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BIOLOGICAL  BULLETIN 


OK    THK 


noannc  Biological  laboratory 

WOODS    II'  '1  1  .    MAS 


Stntt 

1      '  .     '       ,  KI  IN       /''  .  //r. 

[xcqi/BS  LOKB-    /'//'•  A'  /  '        !/••'•   .-   A1- 

T.    I  I  .    Mo|«  ,  A  \         '        It  '>:''••  :     i  :(Y. 

\V.    M.    U'll  I    '  .')'. 

E.  B.  WILSON      '       tmbia  U  .'r. 


.         !i£^ltol 
\\K  K.  I.  ii.  i.  ii       The  University  '<?o. 


\".  >LUM1     XX  II. 


\V.  ••  >DS  I  I'M  1  ,  MA- 
JIM      1"    N«  'VI  MI'.KU.    1912 


PRESS  OF 

THE  NEW  En*  PR:1. ::•,:;  cc"f*'.» 
LANCASTER.  PA 


CONTENTS  OF  VOLUME  XXII 


\".      1.         1  >  l.i    K  \II4KK.     I'll  I. 

PA< 

SlIH.H'Kli.     Yl«    I"K     |  1 

Cini  D,   C.   M..   M«  Kn  .   I  .  V.   M.      7  tral   .\> 

in     I  •  '...'"/>/;///<//'<  mar  in 

I1'  ;<; 

\\  ipoDki  ||.   l.i  i|.  \NM    I  •  ISS 

I  (I    * 

NO.     2 .         I   \  M     \  K  \  .      [912. 

1 1  \M  .1  I  I  .   (    II  \-.    \\  . 

. !  • 
HlGLEY,     K'  -l      M   .     I  ll   \  I  II.     I  I  \KH|  I' 

• 

l\  \\H  \l  I  .      |i  ">l   I  HIM         I  ll    \  I  II.     I  I  \Kiil  I'  .     .1       A 

RIDDLE,  OSCAR.    /I  i 

'"7    S~ 

\  i  • .    ; .       I  RY,  1 91 

McCLENDON,  J.  1 

i  l  > 
Kl  I'M  K.  \\  \l.    \.        /  ':« 

''initory  A  pfxirdtn 
I'M  M  K-I  'N.I      1  in  .\i  ^s.       1  Hi- 

/)(;r(;       .1  >'../"  PI  17.1 

NO.  4-         M   \K<   II.    I'M 

\Virn  M  N  .  I  >    1  '.  '  /»/  11  \ilntiiiti 

STEVENS,  N.  M.  .TV  c'//  /// 

219 
STEVENS,  N    M  .  ''">- 

r 

|IM    ERNEST!        /    •   Relation  of  the  /•'/  •    /'/•.•);'  /.- ///«• 

kntr<nii<:  Pot  "  -  "') 

iii 


iv  CONTENTS   Ol     \  <>l.i  \l  I     XXII. 

\\'(ti>-Ki>A!.KK.  J.  I-"..      Palmen's  Organ  and  its  I-'  unction  in  Xymphs 
tin-  I-.plicmcridn.   lleptagenia  interpiinctata  (Say)  and  f-'.cdy- 
tirns  maculipennis  (  Walsh)  .............................. 


\n.  5.      APRIL,  nil  2. 

KING,  HELEN  DEAN.     'I'  lie  .  Some  Amido-acids  on  the  De- 

•t»neniofth<-  l-'.^"*  <>f  Arlwciu  mid  »f  ('In.'toptfnis  .........    273 

Oi  \\II:KI<^.    KdiiKiM.  JK.     .1  JH.tcussion  of  Cyclops  viridis  Jurine  2<)i  ^ 

I'l  \KI..  K.\v\iiiM>.  \otft  OH  the  History  of  Burred  Breeds  of 

I'l'nltry  ....................................  -''7 

MON  i  n  i  \II-.KV.  T.  II.,  J  k.  Complete  niselnir^e  of  Mi  lot  houdria  from 

the  Sf)en)into:oini  of  I'eri  ptititx  ...........................  .>«") 

\o.  6.      M.\\  .    i<)i2. 

l\i  INKI  ,    l;.n\\i\    I-'..      .1     rrelimituiry   Account  of  the   I'erelopmcnt 
oftlieApyrcnc  Spermatozoa  in  Strombus  and  of  the  Nurse-cells 

in     Litloriiin  ..........................................    ,U  '  i 

I.  il.  1.  1  1-:.    K.M.i-n    S.       Certain  Mean.:  hy  ichieh  Starfish    /v^.v  .\atn- 

rally  Resistant  to  l-'ertilization  m.iy  l>c  Rendered   \ormal  and  the 
I'liysiological  Conditions  of  this  Action  ...................    328  N 

I'onrtcentli  Annual  Report  of  the  Marine    Biological  Laboratory.  .  .    ,U7 


Vol.  XXII.  December,  ryii.  No. 


BIOLOGICAL  BULLETIN 


KCOLOGICAL   SUCCESSION. 


III.    A   ki  <  •  «\\  \i~-  \\CE  oi    ii-  CAUSES   i\    POM*-  \\ini 
I'AK  i  ic  n.AR   Ri-.i  i  KI  \<  i     ro   FlSH. 

Vic  TOR    E.    SHK!  I  <  <\<\> 

I.    IN  imuji  <  1  1 
II     I'k-i-i  -.1    '  H  «    \NU  CONTENT  OK   !'• 

i  .   /'  r  .  .  i 

.;ihy 

tin-  Bottom 
I  '  "lit  .  . 

2.1  <lt      ........ 

i(>arison 

i  _• 
•  ion  u 

..i  ...  i  ^ 

u  .  .  i  i 

KI 

III     I  in    «  ;MI                                                               if, 

i.    v                                       ,-nt  .  ir, 

-•     I  '  >nn  fill                                                        i  s 
!  • 

I  >.  |.ili 
'•'• 

1  -:\    ^.  -'.ill'  \,j 

1-; 

i 

:  i    . 

<lity 
•     ..unity 

Relative  Ii  :  • 

h 

ties  ol    tlii-  I-  i-li 

l\       <  .1  M-.K  \I.    I  M-.  USSH  28 

\        Si    MMAKV 
\l         \i'KNi'\\  I    Mii.MI    M-      \NH     UlUMiM.K  AI'IIY    .  35 

I 


2  V.CI'  >K     I   .    -Ill   I.F<»KI>. 

I.     IXTRODl  '    I  K  >N. 

In  tin-  preceding  paper  we  presented  rrrt.iin  facts  concerning 
ponds,  together  with  a  statement  of  suree^ion  in  tin-  |>ond-  ,u 
the  head  of  Lake  Michigan,  without  entering  into  its  c.uix •-. 
Succession  in  ponds  is  due  to  many  causes.  It  is  only  under  tin- 
most  favorable  conditions  that  we  can  separate  these  causes  one 
from  another  without  long  and  careful  investigation.  The  first 
attempts  of  ecologists  in  this  line  were  considerations  of  tin- 
obvious  general  facts,  such  as  the  accumulation  of  organic  detritus 
and  the  increased  denseness  of  vegetation.  We  can  give  hen  .1 
hint  at  the  more  specific  changes  in  the  ponds  and  the  relations 
of  these  to  fish.  The  subject  is  one  for  cooperative,  research. 
At  present  some  of  the  workers  and  the  necessary  funds  are  not 
available,  and  the  ponds  are  being  destroyed  rapidly.  It  is 
therefore  improbable  that  the  study  can  be  carried  further.  Thi- 
paper  deals  with  the  results  of  a  preliminary  investigation  of  the 
ponds  for  the  purpose  of  learning  something  of  the  causes  of 
distribution  and  succession  of  fish  and  other  organisms  in  ponds. 

II.   THK  PRESENT  CHARACTER  AND  CONTENT  or-~  THE   PONDS. 

The  ponds  with  which  we  are  concerned  are  shown  on  the  maj>, 
p.  1 31,  of  the  preceding  paper  of  this  series.1  This  map  i- e—en- 
tial  to  the  understanding  of  the  data  of  the  present  paper.  Tin 
ponds  here  considered  are  an  ecological  age  series,  ecological 
age  being  determined  by  a  study  of  amount  of  sand  bottom, 
humus,  etc.,  as  shown  in  Table  I.  below.  The  physiographic 
history  of  the  region  is  in  full  accord  with  the  facts  used  in  decid- 
ing age  though  in  this  case  physiographic  hi.-iorv  is  not  essen- 
tial to  the  decision.  The  pond  designated  as  I  is  ecologically 
youngest,  14  the  oldest,  and  the  others  intermediate.  The 
measurements,  analyses,  and  quantitative  study  were  carried 
out  on  Pond  i.  ^c  (west  section),  70,  and  146  of  the  map.  Some 
qualitative  records  from  the  other  parts  of  pond  5r,  from  5/<,  and 
\4d,  are  included  with  those  of  the  ponds  in  which  the  oiln-r 
work  has  been  done. 

See    "Ecological    Succession,"    II.      BIOL.    Hei.i,.,    Aug.,    1911.    pp.    1.27-151. 
Tliese  are  errors  in  the  pond  numbers  of  this  paper  which  shouM  I"    corrected. 
Page  1.52,  line  13,  for  "56"  read  58. 
Pane  133,  Table  I.,  last  line,  last  column,  for  "  15  "  read 


ECOLOGICAL    -ITCESSION.  3 

A-  \ve  have  already  -idled,  the  ponds  which  ha\'c  been  -tudied 
e-pecially,  are  part-  of  the  long  sloughs  which  have  been  long 
enough  i-olated  to  ~ho\\  ihcir  efficiency  in  -upporiing  the  fi-he- 
which  tlu-v  now  contain.  The  ti-hes  found  in  tin-  -eparated 
pond-  are-ho\\n  in  Table  XXI.  '  p.  171  of  the  pre-ent  paper. 

With  the  kno\\n  habits  of  fre-h-water  fi-he-  as  a  ^uide.  the 
pond-  h.i\e  lu-en  roughly  mea-ured  and  area  determined,  depth 
and  angle  of  til-  -lope  of  sides  mea-ured.  the  character  of  bottom 
determined,  -ketr'n.-d,  and  the  area-  of  the  different  kind-  e-g- 
niated  on  the  ba-i-  of  the  sketch--,  and  the  di—  olved  -olid-  and 
lie  \\aier-  have  been  determined  by  chemi-t-.  The 
plant  and  animal  content  of  the  pond-  ha-  been  anab  /ed  <|tialila- 

ti\el\    and  e-timated  quantitatively. 

Tlie-e  re-nlt-  \\ill   be  |»resenled  under  the  main   head-  of.      i 
l'h\ -ii-al  <  har.t.  •    Biological  Content. 

1.     I'liy^iull     ('h>:  -(a)      Topo-r.iphy.       The     chief      topo- 

phii    features  are  -ho\\n  in  Table  I. 

TAHLK  1. 

•-IK  .\\  i  DEPTH  AND  ^ 


,^c 
Depth. 

I 

0.3     • 

0.5 

Mix  1. 

0-S 

h 

[0 

\  cry  lr 

.ill 

The    figure-    repre-enting    depth    ol    water    are    the    re-nlt-    of 
me.i-nn-meiit .  \\ith  e-timatioii  in  th-  -\  averages.       \T 

re-nlt-  o|    r..ii-li    mea-iiring  by   ]  lonntin-    rail-  in   |iarallel 

railroad-,  etc.  \\liile  p^itioii-  -tnditd  dit'ler  in  -i/e,  the\  pi.  -i  nt 
Considerable  nnifo|-mit\  of  other  feature-. 

I    llal.H  'lei'    .-I     Mottoill.        The    bottom    i-  ( •onipn-rd    of     pure 

sand,  or  sand  more  or  less  mixed  with  or  deeply  covered  by  humus. 

The  -and  or  tin-  In  mm-  ha-  a  con -id  era  Me  mi\i  nre  of  marl  at  -ome 
point-  in  the  \oim^er  pond-.  Vegetation  iuarl\  al\\a\-  covers  a 

pure  humus  bottom.     (.'lm><i  -m<l   bnlrn-1^          netimes  gro^  on 

-and  and  marl  bottom-,  but  in  -uch  i  ases  the\  are  -i  altered  and 
in  the  table  -uch  area-  are  included  \\ith  bare  sand,  because  -uch 
sparse  vegetation  does  nol  intei  tfre  \\  ith  the  breeding  of  tish. 


VICTOR  E.  SHELFORD. 
TABLE     1 1 . 

SHOWING   K.INIIS  ANI>  AKKA*  01-    H..IIMMV 


Pond 

Area  Sand  in  Square 
Meters. 

\   •   >   Uiunnv  in  Square 
M.  ; 

1  >>  pill   Hum 
in  Cm. 

i 

1^ 
14* 

I.OOO 
50 

Very  little. 
1               None. 

-'.50O 
3.450 

Nearly  all. 
All. 

-•->  .111. 
2O.  O  i  Hi 
21.0  rm. 

24.11  .  in. 

It  will  be  noticed  that  the  area  of  sand  is  much  less  in  tin-  older 
ponds  and  the  area  of  humus  much  greater,  due  to  accumulation 
of  the  latter  from  the  decay  of  vegetation.  The  depth  of  humus 
does  not  increase  proportionately  with  age  because  it  becomes 
more  compact  with  time.  With  the  exception  of  the  first  pond, 
the  average  depth  of  humu>  \va>  obtained  by  dividing  the  average 
depth  at  the  center  by  two.  In  the  case  of  pond  i,  then-  are 
large  areas  with  only  two  centimeters  of  humus  and  two  deep 
places  which  contain  humus  of  considerable  depth ;  we  give  only 
an  estimate. 

(c)  The  Dissolved  Content  of  the  Water.  For  a  preliminary 
study  of  the  dissolved  solids  of  the  water  we  have  had  a  single 
analysis  of  the  solids  and  four  analyses  of  the  ga^-^  made  by 
chemists,  (i)  Solids.  The  small  value  of  single  analyses  of  solids 
is  well  known;  sanitary  analysts  have  pointed  out  the  danger,^ 
arising  from  conclusions  drawn  from  so  little  data.  Ho\\ever,  in 
this  particular  case,  the  value  of  the  roiilts  is  greater  than  in  tin- 
case  of  single  analyses  of  drinking  water,  because  of  the  following 
conditions  in  and  about  the  ponds. 

(a)  The  ponds  are  without  outlet  and  have  no  >t  i vam>  cmpi  yin.^ 
into  them. 

(b)  During  rain  they  have  little  inwash  because  nearly  all  water 
must  filter  into  them  through  sand;  in  case  rain  falls  in  such  tor- 
rents as  to  actually  run  in  from  the  >ides  tin-  area  of  drainage  is 
small,  being  a  strip  not  more  than  fifteen  meters  wide  on  either 
side  of  each  pond.     The  ponds  than  are  comparable  to  balanced 
aquaria  and  any  variation  of  dissolved  solids  muM  be  due  in  tin- 
main  to  the  effect  of  organisms,  of  evaporation,  and  of   renewal 
from  rain. 

It  -hould  be  noted  that  these  analyses  were  made  at  i  he  end  <>\ 


ECOLOGICAL    SUCCESSION. 


ihc  «lr\-  -t.-uson  and  just  at  the  close  of  tin-  probable  plankton 
maximum  for  the  year. 

TAHLI-:  III. 

I.TS    IN    S«i|.i    [  I'  is'    IN     I'AKI-    I'l.k     M  11.1.  ION. 

Analysis  b]    Mariner  and  Ho<kii  me..  III.  i.      Tlu-  o.Iln  timi-  \\vrr  nuuk- 

mi  i  Wolf  Lake  contain^  all  tin-  -pi-.  i«--  ,>i    ti-h  ..t  all  the 

ami  i-  a'Mi-'l  tut  (  i  iinp.nison. 


!••  ml. 

i 

'A 

•  •-iutn  .  .ir  1"  iii.it>-           .... 

- 

I  I  I.O 

77-J 

96.8 

<  ;ili  iutn  i  ;irl"  iii.it'- 

CA.2 

27.0 

I  14-1 

'47 

.in  -  ulpliati-                    .... 

1  74-3 

.  inn  (  lili  it  i'l'-  .                

i  i  .4 

^'  ill  nun  -ulpl.    '  • 

4vO 

*M  .ilium  .  lili  >i  ji  !<•                    .... 

10.  1 

8  i.o 

1  1.3 

Jri  MI                                          

3.0 



3-0 

' 

1     •  ,il    -..lifl-     in.  Imlinn     tin.-.-    wi\«-n    in 

',  1 

420.0 

Tin-  tal.lt-  -h<>\\-  no  utui-al  <|iialitati\e  features.  Thi-rc  i-  a 
iiotaMi-  di  in  total  solids  in  the  older  pond-.  1  In-  ma\  U- 

dm-  to  tin-  lixiim  ot  tin-  solids  by  organi-m- 

j   Gases       Ph<  gasanalysesweremade  with  two  facts  in  view: 

Water  ma\  I  H-  al  .normal  in  gas  content .  -i  >  a-  to  make  it  impos- 
sible La  ti-h  t,.  live  Marsh,  'oH;  Juda\  and  Wagner,  '08;  Birge 
and  Iuda\.'ii  Phe  eggs  of  all  food  fishes  known  to  be  in 

the  pond-  piol'.il.lv  rest  oil  the  bottom  ..t  \e-etation  during 
inrnl.ation.  Nnnr  ti-h  remove  the  \egi-tatioii  I  mm  tin-  l.otii.m: 
otln-r-  di  po-it  eggs  on  bare  bottom;  a  te\\  ma\  attach  »--L;-  to 
\  .  ^rtation. 

I-  determine  the  general  suitably  of  tin-  pond-  for  tin  1 
place-  of  li-h.  t\\o  determination-  of  the  gas  content  of  the 
\\ater-  \\ere  made. 

TAULI    IV. 

t  >\yi;'-ii  in  cul.i.   .  '•iitiiuf  -t.-rs  per   liter;   collivti'in-   i"    i  J  .in.  ln-li>\v  tin-  -in; 
..!   tin-  fp.-n  \\  a) 


|  'll\      '  '      1  ';  1  i  > 

7-44 

April  26,   i'ji  i 

..           6.- 

7  - 

6.96 

6 

A\  erage  

, 

S 

7.20 

' 

Thr  tal.K-  -ho\\  -  an  oxygen  roiitrnt   in  all  the  pond-.  Hiltirirnt 
-ii|)port  an>   ot    tin-  tislu--. 


6  VICTOR   E.    SHELFORD. 

With  reference  to  fish  breeding  plau->,  the  gas  content  of  the 
water  was  determined  on  tour  occasions.  To  make  collect  ion^  in 
ponds  5r  over  a  sandy  bottom  required  taking  advantage  of  the 
sand  areas  made  by  artificial  filling.  Since  there  is  little  ban- 
sand  in  ponds  ~ja  and  I4/;  the  collections  were  made  over  the 
vegetation. 

TABLK  V. 

Oxygen  content  in  cubic  centimeters  per  liter.  Depth  35-40  centimeters. 
Sample  collected  at  the  bottom  or  among  the  upper  two  inches  of  branches  of 
aquatic  plants  as  indicated. 


Over  Sandy  Bottom. 

Over  Vegetation.           B""om  Materials  1  listurbed, 
|  Vegetation  K                  n  >iinnner 

Date. 

6/27 

7  '22 

4/26 

E/io 

Aver- 
age. 

6.27 

7.22 

V  V  t  •  1 

6/27 

4/26 

1 

5/10 

Aver- 
age. 

I 

Sc 

l<i 
146 

6-57 
7.36 

6.37 
8.18 

5-Qi 
7-31 

6.32 
6.60 

6.28 
7-36 

4.42 
3-33 

2.52 
2.24 

3.34 
6.52 

3.47     o.oo 
2.78  i  o.oo 

5-91 

6.74J 
o.oo  6.26 
o.oo  1.38 

3.36 

1-93 

4.62 
6-63 
2.40 
0.83 

The  table  shows  high  oxygen  content  over  sandy  bottom, 
oxygen  probably  sufficient  for  most  fish  over  vegetation,  and  no 
oxygen  on  the  bottom  where  vegetation  was  removed  in  July  and 
August. 

TABLE  VI. 

SHOWING  CO;  CONTENT  OF  WATER. 

Depth  35-40  cm.  (i)  Over  sandy  bottom.  (2)  Over  vegetation.  (3)  Over 
bottom  with  vegetation  removed. 


>i 


i 

3 

I 

3 

• 

i 

3 

June  27, 

1910. 

o.o 

i 

I 

o  o 

O.O 

1    1 

•  i.o 

i.  ! 

6.6 

Julv  22, 

IQIO  

It,  II 

0 

o 

0.0 

O.O 

2-S 

2    1 

i  • 

Average 

0.0 

2 

o 

0.0 

o.o 

3-4 

i.S-.-i 

J.7 

.v.5 

The  water  was  alkaline  at  points  showing  no  ("()._..  The  tables 
do  not  show  the  uniformity  that  might  be  expected.  This  ma\ 
be  explainable  on  the  basis  of  the  place  of  collection.  (.'/Kirn, 
for  example,  which  was  the  plant  removed  from  the  bottom  ol 
ponds  i.  and  $c,  grows  on  bottoms  of  mixed  sand  and  humus  or  on 
a  bottom  covered  \\iili  humus,  and  sufficient  care  \\a-  not  taken 
in  selecting  places  of  collection,  to  make  the  dillcicncc^  heir  ol 
importance. 


ECOLOGICAL    SUCCESSION.  .  7 

There  are  apparently  numerous  factors  which  influence  gas 
content  Birge  and  Juduy,  '11,  p.  54;.  These  are  temperature, 
light  as  affecting  photosynthesis,  distance  between  point  of  col- 
lection and  plants  which  are  giving  off  o\\  m-n  and  u-in-  CO*, 
and  dirt-ction  and  velocity  of  wind  a-  affecting  circulation  of 
water  Hir^e  and  Juday  (p.  55)  state  thai  Kun.pean  Corker- 
have  noted  marked  diurnal  changes  in  the  amount  of  dissolved 
o\\  vjen. 

I-  nun  three  io  lour  hours  were  required  to  make  our  collections. 
(  )n  Jinn-  2~ .  ihr  collecting  began  at  pond  14''  ai  9:30  A.M.,  and 
ended  ai  pond  5*  at  12:30  P.M.,  temperature:  pond  I,  Jn°  (.'.; 
v  and  ~n.  2~  :  and  I4/;,  25°.  Velocity  of  \\ind  <>  mile-  per  liour. 
Thi-  was  a  <  I'.ndv  <lay.  The  sun  broke  through  the  dm  id-  bet  on 
ihe!a-i  i  olle<  i  i"ii  ua-  made.  July  22  was  a  similar  day.  Col- 
lei  tin n  Li  van  at  |ioild  l^l>  at  8:30  A.M..  and  ended  at  pond  ~n 
at  i-'""  M  .  temperature:  pond  I,  25°  ('.;  5.  and  -<i.  Ji>  ('.. 
and  14'',  -V}  .  Mining  the  forenoon,  the  Mm  came  out  -e\vral 
time^.  I  Hi  i  e\ai  i  n  i  ord  was  not  kept  of  the  time  i>r  len-ih  of  sinh 

|ieri..i|-   n|    MIIIM'lille.        All    t  llC   Other   (  ol  li  i   I  ji  .11  -    \\  el'e    made    ill    full 

Minlight .  \\  ind  and  tem|KTature  Were  a-  !olln\\-:  April  Jo,  tem- 
perature  i,  !;-,'_•  ;  51,  i4l->°',  7a,  15'  _•  :  -md  14''.  14";  \\ind: 
3  miles  per  hour.  May  io,  temperature,  23  .  \\iml:  )6  miles  per 
hour.  Ju^t  \\hat  elTect  distance  from  plain-  \\hirh  \\ere  d.iiiu 
photosynthetic  \M>rk  has  on  gas  conieni  i-  not  kno\\n.  It  i> 
highlv  jn-obable  ih.it  colli-ctions  made  near  to  Mich  plant-  \\ould 
lillereni  Iroiu  those  taken  at  a  greater  di-laiue  1-Jir-e  and 
|uda\  ,  '  I  I  ,  |ip.  54  and  60). 

The  MI  miner  col  led  ions  from  pond  ;./  \\eie  taken  from  beiieaih 

the  \\.iiet    lil\  leaves  at   the  extreme  east  end  \\  here  lilie>  h  i\  «• 
di-placed  i  In-  (  'liiirn. 

Collections  taken  after  scraping  the  \  <•-  elation  fnun  the  bottom 
sho\\  \  a  rim  i-  reMilts  depending  upon  the  character  oi  the  bottom 

licile. illl    the   \  e-elation. 

2.  /•}/(»/(»-/!  •:  :'<»i<l.     (a)  Qualitative  Comparison. 

'1      Specie-    of    Plants   and    their    Abundance.      The    i|iialitali\e 
difference-  in  ]>oiid-  a-  -ho\\  n  in  Table-  VII.  and  \  111. 

(2)    (imuih    li'rm   of   the    Plain-.      Pmid     I    i-   dominated    b\- 
plain-.      There  are  no  bn-ad-leax  e<l  -hade  prodm 


8 


VICTOR    E.    SHELFOKD. 


TABLE  VII. 

SHOWING  THE  PLANTS  OF  THE  CENTERS  OF  THE  PONDS. 
Data  by  Mr.  G.  D.  Fuller.     D  =dominant;  A  =  abundant;  C  =  common;  F  =few. 


Name. 


Scientific  Name. 


1't.ixl  Numbers. 


7" 


Nitflld  batrachosperma    

C 

Stoncwort 

C-hcirn  sp.  i  

D 

D 

( 

Pot&mo^cton  luccns  L  . 

C 

C 

F 

"Ml  'III  ll  'I      IKlUl^. 

Naiasflexilis  Rostk.  &  Schmidt. 

C 

D 

F 

F 

1  *i  <\\(  Iwi'ed 

Potamogeton  peclinatus  L  

A 

? 

C 

F 

Filamentous  green 

alt?;p 

F 

F 

F 

A 

Ceratophyllum  demersum  L.  .  .  . 

A 

F 

•> 

N\lmph(Ea  (idvcnd  Ait 

F 

C 

F 

Potatnogeton  americanus  C.  &  S. 

C 

F 

F 

\fvriophvllum  spicatiim  L  

F 

Utricularia  vulgaris  L. 

C 

A 

Castalia  tuherosa  Greene 

A 

A 

Water  shield 

A 

C 

C 

F 

Duckweed  

Lemna  minor  L  

C 

TABLE  VIII. 

SHOWING  THE  MARGINAL  PLANTS. 

Data  by  Mr.  G.  D.  Fuller. 
Roots  Usually  Submerged. 


Common  Name. 

Scientific  Name. 

Poinl  Nuiiil'crs. 

i 

5<~ 

F 

70 

,4/' 

Bulrush 

Scirpns  validus  Vahl  

F 

F 
F 

C 
C 
C 

Cattail 

Typha  latifolia  L                 

Mermaid  weed   .... 

Proserpinaca  palustris  L  

,,. 



Roots  Submerged  at  High  Water. 


Sedges 

C 

F 

Pines 

Pinus  Banksiana  I>amb  

C 

C 

F 

Shrubs    (other    than 

F 

F 

Button  bush 

Ccphaldnthus  occidcntdlis  I..  .  . 

F 

C 

Sii{  i  v   spn 



F 

C 

F 

Pond  5c  shows  the  beginning  of  shade  producers  such  as  the 
water  lily  and  of  plants  which  reach  above  the  surface  of  the 
water.  Pond  "ja  has  a  large  number  of  emeri;iii^  plants.  In  one 
end  of  this  pond  there  are  many  more  of  these  than  in  the  oilier. 
In  pond  14^  emergents  are  dominant. 

(3)   Animals.     The  different    >pccic>  of  animals  and    their  ar- 


ECOLOGICAL   SUCCESSION. 


TABLE  IX. 

LEECHES. 


'. 


• 
• 


Glos\iph<>nia  fusca  Castle 

puniliit-i  Leidy * 

ft-r:-i'l'i  Y'-rrill .  .  * 

\I-:'r»li,l,-llii  -ay ... 

//.-  '•  •  nill 

I'lijiui'ili-llii  para        -i  Say 
•'nlfllii  rn^»  ii  \Vrrill 

<  ifill<»ll •  :  :ta 

//..  n 

irs  and  letters  see  p.  1 1 . 

TABLI-   X 

SPH^ERID.C  ASP 


• 
• 
• 
• 


• 
• 


S 

M 


i  NIONID/1 

/  afn  1  am 

i  y 

loiltil  H:  

.  •:  ::  /             :tia  Lea.  .  . 
H'll    1    KIN  V 

.\f n  m  Lins 

M  ..  'i  line 

\l  u     :i. '.-..  •'• 


TAHLE  \  I 

ULS. 


ne. 

i 

'     1 

* 

*            * 

t 

... 

* 

*            * 

* 

if 

* 

i-h\ 

F 

1                      ' 

( 

(  '*) 

1   y  IMM.I  i.l.r  : 

1 

1- 

.     

*                    * 

*                    * 

*                    * 

« 

* 

* 

*                    * 

* 



1 

i                      ' 

A 

\ 

* 

* 

( 

A 

"it'ttthtti  tjrw/A,vrti  Say  

* 

1C) 


\  h    ink    E.    Sill   i  r<  IRI). 


TABLE  XII. 


Name. 

1'  .ml  Nuinlii-t-. 

i                 5'' 

7" 

. 

.t° 

11  valellti  KnickcTbockeri  Bate  

c 

C 
F 

i 

C 

c» 

A 

1- 
A 
* 
* 
C 

? 

•  ii  H  ^<  >n  \  v  fffddlis  Smith.             ... 

\fdncdsfllus  ddnitlsi  Rich                               .  . 

Asellus  communis  Sav  

Cambarus  itntnunis  Hagcn  

F 

F 

c 

C 

F 

Cambarus  blandiniti  acutos  Girard.. 

TABLE  XIII. 

AQUATIC  INSECT  LARVAE  AND  NYMPHS. 


Name. 

Pond  Numbers. 

i 

S< 

7" 

I4/- 

3° 

May  flies: 
Ccenis  sp  

* 
* 

* 
* 

* 

* 

Siphlurus  sp.      .                                    

* 
* 

* 

Callibcetis  sp  

Neuroptera  : 
Chauliodes  rasticornis  Ram.        

* 

* 
* 

* 

ifc 

* 

Damsel  flies: 
Lcstes  sp 

l".nallagma  sp  

* 

* 

* 
* 

Ischnura  verticalis  Say.  .  .               

* 

Dragon  flies: 
Tratnea  laccrata  Hagen                                .  . 

* 
* 

* 

* 
* 

Celithctnis  fponinn  Drur\* 

Libellula  pullcella  Drurv  

* 
* 
* 
* 
* 

Gomphus  spicatus  Selvs.         

* 
* 

* 

Leucorhiniu  inlacta  Hagen 

* 
* 

.4  nax  junius  Drury  

* 

Sympcttuni  rubii'unduliim  Sav 

SympelTum  sp.                                           ... 

* 

'• 

* 

Caddice  worms: 
Gcora  sp 

* 
C 

LcplocerincB  sp 

F 

Neuronio  sp.      ...                             ... 

* 
* 

j 

C 

* 
* 
* 
* 

\ 

* 
* 

Diptcra  larva?: 
Chironomid  larv;c  

* 
* 

* 

Stratiomyid  larva?  

Tanvpus  sp 

Tipulid  larva?  .                                       .  .        

* 

Cerutopogon  sp. 

Hemiptera: 
Ranalra  kirkuldvi  Bucn 

* 

* 
* 

* 
* 
* 
* 

* 

* 

* 

* 

F 

Corixa  sp 

Kanalra  fusca  P.B  

* 

i 

* 
* 

* 

Belostoma  Jlntninca  Say.  .  .            ... 

Notonefta  undulnta  Sav. 

Jiuenoa  platycnemis  Firb  

\\'ator  striders: 
Gcrris  rufoscuffllalus  Lat 

* 
* 
* 

* 
* 

Mesovelia  l>i.\ienata  I  "hi.. 

ECOLOGICAL  SUCCESSION.  II 


TABLE  XIV. 

HIGHER  VERTEBRATES. 
The  fish  are  shown  in  Table  XXI.  page  17. 


;  Numbers. 
Name. 


if 


* 
* 


1  /     "  i  iita  Lat * 

Rini,i  f't  i  

nnla  Ag. ...  *  * 

sogtaphica  Les * 

/';.  u;/.  •  »J.V   Raf * 

Hill  '  .'  '  '  ? 


I  h>    :  'lie  muskrat  is  indicate*!  !>y  i;  :  h«>li-».  ii<'xi~.  tt.uk-. 

I  iiit  ii'iiu-  li.t\  ••  Ijcen  seen  except  in  tin-  "M«-«t  p"i; 


with  resjx'ct  to  tin-  ages  "I  tin-  pond-  are  -ho\\  n  in 
Table-  IN.  !••  NI\'.  Letters  indicate  ivl.tii\r  abundance:  l:  — 
few;  (  •  ommuii  ;  A  -  ahund.iin.  The  -i.tr  i-  n-ed  t<>  indi- 
<  .IN  piv-i  in  ••  \\  here  relative  abundance  lia^  not  been  .iMvri.iitird. 
l-i  comparison,  a  fifth  j)oiul  (No.  30  U  .idilrd  ;  tlii--  i-  nldi-r  ih.m 
ill.  others  in  rvery  resfK'ct  and  coni.iin^  certain  species  <>'  ini- 
["'M.iiirr  i<>  fi-li  which  are  not  found  in  .ui\  o!  tin-  ntlu-r--. 

I  I  >i-i  ii—  imi  of  the  Tables.  The  i.il-lr^  rcjur-cin  nut  <»nl\ 
nun  Ii  ..iidul  •  ulleftinjj,  but  long  e\]H-rini»v  \\itli  the  •  "inmuii 
forms  of  the  ponds.  An  inspect  inn  <>i  tin-  t.il>K--  -lu>\\  -  ih.it  tlu-rr 
are  <lilt<  n  u.  es  in  the  species  in  tin-  dilh-n-in  |)umU  .ind  ili.it  tin- 
dilN-n-in  «•-,  .tre  correlated  with  tin-  ages  <>|  tin-  pund-.  l-'m-  ex- 
ample, in  the  case  of  the  let  •  lie-.  T.iMe  IN.,  page  9,  nmie  <>t 
tlie  ~|iet  ie-  ul  the  youngest  pund  i-  !i>iin<l  in  .ill  <>t  the  pmnU  and 
iiuiic  u|'  tin-  -pccies  uf  the  ul<le-t  i->  I'unnd  in  (he  youngest.  \- 

iillvK     .1-    \\e    pass    from    the    \ulll)^e-!     to    the    oldest     \\e    lluli- 

tll.  II    5]  di-.ippear  and  are   replaced   l.y  uther  species.       The 

same  \\ill  be  seen  to  be  true  ul'  the  uther  -ruiip-.  A  -imilar 
relation  i->  illustrated  also  wlu-re  \\  e  have  been  able  to  estimate 
relatixi  ibnndance.  In  -«\\\^  cases  the  number  i-  greater  in 
the  older  ponds;  in  other.-,  le»  in  the  older  jiuiid-  e.  .  1  1  yullelti 
Kni(kt-r!>txkfri,  Table  XII..  p  ;.<  [o). 

The  (  -a  -i  -of  the  caddice  \\urm>  an<l  uther  ai|iiaii<    insects  which 
are  plat  (  d  in  the  water  by  the  laying  tfmale,  i-  u!"  e-pecial  inte: 
as  the  iv-nllin^  distribution  is  jirobabK    either  a  matter  of 


12  VICTOR    E.    SHELFORD. 

tion  on  the  part  of  the  female  during  tin-  breeding  reason  or 
striking  elimination  of  all  eggs  laid  in  tin-  ponds  in  which  the 
larvae  are  not  found. 

It  is  evident  that  ecological  types  (here  represented  by  the 
various  species)  succeed  each  other  as  the  ponds  change  with  age. 
Succession  is  here  as  elsewhere,  a  succession  of  all,  or  at  least  a 
majority  of  the  animals  present. 

(b)  Quantitative  Comparison.  (i)  Vegetation.  Vegetation 
is  evidently  a  good  index  of  the  content,  or  the  relative  numbers 
of  the  different  species  of  plants  and  animals.  In  Table  I.,  page 
3,  we  note  that  more  than  two  thirds  of  the  bottom  of  pond  I 
is  covered  with  humus.  Vegetation  covers  about  70  per  cent, 
of  the  area.  In  pond  5r  vegetation  covers  about  95  per  cent, 
and  in  7  a  about  99  per  cent,  of  the  area  and  in  146  100  per  cent. 
If  the  plants  of  each  unit  area  were  equal  in  volume,  these  per- 
centages would  represent  relative  volume  also.  More  of  the 
plants  of  the  older  ponds  reach  to  the  surface;  plants  are  closer 
together  in  the  older  ponds.  It  is  obvious  from  inspection  that 
the  volume  per  unit  area  is  greater  in  the  older  ponds. 

A  single  test  was  made  with  a  large  tow  net.  The  net  was 
drawn  a  distance  of  40  feet  in  three  of  the  ponds  and  the  volume 
of  vegetation  torn  off  by  the  net  was  measured  by  displacement 
and  reduced  to  terms  of  100.  This  would  give  relative  volume 
if  all  plants  were  torn  with  equal  ease. 

Finally  Mr.  G.  D.  Fuller  and  myself  have  made  an  estimate 
based  on  several  inspections. 

TABLE  XV. 

SHOWING   MEASUREMENTS  AND  ESTIMATES  OF   RELATIVE  V»M  \n    OF    \  i  DICTA- 
TION PER  CUBIC  UNIT. 


fond. 

1 

7" 

'4'' 

On  the  basis  of  areas  of  vegetation  

70 

9S 

99 

IOO 

Tow  net  collections.              ...             .... 

14  c.c. 

30  c.c. 

IOO  C.C. 

Estimate  

!<) 

(0 

60 

I  OO 

(2)  Plant  and  Animal  Food.  The  plant  and  animal  tood  in 
solution  is  expressed  in  a  general  way  by  the  sanitary  analysis. 
The  results  of  a  single  analysis  with  the  total  carbonaies  added, 
arc  given  in  Table  XVI. 


ECOLOf.ITAL    SUCCESSION.  I .} 

TABLT-:  XV  I. 

SHOWING  CONTAMINATION  OF  POND  sc  AND  ELEMENTARY  FOOD  SUBSTANCES  ANP 

<     \KIii>NAII->    IN    Al.L. 

Single  analysis,  Oct.  26.  1909. 


' 

\\ 

(  liliirim-  

JO  7 

90 

i  j  * 

16  T. 

siiiiiii  iiiia  

O  I  OO                O  1  ~  '  ' 

Trace 

o  005 

All>iiiiiit)niil  aiuinonia. 

O   I                     O  I  S" 

O.I7C 

O  2  Su 

o  °oo 

Nitrites. 

1  '  1  •   -»  lit                It 

I  '  ICC 

Nitrati--  

o.i  60           o  030 

o  o^o 

o  040 

o  060 

1  •  i.il  .  ,irl.«.i.    '• 

138.800        139-  s 

160 

if,,, 

[11.500 

'I  In-  chlorine  content  is  regard  »•  I  as  a  good  index  of  the  presence 

or  .ili-fin  i-  nl  sewage  contamination,  f\cn-i.i  lu-in-  hijji  in 
clilnriiif  i  iiinpiiiiixls.  49.7  part-  pi-r  million  in  pond  5.  \\uiild 
such  contamination.  I  "mil  \«i\  iv.vntlv  a  h<m-e  was 
mi  ilu-  margin  of  pond  5*:  tin-  pond  i-  -lill  -ul»ifct  i« 
<  ..iii.iinin.iiion  l.\  domestic  fowls. 

I  ree  .inniiniiia  is  tin-  final  sta^f  in  llx-  l>ii.ikin-  <l<>\\n  «\ 
pn.ii-iiU  .uxl  .i|i|M.-ars  also  in  animal  excreta.  It  is  used  by  plants 
.ind  f  \  iili-nlK  plants  consume  it  in  |m  i|»>rti<m  to  tlu-ir  \  ulun 

\llniiix -imiil  ainmontu  prohabK  n-pn  -cut-  mctalmluid-  in  -ulu- 
li'Ui.  because  tin-  \\atcr  \\as  filtered  l»Hmf  dfic nninatimi-  were 
m.xli  .  Sewage  is  rich  in  such  compi.umU  and  -anii.ir\  analysts 
ha\  i-  I •  ui ml  that  the  number  of  bacteria  is  closely  correlated  with 
.mimim  Hi  albumenoid  antmonia. 

;  I'.aiii-ria.  1  >i  .  I'.  G.  Hfiiifiiiann  and  Mr-  Class,  "I  the 
I  >f  ILII  l  nn  nl  nl  Bacteriology  of  the  l'ni\er^it\  o|'  ("hie.i-o.  \<i\ 
kindU  made  the  counts  of  the  bacteria.  The  re-nil-  an-  i;i\en 

in   fable  X\  II. 

XVII. 


\l  K,  >l.|.       H  M    IKRIA 

I'KR   cc..   CAI-AI                 -                                    \  i 

i 

i 

i  >,  tobei  -•'•.  1909  

April  _•>;.    i<ji  i  

1.  2QO                J.'iOO 
1.1                         SOO                                               4. 

SO? 

I  In-  minil'i-i  In  ir  •!»•-  n«'i  •  "i  i ---I  ii  mil  tn  tin-  .ill'iiini-ii'  '!•!  .umih  mi.i.  hut  iua> 
partially  accounted  for  by  the  fact  that  tin-  Imttli-  \\  •li-ntally  n|ii-in-,l  m-.u 

llir  -in  i.i.-i-        l  in    April    J'i    a    oillrction    at    tin-    -in  I'.iri-  nf    tin-    p»ii'l    -lm\\i-i| 
.it 


14  VICTOR    |  .    -Ill  LI-UKD. 

The  table  shows  that  the  number  of  bacteria  is  greater  in  tin- 
older  ponds,  except  in  5*  which  is  noncomparable  because  of 
contamination. 

(4)  The  Plankton.  The  study  of  the  plankton  has  been  prac- 
tically limited  to  the  Entomostraca — the  most  important  food 
of  young  fishes.  The  presence  of  a  larger  number  of  rotifers 
and  protozoa,  etc.,  is  observable  as  we  pass  from  the  younger 
to  the  older  ponds. 

The  number  of  Entomostraca  in  approximately  90  liters  of 
surface  water,  to  a  depth  of  10-12  decimeters,  is  given  in  the 
table  below.  It  was  thought  best  to  simply  clip  the  desired 
amount  from  the  water  while  walking  and  strain  the  dippings 
through  a  bolting  cloth  strainer.  After  the  first  collection  this 
was  repeated  in  as  uniform  a  manner  as  possible  and  Birge  net 
collections  were  made  at  the  same  time  for  comparison.  There 
was  no  great  discrepancy  in  the  results  of  the  two  methods  of 
collecting,  except  in  the  case  of  Ostracoda  in  pond  i4/>.  As  com- 
pared with  dippings,  some  Birge  net  collections  showed  less 
Ostracoda.  Ostracoda  wrere  probably  started  from  the  bottom 
by  the  feet  of  the  collector  but  were  not  by  the  drawing  of  the 

Birge  net. 

TABLE  XYIII. 

THE   NUMBER   OF   ENTOMOSTRACA   IN   90  LITERS  OF   WATER. 


i 

5<-- 

7" 

146 

September  3,  1909  

5S6 

S  ?9 

2,77  •! 

November  13,  1009.                       ... 

200 

106 

797 

7  SO 

March  26,  1910. 

42 

I1  ' 

I  2 

oo 

Mav  31    1910 

7,407 

1,014 

4,168 

3,600 

Julv  22,  1910  

160 

200 

52O 

''.480 

April  26,  1911  .    .    .  . 

I.2SO 

ISO 

I4O 

=>  ^s 

M  av  101911. 

IOO 

800 

I  2  :; 

s.  1  25 

Total  of  6.  ... 

"5.249 

2,310 

S.v 

16,080 

Average  of  6  

874 

385 

927 

180 

The  table  shows  that  with  the  exception  of  pond  5^,  which 
is  probably  noncomparable  because  of  contamination,  the  older 
ponds  contain  most  Entomostraca  except  in  early  spring  when 
conditions  are  somewhat  reversed. 

A  large  quantity  of  plankton  in  old  ponds  has  been  noted  lor 
sevcral  years  in  connection  with  class  work.  For  comparison 
with  the;  ponds  under  consideration  we  have  studied  \Yoll  Lake, 


ECOLOGICAL    SUCCESSION. 


and  two  small  ponds  near  it.  The  younger  of  the  small  ponds 
will  IK.-  designated  as  I.  and  the  older  one,  II.  They  differ  with 
the  exception  of  the  margin  vegetation)  in  much  tin-  -aim-  manner 
as  (In  ponds  I  and  ~a  of  tin-  -fries  of  special  -tudy.  While  Wolf 
Lake  i-  in.i  -trictly  comparable  to  the  other-,  ii  is  ecologically  the 
youngest,  I- -cause  of  its  greater  area  of  hare  hoi  torn.  The  collec- 
tions made  Sept.  3,  1909)  were  four  jn  numher  in  Wolt"  Lake, 
tour  in  pond  I.,  two  in  pond  II.,  one  half  irom  the  open  water.  ,md 
one  hall  Irom  among  vegetation.  S-\eral  collection-  \\ere  made 
Apr.  The  numbers  given  are  the  ,,\,  of  all  collections 

made  on  t  ho-.-  dates.  They  were  net  collections  made  in  as  uni- 
form a  manner  as  possible. 

TAHI.E  XIX 

OWING  DIFFERENCES  IN  NUMBERS  OF  ENTOMOSTRA<  \> 

\viin  DIFFERENCES  IN  I 


I. 


II. 


Septeml 


. 


•  >cera. 

Total. .  . 

1          |KxJa. 
1         'icera. 


96 

IS 

I  hi-  table  -hows  the  same  feature-  a-  the  pnvedii' 
i)    I'll*    I    irg<  r  Animals.     Little  ha-  been  d"iie  in  e 
the  relaii\e  niimbfr  or  volume  of  the  lai-er  animal-  in   the  dif- 

i  p.,nd-.     A  general  idea  is  given  below  in  Table  XX.      Phis 

Nl'MBER  OF  TH!  HI 

COLLEl  I  l<  ' 


ler. 

i 

(    I.I'  !                                          

i 
1 

371 

[Q 

I.  I 

i.  : 

i.; 
421 

- 

:-  .lur   nuiiiily   tn  -mall   iiniiii  pods. 


16  VICTOR    I£.    SHELFORD. 

is  based  on  the  general  impression  which  ha-  been  acquired  in 
taking  classes  to  these  and  other  ponds  of  similar  character 
several  times  per  year  during  six  years.  Secondly,  by  taking 
the  time  required  to  make  a  representative  collection  from  the 
different  ponds.  On  the  basis  of  this  experience,  the  figures  given 
in  the  table  are  thought  to  be  very  conservative.  That  there  is 
a  far  greater  number  of  animals  and  a  greater  volume  of  animal 
substance  in  the  old  ponds  is  very  easily  demonstrated  to  any 
one  by  inspection. 

TABLE  XX. 

SHOWING  AN  ESTIMATE  OF  THE  RELATIVE  NUMBERS  OF  THE  CHIEF  ITEMS  OF  FISH 

FOOD  IN  THE  DIFFERENT  PONDS. 


i 

$<•' 

7" 

146 

Entomostraca  

T.2 

I  c 

•jc 

IOO 

Chironomid  larva?  

8o7 

80' 

80^ 

IOOJ 

Sphaerida?. 

o 

^O 

^O 

I  OO 

Gilled  snails  

20 

•3Q 

en 

IOO 

Pulmonate  snails  .    .  . 

IO 

-3Q 

^O 

IOO 

Arnphipods  

CQ 

7O 

i  ,.  i 

IOO 

Decapods  

IO 

7O 

ZQ 

IOO 

Insects  

40 

60 

oo 

IOO 

Fish.  . 

80 

100 

7O 

^0 

Previous  to  being  drained  pond  140  should  be  rated  at  70  for  fishes. 

While  the  results  here  presented  are  not  such  as  to  justify 
conclusions  concerning  details,  we  may  state  that  the  amount  of 
life  per  unit  volume  unquestionably  increases  as  the  ponds  grow 
older,  at  least  up  to  stages  like  146.  Qualitative  differences  are 
shown  in  the  Tables  VII.  and  XIV.,  and  the  total  number  of 
species  recorded  in  each  pond  is  about  the  same,  tin-  actual 
quantity  is  far  greater  in  the  older. 

IM.    THE  CAUSES  OF  SUCCESSION  01    FISH. 

A  discussion  of  succession  must  be  made1  with  reference  to  all 
the  organisms  of  the  habitat,  or  at  least  a  large  number  of  them 
considered  in  mass.  Succession  of  one  group  of  organisms  t.ikin^ 
place  without  the  succession  of  others  in  the  ^une  environment 
seems  improbable.  A  discussion  with  reference  to  tish  UHIM 
take  other  organisms  into  consideration. 

i.  Statement  of  the  Problem. — A  rlrar  understanding  of  the 
problem  at  hand  will  perhaps  be  facilitated  bv  a  careful  Mate- 


ECOLOGICAL    SUCCESSK  >N . 


ment  of  the  question  before  us,  after  which  we  shall  di^eu»  tin- 
available  data  with  reference  to  the  relations  of  fn-h  to  the  dif- 
ferent ponds,  from  the  standpoint  of  their  area,  their  depth, 
mineral-  and  gases  in  solution  and  finally  the  available  food  for 
youn.u  and  adults.  Competition,  living  pla<v  and  breeding  place 
(A  the  li-h  will  be  discussed  as  fully  as  data  will  permit. 

TABLE  XXI. 

iM-lklhi    IIOX   OF    THE    FlSH    AND   THEIR    R  KI.A  Tl>  >N    To    Hull 

I  In-  li-tt'-r-  .iiul  numbers  at  the  heads  of  the  column-;  n-tVr  tn  the  various  isnlati-il 
parts  ol  pon  ;  indicates  the  presence  of  the  species;  !'•   that  vi-ry  young 

nii-n     wi-ri-  I'linul  in  numbers  and  the  >;  •:<••!  in   looc;,    in  <ir   n.     The 

ii'iiiifii'  l.itun-  .tii'l  bottom  preference  data  a:  :        i.ud-on. 


Name. 


•    in.. ml.'  '  '  TMS 

'•l.i  .  ••   -ides.  ...        B 

{ill  fnlliJus     B 

Blue-spotted 

-mi  li-li  .us H 

Pumpkin  iced  '.-'tis 

...       H 
\\.iiinouili    I  :»$ 

• B 


V* 

14* 

.iiiil. 

•IH 

iii'l 

I 


b 

(    In. 

bullhead. 

I  ad 

rel 

Mini  ininn... 
i  i.  >l<l<-n  -hincr. 


\i-ll..\\    hllllh 

HI..,  k  bullh.-.i-l 


:  escens  . 
•i   micella 

us 

Its 



•niiiitatiis 

mi 

:iCilS    .  .  . 

natalis 
i .  mrlas  . 


Mud. 
r.  i<  .  k  an  I  sand.      In  ; 

H      B  |... 

In  part,  linn  k 

B      B    .  .  .  Mini   .in-1   -.in. I. 
B     B    ...  . 
B      B     .  .  .   Mud  vAI>l...u  I.     Mu.  k  pi. 

B      B  Mud. 

*     ... 
B      B        " 


Tin-  pmbli-m  of  the  causes  of  sun v  — ion  max   In-  -tau-d  in  tuo 
\\  a  \  3 : 

liiM'Kin-  interpretation:  \\li\  are  the  piniieer  ti-he-  of 
a  (mud  -ncrci  .led  as  the  pond  ",r"\\-  older,  by  ti>lle^  of  dilteiciil 

habits? 

I  in  le  pendent  ol  interpret  a  ti<  m  :  \\  hy  are  tin-  ti-he-  ot  pond 
I.  noi  in  the  older  ponds  and  the  ti>he-  ol  the  older  |>oiid-  not 
in  pond  I ..  u  hen  the  channels  between  I  hem  ha\  e  been  o j  ic' n  until 
the  paM  leu  \  • 


1 8  VICTOR    E.    SHKLFORD. 

2.  The  Cause  of  Succession — Environment. — (a)  Area  of  the 
Ponds.  A  comparison  of  Table  I.,  page  3,  with  Table  XXI., 
page  17,  and  a  comparison  of  Table  I.  of  the  preceding  paper  with 
the  map  (p.  131  of  the  preceding  paper)  show  that  most  of  tin- 
fishes  are  in  ponds  of  all  the  available  areas  of  the  region,  with 
the  exception  of  several  species  which  are  confined  to  pond  I., 
and  which,  on  account  of  their  numbers,  could  find  no  advan- 
tage in  such  close  quarters.  Evidently  no  part  of  the  aiiM\er 
lies  in  the  matter  of  size. 

(6)  Depth  of  the  Ponds.  A  comparison  of  the  records  of  depths 
given  in  Table  II.,  page  4,  with  Table  XXL,  page  17,  shows 
a  situation  parallel  to  the  one  with  reference  to  area.  Species 
are  in  ponds  of  various  depths  and  are  absent  from  ponds  of 
depths  the  same  as  and  greater  than  the  ones  in  which  they  are 
found.  These  ponds  are  shallower  than  the  waters  which  many 
of  the  species  commonly  occupy.  The  matter  of  depth  does  not 
seem  to  be  of  importance  in  the  answer  to  the  question. 

(c)  Minerals  in  Solution.  The  minerals  in  sqlution  in  t In- 
different ponds  on  October  26,  1909,  are  given  in  Table  111. 

(1)  Qualitative  Differences.     The  minerals  represented  in  tin- 
analysis  are  those  normal  to  wraters  inhabited  by  fish  and  probably 
important  to  fish.     No  zinc,  lead,  aluminum,  silver,  or  coppi-r, 
metals  highly  poisonous  to  fish  (Marsh,  '10),  were  found  and  there 
is  no  reason  to  expect  their  presence  at  another  time  of  the  year.1 
From  the  qualitative  standpoint  there  is  no  reason   to  assign 
importance  to  minerals  in  solution. 

(2)  Quantitative  Differences.     The  total  solids  given  in  Table 
III.,  p.  5,  lie  between    the  two  extremes  given    by  Marsh,   '10, 
as  probably  not  affecting  fish  and  as  "normal"  for  waters  which 
are  known  to  support  fish  in  numbers.     He  gives  484  parts  per 
million    for   the    Potomac    River  and   242   for  other  fish   waters. 
Nor  is  a  very  great  seasonal  variation  to  be  expected,  because 
most  of  the  animals  live  through  the  winter  and  the  vegetation 
disintegrates  very  slowly,  especially  through  the  cold  weather, 

1  Because  of  the  small  amount  of  inwash,  this  sc-t  of  ponds  afford  an  IIIIHMM] 
opportunity  for  the  study  of  the  effect  of  a  varying  amount  of  vegetation  »n  ilu- 
chemical  composition  of  the  water.  For  a  statement  of  the  salts  ti«-.l  up  l>\  plant- 
see  Pfeffcr-Ewert,  'oo,  page  410. 


ECOLOGICAL    SUCCESSION.  1«| 

in  the  spring  it-  place  i-  lakc-n  by  iu-\v  vegetation  a-  rapidly 
as  the  decomposition  of  the  old  takes  place. 

Prom  our  knowledge  of  the  composition  of  river  water  in- 
habited liy  all  the  fish,  before  and  after  the  Hood-.  IK.  -real 
importance  could  be  assigned  to  mineral-,  even  though  the  com- 
plexion ot  the  analyses  changed  with  the  season.  However,  no 
positive  '  onclu-ion  could  be  drawn  without  careful  stndv  of 
the  ln'hin'inr  n-nctions  of  fish  to  minute  quantitie-  of  -alt. 

'/  Gas  The  results  of  gas  determination  are  -i\en  in 
Table-  IV..  \'.,  .md  V|.,  pp.  5  and  6.  Table-  IV.  and  V.  -ho\\  the 
'"iitent  «,f  the  open  water,  abo\  e  the  xe^etation  and  -andv 
bottom,  to  be  -ulticient  for  fish  in  all  the  pond-.  Jndav  and 
liir^e.  'n.  p.  iv>.  state:  "  Konig  found  that  he  could  keep  ti-h 
'kind  not  -pei  ilied  i  in  water  which  contained  j.o.S  C.C.  and  i  j8 
ol  di--o|\cd  oxvgcn  per  liter  without  any  apparent  ill  et!<  .  ts. 
'I  horner  loimd  that  a  fish  epidemic  \\a-  i  .m-ed  by  the  ab-ence 
of  free  oxygen.  I  loppe-Seyler  and  1  >mx  an  -t.tte  that  trout  \\  hich 
\\ere  kept  from  one  and  a  half  to  t\\o  and  a  quarter  hour-  in 
\\aier  ha\iiiL;  <>nl\  Irom  o.cjN  to  1.71  c.<  of  o\\-eti  per  liter 
shoued  marke.l  -uiis  of  dyspmra.  r.itoii.  in  experiment-  -MI 
young  i.iiubou  in.  i  it,  found  that  a  fall  in  tin-  amount  of  di— « >\\  e.  1 
o\\-en  be|o\\  one  third  of  the  normal  amount,  /'.  e.t  belo\\  j 
per  liter  ol  \\atei.  is  prejudicial  and  geiieiallx  fatal.  Some  indi- 
\idn.il-  ho\\e\er,  \\i-re  able  to  sustain  life  l.-r  lon^  period-  in 
\\atei  \\hich  (oiitained  only  minimal  tr.ue-  of  <li--ol\ed  oxygen. 

"  l\  nau  i  he  I  ou!  id  that  carp  kept  for  an  hour  and  twenty  minuie- 
in  \\ater  \\hich  i  .  .ntained  1.33  c.c.  "i  o\\-en  per  liter,  did  not 
sh«.\\  an\  -i^n-  ol  dyspild-a,  \\hile  other-  became  d\-pno-ic  in 
\\atei  containinv;  from  2  c.c.  tt)  ,V  I  C.C.  of  tin-  gas." 

I'.ii'^e   and    Juday   state   also   that     Mat  kin, lu    tn.ut    ha\e    been 
taken    tiom    \\ater>  with    I    C.C.   per  liter.       Ti-h   di-ea-e-   .ti\   -aid 
to    be    nion-    pn-xaK-ni    in    low    o\\-eii    i  oim-ni       Knaiithe.    '07   . 
In   thi-  case   I  here  is  no  reason  for  a--i^ninu   importance   to   the 
Oxygen  content   of  the  open  \\ater-  frei|iiented   b\    ti-h.  and   this 
factor    i-    nearly    uniform    in    the   different    pond-.      The   oxyj 
con  t  i'n  t  of  the  Lot  torn  i-  of  great  import  a  IK  e  and  u  ill  be  di-cii- 
laler  in  connection  with  breeding. 

Temperature.      A   -in-le   -el    ..I    reading-    taken   in    the   late 


20  VICTOR    E.    SHELFORD. 

afternoon  of  a  warm  sunny  day  showed  less  than  i  degree  of 
difference  between  the  different  ponds  and  the  readings  \\ere  not 
repeated. 

(/)  Excretory  Materials  in  Solution.  Dacknowski  i'o6)  (see 
Cowles,  'li)  found  that  certain  unknown  water  soluble  sub- 
stances present  in  bog  water  are  poisonous  to  plants.  Colton 
('08),  and  authors  cited  by  him,  found  that  the  excretory  prod- 
ucts of  animals  are  toxic  to  the  producer,  and  sometimes  to 
other  organisms.  This  is  a  physiological  basis  for  succession. 
Knauthe  states  that  the  effect  of  fish  on  their  environments  is 
important,  but  little  of  definite  character  is  known  concerning  it. 

(g)  Food.  The  food  of  the  fishes  from  these  ponds  has  not 
been  studied,  but  knowledge  of  the  food  habits  of  the  same  species 
was  acquired  from  the  study  of  literature,  especially  the  work  of 
Forbes  and  Hankinson.  The  species  found  in  the  ponds  being 
known,  each  pond  was  inspected  with  reference  to  the  things 
eaten  by  each  fish  species.  Forbes  gives  the  percentage  which 
each  item  constituted  in  the  individuals  which  he  studied. 

(i)  Qualitative.  The  method  of  obtaining  the  results  con- 
sisted in  adding  Forbes'  percentages  ['80,  p.  38]  for  the  different 
items  of  food  for  each  species  found  in  each  pond.  For  example, 
take  the  food  of  lake  specimens  of  the  perch.  These  were  found 
to  have  eaten  fish  food  existing  in  pond  i  as  follows:  decapod- 
rated  at  14  per  cent.;  unidentified  fish,  50  per  cent.;  Acan- 
thopteri,  8  per  cent.,  giving  a  total  of  72  per  cent.  Pond  i 
contains  72  per  cent,  of  the  food  of  lake  perch;  Cyprinidae  rated 
at  28  per  cent,  do  not  occur  (see  Table  XXII).  For  the  youngest 
individuals  (under  one  inch)  of  all  the  species,  all  the  ponds  are 
qualitatively  equal.  Hankinson's  data  on  Walnut  Lake  species 
show  that  all  our  ponds  arc  about  qualitatively  equal  tor  the 
fish  which  he  considers. 

An  inspection  of  Table  XXII,  p.  21,  shows  that  in  no  case  an 
the  fish  confined  to  the  place  where  their  food  is  qualitatively  best, 
in  fact,  as  a  rule,  the  fish  are  in  the  pond  where  the  food  is  qualita- 
tively poorest.  The  available  data  on  the  food  of  fishes  shows  that 
the  fish  eat  food  available  where  they  live,  rather  than  that  their 
distribution  is  due  to  the  presence  or  absence  of  certain  foodspecies. 
Excluding  students  of  the  food  of  animals,  the  idea  that  food 
determines  distribution  is  commonly,  though  erroneously,  held. 


TABLE  XXII. 

QUALITATIVE  EXPRESSION  —  VALUE  IN  FISH  FOOD. 

*  indit  Lin-s  presence  of  the  species  being  considered.  The  averages  are  not 
avi-rag'-s  <>f  the  figures  given  here,  but  of  all  Forbes'  iu-ms  taken  <oparau-ly;  their 
number  is  given  in  the  last  column. 


Pond  Numhc 

\ 

hems 

- 

ics. 
IVr 

M 

ptt'ru.    •  •:  :   "                1—2  in.                  98           IOO 

IOO 

IOO 

2-4  in.            100        100 

IOO 

IOO 

Adults. 

Average. 

1-3  in.            100         inn 

ioq 

Adults.            81 

- 

Adults.            60          60 

80 

lltS.                    QI 

91 

91 

Average.        *88 

92 

i  in.                100 

1-4  in.             96 

96 

too 

ilts.            58          71 

7i 

Average.        *88          <;i 

1—4  in.            100 

[   O.I 

.Its.              8l 

Average.        *95          95 

96 

All.               *ioo 

I  .  ... 

1-3  in.            100 

[OO 

3-4  in.              76 

Adults.             72 

Adults.             56          '•] 

Average.       *83 

All.                *io. 

ni'l  mfliis.  Various 

young.          100 

[OO 

Adi 

Average.        *90        *./• 

Various 

young.       100 

[00 

Adults. 

Averag 

-• 

i  '  i  in.             IOO         IOO 

in., 

[00 

A.I  .                     4" 

A\                  *;n        *;o 

*7i 

71 

/  limi.  .  .                                    \  lult.              *.*3         *33 

*33 

68 

Ahr.i"                                                Ailult.               86         *86 

*86 

natiilis  Adult.                               '14 

VICTOR    E.    SIIKLFORD. 

(2)  Quantity  of  Food.  The  quantity  of  food,  like  the  quality, 
is  one  of  the  reasons  assigned  for  the  distribution,  migration,  and 
extinction  of  animals.  Although  my  data  on  quantity  of  food 
in  the  ponds  is  not  as  good  as  that  on  quality,  a  comparison 
is  presented  in  Table  XX IV. 

In  the  case  of  the  young  fishes,  the  table  follows  from  a  com- 
parison of  the  tables  of  Forbes  with  our  own  on  Entomostraca. 
The  quantity  of  food  for  the  youngest  individuals  of  all  species  is 
practically  that  of  the  Entomostraca:  Pond  I,  32;  pond  5<r,  75; 
pond  ja,  jj;  pond  i±b,  wo.  For  the  adults  and  young  from 
one  inch  to  four  inches  in  length,  an  estimate  of  the  quantity  of 
food  in  each  pond  for  each  species  has  been  made  by  averaging 
the  ratings  of  the  principal  articles  of  food  given  for  each  species 
by  Forbes. 

TAULE  XXIII. 

METHOD  RATING  PONDS.     Ameiurus  natalis. 


Diet  According  to  Forbes. 

Rating  in  Table  XX. 

S< 

7" 

14* 

Insects,  30  pe 
Fish,  34  per  c 
Decapods,  17 
Average  

r  cent  

40 
80 
IO 

43 

60 
IOO 

30 
63 

QO 
70 
50 
70 

IOO 

30 

IOO 

ent. 

per  cent. 

The  ratings  being  only  estimates,  a  more  accurate  method  is 
unnecessary. 

An  inspection  of  Table  XXIV  shows  that  the  distribution  of  fish 
is  not  correlated  with  quantity  of  the  foods  known  to  be  eaten 
by  that  species  of  fish  in  other  localities.  The  fish  are  frequently 
found  only  in  the  ponds  where  the  food  is  least  abundant  and  no 
fish  is  found  where  its  food  is  most  abundant.  Are  the  fish 
the  cause  of  the  deficiency  ol  their  own  lood/  To  answer  this 
question  \\Oll  Lake  and  the  small  ponds  were  studied.  \\<>l! 
Lake  contains  many  more  fish  than  any  ol  the  other  bodies  ol 
water  thus  far  mentioned,  but  as  it  is  a  large  body  we  cannot 
compare  it  with  the  ponds.  Pond  I.  (see  p.  15),  which  has  bci-u 
artifically  separated  from  \Volf  Lake,  contains  few  \\^\\  Ahniniis 
crysoleucas,  Umbra  linii,  and  Ameiurus  nebulosus  arc  the  only 
species  and  these  appear  not  to  be  numerous.  Pond  1 1.  contain- 


ECOLOGICAL    SUCCESSION.  23 

TABLE  XXIV. 

.THY    up    FOOD;    THE    RATING    OF    THE    PONDS    FOR    THE    DIFFERENT    SPECIFS. 

*  shows  distribution  of  ti<h. 

i 


pterus  siihnoides  

.  .   Young.  .  . 

*37 

45 

72 

IOO 

Adult  

65 

60 

65 

Lepomis  pallidtf,  

.  .  Young.  .  . 

*^7 

- 

62 

IOO 

Adult  

*33 

77 

IOO 

.  Young.  .  . 

4i 

60 

IOO 

Adult  

62 

us.  . 

.  .   Young.  .  . 

45 

71 

IOO 

A.lult  

50 

73 

IOO 

us  .  . 

.   Young. 

..' 

71 

Adult  

73 

-<• 

Young.  .  . 

... 

71 

IOO 

Adult  

:.... 

.   Young. 

[,,., 

Adult  

... 

*7» 

:id  mtlas.  . 

.  Adult  

... 

*6S 

Si  hill"               '      •<  f  

.  .  Adult  

.... 

.  .   Young.  .  . 

*6i 

*6i 

7" 

Adult  

... 

*4-' 

57 

..  Adult  

if.  .  .  . 

.  Adult  

.  .. 

.  .  . 

.  .  Adult.  .  .  . 

J 

I'tuhrn  .in<l  Esox  vernncnlatus  all  fairly  .ilumdani.      h 
•.idem   ili.it  pond  I.  contains  fewer  fish  per  unit  \olmne,  still 
it   ha-  less  Kntomostraca.     Evidently  con-umpiiun  l.\    \\-\\  <\ 
not    -iv.it!>    .illt-ct   Entomostraca.1      The  i-oudiiimi    \\iih    rc~|H 
to  Kntonn.Mi.il  .1  is  paralleled  by  other  elements  oi  ii-li  loud. 

(//'   (  "omprtition  of  Species.     On  this  poini  \\c  h.i\r  ln-rn  ,i1>l< 
i"  -<  -i  mi  -.tlmo-t  no  data.     The  golden  shiner  is  absent  trum  pund 
I.     Su  I'.ir  .1-  the  conditions  are  concerned,  ii  should  \n- 
in  iiuinlu  i-.      It  is  an  important  article  of  dirt  for  ni.ni\   «•!" 
ti-lir-   fuiind    there,   which  suggests  that    it    h.i->   ln-<  11   cliniin.ued 
I  >\   i  In-  oilier  ti-hes. 

;,.    /\(7.://.-v   Importance  of  the  Brccdi*        '.    'iritics  and  General 
The  activities  will  be  separated  into  ^i  •in-r.il  and  hn-ed- 

(  '.riu-ral  Activities.     This  will  be  taken  up  with  reference 
to  the  depth  of  water,  kind  of  bottom  and  surrounding  \ 


1  My   >t.iti-im-llt   (Slu-llord.   'lO)   to  tin-  rtt.vt    th.it   thf  .ilimuni    • 
xini!  ..ut  tin-  same  in  all  the  i  .       w;i- 

.-  iK-en  (liaiiu-il.      Tal'N-   XX 


24  VICTOR    E.    SHELFORD. 

with  which  the  fish  are  commonly  associated,  according  to  the 
various  writers  cited. 

Microptents  saJmoides. 

Vegetation  of  the  pond  weed  zone  (Hankinson,  '07,  p.  2131; 
3  to  25  feet — plants:  Potamogeton,  Naias,  Myriophyllnni, 
Elodea  (Davis  in  Hankinson's  Report). 

Generally    prefers   still    and    sluggish    waters    (Forbes    and 
Richardson,  '08). 
Lepomis  pallidus. 

5  to  15  feet  of  water,  patches  of  Potamogeton  and  other 
aquatic  plants  (Jordan  and  Everman,  '02). 

Pond  weed  zone,  3  to  25  feet  of  water  (Hankinson,  '07). 
Lepomis  cyaneUus. 

Shoals  where  plants  were  abundant;  bulrushes  and  aquatic 
types  (Hankinson,  '07). 

Small  streams  (Forbes  and  Richardson,  '08). 
Eupomotis  gibbosus. 

Plant  covered  shoals — o  to  3  feet  (Hankinson,  '07). 
Ch&nobryttus  gulosus. 

Shallow  mud  bottomed  ponds  or  lakes  (Jordan  and  Ever- 
man). Still  water,  muddy  bottom,  plenty  of  vegetation 
(Meek,  '08). 

Deep  pools  and  quiet  water  (Henshall,  '03). 
Perca  flavescens. 

Chiefly  an  inhabitant  of  the  pond  weed  zone;  seldom  found 
in  less  than  two  feet  of  water  (Hankinson,  '07). 

Gregarious;  moderate  depths  of  streams  and   ponds  (Hen- 
shall,  '03). 
Erimyzon  sucetta. 

Limited  to  places  where  vegetation  was  abundant  (Hankin- 
son, '07). 
Ameiiirus  nebulosns. 

Loves  mud;  lives  in  weedy  ponds  ami  rivers  without  current 
(Jordanand  Everman,  '02). 

Fond  of  mud;  weedy  ponds  and  rivers  without  current 
(Forbes  and  Richardson,  '08,  p.  206). 

Pond  weed  zone,  shallow  water  at    night   (Hankinson,  '07). 


ECOLOGICAL    SUCCESSION.  25 

Sch  i U>eodes  gyrin  us . 

( '•  mimon  in  dense  vegetation  of  the  shallow,  almost  stagnant 
\\ater  of  bays. 

I  lide-,  under  stones  and  logs  (Ha> .  '141. 
Esox  irnuifnlatus. 

Situations  with  most  aquatic  vegetation  (Jordan  and  Kver- 
inaii.  'oj  . 

I'ret.  nnce  for  quiet  muddy  \\ater:  weedy  streams  Forbe- 
and  l\i<  hard  son,  *o8). 

Grassy  -treamsand  muddy  bayou-    Hen-hall.  '03 
I  'nilini  linii. 

Mevei  seen  swimming  in  the  open  water;  onl\  \\here.iquatie 
plain-  lornit-d  a  dense  growth  in  -hallou  u.itt  r  1  l.mkiu-on, 
'07). 

liiir\  them-elves  in  a  hole  in  tin-  mud  -cooped  out  \\iili  the 
lail:  rest  there  at  an  angle  of  45°  with  tin-  tail  <lo\\n  and  the 
lie-ad  b.tivK  |»n)iruding  (Abbott,  ';• 

Mr.  l>\\igln  L.  Gardner  has  shown  by  experimental  -mdie- 
in  our  laboratory  that  they  avoid  -iron-  li-ln. 

•.'fiicas. 

1   "111111011   iii  all   places  where   tln-n-  an-   mam    \\ait-r   plant- 
I  laiikiii-on,  '07). 

Muddii-i  and  apparently  most  nninxitin^  hole-     ll.i\.  '<ij   . 
.1  niciitrii*  nuttilis. 

'.Mici.ilK  t"re(|iieiiting  the  pond  \\n-d  /on.-  from  \\lii.h  it 
\\t-ni  into  -h.illou  w.iter  at  night.  Yonn^  in  -hallou  \\ait-r 
\\iili  d«-n-«-  \<  Delation  (Hankin-on,  <•; 

Streams  \\ith  mudd\'  bottom     I  itrbc-  and   Kirli.ird-oii.  '. 
Anicitf  'tis. 

^mall  |ioud-  \\ith  muck  bottom  Jordan  and  K\  crnian.  'O2  . 
A. comparison  of  the  data  abo\  »•  \\  ith  that  in  Table  I ..  p.  J,  and 
Table  XXI  .  p.  17,  shows  that  the  lar-c  mouthed  black  ba--.  tin- 
blue  uill.  tin-  \\armouth,  the  perch  and  tin-  \ello\\  .md  -potted 
bullhead-  are  not  in  water  of  the  depth  which  tln-\  pn-l't-r  in  other 
lo(  .ilitii--.  Tin-  other  ti-he-  are  belter  loe.ued  a-  to  the  depth  of 
the  ualei. 

The  lar^e  mouthed  black  ba  — ,  the  blue  gill,  the  perch,  and  the 
-jiotted  and  yellow  bullhead-  are  found  chiclly  in  the  pond  \\eed 


26  VICTOR    E.    SHELFORD. 

zone  of  Walnut  Lake.  This  is  characterized  by  plants  that  do 
not  reach  the  surface.  They  are  Chura,  hormvort,  bladderwort, 
\vater  millfoil,  water  weed,  slender  Xains,  pond  weeds,  etc. 
(Davis  in  Hankinson,  '08).  These  same  plants  grow  also  in  the 
bays  and  coves  in  company  with  the  water  lily  and  othrr  muT^nm 
plants. 

Ponds  i  and  5r  are  dominated  by  submerged  plants.  Here  the 
perch,  bass  and  sunfish  mentioned  above  are  associated,  with  the 
same  species  and  the  same  growth  form  types  as  in  Walnut  Lakr. 
The  bullheads  are  found  common  in  the  ponds  in  which  the 
submerged  and  emerging  vegetation  are  mixed,  and  which  contain 
the  greatest  number  of  species  of  the  pond  weed  zone  of  Walnut 
Lake.  It  seems  impossible  to  draw  any  conclusion  here  as  to  the 
relation  of  these  species  to  either  species  or  growth  form  in  plants. 
The  whole  subject  is  one  for  investigation.  A  comparison  of 
Tables  II.,  p.  4,  and  XXI.,  p.  17,  shows  that  black  bass,  the  sun  - 
fishes  and  pumpkinseed  are  found  only  where  a  considerable 
area  of  their  preferred  bottom  is  present. 

Mud  and  muck  are  evidently  not  distinguished  in  the  tables 
of  Forbes  and  Richardson  ('08)  and  it  is  not  possible  to  make 
much  use  of  their  data  here  for  this  reason.  We  have  noted  in  the 
preceding  paper  thai  the  rliubsucker  prefers  coarse  bottom 
materials.  It  muck  is  included  with  mud  (Forbes  and  Richard- 
son, '08)  with  the  exception  of  the  warmouth  and  chubsucker. 
all  are  well  placed.  The  chubsucker,  the  mudminnow,  ;md  the 
golden  shiner,  tadpole  cats  and  the  bullheads  avoid  strong  li.uht, 
and  their  association  with  dense  vegetation  which  result >,  brings 
them  into  relations  with  bottoms  of  fine  material,  e.  g.,  muck,  because 
they  support  dense  vegetation  (Pond,  '05). 

(b)  Breeding  Activities.  We  give  below  all  that  has  been  found 
regarding  the  location  of  nest  and  eggs. 

Micropterus  salmoides:  Sterile  bottom  of  clay,  sand  or  gravel, 
fibrous  roots  of  the  parrot  feather  preferred  to  others  (Titcomb, 
07,  p.  10  of  separate,  fide  Slranahan);  (b)  blackened  roots  o|' 
waterfoil  i  to  2^/2  feet  of  water,  bulrush  shoals  in  12  to  15  inches  of 
water,  among  conspicuous  growth  of  bulrushes,  eggs  on  rom^ 
(Hankiiison,  '07,  p.  214);  (c)  leaves  of  trees,  gravel;  u.-rd  \\ln-n 
artificial  fibrous  nest  was  present  (Reighard,  '05,  p.  4*1;  ah  sa 


ECOLOGICAL    SUCCESSION.  2~ 

^r.ivi-1  preferred,  mud,  clay,  or  surface  of  plants  in  absence  of 
these  (Henshall,  'o,;  :  e)  gravel,  clay  or  mud  from  which  all 
foreign  materials  have  1  it-en  remo\ed  Smith,  '07,  p.  2^7 

Lepomis  pallidus:  Barren  -hoals;  bottom  pure  marl  or  marl  and 
sand,  bottom  of  marl  or  gravel;  water  5  inches  to  2  feet;  marl 
bottom  with  bulrushes  (Hankinson,  '07,  p.  212  . 

I.<-f>i>nris  cyanellits:  Swamp  loosestrife,  black  bottom,  I  foot  of 
water;  m.irl.  marl  and  sand,  also  root-  I  lankiii-<>n.  '07,  p.2io  , 

Eupoi  ibbosiis:  (a)  Sand  bottom  ;  i  to  2  feet  <>i  water;  -and 

bottom;  in. nl  and  sand  bottom,  -eant  biilru-h  growth;  marl 
bottom,  bulrush  covered  (Hankinson,  '07  sand  and  gravel 

boiiom  not  infrequently  on  roots  (Reighard  in  (till,  '05,  p.  51 
<  lt-.tr  \\ater;  sand  and  gravel  bottom    lien-hall,  'o,; 

Perca  //-.'.-  wr/;s:  (a)  No  nest;  bare  -ami  ami  ^ia\cl  ri\ti  . 
.mum-  a<|iiatic  plants  (Abbott,  '75);  (l»  Stones,  \  (Delation,  other 

objects  or  loose  in  water — no  ne-t  Smith.  '07,  p.  252  . 
nebnlosits:  (a)  Stove  pipe,  etc..  4  5  leet.  -and,  under 
cover,  in  ^  2  \  in.  of  water  (rarely  more  than  24  in.  I -.\  i  !«•  -h\  nu-r, 
'07  vel  and  ac|iiariiun  bottom  Kendall.  '02;  Smith  and 

I  lain.!!,  '<>2 

S<i:i;  zyrinns:   In  tin  can,  marl  bottom.   ^  feet  «\  \\aifi 

1  lankiii-.tii.  '07). 
/  »il>nt  linii:  Stuck  to  a(juatic  plant-    K\d«  i 

TAIU.I-:  XX\  . 

•»IIII\\IM.   mi    RELATION  OK  KNOWN  !•..;••:•      II  M 

IN  THE  SERIES  OF  I'HM>-. 


v 

I'r, 


uitli  I 


ides.  Sand. 

Sand.  o 

-,....  Sand.  i  -•  o 

<us.  .  .  .  Sand.  i  -'    -•  i  o 

•:s Sand  and  vi"_  • 

tation. 
In.  ....  Sand  iiiiclt-i  i  ^ 

.  .  .  .' t.ilinll. 

The  data  on  breeding  habit-  as  -ummari/ed   in    Table   XXV. 
-Ixm    clearlv   that  the  distrihution  of  tin-  species   whoa 
i  Lpparei  -li;illn\\ 


28  VICTOR    E.    SHELFORD. 

habits  are  known  is  correlated  icith  the  distribution  of  the  conditions 
necessary  for  breeding. 

While  our  tables  show  that  there  is  considerable  bare  bottom 
in  the  pond  $c,  there  is  good  evidence  that  this  is  largely  due  to 
building  of  the  road  and  of  the  Lake  Shore  and  Mich.  Southern 
R.  R.  which  separated  this  pond  from  the  others  and  from  the 
lake  and  probably  excluded  fish  since  1851.  The  exposures  of 
bare  sandy  bottom  which  are  due  to  natural  causes  are  usually 
not  covered  with  more  than  six  inches  ol  water. 

Turning  to  the  perch  which  is  abundant  here  we  note  that  the 
eggs  are  extruded  in  the  open  water  or  vegetation  as  well  as  over 
terrigenous  bottom.  Terrigenous  bottom  is  less  necessary  than 
to  the  other  food  fishes. 

Turning  to  the  spotted  bullhead  we  note  that  the  nests  are 
probably  usually  made  in  water  shallower  than  any  of  the  other 
fishes.  Only  one  specimen  has  been  taken  from  pond  I.;  they 
are  numerous  in  pond  $c  and  ja.  There  are  some  old  logs  and 
stumps  and  a  very  narrow  zone  of  bare  sand  in  o  in.  and  less  of 
water  in  these  ponds.  This  is  commonly  shaded  by  vegetation. 

In  connection  with  oxygen  content  we  note  that  it  is  greatest 
in  5c  where  the  first  four  species  of  Table  XXV.  do  not  breed. 
However,  this  pond  must  be  regarded  as  in  a  measure  non- 
comparable  because  of  contamination  and  small  amount  of  plank- 
ton. 

The  low  oxygen  content  on  the  muck  bottoms  of  the  older 
ponds,  at  depths  used  by  the  fishes  present  in  pond  I.,  and  absent 
from  these  older  ones,  certainly  is  a  sufficient  reason  for  their 
absence,  though  it  is  not  to  be  expected  that  this  is  the  sole 
cause.  It  is  apparent  also  that  A.  nebulosits,  which  is  present 
in  the  older  ponds,  not  only  breeds  in  shallower  water  but  also 
has  superior  means  of  aerating  the  eggs  (Smith  and  Harron,  '02). 

Succession  of  fish  then  becomes  succession  of  breeding  condi- 
tions and  breeding  mores.  While  the  major  factors  as  indicated 
here  are  related  to  deptli  and  bottom,  there  are  doubtless  others. 

IV.  GENERAL  DISCUSSION. 

There  is  great  danger  of  error  in  dealing  with  such  complex 
problems  when  compilation  is  necessary  and  especially  when  the 


ECOLOGICAL   SUCCESSION.  29 

point  of  view  of  the  compiler  differs  from  that  of  the  original 
investigator.  To  illustrate  principles  and  methods  we  have  relied 
upon  compilation  far  more  than  could  otherwise  be  justified. 
Still  certain  facts  and  relation-  appear  to  be  clearly  indicated 
by  tin-  reconnaissance.  These  will  be  roughly  grouped  under 
tin-  head-  quantitative,  economic  and  general. 

i.  (Jnantitntive. — As  has  been  pointed  out  in  the  body  of  the 
paper,  the  quantity  of  living  material  in  tin-  form  of  plankton, 
invertebrate-,  and  vegetation  increases  a-  a  pond  '/rows  eco- 
lojcally  older.  In  our  data  there  an-  t\\<>  exception-  to  thU 
which  mu-i  be  noted:  First  the  greater  number  of  Kniomo-traca 
in  the  younger  ponds  in  early  spring  and  the  le— er  number  in 
pom  I  5/  (in  all  occasions.  The  greater  number  in  the  early 
.- prim:  i-  not  ea-ily  explained  but  may  be  due  to  the  better  con~ 
ditioii-  on  the  bottom  where  the  egg-,  etc..  <>f  the  plankton 
Kiiiomo-ir.K  a  art-  found.  Possibly  the  larger  areas  of  clean 
bottom  pre\eiit  their  being  buried  and  shut  a\va\  from  the 
eitei  '  oi  tin  sun's  heat,  oxygen,  etc. 

I'did  5.  i-.  as  we  have  indicated,  probablv  not  comparable  mi 
.K' cunt  of  the  contamination;  also  plankton  production  i-  mca- 
Mired  in  <  iii-tacea  and  Marsh  ('03)  ha-  pointed  cut  pit— ible 
errors  in  thi-  method.  A  study  of  all  the  plankton  <  -cii-t  itueiii- 
mi/In  .-ho\\  a  different  relation  of  5c.  Here,  however,  low  plank- 
tcn  content  is  associated  with  little  COj  (Birge  and  Juday,  'n  . 

The  iccied  /ross  vegetation  secures  necessary  --ib-  Irom  the 
-oil  and  I 'end  '05)  jxiintcd  out  that  it  im  Tea-e-  plankton  bei  ause 
the  foods  absorbed  from  the  soil  are  added  to  thewaterwhen  the 
plain-  decay.  Our  results  are  then  in  full  accord  with  tho-ed 
l'cn-1.  Se<  al-o  Hirge  \'  Juday,  'i  I .  Knauihe,  '07,  p.  57- 

The  greater  number  of  large  invertebrate-,  appears  to  I.,-  gen- 
erally clo.-elv  related  to  the  amount  d"  gross  vegetation.  .\Carl\- 
all  Mich  animals  cling  in  vegetation  and  main  d  the  species 
found  in  tlu-  older  ponds  use  the  \e-ei.uion  a-  a  m.-an-  of  reaching 
the  -nrface  fcr  air,  of  avoidii  -unli-ht,  and  as  breeding 

places.  The  majority  of  such  animal-  plan-  their  into  or 

upon  the  plant-.  Gross  vegetation  i-  al-<>  thickly  covered  \\ith 
minute  organisms  \\hich  afford  Iced  for  many  animal-. 

It   i-  probable  that   the  amount  of  rooted  vegetation  in  i-olated 


3O  V.CTOR    E.    SHELFORD. 

ponds  may  be  taken  as  an  index  of  plankton  production.  It 
appears  that  this  must  be  true  on  tin-  l>a-i-  of  the  conclusions 
of  Pond  ('05)  no  matter  what  factor  is  of  greatest  importance  in 
controlling  the  quantity  of  plankton.  Johnstone  CoS)  pointed 
out  that  the  plankton  production  follows  Liebig's  law  of  mini- 
mum— i.  e.,  quantity  is  determined  by  the  food  substance  present 
in  minimal  quantity.  If  rooted  vegetation  is  the  controlling 
factor  a  deficiency  in  one  food  substance  in  the  soil  would  show 
itself  in  the  rooted  vegetation  and  through  this  affect  the  plankton 
production  of  the  pond. 

The  question  of  the  general  application  of  the  principle  of 
quantitative  increase  with  age  is  important.  It  seems  probable 
that  in  all  bodies  of  water  with  small  outflow  organisms  increase 
with  age  because,  in  addition  to  the  effect  of  rooted  vegetation, 
inwash  continuously  brings  food  substances  which  are  tied  up 
if  not  carried  away  by  extensive  outflow. 

Experimental  study  of  the  quantitative  problem  is  possible  on 
the  basis  of  such  a  set  of  ponds  as  those  at  the  head  of  Lake 
Michigan.  From  such  a  set  all  the  organisms  can  be  trans- 
planted and  most  of  the  conditions  duplicated  where  closer  control 
would  be  possible  than  in  the  natural  ponds.  There  appears 
to  be  no  difficulty  in  such  experimental  study  except  that  it 
requires  extensive  facilities  and  institution  or  government  sup- 
port. Such  ponds  as  ours  and  such  ponds  as  may  be  constructed 
with  them  as  a  basis  give  promise  of  throwing  more  light  on  the 
factors  controlling  the  quantity  of  life  than  do  the  large  and 
complex  bodies  of  water. 

2.  Economic.— The  writer  has  no  practical  knowledge  of  fish 
culture  and  only  the  knowledge  which  has  been  acquired  by  reading 
some  of  the  characteristic  literature.  Apparently  the  economic 
problems  in  fishes  are  concerned  with  questions  of  the  preserva- 
tion of  fishes  in  natural  waters,  and  their  increase  and  main- 
tenance against  the  removal  tor  tood,  which  makes  them  ot 
economic  importance.  With  these  ends  in  view  efforts  have  long 
been  made  mainly  to  increase  fish  by  increasing  food  suppK  ,  to 
care  for  fish  during  the  critical  reproductive  season  by  artificial 
hatching  and  pond  culture,  and  to  decrease  enemies  by  de^t ruc- 
tion of  objectionable  fish  and  fish  parasites.  The  preservation 


ECOLOGICAL    SUCCESSION.  31 

<>\  the  fish  environments  has  received  little  or  no  attention. 
La\\-  have  been  enacted  to  prevent  the  pollution  of  waters,  but 
the-e  have  been  enforced  but  rarely. 

In   practice  the  importance  of  the  breeding   season   ha-  been 

iv<ogni/ed  by  the  culture  worker-  but  appear-  to  have  ivcehcd 

little   attention   from   the   point    of   view   of   the   pre-ervaiion   or 

culti\ation  .•!  fish  breeding  places  in  the  natural  waters.      Chirk 

i<>     i-  one  of  the  few  who  have  empha-i/ed  breeding  Around-. 

1  he   main   emphasis  has  been   laid   on    nutrition      Knauthe.    '07. 
Chap.   1\ 

(  >ur  data  indicate  that  the  breeding  intere-t-  and    the    feeding 
interests  of    ,ij||   water  food  and  gam.-   ti-he-  are  ilistini'tly  an- 
onistic.      I'.irge  Clo)  pointed  out    that   \\here  the  quantity  of 
pl.inkion  t  and  the  fish  food  acconlingU   great,  the  o\\geii 

conteni  i- |o\\  .it  the  bottom  and  the  water  accordingly  un-uited 
i"  the  production  of  certain  of  the  be-t  food  ti-he-  Knauthe 

p.  -,7'»  states  that  a  large  fish  producm  it\  in  a  pond  i-  com- 
nioiiK  indicated  by  large  amount  of  gn--  \egetation,  but  says 
also  that  thf  general  statement  that  Mich  pond-  are  al\\a\-  good 
l»roducer-  ot  ti-h  cannot  be  made.  Thi-  indicate-  that  there 
are  Othei  factors.  lie  makes  no  mention  of  breeding  and  d 
not  state  the  practice  of  pond  owners  as  relating  to  the  breeding. 
In  -landing  and  sluggish  water,  the  problem  of  the  balance  I.e- 
t  \\eeii  the  lood  supply  and  the  fish  prc-ent  -eeiu-  ivlathelv  un- 
important. Cilice  feeding  conditions  of  de-irable  \«,><\  li-lic-  gn>\\ 
better  \\ith  time  at  the  expense  of  the  breeding  condition-,  the 
major  problem  is  that  of  the  halam  •  :/;/^  and  lirccdin^ 

<  <ni(liti(»i.\.      It    appear*   that    such   balance   might    be   maintained 

ea-il\    it   u i    had  an  adequate  knoulrdge  ot   the  environmental 

lelatii'ii-  ol  the  lish.  Definite  knowledge  as  to  -pacing  of  nests 
in  natiin-  -hould  gi\'e  data  as  to  bn-eding  area  re«|iiired  per 
ca|>ita  b\  li-h.  \\'ith  such  knowledge  at  hand,  together  \\ith  the 
e\i-ting  knowledge  of  food  habit-,  it  -hould  not  be  difficult  to 
maintain  adequate  breeding  area-  a<ljai  ent  to  good  feeding  areas 
within  our  \\aters  both  public  and  prhate. 

;v    (/'c;/cn;/.      \\'e   have   noted    the   aspects   of   the   (|uantitali\  e 
and    eci.noiiiic    problem-   \\hich    our    data     I'nable    u-    to   disCUSS. 
The  remaining  indication-  of  the  reconnai— ance  are  tho-e  related 
ictors  governing  distribution  and  metho<l-  of  -tnd\  . 


32  VICTOR    E.    SHELFORD. 

The  study  of  factors  governing  distribution  of  fish  and  other 
animals  has  never  been  reduced  to  an  adequate  working  basis. 
The  problems  are  indeed  complex,  but  the  difficulty  has  arisen 
in  part  from  two  causes,  namely,  (A)  the  lack  of  knowledge  of 
the  activity  which  takes  place  within  the-  narrowest  limits  (Shel- 
ford,  'ii3),  and  (B}  lack  of  recognition  of  the  important  factors 
and  features  of  the  environment. 

The  conclusions  of  workers  on  distribution  often  seem  to  have 
been  to  the  effect  that  the  food  relations  of  fishes  should  stand 
as  first  in  importance,  as  factors  of  distribution.  Hankinson 
('io)  states  that  the  pond  weed  zone,  the  living  and  feeding  place 
of  the  fish  of  \Yalnu t  Lake,  is  probably  the  most  important 
habitat.  Our  evidence  on  the  same  species  points  clearly  to 
the  breeding  grounds.  Indeed  much  careful  work  must  be  done 
before  broad  generalization  should  follow,  but  it  is  evident  that 
here  as  in  birds  (Merriam,  '90;  Adams,  '08)  and  in  the  ti^'-r 
beetles  (Shelford,  '07,  'ii3)  the  breeding  place  and  the  breeding 
activities  are  the  most  important.  (Reighard,  'io,  and  cita- 
tions.) Is  variation  in  nest  building  real  or  only  apparent 
because  we  do  not  know  the  most  important  factors  and  seize 
upon  details  wholly  unessential  to  fish?  What  are  the  la\\> 
governing  the  mores  of  species?  Experimental  work  correlated 
with  field  observations  can  answer  these  questions,  and  it  is  at 
this  point  that  contributions  of  lasting  value  can  be  made.  The 
first  step  in  the  necessary  work  of  raising  natural  history  from  its 
present  state  of  vagary  is  to  determine  what  activity  takes  place 
within  narrowest  limits  and  which  is  least  modifiable  in  as  many 
groups  of  animals  as  possible. 

The  second  difficulty — lack  of  recognition  of  the  important  and 
unimportant  in  en\  inmments — is  one  which  we  have  emphasized 
before. 

The  ecologist  often  uses  vegetation  as  an  index  of  conditions. 
There  is  objection  to  this.  Investigators  have  seen  that  the  same 
species  of  animals  are  not  always  associated  \\itli  a  given  species 
of  plant.  Indeed,  species  of  plants  c.innot  often  and  perhap> 
usually  be  taken  as  an  index,  of  the  environmental  conditions  of 
animals,  especially  in  water,  because  species  ol  plants  an-  not 
necessarily  an  index  of  conditions.  Tin-  physiological  condition 


ECOLOGICAL    SUCCESSION.  V, 

ol  plan1-  i-  the  important  thing  and  is  commonly  indicated  In- 
growth lorm  (superficially  hut  not  finally)  which  is  the  index  of 
internal  physiological  state  induced  by  the  surrounding  condi- 
tion-. IMant  formation-  arc  tlu-  expression  of  the  condition-  ot" 
exi-tence  for  the  plants  of  a  definite  area.  The  formation  i-  the 
fundamental  unit  of  the  ecology  of  communities  and  carries  with 
it  no  consideration  of  species  whatever.  Identical  or  similar  forma- 
tion- ottcn  <lo  not  ha\'e  a  single  specie--  in  common.  A-  \\  e  lia\  e 
pointed  out  I.efore,  species  are  of  importancr  only  in  -o  far  as 
their  e<  ..logical  constitutions  are  specific  character-.  It  i-  not 
s/-"  n-li  that  we  are  to  expect  to  be  a— ociated  with  species 

of  plant-,  hut  mores  of  fish  with  growth  form  in  plant-  or  with 
plain  lormaiions.  Furthermore,  relation-  to  venation  \\hich 
are  ot  importance  are  to  be  expected  primarily  in  connection 
\\  ith  breeding. 

iihjrction  to  ilu-  use  of  vegetation  as  an  index  of  t»ndition-. 
due  ID  mi-apprehension,  is  to  be  expected.  Ho\\e\er.  \\hen  the 
theoretic.il  probabilities  are  understood,  \\  e  have  not  the  data 
in  the  case  ol  li-h,  with  which  to  determine  \\  he t her  or  not  ^roictii- 

"i  and  more*  are  associated.  The  subject  i-  one  for  -pecial 
experimental  and  observational  investigation. 

In  connection  with  the  problems  of  animal  heha\  ior,  (hi-  point 
o|  \ie\\  Dpen-  up  a  field  wherein  the  role  of  the  different  cn\  iron- 
menial  c..iiditioii-  in  the  control  of  behavior  m.i\  he  -!  tidied  in 
naiuie  .1-  \\ell  as  in  experiment.  As  a  background  |..r  the  -tud\ 

"t    all   a-pet  1-   "I    behavior   the   point   of  \ie\\    here   pre-ented   -eelll- 

to  <  'ii«  i  dei  ided  advantages. 

Comparative  study  ol  behavior  from  thi-  point  of  \ie\\  ha- 
heen  impracticable  because  of  a  lack  ot  kno\\led-e  ot  en\  iron- 
mem-.  I  mil  \\e  can  accjiiire  a  knouled-^e  and  a  nomenclature 
that  -hall  he  ^eneralK"  understood  the  \\orker  mu-i  \\rite  exten- 
sive de-criptioit-  of  the  environment,  and  i-  likeK  to  eni])ha-i/e 
detail-  \\lii»-h  are  of  little  important 

The  acti\iiies  of  an  animal  (behavior  are  ..f  greal  economic 
imporian.t.  they  determine  distribution.  The  relation-  ot  the 
hchdrior  prohlcms  and  the  distribution,  the  i/uuntitdtiir  diid  the  eco- 
nomic problems  seem  especially  intimate,  so  that  the  investigation  of 
any  one  from  this  point  of  rie:c  must  contribute  to  all  as  well  a-  to 


34  VICTOR    E.    SHELFORD. 

bring  about  a  better  unification  and  organi/ation  of  biological 
science  as  a  whole. 

V.  SUMMARY  OF  TENTATIVE  "CONCLUSIONS. 

1.  The  quantity  of  bacteria,  plankton,  vegetation  and  large- 
animals  increases  as  a  pond  grows  older. 

2.  Terrigenous  bottom  and  oxygen  content  decrease  as  a  pond 
grows  older. 

3.  The  distribution  and  succession  of  fish  are  not  determined 
by  kind  of  food;  kind  of  food  eaten  is  determined  by  the  availa- 
bility in  localities  suitable  in  other  respects. 

4.  Fish  are  not  necessarily  present  where  food  is  quantitatively 
greatest. 

5.  The  food  and  game  fishes  here  considered  are  closely  as- 
sociated with  their  breeding  conditions  to  the  neglect  of  depth 
of  water,  food,  etc. 

6.  Low  oxygen  content  on   breeding  grounds  is  a  sufficient 
cause  for  their  absence  from  the  older  ponds. 

7.  Conditions   outside   the   breeding   season   are   probably   of 
secondary  importance  in  the  success  of  fish  in  a  given  locality. 

8.  The  food  interests  and  breeding  interests  of  the  food  and 
game  fish  here  considered  are  decidedly  antagonistic.     The  for- 
mer continually  encroaches  upon  the  latter. 

9.  Successful  fish  culture  in   ponds  and  small  lakes  depends 
upon   the   maintenance   of   balance   between   the   breeding   and 
feeding  conditions. 

10.  Animal  succession  in  ponds  is  due  to  an  unused  increment 
of  excretory  and  decomposition  materials  which  causes  an  in- 
crease in  vegetation,  a  decrease  in  Oo,  on  the  bottom  and  a  general 
change  in  surrounding  conditions,  all  primarily  affecting  breeding. 

i  i .  Succession  of  species  is  the  result  of  stability  of  the  mores 
of  species  concerned;  when  mores  are  flexible  species  do  not 
succeed  one  another  but  continue  with  changes  in  behavior  and 
physiological  characters. 

HILL  ZOOLOGICAL  LABORATORIES, 
UNIVERSITY  OF  CHICAGO, 
August  i,  191 1. 


I  i  OLOGICAL    SUCCESSION.  ,^5 

VI.   ACKNOWLEDGE  M-  AND  BIBLIOGRAPHY. 
i.  Acknowledgments.  —  In    the   preparation   of   this   paper    the 
a--istance  of  a  number  of  persons  has  been  nece>s  ary.     A  number 

•  •I  graduate  students  of  the  University  have  -tndied  one  or  more 

•  it"  ilic  ponds  and  have  given  me  the  use  of  their  notes.     The 
following    -liould    be    especially    mentioiu-d:    Mi--    Alma     Bu<h, 
pond  -  i  \   Mr.  \Y.  J.  Saunders,  pond   i;    Mr.  Max    Rohde.  pond 
-a:   Mr.  B.  K.  Isely,  Mr.  \V.  C.  Alice,  Mr.  v  S.  Visher,  Mr.  G.  D. 
.  \lli  ii,  Mr.  I  >.  L.  Gardner,  made  more  general  contribution-..      I 
am  indebted  lo  Dr.  Chas.  C.  Adams  for  reading  the  manii-rript  . 

'Ihr  following  have  rendered  important  service  by  identilyin^ 
the  mail-rial  of  groups  in  which  they  are  speriali-t-:  Mr.  G.  D 
1  nlli  r.  Plants;  Dr.  J.  I'.  Moore.  Leeches;  Mr.  V  .  C.  Raker. 
M'lllu-'  i  I  >r.  ('.  D.  Marsh,  Copepods;  Mr.  k.  Sharpe.  O>tra- 
coda;  Di.  A.  1C.  Ortmann,  Crayfishes;  Mi—  A.  L.  \\irkrl. 
AmpliipixU;  Dr.  J.Ci.  Needham,  Aquatic  insects;  Dr.  (  Drnelius 
r,«iiiii.(  add!.  ••  Hies;  Mr.  W.  J.  Gerhard,  Hemiptera;  Dr.  P.  <•. 
I  Iriiieinaim  and  Mr>.  Hlva  Class,  Bacteria;  Mrs.  Kb  a  (  !lass  and 
Mi.  \\  .  C.  .  \lU-e,  Gases;  Mariner  and  Iloskin-  ("mmeiiial 
(  'hi-mi-i-  .  \\  iter  analysis  without  charge. 


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Cowles,  H.  C. 

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ECOLOGICAL    SUCCESSION.  .^7 

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Vs  VICTOR    E.    SHELFORD. 


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NOTES,    REVIEWS,    ETC. 


tically  all  nucleus.  The  corpuscles  on  the  other  hand  have  lost  their 
nuclei  wholly.  Between  these  extremes  we  have  various  stages/ of 
chromatin  reduction  in  the  development  of  the  specialized  Metaeoan 
tissues.  The  maturation  divisions  in  ova  and  sperm,  the  bodily  ex- 
trusion of  chromatin  observed  on  the  part  of  blood-cells,  etc/ he  re- 
gards as  illustrations  of  the  process. 


THE  RESERVE  OF  FOOD  IN  TREES 

Proton  and\Phillips  (Forest  Quart.,  1911)  agree7  with  the  com- 
mon view  that  staV'h  is  the  principal  form  in  which  reserve  food  is 
stored  in  trees.  Tney  doubt  that  cellulose  is  abl/to  act  at  all  as  a 
reserve  material.  Ti»e  maximum  contained  re/erve  for  deciduous 
trees  occurs  about  theVime  the  leaves  fall,  and  during  the  next  few 
weeks  there  is  a  decidedVeduction  in  its  amount.  The  sugar  content 
in  trees  remains  pretty  constant  <.-xri.pt  f"r  ,'m  incn-a-r  in  -pring  (lur- 
ing the  unfolding  of  the  btn 


ALTERNATION  OF  ''.          N      M 


Lewis  (  Mot.  Gaz.,  Mch..  iQrs^.  by  artificial  plantings  of  tetra- 
spores  and  carj>ospores  of  Polysw1(pnia  and  some  other  genera  of 
red  algae  gets  experimental  re:/ilts\upporting  the  general  conclu- 
sion that  tetrasporcs  produce  only  the  sexual  plants  and  carpospores 
only  the  tetrasporic  plants.  /In  no  instance  was  an  exception  found 
to  the  rule,  although  a  considerable  numbet  of  plantings  developed 
to  maturity.  Tetrasporcs  from  a  given  individual  produced  male 
and  female  plants  in  approximately  equal  numbers.  It  is  also  con- 
cluded that  no  greater  growth  vigor  comes  to  th\  carpospores  over 
the  tetraspores  because  of  the  double  number  of  chromosomes  con- 
tained by  them. 

RELATION    OF   THE    PROTOPLASM    OF    ADJACENT    PROTOPLASTS 


iy  (Ann.  Bot..  1911)  undertakes  to  throw  light  ofa  the  rela- 
tion that  exists  between  protoplasms  of  contiguous  cells,  b\  an  ex- 
amination of  the  relation  between  the  parasite,  Cuscuta,  and  r^s  host. 
She  finds  that  there  is  no  direct  protoplasmic  connection  between  the 
cells  of  Cuscuta  and  the  host,  but  that  the  phloem  cells  of  the  pa^a- 
site  haustoria  apply  themselves  to  the  sieve  plates  of  the  phloem  of 


AMERICAN    MICROSCOPICAL    SOCIETV 
ECOLOGICAL   SUCCESSION    OF    PLANTS   AND   ANIMALS  /  3 

Shelford  i  Biol.  I'.ull..  her..  KM  i  \  concludes  a  series  of  papers 
dealing  with  the  biological  succession  in  ponds  at  the  head  of  Lake 
Michigan.  The  following  are  some  of  the  conclusions  reached  by 
the  author  as  the  result  of  this  series  of  interesting  studies  : 

1.  The  quantity  of  bacteria,  plankton,  vegetation,  and  large  ani- 
mals increases  with  the  age  of  the  pond. 

2.  Terrigenous  bottom  and  oxygen  content  decrease  with  the 
age  of  the  pond. 

3.  Fish  tend  to  adapt  themselves  to  the  type  of  food  rather 
than  to  become  distributed  or  furnish  successions  in  accordance  with 
the  type  of  food.     They  are  not  necessarily  most  abundant  where 
food  is  greatest. 

4.  Small  oxygen  content  of  older  ponds  will  account  for  ab- 
sence of  fish  from  them. 

5.  Conditions  outside  the  breeding  season  are  probably  less  im- 
portant than  those  of  this  season  in  determining  the  success  of  fish. 

6.  The  conditions  most  favorable  to  the  normal  feeding  of  fish 
are  not  only  different  from  those  most  favorable  to  breeding,  but  are 
even  antagonistic  ;  and  the  former  tend  to  encroach  on  the  latter. 
ment,  and  the  preservation  of  balance  between  the  breeding  condi- 
tions and  the  adult  life-conditions. 

7.  Animal  succession  in  ponds  is  due  to  an  unused  increment 
of  excretory  and  decomposition  products  which  causes  increase  in 
vegetation  ;  a  decrease  in  oxygen  at  the  bottom  ;  and  a  general  change 
in  the  conditions  affecting  breeding. 

8.  Succession  of  particular  species,  rather  than  the  continued 
dominance  of  some  when  they  once  become  dominant,  results  from 
the  inflexibility  of  their  standards  of  demands  in  accordance  with 
the  changing  conditions. 

CHROMATIC  REDUCTION  IN  CELL  DEVELOPMENT 

ihde  fZeit.  Wiss.  Zool..  1911)  undertakes  to  showL-tfeat~\a 
marked  clTafarteristic  of  the  (lifferentiatiojr^^-rnSfunng  of  cells  is 
the  reduction  of  chro^latSB^Hthe^uicleus.  He  suggests,  as  illus- 
trative of  trrisj--a-'s£nes  with  bacte?hr-ftt_Qne  end  and  the  red  blood- 
cells  of^fiammals  at  the  other.  The  bacteriaTie-scajfiiders  as  prac- 


THE    CENTRAL    NERVOUS    SYSTEM    IN    TERATOPH- 
I' HALM  1C  AND  TERAT()M<  >KPHIC  FORMS  OF 
PLANARIA   DOROTOCEPHALA. 

C.  M.  CHILD  AND  E.  \'.  M.  McKIE. 

The  -tudv  of  the  nervous  system  in  tin-  teratoplnhalmic  and 
teratomorphic  forms  of  Planaria  dorotocephahi  \\a-  undertaken 
by  i  In-  junior  author  of  this  paper  at  the  senior  author'-  sugges- 
tion. 1  h<-  results  of  this  study  were  accepted  as  a  the-i-  for  the 
M.i-i.  ree  by  the  Department  of  Zoology  of  tin-  l'ni\i T-itv 

o!  (  hi.  .1-0.  Since  the  results  of  the  work  an-  of  ( -on-iderablc 
inten-t  .m<l  since-  Miss  Mi-Kit-  was  prevented  by  \.iriou-  cir- 
ciim-i.iiice-  from  preparing  the  paper  for  publication,  the  -enior 
author  ha-  undertaken,  at  her  express  request,  to  revise  her  manu- 
3<  ript  lor  publication  and  to  add  some  figures  from  her  -lide-;  he 
h.i-  also  added  a  section  on  the  various  method-  b\  \\hich  the 
teratophthalmic  and  teratomorphic  forms  have  been  produced 

and    ha-   e\ii-udrd   sollU'what    the  scope  ot    the   di-cn--ion   ot    the 

results. 

The  primary  object  of  the  work  was  to  determine  the  general 
form  and  tin-  degree  of  development  of  the  cephalic  part  of  the 
central  IK  i  \  oiis  system  in  these  abnormal  form-  as  compared 
uith  normal  animals.  The  observation-  concern  chiellv  the 
teraiomorphii  form*  since  these  repre-eiit  a  more  extreme 
departure  I'mm  the  normal  type  and  afford  more  definite  and 
-tiikin^  re-nit-. 

The  animal-  for  sectioning  were  anesthetized  with  \\eak  alcohol 
beloif  iixation  iii  hot  (Wilson's  fluid  or  -nblimate.  Sections  were 
cm  ten  micra  in  thickness.  Frontal  ami  -au'ttal  as  well  as  trans- 
verse sections  \\i-re  made,  but  all  the  h-uiv-  are  drawn  from  trans- 
\  ei  se  sections  -incc  these  show  the  e--ential  feat  ure-  most  clearK'. 

Ml  li-uie-  of  sections  were  drawn  to  the  same  -calc  with 
the  camera.  They  are  designed  to  -ho\\ .  tir-t  tin-  -eiieral  form 
of  the  nerxou-,  -\-tem  and  second,  the  general  relation^  bet  \\een 
filler  tracts  and  cell>.  The  cell-  are  repre-ented  merel>-  by  small 

39 


4O  C.    M.    CHILD    AND    E.    V.    M.    Mi  KI1  . 

circles  or  ovals  and  the  fiber  tracts  are  filled  in  with  dot-.  except 
where  a  distinct  commissure  or  nerve  is  concerned;  there  the 
direction  of  the  fibers  is  indicated.  Non-nervous  structures  are 
not  shown  except  in  the  case  of  the  alimentary  tract,  which  is 
diagrammatically  indicated  where  it  is  present  in  the  sections 
figured. 

I.    THE  EXPERIMENTAL  PRODUCTION  OF  TERATOPHTHAI.MK    .\\n 

TERAIOMORPFTC  FORMS. 

The  senior  author  has  given  the  names  " teratophthalmic ' 
and  "teratomorphic"  to  certain  types  of  head  which  appear 
under  certain  conditions  in  the  regulation  of  pieces  of  Plannr'ui. 
The  teratophthalmic  head  (Child,  'ua,  pp.  278-9;  'lie)  is  one 
in  which  the  eyes  show  some  departure  from  the  usual  structure 
or  arrangement,  but  the  head  is  otherwise  normal  in  form.  The 
teratophthalmic  forms  may  be  divided  into  several  groups  ac- 
cording to  the  character  of  the  eyes,  for  these  may  be  "abnormal " 
in  position,  size  or  number  or  the  pigment  cups  may  show  tlir 
most  various  degrees  of  fusion  (e.  .(,'.,  Fig.  6  below). 

The  teratomorphic  heads  (Child,  'nr)  represent  a  more  ex- 
treme departure  from  the  norm.  In  these  the  abnormalities 
involve  not  only  the  eyes  but  the  shape  of  the  head  and  the 
position  o!  the  auricles.  The  teratomorphic  head  usually  pos- 
sesses a  single  median  eye  and  the  auricular  sense  organs  appear 
on  the  front  of  the  head,  either  separate  (Figs.  10  and  16)  or 
more  or  less  completely  fused  (Figs.  19  and  2^1.  In  the  senior 
author's  earlier  work  on  Plauaria  the  teratomorphic  hca<l>  \\ere 
not  separated  from  the  teratophthalmic  (Child,  'i  \a),  but  as  the 
degree  of  experimental  control  in  the  production  ot  these  lorms 
increased  it  became  desirable  to  set  these  peculiar  forms  apart 
as  a  distinct  group  and  to  give  them  a  name. 

It  is  possible,  as  the  senior  author  has  shown  in  various  papers 
(Child,  'l  la,  'l  ic,  'lid),  to  control  experimentally  by  a  number 
of  different  methods  the  production  of  these  torms.  In  general 
they  are  the  result  of  conditions  which  decrease  the  rale  ot  the 
dynamic  processes  below  a  certain  level  determined  by  existing 
conditions  which  is  necessary  for  the-  production  of  normal  ani- 
mals. With  the  proper  experimental  conditions  they  can  1  >r 


CENTRAL    NERVOUS    SYSTEM    IN    PLANARIA.  4! 

produced  from  any  region  of  tin-  planarian  body  and  from  piece- 
of  any  size  above  a  certain  minimum,  which  varies  with  region 
ol  tin-  b'ldy,  physiological  condition,  age.  nutrition  and  external 
condition-;.  A  regional  factor  does,  however,  exi-t  Child.  'ii</>: 
in  piece-  of  a  given  length  the  more  posterior  the  level  within  a 
-ingle  /ooid.  the  greater  the  frequency  of  the  abnormal  form-. 
Thu-  far  it  has  been  possible  to  control  experimentally  the 
prodnciion  of  teratophthalmic  and  teratomorphic  as  \\e11  as 
anophthalmic  and  headless  forms  (Child.  'lie)  in  the  t'o'louing 
ways  first,  under  standard  conditions  of  t«-ni|)erature.  ninritit>n. 
eti  .  pi«  <  es  above  a  certain  length  with  anterior  end-  at  a  certain 
le\e|  ,,|  ili,-  l.odv  \vill  produce  normal  whole-,  -horter  piece-  will 
prodiKe  teratophthalmic  forms  and  still  shorter  piece-  terato- 
morphic.  .m<  iphthalmic  and  headless  form-  a-  the  length  de- 
:  'Hid.  in  pieces  of  a  given  length  from  a  gi\en  \-< -ion. 
under  iinit«inn  conditions  of  temperature,  nutrition,  etc..  -tinml.t- 
tion  to  motor  activity  increases  the  frequen<\  ol  normal  anim.t1-. 
uliile  lai  1:  ot  stimulation  increases  the  frequency  of  teratophth.il- 
mic,  teratomorphic,  anophthalmic  and  he, idle--  form-.  Third, 
in  pie.  es  ol  i  Jvc-n  length  from  a  given  re-ion  of  anim.il-  of  the 
ie  -j/e  the  frequency  of  abnormal  and  tioim.il  form-  varies 
\\itli  difference*  in  physiological  age  (C'hi'd.  '  I  I ;"  and  \\ith  dif- 
n.  ex  in  nut  rit ion.  Fourth,  in  pieces  ot  •  -^i\  en  length  Irom  a 
given  ie.  ion  of  animals  of  the  same  size  and  a-  ne.irK  a-  po—i bit- 
in  the  -ame  |  >li\  siological  condition  a  \.uietv  of  external  facior-. 
-uch  as  lo\\  teni|)eratiire,  metabolic  produ.  i-  in  tin  water,  dilute 
al<-ohol.  I'ther,  chloretone,  potassium  c\anide.  etc.,  \\ill  increase 
the  fre(|uenc\  of  abnormal  forms  and  it  i-  po--ible  to  control  to 
a  certain  extent  the  type  of  abnormal  form,  both  through  the 
length  of  the  piece  and  the  intensity  of  the  experimental  factor. 
(  >n  the  other  hand,  the  frequency  of  normal  form-  in  a  -i\  en  -«-t 
of  pieces  can  be  increased  by  good  nutrition,  by  high  temperature 
and  probably  also  by  certain  stimulating  drug-,  'luni-h  as  re- 
gard- the-e  la-t  the  results  are  complicated  l.v  the  fact  that  in 
main  ca-e-  the  stimulating  effect  of  drugs  is  of  relatively  -h..n 
duration  and  i-  fo!io\\ed  by  a  depression. 

Hut  \\hate\  er  may  be  the  results  of  more  ex: ended  experiment, 
the   fact-  alrcadv  e-tabli-hed  demon-irate   that    the  normal   and 


42  C.    M.    CHILD- AND    E.    V.    M.    Me  KIE. 

the  abnormal  forms  described  represent  differences  in  the  dynamic 
processes  which  are  primarily  purely  quantii.itis  e.  The  tem- 
perature experiments  illustrate  this  point  very  clearly.  In  a 
given  set  of  pieces  higher  temperatures  increase  the  frequency  of 
normal,  lower  temperatures  that  of  abnormal  forms.  The  effect 
of  the  anesthetics  and  the  other  external  factors  mentioned  above 
is  probably  also  primarily  quantitative. 

In  these  cases  then  different  morphological  characteristics 
appear  as  the  result  of  primarily  quantitative  changes  in  the 
dynamic  processes  in  the  organism.  This  fact  is  of  considerable 
theoretical  importance,  since  it  can  mean  nothing  else  than  that 
form,  structure,  localization,  number  and  even  presence  or  ab- 
sence of  parts  may  be  determined  by  purely  quantitative  changes 
in  external  factors,  i.  e.,  by  changes  which  alter  primarily  the 
rate  and  not  the  character  of  the  dynamic  processes. 

Certain  external  characteristics  of  the  head  region  of  the  ab- 
normal forms,  viz.,  the  position  and  number  of  the  eyes  and 
auricles,  indicate  that  the  cephalic  ganglia  of  these  forms  must 
show  considerable  departures  from  the  norm.  The  question  as 
to  how  the  form  and  structure  of  the  central  nervous  system 
may  be  altered  by  these  quantitative  changes  in  the  dynamic 
processes  is  one  of  interest  from  various  points  of  view.  The 
data  presented  below  give  a  partial  answer  to  this  question  and 
so  form  a  contribution  to  our  knowledge  of  the  dynamics  of 
morphogenesis. 

The  method  used  for  obtaining  the  teratophthalmic  and  tera- 
tomorphic  forms  described  in  this  paper  was  that  of  cutting 
pieces  of  a  certain  length,  determined  by  previous  experiments 
of  the  senior  author,  from  the  middle  region  of  the  body  of  large, 
well  fed  worms  and  allowing  them  to  undergo  regulation  at  about 
20°  C.  This  method  was  used  merely  because  it  is  the  simplest. 
Teratophthalmic  and  teratomorphic  heads  develop  on  pieces  of 
greater  length  from  the  middle1  region  of  the  body,  /'.  e.,  the 
posterior  region  of  the  first  zooid  (Child,  'i\<i,  'iu;)  than  from 
any  other  region.  This  makes  it  possible  to  use  relatively  long 
pieces  and  the  preparation  and  handling  of  the  material  is  there- 
fore less  difficult.  Of  course  the  abnormal  heads  can  be  obtained 
from  still  longer  pieces  if  regulation  occurs  at  low  teinper.n  mv>. 


CENTRAL    NERVOUS    SYSTEM    IN    PLANARIA.  43 

but  the  length  of  time  necessary  for  regulation  in  such  cases  is  a 
disadvantage.  Abnormal  heads  produced  by  the  action  of  anes- 
thetics and  by  various  other  conditions  were  not  included  within 
i!i«-  scope  of  the  present  investigation.  It  is  not  improbable  that 
compari-on  of  the  heads  produced  by  different  conditions  will 
-how  more  or  less  characteristic  differences  in  the  nervous  system. 
In  all  ca-es  described  the  pieces  were  kept  for  at  least  two 
ueek-  after  -ection.  After  this  length  of  time  the  new  head  is 
well  de\  eloped  and  those  cases  in  which  the  teratomorphic  head 
does  ii"!  UMII,  iin  teratomorphic  but  redifferentiates  into  a  head 
ot  normal  -hape  have  already  undergone  this  further  regulation 
or  -lio\\  unmistakable  indications  of  it.  The  teratomorphic 
head-  \\liiili  persist  as  such  for  two  weeks  at  20°  ('.  almost 
-hou  any  further  changes. 


II      I  in    MEAD  OF  THE  NORMAL  ANIMAL. 
The  form  and  the  chief  external  features  of  the  normal  head 
ot  J'laiHiriti  dorotocephala  are  .,ho\vn  in  Fig.  i.     The  unpigmented 
areas  ••!  the  eyes  and  the  very  slightly  pigmented  sensorj  regions 

ol  iln-  auricle-  are  indicated  by  dotted  lines.  Except  t"i"  these 
the  dor-al  .-mtace  of  the  head  is  usually  rather  deeply  ami  uni- 
loimly  pigmenied.  I  mm  the  ventral  surface  the  outline-  "t 
the  (ephalic  ganglia  and  nerve  cords  are  in  li-iin.  il\  \i-ible  in 
the  li\  in::  animal. 

I  •  j  5  shou    transverse  sections  ol   the  ner\ou-  system 

ai  ilillerent  levels  of  the  head  region.  1  i^.  J  i-  I'  mm  a  K-\el 
al»  nit  half  uav  between  the  eyes  and  the  lip  of  the  head  and 
four  nerves  extending  to  the  anterior  head  region.  Further 
these  ner\es  break  up  and  become  less  di-tin<t  as 
the\  are  ili-nibuted.  l;ig.  3  shows  the  icanulia  at  about  one 
fourth  ot  the  distance  from  the  e\  e-  to  the  tip.  The\  consist 
of  hlier  tracts  including  a  few  i  ell-  .md  -urmnnded  \>\  maii\ 
oilier-.  At  thi-  level  the  chief  fiber  tract  shou-  indication-  of  a 
beginning  -eparation  into  right  and  left  hal\e-.  <  )n  e.ich  side 
of  the  chief  tract  is  a  small  tract  separated  from  it  by  cells: 
the-e  tuo  -mall  tracts  are  cros-  .-ections  of  ner\e-  \\hich  pass 
io  the  anterior  re-ion-  ol  the  head. 

i  i-me  4  shows  th«    ^      Jia  at  the  level  of  the  eyes.     The  t\\o 


44 


C.    M.    CHILD    AND    E.    V.    M.    MCK.IK. 


masses  lie  some  distance  apart  and  are  connected  by  "commissures. 
The  pigment  cups  of  the  eyes  open  laterally  and  the  optic  nerves 
pass  in  a  dorso-ventral  direction.  Between  the  eyes  the  median 
anterior  branch  of  the  alimentary  tract  appears. 


3 


S 

FIGS.   1-5. 


Herr 


Figure  5  is  from  a  section  at   ihe  level  <>l   tin-  aurick>. 
there  is  no  distinct  commissure  between  the  ganglionic 
It  is  quite  possible,  however,  that  they  arc  not  mtiivly  discon- 


i   I.NTRAL    NKRYOUS    ^\>TKM    IN    PLANARIA. 


45 


nected  as  they  appear  in  the  figure;  some  nerve  fibers  may  extend 
aero-,  the  space  between  them.  The  nerves  passing  to  the 
auri<  les  appear  in  the  section  and  the  alimentary  tract  lies  on 
i  IK-  dorsal  side.  Posterior  to  this  level  the  nervous  system  con- 
sists of  the  two  main  nerve  cords,  each  composed  of  a  fiber  tract 
including  -ome  cells  and  surrounded  by  other-  ami  ^i\inu  ri-e 
to  in -r\e-  and  commissures  at  various  level-. 

III.    TKRATOPHTHALMK    Hi  \i>-. 

( »t  i  In.-  \aiious  types  of  teratophthalmic  heads  only  iln>-e  \\hich 
-hou    partial   t'u>ion  of  the  optic  pigment  cup-  were  examined 
Thf   form-    \\ith    unef|ual   or   unsymmetrical   I-NC>   lonstitutr   a 
(littt-rc-nt  type  of  teratophthalmia  and  n-'|tiin-a  niori- 
in\c-ii^ation:  moreover,  the  partial  !ti>i<>M-  ot  ilir  eyes 


9 

FIGS.  c> 

lead   thmui'li  all  possible  stages  to  tin-  -indr  nirdian  r\c  o!   tin- 
ii-raioiiion>hic  head. 

l;i-uir  (>  >ho\\-  tin-  outline  of  the-  body  and  the  condition  of 
tin-  eyes  in  «>iu-  of  the  teratophthalmic  heads  >erti<mcd.     The 


46  C.    M.    CHILD    AND    E.    V.    M.    McKIE. 

two  optic  pigment  cups  are  symmetrically  situated  but  lie  closer 
together  than  in  normal  animals  and  are  united  by  a  continuous 
band  of  pigment. 

In  Fig.  7  a  transverse  section  of  the  nervous  system  from  about 
the  posterior  fourth  of  the  preocular  region  is  shown.  It  consists 
of  a  single  fiber  tract  surrounded  by  cells  and  without  any  trace 
of  division  into  right  and  left  halves.  Comparison  with  Fig.  3 
which  is  from  about  the  same  level  in  the  normal  animal  shows 
a  marked  difference  in  form.  Fig.  8  shows  the-  level  of  the  eye-. 
The  difference  between  this  and  Fig.  4  from  the  normal  animal 
is  striking.  In  Fig.  8  the  fiber  tract  is  partially  divided  into 
right  and  left  halves,  but  the  two  parts  are  close  together  instead 
of  being  widely  separated  and  connected  by  a  long  commissure 
as  in  Fig.  4.  In  Fig.  9  a  section  at  the  level  of  the  auricles  is 
drawn :  much  the  same  differences  from  the  normal  (Fig.  5)  ap- 
pear here.  The  two  ganglionic  masses  are  closely  connected, 
while  in  the  normal  animal  they  are  widely  separated. 

The  figures  from  this  teratophthalmic  head  show  one  other 
point  of  interest.  The  individual  from  which  the  sections  were 
made  was  much  smaller  than  the  full  grown  animal  of  Figs.  2-5. 
Figs.  2-5  and  7-9  are  drawn  to  the  same  scale  and  comparison 
shows  at  once  that  the  ganglia  arc  almost  as  large  in  the  tera- 
tophthalmic as  in  the  normal  animal .  This  is  a  general  character- 
istic of  physiologically  younger  as  compared  with  older  and  of 
smaller  as  compared  with  larger  animals.  In  the  small  young 
animal  the  nervous  system  is  always  of  relatively  large  size  and  in 
small  animals  which  result  from  the  regulation  of  pieces  the 
same  is  true,  except  in  the  more  extreme  abnormal  types,  where 
the  nervous  system  is  often  small.  Thus  as  regards  the  develop- 
ment of  the  nervous  system  as  well  as  its  rate  of  metabolism 
during  development  (Child,  'i  \b)  the  animal  formed  by  regulation 
resembles  a  young  animal. 

In  oilier  teratophthalmic  individuals  with  partially  tused  eyes 
the  general  form  of  the  ganglia  was  found  to  be  murh  the  same 
as  in  the  case  described  and  the  degree  of  fusion  or  separation 
of  the  ganglia  corresponds  rather  closely  with  thedegreeof  fusion 
or  separation  of  the  eyes.  In  these  forms  then  the  eye-  serve 
to  some  extent  as  an  index  of  the  condition  of  the  nervous  -\  stem. 


VTRAL    NERVOUS    SYSTEM    IN    PIANARIA. 


47 


IV.    TERATOMORPHIC  HEADS. 

i  1  lu-  first  case  to  be  described  is  shown  in  Fig.  10.  Here  the 
auricles  appear  on  the  front  of  the  head  and  extend  anteriorly. 
The  anterior  margin  of  the  head  between  them  is  slightly  rounded 
instead  <>t  pointed  as  in  the  normal  animal.  In  tin-  median  line 
is  a  -in.'li-  eye. 

-  tions  of  the  head  region  of  this  animal  are  shown  in  Figs. 
11-15  The  eye  is  situated  almost  at  the  extreme  .interior  end 


to 


FIGS.   10-15. 

"I  i  IK-  \  .ionic  mass  and  a  few  sect  inn-  anterior  io  it  tin- 
in-! -\nu-  -\-tc-ni  appears  as  in  Fig.  11.  Mere  four  filter  tracts 
Mirnumded  by  cells  are  visible  and  are  e\identl\  nerves  t«»  the 
anterior  re-ion-  of  the  head.  The  conditions  ai  the  le\  el  <>!' 
the  eye  are  -ln»wn  in  Fig.  12.  The  -inije  optic  pigment  cup 
open-  anteriorly  instead  of  laterally,  as  the  adjoining  -ectinn- 
on  the  -lide  -ln>\\  .  and  it  is  farther  from  the  dor-al  -urla<  e  ot'  tin- 
head  and  more  nearly  imbedded  in  the  u.uu-!ionie  ma—  than  in 


C.    M.    CHILD    AND    E.    V.    M.    McKIE. 


the  normal  anima!  (Fig.  4).  The  ganglionic  mass  itself  is  some- 
what irregular  in  form  and  shows  no  trace  of  a  division  into 
symmetrical  right  and  left  portions. 

Figure  13  shows  the  condition  of  the  ganglia  six  sections  (sixty 
micra)  posterior  to  the  eye.  Here  the  fiber  tracts  show  indica- 
tions of  a  symmetrical  arrangement,  hut  this  arrangement  is 
widely  different  trom  the  normal.  Three  sections  farther  pos- 
teriorly the  fiber  tracts  are  still  more  broken  up,  as  shown  in 
Fig.  14.  The  level  of  this  section  is  approximately  the  posterior 
end  of  the  anterior  new  tissue  of  the  regenerated  region.  Fig.  15 
shows  a  section  sixty  micra  posterior  to  the  level  of  Fig.  14,  /.  e., 
in  the  old  tissue:  here  tin-  nervous  system  appears  in  the  usual 
form  of  two  ganglionic  ventral  cords,  which,  however,  are  much 
less  widely  separated  than  in  the-  normal  animal  at  this  level. 

2.  The  animal  sectioned  is  shown  in  Fig.  16.  The  head  is 
much  like  that  in  Fig.  10,  but  the  auricles  are  somewhat  closer 
together.  A  single  median  eye  with  a  rather  large  pigment  spot 
is  present. 

Figures   17  and   18  show  sections  of  the  head.     Fig.   17  is  a 


16 


18 


Fics.    16-18. 


section  a  short  distance  anterior  to  the  eyes,  about  the  posterior 
fourth  of  the  preocular  region.  Fig.  iX  shows  the  level  of  tin- 
eyes .  The  single  pigment  cup  appears  in  the  liguiv  io  open 
ventrally,  but  the  opening  is  actually  antero-ventr.il  in  direction. 
The  ganglionic  mass  is  distinctly  double,  /.  c.,  more-  like  the 


CENTRAL    NERVOUS    M>IKM     IN     I'l.ANAKIX. 


49 


normal  than  that  of  Fig.  12.  Two  nerves,  one  from  each  portion 
of  the  ganglionic  mass,  pass  to  the  optic  cup. 

He-re,  as  in  the  preceding  case,  the  eye  is  situated  near  the 
extreme  anterior  end  of  the  ganglionic  re-ion  instead  of  a  con- 
-iderable  distance  posterior  to  it  as  in  normal  forms.  Posterior 
i"  the  eye  tin-  torm  of  the  ganglionic  mas-  i-ontinues  murh  the 
-ame  a-  in  I  i_  18  to  about  the  posterior  end  of  the  ne\\  ii»ue. 
uliere  the  He  lit  and  left  portions  become  more  distinetly  -epa- 
r.iied  \\itli  .1  commissure  between  them  and  then  pa—  into  the 
tuo  ner\  e  e..rds. 

In  general  form  the  nervous  system  is  much  le—  abnormal  in 
i hi-  i hati  in  the  preceding  case.  The  chief  difference-  from  the 
norm  are  the  anterior  position  of  the  eye  on  the  ean-lionic  ma-- 
and  the  partial  !u-ion  of  the  two  ganglia  for  a  considerable  di-- 
tanee  |»i-ierior  to  the  eye. 

v     \-  indicated  in  Fig.  i<),  this  case  shows  a  someu  hat  extreme 


2O 


21 


• 


FlGS.     IQ    22. 

lorm  of  teratomorphism.  The  two  auricle-  are  tn-ed  at  the  lip 
of  the  head,  though  the  sensory  areas  are  in  large  part  separate. 
A  -ingle  median  eve  is  present  as  in  the  preceding  < 

I  ig.  20  sho\\  -  a  t  ransverse  section  <  >t  the  IHT\  <  m-  -\  -tern  .it  the 
level  where  the  in  t  \  e-  to  the  front  of  the  head  ari-e:   rlii-  is  about 


C.    M.    mil, I)    A\l>    I-;.    V.     M.     M,  KII-. 


one  tilth  <>I  the  distance  from  the  cyc>  to  the  tip  of  the  head. 
Five  fiber  tracts  unsymmetrically  situated  an-  indicated  in  the 
section. 

In  Fig.  21  the  level  of  the  eye  is  shown.  The  optic  pigment 
cup  opens  antero-ventrally  and  toward  the  left  side  and  i-  con- 
nected by  a  nerve  with  the  left  side  only  of  the  ganglionic  ma--. 
The  latter  shows  a  distinct  division  into  right  and  left  halves. 

Near  the  posterior  end  of  the  regenerated  region  the  nervous 
system  possesses  the  form  shown  in  Fig.  22  and  a  short  distance 
posterior  to  this  level  and  in  the-  old  tissue  the  two  nerve  conl- 
become  separate  except  for  an  occasional  commissure. 

In  this  case  the  eye,  though  median  in  portion,  evidently 
belongs  to  the  left  half  of  the  ganglionic  mass  and  the  nervous 
system  is  much  less  abnormal  than  in  Case  I.  As  in  the  other 
cases,  the  eye  is  situated  near  the  extreme  anterior  end  of  the 
ganglion. 

4.   In  this  case  (Fig.  23)  the  fusion  of  the  auricles  at  the  front 


24- 


26 


FIGS.   2 ^ 


of  the  head  is  even  more  complete  than  in  Case  ,},  only  the  ba-e- 
ot  the  sensory  areas  being  separated.  The  eye  is  median  and 
apparently  single  and  the  pigment  spot  is  of  rather  large  si/e. 
Anterior  to  the  eye  the  ganglionic  mass  breaks  up  almost  inline- 


CENTRAL    NERVOUS    SYSTEM    IN    PLANARIA.  5! 

diately  into  two  nerves  passing  to  the  front  of  the  head  (Fig.  24). 
In  Fig.  25  it  becomes  evident  that  the  apparently  single  eye  is 
actually  double.  One  of  the  pigment  cups  lies  slightly  anterior 
and  \i-ntral  to  the  other  and  somewhat  to  the  left  of  it.  The 
opening  <>f  the  more  posterior  and  dorsal  cup  i-  -een  in  Fig.  25, 
while  tli«-  other  pigment  cup  appears  here  as  a  complete  circle. 
P.oth  open  antero-ventrally  and  toward  the  right.  The  gan- 
•Jionic  ma—  i-  not  divided  into  right  and  left  halves  and  the 
optic  nerve-  arise  from  its  median  region.  Both  eyes  are  far 
belo\\  i  he  dor-al  surface  of  the  body  and  the  more  ventral  one 
i-  imbedded  in  the-  ganglion. 

The  double  nature  of  the  eye  is  not  apparent  in  the  living 
animal  since  the  two  pigment  cups  lie  so  close  together  and  one 
i-  almo-i  \entral  to  the  other. 

l'<  -icrior  to  the  level  of  the  eyes  the  ganglion  i-  abnormal  in 
lorm  to  about  the  posterior  end  of  the  regenerated  region.  I 
Jo.  .1  -e<  lion  -li-htly  anterior  to  the  boundary  bet  \\een  ne\\  and 
old  tissue,  shous  that  in  general  form  and  arrangement  of  the 
hi  MI  ILK  i-  i  he  ganglion  at  this  level  resembles  that  of  Case  i 
Fig.  13).  \  little  farther  posteriorly,  in  the  old  tissue,  it 
di\ide-  into  lijn  and  left  j>ortions  and  gives  rise  to  t\\o  nerve 
cords  ot  tin-  n-nal  form,  but  somewhat  nearer  together  in  their 
anteHo]  region  than  in  normal  forms. 

.V  Tin-  case  is  like  Case  4  in  external  appearam  <  IK  23 
and  al-o  in  the  number  and  arrangement  of  the  eyes,  but  -onie 
diltereine-  in  the  structure  of  the  nervous  system  e\i-t.  I  ig.  28 
-hou-  the  le\  el  of  origin  of  nerves  to  the  front  of  the  head,  a  l<-\  el 
-liJnK  anterior  to  the  eyes.  In  Fig.  29  the  eyes  an  seen  to  be 
^lightly  larger  than  in  the  preceding  case,  but  other\\i-e  -imilar 
to  it.  Hoth  open  antero-ventrally  and  toward  the  ri^hi  and  one 
lie-  to  the  K  It  of,  \-entral  and  slightly  anterior  to  the  other.  The 
gan^lionie  ma--  is  single,  but  larger  than  in  (  a-e  4  at  tin-  level 
I  igs  Jo  and  25  and  the  optic  nerves  ari-e  Irom  it  -omeuhat 
to  the  ii-ht  of  the  middle. 

Posterior   to   the  eyes  the  ganglion  -ooii   -lm\\  -  di-tinct   right 
and   left    hal\e-   but    these  are  abnormal   in   -hape  and   each    is 
broki'ti   iij)  into  a   number  of  more  or  le--  di-tinct    liber   trai 
\\hich.  however,  are  apparently  symmetrically  ariMtt^ed  in   the 


52  C.    M.    CHILD   AND    £.    V.    M.    McKIE. 

right  and  left  halves  (Fig.  30).  This  peculiar  arrangement  con- 
tinues to  about  the  posterior  end  of  the  regenerated  region  and 
then  changes  into  the  form  shown  in  Fig.  31:  posterior  to  this 
the  two  ventral  cords  appear  in  the  usual  form. 

These  five  cases  give  some  idea  of  the  variations  in  structure  of 
the  eyes  and  the  nervous  system  in  the  teratomorphic  forms.     A 


FIGS.  28-31. 

more  extended  investigation  of  these  forms  will  undoubtedly 
show  other  variations  in  structure  and  examination  of  anoph- 
thalmic  and  headless  forms  will  add  still  further  data  of  interest. 

V.    DISCUSSION. 

In  all  of  the  cases  described,  both  the  teratophthalmic  and 
the  teratomorphic  forms,  the  most  conspicuous  difference  in  the 
nervous  system  as  compared  with  the  normal  forms  is  the  more 
or  less  complete  fusion  in  the  median  line  of  the  two  portions 
of  the  ganglionic  mass,  or  more  correctly,  their  incomplete 
separation. 

It  is  evident  that  to  some  extent  the  condition  of  the  eyes  or 
eye  is  an  index  of  the  condition  of  the  nervous  system.  The 
parallelism  is,  however,  not  complete:  in  Case  I  (Figs.  10-15), 
for  example,  the  nervous  system  is  much  more  abnormal  than 
in  Cases  2  (Figs.  16-18)  and  3  (Figs.  19-22),  though  all  three 
possess  a  single  median  eye. 

Moreover,  the  degree  of  fusion  of  the  auricles  does  not  corre- 
spond exactly  to  the  degree  of  fusion  of  the  ganglia  in  all  cases. 


CENTRAL  NERVOUS  SYSTEM  IN  PLANARIA.          V^ 

In  Case  I  where  the  auricles  are  a  considerable  distance  apart 
(Fig.  10)  the  fusion  of  the  ganglia  (Figs.  11-15)  *s  more  complete 
and  their  structure  is  more  abnormal  than  in  Cases  2  (Figs.  16- 
18)  and  3  I  L-.  19-22),  where  the  auricles  are  nearer  together. 
In  Cases  4  .ni'l  5.  where  Uvo  eyes  develop  close  together,  far 
from  the  -urface  and  in  abnormal  relations  to  each  other  and 
where  the  auricle-  are  partially  fused,  the  ganglionic  region  is 
highly  abnormal. 

\\lien.    ho\\e\er,   we  compare   the   teratophthalmic   with    the 

lomorphic  forms  it  is  evident  that  a  general  parallelism  be- 

tueei!  tin-  external  features  of  the  head  and  the  condition  of  the 

nervous   system  does  exist.     So  far  as  the  observation-  go  at 

•  •in.    tin-   111  r\ oiis  system   is  always  more  abnormal   in    the 

irraioini.rphii     !<>rms   than    in    the   teratophthalmic   form-    \\ith 

partialK    tu-ed  t •• 

\  n  gards  the  eyes  themselves  certain  points  are  of  inttn-t 
In  the  normal  and  partially  fused  eyes  the  pigment  cups  open 
laterally,  \\hile  in  the  teratomorphic  forms  they  open  anteriorly 
or  an  1 1 -i  <  i- \em  rally.  Moreover,  the  eyes  are  usually  farther  from 
tin  doi-s.il  -urlace  of  the  head  in  the  teratomorphic  form-  than 
in  other-. 

1  In    -iiu'le  <-ve  of  the  teratomorphic  head  may  be  connected 
\\ith  both  -ide-  of  the  ganglionic  mass  (Fig.  18)  or  with  only  one 

I  i.  j  i  and  in  case  two  optic  cups  arise  in  the  teratomorphic 
hea-l  I  =,  and  2(>)  they  may  both  be  connected  with  the  -ame 

part  nt   t!  .  limi. 

The  pM-itioiiof  the  eyes  or  eye  on  the  ganglion  may  differ  more 
or  less  ui'lek  in  the  normal  and  abnormal  forms.  In  the  normal 
animal  Figs,  i  5)  the  eyes  lie  dorsal  to  the  posterior  region  of  t  hi' 
^airjioii.  \\liii-h  divides  a  -hort  distance  behind  them  into  the 
t\\o  nerve  cords.  In  the  teratophthalmic  forms  with  partialK 
fn-eil  eyes  a  .  cn-iilerable  portion  of  the  ganglion  lie-  anterior 
to  the  eyes  M^.  7)  but  the  right  and  left  sides  of  the  nervon- 
system  remain  united  farther  posteriorly  than  in  tin-  normal 

I  k-.  5  and  T.  In  the  teratomorphic  forms  the  eye  lies  dor-al 
to  the  extreme  anterior  portion  of  the  ganglion  and  the  t\\<>  eonl- 
do  not  become  -eparated  for  a  considerable  di.-tance  posterior 
to  it 


54  C.    M.    CHILD    AND    E.    V.    M.    McKIE. 

In  the  development  of  the  abnormal  forms  the  eye  undoubtedly 
arises  in  connection  with  the  central  nervou^  system  .1-  it  does 
in  the  normal  animals.  The  position  and  number  of  the  eyes 
must  be  determined  primarily  by  the  condition  of  the  nervous 
system,  though  other  factors  may  play  some  part.  Apparently 
the  median  regions  of  the  nervous  system  are  more  or  less  reduced 
or  fail  to  develop  in  the  abnormal  forms  and  the  lateral  region:- 
consequently  lie  nearer  together  so  that  the  eyes  appear  near 
or  in  the  median  line.  The  approximation  and  fusion  of  the 
auricles  is  also  evidently  due  to  the  reduction  or  absence  of  the 
median  region  of  the  head  and  this  condition  is  undoubtedly 
closely  connected  in  one  way  or  another  with  the  condition  of 
the  ganglia.  There  can  be  no  doubt  that  the  condition  of  the 
nervous  system  is  the  most  important  factor  in  determining  the 
characteristic  features  of  the  teratophthalmic  and  teratomorphic 
heads. 

One  of  the  most  interesting  points  in  connection  with  the 
whole  series  of  forms  is  the  fact  that  in  the  teratomorphic  forms 
the  whole  length  of  the  regenerated  nervous  system  is  abnormal 
(Figs.  13  and  14,  Fig.  26,  Fig.  30).  Not  until  the  level  of  the  old 
tissue  is  reached  do  the  two  cords  appear  in  their  usual  relations 
and  even  there  they  are  commonly  nearer  together  than  in 
normal  animals  (Figs.  15,  22,  27,  31).  This  fact  suggests  that  the 
development  of  the  regenerated  portion  of  the  nervous  system  is 
in  large  measure  independent  of  the  already  existing  portion.  1 1 
the  development  took  place  in  the  anterior  direction  from  the 
cut  ends  of  the  nerve  cords  in  the  old  tissue,  it  is  difficult  to 
understand  how  such  structures  as  those  shown  in  Figs.  13  and 
14,  26  and  30  could  arise  near  the  old  tissue.  Unt  if  the  develop- 
ment of  the  regenerated  part  takes  place  independently  of  the 
old  part,  the  continuation  of  the  abnormal  structure  back  to  the 
level  of  the  preexisting  portion  constitutes  a  less  difficult  problem. 

According  to  recently  published  work  of  the  senior  author 
(Child,  'ii</)  the  formation  of  a  new  whole  from  a  headless  pic  i  e 
of  Planaria  consists  essentially  in  the  formation  tirst  of  all  ot  a 
new  head  region  which  then  reorganizes  the  parts  posterior  to  it 
through  correlation.  The  structure  of  the  regenerated  portion 
of  the  nervous  system  in  the  teratomorphic  lorms  certainly  oller> 


CENTRAL  NERVOUS  SYSTEM  IN  PLANARIA.          55 

mon-  -upport  to  this  conclusion  than  to  that  view  which  maintain-' 
i  hat  the  regenerating  nervous  system  grow-  out  anteriorly  from 
the  cut  ends  of  the  old  nerve  cords.  Apparently  in  tln-r  cases 
,i  neu  central  nervous  system  develops  and  is  abnormal  from 
the  lie-inning,  but  as  its  differentiation  extend-  posteriorly  it 
meet-  the  old  nerve  cords  and  unites  with  them.  In  this  \\ ax- 
il i-  easy  to  account  for  the  relatively  sudden  change  in  the 
-iructure  of  the  nervous  system  as  we  follow  it  posteriorly  from 
the  ne\\  into  the  old  tissue  in  some  of  the  teratomorphic  forms 
Fij  i  }  and  Is,  26  and  27,  30  and  31).  But  ex'en  the  anterior 

ions  oi  the  old  nerve  cords  undergo  reorganization  to  a  greater 

or  le--  extent  under  the  influence  of  the  nexv  region  anterior  to 
them.      In  bigs.  15,  27  and  31  they  are  nearer  together  and  con- 
m  i  ted   1.x    larger  commissures  than   they  were  ori-inallx-   when 
thex   ii.nned  a  part  of  the  posterior  region  of  the  In-t  /ooid. 
The  structure  of  the  nervous  system  in  the  posterior  part  of  the 

ni-rate. 1  tejon  in  such  cases  as  Fig.  14  and  Fig.  $<>  suggests 
a  breaking  up  into  separate  IUTVL-S ,  but  posterior  to  the  lex  els  of 
iln  se  -' ••  tions  where  the  developing  jjortion  meets  the  o'd  cords 
there  i-  a  return  to  something  approaching  normal  structure.  It 
is  possible  that  if  the  old  cords  were  not  present  in  such  cases 
the  m  u  IHTXOUS  system  would  extend  posteriorly  as  a  c,,n-i( Id- 
able  number  of  separated  cords  or  nerves  instead  of  in  the  lorm 
eharai  leii-tic  of  normal  animals. 

It  \\as  pointed  out   in  Section   I.  that  the  teratophthalmic  and 
teratomorphic    forms    can    be    produced    experinientallx     bx     de- 

!-ing  the  rate  of  the  dynamic  processes  in  the  piece  b.  l..\\  ,i 
n  Main  rale  necessary  for  the  production  of  normal  animals 
\\hich  i>  it-elf  not  constant  but  dependent  upon  various  condi- 
tions. |'he-e  abnormal  forms  then  represent  planarian  morpho- 

esis  corresponding  to  certain  rates  ,.i  reaction  belo\\  the 
"  normal "  rate  for  the  existing  conditions.  The  tact  that  change- 
\\hich  are  primarily  quantitative  gixe  rise  to  -uch  dilleivnces  in 
structure  a-  those  recorded  is  important.  A-  the  rate  <.t  reaction 
decreases  we  see  certain  parts,  c.  "...  the  preocnlar  re-ion  of  tin- 
head,  decreasing  in  relative  size  and  finally  disappearing  and 
in  the  nervous  system  the  bilateral  structure  of  the  ganglia 
becomes  lc--  and  less  distinct  in  consequence  of  the  reduction  and 


56  C.    M.    CHILD   AND    E.    V.    M.    McKIE. 

disappearance  of  the  median  regions.  Apparently  \ve  are  jus- 
tified in  concluding  that  the  reduction  and  di-.ippearance  of 
certain  parts  as  the  rate  of  reaction  decreases  is  due  one  of  two 
alternatives:  first,  the  reduced  or  absent  part  may  represent  a 
relatively  low  rate  of  reaction  in  the  normal  animal  and  under 
the  experimental  conditions  the  rate  of  the  reaction  which  i> 
essential  for  its  formation  approaches  or  falls  below  what  m.iy 
be  called  the  morphbgenic  threshold,  i.  e.,  it  does  not  produce 
the  characteristic  morphological  effect.  Second,  a  part  may  be 
reduced  or  disappear  under  conditions  which  decrease  the  rate 
of  reaction,  not  because  the  reaction  concerned  in  its  formation  is 
directly  affected  by  the  experimental  conditions,  but  because 
its  formation  depends  upon  correlation  with  some  other  part 
which  is  thus  affected.  It  is  probable,  for  example,  that  the 
condition  of  the  central  nervous  system  in  the  abnormal  forms 
is  largely,  at  least  in  the  cephalic  ganglia,  a  direct  effect  of  the 
experimental  conditions,  while  the  position,  number  and  presence 
or  absence  of  the  eyes  and  the  degree  of  development  of  the 

* 

preocular  region  are  to  a  considerable  extent  correlative  effects. 

But  however  we  may  account  for  the  results  it  is  a  demon- 
strated fact  that  the  reduction  and  disappearance  of  parts  of  so 
"essential"  an  organ  as  the  central  nervous  system  can  be 
brought  about  experimentally  by  quantitative  changes  in  ex- 
ternal or  internal  conditions.  No  absence  of  chromosomes  or 
determinants  and  no  germinal  variation  is  necessary  for  the 
production  of  these  abnormal  forms,  but  only  a  decrease  in  the 
rate  of  the  dynamic  processes  in  the  piece,  together  with  the 
necessary  correlative  effects  of  such  a  decrease. 

It  is  impossible  to  leave  the  subject  without  some  reference 
to  the  '"cyclopean"  fish  embryos  which  Stockard  ('07,  '09,  '10) 
has  recently  produced  by  means  of  magnesium  chloride  and 
alcohol.  The  resemblance  between  these  forms  and  the  tera- 
tophthalmic  and  teratomorphic  forms  of  Planaria  is  striking.  In 
both  cases  organs  which  are  normally  bilaterally  symmetrical 
in  position  show  various  degrees  of  approach  and  in  the  extreme 
types  a  single  median  organ  develops  in  place  of  the  two.  An- 
ophthalmic  forms  also  occur  in  Planaria  and  under  extreme  ex- 
perimental conditions  completely  headless  forms  also  appe.ir. 


CENTRAL    NERVOUS    SYSTEM    IN    PLANARIA.  57 

Moreover,  in  Planar  ia  the  auricles,  like  the  eyes,  show  various 
degrees  of  approximation  and  fusion  and  in  the  present  paper 
it  has  been  shown  th.it  similar  conditions  appear  in  the  cephalic 
ganglia  them-el\e-.  In  Planaria  these  monstrous  forms  can  be 
produced,  not  inrn-Iy  by  anesthetics,  but  by  a  variety  of  con.li- 
tioii-  tin  essential  efie.-t  of  which  is  a  decrease  in  the  rate  of  the 
reaction-- in  tln-li\iii^  -ystem.  It  seems  probable  that  Stockard's 
<  \.-lope. m  embryo-  .uid  the  other  intermediate  forms  between 
.ui<l  tin-  normal  animals  are  the  result  of  a  deriva-e  in  the 
<>t  reaction  r.iilii-r  than  of  any  specific  anesthetic  effecl  ot 
either  m.mne-inm  -.ilt-,  or  alcohol.  Moreover,  thr  double  or 
p.irti.illv  double  he. id-  which  Stockard  obtained  in  some  ca 
are  also  p-.idiK  .i> -counted  for  on  a  quantitative  b.i-i-:  a  «!>•- 
ised  i. nr  ot  reaction  means  decreased  correlation  and  thi- 
... n<  lit  ion  f.ivor-,  ph\  -iological  isolation  of  parts  and  repr.'.Iuciion, 
;i-  ihi-  -enior  autlior  of  tin-  present  paper  has  shown  i-Ucwlu -n- 

Child,  'n 

1  IK-  problem  ol  ilu-  relation  between  morphogenesis  and  the 
rate  "t  reaction  in  organisms  is  one  of  great  important-,  but  it 
h.i^  received  i-omp.irati\-ely  little  attention.  Current  hypoth. 
ol  .li-\i-lo|uueiit  .in.l  inheritance  scarcely  consider  the  |)o-,i!,ilit\ 
ot  altering  tin-  rhuructeristic  morphological  features  of  t!i«  organ- 
ism bv  i-liaiue>  in  the  rate  of  reaction,  but  of  the  fact  there  <  an 

bi-  no  doubt. 

\'I.    StMMAKV. 

1.  Tin-  ti  i.ito|)hthalmic  and  teratomorphic  forms  of  Planaria 
tlnrot<>iff)liala    can    be    produced    experimentally    by    d..  ic.i-inu 
the  r.ite  of  the  dynamic  processes  in  the  isolated  pieces  below 
.1  certain   \ari.ible  le\el  which  is  necessary  for  the  product!.. n 
o|   not  in.il  I-  'i  in-. 

2.  In   the-e  forms  the  cephalic  region  of  the  IHTVOII-  system 
.litter-   more  or  le^s  widely  from  that  of  normal   .mim.iU.       The 
tu.i    -.nulioiiic    masses    are   always    less  completely   M-p.ir.ited 
than   in    the   normal   animals  and  often   only   a   -in^le   -  m-lion 
de\elop>.     In    the   teratomorphic   form-    the   ganglia    are    more 
.ibnormal  thai;  in  the  teratophalmic  form-. 

V    In  the  normal  animals  the  cephalic  ganglia  extend  a   con- 
-i.lei.tble    (li-i.iiu-e    anterior    to   the   eyes   and    the   l\\o   -eparate 


58  C.    M.    CHILD    AND    K.    V.    M.    McKIE. 

nerve  cords  arise  near  the  level  of  the  auricles.  In  the  teratoph- 
thalmic  forms  with  partially  fused  eyes  the  eyes  lie  nearer 
the  anterior  end  of  the  gangliei  and  the  right  and  left  portions 
are  not  separated  at  the  level  of  the  auricles.  The  eyes  of  the 
teratomorphic  forms  are  situated  at  the  extreme  anterior  end 
of  the  ganglionic  mass. 

4.  The    abnormal    structure    of    the    nervous   system    in    the 
teratophthalmic  and  teratomorphic  forms  continues  posteriorly 
through   the  regenerated  anterior  end   to   the-  level   of   the  old 
tissue  and  even  the  nerve  cords  in  the  old  tissue  may  he  more  or 
U>s  different  from  the  normal.      In  some  teratomorphic  forms 
the    regenerated    nervous  tissue  apparently  begins  to  break  up 
into  separate  nerves  a  short  distance  posterior  to  the  eyes,  but 
resumes  the  form  of  two  nerve  cords  in  the  old  tissue. 

5.  In  the  normal  animal  the  optic  pigment  cups  open  laterally 
and  the  same  is  true  for  the  teratophthalmic  forms  with  partially 
fused  eyes.     In  the  teratomorphic  forms  the  cup  opens  anteri- 
orly or  antero-ventrally  and  in  some  cases  more  or  less  to  one 
side.     The  single  median  eye  may  be  connected  by  two  nerves 
with  right  or  left  portions  of  the  ganglionic  mass,  or  by  a  single 
nerve  with  either  one,  or  the  optic  nerve  may  arise  from  the 
median  region  of  the  ganglion.     The  eyes  are  also  farther  from 
the  dorsal  surface  in  the  teratomorphic  than  in  the  normal  forms 
and   are  sometimes   more  or  less  completely   imbedded   in   the 

ganglionic  mass. 

HULL  ZOOLOGICAL  LABORATORY. 
UNIVERSITY  OF  CHICAGO, 
October,  191 1. 

REFERENCES. 
Child,  C.  M. 

'na  Studies  on  the  Dynamics  of  Morphogenesis  and  Inheritance  in  Experi- 
mental Reproduction.  I.  The  Axial  Gradient  in  Planaria  dorotocephala 
as  a  Limiting  Factor  in  Regulation.  Journ.  Exp.  Zool.,  Vol.  X.,  No.  3, 
191  i. 

'nb     A  Study  of  Senescence  and   Rejuvenescence  Based  on  Experiments  with 
Planaria  dorotocephala.     Arch.   f.    Entwickelungsmech.,    Bd.     XXXI..    II 
A,    ion. 

'lie  Experimental  Control  of  Morphogenesis  in  the  Regulation  of  Planaria. 
Biol.  Bull.,  Vol.  XX.,  No.  6,  1911. 

"nd  Studies  on  the  Dynamics,  etc.  II.  Physiological  Dominance  of  Anti-iini 
over  Posterior  Regions  in  the  Regulation  of  Planaria  dorotocephala.  Journ. 
Exp.  Zool.,  Vol.  XL.  No.  3,  1911. 


CENTRAL    NERVOUS    SYSTEM    IN    PLANARIA.  59 

'ne     Studies  on  the  Dynamics,  etc.     III.  The  Formation  of    New  Zooids  in 

Planaria  and  other  Forms,     Journ.  Exp.  Zool.,  Vol.  XL,  Xo.  3.  ign. 
"nf     Die  physiologische   Isolation  von  Teilen  des  Organismus.     Yortr.  u.  Aufs. 

ii.   EiHvvickelungsmech..  H.  XL,  1911. 
Stockard,  C.  R. 

'07      The  Artificial  Production  of  a  Single  Median  Cyclopean  Eye  inrthe  Fish 

Embryo  by  Means  of  Sea  Water  Solutions  of  Magnesium  Chloride.     Arch. 

:    I  ntwickelungsmech..  Bd.  XXIIL,  H.  2.  1907. 
'09     Th'    I  )••-.. -lupment  of  Artificially  Produced  Cyclopean  Fish        "Tin    M 

mi  Embryo."     Journ.  Exp.  Zool.,  Vol.  VI..  N".  j.   1909. 
'10     Tin-  Intlii'-nce  of   Alcohol   and  other  Anesthetics  mi    Embrynnir 

iiK-nt.     Am.  Juurn.  Anal.,  \*ol.  X.,  No.  3,  1910. 


EVIDENCE   ON   THE   ADAPTATION   OF    PARAM/ECIA 
TO   DIFFERENT   ENVIRONMENTS. 

LORAXDE    LOSS   WOODRUFF. 

The  fact  being  established  that  my  pedigree  culture  of 
Param&cium  aurelia  (I.)  undoubtedly  has  unlimited  power  of 
reproduction  without  conjugation  or  artificial  stimulation,1  a 
culture  of  Paramcecium  caudatum  was  started  for  comparison, 
in  order  to  determine  if  this  animal  would  show  throughout  its 
life  history  characteristics  of  specific  value  and  also  to  determine 
it  it  would  continue  to  live  and  reproduce  indefinitely  without 
conjugation  or  artificial  stimulation. 

The  results  with  this  culture  led  me  to  conclude,  as  did  Jennings 
and  Hargitt,2  that  caudatum  is  a  distinct  species.  This  point  I 
have  discussed  in  a  previous  paper.3  The  results  in  regard  to 
the  second  point  arc  briefly  presented  at  this  time. 

The  pedigree  culture  of  Paramcecium  caudatum  (X.)  was 
started  on  May  14,  1910,  and  has  been  continued  under  observa- 
tion to  the  present  time,  December  i,  i<)ii.  The  methods  em- 
ployed have  already  been  described  in  detail  in  earlier  papers  on 
pedigree  cultures  of  Infusoria.  It  is  only  necessary  to  state  here 
that  the  culture  was  begun  by  placing  a  large  "wild"  individual 
on  a  depression  slide  in  about  five  drops  of  cuhuiv  medium. 
\Yhen  this  individual  had  divided  twice,  producing  four  animals, 
each  of  these  was  placed  on  a  separate  slide,  forming  tin-  four 
lines  of  the  culture.  Thereafter  (until  June  i,  H)ii)  a  single 
cell  from  each  of  the  lines  was  isolated  d.iily  in  fresh  culture 
medium  and  the  number  of  divisions  during  the  previous  twenty- 
four  hour*  was  recorded. 

In   regard    to  the  culture  of    I'arannci  iiini  aurelia    (I.),   which 


1  L.  L.  Woodruff:  "Two  Thousand  <  •<  -m  -i.itiiui^  of  Paramacium."      An'liir  fiir 
Prolistenkunde.  Bd.  21,  3.  1911. 

-II.  S.  Jennings  and  G.  T.  Harbin:  "Characteristics  of  tin-  Divei   •    K.u-i-v  ,,i 
1'aramtrcium."     Journ.  Morph.,  Vol.  21.  n<>.    |.    1010. 

3  L.  I..  Woodruff:  "  Paramiccium  aurelia  and  Paratmecittm  ca  utlatitin  " 
Morph.,  Vol.  22,  no.  2,   10.11. 

,.. 


ADAPTATION'    OF    PARAM.^ECIA    TO    ENVIRONMENTS.  6 1 

served  as  a  control  and  for  comparison  with  the  P.  caudal  um 
culture,  there  are  no  results  to  be  recorded  which  are  not  in 
(in  in-  agreement  with  these  already  published.  The  culture  has 
kept  on  the  even  tenure  of  its  way  and  is  now,  after  over  four  and 
"in-  half  years  of  daily  observation,  at  tin-  2.7051!!  generation, 
and  in  every  way  in  as  normal  morphological  and  physiological 
londition  as  at  the  start.  Given  a  favorable  environment,  thi- 
race  dearly  has  unlimited  power  of  reproduction  without  con- 

•ii»n  or  artificial  stimulation. 

I  In   |  .1  d^rec  culture  of  Paramccc ium  caudatiin:    \       which  \\a- 
-ubi'  -  led  1 1 "in  the  start  to  the  500 th  generations  (twelve  and  one 
halt  montli-i  to  identically  the  same  treatment  and  culture  me- 
dium a-  i  he  Iy.  aitrclia  culture,  showed  during  the  lir-t  350  genera 
lion-     «  K'ln    months)  essentially  the  same  rate  of  repri.diieti.m 

die  <!ur<-!i(i  culture.      However,  an  examin.iiioii   <>l    the  daia 

I  i   and  2)  shows  that  a  slow  decline  in  division  rai. 

in  ai  the  -iart  which  finally  resulted  in  a  race  of  cell-  PD--I-— ini; 
man\  "t  i  lie  morphological  and  physiological  characteri-tii  -  de- 
scribed \<\  ('alkins1  in  liis  careful  study  o!"  pure  line-  of  ihi- 
species  "i  Paramacium.  Alter  about  tin-  4501)1  ^eneiaiinn  it 
became  iin  leasingly  ditVicult  to  keep  the  animal-  ali\e  mi  the 
slide-  iii  the  culture  medium  which  was  supplied  fiv-h  daily. 
llo\\e\er.  the  cells  left  over  from  the  daily  isolations,  which  were 
alli'ued  I"  acru  mil  late  in  the  old  culture  liquid,  appeared  healthv 
and  ii'iit'imed  to  reproduce  slowly.  It  the-e  \\eiv  transferred 

•n  in  lre-h  medium  they  would  di\'ide  a  few  tinii--  and  then 
<lie  l-'inallv  lhe\-  would  not  live  twenty-four  hour-  in  the  I'n  -li 
medium. 

l'»\  -ub-tiiuting  from  the  "stock"  in  thi-  \\a\\  the  direct  line- 
"I  ihe  culture  were  kept  replenished  for  nearK  tlirc'e  month-;  bin 
ImalK  it  uas  evident  that  it  was  impo— ible  i"  cmitinne  the 
culture  b\  ilii-  method,  so  that  the  exact  number  "t  ^<  in-iation- 
could  In-  determined,  and  according  .  at  the  -,o"ih  generation, 
the  method  uas  abandoned,  and  the  animal-  \\eiv  thereafter 
«  aiiicd  iii  -mall  tlasks  of  old  infusions.  /.  e.,  lhe\  were  bred  in  a 
i  "inparati\  cl\  large  volume  of  the  same  type  of  medium  to  \\  hich 

1  (",.     X.    (\ilkin-:    "  Ilir    I   ilr    Cycle    of      l'<ii..  .:i<ltltnm."        \r,lr 

En  nismcn.  Bd.  15,  i,  190.'.     "Death  of  th     V  Series  ol 

t  onclusions."     J»um.  l-.\pfr.    '/..         Vol.  i,  no.  .•;. 


62 


LORANDE  LOSS  WOODRUFF. 


L 

,03  200  300  «°o  son 
FIG.  i.  Paramacium  caitdatnm  (Culture  X.).  Graph  of  the  rate  of  reproduction  for  the  first  500  generations  (May  14.  t<;i< 

June  i,  1  911).  See  text. 
The  average  rate  of  division  of  the  four  lines  of  the  culture  is  again  averaged  for  ten-day  periods.  The  figures  100,  -">...  etc 
present  generations  and  are  placed  below  the  ten-day  periods  in  which  they  were  attained. 

i  — 

1 

J 

r 

i 

- 

I 

1  L.                                                                                   u 

ADAPTATION    OF    I'AKAM.E(  IA    TO    ENVIRONMENTS. 


they  had  been  previously  subjected — but  a  medium  which  was 
from  -•  \ «  ral  days  to  several  w<-k-  old. 

I'nder  these  conditions  this  culture  of  P.  candatum  now 
Hoiiri-hc-,  and  it  is  continued  by  isolating  a  few  cells  every 
feu  \\rrks  and  inoculating  with  them  another  >mall  flask  of  old 
infusion.  I  nder  these  conditions  it  is  impossible,  of  course,  to 
•  It •!<  inline  with  accuracy  the  rate  of  division  or  the  number  ol 
•j<  IK  r.ni'.n-  attained  to  date,  but  the  organi-m-.  are  apparently 
in  .1  normal  physiological  condition.  H<»\\e\er.  it  i>  still  im- 


100 


200 


300 


400 


Fie,  _•.  r.uimtriium  faiiJatum  (Culture  X.).  Graph  of  the  rate  of  reproduc- 
tion I>T  tin-  i.  generations  (May  14.  1910.  to  June  i.  i 

1  In  of  division  of  the  four  lines  of  the  cultun  i-  .1  d  for 

l>iii..'l-  ,.i  on  •:.  The  figures  100,  200.  etc..  represent  &  -m  -t.iti..n-  ;ui«l  are 

IM-|I>\V  tin-  iiniiitlis  in  which  they  were  attaim-'l. 


]Hi^-,iliU-  to  keep  them  alive  on  slides  in  tin-  n-^ulaiion  li\e 
<•!  trc-h  ruhure  medium,  which  has  pr.-M-d  so  highly  fax 
ii  'i  the  nnn-liii  culture. 

\n\\  tin-  i|iie-ii«»n  arises:  Have  the  cells  foniu-aied  in  the 
larger  \-olume  <-t  medium  and  so  been  "rejuvenated."  ^ince  I 
have  been  unable  to  isolate  the  animals  each  day.  I  cannot  pr< 

thai  nmiiiuaiii'ii  ha-  not  occurred,  for  it   i-  ]>'>--  iMe  thai   one  Of  a 

fe\\  pairs  ha\e  ennjugaifil  unobserved  ami  ha\e  given  HM-  to  the 


64  LORANDE  LOSS  WOODRUFF. 

present  generations.  The  only  way  to  prove  that  conjugation 
has  not  occurred  is  to  make  the  conditions  such  that  it  is  an  im- 
possibility for  it  to  occur,  •;'.  c.,  by  daily  Isolations  un<l  record  of 
generations.  Since  the  physiological  condition  of  this  pedigree 
culture  prohibited  this  after  the  5OOth  generation.  1  have  adopted 
the  method  employed  by  many  investigators  of  problems  of  this 
nature  and  allowed  the  Infusoria  to  accumulate  in  considerable 
numbers.  I  have,  however,  in  order  to  increase  the  accuracy  of 
the  method,  confined  the  cells  in  as  small  a  volume  of  old  infusion 
as  possible  and  have  examined  the  flasks  at  frequent  intervals 
for  signs  of  conjugation.  1  have  never  seen  a  single  pair  of  con- 
jugants  in  all  the  multitude  of  cells  which  I  have  examined,  and 
it  seems  highly  improbable  that  conjugation  has  occurred.  It 
should  be  emphasized  that,  if  conjugation  has  taken  place,  it  has 
not  so  altered  the  physiological  condition  of  the  cells  that  they 
will  live  under  the  slide  method  of  culture. 

This  culture,  then,  is  apparently  in  as  healthy  a  condition  as  at 
the  beginning  of  the  work,  but  it  has  become  so  modified  that 
the  animals  are  unable  to  exist  in  small  volumes  of  fresh  infusions. 
This  is  a  decidedly  interesting  result  in  the  light  of  the  work  of 
other  investigators  on  Param&cium  caudatum,  since  it  shows  that 
a  race  of  cells  may  exhibit  all  the  signs  of  "senile  degeneration" 
at  the  end  of  a  typical  "cycle"  of  generations,  and  still  may 
appear  healthy  and  exhibit  a  normal  rate  of  reproduction  when 
put  under  other  conditions  which  approximate  what  is  probably 
the  usual  environment  of  wild  parama>cia. 

In  other  words,  this  culture  of  P.  caudatum  substantiates  the 
conclusion  of  Calkins  that,  under  the  conditions  of  his  experi- 
ments, this  organism  may  pass  through  a  "cycle"  which  finally 
terminates  in  death;  but  it  further  shows  that  this  "cycle"  is 
probably  an  artificial  one  which  is  brought  about  by  the  sub- 
jection of  the  race  to  an  environment  which  is  not  suitable  for 
its  prolonged  existence.  This  culture  also  shows  that  pure  lines 
of  different  species  of  ParamdBcium  (aiirclia  and  caudatum}  are 
adapted  to  different  environmental  conditions,  in  virw  ol  the  tact 
that  the-  race  of  1* .  aitrclia  has  thrived  indefinitely  on  the  same 
culture  medium  which  has  proved  increasingly  unfavorable  for 
the  race  of  P.  caudatnm.  It  may  be  that  I  his  is  actually  a  specific 


ADAPTATION    OF    PARAM.ECIA    TO    ENVIRONMENTS.  65 

difference,  but  I  believe  that  the  fact  that  these  two  races  belong 
to  different  species  is  merely  an  incident  and  that  it  will  be  found 
in  be  equally  a  variation  of  different  pure  races  of  the  same  species 

.1-  tin    n  -nils  of  Jennings  clearly  indicate.1 

CONCLUSIONS. 

1.  The  discrepant  results  of  various  workers  on  the  longevity 
•  it"  p.ti.  i  in.  iii.  i  is  in  all  probability  due  to  variations  in  the  cultural 
driii. md-  of  the  races  isolated  for  study. 

2.  It  is  probable  that  most,  if  not  all,  normal  indi\  idual.-  ha\  e, 
under    suitable   environmental   conditions    unlimited    po\\rr   ot 
i.  prodiK  tion  without  conjugation  or  artificial  stimulation. 

SHIM  ii  ii'   BIOLOGICAL  LABORATORY. 
V.M  i    1  'NIVERSITY. 

I' mi:!',  llar^itt:  loc.  cit..  p.  538.     Jennini:-:   .\m<r.    \atur, i      '.  \.>1.  45, 

,    i<n  i 


Vol.  XXII.  January,  1912.  No.  2 


BIOLOGICAL  BULLETIN 


()\    THE    BEHAVIOR   OF 
TUBICOLOUS    ANNKLIDS. 

111. 

(HAS.   \V.    HARGITT. 

lii  two  earlier  pa|>ers  dealing  with  the  general  subject  of  the 
. i\  i<  'i  <il  tube-dwelling  annelids  the  writer  endeavored  t<>  i;i\e 
in  -onie  ilt  i. ul  .in  account  of  experiments  and  observations  made 
upon  several  species  of  these  worms  available  at  Wood-  1  lole.  and 
in.  Hi •ni.ilK  made  reference  to  a  few  observations  upon  one  »f  the 
Viple-  -pei  ies,  Protula  protula.  During  a  recent  occupancy  of 
the  sniiili-oiiiaM  table  at  the  Naples  laboratory  occa-ion  wa- 
taken  i"  i  xiend  these  observations  to  several  otlu-r  -peeie-.  and 
to  in. ike  .1-  i  ritical  a  comparison  as  might  be  practieable  ol  tlic 
I  K  h.i\  ic  n  M|  i  IK-  la  Her  \v  ilh  that  ol  the  \\oods  HI  >lr  -]  ><  •<  ii  •-.  1 1 
will  IK-  noted  that  in  the  present  account  less  attention  ha-  been 
vjv  en  to  detail-  of  time  reaction,  various  stimuli,  etc.,  than  before, 
and  that  beha\  ior  in  relation  to  light  has  been  enipha-i/ed.  Thi- 
\\.i-  deemed  the  more  important  since  it  was  upon  ihe-e  ^peeie- 
th.it  -nine  o|  the  earlier  work  concerned  with  animal  heliotropism 
\\a-  done.  A-  ma\  be  recalled,  my  \\ork  alread\  ment  i<  >m •<! 
'"«'.  '09  .  did  not  tend  to  confirm  these  \ie\\-;  ,unl  it  \\a-  \\ith 
a  \ie\\  to  te-t  them  \\\  a  repetition  of  tin-  experiment-^  that  1 
underiook  to  i'«  \.miine  the  subject.  In  the  follow  in-  a(e«nmt. 
will  be  found  the  results  and  conclusions  which  my  ob>er\  atioim 
ha\  e  -eemei  1  to  warrant . 

The  following  are  the  species  which  ha\e  been  n-ed:  Protula 
protnhi,  IJydroides  pcctinata  (Serpula  nncinat<D,  Ponuitcxcrn^ 
tru/uctcr,  and  Spirographis  spallanzanii.  The  experiment-  were 
carried  on  from  January  I  to  April  15,  a  period  of  three  ,md  one 
half  month-.  Particular  pains  were  taken  to  vary  the  experi- 

67 


68  <   II  VS.    \V.    HANI-TIT. 

merits  in  every  practicable  way,  and  under  a  range  of  conditions 
which  would  eliminate  as  fully  as  might  IK  errors  of  inference 
based  on  limited  experiments  or  fauhy  en\  ironnu  nt.il  conditions. 
Details  on  these  points  will  be  given  in  later  section;,  of  the  paper. 

PROTULA  PROTULA. 

This  annelid  is  a  very  familiar  element  of  the  fauna  of  the 
Bay  of  Naples.  Its  large  size,  often  175  mm.  in  length  by  ,s  s 
mm.  in  diameter,  its  fantastically  coiled  tube,  and  the  brilliant 
orange-red  gills  which  are  splendidly  displayed  during  expansion 
conspire  to  make  it  a  conspicuous  object.  The  sensitiveness  of 
the  creature  to  differences  of  light  intensity,  such  as  that  involved 
in  the  intervention  of  shadows,  was  one  of  the  first  aspects  of 
behavior  to  engage  my  interest  many  years  ago,  some  brief 
notice  of  which  was  made  in  my  early  paper  ('06,  pp.  311,  314). 
These  observations  I  have  verified  again  and  again  during  tin- 
present  series  of  experiments.  Careful  comparisons  of  many 
specimens  in  their  reactions  reveal  the  fact  of  marked  individual- 
ity as  expressed  in  the  variability  of  behavior  shown  from  day 
to  day.  It  is  not  necessary  to  go  into  details  concerning  this 
point.  What  has  been  pointed  out  in  the  case  of  Ilydroidcs 
dianthus  ('09)  is  confirmed  in  the  case  of  Protula.  Certain  speci- 
mens were  especially  sensitive  and  extremely  active  in  response, 
while  others  would  show  the  very  opposite;  and  it  was  not  un- 
usual to  find  specimens  which  seemed  totally  indifferent  to 
shadow  stimuli.  Again,  specimens  might  prove  quite  sensitise 
at  a  given  time  and  very  indifferent  at  another.  But  let  it  be 
noted  that  some  specimens  seemed  normally  to  be  highly  sensi- 
tive, while  others  seemed  normally  quite  tin-  opposite.  Again, 
the  retraction  aspects  of  behavior,  that  is,  the  time  a  gixen 
specimen  remained  in  the  tube  after  a  given  contraction,  \\.is 
remarkably  variable.  In  some  cases  the  emergence  was  rela- 
tively prompt,  while  in  others  it  was  extremely  slow.  In  this 
matter  Prolnla  differs  materially  from  Ilydroidcs,  whose  retrac- 
tion periods  are  usually  and  normally  very  briel.  Protnla  ohen 
remained  retracted  for  indefinite  periods,  otten  tor  one  or  t\\o 
hours  at  a  time,  in  marked  contrast  to  Hydroides. 

Tubular  Aspects. — The  behavior  of  Protula  as  e\]>iv— ed  in  the 


ON    THE    BEHAVIOR    OF    TUBICOLOUS    ANNELIDS. 


69 


form  <>r  aspects  of  the  tubes  is  noteworthy.  In  my  early  paper 
u.i-  -lunvn  a  figure  which  made  this  very  graphic.  Xo  less  than 
in  tin-  case  of  Hydroides  the  tubes  of  Protuhi  show  the  record  of 
erratic  behavior  in  very  striking  manner.  i.Cf.  '09,  pp.  180- 
[83.  I  Miring  early  life  these  tubes  usually  adlu-rr  very  Mruiisjy 
and  closely  to  the  base  of  support;  but  in  maturity  they  ohm 
in<  line  in-.n-  or  less  toward  the  vertical,  though  in  a  ratlur  sinuous 
fir  - 1  lira  I  din-,  i  ion,  or  may  even  mil  about  rarh  otlu-r  and  assume 


l-ii..    i    -li.,u  .•  colony  of  Protttla.  with    Spirographis'w  tin- 

I  li>-  pi'  •:•  of  the  tubes  is  very  m.ir  kc-<l. 


p.  tit. 


a  do\\nu.ini  a-|>ivt.  This  may  be  seen  nm-t  -trikin^K'  in  the 
l.ii-r  colonies  »i  these  creatures  al\\a\^  ]>IVM-IH  in  tin-  >how 
aijiiaria  ot'  tin-  lal  IOIMI  ory,  where  may  .il>f>  br  M-i-n  to  biM  dtCft 
tlu-  marked  \ariability  as  to  tubular  brhaxior.  Something  of 
tin-  i^  ^iMpliicallN  >ho\vn  in  1'i--.  I  and  ;-,.  ropird  li-oin  |)ln»to- 


JO  CHAS.    W.    HARGITT. 

graphs  taken  by  Dr.  Sobotta,  by  whoso  kind  permission  I  am 
able  to  present  them  here.  As  will  be  seen,  the  aspects  of  the 
tubes  and  of  the  openings  through  which  the  creatures  protruded 
their  heads  are  so  extremely  diversified  as  to  seem  to  be  abso- 
lutely chaotic.  If  one  may  distinguish  any  tendency  toward  a 
given  aspect  of  position,  still  the  departures  are  so  numerous  as 
to  render  it  almost  certain  that  no  single  factor  could  have  been 
determinative.  As  in  the  case  of  Hydroides  ('09,  p.  180.  etc.), 
Pro! nla  has  left  in  ii>  tubes  a  convincing  record  of  the  erratic 
individuality  of  its  behavior  the  significance  of  which  is  ex- 
tremely important. 

Autotomy. — In  this  connection  may  be  considered  a  feature  of 
behavior  more  or  less  unique,  though  not  peculiar  to  Protula, 
since  it  has  been  noted  in  several  cases  of  Spirographis]  namely, 
that  of  autotomy,  or  the  self-excision  of  certain  organs  of  the 
body.  This  was  first  observed  in  the  case  of  Protula.  A  speci- 
men of  this  worm  was  among  the  first  to  come  under  my  observa- 
tion, having  come  to  my  table  almost  the  first  day  in  the  labora- 
tory. It  had  been  placed  in  a  small  aquarium  jar  on  the  table 
for  convenience  of  study.  After  finishing  a  given  series  of  tests 
the  specimen  was  usually  returned  to  the  large  aquarium.  On 
January  7  the  specimen  had  been  under  observation  and  was 
Ic-ft  in  the  table  jar  which  had  a  capacity  of  about  four  or  five 
liters,  while  I  went  out  to  lunch.  This  could  hardly  have  been 
more  than  an  hour  or  so,  but  on  ivturn  I  observed  what  seemed 
strange — the  detached  portion  of  about  half  of  the  gill  mass  lying 
at  the  base  of  the  tube.  An  examination  of  the  gill  failed  to 
reveal  any  signs  of  disease  or  other  abnormality.  My  first  im- 
pression was  that  possibly  the  water  had  become  "bad,"  yet 
other  living  things,  such  as  copepods,  showed  no  signs  of  dis- 
comfort. However  the  water  was  renewed  several  times  during 
the  afternoon  and  the  specimen  finally  left  on  the  table  over 
night,  as  had  been  done  several  times  before.  The  following 
morning  upon  examining  the  jar  I  lound  tin-  oilier  portion  of  the 
gill  in  I  he  same  detached  condition,  lying  at  I  lu-  base  of  t  he  tube, 
but  the  specimen  was  deeply  retracted  within  the  tube.  After 
some  time  it  came  to  the  orifice  and  showed  clearly  that  it  was 
entirely  devoid  of  gill  elements.  It  was  now  transferred  to  the 


ON"    THE    BEHAVIOR   OF    TUBICOL*  >l  -    ANNELIDS.  ~l 

large  aquarium  and  left  to  see  whether  regeneration  would  occur 
and  if  so  at  what  rate.  For  a  few  days  it  frequently  came  to  the 
orifice  and  extended  the  mantle  edge  over  the  margin  of  the 
tube  and  remained  in  that  condition  for  some  time.  Later  it 
again  withdrew  deeply  into  the  tube  and  did  not  .-how  it-elf  for 
-e\eral  da\-,  indeed  for  some  three  weeks  or  more.  Finally,  on 
l-Vbniary  13  it  was  once  more  seen  to  protrude  it-  head,  but 
then-  was  not  the  least  sign  of  any  regeneration  It-  appearance 
m>\\  bee, mie  more  frequent  and  occasion  was  taken  to  test  it  by 
-had'  >u  -,  and  to  my  -urpriM-  it  was  found  to  react  as  promptly  as 
at  tin-  \vr\  first.  These  tests  were  made  main  time-  on  subse- 
quent da\-  with  the  almost  uniformly  prompt  and  po-iii\e  re- 
ai  lion,  but  with  the  variations  observed  at  first,  /'.  c..  -ometimc- 
lc--  -harp,  and  thru  more  so,  and  occasionally  not  at  all. 

Several  interesting  questions  arise-  in  this  connection.      I  ir-i. 

io    i  he    regenerative   capacity   of   Prptnla.     For    lu.uK     tun 

month-  mil   a  sign  of  regeneration  was  distinguishable.      1   had 

pie\iou-ly  recorded  ('06)  tin-  promptness  with  \\hieh   Ilydronlcs 

:ated  ii-  gill-,  and  similar  records  had  also  been  made  1>\ 

/elen\  .      I  inalK    on    March    14    1    found   undoubted   e\  ideiice  ol 

n<  raiion.  and  thi-  went   forward  apparently  rather  rapidlx  . 

ti.i    b\    April    10  the  new  gills  had  become  quite  om-picimu- 

nearK    a   tilth  as  large  as  the  originals,      h    mav  be  noted   here 

that    Liter  I    had   also  a  similar  autotomy   by  another  -pecimeii 

ot  /'"'//i/i/  and  by  two  sjK'cinu'ns  of  Spirogra (this.      In  tin-' 

there  tdiild  not  be  doubt  as  to  any  condition  of  water  inducing 

the   aui»i»my,    for   the  specimens   continued    to    thri\e.   as   did 

man\  otln-r-  in  the  sanu-  lank.      Re-generation  u.i-  very  pn>m]>t 

and  rapid  in  these  cases. 

\  -e.-i.nd  |toint  is  in  relation  to  such  c.ne|.iti..n  of  funcii.m 
.1-  ui.uld  enable  tin-  creature  during  lhi>  IOIIL;  ]>eri'»d  ••('  gill 
depri\aii'>n  to  maintain  the  normal  rc--piratm  \  activity.  It 
n--|iilalii>n  \\ere  restricted  to  the  gill-  almie  nf  course  it  nui-t 
ha\e  i>eri-ln'd.  This  experiment  sho\\  -  «  learU  that  thi-  func- 
tion may  be  taken  up  by  other  organ-  of  the  body  without 
serious  inci>n\enieiuv.  But  the  gills  are  also  concerned  in  the 
process  »i  nutrition,  acting  as  a  medium  for  capture  of  prey. 
How  might  this  function  have  beeen  Mippleim-nu-d.-'  It  has 


72  CHAS.    W.    HARGITT. 

been  said  that  during  this  time  the  specimens  remained  rather 
continuously  within  the  tubes.  Did  they  depend  wholly  upon 
a  reserve  food  supply? 

It  may  not  be  possible  to  answer  these  queries  fully,  but  of 
the  correlation  of  the  skin  in  the  function  of  respiration  there 
can  be  no  serious  doubt.  In  my  earlier  experiments  on  Hydroides 
('06)  it  was  found  that  when  the  gills  wrere  excised  to  test  their 
relations  to  sensory  reactions  the  creature  did  not  seem  to  suffer 
any  serious  inconvenience  as  to  respiration.  So  in  the  case  of 
Protida,  there  was  no  evidence  to  the  effect  that  respiration  wras 
not  normal  during  the  long  period  of  gilless  life.  Bounhiol 
(1900)  has  reached  similar  conclusions  from  experiments  on 
Spirographis.  He  finds  that  respiration  takes  place  through 
both  skin  and  gills,  and  that  they  supplement  each  other  by 
compensatory  interaction.  He  finds  also  that  it  is  apparently 
easier  for  the  gills  to  assume  extra  work  than  for  the  skin,  and 
that  in  excretion  of  COz  the  skin  normally  excretes  about  three 
fourths  of  the  entire  amount. 

In  the  third  place,  there  is  the  interesting  query  as  to  the 
sensory  function.  I  have  shown  that  for  Hydroides  light  per- 
ception is  almost  exclusively  a  function  of  the  gills.  In  Protiila 
this  is  not  so  certain.  Its  behavior  in  this  respect  is  less  easily 
controlled,  owing  to  the  sulking  disposition  of  the  worm.  But 
it  is  quite  certain  that  autotomy  did  not  result  in  entire  inhibi- 
tion of  reaction  to  shadows  and  it  may  not  be  improbable  that 
something  of  sensory  compensation  may  obtain  in  this,  as  in  the 
respiratory  activity;  or  possibly  this  sensory  function  may  be 
shared  in  part  with  sonic  other  head-organ,  possibly  the  mantle 
margin,  which  in  normal  lite  is  often  extended  over  the  orifice  of 
i  he  tube,  hence  in  a  position  admirably  adapted  to  such  a  function. 

Concerning  the  entire  matter  of  the  significance  of  autotomy 
little  can  be  said.  Such  phenomena,  similar  in  many  respect^, 
are  well  known  among  other  animal  groups,  though  not  common 
in  any  case,  unless  we  may  include  phenomena  of  fission  which 
is  a  very  familiar  feature  in  many  annelids;  but  this  MVDIS  to  be 
a  wholly  different  problem.  That  it  is  spontaneous,  hence  not 
attributable  to  the  operation  of  gravity,  contact,  etc.,  seems 
very  evident. 


ON    THE    BEHAVIOR   OF    TUBICOLOUS    ANNELIDS.  73 

POMATOCERAS    TRIQUETER. 

This,  with  an  undetermined  species  of  Serpnla,  are  tubicolous 
annelids  which  much  resemble  in  general  aspect  of  size,  structure 
and  behavior,  Hydroides  dianthus.  Indeed  in  almost  every  par- 
ticular they  might  have  been  substituted  for  the  latter  species 
without  marked  changes  of  results.  In  general  habitat  the  two 
species  arc  much  alike  and  often  found  growing  on  the  same  sub- 
Mr.it urn.  Pomatoceras  is  rather  larger,  and  its  tubes  are  charac- 
teri/ed  liv  rather  sharp  triangularity  with  the  dorsal  angle  form- 
ing a  sharp  crest  along  the  entire  tube.  Mo  attempt  will  be  made 
to  go  into  details  as  to  matters  of  behavior,  since  as  already 
suggested,  they  show  the  same  reaction  phenomena  as  those 
given  in  tin-  accounts  of  Hydroides  dianthus,  and  tin-  growth 
aspects  are  <|»i'«%  as  erratic.  For  the  most  part  the  tubes  adhere 
cloM-|\  to  tin-  substratum,  and  in  some  cases  they  a<lju-t  thcin- 
sel\e-  \\itli  -in  h  nicety  to  grooves  or  similar  depressions  that  one 
might  gin •--  ilit-\  wen-  under  the  control  of  some  such  stimulus  a- 
thigmotaxi- or  stereotropism.  But  when  one  com*-  to  examine 
an\  i  on-idt -rable  number  of  specimens  he  soon  finds  that  in 
l>\  tar  the  larger  number  there  is  absolutely  no  Mich  adjuM- 
ment.  The  -ame  is  the  case  with  Hydroides.  Now  and  then  a 
s|)ci  imi-ii  mav  In-  found  on  a  shell  of  Pecten  in  \\hich  there  i-  a 
verj  tine  illustration  of  such  appearance,  the  creature  ha\ing 
kept  \er\  i  lo-rly  and  exactly  in  the  radial  groo\»-  ,,|  th,-  shell. 
Hut  on  the  same  shell  may  now  and  then  be  found  another 
specimen  \\liich  has  grown  directly  across  the-«  grooves;  and 
ol  course  \>\  far  the  larger  number  have  al>-"lute!v  no  -nnblance 
oi  -neh  re-ponse.  The  conclusion  is  therefore  forced  upon  one 
that  the  operation  of  any  such  factor  must  be,  i!  not  ulmlly  nil, 
\et  ot  onl\  incidental  significance  in  behavior. 

Again,  in  habitat  one  finds  in  the  Mediterranean  species  the 
same  \\ide  r.m-easin  those  of  Woods  Hole.  1  have  dwelt  upon 
tlii-  |)oint  \\ith  some  emphasis  in  a  lormer  pa|>er  ('oi),  |>p. 
i^j  $).  and  need  only  refer  to  the  matter  in  thi-  connection  by 
\\a\-  of  further  emphasizing  a  point  which  I  regard  of  considerable 
significance.  The  growth  of  these  organisms  indiscriminately 
on  a  large  \arietv  of  substrata,  rock-,  shells — the  latter  both 
dead  and  living  nets,  crabs,  lobster^,  etc.,  i-  it-elf  of  no  small 


74  '   "AS.    \V.    IIARC.ITT. 

import  as  to  the  negative  influence  of"  such  factor^  a<  light, 
gravity,  etc.,  in  relation  to  growth.  This  is  further  borne  out. 
by  attention  to  the  aspects  of  the  several  tubes  which  may  com- 
prise a  given  colony.  In  several  such  an  actual  count  of  the 
growth  direction  was  made.  On  a  stone  which  contained  37 
living  specimens  I  found  that  12  had  a  general  upward  direction; 
15  had  just  as  definite  a  downward  course;  and  10  had  a  hori- 
zontal direction.  Another  colony  growing  on  the  inside  of 
an  iron  cup  about  6X10  cm.,  made  up  of  eleven  specimens. 
showed  the  following  disposition  as  to  direction:  4  upward,  5 
directly  down,  and  2  horizontal.  On  the  outside  of  the  same  cup 
were  eighteen  specimens  disposed  as  follows:  8  upward.  7  down- 
ward, and  .}  horizontal.  These  plainly  go  to  confirm  the  con- 
clusions already  drawn,  that  in  the  matter  of  orientation  one  is 
utterly  unable  to  discover  the  operation  of  any  one  or  several 
factors  which  are  in  any  sense  determining. 

HYDROIDES  PECTINATA. 

This  species  and  the  one  described  in  the  following  section, 
Spirogaphis  spallanzanii,  were  the  ones  used  by  Loeb  in  his  well- 
known  experiments  at  Naples  many  years  since,  the  results  of 
which,  including  also  several  species  of  hydroids,  served  as  a 
basis  for  his  far-reaching  theory  of  animal  heliotropism,  especially 
as  it  relates  to  sessile  animals.  Naturally,  therefore,  more  of 
details  will  be  expected  in 'the  following  accounts  than  in  the 
preceding,  and  I  shall  endeavor  to  make  explicit  and  definite 
records  both  of  methods  and  results. 

Ilydroidcs  pectinata  (^Serpiila  uncinata)  is  one  of  the  most 
common  and  abundant  of  the  Naples  annelids.  I  nlike  Spiro- 
graphis,  it  grows  usually  in  immense  colonies,  aggregating  hun- 
dreds or  perhaps  thousands  of  individuals.  Fig.  2  will  give  a 
general  idea  as  to  their  appearance  in  small  colonies.  The  tubes 
of  a  given  colony  form  a  mass  of  more  or  less  parallel  aspect,  the 
individuals  apparently  growing  at  approximately  the  same  rate 
and  in  the  same  general  direction.  When  one  casually  examines 
such  a  colony  it  \\otild  seem  to  afford  a  typical  illustration  of 
orientation  due  to  some  single  constraining  stimulus.  Hut  here 
again,  as  in  the  case  cited  above  as  to  stereo tropism,  extended 


ON    THE    BEHAVIOR    OF    TUHICOl.orS    ANNELIDS. 


75 


ol>MT\ation  brings  to  li^lit  too  many  exception^  to  any  such 
rule,  and  compels  further  inquiry.  Like  other  species  of  7/v- 
<lroide.s  this  one  secretes  a  calcareous  tube,  the  shape  of  which 
<1» -pi -nd-  upon  the  mode  of  growth  of  tin-  individual  constructing 
it.  Hence  in  aspect,  size,  etc.,  these  become  permanent  records 
ol  rvrryduy  behavior,  whether  thi-  be  mf.-hanic.il,  ecological,  <,r 
physiological  in  its  nature.  Several  coloim-  \\i-re  put  under 


-Ih'ws  three  colonies  of    H\dr«i,:  •  :.it<i   whirh   1. 

lixlit  U'-t  i.  n  in.  nr  than  a  month.     As  will  be  seen  the  curvings  of  the  tubes 

iinii  h  u-  in   \  \  +  .    I  . 


i  \  .iiion  c,irl\-  in  January  arid  urn-  u--trd  during  a  period  of 
m«u<-  than  tluve  months;  to  be  more  exact,  lunn  |.iim,ir\  .^  lo 
April  i:r.  'l"he\-  were  tested  as  to  the  po-^ibK-  inlliu-nce  of  both 
li.uht  and  i;ra\it\-.  Loeb  claims  that  in  thi-  -pccif-  tlu-  reaction 
i-  .|iiitr  prompi.  '  I  noticrd  that  in  tin-  course  of  the  nc\i  day 


76  CHAS.    W.    HARGITT. 

the  Scrpulidip,  which  like  Spirographis  presented  only  their  gills 
to  the  light,  bent  them  strongly  upward"   ('90,  "Gen.  Phys.," 
Part  I.,  p.  102),  and  he  continues,  "within  six  weeks  the  entire 
block  was  covered  with  tubes  which  curved  upward;  not  a  single 
individual  had  continued  to  grow  in  the  original  direction,"  and 
presents  a  figure  in  illustration.     There  is  apparent  discrepancy 
between  the  latter  and  the  statement  " not  a  single  individual, 
etc.,"  for  in  the  figure  about  as  many  appear  to  "continue  to 
grow  in  the  original  direction"  as  have  "curved  upward."     My 
own  experiments  show  a  reasonable  conformity  to  Loeb's  figure, 
but  the  ratio  of  tubes  indicating  reaction  is  very  much  smaller. 
Figs.  2,  a,  b,  c,  are  photograph  reproductions,  and  may  therefore 
be  taken  at  their  face  value,  and  they  certainly  fail  to  show  any 
such  response  as  claimed  above.     For  example,  it  w'as  found  by 
actual  count  of  a  colony  comprising  hundreds  of  tubes  which 
had  been  under  test  for  more  than  a  month   that  only  about 
twenty  tubes  had   definite  curves   toward   the   light,   a  similar 
number  had  shown  lateral   curvatures,  and  a  smaller  number 
had  curved  downward;  but  the  larger  number  "had  continued 
to  grow  in  the  original  direction."     A  smaller  colony  which  had 
been  under  test  for  twenty-five  days  under  particularly  favorable 
light  conditions  showed  a  slightly  larger  response  toward   the 
light;  but  here  also  the  number  was  relatively  small.     Another 
colony  was  placed  in  an  aquarium  which  was  covered  on  three 
sides  and  above  with  a  black  hood.     After  a  test  of  nearly  two 
months  (January  23  to  March   18),  it  was  found  by  a  careful 
estimate  from  counting  that  at  most  only  aboni    Jo  per  cent. 
showed  any  possible  light  reaction ;  while  by  far  the  greater  num- 
ber either  continued  to  grow  in  tin-  original  direction  or  showed 
curvature  laterally  or  downward.     Tin-  colony  was  submitted  to 
two  others  working  at  the  laboratory,  I)r.  Butler,  of  University 
College,  Dublin,  and   1  )r.  S.   k.  Williams,  of  Miami  University, 
Ohio,  both  of  whom   made  the  per  cent,  ot    light    reaction   much 
lower  than  my  own. 

A  very  interesting  and,  1  believe,  significant  leature  of  growth 
in  this  species  came  to  light  during  the  observations,  nainelv,  it-- 
very erratic,  or  discontinuous  character.  Some  individuals 
showed  a  very  prompt  and  rapid  growth  at  lir>t  and  later  its 


ON    THE    BEHAVIOR    OF    TUBICOLOUS    ANNELIDS.  J7 

cessation.  In  this  process  of  rapid  growth  some  show  a  bending 
while  others  do  not.  Again,  some  bend  toward  the  light,  others 
away  from  it,  and  still  others  continue  in  the  original  direction. 
The  point  of  importance  here  is  not  the  bending  or  curving,  but 
Dimply  the  tube-extension.  This  extension  is  not,  as  I  interpret 
ii  ,  an  expression  of  growth  at  all,  so  far  as  the  body  mass  of  the 
animal  is  concerned.  Seldom  are  aquarium  conditions  especially 
<  niidiici\c  to  physiological  growth.  What  then  does  such  tube- 
t  -xten-ii  MI  IIH  Mil.-1  Isolated  worms  lying  -ide  by  side,  of  essentially 
similar  age,  state  of  vigor,  under  identical  conditions,  show  the 
most  remarkable  differences  in  relation  to  tin-  matter  of  so-called 
gro\\th.  <  >nr  may  in  the  course  of  a  \\eek  extend  it-  tube  3-5 
mm.;  another  -hows  not  the  slightest  extension  of  its  tube.  <  >n<- 
mav  extend  it-  tube  in  the  line  of  the  body  axis,  /.  «•..  -traight,  the 
oilier  -ho\\  .1  -harp  curvature  from  the  first.  There  has  been 
e.jiial  access  to  food,  air.  liglit.  Why  ha-  not  gro\\th  been  the 
-ame  in  direction  and  amount?  As  a  matter  o|  laci  it  may  In* 
doubted  \\  lieiher  there  has  been  any  appiei  i.iMe  gn>\\  th.  Indeed 
'  mav  not  these  erratic  phenomena  expre—  ju-t  the  oppo-ite, 
namely,  1m  k  of  growth  conditions,  or  some  other  ia<  tors  >  onducive 
to  lointcirt.-'  And  if  so  then  this  erratic  tube-exten-ion  i-  but 
an  expn---ioii  of  such  discomfort,  —  an  expre—  ion  o|  the  elloit- 
iil  th.  .  leatntv  to  seek  better  condition-,  to  n,i<  h  oiii  ,  as  it  \\eiv, 
in  -earc  h  o|  i.i.id,  air,  etc.  Indeed,  it'  my  interpretation  be  cor- 
rect, the-e  (  urvings  are  but  the  natural  expre—  i<  m-  o|  eitort- 

at  (...id-get  tin-^  or  respiration—  adjustments  to  tho-e  particular 

end-  in\ol\ed  in  -nr\i\al  or  selection.  In  the  light  <>l  this 
intei  pieiai  i"ii  the  real  factors  in\o|\e<l  in  these  aspects  ot  bc- 
ha\  ior  are  intrinsic  and  not  cxtrinsit  .  The  lai  ter  are  conditional 
ami  pa--i\  e;  the  t»rmer  are  indi\  idual.  acti\  e.  causative. 


SPA]  I    \\/\II. 

Tin-  species  «lit't'ers  most  markedly  from  those  already  con- 
sidered ill  that  it  possesses  a  very  flexible  tube,  hence  i-  capable 
of  considerable  range  of  movement  within  a  region  mca-nivd 
I  >v  tin-  rad  in-  ot  it-  own  length.  It  i-  a  large  species  at  maturity, 
averaging  perhaps  about  25  cm.  in  length,  by  about  I  cm.  in 
diameter.  In  im  experiment-  care  \\.i-  taken  to  ha\e  -pccimens 


C'HAS.    \V.     HARGITT. 


of  various  sizes,  and  those  actually  used  ranged  from  about  5  to 
,}o  cm.  in  length.  No  less  care  was  exercised  as  to  conditions 
under  which  experiments  were  conducted.  Three  of  the  l.irge 
aquaria  supplied  with  running  water  were  at  my  disposal  during 


Fir,.  3  is  a  portion  of  one  of  the-  large  show-aquaria  containing  Protulu  and 
Spirographis.  The  varied  aspect  of  the  latter  is  quite  marked,  as  will  be  seen  by 
comparing  specimens  in  various  positions. 

tin-  period,  and  in  addition  l\vo  special  experimental  a<|iiari.i  of 
smaller  size,  about  25  X  35  X  45  cm.,  also  supplied  with  running 


OX    Till      hi  IIAVIOR    OF    TUBlM'i.nrs    ANNELIDS.  7<) 

,  were  employed  for  such  experiments  as  called  for  a  critical 
control  of  light,  etc.  Of  the  large  aquaria  two  were  in  a  room 
with  north  exposure,  and  hence  with  diffu-e  light.  Inn  al\\a\- 
adequaie  for  ordinary  observation  and  experiment:  while  tin- 
other  was  in  a  room  with  direct  south  exposure,  In-mv  with  -un- 
liyht  ot  almost  any  degree  of  intensity,  modified  by  -hade-  01 
screens.  I  In-  iwo  smaller  aquaria  \\eiv  in  a  special  room,  the 
light  ol  uhich  was  under  easy  control,  and  tin- aquaria  them-cbe- 
ea-il\  adjusted  to  any  desired  condition.  a-  to  amount  and  direc- 
ii«n  o|  li-ht.  t-ic.  A  still  further  point  i-  \\orthy  of  notice  At 
all  time-  I  had  In-i-  access  to  the  latxe  exhibition  aquari.i,  win-re 
laro-  number-  ol  these  specimens  \\eiv  living  under  condition- 
as  iit-.irlv  natural  a>  the  long  experience  and  painstaking  -kill  of 
tho-e  in  <h.iro  ha\'e  been  able  to  devise.  1  -hall  ha\»-  O(  •  .i-ion 
io  r.i.  r  to  thi>  in  another  connection. 

I  hiring   tin-  pio^ress  of  the  experiment--  ^mie  hlt\    ->|M-cimen-. 

\\  i  te  .  i\  ailal  ile.  .UK  I  tin-  general  healt  h  and  \  i^or  m.i\   In-  intei  "i « •<  I 

lioin  tin-  I. iet   th.n   in  the  three  and  one  half  month-  not  .1  >in-|»- 

-pi -i  imeii  died  oi  e\en  showed  signs  of  detei  i.  n  ,n  ion.  except  .1-  a 

in  I.  id  i  i  re  of  the  brilliance  of  coloration  ma\   ha\e  been  indica- 

ti\  e  ol  -mil.  t  are  was  taken  to  suppK  1 1  from  time  to  time, 

.iluio-t  d.iil\.  -uch  as  came  in  from  plankton  haul-  \\hich  \\eic 
-up|ilied  to  tin-  lo,.!))-  quite  regnlai'K  .  and  this  m.i\  ha\e  con- 
tributed to  tin-  .  \.  ellent  conditions  of  health  already  alluded  to. 

.^[>iri>^nif>hi.\   -eem>  to  take  ratln-r  natnralK    to  the  aquarium 

environment  and  soon  becomes  quite  at  home  so  far  as  one  may 

judge  I  mm  appeal  .nice.      >peiinien-  reqmri-  Irom   I  \\  o  lo  several 

da\-  lirmK    \<>  att.uh    ihem-el\es  to  the  bottom  or  sides  of  the 

• 

aquarium.  Thi-  i-  accompli -lied  b\  an  adhe-i\  e  se<  r<  lion  ol  the 
\\orm  \\hich  i-  di-char^ed  through  a  -mall  pore  at  the  l<i\\er  end 
of  tin-  tube.  Tin-  time  required  for  attachment  max  be  \aried 
\>\  having  the  bottom  of  the  aquarium  covered  \\ith  .1  la\er  ol 
>and  or  b\  |ilacinu  fragments  of  rock  in  contact  \\ith  the  ba-e  o| 
the  tube-.  While  in  most  cases  tin-  -pecimen-  attach  tln-m-el\e- 
\\ln-n-\er  tlu\  hapj>en  to  be  placed,  which  i-  fortunate  in  -uch 
experiment-  a-  tln^e  under  consideration,  -till  it  not  infn-queiuly 
happen-  that  a  specimen  will  go  through  various  irau-lator\ 
movements  before  finally  settling  do\\n.  It  ma\  be  noted  that 


8O  «   II  \S.     \V.     HAKC.II  I  . 

these  locomotor  movements  take  place  usually  during  the  night. 
This  I  have  demonstrated  by  carefully  marking  ldc.it ions  and 
noting  subsequent  changes.  At  no  time  have  1  found  evidence 
of  these  movements  during  the  day. 

In  general  my  experiments  proceeded  along  the  lines  employed 
by  Loeb  ('90,  Arch.f.  ges.  Physiol.,  Bd.  47,  p.  391),  who>c  objec- 
tive aim  was  to  establish  the  essential  identity  of  heliotropism  in 
animals  and  plants,  and  his  experiments  were  directed  to  that. 
end.  Incidentally  it  may  be  observed  that  he  does  hot  hesitate 
to  claim  "I  think  I  have  shown  that  the  heliotropism  of  sessile 
animals  is  essentially  identical  with  the  heliotropism  of  sessile 
plants."  And  still  later  he  asserts  even  more  strongly,  "It  was 
possible  to  show  that  heliotropism  of  animals  agree  in  every  point 
with  that  of  plants"  ("Comp.  Phys.  of  Brain,"  1900,  p.  181). 
It  may  be  doubted  whether,  in  the  light  of  present  knowledge, 
this  would  be  seriously  maintained.  I  shall  not  discuss  the  mat- 
ter here  further  than  to  say  that  my  own  experiments  were 
undertaken  with  a  very  different  aim,  namely,  that  of  ascertaining 
the  questions  of  fact, — Are  these  organisms  heliotropic?  and 
further,  Do  they  exemplify,  or  conform  to  the  mechanical  concept 
of  behavior? 

In  the  following  account  I  shall  present  the  matter  under  some 
three   distinct   series.     First,    those    experiments    made    in    tin 
aquaria  located  in  a  north  room;  second,  those  conducted  in  the 
smaller- experimental  aquaria;  and  third,  those  conducted  in  the 
large  aquarium  located  in  a  room  with  exposure  to  direct  sunlight. 

The  first  series  began  on  January  6  with  some  six  specimens. 
To  these  additions  were  made  from  day  to  day,  till  on  the  i^th 
I  had  twenty,  which  had  been  variously  distributed  in  the  two 
large  aquaria,  some  with  the-  heads  directed  away  from  the  win- 
dows, others  directed  at  right  angles,  and  still  others  facing  the 
windows.  The  aquaria  were  ol  about  the  same  si/e,  probably  1.5 
meters  in  length,  by  about  40  cm.  in  depth  and  width;  the  one 
with  its  end  toward  the-  window,  the  other  with  its  side  toward 
the  light.  It  was  some  time  after  specimens  had  become  at- 
tached before  any  sign  of  orientation  was  discernible.  In  the 
aquarium  (\o.  II  with  the  end  directed  louard  the  light  there 
were  twelve  specimens,  in  the  other  eight.  The  twelve  had 


ON   THE    HKII  \\lok    i  >|      it   BICOLOUS     \N\ELIDS.  8l 

been  distributed  so  that  three  should  face  toward  each  of  the 
-ide-  ot  the  tank;  i.  e.,  three  with  head-  directed  toward  light, 
three  auay  from  light,  and  six  at  right  angles,  three  facing  each 
-ide.  <  )n  January  26  all  specimens  weiv  attached  except  one, 
which  tor  some  reason,  perhaps  injury,  remained  tree  during  the 
entire  course  of  the  experiment,  hence  may  he  disregarded.  A; 
this  date  the  following  is  the  record  of  orientation.  The  three 
facing  tin-  light  continued  in  that  position,  one  of  which  had 
a  — imied  a  nearly  erect  attitude;  the  other  t\\o  had  barely  cle- 
\ati-d  tin-  head  to  a  degree  sufficient  to  allo\\  tin-  gill-  to  clear 
ihe  lioiiom  (jf  the  tank  when  expanded.  Four  specimen-  n<>\\ 
face  tin-  \\all,  and  all  with  barely  sufficient  up-bending  to  tree 
iln-  uilU  Ir-.m  the  bottom.  The  laterally  directed  -pecimciis 
toiitiiiued  as  at  first,  except  thai  one  had  made  a  distinct  up- 
Clirve,  the  hi-.id  elevated  to  an  angle  of  about  .^5  degree-. 

<  MI  I  . -lu-iiary  o  the  record  of  this  tank  is  as  follo\\-:  <  n  speci- 
mens facing  light  two  are  cursed  upward,  one  neaiU  \ertical, 
peihap-  70°,  the  other  about  45°,  while  the  third  remain-,  a- 
beloiv.  and  this  in  spite  of  the  fact  that  direct  light  i-  inter- 
cepted li\  a  tufaceous  mass  bearing  tubes  of  I'rotulu,  etc.  The 
t»in  -pi-<  iini-ns  facing  the  wall  ha\e  made  con-idci  able  char 
•  >IH-  had  rotated  through  an  arc  of  about  loo  d< -gn •« •-,  \\«\\  tai  in- 
ihe  -id<  .  .uid  \\iih  head  elevated  about  35  d«  \notlier 

ha-  aU<>  n>iate«l  to  ne.irly  right  angles  and  cur\ed  ujiuard  50 
degrei  The  other  t\\o  continue  unchanged.  The  specimen^ 
latn.ilK  di -posed  continue  essentially  as  betc.it.  excepl  an  up- 
curve  ol  from  30  to  40  degrees.  Then '"id  for  this  aquarium 

on    I'ebinaix    25   is  as   follows:      Five  spe<  inien-   are    imu    la. 
the  \\all,  three  continue  to  face  the  light,  uhilc  the  others  con- 
tinue e— eniially  as  before. 

I  e  lolliiuing  reconls  of  the  behaxi'T  of  the  other  aiinarium, 
\\hich  ma\  be  called  number  two,  are  inteiv-iing.  In  thi-  were 
placet  1  rigln  -jiecimens,  two  of  which  ueiv  -i i-|  tended  head  down- 
ward, and  in  this  position  they  attach  them-el\e-  ami  continued 
f<.r  nian\  \\eeks.  The  others  were  locateil  \\ith  heads  pre- 
dominantly touard  the  wall,  /'.  <-.,  away  tnun  source  of  light, 
onl\  one  lacing  light.  In  this  tank  but  little  -ign  of  light  rea.  tion 
was  ili-iingni-hable.  The  specimen  originalK  fating  the  light 


82 


CHAS.    \V.    HAKMTT. 


later  curved  to  the  wall  and  remained  in  that  position  during 
the  entire  time,  xvhile  one  of  the  specimens  placed  lacing  the  wall 
later  curved  toward  the  light  >ide. 


FIGS.  .4,  It,  (.',  /)  illustrate  certain  aspects  of  a  specimen  which  was  suspended 
head  downward.  At  .1  is  shown  the  first  indication  of  change  of  position;  a  further 
change  is  shown  at  B;  this  curvature  has  reached  its  limit  in  that  format  ('.  ;m>l 
continued  thus  for  several  days,  oscillating  somewhat  from  side  to  side,  but  with 
no  evidence  of  reaction  to  light.  At  D  the  sickle  shape  is  converted  into  tin-  loose 
spiral,  which  likewise  continued  for  some  days  essentially  as  shown  in  diagram; 
as  in  the  others,  there  was  shifting  and  change  but  with  no  relation  to 


OX   THE    BEHAVIOR   OF   TUBICOLOUS    ANNELIDS.  83 

The  most  interest  attaching  to  this  experiment  is  the  behavior 
of  the  specimens  suspended.  For  several  days  both  remained 
hanging  downward.  Finally  one  began  to  curve,  and  direct  1\ 
by  a  graduated  process  assumed  the  aspects  shown  in  the  dia- 
gram figures  A,  B,  C  and  D.  In  the  entire  course  of  the  exprri- 
incnt  ilx-re  was  not  the  slightest  indication  of  light  response, 
nor  indeed  was  there  more  of  a  geotropic  character.  The  final 
altitude  was  that  indicated  in  D. 

The  «nher  suspended  specimen  attached  itself  to  the  side  of 
thr  M\(  rtlou  tube,  and  has  continued  head  down,  without  appre- 
eiable  change  of  aspect,  the  tube  remaining  almost  perfectly 
-iraii;lii  from  first  to  last.  Both  specimens  seemed  equally  at 
,  both  equally  active;  but  the  one  passed  through  the  series 
dl  tubular  i  "iitortions,  the  other  remained  absolutely  indifferent. 
lin  ideutally  it  may  be  remarked  that  specimens  are  often  found 
in  nature  attached  to  the  under  surface  of  bottom  of  boat-  or 
other  -ubstrata,  much  like  barnacles  or  other  sessile  organism-; 
and  hence  it  mu-t  be  admitted  that  there  is  nothing  especially 
unusual  or  unnatural  in  such  an  attitude.  That  the  beha\  ior 
of  i  he  one  difYered  from  that  of  the  other  is  not  more  str.r 
ill. in  that  differences  likewise  appear  between  others. 

Sf>f<  i<il  Aquaria. — The  second  series  of  experiments  were  con- 
durt. -d  in  t\\o  special  aquaria,  mentioned  above,  and  were 
piompted  by  two  considerations  First,  the  apparently  negative 
ehaiaet.  r  of  the  experiments  began  and  carried  forward  in  the 
lar^e  ai|iiaria.  It  had  seemed  as  if  one  should  have  more  prompt 
and  eoii\  in.  ing  results  than  appeared  in  the  account  just  given. 
"v  .  ou.lly.  it  was  desirable  to  have  aquaria  of  a  size  and  adjti-t- 
meiit  \\hieh  made  possible  ready  and  effective  control  at  all 
time-,  \\ith  such  variation  of  tests  as  seemed  desirable,  lieu.-.- 
the-e  -mailer  aquaria  already  described.  They  were  set  in  a 
room  \\ho-e  light  and  temperature  were  under  easy  control,  and 
\\.re  t  hem-elves  of  a  size  which  enabled  one  to  shift  the  position 
at  anv  time  it  might  seem  desirable.  It  occurred  to  me  that 
po— ibl\  the  fact  that  in  the  first  series  the  light  had  been  ditfu-e 
rather  than  direct  might  have  resulted  in  thr  -omeuhai  ne^ati\e 
beha\ior  already  noted.  Again,  it  seemed  de-irable  to  be  able 
ID  rontrol  both  the  direction  and  inu-n-ity  of  the  li^ht  Ac- 


84  CHAS.    \V.    HARGITT. 

cordingly  the  special  aquaria  were  made  use  of,  and  the  following 
account  is  based  entirely  upon  the  behavior  under  tin-  new  con- 
ditions. Two  were  used  for  the  definite  purpose  of  making  of 
one  a  control  of  the  other.  That  is,  given  identical  conditions 
of  temperature,  food,  etc.,  will  the  mere  difference  of  direction 
or  intensity  of  light  show  itself  in  such  measure  as  to  warrant 
conclusions? 

This  series  was  begun  on  January  15  with  twelve  specimens, 
eight  being  placed  in  the  experimental  tank,  and  four  in  the 
control  tank.  The  bottoms  of  the  aquaria  were  covered  by  a 
layer  of  rather  coarse,  black  sand  to  facilitate  attachment,  and 
at  the  same  time  to  render  any  access  of  light  from  the  bottom 
impossible.  The  test  tank  was  covered  on  three  sides  and  the 
top  with  an  opaque  hood,  painted  black  on  the  inside  and  so 
adjusted  as  to  render  inspection  easy  without  disturbing  the 
specimens.  In  this  tank  the  eight  specimens  were  placed  with 
heads  facing  away  from  the  source  of  light.  Similar  disposition 
was  made  of  the  four  specimens  of  the  control  tank.  In  about 
three  days  the  specimens  had  apparently  attached  themselves, 
and  on  January  19,  four  days  after  beginning,  one  specimen  began 
an  upward  curve.  On  the  2ist  several  had  shown  such  reaction 
and  by  the  25th  several  had  curved  upward  to  from  25  to  50 
degrees.  In  the  control  tank  similar  responses  began  to  appear. 

On  January  25,  ten  days  after  beginning,  the  record  is  as 
follows:  Of  the  eight  specimens  two  have  curved  toward  the  light, 
two  are  nearly  vertical,  two  face  toward  the  side,  while  two  remain 
as  planted.  Essentially  the  same  condition  obtains  in  the  control 
tank.  One  faces  the  window,  one  nearly  vertical,  and  two  as 
originally  located. 

At  the  end  of  four  weeks,  February  i  i,  three  show  apparent 
light  reaction,  two  are  nearly  vertical,  two  remain  facing  away 
from  light,  and  one  shows  an  indifferent  curve  laterally.  The 
positions  in  the  control  tank  remain  as  before.  Repeating  Loeb's 
experiment  at  this  point,  I  no\v  rotated  the  aquaria  through  180 
degrees,  so  that  everything  was  changed  directly  about.  Con- 
ditions went  on  as  before,  the  test  tank  receiving  light  exclusively 
from  one  end,  the  control  receiving  diffuse  light  from  the  room 
as  well  as  the  direct  light  from  tin-  window.  On  February  25, 


ON    THE    I;i;il\\!"K    <  •!•    TUBICOLOUS     \N\KI.IDS.  85 

or  fifteen  days  after  the  aquaria  had  been  rotated,  the  conditioti- 
.in-  ,i-  follow-,:  Five  specimens  now  face  the  light,  while  three 
I". u  i  the  opposite  direction.  But  of  the  five  now  facing  the  light 
three  were  so  placed  in  the  readjustment  made  by  the  rotating. 
or  n Aer-ing  of  the  tank,  so  that  only  two  ha\e  actually  shown 
a  po--il>le  light  reaction.  The  three  specimens  \\hich  had  been 
turned  auav  from  the  window  by  this  reversal  had  not  sho\\  n 
i  li«'  sliijiu-i  response. 

At  i  hi-  i  ime  the  acjuaria  were  again  reversed,  so  that  they  came 
back  to  the  original  positions.  It  should  be  noted  that  in  the 
control  i. ink  there  had  been  no  change  induced  by  the  re\er-al 
ol  tin  pi -i  i  ion,  the  specimens  all  remaining  as  before. 

Another  a-|>ect  of  behavior  may  be  stated  in  thi-  connection. 
n.imel\ .  an  actual  downward  curve  of  several  specimen-.  It  was 
on  in-t  notice  thought  that  possibly  this  might  be  due  to  the 
iin  inn  -in  water,  which  happened  to  be  in  the  region  ot  one  such 
llouever.  it  was  later  observed  that  other  specimens  quite 
remoii-  -houi-d  the  same  thing,  and  on  comparing  similar  con- 
dition- in  the  exhibition  aquaria  it  was  found  to  have  it-  counter- 
part there,  1 1'  nee  it  may  be  regarded  as  only  another  cxpre--ii.n 
«\  the  individuality  of  behavior  which  is  more  or  less  e\ident 

iimler  all  i  "minions. 

The  e\pi  liments  \\ith  these  special  aquaria  \\eiv  continued  to 
M  Mch  J5,  having  thus  been  under  operation  for  about  ten  weeks 
i  JannaiA  i  ;>  to  March  25),  and  have  been  in  the  present  pi  .-it  ion 
loi  e\aitl\  one  month.  During  this  time  there  ha\e  been  inci- 
dental -hillings  on  the  part  of  various  specimens,  a  bending  this 
\\a\  or  that  from  time  to  time,  but  only  to  be  re\«-r-ed  later,  or 
counterbalanced  by  opposite  reactions  of  adjacent  s|iecinien-. 
The-e  ha\  e  been  noted  from  lime  to  time  during  the  o  >ur-e  of  all 
the  experiment-,  and  are  not  to  be  considered  as  orienting  re- 
action-, but  rather  expressions  of  the  individuality  ot  behasior 
charactei  i-t  ic.  a-  I  believe,  of  .ill  grades  ol  animal  behavior. 
Tlu-\  correspond  to  what  Jennings  ha-  de-ignated  as  trial  rc- 
(udons;  and  in  the  present  instances  probably  relate  to  fo.>d- 
seekinj  or  n-piration.  These  statements  refer  directly  to 
condition-  in  the  darkened  aquarium;  but  they  are  quite  as 
applicable  to  those  of  the  control  aquarium,  and  indeed,  the 


86  ('HAS.    \V.    II  \Ki,iTT. 

behavior  of  the-  specimens  in  thi>,  \vhile  differing  in  various 
details,  have  shown  a  striking  similarity  to  that  of  specimens  in 
the  former,  as  well  as  that  of  the  first  series  in  the  large  aquaria. 
As  remarked  in  the  outset,  the  entire  series  of  experiment--  have 
involved  no  appreciable  deterioration  of  the  health  or  vigor  of 
the  specimens.  As  an  evidence  of  this  may  be  mentioned  tin- 
fact  that  one  very  young  specimen  among  those  used  in  tin- 
control  tank  showed  an  apparently  continuous  growth,  haxing 
nearly  doubled  its  original  size.  The  growth  in  this  case  seems 
to  have  been  real  and  normal,  and  not  apparent  as  was  the  case 
with  Hydroides,  mentioned  in  a  previous  section. 

Third  Series. — Early  in  March  it  was  found  desirable  to  change 
rooms   in    the   laboratory,   and    I    came   into   possession   of  one 
admirably  adapted  to  light  experiments.     Advantage  was  taken 
of   this  circumstance  to  continue  the  experiments  with   Spiro- 
graphis  under  light  conditions  which  were  exceptionally  good. 
In  the  room  were  two  large  aquaria,  one  of    which   I  devoted 
exclusively  to  this  experiment.     The  aquarium  was  arranged  with 
its  side  facing  the  window  and  at  a  distance  of  about  two  meter-. 
By  covering  the  back,  ends  and   top  of  the  aquarium  \\ith  a 
black  opaque  screen,  and  with  windows  also  provided  with  ad- 
justable shades,  one  was  able  to  directly  control  the  light  condi- 
tions at  will,  as  to  source,  directness  and  intensity.     The  experi- 
ment was  begun  with  eight  specimens,  all  of  which  \\en-  placed 
with  heads  facing  away  from  the  light,  and  two  other-  suspended 
head  down  by  attaching  them  to  sides  of  the  o\  erllow  pipe,  as 
in    the  similar  experiment  in  series   I.     Other  specimens  were 
added  a  few  days  later  making  a  total  of  twenty  comprising  the 
experiments.     As  before  some  two  to  four  days  were  required 
for  specimens   to   become   attached    to   the   aquarium.      In    tin- 
present  case  to  insure  prompt  and  precise  location  several  were 
secured  to  a  given  place  by  putting  over  the  terminal   base  o| 
the    tubes  a    small    weight,  such   as  a  shell    or  rock    fragment. 
As  before  the  first  indication  of  reaction  was  the  usual  upward 
curve  of  the  oral  end  of  the  tubes,  enabling  the  creature  to  I  reel \ 
expand    the   gills.     This   reaction    has   little,  it    any,  relation    to 
orientation  movements,  as  it  occurs  usually  in  all  cases  and  under 
almost  all  conditions,  whether  in  light  or  darkness. 


ON    THK    BEHAVIOR   OF    TUBICOLOUS    ANNELIDS.  87 

On  March  25,  ten  days  after  the  specimens  were  installed,  only 
had  assumed  a  nearly  vertical  aspect.  Others  showed 
variou-  phases  of  orientation,  from  ten  to  twenty,  or  thirty,  or 
fifty  dej  of  elevation  above  the  bottom. 

<  )n  April  i.  the  following  is  the  record.  Four  -pecimen-  with 
:^ill-  directed  more  or  less  toward  the  light;  two  with  a  vertical 
attitude;  three  oriented  at  right  angles  to  direction  of  light,  and 
facing  darkest  end  of  tank;  nine  remain  oriented  in  original 
po-ition,  /.  i\,  facing  away  from  light.  The  t\\o  -u-pemle<l  -peci- 
ini  11-  lieha\e  almost  exactly  as  in  the  previou-  case;  that  i-.  one 
pcr!e<  il\  unchanged  and  the  other  curved  a\\a\  tn>m  the  pipe. 
Tim-  .tiler  m-arlv  two  weeks  half  of  the  entire  lot  remain  abso- 


l-ii.    i  i-  .in  finl  view  of  an  experimental  aquarium,  tin-  li.^lu  o>min.n 
iiv;lit  -iik  .it  *       '  n  tin-  i-ii;ht  specimens  shown  only  niu-  i-  :.n  inv;  tin-  liKl 
\<-i  tii  .il.  the  others  facing  the  dark  side  of  tank. 


is 


hilelv  unrli.ui-.'d  ;  of  the  others  onl\  ti\e  ^Imu  any  \IT\  clear 
rea(  lion  to  p.^ibk-  light  stimulus.  The  experiment-  o  mi  inued 
under  d.iih  observation  until  April  \2,  a  |)erio<l  ot  one  month, 
\\iili  a  tmal  remnl  as  follows:  Four  -pet  inien-  -lm\\  a  distinct 
curvature  t<>\\anl  the  light;  nine  show  ju-i  a-  distinct  inclination 
a\\a\  tioni  the  light,  in  other  \\oriU  remain  as  ori-inalK  fixed 

except  the  slight  curvature  upward  ;  two  are  almost  vertical  ;  the 

other  three  occup\    portions  at  right  an^le-  t<>  the  line  of  light. 


CHAS.    \V.    HAR(iITT. 

The  two  suspended  specimens  continue  as  before,  one  absolutely 
as  at  first,  the  other  with  a  definite  crescentic  curvature,  but 
forty-five  degrees  away  from  light.  Fig.  4  is  from  a  photograph 
taken  l»y  Dr.  S.  \\.  Williams  and  gives  a  good  impression  of  the 
orientation  of  such  specimens  as  came  within  the  view.  It  is 
taken  from  the  end  in  order  to  show  the  relation  of  the  tubes 
to  light,  which  came  directly  from  the  right  and  into  that  side 
of  the  aquarium.  Of  the  twenty  specimens  only  eight  are  shown, 
and  of  these  only  one  faces  the  light,  one  is  almost  vertical,  the 
other  six  incline  very  definitely  toward  the  dark  side  of  the 
aquarium. 

As  will  be  seen,  nothing  especially  new  has  developed  beyond 
what  has  been  found  in  connection  with  the  earlier  series.  How- 
ever, since  here  the  conditions  of  light,  temperature,  etc.,  have 
been  so  ideal  the  results  not  only  confirm  those  already  given, 
but  render  them  more  certain  and  conclusive.  It  seems  quite 
improbable  that  three  series  of  experiments  directed  to  a  single 
end  should  have  given  uniformly  erroneous  results;  moreover, 
it  is  equally  improbable  that  any  error  of  method  should  have 
vitiated  all  three  series,  varied  as  these  are  shown  to  be,  and 
inspected  as  they  were  by  several  of  my  colleagues  almost  from 
the  beginning.  Nor  is  it  possible  that  the  matter  of  season  could 
have  been  a  modifying  factor,  for  it  coincided  almost  exactly 
with  that  of  Loeb's  experiments.  That  light  has  been  shown  to 
be  a  wholly  negligible  factor  in  relation  to  the  behavior  in  (JIM  -lion 
has  not  at  any  time  been  claimed.  That  it  has  been  shown  to 
have  only  a  minor  influence  I  believe  the  facts  conspire  to  render 
very  certain. 

But  we  are  not  yet  done  with  the  problem.  In  his  original 
account  Loeb  cited  the  behavior  of  Spirographis  in  the  public 
aquarium  as  tending  to  confirm  his  experimental  results  "for  the 
most  part'  I  have  studied  the  problem  in  this  aquarium  with 
especial  care  during  the  entire  course  of  my  own  experiments 
and  have  found  the  behavior  to  confirm  my  experiments,  as  the 
results  will  show.  Let  it  be  expressly  understood  that  in  these 
large  exhibition  aquaria  the  best  efforts  of  many  years  have  been 
directed  to  render  them  as  nearly  natural  as  it  is  possible  to 
have  such  limited  portions  of  the  sea;  and  the  fact  that  some 


OX    THE    BEHAVIOR    OF    TUBICOD  >l-    ANNELID-.  89 

of  their  occupants  have  lived  and  thrived  here  for  more  than 
t \\  enty-five  years  bears  strong  evidence  to  the  measure  of  success 
in  the  effort  to  render  them  natural.  In  the-e  aquuriu  $p:ro- 
•f>his  seems  to  find  a  fairly  congenial  environment,  and  thri\c- 
continuously  in  health  for  many  months.  For  the  sake  of 
exhibition  advantages  the  specimens  have  been  planted,  or  di-- 
po-ed  in  such  ways  as  afford  the  display  of  the  gorgeon-.  tl«>\\rr- 
like  yill-  to  the  best  advantage.  Hence  some  are  located  on  the 
lloor  of  the  aquaria,  others  on  the  back  and  end-  where  rocky 
led^e-  afford  suitable  bases  for  their  support.  It  oujju  aUo  to 
be  -aid  that  in  order  to  render  these  aquaria  the  be-i  po--il>le 
exhibition  cages  the  illumination  is  chiefly,  and  in  -ome  case 
uh'illv,  Irom  above;  while  the  room  itself  is  purposely  kept  dark. 
ex<  epi  |..r  the  light  which  diffuses  outward  from  the  aquaria.  It 
becomes  important  that  in  reference  to  the  problem  before  u- 
thi  >f  the  source  and  direction  of  light  be  borne  in  mind. 

<  in  tin    assumption  of  the  compelling  potency  of  light  it  \\ill  be 

<  le.n  that  in  the  case  under  examination  there  should  be  a  fairly 
unilormlv    vertical   aspect   of   the   various   specimens,   \\hate\er 
m. iv  ha\e  been   their  original  position.     The  following  are   the 
l.it  '       !      nn  several  attempts  it  was  determined  with  approxi- 
in, i;  i racy  that  at  this  time  there  were  about   150  specimen- 

<//>// is  in  the  aquarium.     These  were  di-po>ed,  as  men- 
tioned above,  on  the  bottom,  ends  and  back  of  the  tank.     <  >!  the 
entire  number  about  <)()  were  in  more  or  less  vertical  at  lit  ml 
\\ith  upuard  inclination,  while  60  were  otherwise  inclined,  tli.n  i-, 
the\   were  horizontal  or  inclining  downward.      The  ^n.  ral  t 

well  shown  in  Fig.  ,v  which  is  a  photograph  ol  the 
a(|iiarium  made  by  Dr.  Sobotta,  by  whose  kind  penni--ioii  I  am 
able  to  use  it  in  this  connection.  Of  the  60  specimen-  of  ihU 
ad\  erse  aspect  slightly  more  than  half  were  horizontally  di-po-rd, 
\\liile  the  others,  some  23  specimens,  exhibited  de<  idedl\  <lo\\n- 
\\ard  inclination.  The  picture  will  afford  excellent  illn-t ration, 
though  not  taken  at  the  time  my  observations  \\erc  made. 

I  el  n-  now  attempt  to  analyze  these  facts  and  their  b.-arii 

upon  our  problem.      It  may  be  stated  at  the  out-tart  that  gra\-ity 

ha-  little  or  no  place  in  the  behavior.     I.oeb  ha-  so  concluded 

11   hi-  experiments,  and  my  own  -o  to  confirm   his  venl 


9O  CHAS.    W.    HARGITT. 

Both  in  experiments  and  in  nature  there  seems  to  be  no  evidence 
of  its  operation.  Specimens  attach  themselves  to  the  bottoms 
of  boats,  to  overhanging  rocks,  etc.,  and  seem  quite  indifferent 
to  its  influence.  We  may  therefore  proceed  to  consider  the  main 
question  at  issue,  namely,  that  of  light. 

Of  the  90  specimens  having  a  sub-vertical  attitude  about  (>o 
were  on  the  bottom  of  the  aquarium,  which  leaves  30  of  this 
class  among  those  located  on  the  back  and  end  walls.  In  other 
words,  twice  as  many  of  the  vertical  specimens  were  located  on 
the  bottom  as  on  the  sides.  But  let  it  be  remembered  that  of  the 
total  150  specimens  in  the  aquarium  about  94  were  planted  on 
the  bottom  while  only  56  were  located  on  the  walls.  Further, 
it  is  to  be  noted  that  those  located  on  the  bottom  must  assume  a 
sufficient  degree  of  elevation  to  afford  a  free  expansion  of  the 
gills;  to  those  on  the  walls  this  is  not  essential.  On  the  other 
hand,  of  the  60  specimens  which  had  assumed  a  horizontal,  or 
downward  attitude  about  25  were  among  the  bottom  specimens, 
while  the  other  35  were  among  those  attached  to  the  walls. 
Expressed  in  percentages  we  have  the  following:  Of  the  whole 
number  about  60  per  cent,  showed  a  more  or  less  vertical  aspect, 
while  40  per  cent,  showed  otherwise,  i.  e.,  a  downward  inclination. 
Of  those  planted  on  the  bottom  about  70  per  cent,  showed  a 
vertical  tendency,  and  about  30  per  cent,  were  inclined  downward. 
Of  those  on  the  walls  about  65  per  cent,  inclined  downward, 
while  35  per  cent,  inclined  toward  the  vertical. 

Now,  how  shall  one  interpret  these  varying  aspects?  Accord- 
ing to  theory,  "  If  the  rays  of  light  fall  vertically  from  above  into 
the  aquarium,  Spirographis  directs  its  tube  vertically  upward, 
exactly  as  a  stem  grows  vertically  up  into  the  air."  In  the  case 
before  us  the  light  comes  vertically  from  above,  yet  a  large  per  cent, 
of  the  specimens  fail  to  direct  the  tubes  vertically  upward.  Of 
wall  specimens  65  per  cent,  incline  downward,  or  are  horizontal 
in  relation  to  light.  Of  those  on  the  bottom  the  per  cent,  curving 
downward  is  much  smaller,  but  still  too  great  to  be  explained 
as  merely  incidental,  or  by  the  naive  suggestion  "Here,  however, 
where  free-swimming  forms  easily  disturb  the  orientation  <>t 
Spirographis,  it  is  not  so  perfect  as  when  all  possible-  disturbing 
causes  are  avoided,  as  in  an  aquarium  used  only  for  such  experi- 


<>N    THE    BEHAVIOR    OF    TUBICOLOUS    ANNELIDS.  QI 

ment."  Unfortunately  for  such  explanation  "free-swimming 
form-"  are  rarely  present  in  this  aquarium,  the  only  specimens 
during  my  observations  being  the  slow  ancTdelicate  moving  little 
sea  horse,  Hippocampus,  whose  presence  among  the  relatively 
colossal  Spirographis  could  hardly  be  of  more  influence  as 
,i  disturbing  factor  than  a  few  sparrows  in  an  oak  forest!  In 
fact  -pre  imrns  of  Hippocampus  had  been  kept  for  weeks  in  one 
nl'  tin  aquaria  in  which  my  special  experiments  \\ere  being  made 
.ui'l  \\mild  frequently  attach  themselves  by  their  delicate  pre- 
hrn-ilr  tail-  to  the  tubes  of  Spirographis  but  without  the  le.i-t 
r\idm.  e  ••!"  disturbance  of  any  sort.  One  often  finds  the  tubes 
'  it  these  .imielids  more  or  less  loaded  with  tunicate-,  -p-nu'  - 
li\ilr<ii<l-.  etc.,  but  there  was  never  any  appreciable  sign  "I 
di-iurbaiice  therefrom  so  far  as  their  orientation  was  concerned. 
I  think  it  must  be  rather  obvious  that  the  behavior  exhibited 
b\  these  creatures  under  the  sub-natural  conditions  of  tli.  -<• 
ma^iiiti'  '  nt  aquaria  conforms  in  all  essentials  with  that  found  in 
tin-  r\pi  rimental  tanks,  and  under  both  these  tests  then-  seems 
to  be  a  fair  equivalent  of  that  to  be  observe*  1  in  their  nati\e 

habitat. 

CONCLUMV.  REMARKS. 

Tin  Inn  ^iiing  account,  especially  when  taken  as  a  part  ot  the 
more  extended  observations  already  repeatedly  citr<l  '06,  '09 
must  make  it  more  or  less  evident  that  so  far  from  affording  any 
-iippun  lo  the  sweeping  assumption  of  the  identity  nl  animal 
and  plain  hrliotropism,  based  on  the  behavior  of  these  organisms. 
siKuid  ,-sts,  if  indeed  one  might  not  say  warrants,  tin  \ « -r\ 

opposite.     One  might  even  go  a  step  farther  and    saj    that    ii 

us  extremely  doubtful  whether  the  behavior  of  Hy<lr»iii<-s, 
r«nniti>,  eras,  Spirographis,  or  any  of  the  tubio>lou>  annelids  may 
l.r  interpreted  as  an  expression  of  tropi-m-  at  all.  Without 
-eeking  in  any  way  to  discredit  the  possible  role  of  light  in  rrlaii<m 
to  certain  aspects  of  behavior,  it  may  vet  be  fairly  doubted 
whether  it  sustains  any  such  determining  influence  as  has  been 
claimed  by  the  exponents  of  the  tropism  hypoihesi-.  Indeed  the 
tacts  \\hich  ha\e  been  passed  in  review  show  beyond  rea- -liable 
doubi  that  in  relation  to  these  organisms  it  can  have  but  a  sub- 
ordinate and  incidental  place.  It  seems  perfectly  certain  that 


92  CHAS.    W.    HARC.I  1  1  . 

there  is  not  that  degree  of  constancy,  or  character  of  reaction, 
in  orientation  which  would  warrant  a  tropic  interpretation  of 
any  sort. 

lint  <m  the  other  hand  let  it  not  he  inferred  that  behavior  is 
chaotic  or  beyond  scientific  explanation.  As  I  have  elsewhere 
pointed  out,  reactions  and  adjustments  in  relation  to  food-getting, 
respiration,  etc.,  are  among  the  most  fundamental  of  all  phases  of 
behavior.  These  creatures  must  live,  hence  must  have  food ;  but 
they  are  sessile,  and  therefore  must  utilize  such  as  may  come 
within  reach.  Furthermore,  they  must  respire,  and  hence  must 
have  room  within  which  to  expand  the  gills.  All  this  implies 
that  such  colonial  species  must  of  necessity  frequently  resort  to 
movements  of  readjustment  directed  to  the  above  imperative 
ends.  In  most  of  these  creatures  it  so  happens  that  one  and  the 
same  organ  is  involved  in  this  dual  function  of  food-taking  and 
respiration;  a  fact  of  some  significance  in  simplifying  or  com- 
plicating, according  to  condition,  certain  phases  of  behavior. 
To  the  writer  it  seems  probable  to  the  point  of  certainty  that 
the  aspects  of  behavior  which  have  been  under  review  are  chiefly 
but  varied  expressions  of  these  common  functions.  In  other 
words,  they  are  aspects  of  adjustment  in  the  complex  struggle 
for  existence — varying  modes  in  which  each  species  has  worked. 
out  its  own  special  problem  of  life. 

In  the  light  of  this  mode  of  interpretation  the  complicated 
serpentine  torsions  of  the  tubes  of  Hydroides  and  Pomatoccras 
arc  the  most  natural  expressions  of  just  such  "trial  movements" 
as  one  might  expect.  Likewise  the  bending  aspects  of  the 
flexible  tubes  of  Pot  am  ilia  and  Spirographis  are  not  mysterious 
enigmas  over  which  students  of  behavior  need  array  themselves 
in  warring  camps,  but  rather  the  simple  expressions  of  those 
individual  adjustments  called  for  in  the  varying  struggle  of 
life,  to  the  interpretation  of  which  Huxley  would  have  found 
necessary  only  "trained  and  organized  common  sense" 

I  am  quite  aware  that  to  speak  of  individuality,  or  autonomy, 
or  spontaneity  as  factors  involved  in  problems  of  animal  behavior 
may  to  some  exponents  of  mechanism  seem  "no  explanation," 
and  of  significance  only  to  the  psychologist.  But  as  I  ha\e 
earlier  pointed  out,  they  are  facts,  and  they  bulk  large  in  tin-  sum 


OX    THE    BEHAVIOR    or    TUHK  <  >!.<  >l  -    ANNELIDS.  O.} 

int.il  of  animal  economy  and  behavior.  To  ivco-ni/e  them 
as  facts  is  not  to  imply  thereby  their  explanation;  but  it  does 
imply  that  they  are  no  less  entitled  to  recognition  ami  cxplana- 
t  if  in  than  any  other  classes  of  facts  with  which  we  have  to  deal. 
I  i~  an-  sometimes  characterized  as  "stubborn  thins;-."  they 
ha\e  ways  of  their  own;  they  are  tenacious  fit"  lilV;  and  -o<mcr  or 
later  \\ill  compel  respectful  attention  and  explanation.  A-  i- 
well  known,  in  his  matchless  account  of  the  behavior  ot  earth- 
\\onn-  I'aruin  <lid  not  hesitate  to  employ  a  terminology  which 
iiankK  a->umed  the  presence  in  these  creatures  of  nervou-  and 
p-\rlii(  factors.  While  it  may  not  be  easy  to  pm\e  that  anne- 
lid- ha\e  a  high  degree  of  intelligence,  on  the  other  hand  In  \\lio 

lys  to  prove  that  intelligence  has  no  part  whatever  in  their 
beh.ixior  \\ill  hardly  ha\re  an  easier  problem. 

\t  no  time  has  the  writer  questioned  the  important  relation-, 
ol  ph\  -i«  .-  »  hemieal  factors  to  the  phenomena  of  lit*  .md  beha\  i«'r. 
1  in  tin  i.  hr  has  not  questioned  the  possibility  of  the  correla- 
tion ot  these  phenomena  under  physical  laics,  mm  h  as  ue  n •> 
ni/e  that  |ihenomena  of  electricity  and  magneti-m  and  -ra\  i- 
i.iiion  .in  conserved  under  other  natural  laws.  Hut  tin-  b\ 
n..  mean-  implies  that  these  latter  species  of  ener-\  h.i\e  n-.i 
i  IK  i  o\\n  ''fcial  lau's,  some  of  which  are  alread\  kno\\n  \\hile 
other-  ha\e  thus  far  defied  definition  and  correlation,  ^o.  in 
tlie  in. nter  under  review,  what  he  lias  <|iie-tioiud  i-  the  very 
dillei.nt  po-iilate,  that  knou'n  properties  of  ch<-mi-ir\  of  phy-ie- 
in  an\  ot  their  known  interactions  afford  a<le(|ii.ite  definition 
and  explanation  of  all  the  facts;  or  that  kn»:cn  f>liysi,dl  I: 

ipplied    b\     the    sponsors   of    mechani-m,    an-    convincingl) 
-iiiiuieiu.      It    is  against   the  arrogant  a>-umpiion    that    a    i 
ol"  beha\ior.  .  .r  an  expression  of  emotion  or  atle.  tion.   i-  never 
explaiiu-il  till  cast  into  some  physical  or  mathematical  tormnla, 
that    prote-t    ha-   been   iterated.      In   dire* -tin-    attention    to   the 
po^-ible   interaction   of  well-known   p-ychic    factors   in   belia\  ior 
tlu  re  i-  merely  the  plea  that  similar  re<  •  -^nitif  >n  be  -i\  en  to  them 

to  the  lornier  and,  as  suggested  above,  they  be  included  in 
the  category  of  behavior  calling  for  explanation.  Ho\\e\.-r 
independent  or  unrelated  may  appear  certain  of  their  expressions 
it  i-  not  a--uined  that  in  any  scientific  sen-e  they  are  mutually 


94  CHAS.    W.    HARC.ITT. 

exclusive,  nor  that  the  one  class  of  phenomena  are  any  less 
related  to  causal  antecedents  than  the  other.  But  it  is  main- 
tained that  while  in  some  cases  these  antecedents  may  be  known, 
and  lend  themselves  to  direction  and  control,  on  others  they 
are  as  yet  absolutely  unknown  and  more  or  less  beyond  pre- 
diction or  control.  And  furthermore,  it  is  believed  upon  experi- 
mental evidence  that  certain  aspects  of  behavior  may  be  more 
or  less  variable  under  any  given  set  of  antecedents  or  conditions; 
in  other  words,  given  stimuli  do  not  always  evoke  the  same  response; 
in  fact,  much  of  behavior  is  indeterminate  in  terms  of  existing 
knowledge.  But  so  far  from  implying  a  reactionary  attitude 
toward  the  value  and  importance  of  continued  experimenta- 
tion, the  writer  would  hold  the  very  opposite.  It  is  well  that 
one  pause  now  and  then  and  take  stock  in  science  as  well  as  in 
business.  That  problems  of  behavior  are  complex  beyond 
earlier  anticipation  goes  without  saying.  The  same  must  be 
admitted  of  every  problem  of  biology.  Only  the  biological 
pessimist  will  find  occasion  to  contemplate  intellectual  suicide 
because  he  finds  the  dogmas  of  his  science  in  process  of  revision! 

LITERATURE  CONSULTED. 
Bounhiol 

'oo     Recherches    exper.   sur  la   rcspir.  dcs  annelides    Etude  du   Spirographi-. 

Compt.  Rend.,  T.  132,  1900. 
Hargitt,  Chas.  W. 

'06     Experiments  on  the  Behavior  of  Tubicolous  Annelids.     Jour.  Exp.  Zool., 

Vol.  III.,  pp.  295-320. 
"09     Further  Observation  on  the  Behavior  of  Tubicolous  Annelids.      Ibid.,  Vol. 

VIL,  pp.  157-187- 

Harper,  E.  H. 

'09     Tropic   and    Shock    Reactions  in    Perichceta  and    Lunihriiit^.     Jr.    Conip. 

Xeur.  and  Psych.,  Vol.  XIX..  pp.  569-587. 
Jennings,  H.  S. 

'06     Behavior  of  the  Lower  Organisms.      New  York. 
Loeb,  J. 

'90  \Veitere  Untersuchungen  uber  d.  Ili-lioin>pi>miis  d.  Tiere  u.  sciiu-  t  Ut- 
einstimmirtig  mit  dem  Heliotropismus  drr  PHanzcn.  Arch.  t'.  d.  gi--.ini. 
Physiol.,  Bd.  47,  p.  391. 

'oo     Comp.  Physiol.  Brain  and  Comp.  Psych.      New  York. 
Mast,  S.  O. 

"ii      Light  and  the  Behavior  of  Organisms.      New  York. 
Nagel,  W.  A. 

'96      l)cr  Lichtsin  augenloser  Tiere,  cine  biologische  Studie.     Jena. 
Radl,  Em. 

'03      Untersuchungen  ubcr  den  Photntropismii^  drr    lirir. 


THK    DEVELOPMENT   OF   THK   GONAD    AM)    GONO- 
I)t;CTS    IN    TWO   SPECII->    '  >F    CHITONS. 

ROSE  M.  HK;LEV  AND  HAROI  D  HEATH. 

Tin-  I. HIT  development  of  the  chitons  ha-  never  been  fully 
iim-Mi'-.m-cl.  and  the  fragmentary  obscr\  MI  i«  m-  that  have  been 
in. idi-  relate  .ilmost  exclusively  to  immature  form>  in  very  ad- 
Nam  id  stages.  Accordingly  we  are  at  piv-ein  alim»t  whollv 
ignorant  i.i  tin-  development  of  the  principal  systems  of  organ^ 
.nid  ilu-ir  homologies.  Many  of  the  more  import, uu  i|iie-timi- 
relating  to  'In  se  animals  center  in  the  formatinn  of  tin  i-u-lmn, 
.md  ii  \\.i-  \\iih  the  hope  of  throwing  some  light  <>n  thi>  >ul>iect 
tli.it  tin-  piv-eiit  work  was  undertaken. 

111.  iu<»  -prcies  that  form  the  basis  of  thi>  investigation, 
.•hyiit-nnon  ra ynwndi  and  Xutlallina  tlwmasi,  .\n-  !.iirl\  almn- 
il.mt  li.ini-  in  certain  localities  along  theci>.i-t  <>i  ( '.ilifnrni.i,  and 
n\\iiiv;  tn  tln-ir  Miiall  si/e  are  readily  snt  \<  uinl.  The  tK  i---\\  ini- 
iniii^  young1  were  placed  in  aquaria  together  \\itli  t'lMgnu-nt-  uf 
-ln-ll-  .  'i!ns  californica  on  which  they  Imallv  -i  tiled  alter 

partialK  completing  their  metamorphosis.  Tlu-v  \\(ic  then 
traii-lenvd  to  small  and  protected  tide  pm.U  \\hen-  tln\  de\el- 
"|ird  mu  inalU' and  in  se\-eral  instances  were  allowed  t"  re.n  h  the 
-exualK  mature  condition.  Precautions  \\eiv  taken  to  keep  the 
\  i  ni  nv;  i  >l  eaeh  -pe.  ies  in  separate  pools  and  it  \\  a-  I  mi  ml  that  they 
travel  essen  dally  the  same  developmental  path  tm- a  Ion-  period. 
I  >i-tiiiL;tii-liiiig  characteristics  accordingly  appear  late,  in  I'aet 

roii-idt -ralil\  lievond  the  formation  of  the  L;<'iia<l  ami  it-  dm  I-. 
It  U  to  In-  understood  therefore  that  while  the  tigure>  are  of 
T.  niynn»uii  the\  -erve  equally  \\ell  tor  A",  tlnnintsi. 

\\  a  \ei\  earl\  stage  the  heart  and  peril  ardial  cavity  are 
dexeli.pe.l  troni  (clU,  giving  evidence  of  lu-ing  derixed  exclu- 
>i\el\-  from  the  -e.  .-mlary  mesolila-t  pm-eiu  ot  4/>i,  whieh 
lorm->  an  irregular  la\er  mi  the  poMcro-dm--al  -ide  ot  the  larxa. 
A  rel.n:\el\-  long  period  of  tinu-  then  ensues,  during  which  the 

iii.u  li.iliit-  <>i  tli>  e  /•'.•/.    In       Bd.  XXIX..  \«i.   ij. 

95 


96 


ROSE    M.    HIGLKY    AND    HAROLD    HI  AMI. 


other  systems  of  the  body  develop  to  practically  tin-  same  con- 
dition as  in  tin-  adult,  before  the  gonad  makes  it>  appearance. 
When  the  primitive  sex  cells  become  recognizable  they  usually 
form  two  groups  attached  to  the  anterior  external  surface  of  the 
pericardium  from  which  they  appear  to  be  proliferated.  Very 
soon,  in  rare  instances  at  the  time  of  their  formation,  these  be- 
come so  closely  appressed  as  to  appear  single  though  section^ 
show  them  to  be  distinct  for  a  considerable  time,  frequently  alter 
the  gonoducts  have  formed.  Shortly  after  their  appearance  a 
cavity  forms  within  each  group,  and,  with  the  growth  of  the 
gonad,  soon  becomes  more  or  less  triangular.  In  later  stages. 


A 


FIG.  i.  Gonad  and  ducts  of  Trachydermon  raymondi.  A,  section  through 
animal  about  i  mm.  long,  a,  digestive  tract;  g,  gonoduct  connecting  with  gonad; 
k.  kidney;  /  liver;  n,  lateral  nerve  cord;  s,  shell.  B,  gonad  (o)  and  ducts  in  matuie 
animal,  dorsal  view.  C,  reconstruction  of  same  stage  a»  Fig.  i.  Gonad  with 
ducts  ending  blindly;  kidney  showing  reno-pericardial  and  external  openings. 

generally  about  the  point  of  development  represented  in  the 
figure,  these  cavities  gradually  fuse,  commencing  at  the  posterior 
end  of  the  gonad  and  progressing  anteriorly.  I  n  some  individual- 
a  slight  groove  may  persist  on  the  ventral  surlace  between  the 
halves  of  the  gonad  for  a  considerable  time,  and  in  a  lew  case-  .1 
distinct  cleft  at  the  anterior  end  of  tin-  gland  persists  until  the 
animal  is  half  grown. 

The  aorta  holds  the  normal  position  on  the  dorsal  surface 
of  the  gonad,  and  there  are  slight  evidences  that  a  portion  of 
the  blood  it  carries  makes  its  way  between  the  halves  of  the 


DEVELOPMENT    OF    GONAD    AND    GONODUCTS    IN    CHITON-.      <>7 

organ  as  in  the  solenogastres.  At  all  event-  thefe  are  no  signs 
-.1  di-tinct  branches  penetrating  the  gland  as  in  the  later  sta^ 

About  the  time  of  the  fusion  of  the  gonad  cavities  t when  the 
length  ol  the  body  is  approx'mately  I  mm.',  in  a  stage  slightly 
earlier  than  the  one  represented  in  Fig.  3,  each  gonoduct  ari-c- 
as  a  -lender  evagination  of  the  postero-latcral  walls  of  each  halt 
of  the  reproductive  gland.  These  grow  rapidly,  and  in  contact 
\\iih  the  pericardia!  wall  proceed  laterally  and  vcntrallv  until 
they  come  iii  contact  with  the  ectoderm  of  the  mantle  groove. 
In  the  formation  (A  the  outer  opening  the  ectoderm  cell-  appear 
inereK  to  -eparate:  if  an  ectodermic  diverticulum  i-  formed  it  i- 
evidently  \ery  short  and  transitory. 

In  later  -tages  the  proximal  ends  of  the  gonoduct-  -hilt  lor- 
\\ard  -li'Jitlv  ,  and  are  attached  to  the  dorsal  side  (l-'k.  -  <»t  the 
'^onad  close  to  tin-  mid  line.  During  this  process  ilu-ir  \\all- 
thii  ken,  and  at  the  height  of  the  breeding  season  there  an  -i^n- 
ecretor)  activ  ity  on  the  part  of  the  component  cell-  especially 
in  the  mi-hborho(Ml  of  the  reproductive  organ.  Th  of 

boih  of  these  species  are  held  in  the  mantle  cavity,  and  are  lo.i-dv 

1  it  niiii  1  t'  p'^ei  her  possibly  by  this  secretion  ol  the  o\  id  in  t . 

Ilie  i. nl\  niluT  observations  bearing  on  the  development  ol 
the  ^oimdin  i-  are  those  of  Plate1  who  has  made  the  claim  lh.it 
in  the  \oiiiii;  <»f  Acanthopleura  echinata,  15  mm.  in  length,  the 
gonad  i-  completely  separated  from  the  gonoduct s  that ,  a-  -lender 
divert ieiila.  are  connected  with  the  mantle  cavity  and  are  a< 
cordinglj  «'iodermii-.  drained  that  this  is  the  true  state  o| 
a  Hair-  in  .1 .  ft  hinatu  it  is  unprofitable  for  the  prc-ent  to  attempt 
to  correlate  the  two  t\  pes  of  development  when  only  three  -pe.  n  - 
..I  .hitoii-  have  been  examined  on  this  point.  Hov\ever,  it  i- 
inti-re-tini;  to  note  that  in  several  species  of  California  chiton-' 
three  millimeter-  or  less  in  length  the  gonad  and  it-  duct-  are 
attached  and  open  to  the  exterior.  In  some  species,  -nch  as 
h,  hncuhiton  iun«dalencnsis,  the  ducts  are  highly  glandular  and 
it  i-  1 1..— il.le,  though  it  appears  to  us  improbable,  that  this 
-landulai  -eciion  is  of  ectodermic  origin. 

i/         '  -iippl.  .1  (Fauna  Chilensis,  Vol.  i 

:  Il.atli,  Zoo/.  Jahrh.,  Bd.  21.  p.  -29. 


ASTEROPHILA,    A    \K\V   GENUS  OF   PARASITIC   GAS- 
TROPODS.1 

JOSEPHINE  RANDALL  AND  HAROLD  HEATH. 

During  the  dredging  operations  of  the  U.  S.  F.  C.  Str.  Alba- 
tross in  the  vicinty  of  Japan  (summer  of  1906)  four  specimens  of 
a  starfish,  Pedicellaster  sp.,  were  taken  that  had  been  parasi- 
tized by  a  new  genus  of  gastropods.  All  were  dredged  in  the 
sea  of  Japan  off  the  coast  of  Corea  at  depths  ranging  from  150 
fms.  (sta.  4,867)  to  163  fms.  (sta.  4,861).  In  one  host  three 
parasites  occurred,  while  only  one  was  present  in  each  of  the 
other  three,  but  in  any  event  they  occupied  the  ccelom  in 
the  arm,  and  were  attached  by  connective  tissue  strands  to  the 
body  wall  in  the  vicinity  of  the  ambulacral  ridge.  As  noted 
more  particularly  hereafter,  this  species  is  not  put  in  communi- 
cation with  the  exterior,  the  mouth  and  reproductive  openings 
communicating  with  the  body  cavity  of  the  host.  During  the 
time  that  the  brood  pouch  is  crowded  with  embryos  the 
pseudollapium  becomes  accordingly  considerably  distended  and 
tense,  resulting  in  the  inflation  and  consequent  thinning  of 
the  body  wall  of  the  host  along  the  dorsal  side  of  the  arm 
(PI.  II.,  Fig.  2).  Under  such  circumstances  it  is  possible  that 
the  body  wall  of  the  starfish  finally  ruptures,  causing  a  diminu- 
tion of  the  pressure  on  the  pseudopallium  which  therefore  dis- 
charges the  embryos  into  the  surrounding  medium.  After 
this  process  the  break  in  the  body  wall  is  probably  repaired, 
as  there  are  evidences  that  one  of  the  larger  individuals  has 
recently  discharged  its  brood  though  there  are  no  signs  of  a 
rent  in  the  starfish  arm. 

In  every  case  the  body  resembles  in  form  a  kidney  or  thick-set 
bean,  and  varies  in  size  from  i\\o  to  twenty  millimeters,  this  last 
extreme  being  due  to  some  extent  to  the  large  number  of  embryos 
and  the  fluid  in  which  they  float.  The  ovary  and  the  embryos 
themselves  arc-  light  yellow  in  color  due  to  the  presence  of  yolk, 

1  Published  by  permission  of  Hon.  G.  M.  Bowers,  Commissioner  <>t  I-'Nu-ries. 

98 


ASTEROPHILA.  99 

the  liver  is  of  a  light  brownish  shade  while  the  other  organs  are 
unpigmented  and  more  or  less  translucent,  especially  in  the  case 
"I  i IK  pseudopallium  that  in  life  is  so  thin  and  transparent  that 
tin-  torm  and  movements  of  the  larvae  may  be  readily  observed. 
A-  may  be  seen  in  PI.  I.,  Fig.  I,  there  are  two  openings  into  the 
body,  one  the  mouth,  corresponding  in  position  to  the  hilum  of 
,t  In  -.in  while  the  reproductive  opening  is  placed  laterally  up- 
ward- of  thirty  degrees. 

I  nun  \arious  features  of  its  organization  it  i-  readily  po-sible 
to  orient  this  animal  and  discover  the  axes  of  the  body.  As  in 
-ev.ral  other  parasitic  gastropods  the  body  is  surrounded  by  a 
pseudopallium  that  appears  to  be  a  development  <>f  the  Miout  or 
adjacent  regions  of  the  body.  Growing  upward  it  ha-  en\  eloped 
ilir  body  completely  save  at  one  point,  the  reprodncti\e  and 
excretory  pore.  Considering  the  body  proper,  tin-  foot  i-  seen 
\i-t  in  the  form  of  a  small  though  broad  wedgc--haped  fold 

I'l.  I  .  1  ig.  I.  /)  covered  with  epithelium  of  greater  thickne-- 
than  thai  surrounding  the  body  generally.  In  section-  I'l  11., 
Fig.  i  it  is  a  fairly  conspicuous  object  owing  to  its  affinity  i»i 
-tain-.  Again,  well-defined  pedal  ganglia  and  otocysts,  located 
in  «  lose  proximity  to  the  foot  and  cerebral  ganglia  on  the  oppo-ite 
-ide  of  the  digestive  tract,  demonstrate  the  fact  that  the  antero 
•«  iior  axi-  is  the  shorter  of  the  roughly  cllip-oid  bo  1\  ,  and 
that  the  transverse  axis  is  accordingly  the  longer. 

In  tin-  -|>ecies  the  degenerative  processes  ha\e  a<l\aiurd  to  a 
stage  \\liere  the  mantle  and  mantle  cavity  ha\e  largelv  di-- 
appeared,  and  vet.  though  rudimentary,  they  maintain  tin  ii 
i\  pi.  al  relations.  Since  the  mantle  fold  is  coniparaiixely  narrou 

I'l.  1..  Hg-.  i.  2,  t,')  the  cavity  is  accordingly  -hallow,  as  the 
mantle  i-  closely  applied  to  the  visceral  mas>;  ne\ . -rthele--  the 
epithelial  cells  bounding  the  cavity  are  not  only  higher  than  tin  >se 
cUeuhere  covering  the  body  but  they  stain  more  inten-elv  and 
are  ciliated.  <  Mi  the  left-hand  side  of  the  body  the  mantle  bonlei 
thicki-n-  con-iderably,  and  forms  a  projecting  riilge  th.it  c«»n- 
tinues  until  the  p.illial  cavity  itself  di-appeai>.  In  the  -malle-t 
specimen  the  mantle  and  cavity  are  relatively  larger  and  the 
mantle  fold  is  much  more  glandular,  the  gland  cell-  being  large 
and  conspicuous 


100  JOSEPHINE    RANDALL    AND    HAROLD    1 1  LATH. 

As  noted  in  a  preceding  paragraph,  the  mouth  opening  is 
borne  on  the  summit  of  a  low  papilla  in  the  mid  line.  In  entire 
specimens  it  is  further  distinguished  from  tin-  opening  into  the 
pseudopallium  by  occupying  the  center  of  a  whitish  area,  up- 
wards of  3  mm.  in  diameter  in  the  largest  specimens,  canard  by 
the  compact  feltwork  of  circular  and  radiating  muscles  enveloping 
what  probably  corresponds  to  the  buccal  tube.  In  the  immediate 
neighborhood  of  the  mouth  opening  the  canal  is  comparatively 
slender,  0.28  mm.  in  diameter  in  large  individuals,  and  is  pro- 
vided with  a  lining  of  simple  columnar  cells  whose  distal  portions 
contain  small  quantities  of  a  faintly  staining,  vacuolated  secre- 
tion. Behind  this  point  large  numbers  of  small,  irregularly  dis- 
tributed pyriform  gland  cells  appear  imbedded  in  the  muscular 
meshwork  surrounding  the  digestive  tract,  and  their  darkly 
staining  ductules  may  be  traced  to  intercellular  openings  in  the 
buccal  or  pharyngeal  epithelium,  whose  extent  is  increased  by  two 
symmetrically  placed  diverticula  with  short,  stubby  branches 
(PI.  I.,  Fig.  2)  extending  a  short  distance  into  the  surrounding 
muscle  sheath.  These  paired  glands  probably  correspond  to  the 
ventral  salivary  glands  of  other  molluscs  as  the  buccal  ganglia, 
connected  by  a  commissure,  are  located  in  their  vicinity. 

No  trace  of  a  radula  exists. 

The  buccal-pharyngeal  tube  with  its  enveloping  glands  and 
muscles,  is  relatively  short,  probably  not  over  I  mm.  in  length, 
but  it  spans  a  well  defined  head  cavity  (PI.  II.,  Fig.  3),  which  is  a 
portion  of  the  h?emocele  as  in  other  molluscs.  Curving  gently 
toward  the  ventral  side  of  the  body  the  tube  leaves  the  sinus, 
and  now  devoid  of  gland  cells  and  with  a  comparatively  thin 
sheath  of  longitudinal  and  circular  muscles,  it  passes  back  a 
short  distance  into  the  body  and  unites  with  the  main  portion  of 
the  digestive  tract  (/),  a  spacious  cavity,  lined  with  glandular 
epithelium,  occupying  most  of  the  visceral  mass  not  held  by  the 
gonad  and  its  duct. 

The  pericardial  cavity  (Tl.  I.,  Fig.  2)  is  situated  on  the  anterior 
surface-  of  the  visceral  mass  on  the  right  side.  The  contained 
heart  consists  of  a  single  auricle  and  ventricle,  both  of  large  size 
and  highly  muscular.  The  first-named  receives  the  blood  from  ,1 
broad  sinus,  which  on  one  hand  passes  from  the  liver  surface 


\>TEROPI1ILA.  IOI 

and  tin.-  neighborhood  of  the  accessory  reproductive  glands  in  the 
\  en  t  nil  part  of  the  visceral  mass,  and  by  means  of  another  smaller 
branch  t.ikes  the  blood  from  the  kidney.  The  aorta  is  very  -hort 
and  leads  directly  into  what  may  be  termed  the  head  cavity,  the 
large  space  surrounding  the  pharynx.  From  here  numerous 
1. ranch'-  .\tend  into  the  pseudopallium,  liver  and  between  the 
oxarian  lollieles.  Of  these  the  ones  pa--ing  through  the  pseudo- 
palliuni  probably  function  in  the  interchange  of  gases  as  there 
are  no  trai  es  of  ctenidia  or  branchia. 

One  nephridium  I'l.  I.,  Fig.  2,  n)  is  pre-ent  in  the  form  of  a 
iil\  «on i pressed  sac  covering  the  anterior  surface  of  the 
visceral  ma—  on  the  right-hand  side.  Its  inner  wall-  are  often 
pi<. \idrd  \\ith  lamelke  or  folds,  of  varying  >i/«-.  projecting  into 
ihe  rcniral  lumen.  The  cells  throughout  are  highly  vacuolated 
and  contain  \arying  quantities  of  some  granular  secretion  that 
in  >ome  locaiions  present  the  form  of  concrements.  \\ V  have 
lut  n  unable  to  definitely  locate  any  clearly  defined  ivno-peri- 
•  aidial  opening.  As  shown  in  1M.  11..  Fig.  6,  ;/,  the  kidnev 
in\e-i-  tin  dorsal  pericardia!  wall  but  there  are.  so  tar  as  we 
lia\e  seen,  no  modified  cells  indicating  a  nephro-tonie.  Tlie  ex- 
ternal pore  I'l.  I.,  Fig.  2,  e')  is  situated  on  the  anicrior  lace  of 
tin  visceral  mass  immediately  below  the  margin  of  the  maiulr. 

\\  liilc  the  ganglia  are  fairly  well  denned  and  distinct  tin-  nerve 
liber-  n  M  inble  so  closely  the  connective  tU-ue  and  IHIIM  le 
bundle-  tlii-on^li  \\hich  they  make  their  way  that  it  i-  \  ery  dif- 
in  nit  to  determine  their  course.  The  cerebral  iIM.  I.,  1  i:^.  I,D, 
appan-nil\  a— ociated  with  the  pleural,  are.  in  the  type  -periim-u, 
-ilnated  in  an  asymmetrical  position,  beinv;  placed  mi  the  ri-ht 
>ide  ot  the  pharynx.  From  this  nerve  ma--  connectives  extend, 
on  each  r-ide  o|  tlu-  phar\  nx,  to  the  pedal  uan^lia.  large,  clo-ely 
appn---i-d  ^ioup>  of  ner\'e  cells  placed  -\ninietricall\  \\ith  refer- 
ence to  the  mid  ventral  line.  The  cerebral  uaujia  likewi-e 
originate  buci\il  connectives  that,  extending  along  the  phar\u\. 
unite  \\ith  ganglia  imbedded  in  the  >a!i\ar\  gland-  on  the  dor-al 
and  \entral  -ide.  The  buccal  ganglia  are  further  united  by  t\\o 
c.pinmi  — ure-  that  lorm  a  collar  about  the  pharynx.  In  the 
neighborhood  i.|'  tl,.  opening  of  the  repn>diicti\  e  -\-tem  into 
the  pseudopallium  there  is  a  large  ganglion.  probabK  the  visceralj 


IO2  JOSEPHINE    RANDALL   AND    HAROLD    HEATH. 

that  gives  off  a  strong  nerve  which  may  be  traced  a  short  distance 
posteriorly,  and  in  the  opposite  direction  a  single  connective 
leads  from  it  to  the  cerebro-pleural  ganglia,  as  indicated  in  PI.  I., 
Fig.  i,  v.  At  various  points  throughout  the  body  it  is  possible 
to  discover  nerve  bundles,  but  in  every  case  it  has  been  impossible 
to  determine  their  origin. 

In  the  three  animals  examined  there  is  no  sign  of  a  testis, 
though  the  seminal  receptacle  of  one-  individual  contains  a  con- 
siderable number  of  spermatozoa.  These  last  named  elements 
possess  almost  spherical  heads  measuring  approximately  0.004 
mm.  in  diameter.  Nuclei  of  somewhat  similar  appearance  may 
be  detected  here  and  there  in  the  follicles  of  the  reproductive 
gland,  but  their  close  resemblance  to  those  of  the  connective 
tissue  cells  renders  the  determination  uncertain.  If  self-fertiliza- 
tion does  not  occur  in  this  species  it  is  difficult  to  understand  the 
method  of  sperm  transfer  especially  in  those  examples  where  but 
one  parasite  occurs  in  the  host  which  completely  envelops  it. 

The  ovary,  occupying  fully  half  of  the  visceral  mass,  consists  of 
a  large  number  of  follicles  united  directly  or  indirectly  with  the 
duct  leading  to  the  exterior.  In  a  mature  condition  the  central 
portions  of  each  follicle  are  packed  with  fully  developed  ova, 
while  numerous  cells  in  the  earlier  stages  of  formation  bound  the 
periphery.  About  the  center  of  the  visceral  mass  the  common 
chamber,  communicating  with  the  ovarian  follicles,  narrows 
anteriorly  and  the  short  resulting  tube,  after  a  somewhat  twisted 
course,  unites  (elliptical  stippled  outline,  PI.  I.,  Fig.  2)  with  the 
definite  gonoduct  leading  to  the  exterior.  This  last-named  canal 
consists  of  three  divisions  corresponding  to  the  albumen  and 
mucous  glands  and  the  seminal  receptacle  in  oilier  species  of 
gastropods.  The  canal  from  the  ovary  unites  with  the  albumen 
gland  which  extends  posteriorly  as  a  pouch  of  considerable  size. 
Its  walls  arc  relatively  thick,  and  are  fashioned  into  a  few  promi- 
nent folds,  consisting  of  relatively  slender  cells,  whose  vacuolated 
secretion  stains  lightly  with  Delafield's  hsematoxylin.  Slightly 
anterior  to  the  oviduct  connection,  a  cone-shaped  seminal 
receptacle  (PI.  I.,  Fig.  2,  r)  is  attached  to  the  albumen  secreting 
section.  Its  epithelial  lining  is  developed  into  a  large  number  of 
folds  between  which  there  are  quantities  of  spermatozoa,  that 


ASTKROPH1LA.  IO3 

likewi-e  occupy  the  main  lumen  and  even  extend  in  -mall  quan- 
tities some  distance  into  the  albunu-n  gland.  Anterior  to  the 
-eminal  receptacle  the  walls  of  the  canal  change  abruptly,  be- 
coming thicker  and  the  secretion  stain-  -o  inten-cly  that  the 
cell  outline-  and  nuclei  become  almost  completely  <>l>literau-d 
Thi  <>f  affairs  exists  between  the  seminal  receptacle  and  a 

poim  -lijnly  posterior  to  the  external  repn>ducti\  e  opeiiin^. 
Anterior  to  this  region  the  duct  presents  the  form  of  a  roughly 
i  oni.  .il  sa<  extending  to  a  point  opposite  the  foot.  The  wall-  of 
thi-  |ioin  h  .ire  similar  to  the  darkly  staining  one-  ju-t  described 
save  that  the  secretion  is  more  vacuolatcd  and  accordingly  less 
derpK  -i. lined.  The  duct  leading  from  this  mueou>  -envting, 
in.iin  i  anal  to  the  exterior  is  relatively  short,  thin-walled  and 
passes  into  the  furrow  at  the  right  side  of  the  body  formed  by 
tin  union  oi  the  visceral  mass  with  the  pseudopallium. 

In    t\\o   -pei  imens  whose  pseudopallium  contained   fully   500 
rmbr\o-  the  ovary  held  an  e(|iial  number  of  ova  in  a  fully  de- 

\elop.d  ( lition.      Hence  it  is  probable  that  during  adult  life 

t In    brood  pouch  is  empty  for  short  periods  only. 

The  ;^i -nus  may  be  defined  as  follows: 

.  1  'ild  new  genus.      Body  globular,  2-2O  mm.  in  diameter, 

c.  unpleieK  enveloped  in  the  pseudopallium.  Foot  and  mantle 
nidimentarx  .  liuccal-pharyiigcal  tube,  with  salivary  gland-, 
opens  into  o  unbilled  stomach  and  digestive  gland  that  otheru  i-e 
do  not  open  to  the  exterior.  Xo  radula.  Albumen  and  mucou- 
gland-  on  reproductive  canal  highly  developed,  and  seminal 
i-.epiacle  prominent.  1'arasitic  in  starfish  Pedicellaster  sp., 
>ea  of  Japan.  Type  of  genus  A.japonica. 

.1 .  ju/'onii'ti  new  species.     \Yith  characters  of  the  genus. 


IO4  JOSEPHINE    RANDALL   AND    HAROLD    IIFATH. 


EXPLANATION  OF  FIGURES. 
PLATE  I. 

FIG.  i.  Diagrammatic  view  of  .4 slerophilajaponica,  left  side,  with  the  greater 
portion  of  the  pseudopallium  removed,  a,  albumen  gland;  c,  cerebral  ganglion; 
/,  papilla-like  foot;  g,  mantle  fold;  /,  digestive  gland  or  liver;  m,  mucous  gland; 
oy,  ovary;  p,  buccal  tube  and  pharynx  with  salivary  glands,  buccal  ganglia,  con- 
nectives and  commissures;  r,  seminal  receptacle  under  which  is  dotted  outline  of 
duct  from  ovary;  ur,  urino-genital  opening;  i>,  visceral  ganglion. 

FIG.  2.  Diagram  of  anterior  surface,  e,  e',  openings  of  reproductive  and  ex- 
cretory systems  into  pseudopallial  space;  g,  mantle  fold,  the  depth  of  the  mantle 
cavity  indicated  by  broken  line;  h,  heart;  n,  kidney;  />,  buccal  tube;  r,  seminal 
receptacle. 


BIOLOGICAL  BUUETIN     VOL 


PLATE   I. 


RANDALL   AND  HEATH 


106  JOSEPHINE    RANDALL    AND    II  \ROI.D    Ill-Alll. 


PLATE  II. 

FK;.  i.  Anterior  view  of  Aslerophila  japonica  with  pscudopallium  partially 
removed. 

FIG.  2.     Arm  of  starfish  containing  parasite. 

FIG.  3.  Section  through  pharyngeal  tube,  showing  salivary  glands,  buccal 
ganglia  and  surrounding  head  sinus. 

FIG.  4.     Section  through  foot  and  visceral  mass;  along  line  w  of  PI.  I.,  Fig.  i. 

FIG.  5.  Section  along  line  u,  PI.  I.,  Fig.  i;  the  junction  of  the  oesophagus  and 
stomach-intestine  marked  by  an  arrow. 

FIG.  6.     Section  along  line  /,  PI.  I.,  Fig.  i. 

FIG.  7.     Same  along  line  s,  PI.  I.,  Fig.   i. 

FIG.  8.     Diagram  illustrating  growth  of  pseudopallium  in  Aslerophila. 

FIG.  9.     Same,  Ctenosculiim  hawaiiense. 


BIOLOGICAL  BULLETIN,    VOL     XXII. 


•'    «'•  -'•  ^'J 

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RANDALL  AND   HEATH. 


A    '    \SE    OF    YOLK    FORMATION     NOT    rnNNKiTED 
WITH     IHI-:    PRODUCTION    <  >F    OVA. 

OSCAR   RIDDLE. 

Tli.-  -i-crclion  or  production  of  true  yolk  in  situati.  >n-  other 
th. in  in  ova  or  in  the  follicular  cells  which  surround  ova  is  n»t 
kiniuii  so  far  as  I  am  aware.  'Nurse"  or  "yolk"  cell-  have  of 
course  long  been  known  to  exist  in  several  group-  .'I  animal-; 
the-e.  however,  are  evidently  the  equivalents  of  follicular  cell- 
IT  ol  ova.  It  is  therefore  of  some  interest  to  record  tin-  lindin^ 
ol  i  rue  \olk  in  spaces  within  the  connective  tissues  which  lie 
i  AN  TH. illy  to  the  follicular  membrane  of  capsule-  \\hirh  had 
pn-\  ion-Iv  liberated  ova. 

I  hese  observations  were  made  on  the  ovaries  of  the  r»innion 
fowl  during  the  mid -summer  season.     During  Jul)  and  .\umi-t  ot 
i  In-    ]  in -i  nt    summer   the   writer   had   occasion   to  examine    tin 
ovaries  »t  more  than  one  hundred  full-grown  hens.     Aimm^  ilu--e 
iln  i.  \\  en-  at  least  six  or  eight  ovaries  which  showed  unmi-takablv 
iln-  peculiar  accumulation  and  jJacement  of  yolk  \\hich  i-  In  M 
dr-t  i  il  ii -d. 

In  oidi-r  to  be  Mire  that  one  is  really  di-.ilin-  \\iih  "extra- 
o\  ul.ti  "  .md  "extra-follicular"  formation  of  yolk,  and  not  nuT.-K 
\\iili  ,i  mas(|ue  of  its  usual  source,  it  was  i  r\  \«  deiermine 

three  things  concerning  the  capsules  within  \\hich  the  \.>lk  in 
(|iif-iiou  \\as  found:  (i)  That  an  egg  had  been  -mvly  de\-i-lope<l 
ami  liberated  from  this  capsule.  (2)  That  tin-  >pa<v  in  \\hich 
tin-  \  oik  was  found  is  quite  separate  and  remo\  ( -d  troin  tin-  space 
lornuTK'  occupied  by  the  ovum,  and  like\\i~.-  n  nio\td  from  tin- 
lollicnlar  cells  which  surrounded  the  o\nin.  ;  I  li.u  ih« 
cumulated  substance  is  true  yolk.  I  belit-\e  that  tin-  material 
1  lia\  e  i-xamined  has  enabled  me  satisfactorily  to  dcii-nnine  each 

ol    these   points. 

The  evidence  that  the  yolk-containing  capsiilc->  in  .|iu-.tion 
had  prc-vioii-lv  borne  and  liberated  ova  rests  partially  upon  the 
finding  of  capMilc^  showing  all  the  intermediate  stages  between 

107 


IO8  OSCAR    K1ODI  1  . 

the  recently  broken  capsules  and  the  large.  Hubby,  often  asym- 
metrical, yolk-containing  ones.  Some  of  thor  laitrr  capsules 
might  be  mistaken  for  resorbed  ova,  since  they  too  have  a  closed 
stigma;  that  is,  the  slit  or  splitting  which  occurs  in  the  capsule 
at  the  time  of  ovulation.  and  which  allows  the  escape  of  tin-  <  >vum, 
later  heals  together  and  the  cavity  of  the  follicle  is  once  more 
completely  sealed.  The  chances  for  such  confusion  are  further 
increased  by  the  fact  that  this  central  chamber  may  also  oc- 
casionally re-accumulate  yolk. 

It  is  possible  nevertheless  in  favorable  material  to  be  quite 
sure  that  the  stigma  has  been  broken  and  reunited — a  thickened, 
accentuated,  and  often  more  or  less  ragged  point  of  reunion 
indicating  this.  Furthermore,  a  series  of  follicles  in  the  same 
ovary,  showing  the  most  recent  ones  still  broken  open,  often 
decides  the  matter  at  once  with  certainty.  The  capsule  from 
which  a  sample  of  yolk  for  analysis  was  taken  was  one  of  such  a 
scries.  In  this  case  there  were  nine  yolk-containing  capsules  in 
various  stages  of  extra-ovular  yolk-production;  and  in  addition, 
one  other — the  newest  follicle — plainly  recognized  by  its  whole 
appearance  as  a  recently  emptied  one.  This  follicle,  however, 
showed  the  once  broken  lips  of  the  stigma  now  nearly  completely 
grown  together,  but  with  its  inner  cavity  as  dean  and  free  from 
yolk  as  at  the  moment  of  ovulation.  It  is  certain  that  the  fol- 
licles of  this  ovary  had  liberated  ova,  and  that  instead  of  degen- 
erating thereafter  these  capsules  quickly  closed  tin-  breaches 
formed  in  extruding  the  ova,  and  began  the  production  of  yolk 
in  their  external  walls. 

It  is  easy  to  demonstrate  that  the  yolk-filled  spaces  bulging 
from  the  sides  of  the  capsules  have  no  open  connection  with  the 
central  cavity  of  the  capsule;  that  is  to  say,  these  spaces  are  not 
connected  with  the  former  seat  of  yolk  formation.  Several  times 
I  have  made  a  slit  in  the  scar  or  stigma  and,  finding  the  interior 
clean  and  free  from  yolk,  have  tried  by  squeezing  the  various 
bags  of  yolk  lying  in  the  external  walls  of  the  capsule  to  make 
their  yolk  How  into  the  central  cavity.  In  no  instance  have  1 
succeeded  in  thus  finding  any  connection  whatever  between  thr-.r 
new  yolk-containing  cavities  and  the  old  cavity  lormerly  oc- 
cupied by  the  egg.  On  the  contrary,  careful  di»rnioii>  of 


MILK.  FORMATION.  109 

these  capsules  show  that  the  two  spaces  are  always  separated  by  a 
rather  thick  wall;  certainly  much  thicker  than  that  which 
separates  the  new  yolk  space  from  the  exterior.  This  latter  wall, 
in  fact,  i-  usually  very  thin.  It  consists,  however,  of  an  extremely 
thin  connective  tissue  layer  in  addition  to  the  ovarial  epithelium, 
liv  <  .ireful  handling  the  epithelium  can  be  -tripped  <>tY  and  the 
thin  la\«T  .-iiclosing  the  yolk  space  left  intact. 

"Ilit-  \ery  external  position  of  the  yolk  -pa.  es  of  \\hich  there 
ina\  !  .-r.il  in  a  single  capsule — make-  it  evident  that 

in -in-  1. 1  i  In  cells  of  the  old  follicular  membrane  an-  cn-a-ed  in 
tin-  ]>n  id  in  lion  of  yolk  in  this  new  and  unusual  -ite.  The 
|ini<lm  lion  of  this  yolk  i>  necessarily  accomplished  by  the  cells 
which  form  the  external  theca — a  tissue  from  the  former  ovarian 
stroma,  \\lii-h  in  the  late  growth  stages  of  the  cap-ule  of  large 
Mies  a  very  thick,  firm,  essentially  connect  i\e  ti— ue 
la\er  i  -in  lii-ing  possibly  some  scattered  derivative-  of  the  m-r- 
niin.il  epithelium  -whose  cells  normally  take  no  part  in  yolk 
toriiiaiii.ii. 

|u-t  \\liai  it. is  that  transforms  these  non-yolk-producing  cells 
into  »ell-  ai  ti\ely  engaged  in  yolk  production,  ii  \\mild  lie  nm-i 
inii  n  sting  to  know.  While  confessing  very  complete  ignorance 
as  to  thi-  i  ause,  it  seems  worth  while  to  note  that  the  cells  ichich 
here  hike  n(>  n  new  function  do  so  at  the  time  v//r;/  the  "normal" 
tin  *  -  do  is  to  degenerate  and  be  •  '/. 

In  thi-  connection  it  should  be  stated  that  the  true  lollicular 
«e!l-  iln»-e  \\hich  ha\'e  previously  been  engaged  in  pa--iir^  .-n 
tin-  .  Dii-tiiueiit>  of  yolk  to  the  egg — are  apparently  the  lea-t 
liable  i.t  anv  of  the  capsular  cells  to  take  part  in  any  later  yolk 
production.  <  >nl\  occasionally  in  a  group  of  cap-ule-,  each  of 
\\hich  mav  In-  producing  yolk  at  one  or  more  point-  externally, 
\\ill  one  tin, |  th.it  the  follicular  cells  ha\«-  continued  or  rather 
ha\e  recommenced— to  produce  yolk.  What  I  ha\>-  ob-er\ed 
\\ould  indicate  that  these  follicle  cells  iu-\er  in  an\  case  become 

acti\e  until  after  \  oik  production  ha-  been  initiated  ill  the  more 
exiernal  la\er-;  but  of  this  latter  point  1  ,1111  inn  certain. 

That  the  \ello\\Uh  tluid  enclo-ed  in  the-e  \olk  -|iac.--  i-  true 
\olk  i- indicati'd  b\  its  microscopic  appearance.  The  question 
i-  |io-iti\,-l\  .uul  affirmatively  answered  by  the  chemical  anal 


no 


'  >S<    \K 


of  a  sample.  1.605  grams  of  such  yolk  were  rolltvtrd  from  a 
single  one  of  the  new  yolk  spaces;  this  was  not  all,  but  nearly  all 
of  the  contents  of  the  cavity.  In  order  to  slum  how  closely 
its  chemical  composition  agrees  with  that  of  other  tOnn<  of  true 
yolk,  I  have  added  to  the  table  the  numbers  resulting  from  the 
analysis  of  four  such  samples  of  yolk.  Reference  to  the  table 
readily  shows  the  essential  similarity  of  all  these  substances ;  and 
likewise  a  point  or  two  of  notable  difference. 

In  Per.  Cent,  of  Solids. 


Analysis  ol  : 

Leci- 
thin. 

Protein. 

Neutral 
Fat. 

Total 
Ash. 

anic 
Extractives. 

H,<>. 

Extra-follicular  yolk  . 

IQ  O^ 

26.21 

4S  3Q 

6.6l 

2.6s 

74.22 

Central    "yolk    body"    from    in- 
cubated hen's  egg  

10.68 

28.87 

46.  os 

7..4O 

2.OO 

37.13 

Egg  yolk,  Jungle  fowl  

JQ.OO 

7.O.47 

46.74 

1.7,0 

I.  SO 

48.70 

Contents  of  yolk-sac;  2r  clays  in- 
cubation 

17.62 

-17.24 

47.76 

1.16 

I.  TO 

S6.S2 

Resorbecl  ovum  . 

i<;.7o 

7C.I8 

A2.2S 

1.  71 

•O  ' 

• 

67.20 

It  is  true  that  I  have  selected  for  this  comparison  analyses 
which  most  closely  agree  with  the  analysis  of  the  "cxtra-fol- 
licular"  yolk.  The  high  water  content  of  the  latter  is  of  no  con- 
sequence; an  analysis  of  "white"  yolk  from  the  hen  having 
yielded  more  than  80. 0  per  cent,  of  this  constituent . 

The  high  ash  content,  and  very  low  protein  content,  do  indi- 
cate however  a  species  of  yolk  not  in  all  respects  like  that  pro- 
duced by  the  follicular  cell  and  the  ovum.  In  these  two  respects 
this  yolk  stands  as  a  rather  bold  extreme  in  a  long  series  of 
analyses  of  normal  yolk.  It  can  be  said  therefore  that  though 
this  substance  is  certainly  "yolk,"  its  peculiar  origin  stamps  its 
chemical  composition  with  a  specificity  of  its  own. 

The  foregoing  recital  of  the  facts  is  perhaps  hardly  sufficient 
to  uncover  at  once  to  every  reader  one  of  the  points  of  interest 
in  these  findings;  at  any  rate  it  is  a  point  of  interest  to  the  writer. 
I  refer  to  the  fact  that  in  all  of  the  hitherto  known  cases  of 
yolk  formation  the  whole  process  of  yolk  building  and  stoi  gi 
appears  so  glaringly  and  profoundly  ideological.  The  ovum  pre- 
pares and  stores  food  for  an  embryo  that  is  yet  to  form;  a 
follicular  cell  passes  on  this  rich  material  only  to  an  ovum  \\liich 
in  turn  accumulates  for  a  promised  organism  that  will  arise  and 


FORMATION-.  in 

require  the  store;  ovum  and  ten  thousand  follicular  cells  unite' 
i"  pn-p.ire  and  to  hoard  a  pabulum  for  an  organism  whose 
father  exists  as  yet  only  in  prophecy  and  in  fortune;  a  "nur-e" 
ci-11  arises  in  a  distant  part,  migrate-  with  its  supplie-  and  un- 
erringly delivers  all  to  the  egg — whose  prospective  accomplish- 
ment only  ran  use  or  require  them;  or,  again,  33  in  some  hydroids, 
several  adjacent  ova  laboriously  produce  a  -olden  store  which 

•  tlier  with  their  own  existence  they  place  -acriticially  upon  the 
abar  of  po-terity — giving  all  to  a  more  opulent  neighbor,  who 
through  the  combined  accumulations  of  main  gatherer-  ran  the 
more  adequately  and  assuredly  provide  for  the  beginning  of  an 
indi\  idual  that  is  to  be. 

Nor  i-  Mich  apparent  teleology  ab-ent  from  the\«r\  chem- 
ical composition  of  the  material  that  is  stored.  The  developing 

mi-m  requires  above  all  else  a  store  and   -omve  ,,i   , -m -i 
ami  one  notes  that  yolk — the  material  actually  stored     i-  richer 
in  lecithin  and    fat   than  is  any  other   product  n|    the  bo<l\  ;  and 
fuit  her  that   t  In  -e  constituents  are  the  one-  \\  hii  h  i  arrv  tar  more 
ener-\   pri  unit  of  weight  or  volume  than  do  an\  other-. 

\\lien,  ho \\ever,  one  turns  to  the  sort  of  \..lk  formation  <!<•- 
-..i  i  beil  in  this  paper, — yolk  formation  which  be-in-  in  -ubdiied 
and  a  1 1 .1  i,  f.illii les,  among  cells  largely  "soinati/ed  "  and  doomed 
in  certain  de-i  iteration;  when  one  con-ider-  the  utter  blimlne-- 
in\ol\ed  in  these  ill-conditioned  cell.-  phr  into  a  nio-i 

active  ]>roductioii  of  excessively  rich  loud-,  (,nly  to  cast  them 
into  the  formless  spaces  of  these  spent  cap-ule-.  one  ran  n-ali/e 
that  the  pr. ..  •  ss  of  yolk  building  actually  can  be  as  grotesquely 
ab-urd  and  inappropriate  as  it  has  else\\  here  -eemed  replete  \\  ith 

ill-i-tellt    teleoliigy. 

ORATORY    OF    EXPERIMENTAL    TllKRAPEl'l  l>  -. 

I  i!  u-irv  OF  CHICAGO. 

i<.ii. 


Vol.  XXII.  February,  1912.  No. 


BIOLOGICAL  BULLETIN 


Till.  c-MoTIC  AND  SURFACE  TENSION*  PHI  \OM1.\.\ 
OF    LIVING  ELEMENTS  AND  THEIR  PHYSIO- 
LOGICAL SIGNIFICANCE.1 

J.  F.  McCLENDON. 

CONTENTS. 

I.    I  lit:     i;   tion iij 

1 1.  (  Kinotic  Phenomena  in  Plants 120 

111  Phenomena u  7 

i      In  plants I  J7 

le  and  Nerve \  _•<, 

i-l   Movement i  ;  I 

;>aKation  of  the  Bio-electric  Changes 136 

1\  -is 139 

\  |>crties  of  the  Blood  Corpuscles 142 

VI      V  -i  and  Secretion 

i  •  >n  through  the  Gut i 

Relation  of  Aquatic  Animals  to  the  Medium i 

;i  of  Lymph  and  Tissue  Juice 152 

u 

VII.   (Yll  I>i\i-ion..  : 

PREFACE. 

ThU  paper  formed  the  basis  for  two  lectures  -ivm  In-fore  the 
class  in  ph\-iology  at  Woods  Hole,  July  7  and  8.  i«)ii,  although 
I'uiii-  in  liinii.il  lime,  some  parts  were  omitu-d.  Since  then 
then-  ha^  appeared  a  second  edition  of  Hober's  "  Phy>ik.ili-clu- 
Clu-inir  clt-r  /rile  und  Gewebe,"  which  n-\ir\\>  much  of  tin- 
littT.iiurt  cmi-itlered  in  this  paper.  H<>\\c\cr,  owing  to  an 
t-ntin-K-  (lillncnt  mode  of  presentation,  it  is  hoped  that  the 
pre-ent  treat meiu  of  the  subject  might  be  helpful  to  many 

•ieral  readers,  some  of  whom  would  not  read  HoU-r's  book. 

1  l-iom   tlii-  1-  '.ml. i  \ .  'i.'^ical  Laboratory  of  Cornell  l"nivfrsit>-   Mnlical  College, 
.t\-. 


114  J-    F-    MCCLENDON. 

1  am  indebted  to  several  persons  for  suggestions,  especially 
to  Dr.  Ralph  Lillie1  and  Professor  B.  M.  Duggar. 

I.  INTRODUCTION. 

The  object  of  this  paper  is  to  bring  the  "vital"  phenomena, 
as  far  as  possible,  within  the  scope  of  physics  and  chemistry,  and 
not  to  elucidate  physical  and  chemical  processes.  It  should 
therefore  be  borne  in  mind  that  the  osmotic  phenomena  of 
"dead"  systems  are  not  all  satisfactorily  explained. 

The  Vant  Hoff-Arrhenius  theory  of  osmosis  concerns  itself 
with  the  number  of  particles,  molecules  and  ions,  in  solution, 
and  is  applicable  to  dilute  solutions,  in  which  the  total  volume  of 
the  dissolved  particles  is  negligible.  However,  in  more  con- 
centrated solutions,  the  volume  of  the  dissolved  particles  is  of 
the  same  importance  as  the  volume  of  the  molecules  in  gases,  as 
expressed  in  Van  der  Waal's  equation.  Also  the  dissolved 
particles  bind  molecules  of  the  solvent  and  so  reduce  the  volume 
of  the  free  solvent. 

That  the  molecules  and  ions  of  a  dissolved  substance  bind 
some  molecules  of  the  solvent,  follows  from  the  work  of  Jones 
and  his  collaborators.2  Compare  also  the  work  of  Pickering.3 
Jones  concludes  that  the  larger  the  number  of  molecules  of  water 
of  crystallization,  the  greater  the  hydrating  power  of  a  substance 
in  aqueous  solution.  The  number  of  molecules  of  water  bound 
by  one  molecule  of  the  solute  usually  increases  with  dilution  up 
to  a  certain  point  (the  boundary  between  concentrated  and 
dilute  solutions,  beyond  which  there  is  no  heat  of  dilution). 
The  bond  between  ions  and  the  solvent  is  also  indicated  by  the 
phenomenon  known  as  "electrical  transference."  It  an  elec- 
trolyte and  a  non-electrolyte  be  dissolved  in  water  and  an 
electric  current  passed  through  the  solution,  water  will  be  carried 
along  with  the  ions  to  the  electrodes. 

With  these  corrections,  the  Vant  Hoff-Arrhenius  theory 
accounts  for  osmotic  pressure,  but  does  not  show  why  main 
substances  exert  no  osmotic  pressure,  in  other  words,  why  no 

JCf.  this  journal,  1909.  XVII.,  188. 

2  "Hydrates  in  Aqueous  Solution,"  Pub.  No.  8,  Carnegie  Ins.  Wash.,  1907. 
'  Whetam,  "The  Theory  of  Solution,"  1902,  Cambridge,  p.  170. 


TENSION    PHENOMENA    OF    LIVING    ELEMENTS.  115 

membranes  have  been  found  that  are  impermeable  to  them. 
(Ki-rt'.n  -upposed  that  the  substance,  in  order  to  diffuse,  must 
di--ol\e  in  the  membrane.  Kahlenberg  and  others  consider  a 
solution  as  a  chemical  combination  between  solute  and  solvent, 
and  osmosis  as  a  series  of  chemical  reactions  between  the  mem- 
brane and  the  two  solutions,  continuing  until  equilibrium  is 
established.  The  essential  points  in  the  theory  arc:  that  the 
membrane  is  not  a  molecule  sieve,  but  a  substance  with  specific 
properties  and  the  chemical  characters  of  the  membrane  and  of 
ilie  di—  ol\ed  substances  affect  osmosis. 

Wiflard  <  iibbs  found  that  the  more  a  solute  lower-,  the  surface 
tension  <-t  a  -olution,  the  more  it  tends  to  pass  out  of  the  solution, 
mosis,  or  if  this  is  prevented,  to  collect  at  the  Mirt'ac.-  of 
the  solution.  This  law  has  been  extensively  investigated  ami 
eoiiiirmed  b\  I.  Truube.  For  instance,  in  general,  lipoid-Milnble 
Mib-taiM  c-  lower  the  surf;tcc  tension  of  water  and  tend  to  diiiu-e 
on i  ol  ii.  \\liereas  electrolytes  slightly  raise  the  surfaee  i en-ion 
of  water  and  attract  water  from  the  adjacent  pha-e.  <  Kmo-i- 
ma\  01  <  ur  in  opposite  directions  simultaneously.  (ill>l>-  and 
I'raiibe  -tate  ih.it  the  greatest  osmotic  flow  is  from  the  -olm  i,  ,\\ 
of  louei  -in  face  tension  to  that  of  the  higher,  but  tin-  i-  not 
v.«-ner.ill\  a<iepted.  ( )smosis  consists  of  two  di-i in<  i  pr« 
Irom  our  -oluiion  to  the  membrane,  and  from  the  membrane  to 

the  -eeoinl   -ollltioil. 

In  case  the  membrane  consists  of  two  or  more  ehenhrallv 
dill'etviit  membranes  placed  one  on  another,  osmosis  con-i-t-  of  a 
series  of  Steps;  and  Hamburger1  made  double  membrane-,  through 
\\hieh  en  tain  substances  diffuse  more  rapidly  in  one  direction 
than  in  the  other. 

Tranbe  calls  the  bond  between  solute  and  solvent  the  "attrac- 
tion pie— me."  In  general,  attraction  pre--nre  of  ions  incrc.i 
\\ith  valence.  The  less  the  attraction  pressure  of  the  solute,  the 
more  it  louers  the  surface  tension  and  tends  to  pass  out  of  the 
solution.  The  presence  of  one  solute  louers  tin-  attraction 
pressure  "t  another  in  the  same  solution,  and  the  greater  the 
attraction  prc-.-nrc  of  a  solute  the  more  it  lowers  that  of  another. 
\\  e  mi-lit  e\pre>-  this  idea  by  saying  that  one  substance  takes 

.  7.cit.,  lynS.  XL,  443. 


Il6  J.    F.    MCCLEXDOX. 

part  of  the  solvent  away  from  the  second  and  increases  the  con- 
centration of  the  second  substance.  This  may  explain  the  effect 
of  a  harmless  substance  in  increasing  the  toxicity  of  a  poison. 
Schnerlcn1  observed  that  a  solution  of  phenol  below  the  threshold 
of  toxicity  for  certain  bacteria  is  rendered  toxic  by  adding  NaCl. 
Stockard  showed  that  the  toxicity  of  pure  solutions  of  salts  on  fish 
eggs  is  increased  by  the  addition  of  sugar,  although  the  total 
osmotic  pressure  of  the  mixture  is  less  than  that  of  the  normal 
medium.2 

Just  as  Traube's  precipitation  membranes  are  absolutely 
impermeable  to  certain  substances,  so  do  living  cells  show  this 
selective  permeability.  For  instance,  the  vacuole  fluid  or  cell 
sap  of  certain  plant  cells  contains  colored  substances  which  do 
not  diffuse  into  the  protoplasm  surrounding  the  vacuoles.  If  a 
cell  be  placed  in  a  solution  of  the  pigment,  the  protoplasm 
remains  colorless.  If  the  protoplasm  be  squeezed  out  of  the 
cell  into  a  solution  of  the  pigment,  it  does  not  invariably  become 
stained.  However,  if  the  cell  is  injured  in  certain  ways,  or 
dies  from  any  cause,  the  pigment  diffuses  out  of  the  vacuoles 
into  the  protoplasm  and  thence  into  the  surrounding  medium. 
\Ye  might  conclude  that  the  protoplasm  in  general  is  imperme- 
able to  the  color,  but  at  death  it  becomes  permeable.  On  the 
other  hand,  Pfeffer3  gives  evidence  for  the  existence  of  a  mechani- 
cal membrane  on  the  surface  of  the  cell  and  lining  the  vacuoles. 
De  Vries4  placed  cells  into  10  per  cent.  KNOa  solution  colored 
with  eosin.  The  plasma  membrane  and  granular  plasm  died 
and  stained  long  before  any  dye  entered  the  vacuoles.  How- 
ever, the  granular  plasm  may  have  absorbed  all  the  dye,  thus 
preventing  its  entrance  for  some  time,  without  the  necessity  of 
any  resistance  of  the  vacuole  membrane.  Since  protoplasm  may 
be  squeezed  out  in  the  form  of  droplets  and  still  appears  to  be 
surrounded  by  membranes,  Pfeffer  concluded  that  the  membrane 
was  formed  by  the  contact  of  the  protoplasm  with  the  medium 

i  Arch.  exp.  Path..  1X96,  XXXVII..  84. 

-  However  the.  NaCl  in  Schnerlen's  and  sugar  in  Stockard's  experiment  may 
have  increased  the  permeability  to  the  toxic  substances,  as  discussed  in  later 
chapters. 

"  I'Han/cnphysiologie." 

4Jahrh.  wiss.  Bol..  1885,  XVI.,  465. 


TENSION    PHENOMENA    OF    LIVING    ELEMENTS.  ll"J 

or  with  cell  sap.  He  supposed  these  membranes  to  be  the  semi- 
permeable  parts  of  the  cell,  and  that  they  became  altered  at 
death.  Pfeffcr  called  this  membrane  on  the  cell  surface  the 
"plasma  membrane." 

Whereas  the  nuclear  membrane  and  certain  vacuole  mem- 
branes are  semipermeable,  these  are  lacking  in  crythrocytes, 
which  arc  then-fore  good  objects  for  testing  the  question  whether 
the  protoplasm  in  general,  or  merely  its  surface,  is  semipermeal  >le. 
Hober1  by  two  very  ingenious  but  complicated  methods,  one 
based  on  dielectric  capacity,  determined  the  electric  conduc- 
ti\it\  <>f  the  interior  of  the  erythrocyte  without  rupture  of  the 
pl.i-nia  membrane.  Since  the  conductivity  of  the  interior 
'about  that  of  a  .2  per  cent.  XaCl  solution)  was  found  to  be 
ni.mv  times  greater  than  that  of  the  erythrocyte  as  a  whole,  the 
membrane  must  be  relatively  impermeable  to  ions.  There  is 
much  other,  but  less  direct,  evidence  that  the  semipermeability 
resides  in  the  plasma  membrane,  namely:  the  rapidity  of  change 
in  permeability  of  certain  cells,  the  sudden  innva-e  in  perme- 
ability of  a  cell  after  swelling  to  a  certain  size  idue  presumably  to 
rupture  of  the  plasma  membrane),  the  ease  with  which  mild 
mechanical  treatment  increases  the  permeability,  and  the  locali- 
/ation  of  electric  polarization  at  the  cell  surf 

<  Miim  k«  -upposed  these  membranes  to  be  of  a  fatty  nature. 
This  idea  \\as  carried  further  by  Overtoil,  who  considered  the 
plasma  membrane  to  be  composed,  not  of  neutral  lai-,  but  of 
sub- 1. in.  es  of  the  class  which  are  called  "lipoids."  \\  hich  included 
iion--aponif\  ing  ether  soluble  extracts  of  or-an-,  /.  <-.,  choN-tcrm, 
lecithin,  cuofin,  and  rerebrin.  He  found3  that  all  basic  dyes 
were  ea-ilv  absorbed  by  living  cell.-,  but  not  mo-i  of  the  -ulphonic 
acid  dyes.  This  corresponded  to  their  -olubility  in  melted 
cholesterin.  or  solutions  of  lecithin  and  cholesterin,  or  particles 
of  lecithin,  protagon  or  cerebrin.  His  argument  is  somewhat 
\\eakened.  hou ever,  by  the  fact  that  cholesterin  decomposes 
on  melting,  and  that  if  lecithin  is  allotted  to  absorb  water  its 
s.  .1\  cut  potter  changes. 

\rtlt.  f.  .:  »/.,  1910,  CXXXIII.,  237,  and  Eighth  Internat.   I'hysiol. 

I'mmif  — .  Vi.-mui.    H)IO. 

her.  (/.   K»n.    Pm    f.  Akad.  d.   H  U    l'-rlin,  1888.  B<1.   XXXIN". 

*Jahrh     .          Bo/.,  i«j->n.  X  X  X  l\    .  669. 


II>  J.    F.    MCCLEXDOX. 

Many  of  Overton's  critics  do  not  distinguish  between  lipoids 
proper  and  a  host  of  ether-soluble  substances  which  are  also 
ca'lled  lipoids,  and  of  the  data  which  they  present  we  will  con- 
sider only  that  on  lipoids  proper.  Ruhland1  found  that  certain 
dyes  stain  plant  cells  but  are  not  soluble  in  solutions  of  cholesterin 
(and  vice  versa).  Robertson2  observed  that  methyl  green  freed 
from  methyl  violet  was  insoluble  in  a  nearly  saturated  solution 
of  lecithin  in  benzol,  whereas  it  stained  living  cells.  Hober3 
obtained  Ruhland's  results,  when  using  certain  animal  cells, 
but  found  that  certain  nephric  tubule  cells  absorb  all  dyes  that 
are  not  suspension  colloids. 

Faure-Fremiet,  Mayer  and  Schaeffer1  state  that  pure  choles- 
terin does  not  stain  with  any  dyes  (contrary  to  Overton),  mala- 
chite green  (considered  lipoid-insoluble  by  Ruhland  and  Hober) 
stains  lecithin,  and  Bismarck  brown  (considered  lipoid-insoluble 
by  Ruhland)  stains  lecithin,  cholesterin-oleate  and  cerebrin. 
A  mere  trace  of  free  fatty  acid  greatly  affects  the  behavior  of 
lipoids  toward  stains. 

Mathews5  considers  the  absorption  of  dyes  by  cells  as  a  chemical 
process.  Since  basic  dyes  combine  with  albumin  in  alkaline 
solution,  lipoids  in  the  membrane  are  not  necessary  for  the  ab- 
sorption of  such  dyes. 

Traube  objected  to  Overton's  hypothesis  on  the  ground  that 
Overton's  plasmolytic  series  is  the  same  as  found  by  Brown,  who 
used  the  membrane  of  the  barley  grain,6  and  the  same  as  the 
series  of  the  attraction  pressures  of  the  substances  in  water. 
But  Traube  admits  in  his  later  papers  that  the  chemical  character 
of  the  membrane  affects  osmosis. 

We  may  conclude  that,  although  the  plasma  membrane  of 
some  cells  may  be  lipoid  in  character,  this  lias  not  been  proven, 
but,  in  general,  it  is  more  permeable  the  more  the  diffusing  sub- 
stance lowers  the  surface  tension  of  water. 

1  Juhrb.   wiss.   Bol.,    1908,   XLVI.,    i,   and  Ber.   DeiUsch.   hot.   Gesellsch.,   1909, 
XXVI.,  112. 

2  Jour.  Bio.  Chem.,  1908,  LV.,  I. 

3  Biochem.  Zeit.,  1909,  XX.,  55. 

4  Arch,  d' Anal.  Mic.,  1910,  XII..  19. 

*  Jour.  Pharmacol.  and  Exp.  Tlier.,  1910,  II.,  201. 

6  But  this  is  not  true  of  all  seed  coats.  Atkins,  Sci.  Proc.  Roy.  Dublin  Soc.,  XII., 
n.  s..  No.  4,  p.  35,  observed  that  the  membranes  of  the  bean  seed  are  freely  pcrme- 
at4l,  semipermeable  plasma  membranes  arising  only  after  germination. 


TENSION    PHENOMENA    OF    LIVING    ELI- Ml. NTS.  IK) 

Nathanson1  supposed  the  plasma  membrane  to  be  a  mosaic 
of  lipoids  and  "protoplasm,"  but  it  is  evident  that  if  the  lipoid 
portion  is  not  continuous,  it  can  not  make  the  cell  impermeable 
to  any  -ubstance. 

(  /apck-  states  that  lipoid  solvents  cause  cytolysis  when  the 
surface  tension  of  the  solution  is  reduced  to  .68,  and  concludes 
from  tlii-  that  the  plasma  membrane  contains  glycerine  tri-oleate 
sinci  ii-  emulsion  reduces  the  surface  tension  of  water  to  thi> 
figure. 

Tin-  diffusion  of  water-soluble  substances  through  swollen- 
]il.n  _els"  or  "sols"  of  gelatine,  varies  inversely  with  the 

viscosity  (Arrhenius).  The  great  hysteresis  of  gelatine  gels  i- 
t.il.en  advantage  of  to  show  that  diffusion  depends  on  tin  vis- 
cositj  ami  not  on  the  per  cent,  of  gelatine,  at  the  same  temper- 
aim 

Tin  absorption  of  water  by  a  gelatine  plate  increases  its  per- 
meability.  and  the  temperature  and  therefore  the  presence  of 
MI! .-tain  es  which  affect  this  swelling  of  gelatine  affect  its  perme- 
abilit\.  Impregnation  of  colloidal  membranes  \viih  bile  salts, 
al-oliol,  ether,  acetone  or  sugar  changes  (usually  increases) 
tin  ir  | .ei  mt -ability.  The  effects  of  substances  on  tin-  rate  of 
dilliisioii  through  gelatine  plates,  and  on  their  swelling  (viscositj  • 
and  tin  -It  ing  point  arc  not  always  quite  parallel.4 

In  the  substance  added  to  the  membrane  is  removable, 

(he  i  lian^c  in  permeability  becomes  reversible,  which  is  true 
in  regard  to  many  of  the  substances  mentioned  above.  Changes 
in  non-li\in-  membranes  are  usually  only  partially  reversible  or 
are  irreversible.  I  >enaturalization  of  a  colloid  membrane  by 
lu-ai.  heavy  metals,  or  other  coagulative  agents  which  induce 
chemical  changes  in  the  membrane,  or  the  addition  ot  substances 
which  cannot  be  removed,  produce  irn  -\vr-ible  changes  in 
permeability. 

That  the  permeability  of  the  membrane-  in  living  ti--ne  is 
increased  at  death  is  proven  by  a  host  of  observation-.  The 
electric  conductivity  increases  enormously  at  death.  Contained 

Jahrb    ,  i  r.  Bot.,  1903,  XXXVIII..  284;  190).  XXX  IV..  601,  and  XI...  403- 
/-'.-».  deut  ill.  hot.  Gesell.,  1910.  28,  480. 

r.  Asher  &  Spiro's  Ergeb.  der  Physiol..  1908,  VII.,  99- 
.  loc.  cit. 


I2O  J.    F.    MCCLENDON. 

substances  diffuse  out,  substances  in  the-  medium  (fixing  fluids, 
stains,  etc.)  diffuse  in.  There  is  a  more  general  mixing  of  tissue 
substances.  Enzymes  come  in  contact  with  proteids  and 
autolysis  results. 

Certain  substances  are  known  to  increase  the  permeability 
of  membranes  in  tissues  of  the  body.  Thus  ether,  chloroform, 
etc.,  increase  the  penetration  of  fixing  fluids,  and  the  exit  of 
contained  substances,  and  the  mixing  of  tissue  substances. 
In  this  way  they  increase  autolysis. 

II.  OSMOTIC  PHENOMENA  IN  PLANTS. 

It  is  evident  that  water,  salts,  carbon  dioxide  and  oxygen 
can,  at  least  occasionally,  penetrate  plant  cells,  as  otherwise 
no  growth  could  occur.  In  case  of  the  higher  plants,  the  same 
is  true  of  sugars  and  other  bodies.1  Janse2  found  that  so  much 
KNOs  is  absorbed  by  Spirogyra  cells  in  10  minutes,  that  it  may 
be  easily  detected  microchemically  with  diphenylamin-sulphuric 
acid. 

Osterhout3  grew  seeds  of  Dianthus  barbatus  in  distilled  water. 
The  rate  of  growth  during  the  several  days  of  observation  was 
normal.  In  nature,  calcium  oxalate  crystals  are  found  in  the 
root  hairs,  but  are  not  formed  in  the  distilled  water  cultures, 
showing  that  the  Ca  comes  from  the  medium.  If  placed  in 
calcium  solutions,  crystals  became  large  enough  to  see  with  the 
polarizing  microscope  in  four  hours,  showing  permeability  to  Ca.4 

Nathanson5  found  that  nitrates  and  other  substances  entered 
the  cell.  Ruhland  also  observed  penetration  of  salts. 

Traube-Mengarini  and  Scala6  conclude  that  salts  enter  plant 
cells  only  through  the  partition  walls.  At  these  places  there 
appears  an  "acid  reaction"  (bluing  of  methyl  violet).  They 

1  See  Laurent  in  Livingstone,  "The  R6Ie  of  Diffusion  and  Osmotic  Pressure  in 
Plants,"  1903,  p.  67. 

1  Versl.  en  Medeel.  der  Konikl.  Akad.  van  afdeel.  Naturs.,  3.  Reeks,  IV.  part, 
1888.  p.  333- 

3  Zeits.  f.  physik.  Chem.,  1909,  LXX.,  408. 

4  But  compare  von  Maycnberg,  Jahrb.  f.  wiss.  BoL,  XXXVI.,  381,  who  found 
little  penetration  of  salts  into  fungous  hyphae.     And  see  Demoussy,  Comptcs  Rcnilus, 
CXXVIL,  970. 

1  Jahrb.  wiss.  Bol.,  XXXVIII.,  -'84;    XXXIX..  601;    XL.,  403. 
6  Biochcm.  Zeit.,  1909,  XVII.,  443. 


TENSION    PHENOMi  \A    OF   LIVING    ELEMENTS.  121 

interpret  this  as  showing  that  the  anion  of  the  salt  unites  with 
an  H  ion  of  an  amino  group,  forming  a  free  acid,  and  the  kation 
of  the  -.dt  unites  with  the  protoplasm.  It  appears  to  me  that 
the  ba-i>  of  this  conclusion  is  very  slight. 

Permeability  may  be  investigated  by  a  study  of  plasmolysis, 
which  consists  in  the  shrinkage  of  the  surface  protoplasm  away 
from  tin  (i  llulose  cell  wall,  due  to  the  osmotic  pressure  of  the 
hypertonic  -olution  of  a  dissolved  substance  which  does  not 
I it-iit -irate.  A  regaining  of  turgor  by  the  cell  while  in  the  hyper- 
tonie  -oliitioii  indicates  slow  penetration  of  the  sul»tance.  The 
l>la-im>l\  ii.  method  was  originated  by  Xageli,  who  aUo  noted 
thai  a  shrinkage  resembling  plasmolysis  but  accompanied  by 
omuard  dillu-ioii  of  dissolved  substances,  occur-  at  death  or 
-e\  tic  injury  to  the  cell.1 

The  plain  cell  is  surrounded  by  an  elastic  cell  \\all.  The 
internal  "-m.itir  pressure  may  be  divided  into  three  re-uliant-: 
that  «  aii-inu  rounding  up  of  the  cell  is  called  turgor,  that  re- 
sulting in  stretching  of  the  cell  wall  is  sometimes  distinguished  as 
turgescence,  and  that  resisting  the  surface  ten-i.ni  oi  tin-  cell, 
" .  ential  pressure." 

The  plasmolytic  experiments  of  I  Wrie^-  and  other-'  are 
interpreted  \>\-  them  as  indicating  a  select i\e  impermeability 
"I  tin-  pla-m.i  membrane  to  neutral  salts. 

In  the  plasmolytic  experiments  of  Overtoil'  all  -alt-  pla— 
moly/ed  permanently.  Non-electrolytes  fell  in  four  unmp-, 
thus:  (am  -uijar,  dextrose,  manit,  glycocoll > urea,  ;_;lucenn> 
( -i  h\  K  ne -ali  <  'hi>l,  acetamid> methyl-alcohol,  acetmiitril,  ethyl- 
alt  ..hoi.  phenol,  aniline,  isobutyl-alcohol,  isoamyl-alcohol,  methyl 
acetate,  eth\l  acetate,  butyl  aldehyde,  acetmie.  a<  etald.>\im. 
Diffusion  of  substances  of  homologous  series  in<  reased  \\iih  molec- 
ular \\  eiiLill  t . 

<  >\  ei  inn  a-t  er i. lined  the  permeability  of  plant  cells  to  alkaloids 

1  "  PtUm/rnpliy-i..!.  I  iitersuchungcn."  1885. 

.'.  pliysikul.  cVi.-wi..  iSSS.  II.,  415;  1889.  III..  103. 

3  (I.   l.i\'int;-t«>iir.   "  The   R61e  of  Diffusion  and  Osin»iir   Pressure  in   Plant-." 
Cliu-.ii;.'.    \<j>>i;  J.in-«'.    />')/.  Ccntlb.,  1887,  XXXII.,  21;  Duggar,  Trans.  Acad.  Sc. 
>/.  Lout-*.  1906,  XVI..  473- 

4  \'ifrlfljai.'  ifr  .V aturforschers.  Gesell.  in  Zuriili,  XL IV..  88;  Jahr.  wiss. 
Ji»t..  1900.  XXXH 


122  J.    F.    MCCLKXDOX. 

by  their  precipitation  of  the  tannic  acid  in  the  cell  sap.  Most 
alkaloids  penetrate  rapidly,  but  only  in  the  form  of  the  free 
(undissociated)  base  produced  by  hydrolysis.  Hence  the  pene- 
tration (precipitation  and  toxic  effect)  may  be  prevented  by 
adding  a  little  acid  to  the  medium. 

Pfeffer  had  shown  that  methylene  blue  is  precipitated  by  tannic 
acid  in  the  cell  sap  of  certain  plants. 

Some  discussion  has  arisen  as  to  whether  the  mechanism  of 
the  entrance  of  dyes  into  plant  cells  is  similar  to  that  of  alkaloids. 
Overton  showed  that  lipoid  soluble  basic  dyes  penetrate  easily. 
He  at  first  supposed  that  only  the  free  color  base  (undissociated) 
is  able  to  penetrate  the  cell.1  Overton  found,  however,  that 
triphenylmethane  and  chinonimid  dyes  disprove  his  assumption, 
showing  that  it  is  at  least  not  general.  This  question  was  taken 
up  again  by  Harvey2  who  found  that  neutral  red  or  methylene 
blue,  which  stain  Elodea  leaves  in  tap  water,  do  not  do  so  if  just 
enough  acid  be  added  to  the  water  to  prevent  any  free  color 
base  from  forming. 

He  observed  that,  although  these  dyes  are  not  precipitated 
in  the  cell  sap  of  this  plant,  they  become  more  concentrated  in 
the  cell  sap  than  in  the  medium.  Neutral  red  is  bright  red  in 
the  cell  sap,  indicating  that  the  reaction  is  acid  (no  free  color 
base  is  present).  He  supposes  that  the  absence  of  any  of  the 
dye  in  the  form  of  the  free  color  base  prevents  it  from  diffusing 
out  of  the  cell,  hence  it  becomes  more  concentrated  within  than 
without. 

In  using  the  plasmolytic  method,  if  a  cell  does  not  recover 
from  plasmolysis  in  a  solution"  of  a  salt,  it  is  said  to  be  imperme- 
able to  that  salt.  However,  the  cell  may  recover,  but  may  be 
killed  by  penetration  of  the  salt,  and  shrink  again.  It  is  possible 
that  Overton  and  others  failed  in  some  cases  to  note  this  transient 
recovery.  Contrary  to  Overton,  Osterhout3  found  Spirogyra 
permeable  to  alkali-salts  and  alkaline  earth  salts,  but  more 

1  In  this  connection  it  is  interesting  to  note  that  Robertson  observed  that  free 
color  bases,  and  to  a  less  extent  free  color  acids,  are  much  more  soluble  in  fats 
than  are  their  salts.  This  is  what  we  should  expect,  since  the  salts  dissociate  in 
water,  and  ions  are  insoluble  in  fats. 

*  Science,  1910,  n.s.,  XXX11.,  565. 

3  Science,  1911,  n.  s.,  XXXIV.,  187  ;  XXXV.,  112. 


TENSION    PHENOMENA    OF   LIVING    ELEMENTS.  123 

easily  to  Xa  than  to  Ca.  It  is  plasmolyzed  by  .2. M  CaCl-2  and 
not  by  the  isosmotic  .2<)M  NaCl  but  by  .$x.\f  XaCl.  .195. I/ 
CaClo  and  -375.1/  XaCl  just  failed  to  plasmolyze.  On  mixing 
100  c.c.  -375-^f  XaCl  with  10  c.c.  .I95J/  CaCl2,  thus  decreasing 
the  osmotic  pressure  of  the  former,  marked  pla-mol\ -.-is  occurred. 
Thi-  indicates  that  Ca  decreases  the  permeability  to  Xa.1  From 
further  work  by  the  same  author,  not  yet  published,  it  appears 
th.it  N.i  increases  and  Ca  decreases  the  permeability  of  certain 
marine  plants.  Also  Fluri2  obtained  increase  in  permeability  by 
salt-  of  .ilnniinium,  yttrium  and  lanthanum. 

I  >' \rir-,  plasmolyzed  cells  of  Tradescantia,  containing  blue 
(i-ll  sap,  \\ith  4  percent.  KXO3  solution,  then  added  nitric  acid 
until  tin-  color  changed  to  red.  The  acid  made  the  cells  pcr- 
i-  \»  K\U3  for  they  regained  their  turgor  and  linally  bur-i. 

i-  explain-  the  easy  penetration  of  acids  into  n-11-.  I'tViin 
found  ihat  if  red  beet  cells,  petals  of  Pitlmonariu,  Manu-n  hair- 
oi  Track  'tuitia  and  other  anthocyan-containing  cell-  are  placed 
in  extivmelv  dilute  HC1  or  H-jSO.,,  they  suddenK-  turn  red.  in- 
diiatinii  iniinediate  penetration  of  the  acid.  It  allo\\cd  t<>  re- 
main Inn  a  short  time,  the  cells  are  not  killed,  and  tin-  color 
( lian^c  i-  1 1 -versed  on  returning  the  tissues  to  acid-free  water. 

I  have  n- pealed  these  experiments,  using  cells  of  n-d  Uii, 
ii  d  i.ibb.ige  and  red  nectar  glands  of  Vicia  _,'/;•'•</,  and  tind  that 
mineral  ai  id>  penetrate,  but  that  (the  lipoid  soluble  acetic  acid 
penetrates  much  nion-  rapidly  and  also  more  easily  alters  the 
jila^nia  membrane,  causing  pigment  to  diffuse  out,  if  not  can- 
tii>n-l\  applied.  Alkalis  also  penetrate,  but  'the  Lipoid  soluble) 
ammonia  penetrates  much  more  rapidly  than  the  oiheis.  Am- 
monia doi->  not  so  easily  increase  the  permeability  t<>  the  pi^mt-nt 

a-  dor-  arctic  acid. 

Kuhlaiid1  after  staining  root  hairs  of  Triancn,  etc.,  \\ith  tin- 
indicators,  methyl  orange  and  neutral  re<l,  found  that  mineral 
acid-  a-  well  as  lipoid  soluble  acids  penetrated. 

1  Tin-  \\oik  .-I  Ki.irney,  Report  71,  U.  S.  Dept.  of  Agriculture,  indicates  that 
(a  prevents  tli<-  pl.i-molytic  and  toxic  effect  of  MK.  Imt  thU  i 

follou  illi;  <!i.Mth. 

*  Flora    i      -    XCIX..  81. 

3  "(KiimtUchc  Untersuchungen,"  Leipzig,  1877,  p.  135. 

*Jnlirb.  u-iss.  Bot..  1908.  XLVL.  r. 


124  .!•    F.    MCCLENDON. 

One  defect  in  the  plasmolytic  method  is  the  fact  that  the  cel- 
lulose cell  wall,  if  not  very  thick,  is  elastic,  and  a  slightly  hyper- 
tonic  solution  may  cause  the  cell  to  decrease  in  volume  without 
pressing  the  protoplasm  away  from  the  cell  wall.  This  source 
of  error  may  be  eliminated  by  substituting  calculations  of  the 
volume  of  the  cells  (as  necessary  for  animal  cells)  for  observations 
on  plasmolysis. 

It  is  well  known  that  movement,  and  in  many  cases  increase 
in  size  of  plants  is  due  to  changes  in  turgor  of  the  cells.  If  we 
exclude  the  turgor  changes  in  aerial  plants  produced  by  variations 
in  the  ratio  of  the  water  supply  to  the  transpiration,  turgor 
changes  may  be  due  to  changes  in  the  osmotic  pressure  of  the 
external  medium,  or  of  the  cell  sap  (due  to  metabolic  changes) 
or  to  changes  in  the  permeability  of  the  plasma  membrane. 
Lepeschkin1  has  confirmed  Pfeffer  in  showing  that  changes  in 
permeability  of  stipule  cells  accompany  (or  immediately  precede) 
changes  in  turgor.  By  chemical  analysis  of  the  medium  he  has 
shown  that  an  outward  diffusion  of  dissolved  substances,  from 
the  cells,  accompanies  loss  of  turgor,  and  by  plasmolytic  ex- 
periments, that  the  permeability  to  certain  substances  increases. 

It  is  interesting  to  note  the  force  that  may  be  exerted  by  such 
changes  in  turgor.  From  measurements  of  the  pull  of  a  stamen 
hair  of  Cynara  scolumus  or  Centaurea  jacea  on  loss  of  turgor  fol- 
lowing stimulation,  it  seems  not  improbable  that  the  change  in 
turgor  amounts  to  2-4  atmospheres  (Hober).  This  also  indicates 
the  strength  of  the  cell  wall  necessary  to  prevent  rupture  of  the 
plasma  membrane'.  The  osmotic  pressure  of  the  juices  pressed 
out  of  plants  varies  from  3.5-9  atmospheres.2  The  pressing 
out  of  the  juices  causes  an  error  due  to  chemical  changes;  on 
the  other  hand,  in  taking  the  freezing  point  or  pieces  of  plant 
tissues,  an  error  arises  from  lowering  of  the  freezing  point  by  the 
walls  of  the  capillary  spaces.  Miiller-Thurgau3  found  the  A 
(corrected  freezing  point  lowering)  of  plant  tissues  =.8-3.1°. 
Many  plants  respond  to  light  by  definite  movements,  produced 

1  Her.  deutsch.  hot.  Gesell..  XXVI.  (a).  725. 

2  DeVries,  Pringsheime  Jahrbuchcr  wiss.  Bot.,  1884.  XIV.,  427;  Pantanelli,  ;'/</,/.. 
1904.  XL.,  303. 

3  Landu'irtschaftl.  Jahrb..  1886,  XV.,  490. 


TENSION    PHENOMENA    OF    LIVING    ELEMENTS.  1  25 

by  turgor  changes  in  certain  of  their  cells.     Trondle1  found  that 
light  produced  changes  in  permeability  of  these  cells. 

Changes  in  permeability  may  not  only  affect  the  turgor,  but 
also  the  assimilation  and  excretion,  and  consequently  the 
metabolism  and  growth  of  the  cells.  Chapin2  observed  that 
(  '),  in  certain  doses  is  a  stimulant  to  the  growth,  not  only  of 
green  pl;mt>  but  also  of  moulds.  As  only  ,i  few  saprophytes  can 
decompo-e  ('();,  it  is  not  probable  thai  its  effect  is  nutritive. 
A  ^imilar  simulating  action  of  ether  and  various  salts,  even 
such  toxic  ones  as  those  cf  zinc,  was  previously  knoun.  These 
salt-,  probably  stimulate  without  penetratini;  the  cells,  since 
7.\\,  lor  in-taiice,  is  not  a  constituent  of  protoplasm.  '•  This 
one  to  suppose  that  the  initial  effect  of  .ill  of  these 
i-  on  tip-  surface,  changing  the  permeability  of  the  cells. 

Wachter  l»imd  that  potassium  decreases  the  p'-rmeabilitx  of 
onion  i  ells.  SuL'ar  diffused  out  of  sections  of  Allinm  cc{>n  placeil 
in  distilled  \\ater  or  hypotonic  sugar  solutions,  but  a  trace  of 
potassjuin  s.ilt  entirely  prohibited  the  difliisii.n.  When  the  K 
\\as  n-ino\ed  tin-  diffusion  recommenced. 

Czapek8  determined  increase  in  permeability  by  the  exosmosjs 
ol  tannin  in  cells  of  Echcveria  leaves.  Various  inono\aleni  al- 
c«.h..ls  and  k<  tones,  ether,  ethyl  urethan.  di  and  tri  acetin, 
Na-oleate,  ohic  acid,  lecithin  and  cholesterin  all  \\.\-\  caused 
mosjs  ,,;  i.innin  in  concentrations  (aqueous  solutions)  \\hich 
had  a  siitlacc-  tension  of  about  0.68.  It  \\ould  appear  therel'on- 
that  these  substances,  chiefly  of  the  class  ,,|  indifferent  narcotics, 
alter  ihe  cells  if  tliey  diffuse  into  them,  or  dilhise  into  certain 
Structures  -\\c\\  as  the  cell  lipoids  or  the  plasma  membrane. 
It  seems  more  reasonable  to  suppose  that  the'  plasma  membrane 
is  the  siructure  affected,  and  the  more  the  substance  louers  the 
siirlace  teii-ioii  of  water,  the  more  it  diffuses  into  the  plasma 
membrane,  \\hen  this  membrane  is  altered,  it  allo\\~  escape 
of  tannin.  Some  substances  such  as  chloral  hydrate  are  el  lei  tive 


;  Bot.,  IQIO.  XLVIII..  171. 

X 

•   •    I  "<-\>.  "  I  )\  n.uiiii'-  i.i'  Living  Maiti-i,"  pp.  73,  74. 
1  J.ii-.i-i-  XI.  I.,  165. 

6  "  t  'l.i-r  rim-   Mrtli.i.lt.-  /in  ilirekten  Bestiininung  der  OberMchenspannung  der 
ri.i-iiKili.uit  \"ii  I'll.in/cii/clli-n."  Jena,  ("..  Fi-clitT. 


126  J.    F.    MCCLENDOX. 

in  less  concentration,  and  probably  affect  the  cell  chemically 
as  well  as  physically. 

Mineral  acids  caused  exosmosis  of  tannin  when  the  concen- 
tration just  exceeded  1/6,400  normal,  and  the  effect  is  probably 
due  to  H  ions.  At  this  same  concentration  Kahlenberg  and  True1 
found  the  growth  of  seedlings  of  Lupinus  albns  to  cease.  It 
appears,  therefore,  that  this  cessation  of  growth  is  due  to  in- 
creased permeability,  causing  decreased  turgor  of  the  cells. 

Changes  in  permeability  may  also  affect  secretion  (excretion). 
The  addition  or  formation  of  alcohol  or  acetates  causes  yeast  and 
other  fungi  to  secrete  (excrete)  for  a  short  time,  various  sub- 
stances, especially  enzymes  which  do  not  come  out  in  a  culture 
medium  lacking  the  reagent.2  It  appears  that  the  alcohol  or 
acetates  increase  the  permeability  of  the  fungi  to  these  substances. 

My  own  experiments3  indicate  that  pure  MgClo  solutions 
increase  the  permeability  of  yeast.  A  certain  per  cent,  of  yeast 
and  dextrose  in  .3  molecular  MgClo  eliminated  CO2  more  rapidly 
than  .$M  NaCl  or  .^2^M  CaCl2,  all  which  have  about  the  same 
freezing  points.  Also,  the  CO2  elimination  was  more  rapid  in 
the  magnesium  solution  than  in  a  solution  of  the  same  concen- 
tration of  MgCl2  with  either  of  the  other  salts  in  addition,  or  in  a 
solution  containing  NaCl  and  CaCl2  in  the  same  concentrations 
as  in  their  respective  pure  solutions,  or  in  a  solution  of  all  three 
salts,  or  in  tap  or  distilled  water.  In  order  to  determine  whether 
the  magnesium  entered  the  cells  I  took  two  equal  masses  of  com- 
pressed yeast  and  agitated  one  in  H2O  and  the  other  in  a  molecular 
solution  of  MgCl2  for  5  hours,  i-hen  washed  each  rapidly  in  H2O 
by  means  of  the  centrifuge.  The  ash  of  the  magnesium  culture 
=  .048  gram,  that  of  the  control  =  .0466  gram.  Evidently 
the  Mg  did  not  enter  the  yeast  to  any  great  extent,  and  probably 
acted  on  the  surface,  increasing  the  permeability. 

Ewart4  observed  that  after  placing  plant  tissue  in  2  per  cent. 
HC1  and  washing  in  water  its  electric  conductivity  (ionic  per- 
meability) was  increased.  If  one  portion  of  the  plant  is  stimu- 
lated, the  stimulus  may  be  transmitted  to  other  portions.  '  In 

1  Kahlenberg  and  True,  Botanical  Gazette,  1896,  XXII.,  p.  Si. 

2  Zangger,  "Asher  and  Spiro's  Ergcb.  d.  Physiol.,"  1908.  VII.,  144. 
»  McClendon,  Am.  Jour.  Physiol..  1910,  XXVII.,  p.  265. 

4  "  Protoplasmic  Streaming  in  Plants,"  Oxford,  1903,  p.  96. 


TENSION    PHENOMENA    OF   LIVING    ELEMENTS.  12~ 

this  way  increase  in  electric  conductivity  was  produced  by  stimu- 
lation of  a  point  outside  the  path  of  the  current. 

\Yhereas  many  plants  are  very  sensitive  to  sudden  and  extreme 
changes  in  osmotic  pressure,  Osterhout1  found  that  certain  marine 
alga-  thrived  when  subjected  daily  to  a  change  from  fresh  water. 
to  si  a  water  evaporated  down  until  it  crystallized  out,  and  vice 
versa.  He  does  not  state  whether  these  algae  survive  extreme 
plaHno]v,js:  or  whether  they  are  so  easily  permeable  to  salts 
as  ii"t  t«.  I..-  plasmolyzed  by  the  saturated  sea  water  or  burst 
1>\  tin-  fre-h  water. 

Foi  rej  'il.u ion  to  slight  changes  in  the  osmotic  pressure  of  the 
m<  dium.  .i  change  in  size  of  the  cell  altering  the  turgescence,  or 
ti  nsion  <>!  the  cell  wall,  is  sufficient. 

If  /  .•  iitia  cells  are  placed  in  a  hypotonic  solution,  they 

begin  to  -\\ell.  But  soon  crystals  of  calcium  oxul.tu  are  formed 
in  the  cell  -ap,  and  in  this  way  the  turgor,  due  chielly  to  oxalic 
.K  ill.  i-  redinvd.2  It  would  be  interesting  to  know  wh.it  i-  the 
source  <>f  the  Ca.  Was  it  previously  in  combination  with  pn>- 
teids? 

Tin  nmodation   to  a  hypertonic  medium  take-,  pla<<  ,  ac 

iin^    to    van    Rysselberghe,    parth     through    ab-.o.  pi  inn    of 
Mil>-',c  :  the  medium  and  partly  through  metabolic  produc- 

tion oi  .  •-iiii»tic  substances,  chiefly  the  transformation  of  >uuvh 
into  ox.ilic  .1. 

Ill         BlO-ELECTRICAL    PHENOMENA. 

I.   /;/  P hints. 

(  hai  .<  in  permeability  of  the  plasma  membrane  to  ion-  \\otild 
necessarily  cause  electrical  change  due  to  its  inlhu-iu  •  on  the 
migration  of  ions.  These  electrical  change-  actually  occur,  and 
may  be  ea-ily  studied. 

Stimulation  or  wounding  in  plants  is  accompanied  by  an  elec- 
tronegative variation  of  the  affected  suiface.  This  negative 
region  spreads  in  all  directions  over  the  surface,  but  the  rate  of 

1  I *niv.  of  Cal.  Pub..  Bot..  1906.  II..  227. 

Jic.  Mem.  d.  1'Acad.  royale  de  Belgique,  1899.  LVIII..  i. 
1  Compare  von  Mayenberg  Jahrb.  f.  wiss.  Bot..  XXXVI.,  381. 


128  J.    F.    MCCLENDON. 

propagation1  is  much  slower  than  the  similar  process  in  muscle 
or  nerve.2 

Pfeffer3  supposed  that  the  plasma  membrane  is  normally  per- 
meable to  ions  of  only  one  sign.  Since  the  normal  cell  surface 
is  positive  in  relation  to  the  cell  interior  (cut  surface)  we  may 
conclude  that  the  plasma  membrane  is  normally  more  permeable 
to  kations  (less  permeable  to  anions).  Just  as  the  negative 
variation  of  wounding  is  due  to  the  removal  or  rupture  of  the 
plasma  membrane,  so  the  negative  variation  of  stimulation  would, 
on  the  membrane  hypothesis,  be  due  to  increase  in  permeability 
of  the  plasma  membrane  to  the  confined  anions. 

An  alternative  hypothesis  is  that  these  electrical  changes 
result  from  changes  in  metabolic  activity.  The  production  of  an 
electrolyte  whose  anion  and  kation  have  very  different  speeds 
of  migration  (such  as  an  acid  or  alkali)  would  cause  electrical 
changes.  But  how  are  we  to  account  for  changes  in  metabolic 
activity?  There  exists  varied  evidence  for  changes  in  perme- 
ability, and  it  is  simpler  to  assume  that  changes  in  metabolic 
activity  and  electrical  changes  are  both  the  result  of  changes  in 
permeability. 

Kunkel4  tried  to  explain  the  vital  electrical  phenomena  as  the 
result  of  the  movement  of  fluids  in  the  vessels  of  the  tissues,  but 
bio-electrical  changes  may  occur  without  such  movement  of 
fluids  (Burdon-Sanderson). 

Kunkel  observed  in  i8825  that  the  movement  of  the  leaf  of 

i 

Mimosa  pudica  is  accompanied  by  an  "action  current,"  or  nega- 
tive variation  of  one  surface  of  the  pulvinus.  Similar  results  on 
Dioruza  leaves  were  obtained  by  Munk'1  and  specially  studied 
by  Burdon-Sanderson.7  It  was  stated  above  that  Lepeschkin 
had  shown  that  the  turgor  changes  in  plants  were  accompanied 
or  i  in  mediately  preceded  by  changes  in  permeability  to  certain 
substances.  The  electrical  phenomena  suggest  that  the  turgor 

1  Which  is  in  mimosa  600-1,000  times  as  fast  as  the  geotropic  impulse  in  a  root. 

2  Fitting,  "Ashcr  and  Spiro's  Ergeb.  d.  Physiol.,"  1906,  V.,  155. 
"  Pflanzenphyaiologie." 

*Arch.f.  iL  ges.  Physiol.,  1881,  XXV.,  342. 

s  See  Wintcrstein's  "Ilandbuch  der  vcrgleichenden  Physiologic,"  III.  (2),  2, 
p.  214. 

«  Arch.  f.  Anal.  it.  Physiol.,  1876,  XXX.,  167. 

"  Proc.  Roy.  Soc.  London,  1877,  XXV.,  441;  Philos.  Trans.,  1888,  <  I  XXIX..  417- 


TENSION    PHENOMKNA    OF   LIVING    ELEMENTS. 

change  is  accompanied  for  immediately  preceded)  by  increase  in 
permeability  of  the  plasma  membrane  to  anions.  Burdon- 
Sanderson  states  that,  whereas  the  m<  >\vment  resulting  from 
turgor  change  begins  2.5  seconds  after  stimulation,  the  negative 
variation  reaches  its  maximum  I  second  after  simulation.  This 
max  In-  due  to  the  mechanical  inertia,  or  the  time  required  for 
the  diffusion  of  substances. 

It  ua-  sated  in  the  preceding  chapter  that  light  change-  the 
permeability  of  the  plasma  membrane,  and  Waller1  found  cor- 
responding electrical  changes  due  to  light,  but  not  al\va\-  in  the 
same  direction  in  different  plants.  This  inconstancy  in  direction 
is  probably  due  to  the  fact  that  light  not  only  influences  the 
I -i  i  mi  abilitx ,  but  also  the  assimilation,  and  changes  in  a— imi- 
laiioii  produce  electric  changes.  This  is  supported  by  the  fact- 
thai  i  >IH  rtoii-  found  that  assimilation  as  well  as  electric  chai 
i-  uios  affected  by  the  longer  light  rays. 

2.    In  Muscle  and  Xerve.3 

i  >s  \\ald'  proposed  the  hypothesis  that  the  electric  pin •noniena 
ot  must  le,  nerve  and  the  electric  organs  of  fish  (which  mav  n -at  h 
se\eral  hum lud  volts)  are  produced  with  the  aid  of  semiper- 
meable  membranes.  The  alternative  theory  of  Hermann,  \\hich 
\\oiild  account  for  the  current  of  injury  by  assuming  the  pn>- 
oi  some  electrolyte  (alkali?)  in  the  wounded  region.  \\h"-' 
-  and  kaiions  have  very  different  speeds,  >eem-  le— ,  ]»n»b- 
.ibl\  to  be  i  he  correct  one. 

\.  c.  .j-diii^  to  the  "membrane  theory,"  the  muscle  or  net  \  e 
eleineiii  i--  surrounded  by  a  semipermeablc  membraiu-  allo\\iu^ 
easier  passage  to  kations  than  to  anions.  The  kaii./n-  ua— in- 
through  the  membrane  are  held  back  by  the  ne-ati\e  field  pro- 
duced by  the  confined  anions,  but  owing  to  their  kinetic  energy, 
the  kation-  pa-s  out  far  enough  to  give  the  outside  of  the  cell 
-in  lace  a  i io-i t ive  charge.  Therefore  an \  portion  of  the  siirfai  «• 
that  i-  made  freely  permeable  to  anion>  become-  electronegative 

Jota         1  •l:\aiol..  iSgg-'oo,  XXV.,  18. 

•     ntiilniti.ni  a  lYtude  du  mode  de  la  production  <!'•  l'«  l«-i  trii  it.    'Ian-  <-tres 
\i\anii-.      I  r.i.,nt\  ,1,-  1'hfititnt  Solvay.  1902,  \'. 

R    1  LHie,  .1".-.;.  .'••ur.  Physiol.,  ign.  XXN'III..   1.^7. 

*  /.fit.    /-/IVV/A-.    L'h,-IH..     I  Soc..    \'I..    71. 


130  J.    F.    MCCLENDON. 

in  relation  to  the  remainder  of  the  surface.  This  negative 
variation  may  be  produced  by  artificially  removing  or  altering  a 
portion  of  the  membrane  (producing  the  current  of  injury)  or 
as  the  result  of  normal  stimulation,  making  it  permeable  to  anions 
(action  current). 

Bernstein  resorted  to  mathematical  proof  of  this  hypothesis. 
We  \vill  not  here  go  into  details,  but  the  gist  of  the  matter  is 
that  if  the  process  were  as  we  have  imagined  it,  the  electromotive 
force  of  the  current  of  injury,  or  action  current,  should  be  pro- 
portional to  the  absolute  temperature.  He  found  this  to  be 
true  for  temperatures  between  o°  and  18°,  but  between  18°  and 
32°  the  E.M.F.  was  found  to  be  too  small.  The  muscle  was  not 
permanently  injured  by  exposure  to  the  higher  temperatures 
for  the  length  of  time  necessary  for  the  experiments.  Bernstein 
explained  this  discrepancy  by  the  further  assumption  that  at  the 
higher  temperatures  the  plasma  membrane  became  slightly 
more  permeable  to  anions.1 

Since  the  muscle  contains  a  higher  per  cent,  of  potassium  than 
the  blood  plasma  or  lymph,  it  might  be  supposed  that  K  ions 
passed  outward  through  the  plasma  membrane  and  gave  the 
surface  of  the  muscle  element  the  positive  charge.  But  if  this 
were  the  case,  the  current  of  injury  should  be  reversed  by  placing 
the  muscle  in  a  solution  containing  potassium  in  greater  concen- 
tration than  in  the  muscle.  This  reversal,  howrever,  was  shown 
by  Hober  not  to  occur.  Since  lactic  and  carbonic  acids  are  pro- 
duced by  muscle  and  diffuse  out  in  increased  amount  on  contrac- 
tion, one  might  suppose  H  ions  to  give  the  muscle  surface  the 
positive  charge.  This  is  only  a  guess  (and  a  poor  one,  since  un- 
dissociated  molecules  of  COz  and  lactic  acid  are  lipoid-soluble) 
but  may  be  convenient  until  some  better  one  is  proposed.  Per- 
haps the  carbonic  acid  combines  with  amphoteric  proteids,  which 

1  This  is  similar  to  the  conclusion  reached  by  Biataszewicz,  Bull.  d.  I' A  cad.  d' 
Sc.  d.  Cracovie,  Sc.  Math.  e.  Nat.,  Oct.,  1908,  p.  783,  in  regard  to  the  unfertilized 
frog's  egg.  In  order  to  explain  his  observation  that  the  rate  of  swelling  in  tap 
water  increased  5  times  for  every  10°  rise  in  temperature,  he  assumed  that  heat 
increased  the  permeability  to  H-^O.  This  would  seem  to  be  the  simplest  explana- 
tion, provided  the  swelling  were  not  due  to  chemical  production  of  osmotic  sub- 
stances: and  since  the  A  of  the  ripe  ovarian  egg  is  .48°  but  is  reduced  to  .045°  after 
oviposition,  Biochem:  Zeit.,  1909,  XXII.,  390,  much  if  not  all  of  the  swelling  is 
probably  due  to  the  initial  osmotic  pressure  of  the  egg  interior. 


TENSION   PHENOMENA   OF   LIVING   ELEMENTS. 

tlu-n  set  free  H+  and  HCOs~  ions,  thus  increa-in-  the  ionization 
and  therefore  reducing  the  number  of  undissociated  molecules, 
which  can  escape.1 

Since  Osterhout  showed  that  certain  electrolytes  may  alter 
the  permeability  of  cells,  we  might  expect  to  find,  on  the  membrane 
hypothesis,  an  effect  of  salts  on  the  electric  polarization  of 
imi-rle.  H  ober2  observed  that  a  portion  of  the  surface  of  a 
mu-cle  treated  with  certain  salts,  KC1  for  instance,  becomes 
i  -1.  •  iro-nriMtive  (more  permeable  to  anions)  whereas  a  portion 
n-«l  \\ith  Nal  or  LiCl  becomes  positive  (still  less  permeable 
i"  anions  than  is  the  normal  unstimulated  muscle).  The  order 
of  eite<  ii\  i  ness  of  the  ions  is  as  follows:  Li<Xa<Cs<XHj<  Kb 
<K  ,m<l  (  \S<NO3<  I<  Br<Cl  <valerianate,  bui\rate,  pro- 
pioiiai'  ate,  formate  <SO4,  tartrate.  Similar  ionic  series 

\\en-  found  by  Overtoil,  K.  l.illie,  Schwartz,  Mathews,  C.nii/iur, 
I  loin  i  ,  and  Mayer  in  the  effect  of  salts  on  the  function.il  at  ii\  iiy 
of  inii-(  It  •,  nerve  and  cilia,  but  the  exact  relation  of  tlu--e  phe- 
nomena to  permeability  is  not  understood  in  every  case.  Pure 
-ohnioii^  of  salts  of  alkali  metals  may  "inhibit"  inu-cle  by  in- 
permeability,  but  salts  of  alkali  earth  metals  art  -aid 
to  "inhibit"  by  decreasing  permeability.  .  Mayer  says  that  the 
effect  of  -.ills  on  cilia  is  the  reverse  of  that  of  muscle,  but  the 
relation  (.f  ihis  to  permeability  is  not  known.  Since  ion-  aiit-i  i 
tin  .ition  state  of  hydrophile  colloids  in  the  same  or  ex- 

actl\  n  \«  -i.-ed  order,  and  the  kation  series  is  found  in  no  other 
knoun  physico-chemical  phenomena,  it  might  be  supposed  th.it 
tin  semipermeable  membranes  of  muscle  are  colloitl.il. 

It  -i  t-ni-  probable  that  sugar  solutions  inhibit  the  artivity  of 
nniM  le  l.v  increasing  the  permeability,  but  since  -u-.ir  i-  not  an 
t  K  i  trol\  tt  thi>  question  cannot  be  tested  by  electric  method-. 

A  ne-ati\e  variation  of  muscle  may  also  be  produced  by  the 

•  ailed  "h.emolytic"  substances,  but  i>  inv\  t  -r-ible,  whereas 
that  produced  by  salts  may  be  reversible.  In  this  connection  it 


1  RiMl'.  Q.  J.  Exper.  Physiol..  1910,  III.,  171,  suppo-<  <1  ttio  aninn  to  be  i>n>t--in; 
|II>\MAI-I  H  h.i-  not  been  shown  that  proteids,  or  even  aniino  acids  dilTu-r  nut  nn 
stiniiil.ith.ti.  I  do  not  see  that  the  speculation  of  Galcntti,  Zeit.f.  All  gem.  Physiol., 
1907,  \  1  at  all  explanatory. 

•  r's  Arch.,  1910,  CXXXI\'..  311. 


132  J.    F.    MCCLENDON. 

is  interesting  to  note  that  Overton1  found  the  permeability  of 
muscle  to  be  similar  to  that  of  plant  cells. 

It  might  appear  to  the  reader  that  the  membrane  theory  is 
merely  wild  speculation.  What  proof  have  we  that  on  injury 
or  during  contraction  the  muscle  is  more  permeable  to  any  ion? 

DuBois  Reymond2  and  Hermann3  explained  the  fact  that  living 
muscle  has  a  greater  electric  resistance  than  dead  muscle  on 
the  hypothesis  that  the  resistance  of  living  muscle  is  due  to  the 
presence  of  membranes,  which  become  more  permeable  at 
death.  They  demonstrated  the  resistance  of  muscle  tissue  to 
the  passage  of  ions  by  the  fact  that  electric  polarization  occurs 
in  muscle  tissue  on  the  pasage  of  an  electric  current.  It  seems 
to  me  that  Kodis4  and  Galeotti5  take  a  step  backward,  in  at- 
tributing the  decreased  resistance  of  dead  muscle  to  the  liberation 
of  ions.  Galeotti  tried  to  support  his  view  by  determinations 
of  the  freezing  points  of  the  living  and  dead  muscle,  but  found 
on  the  contrary  that  the  change  in  electric  conductivity  of  the 
muscle  did  not  correspond  to  the  change  in  the  osmotic  pressure. 

Du  Bois  Reymond6  observed  that  the  electric  conductivity 
of  muscle  changes  on  (during?)  contraction  and  Galeotti7  found 
it  to  be  greater  on  strong  contraction  than  on  weak  contraction, 
and  least  on  fatigue-exhaustion  or  cold-anaesthesia.  However, 
the  duration  of  a  contraction  is  momentary  (about  1/5  second  for 
frog's  muscle)  and  it  is  not  clear  that  these  investigators  measured 
the  conductivity  accurately  during  such  a  brief  period,  in  fact 
they  probably  measured  it  after  contraction.  Therefore  I 
decided  to  repeat  these  experiments,  using  a  method  by  which 
I  could  measure  the  conductivity  during  the  actual  contraction 
period,  as  well  as  in  ilic  unstimulntcd  condition.8 

1  PJluger's  Arch.,  1902,  XCII.,  115. 

2  "  Untersuchungen  iiber  thierische  Elcctricitat,"  1849. 

3  PJluger's  Arch.,  1872,  V.,  223,  VI.,  313. 

4  Am.  Jour.  Physiol.,  1901,  V.,  267. 

*Zeil.f.  Biol.,  n.  f..  1902,  XXV.,  289;  1903.  XXVII..  65. 

6  Loc.  cit. 

'  Loc.  cit. 

8  McClendon,  American  Journal  of  Physiology,  1912.  XXIX.,  302. 


TENSION    PHENOMI  NA    OF    LIVING    ELEMENTS. 

Experimental. 

Platinum  electrodes,  platinized  with  platinic  chloride  contain- 
ing a  little  lead  acetate,  and  of  a  form  similar  to  those  designed 
by  r.aleotti,  were  used.  Galeotti  stimulated  tin-  muscle  through 
the  same  electrodes  used  in  measuring  the  electric  conducmity, 
by  -\\iiching  on  a  different  electric  current.  Though  it  were 

-iblc  to  throw  a  switch  quickly  enough  to  have  the  curivm 
for  inea-iireinent  of  conductivity  pass  through  the  muscle 
during  contraction,  it  would  be  necessary  to  use  a  string  u.il- 
\ -.mom! -it -r  to  take  the  reading,  and  this  method  would  probably 
noi  b<-  very  accurate.  A  more  accurate  method  is  that  of  Kohl- 
IMII-I  li,  in  which  a  rapidly  alternating  current  reduces  polari/atioii 
at  the  electrodes  and  in  the  tissue,  but  it  is  necessary  to  throu  the 
nni-cle  into  tetanus  in  order  to  have  time  for  the  reading.  I 
a<  •  Miiipli-lu-d  this  by  using  the  same  current  for  stimulation  and 
inra-mvmein  of  conductivity.  A  very  small  induction  coil  wa- 
luted  \\itli  a  rheostat  in  the  primary.  Another  rheostat  in  the 

-e ilaiA   could  l>e  thrown  out  of  the  circuit  by  a  s\\itch.      By 

adjiMini;  ihr  rheostats,  a  current  strong  enough  to  be  dis- 
tiiu  tlv  heard  in  the  telephone,  yet  too  weak  to  stimulair  the 
nm-cle,  \\a-  obtained.  By  switching  the  resi-tance  out  of  the 

ondarj  >  in  nit,  the  current  could  immediately  be  im-iva-rd  so 
a-  to  thiou  the  muscle  into  tetanus.  Since  the  \Vhrai-tonr 
britlg*  \\a^  u->ed,  the  difference  in  current  strength-  had  no  <lii 
effect  "ii  tht-  readings.  The  conducti\  it\  increa-nl  from  o  to 
28  per  cent,  (being  usually  about  15  per  cent.)  on  stimulation. 
\Ye  ha\c,  then,  evidence  for  the  increase  in  prrnu-ability  of 
muscU-  to  ions  during  contraction,  but  what  relation  has  this 
to  tin-  mechanism  of  the  contractile  process?  It  has  been  su-- 

;rd  by  D'Arsonval,  Ouincke,  Imbert,  P.t  rn-t(  in,  (ialeotti 
ami  others  that  the  increased  permeability  to  ion-,  causes  a  dis- 
appearance of  the  normal  electrical  polari/ation  of  the  elements, 
surface  tension  consequently  increases,  cau-ing  them  to 
up  (shorten).  But  what  are  the  elements  concerned:' 
It  would  be  confu-ing  to  assume  them  to  be  the  fibers,  as  then  the 
function  of  the  complicated  internal  structure  would  be  mu\- 
plained.  They  are  probably  not  the  sarcous  elements  (por- 
tions of  fiber  bet \\een  2  Z-lines)  as  the  rounding  up  of  these  ele- 


134  J-    F.    MCCLENDOX. 

ments  would  elongate  the  muscle.  And  even  though  contraction 
were  produced  by  inequality  in  surface  tension,  as  assumed  by 
Macallum1  the  total  surface  change  would  be  so  small  as  not  to 
account  for  the  energy  liberated  in  contraction.  In  order  to 
avoid  this  last  difficulty  Bernstein  made  use  of  hypothetical 
ellipsoids.  These  were  surrounded  by  elastic  material  to  account 
for  elongation  of  the  muscle.2 

The  great  differences  of  potential  (several  hundred  volts)  that 
may  be  produced  by  the  electric  organs  of  fish,  is  achieved  by 
the  arrangement  of  the  modified  muscle  plates  in  series.  All  of 
the  plates  have  the  nerve  termination  on  the  same  side.  On 
stimulation  of  the  nerve,  each  plate  becomes  negative,  first  on 
the  nerve  termination  side,  and  thus  the  negative  side  of  one 
plate  touches  the  positive  side  of  the  next  plate.  In  this  way 
the  direction  of  the  current  may  be  determined  by  studying  the 
anatomy  of  the  innervation.  This  rule,  discovered  by  Pacini, 
finds  an  exception  only  in  Mahpterurus,  whose  electric  organ 
is  supposed  by  Fritsch  to  be  derived,  not  from  muscle  but  Irom 
skin  glands. 

The  electric  fish  are  relatively  immune  to  electric  currents 
passed  through  the  medium.  This  is  not  merely  an  apparent 
immunity  due  to  the  fish  being  out  of  the  path  of  the  current,  or 
the  current  being  short  circuited  by  sea  water  (in  case  of  marine 
fish).  I  have  received  severe  shocks  from  a  torpedo  that  was 
entirely  submerged  in  sea  water. 

3.  Amoeboid  Movement.3 

The  normal  unstimulated  surface  of  plant  and  animal  tissues 
is  electro-positive  in  relation  to  the  cut  or  injured  surface  of 
the  cells.  We  have  given  reasons  for  assuming  that  this  indicates 
greater  permeability  of  the  plasma  membrane  to  kations  than  to 
anions,  the  latter  accumulating  in  the  cell  interior,  gives  it  a 
negative  charge. 

There  are  two  reasons  for  believing  that  this  is  true  also  of  ihe 
Amoeba: 

1  Science,  n.  s.,  1910,  XXXII..  822. 

2  Meigs.,  Am.  Jour.  Physiol.,  1910,  XXVI.,  191,  supposes  the  rounding  up  of 
muscle  elements  due  to  increased  turgor. 

3  McClendon,  Arch.  f.  d.  ges.  Physiol.,  1911,  CXL.,  271. 


TENSION    PHENOMENA    OF    LIVING    ELEMENTS.  135 

1.  If  a  weak  electric  current  is  passed  through  water  in  which 
an  Amoeba  is  suspended,  it  is  carried  passively  toward  the  anode, 
indicating  that  it  has  a  negative  charge.     This  charge  may  be 
due  to  confined  anions. 

2.  If  a  stronger  electric  current  is  passed  through  an  Anm'ba, 
it  begins  to  disintegrate  first  at  that  surface  nearest  the  anode. 
The  disintegration  is  probably  due  to  the  accumulation  of  ions 
retarded  l>y  the  plasma  membrane.     The  ions  in  the  medium  are 

I- 1  pass  around  the  Amoeba,  but  the  contained  ions  must  pass 
i In-  plasma  membrane  in  order  to  migrate  to  the  fleet  rodes. 
since  tin-  disintegration  is  toward  the  anode,  it  i-  prol.ubly  due 
to  anions  which  cannot  get  out  of  the  Amoeba.  Since  no  corre- 
sponding disintegration  begins  toward  the  kathode,  the  plasma 
membrane  is  probably  more  permeable  to  kations. 

Tin-  Mir  face  tension  of  the  Amoeba  is  very  low,  ami  apparently 
iiKT'M-i  -  on  strong  stimulation  (indicated  by  rounding  up  <>I  the 
ii'<i).  \Ve  saw  that  stimulation  in  plant  and  muscle  alls 
caused  increased  permeability  to  ions,  and  consequently  dis- 
appear.mee  of  the  normal  electrical  polarization,  and  thercbv 
can-iir^.  im  r eased  surface  tension.  \\'e  might  conclude  therefore 
that  the  low  surface  tension  of  the  Amoeba  is  caused  b\  electric 
pulari/ation,  due  to  the  production  of  some  metabolic  elect rol.  te 
\\ho-e  anions  cannot  escape;  and  that  strong  stimulation  causes 
increased  permeability  and  hence  disappearance  of  the  electrical 
polarization. 

This  \\ould  explain  all  negative  tropisms  of  the  Aniii:l><i.  The 
surface  ten-ion  of  the  portion  most  strongly  stimulated  is  in- 
.  reased,  an<l  the  Anucba  Hows  away  from  the  stimulus. 

In  order  to  explain  positive  tropisms  we  would  have  to  make 
another  assumption.  If  the  stimulus  did  not  act  directly  on  the 
plaMiia  membrane,  but  penetrated  the  Anuclm  and  acted  on  the 
protoplasm,  and  increased  the  production  of  the  metabolic 
product  producing  polarization  of  the  plaMiia  membrane,  it 
would  thereby  decrease  the  surface  tension.  The  local  decrease 
in  MII  lace  tension  would  cause  the  Amoeba  to  tlou  toward  the 
source  of  the  stimulus,  just  as  the  quicksilver  drop  in  dilute 
ll\(  '  Hows  toward  potassium  bichromate  in  Bernstein's  experi- 
ment . 


136  J.    F.    MCCLENDOX. 

All  stimuli  producing  positive  tropism  would  then  have  to 
penetrate  to  a  greater  or  less  distance  into  the  Amoeba.  But  the 
same  stimulus  thus  acting  on  the  interior  might,  in  greater 
intensity,  affect  also  the  plasma  membrane,  increasing  its 
permeability  and  changing  the  positive  to  negative  tropism. 
Such  a  change  of  the  sign  of  tropism  has  been  observed. 

Soap  lowers  the  surface  tension  of  fats  and  lipoids,  and  Ouincke, 
Biitschli,  Loeb,  Robertson  and  others  supposed  that  lowering 
of  the  surface  tension  of  living  cells  might  be  due  to  soap.  How- 
ever, I  found  that  soap  always  causes  negative  tropism  in  Amoeba, 
probably  because  it  increases  the  permeability  of  the  plasma 
membrane. 

4.  The  Propagation  of  the  Bio-electric  Changes. 

On  the  hypothesis,  that  the  electric  phenomena  in  muscle  and 
nerve,  as  well  as  other  animal  and  also  plant  tissues,  is  due  to 
change  in  permeability  to  ions,  we  might  hope  to  explain  the 
wave-like  propagation  of  these  changes.  Since  extraneous 
electric  currents  "stimulate"  all  tissues  (presumably  by  in- 
creasing permeability)  thus  causing  them  to  produce  additional 
electric  phenomena,  it  seems  natural  that  these  latter  would  be 
self-propagating.  It  is  probably  the  negative  variation  of  nerve 
which  stimulates  the  muscle,  and  the  negative  variation  of  the 
portion  of  the  muscle  fiber  adjoining  the  nerve  ending,  which 
stimulates  the  adjacent  portions  of  the  muscle.  Ncrnst  found 
mathematical  proof  that  electric  stimulation  is  due  to  change  in 
ionic  concentration  at  the  semipermeable  membranes. 

I  have  found  evidence  that  the  negative  variation  (current  of 
injury)  in  plants,  may  strongly  affect  adjacent  cells.  If  an 
electric  current  of  suitable  density  is  passed  through  plant  or 
animal  tissue,  negatively  charged  colloids  in  the  protoplasm 
migrate  toward  the  anode.  I  have  observed  this  movement  in 
living  cells,  and  the  resulting  displaced  bodies  in  histological 
sections.  In  certain  cases  there  may  be  some  doubt  whether 
the  colloids  moved  toward  the  anode,  or  water  toward  the 
kathode,  but  in  others,  easily  distinguishable  bodies  such  as 
chromatin  granules  or  threads  moved  toward  the  anode. 

If  the  tip  of  a  root  be  cut  oil  we  observe  a  negative  variation 


TENSION    PHENOMENA    OF    LIVING    ELEMENTS. 

of  the  cut  surface.  This  produces  an  electric  current  through 
the  medium  and  surrounding  tissue.  The  fact  that  the  current 
actually  passes  through  adjacent  cells  is  shown  by  a  displacement 
of  their  contained  colloids,  identical  in  appearance  with  the 
displacement  produced  by  the  currents  used  in  the  abo\v  experi- 
ments. V-mec1  apparently  observed  these  changes  but  did  not 
correctly  d< •-<  ribe  or  interpret  them. 

The  fact  that  an  electric  current  on  inert  MM-  make  Cumulates 
inu-cle  .it  the  kathode,  and  the  fact  that  the  muscle  -nrface  is 
ix mil. illy  po-itive  in  relation  to  the  interior  (cut  surface),  prob- 
ably indicates  that  stimulation  is  produced  by  a  rapid  depolari. 
/ation  df  the  muscle  surface. 

It  tlii-  reasoning  be  applied  to  an  individual  contractile 
element,  \\e  may  assume  that  the  current  causes  kations  i<>  leave 
tin  (HINT  surface  of  the  membrane,  and  other  kation-  to  be 
an i -at  ltd  i«. ward  the  inner  side  of  the  membrane,  and  thus  the 
pnl.iri/ation  disappears  or  may  even  be  reversed.  JUM  how  this 
causes  an  increase  in  permeability  of  the  membrane  i-  a  matter 
\\hich  \\«-  \\ill  leave  to  the  future  for  discussion. 

1 1  ha-  b« -i -i)  supposed  that  the  stimulated  region  acts  as  kathode 
it.  tin-  adjacent  portions,  and  these  in  turn  act  as  katln«li •<  to 
tin  in  \t  portions  and  so  the  stimulus  is  propagated. 

^i  imnlai  ii  .11  of  a  part  of  the  surface,  causing  it  t«  >  I  »eo  >me  nx  >n- 
pcinical.lt  in  ions,  depolarizes  the  adjacent  parts  "f  the  -urlai  e 
n\\iii-  tt>  the  fact  that  confined  anions  migrate  through  the 
pcrmcablt  n  and  neutrali/e  the  charge-  t.|  the  kations  on 

adjacent  parts  of  the  impermeable  region  (see  Fig.  i).  1  <>r  ihi- 
iva-oii  the  increase  in  permeability  is  propagated. 

I  hi-  e\|ilanation  of  the  phenomenon  in  a  single  rleim-m  hold- 

lor  a   ti— ue  made  up  of  many  elements  provided   tin -t    are  in 

contact,   as   illustrated   by   the  accompanying   Fig.   2.     Thi-   i- 

]in.babl\-  the  mechanism  of  propagation  of  the  negative  variation 

and  "-tiniulus")  in  many  plant  and  animal  tis-ti> 

This  mechanism  accounts  for  the  movement  of  the  negative 
variation  over  a  -nrface.  But  it  may  be  possible  for  this  electric 
change  to  jump  from  one  element,  to  another  not  touching  it. 
The  observation-  on  the  current  of  injury,  cited  above,  show  that 

1  "Rci/li-itiini;  u    <1.  rd/loitrrulrn  Strukturen  b.  d.  Pflanzen,"  Jena,  1901. 


J.    F.    MCCLEXDOX. 


increased  permeability  of  part  of  a  tissue  surface,  may  cause 
electric  currents  to  flow  through  cells  some  distance  from  the 
wound.  These  currents  probably  stimulate  the  cells  through 
which  they  pass,  which  in  turn  become  permeable  and  produce 
electric  currents.  This  explains  the  propagation  of  stimuli 


4- 


Anions  represented  by  minus  sign,  kations  represented  by  plus  sign.  Arrows 
denote  the  direction  of  migration  of  ions.  The  large,  circle  represents  the  plasma 
membrane,  the  dotted  line  denoting  the  permeable  and  the  continuous  line,  the 
impermeable  portion. 

through  loose  tissues,  and  the  structural  changes,  as  observed 
by  Nemec. 

The  rate  of  propagation  of  the  "wound  stimulus"  is  very  slow, 
whereas  that  of  propagation  of  the  "stimulus"  (negative  vari- 
ation) in  sensitive  plants  is  more  rapid,  and  that  of  the  nerve 
impulse  still  more  rapid.  We  have  not,  however,  sufficient  data 
to  show  whether  this  is  a  mathematical  objection  to  the  hy- 
pothesis. 

The  streaming  movements  in  plants  may  be  stopped  by  a 
strong  stimulus  or  "shock."  This  stimulus  is  usually  propagated 
in  one  or  more  directions.  Ewart1  states  that  the  rate  of  propa- 
gation at  1 8°  in  a  single  elongated  cell  of  Nitella  is  1-20  mm. 

1  Loc.  cit. 


TENSION"    PHENOMENA    OF   LIVING    ELEMENTS. 


139 


per  sec.,  but  where  it  has  to  pass  cell  walls  .001-. 03  mm.  per  sec. 
However,  the  stoppage  of  the  streaming  was  his  criterion  of  the 
presence  of  the  stimulus,  and  probably  the  banking  of  the  stream 

+        -f        +•        + 


•h 
f- 

•h 


1- 

f 
•h 

i~ 
+ 


T 
-h 


-f- 


-1 


FIG.  2. 

I  In-  -|ii.iu-~  represent  the  plasma  membranes  <>:  Fur  further 

explanation  see  Fig.  i. 

at    din    point,  soon  stopped   the  whole  stn-am   thn^  sinnil.uini!, 
tin-  pn 'i Motion  of  the  stimulus. 

\\ .      \AK'  '  i>IS. 

If  si ijnul.it ion  consists  in  increase  in  prrmraln'lity,  we  should 
r\P»-t  i  in.i  -tin  tics  to  prevent  this  change.  The  object  of  this 
c'liauu-r  i-  10  prrsent  evidence  that  may  support  or  refute  such  a 
hypothesis. 

<>\ertoii  i -1  .served  that  warm-  and  cold-blooded  vertebra; 
insects    and   entomostraca,    require   practically    the   same   con- 
centration  of   the  ana-.-t Ill-tic   for  narcosis.     Certain   groups  of 


I4O  J.    F.    MCCLENDOX. 

worms  require  double,  and  protozoa  and  plants  six  times  this 
concentration.  We  might  conclude  from  this  that  nerves  (and 
especially  medullated  nerves?)  are  more  susceptible  to  narcosis 
than  arc  other  cells.  All  groups  of  worms  contain  nerves,  but 
Loeb  has  shown  that  certain  worms  may  perform  coordinated 
movements  after  the  nerves  are  cut,  hence  the  higher  concen- 
tration of  the  narcotic  required  to  quiet  them.  However  it 
should  be  remembered  that  over-stimulation  causes  rounding  up 
and  quiescence  of  Amoeba  and  muscle  may  be  paralyzed  by 
increasing  the  permeability.  The  growth  of  plants  is  increased 
by  a  certain  concentration  of  ether  and  retarded  by  a  greater 
concentration.  It  may  be  that  true  narcosis  (decreased  perme- 
ability) of  protozoa  and  plants  cannot  be  produced  by  such 
substances  as  ether,  etc. 

Vertebrate  nerve  tissues  are  rich  in  lipoids  (which  have  similar 
solubilities  to  neutral  fats)  and  it  is  therefore  significant  that 
Overton  and  also  Meyer1  found  that  the  partition  coefficient  of 
anaesthetic  between  olive  oil  and  water  corresponds  to  its  anaes- 
thetic power.  Meyer2  showed  further,  that  with  change  of 
temperature,  the  change  in  the  partition  coefficient  between  oil 
and  water,  and  the  anaesthetic  power  of  the  substance  were 
parallel.  Pohl,  Frantz,  Grehaut,  and  Archangelsky  found  that 
chloroform,  ether,  alcohol,  chloral-hydrate  or  acetone,  became 
more  concentrated  in  the  central  nervous  system  than  in  other 
tissues.  This  is  probably  due  to  the  absorption  of  the  narcotic 
by  the  lipoids  (especially  the  immense  mass  of  myelin)  in  the 
nerve  tissues. 

It  it  could  be  proven  that  the  plasma  membrane  consists  of 
lipoids,  this  solubility  of  narcotics  might  be  considered  direct 
evidence  for  or  against  the  permeability  hypothec-,  but  lacking 
such  proof  we  must  first  attack  the  subject  from  another  side. 

Hober3  observed  that  ethyl-methane,  phenyl-methane,  chloral- 
hydrate,  chloroform  and  hypnon,  in  low  concentration  prevent 
the  production  by  salts,  of  the  current  of  injury  on  muscle. 
He  showed  that  in  lethal  doses  on  the  contrary  these  narcotics  do 

1  Arch.  exp.  Path.  u.  Pharm.,  1889,  XLII.,  109. 

2  Arch.  exp.  Path.  u.  Pharm.,  1901,  XLVI.,  338. 

3  Pfluger's  Arch.,  1907,  CXX..  492,  501,  508.     Cf.  R.  Lillie,  Am.  Jour.  P/»y> /.»/.. 
1912,  XXIX.,  373. 


TENSION'    PHENOMENA    OF    I.IVIM,    ELEMENTS. 

not  prevent  but  even  produce  a  current  of  injury,  in  this  \vay 
explaining  data  which  might  otherwise  seem  to  contradict  the 
first  statement.  Galeotti  and  Cristina1  observed  that  ether, 
ethyl-chlorid,  and  chloroform  produce  a  current  of  injury  on 
's  nm-cle. 

\\'e  may  conclude,  then,  that  anaesthetic-,  in  the  concentration 
prodiK  ing  narcosis,  so  change  the  pl,t-ma  membrane  as  to 
pri-Miu  -.tits  from  making  it  permeable  to  anions.  This  i- 
pn.bably  a  No  true  of  nerve,  since  Hober  found  th.it  cthyl- 
intili  UK  in  low  concentration  prevented  the-  -cii-iti/ing  of 
in  i  \  e  \\  ith  I\_M)<. 

Hi'lit-r  In-  attempted  to  connect  these  facts  with  tin1  lipoid 
>olul)iliiy  of  narcotics.  Moore  and  Roaf2  h.nl  ob-er\vd  that 
//  i/utinlilics  of  such  narcotics  as  chloroform,  alcohol.  ether. 
"i  brn/ul.  precipitated  lipoids  extracted  from  organ-  and  -u - 
prmlrd  in  \\ater.  Hut  Hober  and  Gordon"*  found  that  colloidal 
solnii.ui-  i.i'  Irdthin  were  not  precipitated,  but  were  made  tran— 
j '.in-lit  b\  i-ilu-r  or  chloroform  in  hi^h  concentration.  SimilarK, 
(  n.ld-(  hmidt  and  1'ribram4  observed  that  lecithin  .-n-])cnilrd  in 
Na<  I  -i.liitioii,  which  is  dissolved  by  chloral  hydrate,  methane. 
or  '  '"  linr.  in  high  concentration,  is  precipitated  by  tin  m  in  1"\\ 
Concentration,  <  >u  the  other  hand,  Koch  and  Mcl...ii, 
that  «  hli.ral.  hypnon,  acetone,  or  pure  ether,  do  nut  change  the 
H/e  i't  ti'llnidal  particle.s  of  lecithin  (i.  e.,  make  them  ta-ii-r  or 
more  diiticult  to  salt  out).  Calugareanu6  explain-  the  mech.m- 
JMU  of  tlu-  precipitation  ol  lipoids  by  anaesthetic-  by  tin  incn 
in  >i/.-  of  the  particles  due  to  absorption  of  the  an.e-thetic. 

Tim-  there  seems  to  be  a  parallel  difference  bet \\een  tin-  action 
of  low  and  high  concentrations  of  anaesthetic-,  <>•  nni-clc  ami 
nerve,  and  the  action  of  the  same  on  lipoid  -u-pi-n-ion-.  but  this 
dm--  not  hold  true  for  all  cases.  Moore  and  l\oa!7  conclude  that 
ana  -thetii  -  are  bound,  not  only  by  lipoids,  but  al-o  by  pn.teids, 

1  ,  •  :ol..  IQIO,  X..  I. 

>ndon.  1904.  LXXIII..  38      .  LXXVII.,  86. 
//.  ••       I^-ilrage,  1904,  \'..  432. 

h.  H.  Ther.,  1909.  \'I..  i. 
Jour.  1'lxirm.  and  Exp.  Ther.,  1910,  II..  9 
.  1910,  XXIX..  96. 


142  J.    F.    MCCLENDOX. 

and  their  charactersitic  action  on  the  permeability  of  the  living 
cell  may  be  due  to  their  action  on  proteids.  In  other  words,  the 
plasma  membrane  may  be  entirely  proteid. 

It  is  well  known  that  during  narcosis  little  or  no  oxygen  is 
absorbed  by  nerve  tissue.  Yerworn  and  his  pupils  assumed  that 
the  narcotic  directly  suppressed  oxidation.  On  the  other  hand 
Mansfeld1  supposed  that  the  narcotic  dissolving  in  a  lipoid  plasma 
membrane  made  it  less  permeable  to  oxygen.  It  would  be  more 
in  harmony  with  the  phenomena  considered  in  previous  chapters, 
to  suppose  that  the  narcotic  in  low  concentration  decreased  the 
permeability  of  the  plasma  membrane  to  the  anions  and  molecules 
of  some  acid  end  product  of  oxidation,  and  thus  stopped  the 
combustion.  An  objection  to  this  hypothesis  is  made  by  War- 
burg2 who  found  that  phenylurethan,  which  only  slightly  re- 
duces oxidation  in  certain  cells,  fertilized  eggs,  delayed  cell 
division  enormously.  With  greater  concentration  of  the  narcotic, 
oxidation  was  greatly  reduced. 

V.    OSMOTIC  PROPERTIES  OF  THE  BLOOD  CORPUSCLES. 

Hamburger  and  Bubonavik3  have  concluded  that  the  ery- 
throcytes  are  permeable  to  K,  Na,  Ca  and  Mg.  However,  the 
opposite  conclusion  was  reached  by  previous  workers. 

Gyrn'?,4  Hedin,8  Traube6  and  others  observed  that  the  ery- 
throcytes  are  relatively  impermeable  to  neutral  salts  (exc.  NH.1 
salts)  amino  acids,  various  sugars  and  hexite,  slowly  permeable 
to  erythrite,  more  permeable  to  glycerine,  and  easily  permeable 
to  monovalent  alcohols,  aldehydes,  ketones,  esters,  ether,  and 
urea.  In  general,  it  may  be  said  that  the  erythrocyte  is  perme- 
able to  lipoid-soluble  substances  or  those  that  lower  the  surface 
tension  of  water.  Such  substances  (for  instance,  ether)  become 
more  concentrated  in  the  corpuscle  than  in  the  serum.  Saponin 
becomes  120,  and  ammonia  880  times  more  concentrated  in 
corpuscle  than  in  serum.7 

1  PJliiger's  Arch.,  1909,  CXXIX.,  69. 

2  Zeit.  physiol.  Chem..  LXVI.,  305. 

3  Arch,  internal,  de  Physiol.,  1910.  X.,  I. 

'  1'flitger's  Arch.,  1896,  LXIII.,  86,  and  Koninkl.  Akad.  von  Wetensch.  Amsterdam, 
1910,  p.  347. 

''PJliiger's  Arch.,  1897.  LXVIII.,  229;  1898,  LXX..  525. 

•  Biochem.  Zeit.,  1908,  X.,  371. 

7  Arrhcnius,  Biochem.  Zeit.,  iQofi,  XI.,  161. 


TENSION    PHENOMENA    OF   LIVING    ELEMENTS.  143 

The  erythrocytes  are  practically  impermeable  to  ions.  Stewart1 
observed  that  they  offered  a  great  resistance  to  the  electric 
current.  It  is  difficult  to  remove  all  of  the  serum  from  a  mass  of 
ervthrocytes,  but  Bugarsky  and  Tangl,  working  independently 
f.t"  Mt -uart,  obtained  sediments  of  corpuscles  having  a  conduc- 
tivity of  only  1/50  that  of  the  serum.  This  indicates  that  the 
corpuscles  are  practically  impermeable  to  both  classes  of  ions, 
for  it  permeable  to  ions  of  one  sign,  they  would  probably  not  be 
-mli  good  insulators.  The  electric  conductivity  of  the  a-h 

made  up  to  equal  volume)  of  the  corpuscles  is  about  that  of  the 
-eriim,  although  the  osmotic  pressure  of  the  solution  of  ash  of 
tin-  latter  i-  greater.2 

llrinr  an   increase  in  electric  conductivity  of  tin-  corpuscles 

as  \\ill  !>••  considered  below)  indicates  increased  prrmrabilit\ 
ion-  After  the  corpuscle  becomes  permeable  to  ion-,  further 
in.  i.  ase  in  conductivity  might  be  due  to  liberation  of  ion-  from 
i .  >ml  'iiiat  i.  nis  with  colloids  in  the  interior.  Howe\  IT  many  i<  uis, 
f.  'i  in-tam  .  I'O^  cannot  be  liberated  without  incineration  or  other 
rigorous  treatment.  Increase  in  conductivity  of  the  blood  by 
lakii  nts  has  been  proven  to  be  chiefly  due  to  increased  per- 

nieabilit\   o!  the  corpuscles,  since  the  conduct i\  ity  of  the  serum 
m MI   -ho\\s  so  great  an  increase  on  the  addition  of  the  1. iking 
in.  ami  is  usually  diminished  (by  the  hicmoglobin    if  the  cor- 
pn-cles  are  present. 

The  portion  of  the  normal  corpuscle  presenting  the  greatest 
resistance  to  the  electric  current  is  the  surface  layer,  since  I  lol»-r 
ol.ser\ed  thai  the  conductivity  of  'the  interior  of  tin  corpn- 

.1.  i«  rmine.l  by  its  dielectric  value)  is  many  time-  greater  than 
thai  of  the  corpuscle  as  a  whole.  Peskind1  cau-ed  bubbles  of 
nitrogen  to  form  \\  i  thin  the  corpuscle  and  observed  that  they  were 
retained  by  a  superficial  membrane.  This  may  be  the  membrane 
\\liicli  n-i-ts  the  electric  current. 

The  chemical  composition  of  the  corpuscle  is  suppo-ed  to  bear 
.-ome  relation  to  its  permeability.     Aside  from  the  haemoglobin, 
and   the  rather  low  water  content  (60  per  cent.i   the  corpuscle 
'  S< .'-'.'.  - .  J.ni.  a,  1897. 

M.Mirr.m'l  Ruaf.  Biochcm.  Jour.,  III.,  155. 
r  •-,..,       \rch.,  1910,  CXXXIII..  237. 
1  .l»i.  Jmtr.  I'hysioL.  VIII. 


144  .!•    F.    MCCLEXDON. 

is  composed  of  lecit-hin  and  cholestcrin  with  a  little  nucleo- 
proteid.  It  is  probable  that  these  lipoids  are  chemically  different 
in  different  species  of  animals,  since  Lefmann1  observed  that  the 
lipoids  of  erythrocytes  of  the  same  species  are  not  toxic-,  whereas 
those  of  another  species  may  be  very  toxic. 

The  distribution  of  these  substances  in  the  corpuscle  has  not 
been  ascertained.  Pascucci2  supposed  the  corpuscle  to  be  a  bag 
of  proteid  impregnated  with  lecithin  and  cholesterin  and  filled 
with  haemoglobin.  He  found  that  artificial  lecithin-cholesterin 
membranes  were  made  more  permeable  to  haemoglobin  by  the 
laking  agents,  saponin,  solanin  and  tetanus  or  cobra  poison. 
Dantwitz  and  Landsteiner  suppose  the  lecithin  to  be  in  com- 
bination with  protein. 

Hoppe-Seyler  assumed  the  haemoglobin  to  be  in  combination 
with  lecithin  in  the  corpuscle,  and  Bang3  has  shown  that  .lipoids 
may  lie  fixed  by  haemoglobin.  It  seems  evident  that  there  does 
not  exist  an  aqueous  solution  of  haemoglobin  within  the  corpuscle, 
since  haemoglobin  crystals  may  be  made  to  form  in  Nectunis 
corpuscles  without  extraction  of  water.  Furthermore,  Traube 
and  Goldenthal4  find  that  haemoglobin  has  a  haemolytic  action, 
and  unless  there  exists  some  body  within  the  corpuscle  which 
antagonizes  this  action  (as  serum  does)  a  haemoglobin  solution 
could  not  be  retained  by  the  corpuscle.  Probably  all  of  the  so- 
called  "stroma"  constituents,  not  in  combination  with  the  hae- 
moglobin, form  the  plasma  membrane  of  the  corpuscle. 

Under  certain  conditions,  the  haemoglobin  comes  out  of  tin- 
corpuscles,  and  the  blood  is  said  to  be  laked.  Laking  of  "fixed" 
corpuscles  occurs  only  after  the  removal  of  the  fixing  reagent. 
Thus,  sublimate-fixed  corpuscles  may  be  laked  by  substances 
which  combine  with  mercury,  such  as  potassium  iodide,  sodium 
hyposulphite  or  even  serum  proteids.  The  fact  that  they  may  be 
laked  by  heating  in  water  is  probably  because  the  nucleo-histone 
is  not  fixed  by  sublimate.  This  process  is  prevented  by  hypertonic 
NaCl  solution,  presumably  on  account  of  its  power  to  precipitate 
nucleo-histone  (Stewart).  Formaldehyde-fixed  corpuscles  m.i\ 

1  Beitrdge  chem.  Physiol.  it.  Path.,  XI.,  255. 

2  Hofmeisler's  Beilrdge,  1905.  VI.,  543,  552. 
'  Ergeb.  d.  Physiol.,  1907,  VI.,  152. 

4  Biochem.  Zeit.,  1908,  X.,  390. 


TENSION    PHENOMENA    OF    LIVING    ELEMENTS.  145 

be  laked  by  ammoniacal  water,  at  a  temperature  which  must  be 
higher,  the  more  thoroughly  they  have  been  fixed.  Ammonia 
combines  with  formaldehyde. 

Sieuart1  supposes  that  the  haemoglobin  must  be  liberated  from 
some  compound  before  the  blood  can  be  laked.  \Ye  cannot  say 
th.it  the  corpuscle  is  always  permeable  to  haemoglobin  from  within 
mi tward.  Ilouever  the  corpuscle  probably  is  impermeable  to  it 
from  \\iihont  inward,  since  it  does  not  take  up  haemoglobin  from 
•Union.  .UK!  alter  the  blood  is  laked  the  serum  contains  haemo- 
globin in  greater  concentration  than  the  "ghosts"  do. 

Ai  .my  rale,  permeability  to  haemoglobin  appears  to  be  inde- 
pendent of  permeability  to  salts,  since  Rollett2  found  that  hiking 
by  coiiden-er  di-rhargcs  may  set  free  the  haemoglobin  \\  iilmut  the 
corpu-e|e  becoming  permeable  to  ions.  Stewart3  concluded  tli.it 
tin  -.line  i-  inn-  of  hiking  with  sodium  taurocholate  even  alter 
i  on-iilriin^  the  depressing  action  of  haemoglobin  on  tin-  con- 
din  ti\  ii\  . 

Stewart4  and  others  had  already  shown  that  blood  laked  by 
minimal  appli<  ations  of  such  hiking  agents  as  free/in^  and  thaw- 
iiu.  Ueaii:  oo°),  foreign  serum,  and  autoly.-U  -poniam-ous 

l.iki  iuse  1'iit  a  Alight  increase  in  the  permeability  to  ion-, 

when-a-  tin  toiitiiuied  application  of  some  of  these  agents,  or 
e-l>e<  i.ill\  -mil  violent  reagents  as  distilled  water  and  saponin, 
cause  a  marked  increase  in  electric  conductivity.  On  the  other 
liaiid  if  saponin  is  added  to  defibrinated  blooil  at  o°,  the  con- 
ductivity "I  ilie  corpuscles  to  ions  begins  to  inert  a-e  before  any 
ha  nio-loliin  escapes  from  the  corpuscles. 

The  liberal ii>n  "f  the  haemoglobin  by  some  lakin^  agents  ma\- 
I'e  <lue  to  i lu  diiet  t  action  of  the  reagent  in  breaking  up  ilie  com- 
ixiiind  in  \\liit-li  ilie  blood  pigment  exists,  but  is  probably  some- 
times a  -ei-oinlary  effect,  following  increase  in  permeability  to 
electrolyt< 

It  ha-  been  shown  that  many  laking  agent.-,  lipoid  soKciu-, 
saponin  nn-atnrated  fatty  acids,  soaps,  and  lurmolysins  (con- 
tainin-  lipa-e  are  such  as  would  alter  lipoids  physically  or 

»i.  ijnd  l-.\f>fr.  Therapeutics,  1909,  I.,  49. 
/'  ' • «.  1  XXX  II.,  199. 

I  ••     .'  .  X. 

1  Jour,  i  XXIV.,  Jii. 


146  J.    F.    MCCLENDON. 

chemically,  whereas  pressure,  trituration,  shaking,  heat,  condenser 
discharges,  freezing  and  thawing,  water,  drying  and  moistening, 
salts  (including  bile  salts),  acids  and  alkalis,  might  act  also  on 
proteids. 

Since  any  treatment  which  causes  great  swelling1  of  the  cor- 
puscle leads  to  loss  of  haemoglobin,  it  is  probable  that  stretching 
or  breaking  of  the  surface  film  increases  its  permeability.  But 
laking  may  occur  without  swelling,  and  even  crenated  corpuscles 
may  be  laked  by  sodium  taurocholate. 

Hober2  observed  that  the  relative  action  of  ions  in  favoring 
haemolysis  is:  salicylate>benzoate>I  >NOs,  Br>Cl>SO4  and 
K  >  Rb  >  Cs  >  Na,  Li.  Since  this  is  the  order  in  which  they  affect 
the  aggregation  state  of  colloids,  their  action  is  probably  on  the 
aggregation  state  of  the  colloids  of  the  corpuscle  (proteids  or  lipoids 
or  their  combinations). 

The  permeability  of  formaldehyde-fixed  corpuscles  to  ions,  is 
greatly  increased  by  extraction  of  the  lipoids  with  ether,  or  by 
treatment  with  substances  such  as  saponin,  which  act  on  lipoids. 
Since  the  proteids  have  been  thoroughly  fixed,  it  is  evident  that 
they  play  no  part  in  this  process,  though  they  may  do  so  in  the 
non-fixed  corpuscles. 

The  relation  of  lipoids  outside  of  the  corpuscles  to  ha?molysis 
has  been  extensively  investigated,  and  cannot  be  fully  treated 
here.  Willstatter  found  that  cholesterin  combines  with  one  of 
the  saponins,  destroying  its  haemolytic  power.  Iscovesco3  con- 
cludes that  cholesterin  combines  with  soap,  and  prevents  its 
toxic  action. 

Changes  in  permeability  of  the  corpuscles  to  ions  were  studied 
chemically  before  the  application  of  the  electrolytic  method. 
Hamburger4  and  Limbeck6  observed  that  when  CO2  is  passed 
through  blood,  chlorine  passes  from  serum  into  corpuscles  and 
the  alkalescence  of  tin-  scrum  is  increased.  On  the  other  hand, 
the  distribution  of  sodium  and  potassium  is  not  changed.6 

1  Roaf,  0-  J-  Exper.  Physiol.,  III.,  75,  supposes  this  swelling  to  be  due  to  ioniza- 
tion  and  hence  increased  osmotic  pressure  of  haemoglobin. 

2  Biochem.  Zeil.,  1908,  XIV.,  209,  and  he.  cil. 

*  Comptes  Rendus,  Soc.  Biol.,  1910,  LXIX..  566. 
4  7,eit.  f.  Biol.,  1891,  XXVIII..  405. 

6  Arch.  exp.  Path.,  1895,  XXXV.,  309. 

*  Giirber,  Sitzungsber.  physik.-med.  Ges.  Wurzlmrg,  1895. 


TENSION    PHI.NMMi  NA    OF    LIVING    ELEMENTS.  147 

Koeppe1  and  Hober2  explain  this  process  in  the  following 
manner:  The  lipoid-soluble  CO2  enters  the  corpuscle,  and  by 
reacting  with  alkali  albuminates  in  the  protoplasm,  gives  off 
more  ,  i  it  ions  than  it  does  in  the  serum.  During  the  presence  of 
CO2,  the  corpuscle  is  permeable  to  anions,  and  the  CO3=  or 
HCO..;-  ions  pass  back  into  the  serum,  brin^  e\chani;i  <1  for  Cl~ 
ion-,  to  equalize  the  electrical  potential.  Sodium  bicarbonate 
being  mop-  alkalescent  than  sodium  chloride,  the  titratable 
alkalinity  ot  the  serum  is  increased. 

Thi-  explanation  is  supported  by  the  follouiii'j  tarts:  \\'hen 
CO  Is  passed  through  a  suspension  of  erythrocyte-  in  cam-  --ugar 
solution  tin-  laiter  does  not  become  alkaline.  If  ( '<  '  i-  pa— ed 
through  a  mass  of  centrifuged  erythocytes,  which  an-  then  added 
to  physiological  salt  solution,  the  latter  become-  more  alkaline 
than  the  M  rum  in  Hamburger's  experiment.  Any  -odium  -.ill 
mav  be  -ub-titiitc-d  for  serum,  and  its  anions  will  pa—  into  the 
corpu-cle-.''  Also  the  number  of  ionic  valence-  pa— in:;  into  the 
coipu-clr  i-  constant,  i.  e.,  if  sulphate  is  used  only  hall  as  many 
ion-  enter  the  corpuscles  as  when  chloride  or  nitrate  i-  n-ed. 
The  pr<  is  reversed  by  removal  of  the  O 

Thi-  -aim-  phenomenon  has  been  observed  in  lencoc\  it-  by 
\  an  del  ^i  hroHT. 

There  -eeins  to  be  some  relation  between  ha-mol\-i-  and 
-liitinaii'iii    of    the   corpuscles.     Arrhenius4   Mippo-cd    thai    ag- 
glutination by  acids  is  due  to  the  coagulation  ot  the  prou  id-  ot 

the     ei;\elnpe.        Ho\Ve\XT,      since     aggllltillat  i<  HI      i-      folloued      b\ 

precipitation,   it  seems  probable  that  the  loss  of  the   negative 

electric  ih.iixe  which  tends  to  keep  the  corpu-cle  in  -n-peii>-iiiii 
and  causes  ii  to  repel  every  other  corpuscle,  i-  partly  rc-pon-ible 

I".  'I    the    |iheli<  Uliena. 

The  fact  that  water-laking  is  preceded  by  agglutination  mi'^ht 
be  explained  if  we  assume  that  increa-e  in  permeability  to  ions 
1« -.til-  to  lo-s  of  electric  charge.  The  char-t  may  be  due  to  the 
charge-  "ii  the  colloids  of  the  corpuscle  or  to  semi-permeability 
to  ion-.  The  corpuscle  is  very  poorly  permeable  to  ions,  but  may 

\rch.,  1897.  LXVII..  189. 

-  ;  >:.,  1904.  CII.,  196. 

3  Il.unluirK'-r  and  van  Lier,  Engelmann's  Arch.,  1902,  492. 

*  Hi,:  >;,»;.  /.,-ii..   1907.  VI..  358. 


148  J.    F.    MCCLEXDOX. 

be  slightly  more  permeable  to  some  one  ion  than  to  others.  If 
this  ion  were  more  concentrated  in  the  plasma  or  in  the  corpuscle, 
the  latter  would  become  electrically  charged,  and  a  general  in- 
crease in  ionic  permeability  would  lead  to  a  reduction  or  loss 
of  this  charge.  The  loss  of  charge  would  favor  their  coming 
in  contact  with  one  another  and  their  precipitation,  but  their 
cohesion  is  probably  due  to  some  other  change,  possibly  the  exit 
of  adhesive  substances,  on  increase  in  permeability. 

VI.  ABSORPTION  AND  SECRETION. 
i.  Absorption  through  the  Gut. 

If  a  live  vertebrate  intestine  be  filled  with  one  portion  of  a 
physiological  NaCl  solution,  and  suspended  in  another  portion 
of  the  same  solution,  fluid  will  pass  through  the  wall  of  the  gut 
from  within  outward.  Cohnheim1  found  that  holothurian  gut 
behaves  in  the  same  way  toward  sea  water,  and  the  absorption 
stops  if  the  gut  is  injured  with  chloroform  or  sodium  fluoride. 

It  might  be  supposed  that  the  hydrostatic  pressure  produced 
by  the  contraction  of  the  musculature,  is  the  driving  force  of 
absorption,  but  on  the  contrary,  Reid2  found  that  the  wall  of 
the  rabbit's  intestine  behaved  in  the  same  way  when  used  as  a 
diaphragm. 

Salt  is  absorbed  by  an  intestine  filled  with  a  very  hypotonic 
solution  of  it,  and  water  may  be  absorbed  when  the  solution  is 
very  hypertonic. 

Blood  salts  enter  the  intestine  when  it  is  injured  by  an  ex- 
tremely hypertonic  solution,  or  sodium  fluoride,  chinin  or  arsenic. 

Grape  sugar  and  sodium  iodide  may  pass  from  without  inwards 
through  the  wall  of  a  normal  holothurian  intestine. 

Traube3  claims  that  absorption  is  explained  by  his  observation 
that  the  surface  tension  of  the  contents  of  the  gut  is  less  than 
that  of  the  blood,  but  this  does  not  apply  to  the  experiments  in 
which  an  identical  solution  was  placed  on  each  surface  of  the 
wall  of  the  gut.  Traube4  found  that  the  addition  of  a  substance 

1  Zeil.  physiol.  Chem.,  1901,  XXXIII.,  9. 
'-Jour.  Physiol.,  1901,  XXVI..  436. 

3  Pfliiger's  Arch.,  1904,  CV.,  559.       Cf.   Iscovesco,  Comptes  Rendus,  Soc.   I 
1911,  I. XXI.,  637. 

4  Biochem.  Zeit.,  1910,  XXIV.,  323. 


TENSION    PHENOMENA    OF    LIVING    ELEMENTS.  149 

lowering  the  surface  tension  increased  the  absorption  of  NaCl 
l>v  the  gut. 

Absorption  is  probably  due  to  irreciprocal  permeability  of  the 
wall  of  the  gut.  Hamburger  showed  that  dead  gut  and  even 
artificial  membranes  showed  irreciprocal  permeability  to  certain 
substances.  These  artificial  membranes  were  of  different  com- 
position on  their  opposite  surfaces  (parchment  paper-chrome 
albumin  or  parchment  paper-collodion)  and  he  assumed  that 
tin  \\all  of  the  gut  is  composed  of  two  osmotically  different  lavers. 
In  reality  tin-re  maybe  more  than  two  such  layers,  and  the  plasma 
membrane-  of  the  individual  cells  of  the  gut  may  show  irreciprocal 
permeability. 

TranU  '  showed  that  the  rate  of  absorption  of  a  snlotance 
by  living  v.ut  is  usually  greater  the  more  it  lower-  the  Mir  face 
tension  of  water.  The  order  of  ions  is:  Cl  >Br>  I  >N()3>SO»I 
Ill'<>:  and  K,  Xa>Ca,  Mg.  The  order  of  non-electrolytes, 
.  i'  <  online  in  Katzcnellcnbogen2  is:  glycocoll  <urca<a(  •<  t'>n<  , 
maun  t  <erythrite<glycerine<acetamidl  methylalcohol,  propyl- 

al<  "h.il.  am\  lalcohol. 

Tin-  rati  <>f  absorption  through  dead  ox  gut  according  to  i  led  in3 
\^:\'n  \(  i  >C1>SO4  and  K  >  Rb>  Na>Li  >Mg  ami  m.mnit 
<er\  thtiti  <  glycerine  <  urethan  <  glycocoll  <  amylenh\  draie 
<  gKtnl  <  urea  <  propylalcohol  <  isolnitylalcohol  <  methyl- 
alcdlmi.  ethylalcohol. 

Tin-  action  of  poisons  on  absorption  may  be  dm-  in  tin-  alter- 
atiiin  i  .t'  tin  plaMiia  membranes  of  the  individual  celU.  Ma\i-r- 
hoi'<  i  and  Mi  in'  state  that  even  sugar  in  certain  concentrations 
inert  -a-ed  the  permeability  of  the  gut. 


2.   '  ^niotic  Relation  of  Aquatic  Animals  to  the  Medium. 

I  redericq    found   that  the  salt  content  of   tin-  body  fluids  of 

marine  in\  .Ttebrates  is  about  the  same  as  that  of  sea  water. 

Henri   and    l.aloii5    showed   that  the   OMii"tic    exchange   bet  \\eeii 

cirl(.m  llu'nl  of  M-a  urchins  and  holothunaii-  and  medium  i-  chielly 


/'  \rch..  CXXXII. 

-  /'  ;        .  i  Xl\'.,  522. 

/'    ,  .  1899,  LXXX'III.,  205. 

1  Hi,;li,-ni.  /.fit..  1910,  XXX'II..  .^76. 
1  NViiitri-t.-iii.  11.  <  .'»,  2. 


I5O  J.    F.    MCCLENDOX. 

water.  If  the  sea  water  was  diluted  with  ]  {  vol.  of  isotonic 
cane  sugar  solution,  the  salt  content  of  the  coelom  fluid  is  very 
little  lowered  in  4  hours,  and  only  traces  of  sugar  appear  in  it. 
The  result  is  the  same  with  isotonic  urea  (which  easily  pene- 
trates most  plasma  membranes).  But  the  salt  content  of  the 
blood  of  elasmobranchs  and  teleosts  is  about  half  that  of  the  sea. 

Botazzi  and  his  colleagues  observed  that  the  osmotic  pressure 
of  the  blood  of  elasmobranchs  is  about  equal  to  that  of  the 
medium,  the  sails  in  the  blood  being  supplemented  by  organic 
substances,  chiefly  urea,  of  which  there  is  2-3  per  cent. 

If  elasmobranchs  are  placed  in  concentrated  sea  water,  the 
osmotic  pressure  of  the  blood  rises,  but  the  ratio  of  urea  to  salts 
remains  the  same.  G.  G.  Scott  found  that  changes  in  the  density 
and  osmotic  pressure  of  the  blood  of  elasmobranchs  accompany 
changes  in  the  salt  content  of  the  medium. 

However,  in  marine  teleosts  as  well  as  all  fresh- water  animals 
which  have  been  studied  in  this  respect,  both  salinity  and  osmotic 
pressure  of  the  body  fluids  are  very  different  from  that  of  the 
medium. 

The  osmotic  pressure  of  the  blood  of  marine  teleosts  is  about 
half  that  of  the  sea,  but  in  fresh-water  teleosts  it  is  still  less 
(but  much  greater  than  the  fresh  water).  This  indicates  that 
there  must  be  a  change  in  the  osmotic  pressure  of  the  blood  as 
the  fish  ascends  a  river.  Greene1  observed  that  it  took  salmon 
30-40  days  to  pass  the  brackish  water,  in  which  time  they  were 
acclimatized  to  fresh  water.  After  being  in  fresh  water  8-12 
weeks,  the  osmotic  pressure  of  the  blood  was  reduced  only  17.6 
per  cent.  This  reduction  may  be  partly  accounted  for  by  the 
absorption  of  the  osmotic  substances  in  the  blood  by  the  sexual 
glands.  In  harmony  with  this  view  is  the  fact  that  the  osmotic 
pressure  of  the  blood  of  the  female  was  reduced  much  more  than 
that  of  the  male.  One  salmon,  that  was  very  weak  and  probably 
dying,  showed  32  per  cent,  decrease  in  A  of  blood.  Sumner2 
observed  that  changes  in  weight  and  salt  content  of  marine  tele- 
osts accompany,  but.  are  not  proportional  to  changes  in  the 
medium. 

1  U.  S.  B.  F..  1904,  XXIV..  445;  1909,  XXIX.,  129;  Jour.  Ex[>.  Zoo!.,  1910. 
IX. 

2  Bull.  U.  S.  B.  F.,  1905,  XXV.,  53,  and  Am.  Jour.  Physiol.,  1907,  XIX.,  61. 


TENSION    PHENOMI  \A    OF    LIVING    I  LI-MI. NTS. 

Ovrrton  observed  that  if  the  cloaca  and  mouth  of  a  frog  in 
ire-h  water  are  closed,  the  frog  constantly  increases  in  weight. 
This  can  be  prevented  by  the  addition  of  .7  per  cent.  XaCl  to 
the  medium.  In  a  hypotonic  solution  water  is  constantly  ab- 
s<>rbed  by  the  skin  and  excreted  by  the  kidney-.  Fischer's1 
experiment,  in  which  ligature  of  the  leg  of  i  frog  caused  great 
i  low  the  ligature  is  probably  to  be  explained  1>\  the 
f.n  i  tin;  v.  ater  was  absorbed  by  the  skin  but  could  not  reach  the 
kidne\~.  -jure  the  blood  circulation  was  stopped.  In  regard  to 
Fi-cher1-  t  \pl. mation,  compare  the  results  of  Sidbury  and  <  ,; 
Sunmi  i  i  .in. Inded  that  in  the  fish,  the  gills  are  the  chief  -eat  of 
\ch.uige. 

It  appear-,  therefore,  that  osmosis  occurs  through  tin   iiiu-ii 
mi-Hi    unhiding  gills),  kidneys  and  gut  simultaneously,  and  since 

« 

the  contents  of  the  gut  and  kidney  tubules  are  not  the  -a me  a-  the 
medium,  u «  -hould  not  expect  an  osmotic  equilibrium  bet \\eui 
the  body  tluids  and  the  medium.  Furthermore,  all  three  .  if  these 
membrane-  m.i\-  show  irreciprocal  permeability. 

I  n  -li-\\.iter  li-li  and  non-migratory  marine  fish  an-  killed  b\ 
ii  changes  in  the  medium,  even  though  it  be  \<r\  ^r.nlual. 

I'.ert  m. tiiii. lined  that  if  fresh-water  fish  are  placed  in  sea  \\.iter. 
ilie  ^ill  i  .ipill.u  ies  contract  and  become  blocked  b\  the  distorted 
i  "i 'liii-t  le-.  lii  naked-skinned  fishes,  not  only  the  v;ills  are 
.itterted.  but  \\.iter  ma\'  be  lost  from  the  tissues. 

lien  .UK!  Simmer  both  agree  that  the  salts  in  sea  water  cannot 
IM  replaced  1  .\  anv  other  substance,  without  can-in-  tin-  death 
oi  certain  marine  fishes.  Mosso3  claimed  that  \\hen  -hark-  are 
placed  in  tn-li  \\.uer,  the  gill  capillaries  become  ><>  1  ilocke<  1  \\ith 
laked  corpu-cles  that  physiological  salt  solution  coiuM  n«»i  be 
1 1 -reed  tlimu^h  ilu-m.  He  observed  that  the  difference-  in  the 
resistance  of  ctiiain  tish  to  changes  in  the  -alt  cmuent  of  the 
medium,  i-oi-re-]ioiided  to  differences  in  the  iv-i-tance  of  their 
bl..i»d  it  11-  to  the  luemolytic  action  of  such  changes.  Simmer.1 
ho\\e\er.  -late-  that  this  blocking  of  gill  capillarie-  doe-  ma 
occur  in  -hark-  or  marine  teleosts  in  fiv-h  water. 

1  Fischer,  M.  H.,     d -l.-nia,"  J.  NViloy  &  Sons,  i<. 
x        Exper.  /•';<>.  and  Mfdicine,  1911.  \'II.,  m.j. 

/.  (,•«///'..  isyo.  N..  570. 
4  /'-         s     >-nth  Inttrnat.  Zoo/.  Congress,  Bost"n, 


152  J.    F.    MCCLENDOX. 

Sumner  showed  that  as  the  fish  becomes  enfeebled  by  the  ab- 
normal medium,  it  becomes  more  permeable  to  salts.1  Whether 
the  direct  action  of  the  abnormal  medium,  or  the  blocking  of 
the  gill  capillaries,  produce  the  increase  in  permeability,  has  not 
been  experimentally  tested.  However,  the  gills  themselves  would 
not  be  asphyxiated  by  blocking  of  their  capillaries,  and  it  seems 
probable  that  the  change  in  permeability  is  due  to  the  direct 
action  of  the  medium. 

We  may  conclude  therefore  that  the  death  of  the  fish  results 
from  the  osmotic  exchange.  This  may  be  sufficient  to  cause  death 
while  the  fish  still  maintains  its  normal  semi-permeability,  or 
death  may  occur  only  after  increase  in  permeability,  due  to  the 
direct  action  of  the  medium  on  the  osmotic  membranes. 

A  similar  increase  in  permeability  may  explain  Wo.  Ostwald's 
observations  on  fresh-water  Gammarus  in  pure  salt  solutions.2  He 
found  that  the  ratio  of  the  rapidity  of  death  to  the  concentration 
is  about  constant  up  to  a  certain  point,  above  which  it  is  much 
greater.  This  critical  concentration  has  nothing  to  do  with  the 
osmotic  pressure,  since  it  is  different  for  different  salts.  Perhaps 
at  this  concentration  the  salt  made  the  membranes  more  per- 
meable. 

Schiicking3  found  that  nicotine  and  strychnine  made  the  skin 
of  Aplysia  more  permeable  to  salts.  Since  cocain  retarded 
shrinkage  in  hypertonic  solution,  he  supposed  that  the  hydro- 
static pressure  produced  by  the  muscles  aided  shrinkage.  How- 
ever the  hydrostatic  pressure  is  probably  very  small,  and  the 
effect  might  have  been  due  chiefly  to  an  increase  in  permeability 
to  salts,  produced  by  the  cocain. 

3.  Secretion  of  Lymph  and  Tissue  Juice, 

Hober  supposes  the  raising  of  the  osmotic  pressure  by  the  kata- 
bolism  of  the  tissues,  causes  fluid  to  be  drawn  out  of  the  blood- 
vessels, and  states  that  the  lymph  in  the  thoracic  duct  has  a 
greater  osmotic  pressure  than  the  blood. 

Traube   states   that   the   surface   tension   of   transudates   and 

1  Cf.  Greene,  above. 

2  PJliiger's  Arch.,  1905,  CVI.,  568. 

*  Arch.  Anal.  Physiol.,  Physiol.  Abt..  1902,  533. 


TENSION    PHENOMENA    OF   LIVING    ELK  MI-NTS.  153 

exudate-  is  always  greater  than  that  of  the  blood.  He  cites  a 
case  in  which  a  transudate  was  caused  to  be  absorbed  by  injecting 
into  it  a  substance  which  decreased  its  surface  tension. 

4.  Excretion. 

Milk  and  bile  have  about  the  same  osmotic  pressure  as  the 
blood,  but  urine  is  almost  dry  in  some  animals:  it  i-  u-ually 
In  I M  rtonic  in  man  but  may  be  hypotonie. 

Traube  maintains  that  the  surface  tension  of  the  normal  urine 
is  alu  r  than  that  of  the  blood,  and  that  thi-  is  the 

dri\  iii;^  Ion  e  in  excretion. 

Houe\<r.  ll<.licr  and  others  suppose  that  tin-  -iib-iam •< -s  to 
•  1  may  be  formed  into  solid  bodies  in  tin-  tubule  cells, 
and  ihroun  out  into  the  hi  men. 

It'  lipoid-iii-oluble  dyes  arc  fed  to  frogs,  granule-  in  tlu-  « •« -11- 
ol  (i-riain  -i  ^mrnts  of  the  kidney  tubule  are  staim-d  \\iih  them. 
Tin-  <l\i  i-  not  tirst  excreted  by  the  glomeruli  and  then  ab-orbed 
from  tin  lumen  by  the  tubule  cells,  for  if  the  vena  Jacol >-.  mi, 
which  -ii|>|>lies  the  tubules,  is  ligatured,  no  staining  occurs,  al- 
though the  renal  arteries  still  supply  the  glomernli. 

Ihi-  -tained  granules  in  the  tubule  cells  are  thrown  out  into 
tin-  lumen  and  pass  into  the  bladder.  The.-c  granule-  u-ually 
di—  ol\  e  to  form  a  slimy  substance  in  the  urine,  bin  -ome  oi  them 
max  remain  intact. 

The  circulation  in  mammalian  kidneys  cannot  In-  controlled 
in  the-  -aim-  \\ay,  but  after  intravenous  injection  .  >f  ,(  certain 
lipoid-in-olublc-  dye,  no  stain  may  be  detected  in  the  \\all-  o| 
the  glomeruli,  although  the  tubule  cells  are  staineil.  The  -tain 
in  the  lumen  does  not  appear  above  the  level  of  the  -tained  tubule 
ci'lls.  In  the  excretion  of  carmine,  it  may  be  found  in  -ranule- 
in  the  tubule  cells  and  lumen,  similar  to  those,  found  in  frog's 
kidtiex  3. 

h  ha-  been  -upposed  that  urea  is  excreted  by  collecting  in 
the-e  granule-,  and  passing  out  with  them,  but  it  would  be  e\en 
sim;iK-r  t«>  a--ume  that  some  substance  is  excreted  into  the  lumen, 
\\hich  combine-  with  urea  and  so  lowers  the  concent  ration  of  that 
in  solution,  thus  accelerating  its  excretion. 

The  chief  recommendation  for  the  granules  is  their  valve-like 


154  J-    Ir-    MCCLENDON. 

action,  which  would  account  for  the  secretion  of  urine  against  a 
concentration  gradient,  but  a  simpler  mechanism  of  such  a  process 
is  shown  in  Hamburger's  double  membranes. 

The  blood  pressure  may  aid  in  the  secretion  of  the  water  of 
the  urine,  which  is  eliminated  chiefly  through  the  glomertili, 
but  its  insignificance  in  the  elimination  of  urea  is  shown  by  the 
fact  that  after  increasing  the  volume  (and  therefore  pressure)  of 
rabbit's  blood  70  per  cent,  by  transfusion,  the  urea  elimination 
was  not  or  only  very  slightly  increased. 

VII.  CELL  DIVISION. 

Various  hypotheses  as  to  the  cause  of  cell  division  have  been 
advanced  by  the  morphologists.  Hertwig,  supposed  that  when 
the  ratio  of  nucleus  to  cytoplasm  is  less  than  normal,  the  cell 
will  divide.1  Gerassimow2  subjected  cells  of  Spirogyra  to  low 
temperatures  and  other  abnormal  conditions  and  obtained  an 
increased  amount  of  chromatin  in  some  of  them.  These  cells 
did  not  divide  until  the  ratio  of  nucleus  to  cytoplasm  was  as 
great  as  at  the  time  of  division  of  a  normal  cell. 

I  found  that  chromatin  is  not  necessary  for  cell  division.3 
After  extracting  the  chromosomes  from  the  starfish  egg,  I  caused 
it  to  divide.  In  this  case  the  ratio  of  nucleus  to  cytoplasm  was 
zero;  however  the  cell  did  not  continue  to  divide  indefinitely. 

There  is  no  easy  method  of  determining  the  ratio  of  nucleus 
to  cytoplasm.  Some  cells  contain  large  vacuoles  \vhose  contents 
are  not  considered  as  cytoplasm.  Eggs  contain  fat  drops  and 
granules  compounded  of  protein  and  lipoids.  These  are  not 
considered  as  cytoplasm  by  all  investigators.  If  the  granules  and 
oil  are  included  as  cytoplasm,  the  ratio  of  nucleus  to  cytoplasm 
is  very  small,  and  yet  the  egg  cell  does  not  divide  unless  "stimu- 
lated" by  the  sperm  or  some  other  means. 

k.  Lillie4  observed  that  chemical  substances,  which  in  low 
concentration  cause  the  Arbacia  egg  to  divide,  in  high  concen- 
tration cause  outward  diffusion  of  the  red  pigment  (echinochrome) 
and  compared  this  to  the  laking  of  erythrocytes. 

1  lie  is  not  confirmed  by  Conklin,  Jour.  Expcr.  Zoo!..  1912,  XII..  i. 
-  Bull.  Soc.  Imp.  Nat.,  Moskau,  I<;<M.  No.  i. 

3  McClendon,  Arch.  f.  Entwicklungsmcch.,  1908,  XXVI,  662. 

4  Bioi..  BULL.,  1909,  XVII.,  188. 


TENSION    PHENOMENA    OF    L1VINC,    KLI.MENTS.  155 

This  is  made  more  striking  by  the  fact,  mentioned  first  by 
Loci),  that  haimolytic  agents  are  effective  in  artificial  partheno- 
ui -nesis.  R.  Lillio  observed  that  pure  solutions  of  sodium  salts 
caused  the  egg  to  divide,  the  order  of  effectiyene-s  of  unions  being 
'Br<ClO3<XO3<CXS<I.  He  also  found  that  these  salts 
could  !>e  inhibited  by  others  (CaCl;,  MgCl2),  as  i-  characteristic 
of  the  antagonistic  effects  of  salts  in  physiological  phenomena, 
and  tin-  pr.-i  ipitation  of  colloids. 

1  found  that  the  sea  urchin's  egg  contains  fatty  sul Mance-,  and 
ielati\el\  large  amounts  of  lecithin  probably  in  combination  with 
pioirid-..  I  found  that  Toxopneustes  eggs  freed  from  tin.-  jclly- 
likc  ci.\crin^s,  contained  about  10  per  cent.  lecithin  'alcohol 
extract  p|it.  with  acetone)  and  about  2  per  cent,  of  an  extract 
-olul'le  in  alcohol  or  acetone  and  containing  rosette-.  <>t  tat -like 
taU.  This  extract  blackened  strongly  with  o-mic  tein>\ide 
and  «-lter\e-ced  on  adding  dry  Xa-carbonate  in  \\atcr,  Hun 
emul-ilied.  probably  it  contained  unsaturated  fatt>  aciil. 

\<.»i<ling  to  a  private  communication  by  Mathews,  tin-  « 
of  i  he  -taiii-h  contains  lecithin  and  an  unsaturated  tatty  acid, 
but  no  (  ho]rMerin.  In  this  last  characteristic  ii  diller-  markedly 
from  the  eiAthrocyte.  There  is  no  way  of  determining  \\hether 
tin -i  -ub-.t.inces  enter  into  the  composition  of  the  plasma 
membrane,  but  the  facts  are  presented  in  order  to  indicate  the 
pi  '--ibiliiies. 

\\  e  haM'seeii  that  the  exit  of  haemoglobin  i>  probably  nm  due 
t.»  in.  i,  MM. I  permeability  to  this  substance.  It  is  po--ible  ihat 
the  same  i-  true  of  echinochrome.  I  found  that  the  echiuochroine 
in  the  e;cu  -hows  a  continuous  spectrum,  when-a-  that  extra<  led 
in  \arioii-  ways  shows  characteristic  band^.  It  ma\  po-^iMy 
In  held  b\  chemical  combination  in  the  e. 

llo\\»-\er  I  found. other  evidence  for  increase  in  permeability  of 
the  sea  urchin's  egg  coincident  with  be^innin^  d«-\<  lopment :' 

t.  1  eiiili/cd  eggs  are  caused  to  shrink  more  quickly  than  un- 
t\-i  tili/i  .1  eggs,  with  isotonic  sugar  solution.  Presumably  the 
fertili/ed  euv;s  are  more  permeable  to  the  substances  exerting 
the  internal  osmotic  pressure. 

j.  Tin-  electric  conductivity  of  the  euy  increases  about  '  \  \\hen 

\1,  Cl. -in Ion.  Attifr.  Jour.  Physiol.,  1910.  XX\  II.,  240. 


156  J.    F.    MCCLEXDOX. 

it  is  fertilized  or  made  parthcnogenetic  with  acetic  acid,  indicating 
increased  permeability  to  ions. 

Lyon  and  Shackell1  and  Harvey2  observed  that  methylene 
blue  and  neutral  red  enter  fertilized  eggs  more  quickly  than 
unfertilized  eggs.  Harvey  supposed  that  only  the  free  color 
base  (undissociated)  entered,  since  the  addition  of  a  little  acid 
to  the  sea  water  prevented  the  staining  of  the  eggs. 

Mathews3  considered  the  penetration  of  stains  into  the  egg 
as  a  chemical  process  (the  stain  forming  a  salt  combination  with 
the  lecithin  or  proteins  of  the  egg  surface). 

Harvey  observed,  further,  that  NaOH  penetrates  fertilized 
more  easily  than  unfertilized  eggs,  but  the  eggs  are  killed  by  the 
alkali. 

The  fact  that  the  unfertilized  frog's  egg  continues  to  swell  for 
a  long  time  in  water  (Biataszewitz)  whereas  the  osmotic  pressure 
of  the  fertilized  frog's  egg  is  quickly  reduced  to  equal  that  of  the 
medium  (Backmann  and  Runnstrom)  indicates  increase  in  perme- 
ability to  osmotic  substances  on  fertilization.  In  this  connection 
it  is  interesting  to  note  that  Bataillon,4  Brachet,  and  myself5 
caused  the  unfertilized  frog's  egg  to  rotate  normally  and  segment 
merely  by  pricking  it. 

1 1  has  been  supposed  by  various  observers  that  the  "forma- 
tion" of  the  fertilization  membrane  in  very  closely  related  to  the 
segmentation  of  the  egg.  Loeb  observed  that  the  sea  urchin's 
egg  may  develop  without  the  formation  of  a  fertilization  mem- 
brane, and  I  have  confirmed  this  observation,  and  shown  that 
it  is  very  probably  wrong  to  suppose  that  this  is  a  case  of  failure 
in  "pushing  out"  of  the  membrane.  Apparently  "membrane 
formation"  is  not  essential  for  the  segmentation  of  the  egg, 
although  by  furnishing  protection  it  may  insure  the  development 
of  the  embryo. 

Loeb  postulated  that  an  osmotically  active  colloid  exists  in 
the  unfertilized  egg,  but  is  so  covered  with  lipoids  that  it  does 
not  absorb  water  until  it  is  squeezed  out  or  otherwise  exposed 

1  Science,  1910,  XXXII.,  250. 
=  Ibid.,  p.  565. 

3  Jour.  Phurmacol.  and  Exp.  Ther.,  1910,  II.,  201. 

4  Arch.  Zoo/.  Exper.,  1910  (5),  VI.,  101. 

8  McClcntlon,  Amer.  Jour.  Physiol,,  1912,  XXIX..  298. 


TENSION    PHENOMENA    OF   LIVING    ELEMENTS.  157 

at  the  surface  of  the  egg,  at  the  beginning  of  development  (when 
it  fills  the  so-called  " perivitelline  space").  I  observed  that  this 
substance  bears  a  positive  charge  (is  basic)  since  it  migrates 
toward  the  kathode  when  an  electric  current  is  passed  through 
water  containing  the  fertilized  egg. 

The  unfertilized  egg  is  imbedded  in  a  mass  of  jelly  which  is 
probably  inucin.  This  jelly  bears  a  negative  charge  (is  acid) 
since  it  combines  with  color  bases. 

When  the  positively  charged  colloid  is  exposed  at  the  Mirface 
ise   in   permeability)   and   come>   in   contact   \\ith    the 
ne^.imeh    (harmed  jelly,  the  two  mum. illy  precipitate  at  their 
MII"  t' »ntuct,    thus   forming   the   fertilization   membrane. 

Mm  it  .ill  "t"  the  jelly  is  washed  off  of  the  egg  before  ihe  l.n  u-r  is 
(.iu-i.1  i"  develop,  no  fertilization  membrane  i-  (Wined  .1-  I 
h,i\r  <ib-er\ed)  because  no  two  oppositely  charged  colloid^  are 
bronchi  in  •  <  nMct,  but  the  basic  colloid  may  with  difficulty  be 
Bi  'ii  as  .1  n  fra<  live  layer,  which  has  been  mi-taken  for  a  p->'>rlv 
developed  "fertilization  membrane." 

flu  oba  i  \  .it  ion  of  Lyon1  makes  it  appear  that  catal.i-e  conu-> 
out  of  fertili/ed  more  quickly  than  unfertilized  •  v. -.  prokil>l\  due 
t"  in-  1 1  .i-ed  permeability. 

l.\i.n  "b-crved  that  C().j  came  out  of  fertili/ed  more  <|ui«  kl\ 
th.iii    iiiilVi  tili/ed    eggs,    and    (  ).    Warburg.     l."cb    .uul    IU\M!! 
ob-iTM-d   that  oxygen  is  absorbed  more  rapidly  by  the  lonui-r. 
\\«-   miiilit    a-k:    I  >oe>   increased   permeability   allow    increased 
o\i<l.i!i"U,  or  is  increased  oxidation  the  primarx    cause  "f   the 
incrc.iM-il  respiration? 

The  p«-rineability  change  is  the  simplest  :  xplaiiatimi,  and  in 
\\liat  other  \\a\-  could  oxidation  be  inciv.i-rd  J  I  .neb  -uppo-i-d 
tin-  >pcnu  carried  an  oxidase  into  the  egg.3  ISut  QO  addition  .  ,t 
oxidate  i-  concerned  in  artificial  parthenogenesis,  and  I  .orb 

Mined  that  the  oxidase  (or  other  en/\  me,  kina-r.-'i  i-  held  in 
the  egg  prriphery  and  cannot  penetrate  the  e;^^  interior  until 
the  permeability  is  increased. 

In  addition  to  oxygen,  oxidase,  and  escape  of  CO2,  hydro\yl 

im.J  .  1909,  IV..  199. 

\1.<   Mini, .n  an.l  M  iti.  hell.  Jour.  Biol.  C  u.  X..    . 

*  In  tin-  « .piiiuvti-'ii  it  K  interesting  to  note  that  M.IMII.U.   '/.fil.   j>hy>i>-     < 
iyi".  1   \\  I      265.  i.iili-il  to  tin«l  inoir  iron  in  s|n-rm  than  in  sea  water. 


158  J.    F.    MCCLEXDON. 

ions  are  necessary  for  the  rapid  oxidation  of  the  sea  urchin  egg 
(Loeb),  and  Harvey  showed  that  the  unfertilized  egg  is  practi- 
cally impermeable  to  OH  ions  of  low  concentration.  The 
increased  permeability  allows  hydroxyl  ions  in  the  sea  water  to 
penetrate  the  egg,  as  shown  by  Harvey,  and,  since  the  sea  is 
always  alkaline,  this  may  explain  the  increased  oxidation. 

Asters  always  develop  in  the  egg  before  segmentation.  In  the 
normal  egg  these  have  some  relation  to  the  division  of  the  nucleus, 
but  even  if  a  nucleus  is  not  present,  1  have  observed  that  the 
cytoplasm  constricts  along  a  line  on  the  surface  farthest  removed 
from  the  centers  of  the  asters. 

The  constriction  of  the  cytoplasm  is  probably  due  to  a  band  of 
increased  surface  tension  (or  to  decreased  surface  tension  at 
the  poles).  This  might  be  caused  by  local  increase  in  perme- 
ability to  ions,  causing  decreased  polarization,  at  the  equator 
(or  increased  polarization  at  the  poles,  clue  to  increased  pro- 
duction of  the  polarizing  electrolyte  in  the  asters). 

The  same  reasons  that  were  given  for  assuming  that  the  surface 
of  the  Amceba  is  electrically  polarized,  hold  good  for  the  egg. 
The  first  change  is  probably  a  general  increase  in  surface  tension, 
indicated  by  rounding  up  of  the  egg.  Later  this  may  become 
localized  from  internal  causes  and  result  in  cleavage. 

Hyde1  observed  local  changes  in  electric  polarization  of 
Fundulus  eggs  during  cleavage,  indicating  that  surface  tension 
changes  and  cleavage  are  due  to  this  cause. 

It  has  been  objected  that  the  segmentation  of  the  egg  is  not  a 
typical  case  of  cell  division,  since  the  egg  cell  is  "wound  up" 
and  ready  for  some  "stimulus"  to  set  it  going,  whereas  tissue 
cells  must  "grow"  or  "rest"  after  each  division  before  dividing 
again. 

1 1  may  be  true  that  growth  is  prerequisite  to  division,  but 
this  cannot  be  formulated  quantitatively.  In  the  spore-forma- 
tion of  certain  organisms,  a  cell  may  divide  in  a  relatively  short 
time  into  myriads  of  almost  ultra-microscopic  cells. 

Hertwig  may  be  right,  in  general,  in  assuming  that  the  relative 
growth  of  nucleus  and  cytoplasm  influences  division,  but  the 
difficulties  in  proving  this  have  been  indicated,  and  this  cannot 

1  Am.  Jour.  Physiol.,  XII..  241. 


TENSION  FHEM'Mi-.NA  OF  LIVING  ELEMENTS.  159 

be  e\pn---ed  in  chemical  terms.  It  is  generally  supposed  that 
nucleic  acid  is  a  more  abundant  constituent  of  the  nucleus  than 
of  the  cytoplasm,  but  much  evidence  ha-  appeared  for  believing 
that  iti-  often  present  in  considerable  quantities  in  the  cytoplasm. 
l.o«-l>  -upposed  that  the  segmentation  of  the  sea  urchin  egg  is 
accompanied  by  an  "autocatalytic"  svntlu--i-  of  nucleic  acid, 
HIM -I-  tin-  nuclei  increased  in  number.  But  Ma-in-1  and  more 
recentlj  ^hackell2  by  chemical  analysis  found  as  much  nucleic 
.11  id  in  the  unsegmented  egg  or  i-cell  st.ro  as  in  the  Ma-tula 
-i.i. 

I  IM n  i-  -omc  indirect  evidence  that  increase  in  permeability 
ina\  e.m-e  an  increased  division  rate  of  tis-ue  cell-.  Though 
ii-ll  Drouth  may  inlluence  division,  it  is  probable  that  permc- 
abiliix  inlliienccs  growth. 

Y.iriou-  "-tiniuli"  cause  increased  proliferation  of  cells  of  the 
-<  Tinin.il  layer  of  the  skin.  It  is  commonly  known  that  mechani- 
•  ,il  -tiimili  increase  growth  of  the  skin. 

I'.tinlian!    1  i.-her    observed   that    Sudan     111.   or  Scharlack    \\ 
cause   iinii-.i^ed  proliferation  of  the  epidermi-.      \\lu-n    iln    d\e 
Is  inje<  i<  d  under  the  skin  of  a  rabbit   tin-  skin  ^r<>\\  -  ti.uard  the 

dye. 

I  m-r  fi'imd  that  gradual  increase  of  temperature  caused  a 
( -.in --ponding  increase  in  proliferation  of  ti— ne  ci-ll-  due  io 
incre.iM-d  chemical  reaction  and  inflammation  <>t  the  ii--n>  . 
Hut  \\hen  a  certain  temperature  was  reached  a  Midden  jump  in 
i  he  incre.i-e  in  proliferation  was  observed  wiihoui  .1  corresponding 
iuciva-e  in  inflammation.  This  is  similar  to  ihe  plieiioineiiini 
-ten  in  iinlei  lili/ed  eggs,  where  a  rise  in  temperature  l.e\nnd  a 
cei  lain  point  causes  segmentation. 

It  h.i-  .ilso  been  observed  that  electrical  Mimulation  max 
can-e  increased  proliferation  of  tissue  cell-. 

\ll  of  these  changes  (electrical,  thermal,  or  mecliaiiic.il  -limu- 
laiion.  or  tn-atment  with  lipoid  soluble  .-ub-tances)  cause  in- 

1  /.,-it.  /•'.\MO/.  Chem..  1910,  LXXVII..  161. 

.  eji  i .  n.  s.,  XXXIX'..  573. 

1  \Vliii-h  an-  practically  insoluble  in  water  but  soluble  in  fats  and  lipoids  and,  as 
1  II.IM-  iili<i-i\t-il.  -lightly  in  lipoid-protein  combinatii'- 

4  S.-r  v.    I  >uni;rrn   u.  Werner,  "Das  Wesen  Bosartigen  Gesch\\  iil-tf,"   I.cip/i.i;, 
.  p.  65. 


160  J.    F.    MCCLENDON. 

creased  permeability  and  segmentation  of  the  sea  urchin's  egg. 
Therefore,  from  analogy,  we  may  conclude  that  increase  in 
permeability  may  cause  tissue  cells  to  divide. 

The  "wound  stimulus"  to  regeneration  of  tissue  may  also 
cause  increased  permeability  of  the  cells. 

In  a  preceding  chapter  it  was  shown  that  the  "current  of 
injury"  produced  by  the  negative  electric  potential  of  a  wounded 
surface  is  common  to  animal  and  plant  tissues.  The  wounded 
cell  acts  as  an  electric  generator  and  a  current  flows  through 
neighboring  cells. 

I  observed  that  if  a  current  is  passed  through  living  tissue, 
which  is  subsequently  fixed  and  stained,  basophile  substances 
will  be  found  displaced  toward  the  anode.  In  sections  of  tissue 
adjacent  to  a  wound  the  extent  of  the  current  is  indicated  by  the 
displacement  of  basophile  granules.  The  current  affects  first 
the  cells  in  contact  with  the  wounded  cells,  then  extends  in  some 
directions  more  than  others.  Electric  currents  ("currents  of 
growth")  continue  for  many  days  after  the  wound  has  healed. 

Since  electric  currents  cause  sea-urchin  eggs  and  tissue  cells 
to  divide  and  proliferate,  probably  these  bio-electric  currents 
constitute  the  so-called  "formative  stimulus"  of  regeneration. 

Embryonic  cells,  cells  of  germinal  regions,  and  cancer  cells 
are  distinguished  by  their  great  power  of  proliferation,  or  rapid 
division.  It  is  probable  that  the  plasma  membranes  of  these 
cells  are  more  permeable  than  those  of  other  tissue  cells  in  the 
same  medium  or  under  the  same  conditions. 

Cancers  have  been  produced  by  the  action  of  X-rays  (electric 
pulsations)  on  the  skin.  The  cells  in  the  skin  were  so  changed 
that  they  proliferated  more  rapidly.  Similarly,  electric  changes 
have  been  observed  to  start  the  egg  cell  to  rapid  proliferation. 
There  is  probably  some  irreversible  change  in  the  permeability 
of  these  cells,  which  does  not,  however,  make  the  plasma  mem- 
brane incapable  of  subsequent  reversible  changes  in  perme- 
ability (i.  e.,  the  change  is  unlike  what  occurs  at  death  of  the  cell). 

The  suggestion  that  cancer  cells  are  more  permeable  than 
tissue  cells  in  general  may  possibly  be  of  therapeutic  importance. 
Loeb  has  shown  that  fertilized  eggs  are  more  sensitive  than  un- 
fertilized eggs  to  various  toxic  substances  (probably 


TENSION    PHKN'iMI  NA    OF   LIVING    ELEMENTS.  l6l 

these  sub-tances  enter  the  fertilized  eggs  more  easily).  The 
same  explanation  may  po--ibly  he  applied  to  the  effect  of  sugar 
on  ten. tin  living  cells.  The-  unfertilized  eggs  of  the  frog,  petro- 
niyzon,  sea  urchin  and  annelid  have  been  caused  to  segment, 
by  placing  them  in  sugar  Dilution-.  Mayerhofer  and  Stein1  ob- 
.  <•'!  that  sugar  in  certain  concentrations  increased  the  per- 
meahility  of  the  gut  to  certain  -.ill-,  ami  in  this  condition  the 
ytn  was  more  easily  injured  by  the  diffusion  of  substances. 

similarly  Mockard  obser\  ed  that  sugar  increased  the  toxicity 
"t  pun-  -oliitions  of  salts  on  the  Fundulus  egg.  Morgan  and 
stiH  k.nd  -hi.ued  that  ih\<  \\a-  not  dm-  to  the  inversion  of  sugar 
or  to  thr  o-motic  pressure,  and  suppn-ed  that  the  sugar  might 
combine  chemically  with  the -alt.  It  >cems  probable  that  the 
ii  increased  the  permeal.ilitx  t"  -.ilt.  The  fact  that  sugar 
in  Ire-h  \\ater  is  toxic  whena-  the  -amc  amount  of  sugar  in 
tin-  n»imal  medium  (sea  water  Uimt  toxic  or  less  toxic,  indicates 
that  tin-  -alts  within  the  l-'nndidit  are  the  same  as  those 

out-idi-  in  sea  water),  and  iiicrea-e  in  permeability  to  them 
d«'<--  nut  lead  to  diffusion  while  they  remain  in  sea  water,  but 
diltii>ioii  takes  place  in  fresh  \\ateiv 

It  it  In  -liiiun  that  cancer  cell-  are  MHHV  permeable,' substances 
Mia\  he  toiiml  which  kill  cam  er  cell-  mure  easily  than  tissue 
i  (II-  as  i  \pl. lined  below. 

\\liena-  a  certain  men  .i-e  in  j HTMU -ability  of  the  cell  seems 
in  cau-e  di\i-imi,  a  very  :^reat  iih  in  permeability  causes 

death  h. i  nicK  -is,  cytoK  -i-.  hai  terii il\  <i- '• .  It  has  been  shown 
that  certain  l\  -ins  are  spt  cilic  t'm-  certain  cells,  probably  because 
the  pla-iiia  Mieinhraiie-  of  ilu-e  cells  differ  chemically. 

I  In-  fertili/ed  e--  is  more  easily  c\  t«|\  /od  than  the  unfertilized 
i  tain  -lib-lances.      It   then-tore  appears  that  the  more 
peimeable   the  cell   is  in   the  beginning,  the  more  easily  is  the 
IH  i  ineahiliiN    brought  to  the  point  \\hit-hcausescytolysis. 

1  It  nee  ii  i-  pmhahle  that  certain  substances  may  be  found  by 
\\  hich  cancer  cell-  tan  1  «e  nn  >\  e  ea-ily  cytolvzcd  than  normal  tissue 
cells. 

•n.  /.fit..   1910,   \\V1I..  376. 

-  BIOL.  r.i  M  ..  [907,  xiii..  - 

3In  tin   .il  -IIU.H  I  have  -tiown  that  no  diffusion  take3  place  in  fresh 

\\.iu-r.      Am fr.  Jour,   i  \.XIX..  295. 


1 62  J.    F.    MCCLENDOX. 

It  has  been  shown  that  narcosis  is  accompanied  by  decreased 
permeability.  On  the  other  hand,  certain  forms  of  inhibition 
of  muscle  are  accompanied  by  an  increase  in  permeability. 
May  certain  cells  be  inhibited  in  proliferation  by  an  increase 
in  permeability,  too  great  for  cell  division  but  not  great  enough 
for  cytolysis?  The  great  oxidation  rate  in  eggs  inhibited  in 
cleavage  by  very  hypertonic  solutions  as  determined  by  Warburg, 
seem  to  indicate  this. 

It  has  been  shown  that  certain  tissue  cells  inhibit  the  pro- 
liferation of  others.  In  the  healing  of  wounds,  the  epidermis 
inhibits  the  growth  of  connective  tissue.  If  a  wound  remains 
uncovered  by  epidermis  for  a  relatively  long  time,  processes  of 
connective  tissue  may  grow  outward,  but  this  is  prevented  by 
the  growth  or  transplantation  of  epidermis  over  the  wound. 

Perhaps  the  proliferation  of  the  connective  tissue  is  due  to 
abnormal  "stimuli"  (bio-electric  currents,  diffusion  of  sub- 
stances) such  as  cause  proliferation  in  regenerating  tissue  gen- 
erally. The  presence  of  epidermis  over  the  wound  might  protect 
the  connective  tissue  from  these  "stimuli." 

The  foregoing  facts  and  the  speculations  based  on  them  may 
not  be  of  far-reaching  importance  in  themselves,  but  they  suggest 
lines  of  research,  which  if  followed,  it  is  hoped,  will  add  a  great 
deal  to  cell  physiology  and  pathology  and  be  an  aid  to  the  under- 
standing of  many  problems  in  therapeutics. 


THE   LARVA   OF  SARCOPHAGA,   A   PARASITE   OF 

CISTUDO   CAROLINA  AND   THE   HISTOLOGY 

OF    ITS    RESPIRATORY  APPARATI  - 

\VM.  A.  KEPNER. 
UNIVERSITY  OF  VIRGINIA. 

The  ^tiulcnt  of  zoology  is  early  impressed  with  the  intcii-ive 
ni.iniuT  in  which  animal  life  has  penetrated  everv  available 
-pace.  Even  so  remote  and  strange  place  as  the  poison  ijand- 
o|  i In-  rattle-snake  have  been  entered,  these  glands  furnishing 
ample  ]  mil  i -id  and  oxygen  supply  fora  little  nematode  ih.it  make- 
i  In  in  it-  habitat.  In  the  example  of  this  Sarcophaga  we  find 
a  lly  that  lias  entered  the-  nucha  of  the  "box-turtle"  —a  region 
oi  i  he  body  where  its  larva  will  not  be  exposed  to  serious  pre— tire 
bet  \\een  parts  of  the  "turtle's"  body  and  where  it  will  al-o  be  five 
from  tin  attacks  of  the  appendages  and  mandibles  of  tin-  ho-t. 
The  occurrence  of  this  parasite  in  Cistndo  was  first  ob-«i\id 
b\  I'ai  kard  ('82).  Packard  described  and  figured  it  as  in  u^'rid 
larva.  Tim-,  so  far  as  I  have  been  able  to  determine,  arose  the 
b.e-i-  for  believing  that  a  "bot-fly"  infested  a  reptile.  Aldrich 
in  n  terring  to  Packard's  paper  states  that  perhap-  it  is 
not  an  o  -trid.  Shar[>e  in  the  Cambridge  Natural  lli-tory  says 
that  <1  -trididie  may  occur  in  the  reptiles.  Wheeler  ('90  next 

>nU  the  occurrence  of  the-  dipteron  lar\^e  on  tin-  nucha  of 
iirolina.  He  succeeded  in  getting  the  larvae  to  pupau- 
and  iii  rearing  imagines  from  the  pupae.  These  adult  Hie-  proved 
to  belon-  to  the  genus  Sarcophaga  and  not  to  be  cestrid  llie-;. 
Thn>  there  appears  to  remain  no  evidence  of  a  " bot-fly  "  infesting 
a  ivpiile. 

In  Octobi-r,  H)io,  a  female  specimen  of  Cistudo  Carolina  was 
brought  into  my  laboratory.  It  was  kept  through  the  winter 
in  a  sink.  January,  1911,  a  student  called  my  attention  to  what 
lu-  called  a  "growth"  in  the  nucha  of  the  ri^ht  >ide.  This,  h<>\\  - 
ever,  proved  to  be  an  insect  larva.  Two  days  later  the  lar\a 

iped    from    the   perforation   made   in    the   skin   of   the   host. 

163 


1 64  WM.    A.    KEPNER. 

Subsequently  two  other  larvae  left  the  excavated  region  of  the 
nucha.  These  specimens  were  preserved  in  alcohol.  May  14, 
the  fourth  and  most  vigorous  larva  dropped  from  the  host. 
This  larva  was  placed  upon  soil  in  a  box  where  it  burrowed  into 
the  earth  and  formed  an  oval,  dark  brown  pupa.  This  pupa 
has  not  yielded  an  imago,  so  that  I  have  been  unable  to  cor- 
roborate Wheeler's  diagnosis  as  based  upon  the  adult  fly. 

Except  for  some  details  which  are  readily  overlooked  in  pre- 
served specimens,  such  as  Packard  had,  the  larvae  I  found  closely 
resemble  the  figures  and  descriptions  given  by  Packard.  With 
the  living  material  which  I  had  at  my  service,  I  was  able  to  see 

| 

details  which  make  these  larvae  correspond  more  closely  to  the 
following  description  of  larvae  of  Sarcophagidae  than  to  that  of 
(Estrididae  larvae.  Brauer  ('83)  says  that  the  larvae  of  Sarco- 
pluigidae  "are  rounded,  thinner  anteriorly  and  amphipneustic. 
The  antennae  are  short,  thick,  cylindrical,  divergent,  wart-like 
tubercles,  each  with  two  ocellus-like  chitinous  rings  at  the  tip. 
The  mouth  hooklets  are  distinct,  strongly  curved  and  separated 
from  each  other.  The  abdominal  segments  are  distinctly  dif- 
ferentiated by  transverse  swellings  and  are  each  provided  with  a 
girdle  of  spines.  The  hind  stigma-plate  is  situated  in  a  deep 
cavity,  which  is  formed  by  the  last  segment  alone.  The  anal 
swelling  is  two-pointed.  The  puparium  is  oval.1"  Thus  I  am 
led  to  infer  that  I  have  the  same  kind  of  larva  that  Packard  had 
figured  and  described  and  am  able  to  corroborate  Wheeler's 
statement  that  this  is  not  a  "bot-fly"  larva  but  a  sarcophagid 
larva. 

Apart  from  this  I  have  been  interested  in  certain  details  that 
no  one  has  recorded  for  this  particular  sarcophagid.  Figure 
I  represents  the  dorsal  aspect  of  the  larva  magnified  ten  diameters. 
Each  segment  is  seen  to  bear  a  band  of  spines.  The  antennae 
are  seen  from  the  ventral  side  (Fig.  6,  ant.}  together  with  the 
strongly  curved,  (list i net  mandibles  (Fig.  6).  On  the  ventral 
side  of  the  posterior  segment  there  is  a  trilobed  disc  armed  with 
stout  spines  (Fig.  3  and  Fig.  5,  d).  This  may  function  as  a 
sucking  disc.  The  posterior  rn<l  of  the  last  segment  is  divided 

1  This  translation  of  Brauer's  description  was  taken  from  Williston's  "N'oitli 
American  Diptera,"  3d  ed.,  page  349,  by  Dr.  J.  M.  Aldrich. 


THE    LARVA    OF    SARCOPHAGA.  165 

into  a  wide,  dorsal  lobe  and  a  narrow,  projecting,  ventral  lobe. 
Between  these  two  lobes  is  a  deep  recess  into  which  the  anus  and 
po-n -rior  -ligmata  open.  The  posterior  stigmata  are  guarded 
by  a  large  -tigma-plate  which  has  two  lobes.  Each  lobe  bears 
three  spatulate  chitinous  bars  (Fig.  4,  c.p.}  which  articulate  with 
six  similar  bars  on  the  ventral  lobe  of  the  segment  (Fig.  4,  c'. /?'.). 
The  -hape  and  relation  of  these  dorsal  and  ventral  rhitim>u>  bars 
i<>  ea<h  "i her  are  such  that  I  am  led  to  believe  that  thev  function 
.1-  pn -In -unle  structures;  the  lower  lobe  of  tin-  se-ment  pressing 
ii-  bar-  .t'_,iinst  the  bars  of  the  stigmatic  plate  can  lay  hold  of 
the  \\all  of  the  excavated  region  in  the  skin  of  the  ho-t  and  thus 
anrhor  tin-  larva.  The  most  striking  feature  to  which  attention 
not  1'i-t-n  called  is  the  presence  of  two  anterior  stigmata 

Fig.  1  These  stigmata  are  fan-shaped  struct  tin--  \\hicli 

bear    -i  \euirrii   or  eighteen    papilhe  along   their   let  initial   edge 

I  i.  I.).      In   a  specimen  cleared  with  xylol   each   ot"   tl 

niata  can  be  seen  to  lead  directly  into  a  large  lateral  tr.u  lua. 
"1 1m-  the\  are  provided  with  an  air-breathing  apparatus  though 
they  li\e  in  a  thick  fluid  of  suppurated  matter  which  make- 
liable  the  i  losing  of  one  or  more  of  these  iracheal  opening  or 
ma\  nei  e--it.ite  the  temporary  closing  of  one  of  them.  In  this 
connection  it  is  interesting  to  find  a  transverse  tra<  heal  commissure 
posterior  to  the  anterior  stigmata  and  another  transverse  tracheal 
( ommi— mv  anterior  to  the  posterior  stigmata.  These  commis- 
sures enable  both  tracheal  trunks  to  get  air  though  for  any  reason 
some  of  the  -ligmata  may  be  closed.  Thu-  the  chief  tracheal 
-\  stem  consists  of  a  pair  of  anterior  and  a  pair  of  posterior  stig- 
mata and  two  lateral  tracheal  trunks  which  are  connected  by 
mean-  of  an  anterior  and  a  posterior  tracheal  commi— ure. 

Nothing  unusual  has  been  noted  concerning  the  histologv  of 
the  tracheal  trunks  and  posterior  stigmata.  The  histology  of 
the  anterior  stigmata  has,  however,  attracted  my  attention. 
These  fan-shaped  structures  are  for  the  mo-t  part  proliferated 
masses  "f  cuticle.  The  anterior  half  of  the  stigma  projects 
be\ond  the  contour  of  the  body  as  a  stigmatic  process.  The 
posterior  half  lies  beneath  the  surface  of  the  body  and  is  covered 
by  an  epithelium  which  represents  the  hypodermis  modified  as 
tracheal  epithelium  (  Fig.  8,  te.}.  l-'mm  the  posterior  margin  of 


166  WM.    A.    KEENER. 

the  stigmatic  process  there  is  a  cuticular  and  hypodermal  in- 
vagination  which  extends  to  near  the  base  of  the  stigma  as  a 
retaining  thread  (Fig.  8,  inv.).  This  retaining  thread  of  cuticle 
and  hypodermal  epithelium  is  seen  in  transverse  section  at  -inv. 
in  Fig.  9.  The  entire  stigma  represents  a  modified  region  of 
hypodermis  and  cuticle.  On  the  mesial  side  of  the  stigma  near 
the  base  of  its  anterior  third  the  hypodermis  becomes  very 
pronounced,  the  cells  becoming  very  large  and  columnar.  These 
cells,  so  far  as  their  form  is  concerned,  are  the  most  conspicuous 
tracheal  cells  (Fig.  7,  te.).  From  them  slender  processes  go  into 
the  cuticular  mass  of  the  stigmatic  process.  These  processes 
and  the  position  of  these  cells  suggest  that  they  not  only  help  to 
elaborate  the  cuticular  substances  of  the  stigmatic  process  but 
that,  also,  they  may  be  able  to  move  the  stigmatic  process. 
Within  the  mesial  wall  of  the  stigmatic  process  no  cytoplasm  ex- 
tends except  that  of  these  cellular  processes;  within  the  lateral 
wall  of  the  stigmatic  process  scattered  hypodermal  cells  are 
found.  There  is  thus  an  indifferent  cellular  supply  to  the  tracheal 
process  of  the  stigma.  Indeed  the  entire  stigma  is  for  the  most 
part  a  cuticular  structure.  The  cuticle  of  the  general  surface 
of  the  body  is  distinctly  two-layered.  The  outer  layer  is  the 
deeper  and  in  hacmotoxylin  stains  the  more  deeply.  The  inner 
layer  is  clearly  a  softer  substance  and  does  not  stain  deeply. 
These  two  strata  are  involved  in  the  formation  of  the  anterior 
stigma.  The  inner  layer,  except  for  becoming  more  abundant 
in  the  stigma,  is  not  modified.  Figure  7  at  c  and  Fig.  8 
show  this  layer  of  the  cuticle  passing  over  into  that  of  the 
stigma.  The  outer  layer  of  cuticle,  however,  is  thinner  over 
the  stigmatic  process  than  over  the  general  surface  of  the  body. 
When  it  reaches  the  tips  of  the  papillae  it  is  invaginated  and  passes 
as  a  series  of  converging  tubules  to  the  bases  of  the  papillae  where 
the  tubules  unite  to  form  a  large  tube  whose  lining  is  confluent 
with  the  lining  of  the  tracheal  trunk.  The  cuticular  lining  of 
the  tracheal  trunk  also  presents  a  deeply  staining  layer  and  a 
layer  that  does  not  readily  stain  (Fig.  11,  //.),  thus  resembling 
the  cuticle,  of  which  I  believe  it  represents  a  modified  region. 
The  inner  denser  layer  of  this  tracheal  lining  gives  rise  to  spiral 
taenidia  as  shown  in  Fig.  u  at  /.  When  this  denser  layer  passes 


THE   LARVA   OF    SARCOPHAGA.  1 67 

into  that  of  the  stigma  very  minute  slender  processes  arise  from 
it  into  the  lumen:  these  processes  branch  and  rebranch  to  form 
a  reticulated  layer  which  takes  the  place  of  the  taenidia  of  the 
trachea  (Fig.  10,  r.).  This  reticulated  layer  is  increased  until  the 
entire  lumen  is  rilled  with  a  reticulated  mass  or  plug  (Figs.  8, 
7  md  9,  rp.).  At  the  base  of  each  papilla  the  reticulated  plug 
branches  and  continues  to  near  the  tip  of  the  papilla  where  there 
is  a  -mall  chamber  into  which  the  branch  of  the  reticulated  plug 
sends  its  terminal  filaments  (text-figure  i).  Thus  we  find 


KrP! 


i      Lou  il  section  of  a  papilla  of  the  anterior  atigma,  showing  the  termi- 

n.il  i  li.iiiil>i-i   mi<»  which  filaments  of  the  reticulated  phi.  t.      X  1.500. 

the  entitle,  trachea!  lining  and  the  cuticular  mas-  of  the  -ti-ma 
lo  he  t\\o-l,i\ercd.  In  all  three  places  the  non-staining  I  i\er  is 
little  iiin.lilied  ;  but  in  the  tracheal  lining  the  deeply  staining  layer 
i-  iiiinliiied  to  form  the  taenidia,  and  in  the  tracheal  pmce--  it 
I"  .....  i«  -  .1  reticulated  plug. 

The  lar\.e  of  blow-lly  and  house  fly  have  likc\\i-e  pn  'thoracic 
siigniatir  processes  with  finger-like  papilla-.  The-e  in  turn, 
a.  •onlini;  to  de  Meijere  ('02),  have  reticulated  plni;-  which  he 
rail-  "felt  -chambers"  (Feltkammern).  What  does  -uch  histo- 
lo-ji-.il  -tructure  mean?  We  see  the  cuticular  hair-  mianlin-  the 
>tumata  ot  ants  or  other  insects  and  we  interpret  them  as  being 
<le\i<  .  ^  to  protect  the  trachea  from  foreign  bodie-.  lint  hen-  \ve 
ha\c  in  ]«lace  of  protecting  hairs  an  exten-i\e.  Imelv  reiiculated 
phi:;  \\hich  resembles  the  cotton  plug  ot  a  bacterial  culture  tube 
.1-  though  it  were  constructed  for  the  purpose  of  protecting  the 
trachea  from  microscopically  minute  bodies.  Tin-  lar\a  feeds 
upon  the  suppurated  fluid  found  within  the  excavated  region 
of  the  nucha  of  the  host,  hence  while  the  larva  is  feeding  these 
bacteria  can  hardlv  be  of  service,  for  the  anterior  end  of  its  body 


1 68  WM.    A.    KEPNER. 

is  bathed  in  the  suppurated  mass.  However,  when  about  to 
pupate  the  larva  reverses  its  position  with  reference  to  the  sup- 
purated mass,  and  lies  with  its  anterior  end  directed  towards  or 
through  the  opening  in  the  skin  of  the  turtle.  The  larva  is  then 
in  a  position  to  breathe  air  through  the  anterior  stigmata.  At 
the  same  time  the  larva  during  the  three  or  four  days  spent  in 
emerging  from  the  host,  frequently  retreats  into  the  excavated 
cavity  when  disturbed,  thus  its  anterior  end  may  repeatedly  be- 
come contaminated  with  the  bacteria  of  the  suppurated  mass. 
I  think,  therefore,  that  the  anterior  stigmata  are  chiefly  functional 
during  the  two  or  three  days  spent  by  the  larva  in  passing  from 
the  turtle  to  the  ground  and  that  the  reticulated  plug  is  a  bacterial 
screen  protecting  the  trachea  from  infection  threatened  by  the 
repeated  retreat  of  the  larva  into  the  excavated  cavity  when  it 
lies  with  its  posterior  end  at  or  within  the  suppurated  mass. 
If  this  conjecture  concerning  the  time  and  character  of  the  func- 
tioning of  the  anterior  stigmata  is  not  warranted,  I  believe  that 
I  am  justified  in  agreeing  with  Hewitt  ('08),  that  the  anterior 
stigmata  of  this  character  are  functional  at  some  stage  in  the 
life  of  the  larva. 


THE    LARVA    OF    SARCOPHAGA.  169 


LITERATURE. 

Aldrich,  J.  M. 

'05  uc  of  the  American  Diptcra.     Smithsonian  M       '  tions,  4''. 

Braucr,  F. 

"83     I1  iuRcr  des  kaisorlichcn  Museums  zu  Wien:   III.     Sy-tnn.iti 

I'litim   auf   Grundlagc   dcr   diptorcn   larvan.  etc.     U«-nk-.  In.  .1.  r    k 

.  math-naturwisse  Classe.  Bd.  47.  s.  i-ioo. 
Brues. 

'oo     '1  i.i.  li.-.il  I  lil.itations.     Biol.  Bui..  Vol.  i.  p.  6. 
Hewitt,  L.  Gordon 
'08     The  Structure.  Development,  and  Bionomics  of  II 

.n.     Part  99.     Quart.  Journ.  Micr.  Sc..  \'ol.  5.-.  I'.nt  IV. 
Krancher.  O. 

'81      I  >-  :    Bau  5l  . ma  l>ci  den  Insecten.     Zeits.  wiss.  Z<»-1..  H<1     |  sos. 

dc  Meijere,  J.  C.  H. 

'02     i  eba  ML   rVothorakalstlgmen  der  Dipterenpuppen.    /.-•!   Jahrb.    \i 
M.I.  \\'..  8.623. 

Packard,  A.  S. 

'8j      M.'t  M.   I  .irvu-  in  a  Turtle's  Neck.     Amer.  Nat..  \'ol.  16. 
Scheiber,  S.  H. 

"62      \'i-iKl'-i<  hi-ndc  Anatomie   und    Physiologie  dcr  Oestri«l<-n-l.iivi-n.      !<•  -jui.i- 
tioi  n.      Sitzb.  Akad.  \Viss.  Wien.  Math-ii.tiurw.   <  I  .   M.I.   -\>.   9.    ~. 

Wheeler,  Wm.  M. 

'90      I'lir  supposed  Bot-Fly  Parasite  of  the  "  Box-Tuitl.-.' 


WM.    A.    KEPNER. 


EXPLANATION  OF  PLATE  I. 

FIG.   i.     Dorsal  aspect  of  larva.     5*.,  stigma.      X  10. 

FIG.  2.  Lateral  aspect  of  anterior  end  of  larva,  mo.,  mouth;  m.,  mandible; 
st.,  stigma.  X  100. 

FIG.  3.  Ventral  aspect  of  posterior  segment,  d.,  tri-lobed  disc  with  stout 
spines.  X  25. 

FIG.  4.  Ventral  aspect  of  posterior  segment.  The  ventral  lobe  is  laid  back  so  as 
to  expose  its  six  chitinous  bars  c'p'.,  and  the  two-lobed  stigma-plate  with  its  six 
chitinous  bars  cp.  X  25. 

FIG.  5.  Lateral  spect  of  posterior  end  of  larva,  d.,  tri-lobed  disc;  cp.,  chitonous 
bar  of  stigma-plate.  X  10. 

FIG.  6.  Ventral  aspect  of  anterior  end  of  larva,  m.,  mandible;  mo.,  mouth; 
st.,  stigma;  ant.,  antenna.  X  25. 

FIG.  7.  Transverse  section  through  base  of  tracheal  process  at  level  indicated 
by  arrow  7  on  Fig.  8.  c.,  cuticle;  rp.,  reticulated  plug;  h.,  hypodermis;  te.,  tracheal 
epithelium.  X  250. 


BIOLOGICAL   BULLETIN      VOL.    IXII. 


PLATE    I. 


St..    ... 


c    .    • 


WH      »     >[fNC*. 


WNf.   A.    KEENER. 


EXPLANATION  OF  PLATE  II. 

FIG.  8.  Reconstructed  drawing  of  anterior  stigma,  h.,  hypodermis;  te., 
tracheal  epithelium;  /.,  tsenidia;  c.,  cuticle;  rp.,  reticulated  plug;  inv.,  invagination 
of  cuticle.  X  200. 

FIG.  9.  Transverse  section  of  trachea  through  level  indicated  by  arrow  9. 
It  shows  the  secondary  invagination  with  its  cuticular  core  inv.,  rp.,  reticulated 
plug;  te.,  tracheal  epithelium.  X  500. 

FIG.  10.  Part  of  trachea  in  the  transitional  zone  between  the  reticulated  plug 
and  the  tsenidia  of  the  trachea,  r.,  reticulated  chitin  arising  from  the  denser  layer 
of  chitin;  te.,  traceal  epithelium.  X  1,500. 

FIG.  n.  Part  of  wall  of  trachea.  /.,  tsenidia;  te.,  tracheal  epithelium;  tl., 
tracheal  lining.  X  1,500. 


BIOLOGICAL    BULLETIN      VOL.    Jin 
A 


V 

i 

I 


8 


9. 


_ 


•' 


*  M  >        .    I    CM   u 


KARLY  DEVELOPMENT  OF  GRAFFILLAGEMELLIPARA 

-A  SI  PPOSED  CASE  OF  POLYKMBRYt  "NY.1 

J.   THOMAS  PATTERSON. 

I.  INTRODUCTION. 

In    th<     Bl    "k-'    Memorial  Volume  of  the  Journal  of  I-'..\-f>eri- 


:<il  /"".'";;  v,  Vol.  o,,  1910,  Professor  Edwin  Linum  rep--n-  the 
•  li-.o\.T\  <-t  a  very  interesting  viviparous  rhabdoccele  commensal 

with  i  In  i  •  unmon  ribbed  mussel,  Modiolus  plicatnlns,  found  along 
tin-  All.  mti<    coast.      Dr.  Linton  refers  this  worm   to  the  ^eiui> 
unl   on  account  of  its  peculiar  method   of   producing 
eml-iAo-  in  pairs,  designates  it  by  the  name  Gra'  nielli  {tarn. 

So  I'.ir  aa  ui   know  the  only  other  statement  in  the  litiT.utiiv  ih.it 
could  l-i-  interpreted  as  referring  to  this  interesting  turbellari.ni 

-nnd   in   .1   -horl   j);i[x?r  by    Nicoll,   '06,  entitli-d    "Notes 
I  I-  in.  it...  I.    I'      twites  of  the  Cockle  (Cardium  eti  id 

(  Mytilu\  filn'. 

Ni«  "II  '  in  his  Fig.  7)  what  he  calls  a  trem.ii'>dr  -\n<\^  . 

i  tin-  li\t  r  i  if  the  cockle,  but  it  is  quite  clear  fmm  Linnm'- 
\\ciik  ih.it  In  i-  in  error  in  calling  this  specimen  .1  sporocyst. 
\\li.n  he  in  .ill  probability  had  was  a  specimen  <-i  .1  species  <>t 
tnrl  ifll.  iri.  in  (  l<i-ely  related  to  if  not  identical  \\ith  (/'.  -i-nifllifHini. 
1  hi-  i-  e\i<  l«-iii  IP-MI  the  fact  that  his  figure  >h.-u-  the  pn-M-n.  r 
nl"  p.iiifd  i-iiil>r\os,  as  well  as  a  pharynx,  \\hich  .ilmu-  umild 
elude  the  case  IP-ID  the  categorx'  of  sporoi\-i-. 

l.iin-iM'-  |M|n-r  gives  an  account  of  the  more  m-n.T.il  t"r,itin 
nl"  tin-  \\onn.  I  nit  leaves  several  important  questions  unanswered, 
.iMioii;^  uhiih  may  be  mentioned  the  fnllt>\\  iiu  :     i     ll.-u    i>  iln 
\t-lk  df]io^ititl  in  the  ova?       (2)  How  do  the  -pi  TIM-  reach   the 
"sperm-sac"?       ;    Is  the  species  protandnm  >.J      4    \\IKTC.  m-  the 
3  lVnili/rtl.J       51  Finally,  and  mo>t  importani  of  all,  How  do 
tin    i\\o  cmlir\  os  in  each  capsule  ari- 

In  p-^.ird  to  thi-  last  point,  Lintt-n  -nggests  that  \\v  may  have  a 

I'K-in  tin-  M.uiii'     •  ':i'l  tin-  /<">l"i;i'a  tin- 

I  ni\  •  ntril-iH:  109. 

173 


174  J-    THOMAS    PATTERSON. 

case  of  polyembryony.  It  was  this  suggestion  that  induced  me 
to  undertake  a  study  of  certain  phases  of  the  development  of 
Graffi.Ua;  and  this  not  only  because  of  my  interest  in  the  general 
subject  of  polyembryony,  but  also  for  the  reason  that  an  oppor- 
tunity seemed  to  be  offered  to  work  out  the  details  of  this  peculiar 
phenomenon.  Furthermore,  if  a  true  gemelliparous  develop- 
ment really  did  exist  in  so  simple  a  fashion  in  a  relatively  low 
organism  like  Graffilla,  it  might  be  possible  to  modify  experi- 
mentally the  process  and  thus  to  be  able  to  get  at  some  of  the 
factors  underlying  it. 

While  the  results  obtained  from  these  studies  have  proved  dis- 
appointing, at  least  so  far  as  the  main  object  for  which  the  in- 
vestigation was  undertaken,  yet  they  are  of  a  character  such  as 
to  warrant  record,  especially  as  they  answer  satisfactorily  some  of 
the  questions  raised  above.  Furthermore,  we  have  as  yet  only  a 
very  few  papers  dealing  with  the  development  of  rhabdocceles, 
and  consequently  there  is  need  of  contributions  along  this  line. 
Methods. — Various  methods  for  preserving  the  material  have 
been  used,  but  the  most  successful  fixing  fluid  has  been  found  to 
be  Benda's  modification  of  Flcmming's  strong  solution.  Speci- 
mens fixed  for  two  hours  in  this  fluid  give  beautiful  results  for 
cytological  study,  especially  when  followed  by  iron-haematoxylin 
stains.  Bouin's  fluid  also  gave  good  preparations,  but  is  much 
less  certain  in  its  results.  In  making  whole  mounts  the  speci- 
mens are  placed  under  slight  pressure  and  killed  over  a  gentle 
flame,  and  then  fixed  in  a  corrosive-sublimate  solution.  If 
followed  by  borax  carmine  such  material  gives  very  clear  figures 
of  many  structures.  However,  I  find  the  same  "indefiniteness" 
I  about  the  reproductive  organs  as  noted  by  Linton,  especially  in 
|  regard  to  the  ducts,  so  that  one  can  not  rely  upon  mounts  for 
'  one's  interpretation  of  the  conditions  of  these  structures. 

Notes  on  the  Habits. — Linton  states  that  G.  gemeUipara  lives 
on  the  gills  of  Modiolits,  but  there  is  some  evidence  that  they 
inhabit  the  kidney.  This  is  brought  out  in  the  following  experi- 
ment. Two  dozen  specimens  of  Modiolus  from  a  lot  yielding  no 
Graffilloe  from  the  gills  were  opened,  care  being  taken  not  to  injure 
any  of  the  tissues,  and  thoroughly  washed  out  in  water.  No 
parasite  was  found.  The  kidneys  of  these  same  individuals 


EARLY    DEVELOPMENT    OF    GRAFFILLA.  1 75 

leased  out  and  the  specimens  again  washed  in  water,  with 
the  rr-nlt  that  thirty-eight  Graffillcc  were  -ecuivd.  I'ndoubtcdlv 
many  individual  parasites  escape  from  tin-  kidiu-y  of  the  ho-t  and 
latrr  found  in  the  mantle  cavity  and  on  the  pill-,  and  this 
would  .Kiount  for  their  discovery  there  by  Linton.  Further- 
nion-  tin-  method  ordinarily  employed  in  opening  the  niolli. 
\\oiild  in •«  e--arily  result  in  injuring  the  kidney,  and  thu-  |u  rinit 
the  < •-(  ape  of  the  parasite  from  that  organ.  Th<  •iinnit 

mentioned  above  would  seem  to  indicate  clearly  that  G.  ^fnn-I- 
li()nr,i  i-  a  true  endoparasite,  but  the  experiment  \\a-  performed 
.it  tin-  c|o-i-  of  the  season  and  the  opportunity  \\a-  IK>I  ofieivd 
to  -i -i  ilt-  the  <|iiestion  conclusively,  as  that  could  onl\  In  done  by 
in. tkin.  I'ul  dissections  of  the  individual  mollu-i •-.  \\ V 

-hould  expect  to  find  this  species  of  parasite  in  the  kidney  or  li\  er 
oi  i  IK  ho-t  -ince  all  of  the  other  species  of  the  genii  ''ilia 

are  found  in  the  same  organs  of  the  various  mollu- 

The  In  -i  M  ,i>on  of  the  year  in  which  to  secure  (  ••llifmni 

.n  \\<H,d-  ||..|r  is  during  August,  from  the  loth  to  ihe  joih  of 
ilie  nioiiili.  ^I'ecimens  may  be  obtained  prior  to  \\i\--,  but  iliev 
.in  u^u.ilK  iininobile  individuals  which  contain  nuinerou-,  \oiin- 
thai  lia\  e  libel. iti-d  themselves  from  their  capsule- and  . m-  -\\ini- 
iniii'c  about  in  the  mescnehyme.  Such  material  i-  valuable  lor 
obtaining  \ei\  \ounganimals.  On  July  5.  1911,  several  of  these 
e\h. ui-ied  niothcrs  wiTi'  placed  separatel\-  in  lian-iii-  drop-  of 
the  iluid  taken  from  the  mantle  cavity  of  Moiliolns.  The  cover 
sli|>  tioin  \\hich  the  drop  was  suspended  wa-  placed  abo\e  the 
cavity  "t  .1  hollow  ground  slide  and  sealed  with  \a-eliin-.  In 
ihi-  \\a\-  the  -[leciniens  could  easily  be  studied  under  the  ini- 
croscope,  <  >n  the  following  day  it  was  noted  that  mo-i  of  the 

\-oiing  ha<l  ruptured  the  wall  of  the  mother  and  were  -\\inuniiiii 
about  in  the  drop.  In  one  case  the  escape  of  tin-  youn^  \\a^ 
actualk  ol,M-r\ed.  \'oung  animals  secured  by  this  method  can 
be  kepi  ali\e  \\iihoiit  much  trouble  for  about  two  days,  and  un- 
doubted lv  \\ould  li\'e  longer  if  proper  care  \\ere  taken.  I  lo\vc\  er, 
it  \\a-  found  unnecessary  to  obtain  material  for  -tudy  in  this 
\\.iv  atier  toii\-ci-ht  hours,  for  the  washings  of  Modinlns  yield 
manv  young  -pecimens  that  correspond  in  -i/e  to  ihe-e  two-day 
old  \\  orm-. 


1 76  J.   THOMAS    PATTERSON. 

An  interesting  periodicity  in  the  reproduction  of  G.  gemellipara 
occurs  at  Woods  Hole.  From  the  2oth  to  the  25th  of  June  (191 1), 
shortly  after  the  writer  arrived  there,  specimens  were  secured 
in  considerable  numbers,  but  from  this  date  until  about  the  loth 
of  August  it  was  extremely  difficult  to  obtain  material,  although 
molluscs  from  many  different  regions  were  examined.  From  an 
entire  bucketful  of  the  Modiolus  not  more  than  a  dozen  would 
be  secured,  and  these  were  either  very  young,  sexually  immature 
animals,  or  very  large  individuals  which  were  about  on  the  point 
of  undergoing  degeneration  and  freeing  their  young.  About  the 
middle  of  August,  both  in  1910  and  1911,  Graffillce  were  secured 
without  difficulty,  but  from  the  25th  of  the  month  until  the  I2th 
of  September,  when  I  left  Woods  Hole,  they  were  extremely 
scarce.  From  this  it  would  seem  that  there  are  two  summer 
periods  of  rapid  multiplication,  one  in  June  and  the  other  in 
August;  and  possibly  a  third  period  occurs  in  October.  Linton 
reports  that  Coe  found  Graffilla  in  abundance  at  New  Haven 
during  the  month  of  October. 

At  no  time  does  one  find  G.  gemellipara  in  such  numbers  as 
reported  by  some  of  the  writers  on  the  other  species  of  the  genus. 
Jameson,  '97,  states  that  from  four  to  several  dozen  individuals 
of  G.  buccinicola,  which  is  parasitic  in  the  kidneys  of  Bnccinum 
undatiim  and  Fusus  antiquus,  are  found  in  every  specimen  of  the 
two  molluscs. 

II.  STRUCTURE  OF  THE  REPRODUCTIVE  ORGANS. 

The  reproductive  organs  of  this  Graffilla  arc  difficult  to  make 
out,  both  on  account  of  the  viviparous  method  of  reproduction  as 
well  as  on  account  of  the  variability  in  the  development  of  the 
different  parts.  G.  gemellipara,  like  certain  other  members  of 
the  genus,  exhibits  successive  hermaphroditism,  but  the  case  is 
not  so  extreme  as  that  described  for  G.  buccinicola  by  Jameson, 
'97.  The  male  organs  develop  first  and  upon  reaching  their 
maturity  at  a  comparatively  early  period  in  the  post-natal  life, 
in  part  atrophy,  and  are  then  followed  by  the  development  of 
the  female  organs. 

The  male  organs  consist  of  the  following  parts:  (i)  a  pair  of 
testes  which  lie  just  posterior  to  the  pharynx,  one-  on  each  side 


EARLY    DEVELOPMENT    OF    GRAFFILI.A.  1 77 

of  the  median  line  somewhat  below  the  central  axis  of  the  animal 
Fig  i  2)  two  very  delicate,  short  sperm  ducts  which  place 
i In-  gonads  in  communication  with  the  seminal  vesicle;  (3)  a 
seminal  vesicle,  which  is  a  rather  large  pear-shaped  sac  situated 
just  In-low  the  genital  pore;  and  finally,  a  plug-like  peni>  arising 
troin  tin-  pointed,  ventrally  directed  end  of  the  seminal  vesicle. 
In  OIK-  ot  the  clearest  specimens  secured  earli  -perm  duct  i-  -mi 
to  ari-i-  troin  the  posterior  median  corner  of  the  te-ti-  ,m<l  to  pass 
inuard  to  the  anterior  face  of  the  seminal  vesicle,  inn  tin-  the 
latii-r  .it  .ilioiit  the  di\'iding  line  between  it-  upper,  bullion- 
|iortioii  and  the  smaller  lower  part.  The  peni-  \\lu-n  contracted 
i-  c\ti«  mel\  difficult  to  make  out,  and  since  in  mounted  prepa- 
ration-, thi-  condition  is  almost  invariably  met  with,  not  m.my  of 
tin-  ili  i, til-  o|  the-  organ  were  studied.  The  penis  \\ln-n  extended 
ol  course  |irotrudes  into  the  common  atrium,  \\hich  in  turn 
communicates  with  the  exterior  by  means  of  the  small  genital 

1 1  he  pore  lies  in  the  median  ventral  line  at  a  point  -i main  I 

•  ibont  one  third  the  distance  from  the  anterior  end  of  tin-  l>od\. 

In  large  individuals  the  testes  are  seldom  found,  ami  \\hrn 
|irr-fin  .in-  mi  i e  degenerating  fragments.  Tin-  seminal  \e-icle, 
ho\\e\er.  p.i-i-t-  .it  least  until  a  late  period  of  tin  \  n  i-t  -naial 
life,  but  iii  many  animals  becomes  reduced  in  si/e.  The  penis 
also  degi  derates  sooner  or  later.  During  this  perio<l  of  degen- 
eration o|  the  male  organs  the  female  reprodin  ti\  i  -titntnie- 
gtadualK  make  their  appearance.  One  occasionally  meet-  \\ith 
specimen-  in  which  the  transition  from  tin  "male"  to  the 
"female"  -tate  is  seen,  and  from  such  individuals  nio-t  of  the 
important  ]n.ints  concerning  the  female  organs  can  be  made  out. 

In  the  t\  pical  "  female"  condition  the  seminal  \e-i<  le  i-  alua\  - 
pn-i  nt.  though  as  stated  above  it  mn\  become  greatly  n-din  ed 
in  >i/e,  and  the  atrium  with  its  genital  pore  still  per-i-t-.  Ju>t 
back  of  the  -eminal  \~esicle  and  (loyally  the  atrium  gives  ri-e 
to  a  small  di\  erticiilum,  which  both  from  it-  po-iti<,n  and  char- 
acter -ugge-t-  its  homology  with  the  receptaculum  >emini>  of 
the  other  members  of  this  genus,  although  in  the  two  clearest 
cases  coming  under  the  observations  of  the  writer  tin-  \e~icle 
contained  no  spermatozoa  (Fig.  2).  If  this  interpretation  is 
correct  then  the  receptaculum  seinini-  i-  in  this  species  clearly  a 
degenerate  -tructure. 


1 78  J.    THOMAS    PATTERSON. 

Posteriorly  the  atrium  is  directly  continuous  with  an  enlarged, 
rather  thick-walled  uterus,  which  in  turn  gives  rise  to  a  duct-like 
structure  that  extends  backwards  and  upwards  (Fig.  5,  •«). 
At  the  point  where  these  two  parts  join,  the  uterus  receives  the 
small  ducts  of  the  many  unicellular  shell-glands  (Fig.  1,5). 

Towards  its  distal  end  the  uterus  bifurcates,  sending  a  branch 
to  each  of  the  bilaterally  arranged  ovaries  (Fig.  3).  The  bi- 
furcated part  of  the  uterus  serves  as  a  receptacle  for  spermatozoa 
—a  condition  that  is  not  entirely  unique  for  this  species — and 
also  performs  the  function  of  insemination.  On  account  of  the 
backward  and  upward  course  taken  by  the  uterus,  the  two  distal 
parts  come  to  lie  just  below  the  ventral  surface  of  the  intestine, 
at  a  place  slightly  posterior  to  the  middle  point  of  the  body 

(Fig.  5)- 

The  development  of  the  uterus  has  not  been  studied  and  I 
can  not  therefore  state  with  certainty  the  exact  nature  of  this 
organ.  Slightly  posterior  to  the  point  where  the  proximal  and 
distal  parts  join  the  duct  is  frequently  very  indefinite  and  difficult 
to  trace.  This,  together  with  the  fact  that  small  yolk  cells  are 
frequently  found  within  its  cavity  (Figs.  4,  5)  has  led  the  writer 
to  believe  that  the  distal  part  of  the  uterus  is  the  product  of 
fusion  between  the  ducts  coming  from  the  reproductive  glands 
and  therefore  should  probably  be  called  the  oviduct. 

The  female  reproductive  glands  consist  of  a  paired  "  germarium  " 
and  a  paired  "vitellarium,"  the  two  glands  on  each  side  being  so 
closely  associated  that  the  compound  structure  might  properly  be 
termed  a  "germ-vitellarium."  The  ovarian  portion  occupies 
the  anterior  part  of  the  body,  while  the  yolk  glands  occupy  the 
posterior  half  mainly. 

The  clearest  idea  of  the  relation  of  these  various  parts  to  each 
other  and  to  the  reproductive  ducts  can  best  be  gained  in  a  study 
of  horizontal  sections  which  pass  just  below  the  ventral  side  of 
the  intestine.  In  such  sections  the  ovary  on  each  side  is  seen  to 
begin  slightly  anterior  to  the  seminal  vesicle,  and  tx>  increase 
gradually  in  diameter  in  passing  backwards  until  it  reaches  the 
region  occupied  by  the  distal  end  of  the  uterus.  Here  it  spreads 
as  a  fan-like  structure,  with  ilu-  inner  margins  of  the  ova  con- 
verging to  meet  the  tip  of  the  uterus  (Fig.  4).  In  composition 


EARLY  DEVELOPMENT  OF  GRAFFILLA.  1 79 

tin-  ovary  is  made  up  of  flattened  cells,  and  one  might  compare  it 
i  rouleau  of  coins  of  gradually  increa-ing  size,  the  smallest 
In-ill.:  !'•'  ,tted  at  the  anterior  end.  The  larger  cells  of  the  ovary 
arc  produced  by  the  absorption  of  nutritive  material-  t'mm  the 
\iiellim-  cells,  in  a  manner  that  will  be  <K --crilied  in  tin-  IH  \t 

ion. 

The  vitellarium  is  an  extensive  organ,  and  in  the  posterior  half 
of  i li<-  body  almost  completely  envelops  tin-  inte-tine  Fig.  6  . 
In  tin-  c.irly  stages  of  its  formation  the  cells  are  very  similar  in 
iliM-i  1,1"  tin-  ovary,  and  even  in  the  definitive  condition  their 
inn  Ic-i  II.IM-  the  characteristics  of  ovarian  nuclei.  Tin-  <>\.iti.m 
and  \itelline  cells  are  in  very  close  association  at  the  middle 
die  body,  and  for  some  little  distance  anteri»r  t<>  thi- 
tin  .  ,\.n\  i-  overlaid  by  the  yolk  cells. 

111.  MARLY  DEVELOPMENT. 

i     \ittr  it  ion  oj  the  Ova  and  the  Formation  of  the  .  :f>sulc. 

In  oidiT  to  be  able  to  understand  clearly  the  mamu  r  in  which 
tin  «>\  i  .ire  nourished  and  the  egg-capsule  is  formed  it  i-  necessary 
in  i  .ill  .mention  to  the  characteristic  condition  in  GraffUla  <>t  the 
duple\it\  of  embryos  in  each  capsule      In  all  of  the  "Id.-r  sta 
the  t\\<>  embryos  are  surrounded  by  a  very  thin  t  ran -parent  niein- 
br.me  m   -hell    inside  of   which  the  two  ciliated  indi\  idnal-  m.i\ 
nm\e  .il.niii  each  other  with  considerable  case.      In  ! 
or  indci  d   in  any  stage  of  segmentation,  this  thin  -hell   in   the 
Mil*  if   the  word  does  not  exist,  though   the  nut'-nin^t 

ii.n  nl  the  yolk  is  of  a  consistency  such  that  it  servee  the 
]>ni|M'-e  nt  a  shell,  and  out  of  this  surface  l.t\er  the  true  -hell 
di'iil'tle—  dillerentiates.  During  the  cleava.  u  i-  seen 

th.u    .1    •  mi-idt  rable   mass  of   yolk   surround--    the    t\\..   enibrxn- 
I  i(     [9         The  two  embryos  may  be  either  clu-i    in-.  iln-r.  \\iih 
onl\    .1  \ir\-  thin  intervening  layer  of  yolk.  i>r  \\idtl\    separated 
and  situated  at  the  extreme  opposite  end- i»t    the  c.ip-nle    I  igs. 
In  either  e\-ent  the  most  pertinent  <|Uc-ii<m  thai  mie  i  .in 
rai-e  i-  hnw  the  two  embryos  have  come  t<>  e\i-t  \\ithin  the  same 

\  i  ilk  ni.i-s. 

\-  \\e  have  pointed   out    in  the  preceding  -ec(i«>n.  the  ovaries 
are  at    their  posterior  ends  somewhat   clo-ely  .iiiprovimated  on 


ISO  J.    THOMAS    PATTERSON. 

the  ventral  side  of  the  intestine,  and  are  intimately  associated 
with  i he  yolk  glands,  being  surrounded  on  the  dorsal  and  posterior 
aspects  by  them.  In  a  longitudinal  section  of  almost  any  indi- 
vidual in  the  egg-producing  stage  one  can  observe  that  the  ova  are 
at  their  upper  margins  absorbing  yolk  from  these  glands,  and  while 
the  nutritive  process  may  involve  the  ova  of  one  half  of  the  ovary, 
yet  it  is  much  more  conspicuous  in  the  posterior  third  of  that  organ 
(Fig.  9).  At  the  extreme  end  of  the  ovary  the  absorption  goes 
on  with  great  rapidity,  the  ova  soon  becoming  gorged  with  nu- 
tritive material.  In  consequence  of  this  rapid  growth  certain 
retrogressive  changes  involving  the  cell  membranes  separating 
contiguous  ova  frequently  make  their  appearance.  As  a  result 
two  cr  even  more  nuclei  may  come  to  lie  within  a  common  yolk 
mass,  which  occupies  the  extreme  tip  of  the  ovary  (Figs.  9,  10). 
In  other  words,  a  syncytium  is  formed  here.  In  the  vast  majority 
of  cases  only  two  ova  are  involved  so  that  the  usual  picture  dis- 
played in  this  region  represents  a  binucleated  yolk  mass  (Fig.  15). 
It  should  be  noted  here  that  in  this  peculiar  method  of  nu- 
trition we  have  a  mechanism  alone  adequate  to  account  fully  for 
the  reason  why  two  embryos  are  habitually  borne  within  a  single 
capsule.  Just  why  two  should  appear  is  difficult  to  answer. 
As  a  matter  of  fact,  however,  twro  are  not  always  present,  for  as 
Linton  has  pointed  out  capsules  are  sometimes  seen  with  three 
embryos,  and  a  few  cases  were  noted  by  him  in  which  only  one 
embryo  is  surrounded  by  the  envelope.  Furthermore,  in  the 
figure  of  Nicoll  referred  to  above,  two  capsules  containing  three 
embryos  each  are  clearly  shown.  In  my  own  material  several 
cases  of  "triplets,"  including  one  with  undivided  eggs,  have  been 
observed,  as  well  as  several  with  one  embryo  each.  \Yhilc  in 
the  light  of  these  facts  the  twin  condition  in  Graffilhi  loses  much 
of  its  apparent  significance,  yet  its  appearance  in  the  great  major- 
ity of  cases  made  it  necessary  to  undertake  a  careful  study  of 
the  histogenesis  of  the  ovary  in  order  to  see  if  any  mechanism, 
other  than  that  of  the  breaking  down  of  intervening  membranes, 
could  be  discovered  that  would  explain  a  potency  to  gemellipa- 
rous  reproduction  on  ihc  part  of  that  organ.  At  first  it  srrmrd 
probable  that  a  binucleated  ovum  was  produced  somewhere  in 
the  oogonial  history.  A  diligent  search  in  the  ovary  fails  to 


EARLY  DEVELOPMENT  OF  GRAFFILLA.  l8l 

n-\cal  any  binucleated  ova,  except  of  course  at  the  extreme  tip, 
nor  has  the  slightest  evidence  been  secured  of  nuclear  divisions 
either  mitotic  or  amitotic  throughout  the  entire  length  of  a  fully 
matured  ovary.  We  are  therefore  forced  to  the  conclusion  that 
what  \\r  have  described  in  connection  with  the  absorption  of 
yolk  furnishes  the  key  to  the  twin  condition  in  Gnifilla.  It  can 
ii"i  IK-  argued  that  the  breaking  down  of  the  membranes  is  only 
apparent  and  therefore  an  artifact  produced  by  reagent-.  I'or 
it  ha-  been  observed  in  preparations  made  from  material  pn - 
-er\e<|  in  a  dozen  different  fixing  fluids,  and  t"«.ll..\\ed  by  as  many 
di Herein  -tain-.  However,  not  in  all  ca-e-  do  the  two  contiguous 
ova  l"-e  their  intervening  membranes,  but  some  become  com- 
pletek  -iirroimc|ed  by  vitelline  cells,  which  through  a  process 
<•!'  di-inii  ^ration  form  the  yolk  mass  of  the  definitive  capsule 
3.  7,  16).  In  such  cases  the  two  ova  d«>  not  lo-r  iheir  "in- 
di\  idnalii\  ."  and  a  subsequent  reorganization  of  ne\\  meinbrane- 
alioin  ihe  i\\o  nuclei  will  not  lake  place.  Considerable  evidence 
ha-  been  se<  nred  which  indicates  that  the-e  tuo  method-  of 
cap-ule  formation  are  but  the  extremes  of  one  and  the  -aim- 

pi  01  ess. 

Throughout  the  entire  history  of  yolk  ab-orption  main  inter- 
e-iing  (han^es,  involving  both  the  nuclei:-  and  c\  topla-m,  are 
seen,  but  \\e  can  not  deal  with  all  of  them  In  r  <  >ur  attention 
mu-t  then-lore  be  directed  to  those  that  stem-  to  u-  to  be  mo-t 
impi  ii  i  ant . 

In  I'ig.  14  is  represented  a  pair  of  nuclei  lying  within  a  single, 
niembraiu  The  lower  of  these  is  immediately  -urroumled  1«\  a 
la\er  ot  tinelv  granular  protoplasm,  about  which  one  can  trace 
another  \et\-  delicate,  but  neverthele—  distinct,  membrane. 
Thi-  condition  has  been  observed  in  a  number  of  ova,  and  may 
begin  before  the  binucleated  stage-  is  reached,  that  i-,  in  ova 
-ituaud  from  two  to  six  cells  from  the  tip  of  the  ovary.  I  have 
not  been  able  to  demonstrate  the  universality  of  thi-  membrane, 
and  I  am  therefore  inclined  to  regard  it  a-  the  intra-cellular  or 
intra-\  itelline  membrane  that  i-  sometimes  laid  down  about 
the  ovarian  nucleus.  It  may  be  that  in  Graffilla  it  marks  the 
beginning  of  the  segregation  of  the  protoplasm  from  the  yolk, 
and  is  then-fore  the  first  step  in  the  reorganization  of  a  cell  about 
each  of  the  nuclei  in  the  capsule. 


1 82  J.    THOMAS    PATTERSON. 

In  Fig.  9  is  seen  the  last  trace  of  the  intracellular  membrane 
in  a  binucleated  mass  that  is  about  ready  to  be  freed  from  the 
ovary.  It  is  possible  of  course  that  the  faint  line  about  the 
large  nucleus  is  not  an  intra-cellular  membrane,  but  only  the 
original  cell-wall  which  has  become  much  attenuated  through 
the  absorption  of  yolk  by  the  ovum.  This  figure  is  of  further 
interest  in  that  it  demonstrates  with  remarkable  clearness  the 
manner  in  which  the  yolk  is  absorbed  by  the  ova.  At  the  ex- 
treme end  of  the  ovary  the  process  is  at  its  height,  and  one  can 
actually  observe  the  configuration  of  the  streams  of  food  material 
extending  from  the  vitelline  cells  to  the  larger  nucleus.  This  is 
particularly  true  in  the  pseudopodial-like  structure  in  the  upper 
median  portion  of  the  figure.  On  the  extreme  right,  near  the. 
section  of  the  tip  of  the  second  nucleus,  the  yolk  cells  are  directly 
open  to  the  ova.  It  is  not  quite  clear  as  to  what  extent  the  yolk 
cells  participate  in  the  formation  of  the  mass  of  yolk  surrounding 
the  eggs,  aside  from  merely  giving  up  their  nutritive  materials; 
but  that  they  do  assist  in  this  formation  is  abundantly  proved 
in  those  capsules  the  yolk  contents  of  which  show  many  degen- 
erating nuclei  of  vitelline  cells.  In  some  cases  these  fading 
nuclei  form  a  complete  row  just  below  the  surface  of  the  capsule. 

Some  hall  dozen  cases  have  been  found  in  which  the  ovum 
apparently  does  not  become  surrounded  by  any  considerable 
amount  of  yolk,  but  after  absorbing  a  small  amount  of  food 
material  is  set  free  from  the  ovary.  These  single  naked  eggs 
float  about  in  the  parenchyma  and  probably  never  succeed  in 
producing  embryos  (Fig.  13). 

Some  time  prior  to  the  liberation  of  the  ova  from  the  ovary  and 
the  yolk-gland,  the  ovarian  nuclei  undergo  marked  changes. 
During  all  of  the  preceding  oogonial  history  the  nucleus  possesses 
that  characteristic  coarse  network  of  chromatin  extending 
throughout  the  nucleoplasm,  and  a  very  large,  deeply  staining 
nucleolus  (Fig.  9);  but  during  the  last  stages  of  yolk  absorption 
the  chromatin  network  becomes  more  or  less  indistinct  (Fig.  7), 
finally  disappearing  altogether,  and  in  its  stead  a  finely  granular 
condition  of  the  chromatin  appears.  At  the  same  time  the 
nucleolus  stains  less  intensely  and  M>OII  becomes  very  irregular 
in  < nitline  (Fig.  10). 


EARLY  DEVELOPMENT  OF  GRAFFILLA.  183 

It  i-  necessary  to  mention  only  briefly  the  manner  in  which 
the  "o\  ulation"  takes  place.  By  the  time  the  absorption  of 
yolk  ha-  reached  the  point  seen  in  the  case  of  tin-  <>\  a  on  the  ridit 
of  Fig.  9  the  formative  capsule  may  be  -aid  to  be  practically 
independent  of  any  ovarian  connections,  and  it  only  remains  for 
the  cap-ule  to  be  freed  from  the  vitellarium.  Ilo\\e\er,  its 
aM.nlinient  with  the  yolk  glands  per-i-t-  f.-r  some  time  after 
thi-.  even  indeed  until  the  two  eggs  reorganized,  if  reorganization 
i-  IM-I  essary.  In  Fig.  10  is  a  capsule  just  about  ivadv  to  be  set 
free  into  tin-  parenchyma;  most  of  the  yolk  cell-  ha\<-  \ieldcd 
11 1  >  i  heir  I'ooii  i  ••ntents  to  the  capsule,  and  the  region  immediately 
-in  roimdin-  its  upper  margin  shows  only  delicate  -trand-  con- 
ii  \\ith  a  few  of  the  remaining  nurse-  cell-.  Shortly 
in.:  this  period  tin-  strands  are  severed  and  the  capsule 
round-  up.  and  as  the  whole  structure  is  pu-hed  about  in  the 
parent  h\  ma  by  the  movements  of  the  mother  \\onn  ill 

Illldel  w.  i    (  |e\  eli  ipmellt  . 

I  p  io  ihe  present  we  have  been  using  the  term  "capsule"  to 
mean  the  \\hole  yolk  mass  surrounding  the  t\v<>  eggs;  and  \\e 
mu-i  now  consider  brielly  the  formation  of  the  thin  cap-ule  <T 
-In  II,  1 iv  \\  hich  we  mean  the  membrane  containing  the  i  u  ••  ciliated 
embryos  ••!  the  later  stages.  Since  the  eggs  with  their  i»llicular 
la\i  i  ..t  \olk  do  not  enter  the  uterus,  it  is  not  probable  that  an\ 
ol  i  In-  -« •,  reiioiis  from  the  unicellular  shell-gland-  reach  the  • 
and  thu-  lake  part  in  the  formation  of  the  -hell,  as  mt  ur-  in 
the  i  ase  "I  "\  i | i.i rous  forms.  I  have  not  followed  all  <>t  the  -tep- 
in  the  li.nnalioii  of  the  shell,  but  it  has  been  ob-er\ed  that  a- 
dexelopmeiu  proceeds  the  outermost  layer  of  the  \.>lk,  \\hich  .it 
lii-t  i-  very  plastic  and  yields  readily  to  any  ob-tnn  i  ii.n  in  the 
pareiich\ma.  gradually  becomes  more  re-i-iant.  linally  taking 
on  (he  thin  elastic  character  met  with  in  all  of  the  advanced 
stages.  It  is  probable  that  the  shell  is  in  part  the  product  of 
the  parenchyma. 

It  remain-  to  say  a  word  about  the  "rcorgani/ation  "  of  cells 
in  those  cases  in  which  the  membrane  in  part  or  completely 
di-appears  from  the  two  ova.  Kven  in  the  extreme  cases  it  is 
doubtful  whether  the  cytoplasmic  part  of  the  cell  becomes  in- 
di-criminaicly  a— ociated  with  the  yolk  portion  of  the  cap-ule. 


1 84  J-    THOMAS    PATTERSON. 

This  part  of  the  study  has  furnished  many  difficulties,  because 
of  the  fact  that  the  capsule  at  this  particular  stage  is  very  plastic 
and  hard  to  fix  properly.  Only  a  few  cases  of  good  fixation  have 
been  secured:  and  in  one  of  the  clearest  of  these  the  nuclei  are 
seen  to  be  surrounded  by  a  finely  granular  protoplasm,  about 
which  a  membrane  must  later  be  secreted. 

2.  The  Aborting  Spindle. — The  study  of  maturation  and  fer- 
tili/ation  was  made-  difficult  by  the  presence  of  a  spindle  which 
appeared  in  the  egg  some  time  before  the  egg  capsule  was  set 
free  into  the  parenchyma.  On  account  of  its  large  size  the  spindle 
was  at  first  taken  to  be  that  of  the  first  cleavage,  but  inasmuch 
as  the  first  division  of  the  fertilized  egg  results  in  cutting  off  a 
small  micromere,  it  soon  became  evident  that  this  interpretation 
was  incorrect.  Furthermore,  in  the  eggs  in  which  the  large 
spindle  appeared  the  most  diligent  search  failed  to  reveal  any 
polar  bodies.  \Yhen  this  fact  once  became  fully  established 
it  was  evident  that  we  had  in  Graffilla  a  display  of  that  remarkable 
phenomenon  of  a  "disappearing"  or  "aborting"  spindle,  first 
discovered  by  Selenka,  '81,  and  to  our  knowledge  of  which 
Wheeler,  Gardiner,  and  others  have  contributed. 

Selenka's  discovery  was  made  in  connection  with  his  work  on 
the  polyclad  Thysanozoon  Diesingli.  He  describes  the  aborting 
spindle  as  appearing  in  the  uterine  eggs.  After  the  egg  has 
reached  its  full  growth,  the  germinal  vesicle  begins  to  make  prepa- 
rations to  divide  in  the  typical  manner;  the  chromatin  forms  a 
spireme,  the  achromatic  spindle  with  its  two  centrosomes  appears, 
and  the  chromosomes  pass  into  the  equatorial-plate  position. 
At  this  point  the  process  stops,  and  the  nucleus  returns  to  a 
resting  condition.  Subsequently  the  egg  throws  off  two  polar 
bodies,  is  fertilized,  and  develops  in  the  normal  manner.  Inas- 
much as  the  yolk  granules  are  evenly  distributed  throughout  the 
egg  at  the  beginning  of  this  peculiar  phenomenon  and  are  col- 
lected about  the  astral  centers  at  its  close,  Selenka  supposes  that 
the  function  of  the  aborting  spindle  is  to  mass  the  granules  at 
the  center  of  the  egg.  But  this  interpretation  fails  to  explain 
the  appearance  of  the  spindle  in  those  eggs  in  which  a  collecting 
of  the  granules  about  the  astral  centers  does  not  take  place,  as 
both  Lang  and  Wheeler  have  observed. 


EARLY    DEVELOPMENT    OF    GRAFFILLA.  185 

Lang,  '84,  next  noted  the  aborting  spindle  in  several  polyclad 
eggs,  and  figures  it  in  the  uterine  egg  of  Thysanozoon  Brocchii. 

\\ "heeler,  '94,  describes  briefly  the  appearance  of  the  uterine 
^pindle  in  the  eggs  of  Planocera  inqiiilina,  a  polyclad  inhabiting 
the  br.iiichi.il  chamber  of  Sycotypus  canalicalalns,  but  does  not 
attempt  to  work  out  the  details  of  the  process.  He  also  noted 
the  spindle  in  the  eggs  of  the  acoelan  Polycfnvrus  cuitdutn*. 

<  .ardiner.  '95  and  '98,  working  on  the  latter  specie-  came  to 
tin  i  •  MM  lu-ion  that  the  aborting  spindle  is  abnormal,  ivpre-enting 
tin-  hr-t  clea\ai;e  spindle  of  eggs  retained  too  long  in  the  HUTU- 
o|  .in  animal  kept  under  abnormal  conditions.  His  point  does 
not  -eem  to  be  well  taken,  as  Surface,  '07,  ha-  -hown  in  hi-  work 
on  I'lniiK,  ,-r<i. 

I  lie  la-t  reference  to  the  aborting  spindle  thai  \\e  may  note 
i-  tint  ot  I..  \  on  ( iraff,  '82,  in  his  monograph  on  the  Khabdocielida. 
Von  (.rail,  although  making  no  reference  to  tin-  -pindle  in  the 
text.  ili.uK  h-ures  one  in  the  uterine  eggs  of  .1  f>li<uio*ti>nui 
<//•.•  •  md  Cyptomorpha  saliens. 

In  our  -pei  ies,  (/.  gemettipara,  the  aborting  -pimlle  appear-  in 

the  <          e  time  before  the  freeing  of  the  egg-capsule  from  the 

\iiellaiinm.  The  spindle  is  really  anticipated  Ion-  belon-  .ill  ot 
the  \ulk  i-  laid  down  about  the  two  eggs,  as  can  be  ->  «  n  in  1  •  iu.  to. 
In  man\  ie-pcvts  the  spindle  is  truly  remarkable.  \\t,\  ,,\\\\  on 
,n  '  omit  ot  its  great  size,  but  also  for  the  iva^m  that  tie,|iieiitl\ 
the  clii-onio-onu>  do  not  appear  upon  it.  <  >ne  ot  the  dearest 
cases  that  ha-  come  under  my  observation  i-  -ho\\n  in  1  i-.  17. 
Thi-  i-  an  e-pei'ially  well  preserved  egg,  \et  one  can  not  detecl 
the  ^li^hte-t  tiare  of  chromosomes  in  the  cell.  HOU.-MT,  it  i- 
probable  that  the  chromatin  is  represented  by  some  of  the  central 
-pimlle  liber-,  \\hich  are  (juite  thick  but  do  not  take  the  -tain 
\\ell.  Thi-  i-  most  certainly  the  case  in  some  eggs  in  which  MT\ 
delicate  i  hroinatin  threads  among  the  spindle  fibers  can  with 
(lillicnlty  be  made  out. 

-•  -int-iime-  the  chromatin  is  in  the  form  of  chromo-<>me.-,  which 
ho\\c\  er  are  not  located  on  the  spindle.  In  Fii;.  I  S  is  -ho\\  n  -nch 
a  case.  Hen-  the  large  conspicuou-  ^pindle  i-  it-ill"  free  from 
chromatin,  but  among  the  astral  ra\>  of  one  end  are  four  chro- 
mo-omr-,  \\  hich  are  of  intcre-t  not  onh  bei  ause  of  their  pi-i-uliar 


1 86  J.    THOMAS   PATTERSON. 

position,  but  also  because  they  are  apparently  bivalent.  They 
are  not  tetrads  in  shape,  as  in  the  characteristic  condition  of  the 
first  maturation,  yet  that  they  are  the  egg  chromosomes  and 
not  those  of  the  sperm  is  evidenced  by  the  fact  that  the  sperm  is 
located  in  another  part  of  the  ovum. 

The  peculiar  behavior  of  this  karyokinetic  figure  is  not  con- 
fined to  the  chromatin;  the  centrosomes  frequently  present  unique 
conditions.  It  is  not  uncommon  to  find  the  centrosome  at  one 
or  both  ends  of  the  spindle  undergoing  division,  but  this  would 
not  be  striking — since  in  many  germ  cells,  both  male  and  female, 
a  precocious  division  occurs — were  it  not  for  the  fact  that  at 
one  end  the  axis  of  the  two  centrosomes  is  at  right  angles  to 
that  of  the  spindle,  while  at  the  other  end  it  is  simply  a  continu- 
ation of  the  spindle  axis.  The  precocious  division  of  the  cen- 
trosome frequently  results  in  the  formation  of  a  double  aster. 

I  have  not  been  able  to  follow  with  certainty  all  of  the  sub- 
sequent steps  in  the.  history  of  this  spindle,  but  the  end  result 
in  all  cases  would  seem  to  be  a  return  to  a  sort  of  resting  stage 
on  the  part  of  the  nucleus.  It  differs  from  the  corresponding 
stage  of  Thysanozoon  Diesingii,  in  that  the  nucleus  instead  of 
being  a  large  vesicle,  appears  in  the  form  of  four  vesicles,  one 
for  each  chromosome  (Fig.  19).  These  may  be  more  or  less 
grouped  together  or  widely  separated,  but  they  later  come  to- 
gether and_  fuse,  producing  a  lobulated  nucleus  which  retains 
this  condition  until  the  onset  of  maturation  (Fig.  21).  It  will 
be  seen  from  this  rather  brief  account  that  the  only  function 
which  one  might  assign  to  the  aborting  spindle  in  G.  gemellipara 
is  that  of  scattering  the  chromosomes  in  the  form  of  vesicles; 
but  since  these  are  later  collected  together  into  a  single  vesicle 
before  maturation,  it  is  difficult  to  attach  any  real  significance  to 
this  whole  peculiar  phenomenon.  Inasmuch  as  several  odd  con- 
ditions have  been  observed,  both  in  the  centrosomes  and  the 
chromosomes,  it  is  not  at  all  improbable  that  the  aborting  spindle 
is  an  abnormal  display.  But  it  can  not  be  the  result  of  placing 
the  animals  under  unfavorable  conditions  because  the  spindles 
are  found  in  worms  killed  immediately  upon  their  removal  from 
the  moll  use. 

It  should  be  pointed  out  here  that  Graffilla  is  not  a  favorable 


EARLY  DEVELOPMENT  OF  GRAFFILLA.  IS; 

form  in  which  to  work  out  the  history  and  significance  of  the 
aborting  spindle,  for  owing  to  the  viviparous  mode  of  repro- 
<lu<  -lion  prevailing  in  this  species  it  is  quite  impossible  to  secure 
a  complete  series  of  stages  showing  the  different  steps.  One 
(,ui  not  he  at  all  certain  that  it  occurs  in  every  egg,  though  the 
frequency  at  which  it  is  met  would  indicate  that  it  did.  Ne\er- 
thele— ,  it  would  seem  that  some  rather  important  function 
-hould  he  aligned  to  the  aborting  spindle;  for  it-  appearance  in 
-ome  do/en  different  species  of  flat  worms  niu-t  exclude  it  from 
the  category  of  abnormal  behavior.  It  i-  therefore  hoped  that 
an  oppori  unity  may  be  offered  to  work  out  it-  history  in  detail 
in  a  !a\orahle  form,  such  as  one  of  the  o\ipan>u-  species  from 
\\hich  a  -eries  of  stages  can  be  secured  from  the  uteru-. 

J,  /•  »'.inatiini. — By  insemination  is  usually  meant  the  act 
ot  introducing  the  spermatozoa  into  the  egg.  In  ('>raflUla  the 
process  »«  curs  during  the  last  stages  of  yolk  ah-orption  \\hilc 
tin-  formative  capsule  is  still  attached  to  the  ovary,  and  consists 
in  the  introduction  of  spermatozoa  into  tin-  cap-ule.  The  in- 
M  ininaiin-  organ  is  the  modified,  or  bifurcated  part  <>l  the  uteru-. 
In  1  is  -hown  a  beautiful  case.  The  -ection  passes  through 

the  di-tal  end  of  the  uterus,  and  the  left-hand  lohe  of  ihat  or^an, 
tilled  \\ith  -pennatozoa,  is  in  direct  contact  with  the  hinucleated 
i  ap-ule.  Any  number  of  similar  fign  n  he  demon-iraied 

in    the   preparations,  so  that   no  doubt  can   exisl    regarding    ihe 
interpretation    which   we   have   placed    upon    Mich    picture-.      It 
\\milil   -eem   that    the  uterus  took  an  acti\e  part    in   the  pn- 
of   in-eminaiii»n.      Linton   reports  an   ob-er\  aii«m    \\hich    points 
to  the  -ame  <  '  inclusion. 

This  method  of  insemination  must  necessarily  permit  a  numher 
•  •f  -permaii>/oa  to  get  into  the  capsule,  hut  o\\in-  to  their  -mall 
-i/e  thev  are  soon  lost  among  the  yolk  granules,  so  that  an 
enunu-ration  of  them  is  impossible.  S- >  far  as  one  can  tell  the 
-perm-  di.  not  at  first  invade  the  immeiliate  neighborhood  of 
the  two  nuclei,  but  remain  in  the  peripheral  portion  of  the  cap- 
Mile,  and  later  penetrate  the  egg-  a  -lion  time  before  the  begin- 
ning of  maturation. 

4.  Maturation. — As  in  the  case  of  all  ova  accompanied  by  the 
proce—  of  feriili/ation.  those  of  Gratfilhi  throw  off  t\\o  polar 


1 88  J.    THOMAS    PATTERSON. 

bodies.  The  first  maturation  follows  immediately  upon  the 
fusing  of  the  chromosome  vesicles  produced  by  the  aborting 
spindle,  and  at  the  time  it  occurs  the  sperm  is  already  present 
in  the  egg  (Fig.  21).  The  demonstration  of  maturation  as  taking 
place  simultaneously  in  the  two  eggs  within  the  same  capsule 
is  the  most  cogent  proof  we  can  offer  against  the  idea  that  this 
animal  exhibits  polyembryony;  because  if  this  is  a  fact,  each  egg 
must  subsequently  be  fertilized  before  it  could  develop,  and  that 
would  at  once  remove  the  case  from  the  category  of  polyembry- 
ony; and  even  though  no  other  proof  could  be  offered,  such  as 
we  have  given  in  connection  with  the  section  on  the  formation 
of  the  capsule,  this  would  be  sufficient  to  establish  our  main 
contention.  As  a  matter  of  fact  we  have  found  two  very  clear 
cases  in  which  each  of  the  two  eggs  is  undergoing  maturescence. 

The  egg  in  one  of  these  shows  the  first  maturation  spindle  in 
the  anaphase  (Fig.  20).  The  spindle  is  extremely  large  and  has 
at  each  end  a  large  aster  with  very  conspicuous  centrospheres, 
in  the  lower  of  which  is  a  single  centrosome  and  in  the  upper  of 
which  are  twro  centrosomes.  The  sperm  head,  already  showing 
signs  of  its  transformation  into  a  pronucleus,  lies  near  the  lower 
aster.  Between  the  upper  pole  of  the  spindle  and  the  egg- 
membrane  is  a  clear  space  due  to  a  depression  in  the  egg  at  this 
point.  In  a  slightly  later  stage  the  egg  elongates  in  the  direction 
of  the  long  axis  of  the  spindle,  taking  on  an  appearance  much 
like  that  of  a  pear,  with  the  smaller  end  representing  the  animal 
pole.  A  very  large  polar  body  is  then  cut  off,  and  the  mate  to 
this  egg  fortunately  shows  this  process  going  on  (Fig.  24).  Since 
the  first  cleavage  division  results  in  producing  a  micromere  of 
about  the  same  size,  opportunity  is  afforded  for  confusing  this 
cell  with  tin-  first  polar  body,  but.  the  difference  can  easily  be 
told  if  the  chromosomes  arc  in  a  condition  that  allows  their 
enumeration  to  be-  made. 

In  the  second  case  one  of  the  eggs  (Fig.  22,  on  the  left)  sho\\> 
the  maturation  spindle  in  prophase  with  four  distinct  tetrad^, 
and  the  other  cell  a  polar  view,  in  which  only  three  chromosomes 
appear.  I  have  been  unable  to  find  a  totinh  tetrad,  and  I  theiv- 
fore  assume  that  it  must  have  been  destroyed  by  the  knite. 

Several  eggs  showing  the  first  polar  body  just  extruded  have 


EABLY    DEVELOPMENT    OF    GRAFFILLA.  I  Si) 

been  found.  In  a  tr\v  <>i  these  the  egg  nucleus  is  in  a  resting 
condition,  thus  indicating  that  the  second  division  may  not 
follow  immediately  upon  the  first.  However,  I  have  not  yet 
succeeded  in  finding  the  spindle  of  the  second  polar  body  divi- 
sion,  but  th.it  a  second  polar  body  is  thrown  off  is  clearly  shown 
in  ai  It -.1-1  one  case  (Fig.  25).  Here  the  constriction  of  the  second 
polar  body  has  just  been  completed,  while  the  first  polar  body 
liaxini:  undergone  division  is  in  the  process  of  disintegration. 
The  rapid  disappearance  of  the  polar  bodies  immcdiatrK  after 
the\  are  vi\«-ii  off  has  added  to  the  difficulty  of  studying  their 
formation,  a-,  well  as  to  the  study  of  the  formation  of  tin-  lir-t 
micromere. 

iVrli.ip-  ihc  most  striking  feature  of  maturation  on  druffilhi 
is  i  In-  large  size  of  the  first  polar  body.  This  i-  not  surprising  : 
foi  it  i-  noi  uncommon  for  a  large  polar  body  to  bi-  :J\en  off  in 

tin  •  i  tain  Hat  worms.     It  was  in  the  egg  of  a  turbellarian, 

Prostth  ,    that    Francotte,    '97,   discovered    the    intere-tin^ 

lai  i   that   tin-  first  polar  body  may  be  nearly  as  lar^c  as  the  < 
it-ell,    ami    may    occasionally    be    fertilized    and    d«-\elop    into    a 
.small  iM-trnla,  after  having  first  formed  a  small  polar  body  like 
the  MI  i  .n<  1  c  tin-  .it  the  egg. 

5.  l-i-rtilizutiini  und  the  First  Cleavage. — Fcrtili/ation  follou- 
alnio-t  immrdialfly  upon  the  throwing  oil  of  the  -,-,-,, D,|  p.ilar 
bi«l\  .  I  ha\e  found  no  exceptions  to  the  rule  that  only  one 
.spn  -niat«  i/oon  i-nters  the  egg.  The  sperm  pem-tratr-  the  • 
in  thr  vegetative  hemisphere  (Figs.  20,  21 ,  24).  and  passes  io\\ard 
tin-  center  \\ln-re  it  remains  while  the  polar  bodie-  an-  bcin- 
^i\rii  ott.  I  'In-  -piTin  nucleus  tlu-n  nm\  i->  to  a  point  mar  the 
animal  ]>oK-  \\here  the  copulation  of  the  t\\o  pi.nmclei  occurs 
(Fig.  26). 

The  fir^t  i  lr,i\a-e  is  unequal  and  iv-nlt-,  in  cutting  off  a  micro- 
mere  at  the  animal  pole.  Any  numhi-r  ot  lir-t  cK-axa-f  -|iindlr- 
ha\e  been  observed,  and  they  are  all  characteri/ed  by  having 
eii;ht  chromo-omi-s.  and  by  having  centn»somes  which  are  much 
more  conspicuous  than  those  ot  the  maturation  spindle--.  In 
this  a-  in  all  of  the  subsequent  early  cleavages,  the  nuclei  enter 
into  a  "rest"  st.i^r  immediately  after  the  completion  of  the 
di\  isji  in  :  and  in-trad  of  forming  a  single  vesicle,  the  chromosomes 


I9O  J.    THOMAS    PATTERSON. 

more  or  less  retain  their  individuality,  thus  producing  a  number 
of  small  vesicles,  some  of  which  may,  ho\ve\vr,  IUM-  together 
(Fig.  8). 

IV.  SOME  GENERAL  CONCLUSIONS. 

We  find  no  evidence  in  Graffilla  that  the  two  embryos  commonly 
found  within  a  capsule  are  the  product  of  a  single  fertilixed  egg. 
On  the  contrary,  it  is  clear  that  they  spring  from  two  ova,  which 
have  become  enclosed  within  a  common  envelope.  In  tlii- 
respect  our  species  does  not  present  anything  unusual;  for  while 
it  is  the  rule  among  the  rhabdocceles  to  have  one  embryo  in  a 
capsule,  yet  there  are  a  number  of  well-known  exceptions  to 
this.  In  his  excellent  monograph  on  the  turbellaria  Von  Graff, 
'08,  has  recently  given  a  list  (p.  2338)  of  these  exceptions,  which 
are  as  follows:  Gyratrix  hermaphroditus,  Provortex,  Collastoma, 
Umagilla,  Polycystis,  Fecampia,  and  Monocells  lineata,  each  has 
two  embryos  in  a  capsule;  Anoplodium,  1-2;  Prorhynchus  stag- 
nalis,  1-3;  P.  balticus,  6;  Graffilla,  2-3;  Promesostoma  marmo- 
ratuw,  4-7;  Dalyellia  truncata,  millportiana  and  viridis,  4-12; 
Plagiostomum  mttatum  and  girardi,  10-12;  and  finally,  Syndesmis, 
2-13.  All  of  this  goes  to  show  that  the  facts  which  we  have 
brought  forward  concerning  the  method  of  reproduction  in 
G.  gemellipara  arc  entirely  in  harmony  with  what  is  known  to 
occur  in  the  other  turbellaria.  Even  the  manner  in  which  the 
two  ova  become  surrounded  by  nurse  cells  within  the  reproductive 
glands  presents  nothing  new  (unless  it  be  in  those  cases  in  which 
the  ova  for  a  while  lose  their  individuality).  Furthermore,  the 
habit  of  directly  freeing  the  ova,  with  their  nurse  cells,  into  the 
mesenchyme  is  also  seen  in  such  forms  as  Dalyellia  viridis  and 
Olisthanella  obtusa.  In  most  forms  in  which  two  or  more  eggs 
are  enclosed  within  a  capsule  the  ova  become  surrounded  by  a 
common  follicle  (A  nurse  cells  before  they  pass  to  the  uterus, 
where  the  shell  or  true  capsule  is  usually  secreted. 

Some  of  the  rather  rare  conditions  seen  in  G.  gemellipara  are 
the  indefiniteness  of  the  reproductive  ducts,  the  rudimnnarv 
state  of  the  reccptaculum  scminis,  the  failure  of  tin-  i-gg^  to 
enter  the  uterus,  and  consequently  the  probable  secretion  of  tin- 
shell  by  the  mesenchyme.  But  all  of  these  conditions  arc  in- 
cident to  the  viviparous  mode  of  reproduction.  Lin  ton 


EARLY   DEVELOPMENT    OF    GRAFFILLA. 

that  this  viviparity  may  be  seasonal  and  parallel  with  the  pro- 
duction of  summer  eggs,  as  is  known  to  be  the  case  in  some  of 
the  Me-o-i,,mata.  Certain  facts  in  Graffilla  might  seem  to 
indicate  that  what  we  have  de-cribed  are  the  conditions  peculiar 
to  a  period  r,f  sUmmer  egg  production.  Thus  the  thin  shell  i-  a 
di-tinctive  characteristic  of  a  typical  >uniiner  egg  (Siibitanei< 
and  the  well  de\ eloped  unicellular  >hell--lands  suggest  at  lea-t 
that  these  organs  could  function  later,  if  the  species  entered 
ii|»on  .1  period  . ,f  winter  egg  production  (I  >auen -ier ••.  However, 
in  the  ab-eiice  ot  any  proof  that  winter  eggs  are  produced,  and 
in  the  li'Jit  ot  the  fact  that  several  of  the  female  reproductive 
ins  s|1(,\v  a  rudimentary  or  degenerate  condition,  we  are 
inclined  to  belie\e  that  what  we  have  described  is  the  e\clu-i\e 
method  of  reproduction  in  this  species.  The  pr--ence  ot  shell- 
•vi 1. 1 nd-,  ot  a  rudimentary  receptaculum  semini-,  and  of  an  indefi- 
nite uterus  and  ducts,  instead  of  indicating  that  t  he  species  could 
Liter  produce  \\  inter  eggs,  may  and  probably  do,  sj-nifv  the  i  1 
relationship  of  this  species  to  the  other  member-,  of  the  -enus  in 
\\hich  ilii--e  -tructures  are  functional. 

(  )f  the  halt  do/en  species  of  Graffilla  described  iii  the  literature, 

'llifxirii  appears  tO  COme  Closest,  in  its  gent  r.il  arrangement 

of  or-. m~,   to  d.    Miiricicohi.      It  also  shows  -oine  -imilarity  to 

•Ha  iniiolii.  but  dilters  primarily  from  the  latter  in  ha\in^  the 

lital  poie  situated  further  back  on  the  bod\  . 

In  conclusion,  we  should  like  to  point  out.  as  a  result  of  our 
studies  on  this  animal,  the  necessity  of  exercising  mv.u  precaution 
in  concluding  that  a  ^iven  species  exhibit  s  p,  ,|\  embr\  on\  .  I  n- 
( lou  bit  i  IK  the  phenomenon  of  polyembryonj  will,  in  the  future,  be 
found  to  be  much  more  extensive  than  we  ha\e  Mi-pecied;  but 
before  coming  to  anv  delinite  conclusions,  the  in\e-ii-aior  should 
trace  the  de\elo].inent  back  to  the  fertili/eil  . 


LITERATI  ki 

Bbhmig,  L. 

'86     1  ntct-iu  luini;' n    uln-r    rhabdocoele  Turbellarien.    I.      Das    genus   Graffilla. 

/fit.  mi  \\  ISB.  /.»>1..  H.I.  43. 
Francotte 

'97      R«-«  In •!.  In •-    ~iir    la    maturation   i-lu-/    l.>    I'nly<-l;i«le<.     Mem.   Cour.   A 
Belg. 


IQ2  J.    THOMAS    PATTERSON. 

Gardiner,  E.  G. 

'95     Early    Development    of     Polychcerus   caudatus    Mark.       Journal    of    Mor- 
phology, Vol.  1 1 . 
Gardiner,  E.  G. 

'98     The  Growth  of  the  Ovum,  Formation  of  the  Polar  Bodies,  and  Fertilization 

in  Polycharrus  caudatits.     Journal  of  Morphology,  Vol.  15. 
v.  Graff,  L. 

'82     Monographic  der  Turbellarien,  I.     Rhabdocoele,  Leipzig. 
'08     Turbcllaria.      Bronn's  Tier-reichs,  Leipzig. 
Hallez,  P. 

'87     Embryogenie  des  Dendrocoeles  d'eau  douce.     Paris. 
Jameson,  H.  L. 

'97     Additional  Notes  on  the  Turbcllaria  of  the  L.  M.  B.  C.  District.     Proc. 

and  Trans,  of  the  Liverpool  Biol.  Society,  Vol.  9,  pp.  160-178. 
v.  Ihering,  H. 

'80     Graffilla  muricola,  eine    parasitische    Rhabdocoele.     Zeit.  fur  Wiss.  Zool., 

Bd.  34- 
Lang,  A. 

'84     Die  Polycladen,  Monographic.     Fauna  und  Flora  des  Golfes  von  Naepel., 

Bd.  ii. 
Linton,  Edwin 

'10     On  a  New  Rhabdocoele  Commensal  with  Modiolus  plicalttlus.      Journal 

of  Experimental  Zoology,  Vol.  9. 
Nicoll,  W. 

'06     Xotcs   on   Trematode    Parasites   of   the   Cockle  and    Mussel.     Annals  and 

Magazine  of  Natural  History,  Ser.  7,  Vol.  17. 
Schmidt,  F. 

'86     Graffilla  braunii  n.  sp.     Archiv  fur  Naturgesch.,  Bd.  i. 
Selenka,  E. 

'81     Ueber  eine  eigentiimliche    Art    der    Kernmetamorphose.     Biol.   Central- 

blatt,  Bd.  i. 
Surface,  F.  M. 

'07     Tin-   Early  Development  of  a  Polyclad,  Planocera  inquilina  Win.     Proc. 

Acad.  Nat.  Sci.  of  Phil.,  Dec.,  1907. 
Wheeler,  W.  M. 

'94      Planocera   inquilina,   A   Polyclad    Inhabiting    the    Branchial    Chamber   of 
Sycolypus  canaliculatus  Gill.     Journal  of  Morphology,  Vol.  9. 


194  J-    THOMAS    PATTERSON. 


PLATE   I. 

FIG.  i.  Horizontal  section  of  a  young  specimen,  showing  the  testes  (<).  seminal 
vesicle  (sv)  which  contains  sperms,  uterus  (K),  unicellular  shell-elands  (s),  and  the 
germ-vitellarium  (v).  X  222. 

FIG.  2.  Anterior  half  of  a  slightly  oblique  section  from  an  adult  individual. 
The  uterus  shows  a  distinct,  but  small  diverticulum  (sr)  which  in  all  probability 
corresponds  to  the  receptaculum  seminis  of  the  other  members  of  the  genus.  Note 
that  the  testes  have  disappeared.  X  222. 

FIG.  3.  Horizontal  section  passing  just  below  the  intestine  of  a  sexually  ma- 
tured individual.  The  section  passes  through  the  distal  or  bifurcated  region  of 
the  uterus  (u),  which  contains  spermatozoa,  o,  ovary;  c,  capsule  containing  two 
eggs,  one  of  which  is  giving  off  the  first  polar  body;  v,  vitellarium.  X  222. 

FIG.  4.  Horizontal  section  of  another  sexually  matured  animal,  but  which 
passes  at  a  slightly  lower  level  than  the  preceding.  It  shows  clearly  the  bifurcated 
region  of  the  uterus;  and  also  the  relationship  existing  between  the  uterus,  ovary 
and  vitellarium.  X  222. 

FIG.   5.     A  longitudinal  median  section  (slightly  schrmuti/i-d)  of  a  rather  old 
individual.     It  shows  an  advanced  stage  of  the  "female"  condition,     m,  mouth; 
pit,  pharynx;  oe,  oesophagus;  a,  atrium;  g,  genital  port-;  />,  penis;  sv,  seminal  ve- 
s,  unicellular  shell-glands;  u,  uterus;  v,  vitelline  cell  in  uterus;  c,  capsules  containing 
embryos;  /',  intestine.      X  117. 


BIOLOGICAL  BULLETIN,  VOL.   XXH 


PLATE  i. 


r-  -e 


^_  _  »j 


\ /  f 


)      '• 


m 


V  C 


J.   T.   PATTERSON. 


J.   THOMAS    PATTERSON. 


PLATE  II. 

FIG.  6.  Transverse  section  taken  through  the  region  of  the  tip  of  the  uterus. 
X  381. 

FIG.  7.  Two  ova  that  are  beginning  to  be  surrounded  by  vitelline  cells  pre- 
paratory to  the  formation  of  a  capsiile.  X  784. 

FIG.  8.     The  two-celled  stage,  showing  a  micromere  and  a  macromere.      X  740. 


BIOLOGICAL  BULLETIN,   VOL.  XXII. 


PLATE  ,1. 


v 


J     T.   PATTERSON. 


198  J.    THOMAS    PATTERSON. 


PLATE  III. 

FIG.  9.  The  posterior  half  of  an  ovary  which  shows  the  process  of  yolk  ab- 
sorption. On  the  right  a  capsule  is  being  formed  about  two  nuclei.  X  650. 

FIG.  10.  A  later  stage  in  the  same  part  of  another  ovary.  Note  that  the  two 
nuclei  are  immediately  surrounded  by  a  finely  granular  protoplasm.  X  650. 

FIGS,  ii  and  12.  Two  eggs  from  the  same  capsule.  This  represents  the  con- 
dition shortly  after  the  disappearance  of  the  aborting  spindle.  The  nucleus  is  in 
the  form  of  faintly  staining  vesicles  which  in  part  are  fused  together.  X  812. 


BIOLOGICAL  BULLETIN,  VOL.  XXII 


ft: 

•  ••  ,  •'    ','/}iir  •  ' 

•  .;,-;     ••/,-    ' 

• 


10 


-'o 


12 


2OO  J.    THOMAS    PATTERSON. 


PLATE  IV. 

FIG.  13.  Two  naked  ova  that  have  not  become  surrounded  by  a  capsule. 
Such  eggs  apparently  float  about  in  the  parenchyma,  but  probably  never  produce 
embryos.  X  543- 

FIG.  14.  A  binucleated  capsule  in  which  the  lower  nucleus  is  surrouned  by  an 
intravitelline  membrane.  X  798. 

FIG.   15.     A  binucleated  capsule.      X  543. 

FIG.  16.  Two  ova  completely  surrounded  by  a  follicular  layer  of  vitelline  cells. 
Only  a  part  of  one  of  the  eggs  is  seen  in  the  section.  X  543. 

FIG.  17.  A  typical  case  of  an  aborting  spindle.  Note  that  chromosomes  are 
absent  from  the  spindle.  X  798. 

FIG.  1 8.  Another  example  of  aborting  spindle,  in  which  the  chromosomes  are 
located  among  the  rays  at  one  end.  X  798. 


BIOLOGICAL   BUILEIIN,  VOL     XXII. 


PLATE   IV 


13 


5 


14 


• 


•"•':-":••    . 

•  -      •  •       . 

5ft 


. .. 


.-;•; 


.•::.v  " 


16 


v 


* 


17 


J     T    PATTERSON. 


2O2  J.    THOMAS    PATTERSON. 


PLATE  V. 

FIG.  19.  This  shows  a  capsule  about  to  be  set  free  into  the  parenchyma.  The 
eggs  exhibit  the  condition  which  immediately  follows  the  disappearance  of  the 
aborting  spindle.  Each  egg  has  four  chromosome-vesicles,  and  in  the  one  on  the 
left  the  centrosome  is  present.  Lying  just  above  this  newly  formed  capsule  is 
another  in  the  process  of  formation.  Only  one  of  the  ova  shows  in  the  section,  and 
in  it  the  centrosome  has  divided  and  the  aster  is  present,  thus  anticipating  the  forth- 
coming aborting  spindle.  X  993. 

FIG.  20.     The  anaphase  stage  of  the  first  polar  spindle.      X  2,394. 


BIOLOGICAL   BULLE'IN,   VOL.    •• 


PLATE  V. 


I  - 


'/I 


20 


• 


J.    T.   PATTERSON. 


2O4  J-    THOMAS    PATTERSON. 


PLATE  VI. 

FIG.  21.  An  ovum  shortly  before  the  formation  of  the  first  polar  body.  The 
nucleus  is  the  product  of  the  fusion  of  the  chromosome-vesicles  of  a  stage  like  that 
in  Fig.  19.  The  section  passes  through  but  one  of  the  two  ova  in  the  capsule.  In 
most  of  the  capsules  of  this  period  the  protoplasm  of  the  eggs  contracts  in  the  re- 
agents more  than  does  the  surrounding  vitelline  material,  thus  producing  a  clear 
space  between  the  two  materials.  X  543. 

FIG.  22.  A  capsule  in  which  both  eggs  are  undergoing  maturation  at  the  same 
time.  X  543- 

FIG.  23.     Two  of  the  tetrads  from  the  preceding  figure.      X  2,394. 

FIG.  24.  The  cutting  off  of  the  first  polar  body.  This  egg  is  a  mate  to  the  one 
shown  in  Fig.  20.  X  798. 

FIG.  25.  This  stage  shows  the  close  of  maturation.  The  first  polar  body  has 
undergone  division  and  is  disintegrating.  X  543. 

FIG.  26.     Fertilization  stage.      X  543. 


BIOLOGICAL  BULLETIN,   VOL.  XXII. 


PLATE  VI 


\ 


21 


23 


22 


24 


25 


£••• 


: 

• 

, 


J.   T.   PATTERSON 


Vol.  XXII.  March,  1912.  No. 


BIOLOGICAL  BULLETIN 


"STRAINS"   IN   IIVDATINA  SENTA 

D.   U.  WHITNEY. 

In  .i  lormer  paper  results  of  experiments  upon  t\\<>  \ 
the  roiiier  Hydatina  scuta  were  given  in  regard  t<>  tin-  production 
ot  OIK-  hundred  generations  of  females  without  the  appearance 
..I  males  in  either  race.  These  experiments  have  In  en  extended 
further  fur  al>uiit  seventeen  months  and  as  they  are  <-onrlnde.! 
it  seems  desirable  to  record  the  results  obtained  partly  a<  a 
•  »iilii  niation  of  the  earlier  conclusions  and  partly  because  they 
turni  »  \  idence  which  shows  that  there  exists  diffrreiit  races  '>r 
strain-  <  >r  lines  within  this  particular  species  of  Hydatina  scntn. 

In  the  furmer  paper  it  was  shown  how  readily  male --prodiu -inu 
females  could  be  produced  in  newly  made  dilute  unconkrd 
hoi -e  manure  cultures  and  also  how  readily  the  male-pro- 
ducing lemales  could  be  repressed  in  newly  made  concentrated 

•  •ked  hoi-e  manure  cultures. 

In  the  piv-eiit  paper  the  parallel  history  of  three  r,(ces  of 
rutiler-  .1,  H,  and  C  is  given.  Races  B  and  C  are  the  same 
races  ii|iun  \\hich  the  former  conclusions  were  based  while  i 
.1  i-  .in  additional  one.  Races  A  and  B  are  si-ter  rate-,  both 
ha\  in-  tle\  eloped  from  one  fertilized  egg  while  race  ( '  is  unrelated 
to  races  .1  and  B  except  in  as  far  as  all  three  races  came  from 
the  s.inie  general  culture  of  rotifers  which  was  originally  collected 
at  ( irantuood.  New  Jersey,  in  1906. 

Race-  .1  and  B  were  always  conducted  in  a  parallel  -,  ries  but 
race  < '  \\as  not  put  into  the  parallel  series  until  it  \va-  in  the  301  h 

Deration.  During  this  early  period  of  the  three  race-  before 
they  \\erc  all  put  into  the  parallel  series  the  food  \\a-  from  mis- 
cellaneous protozoa  cultures  of  various  ages  made  in  dilute  un- 
cooked hor-e  manure  media.  The  summary  ..f  the  early  history 

205 


D.    D.    WH1  PNEY. 


of  these  three  races  before  they  were  all  conducted  in  the  parallel 
series  is  recorded  in  Table  T.  The  percentage  of  male-producing 
females  of  races  A  and  B  are  practically  equivalent,  while  that 
of  race  C  is  much  lower. 

TABLE  1. 

Showing  the  number  of  female-  and  male-producers  in  the  three  races  .4,  B, 
and  C,  from  their  origin  to  the  time  at  which  parallel  records  were  taken.  Female- 
producers  are  designated  9  9  ,  male-producers  c?  9  • 


Race. 

Genera- 
tions. 

No.  of 

9  9- 

No.  of 

<?9. 

Per  Cent,  of 

c?9. 

Time. 

Food. 

A 

I-I44      I,l88        iSl 

13.22  + 

Oct.  6,  1908,  to 

Dilute  uncooked 

Aug.  31,  1909. 

horse  manure 

B 

I-l 

1,224 

167 

12.00  + 

Oct.  6,  1908,  to 

media,  7-28  days 

Aug.  31,  1909. 

old.     Miscella- 

C 

i-  35 

2IO 

10 

4-54  + 

June  16,  1909,  to 

neous  protozoa 

Aug.  31,  1909. 

growing  in  them. 

September  3,  1909,  these  three  races  A,  B,  and  C  were  started 
in  a  parallel  series  under  as  identical  external  conditions  as 
possible.  At  the  beginning  of  this  parallel  series  the  generations 
were  renumbered  and  the  beginning  generation  of  each  of  the 
races  ifi  this  series  is  called  No.  I.  Ten  young  females  from  each 

TAHLI-:  II. 


K: 


No.  of            No.  of 

Per  Cent,  of 

Generation.                  $  Q  .              d"  9  . 

cF9. 

1 

ime. 

Food. 

I 

9                 3 

25 

June 

16. 

1909. 

June 

18, 

1909. 

2 

20                o 

O 

June 

18. 

1909. 

June 

20, 

1909. 

3 

16 

4 

2O 

June 

21, 

1909. 

hH 

June 

22. 

1909. 

w 

3 

4 

8 

2 

20 

June 

24. 

1909. 

rt 
EH 

June 

26, 

1909. 

en 

Partial 

rt 
V 

summary.                   53                 9 

14-51  + 

£ 

summary.                   53                 9 

14 

5H 

E 

& 

5-34                         15"                   0                     D 

Juno 

26, 

i'J  

Aug. 

30, 

1909. 

35 

7 

i 

12. 

5                     Aug. 

30, 

Sept. 

i, 

1909. 

Total 

summary.                 210 

10                           | 

54  + 

Detailed  history  of  race  C  throughout  the  first  35  generations,  wliirh  i 
summarized  in  Table  I. 


"STRAINS"  ix  HYDATINA  SIM  \.  207 

generation  of  each  race  were  isolated  at  the  same  time  and  each 
female  placed  in  a  Syracuse  watch  glass  and  allowed  to  mature 
and  to  product-  daughter  females.  Then  this  pn>cc—  was  re- 
peated tor  .^45  generations.  All  the  females  at  each  isolation 
urn-  placed  in  the  same  quantity  of  tap  water  to  which  \\a- 
added  the  -ame  amount  of  food  culture  that  was  taken  from  one 
food  jar.  The  watch  glasses  in  which  the  rotifers  lived  al\\a\- 
\\en-  in  thn-f  -tacks  side  by  side  at  room  temperature.  IVac- 
ticallv  all  external  influences  were  as  identical  as  it  was  po— ible 
to  make  them. 

Tin-  del  tiled  observations  are  given  in  Table  III.  in  parallel 
column-  and  the  -ummary  is  given  in  Table  IV. 

At   ill'    <nd  of  Table  I.,  races  A  and  B,  which  up  t<>  tin-  time 
were  l<  d  on  \arious  protozoa  cultures,  were  practically  identical 
in  re-ard  to  the  percentages  of  male-producing  females  in  each 
.  but  at   the  beginning  of  Tables  III.  and  IV.  when  the  t  \\  o 
races   \\ere   -ubjected    to.  uncooked   concentrated   food   culture 
media  a  de<  ided  change  occurred.     Race  .1   retained  and  e\en 
•  ede.  I  its  former  rate  of  production  of  male-producing  female-, 
but    in  ra.e  li  the  rate  was  very  perceptibly  lowered.      K 
louered   -liuhilv  its  rate  of  male-producing  females.     Thi- 
runvd  during   the  first  50  generations.     From  the  57th   to  the 
^I5ih  generation  in  races  A  and  6' and  to  the  end  of  race  /•>'.  t  lu- 
ll ;o -iii -ration,  concentrated  cooked  food  media  \\a-  u-cd  ami 
cau-eil  a  decided   lowering  of  the  production  of  male-producing 
female-  in  all  race-.      In  race  t'  this  was  reduced  to  zero,  in  i 
/>'  to  less  than   I   per  cent.,  and  in  race  -1   to  about  3.5  per  cent. 

Tin-  confirm-  the  earlier  results  in  showing  that  it  i-  po--ible 
1>\  external  conditions  to  repress  entirely  the  production  of  male- 
pr.'du.  in.  females  in  some  races  of  this  rotifer  for  a  long  period 
of  time.  In  race  ('  the  male-producing  female-  were  repre— ed 
for  Hi-rations  and  then  reappeared  when  the  food  media 

\\a-  made  too  dilute  accidentally. 

If  the-e  three  races  were  exactly  alike  in  their  power  to  produce 
male-producin-  females  and  all  were  subjected  to  the  -ame  ex- 
ternal conditions  they  ought  to  produce  -uch  male-pro<hn  in- 
female-  at  the  same  rate.  Howe\  er,  as  the  <ibo\e  ob-er\  ation- 
-ho\\  that  the  rates  of  production  of  male-la\  in-  leniale-  vary 


208 


D.    D.    WHITNEY. 


TABLE  III. 

Showing  number  of  female-  and  male-producers  in  a  parallel  series  of  345  gen- 
erations in  the  three  races  A ,  B,  and  C.  Generations  1-56  show  the  detailed  results 
when  the  three  races  were  fed  upon  concentrated  uncooked  food  media  and  gen- 
erations 57-345  show  the  detailed  results  when  the  same  three  races  were  fed 
upon  concentrated  cooked  media. 


Generation. 

Race  A. 

Race  B. 

Race  C. 

No.  of 

9  9- 

No.  of 

c?1?. 

No.  of 

9  9. 

No.  of 

c?9. 

No.  of 

9  9. 

No.  of 

c?9. 

I 

5 

0 

6 

0 

7                  o 

2 

8 

2 

IO 

o 

10 

0 

3 

9 

0 

IO 

0 

9 

0 

4 

9 

o 

9 

o 

9 

0 

5 

9 

o 

8 

0 

9 

0 

6 

9 

o 

9 

0 

9 

o 

7 

9 

o 

9 

0 

9 

I 

8 

10 

o 

9 

o 

IO 

o 

9 

8 

2 

IO 

o 

10 

0 

10 

8 

2                         IO                         O 

9 

1 

1  1 

9 

I                         10                         0 

IO 

0 

12 

10 

O 

IO                         O 

IO 

0 

13 

10 

O 

I  O          .               O 

10 

0 

M 

7 

3 

10                         0 

9 

I 

15 

9 

I 

9 

1 

10 

0 

16 

7 

3 

8 

I 

10 

0 

17 

10 

o 

IO 

0 

10 

o 

18 

9 

I 

10 

0 

10 

o 

19 

10 

o 

9 

0 

9 

I 

20 

9 

I 

10 

0 

IO 

o 

21 

8 

2 

IO 

0 

8 

2 

22 

8 

2 

9 

I 

10 

o 

23 

9 

I 

8                   i 

IO 

0 

24 

8 

2 

IO                         O 

10 

o 

25 

9 

I 

IO                         0 

10 

0 

26 

9 

I 

9                  i 

9 

I 

27 

S 

5 

IO                         O 

10                        0 

28 

10 

o 

1C)                         O 

9 

0 

29 

10 

O'                        M)                         0 

9                 i 

30 

8 

2 

10                         0 

IO                        O 

31 

5 

5 

9                   i 

IO                        O 

32 

10 

0 

8    '                     2 

10                         0 

33 

10 

0 

IO                         O 

10                        0 

34 

4 

6 

IO                         O 

10                        0 

35 

6 

4 

Q                             O                              III                             i> 

36 

6 

3 

10                         0 

IO                        O 

37 

9 

I                           IO                           O 

10                             0 

38 

8 

2 

IO                         O 

10 

0 

39 

9 

I 

IO                         O 

9 

I 

:•' 

i 

9 

6                   4 

6 

4 

41 

7 

3                   "i                   o 

10 

0 

8 

2                         IO                         0 

IO 

0 

43                          7 

3                   '"                   o 

10 

o 

44                          7 

3 

IO                         O 

9 

I 

45                          7 

i                      <i                    »                      <; 

o 

46 

7 

3 

10                         0                         10 

0 

47 

7 

3 

9                  i 

10 

0 

"STRAINS"  IN  HYDATINA  SKNTA. 


TABLE  III.  —  Continued. 

Race  A. 

Race  B. 

Race  C. 

Deration. 
No.  of 

No.  of 

No.  of        of 

No.  of 

\  i.  of 

9  9- 

cf  9. 

99. 

c?9. 

9  9. 

«?9. 

7 

3 

o 

. 

I 

49 

6 

3 

9 

I 

IO 

o 

7 

3 

I 

IO 

0 

51 

7 

3 

IO 

o 

IO 

o 

7 

2 

IO 

id 

o 

IO 

O 

0 

IO 

o 

54 

9 

1 

IO 

o 

IO 

o 

IO 

O 

10 

9 

I 

56 

6 

4 

I 

- 

2 

57 

10 

O 

10 

0 

IO 

IO 

O 

IO 

(} 

IO 

o 

2 

10            .1 

10 

o 

4 

6 

IO          O 

IO 

i.i            8 

2 

IO          O 

10 

62 

4 

6 

IO          O 

10 

0 

63 

) 

y 

IO           O 

10 

o 

64 

to 

o 

10        o 

IO 

o 

65 

10 

O 

IO           O 

10 

0 

66 

9 

i 

1C)         o 

10 

o 

6? 

8 

2 

10        o 

10 

o 

68 

10 

O 

10            O 

IO 

o 

69 

10 

0 

IO          O 

IO 

0 

70 

10 

O 

IO 

o 

10 

o 

7i 

5 

3 

8 

10 

o 

72 

IO 

o 

10 

o 

IO 

o 

7J 

10 

o 

10 

o 

IO 

o 

74          10 

0 

10 

10 

0 

IO 

o 

IO          O 

IO 

o 

76 

10 

o 

10          0 

10 

77 

10 

o 

10       o 

IO 

o 

78 

10 

o 

10       o 

10 

o 

79 

10 

o 

10 

0 

10 

o 

10 

o 

10 

10 

o 

M 

IO 

o 

IO          O 

IO 

o 

1 

IO          O 

10 

o 

I,, 

o 

10          O 

IO 

o 

10 

o 

10          0 

IO 

0 

10 

o 

IO          O 

IO 

o 

10 

o 

IO          O 

10 

o 

10 

o 

10 

o 

IO 

o 

10 

o 

IO 

IO 

o 

IO 

0 

IO          O 

10 

0 

90 

IO 

o 

10          0 

10 

0 

91 

9 

I 

10 

10 

o 

pa 

10 

0 

10 

0 

9 

o 

93 

10 

0 

10 

o 

IO 

0 

<i\         i" 

o 

IO          O 

IO 

o 

95         10 

o 

IO          0 

IO 

o 

96         i" 

10          0 

10 

o 

97 

IO 

IO          0 

IO 

o 

98 

10 

o 

10 

0 

• 

o 

99      1    10 

o 

!   10 

0 

1     10 

0 

<     lurnti.ited  cooked  horse  manure  media  began  to  be  used. 


210 


D.    D.    WHITM.N  . 


TABLK  III. — Continued. 


Generation. 

Race  A  . 

Race  B. 

U.i.  e  C. 

No.  of 

9  9. 

No.  of 

c?9. 

No.  of 

9  9. 

No.  of 

c?9. 

No.  of 

9  9. 

No.  of 

cT9. 

100          9 

I 

IO 

0          IO 

o 

101            10 

0 

10          0 

IO 

o 

IO2 

10 

o 

IO 

0 

IO 

0 

103 

IO          O 

10 

o 

10 

o 

104 

10          O          10 

0 

IO 

o 

105 

9 

I          IO 

0 

10 

0 

1  06 

IO 

o 

IO 

0 

IO 

o 

IO7 

10 

o 

IO 

o 

10 

0 

108 

10 

o 

10 

0 

IO          O 

109 

10 

0          10 

0 

10 

o 

no 

IO 

O           IO 

o 

IO 

o 

1  1  1 

10 

o       10 

o 

6 

o 

112 

10 

o 

10 

o 

10 

0 

H3 

IO           O 

10 

O          IO          O 

114 

IO 

o 

IO 

O          10          O 

"5 

IO 

o 

10 

o 

10 

0 

116 

10          0 

10 

0 

IO 

0 

117 

10          0 

10 

o 

IO 

o 

118 

IO          O 

IO 

o 

10 

o 

119 

IO          O 

IO 

o 

IO 

0 

120 

10 

0 

10 

0 

10 

o 

121 

10 

0 

IO 

o 

IO 

o 

122 

10 

o 

10 

o 

10 

o 

123 

10 

o 

IO 

0 

10 

o 

124 

IO 

0 

10 

0 

IO 

0 

125 

10 

0 

10 

o 

IO 

o 

126 

IO 

o 

10 

o 

10 

o 

127 

10 

o 

10 

0 

IO 

0 

128 

10 

0 

10 

o 

IO 

0 

129 

10 

0 

10 

o 

10 

o 

130 

10 

0 

10 

o 

10 

o 

131 

10 

0 

10 

o 

10 

0 

132 

10 

o 

10 

0 

IO 

o 

133 

IO 

0 

IO 

o 

10 

o 

134 

10 

0 

IO 

o 

10          0 

135 

10 

O          lo 

0 

IO          O 

136 

10 

0 

10 

o 

IO           O 

137 

10 

0 

10 

o 

IO          O 

138 

10 

o 

IO 

0 

10 

0 

139 

10            0 

10 

o 

IO 

o 

I4O 

10            o 

10 

0 

10 

o 

141 

10 

0 

10 

0 

10 

0 

142 

IO 

o 

10 

0 

10 

0 

143 

10 

o 

10 

0 

10 

o 

144 

10 

0 

IO 

0 

IO 

0 

145             M> 

0 

10 

0 

10 

0 

1  46             lo 

o 

10 

o 

10 

0 

147 

IO 

0 

10 

0 

10 

0 

148 

10 

o 

10 

o 

10 

o 

149 

ID 

o 

IO 

I) 

10 

0 

ISO 

Id 

O          lo          O 

ID 

0 

151 

10 

O          10 

o        10 

0 

152 

10 

0           10 

0          IO 

0 

153 

IO            o            in            o            lo 

0 

STRAIN'S        IN    HYDATINA    SENTA. 


21  1 


TAHLK   III. — Continued. 


e  .-J. 

Race  B. 

aeration. 

No.  of 

No.  of 

No.  of 

No 

No 

?. 

c?9. 

99- 

<7  9. 

9  9- 

c?9. 

[0 

o 

10 

10 

0 

I  ii 

I  O 

o 

IO 

i 

In           O 

IO 

IO 

is? 

pi          O 

IO 

0 

IO 

In           O 

IO 

0 

10          O 

159 

!••          0 

IO 

o 

10          0 

160 

pi           O 

10 

0 

IO          ii 

161 

I'l           O 

10 

10          0 

162 

In 

10 

0 

10          O 

163 

10 

o 

IO 

o 

IO 

i64 

to 

(J 

10 

o 

IO          O 

10 

0 

10 

o 

10 

y 

1 

IO 

o 

IO          O 

167 

9 

1 

10 

o 

10          O 

111 

o 

10 

0 

IO          O 

169 

IO 

IO 

o 

10          O 

IT" 

0 

10 

o 

10 

171 

10            0 

IO 

o 

IO 

IO            0 

10 

0 

10            il 

173 

10       o 

IO 

0 

10          0 

10 

o 

IO 

o 

IO          O 

IO 

0 

IO 

o 

10 

'7'' 

10 

0 

IO 

0 

10 

'77 

IO 

0 

o 

10         O 

10 

0 

I  O 

0 

10 

179 

10 

IO 

o 

10 

10       o 

IO 

0 

10 

181 

10          0 

ID 

o 

IO 

IO          0 

IO 

o 

10 

o 

IO          0 

10 

o 

10 

o 

IO 

IO 

0 

10 

o 

10 

o 

IO 

o 

10 

0 

10 

o 

ID 

o 

10 

10 

10 

o 

10 

10 

IO 

0 

111 

o 

10 

o 

IO 

0 

IO 

8 

IO 

0 

10 

o 

191 

10           i' 

10 

0 

10 

o 

10 

o 

10       o 

2 

o 

2 

o 

I 

0 

194 

1 

o 

I 

0 

I 

195 

4 

o 

10 

o 

10          O 

[96 

6 

8 

o 

3        o 

197 

10 

IO 

o 

10 

198 

10 

o 

IO 

IO         0 

199 

10 

o 

IO 

IO          O 

10 

0 

10 

III 

10 

0 

10 

o  . 

In          O 

202 

10       o 

IO 

0 

Hi          0 

9        i 

10 

o 

III 

9 

1 

10 

111 

o 

10 

o 

10 

in          O 

10 

o 

I  II 

0 

Hi          0 

IO 

o 

10 

0 

IO          O 

212 


D.    D.    WHITNEY. 


TABLE  III. — Continued. 


Generation. 

Race.-/. 

Race  B. 

Race  C. 

No.  of 

9  9. 

No.  of 

cT9. 

No.  of 

99. 

No.  of 

d1  9. 

No.  of 

9  9. 

No  of 

c?9. 

208 

10 

0 

10 

o 

10 

0 

209 

IO 

o 

IO 

0 

10 

o 

2IO 

10 

0 

10 

0 

10 

o 

21  I 

10 

o 

10 

0 

10 

0 

212 

10 

o 

9 

1 

10 

o 

213 

10          0 

IO 

0 

IO 

0 

214 

10 

0 

10 

o 

10 

0 

215 

IO 

o 

IO 

o 

10 

0 

216 

8 

0 

8 

0 

8 

0 

217 

9 

I 

10 

o 

10 

o 

218 

10 

0 

10          0 

to 

0 

219 

IO 

o 

10          O 

10 

o 

22O 

10 

o 

10 

0 

10 

0 

221 

10 

0 

IO 

o 

10 

o 

222 

5 

o 

4 

0 

IO 

0 

223 

10 

o 

IO 

o 

IO 

0 

224 

IO 

0 

IO 

0 

10 

0 

225 

IO 

0 

10 

o 

10 

o 

226 

IO 

o 

9 

I 

10 

0 

227 

I 

0 

I 

0 

I 

o 

228 

10 

0 

IO 

o 

IO 

0 

229 

IO 

o 

10 

0 

10 

0 

230 

IO 

o 

4 

6 

10 

0 

231 

10 

0 

10 

0 

10 

0 

232 

10 

0 

5 

0 

10 

0 

233 

10 

o 

10 

o 

10 

o 

234 

7 

0 

4 

0 

9 

o 

235 

i 

0 

2 

0 

10 

0 

236 

4 

0 

I 

0 

3 

0 

237 

10 

o 

I 

0 

10 

o 

238 

IO 

o 

4 

o 

IO 

0 

239 

10 

0 

0 

0 

9 

0 

240 

10 

o 

Died. 

10 

0 

241 

3 

0 

10 

o 

242 

4 

0 

IO 

0 

243 

10 

0 

IO 

0 

244 

8 

2 

IO 

0 

245 

10 

o 

10 

o 

246 

IO 

0 

10 

o 

247 

10 

0 

10 

0 

248 

10 

0 

IO 

0 

249 

IO 

o 

10 

0 

250 

IO 

0 

10 

o 

251 

IO 

o 

10 

0 

252 

9       o 

10 

o 

253 

10 

0 

10 

0 

254 

9 

0 

9 

0 

255 

10 

0 

10 

o 

256 

6 

o 

9 

o 

257 

10 

0 

IO 

0 

258 

10 

0 

IO 

o 

259             ", 

0 

IO 

o 

10 

o 

10 

0 

261         10 

0  '                               10 

0 

STRAINS        IX    HYDATIXA    SEXTA. 


TABLE  III. — Continued. 


Race  A.                                Race  B. 

Xo.  of              Xo.  of            No.  of 

No. 

\ 

d"9.           99.          (79.           99. 

•9. 

7 

IO 

90                                                            10 

o 

90                                                         10 

0 

IO                        O                                                                                  1  'I 

o 

O                                                                                 in 

o 

O                                                                                 IO 

0 

IO 

0 

IO 

o 

IO 

10 

o 

-'71                               I  O                       O 

IO 

o 

in                         O 

IO 

10                         0 

10 

0 

7                 3 

10 

o 

IO                         O 

IO 

o 

IO                         O                                                                                 IO 

10                         0                                                                                 10 

o 

278                                 If)                         0 

IO 

II 

10                         0 

1  II 

0 

8                    o 

IO 

II 

9                     1                                                                    10 

0 

8                     2                                                                  10 

•  1 

8                  a 

IO 

If)                          0 

10 

II 

4                  6 

10 

0 

5                   4 

10 

II 

9                    1 

IO 

II 

8                    2 

10 

II 

6                   o 

10 

7                   o 

7 

-••;!                               6                      1 

9 

•  1 

8                 o 

2 

9                   0 

III 

o 

IO 

pi 

II 

4                  o 

9 

o 

-••/•                           40                                                                 9 

0 

i                   o                                                              v 

o 

4                    « 

o 

4                   5 

4 

II 

8                  o 

- 

0 

70                                                            10 

II 

10                        0                                                                                 10 

o 

9                  o 

o 

10                         0                                                                                  1" 

II 

9                  o 

o 

IO                         0 

- 

o 

80                                                              9 

o 

40                                                              7 

0 

309                          60                                                            i" 

0 

7                                                                                10 

o 

.ill                                 10                                                                                                             1" 

o 

3«                        5                 o 

5 

o 

313                         5                 o 

5 

0 

.i  1  4                           5                   ° 

5 

0 

315                        3                                                                              S 

o 

214 


D.    D.    Will  I  \1   V. 


TAHLK  III. — Concluded. 


Generation. 

Race  A. 

Race  B. 

K.I. 

No.  ot 

9  9. 

;  C. 

No.  of 

99. 

No.  of 

d"9. 

No.  of 

9  9. 

No.  of 

<79. 

No.  of 

d"9. 

316 

4 

I 

5 

0 

317 

5 

0 

5 

0 

318 

5 

o 

. 

5 

0 

319 

10 

o 

10 

0 

32O               S           2 

10 

0 

321 

5 

0 

5       ° 

322 

5 

0 

5 

0 

323 

4 

I 

5 

0 

324 

5 

0 

5 

0 

325 

5        o 

IO 

0 

326 

9        i 

10 

0 

327 

6          2 

IO 

o 

328 

3 

o 

10 

0 

329 

9 

I 

10 

o 

330 

IO 

o 

10 

0 

331 

7 

3 

10 

o 

332 

IO 

o 

10 

0 

333 

8 

2 

IO 

o 

334 

6 

4 

10 

o 

335           8 

0 

10 

o 

336 

8          2 

10 

0 

337 

8          2 

IO 

o 

338 

9 

I 

10 

o 

339 

IO 

O 

10 

o 

340 

10 

o 

10 

0 

341 

10 

o 

10 

0 

342 

IO 

0 

10 

0 

343 

4 

2 

IO 

o 

344 

9 

0 

10 

0 

345 

5 

I 

10 

o 

346 

6 

0 

8 

2' 

347 

8 

o 

10 

0 

348 

5 

0 

9 

I 

349 

3 

1) 

9 

I 

in  the  three  strains,  A,  B,  and  C,  when  all  external  conditions 
are  identical  the  only  conclusion  that  can  he  drawn  is  that  the 
three  strains  differ  at  least  in  regard  to  this  single  characteristic. 
Perhaps  they  all  may  be  potentially  alike  in  their  capacity  to 
produce  male-laying  females  but  some  races  may  be  more  easily 
effected  than  other  races  by  the  influence  which  causes  male- 
producing  females  to  be  produced. 

Whenever  races  A  and  B  were  put  into  newly  made  diluted 
uncooked  culture  media  in  battery  jars  great  numbers  of  fertilized 
eggs  were  produced  in  U>ih  races.  From  general  observations 
they  seemed  to  be  produced  in  equal  numbers,  thus  seeming  to 

1  Food  media  was  diluted  accidentally. 


"STRAINS"  IN  HVDATINA  SHNTA.  215 

form  evidence  that  these  two  races  were  potentially  alike  in  their 
power  to  produce  male-producing  females  but  when  conditions 
were  unfavorable  they  differed,  as  shown  in  the  parallel  series,  in 
their  re-p.  ,ii-iveiiess  to  the  influences  that  so  acted  upon  the 
females  as  to  cau-e  them  to  produce  male-producing  daughter 
fem. ili--.  Ho\ve\er.  when  race  C  was  put  into  newly  made  un- 

ked  culture  media  in  battery  jars  very  few  fenili/ed  eggs 
were  prodm  ed,  thus  K-eming  to  show  either  that  thi-  race  C  was 
potentially  different  from  the  other  two  races  in  it-  rapacity  t» 
produce  male-producing  females  or  that  it  was  not  as  ea-ily  acted 
upon  by  iln-  male-producing  female  influences  as  were  races  .1 
and  /v  \oi\\hh-tanding  this  fact  that  race  C  produced  very 
le\\  male- producing  f. -males  when  put  into  battery  jar-  containing 
dilute  iiiiiooki-d  horse  manure  media  it  should  be  stated  that  in 
the  e.irK  lii.~tor\  of  race  C'  it  had  as  high  a  percentage  of  male- 
producin-  female-  in  the  first  four  generations  as  \\a-  found  in 
either  race  .1  or  B.  Table-  II.  The  race  was  isolated  from  a 

.  r.il  cnli  ure  jar  in  \\hich  an  abundance  of  males  were  appearing 
at  the  time  i >l  i-o|ation.  Beginning  with  generation  ii\e  verj 
le\\  in. ile-  appeared  thereafter.  This  early  history  sh«.\\  -  that 
race  Cat  one  time  was  as  potential  in  its  power  to  produce  male- 
producipv:  it-males  as  races  A  and  B,  but  whether  it  later  lost 
thi-  power  ( -r  never  was  again  subjected  to  as  favorable  intlnem  es 
for  the  production  of  male-producing  females  it  i-  impo--ib!e 
to  state.  \\  ii.ii>  \<r  may  be  the  true  explanation  of  thi-  di\»-r- 
gence  in  the  male-pnKlucing  female  rates  of  the  three  races  it 
-iiteK  indicate-  a  dillerence  in  the  races  either  in  their  capacity 
to  product-  male-producing  females  or  their  responsiveness  to 
tin-  inlluenct  -  thai  cause  male-producing  femak--  to  U-  produ.  .-d. 
I'niinett  coinliidid  that  he  found  "sex  -train-  '  in  Ilydntiiid 
/  \\hich  differed  in  their  power  to  prodnc.-  male-  and  even 
i  on.-ludt-il  that  he  found  some  strains  that  produced  no  male-. 
It  is  verj  po--ible  that  such  maleless  strain-  \\ire  n-alh  like 
rat  ••  c '  in  the  above  experiments.  From  ob-<-r\  atioiis  and  experi- 
ments publi-hed  in  an  earlier  paper  ('07  .  it  was  -lu>\vn  that  no 
pun-  female  -trains  could  be  found.  The  re-ult-  of  the  pie-.nt 
experiment-  o>i roborate  this  earlier  conclusion.  However,  the 
evidence  at  that  time  showed  no  strains  of  anv  kind  but  the 


216 


D.    D.    WHITM.V 


: 


•ss 


'S 

'3 

w 

8 


3 


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STRAINS        IX    HYDATIXA    SI.MA.  21  7 

pre-ent  evidence  collected  from  observations  extending  over  a 
period  of  about  three  years  and  including  300-500  partheno- 

etic  ^em-ration-,  -hows  very  clearly  that  strains  exist  which 
differ  in  their  percenta^-  of  male-producing  females. 

Moreover,  tin-  t\\o  -ister  strains  A  and  B  which  developed 
from  the  -amc  fertili/ed  egg  differed  in  their  longevity.  Strain 
/•!  died  out  from  general  exhaustion  in  the  384th  partheiio-enetic 

•  •ration,  whik-  -train  .-1   is  still  alive  in  the  5041)1   partheno- 
n< -ration,  although  in  a  very  weak  and  exhausted  con- 
dition. 

Shull  lia>  compared  Mime  of  the  New  York  strains  of  Ilydntina 

;  \\itli  a  -train  from  Baltimore  and  has  found  a  divided 
ilitlereiii  «•  in  the  rate  of  production  of  males  in  the  tw»  Miain-. 
II'  'It  is  safe  to  say,  therefore,  that  we  have  lure  t\\o 

pun-  line-  1 1 1,1 1  1 1;  -m  one  another  in  a  fairly  constant  maim-  i . 

and  the  din.  is  an  internal  one." 

SUMMARY. 

i.  The  prudiietioii  of  male-producing  females  can  In-  p.irtly 
or  \\holK  i-epreNM-d  hy  external  conditions  in  parthenogenetic 
iai  es  ot  Ily<!nlinn  *i-nta. 

j.    The  jiartlu  lie  strains  are  shown   to  he  distinct    i 

cause  mi'ltr  identical  external  conditions  they  differ  in  their 
po\\er  t..  produi-i-  male-producing  females.  This  may  indicate 
that  the\  differ  in  their  potentiality  of  producing  male-pn>ducin- 
female-  or  that  they  differ  in  degree  of  respond  cne»  to  the 
inllueiice-  \\hich  cause  male-producing  females  to  \n-  ]>roducei|. 
The  latter  alternative  seems  more  probable. 

j.  The  tuo  -i-ier  parthenogenetic  strains  developing  from  one 
fertili/ed  egg  dilh  red  in  their  longevity.  One  li\ed  almut  a  year 
longer  and  pro.lnced  over  one  hundred  IIIOR  generations  than 
the  other. 

1>I«  'I  '  ".li    \l      I     \Hi>K.VTORY. 

WESLEYAN  I'NIVERSITY. 
MIDIM.KTOWN.  CONN.. 
January  3.  1912. 


21  ^  I).    D.    WHITM  N  . 

LITERATI-RE  CITED. 

Punnett,  R.  C. 

'06     Sex-determination  in  Hydatina,  with  Some  Remarks  on   Parthenogenesis. 

Proc.  Roy.  Soc.,  B,  Vol.  78,  pp.  223-231. 
Shull,  A.  F. 

'n     Studies  in  the  Life  Cycle  of  Hydatina  senta.     Jour.  Exp.  Zool.,  Vol.  10. 
Whitney,  D.  D. 

'07     Determination  of  Sex  in  Hydatina  senta.     Jour.  Exp.  Zool.,  Vol.  5. 
'10     The   Influence  of   External   Conditions   upon   the  Life-cycle  of    Hydatina 
senta.     Science,  N.  S.,  Vol.  32,  No.  819,  Sept.  9. 


-I   PERN1   MI.KAKY    CHROMOSOMES,    AND    SVXAPSIS 

i\  <  1:1  i  Hopmi.rs  (sr 

N.   M.  STEVENS. 

'I'll'  i   (  '-  i<!i:»!>liilus  which  I  have  iist-d  in  this  siudy, 

I  ha\e  ii"!  liccii  able  to  identify.  The  material  st-eiiis  to  be 
homogeneous,  .m<l  i-  the  only  species  of  this  gemi-  that  I  have 
seen  about  Br\  n  M.iur.  The  insects  were  found,  usually  in 
pairs,  in  their  bnrro\\s  under  stones,  and  wen-  i  <  >llected  in 
<  ><tober  and  November,  i<m>  and  i<)ii.  They  are  not  abundant. 
and  onlv  7  m.di  •-  \\  ured  in  1910,  and  5  in  i<ii  i  . 

METHODS. 
In  ill-  h  individual  a  few  aceto-carmine  preparations 

\\ITC    in.  id.  .    .nid    tin-   rcniainder  of   the   testes  fixed    in 
mercuro-nitric    iluid    <>r   in    Remming's  stronger   (-liro 

'ic   niixiiiM         I  In-   \><-[    results  were  obtained    t'rmn    -i-(|i..n< 
"I    I  I*  •ininiii:'   in.iit-ri.il.  <  ut    10/1  thick  and  stained  \\ith  thionin. 


^i  11  K\I  \i  i  KAKV  CHROMOSOMES. 

In  one  "i  tin-  I<H»  insects  one,  and  in  another  \\\«  -uprr- 
A  cliniin..-.!!!!!--  were  found.  Tlu-si-  rhrunn  »-i  >nn--  .ire 
n.idilv  di-iiii'<iii-ln-d  li"in  the  other  chromic-urn  -  b\-  their 
Miialh  r  -i/e  ,unl  i  rralir  brhavior. 

The  -perm.  no:1'  mia!  chromosomes,  like  tho-e  of  Stenopelmatus, 

are  diltn  nil  to  count,  because  they  do  not  form  a  llat  jil.ite  ,u 
an\  stage,  bin  lie  .it  -oinewhat  dilTerent  K-\eU  and  o\eilap. 
The  number  i->  probabk  ,7,  exclusive  <M  -upernuiiierai  ; 

In  the  !ir-t  -|>-  i  niatocytes  there  an  i^  bi\alent-  an<l  the 
uni\-.ilent  A"  1-1  .  \\lu-n  no  supernumeraries  are  preM-m  Fig.  i  . 
Fig.  2  -lio\\-  i  ^  bi\.iK-nts,  the  unpaired  chronio^oiiu-  A",  and  2 
-upernuiiu-rar-i  The  odd  chromosome  A'  i^  u-uall\-  found 

at  one  pole  of  tin-  >pindle  when  the  other  chromosome-,  are  in 
metapliase  Fig  ;  r  but  it  not  infrequently  lags  behind  the  others 
in  the  anapha-e  1  'ijr.  4)  and  is  enclosed  in  a  separate  membrane 

219 


22O 


N,    M.    STEVENS. 


3 


--/-  x 


8 


FIGS.  1-2.  Mctaphasc  of  first  maturation  mitosis,  showing  i8+A'  and  i8+X 
+  25.  (Mag.  1,500  for  all  figures.) 

FIGS.  3-5.     Mctaphase  and  anaphascs  showing  position  of  X. 

FIGS.  60  and  b.     Young  sister  spermatocytes  of  the  second  order. 

FIGS.  7-12.  Variations  in  position  and  division  of  the  two  supernumeraries 
in  first  spermatocytes. 


SUPERNUMERARY   CHROMOSOMES    IX    CKUTHOPHII  221 

in  the  tclophase  (Fig.  5).     Figs.  6a  and  6b  are  sister  spermatocv 
of  the  second  order,  showing  X  in  a  separate  vesicle:  this  does 
not  happen  in  by  any  means  one  half  of  the  second  spermatocytes, 
X  l.rin:/.    I   ^-hould   say,   more  often  included  within   the  same 
nm  Ic-ar  mem! T.ui''  with  tin-  other  chromosomes. 

1  i  7  is  from  the  individual  which  had  one  supernumerary, 
am!  I  )'•<-.  8  to  14  from  tin-  < >ne  that  had  two.  1  ilso  >h-nv- 

a  less  ii-ual  p"-iiioii  of  A",  near  the  equatorial  plate.  As  in  the 
/'/••  5l  the  supernumeraries  mayor  max-  not 

divide  in  the  t"ir-t  maturation  mitosis,  and  they  may,  when  un- 
di\i  >  eiiher  pole  of  the  spindle.  Their  position  in  the 

met.ipha--e  does  not  nec.-^.irily  indicate  whether  they  will  di\  id. 
or  in-!.      1  In-  di'ti-niiiniiig  factor  is  probably  the  attachmen; 
-pindle  ill  MI  OIK-  n  both  poles.    The  supernumeraries 

in  Figs.  7,  >>  and  ')  would  not  divide  in  this  mitosis,  but  the 
piv-en.  i.iry  in  an  equatorial  plate  or  between 

daii;JiiiT  pi. HI ~  d.  -  essarily  assure  its  division.     In  I 

i"  one  i-  di\i<l<-d.  tlie  other  undivided,  while  in  Fie-.  11  and  u 
both  are  divided.  In  the  telophases  shown  in  1  i^-.  i.>  and  14, 
both  -np< •rnumerai :  divided  in  one  case  and  neither  in  the 

othi-t.        I  'hi    p(--ible  c  i.mbinations  of  A'  and  the  2  -npernume- 
raries  in  the  ^pcrmaiid-  are  A",  A"  +  is,  X  +  25,  o,   15  and 
loiiroi  the  possibilities  are  shown  in  Figs.  15  to  [8. 

As  to  the  origin  of  these  supernumeraries,  there  is  little  evidence 
in  tin-  material.  In  MctapodlHS  Wilson  ('09)  disco\end  the 
pmbable  i.ii^in  of  the  -npeniumeraries  in  an  irregular  ^erond 
>pei -maiiH -\  te  miti.~i-  in  which  both  " idiochromosomes "  \\ent 
!•»  the  -ami-  IM.IC  ot  the  >pindle,  and  therefore  to  the  same  -penna- 
lid.  The  supernumeraries  are  thus  shown  \»  be  duplicate-  ..i 
the  -mailer  "  idii  .chromosome"  in  Mctapodius,  or  in  one  case  of 
.in  "  ":-chroMio-i niie"  *io).  In  neither  Diahrotica  nor  in  Ccntlio- 
philn*  i-  there  a  -mailer  mate  for  the  A'  chromosome  pre-ent. 
The  three  supernumeraries  which  have  been  observed  in  (.'cntho- 
philus  are  of  about  the  same  size;  considerably  less  than  mie  half, 
and  app.irciitU  about  one  fourth  the  size  of  A'.  The  behavior 
of  the  -II|H  iniimeraries  in  growth  and  re-t  -tai^e-  of  the  nucleus 
indicate-;  their  probable  relationship  to  A',  and  their  behavior 
in  mito-i-.  dividing  only  once,  either  in  the  lir-t  or  the  -econd 


222 


X.    M.    STEVENS. 


19 


21 


23 


24  26 

FIGS.  13-14.  Telophascs  showing  the  supernumeraries  divided  (13)  or  un- 
divided (1-4). 

FIGS.  15-18.  Spermated  nuclei  showing  variations  as  to  presence  or  absence  of 
X  and  the  two  supernumeraries. 

FIGS.  19-20.     Anaphases  showing  unusual  position  of  X. 


SUPERNUMERARY  CHROMOSOMES    IN    CEUTHOPHELUS.        223 

maturation  mitosis,  shows  that  they  are  univalent.     In  Diabrotica 
soror  I  have  considerable  evidence  that  the  supernumeraries  owe 
their  origin   to  a  transverse  and  a  longitudinal   division  of  X 
('12  .   tii'l  it  seems  probable  that  those  of  Ccnthophilus  have  had 
-'milar   origin.     I    have   occasionally    found    ca-e-   where    A" 
"ied    about    to   divide   late   in    the   first    maturation    mitosis 
I  md  20),  but  I  have  as  yet  no  evidence  of  a  tran-\  < 

di\  i-ion. 

SYNAPSIS. 

The.   material  which  was  collected  in   K)ii   with   the  hope  of 
at  on  the  origin  of  the  Bupernumerari<  \ed 

to  be  l'.i\orable  for  a  study  of  synapsis,  or,  as  I  -hould  prefer  to 
call  the  phenomenon,  conjugation  of  the  chronio-onu  -.     T! 

'  -  uere  all  fixed  in  Flemming  and  stained  with  either  thionin 
or  iron-haematoxylin.     Thionin  gave  the  clearest  figures. 

In  the  re-ting  nuclei  of  the  spermatogonia  the  chromo-ome- 
are  eii  her  \  i-iblr  as  separate  individuals  as  in  Fig.  21,  or  are  m«  >re 
(.1  1  upleiely  resolved  into  rather  fine  spireme  thread-  a- 

in  I  -.      In  sonic  follicles  one  sees  only  such  -pennatogonial 

nuclei  .1-  in  Fig.  21;  in  others  the  various  cyst-  -h»\\  \arimis 
Dilution  into  spireme  threads.  The  former  condi- 
tion I  should  attribute  to  more  rapid  division  of  the  -permato- 
'^onia,  the  time  between  mitoses  being  insufficient  for  complete 
H  -olntion. 

In  the  \oimgcst  spermatocytes,  distinguished  from  the  -p.-rma- 
>nia  by  the  condensed  condition  of  A',  the  -pin -me  thread- 
are  -i  milar  to  those  of  the  spermatogonia,  per  hap-  a  lit  tie  coarser. 
They  are  finely  granular  and  more  or  less  nodular.  There  is  no 
contra,  tion,  or  synizesis,  stage  and  no  complete  pol.iri/atioii  of 
loop-  to  form  a  perfect  bouquet  stage.  The  -pirem-  threads  are 
n-uallv  irregularly  but  rather  evenly  distributed  through  the 
nucleus  as  in  I  ig.  2.},  which  also  shows  A'  in  characteristic  position 
a-ain-t  the  nuclear  membrane.  Fig.  2.\  -ho\\  -  an  extreme  and 


1  i  j.     Spermatogonial  nuclei,  slmwiMi;  1   mm --..Ivr. I   .  liro- 

1  i..  .• ;      Spcrmatocyte  nucleus  before  syna; 

l-'ii..  .'i       Similar  stage  showing  partial  pnlari/.iticn  »\  rhr<>i: 

I  i.  16.     Stages  in  parasynapsi-. 


224 


N.    M.    STEVENS. 


a    » b 


34 


30 


36 


33 


35 


FIG.  27.     Double  spireme  stage. 
FIGS.  28-29.     Prophase  stages. 

FIG.  300-1.     Various  prophase  (a-g)  and  metaphase  (h,  i)  forms  of  the  bivalent 
chromosomes. 

FIGS.  31-32.     Chromosomes  in  metaphase  of  the  first  maturation  mitosis. 


SUPERNUMERARY    CH  K<  >M<  >-<  >M  1  -    IN    i   I  I    1  HOPI11L!   S.          225 

unusual  case  of  polarization,  but  here  only  nine  polarized  loops  are 
-ent  and  the  other  spireme  thread-  extended  in  various  diiec- 
tion-  through  the  nucleus.  In  some  follicles  there  is  a  perfectly 
sharp  divi-ion-line  between  cysts  containing  spermatocvte  nuclei 
of  the  chaiacter  of  that  shown  in  Ficr.  2.^.  and  cysts  in  which  the 
-pin-mi-  i-  about  twice  as  thick  (Fig.  27).  There  is  no  evidence 
of  ti -I..-;.,  nap-is,  and  also  no  evidence  of  any  such  longitudinal 
contraction  of  the  threads  as  would  be  required  to  give  tin-  thicker 
-pin-iii'-  of  I  ig.  27.  In  other  follicles  there  come  in  bet  \\eeii 
tli»  -,  other  cysts  in  which  as  a  rule  the-  chromatin 

thn-.id-  are  \«-ry  irregularly  distributed;  i.  e.,  thru-  i-  a  con- 
cpicnoii-  ab-i-mv  of  the  rather  even  spacing  of  tin-  thn-ad-  ol>- 
-•T\i-d  in  Figs  22  and  23.  Careful  inspection  of  -uch  cysts 
reveals  varioi  .'-sin  pairing,  conjugations,  or  -ynap-i-  "I  the 

chroiiMtin  tlin-ads  (Figs.  25  and  26).  In  tin-  sunc  cyst  "tic 
ofii  n  find-  \arious  stages, — only  one  or  two  pair-  and  the  other 
thread-  -iirJe  as  in  Fig.  25,  and  all  stages  up  to  one  in  which 
all  ot  the  threads  arc  in  pairs.  Occasionally  some  of  the  pair-  in 
a  inn  leu-  -how  as  much  polarization  as  is  indicated  in  I  ig.  J<>, 
I'lit  in  the  -ame  nucleus  other  pairs  are  differently  di-iril.ui.  •!. 
I  Inn  i-  in  \  IT  -uch  complete  polarization  as  i-  sin  >wn  in  ^  >m<  .  >t 
tin  tii'iin--  of  (  ,n'i;oire  Cio),  the  Schreiner-  '"i  •  'n 

and  oilu-i^.  The  occurrence  of  various  stage-  of  p.u\i-\  nap-is 
in  the  -.inn-  cyst,  the  substantial  agreement  in  -i/e  of  the  chn.- 
m.itin  thn-.id-  through  the  stages  indicated  in  I -L-.  2;  2'>,  and 
the  -pirenir  of  double  size  in  the  next  stage,  pn-elude  the  p" 
bilit\  of  iiiirrpreiing  the  paired  condition  seen  in  I  i--.  25  and  2<> 
as  a  longitudinal  split.  Homologous  chronu  in  some  way 

("in.  together,  anil  gradually  twist  up  into  a  tighter  and  ti^hii T 
ro|u--like  -Hand.  A  casual  comparison  of  cysts  in  th' 
sho\\  n  in  I  i-.  j;  with  the  earlier  stages  (Figs.  2,^  to  ji.i,  u-ing  low 
po\\er-  of  the  microscope,  simply  gives  the  impression  that  here 
\\e  ha\e  a  -pitvme,  or  sections  of  a  -pireine,  twice  as  thick  a^  in 
the  |»n-\ious  stages,  but  study  of  such  nuclei  with  /ei--  1.5  nun. 
and  oc.  12  n-veals  the  double  and  twisted  condition  of  the  strands 

Ei  Anaphasc  showing  segregation  of  homologous  chromosomes  and 

Ion.nitmlin.il  splitting. 

1- 1«.-.  .^4-36.  Second  maturation  initosis  -howin^  diinnr|ihi-m  in  nunilx-r  (18 
and  ii;1  .tnd  i-i|ii;U  ional  divisiiin. 


226  X.    M.    STEVENS. 

in  practically  every  nucleus,  indicating  that  the  paired  threads 
are  at  no  time  so  thoroughly  fused  as  even  apparently  to  lose  their 
identity.     This   is   further   indicated   by   the  earliest   prophase 
stage  (Fig.  28)  where  the  paired  strands  begin  to  untwist.     The 
following  prophase  stages  consist  of  further  untwisting  and  longi- 
tudinal contraction  of  the  paired  homologous  chromosomes.     In 
the  synapsis  stage  (Figs.  25  and  26)  it  is  impossible  to  tell  whether 
threads  of  equal  length  form  the  pairs,  but  in  the  prophase  pairs 
this  is  perfectly  evident  (Figs.  29  and  30).     The  untwisting  and 
contraction  frequently   proceed   at   different  rates  in   different 
pairs  in  the  same  nucleus  and  in  different  nuclei  of  the  same  cyst, 
so  that  one  can  easily  compare  the  various  stages  of  the  process 
and  be  perfectly  sure  that  the  untwisting  is  continuous.     There  is 
no  secondary  fusion  of  paired  threads  such  as  frequently  occurs 
in    cases   where   a   precocious   longitudinal   split   appears   in   a 
telosynaptic  bivalent  and  then  closes  up  before  the  rings  and 
crosses  are  formed  (see  Blattilla  germanica,  Stevens,  '05).     Fig. 
30  shows  various  stages  in  the  formation  of  the  definitive  chromo- 
somes of  the  first  maturation   mitosis  from   the  parasynaptic 
threads  of  a  stage  a  little  later  than  that  of  Fig.  28.     The  paired 
chromosomes  untwist  and  contract  simultaneously.     Some  re- 
main united  at  one  end  (b  and  d)  while  in  other  cases  union  of  a 
pair  at  one  or  both  ends  is  a  secondary  phenomenon  and  may 
even  occur  after  the  spindle  has  formed  (a  and  c}.     That  there  is 
much  variation  in  the  form  and  size  of  the  18  bivalents  in  meta- 
phase  is  shown  in  Figs.  3,  7,  8,  9,  30,  31,  32  and  33.     The  most 
frequent  forms  are  rings,  E's  and  crosses,  though  one  or  more 
pairs  of  straight  rods  may  be  found  in  nearly  every  spindle. 
Most  of  the  chromosomes  are  attached  to  the  spindle  fibers  at 
or  near  the  middle  of  each  univalent  member  of  the  pair,  so  that 
the  separated  chromosomes  pass  to  the  poles  of  the  spindle  in 
the  form  of  V's  (Figs.  31  to  33).     In  the  case  of  the  double  rods 
the  fibers  are  attached  at  the  ends.     Many  of  the  chromosomes 
are  partly  or  wholly  split  longitudinally  in  the  anaphase  (Fig.  33). 
There  are  of  course  two  kinds  of  second  spermatocyte  equatorial 
plates  containing  18  and  19  chromosomes  respectively  (Figs.  34 
and  35),  X  appearing  in  the  form  of  a  large  V  (Fig.  35)  in  one 
half  of  the  cells.     Division  of  the  chromosomes  is  here  longi- 
tudinal as  seen  in  Fig.  36. 


-I   IM.KM  Ml   KARY    CHROMOSOMES    IN    CHUTHOPHILUS.         22 


--  i 


In  Ceuthophilus  the  first  maturation  mitosis  is  therefore  a 
segregating  division  of  the  previously  paired  and  united  homolo- 
gous univalent  chromosomes,  while  the  second  mitosis  is  as  clearly 
an  equational  division  of  all  of  the  univalent  chromosomes 
in<  hiding  A". 

DISCUSSION. 

In  an  earli< T  -tudy  ('05)  of  the  spermatogenesis  of  t\v<>  other 
species  of  (  >nln>pirra,  Blattella  (Blatta]  °ennanicti  and  Stcnopcl- 
nnitns  (-p.  I  >und  what  seemed  to  be  good  evidence.  <>t"  telo- 
synapsia  '05,  PI.  II.,  Figs.  55,  56,  58,  59, 62, 63,  64,  and  PI.  III., 
to  115).  That  material  I  have  reviewed  and  compared 
with  th<  '  ''kilns  preparations,  and  I  find  no  such  evidence 

•  it"  parasynapsis  in  fit  her  of  them.  Naturally  I  expected  to  find 
tel"-v  n.tp-i-,  in  Ceuthophilus,  and  was  surprised  on  working  back- 
ward t'n .in  the  maturation  mitoses  to  find  no  evidence  of  telo- 
synapsis  outside  of  the  late  prophases,  and  abundant  evidence  of 
parasynapsis  in  the  young  spermatocytes  at  a  st.'.-^c  \\lure  s\n- 
i/e-i-  i-  frequently  found  in  other  material. 

A  ivc.-nt  review  of  the  literature  on  conjugation  of  chromo- 
somes  ha>  only  strengthened  my  previous  conviction,  based  on 
in\  o\\  n  i  \prricnce  with  the  spermatogenesis  of  a  variety  of 
forms  that  tin-  phenomenon  is  one  which  vari-  aly  in 

ditii  n-nt  L;roiii»  of  organisms,  and  even  in  dinVn-nt  species  of 
the  ^anic  -«  nus,  or  different  sexes  of  the  same  '//a, 

Stevens  '03,  '05;  Btifo,  King  '07,  '08).     Indeed  I  should  not  be 
Mirpri>«-«l  it  the  range  of  variation  should  prove  to  i-\t.-nd  troni 
.  ases  \\lu-re  there  is  nothing  that  could  be  called  conjugation, 
but   nu-n-ly  such  a  pairing,  without  contact  even,  as  will  secure 
ition  of  homologous  maternal  and  patrmul  chromosomes 
to  different  daughter  cells,  through  (b)  an  intermediate  condition 
of  telosynapsis  and  less  intimate  parasynapsis,  to  (c)  cases  where 
hi  Min  )1<  >;^«  »ii»  c  hromosomes  are  so  completely  fused  in  parasynapsis 
that  it  is  impossible  to  tell  whether  the  resulting  chromosomes 
\\  hich  are  segregated  in  mitosis  are  identical  with  those  that  went 
into  synap>is  or  not;  and  the  variation   may  extend  to  cases 
which   may  give  further  support  to  Janssens'  chiasma  theory 
('09)  or  to  Morgan's  modification  of  it  t'i  O  in  which  homologous 
chromosomes   are    supposed   to  be   twisted    tightly    together   in 


228  N.    M.    STEVENS. 

parasynapsis  and  split  across  the  twists  in  preparation  for  mitosis, 
giving  daughter  chromsomes  which  contain  both  maternal  and 
paternal  chromatin. 

In  Ceuthophilus  the  parasynapsis  stage  of  Fig.  27  is  intimate 
enough  and  long  enough  to  favor  the  supposition  that  it  is  a 
true  conjugation  involving  exchange  of  material  particles  or  of 
chemical  substances  (genes),  but  there  is  no  evidence  of  any 
splitting  of  Morgan's  chiasma  type.  All  of  the  evidence  indicates 
that  homologous  paternal  and  maternal  chromosomes  twist 
together  in  parasynapsis  and  untwist  in  the  prophase  of  the 
first  maturation  mitosis.  In  the  flies  and  mosquitoes  (Stevens, 
'08,  '10,  fn)  we  have  examples  of  even  more  pronounced  para- 
synapsis than  in  Ceuthophilus,  but  so  far  as  I  have  seen,  the 
indications  are  that  the  chromosome  pairs  twist  up  in  synapsis 
and  untwist  in  prophase  much  as  in  Ceuthophilus;  i.  e.,  an  op- 
portunity for  interchange  of  genes  between  homologous  maternal 
and  paternal  chromosomes  is  furnished  by  the  observed  phe- 
nomena of  parasynapsis  in  these  forms,  but  no  evidence  of  such  a 
chiasma  type  of  splitting  after  synapsis  as  is  suggested  by  Morgan 
('IT)  to  account  for  the  results  of  his  breeding  experiments  with 
Drosophila.  Such  an  exchange  of  parts  of  chromosomes  as  that 
described  by  Janssens  ('09)  might  of  course  occur  without  being 
detected,  at  almost  any  point  in  the  process  of  twisting  or  un- 
twisting of  the  pairs,  since  the  time  element  is  not  determinate 
in  fixed  preparations. 

Moreover,  it  seems  to  me  that,  in  view  of  the  great  range  of 
variation  in  the  phenomena  of  conjugation  and  segregation  of  the 
chromosome  in  the  maturation  of  germ  cells,  cytological  evidence 
from  one  form  cannot  safely  be  taken  to  serve  as  the  basis  of  a 
theory  or  hypothesis  to  account  for  the  experimental  results  on 
another  form,  but  cytological  and  experimental  work  on  the  same 
form  must  go  hand  in  hand,  in  order  that  any  safe  conclusions 
may  be  drawn  from  the  results. 

There  seems  to  be  no  question  but  that  synapsis,  or  conjugation 
of  the  chromosomes  is  the  most  difficult  phenomenon  connected 
with  the  maturation  of  the  germ  cells,  to  interpret  correctly,  and 
doubtless  earlier  parasynaptic  stages  have  been  overlooked  'in 
some  cases  where  telosynapsis  alone  has  been  described  in  con- 


-I   IMKNUMKK  \KY    CHROMOSOMES    IN    CEl    I  Hnl'HILUS. 

nection  with  the  mitotic  stages  ot  maturation,  but  it  seems  to  me 
quite-  unlikely  that  synapsis  in  all  organisms  follows  one  method; 
and,  moreover,  I  believe  that  the  variations  in  method  of  synapsis 
and  intimacy  of  union  of  homologous  chromosomes  in  different 
forms  will  be  found  to  he  directly  connected  with  variations  in 
method-  i,f  inherit. tin  e  « >t"  unit  characters,  especially  in  relation 
to  inter,  li.m..-  or  Lick  of  interchange  of  maternal  and  paternal 
genes.  If  thi-  i-  true  \\e  should  expect  to  lind  more  cases  of 

•  >f  unit  characters  where  telosynapsis  or  no 
real  svnap  urs.      If  parasynapsis  is  an  adaptation  to  secure 

in!- T<  i  hould  expect  to  find  cas<  <  ot  tcl«»vnap- 

sis  folloucd  IA  parasynapsis,  as  indicated,  but  nor  certainly 
d.-iiion-traied  in  the  guinea-pig  (Stevens, 'l I,  Figs.  9,  10,  11 
In  my  -nnli< •-  on  spermatogenesis  of  the  Coleoptera  1*05,  '06, 
.  I  found  evidence  of  telosynapsis  in  several  cases  and  no 
c\  iilt-iK •«•  of  para^\  nap^is,  but  this  was  only  an  incidental  mat  ter 
at  i  In-  time,  an. I  .  ;  interest  merely  in  relation  to  the  s<  'ion 

oi  uliolr  i  In  -  in  the  maturation  mito-e<.      It  is  ni\   in- 

trntion  to  nine  all  of  my  Coleoptera  and  I  Mptrra  material 

\\itli    refer  ih«-   (juestions   whether   para-vnap^U   occur- 

in  the  Coleoptera,  and  whether  the  Diptera  >ho\\  .m\  evidence 

ot  Janssens1  ehia-ma  types  of  synapsis. 

i  I;E. 

J.i  :  2. 

LITERATURE    CITED. 
Agar,  W.  E. 

1 1  i       i        -  -;is  of  Ltpidosirtn  parad- 

I  I. 
Gregoire,  V. 

'10     I  le  Maturation  dans  les  deux  K   gn<    .     L'un  H  11.-  du 

IM.  tiquo.     La  Cellule,  XXVI. 

Janssens,  F.  A. 
'09  >|ii-iin.  ••   dans   les    Batrachiens.     1 

ictations  des  cindses  de  inatuiatimi.     La  C.-llulc,  XX\  . 
King,  H.  D. 

'07      Ih<-  >|..!  11.. i!. 'genesis  of  Bufo  .  Am.  Jmirn.  of  Anat.,  \"1I. 

'08      I'ln-  '  ••  -  of  Bufo  lentiginosus.     Journ.  of  M«>rpli.,  XIX. 

Morgan,  T.  H. 

'n     An  Attempt  to  Analyze  the  Constitution  of  the  Chromosomes  on  the  H. 

ni  Si-x-limited  Inheritance  in  Drosophilti.     Journ.  Exp.  Zool.,  XI. 
Schreiner,  A.  and  K.  E. 

'04      Dif  Reiiiin.u-teilungen  bei  den  Wirln  Itinon.     Ein  Beitrag  zur  l-'iai;.'  nach 

.ktiiin.      Anat.  An/..  XX1\". 


230  N.    M.    STEVENS. 

Stevens,  N.  M. 

'03     On  the  Oogenesis  and  Spermatogenesis  of  Sagitta.     Zool.    Jahrb.,  XVIII. 

'05     Further  Studies  on  the  Oogenesis  of  Sagitta.     Ibid.,  XXI. 

"05     Studies  in  Spermatogenesis,  I. 

'06     Studies  in  Spermatogenesis,  II.     Carnegie  Inst.,  Pub.  36,  Parts  I.  and  II. 

'08     A  Study  of  the  Germ  Cells  of  certain  Diptera  with  Reference  to  the  Hetero- 

chromosomes  and  the  Phenomena  of  Synapsis.     Journ.  Exp.  Zool.,  V. 
'08     The   Chromosomes  in  Diabrotica  viltata,   Diabrotica  soror  and   Diabrolica 

1 2-punctata.     A  Contribution  to  the  Literature  on  Heterochromosomes  and 

Sex  Determination.     Ibid. 

'09     Further  Studies  on  the  Chromosomes  of  the  Coleoptera.     Ibid.,  VI. 
'10     The  Chromosomes  in  the  Germ  Cells  of  Culex.     Ibid.,  VIII. 
'n     Further  Studies  on  Heterochromosomes  in  Mosquitoes.     Biol.  Bull.,  XX. 
'n     Heterochromosomes  in  the  Guinea-pigs.     Ibid.,  XXI. 
'12     Further  Observations  on  Supernumerary   Chromosomes  and   Sex  Ratios 

in  Diabrotica  soror.     Biol.  Bull. 
Wilson,  E.  B. 

'09     Studies  on  Chromosomes.     V.  The  Chromosomes  of  Metapodius,  a  Con- 
tribution to  the  Hypothesis  of  the  Genetic  Continuity  of  the  Chromosomes. 

Journ.  Exp.  Zool.,  VL 
'10     Studies  on  Chromosomes.     VI.  A  New  Type  of  Chromosome  Combination 

in  Metapodius.     Ibid.,  IX. 


I  I   Kllll.k   «»IM-:KVATIONS  ON   St'PERM  MF.RARY 

(  IIK<  »M<  >-<  >MES.   AND   SEX    RATIOS    IN 

MABROTICA   SOROR. 

X.   M.  STEVENS. 
^i   Tl  KM  MKKARY    CHROMOSOMES. 

lii   iln-   -iiniiiHT  iif   1910  while   I   was  enjoying  the  privile. 
.nnl  ho-pii,ility  uf  ihf  Marine  Biological  Laboratory  at  I. a  Julia. 
<  'alifornia.    I    \<>«k  advantage  of  the  opportunity  to  study   the 
malt   'o-rin  cell-  of  Diabrotica  soror  from  a  new  locality,      Ilaxini; 
pn-\  iou-lv  >und    suj>ernumerary    chromosomes    \ar\in^ 

in  uuiiil.iT  fi« .in  one  to  five  in  about  50  per  cent,  of  the  male 
individuals  of  Diabrotica  soror  at  Mountain  View.  <  'alit»i  nia. 
and  /'.  a  12-punctata  at  Bryn  Mawr,  I'a.,  I  \\a»  inten --led 

\\ln-ther   sujK-rnuineraries  would   be  found   in    ilu-   >aine 
pr-'piinimi  in  a  third  locality. 

Pestes  IK-HI  a  hundred  individuals  were  studii-d  in  aceto- 
(aimiiH  pn  paratimi-.  The  greater  part  of  the  mati-rial  \\a- 
ciillrct«-d  in  a  corn-field  in  the  open  country  between  1. a  Julia 
and  tin  new  laboratory  which  is  two  miles  north  »t  tin-  town. 
A  h  u  \\in-  "litainrd  from  a  rose-garden  in  La  Joll.i  .iiid  one  lot 
o|  os  m.,1,  -,  .mil  females  was  collecteil  for  me  \>\  Mi--  M\nU- 
|<'liii^<ni  on  (..MI  in  a  gardi-n  at  National  <  itv,  ju^t  -omh  of 
San  l>i(^n.  lndi\  idual  records  were  kept  for  earh  loi.  Imt  the 

•  i'iiditi.iii-«  \\iih  respect  to  number  of  supernumeraries  pn«\ed  to 
l-e  aliout  the  same  for  the  three  collecting  Around-.. 

To  m\  .-nrpii-e  I  found  supernumeraries  scarce.     In  the  tir-i 
J5  male-  examinrd,  Ji  had  no  supernumeraries  and  4  one;  while 
out    <.f    tin-   lu-t    25  examined   the  same   >ummer  at    Mountain 
\'ie\\    15  had  no  supernumerary,  7  one  and    ;   two;  and  in   the 
tir-t  25  at  Mountain  View  in  1909,  there  \\.-n    i  ^  with  no  super- 
numerary, ')  with  one,  2  with  two  and    i    \\iih    three.      In   the 
I. a    Jolla    material    the   89th   individual    was    n-.irhed    before   a 
of  two  supernumeraries  was  met  with,  and  in  the  first  100 
male>  ;()  had  no  supernumerary,  20  one,  and  I  two.     The  follow- 
er 


232 


X.    M.    STEVENS. 


ing  table  shows  the  per  cent,   of  supernumeraries  in   the   two 
species  different  years  and  in  different  localities. 


Number  of  Supernumeraries. 

o 

i 

2 

3 

4 

S 

D. 

r>. 

sor.,  Mt.  V.,  '07,  June  23-Aug.  7. 
12—  p.,  B-M.,  '07,  Oct.  3-9  

51 
48 

35 

-17 

II 
I  ? 

2 
-i 

I 
I 

D. 
D. 
D. 
D. 

s.,  Mt.  V.,  '09,  July  lO-Aug.  12.  . 
s.,  Mt.  V.,  '09,  Aug.  2i-Sept.  15. 
s.,  Mt.  V.,  '10,  July  28-Sept.  i..  , 
s..  La  J.,  '10,  June  17-July  4.  ... 

43 
46 
52 
79 

44 
38 
29 
20 

10 
IO 

16 
I 

3 

4 
3 

2 

As  I  had  never  seen  any  signs  of  degeneration  of  the  super- 
numeraries, the  natural  interpretation  of  their  infrequency  at 
La  Jolla  would  seem  to  be  either  that  they  had  originated  here 
more  recently,  or  that  they  had  originally  appeared  in  fewer 
individuals  in  this  locality. 

The  behavior  of  supernumeraries  in  all  cases  where  they  have 
been  shown  to  occur  at  once  classes  them  with  the  hetero- 
chromosomes,  and  in  Metapodius  Wilson  ('09)  has  shown  that 
they  have  probably  originated  in  an  irregular  second  maturation 
mitosis  in  which  both  idiochromosomes  went  to  one  pole  of  the 
spindle  instead  of  separating.  He  therefore  regards  the  super- 
numeraries in  Metapodius  as  duplicates  of  the  smaller  idiochro- 
mosome.  In  1908  I  suggested  that  there  might  be  two  varieties  of 
Diabrotica  soror  and  also  of  D.  12-punctala,  one  having  only  the 
odd  heterochromosome  and  the  other  an  unequal  pair,  and  that 
hybridization  might  have  given  rise  to  the  supernumeraries 
with  their  peculiar  behavior,  dividing  sometimes  in  one  some- 
times in  the  other  maturation  mitosis.  I  have,  however,  been 
able  to  find  no  evidence  in  favor  of  this  view.  In  1910  I  studied 
carefully  the  testesof  many  individuals  where  no  supernumeraries 
were  present,  seeking  some  clue  to  the  origin  of  these  chromo- 
somes. 

As  a  rule  the  odd  chromosome  X  appears  near  one  pole  of  the 
spindle  in  the  metaphase  of  the  first  maturation  mitosis,  but  I 
had  always  noticed  that  occasionally  X  is  in  or  near  the  equa- 
torial plate,  and  in  some  individuals  this  is  quite  common.  At 
La  Jolla  I  found  two  spindles  in  which  X  was  between  the 
daughter  plates  in  the  anaphase,  and  stretched  out  lengthwise 
(Figs,  i  and  2).  In  one  of  these  cases  (Fig.  2)  X  was  split  so 


-I    I'l.KMMI   KARV    CHROMOSOMES    IN    1MAHK«'I1>  233 


f 


f» 

0 


3 


•  *. 


ntt 


8 


B 


9 


1  •  ;•  An.iphases  of  first  maturation  mit»~i-.  -Imwin^  al>mniiial  po-ition 

ami  tian-vfi-i-  .livision  of  X.     (Mag.  1,500  tW  all  ti^iin 

I- i  -i.l  /'.      M  -c  of  first   inatuiatiiui  init"-i-.  -li"\\ini;  t\\"  -upi-rnu- 

•;ii'  . iii.il  in  size. 

Fi<  utlxT  stages  from  same   tr-ii-   -ln.\sin.i;   l»-lia\ii«i    m    A",  ami   the 

Hipernui 


234  N-    M-    STEVENS. 

that  it  was  certain  that  it  was  in  a  position  such  that  it  might 
divide  transversely,  but  I  was  not  able  to  find  any  cases  of  actual 
transverse  division  of  X.  Later  at  Mountain  View  I  did  find 
two  anaphases  where  X  appeared  to  have  divided  transversely 
and  unequally  (Figs.  3  and  5)  and  one  in  which  X  was  caught  in 
the  cell  plate  between  the  daughter  cells  (Fig.  4).  Now  the 
supernumeraries  are  usually  very  uniform  in  size  and  certainly 
less  than  one  half  the  sixe  of  X.  I  have  one  individual  noted  as 
having  an  unusually  large  supernumerary,  about  one-half  as 
large  as  X,  and  a  few  cases  where  an  unusually  small  one  occurs. 
One  of  the  latter  cases  is  shown  in  '08,  PI.  III.,  Figs.  76  to  78. 
From  the  evidence  now  at  hand  I  should  infer  that  the  probable 
origin  of  the  supernumeraries  in  the  Diabroticas  has  been  an 
occasional  transverse  division  of  X  followed  by  a  longitudinal 
division  of  the  two  parts.  Evidently  the  transverse  division  has 
usually  been  an  equal  one,  but  that  it  may  be  unequal  is  showrn  by 
Figs.  3  and  5,  and  the  rather  rare  occurrence  of  unusually  large 
and  unusually  small  supernumeraries.  Figs.  6  to  10  are  from  a 
male  captured  at  Mountain  View,  July  29,  1910.  Here  we 
have  a  large  and  a  small  supernumerary  in  the  same  individual. 
In  the  metaphase  (6a  and  6b)  X  and  the  two  supernumeraries 
were  all  near  one  pole  of  the  spindle,  while  in  Figs.  8  and  9  the 
supernumeraries  are  at  opposite  poles  and  in  Fig.  9,  X  is  near 
the  equatorial  plate.  In  Fig.  10,  X  and  both  supernumeraries 
have  gone  undivided  to  one  second  spermatocyte.  No  cases  of 
the  division  of  either  supernumerary  in  the  first  maturation 
mitosis  were  found  in  this  individual. 

In  Metapodius  Wilson  found  no  somatic  variations  correspond- 
ing to  the  variation  in  the  number  of  supernumeraries.  In  fact 
the  insects  with  X  alone,  X  and  Y,  or  X,  Y  and  I  to  6  super- 
numeraries are  described  as  indistinguishable.  These  speciec  of 
Diabrotica  are  very  variable  in  size,  and  in  regard  to  size  and 
fusion  of  the  12  black  spots  on  the  elytra,  but  as  I  showed  in  1908 
there  is  no  significant  correlation  between  these  somatic  varia- 
tions and  the  presence  or  absence  or  number  of  supernumeraries 
('08,  Tables  I.  and  II.,  and  p.  465,  text).  In  Metapodius  the 
indications  are  that  the  chromosome  Y  is  of  no  hereditary  value, 
and  the  supernumeraries,  as  duplicates  of  Y  would  not  be  ex- 


SUPERNUMERARY   CHROMOSOMES    IN   DIABROTICA. 

pected  to  affect  the  somatic  characteristics  of  the  insects.  If, 
however,  i  he  supernumeraries  of  Diabrotica  come  originally  from 
different  regi<  >ns  -  >f  A',  there  would  seem  to  be  no  reason  why  they 
should  not  bear  functional  genes  for  sex  and  other  characters. 
The  in. ile  alv.  'in. tins  A"  so  far  as  my  experience  goes  (over 

700  male-  ,  but  one  would  suppose,  if  the  supernumeraries  are 
functional  in  heredity,  that  one  A'  and  a  supernumerary  might 
frequently  deii-niiim-  the  development  of  a  female,  and  if  so 
there  -hould  be  males  without  A',  but  with  a  supernumerary 
in  its  place,  h  may.  of  course,  be  true  that  the  abnormal  di\  i-i-ui 
V  pp-dii-  in.  -up.  inumeraries  in  itself  indicates  a  de-.-iu-rati- 
or  non-functional  condition  of  that  particular  A'  chromosome, 
and  ther.  its  progeny — the  resulting  stipernuinera< 

This  \\uiild  tall  in  line  with  Schleip's  ('ll)  s  '  :»n  in  regard 

i"  the  rejected  A"  chromosome  in  the  spcrmato-em -sis  of  the 
lieniiaphiodi  ration  of  .-1  n^iostomum  nigru  ».•.  that  it 

had  ahead\  bec<  une  non-functional  at  an  earlier  stage,  \\  hence  its 
later  beha\ior.  It  is  exceedingly  desirable  that  the  female  sex 
•  elU  of  il  <>ticas  should  be  studied,  but  I  ha\e  not  been 

able   i  ni\'  favorable  mitoses  in   the  adults,   or   to  secure 

lar\  .e  oi  pupa-  I  r  oin  the  soil  or  roots  of  plants  on  u  hit  h  they  live. 
N-\eral  attempts  to  breed  them  have  given  no  iv-uli-,. 

Tin  u  males  each  having  one  supernumerary  \\ere 

studied  from  the  point  of  view  of  tin-  division  of  the  super- 
numerarie-  in  the  first  maturation  mitosis.  All  anaph.tses  and 
metapha-es  in  each  preparation  were  examined  and  all  cases 
\\lure  it  \\.is  possible  to  determine  the  position  and  beha\ior  of 
the  sii].ernnmerar\'  reconled.  In  the  metapha-i-  the  super- 
numerary \\as  in  the  equatorial  plate  in  5}  i  p<  r  cent,  of  <»oi 
cases  and  out  of  the  j)late — nearer  one  pole  of  the  spindle —in 

45.0  per    cent.      Apparently    the    supernumeraries,    \\lnn    they 
di\ide.  do  ~o  later  than  the  bivalent   chromosome-.,  so  all  ana- 
phases  \\ere  examined  on  this  point.      In  55.6  per  cent,  of  the 
anapha-es  found    in  the   12   testes,    the   supernumerary    was    ],e- 
t  \\een  the  daughter  plates,  and  in  44  percent,  it  was  di\  filing  or 
di\  filed.       Here  the  56.6  per  cent,  corresponds  closely  with  the 

54.1  per  cent,  in  the  equatorial  plato  in  the  metaphase,  and  the 
44.4  per  cent,  outside  of  the  daughter  plates  in  anaph.ises  comes 


236 


X.    M.    STKYKXS. 


very  near  the  45.9  per  cent,  out  of  the  equatorial  plates  in 
metaphase.  The  division  of  supernumeraries  or  their  failure  to 
divide  in  the  first  maturation  mitosis  seems  to  be  a  matter  of 
chance,  depending  on  their  position  in  the  spindle  in  the  prophase 
and  on  the  attachment  of  spindle  fibers  from  one  or  from  both 
poles  of  the  spindle.  In  Fig.  n  both  supernumeraries  are  con- 
nected by  fibers  with  both  poles,  in  Fig.  12  the  5-chromosome  is 
connected  with  both  poles  and  is  about  to  divide,  and  in  Fig.  13 
one  5-chromosome  is  connected  with  both  poles  and  will  later 
divide,  while  the  other  will  go  undivided  to  the  upper  pole  of  the 
spindle  and  therefore  to  one  second  spermatocyte.  The  behavior 
of  the  other  chromosomes  indicates  a  more  or  less  definite  attach- 
ment point  for  the  spindle  fibers,  near  the  middle  of  the  chromo- 


11 


12 


FIG.  ii.  Spindle  showing  two  supernumeraries  (s),  each  attached  to  spindle 
fibers  from  both  poles.  Mag.  2,000. 

FIG.  12.     Anaphase  showing  supernumerary  (s)  about  to  divide. 

FIG.  13.  Metaphase  showing  X,  a  supernumerary  (5)  attached  to  one  spindle 
fiber,  and  another  (5)  attached  to  two.  m  =  mitochondria. 

some  in  both  spermatogonial  and  spermatocyte  mitosis  (Figs, 
ii  and  13).  The  supernumeraries  seem  to  be  able  to  make 
connections  with  both  poles  in  most  cases  if  they  are  in  or  near 
the  equatorial  plate  in  late  prophase  stages. 

SEX  RATIOS. 

The  sex  ratios  in  Diabrotica  soror  and  Diabrotica  12-pitnctata 
have  shown  very  peculiar  variations.  In  studying  the  male 
germ  cells  of  D.  soror  in  1907  I  made  no  note  of  the  number  of 
females  found  in  random  collections,  but  in  dissecting  D.  12- 


SUPERNUMERARY    CHROMOSOMES    IN    DIABROTICA. 

punctata  in  October,  1907,  I  found  more  than  two  males  to  one 
female, — in  one  lot  58  males  to  25  females.  In  1909  the  number 
of  males  and  females  was  noted  for  each  lot  dissected.  Between 
July  lo  and  August  12,  107  males  and  102  females  were  counted 
in  random  collections  from  two  neighboring  gardens,  but  it  was 
noticed  that  the  latios  in  the  two  garden-  were  quite  different. 
In  garden  A  there  were  58  males  to  26  females;  in  garden  B, 
4<>  males  to  76  females.  A  second  lot  from  garden  A  collected 
betueeii  August  21  and  September  15  gave  101  male-  to  24 
female-.  The  percentage  of  females  in  garden  .1.  first  lot.  was 
30.9,  -ecnnd  lot  19.2,  average  23.9,  and  for  garden  B  60.8. 
At  I  ..i  J»ll.i  in  1910  the  ratios  ran  more  evenly. 

cf          9 

la,  June  17  and  18 

ioii.il  <  iiy.  June  22 

I  a  Julia.  June  28 14 

l"ll.i.  July  i i; 

I  ..i  J"ll.i.  July  4 


ig 

i  j  i 


Ai  Moiini.iin  View  again  the  ratios  were  peculiar.  I  i\«- 
rand' mi  <  - -llertions  in  Garden  A  gave  loo  male-  to  jo  tnnale-,  and 
i\\o  oilier  later  collections  76  males  to  18  female-.  (  >nl\  a  few 
\\i  M  .  ollrcted  from  garden  B  giving  12  male-,  to  d  female-..  In 
i<n  i  mixed  lots  from  both  gardens  gave  more  male-  than  female-. 
(•I  147.  These  were  recorded  incidentally  while  fixing  a  lot  of 
testes  lor  sections.  By  referring  to  the  table  on  p.rj<  232,  it  will 
be  seen  that  the  numbei  of  supernumeraries  run-  about  the  -aim- 
for  the  lir-t  100  in  1909,  about  one  half  of  which  came  from  each 
warden  .1  51  and  B  49),  and  for  the  second  ion,  .ill  of  which 
came  from  garden  .-1.  It  therefore  seems  unlikely  that  the  -nper- 
mimcrarie-  have  anything  to  do  with  the  dineivn.  .  in  sex  ratio- 
iu  the  t\\o  gardens.  The  soil  in  garden  .1  is  harder  in  -nmmer — 
more  adobe  in  it — and  less  thoroughly  cultivated  than  B.  T\\<- 
possibilities  are  suggested  in  this  connection:  (a)  The  male-  may 
be  more  successful  in  pupating  and  e-c.iping  from  the  hard  -oil 
than  the  females  or  (b)  few  of  either  sex  may  emerge  from  the 
hard  -nil  in  garden  A,  and  the  males  may  be  better  livers  and 
so  come  in  larger  numbers  from  other  neighboring  gardens.  The 
latter  i-  re-aided  as  more  probable. 


238  N.    M.    STEVENS. 

The  Bryn  Mawr  Diabroticas  of  1907  were  all  collected  on  a 
large  clump  of  golden  rod  in  a  pasture  that  had  not  been  culti- 
vated for  many  years,  and  they  may  have  come  out  of  the  ground 
in  the  immediate  neighborhood  or  from  more  recently  cultivated 
fields  near  by. 

These  erratic  sex  ratios  are  probably  merely  another  example 
of  the  interference  of  external  conditions  in  what  would  otherwise 
be  an  equality  of  sexes,  or  in  other  words  a  shifting  of  normally 
equal  sex  ratios,  or  partial  exclusion  of  one  sex  by  peculiarities 
in  the  environment.  The  collections  were  all  random  in  the 
sense  that  all  the  individuals  that  could  be  found  were  collected 

each  time. 

BRYN  MAWR  COLLEGE, 
January  3,  1912. 

LITERATURE    CITED. 
Schleip,  W. 

'n     Uber  die  Chromatinverhaltnisse    bei    Angiosiomum    (Rhabdonema)   nigro- 

venosiim.     Ber.  d.  Naturf.  Gesell.  Freiberg  i/B,  XIX. 

'n      Das   Verhalten    des    Chromatins    bei   Angiosiomum    (Rliabdom-ma)    nigro- 
venosum.     Ein  Beitrag  zur  Kenntnis  der  Beziehungen  zwischen  Chromatin 
und  Geschlechtsbestimmung.     Arch.  f.  Zellforsch.,  VII. 
Stevens,  N.  M. 

"08     The   Chromosomes   in   Diabrotica  vitlala,   Diabrotica  soror  and   Diabrotica 
12-punctata.     A  Contribution  to  the  Literature  of  Heterochromosomes  and 
Sex  Determination.     Journ.  Exp.  Zool.,  \ . 
Wilson,  E.  B. 

'09  Studies  on  Chromosomes,  V.  The  Chromosomes  of  Melapodhis,  a  Con- 
tribution to  the  Hypothesis  of  the  Genetic  Continuity  of  Chromosomes. 
Journ.  Exp.  Zool.,  VI. 


THK    RELATION    OF    THE    FIRST    CLEAVAGE    PLANE 
To    I  Hi;    ENTRANCE    POINT   OF   THE   SPERM.* 

ERNEST  E.  JIM 

I  hiring  the  summer  of  IQII  at  tin.-  Marine  Biological  Labora- 
tory  under  the  direction  of  ProK •— »r  Frank  R.  Lillie,  I  was  cii- 

'•'!  in   tlu-  study  of  the  eggs  of  AY/r/\  of  certain  cvtolo-ical 

problem-  tin-  results  of  which  will  appear  later.     The  question 

ot  the  relation  of  the  entrance-point  of  the  ^perm  and  the  tir-t 

plane  occurred   to  me.     A  verv  pretty   method   made 

-ible  in  .t  satisfactory-  fashion  the  determination  of  this  relation 
the  result-  ol  which  this  paper  embodies.  I  here  take  this  oppor- 
tunity to  express  my  thanks  and  sense  of  gratitude  to  l'n>te— •! 
Lillie  for  hi-  ins[>iring  interest  in  the  work  of  which  this  is  a  part. 

MATERIAL  AND  M  i  i  in  >i>-. 

Tin  of  Xereis  when  shed  are  irregular  in  -h.ipe  din-  i<> 

I  ire— nre  \\hile  in  the  body  of  the  female.  They  soon  till  oni  in 
the  >ea  \\ater,  measuring  about  IOO  /z  eqnatoriallv  and  -oine\\  hat 
less  in  a  polar  direction.  There  is,  ln»\\e\er.  i  dial  of 

indi\  idnal  ~i/e  variation  in  the  «  j  i\  en  female      Tli 

are  almo-i  transparent,  colored  a  pale  ^reen  liy  numerous  deuto- 
plasin  spherules  distributed  throughout  the  endoplasm;  around 
the  eijuator  i-  .m  irregular  double  -irdle  of  14  to  jj  i.il  drops 
I  ig  i  hi  polar  view  the  lar-e  -erininal  ve-icle  appears  to  be 
in  die  center  of  ih.  h  i-.  lio\\t-\cr.  s|j-htl\-  i-lnn^ated  in 

the   pol.ir  diri-ciion.      The  pnlaritx'  of   the  o\  is,   therefore, 

expressed  by  tin-  polar  tlatteninu  already  mentioned,  the  position 
of  tin-  oil  dro|»>.  and  the  form  of  the  nucleus. 

A-  ha>  been  >ln'wn  (^Lillie,  'in  there  are  not  two  membranes 
in  the  unfertili/ed  egg  of  Nereis,  but  rather  a  single  vitellinc 
membrane  external  to  the  radially  striated  cortical  layer  ("zona 
radiata,"  \\ilson)  of  the  egg.  The  ov<>cvte  remains  thus  with 

*A11  ilrawin.ys.  oi  living  t-v;u~.  made  with  the  aid  of  a  camera  luci'la. 


240 


ERNEST    E.    JUST. 


nucleus  intact  until  inseminated  or  otherwise  stimulated — as  for 
instance,  by  squirting  forcibly  through  a  pipette. 

Two  or  three  minutes  after  insemination,  a  jelly  is  rhythmically 
extruded  from  the  cortical  protoplasm.  In  ten  minutes  the 
germinal  vesicle  breaks  down,  development  is  initiated. 

Males  and  females  captured  in  the  evening  while  swimming  at 
the  surface  of  Eel  Pond  were  kept  in  separate  dishes  until  morning 
when  they  were  transferred  to  fresh  clean  sea-water.  To  get  an 
abundance  of  eggs  and  of  sperm  for  an  experiment,  it  was  merely 
necessary  to  cut  open  a  female  and  a  male.  The  cut  animals 


FIG.  i.     Egg  of  Nereis  at  time  of  insemination;  polar  view. 
FIG.  2.     Maturation  stage;  second  membrane  formed;  oil  drops  at  vegetative 
pole. 

were  removed  from  the  dishes  at  once;  moreover,  every  other 
precaution  was  taken  to  avoid  abnormalities  superinduced 
through  toxic  influences,  mechanical  shock,  etc.  In  several 
watch  glasses  of  sea  water  in  which  India  ink  had  been  ground 
up  eggs  were  put  together  with  a  minute  quantity  of  sea  water 
containing  very  few  spermatozoa.  The  time  of  insemination  \\  .1^ 
noted  and  the  numbered  dishes  observed  to  the  second  cleavage. 
This  method  was  varied  somewhat  as  I  shall  later  note. 

Kinged  slides  also  were  used;  eggs  placed  on  these  in  SIM  water 
and  ink  were  inseminated.  Sometimes  a  cover  slip  was  placed 
on  the  eggs.  Finally,  for  the  later  observations  a  very  few  eggs 
were  placed  on  slides  and  the  cover  slips  supported  with  glass  rods. 


CLKAV.U1K  PLANK  AM)  ENTRANCE  POINT  OF  SPERM.      24! 

OBSERVATION-. 
Outline  of  Development  to  First  Clc<: 

Eggs  in  sea-water  in  which  India  ink  has  been  previously 
ground  up  show  clearly  the  formation  of  the  jelly,  the  formation 
of  the  fertilixation  cone,  and  the  entrance  of  the  spermatozoon. 

A  -in-.ik  i if  ink  points  like  a  dagger  or  an  exclamation  point  to 
the  entrance  cone  above  which  on  tin-  membrane  the  spermato- 
/oon  is  .in. iched  (Fig.  4).  This  "exclamation  point"  i-  an  aid 
quickly  to  drtennine  in  a  large  number  of  eggs  the  relation  of 
the  -perm  entrance-point.  The  ^perma to/nun  enter-  the  e^  .n 
any  point .  •->. .  also  Lillie.  'i  I . 


FIG.  |.     Firel  clea> 

I1!''     i.      l  "in-  and  iii'lu.it 
line  Hi.uk-  li"iiii'  •  My. 


t"iiiiati"n.   15  ininutc~  .itu-r   iii-rminut  i"ii.      ()utci 


This  ink  "exclamation  p.-int."  or  ">perm  indicator"  as  I  shall 
call  it.  i-  a  very  in  ten-Mil):;  and  striking  formation  worthy  of 
detailed  smdv.  With  me.  lunvever.  the  interest  lay  not  SO  much 
in  the  lonnation  of  this  indicator  as  in  it-  a\  ailability  to  help 
ans\\cr  the  (itie-iion:  \\hat  is  the  relation  of  the  sperm  entrance 


242  ERNEST    E.    JUST. 

point  to  the  first  cleavage  plane?  I  here,  therefore,  give  only 
as  much  of  an  outline  of  its  formation  and  of  the  development  of 
the  egg  to  the  time  of  first  cleavage  as  will  suffice  to  render 
intelligible  the  subsequent  record  of  observations. 

Almost  at  the  moment  the  spermatozoon  touches  the  egg 
membrane,  the  contents  of  the  cortical  layer  begin  to  flow  out 
as  a  viscid  transparent  substance  of  the  same  refractive  index 
as  water,  leaving  only  radiating  lines  across  the  space  (perivitel- 
line  space)  between  protoplasm  and  membrane  which  represent 
the  walls  of  the  emptied  alveoli.  This  jelly  in  its  flow  carries 
the  ink  from  the  periphery  of  the  egg  so  that  between  each  egg 
and  the  surrounding  ink  is  a  clear  space.  This  outflow  of  jelly 
may  last  for  fifteen  minutes.  The  jelly  forms  about  the  egg  a 
layer  everywhere  continuous  except  along  the  tail  of  the  sperm 
which  thus  forms  a  canal  that  increases  in  length  as  the  jelly 
area  widens. 

Below  the  spermatozoon,  the  protoplasm  of  the  egg  begins  to 
form  a  cone  at  thirteen  to  fifteen  minutes  after  insemination 
which  gradually  increases  in  height  until  it  reaches  the  membrane 
and  then  slowly  retrogresses.  With  this  retrogression,  the  mem- 
brane at  this  point  sinks;  in  this  depression  lies  the  sperm. 
During  this  behavior,  as  the  jelly  area  widens,  the  canal  in  the 
jelly  in  which  the  tail  of  the  sperm  lies  fills  in  with  particles  of 
ink.  This  process  is  a  gradual  one,  the  indicator  reaching  its 
maximum  of  development  fifteen  to  twenty  minutes  after  in- 
semination. The  indicator,  therefore,  is  formed  along  the  tail 
of  the  sperm  and  points  to  the  entrance-point  of  the  sperm. 

Twenty  minutes  after  insemination,  the  spermatozoon  may  be 
seen  attached  to  the  membrane  at  the  end  of  the  indicator.  The 
perivitelline  space  now  becomes  slight.  The  egg  "assumes  an 
amoeboid  appearance"  (Wilson),  changing  its  shape  and  becoming 
very  irregular.  The  sperm  cannot  be  seen  readily  (Fig.  5). 
About  forty  minutes  after  insemination  the  egg  becomes  spherical 
again.  The  sperm  is  easily  visible  on  the  membrane  which  is 
more  widely  separated  from  the  protoplasm  by  the  perivitelline 
space. 

This  condition  is  of  short  duration  for  the  egg  begins  another 
series  of  changes.  The  membrane  appears  everywhere  equi- 


CLEAVAGE    PLANE    AND    ENTRANCE    POINT    OF    SPERM.        2-J.^ 

distant  from  the  egg  except  at  the  point  of  sperm  attachment 
where  it  is  nearer  the  membrane.  Then  gradually  to  the  right 
and  left  of  the  point  of  sperm  attachment  the  perivitelline  space 
becomes  greater;  the  egg  elongates  along  a  line  through  the 
point  of  sperm  attachment  (Fig.  6).  \Yith  the  disappearance  of 
the  sperm  head  within  the  egg  (about  fifty  minutes  after  in- 


5 

l-i-..  5.     Alter  retraction  of  cone;  membraru  -itly 

in  tin-  I'Ki;. 

1-n.    '        1  wo  minutes  before  sperm  is 


this  elongated  appearance  is  lo-  7   :  the  egg 

out.     The  egg  flattens  at  tin.-  animal  ]><>|r  i  Fig.  8)  and 
the  polar  bodies  are  given  off  from  a  clear  apparently  yolk-! 
region  of  the  flattened  pole  (Fig.  2).     Some  little  time  later  the 
tir-a  iK.ivage  furrow  appears  and  the  egg  is  divided  unequally 

I  ig.  3). 

Tlu-  t'l'-rrvations  on  the  relation  of  this  cleavage  to  the  en- 
trance-point  of  tin-  spi-rm  \\ill  be  considered  under  three  heads 
corresponding  to  the  methods  used. 


^44 


KRXEST    E.    JUST. 


Watch  Glass  Series. 

A  female  was  opened  at  9:58,  a  male  at  10:00.  In  five  watch 
glasses  of  india  ink  ground  up  in  sea-water  eggs  and  sperm  were 
mixed  at  intervals  of  two  minutes.  At  10:10  a  few  eggs  were 


FIG.  7.     Just  after  disappearance  of  sperm  within  the  egg. 

inseminated  in  the  ink  solution  on  an  uncovered  slide  (no.  6). 
About  two  minutes  after  an  insemination  the  jelly  began  to  form; 
in  fifteen  minutes  the  sperm  indicator  was  well  developed.  Eggs 


FIG.  8.     First  polar  body  forming. 

inseminated  at  10:15  in  a  watch  glass  (no.  7)  were  washed  at 
10:30:  that  is,  when  the  indicator  had  reached  its  maximum  of 
development. 


CLEAVAGE  PLANE  AND  ENTRANCE  POINT  OF  SPERM.   245 


The  dishes  (nos.  I  to  5)  and  the  slide  (no.  6)  were  examined  as 
the  first  cleavage  furrow  appeared.  In  95  per  cent,  of  the  eggs 
the  first  cleavage  plane  passed  through  the  point  of  sperm 
entrance  (Fig.  9).  Dish  no.  7  showed,  on  the  other  hand,  that  in 
only  50  per  cent,  of  the  eggs  the  first  cle.ixa-e  furrow  passed 
through  the  point  of  sperm  entrance. 


FIG.  g.     First  cl<\i\ 

At  2:45  p.m.  of  the  same  day,  eggs  \\rtv  in>eininate<l  in  watch 
no.  8.     Examination  revealed  that  tin-  fir>t  Hea\a".e  plane 

through   the  point  of  entrance  in  No  prr  cent.  ot   ej 
I  jgs  transferred    from  india  ink  ami   sea-water   to  dean   fiv-h 
MM-u.iit-r  twenty  to  thirty  minute^  alter  insemination  >li<>\\r<l 
per  i «  nt.  of  first  cleavages  through  the  point  of  entrance. 
A  Mimmarv  of  the  results  of  Kxperimein  -   I    to  s  i-,  as  iollo\\>: 

i    >  iiiM-iiiin.itr<l  in  watch  ;.  i   \v.i-ln-'l.  -hn\vi-il   t'u-t   cleavage   tlin>ui;li 

i-nti.uii  e  p<  MII!  .  -i  5  PIT  cent. 

6  iiiM-iiiin.iU'd  on  slide  glass,  not  wa-li>-l.  -li-iwf-l   tit-t  thnni^h  «-n- 

ti.iiu  i-  point .  <>5  per  cent. 

\       -  iii-.-niiii.ucd  in  watch  glass.  wasln-<l,  »ln>\vi-il  tir-t  cli  M\  a.i;<-  thn'iii;h  <-ntrance 
p. -int.  >n  p«-r  cent. 

ii.it.i-il  in  \\.itrh  .ula--.  nut  \va?>lK-i|.  slmwi-il  tir-t  cleavage  thruuKh  en- 
ti.in.  <•  p..int,  80  per  cent. 

No   i)  in-cniinated  in  wat.  h  glass,  ti.ui-i.  rn-<l  to  slide,  slmwc-'l  tir-t  rlrava.ui-  through 
rntr.mre  point,  60  p«-r  o-nt. 

That  tin'  ink  i>  not  to\ic  to  the  eggs  and,  ilietefore,  does  not 
inhiliit  cleavage  I  was  able  to  prove  by  inseminating  at  the  same 
time  t\\o  di-lir-  of  eggs,  one  with  ink  and  one  without;  develop- 


246  ERNEST    E.    JUST. 

« 

ment  in  both  went  on  at  the  same  rate  and  in  prrlVrtly  normal 
fashion.  I  concluded,  therefore,  that  it  was  not  necessary  to 
wash  the  eggs.  Also,  I  found  later  that  the  eggs  \\ere  often  too 
greatly  crowded  and  that  it  was  hard  to  make  counts  unless  the 
eggs  were  in  a  single  layer.  A  trial  made  with  very  few  eggs 
unwashed  in  four  watch  glasses  gave  the  following  result  (actual 
numbers  are  given) : 

FIRST  CLEAVAGE  PLANE. 

Through  Not  Through 

Number.  Entrance  Point.  Entrance  Point. 

1  8  2 

2  16  4 

3  10  I 

4  12  3 

To  what  extent  the  eggs  might  rotate  in  the  jelly  was  yet  to  be 
determined.  It  was  absolutely  necessary  that  the  relation  of  the 
indicator  and  the  sperm  entrance-point  remain  constant;  other- 
wise, the  indicator  would  prove  a  very  pretty  but  useless  phe- 
nomenon. Could  it  be  possible  for  two  spermatozoa  to  reach  the 
egg  and  the  indicator  to  form  along  one  sperm  and  not  the  other? 
How  would  such  an  egg  cleave?  These  points  were  next  to  be 
determined. 

I  found,  first,  that  the  position  of  the  indicator  could  be  altered 
through  tilting  the  watch  glass,  for  the  eggs  would  rotate  in  the 
jelly — especially  when  they  lay  on  the  side.  I  found  later  that 
the  eggs  are  most  liable  to  rotation  after  the  sperm  has  dis- 
appeared. This  might  easily  prove  a  serious  source  of  error. 
Secondly,  I  demonstrated  in  several  experiments  that  polyspermic 
eggs  are  not  apt  to  cleave.  (Professor  Lillie  has  obtained  the 
same  results.)  But  with  fairly  dilute  sperm  and  sea  water, 
polyspermy,  which  merely  cuts  down  the  number  of  cleaving 
eggs,  may  be  avoided. 

In  this  connection  it  will  be  interesting  to  note  the  results 
obtained  with  old  eggs  and  sperm.  On  July  30  eggs  from  a 
female  captured  in  the  evening  of  July  28  were  used  with  fresh 
sperm — of  a  male  captured  in  the  evening  of  July  29.  These  eggs 
proved  very  susceptible  to  polyspermy.  This  proved  true  in 
other  trials.  These  eggs  if  they  segmented  at  all  showed  sixty 
per  cent,  of  first  cleavages  through  the  entrance-point  of  the 


CLEAVAGE    PLANE    AM)    ENTRANCE    POINT    OF    SPERM.        247 

sperm.  In  general,  eggs  that  have  stood  in  sea  water  for  some 
time  after  leaving  the  female,  show  a  low  per  cent,  of  cleavages 
through  the  entrance-point.  Five  hours  after  leaving  the  female 
eggs  fail  to  develop  on  insemination. 

These  results  seem  to  indicate  that  the  first  cleavage  tend-  to 
pa-s  through  the  sperm  entrance  point  —  i.  e.,  through  the  point  at 
the  end  of  the  indicator—  if  the  e---  lie  fresh,  undisturbed  ami 
fertilized  with  a  single  sperm.  Why  then  do  some  first  cleavages 
fail  to  pass  through  this  point?  During  this  time  a  number  of 
experiments  made  by  day  and  often  at  ni^hi  immediately  alter 
the  rapture-  of  the  animals  sho\\ed  e—  eniialK  the  same  propor- 
tion-. 


Ringed  Slide 

It  was  stated  above,  it  will  be  remembered,  that  the  egg  tends 
to  lie  with  either  pole  uppermost.  It.  however,  the  eggs  are 
not  disturbed  those  that  settle  on  the  side  uill  so  remain.  The 
-  are  accessible  to  sperm  at  any  point  it  not  under  pre—  tire 
it  no  time  in  this  study  they  were,  Hie  first  cleavage  always 
rnt^  through  the  animal  pole  near  the  polar  bodie-.  <»b\ioii-ly 
then,  the  question  of  the  relation  of  tin-  hr-t  cleavage  plane  to 
the  entrance-spot  of  the  sperm  cannot  be  -ettled  by  the  «  1«  a\ 
o!  iho-e  eggs  in  which  the  spermato/oa  enter  either  at  the  point 
In-low  which  the  polar  bodies  are  extruded  or  at  a  -p«-i  i 
lr<  mi  ihi-  point. 

In  the  next  trial  with  very  few  eggs  on  ringed  -.li.lt-,  tho-e  e-gs 
in  \\hirh  the  sperm  indicator  pointed  either  to  the  polar  bodies 
or  to  a  point  180°  from  the  polar  bodir-  \\ere  not  counted.  This 
trial  re-ulied  as  follows: 

FIRST  (  ii  \\  AI.M   I'l  A 

Number.  Entrance  I'mm.  MI. 

1  U  4 

2  4 

3  20  9 

()(her  experiment--  with   ringed   -lide-  -ho\\.d   ab-uit    the  same 

proportions. 

l-'or  fear  that  the  rin-ed  -lides  were  toxic  owing  to  the  vaseline 
n-ed  the\  \\ere  abamloned  and  slides  with  cover  >lips  >up|)Oried 


248 


HRNKST    E.    JUST. 


by  glass  rods  as  well  as  the  open  watch  glass  were  used  throughout 
the  next  series  of  observations. 

Slides  ii4th  Glass  Support  for  Cover  Slip. 

Four  or  five  eggs  on  a  slide  were  watched  continuously  through 
the  first  cleavage,  the  indicator  used  merely  to  point  out  quickly 
the  point  on  the  membrane  where  the  sperm  was  attached.  Very 
few  sperm  were  used  in  these  observations,  obtained  through 
diluting  several  times  the  water  which  contained  them.  These 
observations  were  repeatedly  made  at  night  and  at  different 
times  during  the  day.  Some  of  the  eggs  failed  to  show  the  indi- 
cator and  to  develop.  In  all  that  segmented,  the  first  cleavage 
plane  passed  either  directly  through  the  entrance-point  of  the  sperm 


FIG.  10.     First  cleavage. 

or  a  degree  or  so  from  it,  with  the  indicator  parallel  to  the  cleavage 
furrow  (Fig.  10).  It  is  possible,  as  stated  above,  to  keep  the 
spermatozoon  in  view  after  the  amceboid  stage  until  it  disappears 
within  the  egg.  The  middle  piece  is  left  without.  With  the  aid 
of  the  middle  piece,  the  character  of  the  membrane  at  the  en- 
trance point  (Fig.  7),  and  the  oil  drops  near,  it  is  possible  ab- 
solutely to  hold  in  view  the  exact  spot  at  which  the  sperm  was 
engulfed. 

At  intervals  of  two  to  three  minutes,  seven  slides  with  very 
few  eggs  on  each  were  prepared.  Sperm  was  added  and  after 
a  minute  the  eggs  covered  and  every  precaution  taken  to  avoid 


CLI-.\\   \(,I      PLANE    AND    ENTRANCK    POINT   OF    SPERM.        249 


di-turbance.  In  the  sixty  eggs  counted  the  first  cleavage  furr»\\ 
passed  through  the  sperm  entrance-point  in  every  case.  In  some 
cases  the  indicator  appeared  to  be  at  right  angles  to  the  furro\v 
but  in  all  such  it  proved  to  be  aboic  the  egg  and  ended  in  the 
cleavage  plane  (Fig.  n).  This  was  Sunday,  August  20.  The 
laboratory  was  quiet,  the  temperature  conditions  favorable. 
The  results  of  Au.uu-i  23,  24  and  2~  an-  Hinilar.  I  "amera  sketches 


Fit;.  1  1.     First  i 

\\  t  •!•«•  made  of  these  eggs.  Often  I  a-ked  .111  in\  esl  igator,  who  did 
not  know  the  purport  of  the  experiments,  t<>  make  the  sketches; 
the  indicator  without  doubt  was  above  the  e.^  and  pointed  to 
the  elt-avagc  furrow. 

DISCUSSION. 

Tin-   first  cleavage  plane  usually  coinride-  \\iih    tin-   median 
plane  of  the  future  animal  in  the  IV  according  to  Koux, 

\e\\port,  Pfliiger  and  Morgan.     In  the  squid'  I  so,  according 

it  '  \\  .n.i-e,  the  first  cleavage  plane  falls  in  with  the  median  plane 
of  the  embryo.  Agassiz  and  Whitman  (,'^41  nou-d  a  like  co- 
intideiH  e  in  the  teleost  egg;  and  \"an  Beneden  and  Julin,  Castle 
'96)  and  C'onklin  ('05)  found  that  the  first  cleavage  plane  marks 
the  Ion-  axis  of  the  embryo  in  the  a-<  -idian  egg.1 


:.liiiK  to  Harper,  tin-  -pt-rni  •.•ntrr<  tin-  pixoin'-  i-i;.c  pn-vi«m<  ti>  tlic  ogg's 
riui.in.r  into  the  oviduct.  Il«-  In-li.-vc-;  that  th.-  -pi-mi  iiiu-t  <-iuer  as  soon  as  the 
,li~r  i~  i-\p,  .-<-,!  through  rupture  of  the  tollicular  wall.  In  the  p  I..-MII 

i-ntiano-  i-  nu-ir  i«r  I'  ized.     According  to  his  figure,  th(  ige  plane 

in.ik.-  an  .ui^l.-  ..i  45°  with  the  lonp  axis  of  the  embry>       A-  we  know  from  oilier 
j-ches,  the  Iniiv;  axis  "I  the  embryo  is  similarly  placed  in  tlie  egg. 


25O  ERNEST    E.    JUST. 

But  there  are  other  eggs  in  which  the  future  median  plane  does 
not  fall  in  the  plane  of  the  first  cleavage.  In  Xereis  (Wilson, 
'92)  the  second  cleavage  plane,  although  it  does  not  divide  the 
animal  into  "equal  halves,"  coincides  with  the  long  axis.  So  in 
Crepidjila,  the  first  cleavage  plane  is  at  right  angles  to  the  future 
median  plane  (Conklin,  '97).  In  the  newt  (Jordan,  '93)  the 
case  is  the  same.  In  Chatopterus  (Lillie,  '06)  the  axis  of  the 
first  cleavage  spindle  lies  in  the  longitudinal  axis  of  the  embryo. 

There  is  a  third  group  of  eggs  in  which  coincidence  \vith  any 
cleavage  plane  is  wanting  This  is  true  of  the  egg  of  Amia  (Whit- 
man and  Eycleshymer,  '97),  of  the  toadfish  (Clapp,  '91),  and  of 
certain  amphibians  (Jordan  and  Eycleshymer,  '94),  to  name  a 
few.  And  yet  in  most  of  these  eggs  the  symmetry  and  the 
bilaterality  of  the  cleavage  may  be  sharply  marked. 

In  the  frog's  egg  the  first  cleavage  plane  usually  and  the  median 
plane  of  the  embryo  always  (Ran a  fused)  pass  through  the  en- 
trance point  of  the  sperm  (Roux,  '85;  Schulze,  '99;  Brachet). 

In  the  egg  of  Toxopneustes  (Wilson,  '95)  the  first  cleavage  plane 
passes  through  the  entrance-point  of  the  sperm,  "in  the  great 
majority  of  cases,  at  least."  This  plane  of  cleavage  coincides 
with  the  transverse  diameter  of  the  embryo  (Driesch). 

In  the  ascidian  egg,  the  belief  of  Castle  ('96)  is  that  the  first 
cleavage  plane  cuts  through  the  entrance-point  of  the  sperm. 
Conklin  ('05)  says  that  there  is  no  question  but  that  the  first 
cleavage  plane  is  through  the  copulation  path  of  the  germ  nuclei. 
And  indeed  his  figures  show  very  beautifully  that  this  is  actually 
the  case. 

If  now  we  grant  that  in  the  egg  of  the  frog  and  of  Toxopneustes 
as  in  the  egg  of  Nereis  and  of  the  ascidian  the  first  cleavage  plane  is 
determined  by  the  copulation-path,  or  the  entrance-point,  of  the 
sperm  we  have  this  interesting  conclusion :  The  first  cleavage  plane 
in  eggs  whose  cleavages  have  different  values  and  different  rela- 
tions to  the  future  long  axes  of  the  embryos  is  determined  by  the 
entrance  of  the  sperm.  While  the  sperm  entrance  determines 
the  first  cleavage,  the  first  cleavage  does  not  in  all  of  these  forms 
coincide  with  the  median  plane  of  the  future  animal. 

Since  in  the  egg  of  Nereis  the  sperm  may  enter  at  any  point  and 
since  the  first  cleavage  plane  passes  through  this  point,  the  struc- 


CLEAVAGE    PLANE    AND    ENTRANCE    POINT    OF    SPERM.        25! 

turc  of  the  o\  »< -\  tc  of  Xcreis  at  the  time  of  insemination  must  be 
the  same  in  all  meridians.  This,  I  K-lu-ve,  has  an  important 
bearing  on  theories  of  germinal  areas  in  tin-  cytoplasm,  of  pre- 
localization,  and  of  precocious  segregation.  The  determination 
of  bilaterality  follows  fertilization. 

I.ITEKATrKK    <    I  I  FD. 

Agassiz  and  Whitman,  C.  O. 

'84     On    tip    I  )•  •-.  <  lopment  !'•  laui.    I-'i-h    Eggs       I'n-limmaiy    N.>'. 

I':  .  Acad.  An  -.XX. 

Van  Beneden  et  Julin,  J. 

'84     I. a  -I-UIIH  nt.aion  chez  les  A-cidii-:  rapp"i;  .ni-.ition  de 

I. live.     Archive  de  Biologic,  V. 
Castle,  W.  E. 

'96     The  Early  Embryology  of  dona  Flnninv:     1  Hull.    Mus. 

'      mp.  Zool.,  XXVII. 
Clapp,  C.  M. 

'91     Some  Points  in  the  Development  of  the  I ...id-Fi-h.     .|.>m.  M..iph..  \'. 
Conklin,  E.  G. 

'97      I  lie  Embryology  of  Crepidulti.     Jmir.   M»r|>li..  XIII. 

"04     The  Organization  and  Cell  Lineage  of  1        •  I  l^m.  A>al.  x 

<if  Phil..  XIII. 
Harper.  E.  H. 
'04      r*he  Fertilization  and  Early  Development  of  thi    r  i  Am..I"iii. 

it.,  III. 
Jordan,  E.  O. 

'93       I  ![••  II. i1  n>;  and  Development  <>t  t  J"iu.  M-irpli..  \'1II. 

Jordan,  E.  O.,  and  Eycleshymer,  A.  C. 

'94     The  Cleavage  of  Amphibian  Ova. 
Lillie,  F.  R. 

'06     Ol  >ns  and  Experiments  com  c-niinv;  tin-  Kli-iiinituiy   I'lii-nnim-n 

Development  in  Chu  l-m    I  \    /     .1..  III. 

"ii      ^!  idii      "i    Fertilization    in    A  I  rtical    (  h.mui--    in     ili>     1 

II.    I  Fertilization.     Jour.  M.'ipli..  XXII. 

Moszowski.  Max. 

'02      i  i  Einfluss  der  SC!I\M •;  luinu  uml  Eih.iltu 

bilateralen  Symmetric  des  F  h.  Mik.  An.it..  IX. 

Newport,  G. 

'51.  '53.  '54     *^n  lnt-'  Iinpri'Kiuiti'Mi  ni    the  ( >vum  in  tin-  Amphibia.      Phil. 

l\         -.>c.  London. 
Roux,  W. 

'85      Britriige    zur    Enlwickelu:  .uiik     dr-    Embr\n.      Nr.      {.    1   ebei    «lic 

.innuing  der  Hauptrichtungen  -I-      I  ;       licnibryn  im  Ki  uml  ulit-r  die 
erste   lli.-il-,.  i  roschei<.      !'.:•  -lauor  iirztl.  V«-it->  hr. 

'87     Nr.   .}.    I1  iiimiinii  il«T   Mrdianebene  des   Froschembryo  durch    de 

•pulatinii-iii-htuiiK  di_<  Eikcrm-s  und   ilt-s  Sp.-i  inakn  in  •-.       Anhiv   Mikr. 
An.it..  XXIX 


252  ERNEST    E.    JUST. 

Schultze,  O. 

'99     Ueber  das  erste  Auftreten  cler  bilateralen  Symmetric  im  Yerlauf  der  Ent- 

wicklung.     Archiv  Mik.  Anat.,  LV. 
Watase,  S. 

'90     Studies  on  Cephalopods.     I.  Cleavage  of  the  Ovum.     Jour.  Murph.,  IV. 
Whitman,  C.  O.,  and  Eycleshymer,  A.  C. 

'97     The  Egg  of  Amia  and  its  Cleavage.     Jour.  Morph.,  XIII. 
Wilson,  E.  B. 

'92     The  Cell  Lineage  of  Nereis.     Jour.  Morph.,  VI. 
Wilson,  E.  B.,  and  Mathews,  A.  P. 

'95     Maturation,  Fertilization,  and   Polarity  of  the  Echinoderm  Egg.     Jour. 
Morph.,  X. 


PALMEN'S  ORGAN   AND  ITS  FUNCTION   IN   NYMPHS 

OF  THE   EPHEMERID/E,    HEPTA'.IAIA    INTER- 

PUNCTATA   (SAY)   AND   ECDYURUS    MACULI- 

PENNIS   ( WALSH  K 

J.  E.  \VODSEDALEK. 
INTRODUCTORY  RKMARKS. 

Our  knowledge  concerning  the  tracheal  s\  ~u  in  in  tin-  Ephem- 
eii-I.e  .I. acs  baclc  to  the  time  of  Swammerdamm  (1752  ,  luit  the 
existence  of  this  interesting  modification,  Palmcn's  or-an,  found 
only  in  the  tracheal  system  of  this  group  of  insects,  was  not 
knoun  until  comparatively  recent  times.  Swammerdamm  in  his 
"I'.iUI  der  N.itur"  gives  a  large  figure  (Plate  XIV.  .  -howin^  in 
•«ome  detail  the  internal  anatomy  of  a  may-tly  nymph.  Inn  the 
I'.ilmeii's  organ  and  even  the  four  tracheal  tulu  -.  diivnlv  leading 
to  it ,  if  | in-sent  in  that  species,  apparently  e>eaped  his  ob-en  aiion. 
Thi->  oini-sioii  was  no  doubt  due  to  an  imperfeet  di— i •< -lion ;  for, 
upon  do-ely  observing  his  representation  of  the  air  tul.es  in  the 
he.id  of  the  nymph  he  figures,  one  can  detect  .1  nnje  projeetioii 
le.idiiu  fn>m  the  main  tracheal  tube  on  the  left,  \vhieh  eorre-ponds 
Mnne\\  h.it  to  one  of  the  four  tubes  normally  leading  to  \\\\<  -n-.m  ; 
the  other  three  tubes  and  the  organ  itself  \\cre  doubtle^>  de- 
Btroyed  in  his  preparation,  and  hence  not  represented  in  his 
figure. 

The  presence  of  this  chitinous  structure  \\.i-  tir>t  noted  In 
1  >r.  J.  A.  I'.ilmen  (1877),  after  whom  the  or^an  is  named,  and  in 
his  work  he  says:  "Die  vier  im  Schc-itel  zusammenstossenden 
Ae>te  bildeii  in  ilirem  Kreuzpunkt  eincn  rundliehen,  aus  c<m- 
eentri^chen  ( "hitinschichten  besteheiiden  Korper.  dessen  Medeut- 
un.u  iih  nirht  kenne."  On  Plate  I.  (Fig.  7)  he  gives  .1  Imure  of 
the  head  and  thorax  of  the  nymph  of  Clocon  diptcntni  L.,  showing 
tlu-  loeat ion  of  this  organ  in  its  relation  to  the  four  traeheal  tubes 
of  the  head,  without  making  any  attempt  to  dexrilu-  it.  He 
makes  the  statement  that  the  tracheal  >>  stem  is  essentially  the 

253 


254 


J.    E.    WODSEDALEK. 


same  in  the  twenty-three  species  which  he  examined.  It  is  not 
entirely  safe,  however,  to  infer  from  this  that  the  prominence  of 
Palmen's  organ  is  essentially  similar  in  these  various  species. 

The  species  upon  which  the  present  study  is  based  are  Hepta- 
genia  interpundata  and  Ecdyurus  maculipennis.  These  two  forms 
are  very  closely  allied,  not  only  in  matters  concerning  this  organ, 
but  also  in  their  natural  habits  and  general  behavior,  and  the 
present  paper  will  concern  itself  with  nymphs  of  //.  inter  pun  data, 
unless  otherwise  specified. 


FIG.  A.  Head  of  H.  inlerpunctata  nymph.  Basal  joint  of  antennae  only  drawn. 
The  brain  is  drawn,  dotted,  under  the  three  ocelli  just  posterior  to  which  is  shown 
the  Palmen's  organ  and  the  four  tracheal  tubes  leading  into  it. 

Fig.  A  shows  the  relative  position  of  the  organs  in  the  head 
of  a  nymph.  Palmen's  organ  together  with  the  tracheal  tubes 
leading  to  it  can  be  readily  seen  through  the  chitinous  covering, 
especially  in  the  newly  moulted  specimens,  by  placing  them  under 
a  binocular.  It  is  symmetrically  located  between  the  two  large 
compound  eyes  and  a  little  posterior  to  the  brain.  Fig.  I  shows 
the  organ  in  its  relation  to  the  entire  tracheal  system  of  the  head. 
It  has  been  the  fortune  of  the  writer  to  be  able  to  make  a  perfect 
dissection  of  the  system,  the  first  time  merely  through  an  acci- 
dent. Upon  pkicing  a  specimen  which  had  been  dead  for  some 
time  under  a  binocular  almost  the  entire  tracheal  system  of  the 
insect  became  visible  through  the  transparent  chitinous  covering. 
The  muscles  and  all  the  other  soft  tissues  had  sufficiently  de- 
composed to  form  a  sort  of  liquid  mass.  The  thin  hypodermal 
walls  surrounding  the  air  tubes  too  had  disintegrated,  and  prac- 
tically all  that  remained  in  good  condition  was  the  exoskeleton 
and  its  internal  continuation,  the  tracheal  system.  The  location 


PALMEN  S    ORGAN    IN    HEPTAG1-.NIA    AND    ECDYURUS.          255 

and  arrangement  of  the  more  important  parts  of  the  system  were 
carefully  noted  and  a  diagram  indicating  the  relative  position  of 
the  main  tubes  was  sketched.  The  external  covering  was  care- 
fully broken  between  the  pro-  and  mesothorax  and  a  gentle  pull 
on  the  anterior  edge  of  the  head  removed  it,  fully  exposing  the 
air  tubes  which  remained  in  position.  The  macerated  mass  was 
carefully  washed  off  and  the  tracheal  system  being  completely 
filled  with  air  presented  the  most  beautiful  silvery  effect  a-ain-t 
a  dark  back-ground.  Even  the  very  finest  branches  remained, 
but  no  attempt  was  made  to  include  them  in  the  figure. 

DESCRIPTION  OF  PALMEN'S  ORGAN. 

Gross  (1903)  attempts  to  describe  the  organ  in  c<>n-i(lcr,il  >!«• 
detail;  this  description  and  his  ideas  in  general  an  IK-I  fully 
corroborated  by  the  results  of  my  studies.  He  says  in  part: 
"  Reconstruiren  wir  jetzt  aus  den  besprocheaen  Schnittbildern 
ganze  Organ,  so  erhalten  wir  folgendes  Gesammtbild,  Kin 
K'irper,  der  im  Liingsschnitt  kurz  elliptisch,  im  <  >IKT— chnii  i 
ungerfahr  kreisfdrmig  ist,  setzt  sich  aus  14  concentrischen,  an- 
xaitem  Chitin  bestehenclen  Schalen  /u-ammm,  die  an  ilnvr 
Innentluche  mit  feinen  Hiirchcn  dicht  U--rut  -iml.  I  >as  Kllip- 
s<>i«l  i-t  aberkein  vollkommen  geschlosseiie-.  Yielmehr  i-t  es  v>n 
vier  Seiten  her  [sehr]  tief  ausgehdhlt.  Das  uan/c  i-t  in  den  Kn-n/- 
nn^-punkt  von  4  im  Scheitel  des  Hinterkopfs  /n-ainmm'ivtti-n- 
dm  Tracheeniistcn  eingeschaltet,  und  xuar  so,  da-.-  die  Luft 
/\\i-elien  den  Schalen  trei  circuliren  kann,  \veiin  aiich  (lurch  <li<- 
grosse  Xahl  der  Hiirchen  einigermaassen  behindi-rt.  <  ".an/  ahn- 
lich  i^estaltct  wie  bei  Ephemera  vulyitd  L.  fand  irh  da-  I'alinm'- 
selie  (  )r^an  noch  bci  Baetis  rhodani  Piet..  Ht'()ttr^cnia  sulphured 
Miill..  ferner  bei  den  Larvcn  einer  Cacnis  sp.  un<l  eiiur  Chiro- 
les  sp.  Einige  geringe  Abweirhnn-en  in  di-r  Cn--talt  bei 
/•>'<.-(V/.v  rhodani  konnte  ich  nicht  hinreii  IK-IK  1  ^ciiau  fe-istellen, 
nn  -it-  hier  zu  besprechen,  da  ich  inir  nieht  lienii^md  Material 
1'.  ~i  haticii  konnte." 

The  1'almen's  organ  in  both  II.  inter punctata  and  E.  niacnli- 
f>cnnis,  i-  not  composed  of  coneentrie  shells  nor  are  there  any 
hair-  pp  sent  on  the  inner  surface  of  the  scales  which  Gross 
•  Ic-cribes  and  pictures  in  allied  -prcie-.  \\V11  prcparc'l  -tides 


256  J.    E.    WODSEDALEK. 

of  cross  sections  show  that  the  organ  is  not  perforated  with  air 
passages  but  is  a  continuous  mass  of  chitin  in  which  the  differ- 
entiations are  due  mainly  to  variation  in  density  of  color.  Sec- 
tions of  adult  specimens  weie  also  made  and  carefully  examined, 
but  no  difference  in  the  structure  of  the  organ  could  be  detected. 
Dr.  Gross  has  no  doubt  mistaken  the  clearer  areas  or  concentric 
layers  for  air  passages  and  the  darker  layers  for  separate  solid 
areas  forming  the  scales  from  which  the  hairs  protrude. 

Fig.  4  shows  the  external  dorsal  appearance  of  the  organ  and 
its  relation  to  the  four  tracheal  tubes,  the  entire  structure  being 
enveloped  by  the  hypodermal  layer;  Fig.  5  is  a  horizontal  section 
of  the  same.  The  description  of  the  organ  can  be  best  understood 
by  studying  it  in  connection  with  its  development  and  growth. 
It  is  a  well  understood  fact  that  the  tracheal  system  in  insects  is 
formed  by  the  invagination  of  the  ectodermal  layer.  As  to  the 
origin  of  Palmen's  organ  I  am  not  at  all  certain  for  embryonic 
material  has  thus  far  in  this  study  not  been  available.  The 
appearance  of  the  structure  of  the  central  portion  of  the  organ 
suggests  that,  during  the  process  of  the  development  of  the 
tracheal  system,  the  four  large  tubes  leading  to  the  organ  (Fig.  i) 
come  together  at  a  common  point;  here  the  blunt  ends  of  the 
invaginated  portions,  the  tracheae,  surrounded  by  the  hypo- 
dermis,  fuse  and  secrete  this  common  center.  From  the  various 
cross  sections  of  which  Fig.  8  is  typical,  it  can  be  inferred  that  the 
two  posterior  tubes  come  together  first  and  that  a  portion  of  the 
center  is  secreted  before  it  is  met  by  the  two  anterior  tubes. 

In  the  many  sections  of  //.  inter pnnctata  and  E.  niacnlipennis, 
which  were  examined,  the  center  of  the  organ  does  not  show  any 
ring-like  structure,  but  is  an  irregular  mass  which  is  apparently 
M-nvted  Ix-forc  the  hr\.i  c  ists  the  firsl  limii'j  <>i  its  trachea] 
system.  At  the  time  of  this  first  ecdysis  which  is  accompanied 
by  the  shedding  of  the  inner  lining  of  the  air  tubes,  this  central 
mass  is  larger  than  the  openings  in  any  of  the  four  tubes  and  hence 
the  impossibility  of  its  being  cast  out  of  the  body.  Shortly 
after  the  casting  of  the  inner  lining  of  the  trachea?,  the  hypo- 
dermal  cells  surrounding  the  tubes  undoubtedly  begin  to  secrete 
the  new  chitinous  wall.  The  hypodermal  layer  surrounding  the 
central  mass,  the  beginning  of  the  Palmen's  organ,  is  continuous 


PALMEN  S    OKtiAN    IN    HKIMAt,!  MA    AND    ECDYURl   -  257 

with  the  layer  surrounding  the  air  tubes  and  apparently  begins 
active  secretion  at  about  the  same  time.  The  different  con- 
spicuous rings  which  are  shown  ( Fig?.  5-9)  are  sections  through 
the  concentric  layers  of  the  organ  and  are  directly  correlated 
with  the  various  moults.  Further  evidence  of  this  correlation 
is  obvious  from  the  fact  that  the-  number  of  rings  is  directly  in 
proportion  to  the  size  of  the  insects  thcin-elves.  An  examination 
of  the  sections  figured  show-  thai  tin-  hypodermal  cell-  surround- 
ing the  organ  are  much  larger  than  those  enclosing  the  trachea-, 
and  hence,  the  greater  the  secretion  <>f  these  larger  cells;  I  nun  this 
results  the  greater  thickness  of  tlu-  chitinous  layers  of  the  on;. in 
as  compared  with  that  of  their  continuations,  tin-  walls  of  the 
t radical  tubes.  Coincident  with  tin-  increase  of  volume  of  the 

in,  the  cells  surrounding  it  must  necessarily  multiply  as  they 
are  pushed  outward.  Thus,  by  means  of  succe— i\v  periodic 
BC<  ret  ions  the  Palmen's  organ  is  built  up;  the  old  layers  of  the 

m  arc  not  cast  off  as  are  the  walls  of  the  tradieal  -y>tem, 
\\ith  which  they  are  continuous. 

'  .ross  in  commenting  on  the  function  of  Palmcn'.-,  or-an  says: 
"l>h  glaube  deshalb,  dass  fur  das  rath-elhafte  <  )r^an  keine 
KrlJarung  gefunden  werden  kann  oluie  Beriicksichtigung  de- 
\ir\en.  Xehmen  wir  aber  an,  dass  dicker  \\irklieh  /.\\  dnn 

in  gdiort,  so  kann  dieses  nichts  anden--  >ein  aU  t  in  Sim 

m.  Da  es  aber,  wenn  auch  zicmlich  direct  miter  der  Ilypo- 
di TinU  —  von  dieser  nur  durch  wenig  Feitk(")rper  getrennt— doch 
jedeii  1  alls  im  Imiemdes  Korpers  der  Thii n  gelegen  i-t.  k.mn  es 
\  on  alien  uns  %-on  andern  Thiergnippen  bekannteii  Sinne-func- 
lionen  nur  denen  eines  Gleichgewichtssinnes  dienen."  I  p  to  the 
pn-si'iit  study  no  experimental  work  on  the  ori;an  has  lui  n  at- 
tempted with  the  view  of  obtaining  evident---  ,nl-  iis  func- 
lion.  < '.ross  also  says:  "Man  konnte  nieim-n.  der  Heueis  fiir  die 
Kichti^keit  der  \-on  mir  ver>uchten  I'eutun^  (K--  <  )r^an-  \\> 
sich  \  idleicht  durch  zvveckmassig  anuc-tellte  X'ersudie  i-rlirin^en. 
1  >a-  ei-M -heint  mir  aber  ziemlich  an— icht>los.  Es  \\lire  ja  gewiss 
nicht  unmoglich,  das  recht  oberll;ichlidi  gelegene  Organ  zu  zer- 
storon,  nachdem  man  vorher  seine  Lage  so  genau  festgestellt  hat, 
ila-s  man  sie  schon  von  an— en  am  lelu-nden  Thier  angeben  kann. 
. \lier  it  h  fiirchte,  dieses  Experiment  wird  nicht  viel  helfen.  Stellt 


258  J.    E.    WODSEDALEK. 

sich  nach  clem  operativen  Eingriff  irgend  eine  Aenderung  dcr 
Flugweise  ein,  so  kann  diese  auch  durch  die  Verletzung  an  und 
fiir  sich  bewirkt  sein.  \\~ir  wissen  aus  der  experimentellen 
Gehirnphysiologie  der  Vertebraten  zur  Geniige,  in  welche  schwere 
Irrthiimer  man  geraten  kann,  wenn  man  die  Yerletzung  oder 
Zerstorung  eines  Organs  oder  Organtheils  als  reinen  Versuch 
betrachtet.  Wahrend  man  aber  bei  einem  Wirbelthier  wohl 
warten  kann,  bis  die  storenden  Nebeneffecte  des  operativen 
Eingriffs  verschwunden  sind,  so  scheint  mir  das  bein  einer 
'  Eintagsfliege '  kaum  moglich  zu  sein.  Selbst  ein  nicht  zur 
Begaltung  gelangtes  Exemplar  diirfte  in  der  Gefangenschaft  nur 
zu  bald  eingehen.  Auch  wiirden  die  Thiere  wohl  kaum  den  Hoch- 
zeitsflug  aufnehmen,  wenn  man  sie  nicht  in  die  ihnen  zusagende, 
natiirliche  Umgebung  und  unter  Artgenossen  bringt.  Thut  man 
dies  aber,  so  wiirden  einem  die  Versuchsthiere  gar  zu  leicht 
entschliipfen,  nachdem  sie  einmal  aufgestiegen  sind.  Ebenso 
wenig  Erfolg  verspreche  ich  mir  von  dem  Versuch,  die  Function 
des  Organs  durch  Verkleben  der  in  die  Kopftracheen  fiihrenden 
Stigmen  festzustellen." 

REMOVAL  OF  THE  ORGAN. 

Experimental  work  on  the  removal  of  the  organ  did,  as  Gross 
said,  at  first  seem  impossible.  It  is  needless  to  say  that  the 
task  was  very  tedious  and  at  the  outset  far  from  encouraging, 
this  was  mainly  due  to  the  small  size  of  the  organ  and  its  close 
proximity  to  the  brain.  At  first  the  cauterizing  method  was 
used  but  without  satisfactory  results,  then  two  very  fine  platinum 
needles  which  were  attached  to  the  two  wires  leading  from  a 
galvanic  battery  were  employed.  The  apparatus  was  provided 
with  a  resistance  box  so  that  the  voltage  could  be  varied  at  will. 
In  this  method  the  end  of  one  needle  was  turned  into  a  small 
loop  through  which  the  sharp  point  of  the  other  was  inserted, 
thereby  completing  the  current,  heating  the  sharp  point  intended 
for  the  operation,  and  at  the  same  time,  greatly  facilitating  the 
necessary  steady  manipulation  of  the  outfit.  The  hot  point 
of  the  needle  would  be  brought  directly  over  the  organ  and  then 
a  rapid  insertion  and  withdrawal  of  the  point  of  contact  followed. 
It  was  impossible  at  each  attempt  to  destroy  the  organ  owing 


PALMEN  S  ORGAN  IX  HEPTAGENIA  AND  ECDYURl '-.    259 

to  its  natural  instability.  A  few  specimens  from  which  the 
organ  had  been  thus  entirely  removed,  lived  a  sufficient  length 
of  time  to  enable  studies  of  the  behavior  of  the  individuals,  and 
of  the  regeneration  of  some  of  the  destroyed  part  - 

Becoming  more  thoroughly  familiar  with  tin-  structure  and 
exact  position  of  the  organ  in  its  relation  t<»  the  vital  pans  ot  the 
head,  a  more  simple  method  wa-  de\i-ed.  By  mean-  <>f  two 
very  fine  and  sharp-pointed  needles  a  small  slit  can  be  made 
through  the  chitin  above  the  organ  and  thru,  in-crtin-  a  needle 
at  each  side  between  the  posterior  and  anterior  trarlu-al  tubes 
leading  to  the  organ,  it  can  with  some  pra< -tier,  be  entirely 
removed;  this  treatment  apparently  causes  tin-  in-ect-  but  little 
pain.  The  four  tracheal  tubes  were  usually  separated  near  the 
organ  though  sometimes  they  would  break  off  near  their  juncture 
with  the  main  longitudinal  trachea?.  In  special  for  -tudics 

of  regeneration  of  the  organ,  the  four  tubes  were  broken  off 
at  their  immediate  attachment  to  the  organ  or  at  various  drlinite 
di-tances  from  it.  This  was  possible  by  pressing  the  two  point- 
•  •I  the  needles  on  either  side  of  the  place  where  the  break  was 
desired.  Bleeding  was  very  rare  and  usually  the  edges  of  the 
chitinous  slit  were  brought  so  close  together  that  the  detection 
ot  the  wound  was  rendered  almost  impossible. 

After  treatment  by  this  method  the  activity  of  the  nymphs 
\\hcn  placed  back  into  the  water  did  not  set-in  to  lie  impaired 
by  the  operation,  and  the  wounds  healed  over  within  a  leu 
days.  By  this  method  not  only  was  the  remo\  al  of  the  organ 
a— mvd.  but  mortality  was  reduced  to  a  minimum.  In  one  set 
of  experiments  forty-nine  out  of  fifty  specimen-  operated  on 
li\cd  for  more  than  two  months  after  the  operation.  It  might 
be  -aid  in  this  connection  that  no  regeneration  of  the  organ 
takes  place.  The  ends  of  the  broken  tube-  heal  over  within 
t\\o  or  three  weeks  and  with  the  e\eeption  of  a  few  small  air 
tubes  which  grow  out  from  the  blunt  ends  of  the  four  tubes, 
during  the  same  time,  no  further  growth  was  observed  in  any 
of  the  specimens  as  long  as  four  months  after  the  organs  had 
been  removed.  Fig.  3  is  drawn  from  a  nymph  in  which  the 
traehe.e  were  broken  off  at  their  point  of  contact  with  Palmen'- 
,m,  they  almost  touched  but  no  regeneration  of  the  organ 


260  J.    E.    WODSEDALEK. 

took  place,  nor  was  there  a  union  formed  between  the  different 
tracheae  Fig.  2  is  of  a  specimen  in  which  the  trachea?  were 
broken  at  quite  a  distance  from  the  organ;  again,  no  growth 
beyond  the  covering  over  of  the  broken  ends  and  the  formation 
of  a  few  small  tubules  took  place. 

COMPARISON  OF  THE   BEHAVIOR  OF  NORMAL  AND  OPERATED 
SPECIMENS  IN  RELATION  TO  THE  FUNCTION  OF 
PALMEN'S    ORGAN. 

In  my  previous  papers  (Wodsedalek,  'n  and  '12),  the  behavior 
of  H,  interpunctata  nymphs  has  been  discussed  in  considerable 
detail,  and  hence  only  the  more  important  phases  of  the  behavior 
of  this  insect  which  directly  concern  this  problem  will  be  cited 
here.  The  nymphs  are  decidedly  negative  in  their  phototactic 
response  in  all  gradations  of  light,  varying  from  ordinary  day- 
light to  very  intense  electric  illumination.  Their  thigmotactic 
propensity,  or  tendency  to  come  in  contact  with  and  cling  to 
objects,  is  especially  pronounced.  In  their  natural  environ- 
ment the  nymphs  are  never  seen  swimming  freely  about  in  the 
water,  even  when  observed  in  their  favorite  places  in  which 
they  occur  in  great  abundance.  In  their  natural  habitat  they 
are  always  found  clinging  to  the  under  surfaces  of  small  rocks, 
and  this  same  position  is  regularly  assumed  by  all  normal  ones 
in  the  aquaria  of  the  laboratory.  When  a  stone,  to  which  the 
specimens  are  attached  is  inverted  in  the  water,  the  insects 
soon  make  for  its  under  side,  many  of  them  doing  this  as  the 
stone  is  being  turned  over.  This  is  also  true  of  normal  specimens 
in  the  dark-room,  and  hence  it  is  obvious  that  this  tendency  of 
the  nymphs  to  cling  to  the  lower  surfaces  of  rocks,  with  their 
dorsal  side  downward,  is  not  due  entirely  to  their  negative  reac- 
tion to  light.  It  is  unquestionably  due,  in  part,  to  a  definite 
power  of  orientation  independent  of  phototaxis. 

Specimens  from  which  the  Pill  men's  organ  was  removed 
react  to  light  in  practically  the  same  way  as  do  the  normal 
specimens.  Their  thigmotactic  inclinations,  too,  do  not  seem 
to  be  impaired.  However,  when  the  insects  are  taken  into  a 
very  shaded  or  a  dark-room  the  difference  in  orientation  becomes 
quite  obvious.  When  a  stone  to  which  the  insects  are  attached  is 


PALMEX'S    ORGAN    IX    HEPTAGENIA    AND    ECDYURUS.          26l 

inverted  in  the  water,  or  when  the  specimens  are  dropped  on  a 
stone  in  the  water  in  a  dark-room  they  remain  on  the  upper 
surface  or  on  the  sides  of  the  rock  for  a  considerably  longer  time 
than  do  the  normal  individuals  By  the  removal  of  the  organ 
the  nymphs  have  no  doubt  lost,  to  some  extent,  their  usual 
keen  sense  of  orientation,  for  under  such  conditions  they  would 
!'<•  seen  on  the  top,  sides  or  any  part  of  the  rock  for  hours,  days, 
and  even  weeks  after  the  operation  had  been  performed.  The 
same  was  true  of  every  lot  experimented  with.  It  was  also 
noticed,  with  several  lots  of  operated  specimen-,  that  the  tend- 
ency to  remain  on  almost  any  purl  of  the  stone  was  gradually 
dimini-hed  and  that  after  -everal  week-  and  in  some  rases-  about 
two  months  there  were  comparatively  few  individuals  l(,  he  seen 
on  the  upper  surface,  regardless  of  the  fact  that  in  some  -pedal 
experiments  the  stone  would  be  in\  cried  at  e\  cry  »>b-er\  ation 
with  the  view  of  bringing  more  specimen-  to  the  upper  surface 
\\ith  little  disturbance.  This  growing  partiality  to  the  lower 
-nit". ice  of  the  stone  does  not  lessen  the  significance  oi  their  tornu-r 
behavior,  for,  from  my  studies  on  the  po\\er  of  the  tormation 
of  a--odations  in  the  nymphs  of  II.  inter  (nnntata  \  \\ '«<  1-edalek, 
*I2)  it  was  found  that  they  gradually  formed  -e\eral  i\\n><  «\ 
a— •'«  iaiions.  The  associations  formed  in  the-  rimeiu- were 

in  connection  with  their  thi^motactic  inclination-,  which  were 
in  ^ivat  part  responsible  for  the  ^radual  de.  i  !  the  number 

i  on  lop,  and  the  gradual  diminishing  of  ihe  time  the  various 
indi\  iduals  required  to  retreat  to  tiie  l..\\er  surfa 

In  another  paper  (\Vodsedalek,  'u)  on  the  natural  history 
and  general  behavior  uf  these  insects  I  ha\e  di-cu— ed  their 
thigmotaxis  in  considerable  detail.  It  was  le.irned  from  a -imple 
experiment  that  their  thigmotactic  ])ropen-ities  are.  be-,t  -ati-lied 
when  their  dorsal  as  well  as  their  \entral  -urface-  are  in  contact 
\\ith  some  object.  "XYhen  several  specimen-  are  placed  in  an 
aquarium  they  mass  together  into  clusters  where  they  remain  for 
hours,  and  if  recently  collected,  even  days.  As  soon  as  a  rock  or 
any  other  object  is  placed  in  the  water  the  loose  forms  swim  toward 
it,  while  con-iderable  time  often  elapses  before  the  masses  are 
broken  up.  Two  long  bricks  were  placed  one  over  the  other  in  a 
basin  of  water  and  between  them  were  placed  small  pebbles 


262  J.    E.    WODSEDALEK. 

varying  in  size  so  that  the  space  gradually  varied  in  thickness  from 
one  end  to  the  other.  Then  a  large  number  of  specimens  were 
put  in  the  water  and  after  a  short  time  it  was  found  that  nearly 
all  of  the  specimens  were  attached  to  the  lower  surface  of  the 
upper  brick  with  their  dorsal  sides  downward,  and  a  large  major- 
ity of  the  specimens  were  in  that  part  of  the  wedge-shaped  space 
where  their  backs  came  in  contact  with  the  brick  below."  The 
operated  specimens  in  their  wandering  about  over  the  surface 
of  the  stone  accidently  came  into  such  a  place  where  their  backs 
came  in  contact  with  the  floor  of  the  basin.  This  stimulus 
naturally  appealed  to  their  thigmotactic  propensity  and  hence 
the  greater  tendency  to  remain  on  that  portion  of  the  rock.  It 
seems  only  natural,  therefore,  that  an  association  would  be 
formed  between  this  more  satisfactory  environment  and  the 
lower  surface  of  the  stone.  It  is  not  altogether  improbable  how- 
ever, that  such  a  habit  had  already  been  partially  formed  before 
the  operation  took  place. 

Further  evidence  for  the  fact  that  this  thigmotaxis  is  largely 
responsible  for  the  gradual  disappearance  of  the  insects  from  the 
upper  surface,  is  apparent  from  the  results  obtained  in  some 
checking  experiments.  In  those  experiments  the  stone  was  sus- 
pended in  the  water  so  that  the  backs  of  the  nymphs  could  not 
come  in  contact  with  other  objects.  The  results  were  surprising 
and  all  remaining  doubts  as  to  the  function  of  the  Palmen's  organ 
in  the  nymphs  were  resolved.  As  long  as  the  experiment  was 
continued  the  specimens  remained  quite  evenly  scattered  over 
the  entire  surface  of  the  suspended  stone.  A  similar  experiment 
was  tried  with  the  normal  specimens,  also  in  the  dark  chamber, 
and  practically  all  of  the  specimens  remained  exclusively  on  the 
lower  surface.  It  is  only  natural,  then,  to  conclude  that  Pal- 
men's  organ  has  a  great  deal  to  do  with  the  orientation  of  these 
insects.  That  this  unusual  behavior  is  not  due  to  the  shock  the 
insects  receive  from  the  operation  was  proven  by  the  fact  that 
when  other  parts  of  the  head  and  body  were  destroyed  no  com- 
parable results  in  behavior  took  place. 

Although  the  foregoing  results  are  thoroughly  convincing  as 
to  the  function  of  the  organ  in  these  nymphs,  further  results  of 
observations  on  behavior  relative  to  the  role  of  the  organ  may 


• 
PALMEN'S  ORGAN  IN  HEPTAGKNIA  AND  ECDYURUS.       263 

be  cited.  When  the  specimens  are  collected  and  dropped  into  a 
dish  of  water  many  of  the  individuals  fall  to  the  bottom  with 
their  ventral  sides  upward.  This  toppling  over  is  even  more 
obvious  when  the  specimens  are  placed  in  a  dish  of  water  in \ir 
a  light.  In  their  attempts  to  get  away  from  the  light  and 
repeatedly  clawing  at  the  opposite  end  of  the  <li-h  tin-  spedniens 
become  exhausted  and  very  frequently  when  the  clavvin-  move- 
ments cease  the  apparently  lifeless  individual*  fall  to  the  1  >< >\[«\\\, 
dorsal  side  downward.  This  period  of  re>t  t ••  >rr< '-ponds  some- 
what to  the  death-feigning  instinct  of  the  insect.  By  \  i-onm-ly 
stirring  up  the  specimens  or  throwing  them  into  water  havii 
temperature  to  which  the  specimens  are  not  aivn-tonied.  or  into 
relatively  strong  chemical  solutions  of  various  son-,  a-  adds. 
salts,  alcohol,  etc.,  practically  all  of  the  specimen-  tall  into  this 
momentary,  rather  stiff,  inactive  state  and  slowly  <!•  •-< « -ml  to  the 
bottom  of  the  dish.  In  so  doing  almost  all  of  the  specimens 
topple  over  and  fall  down  head-first,  ventral  side  up  ami  on  the 
average,  at  an  angle  of  about  45  degrees.  It  ini^lit  al-o  In- 
mentioned  here  that  nymphs  which  are  found  dead  in  the  aquaria 
lie  almost  invariably  with  their  ventral  side  up.  ( >n  the  oilier 
hand,  the  turning  over  is  under  similar  conditions  far  le--  frequent 
among  the  specimens  from  which  the  organ  had  been  remo\ed. 
It  two  groups  of  freshly  killed  specimens  are  taken,  all  <>l  which 
have  been  cleaned  and  their  appendage-  arranged,  the  one  i^roup 
normal  in  every  way,  the  other  having  the  I'.ilnn-n'-  organ  re- 
move.d.  we  find  by  allowing  them  to  de-rend  through  a  d 
jar  of  water  that  almost  invariably  the  li-rnier  topple  o\cr  and 
-ettle  on  the  bottom  ventral  side  up,  while  the  latter  eqnallv  as 
frequently  reach  the  bottom  and  remain  there  \\iih  thdr  \entral 
side  downward. 

CONCLUDING  RIMAK; 

The  results  of  the  foregoing  experiments  show  conclusively 
that  the  organ,  as  small  as  it  is,  plays  a  very  important  role  in 
the  behavior  of  the  nymphs  upon  which  these  experiments  v, 
performed.  This  is  doubtless  due  to  the  weight  of  the  chitinous 
ma->  whose  pressure  seems,  to  a  large  extent,  to  control  certain 
orientation  of  the  in-ects.  Gross  (1903)  gives  a  figure  of  the 


264  J.    E.    WODSEDALEK. 

cross  section  of  the  head  of  a  may-fly  showing  the  position  of 
Palmen's  organ  in  relation  to  the  other  parts,  and  in  his  discussion 
says,—  "Unter  dem  Palmen'schen  Organ  verlauft  namlich  bei 
alien  5  von  mir  untersuchten  Ephemeridenspecies  ein  starker, 
vom  Gehirn  kommender  Nervenstrang.  Seine  Lagebeziehungen 
ergeben  sich  aus  Fig.  B,  die  einen  Medianschnitt  durch  den  Kopf 
einer  Ephemera  vulgata  bei  schwacher  Vergrosserung  darstellt. 
Der  erwahnte  Nerv  (np)  verlauft  in  der  Medianlinie  vom  Gehirn 
(g)  nach  hinten  unter  dem  Palmen'schen  Organ  (/?)  hindurch  und 
heftet  sich  hinter  ihm  an  der  Korperwand  an.  In  einem  Theil 
seines  Verlaufs  liegt  er  direct  auf  dem  Nervus  recurrens  (nr)  des 
unpaaren  sympathischen  Nervensystems." 

Careful  examination  of  many  nymphs  showed  no  evidence  of 
the  presence  of  the  two  large  nerves  which  Gross  speaks  of  as 
present  in  the  imaginal  species  which  he  examined ;  this  was  also 
true  of  the  adult  specimens  which  I  examined.  It  appears  from 
his  discussion  of  the  subject  and  from  his  figure  (page  98),  that 
what  he  speaks  of  as  nerves  may  possibly  be  the  two  muscles 
which  play  an  important  part  in  the  movement  of  the  head. 
The  posterior  attachment  of  these  muscles  to  the  exoskeleton 
evidently  corresponds  to  the  attachment  of  the  large  nerves  he 
misrepresents .  In  my  preparations  very  thin  sections  were  made, 
but  no  signs  of  nerves  extending  directly  from  the  brain  to  the 
organ  were  detected.  Taking  the  structure  and  function  of  the 
organ  into  consideration  we  should  not  expect  the  presence  of 
such  nerves.  A  mass  of  rather  loose  tissue  exists  between  the 
organ  and  the  brain,  and  the  two  are  loosely  united  by  means  of 
connective  tissue.  It  is  the  writer's  opinion  that  the  chitinous 
organ  being  so  loosely  supported  by  the  four  tracheal  tubes 
exerts  a  pressure  on  the  surrounding  tissues,  whereby  the  dis- 
turbing stimulus  reaches  the  central  nervous  system.  The  ob- 
servations mentioned  on  the  descent  of  nymphs  in  various  con- 
ditions, through  the  water,  particularly  the  death-feigning  and 
the  dead  individuals,  seems  to  indicate  that  the  orientation  is 
also,  in  part,  a  self-directing  process,  that  is,  by  the  presence  of 
the  organ  the  nymph  is  swerved  into  position — a  matter  of 
physical  equilibrium. 

Gross'  theory  that  the  organ  functions  only  in  the  adult  speci- 


PALMEN'S  ORGAN  ix  HEPTAGEXIA  AND  ECDYURUS.       265 

mens  seems  quite  untenable.  Aside  from  the  results  of  my 
experimental  work  arises  another  question.  Why  should  this 
structure  occur  and  persist  in  very  small  nymphs,  and  grow  in 
relative  proportion  during  the  comparatively  long  nymphal  stage 
of  two,  and  in  some  cases  three  years,  for  the  purpose  of  becoming 
functional  only  after  the  nymph  metamorphoses  into  its  short- 
lived adult  stage,  when  all  the  other  modifications  which  are  of 
a  direct  advantage  to  the  adults  develop  during  tin-  comparath  »-ly 
short  time  immediately  preceding  the-  transformation:' 

The  extent  of  the  functions  of  this  organ  in  the  adults  thus  tar 
remains  unknown.  Miall  (1895)  in  >[>.•. ikin-  of  the  Kphenicrid.i' 
gives  the  following  quotation:  "The  n-cently  eim-r^rd  tl\ , "  says 
\><-  Geer,  "settles  on  trees,  plant-,  \\alls  etc.,  lu-.ir  the  water 
wliich  harbored  the  larva.  II-  re  it  iiv-  it-df  by  the  hooks  of 
tin-  feet,  usually  with  the  head  downuanU.  and  rests  until  the 
List  or  sub-imaginal  moult  is  at  hand."  Mv  o|,-(  r\  ation 
tin-  behavior  of  adult  may-flies  are  to  some  extent  in  accord  \\ith 
i he. -i-  of  De  Geer,  however,  no  theory  a>  to  tin-  probable  function 
•  •I  the  organ  in  the  adults  can  be  propounded,  unless  it  can  be 
-upported  by  reliable  results  of  experimental  work.  A  I. 
nuinber  of  nearly  lull  grown  nymphs  from  \\hich  the  1'aliiH  n's 
in  had  been  removed  are  now  in  the  aquaria  \\ith  tin-  \i<-\\  of 
making  a  study  of  their  behavior,  \\hen  they  emero-  .1-  adults  in 
comparison  with  that  of  the  normal  indi\  idual>. 

Among  the  twenty-three  species  in  which  1'almen    1^77    noted 

t  lie  i  ire-nice  of  this  organ,  there  are  se\  «  i  al  tin-  -u  immini;  fi  inns, 
and   at    this  time,  it  is  difficult  to  say   HIM    \\hat   |>art    1'alim-n's 
m  |>la\-s  in  those  forms  during  their  life  history  as  \cr\-  little 
i>  known  of  their  natural  habits. 

1  am  greatly  indebted  to  ProlV--or  \\illiain  S.  Mar-hall  for 
su-v;«-ting  this  problem,  and  also,  lor  his  help  and  earnest  interest 
in  the  progress  of  the  work. 

/<  <•  M  «  K.ICAL  LABORATORY. 

UNIVERSITY  OF  \\'IM  "\-i\. 


266  J.    E.    WODSEDALEK. 

REFERENCES. 
Berger,  E. 

'78     Untersuchungen  iiber  den  Bau  des  Gehirns  und  der  Retina  der  Arthropoden 

Arb.  zool.  Inst.  Wien,  V.,  i. 
Borner,  C. 

'08     Die  Tracheenkiemen  der  Ephemeriden.     Zool.  Anz.,  XXXIII. 
Brehm. 

'92     Thierleben,  3.  Aufl.,  Leipzig  u.  Wien. 
Dewitz,  H. 

'90     Einige  Beobachtungen,   betreffend  das  geschlossene    Tracheensystem  bei 

Insectlarven.     Zool.  Anz.,  XIII. 
Eaton,  A.  E. 

'88     A  Revisional  Monograph  of  the  Recent  Ephemerid;e  or  Mayflies.     Trans. 

Linn.  Soc.  London,  (2),  V.,  3. 
Gerstacker,  A. 

'66     Die  Arthropoden.      Bronn  Class. ( Ordn.  Thierreich,  V.  5,  Berlin  u.  Heidel- 
berg. 
Gross,  J. 

'03     Uber    das    Palmen'sche    Organ    der    Ephemeriden.     Zool.    Jahrb.,    XIX. 

(Anat.). 
Hiibner,  O. 

'02     Neue  Versuche  aus  dem  Gebiet  der  Regeneration  und  ihre  Beziehungen  zu 

Anpassungserscheinungen.     Zool.  Jahrb.,  XV. 
Joly,  N. 

'78     Etudes    sur    les    metamorphoses    et    1'embryogenie    des    Ephemerines,    et 

specialement  sur  celles  de  la  Palingenia  virgo.     Seance. 
Kolbe,  H. 

'93     Einfiihrung  in  die  Kenntniss  der  Insecten.     Berlin. 
Leydig,  F. 

'57     Lehrbuch  der  Histologie.     Frankfurt  a./M. 
Lubbock,  J. 

'63     On  the  Development  of    Chloeon(  (Ephemera)  dimidiatum.     Trans.  Linn. 

Soc.,  Vol.  XXIV. 
Miall,  L.  C. 

'95     Aquatic  Insects.     London. 
Needham,  J.  G. 

'05     May  Flies  and  Midges  of  New  York.     N.  Y.  State  Museum,  Bulletin  86. 
Oppenheim,  S. 

'08     Segmentregeneration  bei  Ephemeriden-Larven.     Zool.  Anz.,  XXX I II. 
Palmen,  J.  A. 

'77     Zur  Morphologic  des  Tracheensystems.     Leipzig. 
Pictet,  F.  J. 

^ 

"43     Famille  Des  Ephemerines.     Paris. 
Stein,  F. 

'47     Yerglcichende  Anatomic  und    Physiologic  der  Insecten.       Monographieen 

bearbeitet,  i,  Berlin. 
Swammerdam,  J. 
1752      Bibel  der  Natur. 
Vayssiere,  M.  A. 

'82     L'organisation  des  Larves  des  Ephcmerines.     Paris. 


PALMEN'S  ORGAN  IN  HEPTAGENIA  AND  ECDYIKI  s.       267 

Wodsedalek,  J.  E. 

'n     Phototactic  Reactions   and    Their    Reversal   in   the    May-fly  Nymph?  //. 

interpunclala.     Biological  Bulletin.  XXI. 

'12     Formation  of  Associations  in  the  May-fly  Nymphs  H.  intfrpundata.     Jour- 
nal of  Animal  Behavior,  Vol.  2,  no.  i. 

'12     Natural  History-  and  General  Behavior  of  the  Ephemerida-  Nymph*  II-  f- 
nia  inlerpunct.it<i.       Annals  of  the  Entomological  Society  of  America, 
Vol.  5,  No.  i. 
Zimmer,  C. 

'98     Die  Facettenaugen  der  Epln-nn-riden.     Z.  wiss.  Zool.,  \" ..  ' 


268  J.    E.    WODSEDALEK. 


EXPLANATION  OF  FIGURES. 
PLATE  I. 

All  drawings  (except  Figs.  2  and  3)  made  with  a  camera-lucida.     X240. 

FIG.  i.  Palmen's  organ  in  its  relation  to  the  tracheal  system  in  the  head  of  the 
nymph  H.  inter punctata.  X  60. 

FIG.  2.  Sketch  drawn  from  a  specimen  which  had  the  organ  removed  and  the 
four  tracheae  broken  off  near  their  juncture  with  the  main  longitudinal  tubes. 

FIG.  3.  Sketch  drawn  from  a  nymph  in  which  the  tracheae  were  severed  at 
their  point  of  contact  with  the  organ. 


BIOLOGICAL  BULLETIN,  VOL.   XXII. 


PLATE   I. 


J.  E.  WOMEDALEK. 


2JO  J.    K.    WODSEDALEK. 


PLATE  II. 

FIG.  4.  Representation  of  the  entire  organ  surrounded  by  hypoderrnis,  as  it 
appears  in  a  mounted  specimen.  Circular  bands  can  be  seen,  especially  at  the 
edges  of  the  organ,  owing  to  the  fact  that  we  look  at  the  vertical  portion  of  each 
deeply  colored  part.  In  this  view  the  large  light  areas  appear  at  the  entrances  of 
the  tracheal  tubes;  this  is  due  to  the  fact  that  we  look  through  a  comparatively 
thin  portion  of  the  chitin  in  those  regions  owing  to  the  direct  extension  of  the  cavities 
of  the  tubes  into  the  organ.  The  darker  areas  appear  as  such  because  of  their 
thickness;  each  is  a  concentric  mass  around  the  organ  and  forms  the  partition  be- 
tween the  cavities. 

FIG.  5.  A  horizontal  section  almost  directly  through  the  center  of  the  two 
posterior  tubes  and  a  little  above  the  center  of  the  two  anterior  ones.  It  is  only 
natural,  therefore,  that  the  two  posterior  tracheae  should  lead  to  the  solid  central 
mass.  The  entrances  of  the  two  anterior  ones  are  not  in  the  same  plane  with  that 
of  the  posterior  pair  and  therefore  the  innermost  portion  of  their  cavities  are  not 
represented  in  this  section.  The  gradually  increasing  diameter  of  each  cavity  is 
understood  when  we  recall  the  development  of  the  organ  and  the  tubes  leading  to  it. 

FIG.  6.  A  horizontal  section  through  the  ventral  projection  of  the  organ  which 
is  apparent  in  Figs.  7  and  8.  The  central  part  of  this  figure  appears  clear  because 
the  section  was  quite  thin  and  the  cut  parallel  with  the  light  portion  of  one  of  the 
concentric  layers. 


BIOLOGICA.  BULLETIN,  VOL.  XXII. 


PLATE   II. 


J.  E.  WOMEDAIEK 


2J2  J.    E.    WODSEDALEK. 


PLATE  III. 

FIG.  7.     A  transverse  section  cut  near  the  center  of  the  posterior  pair  of  cavities. 
FIG.  8.     An  oblique  transverse  section  cut  through  the  front  part  of  the  posterior 
tube  cavities  and  through  the  tips  of  the  anterior  cavities. 

FIG.  9.     A  still  more  anterior  view,  only  the  cross  sections  of  the  two  deeper 
portions  of  the  anterior  cavities  being  in  evidence. 

I 


BIOLOGICAL  BULLETIN,  VOL.  XXII. 


PLATE    III. 


J.   E.    *ODSEDAlE>v. 


Vol.  XXII.  .   April,  1912.  No.  j 


BIOLOGICAL  BULLETIN 


THE  EFFECTS  OF  SOME  AMIDO-ACIDS  OX   THE 

DEVELOPMENT   OF   THE   EGGS   OF   ARBACIA 

AND   OF   CH/ETOPTERUS. 

HKI.KN  DEAN   KING. 
THE  WISTAR  INSTITUTE  OF  ANATOMY  AND  BIOLOGY. 

hi  i<>(>9,  Mathews  published  a  short  account  of  sonic  e\peri- 
mi-nts  which  he  made  to  ascertain  the  effects  of  variou-  amido- 
.11  ids  on  the  development  of  the  eggs  of  Arbacia.  Tin-  re-nlts  of 
these  experiments  have  considerable  theoretical  inteiv-t.  -ince 
i  In -\  -eem  to  show  that  the  course  of  embryonic  development 
( .in  1><  determined,  to  a  greater  or  a  less  extent,  by  tin --e  pn>dnci- 
<>l  pn>iein  digestion. 

\\liilc  I  was  working  in  the  Marine  Biological  I.al>oraii>ry  at 

\\ 1-  llcli,  Mass.,  in  the  summer  of  1909,  Dr.  Mathcu-  kindly 

Inrni-lied  me  \\  iih  a  number  of  amido-acids  in  onlrr  th.it  I  mi^ln 
n-pi-.n  .tml  c\teiid  his  experiments  and  make  a  drt.iilrd  >tml\-ol 
the  diih  ifin  types  of  larvae  that  might  be  obtained.  A~.ii  -•.  nuil 
\\-nili  \\liiK'  to  determine  whether  amido-acids  «.m  .iltcr  the 
course  "t  «1-  \rli>pment  in  various  kinds  of  eggs  or  \\lirtlirr  tlu-\- 
li.i\c  a  -pi  i  ilic  action  on  the  eggs  of  Arbacia,  tin-  tApriiiiifiii- 
\\t-rc  i  ,111  ird  U-vond  the  limits  original ly  intended  and  \\cn-  ni.id<- 
\\ith  tin'  eggs  of  an  annelid,  Chatopterns  pcr^uncntaceits,  .1-  \\cll 
.1-  \\iih  ilu-  *  ^--  of  the  sea-urchin,  Arbacia  pioictiildtd. 

In  addition  to  cystin,  leucin  and  tyrosin,  the  thn •«•  amido-.u -id- 
which  Mat  lieu-  used  in  his  experiments,  both  kind-  «>t  e;^--  \\ 
subjected  to  the  action  of  glutamic  acid,  asjianii-  acid,  a -pa  rapine. 
^l\c(noll  and  alanin.  In  each  series  of  experiment-  eggs  from 
two  or  more  females  were  thoroughly  mixed  and  then  artificially 
lenili/ed  in  -ea-\\ater.  As  soon  as  the  polar  bodie-  had  been 

273 


2J4  HELEN    DEAN    KING. 

extruded,  approximately  equal  portions  of  the  eggs  were  trans- 
ferred into  finger  bowls  which  contained  loo  c.c.  of  the  solution 
to  be  tested.  As  a  control  by  which  to  judge  of  the  effects  of 
the  solutions,  one  portion  of  the  eggs  was  allowed  to  develop  in 
100  c.c.  of  normal  sea-water.  The  various  experiments  were 
made  in  a  similar  manner  and  the  eggs  were  kept  under  like 
conditions  of  light  and  of  temperature  during  their  development 
in  order  that  the  results  of  the  experiments  might  not  be  affected 
by  environmental  conditions  other  than  those  that  were  being 
studied. 

A.    EXPERIMENTS  WITH  THE   EGGS  OF  Arbacia  punctulata. 

As  the  breeding  season  of  Arbacia  is  near  its  close  the  latter 
part  of  July,  only  a  small  number  of  eggs  suitable  for  experimental 
purposes  could  be  obtained.  All  of  the  eggs  used  were  presum- 
ably in  a  normal  physiological  condition,  as  at  least  90  per  cent, 
of  those  in  the  control  cultures  developed  in  a  normal  manner 
and  became  plutei. 

In  each  series  of  experiments  observations  were  made  at  fre- 
quent intervals  on  the  living  embryos.  These  observations  were 
later  supplemented  by  a  microscopic  study  of  various  lots  of 
material  that  had  been  fixed  in  corrosive  sublimate  and  stained 
with  Heidenhain's  iron-haematoxylin  or  with  Delafield's  hacma- 
toxylin  followed  by  eosin. 

Cystin  (C6Hi2OiN2S2). — As  this  substance  is  very  insoluble  in 
cold  sea-water,  the  solution  used  in  the  first  experiment  that 
was  made  was  prepared  in  the  following  way:  A  quantity  of  the 
pure  crystalline  salt  was  placed  in  a  flask  of  sea-water  heated  to 
40°  C.  The  mixture  remained  at  this  temperature  for  one  half 
hour  and  was  then  sealed  and  set  aside.  After  three  days  the 
solution  was  filtered,  to  remove  the  undissolved  cystin,  and  used 
within  a  few  hours. 

A  lot  of  Arbacia  eggs  was  fertilized  at  11.45  A.M.  on  the 
morning  of  July  14,  1909,  and  a  portion  of  them  was  placed  in  tin- 
saturated  solution  of  cystin  at  12.15  I'-M.  These  eggs  were 
found  to  be  segmenting  in  a  normal  manner  when  division  of  tin 
eggs  in  the  control  culture  took  place  at  12.50  P.M.,  and  for  some 
hours  the  eggs  of  both  cultures  seemed  to  be  developing  at  about 


DEVI  I.MpMKNT  OF  EGGS  OF  ARBACIA  AND  CH.ETOPTERUS.  275 

the  same  rate.     If  the  cy>tin  had  any  effect  on  the  segmentation 
it   was  too  slight  to  be  detected  either  in  the-  living  eggs  or  in 
-erved  material. 

On  the  morning  of  July  15,  both  cultures  contained  many 
livin^  embryos;  those  of  the  control  were  well-developed  t 
trul.i-  that  were  swimming  at  the-  -urface  of  the  water  in  a  normal 
manner;  those  in  the  cv-tin  -olmion  were  decidedly  -mailer  than 
the  control  larva?,  and  nio-t  of  them  were  -wininiini;  at  the  bot- 
tom of  the  dish.  Thinv  hours  after  the  experiment  wa-  -tarted 
all  of  the  larva?  in  the  cystin  solution  were  dead,  although  the 
larva-  in  the  control  culture  were  Mill  in  good  condition.  I'iv- 
scrved  material  showed  that  the  development  of  tin-  eggs  that 
had  been  subjected  to  the  action  of  the  cystin  solution  took  p! 
in  a  perfectly  normal  manner,  although  it  was  somewhat  >lo\\er 
than  that  of  the  eggs  in  the  control  lot. 

Mathews  found  that  cystin  produced  a  decided  acivl< -ration 
in  the  development  of  the  eggs  of  Arbacia,  which  \\a-  apparent 
from  the  fourth  division  on.  The  solution  that  In-  u-id  \\a- 
made  as  follows:  "One  hundred  centimeters  of  -e.i-uater  were 
shaken  for  a  moment  with  about  a  centigram  <>\  «  r\ -talline 
i  in  and  the  mixture  poured  into  a  linger  ln.\\l  with  the  uii- 
di-solved  cystin.  The  eggs,  fertili/ed  -omeihin^  K-—  than  an 
hour  before,  were  then  added  and  tin  t\  during  d»-\i-lop- 

iiifiu  .imong  the  crystals  of  c>'stin  at  the  boitom  (.|  the  di-h." 
A-  a  solution  made  in  this  wa>'  i>  tindoul.tedK  much  \\eakcr 
than  that  employed  in  my  fir-t  exjn  riment ,  it  -eenied  ]irobal>U- 
that  the  opposing  results  obtained  \>\  Matheu  -  ,md  m\  -el!  mi-lit 
be  due  to  the  difference  in  the  -iren^th  of  the  -olution^  to  which 
tin  • -gs  were  subjected.  The  experiment  \\a^  therefore  re- 
peated  \\ilh  a  different  lot  of  eggs,  the  -olution  of  c\>tin  that 
\\a-  n-^ed  being  prepared  in  the  manner  described  \>\  M.iiheu-. 

In  tin's  experiment,  also,  the  development  of  the  e^--  appeared 
to  pro^iv-,s  at  about  the  same  rate  in  both  the  c\  -tin  culture  and 
in  the  control.  Some  of  the  e-i;-  in  the  cy-iin  -olution  seemed  to 
Moment  much  more  rapidly  than  other-,  and  a  very  few  of  them 
developed  at  a  faster  rate  than  the  major  portion  of  the  c;^-  in 
the  control  culture.  A  careful  comparison  between  the  \\\o 
cultures,  made  at  intervals  of  about  one  half  hour  during  the 


2j6  HELEN    DEAN    KING. 

entire  day,  failed  to  show  any  marked  acceleration  in  the  develop- 
ment of  the  great  majority  of  the  eggs  in  the  cystin  solution. 
Twenty  hours  after  the  experiment  began  swimming  larva?  were 
found  at  the  surface  in  both  cultures,  so  in  this  instance  the 
development  of  the  blastulae  was  not  retarded  by  the  cystin. 
The  solution  was  ultimately  harmful,  however,  as  all  of  the 
larva?  in  the  cystin  culture  died  within  thirty-six  hours,  while 
those  of  the  control  developed  into  plutei  that  lived  for  several 
days.  No  unusual  types  of  larvae  were  noted  among  the  living 
forms,  and  none  were  found  in  microscopic  preparations  of  the 
older  embryos. 

The  Arbacia  eggs  wvith  which  Mathews  experimented  were 
undoubtedly  in  a  very  different  physiological  condition  from 
those  that  I  used,  for  Mathews  states  that  in  the  control  lots 
for  his  experiments  "hardly  a  pluteus  was  to  be  found  and  these 
few  were  generally  abnormal."  In  both  of  my  control  cultures 
the  great  majority  of  the  eggs  formed  normal  plutei  that  lived 
for  some  days.  With  such  a  great  difference  in  the  lots  of  eggs 
experimented  upon  it  is  not  surprising  that  the  results  do  not 
agree,  since  the  reaction  of  eggs  to  any  external  stimulus  depends, 
to  a  considerable  extent,  upon  the  particular  physiological  con- 
ditions existing  in  the  eggs  at  the  time  that  the  stimulus  is  applied. 

Leucin  (CcHisNOo). — By  the  use  of  a  weak  solution  of  "impure 
leucin"  Mathews  changed  the  course  of  development  of  the  eggs 
of  Arbacia  so  that  many  of  the  embryos  were  totally  unlike 
Arbacia  larva?.  "In  many,  cvagination  of  the  entoderm  instead 
of  invagination,  took  place.  A  few  developed  a  ciliated  band 
in  the  shape  of  the  star-fish  bipinnaria.  .  .  .  Another  form  was 
perfectly  spherical  with  a  single  ciliated  band  about  the  middle. 
It  looked  in  its  external  form  like  a  small  trochophore."  Un- 
fortunately, it  was  not  possible  to  obtain  any  of  the  impure 
leucin  with  which  Mathews  produced  these  remarkable  forms  of 
Arbacia  larva?,  and  the  leucin  with  which  I  experimented  was 
presumably  pure. 

Solutions  of  various  strengths  (2,  I,  \,  yg-  and  •$-$  per  cent.) 
were  used  on  batches  of  eggs  that  were  fertilized  at  11.30  A.M. 
on  the  morning  of  July  16,  1909.  The  eggs  in  all  of  the  cultures 
began  segmenting  at  the  same  time  as  those  in  the  control  lot, 


DEVELOPMENT    OF    EGGS    <>F    ARBACIA    AND    CH^TOPTERT -.     277 

hut  the  stronger  solutions  \vry  soon  proved  toxic  and  greatly 
retarded  development.  None  of  the  eggs  in  the  2  per  cent,  solu- 
tion of  leucin  had  developed  beyond  tin-  2-cell  stage  at  the  time 
ill, it  the  great  majority  of  the  eggs  in  all  of  the  other  solution-, 
as  well  as  in  the  control,  were  in  tin-  S-cell  >tage.  A  solution  of 
this  strength,  however,  does  not  kill  the  eggs  quickly,  a-  twenty 
hours  after  the  experiment  began  this  culture  contained  a  few 
i  iliaied  larva?  that  were  much  -mailer,  and  le—  active,  than  those 
of  the  control  lot.  Within  t\\ent\  -four  hour-  all  of  the  laf\,e 
in  the  2  per  cent,  solution  of  leucin  were  dead. 

A  microscopic  examination  \\a-  made  of  a  large  number  of 
eggs  taken  from  the  2  per  cent,  solution  of  leucin  at  different 
stages  in  their  development.  Manv  of  the  youiu  eggs  uere 
abnormal  in  that  there  was  an  irregular  di-t  ribnt  ion  of  the  chro- 
mosomes to  the  poles  of  the  segmentation-spindle  or  a  very  un- 
equal division  of  the  blastomeres.  Such  abnormal  eggs  e\  identlv 
died  before  reaching  the  bla-tulu  stage,  as  nearly  .ill  of  the  older 
embryos  that  were  examined  were  normal  although  -OHM -\\liat 
Miialler  than  those  of  the  control  culture.  A  fe\\  al'iiormal 
Ma-tula-  were  found  among  the  older  larx.e.  but  a-  tin --e  Iar\.e 
showed  only  such  irregularities  of  form  a>  may  be  Imind  in 
individuals  of  almost  every  control  culiure  of  Ar/xniti  lar\a- 
developing  in  a  small  amount  of  sea-\\ater  under  laboratory 
(onditions,  they  could  not  be  considered  a-  due  to  the  ^p»-(  itic 
ai  tion  ol  the  leucin  in  changing  the  course  ol  de\  elopment . 

The  eggs  in  the  I  per  cent.  ><  'hit  ii  >\\  <  >\  leucin  1  u-gan  to  -In  >\v  t  he 
iniurious  effects  of  the  solution  after  the  !ir>t  hour,  and  from 
tin-  time  on  their  development,  although  normal,  lagged  behind 
that  of  the  control:  the  weaker  -olutioii-  had  apparently  no 
effects  on  the  early  segmentation.  The  bl.iMnl.e  in  the  control 
culture  began  moving  about  lilteen  minute-  -ooiu-r  than  the 
lat  \  a-  in  the  other  culture-,  so  e\  idcntly  all  of  the  leu<  in  -ohnioii- 
retarded  development  some\\hat  after  the  lir-t  tu<»  or  three 
hour-.  Pltitei  that  seemed  perfectly  normal,  and  that  li\cd  for 
several  days,  dexeloped  in  all  of  the  \\caker  -olution-.  An 
examination  of  a  considerable  number  of  the-e  embr\o-,  pre- 
-ei\ed  at  \arion-  -tage-  in  their  development,  tailed  to  >ho\\  any 
la  i  \  .e  that  \\ere  in  an\  \\a\  coni|>arable  to  the  unu>ual  t  \  pe-  that 
Matin  \\-  obtained  with  impure  leucin. 


HELEN    DEAN    KING. 

A  second  experiment  was  made  with  leurin  on  July  24,  1909. 
In  this  instance  a  solution  of  the  strength  of  %  per  cent,  was 
employed,  since  stronger  and  weaker  solutions  do  not  alter  the 
course  of  development.  From  the  beginning  of  the  experiment 
the  segmentation  of  these  eggs  lagged  behind  that  of  the  eggs  in 
the  control  lot,  and  the  retardation  in  development  was  fully 
as  great  as  that  produced  by  the  I  per  cent,  solution  of  leucin  in 
the  former  series  of  experiments.  Later  the  development  of  these 
eggs  progressed  at  a  more  normal  rate,  and  after  seven  hours 
the  embryos  appeared  nearly  as  well  developed,  and  fully  as 
vigorous,  as  those  in  the  control.  The  next  morning  larvae  were 
swimming  at  the  surface  in  both  cultures,  but  those  in  the  leucin 
solution  soon  dropped  to  the  bottom  of  the  dish  and  began  to 
disintegrate.  Microscopic  preparations  showed  that  the  very 
great  majority  of  these  larvae  were  normal  in  every  respect. 

Mathews  states  that  in  the  summer  of  1908,  when  his  experi- 
ments were  made,  the  sea-urchin  eggs  showed  in  many  instances 
the  remarkable  peculiarity,  recorded  by  Mathews  and  YVhitcher 
('03),  that  "a  large  number  of  eggs  while  living  for  several  days 
not  forming  plutei,  or  but  a  small  per  cent,  of  irregular  plutei." 
The  experiments  which  Mathews  made  to  test  the  action  of 
amido-acids  on  the  development  of  the  eggs  of  Arbacia  were  made 
therefore,  wholly  or  in  great  part,  on  eggs  that  were  in  a  peculiar 
physiological  condition  when  experimented  upon:  whether  they 
could  be  considered  as  normal  is  doubtful.  The  unusual  types 
of  larva?  that  Mathews  obtained  by  treating  eggs  with  a  weak 
solution  of  impure  leucin  were  probably  due  to  abnormal  or 
unusual  conditions  existing  in  the  eggs  at  the  time  of  their 
fertilization,  and  not  to  the  specific  actions  of  leucin  in  changing 
the  course  of  development.  The  effects  of  leucin  on  eggs  of 
Arbacia  that  are  in  a  normal  physiological  condition  when  ferti- 
lized depends  chiefly  upon  the  strength  of  the  solution  used:  a 
strong  solution  retards  development  and  causes  the  early  death 
of  the  embryos;  a  weak  solution  permits  of  normal  development 
at  first  and  is  toxic  only  after  many  hours. 

Tyrosin  (CgHnNOs). — This  substance  is  not  very  soluble  in  cold 
sea-water,  and  in  order  to  obtain  a  solution  of  sufficient  strength 
one  gram  of  tyrosin  crystals  was  put  into  looc.c.  of  sea-water  and 


DEVELOPMENT  OF  EGGS  OF  ARBACIA  AM)  CH.ETOP1I-.UI-.  279 

the  mixture  brought  to  the  boiling  point.  The  solution  was  then 
cooled  to  laboratory  temperature,  filtered,  and  used  at  once. 

The  early  development  of  the  eggs  used  in  thi-  experiment  was 
normal,  although  slightly  delayed.  After  twenty  hours  ciliated 
larva-  were  present  in  great  number  in  the  -olmion.  but  they 
were  moving  feebly  and  beginning  to  -how  degenerative  chan. 
Prepared  material  showed  that  tyrosin  had  retarded  the  develop- 
ment of  the  eggs  but  produced  no  abnormalities,  The-c  iv-ults 
agree  with  those  obtained  by  Mathcws  in  a  similar  experiment. 

Glutamic  Acid  (C&HbNOj. — Various  solution-  of  thi-  -ub-tance 
(i,  o,  i\,  and  a'o  Pcr  cent.)  were  used  on  the  e— -  ..I '  .  1  •  and 

all  of  them  proved  to  be  injurious  from  the  lie-inning  of  the  experi- 
ment. The  eggs  placed  in  the  stronger  solution-  \\  and  '  •_.  per 
cent.)  were  killed  at  once.  A  few  of  the  eggs  subjected  to  tin- 
action  of  the  j1,,  per  cent,  solution  began  to  -<  in  .1  normal 
manner,  but  none  of  them  developed  beyond  the  earl\  stages  "I" 
segmentation.  The  eggs  in  the  ^(t-  per  cent,  solution  continued 
to  live  for  some  time,  but  their  development  \\a-  \ei\  -n-.itly 
irded  and  stopped  entirely  when  the  gust  nil  ige  \\a- 
•  hed.  Preparations  of  these  eggs  showed  that  the  <•!" 
the  glutamic  acid  was  to  check  development,  not  t<>  produce 
nmi-ual  types  of  larva?. 

Aspartic  Add  (CA\7\(^). — This  sub-tame  ha-  a  more  dele- 
terious action  on  the  eggs  of  arbacia  than  ha-  glutamic  acid. 
All  of  the  eggs  placed  in  a  i  per  cent,  solution  and  in  a  ^  per  cent. 
-olution  were  killed  at  once;  those  subjected  t<>  the  action  of  a 
,',,  per  cent,  solution  did  not  develop  beyond  the  j-cell  \ 

-olution  of  the  strength  of  ^   per  cent,  aliened  a  con-iderablc 
number  of  the  eggs  to  develop  to  the  bla-iula  stage,  but  -e-  menta- 
tion was  \-er\'  irregular  anel  much  -lo\\er  than  that  of  the  . 
in  the  control  culture. 

Preparations  of  various  lot-  of  eggs  that  had  been  treated 
\\ith  aspartic  acid  solution-  -h<>\\ed  abnormal  condition-  not 
found  in  any  of  the  Arhncin  eggs  subjected  to  the  action  of  other 
amido-acids.  Most  of  th'  that  had  been  subjected  to  the 

at  lion  of  a  -^Q-  per  cent,  -olution  of  a-partic  acid  for  four  hours 
before  fixation  were  found  to  be  -till  unsegmented,  and  main  of 
them  had  been  entered  by  several  -perinato/o.i.  (  )nl\  one  -perm- 


28O  HELEN   DEAN   KING. 

nucleus  had  fused  with  the  egg-nucleus,  however,  and  the  seg- 
mentation-spindle that  was  formed  usually  appeared  normal, 
although  in  many  cases  it  occupied  a  very  eccentric  position  close 
to  the  periphery  of  the  egg.  All  of  the  accessory  spermatozoa 
at  this  time  were  in  the  form  of  a  small,  rounded  nuclei  that  were 
scattered  throughout  the  cytoplasm. 

The  ^  per  cent,  solution  of  aspartic  acid  had  a  different  action 
on  different  eggs,  depending,  doubtless,  upon  the  condition  of 
the  eggs  when  they  were  placed  in  the  solution.  Five  hours  after 
the  experiment  was  begun  about  one  fourth  of  the  eggs  were  still 
unsegmented;  some  of  the  eggs  were  just  beginning  to  segment; 
while  others  were  in  later  stages  of  segmentation,  and  the  cleavage 
planes  were  coming  in  very  irregularly  in  many  cases.  A  very 
few  eggs  had  reached  the  blastula  stage  at  this  time,  but  they 
\vere  not  as  well  developed  as  the  eggs  in  the  control  lot.  After 
twenty-two  hours  the  number  of  eggs  that  had  reached  the 
blastula  stage  was  found  to  be  consideraly  increased.  Develop- 
ment had  been  checked  by  this  time,  however,  and  the  greater 
number  of  larvae  appeared  as  more  or  less  irregular  masses  of 
cells  that  wrere  beginning  to  disintegrate. 

Preparations  of  this  material  showed  many  cases  of  polyspermy. 
Some  of  the  unsegmented  eggs  contained  a  large  multipolar 
segmentation-spindle  formed,  evidently,  by  the  fusion  of  several 
sperm-nuclei  with  the  egg-nucleus:  other  eggs  contained  a  seg- 
mentation-spindle of  the  normal  size  with  the  chromosomes  very 
unequally  distributed  to  the  spindle  poles.  The  condition  of 
these  eggs  greatly  resembled  that  which  O.  and  R.  Hertwig  ('87) 
found  could  be  induced  in  fertilized  echinoderm  eggs  by  subjecting 
them  to  the  action  of  various  chemical  substances  which  pre- 
vented their  normal  development. 

Asparagine  (C4HioNiO.i). — This  amide  of  aspartic  acid  proved 
to  be  far  less  injurious  to  the  eggs  of  Arbacia  than  did  the  latter 
substance,  when  used  in  solutions  of  the  same  strength  (l,  J-,  -^ 
and  3\y  per  cent.).  The  great  majority  of  the  eggs  in  all  of  the 
cultures  began  to  segment  at  the  normal  time  and  in  a  normal 
manner.  After  two  hours  the  eggs  in  the  I  per  cent,  solution 
showed  evidence  of  retarded  development,  but  the  eggs  in  all 
of  the  other  solutions  developed  at  a  normal  rate  for  some  hours. 


DEVELOPMENT    OF    EGGS    OF    ARBACIA    AND    CILETOPTERUS.     28l 

Twenty-four  hours  after  the  experiment  began,  ciliated  larva- 
were  present  in  great  numbers  in  all  of  tin-  solutions,  but  they 
all  died  many  hours  before  tin-  death  of  tin-  lar\a-  in  the  control 
culture. 

Glycocott  (C2H,\<  >.  . — This  -ub-tance,  which  is  the'  -implest 
of  the  amido-acids,  was  much  1<—  h.irmful  to  the  <  ggs  of  Arbucia 
than  wen-  .my  of  the-  other  amido-.u  id-  n-ed  in  the-e  experiment-. 
During  the  first  twenty-four  hour-  the  development  of  tin-  eggs 
did  not  appear  to  be  affected  in  any  wa\  by  the  -..Union-  u-ed 
Ci, .],  -I-Q  and  ;jV  per  cent .),  but  during  the  second  day  the  emlir\  •  is 
began  to  show  degenerative  change-,  ,md  all  of  them  died  about 
fifty  hours  after  the  experiment  be^.m.  Sections  <if  the-e  eggs 
fixed  at  various  stages  of  de\elopment  merely  continued  tin- 
observations  on  the  living  forms,  as  no  unu-ual  t\p<-  of  lar\a- 
were  found. 

Alanin  (CsHyNOO. — This  amido-acid  di--ol\e-  re.idiK  in  cold 
sea-water,  and  it  was  used  in  solutions  of  tin-  toll<i\\in-  -in-n-th-: 
2,  i,  .],  -j1^  and  u'fl-  per  cent.  The  stn-n-cr  -oluiimi-  2,  i  and 
.1  per  cent.)  retarded  development  from  the  l.e-imiin- :  tin- 
weaker  solutions  had  no  apparent  elicit-  on  the  -e^mentarion 
ot  the  eggs.  After  twenty-four  hours  each  of  the  -olntioii-  con- 
tained a  large  number  of  swimming  lar\.e.  and  onl\  iho-e  in  the 
j  l>er  cent,  solution  showed  any  e\  i<K  IK  e  n|  reiaided  de\elop- 
n lent.  The  embryos  in  all  of  the  culture-  died  -c.me  hour-  bet-  >ie 
the  death  of  the  control  larva',  so  weak  i-olution-  ,,f  .ilaniii  cannot 
be  considered  as  favorable  media  in  \\hich  to  rear  the  eggs  of 
Arhiititi.  Preserved  material  -houed  no  .ilniorni.ilitie-  \\orthy 
oi  note  at  an\-  stage  of  development. 

All  of  the  amido-acids  used  in  tin-  series  of  experiment-  with 
the  eggs  of  Arbacia  proved  to  be  toxic,  the  injuriou-  effects  of 
anv  -ub-tance  depending  \t_-r\-  lar^t-K  upon  the  -tren-th  of  the 
solution  u-ed.  In  no  case  was  the  cour-e  of  development  altered 
in  a  definite  direction,  except  in  tin-  very  young  eggs  and  in  these 
the  %ib normalities  produced  were  of  the  types  commonly  found 
when  fertilized  eggs  of  the  -e.i-nrchin  are  treated  with  various 
cheinie.il  -olutions. 


282  HELEN    DEAN    KING. 

B.  EXPERIMENTS  WITH  THE  EGGS  OF  Chatoptems  pergamentaceus. 
'  As  the  eggs  of  Chcctopterus  could  be  obtained  in  considerable 
numbers  at  Woods  Hole  in  the  summer  of  1909,  experiments  wrre 
made  to  study  the  influence  of  amido-acids  on  the  early  develop- 
ment of  this  annelid,  in  the  hope  that  some  definite  alterations 
in  development  might  be  produced  comparable  to  those  obtained 
by  Loeb  ('oi)  and  by  Lillie  ('02)  when  eggs  of  Chcetoptenis  were 
treated  with  potassium  salts.  Material  intended  for  micro- 
scopic study  was  preserved  in  Boveri's  picric-acetic  solution 
and  stained  with  luematoxylin. 

Cyst  in. — On  the  morning  of  August  6,  1909,  a  lot  of  Chcetopterus 
eggs  was  placed  in  100  c.c.  of  a  saturated  solution  of  cystin  as 
soon  as  the  polar  bodies  had  been  extruded.  The  early  develop- 
ment of  these  eggs  was  slightly  accelerated,  and  swimming 
larvae  were  found  in  this  culture  nearly  one  half  hour  before  any 
movement  could  be  detected  in  the  control  larva?.  The  next  day 
the  cystin  solution  was  swarming  with  well-developed  trocho- 
phores,  but  they  all  died  about  fifty  hours  after  the  experiment 
began.  No  abnormal  embryos  were  noted  at  any  stages  of 
development  and  none  were  found  in  preserved  material. 

The  experiment  was  repeated  several  days  later  with  eggs  from 
another  female.  The  results  obtained  were  practically  the  same 
as  in  the  first  experiment,  since  there  was  more  rapid  develop- 
ment during  the  segmentation  period.  The  solution  proved  to 
be  toxic  after  thirty  hours,  however,  killing  the  embryos  without 
producing  any  alterations  in  structure. 

Leiicin. — In  one  series  of  experiments  this  substance  was  used 
on  the  eggs  of  Ch&topterus  in  solutions  of  the  following  strengths: 
2 ,  YJ-  and  TjJ-g-  per  cciu .  None  of  these  solutions  had  any  marked 
effects  on  the  early  segmentation  of  the  eggs,  but  they  evidently 
caused  a  slight  acceleration  in  development  during  a  later  period 
as  the  larva'  in  all  of  the  solutions  began  moving  some  thirty 
minutes  before  there  was  any  movement  of  the  control  larvae. 
Twenty  hours  after  the  experiments  were  started  all  of  the  cul- 
tures were  carefully  examined.  The  majority  of  the  eggs  that 
had  been  treated  with  the  }/£  per  cent,  solution  had  stopped  their 
development  in  the  blastula  stage,  and  were  lying  at  tin-  bottom 
of  the  dish  apparently  dead;  a  very  few  larva?  were  swimming 


M.VELOPMENT   OI     EGGS    « '[     Akl:\<IA    AND    CBLETOPTERUS.     283 

at  the  surface  of  tin-  solution,  hut  tlu-y  had  evidently  reached  their 
maximum  development  and  would  -non  die.  The  ^  per  ivnt. 
solution  contained  a  con-idcrable  number  of  swimming  larvae, 
but  the-e  larvae  wen.-  not  in  good  condition  and  plainly  -ho\\ed 
the  injurious  effects  of  tin-  K-u«-in.  A  lar-c  number  of  ciliated 
embryos  were  found  in  tin-  ^  per  cent,  solution,  and  they  ap- 
peared somewhat  further  adx.mced  in  development  than  tho-e 
in  the  control  culture.  De-eiierative  ch.in-^ •-  appeared  in  the-e 
larva-  in  about  twenty-four  hours,  ho\\e\er,  and  all  of  them 
were  dead  within  thirty  hours.  No  unusual  type-  of  lar\.e  were 
found  in  preparations  of  l\}<  gs  !i\<  d  .it  variou-  stages  in 

their  development. 

As  it  seemed  possible  that  the  solution-  ot  leucin  einplo\ed 
in  the  experiments  described  above  might  h.i\e  been  loo  \\(  ,tk 
to  produce  any  alteration  in  the  development  of  th  .  a 

second  experiment  was  made  in  which  a  batch  <  ub- 

'•d  to  the  action  of  a  I  per  cent,  solution  of  leiicin.  Tin 
inented  at  the  normal  time,  but  two  hours  later  their  develop- 
ment was  found  to  be  lagging  behind  that  of  th-  in  the 
<  '  'iitrol  culture.  After  four  hours  the  rei.n-d.it ion  in  d< -\elopnn  m 
\\.i-  very  marked,  and  in  some  instance-  tuo  m  m  Md 
fu-ed  together.  Loeb  and  Lillie  ha\-c  noted  ih.it  the  I'u-iou  of 
M  \  eral  embryos  into  giant  forms  is  a  phenomenon  of  fiv<|tn-nt 
urrence  when  eggs  of  Chatopterus  are  treated  \\ith  poi.i— iuin 
-alt-.  In  twenty  hours  all  of  the  larva?  \\en-  dr. id,  .md  »>  ili-- 
inti-i.ited  that  it  was  impossible  to  pre-er\i-  ,m\-  mail-rial  lit 
li  'i  -i  udy.  Sections  of  eggs  fixed  in  earlit-r  stages  <  >\  de\  elopmt-in 
tailed  to  show  any  abnormalities  except  the  oec.i-ion.il  fu-ion  of 
i  u  o  or  more  embryos. 

rosin.—  This  substance  was  used  on  the  ej  gs  "f  ('liiitof>ti'rns 
in  a  -.Unrated  solution  which  i-  le—  than    ,',,   per  cent.      <  >nly  a 
\"er\    tew  of  the  eggs  had  segment i-d  when   the  tir-t   di\i-ion 
curred  in  the  control  eggs.     After  four  hours  the  tyn>-in  culture 
shoued   all   stages   in   development    from    the   un-c^mcntcd 
through    to   late   segmentation,    the    nio-l    ad\anced    ci;;^- 
apparently  at  the  same  stage  of  de\elo])iuent  a-  tli<  of  the 

(oiurol.      All  of  tin-  emlir\o-  in  the  tyro-in  solution  died  \\ithin 
twenty-four  hours  after  the  experiment  \\.i-  started.      Pre-erxed 


284  HELEN    DEAN    KING. 

material  showed  that  ty rosin  acts  on  the  eggs  of  Chcctopterus  as 
it  does  on  the  eggs  of  Arbacia,  causing  a  marked  retardation  in 
development  but  producing  no  specific  abnormalities. 

Glntamic  Acid. — Solutions  of  various  strengths  (i,  \  and  yg- 
per  cent.)  were  used,  the  eggs  being  placed  in  the  solutions  about 
three  quarters  of  an  hour  after  their  fertilization.  All  of  the 
eggs  in  the  two  stronger  solutions  were  evidently  killed  at  once 
as  none  of  them  made  any  attempts  to  divide.  Some  of  the  eggs 
in  the  ^  per  cent,  solution  began  to  elongate  after  the  solution 
had  acted  upon  them  for  one  hour,  and  later  many  of  these  eggs 
took  on  an  irregular  shape  as  if  attempting  to  divide  into  several 
cells  at  the  same  time.  None  of  these  eggs  had  segmented  after 
five  hours,  however,  so  they  were  all  returned  to  normal  sea- 
water  in  the  hope  that  they  would  then  be  able  to  continue  their 
development.  There  was  no  segmentation  of  any  of  the  eggs, 
although  they  appeared  to  live  for  some  hours. 

Sections  of  preserved  material  showed  that  the  segmentation- 
spindle  had  formed  in  many  eggs  in  an  apparently  normal  manner, 
but  that  development  had  been  stopped  at  this  point. 

Aspartic  Acid. — Eggs  of  Chcetopterus  fertilized  at  10.55  A.M. 
on  August  8,  1909,  were  placed  in  solutions  of  aspartic  acid  (i, 
~2~>  iV  ar>d  sV  per  cent.)  at  11.25  A.M.  The  eggs  in  the  control 
culture  were  segmenting  at  11.55  A.M.,  but  no  evidence  of  cleav- 
age could  be  detected  in  any  of  the  eggs  in  the  aspartic  acid 
solutions  until  1.30  P.M.,  when  a  few  of  the  eggs  in  the  ^  per 
cent,  solution  began  to  elongate  as  if  about  to  divide.  A  number 
of  these  elongated  eggs  were  isolated  and  carefully  watched  for 
some  time,  but  in  no  case  did  any  division  occur.  Sections  of 
preserved  material  showed  that  some  eggs  contained  a  normal 
segmentation-spindle,  while  others  had  a  multipolar  spindle  that 
occupied  an  eccentric  position  close  to  the  periphery.  The 
stronger  solutions  of  aspartic  acid  killed  the  eggs  before  the 
formation  of  the  segmentation-spindle. 

Aspara°ine. — Solutions  of  this  substance  of  the  same  strengths 
as  those  used  in  the  experiments  with  aspartic  acid  were  tested. 
Normal  cleavage  began  in  the  eggs  of  all  of  the  cultures  at  the 
same  time  as  in  those  of  the  control  lot.  Observations  made  at 
frequent  intervals  durine  the  next  lour  hours  showed  that  seg- 


Itl.VI.I.Mi'Mi.xT    OF    EGGS    OF    ARBACIA    AM)    CH.ETOPTERUS.     285 

mentation  was  progressing  in  a  normal  manner  and  at  about  the 
same  rate  in  all  of  the  solutions. 

Five  hours  after  the  eggs  had  been  fertilized  a  few  larva?  in  the 
YJ  per  cent,  solution  were  moving  slowly:  at  this  time  there  was 
no  movement  of  any  of  the  eml  >ry<  is  in  the  other  cultures  or  in  the 
control  lot.  A  weak  solution  of  asparagine,  therefore,  slightly 
accelerates  the  development  of  the  eggs  of  Chcrtoptcrus,  if  it  be 
ih.it  an  earlier  movement  of  the  embryos  is  indicativ  e  of  a  more 
advanced  stage  of  development.  At  the  end  of  the  sixth  hour 
the  effect's  ot  the  various  solution-  were  MTV  marked:  the 
embryos  in  the  /,;  per  cent,  solution  were  movin-  moiv  actively 
than  those  in  the  control,  and  they  seemed  slightly  better  de- 
veloped; the  larva?  in  the  other  solutions  were  moving  -lowly  and 
their  development  lagged  considerably  behind  that  of  the  control 
larva-.  After  eight  hours  the  larva-  in  the  I  per  cent,  solution 
were  all  at  the  bottom  of  the  dish  and  evidently  d\in^:  no  ab- 
normal types  of  larva?  could  be  detected  amoiu  the  li\  in^  form-, 
and  none  were  found  in  preserved  material  that  was  examined 
Liter.  The  embryos  in  the  other  solution-  \\ere  swimming  at 
the »surface  after  ten  hours,  but  none  of  them  li\ed  more  ihan 
i  \\eiuy-four  hours. 

(ilycocoll. —  In   the  strengths  of    solution-    u-.-d      i,    .1    and    ,' 
per  cent.),   this  substance  did   not   appear  to  ha\e  .my  effects 
whatever  on   the  eggs  during   the   first    twelve   hour-.     <  >n    the 
>nd  day  the  larva?  began  dying,  and  all  of  them  had  1  >eeii  killed 
b\   the  end  of  the  third  day. 

Altinin. — Batches  of  Clh<  eggs  that  had  been  artificially 

fertilized  at  lo.,V>  A.M.  on  the  mornin:;  of  Aui;ii-t  >>,  I«IOM,  were 
put  into  various  solutions  of  alanin  (I,  .', ,  ,',,  and  3',,  p«  i  cent.) 
at  ii  o'clock.  The  eggs  in  all  of  the  cultures,  including  the 
control,  began  segmenting  at  the  same  time,  and  all  of  them 
developed  at  about  the  same  rate  during  the  next  t  \\  o  hour-. 
At  3.30  P.M.  a  number  of  swimming  larvae  were  found  in  the  ,',, 
and  in  the  3^  per  cent,  solutions,  but  at  this  time  there  was  no 
movement  of  the  l.irx.e  in  any  of  the  other  cultures.  At  4..V> 
I'M.  ciliated  larva?  were  present  in  great  numbers  in  all  of  the 
solution-;  but  the  larva-  in  the  I  percent,  -ohition  could  move 
but  -lowly,  and  soon  all  of  them  sank  to  the  bottom  of  the  di-h 
and  disintegrated. 


286  HELEN    DEAN    KIM.. 

At  9  A.M.  on  the  morning  of  August  9,  the  larv.r  in  tin-  ]  •_>  per 
cent,  solution  were  dying,  and  a  number  of  giant  embryos  had 
been  formed  by  the  fusion  of  two  or  more  of  the  larvae:  the 
embryos  in  the  y^  per  cent,  and  in  the  -$-$  per  cent,  solutions  were 
apparently  normal  and  were  moving  vigorously.  All  of  the 
larvae  were  dead  on  the  morning  of  August  10,  although  the  tro- 
chophores  in  the  control  culture  were  still  very  active  at  this 
time.  Preserved  material  showed  no  abnormalities  worthy  of 
note. 

As  weak  solutions  of  alanin  did  not  seem  to  affect  the  early 
development  of  the  eggs  adversely  a  second  series  of  experiments 
was  made  in  which  batches  of  Chcetopterus  eggs  were  treated 
with  4  per  cent,  and  with  2  per  cent,  solutions  of  alanin  as  soon  a 
they  had  extruded  their  polar  bodies. 

None  of  the  eggs  in  the  4  per  cent,  solution  segmented,  and 
sections  of  preserved  material  showed  that  the  eggs  had  been 
killed  before  the  formation  of  the  segmentation-spindle.  When 
cleavage  began  in  the  eggs  of  the  control  lot  at  n  A.M.  a  very 
few  of  the  eggs  in  the  2  per  cent,  solution  were  dividing  in  an 
apparently  normal  manner;  in  the  great  majority  of  the  eggs 
segmentation  was  very  greatly  delayed.  After  four  hours  only 
a  few  eggs  had  reached  the  4-cell  stage,  and  in  these  eggs  the 
cleavage  planes  had  come  in  very  irregularly.  An  hour  later 
development  had  stopped  entirely  and  the  eggs  were  fusing  into 
large,  irregularly  shaped  masses.  At  this  time  the  eggs  were 
transferred  into  normal  sea-water  in  the  hope  that  segmentation 
might  be  resumed,  but  although  the  eggs  seemed  to  live  for  some 
hours,  none  of  them  developed  beyond  the  4-cell  stage. 

In  microscopic  preparations  of  eggs  that  had  been  in  the  2  per 
cent,  solution  of  alanin  for  two  hours  before  fixation  only  a  very 
few  normal  2-cell  stages  were  found,  and  the  great  majority  of  the 
eggs  contained  a  multipolar  spindle  with  tin-  chromosomes  very 
irregularly  distributed  along  the  spindle  fibres.  Material  fixed 
after  the  solution  had  acted  for  five  hours  showed  that  only  the 
first  cleavage  in  any  of  the  eggs  was  normal  and  that  in  most 
eggs  development  had  stopped  at  this  point.  Where  further 
division  had  occurred  the  blaMomcrcs  were  very  irregular  in 
size  and  shape,  and  although  hundreds  of  eggs  were  examined  no 
stage  later  than  an  8-cell  stage  could  be  found. 


DEVELOPMENT  OF  EGGS  OF  ARBACIA  AM)  t  H.E K  H'TKRUS.  287 

When  multipolar  spindles  formed  in  the  eggs  as  a  result  of 
their  treatment  with  a  2  per  cent,  solution  of  alanin  the  eggs, 
apparently,  were  nevi-r  al>le  to  divide,  although  there  seemed  to  be 
a  long  period  during  which  active  .ind  resting  si  -  'Iternatcd 
with  each  other.  In  the-  re-tinu  stages  the  eggs  contained  either 
one  lar^e.  oblong  nu< leu-,  or  -e\  era!  -mailer  ones  that  were  more 
or  less  irregular  in  outline.  In  the  active  period-  one  lai 
multipolar  spindle  \\ith  hundred-  of  chromosome-  scattered 
about  it  wouUl  be  formed,  or  -e\eral  small  spindles,  all  more  or 
less  irregular,  would  be  -cattered  throughout  the  cell.  In  some 
of  these  eggs  a  number  ol  ssory  asters  were  formed,  -imilar 

to  those  that  Morgan  ('96,  '<)<))  found  could  be  produced  in  the 
eggs  of  Arbacia  and  of  variou-  other  lonn-  by  mean-  of  -alt 
solutions. 

A  2  per  cent,  solution  of  alanin  produced  greater  abnormalities 
in  the  eggs  of  Chcctoptcrus  than  did  any  of  the  other  -olutioii-  •>! 
amido-acids  that  were  used,  but  as  the-e  abnormalitie-  were  "i 
the  types  that  can  be  produced  in  dillerent  kind-  ol  by 

itmcnt  with  various  salts  they  cannot  be  cmi-idered  a-  the 
i  ult  of  any  specific  action  on  the  part  of  the  alanin. 

SUMMARY  AND  COM  u  SIGNS. 

With  the  exception  of  cystin,  which  i-  a  sulphur-containi 
compound,  all  of  the  amido-acids  u-ed  in  the-e  e\p< •rimeni- 
com posed  of  the  same  chemical  elemeiu-.   yel    ihe\    diller  to  .1 
marked  extent   in   their  toxic  action  on  de\elopiiu  <  ,lu- 

tamic  acid  and  aspartic  acid  are  by  far  the  nio-t   iiijuriou-,  e\en 
per  cent,  solution  of  the-e  substances  killing  ih<  •!'  both 

Arbacia  and  of  Chcctopterus  at  a  ver\  early  periml.  ( ilyco»-<»II, 
on  the  other  hand,  permits  of  the  de\elopment  of  normal  plutci 
and  t  rochophores,  and  onl\-  injures  the  embryos  after  t  went  y- ton  i- 
hours.  The  other  amido-acid-  u-ed  retard  de\  el.  ipment ,  to  a 
,ier  or  less  extent,  depending  chielly  upon  the  -treii^th  o| 
the  -olution  employed. 

\  brief  summary  of  the  effects  oi  the  \ariou-  -ulution-  ot 
amido-acids  on  the  development  of  the  e^--  of  Ar/xicia  and  of 
Chd'topterus  during  the  tir-t  t\\el\  e  Imur-  i-  ^ivcn  in  the  tollow  iu^ 
table.  I'ltimately  all  of  the  solution-  are  toxic,  even  though 
the\  appi-ar  to  fa\-or  development  during  an  early  period. 


_— 


III  LKX    DKAN    KIN i.. 


Amiilo- 
aciil. 


Solution 
I  "-.L-d 


Leucin. 


tin         saturated 

t  per  cent. 
TV  per  cent. 
|  per  cent 
I  per  cent. 


1  per  cent. 

2  per  cent. 

Saturated. 
3<j  per  cent. 
Y\f  per  cent. 

3  per  cent, 
i  per  cent. 

3\j  per  cent. 


Tyrosin. 

4-  + 

Glutamic 
acid. 

r     -v 


Aspartic 
acid. 


TAHU-:  I. 


Effects  on  Artticia  Eggs. 


Effects  on  Ch<eto/>(crus  Eggs 


No  effects  on  segmentation  : 
later  development  retarded. 

Development  very  slightly  re- 
tarded. 

Development  very  slightly  re- 
tarded. 

.Development  very  slightly  re- 
tarded. 

Development  slightly  retarded. 


Development  retarded  after  i 

hour. 
Development  greatly  retarded ; 

a  few  eggs  abnormal. 

Development  retarded. 

Development  stopped  in  the 
gastrula  stage. 

Eggs  killed  in  early  segmenta- 
tion. 

Eggs  killed  at  once. 

Eggs  killed  at  once. 


Development  accelerated. 


Development  slightly  ac- 
celerated. 

Development  slightly  ac- 
celerated. 


Development  accelerated  at 
first,  but  stopped  in  bias- 
tula  stage. 

Development  retarded  after 
2  hours;  embryos  fused. 


Development  stopped  in  bias- 
tula  stage;  many  eggs  ab- 
normal. 

,'  per  cent,  j  Development  stopped  at  2-cell 
stage;  many  eggs  abnormal. 
2  per  cent.  Eggs  killed  at  once. 
I  per  cent.  Eggs  killed  at  once. 


Aspara- 
gine. 


Glycocoll. 


Alanin. 


3V  per  cent. 
V  per  cent. 

3  per  cent. 


I  per  cent. 


No  effects  noted. 
No  effects  notes. 

\"i>  H'tVrl     anted. 


Development  retarded  after  2 
hours. 


ji'ff  percent.  No  effects  noted, 
r'jj  percent.  No  effects  noted. 

2  percent.jNo  effects  noted. 

i  percent.  No  effects  noted. 


per  cent, 
per  cent. 


Development  retarded. 


Eggs  lived  for  some  time, 

but  no  segmentation. 
Eggs  killed  at  once. 
Eggs  killed  at  once. 

Eggs  lived  for  some  time, 
but  no  segmentation. 

Eggs  killed  at  once. 

Eggs  killed  at  once. 
Eggs  killed  at  once. 


Development  slightly  ac- 

celerated. 
Segmentation  not  affected; 

later     development     re- 

tarded. 
Segmentation  not  affected; 

later     development     re- 

tarded. 

No  effects  noted. 
No  effects  noted. 
No  effects  noted. 
No  effects  noted. 


No  effects  noted. 
No  effects  noted. 


per  cent.  Development     somewhat     re- 
tarded. 


slightly    ac- 
lerated. 
I  >e\  -elo'pment    slightly    ac- 

celerated. 

Segmentation  not  ;ill'ivti-d; 
older  embryos  fused. 


IM.VI.Lf  ;PMENT   OF    EGGS    OF    AK1:\<  I A    AND    CH.ETOPTERUS.     289 


Am 

acid. 

Solution 

Effects  01               '.«  Eggs. 

Effects  on  C/nf!i>/>tc-rus  Eggs. 

Alanin. 

i  per  cent. 
2  per  cent. 
4  per  cent. 

Development  greatly  retarded. 
Development  greatly  retarded. 

Development  retarded  after 
2  hours. 
Development     retarded; 
many  eggs  abnormal. 
Eggs  killed  at  once. 

As  s.ho\vn  in  tin-  aliove  taMe,  all  of  the  stronger  solutions  of 
.iini(|o-.K  ids  that  \\riv  ti-ed  had  much  tlu-  same  effect  on  l>oth 
kinds  of  ei^s  expcrinieiiii  d  upon.  |>ut  several  ot"  tin-  weaker 
solution-,  had  a  much  more  pronounced  action  on  the-  c--s  of 
('l\<ilo|>lcrn^  than  on  tlm-i-  of  Arlmcin.  \\"cak  solutions  of 

leucin,  <,f  asparagine  and  of  alanin  accelerate  the  de\el- 

opment  of  tin-  eggs  of  <  V:,,  toptt'ru*  to  a  noiiceaMc  extent,  yel 
none  of  tlie-r  -olution-  ha\r  .ip|».in.-ntly  an\  i-lii-ci  on  ihc  rarl\- 
d«-\  i-lopini-nt  of  th<  of  Arhaiiti.  Tin-  egga  oi  ''interns 

«  aniioi  segmenl  at  all  \\hcn  |ilaci-d  in  a  ,',,  ]>rr  11  ni.  solution  of 
asp.irtic  acid,  although  this  -oluii"ii  pcnnii-  tin  •!'  Arhncin 

to  dt-M'lop  to  the-  Ma-tula  Staj 

'I'hf    ahiionnalitifs    produced    in    th-          .       of    Ar!><:>-i<i    and    of 

'•terns  by  various  solutions  of  amido-ai  id-  consist   i  hictl\- 

of    po|\  spcTiny,    irn-v:ulariti«>    in    ihc    mitotic    li-un--.    \arialiK- 

and  a  fu-i<>n  of  -.-\rral  t-mln-xo-  into  ijani  form-.      No 

i-ml'iAo^   \vcrc    found    that    -ho\\<d    rillu-r    tlu-    lar\al    i  liaract«-r- 

istic-  of  other   forms  or   marked    peculiarities-   of   structure   that 

mi-lit    hi-    attriliuted    to    the    s|,,-cit'u-   action    of    the    solution    in 

\\hich  they  were  reared. 

The  result-  obtained  in  the-e  e\perinienls  indicate  th.it  -olu- 
tioiis  of  amido-acids  can  alter  the  rate  at  which  tli  %&  of 

add    and    of    ('/'.,,  !u/>tt'rits    di'\i-|op.    but    that     they    have    no 
inlhieme  \\hate\er  in  determining  the  character  of  the  develop- 
ment, \\lien  the  eggs  experimented  u|'oii  are  in  a  normal  physic- 
al condition. 

Ill  l-RAfL'RE    CITKD. 
Hertwig,  O.  und  R. 

'87     I  eber  «h-n  Befruchtungs-  und  Teilungsvorgang  ci  hen  Eies  unter 

di-iu  Kintli.  Xgfn/it-n.      Jen.  Zeitsihr.  Nati:r\\i--.,  B<1.  XX.,  1887. 

Lillie,  F.  R. 

'02      Differentiation  without  Cleavage  in  the  Egg  of  the  Amn-li<l.   Chatopterus 

Arch.  Kntuicklungsmech.,  Bd.  XI\".,  1902. 


2QO  HELEN    DEAN    KINT,. 

Loeb,  J. 

'01     Experiments  on  Artificial  Parthenogenesis  in  Annelids  (Chatopterus)  and 
the  Nature  of  the  Process  of  Fertilization.     Amer.  Jour.  Physiol.,  Vol.  IV., 
1901. 
Mathews,  A.  P. 

'09     The  Influence  of  Some  Amino-Acids  on  the  Development  of  Echinoderms. 

Biol.  Bull.,  Vol.  XVI.,  1909. 
Mathews,  A.  P.  and  Whitcher,  B.  R. 

'03     The  Importance  of  Mechanical  Shock  in  Protoplasmic  Activity.     Amer. 

Jour.  Physiol.,  Vol.  VIII.,  1903. 
Morgan,  T.  H. 

'96     The    Production    of    Artificial    Astrospheres.     Arch.    Entwicklungsmech., 

Bd.  III..  1896. 

'99  The  Action  of  Salt  Solutions  on  the  Unfertilized  and  Fertilized  Eggs  of 
Arbacia,  and  of  Other  Animals.  Arch.  Entwicklungsmech.,  Bd.  VIII.. 
1899. 


A  DISCCSSIOX  OF  CYCLOPS  VIRIDIS  JURIXE. 

ROBERT  CHAMBERS,  JR. 

Bl(II.(H,I<  AI.    I.AIiuK.UiiKY.     CoLCMlilA    UNIVERSITY. 

Cyclops  I'iritlis  Jurinc,  or,  as  it  was  formerly  called,  Cyclops 
l>nri«irnis  Clans,  tin-  object  <>f  tin-  classic  researches  of  Hacckcr. 
i-  de-i  ribed  ,i~  beiny  repre-ented  in  North  America  in  the  form 
ni'  -r\ i-ral  varieties. 

('.  huiuht  Mar-h  i"  .  in  hi- n-\  i-ion  of  the  North  American 
spei  ies  of  ( 'y<  laps.  di\  ide-,  tin-  -|H  ,  ies  :iri<lis  into  four  varieties: 
\ar.  ingens  Ht-rrick.  \ar.  f>r<-:isf)inosiis  Hrrrirk,  \\ir.  fxircns 
Ili-rrick.  and  \  ar.  Amcricanm  M.n-h. 

7;/i'--//N  inrhidr-  tin-  l.ir^c-i  lonn-  of  the  species  and  possiblj 

<<>\\<  -ponds  to  tin-  l-".nro|n-an  \.ir.  >    Ian-. 

Anicricanus  is  the  nm-i  aluindani  \ariei\  of  ::ridis  in  . \ineri- 
t.in  u.iiei>.  It  is  to  lie  HUM  \\ith  in  alnm-i  .ui\  dilch  or  >niall 
|tond.  Pnrcus  i>  nuirh  more  local  in  ii-  haunt-  luit  in  the 
localitii  -  \\  here  it  is  to  be  found  il  may  be  abundantiy  represented. 
[  have  never  found  the  two  \arieiii  her.  run  /o  i-,  on  the 

average,  >maller  than  Amerlcnniis. 

In  the  ,iccipin|ian\  inn  table  I  ha\i-  indicated  the  main  lealnres 
di-linmii-hin^  the  |-airo|»ean  and  the  t\\o  la-l  ineiitioiu-d  Ameri- 
can \  ai  if 

The  inner  margin  of  the  hi  real  rami  o|  I  he  tail  i-  ne\-er  ciliated 
in  the  luo  American  \arietie-  as  it  i>  in  the  |-".nro|>ean  form. 

The    rudimentary    fifth    thoracic    appendage    of    the    Iuin>|iean 

Tiridi*  '  see  laMe  .  i-  described  1>\  Schm^il    '92   as  ha\  in-^  the  -mall 

>pine  on  the  inner  margin  of  the  -I'cond  -eminent  either  a  mere 
culicnlar  ])rotul  icrance  or  connected  \\ith  th.i'  .  ien1  \>\  a  dis- 
I  in.  t  joint.  The  la  Her  feature  is  characteristic  ol  the  t\\o  Ameri- 
can \  arielie-  -ee  tal'K  . 

The  numluT  of  -pine-  on  the  terminal  se-nients  of  the  outer 
rami  of  the  four  -\\imiiiiiii;  appendage-  in  the  luiropean  liridis 
and  in  parcns  are  t  \\  o  for  the  first  pair  of  appendages  and  three 

-  »l 


292 


K<  >HI£RT    CHAMMKRS,    JR. 


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A    DISCUSSION   OF   CYCLOPS   VIRIDIS   JUKI  NT..  293 

for  the  next  three  pairs.  Anicricanus  has  three  on  the  first  and 
four  on  the  other  three  pairs. 

E.  F.  Byrnes  ('09)  considers  parcits  and  AmericuHus  to  be 
het erogenous  *  forms  of  the  same  species.  This  she  assumes  from 
the  fact  that  the  only  fundamental  difference  noted  between 
them  is  tin-  armature  of  the  swimming  appendage-  and  this 
appear-  to  !"•  \ariable,  for  occasionally  one  may  find  an  adult 
('yilops  unmistakably  ('.  Anicricanus,  in  which  most  of  the 
-\\imming  feet  agree  with  ('.  Americanns  in  having  four  spines 
on  tlir  terminal  -egmcnt-  of  tin-  outer  rami,  while  other-  .ire  in 
tin-  condition  characteristic  of  ( '.  f><irc us,  ha\  in-  but  three  -pines 
on  the  tcnnin.il  -cement-  of  the  rami. 

Neither  Byrnes  nor  Mar-h  (*IO  have  remarked  upon  the 
di--imilarity  in  tin-  -hape  of  the  reccpiaculum  -eminis  of  the 
t\\o  forms.  S\  -temati-ts  working  on  ( '>  i -lopid.e  admit  that 
the  most  constant  and  <  haracteri-tic  feature  tor  a  given  Species 
i-  the  -hape  of  that  organ.  It  i-  remarkable,  therefore,  that 
more  care  is  not  taken  in  figuring  the  -eminal  receptacle  of 
de-i  ribecl  forms. 

In    all    three    varieties  tin  ^taciilnm    consists  of    a    lai 

antcio-nu-dian  ])ortion  and  t\\o  narr»\\er  p'^tcro-\ i-nt ral  por- 
tion-, \\hi<  h  are  carried  out  laterally  a<  the  -perm  dm  ts. 

/'iircits  i>osscsscs  a  reci-pt.u-ulum  566  table)  \\hiih  re-embles 
that  of  the  Kuropi-an  riridis  in  that  the  ujiper  portion  i-  concave 
ant.-riorly.  That  of  AnicricdHiis  (see  tabl.  i-  convex. 

\notlit-r  point  which  -eem-  tolea\c  no  room  for  doubt  as  to 

the  di-tinctne--  of   the  I  u  o  varieties  i-  the  constant  dil'ference  in 

their  chromosome  number.  Iha\efound  that  the  somatic  chro- 
mosome number  in  ( '.  .1  nicricanio-  i-  io  \\  hereas  in  (  '.  pure  us  it  i-  6. 

Specimens  colli-cted  in  \\idel\  -eparaied  localities,  as  Toronto, 
\\'o.  ids  Mole,  and  \e\\  \"ork,  ha\e  thu-  far  sho\\  n  tliis  difference 
in  chromo-ome  number  to  be  constant  for  the  t\\o  varieties. 

A  specimen  taken  from  a  pure  culture  of  C.  parcns  showed  a 
variation  in  the  spines  of  its  thoracic  limbs,  the  spine  formula 
being  ,,,  4,  4,  ,}  or  4  (?).  A  systeniatist  would  probably  consider 

1  Tin-  term  kctcro^cny  is  used  here  to  denote  the  existence  of  two  adult  forms 
\\hi.li  represent  sue  .I'm-rations,  both  of  which  are  sexually  mature,  hut 

ii!. -i  |>liii|ni;ii-ally  unlike. 


294 


ROBERT  CHAMIU-:KS,  JR. 


this  a  case  of  interrelationship  between  C.  parcus  and  C.  Ameri- 
can us.  That  this  cannot  he  so  is  to  be  seen  from  the  following: 
I  was  fortunate  enough  to  section  the  specimen  when  the  chromo- 
somes of  its  oviduct  eggs  were  in  the  so-called  "biserial  arrange- 
ment" and  where  the  count  is  particularly  easy.  The  presence 


o 

o 

' 


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h 


0 


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Nucleus  of  oviduct  egg  of  Cyclops  parcus,  showing  the  three  pairs  of  chromosomes 

in  "biserial  arrangement." 

of  three  pairs  of  chromosomes  (see  text  figure)  leaves  no  doubt 
as  to  the  identity  of  the  specimen. 

The  European  Cycl.  viridis  has  12  chromosomes  (Haecker, 
Braun). 

Haecker  ('97)  described  Cyclops  brevicornis  ( 'laus  (viridis 
J  urine)  which  lie  studied  as  being  anywhere  from  3.5  5  mm.  in 
length.  The  size  mentioned  indicates  thai  he  was  probably 
working  with  var.  gigas  Claus.  It  is  remarkable  that  he  gives 
the  somatic  chromosome  number  to  be  24,  although  in  the  ovary 
he  describes  the  chromosomes  as  bivalent,  being  12  in  number. 

Braun  ('09)  also  studied  Cyclops  viridis  Jurine  but  not  the 
variety  gigas.  He  gives  the  somatic  number  of  chromosomes 
for  the  typical  species  as  12.  Unfortunately  he  does  not  mention 
sizes  except  in  stating  that  the  species  varies  between  1.5  5.1 
mm.  in  length. 

Schmeil  ('92)  gives  the  body  length  of  the  typical  I  European 
viridis  to  be  anywhere  from  1.5-3.5  nun. 

Wolf  ('05)  places  the  average  length  at  2.2  mm.  (  Mir  American 
parcus  on  the  other  hand  is  not  more  than  half  the  average  si/e 


A    DISCUSSION   OF    CYCLOP>    YIRI!>I>    J URINE.  2Q5 

of  the  European  form.  The  specimens  I  have  met  with  have 
in-ver  been  o\  <  r  i  .5  mm.  in  length  and  are  much  more  frequently 
between  1.2  mm.  and  1.4  mm.  long. 

1  In-  drawings  in  the  accompanying  plate  also  show  the  dis- 
parity in  sj/e  lit-t ween  the  American  and  the  European  forms, 
that  of  the  fifth  foot  of  the  European  variety,  from  a  specimen 
in  in\  |i<  ^session,  being  drawn  to  a  scale  half  of  that  of  the  other 
drawing 

Is  it  noi  possible  iliat  we  have  here  .1  case  similar  to  that  which 
k  l\.  Gates  ('09)  discovered  \\\  il-jwtlu'ru.'  (Enothcm  zi'^i*.  a 
uiant  mutant  of  (>.  [jinuirckintm,  was  found  to  possess  -s  i  hromo- 
SOmes  or  double  tin-  number  of  tin-  parent  form  (I-J1.  It--  cells 
were  found  to  be  correspondingly  larger.  Gates  su{  1  that 

in  an  egg  of  < ).   I.nnnirck'nuKi  a  double  number  of  chromosomes 

arose  from  a  division  of  the  chromo-ome-  unaccompanied  \<y 

1 1  u<  I  ear  and  cell  di\  i-i<ni  -ooii  after  fert  ili/at  imi  ami   that   thi^  • 

de\  eloped    into    llle   (  >.    ^/\'(/.V    lorill. 

Either  (  '.  f>n  re  its  <>r  the  European  (  '.  ririili*  ma\  conceit  ably  be 
a  m  in  at  ion  one  of  the  other  if.  in  tin-  mu-  ca-e.  all  o|  the  ehrom<  >- 
-ome-  -plit  into  hakes  \\iihout  siib-e<]ueiit  nuclear  dixisjoii.  or, 
in  tin-  other,  go  into  mitosis  without  splitting  SO  as  to  prodm  e 
the  numbiT  12  for  the  Eiiiopi.ui  :iri<Iis  or  (>  for  the  American 
f)(ir<ns.  The  cells  of  the  European  i'iriilis  <  oiiiainin^  tueke 

chromosomes  would  then  be  twice  the  size  ol  those  ot  < '.  f><ir<-ns 

\\hich  ha-  only  H\.  The  .ictual  di-cre| >aiicv  in  -i/e  bi-twi-en  the 
t\\o  forms  could  llnis  be  explained. 

Nob  Since  the  above  was  senl  to  the  printers  I  ha\  e  secured 
specimens  of  a  ( 'y<  !<>t>*  riridi.s  tr.  -m  several  pools  near  Edgewater, 
\  1  l  IHA  are  mostly  from  2-3  mm.  in  length  although  several 

inaiuie  indixiduaU  mea-niv  only   1.4  mm. 

\oi  onk  do  the\  re-emble  I  he  t  \pical  luim]  u-an  riridis  J  urine 
in  si/e;  in  the  ciliated  inner  margin  of  the  furcal  rami;  in  the 
tilth  tool  \\ith  the  \  er\  small  barely  jointed  spine  ,  ,n  the  second 

•IH-IU  ;  but   aUo  in  ilu-  --\n\\c  formula  for  the  four  swimming 

feet  which  is  2,  3,  3,  3;  and.  most  si-m'licant  of  all.  in  the  .shape 
of  the  seminal  receptacle,  the  figure  shown  in  the  table  f.-r  the 
European  I'iricli.s  being  an  exact  picture  of  the  same  organ  in  tin- 
form  under  discussion. 


296  ROBERT    CHAMBERS,    JR. 

And,  lastly,  the  somatic  chromosome  number  I  have  found  to 
be  12,  the  same  as  that  of  the  European  viridis. 

I  see  no  reason  why  this  form  should  not  be  entitled  to  the 
exclusive  rights  of  the  name  Cyclops  riridis  Jurine;  and  C.  Artie  r- 
icanus,  C.  parcus,  and  C.  brevispinosus,  each  with  its  distinctive 
chromosome  number,  spinal  armature  for  the  swimming  feet,-  and 
seminal  receptacle,  be  raised  to  the  rank  of  separate  species. 

That  the  individuals  I  have  just  secured  are  not  to  be  classed  with 
C.ingens  Herrick  I  conclude  from  Herri ck's  statement  ('95)  that  tin- 
latter  is  merely  an  exaggerated  form  of  C.  Americanus.  Neither 
are  they  to  be  compared  with  the  forms  which  Miss  Byrnes 
describes  as  C.  in  gens  (?)  for  this  latter  species  Miss  Byrnes 
distinctly  states  as  possessing  the  brevispinosus  spinal  armature 
of  the  swimming  feet.  My  individuals,  on  the  other  hand, 
possess  the  parcus  spinal  armature  for  both  the  outer  and  inner 
rami  of  the  swimming  feet,  and  this  armature  is  identical  with 
that  of  the  European  viridis. 

LITERATURE    CITED 
Braun,  Alex. 

'09     Die  specifische  Chromosomenzahlen  der  einheimischen  Arten  der  Gattung 

Cyclops.     Arch.  f.  Zellf.,  3. 
Byrnes,  E.  F. 

'09     The  Freshwater  Cyclops  of  Long  Island.     Cold  Spring  Harbor  Monographs, 

VII.     Pub.  by  Brooklyn  Inst.  of  Arts  and  Sc. 
Claus,  C. 

'93     Neue  Beobachtungen  liber  die  Organisation  und  Entwicklung  von  Cyclops. 

Arb.  Zool.  Inst.  d.  Univ.  Wien,  Bel.  10. 
Gates,  R.  R. 

'09     The  Stature  and  Chromosomes  of  CEnolhera  gigas  DeVries.    Arch.  f.  Zellf.,  .}. 
Haecker,  V. 

'97     Die  Keimbahn  von  Cyclops.     Arch.  f.  mikr.  Anat.,  49. 

'03     Uber  das  Schicksal  der  elterlichen  u.  grosselterlichen   Kernanteile.     Jen. 

Zeitschr..  37. 
Herrick,  C.  L.,  and  Turner,  C.  H. 

'95     Synopsis  of  the  Entomostraca  of  Minnesota.    Geol.Nat.  Hist. Surv. of  Minn. 
Marsh,  C.  Dwight. 

'10     A  Revision  of  the  North  American  Species  of  Cyclops.     Trans.  Wis.  Acad. 

Sc.  and  Letters. 
Schmeil,  O. 

'92     Deutschlands    frcilebende    Siisswasser-Copepoden.     I.    Theil,    Cyclopid.r. 

Bibl.  Zool.  (Leuckart  u.  Chun),  Heft  11. 
Wolf.Eugen. 

'05     Die  Fortpflanzungsvcrhaltnisse  unserer  einheimischen  Copepoden.     Zool. 
Jahrb.  Syst.,  22. 


NOTES   OX   THE    HISTORY   OF    BARKED    BREEDS  OF 

POULTRY.1 

RAYMOND   PEARL. 

At  the-  pre-ent  time  modern  Lived-  "f  p»ultrv  are  Lcinsj,  much 
u-ed  as  m, iieri.il  fur  tin-  study  of  vari<>u-  proLlem-  in  -enctic-. 
<  >n  t hi -  ,icc( ,u in  matters  connected  \vith  the  early  hi-tory  of  these 

1  lived-  take  on  a  general  biological  interest  \\hich  they  \\oiild  not 
other\\i-e  possess.  It  i-  the  purpo-e  of  the-e  notes  to  call  atten- 
tion  to  certain  hi-torical  matter-  \\liicli  hear  directlv  ii]»on  -oine 
proLlem<  nou  occupying  the  writer'-  attention. 

I.   Tin   PRODUCTION  01   mi   BARRED  COLOR  PATTERN  FROM  THE 

M.\n\(.  MI   SOLID  BI.MK  \\IIH  SOLID  \Vinn    HIKM-. 

In  an  earlier  paper  from  tlii-  laliorator\-  the  folli  >\\in-  State- 
ment was  made  respecting  the  origin  of  the  l.artvd  color  pattern, 
seen  n-.\\  in  Barred  Plymouth  l\<>ck  and  several  other  kind-  <  >\ 

poultry. 

"  In  regard  to  the  origin  of  thi-  barred  color  pattern  in  poultry 
\ei\  lit  tie  can  he  learned.  It  i-  knou  n  that  the  Barred  Plymouth 
l\o,  k-  o\\e  tlit-ir  Lanin;4  to  the  I  )omini(|tie,  which  \\a-  u-ed  in 
the  i  ro--inji[  which  led  to  the  proiluctioii  of  thi-  Lived.  But  as 
to  the  source  "f  the  Larrinv,  in  the  I  )oininii|iic-  \\c  ha\  e  found  no 

evidence.  Wright  i  says  (p.  301)  that:  'The  col«,ur  (barring) 
it-elf  i-  not  .1  primaiA'  one,  Lut  the  produce  of  \\hite  with  either 
I. lack  or  a  \ei\  dark  color.  Such  colour-,  mated  together, 

produce  as  the  re-nit,  on  a  \\ide  average  of  cases,  more  or  less 

of  Llack-,  white-,  mottle-  or  -pla-he-  with  the  plumage  of  Hoii- 
dans  and  Ancou.i-,  Lines  or  Line  dun-  like  that  of  Line  Lan^-han- 
and  Andalu>ian-,  and  that  Llui-h  Larred  plumage  known  as 
Dominique  in  America  and  Cuckoo  in  En-land.  \\'lu-n  once 
produced,  thi-  la-t  colour  has  however  a  strong  tendenc\-  to 
permanence:  and  in  the  common  nati\e  Dominique  fowl  of  the 

:roin  tin-  Hii>l<ii;ical  Laboratury  nt"  tin- Maine  Expcrinn-nt  Suit  inn,  NTo.  36. 

1  Peat!.  R..  an.  I  Sin  la. .-,  1- '.  M  .      "(  »n  tlu-  Inlu-iitancc  of  tin-  Harn-d  Color  Pattern 

in  I'niiltry."     Ar^'n  .iiklun^mech..  Bd.  XXX.  (Teil  I.),  pp.  45-61.  1910. 

297 


2<),S  RAYMOND    PHARL. 

West  Indies  and  I'nited  States  it  had  been  preserved  and  bred 
so  long  as  to  be  of  a  very  fixed  type  indeed,  though  even  in  these 
fowls  there  was  a  constant  tendency  for  white  or  black  feathers 
of  the  original  components  to  appear,  as  well  as  the  straw  or 
red  which  always  troubles  breeders  of  white  or  black  fowls.' 

"This  is  the  statement  of  a  fancier,  made  without  special 
study  of  the  inheritance  of  barring.  It  is  certainly  correct  in 
the  statement  that  the  barring  has  become  firmly  fixed  in  the 
Plymouth  Rock  at  least.  Such  a  thing  as  a  completely  non- 
barred  bird  appearing  in  any  'pure  bred'  strain  of  Barred  Ply- 
mouth Rocks  no  longer  occurs  and  has  not  for  a  number  of  years. 
The  statement  that  the  barred  pattern  originated  from  a  cross 
between  black  and  white  birds,  as  a  sort  of  intermediate  condition, 
is,  we  feel  certain,  a  mistake.  The  barring  is  a  perfectly  definite 
pattern,  not  simply  a  mixture  of  black  and  white,  or  a  'splashed' 
coloration  such  as  is  seen  in  Houdans.  The  inheritance  of  barring 
is  of  such  character  as  to  indicate  most  strongly  that  we  have  to 
deal  here  with  a  unit  character,  viz.,  a  particular  definite  and 
characteristic  pattern.  Further,  so  far  as  we  are  aware,  none 
of  the  experiments  regarding  the  inheritance  of  color  in  poultry 
carried  out  by  Bateson,  Punnett,  Hurst,  Davenport1  or  the  present 
writers  give  the  slightest  evidence  that  breeding  black  and  white 
birds  together  will  produce  barred  offspring.  Finally,  in  the 
case  of  the  Plymouth  Rocks,  where  this  pattern  reaches  its  most 
perfect  expression,  the  known  history  of  the  breed  makes  it 
certain  that  the  barring  was  not  created  de  noro,  but  wa^  taken 
from  the  Dominique." 

Since  this  was  written  I  have  found  in  the  literature  an  ink-r- 
esting piece  of  definite  circumstantial  evidence  regarding  the 
appearance  of  the  barred  pattern  in  the  offspring  of  a  solid  Mack 
and  solid  white  bird  mated  together.  This  would  seem  at  fn>i 
glance  to  be  clear  proof  for  the  <lc  novo  origin  <•!  the  pattern.  As 
such  it  is  worth  discussing.  The  case  in  point  concern^  the 

1  It  should  have  been  stated  that  Davenport  (Carnegie  Institution,  I'ublicat i"ii 
52.  p.  40)  has  reported  bam-d  nit  -pi  inj;  tmm  crossing  a  White  I.eghoi  n  liantam  cf 
with  a  Black  Cochin  bantam  v.  lli-  H-MIIH  from  pure  matings,  however,  show 
plainly,  as  he  himself  states  (lot.  /:!.,  p.  \»  and  p.  75).  that  the  White  Leghorn 
stock  used  carried  tin-  leaned  pattern  factor.  Tlii-  case  then  evidently  has  no 
critical  bearing  on  the  point  under  di-i  u--ion  here. 


ii INTORY  or  BARRED  BREEDS  OF  POULTRY.  299 

origin  <>\  race  of  bantam  fowl-  known  among  English  fanciers  as 
Cuckoo  IVkins.  The  Cuckoo  Pekin-  were  originated  about  25 
\ears  ago  by  the  well-known  English  fancier  and  authority  on 
the  bantam  breed-.  Mr.  William  Flamank  Knt\vi>k-.  In  his  book  ' 
on  bantams,  which  is  the  standard  work  on  the  subject  in  English, 
he  gives  the  following  statement  re-arding  the  appearance  of  the 
<  'ui-koo  \  ari'-t\  '  loc.  fit.,  p.  40  . 

"\\C  now  pa—  on  to  the  Cuckoo  IVkins.  These  are  a  very 
recent  introduction;  in  fact,  the  tiist  time  that  a  pair  of  this 
variety  \\.i-  e\hiliit«-d  was  at  the  Bautry  Show  in  September. 
[888,  \\hen  Master  I -"rani-.  !  1  nt\\i-le  exhibited  ihree  pair>  of 
them,  \\hich  produced  (]iiitc  a  -en-ation.  one  pair  winning  the 
-il\er  cup.  In  colour  thev  are  <iuite  as  perfect  as  .in\-  -hown 
^inii-,  though  they  are  no\\  much  impro\i-d  in  >hape.  cu-hion. 
Softness  o|  tail,  ami  abundance  o|  foot  an<l  -hank  leather.  \\'e 
fir-i  produced  (  'u<  koo-  in  thi-  \\a\  :  \\hile  cin—ing  Black  Tekin- 
and  \\hite  Hooted  \\ith  the  double  intention  of  strengthening 
the  Blacks,  and  producing  White  I'ekiii-.  \\  e  reared,  amongst 
Others,  one  a  rather  dirtv  looking  white.  SO  verj  excellent  in 
shape,  etc.,  that  u e  thought  it  good  enough  to  >ho\\  as  \\hite 
I'ekill  at  the  Dair\'  Sho\\  ;  SO  \\  e  had  it  (aught  and  \\a-hed.  but 
to  our  siirprisi-  it  \\mild  imi  come  a  better  \\hite  than  \\hen  lir-t 
I  nit  into  the  -,  Mpsiids;  \\e  tried  a  tliorotigli  gof  K  1  soaking,  \\a~h 
and  lin-ing.  and  then  had  her  carefully  dried;  and  mi  the  follou- 
in^  morning  \\  e  h.id  a  carelul  look  .it  her.  when  \\e  di-io\erid 
faint  bin  regular  bar-  of  simu-  colour,  on  a  milk  white  ground. 
\\  e  at  once  sa\\  that  in  this  pullet  \\e  had  a  more  \aluable  pri/e 
than  a  |mre  \\hite  \\oiild  ha\e  been,  and  \\e  mated  her  \\ilh  her 

site,  a  Bla«  k  Pekin  cock,  for  the  next  season.     From  this  mating 

ue  had  di-linct  cuckoo  markings,  and  tlu-si-  pulleis  \\  (  mated 
\\ith  a  (  "uckoo  (o.kere!,  \\hich  Mr.  l.eiio  kindly  s(-nt  u-.  and 
\\  hich  he  bred  from  his  imported  ( 'hiiu-se  Cuckoo  cock,  we  believe 
the  onl\  one  ever  sent  from  C'hina.  Tin  n  ue  bred  in-and-in, 
and  back  to  the  pure  Black  IVkins.  until  they  have  pn>\  cd  them- 
sel\e-  ca]>able  of.  no\\  ami  then,  beating  all  other  colours  of 
Pekins." 

In  considering  this  case  the  lir-t  point  to  be  noted  is  that  there 

\V.  I-"..  "HaiH.iiu-."      NY.ikrtirM.  pp.  1-116. 


3OO  K  \VMOND    PEARL. 

is  every  reason  to  suppose  that  entire  reliance  may  be  placed 
on  the  statements  made,  so  far  as  they  go.  In  other  words, 
Mr.  Entwisle  may  be  regarded  a  reliable  witness  as  to  the  facts. 
He  held  a  distinguished  place  among  British  fanciers,  and  his 
book  furnishes  much  evidence  that  he  was  a  keen  and  careful 
observer.  Of  course,  as  is  usual  in  such  fanciers'  reports,  critical 
evidence  is  lacking  at  important  points  in  the  case  here  under 
discussion. 

One  thing,  however,  seems  clear,  namely  that  while  in  the 
particular  instance  cited,  the  barred  pattern  appeared  in  the 
FI  generation  from  a  mating  of  black  by  white,  it  is  by  no  means 
certain  that  one  or  the  other  of  the  parents  did  not  carry  barring 
latent  (i.  e.,  as  a  cryptomere).  The  difficulty  in  taking  this  case 
as  proof  of  the  de  novo  origin  of  the  barring  lies  in  the  fact  that 
a  "Mr.  Leno"  had  at  that  time  a  cuckoo  cock  "imported  from 
China,"  which  evidently  carried  the  barred  pattern  in  heredi- 
tary form.  If  this  wrere  the  case  it  is  obvious  that  other  color 
varieties  of  Asiatic  bantams  might  carry  the  barred  pattern 
determiner  or  factor  in  their  gametes,  without  its  being1  somat- 
ically  visible.  That  this  is  the  true  explanation  of  the  case  is 
indicated  by  the  fact  that  in  this  pullet  the  pattern  appeared 
at  once  in  apparently  almost  perfect  condition  ("faint  but  regular 
bars").  This  would  scarcely  be  expected  if  what  is  occurring 
here  is  the  beginning  of  the  synthesis  of  a  barred  pattern 
from  pure  black  and  white.  Rather  one  would  suppose  that  at 
the  outstart  the  barring  would  be  irregular  and  indefinite  in 
character. 

This  case  described  by  Entwisle  must,  then,  be  regarded  as 
failing  to  furnish  critical  evidence  of  the  de  novo  origin  of  the 
barred  pattern  in  fowls  from  crossing  solid  black  and  solid 
white. 

Incomplete  as  are  the  data,  however,  the  case  is  of  interest  in 
another  direction.  If  it  be  assumed,  for  the  reasons  set  forth 
above,  that  one  of  the  parents  of  this  faintly  barred  pullet  carried 
the  gametic  determiner  for  barring,  then  one  must  conclude  that 
it  was  the  White  Booted  parent.  The  reasons  are,  (a)  that  in  all 
cases  now  known  at  least  (and  they  cover  in  published  and  un- 
published work  a  fair  number  of  different  breeds  of  poultry),  if 


OF    BARRED    BREEDS    OF    POULTRY.  3OI 

the  factor1  for  black  pigment  and  the  factor  for  the  barred 
pattern  are  present  together  the  zygote  will  show  the  barred 
character.  In  other  words  there  is  no  evidence  that  a  black 
bird  can  carry  barring  latent.  If  such  a  bird  has  the  barring 
factor  at  all  it  will  be  visible  somatically,  so  far  as  present  expe- 
rience goes,  (b)  The  white  of  the  White  Booted  9  parent  must 
have  been  "dominant  white-,"  from  the  account  given.  This  is 
indicated  by  the  fact  that  the  prevailing  color  of  the  FI  bird  from 
the  cross  of  this  white  ?  with  a  black  :?  was  white.  A  "domi- 
nant \\hite"  bird  could  carry  barring  gameticall)  for  an  indefinite 
period  \\ithoni  ij-  >li.i\\in-  -omatically. 

I'rom   tin-  data  ':i\en  ii    i-  po--ib;.-  to  cotiMruct    the  following 
pedign 

I'  k  d*   X  \VI. 


_ 

4, 
I-  1  /  Faintlv  ;  9 


I  l-iilly  1  9    X  K 


4 
1  in--!     •    •  .ind  9  9 

\o\\    in  \  ie\v  of  the  fact   that   the  barred  pat  lern  in  all  C3 

tar  >l  tidied   behaves  as  a   sex-COITelated   character  for  which   the 

leinalc  i-  |]i.-irrn/\  ^i.u-.   thi-  |K-(;  gives  a   remarkable  re-i:lt. 

If  the  baning  was  latent  in  the  original  \\  hite  liootrd     J  ,  which 

ms    probable    for    the    reasons    set    forth    above,    the   ob-nxed 

ills  i,  in  only  be  explained  on  the  assumption  that  th,'  factor  for 

hairing  and  the  v  determiner  .iirried   in   the   same 

gamete.      Tim-  i!   tt  denotes  pre-enci-  <if  factor  for  barred  pattern; 

II'  present  e  of  factor  for  "dominant  \\hiie"  ;  A'  presence  »t  t  H  tor 
for  black;  /•'  presence  of  factor  t.,r   .   sex,  and  ihe  correspond 

^mall    letter-,  the    absriuv   of    the-e    tailor-,    t  lie  |  icdigree  for  this 
black    X  \\hite  CTOSS  \\ould  ^tand  a-  |o!lo\\-. 


1  Or  factors.       I  In.  'Helmut  tlu-  pi  I-M  -in  ili->  u>>iiin  it  will  In-  a  —  unn-'l  f<-r  the  sake 
el    \,-il..il  .  v.,n,,tny  tli.il  tin-  i'luiia>  tera  in  each  instance  ili-privl  iip<>n  tin-  action  of 
•:.v;/i-  K-inu-tu   lait.'t.      It  inak.  -  n»  difference  to  the  arnuiiH-nt  wla-tla-r  this  is 
:n-t  tru<-  in  a  particular  instance. 


302  RAYMOND    PKARL. 


/:ir>;/'  . 

bWnf 

or 

P  Generation   Black 

•yt 

[  bwNf  . 
or 

bwNf} 

\      X 

or 

BWnf 

•  \Vhiti- 

cf 

(  bwNf  . 

l',\\'nF  . 

b-wnf   1       9 

or 

/;ir«/--  . 

Bumf  J 

r  n 

cf  unknown  Faintly  barred  white  9 

Fi  Generation  (i)  bwNf  .  BWnF 

or 
(2)  bwnf    .  BWnF 

Of  these  two  alternative  formulae  in  FI  the  first  is  decidedly 
the  more  probable,  since  there  is  every  reason  to  expect  that  (2) 
would  be  a  pure  white  bird  showing  somatically  no  trace  of 
barring. 

In  the  Fo  generation  got  by  mating  the  faintly  barred  white  9 
of  FI  to  the  black  cf  of  the  P  generation  the  females  were  barred, 
indicating  again  that  one  of  the  gametes  uniting  to  form  these 
individuals  must  have  borne  both  F  and  B,  since  no  gamete  from 
the  sire  could  bear  either  of  these  factors. 

It  is  evident  that  in  interpreting  this  case  we  are  forced  to 
adopt  either  one  or  the  other  of  two  alternatives,  both  of  which 
present  novel  points  in  comparison  with  the  results  of  recent 
experiments  regarding  the  inheritance  of  the  barred  pattern  in 
crosses  involving  Barred  Rocks,  in  which  this  pattern  is  well 
fixed.  On  the  one  hand  we  may  conclude  that  the  White  Booted 
9  original  parent  carried  the  B  factor  in  its  gametes.  This 
interpretation  leads  to  the  results  worked  out  above,  the  novel 
point  in  which  is  that  here  there  is  no  repulsion  between  B  and  F 
in  gametogenesis  (or  coupling  between  B  and  /  if  one  chooses  that 
view)  as  is  the  case  in  Barred  Plymouth  Rocks  of  the  present  day. 
Here  a  non-barred  cf  mated  with  a  female  eariying  barring  (by 
hypothesis)  produces  barred  daughters,  \\here  there  should  be 
produced  (to  accord  with  recent  experiments  on  barring)  barred 
sons  and  non-barred  daughters.  On  the  oilier  hand  it  is  possible 
to  assume  that  the  faint  barring  in  the  FI  9  arose  de  noro,  and 
ih.it  i  he  White  Booted  9  parent  did  not  carry  the  B  factor.  <  hi 
this  view  it  must  be  concluded  that  this  neic  character  burring 
when  it  first  appears  behaves  in  an  absolutely  different  way  in 
inheritance  from  what  it  docs  later.  Either  conclusion  is  sufficiently 
interesting,  and  stimulating  to  further  research. 


HISTORY    OF    BARRED    BREEDS    OF    POULTRY. 


303 


Of  course  a  ihinl  assumption  still  is  possible,  namely  that  the 
barring  of  the  Cuckoo  Pekins  is  a  different  barring  entirely  from 
that  of  Barred  Plymouth  Rocks,  and  therefore  behaves  differently 
in  inheritance.  There  is  no  evidence.  however,  on  which  to 
base  such  an  assumption.  All  of  the  types  of  barring  which  do 
behave  difteremlv  in  inheritance  from  the  Barred  Rock  type 
(e.  .{,'.,  the  Campine  or  the  Pencil  In  I  Hamburgh  barring)  are 
soniatically  di-tinctly  different  from  the  Barred  Rock  type  of 


I  ,; 


/i 


!•!,,.   i.      A    i   uckoo     1'.  kin      -".   \\inn.-i    •  •!     tii-t    and     -pi-ri;il   .11    \Yliilt>y.   tir>t  and 
-i.il.it   H.illain  and  K. -fir-all.  i-U-.,  i-t>-  .   [894.      1- "i  •  'in  Kntu  !-!•  /.,  p.  2O. 

barring.  The  barriu-  <>|  ilic  Curko,,  I'ekin,  h-t\ve\  i-r,  belongs 
to  the  same  i\  pe  -..mati.-ally.  Thi-  i-  -ho\\  n  in  I:ig.  I,  which  is  a 
copy  of  a  picture  of  a  Cuckoo  IVkin  rn  drawn  by  the  well-known 


304  RAYMOND    PKARL. 

English  poultry  artist  Lucllow.  and  published  in  a  plate  facing 
p.  20  of  EntwisleV  book. 

One  of  the  most  important  and  most  baffling  problems  in 
genetics  today  is  the  analysis  of  "  pattern"  inheritance.  There  is 
definite  and  indubitable  evidence  that  certain  plumage  patterns 
in  poultry  behave  as  clean-cut  unit  characters,  dependent  on 
gametic  determiners  or  factors  which  act  precisely  like  pigment 
factors  for  example.  Yet  the  gametic  and  ontogenetic  mechan- 
ism of  pattern  determination  and  development  is  most  difficult 
even  to  imagine.  There  certainly  is  great  need  for  further 
research  in  this  field.  Particularly  it  is  important  to  see  whether 
definite  patterns  may  be  formed  dc  noi'o  from  crosses  of  birds 
which  bear  no  trace  of  the  determiners  of  the  patterns  gametically. 

The  case  here  under  discussion  illustrates  the  difficulties  which 
attend  the  getting  of  really  critical  evidence  on  this  matter  for 
the  barred  color  pattern.  Merely  to  show  that  a  black  and  white 
bird  mated  together  give  barred  offspring  will  not  suffice.  This 
happens  if  one  mates  any  bird  carrying  black  pigment  with  a 
\Yhite  Plymouth  Rock,  but  it  is  merely  (and  obviously)  because 
the  White  Rock  carries  the  barred  pattern  factor  as  a  cryptomere. 
To  get  crucial  evidence  one  must  use  black  and  white  breeds  (a) 
in  which  there  is  no  evidence  of  barred  birds  having  been  used 
in  the  crosses  from  which  the  breeds  were  originated;  (b)  in 
which  there  never  occur  barred  "sports";  (c)  in  which  barred 
varieties  of  the  breed  are  unknown;  and  (d)  in  which  the  white  of 
the  white  parent  is  a  "recessive"  and  not  a  "dominant"  white. 
These  criteria  at  once  exclude  from  experiments  on  synthesi/ini: 
the  barred  pattern  from  black  X  white  crosses,  if  such  experi- 
ments are  to  be  really  critical,  all  Mediterranean  breeds  (so  far  at 
least  as  these  breeds  are  known  to  the  writer).  Further  the  >tock 
used  must  be  given  a  thorough  preliminary  test  in  Barred  Rock 
crosses  to  determine  whether  it  docs  or  does  not  carry  the  B 
factor.  Experience  indicates  that  it  is  difficult  to  settle  this 
point  if  the  white  of  the  white  race  belongs  to  the  "dominant 
white"  category.  There  are,  however,  certain  races  of  poultry 
which  seem  to  fulfil  the  requirements  for  a  crucial  test  of  the 
fundamental  question  of  the  de  iioro  formation  of  the  barred 

1  Loc.  cit. 


HISTORY    or    |;.\RRI  I)    KKU  1>-    OF     Poll   1RY. 

pattern  from  a  b!a'-k  X  white  <  ross.     Ex]  crimcnts  an-  now  beini; 
carried  on  in  this  direction  by  the  writer. 


I!.    Tin.   COLOR    \\i>   I'\IIIK\   01     EARL^    BAKKI  i>   IM\M»I  in 

Ri  '(  K.S. 

"I'll:-  In-:  Barred  I'lynioutli  Rock  fowl-  to  In-  entered  under  this 
n.  i  n  K-  .ii  .1  j  "ul;  r\  -ho\\  \\en-  e\  hi  Li  led  in  I  *<><)  by  I  ).  A.  I  'pham 
at\Yoi.  Ma—.1  They  had  first  been  bred  some  four  or  five 

irs  I  .el  on-  that  time.  So  far  as  I  ha\e  be.  n  alile  to  learn  the 
earlie-t  ptibli-hed  picture  of  foul-  of  thi-  new  l»n-ed  nf  fo\\|-, 
which  \\a-  in  any  d(  MI  accurate  or  adequate  repiv-cniat  ion 

(A  the  actual  bird-.  fir-t  .ijtpeared  in  the  .I//  .\.-ricultnrist 

in  Jaiuiar\-,    [87  The  \\ell-kno\\n   |X'iiltr\    arti-t    Mv.    I  r.'iik- 

lane  L.  Sewell  siaiev1  (hat  he  kn«-\\-  of  no  earlier  picture  than  ihU 
and  his  experience  in  ;  hi-  held  i-  extensive.  \\  IH-I  her  t  hi-  pic  t  urc 
is  absolutely  the  lir-t  '.I  the  Im-ed  t«i  a  is  not  e--eiuial. 

nor  of   any   intere-t    other    than    pureK    anlii|iiariaii.      The    liju- 
cal  interest  of  the  picture  lie-  hr-t  in  the  fact  that  it  -h«>\\  -  the 
appearance  of  bird-  of  thi-  l.reei]    \er\    .  arl\    in   it-  hi-tory.  and 

••lid    in    the  fact    that    tin    drauin^   j^   \\d|   done,   and    nia\    be 

en  with  considerable  confident  «  as  an  aC(  male  i  •«•(  >i  •  -t  illation 
of  the  appearance  of  tin  I  (  si  .if  the  I'K  tin  •nth  Rock  f<>\\  1-  .if  that 
time.  This  wood-cut  ,  \\  Inch  i-  hen  reproduced  as  l-'i^.  2,  I 

Anally  the  initials,  "  E.  I  .."\\hich\\ereiln  d\\in  l-'m-l 

a  rather  clever  delineator  «>f  pmiltrv  .  \\lio  \\orked  in  \t  u  York. 
The  picture  was  copied,  or  rather  apparently  printed  from 
the  same  block,  without  credit  for  a  prior  appearance,  ami  \\ith 
the  artist's  initials  cra-ed.  in  the  Poultry  \Vorld'  ft.r  |u!\.  [872. 
hi-  from  this  publication  that  the  pic  u  ire  ha-  been  chieily  knou  n 
to  poultry  fanciers.  N-\\elb  ^i\e-  the  I'.-nltry  ]\'or!<l  a-  the 

_inal  place  of  publication. 

Kubinson.    J.    H.      "  Piiiu-iplf-    ami    I'KI  I. 

Ginn  &  Co.,  p. 
*Vol.  31- 

3  Sewell.  F.  L.      "(.'l.'i   .,|   1'luinai;.-  i.l    th.-   I',.ui.-.;  <il>l,- 

•..;/.  \'nl.  XV..  p.  5J.i,   Iul\-,  1908. 
1  \"ol.  I.,  p.  85.       I  ii'-  i"Uinul  \\liirh  lia<  loii.u  -in. 

!.ll  tl-'I.|.    (.'.Mill. 

Lot  .  ,  ;/. 


306 


RAYMOND    IMARL. 


The  drawing  was  made  from  actual  birds,  and  is  therefore  not 
wholly  imaginary  or  idealistic.  The  pair  of  birds  figured  belonged 
to  a  Mr.  C.  C.  Corbett,  of  Norwich,  Conn.,  and  represented  a 
high  degree  of  excellence  for  the  time. 

The  chief  points  of  interest  in  this  picture  for  the  student  of 
genetics  are  the  relatively  dark  color  of  the  birds  and  the  indis- 
tinctness of  the  pattrrn.  In  both  respects,  of  course,  these  birds 


^y 


FIG.    2.     Plymouth   Rock  fowls  as   they  appeared  in    1872.     This  is  believed  to 
be  the  earliest  adequate  picture  of  birds  of  this  breed. 

stand  in  marked  contrast  to  the  Barred  Rocks  of  the  present 
day.  While  a  generally  barred  effect  is  evident,  the  pigment 
obviously  spreads  over  a  great  deal  of  the  feather,  encroaching 
on  the  areas  which  are  white  in  a  modern  Barred  Rock. 

The  fact  to  which  I  wish  especially  to  call  attention  is  that 
these  early  Plymouth  Rocks  were  evidently  very  similar  indeed  in 
color  and  color  pattern  to  the  F\  birds  obtained,  by  crossing  a  modern 
Barred  Rock  cf  with  a  9  belonging  to  some  heavily  pigmeutcd 
breed,  such  as  for  example  the  Cornish  Indian  Game.  Allotting 


HISTORY    OF    BARRED    BREEDS    OF    POULTRY. 


307 


for  differences  between  modern  photographic  technique  and  a 
wood -cut  printed  on  poor  paper,  and  for  differences  in  shape  of 
body,  due  to  the  game  blood,  the  similarity  between  the  birds  in 
Fig.  2  and  the  barred  cf  and  9  of  the  FI  generation  of  the  cross 
B.  P.  R.  cf  X  C.  I.  G.  9  published  in  Roux's  Archiv1  is  striking. 
The  male  plumage  is  relatively  dark  in  both  cases  as  compared 
with  that  of  the  modern  pure  B.  P.  R.  cf .  In  the  female  there  is 
al-»  .in  excess  of  pigment,  seen  not  only  in  the  general  color  tone 


Anally  published  in  1870. 


I-'  n.    |      Reproduction  of  a  wood-cut  of 

.»t'  the  bird,  but  in  the  .i|>[)arent  "Mimkine—  "  due  l»  the  en- 
i-n>ai  huient  of  the  blark  bars  on  to  the  white  areas  of  the  indi- 
\  idual  featht  I  . 

\..\\  in  the  rase  of  the  l:i  birds  it  i>  clear  what  causes  (or  is  at 
lea-i   a-->riated  with)   the  det'ecti\e  development  or  expression 

• 

I'.  ,nl  anil  Surface,  loc.  cit. 


308  RAYMOND    PEARL. 

of  the  pattern.  The  pure  B.  P.  R.  c?  of  today  is,  so  far  as  is 
known,  always  homozygous  with  reference  to  the  barring  factor, 
whatever  that  may  be.  He  carries  two  "doses"  of  B.  Somat- 
ically  he  is  light  in  color  with  narrow  clean  cut  bars.  When  by 
crossing  a  male  is  made  carrying  but  one  "dose"  of  B  (i.  e., 
heterozygous  in  relation  to  the  barring  factor)  the  somatic  pig- 
mentation is  markedly  changed,  and  becomes  like  that  of  the 
earliest  Barred  Rocks  known.  This  obviously  suggests  that  in 
the  early  history  of  the  breed  the  males  were  regularly  hetero- 
zygous with  reference  to  barring.  If  so  they  should  have  pro- 
duced, with  considerable  regularity,  non-barred  (black)  daugh- 
ters. As  a  matter  of  fact  this  was  probably  the  case.  Up  until 
20  years  ago,  and  even  later  in  some  localities,  one  would  judge 
from  various  statements  to  be  found  in  poultry  journals,  agri- 
cultural papers  and  the  like,  that  it  was  not  a  particularly  un- 
common occurrence  for  a  Barred  Rock  mating  to  throw  some 
solid  black  chickens. 

It  is  of  interest  to  note  that  at  the  time  of  the  original  founda- 
tion of  the  Plymouth  Rock  breed  the  Dominiques,  from  which 
the  barred  pattern  was  derived,  had  the  same  type  of  pigmenta- 
tion. This  is  shown  in  a  contemporary  wood-cut  of  the  latter 
breed,  reproduced  in  Fig.  3. 

This  picture  of  Dominique's  was  published  in  the  American 
Agriculturist,  Vol.  29,  p.  13,  1870.  It  was  drawn  by  Edwin 
Forbes  from  a  pair  of  birds  owned  by  Col.  Henry  Howland  of 
Chicago.  These  birds  were  prize  winners  in  their  time.  This 
cut,  in  a  very  much  garbled  form,  was  reproduced  in  the  Fanciers 
Journal  in  1876,  from  which  source  it  has  been  copied  by  Sewell.1 

1  Loc.  cit. 


COMPLETE    DISCHARGE   OF   MITOCHONDRIA   FROM 
THE   SPERMATOZOON   OF   PERIPATUS.1 

THOMAS  H.   MONTGOMERY,  JR., 
I'sivKKMTY  OF  PENNSYLVANIA,  PHILADELPHIA. 

The  follotting  account  presents  the  unique  case  of  a  spermato- 
x"«",n  that  loses  all  its  mitochondria  with  the  abstriction  of  the 
cytoplasm. 

In  my  paper  ('oo)  on  tin-  -pfMiiain-i-nr-i-  of  Periputns  hulfonri 
tin-  history  of  the  germinal  cycle  \\a-  de-scribed  from  tin-  sperma- 
tOgonia  through  tin-  m.ii  ur.it  ion  division-,  and  that  is  now  com- 
pleted by  a  description  of  tin-  -permio^ene-i-.  In  that  account 
(criain  di-t  in<  t  j\ ,•  bodies  uerc  figured  and  di-cu— cd  under  the 
n. inn-  «•!'  "\olk  -pin-rules."  Tin -e  were  in>ird  to  occur  only 
spar-fly  in  the  spe  rmatogonia.  Inn  to  become  almndant  during 
tin-  Drouth  period  of  tin-  -pf rmatoc\  !<•-,  and  in  all  mito>fS  to 
lie  mil-idf  of  tin-  -pindlr-.  Similar  hodic-  \\cn-  -ct-n  in  tin- 
sln-ath  ce|]>  of  tin-  tCStlS,  and  I  •  .included  the-e  1.1  l.e  nni-e  cells, 
flaln-rat.irs  of  the  suppc^-d  \»lk  spin-rule^,  and  that  the  sprrma- 
toi;onia  received  tln-ir  "\.-lk  -phfruK-s"  from  ihe-e  nnr-e  celN. 
.\i  the  time  wln-n  that  paper  \\a-  -ent  to  press  I'l.-nda  had  not 
\et  puMi-hed  hi-  term  mitochondria,  and  in  coiiformit\-  \\ith 
the  rc-nlt-  of  other  in\e-.ii-at.ir-  «.f  that  dav  I  -ti|>p.i-ed  true 
y.ilk  to  I  if  lurnifd  \\iihin  -pc  rmal<  •(  \  t>--. 

\.i\\  I  amalile  n  >  dem.  HIM  rate  that  the  lux  lie-,  in  <|iie-tion  an- 
not  >.>lk  xpln-rnle-.  rather  chemically  quite  different  from  these, 
luit  are  mitochondria  according  to  their  l>eha\i»r  and  -laining 
reaction-.  Indeed,  it  \\'ill  |)rolial>l\  1  u-  found  that  nio-t  bodies 
(K-scriln-d  as  \olk  spherules  in  sperm. iioc\  te-  are  re.ill\-  mito- 
i-hoinlria.  My  early  account  was  therefore  one  of  tin-  tir-t  to 
de-crilie  mitochondria  through  successive  cell  generations  of 
spermaio^cin-sis.  But  that  account  \\.i-  wrong  in  its  inference 
that  those  of  the  germ  cells  proper  an-  derived  from  those  of  the 
nurse  cells;  on  the  contrary,  they  occur  independently  in  the 

'.Vi'/c-  l>y  tin-  Editor.  Professor  Montgomery  died  \vhili-  this  papt-r  was  in  press. 
Hi-  h.i.l  tln-r«-t.iri-  no  opportunity  to  make  any  changes  in  the  proof,  and  the  paper 
i-  printed  exactly  as  the  manuscript  left  his  hands. 

3°9 


310  THOMAS    M.    MONTdoMKRV,    JR. 

two  classes  of  cells,  just  as  they  do  in  the  Sertoli  cells  and  sperma- 
tocytes  of  mammals. 

The  material  consisted  of  testes,  seminal  vesicles,  vasa  defer- 
entia,  and  oviducts,  some  fixed  in  strong  Flemming's  fluid  diluted 
with  an  equal  part  of  distilled  water,  and  others  preserved  in 
corrosive  sublimate-acetic;  all  were  originally  received  from  my 
friend,  Dr.  Purcell,  of  Cape  Town.  The  mitochondria  appear 
pale  red  after  Hermann's  safranine-gentian  violet,  and  after 
the  Ehrlich-Biondi-Heidenhain  method;  shades  of  gray  or  black 
after  iron  hrematoxylin,  according  to  degree  of  destaining;  and 
after  Benda's  stain  they  are  deep  violet,  while  the  chromatin  is 
brownish  and  the  centriole  red — the  typical  reaction  to  this  stain. 
There  is  only  one  other  object  known  to  me  on  which  they  are 
equally  readily  demonstrated,  namely,  the  spermatocytes  of 
A  scar  is. 

Fig.  I,  PI.  I.,  exhibits  the  position  of  the  mitochrondia  at  the 
end  of  the  second  maturation  mitosis  where,  as  after  the  first  also, 
they  lie  at  the  distal  poles  (equatorial  ends)  of  the  daughter  cells. 
Until  their  later  fusion  takes  place  they  are  chiefly  peripheral, 
next  the  cell  wall,  spherical  or  slightly  elongate,  and  in  the  form 
of  hollow  vesicles.  In  the  earliest  spermatids  they  always  form 
a  layer  at  the  distal  pole,  but  sooner  or  later  move  forward,  along 
the  cell  membrane,  so  as  to  take  a  position  on  the  side  of  the 
nucleus  (Figs.  2-16);  at  the  same  time  the  sphere  (s)  always  ad- 
vances from  its  original  position  and  the  cytoplasm  comes  to 
make  a  lobe  around  the  nucleus  and  entirely  in  front  of  the  cen- 
triole (c).  These  movements  do  not  occur  synchronously  on 
cells  of  the  same  stage,  there  is  much  variation  in  the  process,  yet 
the  end  result  is  the  same  in  all.  By  reason  of  tin-  mitochondria 
remaining  generally  in  a  single  layer  they  may  be  readily  counted, 
and  their  number  is  found  to  differ  in  different  spermatids,  which 
shows  their  mass  cannot  be  accurately  quartered  by  the  matura- 
tion divisions.  In  Figs.  4,  6,  11-13,  a"  ()'  each  cell  are  drawn 
with  care,  and  their  numbers  here  are  respectively:  33.  45.  ()S- 
64,  49.  Their  volumes  also  differ  considerably,  as  the  figures 
show.  They  do  not  blacken  with  osmic  acid. 

In  the  nucleus  the  chromosomes  are  at  first  peripheral  and 
quite  distinguishable  (Fig.  2),  then  coalesce  to  produce  a  hollow 


DISCHARGE   OF    MITOCHONDRIA.  311 

chromatin  sphere  enclosing  clear  karyolymph  (Fig.  3).  Next  the 
nucleus  lengthens,  and  distally  its  chromatin  border  becomes 
very  thin  (Figs.  4,  5,  7,  8);  but  it  could  not  be  determined  that 
at  this  thin  region  nuclear  sap  passes  out  of  the  nucleus  in  the 
way  I  have  described  (1911)  for  Euschistns.  This  thin  area  of 
the  nuclear  wall  later  becomes  as  thick  as  the  remainder  Figs.  9, 
10).  Then  the  proximal  end  of  the  nucleus  becomes  pointed 
(Fig.  loj.  With  its  later  great  elongation  (Figs.  11-17.  and 
Plate  II.)  the  nucleus  changes  ii-  appearance,  due  apparently 
to  it-  interior  becoming  more  chromatic,  so  that  on  -urface  \ie\\  9 
it  appears  nearly  homogeneous  throughout;  it  continues  the  same 
allmity  lor  ba-ic  -tain-,  and  from  tin-  -tage  of  Fig.  II  onwards  I 
ha\e  drawn  it  brown  and  not  deep  black  -imply  in  order  to 
repre-ent  the  mitochondria  more  distinctly.  Yet  CP>--  sections 
show  that  even  in  the  mature  -perm  the  chromatin  make-  a 
hollou  i  \  linder  and  not  a  solid  rod. 

\o\\  to  return  to  the  mitochondria,  to  de-cribe  their  particu- 
larly notable  phenomenon.  After  all  of  them  have  mo\  ed  for- 
ward from  the  di-tal  pole,  carried  probably  by  Currents  in  the 
lobe  ol  <  \  lo|i|a-m.  t  hey  fll-e  together  to  produce  a  true  Nebellkern 

01  •(  hondrio-omi-  Me\e-,'oo).  Figs.  I  l  MI -h.  .w  them  becoming 
agglomerated,  and  in  Fig.  [7  they  are  seen  to  be  In -ing  to  produce 
compound  vesicles.  Fig.  IN.  PI.  II.,  exhibit-  them  on  the  -idi- 
ot the  niicltu-,  \\ith  tii-ion  far  advanced,  and  Fig.  MJ,  their  con- 
solidation into  a  chondrioSOme.  A-  the  process  of  fn-ion  ad- 
Nance-  thev  -tain  more1  deeply,  so  that  the  chondrio-oines  when 
completed  appear  den-ely  chromatic  i  Fig-.  20  22).  Some- 
time- a  few  mitochondria  remain  isolated  \\ithout  joining  with 

the  others,  sometime-  all  fuse  together  Figs.  i>  21).  Simul- 
taneou-l\  the  c\  topla-mic  lobe  mo\  e-  foruard  along  the  nucleus, 
01,  probably  more  correctly,  the  nucleus  move-  backward 
through  it;  and  in  its  substance  appear  denser  strands  and 
minute  granules  (Figs.  18,  19)  which  may  be  degeneration 
products  comparable  with  the  "tingible  granules"  of  mammal-. 
( "on-equently,  each  nearly  mature  sperm  of  the  seminal  vc-icle 
(\ ;ig-.  20,  21),  as  all  of  the  vas  deferens  (Fig.  22),  carries  near 
the  anterior  end  of  the  nucleus  a  cytoplasmic  lobe  with  a  densely 
staining  chondriosome;  there  appears  to  be  no  cytoplasm  at  all 


312  THOMAS    H.    M<  >N  I '<  .<  >M  KRY,    JR. 

in  the  region  of  the  centriole  and  the  rlagellum.  \Verc  the  his- 
tory of  the  spermatozoa  unknown  beyond  their  conditions  in  the 
vasa  deferentia,  there  would  be  no  evidence  of  the  fate  of  the 
cytoplasmic  lobes  and  chondriosomes.  But  fortunately  I  have 
numerous  preparations  of  oviducts  from  female  individuals, 
all  crowded  with  spermatozoa,  and  in  these  all  the  spermatozoa 
lack  entirely  the  cytoplasmic  lobes  and  chondriosomes  (Figs.  23, 
24);  in  not  a  single  case  was  a  cytoplasmic  lobe  observed  upon 
a  spermatozoon  when  within  an  oviduct. 

Peripatus,  ^accordingly,  has  for  us  more  than  a  phylogenetic 
interest,  it  has  a  high  cytological  importance.  The  sperma- 
tozoon during  its  development  casts  off  its  cytoplasm,  and  evi- 
dently all  of  it.  But  this  abstriction  of  the  cytoplasm,  or  a 
portion  of  it,  is  now  known  to  be  a  quite  general  phenomenon 
in  animals,  and  only  amphibians  and  certain  insects  appear 
to  furnish  exceptions  to  it.  Much  more  important  is  that  all 
the  mitochondrial  substance,  in  the  form  of  a  compact  chondrio- 
some,  is  cast  away  with  it.  Further,  I  had  previously  described 
the  spermatozoon  as  possessing  a  lance  or  perforatorium,  staining 
differently  from  the  nucleus.  Now  I  can  demonstrate  that  this 
supposed  perforatorium  stains  differently  only  on  account  of  its 
excessive  tenuity,  that  it  is  only  the  narrowed  proximal  end 
of  the  nucleus,  and  that  it  has  no  connection  with  the  sphere. 
We  have  seen  that  the  sphere  arises  just  behind  the  nucleus 
(Figs.  2,  3),  and  moves  forward  into  the  cytoplasmic  lobe  (Figs. 
5,  7,  8,  10,  12,  14,  16-19).  When  the  chondriosome  is  fully 
developed  the  sphere  lies  still  in  the  cytoplasmic  lobe,  separated 
from  the  nucleus  (Fig.  20),  and  no  evidence  was  observed  that 
it  moves  along  the  latter  to  constitute  a  perforatorium.  There- 
fore it  is  certain  that  the  sphere  as  well  as  the  chondriosome 
becomes  thrown  off  with  the  cytoplasm. 

The  history  of  the  centriole  was  not  followed  in  detail.  In  the 
telophasc  of  the  secondary  spermatocytes  a  minute  centriole  is 
present  at  each  pole  (Fig.  i).  At  the  next  stage  when  it  was 
noticed  (Fig.  3)  it  appeared  as  a  much  more  voluminous  body  a  I 
the  distal  pole  of  the  nucleus,  and  it  retains  this  position  there- 
after. Later  it  becomes  discoidal  with  indication  of  subdivision 
into  two  parts  (Figs.  4-10),  and  afterwards  lengthened  in  tin- axis 


DISCHARGE    OF    MITOCHONDRIA.  313 

of  the  spermatid  (Figs.  11-19),  reaching  its  maximum  size  at  the 
stage  of  Figs.  13,  14.  In  the  mature  and  nearly  mature  sper- 
matozoa it  makes  a  slender  rod,  joining  nucleus  and  flagellum, 
and  then  is  seen  to  have  decreased  in  volume  (Figs.  21-24). 
The  flagellum  connected  with  it  is  a  delicate,  flattened  thread, 
evidently  without  spiral  membrane  or  cytoplasmic  sheath;  in  the 
figure-  only  its  proximal  portion  is  shown.  In  some  cases  there 
appeared  to  be  a  spiral  skeleton  around  the  nucleus,  such  as 
Koltzoff  (1908)  has  recently  described  in  other  species:  but 
examination  proved  that  in  Peripatns  this  is  occasioned  simply 
by  chance  wrapping  »t  a  tla^dluin  around  tin-  nucleus. 

In  the  mature  e^i;  and  in  deavag  .  -  m>  structures  were 

tonml  in  any  way  resembling  the  mitochondria  of  the  sperm  cells. 

.\b\i--  [QOS,  and  later  paper-)  dra\\s  the  <  <  mclu-ii  >n  that 
mitochondria  are  important  hereditable  elements,  directing 
Cytoplasmic  acii\iiies  as  the  chnmios,  ,nu..  direct  tho-e  of  the 

inn  leu-,  self-perpetuating  bodies  differentiating  during  ontogeny 

in  I -t  of  the  tibrillar  M  ruct  tires  of  the  b<  -d\  .     \\"\\  \\«\\\  entering 

into  the  rapidly  i^rowini;  literature  no\\ ,  \ve  will  be  content  with 
the  Matemeiu  that  a  considerable  number  of  investigator-  cor- 
roborate the-e  \ie\\s,  and  that  the\'  have  been  especially  elabor- 
ated b\  (  iiiJio-Tos  and  <  .ranat.;  'ox  .  This  hypothe-is  more 
than  anv  other  has  dire<  ted  attention  to  these  bodies.  V 
they  are  far  less  conservative  and  regular  than  tin-  chromosomes 
in  number,  form  and  beha\ior  and  there  is  e\  idence  that  occa- 
sionally some  of  them  are  eliminated  during  spermio-rnesis. 
Thus  l-'aure-l-'remiet  has  distinguished  tour  t  \-pe-  o|  them: 
i  Those  that  undepco  changes  of  position  without  profound 
morphological  chanu«  s.  as  in  mammalian  spermiogenesis. 
Those  that  at  the  same  time  undergo  -real  structural  changes, 
a-  in  insect  sprrmatids.  131  Those  of  which  oiil\  a  part  change 
into  the  i  hondriosome  or  Nebeiikern  of  the  spcnuatid,  while 
others  degenerate,  a-  in  spermatogenesis  of  certain  gastropods. 
And  4)  those  that  transform  wholly  or  partly  into  deutoplasmic 
bodies,  some  cas,s  of  oogenesis.  The  fourth  of  these  classes 
cannot  be  said  to  be  definitely  established,  but  there  can  be  little 
doubt  about  the  evidence  for  the  third.  Thus,  besides  the 
in  -astro pod  spermatogenesis  studied  by  Faure- 


314  THOMAS    H.    MONTGOMERY,    JR. 

Fremiet,  Retzius  noted  in  mammals  a  "considerable  reduction 
in  their  substance  as  they  enter  into  the  formation  of  the  spiral 
thread."  Jordan  ('11)  found  that  in  spermatids  of  the  opossum 
a  considerable  number  of  mitochondria  are  cast  off  with  the 
cytoplasm;  and  while  Duesberg  ('10)  maintains  that  in  the 
guinea  pig  all  take  part  in  forming  the  spiral  thread  of  the  sper- 
matozoon, yet  he  figures  granules  with  similar  staining  reactions 
in  the  dehiscent  cytoplasmic  lobe.  The  parallel  does  not  seem 
yet  to  have  been  made,  yet  may  not  the  "tingible  corpuscles" 
of  mammalian  spermatids  be  metamorphosed  mitochondria,  ones 
that  have  nothing  to  do  with  the  spiral  threads?  And  now  we 
are  able  to  adduce  the  positive  case  of  Peripatits,  in  which  all  the 
mitochondria  become  removed  from  the  spermatozoon. 

In  view  of  these  facts  it  seems  to  me  we  should  be  very  cautious 
in  attributing  to  the  mitochondria  a  role  in  cellular  activity  at 
all  equal  to  that  of  the  chromosomes.  No  spermatozoon  ever 
discards  chromosomes,  but  that  of  Peripatns  throws  off  all  its 
mitochondria. 


]>!-<  H.\K<;E  OF  MITOCHONDRIA.  315 


LITERAl  I    Kl     i    I  I  ID 
Duesberg. 

'10      N'.uvi-llf-    i«-i  ln-ii  In--    -in    1'app.iifil    mil.*  IioMdiial    >\<-~    ci-Uuli--.    -•'•  min.ilf-. 

Arch.  /.-Hi.,  6. 
Faure-Fremiet. 

*IO      Ktiidi-    -nr    If-    mum  limi'liii--    >!••-    I'liiti'/i'.iin--    .-t    'If-     relink-- 

Ai<  h    An.it.   Mi.  i   .   II. 
Giglio-Tos  e  Granata 

'08     J    iiiitn.  inxli  i    IK-MI-    n-lluli-    -i-inin.ili    nui-i  liili    <li    I'nin  flni^us 

i  Miinn.i.      Hii>lt)Kii-a.  i. 
Jordan. 

"u       I  In-  S|H-iin.ili'v;i-iii--i-  "1   tin-  i  )|iii-.-uni.  i-ti  .      Aii  h.  /•  111..   7. 
Meves. 

'oo      I'l-ln-i  ili-n  \iiii  v.  l.i  \  .ill  td    >t .  '  ..  ••  .1-^1  •  i-ntili-i  kli-ii  N  flu-ilk. -in  ..  Mit.>.  li.  'ii- 

i|in-iik..i ;..  i       '•  i   ^.imi-n/i- lli-n.      Aii  h.  mikr.  An.it..  56. 
"08      l)ii-(  hiiinliiii-Kiii'-M  .il-    li.iu'i  fiMiiln-1   Anl.iwfii.      Ibid..  72. 
Montgomery. 

'oo      Tin-    >|if[iii.itM.i;fiif-i<   nl    I't-riputus   baltouri    up   to   tin-    1- .  .1  in.ui.  >n    .it    tin- 

^|M-I  in.ili'l.      /mil.  J.iln  1'.,    14. 
Montgomery. 

'ii       11,,    --],. •iiiiatiii;t-i!.  .11   llfiiiipt.-mn.    1   >     '••:  tus.     Journ.   M.irph.    22. 


316  THOMAS    H.    MONTGOMERY,    JR. 


EXPLANATION  OF  PLATES. 

AH  figures  were  drawn  with  the  cameral  lucida  at  the  level  of  the  base  of  the 
microscope,  Figs.  13,  20  with  Zeiss  apochr.  1.5  mm.,  oc.  XII.,  the  others  with  Zeiss 
achr.  1/12  mm.,  oc.  XII.  Figs.  1-3,  5,  7-12,  14-19  from  a  seminal  vesicle,  Flem- 
ming's  fluid,  iron  haematoxylin;  Figs.  4,  6,  21  from  a  seminal  vesicle,  corrosive 
sublimate-acetic,  iron  haematoxylin;  Figs.  13,  20  from  a  seminal  vesicle,  Flemming's 
fluid,  Benda's  stain;  Fig.  22,  from  a  vas  deferens,  Flemming's  fluid,  safranine- 
gentian  violet;  Fig.  23,  from  an  oviduct,  treated  like  the  last;  Fig.  24,  from  an 
oviduct,  corrosive  sublimate-acetic,  iron  haematoxylin. 

c,  centriole. 

5,  sphere. 

PLATE  I. 

FIG.  i.     Telophase  of  second  maturation  mitosis. 

FIGS.  2-17.  Successive  stages  of  spermiogenesis  from  the  seminal  vesicle. 
Fig.  6  is  an  apical  view  of  the  stage  of  Fig.  5. 


BIOLOGICAL  BULLETIN,  VOL.   XXII. 


T.   H.    MONTGOMERY. 


31 8  THOMAS   H.    MONTGOMERY,    JR. 


PLATE  II. 

FIGS.  18-21.     Later  stages  of  spermiogenesis  from  seminal  vesicle.     In  Fig.  20 
only  the  cytoplasmic  lobe  and  the  proximal  portion  of  the  head  are  shown. 
FIG.  22.     Spermatozoon  from  vas  deferens. 
FIGS.  23,  24.     Spermatozoa  from  oviduct. 


BIOLOGICAL  BULLETIN,   VOL.  XXII. 


PLATE  II. 


18 


C- 


T.   M.   MONTGOMERY. 


Vol.  XXII.  May,  1912.  No.  6 


BIOLOGICAL  BULLETIN 


A   PRELIMINARY  ACCOUNT  <  T  THE  DEVELOPMENT 

I   THF.  APYRENE  SPERMATOZOA  IX  STRo.MHl'* 
AND   OF    Till-.    M   RSE  CELLS    IX    LITTORIXA 

KIAYIN    I       Kl   !\KI  . 
-\i\. 

Tin-  dimorphic  >j>rnn.n<  >/<  >.i   in  nbus  wen-   fir-t   de-cril  >rd 

l»y  Brock  in  INS;  in  th*  v  Lt-nti^inosns.     While  accurate 

enough  in  .general,  hi-  account  i-  lacking  in  certain  interesting 
details  and  he  al-o  made  no  attempt  to  trace  the  de\  elopmental 
Stages  of  cither  kind  of  -perma!*  I',  'ill  his  description 

ami  hi-  liv.mv-,  however,  are  -nllicienl  to  -hu\v  h"\\  marked  and 
striking  i->  the  diniorphi-in  e\i-tiir^  here  .md  it  i-  Mirpri-in.^  that 
thi-  ha-  not  bei-n  made  the  -iil.jecL  of  further  Hive-libation  \>\ 
m«. i.-  M  <  <  in  workers. 

Mv  attention  Was  attracted  t"  X'/^;;."'./r.  in  Ma\.  I'd  I,  \\hile 
at  tlie  ].-mporar\  lal ..  .rator\  i-tal  'li-hed  l»\  'I'he  (  lamegie  Institu- 
tion "t  \\  a-liinul"ti  at  l'»rt  l\..\al.  Jamaiia.  \\'.  I.  There  I  had 
tlie  o|)portunit\-  of  .il)-er\in^  the  li\  in-  -permai'  'iid  also  ot 

-eciirin-  a  1m  of  material  for  further  -tud\  .  The  species  -uidud 
\\.i-  .Y  bituberculatus.  M\-  thank-  are  due  t.i  1  >r.  II.  A.  Til-hry 
ot  The  Ac.idrnu  of  Natural  Scieiic.--  of  1'hiladelphia.  who  kindly 
identilied  it  for  me. 

Ad"])tin:4   the    terminoln-y   -u-^e-ttd    l.v    \\'alde\ir   and   u-ed 

iii-~i   |.\    Meves  the  two  kind-  of  spermatozoa  found  in 

Stronilms  are  the  eiipyivnc.  /.  <•.,  tho-u  that  function  in  the  ordi- 
nary way,  and  the  ap\  rene  whose  function  is  unknown  and  in 
who.-e  adult  -tnicture  there  i>  no  at'tixe  nuclear  material.  The 
cupyrene  spermatozoa  do  not  pre-ent  aii\  s(rikinj;  diften  ; 
from  those  found  in  other  form-  \\hich  ha\e  the  same  -exual 
dimorphism,  Palndiini  for  example,  but  they  lack  tlie  tenuous 

3'9 


320  EDWIN   E.    REINKE. 

perforator! um  and  the  very  long  tail-piece  of  the  spermatozoa 
found  in  forms  like  Littorina  and  Fasciolaria  (Fig.  3).  The 
apyrene  spermatozoa,  on  the  other  hand,  bear  very  little  re- 
semblance to  those  described  in  other  forms  and  to  the  eupyrene 
spermatozoa  they  bear  absolutely  none  at  all.  They  are  vrry 
much  larger  than  the  latter  and  are  outnumbered  by  them, 
according  to  Brock's  estimate,  about  I  to  500;  the  correct  numer- 
ical relation  existing  between  them  in  S.  bituberculatus  has  not 
been  ascertained  but  it  is  probably  the  same  as  in  S.  lentiginosiis. 
In  length  the  apyrene  spermatozoa  average  somewhat  over  90 
micra. 

The  adult  apyrene  spermatozoon  is  composed  of  a  central 
spindle-shaped  cell  body,  which  is  long  and  narrow  and  slightly 
flattened  dorso-ventrally,  and  two  undulating  membranes  which 
pass  down  either  side  of  the  cell-body  (Fig.  2).  At  the  anterior 
end  of  the  spermatozoon  the  membranes  round  out  sharply  to 
their  maximum  width  while  posteriorly  they  narrow  more 
gradually  and  finally  end  in  a  short  sharply  pointed  tail-piece. 
The  interior  of  the  cell-body  is  filled  with  a  number  of  large  poly- 
gonal bodies  composed  of  an  albumen,  probably  a  nutritive 
material.  These  bodies  are  more  or  less  regular  in  shape  and 
position  but  they  decrease  in  size  at  either  end  of  the  cell. 

The  living  spermatozoa,  as  they  leave  the  sperm-ducts,  do 
not  show  any  violent  movements;  at  first  long  slow  contraction 
waves  pass  alternately  down  the  two  membranes  in  a  postero- 
anterior  direction,  propelling  the  spermatozoon  in  the  opposite 
direction,  that  is,  the  posterior  end  is  directed  forward.1  Occa- 
sionally a  spermatozoon  is  seen  moving  with  its  anterior  end 
directed  forward.  The  movement  of  the  spermatozoon  is  com- 

1  In  Paludina  that  end  of  the  spermatozoon  which  contains  the  remains  of  the 
nucleus,  i.  e.,  the  head,  has  been  designated  as  the  anterior  end  and  this  is  the  end 
which  is  directed  forward  in  movement;  it  is  also  the  end  toward  which  the  axial 
fibers  have  grown.  In  Strombus,  on  the  other  hand,  there  is  no  nuclear  head  in  the 
spermatozoon  and  therefore,  following  the  precedent  established  above,  I  have 
designated  as  anterior  that  end  toward  which  the  axial  fibers  have  grown.  It 
happens  as  a  rule  that  in  movement  this  end  is  dinvtrd  backward.  It  was  thought 
better  to  orient  the  spermatozoon  morphologically  rather  than  by  the  direction  of 
movement.  To  be  correct,  the  orientation  should  be  reversed  in  both  cases  as  the 
end  of  the  eupyrene  spermatozoon  toward  which  the  axial  fiber  has  grown  is  the 
posterior  one. 


DEVELOPMENT    OF   APYRENE    SPERMATOZOA. 


paratively  slow  and  is  not  long  continued  as  it  soon  attaches 
itselt  by  means  of  its  tail-piece  to  the  glass  slide  or  other  object 
upon  which  it  is  being  observed.  As  soon  as  this  occurs  the 
contr.H  -lion  waves  pass  down  both  the  membranes  simultaneously 


v 


Initial  magnification  <>f  1.850  diamrt'i-.  ii-.luo-cl  one  third.     Fig. 

^:n>mbus  bitubercttlatus.     Fig.  2,  apyrene  spermato- 

ie  form.     Fii:.   3,  -prrmatozoun  of  Lilturina  nehulosj  >lu .\ving  the 

lout;,    thin    |n-iii«iati>rinin.     Fig.    4,    spermatozoon   of   L.    anguliffm    rho\\ing    the 

1"  MI 'i.it"iiiiiii  -\vnlli-n  aiti-r  hi-ing  in  sea-water  lor  some  time.     Fig.  5.  nur<e-cell 

1.1  /..  Mt-/»«/i>.v(j  with  attaih'-il  spermatozoa;  drawing  made  from  a  living  cell. 

and  they  Urome  much  shorter  and  faster.  With  tin-  spermato- 
zoon attached  in  this  way,  the  membranes  may  continue  to  be 
active  for  an  hour  or  two.  It  very  frequently  happens,  however, 


322  EDWIN    E.    REINKE. 

that  the  tail-piece  breaks  off  and  the  spermatozoon  swims  a  way 
with  a  much  more  rapid  movement  than  it  had  at  first.  When 
the  tail-piece  begins  to  break  off  it  can  be  seen  to  be  composed 
of  a  number  of  fused  flagella;  sometimes  as  the  spermatozoon 
moves  away  one  or  more  of  the  flagella  may  be  seen  still  adhering 
to  it.  This  explains  the  statement  of  Brock  to  the  effect  that  a 
tuft  of  flagella,  which  is  invisible  at  first,  is  to  be  seen  after  the 
spermatozoon  has  been  swimming  about  for  a  while.  It  very 
frequently  happens  that  long  before  the  undulations  of  the  mem- 
branes have  ceased,  the  spermatozoon  flattens  out  and  the 
albuminous  bodies  break  down,  leaving  in  their  place  a  brownish, 
semi-fluid  substance  in  which,  however,  may  still  be  seen  the 
outlines  of  those  bodies. 

As  in  Pahidina,  the  apyrene  spermatocytes  of  Strombus  are 
easily  recognized.  They  are  large  pear-shaped  cells  provided 
with  a  nucleus  of  regular  pattern  and  a  large  centrosome  about 
which  may  be  seen  an  inner  clear  court  and  an  outer  dark  court. 
Until  a  very  late  stage  in  their  growth  period  they  retain  a  con- 
nection with  the  cyst-wall  of  the  testis  by  means  of  a  short  stalk; 
later  they  lose  this  attachment  and  become  spherical.  By  this 
time  the  chromatin  has  begun  to  form  in  lumps  beneath  the 
nuclear  membrane  and  from  a  large  number  of  centrioles  lying 
at  the  periphery  of  the  centrosome  strong  radiations  may  be 
seen  to  pass  out  in  all  directions  (Fig.  6).  In  the  outer  court, 
but  away  from  the  nucleus,  lies  a  mass  of  mitochondria.  A  divi- 
sion of  the  nucleus  and  cell  never  follows;  instead,  the  nuclear 
wall  breaks  down  and  the  centrosome  with  its  radiations  dis- 
appears. A  little  later  the  chromatic  masses  are  seen  scattered 
through  the  cell  while  the  centrioles  have  moved  to  the  periphery 
of  one  half  of  the  cell  where  they  are  easily  recognized  by  their 
radiations  (Fig.  7).  The  cell  now  begins  to  develop  directly  into 
the  spermatozoon. 

The  chromatin,  as  such,  takes  no  further  active  part  in  the 
development  of  the  spermatozoon;  the  fragments  very  soon 
begin  to  become  vesiculated  and  to  degenerate.  The  centrioles 
mass  together  at  a  point  just  beneath  the  cell  membrane  where 
they  divide  (Fig.  8).  One  half  of  the  number  of  daughter  or 
secondarv  centrioles  remain  attached  to  the  cell-membrane  and 


DEVELOPMENT    OF    APYRENE    SPERMATOZOA. 


J23 


In. m  tin-in  -pi\\-  out  tla^rlla  which  ultimately  fuse  to  form  the 

tail-pirn-.     The  other:-  move  across  the  cell  forming  a  bundle  of 

•I  fibers.     At  its  ba-r  the  bundle  i-  round  hut  it  becomes  more 

and   more  flatly  <iv.il  as  thr  iihrr-  nio\«    across  the  cell     Fi-.  u). 


I*    '•    * 

J  r»»    - 


> 


1  :  Initial   i  ttiii-1. 

•  in  oi  i 

l'itnl>,r<  ul, UK          I  m>4   ilowu  nl 

tli--  IMI.  li-u-  aii'l  tin-  !                                              .\  ilii; 

kini;  i|n\vn   ni    tin-  luii-lt-u-;   tin-  ii-nt:  :it  mily   a   \><  •: 

.•I  tli<-  t-.t.il  niiinln-r  in  tin-  o-ll.                     bowe  tin-  In-^inn 

fibers.     1                                               '\MI  partly  a  a  an 

•:.i|ll    til.;'  ;|)'l     I". 

The   mitochondria   may   !>»•  -mi   to  tOrm   the  imu-r   margin   of  a 
rini:  of  differentiated  c\  topla-m  \\hich  surround-  the  ba-r  <  >l"  thr 
bundle.      The>-  pn-i-t   hnv  until  the  -permah  •/<  ><  MI  ha-  n-arhrd 
nhilt  form     1  igs.  ^  ami  10). 


324  EDWIN    E.    REINKE. 

\Yhen  the  bundle  of  axial  fibers,  by  its  growth,  has  increased 
the  length  of  the  cell  by  about  one  third,  it  splits  and  the  halves 
begin  to  move  to  either  side  of  the  cell  (Figs.  10  and  u).  This 
movement  is  caused  by  a  twisting  of  the  secondary  bundles,  due 
probably  to  the  unequal  growth  of  the  fibers.  This  process 
continues  until  they  push  out  the  cell  membrane  on  either  side  to 
form  the  undulating  membranes  (Figs.  12  and  13).  Fig.  12  is  an 
oblique  section  through  the  middle  of  a  spermatozoon  which  has 
not  quite  matured;  later  the  membranes  become  flatter  and 
wider  as  shown  in  Fig.  13,  which  is  a  more  anterior  section  of  a 
still  older  spermatozoon.  As  is  indicated  in  Fig.  12,  the  con- 
tinued growth  of  the  fibers  without  a  compensating  increase  in 
the  length  of  the  cell  causes  several  even  folds  to  occur  throughout 
the  length  of  the  membranes.  Some  of  the  axial  fibers  do  not 
participate  in  the  formation  of  the  membrane.  As  the  bundles 
begin  to  evaginate  the  cell  membrane,  a  few  of  the  fibers  begin 
to  migrate  and  finally  come  to  lie  longitudinally  across  the  cell 
and  just  beneath  its  membrane  (Figs.  12  and  13). 

The  albuminous  bodies  are  formed  in  the  same  way  as  Kuscha- 
kewitsch  ('n)  has  briefly  described  in  Vermetus  gigas.  Large 
vacuoles  appear  in  the  cytoplasm,  first  in  the  anterior  portion  of 
the  cell,  and  these  gradually  become  filled  with  an  albuminous 
substance.  When  such  a  vacuole  has  been  almost  filled  but 
before  a  membrane  is  formed,  narrow  strands  may  be  seen 
connecting  the  albumen  with  the  surrounding  cytoplasm  (Fig.  1 1). 
The  cytoplasm  which  is  not  displaced  by  the  formation  of  these 
bodies  becomes  fibrillar,  the  greater  part  of  it  forming  a  core  down 
the  center  of  the  spermatozoon  (Figs.  12  and  13). 

As  the  spermatozoon  develops,  the  vesiculated  nuclear  frag- 
ments which  were  scattered  throughout  the  cell  continue  to 
degenerate.  They  gradually  become  more  condensed  and  darkly 
staining  and  undergo  further  fragmentation.  They  may  dissolve 
in  situ,  but  they  may  also  go  to  form  the  many  small  granules 
that  lie  in  amongst  the  fibers  composing  the  undulating  mem- 
branes (Fig.  13).  These  granules  are  probably  mitochondria 
and  while  at  present  it  cannot  be  definitely  asserted  that  they 
are  thus  of  a  direct  nuclear  origin,  there  is  considerable  evidence 
in  favor  of  this  view.  The  mitochondria  which  originally  sur- 


DEVELOPMENT    OF   APYRENE    SPERMATOZOA. 


325 


14- 


l-'ir.s.   10  to  14.     Initial   magnitii  ation  of  3.450  diameters,  reduced  "ii<-  tliird. 

in  t<>  13.  vatiou-  -tages  in  the  lutrr  d<-\  rl<  .pment  nf  tin-  apyn-nr  -p.-nnai 
of  Strombus  bitubcnnliitus.     Fig.  10,  bundle  of  axial  fibers  beginning  to  split;  a  ir\v 
albuminous  bodif~.  not  figured,  havr  IH-.-II  lorun-d   in  th«-  ant>-ri,  a   i-oition  oi   tin- 
cell.     I'i.i;.   ii   -how-  the  completed  splitting  of  the  Imndl  •  of  axial  Iil>i-i-;  the  cell 
i-  not  cut  through  it-  h.      l-"ig<.  u  and  13  are  section-  through  two 

neatly  adult  -pet  tnato/oa.     Fig.  14  is  a  nurse-cell  of  Littorina  n?t>ul<>sa  still  attached 
to  the  \\all  of  tin-  tCStia;  the  <talk  wa<  about   tO  !•«•  severed   iu-t  lielow  tin-  d> 
eiating  nucleii-. 


326  EDWIN    E.    REINK!.. 

rounded  the  centrosome  remain  at  the  posterior  end  of  the  sper- 
matozoon. 

The  species  of  Littoritia  in  which  have  been  found  a  free  nurse- 
cell  to  which  the  spermatozoa  are  attached  are  L.  an^ulifera,  L. 
nebulosa  and  L.  rndis.  The  first  two  species  were  studied  at 
Port  Royal,  Jamaica,  along  with  Strombus  bitnbcrcnlatits,  and 
they  too  were  identified  by  Dr.  Pilsbry. 

If  the  sperm-ducts  of  L.  nebulosa  or  L.  rudis  are  ruptured, 
the  contents,  when  diluted  with  sea-water,  will  appear  under  the 
microscope  as  a  great  number  of  spheres  to  each  of  which  is 
attached  a  tuft  of  spermatozoa.  The  spheres  are  nurse-cells 
composed  of  vacuolated  yolk  bodies  and  a  degenerate  nucleus 
(Fig.  5).  The  nurse-cells  of  L.  angulifera  differ  from  those  of 
the  other  two  species  in  that  here  the  yolk  bodies  arc  not  vacuo- 
lated and  they  partly  enclose  a  long  thick  cytoplasmic  rod  to 
one  end  of  which  are  attached  the  spermatozoa.  In  all  three 
species  only  the  perforatoria  and  possibly  the  tips  of  the  heads 
of  the  spermatozoa  are  inserted  into  the  cell. 

The  first  movement  to  be  seen  is  a  rhythmical  and  uniform 
beating  of  the  tuft  of  spermatozoa  which  sends  the  nurse-cell 
rapidly  forward.  Later  the  spermatozoa  beat  independently  and 
the  tuft  spreads.  Very  frequently  the  spermatozoa  of  one  nurse- 
cell  become  entangled  with  those  of  another;  in  that  event  the 
nurse-cells  are  drawn  together  and  held  by  an  agglutinous  sub- 
stance forming  the  pabulum  in  the  cell  into  which  the  spermatozoa 
are  inserted.  In  this  way  a  great  many  nurse-cells  are  drawn  to- 
gether and  from  such  a  mass  the  spermatozoa  may  later  be  seen 
protruding  on  all  sides  and  beating  iv.nularly  like  cilia.  This 
beating  of  the  spermatozoa  will  continue  for  several  hours. 

In  case  a  nurse-cell  has  not  become  entangled  with  others,  the 
spermatozoa  soon  free  themselves;  their  heads  become  further 
and  further  separated  from  the  nurse-cell  until  the  attachment 
is  completely  lost.  A  sperm  thus  freed  is  seen  to  h.i\c  a  long, 
thin  perforatorium  behind  which  is  the  head;  tin-  latter  passes 
almost  imperceptibly  into  a  very  long  tail  (Fig.  3).  After  tin- 
spermatozoon  has  been  swimming  about  in  the  wau-r  for  a  time 
the  perforatorium  becomes  swollen;  this  is  what  has  usually  been 
figured  as  the  sperm  head.  The  whole  process  can  be  seen  to 
better  advantage  in  Fasciolaria. 


DEVELOPMENT    OF   APYRENE    SPERMATOZOA.  ^27 

The  nurse-cells  develop  from  large  cells  which  are  attached 
to  the  walls  of  the  testis  by  a  long  stalk.  These  cells  closely 
resemble  the  apyrene  spermatocytes  of  Strnnifms  except  that  they 
lack  the  pronounced  centrosome  of  the  latter.  The  only  indica- 
tion of  -uch  a  structure  in  the  case  of  L.  ncfntlosa  is  a  series  of 
fiber-  running  up  one  -ide  of  the  cell.  Thi-  later  di-appcar- 
ancl  probably  forms  the  portion  of  the  cell  to  which  the  sper- 
m.iio/oa  are  attached  Fig.  141.  In  L.  angulifera,  in  the  early 
stages  of  the  nur-e-cell,  tin- re-  i-  a  darkly  -taining  body  lying 
in  the  c\topla-m  \\hich  grow-  to  form  the  rod  -poken  of  above; 
thi-  al-o  may  !>»•  of  a  run  manual  origin.  The  formation  of  the 
\olk  bod  it--,  i-  much  thr  -ame  as  that  of  thr  bodir-  dc-rri!  n-.l  in 
Strnnilms.  Tlu-y  diffrr.  ho\\r\rr.  in  that  huv  the\-  reach  vi  much 
larger  -i/<-  and  lliui  fragment  into  t\\<>  or  more  parH.  Coincident 

\\  itli  this  cytoplasmic  dit'fen-ntiation  the  nucleus  undergoes  partial 

.-neration;   it   -iniph    becomes  m.T<-  and  more  vacuolated  but 

lle\  er  '  '  'IllpleteU    d  i  -a  p]  lea  I'-. 

Before  ihe  nur-e-cell  ha-  reached  it-  full  di-\-elopiiu-nt  it  loses 
it-  connection  with  the  \\all  and  moves  into  the  lumen  of  the 
testis.  Mere  I  lie  -permato/oa  become  attached.  The  nur-e-cell- 
IniK  lion  as  -iii-h  in  the  -perm-duct-.  Section-  ot  /..  rmlis,  made 
from  a  -pecimen  killed  in  Kebruar\  .  -ho\\cd  the  nur-e-cdl-  in 
the  -perm-duct-  to  be  in  a  more  or  le—  iU-])lete.l  comlitiou. 

LITE  1<A  PURE    CITED 

Brock. 
"87     LJebei    die     :"i>i»-lt'''i    S|><TIH.U.I/O.-II    <-in:  luer. 

'     J.lllli...    H.I.    2. 

Meves,  Fr. 

"03      IVI'ri  oil]  uii'l  >M  .ii-n  uiul  iibcr  ihr--  Kiit-ti-lnin.;.  nach 

iiiiiK'-n    an    /'ji':«//«ij    uiul    I'y^iri.i.       Arrhiv    I.    inikr.    An.it.    uml 

'.i. 
Kuschakewitsch,  Sergius. 

'n      I  l-:m\vi.  killing  '!i-r  >i'i-iiiiii-n  bei  Conns  mfJit<rninfns  Brug.  uml 

\',-r>n,-tns  gi'i'(J5  Biv.     Biol.  Centralblatt,  Bd.  31. 


CERTAIN  MEANS  BY  WHICH  STARFISH  EGGS  NATU- 
RALLY RESISTANT  TO  FERTILIZATION  MAY  BE 
RENDERED  NORMAL  ANT)  THE  PHYSIOLOG- 
ICAL CONDITIONS   OF  THIS  ACTION. 

RALPH  S.  LILLIE. 

(From   the   Marine   Biological  Laboratory,    Woods   Hole,   and   the   Physiological 
Laboratory,  Department  of  Zoology,  University  of  Pennsylvania.) 

Last  summer  at  Woods  Hole,  while  investigating  the  influence 
of  ether  and  other  anaesthetics  in  protecting  the  unfertilized  eggs 
of  starfish  against  the  cytolytic  action  of  pure  isotonic  sodium 
chloride  solution,1  I  observed  that  toward  the  end  of  the  breed- 
ing season  the  eggs  often  proved  .abnormally  resistant  to  this 
solution,  withstanding  in  some  instances  exposure  of  more  than 
three  hours  to  0.5577*  NaCl  without  losing  the  power  of  develop- 
ment on  fertilization;  in  normal  eggs  this  solution  typically 
causes  complete  cytolysis  and  coagulation  of  the  protoplasm  in 
two  hours  or  less.  Other  characteristic  abnormalities  of  be- 
havior were  found  to  be  associated  writh  this  abnormal  resistance 
to  salt  solutions.  Usually  a  large  proportion  of  such  eggs  failed 
to  undergo  maturation  in  sea-water,  and  of  those  which  matured 
only  a  small  proportion  developed  to  a  free  swimming  stage  on 
fertilization,  and  the  resulting  larvae  were  largely  abnormal.  Also 
the  mature  eggs,  if  left  unfertilized  in  sea-water,  frequently  failed 
to  die  and  break  down  within  the  usual  time,  but  remained  clear 
and  apparently  normal  in  appearance  for  an  unusually  prolonged 
period.  It  is  well  known  that  unfertilized  mature  starfish  eggs 
undergo  spontaneously  a  characteristic  cytolytic  alteration,  ac- 
companied by  a  darkening  or  coagulation  of  the  protoplasm, 
which  is  typically  complete  within  twelve  to  fifteen  hours  after 
deposition;2  in  contrast  to  this  behavior  a  considerable  propor- 
tion of  the  eggs  under  consideration  often  remained  clear  and 
uncoagulated  in  sea-water  for  twenty-four  and  in  some  cases  for 
forty-eight  hours.  These  several  peculiarities,  (i)  failure  of 

1  American  Journal  of  Physiology,  1912,  Vol.  30,  p.  i. 

2  Cf.  J.  Loeb,  Archiv  fur  die  gesammte  Physiologic,  1902,  Vol.  93,  p.  59. 


RENhKKING    RESISTANT    STARFISH    EGGS    NORMAL. 

maturation  in  a  large  proportion  of  eggs,  (2  i  failure  of  those  eggs 
which  did  maturate  to  develop  after  fertilization.  (3  marked 
delay  in  the  typical  post-maturational  cytoly-is  of  unfertili/ed 
eggs,  and  (4)  unusual  n--i-tance  to  the  cytolytir  action  of  >ah 
-olutioi:-.  \\ere  found  in  more  or  less <  "ii-tam  a--oeiation  with  one 
another  in  numer<ui>  lot-  of  ci^-.  All  nf  these  pcculiaritie-  are 
e\ideme  of  ,i  certain  inertia  or  rc-i-tance  to  change  in  the  «  gg 

•ail   to  react,  or  react    -lowly  and   imperfectlv 

to  condition-  \vhich  call  forth  a  definite  and  regular  re-pon-c  in 
normal    eggs.      Tin-    fact    of    their   concurrence,    as    \\ell    a-    their 
•T.il  natui  tS  that  -OUR-  -in;Je  -tructural  or  metabolic 

al. normality,  \vh«»e  general  i-ftect  i-  to  lo\\er  reactions  (  1.  .cit  ie- 
in  the  •  -tern,  i-  re-pon-iLlr  f,  ,r  all  of  tlu-e  peculiarities  of 

lieha\  ior. 

The  lurther  in<  idental  ol  .MTN  alion  \\a-  made  in  -e\ei'al  in- 
^tance-.  that  >uch  eggs  after  treatment  for  t\vo  or  three  hour- 
wit  h  -odium  chloride  -oluti<.n  containing  a  little  ether  or  chloral 

h\dra''          vered  to  a  considerabl<  e  the  power  of  develop- 

ing* to  a  normal  larval  'ii  fertili/ation:  /.  <  treated 

\ielded  more  numeioii-  ami  mote  acti\e  lar\.e  than  the  un- 
treated  of  the  -am«-  \»[.  fenili/ed  at  the  -ame  time.  Pure 

-odium  (  hloride  -olution  -ho\\ed  i(  ^imilar  though  K-—  marked 
action.  The  siirpri-in^  re-ult  thn-  ajipeared  that  treatment  \\ith 
-olution-  \\hich  are  markedly  injurioii-  to  norm.:  -na\  lnin^ 

a  certain  proportion  of  the-e  almormal  or  "over-ripe"  nto 

a  condition      as  te-ard-  |>o\\er  of  de\  i  lopnn  nt  <m  fertili/ation 
clo-ely  a]ipfoachin;4  the  normal. 

The  follouitu  icconl  will  illu-trate  the  al>o\  c-de-cril -ed  condi- 
tion^ in  detail. 

Juno  -MJ.  njii.      1:  !  imiii  ;i  ial«li.-  luiiiili'  •  -ii-h 

at    tO.OO    A  M.      A   lai>;<-    pr  r.i.iim-.l    p,  r  ui.iiii-iuly 

iimiKitiiii-  in  -i.i-uatcr  tuitli  int.  ->,  hut  ahout  half  un<l'-r\vcnt 

api'.urntly  normal  maturation.     ( >n  u-rtili/atinn  tat  3:30  P.M  v  ,.i   the 

a   n.ii-tiiin  of  one  per  cent. — lurninl   lila-tul.r.   nm-t  difl   in  --arly  clt-a 
-.   but   many  failed  In  cleave  or  even  t<  n-iiH-inlnan' 

unli-rtili/e.l    inattiu-  e.i;v;<  after   t\venty-l"i>ur   hour-   in   -i\i-uat<r    I'-niaiii'-d    i^r   th< 
ni"-t  part  clear  aiul  tran-lucent  with  no  sign  of  coagulation;  a  good  many,  !. 
slio\\.-,l    appai'-iuly    normal    coagulation,    while    other-    -Imwed    an    intei  inediat"- 
con.lition.     After  forty-eiv:ht   hours  nian>-  unfl•rtili^e<l   mature  ex-  -till   un- 

.  Ill.ltecl. 

Al-out    two  hours  after   removal  from   the  animals  tlie  unfertili/ed   e.u.u-   \>. 


330  RALPH    S.    LILLIE. 

placed  in  the  following  solutions:  (i)  pure  0.55™  NaCl,  and  (2-10)  o.55m  NaCl 
containing  anaesthetics  as  follows:  (2-4)  ether,  0.6,  0.45,  and  0.3  volumes  per  cent., 
(5-7)  chloral  hydrate,  0.6,  0.4  and  0.2  per  cent.,  (8-9)  chloroform,  one  sixth  and 
one  tenth  saturated,  and  (10)  ethyl  alcohol,  5  volumes  per  cent.  After  three  hours 
and  fifteen  minutes  in  these  solutions  the  eggs  were  transferred  to  normal  sea-water 
and  washed  free  of  the  anaesthetics  by  two  changes  of  sea-water;  spermatozoa  were 
then  added.  At  the  same  time  spermatozoa  were  added  to  the  untreated  eggs 
which  had  remained  in  sea-water.  The  result-  were  as  tollows:  of  the  control  eggs, 
fertili/ed  in  sea-water  without  treatment,  almost  all  died  before  reaching  the  blastula 
stage;  only  a  few  feeble  blastulae  (a  fraction  of  i  per  cent.)  were  found  on  careful 
search;  many  eggs  failed  to  cleave  or  even  to  form  membranes.  The  eggs  exposed 
to  pure  0.55/n  NaCl  also  formed  few  blastulae,  but  these  were  relatively  somewhat 
more  numerous  as  well  as  more  active  than  in  the  control;  the  eggs  treated  with 
0.55JW  NaCl  containing  0.45  and  0.3  vol.  per  cent,  ether,  especially  the  latter,  showed 
a  more  decided  improvement  over  the  control,  though  the  proportion  of  blastulae 
was  still  small.  The  eggs  from  the  other  solutions  showed  no  improvement. 

A  similar  result  was  observed  in  a  second  series  of  experiments 
with  eggs  which  showed  similar  peculiarities.  Eggs  treated 
for  three  hours  with  o.55w  NaCl  containing  0.3  vol.  per  cent, 
ether  gave  about  5  per  cent,  of  blastula,  while  of  the  control 
untreated  eggs  less  than  one  per  cent,  reached  this  stage.  Eggs 
similarly  treated  with  solutions  containing  a  higher  proportion 
of  ether  (0.75,  0.6,  and  0.45  vol.  per  cent.)  showed  no  improve- 
ment over  the  control.  In  another  series  eggs  exposed  for  3  h. 
45  m.  to  o.55ra  NaCl  containing  o.i  per  cent,  chloral  hydrate 
gave  considerably  more  blastulse  than  the  control  eggs. 

It  is  to  be  noted  that  the  improvement  in  the  developmental 
power  of  these  abnormal  or  resistant  eggs  was  produced  only 
by  the  pure  salt  solution  or  by  solutions  with  a  low  concentration 
of  anaesthetic.  The  concentration  of  ether  most  favorable  for 
retarding  the  cytolytic  action  of  0.55?;?  NaCl  is  considerably 
higher — from  0.5  to  0.6  vol.  per  cent.1  The  present  effect,  how- 
ever, is  not  due  to  a  simple  prevention  of  cytolysis;  the  improve- 
ment over  eggs  left  in  sea-water,  none  of  which  undergo  cytolysis 
within  the  time  of  exposure,  cannot  thu>  U  explained.  The  effect 
is  different  from  a  simply  protective  action;  and  since  it  seemed 
to  be  favored  by  weak  solutions  of  ether,  the  experiment  was 
tried  of  exposing  a  batch  of  similarly  abnormal  eggs  to  sea-water 
containing  0.3  vol.  per  cent,  rtlu T.  After  three  hours  the  eggs 
were  returned  to  normal  sea-water  and  fertilized.  Next  day 
it  was  found  that  the  great  majority  of  mature  eggs  had  formed 

i  Cf.  R.  S.  Lillie,  loc.  cit.,  p.  6. 


RENDERING    RESI-IAM     -TAKII-H    EGGS    NORMAL.  .VU 

active  and  \  i^op.u-  bla-tula-  and  gastrula-,  while  of  the  untreated 
control  >  _-  left  in  -ca-water  and  fertili/ed  at  the  -ame  time  as 
tin-  other-  It---  than  one  per  cent,  formed  larva-  and  the-e  \\en 
tcel.le  and  .iliiiormal.  It  thu-  appeared  that  the  abnormal  condi- 
tion \\hirh  render-  the  egg  inca]  >al  ile  of  re-ponding  fully  to  the 
tertili/i  .  :ioii  of  the  -permato/oon  mi^ht  In-  removed  by 
treatment  \\ith  sea-water  containing  ether  in  certain  concentra- 
tion-. which  are  con-ider.ibly  lower  than  tho-e  required  for 
typical  an.i-theiic  or  protective  action. 

What  are  the  condition-  of  thi-  effect?  In  a  recent  paper1  I 
ha\f  pre-ented  evidence  indicatm-  that  the  pr"iecii\c  anil 
an.  i  -tin  tie  a<  lion-  exerted  by  ether  and  other  lipoid-solvents  in 
certain  (  OIK  duration-  .in-  due  primariU  to  i  the 

pltuniii  iiif)nl>rnnt^  of  the  <  ell-  .  ,r  irritable  i-!einent-.  of  Mich  a 
kind  .1-  to  render  the-e  membrane-  more  n-i-tant  toward 
ncies  that  under  the  n-n.il  condition-  rapidK  imie.i-e  their 
|M  |-me.diilit\  :  cytolysis  ami  -t  imnlat  ion,  both  of  \\hit  h  di-pi-nd 
on  -IH  h  ii,  ibility.  an-  hence  clu-(  ke«l  or  |.re\eiited. 

I1'  in    the    re.idine--    \\ith    \\hich    the    |)erme.ibilit  \    i-   in- 

I  thus  involves  for  an  irritable  tissue  d«  d  irritabilit\  ; 

thi-  ellei  i   i-  (iroduced  by  \ariou-  salts,  of  ma^ne-ium.  and 

b\  ether  and  other  lipoid-solvent  anaesthetics  in  certain    not  too 

hi^hi  concentrations.  In  lo\\er  concentration-  it  ha-  been  ob- 
-i  i  \  ed  that  i-tlu-r  and  other  lipoid--i  >1\  ent-  frequently  hci-Jitcn 
irritability;7  .  •  .  expressed  in  term-  of  the  membrane  theory 
of  -ti  mu  la  tion.  they  incn-a-e  the  readme--  \\ith  \\hich  the  |n-rmea- 
bilit\  and  hence  tlu-i-lectric.il  ]iol.iri/.ition  of  the  pla-ma  mem- 
brane line1  change.  It  -eem-  clear  that  for  irritable  ti--ue- 

the  -tale  of  the  li]>oid-  in  the  pla-ma  membrane  largely  ilelermiiu  - 
the  readiiu—  \\ith  \\hich  change-  of  permeability-  -and  of  the 
dependent  electrical  polarization—  are  induced  by  external  agen- 
cies. Mijn  perme.  ition  of  the-  lipoid-  \\ith  a  lipoid--ol\  cm  like 
ether  a]>parenil\  often  facilitate-  -nch  change-  and  hence  in- 


K     -     I    illi         I":   '       HI  .'     ./•:;.         /'•    |    .    .'    ,\,   ]ijI-\  \"i«l.  JO.  P-  .i7-'- 

1  in  my  roeciu  | 
ill    .  I  '  P  .'.    \  •  '      -  -im-rul.u 

in  i)l  iiiariiu-  aninuils  n\~«  >li.«\v-  it;  r'.  .1;..  Hrtln  lOund  that  aloili"!  (o  5  |»  i 
in  -.-a-u.itri  i  ili-i-iilr.lly  in>  .  MMM--!  tin-  iin-i-hanii-al  irritability  of  tin-  i-i>l.iti--l  o-ntral 
l>"iti<>n  Hi  tin-  inrtlu  t.  ".  \ll.m-i7H-iiu-  Anatoinii-  uinl  I'liysi«ilo.i;i' 

1  -'(M-  I'-  359- 


332  RALPH    S.    LILLIE. 

creases  irritability;  the  presence  of  more  lipoid-solvent  renders 
a  change  of  permeability  difficult,1  hence  the  protective  or  an- 
aesthetic action;  while  concentrated  solutions  of  lipoid-solvents 
disrupt  the  membrane  and  produce  cytolytic  or  irreversible 
alterations  in  the  cells;  hence  such  substances  in  higher  concen- 
trations are  markedly  toxic. 

On  the  assumption  that  lipoid-solvents  influence  the  plasma 
membranes  of  egg  cells  in  essentially  the  same  manner  as  those  of 
irritable  tissues,  the  above  action  of  ether  on  abnormal  egg  cells 
becomes  more  readily  intelligible;  it  falls,  in  fact,  into  the  same 
essential  category  \vith  the  facts  just  cited.  There  is  a  close 
analogy  between  the  stimulation  of  irritable  tissues  and  the  initia- 
tion of  cleavage  in  egg  cells;  the  primary  or  critical  change  in 
both  cases  appears  to  be  a  temporary  and  reversible  increase  in 
the  permeability  of  the  plasma  membrane,  with  accompanying 
changes  in  the  electrical  polarization  of  the  latter.2  This  analogy 
suggests  that  the  irresponsive  condition  of  the  above  "over- 
ripe" starfish  eggs  is  essentially  the  symptom  or  expression  of  an 
abnormal  condition  of  the  plasma  membrane.  Apparently  the 
latter  has  in  these  eggs  become  abnormally  resistant  to  changes  of 
permeability;  hence  the  eggs  are  irresponsive  to  the  spermato- 
zoon (whose  primary  action  is  to  increase  permeability);  hence 
also  they  show  heightened  resistance  to  cytolytic  action — which 
also  depends  on  increase  in  surface  permeability;  this  is  shown 
by  the  slowness  with  which  they  undergo  the  typical  post- 
maturational  cytolysis,  and  also  by  their  increased  resistance  to 
pure  isotonic  sodium  chloride  solution.  If  this  interpretation  is 
correct,  the  favorable  action  of  weak  ether  solution  consists  es- 
sentially in  altering  the  plasma  membrane  and  rendering  it  more 
susceptible  to  the  action  of  permeability-increasing  (and  hence 
depolarizing)  agencies — i.  e.,  more  irritable,  on  the  above-men- 
tioned analogy  with  irritable  tissues.  Through  this  means  the 
plasma  membrane  is  restored  to  an  approximately  normal  condi- 

1  This  is  very  clearly  shown  in  the  larva  of  Arenicola;  cf.  the  paper  just  cited, 
p.  380  ff. 

2  I  have  discussed  the  probable  basis  of  this  resemblance  at  some  length  in  an 
earlier  paper  in  the  BIOLOGICAL  BULLETIN,  1909,  Vol.  17,  pp.  20  ff.     The  title  of 
Loeb's  recent  book,  "Entwicklunjjserregung  des  tierischen  Eies,"  also  emphasizes 
this  analogy. 


RENDERING    RESISTANT    STARFISH    EGGS    NORMAL. 

tion  of  responsiveness;  the  sperm  then  exhibit-  its  normal  action. 
It  i-  evident  that  this  hypothesis  also  implies  that  the  other 
change-  in  the  egg  e\pre--ive  of  increased  permeability  should, 
after  the  etht-r  treatment,  also  follow  an  approximately  normal 
eotir-e.  This  in  in  ta<t  tl  as  regard-  the  post-maturational 

cytolysis;  thi-  change  i-  delayed  in  the  above  abnormal  eggs,  as 
already  de-cribed  :  but  it  i-  found  to  t.ike  place  in  a  normal 
manner  in  the  ether-treated  nnfert  ili/ed  eggs.  The  follo\\in- 
de-( Tipn'oii-  will  illustrate  both  of  the-e  effe< 

It  -hould  lir-t  be  noted  that  tlie  •  to  which  .1  •nor- 

mally re-Utant  to  fertili/ation  may  be  rendered  normal  by  the 
el  her  t  real  men  t  i-  variable.  In  -<>me  of  my  la-t  -u  miner'-  experi- 
ment- the  difference  between  the  ether-treated  and  the  untreated 

-  of  tin-  -aine  lot  \\,i-  -lijn;   in  other-  the  contra-t  \\a-  mo-t 

-inking.     The  degree   of   resistance   to   the   post-maturational 

cytolysis  i-  -imil.irly  variable.      In  general  it   \\a-  "b-er\ed  that 

-  \\hich   -ho\\ed   the  mo-t   pronounced  delay  in   the  oii-t-i   of 
ilii-   Kilter   change   wen-   mo-t    readily   brought    into  a   normally 

ponsive    condition  —or    "  rcju\  eiiatt -d  "     by    ether.      The    fol- 
louiiu  -.  ries  of  experiment-  \\ith   three  -ejiarate  lot-  of  -iar!i-li 
.ill   of   \\hich    failed    \\ith   a   feu    exceptions   1"  de\elop   t..  .1 
bla-tula  MI  -ini|'le  fertili/ation  \\ithoiu  ether  treatment 

illu-lrale-  thi-  \ariability,  a-  \\ell  a-  the  e.  n  relation  bi-tueeii 
i|ela\-  in  the  |io-t-mat  nrational  (\toly-i-  and  the  po— ibility  of 

i\« nation1  b\-  the  ether  treatment. 

JlMH-   3'1,    I(;I  I.         I!  lotS  Of   <  .  .          A,  B,  (  •!)- 

1  ,.i  tli<-  mix  .     Al't'-r  n-iiiaiiiinn  alxmt  mi--  .m<l  .1 

hall   In-ill-   in   ii.-iiii.il   -c.i-\va:  .-\\   Jut   \\cn-   tr.e 

r.-nt. lining  "..i  vol.  i"-i  n-nt.  rtlu-r.       i  kept  in  Miiall  ti>;litl\-  n-rknl 

il.i-k-        •.      :  [  hour  and  xo  minutes  in  thic  n  p.nt  m  tlu-i-xi;- \M-I             •  :n-«l 

Iti-in  rarli  !l.i-k  to  iii-nnal  -i-.i-\\.itrr  in   :  -r  \\.i-liini;  tin-  • 

ir<'in   cth'-i    -pci  in. it"/' '.i   \\iic   ail' It-il.       1  :    tin-  <'KX-   in   <  .u'li   ll.i-k 

-iinilail>  tian-ii-rri-':  1  a  quarter  ln-urs 

in  tin-  i-tli'-r  M-lutimi.      1  •  i  •  -n'li  I"t  t:  -  a  fertilized  tontrol  c<-n-i-ting  of  eggs 

\\hii_h  had  lain  untrraU-il  in  sea-water  for  about  2  ln-ur>  and  45  minutes  before 
fertilization. 

1  I  use  this  term  because  of  the  analogies  it  suggests.  The  eggs  are  in  fact 
brought  by  the  ether  treatment  into  a  condition  which  is  characteristic  of  eggs 
pii-'lueeil  in  the  earlier  portion  of  the  reproductive  cycle.  The  production  of  \\\ 

ant  eggs  like  the  above  occurs  late  in  the  breeding  season,  and  the  phenomcn-  <n 
bears  certain  analogies  to  senescence.     See  below,  page  345- 


334  RALPH    S.    LILLIE. 

1  he  characteristics  and  behavior  of  the  eggs  from  tlu-sr  three  lots  were  respec- 
tively a-  follow-: 

LOT  A. — The  great  majority  oi  these  eggs  fail  to  maturate.  A  small  proportion 
undergo  apparently  normal  maturation 

Unfertilized  Eggs. — 22  hours  after  removal  almost  all  of  the  mature  eggs  show 
the  typical  opaque  and  coagulated  protoplasm;  i.  e.,  post-maturational  cytolysis 
appears  normal. 

Fertilized  Eggs. — Condition  ca.  20  hours  after  fertilization. 

1.  Untreated  (Control)  Eggs. — Many  of  the  immature  eggs  have  typical  fer- 
tilization membranes;  but  are  otherwise  unchanged.     The  few  mature  eggs  are 
mostly  dead;  only  one  abnormal  blastula  was  found. 

2.  Ether-treated  Eggs. — (a)  Exposed  i  hour  10  minutes.     Four  or  five  blastulse 
are  found  in  some  hundred  eggs;  little  difference  from  control,     (b)  Exposed  .•; '  t 
hours.     Little  or  no  improvement  over  control;  a  few  blastula.1  as  in  (a). 

LOT  B. — Most  of  these  eggs  remain  immature,  but  about  20  per  cent,  undergo 
apparently  normal  maturation. 

Unfertilized  Eggs. — 22  hours  after  removal  from  the  animals  most  of  the  mature 
eggs  are  opaque  and  coagulated,  but  in  many  the  coagulation  is  less  advanced  than 
in  normal  eggs,  and  in  some  the  protoplasm  remains  semi-translucent. 

Fertilized  Eggs. — Condition  ca.  20  hours  after  fertilization. 

1.  Untreated  (Control)  Eggs. — Almost  all  of  the  mature  eggs  are  dead.     Many 
immature  eggs  have  fertilization  membranes.     Only  two  abnormal  blastulae  are 
found  in  several  hundred  eggs. 

2.  Ether-treated  Eggs. — (a)    Exposed    i    hour   10   minutes.     Improvement  over 
the  control;  a  large  proportion  (about  one  third)  of  the  mature  eggs  have  formed 
blastulae,  many  of  which  have  begun  to  gastrulate.      (b)   Exposed  3*4,  hours.     Also 
shows  a  marked  improvement  over  the  control,  but  the  larva?  are  fewer  and  less 
active  than  in  20. 

LOT  C. — In  this  lot  of  eggs  the  majority  show  normal  maturation,  though  a  few 
remain  immature. 

Unfertilized  Eggs. — After  22  hours  in  sea-water  most  of  the  eggs  are  more  or  less 
coagulated,  but  the  degree  of  opacity  is  distinctly  less  than  in  normal  eggs,  and  a 
considerable  proportion  remain  translucent — almost  like  freshly  shed  eggs. 

Fertilized  Eggs. — Condition  ca.  20  hours  after  fertilization. 

1.  Untreated  (Control)  Eggs. — Nearly  all  are  dead.     Most  have  membranes  and 
show  evidence  of  having  cleaved  or  fragmented,  but  many  have  failed  to  cleave  or 
even  to  form  membranes.     A  small  proportion  of  eggs  have  formed  larvae  some  of 
which  appear  normal:  the  larvae  though  few  are  more  numerous  than  in  the  controls 
of  A  and  B. 

2.  Ether-treated  Eggs. — (a)  Exposed   i  hour  10  minutes.     Striking  contrast   to 
control.     Almost  all  of  the  mature  eggs  have  formed  active  larvae,  many  in  tin- 
early  gastrula  stage  and  swimming  at  the  surface  of  the  water,     (b)  Exposed  .i '  i 
hours.     Here  also  the  majority  of  eggs  form  larva;,  but  these  are  largely  abnormal, 
and  relatively  few  gastrula?  or  surface  swimmers  are  present. 

The  power  of  development  after  fertili/ation  is  thus  ^really 
increased  after  ether-treatment  in  Lots  B  and  C,  l>ut  not  in  Lot  A . 
The  mature  eggs  of  Lots  B  and  Cshow  marked  delay  in  the  post- 
maturational  cytolysis;  in  Lot  C  this  delay  is  greater,  and  the 


RENDERING    RESI>TANT    STARFISH    EGGS    NORMAL.  $35 

action  of  the  ether  is  correspondingly  more  favorable,  than  in  Lot 
B.  I  have  already  presented  evidence  that  eggs  showing  this 
abnormal  behavior  are  characterized  by  the  posse— ion  of  hyper- 
re-i-tant  plasma  membranes.  It  should  be  noted  that  variations 
in  the  degree  of  resistance  of  thi-  membrane  occur  regularly  in 
normal  egg-.  Various  fact-  indicate  that  the  process  of  matura- 
tion i-  constantly  associated  with  a  change  in  the  properties  of 
tin-  pla-ma  membrane.  Thi-  i-  shown  by  tin-  fact  that  mature 
eggs  undergo  cytoly-is  in  0.55/77  Na('l  solution  more  rapidly 
than  immatu:  :'  al-o  b\  the  f.tcl  that  contact  with  sperma- 

tozoa  .mil  \arioii-  tomi-  "I  artificial  treatment  cause  the  -epara- 
lioii  of  the  -urfact  -film  of  ma  tun-  eggs  in  tin-  f"rm  of  a  fert  ili/at  ion 
membrane,  ;t  change  indii  at  ing  a  -.u]K-rtii-ial  cytolylic  or  pi-rme- 
abilii\  -int  rca-iir^.  acti-m;  \\hili-  immatiin  .  ggs  ire  not  normalK' 
t-i  thi-  i  lian  Thi-  dil'fi -rnn v  brt \\i-ni  immaturr  and 

eggs    i-   .1    foil-taut    or    ph\  -iolo-ic.il    Iraturr    in    the    lite 
hi-tor\    of  th.  Tin-  diltrrriifr  lu-turcii  normal  .  ggs  and 

tin-  iv-i-taut  i-gg-  undi-r  fon-idt-ratiou  i-  in  man\   rr-prc  t-  similar. 
I  or  5Om<    reason  tin-  maturation  profr—  tail-  to  briu-  tin 

into  the  normalU  sensitive  fondition  in  \\hifh  tin-  permeability 

of    tin-    membrane    i-    readily    imiv.i-ed.      1  lenee    lert ili/at ion    i- 

impi-rfeci.  e\en  fertilization-membranes  failing  to  lorm  in  -ome 
cases;  in  other-  membrane-  are  formed  and  cleavage  be- in-,  but 

the  latter  is  i  harai  teri-t  iealK'  irregular  and  tail-  to  proteed  tar. 
According  to  thi-  \ie\v  the  failure  of  de\elopmeiit  i-  due  not  [<> 
defectixe  or-a  ni/al  ion  of  the  ]  n'ot.  .pla-m.  but  -impK'  to  the 
existence  of  an  abnormal!)  re-i-tant  pla-ma  membrane.  The 
action  of  ether  consists  iu  re-toriu-  the  membrane  to  it-  normal 
tondiiioii.  Tlu-  re-pon-e  to  the  -permato/ooii  then  becomes 
normal. 

The  experimental  i-videnre  in  favor  of  this  h\  pothe-i-  < -oii-i-t- 
at  prc-cnt  simply  in  the  fact  that  such  abnormal  e;.;--  are  rendi  n  d 
normal  b\  ether  treatnu'iit  not  only  in  regard  to  their  re-pon-e 
to  tertili/ation.  but  also  in  ic-ard  to  the  rate  and  character  of 

'  R.  S.  I  illir.  .\>ni-rifini  Journal  ><f  1'h y--:  >\2.  Veil.  — . 

i   :      1     I  oeb's  i-\i'i-iii)i«-nt~  .'ii   tli  •     \-tfrina,   I  "niv.-i  -ity  of   California 

l'ul.li> Mti.'ii-.  I'liy-inlniLty.  i<;(>5.  \'nl.  J.  p.  i  ^,.    Sp«-r  inato/,,a  ami  ai  tifu  ial  iiic-tnl.' 
II.IIUMU  may  linurvrr  \  -\jiiial  iiH-nihniin--' in  alninrmal  immature 

(   •     J(  •••'  .    '  ft-ritni'tilal  /  [908,   \    •!.  5.   p.  _)"7. 


336  RALPH    S.    LILLIE. 

the  spontaneous  post-maturational  cytolysis;  i.  e.,  they  undergo 
complete  coagulation  within  eighteen  hours  or  less,  precisely  as 
do  normal  eggs.  Whether  the  resistance  to  cytolysis  by  salt 
solutions  is  also  decreased  I  have  not  yet  determined;  but  the 
decrease  in  the  resistance  to  the  post-maturational  cytolysis— 
a  change  supposedly  due  to  the  action  of  certain  protoplasmic 
oxidation  products  upon  the  plasma  membrane1 — is  clear  evidence 
that  the  membrane  has  been  brought  into  a  condition  more 
nearly  approaching  the  normal .  The  experiments  to  be  described 
show  that  a  return  of  the  normal  responsiveness  to  the  spermato- 
zoon is  closely  correlated  with  a  return  of  the  normal  behavior 
with  respect  to  this  spontaneous  oxidative  cytolysis.  In  other 
words,  the  plasma  membranes  of  the  ether-treated  "reju- 
venated" eggs  undergo  breakdown  in  the  manner  and  at  the 
time  characteristic  of  normal  eggs. 

The  following  record  gives  the  description  of  two  typical 
experiments. 

July  6,  1911.  Eggs  were  removed  at  11:00  A.M.  from  two  lots  of  starfish,  A 
and  B.  In  both  lots  a  good  proportion  of  eggs  underwent  apparently  normal  mat- 
uration. Eggs  from  each  lot  were  divided  into  two  portions.  One  portion  re- 
mained in  sea-water;  and  about  four  hours  after  removal  from  the  animals  part  of 
these  eggs  were  fertilized;  the  rest  remained  unfertilized.  The  other  portion  was 
transferred,  two  hours  after  removal,  to  sea-water  containing  0.3  vol.  per  cent, 
ether;  in  this  solution  they  remained  for  one  hour  and  thirty-five  minutes;  they  were 
then  returned  to  sea-water;  to  part  of  these  eggs  spermatozoa  were  added,  the  rest 
remained  unfertilized.  The  ether-treated  and  the  untreated  eggs  were  fertilized 
at  the  same  time.  The  two  lots  -4  and  B  were  treated  alike  so  far  as  possible.  The 
results  of  these  experiments  were  as  follows: 

LOT  A.     The  following  was  the  condition  of  the  eggs  ca.  22  hours  after  removal: 

1.  Untreated  Eggs,     (a)    Unfertilized. — Most   mature  eggs  are  coagulated   hut 
to  a  varying  degree;  some  are  only  slightly  darkened,  and  in  a  fair  proportion  the 
protoplasm  remains  semi-translucent. 

(b)  Fertilized. — All  of  the  mature  eggs  have  formed  membranes  and  most  have 
undergone  cleavage  or  irregular  fragmentation;  but  many  remain  uncleaved;  no 
blastulae  are  present. 

2.  Ether-treated  Eggs. — (In  0.3   vol.   per  cent,   ether   from   i.oo  to  2.35    P.M.) 

(a)  Unfertilized. — All  mature  eggs  are  completely  and  uniformly  coagulated; 
there  are  no  partly  coagulated  or  semi-translucent  eggs. 

(b)  Fertilized. — Decided  contrast  to  ib.     Most  eggs  are  dead,  but  among  these 
there  are  no  uncleaved  eggs;  numerous  blastulae  and  gastrulse  are  present,  many 
swimming  at  the  surface. 

LOT  B. — (The  condition  of  the  eggs  ca.  22  hours  after  removal.) 

1  Since  the  change  is  greatly  retarded  in  oxygen-free  or  cyanide-containing  sea- 
water.  Cf.  J.  Loeb,  Archiv  fur  die  gesammte  Physiologic,  1902,  loc.  ,  it. 


RENDEKIN".    RH-I-TANT    STARFISH    EGGS    NORMAL.  33J 

1 .  Untreated  eggs. — (a)   Unfertilized. — The  degree  of  post-maturation  coagulation 
varies  here  as  in  Lot  A,  but  a  larger  proportion  of  eggs  remain  semi-transclucent, 
an<l  many  are  virtually  unchanged  in  appearance. 

/  ,rtilized.-  Mi  •  ggs  have  formed  membranes  and  cleaved  or  fragmented; 
.1  mmitii-r  form  membranes  but  fail  to  cleave.  A  \>-\\  feeble  blastula?  are  present — 
a  fraction  of  i  per  o-nt.;  no  -urtaoe  swimni- 

2.  l:th>-r-tr-->ileii  eggs. — (In  ».\  vol.  per  cent,  ether  from  i.oo  tn  2..;_s  P.M.) 

•lizeil. — Markt-d  .\    t"    i<i.     All    mature  eggs  are  completely 

CoaKiila1-  'apart  looking,  without  nu-rnhra:. 

/  -rtilizfJ. — A  large  nun  bla-iula?  and  gastrula?  are  present,  many 

-wimining  at  the  surface.     \'i  uni  leaved  eggs  are  present,  though  a  good  many 
died  in  •  LgC8.      M  '     - 

The  return   ct    tin-   normal   re-| » >n-i\  cue—   to  trrtili/atiini  and 
iiorinal   j)o\v(.-r  <>l   <U-\  »•!<  .pnn-nt    i-   \}\u<  .1— •  u-i.iu-il  \\ith   a   muni 

•  •I    ihr   iinnii.il   rate  <>t"  post-maturational  i-\t<il\-i-.     Thi^  iil>- 

SCrvation  \va-  made  in  -i\  "in  i.t'  nine  e\])eriini-nt-,  at  dilYrrein 
time-  and  \\itli  ditterent  l<«t-  ol  in  \\hiih  rtlu-r-t  real  nieiu 

led  tn  marked  Increase  in  the  proportion  <>|  eggs  under^oin^ 
ta\oral»le  de\  elopmen  t .  In  three  <>!  the  earlier  i-\[ierimein-  the 
lieha\  io|-  of  tin-  ether- treated  ii nter l ili/i  d  eggs  \\a-  imt  nl>-er\  ed  ; 
Inn  in  .dl  nl  the  BU  -  \\heie  luith  uli-er\  ation-  \\eie  made 

•  oiirurrenlly    tin-  •  «\  1 1  lal  imi    held.      A-  already    mentioned,    the 

decree  «.|    impr<  A  eineiil    ellei  led    |.y    the  ether   ha-   lueli    \aiialile, 

in  correspondence  with  the  def         f  abnormality  in  the <  In 

all  ol  the  <  asCS  in  \\hicli  the  improvement  \va-  derided,  a-  in  -i  H  ne 
ol  those  de-crilied  aluAe,  the  nntreati'd  mature  eggs  ;>ro\cil 
lar-^eK  refractory  touard  Imth  r\  tol\  --i-  .nid  iCrtili/alion  ;  \\hik- 
alter  the  treatment  \\ith  ether  the  eggs  -ho\\i-d  in  hoth  respects  a 
luhaxior  ,i|iproai-hini;  the1  imrmal.  In  three  other  1m-  ot  ab- 
normal eggs  treatment  \\ith  ether  had  no  appreciable  effecl 
1'itlu-r  in  accelerating  cytolysis  or  in  increa-in;^  the  proportion 
ol  l'a\orabl>-  de\i'loi>in.  ;  \\hile  in  <>i  •  in  which  a  con- 

siderable   proportion    ol     untreated    CggS    dc\eloped    l'a\orably 
about    one    third    furmini;    larxa—  tin-    ether-treati'd    t-y^>    WCT( 
~ome\\hat    /t.vv   ta\orable   than   the  untreated ;   in   thi-  -i-ries   the 
unfertili/ed   eggs,   both    tn.\itcil   and    untreated,    -ho\\ed   an   ap- 
pan-ntly    normal    -poiuanemis   rytoly-i~.      I'mbably    the   plasma 
membranes    of    the-e    eggs   were    over-suxvpiible    rather    than 
under-susceptible   to   increase   of   permeability.     The    po--ible 
e\i-tenre   of   both   kinds  of   abnormalities   must    be   reco-ni/ed. 


338  RALPH    S.    LILLIE. 

Deviation  in  either  direction  from  the  physiological  norm  would 
presumably  impair  the  power  of  development. 

GENERAL  DISCUSSION. 

I  shall  now  discuss  somewhat  more  fully  the  general  physio- 
logical significance  of  the  above  abnormalities  and  their  rela- 
tions to  analogous  conditions  elsewhere.  The  above  condition, 
described  in  general  terms,  is  essentially  one  of  lowered  suscep- 
tibility to  agencies  which  ordinarily  call  forth  a  definite  re- 
sponse. Similar  conditions  exist  in  other  cells  and  tissues. 
There  are  also  cases  wrhere  a  tissue  is  normally  irresponsive  to 
certain  agencies  or  conditions,  to  which  however  it  may  be 
rendered  responsive  by  certain  forms  of  artificial  treatment.1 
It  seems  probable  that  in  all  of  these  cases  the  condition  of  the 
plasma  membrane  is  the  essential  factor  which  determines 
whether  the  cell  or  tissue  responds  to  the  agency  in  question  or 
not.  This  structure,  as  the  most  external  layer  of  the  cell, 
is  the  part  most  accessible  to  artificial  modification;  and  if  its 
condition  of  permeability  and  electrical  polarization  plays  the 
controlling  role  in  cell-processes  which  modern  investigation 
tends  more  and  more  to  indicate,  knowledge  of  the  means  by 
which  its  properties  may  be  altered  at  will  becomes  a  matter 
of  the  highest  importance  for  both  the  theoretical  and  the  prac- 
tical aspects  of  biology. 

The  abnormalities  under  consideration  appear  typically 
in  the  eggs  of  Asterias  forbesii  toward  the  close  of  the  breeding 
season.  Eggs  are  abundant  at  Woods  Hole  in  early  June.2 
During  the  greater  part  of  this  month  they  exhibit  as  a  rule 
a  normal  response  to  fertilization;  and  if  left  unfertilized  in 
sea-water  at  20°  they  undergo  the  above  described  coagulativc 

1  Instances  of  this  are  seen  in  various  phenomena  of  sensitization.     A  good  in- 
stance is  the  hypersensitiveness  to  contact  induced  in    frog's  skeletal  muscle  by 
isotonic  solutions  of  sodium  citrate,  tartrate,  sulphate,  and  certain  other  salts. 
Cf.  J.  Loeb,  American  Journal  of  Physiology,  1901,  Vol.  5,  p.  362. 

2  In  former  years  a  considerable  proportion  of  starfish  collected  in  August  and 
September  have  yielded  numerous  normal  eggs.     Probably  these  starfish  \vi-re  of  a 
different  species  from  the  above — presumably  A.  rnl^nris.     During  the  last  few 
years  this  form  seems  to  have  become  rare  in  the  neighborhood  of  Woods  Hole, 
and  eggs  have  been  difficult  to  obtain  later  than  June.     Two  species  of  Astri-ia*, 
forbesii  and  vulgaris,  are  recognized  as  occurring  in  thi-  region;  cf.  II.  L.  (.'lurk, 
Bulletin  of  the  U.  S.  Fish  Commission,  1902,  p.  552. 


kI-:.\I)KRIN<;    KK-I-TANI     -T. \KI1-H    EGGS    V'KMAL  ;  v  - 

eytoly-i-  within  12  to  15  hours  or  less.  T"\\,inl  the  end  of 
June  r;^>  become  fewer  and  more  variable  in  quality,  many 
fail  to  maturate  and  the  mature  e;^-on  fertilization  tend  to  dea\  e 
irregularly  and  largely  die  before  rearhini;  the  Mastula  Stage;  a 
vop.  inion  of  evji:-  -how  the  abiiormalil:  ribed  al><>\e; 

iin  n-a-ed    re-i-ian<  e   to   fertili/atii  >n   ami    t«>   r\  tolvnr  alteration 
••  iallv  character!-:  :<    and  indicates  that   the  pla-ma  mem- 
brane li.i-  In-come  aluiormalK-  reM-tant  l«>  change-  in  permeal 'ilit\\ 
I    have   dc-criliril    tin-   jieculiariiii  -  of    thes<     eggs    in   -uMirient 
detail    above,    and    ha\e    aln-ady    brietK"    <lixil—  ed     the    phy-in- 

cal    n.itnn-    of    the    al'iiormalitii •-.       I'he    failure    to    re-pond 
normally    in   |Crt  ili/alion   ln-r<ime-   intelli^il  >Ie  on    the   h\pot!; 
that  the  essential  Or  critical  event  in  the  initiation  of  tvll-di\  i-i«»n 
icmpor.ir\    and  r«  \ti~iblr  increase  in  the  ioni,-  ju-rnu-al  -ilitx 
Ol    tin-  pla-ma    nieml  ifaiu-.      ^u.  h    a    .1  ;n\ol\e-   a    il. 

in    the  rlt-i  tiital    pdlari/atimi   of   the   nu-mbrane.  and   it    appeal-- 
pr-'baHr       as    ill    the    allalo^ni-    (  a-r    of    mil-(le    and    ner\  i-       that 
tin-  c  hair^i-  <•!    polai  i/at  imi.   and    not    ihr   men-   in> 
mcabilitN   a-  -mil.  i-  tlu-  rritiral  i\i-nt  \\hich  initiate-  the  rh\th- 
mii-.il  -•  :   |'h\-iial  and  (  hemical  p-  :   \\hich  rKa\ 

i-   tin-   normal   c\|.|,  In  md»-r   that    il:<  -na\    n -pond 

he   '  "in. ui    and   entra:  Mialo/oi'm    in    a    normal 

manner,     it-     |>la-ma     membrane     mn-t    ha\c    a    '(ilain    delimit- 
ph\  lemii  al    i  on-iiuitii.n    -uch    that    the    -nb-tan  ins- 

mitttd   b\    thi  rmatOZOdn1   max    t  lit  •  t    an   im  rea-i-  of   perme- 

ability  uhiih   in   rate  and   derive  a|  >pro\imaie-  a   <«riain    norm. 
Thi-  imp!'  istance  \<  >  ehan^e  oi   jn-rn 

bilit\  :    if    thi-  resistance  i-  abnormal!'.  .   tin-  iv-pon-e  tail- 

OCCUr   or   i-   imperlet  t  ;   it    abnonnalU    1«.\\  .    the   -pi-rmaio/' -on 
effects    tOO    ^reat    and    tOO    la-iin^    an    increase    in    permeability 

1  M\-  tlif  lil- 

'.1.   ;iri-i;.. 
|K>l.iii/.itii>ii  MI  tin-  limitm.i:  n  :  i!i-.ii'; 

itimi^  in  :  rlnilui:  :i  "I   tin-  liiniiiiiK 

IIU-H.  :     .1    lllltllcr  UN. 

.    1  tn,-r.  J 

]••  7'  i- 

.iin    ly-in-lik. 
Iling  these  " membranol)  miul 

Il.ttllK-  "I    t:  .;i. 


340  KALPH    S.    LILLIE. 

resulting  in  early  death  or  cytolysis — just  as  occurs  in  eggs  sub- 
jected to  a  simple  membrane-forming  treatment  without  sub- 
sequent exposure  to  hypertonic  sea-water.1  Eggs  which  begin 
cleavage,  but  fragment  and  break  down  before  proceeding  far 
in  development,  possibly  belong  to  this  latter  class.  If  normal 
development  is  to  follow  fertilization,  the  properties  of  the 
plasma  membrane  cannot,  on  the  present  hypothesis,  depart 
\vidrly  from  a  constant  mean  or  physiological  norm. 

The  failure  of  the  above  eggs  to  respond  normally  to  fer- 
tilization, as  also  their  resistance  to  cytolysis,  is  thus  to  be 
regarded  as  the  expression  of  a  highly  resistant  condition  of 
i  In-  plasma  membrane.  The  latter  fails  readily  to  undergo 
the  increase  of  permeability  essential  to  these  changes, — probably 
because  of  abnormalities  in  the  nature,  state,  or  proportions 
of  its  chemical  constituents.  The  essential  effect  of  the  treat- 
ment with  ether  is  to  restore  the  normal  properties  of  the  mem- 
Inane.  There  is,  however,  no  reason  to  believe  that  this  effect 
is  specific  to  ether.  In  some  of  my  last  summer's  experiments 
a  similar  though  less  favorable  effect  was  produced  by  exposure 
to  isotonic  sodium  chloride  solution  and — in  one  case — to  a  o.i 
per  cent,  solution  of  chloral  hydrate  in  sea-water.  In  its  general 
form  the  problem  relates  to  the  essential  nature  of  the  modifi- 
cation which  these  substances  induce  in  the  egg,  and  by  which 
the  latter  is  brought  from  an  irresponsive  condition  into  one 
in  which  it  shows  a  normal  response.  Light  is  thrown  on  this 
problem  by  the  conditions  in  irritable  tissues  such  as  muscle 
and  nerve. 

A  close  analogy  exists  between  the  initiation  of  cell-division 
in  eggs  or  other  resting  cells,  and  the  response  of  an  irritable 
tissue  to  stimulation.  In  both  cases  the  initial  or  critical  event 
is  apparently  a  temporary  increase  in  surface-permeability, 
with  accompanying  changes  in  the  electrical  polarization  of  the 
limiting  membranes.  The  means  by  which  refractory  eggs  may 

1  The  second  part  of  the  treatment  appears  to  effect  a  return  of  tin-  permea- 
bility— which  has  been  increased  by  the  membrane-forming  treatment — to  the 
normal  (cf.  Amcr.  Jour.  Pliysiol.,  1911,  Vol.  27,  p.  289).  Godlewski  (Arthi, ••  liir 
Entu'icklung^mechanik,  1911,  Vol.  33,  p.  225)  has  independently  reached  a  similar 
conclusion  with  regard  to  the  essential  nature  <>t  the  eiiei-t  pn»liu-ed  by  the  hyper- 
tonic  sea-water. 


RENDERING    RESISTANT    STARFISH    EGGS    NORMAL.  34! 

he  rendered  normally  responsive  i-  thus  analogous  to  that  hy 
which  the  responsiveness  of  muscle  and  nerve  to  stimulation 
may  he  increased.  An  irresponsive  condition  in  living  muscle 

and  nerve  may  he  due  to  aiuc-the-ia.  fatigue,  electrotonus,  toxic 
act  inn,  or  other  changes  of  state.  The  stimulating  action  of 
-in, ill  doses  ot"  alcohol1  and  other  narcotics  during  fatigue  -ug- 
ts  an  analogy  which  i-  prohahly  not  without  -igniticancc.  It 
is  known  that  traces  of  various  lipoid-solvent  >uh-tance-  very 
.>  iH-rallv  increase  irritahility.  or  the  rate  of  spontaneous  activity, 

in  thcmo-t  various  cells  and  tissues  leucocytes,  cilia,  the  heart, 

eli  The     incrca-cd     re-pi.  Il-i\  ein  •--    nf     the    al»'\e    egg--    after 

etlier  treatment  is  a  phenomenon  ,,f  the  -ame  general  kind.  It 
\\oiild  appear  that  the  condition  of  the  lipoid-  in  cell-  detcrmine- 
llie  readme--  of  response  or  tin-  raU'  of  ^pontaiicoii-  acti\iiy; 
and  that  -light  impregnation  of  the  lipoid-  in  the  niemhrane  with 
.1  lipoid--ol\ent  tat  ilitatc-  :n  thi-  -trm  ture  the  alteration  which 
londition-  tin-  pcrmcahiliu  -im  i  •  ml  jiolari/ation-chairge  of 

stimulation. 

Certain  -alt-  markedly  incn  a-e  the  irritahility  of  mn-(  K    and 

IHT\  •          e.,  induce  sensitization.3     Treatment  with  sail  -olution- 

max  al-o  re-tore  taiigiicd  mu-i  le-  to  an  irritahle  condition. 
Frog's  -kelctal  inii-t  lr-  immer-ed  in  i-otonic  -odium  chloride 
-olntioii  and  made  to  contract  hy  successive  electrical  -timuli 
until  irre-]ion-i\  c  iiromi»tl\-  recover  irriiahilily  if  immer-ed  in 
i-oioiiir  -odium  hromide,  nitrate  or  iodide  -ohilion-.  Sodium 
iodide  re-tore-  irritahility  to  inu-i  le-  \\hich  ha\e  heen  fatigued 
in  -odium  hromide  or  nitrati-  -olntioii-,  hut  chloride  ha-  im  -uch 
action;  ;.  ,..  the  order  of  the  -alt-  cannot  he  reversed.  The 
re-torati\e  eltect  is  rapid,  and  evidently  depend-  t>n  a  >lli  ! 
action,  the  colloids  ot  the  nicmhraiie  heing  ap|iareiitl\-  hroiight 
into  a  condition  la\orahle  for  -timulation  -ajiparently  a  condi- 
tion of  iiuTea-ed  di>per-ioii.;  A-  alread\  ile-crihed,  sodium 
chloride  -ohition  ma\-  produce  an  analogous  increase  of  iv-pon- 

1  Tin-  .i>ti,.n  i if  ^inall  (iu;uuitir<  . ,1'  alo -h. >1  in  o>uiUi-i.i.  ting  fatigue  in  excised 
frog  inn  11  \\vll  .-Imun  ]>\    I  '      and  S.ilant.  A>n<r.  J.<:ir.  1'iiy  /.•:'.,    i 

.I.  s.  |>.  l>i. 

131  . 
s  Cf.  iM..tM<>i.-  i  .  p.m>-  ,uS  . 

Sv.-li\\aiv.  Aril;;:    >nr  die  gftaninili    l'<  .    :<>logie,  1907,  \'ul.   117.  p.  l6l. 


RALPH    S.    LILLIE. 

siveness  in  refractory  starfish  eggs.  Schwar/'s  observations,  as 
\\cll  as  my  own  with  abnormal  starfish  eggs,  thus  belong  to  that 
general  class  of  cases  in  which  the  responsiveness  of  cells  is 
increased  by  treatment  with  salts  or  low  concentrations  of  lipoid- 
solvents.  The  response  of  voluntary  muscle  to  various  forms  of 
chemical  stimulation  may  be  increased  by  brief  immersion  in 
isotonic  solutions  of  various  sodium  salts;  in  the  case  of  salts 
which  do  not  precipitate  calcium,  this  sensitizing  action  increases 
with  variation  in  the  nature  of  the  anion  in  the  following  general 
order:  Cl  •  :  Br  :  XO3  <  C1O3  <  CNS  and  I,  an  order  corre- 
sponding to  the  order  of  increasing  effectiveness  in  promoting 
colloidal  dispersion.1  Interpreted  in  terms  of  the  membrane 
theory,  these  facts  mean  that  the  readiness  with  which  the  plasma 
membrane  undergoes  increase  in  permeability  may  be  increased 
either  by  altering  the  general  state  of  the  colloids  in  the  mem- 
brane, or  by  slightly  altering  that  of  the  lipoids  alone. 

\Ve  conclude  that  the  effect  produced  by  salts  and  weak  ether 
solutions  in  increasing  the  responsiveness  of  refractory  eggs  t<» 
fertilization  is  comparable  with  the  sensitization  of  irritable  tis- 
sues by  these  substances;  also  that  in  both  cases  the  essential 
change  consists  in  an  increase  in  the  readiness  with  which  the 
plasma  membrane  undergoes  the  critical  change  of  permeability 
and  of  electrical  polarization. 

It  is  to  be  noted  that  the  resistance  of  eggs  to  fertilization  by 
foreign  sperm  may  also  be  decreased  by  chemical  treatment,  as 
Loeb  discovered  several  years  ago.2  Heightening  the  alkalinity 
of  the  medium  has  this  effect.  This  characteristic  and  striking 
effect  is  probably  an  expression  of  a  very  general  action  of  weak 
alkali.  Many  facts  indicate  that  slight  increase  in  the  alkalinity 
of  the  medium  usually  increases  the  readiness  with  which  the  per- 
meability of  cells  is  altered:  cell-division  is  accelerated,  the  irritabil- 
ity of  irritable  tissues  and  the  rate  of  activity  of  automatic  tissues 
is  increased,  the  cytolytic  action  of  salt  solutions  is  accelerated, 
and  in  unfertilized  eggs  membrane  formation  and  the  initiation  of 
cleavage  may  be  induced.3  It  remains  to  In  determined  whether 

1  R.  S.  Lillie,  Proceedings  of    the  Society  '•rimoitnl  ttiology  and  Mcdicin<-, 

New  York,  1910,  Vol.  7,  p.  170. 

•  J.  Loeb,  University  of  California  Publication*.  Physiology,  i<;<>3,  Vol.  i,  p.  i. 
3  Cf.  J.  Loeb,  Archiv  fur  die  gesammle  Physiologic,  1907,  Vol.  118,  p.  7. 


RENDERING    RESISTANT    STARFISH    EGGS    NORMAL. 

alkali  a>  wt-11  asethi-r  can  o\  ercome  the  resistance  of  over-ripe  star- 
ti-h  e^-tofertili/ation  by  their  o\\n  sperm;  also  whether  treatment 
\\ith  \\eak  Dilutions  of  ether  or  other  lipoid-modifying  -ub-tar 
as  \\ell  ,t~.  with  weak  alkali,  can  render  en  >— fcrtili/ati<  MI  po— ible. 
Thr  case  of  tin  .  of  hermaphrodite  animal-,  xvhich  are  irro 
-pon-i\e  i.i  -perm  from  tin-  -aim-  individual  but  not  t<>  that  of 
other  individual-.  may  po--ibly  belon-  in  p. in  to  the  prc-eiit 
•\  Morgan1  found  that  tin-  <  ggs  •  >f  (';<>;/./,  which  furni-h 
a  typii  al  in -tame  of  tin-  beha\  i<>r.  could  be  lertili/ed  b\  -perma- 
IO/.M  from  the  -aim-  individual  in  \\eak  -olution^  of  ether,  ani- 

inoni.i  .,r  ali  <.ho| ;  but  he  is  inclined  to  attribute  th<  to  tin- 

-liinulatin'<  action  of  the-e  -ub-tanct  -  on  tin-  -perm,  rather  than 

to  an  alteration  of  tl  In  one  experiment,  however    p.  147  . 

in   \\hich   the  -permato/.i.i  alone  \\ere   treated   \\ith  ether  before 
adding  t"  Hi'  tertili/ed.  \\hile  \\heii  both  . 

and    -perm    \'.  •    treated    the    percentage   .  if    fenili/ati"ii    \\a- 

hi'^h      in    om  -5    per   cent,   a-   c.nnpared    \\ith    4    ; 

I'hi-  (  \]ietimeiit    -i;  that    the  ether   produce-  it-  effecl    not 

mereh     by    increa-in^    the    motility    nt    tin-    -perm,    but    al-<>    b\- 
altering  the  t  ondition  .,)   the  egg,  as  in  tin  '  An- 

other intere-tiiu  :n  \\hich   the  •  rendered  refractoiA    tn 

;ili/ati"ii   ha-  r.-ieiitU    been  de-i  ribed   b\    (.odle\\-l  If  the 

-permato/.M  ..i  >  ;iidoi    -  ruts  are  mixed,  both 

are   toliml   alter  a    le\s-    minute-   to  ha\e  iiUll|ileU-|\    lo-l    the   po\\er 

o|  lertili/in^  tin  Eggs  left  <  \poM-d  t'  •  thi- 

M  organ ,  . > . 

/i  i.  \'..l.  u.  p.  . 

1  Tin-  -|»-i  :  n>tilily  in  -;  li.ivinn 

kin.u   ii  tin-  iimtility  un.!  mly  tli> 

•i  i~  nioililic'!  l   iiii-inliiaii"-l\  -in-  t"  tin- 

."llld  illl|i!>-  liri^lltdl' 

inrinl'i.iin-   ol    ili--   -i-«-i!!. 

I  iiintik1  .ii'tivity  —  (.  :hytliin  in  ti 

liilit>    ;uiil   |)"l.u.  nilitiiiiiim;  tin-  umvciin-i:: -.      Vari 

in   nciiii.il   iritili/.iti»n   t!  mutually 

in   a   ~.'iii'-\\h.it    -iinilar   inanin-r.   .in<l    that   a   i-yt-'lyt:  ibility-ii. 

I   liy  tin-  ri;v;  ii|i"ii   tin'  -IM-IIII  ,i-  \\'-!l  a-   liy   -|»-!in   U] 

Thu-i  tlu-  -pi-i  in  u-ually  .  iiiiivrnit-nt*  -•"ii  a:j  ..'t  with  •  ami 

i.ltrn  nnly  a  p.irtion  i-ntrrs  the  latt<  •  lytic  or  <li-intri;rati  .  tin- 

thiH  iiiiluati-«l.      Tli<-  I'la-ina  iiiciiihram-  ni   t  m   iniuht — as 

apparently   in    (ioilk-\v-ki'-   'Xp'-riim-.  ;itly   hy;  --.ant 

to  prcvi-nl  l\\\<  mutual  (.-ytnlytic  action  without  entir  i   motility. 


RALPH    S.    LILLIE. 

sperm  mixture  arc-  at  first  unaffected,  luit  in  the  course  of  half 
an  hour  they  become  so  modified  that  fertili/ation  with  normal 
Spharechinus  sperm  is  impossible.  The  egg  is  thus  deprived  of 
the  power  of  response  to  its  own  spermatozoa.1  Whether  this 
condition  of  irresponsiveness  (which  is  comparable  to  paral>>i>) 
may  be  removed  by  artificial  treatment  such  as  the  above  has 
apparently  not  been  determined;  but  from  the  analogies  with 
the  conditions  just  described  there  is  every  reason  to  believe  that 
this  would  readily  be  found  possible.2 

In  conclusion  I  wish  briefly  to  indicate  the  bearing  of  the  above 
observations  on  the  general  theory  of  pathological  alterations  in 
cells.  The  conclusion  that  many  pathological  conditions  have 
their  primary  origin  in  abnormalities  of  the  limiting  membranes 
of  cells  is  an  obvious  corollary  of  any  view  that  regards  such 
membranes — which  are  essentially  insulating  surface-films  of 
varying  ionic  permeability  and  electrical  polarization — as  largely 
controlling  the  rate  and  character  of  the  cell-processes.  If  stimu- 
lation depends  primarily  on  altered  polarization  of  the  plasma 
membrane  due  to  increased  ionic  permeability,  it  is  clear  that  a 
normal  response,  in  the  case  of  any  cell,  implies  a  definite  condi- 
tion of  the  membrane.  If  this  condition  is  permanently  altered 
the  cell  processes  inevitably  undergo  derangement,  and  patho- 
logical changes  follow.  Such  a  deranged  condition,  if  not  too 
far  advanced,  may  be  rectified  by  restoring  the  membrane  to  its 
normal  condition.  How  this  may  be  accomplished  is  illustrated 
by  the  case  of  the  abnormal  starfish  eggs  described  above.  It 
is  clear  from  the  cytolytic  effects  produced  by  many  toxine- 
that  they  cause  abnormal  increase  in  the  permeability  of  the 
membranes;  and  in  all  probability  their  destructive  action  is  in 
many  cases  directly  due  to  this  surface  action.  The  alteration 
caused  by  a  toxic  agent  may  consist  primarily  either  in  increasing 
or  in  decreasing  the  permeability  normal  to  the  membrane,  or 
in  altering  in  either  direction  the  readiness  with  which  the  latter 
undergoes  change.  Evidently  the  plasma  membrane,  as  an 

1  A  similar  and  reversible  effect  may  be  produced  by  treating  tin-  »-i;.u  with  cei  tain 
salt  solutions,  as  Mathews  and  Newman  sh<>\\<  years  ago  for  Fan  tin  I  us 
eggs;  cf.  BIOLOGICAL  BULLETIN,  1905,  Vol.  9,  p.  378. 

2  Evidence  that  this  change  is  in  fact  reversible  i*<  *rrn  in  the  effects  of  washing 
the  eggs  thoroughly  in  sea-wut<  i  alter  the  treatment  with  the  -perm  mixture.     A 
partial  return  of  respond  \ -c-ness  to  the  -pei  matn/oi'in   \\a-  observed   '  p-   -36). 


RENDERING    RESISTANT    STARFISH    EGGS    NORMAL.  345 

insulating — and  hence  semi-permeable — layer  on  the  integrity  of 
which  the  normal  composition  of  the  living  substance  depends, 
cannot  undergo  marked  and  prolonged  increase  of  permeability 
without  alteration  in  the  nature  and  proportion  of  the  cell- 
con-t  ii  nent-;  thi-  involve-  altered  chemical  organization  and 
exentual  derangement  of  the  cell-processes.1  In  most  of  the 
abnormal  conditions  considered  above  the  membrane  appears  to 
ha\e  nn<!'  the  oppo-iu-  kind  of  modification,  becoming 

abnormally  impermeable  and  re-i-tant  I"  changes  of  permeability. 
Such  a  condition  i-  e--entially  one  of  irre-p<  >n-i\  cue— ,  and  i-  in 
ense  pathological,  although  it  differ-  fnun  a  condition  of 
permanently  increased  pi •rmi-abilit>-  in  inxoKin-  no  loss  of 
material  fr»m  the  cell;  hence  tin-  po--ibility  of  rc-iorin^  the 
n«  ir ma  1  proper  tie-  of  the  cell  by  brin^in^  the  permeability  aiiain 
to  the  normal-  -hould  theoretically  1  ter  in  tin-  class  of 

cases  than  iii  the  other.2 

IVrhap-  the  coin  lit  imi  of  the  above  iv-i-tant  -tarti-h  eggs  i-  not 
properly  to  be  (ailed  pathological,  -ince  the  change  in  tile  pn>p- 
ertie-  of  tin-  eggs  lo\\ard  the  close  of  tin-  breeilii  •  >u  i-  \>vc- 

-ninabK  a  con-t.mt  one.  ami  hence  normal  in  a  ph\ -i»l<  njral 
sense.  I  in-  eggs  meivlv  bt  come  h>  perre-i-tant  to  ferl  ili/at  i<  >\\ ; 
/.  f.,  \\ilh  inci-ea-in^  a;^e  the  inetaboli-in  of  thi'  o\  arie-  mider^..  - 
alteration  lo  Kail  to  the  production  ,  ha\in^  more 

re-i-tant  membrane-  than  before.  The  cycle  of  e-^-|)roducti- >n 
>hortl>-  al'teruard-  COmes  to  a  clo-i-.  The  phenomenon  bear-  in 
Certain  respects  a  marked  re-cm blam  e  lo  -eiie-ceiice.  and  it-  con- 
dition-, max  throu  li^ht  on  the  ph\-iol"-\  (,f  ih,  latter  process. 
In  old  .i-e  the  irritable  dssues  became  le—  an<l  less  responsive, 

and    the   rate   of   mctab<>li-m    i-   corn  -p<  .ndin-ly   lo\\rred.      Irre- 
1  I  h.ui-  iludt  "ii  ;:  tations  at  Sdincwhat  ^rt-ati-r  k-n.uth  in  iu\-  i-arlirr 

i  in  I  hi-  jniunal.  I  <)•><},  \'<>\.  17,  p.  \<i-  i-t  seq.  I  In-  tiindann-iUa!  iinpi  PI  ! 

>.|  tin-  p. ut  uliiih  a!;  •  .I'nihraiu-s  play  in  pathological  pnio-—«-<  \\a- 

•  .1  l.y  /ani;K<-T.     Tin-  tollowing  quotation  will  ilhir-uai'  :  "  I  >i.-  n..niiah- 
t\-pi~.  !,•      r>  i  ini-.il'ilitat    ih-r    Mrinlnani-n    i<t    al->    \'"iau--i-tzung    dcr    norinalcn 
ii-tiinktioiu-n.       I)aurmil  v«-ran«li-rtu  IVrmeabilitat  ilt-r  Muniliraiu-n  hcdi-utet 
l'ath«.lfxi.\   path..U'.i;i~i  hen  Stoff\v<\ -h-i  1."  i-tc.:  Yierteljahrs#chrift  d.  Xaturf.   < 

h,   1906,  Vol.  51,  p.  4,^.     Cf.  also  ibid.,  1907,  1908,  and  the  other  papers  of 
Zangger  and  his  students,  e.^x-dally  Fn-i  and  Stoffel,  for  a  fuller  <l<  .  at  "f 

;ogether  with  experimental  data  bearing  on   the  relations  of  membrane 
changes  to  pathological  processes,  immunity,  and  related  phenomena. 

'For  further  discussion  of  this  subject  cf.  my  earlier  paper  in  American 
IIJKI,  Vol.  jr>.  pp.  nj  et  ~eq. 


346  RALPH    S.    LILLIE. 

spon>i\eness,  as  already  pointed  out.  implies  a  state  of  the  plasma 
membrane  in  which  changes  of  permeability  and  of  electrical 
polarization  are  produced  with  difficulty.  The  increasingly 
resistant  character  of  the  membranes  implies  altered  composition. 
Possibly  the  condition  in  over-ripe  eggs,  as  well  as  in  senescence, 
results  from  a  progressive  accumulation  or  adsorption  at  the 
phase-boundaries,  i.  e.,  in  the  membranes,  of  materials  which  for 
some  reason  are  not  readily  eliminated  by  the  organism.1  A 
^imilar  view  has  recently  been  expressed  by  Child.2  According 
to  his  theory  "senescence  in  nature  consists  physiologically  in  a 
decrease  in  the-  rate  of  metabolism,  and  this  is  determined  mor- 
phologically by  the  accumulation  in  the  cell  of  structural  ob- 
stacles to  metabolism,  e.  g.,  decrease  in  permeability,  increase  in 
density,  accumulation  of  relatively  inactive  substances,  etc." 
Minot's3  theory  that  senescence  is  the  expression  of  a  progres- 
--ively  increasing  differentiation  of  cells,  i.  e.,  of  an  increase  in  the 
proportion  of  separated  solid  structural  material,  bears  a  certain 
resemblance  to  this  view.  The  view  which  I  have  suggested 
above  is  distinctive  only  in  so  far  as  it  attributes  the  essential 
change  to  a  modification  of  the  membranes.  It  is,  theoretically  at 
leaM,  within  the  possibilities  of  physiological  science  to  prevent 
or  retard  this  accumulation  of  inert  materials  in  the  membranes 
and  so  to  delay  senescence.  Or  the  already  modified  membranes 
might,  it  not  too  profoundly  altered,  be  restored  to  a  normal 
condition  by  certain  forms  of  treatment.  The  simpler  the  metab- 
olism and  the  less  widely  differentiated  the  tissues,  the  greater 
would  appear  to  be  the  possibility  of  such  "rejuvenescence"  <>l 
the  organism  as  a  wrhole.  In  one  large  group,  tin-  Protozoa 
senescence  seems  not  to  be  an  inevitable  occurrence;  the  earlier 
interpretation  of  conjugation  as  a  process  whose  essential  role 
is  to  counteract  an  innate  tendency  to  senescence  has  been 
discredited  by  the  work  of  Calkins  and  Woodruff.  The  condi- 
tions in  Mcta/oa  differ  from  those  in  Protozoa  chielly  in  their 
greater  complexity,  but  probably  in  no  other  essential  respect. 

'Probably  certain  colloidal  (i.  e.,  iixliitu-iblr i   ,m<l  . -li. •mirully  n-tractory  by- 

l>i''xlucts  of  nn-uilinli-iii. 

2  Cf.  Child,  Ariliir  ftir  Enlwicklungsmechanik,   HJM.  Y<>1.  .u.  !>•  5,i7- 

3C.  S.   Minot,   "The  Problem   ol    Age,   Growth  .in<!    D.-.uli."      NYw    York  ami 

London,  1908. 


THK  MARIXK  BIOLOGICAL  LABORATORY 

FOURTEENTH  Ri  I-ORT 

FOR    Till     YEAR     I'll  I 

I.  I.I--I    MI-    Tki  5TBBS 

II.  Ac   :  •  iki-iiK  \  Il"N  S49 

III.  Bv-I.A 

I  \  l  i.  >  \  -  •  i.  i  i  -   k  •• 

V         I   ill       I   MUI •<    |.  IR'S     Kl!''  II 

Inti'i.lnriinn 

I.I  :  ! 

1  Mu-l«  '  l 

1  alnil.ir  \'ii-w  ..I  Attni'l.in   • 
4.    Sul>->  iil>in«  In-titilti    • 

-M  ....  Mi 

•     a 

1.   TRt  STI  ES 

l  \    OFFICIO 

I  .   K.  I  ii  i  11  l'iii\iT>ity  <  t'  Cl 

(.UMAX  A.  I'KI.U.  Assistant  /'.  !  Maim-. 

I  >.  r,i  \KIIY  ll«>\k.   Treasurer,  \<-\  I  ><-\..n-hi      -      et,  Boston,  Mass. 
Tu«»v   II.   Mosn.uMKKV,  JK..'   CU-rk  of  the  CV>r/>  I   'liMTMi- 

l\-nn>\  Iv.iniu. 

rO    SERVE    t  MIL    I  Ml  5 

II.  (.   I'.i  \M  ....  University  of  Wiscoi 

K.   A.   1 1  \KI-IU      Colunil)ia  I'niversity. 

\\ .    \.   Loc\  Northwestern  University. 

|  \.  ..i  i  -    I  I\.ii-ki-li-lli-r  In-tituu-  f«ir  Moli<  al   l\«--  .in  li. 

I",   r.   M  \i  i Johns  Hopkins  University. 

rge    I-    Mi'"Ui  Mi-M'iiri  Mi)ianit\il  (.ianlt-n.  .v  '  the 

Board. 

[..   L.   NUNN  'lYlluridf,  (".-In. 

|.»n\  c.   I'liii.iii's  .M')  B<T!;I-|-     5ti  ton,  Mass 

i  >. 

54- 


348  MARINE    BIOLOGICAL   LABORATORY. 


TO    SERVE    UNTIL 

CORNELIA  M.  CLAPP.  .  .  .Mount  Holyoke  College. 

V..   G.   CONKLIN  .........  Princeton  University. 

Ross  G.  HARRISON  .....  Yale  University. 

CAMILLUS  G.   KIDDER  .  .  .27  William  Street,  New  York  City. 
M.   M.  METCALF  .......  Oberlin  College. 

WILLIAM  PATTEN  .......  Dartmouth  College. 

JACOB  REIGHARD  .......  University  of  Michigan. 

W.   B.  SCOTT  ...........  Princeton  University. 

TO    SERVE    UNTIL    1  913 

S.  F.  CLARKE  ..........  Williams  College. 

CHARLES  COOLIDGE  .....  Ames  Building,  Boston,  Mass. 

C.  R.  CRANE  ..........  2559    Michigan    Boulevard..    Chicago,    III., 

President  of  the  Board. 
ALFRED  G.   MAYER  .....  Carnegie  Institution. 

T.   H.   MORGAN  .........  Columbia  University. 

ERWIN  F.  SMITH  .......  United  States  Department  of  Agriculture. 

E.   B.  WILSON  ..........  Columbia  University. 

TO   SERVE    UNTIL    1  9!  2 

M.  J.  GREENMAN  .......  Wistar  Institute  of  Anatomy  and  Biology. 

C.  W.  HARGITT  ........  Syracuse  University. 

H.  S.  JENNINGS  ........  Johns  Hopkins  University. 

GEORGE   LEFEVRE  ......  University  of  Missouri. 

A.   P.   MATHEWS  ........  The  University  of  Chicago. 

G.  H.  PARKER  .........  Harvard  University. 


II.   ACT  OF  INCORPORATION 

No.  U70. 

COMMONWEALTH  OF  MASSACHUSETTS 

Be    It    Known,    Thai    whereas    Alpheu-    Hyatt,    William    San  ford 
.•II-.   William  T.   "-id-wick,    Kdward   G.   C.ardiner,  Susan   Minn?-. 
Charles  Sed^uii  k  Minot.  SamiK-1  Well-.  William  G.  l'arlow,  Anna  D. 
Phillips  ami    1'.    II.    \'an   \'l«-ck   hav(  '•<!    them-eh  c-   \\ith   the 

intention   of   forming  a   Corporation   under   the   name  of   the    Marine 
liioln-jcal  Laboratory,  for  the  pur]  -tabli-hini;  and  maintaining 

a   lalior  nation   for  M  ientitic   -tudy  and   in\-  ion.   and   a 

Bi  h-i-'l  for  in-tructioM  in  !•!•  :id  natural   hi-t.>r\,  ami  have  coni- 

|,lied    \\ilh    the    ]>ro\  i-ii.n--   "I    the   Matnte-   •  I    thi-    Commonwealth    in 
Hich    Case    made  ami    ]  -n  >\  id>  d .  .i~    appears  from    the  i  i-rtil'u-ate   o|    the 

President,  Treasurer,  and  Trust  '     rporation,  duly  approved 

the  Commi  — ir.ner  o|   C,.r|ii  rations,  and  recorded  in  this  ot; 

\<>ic,  !'  .  I.  HENRI    P-.   PIERCE,  -         arj   . -i  the  Common- 

wealth  "i  Massachusetts,  •/"  /;.  .'.•'">'  '"-lt  --l'(1  -\-  Hy-nc  \\     S 

Stevens,  \\ .  'I.  Sedgwick,  E.G.  Gardiner,  S.  Minn     <     5.  Minor    - 

Well-.  \\  .  (',.   Farlow,  A.   1  >.  Phillips,  ami   I'..  II.  Van  Vle.-k.  their  a 
(  iate.-  and  MH  •  -  ani2(  d  ami  i  -laMi-hrd  as,  and 

hereby  made,  an  e\i-iim.;  Ci>r|ioration.  under  the  name  ->l  tin-  M  A  KIM: 
I',! i  .i.tM.i.   \|.  1.  \lioK.\l"K\  .  \\ith  the  |.o\ver>.  riijit-.  and  |>ri\  and 

-ul'ject  to  the  limitation.-,  duties,  and  re-tri<  tidi-,  \\hich  hy  law  a| 
tain   thereto. 

ll';/»;i-.vv   my  ol't'u  ial   -^nature  hereunto  -nh-t  i  ilied.   and    the  -eal   "I 

the  Commonwealth  of  Massachusetts  hereunto 'affixed,  thi-  t\\<-iuieth 

day  of   March,  in  the  year  of  OW  LORD  ONE  THOUSAND,  ElGHl    HUN- 

MKI  I'    AM'    lilt. II  1  V-l.It.ilT. 

lll.NRV   B.   1'IKUCI  . 
•r/iirv  c/  tilt-   C"»;>'  .'////. 

\l..| 


349 


III.    BY-LAWS    OF    THE    CORPORATION    OF 
THK  MARINE  BIOLOGICAL  LABORATORY 


I.  Tlu-  annual  meeting  of  the  members  shall  be  held  on  the  second 
Tuesday  in  August,  at  the  Laboratory,  in  Woods  Hole,  Mass.,  at  12 
i ''clock  noon,  in  each  year,  and  at  such   meeting  the  members  shall 
choose  by  ballot   a  Treasurer  and  a  Clerk,   who  shall   be,  ex  officio, 
members  of  the  Board  of  Trustees,  and  Trustees  as  hereinafter  pro- 
vided.    At  the  annual   meeting  to  be  held  in   1897,   not  more  than 
twenty-four  Trustees  shall  be  chosen,  who  shall  be  divided  into  four 
classes,   to  serve  one,   two.   three,  and  four  years,   respectively,   and 
thereafter  not  more  than  eight  Trustees  shall  be  chosen  annually  for 
the   term   of   four   years.     These  officers  shall   hold    their  respective 
offices  until  others  are  chosen  and  qualified  in  their  stead.     The  Direc- 
tor and  Assistant  Director,  who  shall  be  chosen  by  the  Trustees,  shall 
also  be  Trustees,  ex  officio. 

I 1.  Special  meetings  of  the  members  may  be  called  by  the  Trustees, 
to  be  held  in  Boston  or  in  Woods  Hole  at  such  time  and  place  as  nun- 
be  designated. 

III.  The  Clerk  shall  give  notice  of  meetings  of  the  members  by 
publication   in  some  daily   newspaper  published   in   Boston  at  least 
fifteen   days  before  such   meeting,  and  in  case  of  a   special   meeting 
the  notice  shall  state  the  purpose  for  which  it  is  called. 

IV.  Twenty-five  members  shall  constitute  a  quorum  at  any  meeting. 

V.  The  Trustees  shall  have  the  control  and   management  of  the 
a  flairs  of  the  Corporation;  they  shall  present  a  report  ot  its  condition 
at  every  annual  meeting;  they  shall  elect  one  of  their  number   Presi- 
dent and  may  choose  such  other  officers  and  agents  as  they  may  think 
best  :  they   may  fix  the  compensation  and  define  the  duties  of  all   the 
officers  and   agents;  and    may  remove,   them,   or  any  of   them,   except 
tln>-«    chosen    by   the   members,   at   any   time;   they   may   till    vacancies 
Oi  •  Hiring  in  any  in. inner  in  their  own  number  or  in  any  of  the  offices. 
They  shall  from  time  to  time  elect  members  to  the  Corporation  upon 
such  term-  and  (onditions  as  they  may  think  best. 

VI.  Meetings  of  the  Trustees  sh.ill   be  called   by   the    President,   oi 
by  any  two  Tru-'  :id  the  Secret. ir\    shall  gi\c  notice  thereof  by 
written    or   printed    notice    sent    to   each    Trustee    by    mail,    postpaid. 
Seven  Trustees  shall  constitute  a  quorum  for  the  transaction  of  busi- 

350 


BY-LAWS   OF   THE   CORPORATION.  351 

ness.  The  Board  of  Trustees  shall  have  power  to  choose  an  Execu- 
tive Committee  from  their  own  number,  and  to  delegate  to  such  Com- 
mittee such  of  their  own  powers  as  they  may  deem  expedient. 

VII.  The  President  shall  annually  appoint  two  Trustees,  who  shall 
constitute  a  committee  on  finance,  to  examine  from  time  to  time  the 
books  and  accounts  of  the  Treasurer,  and  to  audit  his  accounts  at  the 
close  of  the  year.     No  investments  of  the  funds  of  the  Corporation 
shall  be  made  by  the  Treasurer  except  approved  by  the  finance  com- 
mittee in  writing. 

VIII.  The  consent  of  every  Trustee  shall  be  m -i -es-ary  to  .1  <li-<olu- 
tioii  of  the  Marine  Biological  Laboratory.      In  case  of  dissolution,  the 
property  shall  be  given  to  the  Boston  Society  of  Natural  History,  or 
some  similar  public  institution,  on  such  term>  as  may  then  be  agreed 
upon. 

IX.  These  By-I.au>  may  be  altered  at  any  meeting  of  the  Trtiste.  -. 
l>ro\i<led  that  the  m  -uch  meeting  >hall  Mate  that  an  alteration 
of  the  Ii\ -I.au-  will  be  acted  upon. 

X.  Any  member  in  good  standing  may  \ote  at  any  meeting,  either 
in  person  or  by  proxy  duly  executed. 


IV.   TREASURER'S  REPORT 


FOR  THE  YEAR  ENDING  DECEMBER  31,  1911 

INCOME 

Annual  dues S      728.00 

Donations 13,412.00 

Homestead,  general  account  (net) 1,111.93 

Miscellaneous: 

Interest  on  deposits $97.06 

Rent  of  microscopes 9.90 

Use  of  drain  (4  years) 16.00  122.96 

Supply  department 10,303.61 

Tuitions 4,574-99  #30,253.49 

EXPENSES1 

Administration $3,044.22 

Advertising  and  printing 137-26 

Bath  house 96.91 

Biological  Bulletin  (net) 1,082.66 

Boats 6,003.45 

Chemical  department 838.44 

Dormitories 72.97 

Fish-trap 239.66 

Homestead  icehouse 609.92 

Homestead  shop 262.91 

Instructors'  salaries 3,i75-oo 

Interest 1 50.00 

Lectures 25.96 

Library 1,297.71 

Maintenance  of  buildings  and  grounds.  .  .  1,879.00 

Mosquito  fund 280.47 

Real  estate 5,100.00 

Scientific  instruments 504.19 

1  Owing  to  a  change  in  classification  of  expenses,  the  amounts  charged  the  various 
accounts  are  not  comparable  with  the  reports  of  previous  years. 

352 


TREASURER'S  REPORT.  353 

Sundries  .............................  2IQ.81 

Supply  department  ...................      8.<>ov;;   S33.O29.Q71 

I  n  MI/I.  i)  Li-i  "i   SUNDRY  EXPENSES  I->R  mi-:  Yi  AR  i<m 
I  spenses  of  W.  C.  Curtis  ................   ^2^.~^ 

Howes'  l.ills  .......  i>4 

1  "i  aming  .md  freight  .....................  -.57 

...............................  19.00 

KM  -li.iirc'-  "ii  i-li.-.  k-     ..................  2I..V> 

(  !are  »\  lot  in  crnn-u-ry  ...................  2.00 

-ohm-  f.  ir  ;.;.i-  machine  .................  3I-9O 

I'.ilint  i  ,\  l..nrjilin  (order  \\'.  (  '  .  <  'uni-     .  .  .  6.o<> 

5.  I.  sii"\v    -undry  bill-)  .................  $.67 

Hl.id.-     ..................  -SO 

I    .  5.  Powei  -    ~iindr\-  l.ills)  ........  j.io 

^.  r\  ices  J"hn  <  '..  HiiMurd  ......                     .  .  6O.OO 

Ice  ........  .1  1  --5 

John  J.  Veed<  i          ......  .50 

frusta       diiiiu-r  ..........  [7«OO 

i      I   .  >\\il't  v\  ^"ii  ............  2.57 

1    iiinnirr  .......  1.^-74 

(  h.nl.  J  J.  (  -riniK-11  ........  I  .  V5 

John  !•'.  I'hillip-.  <  ti-.tniin^  i  .........  5.30 

I'rriniuin  on  liond  on  .ili'ohol  .  .  7.50 

drcil  ..................  .x'i 

ilr<  l.ir.itioji  o|   iru-t  ............  -7') 

M\|'n  --  "ii  p.i\  -foil-  I"  \\  oo.  U  Holr     ......  5.J5 

r,i\  -foil  nuriopi--  ............  .29 

.30 

p.i|H-f  .....................  o/X) 

Carpenter    botany  department)  ...........  .45            ^1.51 

Credit 

\\-\i\-  r.i-h  at  \Yoo.U  Hole  i'of  i«jn>  ........  S      i  .00 

Ki-print-  ...............................  -.30 

1  laf\  .ird  Appar.itu-  roniiviny 

.-lurk  -cm  l.y  cffof  l,i-i  \r-ar)  ........  25.40              .U.7" 


--MO.SI 

1  On  January  i.  lyii.  tin-  Laboratory  had  cash  on  hand  $2.719.17;  the  ovcnlrait 
r  tin-  yat  ua-  tin  :  6.41,  which  has  since  been  adjusted. 


354  MARINE  BIOLOGICAL  LABORATORY. 

MOSQUITO  FUND 
Receipts 

1910  Miss  S.  B.  Fay $100.00 

Joseph  Fay,  Jr 100.00 

A.  C.  Harrison 100.00 

H.  K.  Dyer 100.00 

1911  Charles  R.  Crane 100.00        $500.00 

Payments 

1910  Services  H.  H.  Brehme $  50.00 

3  pair  rubber  boots 19-50 

Labor  (regular  pay-roll) 30.00 

Nets 1.47 

1911  Oil 5.40 

Labor  digging  ditch 19740 

Carting 73.20 

Spray  for  hose 9.72 

Dr.  Drew's  expenses 11.13 

T.  E.  Howes,  June  account 6.50 

E.  C.  Brown  Co .42 

John  F.  Phillips 1.70 

Labor  (regular  pay-roll) 75-OO          481.44 

"$18.56 


MARINE   BIOLOGICAL   LABORATORY 
I! X  VESTMENTS 


JANUARY   i.   [912 
Ri->iK\r  FUND 

Ann'iini  "f  fund  I  K'crniU-r  l  ,   i  -*•><>  ............  $4,  553.14 

Received  i>"in  life  nK-mliiT.-hi]>-  ...............        6oo."<> 

Income  to  January   i,  PUJ  ...................    2,292.15 

<  i.iin  I'n.m  -.ili-  -'I   sei  untie-  ,in«l  ri^lit-  ......... 


:  817.67 

1'aid  !»r  current  e\|M  I  .al>-  -r.it.  TV  ........     6.OOO.OO    $I,8l7 

Reserve  I  un<l  n<>\v  •  "ii-i  the  following: 

.....  i  Am.  Tt-l.  i\  Tel.  Co.  45  cost  .........  »2.'  121.25 

5  >li~.  AIM.  SniL-ltini;  «V   Ki-tinin^  Co.  Efd.  COSl        ~  ;.' 

hs.  '  .fin  r.il   l-'.li-i-iric  d>  ................        75^.25 

14   >h>.    riiiti-d    Slim-    M.Kliiix  -ry    Corp.    Pld. 

•  ........................  1-75 

'    i-h  ..........  14-4- 

.    i;.': 

i.f    tlir   above  .md    iM.nd-    .irr   lu-ld 

collatir.il  I'.T  Inan  ••!  ..........     .^noo.oo    $1,817.67 

l.IHKAKV     1   IM) 

A  nil  'ii  ii  i  "1  fund   1  )rrcni|iiT  I  .   i  -')•)  ............  $    S6(>.  I  =, 

Ini-i'inc  In  J.iiiuar\    I.   I'M-'  ...................  -.47 

d.iin  frmii  >.ilr  "I  -mirities  and  rights  .........          96.39    $1,74;."! 

Library   Fund  im\v  mn-ists  of  tlie  f"ll'»\vinu: 

;  shs.  Am.  "1\-1.  Ov:  Tel.  Co.  cost  ............  S     $83.25 

4  >   of    ^1,000  Am.   Tel.   vS;   Tel.   Co.   4s  cost       77-).  OO 

1  -h.  Am.  Smdtiiu  A   lM-tining  Co.  Pfd.  cost        122.00 

2  -hs.  (".riu'ral  Klt-rtric  Co.  cost  ............        302.50 

5  shs.  I'niti-d  Sin  >«  •  Marh.  Corp.  Pfd.  cost.  .  .  .        140.63 

C  ish  ..................................  [5-63    Si.74VOi 

355 


356  MARINE    BIOLOGICAL   LABORATORY. 

LUCRETIA  CROCKER  FUND 

Amount  of  fund  December  i,  1899 $2, 500.00 

Income  after  paying  students'  fees 557-91 

Sale'of  rights 8.79  #3,066.70 

Lucretia  Crocker  Fund  now  consists  of  the  following: 

18  shs.  Vermont  &  Mass.  R.  R.  Co.  cost.  .  .  .  #2,416.50 

I  sh.  West  End  Street  R'y  Co.  cost 83.00 

I  sh.  Am.  Tel.  &  Tel.  Co.  cost 127.75 

1/5  of  #1,000  Am.  Tel.  &  Tel.  Co.  45  cost.  .  .       194-75 

I  sh.  General  Electric  Co 151-25 

Cash 93.45   #3,066.70 


V.   THE  DIRECTOR'S  REPORT 


I  "  CHE  TRUSTEES  OF  mi.  MARINE  BIOLOGICAL  LABORATORY: 

Gentlemen:  Tin    session  just  clo-rd  is  the  twenty-fourth  o>n- 
timioii  'ii  «>t"   tlu-   L.tlioraiory.      Tin-  attendance  of  inve-ti- 

ors  u.i-  larger  th.  in  »-\  t-r  befi  >re  in  tin-  hi-torv  of  the  In -lit  in  ion, 
and  the  total  at  tendance  the  greatest  sina  [QO2  when  the  policy 
ol  re-trilling  instruction  wa-  fir-t  definitely  e-tabli-hed.  The 
principle  ••!  •  ••  ><>peration.  placed  at  the  foundation  of  the  Labora- 
tory |..y  Prole—or  \\'hitinan.  ha-  ne\  er  more  fully  justified  it-elf; 
and  it  i-  a  pleasure  to  testify  to  tin-  ^ -iieioii-  »|>irit  in  wliii'h  the 
corporation,  the  hoard  of  tru-tre-.  the  -tatt  and  tin-  eni]>lo\ ,-,--  of 
the  institution  ha\e  lalmreil  tor  it-  \\ellare.  Through  Mr. 
Crane's  generosity  the  Laboratory  ha-  ln-eii  pre-ented  \\ilh  the- 
Kidder  Annex  |ini|n-rt\-,  a  lot  of  land  -ituated  1-et  \\eeii  the 
liotanical  lal  ior.it,  ,r\  and  the  main  building;  and  \\ith  more  than 
three  fourths  Ol  the  -hare-  <.|  the  \V(  -oiU  Hole  ^"acht  Chili  prop- 
cm  adjoining  and  continuing  our  trouta^e  on  the  harbor. 
The-e  ailditioii-  practicalK'  I'oinplete  the  arrangement-  n« ••  .  --ary 
I.  if  tin-  next  forward  Mep. 

\\'e  note  \\ith  regret  the  re-i^nat  i.  >n  of  ProfeSSOr  Nathaniel  L. 
liritton  Irom  the  board  of  trn-tee-.  ]>re-enteil  at  the  -umnier 
meeting,  ouin^  to  hi>  inabilit\'  to  take  an  active  -hare  in  attair-. 
and  \\i  extciul  cordial  ^teetin--  t<>  Professor  l\.  A.  Harper,  ol 
Columbia  I 'ni\  rr>it\'.  a  newly  elected  member  of  the  board, 
\\ho-e  co.'iper.tiioii  \\ill  -n-atU'  -treii-then  our  work,  e-pecially 
on  the  botanical  -ide.  The  board  of  tru-tei--  remain-  other\\i-« 
unclKiniicd.  There  an-  three  \acaiu  it>  in  the  member-hip  which 
it  i-  the  pro\ince  of  the  board  to  till.  Mep-  should  U-  taken  to 
secure  the  be-t  nietl  to  till  the-e  vacancies. 

Prolfs-or  C'urti-  re-i-ned  as  head  of  instruction  in  invertebrate 
/o()lou\  at  the  close  of  the  summer,  after  four  years  of  service, 
during  which  he  successfully  maintained  the  be-t  traditions  of 
the  course.  Particular  importance  attache-  to  thi-  course  as  the 

357 


MARINE    BIOLOGICAL    LABORATORY. 


first  to  be  established  in  the  Laboratory  and  as  lying  at  the 
foundation  of  the  more  advanced  work.  The  directors  have 
therefore  considered  the  question  of  a  successor  to  Professor 
Curtis  with  great  care,  and  they  are  happy  to  be  able  to  announce 
that  Professor  Caswell  Grave,  of  Johns  Hopkins  University,  has 
accepted  the  appointment  as  Professor  Curtis'  successor.  We 
believe  that  no  better  selection  could  have  been  made.  Professor 
Grave  is  given  a  free  hand  in  the  selection  of  other  instructors  in 
the  course. 

The  attendance  in  1911  taxed  the  resources  of  the  Laboratory 
to  the  utmost.  The  number  of  investigators  was  82  during  the 
entire  season  and  of  students  65,  a  total  of  147.  For  comparison 
I  give  the  figures  since  1903: 


1903 

1904 

1905 

1906 

1907 

tgt  18 

1909 

1910 

1911 

Investigators.  . 
Students  

76 

S4 

51 
=51 

68 

,7 

68 

41 

60 

47 

52 
48 

66 
63 

62 

64 

82 
65 

Total  . 

no 

IO2 

12Z 

IOO 

107 

IOO 

120 

126 

147 

But  the  actual  increase  in  the  number  of  investigators  does  not 
tell  the  full  story.  For  several  years  there  has  been  a  steadily 
growing  tendency  on  the  part  of  workers  at  the  Laboratory  to 
make  Woods  Hole  their  regular  summer  home  and  to  purchase 
houses  there.  The  body  of  workers  at  the  Laboratory  has  thus 
become  not  only  larger,  but  more  constant  in  attendance;  the 
increase  of  attendance  this  year  is  therefore  to  be  regarded  as 
normal,  not  due  to  exceptional  causes.  That  the  Laboratory 
should  come  to  be  regarded  as  their  regular  summer  home  and 
\\orking  place  by  so  large  a  number  of  prominent  naturalists 
must  be  a  source  of  gratification  to  all  the  members  of  the  board; 
while  it  serves  at  the  same  time  to  emphasize  anew  the  need  of 
greatly  increased  accommodations.  Such  over-crowding  as  ex- 
isted for  a  considerable  part  of  last  summer  is  certainly  undesir- 
able and  its  continuance  for  any  considerable  length  of  lime 
rmild  not  be  anticipated  with  composure.  Neither  do  we  wish 
to  restrict  the  policy  of  hospitality  which  has  been  so  character- 
i-tic  of  the  Laboratory  in  the  past. 

The  proposed  new  building  is,  therefore,  a  necessity  for  working 
space  alone.     And  it  is  equally  important  for  two  other  reasons: 


THE  DIRECTOR'S  REPORT.  $59 

first,  to  provide  for  the  development  of  a  more  adequate  library, 
which  will  remove  one  of  the  most  serious  limitations  of  our 
re-earch  facilities,  and  second,  to  provide  more  commodiou- 
quarter-  for  <  i -rtain  types  of  research,  especially  in  experimental 
line-.  For  the  season  of  1912  some  additional  working  space 
will  lie  .ivailahle  iii  the  Kidder  Annex,  so  that  it  may  lie  hoped 
thai  the  i  rowdinii  will  be  no  wor-c  next  year  than  it  wa-  this. 
During  the  la-t  ti\e  years  there  ha-  been  a  -teady  increase  in 
the  nninlier  of  -nl i-<  ril iin-  in-titution-.  from  K>  in  1007  to  25 
in  [•)[  i.and  on  the  u  hole  there  is  reason  to  believe  that  the  pres- 
ent li-l  (Min, (in-  fewer  -nh-criptions  for  the  \ear  only  than 

ever  l>efore;  there  are,  moreover,  indication-  that  the  increase  in 

number  ma\  In-  e\pei  led  t<i  continue  f<>r  -miie  years  io  come.  A 
neu  lorn)  of  arrant-mem  u.i-  intend  into  tin-,  \ear  \\ith  the 

K".  keleller     Ill-titUtc    for    Medic. il     Kr-eaivh    of    \e\\     York    City, 

under   the  term-  ol   uhich   the   Km  kefeller   hi-tituie  ha-  ere< 
a    -MI. ill    laboratory    |,,r    llie    u-e    o|     Professor    Jacquc-    I.oeh   on 
land    ol    the    Marine    Bioloji  .d    I  -d  'oratory,    Inn    receives   other 
lacililie-  and    service   -MI    |  !y    the   -ami-    lenn-  a-  other   CO- 

opi-r.nin-  iii-iiintion-.  \\itlnnit  the  additional  lalmratory 
-pace  thtl-  pio\ided,  il  \\»uld  ha\e  lieeil  Mecr— ar\'  to  retu-e 
accommodations  to  a  numl'ei-  o|  in\  tors.  The  direcior 

ha-  al-o  lu-i-n  notitied  o|  the  endowment  of  a  -clmlar-hip  in 
Normal  (  'olli -^i  .  \>  u  ^'o|  k  (  "ii\  .  in  nn-nior\  of  |-'.Ur  ^.  i  in-haii-, 
!"iineil\  a  -Indent  at  the  Marine  Iiiolo-i,  al  Lalmratory  for 
-e\e|-.d  yeai  1  he  income  of  the  fund  o|  Si. ooo. oo,  to  be 

kno\\n  a-  the  F.l-e  Serin^hau-  Scholar-hip,  i-  to  he  applied  to 
tlu-  p.i\  nieiit  o|  uiitioii  fee-  |o|  one  -indent  each  \ car  at  the 
Marine  Hiolo^ical  Lal>oiatoi\  .  ,j-  el-i-\\  here,  as  ma\-  lie  desig- 
nated liy  the  committi-i-  of  auanl. 

I'ollouin^    the    recommendation    in    the    director's    report    for 

ioio.  Professor  Gilman  A.  1  >n\\  was  appointed  resident  assistant 

direcior  al  the  -nninier  meelini;  o|  the  Hoard  of  Tru-ti-es.  This 
-tep  ua-  taken  none  tOO  SOOH,  a-  the  burden  of  admini-tration 
ot  Laliorator\-  affair-,  whii'h  has  been  largely  carried  by  Pro- 
»or  1  h'ew  for  -t  \eral  years,  in  addition  to  regular  univer-ity 
work,  u.i-  already  much  too  j;reat ;  the  Laboratory  needs  such 
service  as  I'rote— or  Pivu  '-  e\])erience  and  charai'ter  lit  him  so 


360  MARINE    BIOLOGICAL    LABORATORY. 

eminently  well  to  render,  and  we  can  now  face  the  growing 
complexities  of  the  Laboratory  with  confidence  and  more  ease. 

Among  additions  to  the  equipment  of  the  Laboratory  during 
the  year  may  be  noted  a  large  motor  boat,  a  Zeiss  microscope 
of  the  best  and  newest  model  and  a  new  microtome.  The  mess 
did  an  unusually  large  business  and  exhibits  an  unexpectedly 
large  surplus,  all  of  which  is  to  be  turned  back  into  improve- 
ments in  its  equipment  and  service.  Among  these  may  be 
mentioned  a  new  ice-house  costing  $600.00  and  a  work-shop 
costing  ^250.00  already  completed.  The  supply  department 
has  made  its  usual  gain  in  business  transacted,  and  it  is  worth 
noting  that  under  Mr.  Gray's  management  this  business  has 
increased  from  $5,616.54  in  1906  to  $10,303.61  in  1911. 

Our  thanks  are  again  due  Mr.  Crane  for  the  donations  which 
have  enabled  us  to  maintain  the  work  of  the  Laboratory  on  an 
efficient  basis;  and  for  the  presentation  of  additional  stock  of 
the  Yacht  Club,  and  the  Kidder  Annex  property. 

There  are  appended  as  parts  of  this  report  the  names  of  the 
staff  for  1911,  a  list  of  investigators  and  students  with  a  tabular 
view  of  attendance  since  1908,  and  lists  of  subscribing  institu- 
tions, of  the  evening  lectures  for  1911,  and  of  the  members  of 
the  corporation. 


i.     Till:   STAFF. 


V.     R.     I.  II.  I.  IK.     I  'IKK.  TOR. 

Prof.  -~op  of  Kmlir\  i>lo-y  and  Chairman  of  tl.-    1  '•     .iruiK-iu  of 
y.  Tin-  l"niver>it  y  of  < 


c,!l.  MAX  A.  I'klAV.  ASSISTANI   DIRECTOR, 
I1-  r  of  Biol   gy,  University  of  Mail 


ZOOLOGY 

I.        IN\  • 

Zoology  and  Embryology 
(,.\i<\    N".  CALKINS  Prof<  '•  Colmnliia    I'ni- 

vereity. 

I      G.  CONKLIN  I'  .  v.  Princeton  rnivrr-it  v. 

(.11  MAN  A.  I»K-I\\  I'  r  ol   Biology,  I  niversit^  <-f  Maine. 

GEORGI   I  i  i  i  \  \<\-\  .....  I  '  r  of  Zoology,  University  of  Missouri. 

FRANK  K.  In  in  Professor  ol    Embryology,   Tlu-   rni\cr~it\- 

of  C'l.i      . 

T.  ii.  MONTGOMERY,  IK     IY.<U  —  r  ol  /  ••••'  gy,  rni\t-:  i'.-nn- 

>\  1\  ania. 

T.  II.  MORGAN  I'mu-ssor   of    I'.xi'criincni.il    Zo6logy,   <     • 

luiuliia  rnivcrsit\'. 
K.  l>.  \\  n  ~"\          .  I'p'h  -----  r  of  Z          .;.  .  (^'Iiiiiil'ia  rni\iT-it\  . 

II.         IN^I  Kit    I  ! 


WlNTERTON  C.  CURTIS        I'r-  't\»iT  <>f  XniVil..-y.  rni\tT-it\  of  Mi--oiiri. 
I'\t  i    M.  Ki  \  ......          Professor  of  Biology,  College  of  Charleston, 

and  Director  of  the  Charleston  MUM-UIH. 

I  n\\.\Ki>  I  .  \\  n  i'M.\N      .  .  Central  Ilii;h  Si'houl,  Philadeljihia. 
IOIIN  W.  SCOT!  .........  U'e-iptirt  High  School,  Kan-a=.  City. 

G,  S.  1  '"i>ns  ............  Professor  of  Biology,  St.  Louis  I'niversity. 

1.  I'.  .  \HHOTT  ...........  Professor  of   ZoSlogy,  \Va~hinicton    I'niver- 

-i- 

361 


362  MARINE    BIOLOGICAL    LABORATORY. 

EMBRYOLOGY 
i.     INVESTIGATION.     (See  Zoology) 

II.        INSTRUCTION 

OILMAN   A.    DREW Professor  of  Biology,  University  of  Maine. 

LORANDE  L.  WOODRUFF.  .Assistant  Professor  of  Biology,  Vale  Uni- 
versity. 

WILLIAM  E.  KELLICOTT.  .Professor  of  Biology,  Goucher  College. 

ROBERT  A.  BUDINGTON.  ..  Associate  Professor  of  Zoology,  Oberlin 

College. 

PHYSIOLOGY 

I.       INVESTIGATION 

ALBERT  P.  MATHEWS.  ...  Professor  of  Physiological  Chemistry,  The 

University  of  Chicago. 

R.  S.  LILLIE Instructor  in  Comparative  Physiology, 

University  of  Pennsylvania. 

HAROLD  C.  BRADLEY Assistant  Professor  of  Physiological  Chem- 
istry, University  of  Wisconsin. 

II.       INSTRUCTION 

H.  H.  NEWMAN Professor  of  Zoology,  University  of  Texas. 

CHARLES  G.  ROGERS Associate  Professor  of  Physiology,  Syracuse 

University. 
F.  H.  PIKE Instructor  in  Physiology,  The  University  of 

Chicago. 

PHILOSOPHICAL    ASPECTS    OF    BIOLOGY    AND    ALLIED    SCIENCES 

LECTURES 

EDWARD  G.  SPAULDING..  .Assistant  Professor  of  Philosophy,  Princeton 

University. 

BOTANY 
GEORGE  T.  MOORE Professor  of  Plant  Physiology  and  Applied 

Botany,  Washington  University. 
GEORGE  R.  LVMAN Assistant  Professor  of  Botany,  Dartmouth 

College. 
15.  M.  Dn.i.AK Professor     of     Plant     Physiology.     Cornell 

University. 
IVKY  F.  LEWIS Professor     of     Biology,      Randolph-Macon 

College. 
LEV,  i>  KM  DSON Instructor    in     Plant     IMi\>iology,     Cornell 

University. 


THE  DIRECTOR'S  REPORT.  363 

LIBRARY 
II.  Mr (•;.  KNO\VER University  of  Cincinnati,  Librarian. 

CHEMICAL  SUPPLIES 

OLIVER  S.  SII«>M, College  of    Ph\>u-ians  ;md  Surgeons.    NY\v 

V-irk  City,  Chemist. 


(  ,.  \|.  (  ,K  \y Curator  of  Supply  Department. 

TllnMAs    M.    I)oriIIAkT 

and  JOHN  J.  M<>KK>N    .Collectors  in  Zoology. 
J.  M.  Ik\viN Collector  in  Hotany,  hartmouth  Col 

[r ,n\  VEEDEB Co. -k-\vain. 


2.    INVESTIGATORS  AND  STUDENTS 

IQII 


INVESTIGATORS  —  OCCUPYING}ROOMS. 


ZOOLOGY 

ABBOTT,  JAMES  FRANCIS,  Professor  of  Zoology,  Washington  University. 
Amu-Mix.  W.    II.   F.,   Demonstrator  of  Histology  and  Embryology,   University  of 

Pennsylvania. 

BARTELMEZ,  GEORGE  W.,  Associate  in  Anatomy,  University  of  Chicago. 
BECKWITH,  CORA  J.,  Instructor  in  Biology,  Vassar  College. 
BUDINGTON,  ROBERT  A.,  Associate  Professor  of  Zoology,  Oberlin  College. 
CALKINS,  GARY  X.,  Professor  of  Protozoology,  Columbia  University. 
CHAMBERS,  ROBERT,  Columbia  University. 

CLAPP,  CORNELIA  M.,  Professor  of  Zoology,  Mount  Holyoke  College. 
CONKLIN,  E.  G.,  Professor  of  Zoology,  Princeton  University. 
CRAIG,  WALLACE,  Professor  of  Philosophy,  University  of  Maine, 
('runs,  W.  C.,  Professor  of  Zoology,  University  of  Missouri. 
DODDS,  GIDEON  S.,  Instructor  of  Zoology,  University  of  Missouri. 
DREW,  GILMAN  A.,  Assistant  Director,  Marine  Biological  Laboratory,  Woods  Hole, 

Mass. 

DI  NX,  ELIZABETH  HOPKINS,  Instructor  in  Anatomy,  University  of  Chicago. 
Fox,  HENRY,  Professor  of  Biology,  Ursinus  College,  Collegeville,  Pa. 
GOLDFARB,  A.  J.,  Instructor,  College  of  the  City  of  New  York. 
HARVEY,  BASIL  C.  H.,  Assistant  Professor  of  Anatomy,  University  of  Chicago. 
HARVEY,  E.  NEWTON,  Instructor  in  Physiology,  Princeton  University. 
HOGUE,  MARY  J.,  Instructor  in  Zoology,  Mount  Holyoke  College. 
KELLEY,  FRANK  J.,  Assistant  in  Experimental  Breeding,  University  of  Wisconsin. 
KKI.LICOTT,  WILLIAM  E.,  Professor  of  Biology,  Goucher  College. 
KNOWER,  H.  McE.,  Professor  of  Anatomy,  University  of  Cincinnati. 
LEFEVRE,  GEORGE,  Professor  of  Zoology,  University  of  Missouri. 
1. 1 i.i. IE,  FRANK  R.,  Professor  of  Embryology,  University  of  Chicago. 
LYON,  MARY  B.,  Instructor  in  Zoology,  Mount  Holyoke  College. 
\I<  (  LUNG,  C.  E.,  Professor  of  Zoology,  University  of  Kansas. 
M  \VKR,  A.  G.,  Director,  Department  of  Marine  Biology,  Carnegie  Institution. 
MONTGOMERY,  T.  H.,  Jr.,  Professor  of  Zoology,  University  of  Pennsylvania. 
MORGAN,  T.  H.,  Professor  of  Experimental  Zoology,  Columbia  Univrr.-ity. 
I'M  KAKD,  CHARLES,  Assistant  in  Zoology,  Columbia  University. 
I 'AH  IN,  STEWART,  Lecturer  in  Biology,  Princeton  University. 
PATTEN,  WILLIAM,  Professor  of  Biology,  Dartmouth  College. 
PATTERSON,  J.  T.,  Adjunct  Professor  of  Zoology,  University  of  Texas. 

' .  i  i  '  SH,  L.  S.,  27  West  73d  Street,  New  York  City. 
KI.A,  PAUL  M.,  Professor  of  Biology,  College  of  Charleston. 
SCOTT,  JOHN  W.,  Westport  High  School,  Kansas  City,  Mo. 

364 


THE  DIRECTOR'S  REPORT.  365 

>xc,  OLIVER  S.,  Instructor  in  Anatomy,  College  of  Physicians  and  Surgeons, 

New  York  City. 
WHITNEY,  D.  D.,  Associate  Professor  of  Zoology,  Wesleyan  University.  Middletown. 

Conn. 

\VIEM.\N,  H.  L.,  Assistant  Professor  of  Zoology.  University  «n"  Cincinnati. 
WiuiMAN,  E.  E.,  Professor  of  Zoology,  Central  High  School,  Philadelphia.  Pa. 
\Vii.sos,  E.  B.,  Professor  of  Zoology,  Columbia  University. 
W.  MILK'  i-K,  L.  L.,  Assistant  Professor  of  Biology.  Yale  University. 

PHYSIOLOGY 

AMHKRL,  SAMUEL,  Associate  Professor  of  Pediatrics  John-  ll-;.kins  University. 

•  ROFT,  FRANK  \V..  Associate,  Rockefeller  In-titute  for  M.-<li>al  Re--  arch,  New 

York  City. 
Hi  '  i  MR.  Ki.iNHAki'.  A--i-tant,  Ro.  In-titute  foi  M.-.lical  Ke-\u.h.  New 

York  City. 
l',u  \iii.i.v,  H.  C.,  A--i-taiit  I':  '••  '  heinistry.  University  of 

\Vi-.  i  .n-iii. 

II.  II..  P  .  logy,  \Vi-tar  In-titut'-  1  Anatomy  and 

Biol 

I.  s,  A--i-tant    :  Cornell  University   M.-dical 

tool. 

-I.R,  i  >.  •  taut   1';  -y.  Uim  '  '  .111. 

I  ii  in  .   K.  S.,  Iii~trin  -t..r  in  I'liy-i'  .1.  .^i.  al  2  ,  University  of  lVmi->  I  \ania. 

LOBB,  JACQI  .i-T  In-tiiuti-  :•  :   M>  lical  Research,  IS  -.ty. 

MATHEWS,  A.  P.,  1  r  ..i  I'h-  ?.  University  of  < 

M  \MII-.\\ •>.  >\M'II.  A..    .  :iineiual    Pherapi  itics,    I'ni- 

Mi  IGS,  1      H  .  l-.-IltJW  in  /..«..l.ii;y.  \Vi?t.ir  In-titute  ut  An.it. -my  and  Hi.-l.  . 

NEWMAN,  H.  H.,  Ai  '        •  1:0. 

I'lki.  I-  i;  \NK  \\..  In-tin*  L>r  in  1'i.  .1    nivei  '    liieago. 

ROGERS,  CHARLES  G.,  Pi  ;ty. 

SPAULDING,  1     .  :.uu  l'i  •  phy.  Princeton  Univi 

H  \ki" < i.ni.  Assistant,   i<  ckefeller  In-titnt<-  !<T  M«-di,_al  k---.-aich, 

Ni-w  \'.uk  City. 

BOTANY 

K,   (    \KKII     M  :  ;-.  .    M.I, ill   University, 

in  I.I.AR,   H     M..   Prof(  »OI  .'i   Plant  l'h\  :..-ll  University. 

KM  DSON,   I  i\\i>.   In.-tnictiir  in  IMant  1'lr  '      inell  Univer-ity. 

1  i  \MV.  i\i  N  i -.,  T:  :  -,>r  of  Biology,  Randolph-Macon  d. liege. 
I.VMAN.  GEORGE  K..  A--i.-iaiu  1':  :;>.  Daitiiii-uth  College. 

M.'.'Ki,  «,II.K<.K  T.,  Professor  of  Botany.  \\\i-hini;t<'n  University.  M.  I      :       M 
<  i-ii  kin>i  r.  \\'.  J.  Y.,  Assistant  Professor  of  Botany,  Harvard  University. 
lii..\i\v.  MASON   I:  .  Plofi    -or  of  Botany.  WaNash  College. 

OCCUPYING  TABLES 
ZOOLOGY 

ABBOTT,  MARGARET  B.,  Bennett  School,  Millbrouk,  New  York. 
Ai  i  vs.  HAKKII  i  M..  Fellow  in  Zoology,  University  of  Chicago. 
BK«>\VNE,  ETHEL  N.,  Graduate  Student,  Columbia  University. 


366  MARINE    BIOLOGICAL   LABORATORY. 

DAVIS,  SARAH  ELLEN,  512  West  i32d  Street,  New  York  City. 

I  M  M.AY,  NEIL  S.,  Professor  of  Biology,  Carleton  College,  Northfield,  Minn. 

EDDY,  MILTON  YV.,  Northwestern  University,  Evanston,  111., 

ENNIS,  AGNES,  453  Convent  Avenue,  New  York  City. 

JUST,  E.  E.,  Instructor  in  Biology,  Howard  University. 

MACKENZIE,  MARY  D.,  Associate  Professor  of  Biology,  Western  College,  Oxford, 

Ohio. 

SPENCER,  HENRY  J.,  Graduate'Student,  Columbia  University. 
SIXK,  EMORY  W.,  Assistant  in  Zoology,  University  of  Michigan. 
WALLACE,  EDITH  M.,  Columbia  University. 

PHYSIOLOGY 

MORSE,  MAX  W.,  Professor  of  Biology,  Trinity  College,  Hartford,  Conn. 
TASHIRO,  SHIRO,  Student,  University  of  Chicago. 


STUDENTS 

ign 

INVERTEBRATE  ZOOLOGY 

BAII  i.   |  .  608   M.itli'-\\-  Av.-nue.  I'rhana.  111. 

BOI.A-.I..   Mil  DR1  D,  \\V-t.-rii  Col  '"rd.  Ohio. 

BROWN.  Vik<.!Ni\  R..   ui  l'p-o>ti  Stn-.-t.  T..1, •<!'>.  <  >hi.>. 
HI   MM-  -i  I'll  K..   17.'';  Turn.T  Stn-<-t.  All«-m-,\vn.   I '  •- 

COPKMIX-  M     N    .   I!      Bristol,  Ti-nn. 

IIIXIIK.    |,.ir.   S.,  P  B  il  St-ii-ii<  ••-.   Northland  <  A-hland, 

\Vi-<  nii-iii. 

ii.  Hi-.i  i  ,  ;.!,  t'.n  1  -    .1. 

:k  Cits-. 

•  \  ol  M.I:M.  .  '  Irono,  M.'. 

KI   in.  U'illi.im  I'-'iin  Hinli  S'li..<.l.  I'liil.i.l.-lplii.i 
<  ,ini.-.   I-  i  n  '  -  'fk. 

M-.  A  .    •  '  I  •'••» 

|,,M-.  .    ,.  in  \    I    .  i  >'  •  •  .rilm.  «  >hi.>. 

I  -, 

NI   ii  .   I  IM  •  .  B  M 

Mi  KK  \v.   M  \k  rORlE  1  \I.i\vr  ( loili 

KMUI.  1  'ii  A.   [fl       1      lUltim.'i-  M.iltim..re.   M! 

^i    1111   I  .    I  >•  M.illMt    H 

SMiiiN.t    UlOLYN    I'.   M.'imt   II.ily.ik«-( 

J- 

^Pk  \\.    K'    :  n  '  .  .   I    :       • 
Ml!.  •  lin  ('••!;• 

:lv. 

\\vKki--.H  •      t6  Brent  Street,  Dorchester,  M 

\\  mil  .  I  -mi  K  I       M. -ri.li.in  <  "II.-. 

EMBRYOLOGY 

Ai  ii-  :  1803    Il.n.l  Av.-nii'-.   N.-w  \"..rk  I'ity. 

\M.I  KI  GG,  LOUIS  T.,  I  lb(  iliu  (',.11, 

M  .  ln-tnhi,,r.  1'iiivi-i-ity  •>!  ("inrimiuti. 

Mi -KKI-.  I  i. MI  M>  J..  Iii>truct,.r  in  Biology.  H..I-.  '  ..lli-i;.-.  \\  Mass. 

t    \i  i  i.i.i..  M>  K  •!!.  N«-w  Y.,rk. 

MM.  M  \KV  K..  gi5  North  51)1  Mnvt.  A.tchinson,   K 
•IK.  GEORGIA  M..  i.i  llit;lilan.l  Avt-nuf.  Auburn.  Main--. 

k.S<  ,  H  K.  HAKIMS-  K..  »>hi.-  \V,--l,-\.in  University,  1  ><  l.iwarc,  Ohio. 
i.i  wi.K.  kri.ui.i-ii  \V..  1'niv.Tsity  .-I  Mirhijjan. 
H.M.I  .  Mn  PKI  D  A.,  \\.-t,  in  Hi«h  Sch.H.l.  B.ikiinnn-.  Md. 
KKI.LV.  J  VMI->  I'..  Bl.uk  kin-k  A\  riuic.  I'ni,  mpm  t.  Xr\v  \'.>rk  City. 
M.-kki--.   MARGARET,  >j  Edsehill  Road.  NVw  Havt-n.  Conn. 

367 


368  MARINE    BIOLOGICAL    LABORATORY. 

Mi  I.LER,  HERMANN  J.,  562  West  i5oth  Street,  New  York  City. 

I1  \INTKR,  TnKormi  i  S  S.,  Vale  University. 

PATTEN,  HAZEL,  1719  McCulloh  Street,  Baltimore,  Md. 

Rn  HARDS,  ETHEL  L.,  Washington  I'nivcrsity,  St.  Louis,  M<>. 

S\i:\x,  MOIM>.  Washington  and  Jefferson  College,  Washington,  Pa. 

SCHUII ALI.A.  Ai.rnoNsic  M.,  Instructor  in  Biology,  St.  Louis  University. 

WHEELER,  THEODORA,  Fairfield,  Conn. 

WALKER,  GEORGE,  Associate  in  Surgery,  Johns  Hopkins  University. 

NORTIIRI  r.  J»IIN  II.,  500  Washington  Avenue,  Yonkers,  \V\\-  York. 

PHYSIOLOGY 

BIGNKV,   ANDREW  J.,   Professor  of   Biology  and   Geology,    MOOP  -    Hill   College, 

Indiana. 

CARROLL,  ALBERT  H.,  "Evergreen,"  Hampden,  Baltimore,  Md. 
ICKKS,  MARGARET,  Smith  College,  Northampton,  Mass. 
KELLERSBERGER,  EUGENE  R.,  University  of  Texas. 
N'iRCROSs,  KATHARINE,  University  of  Chicago. 
OLIVER,  WADI-:  \V.,  University  of  Michigan. 

BOTANY 

BOSSON,  RICHARD  M.,  Wabash  College. 

CAREY,  CORNELIA  L.,  Box  956,  Orange,  X.  J. 

CURTIS,  OTIS  F.,  163  North  Main  Street,  Oberlin,  Ohio. 

DA\VM>N,  AVA  H.,  <>-  Mountfort  Street,  Boston,  Mass. 

K.\rsT,  ERNEST  C.,  Oberlin  College. 

GROSS,  BEATRIX  H.,  Normal  College,  New  York  City. 

1 1  ILL,  ALBERT  F.,   192  South  Main  Street,  Attlebom,    Mass. 

JENNISON,  HARRY  M.,  Auburn,  Mass. 

KENOYER,  LESLIE  A.,  Leandcr  Clark  College,  Toledo,  Iowa. 

NOHI.E,  ARLYLE,  Smith  College. 

RICHARDS,  ANNA HM. LA  E.,  Ventnoe  City,  N.  J. 

\\  i  i  -H.  ('ARLTON  K.,  Dartmouth  College. 

l'i  NNELL,  FRANCIS  \\'.,  Harrison  Fellow  in  Botany,  University  of  Pennsylvania. 


TABULAR  VIEW  OF  ATTENDANCE 


1909 

INVESTIGATORS  -Total.  -- 

i  i.  i  ii|>\  in^  K  .....  11- 

Zooiog:  .......  4- 

IM  v  .....  9         18 

Botai  4 


Zoeiog>  ''  1J 

IM 

tany  ..... 

DENTS      rotal....  *8 

ZoSlog:  ......  '' 

Embr5  ]i         '  -         "' 

I'l 

11  17         [3 

IN-.IMI   flONS    Ki  I'Kl  -l  \  Ml' 
I1.-.    I' 

......  -"  J4  ;1 

S,   H,,..|  S     AND     A-   AIM.  Mil  -     Ul   PR1   -IMED 
Bj    i:1 

l'.\  students.  '  l  '' 


4.     SUBSCRIBING  INSTITUTIONS,  1911 


BRYN  MAWR  COLLEGE. 
COLUMBIA  UNIVERSITY. 
DARTMOUTH  COLLEGE. 
GOUCHER  COLLEGE. 
LUCRETIA  CROCKER  SCHOLARSHIP. 
MOUNT  HOLYOKE  COLLEGE. 
NORTHWESTERN  UNIVERSITY. 
OBERLIN  COLLEGE. 
PRINCETON  UNIVERSITY. 

ROCKEFELLER  INSTITUTE  FOR  MEDICAL  RESEARCH. 
SHEFFIELD  SCIENTIFIC  SCHOOL  OF  YALE  UNIVERSITY. 
SMITH  COLLEGE. 
SYRACUSE  UNIVERSITY. 
TRINITY  COLLEGE,  HARTFORD,  CONN. 
UNIVERSITY  OF  CHICAGO. 
UNIVERSITY  OF  CINCINNATI. 
UNIVERSITY  OF  ILLINOIS. 
UNIVERSITY  OF  KANSAS. 
UNIVERSITY  OF  MICHIGAN. 
UNIVERSITY  OF  PENNSYLVANIA. 
VASSAR  COLLEGE. 

WASHINGTON  UNIVERSITY  ALUMNI  ASSOCIATION. 
WESTERN  COLLEGE  FOR  WOMEN. 
WELLESLEY  COLLEGE. 

WISTAR  INSTITUTE  OF  ANATOMY  AND  BIOLO<.\. 
SCHOLARSHIP  OF  #100.     SUPPORTED  HY  A  FRIEND  OF  THE  LABORATORY 
SINCE  1898. 


KVKXINV,  LECTURES,  1911 


i  .  \RY  N.  CAI.KINS "The  Scope  of  Protozoology" .  •    .Junr  30. 

G.  II.  PAKKKK "Some    Recenl    Work    mi    Animal 

Reaction-    tn    (."dm-rd    I.i-ht  "  .  .  July   ;v 
T.  H.  MoKiiAN "What  i>  the  Mtvhani-m  of   Mi-n- 

drlian  Segregation  in   tin-   <  ii-nn 

July   7- 

I.   I'.  LEWIS     "  Alti-rnatimi    of    (  'UMKT.II  i<  MI-    and 

IVriodicity  in  the  Marine  .\k.i-"July    II. 
SIM.  .\    I  |  |  \\i;i< "The-  I1.:  J   I'.a-i-  «\  the    l"i«  at- 

nient  '  ;  I  'i sease" July  '4 

\\  \i  i  \.  i     CB  \i "Why  I)..  Hir.U  Siim?" July    i  3. 

J.  McKEEN  CATTELI "S<  ience  and  I  Democracy" July  21. 

I'.K  \HII-.N  M.  |)\\i- "Thf  Synth<-i-  and  I'.'-!ia\icr  of 

-  me  IM'rids  that  Rf-rinlilc 

CEnot'  ninrckitinn" July  2$. 

JA(  ''.'I  i  -  I  "i:» "The  I.it"r-Pre>er\  int:  Action  <•! 

Its" July  28. 

I   .  (  ,.   SPA!  l  DING  "  I'-'T^MHi's  CVt-aLi\e   I-Ivnlutinn  "  .  .  Aui;.  4. 

W.  M.  Wm.i  i  i  u  .  "  Insect  Para-iti-in  and  I-  -  P.  .  uli- 

arities" Aug.  8. 


6.     MEMBERS     OF    THE     CORPORATION     OF 
THE  MARINE  BIOLOGICAL  LABORATORY 

AUGUST  8,    1911 


LIFE  MEMBERS 
ALLIS,   MR.   EDWARD   PHELPS,   JR.,    Palais   Carnoles,   Menton, 

France. 
ANDREWS,  MRS.  GWENDOLEN  FOULKE,  821  St.  Paul  St.,  Balti- 

more-. Md. 

BILLINGS,  MR.  R.  C.,  66  Franklin  Street,  Boston,  Mass. 
CAREY,  MR.  ARTHUR  ASTOR,  Fayerweather  Street,  Boston,  Mass. 
CLARKE,  PROF.  S.  F.,  Williams  College,  Williamstown,  Mass. 
Ci'NKLiN,    DR.    E.    G.,    Princeton    University,    Princeton,    New 

Jersey. 

CRAM:,  MR.  C.  R.,  2559  Michigan  Boulevard,  Chicago,  111. 
I  )AVIS,  MAJOR  HENRY  M.,  Syracuse,  New  York. 
KNDICOTT,  WILLIAM,  JR.,  31  Beacon  St.,  Boston,  Mass. 
I;.\  ANS  MRS.  GLENDOWER,  12  Otis  Place,  Boston,  Mass. 
FARLOW,  PROF.  \\".  G.,  Harvard  University,  Cambridge,  Mass. 
FAY,  Mi-s  S.  B.,  88  Mt.  Vcrnon  Street,  Boston,  Mass. 
FOLSOM,  Miss  AMY,  88  Marlhorough  St.,  Boston,  Mass. 
FOOT.  Mi->  KAIIIAKINI:,  80  Madison  Avenue,  New  York  City. 
(  '.AKDINER,  Miss  Kri.ENiA,  15  West  Cedar  Street,  Boston,  Mass. 
HAMMOND,  MR.  G.  W.,  Hotel  Hamilton,  Boston,  Mass. 
HANNAMAN,  MR.  CHARLES  E.,  103  First  Street,  Troy,  New  York. 
HARRISON,  PROVOST  C.  C.,  University  of  Penn>yl\.iniu,   Phila- 

delphia, Pa. 
HERN  R.  DR.  C.  A.,  MQ  Madison  Avenue,  New  York  City. 

.N,  MR.  Hi  NKvL.,  191  Commonwealth  Avenue,  Boston, 


JACKSON,  Miss  M.  C.,  88  Maryborough  Street,  Boston,  Mass. 
I  \«  KSON,  MR.  CiiAki.i.>  C.,  24  Congress  Street,  Boston,  Ma--. 
KENNEDY,  MR.  C,i  ORC.E  G.,  284  Warren  Street,  l\<>\l>ury,  Mass. 
KIDDI.K,  MR.  C.  ('..,  2~  William  Street,  New  York  City. 
KIDDI  K,  MR.  NA  i  ii  \NII-.I.  T.,  Milton,  Ma--. 


HIM    DIRECTORS    REPORT.  37;, 

KlN(,,    MR.   (  "HARI.I>   A. 

Li  E,  MR-.  1  Ki.ni.RK    S.,  270  Madison  Avnuu-,  \\-\v  York  City. 
L"\VI.LL.   MR.  A.   LAURI.NIK.    171    Marlliorouuh  Stnvt,   Bo-ton, 

M 

MASON,  Mi--  K.  I'..  I  \\"alnnt  Stnvt.  Bo-ton.  Ma--. 
M\-«'N.  Mi--  IhA  M.,  i  \\"alnnt  Stnvt.  Bo-ton.  M 
Mi  AN-,  MR.  JAMI  -  HMUARD.  i<><>  Beacon  Stnvt.  Bo-ton.  Ma  —  . 
Mi  RRIMAN,  MR-.  PANII.I..  Worcester,  M 
MINN-,  Mi--  Si  -AN.  14  Loni-lnir;4  Sijiian-.  B.-t«.n.  Ma  —  . 
MINN-,  MR.  THOMAS,   14  Loiii-Uin;  S.|ii.uv.   B..-ton.  Ma  —  . 
M  INC  ,i ,  I  )R.  (  "HARM  -  "v.  Harvard  M  School,  Bo-ton.  M 

MIXIIR,   Mi--   M.  C..  241    Marl|porou-h  Sti.--.    Boston,   M 
MI.RI.NN,   MR.  J.    I'll  RPONT,   JR..   \\"all  and    Broad  S  .    \r\\ 

Y«>rk  ( 'ity. 

MI  IRI  -AN.  I'R'  'i  .    I  .  I  I  .  <  oluml'ia  lni\  rr-ity.  \C\\   York  Cii\ 
MMR(,  \\,  M  R-.   1  .  1 1.,  \,  \\  York  i  )ity. 
NORCl  Mi--     I.AlRA.      .    (   •  'iniiioiiucalth    .\\nnir.     Boston, 

Ma--. 

\''',  i  5,    Mi--    l.\  \  J..  ill   Willov     -  Monti-lair.    \.  J. 

NUNN,  MR.  LUCl  \".    I     .     Irlluridr.  (  !ol 
OSB(  1 '      i  .  Hi  NR\  1  .,  An  mil  an  Mn -nun  of  Natural  I  li-tor\  . 

\i\\    ^"«  -rk  (   il  v. 

I'l  M,  MR.  A  i.i  R  i  i>.  Highland  1'all-.  <  >i  '  «\\\\[\  .  N.  J  . 

I'HII  i  IP-.  I  >R.  Ji  MI     '  I '.-  rkelej   Street,  I  '••  is    >n,  M.' 

Tun  i.ii1-.  MR-.  JMIIN  (  BrrkrK-\    -  B         n.  M. 

n  R.  I  >R.  1 1.  ( '.,  I  'ni \n--ity  of  I'nin-\  1\  ania.  l'hila(K-l|>liia.  ! 
I'l  i  -n  i  R,  MR.  W.  II..  \f\\ton  (Ymn-.  M 

ROGERS,  Miss  A.  P.,  5  Joy  Stri        l  M 

ROGERS,    MRS.    \\llll\\I    B..    117    Marll.oroii^li   Sin  i  ;  .    BO-I.III, 

Mass. 

^i  \R-.  I  >R.  Hi  NR\  F.,  420  !'•    icon  Street,  Bo-ton.  M 
SiiM'i«.  M  R.  I  .  A. 

>MIIII.  MR-   i     »      286  Marll»oroii-li  Sin-i-t.  Boston,  Mas 
SiRor.M  i  .  Mi--  L.  C.,  80  Madi-on  Axnuir.  \\-\v  \'ork  City. 
TiioKNMKi  .  PR.  |-]D\VARI)  I...    I',   tchers  *  olli  •lunihia    Cni- 

\  i-r-itx .  \\-\\  N'ork  ( "it\ . 
TRI  i.i  A-I.    TROI'.    WILLIAM.    Mi— onri    Botanical    C.ank'ii-.    St. 

l.oni-.  M 


374  MARINE    BIOLOGICAL   LABORATORY. 

WARE,  Miss  MARY  L.,  41  Brimmer  Street,  Boston,  Mass. 
\YARREN,  MRS.  S.  D.,  67  Mt.  Vernon  Street,  Boston,  Mass. 
WHITMAN,  DR.  C.  O.,  University  of  Chicago,  Chicago,  111. 
WHITNEY,  MR.  HENRY  M.,  Brookline,  Mass. 
WILLCOX,  Miss  MARY  A.,  Wellesley  College,  Wellesley,  Mass. 
WILMATH,  MRS.  H.  D.,  Elliott  Street,  Jamaica  Plain,  Mass. 
WILLIAMS,  MRS.  ANNA  P.,  505  Beacon  Street,  Boston,  Mass. 
WILSON,  DR.  E.  B.,  Columbia  University,  New  York  City. 
WILSON,  PROF.  W.  P.,  Philadelphia  Museum,  Philadelphia,  Pa. 


MEMBERS  OF  THE  CORPORATION'  OF  THE 
MARINE  BIOLOGICAL  LABORATORY, 


DECHMHI  K.   1911 

Ai:i:''i  i.  Pk<>i;.  J.  F.,  \Ya-hin;;ton  lni\er-ity.  St.  Louis.  Mo. 
ABBOTT,  M\K>,  \\<\.i   B..  Bennett  Si-lux .1.  Milll>r<M,k.  New  York. 
ADAMS,  DR.  (\  K..  I'niver-itv  of  Ark.in-.i~.  F.t\  ritr\  ilk-.  Ark. 
Ahi>i-<>\.   I)K.  \Y.   II.   1  •'..   I   ni\er-it\    of  IYnn-\  1\  ania,   Philailel- 

|)hia,  I'. i. 
ALLYN,   Ml--   HARRII.I    M..  rni\cr-ity  of  Chicago.  1  )r]>.irtnu-nt 

of    /'    olo-y,    (  'llic. l^o.     111. 

A  i  -1:1  k< ..  I  >k.  (  'ARI.  S..  LJ.  S.  1  >e|>ai  •tinriii  ot  Agriculture.  \Ya-h- 

in^toii.  I  >.  < 

BAKI  K.  K.  1 1..  5444  ( '.itln-riiu-  Strn-t,  I'hiKulrlphi.i.  P.i. 
H\\<KI>M,    Pki'i.    1  .'  \\  ..    K.,.  k!illi-r    lu-tittiti-  for   Mi-ilical    l\.  - 

•    in  h.  Nr\\   \'i  >rk  ( 'it\  . 
Pi  \  kin  i  \.  I  >k.  C,   K  . .  I   11  i\  n-ii\   of  \\  i -con -in,  M.uli-on,  \Yi-. 

I'.i  i  K\M  in.  Mi--  CORA  J..  Vassar  (  oil,--,-,  pou-hki  rp-ic.  N.\\ 

York. 
I'.h.i  1 1  .\\ .  M  \i  KI<  i   A  .   feachers  College,  Ni  \\  \«\k  ("\\y. 

BlGELOW,    K.    P..    Massachusetts    In-titlUe-  of   'IVrhnolo^y.    Bos- 
ton, M.i--. 

lii  \  1 1  ill  i  iRii.  I-"..  \\'..   I  l  I  I    I  aSalle  A\  nun-,  Chicago.  111. 
liki'\\\r.     (.mil.     \.,     Coluinl'ia     I  'ni\  rr-itv,     1  Jrp.irt  nu-nt    of 

Xoolo-\  .  Nr\\  \'ork  C'it\\ 

P.I  i  KIM.IIAM.   MlMIII    \.,  .VJJ   Marll-oro  Street,   Ho-ton.   Mass. 
I'.i  DINGTON,    l\i'i:i  ki    A..  (M.rrlin  ('ollr.ui-,  ()ln-rlin,  (  )hio. 
Hi  \in  -,  l>k.  II.  C.,  liii\er-it\   of  \\"iscon-in,  Madi-on,  Wis. 
H\k\i  -.  l>k.  l;.-nu  k   1    .   [93  Jetieixm  .\\enue,  Brooklyn,  Xe\v 

York. 

CALKINS,  I>k.  »'.Ak\    N.,  C"olunil>ia  I'liivLTsity,  Ne\v  York  City. 
CALVERT,    1>R.    PuiLir   P.,    rni\er.-ity   of   Pcnn-\  hania,   Phila- 

delphia.  Pa. 

CARLSON,  I'K.  A.  J.,  I'niversity  of  Chicago,  Chicago,  111. 
("\kv,  I)K.  L.  R.,  Princeton  I  'imvr-itv,  Princeton,  X.  J. 

375 


MARINE    BIOLOGICAL    LABORATORY. 

CATTELL,  DR.  J.  McKEEX,  Garrison-on-Hudson,  New  York. 
CHAMBERS,  DR.  ROBERT.  Columbia  University,  Department  of 

Zoology,  New  York  City. 

CHESTER,  PROF.  WEBSTER,  Colby  College,  \Yaterville,  Maine. 
CHIDESTER,  DR.  F.  E.,  Rutgers  College,  New  Brunswick,  X.  J. 
CHILD,     DR.    C.    M.,     University    of    Chicago,    Chicago,    111. 
CLAPP,  PROF.   CORNELIA   M.,   Mount   Holyoke  College,  South 

Hadlcy,  Mass. 
CLARK.   DR.  ELIOT  R.,  Johns  Hopkins  University,   Baltimore, 

Md. 

COE,  DR.  \Y.  R.,  Yale  University,  New  Haven,  Conn. 
COLTON,  DR.  H.  S.,  3409  Powellton  Avenue,  Philadelphia,  Pa. 
COMSTOCK,  DR.  J.  H.,  Cornell  University,  Ithaca,  New  York. 
COOLIDGE,  MR.  CHARLES  A.,  Ames  Building,  Boston,  Mass. 
(  KAIG,  PROF.  WALLACE,  University  of  Maine,  Orono,  Maine. 
CURTIS,  DR.  W.  C.,  University  of  Missouri,  Columbia,  Mo. 
DEXTER.  PROF.  J.  S.,  Northland  College,  Ashland,  \Yisconsin. 
I  )IMON,  Miss  ABIGAIL  C.,  367  Genesee  Street,  Utica,  New  York. 
DODDS,  DR.  G.  S.,  University  of  Missouri,  Columbia,  Mo. 
DONALDSON,  DR.  H.  H.,  Wistar  Institute  of  Anatomy  and  Biol- 
ogy, Philadelphia,  Pa. 
1  )MRRAVE,  Miss  ANN,  Dorranceton,  Pa. 
DOR  RANGE,  Miss  FRANCES,  Dorranceton,  Pa. 
DREW,  DR.  GILMAN  A.,  Marine  Biological  Laboratory,  Woods 

Hole,  Mass. 

I  >(  GGAR,  DR.  B.  M.,  Cornell  University,  Ithaca,  New  York. 
EATON,  PROF.  E.  H.,  Hobart  College,  Geneva,  New  York. 
Kn.i  NMANN,  PROF.  C.  H.,  University  of  Indiana,  Bloomington, 

Ind. 
1  i  i«,i  SON,  DR.  J.  S.,  Cornell  University  Medical  School,  New 

York  City. 

I-ILLD,  DR.  IRVING  A.,  Clark  University,  Worcester,  Mass. 
1  i  RST,  MR.  CLYDE,  Carnegie  Foundation,  ,s;<>  Fifth  AM-.,  New 

York  City. 

GA'GE,  PROF.  S.  H.,  Cornell  University,  Ithaca,  New  York. 
GlES,    DR.    WILLIAM     J.,     Director,     Department    of    Biological 

Chemistry,  Columbia  University. 
'  .1  \-I-.R.  DR.  (  ).  ('.,  University  of  Michigan,  Ann  Arbor,  Mich. 


THE    DIRECTORS    REPORT.  377 

hi  A.RB,  I  )R.  A.  J.,  College  of  the  City  of  \e\v  York.  Xe\v  York 

City. 
( iki.i.NMAV  I  )k.  M.  1 ..  \Yi-tar  In-titnte  of  Anatomy  and  Biol.  \ 

Phila<lel])hia.  Pa. 

C.ku.nkV  Ml--  PI  'i  i -I.  IP.  Barnard  Coll.  ge,  V  v.   Y.-rk  City. 
HAI.I..  I)K.  K"i;i.ki  \Y..  152  South  Pinden  Street.  Bethlehem.  P 
I  lAki.l  M.  C.  \\'..  Syracu-e  1   ni\  er-itv.  Syracu-e.  Ne\\   York. 
IP\kki-''\.  MK.  A.  C.,  \V«"'d-  H.ile.  M 

llAkki-"\.   I  >k.   !<<>--  «...  Yale  Pni\er-it\.   New   Ha\en.  Conn. 
H\k\l  y.   l>k.   P.A-II    ('.   IP.   Pniver-iiY  of  Chicago.  Chicago.   111. 
ll\k\i  N.  MK.  I-'..  N.,  Princeton  Pni\er-iiy.  Princeton,  N.  |. 
HANI-.    Pk'ii.  S.    P.    Mount    ll<>l\oke  Collegi     S  lladk-y. 

M.i--. 

rn,  DR.  HAROLD,  Si  'nfoi-d  I  University,  Calif. 
1 1 1  >\K.  MK.  I  >.  P.I  VKELY,  i»ii  Devonshire  Street,   PO-I.UI,  M 
HOLMES,  DR.  S.  J.,  133  1  '      rham  Sir, ,  i .  Maili-on,  \Yi-. 

[SELEY,    MK.    I.    P..   <  iklahoma   Acadenn  -  Toiikaua, 

la. 

I  .I'K.MIKKII   I  P.  Pni\er-ii-        Pennsylvania,]  ;cal 

I    ilior.iioiA  .  Philailelphia.  P 

|  M  KSON,  I  >1     '      M  .  I   iii \  i  •  -it\   of  Mi--"iiri.  Columbia.  M,,. 
|\N  \i  .   I  >K.   Ili'k  \>  l  .   Philadelphia.  P 

\i  K.  ['km  .  P.  A..  Simp-oii  Colleui-.  IndianoKi.  lo\\ 

JENNINGS,  DF    H.  S.,  Johns  Hopkins  1  niversit) .  l-Jahimore.  Md. 

JONES,  DR.  LYNDS,  ( »lieilin  Col'.       01  'erlin.  <  >hio. 

Ki  i  i  i  N  .  M  K.   I  .  J  ..  Pni\  er-iiy  of  \\  "\-<  mi -in.  Madi-on.  \\  i~. 

KELLICX>TT,  PROF.  W.  E  ,  Goucher  College,  Baltim-n-e.  Md. 
KIII.N.  MK.   |.  P..  Black  Rotk  A\enue.    I  nionport,    Ne\\    \«vk. 
Ki  \\i  \>\ .  MK.  MAKKI-.  l\.    <!\ille,  M. 
Kl\t,.  1  >K.  Hi  i  i  \  I  >..  \\  i-tar  In-lit nte  of  Anatomy  and  Biol. 

Philadelphia.  Pa. 
Kl\i.-i:t  KV,    PKIII.     I',      1   ..Cornell     I   ni\er-it\     Medical    School, 

\e\\    \'i  >rk  (  it\  . 

KI\(.-I  i  N  .  I  'K.  P  s..  Tuft-  College,  M 

KikKHAM.  I)K.  \\".  P.,  N'ale  Pni\vr-ity.  New  IPi\cn,  Conn. 
K\M\\IK,    Hk.    11.    M*l'...    1   ni\ti-ii\    of   Cincinnati.   Cincinnati. 

Ohio. 
KNOWLTON,   I>K.   l;.    P..   Syracus)     I  niversity,   Syr.acUse,   Ne\\ 

York. 


378  MARINE    BIOLOGICAL   LABORATORY. 

KXUDSOX,  PROF.  LEWIS,  Cornell  University,  Ithaca,  New  York. 
KRIBS,  DR.  HERBERT,  University  of  Pennsylvania,  Phikidelphia, 

Pa. 

LEE,  DR.  F.  S.,  437  West  59th  Street,  New  York  City. 
LEFEVRE,  DR.  GEORGE,  University  of  Missouri,  Columbia,  Mo. 
LEWIS,  DR.  I.  F.,  Randolph-Macon  College,  Ashland,  Va. 
LEWIS,   DR.  WARREN  H.,  Johns  Hopkins  University  Medical 

School,  Baltimore,  Md. 

LILLIE,  DR.  F.  R.,  University  of  Chicago,  Chicago,  111. 
LINTON,    PROF.    EDWIN,    Washington    and    Jefferson    College, 

Washington,  Pa. 
LOEB,  DR.  JACQUES,  Rockefeller  Institute  for  Medical  Research, 

New  York  City. 
LOEB,  DR.  LEO,  St.  Louis  Skin  and  Cancer  Hospital,  St.  Louis, 

Mo. 

LUSCOMBE,  MR.  W.  O.,  Woods  Hole,  Mass. 
LYMAN,  DR.  GEORGE  R.,  Dartmouth  College,  Hanover,  N.  H. 
LYON,  DR.  E.  P.,  St.  Louis  University  Medical  School,  St.  Louis, 

Mo. 
McCLENDON,  DR.  J.  F.,  Cornell  University  Medical  School,  New 

York  City. 

McGiLL,  DR.  CAROLINE,  University  of  Missouri,  Columbia,  Mo. 
McGREGOR,  DR.  J.  H.,  Columbia  University,  New  York  City. 
MclNDOO,  MR.  N.  E.,  University  of  Pennsylvania,  Department 

of  Zoology,  Philadelphia,  Pa. 

MACKENZIE,  PROF.  MARY  D.,  Western  College,  Oxford,  Ohio. 
McKiBBEN,  MR.  PAUL  S.,  University  of  Chicago,  Chicago,  111. 
McMuRRiCH,  DR.  J.  P.,  LTniversity  of  Toronto,  Toronto,  Canada. 
MALL,  DR.  F.  P.,  Johns  Hopkins  University,  Baltimore,  Md. 
MAST,  DR.  S.  O.,  Johns  Hopkins  University,  Baltimore,  Md. 
MATHEWS,  PROF.  A.  P.,  University  of  Chicago,  Chicago,  111. 
MAYER,  DR.  A.  G.,  Carnegie  Institution,  Washington,  D.  C. 
MEIGS,  DR.  E.  B.,  Wistar  Institute  of  Anatomy  and  Biology, 

Philadelphia,  Pa. 

MELTZER,  DR.  J.  S.,  13  West  I2ist  Street,  New  York  City. 
METCALF,  DR.  M.  M.,  Oberlin  College,  Oberlin,  Ohio. 
MINOR,  Miss  MARIE  L.,  Wadleigh  High  School,  H4th  St.  and 

7th  Avenue,  New  York  City. 


i  Hi.  i'iki  •  r<  »RS  EU  P  •;•  ; . 

MOI.NKHAI  -,  Pk<>r.  \\".  J.,  University  of  Indiana.  Bloomington. 

[nd. 
MONTGOMERY,  PK<>F.   T.    H..  Jk.,1  University  of   IYnn-\  l\ania. 

Philadelphia,  Pa. 

Monk!-:.  Pk<>]  .  (',.  T..  Washington  l"ni\  vr-ity,  St.  I.oiii-.  M<> 
Mnnki:.  Pkoi  .  J.   I'l  M  \.  l'ni\vrMty  of  IVnn-vU  ania.  Philadel- 

|)l)i.t.    Pa. 

M»KI.AN,  Pi<i'i  .  II.  A..  Agricultural  Kxpfriim-m  Station,  Km>\- 

\  illr.  Tt-nn. 

M-.KKII.I  .  I'koF.  A.  I)..  Hamilton  ColK-r.  ('lintoii.  \.  \". 
M"KKii  I  .    I  )k.  ( 'n  \KI.I  -    \'..    rimrr-ilv   and    Hi-lk-viu-    Mc-dical 

( lollege,  \i-\v  \"ork  ( 'it\ . 

M«  'k-i  .  1  IK.  M  \\  \\  ..  Trinity  College  .  1 1  art  lord.  I'onn. 
Mi  Kii.M  n.  |)K.  I.MI  i-.  |'..iiltiu-\  .  \\-rmont. 
N.Miiikiiu.   I'K.    II.   1"..   rni\cr-it\    of   Minnr-ota,   Mimu-apoli-. 

Minn. 

MEAL,  DR.  H.  V.,  Knox  College,  <  .  iK--l.ur^.  111. 

\i  \\ M  \\,  1  )i<.  1 1.  II  .  I  niversity  of  Chicago.  C\\  111. 

Nn  HOLS,  Ml--  M.  I.  .  ;-,Jo;  sun unt  i   Street,   Philadelphia,  Pa. 

'  ''.II  \  i  i  .  I  IK.  C.  S.,  I. inc. .In.  111. 

(>KIM\\\.  I)K.  A.  I-"...  ( 'ariu-^ic-  Mu-rnin.  Pitt-l»uri;h,  Pa. 

(  Km  u\.  1  IK.  RAYMOND  C.,  Barnard  College,  New  York  city. 

(i-MKIH'i   1.    1>K.    \Y.    J.    \'.,    Har\ard     Tnixcr-iu,    CaniLri. 

Mass. 
PACKARD,  MR.  CHARLES,  Columhia  I "ni\vr-it\ ,  l  >c|ianmcin  of 

/•  •.  il.  .-^\  .  N.  u   \  i  irk  (  it  y. 

I'M  K\KI>.  iMc.  \\  .  H..  BardlrN    P<  •!>  tcchnic  In-tiiuti-,  Pi-oria.  111. 
P  \i\  1 1  K.  M  K.  T.  S.,  Y.ilr  I  "ni\».-r-it\  .  Nc\\   1  la\  en,  ( 'min. 
PAKKIK.  I>K.  c,.  II.,  i<>  Berkeley  Street,  Cambridge,  Ma~-. 
PATON,    MK.   STEWART,    Princeton    1  Hi\rr-it\  ,    Princrton,    N.   J. 
P\i  n  \.  Mi--  J.  B.,  Simmons  ColK-^i-,  Boston,  Ma--. 
P  \  i  n  \,  1  >K.  \Yii.i.i.\M.  1  >art mouth  College,  Hano\,-r,  N  .  II. 
PviiiK-tiN.  |)K.  J.  1'.,  University  of  Texas,  Austin,  Texas. 
PAN\I.    I>K.   pERNANDUS,   I  ni\«.-r-ii\    of   Indiana,  Bloomington, 

[nd. 

Pi  \K-I  .  I>K.  A.  S.,  1'nivi-r-ity  of  Michi-an.  Ann  Arbor,  Mich. 
PIKI  .  1>K.  1  KANK.  H..  I'niversity  of  Chicago,  C'hicago.  111. 
1  Deceased. 


380  MARINE    BIOLOGICAL    LABORATORY. 

PKKNTISS,  Miss  HENRIETTA,  Normal  College,  New  York  City. 

<  M  \<  KENBUSH,  MR.  L.  S.,  2~  West  73cl  Street,  New  York  City. 

RANDOLPH,  DR.  HARRIET,  Bryn  Mawr  College,  Pa. 

RANKIN,  PROF.  \V.  M.,  Princeton  University,  Princeton,  N.  J. 

REA,  PROF.  PAUL  M.,  College  of  Charleston,  Charleston,  S.  C. 

REIGHARD,  PROF.  JACOB,  University  of  Michigan,  Ann  Arbor,- 
Mich. 

RICE,  PROF.  EDWARD  L.,  Ohio  Wesleyan  University,  Delaware, 
Ohio. 

ROGERS,  DR.  CHARLES  G.,  Syracuse  University,  Syracuse,  New 
York. 

ROMINE,  MR.  A.  P.,  1801  "I"  Street,  Bellingham,  Wash. 

SCOTT,  DR.  G.  G.,  College  of  the  City  of  New  York. 

SCOTT,  DR.  J.  W.,  Westport  High  School,  Kansas  City,  Mo. 

SCOTT,  DR.  YV.  B.,  Princeton  University,  Princeton,  N.  J. 

SHOREY,    DR.    MARIAN    L.,    Milwaukee-Downer    College,    Mil- 
waukee, \Vis. 

SMITH,  DR.  BERTRAM  G.,  University  of  Wisconsin,  Madison,  \Vis. 

SMITH,  DR.  ERWIN  F.,  U.  S.  Department  of  Agriculture,  Wash- 
ington, D.  C. 

SOLI. MAN.  DR.  TOR  A  LI>,  Western  Reserve  University,  Cleveland, 
Ohio. 

SPAVLDING,  PROF.  E.  G.,  Princeton  University,  Princeton,  N.  J. 

SPENCER,  MR.  HENRY  J.,  Columbia  University,  New  York  City. 

SPOONER,  Miss   GEORGINA  B.,  161    Bryant  Street,  Palo  Alto, 
Calif. 

STOCKARD,  DR.  C.  R.,  Cornell  University  Medical  School,  New 
York  City. 

STREETER,  DR.  GEORGE  L.,  University  of  Michigan,  Ann  Arbor, 
Mich. 

Si  RUNG,  DR.  O.  S.,  College  of  Physicians  and  Surgeons,  New 
York  City. 

S-IK<>\G,  DR.  R.  M.,  University  of  Chicago,  Chicago,  111. 

Si  MNER,  DR.  F.  B.,  Woods  Hole,  Mass. 

TAYLOR,   Mi-^   K.vi  HAKIM    A.,   Cascade,   Washington  County, 

M.IIA  1,111(1. 

TENNENT,  DR.  1).  H.,  \\\-\\\  Mawr  College,  I'a. 
TKKRY,  DR.  ().  P.,  Purdue  University,  L.il".i\rtte,  Ind. 


DIRECTOR'S  REPORT.  581 

Tll'iMI'-nN.     Ml--    r.\K<>!.lM.     IV,     In^    \\Y-tnll     Road,    \\VlU-lrY. 

Mass. 

TINKHAM.    Ml--    FLORENCE    I..,    -<>   "lYmpK-  Street,   Sprinutk-M, 

M 

TiK'MA-.'  Dk.  M  \-M\  IV.  \V.ilM-h  College,  l"r.i\vt"onl-\  illc.  Iml. 
T'lMi-Kis-.  Mi--  KI.I/AHKTH  M.,  1770  K.  I4th  Sm-ri.  Bn>,,klvM. 

\Y\v  Y'.rk. 

I     •.  i  K.  l'i-"i    W.  L.,  University  of  Chi     -      i  "hir,i-<>,  111. 
TKI  \n\\  i  i  i.,  I'K'.I  .  A.  I,.,  Vassar  College,  Pi'imlikn-p-Y.  NY\\ 

Y..rk. 

R,  SUSANNAH,  1008  W.  ( Oregon  Street,  I  "HMMJ,  III. 
\\'\in.   I'K'ni.   I  .  '  ..  Western   Reserve  University,  I' 

Ohio. 

\\'\lKlk.    I>K.   GEORGE,   <   li.uh  -  and   lYiiu-r  Stivrt-.    H.i 

Md. 
\\  \l  LACE,     Pivi'i  .    l.'-i  l-l      IV.    M"Uiit     ll'>l\"k«     1'i'llr^i  . 

1   l.ldli    \    ,     M     : 

WATSON,  I  >K.  I  K  \\K  I-'...  218  \\  .i-hiiui'-n  Street,  Geneva,  \    Y. 

\Yi  KI:I  K,     I  )i-      I        I   .     [ohns     ll'ipkin-    I  "ni\  n -ii\  ,    Aii.ii«  unii  ,d 

l..ili<nMi"iA  .  IVih inn »re,  Md. 
\Yiii  i  i  i  K.  I'KMI  .  \\  .  M..  Hu— 1-\  hi-iiiutiiiii.  !•".  in-t  Hill-.  IJc.-tnii, 

Mass. 

\\  in  i  \i  \ .  I  >i-:.  I  >  \\  in  1  >..  \\  •  -1-  \  in  ( 'oil i xr.  Middletown,  Conn. 
\\n  \i\\.  1>K.  II.  I...  1  ni\«i-ii\  «'|  ( "iin  iiin.iti,  ( "iiiciiiii.ui.  ()liii». 
\\'n  •  •  i\.  M  I--  \i.n  i  \\  ..  5<«  Al ii inni  A\r..  l'r<i\  i.lriu  »•.  l\.  1. 

\\ni'M\\,  hiv.  1     I    .  ^331  Os  igi    Vvenue,  riiil.idi-lplii.i.  I'.i. 

\\  li  l  i  \M-,  I  >K.  ANNA  \\ '..  ^\<>  l\i\<  i  -id»-  I  >ri\  e,   \«\\    Y<>rk  (  ity. 
\\  n  -»\.  I'KMI     1 1    \  ..  I  'ni\rr-it\  .  •!'  \«.nh  ( '.imliii.i.  ( 'h.iprl  Hill, 
\    < 

• 

\\'.  >i  i  i  .  1  >K.  |  \MI  -  J ..  Trinity  College,  1  Durham,  \    ' 

\\'<  H  >DKI  i  i  .  PK.  L.  I...  ^^  .ilr  t  ni\  rr-it\  .  NtA\   I  l.i\ »  n.  ( '« nin. 

\\KH, HI,    I'KOI-".    1\.    K\M-\N.    I  ni\»T-it\    "I    T<  ir- mi 

(  '.in. id. i. 
YERKES,    PK.     K-T.IKI     M.,    ll.ir\.ird     I'liivrr-iiy. 

Mass. 

i  > 


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