance the accidental 
presence of some toxic substance in one of the dishes of a series, 
though painstaking care was used to avoid such sources of error. 
The failure to obtain exactly the same curve in different experi- 
ments is no doubt also due in part to the natural variability of dif- 
ferent lots of eggs and sperm. 

In an attempt to discover the sources of variation and error, 
the effect of egg concentration, 7. e., the absolute quantity of 
eggs in a given bulk of a sperm suspension of given strength was 
tested. On the whole the effect of egg-concentration was found 
to be relatively small within so wide a range that it cannot be 
regarded as a large factor in the variability of the curves; because 
the egg-concentration of the curves was always below the point 
where it was demonstrably a limiting factor. Tests were made 
of sperm suspensions ranging from 1/62.5 per cent. to 1/8,000 


STUDIES OF FERTILIZATION. 245 


per cent. But it was only from about 1/500 per cent. down that 
any considerable effect was observed within the range of egg 
concentration employed. 

- The method of the experiments tabulated (Table IV.) may be 
given for 1/500 per cent. sperm as it was the same for the others 
August 31: A quantity of 1/500 per cent. sperm suspension was 
freshly prepared 10.54.30 A.M., 2 c.c. of this was then placed 
in each of seven crystals (1-7). From 10.56.30 to 10.59 A.M. 
eggs were added as follows: to 1, one drop of a 1.75 per cent. 
egg-suspension, to 2 two drops, to 3 four drops, to 4 eight drops, 
to 5 sixteen drops, to 6 one c.c., to 7 twoc.c. The numbers in 
the table give the percentages of segmented eggs. The tests 
with 1/1,000 per cent. and 1/2,000 per cent. sperm were made with 
the same egg suspension. For the tests with 1/4,000 per cent. 
and 1/8,000 per cent. sperm a 3.3 per cent. egg-suspension was 
used. Thus for each series the egg-concentration is approxi- 
mately doubled in successive numbers of the series (in No.7 = 64 
times No. I). 


TABLE IV. 
EFFECT OF EGG-CONCENTRATION ON THE FERTILIZING POWER OF SPERM SUS- 
PENSIONS. 
Sperm Egg-suspensions. 
Suspen- 

SOS it 2. Sy. 4. | Ss 6. Ts 
t/500% | 100 99 99.5 07 ay n93s5 82.5 56 
1/1000%| 97.5 94.5 93 7Go5 1 9 ASE 76.5 83-5 
1/2000% 96.5 83.5 75 WBa5 | 42.5 36 32 
1/4000% 719.5 66.5 42.5 47-5 | 72.5 16 4.5 
1/8000% 46.5 52 80? 30.66 Do WES 7.5 


The percentages of fertilization fall off in each of these sperm 
suspensions with increase of egg-concentration, and the amount of 
falling off increases in general with the dilution of the sperm. 
There was certainly no numerical deficiency of spermatozoa in 
the highest egg-concentrations; the reason for the falling off 
therefore appears rather obscure, and as it is not involved in the 
present problem, I shall not discuss it here. But as the egg- 
concentration employed in any of the preceding experiments 
did not exceed that of column 3, and the same egg-concentration 


246 FRANK R. LILLIE. 


was always employed throughout any experiment, it is obvious 
that the effect to be attributed to the egg-concentration employed 
in the preceding experiments is very small. 


III. Discussion. 


Within a wide limit of egg-concentration the important factors 
in fertilizing power of sperm suspensions are: (1) concentration, 
(2) time. A third factor, which is not of equal significance to the 
other two, is the given variability of the reproductive elements. 
Such variability attaches of course both to ova and spermatozoa; 
in general it will affect only absolute values for given combina- 
tions, and not at all the relative values found in any single experi- 
ment. Moreover, as it is a chance factor, it will tend to be 
eliminated in a series of determinations. Fortunately both eggs 
and spermatozoa of Arbacia are relatively very constant mate- 
rials if care be taken to wash the eggs thoroughly, and if the 
factors of concentration and time are fairly constant for the 
sperm. For the eggs these two latter factors are of such slight 
importance within the given limits as to be practically negligible. 
The significance of the concentration factor for the fertilizing 
power of sperm is of course obvious without discussion. We 
therefore turn to the time factor. . 

The most significant aspects of the time factor are, first, the 
unexpectedly rapid rate of loss of fertilizing power of sperm 
suspensions, and second the increase of rate of loss with dilution. 
There are but two ways of explaining these facts: either (1) the 
motility of the spermatozoa is quickly reduced in sperm suspen- 
sions to such an extent that they cannot bore into the egg or (2) 
the spermatozoa lose some substance essential for the fertilization 
reaction. 

The following are the objections to the first alternative: (a) 
Microscopical examination lends it no support; I have repeatedly — 
observed, that fertilizing power of sperm cannot be expressed 
either in terms of motility, or of success in penetrating the jelly 
of the egg and coming in contact with the membrane. In the 
experiments on successive half dilutions (p. 238) I kept records, 
in several series, of the numbers of spermatozoa in the jelly of 
unfertilized eggs, and found in some cases an average of 9 


STUDIES OF FERTILIZATION. 247 


spermatozoa visible in the jelly, or on the membrane of certain 
lots of eggs none of which had fertilized; this could not be more 
than half of the spermatozoa in association with such eggs; and 
other observations made immediately after insemination demon- 
strated the high degree of motility of spermatozoa of entirely 
barren sperm suspensions. 

These observations contrast in the most striking manner with 
the fact that not a single spermatozoén can be seen in the jelly 
of eggs fertilized with highly dilute fresh sperm suspensions, 
where, nevertheless, nearly every egg may be fertilized. 

(b) Penetration of the egg is not solely a function of motility 
of the spermatozoén. Penetration follows, as a matter of fact, 
after the fertilization reaction has begun, and it is due to the 
inception of such reaction, not the reverse as is commonly 
assumed.2 In Nereis, as I have previously described, penetration 
does not take place until 45 to 50 minutes after insemination 
and the initiation of the fertilization reaction. The facts 
described in this paper show that in Arbacia no penetration takes 
place unless the sperm has started the fertilization reaction; if 
this does not take place, the spermatozoén remains external, 
however active it may be. And if it does occur the initiating 
spermatozo6n is speedily engulfed by the egg. 

(c) It is not easily understood on this theory why dilute sperm 
suspensions should lose their fertilizing power more rapidly than 

1 Glaser’s experiments (1915) would bear the interpretation that, in those cases 
of normal insemination described by him in which fertilization does not occur except 
in the presence of several spermatozoa for each egg, the time factor which I have 
just described was operative. In other words that the majority of the spermatozoa 
in question had lost their receptors. But in the absence of exact data as to age and 
concentration of the sperm suspension, it cannot be asserted that this is the correct 
interpietation although I obtain exactly the same results in my time series (p. 238). 
My dilution experiments prove beyond a doubt that a single spermatozoon 
suffices for the whole process of fertilization under optimum conditions 
(defined on p. 233). Glaser’s experiments, however, raise the question whether 
the efficacy of heavy insemination in the case of a stale sperm suspension is 
due to mass action, or to the survival of a small percentage of effective spermato- 


zoa? So far as I can see this question can not be answered on the basis of our 
present information. 

2 Spermatozoa may penetrate into unripe ovocytes in some cases, as has been 
noted by several observers; in such a case there is no fertilization reaction. In the 
present experiments the unfertilized eggs were not penetrated by the spermatozoa. 


248 FRANK R. LILLIE. 


more concentrated suspensions;! the relative freedom from CO, 
and other sperm excreta should favor a longer continuation of 
their motility in the dilute suspensions rather than the reverse. 

(d) Moreover, in general the results of recent fertilization 
studies such as the antagonistic action of sperm suspensions 
of different phyla, inhibition of fertilization in the presence 
of blood of the species, or in the absence of certain ions (Loeb, 
14), or again the sterility in certain self-fertilizations, and finally 
the inability of spermatozoa to penetrate fertilized eggs, unite 
in demonstrating the relative lack of significance of motility 
as such. | 

We come therefore to the conclusion that the individual spermatozoa 
im suspension tend to lose their fertilizing material, so that an 
increasing proportion of these spermatozoa become absolutely in- 
effective whatever their motility. This conclusion is in agreement 
with all the data of the foregoing experiments, and seems to 
be the only one competent to explain the results. 

The following questions arise: (1) Whether the loss of this 
substance by the sperm is a mere process of diffusion or an active 
secretion? (2) Can the substance be recovered from the fluid 
of the suspension, or can its presence in the fluid be demonstrated 
in any way? 

As regards the first question: In the case of the ova we know 
that the external jelly-covering is loaded with sperm-agglutinat- 
ing substance which diffuses into the sea-water continuously. 
It is theoretically possible, at least, to apply a similar conception 
to the spermatozoén, although no such covering is demonstrable. 
The more rapid: loss of fertilizing power in the greater dilutions 
would be consistent with this interpretation. From this point 
of view we would have to regard the sperm head as covered 
superficially with a layer of fertilizing material, like the phos- 
phorus on a match. Such a conception is by no means im- 
possible. On the other hand the fact that dilutions reached by 
a series of successive half-dilutions from I per cent. lose their 


1Gemmill (1900) observed the same phenomenon and concluded that the more 
rapid exhaustion of spermatozoa in dilute suspensions is due to dilution of a 
hypothetical nutritive medium which keeps the spermatozoa of concentrated sus- 
pensions in a vigorous condition. This explanation comes back to the principle 
of loss of motility, so far as it relates to fertilizing power. 


STUDIES OF FERTILIZATION. 249 


fertilizing power more rapidly than the same dilutions made in 
one stroke, indicates that successive stimulation hastens the 
loss, which therefore appears more in the nature of a secretion 
or a discharge than mere diffusion. The source of the substance 
must ultimately be the sperm cell itself, and it is quite possible 
that, as in the case of the egg, there is both a superficial layer 
and an internal supply. 

It must be admitted that the data are inadequate to answer this 
problem. The statement of the problem can therefore serve 
only to bring out the resemblance between the spermatozo6n 
and the ovum in respect to the existence of a fertilizing substance 
in each, the fertilizin in the case of the ovum and the sperm 
receptors in the case of the spermatozo6n, and also the possible 
resemblance in respect to the disposition of the substances in 
each. It certainly is an interesting parallelism that both cells 
contain a substance necessary to fertilization, which may be 
lost in the sea-water. 

The most interesting and crucial question of course concerns 
the possibility of detecting this lost substance in the fluids of the 
suspensions. If such a substance actually occurs in the fluid it 
should have the property of fertilizing ova; unless it can be 
detected by this property, we have no other indicator for it. 
So far I have not been able to make even a beginning on this 
problem. As is well known a number of experimenters have 
attempted without success to derive a fertilizing medium from 
spermatozoa. It has been suggested by Loeb that the reason 
for the failure to secure an extract of spermatozoa that will 
fertilize is that the motile power of the spermatozo6n is needed 
to carry the effective substance into the egg. But it may equally 
well be that the methods hitherto employed have been too brutal; 
the substance may well be too labile to withstand extraction 
by ether, etc. 

My results strongly suggest, if they do not prove, that such a 
substance must be present in the fluid of sperm suspensions of 
Arbacia, and they therefore suggest other methods for securing 
it for testing. We must bear in mind that it can form only 
an extremely small proportion of the entire spermatozo6n, as 
proved by morphological considerations alone, and that it must 


250 FRANK R. LILLIE. 


be superficial in position and easily detached as proved by its 
effectiveness before the spermatozo6n penetrates. Extracts of 
the entire spermatozo6én must contain numerous other substances 
which may neutralize its effectiveness. 

The difficulty of the investigation as shown by my experiments 
is that it is liberated only very slowly in concentrated suspensions 
and that its amount in dilute suspensions would presumably be 
too slight to be effective. Some means can probably be devised 
for liberating it in concentrated sperm suspensions and freeing it 
of the spermatozoa for testing. 

Finally I may point out that the conclusion that spermatozoa 
lose a substance necessary for the exercise of their fertilizing 
power is consistent with my own point of view of the mechanism 
of fertilization as well as with Loeb’s. From my point of view 
the spermatozoén loses its receptors, viz., the substance that 
activates the fertilizin of the egg; from Loeb’s point of view the 
spermatozoon loses its lysin, the substance that corrodes (cy- 
tolyzes) the egg. 

My previous experiments had shown that eggs produce a 
certain substance in sea-water (fertilizin) which is necessary 
for their fertilization; fertilized eggs no longer produce this 
substance and are incapable of fertilization. Both eggs and 
spermatozoa therefore contain substances, more or less liable 
to loss, which are necessary for fertilization. The mechanism 
of fertilization cannot possibly, therefore, be regarded in the 
simple manner postulated by Loeb’s theory. The existence of 
parthenogenesis demonstrates the efficacy under given condi- 
tions of the egg-substance alone; we must therefore regard the 
spermatic substance essentially as an activator of the fertilizin 
of the egg. 


LITERATURE. 
Gemmill, James F. 


700 On the Vitality of the Ova and Spermatozoa of Certain Animals. Journ. 

of Anat. and Physiol., Vol. 34 (N. S., Vol. 14), pp. 163-181. 
Glaser, Otto. 

713. On Inducing Development in the Sea-urchin, together with Considerations. 
on the Initiatory Effect of Fertilization. Science, N. S., Vol. XX XVIII, 
Pp. 446-450. 

14 The Changein Volume of Arbaciaand Asterias Eggs at Fertilization. BioL. 
BULL., Vol. XXVI., pp. 84-01. 


OE a 


STUDIES OF FERTILIZATION. 251 


"75 Cana Single Spermatozo6n Initiate Development in Arbacia? Bio. BULL., 
Vol. 28, pp. 148-152. 
Lillie, Frank, R. 
713) The Mechanism of Fertilization. Science, N.S., Vol. XX XVIII, pp. 524—- 
528. 
’r4 Studies of Fertilization VI. The Mechanism of Fertilization in Arbacia. 
Journ. Exp. Zoél., Vol. 16, pp. 523-590. 
Loeb, Jacques. 
’r4 On Some Non-specific Factors for the Entrance of the Spermatozo6n into the 
Egg. Science, N. S., Vol. XL., pp. 316-318. 


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mOLOGCICAL BULLETIN 


oan Institon~ 
ON THE RHYTHMICAL SUSCEPTIBILITY OF DE- fae “ oN 


< 


VPLOPING SEA URCHIN EGGS TO foo i an ee 0 1915 
HYPERTONIC SEA WATER. ( Mie ag ae 
x ry) ie 4 
ARTHUR RUSSELL MOORE. tational Mus? . 


(From the Biological Laboratory of Bryn Mawr College and the Marine Biological 
Laboratory at Woods Hole.) 

In a recent communication M. Herlant! attempting an 
analysis of Loeb’s method of artificial parthenogenesis concludes 
that (1) the fatty acid treatment gives rise to the rhythmical 
activity of the centrosome but never to normal divisions of the 
egg; (2) the treatment of the eggs with hypertonic sea water 
causes the formation of accessory asters and is necessary to 
complete the causes for normal division; (3) the optimum results 
are obtained by applying the hypertonic solutions at certain 
intervals after fatty acid treatment, viz., 30 and 70 minutes and 
possibly 115-120 minutes, while with 40-50 and 95-100 minute 
intervals marked minima are shown. 

As to the first generalization, Herlant ignores the fact that 
in Strongylocentrotus and Arbacia the fatty acid treatment alone 
may cause normal segmentation. If the eggs of S. purpuratus 
are kept at a low temperature (5°-10°) after acid treatment 
alone, they divide regularly and may reach the morula stage.’ 
In Arbacia eggs, normal segmentation may take place after 
acid treatment without subsequent treatment with the hyper- 
tonic solution, but does not asa rule proceed beyond the two- 
cell stage. On the other hand hypertonic treatment alone may 
cause Arbacia eggs to segment and develop into swimming larve, 
while it brings about only early segmentation stages in the eggs 
of Strongylocentrotus. . 


1M. Herlant, Comptes Rendus de l’ Academie, T. 158, p. 1531. 
2]. Loeb, ‘‘ Artificial Parthenogenesis and Fertilization,’’ p. 76. 


253 


254 ARTHUR RUSSELL MOORE. 


Herlant apparently assumes that the action of the hypertonic 
solution must be subsequent to the acid treatment, that to be 
effective such action must occur in a certain phase of the rhyth- 
mical activity of the centrosome. Since in Strongylocentrotus 
purpuratus treatment of the eggs with the hypertonic solution 
may precede that with acid sea water by as much as a forty-eight 
hour interval with the result that normal parthenogenetic larve 
are formed,! Herlant’s contention is not justified. Furthermore, 
treatment of the eggs with small quantities of KCN or depriving 
them of oxygen, may replace hypertonic treatment after fatty 
acid. Now lack of oxygen or the repression of oxidations does 
not cause aster formation, but on the contrary suppresses it. 
In fact it has even been shown that in Arbacia the first steps in 
development induced by the acid treatment may be reversed 
and the egg returned to its resting stage with its original possi- 
bilities of fertilization, simply by withholding oxygen from such 
an egg or by treating it with KCN In view of these facts, 
it seems evident that Herlant’s conclusion that the hypertonic 
solution is a necessary factor in artificial parthenogenesis because 
it controls aster formation, does.not hold. 

As to the rhythmicity in effectiveness of the hypertonic treat- 
ment which Herlant found in his experiments, it seemed possible 
that the relation between the time spent in normal sea water 
after acid treatment and the time in the hypertonic solution 
might have a bearing upon the question. Six years ago the 
present writer found, in working with the eggs of Strongylocen- 
trotus purpuratus, that if the exposure to normal sea water in 
such an experiment be lengthened, the subsequent treatment 
by hypertonic sea water must be shortened to secure optimum 
results.2 Repetitions of the experiment at Woods Hole during 
the past summer, however, have indicated that the relation does 
not exist for Arbacia. 

Table I. shows the results of ‘dividing a lot of Arbacia eggs, 
after fatty acid treatment, into three parts which remained in 
normal sea water, 5, 25 and 90 minutes respectively, before 
being put into hypertonic sea water. In each case a portion 


1J. Loeb, Journ. Exp. Zool., vol. 15, p. 201. 
2 J. Loeb, Science, N. S., Vol. 38, p. 740. 
3 J. Loeb, ‘‘ Artificial Parthenogenesis and Fertilization,” p. 96. 


RHYTHMICAL SUSCEPTIBILITY OF SEA URCHIN EGGS. 255 


of each lot was removed from the latter solution after 17%, 
20, 25, 30, 35, and 40 minutes, and allowed to develop in normal 
sea water. The percentages given in the table show the degree 
of blastula development in each culture. Repetitions of the 
experiment showed no significant variation in the optimum 
exposure to the hypertonic solution with changes in the time 
the eggs remained in the normal sea water after acid treatment. 


SABE le 


LO? = BOP = 2e® 


Eggs Remained in Percentages of Eggs Develop into Blastule After Exposure to Hypertonic 
Normal Sea Water, Sea Water for 
After Butyric ; 
Acid Treatment 17% Min. 20 Min. | 25 Min. 30 Min. 35 Min. 40 Min. 
5 minutes...... — 1% | 5% 13% 24% 18% 
DIST ae = 8% | 18% 24% 20% 8% 
QOm me ip is fous — — | 1% 3% 1% 


In order to determine if the eggs of Arbacia punctulata which 
had been treated with acidulated sea water, showed a rhythmicity 
in sensitiveness to the hypertonic solution, such as Herlant’s 
experiments with the eggs of Paracentrotus lividus indicated, 
the following experiments were carried out. The eggs of several 
sea urchins were collected, treated with sea water made acid by 
the addition of 2 c.cm. N/1Io butyric acid to 50 c.cm. of sea water. 
After remaining in this solution for from 2 to 24% minutes the 
eggs were transferred to normal sea water. At the end of 5- 
minute intervals lots were removed to finger bowls containing 
hypertonic sea water [50 c.cm. sea water + 8 c.cm. 24 M 
(NaCl + CaCl, + KCl)]. After remaining in the hypertonic 
sea water for 25 minutes the eggs were put into normal sea water 
and allowed to develop. The percentages of advanced morule 
or non-swimming blastule were determined by counting random 
fields. The following table (Table II.) gives a typical result, 
showing optimum effects when the eggs were put into the hyper- 
tonic solution 40, 60, 90-100 and 115-125 minutes after acid 
treatment. The rhythmical character of the result is obvious 
from Curve I., where the ordinates indicate the percentage of 
larvee formed, while the abscisse indicate the time which elapsed 
between acid treatment of the eggs and their exposure to hyper- 
tonic sea water. 


ARTHUR RUSSELL MOORE. 


256 


soynurpy SST 


SVI Cer 


"II @AunD 
Sor S6 Sg SL 


U01}e}USUISOS 
P4IGL 


UO01}e}JUIUISOS 
puoses 


‘] aAuaAD 


UO}e} UNOS 
4ST 


RHYTHMICAL SUSCEPTIBILITY OF SEA URCHIN EGGS. 257 


TABLE II. 
T° = 19°— 21°. Hypertonic exposure =25 minutes. 


Time in normal sea 

water after acid 

(IRE VOMEM, Ao bo ode 5 15 25 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 
Blastule per cent.... — 5 1623 301I QI012 9 5 6 7 91316 12 5 4 
Time in normal sea 

water after acid 

treatment......... II5 120 125 130 140 150 165 175 185 
Blastule per cent. ... 8 1 & A O<r © To 


It seemed possible that the normally fertilized eggs also might 
show a rhythmical susceptibility to hypertonic sea water. 
Especially did this seem probable in view of Lyon’s experiments. 
He found that normally fertilized eggs of Arbacia gave alter- 
nating maxima and minima of susceptibility to heat, cold and 
lack of oxygen; and that COs, production was greatest at the 
time of cytoplasmic division! E. G. Spaulding has shown a 
rhythmical susceptibility and immunity of fertilized Arbacia 
eggs to the effects of ether, HCl, KCl and NaCl. He found a 
rise in immunity up to the time segmentation begins, followed 
by a sharp decrease during cleavage, with a marked rise at the 
end of cleavage.2, A. P. Mathews found an approximate rhyth- 
micity in the behavior of Asterias eggs toward KCN solutions.* 

In my own experiments the Arbacia eggs were fertilized in 
one finger bowl and at the end of each ten-minute interval a lot 
was removed to the hypertonic solution and kept there for 4o 
minutes, after which the eggs were returned to normal sea water 
to develop. The results of a number of such experiments indi- 
cate (Table III., Curve II.) that the maximal susceptibility 
occurs just after fertilization (5-15 minutes) and immediately 
before and during each cytoplasmic division, and that the maxi- 
mal resistance is shown 35-45 minutes after fertilization and 
just after each division. This corresponds to Lyon’s statement 
regarding the effects of heat upon dividing eggs, viz., that 
Arbacia eggs are especially sensitive to heat just before division 
and that they are most resistant after division. 

1E. P. Lyon, Am. Journ. Physiol., Vol. 7, p. 56, and Vol. 11, p. 52. 


2E. G. Spaulding, Brot. BULL., Vol. 6, p. 224. 
2A. P. Mathews, Biot. BULL., Vol. 11, p. 137. 


258 "ARTHUR RUSSELL MOORE. 


TABLE III. 
To = 26). 
Time in normal sea water 
after fertilization....... 5 15 25 35 45 55 65 75 85 95 105 I15 125 135 145 155 
Blastule per cent........ 50 5095 95 90 72 74 87 91 73 80 OI Or 7I 86 89 


The character of the curve constructed from Table III. 
differs from that made from the data of Table II. It will be 
noted that in the latter case there is an early maximum and 
minimum, and a slight secondary maximum reached at about the 
time cleavage would take place if the eggs had been normally 
fertilized. The dissimilarity may be due to the fact that the 
hypertonic solution may have two effects on developing eggs, 
viz., beneficial and injurious. We have seen that the acid 
treatment alone leads to the early stages of development, and 
that if the temperature is kept low the eggs of S. purpuratus 
develop to the early blastula stage. But before the gastrula 
stage is reached, the embryos go to pieces. To all appearances 
the early morula stages are quite normal. Loeb has suggested 
that cleavage may be accompanied by the production of toxic 
substances which, accumulating with each successive division, 
cause the larve to sicken and die. The injurious effects of these 
substances may be prevented by treatment of the egg with a 
hypertonic solution, or the formation of the injurious substances 
may be inhibited by long hypertonic treatment before membrane 
formation. It has been shown that simply by preventing oxida- 
tions for a time instead of treating the eggs with hypertonic 
sea water, normal development may be secured. Hence, the 
hypertonic solution or lack of oxygen exercises a beneficial or 
curative effect on parthenogenetically developing eggs and allows 
the embryo to develop to maturity. 

On the other hand there is an optimum time for the continuance 
of the action of the hypertonic solution, and if the exposure is 
continued longer the solution acts deleteriously and as a result 
development stops and the egg disintegrates. It becomes clear 
then that the hypertonic treatment may have one of the two 
opposite effects upon the egg, 7. e., beneficial or injurious, depend- 
ing upon the duration of the treatment, or in other words upon 
the condition of the egg when treated. 


RHYTHMICAL SUSCEPTIBILITY OF SEA URCHIN EGGS. 259 


In normal fertilization the sperm cell which fertilizes the egg 
accomplishes two things of immediate importance, viz., starts 
division and prevents the production of toxic substances during 
cleavage or inhibits their action. It is therefore impossible 
for the hypertonic sea water to exercise its protective action 
upon the normally fertilized egg. It can affect the egg only 
injuriously. Our experiments show that this injurious action of 
the hypertonic solution is most pronounced just preceding and 
during cytoplasmic division, and that such action is very slight 
immediately afterward. 

In the case of artificial parthenogenesis the hypertonic treat- 
ment is much shorter than in the experiments just described, 
and hence we may consider its injurious effects excluded. The 
Curve I. representing the results of Table II. shows the rhyth- 
micity of the beneficial effect of the hypertonic treatment, 
while the curve constructed from Table III. indicates only 
injurious effects. There is therefore no reason why the two 
curves should be identical in character, although each shows a 
rhythmicity of susceptibility to the action of hypertonic sea 
water. 

According to Loeb the artificial membrane’ formation in 
artificial parthenogenesis starts the chemical phenomena which 
give rise to the process of cell division and development; but 
the process is incomplete or abnormal and leads to the disintegra- 
tion of the egg unless a second treatment is added, usually a 
treatment with hypertonic sea water. Since, by the membrane 
formation, chemical or physico-chemical changes induced in the 
egg are rhythmical, it is intelligible that it should make a differ- 
ence in which stage of the cycle the treatment with the hypertonic 
solution is supplied. This is presumably the explanation of 
Herlant’s observation. 

In conclusion I wish to express my best thanks to Dr. Frank 
R. Lillie for so generously giving to me the privileges of the 
Marine Biological Laboratory at Woods Hole, and to Dr. 
Jacques Loeb for much helpful advice and criticism. 


ON THE CONDITIONS OF ACTIVATION OF UNFER— 
TILIZED STARFISH EGGS UNDER THE INFLUENCE 
OF HIGH TEMPERATURES AND FATTY ACID 
SOLUTIONS .1 


RALPH §S. LILLIE. 


INTRODUCTORY. 


In a former paper? I showed that brief exposure of the un- 
fertilized eggs of Asterias forbesiit to temperatures of 32° to 38° 
resulted in membrane-formation, cleavage and development. 
With normal eggs and the proper times of exposure almost every 
egg developed to a free-swimming larval stage; this treatment 
thus forms a highly effective parthenogenetic method. The 
time of exposure to the warm sea-water required to produce these 
effects is definite (within a certain slight range of variation) for 
any given temperature and decreases rapidly as the temperature 
rises. Thus, as regards the least exposure necessary for the 
formation of typical fertilization-membranes: ‘At 33° exposure 
must be prolonged to two minutes; at 34° the minimum lies 
somewhere between 30 and 60 seconds, at 35° between 15 and 
30 seconds, at 37.5° between 5 and 15 seconds, and at 40° momen- 
tary exposure (5 seconds) produces membranes in practically 
all eggs.’ The exposure required to induce development to 
larval stages was found to be considerably longer than for 


simple membrane-formation; at 35° from 70 to 90 seconds was 


required, at 36° from 50 to 60 seconds, at 37° from 30 to 35 
seconds, and at 38° about 20 seconds. The responsiveness of the 
eggs to this form of treatment was found to depend on the stage 
of maturation; warming before the dissolution of the germinal 
vesicle had begun was ineffective and in fact inhibited matura- 
tion entirely; the most favorable period lay between the break- 

1from the Marine Biological Laboratory, Woods Hole, and the Biological 
Laboratory, Clark University. 

2 Journal of Experimental Zoology, 1908, Vol. 5, p. 375- 

3 Loc. cit., p. 384. 

260 


2 
| 
2 


ACTIVATION OF UNFERTILIZED STARFISH EGGS. 261 


down of the germinal vesicle and the separation of the first 
polar body; after both polar bodies had separated development 
was imperfect and never proceeded far,—even membrane-forma- 
tion then failed in many eggs. 

Recent advances in the physiology of fertilization and artificial 
parthenogenesis have made it desirable to examine these effects 
of temperature in greater detail and to correlate them with the 
similar effects produced by other agents. During the past 
summer at Woods Hole I have accordingly re-investigated the 
changes in unfertilized starfish eggs following exposure for 
different periods to temperatures ranging from 28° to 36°, with 
especial reference to the differences in physiological effect result- 
ing from differences in time of exposure to a given temperature 
(e. g., 32°), and also with reference to the manner in which the 
time of exposure required to produce a given effect (e. g., mem- 
brane-formation) varies at different temperatures. Determina- 
tion of the temperature-coefficients of the processes underlying 
these effects is likely to afford indications of the nature of the 
fundamental changes concerned in the activation of the egg. 
Experiments on the effects of exposure to weak fatty acid 
solutions for different periods were also carried out; and on the 
action of high temperatures (32° to 34°) and fatty acid solutions 
on eggs which had previously been subjected to a membrane- 
forming treatment. 

It is well known that the temperature-coefficients of a large 
number of physiological processes have been found similar to 
those of chemical reactions in general.! This result is to be 
regarded simply as an expression of the fact that the energy for 
such processes is usually chemical energy freed by oxidations or 
other reactions, whose rate accordingly determines that of the 
process in question. There are, however, many instances in 
which rise of temperature produces an entirely different kind of 
effect. Often a process exhibits a critical temperature below 
which it entirely fails to take place.2, In such instances the 

1 For a summary account of researches in this field cf. C. D. Snyder: American 
Journal of Physiology, 1908, Vol. 22, p. 309. 

2 Examples of such processes are: inactivation of enzymes and toxines or de- 


struction of microorganisms by heat; heat-coagulation of proteins, and dependent 
processes like injury or destruction of various cells by heat; onset of heat-rigor; 


262 RALPH S. LILLIE. 


process may show a very rapid acceleration through a range of a 
few degrees above the critical temperature; it is then clear that 
the change of temperature acts in some other way than simply 
by accelerating an already existing chemical reaction. This is 
the class of cases to which belongs the influence of higher tem- 
peratures in initiating development in starfish eggs. Such 
departures from the usual temperature-coefficients of physio- 
logical processes indicate the entrance of other factors, the nature 
of which may be partly inferred from the character of the tem- 
perature-coefficient. Thus, to take the case of the starfish egg: 
in order to induce development of all eggs to a larval stage by 
exposure to a temperature of 31° it is necessary to keep them 
at this temperature for a period of about 15 minutes; at 36° an 
exposure of only one minute is necessary. The physiological 
process, whatever its nature, which renders the egg capable of 
proceeding with its development, thus takes place about fifteen 
times as rapidly at 36° as at 31°. This high temperature- 
coefficient indicates that a physical rather than a purely chemical 
change—possibly a change of the same nature as that determin- 
ing the liquefaction of a warmed gel—is responsible for the 
altered behavior of the egg. The time-relations show that some 
definite and progressive process, the end-effect of which is to 
remove the conditions hindering further development, is taking 
place in the egg during the entire 15 minutes at 31°. Exposure 
for the full period of fifteen minutes is necessary to bring this 
process to its completion, 7. e., to a stage at which the egg is 
in a position, when returned to sea-water, to continue auto- 
matically its development to a larval stage. If the exposure is 
only 5 minutes there is also a definite change in the egg; a 
typical fertilization-membrane is formed and there may be 
some irregular change of form or possibly a few abnormal 
cleavages, but the egg never develops far and soon dies. In this 
case the process of activation is evidently incomplete, and only 
a few of the early steps in development are carried out. If the 
exposure is too long (20 to 25 minutes) the egg also fails to 
develop; the process initiated by the higher temperature thus 


excitation of thermal sense-organs (e.g., of frog’s foot) and of certain vaso-motor 
and other temperature-regulatory mechanisms by heat; thermotactic responses. 


ACTIVATION OF UNFERTILIZED STARFISH EGGS. 263 


gives rise to injurious conditions if it continues beyond a certain 
time. For each temperature, in fact (from 30° to 38°), there is 
a well-defined optimum duration of exposure which initiates 
favorable development in all normal eggs; also a briefer exposure 
which results in simple membrane-formation followed by break- 
down; and a more prolonged exposure which renders the egg 
incapable of development. It is noteworthy that at each tem- 
perature the ratios of the durations required for these several 
effects are closely similar,—the optimum exposure being typically 
from two to three times that required for simple membrane- 
formation, and the maximum exposure (at which development 
to a larval stage just fails) about one and a half times the 
optimum.! This indicates that some single process, involving 
a critical change in the condition of the egg-protoplasm and 
having a characteristically high temperature-coefficient, under- 
lies and conditions all of these effects. This process does not 
begin until a temperature of about 29° is reached, and proceeds 
slowly at that temperature, taking approximately 30 minutes to 
attain its completion. A rise of eight degrees accelerates it 
some hundred times. Such facts appear to narrow the range of 
possibilities very materially; they point clearly to some physical 
change,—of structure, colloidal aggregation-state, viscosity, etc. 
—rather than to one of a purely chemical kind, as constituting 
the critical process underlying the activation of the egg. 

The experiments of the past summer have shown further that 
exposure to weak fatty acid solutions produces in the egg effects 
which are in all essential respects identical with those resulting 
from exposure to the above temperatures. Starfish eggs placed 
for one minute in sea-water containing 1/260 butyric acid (2 c.c. 
n/to butyric acid plus 50 c.c. sea-water).all form fertilization- 
membranes on return to normal sea-water; but if left without 
further treatment the eggs typically fail to cleave and soon 
break down without further development. Precisely the same 
effect is produced by brief exposure to warm sea-water, e. ¢g., 
three or four minutes at 32°. In either case it is necessary, in 
order to induce complete development of such eggs, to subject 
them to some second or supplementary treatment, such as 


1Cf. below, page 279. 


264 RALPH S. LILLIE. 


exposure to hypertonic or cyanide-containing sea-water. The 
starfish egg can, however, be made to develop completely without 
the necessity of any such after-treatment, simply by sufficiently 
prolonging the exposure to the membrane-forming agent. An 
exposure of 8 minutes to 32° is followed not only by membrane- 
formation, but by cleavage and development of all normal eggs 
to larval stages (cf. page 271). Similarly, exposure to 2/260 
butyric acid for a sufficient period—varying from 6 to 10 minutes 
—also causes all eggs to cleave and develop to larve (cf. p. 282). 
Over-exposure, if slight, is followed in both cases by a decrease 
in the proportion of favorably developing eggs; and if well- 
marked, by complete failure of development and early break- 
down. The only noteworthy difference that I have observed 
between the effects of the two agents is that the time-relations 
in the case of exposure to fatty acid have been somewhat more 
variable than in the case of exposure to a definite temperature 
such as 32°. Thus in some experiments eggs have exhibited a 
considerable proportion of favorable developments after only 
one minute’s exposure to weak fatty acid solutions.t In such 
cases however the concentration of acid was somewhat higher 
(3 c.c. n/I0 fatty acid plus 50 c.c. sea-water) than in the experi- 
ments described above. In last summer’s experiments (in which 
the fatty acid was always used in 7/260 concentration) the curves 
relating time of exposure to the proportion of eggs forming 
larvee were virtually identical in form with the two agents,—a 
fact showing that the essential effects produced by both types 
of treatment are the same. 

The fact that a properly timed single exposure to warm sea- 
water or fatty acid solution causes complete development 
suggests that the necessity for a supplementary after-treatment 
(e. g., with hypertonic sea-water), in the case of eggs in which 
fertilization-membranes have been formed by brief preliminary 
exposure to a cytolytic agent, depends simply on the incomplete- 
ness of the change induced in such eggs by the membrane-forming 
treatment. The fact that by sufficiently prolonging this treat- 


1 See the experiments described in my recent paper in the Journal of Experi- 
mental Zoology, 1913, Vol. 15, pp. 41, 42. Starfish eggs exposed for 1 minute toa 
mixture of 3 c.c. m/10 acetic or butyric acid plus 50 c.c. sea-water (7/176 acid) gave 
in several cases 20-30 per cent. of larve and in one case 70-80%. 


ACTIVATION OF UNFERTILIZED STARFISH EGGS. 265 


ment one can induce complete development in all eggs indicates 
clearly that the after-treatment produces in the egg effects which 
are physiologically of the same kind as those resulting from the 
membrane-forming treatment, and not qualitatively different 
as has usually been supposed. If this is so, we must conclude 
that hypertonic sea-water is favorable not because it exerts a 
“‘corrective’’ action different from that of the membrane-forming 
agent, but simply because it enables the process started by the 
first treatment and arrested at an unfinished stage to proceed 
to its completion. On this view the effects of the two successive 
treatments are simply additive. Apparently under the influence 
of the higher temperature or the fatty acid a certain definite 
process, which we may call the activation-process, is started 
in the egg. This process, if it proceeds to a certain definite 
stage, puts the egg in a condition to continue automatically its 
development to the formation of larvee; but if the process is 
arrested too soon (by the return to sea-water), the egg is able 
to carry out only a few of the early steps of development, includ- 
ing membrane-formation and perhaps a few cleavages. The 
after-treatment merely causes the resumption of the process 
and carries it to its completion. The unitary character of the 
activation-process is further indicated by the fact that the tem- 
perature-coefficients for simple membrane-formation and for the 
complete initiation of development are the same, as will be shown 
below. If this conclusion is correct, it should be a matter of 
indifference whether the exposure for the required period to 
the high temperature or the fatty acid solution is continuous or 
discontinuous. It ought to be possible to form fertilization- 
membranes by brief exposure to warm sea-water or fatty acid 
followed by a return to normal sea-water, and then later to 
complete the activation-process by a second exposure to either 
agent for an appropriate time. This is in fact the case; all of 
the four combinations have been tried: brief treatment with 
warm sea-water followed by after-treatment for several minutes 
with either warm sea-water or butyric acid solution; and mem- 
brane-formation by butyric acid followed by warming or a 
second treatment with acid. All four methods give the same 
result, namely the development of a high proportion of eggs to 


266 RALPH S. LILLIE. 


larval stages. The effect of such second treatment is in fact 
indistinguishable from that of exposure to hypertonic sea-water 
or cyanide. 

The problem of the nature of the effect produced on the egg by 
hypertonic sea-water, or the other corrective agent employed 
to supplement the membrane forming treatment, thus appears 
in a simpler light. In the starfish egg, after membranes have 
been formed as above, an exposure to (e. g.) 32° or to weak 
butyric acid solution for several minutes constitutes a highly 
favorable form of after-treatment, producing the same effect on 
development as hypertonic sea-water or cyanide.! This makes 
it appear doubtful that two qualitatively distinct processes are 
concerned in the activation of other eggs like the sea-urchin egg, 
where some form of after-treatment, different from the mem- 
brane-forming treatment, has hitherto proved necessary in order 
to induce development in a high proportion of eggs. The condi- 
tions are unlikely to be fundamentally different in the two 
animals. In the starfish egg the “corrective” effect resulting 
from after-treatment by heat has the same high temperature- 
coefficient as the initial change underlying simple membrane- 
formation by heat.?, This could hardly be the case if the two 
processes were qualitatively dissimilar; it indicates clearly that 
the same fundamental change in the egg-protoplasm furnishes 
the conditions for both the membrane-forming process and the 
“corrective’’ process.. I have found that in the Arbacia egg 
temporary warming (1 to 6 minutes at 32°, 34° and 35°) does 
not cause development (except in very few cases) even if followed 
by hypertonic sea-water;? and there is no evidence that pro- 
longed treatment with weak fatty acid solutions will cause com- 
plete development in this egg.t. The only highly and invariably 

1Cf. the experiments summarized in Tables XIII to XVII below. 

2 Compare the experiments of Tables XIV and XV below. 

3 Unpublished experiments performed last summer. An occasional egg may 
form a larva under this treatment, but the great majority remain unaltered. 

4In the case of Strongylocentrotus purpuratus Loeb found that eggs exposed to 
butyric acid solutions of the concentrations 2/250, 2/166, and ~/125 for more than 
2 minutes failed to form membranes (“‘ Artificial Parthenogenesis and Fertilization,” 
p- 141). Herbst found that eggs of Spherechinus treated for 2, 5, and 8 minutes 


with a mixture of 50 c.c. sea-water plus 3 c.c. n/10 acetic acid gave only occasional 
larve (Roux’s Archiv, 1906, Vol. 22, p. 473). Apparently no systematic experi- . 


ACTIVATION OF UNFERTILIZED STARFISH EGGS. 267 


effective after-treatment hitherto discovered for the sea-urchin 
egg is hypertonic sea-water.1 It would thus appear that the 
conditions in this egg differ considerably from those in the 
starfish; but the fact that a simple exposure to hypertonic sea- 
water, if sufficiently prolonged, has the same effect in inducing 
development as a briefer exposure to the same agent combined 
with membrane-formation by fatty acid, seems to indicate that 
the conditions are fundamentally similar in both types of egg, 
and that a unitary process underlies activation in both cases. 
The remarkable effectiveness of hypertonic sea-water with the 
sea-urchin egg would seem to be due to certain special largely 
incidental pecularities; temporary abstraction of water appears 
for some reason to render this egg more resistant to the dissolu- 
tion that otherwise results from the membrane-forming treat- 
ment. In other eggs, however, like those of the starfish or 
Nereis, hypertonic sea-water shows no special advantages over 
a number of other forms of after-treatment. The fact that a 
double form of treatment has hitherto proved especially effective 
with the sea-urchin egg is thus not inconsistent with the view 
that the activation-process is essentially unitary in character 
in all eggs. 


EXPERIMENTAL. EFFECTS OF SIMPLE EXPOSURE TO WARM 
SEA-WATER. 


In these experiments the procedure was similar to that de- 
scribed in my earlier paper... Sea-water at a temperature slightly 
above that chosen for the experiment was added rapidly to the 
small beaker containing the eggs (with a thermometer) until the 


ments of this kind have yet been performed with Arbacia. At Naples, using 
Arbacia pustulata, Lyon was able to cause development to larvee in ca. 10 per cent. 
of eggs by exposure to sea-water acidulated with HCl, but he did not try fatty 
acids (Amer. Journ. Physiol., 1903, Vol. 9, p. 310). 

1 Cyanide is only slightly effective with Arbacia punctulata (cf. my experiments 
described in Journal of Morphology, 1911, Vol. 22, page 703); it is more so with 
Strongylocentrotus, according to Loeb’s results (cf. “‘ Artificial Parthenogenesis and 
Fertilization,’ p. 80), but even here it is less uniformly favorable than hypertonic 
sea-water. 

2 Cf. the experiments of Loeb (loc. cit., Chapter XI; also Archiv fiir Entwick- 
lungsmechanik, 1914, Vol. 38, p. 409). It is probable that hypertonic sea-water 
has another and more distinctive mode of action (see below, p. 300). 

3 Journal of Experimental Zoology, 1908, Vol. 5, p. 379. 


268 RALPH S. LILLIE. 


required temperature was reached; this temperature was then 
kept constant during the period of the experiment by immersing 
the beaker in a water-bath at the same temperature. At inter- 
vals eggs were transferred to sea-water at room-temperature 
contained in finger-bowls. The exposure to the warm sea-water 
always took place during the interval between the complete 
disappearance of the germinal vesicle and the formation of the 
first polar body. 

Exposure to 28°, even if prolonged to 45 minutes, proved 
almost entirely ineffective in forming membranes in starfish 
eggs. With exposures of 30 minutes or more an occasional egg 
may form a membrane, but the great majority always remain 
unaltered. 

At 29° membranes appear in a considerable proportion of eggs 
after exposures of 12 to 15 minutes. With longer exposures 
(25 to 30 minutes) a majority in some cases (not always) may 
form membranes, and a considerable number may develop to 
larval stages. Table I. summarizes the results of two series of 
experiments in which eggs were exposed to 29° for periods ranging 
from 2 to 40 minutes. In both lots of eggs the great majority 
underwent normal maturation, and a large proportion developed 
normally to larve after sperm-fertilization. 


TABLE I. 
RO 
Approximate Proportion of Eggs Forming Fertilization-membranes and Larve. 
Duration of 
Exposure Series of June xo. Series of June rr. 
in Minutes. 
Membranes. Larve. Membranes. Larve. 
2to 10m. (0) (0) O (0) 
I2 m. ca. 2-3% (0) ca. 1% to) 
I4 m. Io— 15% fo) ca. 3-4% fo) 
I7 m. 25- 30% to) ca. 10% *o 
20 m. ca. 50% <7, ca. 20% (0) 
25 m. 70-— 80% ca. 2-3% 5-10% ca. 1% 
30 m. 30-40% 15-20% few ca. 5% 
40 m. ca. 1-2% ca. I-2% (0) (0) 


The two series show some minor differences, but in both the 
number of eggs forming larve is small, and a certain proportion 
fail to form membranes even with the optimal exposures. This 
temperature is near the lower limit below which the eggs show 


no response to this form of treatment. 
> 


ACTIVATION OF UNFERTILIZED STARFISH EGGS. 269 


At 30° the proportion of eggs forming membranes and develop- 
to larval stages is higher than at 29°, although considerable 
variability is still shown. Five series of experiments were 
performed at this temperature. Table II. summarizes the 
results of four of these.!_ Each lot of eggs was favorable, matura- 
tion and development to larve after sperm-fertilization taking 
place in nearly all. Table II. gives the approximate proportion 
of eggs forming membranes and developing to blastule after 
exposure for the periods given in the first column. 


TABLE II. 
30°. 
Proportion of Eggs Forming Fertilization-membranes and Larve. 
Duration 
of Ex- June 7. June 8. June re. June 13. 
‘unapnes. [os | | | 
; eee | Larve. nae Larve. eee | Larve. | Membranes.) Larve. 
Up to | | 
3 ida, (0) (0) (0) (o) O 0) 
Aim, ee, 596| Oo (0) (0) O | .@ 
Galil a |P5— 2095) 0 fo) (0) (0) (0) Ki | o 
6m. |ca. 20%| 0 0) ) | 
“i. |ca. 30%) ° 0 | GQ, 39)| © <1% fe) 
Sims ia, SOI) O len, 19%) © | 5-10% (0) 
9-10 m.|ca. 100% ca. 2-3 |20-25%| 0 40-50%| oO I0-15% (0) 
| A || | 
I2 m. 50-60% ca. I % 70-80% (o) 20-25% <1% 
I4-1I5 m.|ca. 100% ca. 5% |80-90%| 2— 3%| >90% 10-15% 
17-18 m. | ca. 90%| 5-10% | 30-40% |ca. 5% 
20-21 m. <50%| 5-10% | ca. 40% ca. 30% 
24m. | | | 30-40% |30-40% 
28 m. | ca. 40% ca. 40% 
30 m. | ca. 10%| <5% | ; 
34 m. | ca. 15-20%| <1% 


It will be noted that in four out of the five series at 30° an 
exposure of 8 to 10 minutes was required to cause membrane- 
formation in IO per cent. or more of the eggs; in the fifth series 
(June 7) 5 minutes was sufficient. The proportion of eggs 
developing to larvae was comparatively low in all series; the 
optimum exposure lay between 24 and 28 minutes in the only 
series (June 13) in which the proportion of larve was consider- 
able. With longer exposures membranes become fewer and there 
is a rapid decline in the proportion of eggs forming larva. 


1Tn the remaining series the longest exposure was 10 minutes, at which about 
two thirds of the eggs formed membranes and a small number developed to larve. 


270 RALPH 5S. LILLIE. 


At 31° the-conditions become more favorable and with the 
proper times of exposure practically all mature eggs form fertiliza- 
tion-membranes, and in favorable cases the great majority 
develop to larve. Four series of experiments were performed 
at this temperature; in one of these (August 28) only about half 
the eggs underwent maturation, and with 15 minutes’ exposure 
(approximately the optimum) only Io to 15 per cent. of all eggs 
formed larve. In the other three series the eggs were normal. 
The proportions of eggs forming membranes and larve in these 
series with the different times of exposure are given in Table III. 


TasBLe III. 


eiiee 


Proportion of Eggs Forming Fertilization-membranes and Larve. 


Duration of 
Exposures June 8. June 12. June 13. 
in Minutes. : 
Membranes, Larve. Membranes. Larve. Membranes, Larve. 
I-2 m. (0) @ (0) 

24 m. Oo (0) O 
3m. Co) fo) ca. 10-15% ca. 5% (0) 

33 m. 30- 40% ca. 50% 0 
4m. | Few (<1%) o 70-— 80% 60- 70% Co) 
5m.| 10-15% ) ca. 90% >95% Oo 
6m. ca. 20% (0) S90% ca. 100% fo) 

8 m. 80-90% ca. 2-3%| > 90% | ca. 1% | ca. 100% ca. 1% 

Io m. ca. 90% 15-20% | ca. 100% | 20-30% | ca. 100% ca. 20% 

I2 m. ca. 90% 40-50% ca. 100% ca. 60% 

I4-I5 m. 70-80% 40-50% ca. 90% 80-90% 
I7-18 m. ca. 50% ca. 40% ca. 75-80%| 50-60%; 
20-21 m. 15-20% Bt 5% 40-50% |ca. 10-15% 
25-30 m. ca. 20% (0) 


At this temperature an exposure of 3 to 4 minutes is required 
to cause membrane-formation in Io per cent. or more of the eggs; 
exposure must be prolonged to ca. 8 minutes before any eggs 
form larve; 14 to 15 minutes is the approximate optimum. In 
the series of June 12 this optimum was not reached. 

At 32° a larger number of experiments were performed than 
at any other temperature, and their results show a decidedly 
greater uniformity than at lower temperatures. With the 
optimal times of exposure (from 7 to 8 minutes) the proportion 
of larve yielded by normal eggs is always high,—usually over 
go per cent. This is illustrated by Table IV., which summarizes 
the results of six successive series performed during June at a 


a 


ACTIVATION OF UNFERTILIZED STARFISH EGGS. 271 


time when starfish eggs were unusually abundant and favorable. 
On account of the relative completeness of my observations at 
this temperature, the general results of these experiments will be 
described in some detail. 

‘The exposure required for membrane-formation is about half 
that at 31°. An exposure of 3 minutes typically forms mem- 
branes in all normal eggs, and one of 2 minutes is usually sufficient 
to produce this effect in a minority and sometimes in a majority 
of eggs. From 3 minutes on the conditions remain normal for 
membrane-formation until the exposure is prolonged to 12 or 
15 minutes, after which in a certain proportion of eggs membranes 
tend to separate imperfectly or even fail to form. In most series 
exposures longer than 15 minutes were not used, since eggs so 
treated never form larve; in one series, however, eggs were 
exposed for 27 minutes, at which exposure nearly half failed to 
form membranes. This decline in membrane-formation when 
exposures are prolonged beyond a certain maximum is general 
for all temperatures (cf. also the series at 30°, 31° and 33°); the 
fact is interesting since it indicates that the process is not a direct 
effect of the high temperature but constitutes an active response 
—probably in the nature of a secretion—on the part of the egg. 

The optimum exposure for inducing complete development at 
32° varies between 6 and 8 minutes, and with this exposure the 
great majority of normal eggs cleave and develop to larval stages. 
Many of the gastrule and Bipinnariz thus obtained are appar- 
ently quite normal and swim freely at the surface of the water. 
The rate of development is, however, always slower than that 
of sperm-fertilized eggs; relative slowness of development seems 
in fact to be a constant peculiarity of parthenogenetically acti- 
vated eggs.! Exposures well above the optimum are followed 
by imperfect or delayed cleavage and failure to develop beyond 
early stages. 

Table IV. gives a summary of the results of the six successive 
series referred to above. The approximate proportions of eggs 
forming free-swimming larve are given; the conditions of mem- 
brane-formation have already been sufficiently described. 

1 This has been my uniform experience since I began studies of this kind, and 


apparently the experienceis general. This suggests.strongly that the spermatozoon 
contributes to the egg material which is utilized in normal development. 


22 RALPH S. LILLIE. 
TABLE IV. 
Ray?) 
SORE Proportion of Eggs Forming Free-swimming Larvz. 
eeeaaey June 12 June 13. June 18. June 24. June 25. June 26. 
I-3 m. 0) 0) 0 (o) Ce) Co) 
4m. Ba, u% KG, | ets G=B% || a= BIA || Ga, 3% Co) 
& io. ca. 3- 4% 2— 3% | 15-20% | 25-35% | ca. 50% | 10-15% 
6 m. ca. 35-40% | 20-30% | 55-60% | 60-70% | 80-90% | 25-35% 
7 OD, 70-80% =90% ca. 60% | 50-60% 
8 m. >90% ca. 95% | Ca. 957% | 90% | 25-35% | 80-90% 
ro m. 85-90% 50-55% | 75-85% | 50-60% | < 5% | 80-90% 
I2 m. 15-20% | 25-35% (0) ca. 20% 
I5 m. fo) <1% 
18, 22, and 
27 m. ) | (0) 
These results may safely be regarded as typical. Six other 
similar series were carried out at this temperature. In two of 


these the eggs were unfavorable or the treatment was applied 
too late. In the four others—two in early June and two in late 
August—the results were similar to the above, although fewer 
eggs formed larve; the optimum exposures ranged from 6 to 8 
minutes, with respectively 20, 20, 40 and 50 per cent. of mature 
eggs forming larve. It will be noted that the optimum exposure 
is approximately 8 minutes in five out of the six series in Table IV. 
Different lots of eggs vary somewhat in the duration of this 
optimum; thus in the series of June 25 half of the eggs formed 
larve with only five minutes’ exposure and the optimum was 6 
minutes, and on June 26 the eggs showed almost equally good 
development with the 8- and the 10-minute exposures. In the 
majority of series, however, there was a well-defined optimum at 
7 or 8 minutes. 

The physiological effects following exposure to 32° vary in a 
constant and highly characteristic manner with the duration of 
the exposure. Eggs exposed for a period insufficient to induce 
membrane-formation show no apparent change on return to 
sea-water and later break down without development. Such 
eggs, however, can be shown to have undergone some internal 
change similar in kind to that following longer exposures; thus 
if later they are again exposed to 32° they are found to require, 
in order to induce favorable development, a shorter exposure 


ACTIVATION OF UNFERTILIZED STARFISH EGGS. Dis 


than previously untreated eggs (cf. below, p. 288). Exposure 
for 3 to 4 minutes induces typical membrane-formation in all 
eggs, followed, however, not by cleavage and further develop- 
ment but by irregular changes of form, fragmentation, and 
eventual breakdown. With somewhat longer exposures (4 to 5 
minutes) membrane-formation is followed by symmetrical cleav- 
age in a certain proportion of eggs; and the proportion of such 
cleavages, and also their approximation to the normal in rate 
and character, show a progressive increase with increasing length 
of exposure up to the optimum of about 8 minutes. With still 
longer exposures the response again becomes unfavorable, and 
eventually the eggs entirely fail to develop and even to form 
membranes. We have here an apparent reversal of the rule 
enunciated by Loeb with reference to the action of membrane- 
forming agents on the sea-urchin egg. ‘‘A relatively brief 
exposure to a cytolytic agent leads only to membrane-forma- 
tion, while a longer exposure causes cytolysis.’ In the star- 
fish egg a relatively brief exposure to warm sea-water (one just 
sufficient for membrane-formation) is followed by an early 
cytolysis, while a longer exposure results not only in membrane- 
formation but in an approximately normal development; still 
longer exposures again cause cytolysis without development. 
This rule applies to the action of cytolytic substances like fatty 
acid, as well as to high temperatures (cf. below, p. 282). 

To illustrate the effects of exposures of different duration on 
cleavage the following record is given (Table V.) describing the 
condition of the eggs about four hours after exposure to 32° for 
the times given. 

It will be noted that with brief exposures (3 to 4 minutes) 
membrane-formation is typical, but the eggs are unable to cleave 
normally and undergo irregular change of form followed by 
breakdown. As the time of exposure increases, an increasing 
proportion of eggs cleave, until the optimum (6 to 7 minutes) 
is reached at which cleavage approaches the normal in rate and 
character, and the great majority develop to larval stages. 
Over-exposure (10 minutes) is again followed by failure of 
cleavage and development. Similar observations were made 


1“ Artificial Parthenogenesis and Fertilization,” 1913, p. 8. 


274 RALPH S. LILLIE. 


in experiments at other temperatures; in all cases the exposure 
which induced the largest proportion of regular cleavages was 
found to correspond with that at which the largest proportion 
of eggs formed larve. In the series of Table V. the optimum, 
6 minutes, is somewhat shorter than usual; on June 24 the 
optimum of cleavage was found at 7 minutes, and on June 26 
at 8 minutes, with 10 minutes somewhat less favorable. 


RABE Ve 
JUNE 25. 32°. 


Condition of Eggs 4 Hours after Exposure, and Proportion 


Time of Exposure. of Eggs forming Larve. 
TH SME T SS Oa e ear Mealy clay TS ete ts Great majority are unchanged; a few have membranes. 
No larve. 
Dee Tha cS leeway ee aaa Most eggs have typical membranes and are irregular 
or amceboid in form; a few show irregular cleavages. 
No larve. 
BMA EYL ste sa eerepeece elope Antaris Almost all eggs have membranes and exhibit irregular 


forms; a few have cleaved symmetrically. Ca. 
5 per cent. form larvae. 

AISMISP OO ieueeter en EN ots Gis eecL as 0 Marked improvement over Experiment 3: most eggs 
have cleaved, and many are in regular 4- and 8-cell 
stages. Ca. 50 per cent. form larve. 

ReeOMIIs 5 cea ens cies ymeeen eee Almost all eggs are cleaved; cleavages are more regular 
and advanced than in Exp. 4; 16-cell stages are 
frequent. 80-90 per cent. form larve. 


On 7m Bae a aetna eee eae The proportion of regular cleavages is also high, but 
rather less than in Exp. 5. Ca. 60 per cent. form 
larve. 

iat an beaietea her tyeteneosste: Uo. aie eb Cleavages are fewer and less advanced than in Exp. 6. 
Ca. 25-30 per cent. form larve. 

SD LOM cc eee eee eee Great majority are uncleaved; many are irregular in 
form or fragmented. Larve are few: < 5 per cent. 

EMME Qe Tihs ean canieaetaaye eer Almost none have cleaved. The eggs are largely 


irregular or with small surface-vesicles detached. 


While an exposure just long enough for membrane-formation 
is insufficient by itself to induce normal cleavage and develop- 
ment, it is possible, after forming membranes in this way, to 
make the eggs cleave and develop to larval stages by subjecting 
them to a second treatment with warm sea-water, or by after- 
treatment with fatty acid (m/260 butyric acid in sea-water), 
hypertonic sea-water, or cyanide (7/1000 KCN in sea-water). 
These effects will later be described in detail (cf. Tables XIII., 
XVIL.). 


‘| 
| 


ACTIVATION OF UNFERTILIZED STARFISH EGGS. 275 


It is interesting to note that the effects produced by weak fatty 
acid solutions (7/260 butyric acid) on unfertilized starfish eggs 
also vary with the time of exposure in a manner closely similar 
to that just described. Brief exposure causes membrane-forma- 
tion followed by irregular change of form and breakdown without 
development, while longer exposure induces not only membrane- 
formation but cleavage and development to larval stages; still 
longer exposure is again unfavorable. Eggs in which membranes 
have been formed by the minimal exposure to fatty acid may be 
made to develop by the above forms of after-treatment. The 
effects of the two agents, warm sea-water and weak butyric 
acid solution, seem in fact to be identical in every essential 
particular, and the one may be substituted for the other without 
altering the effect on the egg (cf. Tables XIV. to XVII.). Experi- 
ments showing this parallelism will be described in detail later. 
There is in fact every indication that the underlying physio- 
logical process which enables the egg to continue normal develop- 
ment is of the same nature as that which induces simple mem- 
brane-formation, the only difference being that the duration of 
the process must be considerably longer in the second case than 
in the first. The temperature-coefficients of both effects indicate 
the same, as will appear below (cf. Table X.). The possible 
nature of this process will be discussed in the concluding section 
of this paper. 

Treatment with sea-water at 33° gives similar results to those 
above described, except that the times required to produce a 
given physiological effect are only a little more than half as 
long as at 32°. An exposure of from one to one and a half 
minutes is needed to call forth membrane-formation in the 
majority of eggs. Four series of experiments with normal eggs 
were performed at this temperature, and in every series the 
great majority of eggs formed larve with the optimal times of 
exposure. In these series the earlier transfers from the warm 
sea-water to normal temperature were made at half-minute 
intervals. The results are summarized in Table VI. 

In all of these series the proportion of favorably developing 
eggs is high with the optimum exposures of 414 to 5% minutes. 
The series of June I5 is unusual in that nearly all of the eggs 


276 


RALPH S. LILLIE. 


form larvee with exposures varying in length from 4 to 6 minutes. 
The optima seem to be less sharply defined when the eggs are 
in the best of condition, probably because then the power of 
regulatory adjustment to environmental variations is at its 
maximum, and slight deviations from the optima are auto- 


matically corrected. 


TABLE VI. 


Bon. 


Time of Exposure. 


1 
iS} al 


eon a ees NIE NY NIH 
PE, BEEEESE 


6 m. 


I2 m. 
I5, 18, 21 and 25 m. 


Proportion of Eggs Forming Larve (Blastule and Gastrule). 


June 9. 
10) 


(0) 
Few (<1%) 
IO-15% 
ca. 50% 
80-90% 
60-70% 
40-50% 


20-25% 
ca. 2-3% 


June zo. June 15. 
(0) (0) 
Very few (<0.1%) | (0) 
a, 2%, << 1% 
ca. 10% CG. 8% 
50-60% 20-30% 
80-90% ca. 90% 
90% ca. 90% 
90-95% ca. 95% 
| 90-95% 
>90% | ca. 90%. 
75-85% 
70-80% 10-15% 
15-20% (o) 
(0) 
(o) 


June 17. 


(0) 
Oo 


25-30% 
55-65% 
90-95% 
ca. 95% 


70-80% 
30-40% 


At 34° the majority of eggs form membranes with one minute’s 
exposure, and 30 seconds is sufficient for a minority. A few eggs 
form larve after 2 minutes’ exposure; the optimum is 3 to 4 


minutes; longer exposure is injurious. 


Table VII. summarizes 


Time of Exposure, 


2m. 


Ny 


QdikU1 diH BE NIKO bi 


Ww 
HHESH ESS 


aS 


On 


Proportion of Eggs Forming Larve. 


TABLE VII. 
34%. 

June ro. June 5. 

to) (0) 

gn, 2%, (0) 

25-35% ) 
50-60% 20-30% 
65-75% ca. 90% 
70-80% ca. 90% 
50-60% ca. 90% 
: ca. 90% 
5-10% ca. 10% 
cay 2% 

<1% (0) 

(0) 


7, 8, 10 and 12 m. (0) 


August 26, 


August 27. 


eal eae 


ACTIVATION OF UNFERTILIZED SATRFISH EGGS. PTT 


the results of four series of experiments at this temperature. 
The August eggs were less favorable than the June eggs. 

Two similar series at 35° and one at 36° were carried out in 
June at a time when starfish eggs were unusually favorable. The 
results were similar to those at 33° and 34° except that the physio- 
logically equivalent exposures were shorter. At 35° an exposure 
of 30 seconds induces membrane-formation in many but not in 
all eggs, and one of 45 seconds in practically all. At 36° 15 
seconds is sufficient to form membranes in about half the eggs 
and 30 seconds in all. Longer exposures eventually interfere 
with membrane-formation; thus after 6 to 8 minutes at 36° mem- 
branes failed to form or were imperfect in 40 to 50 per cent. of 
eggs. Table VIII. gives the proportions of eggs forming swim- 
ming larve in these experiments. The transfers from warm to 
normal sea-water were made at first at intervals of fifteen seconds. 


TaBLeE VIII. 
35° and 36°. 
Proportion of Eggs Forming Larve. 
Time of Exposure. |—_ aa 
June 15 (35°). June 16 (35°). June 17 (36°). 
Tne, BO (0) (e) (0) 
asl” (0) ) 20-30% 
Im. Gi, UGG fo) 85-90% 
ay ale 57 ca. 5% ca. 4-5% 95% 
I m. 30” 80-90% 35-45% 70-80% 
i Io ARY >95% 70-80% ca. 10% 
2m. ca. 90% =90% none free 
24 m. ca. 50% 40-50% oO 
Bere CO, B%, 5-10% (0) 
33 Mm. ) 0 Oo 
4m.—-I0om (0) () fo) 


The rapid decrease in the optimum exposures as the tempera- 


ture rises is to be noted; the optima are respectively 1144 to 2 
minutes at 35°, and I to 144 minutes at 36°. 


VARIATION WITH TEMPERATURE IN THE RATE OF THE PROCESS 
UNDERLYING ACTIVATION BY HEAT. 


The foregoing results show that the process, whatever its 
nature, which is initiated in the mature unfertilized starfish egg 
by temperatures of 30° to 36°, and which brings the egg into a 
condition to form membranes cleave, and develop, proceeds 


278 RAPLH S. LILLIE. 


twenty or twenty-five times as rapidly at 35° as at 30°. Thus 
at 30° the minimum exposure for membrane-formation is about 
IO minutes, and at 35° 30 seconds or less; similarly at 30° the 
minimum exposure needed to induce even a few eggs to form 
larve is about 20 minutes, at 35° it is about I minute; at 30° the 
optimum exposure is ca. 30 minutes, and at 35° ca. 1% minutes. 
For each temperature it is possible to assign a definite length of 
exposure which produces a definite effect on the egg. The 
manner in which these times of exposure vary at different 
temperatures may be seen by reference to Table IX. Here are 


TABLE IX. 
s Minimum for ae 
Temperature Membrane- Minimum for Optimum for Larve. | Maximum for Larve. 
and Series. fOCmnOne arve. 
29° (June 10) I4 m. AS ian. 
30° (June 13 8-10 m. 12-18 m. 24-28 m. 
an® Mee 5 intl, 8 m. 12-14 m. 20 m. 
June 12 3 m. 8 m. 
; Ce 33 m. 8 m. I4-I5 m. 21 m. (<25 m.) 
32° (June 12 25 mM. 4m. 8 m. 
(June 13) 2m. 4-5 m. 8 m. I2 m. (<I5 m.) 
(June 18) 2m. 4m. 8 m. I2 m. (<I5 m.) 
(June 24)| 2-3 m. 4m. 47-8 m. ? (>I10 m.) 
(June | 2m. 4m. 6 m. Io m. (<12 m.) 
(June 26)) 2 ig). 5 m. 8-10 m. I2 m. 
33° (June 9) 2m. 3m. 4m. 7m. 
(June 10), Im. 24 m. 43-6 m. IO m. 
(June a Im. 3m. 4-6 m. 8 m. (<12 m.) 
(June 17)) Im. 3 m. 5-6 m. Io m.+ 
34° (June 10) 30 sec. 134m. 3-33 m. 5 m. (<6 m.) 
(June 15) I m. 24 m. 3-4 m. 53 m. (<6 m.) 
35° (June 15) 30 sec. Im. |r m.30/—r m. 45” 3 m. (<3% m.) 
(June 16) 30 sec. i fia, eR” ca.2m . 3m. (<3% m.) 
36° (June 17) I5 sec. As! ( S30”) Im.-rm.15” | ca.2m. (<23 m.) 


tabulated the observations made in all of those series of experi- 
ments in which a large proportion of eggs formed larve,—in 
which, therefore, the conditions may be regarded as essentially 
normal. In the series at 29° few eggs formed larvee; at 30° only 
one series out of five gave a considerable proportion of larve (ca. 
40 per cent.) with ca. 30 minutes’ exposure; in all of the other 
series in the table, except one at 31°, the great majority of eggs— 
usually over 90 per cent.—formed larve with the optimal 
exposures. In the first column is given the least time of ex- 
posure required for membrane-formation in a significant pro- 


ACTIVATION OF UNFERTILIZED STARFISH EGGS. 279 


portion of eggs—1Io per cent. or more; in the second column the 
least exposure at which any eggs (> I per cent.) formed larve; 
in the third the optimal time of exposure; and in the fourth the 
longest observed exposure at which any eggs (> 1 per cent.) 
formed larve. 

If the several observed durations at each temperature are 
averaged, the following results are obtained (Table X.); the 
values are given in approximate terms rather than strict arith- 
metical averages, to emphasize the fact that the precise durations 
vary to a certain degree, even in normal eggs. There is, however, 
for each temperature a well-defined modal duration of exposure 
for producing a definite physiological effect such as membrane- 
formation or complete activation. 


TABLE X. 


APPROXIMATE TIMES OF EXPOSURE REQUIRED TO PRODUCE THE FOLLOWING 
EFFECTS AT DIFFERENT TEMPERATURES. 


Formation of Minimum for Optimum for Maximum for 
Temperature, Membranes. Larve. Larve. Larve. 
Bow ca. 12-14 m. 20-25 m. 30-40 m. 
30° 8-10 m. ca. 18 m. | ca. 28 m. ?>30 m. 
Biles ca. 4m. ca. 8m. ca. 15 m. 21-25 m. 
Boe ca. 2 mM. 4— 5 m. 7—- 8m. IO-12 m. 
Bee ca. I m. 24-- 3m. 43-53 m 8-10 m. 
34° 30” to I m. Ga. 2) ms 3-34 Mm. ca. 5 m. 
25° ca. 30” I-I}t m. 13-23 m. ca. 3 m. 
36° Gan TY” 30-45” I-1I¢ m. ca. 2 m. 
Bin 30-35 sec. 
38° ca. 20 sec. 


It will be noted (1) that for each temperature there is a 
minimum effective exposure which induces membrane-formation 
followed by failure to develop and early breakdown; and (2) 
that an exposure of approximately twice the minimum for 
membrane-formation is required to enable even a few eggs to 
develop to larval stages, and an exposure of three or four times 
this minimum to enable development to proceed normally in 
all eggs; and (3) that if the exposure is prolonged to about one 
and a half times this optimum the eggs are again incapacitated 
from further development. The fact that the ratios of the dura- 
tions required to produce these several effects are approximately 
the same at any one temperature indicates that a single process 


280 RALPH S. LILLIE. 


of a definite kind forms the determining condition of all. This 
process is peculiar in undergoing marked acceleration by slight 
rise of temperature; it is also clear, from the fact that an effective 
exposure must last for a certain minimal time at any temperature, 
that the process must proceed to a definite stage before the egg 
is rendered capable of continuing its development to advanced 
stages; if the process is arrested before its completion, only the 
earlier developmental changes can be carried out (membrane- 
formation, early cleavage or change of form); if, on the other 
hand, it is allowed to proceed too far, injurious conditions arise 
which eventually prevent all development; a sufficiently pro- 
longed exposure to high temperature renders the egg incapable 
even of membrane-formation. | 

In endeavoring to form some consistent conception of the 
nature of this process the following facts have to be considered. 
It exhibits a high temperature-coefficient: from fifteen to twenty 
times the duration of exposure is required to induce membrane- 
formation at 30° as at 35°; the ratios between 29° and 34° and 
between 31° and 36° are the same. At each temperature the 
proportionate durations of the minimum, optimum, and maxi- 


mum exposures for forming larve are approximately the same. 


In other words, the critical change underlying simple membrane- 
formation is affected by temperature in the same way as that 
underlying complete activation of development: 7. e., the pro- 
portionate increase in velocity by rise of temperature is the same 
in both cases, a fact which can only indicate that one funda- 
mental process—and not two—is concerned in producing both 
effects. If we assume that the above proportionate increase in 
velocity prevails through a rise of 10°, a Qio value of from 225 to 
400 is indicated, as against the 2 to 3 characteristic of chemical 
reactions in homogeneous media. Activation by heat thus 

1 The temperature-coefficients of the rate of cytolysis of sea-urchin eggs and of 
the duration of life of sea-urchin larve and of Tubularia stems at temperature of 
25° to 40° show similarly high values. In these cases the direct effect produced on 
the cell by the high temperature is probably of the same kind as that underlying 
the above activation-effect; this appears to be a change in the colloids of the plasma- 
membranes, leading to an increase of permeability. (See below, p. 296.) Such 
a change if not reversed within a certain time results in cytolysis. In the unfer- 


tilized starfish egg temporary increase of permeability involves activation. For 
‘data on the temperature-coefficients of cytolysis and heat-death, cf. J. Loeb, 


srt wel 


ACTIVATION OF UNFERTILIZED STARFISH EGGS. 281 


depends on some critical change in the egg which does not 
begin until a temperature of about 29° is reached, but which 
undergoes very rapid acceleration with further rise of tempera- 
ture. The liquefaction of gels by heat seems to be the only 
relevant process which shows these characteristics. The change 
in viscosity preceding the gelation of a gelatine sol undergoes 
very rapid acceleration with lowering of temperature, within a 
few degrees of the temperature of gelation. The inverse process, 
melting of gels, has a similarly high temperature-coefficient (cf. 
below, p. 295). In general the facts suggest that the direct effect 
of the high temperature is to cause a change in the colloidal 
system of the egg, of such a kind as to render possible a chemical 
interaction between substances which in the normal condition 
of the resting egg are kept apart. *This restraining condition 
may be some physical barrier like a membrane, impermeable to 
the diffusion of the substances concerned, or it may be a certain 
state of electrical polarization of the general cell-surface, as 
suggested below (p. 299). It is also important to note that the 
activation-process may be arrested by a return of the eggs to 
sea-water at ordinary temperatures, and renewed after an interval 
without interfering with its effect. A reversibility of the physico- 
chemical change forming its basis is thus indicated. It should 
further be noted that cytolytic agents like butyric acid not only 
have the same general physiological effect as brief warming, but 
that the relations between time of exposure and physiological 
effect produced are the same in both cases. Some process which 
is affected similarly by these two dissimilar agents is thus to be 
sought. In the following section the results of experiments with 
weak butyric acid solution are described in greater detail. 


EFFECTS OF EXPOSURE TO ButTYRIC ACID SOLUTION FOR 
DIFFERENT PERIODS. 


As already stated, treatment of starfish eggs during the early 
maturation period with weak solutions of butyric acid in sea- 
water (7/260) produces the same effects as temporary warming, 
Archiv f. d. ges. Physiologie, 1908, Vol. 124, p. 411; A. R. Moore: Quarterly Journal 


of Experimental Physiology, 1910, Vol. 3, p. 257; Arch. f. Entwicklungsmech., 1910, 
Vol. 20, pp. 146, 287. 


282 


RALPH §. LILLIE. 


and the time-relations of the exposures necessary for these 


effects are closely similar with both methods. 


Table XI. 


summarizes the results of five series of experiments with separate 
lots of eggs. The eggs were exposed at normal temperatures 
(20° to 22°) to an 2/260 solution of butyric acid in sea-water 
(50 c.c. sea-water plus 2 c.c. n/10 butyric acid), and portions 
were transferred to normal sea-water at the intervals named. 
The approximate proportion of mature eggs developing to free- 


swimming larve (blastule and gastrule) is given. 


RhApre XU: 


N/260 Butyric ACID.) 


Proportion of Mature Eggs Developing to Larve. 


Time of : : 
Reporte: Series 1 (Aug. 31.) ERS Series 3 (Sep. 1). |Series 4 (Sep. 2.) ee 
I m. (0) (0) I or 2 larve t blastula <1% 
2m. 2 or 3 larve fo) GG, Wf ca. 1% 2-3% 
A ia, <1% <1% ca. 4-5 % I- 2% 20-30% 
4m. <1% ca. 1% ca. 10% 5-10% 55-60% 
5m. ca. 1% ca. 10% IO-15% 20-30% | 75-85% 
6 m. ca. 5% ca. 50% 20-30% 30-40% 80-90% 
7m. 20-30% 70-80% 40-50% 20-25% 35-40% 
8 m. ca. 50% 65-75% 50-60% 15-20% 20-30% 
IO m. 80-90% 65-75% ca. 60% ca. 1% 10-15% 
I2 m. 30-40% 25-35% 40-50% t blastula (0) 
I5 m. <1% Gn, WG 20-30% (0) (0) 


The close parallelism between these experiments and those of 
warming to 32° or 33° will at once be noted. With brief exposure 
there is the same simple membrane-formation followed by break- 
down without development; as the exposure is prolonged there 
is a progressive increase in the proportion of favorably develop- 
ing eggs up to an optimum; then follow a decrease and eventual 
failure to develop. More detailed observations show that the 
rate and regularity of cleavage show a corresponding steady 
improvement up to an optimum which is again followed by a 
decline. } 

The following observations show the condition of the eggs in 
the second series of September 1, at about four hours after the 
treatment with butyric acid (Table XII.). 

The optimum time of exposure shows somewhat more vari- 
ability in these series than is usually the case with exposure to 


ACTIVATION OF UNFERTILIZED STARFISH EGGS. 283 


warm sea-water (32°); in all five, however, the optimum lay 
between five and ten minutes.! There is thus an approximate 
constancy in the time of exposure required to induce complete 
development with solutions of this concentration. Probably 


TABLE XII. 


N/260 Butyric AcID. 


Condition of Eggs 4 Hours after Treatment, and Proportion 


Time of Exposure, forming Larve. 
TWIP eee ane ae er eae wane All eggs have membranes; most are irregular or 
amoeboid in form; none are cleaved. No larve. 
DAIS Osa DECOR OLR OTe Similar to r m. lot, but a few eggs (ca. 2-3 per cent.) 
are in the 2-cell stage. No larve. 
B TODS sy carceb CRI ETERS Generally similar to the 2 m. lot, but the cleavages 


are more numerous (ca. 10-15 per cent.), mostly 
2-cell with a few 4-cell stages. Very few larve 
(< I per cent.). 

Gl, Sai dps ie ee sets dem ree ear Cleavages are more numerous and advanced; 40-50 
per cent. are cleaved, mostly 2 and 4-cell, with a 
few 8-cell stages. Larve still few (ca. I per cent.). 

pla Meeet oyes cane sesalece iy a eapaiseaie Cleavage is more advanced than in the 4 m. lot; ca. 
50 per cent. are cleaved, largely 8- and 16-cell 
stages. Ca. Io per cent. form larve. 


Gaderer pree se oe ey eee RN Most eggs are cleaved (ca. 70-80 per cent.), many in 
16- to 32-cell stages. Ca. 50 per cent. form larve. 
GT IODA hoeee eect Ree tv er a Almost all eggs are cleaved (90 per cent. or more), 


many in normal-looking 16- to 32-cell stages. 
470-80 per cent. form larve. 


RTI Re in ee ne as i tlre Similar to 7 m. lot; most eggs are in 16- to 32-cell 
stages. 65-75 per cent. form larve. 
MOpMbey tre caepewe a aseaw eke ee eee alec Cleavages are fewer and less advanced; ca. 70-75 


per cent. are cleaved, mostly 4- and 8-cell stages. 
65-75 per cent. form larve. 

TD! Weoley echelons OeRCRe Ve ee Te Nee ace Comparatively few cells are cleaved; ca. 10 per cent. 
are in 2- or 4-cell stages, largely irregular; the rest 
uncleaved. 25-35 per cent. form larve. 

TES) SRG 3:3 dye sai cae Tee ane oes pace Almost all eggs remain uncleaved, and many show the 
beginnings of surface-disintegration. Few form 
larvee,—ca. I per cent. 

Controls: Unfertilized eggs disintegrate without membrane-formation or de- 
velopment. Nearly all sperm-fertilized eggs develop to larve. 


1 This variability may be due partly to the fact that on account of the lateness 
of the season and consequent scarcity of ripe starfish the eggs used in these experi- 
ments came from fewer animals; thus in Series I, 4, and 5, eggs from only one star- 
fish were used in each case, and in Series 2 and 3fromthree. Inthe earlier experi- 
ments with warm sea-water the mixed eggs from several animals were used in 
each series. 


284 UNIEIZIEL Sj ILIDLILIIS, 


an inverse relation exists between the concentration of fatty acid 
and the time of exposure required to produce a given effect. 
Systematic experiments to determine the character of this rela- 
tion have not yet been carried out, but there are some observa- 
tions bearing on this question. In several of my experiments in 
the summer of 1912 starfish eggs exposed for only one minute to 
acetic or butyric acid of ca. /176 concentration (6 c.c. 1/10 
acid plus 100 c.c. sea-water) formed a large proportion of larvee.! 
Lyon observed some years ago that the exposure required to 
induce parthenogenesis in Arbacia pustulata by means of weak 
solutions of HCl in sea-water decreased with increase in the con- 
centration of acid up to a certain point.2, The minimum exposure 
to n/260 butyric acid required to form membranes is very brief 
in starfish eggs. Experiments last summer showed that while 
10 seconds was insufficient to form membranes in more than a 
few eggs (ca. 10 per cent.), with 20 seconds all formed membranes, 
followed by the typical irregular changes of form and breakdown. 
After one minute’s exposure to 2/260 butyric acid an occasional 
egg may form a blastula; yet in the series showing the shortest 
optimum exposure of any performed last summer (No. 5, Sept. 2) 
at least 3 minutes was required to enable any considerable 
proportion of eggs to develop to a larval stage. The parallelism 
between the effects of high temperature and of weak fatty acid 
solutions indicates that the two agents act by producing the same 
kind of change in the egg-system. More detailed experiments 
to determine the influence of concentration as well as time on the 
action of this and other cytolytic substances remain to be carried 
out, and their results will probably throw further light on the 
nature of this change. 


EFFECTS OF MEMBRANE-FORMATION BY HEAT oR Fatty ACID 
COMBINED WITH AFTER-TREATMENT BY THE SAME AGENT. 


The fact that a longer treatment with the membrane-forming 
agent produces the same effect as a short treatment combined 
with after-exposure to hypertonic sea-water or cyanide suggests 
that a suitable after-treatment with the membrane-forming 


1Cf. Journal of Experimental Zoélogy, 1913, Vol. 15, pp. 41, 42. 
2Lyon, American Journal of Physiology, 1903, Vol. 9, p. 310. 


ACTIVATION OF UNFERTILIZED STARFISH EGGS. 285 


agent itself should have a corrective effect similar to that exerted 
by the agents just named. If the effect of the initial or mem- 
brane-forming treatment is to cause a partial activation which 
requires later to be completed by the after-treatment, we should 
expect it to be a matter of indifference (within certain limits of 
time) whether the activation is completed in one stage—e. g., by 
a continuous warming to 32° for 8 minutes—or in several; 
development ought to follow equally well if the eggs are returned 
to sea-water after an exposure just sufficient for membrane- 
formation, and afterward again exposed to the same treatment 
for an appropriate length of time. Experiment shows that it is 
in fact possible to substitute for the after-treatment with hyper- 
tonic sea-water or cyanide a brief exposure either to warm sea- 
water or to 2/260 butyric acid. We have here clear indication 
that the essential changes produced in the egg by after-treatment 
with an agent like hypertonic sea-water are not qualitatively 
different from those caused by the first or membrane-forming 
treatment, but serve simply to renew and bring to its completion 
a process which has been initiated by the first treatment but 
prematurely arrested by the early return to normal sea-water. 
According to this conception the whole activation-process is 
unitary in nature and does not consist of two qualitatively 
distinct and mutually complementary processes, as Loeb has 
maintained on the basis of his experiments with sea-urchin eggs. 

The following series (Table XIII.) illustrates the effects of 
treating eggs, in which membranes have been formed by 3 
minutes’ exposure to 32°, a second time with sea-water at 32° 
for 4. minutes; the second exposure was made at varying intervals 
after the first, ranging from 9 minutes to nearly 4 hours. 


TABLE XIII. 


AFTER-TREATMENT WITH SEA-WATER AT 32°. 


June 24. Eggs from several starfish were exposed, about 35 minutes after 
removal from the animals, to sea-water at 32° for 3 minutes (11.12-11.15 A.M.), 
and then returned to sea-water. Part of these eggs were left permanently in sea- 
water for control; the rest were again exposed to 32° for 4 minutes, successive portions 
being thus treated at 10-minute intervals until well after the separation of the second 
polar body. The condition of the maturing eggs at the time of the second treatment 
is indicated in the first column. 


286 RALPH S. LILLIE. 


Control lots of eggs were exposed (for purposes of comparison) to 32° for single 


continuous periods ranging from 2 minutes to 10 minutes. 


After-exposures to 32° at Follow- Results (Condition of Eggs ca. 4 Hours Later, and 


' ing Times after First Exposure. 


Proportion Forming Larve. 


I. Control: no second ex- 


iS) 


DOSUTE Aj sere All mature eggs have membranes but are uncleaved 
and largely irregular in form. None form larve. 


- 9 Mm. (II.24—11.28) (no 


polar bodies at 11.28)... Marked contrast to control; almost all eggs are 
cleaved, largely to 32- or 64-cell stages. Ca. 70-80 
per cent. form larvae many of which swim at the 
surface of the water. 


. IQ m. (11.34-11.38) 


(first polar bodies be- 

ginning to separate at 

Tet? 3'8)\ agence meade eee cetas Cleavage is less advanced than in Experiment 2, and a 
minority are uncleaved. Somewhat fewer larvee 
(ca. 65-75 per cent.). 


. 290 m. (II.44-11.48) 


(first polar bodies in all 

CEES) ane MIWA) oooncoese Cleavages are fewer and less advanced than in Ex- 
periment 3. Most eggs are uncleaved. Larve 
fewer (ca. 30—40 per cent.). 


2 20) me) Gr54—T1 58) 


(all with first polar 

bodies, none with 

second at 11.58)........ Contrast to Exp. 4; great majority are uncleaved and 
largely irregular; a few 2- and 4-cell stages present; 
few form larve (ca. 5 per cent.). 


. 49 m. (12.04—12.08) (ca. 


50 per cent. have 

second polar body at 

TOS Nise cen cee ar oeerees Similar to Exp. 5 but with fewer cleavages. Larve 
also are fewer (ca. 2-3 per cent.). 


> 5G) sin, (12.14-12.18) 


(all eggs have second 

polar bodies).......... Nearly all are uncleaved; largely irregular or frag- 
mented. Almost no larve (only one feeble blas- 
tula seen). 


. Th. gm. (12.24-12.28)...All are uncleaved but irregular forms are fewer. No 


larve. 


» Gs B lls (AAGO=BOB) ooo coc Similar to Exp. 8. 


Controls with one exposure to 32°: eggs exposed 7 minutes continuously (11I.12— 


II.19) gave ca. 90 percent. larve. With 4 minutes’ exposure few eggs (ca. 2-3 per 
cent.) formed larve. Controls of unfertilized and sperm-fertilized eggs were normal. 


ACTIVATION OF UNFERTILIZED STARFISH EGGS. 287 


Experiments similar to the above were performed with pre- 
liminary exposures to 32° of 2, 3 and 4 minutes, followed by 
after-exposure to 32° as above for 4 minutes (in one series for 
five minutes), all of which gave the same general result. Appar- 
ently it is a matter of indifference whether the second exposure 
to 32° follows immediately after the first or at an interval, 
provided that the second exposure takes place before the separa- 
tion of the first polar body. After this event there follows a 
decided and rapid decline in the favorability of the response to 
the after-warming treatment, and after the separation of the 
second polar body after-warming is apparently quite ineffective. 
As I described in my former paper on this subject, the suscepti- 
bility to parthenogenesis by temporary continuous warming 
always undergoes marked and rapid decrease at the time of the 
maturation-divisions... The above decline in the response to 
after-warming is evidently the same phenomenon. A similar 
decrease in the susceptibility of the eggs to sperm-fertilization 
also takes place at about the same time, although this decrease 
is not so pronounced as in the case of parthenogenesis; thus it is 
usually possible to fertilize a certain variable proportion of 
starfish eggs (not all) after maturation has been complete for 
some hours.” The fact that the general responsiveness of the 
egg to any activating agent undergoes a sudden decline at the 
time of separation of the polar bodies suggests either that some 
material necessary to development is then lost, or that a refrac- 
tory state conditional on some other kind of change (possibly a 
change in the plasma-membrane) then develops. As already 
pointed out, the fact that sperm-fertilization is possible (although 
less favorable) at a time when the egg fails to respond to the 
parthenogenetic treatment suggests that some definite material 
playing an important part in development is introduced into the 
egg by the sperm. This is also indicated by the general fact 
that sperm-fertilization induces a more favorable development 
than artificial activation. It may be that this material is the 
Same as some substance lost from the egg at the time of the 
maturation-divisions. Further research has to decide between 
these possibilities. 


1 Loc. cit., 1908, p. 400. 
2 IL GG, (icy 185 Alike 


288 RALPH S. LILLIE. 


In the above described experiments the total optimum period 
of exposure to 32° is about the same (ca. 7 to 8 minutes) whether 
the exposure is continuous or in two stages. No doubt it would 
be possible to increase the number of stages to three or more, 
especially if lower temperatures (31° or 30°) were used, but no 
experiments of this kind have so far been attempted. Appar- 
ently what is essential is that the critical process begun by the 
warming should continue, at the given temperature, for a 
certain definite length of time, sufficient presumably to allow 
some critical chemical interaction to proceed to its completion. 
It is interesting to note that a preliminary warming which is 
too brief in itself to cause membrane-formation may nevertheless 
have the effect of shortening the period of after-warming neces- 
sary to cause complete development. In one experiment the 
preliminary exposure to 32° was only 2 minutes, a time insufficient 
for membrane-formation in more than very few eggs (ca. I per 
cent.); these eggs, however, when again exposed to 32° for 4 
minutes, gave a considerable proportion of larve (5 to 10 per 
cent.); while eggs exposed to 32° for 4 minutes without any 
previous treatment formed membranes, but none developed to 
larve. A continuous single exposure of 6 minutes gave 25 to 35 
per cent. of larve; this exposure was well below the optimum 
of 8 to 10 minutes at which 80 to 90 per cent. formed larve. 
This effect of the four minutes’ after-exposure on eggs which 
otherwise showed no external change indicates that membrane- 
formation is not in itself a critical event, but simply an expression 
of a partial initiation of the general developmental process: 
i. €., a partial activation has been accomplished, enabling the 
egg to carry out a few of the early steps in development. 

Since brief exposure to weak fatty acid solution has the same 
physiological effect on the egg as brief warming, it would appear 
that the essential change produced in the egg-protoplasm by 
either form of treatment is the same; if so, after-treatment with 
warm sea-water should have a similarly favorable effect on eggs 
in which membranes were formed by fatty acid. The following 
series of experiments shows that this is the case (Table XIV.). 
The eggs, after membrane-formation by butyric acid, were after- 
treated with warm sea-water (32°) for periods ranging from 2 


ACTIVATION OF UNFERTILIZED STARFISH EGGS. 289 


to I2 minutes. For comparison part of the eggs were after- 
treated with hypertonic sea-water and cyanide. 


TABLE XIV. 


n/260 ButTyrRic ACID WITH AFTER-TREATMENT WITH SEA-WATER AT 32°. 


August 24. Eggs from one starfish were used. These eggs were not very 
favorable and a rather small proportion underwent maturation. They were 
exposed, about 45 minutes after removal, to 7/260 butyric acid solution for one 
minute and then returned to normal sea-water. Twelve to sixteen minutes later 
they were aiter-treated as follows, with the results indicated. 


ieee Mee Results iatavss ener ee oe Proportion 
rt. None (control treated 
with butyric acid 
AONE) ernest Typical fertilization-membranes in all mature eggs; 
later the eggs assume irregular forms and break 
down. None form larve. 
2. Hypertonic sea-water 
(250 c.c. Ss. w.+40 
c.c. 2.5 m NaCl) for 


SO MTEUIA ee eyn ee ester Markedly favorable effect: most mature eggs are 
cleaved to ca. 32-cell stage. 20-30 per cent. form 
larve. 


3. M/tooo KCN in sea- 
water for 30 min. ....Eggs cleave as in Experiment 2. Ca. 25-30 per cent. 
form larve. 
4. Sea-water at 32° for 2 
LTT TA es EU EWG Fah ne wae After four hours most eggs are irregular and un- 
cleaved; a few are cleaved. Very few form larvee 
(< I per cent.). 
5, QO° tigi @ imi, ocaono0s Like Exp. 4, but more eggs are cleaved. Few larve, 
—1I per cent. or less. 


OG, 32° ior Al WA. Gono ane Cleavages are more numerous than in Exps. 4 and 5. 
Ca. 5 per cent. of mature eggs form larve. 
Fo BEC TOr FR WMDs 5 a5 o00cc Ca. 20-30 per cent. are cleaved. Ca 5 per cent. form 
larve. 
So BO? tow © WM boo cop ad Cleavages are more numerous than in Exp. 7. Ca. 
40-50 per cent. larve. 
OQ. BAe tow G/ whe. bs oonoece Most eggs in 16- to 64-cell stages. 50-60 per cent. 
form larve. 
10. B@° iow 8 imine. po dono06 Like Exp. 9, but somewhat less favorable. Ca. 50 
per cent. form larve. 
II. 32° for 10 min.........After five hours few eggs are cleaved and cleavages are 


less advanced than in Exps. 9 and 10. Ca. 10-15 
per cent. form larve. 

TA, QO nohe wy inshngs 6! og oo oe Almost none have cleaved after five hours. Practic- 
ally none form larve (one blastula seen). 


290 RALPH S. LILLIE. 


For comparison eggs were exposed to 32° without previous membrane-formation 
for 4, 5, 6, 7, 8, and 10 minutes; the optimum exposure was 8 minutes at which 
50-60 per cent. of the mature eggs formed larve. A sperm-fertilized control also 
yielded numerous larve. 


After-exposure to 32° for the proper time thus greatly increases 
the proportion of favorably developing eggs. No marked im- 
provement is seen until the duration of after-exposure reaches 
four minutes; with longer exposures the proportion of eggs 
forming larve shows progressive increase up to an optimum at 
about seven minutes; a decline then follows; an exposure of 10 
minutes effects only slight improvement, and one of 12 minutes 
appears ineffective. Similar results, differing slightly in detail 
in different series, were obtained in eight other series of experi- 
ments. In general, after the preliminary membrane-formation 
by one minute’s exposure to 2/260 butyric acid, the time of 
exposure to 32° required for optimal development was found to 
range from 5 to 7 minutes; one minute’s exposure to 2/260 
butyric acid appears thus physiologically equivalent to warming 
at 32° for the same or a somewhat longer period. After-treat- 


ADATEILID OVE 


n/260 BUTYRIC ACID WITH AFTER-TREATMENT WITH SEA-WATER AT 34°. 


August 27. The eggs from one starfish were used. The eggs were few in 
number, but the majority showed normal behavior. They were exposed to 1/260 
butyric acid for one minute and then returned to sea-water. Later (within 20 
minutes) portions were exposed to hypertonic sea-water, cyanide, and warm sea- 
water as indicated. 


After-treatment. Results. 


Te NOnen(COntLOl) peer Typical membrane-formation, followed by breakdown 
of almost all eggs. One blastula found. 
2. Hypertonic sea water 


POTS O} TS Prose spoon ere oes 35-45 per cent. of the eggs form larve. 
3. n/t1000 KCN for 30m. . Ct, 50 per cent. of all eggs form larve. 
Ay Bao nope in Tb Spo Goa 6 Only a few eggs form larve: < I per cent. 
a Syl Noe A Me Sb 6 bin 68 oo Marked improvement: 20-30 per cent. form larve. 
(Gs Gal NOW B WMNbNes oo bond 6 Larvee are fewer than in Exp. 5: ca. 20 per cent. 
7. 34° for 4min...........Few eggs form larve: < I per cent. 
Go GVO Oe Ginins 55 oboe + ae Most eggs fail to divide; none form larve. 


Warming at 34° without previous membrane-formation: Eggs were exposed to 34° 
in the usual manner for 2, 3, 4, 5,6, and 7 minutes. The best development resulted 
from the 2- and 3-minute exposures, with respectively 25-35 per cent. and 35-40 
per cent. of eggs forming larve; with the 5-minute exposure only 5 per cent. formed 
larvee. 


ACTIVATION OF UNFERTILIZED STARFISH EGGS. 291 


ment with sea-water at 31° and at 34° was also tried; the results 
were the same except that the after-exposure required at 34° 
was only a half to a third as long as at 32°, and at 31° about 
twice as long. The following series at 34°(Table XV.) is 
typical. 

These results show that the effective duration of after-exposure 
at 34° is about one third of what it is at 22°22 aie Bins We ESE 
results were gained with after-exposures of 8 to 10 minutes. 
The temperature-coefficient of the physiological change resulting 
from the after-warming treatment is thus evidently of the same 
order as in the case of simple warming without previous mem- 
brane-formation. This of course is not surprising, since un- 
doubtedly the same process is concerned in activation by heat 
whether this is preceded by another treatment or not. 

It is thus plainly a matter of indifference, as regards the effect 


AUgsieis OVAL. 


BotH MEMBRANE-FORMATION AND AFTER-TREATMENT BY n|/260 Butyric ACID 


September 6. The eggs from one starfish were used; these were few in number, 
but almost all (ca. 90 per cent.) showed normal maturation, and in the sperm-fer- 
tilized control almost all formed larve. The eggs were exposed for one minute to 
n/260 butyric acid and returned to sea-water; part were left in sea-water as control; 
the remainder were again placed, 18 minutes later, in n/260 butyric acid, from which 
portions were returned to normal sea-water at the intervals indicated. These eggs 
developed as follows: 


Results (Condition of Eggs after 4 Hours and 


fter- t 5 A : 
ASSES Sie ee Proportion forming Larvz). 


TeNonen(control) pene eee All show typical membrane-formation followed by 
irregular change of form and breakdown in nearly 


all eggs. Only one larva seen. 
2. N/260 Butyric acid: 2 


TLIO eS alone ToeWe were A few eggs are cleaved. Ca. 10-15 per cent. form 

larve. 

3. Butyric acid: 4m. ......Cleavages more numerous and more regular than in 
Exp. 2. Ca. 40-50 per cent. of eggs form larve. 

4. Butyric acid: 6 m....... Cleavages still more numerous: Most eggs form larve 
(70-80 per cent.). 

5. Butyric acid: 8m....... Like Exp. 4, but fewer eggs form larve (50-60 per 
cent.). 

6. Butyric acid: 10 m...... Cleavages are fewer and slower. 25-35 per cent. of 
eggs form larve. 

4. Butyric acid: 12 m...... Cleavagesare stillfewer. Ca.10 percent. of eggs form 
larve. 


8. Butyric acid: 15 m...... Practically none are cleaved. No larve. 


292 RALPH S. LILLIE. 


produced by this form of after-treatment, whether the membrane- 
formation is induced by heat or by fatty acid; in either case 
warming for a few minutes completes the process of activation 
and enables the eggs to develop favorably. Precisely the same 
effect is gained by after-exposing eggs, in which membranes 
~have been formed by either method, to weak solutions of fatty 
acid for a brief period; the effects of such treatment are in all 
respects similar to those of after-warming. This is illustrated 
by the following experiment (Table XVI.). 

It is clear that in the time-relations of its action as well as in 
its other characteristics, this form of after-treatment resembles 
closely that with warm sea-water. It is also possible to treat 
the eggs first with warm sea-water and then after-treat with 


butyric acid solution; precisely the same results follow as in | 


the experiment just described. Thisis illustrated by the following 
series (Table XVII.). ? 
TaBLeE XVII. 


BRIEF EXPOSURE TO 32° WITH AFTER-TREATMENT BY 7/260 BuTYyRIc ACID. 


September 7. The eggs from one starfish were used; eggs were few but appar- 
ently normal, over 90 per cent. showing normal maturation, and sperm-fertilization 
resulting in a large proportion of larve. The eggs were exposed to sea-water at 32° 
for 3 minutes, then returned to sea-water at normal temperature, and 16 minutes 
later placed in 2/260 butyric acid solution, from which they were again returned 
to sea-water after the times indicated. 


After-treatment. Results. 


To Nome (@2a° jie BS. i, 


alone) ee ae cere No development; only a small proportion form mem- 
branes. 
2. N/260 butyric acid: 
DIN Se Servite eueeskeiene eacnmaren aus All form membranes but few are cleaved after three 
hours. Ca. 5 per cent. form larve. 
33, Biba BXCNGIS 4) Ts 5 sao oc A large proportion (50-60 per cent.) are cleaved after 
three hours. More than 50 per cent. form larve. 
Al, sie PAS ACIS © 7, 555000 Most eggs are cleaved after three hours. 70-80 per 
cent. form larve. 
Be Bltyricacid @oms sane In contrast to Exp. 4, few eggs are cleaved after three 
hours, and only 1-2 per cent. form larve. 
6. Butyric acid: 10m...... No eggs cleave within three hours. None form larve. 


The favorable effect of this after-treatment is evident. It 
will be noted that the three minutes’ exposure to 32° was in- 
sufficient for membrane-formation in most eggs; but the effect 


ACTIVATION OF UNFERTILIZED STARFISH EGGS. 293 


of this preliminary treatment is seen in the fact that an after- 
exposure of only 4 minutes was sufficient to induce development 
to larval stages in more than half of the eggs. After-exposure 
to butyric acid solution has the same favorable effect when the 
preliminary warming is sufficient to form membranes in all eggs; 
in a second similar series on September 12 the eggs were exposed 
for 4 minutes to 32° and all mature eggs thus treated formed 
membranes; without any after-treatment almost none (less than 
I per cent. formed larve, but with an after-treatment of 4 to 8 
minutes with 2/260 butyric acid favorable development took 
place in a large proportion of eggs. 


GENERAL DISCUSSION AND CONCLUSION. 


The interchangeability of the treatments with warm sea-water 
and butyric acid solution indicates that both agents produce 
their effect by inducing the same kind of change in the egg- 
system. This change is evidently of a ‘‘releasing’’ kind, and 
initiates the sequence of developmental processes; these, once 
started, continue automatically to their conclusion. Probably 
their most distinctive peculiarity is the highly specific character 
of the chemical transformations that take place. From the food 
contained as reserves in the egg, or taken in from the surround- 
ings, the developing germ builds up the specific compounds 
which form the structural basis of the organism; this synthetic 
process, in the case of the chief structure-making compounds, the 
proteins, undoubtedly starts—as in the constructive metabolism 
of the adult animal—with the amino-acids, which are recombined 
in the specific manner predetermined by the chemical organiza- 
tion of the germ. Bodies of the most highly specific and indi- 
vidualized physical and chemical properties are thus built up 
and laid down in definite positions as development proceeds. 
Their properties and their spacial disposition determine at any 
time the character of the transformation undergone by the 
building material which is being incorporated. According to 
this conception it is the chemical specificity of these substances 
that determines the specific character of development in the 
more evident or morphological sense,! 7. e., why the egg gives 


1 Reichert’s work on the crystal-forms of haemoglobin and other complex 
compounds from different species of animals and plants constitutes perhaps the 


2904 RALPH S. LILLIE. 


rise to an individual of the same species; and we must therefore 
be prepared to find among the earliest chemical changes asso- 
ciated with development, interactions of a specific kind— . e., 
specific in the sense in which the interaction of antigen and anti- 
body is specific—between complex substances already present in 
the egg. There is now definite experimental evidence that such 
reactions do in fact constitute an essential part of the fertilization- 
process. Specific substances which apparently unite in fertiliza- 
tion (since after fertilization they are no longer demonstrable) 
are present in the unfertilized mature egg; one of these (‘‘fer- 
tilizin’’) may be largely removed from the egg by washing, and 
when this is done fertilization is prevented. If such specific 
unions are essential to fertilization, we must conclude that the 
specific substances concerned in this process are in some way 
kept from interaction in the resting mature egg, and that the 
activating agent removes this hindrance to interaction. The 
question which I wish briefly to discuss in this section relates 
to the nature of this inhibiting condition, and the manner in 
which the activating agent effects its removal. 

The nature of the effects following exposure of unfertilized 
eggs to temperatures of 30°-35° indicates clearly that activation 
does not depend on simple acceleration of some chemical process, 
é. g., oxidation, which is already proceeding in the egg, since in 
this case the temperature-coefficient of the activation-process 
would presumably show the usual value of Qi = 2-3. It is 
also evident that heat-coagulation is not concerned, since these 
temperatures are too low, and the readiness with which the 
activation process can be arrested by cooling and renewed by a 
second warming shows that its basis is some effect which is com- 
pletely reversible by change of temperature. These character- 
istics, high temperature-coefficient and reversibility with change 
of temperature, are however shared by the typical melting and 
gelation (sol-gel transformation) exhibited by solutions of gela- 
best evidence of this. The morphological characters of crystals and crystal-aggre- 
gates varies with their chemically specific (‘‘species-specific’’) character in a 
definite and constant manner. It is fair to assume that the influence of these 
compounds in determining organic structure depends largely on the kind of aggre- 


gates they form. Cf. Reichert: Science, 1914, N. S., Vol. 40, page 649. 
1Cf. F. R. Lillie, Journal of Experimental Zoology, 1914, Vol. 16, p. 523. 


ACTIVATION OF UNFERTILIZED STARFISH EGGS. 295 


tine, agar, soaps, lipoids and other hydrophilous colloids. The 
relations of temperature to this process show in fact a close 
resemblance to those described above for the activation-process. 
One striking peculiarity of melting and gelation is that both 
processes take place gradually; when (e. g.) a gelatine sol is 
brought below the gelation-temperature and the conditions are 
then kept constant, the actual solidification takes place only 
after the lapse of a considerable period of time. The time re- 
quired to reach the gelation-stage decreases rapidly as tempera- 
ture is lowered; thus Levites found that a gelatine sol kept un- 
disturbed at 26° took 26 hours to gelatinize, at 25° only 11 hours.! 
The first observable change in the solution is an increase in 
viscosity; this continues until the system sets; the setting repre- 
sents the end-stage of the whole process, whose course can thus 
be traced by successive viscosity-determinations. Gelation is 
thus equivalent to a progressive increase in viscosity to a final 
stage at which the ordinary fluid mobility is lost.2. It is found 
that above a certain temperature the viscosity of the hydrosol 
undergoes no change with time; but if the temperature is 
lowered a critical point is eventually reached below which the 
viscosity undergoes steady increase (at a rate dependent on 
temperature, presence of salts, reaction) until gelation occurs. 
The rate of this increase in viscosity (i. e., of the gelation-process), 
An/At, shows a high temperature-coefficient. With a 1 per cent. 
gelatine solution Schroeder? obtained the following values for the 
viscosity at 21°, 24.8°, and 31° at different intervals after bringing 
the warm gelatine solution to the temperature of observation: 


Viscosity Observed at 
Interval. 

210% 24.89. Bro. 

ETI ATA pene ey ate mies aus iss, Cae 1.83): T.05 I.41 
HG) Wahea sy cle osteo eH GI eRe 2.10 I.69 I.41 
IG AUGDUT Ae cnc ceeee eee Rc ee 2.45 1.74 I.42 
OMIM Rea yokes haat Arle 4.13 1.8 1.42 
OOM recy Sie seve uees alteate 13.76 1.9 I.42 


Thus while at 31° the viscosity undergoes no change with time, 


1Levites, Kolloid-Zeitschrift, 1907, Vol. 2, p. 211. 

2 Cf. Schroeder, Zeitschrift fiir physikalische Chemie, 1903, Vol. 45, p. 75; Levites: 
loc. cit., p. 209; Freundlich, “‘Kapillarchemie,’’ 1909, pp. 416 ff. 

3 Schroeder, loc. cit., p. 88. 


296 UNLESS, LULL, 


at the lower temperatures there is a steady increase. If we 
take comparatively short time intervals, e. g., Io minutes, we 


Bilis = Th foe 
10 


ey == 1405; 
10 


find that the value of Ay/At at 21° ( = 0.062 ) is 


about seven times greater than at 24.8° ( = 0.009 ) ; 


In other words, a difference of about 4° increases the average 
rate of the gelation-process from six to seven times. What is 
true of the gelation-process is also true of the inverse degelation 
or melting process, whose rate increases at a similarly rapid rate 
with rise of temperature above the critical maximum at which 
the system remains permanently in the gel state.! 

In starfish eggs the rate of the activation-process, at tempera- 
tures between 30° and 36°, shows a similar proportionate increase 
with a given rise of temperature, as will be seen by reference 
to Table X.; 7. e., the temperature-coefficients of the two proc- 
esses, gel-sol transformation, and activation of the egg under 
the influence of high temperatures, are similar in their order 
of dimensions; thus a rise of 4° shortens the time of exposure 
necessary to cause membrane-formation or development by six 
to ten times. On the assumption that some specific chemical 
interaction is the essential change in the initiation of develop- 
ment, such a result indicates that the rate of this interaction is 
dependent, in the case of parthenogenesis by warming, on the 
rate of some process involving either degelation or decrease in 
the viscosity of some portion of the colloidal system of the egg. 
This is as much as can be inferred on the basis of these facts alone. 
If we also take into account the other methods by which mem- 
brane-formation and activation can be induced, we are led to the 
further inference that this colloidal change affects chiefly if not 
exclusively the surface-layer (cortical zone or plasma-membrane) 
of the egg. Thus typical membrane-formation can be induced 
by brief treatment with pure isotonic solutions of neutral salts.? 


1On account of the hysteresis of the gelatine system, the melting temperature 
is typically several degrees higher than the solidification-temperature; it is also 
higher after the gel has stood some time than immediately after solidification. Cf. 
Pauli (Pascheles): Archiv f. d. ges. Physiologie, 1898, Vol. 71, p. 336. 

2R.S. Lillie: American Journal of Physiology, 1910, Vol. 26, p. 106. The fact 


CAGE ete tie 


ee aa 


ACTIVATION OF UNFERTILIZED STARFISH EGGS. 297 


whose action is certainly superficial, as well as by substances like 
fatty acids, weak bases, and lipoid-solvents, which readily 
penetrate the plasma-membrane. ‘Those neutral salts of sodium 
and potassium which are the most effective in inducing mem- 
brane-formation, iodides and thiocyanates, are also the most 
effective in lowering the melting points of protein gels and in 
promoting water-absorption by such gels.'. Such facts suggest 
that the salts act in a way similar to that of high temperatures, 
i. e., by furthering degelation of surface-structures or absorption 
of water in the surface-layer of the egg. The effect of such an 
increase in water-content would be to increase the general 
permeability of this region, since according to the experiments 
of Bechhold, Ruhland, and others? the permeability of gels to 
diffusing substances, especially to colloids, is a direct function of 
their water-content. 

High temperature, according to this interpretation, acts like 
other parthenogenetic agents, by increasing the permeability of 
the surface-layer,—this effect resulting directly from some change 
in the nature of a degelation or decrease in the viscosity of the 
colloidal system in this region. Apparently. the immediate 
effect .of this change is to allow a chemical interaction to“take 
place between substances which in the normal resting state of the 
surface-layer are kept apart. The general fact that identical 
physiological effects may be produced by lipoid-solvents, and 
by substances which appear to alter the membrane by interacting 
chemically with its constituents,’ indicates that the integrity 
of the plasma-membrane as a semi-permeable partition is the 
essential factor in preserving the resting condition of the egg.* 


that this action can be prevented by anesthetics confirms the view that it depends 
on an increase in the permeability of the plasma-membrane: cf. my recent paper in 
the Journal of Experimental Zoology, 1914, Vol. 16, p. 591. 

1 Cf. Pauli (Pascheles): Archiv f. d. ges. Physiologie, 1898, Vol. 71, p. 333; Levites: 
loc. cit.; Pauli and Rona, Beitriéige zur chemischen Physiologie u. Pathologie, 1902, 
Wolk, By Ds Ala 

2 Bechhold u. Ziegler, Zeitschr. f. physik. Chem., 1906, Vol. 56, p. 105; also, “die 
Kolloide in Biologie u. Medizin,’ 1912, p. 48. Ruhland: Biochemische Zeitschrift, 
1913, Vol. 54, p. 59; Freundlich, Kapillarchemie, pp. 515 seq. 

3 When membrane-forming substances act by combining chemically with egg- 
constituents, it is to be expected that the rate of action will vary with temperature 
in accordance with the chemical temperature-coefficient. Cf. the experiments of 
Loeb and Hagedoorn, ‘‘ Artificial Parthenogenesis and Fertilization,’ page 146. 

4Cf. my paper, Amer. Journ. Physiol., 1911, Vol. 27, p. 289. 


298 LUNIEI2IEL Sip IED, 


Hence it is a matter of secondary importance in what manner 
this semi-permeability is temporarily destroyed, provided that 
the condition of increased permeability lasts long enough—not 
too long—and is not associated with irreversible changes making 
recovery impossible. It is presumably during this stage of 
increased permeability that the above specific interaction takes 
place; this process requires time, and its rate will be a function 
of the rate at which the two interacting substances can come 
together; this second rate will be a function of the viscosity 
or gelation-state of the protoplasmic system at the site of inter- 
action,—hence its dependence on temperature, as seen above. 
When this critical interaction has taken place, there follows at 
once the characteristic change of physiological activity normally 
resulting from fertilization; membrane-formation and the other 
events preparatory to cell-division occur and the developmental 
process proper is initiated. How far development proceeds, 
however, depends on the degree of completion of the primary 
specific reaction; hence for complete activation the exposure to 
the membrane-forming condition must have a certain minimal 
duration, and in case the preliminary exposure is insufficient 
some after-treatment may be necessary to complete the process. 
This after-treatment may be of the same kind as the preliminary 
membrane-forming treatment, or it may be of entirely different 
kind—e. g., hypertonic sea-water, cyanide, an anaesthetic, etc. 
But there seems to be no need of assuming that its direct physio- 
logical effect is qualitatively different from that of the membrane- 
forming agent.1 It merely renews and brings to completion a 
process already initiated by the first treatment. 

Comparative study of the conditions of both normal and 

1 The above experiments are a sufficient justification of this contention. But 
they do not explain why, for instance, after-treatment with cyanide, which by itself 
does not induce membrane-formation in starfish eggs (cf. Journal of Experimental 
Zoology, 1913, Vol. 15, p. 38), is so effective. Clearly the condition of the egg after 
membrane-formation is altered so that the activation-process may then be influ- 
enced by agents which previously had no effect upon it (as cyanide, alcohols, or 
hypertonic sea-water in brief exposure). Sensitization to these agents seems to be 
involved in the process of membrane-formation, but the basis of this effect can not 
be defined at present. There is, however, no necessary inconsistency between these 
facts and the conception that the activation-process is essentially unitary in char- 


acter in the above sense. The case of hypertonic sea-water offers certain special 
problems, which are partly discussed below. 


ACTIVATION OF UNFERTILIZED STARFISH EGGS. 299 


artificial activation ought to yield data from which by elimina- 
tion the essential factors common to the two processes may be 
determined. Judging from the data available at present, the 
most general common feature appears to be the initial increase 
in permeability.t It is not yet clear, however, how this change 
can be the means of initiating the specific interaction assumed. 
The substances which interact are assumed to be present in 
advance in the egg; how is their interaction prevented by the 
existence of a semipermeable surface layer? The connection 
between change of permeability and activation is probably 
indirect; and the analogy to stimulus and response in the 
general stimulation-process of irritable tissues still seems the 
best adapted to throw light on this question.? In stimulation 
an electrical depolarization of the plasma-membranes of the 
irritable elements is apparently the critical event; in some way 
this change enables the characteristic response of the irritable 
system to take place. Similarly in the initiation of development 
in the unfertilized egg. The agents which induce membrane- 
formation in eggs have typically a depolarizing action on irritable 
cells like muscle-cells—4. e., cause a negative electrical variation.’ 
Such a change appears to result whenever surface-permeability 
is increased; and it seems therefore probable that this depolariza- 
tion, as such, is what enables the union of specific substances— 
the first step in activation—to take place. We may assume 
that one of the interacting substances is situated immediately 
beneath the electrically polarized surface-film of the egg, that it 
is a negative colloid, and that its tendency to unite with some 
amboceptor-like body also present in this region is compensated 
by the electrostatic attraction between it and the layer of 


1 Cf. my paper just referred to. Inarecent paper Gray confirms McClendon in 
finding a temporary increase in the electrical conductivity of sea-urchin eggs 
immediately after sperm-fertilization. Cf. Gray, Journ. Mar. Bicol. Ass., 1913, 
Vol. r0, p. 50; McClendon, American Journ. Physiol., 1910, Vol. 27, p. 240. 

2 T have discussed this analogy in more detailin the paper above cited (footnote 
2, p. 296); also in the Journal of Experimental Zoology, 1913, Vol. 15, p. 23. 

3 For the action of cytolytic substances in producing local negative variation, 
cf. Straub, Archiv f. exp. Path. u. Pharm., 1902, Vol. 48, p. 1; Zeitschr. f. Biol., 
1912, Vol. 58, p.'251; Henze: Arch. f. d. ges. Physiol., 1902, Vol. 92, p. 451; Hermanns: 
Zeiischr. f. Biologie, 1912, Vol. 58, p. 261; Allcock; Proc. Roy. Soc., B, 1906, Vol. 77, 
p-. 267; Journal of Physiology, 1906, Vol. 33, p. xxviii; Evans, Zeitschr. f. Biol., 1913, 
Vol. 59, Dp. 397. 


300 RALPH S. LILLIE. 


positive ions immediately external to the egg-surface. De- 
polarization would then permit interaction to take place. Such 
a conception, while in a sense diagrammatic, helps at least to 
explain how a non-specific agency, provided it only alters 
sufficiently the boundary-layer of the egg, can be the means 
of initiating such a highly specific process as development. 

The discussion of this question can hardly be considered 
complete without some reference to the case of hypertonic sea- 
water. As Loeb has shown, exposure to this agent forms a 
supplementary treatment which is remarkably favorable with 
some eggs, especially sea-urchin eggs. This treatment seems to 
occupy a special position among the parthenogenetic agents. 
It may either precede or follow the membrane-forming treat- 
ment,” and in some way it puts the egg into a condition which is 
favorable to subsequent development; this action seems quite 
independent of the nature of the membrane-forming or activating 
agent, and so far it has received no satisfactory explanation. 
Loeb has shown that a purely physical abstraction of water is 
not the only factor concerned; a chemical factor, apparently 
involving oxidation, is essential; free oxygen must be present 
during the treatment, and the effective times of exposure vary 
at different temperatures according to the chemical temperature- 
coefficient.2 Some hypothesis as to its mode of action seems 
required; and I suggest the following, which is consistent with 
the foregoing point of view, and has not, to my knowledge, yet 
been put forward. 

1 The inorganic analogy would be, e. g., the interaction between solution and 
metal at the surface of the plate in a battery when the circuit is closed. While the 
battery is at rest (with open circuit), interaction between (e. g.) sulphate ions and 
zinc is prevented by the polarization at the surface of the zinc plate. The tendency 
to this ionic interaction is compensated by the polarization, the zinc ions being 
held back by the negatively charged plate. Similarly, mutatis mutandis, with the 
reactions at the cell-surface, or other surfaces (adsorption-surfaces) within the cell. 
The facts of stimulation afford in general strong evidence that the chemical proc- 
esses in the living cell are largely dependent on changes in the electrical polar- 
ization of the limiting membranes. Cf. my paper in the Journal of Biological 
Chemistry, 1913, Vol. 15, p. 237. Also, for a more general discussion of this ques- 
tion, the article entitled ‘“‘The Physico-chemical Conditions of Stimulation,” in 
the Popular Science Monthly, 1914, p. 579. 

2 Cf. Loeb, “‘ Artificial Parthenogenesis and Fertilization,’ Chapter 11; Archiv 


fiir Entwicklungsmechanik, 1914, Vol. 38, p. 409. 
3 “* Artificial Parthenogenesis and Fertilization,’’ Chapter 11. 


ACTIVATION OF UNFERTILIZED STARFISH EGGS. 301 


It is to be assumed that the activation-process—as the earliest 
step in development, an essentially constructive process—in- © 
volves syntheses of some kind. Now the intracellular as well as 
other organic syntheses consist as a rule, in the union of two or 
more molecules, with loss of water, to form larger molecules,—as 
in the formation of fats from glycerol and acids, of starch and 
glycogen from sugar, of polypeptides and proteins from amino- 
acids, etc. In order to account for the readiness with which 
these condensations occur in cells, it seems necessary to assume 
that the protoplasm is the seat of energetic dehydrations, prob- 
ably in certain localized situations (possibly at membranes or 
other adsorption-surfaces). The artificial enzymatic synthesis 
of triolein from glycerol and oleic acid has been found to take 
place readily only when water is removed as completely as 
possible from the reacting mixture.’ Hence the synthesis of 
fats by enzyme action in cells is intelligible only on the assump- 
tion that in the region of their formation there is energetic 
abstraction of water or dehydrolysis. Certain biological facts 
indicate that partial removal of water from cells is favorable to 
syntheses of the above kind. According to Overton, plasmolysis 
of plant-cells furthers the formation of starch in chloroplasts.” 
Butkewitsch also finds that the formation of starch in the 
amylase-rich cortex of certain plants (Sophora, Robinia) is 
promoted by placing in strong sugar-solutions (10-20 per cent. 
dextrose and saccharose).2 The observations of Pavy and 
Bywaters and of Rubner on the formation of glycogen by yeast 
cells in strong sugar solutions constitute probably a further 
instance of the same phenomenon.? In general loss of water will 

1Cf. the papers of Pottevin: Comptes rendus de l Académie, 1903, Vol. 136, 
p. 1152, and 1904, Vol. 138, p. 378; Taylor, Journal of Biological Chemistry, 1906, 
Vol. 2, p. 87; Hamsik, Zeitschr. f. physiol. Chemie, 1909, Vol. 59, p. 1; Armstrong and 
Gosney, Proceedings Roy. Soc., Ser. B, 1914, Vol. 88, p. 176. 

2 Overton, Vierteljahrsschrift d. naturf. Ges. in Ziirich, 1899, Vol. 44, pp. 131-2. 

3 Butkewitsch, Biochem. Zeitschr., 1908, Vol. 10, p. 314; of. pp. 336 seq. 

4Pavy and Bywaters, Journal of Physiology, 1907, Vol. 36, p. 149; Rubner, 
Archiv fiir Physiologie, Suppl. 1912, p. 252, and zbid., Vol. for 1913, p. 244. 

Pavy and Bywaters found that in pure dextrose solutions the deposition of 
glycogen in yeast cells increased rapidly with increase in the concentration of dex- 
trose up to an optimum. In 2 per cent. solutions there was little effect; in 4 per 


cent., 8 per cent., and 16 per cent. solutions there was a rapid progressive increase 
in the quantity of glycogen laid down in the cells toa maximum of over 13 per cent. 


302 RALPH 5S. LILLIE. 


be favorable to—since it will supplement—the action of any 
dehydrating mechanism; and it is possible that in the sea- 
urchin egg after membrane-formation the intracellular dehydra- 
tion-processes are by themselves not quite energetic enough to 
effect the syntheses necessary for initiating development, but 
become so when supplemented by the action of the hypertonic 
sea-water; 7. e., this agent has the effect of reducing the con- 
centration of water at the locus of the reactions sufficiently to 
enable syntheses to take place which otherwise are impossible 
under the conditions. It is significant that cell-division is 
started in the sea-urchin egg by simple membrane-formation, 
but fails to continue,—just as if there were some failure in the 
supply of the necessary constructive materials; partial abstrac- 
tion of water rectifies this condition. Since oxygen is necessary 
to this corrective process, we may assume that the syntheses 
belong in part to the class designated by Schmiedeberg! as 
oxidative syntheses. 

From this general point of view the action of hypertonic 
sea-water becomes in a measure theoretically intelligible and 
ceases to be merely a detached empirical fact. Certain avenues 
of experimental approach to the problem are also suggested. 


SUMMARY. 


§1. The effects following exposure of maturing unfertilized 
starfish eggs to high temperatures (29-36°) vary in a constant 
manner with the times of exposure as follows. Below a certain 
minimal duration of exposure to any given temperature (e. g., 
32°), no visible change is produced in the egg; slightly longer 
exposures induce the formation of typical fertilization-mem- 


(as compared with about 5 per cent. under normal conditions) ; in more concentrated 
solutions there wasa decline. They also found that too long exposure to a favorable 
solution (10 per cent.) was unfavorable; thus yeast incubated in ro per cent. dextrose 
for 25 hours showed an increase in glycogen-content from 4.84 per cent. to 11.66 
per cent.; four hours later there was a decline to 9.33 per cent. These facts show a 
suggestive parallel with the effects of hypertonic sea-water on sea-urchin eggs; 
here also there is no effect until a certain minimal osmotic pressure is reached; 
with further increase in osmotic pressure there is a rapid increase in favorability 
up to an optimum; still further increase is unfavorable. Also for a favorable con- 
centration there is at any temperature a definite optimum time of exposure. 

1 Cf. Schmiedeberg, Archiv f. exper. Pathologie u. Pharmakologie, 1893, Vol. 31, 
p. 281. 


Se 


ACTIVATION OF UNFERTILIZED STARFISH EGGS. 303 


branes, but the eggs fail to cleave and soon break down without 
development; in order to induce favorable development an 
exposure of three to four times the minimum for membrane- 
formation is required (e. g., 7-8 minutes at 32°); more prolonged 
exposures are again followed by failure to develop. 

2. Between 29° and 38° the times of exposure required to 
produce these effects decrease very rapidly with rise of tempera- 
ture; on the average a rise of 1° approximately halves the 
exposure required for a given physiological effect (such as 
membrane-formation, or complete activation, or heat-inactiva- 
tion). The activation-process thus exhibits a characteristically 
high temperature-coefficient (Qi9 = 200-400). 

3. The effects of exposure to weak butyric acid solution (7/260) 
vary with time of exposure in a similar manner,—brief exposure 
causing membrane-formation followed by breakdown, longer 
exposures causing cleavage and development to larval stages, 
and still longer exposures causing cytolysis without development. 

4. The inference is that the same process is initiated in the 
eggs by exposure to warm sea-water as by fatty acid solution. 
This process must proceed to a certain stage in order that 
activation may be complete; if arrested too soon (brief exposure) 
only partial activation (membrane-formation followed by break- 
down) results. 

5. Eggs in which membranes are formed by minimal exposure 
to warm sea-water or 2/260 butyric acid, followed by return to 
sea-water, may be made to develop favorably by a second 
treatment with either warm sea-water or fatty acid solution, as 
well as by after-treatment with cyanide-containing or hypertonic 
sea-water. A favorable after-treatment may thus be of the 
same kind as the membrane-forming treatment. 

6. The temperature-coefficient of activation by high tempera- 
tures is of the same order as that of the melting of gels or the 
decrease in the viscosity of gelatine solutions. The above high 
temperatures thus probably act by producing degelation-effects 
in the surface layer of the egg; increase of permeability, with 
consequent depolarization, is the result of this change. 

7. A new hypothesis of the mode of action of hypertonic 
sea-water is put forward. 


DIVISION RATE. IN SCILIADE~ PROMOZOAR AS 
INFLUENCED BY THYROID CONSTITUENTS? 


ROBERT A. BUDINGTON AND HELEN F. HARVEY. 


INTRODUCTION. 


In the very numerous studies which have been made to ascer- 
tain the effect of thyroid tissues and extracts on growth and 
differentiation, the material employed, whether used as a food 
for large organisms or as a component of a medium in which to 
breed smaller forms, has been taken in a very large proportion 
of instances, if not always, from some mammal, e. g., cow, horse, 
or sheep. This has been the case even though the animal under 
observation may have been a mammal, a bird, an amphibian, 
or a protozoan. 

Assuming that the doctrine of evolution is a fairly probable 
hypothesis it is only a natural if not necessary corollary that 
each of the several organs involved, as well as the organism as a 
whole, has experienced its own successive changes, its own evo- 
lutionary modifications. Variations, ‘‘continuous’” and ‘‘dis- 
continuous,’ have occurred in internal as well as in external 
organs, and these variations have involved the physiological 
value of the organs concerned, as well as their anatomy; so that, 
of glandular tissues, for example, the composition of the output 
has undergone phylogenetic changes, so to speak, during the 
process of descent of one phylum from another. It is a prior. 
improbable, of course, that the chemical composition, and conse- 
quent stimulating potency, of the thyroid secretion is the same 
throughout the entire vertebrate phylum. 

Apparently the earliest experimentation along the line with 
which this paper deals was carried out by Nowikoff ('08), who 
found that one effect of putting sheep thyroid into the medium 
in which Paramecium was living was to cause it to divide more 
rapidly than normally. 

Recently, Shumway (14) has published a paper in which he 
verifies Nowikoff’s contentions. Both these investigators, how- 
ever, employed mammalian thyroid; and, since our results agree 

1Hrom the Department of Zoology, Oberlin Colleg>. 

304 


DIVISION RATE IN CILIATE PROTOZOA. 305 


with theirs, the question for mammalian thyroid, at least, seems 
fairly well settled. 

Nowikoff’s work suggested to us the query whether or not his 
results could be taken as widely significant.!_ Our purpose has 
been, therefore, to add to the known facts along this line by 
ascertaining the influence of glands taken from each of the five 
main subdivisions of vertebrated animals, so far as they or sub- 
stances derived from them, affect certain protozoa; and, using 
division-rate as an index, to thus get a line on the comparative 
physiology of this gland. 


MATERIALS AND METHODs. 


Perfectly fresh thyroid glands were taken from the fresh-water 
sucker (Catostomus teres), the frog (Rana pipiens), the turtle 
(Cistudo carolina), the chick (Gallus domesticus), and the cat 
(Felis domestica), dissected as cleanly as possible from surround- 
ing tissues, and then dried by moderate heat; each was then 
ground to powder in a mortar, and the material then kept in 
vials till used. In the instance of the mammalian gland, fatty 
tissue was present in such amount that this was dissolved away 
by repeated washing in ether to bring the gland to such condition 
that it could be finely pulverized. In supplying thyroid material 
to protozoa in this form, we endeavored to avoid any alteration 
in its character such as might result in the making of glycerin or 
alcoholic extracts. This seems a point which should be rather 
carefully guarded. 

The forms employed for experimentation were Stylonichia and 
Paramecium. ‘Yo familiarize ourselves with a method of hand- 
ling such organisms, and also for the purpose of securing indi- 
viduals whose ancestry would be known to us, we first carried 
isolated “‘wild”’ forms through a considerable number of genera- 
tions (in the case of Stylonichia, seventy-four); we employed 
depression slides, kept in a moist chamber, each slide carrying 
four drops of bacterial hay infusion made up in the manner of 
that used by Woodruff (’05) in much of his work. The “wild”’ 
specimens were taken from ordinary laboratory cultures, but the 
particular individuals used in any given experiment were taken 
from the pedigreed lines descended from a single parent. The 


1 Qur work was completed before Shumway’s article appeared. 


306 ROBERT A. BUDINGTON AND HELEN F. HARVEY. 


protoplasm of the line treated with thyroid was identical with 
that of the control carried beside it. 

The procedure in any experiment was this: two protozoa of 
common parentage were isolated, each in four drops of the same 
culture medium. To one of the slides was added a minute mass 
of pulverized gland, which would thus influence the protozoan 
either as a food, or as a factor in the environing medium so far as 
this acted as a solvent.! The actual amount of each pulverized 
gland thus used was small, and a like amount of each was deter- 
mined as closely as possible by careful subdivision of a slightly 
larger mass on a clean paper surface. To weigh out the powder 
would give no more equal amounts, inasmuch as the glands are 
so invaded by vascular and connective tissue that any moiety 
taken might easily contain more or less of other than glandular 
material. A slight amount of fresh hay infusion was added to 
each slide each day, and the experiment continued six days or 
more. The results given in this account are limited to those 
obtained during the first six days only; to keep track of the off- 
spring of even a single protozoan longer than this is extremely 
difficult, as many know. The effect of each different gland was 
tested by three trials. 

Circumstances were such that it was not always convenient 
or possible to run experiments with all five different thyroids at 
one time, so a control was carried along beside the gland-fed 
individual in each case. This assured that the same conditions 
of every sort attended both experimental and control lines, no 
matter when the observations were made. If any circumstance 
favored or interfered with either, the same was true for the other. 


EXPERIMENTAL FINDINGS. 


The following tables show the exact results, so far as number 
of individuals resulting from division of the original one goes, 
this rate of cell division being the only index of thyroid effect at 
present ready for presentation. The ciliate used in the first 
series of experiments was Stylonichia; in the second and third 
series we used Paramecium. While the evidence is too limited 
to permit any rigid conclusion of the kind, the data at hand seem 


1 Shumway states in his recent paper, loc. cit., that more or less of the material 
thus offered Paramecium is actually ingested and digested. 


DIVISION RATE IN CILIATE PROTOZOA. 


307 


to indicate that Paramecium is rather more susceptible to thy- 
roid ingredients than is Stylonichia. 


RESULTS FROM USE OF FISH THYROID. 


First Experiment, Second Experiment, Third Experiment, 
No. of Individuals. No. of Individuals. No. of Individuals. 
Control. |Thyroid-fei.) Control. |Thyroid-fed.| Control. |Thyroid-fed. 
Ist day T I I I I I 
2d day 4 2 I 2 I I 
3d day 6 6 3 2 6 6 
Ath day 15 15 5 7 TA 16 
5th day Dy Ba 7 50 18 BE 
6th day 43 49 15 90 52 73 
RESULTS FROM USE OF AMPHIBIAN THYROID. 
First Experiment, Second Experiment, Third Experiment, 
No. of Individuals, No. of Individuals. No, of Individuals. 
Control. |Thyroid-fed.| Control. |Thyroid-fed.| Control. |Thyroid-fed. 
Ist day I I I I I I 
2d day I it I 2 I 2 
3d day 6 5 2 4 8 18 
Ath day 7 Tit 8 9 16 76 
5th day 7 12 a0) 30 25 127 
6th day 12 30 12 (op 36 243 
RESULTS FROM USE OF REPTILIAN THYROID. 
First Experiment, Second Experiment, Third Experiment, 
No. of Individuals. No. of Individuals. No. of Individuals. 
Control. |Thyroid-fed.} Control. |Thyroid-fed.} Control. |Thyroid-fed 
Ist day I I it I I I 
2d day 4 4 4 5 I 2 
3d day 8 Io 8 23 3 a 
Ath day 8 Io 8 47 I5 42 
Sth day 8 21 14 148 23 80 
6th day 8 38 15 362 50 276 
RESULTS FROM USE OF AVIAN THYROID. 
First Experiment, | Second Experiment, Third Experiment, 
No. of Individuals. No. of Individuals. No. of Individuals. 
Control. | Thyroid-fed. Control. |Thyroid-fed.} Control. |Thyroid-fed. 
Ist day I I I I I I 
2d day I 4 if 2 2 I 
3d day 2 9 3 7 8 16 
4th day 2 a0) 4 45 23 32 
5th day 4 15 15 63 59 141 
Cult Gene A 24 35 243 QI 399 


FIsH. 


450 


308 ROBERT A. BUDINGTON AND HELEN F. HARVEY. 


RESULTS FROM USE OF MAMMALIAN THYROID. 


First Experiment, | Second Experiment, Third Experiment, 
No. of Individuals. No. of Individuals. No. of Individuals. 
Control. |Thyroid-fed.| Control. |Thyroid-fed.{ Control. |Thyroid-fed. 
Ist day I I I I I I 
2d day 13 I5 4 8 2 2 
3d day 16 18 6 18 9 18 
Ath day 18 20 IO 48 34 590 
5th day 30 132 I4 60 56 129 
Gt hidays ees 306 29 use) i G0 487 


For convenience in seeing at a glance the relation which held 
between the experimental lines and the controls during the use 
of any particular kind of thyroid, the data for the three experi- 
ments with each thyroid have been averaged, the controls for 
the same averaged, and the results placed in curve form. These 
follow: 


° ° ° ° ° ° ° ° 

° 1n ° 1n (o} Ye} to) In 

+ ise) Sp) N NQ H 4 
STeNprAIpuy 


1 An erroneous conclusion is rather easily drawn from these charts, for at first 
glance it appears that the potential of the gland increases by steady gradation from 
the fish up to the mammal. When figured as percentage increases of the experi- 
mental over the control lines, it is found that such is not the case. Data pertaining 
to this relation are now being collected. 


Day 1 


those treated with fish thyroid; 


Showing number of individuals at end of each 


CHART I. 


309 


DIVISION RATE IN CILIATE PROTOZOA. 


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DIVISION RATE IN CILIATE PROTOZOA. 3II 


A curve plotted from the averages of all five of the different 
experimental lines and drawn beside a curve portraying the 
averages of the control lines for the same periods, each figured 
day by day for the six days, represents, in a manner, a generalized 
curve of the effect of vertebrate thyroid constituents on protozoa, 
as based on our data. This curve takes the following form: 


300 


iS) 
On 
(o} 


Individuals 


150 


Too 


50 


CuaArt 6. Acurve plotted from the averages of Charts 1, 2, 3, 4 and 5, and thus 
representing a composite of results obtained from use of thyroids from each of the 
five main classes of vertebrates. 


DISCUSSION. 


The number of papers which have hitherto been published 
along this line seems to be limited to those already mentioned; 
so that conclusions have to be drawn from a comparatively 
meager literature, and to gather largely around the investigator’s 


312 ROBERT A. BUDINGTON AND HELEN F. HARVEY. 


own experience. There is essential agreement between the 
findings of Nowikoff, Shumway, and ourselves as to the constant 
effect of thyroid ingredients in increasing the division rate of 
protozoa beyond the normal, at least so far as Paramecium is 
concerned. 

The work of others, notably Gudernatsch (12, ’13), on the 
feeding of amphibian embryos, seems to indicate that the effect 
there observed is mainly one of acceleration of differentiation of 
tissues in the growing organism; at least this is the interpretation 
given their findings. West (14) has verified certain features of 
Gudernatsch’s results. A similar betrayal of specialization in 
function would, of course, not be possible within the limits of 
a unicellular organism. It seems entirely probable, however, 
that intra-cellular modifications of the Paramecium protoplasm 
does accompany its feeding upon and living in a medium which, 
among other things, brings it hurriedly to its most crucial ex- 
perience, self-division. The fact that rapid fission of thyroid-fed 
Paramecia is accompanied by their increased activity and trans- 
parency, and by smaller size,! indicates that very important in- 
ternal modifications doubtless occur. Careful study of protozoa 
exposed to exigencies of this sort should be made. 

If cell division in protozoa is to be compared with anything in 
the life history of metazoa, it should certainly be considered be- 
side the early development of the metazoan egg. If the egg has 
already advanced to the proportions of an embryo or larva, and 
the precocious differentiation of tissues and organs in such is 
under consideration, the question arises: Is this differentiation 
at all explained in the same terms as is protozoan cell division, 
or does it involve the same basic factors? It seems to us that 
this query may very possibly be answered in the affirmative, for 
the reason that the sprouting out of legs from the tadpole and 
establishment of other organs characteristic of the adult, is surely 
not due to mere unusual division of labor among the young 
cells generally acting as little more than unit components of the 
infant tadpole body; but that these latter have been provoked 
(by thyroid ingredients?) to abnormally rapid division, probably 


1 Shumway mentions these alterations to occur in thryoid-fed Paramecia, and 
we have found such to be practically always observable. 


DIVISION RATE IN CILIATE PROTOZOA. 313 


with accompanying abnormally small size, and that entirely 
normal differentiation has set in among cells which have been de- 
rived by the abnormally early (rapid) multiplication of their 
_ ancestors. 

We would suggest, therefore, that there may be, at bottom, 
not any great difference between what shows itself in Guder- 
natsch’s work as differentiation, and the result which shows 
itself as cell division in an animal where differentiation, so far 
as it exists, can assert itself only intra-cellularly, and thus in a 
very obscure manner. 

It is certainly entirely unnecessary to dwell upon the obvious 
fact that the more nearly adult a metazoan animal is, the more 
difficult it becomes to even suggest parallelisms which may exist 
between it and unicellular organisms; so that, to discuss the 
numerous physiological effects which have been obtained from 
feeding thyroid tissues to various vertebrata, or from grafting 
and transplantation experiments, or to examine the studies of 
conditions in higher types provoked by pathological thyroid 
growth and disease, is quite beyond the scope, if not impossible 
in connection with the subject, of this paper. 


SUMMARY. 


The conclusion to which the foregoing experimental results 
point is that thyroid ingredients, no matter from what class of 
vertebrates the gland be taken, produce essentially the same 
result when given to ciliate protozoa (Paramecium and Sty- 
lonichia) as a food or as a factor in the medium in which they 
live, viz., increased division rate. : 

The tissue which has hitherto been used in experimental work 
along this line has, we believe, always been taken from mammals. 
We think it safe to say that, no matter how far apart taxonomi- 
cally, or how distantly related phylogenetically the “higher”’ 
and “‘lower’’ members of the vertebrate phylum may be, certain 
physiological qualities in the thyroid glands are constant and 
similar in all. 

Sufficient difference exists between the potential of the thyroid 
secretion of one vertebrate class and that of another, so that, if 
studies of the normal value of this gland are being made, glands 


314 ROBERT A. BUDINGTON AND HELEN F. HARVEY. 


from the same class, if not from the same genus and species of 
animal as the one under observation, may well be employed. 


BIBLIOGRAPHY. 
Gudernatsch, J. F. 
12 Feeding Experiments on Tadpoles, I. Arch. f. Entwick., XXXV., 3. 
’13. Feeding Experiments on Tadpoles, II. Am. Jour. Anat., XV., 4. 
Nowikoff, M. 
’08 Die Wirkung des Schilddrusenextracts auf Ciliaten. Arch. f. Prot., XI., 2. 
Shumway, W. 
"14 Effect of Thyroid on Division Rate of Paramcecium. Jour. Exper. Zool., 
SOWA 5 Be 
Woodruff, L. L. 
705 An Experimental Study on the Life History of Hypotrichous Infusoria. 
Jour. Exper. Zool., II., 4. 
West, P. A. 
’14 Experiments in Feeding Tadpoles. Science, N. S., XX XIX., 106, p. 918. 


AN EXPERIMENTAL STUDY OF THE MOVEMENTS 
OF HERRING AND OTHER MARINE FISHES! 


VICTOR E. SHELFORD AND EDWIN B. POWERS. 


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I. INTRODUCTION. 


The general problem of increasing the supply of any species 
of fish or any other aquatic food animal, or of maintaining such 
species against extensive catch and against pollution of waters 
with sewage and the waste products of manufacturies, is very 
complex. The older methods of study are as important now 
as ever. The study of the food of an animal, its relation to its 
natural enemies, and its breeding habits still must receive their 
proper share of attention. In addition to these we now know 
that attention must be given to the chemical condition of the 
water, its effect on the movements and migrations and general 
health of the animals. Likewise it is especially important to 
study the physical and chemical conditions in which the animals 
breed and to look especially into the matter of the preservation 
of the natural breeding grounds. It is well known that one of the 
reasons for the depletion of the white-fishes in Lake Michigan 
is the destruction of their breeding grounds by the addition of 
sewage, saw-dust and other refuse to the water, which has 
settled on the breeding grounds and rendered them uninhabitable 


1 Contribution from the Puget Sound Marine Station. 
315 


316 VICTOR E. SHELFORD AND EDWIN B. POWERS. 


by the lowering of the oxygen content and covering the surface 
with materials which bury and tend to smother the eggs during 
development. The number of individuals of a species is never 
any greater than the breeding grounds can support. Finley 
(13) has shown that the number of prairie chickens in certain 
counties of Illinois is directly proportional to the area of breeding 
grounds. Likewise the senior author (Shelford, ’11) has shown 
that in a series of ponds at the head of Lake Michigan, food 
fishes are absent where their food is greatest in quantity because 
the breeding conditions are absent, due to the covering of the 
bottom with the decaying food of fishes. It is especially note- 
worthy that the food of the youngest fishes is especially abundant 
in ponds where the best food fishes cannot breed. This is not 
due to the failure of young fishes to destroy the small crustacea, 
because the same principle holds for ponds in which there are 
as many crustacea-eating fishes in stages suitable for food fishes 
as in stages suitable for only non-food fishes. 

The economic justification for the study of the movements of 
fishes is two fold. First experimental studies are concerned with 
the question of the conditions which the fishes select or reject when 
presented with two or three kinds of water to which they have 
free access under experimental conditions. Their importance 
in this connection is based upon the fact that so long as we are 
concerned with conditions which the fishes habitually encounter 
in nature, the selections or rejections represent in a general way 
the physiological character of the fishes and as a rule conditions 
which fishes reject are detrimental if continued for a long time. 
Thus, as we shall see later, fishes turn away from water contain- 
ing hydrogen sulfide and we will show further that they die very 
quickly when exposed to only a small excess of this gas in the 
sea water. Here then the fish is so constituted that its behavior 
and safety are intimately linked. Of course there are exceptions 
to this rule and it does not hold when we are concerned with 
changes in conditions which are not commonly encountered in 
nature. Thus we learn something of the conditions that are 
probably deleterious to the animals without either killing them 
or breeding them continuously under the modified conditions. 
The second justification lies in the fact that we can learn by 


MOVEMENTS OF HERRING AND OTHER MARINE FISHES. 317 


such experiments the effect of advancing civilization and industry 
upon the presence or absence of a species in any locality. The 
movements of the fishes must be known as well as the cause 
-therefor, before we can intelligently approach the question of 
capturing them in quantities. 


Il. MATERIAL AND METHODS. 


1. Stock of Fishes. 


The material used in these experiments was chiefly the fry 
of the herring (Clupea pallast Cuvier) 6 cm. (2% in.) common 
in Puget sound. The fry were caught on July 2 and were kept 
in a float car anchored ina good tide and until July 22 practically 
none of the fish died except during the first few days when those 
probably injured in catching were the chief victims. A few soles 
(Lepidopsetta bilineata Ayres) secured on July 4 at Fisherman’s 
bay, Lopez Island, were keptin the same car. A few young hump- 
back salmon (Oncorhynchus gorbuscha Wal.) 7 cm. (234 in.) long 
were secured at sea, through the courtesy of Dr. E. Victor Smith 
on June 30, at Turn Island and were not used after July 8 as they 
did not appear to be in normal condition after that date. A 
single Cottid (Oligocottus maculosus Girard) was used in killing 
experiments. The soles and herring appeared to be in essentially 
as good condition at the end of the period of work as at the 
beginning. 

2. The Water Supply of the Station. 


Experiments were run in which both fresh and salt water were 
used. Thus it is necessary to consider the character of both. 
The frésh water in use during the summer of 1914 was supplied 
by the village of Friday Harbor and came from deep wells. 
Owing to the rocky character of the ground in the vicinity, it 
was impracticable to bury the pipes and the temperature varied 
greatly with the weather, night, day, etc. The highest tem- 
perature noted was 24° C. The water contained an excess of 
gas which escaped in a cloud of bubbles when it was withdrawn 
from the tap. This was neither oxygen nor carbon dioxide and 
gave no odor which points to the conclusion that it was nitrogen. 
The water was distinctly alkaline to phenolphthalein, free carbon 


318 VICTOR E. SHELFORD AND EDWIN B. POWERS. 


dioxide being wanting. The half bound carbon dioxide was 
24.2 c.c. per liter and the fixed 28.6 c.c. The oxygen was less 
than 0.5 c.c. per liter (for methods see Birge and Juday, ‘11, 
pp. 13-21). Such water is unsuitable for biological purposes and 
was used in these experiments only after aération by running it 
slowly over an inclined board ten inches wide and four feet long. 
After this aération the oxygen content was, at 13° C., 4.9 c.c. 
per liter and the excess of other gas was removed, but the water 
still remained alkaline. 

The sea water supplied at the station building was pumped 
from a depth of about four feet below mean tide. It was retained 
in a wooden tank, being pumped twice per day, in the evening 
and in the morning. Upon standing in the tank the temperature 
rose from 11° to nearly 15° on warm days. 

The oxygen was determined by the Winkler method. In no 
case was the sea water from the tank or from the bay from which 
it was pumped, saturated with oxygen even in samples collected 
at the surface. The only surface collection made that showed 
saturation according to the tables of Fox (see Murray and Hjort, 
"12, p. 254) was from the strong tide rips off Point Caution at 
5:30 P.M. Collections from the bottom of sandy shores among 
Ulva were super-saturated. , 

Chlorine was determined by titrating with silver nitrate. It 
usually amounted to about 16.93 grams per liter. It was usually 
a little higher in water from the tank than in water collected 
from the sea. The determination of carbon dioxide was made 
by the method in common use in fresh water. The sea water 
was titrated with 3'5 normal solution of sodium carbonate, with 
phenolphthalein as an indicator. The water was usually ‘acid in 
reaction indicating about 1.7 c.c. per liter of free carbon dioxide. 
The half bound and bound carbon dioxide as indicated by the 
method used by Birge usually amounted to 25.3 c.c. per liter 
each. There was considerable uniformity in the results of such 
titrations and while the method is not especially accurate the 
lack of oxygen common in the water would indicate an excess 
of free carbon dioxide over that commonly reported for sea 
water. The correctness of these figures is further suggested 
by the slight alkalinity of the water taken from the vicinity of 
green alge and containing an excess of oxygen. 


MOVEMENTS OF HERRING AND OTHER MARINE FISHES. 319 


Hydrogen sulfide is very commonly present in sea water when 
decomposition is taking place. This was determined by titration 
with iodine which was the only method we were equipped to 
employ. It is never present in any quantity in freely circulating 
waters. The highest records are for collections made near the 
bottom under Ulva, where the odor is often quite distinct. On 
account of the probable presence of other substances which may 
absorb iodine the determinations may be slightly too large 
(Birge and Juday, ’11). 

MABLE I: 


THE DISSOLVED GASES OF THE SEA WATER ABOUT F RIDAY HARBOR, 


WASHINGTON. DATA IN C.C. PER LITER. 
Date. Place. Hour. Tide. Collected. | COg.| Og. | HoS.| Temp. 
Point Caution 5:30 P.M.| Low, in | Surface 5.6 
7/23 | N.E. Brown’s Id. |10:10 A.M.| Low, out | Surface 1.76] 4.9 |.187| 11.6 
7/25 Do. 11:10 A.M.| Low, out | Surface |1.64| 4.6 |.237 10.7 
4/25 Do. 7:15 P.M.) High, Surface [1.91] 4.6|.268] ro.5 
7/26 | S. Brown’s Id. 10:45 A.M.| Low, in | 8’ under |o.00| 9.2 |.536 10.5 
Ulva 

7/23 Do. 12:00 M. | Low, in Do. 0.00 |10.8 |.536| 13.2 
7/25 Do. 12:00 M. | Low, in | 18’ Do. |o.00| — -339| 13.2 
7/25 | Station dock tr:10 A.M.| Low, in | Surface [1.86] 5.2 -149|] 11.6 
7/25 Do. 12:45 P.M.| Low, in | Surface |3.10| 4.8 -205 

7/26 Do. 9:30 A.M.} High, out} Do. 1.81] 4.2|.205|} 10.6 
7/26 | Tap-pumped at 6:30 A.M.) Med. low| 4’ deep |1.76| 4.7 .223 j 


It will be noted from a study of the table that the water from 
Point Caution where the tide has full sweep is the only water 
saturated with oxygen at the surface. In other places the sea 
water at the surface is about 1 c.c. less than the amount given 
by Fox (see Murray and Hort, ’12, p. 254). Aérating the sea 
water increased the oxygen. The water from the tank did not 
seem to have been modified by standing for sixteen hours or more. 
On the whole there must be much decomposition in Puget Sound 
waters. There was no constant difference between the water 
from outside and inside the side of the island which encloses 
Friday Harbor. The COs is a little higher except at low tide 
in the sample taken near the Ulva; the oxygen remains about the 
same. The hydrogen sulfide does not average appreciably higher. 

The explanation for the alkaline character of the water under 
the Ulva is that the plants take up the CO, and give off oxygen 
and thus remove the excess which occurs in other localities. 


320 VICTOR E. SHELFORD AND EDWIN B. POWERS. 


The absorption of oxygen in connection with the development 
of the hydrogen sulfide probably prevents any very great excess 
of CO, from accumulating (Lederer ’12). 


III. THE RESISTANCE OF FISHES TO CONTAMINATION AND 
DECOMPOSITION PRODUCTS. 


It was not possible to try the resistance of the fishes (Wells, 
13) to the effect of the lack of oxygen either separately or in 
combination because no means of removing it was at hand. It 
was possible only to add gases to the water. Hydrogen sulfide 
and carbon dioxide were used. 


1. Herring (Clupea pallasit Cuvier). 


Hydrogen sulfide is extremely poisonous to the fishes (Weigelt, 
03). In the first attempted gradient experiments where the 
water at one end contained only a little of the gas the fishes 
turned on their backs in two or three minutes when the one 
inflow was showing 8.3 c.c. per liter and the other was pure sea 
water. This happened in spite of the fact that more than half 
of the time was spent in the end with least H2S. The experiments 
were performed in the manner described by Wells. When placed 
in a solution of 7.6 c.c. per |. the herring gasped after 1 minute 
and 45 seconds, turned over after 5 minutes, and were apparently 
all dead in 6 minutes. In carbon dioxide of about 20 c.c. per |. 
the herring showed evidence of loss of equilibrium after three 
minutes. Some of them sank to the bottom after 12 minutes. 
After 39 minutes to 62 minutes herrings turned on their sides 
on the bottom, resting in this position for a time and then 
swimming more nearly normal for a time again. One died after 
102 minutes, the others after 159 minutes’ exposure. The 
oxygen was about 5.5 c.c. per |. and varied directly with the 
amount of tank COs: used, indicating that the carbon dioxide 
contained much oxygen. 

When carbon dioxide and hydrogen sulfide were used together 
the carbon dioxide was about 30 c.c. per 1. and the hydrogen 
sulfide 2.9 c.c. per 1. The amounts were controlled with some 
difficulty and thus the experiments are not alike in the matter of 
concentration. Herring were much stimulated at the beginning. 


ain, 


MOVEMENTS OF HERRING AND OTHER MARINE FISHES. 321 


After I minute and 30 seconds there was a general loss of correla- 
tion of movements. At the end of four minutes all the herring 
were dead. Thus we note that the combination of hydrogen 
sulfide and carbon dioxide is exceedingly deadly. In alkaline 
partly aérated fresh water herring showed loss of equilibrium in 
from 10 to I4 minutes. They nearly succumbed and then 
recovered a few times, the first one dying after 30 minutes and 
all being dead in 44 minutes. 


2. Soles (Lepidopsetia bilineata Ayres). 


In the hydrogen sulfide (7.6 c.c. per 1.) the soles showed some 
signs of loss of equilibrium at the end of one minute. In 5 
minutes they were on their backs. After 13 minutes they had 
revived again. They were nearly dead after 16 minutes and all 
dead at the end of 24 minutes. 

In the carbon dioxide (20 c.c. per 1.), after 45 minutes one sole 
gasped, which was the first sign of any disturbance and one turned 
on its back after 54 minutes. For three hours this was repeated 
at intervals and each gasping time was followed by recovery. 

In the combined carbon dioxide (30 c.c. per 1.) and hydrogen 
sulfide (2.9 c.c. per 1.) the soles lost equilibrium after 2 minutes 
and 30 seconds. In I1 minutes they were barely alive and in 
13 minutes were dead. In fresh water the soles showed stimula- 
tion at the end of 3 minutes. They died in from 48 minutes to 
one hour. 


3. Cottid (Oligocottus maculosus Girard). 


One fish of this species was added from curiosity but the results 
were sufficiently surprising to record. In the hydrogen sulfide 
the cottid seemed unaffected until the end of 6 minutes, after 
the herring were all dead. It breathed heavily after 16 minutes. 
The fish was alive at the end of three hours when it was returned 
to running salt water, and allowed to recover, after which it was 
used in the carbon dioxide experiment, with similar results. In 
the combined carbon dioxide and hydrogen sulfide it was not 
visibly affected and in fresh water there was no evidence of any 
disturbance. These fishes were seined from the sandy bottom 
among the Ulva, coming in with numbers of the small soles. 


322 VICTOR E. SHELFORD AND EDWIN B. POWERS. 


4. Summary. 


We note that on the whole the presence of a quantity of 
carbon dioxide in the water affected the fishes less than a 
smaller amount of hydrogen sulfide. The combination of hydro- 
gen sulfide and carbon dioxide was most rapidly fatal. Since 
decomposition yields CO: and consumes oxygen and is accom- 
panied by the production of hydrogen sulfide which is also accom- 
panied by the consumption of oxygen, it is reasonable to suppose 
that on a bottom from which vegetation is absent and decom- 
position actively takes place a fatal combination of lack of 
oxygen, and presence of hydrogen sulfide and probably carbon 
dioxide can develop quickly. 

Considering the fishes tested we note that the herrings were 
most sensitive. They were sharply marked off from the bottom 
species which are resistant to a marked degree. This resistance 
is in a very general way associated with the habitat preference 
of the species. Still the marked resistance of the small cottid 
is not quite explicable on this or any other basis. 

The importance of factors which kill fishes is greatest in the 
early stages for two reasons. First the small size of the eggs 
and embryos makes the ratio between volume and surface 
smallest and thus any substance in solution will reach all parts 
of the organism at a more rapid rate. Secondly the inability 
of the eggs and embryos to move about makes them the easy 
victims of any adverse conditions that may occur. The eggs 
of the herring are deposited on the bottom. Nelson mentions 
rocks only (Marsh and Cobb, ’10, p. 46) and rocks are usually 
swept fairly clear of organic matter and the water well aérated 
down to the depth of one fathom where the fishes breed. If this 
means that sandy bottoms of bays are avoided it probably means 
the avoidance, during the breeding, of water high in hydrogen 
sulfide (see table) which would be fatal to the eggs and small herring 
fry to a greater degree than to those studied, which were 6 cm. 
long. Sensitiveness to hydrogen sulfide is a matter of much 
importance from the standpoint of the suitability of a given arm 
of the sea for herring and the influence upon fishes of contamina- 
tion of the shores with refuse from the land. 

Carbon dioxide is not high in such shallow water on account 


Pe 


MOVEMENTS OF HERRING AND OTHER MARINE FISHES. 323 


of the presence of so many green plants. Carbon dioxide is 
probably more important in connection with movements of the 
fishes than in the matter of restricting their breeding places. 


IV. REACTIONS OF FISHES TO CHEMICAL CONDITIONS IN 
SEA-WATER. 


1. Conditions and Methods of Study. 


The experiments were performed in a gradient tank. Water 
of two kinds was used in the experiments. One kind was 
allowed to flow into one end at a definite rate and another kind 
into the other end at the same rate. It flowed out at the middle 
at the top and at the bottom so that the two kinds of water met 
at the center. The outflow at the center did not of course 
prevent the mixing of the two kinds of water and thus the middle 
section, equal to one half or one third of the tank was a gradient 
between the two kinds of water. The tank used in these experi- 
ments was 122.3 cm. by 15 cm. by 13 cm. deep. The front wall 
was of plate glass and a plate glass top was used at times. Water 
was allowed to flow in at both ends at the same rate (usually 
600 c.c. per minute) through tees the cross bars of which con- 
tained a number of small holes. The cross bars of the tees were 
at the center of the ends of the tank behind screens. The drain 
openings were located at the center near the top and in the 
bottom. The outer openings of the drain tubes were at the 
level of the water in the tank. The water flowed in at the ends 
and drifted toward the center and flowed out through the drains. 
We found no evidence that fishes react to the slight current thus 
produced. Since each half of the tank held about 9 liters, it 
required 15 minutes to fill it or to replace all the water in 
one of the halves. The tank was enclosed under a black hood. 
Two candles (electric lights being wanting) were fixed in the 
rear and above the center of the two halves, 7. e., above a point 
midway between the screen partition and the center drain. 
The light was 15-20 cm. above the surface of the water which was 
13 cm. deep. The room was darkened during the experiments 
which were observed through openings in the hood above the 
lights or through the glass side late at night. Fishes do not 
usually note objects separated from them by a light. 


324 VICTOR E. SHELFORD AND EDWIN B. POWERS. 


Water differing as little as possible from that in which the 
fishes usually live was used for control readings. Controls were 
observed and the conditions in the two ends of these were the 
same either because the water introduced at the two ends was 
alike or because no water was run into either end (standing 
water). 

In the controls the fishes usually swam from end to end in a 
rather symmetrical fashion, and thus by comparing these move- 
ments with those occurring when the fishes encountered differ- 
ences in water, we are able to determine the reactions of the 
fishes to the differences. Various kinds of water were used at 
one end as follows: (1) water with varying amounts of carbon 
dioxide added; (2) water with oxygen added; (3) water with 
hydrogen sulfide added; (4) fresh water. 

When the difference between the solutes at the two ends of the 
tank was not great we found by chemical tests that the central 
portion of the tank was a gradient between the characteristic 
waters introduced at the two ends. Usually the end thirds were 
essentially like the inflowing water. When the difference in con- 
centration was great the region of the gradient was propor- 
tionally longer and the ends with the inflowing concentrations 
correspondingly shorter. When the difference in concentration 
was very great the entire tank was gradient. For an experiment 
a fish was placed in a dish containing enough water to barely 
cover it and set above the tank. When all was in readiness 
the fish was emptied into the center of the tank. Marks on the 
sides divided the tank into thirds. The fish nearly always swam 
back and forth, apparently exploring the tank. The movements 
of the fish were recorded graphically as‘shown in Chart I. For 
this purpose sheets of ruled paper were used. Four vertical 
double rulings corresponded to the thirds and two ends of the 
tank. Distance from right to left was taken to represent the 
length of the tank, vertical distance to represent time and the 
graphs drawn to scale. The width of the tank was ignored. 
THe graphs on the following pages are copies of the originals. 

Before or after the experiment, the headings of the sheets 
were filled with data regarding the kind, size, and previous 
history of the fish, the conditions in the tank, concentration of 


MOVEMENTS OF HERRING AND OTHER MARINE FISHES. 325 


the solutes and other significant data. The fish was observed 
continuously for twenty or more minutes. 

In order to maintain a constant flow the water was introduced 
into the tank by means of siphons from cans on the top of the 
hood with a 74 cm. head. Connected with one of the two cans 
was an inclined plank trough 420 cm. by 25 cm. for the purpose 
of aérating water before it entered the can if so desired. 

By the method just described it is possible to obtain unusually 
accurate data on the factors influencing the movements of 
fishes. According to Marsh and Cobb ('07) a great difficulty 
in the herring fishery is the erratic movements of the fish. 
Schools may visit a bay for three or four years, in succession, and 
then without any apparent reason, avoid it for a season or two 
_or altogether. Bertham (97) noted a possible relation between 
the abundance of these fishes and weather and suggests that 
climatic cause may have more to do with the failure of some 
branches of the fisheries than is generally believed. He attri- 
buted the failure of the fisheries of Cape Benton to the occurrence 
of severe east and northeast storms during the running season. 
It is not clear what the effect of such storms may be, but they 
chiefly affect the dissolved content of the water. Johnstone, ’08, 
page 246, says that it is now nearly certain that the shoaling 
migrations of the herring of Europe are to be associated with the 
salinity and temperature of the sea. It is évident from these 
experiments that acidity and alkalinity are more important than 
salinity and the solution of the problem will come from a careful 
study of the reactions of fishes along with a similar study of 
hydrographic conditions. 


2. Reactions to Temperature. 


These fishes are remarkably sensitive to differences in tem- 
perature. We obtained good reactions with a difference of 
0.6° C. in the length of the tank. Fair reactions were obtained 
with differences of 0.5° C. and since the fishes often turned around 
near the center it appears that they recognized a difference of 
0.2° C. In graph 1, Chart I., we show the reaction of fish in a 
gradient of 0.6° C. (compare with graph 2—control). The fish 
was taken from sea water at 10.9° and the experiment performed 


326 VICTOR E. SHELFORD AND EDWIN B. POWERS. 


at 12.8° and 13.2°. It will be noted that the fish showed a prefer- 
ence for the higher temperature. Eleven experiments were 
performed with herring and in seven cases the fishes showed a 
preference for the warmer water and in three cases for the colder. 
One did not show any marked preference. The differences were 
too slight to be of great significance in determining whether the 
fishes move into warmer or colder water but show a great sensi- 
tiveness. Thus temperature may play an important rdle in the 
movements of fishes. 

It will be noted by reference to the graph, that the fish moved 
into the colder water several times as if trying out the entire 
tank and then turned back periodically from the colder end. 
In the control where there was no flow or difference in tempera- 
ture the fish turned back from both ends at times but by chance 
as shown by other controls, turned a little more often from the 
end corresponding to the cold end of the experiment due perhaps 
to difference. 

3. Hydrogen Sulfide. 


The animals turned back sharply from all concentrations not 
great enough to cause intoxication as shown in graph 3, Chart I. 
(compare with control graph 4). In this experiment the hydrogen 
sulfide was only 4.5 c.c. per |. and the fishes avoided it sharply 
and after trying out the tank turned about at a point where the 
concentration could not be more than one tenth of that at the 
‘treated water end or about equal to that under the Ulva on the 
south side of Brown’s Island (p. 319). 

This experiment is typical of several and the fishes are thus 
seen to be able to orient with reference to an increase in the 
solute and to turn back from it very sharply. 

The control (graph 4) to this experiment is symmetrical, 
there being turning from both ends in equal number. It shows 
the reaction of the fishes when no stimuli are encountered in the 
tank. 


4. Reactions to Salinity, Acidity and Alkalinity. 


As noted above, the fresh water of the laboratory was from 
deep wells and not good for biological work. It was alkaline, 
containing no free carbon dioxide, 24.2 c.c. per |. half bound and 


ihe 


MOVEMENTS OF HERRING AND OTHER MARINE FISHES. 327 


28.6 c.c. per |. of bound carbon dioxide. There was a deficiency 
of oxygen and what was present was probably due to leaky pipes. 
It was only 0.5 c.c. per liter. It contained a large excess of some 
odorless gas which escaped in bubbles and was probably nitrogen. 
This water was aérated by running it over a board 420 cm. long, 
into the siphon bucket. This reduced the gas in excess to air 
saturation and raised the oxygen to 4.8 c.c. per liter. 

In the experimental tank the difference between the density 
of the fresh and salt water was so great that the fresh extended 
nearly to the opposite end at the top with very little mixing and 
the salt water occupied a corresponding place on the bottom. 
Thus there was a sharp gradient from top to bottom, but a very 
imperfect one from end to end. To avoid this siphons were 
inserted which withdrew water from each side near the bottom 
at a point one third the length from the salt end and from near 
the top at the same distance from the fresh end. This was found 
not to remedy the difficulty sufficiently and so a screen incline 
which extended from bottom at the salt end to the height of 
8.5 cm. at the fresh end. Above this was another screen which 
was 8.5 cm. at the salt end, and which ran up to the surface of 
the water at the fresh end. This enclosed the fish in an inclined 
cage 8.5 cm. deep at the salt water end and 5.0 cm. deep at the 
fresh end. The fish moved back and forth in this at a distance 
of about 4 cm. from the lower screen. The gradient of salinity 
between the acid sea water and the alkaline fresh water was 


essentially as perfect as shown in the accompanying Fig. 1. By 


AS 
4,41 ve Me Ss 4 


Fic. I. 

Fic. 1. Showing the distribution of salinity in terms of grams of chlorine per I. 
in Roman and oxygen content in c.c. per 1. in italics; Al, alkaline; N, neutral; Ac, 
acid. 
consulting this figure it will be seen that the oxygen content 
was essentially the same throughout. ‘The salinity corresponded 
to 10.561 grams of chlorine in the salt water end to 6.45 grams 


in the’ fresh water end. The acidity to phenolphthalein reached 


328 VICTOR E. SHELFORD AND EDWIN B. POWERS. 


1 TG ee 3 Hos 4 5 Hons 6 v8 ae 


I = SS — == poss 
‘ = a= \ == 1 = =} 
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== == SS — = = = = 
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== ; == == 1 = =) 2) i 
25 a= Eee — ae (= Se =i 
= eer ; == == ——— —— = =" See ecg 
25 iS SS — Ze Peet 0 ESE 
== : Sis ————— = 52) == 
= == 2 — z Ee 22 (22 2.2 
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bi ae a ee 
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ee = 1 9 2 Ce wi 2 SF: 22) = 
== =e So) = SS BS eee 
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S55 == Se SS SS eee 
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== _—— = 5 ae aaa Be c Bs _—S— ———— ——— 
ails NE a S02 SSE 
== = == A 22 SS SS SS 22 1S == 
= = = == 1 == ae es f — 2 i == 
i =S wen3: —— 
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aye == = f == ray! m=  |@22 22 
== H == 1 =e ' == ne == 
== = i = 1 == a0 == ' = 2 ee =s 
== == == ty Sees == 4 ae === == 
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a= : f= = H == 22 4 paper ily = == 
= = phe = == roe Sa ==} == 
=15=- =15= = i S= SS S5— 
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== ——— 1 _ 1 = ft ore eo = 
at == = 1 = bh 0 SOS <a == 
= = 1 = 1 = = tooF == U == 1 == 
aS =e in t == Wares ean) =e Ga ag = 
— = == 1 Sf SE g =e == 
Cuart I. 
\ 


E, experiment; C, control; f, fresh water, T, and figures following show temper- 
ature difference. 

Graph 1 shows the reaction to a difference of 0.6 of a degree the lower avoided 
tempcrature being on.the left. Graph 2 shows the movement of the fishes in the 
tank when there is no difference in temperature (see also graph 6 and other graphs 
marked C). 

Graph 3 shows the avoidance of hydrogen sulfide introduced at the right. After 
a few trials the avoidance became very sharp. Graph 4 is the control, 7. e., with 
no difference between the ends. 

Graph 5 shows the reaction of a fish to fresh water introduced at the right 
showing the avoidance of the acid salt water and selection of the alkaline fresh water 
with the incline described on p. 327 in position. Temperature the same at the two 
ends. Graph 6 is the control of the same. 

Graph 7 shows the selection of lower temperature with the incline screen cage 
in position; difference in beginning .55° C., at end .25° C. A difference in tem- 
perature occurred in some of the incline experiments but lower at the salt end. 
The graph shows that the fishes would have selected the salt water end where the 


. 


temperature was a little lower if they had been reacting to temperature. 


ch 


MOVEMENTS OF HERRING AND OTHER MARINE FISHES. 329 


almost to the center while the central region was essentially 
neutral. Consulting graph 5, Chart I., we note that the fish 
moved the entire length of the tank for two minutes and then 
began to turn back before the highest salinity was reached. 
After a few such turnings it went the entire length of the tank 
for a short period with one exception. Between the 7th and 14th 
minutes the excursions into the salt water were gradually 
shortened. In other words after a few brief entrances into the 
salt water the fish gradually shortened its invasions of the salt 
water until it was turning rather regularly just on the alkaline 
side of neutrality, which continued to the end of the observation. 
It will also be noted that the fish turned back twice from the 
fresh water end, which is significant because in other cases the 
fishes selected this region. This was true in four other experi- 
ments with the incline and in six out of eleven performed without 
the incline. It appears that the herring select either brackish 
or slightly alkaline water. The control, graph 6, is symmetrical. 

In some of the experiments performed with the incline there 
was a slight difference in temperature between the two ends, 
the fresh water being a little higher. To check this source of 
error, the experiment was performed with the incline but with 
the difference in temperature reversed, and the fishes selected 
the opposite end of the tank, showing that this was not only a 
reaction to solutes but that the solutes inhibited any reac- 
tion to temperature that might otherwise have taken place 
(graph 7). In the temperature experiments the fishes selected 
the higher temperature when the stock was fresh and the lower 
temperature near the close of the work showing that the fishes 
had undergone some slight physiological change during their 
stay in the float-tank. 

The tendency to come to rest in the region on the alkaline 
side of neutrality was very clearly shown in all the experiments 
except one. The salmon oriented with their heads toward the 
fresh water end, drifting very slowly back, probably floating 
in a current and then swimming up again to the same point. 
This was very striking and constituted an unmistakable difference 
between the experiment and control. Chart II., graph 13 and 
14, show such an experiment and control. The swimming up 
occurred notably in the 13th and 18th minutes. 


330 VICTOR E. SHELFORD AND EDWIN B. POWERS. 


To determine whether or not this peculiarity is a reaction to 
salinity or alkalinity, the experiment with herring was repeated 
and carbon dioxide to which the fish are negative (graphs 8 and 
9) run in the fresh water, to neutralize the alkalinity. At the 
beginning of the experiment shown in Chart II., graphs to and 
11, the carbon dioxide content of the fresh water was 26.5 c.c. 


8 8 10 HIoNs It 2 13. Hions = 14 
E c Bae E ¢ Fe E C 


_ fT an 
= 2= == + == == SS: a = == 
Ze Ss Se — = = 2 SS 5 Hl = =| 
Ene =0S == 1 Se = 2 == ifs = Sees 
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= ae 2 SS StS 2 SS hatin BES 
: = = SOS — == | [JS=fi ! Ze 
= Z°= SVS Sis 22 4 te 2 Slee 
= == == SS ae Ser 5 Wi = ea, 
= == = == ee = a 
= =iE =u= TR = 55 Ser acd = ' Sze 
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= == == — == == en 0 SUS 
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= Sr= Sees pees == | 3 = , = 
= == — So SS SS == ee a =e 
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= SS == et e255 ,) == 
= ' =a a q S/= Sf ——— = i 4 ' Se} 
= 22 SS 2 SSS Ee 22 = 2: | =: Mes 
= = = S= == ES SS i ee == i) = 
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2225 22 J Ne = 2 eee —— =e My) wets 
S25 Se =— = 2) == Sue — i ft alts = 
a == } £2 z= —— = 
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= \ == Sas att West 9 GEIS es = See i BS 
== SSS == ob hh 0 == SSS] | ie Sa 
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=-- — - - . 7 - - ' -- i) { --=- ——— = ' a 
n= : SES pee SNe ee =; Ue yee. e e=ygee Sel p- 105 , == 


Cuart II. 


Graph 8 shows the avoidance of carbon dioxide in sea water introduced at the 
right. Graph 9 is the control of the same. 

Graphs 10 and 11 show thereaction to fresh water rendered acid by the addition 
of 26.5 c.c. per liter of carbon dioxide and the reversal of the reaction when the 
carbon dioxide fell to 8.1 c.c. and finally the gradual reversal to a preference for 
the fresh water when it became less acid than the salt. 

Graph 12 shows the preference for sea water with oxygen added (right end.) 

Graph 13 shows the selection of essential neutrality by a small salmon. 14 is 
the control] of the same. 


MOVEMENTS OF HERRING AND OTHER MARINE FISHES. 331 


per I. and the reaction was very sharply negative to fresh water. 
The concentration of the carbon dioxide in the fresh water was 
gradually lowered and the avoidance fell off as is shown in graph 
12 which was really only a continuation of 11 interrupted to take a 
sample which showed the carbon dioxide content to be 8.1 c.c. 
per 1. During the period represented by 11 the negative re- 
action decreased gradually until a point was reached when the 
tank was probably about equally acid throughout, after which 
the fish became negative to the sea water at the end of 13 minutes 
when on the basis of a uniform decrease the sea water which 
usually contained a little less than 2 c.c. per 1., became more 
acid than the fresh. Thus it appears that these fish are as 
sensitive to acidity as litmus paper. 

The relation of the two species of fishes to salinity is inter- 
esting in this connection. The salmon goes into fresh water to 
breed and some may reach maturity there or they may return 
to salt at varying ages. In connection with the entrance of 
salmon into fresh water, the orientation of these specimens with 
head in the fresh water is of interest but it is evident that the 


orientation is with reference to acidity and alkalinity rather than 


salinity. Sea water is less acid than fresh and the reactions of 
the salmon accord with their recent entrance into salt water. 
In the case of the herring, they are known to enter fresh water 
and some remain there permanently. Lydekker! states that 
some of them will live in brackish water and become dwarfed. 

When carbon dioxide was used in sea water the avoidance of 
the higher concentration was very striking, in all concentrations 
tried, up to 70 c.c. per 1. The avoidance was usually propor- 
tional to the concentration with staggering in the very high ones 
just as is the case with the fresh water fishes. 


6. Oxygen. 


The oxygen in the sea water in use at the station never reached 


saturation. One experiment was tried with water drawn directly 


from the tap, against water aérated by running over a board. 
The fishes selected the aérated water. When oxygen was 
added to the water used in opposition to that drawn directly 


1 New N. H., Vol. V., p. 489. 


332 VICTOR E. SHELFORD AND EDWIN B. POWERS. 


from the tank the preference for the higher oxygen content was 
decided (graph 12). 


V. SUMMARY AND DISCUSSION OF CONCLUSIONS. 


In these brief experiments we have only outlined the possi- 
bilities of much more extensive work along similar lines. Such 
experimental study alone can of course not solve the problems 
of migration but the extreme sensitiveness of the fishes studied, 
as shown by their detection of slight deviations from neutrality, 
temperature differences as small as 0.2 of a centigrade degree, of 
small fractions of a cubic centimeter per liter of hydrogen 
sulfide, etc., makes it very clear that there is no difficulty in 
fishes determining the direction to large rivers from hundreds 
of miles out at sea or of finding their way into any bay or harbor 
or river or other arm of the sea which their particular physio- 
logical condition at a given time demands. It is not necessary 
to appeal to “‘instinct”’ to explain the return of certain salmon 
to certain rivers, or the running of herring in certain localities. 
The mere fact of their origin in the region, the probable limited 
tendency to leave it (Johnstone, ’08), coupled with their ability 
to detect and follow slight differences in water is a sufficient 
explanation of all their peculiar migrations. The close way in 
which animals stay about certain localities from generation to 
generation is hardly appreciated. Thus as Johnstone points 
out, the herring of the east coast of Britain are largely local, 
having formerly been assumed to belong to shoals that came 
from distant points. ; 

The experimental method cannot of course determine the 
cause for the absence of fishes from any given point but must be 
accompanied by hydrographic studies. Such combined efforts 
must give very trustworthy results; hydrographic studies alone 
may lead to entirely erroneous assumptions because of the lack 
of knowledge of the sensibilities of the fishes concerned and the 
selection of some insignificant factor correlated with their 
absence or presence, as‘an explanation. Such correlates, offered 
as explanations, become the basis of erroneous remedial measures. 

Noting the remarkable discriminations of fishes for differences 
in alkalinity, acidity and neutrality, a note of warning may be 
sounded in regard to the relation of pollution to the run of her- 


ite 


MOVEMENTS OF HERRING AND OTHER MARINE FISHES. 333 


ring, and the presence in valuable numbers of many other fishes. 
Their tendency to avoid acid waters, hydrogen sulfide, etc., 
which result from decomposition and are increased by the pres- 
ence of refuse of fish canneries, sewage, etc., makes diversion of 
such refuse from the sea an important consideration.. The 
Baltic towns of the Hanseatic League were dependent in part 
upon the herring industry and after a century of great growth 
and prosperity fell into decline at the middle of the fourteenth 
century. Their prosperity was the accompaniment of the pres- 
ence of great shoals of herring off the Island of Riigen in the 
Baltic. Their decline was caused in part by the failure of the 
herring industry and the supposed migration of the herring to 
the North Sea which has since been the center of the industry. 
Schouwen (on the Netherland coast of the North sea) appears 
in the fourteenth century to have been frequented by the her- 
ring shoals in preference to Riigen (Yeats, ’86). The rapid 
growth of the Netherland cities, their supremacy and final sepa- 
ration from the Hanseatic league followed. A little later the 
herring again changed their haunts choosing the coast of Norway 
where both Norsemen and Netherlanders caught them. The 
Beukelszoon method of curing herring having come into use 
nearness to home was no longer a necessity. The Norse fisheries 
flourished until 1587 when an “‘ apparation of a gigantic herring 
frightened the shoals away.”’ Thus it appears that the develop- 
ment of the herring industry in each locality led to the apparent 
desertion of the locality by the fish, though the migrations 
assumed by historians may be doubted (Yeats) (Putzger ’or, 
p. 17a). Was this due to the contamination of the sea by 
the cities, or merely to over catch? Whichever may have been 
the case it is certain that contamination will not invite runs of 
the herring. The common assumption that the sea is so large 
that pollution cannot have a significant rdle is rendered entirely 
untenable by the greatly increased sensitiveness of the marine 
fishes as compared with fresh water ones. 


VI. ACKNOWLEDGMENTS AND BIBLIOGRAPHY. 


One hundred and twenty-five dollars of the expense of this 
research was borne by the University of Chicago, the remainder 
by the Puget Sound Marine Station. 


334 VICTOR E. SHELFORD AND EDWIN B. POWERS. 


Bertham, A. C. 
’96—7 Annual Report; Canada Department of Marine and Fisheries. 1896-7. 
Birge, Edward, A. and Juday, Chauncey. 
’71 Dissolved Gases. Wisconsin Geological and Nat. Hist. Survey Bull. No. 22. 
Finley, C. W. 
’r3 Natural Nesting Sites as a Factor in Bird Abundance. Nature Study 
Review, Dec., 1913, pp. 79-81. 
Henderson, L. J. 
173. The Fitness of the Environment. New York. 
Johnstone, James. 
08 Conditions of Life in the Sea. Cambridge. 
Lederer, A. 
%72 Some Observations on the Formation of Hydrogen Sulfide in Sewage. Am. 
Jour. Pub. Health, Vol. 3, pp. 552-61. 
Lydekker. 
’03 The New Natural History, Vol. V. 
Marsh, Millard C. and Cobb, John N. 
’97 The Fisheries of Alaska in 1907. Rep. U.S. Com. Fish 1907, p. 53. 
’t0 The Fisheries of Alaska in 1910. Rep. U.S. Com. Fish 1910, p. 45. 
Murray, John and Hjort, Johan. 
’12 The Depths of the Ocean. Lond. 
Putzger, F. W. 
’91 Historischer Schul-Atlas. Leipzig. 
Shelford, Victor E. 
’1z Ecological Succession, III. A Reconnaissance of its Causes in Ponds with 
Particular Reference to Fish. Br1or. BULL., Vol. 22, No. 1, pp. 1-38. 
Shelford, V. E. and Allee, W. C. 
’13 Reactions of Fishes to Gradients of Dissolved Atmospheric Gases. Jour. 
Expt. Zool., Vol. XIV., pp. 207-266. 
14 Rapid Modification of the Behavior of Fishes. Jour. An. Beh., Vol. IV., 
(Ds L—BOe 
Tittmann, O. H. 
’r3 Tide Tables for the Pacific Coast of the U. S. together with a number of 
Foreign Ports in the Pacific Ocean. Reprinted from Tide Tables for 1914. 
Dept. of Commerce Coast and Geodetic Survey, Washington, D. C. 
Weigelt, C. 
203 ~L’assainessement et le repeuplement des rivieres. Memoires courounes. 
(Translated from German) Brussels. 
Wells, Morris, M. 
713 The Resistance of Fishes to Different Concentrations and Combinations of 
Oxygen and Carbon dioxide. Biot. BULL., Vol. 25, No. 6, pp. 323-347. 
’r4 Resistance and Reactions of Fishes to Temperature. Transactions of the 
Illinois. Acad. Science, Vol. VII., 1914. 
Yeats, J. 
86 The Growth and Vicissitudes of Commerce. London (Philip and Son) 
(bears no date, ’86 date of latest statistics given). 


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Now, therefore, I, HENRY B. PIERCE, Secretary of the Common- 
wealth of Massachusetts, do hereby certify that said A. Hyatt, W. S. 
Stevens, W. T. Sedgwick, E. G. Gardiner, S. Minns, C. S. Minot, S. 
Wells, W. G. Farlow, A. D. Phillips, and B. H. Van Vleck, their asso- 
ciates and successors, are legally organized and established as, and are 
hereby made, an existing Corporation, under the name of the MARINE: 
BIOLOGICAL LABORATORY, with the powers, rights, and privileges, and 
subject to the limitations, duties, and restrictions, which by law apper- 
tain thereto. 

Witness my official signature hereunto subscribed, and the seal of 
the Commonwealth of Massachusetts hereunto affixed, this twentieth 
day of March, in the year of our LoRD ONE THOUSAND, EIGHT HUN- 
DRED and EIGHTY-EIGHT. ISUINIRAY 1B, JPWIBIRCIE, 

Secretary of the Commonwealth. 

[SEAL. | 


NUL EB WLANWS (Ole Isle, (CORIO RAIN IOI, Ola 
THE MARINE BIOLOGICAL LABORATORY 


I. The annual meeting of the members shall be held on the second 
Tuesday in August, at the Laboratory, in Woods Hole, Mass., at 12 
o’clock noon, in each year, and at such meeting the members shall 
choose by ballot a Treasurer and a Clerk, who shall be, ex officio, 
members of the Board of Trustees, and Trustees as hereinafter pro- 
vided. At the annual meeting to be held in 1897, not more than 
twenty-four Trustees shall be chosen, who shall be divided into four - 
classes, to serve one, two, three, and four years, respectively, and 
thereafter not more than eight Trustees shall be chosen annually for 
the term of four years. These officers shall hold their respective 
offices until others are chosen and qualified in their stead. The Direc- 
tor and Assistant Director, who shall be chosen by the Trustees, shall 
also be Trustees, ex officio. 

Il. Special meetings of the members may be called by the Trustees, 
to be held in Boston or in Woods Hole at such time and place as may 
be designated. 

III. The Clerk shall give notice of meetings of the members by 


338 MARINE BIOLOGICAL LABORATORY. © 


publication in some daily newspaper published in Boston at least 
fifteen days before such meeting, and in case of a special meeting 
the notice shall state the purpose for which it is called. 

IV. Twenty-five members shall constitute a quorum at any meeting. 

V. The Trustees shall have the control and management of the 
affairs of the Corporation; they shall present a report of its condition 
at every annual meeting; they shall elect one of their number Presi- 
dent and may choose such other officers and agents as they may think 
best; they may fix the compensation and define the duties of all the 
officers and agents; and may remove them, or any of them, except 
those chosen by the members, at any time; they may fill vacancies 
occurring in any manner in their own number or in any of the offices. 
They shall from time to time elect members to the Corporation upon 
such terms and conditions as they may think best. 

VI. Meetings of the Trustees shall be called by the President, or 
by any two Trustees, and the Secretary shall give notice thereof by 
written or printed notice sent to each Trustee by mail, postpaid. 
Seven Trustees shall constitute a quorum for the transaction of busi- 
ness. The Board of Trustees shall have power to choose an Execu- 
tive Committee from their own number, and to delegate to such 
Committee such of their own powers as they may deem expedient. 

VII. The President shall annually appoint two Trustees, who shall 
constitute a committee on finance, to examine from time to time the 
books and accounts of the Treasurer, and to audit his accounts at the 
close of the year. No investments of the funds of the Corporation 
shall be made by the Treasurer except approved by the finance com- 
mittee in writing. 

VIII. The consent of every Trustee shall be necessary to dissolu- 
tion of the Marine Biological Laboratory. In case of dissolution, the 
property shall be given to the Boston Society of Natural History, or 
some similar public institution, on such terms as may then be agreed 
upon. 

IX. These By-Laws may be altered at any meeting of the Trustees, 
provided that the notice of such meeting shall state that an alteration 
of the By-Laws will be acted upon. 

X. Any member in good standing may vote at any meeting, either 
in person or by proxy duly executed. 


TREASURER’S REPORT. _ 339 
IV. TREASURER’S REPORT 


CasH RECEIPTS AND DISBURSEMENTS FOR THE YEAR ENDING 
DECEMBER 31, 1914 


RECEIPTS 
Casin @in Ineveral laimeiny i, WOW so sins eee os $ 4,663.05 
PAM UES ewe nnn May te Le ey aa 1,024.00 
EKO OGICA M I WIPTEESTIN: 2 ee a ce. tok 122 Tee 
Dexcet MOUSER nia elses es Gis es ee 4,151.18 
CGlinanlespRes Cramer ee OrGe ee cle 34,200.00 
DONO Sear Hane CMM Re Oy) ee 35.14 
Wornitony stone building. 2.......55.. 424.60 
Dornitony, \Winitaman Cottage... .+.-.- 252.75 
Fish Trap. Be ae gt 1 ee eas 995.24 
MSPUCOMy ONE SONs eis ay so Saas oo ee 750.00 
IMSERUEHOM RemTyOlOgy 2. .4555---.542 - 1,000.00 
Instruction, physiology................ 500.00 
IistquUetionyZOOlOSy ae e562. a 2s 2,100.00 
IWIGSS IE ia rnrse Cee nie trae ch. oa cu bs 12) 7/0). 22 
MKS cella COUSt eae eho cme aves 620.15 
TRESSAINGIOL Fai ss SO aes ene elena le Sr ae 2,950.00 
moupplyadepantiment. 4.8 4.525025 -ss65 5 14,003.35 $81,639.80 
PAYMENTS : 
PNG MIMIINIS CeO ee ecety ole ceciats ee cu sO $ 7,202.56 | 
PIOUOGICAT BOMMETIN. Arle koe oe a a7 7 Oul 
IBYORUES 3.14 4 ilies sete is et ceo Rs ra eee eae aR 5,604.42 
Carpenter SMOPme Gl as ee ee De 288.27 
@hemical department. 9.02... 2... 2,640.20 
IDexitee THIG WIS ay hens Alou cng ye olen eee Sel eee 4,104.87 
Wornimtoryea stone buildings sy. 4.5.4... 2: 204.06 
Dormitory evVviateman) Plouses: 25.5... 148.37 
ID iReCleae:) TE NY: Rte Ra Sane ccna Og PE 1,751.00 
IETIGloy “TETE2 Oy AR at iy eM NR GUS Ante ahr or nS Re eno eR 1,206.41 
as rGUCiOnlbOtanvey gaa. sso). ais 950.00 
imistruetion, emibryology.2....... 5224: 3% 475-00 


ISERUctions pliyslOlogy seh. --- soe. 650.00 


340 MARINE BIOLOGICAL LABORATORY. 


Instruction, zoology:) 7) seeds ees = ae 30.00 
ie CtUnGs spew nti: RE ete etek ee re oe 18.85 
Liban. einen ae CNG CR ane 3,000.88 
Maintenance of buildings and grounds... 4,573.61 
1 Fe EE MER Oia cP on) Lr 17,665.68 
Miscellancousme rete i eere o 1,569.59 
New laboratory (furniture and equipment) 10,263.10 
Bhilosophicalilecturess =) ye ne 100.00 
[EVUNOOy OMANS? SHENTON 5 So ob no doe bake se dasie 3,892.38 
Sip plyad coat cnt ena ae ere D2 LO2O5e75 
Supply department improvements....... 437.48 
(Ozigha Gin Inenacl AMMeINy 1, TORS... 255508. 1,686.68 $81,639.80 


CasH RECEIPTS AND DISBURSEMENTS ON ACCOUNT OF FUNDs, 
AUGUST I, 1914 TO JANUARY I, I9QI5 


RESERVE FUND 


(Caislal' Orn levocl Ae WSIE I, WOM... .25..¢8s65- Si Oey ae 

Div. 14 shs. United Shoe Mach. Corp. Pfd... 5.25 

Div. 6 shs. Am. Smelting & Refining Co. Pid. 21.00 

Divesishs Gene lec. Comer ae) a 16.00 
Interestion depositary tee eer oe .28 $150.30 


LUCRETIA CROCKER FUND 


(Cacia Oia Inevacl AMIDE 1, WOM. +, 6-540ecsece $136.29 
Dive ish WestsemdyoumRy. Conn. 0) yee: 1.75 
Div. 18 shs. Vermont & Mass. Ry. Co...... 54.00 
Div. 2/o she) Gent BlecCon ye eee 5.00 
Diver she AmieeMelycemlicla(Gouaege ca. ee 2.00 
$199.04 
LOTASChOlarslip et Mieewe eee hk ek. eke 100.00 99.04. 


LIBRARY FUND 


Cash on hand August 1, 1014. ....-..25.2.08 $157.79 
Div. 5 shs. United Shoe Mach. Corp. Pfd... 1.87 
Div. 1 sh. Am. Smelting & Refining Co. Pfd. 3.50- 
Divas shs Atm Mel sGcmbela@ouss... ose cre 6.00 
Diy.) 214 shs.’GensHleciCon ys 2. 2 14.1 ee 5.00 174.16 


Cash’ on hattd: January 1, -1o1s4..... 5.1.1.8 eee $423.50 


LIBRARIAN'S REPORT. 341 


HARVEY S. CHASE AND COMPANY 
Certified Public Accountants 
84 State Street 
Boston . 
December 18, 1914. 
D. BLAKELY Hoar, Esq., 
Treasurer, Marine Biological 
Laboratory at Woods Hole, Mass., 
161 Devonshire Street, Boston. 

Dear Sir: We have audited the accounts of the Marine Biological 
Laboratory as kept at Woods Hole, and of the Trust Funds and 
accounts as kept at your office, 161 Devonshire Street, for the year 
ended December 31, 1913. 

We have checked the report of the Treasurer, submitted to us, 
and find it correct and in accordance with the books and accounts of 
the Laboratory. The extent of the audit is set forth in a detailed 
report under date of November 25, 1914. 

Very respectfully, 
Harvey S.. CHASE & Co. 


Cost OF THE NEw LABORATORY, BY CONTRACT, PAID DIRECTLY FROM 
Mr. CRANE’S OFFICE 


Pa aNVillcuteSonsiCombuilderss.....i6e. os. Sa ene $77,810.32 
(Ce 12g (Citoraitinty TEA huiraal oF neg Uh a are rene ens eau anat rene SI BI non 4 9,482.52 
RaiwinwleewiseMOlectmClaman oar sa. s- ose ass ee 3,479.00 
lechorneleatimor Corp Mleating 4.8. te. eth ee: 3,132.55 
OricHlevatorn Com levator. 23.5... sce as ce oot anes 1,485.00 
Simnpsonmnosmecmusce ese e Sle Oe EER Beer 360.00 
Monnoenvetmeerdtor Goss son 5. Wes Me hee. 195.00 
Nclnipe ces COMMMSSOMes Pee cao. cis s Lela oe same tranaeate 4,842.61 

$100,787.00 


Vv. LIBRARIAN'S REPORT 


AUGUST, 1914 
All who have worked in Woods Hole for any length of time 
must appreciate how grateful we are to have the library so well 
cared for; now, in the new fireproof building. It is certainly a 
great relief to fear no longer its sudden destruction. In addition 


342 ® MARINE BIOLOGICAL LABORATORY. 


to this, the librarian may here announce the appointment of an 
assistant librarian to be in charge at the Laboratory throughout 
the year. This has been a pressing need for several years. It 
assures continuity of the work and has made possible many 
important improvements. We were fortunate to secure for this 
position Miss May E. Scott, who started work in April, after 
some weeks devoted to special training. In the meantime Dr. 
Drew supervised the moving and installation of the books in the 
new library. 

The improvements noted below have been accomplished 
through Miss Scott’s skillful management and very great indus- 
try. Unfortunately she has been ill since the middle of June, 
but her work was already so well in hand that it has been possible 
to continue most of it during this season. We must thank Miss 
Elizabeth Dunn for her valuable services in this emergency 
during the summer. Without her generous efforts in behalf of 
Miss Scott and the library we should have been seriously em- 
barrassed. In addition to this we have been much helped by 
having extra assistance for two hours a day for the heavy summer 
routine. 

The entire library has been re-accessioned, and new catalogs 
made. There are over 3,300 volumes exclusive of the reprints, 
which number over 1,500. A modern system has been intro- 
duced throughout, which will make the library much more access- 
ible next year. Many missing parts were secured; and more 
than 500 volumes were bound, necessitating an extra expenditure 
of $500. Several notable additions have been made to the 
library: 

The American Museum of Natural History has loaned us 
indefinitely a number of their duplicate sets and books which 
will be of great assistance. These works are not yet catalogued, 
but there will be over 2,500 volumes, among which is a number 
of useful sets of journals, and of memoirs, transactions, and 
proceedings, of academies, etc., hitherto not on our shelves. 

The Journal of Biological Chemistry was added, the back 
volumes being a gift from the editors. 

Dr. Beyer presented several boxes of books containing among 
other things a duplicate set of the American Journal of Physiology, 


LIBRARIAN'S REPORT. 343 


and a set of the Journal of Medical Research; also Zeitschrift fir 
Hygiene, and the Archiv fiir Schiffs- und Tropenhygiene. 

Dr. Kellicott gave, in addition to his own text-books, about 
100 volumes. 

' Dr. Otto Glaser sent a set of the Proceedings of the Society of 
Experimental Biology and Medicine, with other volumes. 

Dr. Duggar has obtained a lot of much needed pamphlets 
missing from sets. 

Mrs. Edward Gardiner added a number of volumes to her 
former gift. 

Drs. Loeb, Minot, Hegner, McFarland, and Mrs. Agassiz and 
Mr. Crane presented books recently published. 

The book-publishing companies have continued to present new 
books, Blakiston forwarding this week 14 volumes on chemistry, 
etc. 

Stechert has inaugurated a new plan of sending books recently 
issued for examination. This was done at Dr. Oliver Strong’s 
request, and has proved to be a great convenience. 

Dr. H. F. Osborn gave a volume, and has turned over to us 
the premium on sales of one of his books. This will be continued. 

Dr. E. E. Just has offered $5.00 for five years to assist us in 
securing more journals. Dr. Rice has promised $10 per year 
for the same purpose; Dr. E. B. Meigs gave $10.00 also, and the © 
amount already on hand, announced last year for this fund, now 
totals $50.00, which will be expended for certain missing volumes 
of our files. We have acknowledged a number of reprints and 
books, but feel that here is a place where biologists may help 
us still more. A word to others and to publishers may greatly 
aid us. 

The library has now reached a stage of stability. It is well 
housed, well cared for, of great use, and rapidly growing. We 
should be spending at least twice the present amount (that is 
about $1,000 a year more for books); especially for a number of 
journals, now much in demand. There should be a few more 
works of general character. Members of the corporation might 
greatly extend the library with a little effort. The exchange 
list might be further extended as soon as conditions become 
quiet abroad. 


344 MARINE BIOLOGICAL LABORATORY. 


There is little to be added to the material equipment; beyond 
a truck with shelves for shifting volumes, and some suitable 
arrangement, possibly glass gallery platforms, to make the upper 


shelves readily accessible. 
. H. McE. KNOwWER, 


. Librarian. 
Aug. II, 1914. . 


Vir THE DIREGROR’S REPOR® 


To THE TRUSTEES OF THE MARINE BIOLOGICAL LABORATORY: 
Gentlemen: I have the honor to transmit herewith a report of 
the twenty-seventh session of the Marine Biological Laboratory, 
for the year 1914. The number of investigators in attendance 
was 128, as compared with 122 in 1913, 93 in 1912, and 82 in 1911. 
The number of students in the courses was 89 as compared with 
69 in 1913, 67 in 1912, and 65 in 1911. ‘The total attendance 
was 217 as compared with I9I in 1913, 160 in 1912, and 147 in 
1911. The number of subscribing institutions was 40 in 1914, 
as compared with 30 in 1913, 29 in 1912, 25 in I9II, 24 in 1910, 
20 in 1909, 18 in 1908, and 16 in 1907. The list is given on 
p- 356. Ambherst College, Beloit College, Johns Hopkins 
University, Rutgers College, and the University of Wisconsin, 
are among the new subscribing institutions. The total member- 
ship of the Corporation is now 327 as against 303 in 1913. The 
receipts from subscribing institutions and students’ fees were 
$7,300.00 as compared with $6,160.00 in 1913, $5,175.00 in 1912, 
$4,574.99 in I9II, $4,150.00 in 1910, and $3,700.35 in 1909. 
The receipts from the supply department were $14,003.35! in 
1914, as compared with $14,554.90 in 1913, $13,966.35 in 1912, 
$10,303.61 in 1911, $9,300.58 in 1910 and #8,549.55 in 1909. 
The main event of the year was the occupation and dedication . 
of the new permanent laboratory building, the gift of the presi- 
dent of the board of trustees, Mr. Crane. It has satisfied the 
pressing need of space for investigation which has been felt so 
keenly in the past two or three years; it has also provided 
improved facilities so greatly needed for certain types of research; 
and the new library room contained in it has enabled us to 


1 The business transacted in 1914 was actually $1,100 more than in 1913, but 
collections were slow. 


THE DIRECTOR'S REPORT. 345 


undertake a definite policy of library expansion. The building 
proved to be perfectly adapted for the purposes for which it 
was erected. 

The exercises in dedication of the new building held on July 10 
‘were attended by representatives of universities, members of the 
staff, the investigators and students working at the Laboratory, 
and by many friends. The buildings were open to inspection in 
the morning, and demonstrations of sea-animals and of research 
in progress were made, and the laboratory steamer made a 
collecting trip. Lunch was served to the invited guests. The 
formal exercises were held at 2 P.M., in a tent erected for the 
occasion, with an attendance of about 800. The addresses were 
interspersed with music by the Russian Balalaika Orchestra. 

Mr. C. R. Crane, president of the Board of Trustees, and 
donor of the building presided. In opening the exercises, he said: 

“T think we have come here particularly to celebrate the 
wonderful spirit that is back of the Woods Hole Biological 
Laboratory. It is very difficult to define that spirit, but I think 
we all know something of it and something is also known all 
through the scientific world. Without that spirit no amount of 
bricks and mortar and organization would be of any great 
service, but with that spirit the laboratory has been able to 
accomplish a very great deal with very simple means. 

“For some time back it has seemed to be worth while to give 
this spirit a more substantial body. This spirit, as I see it, is 
very much like the spirit that President Wilson speaks so much 
of, the spirit of freedom and of codperation, the fundamental 
spirit of democracy. In giving this spirit a more substantial 
body, we have been very fortunate in having with us Dr. Drew. 
I think we are all very happy at the wonderful result of his year’s 
work. There is a rumor in circulation around here that Dr 
Drew is a zoologist. I believe that rumor has spread into the 
outside world, but I am very certain that we must all feel, after 
looking over the new laboratory, that Dr. Drew would have made 
his reputation as an engineer if he had a chance.” 

Short speeches were made by the Director, by Professor 
Conklin, and by the head of the U. S. Bureau of Fisheries, the 
Hon. Hugh M. Smith. Dr. R. M. Woodward, the Director of 


346 MARINE BIOLOGICAL LABORATORY. 


the Carnegie Institution of Washington, then delivered the main 
address on the Needs of Research. These addresses were 
published in full in Science.! 

Extra provision was made this summer for the expected large 
attendance, by the lease of the Dexter House, which was run 
by the laboratory, and was made to pay expenses, including rent. 
With this increase the mess was able to accommodate the 
students and investigators comfortably. The Dexter House is in 
bad repair, and no lasting arrangement with the owner seems 
possible. We have therefore been casting about for other 
arrangements. The ‘‘Homestead’’ used for the help of the mess 
has long been overcrowded, and for some time we have regarded 
it as unsafe. Mr. Crane therefore offered to build on the home- 
stead site a new dwelling house with accommodations for about 
forty people, which will be 10 to 12 in excess of the number 
employed in the Mess, and will therefore furnish some available 
space for women of the laboratory. This work is already nearly 
finished. Mr. Crane has also presented this autumn (1914) 
funds for improvement of the Cayadetta and for other 
purposes including the completion of the stone wall on the 
Yacht Club frontage and filling in behind it. This work is far 
advanced; when it is finished the building will be moved from 
in front of the new laboratory to the east end of the lot. 

We shall thus begin the new year with most of our material 
needs satisfied to an extent that will probably be adequate for 
several years. The estimate of running expenses for 1915 shows 
a deficit of $20,000 above receipts, as in 1914. Mr. Crane has 
again most generously promised his support to this extent. 
This brings up again the need of an endowment, which I think 
we should keep constantly before us until attained. The 
flourishing condition of the Laboratory constitutes a strong 
argument for its endowment; the Laboratory represents no new 
experiment, but a demonstrated success, and the fulfilment of 
one of the greatest needs of American biology. The codperation 
of forty American universities and the attendance of representa- 
tives of thirty-seven more proves that we are supplying a want 
that is felt by all the institutions of higher learning. The 

1Vol. XL., No. 1024, pp. 217-232. 


THE DIRECTOR’S REPORT. 347 


attendance of the largest body of scientific investigators ever 
gathered for work at one place and time also proves the magnitude 
of the want that we supply. Not only so but it demonstrates 
the great influence over the progress of research which the Marine 
Biological Laboratory exerts. We can feel justified in using all 
the means in our power to secure the funds that will place this 
great organization beyond the stress of ordinary vicissitudes. 

Provision has been made for continuation of the students’ 
courses as in 1914, with one exception. Dr. Drew has felt for 
some years that the burden of executive work during the summer 
is so great that it is undesirable for him longer to retain charge 
of the course in embryology. The directors have therefore 
requested Professor Wm. E. Kellicott, who has been associated 
with Dr. Drew in this course for several years, to assume charge 
of it. He has consented, and we may feel confident that he will 
maintain its best traditions. We must all feel nevertheless a 
sense of loss in the relinquishment by Dr. Drew of this important 
course. For many years in charge of the course in invertebrate 
zoology, and then of the course in embryology, Dr. Drew has 
impressed the lessons of our science on students to an extent 
which few teachers can equal. I am sure that all will join in 
congratulating Dr. Drew on his great success as a teacher, and 
in the hope that the future will yield him more leisure again to 
resume this cherished part of a scientist’s work. 

There are submitted as parts of this report lists of the staff of 
I914, investigators and students in attendance, subscribing 
institutions, evening lectures and of members of the corporation. 


te IGS Si Vals 
1914 


FRANK R. LILLIE, Director, 


Professor of Embryology, and Chairman of the Department of 
Zoblogy, The University of Chicago. 


GILMAN A. DREW, Assistant DIRECTOR, 


Marine Biological Laboratory. 


348 MARINE BIOLOGICAL LABORATORY. 


ZOOLOGY 


I. INVESTIGATION 


GARWaIN | @ATIKGINGE eee Professor of Protozodlogy, Columbia Uni- 

: versity. 

E. G. ConKLIN.:.........Professor of Zodlogy, Princeton Univer- 
sity. i 

GIL MAND AS DREW nee Assistant Director, Marine Biological 
Laboratory. 

GEORGE LEFEVRE.......... Professor of Zodlogy, The University of 
Missouri. ; 

FRANK R. LILLIE.......... Professor of Embryology, The University 
of Chicago. 

(CB IMMCGie OH a6 oe b og ek Professor of Zodlogy, University of 
Pennsylvania. 

Aiello ORIG AINE aries ere Professor of Experimental Zodlogy, Co- 
lumbia University. 

1D Bian WIMESOINS 4 30 Gols oa a Professor of Zodlogy, Columbia Univer- 
sity. 


II. INSTRUCTION 


(CASIDIA, (Greys 5 ooo po bes Associate Professor of Zodlogy, Johns 
Hopkins University. 
WHC WALT EE | ite euesenate Instructor in Biology, Williams College. 
GrEorGE A. BAITSELL......Fellow in Zodlogy, Yale University. 
RAYMOND BINFORD........ Professor of Biology, Earlham College. 
EV GeUND aa meniae ay ee Bruce Fellow in Zoélogy, Johns Hopkins 
University. 
WSs RAINT ER A earl Dees Graduate Student, University of Wiirz- 
burg. 
EMBRYOLOGY 


I. INVESTIGATION (See Zodélogy) 


II. INSTRUCTION 


GimmAny Ac DREW «2 an aoe Assistant Director, Marine Biological 
Laboratory. g 

LoranDE L. WoopruFF....Assistant Professor of Biology, Yale 
University. 

WituiaM E. KELLIcoTT.... Professor of Biology, Goucher College. 


RoBerT A. BUDINGTON..... Professor of Zoédlogy, Oberlin College. 


THE 


EDWARD B. MEIGS...... 


DIRECTOR’S REPORT. 349 


PHYSIOLOGY 


I. INVESTIGATION 


.. Professor of Physiological Chemistry, 
The University of Chicago. 

.. Professor of Biology, Clark University. 

.. Assistant Professor of Physiological 
Chemistry, University of Wisconsin. 


II. INSTRUCTION 


.. Professor of Biology, Clark University. 
..Associate Professor of Physiology, Wash- 
ington University Medical School. 
..Professor of Physiology, Syracuse Uni- 

: versity. 
.. Associate in Physiology, Wistar Institute 
of Anatomy and Biology. 


PHILOSOPHICAL ASPECTS OF BIOLOGY AND ALLIED SCIENCES 


EDWARD G. SPAULDING... 


GEORGE. VIOORE. 4. 


GEORGE R. LYMAN...... 


Bee View DinG GAR oe 


IAIN? 19, ILIBWWAIS so 6c oh oo 6 
WE eINOBBINS nucle 


PABLO AAS. 2 cele Tine 


H. McE. KNower....... 


NAR Vay COM: 751. a. 


LECTURES 


.. Assistant Professor of Philosophy, Prince- 
ton University. 


BOTANY 


.. Director, Missouri Botanical Garden and 
Professor of Botany, Washington Uni- 
versity. 

..Assistant Professor of Botany, Dart- 
mouth College. 

.. Physiologist, Missouri Botanical Garden 
and Professor of Plant Physiology, 
Washington University. 

.. Assistant Professor of Botany, University 
of Wisconsin. 

.. Instructor in Plant Physiology, Cornell 
University. 

.. Lackland Research Fellow, Shaw School 
of Botany. 


LIBRARY 


.Professor of Anatomy, University of 
Cincinnati, Librarian. 
.. Assistant Librarian. 


350 MARINE BIOLOGICAL LABORATORY. 


CHEMICAL SUPPLIES 


OLIVER) Si STRONG... 2 eee Instructor in Anatomy, College of Phy- 
sicians and Surgeons, New York City, 
Chemist. 


SUPPLY DEPARTMENT 


Ge INIEAG RAW Pe 8 oo ccna Curator. 

Joun J. VEEDER.........: Captain. 

FNS WHS aed: eect Smee we Engineer. 

OnE) CURTIS) 3 Gio ke eee Collector and Assistant Curator of Botan- 
ical Supplies. 

A WiLL EATHERS 4). eae. Collector. 

ARV EIMERONG: 2 cake ere Collector. 

IBID Is WWBILIUS) 6 oo 5 oo 05 oo CGF 

F. M. MacNauecut........ Business Assistant. 


2. INVESTIGATORS AND STUDENTS 


INVESTIGATORS 


1914 


ZOOLOGY 


Independent Investigators 


ALLEE, W. C., Assistant Professorin Zodlogy, University of Oklahoma. 
BAITSELL, GEORGE A., Instructor in Biology, Yale University. 

Bcc, A. S., Instructor in Comparative Anatomy, Harvard Medical School. 
BINFORD, RAYMOND, Professor of Zodlogy, Earlham College, Richmond, Ind. 
BROWNE, ETHEL N., 510 Park Ave., Baltimore, Md. 

BUDINGTON, ROBERT A., Professor of Zodlogy, Oberlin College. 

CALKINS, Gary N., Professor of Protozodlogy, Columbia University. 
CHIDESTER, FLoyp E., Assistant Professor of Zodlogy, Rutgers College. 
CHILD, C. M., Associate Professor of Zoédlogy, University of Chicago. 

CLapp, CORNELIA M., Professor of Zodlogy, Mount Holyoke College. 
CLARK, ELioT R., Professor of Anatomy, University of Missouri. 

CLARK, ELEANOR L., Johns Hopkins Medical School. 

Cor, WESLEY R., Professor of Biology, Yale University. 

ConkKLIN, E. G., Professor of Biology, Princeton University. 

Cownpry, EDMUND V., Associate in Anatomy, Johns Hopkins Medical School. 
Cowpry, N. H., Johns Hopkins Medical School. 

Crampton, H. E., Professor of Zodlogy, Barnard College, Columbia Univ. 


THE DIRECTOR’S REPORT. 351 


DANCHAKOFF, WERA, Director of laboratory for medical research, Moscow, Russia. 

DoL.LeEy, DaAvip H., Professor of Pathology, University of Missouri. 

Dona.pson, H. H., Wistar Institute of Anatomy and Biology. 

Drew, Gitman A., Assistant Director, Marine Biological Laboratory. 

DuNN, ELIZABETH H., Woods Hole, Mass. 

ERDMANN, RuopA, Theresa Leesell Research Fellow, Yale University. 

GOLDFARB, A. J., Professor of Biology, College of the City of New York. 

GRAVE, CASWELL, Associate Professor of Zodlogy, Johns Hopkins University. 

HEGNER, ROBERT W., Assistant Professor of Zoélogy, University of Michigan. 

HocGueE, Mary J., Instructor in Zodlogy, Wellesley College. 

JACKSON, FREDERIC S., Lecturer in Histology and Embryology, McGill Univ. 

JoRDAN, Harvey E., Professor of Histology and Embryology, University of 
Virginia. 

KeE.iicott, W. E., Professor of Biology, Goucher College. 

KINGSBURY, FRANCIS B., Instructor, University of Minnesota. 

KNOWER, HENRY MCE., Professor of Anatomy, University of Cincinnati. 

KUNKEL, BEVERLY W., Professor of Zodlogy, Beloit College. 

LEFEVRE, GEORGE, Professor of Zodlogy, University of Missouri. 

LEwis, MARGARET R., Johns Hopkins Medical School. 

LEWIS, WARREN H., Professor of Physiological Anatomy, Johns Hopkins Medical 
School. 

LILLIE, FRANK R., Professor of Embryology, University of Chicago. 

Loomis, FREDERIC B., Professor of Comparative Anatomy, Amherst College. 

Lunp, E. J., Instructor in Protozoélogy, University of Pennsylvania. 

MacDowE tt, E. C., Instructor, Yale University. 

MALONE, E. F., Assistant Professor of Anatomy, University of Cincinnati. 

McC.iunge, C. E., Director of the Zodlogical Laboratory, University of Pennsyl- 
vania. 

Morean, T. H., Professor of Experimental Zodlogy, Columbia University. 

Newman, H. H., Associate Professor of Zodlogy, University of Chicago. 

NOwLtin, NADINE, Assistant Professor of Zodlogy, University of Kansas. 

PAINTER, T. S., Instructor, Yale University. 

PATTERSON, J. T., Professor of Zoédlogy, University of Texas. 

PEARL, RAYMOND, Biologist of Maine Agricultural Experiment Station. 

PEEBLES, FLORENCE, Lecturer, Bryn Mawr College. 

PINNEY, Mary E., Demonstrator in Biology, Bryn Mawr College. 

RicHArps, A., Instructor in Zodlogy, University of Texas. 

SHOREY, MARIAN L., Professor of Biology, Milwaukee-Downer College. 

SPAULDING, E. G., Professor of Philosophy, Princeton University. 

STOCKARD, CHARLES R., Professor of Anatomy, Cornell Medical College. 

STRONG, O. S., Instructor in Anatomy, Columbia University. 

STRONG, R. M., Instructor in Zoélogy, University of Chicago. 

TENNENT, Davip H., Professor of Biology, Bryn Mawr College. 

WIEMAN, H. L., Assistant Professor of Zoédlogy, University of Cincinnati. 

WILpMAN, E. E., University of Pennsylvania. 

Witson, E. B., Professor of Zodlogy, Columbia University. 

WooprurfF, L. L. Assistant Professor of Biology, Yale University. 

ZELENY, CHARLES, Associate Professor of Zodlogy, University of Illinois. 


352 MARINE BIOLOGICAL LABORATORY. 


Beginning Investigators 


ADKINS, W. S., Graduate Student, Columbia University. 

ALLEN, EzrA, Professor of Biology at Philadelphia School of Pedagogy, University 
of Pennsylvania. 

BRIDGES, CALVIN B., Fellow in Zodlogy, Columbia University. 

BULLOCK, FREDERICK D., Instructor in Cancer Research, Columbia University. 

CAROTHERS, E. ELEANOR, University of Pennsylvania. 

Cops, MARGARET V., University of Illinois. 

DEXTER, JOHN S., Graduate Student, Columbia University. 

FIELD, HAZEL E., Graduate Student, University of Chicago. 

GoopricH, H. B., Fellow in Zodlogy, Princeton University. 

GOULD, HaArRLeEy N., Fellow in Biology, Princeton University. 

GUNTHER, MAUDE C., Instructor in Biology, Eastern High School, Washington, 
1D), Ce 

HEILBRUNN, L. V., Assistant in Zodlogy, University of Chicago. 

HocGe, Mitprep A., Graduate Student, Columbia University. 

Hoy, WILLIAM E., JR., Fellow in Biology, Princeton University. 

Key, J. A., Student, Johns Hopkins Medical School. 

LARRABEE, AUSTIN P., Professor of Biology, Fairmount College. 

LEATHERS, A. L., Instructor in Zodlogy, Northwestern University. 

MEDES, GRACE, Graduate Student, Bryn Mawr College. 

Metz, CHAs. W., Carnegie Institution Staff, Cold Spring Harbor, L. I. 

Moore, Cart R., Fellow in Zoédlogy, University of Chicago. 

Morris, MARGARET, Osborne Zoélogical Laboratory, Yale University. 

OKKELBERG, PETER, Instructor in Zodlogy, University of Michigan. 

PACKARD, CHARLES, Instructor in Zodlogy, Columbia, University. 

SHUMWAY, WALDO, Assistant in Zodlogy, Columbia University. 

STARK, Mary B., Graduate Student, Columbia University. 

_ STOCKING, RuTH J., Student, Johns Hopkins University. 

STURTEVANT, A. H., Graduate Student, Columbia University. 

WEINSTEIN, ALEXANDER, University Scholar in Zodlogy, Columbia University. 

WoOoDWARD, ALVALYN E., Fellow in Zoédlogy, University of Michigan. 

Yocum, H. B., Instructor in Zodlogy, Kansas State Agricultural College. 

YOUNG, DONNELL B., Laboratory Assistant, Columbia University. 


PHYSIOLOGY 


Independent Investigators 


BRADLEY, H. C., Associate Professor of Physiological Chemistry, University of 
Wisconsin. 

CHAMBERS, ROBERT, JR., Assistant Professor of Histology and Comparative 
Anatomy, University of Cincinnati. 

GARREY, W. E., Associate Professor of Physiology, Washington University Medical 

School. 

Harvey, E. N., Instructor in Physiology, Princeton University. 

Hype, IpA H., Professor of Physiology, University of Kansas. 

Just, E. E., Professor of Physiology, Howard University. 

KANDA, SAKYO, Research Assistant, University of Minnesota. 

KITE, GEORGE D., Assistant in Physiology, Henry Phipps Institute. 

KNOWLTON, FRANK P., Professor of Physiology, Syracuse University, College of 
Medicine. 


it, 


THE DIRECTOR’S REPORT. Se 


LILLIE, R. S., Professor of Biology, Clark University. 

LorEsB, JACQUES, Head of Department of Experimental Biology, Rockefeller In- 
stitute for Medical Research. 

MatHews, A. P., Professor of Physiological Chemistry, University of Chicago. 

MattTILL, HENrRy A., Professor of Physiological Chemistry, University of Utah. 

Meics, Epwarp B., Fellow in Physiology, Wistar Institute of Anatomy and 
Biology. 

Moore, A. R., Associate Professor of Physiology, Bryn Mawr College. 

OLIVER, WADE W., Student, Ohio-Miami Medical College. 

TASHIRO, SHIRO, Instructor in Physiological Chemistry, University of Chicago. 

UHLENHUTH, EDUARD, Rockefeller Institute for Medical Research. 

WARREN, HowarbD C., Stuart Professor of Psychology, Princeton University. 

WASTENEYS, HARDOLPH, Associate in Experimental Biology, Rockefeller Institute 
for Medical Research. 

WERBER, ERNEST I., Instructor in Anatomy, Northwestern University. 

WHeERRY, W. B., Associate Professor of Bacteriology, University of Cincinnati. 


Beginning Investigators 


HyMaAn, LisBiE H., Laboratory Assistant in Zodlogy, University of Chicago. 


BOTANY 
Independent Investigators 

BLAKESLEE, A. F., Professor of Botany and Genetics, Connecticut Agricultural 
College. 

Co.iey, R. H., Instructor in Botany, Dartmouth College. 

Davis, A. R., Lackland Research Fellow, Shaw School of Botany, Washington 
University. 

DuGear, B. M., Physiologist in charge of Graduate Laboratory and Professor of 
Plant Physiology, Missouri Botanical Garden. 

GATES, R. R., University of London, England. 

Lewis, I. F., Professor of Botany, University of Missouri. 

LYMAN, GEORGE R., Assistant Professor of Botany, Dartmouth College. 

Moore, GEo. T., Director, Missouri Botanical Garden. 

RUMBOLD, CAROLINE, 3824 Locust St., West Philadelphia, Pa. 

STOKEY, ALMA G., Associate Professor of Botany, Mount Holyoke College. 


Beginning Investigators 


STEWART, Mary W., Assistant in Botany, Barnard College, Columbia Univesity. 
SWEATMAN, ELIZABETH A., 3032 Parkwood Ave., Toledo, Ohio. 
WUIST, ELIZABETH D., Instructor in Botany, Milwaukee State Normal School. 


SO OIDIE IES) 


1914 


ZOOLOGY 
ALLEN, WILLIAM Ray, Assistant in Kansas State Agricultural College, Manhattan. 
Kans. 
APPLEGATE, ANNE G., Student, Western College, Oxford, Ohio. 


354 MARINE BIOLOGICAL LABORATORY. 


APPLEGATE, ELEANOR, Western College, Oxford, Ohio. 

ASHMAN, RICHARD, Student, Rutgers College, New Brunswick, N. J. 
BALitou, Marion M., Mount Holyoke College. 

BATCHELDER, CHARLES H., Assistant, New Hampshire College, Durham, N. H. 
BEHRE, CHARLES H., Jr., Student, New Orleans High School. 
CARROLL, MITCHELL, 617 South 16th St., Philadelphia. 

CuHILps, HENRY E., Student, University of Rochester. 

CLARK, KATHARINE E., Assistant in Zodlogy, Mt. Holyoke College. 
CLosson, J. HARWoopD, 53 W. Chelten Ave., Germantown, Pa. 

CoHN, EpwIN T., University of Chicago. 

CONNET, HELENE, Student, Goucher College, Baltimore. 

DICKINSON, CLAIRE, Teacher in elementary schools, New York University. 
ELLIOTT, MARGUERITE, Vassar College. 

GREENE, PHILLIPS F., Biology Laboratory Assistant, Amherst College. 
GREENE, WALTER F., Amherst College. 

HALSTED, MARGIE H., Instructor in Biology, West High School, Rochester, N. Y; 
HAMILTON, FLORENCE N., Student, Vassar College. 

JACKSON, FRANCES E., Student, Mt. Holyoke College. 

JANNEY, Marion, Goucher College. 

KELLOGG, EMILIE, Student, Mt. Holyoke College. 

LaporFr, SontA, Assistant in Biology, State Normal School, La Crosse, Wis. 
LANCASTER, DEWItT B., College of Charleston, S. C. | 
LoEB, ROBERT F., Student, University of Chicago. 

LOovETT, J. ELIZABETH, Student, Goucher College. 

MANNHARDT, L. ALFRED, Student, Yale University. 

McWILLIAMS, MINNIE R., Student, University of Chicago. 
MONTGOMERY, PRISCILLA B., 105 S. 41st St., Philadelphia. 

Moses, BEssiE L., Student, Goucher College. 

MULLIKIN, JEANNETTE, Vassar College. 

PAPPENHEIMER, BEATRICE L., 309 W. 99th St., New York City. 
PLACE, JESSE A., Instructor in Biology, Ohio University, Athens, Ohio. 
REDFIELD, ELIZABETH S., Student, Radcliffe College. 

RONES, MARGUERITE T., Teacher in elementary schools, Boston. 
Rusu, J. E., Assistant Professor, Wisconsin. 

SCHMOLL, HAzeEL M., Assistant in Biology, Vassar College. 

STOCKING, BESSE E., Student, Goucher College. 

STRONGMAN, BeEssiE T., Student, University of Colorado. 

THOMAS, ANNA M., Student, Carnegie Institute of Beene 
TIESING, PAUL E., Student, Yale University. 

WARREN, CATHERINE C., Princeton, N. J. 

WAYMAN, MARGUERITE, Student, Hunter College, New York City. 


EMBRYOLOGY 


ALLEN, CHARLES E., Assistant Instructor, Wabash College. 

BALDWIN, FRANCIS M., Instructor in Biology, Western Maryland College. 

BINKLEY, LELIA T., University of Texas. 

CHAMBERLAIN, MAry M., Student, Bryn Mawr College. 

DAvis, CARL L., Professor of Anatomy, George Washington University, Washing- 
ton, D. C. 

DIEHL, JANE K., Student, Wellesley College. 


THE DIRECTOR’S REPORT. 355 


GOVER, Mary, Goucher College, Baltimore, Md. 

Hess, WALTER N., Instructor, Pennsylvania State College, State College. 

HunrtTER, OSCAR B., Professor of Histology and Embryology, Associate in Anatomy, 
George Washington Univers ty, Washington, D. C. 

Mann, Mary LEE, Barnard College. 

McFARLAND, HELEN J., Student, Bryn Mawr College. 

MILLS, FRANCES A., Barnard College. 

NELSON, THURLOW C., Assistant in Zodlogy, University of Wisconsin. 

OGDEN, WARNER, Student, Carleton College, Northfield, Minn. 

PLouGH, HAROLD H., Assistant in Zodlogy, Amherst College. 

Potter, Bess, Student, Doane College, Crete, Neb. 

PREBLE, JESSIE L., Student, Bryn Mawr College. 

Tart, ANNIE E., Curator Dept. of Neuropathology, Harvard Medical School. 

WALTON, ARTHUR C., Student Assistant, Northwestern University. 

WarRE, CLARA C., Graduate Student, Columbia University. 

Younc, Tuomas O., Student, Carleton College, Northfield, Minn. 


PHYSIOLOGY 


ATWOOD, WARREN G., Student, Dartmouth College. 

BENJAMIN, BLANCHE M., Student, Radcliffe College. 

FREDERICK, NorA, Teacher in Biology, Lewis Institute, Chicago. 

HyMAN, LIBBIE H., Laboratory Assistant in Zodlogy, University of Chicago. 
LINTON, EDWIN S., Graduate Student, Washington and Jefferson College. 
LYNCH, VERNON, Graduate Student, Johns Hopkins University. 

‘OLIVER, SYMMES F., University of Michigan, Ann Arbor. 

REDFIELD, ALFRED C., Assistant in Zodlogy, Harvard University. 
VANNEMAN, AIMEE S., Vassar College. 

WALLING, LALIA V., Instructor in Physiology, University of Kansas. 


BOTANY 


ALLARD, ANNE D., Teacher in Boston Normal School, Boston. 

BuRKS, GEORGE PAUL, Student, Wabash College. 

CARROLL, FRANKLIN B., 617 South 16th St., Philadelphia. 

Coss, RutTuH, Smith College. : 

FINE, SOLOMON, Student, Rhode Island State College, Kingston, R. I. 

FORBES, WILLIAM T. M., 23 Trowbridge Road, Worcester, Mass. 

Fritz, CLARA W., McGill University. 

GREEN, NEWTON B., Student, Oberlin College. 

HERRICK, JOSEPH C., Professor of Biology, St. Joseph’s Seminary, Yonkers, N. VY. 
MALLARD, AGNES K., Teacher, Boston Normal School, Boston. 

McLAUGHLIN, FREDERICK A., Instructor in Botany, Mass. Agricultural College. 
SEVERY, J. WARREN, Student, Oberlin College. 

STEARNS, FRANCES L., Teacher, Central High School, Grand Rapids, Mich. 
WANN, F. B., Missouri Botanical Garden, St. Louis, Mo. 

WILLEY, ALBERT G., Student, Dartmouth College. 


356 MARINE BIOLOGICAL LABORATORY. 


3. TABULAR VIEW OF ATTENDANCE 


IQIT 
EN VESEIGATORS— slice eastern ee 82 
Independent: 
LOLOL Yee fA Se eRe gen ee 42 
Physiology. 2158 namie pameint cr 18 
Botansyie. Yasir OR Oe miei pte 8 
Under Instruction: 
LOONOBY Seo ais Cee mo ee 12 
PV SIOLOY, csi os Oy eran en elena erg 2 
Botatty (G6 sos Sine Angee ween 
STUDENTS — TI otal... eae eee ce cer 65. 
ZOOLOGY 79 SE SANE A Coe AE 26 
Embryology ie a0. eee eee rie 20 
Phiysi@loewe.iie is ey crue ue near aa 6 
Botany sich Se aan Aen ere es 1 
LOTAT AGEN DANG hirer Mei ea 147 
INSTITUTIONS REPRESENTED—Total.... 
Byaitiviesti@ators ane ni: ee err ek 87, 
Bayes tiem tS,0 Oa See ae iene epee gee 31 
SCHOOLS AND ACADEMIES REPRESENTED 
Byatlvestigatonsre taal seer ee 8 
By Studentctrn sree ee cen ea os 9 


I9gi2 


93 
44 
14 


IO 


21 


17 


160 


1913 
122 


58 
17 
iil 


4. SUBSCRIBING INSTITUTIONS 


AMHERST COLLEGE. 
BARNARD COLLEGE. 
BELOIT COLLEGE. 
Bryn MAwr COLLEGE. 
CARLTON COLLEGE. 
COLUMBIA UNIVERSITY. 


CARNEGIE INSTITUTE OF TECHNOLOGY. 


DARTMOUTH COLLEGE. 
DOANE COLLEGE. 

GOUCHER COLLEGE. 
HARVARD UNIVERSITY. 
HARVARD MEDICAL SCHOOL. 
HUNTER CormEce, N. Y. C. 


IQI4 
128 


217 


b) 
THE DIRECTOR S REPORT. 357 


KANSAS STATE AGRICULTURAL COLLEGE. 
Jouns Hopkins UNIVERSITY. 
LUCRETIA CROCKER SCHOLARSHIPS. 
McGiL_L UNIVERSITY. 

Mount HoLyoKE COLLEGE. 
NORTHWESTERN UNIVERSITY. 
OBERLIN COLLEGE. 

PRINCETON UNIVERSITY. 
RADCLIFFE COLLEGE. 

RHODE ISLAND STATE COLLEGE. 
ROCHESTER UNIVERSITY. 
ROCKEFELLER INST. FOR MED. RESEARCH. 
SMITH COLLEGE. 

RUTGERS COLLEGE. 

UNIVERSITY OF CHICAGO. 
UNIVERSITY OF CINCINNATI. 
UNIVERSITY OF ILLINOIS. 
UNIVERSITY OF KANSAS. 
UNIVERSITY OF MICHIGAN. 
UNIVERSITY OF PENNSYLVANIA. 
UNIVERSITY OF WISCONSIN. 
VASSAR COLLEGE. 

WELLESLEY COLLEGE. 

WESTERN COLLEGE. 

WISTAR INSTITUTE. 

WABASH COLLEGE. 

YALE UNIVERSITY. 


5. EVENING LECTURES, 10914 


Friday, July 3, 
BRO la VIORG AN fase, «0: ‘‘Chromosomes and Mendelian 
Heredity.” 
Tuesday, July 7, 
Dre BPRwin b. SMInEH oo... .. “Crown Gall in Plants, with 
Reference to the Nature and 
Origin of Cancer.” 
Tuesday, July 14, 
Dis (Ci ae) Gia oe ae “The Colloidal Structure of Living 
Matter as Determined by Micro- 
dissection.” 


358 


Friday, July 17, 
Dr. LAWRENCE J. HENDERSON.. 


Tuesday, July 21, 

Pror. D. H. TENNENT........ 
Friday, July 24, 

Prom, Jal, Co VWARIRIBIN. oc coo coe 
Tuesday, July 28, 

Dr. FRANK M. CHAPMAN..... 


Friday, July 31, 
Dr. RAYMOND PEARL........ 


Tuesday, Aug. 4, 
Dr. ALFRED G. MAYER....... 
Friday, Aug. 7, 
Dr. Epwin LINTON 


Friday, Aug. 14, 
Dr. R. R. GATEs 


MARINE BIOLOGICAL LABORATORY. 


“The Functions of the Environ- 
ment.” 


“Hybridization in Sea-urchins.” 


‘Freedom of Teaching in Amer- 
ican Colleges.” 


.‘An Ornithological Expedition to 


Colombia.” 


.““The Physiology of Reproduction 


in the Domestic Fowl.” 


‘““The Coral Reefs of Torres 
Straits.”’ 
“Reminiscences of the Woods 


Hole Laboratory Oi ule Ws Sr 
Fish Commission, 1882-1889.” 


“Recent Aspects of Mutation.” 


6. MEMBERS OF THE CORPORATION. 


Ic 


LIFE MEMBERS 


Auuis, Mr. E. P., Jr., Palais Carnoles, Menton, France. 
ANDREWS, Mrs. GWENDOLEN FOULKE, Baltimore, Md. 
BILLInGs, MR. R. C., 66 Franklin St., Boston, Mass. 

Carey, Mr. ArtHur Astor, Fayerweather St., Boston, Mass. 
CLARKE, Pror. S. F., Williams College, Williamstown, Mass. 
CONKLIN, PRroF. EDWIN G., Princeton University, Princeton, N. J. 


CRANE, Mr. C. R., Woods Hole, 


Mass. 


Davis, Major HEnry M., Syracuse, N. Y. 

Evans, Mrs. GLENDOWER, 12 Otis Place, Boston, Mass. 
FaRLOw, Pror. W. G., Harvard University, Cambridge, Mass. 
Fay, Miss S. B., 88 Mt. Vernon St., Boston, Mass. 

Foitsom, Miss Amy, 88 Marlboro St., Boston, Mass. 


THE DIRECTOR’S REPORT. 359 


Foot, Miss KATHERINE, 80 Madison Ave., New York City, N. Y. 

GARDINER, Mrs. E. G., Woods Hole, Mass. 

GARDINER, Miss EUGENTA, 15 W. Cedar St., Boston, Mass. 

HANNAMAN, Mr. Cuar Es E., 103 Ist St., Troy, N. Y. 

HARRISON, Ex-Provost C. C., University of Pennsylvania, 
Philadelphia, Pa. 

Jackson, Miss M. C., 88 Marlboro St., Boston, Mass. 

Jackson, Mr. Cuas. C., 24 Congress St., Boston, Mass. 

KENNEDY, Mr. GEo. G., 284 Warren St., Roxbury, Mass. 

Kipper, Mr. C. G., 27 William St., New York City, N. Y. 

KIppER, Mr. NATHANIEL T., Milton, Mass. 

Kinc, Mr. Cuas. A., 

LEE, Mrs. FREDERIC S., 279 Madison Ave., New York City, N. Y. 

LowELL, Mr. A. LAWRENCE, 171 Marlboro St., Boston, Mass. 

Marrs, Mrs. LAurRA Norcross, 9 Commonwealth Ave., Boston, 
Mass. 

Mason, Mr. E. F., 1 Walnut St., Boston, Mass. 

Mason, Miss Ipa M., 1 Walnut St., Boston, Mass. 

Means, Mr. JAMES HOWARD, 196 Beacon St., Boston, Mass. 

MERRIMAN, Mrs. DANIEL, Worcester, Mass. 

Minns, Miss Susan, 14 Louisburg Square, Boston, Mass. 

Minns, Mr. Tuomas, 14 Louisburg Square, Boston, Mass. 

MrxtTer, Miss M. C., 241 Marlboro St., Boston, Mass. 

Morcan, Mr. J. PIERPONT, JR., Wall and Broad Sts., New York 
City NEY 

Morean, Pror. T. H., Columbia University, New York City, 
INE SY 

MoRcANS Wins iE New York City, N.Y: 

Noyes, Miss Eva J., 28 South Willow St., Montclair, N. J. 

Nunn, Mr. LuctAn L., Telluride, Colo. 

OsBorN, Pror. HENRY F., American Museum of Natural History, 
New York. 

PHILLiIes, Dr. JOHN C., Windy Knob, Newham, Mass. 

Puitiies, Mrs. JoHN C., Windy Knob, Newham, Mass. 

Porter, Dr. H. C., University of Pennsylvania, Philadelphia, Pa. 

PuLsIFER, Mr. W. H., Newton Center, Mass. 

Rocers, Miss A. P., 5 Joy St., Boston, Mass. 

SEARS, Dr. HENRY F., 420 Beacon St., Boston, Mass. 


360 MARINE BIOLOGICAL LABORATORY. 


SHEDD, Mr. E. A., 

SmitH, Mrs. C. C., 286 Marlboro St., Boston, Mass. 

STROBELL, Miss E. C., 80 Madison Ave., New York City, N. Y. 

THORNDIKE, Dr. Epwarp L., Teachers College, Columbia 
University, New York City, N. Y. 

TRELEASE, ProFr. WILLIAM, University of Illinois, Champaign, II]. 

Ware, Miss Mary L., 41 Brimmer St., Boston, Mass. 

Warren, Mrs. S. D., 67 Mt. Vernon St., Boston, Mass. 

Wuitney, Mr. Henry M., Brookline, Mass. 

WILLcox, Miss Mary A., Wellesley College, Wellesley, Mass. 

WitmartTH, Mrs. H. D., Elliott St., Jamaica Plain, Mass. 

WiiiaMs, Mrs. ANNA P., 505 Beacon St., Boston, Mass. 

Witson, Dr. E. B., Columbia University, New York City, N. Y. 

Witson, Pror. W. P., Philadelphia Museum, Philadelphia, Pa. 


2. MEMBERS, JANUARY, I9QI5 


AxssotTt, Pror. J. F., Washington University, St. Louis, Mo. 

Appott, Miss MarGAret B., The Bennett School, Milbrook, 
IN, We 

AppIson, Dr. W. H. F., University of Pennsylvania, Medicy 
School, Philadelphia, Pa. 

Apkins, Mr. W. S., Texas Christian University, Fort Worth 
Texas. 

ALLEE, Dr. W. C., University of Oklahoma, Norman, Okla. 

ALLEN, Pror. Ezra, 413 Lancaster St., Ardmore, Pa. 

Atityn, Miss Harriet M., Hackett Medical College, Canton, 
China. 

ALsBuRG, Dr. C.S., U.S. Dept. of Agriculture, Washington, D.C. 

BAITSELL, Dr. GeEorGE A., Sheffield Scientific School, Yale 
University, New Haven, Conn. 

BAKER, Dr. E. H., 5436 University Ave., Chicago, Il. 

BANCROFT, Pror. F. W., Aloha Farm, Concord, California. 

BARDEEN, Pror. C. R., University of Wisconsin, Madison, Wis. 

BeckwitH, Miss Cora J., Vassar College, Poughkeepsie, N. Y. 

BrenHre, Miss Etinor H., Sophie Newcomb College, Tulane 
University, New Orleans, La. 

BEYER, Dr. H. G., Stoneleigh Court, Washington, D. C. 

BiGELow, Pror. M. A., Teachers College, Columbia University, 
New York City, N. Y. 


THE DIRECTOR'S REPORT. 361 


BIGELOW, PROF. R. P., Mass. Institute of Technology, Boston, 
Mass. 

BINFOoRD, Dr. RAYMOND, Earlham Colleee. Richmond, Ind. 

-Brngxey, Miss Lexta T., University of Texas, Austin, Texas. 

BLAKESLEE, Pror. A. F., Connecticut Agricultural College, 
Storrs, Conn. 

Box, Miss Cora May, University of Cincinnati, Cincinnati, 
Ohio. 

BRADLEY, Dr. HAROLD C., University of Wisconsin, Madison, 
Wis. 

Browne, Miss EtHeL N., East Hall, Univ. of California, 
Berkeley, Cal. 

BUDINGTON, ProF. R. A., Oberlin College, Oberlin, Ohio. 

Bumpus, Dr. H. C., Tufts College, Mass. 

Byrnes, Dr. EsTHER F., 193 Jefferson Ave., Brooklyn, N. Y. 

BUCKINGHAM, Miss EpitH N., 342 Marlboro St., Boston, Mass. 

CALKINS, Pror. Gary N., Columbia University, New York City, 
ING AG 

CALVERT, Pror. Puiuip P., Univ. of Pennsylvania, Philadelphia, 
Pa. 

Carson, Pror. A. J., University of Chicago, Chicago, III. 

CARVER, Mr. Gait L., 307 Adams St., Macon, Georgia. 

Cary, Dr. L. R., Princeton University, Princeton, N. J. 

CATTELL, Pror. J. MCKEEN, Garrison-on-Hudson, N. Y. 

CHAMBERS, Dr. ROBERT, JR., eae of Cincinnati, Cin- 
cinnati, Ohio. 

CHESTER, Pror. WEBSTER, Colby Ogee Waterville, Me. 

CHIDESTER, Dr. F. E., Rutgers College, New Brunswick, N. J. 

CHILD, Pror. C. M., University of Chicago, Chicago, III. 

Ciapp, Pror. CornELIA M., Mount Holyoke College, South 
Hadley, Mass. 

CLARK, Dr. E. R., University of Missouri, Columbia, Mo. 

Cor, Pror. W. R., Yale University, New Haven, Conn. 

CoLiEy, Dr. R. H., Dartmouth College, Hanover, N. H. 

CoLton, Pror. H.S., Ardmore, Pa. 

CooLipGE, Mr. C. A., Ames Bldg., Boston, Mass. 

CopPpELAND, Dr. Manton, Bowdoin College, Brunswick, Maine. 

Cowpry, Dr. E. V., Johns Hopkins Medical School, Baltimore, 
Md. 


362 MARINE BIOLOGICAL LABORATORY. 


CRAMPTON, PRoF. H. E., Barnard College, Columbia University, 
New York City, N. Y. 

CRANE, Mrs. C. R., Woods Hole, Mass. 

Curtis, Pror. W. C., University of Missouri, Columbia, Mo. 

DERICK, Pror. Carrie M., McGill University, Montreal, 
Canada. 

DEXTER, MR. J. S., Oliver College, Olivet, Mich. 

Dopps, Pror. G. S., University of Missouri, Columbia, Mo. 

DoNALDSON, Pror. H. H., Wistar Institute of Anat. and Biol., 
Philadelphia, Pa. 

DorRRANCE, Miss ANN, Dorranceton, Pa. 

DorRANCE, Miss FRANCES, Dorranceton, Pa. 

Drew, Pror. GILMAN A., Marine Biological Laboratory, Woods 
Hole, Mass. 

Duccar, ProF. B. M., Missouri Botanical Garden, St. Louis, Mo. 

Duncay, Dr. NEIL S., Carleton College, Northfield, Minn. 

EATON, Pror. E. H., Hobart College, Geneva, N. Y. 

Epwarps, Dr. D. J., College of the City of New York, New York 
Cry BNaNe 

EIGENMANN, Pror. C. H., University of Indiana, Bloomington, 
Ind. 

EwALp, Dr. W. F., Kaiserin Augustastr. 78, Berlin, W to, 
Germany. 

FarNnAM, Miss Louise W., 43 Hillhouse Ave., New Haven, Conn. 

FERGUSON, PRoF. J. S., Cornell Univ. Medical School, New York 
City NaNe : 

FreLp, Miss HAzeE E., University of Chicago, Chicago, III. 

FIELD, Pror. Irvinc, Auburn, Mass. 

Fiso, Mr. J. Burton, 883 Freeman St., New York City, N. Y. 

FLANIGEN, Miss Rutu, Woodbury, N. J. 

GaGE, Pror. S. H., Cornell University, Ithaca, N. Y. 

GARREY, Pror. W. E., Washington University Medical School, 
St. Louis, Mo. 

Gigs, Pror. W. J., Columbia Univ., Dept. Physiological Chem- 
istry, New York City, N. Y. 

GLASER, Pror. O. C., University of Michigan, Ann Arbor, Mich. 

GLASER, Dr. R. W., Bussey Institution, Forest Hills, Mass. 

GOLDFARB, ProF. A. J., College of the City of New York, New 
Wok City, IN MW. 


THE DIRECTOR’S REPORT. 363 


GoopricH, Mr. H. B., Princeton University, Princeton, N. J. 

GRAVE, Dr. CASWELL, Johns Hopkins University, Baltimore, Md. 

Grecory, Dr. Louise H., Barnard College, Columbia Univer- 
sity, New York City, N. Y. 

GREENMAN, Dr. M. J., Wistar Institute of Anat. and Biol., 
Philadelphia, Pa. 

GUNTHER, Miss Maup C., Eastern High School, Washington, 
Da Ge 

Haun, Dr. C. W., High School of Commerce, New York City, 
NEY: 

HALL, Pror. Ropert W., Lehigh University, South Bethlehem, 
ae 
Hance, Mr. Rosert T., Univ. of Cincinnati, Cincinnati, Ohio. 

Hareitt, Dr. C. W., Syracuse University, Syracuse, N. Y. 

HarMAN, Dr. Mary T., Kans. State Agricultural College, 
Manhattan, Kans. 

Harper, Pror. R. A., Columbia University, New York City, 
Nees 

Harrison, Mr. A. C., 660 Drexel Bldg., 5th and Chestnut Sts., 
Philadelphia, Pa. 

Harrison, Pror. Ross G., Yale University, New Haven, Conn. 

Harvey, Pror. B. C. H., University of Chicago, Chicago, Ill. 

Harvey, Dr. E. N., Princeton University, Princeton, N. J. 

Haucuwont, Mr. F. G., Columbia University, New York City, 
INS AG 

HaypEN, Miss MarGareT A., Carnegie Inst. of Technology, 
Pittsburgh, Pa. 

Haves, Pror. S. P., Mount Holyoke College, South Hadley, 
Mass. 

Heatu, Pror. HAROLD, Stanford University, San Francisco, Cal. 

HEGNER, Pror. R. W., University of Michigan, Ann Arbor, Mich. 

HEILBRUNN, Mr. L. V., University of Chicago, Chicago, III. 

Hoar, Mr. D. BLAKELY, 161 Devonshire St., Boston, Mass. 

HocueE, Dr. Mary J., Wellesley College, Wellesley, Mass. 

Hoce, Miss Mitprep A., Univ. of Indiana, Arbutus Apts., 
Bloomington, Ind. 

HoimeEs, Pror. S. J., University of California, Berkeley, Cal. 

Isaacs, Mr. RAPHAEL, University of Cincinnati, Cincinnati, 
Ohio. 


ZOAg a MARINE BIOLOGICAL LABORATORY. 


IsELEY, Pror. F. B., Central College, Fayette, Mo. 

Jackson, Pror. C. M., University of Minnesota, Minneapolis, 
Minn. 

Jacops, Mr. Murxket H., Univ. of Pennsylvania, Zoél. Lab., 
Philadelphia, Pa. 

JENNINGS, PRor. H. S., Johns Hopkins University, Baltimore, 
Md. 

JENNER, Pror. E. A., Simpson College, Indianola, Iowa. 

JEWETT, Pror. J. R., Harvard University, Cambridge, Mass. 

Jones, Pror. Lynps, Oberlin College, Oberlin, Ohio. 

JorDAN, Pror. H. E., University of Virginia, Charlottesville, Va. 

Just, Pror. E. E., Howard University, Washington, D. C. 

KanbA, Dr. Saxyo, University of Minnesota, Minneapolis, Minn. 

KELLEY, Mr. F. J., University of Wisconsin, Madison, Wis. 

KELLICOTT, Pror. W. E., Goucher College, Baltimore, Md. 

KELLY, Mr. J. P., 2163 Gleason Ave., Unionport, N. Y. 

KENNEDY, Dr. HARRIS, 286 Warren St., Roxbury, Mass. 

Key, Dr. J. A., Johns Hopkins Medical School, Baltimore, Md. 

Kinc, Dr. HELEN DEAN, Wistar Institute, Philadelphia, Pa. 

KincsBury, Pror. B. F., Cornell University, Ithaca, N. Y. 

KINGSLEY, Pror. J. S., University of Illinois, Urbana, III. 

KirKHaAm, Dr. W. B., Yale University, New Haven, Conn. 

Kite, Dr. G. L., Henry Phipps Institute, Philadelphia, Pa. 

Kwnicut, Miss Marian V., 36 Bedford Terrace, Northampton, 
Mass. 

Knower, Pror. H. McE., University of Cincinnati, Cincinnati, 
Ohio. 

KNOWLTON, Pror. F. P., Syracuse University, Syracuse, N. Y. 

Knupson, Pror. Lewis, Cornell University, Ithaca, N. Y. 

Kriss, Dr. HERBERT, University of Pennsylvania, Philadelphia, 
Pa. 

Lee, Pror. F. S., 437 West 59th St., New York City, N. Y. 

LEFEVRE, Pror. GEORGE, University of Missouri, Columbia, Mo. 

Lewis, Pror. I. F., University of Missouri, Columbia, Mo. 

Lewis, Pror. W. H., Johns Hopkins University, Baltimore, Md. 

LILLIE, Pror. FRANK R., University of Chicago, Chicago, IIl. 

Lituie, Pror. R. S., Clark University, Worcester, Mass. 

Linton, Pror. Epwin, Washington and Jefferson College, 
Washington, Pa. 


THE DIRECTOR’S REPORT. 365 


Loes, Pror. Jacques, Rockefeller Institute for Medical Re- 
search, New York City, N. Y. 

Loes, Dr. Leo, Barnard Free Skin and Cancer Hospital, St. 
Louis, Mo. 

LowTHer, Mrs. FLORENCE DEL., Barnard College, Columbia 
University, New York City, N. Y. 

LucomsBeE, Mr. W. O., Woods Hole, Mass. 

Lyman, Pror. GEORGE R., Dartmouth College, Hanover, N. H. 

Lyncu, Miss CLARA J., Smith College, Northampton, Mass. 

Lyon, Dr. E. P., University of Minnesota, Minneapolis, Minn. 

Lunp, Dr. E. J., University of Pennsylvania, Philadelphia, Pa. 

McC Lenpbon, Dr. J. F., University of Minnesota, Minneapolis, 
Minn. 

McCiunc, Pror. C. E., University of Pennsylvania, Phila- 
delphia, Pa. 

McGILt, Dr. CAROLINE, Murray Hospital, Butte, Montana. 

McGrecor, Dr. J. H., Columbia University, New York City, 
ING AS 

MclInvoo, Dr. N. E., Bureau of Entomology, Washington, D. C. 

MACKENZIE, Pror. Mary D., Carnegie Institute of Technology, 
Pittsburgh, Pa. 

McMuraricu, Pror. J. P., University of Toronto, Toronto, Can- 

ada. 

Matt, Pror. J. P., Johns Hopkins University, Baltimore, Md. 

Ma tong, Dr. E. F., University of Cincinnati, Cincinnati, Ohio, 

Martin, Miss Bertua E., University of Chicago, Dept. of Zodl., 

Chicago, II. 

MARQUETTE, Mr. WILLIAM, Columbia University, New York 

CisyANe Ne 

Maruews, Prop. A. P., University of Chicago, Chicago, Ill. 

Maver, Dr. A. G., Maplewood, N. J. 

Metcs, Dr. E. B., Wistar Institute of Anat. and Biol., Phila- 
delphia, Pa. 

MELTZER, Dr. S. J., 13 West 121st Street, New York City, N. Y. 

Mercatr, Pror. M. M., 128 Forest Street, Oberlin, Ohio. 

Minor, Miss Marte L., Bryn Mawr College, Bryn Mawr, Pa. 

MitTcHELL, Dr. Puitip H., Brown University, Providence, R. I. 

Morean, Pror. H. A., Agricultural Experiment Station, Knox- 
ville, Tenn. 


366 MARINE BIOLOGICAL LABORATORY. 


Moore, ProFr. GEORGE T., Missouri Botanical Garden, St. Louis, 
Mo. 

Morr iL, Pror. A. D., Hamilton College, Clinton, N. Y. 

MorriL1, Dr. C. V., 338 East 26th St., New York City, N. Y. 

Morris, Miss MARGARET, Yale University, New Haven, Conn. 

Mursacu, Dr. L., Central High School, Detroit, Mich. 

Moore, Pror. J. Percy, University of Pennsylvania, Phila- 
delphia, Pa. 

NACHTRIEB, Pror. HENRY F., University of Minnesota, Minne- 

apolis, Minn. 

NEAL, Pror. H. V., Tufts College, Mass. 

NEwMaAN, Pror. H. H., University of Chicago, Chicago, Ill. 

NicHots, Dr. M. Louise, 3221 Race St., Philadelphia, Pa. 

OLIVER, Mr. WADE W., Ohio-Miami Medical College, Cincinnati, 
Ohio. 

OsBuRN, Pro. R. C., 557 West 124th St., New York City, N. Y. 

OsTERHOUT, Pror. W. J. V., Harvard University, Cambridge, 
Mass. 

PACKARD, Dr. CHARLES, Columbia University, Dept. Zodl., 
New York City, N. Y. 

PACKARD, Dr. W. H., Bradley Polytechnic ee Peoria, Ill. 

PAINTER, Mr. T.S., Yale University, New Haven, Conn. 

PAPPENHEIMER, Dr. A. M., Columbia University, 28s Path- 
ology, New York City, N. ve 

PARKER, Pror. G. H., 16 Berkeley Street, Cambridge, Mass. 

Paton, Dr. STEWART, Princeton University, Princeton, N. J. 

PATTEN, Miss J. B., Elm Brook, South Natick, Mass. 

PATTEN, Dr. WILLIAM, Dartmouth College, Hanover, N. H. 

PATTERSON, Pror. J. T., University of Texas, Austin, Texas. 

PAYNE, Pror. F., University of Indiana, Bloomington, Ind. 

PEARSE, Pror. A. S., University of Wisconsin, Madison, Wis. 

Puituies, Miss Ruts L., Western College, Oxford, Ohio. 

PIKE, Pror. FRANK H., 437 West 59th Street, New York City, 
INERYE 

PInNEy, Miss Mary E., Bryn Mawr College, Bryn Mawr, Pa. 

PRENTIss, Miss Henrietta, Normal College, New York City, 
INE NG 

QUACKENBUSH, Mr. L. S., 27 West 73d Street, New York City, 
INPSYece 


THE DIRECTOR’S REPORT. 367 


RANKIN, Pror. W. M., Princeton University, Princeton, N. J. 

Rea, Dr. Paut M., Charleston Museum, Charleston, S. C. 

REIGHARD, Pror. JAcos, University of Michigan, Ann Arbor, 
Mich. 

REINKE, Mr. E. E., Princeton University, Princeton, N. J. 
Rice, Pror. EpwArD L., Ohio Wesleyan University, Delaware, 
Ohio. 

RicHarps, Dr. A., University of Texas, Austin, Texas. 

Ropsins, Mr. W. J., Cornell University, Ithaca, N. Y. 

Rosperts, Miss EpitaH A., Mount Holyoke College, South 
Hadley, Mass. 

Rospertson, Miss AxiceE, Wellesley College, Wellesley, Mass. 

ROBERTSON, Pror. W. R.B., University Club, Lawrence, Kansas. 

RoGeErs, Pror. CHARLES G., Oberlin College, Oberlin, Ohio. 

Rosenow, Dr. E. C., People’s Gas Bldg., Chicago, II. 

RuDDIMAN, Miss MARGUERITE, 441 Senator Street, Brooklyn, 
Ni. 

Sanps, Miss ADELAIDE G., 348 N. Main St., Port Chester, N. Y. 

SANDS, Dr. GEORGIANA, 348 N. Main St., Port Chester, N. Y. 

Scott, Pror. G. G., College of the City of New York, New York 
(Cine, IN. NE 

Scor, PROF- JouN W., University of Wyoming, Laramie, Wyo. 

SHOREY, Dr. Marian L., Milwaukee-Downer College, Mil- 
waukee, Wis. 

SHULL, Dr. A. FRANKLIN, University of Michigan, Ann Arbor, 
Mich. 

SHUMWAY, Mr. Waxpo, Columbia University, New York City, 
IN Ys 

SMITH, Dr. BERTRAM G., State Normal College, Ypsilanti, Mich. 

SOLLMAN, Dr. ToRALD, Western Reserve University, Cleveland, 
Ohio. 

SPAULDING, Dr. E. G., Princeton University, Princeton, N. J. 

SPENCER, Dr. H. J., Cornell University, Ithaca, N. Y. 

STEWART, Miss Mary W., Barnard College, Columbia Univ., 
New York City, N. Y. 

STOCKARD, ProF. C. R., Cornell Medical College, New York City, 
INES 

STREETER, Dr. GEORGE L., Johns Hopkins Medical School, 
Baltimore, Md. 


368 MARINE BIOLOGICAL LABORATORY. 


STRONG, Dr. O. S., 437 West 59th St., New York City, N. Y. 

STRONG, Dr. R. M., P. O. Box 58, University, Miss. 

STURTEVANT, Mr. A. H., Columbia University, Dept. Zoél., New 
Vork City. sNae 

TAsHIRO, Dr. SHIRO, University of Chicago, Chicago, IIl. 

TayLor, Miss KATHERINE A., Cascade, Washington Co., Mary- 
land. 

TENNENT, Prof. D. H., Bryn Mawr College, Bryn Mawr, Pa. 

- THOMAS, Dr. ADRIAN, 23 King St., Worcester, Mass. 

THOMPSON, PRoF. CAROLINE B., 195 Weston Road, Wellesley, 

Mass. ) 

TINKHAM, Miss FLORENCE L., 71 Ingersoll Grove, Springfield, 

Mass. 

TOMPKINS, Miss ELIzABETH M., 2019 Bedford Avenue, Brooklyn, 

IN. YW. 

TREADWELL, Pror. A. L., Vassar College, Poughkeepsie, N. Y. 

TuRNER, Mr. C. L., Ohio Wesleyan University, Delaware, Ohio. 

UHLENHUTH, Dr. EpuarD, Rockefeller Institute for Medical 
Research, New York City, N. Y. 

UsHER, Miss SUSANNAH, 9 Kirkland Place, Cambridge, Mass. 

VAN CLEAVE, Dr. H. J., University of Illinois, Urbana, IIl. 

VAUGHAN, Dr. T. W., U.S. Geological Survey, Washington, IDEG. 

WalITE, Pror. F. C., Western Reserve Univ. Medical School, 
Cleveland, Ohio. 

WALKER, Dr. GEORGE, Charles and Center Streets, Baltimore, 
Md. 

WaLtace, Pror. Louise B., Mount Holyoke College, South 
Haldley, Mass. 

WaARBASSE, Mrs. J. P., 384 Washington Ave., Brooklyn, N. Y. 

Warp, Pror. H. B., University of Illinois, Urbana, III. 

WARDWELL, Mr. E. H., New Canaan, Conn. 

WarRREN, Pror. Howarp C., Princeton University, Princeton, 
Ne) 

WASTENEYS, Mr. Harpoupu, Rockefeller Institute, New York 
City, N.Y. 

Watson, Mr. FRANK E., Hobart College, Geneva, N. Y. 

WHEELER, Miss ISABEL, Dana Hall, Wellesley, Mass. 

WHEELER, Pror. W. M., Bussey Institution, Forest Hills, Mass. 


THE DIRECTOR’S REPORT. 369 


Wouerry, Dr. W. B., Cincinnati Hospital, Cincinnati, Ohio. 

WuitNnEy, Dr. Davin D., Wesleyan University, Middletown, 
Conn. 

WIEMAN, Pro. H. L., University of Cincinnati, Cincinnati, Ohio. 

Witcox, Dr. ALICE W., 56 Alumni Avenue, Providence, R. I. 

WILpMAN, Dr. E., 4331 Osage Avenue, Philadelphia, Pa. 

WILLIAMS, Dr. ANNA W., 549 Riverside Drive, New York City, - 
INGE 

WiLson, Pror. H. V., University of North Carolina, Chapel! 
Jeti, IN|. (C- 

Wociom, Dr. WiLLtIAmM H., Columbia University, New York 
GityeeNaNe 

WoLFE, Pror. JAMES J., Trinity College, Durham, N. C. 

Wooprurfr, Pror. L. L., Yale University, New Haven, Conn.. 

WricHT, Pror. R. Ramsay, Red Gables, Headington Hill,. 
Oxford, England. 

Younc, Mr. D. B., Hartley Hall, Columbia University, New 
Worl CityA Ne Ye 


REGENERATIVE POTENCIES OF DISSOCIATED CELLS 
OF HYDROMEDUS-. 


CHAS. W. HARGITT. 


INTRODUCTORY. 


At various times during my earlier work on the development 
and regeneration of hydromeduse, especially that dealing with 
the developmental capacity of egg fragments, there had grown 
the conviction of the remarkable potencies of the various tissue 
elements of these organisms. So strong had this impression 
become that the desirability of repeating Trembly’s picturesque 
experiment of turning Hydras inside out and‘testing again the 
possibilities that ectoderm and entoderm might really exchange 
functions under the new conditions involved was entertained. 
While admitting the rather convincing results of the experiments 
of Ischikawa (90), touching this feature it still seemed that 
there might be some warrant that under certain conditions 
Trembly’s conclusions might find confirmation. However, the 
writer never found the convenient season for trying out the 
experiment, though other correlated features were observed at 
various times in connection with the work above cited, especially 
in the summer of 1908 while working on the development of 
Clava and Hydractinia. This was further incited by the work 
of H. V. Wilson, ““On Some Phenomena of Regeneration in 
Sponges,” ’07. Therefore with the opportunity for investigation 
at the Naples Laboratory I set about a series of experiments 
with a view to settle some of the problems concerned. My work 
at Naples began in December, 1910, and continued till mid-April 
following, and during this period systematic experiments were 
made upon the regenerative potencies of somatic cells of about 
a dozen different species, among which the following may be 
named: two species of Eudendrium, two species of Tubularia, 
one each of Hydractinia, Podocoryne, Campanularia, Obelia, 
Halecium, Sertularia, and a medusa, Liriope exigua. 

In Science of March 10, 1911, appeared a preliminary report 

370 


REGENERATIVE POTENCIES OF DISSOCIATED CELLS. 371 


of similar experiments by Dr. Wilson, which reached Naples 
about April 1, just when my own experiments were being con- 
cluded, results of which had already been written and some of 
which will appear in the following sections just as originally 
prepared. I immediately wrote Professor Wilson, giving a brief 
account of my work and stating that my results would be held 
pending the appearance of his completed report. This appeared 
in due course (11). While dealing in the main with different 
material yet he had employed essentially the same methods 
which I had followed and they seemed so conclusive that I had 
laid my own paper aside, deeming it unnecessary. However, 
the report in the Journal of the Marine Biological Associa- 
tion of similar experiments by DeMorgan and Drew (Oct., ’14) 
which seemed to express some doubt as to the conclusiveness of 
Wilson’s results, prompts me to submit even at this late date 
my own results, though in somewhat abbreviated form. 


MATERIAL AND METHODS. 


Concerning material employed in the experiments mention 
has been made in the previous section as to the several species 
used, though two species of medusa instead of one were experi- 
mented upon. For the most part particular care was taken to 
have perfectly fresh and vigorous specimens, but it was later 
found that this precaution was not absolutely essential in all 
cases, some of my best hydranths having been reared from mater- 
ial which had been several days in the laboratory before being 
used. Later mention will also be made of a probable reason why 
this may happen. One point however calls for specia! emphasis, 
namely, that of the freshness and purity of the water used in the 
experiments. In my work several expedients were employed 
to guard against the presence of parasitic organisms, especially 
predatory protozoa. Of most efficiency was that of having 
water fresh from the open sea. Another expedient was that of 
sterilizing water of the aquaria, and still a third was that of 
using synthetic, that is, artificial sea-water. But of all these 
the first was found to be most satisfactory. 

Concerning methods much might be said, though only the 
briefest reference will be made of those employed by me. Among 


272 CHAS. W. HARGITT. 


the several modes of isolating the tissue cells the following were 
employed: With hydroids the ccenosare may be forced out of 
the perisarc by clipping off the hydranths and then stripping 
the stems through clean fingers, or the ends of smooth forceps, 
or similar device. Again, one may finely clip up the stems with 
scissors and then still further reduce the cells by continuing the 
operation in deep watch glasses or small beakers. A still further 
mode was used, that of clipping up the stems with scissors and 
later grinding the tissues under a smooth glass rod whose end 
had been rounded in the flame, employing it as a pestle and a 
watch glass asa mortar. One objection to this was the crushing 
of the individual cells in many instances, and otherwise injuring 
them. On the other hand there may be reason to believe that 
such treatment is not really so serious an injury as might at 
first sight appear, for as will be seen in some later discussion, the 
shock may actually serve as a stimulus to hasten cellular de- 
specialization and hence initiate regenerative processes. 

Cell dissociation having been effected the next method is to 
arrange them in dishes where aggregation may take place. First 
in this operation is the straining or filtering out of debris and such 
fragments as are undesirable. This was done chiefly by pressing 
the cells through bolting cloth. In my experiments it was found 
that a better medium than silk bolting cloth was a fine meshed 
cheese cloth, or a coarse meshed linen or cotton fabric, which was 
softer and apparently more efficient. In a few cases in the earlier 
experiments I merely placed the entire mass in watch glasses 
and with a pipette carefully drew off most of the coarser stuff 
and left the cells as free as such a process might leave them. On 
the whole, the pressing process worked better and was more 
expeditious. Following this operation the cells were left for a 
time to settle and then the milky sediment was carefully drawn 
off, when fresh water was added and the dishes set aside in 
bowls surrounded by running water to ensure as constant a 
temperature as could be had. 


THE EXPERIMENTS. 
Podocoryne carnea.—This was the first species which came to 
hand and proved one of the most responsive and convincing 


REGENERATIVE POTENCIES OF DISSOCIATED CELLS. 373 


of the entire series. Several colonies were brought in, all occupy- 
ing shells inhabited by hermit crabs, the usual habitat of this 
species. In one respect the species is rather difficult to operate 
on owing to the spinous condition of the basal coenosarc, which 
made it hard to obtain enough of the polyps to make the desired 
culture. By allowing the colony to expand fully in shallow 
dishes it was possible by a dextrous sweep of the scissors to cut 
off quite a bunch at a single time, and by allowing others to 
expand in the same way and repeat the operation it was found 
possible to secure sufficient material for the culture. The speci- 
mens were finely cut or ground into a pulpy mass, filtered through 
the sterilized cloth, and thus fitted to undergo later changes. 
It may as well be stated here that among hundreds of prepara- 
tions relatively few gave completely successful results in the 
regeneration of new polyps. My first surprise was not that many 
of the preparations ‘‘went bad,” but that any survived the opera- 
tion and went forward in regeneration. Here as in most experi- 
ments on regeneration a large mortality occurs in the prepara- 
tions. 

Character of the Dissociated Cells——Ilf{ examined soon after 
their dissociation one may easily distinguish the several sorts of 
cells even under a magnification of three hundred diameters, 
that is, ectoderm, entoderm, nematocyst, interstitial, etc. The 
very minute ectoderm cells are in striking contrast with the 
large flagellated cells of the entoderm. In the course of an hour, 
sometimes less, these differences become less marked, and ulti- 
mately almost disappear. They have become despecialized into 
potentially embryonic cells, and probably from this change have 
acquired their regenerative capacities. A careful study of such 
dissociated cells from various species has strongly suggested the 
probability that some such cytomorphic process is involved in 
most regenerative phenomena, and leaves little doubt that the 
features under consideration here are positively brought about 
through such a process. 

Cell Aggregation——Examination of a culture within a few 
hours, three to five, will show that a remarkable change has 
taken place among the cells in their relations to each other. They 
will be found to have formed numerous small nodular groups 


374 CHAS. W. HARGITT. 


having the appearance in many cases of embryonic morulz, or 
blastule. It was this phenomenon among others already men- 
tioned which first raised the question in my mind as to their 
regenerative possibilities many years ago. Concerning the mode 
by which this process of aggregation is brought about there is 
some doubt. The attempt was made to actually observe it by 
carefully keeping a fresh culture under direct observation with 
the microscope. It was thought that the action of the flagellated 
cells of the entoderm might act as a means by causing vortices 
in the water, but careful study failed to show that this was a 
factor of any direct value. Such action of these cells may be 
easily seen but its effects are as often repellent as attractive. 
The fact that certain of the cells show amceba-like aspects sug- 
gested a possible amceboid action in the process. But here again 
no evidence whatever was found to prove the suggestion. One 
might imagine some chemotropic influence, but no evidence was 
found that such was the case. I am inclined to the view that 
chance contact is perhaps the chief factor in the process. This is 
made probable by the fact that such aggregation may be greatly 
facilitated by mechanical agitation of the cells, and by a gentle 
rotary motion of the dishes. In the earlier experiments con- 
siderable care was taken to handle the dishes as little as possible 
during the early stages of an experiment, thinking such might 
be undesirable, but later the opposite view was taken, and the 
dishes often rotated to hasten the process. It must be admitted, 
however, that there seemed to be other factors involved, for 
even when a considerable mass of cells had been brought together 
by this means there was later found to have been a sort of segrega- 
tive process at work, for the mass had been more or less broken 
up into sections or lobes which later behaved as entirely inde- 
pendent bodies. , 

The cell aggregates, while rather predominantly sub-spherical 
in shape, showed considerable variation. Some were flattish, or 
disk-like, and some were somewhat lobulated and irregular in 
shape. But throughout a series of such aggregates one of the 
most conspicuous features was that already referred to above, 
namely, the resemblance to an embryonic blastula or morula, 
especially a hydroid morula; and if one were to take account of 


REGENERATIVE POTENCIES OF DISSOCIATED CELLS. 375 


such morule as those of Pennaria or Turritopsis or Hydractinia 
it would include practically the entire range of shape exhibited 
by these regeneration aggregates, and one might designate them 
as regeneration morulz, for such they really seem to be. 

Encystment.—Following the process of aggregation there oc- 
curred in those vitally active a process of encystment, that is, 
the secretion of a definite perisarc about the entire mass, and 
its adhesion to the bottom of the glasses. Lest this feature be 
regarded as peculiar to these particular cases it should be pointed 
out that the phenomenon is often shown at a certain stage in the 
normal development of the hydromedusz, and indeed in some 
scyphomeduse as well. The writer has directed attention to 
this in the case of Cyanea (’02, ’10) and it is doubtless shared by 
many others. Its function is doubtless protective, just as is 
that of the perisarc in the adult hydroid. Encystment usually 
occurs shortly following the completion of the phase of aggrega- 
tion just described. This encysted stage may continue for an 
indefinite time, or it may be of short duration. The latter was 
more frequently the case with Podocoryne than with some others. 
In the present case the cyst was frequently ruptured for the up- 
growth of the hydranth within a comparatively short time, say 
two days; but in many cases this stage persisted for a week or 
even more, and indeed in certain cases the cyst became a prison, 
being so dense as to become impenetrable from within as well as 
without. This again is comparable with what may happen in 
such stages in normal development (vide supra). This process 
of perisarc formation often takes various forms, following the 
phases of growth. In Podocoryne there was frequently the 
development of a reticulated hydrorhiza before the appearance 
of a hydranth, and later there appeared nodular enlargements of 
these stolon-like tubes and from these points would occur the 
upgrowth of a series of polyps. In one such preparation I 
obtained three vigorous young hydranths. 

What has been stated in this connection as to Podocoryne is 
likewise true of other species experimented with. The behavior 
of the encysted aggregation morule is quite like that of the grow- 
ing stolons of the hydrorhiza. Both may live for weeks under 
these conditions without any signs of further development. 


376 CHAS. W. HARGITT. 


Again, after such prolonged periods there may come about 
another direction of regenerative activity and a hydranth may 
arise. Aside from the evidence of life observed in the active 
circulation within the ccenosarc of stolons it becomes easy for 
one to recognize in the character of the cells of the various 
structures the evidences of life or death, and furthermore death 
of any portion is rapidly followed by disintegration brought 
about by microérganisms. It should have been stated in the 
earlier part of this section that the process of encystment usually 
begins soon after the aggregation phase is complete, which may 
be within twelve to twenty hours, though it may not become 
evident until much later, thirty to forty hours. The first 
evidence of its formation is the adhesion of the mass to the 
bottom of the glass, and somewhat later may be distinguished as 
a very delicate transparent film covering the entire mass. Its 
later extension may be easily followed as the growth of stolons 
_takes place, which may be quite rapid in some cases, or in others 
very slow. Here again as was pointed out in an earlier connec- 
tion, there is a marked similarity in the aspects of regenerative 
growth and those of embryonic development which further 
emphasizes the probability that they are fundamentally identical, 
having their initiative in potentially embryonic cells. 

Polyp Formation.—In Podocoryne the first evidence of definitive 
hydranth organization was found during the second day following 
the experiment. This consisted in the dissolution of the cyst 
at its upper surface and the protrusion of a bud-like upgrowth. 
At first these were barely distinguishable, but during the third 
day they had become large enough to be seen with che unaided 
eye. The first fully formed hydranth appeared /early on the 
fifth day, when a polyp having the distinctive art of hypostome 
and three tentacles was noted. This was followed by further 
growth of the young specimen in allits parts. The movement of 
the tentacles and their growth in length was interesting and strik- 
ing, leaving not the least doubt as to the genuineness of the 
regenerative process. Usually the first three tentacles appeared 
at about the same time, but in a few cases it was noted that when 
first observed there were but two, though a third appeared 
rather soon after. The full six tentacles of the new polyp were 


REGENERATIVE POTENCIES OF DISSOCIATED CELLS. 377 


developed within the next two or three days, and conformed 
exactly to the phases of the growth of an embryonic specimen. 
It should be emphasized in this connection that in the rate of 
growth in these specimens, as in the entire regenerative process, 
there was great individual difference. Apparently this was 
dependent upon the state of vitality of the underlying organiza- 
tion. For example, it was found that development was slower, 
and the resulting polyps smaller when arising from small cell 
aggregates, and in cases where there had been an excessive 
stolonization prior to polyp formation. In the one case it would 
seem as if the store of energy was smal! to begin with, and in the 
second that it had been depleted by excessive stolon formation. 

The young polyps continued to live for several weeks, much 
longer than would have seemed probable when the highly 
artificial conditions, and the very limited food supply are taken 
into consideration. During the course of the experiments more 
than a dozen of these polyps of Podocoryne were reared to func- 
tional maturity and many others to such stage as to leave no 
trace of doubt as to the validity of the results. 

Let it be remarked here that in this species all the eral 
was in the asexual condition, that is, there were polyps only, no 
signs whatever of medusz, which are the sexual stage in the life 
cycle of Podocoryne. Other experiments go to show that so far 
as use of material of asexual or mixed condition no difference as 
to regenerative potency could be distinguished. In Eudendrium 
where the medusa stage is absent, and where one finds sex cells 
in various stages of growth, the experiments were apparently 
not thereby influenced at all. Indeed, in those cases in which 
egg cells were present they took no part whatever in later re- 
generative activity, either degenerating or being absorbed as 
yolk material. 

Eudendrium.—tin experiments upon Eudendrium two species 
were used, E. rameum, and E. racemosum, both very common at 
Naples. Methods of treatment were the same as in the case 
already described. The promptness with which these hydroids 
had responded in the numerous previous experiments by the 
writer! and others in regeneration and regulation led me to 

1 Biol. Bull., Vol. I., p. 35. 


378 CHAS. W. HARGITT. 


anticipate that a similar type of reaction might be anticipated in 
this connection, but as will be seen this expectation was not 
realized fully. The early reactions in aggregation, encystment, 
etc., were quite as prompt and promising as in Podocoryne. And 
in these features the species showed nothing peculiar. But 
beyond the initial stages the results were disappointing. The 
mortality was much greater and the growth reactions much less 
energetic. Experiments were varied in every way practicable, 
hydranths alone being used for obtaining disorganized cells, 
coenosarc alone, male colonies alone and female colonies alone. 
There seemed to be no very marked differences in results, though 
the cells obtained from crushing hydranths gave the least satis- 
factory results. As already stated the early stages followed 
quite as in Pocodoryne, encystment, and stolonization, but beyond 
these my experiments were far less satisfactory than in the former. 
In only a few cases was I able to obtain polyps, and these were 
small and very weak. A few developed tentacles, but never the 
usual number, nor were they more than buds on the base of the 
hydranth. The few polyps which developed secreted the usual 
perisarc, which was indistinguishable from that of an embryonic 
Eudendrium. 

Tubularia.—As in the former I employed two species, T. 
mesembryanthemum, and T. larynx. As in the former the early 
reactions were prompt and quite like the others. But unlike 
the others my experiments never afforded a single polyp. The 
massing of dissociated cells was quite as prompt and the resulting 
morula-like embryo as promising as in ¢ither of the others. 
The encystment of perisarc followed in dué order, and these lived 
for many days, but they never showed (urther signs of develop- 
ment. Perhaps no hydroid genus has/ had so large a place in 
experimental work as has Tubularia. If therefore my anticipa- 
tions as to the behavior in cellular regeneration of Eudendrium 
were disappointing, those concerning Tubularia were really per- 
plexing, at least for the time being. I think an explanation may 
be ventured which, though not absolutely convincing, may relieve 
a measure of the perplexity. It is to the effect that regenerative 
potency in an organism is more or less conditioned by its state 
of vitality, or in still more suggestive phrase, its physiological 


REGENERATIVE POTENCIES OF DISSOCIATED CELLS. 379 


state, at the time it is subjected to the test. This has been 
recognized in principle in experiments on ordinary regenerative 
processes, in that only especially vigorous specimens are used. 
Further discussion of this point will be deferred to another section 
of the paper. 

Other Species—As indicated in the outstart, about a dozen 
different species were tried in the course of the investigation. 
Among these were several campanularian hydroids and two 
species of meduse. The hydroids tested gave the same initial 
responses as those just described for Tubularia, but beyond 
that the results were likewise negative. In all cases the phase 
of cell-aggregation was essentially the same as in the former cases. 
The same was likewise true of the internal organization of the 
morula-like embryo, and in the perisarc formation, but beyond 
this there was no development. 

Species of sertularian hydroids and also of Haleciwm were 
tested and gave exactly the same initial responses, including 
encystment of the embryonic mass which lived for a time but 
soon showed signs of disintegration and death. The reactions 
of the last species were the least satisfactory of any tested. 

Meduse.—Two species of medusz were tried, though with 
hardly any hope of getting any regenerative responses. They 
were prepared just as had been the hydroids, strained through 
the bolting cloth and set aside after addition of fresh water. 
An examination of the dissociated cells showed about the same 
condition of the other preparations, and further inspection in 
about an hour showed a series of the most beautiful cell-aggre- 
gates found in any of the experiments. When it is recalled that 
Meduse represent the most highly specialized group of Hydrozoa 
it will seem strange to find cells thus organized after having been 
dissociated in the manner indicated. In all my observations 
upon coelenterate development I have seldom seen more typical 
blastula-like embryos than those under review. Unless one were 
actually aware of their source he could hardly have been con- 
vinced that they were not genuine embryos in process of develop- 
ment. However, so far as my experiments show the regenerative 
process does not go farther. Moreover, the organism thus formed 
is very short lived, and devoid of further significance so far as our 
problem is concerned. 


380 CHAS. W. HARGITT. 


ADDENDA AND DISCUSSION. 


As mentioned in the introduction the immeditae occasion 
leading to the publication of this paper at this time after having 
been laid aside for four years was the appearance in the Journal 
of the Marine Biological Association, October, 1914, of a paper by 
DeMorgan and Drew, setting forth the results of similar experi- 
ments, all of which had given generally negative results. More- 
over, certain of their conclusions seemed to leave a measure of 
doubt concerning the conclusiveness of certain of Wilson’s 
experiments, and phases of their discussion involved assumptions 
which are at variance with those which my own work had 
rendered very convincing. 

In the first place I desire to refer briefly to Wilson’s methods 
and results with most of which my own are in accord. His 
experiments on Eudendrium seem to have been much more 
successful than my own, for which I am very glad, since it confirms 
with great certainty points which in my own experiments were 
incomplete, though sufficiently complete to warrant definite 
conclusions. In another point Wilson’s work goes beyond my 
own, namely, in the admirable demonstration which his actual 
sections of various stages affords as to the precise features 
involved in the regenerative process at given times. Further- 
more, the excellent series of drawings and photographs illustrating 
his results leave nothing to be desired in that respect, and I am 
purposely omitting any of my own, the only series of which not 
better covered are those relating to Podocoryne, and in these 
nothing essentially different occurs. 

The work of DeMorgan and Drew covered experiments on 
two species of Antennularia and are restricted to these only. 
In order to consider certain of their views it may be well to first 
quote certain specific statements in their own words. “Our 
results largely bear out his (Wilson) contentions, though we 
were not successful in carrying the regenerative process as far 
as the production of new hydranths, and the histological struc- 
ture of the restitution masses we obtained differed in many ways 
from that described in Wilson’s paper. ‘These differences are 
probably due to the fact that we experimented with other species 
of hydroids to those used by Wilson. The especial interest of 


REGENERATIVE POTENCIES OF DISSOCIATED CELLS. 381 


our investigations lies in the rather anomalous fact that we have 
not been successful in obtaining regeneration of the complete 
organism from the dissociated cells. In our experiments the 
restitution masses, by some rearrangement or metaplastic process 
taking place among their conglomerated cells, formed tissue 
aggregates histologically reduplicating the structure of the parent 
organism, but in a quite irregular and apparently meaningless 
manner.” 

Two features in this quotation call for brief consideration, 
that included in the first sentence, and that which I have itali- 
cized in the last. It will have been noted in the accounts given 
in the earlier sections of this paper that I have given a number of 
cases comprising the exact equivalent of the failure they mention. 
This point will be further noted in a later paragraph. To the 
second feature it is only necessary to state that in normal hydroid 
development the entire process is often “quite irregular and 
apparently meaningless,” frequently more so than they found in 
the cases concerned. Ina final paragraph the authors say: ‘Our 
experiments have resulted in the production of masses that are 
certainly abnormal and pathological, but nevertheless we would 
submit that the segregation and rearrangement of the cells after 
isolation, and the comparatively long duration of life of the 
tumor-like masses to which they give rise are facts of considerable 
theoretical interest.” 

In this quotation I have italicized the points to which it seems 
necessary to make some reference. It may be admitted that in 
some sense such restitution masses are abnormal, in that the very 
process by which their dissociation was brought about was 
presumably abnormal. But that the resulting restitution masses, 
involving as they have the regenerative potencies of the com- 
ponent cells, is abnormal I must seriously challenge. Again, the 
assumption that they are pathological | should emphatically 
doubt. The writer once submitted a series of preparations of 
embryological material to a well-known cytologist and received 
the (at that time) very disconcerting comment, “your prepara- 
tions appear to have been made from pathological material.”’ 
Yet from that very material I had been getting living embryos by 
the hundred! So in the present case to designate as pathological 


382 CHAS. W. HARGITT. 


cell aggregates which are producing right along perfectly normal 
and healthy polyps is to use a term whose significance implies 
the very opposite. It is admitted in the above citation that the 
tumor-like masses continued to live for a long time, as much as 
sixty days according to a preceding sentence, which shows a 
degree of vitality greater than that of colonies of the hydroid 
when placed in the aquarium. This fact of itself should prompt 
serious hesitation as to an assumption of a pathological condition. 

It may throw some light upon the problem if attention is 
directed to conditions involved in the life history of many of 
these organisms. It is well known that many hydrozoa have 
alternating periods of activity and repose—growth, reproduction, 
etc., followed by corresponding periods of decline and more or 
less degeneration. In some these periodic alternations are 
correlated with seasonal changes in which temperature is an 
important factor. In others it is directly correlated with repro- 
ductive activities and has apparently little relation to season or 
temperature. What is of immediate importance in this connection 
is the fact of rather evident degenerative phases. For example, 
it is well known that in the spring, following the active reproduc- 
tive period in several species of Tubularia, there is a marked 
degenerative phase, first evident in the casting off of the hy- 
dranths of almost the entire colony, then the gradual disintegra- 
tion of the whole trophosome, till within a period of a few weeks 
it is difficult to find an entire and vigorous vegetative colony. 
An examination of the histological condition of the degenerative 
coenosarc reveals the fact of positive decline marked by cytolytic 
conditions which might really be designated as pathologic for 
the time being. But even here a continued study would probably 
reveal the fact of its being associated with perfectly normal 
cyclic phases of life, being in fact phases of varying physiological 
states to which reference has already been made in an earlier 
connection. Similar facts of degeneration phases have also 
been described as associated with regenerative activity in hy- 
droids. In experiments on Tubularia Stevens states in so many 
words: “The red granules seen in the circulation of regenerating 
pieces of Tubularia are derived from the disintegrating ento- 
dermal ridges, and are ejected by the young hydranth soon after 


REGENERATIVE POTENCIES OF DISSOCIATED CELLS. 383 


it emerges from the tube. They are waste material rather than 
formative substance”’ (oI, p. 414). The writer himself has 
made similar observations in several cases, and has demonstrated 
the regressive condition in the coenosarc of hibernating specimens. 
A most interesting study of a series of changes of apparenly- 
similar character has been made by Schultz, ‘‘ Uber Hungerer- 
scheinungen bei Hydra fusca’’ (’06), discussed under the larger 
topic of ‘‘Reductionen,” under which are considered a series of 
marked phenomena observed in organisms of various grades of 
complexity, including Planaria, Lumbricus, AAolosoma, etc. 
Similar experiments by Greeley (’03), on ‘‘Effects of Variations 


7 


of Temperature on Animal Tissues,’”’ show essentially the same 
phenomena. Among these experiments some made on Hydra 
are especially pertinent in this connection. To quote, “It was 
at once observed that whenever a Hydra is exposed to a tempera- 
ture of 4° to 6° C. the tentacles gradually become thicker and 
shorter, and finally are completely absorbed into the body. As 
the absorption goes on, the ectoderm and entoderm cells of the 
tentacles lose their individuality and form an undifferentiated 
mass of protoplasm, which is slowly resolved into the body of the 
Hydra. The tentacleless body of the Hydra becomes slowly 
resolved into a dense spherical mass of coagulated protoplasm, 
in which no distinction between individual cells can be made out, 
and remains in this condition as long as it is kept at a low tem- 
perature, but quickly forms tentacles and a double layer of cells 
again when it is returned to the temperature of the room”’ (p. 43). 

Enough has now been said to show, I think, that only in some 
qualified sense can one use such terms as irregular, meaningless, 
abnormal, pathological, etc., in describing phenomena such as 
those involved in the experiments and results under review. 

As a final note it may be stated that in my experiments no 
attempt was made to detach and isolate the several cell-aggre- 
gates such as was done by Wilson. Neither did I attempt to 
augment the masses by artificially bringing several masses into 
contact as he had done. Attention has already been directed 
to the fact that there was some evidence that from larger masses 
were derived larger polyps, and that below a certain minimum 
size there was no evidence of growth. 


384 CHAS. W. HARGITT. 


It need hardly be stated that my experiments add little essen- 
tially new to those of Wilson; yet they seem to afford valuable 
confirmation of some importance which may add to the con- 
clusiveness of his admirably conceived and conducted research. 

I desire also to express my gratification in the experiments of 
DeMorgan and Drew, which seem to me to have been admirably 
done and contribute to the value of the investigation as a whole. 


SYRACUSE UNIVERSITY, 
Jan. 20, 1915 


LITERATURE CITED. 
DeMorgan, W., and Drew, G. Harold. 

’t4 A Study of the Restitution Masses Formed by the Dissociated Cells of the 
Hydroids Antennularia ramosa and A. antennina. Jour. Marine Biol. 
Assoc., Vol. X., pp. 440-463. 

Greeley, A. W. 
’03 Studies of the Effect of Variation in the Temperature on Animal Tissues. 
BIOL. BULL., Vol. V., p. 43. 
Hargitt, Chas. W. 
’02 Notes on Cyanea Arctica. Science, Vol. XV., p. 571. 
’99 Experimental Studies upon Hydromeduse. BIOL. BULL., Vol. I., p. 35. 
Hargitt, Chas W. and G. T. 

’r0 Studiesin the Development of Scyphomeduse. Jour. of Morph., Vol. XXI., 

pp. 217-262. 
Ischikawa, C. 
¥90 Trembley’s Unkehrungsversuche an Hydra nach Neuer Versuchen erklart. 
Zeits. f. wiss. Zool., Bd. XLIX. 
Stevens, N. M. 
’91 Regenerationin Tubularia. Arch. f. Entwicklungsmechanic, Bd. 13, p. 414. 
Schultz, Eugen. 
204 Uber ReductionenI. Arch. f. Entwicklungsmechanic, Bd. 18. 
204 Uber Reductionen, II. Arch. f. Entw., Bd. 21, p. 703. 
Wilson, H. V. 

’07_ On Some Phenomena of Coalescence and Regeneration in Sponges. Jour. 
Exp. Zool., Vol. V. 

*zr On the Behavior of Dissociated Cells in Hydroids, Aleyonaria, and Asterias. 
Jour. Exp. Zool., Vol. 11. 


THE EYES OF CAMBARUS SETOSUS AND CAMBARUS 
EELEUCIDUS? 


CHARLES H. SPURGEON. 


(Department of Biology, Drury College.) 


Cambarus setosus Faxon inhabits the caves of southwestern 
Missouri. It was described by Faxon in Garman’s account of the 
“Cave Animals of Southwestern Missouri.’’ An account of the 
eyes of this species was published by Parker. 

In June, 1911, I secured an abundance of fresh material which 
made a reéxamination of the eyes desirable. I found it in the 
caves about Springfield, Sarcoxie and Ozark, Missouri. From: 
Smallen’s cave near Ozark I secured seventy-five specimens from: 
20 to 110 mm. in length. In addition to these, twenty-seven. 
specimens of C. pellucidus testi Hay were taken from Mayfield’s. 
cave near Bloomington, Indiana. Also thirty-three C. pellucidus 
(Tellkampf) from Shawnee cave, Indiana University farm, near 
Mitchell, Indiana. The specimens from Indiana were collected! 
during the fall and winter of 1911 and 1912. The blind crayfish: 
from Indiana are smaller than those from Missouri. The 
smallest taken from Indiana caves were 9 mm. in length. Others 
varied from 15 to 60 mm. The largest specimen of C. setosus 
taken was 120 mm. in length. 

C. setosus has been found only west of the Mississippi river, 
while C. pellucidus has been found only east of the Mississippi 
both north and south of the Ohio river. C. pellucidus testit 
Hay has been recorded only from Mayfield’s Cave, Bloomington, 
Indiana. C. pellucidus has probably the widest distribution of 
any of the blind crayfish. It has been taken from Mammoth and 
other caves of Kentucky and from Wyandotte and other caves 
in Crawford county, from Clifty cave, Washington county, from 
Lost river, Orange county, from Shiloh, Down’s, Donnehue’s 

1 Contribution from the Zodlogical Laboratory of Indiana University, No. 146. 
This work has been carried on under the direction of Professor Carl H. Eigenmann, 


to whom I am thankful for advice and criticism. 


385 


386 CHARLES H. SPURGEON. 


and Donnelson’s caves in Lawrence county, Indiana. Strong’s, 
Truitt’s, Kuntz’s, Marengo, Little Wyandotte or Seibert’s and 
several smaller caves of Indiana were examined for blind crayfish 
but none were found. 

The cavesfof Indiana are in the same general geological forma- 


Fic. 1. Dorsal view of C. setosus 110 mm. long, with small, inconspicuous eyes 
extending slightly from under the rostrum. (Life size—reduced 144.) Drawn by 
Miss Dorothy L. VanDyke. 


tion as those of Missouri. For an account of the Missouri caves 
see Shepard, in the Missouri Geological Survey, Vol. XII., 1898. 
For the fauna and geology of Indiana caves see Green, Indiana 
Academy of Science, 1908, and Blatchley, Indiana Department 
of Geology and Natural Resources, Twenty-first Annual Report, 
1896. 


387 


EYES OF CAMBARUS SETOSUS AND CAMBARUS PELLUCIDUS. 


The structure of the eyes in the blind crayfish, C. pellucidus, 


Concerning his*specimens 


which were taken from Mammoth cave, Kentucky, he states: 


was first noted by Newport (1855). 


Dorsal views of the anterior ends of C. setosus and C. propin- 


FIGS. 2 AND 3. 
quus(?) 36 mm. long. 
of the two species. 


They show the relative size and conspicuousness of the eyes 


“The hardened tegument which clothes the entire organ is 


thinnest and most transparent in that part of the eye which forms 


388 CHARLES H. SPURGEON. 


the cornea in other Crustaceans; so that the eye may be unfitted 
for distinguishing form, the creature may yet possess the faculty 
of perceiving the small amount of actinic rays of light which 
might penetrate into its subterranean abode . . . ; the cornea 
also exhibits an appearance of being divided into a few imperfect 
corneals (facets) at the apex of the organ, and the structures 
behind these into chambers, to which a small but distinct optic 
nerve is given.”” He also noted that the eyes are not pigmented. 

I find that the eyes of C. pellucidus from Indiana caves and of 
C. setosus from Missouri caves show neither ‘“‘corneals’’ (facets) 
nor “chambers, to which a small but distinct optic nerve is 
given.” 

The next writer on the eyes of blind crayfish was Leydig (1883). 
He stated that the cornea in C. pellucidus is lamellated, without 
pigment and without facets. His description of the internal 
Structures of the eye is very general and indefinite. 

Packard (1888) in his memoir on ‘‘The Cave Fauna of North 
America’’ describes and illustrates the form and structure of the 
eyes of C. pellucidus from Indiana and Kentucky caves and C. 
hamulatus Cope and Packard from Nickajack cave, Tennessee. 
He found that in both species the cornea is without facets and 
that the hypodermis is of the same thickness in the retinal region 
as in other parts of the eye; also that the optic nerve and optic 
ganglion are present. 

The following year (1889) Garman published Faxon’s descrip- 
tion of C. setosus to which reference was made in the opening 
paragraph. 

Parker (1890) published a paper on ‘The Eyes in Blind Cray- 
fishes.’”’ He had access to C. hamulatus and C. setosus. The 
major part of his paper deals with C. setosus. He emphasizes the 
uniform thickness of the cuticula, the nearly uniform thickness 
of the hypodermis and the relation of the size and conical shape 
of the optic stalk to the amount of degeneration, as well as the 
histological structure of the degenerated eye. He also called 
attention to the relation of the axis of the cone, which is the 
terminal part of the optic stalk, to the axis of the stalk itself. 

The most striking characteristics of the gross anatomy of the 
eyes of C. setosus and C. pellucidus are smallness, lack of pigmen- 


EYES OF CAMBARUS SETOSUS AND CAMBARUS PELLUCIDUS. 389 


tation and inconspicuousness. These features are shown in 
Figs. I and 2. 

The eyes of all the blind crayfish examined are nearly covered 
by the rostrum, from a dorsal view, while the eyes of normal 
crayfish are only slightly concealed by the rostrum. However 
the eyes of the young blind crayfish are relatively larger than 
they are in the adult. The relative size of the eyes of C. setosus 
and C. propinquus (2) is shown in Figs. 2 and 3. 

Size and pigmentation make the eyes of normal crayfish con- 
spicuous. The eyes of blind crayfish are smaller and without 


ox 4 


4 


os 


v Lae 


4 5 


Fic. 4. Outline of the eyes of C. pellucidus 10 mm. long. 
Fic. 5. Horizontal (longitudinal) section of the eye of C. setosus, with the cuti- 
cula removed. 


pigment. The relative shortness of the optic stalk in the eyes of 
blind crayfish tends to make them inconspicuous. 

The distal end of the optic stalk of the eyes of the blind cray- 
fish examined is roundish or almost hemispherical in shape. 
Fig. 4 which shows this was made with the aid of a camera 
lucida, from a fresh specimen of C. pellucidus, 10mm. long. The 
same general form of the eye is shown in photomicrographs A and 
B, which were made from a horizontal section of the eyes of C. 
setosus. In no case out of fifteen series of sections of the eyes of 
C. setosus and C. pellucidus have I found the exaggerated conical 
form figured by Packard and by Parker. Crayfish killed in 
Perenyi’s fluid and kept in 85 per cent. alcohol for a few weeks 


, 


390 CHARLES H. SPURGEON. 


show considerable shrinkage of the connective tissue and after 
some months the cuticula shrinks. Dehydration and embedding 
also cause further shrinking. See photomicrographs A and B. 
The optic stalk may then approach the conical form observed 
by Packard and by Parker. 

The cuticula of the eyes of C. setosus and C. pellucidus is 
usually smooth. Sometimes it is wrinkled by the fixer or pre- 
servative. These wrinkles may have led Newport to the con- 
clusion that itis faceted. The main points of interest concerning 
the cuticula are; first, it is thinnest in that part of the optic stalk 
occupied by the cells of the vestigeal eye; second, it is laminated. 


Fic. 6. Horizontal section of the eye of C. setosus, with the cuticula removed 
Made from a section near the one shown in photomicrograph A. 


Sometimes the cuticula in C. setosus and in C. pellucidus is from 
two to three times as thick on the sides as it is on the anterior end 
or retinal region of the optic stalk. My photomicrographs A 
and B and Fig. 6 show that the cuticula is thinnest in the retinal 
region. According to Gilbert the average thickness of the retinal 
cuticula is 3.41 mm., while that of the sides of the stalk is 12.41 
mm. This is quite contrary to Parker’s statement that “The 
optic stalk is covered with a cuticula which is of uniform thick- 
ness.” 

It will be recalled that the cuticula is secreted by the hypo- 
dermis. The hypodermis which is of ectodermal origin becomes 
differentiated into the visual organ in Arthropoda. This dif- 
ferentiation consists of the thickening and invagination of the 
hypodermis. 


EYES OF CAMBARUS SETOSUS AND CAMBARUS PELLUCIDUS. 391 


The part of the degenerated eye of the blind crayfish which is 
of chief interest is the retinal hypodermis. Here the largest 
amount of degeneration has taken place. The optic ganglion, 
optic tract and brain show little or no signs of degeneration. 
The condition of these structures is well illustrated by photo- 


CB oR) 


2) 


Fic. 7. A secondary thickening of the hypodermis from the antero-median 
part of an eye of C. setosus. 


micrographs B, C and D. The optic nerves (Fig. 8 and photo- 
micrographs C and £) connecting the retinular cells with the 
optic ganglion are also present but they are not as well developed . 
as the above mentioned structures. 

It is probable that when the embryology of the eye of the 
blind crayfish is worked out, we may be able to definitely identify 
the retinal cells shown in Fig. 8. The eyes of a C. setosus 15 mm. 
long show about the same amount of degeneration as the eyes of 
adults. The arrangement and general appearance of the retinal 
cells of the hypodermis, shown in Fig. 8, is comparable to an 
early embryonic condition found in the developing eyes of 
many Crustacea, the bee and other Arthropoda. 

Parker states that the hypodermis in C. setosus is “very nearly 
uniform in thickness.” I find that the retinal hypodermis in C. 
setosus and C. pellucidus is quite irregular as to thickness. Some- 
times there are as many as three and four different thickened 
regions in a single section. These thickened places in the 
hypodermis are found at various places around the anterior end 
and the sides of the optic stalk. The principal and most common 
thickening is in the anterio-lateral part of the optic stalk, as is 
shown in Fig. 5 and photomicrographs B, C and E. Secondary 
thickenings are sometimes low on the sides of the optic stalk, as 
illustrated by Fig. 6 and photomicrograph A. Fig. 7 represents 
a type of the secondary thickenings. It was taken from a portion 
of the hypodermis along the anterio-median part of the right 
eye of C. setosus. The most highly developed or the least 


392 CHARLES H. SPURGEON. 


degenerated of these thickenings are in the anterio-lateral or 
retinal portion of the optic stalk. This point is well illustrated 
in Figs. 5 and 8 and photomicrographs B, C and E. ! 

The retinal region of the hypodermis has two or three distinct 
layers of cells, while the rest of the hypodermis has only one 
layer of cells, except in secondary thickenings such as are shown 
in Figs. 6 and 7 and photomicrograph A. The cells of the retinal 
hypodermis in C. setosus and C. pellucidus differ in size, shape and 
staining properties from the other cells of the hypodermis. My 
drawings and photomicrographs show that the hypodermis is 
considerable thicker in the retinal region than elsewhere. Here 


Fic. 8. Cell structure of the retinal region of the eye of C. setosus from a section 
similar to those of photomicrographs B, C and E. 


again I differ from Parker who said of the hypodermis in C. | 


setosus: ‘“‘At least it is not thicker in the region of the retina 
than at many other places.” 

The ommatidium or ocellus (Fig. 9) is the unit of structure of 
the compound eyes of Arthropoda. The functional eye of 
Cambarus has four layers of cells in the developing retinal 
hypoderinis; the corneagen, the vitrelle, the retinule and pig- 
ment cells. 

The diopteric structures of the eye, such as the lens, cone and 


: 
‘ 
5 


EYES OF CAMBARUS SETOSUS AND CAMBARUS PELLUCIDUS. 393 


rhabdom are absent in the eyes of C. setosus and C. pellucidus. 
Some of the sensory cells are present even though they may be 
no longer functional. I believe we may feel reasonably sure of 
the identity of some of the cells found in these degenerated eyes. 

In Fig. 8 there are at least three kinds of cells 
shown; (1) the large, oval, dark staining, granu- 
lar cells with several nuclei, the “granular bod- 
ies,’ ‘‘degenerated representatives of the cones 
in the normal eyes’”’ of Parker, (2) elongated cells 
or nuclei with fibers, and (3) small, round, gran- 
ular cells with clear nuclei. 

The large, oval, dark staining, granular cells 
with several nuclei are probably pigment cells 
as they are the only similar cells found on both 
sides of the basement membrane in the func- 
tional eyes as well as in these degenerated ones. 
Parker found ‘granular bodies”’ on the distal 
side only of the basement membrane and called 
them “degenerated representatives of the cones 
in the normal eye.” 

The elongated cells with fibers compare with 
the retinulz of the functional eye. The retin- - 


ulz are the only cells with fibers in retina. 

The small, round, granular cells with clear Fic. 9. The om- 
matidium of Cam- 
barus. (After Wa- 
tase.) 


nuclei may be degenerated cone cells or the 
“undifferentiated hypodermal cells” of Parker. 
But most of these cells are too far removed from 
the cuticula to be “ undifferentiated hypodermal cells”? which have 
secreted the cuticula. The cuticula is thin in the retinal region. 
According to Watase, the sensory cells of the ommatidium secrete 
the cuticula. I am inclined to believe that the small, round, 
granular cells are degenerated cone cells. 

Comparing the sections of the eyes of C. setosus and of C. 
pellucidus it is found that the eyes of setosus are little if any 
more degenerated than those of pellucidus. Irregularities are 
common in the retinal hypodermis of the eyes of these crayfish. 

Whether the eyes of the blind crayfish have passed through a 
higher stage of development and then degenerated or whether 


394 CHARLES H. SPURGEON. 


their development has been arrested at this stage, can be deter- 
mined only by a study of the developing eyes. 

However I believe that the degenerated eyes of C. setosus and 
C. pellucidus are instances of arrested development rather than 
examples of degeneration. The reasons for this belief are that 
the eyes as found in the adult have a cell structure which appears 
to be comparable to the developing eyes of Crustacea. The 
radiate arrangement of the retinal cells of the hypodermis, is 
suggestive of the developing ommatidium. Also the eyes of 
the young blind crayfish, C. setosus and C. pellucidus, show about 
the same amount of degeneration as the eyes of the adult. 


EYES OF CAMBARUS SETOSUS AND CAMBARUS PELLUCIDUS. 395 


LITERATURE CITED. 
Garman, Samuel. 


89 Cave Animals from Southwestern Missouri. Bull. Mus. Comp. Zodl. at 
Harvard College, Vol. XVII., No. 6. 
Gilbert, Abigil. 
’97 A Study of the Degenerated Eye of the Blind Crayfish. (Made at Indiana 
University—Unpublished.) 
Leydig, F. 
783 Auge und Antennen des Blinden Krebses der Mammuth-Héhle. Bonn. 
Newport, George. 
*55 On the Ocelli in the Genus Anthophorabia. Trans. Linn. Soc. London, 
Vol. XXI. 
Neher, Edwin M. 
?0r The Eye of Palemonetes Antrorum. Proceedings of the Indiana Academy 


of Science. 
Packard, A. S. 
73 On the Cave Fauna of Indiana. Fifth Report of the Peabody Acad. Sci., 
Salem. 


788 The Cave Fauna of North America, with Remarks on the Anatomy of the 
Brain and Origin of the Blind Species. Mem. Nat. Acad. Sci., Vol. IV. 
Parker, C. H. 
’90 Histology and Development of the Eye in the Lobster. Bull. Mus. Comp. 
Zool. at Harvard College, Vol. XX., No. fr. 
790 ©The Eyesin Blind Crayfishes. Bull. Mus. Comp. Zoél. at Harvard College, 
Vol. XX., No. 5. 
Shafer, G. D. 
’07 Histology and Development of the Divided Eyes of Certain Insects. Pro- 
ceedings of the Washington Academy of Sciences, Vol. VIII. 
Grenacher. 
’79 Untersuchungen uber das Sehorgan der Arthropoden, Insbesondere der 
Spinnen, Insecten und Crustacean. Gottingen. 
Pike, F. H. 
’06 The Degenerate Eyes in the Cuban Cave Shrimp, Palaemonetes Eigenmani 
Hay. Butov. BULL., Vol. XI., No. 5. 
Watase, S. 
790 ©=On the Morphology of the Compound Eyes of Arthropods. Studies from 
the Biological Laboratory of Johns Hopkins University, Vol. IV., No. 6. 


396 . CHARLES H. SPURGEON. 


EXPLANATION OF FIGURES AND ABBREVIATIONS. 


The tissue was fixed with Perenyi’s fluid. The sections from which the drawings 
and photomicrographs were made were cut eight microns in thickness and stained 
with Haidenhain’s iron hematoxylin. The drawings were made with the aid of a 
camera lucida. 


bm., basement membrane. opt. g. f., optic ganglion fibers. 

br., brain. opt. n., optic nerve. 

c., cornea. : opt. tr., optic tract. 

cc, crystalline cone. pg. c., pigment cell. 

cg., corneagen cell. ve., retina. 

con., connective tissue. ret., retinule. 

cu., cuticula. rha., rhabdom. 

hy. c., hypodermal cells. sec. th., secondary thickenings of the 
mus., muscle. hypodermis. 

opt. g., optic ganglion. v., Vitrella. 


PHOTOMICROGRAPH A. Horizontal section of the eyes of C. setosus. 

PHOTOMICROGRAPHS B AND C. Horizontal sections of the eyes of C. sefosus, 
C has the cuticula removed. The cuticula in B is thinner in the retinal than it is 
on the sides of the optic stalk. It is not as thick as it appears to be in the retinal 
region, due to the fact that it is wrinkled. It is also pulled loose from the under- 
lying hypodermis, caused by the shrinking of the more delicate tissues. 

PHOTOMICROGRAPH D. Horizontal section of the brain of C. setosus. 

PHOTOMICROGRAPHS E aND F. Horizontal sections of the eyes of C. pellucidus. 
The cuticula has been removed. F has a tract of nerve fibers extending anterio- 
medianly towards the hypodermis, but there is no thickened portion of the hypo- 
dermis with which it is connected. : 


BIOLOGICAL BULLETIN, VOL. XXVIII. 


CHARLES H. SPURGEON. 


PLATE I. 


eee SLA a tha te eee 


STUDIES ON TISSUES OF FASTING ANIMALS. 
S. MORGULIS, PAUL E. HOWE AND P. B. HAWK. 


The changes rendered in the finer structure of tissues of fasting 
animals have been extensively studied and the results of these 
investigations have an important bearing upon our understanding 
of the inanition phenomena in general. Apart from the interest 
which the subject presents from a purely histological point of 
view, it throws light on many obscure problems regarding the 
transformation of materials within the organism occasioned by 
the fast. 

The account here presented is based upon an examination of 
tissues from several dogs and one fox which had died of pro- 
tracted inanition, having previously suffered a very large loss 
in body weight. These animals had been used in a number of 
metabolism experiments! conducted some years ago in the 
University of Illinois? The tissues were removed immediately 
after the animal’s death and fixed in Teleschnitzky’s and Zenker’s 
fluids. The material was carried-through graduated alcohols 
and then preserved in eighty per cent. alcohol. It was embedded 
in paraffin, sectioned and stained in Delafield’s hemotoxylin, with 
eosin as a counterstain. 

A superficial examination of the sectioned material, except in 
a few instances, reveals nothing abnormal. Buta little attentive 
study is sufficient to appreciate the different ways in which the 
effect of prolonged inanition is stamped upon the histological 
elements of the organism. 

Looking at the smooth muscles in the intestinal tract of 
every one of the animals which died of fasting the cells appear 
turbid and without a trace of longitudinal fibrillation. The 


1 Howe, Mattill and Hawk, Jour. Biol. Chem., 10, 417, 1911 and 11, 103, 1912. 
Howe and Hawk, Jour. Am. Chem. Soc., 33, 215, 1911; Am. Jour. of Physiology, 
29, Xiv., 1912, and 30, 174, I9I2. 

2 We take this opportunity to acknowledge the material assistance which we 
received from the department of chemistry of the University of Illinois in defraying 
the expenses of the research. 


397 


398 S. MORGULIS, PAUL E. HOWE AND P. B. HAWK. 


fibers seem widely separated from each other, giving the entire 
muscle a very loose appearance. In cross section they are seen 
to consist of a dense central portion, which stains more or less 
strongly, surrounded by a colorless material. There is, however, 
no indication of a swelling of the muscle fibers as there is likewise 
no evidence of fatty degeneration, but they apparently undergo 
a process of liquefaction similar to that described by Miescher 
in the Rhein Salmon occasioned also by protracted fasting while 
it remains in fresh water. The nuclei are extremely irregular 
in outline and stain faintly. 

In the voluntary, or striated muscle fibers, the cross markings 
lack the usual distinctness. Swelling or granular degeneration, 
such as described by Statkewitch, was never seen in our material. 

Of all the organs of the body the liver is taxed most heavily 
during inanition inasmuch as it must take care of the products 
of metabolic activity of all other organs besides sustaining itself. 
It is natural to expect, therefore, to find the changes in the 
structure of the liver cells of a most pronounced character. 
Indeed, in the material under our examination a variety of 
degenerative phenomena has been observed. Considering the 
great difference in the degree of degeneration of the liver from 
animals which have all died of starvation it follows that death is 
not necessarily preceded by extreme cellular transformation. 
In our material every gradation from very slight changes to 
complete fatty degeneration of the liver cells could be observed. 
In two dogs which fasted 30 and 48 days respectively, whereby 
they lost 46 and 53 per cent. of their weight, there has been very 
little fatty degeneration in the liver. Some cells, however, were 
coarsely granular and others were riddled with vacuoles. In the 
case of the fox, which in 13 days of absolute fasting lost only 
13 per cent. of its weight, the cells were found to be hollowed out 
by vacuoles of various sizes. These frequently encroach upon 
the nucleus and distort its shape as may be seen in Fig. 1. The 
vacuoles never show a very sharp outline, their boundary being 
more commonly diffuse and indefinite. In one extreme case of 
degeneration the liver presented complete transformation of its 
cells into typical fat cells. The polygonal shape of the cells was 
retained but the protoplasm was reduced to a mere band enclosing 


STUDIES ON TISSUES OF FASTING ANIMALS. 399 


a mass of fat. The cells seemed rather distended. The nuclei, 
pushed out to the periphery and usually into a recess of a corner, 
were flattened against the wall. Their staining capacity as well 
as that of the protoplasm was very feeble. The fatty degenera- 
tion was not equally intense in every portion of the liver, and 
here and there groups of intact liver cells could be seen whose 
poor staining power was the only evidence of degeneration. 

No particular changes have been observed in nuclei. Cells 
with more than one nucleus are not uncommon, but these are 
found also in the normal liver. Phenomena of chromatolysis 
and vacuolization of the nuclei described by Statkewitch were 
never observed by us. 

The histological structure of the stomach and intestine shows 
no striking changes. In sections of the stomach the oxyntic or 
parietal cells of the fundus glands are most conspicuous owing 
to their relatively large size and deep staining capacity. Their 
protoplasm is very granular. The other cells of the gland are 
small and their protoplasm is thin and practically colorless. The 
nuclei are usuaily normal, but in some portions, especially near 
the proximal end of the gland, they are much elongated and 
pressed against the cell wall adjoining the basal membrane. 
The two figures in the plate, 2 and 3, one a cross section of the: 
upper region of the gland, the other a longitudinal section 
through the base of the gland, show these points. The clear, 
transparent character of the protoplasm is very well seen in 
the former, Fig. 2. The nuclei are always near the basal mem- 
brane. 

The points brought out in the study of the fundus glands are 
also essential for all other glands as well as the mucous membrane 
of the intestine. The cells stain very feebly, their protoplasm 
being free of any granules. The nuclei migrate toward the basal 
membrane. 

The phenomenon of particular interest, especially when viewed 
in the light of certain results of bacteriological studies on the 
permeability of the intestinal canal, is the invasion of the tissue 
underlying the mucous membrane as well as of the cells of the 
mucous membrane itself by numerous leucocytes. These occur 
not only singly but in groups of several cells together and occa- 
sionally accumulate in masses resembling solitary glands. 


400 S. MORGULIS, PAUL E. HOWE AND P. B. HAWK. 


The submaxillary gland presents a few changes which are 
worth pointing out. The protoplasm of its cells, as was seen 
also in other gland cells, is thin in character and fails to take up 
the stain. Many cells are without nuclei, and the darkly stained 
crescent cells though present are generally flattened out and cap 
the outside of the alveoli like a narrow band. Fig. 4 shows that 
the submaxillary gland at the fatal termination of a protracted 
fast has all the appearance of a resting gland. 

The most interesting set of modifications is to be observed in 
the kidneys. There one encounters various forms of degenera- 
tion and their distribution in the kidney is quite significant. 
We have already mentioned in discussing the changes in the 
liver that the extent of the degeneration of the histological 
elements apparently bears no relation to the death of the fasting 
animal as one of its direct causes. This statement holds equally 
true for the kidneys, where we found likewise a very wide range 
of modifications at the time of the animal’s death. 

The glomerulus has the usual lobulated structure but the 
Bowman capsule enclosing it is invariably thickened as in the 
case of nephritic kidneys. The cells of the convoluted tubules 
have a coarsely granular content and are invariably vacuolated. 
In some instances the vacuolization is so extensive as to give 
the tubule a striking honeycombed appearance. In Fig. 5 
which is from a section of the kidney of the fox, this is shown 
very clearly. Similarly Fig. 6, which represents a section of a 
tubule in the kidney of a fasting dog, shows extensive vacuoliza- 
tion and the absence of boundaries between the cells. This last 
phenomenon, namely, the formation of a syncitium is char- 
acteristic not only of the kidney but also of the liver where the 
cells seem to melt together. In the ascending and descending 
limb of Henle’s loop, however, vacuolization is a very rare 
occurrence, particularly in the later. The tubules were generally 
very granular in structure and contained frequently casts of 
various kinds, cellular, hyaline, etc. Fig. 5 is interesting further- 
more on account of the well-preserved ciliated band lining the 
lumen of the tubule. The nuclei of the tubules are small and 
more or less irregular in shape. The cells of the collecting tubules 
show hardly any effect. The protoplasmic content is very clear 


STUDIES ON TISSUES OF FASTING ANIMALS. 401 


and free of granules. Here and there cells are found which have 
no nuclei. But when present the nuclei are relatively large and 
round, frequently bulging out into the lumen owing to the diminu- 
tion of the cubical cells. 

Before concluding this description of the changes which were 
observed in the tissues of fasting animals, a few words may be 
said concerning the condition of the testes and ovaries. In the 
former we failed to find any dividing cells. The nuclei were of 
the characteristic large round shape, whose chromatic content was 
intensely stained. It is noteworthy that ina very large proportion — 
of the tubules the chromatic substance of all the nuclei was 
massed together to one side, as in the case of synizesis. It is 
hardly possible that this should be due to an artifact, as the 
nuclear condition varied in different tubules, but the former was 
found in most of them. The ovary which was examined seemed 
normal in every respect with numerous eggs in all stages of 
growth. We examined a number of fully developed eggs which 
were perfectly normal in every detail of their structure. 

Bearing in mind that in inanition the organism is obliged to 
draw upon its own resources to derive the energy necessary for 
its maintenance, the metaplasmic material stored up in its cells 
and the dep6ts of fat are first to yield their quota to this stringent 
need. With the prolongation of the fast, as these reserve ma- 
terials become reduced in quantity and at last disappear alto- 
gether, the substance of the cell body proper must contribute 
to the organism’s demand for nourishment. It is now a well- 
established fact that various organs and tissues share unevenly 
in the support of the starving organism. As would be expected 
a priort, those elements of the organism the integrity of which is 
indispensable to its continued existence resist the pressure of the 
unfavorable conditions longest. This is true not only for the 
different systems of organs, but also for the minutest element of 
the organism, the cell, where the nucleus is usually the last part 
to fall prey to the exhausting effect of the fast. The nervous 
system likewise maintains its weight practically at a constant 
level as well as it preserves its morphological integrity until a 
very advanced stage in the fast. 

Degenerative changes do not, as a rule, occur in any of the 


402 S. MORGULIS, PAUL E. HOWE AND P. B. HAWK. 


tissues so long as the reserves of the body have not yet been 
entirely exhausted. The early appearance of fat globules in the 
liver of fasting animals led Mottram to believe that this process 
must be a physiological and not a pathological one. A similar 
opinion was likewise expressed earlier by Gilbert et Jomier. 
Mottram showed by means of histological examination of the 
liver of rabbits and guinea-pigs as well as by actual chemical 
investigation that with the advance of the fast an infiltration of 
the liver cells with fat from the depéts does take place. None 
of the authors who gave attention to this matter studied the 
liver of animals in very advanced stages of a fast. In our own 
case the animals succumbed after a loss of about 50 per cent. of 
their weight. There. was very little histological evidence of a’ 
fat accumulation, but vacuolization of the cells was most promi- 
nent. It is hardly conceivable that the vacuoles were produced 
by the removal of the fat content by the reagents used in pres- 
ervation in as much as it has been shown above that in one 
instance a liver was observed the cells of which in certain localities 
have undergone complete fatty degeneration. We are aware of 
the fact, of course, that a parallelism does not exist between 
histologically and chemically demonstrable fat in tissues. It 
may be that the infiltration of the liver with fat, especially in 
the early period of fasting, which is now proven beyond reason- 
able doubt (Mottram, Smirnow) and is very properly considered 
a physiological phenomenon, is concerned with the transfer of 
depdét fat to the rest of the tissues as food, while fatty degenera- 
tion of the liver cells, such as we observed over certain areas, 
is an independent phenomenon and is accompanied by the loss 
of the normal functional power of the cells. The view expressed 
here that the infiltration of the liver with fat may have to do 
with the conveying of the fat as nutriment to the starving 
tissues is borne out by Mottram’s interesting observation on 
the qualitative change of the liver fat on different days of a short 
fast. There is a striking parallelism between the pure fatty 
acids present in the liver and the fat-quotient, 7. e., the ratio of 
the total fat of the liver to the initial body weight, showing that 
whenever an increase in fat content, 7. e., an infiltration, occurs 
it is due to an accumulation of fatty acids. If this view of the 


STUDIES ON TISSUES OF FASTING ANIMALS. 403 


role of the liver is correct it may also clear up the problem of the 
cause of the premortal rise in the nitrogen elimination. The 
latter was thought to be due either to an exhaustion of the entire 
supply of fat or to an excessive disintegration of cells. Neither 
the one nor the other of these hypotheses can be considered 
beyond criticism, because even in animals succumbing to a 
much protracted fast there is still sufficient fat present,! whereas 
there is no histological evidence of an unusual cellular destruction 
towards the end of the fast. A morphological and physiological 
degeneration of the liver interfering in some manner unknown to 
us with the endogenous fat metabolism probably results in an 
increased demand upon the body proteins which hastens the 
death of the animal. 

In this connection it is interesting to point out that fat has 
never been demonstrated in tissues which in the fasting organism 
are among the strongest consumers, such as nervous and muscular 
tissues. The glandular tissues on the other hand, which are 
more or less deprived of their proper activity during a fast 
invariably show the presence of fat globules, according to 
Nicolaides. Later these fat globules disappear, leaving ‘empty 
spaces’’ which evidently correspond to our vacuoles. Nicolaides 
observed that in the gland cells of the duodenum and the pylorus 
small fat globules appear as soon as the animals commence to 
fast, whereby they invariably assume a regular arrangement in 
two parallel rows. We cannot agree with Nicolaides who con- 


b) 


siders the fat globules as “degenerative,” and certainly see no 
reason for his assumption,—since their arrangement within 
the cell points against the supposition of a migration from fat 
depé6ts,—that they are formed from the protein constituents. 
The fact that in the submaxillary gland the fat globules appear 
only in the albuminous cells but never in the mucous or crescent 
cells, which has been observed by both Statkewitch and Nico- 
laides, cannot be taken as good proof of a formation of fat from 
protein. Also these facts become plain in the light of our 
hypothesis that the early appearance of the fat globules speaks 
decisively against any supposition that they result from degenera- 
tive transformation. We believe, on the contrary, that this is 


1 Tn the case of a dog which fasted 117 days and subsequently 104 days there were 
large masses of fatty tissue in the abdominal cavity at the time of death. 


404 S. MORGULIS, PAUL E. HOWE AND P. B. HAWK. 


simply due to a qualitative transformation of the fat present in 
the cells whereby it becomes histologically visible. Since the 
staining reagents which are used in demonstrating the presence 
of fat globules are such that they react only with unsaturated 
fats, the appearance of the globules indicates that with the 
beginning of the fast a process of desaturation and probably the 
formation of fatty acids is started preliminary to the absorption 
of this fat to serve the nutritional needs of the organism. 

The results of studies on fasting unicellular organisms where 
the conditions are simpler and easier to be appreciated support 
the view that vacuolization is one of the earliest and the most 
common degenerative process which ensues with the exhaustion 
of the reserves of the cell. Wallengren indeed in his most valu- 
able research on inanition of infusoria distinguishes two periods, 
before and after the reserve material is exhausted. The former 
is accompanied by a gradual diminution of the animal, while in 
the second period the endoplasm becomes honey-combed with 
vacuoles of various sizes. 

Vacuolization has been observed in various tissues: in gangli- 
onic cells of the heart (Statkewitch), in the cells of the motor 
ganglia of the anterior horn (Schaffer) in bone marrow (Soltz), 
in the nephridial epithelium, etc. 

Before concluding the paper mention should be made of an- 
-other degenerative process which develops in the course of 
inanition. We refer to the gradual melting away of the cell 
boundaries which, with their complete disappearance, may even 
result in the formation of a syncytium. One of us described 
this condition in the liver of fasting salamanders. We also 
observed this phenomenon in our liver and kidney preparations. 
Similar observations have been made in fasting lower animals 
(Schultz). In the study of the salamanders it was shown how 
rapidly the cell walls are built up again around the intact nuclei 
as soon as the emaciated animals are once more given food. 

At last the more or less universal loss of staining capacity due 
to the degenerative transformation produced by inanition and 
described by practically all authors must be pointed out. 


STUDIES ON TISSUES OF FASTING ANIMALS. 405 


LITERATURE CITED 
Gilbert, A. et Jomier. 
96 Etude histologique du foie pendant Vinanition. Bull. et Mém. Soc. anat. 
de Paris, 81, 301-314. 
Nikolaides, R. 
’99 Ueber den Fettgehalt der Driisen im Hungerzustan de und iiber seine 
Bedeutung. Arch. f. Physiol., 518-523. 
Morgulis, S. 
’z1 Studies of Inanition in Its Bearing Upon the Problem of Growth. Arch. f. 
Entwicklungsmech, 32, 169-268. 
Mottram, V. H. 
’09 ©Fatty Infiltration of the Liver in Hunger. Jour. of Physiol., 38, 281-314. 
Schaffer, K. 
’97 Ueber Nervenzellenveranderungen wahrend der Monition. Neurol. Cen- 
tralbl., 16, 832-837. 
Schultz, E. 
%08 Ueber Hunger bei Asterias rubens und Mytilus bald nach der Metamorphose. 
Arch. f. Entwicklungsm., 25, 401-407. 
Smirnow, M. R. 
’13. The Effect of Water Ingestion on the Fatty Changes of the Liver in Fasting 
Rabbits. Amer. Jour. Physiol., 32, 309-314. 
Soltz, O. S. : < 
’94 The Anatomical Changes in the Bone Marrow of Animals Subjected to 
Complete Inanition and Subsequently Fed Again. An experimental study. 
Diss. No. 27 of the Imperial Military Medical Acad. St. Petersburg (Russian). 
Statkewitch, P. 
’94 Ueber Veranderungen des Muskel- und Driisengewebes, sowie der Herz- 
ganglien beim Hungern. Arch. f. exper. Pathol. u. Pharmakol., 33, 41 5-462. 
Wallengren, H. 
’o92 Inanitionserscheinungen der Zelle. Zeitschr. Allgem. Physiol., 1, 67-129. 


406 S. MORGULIS, PAUL E. HOWE AND P. B. HAWK. 


EXPLANATION OF FIGURES. 


All figures have been made with the camera lucida and under the same magni- 
fication, using an objective No. 6 with an ocular piece No. 4 at normal tube length. 

Fic. 1. A group of liver cells of a fasting fox. 

Fics. 2 AND 3. Cross-section and longitudinal section of fundus gland of a 
fasting dog. 

Fic. 4. Section of submaxillary gland of fasting dog. 

Fic. 5. Section of tubules in kidney of fasting fox. 

Fics. 6 AND 7. Section of convoluted tubule and of Henle’s loop in kidney of 
fasting dog. 


BIOLOGICAL BULLETIN, VOL. XXVII._ . PLATE 1. 


MORGULIS, HOWE AND HAWK. 


THE OLFACTORY SENSE OF COLEOPTERA 


N. E. McINDOO, Pu.D., 


BUREAU OF ENTOMOLOGY, WASHINGTON, 1DEXGH 


CONTENTS. 

PAGE 
nNOS TOM ANG! MMBUNOESs o os0o5accns0csa0s0 ess suc dnc Hoaso Don Oe HOH O I: 408 
Morphology of the olfactory pores. ......--- +++ +++ sees etree sees t tees 410 
TOVynoamttilovi Ug b nm de clee2 ae ore any See ia a Simao cee by Fea o or pda tc 410 
(@) IBGE Lae: WeRAHISs . o6 o> Sonat OD ee Be Roane Deeb OouO POPS 0 0552 AII 
(D) Oenae SOLIS a ooo s 05s So aemeaN ae aocsceU Mer soo Gnd op le os Fer AT4 
(c) Individual and sexual VAT a tLLOTS Si pn ee Oe Ee een ae A418 
Siro AS) gin sk bo 0 ohne 0 yb eid or Dae NORTE oie Ga a chioneeapeoie cio eee Go) 3 ccika etosare hoa 7c 420 
(@) Dsxiearall GuANCHURS. oo 6c (Pe pome ne Bap se Reo eS Reo Das R cae oes eee 420 
(OD) Thartennell SienCHwe. . soon eodaaedeee oles ued mens Nebo Gaede ste R a 420 
Experiments to determine the location of the olfactory oreans see a) 420 
The olfactory sense of Harpalus pennsylvanica.......-.++-+---2+2+ 2227s A28 
(a) Effects with antennee UIE! Otte aiem seh ab eaduime danse >e2ssedeo0 430 
(b) Effects with elytra and ebavers) UIUC! Ott; wo ono caer ooee area yp sce A31 

(c) Effects with elytra and wings pulled off and pores on legs covered with 
PART > oc cad oro pn odo Seles Scola Sheets Bo.c oe eRe re 432 
The olfactory sense of Harpalus caliginosus......-------++++2s seer trees 433 
(Gy ibabectsiwithyancvennes pullediofi 2). 0. 433 
The olfactory sense of Epilachna borealis.....---------- ++ ++ -22 ese s ste A34 
(a) Effects with antenne pulled off........----------++s sets s scenes 434 
(b) Effects with elytra and wings plilledtotie te aae cee 435 
The olfactory sense of Chaulcognathus pennsylvanica.........--------+--- 436 
(@mbtectsswitheantennas pulledt of yoo x. oy 5) Sol ea as 436 
The olfactory sense of Passalus cornutus.....---+-++-+-+++2+s 0 soos a 436 
(a) Effects with antenne pulled Off eR eae ea ees te mam Ons clo ors, A e-or5 0 437 
The olfactory sense of Cotimis nitida.........---+- +++ ene 437 
@) Effects with antenna pulled off... .. 2. 2+ +32 - 2H ae A437 
The olfactory sense of Euphoria Sepulohralis. «2 aj.6 2 sai A437 
(@\Etects withvantenne: pulled othe) -)-7.° 5-5 438 
The olfactory sense of Cyllene robini@......--- +++ 2222 ttt tts 438 
@) Effects with antenne pulled off.....-.-.------2 0223s ae 438 
(b) Effects with elytra and wings plilled\ oft sclerotic 438 
The olfactory sense of Leptinotarsa ro-lineala.....--- +--+ +000 439 
(@)) Bifects with antenne pulled off2.-...--.-- 4... --) os. 440 
(b) Effects with elytra pulled off and wings cut off...2....--:2-.---.-- 440 

(c) Effects with elytra pulled off, bases of wings glued and pores on legs 
covered withivascliness ae seco: = cis noone 441 
The olfactory sense of Epicauta marginata.......------+++s sess ttt tes A42 
(a) Effects with antenne cut off......... es BRU ane am ge Mt 2s 443 
(@ywEtectstwithvartemnce pullledl oft 9-55. <i yy i a ia 443 

407 


408 N. E. MCINDOO. 


(c) Effects with elytra and wings pulled off.......................... 443 

The olfactory sense of Epicauta pennsylvanica.......................... 443 
(@)eEittects withvantenncespulledtocieeie eee ene nee 444 

(2) Pe Eittectsiwithvelytravane mwanecE pple corinne eier liane ee ae AAA 
SUUMUTTA TVs: 6 fed Sa ae a a Roe oo tod te ete eg 447 
TD ISCUSSTOM scoot Bh ae eae cau a lee RIS Oe Sea oc chs ns 6 lence RR 451 
Titeraburé: Cited si. cee a) 2 2s oie eee ae AES cae) ae ae 455 
Explanation of plates Mr. and) lie vis saves ee eas coin 6) Ae set cas ee 456 


INTRODUCTION AND METHODS. 


In the investigation here recorded two objects have been 
kept in view: (1) To make a careful study of the morphology and 
physiology of the olfactory pores of beetles, and (2) to determine 
experimentally whether or not the olfactory organs lie in the 
antenne. 

Since those investigators, who have performed experiments on 
beetles with mutilated antenne, have failed to study sufficiently 
the behavior of the insects investigated, the responses observed 
have misled them in determining the seat of the olfactory organs. 
Entomologists are generally agreed that the organs of smell in 
beetles lie in the antenne, but when the results of those who have 
performed experiments on beetles are carefully considered, it 
is seen that some beetles with amputated antenne smell prac- 
tically as well as unmutilated ones, while other beetles are 
materially affected when the antenne are mutilated. Hicks 
(1857 and 1860) discovered some peculiar organs (called olfactory 
pores by the present writer) on the wings and legs of beetles 
and he suggested that they have an olfactory function. Lehr! 
discovered the same organs on the peduncles of the elytra of 
Dytiscus marginalis. The present writer (14a and 6) made a 
comprehensive study of the olfactory pores in Hymenoptera and 
he ('14c) gives a complete review of the literature pertaining to 
the sense of smell in insects. The present paper embodies the 
results of a careful study of the olfactory pores in Coleoptera in 
much the same manner as pursued on those in Hymenoptera. 

To obtain material for the study of the disposition of the 
olfactory pores, adult specimens were used. In regard to prepar- 


1 Lehr’s paper, which deals only with the morphology of these organs, was over- 
looked until after my paper had been sent to press. Lehr has not seen any of my 
papers on this subject because my first one (’14a) appeared only three months 
before his, and my second one (’14b) appeared in the same month as his. 


ae 
ee ae z 


' 
f 


en oe a 


—— 


THE OLFACTORY SENSE OF COLEOPTERA. 409 


ing the specimens with caustic potash and to bleaching them 
with chlorine gas, the reader is referred to the writer’s work on 
Hymenoptera (14), p. 295). 

To obtain material for the study of the internal anatomy of 
the organs herein discussed, beetles just emerging from the last 
pupal stage were mostly used. At this stage the chitin is soft, 
the wings are usually expanded, and the sense organs are fully 
developed. In order that the desired stages of beetles might be 
had, many larve and pupe of various Coleoptera were collected 
on plants and in rotten stumps and logs. These immature 
insects were reared in the laboratory. When each one of them 
had reached the proper stage, it was killed and parts of it were 
put into a fixing fluid. 

The writer (14a, p. 268) describes the usual method of em- 
bedding with celloidin and paraffin. Since then, a rapid method 
has been used which is described in detail as follows: The various 
appendages of the insects are removed, and are cut into small 
pieces, which are immediately dropped into a modification of 
Carnoy’s fixing fluid. This fluid, containing equal parts of 
absolute alcohol, chloroform, and glacial acetic acid, with corro- 
sive sublimate to excess, should be kept in a glass-stoppered 
bottle so that it may not lose its fixing ability by air being mixed 
with it. Also, while dropping material into vials containing this 
fluid, the stoppers of the vials. should not be removed longer than 
absolutely necessary. When the material sinks to the bottom of 
the vial, it is removed and is thoroughly washed in 85 per cent. 
alcohol. It is then preserved in 85 per cent. alcohol. When 
ready for embedding, the material is cut into pieces from two to 
four millimeters in length. These pieces are then put into 95 per 
cent. alcohol containing eosin. When sufficiently stained, they 
are placed in a vial containing absolute alcohol and cedar oil. 
As soon as they sink through the alcohol into the oil and lie on 
the bottom of the vial, the alcohol and oil are removed. A small 
amount of ether is then poured into the vial. Five minutes later 
the ether is removed, and thin celloidin is poured into the vial. 
Ten minutes still later the thin celloidin is exchanged for thick 
celloidin. After remaining in the thick celloidin five minutes, 
the pieces of material are removed and are put into a vial of 


AIO N. E. MCINDOO. 


chloroform where they remain five minutes. They are then em- 
bedded in 55° M.P. paraffin for five minutes. The sections were 
cut from five to ten microns in thickness and when they failed 
to ribbon the microtome knife was warmed. From this stage 
on the sections are treated like ordinary paraffin sections with 
the following exceptions. A rather thick film of fresh Mayer’s 
albumen is spread upon each slide. After drawing the water 
from the slide upon which are mounted the sections, the latter 
are flattened to the slide by using a piece of wet tissue paper. 
No heat is used for straightening the ribbons on the slides because 
the least amount of heat blisters the celloidin. After drying 
over night, most of the sections adhere to the slides while being 
passed through the reagents, but to be sure of not losing any 
sections, the slides were sometimes: wrapped in tissue paper and 
thread was then firmly wound around the paper. Instead of 
using absolute alcohol a mixture of equal parts of absolute 
alcohol and chloroform is employed so that the celloidin may 
not be dissolved, and instead of using eosin in 95 per cent. alcohol 
as a counter stain, the eosin is put into a mixture of the absolute 
alcohol and chloroform. The sections were stained in Ehrlich’s 
hematoxylin from 10 to I5 minutes, the time depending on 
their thickness and whether or not they were wrapped in tissue 
paper. 

The writer is grateful to Mr. H. S. Barber of the Bureau of 
Entomology for most of the dried specimens used which belonged 
to the collections of the U. S. National Museum. Mr. Barber 
is also to be thanked for the identification of all the beetles used 
in the experimental part of this work. 


MORPHOLOGY OF THE OLFACTORY PORES. 


Before experimenting to determine the function of the organs 
called the olfactory pores by the writer (14a), the distribution 
and number of these pores in many beetles were studied. 


DISPOSITION. 


In making a comparative study of the disposition of the ol- 
factory pores in beetles, 50 species, belonging to 47 genera and 
representing 34 families, were used. With the exception of two 


THE OLFACTORY SENSE OF COLEOPTERA. All 


species used for individual and sexual variations, only one speci- 
men of each species was studied. Whenever a portion of an 
appendage or an entire appendage was missing or was badly 
mutilated in being prepared for study, the number of pores on 
this portion or entire appendage was regarded the same as the 
number found on the corresponding portion or entire appendage 
on the opposite side of the body. Since the pores on only one 
specimen for each species were counted, the total number of 
pores recorded can not be a fair average. Besides this error, 
there is also a probable error of not less than Io per cent. on an 
average for all the specimens. In the smaller specimens the 
probable error is perhaps not more than two or three per cent., 
but in some of the larger ones, this error is probably more than 
Io per cent. The pores on only the legs, elytra and wings are 
- included in the total numbers. Other parts of the insects were 
not examined, and it is quite possible that olfactory pores may 
be found on some of the parts not examined, particularly on the 
mouth parts. 


(a) Epilachna borealis. 


Since the lady beetle, Epilachna borealis, is most conveniently 
studied and as its pores are typical for most of the smaller beetles, 
the disposition of its pores will be described in detail, and then 
the variations found in the other species will be given. 

The elytra and wings have dorsal and ventral surfaces, and the 
legs may be divided for description into two surfaces. The inner 
surface faces the body of the beetle and the outer surface is 
directed from the body. On the specimen examined, one group 
of pores was found on the peduncle of each elytron; three groups 
besides a few scattered pores on each wing; and two groups 
besides a few scattered pores on each leg. The groups and 
scattered pores are located as follows: Group No. I lies on the 
dorsal surface of the peduncle of the elytron with its distal or 
broader end against or just beneath the basal margin of the 
elytron (Text-fig. 14, BM). Under a high-power lens, it is seen 
that this group lies on the radial plate (Text-fig. 1B, RP) between 
the muscle disk (VD) and the subcostal head (ScH). The distal 
ends of these heavy chitinous plates sometimes lie beneath the 


412 N. E. MCINDOO. 


basal margin (BM) of the elytron so that all or a portion of the 
group may be concealed. In such a case it is necessary to pull 
the peduncle from beneath the base of the elytron in order to 
count the pores. Group No. 1 on the left elytron consists of 
71 pores (Plate I., Fig. 4), while the same group on the right 
elytron has 78 pores. 

Groups Nos. 2, 3 and 4 lie on the dorsal surface of the wing on 
the radius (Text-fig. 1C, R). No. 2 lies on the extreme anterior 


Fic. 1. Portion of left elytron and left wing of the lady beetle, Epilachna 
borealis, showing groups I to 4 of olfactory pores, as indicated by the numbers I to 
4; A shows relative sizes of peduncle of elytron and group of pores on peduncle 
when compared with size of basal margin (BM) of elytron; A and B, dorsal surface 
of peduncle of elytron, showing position of group 1 of olfactory pores on radial 
plate (RP) between muscle disk (MD) and subcostal head (ScH). The lower side 
of each drawing is the outer margin of the elytron. A, X 8; B, X 45; C, dorsal 
surface of wing, showing position of groups 2 to 4 of olfactory pores on radius (R), 
X 8; a, position of scattered pores on ventral side of wing on union of costa (C) 
and subcosta (Sc) near fold of wing (Fo). Sometimes a group is found on the 
media (Me) just below group 4. 


end of the radius and it is usually difficult to count its pores, 
because the surface of the radius at this place is greatly arched 
causing some of the pores to lie on the top of the arch while the 
remainder of them lie on the side of the arch facing the anterior 
margin of the wing. Nos. 3 and 4 are found on the radius where 
the media (Me) joins the radius. On the right wing, No. 2 


THE OLFACTORY SENSE OF COLEOPTERA. AI3, 


Fic. 2. Position of olfactory pores on legs of beetles, X 8; A—F, legs of lady 
beetle, Epilachna borealis, showing position of groups 5, 6, band ¢ of olfactory pores. 
The drawing of each leg in which the tarsus (Tar) is shown represents the outer 
surface of that leg, and the drawing not showing the tarsus represents the inner 
surface of the same leg. A, right front leg; B, left front leg; C, right middle leg; 
D, left middle leg; E, right hind leg; F, left hind leg. G, distal end of tibia from front 
leg of Epicauta marginata, showing five olfactory pores on one of the two tibial 
spines. 


consists of 55 pores; No. 3 of 43 pores; and No. 4 of 43 pores. 
On the left wing, No. 2 consists of 50 pores; No. 3 of 43 pores; 
and No. 4 of 46 pores. 

Group a of the scattered pores lies on the ventral surface of the 


414 N. E. MCINDOO. 


wing near the anterior margin of the wing a short distance from 
the place where the wing folds (Text-fig. 1C, a). On the right 
wing it consists of three pores and on the left wing of five pores. 

Groups Nos. 5 and 6 are located at the proximal end of the 
trochanter (Text-fig. 2A—-F, Tro), No. 5 lying on the outer 
surface and No. 6 on the inner surface. No. 5 usually extends 
only about half way across the leg, while No. 6 extends nearly all 
the distance across the leg. No. 5 on each leg consists of five 
pores, except on the left front leg where there are seven init. On 
the right side No. 6 on each leg consists of seven pores, whereas 
on the left side on each of the middle and hind legs it consists of 
eight pores but of only six on the front leg. 

Groups 0 and c of the scattered pores lie at the proximal end of 
the tibia (Text-fig. 2A—F, Tb), group 6 being located on the outer 
surface and group ¢ on the inner surface. Group 0 on each front 
leg consists of only one pore; on the right middle leg it has one 
pore, but on the left middle leg it has two pores; on the right 
hind leg it has three pores while on the left hind leg it consists of 
two pores. Group c on each front leg has three pores, whereas 
on each of the middle and hind legs it has only one pore. 

All six legs of the specimen of Epilachna borealis examined 
bear 95 olfactory pores; both elytra carry 149 pores, and both 
wings carry 288 pores. All of these combined make 532 olfactory 
pores. 


(b) Other Species. 


The greatest variation found in the olfactory pores of the 
other species examined is in regard to the total numbers of the 
pores. The second greatest variation is in regard to the distribu- 
tion of the pores on the wings. This variation and other minor 
ones will now be given and a discussion of the total numbers of 
the pores will be presented last. For sake of brevity, instead of 
using the long scientific names of the beetles, the species will be 
numbered from 1 to 50, and those interested in associating the 
names of the species with the variations described may do so by 
referring to the namesand numbers of the species in the table on 
page 419. 

A group of pores (No. 1) was found on the peduncle of each 
elytron. This group in 46 species is definite, that is, the pores 


THE OLFACTORY SENSE OF COLEOPTERA. AI5 


are close together and are not scattered as they are in the other 
four species (Nos. 2, 34, 35, 44). In some beetles it is almost 
impossible to identify the various chitinous plates in the peduncles 
of the elytra, but as far as can be ascertained the definite groups 
of pores are located on the radial plates, while the scattered 
groups may spread over two or more of the plates. In shape 
these groups are round, oblong and triangular. The triangular- 
shaped ones are most common. Asarule, the more pores in this 
group, the smaller they are and the closer they are together. 
In three species (Nos. 23, 29, 36) the pores in this group are com- © 
paratively large, while those in the lady beetles are medium in 
size. Osmus with 12 pores on both elytra has the least number 
and Hydrophilus with 310 pores on both elytra has the largest 
number. In regard to the total numbers of pores on the elytra 
for the 50 species, the reader is referred to the table on page 
A419. 

The three beetles, Osmus, Clinidium and Cysteodemus, are 
wingless. No rudiments of the wings were even found. The 
number of groups of pores on each wing of the other species 
Watessironi® tor4- den species (Nos. 1, 10, 12,914.16) 17, 1S 
21, 22, 47) have only one group on each wing. One wing of 
Lucidota has one group while the other wing has two groups. 
Twenty-one species (Nos. 4, 5, 8, II, 20, 24, 25, 26, 28, 30, 31, 
32, 36, 38, 40, 42, 43, 45, 46, 49, 50) have two groups on each 
wags.  IWyvelhye species (INOSs Bp Oy 75 Oh US) Zon 2On Bon V7 Os Ay 
48) have three groups on each wing. Three species (Nos. 12, 34, 
35) have four groups on each wing. When only one group is 
present on each wing it usually occupies the position of Nos. 3 
and 4 of Epilachna borealis on the radius (Text-fig. 1C). It may 
be no longer than No. 4 of Epilachna, or it may extend nearly 
all the distance to the fold of the wing (Fo). When two groups 
are present on each wing, one is similar to No. 2 of Epilachna 
and the other is similar to Nos. 3 and 4 united. The latter 
group may or may not extend all the way to the fold of the wing. 
In Collops, 20 pores were found on the ventral side of one wing 
besides the two groups on the dorsal surface. When three 
groups are found on each wing, they may be located like those of 
Epilachna, or two of them may lie on the radius and the third 


416 N. E. MCINDOO. 


one on the media. The largest one is similar to No. 4 of Epi- 
lachna and it may or may not extend all the way to the fold of 
the wing. When the third group lies on the media, as in Ortho- 
soma (Plate II., Fig. 31), it occupies a position just beneath the 
larger group on the radius. Its pores are generally scattered 
considerably. When four groups are found on each wing, one 
of them lies on the subcosta, two on the radius and one on the 
media. It is common for the distal end of the largest group on 
the radius of any wing to become attenuated so that a row of 
“pores may extend nearly, if not all, the way to the fold of the 
wing. The farther this row of pores extends along the radius the 
farther apart are the pores. It is also common for the largest 
group on the radius to consist of pores of two sizes. The diam- 
eters of the larger pores may be two or three times those of the 
smaller ones. The larger pores extend lengthwise through the 
center of the group. Eight species (Nos. I, 3,4, 5,6, 33, 36, 38) 
have pores as just described. The pores in this group of seven 
other species (Nos. 10 to 14, 23, 30) are also of two sizes, but 
there is not such a great difference in the sizes of the smaller and 
larger pores, as in the pores of the preceding eight species. 
These pores are also comparatively larger. All the pores on the 
wings of nine species (Nos. 7, 9, 16, 17, 18, 20, 22, 31, 40) are of 
about the same size and they are comparatively large. All the 
pores on the wings of the remaining species are of about the same 
size, but they are comparatively small. Coxelus, the smallest 
beetle examined, with 130 pores on both wings has the least 
number, while Orthosoma, perhaps the largest beetle examined, 
with 982 pores on both wings has the greatest number. 

The trochanters never fail to possess at least a few pores. The 
trochanter with the fewest pores has two, whereas the one with 
the most has 59. Asa rule, the more pores on a trochanter, the 
smaller they are. The pores are generally located at the proximal 
end of this segment in about the same arrangement as represented 
in Epilachna (Text-fig. 2A—F), but occasionally they are con- 
siderably scattered, and a few may be found at the distal end 
of the segment. 

A pore was found at the proximal end of one or more femurs 
belonging to each of 18 species (Nos. 1, 2, 4, 7, 8, 10, II, 13, 16, 


THE OLFACTORY SENSE OF COLEOPTERA. 417 


17, 18, 20, 21, 22, 24, 31, 48, 49), and from one to three pores were 
found at the proximal end of each femur of Flater. 

While it is common to find one or more pores at the proximal 
end of a tibia, many of these segments are entirely devoid of 
olfactory pores. The greatest number of pores found on any 
tibia at this placeisnine. In each of the tibio-tarsal articulations 
of the front and middle legs belonging to Cotinis from 7 to II 
pores were found. Pores were found in the tibial spines (Text- 
fig. 2G and Plate II., Fig. 27) of 15 species (Nos. 9 to I1, 20 to 
25, 31, 32, 34, 35, 45, 48). The pores usually lie on the bases of 
the large spines. The largest number of pores found on a single 
tibial spine is 12. Of the 50 species examined, Passalus has 
the most pores on these spines. 

Pores were found on the tarsi of 13 species (Nos. I, 2, 4, 10 to 
12, 16, 18 to 21, 25, 31). The greatest number found on a 
single tarsus was 37. Osmus, one of the three apterous species, 
has the most pores on its tarsi. 

Bleven species (Nos. 6, 26, 27, 29, 31, 32, 33, 38, 42, 44, 47) 
were found with no pores on the legs except those on the tro- 
chanters and on the tibial spines. Cybister with 49 pores on all 
six legs has the least number on these appendages, while Podabrus 
with 341 pores on all six legs has the largest number. 

No special examination was made to find any structure other 
than the olfactory pores, nevertheless, minute pores were seen 
Mons species (Ni@Sa7410,004+ 15, 16, 20, 25,27 20,20, 405 44-457 
46, 48). These pores were seen on various parts of the beetles, 
but particularly on the legs and elytra. They usually lie near 
the bases of the hairs, but sometimes they lie a considerable 
distance from the hairs. Since they are many times smaller 
than the olfactory pores, without exception they are. probably 
the pores belonging to hypodermal glands, as will be shown for 
those of Epilachna on page 423. However, a careful comparative 
study of these pores is needed before anything definite can be 
said about them. 

Coxelus, the smallest species examined, has a total number of 
273 pores which is the smallest number of all the winged species, 
while Orthosoma, perhaps the largest species examined, has a total 
number of 1,268 pores, which is the largest number of all the 


418 N. E. MCINDOO. 


species examined. As a rule the smaller the species, the larger 
are the pores, comparatively speaking, and the fewer they are. 
Likewise, the larger the species, the greater is the number of its 
pores and the smaller they are. As a rule there are no generic 
and specific differences, except variations in number of pores, 
the amount of variation depending on the size of the individuals 
compared. Judging from the sizes of the four water beetles 
examined, the pores on their legs are fewer and smaller than 
those on the legs of any other beetle examined. Pores were 
found only on the trochanters of Cybister, while a few were also 
seen on the femurs and tibiz of the other three water beetles. 
The number of pores on the legs of these beetles are as follows: 
Cybister—49, Dineutes discolor—65, Dineutes vittatus—o98, and 
Hydrophilus—93. These numbers indicate that the better the 
legs are adapted for locomotion in water, the fewer pores they 
have. 

The small total numbers of pores of Osmus, Clinidium and 

Cysteodemus are due to the absence of wings. In Osmus and 
Clinidium more pores are found on the legs than might be sus- 
pected. The tarsi of Osmus have more than the tarsi of any 
other beetle while the tarsi of Clinidium have more than sus- 
pected. 
. The following table (p. 419) includes the family, name and 
number, the olfactory pores on the legs, elytra, wings, and the 
total number of pores of each of the 50 species examined. In 
the preceding pages the beetles are usually referred to in this 
table by their respective numbers. 


(c) Individual and Sexual Variations. 


For this study five males and five females each of Harpalus 
pennsylvanica and Leptinotarsa to-lineata were used. No indi- 
vidual and sexual variations were found, except slight variations 
in the number of pores. The total numbers of pores of the males 
of Harpalus vary from 550 to 580 with 570 as an average; those 
of the females of Harpalus from 575 to 699 with 628 as an average. 
The average number of pores for males and females of Harpalus 
is 599. The total numbers of pores of males of Leptinotarsa 
vary from 665 to 780 with 722 as an average; those of the females 


THE OLFACTORY SENSE OF COLEOPTERA. 


419 


TABLE I. 
THE NUMBER OF OLFACTORY PORES ON THE LEGS, ELYTRA AND WINGS OF 
COLEOPTERA. 

No. of | No. of | No. of | Total 
Family. Name and Number of Species. toss zones ee | No. of 
| Legs. | Elytra.| Wings.) Pores. 
Ciemdelidce tee er I. Cicindela vulgaris 180 20 924 | 1,133 
2. Osmus sp. 290 12 | 302 
Carabidcer eee 3. Calosoma scrutator T40 62 869 | 1,071 
4. Harpalus caliginosus 180 69 600 | 849 
5. Harpalus pennsylvanica 107 39 453 | 5990 
Dytiscide....... 6. Cybister fimbriolatus 49 | 180 | 917 | 1,046 
(GayintionGleS 5 6 3 0 0 7. Dineutes discolor 65 46 ARS | §AG 
8. Dineutes vittatus 98 42 532 | O72 
Hydrophilide....| 9. Hydrophilus triangularis 03 310 662 | 1,065 
Silica err 10. Necrophorus marginatus IIl 60 652 823 
11. Silpha inequalis 118 30 664 812 
Staphylinide ...|12. Staphylinus macrulosus I10 23 493 626 
Scaphidiide..... 13. Scaphidium quadguttatum TOT 96 3S || Box 
Coccinellide.....'14. Coccinella 9-notata 93 132 BON Ses 
15. Epilachna borealis 95 wiKo) ASS || 5 Qa 
Endomychide...|16. Endomychus biguttatus 144 Rit 178 353 
IDIAOUWANCED> 5 3 oc 17. Megalodacne heros 102 120 383 605 
Colydiide...... 118. Coxelus guttulatus 93 50 LA® || BAS 
Rhyssodide .....|19. Clinidium sculptile I31 4o | aie 
Cienyicceane ak 20. Cucujus clavipes 107 104 aR || sa 
Mycetophagide .|21. Mycetophagus punctatus 165 135 379 679 
Dermestide..... 122. Dermestes marmorata 90 80 570 740 
Inistenidan. a. |23. Hister depurator 74 80 240 403 
Trogositide..... 24. Tenebroides castenea 96 Lis 200 505 
Eilateniciaen «alae: 25. Elater apicatus III 130 365 | 606 
Buprestide ..... 26. Melanophila longipes 186 IIo Dis || Bird 
Lampyride..... 27. Lucidota californica it 7G) 123 AS || Sz 
Telephoride...../28. Chaulcognathus pennsylvanica | 308 157 445 | 910 
\29. Podabrus comes 341 157 280 | 778 
Malachide...... 30. Collops bipunctatus 120 IOI 248 | 469 
Wucanidaesneeee 31. Platycerus quercus 158 160 536 | 854 
32. Passalus cornutus 203 184 782 | 1,169 
Scarabeidz..... 33. Canthon levis 62 180 724 | 966 
34. Cotinis nitida 162 39 934 | 1,135 
35. Euphoria sepulchralis 90 36 Org i) Wao 
36. Osmoderma scabra I27 182 782 | I,091 
Cerambycide....|37. Orthosoma brunneum 79 207 982 | 1,268 
i38. Callimoxys fuscipennis 87 175 BuO | 97/2 
39. Cyllene robinie IIo 40 620 779 
Chrysomelide...!40. Leptinotarsa ro-lineata II5 130 A476 721 
IBiuchicdaersa ae ee 41. Bruchus pist 65 137 352 554 
Tenebrionide....|42. Tenebrio molitor 66 I45 462 673 
43. Uloma impressa 80 im ge) 361 560 
IMIGIONGBES se Gooe 6 '44. Cysteodemus armatus 133 39 172 
45. Epicauta marginata 157 94 504 755 
46. Epicauta pennsylvanica 125 Too 440 665 
Rhipiphoride....'47. Myodites scaber mo || AGO) || Bee) |). Fon 

Rhynchophora: | 
Rhynchitide. .'48. Rhynchites bicolor 84 148 480 412 
Otiorhynchide |49. Cephus latus 41 IIL 382 | Sod 
Curculionide .|/50. Zygops seminivius 65 116 BO2n oS 
49- 12— UZO= || - 27 Z= 
Variation 341 310 982 | 1,268! 


1 The total number of pores of apterous species are not included. 


420 N. E. MCINDOO. 


of the same species from 661 to 785 with 720 as an average. It 
is thus seen that the females of Harpalus have a few more pores 
than the males, while the males and females of Leptinotarsa 
have the same number of pores. ; 


STRUCTURE. 


In the preceding pages it has been shown that most of the 
variations in regard to the disposition of the olfactory pores are 
slight. In the following pages it will be shown whether or not 
this is true for the structure of these pores. 


(a) External Structure. 


When examined under a low-power lens, the olfactory pores 
may be easily mistaken for hair sockets from which the hairs 
have been removed. When more carefully observed under a 
high-power lens, a striking difference in external form is usually 
seen, but sometimes it is difficult to distinguish the pores from 
hair sockets. The pores appear as small bright spots when a 
strong transmitted light is used. Each bright spot has a dark 
boundary or pore wall (Plate I., Fig. 1, PorW).1 Near the 
center of this boundary is a transparent spot, the pore aperture, 
which may be round, oblong, slit-shaped, or club-shaped. On 
the legs the pore apertures may be round (Fig. 2, PorAp), 
oblong (Fig. 3, PorAp), slit-shaped or club-shaped (Fig. 1, 
PorAp). On the elytra and wings they may be round or oblong 
(Figs. 4 to 8). The hair sockets (Figs. 1 and 2, PorWHr) are 
generally smaller than the olfactory pores and the pores of the 
hypodermal glands (Figs. 1 and 2, PorWGl) are easily dis- 
tinguished from the hair sockets and olfactory pores by their 
small size. 


(b) Internal Structure. 


All the olfactory pores studied are more or less flask-shaped 
structures. They are of three general types. In the most 
common type, as found in Uloma, the mouth of the pore (Figs. 
9-12, Mo) is flaring and the sense cell (Fig. 12, CS) lies in the 
lumen of the appendage outside the pore cavity. The chitinous 


1 All figures, except Text-figs. 1, 2, and 3 are numbered consecutively on Plates 
I. and II. , 


‘ THE OLFACTORY SENSE OF COLEOPTERA. A2l 


cone (Fig. 9, Con) never occupies more than one fourth of the 
pore cavity and usually much less (Fig. 12, Con). The cone 
always stains less deeply than the surrounding chitin, and it is 
common to see a hypodermal secretion (Figs. 9 and 10, Hyp) 
inside the pore cavity. The sense fiber (Fig. 9, SF) pierces the 
cone, and the chitin between the pore aperture and the cone, and 
it ends in the bottom of the pore aperture or pit (Figs. 9-12, P) 
with its peripheral end exposed to the air in the pit. 

The second type of pores is found in the legs of Orthosoma 
(Figs. 13-15), although the pores in the elytra (Fig. 21) and 
wings (Fig. 31) of the same beetle belong to the first or most 
common type. The chitinous integument of the legs of Ortho- 
soma is thicker than that of the legs of any other beetle examined. 
Instead of the sense cells (Fig. 13, SC) lying in the lumen of the 
legs outside the pore cavities, in this type they lie inside the 
pore cavities. When the chitin forming the wall of the pore is 
not thick enough to protect the entire sense cell, the wall of the 
pore projects flange-like (Fig. 14, Fl) into the lumen of the leg. 
In Fig. 14 only about one third of the sense cell (SC) is shown. 
Studies of the olfactory pores in various hymenopterous insects 
made by the writer have shown that the sense cells begin to 
differentiate at the time when the chitin is beginning to be 
formed. From this fact, it is quite probable that the sense cells 
found in the second type of pores have not migrated into the 
pore cavities, but they now remain in approximately the same 
position as when the chitin was being formed. 

The third type of pores is found in the legs of the lady beetle, 
Epilachna borealis. Instead of the chitin over the external end 
of the pore being depressed to form a pit, it is elevated dome-like 
above the surface of the leg. In the center of the dome lies 
the pore aperture (Fig. 16, PorAp). All the pores in the tro- 
chanters and most of those in the tibiee (Fig. 17, PorAp) are of 
this type. Sometimes in the tibia is found a pore whose aperture 
is on a level with the surface of the tibia. The apertures of all 
the pores in the elytra (Fig. 18, PorAp) and wings (Fig. 19) of 
this beetle are on a level with the surfaces of the appendages. 

As already stated, the olfactory pores of beetles are more or 
less flasklike as a rule, but there are many variations among 


422 N. E. MCINDOO. 


them. ‘They may be inverted flask-shaped as found in the legs 
of Epilachna (Figs. 16 and 17) and in the wings of Passalus 
(Fig. 20). ‘Some have the shape of a flask without the neck 
(Figs. 9, 10 and 12). Some are long and slender like fingers or 
test tubes (Figs. 11, 18, 19 and 21). 

Their sizes also vary much. The length of a pore always 
depends on the thickness of the chitin. The diameters of the 
pores of a small beetle (Fig. 25) may be as large, or even larger 
(Figs. 9 and 10) than the diameters of the pores of a large beetle 
(Figs. 13-15). 

A chitinous cone is always present, although it may sometimes: 
be almost indiscernible. It invariably has the same shade of 
coloration (Fig. 17, Con) as the remaining chitin (Fig. 17, Che) 
which is formed after the insect has emerged into the imago stage. 
This is the first time that the writer has been able to determine 
definitely the formation of the cones. In all the hymenopterous 
insects studied by the writer, the chitinous integument is prac- 
tically developed when the insects emerge, but in most beetles 
only about one third of the chitin is formed when the insects 
emerge. Since this is true the hypodermal cells are still large 
and they are rapidly secreting a substance which forms new chitin. 
Their external ends stand in contact with the chitin, and when 
no chitin is present they send processes into all holes or cavities 
in the chitin. Thus the hypodermal cell (Fig. 23, HypC) at 
the mouth of each olfactory pore sends a process into the pore. 
Since the sense fiber has entered the pore aperture before the 
cone is formed, the latter is formed at the external end of the 
pore around the sense fiber. When the chitinous integument 
(Fig. 17) is fully developed no hypodermal processes run into 
the pores and the hypodermal cells are very small. 

The sense cells are always spindle-shaped (Figs. 12, 13, 16-19 
and 23, SC). Only occasionally is an entire sense cell seen in a 
cross section, because the entire cell seldom lies in the same plane 
as that of the section. More entire sense cells may be seen in 
longitudinal sections, but even in these the cells are usually eut 
in two. Entire sense cells were best seen in the oblique sections 
through the peduncles of the elytra of Passalus and Epilachna. 
The nucleus (Figs. 13 and 23, SC Nuc) of the sense cell is always 


THE OLFACTORY SENSE OF COLEOPTERA. 423 


conspicuous. It may be darker (Fig. 13, SCNuc) or lighter 
(Fig. 23, SCNuc) in color than the cytoplasm in the cell. The 
nucleoli (Fig. 23, SCNuc) are also conspicuous. 

Smaller sense cells may be seen in the sections through the 
proximal ends of the trochanters and through the proximal ends 
of the tibia. These (Fig. 17, SC:) belong to tactile hairs (Fig. 
7, JBkAe 

In the sections through the legs and elytra of Epilachna, 
gland cells (Fig. 17, GIC) are plainly seen in the hypodermis 
(Hyp). These are equally as large as the olfactory sense cells, 
-but they are quite different in structure. The diameters of the 
pores of the glands (PorGl) are slightly smaller than those of the 
hairs (PorHr), and they are much smaller than those of the 
olfactory pores (Por). The morphology and physiology of these 
gland cells will be given in another paper. 

The shapes of the external ends or tops of the pits depend on 
the shapes of the pore apertures when seen in superficial views. 
That is, they are round, oblong, slitlike or clublike. The interna! 
ends or bottoms of the pits are always round. The pore aperture, 
proper, is the round opening leading from the bottom of the pit 
to the external end of the pore. This aperture is closed by the 
peripheral end of the sense fiber. The shapes of the pits in cross 
sections, therefore, depend on the directions in which the micro- 
tome knife passes through the pits. The most common shape 
of a pit in cross section is that of an urn (Fig. 9, P). Pits includ- 
ing the pore apertures may be likened to round funnels, or to 
funnels slightly flattened, or to funnels considerably flattened, 
or to funnels so flattened that their tops would be club-shaped. 
In spiders the pits are slits which pass entirely through the 
cuticula. The sense fibers enter the pore apertures at the bot- 
toms of the slits. The pits or slits in spiders, therefore, may be 
likened to funnels considerably flattened. When just emerged 
into the imago stage the pits (Fig. 9, P) in the legs generally 
extend about one-third the distance through the chitin, but 
when the chitin is fully developed, the pits extend perhaps from 
one fifth to one eighth the distance through the chitin. In all 
the figures showing two shades in the chitin, the darker one 
(Fig. 17, Ch,) represents the chitin formed at the time when the 


424 N. E. MCINDOO. 


insect emerges from the last pupal stage, and the lighter one 
(Che) represents the chitin formed after emerging into the imago 
stage. 

As already stated, instead of the olfactory pores of the lady 
beetle, Epilachna, having pits, the chitin over each pore in the 
legs is elevated domelike above the surface of the leg. The 
olfactory pores (Figs. 24 and 25) in the legs of the two blister 
beetles, Epicauta marginata and Epicauta pennsylvanica, have 
only indications of pits. Their pore apertures are therefore on 
a level with the surface of the legs. The olfactory pores in the 
legs of the potato beetle, Leptinotarsa to-lineata, have shallow 
pits (Fig. 26, P). All four just enumerated species have hypo- 
dermal gland pores distributed over the entire body except the 
wings. These pores are perhaps most abundant on the elytra, 
but they were never seen on the peduncles of these appendages, 
and it is quite probable that the secretion from their glands 
never covers the olfactory pores found on the wings and on the 
peduncles of the elytra. Judging from the gland pores, the 
hypodermal glands in the legs of Epilachna are more highly 
developed than are those of the other three species. The gland 
pores (Figs. I, 2 and 28, PorWGl) on the legs of Eptilachna lie 
on all sides and even among the olfactory pores, but in the legs 
of the other three species the gland pores never lie near the 
olfactory pores. When examined under a low-power lens the 
legs and elytra of Epilachna appear wet, and many small yellow 
flakes may be seen on them. The wet appearance is certainly 
due to the secretion from the hypodermal glands and the flakes | 
are the remains of the secretion after it becomes dry. Thus 
in Epilachna there seems to be a direct correlation between the 
olfactory pores and the gland pores. Since the pore apertures in 
the legs lie above the surface of these appendages, the secretion 
from the hypodermal glands runs away from the pore apertures 
instead of into them. Such a device enables both sets of organs 
to function normally without the one hindering the other. 

In the legs the sense cells always lie in a blood sinus (Figs. 16 
and 17, BlSin) some distance from the muscles (Fig. 28, M). 
The nerves (J) are easily seen and branches (VB) are given off 
which run to the sense cells (SC). The neurilemma (Fig. 17, 


THE OLFACTORY SENSE OF COLEOPTERA. 425 


Neu) of the nerve is usually distinct. In the cross section of a 
nerve, the nervous substance appears more or less netlike and 
nuclei, probably neuroglia nuclei (Fig. 17, NeurNuc), stand out 
conspicuously in the network. The trachea (Figs. 16, 17 and 
28, Tr) and nerves (N and NB) are firmly suspended by the 
connective tissue whose nuclei (Con7TNuc) are seen only occa- 
sionally. The lumen of the leg at the proximal end of the tibia 
of Epilachna seems to be divided into two chambers by a mem- 
brane (Fig. 17, Hyp:) which resembles hypodermis. This 
structure has never been seen before by the writer and nothing 
can be said about its function. 

The hypodermis (Fig. 18, Hyp) beneath the olfactory pores in 
the peduncles of the elytra is much thicker than elsewhere. It 
usually contains all the sense cells (SC), but in the elytra of 
Passalus the hypodermis is thinner and since the sense cells are 
so large and so numerous there is not enough room for all of 
them in the hypodermis. For this reason only a few of them 
lie among the hypodermal cells and the remainder of them lie 
in the lumen of the peduncle between the hypodermis and nerve. 
As usual they are surrounded by blood. In only one instance 
was the writer able to trace a sense cell all the way from the 
pore aperture to the nerve. Fig. 23 represents this sense cell 
connecting with the pore aperture (PorAp) and with the nerve 
(NV). The trachea (Tr) lies by the side of the nerve. A large 
nerve (Fig. 29, N) and a large trachea (77) run through the 
radial plate (RP) of the peduncles beneath the olfactory pores. 
From the nerve many branches are given off which connect with 
the sense cells. 

The hypodermis (Fig. 22, Hyp) beneath the olfactory pores 
in the wings is usually much thicker than elsewhere, but it does 
not contain the sense cells (SC). These cells lie in a blood sinus 
(Fig. 22, BlSin) between the hypodermis (Hyp) and the trachea 
(Tr), nerve (NV) and nerve branches (VB). In the wings it is 
usually difficult to trace a sense fiber all the way to the pore 
aperture, but in oblique superficial sections this is easily done 
(Fig. 30). A large nerve and a large trachea run into each wing. 
These divide so that a smaller nerve and a smaller trachea run 
through each main vein. The largest trachea (Fig. 31, Tr) runs” 


426 N. E. MCINDOO. 


through the subcosta (Sc) while the largest nerves (VV) pass through 
the veins bearing the olfactory pores. The nerve and trachea 
run directly beneath the sense cells (SC) and from the nerve 
pass off many branches which connect with the sense cells. In 
the costa (C) and subcosta (Sc) where there are no sense cells, 
only a few nerve fibers can be seen. 

In the preceding pages it has been shown that there are many 
variations in the structure of the olfactory pores of beetles, and 
that these organs are very similar to those of hymenopterous 
insects. On the basis of the location of the pore apertures in 
the integument, the olfactory organs in beetles are intermediate 
between those of spiders and those of Hymenoptera. 


EXPERIMENTS TO DETERMINE THE LOCATION OF THE OLFACTORY 
ORGANS. 


Since it is now generally believed that the olfactory organs of 
beetles are borne by their antenne, these appendages of many 
individuals were pulled off. From one to seven days later, the 
mutilated insects were tested with odors. In the preceding pages 
it has been shown that the olfactory pores of Coleoptera are 
located on the peduncles of the elytra, on the wings and on the 
legs. In order to ascertain if these structures receive odor 
stimuli, the elytra, wings and legs were mutilated. One or more 
days later these mutilated beetles were tested with odors. In all 
the experiments with unmutilated and mutilated beetles, 434 
individuals have been tested. These belonged to I1 species 
representing eight families. 

In order that the behavior of the mutilated beetles would be 
correctly interpreted, the behavior of unmutilated beetles under 
experimental conditions was first studied. Since it was not 
desired to ascertain the relative sensitiveness of males and 
females, both sexes were used indiscriminately. To determine 
the relative sensitiveness of unmutilated and mutilated indi- 
viduals under conditions which permitted of their close observa- 
tion, triangular experimental cases were employed. These were 
made of three narrow wooden strips, two of which were five and 
the third four inches long, each strip being half an inch thick. 
Wire screen served as a bottom and glass as a top for the case. 


THE OLFACTORY SENSE OF COLEOPTERA. 427 


The apices and bases of these cases rested on two supports above 
a rigid table near a window. No screen was used to prevent 
the beetles from seeing the observer because they never showed 
any responses to the movements made by the observer. 

' The following sources of odors were used for determining the 
reactions of the beetles in the experimental cases; chemically 
pure essential oils of peppermint, thyme, and wintergreen; parts 
of plants—leaves and stems of pennyroyal (Hedoma pulegi- 
oides?), and of spearmint (Mentha spicata); decayed matter 
—parts of decayed beetles (Harpalus pennsylvanica). All 
these substances were kept in stoppered vials of the same shape 
and size. The leaves and stems of the pennyroyal were dried, 
but they still gave off a strong odor when the vial was uncorked. 
The leaves and stems of the spearmint were fresh and they did 
not emit as strong an odor as did the other substances used. 
Beetles were killed and were torn to pieces. The pieces were put 
into a vial and after two or three days they emitted a foul and 
sickly odor. 

A beetle was carefully placed into one of the experimental 
cases. When first put into the case the insect usually wandered 
about for several minutes, but finally it became quiet. The 
insect was tested with the above odors only when it had become 
perfectly quiet, without the antennz being moved in the least. 
The stopper of a vial was quickly removed and the vial was gently 
and slowly placed under the experimental case directly beneath 
and within one half inch of the individual being tested. When 
all of these precautions are taken, a normal beetle generally 
responds to anyone of these odors within 60 seconds, but when 
all the reaction times are counted, it is seen that several of them 
failed to respond within 60 seconds. If a beetle when tested fails 
to react to an odor within 60 seconds, the response may be 
regarded as negative, and when it reacts to an odor within 60 
seconds, the response may be called positive. As a control, an 
empty and odorless vial was now and then placed under the 
insects in the same manner. If by chance a beetle moved while 
the control test was being made, its behavior was different from 
that observed when odors were used. Only the first responses 
have been recorded and in all cases where there was the least 


428 N. E. MCINDOO. 


doubt as to whether the insect moved for any reason other than 
the olfactory stimulus, such movements were never recorded. 
The reaction time was counted in seconds. With an ordinary 
watch the minimum time which can be definitely recorded is two 
seconds, although many of the individuals responded to some of 
the odors much more promptly. Owing to this source of error, 
the average recorded time is probably double what it should be 
in the cases where all the responses for the same insect were 
prompt. An intermission of 10 minutes elapsed between any 
two tests in the same experimental case. Each individual was 
tested only once with the same odor. 

In recording the responses the term “‘vibrated’’ is used to 
describe the rapid movement of the antennz or legs up and down: 
or from side to side. When this movement is slow, these ap- 
pendages are described simply as having “moved.” When the 
antenne, legs or mouth parts are moved so that they are quickly 
bent at their articulations, they may be described as being 
‘“worked.’’ When at rest a beetle usually lies flat on its thorax 
and abdomen, so the word ‘“‘arose’’ means that the insect gets 
up and stands on its feet. In the averages of reaction times the 
probable error is presumably high. It has not been calculated 
since slight differences in reaction times are not considered as 
Significant in the discussion of results, All anthropomorphic 
terms are put in quotation marks. 


CARABID. 
Tue OLFACTORY SENSE oF Harpalus pennsylvanica. 


Many ground beetles (Harpalus pennsylvanica) were caught 
under flat stones in a corn field near the laboratory. As soon as 
brought to the laboratory, 25 of them were placed singly into 
the experimental cases. As they were being placed into the 
cases, some of them discharged a substance, presumably from the 
anal glands, which gave off an odor similar to that from formic 
acid. Confined in these cases, they sought the dark corners of 
the cases and did not wander about much inside the cases unless 
irritated. When half hidden in the dark corners, they rarely 
responded to odors, so it was necessary to keep them out of the 
corners while they were being tseted. The longer they remained 


THE OLFACTORY SENSE OF COLEOPTERA. 429 
ha 
in the light and the more they were handled, the more satisfactory 


they were to experiment with. Owing to this kind of behavior, 
this species and several others used responded more slowly to 
odors a short time after being caught than they did a few days 
after being kept in confinement. This fact will explain why some 
unmutilated beetles just caught respond to odors more slowly 
than they do two or three days later after having had their 
antenne pulled off. The following are the responses of this 
eround beetle to the odors from the six different substances and 
the average reaction times in seconds. 


Oil of peppermint: 


5 moved away quickly. rt worked legs. 

5 vibrated antenne. I kicked quickly. 

4 arose quickly. I vibrated antenne and legs. 
4 moved slightly. _ I vibrated legs. 

2 moved antenne and legs. rt jumped slightly. 


Reaction time 2 to 10 seconds, average 3.6 seconds. 


Oil of thyme: 


6 moved away quickly. I arose slowly. 

5 moved quickly. I vibrated antenne. 

5 moved slightly. — I moved backward slowly. 
2 worked antenne. rt worked legs. 

2 moved antenne and legs. t did not respond. 


Reaction time 2 to 60 seconds, average 8.5 seconds. 


Oil of wintergreen: 


5 moved away quickly. rt stroked antenne. 

5 moved slightly. I vibrated antenne and legs. 
4 moved away slowly. I worked antenne. 

3 moved quickly. rt worked legs. 

2 vibrated legs. I did not respond. 


1 arose slowly. 
Reaction time 2 to 60 seconds, average 16.4 seconds. 


Leaves and stems of pennyroyal: 


Io moved away quickly. I moved away slowly. 
6 moved slightly. I vibrated legs. 
3 vibrated antenne. 1 did not respond. 


3 worked antenne. 
Reaction time 3 to 60 seconds, average 21.1 seconds. 


Leaves and stems of spearmint: 


5 moved away slowly. 2 did not respond. 
5 moved slightly. t worked mouth parts. 
3 moved antennee and legs. I vibrated legs. 


2 moved away quickly. I vibrated antenne and legs. 


430 N. E. MCINDOO. 


2 worked antenne. I moved antenne. 
2 jumped slightly. 
Reaction time 3 to 60 seconds, average 21.8 seconds. 


Parts of decayed beetles: 


7 moved slightly. I moved legs. 

5 did not respond. t worked antenne. 

4 moved away quickly. I vibrated legs. 

3 moved away slowly. I vibrated antenne and worked 
2 jumped slightly. mouth parts. 


Reaction time 5 to 60 seconds, average 28.1 seconds. 


The general average reaction time of the 25 beetles tested 
to the six odors is 16.5 seconds. As a possible reason why one 
fifth of the individuals tested failed to respond to the odor 
from the decayed beetles is that these insects probably do not 
respond to decayed matter unless they are hungry. The 25 
beetles tested were put into a wooden box four inches wide, 
seven inches long and two inches deep. One half inch of moist 
earth was also put into the box. The beetles soon buried in 
the earth and from that time on they appeared quite “at home.” 
The box was put into a table drawer where it was more or less 
dark. About twice each week water was poured upon the earth 
and the beetles were fed earthworms and various insect larve. 
They drank some of the water and always greedily ate the food 
given to them. Up to the time of this writing (Jan. 15), 24 
of these beetles have died. These lived from 18 to 180 days with 
61 days as an average. All the beetles confined in the laboratory 
have not been fed since Oct. 15, but they have been given water 
once or twice a week. A few of the dead beetles when removed 
from the box had been partially eaten, but these insects were 
never seen fighting one another. While collecting this species 
in the corn field, a dead one was now and then found. 


(a) Effects with Antenne Pulled Off. 


The antenne of 25 Harpalus pennsylvanica were pulled off at 
their bases. These insects were then put into a wooden box 
similar to the one containing the unmutilated individuals just 
described. This box, also containing moist earth, was placed 
into the table drawer. The beetles appeared normal in all 
respects for they drank and ate as greedily as the unmutilated 


THE OLFACTORY SENSE OF COLEOPTERA. 431 


ones and buried in the earth as usual. Seven days later they 
were placed singly into the experimental cases and were tested 
with the six odors as usual. They wandered about in the cases. 
slightly more than did the unmutilated ones, but when tested 
they gave similar responses and reacted just as promptly. 

Their reaction times are as follows: Oil of peppermint, 2 to 15; 
seconds, average 3.8 seconds; oil of thyme, 2 to 25 seconds,. 
average 4.7 seconds; oil of wintergreen, 2 to 25 seconds, average 
6.9 seconds; leaves and stems of pennyroyal, 3 to 50 seconds, 
average 14.4 seconds; leaves and stems of spearmint, 3 to 60 
seconds, average 34.9 seconds. Ten failed to respond to this 
odor. Parts of decayed beetles, 3 to 60 seconds, average 32 
seconds. Eight failed to respond to this odor. The general 
average reaction time of the 25 beetles tested to the six odors 
is 16.1 seconds. Up to the time of this writing (Jan. 15), 23 
of these beetles have died. They lived from 19 to 171 days 
with 58 days as an average. 


(b) Effects with Elytra and Wings Pulled Of. 


The elytra and wings of 25 Harpalus pennsylvanica, just 
collected from the cornfield, were pulled off at their articulations.. 
These mutilated insects were then put into a third box, similar 
to the two already described. The box was kept in the table: 
drawer with the others. On the following day after mutilating: 
the beetles, they were placed singly into the experimental cases: 
and were tested with the six odors as usual. They seemed 
normal in all respects except they were extremely restless. 
Their responses to odors were similar to those of unmutilated 
ones, except they were slower. 

Their reaction times are as follows: Oil of peppermint, 3 to 45 
seconds, average 10.7 seconds; oil of thyme, 5 to 50 seconds, 
average 10.2 seconds; oil of wintergreen, 5 to 60 seconds, average 
18 seconds. Two failed to respond to this odor. Leaves and 
stems of pennyroyal, 5 to 60 seconds, average 29.2 seconds. 
Seven failed to respond to this odor. Leaves and stems of spear- 
mint, 5 to 60 seconds, average 24.7 seconds. Four failed to 
respond to this odor. Parts of decayed beetles, 5 to 30 seconds, 
average 13.4 seconds. The general average reaction time of the 


432 N. E. MCINDOO. 


25 beetles tested to all six odors is 17.7 seconds. These mutilated 
insects lived from 2 to 21 days with 9 days as anaverage. All the 
time they were confined in the small box, they drank, ate, and 
buried in the earth normally, but many times one was seen biting 
the soft dorsal portion of the abdomen of another. With the 
elytra and wings removed, the abdomens were unprotected and 
many of them shrank considerably in size before the beetles died. 
Some of these beetles were certainly killed on account of the 
dorsal sides of their abdomens being bitten, because nearly 
every one found dead had been entirely eaten except the chitinous 
parts. In the other two boxes as already mentioned, only 
occasionally was a dead beetle found that had been eaten. 


(c) Effects with Elytra and Wings Pulled Off and Pores on Legs 
Covered with Vaseline. 


The elytra and wings of 18 Harpalus pennsylvanica were pulled 
off at their articulations. Four days later the trochanters, 
femurs and proximal ends of the tibize of these mutilated beetles 
were covered with a vaseline-beeswax mixture, consisting of 
three fourths yellow commercial vaseline and one fourth beeswax. 
An hour after the legs had been vaselined, the beetles were placed 
singly into the experimental cases and were tested with the six 
odors as usual. Most of them were comparatively quiet, but a 
few were extremely restless. Their responses to odors were not 
pronounced and were slow, otherwise they were similar to those 
of unmutilated beetles. 

Their reaction times are as follows: Oil of peppermint, 3 to 60 
seconds, average 19.5 seconds. Three failed to respond to this 
odor. Oil of thyme, 3 to 60 seconds, average 12.5 seconds, 
‘Two failed to respond to this odor. Oil of wintergreen, 3 to 60 
seconds, average 18.7 seconds. Four failed to respond to this 
odor. Leaves and stems of pennyroyal, 5 to 60 seconds, average 
38.6 seconds. Nine failed to respond to this odor. Leaves and 
stems of spearmint, 3 to 60 seconds, average 32.9 seconds. 
Seven failed to respond to this odor. Parts of decayed beetles, 
4 to 60 seconds, average 22.1 seconds. Two failed to respond to 
this odor. The general average reaction time of the 18 beetles 
tested to all six odors is 24.1 seconds. Confined in a box similar 


THE OLFACTORY SENSE OF COLEOPTERA. 433 


to the other three already mentioned, these mutilated beetles 
drank, ate and buried in the earth normally, but they were less 
active than unmutilated ones. It was common to see them biting 
the dorsal sides of the abdomens. Before they died several of 
their abdomens had shrunk considerably in size. When found 
dead several of them had been entirely eaten except the chitinous 
parts. Counting from the time the elytra and wings were pulled 
off, they lived from 5 to 21 days with ro days as an average. 


THE OLFACTORY SENSE OF Harpalus caliginosus. 


Eight ground beetles (Harpalus caliginosus) were caught under 
flat stones. They were tested with the odors from only the three 
essential oils. In behavior, they were comparatively quiet. 
When tested, many of them moved away quickly; a few vibrated 
the antenne, and a few moved their legs. 

Their reaction times are as follows: Oil of peppermint, 2 to 10 
seconds, average 4.4 seconds; oil of thyme, 2 to 8 seconds, aver- 
age 4.1 seconds; oil of wintergreen, 2 to 8 seconds, average 4.1 
seconds. The general average reaction time to all three odors is 
4.2 seconds. The antennz of these beetles were pulled off and 
the insects were then kept in a small box containing earth in the 
table drawer. 


(a) Effects with Antenne Pulled Off. 


Eight days after the antenne of the eight preceding Harpalus 
caliginosus had been pulled off, the remaining six live ones were 
again tested with the same odors in the usual way. Their 
responses were similar to those given before they were mutilated, 
but were not so pronounced. When tested with the oil of thyme, 
one beetle rubbed a hind leg on an elytron for a half minute. 

Their reaction times are as follows: Oil of peppermint, 3 to 25 
seconds, average 12.5 seconds; oil of thyme, 4 to 60 seconds, 
-average 14.3 seconds. One failed to respond to this odor. 
Oil of wintergreen, 10 to 35 seconds, average 22.5 seconds. The 
general average reaction time to all three odors is 16.4 seconds. 
These mutilated beetles were quite inactive and sometimes 
scarcely moved when touched with a pencil. They did not eat 
as greedily as before being mutilated. They lived from 2 to 65 
days with 18 days as an average. 


434 N. E. MCINDOO. 


COCCINELLID&. 
THE OLFACTORY SENSE OF Epilachna borealis. 


Many lady beetles (Epilachna borealis) were caught on pumpkin 
vines in the corn field. When brought to the laboratory, they 
were put into a large glass jar near a window. The jar was II 
inches tall and 9 inches in diameter. It was covered with cheese-_ 
cloth. Since this lady beetle feeds upon the leaves of pumpkin 
and of allied plants, several pumpkin leaves were put into a wide- 
mouthed bottle containing water. The bottle with contents 
was then put into the jar. The beetles soon found the leaves 
and from that time on, they appeared ‘‘at home” as much as 
they do in corn fields on pumpkin leaves. They were regularly 
provided with a fresh supply of food. Occasionally they were 
seen copulating. 

On the following day after being caught, 18 of them were 
removed from the jar and were put singly into the experimental 
cases. When mechanically irritated they draw in the antennz 
and legs, usually eject a small drop of yellowish liquid from each 
femoro-tibial articulation, and feign death. They may lie 
apparently lifeless for several moments and when tested with 
odors they may or may not respond. Owing to this peculiar 
behavior, they were unsatisfactory to experiment with and 
their average reaction times are slower than might be expected. 
They were extremely quiet and when tested they generally moved 
away slowly. They often vibrated the antenne and mouth 
parts, and sometimes the legs. 

Their reaction times to the odors from the three essential oils 
are as follows: Oil of peppermint, 2 to 55 seconds, average 12.4 
seconds; oil of thyme, 2 to 20 seconds, average 6.8 seconds; oil of 
wintergreen, 3 to 60 seconds, average 22.2 seconds. Three failed 
to respond to this odor. The general average reaction time to all 
three odors is 13.8 seconds. Sixteen of these insects were muti- 
lated for other experiments. The seventeenth lived only 3 days 
and the eighteenth is still living at this writing (Jan. 15). 


(a) Effects with Antenne Pulled Of. 


The antenne of 25 Epilachna borealis, just caught, were pulled 
off at their bases. A small drop of yellowish blood exuded from 


THE OLFACTORY SENSE OF COLEOPTERA. 435 


each wound. On the following day the beetles were tested with 
odors. Asa rule they were so inactive that they appeared life- 
less. If touched while moving they feigned death and remained 
inactive for several moments. When tested with odors most of 
them worked the mouth parts; some moved away slowly; a few 
vibrated one or more legs, and some failed to respond. 

Their reaction times to the odors from the three essential 
oils are as follows: Oil of peppermint, 2 to 60 seconds, average 
18.6 seconds. Three failed to respond to this odor. Oil of 
thyme, 2 to 60 seconds, average 38.7 seconds. Fourteen failed 
to respond to this odor. Oil of wintergreen, 3 to 60 seconds, 
average 35.1 seconds. The general average reaction time to all 
three odors is 30.8 seconds. Up to the time of this writing 
(Jan. 15), 15 of these mutilated beetles have died. They lived 
from I to 96 days with 22 days as an average. 


(b) Effects with Elytra and Wings Pulled Off. 


The elytra and wings of 10 Epilachna borealis were pulled off 
at their articulations. A small drop of yellowish blood exuded 
from each wound. A liquid of the same color is also present 
throughout the elytra and in the veins of the wings. On the 
second day after being mutilated, the four remaining live beetles 
were tested as usual. They were very quiet, but appeared 
normal in all respects except they responded to odors more 
slowly than unmutilated ones. 

Their reaction times to the odors from the three essential oils 
are as follows: Oil of peppermint, 10 to 60 seconds, average 25 
seconds. One failed to respond to this odor. Oil of thyme, 5 to 
60 seconds, average 33.5 seconds. Two failed to respond to this 
odor. Oil of wintergreen, 7 to 60 seconds, average 35.5 seconds. 
Two failed to respond to this odor. The general average re- 
action time to all three odors is 31.3 seconds. Up to the time of 
this writing (Jan. 15), I of these beetles has died. Counting the 
7 mutilated beetles that died, they lived from 2 to 3 days with 
2 days as an average. 


436 N. E. MCINDOO. 


TELEPHORID#. 
THE OLFACTORY SENSE OF Chaulcognathus pennsylvanica. 


Many fireflies (Chaulcognathus pennsylvanica) were caught on 
goldenrod (Solidago). They were put into a cage 20 inches long, 
16 inches tall and 12 inches wide. The sides and top of the cage 
were cheesecloth while the ends and bottom were wood. The 
cage was kept in the light near a window and a fresh supply of 
goldenrod was constantly kept in the cage. On the goldenrod 
in the cage, these insects appeared quite ‘‘at home.’’ Twenty- 
five of them were tested with the odors from the three essential 
oils. When tested most of them moved away quickly; a few 
vibrated antennz; a few vibrated legs, and a few arose slowly. 
They were extremely restless at all times. In the cage they 
copulated as freely as they do out-of-doors. 

Their reaction times are as follows: Oil of peppermint, 2 to 12 
seconds, average 2.6 seconds; oil of thyme, 2 to 10 seconds, 
average 3 seconds; oil of wintergreen, 2 to 10 seconds, average 3 
seconds. The general average reaction time to all three odors is 
2.8 seconds. They lived from 3 to 7 days with 3.2 days as an 
average. 


(a) Effects with Antenne Pulled Of. 


The antennz of 27 Chaulcognathus pennsylvanica were pulled 
off at their bases. A day later only three were alive. When 
tested these three responded as promptly as unmutilated ones. 
The general average reaction time to the odors from the three 
essential oils is 2.8 seconds. Counting all 27 beetles, they lived 
from I to 5 days with 1.3 days as an average. 


LUCANID. 
THE OLFACTORY SENSE OF Passalus cornutus. 


Four stag beetles (Passalus cornutus) were removed from rotten 
stumps. While being tested with odors they were compara- 
tively quiet and responded promptly. Their most common 
response was to draw in the antenne and to move away slowly. 
The general average reaction time to all six odors is 3.2 seconds. 
The antenne were pulled off at their bases. A small drop of 


THE OLFACTORY SENSE OF COLEOPTERA. 437 


blood exuded from each wound. The beetles were kept in a 
small box filled with moist rotten wood. 


(a) Effects with Antenne Pulled Of. 


Two days after pulling off the antennae, the four preceding 
mutilated beetles were again tested with the same odors. They 
were more quiet than before being mutilated. Their responses 
were just as prompt but were less pronounced than before they 
were mutilated. Their most common response was to work the 
mouth parts and to move away slowly. The general average 
reaction time to all six odors is 3.3 seconds. They lived from 
4 to 20 days with 12.5 days as an average. 


SCARABAID. 
THE OLFACTORY SENSE OF Cotinis nitida. 


One lamellicorn beetle (Cotinis nitida) was tested with the six 
odors. The most common response was to stretch out its head, 
and to move its antenne and front legs. Once it drew in the 
antenne and moved the front legs. The average reaction time 
is 8 seconds. The antenne were pulled off at their bases. A 
small drop of blood exuded from each wound. 


(a) Effects with Antenne Pulled Off. 

A day after pulling off the antenne, the preceding Cotinis 
nitida was again tested with the same odors. It responded as 
promptly as before being mutilated. The most common re- 
sponse was to work the mouth parts and to move away slowly. 
The average reaction time is 8.3 seconds. It lived 12 days after 
being mutilated. 


THE OLFACTORY SENSE OF Euphoria sepulchralts. 


Five lamellicorn beetles (Euphoria sepulchralis) were caught 
on goldenrod (Solidago). While being tested with the odors 
from the three essential oils, they were extremely restless. 
They generally moved away slowly and drew in the antenne 
when tested with an odor. The general average reaction time 
is 3.6 seconds. After the antenne had been pulled off at their 
bases, the beetles were put into the cage described on page 436. 


438 N. E. MCINDOO. 


(a) Effects with Antenne Pulled Of. 


A day later the five preceding mutilated insects were again 
tested with the same odors. They were quiet and their responses 
were similar to those before being mutilated, except, of course, 
there were no antennal movements. The general average re- 
action time is 4.3 seconds. These beetles lived from 9 to 42: 
days with 20 days as an average after being mutilated. 


CERAMBYCID&. 
THE OLFACTORY SENSE OF Cyllene robinie. 


Eighteen wood-boring beetles (Cyllene robinie) were caught on 
goldenrod (Solidago). While being tested with the odors from 
the three essential oils, they were extremely restless. When 
tested, most of them moved away quickly; a few arose quickly, 
and a few vibrated the antenne. The general average reaction 
time is 5.4 seconds. These beetles were confined in the cage 
described on page 436. They were regularly given a fresh supply 
of goldenrod. They seemed ‘‘at home”’ and copulated as freely 
in the cage as they do out-of-doors. They lived from I to 17 
days with 10.4 days as an average. 


(a) Effects with Antenne Pulled Of. 

Eighteen more Cyllene robinie were collected from goldenrod. 
Their antennz were pulled off at the bases. A small drop of 
blood exuded from each wound. These beetles were placed into 
the cage with the unmutilated ones. Two days later the 15 
remaining live ones were tested with the odors from the essential 
oils. They were very quiet and their responses were similar to 
those of unmutilated individuals, except as a rule they were more 
prompt. The general average reaction time is 3 seconds. In 
the cage it was common to see the unmutilated and antenneless 
cerambycids copulating. The former were very active and flew 
out of the cage whenever the door was opened, but the latter 
seldom flew and they were not so active. The mutilated ones 
lived from I to 11 days with 5 days as an average. 


(b) Effects with Elytra and Wings Pulled Off. 
Eighteen more Cyllene robinie were collected. Their elytra 
and wings were pulled off at the articulations. A small drop 


THE OLFACTORY SENSE OF COLEOPTERA. 439 


of blood always exuded from each wound caused by the elytron 
being pulled off, but only occasionally was blood seen where a 
wing had been pulled off. A day later when tested with the 
odors from the three essential oils, these beetles were compara- 
tively quiet and they appeared normal in all respects except in 
their slowness in responding to odors. 

Their reaction times are as follows: Oil of peppermint, 2 to 30 
seconds, average 7.1 seconds; oil of thyme, 3 to 20 seconds, 
average 8.9 seconds; oil of wintergreen 3 to 55 seconds, average 
13.4 seconds. The general average reaction time to all three 
odors is 9.8 seconds. In the cage with the other beetles, these 
mutilated ones were as active as the unmutilated cerambycids 
and they were often seen copulating with each other, and with 
the unmutilated and antenneless ones. They lived from I to II 
days with 4.2 days as an average. 


CHRYSOMELID. 
THE OLFACTORY SENSE OF Leptinotarsa ro-lineata. 


Forty-five Colorado potato beetles (Leptinotarsa 1o-lineata) 
were collected in a potato patch near the laboratory. While 25 
of them were being tested with the six odors, they were com- 
paratively quiet as a rule, but five were so restless that they 
were discarded and others were used. Their responses were 
similar to those of Harpalus pennsylvanica, described on page 429. 

Their reaction times are as follows: Oil of peppermint, 2 to 7 
seconds, average 3.3 seconds; oil of thyme, 2 to 5 seconds, average 
3.1 seconds; oil of wintergreen, 2 to 12 seconds, average 5 seconds; 
leaves and stems of pennyroyal, 4 to 60 seconds, average 26.7 
seconds. Six failed to respond to this odor. Leaves and stems 
of spearmint, 2 to 60 seconds, average 25.6 seconds. Seven failed 
to respond to thisodor. Parts of decayed beetles, 5 to 60 seconds; 
average 27.9 seconds. Seven failed to respond to this odor. 
The general average reaction time of the 25 beetles tested to all 
six odors is 15.4 seconds. These insects were confined in a 
cage in the light near a window. This cage is 30 inches long, 
30 inches high and 4% inches wide. A\ll six sides are wire-screen. 
A fresh supply of potato plant leaves was constantly kept in the 
cage. The beetles confined in this cage on the potato plant 


440 N. E. MCINDOO. 


leaves appeared “‘at home”’ just as much as they do in potato 
patches. They ate the leaves, copulated and laid eggs as usual. 
Up to the time of this writing (Jan. 15), 28 of the 45 beetles have 
died. These lived from 14 to 151 days with 69 days as an 
average. , 


(a) Effects with Antenne Pulled Of. 


Twenty-nine more potato beetles were collected from the 
potato patch. Their antennze were pulled off at the bases. A 
small drop of blood exuded from each wound. These insects 
were put into the wire-screen cage with the unmutilated ones. 
Two days later the 23 remaining live ones were tested with only 
the odors from the three essential oils. All of these beetles were 
quite inactive and three failed to respond when tested. These 
three also failed to respond when touched with a pencil. For this 
reason they were discarded. The general average reaction time 
of the 20 beetles tested is 3.5 seconds. Asa rule these mutilated 
insects appeared normal in all respects several days after having 
the antennz pulled off, because they ate, copulated and were as 
active as ever. They lived from 2 to 140 days with 38 days as 
an average. 


(b) Effects with Elytra Pulled Off and Wings Cut Off. 


Thirty-one more potato beetles were collected. Their elytra 
were pulled off at the articulations and the wings were cut off as 
closely as possible to the articulations. A small drop of reddish 
or yellowish blood exuded from each wound. The heavy veins, 
extending from the base of the wing to where the wing folds, 
contain most of the blood found in these wings. The elytra are 
also filled with blood. The amount of blood in them gradually 
diminishes from the base to the distal end. A day after being 
mutilated 25 of these insects were tested with the six odors. 
They were apparently normal in all respects except in their 
slowness in responding to odors. They were as active as un- 
mutilated ones and eight were extremely restless. Their re- 
sponses were similar to those of unmutilated beetles, except 
they were not pronounced. 

Their reaction times are as follows: Oil of peppermint, 2 to 40 


THE OLFACTORY SENSE OF COLEOPTERA. AAT 


seconds, average 7.8 seconds; oil of thyme, 2 to 15 seconds, aver- 
age 4.8 seconds; oil of wintergreen, 3 to 60 seconds, average 21.1 
seconds. Five failed to respond to this odor. Leaves and stems 
of pennyroyal, 5 to 60 seconds, average 32.2 seconds. Ten failed 
to respond to this odor. Leaves and stems of spearmint, 3 to 60 
seconds, average 29.8 seconds. Eight failed to respond to this 
odor. Parts of decayed beetles, 3 to 60 seconds, average 30.4 
seconds. Eight failed to respond to this odor. The general 
average reaction time of the 25 beetles tested to the six odors is 
22.7 seconds. In the wire-screen cage with the other potato 
beetles already tested, these mutilated ones appeared normal, 
because they ate normally and copulated as much as usual. 
Since the soft dorsal sides of their abdomens were unprotected, 
many of them soon began to sink, so that by the time a beetle 
died, the abdomen had shrunk to about one-fourth its original 
size. Up to the time of this writing (Jan. 15), 29 of these 31 
mutilated insects havedied. They lived from 3 to 140 days with 
52 days as an average. 


(c) Effects with Elytra Pulled Of, Bases of Wings Glued and Pores 
on Legs Covered with Vaseline. 


Twenty-nine more potato beetles were collected. Their elytra 
were pulled off at the articulations. Two days later the upper 
surfaces of the bases of the wings of the 26 remaining live ones 
were covered with liquid glue. Since the olfactory pores extend 
a considerable distance from the base of the wing along the 
radial vein, the glue applied probably did not cover more than 
90 per cent. of the pores on each wing. Three hours after apply- 
ing the glue, the trochanters, femurs and proximal ends of the 
tibiz of these beetles were covered with the vaseline-beeswax 
mixture. An hour still later the insects were tested with the six 
odors. They were as active as unmutilated ones and appeared 
normal in all respects except in their responses to odors. Their 
responses were never pronounced and seldom prompt. 

Their reaction times are as follows: Oil of peppermint, 3 to 60 
seconds, average 10.7 seconds. One failed to respond to this 
odor. Oil of thyme, 3 to 60 seconds, average 9 seconds. One 
failed to respond to this odor. Oil of wintergreen, 5 to 60 seconds, 


442 N. E. MCINDOO. 


average 35.9 seconds. Eleven did not respond to this odor. 
Leaves and stems of pennyroyal, 3 to 60 seconds, average 35.2 
seconds. Twelve did not respond to this odor. Leaves and 
stems of spearmint, 5 to 60 seconds, average 42.6 seconds. Four- 
teen failed to respond to this odor. Parts of decayed beetles, 
5 to 60 seconds, average 40.3 seconds. Fourteen failed to 
respond to this odor. The general average reaction time of the 
26 beetles tested to the six odors is 29 seconds which is twice the 
reaction time of unmutilated potato beetles to the same odors. 
When the reaction times to the odors from only the three essential 
oils are considered, these mutilated insects responded only one 
fifth as rapidly as did the unmutilated ones. In the wire-screen 
cage with the other potato beetles already tested, they were 
apparently normal as long as they lived, because they ate and 
copulated as usual and were always as active as the unmutilated 
ones. Before they died their abdomens shrunk considerably in 
size. Up to the time of this writing (Jan. 15), 28 of the 29 have 
died. These lived from 2 to 151 days with 61 days as an average. 


MELOID. 
THE OLFACTORY SENSE OF Epicauta marginata. 


Twenty blister beetles (Epicauta marginata), commonly known 
as the “old-fashioned potato bugs,’ 
When mechanically irritated, they fold the antenne and legs 


’ 


were caught on clematis. 


against the body, usually eject a small drop of amber-colored 
liquid from each femoro-tibial articulation, and feign death. 
On account of this behavior, they were unsatisfactory to experi- 
ment with. When put into the experimental cases, some of them 
lay apparently lifeless for almost a half day. In this state they 
never respond to any odor, and after becoming as active as usual, 
they may or may not respond to odors. 

When tested with the odors from only the three essential oils, 
a general average reaction time of 13.9 seconds was obtained. 
Two of them failed to respond to each of the oils of peppermint 
and wintergreen. These insects were confined in the cage 
described on page 436. They were regularly provided with a 
fresh supply of clematis. In this cage on the clematis they 
seemed ‘‘at home,’’ but they flew out at every opportunity. 


THE OLFACTORY SENSE OF COLEOPTERA. 443 


They copulated as usual. They lived from 11 to 40 days with 
27.6 days as an average. 


(a) Effects with Antenne Cut Off. 


_ Eight more Epicauta marginata were collected. Their antennze 
were cut off at the bases. A small drop of amber-colored blood 
exuded from each wound. Seven days later the two remaining 
live ones were tested with the odors from the three essential oils. 
The general average reaction time is 5 seconds. All these beetles 
were abnormal in behavior. They lived from 1 to 8 days with 
3.4 days as an average. 


(b) Effects with Antenne Pulled Of. 


The antenne of 12 more Epicauta marginata were pulled off 
at their bases. A small drop of blood exuded from each wound. 
When tested with the odors from the essential oils three days 
later, the eight remaining live beetles gave a general reaction 
time of 5.9 seconds. They were less abnormal in behavior than 
those with the antennz cut off. They lived from 2 to 13 days 
with 5.5 days as an average. 


(c) Effects with Elytra and Wings Pulled Of. 

The elytra and wings of nine Epicauta marginata were pulled 
off at their articulations. A small drop of blood exuded from 
each wound. When tested with the odors from the essential 
oils two days later, the seven remaining live beetles gave a gen- 
eral reaction time of 25.7 seconds. Two of them failed to re- 
spond to each of the oils of peppermint and wintergreen. These 
mutilated insects appeared normal in behavior and in confine- 
ment they copulated as usual. They lived from 2 to 14 days 
with 8 days as an average. 


Tuer OLFACTORY SENSE OF Epicauta pennsylvanica. 


Twenty-five blister beetles (Epicauta pennsylvanica) were 
caught on golden rod (Solidago). This species has the same 
habit of feigning death when mechanically irritated as has Hp- 
cauta marginata. When tested with the odors from the essential 
oils, they gave a general average reaction time of 11.5 seconds 


444 N. E. MCINDOO. 


which is only one-half as rapid as the reaction time of the same 
species devoid of antenne. Three failed to respond to the oil 
of peppermint, one to the oil of thyme and two to the oil of 
wintergreen. A common response was to vibrate the legs. They 
were placed into the cage with the other species of blister beetles. 
They were regularly provided with a fresh supply of goldenrod. 
In the cage they appeared normal, and they copulated as much 
as usual. They lived from 2 to 25 days with 11.2 days as an 
average. 
(a) Effects with Antenne Pulled Of. 

The antenne of 30 Epicauta pennsylvanica were pulled off at 
their bases. When tested with the odors from the essential oils 
three days later, the 22 remaining live beetles gave a general 
reaction time of 5.3 seconds. They were only slightly abnormal 
in behavior. They lived from 2 to 25 days with 8.7 days as an 
average. 


(6) Effects with Elytra and Wings Pulled Off. 


The elytra and wings of 21 Epicauta pennsylvanica were pulled 
off at their articulations. A small drop of blood exuded from 
each wound. Blood was also seen in the distal ends of the elytra. 
When tested with the odors from the essential oils two days later, 
the 17 remaining live beetles gave a general reaction time of nine 
seconds. One of them failed to respond to the oils of thyme and 
wintergreen. These insects appeared normal in confinement 
with the other blister beetles. They copulated as usual. They 
lived from 1 to 33 days with 10.7 days as an average. 


A summary of all the preceding experiments to determine the 
location of the olfactory organs in beetles is best presented in a 
tabulated form. The following table is such asummary. Since 
a comparison of the behavior of unmutilated and mutilated in- 
sects alone is not always a safe criterion for judging the general 
behavior of mutilated beetles, the behavoir of the mutilated 
beetles recorded in this table is based mostly upon a comparison 
of the longevities of unmutilated and mutilated individuals of 
the same species. A “+ ”’ after a figure in the last column means 
that all the insects used in the experiment have not yet died. 
The longevity is based only on those that have died up to the 
time of this writing (Jan. 15). 


THE OLFACTORY SENSE OF COLEOPTERA. 


divers: JL 


445 


SUMMARY OF EXPERIMENTS TO DETERMINE THE LOCATION OF THE OLFACTORY 
ORGANS IN COLEOPTERA. 


Average 


Reaction Time. 


3| Average Length 


Ey Average Le 
6 ; nS) of Life in 
Species Se ee ae lace ce eel, Cees 
Odors. oe 
| os 
| Sec. Sec. A Days. 
Harpalus Unmutilated. Normal in be- 
DAMS ACEH: | IANO co noo so Gade ao oar Ge O.§ || TO5 | as 61.0+ 
Antenne pulled off. Normalin 
De AVM OTE Se ihe e ialav ede saceranane Sot | mOsw || Bs 58.0+ 
Elytra and wings pulled off. 
\Slightly abnormal in behavior.| 13.0 | 17.7 | 25 9.0 
‘Elytra and wings pulled off and 
pores on legs covered with 
vaseline. Slightly abnormal 
MINE AO Ioan ahs. cm, sinclar eqenar ene 16.9 | 24.1 | 18 10.0 
Harpalus Unmutilated. | Normal in be- 
caliginosus. IOEINAUGI Cd Gin Rectete piece ements 4.2 8 |Used below 
Antenne pulled off. Slightly 
abnormal in behavior....... 16.4 6 18.0 
Epilachna Unmutilated. _ Normal in be- 
borealis. IN AWAO Tey dete cues tens). ote ss, 4s Gee 13.8 18 | Used below 
Antenne pulled off. Slightly 
abnormal in behavior....... 30.8 AS 22.0+ 
Elytra and wings pulled off. 
Slightly abnormal in behavior) 31.3 4 2.0+ 
Chaulcognathus |\Unmutilated. Normal in be- 
PELMOSSMTCOEE. || WENO, cocoscasnoadcecoou™ 2.8 25 Baz 
Antenne pulled off. Slightly 
abnormal in behavior....... 2.8 3 I.3 
Passalus Unmutilated. | Normal in be- 
cornutus. IDERGKOIES oro. ec rote Ol creer ee 3.0 Bho 4| Used below 
Antenne pulled off. Slightly 
abnormal in behavior....... 3.0 3.3 4 12.5 
Cotinis nitida...|Unmutilated. Normal in be- 
INEINVAVONES oitib.5s5 ar Cho CE RENE ROR Ge RCI 5.0 8.0 1 | Used below 
Antenne pulled off. Normal 
ime belasylOte ayes nies s+ <0 wos 5.6 8.3 I 12.0 
Euphoria Unmutilated. Normal in be- 
sepulchralis. IGEN AOS 5 Gd ose biol iene RE 3.6 5 | Used below 
Antenne pulled off. Normal in 
behavior saeco. osisa ce. 4.3 5 20.0 
Cyllene robinie..|Unmutilated. Normal in be- 
MAW Oren Verne ss oleic aes 5.4 18 10.4 
Antenne pulled off. Slightly 
abnormalin behavior....... 3.0 15 5.0 
Elytra and wings pulled off. 
Slightly abnormalin behavior! 9.8 18 4.2 


446 N. E. MCINDOO. 


Average ay 
NeaeioNn Biases = Z Average Length 
Sage Experiment and Behavior of For For E 4 oh ene 
ee? Insects Tested. Three Six |aa Capt. 
Odors. | Odors. oe 
2s 
Sec. Sec. Bi Days. 
Leptinotarsa Unmutilated. Normal in be- 
ro-lineata. RAVAGE case ee cole e eae Bn | ws o4) || 2s 69.0 + 
Antenne pulled off. Normal in 
behavior sscee eae: 3.5 20 38.0 
Elytra pulled off and wings cut 
off. Normal in behavior...| I1.2 | 22.7 | 25 52.0+ 
Elytra pulled off, bases of wings 
glued and pores on legs 
covered with vaseline. Nor- 
maleinebehavloissaeieneenon 18.5 29.0 | 26 61.0+ 
Epicauta Unmutilated. Normal in be- 
marginata. NAVIOK ties. cane oe eee 13.9 20 DL) 
Antenne cut off. Considerably 
abnormal in behavior....... 5.0 2 3.4 
Antenne pulled off. Slightly 
abnormal in behavior....... 5.9 8 5.5 
Elytra and wings pulled off. 
Slightly abnormalin behavior] 25.7 7 8.0 
Epicauta Unmutilated. | Normal in be- 
pennsylvanica. HaviOrss o..0 ne eee or II.5 25 Rit, 
Antenne pulled off. Slightly 
abnormal in behavior....... 5.8 22 8.7 
Elytra and wings pulled off. 
Normal in behavior........ | 9.0 ie 10.7 


A summary of the preceding table shows the following: After 
the antenne were pulled off, four of the 11 species tested were 
normal and seven were slightly abnormal in behavior. After 
the elytra and wings were pulled off one species was normal while 
four were slightly abnormal in behavior. After the elytra were 
pulled off and the wings were cut off, the one species tested was 
normal in behavior. After the elytra and wings were pulled off 
and the pores on the legs were covered with vaseline, the one 
species tested was slightly abnormal in behavior. After the 
elytra were pulled off, the bases of the wings glued and the pores 
on the legs covered with vaseline, the one species tested was 
normal in behavior. 

Four unmutilated species responded to odors more slowly than 
did the same species after the antenne had been pulled off. This 
is explained by the fact that most beetles are more or less “‘ timid” 


THE OLFACTORY SENSE OF COLEOPTERA. 447 


for some time after being caught, and some feign death. Asa 
rule the longer they are confined and the more they are handled, 
the more satisfactory they are to experiment with. Five species 
without antennz responded to odors as promptly as did the same 
species unmutilated. Two species without antenne responded 
to odors more slowly than did the same species unmutilated. 
Since these were abnormal in behavior and judging from the 
reaction times of the other nine species with antennz pulled off, 
it is only reasonably to attribute the slow reaction times of these 
two species to their abnormal condition caused by the antennz 
being pulled off. The six species so mutilated that most of their 
olfactory pores on the elytra and wings were prevented from 
functioning responded from two to five times more slowly than 
did the same species unmutilated or with the antennez pulled 
off. The two species so mutilated that most of their olfactory 
pores on the elytra, wings and legs were prevented from func- 
tioning responded from two to six times more slowly than did 
the same species unmutilated or with the antenne pulled off. 

From all the preceding results, it seems that the antenne do 
not carry any of the olfactory organs, while the olfactory pores 
found on the peduncles of the elytra, on the dorsal surfaces of 
the wings, on the trochanters, tibia, sometimes on the femurs 
and tarsi, and perhaps on the mouth appendages, are the true 
olfactory organs in beetles. 


SUMMARY. 


In making a comparative study of the olfactory pores in 
beetles, 50 species belonging to 47 genera and representing 34 
families were used. A group of pores is always present on the 
peduncle of each elytron. It lies on the dorsal side of the well- 
exposed radial plate. The number of pores on a pair of elytra 
varies from 12 to 310. Asarule, the more pores in the group the 
smaller they are and the closer they are together. 

Of the 47 winged species examined, 11 have only one group of 
pores on each wing, 21 have two groups on each wing, 12 have 
three groups on each wing, and 3 have four groups on each wing. 
These groups are always located on the dorsal surface. Only 
occasionally are a few scattered pores found on the ventral side 


448 N. E. MCINDOO. 


of a wing. When one or two groups are present, they lie on the 
radius. When three groups are present, all three may lie on the 
radius, or two may lie on the radius and the third on the media. 
When four groups are present, one lies on the subcosta, two on 
the radius and one on the media. The largest group on the 
radius usually extends nearly all the way to the fold of the wing 
and sometimes all the distance to the fold. The number of 
pores on a pair of wings varies from 130 to 982. 

There are usually two groups of pores at the proximal end of 
each trochanter. Sometimes a pore is found at the proximal 
end)'of the femur. It is common to find a few pores at the prox- 
imal end of each. tibia; and sometimes pores are found in the 
tibial spines and on the tarsi. The number of pores on all six 
legs varies from 49 to 341. 

In regard to water beetles, the better the legs are adapted for 
locomotion in water, the fewer pores they have. The smallest 
winged species (Coxelus) examined has 273 pores, which is the 
smallest number of all the species, and the largest species (Ortho- 
soma) has 1,268 pores which is the largest number of all the species 
examined. The apterous species have more pores on the legs 
than usual. Asa rule, the smaller the species, the fewer its pores 
and the larger they are, comparatively speaking. As a rule, 
there are no generic and specific differences, except variations in 
number of pores, the amount of variation depending on the sizes 
of the individuals compared. There are no individual and sexual 
differences other than slight variations in number of pores. 

The pore apertures or pits are round, oblong, slitlike or club- 
shaped. On the elytra and wings they are always round or 
oblong. On the legs they have all four of the enumerated shapes. 

The spindle-shaped sense cells of most beetles lie in the lumens 
of the appendages outside the pore cavities, but in the legs of 
Orthosoma the sense cells lie inside the pore cavities. A small 
chitinous cone is always present. It is formed by the hypodermal 
cell at the mouth of the pore after the insect has emerged from 
the last pupal stage, and at the same time when the chitinous 
integument is being considerably thickened. The sense cells 
are fully developed when the insect emerges into the imago stage. 
The sense fiber pierces the cone and the layer of chitin between 


THE OLFACTORY SENSE OF COLEOPTERA. 449 


the pore aperture and cone, and it enters the bottom of the pore 
aperture or pit where its peripheral end comes into direct contact 
with the outside air. In Hymenoptera the sense fibers enter the 
pore apertures which are almost on a level with the external 
surface of the chitin. In Coleoptera, with a few exceptions, the 
sense fibers enter the bottoms of pits which lie in the chitin one 
third (at time of emerging into imago stage) the distance from 
the external surface. In the legs of the lady beetle, Epilachna 
borealis, instead of the chitin which surrounds the pore apertures 
being depressed, it is elevated so that the pore apertures lie in 
the center of domes above the general surface of the legs. In 
the legs of the blister beetles, Epicauta marginata and E. penn- 
sylvanica, the pore apertures lie on a level with the surface of 
the legs. In the legs of the potato beetle, the pore apertures lie 
at the bottoms of shallow pits. All four preceding species have 
hypodermal gland pores over the entire body, except the wings. 
These pores in the lady beetle are perhaps the most highly 
developed. They lie on all sides and even among the olfactory 
pores on the legs. In the other three species they are less highly 
developed on the legs near the olfactory pores and none is found 
very close to an olfactory pore. This correlation between the 
hypodermal gland pores and the olfactory pores is certainly a 
means of preventing the secretion from the gland cells from 
running into the pore apertures. 

A large nerve and a large trachea run into oe elytron and 
wing. In the peduncle of the elytron they run through the 
radial plate just beneath the group of olfactory pores. Branches 
from the nerve are given off which connect with the sense cells. 
The large nerve and trachea passing into the wing soon divide 
so that a smaller nerve and a smaller trachea run through each 
main nerve. The largest trachea passes through the subcosta, 
and the largest nerves pass through the veins carrying the ol- 
factory pores. These nerves give off branches which connect 
with the sense cells. The sense cells wherever found are always 
surrounded by blood. 

In the experiments to determine the location of the olfactory 
organs, 434 individuals were tested. These belonged to I1 
species representing 8 families. After the antenne were pulled 


450 N. E. MCINDOO. 


off, 4 of the 11 species tested were normal and 7 were slightly 
abnormal in behavior. After the elytra and wings were pulled 
off I species was normal while 4 were slightly abnormal in 
behavior. After the elytra were pulled off and the wings were 
cut off, the 1 species tested was normal in behavior. After the 
elytra and wings were pulled off and the pores on the legs were 
covered with vaseline, the I species tested was slightly abnormal 
in behavior. After the elytra were pulled off, the bases of the 
wings glued and the pores on the legs covered with vaseline, 
the I species tested was normal in behavior. 

Four unmutilated species responded to odors more slowly than 
did the same species after the antenne had been pulled off. This 
is explained by the fact that most beetles are more or less “timid” 
for some time after being caught, and some feign death. Asa 
rule, the longer they are confined and the more they are handled, 
the more satisfactory they are to experiment with. Five species 
without antennz responded to odors as promptly as did the same 
species unmutilated. Two species without antennz responded 
to odors more slowly than did the same species unmutilated. 
Since these were abnormal in behavior and judging from the 
reaction times of the other 9 species with antennz pulled off, it is 
only reasonable to attribute the slow reaction times of these 
two species to their abnormal condition caused by the antenne 
being pulled off. The 6 species so mutilated that most of their 
olfactory pores on the elytra and wings were prevented from 
functioning responded from 2 to 5 times more slowly than 
did the same species unmutilated or with the antenne pulled off. 
The two species so mutilated that most of their olfactory pores 
on the elytra, wings and legs were prevented from functioning 
responded from 2 to 6 times more slowly than did the same 
species unmutilated or with the antenne pulled off. 

From all the preceding results, it seems that the antenne do 
not carry any of the olfactory organs, while the olfactory pores 
found on the peduncles of the elytra, on the dorsal surfaces of 
the wings, on the trochanters, tibia, sometimes on the femurs and 
tarsi, and perhaps on the mouth appendages, are the true ol- 
factory organs in beetles. 


THE OLFACTORY SENSE OF COLEOPTERA. A5I 


DISCUSSION. 


Since the writer (’14c) has already written a complete review 
of all the literature available concerning the sense of smell in 
insects, only a brief discussion is necessary in this paper. 

Hicks (’57) says that the olfactory pores in Coleoptera are 
arranged in long rows along the subcostal nerves. The same 
author (’59) states that in Coleoptera these organs are highly 
developed and occur in numerous groups on the subcostal vein, 
mostly at the widest part, but are also scattered along it to the 
fold of the wing. In Carabus they are found on veins other than 
the subcostal. In many beetles the pore is overarched by a hair, 
which probably protects the organ. He could distinguish no 
sexual differences in these organs, except the pores are slightly 
larger in the females, due to their greater size. Hicks (’60) 
first found the olfactory pores on the legs of beetles. The 
present writer has never seen a hair overarching an olfactory pore. 

Hochreuther (12) seems-to be the first to study the internal 
anatomy of the olfactory pores in beetles. Since he used only 
Dytiscus marginalis and perhaps because he did not have enough 
sections through these organs, he failed to understand their 
anatomy. He states that each dome-shaped organ is located at 
the bottom of a chitinous flask, the mouth of which communicates 
with the exterior. Instead of the peripheral end of the sense 
fiber coming into direct contact with the air in the flask, it 
apparently stops just beneath the chitinous dome at the top of 
the organ. His terminal strand (Terminalstrand) may be the 
same as the hypodermal secretion forming the cone described 
by the writer. Hochreuther found a few of these dome-shaped 
organs on the epicranium near the margin of the eyes, nine on the 
proximal end of the first antennal segment, two on the distal end 
of the second antennal segment, a few on the dorsal side of the 
labrum, a very few on the dorsal side of the mandible, several 
on each maxilla, about 18 on the first four segments of the front 
legs, about 10 on the first three segments of the middle legs, and 
a few on the trochanters of the hind legs. He evidently did not 
examine the wings. Thus according to Hochreuther these 
organs are rather widely distributed. Since the peripheral ends 
of the sense fibers do not come into contact with the outside air, 


452 N. E. MCINDOO. 


but connect with the tops of the domes, he suggests that they 
receive some kind of mechanical stimuli, although he performed 
no experiments to determine their function. 

Lehr (14), resuming the search for sense organs in Dytiscus 
marginalis where left off by Hochreuther, found dome-shaped 
organs on the elytra and wings. He found three main groups 
in identically the same places as described by the present writer. 
The number of pores in the group on the elytron varies from 130 
to 150. The two main groups on the radius (his subcosta) of 
the wing are large, but he did not count the pores in them. He 
found a fourth group, consisting of about 30 pores, on the ventral 
side of the costa near the base of the wing. He also found a few 
scattered pores on the dorsal side of the costa just distal to the 
fold of the wing, a few on the second cubitus, and a few irregularly 
scattered along the full length of the media. Lehr has described 
the anatomy of these organs almost identically as seen by the 
present writer, but it seems that he has not correctly interpreted 
some of the structures. He seems to think that each sense cell 
is surrounded by another cell, but the latter cell is perhaps 
nothing more than coagulated blood and the portion of it extend- 
ing into the pore is certainly a hypodermal secretion forming the 
cone as described in the preceding pages. His neurilemma 
nuclei are perhaps hypodermal nuclei. He is able to trace the 
sense fiber through the cone, but he has not recognized the small 
opening through the dome. ‘This is not surprising, because the 
pores in the wings as so small that the openings or pore apertures 
are never noticed unless first seen in the largest pores in the legs 
or mouth parts. In the thinnest sections, the chitin forming the 
dome is so thick as compared to the diameter of the pore aperture 
that the aperture appears only as a streak slightly lighter than 
the other chitin in the dome. Lehr has nothing to say about the 
physiology of these organs. 

In experimenting with mutilated beetles, Hauser (’80) seems 
to be the only one who has taken their longevity into considera- 
tion. And even he has not kept an accurate record of their 
behavior and longevity. He claims to have studied the behavior 
of beetles before and after the removal of the antenna. When 
the antennze were removed he ascertained that many beetles 


THE OLFACTORY SENSE OF COLEOPTERA. 453 


soon became sick and died, while others lived thereafter for 
many days. When tested with odors, most of the beetles without 
antennz failed to respond, but Hauser states that Carabus, 
Melolontha and Silpha still responded to odors, although more 
slowly. 


PorPI MPPg PeorP] 


P| a ' 


Fic. 3. Antennal organs of the water beetle, Dyliscus marginalis, copied from 
Hochreuther (1912). A, small tactile hair (Sinneshaar) from first segment of an- 
tenna, total preparation (Fig. 1 from Hochreuther), X 330; B, portion of Fig. 12 
from Hochreuther, showing four small sense bristles (Sinnesborsten) from proximal 
end of second segment of antenna, X 265; C, longitudinal section (Fig. 48 from Hoch- 
reuther) through a hollow pit peg (hohlen Grubenkegel), X 470; D, longitudinal 
section through a small massive pit peg (massiven, grubenstandigen Zapfen) and 
two pore plates (kelchférmige Organe), X 590. This drawing is a combination of 
Figs. 32 and 58from Hochreuther. Only the pore plates (PorPl) are taken from Fig. 
58. Hochreuther gives a drawing of only one perfect pore-plate organ, or cup- 
shaped organ, and it is from the maxillary palpus. CM, cup-shaped membrane; 
HPPg, hollow pit peg; MPPg, massive pit peg; PorPl, pore plate; SB, sense bristle; 
THr, tactile hair. See page 456 for other abbreviations. 


A54 N. E. MCINDOO. 


For the purpose of judging whether the antennal organs are 
better adapted anatomically than the olfactory pores for receiving 
odor stimuli, the former organs (Text-fig. 3, p. 453), of Dytiscus 
marginalis have been copied from Hochreuther (12). This work 
of Hochreuther is a comprehensive study of the morphology of 
all the chitinous sense organs of Dytiscus. Since it is perhaps 
the latest and certainly the best study on the antennal organs 
of beetles, these organs shall be briefly described. 

Each of the 11 segments in the antenna of Dytiscus carries a 
number of sense organs. The farther from the base of the 
antenna the more numerous they are. The distal half of the 
antenna is covered abundantly with sense organs, while the 
proximal half is sparingly covered with them. The first and 
second segments are well provided with slender tactile hairs 
(Text-fig. 34, THr) which have been called Sensilla trichodea 
by Schenk. These hairs are also found on all the other appen- 
dages and even on the head, thorax and abdomen. Two groups 
of sense bristles (Text-fig. 3B, SB), called Sensilla chetica by 
Schenk, lie at the proximal end of the second segment. These 
hairs are also common on most of the other appendages, on the 
head, thorax and abdomen. All segments, except the first one, 
are well provided with small massive pit pegs of the thick-walled 
type (Text-fig. 3D, MPPg). All segments, except the first one, 
are only sparingly provided witha second type of pit pegs. This 
one is the hollow or thin-walled type (Text-fig. 3C, HPPg). 
Only about six of these were found on each segment. Besides 
being found on the antenne, both types of pit pegs are common 
on all the mouth parts, on the mesothorax, around the spiracles, 
on all the legs, and on the sexual apparatus. Pit pegs have 
been called Sensilla coelloconica by Schenk. All segments, 
except the first two, are abundantly supplied with the cup- 
shaped or pore-plate organs (Text-fig. 3D, PorPl). For both 
antenne they are estimated between 4,500 and 5,000. These 
organs are also common on the palpus of the first maxilla. They 
were first studied by Nagel on the antenne and maxillary palpi 
of Dytiscide. In the honey bee Schenk has called them Senszlla 
placodea. Of the five antennal organs of Dytiscus, only the 
hollow pit pegs are regarded by Hochreuther as probably ol- 


THE OLFACTORY SENSE OF COLEOPTERA. 455 


factory in function. If they really act as olfactory organs, then 
the mouth parts, thorax, legs and sexual organs must aid in 
receiving odor stimuli. Hochreuther considers the antennze more 
important as appendages for carrying organs for receiving 
mechanical stimuli rather than those receiving chemical stimult. 

According to various authors the antennal organs of different 
beetles vary only slightly. The antennal organs of Dytiscus 
are also similar to those of the honey bee. In both of these 
snsects the tactile hairs are of the same type. The Forel flasks 
and pit pegs of the honey bee are two types of pit pegs which 
are perhaps rudimentary, because the tips of the hairs do not 
come to the exterior of the chitin. The massive pit pegs, 
hollow pit pegs, and the sense bristles of Dytiseus are certainly 
nothing more than three types of tactile hairs. The hollow 
pit pegs compare closely with the pegs of the honey bee, except 
the pegs have thinner chitin at’ the tips. This is probably on 
account of more acute sense of touch in the honey bee. The 
pore-plate organs of the honey bee and the cup-shaped organs 
of Dytiscus are also quite similar. ; 

One or more of the antennal organs of every insect studied 
have been called olfactory organs, and it is possible that most of 
these organs may be fourd on other appendages, besides the 
antenne, as already seen in Dytiscus. 

In conclusion it seems beyond a doubt that none of the antennal 
organs of beetles shown in Text-fig. 3 serves as an olfactory organ, 
and that the olfactory pores are well adapted anatomically for 
receiving odor stimuli, because the peripheral ends of their sense 
fibers come into direct contact with the external air. 


LITERATURE CITED. 
Hauser, Gustav. 

*80 Physiologische und histologische Untersuchungen iiber das Geruchsorgan 

der Insekten. Zeitsch. f. wiss. Zool., Bd. 34, Heft. 3, pp. 367-403, with 2 pls. 
Hicks, J. B. 

’57 On a New Organ in Insects. Jour. Linn. Soc. London, Zool., Vol. 1, pp. 
136-140, with r pl. 

’e9 Further Remarks on the Organs Found on the Bases of the Halteres and 
Wings of Insects. Trans. Linn. Soc. London, Zool., Vol. 22, pp. 141-45, 
with 2 pls. 

760 On Certain Sensory Organs in Insects, Hitherto Undescribed. Jbidem, 
Vol. 23, pp. 139-153, with 2 pls. 


456 N. E. MCINDOO. 


Hochreuther, Rudolf. 
212 Die Hautsinnesorgane von Dytiscus marginalis L., ihr Bau und ihre Ver- 
breitung am Ké6rper. Zeitsch. f. wiss. Zool., Bd. 103, pp. I-114. : 
Lehr, Richard. 
’t4 Die Sinnesorgane der beiden Fliigelpaare von Dytiscus marginalis. Zeitsch. 
f. wiss. Zool., Bd. 110, Heft. 1, pp. 87-150, with 45 text figs. 
MclIndoo, N. E. 
’t4a The Olfactory Sense of the Honey Bee. Journ. Exp. Zool., Vol. 16, no. 3, 
April, pp. 265-346, with 24 text figs. 
’t4b The Olfactory Sense of Hymenoptera. Proc. Phila. Acad. Nat. Sci., 
Vol. 66, pp. 294-341, with three text figs, and 2 pls. : 
’14c The Olfactory Sense of Insects. Smithsonian Misc. Collec., Vol. 63, 
no. 9, Nov. (Publication 2315), pp. I-63, with six text figs. 


EXPLANATION OF PLATES I. AND II. 


All figures including: Text-figs. I and 2 are from camera lucida drawings made at 
the base of the microscope. Figures 1 to 8 inclusive and 22 on the plates are 
enlarged 465 diameters. All the remaining figures on the plates, except the dia- 
grams 28, 29 and 31, are enlarged 580 diameters. 


ABBREVIATIONS. 
WE SCM esse blood sinus. 
BMn ee ee basal margin of elytron. 
Ge otan seie costa. 
(Gl etn blaerccohg chitin. 
Chie aera chitin formed before insect emerges into imago stage. 
Ghia meee oaaheeees chitin formed after insect emerges from last pupal stage. 
(Gls Sani aes chitinous membrane of pore plate. 
GME IBN acres: cup-shaped membrane of tactile hair on antenna. : ; 
COne ne ates chitinous cone. 
Conds cun = 2 connective tissue. 
ConT Nuc. ...nucleus of connective tissue. 
Cx AR ee coxa. 
Y ASN eae J oe aks femur. 
TE Dre trac is opti, bes flange of olfactory pore. 
EO bate pe ete te: where wing folds. 
GIO Aeon Pee. gland cell. 
EAP ORS ir sae hollow pit peg on antenna. 
Ui i Reeton eee ae hair. 
EBV Ds 3 tic a6, hypodermis. 
YE bihen eave ears membrane resembling hypodermis which divides the lumen of proxi- 
mal end of the tibia of Epzlachna into two chambers. 
FA PEG ees hypodermal cell. 
HypNuc..... hypodermal nucleus. 
IES. oa ob dioe hypodermal secretion. 
MESS A casein: muscle. 
VID) nye eco muscle disk. 
MCUs netets media 


THE OLFACTORY SENSE OF~COLEOPTERA. A57 


WWPIP LASS 5 5 Be small massive pit peg on antenna. 
INS cic Been eR CONE nerve. 

IN(IB} 3 Seep nerve branch. 

UNI U hte variety ee neurilemma. 

NeuNuc..... nucleus of neurilemma. 
NeurNuc....neuroglia nucleus. 

Bre Rats ctisicts: boli pit of pore. 

IACPAMay's baa pore aperture. 

POvV.Gline oh. 4 pore of gland. 

UZ OVEL IA eee pore of hair. 

LOY Zieh eererene pore plate on antenna. 

J ENOV? Ls ne OR pore of olfactory organ. 
OTe a ale. pore wall. 

PorWGl.....pore wall of gland. 
PorWHr.....pore wall of hair. 

Je elaine radius. 

REIS oe ees radial plate. 

AES beste Uyak small sense bristle on antenna. 
Oars sense cell. 

IS Gress sie avon sense cell of tactile hair 

SS GG tks ae sense cell group. 

SXCINICS bo p00 sense cell nucleus. 
SCNucl......sense cell nucleolus. 

SG oie eee subcosta. 

Sal Eleca Bue peor subcostal head. 

SV AL aay cee sense fiber. 

TPG ted ost tarsus. 

TE Die ag Sea Me tibia. 

TbSp........tibial spine. 

BELPER ohh lS tactile hair. 

IE? soe EEG trachea. 
TrNuc.......Nucleus of trachea. 

IR Oe eee es trochanter. 

THEO OMe), 3 aysus a groups Nos. I to 6 of the olfactory pores. 
(ie, eee ONT location of scattered pores on ventral side of wing. 


) Bal @oicdcoc location of scattered pores on tibia. 


458 N. E. MCINDOO. 


PLATE I. 


Fic. 1. Six of the eight olfactory pores (PorW) in group 6 on inner surface of 
right hind leg of Epzlachna borealis; also one hair (Hr), one hair socket (porWHr) 
and two hypodermal gland pores (PorWGl). 

Fic. 2. Two olfactory pores (PorAp), five hairs (PorWHr) and 19 gland pores 
(PorWGl) on outer surface at proximal end of right hind leg of Epilachna. 

Fic. 3. Five olfactory pores from tibial spine of Epicauta marginata (same as 
shown in Text-fig. 2G). 

Fic. 4. Group r of olfactory pores on peduncle of elytron of Epilachna (same as 
shown in Text-fig. 1B). 

Fic. 5. Seven of the olfactory pores in group 2 on wing of Epilachna. 

Fic. 6. Eleven of the olfactory pores in group 3 on wing of Epilachna. 

Fic. 7. Ten of the olfactory pores in group 4 on wing of Epilachna. 

Fic. 8. Four of the five olfactory pores on ventral side of wing of Epilachna. 
Figs. 5 to 8 represent some of the pores as shown in Text-Fig. 1C. 

Fic. 9. Olfactory pore from trochanter of Uloma. 

Fic. ro. Olfactory pore from tibia of Uloma. 

Fic. 11. Three olfactory pores from elytron of Uloma. 

Fic. 12. Olfactory pore and sense cell from wing of Uloma. 

Fic. 13. Olfactory pore and sense cell from trochanter of Orthosoma (cut slightly 
obliquely). 

Fic. 14. Olfactory pore and about one third of sense cell (SC) from trochanter 
of Orthosoma, showing pit (P) and flange (Fl). 

Fic. 15. Olfactory pore from tibia of Orthosoma. 

Fic. 16. Oblique section through trochanter of Epilachna, showing anatomy 
of leg. It was cut in such a manner that no muscles are shown in the section and 
that the nerve (VV) is severed in two places. 

Fic. 17. Cross section through proximal end of tibia of Epilachna, showing 
anatomy of leg at this place. The gland pore (PorGl), hair pore (PorHr) and sense 
cells (SC), belonging to the tactile hairs (Hr) were taken from two other sections, 
and the gland cell just beneath the gland pore was taken from the other end of this 
section. 

Fic. 18. Four olfactory pores and a small portion of hypodermis from elytron 
of Epilachna. The material used for Figs. 17 and 18 was from an old adult beetle 
that had been confined in the laboratory nearly all summer. 

Fic. 19. Four olfactory pores, sense cells and nerve (NV) from wing of Epilachna. 


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BIOLOGICAL BULLETIN VOL. XXVIII. 


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Fic. 20. Three olfactory pores from wing of Passalus. 

Fic. 21. Six olfactory pores from elytron of Orihosoma. 

Fic. 22. Cross section through wing of Orthosoma, showing anatomy of wing 
beneath olfactory pores. 

Fic. 23. Olfactory pore from elytron of Passalus, showing sense cell (SC) 
connected with pore aperture (PorAp) and with nerve (NV); also hypodermal cell 
(HypC) that forms the cone (Con). 

Fic. 24. Olfactory pore from trochanter of Epicauta marginata. 

Fic. 25. Olfactory pore from trochanter of Epicauta pennsylvanica. 

Fic. 26. Olfactory pore from trochanter of Leptinotarsa ro-lineata. 

Fic. 27. Three olfactory pores from tibial spine of Epicauta marginata. The 
material used for Figs. 24 to 27 had been treated with caustic potash. 

Fic. 28. Transverse-longitudinal diagram of proximal end of trochanter be- 
longing to right hind leg of Epilachna, showing internal anatomy of leg and super- 
ficial view of hairs, hair sockets, gland pores and olfactory pores. The four pores 
at the right belong to group 6 and the three at the left belong to group 5. 

Fic. 29. Oblique transverse-longitudinal diagram of portion of peduncle 
belonging to Epilachna, showing internal anatomy of radial plate (RP), innervation 
of olfactory pores and a superficial view of a few of the pores in group 1. The 
transverse portion of the diagram passes through the radial plate in the direction 
of the line marked “‘a”’ in text Fig. 1B. 

Fic. 30. Oblique superficial view of olfactory pores on wing of Epilachna, 
showing sense fibers (SF) connected with pore apertures (PorAp). 

Fic. 31. Transverse-longitudinal diagram of portion of wing belonging to 
Orthosoma, showing internal anatomy of wing, innervation of olfactory pores and 
a superficial view of a few of the pores on radius (R) and media (Me). 


BIOLOGICAL BULLETIN, VOLe XXVille PLATE ll. 


N. E. MCINDOO DEL. 


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