Lt collet eigheeoteh Maem iclaeanaap ete Pea rwer gem Ow a ct eee be ee ae eee see pareticor drow Spay sraine Cen TTR Oe WR eee aoe pad one b spo - Fe Poe Te pO A Nig eet ae cme aD ak ey Se eegtecterenen are irn ee a en eel , ae Al », Ley icy Mite See oS Saspihe nas ; ee wnt apes - Ay SRY A gl or re * - j Wea Sally: A tele As) i ae th ress 4 Binh Digitized by the Internet Archive in 2009 with funding from University of Toronto http://www.archive.org/details/journalofexperim25broo THE JOURNAL OF EXPERIMENTAL ZO0OLOGy EDITED BY Wiuiiam E. Castie Jacques LoEB Harvard University The Rockefeller Institute EpWwIN (Gi. CoNKLIN EDMUND B. WILSON Princeton University Columbia University THomas H. MorcGan Cuarues B. DAVENPORT ; eh Columbia University Carnegie Institution GEORGE H. PARKER HERBERT S. JENNINGS Haeard Unseiunty Johns Hopkins University RAYMOND PEARL FRANK R. LILLIE Maine Agricultural University of Chicago Experiment Station and Ross G. HARRISON, Yale University Managing Editor HO aa VOLUME 25 | 1918 | 6 1 /17 THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY PHILADELPHIA, PA. CONTENTS No. 1. FEBRUARY Auice M. Bortna AND RAayMonD PzarRL. Sex studies. XI. Hermaphrodite birds. Nine text figures and nine plates............--.-+--+++-s++-+> Ropert Stantey McEwen. The reactions to light and to gravity in Droso- phila and its mutants. Three figures...........----------+seeeeceees CG. V. Morritu. Some experiments on regeneration after exarticulation in Diemyctylus viridescens. Ten figures (three plates). 282. .c ae tele - Epuvarp Usntenuutu. Is the influence of thymus feeding upon develop- ment, metamorphosis and growth due to a specific action of that gland? J.M. D. Otmstep. The regeneration of triangular pieces of Planaria macu- lata. A study in polarity. Fourteen figures........-.-.-.-+--+--+++-- Manton Coretanp. The olfactory reactions of the marine snails Alectrion obsoleta (Say) and Busycon canaliculatum ((Bamitis eee xc eehet aah Sener Setic Hecut. The physiology of Ascidia atra Lesueur. I. General physi- ology. Witteemeneures).... 2.52.2 Hee. 2 ee ee ee ye 2 a Sexic Hecut. The physiology of Ascidia atra Lesueur. II. Sensory physi- OLOGYs | AOMINENIEES 005.2 Fen oe Fee soe nina re neeintnin cin ole ee atin maine No» 2 APRIL Cuester A. Stewart. Changes in the relative weights of the various parts, systems and organs of young albino rats underfed for various periods. Orie pe IR A ts ga ee c eRe aye coke a encase cine»
“(4a ies) V84e3) Sile2) OS OO-2 OO es
Mests7..-: 21 22 23 24 25 26 27 28 29 30
Indices... 66.8 65.6 81.8 68.7 61.2 64.9 58.7 59.3 60.6 61.6
Mests: x-c: 31 32 33 34 35 36 37
Indices... 58.1 58.0 59.3 61.2 58.0 53.1- 55.5 48.0 50.6 46.8
Testsper 41 42 43 44 45 46 47 48 49 50
Indices... 45.6 52.4 49.9 52.4 49.3 52.9 48.1 55.3 45.6 43.0
ing all the groups in each series. This was done by averaging
the corresponding tests in each group of the same age and sex
(table 1, graph 1).
Fatigue curves for flies of different ages
Average of seven cards of flies 4-6 days old
ote ee toe es ea ee Average of seven cards of flies 4-6 days old
~...Average of five cards of flies 16-18 hours old
+ Average of five cards of flies 16-18 hours old
Per cent of phototropism
7] > 10 St 20 aoe 30 3S, 40 45 ar)
Graph 1
It appears that the younger flies are rather less phototropic,
or at least less active, than those 4 or 5 days old. Also they tire
more easily. Furthermore, although in the younger groups the
females are consistently above the males, in the older groups the
males, after the first few tests are quite the equals of the females.
This result is substantially in accord with that obtained in my
preliminary tests.
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 59
As has been noted, the older females gain rather less rela-
tively in speed than the older males. This, it appears, might
very likely be due to the fact that the female is gradually weighed
down by the growth of her eggs. Indeed, it is known that these
do not reach their full size for 4 or 5 days, the time varying some-
what with the abundance of food. This suggested the experi-
ment of running some insects of both sexes through a series of
tests to be made daily for a period of two weeks, during which
time they would be given fresh food each day. Three groups of
flies with ten males and ten females in each group were selected
for this purpose. Five trials were given to every set of ten flies
at each of the testing periods, and the results of these five trials
averaged for the period in question. This average has been
taken as the index for the set of ten flies for this test. It is from
an average of these averages that table 2 is made up. The tests
for the first day were given at the ages of 4, 12, 15, 18, 21, and
24 hours. Afterward there was one test a day consisting of the
usual five trials. The results indicated in table 2 and the cor-
responding graph show that the females, though starting ahead
of the males, fell away much more rapidly than usual, thus tend-
ing to confirm the conclusion that a good share of their falling
off with age is due to the increased weight of their ovaries. In-
cidentally, it is again evident that the strongest reaction does
not come at 18 hours.
The next step was to run a similar series of daily tests for flies
whose food was not changed daily. In this case a small amount
of banana food was put in the vial in which a group of flies was
kept and allowed to remain there. It gradually dried up so that
the flies could derive less nourishment from it each day. That
they must have derived some is certain, for Drosophila can not
live 24 hours without food. Besides the drying, however, there
is also a chemical change in food in which larvae are not work-
ing. This, as well as the drying, tends after a few days to make
the food unfit for the insects. This fact accounts for the death
of those flies to which a little fresh food was not given on the
sixth day. From this, as well as other experiments, it appears
that 6 days is about the average time that flies will live under
60 ROBERT STANLEY McEWEN
such conditions, though I have not infrequently had them live
longer. For some reason not recorded no B and C groups of
males were started. Hence this experiment is not particularly
satisfactory. However, so far as it goes it tends to uphold the
views already stated. The males gain on the females as age in-
creases, and both sexes show a general increase up to the time
when feeding was necessary. The results are summarized in
table 3 and the accompanying graph.
There remain to be described a couple of experiments which
throw more light on the fatiguing effect of frequent tests given
TABLE 2
Males
themperavuressa-seeeeeres oie 20° mile Oe D3 2e 24° 24.1° PB | 24° | 22.5°
Hours Days
Age in hours or days.... 4 12 15 18 21 24 2 3 4
Group
TNb, eater 8-6 ocrchatie 82.5 | 60.5 | 57.5 | 62.0 | 69.5) 72.5 | 71.0 | 84.0 | 83.0
BE e:, one ee ee eae es (SROM OS LOE EON O4eOR ECD eou| OS.Obln@2 Onl Sleds leo
[ Or o> aR bake ga So20) old) | G2.571Ga20 5 | 89.5 | 95.0 | 96.0 | 92.0
AVC AL Che ashes (OES Obes GOO eI a 5 OeSaleniticor| (6.6 |e (Qual Sy sll oaee
Temperature..... 21.0° 21.6° | DE yin 24.6° | 22.8° 215° PPG 20.0° 23.0° 24.5°
Days
Age in days..... 5 6 7 8 9 10 11 12 131 14
Group:
Atel MOO SON OES GOROM IES Hal llviotas I iG2) i ILS. Sill zaesl a ets lean lm SG)
Be. eS leon CORON eile On sOS250 6565146920) || 56500 c44e0n oS om mos
Ceee ee SOLO AGIe on Ole On|nS9e5) |) 82-00 (he 8520) | SSei ez Geon eet OmmesonO
Average...... COLON SLe Os eiGe2alShe9! |c7325 7220) | G424s e56eSalnioeD alee
1 On this day one fly from the A group of males was lost. As the records
indicate that very probably the fly lost was a slow one, a new calculation was
made on the assumption that had this fly been present it would always have
remained in the zero section. The figures resulting from this calculation are
72.1 for the thirteenth day and 73.8 for the fourteenth. This changes the aver-
ages for these days to 70.5 and 69.6 respectively.
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 61
TABLE 2—Continued.
Females
Temperature..... Tie 20° 21252 23-27 24.0° | 24.1° 23.0° 24.0° PPL S 21.0°
Hours Days
Age in hours or
VSien caganeice 4 12 15 18 21 24 Z, 3 4 5
Groups
Acs. 92.5 |.69.0 | .90.0)| 7625.) 84.0 | 84.0 | 75.5 |.76.5 | 70.5 |.72-0
1B EN Goes 95.0 | 79.5 | 77.0 | 77.5 | 90.0 | 87.0 | 83.0 | 83.0 | 76.5 | 65.5
Gr O4IS 7 Ser lanOsoL ll eiSeor lkOdeos aoe. Ol meneon || Slo) (o-o I OlLeo
Average...... 94.0 | 73.8 | 79.1 | 76.8 | 90.5 | 87.6 | 81.6 | 80.3 | 74.1 | 69.6
‘emperaturesc.s s2ose-1 5 21.6° Date 24.6° | 22.8° | Pa ase Dee 20.0° | 23.0° 24.0°
Days
fAlreninidayssuts ses cee 6 7 8 9 10 11 12 13 14
Groups
Lin 6 {re 65.0 | 58.5 | 56.5 | 51.0 | 38.0 | 33.5 | 33.5 | 44.0 | 43.5
13. eso Se ee 655m os ON eAteOL le S8e5r |) So20) eobron eaten! oS sonon -O
CL. 5 eee 63.5 | 43.5 | 43.5 | 32.5 | 26.51 37.0] 18.5 | 29.0 | 29.5
Averipewers.......| 64.6.1-52.0 | 47.0 | 40.6 | 33.1 | 35.6] 26.5 [3711 | 36.0
Showing change in phototropism of wild flies when fed daily with fresh food
Per cent of phototropism
Hours °9 827% #
Days A ~
Temperature at each
test dlso indicated,
8
7 b
Age of flies
Graph 2
62 ROBERT STANLEY McEWEN
TABLE 3
Males
Temperature....| 22.7° leo Deve 215° Dy hs 24.5° PBL 21:6° Zi e1e | PP BIG
Hours Days
Age in hours or
AYS ence 4 12 15 18 21 24 2 3 4 5L
Group A: ;
7 flies)... 91.4 | 80.6 | 69.9 | Sil aey Hi SAP eteytall |) Pane) || LekDacch || Acc) |) We atss
Temperature ss -7seeeer 23.0° 22.6° 21.8° | 23.0° 23.0° 22.6° PAGS PA pA? 24.7°
Days
Aven daysieeeeeeeeeeen: 6 7 8 gz 10 11 12 13 14
Group A:
(eieSece.: eee GUSH Wisi 93.3 | 94.9 | 92.4 | 82.4 | 88.3 | 80.1
Females
Temperature.....| 23.5° 22.4° 221° Qo 22.0° 23.0° 23.0° Deelie 23.0° 21.6°
Hours Days
Age in hours or
RYS vce 12 15 18 21 24 2 3 4 51
Giflies Aes al S028) BOFS | iGEG Nie | 642986823) | 85.8) | 9626 |e 93esntOres
9 flies B.....| 99.4) 96.6 | 85.5 | 92.1 | 77.4 | 93.8 | 97.7 | 98.8 | 94.9 | 97.7
10 flies C.....| 100.0} 96.5 | 95.5 | 98.0 5.3 | 98.0 | 87.5 | 96.0 | 94.0] died
Average......| 93.4) 87.9 | 85.8 | 87.5 | 79.2 | 88.3 | 90.3 | 97.1 | 94.0) 97.5
Memperatures.-.eeeeeeeer 21.2° (for B and C females only).
Days
Age LINGSVS 2254 eee 6 7 8 9 10 ll 12 13 14
(Gabi A oes ee ocacec| Sl@ |) 7e.83 CoO yee eel oils) 1) 837/44 |] SHS | che!
Olflres Breese eee sell OGL OR aired
IAVCLAPE CHEE Ee eee RSSaS
1 Indicates that food was changed at this point to prevent flies from dying.
The drop on the sixth day is probably accounted for by this fact.
? Indicates only six flies in this group from the ninth day on.
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 63
1a0
een s dad 6” Jey.”
asl =~
A y skipped “
g \
ye ws
2 \ ~ 2 \
Bor \ \
a belenging 1 \
2, 80 o. A y
° from th F)
45;
i 2
2
= 70 \ SSS Ses 9
Q
a 6s
°
Z «|
ed '
S '
5 1
' A
' — ‘
'
48 p ‘
v u
AS \
] Died
74 “a IS 18 12). a+ 2 3 4+ 5 ‘ 7 3 y io “ut f2 1s (O/4#
Age in hours Age in days
Graph 3
to flies of different ages, as well as on the problem of time of
maximum activity.
The first of these experiments was designed to show the effect
of testing flies aged 18 to 24 hours three times with an interval
of 2 hours between the tests. Seven groups of insects were
thus tested, each group save three containing sixteen flies, eight
male and eight female. Groups A and B only contained six
flies of each sex, and Group E only five. The usual three trials
constituted a test for each group, the sexes as always being
tested separately. The average of the three trials is given as the
index for the test in question.2. The following summary of the
results was obtained by averaging the indices of the seven groups
(table 4).
Along with this series of tests there was run a parallel series
similar in every respect except that the insects were 9 days
old instead of 18 hours. Only six groups were used in this case,
but there were eight flies in every group. A separate single
* It is to be noted that in this experiment as well as in the one on fatigue,
the testing tube was only divided into four sections instead of five. The sec-
tions were then valued as follows: 100, 75, 25 and 0. With this variation ealcula-
tions were made as described under method II.
64 ROBERT STANLEY McEWEN
TABLE 4
MALE FEMALE TEMPERATU RE
First testi sce.c..ahc eee ec eee 68.9 Sie 243°
Second! test:2.c aa aon ee eee 59.9 85.3 DAA
Third testes. eee ee 55 83.5 DAR ie
Average: see teen emer ene Sek eee 61.4 85.5
Difference betweenmmalesvand temaleshe 4]. oe ase eee eee ee oe eo all
Rotalentimberkotmalestusedss.4- eee eee eee 47
Motalenumibercottemaleshused..... ae. aan ee eee 47
SECON Fea a ve ADR ee Aee Cis, Ae fs ais ties SRS ood a teehee a tC 94
group was put through the series at 6 days, and the results
were practically similar to those obtained from the 9 day flies.
For the sake of uniformity, however, they are not included in
the following summary (table 5).
TABLE 5
MALE FEMALE TEMPERATU RE
RiTsiti GEsthr cc taot eo ae one Sere ee 71.6 78.3 PIs 1
Second testacncckec cine ee eee ee eee 74.8 83.9 sy Al
EHindsGesittemosccsct< eee tata eee 76.8 83.3 DAE
ANGLER Coen ais iss aiede a OE cian 74.4 81.8
Differencesbetween malestand temales:+2a-....c.s0-21--s lose eee. 7.4
Notalenum|berrotemsleswused sweeeiaretes 4 415 lcs joel ane 48
TNotalinumberottemalessusedsasneeec.. sc. sss.e ecco 48
FOUAR ee ae Re aera: todd dhcelnw site deena 96
A glance at tables 4 and 5 is sufficient to indicate the general
results. It again appears that the younger flies, both male and
female, are fatigued by successive tests, whereas the older in-
sects actually improve. Also it is clear that although the fe-
males are more active than the males in both cases, they are
relatively less so in the older groups. Not only this, but the
older females are absolutely slightly less active than are the
females of the younger group. In this particular instance, there-
fore, 18 hours is actually the maximum age of activity. It may
be added, however, that were the results from the 6 day flies
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 65
TABLE 6
| MALE | FEMALE
X | ss
TTS hei MM SON eRe Rune oe a cea IC Lo eiaick | Sos
(Sey evap 0G [GLASS Pe Lies se Le SR party IS Ns Se oe eae anne | 90.0 aalO0Z0
ARIE GES tae nos ee Aone oe a a Ae aoe | 93.1 | 97.8
included in this. average this statement would no longer hold.
The indices for these insects were as follows (table 6): The
average male index is 84.9 and the female, 98.5, with a difference
of 13.6. Thus while the males have again gained relatively, the
females are also absolutely much faster than are younger females.
Finally, a series of tests were run on flies 4 to 6 days old as fol-
lows. An initial test was run at the same time of day at which
it had been the custom to remove newly hatched flies from their
bottles. They were then tested at the same relative intervals
as the newly hatched insects had been in the experiments de-
seribed above. In this case ten males and ten females were
used in each group, and the number of trials constituting a test
was raised to 5. The results were as follows (table 7):
TABLE 7
Male indices
0 HRS. 4 HRS. 12 HRs. 15 HRs. 18 HRs. 21 HRS. | 24 HRs.
83.6 86.5 88.5 86.6 86.8 96.6 | 98.8
Female indices
96.8 98.3 97.6 97.1 G6USIG ir 2S6eI ay GUa0
Temperatures
|
BIKE | 99° Die | D2 | Pile | De 99°
The main feature of this series is that there is no drop occur-
ring in the middle of the series, such as was the case with the
young flies tested at similar intervals. It is thus made more
likely that the falling off in question was due, as suggested, to
the more rapid fatigue of newly hatched insects. Incidentally,
it will be noted that the males are not far behind the females,
and that they gain on them during the series.
THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 25, NO. 1
66 ROBERT STANLEY McEWEN
In conclusion, it may be said that females are never twice as
active as males. They are, however, somewhat more active,
particularly when only 1 or 2 days old. As age advances the
difference between the sexes decreases until in some cases at 8 or
10 days the males actually surpass the females. Moreover, in-
stead of the maximum period of phototropic response occurring
at 18 hours, it would seem rather that both males and females,
if not too heavily fed, increase their response with age, reaching
a maximum in the neighborhood of 4 or 5 days. After this
point both sexes tend to become less active, the females more
rapidly than the males. It may also be added that the young
flies fatigue much more rapidly than do older insects.
EFFECTS OF OPERATIONS ON THE REACTIONS TO LIGHT
a. Removal of wings
The operation of removal of the wings was suggested by Dr.
T. H. Morgan as a laboratory experiment for one of his classes.
Mr. 8. Safir was the first to try the experiment, and obtained
the rather surprising result that flies so treated no longer showed
any response to light. This effect was so unexpected that it
was determined by the writer to investigate the matter as thor-
oughly as possible.
The first experiments performed in this connection were under-
taken with a view to determining whether the insects would
recover their normal response if kept a sufficient length of time
after the operation. As these tests were made at the beginning
of the work no apparatus was employed except the tube and
light from the north window. One fly was tested at a time and
its record calculated according to method I, for instance, the fly
was placed alternately in the end of the tube toward the light
and in the end away from the light, and the algebraic sum of
the average of the two sets of records in inches crawled was
taken. as the index of the fly in question. Five groups of animals
were tested, in which the number of insects varied from one to
five. When there was more than one fly in a group, the index
of the group as a whole was computed by adding algebraically
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 67
the indices of the individuals constituting the group. It is the
indices thus obtained that are set down in table 8. From these
results it would appear that there is a slight recovery of photo-
tropism. Nevertheless, in view of what occurs in normal flies
TABLE 8!
DAYS
GROUP 1 2 3 4 5
tl. fl. tl. fi. tl. fi. tie fl. tl. fl.
eee eS 24.0 W036 9.3 8.6 4.3
ETE Ue Si le 00.7 155.63 12.8 2.0 Bee
IL aed Sa Cope eee 10.0 8.9 es 18.5 9.9
IES i ee eee 4.3 1.0 00.7 00.9 3.0
Ein eon Munna 00.4 00.3 2.0 Al Tf 0.4
WHotalSinge nee 00.7| 38.7 43.1 42.1 2.0 | 32.7 19.9
Atverage. 2.0.2.2. 00.7) 9.6 8.6 Ate 2 (ileal. 3.9
Differences..... 8.9 8.6 8.4 6.1 3.9
DAYS
GROUP 6 if 8 9 10
tl. fl. tl: fl. tl. fl. tl. fl. tl. fi.
Li. oe ed 7.0 15.0 18.4 10.0
T«.....0 see 10.3 3.6 16.0 Ae ey
eo eee Bs revel cs Vato) 4.2 1A Sol 00.7
LV = 2. 2h eee 10.3 Ye 5a aaa 18.6
Vl, tele ie ee 4.4 1.7 4.3 3.6
otal See 24.5) 6.1 | 10.6) 8.8 | 41.0 | 14.2 | 38.5 42.3 | 00.7
A:vverage........ 8.1] 3.0 Oeoleeeon | LOe2 |) 14.2 Teeth 14.1 | 00.7
Differences.....}| 5.1 yy a! 4.0 Meal 13.4
' No temperature was taken in these early experiments, but later tests made
in the same room under similar conditions showed a variation of less than a
degree during a period of two weeks.
tl. indicates excess of inches crawled toward light, resulting from the alge-
braic sum of indices in the group. fl. indicates excess of inches crawled away
from the light, calculated in the same manner. Diff’s. indicates the algebraic
sum of the fl and tl averages.
68 ROBERT STANLEY McEWEN
TABLE 8—(Concluded)
DAYS
GROUP 11 12 13
tl fl tl. fl tl fl
To 2 SR eee ae 113)-3 22.9 27.3
Ls. ER ee ee reser eer 25.0 22.6
TLE 2 See eee ee ero 4.3
LV xs. ee ee eee ee eee ae
NW ee dari ead is 6 ha eteue cue ous oaco or ame
AG Calls ee: Aas Maisie cies eecnerarersietck 2 38.3] 4.3 | 45.5 27.3
AVGRA GES 7 tame aa sere hans Aes cence slay: 19.1) 4.3 | 22.7 27.3
Differencestarne ewer e ear aes tae. 14.8 22.7 27.3
with advancing age, it seems likely that this recovery represents
nothing more than the usual increase. It should be noted that
the usual index for unmutilated flies under this system of record-
ing is from 30 to 36. This will appear in the next experiment.
In this experiment, also under method I, five groups of flies
were used. In the first group the wings were not removed until
the sixth day after hatching, while in the fifth group they were
removed on the day of hatching. The results were the same in
every case. The insects showed the usual positive reaction to
light until the day the wings were removed. At this point the
positive reaction disappeared, the insects being indifferent to
light and remaining substantially so for the rest of the tests.
This occurrence was perfectly regular and very striking. It will
therefore suffice to give only a couple of illustrations. In Group
I there was only one fly. On the day after hatching, a t. |. in-
dex of 30.4 was recorded. Its record was about this each day
until the sixth, when the wings were removed. For the subse-
quent five tests its average was f. 1. 1.7. On the sixth test it
went up to t. |. 4.7, but on the seventh it dropped to t. 1. 2 and on
the eighth and last the record was f. 1. 1. In Group IV there
were five flies. On the day after hatching they averaged t. 1.
33.4. The day following their wings were removed, one insect
having been lost in the meantime. On this, the third day after
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 69
hatching, they now averaged f. 1. 3.1. Thus, it does not ap-
pear that the age at which the wings are cut off has anus to
do with the effect.
It should be added that the slight f. 1. excesses recorded in
the above experiments are probably not significant. From
watching the actual behavior of the flies it did not appear to me
that the operation did any more than to render them practi-
cally indifferent to light. Indeed, I have never observed a
clearly negative reaction in Drosophila.
So far the apparent loss of phototropism might mean merely
that the operation had made the insects inactive. However,
since Drosophila is strongly negatively geotropic it was possible
to use this reaction as a measure of general activity. For
this purpose the system of testing several insects at a time, known
as method II was used. ‘The flies were introduced into the
usual testing tube and given one trial for the reaction to light
in the regular manner, except that no agitation was employed.
Following this the tube was fastened in a vertical position with
the flies at the bottom, and at a distance of 41 ems. from a 100
watt tungsten lamp hung so that its tip just touched the table.
Three such tests were given, alternating with three light tests,
and the indices for the two sets calculated as usual for the above
method. The elimination of agitation in these tests was made
necessary in order to make comparable the records of the flies
with and without wings. When agitated the former move to-
® Regarding the relative strength of the two stimuli, light and gravity, Cole
decides in favor of the latter. He found that when flies were placed in a ver-
tical cylinder illuminated from below the larger per cent went to the upper-
most third. Carpenter, on the other hand, was able to attract the insects to
the bottom of a similar cylinder without using as strong a light as did Cole.
On account of the great variability of Drosophila, I suggest that this diserep-
ancy may be due to the small number of flies used, Cole employing only twenty-
one and Carpenter only six.
My own results are not strictly comparable with those of either of these
authors, because I used a type of apparatus which did not directly oppose the
two stimuli, but such evidence as I have agrees with that of Carpenter. Thus,
a reference to any of the tables where the light and gravity indices of normal
insects are recorded will show that the light-index in any given case always ex-
ceeds that for gravity. In any event, the matter of which stimulus is the stronger
is not one of any great significance.
70 ROBERT STANLEY McEWEN
ward the light both by crawling and flying, whereas the latter
can only crawl. When not agitated, however, the response even
of winged insects is almost purely a crawling reaction.
Three groups of flies containing 20 insects each were now se-
lected at random from the stock bottles. They were placed in
vials in the morning and tested according to the above plan in
the afternoon. After this first test all the insects were etherized
and the wings removed from the two groups which had made
the best record. These groups will be designated as B and C.
The following afternoon all three groups were tested again.
Following are the records of Groups B and C before and after
the wings were removed, and also the two records of the control
Group A (table 9).
TABLE 9
Before removal. Temperature 24°
A B G
Gravity Light Gravity Light Gravity Light
(First test)
Males Males Males Males Males Males
29.1 57.6 28.3 75.0 21.6 65.0
Females Females Females Females Females Females
316 81.6 45.0 90.8 32.5 85.0
After removal
(Second test)
Males Males Males Males Males Males
50.0 86.6 35.0 5.8 39.1 20.8
Females Females Females Females Females Females
41.6 90.8 30.0 Thea 25.0 SED
As a further check, Group A, the control, was tested a third
time 48 hours after the second test. The wings were then re-
moved and eight hours later a fourth test was given (table 10).
It is evident from this data that the removal of the wings affects
the light reaction specifically, and does not merely reduce the
activity of the insects.
The next point investigated was the effect of the removal of
parts of these appendages. The first experiment was done un-
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA iol
TABLE 10
Group A. Temperature 23°
GRAVITY | LIGHT
Males Females Males Females
Third test, before wing removal......... 46.6 30.0 88.3 84.1
Hourthr test atterenemovalesssreec rece ce 43.3 40.0 116 6.6
der method I, the age of the insects being unknown. The appa-
ratus was exactly the same as that described in connection with
the effect of removing the wings at different times after hatching.
Five groups of flies were employed in the first series of tests.
In the case of the first two groups only half of the wings were
removed throughout the tests, while in the case of the last three
groups one or two tests were given with half the wing removed,
and then a second operation was performed in which three-fourths
of the total wing was taken. Table 11 summarizes the results.
It appears that though the insects are very erratic and vary
much from time to time, those animals which had had the wing
completely removed, with one single exception, made lower rec-
ords than any made by flies with only half of the wings removed.
These experiments are unsatisfactory, however, in failing to show
what, if any, effect the removal of half the wing has. Later on,
therefore, another set of experiments was devised to answer this
question. In this case method II was used with the improved
apparatus. Likewise, the alternating gravity trials were intro-
duced as a control. In short, the general method was precisely
similar to that employed in the proof that wing removal has
a specific effect on the reaction to light (table 9).
Four groups of flies were selected at random from the bottles
and run through the tests. The flies were then etherized and
treated in the following way. Group I, which contained nine
males and ten females, had made the lowest record and was re-
stored to the vials without operation. Group II, which con-
tained ten males and eleven females, and had made next to the
lowest record, had only the tip ends of the wings removed.
Group III, which contained ten males and nine females and had
the second best record, had one-half of the wings cut off, and
V2 ROBERT STANLEY McEWEN
TABLE 11
Rot sy ee cae ONE-HALF WING REMOVED eoeheata eka Meeks
OF FLIES ;
itsale fel. Galle felis
6 I 1 144.4
6 I 2 28.8
6 I 3 129.1
6 I 4 61.3
AV GRAS C: Acme the roe err: 90.9
6 IT 1 57.0
6 II 2 8.9
6 Ji 3 52.3
AWVCTODC 4 2 nue eoe temenras toe Ss 39.4
6 III 1 104.3
6 Itt 2 38.6
6 Til 3 8.8
6 INGE 4 24.2
AViCRAC CUR errr: hee eee rae 104.3 = lets! 31.4
6 IV 1 69.0
5 JY, 2 20.3
IV 3 B85
IA VIET ALE seh icy eee oe ee ae 69.0 26.3
6 V 1 48.1
6 Vv 2 46.4
6 Vv 3 9.1
6 V 4 10.2
4 Vv 5 tel
IAWER ACOA. «2% ..5 eee eee 47.2 6.8
Averacestorone=lhaliimvyal eSpace reser td erici sci) ciel «Re ee eto ual eameZ OR
A-verapertor three tOUnbnGnWwANGS. i826. 5. oss A. «ways celeateoe dekeee flee 127
Group IV, which had made the best record and contained ten
males and six females had three-fourths of every wing removed.
The next day all groups were retested with the following results
(table 12):
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 73
TABLE 12
GROUP II. WINGS CUT
GROUP I. WINGS NOT CUT ONE-FOURTH
Light Gravity Light Gravity
MiAlapi|fiartals | Medio" |:arills ||) Malos)| Boranla pileden lepers
Before cutting: Le) LoL. 66.6 | 82.5 | 27.7 | 26.6 | 80.0 | 88.5 | 27.5 | 24.9
NemperacGueesss- seca ee | | SPE 23.5°
Avgen canbe. 4. 0..0hy 911.6) /-9205)\-48 00 | 4803 | 76.6 | 72.5 | 40.0 | 36.6
Mempersturess-- eee 24° | 24°
GROUP IV. WINGS CUT
GROUP III. WINGS CUT HALF THREE-FOURTHS
Before cutting.............| 80.0 | 95.3 | 41.6 | 42.5 | 90.8 | 95.8 | 49.8 | 38.3
Memperatures.-..2-5-552-- 23.52 | a ae
After cutting......./......] 19.1 | 21.2 | 65.0 | 49.0 | 25.8 | 26.3 | 61.6 | 49.9
sllemiperaturess.a-a.e 2s. oe 24° 24°
It is evident from these figures that with a single exception,
such as the case of the groups in which three-fourths of the wing
was removed, these results support the conclusion that the de-
crease in phototropism is directly proportional to the amount of
the wings removed. Moreover, in considering this result and
particularly the one exception, it must be remembered that the
amount of wing cut off in each group was directly proportional to
the height of the index originally scored by that group in the
initial tests. Thus, the group having three-fourths of the wing
removed was originally the fastest of all, and this may account
for its still retaining enough speed to win out over the group
with only one-half of the wing removed. This seems particu-
larly probable when this experiment is considered together with
the previous one. Taking the two together, I believe we are
justified in the conclusion that at least roughly speaking the pro-
portion between phototropism and wing length holds good.
74 . ROBERT STANLEY McEWEN
b. Gluing the wings
Several attempts were made to glue the wings in such a way
that though uninjured, the insect could not use them. These at-
tempts were made fruitless, however, by the fact that a fly whose
wings are stuck thoroughly enough so that they can not’be freed,
will spend all its time in an effort to do so, and will scarcely re-
spond to any other stimulus during the process. This experiment,
therefore, had to be given up.
There remained two other possibilities. First, the effect of —
operations as such could be determined, by operating on other
parts of the insect. Secondly, the existence in this laboratory
of mutations of all degrees of winglessness made it possible to
discover the effect of the absence, or partial absence, of wings in
Drosophila upon which no operation had been performed. The
effects of other operations will be considered first.
c. Cutting off legs
For this purpose eleven males and ten females, newly hatched,
were selected and kept in vials until five days old, this being the
usual procedure when records comparable with those made by
other groups were desired.. Before placing in the vials each in-
sect was operated on, and the tarsus and tibia of the middle
pair of legs cut off. It was thought that removal of the middle
pair in this manner would interfere least with the animal’s
balance and ability to crawl. On the fifth day these flies were
tested with a resulting index of 53.1 for the males and of 86.3
for the females. Under similar conditions it will be recalled that
a normal index would be approximately 95 and 97, though I
have cases where it was considerably lower. Thus, though there
may be a slight effect from this operation, it is obviously not
very great. Furthermore, it must be remembered that however
quickly and accurately these flies might orient, they were neces-
sarily handicapped in their speed of movement except when they
took to wing. Since this experiment was performed under agi-
tation this was frequently the case. As a matter of fact, orien-
tation and movement toward the light was perfectly constant
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA t
with these insects, a statement that does not hold at all for flies
without wings.
d. Removal of antennae
The other type of operation which was attempted was the
removal of the antennae. Inasmuch as these organs in Dro-
sophila lie close against the head, considerable difficulty was ex-
perienced in cutting them off. At length, however, a technique
was developed which though tedious was successful in almost every
case in removing the entire organ close up to the head. In this
experiment four sets of tests were run which may be designated
as A, B, C, and D. Since the method varied slightly in each of
TABLE 13
Controls without antennaz removed
LIGHT GRAVITY
SINS Vertical Horizontal
Male | Female
Male |Female} Male |Female
1 SiO ole bl 27 Ole 2o20
SGOPA 2.2 --: eam cere etn: 2
[ 3
shemperature tren eee oe. ner te dees Dow Doe
. 1 100 | 97.5 20.0} 48.0} 20.5) 35.0
Set B.. SARA Aaa eee ort a ( 1 41.5] 48.01 29.0 27.5
TemPerahUnegerae eet ees eee sc 23° 232
an ie te
Set Co 24 nae eee ee rae ( ! a ice 33.0 sie
2
Temperature (Group 4) en aoe. 4 23° 23°
|
‘ 1 36.6 Sees
CUAL cn Mei ena Sra oa ae { 99.7 10.5
tpiaMye ceo2x). <2: iaeee ee ae 100 | 97.5 | 166.8| 135.5| 105.3] 82.5
Averavest 0 7 1) re 100 | 97.5 | 33.3) 44.5! 21.0] 27.5
Wierticalvexcessy, mailed. wud tid Some RD, © s:)6 5s autre nitetad ran elg cies Se 12.3
Wertichilsexcess: female... tas aeteeras erties Sac. Seether lenin 17.0
76 ROBERT STANLEY McEWEN
TABLE 13—Concluded
With antennae removed
LIGHT GRAVITY
eS Vertical Horizontal
Male | F emale}
| Male | Female} Male | Female
1 91.0) 66.5 0:5 1.0 6.0
Set Ac uikce Seu 2 86:0). 81.5). 11.0) 6.0)7 2725), 6:0
Ue 11.0 17.0
Temperstureisas ore oo cera eee 23° 23° ;
detest a) i 1 | 92.5] 99.5] 21.5] 17.5] 17.0| 27.0
ee \ fi
Temperature Be iad coe Ske Ao ae fe%c 24° DAS
1 77.5| 100.0 fa20) 1025) 12-0
Sei Osea | 2. 0).210s0
Be Se | 2 | 71.0| 90.1} 15.5| 16.5] 14.0
Temperature (Group b)...-.... 2B 230
. | } 2
1 | Also wings ; 16.0 13.5
Sethe) hic 3: Seon ee ee { 9 élipped 12.1 55
‘Temperatureseoo 0. sees eee eee 23°
Totals .csesesee os ee eee 418.0} 427.6} 142.1} 104.0) 140.0) 85.5
Averages). Beth aet est sean eee 83268525)! 1527) = Lied), oso
Wierticalsexcessh an alle. a ree eee wn EPs ius nick. 2 has es meuorenens 0.2
Vertical,excess iemales.- sere one eee Plo. ot oe te eee 0.2
these tests, each will have to be described separately. The re-
sults, however, will be found summarized in table 13.
In Set A, two groups of twenty flies were removed from the
bottle shortly after hatching. One group was operated on at
once, while the other was kept as a control. Five days later the
group from which the antennae had been removed was tested
for light reaction. Three hours later both groups were given a
gravity test. In this case this test consisted of two series of five
trials each. In the first series the tube was held vertically, and
the index calculated as usual, the result being designated as the
vertical index. The second series acted as a control, the tube
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA as
being placed horizontally and the result designated as the hori-
zontal index. The difference between these indices may thus be
taken as the index of the animal’s reaction to gravity. After
these tests the flies with the antennae removed were given fresh
food and kept for 3 days. The light and gravity tests were
then repeated. On the following day, for instance, 9 days
after hatching, the males of this group were given one more
gravity test. At this point the alternating system for the ver-
tical and horizontal positions of the tube was introduced, and
used in all the subsequent tests of this experiment.
In Set B three groups of flies were taken and from one group the
antennae were removed. The other two were used as controls.
Five days later one control group and the one from which the
antennae had been cut were given the light and gravity tests as
in Set A. The second control group was given only a gravity
test.
In Set C two groups of twenty insects were taken and from one
group the antennae were removed as usual. At 5 days the
animals which had been operated on were tested for ight and for
gravity. The control was tested for gravity only. Three days
later the former’ group was again subjected to both light and
gravity tests.
Set D consisted of two groups of male flies only, each having
been used previously in tests on the effect of wing removal. One
group, which we will designate as a, had been used as a control
and had not had the wings removed. In the other group, b, the
wings had been cut off. This latter group was now etherized
and the antennae as well as the wings were taken off. The con-
trol group was etherized at the same time but no operation per-
formed... Four hours later a gravity test was given to each
group, and 3 days later these tests were repeated.
From the results of this experiment it appears that there has
possibly been a very slight reduction in phototropic response.
However, it is certainly in no way comparable with the reduction
which occurs regularly as the result of wing removal. Further-
more, a study of the light responses of normal insects contained
in other tables, shows such variation that it is extremely doubtful
78 ROBERT STANLEY McEWEN
if the slight falling-off of some of the antennaeless groups in
table 13 is of any significance at all. From this result, therefore,
as well as that obtained by the removal of legs we are led to con-
clude that any operation as such is not sufficient to cause a loss
of phototropism. Incidentally, however, a rather interesting re-
sult does appear here as to the specific effect of the removal of
the antennae and reaction to gravity. From the small amount
of data on hand, it- appears that the loss of these organs greatly
reduces a fly’s negative geotropism. It also seems to produce a
slight reduction in general activity. There are not, however,
sufficient data collected on these particular points to do more
than suggest a line for further investigation.
EXPERIMENTS ON MUTANT WING-CHARACTERS
We are now in a position to attempt the second method of
analyzing our problem by testing the various sorts of wing mu-
tants which have arisen in this laboratory. These mutants vary
all the way from vestigial, in which the wings are mere stubs to
curled, in which the wings though of normal length are turned
upward at the end and are not very effective in flying. There
are many other variants between these two, one of which is desig-
nated as strap. Strap has wings almost as long as normal, but
they are narrow, often cleft at the end, held off from the body
at a peculiar angle and are useless for flight. These three mu-
tants therefore were selected as bearing the closest approximation
to insects with one-fourth, one-half and three fourths of the wing
removed. ‘The flies in question are represented in figure 2. A
normal insect is also included for the sake of comparison.
The tests on the above mutants have all been made ac-
cording to the plan already outlined in one of the experiments
for testing the effect of the removal of wings on light and gravity
(p. 69). In brief, three tests were made for light, alternating
with three tests for gravity, with the tube in the vertical posi-
tion only. No agitation was used during any of these tests.
Only one new point needs to be mentioned and that is in regard
to an improvement in the apparatus. Under the former system
of testing for geotropism the lamp whose rays struck the tube at
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 79
right angles was nevertheless near its foot. It was recognized
that this method was unreliable when we attempted to compare
the gravity reaction of flies which were phototropic with that
of those which were not. Thus, two sets of flies, one photo-
tropic and the other not, but possessing an equal amount of
Fig. 2. A, Normal insect. B, Specimen of ‘curled’ stock. C, Specimen of ‘vestigial’
stock. D, Specimen of ‘strap’ stock.
SO ROBERT STANLEY McEWEN
negative geotropism would show the latter more negatively
geotropic than the former. This would result from the fact
that though equally impelled to move upward, the phototropic
animals would be constantly handicapped by the attraction of
the light from below. In order to remedy this difficulty, there-
fore, three lamps of the same candle power were arranged in a
vertical line, so that one came opposite the foot of the tube, one
opposite the center, and one opposite the top. The end of each
bulb was a distance of 41 em. from the tube. The latter, more-
over, was now held in its vertical position by a wire support, so
that there was no danger of its wabbling. With these improve-
ments, the following experiments were undertaken.
In the first place, it was decided to run a test on some wild
flies in order to get some data which should be comparable with
that obtained by the same system for the mutants. Also in mak-
ing these tests it was decided incidentally to run a few checks
on the effect of wing removal, in order to make sure that the
former tests were not invalidated by the position of the single
lamp. For this purpose three groups of insects, each contain-
ing ten males and ten females, were selected, and kept in the
usual manner until 5 days old. They were then tested as de-
scribed above. After the test, the males in the groups which
we shall designate as A and B were etherized and in the case of
group B the wings were removed. Eight hours later both groups
were retested. The control males in Group A were now also
operated on, and tested for the third time 10 hours later. In
the case of the females, Group A was operated on after the first
test and Group B kept as a control. Twenty-four hours later
both these female groups were tested again. Group B wasnow
operated on and tested after eight hours. The results of these
tests are summarized below (table 14).
This experiment confirms the conclusions alr eee set forth re-
garding the effect of wing removal. In other words, the re-
arrangement of the lights has produced no significant effect. The
only thing of note in the record is the extremely high gravity index
registered by the males of Group B after being operated on. To
determine whether this is of any significance or not will require
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 81
TABLE 14
BEFORE WING REMOVAL AFTER WING REMOVAL
: Gravity ; Gravity
TESTS y
Light (vertical) Light (vertical)
|
Male | Female} Male | Female} Male | Female) Male | Female
1 96.6 | 98.3 | 68.3 | 77.5
Group! Aa. ease 2 96.6 66.6 41.6 50.0
3 5.8 45.0)
Mempenrabunese smear ee s: BB 8" 23.5°
1 98.3} 99.1) 67.5) 64.1
Group Bae Bt 2 97.5 55.0] 52.5 91.6
3 35.8 | 10.1 80.0} 42.5
Temperature........ te eee 23.5° 23 Oe
Soup Gee | i) Usa G4itiles5(0| 2508 | |
Memiperature...2.. sh.) las. 24° 24°
Totals...................| 375.6| 389.0| 237.4| 222.4] 94.1 | 51.7 | 216.6| 92.5
VENA ESHA eae) 95-9) 9722) 59F3) 5526) Sirs 2578 | 72.2 | 46.2
more data. It is true that a somewhat similar tendency is man-
ifest among the males in table 9, but the poor light arrangement
in the earlier experiments makes the records of doubtful validity
on this particular point.
Let us now consider the reactions of vestigial flies. Three
groups of insects, half male and half female, were kept for the
usual length of time after hatching, and then subjected to the
test just described for wild flies. The only difference was that
in this case the single lamp was used in the gravity trials. As
will appear from the results, however, this feature was of no
consequence in this instance because the insects were only very
slightly phototropic. Also since the wings were already only
stubs, nothing was cut off. Table 15 summarizes the results.
Strap stock was next tested. Three groups, constituted asin
vestigial and wild were kept as usual till 5 days old. The only
irregularity in this connection was the use of only nine instead
of ten females in Group A. As to the apparatus, the single
THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 25, NO. 1
82 ROBERT STANLEY McEWEN
TABLE 15
Temperature 24°
LIGHT GRAVITY (VERTICAL)
Male | Female | Male | Female
Group tA... eee ee ee 20.0 7.9 31.6 24.1
Grotp: Bosc. aah ere aeieees eee oe ae 13.3 10.0 24.1 20.8
Group Spec a ecae nce oe a eee 10.0 11.6 42.5 35.0
Totalsteer #ycrn-. bees Ser oe cere. fs 43.3 29.1 98.2 Ty a)
ANGTAT Oh crties soca oN Ae a Re ee 14.4 Vel 32.7 26.6
lamp in the gravity trials was used in the first tests of the males
in Groups A and B. After the first test the males of Group A
were kept as a control, while those of Group B suffered the re-
moval of the rather poorly developed wings which they possessed.
Both sets were retested 8 hours later. Table 16 gives the
results:
The results from this experiment are enough to suggest strongly
the slight increase in phototropic response which might be ex-
pected to distinguish these flies from the vestigials. The most
TABLE 16
| BEFORE WING REMOVAL AFTER WING REMOVAL
‘ ate ( | ss
22) | 2a pe a oe
Male | Female} Male | Female) Male | Female} Male | Femaie
Temperature 24°
. 1 32.5] 17.6 | 45.0} 30.8
Ce eT ae { ibs BOND 71.2
é 1 42.5 45.0
Group Bee. f 9 63.8 68.4
Temperature 23°
Group) Czes ee 1 10.0) 24.1 | 41.6} 48.3
ARoGal eee eee 147.0} 41.7 | 202.8] 79.1 | 63.8 68.4
Averages......... 3084 2028 | 50.4) 39-5) 6858 68 .4
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 83
striking point which distinguishes these data, however, is the
failure of wing removal to affect light reaction in Group B.
Indeed, the test after the operation shows an actual increase in
the reaction to light. In view of the rather low light indices in
all the flies of this variety, I am inclined to explain this as fol-
lows: Even normal insects whose wings have been removed,
vary a good deal from time to time in their degree of response
to light. Also the general variability is such that a fly with
three-fourths of its wings gone will not always show a lower re-
_ sponse than one with only one-half gone. I therefore suggest
that since these flies already have a low index on account of their
imperfect wings, the removal of the remainder of the wing might
not have sufficient additional effect to counterbalance some un-
known change in the physiological condition of the animal.
This statement is partially borne out by the fact that the males
in Group A which were not operated on, also showed a markedly
higher index for both light and gravity in the second test.
Further experiments now under way will serve to show whether
this is the true explanation or not. If it is not, we should have
to accept the rather astonishing hypothesis that a fly with short
wings and a low index to begin with, actually has its photo-
tropism increased instead of diminished by the removal of such
wings as it has.
Finally, we have to consider the reaction of the flies desig-
nated as curled. As usual, three groups of insects, constituted
as in the previous tests of this series, were tested when 5 days
old. As this particular group was really the first of the series
the use of the single lamp in the gravity test was still customary.
The change to the new system was, indeed, made during the
work on this group, which accounts for the fact that only the
males in Group A were subjected to the improved treatment.
That this feature was really of no great significance, at least in
the case of these flies can be told by comparison of Group A’s
record with those of the others. The males of both Groups A
and B had their wings removed after the first test and were re-
tested eight hours later. Group C males were not operated on,
but were retested after the same interval of time as a control.
84 ROBERT STANLEY McEWEN
In both Groups A and B one fly was lost before the re-test. Table
17 shows the results.
For the sake of comparison the average indices for light and
gravity obtained from the above experiments on wild, vestigial,
TABLE 17
Temperature 24°
‘
BEFORE WING REMOVAL AFTER WING REMOVAL
TESTS Light Geer Light eae
Male | Female} Male | Female} Male | Female| Male Female
: 1 83.3} 34.1] 86.6} 40.0
GroupgAea cee: i > 55.5 83.3
ee f 1 83.3] 51.6} 80.8] 45.0
Group Beas \| 2 69.4 74.0
CG 1 75.8) 48.3) 81.6) 49.1
wroup U......... 2 83.3 85.0
Potala 325.7| 134.0] 334.0} 134.1] 124.9 157.3 ;
Averages......... 81.4; 44.6) 83.5) 44.7; 62.4 78.6
strap and curled flies have been brought together in table 18.
From this table it is evident that there is a steady drop in pho-
totropic response which in a rough way is directly proportional
to the lack of development of the wings.
The curious effect of defective wings upon light reaction as in-
dicated by the above experiments made it desirable to examine
TABLE 18
Temperature 23°-24°
BEFORE WING REMOVAL AFTER WING REMOVAL
Gravity Light Gravity
AVERAGES FOR I ight
Male | Female| Male | Female| Male | Female} Male | Female
NST Le eri aes ican anes 8 sitios 93.9 | 97.2 | 59.3 | 55.6 | 31.3 | 25.8 | 72.2 | 46.2
Cuplediss: 4 :4:98: eesoeee 81.4 | 44.6 | 83.5 | 44.7 | 62.4 78.6
SRD! 2s Geoiae d eae 36.7 | 20.8 | 50.7 | 39.5 | 63:8 68.4
6
Vestigial..................| 14.4 ath Ways CO |) Ao):
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 85
these organs carefully in order to see if they might possibly contain
any sort of light receiving structures. A number of minute or-
gans were found (fig. 3). Except for the seven larger ones which
occur well out on the veins, the majority are arranged in groups
near the base. They have in fact very much the same arrange-
ment and appearance as have the so called olfactory organs de-
scribed by McIndoo for the honey bee (14).
In order to discover whether these organs have anything to
do with the reaction to light three groups of twenty flies each
were selected and kept for the usual 5 days. In one group the
Fig. 3. A B, Line of cut made to isolate the larger sense organs. E F, (OLD)
Line of cuts made as a control, on veins which contained no sense organs.
vein upon which occured the largest number of organs was cut
as shown by the line AB. In the other group the veins were
cut along the lines CD and EF, while in the third group no
operation was performed, though the flies were etherized as in
the first two cases. Table 19 gives the result.
These operations were performed on the assumption that if
the structures on the veins were light receiving organs, the nerv-
ous connection for such organs must pass along these veins. If
such were the case, then the insects which had those veins cut
on which some of the organs occurred, should have been most
affected by the operation. As a matter of fact, however, flies in
which the veins were cut which contained no sense organs were
as much affected as the others. Furthermore, it should be noted
86 ROBERT STANLEY McEWEN
TABLE 19
La
VEIN CUT
NO OPERATION
A B. (O/ID)5 913} 1045
Males Females Males Females Males Females
Before operation..... 100.0 99.1 93.3 95.8 93.3 80.8
After operation...... * 43.1 41.6 40.8 46.6 92.5 83.3
that in neither case was that part of the wing injured where the
chief groups of organs occur. ‘These points make it pretty clear
that the effect produced on light reaction due to injuring the
fly’s wing is not the result of injury to these particular organs.
The fact that the stimulus for the light reaction is received
in large part at least through the eyes and not through the wings
or other organs is attested by the following experiment. In one
of the mutant stocks known as eyeless, the compound eyes are
very poorly developed, and in the case of many of the females
are entirely lacking. Two groups of twenty flies each were,
therefore, selected, one group containing only the individuals
with the best developed eyes, and the other only those males
with poorly developed eyes, and those females with no eyes at
all. At the age of 5 days a light and gravity test was given
these insects with the following results (table 20).
TABLE 20
EYES PRESENT EYES POORLY DEVELOPED OR LACKING
Light Gravity Light Gravity
Male Female Male Female Male Female Male Female
97.5 65.8 65.8 46.6 74.1 25.0 70.8 39.1
The data seems to indicate that at least a large share of the
stimulus causing the light reaction is received through the com-
pound eyes, since when these are undeveloped the response is
greatly reduced, whereas that for gravity remains approximately
the same.
We may now summarize the work which has been done on
the relation of the fly’s wing to its phototropic response as follows.
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 87
If the wings of Drosophila are removed, the insect’s response to
light is greatly reduced. Furthermore, if they are partially re-
moved, the reduction in response is roughly proportional to the
amount taken off. That such a reduction is really due to a loss
of phototropism and not to a general decrease in activity is
proven by the fact that the insect’s response to the stimulus of
eravity is reduced, very slightly, if at all. It now remained to
show that the effect was directly due to the loss of the wings
and not to the operation in itself. This has been accomplished
first by performing other than wing operations and noting their
effect and, secondly, by using breeds of insects which are hatched
with imperfect wings. The operations performed involved the
removal of legs and antennae. However, except in so far as
general speed of locomotion was affected by the former operation,
it could not be concluded that such injuries specifically affected
the response to light. One incidental suggestion arising from
these operations, however, is to the effect that removal of the an-
tennae may materially affect the reaction to gravity. There is
no obvious explanation for this, since Colet has shown that the
stimulus of gravity is probably received through the leg muscles.
The second method, namely, the use of vestigial, strap and curled
wing flies gave results which still further bear out the hypothesis
that it is the condition of the insect’s wing as such that in some
way directly affects the response to light. The possibility that
sense organs on the wings were responsible for this peculiar re-
sult was tested by injuring the wing so as to break the nerve con-
nection with some of these sense organs. It was found, however,
that these organs had nothing to do with the response to light.
That the stimulus for this response is received chiefly through
the compound eyes was proved by testing eyeless stock contain-
ing individuals with and without these organs. Finally, the
notion that the effect may be due to a variation in the weight
of the wing is made very improbable by the fact that the wings
of curled insects, though deformed, are apparently just as large
as those of normals.
‘W. H. Cole, The reactions of Drosophila ampelophela Loew to gravity and
air currents. Jour. Animal Behavior, Jan., Feb. 1917.
88 ROBERT STANLEY McEWEN
INHERITANCE OF PHOTOTROPISM IN DROSOPHILA
In the Biological Bulletin, 1911, Dr. Fernandus Payne gives
the results of some phototropic tests made upon Drosophila
which had been bred for 69 generations in the dark. In the
course of the work he discovered, as I have done, that there
was great variation among individuals and he therefore made an
attempt to test the inheritance of the reaction. He was unable,
however, to obtain any significant results and this, so far as I
know, is the only effort to study the inheritance of this reaction
in Drosophila that has ever been made.
It was with great interest, therefore, that I discovered that a
certain stock of flies in this laboratory showed very little re-
sponse to light. This stock was a combination of three separate
mutants which were being carried along together for the sake of
convenience. It is known as eosin, tan, vermillion. Eosin and
vermillion are eye colors while tan refers to a slightly tan tinge
to the body and a clear tan in the antennae. The antennae of
the wild flies are gray. At first thought, of course, it appeared
likely that the peculiar reaction to light was due to the light
eye color. Stock in which these eye colors occurred without the
tan were therefore secured and tested. Neither of these eye
colors, however, were any less phototropic than normal. It is
unnecessary to give the figures for the tests here, since experi-
ments performed later in connection with colored lights amply
demonstrate the phototropism of these breeds. The fact still
remained that the peculiar reaction of eosin and vermillion might
be due to a combination in one fly of the factors producing these
characters. As tan had arisen as a mutation in eosin vermil-
lion stock, the only way to test this was to get the tan separated
from the two eye colors. By a suitable series of crosses this was
ultimately accomplished. It was then possible to test tan by
itself. The test immediately showed that either the factor for
tan itself or some other factor very closely linked with tan, was
responsible for the peculiar light reaction. Whenever an insect
was homozygous for tan it failed to react to light. It may be
stated at this point that tan is a recessive sex linked character.
That is, the daughters inherit the factor for the character from
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 89
their fathers, but do not show the character, while sons inherit
the factor from their mothers and do show the character.®
The independence of the light reaction from the color as such
may be realized from the following facts. Tan, like most other
characters, varies about a mode. Furthermore, this particular
character is so delicate, that at the extreme of-variation toward
the normal color it is quite impossible to distinguish the indi-
vidual possessing it from wild stock. This being the case, it
sometimes happened that an insect whose genetic constitution was
really tan would be accidentally mixed with flies which were
supposed to be normal and vice versa. For example, one case
occurred of a male fly which was apparently tan. Its light test,
however, proved to be that of a normal. To test it, therefore,
it was bred to a tan female. If the father were really tan, as it
appeared, its daughters would all be tan. If, however, it were
really normal as its light reaction indicated, then all its daugh-
ters would be normal. The latter turned out to be the case.
The daughters were. normal both in appearance and in light re-
action. Later a case arose where several normal appearing fe-
males reacted as though tan. They were bred to a normal
male. If they had been normal then all of the offspring should
have been normal; if they were heterozygous then all the females
would have been normal but half the males tan. What hap-
pened, however, was that all the females were normal, since tan
is recessive, but all the males were tan. This proved that the
females were really all homozygous tans as they had indicated
by their light reaction, though their appearance had belied the
fact. Thus it developed that light reaction was a surer test for
the character of tan than was the color itself.
It remains merely to give a table showing the records of
three -groups of twenty tan flies each. They were tested ac-
cording to the most recent system of light and gravity tests
(table 21).
Table 21 offers conclusive evidence that the failure of tan flies
to respond to light is not due to any general inactivity. This is
5 For a full discussion of Mendelism in Drosophila, see the Mechanism of
Mendelian Heredity by Morgan, Sturtevant, Muller and Bridges.
90 ROBERT STANLEY McEWEN
TABLE 21
LIGHT GRAVITY (VERTICAL)
Male Female Male Female
Temperature 24°
Group Arts. Ie eae ee 16.6 7.5 53.3 41.6
Temperature 23°
Group {Binns cece ie pace ee 1.9 5.8 78.3 44.1
Group iG cates si ee fae eee ro 18.3 20.8 81.6 70.8
otal sarees Sete eee re ae ae 42.4 34.1 213.2 156.5
AV. GTARESE hs) Sea those aaa 14.1 11.3 iW 52.1
further borne out by observation of the insects. They are fully
as active as are those from normal stock.
Finally, a number of normal, white and vermillion eyed and
tan flies have been sectioned and examined, both with and with-
out staining. So far, no histological abnormality has been dis-
covered in the eyes of the tan insects, to account for their peculiar
lack of response to light.
EFFECT OF COLORED LIGHTS ON. NORMAL AND MUTANT
EYE COLORS
A very considerable amount of work has been done by various
investigators upon the effects of different wave lengths on or-
ganisms which respond either positively or negatively to light.
Though it has generally been found that animals as well as plants
respond more readily to the more refrangible rays of the spec-
trum, such is by no means invariably the case. In Daphnia,
for instance, Lubbock (Journ. of the Linnean Society, 1881) and
others have found that the green and yellow rays are more effec-
tive than any of the others, including the blue and violet... Also
in the case of simpler organisms, it has been shown by Engel-
mann (Mast’s account, Mast ‘‘ Light and the Behavior of the
Lower Organisms’’) that Bacterium photometricium tends to
form aggregations in the infra red.
The general apparatus used in this work has already been de-
scribed. The composition of the liquids used, together with the
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 91
wave lengths transmitted by each, were as follows (table 22).
The spectrum tests were made often enough to make sure that
no fading of the colors was taking place.
TABLE 22
FORMULA WAVE LENGTHS
\WERWSIE, co ansas sone .285.0 ce. 5380 A° (green)—4240 A° (violet
Violet Ammonia. 47...- - 15.0) Ce: Green strong at 4950 A°
ee eS Copper sulphate Violet strong at 4510 A°
(HSA) pesscodode 7.5 grams | Blue, weak
Waiter ttets sien oe 30020)Acec: 5660 A°-5050 A°
; ichitierunes sere 0.03 grams | Strongest at 5320 A°
Green...... = ae
Napthol yellow... 0.25 grams
Napthol green.... 0.03 gram
Red Waters. emcees 300.0 ce. 7200 A°-6325 A°
ars Ponceau Red..... 3.0 grams | Strongest at 6570 A®°
The above formulae were only selected after a long series of
experiments, and are for the most part modifications of formulae
contained in the ‘“‘ Methods of Studying Vision in Animals” by
R. M. Yerkes and John B. Watson, Behavior Monographs,
1911. The red and the green are very satisfactory for colors
obtained by ray filters, while the so-called violet is evidently
not so good. It is, as a matter of fact, continuous from green to
violet. The blue, however, is very weak, the green moderate
and the violet band very strong and wide. ‘The results of the
experiments show that it is probably not the green to which the
effectiveness of this filter is due, and since the blue band is so
slight, the probabilities are that violet is the effective stimulus.
It is practically impossible to get a strictly violet filter. We
find, however, that blue is obtainable, and it is intended to use
such a filter in analyzing our results further at the earliest
opportunity.
Besides the wave lengths, the relative energy transmitted by
the filters was also measured by means of a thermocouple, using
the same source of light employed during the experiments. The
results are indicated in graph 4. From this it appears that if the
energy transmitted by the colorless flasks be represented by 100
92 ROBERT STANLEY McEWEN
per cent, then the red flask transmits 103 per cent, the green 64
per cent and the violet 51 per cent. The fact that the red flask
actually transmits more than the white is explained by the fact
that the layer of clear water was slightly thicker than the red
solution. It will be noted, however, that in the visible spectrum
the red is somewhat less than the white, while the green and the
violet are approximately equal.
The first colored light experiments performed were under-
taken in the Zoological Laboratory of Western Reserve Univer-
sity, Cleveland, Ohio, during the summer of 1916. After some
preliminary experimentation it was decided to make use of
so) Curves showing relation between galvanometer
deflections and scale readings
wanes Bortle , Area~—635.4 cm
\
evi. te Light, Area—Sl.7 cr7>
130
420| Areagsinyisible Spectra
White Lignt—/3 cin
Red Bottle — 9.8cm*
Green Bottle—/.7 com>
Ui Blue Bott/e—/-4¢m*
9o
Blue Bottle, Area—3!.5c7n
go
4o
re
Ss
60| >
vy
u
= ~
U =
50 Ps 5
so
a
a
40
uv
~
Q
5
re) ~
3 S
20
adings
Graph 4
Seale re
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 93
method II.° Four sets of flies were employed, each set consist-
ing of six males and six females, the sexes being tested separately
as usual. Every test consisted of three trials which were aver-_
aged to obtain the index for that test. Each of the four sets of
flies was tested once for each of the three colors, the successive
tests coming at intervals of 2 hours. For each set, however, the
arrangement of the colors in the series was varied. Thus for set
A it was red, green, violet; for set B, red, violet, green; for set C,
violet, green; red; and for set D, green, red, violet. The results
of these tests when averaged together for the four groups were as
follows: males—violet, 64, red, 29.8, green, 24.5; females—violet,
81.8, red, 64.6, green, 57.2. In every one of the four sets violet
was first in each complete test for males and females. As be-
tween green and red, red won in three out of the four sets for
both males and females. Thus it would appear from this ex-
periment that the colors are effective in the order—violet, red,
green.
A further and better test was later made according to the fol-
lowing method. Two groups of insects each consisting of ten
males and ten females, were picked out and designated as Group
A and Group B. Each group was now tested six times at two
hour intervals, with three trials to a test. In this case, however,
the three trials constituting a test were not all of one color.
Instead, there was a single trial for each of the three colors in
every test. Furthermore, in each of the six tests the arrange-
ment of the colors was altered according to a set plan. Group
B was treated in exactly the same manner, except that the se-
quence of the color arrangements for each test in the series was
reversed. Thus for test one the Group A arrangement was V,
G, R; for test 2 V, R, G; for test 3 ha GeV ofortestetuney Ve Gs
for test 5 G, V, R; and for test 6G, R, V. For Group B, the
series began G, R, V and ended with V, G, R. At the end of
the tests the indices for all the trials of a given color were aver-
aged together in Group A and Group B. Finally the averages
thus obtained for Group A and for Group B were averaged.
* The tube in this experiment was only divided into four sections instead of
the usual five.
94 ROBERT STANLEY McEWEN
Furthermore a record was kept in such a way that it was possible
to see in how many trials a given color came out first, second,
or third. It is evident that in this scheme, since every color was
used in every test, the effect of previous tests would not change
the relative value for any color in any given test. On the other
hand the possible effect of the arrangement of colors in any given
test is overcome by altering the arrangement every test. Finally,
the method of recording gives not only the average of all the trials,
but an analysis of individual trials. It, therefore, seems that a
tolerably clear-cut result obtained in this way may reasonably
be supposed of some significance.
This method was now applied in testing a series of mutant eye
colors as well as the normal stock. The eye colors were as fol-
lows: white, an eye entirely lacking in pigment; tinged, almost
white, but containing the lightest shade of red; eosin, a reddish
yellow somewhat darker in the females; vermilion, a very good
sample of this color; normal; and sepia. The last named color
is virtually maroon on hatching, but grows darker with age until
at five days it is practically black. The results from the tests
on these stocks are summarized in tables 23 to 28. Graph 5 is
based on the results for each eye color as indicated in groups A
and B combined. Since there is no apparent sex differentiation
as regards reaction to varied wave lengths, the graph has been
constructed from the average male and female indices in every
case.
It is evident from these tables that in the case of all colors
lighter than normal, the general tendency is for the order of ef-
fectiveness to be violet, green, red. There are, however, three
exceptions. First in group A of white eyed males, the red is
ahead of the green both in the average index and in the number
of tests in which this occurs. This case is more than oftset,
however, by group B, so that in the average of the two the order
of colors is as stated above. It should be mentioned, moreover,
that white eyed insects are extremely erratic even for Dro-
sophila. It is quite usual for them after making a few con-
sistent trips up the tube to become very much excited and to
simply buzz about convulsively. The second exception is that
Male.....
Female..
Male.....
Female. .
Male.....
Female. .
of group A tinged females.
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA
TABLE 23
White eyed flies
Group A
AVERAGE
R
Sex Violet Green Red
2 Male...... 72.9 49.9 52e5
1 | Female... 48.7 38.7 29.1
3
il :
0 Renae a. Tied once
5 b. Tied once
Group B
0 IMG escce 61E3 282, 155-1
0 Female... 64.7 AY) al 43.2
6b
1 ; ; :
1 Mate a. Tied twice. Watanls a. Tied once
4b 7 b. Tied twice b. Tied once
Groups A and B combined
2 Malena 67.1 39.0 33.8
1 Female... 56.7 42.9 36.1
9b
2 a. Tied once
1 Male /2 Tied twice Henle b. Tied once
Odulses sr \b. Tied twice '~ \e. Tied once
95
d. Tied once
slightly exceeds the violet. The table indicating the number
times each color won, however, shows that from this standpoint
violet is still well in the lead. The third exception is found
in group B vermilion females where red very slightly exceeds
green both in the average index and in the number of times
which it was ahead. There is no special explanation for this ex-
cept the fact that the eye color is approaching that of normal.
In any case group A more than overbalances it.
Here the average green index very
of
96 ROBERT STANLEY McEWEN
TABLE 24
Tinged eyed flies
Group A
| AVERAGE
SEX Vv G R
Sex Violet | Green | Red
‘| Ist] 6 0 0 Male...... 100.0 , 100.0 79.1
Male..... 2d | 0! 6 0 Female... 98.3 99.1 97.0
[| Say OM eorulee |
Ist | 4 asta 0 (a. Tied twice
Female.. ;|2d |0 | 2 | 1 Female < b. Tied twice
adel e224 eabaeoe _c. Tied three
Group B
fst | 40n20. 100 | Males-e- 100.0 GSi7aaal\ (Per Oad
Male..... ¢| 2d | 2a| 6b| 0 | Female... 100.0 97.9: | 97.5
i Se tans eon 86 |
Ist| 6 | 0 a ae Re ras : ;
Female..{/2d|0 | 2 |1 | Male{® 764 UV Female (#7186 Free
3d |0 | 4a] 5b Si
Groups A and B combined
ist}10 |0 |0 | Male....., 1000 | 99.3 | 74.6
Male.....{|2a | 2d|12b|0 | Female..| 99.1 | 98.5 | 97.2
[|3a}o | 0 [12 |
Est 10) 4] 080 (a "Tied twic ‘a. Tied twice
Female..4| 2d |0 | 4 | 2 Male ! = = j as © Female , b. Tied five
3d | 2a | 7b |10c Seago Le. Tied six
Turning now to the results obtained from the tests on normal
and sepia, we find that the early records for normal made during
the summer of 1916 have been confirmed. The order of effec-
tiveness is not violet, green, red, but violet, red, green. There is
one exception to this in the group A males where the green
slightly exceeds the red. Finally in the case of sepia, there is
the same reversal of the relative effectiveness of red and green.
This instance, however, is more clear-cut than is the case with
normal, for with sepia the red exceeds the green in all respects
with absolutely no exceptions.
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA Q7
TABLE 25
Eosin eyed flies
Group A
AVERAGE
SEX VA G R
Sex Violet Green Red
Ist | 4 0 | Male..... g 93 .5 87 .9 70.0
Male..... 2do 2a) e510 Female... 89.5 85 .7 70.0
Sys) @) WO. We |
SiG) 5, 0) 0 : :
Female..<| 2d} la} 6b] 0 Male Ve Foie Mia Female ee aoa ae
Bar Oncor 6 ue i
Group B
iste 0 .| OAMMale... |) £2100.0 948 | 71.8
Male..... 2d | 2a | 6b} 0 Female... 96 .2 92 .0 70.8
Bol) ©, PO) WG
Ist | 5 1 0 : Be
Female..{|2d}1 |5 | 0 Maley Pears
Bralp OFF nOa | 6 Ue
Groups A and B combined
Isr} 8 if 0 Male..... : 96 .7 91 3 70.9
Male..... 2d | 4a |1lb | 0 Female... 92.8 88.8 70 .4
SanieOr | O° 112
Ist |10 1 0
Female... | 2d | 2a |11b | 0 Male
3d | 0 ONL
fa. Tied three
\b. Tied three
fa. Tied once
Henle \b. Tied once
In order to discover a possible cause for the phenomenon just
described, sections of the eye were made and examined micro-
scopically. As expected, these sections showed that the pigment
which imparts to the organ its color, is simply the pigment
usually found surrounding the rhabdomes in the compound eye
of arthropods. This pigment so far as its function is known, is
supposed to be of a protective nature, placed so as to absorb all
rays of light which do not fall directly parallel to the axis of the
rhabdome in question. Of course, in cases such as we have un-
der consideration the pigment is not black but colored, and will
consequently reflect light of a certain wave length. At first,
THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 25, No. 1
98 ROBERT STANLEY McEWEN
TABLE 26
Vermilion eyed flies
Group A
AVERAGE
3EX Vv G R
Sex Violet Green Red
sta (Gee Ole Omala Viale seser ; 90 .4 72 .0 59 .6
Male..... Jd OF 4 1 Female... 94.1 94.1 60.8
3d | 0 2a | 5b
MSE @ NeOr |-o :
Female..<| 2d |0 | 6 | 0 Male fa ae ee
eG au . Tied once
Group B
TSS NO! WO: |) MIE 6 : 100.0 97 .5 90 .0
Male..... Dag ssanleoby|) Ld |i@kemalei a: 95 .0 72.0 76.6
3d.) 0) | 3¢ |"5e
ist] 6 |0 | 0 (a. Tied three
Female..<| 2d | 0 4 N83 b. Tied twice i Tied
3d 10 | 4a/] 38b| Male;c. Tied twice Female : pie ae
| Ato enies \b. Tied once
le. Tied twice
Groups A and B combined
(|ast| 9 | 0 [0 | Male...... 95 .2 84.7 74.8
Males.... 2d | 3a | 7b | 2d | Female... 94.5 78 .0 68 .7
3d |0 | 5e |10e
(| Ist}12 | 0 | 0 a. Tied three
Female.. {| 2d | 0 Salto b. Tied twice (a. Tied
3d | 0 4a | 9b| Male 4 ec. Tied three Female een ht
b. Tied once
d. Tied once
e. Tied three
therefore, when normal eye color was thought to be the only one
with an increased effectiveness for red, it seemed possible that
this might be explained by assuming the red of the light to be
exactly the same shade as that of the eye. This would then
mean that a larger percentage of the red light entering the eye
would be reflected and therefore effective, than would be the
case with any other color. When it was discovered, however,
that a still darker shade of red still further increased the effect-
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA
99
}
TABLE 27
Normal eyed flies
Group A
AVERAGE
SEX Vv R G
Sex Violet Red Green
1atnie4s, 10) ~ 10: ae Male... , 100 .0 96 .2 97 .0
Male.....2| 2d | 2a} 2b | 1d.) Female. .. 100 .0 99 .5 95 .4
|| 3d | 0 | 4e | 5c
sti On LO (a. Tied twice (a. Tied three
Female.. {| 2d | 8a | 4d | 0 | b. Tied once b. Tied twice
3d | 2b) 2e | 6c Male /c. Tied three Female { c. Tied twice °
d. Tied once | d. Tied three
le. Tied three Le. Tied twice
Group B
Tet 2.24 lO |e Mater: ..<.- 93.7 93.3 86.6
Male:...- 2d | 3a | 4d | 0 Female... 97 .0 96.6 83.7
3d |1b|0 | 6e
1st | 2 LNW ( a. Tied twice
Female.. {| 2d | 4a | 5b | 0 Male: bs Liedionces ae ale Tied three
Sel C1 O We ) ce. Tied once 3 b. Tied three
| d. Tied twice
Groups A and B combined
StiGe i 2.) (WON WeMiale:ce a: 96.8 94.7 91.8
Male..... 4| 2d | 5a | Ge | Ic | Female... 98.5 98.0 89.5
3d | 1b | 4f |11ld
Stileomel ele fa. Tied four Tied six
Female.. {| 2d | 7a | 9d | 0 |b. Tied once Tied t
|| 8d | 2b | 2e |12c |e. Tied once iar ae
Male : Female 4c. Tied two
\d. Tied four : ?
3 d. Tied six
|e. Tied three le. Tied two
\f. Tied three ; .
‘veness of that color this theory had to be given up. Thus, at
the present time I have no explanation to offer for the increased
effectiveness of red light which appears to accompany the dark-
ening of pigment in the eye, other than the vague assumption
that it may be due to some physiological difference which occurs
in connection with this change of pigmentation.
100° ROBERT STANLEY McEWEN
TABLE 28
Sepia eyed flies
Group A
AVERAGE
SEX Vv R G
Sex Violet Red Green
stro 1 0 Male. =sea- 93.9 87.4 66.0
Male..... Zama 5 0 Female... 93.9 91.0 68.0
Srl Oy PO |e
Ist 3 0 :
Female.. {| 2d |2 |3 | 0 Female ia Eee Ong:
; aq | dastton |b b. Tied once
Group B
Ist | 6 0 | 0 Males 2a 9353 70.4 54.5
Male..... Pxol--{| 6 0 Female... 96.6 86.6 55.0
3d /08 ONG
iE OO IC
Female. . 2d | 0 6 | 0
\}3d | 0 |0 |6
Groups A and B combined
Ist |11 1 0 Male...... 93.6 78.9 60.2
Male..... 2 el a aie 0 Female... 95.2 88.8 61.5
3d | 0 0 {12
Ist | 9 3 0 :
Female... ;|2d |2 |9 |0 Female [a. Tied paps
a Pa as \b. Tied once
SUMMARY AND CONCLUSIONS
1. Females of Drosophila ampelophila react to light somewhat
more readily than do the males. This difference is most marked
in young insects and steadily decreases with age, until at 8 or 9
days it has almost vanished. The time of maximum activity for
both sexes does not seem to come at 18 hours, but more probably
at from 3 to 5 days.
2. The removal of the wings causes the fly to lose most of its
phototropism. The effect is specifically on the tendency to re-
act to light, as is shown by the fact that such an operation
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 101
affects little if at all the response to gravity. The effect is
roughly proportional to the amount of the wing cut off. It is
not a result of the operation as such, since other operations do
not produce it, and because wingless flies and flies of other
stocks with defective wings show the same deficiency of response.
Certain organs (fig. 3) occur on the wings of Drosophila, but
operations fail to show that they are connected with the response
Showing relative tropism of flies of varied eye colors toward lights of different
wave lengths
White Eyed RAG
tf
recon aa
Red Pee igi aa
Tinged Eyed Flies
Violet
Cc.
Red
Eosin Eyed Flies
Violet
. aa
Red Ree Bernese
Vermilion Eyed Flies
| ee,
Gen
Red [SSR Semone eae ay
Normal Eyed Flies
PZ Eco <
Red De
Sepia Eyed Flies
.-' TE er
Cn
Red ee
Graph 5
102 ROBERT STANLEY McEWEN
to light. It appears fairly certain on the other hand that the
chief light receiving organs are the compound eyes as shown by
experiments with eyeless stock.
3. Operations on the antennae may produce a weakening of
the response to gravity, though they have little effect on the
reaction to light.
4. In a mutant stock of flies known as tan, there is clear-cut
evidence for the sex linked inheritance of a character which may
be described as indifference to light. It is apparently not due
to any structural defect in the eye.
5. Colored lights which may be conveniently described as vio-
let, green and red, are effective in the order named upon insects
whose eye color is lighter than the red eye of the wild fly. In
the case of wild flies, and flies whose eyes are of a still darker
shade called sepia, red is more effective than green.
GENERAL DISCUSSION
In most of the earlier work on various organisms, both animals
and plants, the conclusion generally reached was that the blue
and violet rays possessed much more stimulating value than
uid those which are less refrangible, particularly the red and
orange. Thus Payer (’42) using both the solar spectrum and col-
ored media found that seedlings turned toward blue and violet
light but not toward red, yellow, orange or green. Sachs in
1864 obtained similar results, using coléred solutions and glass.
Also in the case of animals Engelmann (’82) found that Euglena
viridis collected in the blue of a solar and gas spectrum, while
E. B. Wilson (91) working on Hydra viridis with colored glasses
again found the maximum effect in blue. Finally, Loeb’s ear-
lier work (90-93) led him to conclude that as between red and
blue, the latter color was the more effective for fly larvae, plant
lice, caterpillars of Porthesia chrysorrhoea, moths of Sphinx
euphorbia, Geometra piniaria, various copepods, the meal worm
Tenebrio molitor, the larvae of Polygordius, Limulus polyphemus,
and the June bug Melolontha vulgaris. |
Even some of the early investigators, however, found cases in
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 103
which the above condition did not hold. Thus Kraus (’76) using
colored media, discovered that in Claviceps, a fungus, red light
was nearly as effective as blue, while Engelmann (’82) showed
by the use of a solar and gas spectrum that Bacterium photo-
metricum actually collects most readily in the infra red. Further-
more, the study of various other animals with more refined
methods began to show that many forms were most affected by
intermediate points in the spectrum. “Thus Yerkes (’99) has
shown that for Simocephalus the point of maximum efficiency
in a Welsbach gas prismatic spectrum is in the yellow. Bert
(69) and Lubbock (’81) located this point for Daphnia in the
green, while recently Loeb and Wasteneys (716) using the spec-
trum from a carbon arc, have found the most effective point for
Balanus larvae in the yellow and that for Chlamydomonas
pisiformis in the yellow-green. Likewise, Hess, an opthalmolo-
gist, (10) using the spectrum from a Nernst glower has fixed
ereen or yellow-green as the maximum stimulating point for a
variety of forms, including ichneumon flies, Culex pipiens, adults
and larvae, Coccinella septenpunctata, Dasychira fascelina and
cephalopods. In the last instance the reaction of the pupil of
the eye is taken as a criterion of response. Lastly, 8. O. Mast
has recently given an excellent summary of work previously
done and the results of a recent series of experiments of his own
on Arenicola larvae, blowfly larvae and a number of. unicellular
forms. For the blow-fly larvae the maximum is in the green,
while for Arenicola it is in the blue.
From these results it is apparent that the variation in the
point of maximum response for different animals and plants is
very wide. To explain this divergence, the existence in differ-
ent organisms of different chemical compounds varying respec-
tively in the degree to which they are altered by light of differ-
ent wave lengths has been suggested. That there are com-
pounds of this kind we know, but their presence in phototropic
organisms has not yet been proved: Aside from this view Hess
believes that phototropic animals are all color-blind, and that,
they go to the part of the spectrum which seems to them bright-
est. He apparently gets this idea from the fact that he found
104 ROBERT STANLEY McEWEN
so many organisms for which yellow-green is the most effective
part of the spectrum, this being also the brightest part for color-
blind men. This notion, has been criticized by Ewald (15)
and Loeb (16).
The peculiar fact about Drosophila is the reversal in the ef-
fectiveness of red and green as the insects’ eye color grows darker.
Thus for eye colors lighter than normal the order of effectiveness
is violet, green, red, while probably for normal, and certainly for
sepia, the order is violet, red, green. This case besides showing
the peculiar reversal is remarkable as being the only instance so
far discovered among the lower animals in which red is more
effective than green, with the possible exceptions of Daphnia
(Frisch and Kupelwieser, 713; Ewald, 714), and paramoecium
bursaria (Engelmann, ’82). How to account for this phenomenon
of reversal it is difficult to say. Were it not for the case of sepia
it might be explained on the basis of the changed amount of red
reflected by the normal colored rhabdomes as compared with that
reflected by those of lighter shades. When two shades produce
the same effect, however, it is difficult to see how this will suffice.
It would thus seem as though we must fall back on the assump-
tion that as the eye grows darker, the supposed sensitive chemi-
cal substances on which the light has its effect change also.
What this change could be, it is hard to imagine from what we
now know of photo-chemical reactions. I am inclined to think,
therefore that the explanation may yet be found in connection
with some sort of differential absorption.
It may be noted that my results with colored lights do not
agree in one respect with those of Dr. A. O. Gross who also
worked on Drosophila This writer found green more effective
than red for flies with normal eyes, while my experiments re-
versed this order. I suggest, however, that this descrepancy is
due to the fact that Dr. Gross used lights which were equated
in energy, whereas in the case of my filters, as is also true for the
normal spectrum, the energy of the red is much greater than
that of the green. This fact, nevertheless, does not invalidate
or make less interesting the very evident increase in the effective-
ness of red in the case of the darker eye colors, since whatever
REACTIONS TO LIGHT AND GRAVITY IN DROSOPHILA 105
the relative difference in energy content, that difference remained
constant for all the eye colors tested in my experiments.
' Lastly, it may be well to emphasize the peculiar relation
which exists in Drosophila between general activity and photo-
tropism This phenomenon has been clearly recognized by
Carpenter and in general I agree with this author’s conclusions.
The fact seems to be that this insect is not phototropic unless
it 1s in a certain physiological state brought on by, or at least
accompanied by, activity. When the fly reaches a certain de-
gree of activity, induced by various means, it suddenly becomes
phototropic. When it quiets down, however, it may still crawl
about but ceases to be phototropic. Thus, when an insect has
been exposed to constant illumination for some time, it no
longer orients to light but wanders aimlessly up and down the
tube. Eventually such an animal may even come to rest with
its head away from the source of light. This phenomenon, Car-
penter suggests, is probably due to slight fatigue. However
this may be, it is certain that without a continuance of the me-
chanical agitation or sudden increases in light intensity, the ani-
mal’s general activity soon falls to the point where phototropic
_Tesponse ceases. \
‘
106 ROBERT STANLEY McEWEN
BIBLIOGRAPHY
Carpenter, F. W. 1905 The reactions of the pomace fly (Drosophila ampelo-
phila Loew) to light, gravity and mechanical stimulation. Amer.
Nat., vol. 39, pp. 157-171.
1908 Some reactions of Drosophila, with special reference to con-
vulsive reflexes. Jour. Comp. Neur. and Psych., vol. 18, pp. 483-491.
Coir, W. H. 1917 The reactions of Drosophila ampelophila Loew to gravity
centrifugation and air currents. Jour. Animal Behav., vol. 7, no. 1,
pp. 71-80. ‘
Gross, A. O. 1913 The reactions of arthropods to monchromatie lights of
equal intensities. Jour. Exp. Zodl., vol. 14, pp. 467-514.
Hess, C. 1910 Neue Untersuchungen iiber den Lichtsinn bei wirbellosen
Tieren. Arch. f. d. ges. Physiol., Bd. 136, pp. 282-367.
Loxrs, J. 1906 The dynamics of living matter. New York, 233 pp.
Lors, J. AND WAsTENEYs, H. 1915 The relative efficiency of various parts of
the spectrum for the heliotropic reactions of animals and plants. Jour.
Exp. Zoél., vol. 19, pp. 23-35.
1916 The relative efficiency of various parts of the spectrum for
the heliotropic reactions of animals and plants. Jour. Exp. Zodl.,
vol. 20, pp. 217-236.
Lutz, F. E. 1914 Biological notes concerning Drosophila ampelophila. Jour.
New York Entomol. Soc., vol. 22, no. 2.”
Mast, 8. O. 1911 Light and the behavior of organisms, New York, 410 pp.
1917 The relation between spectral color and stimulation in the
lower organisms. Jour. Exp. Zoél., vol. 22, no. 3, pp. 471-528.
McInpoo, N. E. 1914 The olfactory sense of the honey bee. Jour. Exp.
Zool., vol. 16, no. 3, April, pp. 265-346.
1916 The sense organs on the mouth parts of the honey bee. Smith-
sonian Miscell. Coll., vol. 65, no. 14, Jan.
Payne, F. 1911 Drosophila ampelophila loew bred in the dark forsixty nine
Payne, F. 1911 Drosophila ampelophila loew bred in the dark for sixty-nine
generations. Biol. Bull., vol. 21, pp. 297-801.
AUTHOR’S ABSTRACT OF THIS PAPER ISSUED BY
THE BIBLIOGRAPHIC SERVICE, DECEMBER 22
SOME EXPERIMENTS ON REGENERATION AFTER
EXARTICULATION IN DIEMYCTYLUS
VIRIDESCENS
C. V.. MORRILL
Department of Anatomy, Cornell University Medical College, New York City
TEN FIGURES (THREE PLATES)
The earlier writers (Phillipeaux and Fraisse)! on regeneration
in urodeles seem to have held the opinion that the extremities of
adult animals are completely replaced only when one or more
bones are injured in the amputation, that is to say, not after
total extirpation (exarticulation). Wound-irritation from an
injured bone was considered necessary as a stimulus to the re-
placement of a missing part. Also the bone supplied a ‘tissue-
rest? to serve as a matrix. However in young salamanders and
especially in their larvae, it was found that the regeneration of
extremities takes place very readily since here the joints are
only partially formed and wounding of the bones always occurs
in amputations. This general conclusion, that in adults re-
generation does not occur after complete extirpation, seems to
have been shared by a number of the more recent investigators,
some, Kochs (97) and Wendelstadt (01 and ’04) expressly
confirming it, others, Towle (01), Morgan (’03), Reed (’03) and
Glaeser (710), while not putting it to the test, seem to have taken
care in their experiments to amputate through a bone.
Kurz (’12) in the course of his experiments on transplantation
of entire limbs in Triton, found that if the limb is completely
extirpated (exarticulated) at the hip- or shoulder-joint, a new
limb regenerates. Presumably no wounding of the bones of the
hip- or shoulder-girdle took place although Kurz does not state
1 The works of Phillipeaux and Fraisse were not accessible to the writer.
Their conclusions were obtained from Barfurth’s review in Merkel and Bonnet’s
Ergebnisse, vol. 1, 1891.
107
108 C. V. MORRILL
what precautions were taken to avoid this. The writer using
the American salamander, Diemyctylus viridescens, obtained
similar results some years previous to Kurz’s report but for
various reasons they were not published.? Recently a new series
of experiments were made to work out the histological details
of the process and to determine how it differs, if.at all, from
regeneration after Injury to remaining bones or cartilages. In
addition, a number of more complicated operations were made
to analyze further, if possible, the extent and power of regenera-
tion after losses not usually met with in nature.
MATERIAL AND METHODS
A large supply of adult Diemyctylus was obtained through the
kindly assistance of Prof. A. Treadwell, of Vassar College. Since
many of these animals were in a weak, semi-starved condition
when brought to the laboratory, they were kept for a month in
glass aquaria before using and were fed on fresh liver. Under
these conditions the animals became very vigorous, and with-
stood the operations well. All operations were done under
narcosis. At first ether was used, but this, owing to its irritating
effect on the skin and to a certain percentage of mortality which
followed its use, was soon discarded. Much better results were
obtained by using a solution of chloretone, of 1: 2000, in which
the animals were immersed. This acts very gently. After
swimming around rapidly for a few minutes, the salamanders
slowly come to rest and in about ten minutes are completely
narcotized. The animals recover readily, though sometimes
slowly after this treatment. There is no irritation of the skin
and no mortality.
After the amputations, to be described in detail beyond, the
best results were obtained by closing the wounds with a stitch
or two of fine silk thread. Although this is not absolutely
necessary to the success of the experiment, healing then takes
place more rapidly and there is less danger of fungoid growths.
Immediately after operation, the animals were placed in a dark
2 The experiment was made at the suggestion of Prof. T. H. Morgan, in 1907.
REGENERATION AFTER EXARTICULATION 109
chamber lined with moist filter paper for two days as recom-
mended by Reed (’03). During this period the operated ex-
tremity was moistened from time to time with a solution of
permanganate of potash 1:1000. The animals were then
returned to the aquaria. The above precautions almost entirely
prevented the growth of fungus and consequent failure of the
experiments.
For microscopic study, the regenerating regions were removed
and fixed for the most part in sublimate acetic or Gilson’s mer-
curo-nitric fluid. Other fixatives, such as Zenker’s fluid, Bouin’s
fluid and ten per cent formalin were occasionally used but on
the whole the sublimate mixtures proved the most satisfactory.
After hardening in alcohol for a few days, the objects were
decalcified in a mixture of four per cent nitric acid in seventy
per cent alcohol for three or four days. They were then imbedded
in paraffin and sectioned. As a rule, good series were obtained,
seven or eight micra thick, although the rather tough bone and
cartilage from large specimens sometimes gave trouble. For
staining Mayer’s haemalum followed by picro-acid fuchsin was
most frequently employed. This gives a brilliant differentiation
of tissues but is not always permanent. Other stains such as
Mallory’s connective tissue stain, borax carmine and Lyons blue,
haemalum and congo red were also used but none proved as
satisfactory for most purposes as the haemalum and _picro-
acid fuchsin combination.
EXPERIMENTS
Pari tf
The fact that regeneration does occur after complete extirpa-
tion (exarticulation) has been established by the observations
of Kurz and the writer as stated above. In order to work out
the detail of this process, two sets of operations were made, the
hind limbs being used in both cases. In the first set the limb
was amputated at the hip-joint, in the second at the knee-joint. .
Great care was exercised in making these amputations. The
skin and muscles were first carefully divided with a small sharp
110 Cc. V. MORRILL
scalpel. Then the part to be removed was grasped with the
forceps and slight traction employed to draw the joint surfaces
apart. The capsular ligaments were then divided with the
scalpel and the limb removed, care being taken not to touch the
skeletal parts remaining (hip bones or femur according to the site
of operation). A flap of skin and muscle was drawn over the
wound and a couple of stitches taken. There was very little
trouble from bleeding, but in cases where it was profuse, the
specimen was discarded.
1. Amputation at the hip-joint. Nineteen animals were used
for this operation divided into groups of eleven, six and two.
All of the first group were killed and examined between thirty-
nine and forty-six days after operation. Externally each showed
a small bud at the site of amputation. Microscopically the bud
was composed of a dense mass of indifferent cells with small
round nuclei. No change in the hip-girdle was observed. The
second group of six were kept for six months. At this time all
had regenerated a new limb about three-fourths the normal size.
Microscopic examination showed the normal number of skeletal
elements in the limb, each represented by a bar of cartilage.
There was a well developed narrow cavity in the femur and
peripheral ossification had begun in all the cartilages except
the tarsals which do not ossify in these animals.*? Joint cavities
were well marked at this time. The third group of two animals
_was lost. Owing to the small number of specimens and the
lack of intermediate stages, the successive steps in this type
regeneration could not be made out. The detailed account of
this process will therefore be based upon the larger and more
complete series of operations at the knee-joint (vid. infra).
2. Amputation at the knee-joint. About seventy-five opera-
tions of this kind were made. Most of the specimens obtained
were fixed at intervals, of from ten to fifty days and sectioned.
The remainder were allowed to complete their regeneration to
3’ While it is true that peripheral ossification does not occur in the tarsalia,
nevertheless an extensive marrow cavity is normally present and the irregular
trabeculae of cartilage bordering it generally become calcified, if not actually
bony.
REGENERATION AFTER EXARTICULATION MOE
determine whether the new part exactly resembles the old both
in size and gross structure. In addition fifteen amputations
were made through the distal end of the femur for comparison
with the exarticulation experiments.
Descriptive. Wendelstadt (’04) and Glaeser (10) have given
very detailed accounts of regeneration in the limbs based chiefly
on species of the European salamander Triton and on the Axolotl.
In Diemyctylus the process is quite similar to that observed in
Triton. A study of the specimens in which amputation was
made through the distal end of the femur, ie., an operation
corresponding to those of Wendelstadt and Glaeser, showed that
the descriptions given by these writers for Triton, apply almost
equally as well to Diemyctylus. It is true there is some slight
discrepancy in their accounts but this can be discussed more
conveniently when comparing the exarticulation experiments
with those previously made.
The earlier changes which take place in the stump may be
passed over briefly here. They are concerned chiefly with the |
over-growth of the integument, the breaking down of the soft
parts, notably the muscle and the formation of a dense mass or
bud of small cells with round, deeply staining nuclei over the
distal end of the bone (femur). This mass lengthens out and
forms a projection when seen externally but does not always lie
in the axis of the limb. The origin of these cells could not be
determined with exactness. Wendelstadt (704) encountered
the same difficulty. Towle (’01) however, states that the
accummulation of ‘nuclei’ in the bud is due to rapid (direct)
division of nuclei in the old muscle fibers and the disintegration
of these fibers. Undoubtedly the degeneration of muscle-
fibers is largely responsible for the accumulation, but whether
exclusively so or not, is difficult to decide. Connective tissue
elements may also contribute something.
Turning now-to the changes in the bone and cartilage with
which this paper is chiefly concerned, it is here that an essential
difference appears between regeneration after exarticulation and
4 Towle’s experiments are concerned almost entirely with the regeneration of
muscle.
142 Cc. V. MORRILL
regeneration after amputation through a bone. This difference
has to do principally with the behavior of the distal epiphyseal
cartilage of the femur. This epiphysis (fig. 1, Hp.f.) becomes
slowly detached from the shaft by resorption of the bone and
calcified cartilage (C.c.) proximal to it. The resorption seems
to be brought about largely by the action of cells which arrange
themselves along the surface of the bone or calcified cartilage
and erode it. Many of them are giant cells. Their origin was
not determined. Both Wendelstadt (04) and Glaeser (10)
have described this resorption process by giant cells. Figure 1
shows the distal end of the shaft (£.f.) broken up into irregular
fragments of bone and calcified cartilage.
Coincident with the resorption process, a change takes place
in the epiphysis itself. The cartilage matrix begins to break
down. This occurs first on its distal and proximal surfaces
(fig. 1, Hp.f.). There is no evidence, however, that the cartilage
cells themselves undergo degeneration. Indeed, in many in-
stances they have been seen dividing mitotically and further,
as the lacunae are opened by the degeneration of the matrix, the
cells pass out and mingle with the surrounding tissues. Those
liberated on the distal surface could not be further traced but
on the proximal surface, that is facing the marrow cavity (figs.
1 and 2, M.C.), they contribute to a mass of tissue (Az.C.)
which is forming between the old epiphysis and the shaft. This
mass which quickly takes on the appearance of young cartilage
may be ealled axial cartilage adopting Glaeser’s (’10) term.
This axial cartilage appears to have a two-fold origin: (a)
from the liberated cells of the old epiphysis as stated above and
(b) from the cells lining the marrow cavity and covering the
trabeculae of bone and calcified cartilage at the distal end of the
shaft; in other words from the endosteum. These latter cells
appear to increase in number and, streaming out from the interior
of the femur (fig. 1) become massed under the old epiphysis
where they form the main contribution to the new axial cartilage.
5 It may be stated that there is normally a considerable amount of calcified
cartilage at the plane of union of shaft and epiphysis. This is distinct from the
uncalcified, hyaline cartilage of the epiphysis itself.
REGENERATION AFTER EXARTICULATION i13
This substantiates Wendelstadt’s conclusion that ‘osteoblasts’
(in reality chondroblasts) arising from the lining of the marrow
cavity contribute to the new formation (of cartilage). Glaeser,
however, derives the axial cartilage from periosteal and connec-
tive tissue fibrillae exclusively. He thinks that cartilage may oc-
casionally arise from bone-marrow though this is doubtful. The
origin of a part of this cartilage from cells of the old (distal
epiphyseal) cartilage naturally could not occur in the experiments
of Wendelstadt and Glaeser since the distal epiphysis was always
removed in their operations. Glaeser nevertheless states that
regeneration from old cartilage, either from the cells or the matrix
does not occur. The writer is unable to determine from Glaeser’s
account upon what experiment this conclusion is based. That
giant-cells in certain regions break down the matrix and open up
the capsules is true but in Diemyctylus, at least, the cartilage
cells certainly do not degenerate. Evidences of further activity
are abundant in this material including the frequent presence
of mitosis.
During the early stages in the formation of axial cartilage,
the cells of the periosteum also begin to form cartilage. The
site of this new formation is at first some distance from the distal
end of the femur and entirely distinct from that of the axial
cartilage. It may be called periosteal cartilage, or using Glaeser’s
term, peripheral cartilage. An early stage of its growth is
shown in figure 1, Per.C; a later one in figure 2, Per.C. The
formation of peripheral (periosteal) cartilage is mentioned by
most workers on regeneration and there is no need to discuss
it at length. In all cases it first appears at some distance proxi-
mal to the wound and gradually spreads distalward until it
forms a continuous collar around the shaft reaching to the distal
end (fig. 3, Per.C.). Cornil and Coudray (’03) described a
similar formation in the healing of experimentally produced
fractures in a mammal (rabbit). The first phenomena of repair,
they state, are to be found at some distance from the fracture,
that is the formation of peripheral (periosteal) cartilage.
Whether or not the axial cartilage always appears first could
not be determined. In some eases the peripheral cartilage was
THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 25, No. 1
114 Cc. V. MORRILL
distinguishable earlier while in most the two appeared at about
the same time. Glaeser seems to think the axial cartilage ap-
pears before the peripheral, while Wendelstadt describes activity
in the periosteum leading to the formation of peripheral cartilage
before there is any outgrowth of cells from the marrow cavity
to form axial cartilage. In the experiments on Diemyctylus
the presence of an epiphysis may have had a modifying effect
on the order of appearance. This point is probably not of any
great importance.
Soon after the appearance of axial and peripheral cartilage
the tissue of the bud which has formed distal to the femur, begins
to show signs of differentiation. This tissue as mentioned
previously is at first composed of apparently indifferent cells.
Cartilaginous masses now appear in it and, since they are formed
in the same manner as in normal development, they may be
spoken of collectively as embryonal cartilage following Glaeser
(10). In figure 2, a mass of this cartilage (Hm.C.) can be seen
lying distal to the old epiphysis (Ep.f.). It is at first entirely
distinct from the axial cartilage.’ As the limb bud enlarges
rapidly the growth of the embryonal cartilage keeps pace with it,
the cartilage extending right into the growing tip (fig. 3, Hm.C.)
In this way the skeleton of the new leg and foot is blocked out
in cartilage at a young stage, as the early experimenters found.
The formation of new skeletal parts is due chiefly to concentra-
tions of cells in situ rather than to growth from the first formed
mass or masses. There is, it is true, a continuous substratum or
core of tissue running through the center of the bud from which
the skeleton arises but it does not give rise to one or two con-
tinuous bars of cartilage. There are always interruptions at
points where joints are to be formed.
During the growth of the embryonal cartilage, the axial
cartilage continues to enlarge. This is due in part to growth
at the expense of the old epiphyseal cartilage. The latter
remains, however, for some time partly imbedded in the axial
cartilage (fig. 3, Hp.f) where it can be distinguished by its different
° The space seen in figure 2, between the embryonal cartilage and the old
epiphysis is probably an artifact.
REGENERATION AFTER EXARTICULATION 115
staining reaction. The expansion of the axial cartilage finally
brings it into contact with the embryonal cartilage and they
become so closely united as to appear almost continuous (fig. 3).
The peripheral cartilage (fig. 3, Per.C’.) meanwhile has spread
to the distal end of the bone (B.f.) and there unites with the
axial cartilage (Az.C). This results in a continuous cap of
cartilage covering the distal end of the femur, and extending
proximally for some distance. Figure 4, E p.f.n. from a much
later stage than figure 3, shows this new cap. From it, the new
epiphysis of the femur is formed and also the new bone of the
intermediate zone between epiphysis and shaft which was lost
in the early resorption process. A portion of the marrow cavity
M.c.n. can be seen extending into it.? At Os.n., new bony
tissue is spreading into the cartilage. This new ossification
is shown under high magnification in figure 10, which is taken
from the same region of a neighboring section. The new bone
(Os.n.) appears as a mass of interlacing fibers extending through
the matrix of the cartilage (C.n.). In this process, the cartilage
cells are gradually enclosed by the newly formed bone and eon-
verted directly into bone cells. Three such cells are shown at
Os.c. Portions of the marrow cavity (M.C.) and bone of the
shaft (B/f.) are included in the figure. Between the shaft and
the marrow cavity some of the old calcified cartilage remains
(compare fig. 3, C.c.). A direct transformation of cartilage into
bone was noted by Cornil and Coudray (03) in the healing of
fractures in the rabbit. They state that in most cases the car-
tilage bordering the bone first ossifies along its edges while later
the cartilage capsules themselves ossify, the cells being directly
transformed into bone cells.
It will be recalled that a portion of the distal end of the shaft
is destroyed in the resorption process by which the old epiphysis
is detached from the bone. This is replaced by ossification of
a part of the new cartilage in the manner just described. The
further history of this process was not followed owing to lack of
material in the advanced stages.
’ Through an error, the leader from the letters M.c.n. does not point to this
extension.
116 Cc. V. MORRILL
Distally the new formed epiphysis comes into relation with
the embryonal cartilage which forms the new fibula and tibia
(figs. 4 and 9) and later a joint appears at this level. As pre-
viously stated, before the new epiphysis is completed and while
a portion of the old epiphysis is still present (fig. 3) a close
attachment is developed between the axial cartilage (Az.C.) and
the embryonal cartilage (Hm.C.). It is difficult to say whether
or not the two cartilages become actually continuous. The
tissue uniting them is composed of cells without definite bounda-
ries and with small elongated nuclei. It does not appear to be
cartilaginous. Small cavities soon appear in this connecting
tissue, beginning usually at the circumference and spreading
toward the interior. An early stage in the joint formation is
shown in figure 9. The new joint cavity (J.c.n.) is Just appearing
between the new femoral epiphysis (Hp.f.n.) and the new fibula
(Fib.n.). In a later stage (fig. 4) the joint cavity has enlarged
at the circumference and a definite capsule has been formed, the
latter being continuous with the perichondrium of the new
cartilages (epiphyses). A small joint cavity has also developed
between the fibula and a tarsal cartilage (7.c.n.). At this
stage the fibula itself has begun to show evidences of subdivision
into shaft and two epiphysis. In the central portion, the car-
tilage areolae are enlarging and becoming more spherical pre-
paratory to the formation of a marrow cavity, while at the sur-
face a thin layer of subperiosteal bone has been laid down. In
the epiphyseal portions, the cartilage cells have a tendency to
arrange themselves in concentric ares, a characteristic of epi-
physes in general.
No attempt has been made in this study to arrange the speci-
mens in a series based on time after operation. The stages of
regeneration .do not necessarily correspond to the intervals of
time. For example figure 3 shows a stage obviously more ad-
vanced than that shown in figure 2, vet it was taken from a
specimen killed thirty-eight days after operation, while the latter
came from one killed at forty days. This is very probably due
partly to difference in time of healing of the wound and partly
to temperature. It was noticed that among animals operated
REGENERATION AFTER EXARTICULATION 7,
on at the same time and as far as possible treated in the same
manner, some always healed more readily than others. Re-
garding temperature—the higher degrees were in general more
favorable to rapid regeneration than the lower as might be ex-
pected, but there was so much individual variation due to time
of healing that no great reliance can be placed on this statement.
Comparing, from the standpoint of time, regeneration after
exarticulation with regeneration after wounding a bone, one finds
that the former is appreciably slower. On the average, there is
about ten days difference in corresponding stages.
With regard to regeneration of soft parts (muscle, nerve, blood-
vessels, etcetera), there is nothing to add to the accounts already
published. Muscle regeneration has been very carefully worked
out by Towle (’01) in Plethodon and Schminke (07) in Triton
taeniatus and T. cristatus. These writers agree that the new
muscle is formed chiefly by isolated cells (sarcoplasts) which
arise from degeneration of the old muscle of the stump. The
sarcoplasts are small masses of cytoplasm which contain at first
several nuclei. They usually break up into small cells which
are responsible according to Towle (’01) for the accumulation
of nuclei (cells) in the growing bud distal to the stump of the
old bone where they give rise to new muscle fibers. It was
previously mentioned that the exact origin of this entire mass of
small cells is somewhat uncertain (cf. Wendelstadt). Probably
most of the cells originate from degenerating muscle and some
perhaps from other soft tissues. In any event, there is no evi-
dence that any are derived from bone or cartilage. Histologically
there is at first no sign of differentiation in these cells, and it
seems useless to assume that such exists. It is from a part of
them, however, that new embryonal cartilage is developed in
the midst of the bud. This seems to be an example of dediffer-
entiation followed by redifferentiation in the sense of Child (’15).
From the apparently indifferent mass both cartilage and muscle
are formed, the cartilage showing the typical embryonic type of
development.
A somewhat similar process is to be seen in the behavior of the
old epiphyseal cartilage of the femur. Here the matrix breaks
118 C. V. MORRILL
down, liberating the cells which again become active and form
new matrix. In this case, however, the cells do not dedifferenti-
ate so far as to become indifferent; they remain cartilage forming
cells. The formation of the peripheral cartilage and that part
of the axial cartilage derived from endosteal cells is of course
quite different in nature. Here the more or less undifferentiated
cells of the periosteum and endosteum which have lain gormant,
are suddenly stimulated to activity by the amputation. They
form cartilage first and later a portion of the cartilage is trans-
formed into bone as described on page 115.
It was stated at the beginning of this account that some animals
were allowed to complete their regeneration to determine whether
the new skeleton was like the old. There seems to be no dif-
ference whatever provided sufficient time is allowed for devel-
opment. Externally, a slight deformity sometimes appears at
first, since the new bud does not always le in the longitudinal
axis of the limb. This is more common after operations at tbe
knee-joint than at the hip-joint. In the course of time this
irregularity disappears and the limb becomes normal in shape
and position. Complete ossification, however, may take almost
a year and sometimes even longer. .
A glance over the literature of regeneration in amphibia shows
that the power to regenerate a new normal skeleton does not
extend to all animals of this class. Morgan (’03) found that in
Amphiuma the new skeleton was abnormal and deficient al-
though some specimens were kept under observation for nearly
a year. Certain results which were obtained by the earlier
experimenters Goette and Fraisse seem to indicate that some of
the European urodeles (Triton marmoratus and Proteus) lack
the power of complete regeneration but Kammerer (06) states
that this is not the case if the animals are kept under favorable
conditions and for a sufficient length of time.
In the Anura the power is much more limited. New limbs will
regenerate only if amputation is made in the tadpole stage.
Barfurth (’94) was the first to find that the limbs of frog-larvae
(Rana fusca) are capable of regeneration, but this power disap-
pears in the progress of development. Ridewood (’98) obtained
REGENERATION AFTER EXARTICULATION 119
regeneration of posterior limbs in the tadpole of the midwife-
toad (Alytes obstetricans). The new skeleton was “normal
or nearly so” in five cases. Byrnes (’04) using frog-tadpoles
showed that the anterior limbs would regenerate while still
under the operculum but the new limb is invariably smaller than
normal and there is a tendency to reduction in the skeletal
elements. Morgan (’08) (and Goldfarb) attempted to induce
regeneration in the fore-leg of the adult frog by artificial means.
Pieces of the leg, muscle and other tissues from the tail of the
tadpole were grafted into the stump but with only small success.
In some cases, however, incomplete regeneration of the leg with
rudimentary toes was obtained, or a broad flat ‘foot’ with scant .
toes. Histological details were not given. Glaeser (’10) more
recently tested the power of regeneration in the hind limbs of
adult frogs but found none except in two cases where a ring of
peripheral (periosteal) cartilage developed around the stump of
the femur. No artificial means were used to induce regeneration
in this case.
Rar TT
To test further the power of regeneration in Diemyctylus a
series of more complicated operations were made, involving
losses not usually met with under natural conditions. These
are to some extent a repetition of. those of Wendelstadt (’01)
and Reed (’03) but with certain modifications.
Expervment 1. Extirpation of the fibula and a portion of the
femoral epiphysis but without injury to the tarsus. Number of
animals, ten:—Of these two. escaped and one lost the foot. The
remaining seven were killed at intervals of from forty-eight
days to one year. There was no indication of regeneration of
a new fibula, but the lost portion of the femoral epiphysis was
restored.
Experiment 2. Eextirpation of the fibula without injury to
either femoral epiphysis or tarsus. Number of animals, ten:—
These were killed at intervals of from thirty-three days to one
year. For the most part no indication of regeneration was
observed but in two specimens there was a narrow mass of eal-
120 Cc. V. MORRILL
cified fibrous tissue or bone in the place occupied by the old
fibula. This mass may have developed from fragments of the
old fibular epiphysis which were accidentally left in the wound
in two of the operations.®
The results of experiments 1 and 2 substantiate the conclusion
of Wendelstadt and Reed that regeneration in a lateral direction
(in the limb) does not occur.
Experiment 3. Extirpation of both leg bones and a portion of
the femoral epiphysis but without injury to the tarsus. Number
of animals, five-—Considerable shortening occurred but in no
case did the foot drop off. One was killed after sixty-three days
and the remainder after one year. ‘Two of the latter showed some
indication of regeneration. In these there were one or two car-
tilaginous nodules, in one case fairly extensive, connected with
the femoral epiphysis by fibrous tissue. The femoral epiphysis
itself was restored in all.
Experiment 4. Extirpation of both leg bones but without injury
to either femoral epiphysis or tarsus. Number of animals, seven:—
Shortening of course occurred. Two lost the foot subsequently
and were discarded. Of the five remaining, one was killed at
sixty days and the remainder after one year. All showed def-
inite attempts at regeneration, in some cases quite well marked.
Figures 5 and 6 from the same specimen cut in the dorsi-ventral
plane show the extent of regeneration in the best marked case.
The time elapsed was sixty days. Only a small portion of the
femoral epiphysis (Hp.f.) and one tarsal cartilage (T.c.) appear
in figure 5, since the section lies near the border of the limb.
The new skeletal element of the leg (L.s.n.) consists of an epi-
physeal portion which articulates with the femur and a long,
narrow bar of cartilage which is partly overlaid by bone at either
end. (The bone is darkly shaded in the figure.) At the proxi-
mal end a distinct joint capsule with cavity (J.c.n.) has developed.
Figure 6, from a section near the median plane, shows another
portion of the new skeletal element (/.s.n.). This portion is
8 In the experiments described in part II, all bones removed were examined
under a binocular microscope to determine whether any part of them had been
accidently left in the wound.
REGENERATION AFTER EXARTICULATION 121
a solid mass of cartilage united to the femoral epiphysis (/’p.f.)
by a capsule with joint cavity (J.c.n.) as before. Distally it is
in contact with a tarsal cartilage (7’.c.) but no joint capsule has
developed. The two portions of the new element are continu-
ous proximally when traced through the series. Unfortunately
intermediate stages in the formation of the new skeletal element
were not obtained. The epiphysis of the femur (fig. 6, Hp.f.)
has the appearance of new cartilage similar to that of the new
element (L.s.n.). This seems to indicate that the regeneration
was centrifugal in direction and probably occurred in the same
manner as described in the first section of this paper. _No
changes were observed in the tarsal cartilages.
Figure 7 is from another specimen killed after one year and
cut in a plane passing through the borders of the limb. The
new elements here consist of two large masses of cartilage (L.s.n.)
united by fibrous tissue and connected with femoral epiphysis
(Ep.f.) by a capsule containing a joint cavity (J.c.n.). Distally
the new cartilages fall short of the tarsus. The tarsal cartilages
themselves (7.c.) show signs of growth in a proximal direction
(centripetal regeneration). They have become united proximally
by a mass of cartilage which, however, has no connection with
the new skeletal elements. The. arrangement produces what
may be called a soft joint.
Other specimens in this experiment showed the formation of
irregular masses or nodules of cartilage but not so extensively
as the two described above. There appears, then, to be a limit
to the power of regeneration under the conditions of the experi-
ment. This may be due to an inhibiting influence from the
presence of the foot and to shortening of the limb which leaves
very little room for the new growth. It may be well to state
that as soon as the wound heals, the animal uses the limb con-
stantly when creeping over the bottom of the aquarium. Wendel-
stadt (’01) performed a similar experiment upon the anterior
limb of the Axolotl but with entirely negative results though
he kept the animals under observation for ten to fifteen months.
The limbs shortened as in the case of Diemyctylus but the animals
apparently made no attempt to use them. It is just possible
122 Cc. V. MORRILL
that a certain amount of activity in the limb is necessary to
start the regenerative process. Wendelstadt also tried the
effect of leaving a small piece of one of the bones (ulna) in situ.
For this operation he used one Axolotl and one Triton. In the
latter the humerus was also wounded. The axolotl regenerated
a new ulna which was shorter than normal while in the Triton
a whole new forearm and a second hand were formed. This
peculiar malformation in the Triton was never duplicated in
any of the writer’s experiments on Diemyctylus, although in
some cases the femur was purposely wounded. It is improbable
that there is any difference in the power of regeneration of fore
and hind-limbs in these animals.
Experiment 5. Eextirpation of the fibula and removal of the
foot entire without injury to the femoral epiphysis or tibia. Num-
ber of animals, five:—In this lot two were killed at sixty-six
and ninety-five days respectively and the remainder at the end
of a year. ‘The first of these had regenerated a well-marked foot
when killed and a new fibula. The latter consisted of a solid bar
of cartilage with a layer of subperiosteal bone surrounding its
proximal two-thirds. It was attached to the femoral epiphysis
by a capsule, common to it and the tibia. Distally it was
connected with the new tarsalia by ligaments, in places showing
the beginning of a joint cavity. In the specimen killed at
ninety-five days, the foot had regenerated but there was scarcely
any indication of a new fibula. The remaining three specimens
killed after one year regenerated a new complete foot including
tarsalia and a new but incomplete fibula. A section through one
of these is shown in figure 8. The old tibia (77b.) articulates
with the femoral epiphysis (/p.f.) while the new fibula (F7b.n.)
falls short proximally. Peripheral ossification has started in
the new element but there is no marrow cavity as yet. The new
tarsalia are seen at 7.c.n. The distal epiphysis of the tibia
seems to be composed of new cartilage like that of the tarsals
and fibula. This is to be expected since it was shown that the
old epiphysis in a stump is always replaced by new cartilage
(vid. Part I). In the present experiment one would expect
first a new formation of cartilage from which a new tibial epi-
REGENERATION AFTER EXARTICULATION 123
physis and the skeleton of the new foot is formed. This is
followed by growth in a proximal direction to form the new fibula
(centripetal regeneration). Apparently the energy of regenera-
tion is not always sufficient to produce a complete fibula even
in a year’s time although it may do so in two months as in the
first specimen described (sixty-six days).
A tendency to regenerate centripetally was also noted in
experiment four, where both leg bones were removed and the
foot allowed to remain. In this case however it was limited to
the formation of a mass of cartilage (fig. 7, 7.c.) uniting the proxi-
mal surfaces of the tarsalia. Wendelstadt (’01) obtained centri-
petal regeneration in the axolotl by extirpating the upper ends
of the radius and ulna. In three animals, the bones were com-
pletely restored after fifteen to eighteen months. There was
apparently no tendency to regenerate centrifugally from the
femoral epiphysis.
Morgan (’08) using Plethodon and Diemyctylus tried to dis-
cover what kind of a structure would regenerate from the proxi-
mal end of a limb. For this purpose the limb was cut off and
grafted onto the stump in reverse position. Regeneration oc-
curred but results were complicated, due to mixing of old and
new material and to the turning of the graft in the skin-pocket.
This method was discarded in favor of the following: The
hind leg was cut off at the knee. Then the femur was cut off
high up in the thigh and the distal portion reversed in position.
A new limb regenerated. Its skeleton was composed of (1)
proximal stump of femur, (2) connecting cartilage, (3) piece of
reversed femur, (4) new tibia and fibula, and (5) foot. There
was considerable evidence of absorption and in only a few cases
did it seem probable that the material for the new limb came
from the exposed proximal end of the grafted piece. In some
cases the new cartilage from the proximal stump grew past the
graft. On the whole it is not quite clear from Morgan’s account
what part the graft played in regeneration, as histological de-
tails are not given.
Reed (’03) performed a series of experiments somewhat similar
to those just described (Exp. 5) except that the distal end of the
124 Cc. V. MORRILL
tibia was removed with the foot. Spelerpes ruber was used for
these experiments. The results were, regeneration of the distal
end of the tibia, a new foot and a new fibular element. The
latter was usually incomplete but in one case it almost completed
itself proximally, that is, reaching the femoral epiphysis. As
in the present experiments there was no tendency to regener-
ate from the femur. Wendelstadt, in his later paper (04)
states that the experiments of Reed confirm his general conclusion
that wounding of the skeletal elements is necessary for regenera-
tion. The experiments described in the present paper show that
this conclusion is too sweeping. It is true that if one bone only
(fibula or radius) is removed (Wendelstadt, Reed and the writer)
or if the proximal parts of two bones are removed (Wendelstadt)
no regeneration occurs from the uninjured epiphysis of the femur
(or humerus). In the first case, the pressure of the remaining
bone against the joint surface of the femur (or humerus) and the
tarsals (or carpals), that is the presence of a functional joint may
inhibit regeneration from these points. In the second case the
new growth centripetally from the remaining injured bones,
which is always more rapid than from uninjured ones, may make
up the deficiency in time to check any tendency to regenerate
from the epiphysis of the humerus.’ Shortening of the limb
which must occur in this ease would also be a factor. These, of
course, are mainly suggestions. Further experiments are neces-
sary before definite explanations can be made.
SUMMARY AND CONCLUSIONS
1. In Diemyctylus regeneration takes place readily after com-
plete extirpation (exarticulation) whether the operation is made
at the hip- or knee-joint (Part I), or at the ankle-joint (Part I,
Exp. 5). The time elapsed is somewhat longer than when a
skeletal element is injured.
2. The new skeletal elements are similar to the old. There is
no tendency to reduction.
3. The essential difference between regeneration after exarti-
culation and regeneration after wounding a skeletal element lies
in the behavior of the cartilaginous epiphysis which is present
REGENERATION AFTER EXARTICULATION 125
in the stump in the former case. This cartilage becomes de-
tached from the shaft, gradually breaks down and is, partly
at least, reconverted into cartilage which assists in the formation
of a new epiphysis.
4. The new cartilage which forms the basis for the skeletal
elements appears independently in three localities:
a) Around the shaft of the bone proximal to the epiphysis
(peripheral cartilage). This cartilage is periosteal in origin.
b) In the axis of the bone and in contact with the marrow
subsequent to detachment of the epiphysis (axial cartilage).
The origin in this case is twofold: (1) From the cells of the old
epiphyseal cartilage and (2) from the lining of the marrow cavity
(endosteum).
c) In the tissue of the bud distal to the epiphysis (embryonal
cartilage). Here dedifferentiation appears to have taken place
forming a substratum of indifferent cells from which in turn
new cartilage is formed as in early development of the limb.
5. If a single bone (fibula) is removed completely from the
leg, it is not replaced either by proliferation from its fellow
(lateral regeneration) or from the skeletal elements lying proximal
and distal to it even when one of the latter is injured.
6. When both leg bones are completely removed they are
replaced to some extent by new elements which, however, are
always irregular and incomplete. The origin of the new parts
was not definitely determined.
7. When one leg bone (fibula) and the foot are removed with-
out injuring any of the remaining skeletal elements, a new
complete foot is regenerated from the distal end of the remaining
leg bone (tibia). This is followed by a slow and often incomplete
regeneration of the lost leg bone (fibula) in a proximal direction
(centripetal regeneration).
126 ' ©. V. MORRILL
LITERATURE CITED
Barrurtu, D. 1894 Sind die Extremititen der Frésche regenerationsfihig?
Arch. f. Entw.-Mech., Bd. 1.
Byrnes, E. 1904 Reconenanion of the anterior limbs in the tadpoles of frogs.
Arch. f. Entw.-Mech., Bd. 18.
1904 On the skeleton of regenerated anterior limbs in the frog. Biol.
Bull., vol. 7.
Cuitp, C. M. 1915 Senescence and rejuvenescence. Univ. of Chicago Press.
Corniu. V. et Coupray, P. 1903 (Note) De la formation du cal. Comptes
rendus de l’Academie des Sciences, Tome 137, p. 220.
GLAESER, Kart 1910 Untersuchungen iiber die Herkunft des Knorpels an
regenerierenden Amphibienextremititen. Arch. f. mikr. Anat., Bd.
75.
KAMMERER, P. 1906 Die angeblichen Ausnahmen von Regenerationsfaihigkeit
bei den Amphibien. Zentralbl. f. Phys., Bd. 19.
Kocus, W. 1897 Versuche iiber die Regeneration von Organen bei Amphibien.
Arch. f. mikr. Anat., Bd. 49.
Kurz, Oskar 1912 Die beinbildenden Potenzen entwickelter Tritonen. Arch.
f. Entw.-Mech., Bd. 34.
Moraan, T.H. 1903 Regeneration of the leg of Amphiumameans. Biol. Bull.,
viol
1908 Experiments in grafting. Amer. Nat., vol. 42, No. 493.
Reep, M. 1903 Regeneration of a whole foot from the cut end of a leg contain-
ing only the tibia. Arch. f. Entw.-Mech., Bd. 17.
Ripewoop, W. G. 1898 On the skeleton of regenerated limbs of the midwife-
toad (Alytes obstetricans). Proc. of Zoél. Soc. of London.
ScumincKke, A. 1907 Die Regeneration der quergestreiften Muskelfasern bei
den Wirbeltieren. Verhandl. d. phys.-med. Gesell. zu Wiirzburg.
Bd. 39.
Towts, E. 1901 On muscle regeneration in the limbs of Plethodon. Biol.
Bull., vol. 2
Wenvetstapt, H. 1901 Uber Knochenregeneration. Arch. f. mikr. Anat.,
Bd. 57.
1904 Experimentelle Studie iiber Regenerationsvorginge am Knochen
und Knorpel. Ibid., Bd. 63.
PLATES”
~
PLATE 1
EXPLANATION OF FIGURES
Figures 1 to 4 are from specimens in which complete amputation of the limb
was made at the knee-joint.
1 Longitudinal section of a limb 30 days after operation. Ep.f., femoral
epiphysis; Ax.C., axial cartilage; Per.C., peripheral cartilage; C.c.,calcified
cartilage of the shaft; B.f., bone of the shaft (femur); M@.c., marrow cavity. The
narrow space distal to the epiphysis is an artifact. Magnified about 30 diameters.
2 Longitudinal section of a limb 40 days after operation. Em.C., embryonal
cartilage. Other abbreviations as in figure 1. The space between the femoral
epiphysis, Ep.f., and the embryonal cartilage Em.C., is probably an artifact.
Magnified about 30 diameters.
3 Longitudinal section of a limb 38 days after operation. Abbreviations as
in figures 1 and 2. Magnified about 30 diameters.
4 Longitudinal section of a limb 48 days after operation. T.c.n., new tarsal
cartilage; Fib.n., new fibula; J.c.n., new joint-cavity; Ep.f.n., new femoral epi-
physis; Os.n., bone formation in new cartilage; W.c.n., extension of the marrow
cavity into the new cap of cartilage (see foot-note on p. 115); B.f., bone of the
femur. Magnified about 30 diameters.
PLATE 1
REGENERATION AFTER EXARTICULATION
Vv. MORRILL
Cc.
H. Murayama del.
129
1
THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 25, NO.
PLATE 2
EXPLANATION OF FIGURES
5 and 6 Longitudinal sections of a limb from which both tibia and fibula
were completely removed. ‘Time after operation, 60 days. T.c., tarsal cartilage
showing normal marrow cavity and partly calcified lining; L.s.n., new skeletal
element of the leg, in figure 5, partly ossified; Hp.f., epiphysis of the femur;
J.c.n., new joint cavity; W.c., marrow cavity and B.f., bone of the femur. Magni-
fied about 30 diameters.
7 Longitudinal section of another specimen after same operation as above.
Time one year. ‘Tarsal cartilages blended proximally, 7’.c.; new skeletal elements
L.s.n. Other abbreviations as in figures 5 and 6. Magnified about 30 diameters.
8 Longitudinal section of a limb from which the foot and the fibula were
completely removed. Time after operation, one year. 7'.c.n., new tarsal ear-
tilages; Fib.n., new fibula (incomplete); 7%b., tibia; Hp.f., femoral epiphysis.
Magnified about 30 diameters.
130
REGENERATION AFTER EXARTICULATION PLATE 2
Cc. V. MORRILL
H. Murayama del.
131
PLATE 3
EXPLANATION OF FIGURES
Figures 9 and 10 are from specimens in which complete amputation of the
limb was made at the knee-joint (exarticulation).
9 From a longitudinal section of a limb 41 days after operation. Ep.f.n.,
new femoral epiphysis; Fib.n., new fibula, proximal end; J.c.n., new joint-cavity
forming. Magnified about 100 diameters.
10 From a longitudinal section of a limb 48 days after operation. C.n.,
new cartilage; Os.n., new bone spreading through the cartilage; Os.c., new car-
tilage cells transforming into bone-cells; B.f., old bone of the shaft (femur) ;
M.c., portions of the marrow-cavity. Magnified about 420 diameters.
132
REGENERATION AFTER EXARTICULATION PLATE 3
C. V- MORRILL
H. Murayama del.
AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, JANUARY 19
IS THE INFLUENCE OF THYMUS FEEDING UPON
DEVELOPMENT, METAMORPHOSIS AND GROWTH
DUE LO A. SPECIBIC- ACTION, OF THAT GLAND?
EDUARD UHLENHUTH
The Rockefeller Institute of Medical Research, New York City
The experiments on thymus feeding thus far reported in the
literature have given results sufficiently different to prevent the
formation of a definite idea as to the réle of the organ in these
experiments. This is even true if one has in mind only one of the
various groups of animals which have been studied in such
experiments. Concerning the Amphibia among which only the
larvae of Anura have been studied carefully regarding their
reaction to thymus feeding, it seems that most of the experiments
showed a retarding influence of the thymus upon development
and metamorphosis although some exceptions are reported.
With respect to growth however, the results are so lacking in
uniformity, that Gudernatsch as well as Romeis who studied the
effect of thymus feeding in tadpoles doubted whether the effect
produced by this organ was due to a specific action or only to
quantitative conditions. Gudernatsch in his experiments on
tadpoles noted accelerated growth leading to enormous size;
but Romeis obtained completely normal growth in various series
of thymus-fed tadpoles and pointed out that the thymus feed-
ing never produces abnormally large animals.
The following experiments which will be reported elsewhere
in detail, seem to indicate that the accelerated growth of thy-
mus fed Amphibian larvae is merely the effect of quantitative
conditions and not the result of a specific quality of the organ
such as a specific growth-stimulating agent. Furthermore, they
yielded some very interesting results concerning development
and metamorphosis although these are still difficult to explain.
Finally they showed that in each thymus-fed larva severe tetany
135
136 EDUARD UHLENHUTH
is produced. The last mentioned phenomenon will be discussed
in another article; the effects of thymus upon development,
metamorphosis and growth will be outlined briefly in the follow-
ing pages.
In the experiments to be reported, only larvae of Urodela were
studied (Amblystoma punctatum and A. opacum). The ad-
vantage of using Salamander larvae is that the quantity of food
given to them can be controlled and measured with exactness,
up to a certain degree, which cannot be done if tadpoles are used;
and it should be emphasized that in order to avoid errors the
possibility of measuring the quantity of food is very desirable
and even should be demanded in experiments in which it is
suspected that qualitative relations are involved.
1. DEVELOPMENT AND METAMORPHOSIS
Gudernatsch found that the development of tadpoles was
delayed if the animals were fed on thymus. Similar results
were obtained by Romeis in his first experiments. This led to
the inference that thymus contains a substance whose specific
property is a retarding effect upon development. Only recently
Gudernatsch again published a paper based on this hypothesis.
However, in his work published in 1915 relative to the influence
of the. glands.of internal secretion on Anura larvae, Romeis
reports upon two series of experiments, which cannot be ex-
plained from the above-mentioned standpoint, and which caused
the author himself to doubt whether inhibition of development
were indeed a specific function of the thymus. ‘The first series
consisted of fairly old larvae of the species Rana esculenta,
the individuals of which developed in a perfectly normal manner,
in spite of being fed with thymus; but in this case it might have
been supposed that the thymus feeding had been started too
late. In the second series, however, in which larvae of Rana
temporaria were employed, the thymus feeding was commenced
at a very early stage, in spite of which fact the development of the
larvae was not delayed. On the contrary, they underwent
metamorphosis at an earlier stage than did the larvae fed with
INFLUENCE OF THYMUS FEEDING UPON DEVELOPMENT 137
muscle tissue, and before the latter had developed front limbs.
This last series of experiments suggests the assumption that the
influence exerted by the thymus on development must be de-
pendent on factors not specific for the thymus. The experi-
ments on Salamander larvae now to be described led to similar
conclusions.
Both Gudernatsch and Romeis took as index of the rate of
development, the growth of the hind and front limbs, the
absorption of the tail and the abandonment of the water. The
latter phenomenon however, which we will refer to as meta-
morphosis seems in Salamanders, to be dependent on a mechan-
ism different in many respects from that which controls develop-
ment, such as the growth of limbs, etc.; for in the first place
even under conditions of normal feeding, different individuals
show a different stage of development when they leave the water,
and secondly the effect of thymus upon development and upon
metamorphosis does not seem to be the same in the Salaman-
der larvae examined. Therefore we shall distinguish between
development and metamorphosis; growth and differentiation
of the limbs, certain changes of the fin and gills not being in
direct relation to the abandonment of the water, and the changes
of the color pattern of the skin which finally lead to the definite
coloration of the skin, will be referred to as development; while
the abandonment of the water together with the sudden reduc-
tion of the gills to mere stumps and the complete absorption of
the fin will be called metamorphosis.
In a group of eight series, O 1916, in which larvae of Ambly-
stoma opacum were used, four series were fed with small frag-
ments of earthworms and four series with equal sized pieces of
thymus. As will be explained later on, these experiments were
conducted with the intention of feeding the respective animals
with equal quantities of worms and thymus. So far as develop-
ment and metamorphosis is concerned, it would seem at least
possible, that besides the quality of food, the amount of food may
also have some influence upon these two phenomena; but at any
rate only if we make the quantities of food alike in the experi-
mental series and the controls, can we be sure that the differences
138 EDUARD UHLENHUTH
obtained in both series are the expression of the quality of the
food. ;
The development of the legs and toes was carefully noted, and
the development of these organs was seen to occupy a period of
from 6 to 9 weeks. As the thymus feeding began as early as the
second week, the development of the legs took place under the
influence of thymus feeding during 4 to 7 weeks. According to
Gudernatsch and Romeis this length of time sufficed in the case
of tadpoles to produce the retarding effect upon development of
the thymus; but in the case of the Salamander larvae absolutely
no retardation could be noted as a consequence of the thymus
feeding. Indeed, in those series which as a result of the simul-
taneous effect of a lowered temperature necessitated a longer
period of time in order to attain complete development, a change
in the contrary direction could even be noted in the latest stages,
the thymus animals attaining complete development of their
limbs more quickly than the worm-fed animals.
Thus it can hardly be argued in this connection that feeding
had not lasted long enough to produce a result, in view of the
fact that in the latest stages of development of the legs when the
feeding had lasted a- longer period than in the first stage, the
opposite result was obtained. This result then indicates that a
distinct difference exists between the Anura and Urodela with
respect to the effect of thymus-feeding upon development.
The development of the legs in such animals to which instead
of equal quantities of food as much food was given as each animal
was able to take, was not studied in sufficient detail. In one
group of experiments (P 1719) which consisted of A. punctatum
larvae, development of the legs was recorded during 14 days
after the beginning of the feeding; in this group the relation be-
tween the thymus-fed animals and the controls was the same as
in the above experiments on A. opacum.
The differences between the Anura and Urodela become
even more accentuated as development proceeds. But a
careful distinction must be made between animals fed on
equal quantities and those which obtain as much food as they
will take.
INFLUENCE OF THYMUS FEEDING UPON DEVELOPMENT 139
In the above mentioned group (O. 1916) which consisted of
larvae of Amblystoma opacum fed with equal quantities, the
development of further advanced characteristics of the legs,
of the shape of the head, of the gills and of the color of the skin
proceeded much more rapidly in the thymus-fed animals than
in those fed on worms.
With respect to the development of the gills, the following
should be remarked: In larvae of Amblystoma opacum fed with
worms and kept at an average temperature of 22.6°C. as late as
one or more days before metamorphosis the gills attain a stage
not only of considerable size, but one in which they are char-
acterized by considerable redness and above all by the fact that
they are bent upwards in a crescent shape. The long well
developed branches are widely extended and the points of the
stem inclined forwards so as to bend over. In worm-fed
animals kept at a high temperature (22.6°C.) this condition of
the gills was only attained in the 28rd week, but in thymus-fed
animals kept at the same temperature as early as the 11th week;
in worm-fed animals kept at a low temperature (in average 14.8°C. )
only in the 29th week; in thymus-fed larvae kept at the same
temperature as early as the 11th week (although in the latter
case the gills were less developed than in the case of the high
temperature thymus-fed animals).
A similar relationship is observed with respect to the color of
the skin. In the case of the warm worm-fed animals the melano-
phore spots only began to develop in the 13th week, at which
time they had already attained very considerable development
in the case of the warm thymus-fed animals; in the warm worm-
fed individuals the blue-grey pigment did not appear until the
24th week, but in that of the warm thymus-fed animals as early
as the 12th week. In the cold worm-fed animals the fusion of
the melanophore spots into a uniform black-brown coat only
began in the 30th week, and occurred as early as the 13th week
in the case of the cold thymus animals; but in the cold worm-fed
animals no trace of a silver-grey pigment can be detected after
32 weeks, although this appeared in the cold thymus-fed in-
dividuals as early as the 13th week. These differences in the
140 EDUARD UHLENHUTH
rate of development are doubtless sufficiently great to indicate
distinctly the differences existing between the Anura and Urodela.
Under conditions of quantitatively equal feeding (which alone
can be taken into consideration in a study of qualitative effects)
the feeding of thymus to larvae of Amblystoma opacum causes
accelerated development.
Nevertheless, the above mentioned experiments become even
more clear if the results obtained by them are compared with the
result in experiments made by a different method; for the fac-
tor to be emphasized is not the time elapsed since the hatching
but the size of the animals. The thymus-fed individuals attain
the stated conditions of development while much smaller in size
than the worm-fed animals. The latter must attain much greater
size than the thymus-fed animals, in order to acquire the same
degree of development.
In a group of A. punctatum (P. 1916) kept at a high tempera-
ture, one series was fed with pieces of thymus and another with
Tubifex. In both series the animals were allowed to eat accord-
ing to their inclination as a result of which the worm-fed animals
consumed a considerably larger quantity of food than did the
thymus-fed animals, and consequently grew much more rapidly.
The development of the skin pigmentation also proceeded more
quickly than in the case of the thymus animals, although the
latter attained these various stages while much smaller in size.
At the time no exact drawings were made to show the relation-
ship between the size and stage of development. This will be
taken up in a recently initiated experimental series of A. puncta-
tum (P. 1917) as yet incomplete, in which the same system of
feeding is being maintained as in Group P. 1916. Meanwhile
it can already be noted that the worm-fed animals do not de-
velop the yellow network until they have attained the average
size of 62.91 mm., the minimum length being 59 mm. The
thymus animals, which have only attained an average length
of 32.22 mm., with a maximum length of 36 mm., have not yet
shown signs of this network. In group P. 1916 of A. punctatum
in which the worm-fed animals behaved like the worm-fed animals
in Group P 1917 regarding the relation between size and de-
INFLUENCE OF THYMUS FEEDING UPON DEVELOPMENT 141
velopment of network, the first thymus animal attained the
network stage when only 41.3 mm. in length.
We thus see that the time at which the various phases of
development are attained varies according to the quantity of
food, with the result that sometimes the thymus animals, at
other times the worm-fed animals appear to lead. But the
constant factor is the size at which the various stages are at-
tained; that is, constant to the extent that the thymus animals
always develop more quickly than do the worm animals, if the
various stages are referred to the size of the animals. This
relationship is directly opposed to that of the Anura larvae, for
in these animals the thymus-fed individuals must usually attain
a considerably greater size than the worm-fed animals in order
to arrive at the same degree of development.
Identical relationships as occur in the development are also
found in the metamorphosis; but in this case one or more addi-
tional factors seem to play a réle to complicate considerably the
phenomena, as we shall see.
Here again we must differentiate between the experiments in
which the food was quantitatively equal and those in which each
animal was allowed to eat to the point of satiety. But it should
be emphasized that only the first method permits of a correct
comparison. For when the worm animals feed at will they eat
approximately 10 to 20 times the quantity of food that is con-
sumed by the thymus animals when the latter begin to suffer
from tetany; as the worm animals also grow much more rapidly
as a result, it would not be surprising that they also metamorphose
earlier, since we might expect that if a definite size of the animal
is indispensable to metamorphosis, metamorphosis will be ac-
celerated if we accelerate growth by some external conditions.
We will now turn our attention again to the group O 1916 of
A. opacum in which each series was given approximately the
same quantity of food. In this group the warm thymus animals
were the first to undergo metamorphosis; thus in the warm thy-
mus series (22.6°C.) the first animal underwent metamorphosis
in the 13th week, in the warm worm series only in the 24th week;
in the cold thymus series (14.8°C.) the first animal left the water
142 EDUARD UHLENHUTH
in the 24th week; whereas in the cold worm series no animal
has yet undergone metamorphosis (in the 32nd week). Thus,
thymus-fed animals are seen to metamorphose earlier than worm-
fed animals; that is, provided they receive equal quantities of
food.
The relationship of time however becomes inverted if the worm
and thymus animals, instead of receiving equal quantities of
food are allowed to eat at will. In a group of A. punctatum
(P 1916) consisting of two series, which had been kept at a high
temperature and in which the last-mentioned mode of feeding
was adopted, the first animal of the thymus series underwent
metamorphosis after five months, whereas the first of the worm
series did so after only 35 month
As in development, so also in metamorphosis, the relationship
of time is seen to be inconstant and depends on the amount of
food given to the animals. But a constant factor exists in the
relationship between size of the animal and metamorphosis.
Whatever method of feeding may be adopted, the thymus-fed
individuals are always much smaller when they undergo meta-
morphosis than are the worm-fed ones. In the Opacum group
(O 1916) consisting of equally fed animals, the warm thymus
animals averaged only 47.8 mm. in length at the time that the
first individual underwent metamorphosis, whereas in the worm
series at the beginning of metamorphosis the average size was
53.5mm. The same relationship can be observed at a low tem-
perature; the average size of the thymus animals being only 57.5
mm. at the beginning of metamorphosis, whereas the worm
animals had not yet begun to metamorphose when their average
length was 65.1 mm. The same conditions apply in the above-
mentioned Punctatum series (P. 1916); the thymus animals begin
to metamorphose when their average size is 41.9 mm., but the
worm animals only at an average size of 50.0 mm.
As in the case of development, so in metamorphosis the rela-
tionships obtaining in A. opacum and punctatum are exactly
the reverse of those found in the Anura larvae, for in the former
the worm-fed animals must attain a much greater size than the
thymus-fed individuals before they can undergo metamorphosis
INFLUENCE OF THYMUS FEEDING UPON DEVELOPMENT 143
whereas in the case of the Anura larvae the thymus animals
must be larger than the worm animals before metamorphosis
can occur.
However, in addition to the facts mentioned above, still an-
other phenomenon must be described which seems to aid greatly
our understanding of the relation between development and
metamorphosis. If we refer metamorphosis neither to the time
which has passed since hatching nor to the size of the animals but
to the stage of development of certain structures, metamorphosis
does not appear to be accelerated in the thymus animals but
rather retarded.
For example, when comparing the warm worm animals with
the warm thymus animals of the Opacum group (O 1916) we
see that as early as the 11th week the warm thymus animals
attained the same stage of development at which the warm worm
animals commenced to metamorphose. At this stage, however,
a remarkable phenomenon is noted; the warm thymus animals
fail to metamorphose while some of their organs continue to
develop; the structures of their skin, which are responsible for
the development of the color of the skin attain, while the animal
is still larval a phase of development reached by the worm animals
only some time after metamorphosis has been accomplished.
After the warm thymus animals have entered upon the stage
characterized by the crescent-shaped gills and the fusion of the
melanophore spots, they should, if compared with controls,
undergo metamorphosis, but instead they develop the silver-
erey pigment and undergo reduction of the size of the fin. Simul-
taneously (a point to be specially emphasized) they stop growing
and become reduced in length, a condition which also occurs in
the case of worm animals before metamorphosis. They assume
an aspect which on the whole resembles that of a worm-fed
animal which had undergone metamorphosis about two weeks
previously. As can already be seen, these relationships can
be noted much more distinctly in the cold Opacum series; but
as the animals of these series have not yet all undergone meta-
morphosis and the worm animals have not yet begun to meta-
morphose, we will not describe the phenomena already noted.
144 EDUARD UHLENHUTH
Exactly the same phenomenon can be seen in agroup of Punctatum
(Group P 1916) maintained at a high temperature, such as
the development of definite characteristics of a metamorphosed
animal during the larval stage. In this case the yellow network
was separated into yellow spots during the larval stage—a
phenomenon which does not occur in the case of the worm animals
before they have left the water.
From what has been stated above we can see that even in those
animals which metamorphosed first and, in the series of Opacum
larvae (O 1916) fed with equal quantities, metamosphosed 11
weeks earlier than the worm animals, the process of metamor-
phosis was disturbed. This becomes much more apparent. if
for the date at which the first animal underwent metamorphosis
we substitute that of the last animal metamorphosed. In that
case we obtain the following relationship: In the series of Opacum
larvae (O 1916) after 32 weeks have passed, 12 per cent of the
thymus-fed animals are yet in a larval stage, whereas the worm-
fed individuals all had metamorphosed as early as the 29th week.
Thus, the period of metamorphosis in the worm series extended
only over 5 weeks, whereas in the case of the thymus animals
it has already lasted 19 weeks. In the repeatedly mentioned
group of A. punctatum larvae (P1916) kept at a high tempera-
ture, the last thymus fed animal had not left the larval stage
even after 8 months, whereas the last worm-fed animal had
metamorphosed after only 53 months; thus in the worm-fed
animals, metamorphosis covered a period of only 24 months,
whereas in the thymus animals it lasted 5 months. In a group
of A. punctatum (P 1916 C) kept at a low temperature, a worm-
fed series comprising individuals which out of a number of 300
larvae had not yet undergone metamorphosis was added to a
thymus-fed series which had been under observation for about
5 months. In other words, this worm-fed series consisted of
larvae which were abnormally late in undergoing metamorphosis.
The first of these worm-fed animals left the water 5% months
after hatching, the last 7} months after hatching, -the period of
metamorphosis extending in this series over 2 months. Of the
thymus-fed animals the first metamorphosed after 43 months,
INFLUENCE OF THYMUS FEEDING UPON DEVELOPMENT 145
the last (leaving two animals out of consideration) after 64
months. In the case of these thymus-fed animals the period
of metamorphosis lasted 2 months, for instance, not longer than
in the case of the worm animals; but 2 of these thymus animals
not yet mentioned, behaved very differently from all the other
animals. They both remained at a low stage of development,
so far as coloring was concerned, and their tails underwent but
slight reduction in size. On the other hand, the gills were re-
duced to short stumps. Although neither of these 2 animals
was shedding its skin (which should take place before meta-
morphosis) at the time of the reduction of the gills, they were
taken out of the water and placed in a vessel, the bottom of
which was covered with filter paper and just enough water to
keep the vessel wet. But neither of the animals showed any
further change, until finally 123 months after hatching one of
them shed its skin and its gills became completely atrophied,
while at the same time the skin became darker in color although
the yellow network failed to develop. The other animal is still
in the larval stage, 133 months after hatching.
We must not fail however to mention that it still appears very
doubtful whether this is a direct effect of thymus, fora similar
phenomenon was also noted in the case of worm-fed animals,
although not to so extreme a degree. Out of approximately
300 worm-fed animals, only 1 individual showed such a condition;
after more than 8 months it was still in a larval condition and
had not developed a trace of the yellow network. The fin of
its tail was but slightly reduced; its gills were more reduced
and the animal was still undergoing growth and taking food
spontaneously. It was used for the purpose of an operation, in
the course of which it died. However, as has been said, at this
stage it showed no trace of approaching metamorphosis. From
this it seems very doubtful that the delay of metamorphosis
in the two last mentioned thymus animals was actually due to
the action of thymus and we must exclude them from discussion
until the same phenomenon is obtained in a greater number of
cases.
TdE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 25, No. 1
146 EDUARD UHLENHUTH
2. GROWTH
In one series of experiments (P 1917) for which purpose
larvae of A. punctatum which had hatched on the same day and
were the offspring of the same mother were employed, it was
assumed that where there is an unlimited supply of food, the
amount spontaneously taken up by each animal is a function of
growth, and that growth is not a function of the food quantity.
For that reason in these experiments which were carried out at
an average temperature of about 22°C., the animals were allowed
as much food as they felt inclined to take.
The group consisted of three series. The animals of the first
series were given small equal-sized fragments of thymus with a
pair of forceps, until each animal was satisfied. They took the
pieces easily and owing to the softness of the material had no
difficulty in swallowing them. The second series received frag-
ments of earth-worms. Owing to the hardness of this food,
however, the animals found great difficulty in swallowing it,
and it took several minutes, or even hours for each piece to be
swallowed. As they were fed only once a day, these worm ani-
mals remained hungry and consequently were soon backward
in growth, as compared with the thymus-fed animals. The
latter finding coincided with the observations made in the case
of the Anura; ie., that the thymus stimulates growth; but it
failed to prove a specific influence of thymus, for the reason that
the animals which were fed in a normal manner were found to be
starving. In a third series the animals were fed with small
worms (Enchytraeus), which were at first given in small pieces;
these worms were thrown into the containers in such large quan-
tities that the animals never lacked food. Besides this, each
animal was fed on pieces of earthworms which the fast-growing
animals soon took readily and in large quantities. The individ-
uals of this series grew faster from the very outset than did the
thymus animals. As the latter did not develop tetany until the
5th week and were in a completely normal condition until the
end of the 4th week, we may look upon the result attained up
to that time as the pure effect of nutrition. The salamander
INFLUENCE OF THYMUS FEEDING UPON DEVELOPMENT 147
larvae failed to show that the thymus had exerted any growth-
accelerating influence. On the other hand, the quantity of food
given plays an important part in this connection, for the animals
react in a highly sensitive manner to relatively slight differences
in food quantities. From these experiments it would seem that
in the experiments on tadpoles conducted by Gudernatsch and
Romeis the factor revealed is not a specifically growth-promoting
influence, but that the accelerated growth of the thymus animals
should be attributed to the fact that the jaws of the tadpoles,
although adequate to supply the body with a quantity of the
soft thymus material corresponding to the needs of the organism,
were nevertheless not the most appropriate instrument for pre-
paring from the hard beef muscle sufficient nutriment for the
purpose of keeping up normal growth. The very fluctuating
results which Romeis obtained in his experiments indicate pro-
nounced sensitiveness on the part of the tadpoles to small quanti-
tative differences of food which often completely escape control,
rather than the presence in the thymus of a specific growth-
promoting influence. It should also be remarked that in the
above-mentioned experimental group it was also noted that the
animals fed with worms must consume a much greater quantity
of earth-worms than the thymus-fed animals consume of thy-
mus in order to grow equally quickly; the supply of earth-worm
fragments which the second series consumed was only slightly
smaller than that of the first series fed with fragments of thymus.
This fact speaks in favor of relatively high nutritive value in the
thymus. It should also be taken into consideration that in the
fragments of earth-worms a not inconsiderable part of the volume
consumed consists of indigestible substance (chitin, earth) which
are later eliminated in the feces.
In the preceding order of experimentation it is seen that at the
moment that the tetany period begins in the thymus-fed animals
we are confronted by an obstacle which prevents any quantitative
judgment from being formed; for from this time on the thymus
animals are seen to be abnormally placed and the amount of
food taken in by them becomes abnormally low. This is all
the more disturbing for the reason that it is uncertain whether
148 EDUARD UHLENHUTH
under these conditions the quantity of food spontaneously taken
is really a function of growth. On the contrary it appears very
probable that the reduced amount of food taken must be at-
tributed to disturbances caused in the swallowing apparatus by
the convulsions. In such a case the animals would be in a
condition of starvation and in contradiction to the idea of the
experiment the rate of growth would be the function of the food
quantities introduced into the organism. I thought to be able
to overcome this obstacle in another group of experiments, in
which I proceeded from the fact that when food is present in
sufficient quantities equal amounts of food produce an equal
rate of growth.
In a group consisting of 4 series (O 1916) for which larvae of
A. opacum were used, the food was given in small fragments
at the point of the forceps in all the series; an attempt was made
to make all the pieces of approximately the same size on the same
day of feeding. The number of pieces given to each individual
animal was noted, and on each feeding day approximately (for
the week) the same number of pieces was given, so that all the
animals of these 4 series received approximately the same
number of pieces, the series comprising one thymus and one worm
group at an average temperature of 22.6°C., and one thymus
and one worm group at about 14.8°C. An effort was hereby
made to distribute a quantitatively equal amount of food among
the 4 series; but it must be remarked that this can only be
roughly attempted and cannot be exactly carried out. As it
can never be known beforehand how much food the animals
may need on a given day in order to be satisfied, it would also
be quite impossible to weigh the food. But even if this were
possible, the distribution of equal quantities according to weight
would not lead to the distribution of equal nutritive quantities
as a given volume of thymus contains a larger quantity of sub-
stances available for metabolism than does the same quantity
of fragments of earth-worms, as has been shown in the first
experimental group. Although this method is not exact, it
has at least furnished an approximate idea as to how important
it is to control the quantity of food in such experiments.
INFLUENCE OF THYMUS FEEDING UPON DEVELOPMENT 149
Of course the quantity of food to be given each day was always
standardized from the series which desired the smallest amount
to eat. At the beginning these were the worm series, and of
these the cold worm series showed less avidity for food than did
the warm worm series. As a result the thymus series at first
received less than they would have liked to eat. The reasons
for this comparatively small appetite in the worm animals
have been specified above when discussing the first experimental
group. From the time that the series of warm thymus animals
began to undergo metamorphosis, the animals of this series
showed the least desire to eat; after that it was the worm animals
in general, and the warm worm series in particular which received
less than they could have consumed.
It may be emphasized at this point that when this method of
distributing quantitatively equal amounts of food is followed,
tetany exerts a very slight influence on growth. Sometimes the
rate of growth is reduced at such points where the tetany curve
reaches its apex, but in other cases, on the contrary it increases
or reaches even a maximum when the tetany curve does.
The condition which exerts an influence on growth in compari-
son with which all other influences are reduced to insignificance,
is metamorphosis, as will be apparent from the following
description.
During the first few weeks the warm thymus animals are seen
to lead in size; next in order come the cold thymus animals,
then the warm worm series, and finally the cold worm series.
Nevertheless no special importance must be attributed to this
relationship, for as has already been stated, given an equal volume
of food, the thymus animals probably obtain more nourishment
frem their pieces of thymus than do the worm animals from an
equal quantity of worm fragments. T he relationship of size
which has just been mentioned lasts until the 10th week, and the
acute tetany which has meanwhile set in among the warm
thymus animals and reached its climax has failed to influence
this relation at all. In the 11th week a pronounced change sets
in; at this stage the warm thymus animals are all ready for
metamorphosis, the first individuals being 14 days removed from
150 EDUARD UHLENHUTH
this step. During this week the curve of the body size of the
cold thymus animals, which up to that time occupied the second
position, can be seen to cross that of the warm thymus animals.
From the time that the first animal of the warm thymus series
entered upon metamorphosis, the warm thymus animals com-
pletely stopped growing. Their curve, which of course does not
include the metamorphosed animals, is soon after crossed by
those of the two series of worm animals, and the warm thymus
animals remain smallest in size for the rest of the experiment.
The cold thymus series, the first individuals of which under-
went metamorphosis in the 24th week, also increase in size only
a little from the time of metamorphosis on; but as the first
animals of the warm worm series which is most proximate to
the cold thymus curve similarly undergo metamorphosis in the
24th week, and also because the curve of the cold thymus animals
is higher above that of the warm worm animals than the curve
of the warm thymus animals is above the cold thymus animals,
the curve of the latter remains the first at the beginning; it is
not crossed by that of the warm worm animals until the latter
have all metamorphosed. Finally, in the 29th week, together
with the curve of the warm worm-fed animals, it is crossed by
the curve of the cold worm series, which now occupies the first
position. As early as the end of the 29th week the largest animals
of the cold worm series have attained a size greater than that of
each non-metamorphosed (and of course of each metamorphosed)
individual of the three remaining series. As for the present
(after the 32nd week) the animals give no sign of ee
metamorphosis and continue to grow.
The above-reported circumstance appears to. us to a the
most instructive with reference to the statement that thymus-
fed anuran larvae attain a size by many denoted as abnormally
large but stated by Romeis never to exceed normal limits, al-
though sometimes exceeding the size of the muscle-fed animals.
If we begin by comparing each of the two thymus series with the
corresponding worm series, we see that the same relation exists
between them as between muscle and thymus-fed tadpoles, inas-
much as the animals which metamorphose later attain greater
INFLUENCE OF THYMUS FEEDING UPON DEVELOPMENT 151
dimensions than do those which first underwent metamorphosis.
It can be seen that this is not connected with a specific influence
of thymus feeding, from the fact that exactly the same relation
exists between the warm and the cold worm series, the warm
worm-fed series which first underwent metamorphosis meta-
morphosing while smaller in size than the cold worm series, and
the latter continuing to grow after the former has metamorphosed.
While yet in the larval stage the cold worm-fed animals attain a
size which when the largest animals of both series are used for
comparison, already exceeds that of the largest warm worm-fed
larva by 12.5mm.' From another point of view the salamander
larvae of those species so far examined show the very opposite
characteristics from those possessed by the anuran larvae; for
it is not the worm-fed salamander larvae which first undergo
metamorphosis but the thymus-fed individuals. Thus, the
point to be primarily emphasized is not the greater size ulti-
mately attained by the worm-fed salamanders and thymus-fed
tadpoles, for we have seen that this does not depend upon the
specific qualities of the thymus, but that it is a general phe-
nomenon peculiar to amphibia and one dependent upon the
time at which the animals undergo metamorphosis. The point
of importance in both cases—the larvae of Anura as well as of
A. opacum and A. punctatum is the circumstance that thymus-
feeding produces metamorphosis in the Anura only when con-
siderable size has been attained, whereas in the Urodela, on the
other hand, this occurs while the animal is but small in size.
To summarize, we may make the following statement: The
differences in the rate of growth to be noted before metamorphosis
are not the result of a specific growth-promoting influence of the
thymus; they are based on the circumstance that animals which
are better fed grow more quickly. In the experimental group of
A. punctatum (P 1917) discussed in the preceding section, these
‘Although the cold worm larvae are at the time of writing larger than the
largest metamorphosed warm worm animals, we do not here intend to take up
the question of this relation; moreover, a comparison of the experiments
hitherto conducted in connection with the Anura shows this not to be possible,
as the respective authors never observed their experimental animals beyond
the period of metamorphosis.
152 EDUARD UHLENHUTH
individuals are obviously the worm-fed animals of the third
series, which are allowed to have as much food as they wish; in
the experimental group with A. opacum (O 1916) it is the thymus
animals which take in a greater quantity of nutritive material
through eating thymus. The experiments furthermore show
that qualitative influences exerted on the rate of growth would
have to be very considerable in order that they can be experi-
mentally tested in the case of amphibia, for in these animals the
slightest quantitative differences, such as can hardly be controlled,
would bring about very misleading differences in growth.
With respect to the ultimate size attained by the animals,
Salamander larvae resemble tadpoles in the fact that under
certain conditions the later they metamorphose the greater is
their final size; this is not only true for thymus-fed animals in
comparison to worm-fed animals, but also for worm-fed animals
kept in high temperature in comparison to worm-fed animals
kept in low temperature.
The action of thymus on development and metamorphosis
may be summarized in the following way:
In animals fed on thymus the development presumably of the
organism as a whole but certainly of the legs, gills, shape of the
head and color of the skin, is greatly accelerated during the
larval period. The thymus-fed animals, therefore, reach the
stage at which worm-fed animals are ready for metamorphosis,
much more quickly than worm-fed animals. As development at
least to some degree may be dependent on growth, on the rate of
growth and on size, it is impossible to examine the specific
influence upon development of any substance without keeping
alike the conditions of growth in both the experimental and
control series; such was attempted by admitting an equal amount
of food to both series. ;
When the thymus animals have reached the stage at which
worm-fed animals go into metamorphosis, the development of
most organs seems to stop, while certain characteristics of the
skin continue to develop; the skin of such animals then behaves
very similarly to the sex-organs of neotenic larvae, since the skin
at least with regard to the structures determining pigmentation,
INFLUENCE OF THYMUS FEEDING UPON DEVELOPMENT 153
develops characteristics of a metamorphosed animal, while the
animal as a whole still is in a larval stage. At the time when
metamorphosis should occur disturbances in the course of de-
velopment begin to appear evidently due to the suppression of
the development of some factor, without which further develop-
ment is impossible. In most of the animals of a thymus-fed
series this factor still develops much earlier than in the controls;
but even in these individuals metamorphosis becomes a grave
danger to the animals’ life. In high temperature some animals
die during metamorphosis and those which survive metamorpho-
sis die a relatively short time after metamorphosis. In some in-
dividuals the development of the factor necessary for meta-
morphosis is still more disturbed and becomes delayed in com-
parison with the controls; at high temperature all individuals in
which this is the case die on the day when the gills and the rest
of the fin undergo the sudden reduction in size, characteristic
of the entrance into metamorphosis. In low temperature they
may survive metamorphosis. In low temperature a very small
percentage of the thymus-fed animals may remain at a low stage
of development and not metamorphose for more than a year;
but whether this is due to the action of the thymus diet is not
yet certain, as a similar phenomenon was observed in one worm-
fed animal of the stock.
It seems that we cannot understand the results reported in
thymus feeding experiments if we assume that they are the pure
expression of the influence of the thymus substance. The rather
great fluctuations reported in individuals of the same species
as well as the surprising differences between larvae of Anura and
Urodela when fed on thymus, indicate that quite a number of
factors are involved in metamorphosis, some of which were not
controlled in the experiments. It is of course clear, that differ-
entiation of the organism is one of these factors; that a certain
degree of differentiation is indispensable for metamorphosis, or
at least to facilitate it, was shown by Gudernatsch in some recent
experiments on the influence of thyroid. That some of the
individuals among a thymus-fed series of Salamander larvae
metamorphose earlier than the controls may be due in some degree
154 EDUARD UHLENHUTH
to the fact that in the thymus-fed Salamander larvae develop-
ment and differentiation and consequently metamorphosis also
depend on the general conditions of growth; the experiments on
Salamander larvae reported suggest that rate of growth and size
play an important réle in metamorphosis. The difference noted
between Anura and Urodela when fed on thymus can be ex-
plained only by assuming a fundamental difference between
the organization of these two groups of animals. It will be
pointed out in another article that such a difference, namely
the absence in the Salamander larvae and the presence in the
anuran larvae of the parathyroids, seems to explain why thymus-
feeding should develop tetany in Salamander larvae and should
not in anuran larvae. It suggests itself that metamorphosis
in part must depend on a factor similarly being present in one
eroup but absent in the other group. The development of that
factor may be induced primarily by processes occurring in a
- certain stage of differentiation, but also may be influenced and
inhibited or disturbed by thymus diet. The action upon this
factor of the thymus may be widely different from that upon
developmental processes preceding its development; this is
indicated by the fact that development while accelerated during
the larval period is on the contrary retarded from the time at
which metamorphosis should occur. It is this phenomenon
which emphasizes the fact that metamorphosis to some degree
must occupy a particular place among the processes of develop-
ment. In this connection, finally, frequent reports may be
remembered according to which thymus causes disturbances of
the blood circulation; in metamorphosis of the Amphibians the
blood circulation undergoes a fundamental change in the course
of which the gills are absorbed, and in Salamanders, the absorp-
tion of the gills according to Maurer, is a prerequisite for the
formation of the parathyroids. It may be worth while to keep
these facts in mind during further studies of the influence exerted
upon metamorphosis by the thymus.
Though the effect of thymus feeding on development and meta-
morphosis is very evident, it appears to the writer that similar
effects may be produced by other and purely quantitative exter-
INFLUENCE OF THYMUS FEEDING UPON DEVELOPMENT 155
nal conditions, such as temperature and quiéntity of food and in
general by all factors which modify growth, rate of growth, size
and velocity of development. No doubt such factors are of
great importance in determining at what time, at what size and
developmental stage of the animal, metamorphosis will occur.
Since the relations between these different factors are very
complicated and the number of experiments relative to them is
rather small, discussion of these conditions must be postponed.
Finally it should be mentioned that the thymus gland appar-
ently contains all substances which are necessary to build up the
substance of an Amphibian organism to maintain the animal
growing and to sustain life permanently. This is demonstrated
by a number of specimens of A. punctatum kept at low tem-
perature which have been fed on thymus since about the 14th
day of their life and are now about 14 months old; they are in-’
creasing in size.
AUTHOR'S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, JANUARY 19
THE REGENERATION OF TRIANGULAR PIECES OF
PLANARIA MACULATA. A STUDY IN POLARITY!
J. M. D. OLMSTED
FOURTEEN FIGURES
Morgan (’98), in his studies on the regeneration of Planaria
maculata, describes two types of operation by which he was able
to obtain regenerated pieces in which “the long axis of the new
head” was ‘‘at right angles to the long axis of the original worm.”’
When he cut narrow strips from the side of a planarian, he found
that the piece, through contraction, assumed the shape of a
crescent, the cut edge forming the concave margin. In certain
cases all the new tissue which formed in the concavity of the
crescent was used in the production of a head. A similarly
shaped worm was formed in several cases when he cut from the
side of a planarian a triangle the apex of which lay within the
body. Both these methods of cutting, however, produced other
pieces, which upon regeneration nearly or quite retained their
original polarity. Morgan remarks (p. 373) ‘The experiments
do not show clearly, why, at one time pieces cut from the side
give rise to new worms having the long axis in the direction of
the original long axis, and at other times at right angles to the
original long axis.’
Child (715, p. 165) states that in triangular pieces cut from the
side of Planaria dorotocephala the regenerated “head often devel-
ops nearly or quite in the direction of the transverse axis.”’
The possibility of producing regenerated planarians whose polar-
ityshas apparently been so changed that their chief axis is at right
angles to the chief axis of the worm from which they were taken
having been demonstrated, at Dr. H. W. Rand’s suggestion a
more detailed study of the regeneration of such pieces was under-
taken, the results of which are given in this paper.
‘Contributions from the Zoological Laboratory of the Museum of Compara-
tive Zoology at Harvard College, No. 302.
157
158 J. M. D. OLMSTED
The species of planarian used in these experiments was Plan-
aria maculata Leidy, and the specimens were taken from Fresh
Pond near Cambridge, Mass. Worms of various sizes, from 12
to 5 millimeters in length, were used. Some specimens, after
being brought into the laboratory, were fed on liver until at the
time of operation they were of the maximum size. Others,
medium and small worms, were kept without food for several
weeks. Neither the condition of satiety nor of starvation
noticeably influenced regeneration. At one time the mortality
of one lot would be greater, at another time, that of the other.
In the fed worms, however, it was found best to allow one week
to elapse after the last feeding before the operation was performed.
To prepare the planarians for operation, they were narcotized
in a 0.1 per cent solution of chloretone until they ceased to move.
Cuts were then made with a sharp scalpel, care being taken to
have the cut edges as nearly straight as possible. Triangular
pieces were taken from all regions of the body, each triangle
having for one of its sides a portion of the origina! uncut right
or left margin of the worm, and, for the other two sides, cut edges
which intersected near the original median axis of the worm (fig.
2a, 5a, 7a). The two cut edges, intersecting at a point which
I shall refer to as the vertex of the triangle, are distinguished in the
following account as the anterior and posterior edges.
It was only towards the end of experimentation that the im-
portance of fairly exact measurements of the lengths of the cut
and uncut edges, the angle where the cut edges meet, the distance
of the vertex of this angle from the median axis of the worm
from which the piece is taken, and the size of the piece, was
realized. In the earlier part of the work, no camera drawings
were made until the day following the operation. Because of
the decided contraction of the pieces at this time and the con-
sequent distortion of their original shape, it was possible to
estimate only rather roughly their original measurements. Later
in the work, however, camera drawings were made immediately
after operation while the pieces were still in chloretone, the very
slight contraction in this condition being negligible; a second
drawing of each piece was made on the day following, when they
A STUDY IN POLARITY 159
were in the contracted state. The drawings made while the
pieces were still in chloretone formed the basis for classification
into groups, according to the relative lengths of the cut ‘edges,
the size of the angle at the vertex, etcetera. The drawings
made on the day after the operation during the earlier experi-
ments were compared with those of the later work and each of
the earlier ones was placed in that group which it most resembled.
One may fairly assume that pieces which resemble one another
on the day after operation would also have been similar immedi-
ately after the operation. Thus it was possible to estimate with
some degree of accuracy the measurements which the triangular
pieces in the earlier work had immediately after operation.
In the following account it was thought best, however, to enumer-
ate the cases separately; hence the earlier experiments, in which
the original measurements are estimated merely, are referred to
as Series I, whereas the later ones, in which the pieces were
drawn while still in chloretone, are designated as Series II.
The mortality of such triangular pieces is very great. Less
than one-fifth of them survive the operation and regenerate.
Pieces taken from the region of the pharynx (fig. 5a) had the
greatest vitality, though regeneration of pieces from other regions
of the body, if accomplished, proceeded along exactly the same
lines as in the pieces from near the pharynx. Bardeen (’03)
found that in Planaria maculata he could more frequently obtain
double-headed worms from cross-pieces when they were taken
from the pharyngeal region than when from any other region
of the body. Morgan (’04) was also more successful in getting
pieces from this same region to regenerate, but he remarks,
“Whether this is only because shorter pieces are more easily
obtained here, or because the very short pieces from this region
survive the operation, remains an open question.”’ The latter
explanation seems to be the true one, since in many cases In My
experiments the same sized pieces were taken from all regions
of the body and only those from near the pharynx survived.
When, in the operation of cutting, the epidermal layer is
broken, a great mass of loose parenchyma cells flows out from
the wound, and if the two cuts form a very acute angle, the
160 J. M. D. OLMSTED
projecting point on the triangular piece becomes rounded off by
loss of material (cf. Morgan, ’98, p. 393). Immediately after
the operation there is always a very slight contraction of the cut
edges, even though the piece is still immersed in chloretone.
This, no doubt, is due to the direct stimulation of the muscle
fibers. As soon as the effect of the narcotic is gone, the piece
contracts ¢reatly, often assuming the form of a hollow cone, the
apex of which lies approximately at the center of the dorsal
surface of the piece. Epithelial cells soon cover the wound
(Lang, 712, p. 272), and after twenty-four hours new white tissue
can be seen along the cut edges. This new material is never
evenly distributed along the cut edges, but (figs. 2b, 5c, 6c)
a greater amount of it appears near the center of the anterior
edge, a less amount along the posterior edge, and very little at
the vertex (cf. Morgan, 798, p. 378). cosr eae oon 178
PNIECERIONRODSOLe uate RUNS Some NC Saari. tated ERR ORR. 178
Il, INGACITOMNE tO OlinCuorry Suinomtlbl, oj shcekeoasbbeseconcedcepocousou- 179
Za OrcancEschisithve suOMOOGHexGRaCiSe =... : hese eG acece etre sees - 184
ALOU STOLEN GIA) Manne Ga hwals Ge Low Sens Om Poeiras Ue oo ae artaeone 184
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IMaVED Sitio) cKO ce Mie ere Rome Nee ote ork iris A Reis Cnet ne nee a 187
COME USTONSNes mepess « vHORES oak Ske Ee RC as HAE ARS ase eyo 191
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[PORcansesensitivertO LOOd exXtnacts. 4.6. oneness oe al 192
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PLDSRD IS CUSSION MERE wey shine cts Sane wean cc a teers inte: yen te ens, Sie 216
1. Movements resulting from olfactory stimulations................. 216
Dre AS COHAN OMSINe lll pee ae Pe teers Auch ete oi Ao Mele ciate ole ole ees 221
TW MSU TT aT yar eee ee En EINE ECT TIN SES oe Se scp os sits S Seeds sie ohsts 224
We oiteratinerc ited peewwaa ext ain Ps ae atc eames hye tery oo Aisi s aierele es 227
I. INTRODUCTION
It has long been recognized by fishermen and naturalists
that many species of marine carnivorous snails are conspicuously
successful in finding food. ‘To the former they may be a source
of considerable annoyance through their habits of entering lobster
pots, eating fish entangled in nets and feeding upon bivalve
mollusks, sometimes doing so much damage that like the whelk
and oyster drill they become serious pests. The extensive
177
THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 25, NO. 1
178 . MANTON COPELAND
literature devoted to mollusks contains numerous references to
these gasteropods congregating in great numbers about dead
animals, and several observers have described buried snails
coming out of the sand when food was placed in the water near
them. This ability of the carnivorous snails to find food has
quite generally been attributed to a well developed sense of
smell, but very little definite information concerning their
olfactory reactions and organs appears to have been obtained
by experimental methods. Most recent writers in discussing
the sense of smell in this group refer to the work of Nagel (’94),
whose studies on marine gasteropods however were evidently
limited, and who, by stimulating them with strong or irritating
substances, used stimuli which were inappropriate for calling
forth olfactory reactions, at least so they could be distinguished
from other types of chemical responses. Accordingly an experi-
mental investigation of olfaction in two species of snails was
undertaken in view of determining particularly the sensitiveness
of the animals and the characteristics of their responses to
olfactory stimuli, whether they are directed to food or find it
by random movements, and the location of the receptors con-
cerned in the reactions.
A part of the work was carried on at the United States Fisher-
ies Biological Station at Woods Hole, and I wish to express my
gratitude to the resident Director, Dr. P. H. Mitchell, for many
favors received during my stay.
¢
II. EXPERIMENTS
ALECTRION OBSOLETA
An investigation of olfaction in marine gasteropods was begun
by a study of the food reactions of the mud snail, Alectrion
obsoleta (Say), formerly included under the genus Nassa or
Ilyanassa. It is a rather small species, the expended foot
measuring from fifteen to eighteen millimeters in length, and
about ten millimeters in width in its broadest part. The an-
terior border of the foot is extended on both sides so as to pro-
duce recurved processes, approximately one millimeter long,
OLFACTORY REACTIONS OF MARINE SNAILS 179
provided with sharply pointed tips. The slender tapering tenta-
cles, about five. millimeters long, are borne on stout processes
projecting in an antero-lateral direction three millimeters from
the head, and the eyes are situated at the base of the tentacles
on the outer side. The siphon, which runs forward above the
head, represents a prolongation of the mantle. The ventral
wall is split its entire length, but when the edges are brought
together a tube is formed, open at the tip, through which water
passes to the gill within the mantle chamber. The organ pro-
trudes from ten to fourteen millimeters beyond the anterior
border of the shell, and can be swung about in various directions.
Alectrion occurs commonly on tide flats, where it is a thorough
scavenger, feeding on a variety of food substances, but often
to be seen collected in great numbers on some dead animal such
as a fish or- a crustacean. Dimon (’05) writes of its food as
follows:
Among the various food materials upon which Nassa was seen to
feed were hen’s egg shells, dead hermit crabs and Squilla, live Nereis,
ulva, the alga that grows upon the shell of Nassa itself, the thick black
mud of the inner harbor, and the alga that gathers on the glass of
aquaria. . . . . Nassa did not usually attack a live uninjured
clam, though I have seen the snails collected at the edge of the mantle
of a clam that was apparently alive, devouring it and preventing the
shell from shutting by pushing themselves into the opening between the
valves.
Belding (710) has shown that Alectrion occasionally feeds
upon the living scallop, gaining access to the soft parts of the
mollusk by entering between the open valves.
1. Reactions to olfactory stimula
That an olfactory sense plays an important part in the dis-
covery of food by Alectrion seemed probable. Dimon (’05)
reports finding a Squilla about six inches in length
on the mud, with ninety-eight mud-snails crowding about it. It
was then taken away from these and put into a pool about eighteen
inches in diameter, which was full of quiet mud-snails. These snails
immediately became active, but without moving definitely toward the
Squilla. When one happened to reach it, it stopped and began to
180 MANTON COPELAND
eat, so that in a short time they were once more gathered thickly about
the Squilla. The same test was repeated in another pool, with the
same aimless wandering and gradual collecting of the snails about the
Squilla. In the laboratory the Squilla was put into an agate pan
about sixteen inches square, in which were thirty-five mud-snails.
The effect was the same as in the pools out of doors. The snails moved
about actively at first, and in the course of twenty minutes, twenty of
them had collected on the Squilla and the others had quieted down.
The Squilla was large and had probably been dead for some time,
so that its odor or taste was quickly diffused through the pool. Towards
smaller, live creatures, such as Mya arenaria with a cracked shell, the
response of Nassa was less rapid, and was not at all definite, unless the
snail came very near the dam. The definiteness and promptness of
response seemed to vary somewhat with the nearness of the animal
to the stimulus, and also with the individual snail.
This description gives the impression that the food was scented
by the snails, but that its discovery was perhaps largely a matter
of chance. In the hope of gaining more detailed information on
the behavior of Alectrion in the presence of food, the following
experiment was tried:
A shallow rectangular glass dish, measuring approximately
thirty-eight by twenty-three centimeters, was filled with sea
water. At one end of the dish was placed a ball of cheese cloth
about three centimeters in diameter, and at the opposite end
a cheese cloth bag of the same size Gontaining fresh fish (Fundulus)
meat. Both packets were weighted with a pebble to hold them
in position. Ten snails were then placed along a line midway
between the two packets and their behavior noted for one hour.
That the snails scented the fish when some distance from the
baited bag was soon made evident. The bottom of the dish was
slightly concave at the margins, so that the fish juice slowly
drained toward the two corners nearer the baited bag. The
snails which moved into the region of the juice extended their
probosecides, and worked them over the surface of the glass.
This proboscis reaction, which later was carefully studied and
found to be characteristic of snails stimulated with dilute food
materials, often beginning when the animal was several centi-
meters away from the packet, was particularly marked where the
juice drained toward the corners of the dish, and sometimes
occurred in the corners, which were about ten centimeters from
OLFACTORY REACTIONS OF MARINE SNAILS 181
the tish meat. The proboscis was extended farther when the
snail approached the source of the stimulating material closely,
and two or three of them chimbed upon the bag in their attempts
to secure the food. Sometimes the snail’s siphon was brought
within one or two millimeters of the bag before the proboscis
was protruded. This was especially true when the bag was
approached from the center of the dish toward which less Juice
was spreading. Eight of the ten individuals came near to the
bag and extended their proboscides, the latter reaction always
occurring before the siphon touched the cheese cloth. No
behavior of this sort was observed at the opposite end of the
dish, although six individuals came into contact with the un-
baited packet, touching it ten times during the hour. They
always moved away from it and no proboscis activity took place.
Other experiments of a similar kind, varying somewhat in
details, were carried out, and gave essentially the same results.
That dilute materials emanating from fish meat stimulate
Alectrion and cause the animal to thrust its proboscis, in short,
that’ the snail scents distant food, was clearly demonstrated.
Whether the final discovery of the food is the result of chance
movements, or of some directive influence of olfaction, is a prob-
lem which will be discussed later, particularly in connection with
the reactions of Busycon.
The responses of Alectrion to distant food in a current of water
were next investigated. Dimon (’05) describes the activities
of the mud snail when in a stream containing food juices which
flowed down a beach, and concludes that “if a current flows
from the food to a snail, the animal will crawl up toward the
food.”” The same author also finds, however, that the snail
“shows a tendency either to move against a moderately strong
current, or to orient itself when at rest with its head pointing
against the current.’ The reader, therefore, is left somewhat
in doubt as to the significance of the snail’s movements against
the current in the first instance, and may ask: Were they affected
by the presence of food up the stream?
In order to determine whether food juices added to the water
caused Alectrion to move more frequently against the current,
182 MANTON COPELAND
the following tests were made: A wooden box open at the top,
nearly one hundred and seventy centimeters long and about
twelve centimeters wide, was lined with glass, and placed in a
horizontal position on a table. A rubber tube for conducting
sea water was inserted in one end of the box near the bottom.
The water flowed the length of the box and out an opening at
the opposite end. The current was moderately strong, and care
was taken to keep it as uniform as possible throughout the
experiment.
A snail whose reactions were to be studied was placed in a
position near the middle of the box, and all its movements with
or against the current recorded until it passed a line seventy-five
centimeters, either down or upstream, from the starting point.
Each animal was first tested four times in the current without
food. Two, which moved against the current to the seventy-
five centimeter line in all their trials, were discarded, as the
experiment necessitated the selection of individuals which
showed at least some tendency to move in the direction of the
current. Five animals were finally selected which answered the
requirements. After determining their responses to the current
alone, they were tested again with two fish (Fundulus) placed
near the head of the stream above the seventy-five centimeter
line. The fish were constantly bathed by the water after it
left the tube, but were so situated on the right and left margins
of the stream as not to interfere with the force of the current.
They were cut open, and were turned over or moved from time
to time during the tests in order to disperse the juices more
freely. In trial one, throughout the experiment, the snail
was placed heading across the current, so that its initial impact
was on the animal’s left side. It was started in the opposite
direction in trial two; in trial three it was headed down stream
and in trial four, upstream. The position of the snail at the
beginning of the test, however, seemed in no way to affect its
later activity and the final result. A single snail was given but
one trial a day, and all were kept in good physiological condition
by occasional feeding. .
Table 1 shows the result of the experiment.
OLFACTORY REACTIONS OF MARINE SNAILS 188
TABLE 1
Showing the distances in centimeters moved by mud snails and their arrivals up and
down stream, when started midway between two lines 150 cm. apart
in a current with and without food juices
IN CURRENT WITHOUT FOOD JUICES IN CURRENT WITH FOOD JUICES
ANIMAL TRIAL Distances moved Arrivals Distances moved Arrivals
NUMBER |NUMBER
Up Down Up Down Up Down Up Down
stream stream stream stream stream | stream | stream | stream
Ags 75 0 + 80 5 a
1 2 0 75 Be 75 0 --
3 0 75 + 75 0 —
4 0 75 aa 81 6 ~
1 0 75 a 75 0 a
9 2 90 15 SF ilile/ 42 —
3 120 45 + 119 44 +
4 51 126 se 134 59 +
1 18 93 + i aa +
3 2 116 41 =e 1D 37 os
3 0 75 + 63 138 a
4 7 82 + 173 98 +
1 0 75 + 61 136 a
4 2 75 0 =- 4 79
3 0 75 Se 80 5 a
4 15 90 a 10 85
1 53! 0 = 77 2 a.
5 2 632 0 = 75) | 0 os
3 87 12 + (fa eee ee =
bl aa 0 75 + aoe Oy. ee |
| —| -
Gta 770 1104 6+ | 12 1636 | 736 | 16 4
1 Closed trial with snail resting at 53 em. line.
2 Closed trial with snail resting at 63 em. line after starting it once by touching
it.
Of the twenty preliminary trials in the current alone, there
were eight in. which the snails either arrived at the seventy-five
centimeter line upstream, or showed a marked tendency to
move against the current. The number of arrivals upstream
was doubled, however, when the fish were placed near the head
of the current. The five snails traveled 770 centimeters against
184 MANTON COPELAND
the current without food and 1636 centimeters with the food
present. The distances moved across the current are not recorded.
A snail failed in but one case to increase its arrivals upstream
when the fish were there (Animal number four). It should be
noted, however, that this individual showed a greater tendency
to move against the current when food was present than it did
in its,absence. The force of the current infrequently caused a
snail to lose its foothold and slip a short distance on the glass,
but it soon recovered itself and continued its locomotion. The
total distance moved as a result of slipping in the forty trials
was but little over fifty centimeters, over forty of which were
recorded for this same animal in its trials with food juices in the
water.
The average time taken by the snails in reaching the seventy-
five centimeter line up the current without food, in six trials, was:
approximately twenty-seven minutes, and with food present, in
sixteen trials, twenty-nine minutes.
The experiment indicates that snails which are inconstant in
their reactions to a current, or more often go downstream, move
more frequently against a current when it carries dilute food
juices; in truth, they exhibit olfactory responses leading to the
discovery of distant food. When the animals tested were al-
lowed to continue their progress against the current beyond the
seventy-five centimeter line, they usually arrived at the fish
and began feeding.
2. Organs sensitive to food extracts
Having made certain that Alectrion responds actively to
dilute food stimuli, experiments were begun to determine the
sensitiveness of the external parts of the snail to odorous material,
in the hope of discovering the olfactory receptor.
An extract of fish meat was prepared by grinding muscle tissue
of Fundulus in sea water and filtering the product. A small
amount of dry carmine was added to the filtrate, in order to
make it visible in water.
The tentacles. The tentacles were first tested. When the
fish extract was squirted over the tentacle of a moving snail by
OLFACTORY REACTIONS OF MARINE SNAILS 185
means of a finely drawn out pipette, a marked reaction followed.
The tip of the tentacle coiled rather violently, the animal stopped,
the siphon was swung into the stimulating material and the
proboscis extended and worked over the bottom of the glass
dish in which the tests were made. Numerous animals were
tested, and although considerable individual variation in respect
to sensitiveness to the stimulus was noted, the reactions were
remarkably constant, as the following responses of five snails
will show. Sea water mixed with carmine was always applied
with a special pipette to the tentacle before the fish juice in
order to make sure that the reactions observed were due to chemi-
cal rather-than tactile stimulation. The snail was moving when
the test was made, and at least a minute elapsed between each
trial with the fish juice. The material was applied five times
to the right tentacle, and then five times to the left one, and
care was taken to have the snail in water freé from the stimulat-
ing substance when the test occurred. In fifty trials, ten with
each individual, there were forty-seven reactions as described
above. One failure to respond was noted, and two cases where the
proboscis was extended but locomotion continued. ‘The animals
scented the juice, therefore, forty-nine times in fifty trials.
To sea water and carmine the snails usually responded, if at
all, by twitching or coiling slightly the tips of their tentacles
without cessation of locomotion. With the exception of one
doubtful case, the proboscis was protruded but three times in
the fifty trials, and then without the animal stopping as it
characteristically did when stimulated with fish juice.
Another method for comparing the effect of a pure tactile
stimulus with that of one accompanied by a chemical stimulus was
as follows: A small piece of cotton was rolled into a ball and
placed in the open end of a pipette. Some filtered Fundulus
extract was then put into the pipette back of the cotton. By
exerting slight pressure on the bulb it was possible to flood
the cotton with juice, which then could be applied locally to
any part of the snail’s body. JUMPROG LIKCUNO) Neo ye Pro aeie abc o Bodao De OD COOaC cnt CPA O ESO co IoC e Ger mOre 229
likeli xternaleappearan cen... verse ere ence racine Gieicionieis Sc eke aes es elles 230
OTIC GL ON yes TC PoE Corea ste miso late Seis ee eis Seas 230
Dee CC OVOTE SL 5 eben RO ae ec hide aeitis Fae aie ote ve 232
3), IM oIMIMPR AKON’ LSE Se ercoctS Hood bbe buced Sma at ers SUED apoE Cab eece 234
ASP Ain phi pod eCOMMVeEn salle everett ey aes n ears even yar oleseot ax oockeh al olen oie 237
ELE Hood and energy supplys- ca. cerreetr sar eters ns ate wave sche ie's state w Slay vietelera 237
LER WiateTACUrrenit crite vat cree eee Te ee eee eT ee ene hal or leverer 238
FAS VOLE HIV EYE Bye Soe Reon bho bts GAs GiB eR ERS CRI GAO backs Bieta 6 Eten es Ee 240
IWarEheemovementsy osc crditas see rer vaio creo okeke ier e ede okey e a aieke ces lets 243
is Biola TerbaOlOSUas esndsens dogccns Uda pbobe Aeon Coes coe oe Coe damone 243
Dees OMANI VEMEMNLG sree crn tarrme: Someries ckarsrocrarste cic ce ete eneyet a eucte 248
SR OPOMUANCOUS HIN OVEIMEN Use aerate s. Muet. eter eee EN eee eiees = 253
Vip Suan nvpyrr artes 2.043 fer PHN ees aes ak edge bigtamsoersy: Feqaeeeias 257
Vile {Bil lyo grap liye en teara an ne oe area oe cue Disb Se wiee Basisc ic + cehieesiseit ers 258
I. INTRODUCTION
Ascidia atra is a large tunicate common in Bermuda and the
West Indies. It was first described from Guadaloupe in 1823
by Lesueur. Many years later it was found in Bermuda by the
Challenger Expedition, and figured by Herdman (’82), who,
however, confused it with the European form Ascidia nigra Sav.
The distinction between the two is that the European species
possesses intermediate papillae on the longitudinal bars of the
branchial sac, while the American species does not (VanName,
02).
! Contributions from the Zodlogical Laboratory of the Museum of Compara-
tive Zodlogy at Harvard College, No. 303, and contributions from the Bermuda
Biological Station for Research, No. 78.
229
230 SELIG HECHT
Ascidia atra lives attached to rocks, in most cases well under
low water. The attachment of the larger animals is by the
posterior edge; in the smaller individuals a portion of the left
side is frequently attached as well. The species is well distributed
throughout the Bermuda Islands. Although animals for this
research were obtained from many regions, the main supply
came from localities very near Agar’s Island.
Ascidians first became of interest during the last half of the
nineteenth century. Their significance in relation to the origin
of vertebrates, which was first made apparent by the work of
Kowalevsky (’67), resulted in innumerable researches on the
anatomy and embryology of the group. Since that burst of
activity, sixty years ago, the knowledge of ascidians has not
kept pace with the newer points-of-view. As a consequence,
little, indeed, is known of the life and activities of these animals.
It is with the hope of supplying this deficiency that the present
series of papers is presented.
The work was done at the Bermuda Biological Station with
the assistance of a grant from the Humboldt Fund. I wish to
acknowledge the kindness of Prof. E. L. Mark, who made this
grant possible, and at whose invitation the experiments were
performed in Bermuda. To the Resident Naturalist, Dr. W. J.
Crozier, the depth of whose friendship was manifest in many
ways, I extend my frank admiration and thanks.
My chief indebtedness, however, is to Prof. G. H. Parker.
His teachings and researches, which have the “rare merit of
combining both anatomical and physiological view-points,”’
have influenced my work and thought. It is a privilege to
express my gratitude for the inspiration which he has given me.
Il. EXTERNAL APPEARANCE
1. Orientation
At the outset of this account of the physiology of Ascidia
atra, it is necessary to define the various surfaces and planes of
the body. The earlier writers on ascidian anatomy were far
from agreed on the application of such terms as anterior, poste-
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR Zot
rior, dorsal, and ventral. As a result, however, of the embryo-
logic investigations of his time, Kupffer (75) applied these desig-
nations correctly. The nomenclature suggested by him has been
accepted by all of the later workers (Herdman, ’82), and will be
used in the description of the present species.
Fig. 1 Medium sized specimen of Ascidia atra, life size, showing view of right
side.
The large opening of the animal is within the rim of the oral
siphon, and the smaller one is within that of the atrial siphon.
These two openings are at the anterior end, the oral being to-
ward the ventral edge, and the atrial toward the dorsal edge.
The place of attachment is at the end opposite the siphons, and
constitutes the posterior part (fig. 1). Consequently, the side
of the animal which shows many creases and irregularities in
the test is the right side, and the smooth face forms the left
side.
DSP, SELIG HECHT
According to this scheme, the pharynx, or branchial sac, of
Ascidia covers the right side almost completely, whereas the
renal body and the intestine lie on the left side. The atrial
cavity extends along the dorsal edge and terminates anteriorly
in the atrial siphon. Quite unusually for this genus, the heart
lies on the right side along the ventral edge.
2. Color
On first sight Ascidia atra appears to be of a dead black color.
Closer examination shows that it is really a very deep blue. The
color is located in the test, on the inner surfaces of the siphons,
and on the outer faces of the oral tentacles; it may even extend
beyond the tentacles into the anterior part of the branchial
sac. In sections of the animal, the blue pigment is shown as a
thin rim marking the outer edge of the test.
The coloring matter is insoluble in water. Acetone extracts
of the test are reddish in appearance, and do not resemble the
opaque purplish blue seen in sections of the test. The extract
has the properties of an indicator; it is red with acids and green
with alkalies, the color change occurring near the neutral point
(Crozier, 716).
The blue pigment is contained in spherical granules, which
are nearly all of the same size: approximately 3 micra in di-
ameter. They occur in very compact groups of four to six.
Although each group may represent a cell containing the pig-
ment granules, it is difficult to make out any cell substance or
cell boundaries in thin sections of fresh and fixed tissue. There-
fore, it seems improbable that the granules as they are found in
the test are within the living substance of a pigment cell.
The groupings may, however, represent the remains of meta-
morphosed cells whose cytoplasm has disintegrated. On the
basis of such an idea, there should be present in the body of
Ascidia some living cells which would be the precursors of the
pigment groups; and, moreover, it should be possible to find
intermediate stages between the two.
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR Dae
It was not difficult to satisfy the first of these requirements.
The blood contains cells whose volume is the same as that
occupied by the compact group of pigment bodies. In addition,
these blood cells are packed full of spherical granules whose size
and number correspond to the pigment granules in the test.
Most of the blood cells are of a rich green appearance, the color
being resident in the granules. There are also to be found, in
much less abundance, similar blood cells whose granules instead
of being a transparent green, are an opaque, dark blue, which
to all appearances is identical with the color of the test. It was
possible to observe the change from green to blue in individual
cells in drawn blood under the microscope. I considered it,
therefore, extremely likely that the blue blood cells, representing
the later stages of the green cells, are the forerunners of the groups
of pigment granules in the test.
In order to prove this satisfactorily, it was necessary to find
a stage between the free, blue cell and the group of pigment gran-
ules imbedded in the cellulose of the test. Examination of
sections of the test brought out only a few, and these doubtful,
instances, indicating that pigment deposition in an adult Ascidia
is probably not a very active process.
The evidence came when it was found that an animal would
regenerate its test. An individual denuded of a portion of tbe
test began almost at once to secrete a new one. At the end of
one day, a thin layer of cellulose of the characteristic color had
been formed over the denuded portion. When this delicate
layer was removed and examined with the microscope, there
were found hundreds of definitely shaped, blue blood cells
imbedded in the cellulose, imparting to it the usual color of the
test. Asa result of this, it seems safe to conclude that the blue
pigment granules in the test of Ascidia are the remains of the
metamorphosed green cells of the blood.
In this connection the observations of Caullery (95) are of
interest. Botrylloides cyanescens, which in nature is yellowish
green, turns blue after remaining in the laboratory. Caullery
found that the green color was due to cells which contained a
number of colored granules, and that the blue appearance in
234 SELIG HECHT
captivity was the result of the change of these granules to a
deep blue. The figures which he gives (Caullery, ’95, fig. 52)
for these cells resemble the blood cells of Ascidia and of ascidians
in general (Cuénot, 91). The color change in Botrylloides is
artificial; in Ascidia atra it is in the regular course of events.
3. Formation of the test
Freshly collected specimens of A. atra, as well as animals in
their natural surroundings, possessed a bright and clean appear-
ance, which was often lost in the laboratory in a short time. In
confinement, the outer surface of the test soon changed to a dull
gray. The gray material was gradually sloughed, coming off
in shreds, and resembling a human skin peeling after a sunburn.
Although the layers were removed by the movement of the water,
more appeared in a short time, and the animals continued to
shed the outer portion of the test as long as they remained in the
laboratory.
The animals were kept in battery jars of about ten liters
capacity, into which the seawater flowed in a gentle stream.
Under such circumstances, the water surrounding the animals
had but little motion. This is quite in contrast to the compara-
tively turbulent conditions to which the species is normally
subjected. Consequently, it seemed probable that the ap-
pearance of the test was merely a superficial laboratory product,
and not due to any real effect on the animal. Indeed, individuals
kept in smaller jars, in which a more vigorous current was
present, showed little sign of this surface change. The sloughing,
therefore, merely indicates that Ascidia renews its test continu-
ally by secreting fresh test material on the inside, and allowing
the outside surface to disintegrate and to be removed by the
action of the waves.
This conclusion is strengthened by the phenomena which
attend the regeneration of the test. Occasionally animals were
collected which showed an appearance that can be interpreted
only as a regeneration of the test and perhaps of other structures
(Hirschler 14). Figure 2 is a sketch of such an individual.
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR Zoe
The test shows a ragged surface undoubtedly representing the
places where it had been torn. Such a test is highly instructive.
In cross section (fig. 2) it can be seen that regeneration had not
taken place by the mending of the injured edges, but by the
growth of the new test around the body tissue. It does not
seem as if the injured portion had been specially reconstructed ;
but rather as if the test material had been secreted by the sur-
face generally, and only incidentally covered the injured part.
These appearafices may be duplicated experimentally. Asci-
dians present varying degrees of ability to regenerate the test.
Some, like Cynthia and Phallusia, seem incapable of surviving
Fig. 2 Sketch of regenerated animal. The transverse section of the test
shows the mode of regeneration.
even partial removal of it (Fol, 08). Ascidia atra, however,
not only recovers rapidly after portions of the test have been
removed, but it makes good such deficiences in a short time.
It has been described how, within a day after the removal
of a section of the test, there has already been secreted a thin
layer of cellulose. This film is continuous with the innermost
surface of the uninjured test, and adheres closely to the soft
tissue which has formed it. Animals in this condition, when
placed in a sheltered position in the natural environment of the
species, continue to thicken the film until it assumes the ordinary
dimension of the test. In one animal, for example, a hole several
centimeters square was mended in two weeks. Sections of such
tests are identical with those of animals like the one in figure 2.
236 SELIG HECHT
The edges of the old test gape at the injured region, and the new
test which is formed underneath them is continuous with the
old test where the two meet close to the mantle tissue of the
animal.
It was impossible to secure complete regeneration in an animal
whose entire test had been removed. ‘This was due solely to a
deficiency in technic, and not to an incapacity on the part of the
animal. The seawater supplied to the laboratory contains
almost no plankton organisms, and since thesé furnish the food
supply of Ascidia, even perfectly normal animals died after a
week or more of laboratory confinement. I was unable to devise
a method of keeping animals without a test out in the open water,
because they could not be attached to anything. Consequently
it was possible to observe them only for a few days in the labora-
tory. Here they showed the usual beginnings of regeneration.
After a day, a thin layer of test material was formed on the right
side of the body and on the entire surface of the siphons, but
not on the surface of the renal body. Soon the cellulose began
to extend over to the left side, making the bare region smaller
and smaller. Undoubtedly, under better conditions, a complete
test would have been regenerated.
The newly formed test material is pigmented in the usual way.
The pigmentation is not dependent on the presence of light.
Animals whose tests had been removed at night, were kept in
complete darkness for several days. They regenerated as usual,
and the fresh test contained the blue pigment. Moreover, a
new, pigmented test will form on the right face of the animal
under the intact, opaque, old one, when the latter has been
accidentally separated from the ectodermal surface which secretes
it. Therefore the formation of a pigmented test is not the result
of a photic stimulus only.
These regeneration experiments, as well as the phenomenon
of sloughing, indicate that normally there is a continuous addi-
tion to the thickness of the test, in order to compensate for the
disintegration of the exterior, and for the changing size of the
animal.
PHYSIOLOGY OF ASCIDIA ATRA LESUBUR ae
“
4. Amphipod commensal
One of the striking things associated with the external ap-
pearance of Ascidia is the frequent presence of the young of a
species of Orchestia within the cavity of the oral siphon. The
occurrence of crustacean messmates in ascidians has long been
the subject of comment, and many species of copepods have been
described for ascidians all over the world (Scott, 07). However,
with the probable exception of Verril’s (’70) report of an amphipod
in the ‘interior’ of Ascidia callosa, I have found no previous record
of the free association of an amphipod in the branchial cavity
of an ascidian.
The species in A. atra is a pretty animal varying in size from
two millimeters to nearly a centimeter. It possesses bright red
eyes and a dark band across the middle of the back, both struc-
tures showing conspicuously against the whiteness of the body.
In the oral cavity of an Ascidia which has not been disturbed
for a time, the amphipods are arranged near the rim of the siphon
with the anterior end facing outward. Frequently as many as
ten may be found in this position in a single siphon. It is a
startling sight, when the blackness of the interior of the siphon
is illumined, to see the brilliant red eyes of the creatures arranged
in a circle a few millimeters within the cavity.
The amphipods are capable of rapid locomotion when forced
to leave their host, and may perhaps be free living at times.
Their position in the oral siphon of Ascidia, however, is of dis-
tinct advantage to them. The water current entering the oral
siphon brings with it a host of small organisms to serve as food
for Ascidia. The amphipods share this with their host, and,
therefore, furnish an example of real commensalism.
III. FOOD AND ENERGY SUPPLY
The only source of metabolic and growth materials which is
available to Ascidia is the surrounding seawater with its sus-
pended and dissolved content. In order to utilize this supply,
the animals perform certain activities whose function it is to
furnish quantities of fresh seawater continuously, and to remove
238 SELIG HECHT
therefrom the substances necessary for the existence of the spe-
cies. Both of these processes are accomplished in the branchial
sac. ‘The enormous development of this structure testifies to
the necessity of working on a large scale in order to abstract
the relatively meager proportion of food and energy contained in
the seawater.
1. Water current
A study of the water current (Hecht, 716) has already shown
that this form of activity has the following properties. The
current is produced by the cilia of the branchial sac. It is
maintained under a low pressure of 1.7 mm. of seawater. The
quantities of water moved are large; in a medium sized individual,
173 liters of seawater are transported in a day. The volume of
water moved per unit body weight varies inversely as the size
of the animal.
Since the water enters by way of the oral siphon, and leaves
through the atrial siphon, it is of primary importance to Ascidia
to avoid a mixing of the incoming and outgoing currents. In the
open water the movements of the sea undoubtedly change the
water immediately surrounding an individual, so that a fresh
supply of seawater is frequently available. Ascidia, however,
does not rely on such a chance renewal of its food and energy
supply, because even in very quiet water, such as that in a large
dish in the laboratory, the two water currents are definitely iso-
lated from each other.
If, in such a dish, particles of carmine are floated near the
atrial and oral siphons, it is at once apparent that the outgoing
current is considerably stronger than the incoming current.
Figure 3 shows a drawing of a small specimen of Ascidia life size.
The arrows near the oral siphon indicate the range of its activity,
that is, the distance from the opening within which a particle
of carmine was sucked into the cavity. For this specimen the
distance was at most 5 millimeters. The arrow pointing away
from the atrial siphon represents the distance within which a
particle was deflected by the outgoing current. This range was
ca. 65 millimeters. Other individuals showed similar relations
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR 239
for the range of activity of the two currents. In columns 2 and
3 of table 1 are given the values obtained for two addition&l
animals. Record VI.20.3 is of an individual about twice, and
VI.22.1 of an individual about four times, the size of the speci-
men in figure 3. g
202
Fig. 3 Range of the incoming and outgoing streams of the water current.
‘
The maintenance of such a contrast in the force of the two
branches of the water current is accomplished by a difference
in the size of the openings of the oral and atrial siphons. The
cilia furnish the motive power, and the same quantity of water
which they move passes through both openings in the same time.
240 SELIG HECHT
TABLE 1
RANGE DIAMETER ANGLE
ANIMAL BETWEEN
Incoming Outgoing Oral Atrial EOIN
mm. mm. mm. mm.
ViAE2082 5 65 4.0 220 43°
V1I.20.3 HZ, 110 ono 3:5 65°
WALPPAI 15 230 13.0 6.0 35°
The oral orifice is large, whereas the atrial is small. Therefore,
the velocity, and consequently the momentum, of the water in
the atrial current is greater than in the oral current. The di-
ameters of the siphon rims of the three animals mentioned are
given in columns 4 and 5 of table 1. The figures are not very
accurate, because of the difficulty of maintaining the living animal
in a constant state of expansion. They show unmistakably,
however, that the difference in the force of the two currents
depends, in the main, on the size of the siphon orifices.
A second significant factor concerned with the separation of
the two currents is the angle formed by the diverging axes of
the two expanded siphons. In the last column of table 1,
this angle is recorded for the same three animals. The individual
variation in the extent of the divergence of the siphons is sur-
prising; the net result, however, is that the currents are prevented
from mixing. The combination of a difference in range with a
difference in direction of the two streams of the water current
makes Ascidia independent of the fortuitous movements of the
surrounding sea.
2. Feeding
The stream of seawater which passes through the branchial
sac of Ascidia brings with it a supply of solid food in the shape
of plankton organisms. The exact method which is used in the
collection of these organisms has been the subject of conflicting
statements.
Earlier writers, such as Roule (’84), described it as follows.
The mucus secreted by the endostyle is spread over the inside
of the branchial sac by the ciliary activity of the gill bars. This
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR 241
mucus catches the food particles which come in with the water,
and the mixture of food and mucus is transported across the
face of the branchial sac, dorsally and posteriorly into the
oesophagus. In recent textbooks (Herdman, ’99) the process
is described in a totally different way, somewhat like the fol-
lowing. The mucus from the endostyle passes anteriorly to the
peripharyngeal grooves. Here the food particles are caught at
their very entrance to the branchial sac, and carried by the
mucus on its way along the dorsal lamina to the oesophagus.
Delage et Herouard (’98, p. 144) point out the differences in
these descriptions, but cautiously avoid anything but a general-
ized account of feeding.
The matter has been recently investigated on many transparent
ascidians by Orton (713), who has proved very clearly that the
earlier accounts are correct. I have examined the process of food
collection in Ascidia and in the transparent Ecteinascidia tur-
binata, and my observations are in complete agreement with those
of Orton. Occasionally specimens of A. atra are found which
are quite translucent. By feeding carmine to such animals, it
is possible to see the red band of mucus-entangled material
swept along the branchial sac, upward and backward into the
oesophagus.
The mechanism by means of which the transportation is
accomplished deserves a closer scrutiny. At regular intervais
along the junctions of the transverse and longitudinal vessels
of the branchial sac, there are present small papillae which pro-
ject into the cavity of the sac. A papilla is really the wall of
a blood sinus, and in A. atra its ventral wall is composed of a
ciliated epithelium (fig. 4, A). At its junction with the inter-
secting vessels, I have always found a flat semicircle of what
seems to be smooth muscle cells (fig. 4, B). The location of
the ciliary surface of the papilla and the muscle at its base are
intimately concerned with the collection and movement of the
food.
By removing a part of the test and branchial sac, it is easy
to observe with a binocular microscope the function of the
papillae. Food particles which are filtered by the meshes of
THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 25, No. 1
242 SELIG HECHT
the branchial sac are rapidly lashed to the tips of the papillae
by their ventral cilia. Here they are caught by the mucus, and
incorporated into the thread of food which is passing across the
branchial sac. This cord of mucus and food is transported by
the papillae. Waves of contraction bring two rows of papillae
together, and by the action of the cilia the food cord is passed
from one row to the next, until it reaches the oesophagus.
Fig. 4 Papilla of the branchial sac. A, median section; B, section at the base
of the papilla.
The mechanism for these papillary movements is probably
local, because touching a papilla with a glass rod causes a con-
traction to appear. This would indicate that the waves are the
result of a series of stimulations of the papillae by the contact
of the food mass.
The food as it enters the oesophagus is in the shape of a cord,
and in this manner it is passed along the digestive tube. With
the food also goes the mucus. Although the food is digested
and absorbed, the mucus is probably not affected at all. When
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR 243
the feces come out in neat, flat, oblong packets, they are incased
in a thin layer of gelatinous material, which is probably the
mucus. The presence of the gelatinous covering of the feces
is best seen in animals which have been in the laboratory for
some days. In such cases the feces contain but little excrement,
and are composed mainly of a transparent mass of mucus.
The seawater in the laboratory is coarsely filtered, and con-
tains very few organisms. In this way the food supply of Ascidia
is cut off; consequently, it does not live long in confinement.
This shows that the dissolved organic content of seawater, to
which Piitter (’07) has attributed such great importance, is of
little significance in maintaining the metabolic balance of Ascidia.
The species has developed an elaborate mechanism for capturing
the organisms in seawater, and without them it slowly starves to
death.
IV. THE MOVEMENTS OF ASCIDIA
Ascidia atra, though permanently attached to the rock, is
capable of moving not only certain of its structures, but also of
bending and contracting its body in relation to its base of attach-
ment. The siphon rims can close and open, the body can con-
tract along the dorso-ventral axis, and the entire animal can
bend with a surprising degree of vigor. When arranged in
certain combinations and sequence, these activities form the
reflexes with which the animal responds to stimulation. From
such an aspect they will be considered in the description of the
sensory reactions of the species. At present, however, my object
is to present the physiology of these movements in themselves
by examining the factors which are concerned in their production.
1. Stphon rim closure
Ascidia is usually described as possessing eight lobes on the
rim of the oral siphon and six on the atrial. These are shown in
figure 1. All such photographs and descriptions are of dead
animals and tell only a partial truth. In the normal, living
animal under water, these lobes are not shrunken and collapsed,
244 SELIG HECHT
but stand out expanded on the siphon in the form of thin lappets
(fig. 5).
Under special circumstances it is possible to secure a local
contraction of the region near an individual lappet. Ordinarily,
however, the entire siphon rim shuts as a unit. This closure is
Fig. 5 Sketch of living, expanded Ascidia, to show the cheeks on the right
side and the protruding lappets on the siphon rims.
conditioned by the presence of well-defined ridges and folds in
the test, along which the contraction takes place. An end-on view
of a nearly closed oral siphon (fig. 6) shows that the alternation
of folds and ridges depends on a surprisingly accurate pattern,
which involves thick and thin portions of the supporting test.
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR 245
The closing of the siphon rim, however, is more than a mere
puckering together of its edge due to the action of circular muscle
fibers. The rim is not only pulled together, but is also drawn
down toward the body of the siphon. This is due to the action
of longitudinal muscle fibers which, in the.siphon, lie nearer the
cavity of the siphon than do the circular muscles.
The siphon rim in Ascidia is so opaque that it was impossible
actually to observe the action of the two sets of muscles. A
transparent species Ecteinascidia turbinata, furnished the desired
opportunity. Individuals two or three days old, measuring
three to four millimeters in length, can readily be examined
Fig. 6 End-on view of a partially closed oral siphon, showing the geometric
arrangement of the folds in the test. The left side of the body is uppermost.
with the low power of the microscope. These animals show the
two factors of siphon closure beautifully. At first the circular
(sphincter) muscles contract and partially close the rim. This
is followed by a contraction of the longitudinal fibers, which
results in a drawing in of the rim, thereby completing the closure.
In these young individuals I have frequently observed the
longitudinal muscles of the oral siphon contract so vigorously
that the upper portion of the siphon was completely inverted and
tucked into the branchial cavity. In Ascidia this retraction is
provided for by a sudden decrease in the thickness of the test
near the rim (fig. 10). The combined action of the two sets of
muscles results in a closure which is really complete. No trace
of a water current can be demonstrated after the siphons have
been shut.
246 SELIG HECHT
Fig. 7 Apparatus to record the movements of Ascidia.
In order to analyze the movement of the siphon rims, I secured
kymographic records of their activity. The apparatus which
was employed is represented in all essentials in figure 7. The
drawing needs little explanation. The long, light, aluminum
lever was nearly, but not quite, balanced by the weight on the
short arm. ‘This slight excess on the long arm kept the pendant
vertical rod in continual contact with the right edge of the siphon
rim. Such a procedure proved more effective and less disturb-
ing to the animal than actually attaching the lever to the tissue.
In addition the vertical arm was curved so as to fit into the
cavity of the siphon. To keep the animal in place, it was fixed
to a piece of plate glass, which was heavy enough so that even
a vigorous movement of the entire animal did not change its
position.
Fig. 8 Record of siphon rim contraction, and record of body bending. The
base line marks one minute intervals.
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR 247
The smaller contraction in figure 8 represents a siphon rim
movement; all the other records are almost identical with it.
Curve 7 in figure 11 is another example, and represents in addi-
tion an analysis of the movement. The record is divisible into
four distinct phases. The first two are the phases of contraction;
the second two those of recovery.
The first phase is an almost straight line, and represents,
therefore, a comparatively rapid contraction of the siphon rim.
Although of short duration, lasting about a second, this por-
tion of the movement accomplishes nearly three-fourths of the
total contraction. The remaining closure is made at a much
slower rate, as is shown by the amplitude and duration of the
second phase. The condition of maximum contraction is reached
in approximately four seconds. Recovery begins almost im-
mediately, and in the beginning is comparatively rapid. The
third phase of the record may be defined as that portion which
lies between the position of maximum contraction and the point
where the curvature of the line changes so as to be convex to
the time axis. It lasts nearly twice as long as the second phase,
and includes about one-half the recovery of the siphon rim. The
last phase of the movement is the longest, occupying nearly
three-fourths of a minute. At the end of it the siphon rim has
assumed the diameter which it had at the beginning of the
contraction.
An analysis of the time relations of the phases of two separate
records of the same siphon rim is given in the accompanying
table (table 2). The two movements were made under the
same conditions within a few minutes of each other, and were
produced by the same intensity of mechanical stimulus. The
similarity in the resulting records is very evident.
TABLE 2
Siphon rim closure. Exp. VI.23.1
DURATION OF PHASES, SECONDS
1 2 3 4
I ee} 3.4 5.3 36.8
II 1.5 3.4 9.2 | 40.5
248 SELIG HECHT
It will be noticed that the general shape of the curves produced
by the closing and opening of the siphon rims resembles that of
the contraction and recovery of smooth muscle (Winkler, ’98).
The effective agent in the closure is, indeed, the sphincter of
smooth-muscle cells in the siphon working against the elasticity
of the tissues and the test. Although the presence of the test
undoubtedly helps in the opening of the rim, the recovery from
the contracted condition can occur without the test. Animals
Fig. 9 Right side of animal with the test removed. h., heart; /.m., longitudinal
muscles; ¢.m., transverse muscles.
from which the test had been removed were still capable of
closing and opening the siphons. It seems reasonable to suppose
that the elasticity of the tissues which is responsible for this
recovery is due to the presence of spaces filled with blood under
pressure.
2. Body movemenis
The bending of the body on its long axis occurs so that the
right side of the animal always forms the concave surface of the
bend. This is accomplished by the contraction of longitudinal
muscle strands which lie on the body wall of the right side only
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR 249
(fig. 9). In the living animal this side of the body adheres firmly
to the test. The left side, however, which includes the renal
organ, parts of the intestine, etc., is free from the test, the two
being connected only by a single blood vessel. Consequently
any contraction of the long muscle strands on the right side will
result in a bending of the body and test in that direction. This
is facilitated by the fact that the right side of the test is much
thinner than the left (fig. 10).
The bending of the body on its long axis is always associated
with a movement of the siphon rims. This becomes clear when
Fig. 10 Longitudinal section of test, passing through oral siphon.
its kymographic record is examined (fig. 8). The kink in the
very first part of the curve denotes a siphon rim closure. It will
be seen that the movement of the siphon precedes the vigorous
activity of the body as a whole.
The curve made by the bending of the body (fig. 11, curve 6)
‘resembles in all essential features the one made by the siphon
rim contraction (curve 7). It may be divided into four phases,
the durations of which are relatively the same as in the activity
of the siphon. The first phase is short, and accomplishes the
main extent of the contraction. During the longer, second, phase
a slower activity brings about the maximum point in the curve.
The resumption of nearly the normal position of the body is
accomplished in the third phase, whereas the complete relaxation
250 SELIG HECHT
L25/
Curve 6
STW DUTES
Fig. 11 Analysis of records. Curve 7, siphon rim closure; curve 6, body
bending.
takes place slowly during the much longer period of the fourth
phase. The actual time occupied by the different periods is
given in the accompanying table. In table 3 there is represented
an analysis of three records of the movements of one animal
produced under the same conditions and intensity of stimulation.
The appearance and duration of the second and third phases of
the record depend in the main on the intensity of the stimulus
which produces the body contraction. The more intense the
stimulus, the longer do the two phases last, their greater duration
showing itself in the maintenance of the condition of maximum
contraction. Compare, for example, the values in table 3
(also fig. 8) with curve 6 of figure 11. Although both were
TABLE 3
Body Contraction. Exp. VI.28 .1
DURATION OF PHASES, SECONDS
_
bo
oo
cs
—
—_
oan
cow
_—
~J
Co
mm bo
COW
wm > CO
_
—
ie)
on
III
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR 251
obtained from the same individual, curve 6 is the result of a
stimulus twice as intense as those whose records are given in the
table, and consequently, the two phases last considerably longer.
Whereas the actual bending of the body involves merely the
action of the longitudinal muscles, the recovery to the normal
shape depends upon several factors. One of these is the activity
of a set of muscles situated on the left side of the siphons. Al-
though these muscles are not well developed, and extend for a
short distance only, they act probably like extensor muscles,
and tend to antagonize the action of the long muscle strands of
the right side. A second factor is the elasticity of the soft tissue
of the body. That these two agencies alone are capable of
bringing an animal back to normal shape is shown by the com-
plete, though slower, recovery of animals with the test entirely
removed.
The test, however, is a structure of considerable significance
in the resumption of the normal form after the bending of the
animal. Although apparently homogeneous, the cellulose ma-
terial of ascidians has been shown to possess a fibrillar structure
visible in polarized light (Schulze, ’63). Probably this hetero-
eeneity, which is also to be seen in some stained preparations of
A. atra, is of importance for the elastic properties of the test.
Its peculiarities resemble those of a viscous solid. To a sudden
distortion, the test will respond in a manner comparable to most
elastic bodies. If, however, it is subjected to a slow distortion,
it will partially accommodate itself to the new form, and never
return to the original shape.
In the bending of Ascidia the activity is sufficiently rapid to
cause an immediate elastic recoil on the part of the test. Sec-
tions of the body show that the resilience of the test is utilized |
to good advantage (figs. 10 and 12). The left side is noticeably
thicker than the right and, consequently, serves as an elastic
back which antagonizes the muscles of the right side. Although
controlled by the relaxation of these muscles, the elastic rebound
of the left side probably serves in a large measure to straighten
the curved body.
252 SELIG HECHT
The difference in the elastic response of the test to strains of
short and long duration may be the explanation of the distorted
appearance of many laboratory and museum specimens. The
collection and transportation of living animals involve a continued
stimuiation. This results in the maintenance of the curved
condition for a long time, until finally the elastic limit is passed,
and the animal remains permanently abnormal in appearance.
The elasticity of the test is further made use of in the third
type of movement of which Ascidia is capable. This is a con-
traction of the body along its dorso-ventral axis, in such a way
that the right side forms the concavity. The muscles which
are concerned are the transverse fibers on the right side (fig. 9).
In order that they may exert their influence, the test is thinned
&
a c
Fig. 12 Transverse sections of test. a, near tip of oral siphon; b, near base
of oral siphon; c, through middle of body.
out along a narrow line, in the middle of the right side, running
parallel to the long axis. The contraction of the transverse
muscles bends the test along this line, with the consequent forma-
tion of two prominent cheeks (figs. 5 and 12c).
The contraction of the body on its dorso-ventral axis extends
in some cases well into the oral siphon. ‘There are circular mus-
cles present in the siphon, which by their contraction can de-
crease its diameter. Here also the test shows an arrangement
of thick and thin portions on the right side, whereas the left
side still maintains its uniform thickness in order to aid in the
recovery (fig. 12b).
It is consequently obvious that the test is intimately concerned
in the contraction of the body on its short axis. The same is
true for the siphon rim movements and especially for the bending
of the body on its long axis. Definite structures in the test go
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR 253
with definite sets of muscles. Therefore, besides serving as an
excellent covering for the soft internal parts, the test in addi-
tion functions as an exoskeleton, on which depends the proper
execution of the movements of the animals (Fol. ’08).
3. Spontaneous movements
In the continued observation of Ascidia under all sorts of con-
ditions, it became evident that complete movements of the body
and siphons often occurred when no apparent external stimulus
was present. The animal is extremely sensitive to mechanical
stimulation, and at first I was inclined to attribute these aberrant
contractions to very sight movements of my body or of other
people in the laboratory. Such an explanation was, however,
abandoned when the same movements occurred under con-
ditions which precluded this source of stimulation.
Somewhat similar spontaneous contractions have been de-
seribed for the cirri and oral hood of Amphioxus as the result
of the mechanical stimulation of the cirri by the accumulation
of particles of sand (Parker, ’08, p. 481). This explanation does
not hold in the case of Ascidia. When animals which had been
carefully washed were placed in filtered seawater, they continued
to perform spontaneous movements. Moreover, animals with
the test entirely removed and with the greater part of the siphon
cut away, and consequently deprived of their sensory apparatus,
still exhibited frequent contractions. The factors for their produc-
tion, therefore, rest within the organism itself.
The solution of the difficulty came when a record was kept of
the appearances of the spontaneous contractions under con-
ditions which excluded external stimulation. Several animals
were placed in individual battery jars containing about five
liters of filtered seawater. The jars rested on a heavy table
placed on the concrete floor of an isolated house built directly
on the rock of Agar’s Island. The animals were observed con-
tinuously for an hour, and the time of each spontaneous con-
traction was noted. Figure 13 gives a graphic account of two
such animals. It is very evident that there is a rhythmic
occurrence of the spontaneous movements.
254 SELIG HECHT
By means of the apparatus which has been previously de-
seribed (fig. 7), kymograph records were made of this rhyth-
micity. Animals were allowed to register their activity at times
of the day and night when Agar’s Island was deserted except
for the presence of two people in a house more than a hundred
yards from the laboratory. The curves in figures 14 and 15
show the movements of two animals which are entirely typical
of all the others. There can, therefore, be no doubt of the
- rhythmic character of the spontaneous contractions exhibited
by Ascidia.
The rhythmicity of the movements possesses a peculiarity
which resemble the refractory properties of the vertebrate
heart (Woodworth, ’02). It is well known that immediately
after a contraction of the ventricle, it fails to respond to stimula-
tion. After this refractory period, an external stimulus will
cause a pulsation even before the expected rhythmic contraction
is due. Similarly in Ascidia a stimulus which is so slight that
it causes merely a siphon rim movement will, when applied
regularly at intervals of a minute, call forth complete body
movements at approximately the periods when they are expected
to occur rhythmically. The following record is chosen as an
example because the spontaneous contractions of this animal
have already been recorded (figs. 13 and 14).
Exp. VI.27.3. Animal in a nine liter battery jar. Stimulated every minute
by the impact against the jar of a pendulum bob swinging from a distance of five
centimeters.
12:21 Siphon rim movement
22 “ “cc “ec
93 “cc “ce ““
94 “ce iT9 “ce
25 Complete body movement
26 Siphon rim movement
2h “ “cc “
28 “ “ “
29 Complete body movement
30 Siphon rim movement
31 “ce “ec “cc
32 “cc “ “
33 Complete body movement
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR 255
I-30 11-40 W-50 12.00 12.10 12:20 12:30
Fig. 13 Graphic record of occurrence of spontaneous movements.
Comparison with the other records (figs. 18 and 14) shows
that the complete body movements occur at intervals similar
to their rhythmic appearance spontaneously. The significance
of this type of experiment I believe to be as follows. After the
Fig. 14 Records of rhythmic, spontaneous movements. The base line
marks five minute intervals.
ls EN a a Ds a OU ed a a SG la Se Be Re aE i a TA INS Fe eH
24.) C WRA/A
Fig. 15. Records of rhythmic, spontaneous movements. The base line
marks five minute intervals.
256 SELIG HECHT
performance of a rhythmic contraction, Ascidia passes through
a period during which a certain strength of stimulus fails to call
forth a movement of the body. However, as this refractory
period passes by and the time approaches for the culmination
of the next rhythmic movement, this same sub-liminal stimulus
will call forth not only the usual siphon contraction, but also the
body contraction in advance of its scheduled time.
The function of these rhythmic movements is by no means
clear. Their accomplishment of a partial discharge of the water
in the branchial sac would be intelligible if something else be-
sides the water were also expelled. Ascidia, however, possesses
an effective mechanism for avoiding just such a necessity. The
tentacles in the oral siphon screen out all but the smallest particles
which come in with the water current. Everything that passes
beyond them is incorporated into the food cord in the branchial
sac. The particles which are large enough to touch the tentacles,
set off a reaction that drives the water and the particles out of
the cavity of the siphon. Substances in suspension either get
into the branchial sac and stay there, or they are forced out at
once. Therefore, the function commonly attributed to rhythmic
movements in other animals (Redfield, ’17) cannot apply in the
case of Ascidia atra.
To call the movements a respiratory rhythm would also fail
to explain their existence. Ascidia has a highly efficient respira-
tory mechanism which moves large quantities of seawater. The
renewal of the contents of the branchial sac by the rhythmic
discharges would be of no significance compared to the continuous
stream of water produced by the cilia. The relative infrequency
of the discharge would also argue against a respiratory rhythm.
The spontaneous movements in Ascidia are not an isolated
instance. I have observed them in a colonial species, Ectein-
ascidia turbinata, the individuals of which are about two centi-
meters long and quite light in weight. When the animals are
attached, the effect of the body contraction is solely to discharge
the water. If, however, an individual is removed from its attach-
ment and placed in a large jar of seawater, it Jerks itself along
the bottom in a manner that vividly recalls the behavior of Salpa
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR Zot
under similar conditions. The frequency with which Salpa
pulsates is several hundred times as great as those with which
Ascidia and Ecteinascidia perform their spontaneous contractions.
These facts as well as the apparent lack of function of the
rhythmic movements have led me to suggest that perhaps the
rhythm is the degenerate remains of a once vigorous activity.
The ascidians are generally supposed to have originated from the
free swimming appendicularians. These possess no tentacles,
and most probably the earliest ascidians did not possess them
either. It is, therefore, entirely intelligible that the rhythmic
discharge of the water from the branchial sac of these ancestral
ascidians was of considerable value as a cleansing process. More-
over, the salpas are derived from the early ascidians. With
their specialization for a pelagic existence, the rhythmic move-
ments were developed into a mechanism for respiration, feeding
and locomotion. The stem line of ascidians, however, soon
developed tentacles, and the rhythmic discharge of the bran-
chial sac contents, therefore, decreased in importance. The
frequency with which it occurred probably also decreased. On
the basis of this hypothesis, there exist at present two divergent
lines of development of the spontaneous movements. One of
these constitutes the salpas, whose frequency of contraction is
several hundred times greater than that of the ascidians, which
constitute the other line.
V. SUMMARY
1. The blue-black color of Ascidia is: due to the presence of
spherical pigment granules, which are the metamorphosed re-
mains of the green blood cells. Before becoming imbedded in
the test, the green cells turn blue, and may be found as such in
the blood stream.
2. Ascidia is capable of regenerating its test. The process of
regeneration and the normal sloughing of the test show that there
is a continuous secretion of material on to the inner face of the
test.
3. A species of amphipod lives commensally in the branchial
sac of Ascidia.
THE JOURNAL OF EXPERIMENTAL ZOOLOGY, VOL. 25, No. 1
258 SELIG HECHT
4. Ascidia maintains a seawater current of large volume and
low pressure. The volume of the water moved per unit weight
decreases as the size of the animal increases. The difference in
intensity and direction between the incoming and outgoing
currents enables Ascidia to secure a continuous supply of fresh
seawater independent of the movements of the sea.
5. Food collection is accomplished in the branchial sae with
the aid of the papillae and the mucus secreted by the endostyle.
The food particles are transported in a mass across the face of
the branchial sac, dorsally and posteriorly into the oesophagus.
6. Ascidia cannot survive solely on the dissolved organic
contents of the seawater. It must be furnished the suspended
contents as well.
7. The species is capable of three kinds of movement: the
siphon rims can close and open, the body can contract along the
dorso-ventral axis, and the entire animal can bend toward its
base of attachment. The movements are accomplished by
several sets of smooth muscle, which depend for their proper
action on the function of the test as an exoskeleton.
8. Ascidia performs rhythmic movements to discharge the
contents of the branchial sac. No function can be ascribed to
this rhythmic occurrence, and a suggestion is made that it may
represent the degenerate remains of an activity homologous with
the rhythmic pulsation of the salpas.
VI. BIBLIOGRAPHY
Cauutery, M. 1895 Contributions a l’étude des ascidies composées. Bull.
sci. France et Belg., T. 27, p. 1-158.
Crozier, W. J. 1916 Some indicators from animal tissues. Jour. Biol. Chem.,
vol. 24, p. 443-445.
Cuénort, L. 1891 Etudes sur le sang et les glandes lymphatiques dans la série
animale. Arch. Zool. Expér., Sér. 2, T. 9, p. 138-90.
Detace, Y., ET Hrovarp, E. 1898 Les Procordés. Traite de Zoologie
concréte, T. 8, 379 pp.
For, A. 1908 Note sur la régénération de la tunique chez les Tuniciers. Bull.
Soc. Zool. France, T. 33, p. 79-81.
Hecut, 8. 1916 The water current produced by Ascidia atra Lesueur. Jour.
Exp. Zodl., vol. 20, p. 429-434.
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR 259
Hrerpman, W. A. 1882 Report on the Tunicata collected during the voyage
of H. M. 8. Challenger during the years 1873-76. Part I. Ascidiae
simplices, 293 pp.
1899 Ascidia. Liverpool Mar. Biol. Committee Memoirs, I. Liver-
pool, 52 pp.
Hirscuuer, J. 1914 Ueber die Restitutions- und Involutionsvorginge bei
operierten Exemplaren von Ciona intestinalis Flem. nebst Bemer-
kungen ueber den Wert des Negativen fiir das Potenzproblem. Arch.
mikr. Anat., Bd. 85, p. 205-227.
Kowatevsky, A. O. 1867 Entwickelungsgeschichte der einfachen Ascidien.
Mem. Acad. Sci. St. Pétersb., T. 10, 19 pp.
Kuprrer, C. 1875 Tunicata. Jahresb. Comm. wiss. Untersuchung deutschen
Meere in Kiel., Bde. 2 u. 3, p. 197-228.
Lesueur, C. A. 1823 Descriptions of several new species of Ascidia. Jour.
Acad. Nat. Sei. Phila., vol. 3, pt. 1, p. 2-8.
Orton, J. H. 1913 The ciliary mechanisms on the gill, and the mode of feeding
in Amphioxus, Ascidians, and Solenomya togata. Jour. Mar. Biol.
Assoc., N.S8., vol. 10, p. 19-49.
Parker, G. H. 1908 The sensory reactions of Amphioxus. Proc. Amer.
Acad. Arts. Sci., vol. 43, p. 415-455.
Pirrer, A. 1907 Die Ernihrung der Wassertiere. Zeit. allg. Physiol., Bd. 7,
p. 283-320.
RepFieLp, E. 8. P. 1917 The rhythmic contractions in the mantle of Lamelli-
branchs. Jour. Exp. Zodél., vol. 22, p. 231-239.
Rovtez, L. 1884 Récherches sur les Ascidies simples de cétes de Provence.
Ann. Mus. Hist. nat. Marseille., Zool. T. 2, p. 1-270.
Scuutze, F. E. 1863 Ueber die Struktur des Tunikatenmantels und sein
Verhalten im polarisierten Lichte. Zeit. wiss. Zool., Bd. 12, p. 175-
188.
Scorr, T. 1907 Observations on some copepoda that live-as messmates or
commensals with ascidians. Trans. Edinb. Field Nat. and Micr.
Soe. vol. 5, p. 357-372.
Van Name, W.G. 1902 The Ascidians of the Bermuda Islands. Trans. Conn.
Acad. Arts Sci., vol. 11, p. 325-412.
VerRRILL, A. E. 1870 Parasites of Ascidians. Amer. Nat., vol. 3, p. 383.
Winker, H. 1898 Kin Beitrag zur Physiologie der glatten Muskeln. Arch.
ges. Physiol., Bd. 71, p. 357-398.
Woopworti, R.S. 1902 Maximal contraction, staircase contraction, refractory
period, and compensatory pause of the heart. Amer. Jour. Physiol.,
vol. 8, p. 2138-249.
AUTHOR'S ABSTRACT OF THIS PAPER ISSUHD BY
THE BIBLIOGRAPHIC SERVICE, DECEMBER 22
THE PHYSIOLOGY OF ASCIDIA ATRA LESUEUR!
II. SENSORY PHYSIOLOGY
SELIG HECHT
TWO FIGURES
CONTENTS
I. The responses of Ascidia and their nervous relations.................. 261
i Descripbion Ol TEACtiONn sn Jo5s onan sae oo Re sich e wale wast cue bee 262
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I. THE RESPONSES OF ASCIDIA AND THEIR NERVOUS RELATIONS
The assumption of a sessile mode of life involves a sacrifice
in the number and kinds of responses of which an animal is
capable. The comparatively few reactions exhibited by the
sessile tunicates are undoubtedly accountable for the almost
complete absence of our knowledge of their sensory physiology.
The common European ascidian, Ciona intestinalis, is the only
one in which anything is known of the behavior under stimulation.
‘Contributions from the Bermuda Biological Station for Research, No. 79,
and contributions from the Zodlogical Laboratory of the Museum of Compara-
tive Zodlogy at Harvard College, No. 304.
261
262 SELIG HECHT
Even here, however, the data are meager and scattered, and con-
sist largely of incidental observations. This lack of knowledge
and the abundant presence of Ascidia atra at Bermuda served
as incentives for the following investigation of the sensory re-
actions of this species.
1. Description of reactions
Jordan (’07) on the basis of his observations has called the
ascidian Ciona, an animal poorinreflexes (reflexarmes Tier). With-
out subscribing to any of his theoretical generalizations, which
Baglioni (713) has justly criticized, I have no hesitation in simi-
larly describing the behavior of Ascidia atra. Tests with a
variety of conditions of stimulation have revealed very definite
activities, by means of which the animals respond to changes
in the environment. The number of these activities, however,
is small.
The structures and movements which are involved in their
execution have already been described (Hecht, 717). It remains
to explain their relation to one another and to the source of
stimulation. The presence of the open siphons and of the water
current makes it possible for Ascidia to receive indica'tions of
changes of the environment not only on its exterior, but also on
its interior surfaces. This distinction is of fundamental im-
portance, because the place of reception of the stimulus deter-
mines the kind of movement which the animal executes. As a
result there are manifested two qualitatively distinct groups of
reactions. Each of the groups consists of three responses, which
involve the use of different combinations of muscles.
The group of direct responses depends for its origin on a source
of stimulation which affects the external surface of the animal.
This group of responses is concerned mainly with mechanical
stimuli. Although the three reactions included under this head
result from different intensities of the same outside disturbance,
the reactions themselves involve an activity of different muscles,
and not a different degree of activity of the same effectors.
1) If the test of Ascidia be touched very lightly, the siphon
nearer the point of stimulation will contract. The extent of the
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR 263
resulting closure depends on the intensity of the stimulus and on
its distance from the siphon. After a short interval the siphon
rim opens and the animal is normal again.
2) If, however, the stimulus has been stronger, not only does
the siphon rim nearer the stimulated area close, but the other
siphon rim also closes. A new set of muscles has been called into
play.
This reaction is to be differentiated from the one in which both
siphons are stimulated, such as when a drop of water is allowed to
fall on the surface of the water in the aquarium. In this case
each siphon is independently stimulated by the same disturbance.
Such a reaction persists when all nervous connections between
the two siphons have been cut (cf. Loeb, ’92 and Magnus, ’02).
It is otherwise with the response which I have described. Nor-
mally, if one siphon is touched so carefully that the animal is
not jarred, both siphons will close provided the proper intensity
of stimulus is used. When, however, the nervous connections
between the two siphons is severed, only the stimulated siphon
rim contracts.
3) In response to an ordinarily vigorous mechanical stimulus,
A. atra reacts by the employment of still an additional set of
effectors, the longitudinal muscles of the body. Not only do
both siphons close, but the body bends on its long axis toward
the right side.
This bending toward a structurely determined side is of signifi-
cance in the ecology of Ascidia. Most individuals of the species
are attached with the body projecting at any angle, but mainly
in a nearly horizontal plane. All such animals which I examined,
were found with the left side of the body uppermost. Conse-
quently, the curving toward the right side results in bringing the
siphons into such a position, that a disturbing body on the outside
will roll off, and one on the inside will fall out.
In the previous work on ascidians the reaction which involves
the bending of the body has been the only one which has received
any adequate attention. It has been generally regarded as the
only reflex of which this group of animals is capable, and there-
264 SELIG HECHT
e
fore called ‘the reflex’ (Loeb, ’02). Jordan (’07) has more appro-
priately called it the protective reflex (Schutzreflex).
The reactions which are comprised in this direct group show
individual variations depending upon the intensity of the stimulus
which sets them off. They can, however, be very definitely
separated from one another when the animal is observed with
any degree of care, or when graphic methods are employed. (See
for example, figure 8 of the first paper of this series: Hecht, ’17.)
All these reactions are to be kept apart from those which are
in the group of crossed responses. The stimuli which result in
the reactions of this second type are all localized on the interior
surfaces of the siphons, of the atrial cavity, and of the branchial
sac. They include changes in the environment not only of a
mechanical nature, but of a thermal, photic, and chemical kind
as well.
Although they may be produced by the same kind of stimulus
varying in intensity, the three reactions included in this group
are, nevertheless, the result of different combinations of effectors.
As in the case of the direct reflexes, they must, therefore, be
sharply distinguished one from another. To make this clearer,
I may refer to the behavior of a human being suddenly exposed to
a bright light. The person will reflexly close his eyes. If, how-
ever, the light be made excessively bright, he will not only close
his eyes but also place his hand over them. The nature of the
stimulus is the same, but the greater intensity of the stimulus
brings forward a new activity superimposed upon the simple eye
closure. The same is true of the following reactions of Ascidia.
1) An exceedingly delicate stimulus on the inside of one siphon
results in a closure of the other siphon. The stimulated siphon
remains wide open, while the sphincter of the other siphon is
called into play. This kind of response can be secured only
under very carefully controlled conditions. The animal must.
not be jarred and the stimulus must be a delicate one. It is
best to use large animals because they are not as sensitive as the
smaller ones.
2) An increase in the intensity of the stimulus produces a re-
action which does more than merely stop the water current; in
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR 265
addition, it brings about a discharge of the water present in the
branchial sac. The stimulated siphon remains open, the other
siphon closes tightly, and the animal contracts vigorously along
its dorso-ventral axis, resulting in a sudden decrease in the ca-
pacity of the respiratory chamber. Occasionally the stimulated
siphon may also contract partially so as to decrease the size of
its opening. This gives the ejected water a greater momentum.
3) The last reaction of this group combines a bending of the
body on its long axis with the movements of the previous response.
This is the usual reaction which A. atra gives under ordinary
conditions of stimulation of its internal surfaces.
The last two reactions probably correspond to what Jordan
(07) has described in Ciona as the ‘Ejektionsreflex’: “Closure
of one siphon, rapid contraction of all muscles, other siphon
(most frequently, but not always, the anal siphon) remaining
open” (’07, p. 98). This description is repeated: by Polimanti
(11), who, however, added nothing to it. Jordan did not study
this reflex at all, but contented himself with the statement that
it serves to throw out foreign bodies, and that the causes for its
appearance are not clear.
In Ascidia there is no doubt about the nature of the stimulus
which will produce any of these three crossed reactions. It is
always a disturbance on the interidr surfaces of the body. I have
observed the same ‘Hjektionsreflex’ in the common Ecteinascidia
turbinata of Bermuda under the same conditions of stimulation
as in Ascidia atra. Jordan’s statement of its function is correct;
it must, however, be broadened to include not only the ejection
of foreign particles, but also the response to any internal irrita-
tion, such as strong light or chemicals.
The point of special significance is the crossed behavior of the
siphon rims. Stimulation of the outside of a siphon causes that
siphon rim to close. Stimulation of the inside of a siphon results
in that siphon remaining open while the other siphon rim con-
tracts. This points to the presence of a complexity of innervation
in ascidians of which there has previously been no suspicion.
The one factor which the six reactions of Ascidia possess in
common is their negative character. A source of stimulation
266 SELIG HECHT
is either excluded by the closing of the entrances to the body, or
it is thrown out by a discharge of water. I have never observed
any positive response to a stimulus in this species. This is not
unexpected from its mode of existence. The animals are entirely
dependent for their supply of energy on what is brought in by the
water current, and they merely exercise a choice by rejecting
anything which acts as a stimulus.
2. Nervous relations
Among higher animals, the tunicates are peculiar in the
concentration of the entire central nervous system into a single
inter-siphonal ganglion. In Ascidia atra, according to Hilton
('13),? this is a roughly cylindrical mass, on one side of which is
to be found a rather unusual neural gland (Metealf, ’00). It
gives off many more nerve trunks than are usually described for
this genus of ascidians. From the oral end there arise three large
nerves, which go to the region of the oral siphon. Several nerves
leave the atrial end, while from the middle of the ganglion there
emerge three large nerves, four smaller ones, and many minute
ones. It is significant that all the nerves contain both afferent
and efferent fibers (Hilton, 713, p. 116).
Practically nothing is known of the nerve endings in ascidians.
The same may bé said of the presence of sense cells. Hilton
describes the fibers of the oral nerves as ending in the oral tenta-
cles, but fails to state whether they form free nerve terminations
or arise from sense cells. Lorleberg (’07), after prolonged in-
vestigation of the nervous system of Styelopsis, concludes that
there is a complete lack of sense cells, but that there are un-
doubted free nerve terminations present.
In relation to the reactions of ascidians, one point Is clear:
the only demonstrated means of direct nervous communication
between the siphons is by way of the ganglion. The ganglion,
however, has more than the mere conducting function supposed
2 This author refers to the species as Tunica nigra. I have it from Professor
Mark that Hilton’s work was done on Ascidia atra. Moreover, his description
of the species as the ‘‘ascidian very abundant on Agar’s Island’’ leaves no doubt
as to its identity.
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR 267
by Loeb (92). Although some of the results of Fréhlich (’03)
on the removal of the ganglion of Ciona have been questioned by
later authors (Jordan ’07, and Kinoshita 710), the combined
work of all the investigators on Ciona proves that ganglion re-
moval affects at least the threshold sensitivity, the tonus, and the
rate of recovery after stimulation.
Ascidia does not remain normal in the laboratory long enough
to permit of a study of the quantitative effects of ganglion re-
moval. I had, therefore, to content myself with a determination
of the qualitative results produced by the mere nervous isolation
of the two siphon regions from each other. This was accom-
plished by means of a rapid incision into the test and mantle so
directed as to result in the severing of the nervous mass into two
parts. The animal recovered from this slight operation in a
few minutes.
The behavior of individuals under such nervous conditions was
very instructive. Of the group of direct reactions, the first
persisted, and seemed, qualitatively at least, to be normal. The
second reaction, that is, the closure of both siphons, disappeared
at once. As long as the whole animal was not jarred, no amount
of contraction of one siphon called forth a similar response of the
other siphon. The reaction involving the body flexure depends
mainly on the bending of the oral siphon. Therefore when
this siphon was stimulated the bending occurred, but the atrial
siphon still remained unaffected.
The essential element of the group of crossed responses is the
closure of the siphon which is not stimulated. This element .
completely disappears after the operation. Stimulation of the
inside of the oral siphon, frequently even when strong enough to
involve the dorso-ventral contraction and the body bending, fails
to affect the atrial siphon, and only causes a partial contraction
of the oral one. Irritation of the inside of the atrial siphon
brings about no change at all in the oral.
These experiments leave no doubt of the ability of each portion
of the animal to perform its part of a reaction even though it is
isolated nervously from the rest. The reaction of the animal
as a whole, however, depends on its nervous system being intact.
268 SELIG. HECHT
Il. MECHANICAL STIMULATION
1. Touch
Ascidia atra is an animal that under normal conditions is
stimulated preéminently by mechanical means. This is the only
variety of stimulus which is capable of calling forth all the possible
responses of the species. The selection of its food—if mere ex-
clusion may be called selection—is made on the basis of size, and
rejection depends on the mechanical stimulation by the larger
particles. The remarkable sensitivity to touch was known to
even the oldest zodlogists who concerned themselves with the
study of the large monascidians. Its very delicacy in Ascidia
atra was a stumbling block to locating precisely the sensitive
regions. |
The presence of a heavy cellulose test would suggest an in-
sensitivity of the exterior to any stimulation. Yet, even a gentle
touch on the surface of the body results in a reaction of the direct
type. Careful experimentation has convinced me that this is
not due mainly to an irritability of the test to mechanical stimula-
tion. An individual normally attached to a rock, and removed
to the laboratory with its attachment intact, serves best for this
type of experimentation. Moreover, if the substrate be securely
clamped in the aquarium, the accidental jarring of the animal
may be almost completely eliminated. Under these conditions
a gentle touch with a glass rod on the test surface leaves the
animal undisturbed. A coarser application at once stimulates.
I am not prepared to deny the presence of touch receptors on
the surface of the test. But I am convinced that most of the
results of mechanical stimulation of the test are not due to
sense organs within it, but to the passage of the stimulus through
the elastic material to the more sensitive region of the siphon rim.
In favor of this view is the lack of any demonstrable nerve con-
nection between the test and the tissue underneath it. More-
over, sources of stimulation, such as light, heat, and chemicals,
which cannot easily be transmitted along the test substance, fail
to be effective when applied to the outside of the test; whereas
in all other regions, they are just as effective as touch.
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR 269
The reaction to mechanical stimulation of the test is not due
to an irritation of the underlying mantle tissue. Individuals
whose tests have been removed from a portion of the body show
that the mantle is insensitive to touch. It is interesting to ex-
plore the sensitivity of such an animal. Even vigorous poking
of the mantle (the animal must be rigidly clamped, of course)
is followed by no effect. One may approach to within one
millimeter of the cut test and produce no stimulation. But
once the test is touched, the animal immediately gives its char-
acteristic respopse. An animal wholly denuded of its test is
insensitive to touch on the outside except near the rims of the
siphons.
In the normal animal as one approaches the region of the
siphons the sensitivity to mechanical stimulation rises rapidly,
and at the rim of the siphons the irritability is very great. The
rim of the oral siphon is usually divided into eight lobes, and the
atrial into six lobes. These thin lobes are the most sensitive
portions of the outside of the body. By using large animals
that have been a few days in the laboratory, and stimulating
the individual lobes with a fine glass rod, I have secured local
contractions of the portion of the rim contiguous to the stimu-
lated lobe. The folds between the lobes are only slightly less
sensitive than the lobes themselves.
On theinside of the siphons below the lobes, a similar degree of
sensitivity exists.» Inside the atrial siphon the irritability is
greatest near the rim, but the entire atrial cavity is also sensitive
to touch. Within the oral siphon the surface is extremely sen-
sitive, and remains so as far down as the ring of oral tentacles.
Beyond this the sensitivity falls off rapidly.
Of the surfaces which produce the group of crossed reactions,
the tentacles are probably the most sensitive. The prettiest
automatic response of Ascidia results from their stimulation.
By illuminating the inside of the oral siphon it is possible to touch
a single tentacle with a fine glass rod. If a delicate stimulus be
applied carefully, it is most interesting to see the atrial rim close
quietly while the oral siphon remains undisturbed. If the stim-
ulus is more intense the ‘Ejektionsreflex’ is produced. When a
270 SELIG HECHT
small particle of sand is dropped carefully upon the tentacles,
the slight back pressure produced by the closing of the atrial
rim at once squirts the particle out of the oral siphon.
In view of the certainty and ease with which these reactions
may be demonstrated, not only in A. atra, but also in Ecteinas-
cidia turbinata and in another unidentified species, it is difficult
to understand why some authors have reported that the tenta-
cles are practically insensitive to mechanical stimulation. Thus
Roule (’84, p. 37), who studied Phallusia, and Lacaze-Duthiers
et Delage (99), who observed Cynthia, state that no noteworthy
reaction occurs when the tentacles are touched in this way.
This is all the more strange because it is precisely here that See-
higer (1893-11, p. 323) has found most of the bristle cells to which
he rather doubtfully ascribed the réle of touch receptors.
The perception of mechanical irritation by the internal surface
of the atrial siphon is of significance in the daily routine of the
species. A decidedly sensitive area is at the bottom of the atrial
cavity near the anus. The feces are discharged into this cavity.
Here they furnish the mechanical stimulus for a reflex of the
crossed type: the oral siphon closes and the body contracts,
squirting the water and the feces out through the atrial siphon.
To one unacquainted with the presence of the group of crossed
reflexes, the defecation of Ascidia seems almost a conscious pro-
cedure. It ‘tries’ to force out the feces, and if a piece becomes
caught in the siphon rim or in the atrial cavity, it ‘tries’ again
to dislodge it by means of the ejection reflex, until finally it
succeeds. The whole process can, however, be called forth by
placing a glass bead ora pebble in the atrial cavity, or by repeat-
edly stimulating it with a glass rod.
2. Vibration
The extreme sensitivity of Ascidia to mechanical stimulation
is manifested in its ability to respond to vibrations (compare
Marage, ’05). Ascidia lives in shallow water, and if the rocks
within two or three meters of an individual are stamped upon
with even a modicum of vigor, it closes its siphons. NH.>Na
This parallels the stimulating strengths of these cations found by
Cole (10, p. 607) for the common chemical sense in the frog.
In order to determine the effects of a group of anions, the
following salts were used: KCl, KBr, KNO;, KI, CH;COOK,
and KSCN. The first experiments were made on small, and
consequently very sensitive, animals. By this means large
differences in stimulating power became evident; this is typified
by Exp. VIII.3 of which the following table is a summary (table 6).
Later, in ordef to separate KCl, CH;COOK and KSCN,
larger, and therefore less sensitive, animals were used. Exp.
VIII.4 was of this type and gave the results shown in table 7.
THE JOURNAL OF EXPERIMENTAL ZOOLOGY. VOL. 25. No. 1
290 SELIG HECHT
TABLE 6
Liminal concentrations of a series of potassium salts
SALT CONCENTRATION
KCl : 0.075 N
KBr 0.050 N
KI 0.010 N
KNO; 0.15 N
CH;COOK 0.075 N
KSCN 0.075 N
TABLE 7
SALT CONCENTRATION
KCl 0.20 N
CH;,COOK 0.15 N
KSCN 0.10 N
A combination of the two tables gives an anion series of
stimulating power as follows:
Br SCN =CEACOO > ClS=NO;
Excepting SCN and NOs, which are not in the usual positions,
this order agrees with the familiar Hofmeister series (Hober, 714,
p. 309). An absolutely complete agreement is hardly to be
expected, because my tests were made in seawater. Hd6ber (’14,
p. 323) has constructed ‘Uebergangsreihen,’ in which he has been
able to change the position of some members of this lyotropic
series by altering the milieu in which the experiments were per-
formed. Analogous to this is Cole’s (10) observation for the
stimulation of the frog foot, in which the positions of NH, and K
were reversed by an increase in the concentration of the solutions.
Acids. , Seawater to which acid is added, gradually returns
to its normal hydrogen-ion concentration. Therefore, the solu-
tions to be tested were freshly made up immediately before being
applied to the animal. This was accomplished by having a
stock 0.1 N solution made up in rain water, and diluting it to the
desired concentrations with seawater. The effect of the dilution
of the seawater is insignificant. Three acids, hydrochloric, formic
and acetic, were tested. The following table gives the values
which were obtained in Exp. VIII.9, typical of the others (table 8) -
PHYSIOLOGY OF ASCIDJA ATRA LESUEUR 291
TABLE 8
Liminal strengths of acids for the stimulation of Ascidia
ACID CONCENTRATION
HCI 9.0016 N
HCOOH 0.0018 N
CH,COOH 0.010 N
The order of the stimulating efficiency of the acids is, therefore,
HCl > formic > acetic
Bases. As representatives of this group of substances, I used
NH.OH and NaOH. Exp. VIII.9.1 gave the liminal values shown
in table 9. This places them in the order,
NaOH >NH,OH
TABLE 9
BASE CONCENTRATION
NaOH 0.010 N
NH,OH 0.015 N
Sugars. Both glycerin and sucrose did not stimulate until
they reached a concentration of 1 M. This quantity of solute,
plus the salts of the seawater in which these substances were
dissolved, brought the concentration of the stimulating solution
to just that equivalent of concentrated seawater which irritated
Ascidia osmotically. We can, therefore, conclude that Ascidia
is not sensitive to these two substances.
This has been found to be generally true for aquatic animals
(Parker, 12). Crozier (15a), however, has shown that glycerin
and maltose can stimulate Holothuria.
Alkaloids. The sulphates of quinine, strychnine and morphine
were tested. The order of their effectiveness,
strychnine > quinine >morphine
was found in Exp. VIII.1, the results of which are given in
table 10.
These values show a surprising sensitivity of the species to
alkaloids. A bitter taste in man may be secured from 0.00004 M
quinine sulphate. This amounts to one-tenth of the uncorrected
concentration to which Ascidia reacts.
292 SELIG HECHT
TABLE 10
ALKALOID CONCENTRATION
Strychnine 0.00005 M
Quinine 0.0004 M
Morphine 0.001 M
Anesthetics. Ether, chloral hydrated, ethyl aleohol and amy]
aleohol all caused reactions which were very pronounced. The
order of their effectiveness, taken from the values obtained in
Exp. VIUI.1.1 and VIII.2.2 and given in the accompanying table
(table 11), is
amyl ale. >chloral, ether >ethyl] ale.
TABLE 11
ANESTHETIC CONCENTRATION
- Ether 0.02 M
Chloral hydrated 0.02 M
Ethyl alcohol 0.75 M
Amyl alcohol | 0.001 M
6. Nature of the sense organs
The morphological nature of the chemical, and indeed of any
other kind of receptors in ascidians, is practically unknown.
Seeliger (93-11, p. 323) has described, rather doubtfully and
with much reserve, the presence of bristle cells on the tentacles
of Ciona. Lorleberg (07), however, failed to secure any trace
of such structures in Styelopsis; although he found many regions
richly supplied with nerve endings. It may then be that the
organs of chemical sense in Ascidia are similar to those which
underlie the common chemical sense of vertebrates (Parker, ’12).
The physiology of the receptors would seem to favor such an
assumption. The problem of the physiological nature of the
chemical sense organs is simplified in Ascidia atra by the mo-
notony of response to all classes of substances. This negative
reaction of the crossed type and its variation with the intensity
of the stimulus have already been made clear. We are, there-
fore, dealing apparently with an automatic reflex, of which the
receptor and effector mechanisms are all set, and the conduction
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR 293
provided for. The application of the stimulating substance to
the sense organ merely starts a prearranged process of response.
In order to understand the nature of the process set up in the
receptor, it will be necessary to consider more closely the phys-
iological effects of the substances used in the stimulation of
Ascidia. It was on the basis of the action of the salts of the alkali
metals that Héber (’14) first pointed out the relation between
irritability and colloidal constitution of the plasma membrane.
Since then the ubiquity of the cation and anion series has been
demonstrated for such diverse processes as melanophore con-
traction (Spaeth, ’13), hemolysis (Héber, *14) and rhythmic
pulsation (Crozier, 16a). The presence of these ionic series in
the sensory stimulation of Ascidia indicates that the significant
process which underlies it, resembles, if it is not identical with,
the determining reactions of the other physiological phenomena.
The acids have already received attention in regard to their
sensory effects (Richards, ’00; Kahlbaum, ’00). The anomalies
which are exhibited by the acid taste in man are typified in the
behavior of the three acids which were used in these experiments.
Although HCl is more effective than formic acid, the difference —
between them is not great. They both, however, are much more
powerful than acetic. In the penetration of cells by acids
(Crozier, ’16b), we find the same order of effectiveness. The
anomalies which were referred to are as follows. When the dis-
sociation constants of the acids are taken into account, it is found
that the same effect is produced by acetic acid with a lesser
quantity of hydrogen ions than by formic acid; and less in turn
by formic than by hydrochloric acid. In Ascidia the liminal
concentrations of the acids contain the following quantities of
hydrogen ions: acetic, 4.1x10-‘N; formic, 6.0X10-!N; and
hydrochloric, 1.6 10-N.
An analogous difficulty exists in the effects of NaOH and
NH,OH. Experiments on penetration have shown that NH,OH
enters tissue rapidly, whereas NaOH may hardly be said to pene-
trate living tissue at all. Still, NaOH is more toxic than NH,OH
(Harvey, 713). Similarly it is a more effective sensory stimulant
than NH,OH.
294. SELIG HECHT
The physiological inertness of the sugars is known only too
well to require more than mention. Their ineffectiveness has
made their use possible in experiments where the effect of osmotic
pressure only is desired (Hoéber, ’14, p. 496). It is therefore
altogether in keeping with the parallelism between genera! phys-
iological activity and sensory stimulation that Ascidia fails to
be stimulated by even high concentrations of glycerin and sucrose.
It has been suggested that the sensory inactivity of the sugars
may be due to the lack of these substances in an aquatic environ-
ment (Parker, ’12). The improbability of the occurrence of
saccharin in the seawater, however, does not prevent its chemical.
stimulation of Ascidia. The liminal concentration of a commer-
cial preparation was 0.025 M, to which the usual negative re-
sponse was given.
It is necessary, similarly, to look in a different direction for the
explanation of the sensitivity of Ascidia to alkaloids and anes-
thetics. The minute quantities of alkaloids which are effective
in stimulation find their counterpart in the extremely low con-
centrations in which they penetrate cells (Overton, ’97).
As a consequence of these results there can be no doubt of the
essential similarity between the general physioiogical reactions
of chemical substances and their effects on the sensory processes
in Ascidia. This indicates that the action of the stimulating agent
on the sense organ involves an effect of the same nature as the
action of these substances on other cells and tissues. Moreover,
it shows that the effect of a chemical on the receptor concerns
that structure primarily as a cell, and only secondarily as an
organ for receiving stimuli.
It must be emphasized that these generalizations are not
intended for the sense of taste in vertebrates, but solely for the
sensitivity of animals, like Ascidia and Holothuria, which possess
a general chemical sense. This type of irritability corresponds
in many ways to the common chemical sense of vertebrates
(Parcer, 712), although the two need not necessarily be homolo-
gous. ‘Lhe problems involved in the higher organs of taste, par-
ticularly the sweet taste, do not concern us here. They represent
specialization for certain needs; and in the present condition
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR 295
of our knowledge it is futile to attempt an explanation of their
physiology.
It has been tacitly assumed that chemical sense organs are
capable of detecting substances in concentrations which fail to
affect the ordinary cells of the body. This is largely because
the effects on the sense organs become evident through certain
effectors, whereas the action on other tissues must be noted by
special, indirect means on the cells themselves. When, however
other tissues are studied, it is seen that they are influenced
by concentrations of the same magnitude as sense cells. The
effect of minute changes.of the hydrogen and hydroxyl ions on
the permeability of eggs and blood corpuscles need only be men-
tioned. Acids and bases enter cells in concentrations like those
which stimulate animals. The poisoning effects of extremely
low concentrations of alkaloids are also familiar.
The modifications produced by these various substances are
more or less the same for all cells and tissues: witness the simi-
arity of effects produced on egg cells, sperm cells, fronds of algae,
blood corpuscles, chromatophores, hearts, medusa bells and a
host of others too numerous to mention. The concepts of ionic
antagonism and salt balance apply not only to these tissues, but
to sensory stimulation as well (Crozier, 715 b). It is therefore
clear that chemical sensitivity is merely one of a large numer of
similar manifestations of the fundamental nature of cells
The explanation which seems to me to account for all the
phenomena o this sensory activity, in Aszidia at least, is that the
factor which primarily converts a group of cells nto chemica!
sense organs 1s not any special modification of their structure or
sensitivity, but rather their connection, directly or indirectly,
with an effector system.
In this way the problem of the chemical sensé@ of such aquatic
forms is linked with the general problems of the physical chemis-
try of cells and tissues. Our present knowledge, in this respect,
of the chemical senses is, however, extremely meager. The time
is therefore not ripe-for any adequate explanation of the proces:
in the receptor cell which results from the contact with a sa>-
stance in solution.
296 SELIG HECHT
One such attempt has been made. On the basis of his work
on echinoderm eggs and Arenicola larvae, Lillie (11) has proposed
an explanation for general irritability. It is, that sensory stimula-
tion means an Increase in the permeability of the irritable element.
Lillie’s explanation is based on the assumption that the de-
marcation current and kindred phenomena are functions of the
differential permeability of the cell membrane to certain sub-
stances, notably H and OH ions. The work of Loeb and Beutner
(14) has, however, shown that this bioelectric potential is due
on the contrary to the presence of certain lipoid materials in the
protoplasm. It is still uncertain to what extent differential
solubility and the effect of interphase’ boundaries are concerned
in the interpretation of these results. It ismuch to be regretted
that the experiments were discontinued.
There is, moreover, another and more significant objection to
Lillie’s idea. All the substances which increase permeability
undoubtedly do stimulate. But many substances, like Ca and
the anesthetics in general, all of which have a decreasing action
on permeability (Osterhout, ’16), also serve as vigorous stimu-
lants to Ascidia and other aquatic organisms.
The theory in its present form can therefore not be accepted
as an adequate explanation. However, the attempt at an inter-
pretation along the lines of permeability and similar concepts
is entirely in the right direction.
VI. SUMMARY
1. Ascidia possesses six distinct reactions to stimuli, all of
them negative in character. They may be divided into two
groups of three each: the direct refiexes, which depend upon a
stimulation of the exterior of the body, and the crossed reflexes,
which depend upon a stimulation of the interior of the body.
2. Theintersiphonal ganglion connects the two siphons. Sev-
ering this nervous mass completely abolishes the crossed reactions,
and interferes with the direct ones. Nevertheless, each portion
of the animal is able to perform its part of a reaction, even though
nervously isolated from the rest. :
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR 297
3. Ascidia is sensitive to tactile stimulation. The regions of
greatest sensitivity are the siphon rims and the oral tentacles.
4. Vibrations through solid and liquid media affect Ascidia,
although transmission through the seawater is the normal method
of stimulation. The receptors are located in the lobes of the
siphon rims.
5. The records of the amplitude of contraction to regularly
repeated mechanical stimulation show that the cessation of
response after a time is due mainly to a fatigue of the receptor
mechanism.
6. The ‘ocelli’ of Ascidia are not organs for photo-reception.
The animals are sensitive to light of very high intensity only,
and the sense organs are located within the siphon near the
oral tentacles.
7. Ascidia is thermosensitive. It reacts to temperatures above
32°C. and below 20°C.
8. Its test is insensitive to light, heat and chemicals.
9. The animals react to large changes in osmotic pressure,
and to the presence of the following classes of substances in solu-
tion: salts, acids, bases, alkaloids and anesthetics. Solutions
of sugars do not stimulate, but saccharin gives a decided reaction.
10. The liminal concentrations and the relative effectiveness
of all these stimulating substances are very similar to those which
have been demonstrated for other physiological activities. It is,
therefore, suggested that the primary factor which converts a
group of cells of Ascidia into chemical sense organs is their con-
nection with an effector system.
298 SELIG HECHT
_ VI. BIBLIOGRAPHY
Baauiont, S. 19138 Die Grundlagen der vergleichenden Physiologie des Nerven -
systems und der Sinnesorgane. Winterstein’s Handbuch vergl.
Physiol., Bd. 4, p. 1-450.
Coin, L. W. 1910 Reactions of frogs to chlorides of ammonium, potassium,
sodium and lithium. Jour. Comp. Neur., vol. 20, p. 601-614.
Crozier, W. J. 1915 a Thesensory reactions of Hologhutia surinamensis Ludw.
Zool. Jahrb., Allg. Zool., Bd. 35, p. 233-297.
1915 b Ionic anipeenisn in sensory stimulation. Amer. Jour. Phys-
iol., vol. 39, p. 297-302.
1916. a The rhythmic pulsation of the cloaca of Holothurians. Jour.
Exp. Zoél., vol. 20, p. 297-356.
1916 b Cell penetration by acids. Jour. Biol. Chem., vol. 24, p. 255-
279.
Cusuny, A. R. 1916 On the analysis of living matter through its reactions to
poisons. Science, N.S., vol. 44, p. 482-488.
Frouuicu, A. 1903 Beitrige zur Frage der Bedeutung des Zentralganglions bei
Ciona intestinalis. Arch. ges. Physiol., Bd. 95, p. 609-615.
Harvey, E. N. 1913 A criticism of the indicator method of determining cell
permeability for alkalies. Amer. Jour. Physiol., vol. 31, p. 335-342.
Hecut, 8. 1917 The physiology of Ascidia atra Lesueur. I. General Physiol-
ogy. Jour. Exp. Zoél., vol. 25, p. 229-259.
HerpMan, W. A. 1904 Ascidians and Amphioxus. Cambridge Natural His-
tory, vol. 7, p. 33-138.
Hinton, W.A. 1913 The central nervous system of Tunicanigra. Zool. Jahrb.,
Anat. Abt., Bd. 37, p. 113-130.
Hoser, R. 1914 Bieialeehe Chemie der Zelle und der Gewebe. cyeabee
und Berlin, xvii + 808 pp.
Howetz, W.H. 1912 Text Book of Physiology. Phila., 1018 pp.
Jorpan, H. 1907 Ueber reflexarme Tiere. Zeit. allg. Physiol., Bd. 7, p. 86-135.
Kanipaum, L. 1900 The relation of the taste of acid salts to their degree of
dissociation, II. Jour. Phys. Chem., vol. 4, p. 533-537.
Krnosuita, T. 1910 Ueber den Einfluss mehrerer aufeinanderfolgender wirk-
samer Reize auf den Ablauf der Reaktionsbewegungen bei Wirbellosen.
I. Versuche an Tunicaten. Arch. ges. Physiol., Bd. 134, p. 501-530.
1911 Ueber den, ete. III Mitteilung. Arch. ges. Physiol., Bd. 140,
p. 198-208.
Lacaze-Dururers, H. pr, et Devacn, Y. 1899 Etude sur les Ascidies des cétes
de France. Mém. Acad. Sci. Inst. France, T. 45, p. 1-323, 20 pl.
Linturn, R. 8. 1911 The relation of stimulation and conduction in irritable
tissues to changes in the permeability of the limiting membranes.
Amer. Jour. Physiol., vol. 28, p. 197-222.
Lors, J. 1892 Untersuchungen zur physiologischen Morphologie der Tiere.
II. Organbildung und Wachsthum. Wiirzburg, 82 pp.
1902 Comparative physiology of the brain and comparative psychol-
ogy. N. Y., x + 309 pp. '
PHYSIOLOGY OF ASCIDIA ATRA LESUEUR 299
Lors, J., AND BeutrNer, R. 1914 Ueber die Bedeutung der Lipoide fiir die
Entstehung von Potentialunterschieden an der Oberfliche tierischer
Organe. Biochem. Zeit., Bd. 59, p. 195-201.
LorLeBerG, O. 1907 Untersuchungen ueber den feineren Bau des Nerven-
systems der Ascidien. Zeit. wiss. Zool., Bd. 88, p. 212-248.
Maaenus, R. 1902 Die Bedeutung des Ganglion bei Ciona intestinalis. Mitt.
zool., Stat. Neapel, Bd. 15, p. 483-486.
Maracas, M. 1905 Pourquoi certains sourd-muets entendent mieux les sons
graves que les sons aigus. C. R. Acad. Sci., T. 141, p. 780-781.
Mercatr, M.M. 1900 Notes on the morphology of the Tunicata. Zool. Jahrb.,
Anat. Abt., Bd. 13, p. 495-602.
Nace, W. A. 1894a Ergebnisse vergleichend-physiologischer und anatomis-
cher Untersuchungen ueber den Geruch- und Geschmacksinn und
ihre Organe. Biol. Centralbl., Bd. 14, p. 543-555.
1894 b V Bealcichendipbeiolocisctie und anatomische Untersuchungen
ueber den Geruch- und Geschmacksinn und ihre Organe. Bibl. Zool.,
Bd. 7, Heft 18, viii + 207 pp.
1896 Der Lichtsinn augenloser Tiere. Jena, 120 pp.
OstrerHOUT, W. J. V. 1916 The decrease of permeability produced by anes-
thetics. Bot. Gaz., vol. 61, p. 148-158.
Overton, E. 1897 Ueber die osmotischen Eigenschaften ae Zelle in ihrer
Bedeutung fiir die Toxicologie und Pharmakologie. Zeit. physik.
Chem., Bd. 22, p. 189-209.
Parker, G.H. 1908 The sensory reactions of Amphioxus. Proc. Amer. Acad.
Arts and Sci., vol. 43, p. 415-455.
1912 The relation of smell, taste, and the common chemical sense in
vertebrates. Jour. Acad. Nat. Sci., Phila., vol. 15, Ser. 2, p. 221-234.
1917 Actinian behavior. Jour. Exp. Zoél., vol. 22, p. 193-229.
Pouimmanti, O. 1911 Beitriige zur Physiologie des Nervensystems und der
Bewegung bei niederen Tieren. II. Arch. Anat. Physiol., Physiol.
Abt., Suppl. 1910, p. 39-152.
Ricuarps, T. W. 1900 The relation of the taste of acids to their degree of dis-
sociation. Jour. Phys. Chem., vol. 4, p. 207-211.
Rove, L. 1884 Recherches sur les Ascidies simples de cétes de Provence.
Ann. Musée Hist. Nat. Marseille, Zool., T. 2, p. 1-270.
SEELIGER, O. 1893-1911 Tunicata. Bronn’s Klassen und Ordnungen des
Tier-Reichs, Bd. 3 (Suppl.), Abt. 1, p. 1-1280.
Spagtu, R. A. 1913 The physiology of the chromatophores of fishes. Jour.
Exp. Zodl., vol. 15, p. 527-585.
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AUTHOR’S ABSTRACT OF THIS PAPER ISSUED
BY THE BIBLIOGRAPHIC SERVICE, FEBRUARY 2
CHANGES IN THE RELATIVE WEIGHTS OF THE VARI-
OUS PARTS, SYSTEMS AND ORGANS OF YOUNG
ALBINO RATS UNDERFED FOR VARIOUS
PERIODS
CHESTER A. STEWART
Institute of Anatomy, University of Minnesota, Minneapolis
ONE FIGURE AND FOUR TABLES
CONTENTS
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