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BIOLOGICAL  BULLETIN 


OF  THE 


HDarine  Biological  Haborator? 


WOODS  HOLE,  MASS. 


lEMtorial  Staff 

GARY  N.  CALKINS  —  Columbia    University. 
E.  G.  CONKLIN  —  Princeton  University. 
M.   H.  JACOBS  —  University  of  Pennsylvania. 
FRANK   R.  LILLIE  —  University  of  Chicago. 

GEORGE  T.  MOORE  —  The  Missouri  Botanic  Garden. 
T.  H.  MORGAN  —  Columbia  University. 
W.  M.  WHEELER  —  Harvard  University. 
E.  B.  WILSON  —  Columbia  University. 


JEMtor 

C.   R.   MOORE  —  The    University  of  Chicago. 


VOLUME  LV. 


WOODS  HOLE,  MASS. 


JULY  TO  DECEMBER,   1928 


LANCASTER   PRESS, INC. 
LANCASTER,  PA. 


Contents  of  Volume  LV 


No.  i.    JULY,  1928. 
Thirtieth  Annual  Report  of  the  Marine  Biological  Laboratory.        I 

No.  2.     AUGUST,  1928. 
KING,  ROBERT  I.     The  Contractile  Vacuole  in  Paramecium 

trichium 59 

MAIN,  HOLLAND  J.     Observations  of  the  Feeding  Mechanism 

of  a  Ctenophore,  Mnemiopsis  leidyi 69 

AMBERSON,  WILLIAM  R.     The  Influence  of  Oxygen  Tension 

upon  the  Respiration  of  Unicellular  Organisms 79    « 

BOYD,  MARJORIE.     A  Comparison  of  the  Oxygen  Consumption 

of  Unfertilized  and  Fertilized  Eggs  of  Fundulus  heteroditus     92  * 
CALKINS,  GARY  N.,  AND  BOWLING,   RACHEL.     Studies  on 

Dallasia  frontata  Stokes 101 

PARPART,  ARTHUR  K.     The  Bacteriological  Sterilization  of 

Paramecium 113* 

HUESTIS,  R.  R.  The  Effect  of  Maternal  Age  and  of  Temper- 
ature Change  in  Secondary  Non-Disjunction 121 

MELVIN,  ROY.     Oxygen  Consumption  of  Insect  Eggs 135  • 

No.  3.     SEPTEMBER,  1928. 

HILL,  SAMUEL  E.  The  Influence  of  Molds  on  the  Growth  of 
Luminous  Bacteria  in  Relation  to  the  Hydrogen  Ion 
Concentration,  Together  with  the  Development  of  a 
Satisfactory  Culture  Method 143 

IVAROL,  JOHN  J.     The  Sex  Ratio  in  Peromyscus 151 

PAYNE,  NELLIE  M.  Cold  Hardiness  in  the  Japanese  Beetle, 

Popillia  japonica  Newman 163 

NELSON,  THURLOW  C.  Pelagic  Dissoconchs  of  the  Common 
Mussel,  Mytilus  edulis,  with  Observations  on  the  Behavior 
of  the  Larv  >  of  Allied  Genera 180 

TURNER,  C.  L.  Studies  on  the  Secondary  Sexual  Characters 
of  Crayfishes. —  VI.  A  Female  of  Cambarus  immunis 
with  Oviducts  Attached  to  Openings  of  Sperm  Ducts.  ...  193 


^(LIBRARY 


.'51570 


IV  CONTENTS    OF   VOLUME    LY. 

TURNER,  C.  L.  Studies  on  the  Secondary  Sexual  Characters 
of  Crayfishes. —  VII.  Regeneration  of  Aberrant  Secon- 
dary Sexual  Characters 197 

SAYLES,  LEONARD  P.  Regeneration  of  Lumbriculus  in 

Various  Ringer  Fluids 202 

ALPATOV,  W.  W.  Variation  of  Hooks  on  the  Hind  Wing  of 

the  Honey  Bee  (Apis  mellifera  L.} 209 

No.  4.     OCTOBER,  1928. 

HARMAN,  MARY  T.,  AND  ROOT,  FRANK  P.  The  Development 

of  the  Spermatozoon  in  Cavia  cobaya 235 

TURNER,  C.  L.  Studies  on  the  Secondary  Sex  Characters  of 
Crayfishes,  VIII.  Modified  Third  Abdominal  Ap- 
pendages in  Males  of  Cambarus  virilis 255 

GRAVE,  B.  H.  Natural  History  of  Shipworm,  Teredo 

navalis,  at  Woods  Hole,  Massachusetts 260 

NEWMAN,  H.  H.  Studies  of  Human  Twins,  I.  Methods  of 

Diagnosing  Monozygotic  and  Dizygotic  Twins 283 

NEWMAN,  H.  H.  Studies  of  Human  Twins,  II.  Asym- 
t  metry  Reversal,  of  Mirror  Imaging  in  Identical  Twins.  .  298 

No.  5.     NOVEMBER,  1928. 

HUMPHREY,  R.  R.  Sex  Differentiation  in  Gonads  Developed 
from  Transplants  of  the  Intermediate  Mesoderm  of 
Amblystoma 317 

MOORE,  CARL  R.  On  the  properties  of  the  Gonads  as  Con- 
trollers of  Somatic  and  Psychical  Characteristics,  XI.  .  . .  339 

JUST,  E.  E.  Initiation  of  Development  in  Arbacia,  VI.  The 
Effect  of  Slowly  Evaporating  Sea-Water  and  its  Signifi- 
cance for  the  Theory  of  Auto-Parthenogenesis 358 

CHAMBERS,  ROBERT.  Intracellular  Hydrion  Concentration 
Studies,  I.  The  Relation  of  the  Environment  to  the  pH 
of  Protoplasm  and  of  Its  Inclusion  Bodies 369 

REZNIKOFF,  PAUL,  AND  POLLACK,  HERBERT.  Intracellular 
Hydrion  Concentration  Studies,  II.  The  Effect  of  In- 
jection of  Acids  and  Salts  on  the  Cytoplasmic  pH  of  Amoeba 
dubia 377 

POLLACK,  HERBERT.  Intracellular  Hydrion  Concentration 
Studies,  III.  The  Buffer  Action  of  the  Cytoplasm  of 
Amoeba  dubia  and  Its  JJse  in  Measuring  the  pH 383 


CONTENTS    OF   VOLUME    LY. 

GREGORY,  LOUISE  H.  The  Effects  of  Changes  in  Medium 
during  Different  Periods  in  the  Life  History  of  Uroleptus 
mobilis  and  Other  Protozoa 386 

No.  6.     DECEMBER,  1928. 

BODINE,  JOSEPH  HALL.     Insect  Metabolism 395  • 

LLOYD,  FRANCIS  E.,  AND  BEATTIE,  J.  The  Pulsatory 

Rhythm  of  the  Contractile  Vesicle  in  Paramecium 404 

THRELKELD,  W.  L.,  AND  HALL,  S.  R.  Observations  on  Hydra 
and  Pelmatohydra  Under  Determined  Hydrogen  Ion 
Concentration 4J9 

MAN  WELL,  REGINALD  D.  The  Occurrence  of  Nuclear  Vari- 
ations in  Pleurotricha  lanceolata  (Stein) 433 

QUIGLEY,  J.  P.  Observations  on  the  Life  History  and  Physio- 
logical Condition  of  the  Pacific  Dog  Fish  (Squalus  sucklii)  439 

FARLOWE,  VIVIAN.  Algce  of  Ponds  as  Determined  by  an 

Examination  of  the  Intestinal  Contents  of  Tadpoles 443 

PAGE,  IRVINE  H.  Further  Observations  on  the  Chemical 
Composition  of  Woods  Hole  Sea  Water — The  Chlorine 
Content  and  Salt  Analysis 449  • 

KAPP,  ELEANOR  M.  The  Precipitation  of  Calcium  and 

Magnesium  from  Sea  Water  by  Sodium  Hydroxide 453 

HARVEY,  E.  NEWTON,  HARVEY,  ETHEL  B.,  AND  LOOMIS, 
ALFRED  L.  Further  Observations  on  the  Effect  of  High 
Frequency  Sound  Waves  on  Living  Matter 459 


Vol.  LV 


July  1928 


No.  i 


BIOLOGICAL  BULLETIN 


THE  MARINE  BIOLOGICAL  LABORATORY. 


.1. 


II. 
III. 

IV. 

V. 

VI. 


THIRTIETH  REPORT  FOR  THE  YEAR  1927— 
FORTIETH  YEAR. 

TRUSTEES  AND  EXECUTIVE  COMMITTEE  (AS  OF  AUGUST 

9>  1927)    * 

LIBRARY  COMMITTEE   3 

ACT  OF  INCORPORATION  3 

BY-LAWS  OF  THE  CORPORATION  4 

REPORT  OF  THE  TREASURER  5 

REPORT  OF  THE  LIBRARIAN  1 1 

REPORT  OF  THE  DIRECTOR  17 

Statement    17 

Addenda : 

1 .  The  Staff,  1927 27 

2.  Investigators  and  Students,  1927 30 

3.  Tabular  View  of  Attendance 41 

4.  Subscribing  and  Cooperating  Institutions,  1927  42 

5.  Evening  Lectures,  1927 43 

6.  Members  of  the  Corporation   44 


I.     TRUSTEES. 

EX    OFFICIO. 

FRANK  R.  LILLIE,  President  of  the  Corporation,  The  University  of 

Chicago. 

MERKEL  H.  JACOBS,  Director,  University  of  Pennsylvania. 
LAWRASON   RIGGS,  JR.,   Treasurer,  25  Broad  Street,   New  York  City. 
L.  L.  WOODRUFF,  Clerk  of  the  Corporation,  and  Secretary  of  the  Board 

of  Trustees  pro  tan,  Yale  University. 

EMERITUS. 

CORNELIA  M.  CLAPP,  Mount  Holyoke  College. 
OILMAN  A.  DREW,  Eagle  Lake,  Florida. 

TO    SERVE    UNTIL     IQ3I. 

H.   C.   BUMPUS,   Brown   University. 
W.  C.  CURTIS,  University  of  Missouri. 
1  i 


2  MARINE    BIOLOGICAL    LABORATORY. 

B.  M.  DUGGAR,  University  of  Wisconsin. 

GEORGE  T.  MOORE,  Missouri  Botanical  Garden,  St.  Louis. 
W.  J.  V.  OSTERHOUT,  Member  of  the  Rockefeller  Institute  for  Med- 
ical Research. 

J.  R.   SCHRAMM,  University  of  Pennsylvania. 
WILLIAM  M.  WHEELER,  Bussey  Institution,  Harvard  University. 
LORANDE  L.  WOODRUFF,  Yale  University. 

TO    SERVE    UNTIL    I93O. 

E.  G.  CONKLIN,  Princeton  University. 
OTTO  C.  GLASER,  Amherst  College. 
Ross  G.   HARRISON,  Yale  University. 

H.  S.  JENNINGS,  John  Hopkins  University. 

F.  P.  KNOWLTON,   Syracuse  University. 

M.    M.   METCALF,   Johns   Hopkins   University. 
WILLIAM  PATTEN,  Dartmouth  College. 
W.  B.  SCOTT,  Princeton  University. 

TO   SERVE   UNTIL    1929. 

C.  R.   CRANE,  New  York  City. 

I.   F.   LEWIS,   University  of   Virginia. 

R.   S.  LILLIE,  The  University  of   Chicago. 

E.  P.  LYON,  University  of  Minnesota. 

C.  E.   McCLUNG,   University  of   Pennsylvania. 
T.  H.  MORGAN,  Columbia  University. 

D.  H.  TENNENT,  Bryn  Mawr  College. 

E.  B.   WILSON,   Columbia   University. 

TO    SERVE    UNTIL    1928. 

H.   H.   DONALDSON,  Wistar   Institute   of   Anatomy   and   Biology. 

W.  E.  GARREY,  Vanderbilt  University  Medical    School. 

CASWELL  GRAVE,  Washington  University. 

M.  J.  GREENMAN,  Wistar  Institute  of  Anatomy  and  Biology. 

R.  A.  HARPER,  Columbia  University. 

A.  P.  MATHEWS,  The  University  of  Cincinnati. 

G.  H.  PARKER,  Harvard  University. 

C.  R.   STOCKARD,  Cornell  University  Medical   College. 

EXECUTIVE  COMMITTEE  OF  THE  BOARD  OF  TRUSTEES. 

FRANK  R.  LILLIE,  Ex.  Off.  Chairman. 
MERKEL  H.  JACOBS,  Ex.   Off. 
LAWRASON   RIGGS,  JR.,  Ex.   Off. 
OTTO  C.  GLASER,  to  serve  until  1928. 
CASWELL  GRAVE,  to   serve  until    1928. 
E.  G.  CONKLIN,  to  serve  until   1929. 
C.  R.  STOCKARD,  to  serve  until   1929. 


ACT   OF    INCORPORATION. 


THE  LIBRARY  COMMITTEE. 


C.   E.   McCLUNG,   Chairman. 
ROBERT  A.  BUDINGTON. 
B.  M.  DUGGAR. 
E.  E.  JUST. 
FRANK  R.  LII.LIE. 
M.   M.   METCALF. 
ALFRED  C.  REDFIELD. 
A.  H.   STURTEVANT. 
L.  L.  WOODRUFF. 


II.     ACT  OF  INCORPORATION. 

No.  3170 

COMMONWEALTH  OF  MASSACHUSETTS. 

Be  It  Known,  That  whereas  Alpheus  Hyatt.  William  San  ford  Ste- 
vens, William  T.  Sedgwick,  Edward  G.  Gardiner,  Susan  Minns. 
Charles  Sedgwick  Minot,  Samuel  Wells,  William  G.  Farlow,  Anna  D. 
Phillips  and  B.  H.  Van  Vleck  have  associated  themselves  with  the 
intention  of  forming  a  Corporation  under  the  name  of  the  Marine 
Biological  Laboratory,  for  the  purpose  of  establishing  and  maintaining 
a  laboratory  or  station  for  scientific  study  and  investigation,  and  a 
school  for  instruction  in  biology  and  natural  history,  and  have  complied 
with  the  provisions  of  the  statutes  of  this  Commonwealth  in  such  case 
made  and  provided,  as  appears  from  the  certificate  of  the  President, 
Treasurer,  and  Trustees  of  said  Corporation,  duly  approved  by  the 
Commissioner  of  Corporations,  and  recorded  in  this  office; 

Now,  therefore,  I,  HENRY  B.  PIERCE,  Secretary  of  the  Common- 
wealth of  Massachusetts,  do  hereby  certify  that  said  A.  Hyatt,  W.  S. 
Stevens,  W.  T.  Sedgwick,  E.  G.  Gardiner,  S.  Minns,  C.  S.  Minot,  S. 
Wells,  W.  G.  Farlow,  A.  D.  Phillips,  and  B.  H.  Van  Vleck,  their  asso- 
ciates and  successors,  are  legally  organized  and  established  as,  and  are 
hereby  made,  an  existing  Corporation,  under  the  name  of  the  MARINE 
BIOLOGICAL  LABORATORY,  with  the  powers,  rights,  and  privi- 
leges, and  subject  to  the  limitations,  duties,  and  restrictions,  which  by 
law  appertain  thereto. 

Witness  my  official  signature  hereunto  subscribed,  and  the  seal   of 
the  Commonwealth  of   Massachusetts  hereunto  affixed,  this  twentieth 
day  of  March,  in  the  year  of  our  Lord  One  Thousand,  Eight  Hundred 
and  Eighty-Eight. 
[SEAL]  HENRY  B.  PIERCE, 

Secretary  of  the   Commonwealth. 


4  MARINE    BIOLOGICAL    LABORATORY. 

III.     BY-LAWS    OF    THE    CORPORATION    OF 
THE  MARINE  BIOLOGICAL  LABORATORY. 

I.  The  annual  meeting  of  the  members  shall  be  held  on  the  second 
Tuesday    in   August,   at   the   Laboratory,    in   Woods   Hole,   Mass.,   at 
12  o'clock  noon,  in  each  year,  and  at  such  meeting  the  members  shall 
choose  by  ballot  a  Treasurer  and  a  Clerk,   who   shall  be,   ex  officio, 
members  of  the  Board  of  Trustees,  and  Trustees  as  hereinafter  pro- 
vided.    At   the   annual    meeting   to   be   held    in    1897,    not   more   than 
twenty-four  Trustees  shall  be  chosen,  who  shall  be  divided  into  four 
classes,   to   serve   one,   two,   three,   and    four   years,    respectively,   and 
thereafter  not  more  than  eight  Trustees  shall  be  chosen  annually  for 
the   term   of    four   years.     These   officers   shall   hold   their    respective 
offices  until  others  are  chosen  and  qualified  in  their  stead.     The  Presi- 
dent of  the  Corporation,  the  Director  and  the  Associate  Director  of 
the  Laboratory,  shall  also  be  Trustees,  ex  officio. 

II.  Special  meetings  of  the  members  may  be  called  by  the  Trustees  to 
be  held  in  Boston  or  in  Woods  Hole  at  such  time  and  place  as  may  be 
designated. 

III.  The  Clerk  shall  give  notice  of  meetings  of  the  members  by  pub- 
lication 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  af- 
fairs of  the  Corporation ;  they  shall  present  a  report  of  its  condition  at 
every  annual  meeting;  they  shall  elect  one  of  their  number  President  of 
the  Corporation  who  shall  also  be  Chairman  of  the  Board  of  Trustees ; 
they  shall  appoint  a  Director  of  the  Laboratory;  and  they  may  choose 
such  other  officers  and  agents  as  they  may  think  best ;  they  may  fix  the 
compensation  and  define  the  duties  of  all  the  officers  and  agents ;  and 
may  remove  them,  or  any  of  them,  except  those  chosen  by  the  members, 
at  any  time;  they  may  fill  vacancies  occurring  in  any  manner  in  their 
own  number  or  in  any  of  the  offices.     They  shall   from  time  to  time 
elect  members  to  the  Corporation  upon  such  terms  and  conditions  as 
they  may  think  best. 

VI.  Meetings  of  the  Trustees  shall  be  called  by  the  President,  or  by 
any  two  Trustees,  and  the  Secretary  shall  give  notice  thereof  by  written 
or    printed    notice  sent     to    each    Trustee    by    mail,    postpaid.     Seven 
Trustees   shall   constitute   a   quorum   for   the   transaction   of   business. 
The  Board  of  Trustees  shall  have  power  to  choose  an  Executive  Com- 


TIFK    KKPORT    OF    THE    TREASURER.  5 

mittee  from  their  own  number,  and  to  delegate  to  such  Committee  such 
of  their  own  powers  as  they  may  deem  expedient. 

VII.  The  accounts  of  the  Treasurer  shall  be  audited  annually  by  a 
certified  public  accountant. 

VIII.  The  consent  of  every  Trustee  shall  be  necessary  to  dissolution 
of  the  Marine  Biological  Laboratory.     In  case  of  dissolution,  the  prop- 
erty shall  be  disposed  of  in  such  manner  and  upon  such  terms  as  shall 
be  determined  by  the  affirmative  vote  of  two-thirds  of  the  Board  of 
Trustees. 

IX.  These  By-laws  may  be  altered  at  any  meeting  of  the  Trustees, 
provided  that  the  notice  of  such  meeting  shall  state  that  an  alteration 
of  the  By-laws  will  be  acted  upon. 

X.  Any  member  in  good  standing  may  vote  at  any  meeting,  either  in 
person  or  by  proxy  duly  executed. 


IV.  THE  REPORT  OF  THE  TREASURER. 

To  THE  TRUSTEES  OF  THE  MARINE  BIOLOGICAL  LABORATORY. 

Gentlemen:  As  Treasurer  of  the  Marine  Biological  Laboratory, 
I  herewith  submit  my  report  for  the  year  1927. 

The  books  have  been  audited  by  Messrs.  Seamens,  Stetson  & 
Tuttle.  A  copy  of  their  report  is  on  file  at  the  laboratory  and  is 
open  to  inspection  by  any  member  of  the  Corporation. 

There  were  no  changes  in  the  investments  in  the  Endowment 
Fund  and  that  Fund  at  the  close  of  the  year  consisted  of  securi- 
ties of  the  book  value  of  $906,337.50  and  cash  of  $112.  The  in- 
come from  the  Endowment  Fund  for  the  year  was  $47,583  and 
the  fee  of  the  Trust  Company  as  Trustee  was  $787.50,  leaving  a 
net  income  from  the  Endowment  Fund  of  $46,795.50.  The  full 
list  of  the  securities  will  be  found  in  the  Auditors'  report. 

At  the  end  of  the  year  the  Lucretia  Crocker  Fund  consisted  of 
securities  of  the  book  value  of  $3,590.59  and  cash  of  $1,093.17. 

During  the  year  a  fund  of  Two  Thousand  Dollars  ($2,000) 
invested  in  a  note  secured  by  Chicago  real  estate  was  presented  to 
the  Laboratory  to  found  the  IDA  H.  HYDE  SCHOLARSHIP  and  the 
fund  remained  invested  in  this  security  at  the  end  of  the  year. 

The  Retirement  Fund  consisted  of  Seven  Thousand  Dollars 
($7,000)  invested  in  participations  in  bonds  secured  by  mortgages 
on  New  York  City  real  estate  and  $147.76  in  cash. 


6  MARINE    BIOLOGICAL    LABORATORY. 

The  land,  buildings,  library  and  equipment  of  the  Laboratory 
including  the  new  apartment  house  and  dormitory  but  excluding 
the  Gansett  and  Devils  Lane  property  represents  an  investment  of 
$1,545,616.36,  and  after  deducting  $136,181.18  for  depreciation, 
a  book  value  of  $1,409,435.18. 

During  the  year  the  following  donations  were  received : 

From   General   Education   Board   for   improving  the   facilities  of  the 

Library     $15,000 

From  Dr.  Frank  R.  Lillie  for  grading  and  planting 1,500 

During  the  year  Two  Thousand  Dollars  ($2,000)  was  paid  off 
on  account  of  the  Danchakoff  mortgage,  and  the  indebtedness  of 
the  Laboratory  at  the  end  of  the  year  consisted  of  $6,542.83  in 
accounts  payable,  and  $42,500  in  mortgages  on  its  real  estate. 

The  expenditures  closely  approximated  the  estimates  for  the 
year  and  including  an  item  of  depreciation  of  almost  $29,000 
exceeded  the  income  for  the  year  by  $91.06.  Against  this  item  of 
depreciation  the  sum  of  almost  $20,000  was  expended  out  of 
current  funds  upon  permanent  improvements  and  equipment. 

Since  January  I,  1916,  the  Laboratory  has  adopted  the  policy 
of  charging  income  and  crediting  reserve  for  depreciation  each 
year  with  2  per  cent,  of  the  book  value  of  the  buildings  and  5 
per  cent,  of  the  book  value  of  equipment  and  library.  This  de- 
preciation at  the  end  of  the  year  1927  amounted  to  $136,181.18. 
It  is  interesting  to  note,  however,  that  against  this  the  Laboratory 
has  spent  from  current  cash  approximately  $141,000  in  perma- 
nent improvements,  thus  more  than  meeting  the  depreciation 
charge  by  improvements  paid  for  out  of  income. 

Following  is  the  balance  sheet  at  the  end  of  the  year  and  the 
condensed  statement  of  income  and  outgo  for  the  year,  also  the 
Surplus  Account.  The  figures  are  those  reported  by  the  Auditors, 
arranged  in  the  case  of  Exhibit  B  so  as  to  conform  to  the  system 
followed  in  previous  reports. 


THE   REPORT    OF    THE    TREASURER. 

EXHIBIT  A. 

MARINE  BIOLOGICAL  LABORATORY  BALANCE  SHEET, 
DECEMBER  31,  1927. 

Assets. 
Endowment  Fund  Assets : 

Securities  in  Hands  of  Trustee — Schedule  I.  $    906,337.50 
Investment  Cash  in  Hands  of  Trustees   .  112.00 


$    906,449.50 
Lucretia  Crocker  Fund  Assets, 

Securities — Schedule    II $     3,590.59 

Cash — Schedule  II 1,093.17  4,683.76 

Ida  H.  Hyde  Fund  Assets, 

Securities    $        2,000.00    $    913,133.26 

Plant  Assets : 

Land — Schedule   III $i  13,603.05 

Buildings — Schedule   III 966,279.78 

Equipment — Schedule   III 126,197.40 

Library — Schedule    III 90,682.87  $1,296,763.10 

Less  Reserve  for  Depreciation   136,181.18 

$1,160,581.92 

Cost    of    New    Dormitory    and    Apartment 
House  Buildings  to  December  31,    1927— 

Schedule  IV   $    248,853.26 

Cash  in  Dormitory  Building  Fund   3,590.31    $1,413,025.49 

Current  Assets : 
Cash, 

In   New   York   Bank    $     2,335.99 

In  Hands  of  Trustee   2,200.00 

In    Falmouth    Bank    1,558.19 

Petty  Cash    500.00  $       6,594.18 

Accounts — Receivable    20,440.55 

Inventories, 

Supply    Department    $  30,802.33 

Biological    Bulletin     6,237.30         37,039.63 

Investments, 

Devil's    Lane    Property $  33,395.51 

Gansett    Property    1,769.35 

Stock    in    General    Biological 

Supply  House,  Inc 12,700.00 

Retirement  Fund  Assets   ...         7,147.76         55,012.62 

Prepaid    Insurance    ...  4-3/8.33         123,465.31 

$2,449,624.06 


\u~l    L 

',  -*-«••*• 


8  MARINE    BIOLOGICAL    LABORATORY. 

Liabilities. 
Endowment  Funds : 

Friendship  Fund,   Inc $    405,000.00 

John  D.  Rockefeller,  Jr 400,000.00 

Carnegie   Corporation    100,000.00 

Gain  on  sale  of  Securities   1,449.50 


906,449.50 

Lucretia  Crocker  Fund   4,683.76 

Ida   H.   Hyde   Fund    2,000.00   $    913,133.26 


Plant  Funds  : 

Rockefeller    Foundation    500,000.00 

Friendship  Fund  Gift  of   1925    221,608.61 

General  Education  Board  for  Buildings   ....        250,000.00 
General     Education     Board      for 

Books    $25,000.00 

Less   Unexpended   in   Current 

Cash    1,300.50         23,699.50 


Other    Investments    in    Plants     from    Gifts 

and  from  Current  Funds    399,525.38 


$1,394,83349 

Mortgages  on  Drew  and  Danchakoff  Estates          17,500.00 
Suspense — Interest  on  Building  Fund  Cash  ;  692.00      1,413,025.49 


Current  Liabilities  and  Surplus : 

Mortgage  Note  on  Devil's  Lane  Property..  25,000.00 

Accounts — Payable     6,542.83 

Items    in    Suspense    (net)     222.47 


$      31,765-30 
Current    Surplus— Exhibit    C    91,700.00         123,465.31 


$2,449,624.06 

EXHIBIT  B. 

MARINE   BIOLOGICAL   LABORATORY,   INCOME  AND  EXPENSE, 
FOR  THE  YEAR  ENDED  DECEMBER  31,  1<)2"J . 

Total.  Net. 

Expense.         Income.         Expense.        Income. 

Income,    Endowment    Fund  $  47,583.00  $47,583.00 

Donations       (See      Current 
Surplus) 

Instruction    7,829.30         10,640.00  2,810.70 

Research    3,598.86         14,525.00  10,926.14 

BIOLOGICAL    BULLETIN    and 
Membership   Dues    7,117.54          7,659-51  54J-97 


THE    REPORT    OF   THE    TREASURER. 


Supply  Department,  Sched- 
ule IV  52,174.46  59,820.90  7,646.44 

Mess,  Schedule  V    33,08542        36,180.30  3,«94-88 

Dormitories,     Schedule     VI       25,870.11         12,865.29       13,004.82 

Interest  and  depreciation 
charged  to  above  three  de- 
partments, See  Schedules 
IV,  V  and  VI 29,719.11  29,719.11 

Dividends  on  Stock,  Gen- 
eral Biological  Supply 
House,  Inc 2,540.00  2,540.00 

Rent  of  Danchakoff  Cot- 
tages    449-99  750.00  300.01 

Rent  of   Microscopes    3?o.oo  370.00 

Rent    of    Garage,    Railway, 

etc 356.91  356.91 

Rent    of    Newman    Cottage  164.08  150.00  14.08 

Interest  on  Bank  Balances.  .  I37-2O  I37-2O 

Medical    Fees    114.00  114.00 

Sundry   Items    15-31  15-31 

Maintenance  of   Plant : 

New  Laboratory  Expense       15,044.43  15,044.43 

Maintenance  of   Buildings 

and    Grounds     13,296.19  13,296.19 

Chemical  and  Special  Ap- 
paratus   Department    . .         9,088.83  9,088.83 
Library    Department    Ex- 
penses           7,958.73  7-958.73 

Carpenter  Department  Ex- 
penses   1,123.04  1,123.04 

Truck    Expenses     1,203.52  1,203.52 

Sundry    Expenses     814.22  814.22 

Bar  Neck  Property  Ex- 
penses    405.00  405.00 

Evening    Lectures    159-51  I59-5I 

Workmen's    Compensation 

Insurance    627.43  627.43 

General  Expenses  : 

Administration   Expenses.        12,335.00  12,335.00 

Interest  on   Loans    1, 168.00  1, 168.00 

Endowment  Fund  Trustee  787.50  787.50 

Bad    Debts     230.20  230.20 

Contribution  for  Research 
Space,  Naples  Zoolog- 
ical Station  250.00  250.00 

Reserve    for   Depreciation..       28,736.23  28,736.23 

Excess  of  Expense  over  In- 
come carried  to  Current 
Surplus— Exhibit  C  91.06  91.06 


$193.798.48     $193.798.48  $106,246.73  $106,246.73 


IO  MARINE    BIOLOGICAL   LABORATORY. 

I 

EXHIBIT  C. 

MARINE  BIOLOGICAL  LABORATORY,  CURRENT  SURPLUS  ACCOUNT, 
YEAR  ENDED  DECEMBER  31,  1927. 

Balance,  January    i,    1927    $  83,503.64 

Add: 

Donations  Received, 

From  General  Education  Board  for  purchase  of  Books 

for   Library    15,000.00 

From  Dr.  Frank  R.  Lillie  for  Grading,   Planting,  etc. 
on    Laboratory   Grounds    and   around    Drew    House, 

Apartment   House,   and  Whitman   House    1,500.00 

Income  of  Retirement  Fund  Assets    222.82 

Reserve   for   Depreciation  charged  to   Plant   Fund    28,736.23 


$128,962.69 
Deduct : 

Payments  from  Current  Funds  during  Year 
Plant  Assets  as  shown  in  Schedules  III 
and  Ill-a, 

Cost  of  completing  Sea  Wall    $     435.50 

Buildings  7,303.90 

Equipment  4,312.23 

Library  Books,  etc 7,623.94 

New  Dormitory  and  Apartment  House   ....          251.57 


$19,927.14 

Payments    from    above    Donations    charged    to 
Plant  Assets   General   Education   Board, 

Purchase  of  Books 13,744.48 

Dr.  Frank  R.  Lillie,  Grading,  etc 1,500.00 

Payment  on  Danchakoff  Mortgage   2,000.00 

Balance  of  Income  and  Expense  Account — Exhibit  B  91.06        37,262.68 


Balance,  December  31,  1927 — Exhibit  A   91,700.01 

Respectfully  submitted, 

LAWRASON  RIGGS,  JR., 

Treasurer. 


REPORT    OF   THE    LIBRARIAN.  II 

V.     THE  REPORT  OF  THE  LIBRARIAN, 
DECEMBER  31,   1927. 

The  expenditures  of  the  Library  during  1927  totalled  $26,- 
039.67;  segregated  under  the  following  headings:  books,  $527.20; 
serials,  $3,325.57;  binding  of  current  serials,  etc.,  $997.25;  sup- 
plies, $541.88;  express,  $253.53;  salaries,  $5,650.00;  miscellaneous 
salaries,  $1,550.50;  and  General  Education  Board  Fund,  appropri- 
ated for  back  sets,  $14,093.74.  The  total  appropriation  was  $27,- 
400.00,  apportioned  as  follows:  books,  $500.00;  serials,  $3,000; 
binding,  $1,000;  supplies,  $500.00;  express,  $200.00;  salaries, 
$5,650.00;  miscellaneous  salaries,  $1,550.00;  and  General  Educa- 
tion Board  appropriation,  $15,000.  It  will  be  noted  that  the  great- 
est part  of  the  unused  total  occurred  under  the  fund  for  back 
sets.  The  sum  of  $906.26  was  carried  on  into  1928  and  was  in 
fact  expended  before  January  I5th,  so  that  the  expenditures  all 
along  the  line,  show  a  slight  over-running  of  the  appropriation. 
The  most  interesting  item  in  this  respect  is  that  for  serials.  A 
special  point  of  this  condition-  was  made  by  the  Librarian  in  the 
1926  Annual  Report  and  in  the  informal  report  given  to  the  cor- 
poration last  August.  Either  an  increase  in  appropriations  will 
have  to  be  made  for  this  purpose,  or  some  of  the  current  serials 
must  be  dropped.  For  1928,  an  increase  was  granted  by  the  Ex- 
ecutive Committee  and  this  will  have  to  be  further  increased  for 
1929.  It  will  be  noticed  that  of  the  total  $26,939.67,  $19,739.17 
was  expended  on  material  and  acquisitions,  and  $7,200.50  on  sal- 
aries to  carry  on  the  work  of  the  Library. 

The  Library  now  contains  22,762  bound  volumes,  most  of  these 
coming  under  the  category  of  serials  and  books,  and  in  about  the 
proportion  of  8  to  I.  Of  these,  4,154  were  acquired  this  year, 
657  by  binding  current  serials ;  674  by  binding  back  sets ;  and  the 
others  by  new  back  sets  and  books.  Besides  these  volumes,  the 
reprints  number  43,000.  Only  5,000  of  these  were  catalogued 
and  filed  during  this  year. 

The  Library  receives  764  serials  currently  and  in  addition  to 
these  periodicals,  subscribes  to  36  books  and  monographs  that  are 
being  issued  serially,  800  serial  publications  in  all.  Of  these  764 
periodicals,  136  were  new  this  year;  and  of  the  36  books  and 


12  MARINE    BIOLOGICAL    LABORATORY. 

monographs,  5  are  new.  The  new  periodicals  for  1927  are  not 
all  by  paid  subscription,  but  87  were  acquired  by  exchange  with 
the  BIOLOGICAL  BULLETIN  (67  copies  of  the  BIOLOGICAL  BULLE- 
TIN being  sent  out)  ;  and  19  by  gift,  while  only  30  new  paid  sub- 
scriptions have  been  added.  The  total  number  of  paid  subscrip- 
tions including  the  serially  issued  books  is  now  270 ;  and  the  total 
number  of  exchange,  274 ;  and  the  gifts,  256.  We  have  pur- 
chased 180  new  books,  and  have  received  from  publishers,  112, 
and  from  authors,  24,  and  from  other  sources,  144;  460  in  all. 
These  gifts  were  as  follows: 

From  the  publisher,  P.  Blakiston's  Son  &  Co.,  Craigie,  E. 
Home :  An  Introduction  to  the  Finer  Anatomy  of  the  Central 
Nervous  System  Based  upon  That  of  the  Albino  Rat;  Evans,  C. 
Lovatt:  Recent  Advances  in  Physiology;  Gould,  George  M. : 
Medical  Directory;  Hawk,  Philip  B. :  Practical  Physiological 
Chemistry;  Lewis,  F.  T.  and  Bremer,  J.  L. :  A  Text-book  of  His- 
tology arranged  upon  an  Embryological  Basis ;  Meyers,  Milton 
K.,  Editor :  Lang's  German- English  Medical  Dictionary ;  Pryde, 
John:  Recent  Advances  in  Biochemistry;  Stitt,  E.  R. :  Practical 
Bacteriology ;  Youngken,  Heber  W. :  Pharmacognosy. 

Gebruder  Borntraeger :  Diirken,  Bernhard :  Allgemeine  Ab- 
stammungslehrc ;  Herter,  Konrad :  Tastsinn,  Stromungssinn  und 
Temperatursinn  der  Tiere  und  die  diesen  Sinnen  sugeordneten 
Rcaktionen ;  von  Buddenbrock,  W. :  Grundriss  dcr  vergleichenden 
Physiologic. 

Chicago  University  Press:  Newman,  H.  H. :  Evolution,  Gen- 
etics, and  Eugenics;  Newman,  H.  H.,  et  al :  Nature  of  the  World 
and  of  Man. 

Columbia  University  Press :  Chandler  Chemical  Laboratories : 
Contemporary  developments  in  Chemistry. 

Detroit  Digestive  Ferments  Co. :  Manual  of  Dehydrated  Cul- 
ture Media  and  Reagents. 

E.  P.  Dutton  Co.:  Einstein,  Albert:  The  Theory  of  Brownian 
Movement;  Freundlich,  Herbert:  New  Conceptions  in  Colloidal 
Chemistry;  Nernst,  W. :  The  New  Heat  Theorem:  its  Founda- 
tions in  Theory  and  Experiment',  Ostwald,  Wolfgang:  Practical 
Colloid  Chemistry;  Stock,  Alfred:  Structure  of  Atoms. 

Friederichsen   &   Co. :    Michaelson,   W.,    Editor :   Beitrage  sur 


REPORT    OF    THE    LIBRARIAN.  13 

Kenntnis  dcr  Land-und  Siisswasserfauna  Deutsch-Sudwestafri- 
kas;  Michaelsen,  W.,  Editor:  Beitrdgc  zur  Kcnntiiis  dcr  M ceres- 
fauna  irestafrikas. 

Ginn  &  Co. :  Miller.  Dayton  C. :  Laboratory  Physics. 

Harcourt,  Brace  £  Co. :  von  Uexkiill,  J. :  Theoretical  Biology. 

Hokuryukwan  £  Co.  Ltd. :  Hirase,  S.  et  al :  Figuraro  dc  Jap- 
ana]  Bostoj. 

Alfred  A.  Knopf,  Inc. :  Pearl,  Raymond :  Biology  of  Popula- 
tion Growth ;  Perrier,  Edmond :  The  Earth  before  History,  Man's 
Origin  and  the  Origin  of  Life;  Wheless,  Joseph:  Is  it  God's 
Word? 

Lea  and  Febiger :  Berkeley,  W.  N. :  The  Principles  and  Prac- 
tice of  Endocrine  Medicine ;  DuBois,  E.  F. :  Basal  Metabolism 
in  Health  and  Disease ;  Wiggers,  Carl  J. :  Modern  Aspects  of  the 
Circulation  in  Health  and  Disease. 

Lewis,  H.  K.  £  Co.:  Boes,  P.  K. :  X-ray  Apparatus;  its  Ar- 
rangement and  Use. 

J.  P.  Lippincott  Co.:  Addison,  W.  H.  F. :  Piersol's  Normal 
Histology;  Craig,  C.  F. :  Manual  of  the  Parasitic  Protozoa  of 
Man;  Meyer,  H.  H.  and  Gottlieb,  R. :  Experimental  Pharma- 
cology. 

Longmans,  Green  &  Co. :  MacLeod,  John  J.  R. :  Carbohydrate 
Metabolism  and  Insulin. 

McGraw-Hill  Book  Co.:  Allen,  E.  S.:  Six-place  Tables;  Ban- 
croft, Wilder  D. :  Applied  Colloid  Chemistry;  Fernald,  H.  T. : 
Applied  Entomology — an  Introductory  Text-book  of  Insects  in 
their  Relations  to  Man;  Pearse,  A.  S. :  Animal  Ecology;  Rogers, 
Charles  G. :  Textbook  of  Comparatiz'e  Physiology ;  Shull,  Charles  : 
Heredity. 

The  Macmillan  Co.:  Adams,  L.  A.:  Necturus;  A  Dissection 
Guide;  Baitsell,  Geo.  A.:  Manual  of  Biological  Forms;  Bernard, 
Claude:  Introduction  to  the  Study  of  Experimental  Medicine; 
Billroth,  Theodor:  Medical  Sciences  in  the  German  Universities; 
Brinkley,  Stuart  R.  and  Kelsey,  E.  B. :  Laboratory  Manual  ar- 
ranged to  accompany  "Principles  of  General  Chemistry";  Cahn, 
Alvin  R. :  The  spiny  dogfish ;  A  Laboratory  Guide ;  Creaser, 
C.  W. :  The  Skate;  A  Laboratory  Manual;  Frazer,  James  G. : 
The  IVorsIiip  of  Nature,  vol.  I ;  Jeffrey,  Edw.  C. :  Coal  and  Civi- 


14  MARINE    BIOLOGICAL   LABORATORY. 

lication;  Kerr,  J.  Graham:  Evolution;  Needham,  Joseph:  Science, 
Religion,  and  Reality;  Newman,  Horatio  H. :  The  Gist  of  Evo- 
lution; Smuts,  J.  C. :  Holism  and  Evolution;  Woodruff,  L.  LL. : 
Foundations  of  Biology. 

Open  Court  Publishing  Co. :  Brodetsky,  S. :  First  Course  in 
Nomography;  Friess,  Horace  Leland :  Schlciermacher's  Solilo- 
quies; Leathern,  J.  G. :  The  Mathematical  Theory  of  Limits; 
Piaggio,  H.  T.  H. :  Elementary  Treatise  on  Differential  Equations 
and  their  Application ;  Silberstein,  L. :  Protective  Vector  Algebra. 

Oxford  University  Press:  de  Beer,  G.  R. :  An  Introduction 
to  Experimental  Embryology ;  Dobson,  G.  M.  B.,  Griffith,  I.  O. 
and  Harrison,  D.  N. :  Photographic  Photometry;  Goodrich,  Ed- 
win S. :  Living  Organisms,  an  Account  of  their  Origin  and  Evo- 
lution; Heresy,  George  and  Panetti,  Fritz:  Manual  of  Radio- 
activity; Hinshelwood,  C.  N. :  Kinetics  of  Chemical  Change  in 
Gaseous  Systems;  Smith,  G.  Eliot:  The  Evolution  of  Man;  Col- 
well,  H.  C. :  Introduction  with  Study  of  Roentgen  Rays  and  Ra- 
dium; Cooper,  Eugenia  R.  A.:  The  Histology  of  the  More  Im- 
portant Human  Endocrine  Organs  at  Various  Ages ;  Dakin,  W. 
J. :  The  Elements  of  General  Zoology ;  Dodds,  E.  C.  and  Dick- 
ens, F. :  The  Chemical  and  Physiological  Properties  of  the  In- 
ternal Secretions. 

Presses  Universitaires  de  France :  Problemes  Biologiques,  4-6. 

Princeton  University  Press :  Conklin,  E.  G. :  A  Synopsis  of  the 
General  Morphology  of  Animals;  More,  Louis  T. :  The  Dogma 
of  Evolution ;  Morgan,  T.  H. :  Evolution  and  Genetics. 

W.  B.  Saunders  Co. :  Arey,  Leslie  Brainerd :  Developmental 
Anatomy;  Castle,  W.  E.  et  al :  Our  Present  Kno^vlcdge  of  He- 
redity; Cecil,  R.  L. :  A  Text-book  of  Medicine  by  American  au- 
thors; Borland,  W.  A.  N. :  American  illustrated  Medical  Dic- 
tionary; Falk,  I.  S.:  Principles  of  Vital  Statistics;  Friedenwalt, 
J. :  Diet  in  Health  and  Disease ;  Herrick,  C.  J. :  Neurology ;  Her- 
rick,  C.  Judson:  An  Introduction  to  Neurology;  Kolmer,  John 
A. :  A  Practical  Text-book  of  Infection,  Immunity  and  Biological 
Therapy ;  Stollmann,  Torald :  A  Manual  of  Pharmacology ;  Sten- 
gel, Alfred  and  Fox,  Herbert :  Text  of  Pathology ;  Stevens,  A.  A. : 
The  Practice  of  Medicine ;  Stiles,  Percy :  Human  Physiology ; 
Todd,  J.  C. :  Clinical  Diagnosis  by  Laboratory  Methods;  Wells, 
H.  Gideon:  Chemical  Pathology. 


REPORT    OF   THE    LIBRARIAN.  15 

D.  Van  Nostrand  Co.:  Baker,  A.  L. :  Thick  Lens  Optics; 
Brownell,  B. :  The  New  Universe ;  Cathcart,  W.  L.  and  Chaffee, 
J.  I.:  The  Elements  of  Graphic  Statics;  Howe,  Harrison  E. : 
Chemistry  in  the  World's  Work ;  Lee,  W'illit,  T. :  Stories  in  Stone ; 
Ireland,  L.  T. :  The  Mystery  of  Mind. 

Yale  University  Press:  Lewis,  Gilbert  N. :  The  Anatomy  of 
Science;  Millikan,  R.  A.:  Evolution  in  Science  and  Religion. 

From  the  authors :  Bailey's  Text-book  of  Histology,  revised 
by  O.  S.  Strong  and  Adolph  Elwyn;  Conklin,  Edwin  G. :  A 
Synopsis  of  the  General  Morphology  of  Animals;  Curtis,  Win- 
terton  C. :  Textbook  of  General  Zoology  (2  copies)  ;  Harvey,  E. 
Newton :  Laboratory  Directions  in  General  Physiology ;  Herrick, 
C.  Judson :  Brains  of  Rats  and  Men ;  Johnson,  Chas  W. :  The 
Insect  Fauna  (Biological  Survey  of  the  Mt.  Desert  Region) ; 
Mathews,  A.  P. :  The  Nature  of  Matter,  Gravitation  and  Light ; 
McKeough,  Rev.  Michael  J. :  The  Meaning  of  the  Rationes  Sem- 
inales  in  St.  Augustine;  Means,  James  H. :  Dyspnoea;  Patten, 
Bradley  M. :  The  Embryology  of  the  Pig;  Pratt,  H.  S.:  A  Lab- 
oratory Course  in  General  Zoology  ;  Warbasse,  James  P. :  Surgical 
Treatment;  Wieman,  H.  L. :  General  Zoology;  Woodruff,  L.  L. : 
Foundations  of  Biology ;  Workman,  F.  B.  and  Workman,  W.  H. : 
Algerian  Memories;  The  Call  of  the  Snowy  His  par;  Ice-bound 
Heiglits  of  flic  Mustagh;  Illustrations  of  Ice  JTilds  of  Eastern 
Karakoram ;  In  the  Ice  World  of  Himalaya ;  Peaks  and  Glaciers 
of  Nun  Kun;  Sketches  Awheel  in  Fin  de  Siecle  Iberia;  Through 
Town  and  Jungle;  Two  Summers  in  the  Ice-Wilds  of  Eastern 
Karahoram. 

An  especially  notable  gift  this  year  is  that  of  the  Library  on 
Coelenterates  of  Professor  Charles  Wesley  Hargitt,  presented 
to  the  Library  of  the  Marine  Biological  Laboratory  by  his  son. 
Professor  George  T.  Hargitt.  The  volumes  and  pamphlets  num- 
ber about  500  in  all.  They  will  not  be  catalogued  and  shelved 
as  a  special  library,  but  each  will  be  marked  by  a  special  book- 
plate, and  in  addition,  the  collection  as  a  whole  will  be  marked 
by  a  small  brass  tablet  with  an  inscription  showing  that  it  is  in- 
corporated with  the  other  books  of  the  Library.  This  commem- 
oration tablet,  in  size  about  7"xQ",  will  be  placed  on  the  walls 
in  the  stack-room  of  the  reading-room  floor. 


16  MARINE    BIOLOGICAL    LABORATORY. 

A  special  acknowledgment  should  be  made  of  a  gift  of  reprints, 
books  and  pamphlets  sent  by  Dr.  Ida  H.  Hyde  who  had  already 
presented  to  this  Library  in  1917-18,  the  main  collection  of  her 
reprints.  The  library  wishes  to  acknowledge  another  gift  from 
Mrs.  Edward  G.  Gardiner,  of  reprints  from  Dr.  Gardiner's  li- 
brary. Dr.  Louis  Murbach  has  presented  a  number  of  his  books 
and  pamphlets  that  will  be  of  use  in  the  Library  here.  And  very 
especially,  the  Library  makes  acknowledgment  of  a  gift  of  books 
and  other  interesting  pamphlets  which  were  sent  to  us  by  Dr. 
Elizabeth  H.  Dunn  when  she  closed  her  shop  in  Woods  Hole. 

The  sum  granted  by  the  General  Education  Board  for  back 
sets  was  $5000  more  this  year  than  in  1926,  and  the  number  of 
sets  completed  for  the  library  was,  therefore,  correspondingly 
larger.  84  back  sets  were  completed  and  30  partially  completed. 
The  most  interesting  of  these  are : 

Periodicals :  Jahrbiichcr  fiir  ivissenschaftliche  Botanik ;  Journal 
of  the  Franklin  Institute ;  Philosophical  Transactions  of  the  Royal 
Society  of  London ;  Proceedings  of  the  Zoological  Society  of 
London;  Transactions  of  the  Zoological  Society  of  London;  Zcit- 
schrift  fiir  iwissenschaftliche  Zoologie ;  Zoologica. 

Serially  issued  monographs :  Bijdragcn  tot  de  Dierkunde  K. 
soologisch  genootschap  Natura  Artis  Magistra  te  Amsterdam ; 
Oppel,  Albert :  Lehrbuch  dcr  vcrgleichendcn  Mikroskopischen 
Ana-tomie  der  Wirbeltiere ;  Oppenheimer,  C. :  Handbuch  dcr  Bi- 
ochemie  des  Mcnschen  und  der  Ticrc ;  Chun,  Carl :  Valdivia  Ex- 
pedition. 

The  completion  of  back  sets  of  serials  has  not  been  confined 
to  those  purchased.  We  owe  grateful  acknowledgment  for  7  sets 
completed  by  gift  and  19  partially  completed.  The  back  sets  of 
the  Biological  Bulletin  have  also  been  used  to  secure  missing  sets 
of  serials;  19  having  been  completed  by  this  method,  and  21  par- 
tially filled  in.  In  this  connection,  there  should  be  mentioned 
also  the  use  made  of  the  duplicate  material  which  was  listed  and 
arranged  alphabetically  in  1925-26.  Several  important  gaps  have 
been  filled  through  exchange  for  these  duplicates.  It  is  greatly 
to  be  regretted  that  lack  of  time,  both  on  our  part,  and  the  part 
of  other  libraries,  hinders  a  freer  advertisement  of  duplicate  ma- 
terial. 


REPORT    OF    THE   DIRECTOR.  l"J 

The  financial  statement  and  the  acquisitions  of  the  year  are 
simple  to  enumerate.  The  uses  made  of  the  Library  during  the 
year  cannot  so  easily  be  appraised.  This  is  especially  true  be- 
ginning with  this  year  when  new  books  and  the  serial  publica- 
tions were  for  the  first  time  restricted  in  place  of  use  to  the  Li- 
brary itself.  1,846  loans  were  charged  out,  however,  during  the 
year.  The  out-of-town  loans  were  greater  than  in  any  previous 
year,  although  but  1 1 .  Also  more  volumes  were  borrowed  from 
other  libraries,  37  in  all,  one  more  than  in  1926  and  five  more 
than  in  1925. 

VI.     THE  REPORT  OF  THE  DIRECTOR. 

To  THE  TRUSTEES  OF  THE  MARINE  BIOLOGICAL  LABORATORY. 

Gentlemen:  I  beg  to  submit  herewith  a  report  of  the  fortieth 
session  of  the  Marine  Biological  Laboratory  for  the  year  1927. 

i.  Attendance. — In  the  Tabular  View  of  Attendance  on  page 
41  a  departure  has  been  made  from  the  previous  customs  of  list- 
ing the  investigators  under  the  three  headings  of  Zoology,  Physi- 
ology and  Botany.  This  change  has  appeared  advisable,  first, 
because  the  broadening  of  the  activities  of  the  Laboratory  in  re- 
cent years  has  brought  to  it  an  increasing  number  of  persons 
whose  work  does  not,  strictly  speaking,  fall  into  any  of  these 
categories,  and,  second,  because  a  considerable  number  of  inves- 
tigators in  filling  out  their  registration  blanks  have  indicated  that 
in  their  own  opinion  their  work  belongs  equally  to  Zoology  and 
Physiology  or  to  Botany  and  Physiology.  For  this  reason  in- 
vestigators have  been  classified  merely  as  "independent"  or  "un- 
der instruction."  Following  the  custom  inaugurated  in  1926  a 
separate  class  has  been  provided  for  research  assistants,  whose 
number  during  the  past  few  years  has  been  rapidly  increasing. 

An  examination  of  the  figures  for  the  attendance  during  the 
years  1923-7,  inclusive,  shows  that  the  number  of  students  in 
the  courses  has  remained  nearly  stationary  owing  to  the  strict 
limitation  of  the  sizes  of  our  classes.  The  attendance  of  investi- 
gators, on  the  other  hand,  being  subject  to  no  such  restriction 
has  shown  a  remarkable  growth.  For  1927  the  increase  over  the 
preceding  record-breaking  year  of  1926  was  approximately  thir- 
2 


l8  MARINE    BIOLOGICAL    LABORATORY. 

teen  per  cent.  This  increase  is  especially  remarkable  in  view 
of  the  fact  that  for  a  considerable  time  during  the  previous  sum- 
mer every  room  in  both  the  brick  and  the  wooden  buildings  was 
occupied.  The  accommodation  of  over  40  additional  investigators 
under  these  circumstances  was  made  possible,  first,  by  the  con- 
version into  laboratories  of  several  rooms  formerly  used  for  other 
purposes,  second,  by  an  extensive  sharing  of  the  larger  rooms  by 
two  or  more  workers,  and,  third,  by  a  considerable  lengthening 
of  the  season  of  greatest  activity.  To  encourage  the  attendance 
of  investigators  during  the  less  crowded  parts  of  the  summer,  the 
Mess  in  1927  was  opened  about  two  weeks  earlier  and  closed  about 
five  days  later  than  in  previous  years.  This  policy  has  proved  to 
be  so  successful  that  it  is  planned  to  continue  it. 

A  consideration  of  the  unprecedented  increase  in  the  number 
of  investigators  during  the  past  two  years,  which  considerably 
exceeds  that  for  the  previous  thirteen,  raises  the  question  of  the 
maximum  capacity  of  the  present  laboratory  buildings.  It  may 
be  said  in  this  connection  that  for  the  months  of  July  and  August 
the  limit  has  already  been  almost,  though  not  quite,  reached.  A 
further  sharing  of  rooms  during  this  period  will  make  possible 
the  accommodation  of  a  small  additional  number  of  investigators. 
However,  chief  reliance  in  the  future  must  be  placed  upon  a 
further  extension  of  the  working  season.  With  the  Laboratory 
at  present  occupied  to  its  full  capacity  for  only  two  months  of 
the  year  it  is  evident  that  it  is  still  far  from  having  reached  the 
condition  of  its  greatest  usefulness. 

The  possibilities  for  extending  the  season  in  both  directions 
are  strikingly  shown  by  the  following  tabulation  of  the  numbers 
of  investigators  and  research  assistants  in  attendance  on  selected 
clays  throughout  the  summer  of  1927 : 

April  20    None 

"      30 i 

May  10     3 

"        20      6 

"     30  7 

June  10  50 

"      20  114 

"      30  212 


REPORT    OF    THE   DIRECTOR.  K) 

July   10     247 

"       20      247 

"       30      245 

August  10    234 

"  20     208 

30    168 

September   10    no 

20    50 

30    12 

October  10    8 

20    2 

30    2 

November  10     None 

2.  The  Nciu  Dormitory  and  Apartment  House. — In  the  report 
of  the  Directors  for  1926  mention  was  made  of  the  generous  gift 
of  $250,000  by  the  General  Education  Board  for  the  purpose  of 
erecting  a  Dormitory  and  an  Apartment  House.  Both  of  these 
buildings  were  completed,  except  for  certain  minor  details,  early 
in  June,  1927.  The  first  family  moved  into  the  Apartment  House 
on  June  I  and  the  first  persons  into  the  Dormitory  a  few  days 
later.  From  that  time  until  early  in  September  the  buildings 
were  occupied  to  almost  their  full  capacity  and  were  not  entirely 
vacant  until  the  first  of  November. 

The  new  buildings  fill  admirably  the  long-felt  need  of  the  Lab- 
oratory for  suitable  accommodations  for  investigators  with  fam- 
ilies. Together  they  provide  six  large  apartments,  fully  equipped 
for  housekeeping,  each  consisting  of  a  living-room,  two  bed-rooms, 
screened  porch,  kitchenette,  and  bath ;  two  smaller  apartments  of 
similar  character  but  with  only  one  bed-room  and  without  a  porch ; 
nine  suites  of  two  rooms  with  bath ;  sixteen  double  rooms  which 
can  be  combined  in  various  ways  into  suites ;  nineteen  other  double 
rooms  and  eighteen  single  rooms.  Each  of  the  rooms  which  is 
not  part  of  a  suite  containing  a  bath  is  provided  with  hot  and 
cold  running  water.  For  the  use  of  those  persons  who  do  not 
occupy  the  furnished  apartments  there  is  provided  in  each  of  the 
buildings  a  large  and  comfortable  social  room  and  in  the  basement 
facilities  for  laundry  work  and  simple  cooking.  An  especially 
valuable  feature  of  the  Apartment  House  is  that  it  can  be  heated, 


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MARINE    BIOLOGICAL    LABORATORY. 


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22  MARINE    BIOLOGICAL   LABORATORY. 

thus  making  it  possible  for  the  working  season  of  the  Laboratory 
to  be  extended  into  the  colder  months  of  the  year. 

According  to  the  original  plans,  the  Apartment  House  was 
to  be  used  for  investigators  and  the  Dormitory  for  students,  but 
the  experience  of  the  first  year  of  operation  has  shown  that  the 
demand  from  the  investigators  alone  will  be  more  than  sufficient  to 
fill  both  buildings,  and,  on  the  whole,  it  seems  fairer  to  give  pref- 
erence in  the  assignments  to  this  class  of  workers,  both  because 
their  stay  at  the  Laboratory  is  longer  and  because  they  have 
greater  difficulty  on  account  of  their  families  in  finding  suitable 
accommodations  in  the  village.  The  usefulness  of  the  new  build- 
ings to  investigators  with  families  is  indicated  by  the  fact  that 
during  the  first  year  of  their  operation  they  accommodated  a  total 
of  38  children. 

The  following  is  a  classification  of  the  occupants  of  the  two 
buildings  for  1927: 

Dormitory  Apartment  House     Total 

Investigators     52  28                     So 

Members   of    familes   of   inves- 
tigators      47  41                    88 

Students    7  7 


Total    106  69  175 

The  cost  of  the  buildings,  in  spite  of  various  unforseen  diffi- 
culties which  arose  during  their  construction  has  to  the  time  of 
this  report  come  well  within  the  appropriation  of  $250,000, 
though  a  certain  amount  of  interior  painting  still  remains  to  be 
done.  This  very  favorable  financial  showing  would  have  been  im- 
possible, however,  without  the  generous  assistance  of  Dr.  Frank 
R.  Lillie  in  connection  with  the  grading  of  the  grounds  and  the 
planting  of  shrubbery  which  have  added  greatly  to  the  appearance 
of  the  buildings.  The  following  figures  taken  with  slight  sim- 
plification from  the  Auditors'  Report  for  1927  show  the  status 
of  the  building  fund  on  December  31,  1927: 


REPORT    OF    THE    DIRECTOR.  23 

ANALYSIS    OF    COST    OF    DORMITORY    BUILDING 
AND    APARTMENT    HOUSE 

Dormitory  Apartment  House 

General   Contractor    $  90,016.70  $  72,086.88 

Plumbing     16,451.00  13,170.00 

Electrical  Work    4,657.98  5,378-73 

Heating    649.00  5,800.00 

All   other   Building  Expenses...      14,806.75  13,185.18 

Total  Building  Expenses   .  .$126,581.43  $109,620.79 

Furnishings     7,266.26  6,067.33 

Total     $133,847.69  $115,688.12 

Summary 

Total   New  Dormitory    $126,581.43 

Apartment   House    109,620.79  $236,202.22 

New  Dormitory  Equipment   ....       7,266.26 

Apartment  House  Equipment   .  .       6,067.33  13-333-59 

Total  Cost  to  Dec.  31,  1927  $249,535.81 

Accounted  for  as  follows : 

Gifts  of  General   Education 

Board    $250,000.00 

Less  Cash  on  Hand   2,898.31 

$247,101.69 

Gift  of  Dr.  Frank  R.  Lillie 

for  Grading,  etc 1,500.00 

Payments  from  Current 
Funds  25I-57 

Unpaid  Balance  of  Archi- 
tects' Commission  682.55  $249,535.81 


3.  Other  Buildings. — Among  the  additions  to  the  permanent 
equipment  of  the  Laboratory  during  1927,  one  which  would  have 
received  more  notice  than  it  has  in  a  year  not  marked  by  such 
extensive  building  operations  is  the  new  carpenter  shop  and  boat 
house,  situated  beside  the  Eel  Pond  near  the  Marine  railway. 


24  MARINE    BIOLOGICAL    LABORATORY. 

The  erection  of  this  building  involved  the  previous  preparation 
of  the  site  by  extensive  rilling  with  material  from  the  excavations 
for  the  Dormitory  and  the  construction  of  a  substantial  retaining 
wall.  The  building  itself  measures  66  by  44  feet  and  is  two 
stories  in  height.  The  first  story  includes  a  carpenter  shop  33 
by  44  feet  and  a  boat  shop  of  the  same  size ;  the  latter  is  on  the 
side  of  the  building  adjacent  to  the  Marine  railway  and  is  pro- 
vided with  doors  of  sufficient  size  to  admit  large  boats.  The 
second  story  consists  of  a  single  large  room,  used  at  present 
chiefly  for  storage  purposes  but  suitable  for  meeting  a  variety 
of  needs  which  may  arise  in  the  future.  The  cost  of  the  building 
with  the  retaining  wall  was  approximately  $7,500. 

While  the  new  Dormitory  and  Apartment  House  were  under 
construction  the  Dexter  House,  which  for  some  years  has  ac- 
commodated a  large  number  of  our  students  and  younger  in- 
vestigators was  removed.  Many  of  its  former  occupants  were 
cared  for  during  the  past  summer  in  the  new  buildings  and  an 
additional  number  in  the  Drew  House,  whose  capacity  has  been 
considerably  increased  by  a  more  economical  use  of  space,  and 
which  is  now  used  exclusively  as  a  men's  dormitory. 

4.  The  Report  of  the  Treasurer  shows  an  increase  in  the  total 
assets  of  the  Laboratory  from  $2,281,219.79  in  1926  to  $2,449,- 
624.06  in  1927,  the  largest  single  item  in  this  increase  being  ac- 
counted for  by  the  completion  during  the  past  year  of  the  new 
dormitory  and  apartment  house  buildings.  The  income  for  1927 
was  $193,707.42  and  that  shown  in  the  Auditors'  Report  for  1926 
was  $187,979.11 — an  apparent  increase  of  approximately  $6000. 
The  actual  increase,  however,  was  in  reality  nearly  $16,000,  since 
the  gift  of  $10,600  from  the  General  Education  Board  in  1926 
was  listed  under  income  and  the  corresponding  gift  of  $15,000 
in  1927  under  current  surplus.  By  the  same  system  of  book- 
keeping as  that  adopted  in  1927  the  income  for  the  previous  year 
would  be  $177,979.11  instead  of  the  figure  mentioned  above. 
The  expenses  for  the  two  years  were  $180,182.80  in  1926  and 
$193,798.48  in  1927,  giving  apparent  deficits  of  $2,203.69  and 
$91.06,  respectively.  These  deficits  are  at  present  on  paper  only 
and  are  due  to  the  depreciation  charged  against  the  plant  which 
the  Treasurer  discusses  in  his  report.  It  may  be  mentioned  in 


REPORT    OF    THE   DIRECTOR.  25 

this  connection  that  no  charge  for  depreciation  was  made  for  1927 
in  the  case  of  the  new  Dormitory  and  Apartment  House  build- 
ings, since  some  work  still  remained  to  be  done  upon  them  and 
the  construction  account  had  not  therefore  been  closed  into  the 
general  plant  account.  In  future  years,  however,  an  additional 
annual  depreciation  charge  of  $5,000  on  these  buildings  will,  un- 
less additional  sources  of  income  are  found,  still  further  increase 
the  unfavorable  balance  of  the  past  two  years.  In  this  connection 
it  is  well  to  remember  that  depreciation  in  the  case  of  the  Lab- 
oratory buildings  occurs  during  twelve  months  of  the  year  while 
most  of  the  income  derived  from  them  is  at  present  limited  to 
three  months.  This  is  an  additional  reason  for  making  every 
effort  to  increase  the  length  of  the  active  season  of  the  Laboratory. 

5.  The  Report  of  the  Librarian  shows  a  very  substantial  in- 
crease in  our  library  facilities  during  the  past  year.     The  greater 
part  of  this  increase  has  been  made  possible  by  a  second  install- 
ment of  $15,000  from  the  gift  of  $50,000  appropriated  by  the 
General  Education  Board  in   1925.     This  sum  was  used  chiefly 
for  the  purchase  of  back  sets  of  serials  and  the  Librarian  was 
fortunate  in  being  able  to  fill  some  of  the  most  serious  of  the 
existing  gaps  in  these  sets.     Another  noteworthy  addition  to  the 
library  during  1927  was  the  valuable  collection  on  Coelenterates 
of  the  late  Professor  Charles  W.  Hargitt,  generously  presented  to 
the  Laboratory  by  his  son,  Professor  George  T.  Hargitt.     The 
total  number  of  bound  volumes  on  our  shelves  at  present  is  over 
22,000  and   of   reprints   over  43,000.     Of   the    former,   approxi- 
mately 4,000  and  of  the  latter  approximately  5.000  were  acquired 
during  the  past  year.     The  number  of  current  serials  regularly 
received  by  the  Library  is  now  nearly  800. 

6.  The  Loeb  and  Gardiner  Memorial  Tablets. — On  August  4 
there  was  unveiled  a  tablet  to  Jacques  Loeb,  whose  work,  carried 
on  at  Woods  Hole  over  a  period  of  more  than  twenty  years,  has 
been  one  of  the  outstanding  contributions  to  science  of  the  Ma- 
rine Biological  Laboratory.     Addresses  were  delivered  in  this  oc- 
casion by  Professor  Frank  R.  Lillie,  Doctor  Simon  Flexner  and 
Professor  Hardolph  Wasteneys. 

The  tablet  bears  the  following  inscription : 


26  MARINE    BIOLOGICAL    LABORATORY. 

JACQUES  LOEB 

1859—1924 

BRAIN  PHYSIOLOGY 

TROPISMS,  REGENERATION 

ANTAGONISTIC  SALT  ACTION 

ARTIFICIAL  PARTHENOGENESIS 

DURATION  OF  LIFE 
COLLOIDAL  BEHAVIOR 

A  similar  tablet  was  unveiled  on  September  10  to  Edward 
Gardiner  Gardiner,  one  of  the  founders  of  the  Laboratory  and 
until  his  death  one  of  its  most  loyal  supporters.  Addresses  were 
delivered  by  Professor  E.  G.  Conklin  and  Professor  Frank  R. 
Lillie. 

The  tablet  is  inscribed  as  follows : 

EDWARD  GARDINER  GARDINER 

ZOOLOGIST 
INCORPORATOR  OF  THE  MARINE  BIOLOGICAL 

LABORATORY 
AND  MEMBER  OF  THE  ORIGINAL  BOARD  OF 

TRUSTEES 
FOR  MANY  YEARS  CLERK  OF  THE  CORPORATION  AND 

SECRETARY  OF  THE  BOARD  OF  TRUSTEES 
A  MAN  WHOSE  FINE  SENSE  OF  HONOR  AND  LOYALTY 

SHONE  IN  HIS  DEEDS 
A   SCIENTIST  TO  WHOM  THE   SUCCESS   OF  THE 

INSTITUTION 

WAS   THE  FULFILLMENT   OF  HIS   LIFE  INTEREST 

1 854 --1907 

/.  Changes  in  Personnel. — At  the  close  of  the  courses  in  10,27 
the  resignation  of  Dr.  Ivey  F.  Lewis,  who  for  twenty  years  has 
been  in  charge  of  the  Botany  Course,  was  received  and  accepted 
with  regret.  The  valuable  services  to  the  Laboratory  of  Dr. 
Lewis  were  recognized  in  the  following  resolution  of  the  Board 
of  Trustees : 

Voted:  That  the  Board  of  Trustees  learns  with  regret  of  the  res- 
ignation of  Doctory  Ivey  F.  Lewis  from  the  headship  of  the  course 
in  Botany  at  the  Marine  Biological  Laboratory,  and  expresses  to  Doc- 
tor Lewis  its  keen  appreciation  of  his  highly  efficient  and  constructive 
conduct  of  the  work  during  his  long  service  of  twenty  years. 


REPORT    OF    THE   DIRECTOR.  2/ 

Dr.  Lewis'  successor  is  Dr.  W.  R.  Taylor,  Professor  of  Bot- 
any at  the  University  of  Pennsylvania,  who  both  by  his  scien- 
tific attainments  and  by  his  long  experience  with  the  work  of  the 
course  in  excellently  fitted  for  the  position. 

8.  The  Board  of  Trustees. — At  the  annual  meeting  of  the 
Board  of  Trustees  held  August  9,  1927,  Professor  B.  M.  Duggar 
of  the  University  of  Wisconsin  was  elected  to  fill  a  vacancy  in 
the  Class  of  1931  of  the  Board. 

There  are  appended  as  parts  of  this  report : 

i.  The   Staff,    1927. 

3.  Investigators  and  Students,   1927. 

3.  A  Tabular  View  of  Attendance,   1923-1927. 

4.  Subscribing  and   Cooperating   Institutions,    1927. 

5.  Evening  Lectures,  1927. 

6.  Members  of  the  Corporation,  August,  1927. 


i.     THE   STAFF,   1927. 

MERKEL    H.    JACOBS,    Director,    Professor    of     General     Physiology 

University  of  Pennsylvania. 
Associate  Director  :  —  — . 

I.    INVESTIGATION. 

GARY  N.  CALKINS,  Professor  of  Protozoology,  Columbia  University. 

E.  G.  CONKLIN,  Professor  of  Zoology,  Princeton  University. 

CASWELL  GRAVE,  Professor  of  Zoology,  Washington  University. 

H.  S.  JENNINGS,  Professor  of  Zoology,  Johns  Hopkins  University. 

FRANK  R.  LILLIE,  Professor  of  Embryology,  The  University  of  Chi- 
cago. 

C.  E.  McCLUNG,  Professor  of  Zoology,  University  of  Pennsylvania. 

S.   O.   MAST,   Professor  of  Zoology,   Johns   Hopkins   University. 

T.  H.  MORGAN,  Professor  of  Experimental  Zoology,  Columbia  Uni- 
versity. 

G.   H.   PARKER,  Professor  of  Zoology,   Harvard  University. 

E.   B.   WILSON,   Professor  of  Zoology,   Columbia   University. 

LORANDE  L.  WOODRUFF,  Professor  of  Protozoology,  Yale  University. 


28  MARINE    BIOLOGICAL    LABORATORY. 

II.    INSTRUCTION. 

J.   A.   DAWSON,   Instructor    in  Zoology,   Harvard   University. 
RUDOLF  BENNITT,  Instructor  in  Biology,  Tufts  College. 
E.  C.  COLE,  Assistant   Professor  of  Zoology,  Williams   College. 
T.   H.   BISSONNETTE,   Professor   of   Biology,   Trinity   College. 
MADELEINE  P.  GRANT,  Assistant  Professor  of  Zoology,  Mount  Holy- 

oke  College. 
E.  A.  MARTIN,  Assistant  Professor  of  Zoology,  College  of  the  City 

of  New  York. 

A.  E.  SEVERINGHAUS,  Instructor  in  Zoology,  Columbia  University. 
DONNELL  B.  YOUNG,  Associate  Professor  of  Biology,   University  of 

Arizona. 

PROTOZOOLOGY. 

I.    INVESTIGATION. 

(See  Zoology.) 

II.    INSTRUCTION. 

LORANDE  Loss  WOODRUFF,  Professor  of  Protozoology,  Yale  Univer- 
sity. 

GARY  N.  CALKINS,  Professor  of  Protozoology,  Columbia  University. 
(Absent  in  1927.) 

MARY  STUART  MACDOUGALL,  Professor  of  Zoology,  Agnes  Scott  Col- 
lege. 

W.   B.   UNGER,  Assistant  Professor  of   Zoology,   Dartmouth   College. 

MARY  STUART  MACDOUGALL,  Professor  of  Zoology,  Agnes  Scott  Col- 
lege. 

EMBRYOLOGY. 

I.    INVESTIGATION. 

(Sec  Zoology.) 

II.    INSTRUCTION. 

HUBERT   B.   GOODRICH,   Professor  of   Biology,   Wesleyan   University. 

BENJAMIN  H.  GRAVE,  Professor  of  Biology,  Wabash  College. 

CHARLES  PACKARD,  Associate  in  the  Institute  of  Cancer  Research,  Co- 
lumbia University. 

HAROLD  H.  PLOUGH,  Professor  of  Biology,  Amherst  College. 

CHARLES  G.  ROGERS,  Professor  of  Comparative  Physiology,  Oberlin 
College. 


REPORT    OF    THE   DIRECTOR.  2Q 

PHYSIOLOGY. 

I.    INVESTIGATION. 

HAROLD  C.  BRADLEY,  Professor  of  Physiological  Chemistry,  Univer- 
sity of  Wisconsin. 

WALTER  E.  GARREY,  Professor  of  Physiology,  Vanclerbilt  University 
Medical  School. 

RALPH  S.  LILLIE,  Professor  of  General  Physiology,  The  University 
of  Chicago. 

ALBERT  P.  MATHEWS,  Professor  of  Biochemistry,  The  University  of 
Cincinnati. 

II.    INSTRUCTION. 

MERKEL  H.  JACOBS,  Professor  of  General  Physiology,  University  of 
Pennsylvania. 

WALLACE  O.  FENN,  Professor  of  Physiology,  University  of  Rochester. 

LEONOR  MICHAELIS,  Professor  in  the  University  of  Berlin  and  Resi- 
dent Lecturer  in  the  Johns  Hopkins  Medical  School. 

H.  K.  HARTLINE,  Department  of  Physiology,  Johns  Hopkins  Univer- 
sity. 

CHARLOTTE  HAYWOOD,  Department  of  Physiology,  University  of 
Pennsylvania. 

BOTANY. 

I.    INVESTIGATION. 

B.  M.  DUGGAR,  Professor  of  Plant  Physiology,  Washington  University. 

C.  E.  ALLEN,  Professor  of  Botany,  University  of  Wisconsin. 

S.  C.  BROOKS,  Department  of  Public  Health,  Washington,  D.  C. 
WM.  J.  ROBBINS,  Department  of  Botany,  University  of  Missouri. 
J.  R.  SCHRAMM,  Editor-in-Chief;  Biological  Abstracts,  University  of 
Pennsylvania. 

II.    INSTRUCTION. 

IVEY  F.  LEWIS,   Professor  of  Biology,  University  of  Virginia. 
WILLIAM   RANDOLPH   TAYLOR,  Assistant  Professor  of   Botany,  Uni- 
versity of  Pennsylvania. 
JAMES  P.  POOLE,  Professor  of  Evolution,   Dartmouth  College. 

LIBRARY. 

PRISCILLA  B.  MONTGOMERY,  (Mrs.  Thomas  H.  Montgomery,  Jr.),  Li- 
brarian. 

KATHERINE  UNDERWOOD,  Assistant  Librarian. 
DEBORAH  LAWRENCE,  Secretary. 


3O  MARINE    BIOLOGICAL    LABORATORY. 

CHEMICAL  SUPPLIES. 

OLIVER  S.  STRONG,  Associate  Professor  of  Neurology,  Columbia  Uni- 
versity, Chemist. 

APPARATUS  ROOM. 

SAMUEL  E.  POND,  Assistant  Professor  of  Physiology,  Medical  School, 
University  of  Pennsylvania,   Custodian  of  Apparatus. 

SUPPLY  DEPARTMENT. 

GEORGE  M.  GRAY,  Curator.  A.  W.  LEATHERS,  Head  of  Ship- 
Assistant    Curator :              .          ping   Department. 

JOHN  J.  VEEDER,   Captain.  A.  M.  HILTON,  Collector. 

E.  M.  LEWIS,  Engineer.  J.   MC!NNIS,   Collector. 


F.  M.  MACNAUGHT,  Business  Manager. 

HERBERT  A.  HILTON,  Superintendent  of  Buildings  and  Grounds. 

THOMAS  LARKIN,    Superintendent   of   Mechanical    Department. 

RAYMOND  E.  PHIPP,  Mechanician. 

WILLIAM  HEMENWAY,  Carpenter. 

ARNOLD  H.  Bisco,  Storekeeper  and  Head  Janitor. 


2.     INVESTIGATORS  AND   STUDENTS,    1927. 

Independent  Investigators. 

ADDISON,  W.  H.  F.,  Professor  of  Normal  Histology  and  Embryology,  University 
of  Pennsylvania. 

ALLEE,  W.  C.,  Associate  Professor  of  Zoology,  University  of  Chicago. 

ALLEN,  EZRA,  Research  Associate,  Carnegie  Institution  of  Washington. 

AMBERSON,  WILLIAM  R.,  Assistant  Professor  of  Physiology,  University  of  Penn- 
sylvania. 

ARMSTRONG,  PHILIP  B.,  Instructor  in  Anatomy,  Cornell  University  Medical  College. 

AUSTIN,  MARY  L.,  Lecturer  in  Zoology,  Barnard  College. 

BAITSELL,  GEORGE  A.,  Associate  Professor  of  Biology,  Yale  University. 

BAKER,  LILLIAN  E.,  Assistant  in  Department  of  Experimental  Surgery,  Rockefeller 
Institute  for  Medical  Research. 

BELLING,  JOHN,  Investigator,  Carnegie  Institution  of  Washington. 

BENNITT,  RUDOLF,  Instructor  in  Zoology,  Tufts  College. 

BERRILL,  N.  J.,  Assistant  of  Zoology  Department,  University  College,  London, 
England. 

BIGELOW,  ROBERT  P.,  Professor  of  Zoology  and  Parasitology,  Massachusetts 
Institute  of  Technology. 

BISSONNETTE,  THOMAS  HUME,  Professor  of  Biology,  Trinity  College. 


REPORT    OF    THE   DIRECTOR.  3! 

BOWEN,  ROBERT  H.,  Assistant  Professor  of  Zoology,  Columbia  University. 

BRADLEY,  HAROLD  C.,  Professor  of  Physiological  Chemistry,  University  of  Wis- 
consin. 

BREITENBENBECHER,  JOSEPH  K.,  Lecturer  in  Zoology,  McGill  University. 

BRIDGES,  CALVIN  B.,  Research  Assistant,  Carnegie  Institution  of  Washington. 

BRONFENBRENNER,  JACQUES  J.,  Associate  Member,  Rockefeller  Institute. 

BRONK,  DETLEV  W.,  Assistant  Professor  of  Physiology  and  Biophysics,  Swarthmore 
College. 

BROOKS,  MATILDA  MOLDENHAUER,  Associate  Biologist,  Hygienic  Laboratory, 
Washington. 

BROOKS,  SUMNER  C.,  Professor  of  Physiology  and  Biochemistry,  Rutgers  Uni- 
versity. 

BUDINGTON,  ROBERT  A.,  Professor  of  Zoology,  Oberlin  College. 

CAMPBELL,  CLARENCE  JAMES,  Assistant  Professor  of  Physiology,  Syracuse  Uni- 
versity. 

CAROTHERS^  ELEANOR  E.,  Lecturer  in  Zoology,  University  of  Pennsylvania. 

CATTELL,  MCKEEN,  Instructor  in  Physiology,  Cornell  University  Medical  College. 

CATTELL,  WARE,  Research  Fellow  in  Biology,  Memorial  Hospital. 

CHAMBERS,  ROBERT,  Professor  of  Microscopic  Anatomy,  Cornell  University 
Medical  College. 

CHEN,  C.  C.,  Professor  of  Biology,  Shanghai  College. 

CHIDESTER,  FLOYD  EARLE,  Professor  of  Zoology,  West  Virginia  University. 

CHRISTIE,  JESSE  R.,  Associate  Nematologist.  U.  S.  Department  of  Agriculture, 
Bureau  of  Plant  Industry. 

CLARK,  ELEANOR  LINTON,  Private  Investigator,  University  of  Pennsylvania. 

CLARK,  ELIOT  ROUND,  Professor  of  Anatomy,  University  of  Pennsylvania,  Medical 
Department. 

CLOWES,  G.  H.  A.,  Director  of  Lilly  Research  Laboratory,  Eli  Lilly  &  Co. 

COBB,  NATHAN  A.,  Technologist  and  Nematologist,  U.  S.  Department  of  Agri- 
culture, Washington,  D.  C. 

COHEN,  BARNETT,  Chemist,  Hygienic  Laboratory,  Washington,  D.  C. 

COHN,  EDWIN  J.,  Assistant  Professor  of  Physical  Chemistry,  Harvard  Medical 
School. 

COLE,  ELBERT  C.,  Assistant  Professor  of  Biology,  Williams  College. 

COLE,  KENNETH,  National  Research  Fellow,  Harvard  University. 

CONKLIN,  EDWIN  G.,  Professor  of  Biology,  Princeton  University. 

COPELAND,  MANTON,  Professor  of  Biology,  Bowdoin  College. 

CORDIER,  DR.  ROBERT,  Assistant  Professor  in  Histology,  University  of  Brussels, 
Brussels,  Belgium. 

COVELL,  WALTER  P.,  Associate,  Rockefeller  Institute. 

COWDRY,  E.  V.,  Associate  Member,  Rockefeller  Institute. 

COWLES,  R.  P.,  Associate  Professor  of  Zoology,  Johns  Hopkins  University. 

CRABB,  EDWARD  D.,  Instructor  in  Zoology,  University  of  Pennsylvania. 

CRAMPTON,  HENRY  E.,  Professor  of  Zoology,  Barnard  College,  Columbia  Uni- 
versity. 

CROCKER,  WILLIAM,  Managing  Director,  Boyce  Thompson  Institute  for  Plant 
Research. 

CURTIS,  WINTERTON  C.,  Professor  of  Zoology,  University  of  Missouri. 

DARBY,  HUGH  HACKLAND,  Instructor,  New  York  University. 

DAVVSON,  JAMES  A.,  Instructor  in  Zoology,  Harvard  University. 


32  MARINE    BIOLOGICAL   LABORATORY. 

BELLINGER,  S.  C.,  Professor  of  Zoology,  University  of  Arkansas. 

DISALVO,  MRS.  BEATRIX,  Assistant  Teacher,  Biology  Department,  George  Wash- 
ington High  School,  New  York. 

DOLLEY,  WILLIAM  L.,  JR.,  Professor  of  Biology,  University  of  Buffalo. 

DONALDSON,  HENRY  H.,  Professor  of  Neurology,  The  Wistar  Institute  of  Anatomy 
and  Biology. 

DREW,  KATHLEEN  MARY,  Lecturer  in  Botany,  The  Victoria  University  of  Man- 
chester, England. 

DUGGAR,  B.  M.,  Professor  of  Plant  Physiology,  Missouri  Botanical  Garden  and 
Washington  University. 

DURRANT,  EDWIN  POE,  Assistant  Professor  of  Physiology,  Ohio  State  University. 

EDWARDS,  DAYTON  J.,  Associate  Professor  of  Physiology,  Cornell  University 
Medical  College. 

EMMART,  EMILY  WALCOTT,  Associate  Professor  in  Biology,  Western  Maryland 
College. 

ESAKI,  SHIRO,  Department  of  Zoology,  University  of  Chicago. 

FARR,  CLIFFORD  H.,  Associate  Professor,  Washington  University. 

FENN,  WALLACE  O.,  Professor  of  Physiology,  Rochester  University.  Medical  School. 

FRY,  HENRY  J.,  Assistant  Professor,  Washington  Square  College. 

GARREY,  W.  E.,  Professor  of  Physiology,  Vanderbilt  University  Medical  School. 

GATES,  FREDERICK  L.,  Associate  Member,  Rockefeller  Institute  for  Medical 
Research. 

GLASER,  OTTO,  Professor  of  Biology,  Amherst  College. 

GLASER,  RUDOLF  W.,  Associate  Member,  Rockefeller  Institute  for  Medical  Re- 
search. 

GOLDFORB,  A.  J.,  Professor  of  Biology,  College  of  the  City  of  New  York. 

GOODRICH,  H.  B.,  Professor  of  Biology,  Wesleyan  University. 

GORDON,  ISABELLA,  n  Balloch  Road,  Keith,  Banffshire,  Scotland. 

GRAHAM,  JOHN  Y.,  Professor  of  Biology,  University  of  Alabama. 

GRANT,  MADELEINE  P.,  Assistant  Professor,  Mount  Holyoke  College. 

GRAVE,  BENJAMIN  H.,  Professor  of  Zoology,  Wabash  College. 

GRAVE,  CASWELL,  Professor  of  Zoology,  Washington  University. 

GRUENBERG,  BENJAMIN  O.,  Managing  Director,  American  Association  for  Medical 
Progress. 

HAGUE,  FLORENCE,  Assistant  Professor  of  Biology,  Sweet  Briar  College. 

HALL,  RICHARD  P.,  Assistant  Professor,  New  York  University. 

HANCE,  ROBERT  T.,  Associate,  Rockefeller  Institute  for  Medical  Research. 

HANN,  HARRY  W.,  Instructor  in  Embryology,  University  of  Illinois. 

HARTLINE,  H.  KEFFER,  Johns  Hopkins  Medical  School. 

HARVEY,  ETHEL  BROWNE,  Princeton,  New  Jersey. 

HARVEY,  E.  NEWTON,  Professor  of  Physiology,  Princeton  University. 

HAYWOOD,  CHARLOTTE,  Graduate  Student,  University  of  Pennsylvania. 

HECHT,  SELIG,  Associate  Professor  of  Biophysics,  Columbia  University. 

HEILBRUNN,  L.  V.,  Assistant  Professor  of  Zoology,  University  of  Michigan. 

HEYROTH,  FRANCIS  F.,  Research  Fellow,  Harvard  University  Medical  School. 

HIBBARD,  HOPE,  Preparateur,  The  Sorbonne,  Paris,  France. 

HILLER,  S.,  Assistant,  Biological  Laboratory,  Cracow,  Poland. 

HOADLEY,  LEIGH,  Assistant  Professor,  Brown  University. 

HOSKINS,  MARGARET  MORRIS,  Assistant  Professor  of  Microscopic  Anatomy,  New 
York  University  Dental  College. 


Kl  I'ORT    OF    THE    DIRECTOR.  33 

HOSKINS,  R.  G.,  Research  Associate  in  Physiology,  Harvard  University  Medical 
School. 

HOWE,  H.  E.,  Editor,  American  Chemical  Society. 

HOWE,  THOMAS  D.,  Instructor  in  Biology,  James  Millikin  University. 

HOWLAND,  RUTH  B.,  Assistant  Professor  of  Biology,  New  York  University. 

HUETTNER,  ALFRED  F.,  Assistant  Professor  of  Zoology,  Columbia  University. 

HLIGGINS,  JOHN  R.,  Assistant  Instructor,  University  of  Pennsylvania. 

HUGHES,  THOMAS  P.,  Assistant,  Rockefeller  Institute  for  Medical  Research. 

INMAN,  ONDESS  L.,  Professor  of  Biology,  Antioch  College. 

IRWIN,  MARIAN,  Associate  in  General  Physiology,  Rockefeller  Institute. 

JACOBS,  MERKEL  H.,  Professor  of  General  Physiology,  University  of  Pennsylvania. 

JENNINGS,  H.  S.,  Professor  and  Director  of  the  Zoological  Laboratory,  John  Hopkins 
University. 

JOHLIN,  J.  M.,  Associate  Professor  of  Biochemistry,  Vanderbilt  Medical  School. 

JUST,  E.  E.,  Professor  of  Zoology,  Howard  University. 

KAUFMANN,  BERWIND  P.,  Professor  of  Biology,  Southwestern,  Memphis,  Tennessee. 

KEEFE,  Rev.  ANSELM  M.,  Professor  of  Biology,  St.  Norbert's  College. 

KINDRED,  JAMES  E.,  Associate  Professor  of  Histology  and  Embryology,  University 
of  Virginia. 

KLEINER,  ISRAEL  S.,  Professor  and  Head  of  the  Department  of  Chemistry,  New 
York  Homoeopathic  Medical  College. 

KNOWLTON,  FRANK  P.,  Professor  of  Physiology,  College  of  Medicine,  Syracuse 
University. 

KUNITZ,  MOSES,  Associate,  Rockefeller  Institute  for  Medical  Research. 

LANCEFIELD,  D.  E.,  Assistant  Professor  in  the  Zoology  Department,  Columbia 
University. 

LANCEFIELD,  REBECCA  C.,  Assistant  in  Bacteriology,  Rockefeller  Institute. 

LANDIS,  EUGENE  M.,  University  of  Pennsylvania. 

LEE,  MILTON  O.,  Research  Associate,  Harvard  Medical  School. 

LEWIS,  IVEY  F.,  Professor  of  Biology,  University  of  Virginia. 

LILLIE,  FRANK  R.,  Chairman  of  the  Depaitment  of  Zoology,  University  of  Chicago. 

LILLIE,  RALPH  S.,  Professor  of  General  Physiology,  University  of  Chicago. 

LOEB,  LEO,  Professor  of  Pathology,  Washington  University  Medical  School. 

LOWTHER,  FLORENCE  DEL.,  Assistant  Professor  of  Zoology,  Barnard  College. 

LUCAS,  CATHERINE  L.  T.,  Travelling  Fellow,  London  University. 

LUCKE,  BALDWIN,  Assistant  Professor  of  Pathology,  University  of  Pennsylvania. 

LYNCH,  RUTH  S.,  Instructor,  The  Johns  Hopkins  University. 

McCLENDON,  J.  F.,  Professor  of  Physiological  Chemistry,  University  of  Minnesota. 

McCLUNG,  CLARENCE  E.,  Director  of  Zoological  Laboratory,  University  of  Penn- 
sylvania. 

McCuTCHEON,  MORTON,  Assistant  Professor  of  Pathology,  University  of  Penn- 
sylvania. 

MACDOUGALL,  MARY  STUART,  Professor  of  Zoology,  Agnes  Scott  College. 

MANWELL,  R.  D.,  Rockefeller  Special  Fellow,  School  of  Hygiene  and  Public  Health. 

MARTIN,  EARL  A.,  Assistant  Professor,  College  of  the  City  of  New  York. 

MAST,  S.  O.,  Professor  of  Zoology,  Johns  Hopkins  University. 

MATHEWS,  ALBERT  P.,  Professor  of  Biochemistry,  University  of  Cincinnati. 

MAYOR,  JAMES  W.,  Professor  of  Biology,  Union  College. 

MAY,  DR.  R.  M.,  Research  Fellow,  American  Field  Service  Fellowship. 

METCALF,  MAYNARD  M.,  Research  Associate  in  Zoology,  Johns  Hopkins  University. 


34  MARINE    BIOLOGICAL    LABORATORY. 

METZ,  CHARLES  W.,  Member  Staff,  Department  of  Genetics,  Carnegie  Institution 
of  Washington. 

MICHAELIS,  LEONOR,  Resident  Lecturer  in  Medical  Research,  Johns  Hopkins 
University  School  of  Medicine. 

MITCHELL,  PHILIP  H.,  Professor  of  Physiology,  Brown  University. 

MORGAN,  LILIAN  V.,  409  West  117  Street,  New  York  City,  N.  Y. 

MORGAN,  T.  H.,  Professor  of  Experimental  Zoology,  Columbia  University. 

MORGULIS,  SERGIUS,  Professor  of  Biochemist:y,  University  of  Nebraska. 

MORRILL,  CHARLES  V.,  Associate  Professor  of  Anatomy,  Cornell  University 
Medical  College. 

NEWMAN,  H.  H.,  Professor  of  Zoology,  University  of  Chicago. 

NOBLE,  G.  KINGSLEY,  Curator,  American  Museum  of  Natural  History,  New  York. 

NONIDEZ,  JOSE  F.,  Associate  in  Anatomy,  Cornell  University  Medical  College. 

PACKARD,  CHARLES,  Associate,  Institute  of  Cancer  Research,  Columbia  University. 

PARKER,  GEORGE  HOWARD,  Professor  of  Zoology,  Harvard  University. 

PARMENTER,  CHARLES  L.,  Assistant  Professor  of  Zoology,  University  of  Pennsyl- 
vania. 

PATTERSON,  WILLIAM  MORRISON,  University  Club,  5th  Avenue  and  54th  Street, 
New  York  City. 

PEARSE,  A.  S.,  Professor  of  Zoology,  Duke  University. 

PERLZWEIG,  W.  A.,  Associate  in  Medicine,  Johns  Hopkins  University  Medical 
School. 

PHELPS,  LILLIAN  A.,  Instructor  in  Zoology,  Cornell  University. 

PINNEY,  MARY  EDITH,  Professor  of  Zoology,  Milwaukee-Downer  College. 

PLOUGH,  HAROLD  H.,  Professor  of  Biology,  Amherst  College. 

POND,  SAMUEL  E.,  Assistant  Professor  of  Physiology,  School  of  Medicine,  Uni- 
versity of  Pennsylvania. 

POOLE,  JAMES  PLUMMER,  Professor  of  Evolution,  Dartmouth  College. 

RAND,  HERBERT  W.,  Associate  Professor  of  Zoology,  Harvard  University. 

REDFIELD,  A.  C.,  Assistant  Professor  of  Physiology,  Harvard  Medical  School. 

REDFIELD,  HELEN,  National  Research  Fellow  in  Zoology,  Columbia  University. 

DE  RENYI,  GEORGE  ST.,  Assistant  Professor  in  Department  of  Anatomy,  Uni- 
versity of  Pennsylvania. 

REZNIKOFF,  PAUL,  Associate  in  Anatomy,  Cornell  University  Medical  College. 

RICE,  KENNETH  S.,  Brown  University. 

RICHARDS,  A.,  Professor  of  Zoology,  University  of  Oklahoma. 

RICHARDS,  MILDRED  HOGE,  University  of  Oklahoma. 

RINGOEN,  ADOLPH  R.,  Assistant  Professor  of  Zoology,  University  of  Minnesota. 

ROBBINS,  WILLIAM  J.,  Professor  of  Botany,  University  of  Missouri. 

ROGERS,  CHARLES  G.,  Professor  of  Comparative  Physiology,  Oberlin  College. 

ROMER,  ALFRED  S.,  Associate  Professor  of  Vertebrate  Paleontology,  University  of 
Chicago. 

SANDISON,  JAMES  CALVIN,  Instructor  in  Anatomy,  University  of  Pennsylvania. 

SAYLES,  LEONARD  PERKINS,  Assistant  Professor  of  Biology,  Norwich  University. 

SCHAEFFER,  ASA  ARTHUR,  Professor  of  Zoology,  University  of  Kansas. 

SCHMITT,  FRANCIS  O.,  National  Research  Fellow,  Washington  University. 

SCHRADER,  FRANZ,  Associate  Professor,  Bryn  Mawr  College. 

SCHRADER,  SALLY  HUGHES,  Instructor,  Bryn  Mawr  College. 

SCHULTZ,  JACK,  National  Research  Fellow,  Columbia  University. 

SCOTT,  MIRIAM  J.,  University  of  Pennsylvania. 


REPORT    OF   THE   DIRECTOR.  35 

SEVERINGHAUS,  AURA  E.,  Instructor  in  Anatomy,  Columbia  University. 
SHAFTESBURY,  ARCHIE  D.,  Associate  Professor  of  Zoology,  The  North  Carolina 

College  for  Women. 

SIMPSON,  GEORGE  E.,  Assistant  Professor,  University  of  Pennsylvania. 
SMITH,  SEPTIMA  CECILIA,  Fellow  in  Medical  Zoology,  Johns  Hopkins  School  of 

Hygiene. 

SMITH,  WILBUR  A.,  Assistant,  University  of  Pennsylvania. 
SRIBYATTA,  DR.  L.,  Instructor  in  Physiology,  Chulalongkora  University,  Medical 

School,  Bangkok,  Siam. 
STARK,  MARY  B.,  Professor  of  Embryology  and  Histology,  New  York  Homoeopathic 

Medical  College. 

STEGGERDA,  F.  R.,  Teaching  Fellow,  University  of  Minnesota. 
STIER,  T.  J.  B.,  Graduate  Student,  Harvard  University. 

STOCKARD,  CHARLES  R.,  Professor  of  Anatomy,  Cornell  University  Medical  College. 
STOKEY,  ALMA  G.,  Professor  of  Botany,  Mount  Holyoke  College. 
STRONG,    OLIVER    S.,    Professor   of    Neurology   and    Neuro-Histology,    Columbia 

University. 

STUNKARD,  HORACE  W.,  Professor  of  Biology,  New  York  University. 
STURTEVANT,  A.  H.,  Member  of  Staff,  Carnegie  Institution. 
SVVETT,  F.  H.,  Associate  Professor  of  Anatomy,  Vanderbilt  School  of  Medicine. 
TAYLOR,  W.  RANDOLPH,  Professor  of  Botany,  University  of  Pennsylvania. 
TENNENT,  DAVID  H.,  Professor  of  Biology,  Bryn  Mawr  College. 
TERAO,  ARATA,  Professor  of  Zoology,  Imperial  Fisheries  Institute,  Tokyo,  Japan. 
TRACY,  HENRY  C.,  Professor,  University  of  Kansas. 

TURNER,  ABBY  HOWE,  Professor  of  Physiology,  Mount  Holyoke  College. 
UHLENHUTH,  EDUARD,  Associate  Professor  in  Anatomy,  University  of  Maryland 

Medical  College. 

UNGER,  W.  BYERS,  Assistant  Professor  of  Zoology,  Dartmouth  College. 
VISSCHER,  J.  PAUL,  Associate  Professor  of  Biology,  Western  Reserve  University. 
WARREN,  HOWARD  C.,  Professor  of  Psychology,  Princeton  University. 
WEECH,  ALEXANDER  ASHLEY,  Instructor  in  Research  Medicine,  Johns  Hopkins 

University. 

WENRICH,  D.  H.,  Assistant  Professor  of  Zoology,  University  of  Pennsylvania. 
WHITAKER,  DOUGLAS  M.,  Assistant  in  Zoology  and  Graduate  Student,  Stanford 

University. 

WILSON,  J.  WALTER,  Assistant  Professor  of  Biology,  Brown  University. 
WOLF,  ERNST,  University  of  Heidelberg,  Germany. 
WOLF,  E.  ALFRED,  Instructor  in  Zoology  and  Comparative  Physiology,  University 

of  Pittsburgh. 

WOODRUFF,  LORANDE  Loss,  Professor  of  Protozoology,  Yale  University. 
WOODWARD,  ALVALYN  E.,  Associate  Professor,  University  of  Maine. 
WRIGHT,  SEWALL,  Associate  Professor,  University  of  Chicago. 
WYMAN,  JEFFRIES,  JR.,  Instructor  and  Tutor  in  Biology,  Harvard  University. 
YAGI,  NOBUMASA,  Assistant  Professor  of  Entomology,  Kyoto  Imperial  University, 

Kyoto,  Japan. 
YOUNG,  DONNELL  BROOKS,  Professor  of  Biology  and  Head  of  Biology  Department, 

University  of  Arizona. 

Beginning  Investigators. 
1927. 

ALLEN,  ELEANOR,  Graduate  Student,  Brown  University. 


36  MARINE    BIOLOGICAL   LABORATORY. 

ARNOLD,  CONSTANCE  W.,  Demonstrator,  Brown  University. 

EARTH,  L.  G.,  Graduate  Assistant  in  Zoology,  University  of  Michigan. 

BASKERVILL,  MARGARET,  Adjunct  Professor,  University  of  Texas,  Medical  School. 

BLUMENTHAL,  REUBEN,  Graduate  Student,  University  of  Pennsylvania. 

CANAVAN,  WILLIAM  P.,  Instructor  in  Zoology,  University  of  Pennsylvania. 

CARPENTER,  ESTHER,  Assistant  in  Zoology  Department,  University  of  Wisconsin. 

CARVER,  GAIL  L.,  Professor  of  Biology,  Mercer  University. 

CHEER,  SHEO-NAN,  Fellow  of  the  Rockefeller  Foundation,  Peking  Union  Medical 
College,  Peking,  China. 

CLARK,  L.  B.,  Graduate  Student,  Johns  Hopkins  University. 

CRAWFORD,  WILEY  W.,  Fellow,  University  of  Missouri. 

ELFTMAN,  HERBERT,  Assistant  in  Zoology,  Columbia  University. 

FISH,  H.  D.,  Student  Investigator,  Columbia  University. 

FREEMAN,  LEO  BOYES,  University  of  Pennsylvania. 

GOODKIND,  ROBERT,  Student,  Harvard  University  Medical  School. 

GRISWOLD,  SYLVIA  M.,  Instructor  of  Botany  and  Bacteriology,  Pennsylvania 
College  for  Women. 

GRUNDFEST,  HARRY,  University  Fellow,  Columbia  University. 

HADLEY,  CHARLES  E.,  Harvard  University. 

HOP,  ANNE,  Student,  Radcliffe  College. 

HOFKESBRING,  ROBERTA,  Instructor  in  Physiology,  Tulane  University. 

HOLMES,  GLADYS  E.,  Graduate  Assistant,  Brown  University. 

JOHNSON,  Percy  L.,  Graduate  Assistant,  Johns  Hopkins  University. 

KAPP,  ELEANOR  M.,  Assistant  in  Biology,  New  York  University. 

KLEIN,  H.,  University  of  Pennsylvania. 

KOEHRING,  VERA,  Fellow,  University  of  Pennsylvania. 

KROPP,  BENJAMIN,  Graduate  Student,  Harvard  University. 

LIGHT,  V.  EARL,  Student  Technician,  Johns  Hopkins  University. 

LUCAS,  ALFRED  M.,  Instructor,  Washington  University. 

LUCAS,  EMILIO  R.,  Instructor,  University  of  Kansas. 

Lu,  HWEI-LING,  Graduate  Student  of  Zoology,  Columbia  University. 

McCARDLE,  Ross  CLAYTON,  University  of  Michigan. 

MACNAB,  ALLEYNE,  Technician  in  Department  of  Experimental  Surgery,  Rocke- 
feller Institute  for  Medical  Research. 

MATTHEWS,  SAMUEL  A.,  Student,  Harvard  University. 

MITCHELL,  WILLIAM  HINCKLEY,  JR.,  Thayer  Fellow,  Harvard  University. 

MONTGOMERY,  HUGH,  Student,  Harvard  University  Medical  School. 

MORRISON,  MARY  ELINOR,  University  of  Pennsylvania. 

MOSES,  MILDRED  S.,  Research  Assistant,  Carnegie  Institution  of  Washington. 

NELSON,  OLIN  E.,  Instructor  in  Zoology,  University  of  Pennsylvania. 

NOMURA,  SHICHIROKU,  Assistant  Professor  of  Zoology,  Tohoku  Imperial  University. 

PIERCE,  MADELENE  E.,  Graduate  Student,  Radcliffe  College. 

POLLACK,  HERBERT,  Cornell  Medical  College. 

RITTER,  RAYMOND  A.,  Assistant  in  Zoology,  University  of  Missouri. 

RUNYON,  ERNEST  H.,  Instructor,  Washington  University. 

SEARS,  MARY,  Research  Student,  Radcliffe  College. 

SHLAER,  SIMON,  Student,  Columbia  University. 

SHOUP,  CHARLES  S.,  Assistant  in  Instruction,  Princeton  University. 

SICHEL,  FERDINAND  J.  M.,  Student,  McGill  University,  Montreal,  Canada. 

SMITH,  GEORGE  HUME,  Instructor,  University  of  Illinois. 


REPORT    OF    THE   DIRECTOR.  37 

SONNEBORN,  TRACY  MORTON,  Graduate  Student,  Johns  Hopkins  University. 

STEEN,  EDWIN  B.,  Instructor  in  Zoology,  Wabash  College. 

STEWART,  DOROTHY  R.,  Instructor  in  Biology,  Lake  Erie  College. 

SUMWALT,  MARGARET,  Instructor,  University  of  Pennsylvania. 

TAFT,  CHARLES  H.,  JR.,  Student,  Columbia  University. 

TAYLOR,  MRS.  JEAN  GRANT,  3454  N.  23d  Street,  Philadelphia,  Pennsylvania. 

TITLEBAUM,  Albert,  Assistant  in  Zoology,  Columbia  University. 

WILLEY,  CHARLES  H.,  Instructor  in  Biology,  New  York  University. 

YOUNG,  R.  A.,  Assistant  Professor  of  Zoology,  Howard  University. 


RESEARCH  ASSISTANTS— 1927 

ARZBERGER,  E.  G.,  Pathologist,  Bureau  of  Plant  Industry,  U.  S.  Department  of 

Agriculture. 

BARTHOLOMEW,  THOMAS  HAYWARD,  Columbia  University. 
BARTHOLOMEW,  WILLIAM  WEST,  Columbia  University. 
DOWNING,  R.  C.,  Student,  Wabash  College. 

FIELD,  MADELEINE  E.,  Assistant  in  Physiology,  Mount  Holyoke  College. 
GENTHER,  IDA  T.,  Graduate  Assistant  in  Zoology,  University  of  Wisconsin. 
GREENE,  EUNICE  CHASE,  Medical  School,  Syracuse  University. 
HANSEN,  IRA  B.,  Assistant  in  Zoology,  Wesleyan  University. 
HARROP,  GEORGE  A.,  JR.,  Associate  Professor  of  Medicine,  Johns  Hopkins  Medical 

School. 
HIDALGO,    FRANCISCO,    Technical    Assistant,    Rockefeller    Institute    for    Medical 

Research. 

HOLMES,  W.  C.,  Rockefeller  Institute  for  Medical  Research. 
HOSKINS,  FRANCES,  Research  Assistant,  Columbia  University. 
JOHNSON,  ROSVVELL  HILL,  Columbia  University. 
KALTREIDER,  NOLAN  L.,  Swarthmore  College. 

KELTCH,  Anna  K.,  Research  Assistant,  Lilly  Research  Laboratory,  Indianapolis. 
LORBERBLATT,  ISAAC,  Chemist,  Harriman  Research  Laboratory,  New  York. 
MCNAMARA,  HELEN,  Technician,  Rockefeller  Institute. 
MORGAN,  EDITH,  409  West  nyth  Street,  New  York  City. 
PARPART,  ETHEL  ROBERTA,  Assistant,  Amherst  College. 
REYNOLDS,  SAMUEL  R.   M.,  Assistant  in  Physiology  and  Zoology,  Swarthmore 

College. 
RIOCH,    DAVID    MCKENZIE,    Instructor    in    Medicine,    University    of   Rochester, 

Medical  School. 

SANDERS,  GERTRUDE  B.,  Swarthmore  College. 
SCHAUFFLER,  WILLIAM  GRAY,  Private  Practitioner  of  Medicine,  Princeton,  New 

Jersey. 

ULLIAN,  SILKA  STOCKER,  Research  Assistant,  Carnegie  Institution  of  Washington. 
WALDEN,  EDA  B.,  Research  Assistant,  Lilly  Research  Laboratory. 
WALLACE,    EDITH    M.,    Artist   and    Research   Assistant,    Carnegie    Institution    of 

Washington. 

WATERMAN,  HARRIET  C.,  Research  Assistant,  Carnegie  Institution  of  Washington. 
WEARE,  J.  H.,  Research  Assistant,  Harvard  University  Medical  School. 


38  MARINE    BIOLOGICAL    LABORATORY. 

STUDENTS 

Botany. 

BAKER,  CAROLYN,  Bellair  Drive,  Dobbs  Ferry,  New  York. 

BOWERS,  W.  B.,  Student,  Harvard  University. 

DUNBAR,  FRANCIS  F.,  Student,  Harvard  University. 

FORT,  IRENE,  University  of  Pennsylvania. 

HOPPAUGH,  KATHERINE  W.,  1176  East  South  Temple  Street,  Salt  Lake  City.  Utah. 

HUSTED,  DON  L.,  Student,  Oberlin  College. 

JEWETT,  FRANCIS  L.,  273  Woodland  Road,  Ravinia,  Illinois. 

KEITH,  BERNICE,  Hastings,  Nebraska. 

MACFARLANE,  CONSTANCE,  87  Upper  Prince  Street,  Charlottetown,  Prince  Edward 

Island,  Canada. 

McCLiNTOCK,  BARBARA,  Instructor  in  Botany,  Cornell  University. 
NAYLOR,  ERNST,  Instructor  in  Botany,  University  of  Missouri. 
PATRICK,  RUTH  M.,  Student,  Coker  College. 
PINSDORF,  KATE,  Smith  College. 
PYLE,  THERESA  P.,  Smith  College. 

WELLS,  EVELYN  CLARE,  Teaching  Fellow,  University  of  Tennessee. 
ZIMMERMANN,  RUTH  HELEN,  Teacher,  Brockton  High  School. 

Embryology. 

BAILY,  JOSHUA  L.,  JR.,  Institute  for  Biological  Research,  Baltimore,  Md. 

BAILEY,  PERCY  L.,  JR.,  Graduate  Student,  Brown  University. 

BEYER,  KATHE  M.,  93  Benefit  Street,  Providence,  Rhode  Island. 

BOSWORTH,  EDWARD  B.,  Assistant,  Yale  University. 

BOUGHTON,  ESTHER  MARIE,  Box  339,  Poughkeepsie,  New  York. 

CHASE,  AURIN  M.,  JR.,  Assistant,  Amherst  College. 

CHEN,  NELSON  S.,  University  of  Pennsylvania. 

CRANE,  NORMAN  F.,  Bowdoin  College. 

CURTIS,  MARY  ELIZABETH,  Assistant  in  Biology,  Wilson  College. 

DALTON,  ALBERT  JOSEPH,  Wesleyan  University. 

DAVIDSON,  MARGARET  H.,  North  Carolina  College  for  Women. 

DEICHMANN,  ELIZABETH,  Radcliffe  College. 

FLETCHER,  LYDIA  M.,  Brown  University. 

GRIZZLE,  LUCILE  A.,  University  of  Southern  California. 

HAMILTON,  SALLY,  Elmira  College. 

H.ARDESTY,  MARY,  Teaching  Fellow  in  Biology,  Newcomb  College. 

HARLAND,  MARGARET,  North  Carolina  College  for  Women. 

HERSKOWITZ,  ISIDOR  A.,  Columbia  University. 

HIRAIWA,  YOSHI  KUNI,  University  of  Chicago. 

HOLLINSHEAD,  WILLIAM  HENRY,  Instructor,  Vanderbilt  University. 

JANSEN,  JAN  BIRGES,  Royal  Fredericks  University,  Oslo,  Norway. 

LICHTMAN,  FRIEDA,  Student,  New  York  University. 

LUCE,  WILBUR  M.,  University  of  Illinois. 

McGouN,  RALPH  C.,  JR.,  Assistant,  Amherst  College. 

MILLER,  RUTH  A.,  13  Poplar  Avenue,  Woodlawn,  Wheeling,  West  Virginia. 

NABRIT,  SAMUEL  M.,  Instructor  in  Zoology,  Morehouse  College. 

PARSONS,  ELIZABETH  H.,  Graduate  Student,  Oberlin  College. 

ROWELL,  LYMAN  S.,  Instructor,  University  of  Vermont. 


REPORT    OF    THE   DIRECTOR.  39 

TRACY,  BARBARA,  Connecticut  College. 

WATERMAN,  ALLYN  JAY,  Graduate  Assistant,  Western  Reserve  University. 

WEN,  I.,  Medical  College,  Peking,  China. 

WOODARD,  THOMAS  M.,  JR.,  Instructor,  Vanderbilt  University. 

Physiology. 

BAHRS,  ALICE  M.,  Assistant  in  Physiology,  University  of  California. 
BARRON,  E.  G.,  802  North  Washington  Street,  Baltimore,  Maryland. 
BORQUIST,  MAY,  Research  Fellow,  Cornell  Medical  College. 
CLARKE,  ROBERT  W.,  New  York  University. 
DEBRUE,  GEORGES  H.,  Louvain  University. 

DOWNEY,  HAROLD  R.,  Student,  Johns  Hopkins  Medical  School. 
FRANK,  RICHARD  L.,  Student,  Cornell  Medical  College. 
HENDERSON,  JEAN  T.,  Lecturer,  McGill  University,  Montreal,  Canada. 
HOWLAND,  ESTHER,  107  East  64th  Street,  New  York  City,  N.  Y. 
LIGHT,  FREDERICK  W.,  JR.,  Student,  Johns  Hopkins  Medical  School. 
MILLER,  EVELYN  H.,  Graduate  Student,  Stanford  University,  California. 
NEWTON,  ISABEL  M.,  Assistant  in  Physiology,  Mount  Holyoke  College. 
OLCOTT,  CHARLES  T.,  Instructor  in  Pathology,  Cornell  Medical  College. 
PANKRATZ,  DAVID  S.,  Instructor,  University  of  Kansas. 
PARPART,  ARTHUR  R.,  Instructor,  Amherst  College. 
STEELE,  CHARLES  W.,  University  of  Missouri. 
TEWINKEL,  HELEN,  Assistant  in  Zoology,  Oberlin  College. 

TURNER,  EDNA  M.,  Assistant  in  Biology,  Washington  Square  College,  N.  Y.  U. 
UHLENHUTH,    EDUARD,    Associate    Professor,    University   of    Maryland,    Medical 
School. 

Protozoology. 

ADAMS,  THEODORE  G.,  College  of  the  City  of  New  York. 

ALEXANDER,  ELEANOR  G.,  Graduate  Student,  Columbia  University. 

DEBONE,  FRANCES  M.,  Student  Assistant  in  Anatomy,  University  of  Pittsburgh. 

DETTMER,  CLARA  ROWENA,  Columbia  University. 

GOODLOW,  SARA,  Goucher  College. 

HETHERINGTON,  WILLIAM  A.,  Assistant  in  Zoology,  Columbia  University. 

HOLLIDAY,  GAIL  H.,  Teacher  of  Biology,  Wheeling  High  School. 

HUBBARD,  CATHERINE  E.,  Cromwell,  Connecticut. 

KINNEY,  ELIZABETH  T.,  Graduate-assistant,  University  of  Pittsburgh. 

MORRIS,  HELEN  S.,  Graduate  Student,  Columbia  University. 

NELSON,  GEORGE  E.,  3038  Hull  Avenue,  Bronx,  New  York  City. 

RICHTER,  MARION  C.  R.,  Columbia  University. 

ROBERTSON,  GEORGE,  Instructor,  Dartmouth  College. 

SHIELDS,  LAWRENCE  M.,  Instructor,  Phillips  Academy. 

VAN  RHYN,  ELSIE  A.,  Instructor  in  Biology,  University  of  Porto  Rico. 

Wu,  CHAO-FA,  Assistant,  University  of  Wisconsin. 

ZIMMER,  DOROTHY  K.,  Columbia  University. 

Zoology. 

ABELL,  RICHARD  G.,  Instructor  in  Biology,  Hampton  Institute. 
ANDREWS,  AVA  LEE,  North  Carolina  College  for  Women. 
APGAR,  GRACE  M.,  University  of  Pennsylvania. 


AQ  MARINE    BIOLOGICAL    LABORATORY. 

BALLARD,  WILLIAM  W.,  Student,  Dartmouth  College. 

BEEBE,  MARY  ELIZABETH,  Oberlin  College. 

BILSTAD,  NELLIE  MAE,  Assistant,  University  of  Wisconsin. 

BLOUNT,  RAYMOND  F.,  Instructor,  University  of  Arizona. 

BOND,  EVELYN,  University  of  Pennsylvania. 

BRADLEY,  MARY  A.,  Wabash,  Indiana. 

BROWN,  DUGALD  E.  S.,  Instructor  in  Biology,  New  York  University. 

BUTLER,  ELIZABETH,  257  Newbury  Street,  Boston,  Massachusetts. 

CLINE,  ELSIE,  Teacher,  Baltimore  Public  Schools. 

CLOUDMAN,  ARTHUR  M.,  Instructor,  University  of  Vermont. 

COLDWATER,  KENNETH  B.,  University  of  Missouri. 

DRUMTRA,  ELIZABETH,  5  Curran  Avenue,  Binghamton,  New  York. 

ELFTMAN,  HERBERT  O.,  Assistant  in  Zoology,  Columbia  University. 

ELLIS,  MARJORIE  F.,  Dalhousie  University,  Halifax,  Nova  Scotia. 

FERRIS,  FRANCES  R.,  Assistant  in  Zoology,  Washington  University. 

FRAME,  ELIZABETH  G.,  Dalhousie  University,  Halifax,  Nova  Scotia. 

FURTOS,  NORMA  C.,  Graduate  Assistant,  Western  Reserve  University. 

GREGG,  WARD  I.,  Harvard  University,  Cambridge,  Mass. 

GREGORY,  PAUL  W.,  Harvard  University. 

HALL,  EDMUND  K.,  Assistant,  Yale  University. 

HAMPEL,  CHESTER  W.,  Wesleyan  University. 

HARE,  LAURA,  DePauw  University. 

HOPKINS,  SEWELL  H.,  William  and  Mary  College. 

HUSTED,  CLARA  M.,  Graduate  Student,  University  of  Rochester. 

JOHNSON,  PORTEOUS  E.,  Amherst  College. 

KERRIGAN,  ALICE  M.,  Teacher  of  Biology,  Teachers  College  of  the  City  of  Boston. 

LANE,  ELINOR  M.,  Assistant  in  Biology,  Goucher  College. 

LEONARD,  SAMUEL  L.,  Rutgers  University. 

LOVELL,  HARVEY  B.,  Harvard  University. 

MCCLURE,  GEORGE  Y.,  Student,  Dartmouth  College. 

MCCLURE,    KATHERINE  L.,   Instructor  in   Biology,   Morningside   College,    Sioux 

City,  Iowa. 

MACCOY,  CLINTON  V.,  Harvard  College. 
MclNERNEY,  KATHRYN  M.,  Tufts  College. 
McNuTT,  DOROTHEA,  Illinois  Wesleyan  University. 
MARTIN,  STEVEN  J.,  University  of  Wisconsin. 

MARTINOVITCH,  PETAR  N.,  Graduate  Student,  Syracuse  University. 
MILLIKEN,  ELEANOR,  Wellesley  College. 
MOLINA,  ANA  M.,  University  of  Porto  Rico. 
NEWCOMER,  A.  VIRGINIA,  Goucher  College. 
PFEIFER,  KATHERINE  M.,  Washington  University. 
PICKETT,  NATHAN  W.,  Wabash  College. 
PREFONTAINE,  GEORGE  H.,  Assistant  in  Biology,  University  of  Montreal,  Montreal, 

Canada. 

RECK,  VIRGINIA  D.,  Assistant  in  Biology,  Yale  University. 
SCHMIDT,  LORRIMER  M.,  Treble  Cove  Road,  North  Billerica,  Massachusetts. 
SHINAR,  CATHERINE,  Hunter  College. 
SHOREY,  DOROTHY  E.,  Radcliffe  College. 
SMALL,  VIRGINIA,  Butler  University. 
SMELSER,  GEORGE  K.,  Studer';,  Earlham  College. 


REPORT    OF   THE   DIRECTOR.  4! 

SNELL,  GEORGE  D.,  Graduate  Student,  Harvard  University. 

STABLER,  ROBERT  M.,  Swarthmore  College. 

STEHR,  WILLIAM  C.,  Assistant  in  Zoology,  University  of  Minnesota. 

SUN,  TSON  P.,  Wusih,  Kiangsu,  China. 

TURNBULL,  VIRGINIA  E.,  Teacher,  Dorchester  High  School  for  Girls. 

WILDE,  FRANCES,  Student,  Radcliffe  College. 


3.     TABULAR  VIEW   OF  ATTENDANCE. 

1923  1924  1925  1926  1927 

INVESTIGATORS — Total    176  194  207  252  294 

Independent   126  124  135  156  209 

Under  Instruction    50  70  72  84  57 

Research   Assistants    12  28 

STUDENTS — Total     146  134  132  141  141 

Zoology    59  50  54  56  57 

Protozoology    16  17  17  19  17 

Embryology     31  29  29  28  32 

Physiology     22  18  19  18  19 

Botany    18  20  13  20  16 

TOTAL  ATTENDANCE    322  328  339  393  435 

Less  persons  registered  as  both 

Students  and   Investigators    8  I 


385  434 

INSTITUTIONS   REPRESENTED — Total    ..    107  no       112       119  in 

By    investigators    62  69         74         84  89 

By  students   73  68        65         60  63 

SCHOOLS  AND  ACADEMIES  REPRESENTED 

By  investigators   I  I 

By  students   4  4           4  4 

FOREIGN  INSTITUTIONS  REPRESENTED 

By  investigators   17  15 

By  students   3  8 


MARINE    BIOLOGICAL   LABORATORY 


4.     SUBSCRIBING    AND    COOPERATING 
INSTITUTIONS,    1927. 


Amherst  College 

Antioch   College 

Barnard  College 

Bowdoin   College 

Brown  University 

Bryn    Mawr   College 

Butler  College 

C.  R.  B.  Educational  Foundation 

Carnegie  Institution,  Cold  Spring 
Harbor 

Carnegie  Institution  of  Washing- 
ton 

Columbia  University 

Commonwealth  Fund 

Connecticut    College 

Cornell  University 

Cornell  University  Medical  Col- 
lege 

Dalhousie  University 

Dartmouth    College 

De  Pauw  University 

Duke   University 

Elmira  College 

T.  W.  Evans  Dental  Museum  and 
School  of  Dentistry 

General    Educational    Board 

Goucher  College 

Harvard  University 

Harvard       University       Medical 

School 

Howard  University 

Hunter  College 

Industrial  &  Engineering  Chem- 
istry, of  the  American  Chem- 
ical Society 

International  Education  Board 

Johns  Hopkins  University 


Johns    Hopkins   University   Med- 
ical  School 

Knox  College 

Eli  Lilly  &  Co. 

Massachusetts  Institute  of  Tech- 
nology 

McGill   University 

Morningside  College 

Mount  Holyoke  College 

National    Research   Council 

New  York  Homeopathic  Medical 
College 

New  York  University 

New     York     University     Dental 
School 

North      Carolina      College      for 
Women 

Norwich  University 

Oberlin   College 

Princeton  University 

Radcliffe   College 

Rockefeller  Foundation 

Rockefeller  Institute  for  Medical 
Research 

Rutgers   University 

Smith  College 

Sophie  Newcomb  College 

Southwestern 

Swarthmore  College 

Tufts  College 

Union  College 

United   States   Dept.   of   Agricul- 
ture 

University  of  Alabama 

University  of  Arkansas 

University  of  Chicago 

University  of   Illinois 

University  of   Kansas 


REPORT   OF   THE   DIRECTOR. 


43 


University   of 

School 

University  of 
University  of 
University  of 

School 

University  of 
University  of 
University  of 

ical  School 
University  of 
University  of 
University  of 
University  of 


Maryland   Medical 

Michigan 
Minnesota 
Minnesota  Medical 

Missouri 
Pennsylvania 
Pennsylvania  Med- 

Pittsburgh 
Rochester 
Vermont 
Virginia 


University  of  Wisconsin 
Vanderbilt      University     Medical 

School 

Vassar    College 
Wabash  College 
Washington   University 
Washington    University    Medical 

School 

Wellesley  College 
Wesleyan  University 
Western   Reserve  University 
Wistar  Institute  of  Anatomy  and 

Biology 
Yale   University 


SCHOLARSHIP  TABLES. 


Ida  H.  Hyde  Scholarship  of  the  University  of  Kansas. 

Lucretia  Crocker   Scholarships   for  Teachers  in  Boston. 

Scholarship  of  $100.00  supported  by  a  friend  of  the  Laboratory  since 

1898. 
The    Edwin    S.    Linton    Memorial    Scholarship    of    Washington    and 

Jefferson  College. 

5.     EVENING  LECTURES,   1927. 

Tuesday,  June  28, 

PROFESSOR  S.  O.  MAST "Structure,  Locomotion  and  Stim- 
ulation  in  Ameba   Proteus." 
Friday,  July   I, 

PROFESSOR  L.  V.  HEILBRUNN "The  Colloid  Chemistry  of  Proto- 
plasm." 
Tuesday,  July  5, 

PROFESSOR  ALEXANDER  FORBES.  .  ."Implications  of  the  All-or-none 

Principle  in  the  Physiology  of 
the    Central    Nervous    System." 
Friday,  July  8, 

PROFESSOR  B.  M.  DUGGAR "The    Physiological    Behavior    of 

Certain  Virus  Agencies." 
Tuesday,  July  12, 

PROFESSOR   E.   G.   CONKLIN "Localization  Phenomena  in  Em- 
bryology." 


44  MARINE    BIOLOGICAL    LABORATORY. 

Friday,  July   15, 

DR.  E.  M.  LANDIS   "The   Permeability   of  the   Capil- 
lary Wall." 
Tuesday,  July  19, 

DR.  JACQUES   BRONFENBRENNER.  .  "The    Nature    of    the    Bacterio- 

phage  Phenomenon." 
Friday,   July   22, 

PROFESSOR  FRANK  R.  LILLIE "The   Gene   and   the    Ontogenetic 

Process." 
Tuesday,   July  26, 

PROFESSOR  R.  M.  YERKES "The  Psycho-biology  of  the  Go- 
rilla." 
Friday,  July  29, 

DR.  HENRY  B.  BIGELOW "Oceanographic      Problems     and 

Opportunities." 
Tuesday,   August   2, 

PROFESSOR  J.  C.  DRUMMOND "The    Chemistry    of    Vitamines." 

Wednesday,      August      3,      (Special 
Lecture) 

PROFESSOR  H.  D.  FISH "Life     along     the     Trails     and 

Streams  of  British  Guiana." 
Friday,  August   5, 

DR.  WILLIAM  MANSFIELD  CLARK.  "A  Restricted  but  New  Approach 

to    Oxidation-reduction    in    the 
Living   Cell." 


6.     MEMBERS  OF  THE  CORPORATION. 

LIFE  MEMBERS. 

ALLIS,  MR.  E.  P.,  JR.,  Palais  Carnoles,  Menton,  France. 
ANDREWS,  MRS.  GWENDOLEN  FOULKE,  Baltimore,  Maryland. 
BILLINGS,  MR.  R.  C.,  66  Franklin  Street,  Boston,  Mass. 
CLARKE,  PROF.  S.  F.,  Williamstown,  Mass. 
CONKLIN,   PROF.   EDWIN  G.,   Princeton   University,   Princeton, 

New  Jersey. 

COOLIDGE,  MR.  C.  A.,  Ames  Building,  Boston,  Mass. 
CRANE,  MR.  C.  R.,  New  York  City. 
EVANS,  MRS.  GLENDOWER,  12  Otis  Place,  Boston,  Mass. 
FAY,  Miss  S.  B.,  88  Mt.  Vernon  Street,  Boston,  Mass. 


REPORT    OF    THE   DIRECTOR.  45 

FOOT,   Miss   KATHERINE,   Care  of  Morgan   Harjes   Cie,   Paris, 

France. 

GARDINER,  MRS.  E.  G.,  Woods  Hole,  Mass. 
HARRISON,    EX-PROVOST    C.    C.,    University   of    Pennsylvania, 

Philadelphia,  Pa. 

JACKSON,  Miss  M.  C.,  88  Marlboro  St.,  Boston,  Mass. 
JACKSON,  MR.  CHAS.  C.,  24  Congress  St.,  Boston,  Mass. 
KIDDER,  MR.  NATHANIEL  T.,  Milton,  Mass. 
KING,  MR.  CHAS.  A. 
LEE,  MRS.  FREDERIC  S.,  279  Madison  Ave.,  New  York  City, 

N.  Y. 

LOWELL,  MR.  A.  LAWRENCE,  17  Quincy  St.,  Cambridge,  Mass. 
MARRS,  MRS.  LAURA  NORCROSS,  9  Commonwealth  Ave.,  Boston, 

Mass. 

MASON,  Miss  E.  F.,  i  Walnut  St.,  Boston,  Mass. 
MASON,  Miss  IDA  M.,  I  Walnut  St.,  Boston,  Mass. 
MEANS,  DR.  JAMES  HOWARD,  15  Chestnut  St.,  Boston,  Mass. 
MERRIMAN,  MRS.  DANIEL,  73  Bay  State  Road,  Boston,  Mass. 
MINNS,  Miss  SUSAN,  14  Louisburg  Square,  Boston,  Mass. 
MORGAN,   MR.  J.   PIERPONT,  JR.,  Wall  and   Broad  Sts.,   New 

York  City,  N.  Y. 
MORGAN,  PROF.  T.  H.,  Columbia  University,  New  York  City, 

N.  Y. 

MORGAN,  MRS.  T.  H.,  New  York  City,  N.  Y. 
No  YES,  Miss  EVA  J. 
OSBORN,    PROF.    HENRY    F.,    American    Museum    of    Natural 

History,  New  York  City,  N.  Y. 

PHILLIPS,  MRS.  JOHN  C.,  Windy  Knob,  Wenham,  Mass. 
PORTER,  DR.  H.  C.,  University  of  Pennsylvania,  Philadelphia,  Pa. 
PULSIFER,  MR.  W.  H.,  Newton  Center,  Mass. 
SEARS,  DR.  HENRY  F.,  86  Beacon  St.,  Boston,  Mass. 
SHEDD,  MR.  E.  A. 
THORNDIKE,    DR.    EDWARD    L.,    Teachers    College,    Columbia 

University,  New  York  City,  N.  Y. 

TRELEASE,  PROF.  \VILLIAM,  University  of  Illinois,  UYbana,  111. 
WARE,  Miss  MARY  L.,  41  Brimmer  St.,  Boston,  Mass. 
WILLIAMS,  MRS.  ANNA  P.,  505  Beacon  St.,  Boston,  Mass. 
WILSON,  DR.  E.  B.,  Columbia  University,  New  York  City,  N.  Y. 


46  MARINE  BIOLOGICAL  LABORATORY. 

CORPORATION  MEMBERSHIP  LIST, 
AUGUST  1927. 

ADAMS,    DR.   A.    ELIZABETH,    Mount   Holyoke    College,   South 

Hadley,  Mass. 
ADDISON,  DR.  W.  H.  F.,  University  of  Pennsylvania  Medical 

School,  Philadelphia,  Pennsylvania. 
ADOLPH,  DR.  EDWARD  F.,  University  of  Rochester,  School  of 

Medicine  and  Dentistry,  Rochester,  N.  Y. 
AGERSBORG,  DR.  H.  P.  K.,  James  Millikin  University,  Decatur, 

Illinois. 

ALLEE,  DR.  W.  C.,  University  of  Chicago,  Chicago,  Illinois. 
ALLEN,    PROF.    CHAS.    E.,   University  of  Wisconsin,    Madison, 

Wisconsin. 
ALLEN,    PROF.    EZRA,    1003   South    46th    Street,    Philadelphia, 

Pennsylvania. 

ALLYN,  DR.  HARRIET  M.,  Vassar  College,  Poughkeepsie,  N.  Y. 
AMBERSON,    DR.    WILLIAM    B.,    University    of    Pennsylvania, 

Philadelphia,  Pa. 
ANDERSON,    DR.   E.   G.,   University  of   Michigan,   Ann   Arbor, 

Michigan. 

ATTERBURY,  MRS.  RUTH  R.,  Great  Neck,  Long  Island,  New  York. 
BAITSELL,    DR.    GEORGE    A.,    Yale    University,    New    Haven, 

Connecticut. 
BAKER,   DR.   E.   H.,   5312   Hyde   Park  Boulevard,   Hyde   Park 

Station,  Chicago,  111. 
BALDWIN,  DR.  F.  M.,  University  of  Southern  California,  Los 

Angeles,  Calif. 
BASCOM,   DR.   K.   F.,   Medical   School   of  Virginia,   Richmond, 

Virginia. 

BECKWITH,  DR.  CORA  J.,  Vassar  College,  Poughkeepsie,  N.  Y. 
BEHRE,    DR.    ELINOR   H.,    Louisiana   State    University,    Baton 

Rouge,  La. 
BENNITT,    DR.    RUDOLF,    University    of    Missouri,    Columbia, 

Missouri. 
BIGELOW,  PROF.  R.  P.,  Massachusetts  Institute  of  Technology, 

Cambridge,  Mass. 
BINFORD,  PROF.  RAYMOND,  Guilford  College,  Guilford  College, 

N.  C. 


REPORT    OF    THE   DIRECTOR.  47 

BISSONNETTE,  DR.  T.  H.,  Trinity  College,  Hartford,  Connecticut. 
BODINE,  DR.  J.  H.,  University  of  Pennsylvania,  Philadelphia,  Pa. 
BORING,  DR.  ALICE  M.,  Yenching  College,  Peking,  China. 
BOWEN,  DR.  ROBERT  H.,  Columbia  University,  New  York  City. 
Box,  Miss  CORA  M.,  University  of  Cincinnati,  Cincinnati,  Ohio. 
BRADLEY,  PROF.  HAROLD  C.,  University  of  Wisconsin,  Madison, 

Wisconsin. 

BRAILEY,  Miss  MIRIAM  E.,  800  Broadway,  Baltimore,  Maryland. 
BRIDGES,  DR.  CALVIN  B.,  Columbia  University,  New  York  City. 
BROOKS,  DR.  S.  C.,  University  of  California,  Berkeley,  California. 
BUCKINGHAM,  Miss  EDITH  N.,  Sudbury,  Massachusetts. 
BUDINGTON,  PROF.  R.  A.,  Oberlin  College,  Oberlin,  Ohio. 
BULLINGTON,   DR.  W.   E.,   Randolph-Macon  College,  Ashland, 

Virginia. 

BUMPUS,  PROF.  H.  C.,  Duxbury,  Massachusetts. 
BYRNES,  DR.  ESTHER  F.,  1803  North  Camac  Street,  Philadelphia, 

Pa. 

CALKINS,  PROF.  GARY  N.,  Columbia  University,  New  York  City. 
CALVERT,  PROF.  PHILIP  P.,  University  of  Pennsylvania,  Phila- 
delphia, Pa. 

CARLSON,  PROF.  A.  J.,  University  of  Chicago,  Chicago,  Illinois. 
CAROTHERS,    DR.    ELEANOR    E.,    University    of    Pennsylvania, 

Philadelphia,  Pa. 
CARROLL,    PROF.    MITCHEL,    Franklin    and    Marshall    College, 

Lancaster,  Pa. 

CARVER,  PROF.  GAIL  L.,  613  Orange  Street,  Macon,  Georgia. 
CASTEEL,  DR.  D.  B.,  University  of  Texas,  Austin,  Texas. 
CATTELL,  PROF.  J.  McKEEN,  Garrison-on-Hudson,  New  York. 
CATTELL,   DR.   McKEEN,   Cornell  University  Medical   College, 

New  York  City. 

CATTELL,  MR.  WARE,  Garrison-on-Hudson,  New  York. 
CHAMBERS,   DR.  ROBERT,  Cornell  University  Medical  College, 

New  York  City. 
CHARLTON,  DR.  HARRY  H.,  University  of  Missouri,  Columbia, 

Missouri. 
CHIDESTER,  PROF.  F.  E.,  West  Virginia  University,  Morgantown, 

W.  Va. 

CHILD,  PROF.  C.  M.,  University  of  Chicago,  Chicago,  Illinois. 
CLAPP,  PROF.  CORNELIA  M.,  Montague,  Massachusetts. 


48  MA  KIM.    lilOLOGlCAL    LABORATORY. 

CLARK,  PROF.  E.  R.,  University  of  Pennsylvania,  Philadelphia, 
Pa. 

CLELAND,  PROF.  RALPH  E.,  Goucher  College,  Baltimore,  Mary- 
land. 

CLOWES,  PROF.  G.  H.  A.,  Eli  Lilly  &  Co.,  Indianapolis,  Indiana. 

COE,  PROF.  W.  R.,  Yale  University,  New  Haven,  Connecticut. 

COHN,  DR.  EDWJX  J.,  19  Ash  St.,  Cambridge,  Mass. 

COKER,  DR.  R.  E.,  University  of  North  Carolina,  Chapel  Hill, 
North  Carolina. 

COLE,  DR.  LEON  J.,  College  of  Agriculture,  Madison,  Wisconsin. 

COLLETT,  DR.  MARY  E.,  Western  Reserve  University,  Cleveland, 
Ohio. 

COLLEY,  MRS.  MARY  W.,  1712  Madison  St.,  Madison,  Wisconsin. 

COLTON,  PROF.  H.  S.,  Box  127,  Flagstaff,  Arizona. 

CONNOLLY,  DR.  C.  J.,  Catholic  University,  Washington,  D.  C. 

COPELAND,  PROF.  MANTON,  Bowdoin  College,  Brunswick,  Maine. 

COWDRY,  DR.  E.  V.,  Rockefeller  Institute,  New  York  City. 

CRAMPTON,  PROF.  H.  E.,  Barnard  College,  Columbia  University, 
New  York  City. 

CRANE,  MRS.  C.  R.,  Woods  Hole,  Mass. 

CURTIS,  DR.  MAYNIE  R.,  Crocker  Laboratory,  Columbia  Uni- 
versity, New  York  City. 

CURTIS,  PROF.  Wr.  C.,  University  of  Missouri,  Columbia,  Mo. 

DANCHAKOFF,  DR.  VERA,  Timiriaseff  Research  Institute,  Mos- 
cow, Russia. 

DAVIS,  DR.  DONALD  W.,  College  of  William  and  Mary,  Williams- 
burg,  Va. 

DAVIS,  DR.  ALICE  R.,  19  Ash  St.,  Cambridge,  Mass. 

DAWSON,  DR.  J.  A.,  Harvard  University,  Cambridge,  Mass. 

DEDERER,  DR.  PAULINE  H.,  Connecticut  College,  New  London, 
Connecticut. 

DELLINGER,  DR.  S.  C.,  University  of  Arkansas,  Fayetteville,  Ark. 

DETLEFSEN,  DR.  J.  A.,  Swarthmore,  Pennsylvania. 

DEXTER,  DR.  J.  S.,  University  of  Porto  Rico,  Rio  Piedras, 
Porto  Rico. 

DODDS,  PROF.  G.  S.,  Medical  School,  University  of  West  Virginia, 
Morgantown,  WTest  Virginia. 

DOLLEY,  PROF.  WILLIAM  L.,  University  of  Buffalo,  Buffalo, 
N.Y. 


REPORT    OF    THE   DIRECTOR.  49 

DONALDSON,  PROF.  H.  H.,  Wistar  Institute  of  Anatomy  and 
Biology,  Philadelphia,  Pennsylvania. 

DONALDSON,  DR.  JOHN  C.,  University  of  Pittsburgh,  School  of 
Medicine,  Pittsburgh,  Pa. 

DREW,  PROF.  GILMAN  A.,  Eagle  Lake,  Florida. 

DUGGAR,  DR.  BENJAMIN  M.,  University  of  Wisconsin,  Madison, 
Wisconsin. 

DUNGAY,  DR.  NEIL  S.,  Carleton  College,  Northfield,  Minn. 

DUNN,  DR.  ELIZABETH  H.,  105  North  5th  Ave.,  La  Grange, 
111. 

EDWARDS,  DR.  D.  J.,  Cornell  University  Medical  College,  New 
York  City. 

ELLIS,  DR.  F.  W.,  Monson,  Mass. 

FARNUM,  DR.  LOUISE  W.,  43  Hillhouse  Ave.,  New  Haven,  Conn. 

FARR,  DR.  C.  H.,  Washington  University,  St.  Louis,  Mo. 

FENN,  DR.  W.  O.,  Rochester  University  School  of  Medicine, 
Rochester,  N.  Y. 

FIELD,  Miss  HAZEL  E.,  Occidental  College,  Los  Angeles,  Cali- 
fornia. 

FORBES,  DR.  ALEXANDER,  Harvard  University  Medical  School, 
Boston,  Mass. 

FRY,  DR.  HENRY  J.,  Washington  Square  College,  New  York  City. 

GAGE,  PROF.  S.  H.,  Cornell  University,  Ithaca,  N.  Y. 

GARREY,  PROF.  W.  E.,  Vanderbilt  University  Medical  School, 
Nashville,  Tennessee. 

GATES,  DR.  F.  L.,  Rockefeller  Institute,  New  York  City. 

GATES,  PROF.  R.  RUGGLES,  University  of  London,  London, 
England. 

GEISER,  DR.  S.  W.,  Southern  Methodist  University,  Dallas, 
Texas. 

GLASER,  PROF.  O.  C.,  Amherst  College,  Amherst,  Mass. 

GLASER,  PROF.  R.  W.,  Rockefeller  Institute  for  Medical  Re- 
search, Princeton,  N.  J. 

GOLDFORB,    PROF.   A.   J.,    College   of   the   City  of   New  York, 

New  York  City. 
GOODRICH,    PROF.    H.    B.,    Wesleyan    University,    Middletown, 

Conn. 

GRAHAM,  DR.  J.  Y.,  University  of  Alabama,  University,  Alabama. 
GRAVE,  PROF.  B.  H.,  Wabash  College,  Crawfordsville,  Indiana. 


5Q  MARINE    BIOLOGICAL    LABORATORY. 

GRAVE,    PROF.    CASWELL,    Washington    University,    St.    Louis, 

Missouri. 
GREENMAN,    PROF.   M.   J.,   Wistar  Institute   of  Anatomy  and 

Biology,  Philadelphia,  Pennsylvania. 

GREGORY,    DR.   LOUISE   H.,    Barnard   College,    Columbia   Uni- 
versity, New  York  City. 
GU*THRIE,    DR.    MARY   J.,    University   of   Missouri,    Columbia, 

Missouri. 

GUYER,  PROF.  M.  F.,  University  of  Wisconsin,  Madison,  Wis- 
consin. 
HAGUE,    DR.    FLORENCE,    Sweet    Briar    College,    Sweet    Briar, 

Virginia. 

HALSEY,  DR.  J.  T.,  Tulane  University,  New  Orleans,  Louisiana. 
HANCE,  DR.  ROBERT  T.,  University  of  Pittsburgh,  Pittsburgh, 

Pennsylvania. 
HARGITT,    PROF.    GEORGE  T.,   Syracuse   University,   Syracuse, 

New  York. 
HARMAN,    DR.    MARY  T.,   Kansas  State   Agricultural    College, 

Manhattan,  Kansas. 

HARPER,  PROF.  R.  A.,  Columbia  University,  New  York  City. 
HARRISON,    PROF.    Ross    G.,    Yale    University,    New    Haven, 

Connecticut. 

HARVEY,  PROF.  E.  N.,  Princeton  University,  Princeton,  N.  J. 
HARVEY,  MRS.  E.  N.,  Princeton,  New  Jersey. 
HAYDEN,    DR.    MARGARET    A.,   Wellesley    College,   Wellesley, 

Mass. 
HAZEN,  DR.  T.  E.,  Barnard  College,  Columbia  University,  New 

York  City. 

HEATH,  PROF.  HAROLD,  Pacific  Grave,  California. 
HECHT,  DR.  SELIG,  Columbia  University,  New  York  City. 
HEGNER,  PROF.  R.  W.,  Johns  Hopkins  University,  Baltimore, 

Maryland. 
HEILBRUNN,   DR.  L.  V.,  University  of  Michigan,  Ann  Arbor, 

Michigan. 
HESS,    PROF.    WALTER   N.,.  DePauw    University,    Greencastle, 

Indiana. 
HINRICKS,    DR.    MARIE   A.,    University   of    Chicago,    Chicago, 

Illinois. 
HISAW,  DR.  F.  L.,  University  of  Wisconsin,  Madison,  Wisconsin. 


REPORT    OF   THE   DIRECTOR.  5! 

HOADLEY,  DR.  LEIGH,  Harvard  University,  Cambridge,  Massa- 
chusetts. 

HOGUE,  DR.  MARY  J.,  503  North  High  Street,  West  Chester; 
Pennsylvania. 

HOLMES,  PROF.  S.  J.,  University  of  California,  Berkeley,  Cali- 
fornia. 

HOOKER,  PROF.  DAVENPORT,  University  of  Pittsburgh,  Pitts- 
burgh, Pa. 

HOPKINS,  DR.  HOYT  S.,  New  York  University  College  of  Den- 
tistry, New  York  City. 

HOSKINS,  MRS.  ELMER  R.,  New  York  University,  College  of 
Dentistry,  New  York  City. 

HOWE,  DR.  H.  E.,  2702— 36th  Street,  N.  W.,  Washington,  D.  C. 

HOYT,  DR.  WILLIAM  D.,  Washington  and  Lee  University, 
Lexington,  Virginia. 

HUMPHREY,  DR.  R.  R.,  University  of  Buffalo  School  of  Medicine, 
Buffalo,  N.  Y. 

HYMAN,  DR.  LIBBIE  H.,  University  of  Chicago,  Chicago,  Illinois. 

INMAN,  PROF.  ONDESS  L.,  Antioch  College,  Yellow  Springs,  Ohio. 

IRWIN,  DR.  MARIAN,  Rockefeller  Institute,  New  York  City. 

JACKSON,  PROF.  C.  M.,  University  of  Minnesota,  Minneapolis, 
Minnesota. 

JACOBS,  DR.  MERKEL  H.,  University  of  Pennsylvania,  Phila- 
delphia, Pennsylvania. 

JENNINGS,  PROF.  H.  S.,  Johns  Hopkins  University,  Baltimore, 
Md. 

JEWETT,  PROF.  J.  R.,  Harvard  University,  Cambridge,  Massa- 
chusetts. 

JOHNSON,  PROF.  GEORGE  E.,  State  Agricultural  College,  Man- 
hattan, Kansas. 

JONES,  PROF.  LYNDS,  Oberlin  College,  Oberlin,  Ohio. 

JORDAN,  PROF.  H.  E.,  University  of  Virginia,  Charlottesville, 
Virginia. 

JUST,  PROF.  E.  E.,  Howard  University,  Washington,  D.  C. 

KEEFE,  REV.  ANSELM  M.,  St.  Norbert's  College,  West  Depere, 
Wisconsin. 

KENNEDY,  DR.  HARRIS,  Readville,  Massachusetts. 

KINDRED,  DR.  J.  E.,  University  of  Virginia,  Charlottesville, 
Virginia. 


52  MARINE    BIOLOGICAL    LABORATORY. 

KING,  DR.  HELEN  D.,  Wistar  Institute  of  Anatomy  and  Biology, 
Philadelphia,  Pa. 

KING,  DR.  ROBERT  L.,  University  of  Pennsylvania,  Philadelphia, 
Pennsylvania. 

KINGSBURY,  PROF.  B.  F.,  Cornell  University,  Ithaca,  New  York. 

KINGSLEY,  PROF.  J.  S.,  2500  Cedar  Street,  Berkeley,  California. 

KIRKHAM,  DR.  W.  B.,  Springfield  College,  Springfield,  Mass. 

KNAPKE,  REV.  BEDE,  St.  Bernard's  College,  St.  Bernard,  Ala- 
bama. 

KNOWER,  PROF.  H.  McE.,  University  of  Alabama,  University, 
Ala. 

KNOWLTON,  PROF.  F.  P.,  Syracuse  University,  Syracuse,  New 
York. 

KOSTIR,  DR.  W.  J.,  Ohio  State  University,  Columbus,  Ohio. 

KRIBS,    DR.    HERBERT,    Ewing    Christian    College,    Al'ahabed, 
North  India. 

KUYK,  DR.  MARGARET  P.,  Westbrook  Ave.,  Richmond,  Va. 

LANCEFIELD,  DR.  D.  E.,  Columbia  University,  New  York  City. 

LANGE,  DR.  MATHILDE  M.,  Wheaton  College,  Norton,  Mass. 

LEE,   PROF.   F.  S.,   College  of  Physicians  and  Surgeons,   New 
York  City. 

LEWIS,  PROF.  1.  F.,  University  of  Virginia,  Charlottes ville,  Va. 

LEWIS,  PROF.  W.  H.,  Johns  Hopkins  University,  Baltimore,  Md. 

LILLIE,    PROF.    FRANK    R.,    University    of    Chicago,    Chicago, 
Illinois. 

LILLIE,  PROF.  R.  S.,  University  of  Chicago,  Chicago,  Illinois. 

LINTON,  PROF.  EDWIN,  University  of  Pennsylvania,  Philadelphia, 
Pennsylvania. 

LOEB,  PROF.  LEO,  Washington  University  Medical  School,  St. 
Louis,  Mo. 

LOEB,  MRS.  LEO,  6803  Kingsburg  Boulevard,  St.  Louis,  Missouri. 

LOWTHER,  MRS.  FLORENCE  DEL.,  Barnard  College,  Columbia 
University,  New  York  City. 

LUCRE,    PROF.    BALDWIN,    University  of   Pennsylvania,    Phila- 
delphia, Pa. 

LUND,  DR.  E.  J.,  University  of  Texas,  Austin,  Texas. 

LUSCOMBE,  MR.  W.  O.,  Woods  Hole,  Mass. 

LYNCH,  Miss  CLARA  J.,  Rockefeller  Institute,  New  York  City. 

LYON,  PROF.  E.  P.,  University  of  Minnesota,  Minneapolis,  Minn. 


RKPORT    OF    THE    DIRECTOR.  53 

MACCALLUM,  DR.  G.  A.,  925  St.  Paul  St.,  Baltimore,  Md. 

MACDOUGALL,    DR.   MARY  S.,   Agnes  Scott   College,   Decatur, 
Georgia. 

McCLUNG,    PROF.    C.    E.,    University   of    Pennsylvania.    Phila- 
delphia, Pa. 

McGEE,  DR.  ANITA  NEWCOMB,  2707  Adams  Mill  Road,  Wash- 
ington, D.  C. 

MCGREGOR,  DR.  J.  H.,  Columbia  University,  New  York  City. 

MclNDOO,  DR.  N.  E.,  Bureau  of  Entomology,  Washington,  D.  C. 

McMuRRiCH,    PROF.    J.    P.,    University   of   Toronto,    Toronto, 
Canada. 

McNAiR,  DR.  G.  T.,  1909  Ohio  St.,  Lawrence,  Kansas. 

MACKLIN,  DR.  CHARLES  C.,  School  of  Medicine,  University  of 
\Vestern  Ontario,  London,  Canada. 

MALONE,  PROF.  E.  F.,  University  of  Cincinnati,  Cincinnati,  Ohio. 

MARTIN,  MR.  E.  A.,  College  of  the  City  of  New  York,  New 
York  City. 

MAST,  PROF.  S.  O.,  Johns  Hopkins  University,  Baltimore,  Md. 

MATHEWS,   PROF.  A.   P.,  University  of  Cincinnati,  Cincinnati, 
Ohio. 

MATSUI,  PROF.  K.,  Imperial  College  of  Agriculture  and  Den- 
drology, Morioka,  Japan. 

MAVOR,   PROF.  JAMES  W.,   Union   College,  Schenectady,   New 
York. 

MEDES,    DR.    GRACE,    University  of   Minnesota,    Minneapolis, 
Minn. 

MEIGS,  DR.  E.  B.,  Dairy  Division  Experiment  Station,  Beltsville, 
Md. 

MEIGS,  MRS.  E.  B.,  1736  M  St.,  N.  W.,  Washington,  D.  C. 

METCALF,  PROF.  M.  M.,  Johns  Hopkins  University,  Baltimore, 
Md. 

METZ,  PROF.  CHARLES  W.,  Carnegie  Institution  of  Washington, 
Cold  Spring  Harbor,  Long  Island. 

MINER,  DR.  ROY  W.,  American  Museum  of  Natural  History, 
New  York  City. 

MITCHELL,  DR.  PHILIP  H.,  Brown  University,  Providence,  R.  I. 

MOORE,  DR.  CARL  R.,  University  of  Chicago,  Chicago,  Illinois. 

MOORE,    PROF.    GEORGE   T.,    Missouri    Botanical    Garden,    St. 
Louis,  Mo. 


-^  MARINE    BIOLOGICAL    LABORATORY. 

MOORE,  PROF.  J.  PERCY,  University  of  Pennsylvania,  Phila- 
delphia, Pa. 

MORRILL,  PROF.  A.  D.,  Hamilton  College,  Clinton,  N.  Y. 

MORRILL,  PROF.  C.  V.,  Cornell  University  Medical  College, 
New  York  City. 

MULLER,  DR.  H.  J.,  University  of  Texas,  Austin,  Texas. 

NABOURS,  DR.  R.  K.,  Kansas  State  Agricultural  College,  Man- 
hattan, Kansas. 

NACHTRIEB,  PROF.  HENRY  F.,  2448  Cedar  St.,  Berkeley,  Cali- 
fornia. 

NEAL,  PROF.  H.  V.,  Tufts  College,  Tufts  College,  Mass. 

NEWMAN,  PROF.  H.  H.,  University  of  Chicago,  Chicago,  Illinois. 

NICHOLS,  DR.  M.  LOUISE,  Powelton  Apartments,  Philadelphia, 
Pa. 

NONIDEZ,  DR.  JOSE  F.,  Cornell  University  Medical  College, 
New  York  City. 

OKKELBERG,  DR.  PETER,  University  of  Michigan,  Ann  Arbor, 
Michigan. 

OSBURN,  PROF.  R.  C.,  Ohio  State  University,  Columbus,  Ohio. 

OSTERHOUT,  PROF.  W.  J.  V.,  Rockefeller  Institute,  New  York 
City. 

PACKARD,  DR.  CHARLES,  Columbia  University,  Institute  of 
Cancer  Research,  1145  Amsteidam  Avenue,  New  York  City. 

PAGE,  DR.  IRVINE  H.,  Presbyterian  Hospital,  New  York  City. 

PAPANICOLAOU,  DR.  GEORGE  N.,  Cornell  University  Medical 
College,  New  York  City. 

PAPPENHEIMER,  DR.  A.  M.,  Columbia  University,  New  York 
City. 

PARKER,  PROF.  G.  H.,  Harvard  University,  Cambridge,  Mass. 

PATON,  PROF.  STEWART,  Princeton  University,  Princeton,  N.  J. 

PATTEN,  PROF.  WILLIAM,  Dartmouth  College,  Hanover,  N.  H. 

PATTERSON,  PROF.  J.  T.,  University  of  Texas,  Austin,  Texas. 

PAYNE,  PROF.  F.,  University  of  Indiana,  Bloomington,  Ind. 

PEARL,  PROF.  RAYMOND,  Johns  Hopkins  University,  Baltimore, 
Md. 

PEARSE,  PROF.  A.  S.,  Duke  University,  Durham,  North  Carolina. 

PEEBLES,  PROF.  FLORENCE,  1095  Marengo  Avenue,  Pasadena, 
California. 

PHILLIPS,  DR.  E.  F.,  Cornell  University,  Ithaca,  New  York. 


REPORT    OF    THE   DIRECTOR.  55 

PHILLIPS,  DR.  RUTH  L.,  Western  College,  Oxford,  Ohio. 

PIKE,  PROF.  FRANK  H.,  437  West  59th  St.,  New  York  City. 

PINNEY,  DR.  MARY  E.,  Milwaukee-Downer  College,  Milwaukee, 
Wisconsin. 

PLOUGH,  PROF.  HAROLD  H.,  Amherst  College,  Amherst,  Mass. 

POND,  DR.  SAMUEL  E.,  University  of  Pennsylvania,  School  of 
Medicine,  Philadelphia,  Pennsylvania. 

PRATT,  DR.  FREDERICK  H.,  Boston  University  School  of  Medi- 
cine, Boston,  Mass. 

RANKIN,  PROF.  W.  M.,  Princeton  University,  Princeton,  N.  J. 

RAPPORT,  DR.  ANNA  YATES,  Bryn  Mawr  College,  Bryn  Mawr, 
Pennsylvania. 

REDFIELD,  DR.  ALFRED  C.,  Haivard  University  Medical  School, 
Boston,  Mass. 

REESE,  PROF.  ALBERT  M.,  West  Virginia  University,  Morgan- 
town,  W.  Va. 

REINKE,  DR.  E.  E.,  Vanderbilt  University,  Nashville,  Tennessee, 

REZNIKOFF,    DR.    PAUL,    Cornell    University    Medical    College. 
New  York  City. 

RHODES,  PROF.  ROBERT  C.,  Emory  University,  Atlanta,  Georgia. 

RICE,  PROF.  EDWARD  L.,  Ohio  \Vesleyan  University,  Delaware, 
Ohio. 

RICHARDS,  PROF.  A.,  University  of  Oklahoma,  Norman,  Okla- 
homa. 

RIGGS,  MR.  LAWRASON,  JR.,  25  Broad  St.,  New  York  City. 

ROBERTSON,  PROF.  W.  R.  B.,  Columbia,  Missouri. 

ROGERS,  PROF.  CHARLES  G.,  Oberlin  College,  Oberlin,  Ohio. 

ROMER,  DR.  ALFRED  S.,  University  of  Chicago,  Chicago,  Illinois. 

RUDISCH,  DR.  J.,  Fifth  Avenue  Bank,  44th  St.  and  Fifth  Ave., 
New  York  City. 

SAMPSON,  DR.  MYRA  M.,  Smith  College,  Northampton,  Mass. 

SANDS,   Miss  ADELAIDE  G.,   348   N.   Main  St.,   Port  Chester, 
New  York. 

SCHRADER,  DR.  FRANZ,  Bryn  Mawr  College,  Bryn  Mawr,  Pa. 

SCHRAMM,  PROF.  J.  R.,  University  of  Pennsylvania,  Philadelphia, 
Pa. 

SCOTT,  DR.  ERNEST  L.,  Columbia  University,  New  York  City. 

SCOTT,   PROF.  G.  G.,  College  of  the  City  of  New  York,  New 
York  City. 


56  MARINE    BIOLOGICAL    LABORATORY. 

SCOTT,    PROF.    JOHN    W.,    University    of    Wyoming,    Laramie, 

Wyoming. 

SCOTT,  PROF.  WILLIAM  B.,  7  Cleveland  Lane,  Princeton,  N.  J. 
SHULL,  PROF.  A.  FRANKLIN,  University  of  Michigan,  Ann  Arbor, 

Michigan. 

SHUMWAY,  DR.  WALDO,  University  of  Illinois,  Urbana,  Illinois. 
SIVICKIS,  DR.  P.  B.,  University  of  the  Philippines,  Manila,  P.  I 
SMITH,  DR.  BERTRAM  G.,  119  Grove  St.,  Tarrytown,  N.  Y. 
SNOW,  DR.  LAETITIA  M.,  Wellesley  College,  Wellesley,  Mass. 
SNYDER,  PROF.  CHARLES  D.,  Johns  Hopkins  University  Medical 

School,  Baltimore,  Md. 
SOLLMAN,  DR.  TORALD,  Western  Reserve  University,  Cleveland, 

Ohio. 
SPEIDEL,  DR.  CARL  C.,  University  of  Virginia,  Charlottesville, 

Va. 

SPENCER,  PROF.  H.  J.,  24  West  loth  St.,  New  York  City. 
STARK,  DR.  MARY  B.,  N.  Y.  Homeopathic  Medical  College  and 

Flower  Hospital,  New  York  City. 
STOCKARD,   PROF.   C.   R.,   Cornell  University  Medical   College, 

New  York  City. 
STOKEY,  DR.  ALMA  G.,  Mount  Holyoke  College,  South  Hadley, 

Mass. 
STREETER,  PROF.  GEORGE  L.,  Carnegie  Institution,  Baltimore, 

Maryland. 

STRONG,  PROF.  O.  S.,  Columbia  University,  New  York  City. 
STUNKARD,  PROF.  HORACE  W7.,  New  York  University,  University 

Heights,  New  York. 
STURTEVANT,  DR.  ALFRED  H.,  Columbia  University,  New  York 

City. 

TASHIRO,  DR.  SHIRO,  Medical  College,  University  of  Cincinnati, 
Cincinnati,  Ohio. 

TAYLOR,  Miss  KATHERINE  A.,  Cascade,  Washington  Co., 
Maryland. 

TAYLOR,  DR.  WILLIAM  R.,  University  of  Pennsylvania,  Phila- 
delphia, Pa. 

TENNENT,  PROF.  D.  H.,  Bryn  Mawr  College,  Bryn  Mawr,  Pa. 

THARALDSEN,  PROF.  C.  E.,  618  Church  St.,  Evanston,  Illinois. 

THATCHER,  MR.  LLOYD  E.,  University  of  Mississippi,  University, 
Mississippi. 


REPORT    OF    THE    DIRECTOR. 


57 


TIXKHAM,  Miss  FLORENCE  L.,  71  Ingersoll  Grove,  Springfield, 

Mass. 
TOMPKINS,  Miss  ELIZABETH  M.,    134  Linden  Ave.,   Brooklyn, 

N.  Y. 
TRACY,    PROF.    HENRY    C.,    University   of    Kansas,    Lawrence, 

Kansas. 

TREADWELL,  PROF.  A.  L.,  Vassar  College,  Poughkeepsie,  N.  Y. 
TURNER,  PROF.  C.  L.,  Beloit  College,  Beloit,  Wisconsin. 
LTHLEMEYER,  Miss  BERTHA,  Washington  LTniversity,  St.  Louis, 

Mo. 
UHLENHUTH,  DR.  EDWARD,  University  of  Maryland,  School  of 

Medicine,  Baltimore,  Maryland. 
UNGER,    DR.   W.    BYERS,    Dartmouth    College,    Hanover,    New 

Hampshire. 

VAN  DER  HEYDE,  DR.  H.  C.,  Galeria,  Corse,  France. 
VISSCHER,  DR.  J.  PAUL,  Western  Reserve  University,  Cleveland, 

Ohio. 
WAITE,  PROF.  F.  C.,  Western  Reserve  University  Medical  School, 

Cleveland,  Ohio. 
WALLACE,   DR.   LOUISE   B.,   Constantinople  Woman's   College, 

Constantinople,  Turkey. 

WAXX,  PROF.  FRANK  B.,  Utah  Agricultural  College,  Logan,  Utah. 
\VARD,  PROF.  HENRY  B.,  University  of  Illinois,  Urbana,  Illinois. 
WARDWELL,  DR.  E.  H.,  Chappaqua,  New  York. 
WTARREN,  PROF.  HOWARD  C.,  Princeton  University,  Princeton, 

New  Jersey. 

WARREN,  DR.  HERBERT  S.,  White  Plains,  New  York. 
WENRICH,  DR.  D.  H.,  University  of  Pennsylvania,  Philadelphia, 

Pa. 
WHEDON,  DR.  A.  D.,  North  Dakota  Agricultural  College,  Fargo, 

No.  Dakota. 

WHEELER,  PROF.  W.  M.,  Bussey  Institution,  Forest  Hills,  Mass. 
WHERRY,  DR.  W.  B.,  Cincinnati  Hospital,  Cincinnati,  Ohio. 
WHITE,  DR.  E.  GRACE,  Wilson  College,  Chambersburg,  Pa. 
WHITESIDE,   DR.   BEATRICE,   Detroit  College  of  Medicine  and 

Surgery,  Detroit,  Michigan. 

WHITING,  DR.  PHINEAS  W.,  Bussey  Institute,  Forest  Hills,  Mass. 
WHITNEY,  DR.  DAVID  D.,  LTniversity  of  Nebraska,  Lincoln,  Neb. 
WIEMAN,  PROF.  H.  L.,  University  of  Cincinnati,  Cincinnati,  Ohio. 


f<^* 

LjlLIBRARY 


,<*V^!A 


58  MARINE   BIOLOGICAL    LABORATORY. 

• 

\YILLIER,  DR.  B.  H.,  University  of  Chicago,  Chicago,  Illinois. 
WILSON,   PROF.  H.  V.,   University  of  North  Carolina,   Chapel 

Hill,  N.  C. 

WILSON,  DR.  J.  W.,  Brown  University,  Providence,  R.  I. 
WOGLOM,  PROF.  WILLIAM  H.,  Columbia  University,  New  York 

City. 

WOODRUFF,  PROF.  L.  L.,  Yale  University,  New  Haven,  Conn. 
WOODWARD,    DR.   ALVALYN   E.,   5507   New   Medical   Building, 

Ann  Arbor,  Michigan. 

YOUNG,  DR.  B.  P.,  Cornell  University,  Ithaca,  New  York. 
YOUNG,  DR.  D.  B.,  University  of  Maine,  Orono,  Maine. 
ZELENY,  DR.  CHARLES,  University  of  Illinois,  Urbana,  Illinois. 


Vol.LV. 


August,  1928. 


No.  2. 


BIOLOGICAL  BULLETIN 


THE  CONTRACTILE  VACUOLE  IN  PARAMECIUM 

TRICHIUM. 

ROBERT  L.  KING, 
ZOOLOGICAL  LABORATORY,  UNIVERSITY  OF  PENNSYLVANIA. 

INTRODUCTION. 

The  contractile  vacuolar  apparatus  of  Ciliata  seems  to  be  more 
highly  specialized  than  that  of  other  Protozoa.  As  to  the  form 
of  the  vacuole  itself,  we  may  recognize  for  convenience  two  gen- 
eral types  among  the  Ciliata:  (a)  Vesicle-fed  vacuoles,  in  which 
the  contracting  vacuole  is  surrounded  by  a  series  of  small  vacuoles 
(called  vesicles  in  this  paper)  which  seem  to  coalesce  and  form 
a  new  contracting  vacuole  after  systole,  new  smaller  vesicles  ap- 
pearing around  the  contracting  vacuole  after  systole,  and  growing 
larger  during  diastole  as  described  by  Taylor  ('23)  in  Euplotes; 
(b)  Canal-fed  vacuoles,  in  which  the  vacuole  is  fed  by  canals, 
which  vary  in  number  from  one  in  Spirostomum  and  Stcntor 
(Roux,  '01)  to  thirty  in  Ophryoglena  (Biitschli,  '87-' '89).  A 
condition  which  seems  to  be  intermediate  has  been  described  by 
Schewiakoff  ('89)  for  Prorodon  tercs.  Here  the  vacuole  is  fed 
by  four  radial  rows  of  vesicles  which  decrease  in  size  distally  from 
the  contractile  vacuole. 

The  contractile  vacuole  usually  communicates  with  the  exterior 
by  a  tubule  through  a  pore.  This  excretory  tubule  leading  from 
the  vacuole  to  the  pore  may  be  very  short  as  in  Nassula  and  Fron- 
tonia  relatively  long  as  in  Urocentrutn,  or  very  long  as  in  Lemba- 
dion  (Schewiakoff,  '89)  where  the  vacuole  is  located  near  the 
central  part  of  the  body  and  the  excretory  tube  leads  to  the  pore 
which  is  about  one  quarter  of  the  body  length  posterior  to  the 
vacuole. 

5  59 


6o  ROBERT    L.    KING. 

The  purpose  of  this  paper  is  to  describe  the  contractile  vacuolar 
apparatus  of  Paramecium  trichium  which  has  been  described  by 
Stokes  ('88)  as  consisting  of  two  vacuoles  "close  together,  con- 
tracting quickly,  the  one  beginning  to  again  form  almost  before 
the  completion  of  the  other's  systole  "  and  by  Wenrich  ('26)  who 
was  unable  to  reach  a  satisfactory  conclusion  as  to  "  whether  the 
two  main  vacuoles  empty  alternately  into  the  same  outlet  tube  or 
whether  there  is  an  auriculo-ventricular  relationship  between 
them." 

Without  the  use  of  methods  introduced  by  Bresslau  ('21)  as 
modified  in  this  paper,  the  structures  to  be  described  could  not 
have  been  studied.  I  am  also  particularly  indebted  to  Doctor  D. 
H.  Wenrich  who  first  pointed  out  the  need  for  further  study  of 
the  contractile  vacuoles  of  Paramecium  trichium. 

MATERIALS  AND  METHODS. 

The  Paramecium  trichium  used  in  this  series  of  observations 
were  obtained  from  two  sources  in  the  vicinity  of  Swarthmore, 
Pa. :  from  the  east  branch  of  Crum  Lynne  Creek  where  they  were 
found  among  the  jelly  of  toads  eggs  and  from  the  west  branch  of 
Stony  Creek,  the  source  of  Wenrich's  ('26)  Swarthmore  race. 
The  material  was  first  studied  in  mixed  cultures  but  later  pure 
lines  were  established.  For  purposes  of  comparison,  Colpidium 
colpoda  from  the  same  sources  was  studied. 

Observations  on  the  contractile  vacuolar  apparatus  of  Parame- 
cium trichium  were  first  made  on  material  prepared  according  to 
the  methods  described  by  Bresslau  ('21)  and  later  those  described 
by  Coles  ('27).  These  methods  consist  essentially  of  drying  the 
protozoa  in  concentrated  solutions  of  stains,  which  are  relatively 
low  in  toxicity.  The  dye  is  precipitated  in  and  on  surface  struc- 
tures such  as  the  depressions  from  which  cilia  arise,  the  cilia 
themselves,  the  mouth  and  the  cytopharynx.  The  routine  used  is 
essentially  as  follows :  A  small  drop  of  concentrated  culture  of 
the  protozoa  to  be  examined  is  placed  upon  a  perfectly  clean  glass 
slip  and  mixed  with  a  similarly  sized  drop  of  the  stain.  The  two 
are  then  mixed  and  spread  evenly  over  the  slip  with  a  needle  or  a 
glass  spreader.  The  slides  are  then  allowed  to  dry  in  the  air  and 
are  examined  under  oil  or  mounted  in  damar. 


CONTRACTILE    VACUOLE    IN    PARAMECIUM.  6 1 

Bresslau  ('21)  has  used  the  following  staining  solutions:  (a) 
Three  parts  saturated  aqueous  solution  China  Blue  to  one  part 
saturated  aqueous  solution  Cyanosin  (a  mixture  previously  used 
in  bacteriological  technique)  and  (&)  one  cc.  10  per  cent,  aqueous 
solution  Opal  Blue  to  4-6  drops  6.5  per  cent,  aqueous  solution 
Phloxinrhodamin. 

Coles  ('27)  has  used  numerous  stains  for  Protozoa  in  the  same 
manner,  obtaining  his  best  results  with  a  saturated  aqueous  solu- 
tion Nigrosin. 

The  stains  recommended  by  Bresslau  are  very  toxic  to  Para- 
mecium  trichium  which  are  killed  quickly  and  usually  burst  open 
before  they  become  dry.  However,  with  solutions  of  (a)  10  per 
cent.  China  Blue  (Coleman  Bell),  (b)  10  per  cent.  Nigrosin 
(Coleman  Bell  or  Griibler),  (c)  Mixture  of  equal  parts  of  above, 
and  (rf)  10  per  cent.  Opal  Blue  (Coleman  Bell),  beautiful  prepara- 
tions have  been  made.  The  above  methods  have  been  placed  in 
order  of  their  apparent  usefulness  for  the  structures  studied  in 
Paramecium  trichium. 

Observations  made  on  such  dried  preparations  have  been  veri- 
fied on  material  fixed  with  Bouin's  or  Schaudinn's  and  stained 
with  hsemalum  and  on  living  animals  in  hanging  drops  of  the 
culture  medium  or  of  the  culture  medium  mixed  with  China  Blue, 
Opal  Blue  or  Nigrosin.  Paramecium  trichium  will  survive  over 
two  hours  in  such  hanging  drops  containing  stains  and  remain 
apparently  typical  during  that  time. 

OBSERVATIONS. 

There  are  two  contractile  vacuoles  in  Paramecium  trichium. 
one  located  in  the  anterior  and  one  in  the  posterior  part  of  the 
animal  (Wenrich,  '26).  When  dried  in  the  staining  solutions 
described  above  the  coloring  matter  collects  in  the  contractile 
vacuolar  apparatus  sometimes  penetrating  into  the  vacuole  itself. 
The  contractile  vacuole  is  seen  to  be  connected  with  the  exterior 
by  a  long  convoluted  tubule  (Figs.  2,  3,  4  and  9)  which  terminates 
in  a  small  pore  (Figs,  i,  2  and  4)  located  between  the  longitudinal 
rows  of  cilia  on  the  dorsal  surface  of  the  body  opposite  the  mouth. 
The  pore  of  the  anterior  vacuole  and  that  of  the  posterior  open 
to  the  exterior  between  the  same  or  adjacent  rows  of  longitudinal 


62  ROBERT    L.    KING. 

rows  of  cilia  (Figs.  8,  15  and  14).  The  vacuoles  themselves  do 
not  seem  to  be  fixed  in  position  in  living  animals  but  move  about 
more  or  less  in  the  endoplasm;  this  apparently  is  made  possible 
by  the  presence  of  the  convoluted  tubule.  The  inner  end  of  the 
tubule  terminates  in  a  cup-like  valve  (Figs.  7  and  9)  with  which 
the  vacuole  is  in  contact  when  undergoing  systole.  This  cup-like 
valve  has  been  observed  both  in  prepared  slides  and  in  living 
animals;  while  it  seems  always  to  be  in  contact  with  the  vacuole 
it  may  be  exterior  or  interior,  anterior  or  posterior  in  relation  to 
the  vacuole.  In  fact  the  whole  structure  seems  to  be  continually 
in  active  movement.  The  proximal  portion  of  the  tubule  shows 
the  effect  of  increased  pressure  at  the  beginning  of  systole,  tend- 
ing to  straighten  out  as  does  a  coiled  hose  when  water  is  first 
turned  into  it  under  pressure. 

In  living  animals  each  of  the  contractile  vacuoles  appears  to 
be  made  up  of  two  vacuoles  which  contract  alternately.  However, 
if  they  be  carefully  compared  with  a  typical  vesicle-fed  vacuole 
such  as  that  of  Colpidium  colpoda,  it  is  found  that  the  two  are 
fundamentally  alike.  Observations  made  with  a  stop-watch  show 
that  the  time  relations  of  various  parts  of  the  cycle  of  systole  and 
diastole  in  the  two  species  are  quite  different.  The  table  gives  a 
set  of  typical  observations  on  Paramecium  trichiuni  and  Colpidiuin 
colpoda.  These  observations  were  made  upon  a  single  individual 
of  each  species  on  the  same  slide.  For  the  interval  between  con- 
tractions seven  groups  of  three  contractions  each  were  timed  in 
Colpidiiim  colpoda;  ten  groups  of  three  contractions  each  in  Para- 
mecium trichiuni  (anterior  vacuole).  A  number  of  separate  ob- 
servations of  the  time  from  beginning  to  end  of  systole  were 
made  on  the  same  individuals. 

TABLE  I. 

TIME  RELATIONS  IN  SECONDS  OF  CONTRACTILE  VACUOLES  OF  Paramecium 
trichiuni  AND  Colpidiiim  colpoda  AT  25°   C. 

Time  between  Completion      Time 
of  Two  Systoles.          for  Systole. 

Paramecium  tricliium    3.1  2.0 

Colpidium  colpoda    7.4  0.4 

In  Colpidiiim  colpoda  the  vacuole  empties  rapidly  (about  0.4 
seconds)  ;  the  small  feeding  vesicles  gradually  enlarge,  coalesce 


CONTRACTILE    VACUOLE    IN    PARAMECIUM.  63 

into  one  vacuole  which  finally  reaches  its  maximum  size  about 
seven  seconds  after  the  last  contraction,  in  the  meantime  there 
have  appeared  more  tiny  feeding  vesicles  around  its  periphery. 
The  vacuole  then  contracts  rapidly  and  the  cycle  is  repeated. 

In  Paramecium  trichium  the  vacuole  (Fig.  120)  empties  slowly 
(about  2  seconds).  As  it  contracts  the  feeding  vesicles  grow 
(Fig.  126)  and  by  the  time  the  vacuole  has  half  completed  systole 
(Fig.  I2c)  the  feeding  vacuoles  coalesce  with  an  apparent  passing 
of  membranes  over  their  surface  marking  their  fusion  [called  by 
Taylor  ('23)  "vestiges  of  the  adjacent  walls"  in  Euplotes]. 
When  systole  is  completed  (Fig.  i2cQ  the  new  contractile  vacuole 
has  reached  its  maximum  size ;  as  it  comes  in  contact  with  the  cup- 
like  valve  a  membrane  (as  described  by  Wenrich,  '26)  appears 
to  pass  over  its  surface,  and  it  begins  to  contract  (Fig.  I2c). 

The  two  processes  are  fundamentally  alike;  in  Paramecium 
trichium  the  systole  of  the  old  contractile  vacuole  and  the  diastole 
of  the  new  occur  almost  simultaneously  and  are  of  approximately 
the  same  duration  while  in  Colpidium  colpoda  they  occur  consecu- 
tively with  systole  very  brief  and  diastole  protracted. 

SUMMARY. 

1.  There   is   an   anterior   and   a   posterior   contractile   vacuolar 
apparatus  in  Paramecium  trichium. 

2.  Each  is  permanent  and  consists  of  feeding  vesicles,  contrac- 
tile vacuole,  excretory  tube  and  pore. 

3.  The  contractile  vacuoles  of  Paramecium  trichium  are  vesicle- 
fed,  differing  here  from  those  of  other  well  known  species  in  the 
same  genus  which  are  canal-fed. 

4.  Diastole  of  the  new  vacuole  is  practically  complete  before  the 
prolonged  systole  of  the  old  is  over.     This  gives  the  appearance  of 
two  vacuoles  contracting  alternately. 

5.  The  excretory  tube  is  long  and  convoluted  with  its  flattened 
cup-like  end  in  contact  with  the  contractile  vacuole. 

6.  The  excretory  tube  opens  as  an  excretory  pore  on  the  surface 
of  the  body  opposite  the  mouth. 

7.  The  pore  of  the  anterior  and  that  of  the  posterior  apparatus 
are  located  between  the  same  or  adjacent  longitudinal  rows  of 
cilia. 


64 


ROBERT    L.    KING. 


8.  The  long  duration  of  systole  may  be  correlated  with  the 
presence  of  the  convoluted  excretory  tube. 

9.  The  excretory  tube  and  pore  were  first  demonstrated  by  the 
use  of  Bresslau's  relief  staining  method. 

LITERATURE  CITED. 

Bresslau,  E. 

1922  Die    Gelatinierbarkeit   des   Protoplasmas   als    Grundlage   ernes 

Verfahrens   zur    Schnellanfertigung   gefarbter    Dauerprapar- 
ate  von   Infusorien.     Arch.  f.   Protistenk.,  43:  467-480. 
Butschli,  A. 

1887-89     Infusoria.     Bronn's   Thier-reichs,   I,   III   Abt. 
Coles,  A.  C. 

1927     Relief    Staining    of    Bacteria    Protozoa,    Infusoria.     Watson 

Microscope  Record,  No.  10:  23-25. 
Roux,  J. 

1901     Faune  infusorienne  des  eaux  stagnantes  des  environs  de  Gen- 
eve.    Geneve:  Henry  Kiindig. 
Schewiakoff,  W. 

1889     Beitrage  zur  Kenntniss  der  holotrichen  Ciliaten.     Bibliotheca 

zoologica,  Heft  5. 
Stokes,  A.  C. 

1888     Fresh-water    Infusoria.     Jour.    Trenton    Nat.    Hist.    Soc.,    i: 

7I-344- 
Taylor,  C.  V. 

1923  The    Contractile   Vacuole   in   Euplotes.    Jour.    Exp.   Zool.,   37: 

259-289. 
Wenrich,  D.  H. 

1926     The    Structure    and    Division    of    Paramecium    trichium.    Jour. 
Morph.  and   Physiol.,  43:  81-103. 


66  ROBERT    L.    KING. 


PLATE  I. 
Explanation  of  Figures. 

Figs.  i-n.  Contractile  vacuolar  apparatus  of  Paramccium  trichuun, 
dried  in  10  per  cent.  China  Blue  unless  otherwise  stated  X  1000. 

1.  Posterior  apparatus  of  animal  photographed  for  Fig.   13. 

2.  Anterior  apparatus  of  animal  photographed  for  Fig.  14. 

3.  Posterior  apparatus. 

4.  Posterior  apparatus. 

5.  Anterior  apparatus. 

6.  Posterior  apparatus  of  same  animal  as  Fig.  5. 

7.  Posterior  apparatus  of  animal  photographed  for  Fig.  16. 

8.  Entire   animal   showing  cytopharynx,   anterior  and  posterior  vacuolar 
apparatus.     Same  as  photograph  Fig.  15. 

9.  Anterior  apparatus. 

10.  Anterior  apparatus  (10  per  cent,  nigrosin). 

u.  Two  tubules  and  pores  from  anterior  end  of  same  animal. 

Fig.  12.  Diagrams  of  various  stages  of  contracting  vacuole.  a.  Begin- 
ning of  systole,  b.  Systole  half-completed,  feeding  vesicles  grow  larger. 
c.  Fusion  of  feeding  vesicles,  d.  Systole  complete.  Cup-like  inner  end  of 
tubule,  e.  Fusion  of  new  contractile  vacuole  with  cup-like  inner  end  of 
tubule  preparatory  to  systole. 


BIOLOGICAL  BULLETIN,    VOL.    LV. 


PLATE  I. 


*    i 


II 


12      I  f        a 

ROBERT   L.    KING. 


68  ROBERT   L.    KING. 


PLATE  II. 
Photographs  X  750, 


BIOLOGICAL  BULLETIN,   VOL.    LV 


PLATE     II. 


ROBERT  L.    KING. 


OBSERVATIONS  OF  THE  FEEDING  MECHANISM  OF' 
A  CTENOPHORE,  MNEMIOPSIS  LIIIDYI.1 

ROLLAND  J.  MAIN. 

Observations  of  the  feeding  habits  of  ctenophores  are  scattered 
through  the  literature  dealing  with  these  organisms  (Bigelow, 
'15;  Lebour,  '22-23;  Mayer,  '12;  Nelson,  '25),  but  as  yet  no 
detailed  study  of  the  feeding  mechanism  has  appeared. 

The  ciliation  of  a  hydromedusa  has  been  studied  ( J.  F.  Gemmill, 
'19),  but  this  compares  in  no  way  with  the  complex  food  catching 
apparatus  of  a  ctenophore  such  as  Mnemiopsis  Icidyi.  The  mor- 
phological work  done  upon  this  ctenophore  is  incomplete,  for 
neither  Agassiz  (1849),  Fewkes  (1881),  nor  Mayer  ('12),  men- 
tion the  presence  of  its  remarkable  mechanism  for  the  capture  of 
food. 

Mnemiopsis  Icidyi  through  its  habit  of  devouring  the  free- 
swimming  larvse  of  the  oyster  and  of  other  molluscs  becomes  of 
such  economic  importance  that  it  is  of  interest  to  determine  by 
what  means  these  organisms  are  captured  and  carried  into  the 
stomodseum,  and  how  the  undigested  residues  are  discharged.2 

MATERIALS  AND  METHODS. 

The  specimens  of  Mnemiopsis  Icidyi  were  obtained  in  the 
northern  half  of  Barnegat  Bay,  N.  J.,  a  shallow  estuary,  in  water 
of  a  specific  gravity  approximating  i.oio,  with  temperatures  close 
to  20°  C,  during  August  and  the  first  part  of  September,  1926. 
The  animals  were  caught  in  a  net,  placed  in  jars  without  injury 
and  within  ten  minutes  after  capture  they  were  being  examined 
under  the  binocular. 

Living  plankton  was  used  to  determine  the  feeding  mechanism, 
and  it  is  felt  that  to  this  the  success  of  the  experiment  is  pri- 

1  From  the  Zoological  Laboratory  of  Rutgers  University,  Publication 
No.  n,  New  Jersey  Oyster  Investigation  Laboratory. 

-  The  writer  is  indebted  to  Dr.  Thurlow  C.  Nelson  of  Rutgers  University 
for  aid  and  advice  during  this  investigation  and  for  reading  the  manuscript. 

69 


JO  ROLLAXD    J.    MAIN. 

marily  due,  since  it  is  doubtful  for  reasons  given  below  whether 
any  other  material  could  have  been  successfully  used.  The  plank- 
ton was  secured  by  pouring  sea  water  through  a  200  mesh  plankton 
net,  and  concentrating  the  organisms  in  a  small  amount  of  water. 
A  Mncmiopsis  was  placed  in  a  watch  crystal  under  the  binocular, 
a  little  of  the  plankton  culture  was  added,  and  the  reactions  of  the 
ctenophore  noted. 

STRUCTURE  AND  OPERATION  OF  THE  FOOD  CATCHING 

MECHANISM. 

To  understand  the  mechanism  of  the  food  catching  apparatus, 
it  is  first  necessary  to  have  a  clear  idea  of  the  gross  anatomy  of 
Mncmiopsis,  Fig.  I.  Although  considerable  work  has  been  done 


FIG.  I.  Adult  Mncmiopsis  Icidyi  from  Barnegat  Bay.  Photographed  im- 
mediately after  fixation  in  10  per  cent,  hydrochloric  acid.  The  oral  lobes 
have  contracted  to  approximately  2/3  the  length  characteristic  of  the 
living  animal.  Photographed  by  T.  C.  Nelson. 

on  the  morphology  of  the  animal,  all  the  writers  have  apparently 
disregarded  the  presence  of  a  definite  ridge,  an  extension  of  the 


FEEDING    MECHANISM    OF    M  XKM  IOPSIS.  yi 

lips  of  the  mouth,  which  the  writer  has  named  the  "  labial  ridge." 

There  are  four  furrows  formed  by  the  juncture  of  the  oral  lobes 
with  the  body.  In  each  furrow  along  the  inner  side  of  the  labial 
ridge  is  a  line  of  tentacles.  Through  the  base  of  this  labial  ridge 
runs  a  branch  of  the  paragastric  canal,  which  finally  unites  with 
the  auricular  canal.  On  the  opposite  side  of  this  ridge  is  the 
ciliated  channel  for  conveying  food  to  the  mouth,  Figs.  2  and  3. 


FIG.  2.  Adult  Miiciniopsis  Icidyi.  Part  of  the  right  lobe  and  the  tip  of 
the  right  auricle  have  been  omitted.  It  is  difficult  to  represent  the  turning 
of  the  labial  ridge.  The  lips  are  in  the  plane  of  the  paper.  As  the  lip  be- 
comes the  labial  ridge,  it  turns  so  that  it  lies  in  a  plane  at  right  angles  to 
the  paper,  i.  The  tentacular  bulb.  2.  The  tentacular  ridge,  with  ten- 
tacles. 3.  The  labial  ridge,  along  which  runs  the  tentacular  ridge.  4.  Lip. 
5.  Auricular  groove.  6.  Cilia  of  auricle. 

To  this  channel,  or  trough,  will  be  applied  the  term  "  labial  trough." 
It  is  formed  by  the  labial  ridge  on  one  side,  and  the  oral  lobe  on 
the  other.  It  runs  along  the  ridge  to  the  point  where  the  ridge 
becomes  the  lip,  and  here  the  trough  runs  directly  into  the  corner 
of  the  mouth,  Fig.  4.  The  labial  ridge  is  separated  from  the  cilia 
of  the  auricles  by  the  auricular  groove  in  which  the  cilia  of  the 
auricle  beat,  and  at  the  bottom  of  which  lie  the  tentacles  stretched 
out  in  the  current. 

3  The  writer  calls  attention  to  some  apparent  discrepancies  in  earlier  work 
on  Mnciniopsis  Icidyi.  Fewkes  pictures  an  adult  of  this  species  which  differs 
widely  from  the  type  obtained  from  Barnegat  Bay.  The  latter,  save  for 
the  contraction  of  the  oral  lobes,  is  well  illustrated  in  Figure  I.  Fewkes' 
figure  shows  the  surface  of  the  animal  covered  with  discoidal  warts  which 
are  claimed  by  Mayer  to  be  present  in  M.  inccradyi  and  in  .!/.  f/ardcni  but 
absent  in  M.  Icidyi.  Fewkes'  figure  differs  also  in  the  shape  of  the  body. 


72  ROLLAND   J.    MAIN. 

Near  the  mouth  the  line  of  tentacles  curves  away  from  the 
labial  ridge  up  to  the  tentacular  bulb.  The  tentacles  are  placed 
irregularly  along  this  line,  usually  in  groups,  some  animals  having 


8 


FIG.  3.  A.  Cross  section  of  auricular  groove.  The  cilia  of  the  auricle 
(i)  beat  up  and  down  as  indicated  by  the  arrow  and  dotted  line.  The 
other  two  arrows  show  the  direction  of  the  current  produced  by  the  cilia. 
I.  Cilia  of  auricle.  2.  Auricular  canal.  3.  Auricular  groove.  4.  Tentacle. 
5.  Tentacular  ridge.  6.  Labial  ridge.  7.  Labial  trough.  8.  Branch  of  para- 
gastric  canal.  9.  Oral  lobe.  B.  View  of  auricular  groove  from  above. 
The  oral  lobe  has  been  laid  back.  Parts  correspond  to  Fig.  ^A.  Three  ten- 
tacles are  here  shown  putting  food  in  the  labial  trough,  where  it  will  be 
drawn  off  and  conveyed  to  the  mouth. 

many  more  tentacles  than  others.  This  may  be  due  to  the  fact 
that  they  have  been  broken  off  in  securing  food,  for  often  food 
may  be  seen  entering  the  stomoclaeum  with  portions  of  tentacles  at- 
tached. 


FIG.  4.  Oral  view  of  adult  Mncmiopsis  leidyi.  This  shows  how  the  lips 
are  continued  into  the  labial  ridge  and  how  the  trough  runs  into  the  mouth. 
I.  Lip.  2.  Mouth.  3.  Labial  ridge.  4.  Labial  trough. 

When  a  particle  of  food  is  caught  in  the  current  produced 
by  the  cilia  in  the  grooves  it  is  whirled  about  until  it  finally 
touches  a  tentacle.  This  entangles  it,  often  with  the  aid  of 
several  other  tentacles.  These  tentacles  then  contract,  and 


FEEDING    MECHANISM    OF    MNEMIOPSIS.  73 

apparently  are  drawn  over  the  labial  ridge  into  the  labial 
trough,  presumably  by  cilia,  Fig.  ^B.  Here  they  stretch  out  in 
the  direction  of  the  mouth,  the  food  is  drawn  off,  and  passes  down 
toward  the  mouth.  The  tentacles  then  relax,  and  resume  their 
normal  position.  Often  several  pieces  of  food  are  beaten  about 
for  some  time  in  the  groove.  Dirt  in  the  groove  is  gradually  en- 
tangled in  mucus  into  a  long  thread  which  slowly  passes  out  at  the 
aboral  end  of  the  groove.  If  much  dirt  be  present,  the  whole 
animal  pulsates,  contracting  the  groove  and  forcing  out  all  material 
present.  The  tentacles  were  never  seen  placing  any  foreign 
material  into  the  labial  trough,  unless  a  little  happened  to  be  caught 
up  with  the  food.  Possibly  it  is  for  these  reasons  that  Muciniop- 
sis  Icidyi  is  not  found  in  muddy  waters,  since  it  will  not  seize 
food  if  much  dirt  be  present.  Carmine  introduced  directly  into 
the  labial  trough  is  drawn  along  but  for  a  short  distance,  and  then 
is  passed  out  over  the  labial  ridge.  For  this  reason  the  use  of  the 
natural  plankton  food  organisms  in  studying  the  mechanism  is 
imperative. 

It  is  here  that  we  must  search  for  the  explanation  of  why 
Mncniiof>sis  Icidyi  lives  so  largely  upon  bivalve  larvae,  in  spite  of 
the  great  preponderance  of  other  plankton  in  the  water  (Nelson, 
'25).  The  writer  has  observed  that  often  the  ctenophore  is  un- 
able to  hold  an  active  copepod.  Possibly  the  stronger  swimmers 
are  able  to  escape  the  ciliary  currents,  whereas  the  young  oyster 
shuts  its  shell  on  contact  and  is  therefore  an  easy  prey.  Poly- 
chaste  larvae  were  found  in  Mncnnopsis  at  this  time,  although  never 
more  than  one  or  two  per  animal.  This  is  contrary  to  Nelson's 
('25)  belief  that  it  would  be  almost  impossible  for  this  ctenophore 
to  ingest  such  a  prey. 

Food  captured  by  the  tentacles  about  the  mouth  was  passed 
down  directly  over  the  lips  into  the  mouth,  often  aided  by  a  con- 
traction of  the  lips,  bringing  them  near  the  tentacular  bulb.  After 
the  food  has  passed  into  the  stomodaeum,  it  usually  proceeds  slowly 
to  the  center,  between  the  two  paragastric  canals,  close  to  the  con- 
voluted tubules  which  probably  secrete  the  digestive  juices.  It 
may,  however,  lodge  below  this  point,  Fig.  5.  Sometimes  it  is 
caught  in  the  swifter  current  at  the  very  edge  of  the  stomodaeum, 
and  is  whirled  up  to  the  beating  cilia  at  the  aboral  end.  Here  it 


74 


ROLLAND    J.    MAIN. 


is  usually  turned  back,  for  these  cilia  seem  to  act  partly  as  filters. 
At  times,  however,  a  particle  may  be  squeezed  through  and  enter 
the  funnel  to  pass  around  in  the  food  canals. 


FIG.  5.  The  stomodfeum  of  Mnemiopsis  Icidyi.  In  order  to  avoid  con- 
fusion, the  paths  taken  by  ingested  food  are  shown  on  the  right  side  only. 
The  larger  arrows  are  the  more  usual  paths.  The  smaller  arrows  on  the 
extreme  right  denote  a  swifter  current,  in  which  the  food  sometimes 
travels.  On  the  left  half  only,  are  shown  the  paths  taken  by  the  excreted 
materials.  I.  Mouth.  2.  Paragastric  canal.  3.  Digestive  glands?  4.  Cilia. 
5.  Funnel. 

The  undigested  material  in  the  stomodaeum  is  passed  down  as 
indicated,  and  ejected  through  the  mouth.  These  paths  are  not 
definite,  for  incoming  food  will  pass  a  certain  spot,  and  immediately 
afterwards  outgoing  wastes  will  cross  the  same  spot  going  in  the 
opposite  direction.  Those  particles  which  have  passed  through 
into  the  food  canals  may  reenter  the  stomodaeum  and  pass  out 
through  the  mouth,  or  they  may  follow  the  usual  procedure  for 
material  in  the  canals,  and  be  voided  at  the  anus. 

Just  before  defecation  occurs,  particles  may  be  seen  gathering 
about  in  the  funnel  and  in  the  axial  funnel  canal.  Then  one  of 
the  branches  of  this  canal  elongates  above  the  surface  and  the 
particles  are  forced  out  through  the  pore.  The  current  in  all  the 
food  canals  now  seems  to  be  in  the  direction  of  the  funnel.  By 


FEEDING    MECHANISM    OF    MNEMIOPSIS. 


75 


this  time  the  cilia  of  the  aboral  end  of  the  stomodseum  have  ceased 
beating,  and  the  whole  upper  end  of  the  stomodseum  presents  a 
contracted  appearance,  Fig.  6.  After  the  particles  of  waste  have 


FIG.  6.  Aboral  portion  of  stomodaeum,  and  axial  funnel  canal  of  Mnciiri- 
opsis  leidyi.  A.  Before  defecation.  I.  Paragastric  canal.  2.  Cilia.  3. 
Food  canals.  4.  Funnel.  5.  Axial  funnel  canal.  6.  Sense  organ.  7.  Ex- 
cretory pore.  B.  During  defecation,  arrows  showing  direction  of  waste. 
Note  shrunken  appearance  of  stomodseum. 

all  passed  out  the  cilia  begin  beating  again,  and  the  branch  of  the 
funnel  canal  slowly  retracts.  Although  several  successive  defeca- 
tions of  specimens  have  been  observed,  only  one  branch  was  used, 
and  in  no  specimen  was  the  use  of  both  branches  observed. 

THE  EARLY  DEVELOPMENT  OF  THE  FOOD  CATCHING 

MECHANISM. 

Since  the  complex  food  catching  apparatus  is  present  only  in 
the  adult  Mnemiopsis,  the  question  of  its  ontogeny  naturally 
arises.  The  young  were  plentiful  at  the  time  of  this  study,  and 
various  stages  were  examined. 

The  smallest  specimens  obtained  were  in  the  Cydippidse-stage, 
approximately  2  mm.  high  and  2  mm.  broad.  Fig.  7.  These  pos- 
sess two  long  branching  tentacles  with  no  trace  of  the  tentacular 
ridge,  labial  ridge,  or  labial  trough.  They  feed  by  capturing  the 
food  with  the  tentacles,  retracting  them,  and  drawing  them  down 
over  the  lip  and  into  the  stomodseum,  where  the  food  is  drawn 
off.  Another  contraction,  and  the  tentacles  emerge,  to  again  float 
up  above  the  animal. 


j6  HOLLAND    J.    MAIN. 

The  next  step  in  development  was  found  in  a  6  mm.  specimen, 
Fig.  8.     This  stage  has  still  the  two  compound  tentacles. 


FIG.  7.  Young  Mnemiopsis  Icidyi,  2  mm.  high.  i.  Branching  tentacle, 
partially  contracted.  2.  Paragastric  canals,  only  unbranched  terminations 

shown.     3.  Mouth. 

\ 

The  8  mm.  specimens  are  much  further  advanced,  Fig.  9.  The 
auricles  are  now  forming,  and  the  tentacular  ridge  has  appeared 
as  a  slight  fold  or  line  as  shown,  but  it  is  not  connected  to  the 
tentacular  bulb,  and  possesses  no  tentacles.  It  was  observed  that 
tentacles  never  appeared  along  the  tentacular  ridge  until  it  had 
joined  the  tentacular  bulb. 


FK;.  8.  Young  Mnemiopsis  Icidyi,  6  mm.  high.  i.  Tentacular  bulb. 
(Tentacle  omitted,  being  same  as  in  Fig.  7.)  2.  Juncture  of  paragastric 
and  auricular  canals.  3.  Mouth. 

It  is  now  easy  to  see  how  the  adult  structures  are  completed. 
As  the  junction  of  the  paragastric  and  auricular  canals  moves  up- 
ward forming  the  auricular  groove,  the  tentacular  ridge  and  labial 
ridge  grow  with  it.  The  large  branched  tentacle  disappears,  and 
small  tentacles  appear  along  the  tentacular  ridge. 


FEEDING    MECHANISM    OF    MXEMIOPSIS. 


77 


This  remarkable  food  catching  apparatus  of  Mnemiopsis,  in 
which  the  conveying  system  seems  to  foreshadow  that  of  the 
bivalves,  is  certainly  a  great  advance  over  that  of  the  Scyphozoa. 


FIG.  9.  Young  Mnemiopsis  Icidyi,  8  mm.  high.  i.  Branched  tentacle  en- 
tirely retracted,  but  same  as  in  Fig.  7.  2.  Tentacular  ridge.  3.  Paragastric 
canal,  termination  shown  with  branches.  4.  Mouth.  5.  Beginning  of  labial 
ridge.  6.  Developing  auricles. 

Of  its  efficiency  there  can  be  no  doubt,  for  compare  Bigelow's 
('15)  statement  that  the  plankton  was  greatly  diminished  in  a 
swarm  of  ctenophores.  Nelson  ('25)  also  brings  forth  evidence 
of  a  correlation  between  the  abundance  of  Mnemiopsis  lcld\i 
and  the  intensity  of  shipworm  infestation  and  oyster  sets.  More- 
over, the  fact  that  the  ctenophores  are  usually  found  in  such  vast 
and  dense  swarms,  argues  well  for  their  ability  to  obtain  food. 
Possibly  it  is  due  to  this  efficient  apparatus  that  we  find  in  many 
species  of  ctenophores  the  small  compact  bodies  and  absence  of 
long  trailing  tentacles. 

SUMMARY. 

The  mode  of  feeding  was  studied  in  young  tentacled  forms  and 
in  the  adult  Mnemiopsis  Icidyi.  The  young  capture  food  with 
their  branched  tentacles,  and  deposit  it  in  the  mouth.  The  adults 
entangle  the  food  with  the  small  tentacles  along  the  tentacular  ridge, 
and  deposit  it  in  the  labial  trough,  whence  it  is  carried  to  the  mouth. 


78  HOLLAND    J.    MAIN. 

Food  enters  the  stomodaeum  and  after  digestion  is  cast  out  of 
the  mouth,  or  it  may  enter  the  food  canals  and  pass  out  of  the 
anus. 

BIBLIOGRAPHY. 
Agassiz,  A. 

'65     North  American  Acalephae.     111.   Cat.   Mus.    Comp.   Zool.,   No. 

II.     Harvard. 
Bigelow,  H.  B. 

'15     Exploration    of   the    Coast    Waters    between    Nova    Scotia   and 
Chesapeake  Bay,  July  and  August,  1913,  by  the  U.  S-.  Fisheries 
Schooner,  Grampus,  Oceanography  and  Plankton.     Bull.  Mu- 
seum of  Comp.  Zool.     Cambridge,  Vol.  LIX.,  No.  4. 
Fewkes,  J.  W. 

'81     Studies  of  the  Jelly-fishes  of  Narraganset  Bay.     Bull.  Museum 
Comp.    Zool.     Harvard,    Vol.    IX.     On    the    Acalephae   of   the 
East  Coast  of  New  England.     Ibid.,  Vol.  VIII. 
Gemmill,  J.  F. 
'19     The   Ciliation  of  the  Leptomedusan  Mclicertidium  octocostatum. 

Proc.  Zool.  Soc.,  1919. 
Kincaid,  T. 

'J5     Oyster    Culture    in    Washington.     Trans.    Second    Ann.    Meet- 
ing Pacific  Fisheries,  San  Francisco,  p.  4. 
Labour,  M.  V. 
'22     The    Food    of    Plankton    Organisms.     Journ.    Mar.    Biol.    Assn. 

Plymouth,  N.  S.,  Vol.  XII.,  No.  4,  p.  644. 
'23     Ibid.,  Vol.  XIII.,  No.  I  p.  70. 
Mayer  A.  G. 

'12     Ctenophores  of  the  Atlantic  Coast  of  North  America,  Publ.  No. 

162.     Carnegie  Inst.  of  Washington. 
Nelson,  T.  C. 
'23     On   the   Occurence  and   Food   Habits  of   Certain   Ctenophores. 

Anat.  Rec.,  Vol.  26,  No.  5,  p.  381. 

'25     On  the  Occurrence  and  Food  Habits  of  Ctenophores  in   New 
Jersey  Inland  Coastal  Waters.     BIOL.  BULL.,  Vol.  XLVIIL,  No.  2. 


THE  INFLUENCE  OF  OXYGEN  TENSION  UPON  THE 
RESPIRATION  OF  UNICELLULAR  ORGANISMS. 

WILLIAM   R.   AMBERSON. 

(From  the  Department  of   Physiology,  School  of  Medicine,  University  of 
Pennsylvania,  and  the  Marine  Biological  Laboratory,  Woods  Hole,  Mass.) 

Our  knowledge  of  the  influence  of  oxygen  tension  upon  the 
oxygen  consumption  of  unicellular  organisms  is  quite  incomplete. 
The  literature  contains  many  studies  of  the  influence  of  oxygen 
tension  changes  upon  growth  and  activity  of  such  forms,  but 
relatively  few  direct  measurements  of  oxygen  consumption  have 
been  made.  In  some  studies  in  which  the  consumption  has  been 
measured  the  problem  has  been  complicated  by  changes  in  the 
number  of  respiring  cells  during  the  course  of  the  experiment. 
This  would  appear  to  be  true  of  such  observations  as  those  of 
Stephenson  and  Whetham  (1924)  who  have  found  that  the  oxygen 
intake  of  B.  coli  is  much  greater  in  pure  oxygen  than  in  air,  and 
of  Novy  and  Soule  (1925)  who  report  that  the  tubercle  bacillus 
grows  best  in  an  atmosphere  containing  40-50  per  cent,  oxygen, 
the  growth  and  the  oxygen  consumption  falling  off  progressively 
above  and  below  this  value.  It  is  not  possible  to  infer  that  a 
change  in  division  rate  indicates  a  change  in  the  oxygen  intake  of 
the  individual  bacterium.  The  influence  of  the  oxygen  tension 
may  be  more  indirect,  possibly  through  the  formation  of  such 
growth-promoting  substances  as  Burrows  (1924)  has  described, 
whose  production  is  increased  by  an  increased  oxygen  supply. 

In  other  studies  of  bacterial  respiration  in  which  there  has 
probably  been  no  significant  change  in  the  number  of  respiring 
cells,  Piitter  (1924)  and  E.  N.  Harvey  (1926)  have  secured 
evidence  that  the  respiratory  rate  is  not  influenced  by  changes  in 
the  oxygen  tension.  In  unicellular  animal  organisms  the  weight 
of  the  somewhat  meagre  evidence  so  far  secured  indicates  that 
oxygen  consumption  is  independent  of  oxygen  tension  over  a  wide 
range.  Lund  (1918)  found  this  to  be  true  for  I\innnccinm. 
Henze  (1910)  and  Warburg  (1908)  found  a  similar  situation  in 

79 


So  WILLIAM    R.    AMBERSON. 

sea-urchin  eggs,  in  which  there  was  little  change  in  oxygen  intake 
when  the  oxygen  tension  varied  from  double  that  in  air  to  one- 
fourth  of  the  same  value. 

In  all  of  the  studies  in  this  last  group  in  which  oxygen  has 
actually  been  measured,  the  Winkler  method  has  been  employed. 
It  is  well  known  that  this  method,  while  very  satisfactory  for  the 
determination  of  dissolved  oxygen  in  pure  water  or  in  salt  solu- 
tions, becomes  untrustworthy  when  organic  material  is  present  in 
the  fluids  tested.  Heilbrunn  (1915)  and  others  have  objected  to 
the  use  of  the  method  in  the  study  of  heavy  suspensions  of  pro- 
tozoa and  marine  eggs.  The  presence  of  iron,  found  by  Warburg 
(1914)  to  be  contained  in  sea-urchin  eggs  in  considerable  amounts, 
is  known  to  introduce  large  errors  in  the  titration.  (See  Alster- 
berg,  1926.) 

I  became  interested  in  this  problem  after  making  the  observa- 
tion (1924)  that  the  oxygen  consumption  of  a  number  of  marine 
invertebrates  is  directly  proportional  to  the  oxygen  tension  in  the 
sea  water,  over  a  considerable  part  of  the  normal  physiological 
range.  This  observation  has  led  me  to  a  reexamination  of  the 
problem  in  other  forms.  The  present  communication  deals  with 
some  results  obtained  on  unicellular  materials  in  an  attempt  to 
confirm  the  conclusions  of  previous  workers  by  methods  not  open 
to  the  criticisms  which  can  be  leveled  against  the  Winkler  technique. 
This  confirmation  has  been  secured.  The  data  are  submitted  in 
support  of  the  older  observations,  and  as  giving  a  more  complete 
account  of  the  oxygen  tension  relationships  in  the  Arbacia  egg 
than  has  previously  been  published. 

On  the  technical  side  an  attempt  has  been  made  to  apply  stand- 
ard methods  of  gas  analysis  to  the  study  of  the  problem.  Novy 
and  his  collaborators  have  previously  successfully  used  such  meth- 
ods in  their  study  of  bacterial  respiration.  I  find  that  the  oxygen 
consumption  of  unicellular  animal  organisms  can  be  similarly 
followed  by  such  methods,  with  an  accuracy  at  least  as  good  as 
that  possible  in  human  and  mammalian  metabolic  studies.  The 
carbon  dioxide  production  is  more  difficult  to  determine  because  of 
the  high  solubility  of  the  gas  in  the  liquid  phase,  and  the  possi- 
bility of  its  chemical  fixation.  No  great  reliance  can  therefore  be 
placed  upon  the  carbon  dioxide  values  given  below,  or  upon  the 


INFLUENCE   OF    OXYGEN    TENSION    UPON    RESPIRATION.          8l 

respiratory  quotients  calculated.  The  large  variations  in  the  value 
of  the  quotient  is  sufficient  to  indicate  the  magnitude  of  the  errors 
which  must  be  present  in  the  determination  of  carbon  dioxide. 
My  main  concern  has  been  to  study  the  oxygen  consumption. 

EXPERIMENTS  WITH  Panuncciitin. 

A  group  of  experiments  was  first  carried  out  with  Parameciuin, 
in  an  attempt  to  develop  a  satisfactory  technique.  For  several 
reasons  the  data  obtained  are  not  as  complete  or  accurate  as  the 
values  secured  later  on  Arbacia  eggs.  The  results  are,  however, 
fairly  consistent  and  give  a  satisfactory  confirmation  of  Lund's 
report  on  this  organism. 

A  thick  suspension  of  the  protozoa  was  prepared  by  centrifug- 
ing  several  liters  of  fluid  from  a  number  of  cultures.  The  or- 
ganisms were  then  washed  through  several  changes  of  tap  water, 
being  concentrated  with  the  centrifuge  after  each  washing.  The 
suspension  in  its  final  form  was  practically  free  from  bacteria. 
The  cultures  were  never  entirely  pure,  but  P.  caudatum  always 
constituted  at  least  95  per  cent,  of  the  protozoa  present.  The 
presence  of  other  unicellular  organisms,  either  animal  or  plant, 
cannot  appreciably  have  modified  the  results. 

A  preliminary  obstacle  was  encountered  when  it  was  observed 
that  it  is  exceedingly  difficult  to  secure  two  samples  of  such  a 
suspension  which  will  contain  the  same  number  of  animals.  This 
difficulty  arises  from  the  high  mobility  of  the  organisms  which  are 
negatively  geotropic,  and  tend  to  rise  to  the  surface  even  while  the 
sample  is  being  drawn.  After  many  unsuccessful  attempts  to 
secure  two  identical  samples,  the  procedure  was  abandoned.  In 
its  stead  it  was  found  possible  to  carry  out  two  consecutive  meas- 
urements of  respiration  upon  the  same  suspension,  the  first  at 
atmospheric  pressure,  the  second  at  some  lower  or  higher  pressure. 
Under  the  conditions  of  the  experiments  division  was  absent,  yet 
the  measurements  were  completed  before  starvation  intervened. 

20  cc.  of  the  suspension  finally  obtained  were  introduced  into  a 
cylindrical  glass  vessel,  of  about  the  size  and  form  of  a  Haldane 
gas  collecting  tube.  This  tube  was  fitted  with  three-way  stopcocks 
at  both  ends.  The  volume  was  80.85  cc.  After  the  introduction 
of  the  suspension  the  volume  of  gas  in  the  tube  was,  therefore, 


82  WILLIAM    R.    AMBERSON. 

60.85  cc.  Air  delivered  by  a  pump  under  a  small  pressure  was 
now  bubbled  through  the  suspension  for  five  minutes.  This  air 
was  taken  by  the  pump  from  a  large  room  in  the  basement  of  the 
medical  building;  its  oxygen  content  was  slightly  lower,  and  its 
carbon  dioxide  content  slightly  higher,  than  in  outside  air.  The 
actual  percentages  were  determined  by  later  analysis.  At  the  end 
of  the  equilibration  period  the  tube,  completely  filled  with  the  room 
air,  and  with  the  suspension,  in  gaseous  equilibrium  with  this  air, 
was  closed  off,  leaving  the  contained  gas  completely  saturated  with 
water,  at  atmospheric  pressure,  and  at  approximately  25°  C.,  the 
temperature  of  the  room.  The  tube  was  then  placed  horizontally 
within  a  water  bath  at  a  temperature  of  25°  C.  ±  .2°.  From 
time  to  time  the  tube  was  gently  rocked  by  hand  to  keep  the  sus- 
pension approximately  in  gaseous  equilibrium  with  the  air  above 
it.  At  the  end  of  three  hours  the  tube  was  removed  and  the  sus- 
pension vigorously  shaken  into  complete  equilibrium  with  the 
gaseous  phase.  A  sample  of  the  contained  gas  was  now  withdrawn 
into  a  Bailey  collector,  and  set  aside  for  later  analysis. 

As  quickly  as  possible  the  same  suspension  was  again  equi- 
librated with  room  air.  The  tube  was  then  partially  exhausted  by 
a  water  pump,  the  residual  pressure  being  measured  by  a  mer- 
cury manometer  connected  with  one  inlet.  Upon  the  attainment 
of  the  desired  low  pressure  the  stopcocks  were  closed,  and  the 
tube  placed  again  within  the  water  bath.  At  the  conclusion  of  a 
second  three  hour  period  the  gas  in  the  tube  was  brought  to  at- 
mospheric pressure  and  a  sample  collected.  At  the  end  of  this 
second  period  the  organisms  were  alive  and  active. 

The  gas  samples  were  now  analyzed  by  the  use  of  a  Haldane- 
Henderson  gas  analyser.  Whenever  possible  duplicate  or  tripli- 
cate determinations  were  made,  and  the  results  averaged.  As- 
suming the  gaseous  solubilities  to  be  those  given  by  the  standard 
tables  for  pure  water  at  this  temperature,  the  total  oxygen  and 
carbon  dioxide  present  at  the  beginning  and  at  the  end,  in  both 
air  and  water,  were  now  calculated,  the  usual  corrections  for  ba- 
rometer, water  vapor,  etc.,  being  applied. 

The  results  obtained  in  fovirteen  experiments  are  given  in  Table 
I.  It  is  seen  that  the  oxygen  intake  is  practically  constant  from 
200  to  50  mm.  Hg  partial  pressure  of  oxygen.  Below  50  mm.  the 


INFLUENCE   OF    OXYGEN    TENSION    UPON    RESPIRATION. 


values  are  somewhat  reduced,  but  down  to  n  mm.  the  intake  is 
still  at  least  80  per  cent,  of  that  at  atmospheric  pressure.  Since, 
in  these  experiments,  an  oxygen  gradient  must  have  been  present 
from  air  to  water,  the  actual  tensions  in  the  water  were  somewhat 
lower  than  those  given  in  the  table,  which  represent  the  tensions  in 
the  air.  The  ability  of  these  organisms  to  utilize  oxygen  at  low 
tensions  therefore  becomes  even  more  evident. 

TABLE  I. 
RESPIRATION  OF  Paramcchim  AT  DIFFERENT  OXYGEN  TENSIONS. 


Respiration  in 

Respiration  in 

Ratio  be- 

Oxygen 

First  Period. 

Second  Period. 

tween  C>2 

Ex- 

Pres- 

Consumption 

peri- 

sure in 

in  Second 

ment. 

Second 
Period. 

O2 
Cons. 

CO2 

Prod. 

R.  Q. 

02 
Cons. 

CO2 

Prod. 

R.  Q. 

Period  and 
that  in  First 

Period. 

mm.  Hg. 

*c.c. 

c.c. 

c.c. 

c.c. 

I 

208-192 

1.030 

•  703 

.683 

1.027 

•  753 

•  733 

•997 

2 

211-195 

1.107 

•663 

•  598 

1.167 

.762 

•653 

1.054 

3 

IS4-I39 

.969 

•  565 

.583 

1.025 

.640 

•625 

1.058 

4 

IS4-I35 

1-345 

.849 

.632 

1.390 

•903 

.649 

1-033 

5 

122-109 

•933 

.763 

.817 

1.029 

1.016 

.986 

1.103 

6 

92-68 

2.088 

1.490 

.714 

2.  OO2 

1.446 

.722 

•952 

7 

Qi-74 

.216 

i.  086 

.893 

1-245 

1.204 

.967 

1.024 

8 

70-48 

•654 

1.302 

.787 

1.612 

1.448 

.898 

•975 

9 

70-60 

.698 

•390 

•559 

.724 

•458 

•633 

1-037 

10 

70-57 

•131 

.676 

.598 

•973 

•553 

•  568 

.860 

ii 

60-42 

•045 

1.028 

.686 

1.440 

1.008 

.699 

•  875 

12 

49-28 

•592 

1.093 

.686 

1.546 

l.«5 

.721 

.971 

13 

28-n 

1.146 

.766 

.668 

•977 

•638 

.652 

-853 

14 

28-11 

1.642 

I-I34 

.691 

1.290 

1.038 

.804 

.786 

Average  R.  Q.         .685                                       .736 

*  Volume  measured  at  760  mm.  Hg  and  O°  C. 

The  average  of  the  respiratory  quotients  obtained  in  twenty- 
eight  determinations  comes  out  to  be  .710.  Considering  the  wide 
range  of  the  individual  values  it  is  hardly  possible  to  attach  any 
great  significance  to  this  figure,  although  it  may  be  taken  to  suggest 
the  presence  of  a  fat  metabolism  under  the  conditions  of  the  ex- 
periment, when  the  normal  food  supply  is  absent. 

These  preliminary  experiments  indicated  that  the  method  is  ap- 
plicable to  such  problems,  but  certain  difficulties  were  encountered 
which  made  it  advisable  to  complete  the  study  on  another  material. 


84 


WILLIAM    R.    AMBERSON. 


These  consisted  in  (i)  the  impossibility  of  controlling  the  activity 
of  the  organisms,  (2)  the  manipulation  of  gases  at  pressures  very 
much  below  atmospheric,  which  prevented  the  exploration  of  very 
low  oxygen  tensions,  and  (3)  the  lack  of  complete  gaseous  equi- 
librium between  air  and  water  during  the  course  of  the  experiment. 
The  study  was  therefore  continued  with  a  modified  method  at 
Woods  Hole  on  fertilized  Arbacia  eggs,  which  have  no  independent 
motility  during  the  first  hours  of  their  development. 

EXPERIMENTS  WITH  FERTILIZED  Arbacia  EGGS. 

In  these  experiments  it  has  been  found  possible  to  secure  two 
suspensions  of  eggs  containing  equal  numbers  of  cells,  whose 
respiratory  exchanges  check  well  with  each  other  when  the  two 
are  studied  simultaneously  under  identical  conditions.  The  eggs 
were  freed  from  ovarian  debris  and  body  fluid  and  washed  through 
several  changes  of  sea  water.  A  heavy  suspension  of  cells  was 
secured  by  permitting  the  eggs  to  sediment  in  a  large  beaker  and 
then  pouring  off  the  greater  part  of  the  supernatant  sea  water. 
These  were  then  fertilized.  About  ten  minutes  after  fertiliza- 
tion two  60  cc.  samples  of  this  suspension  were  taken  up  by  pipette 
and  introduced  into  two  tubes  similar  to  that  used  for  Paranic- 
chtin  but  of  a  somewhat  larger  volume. 

The  lower  oxygen  tensions  were  secured  by  mixing  oxygen  and 
nitrogen,  or  air  and  nitrogen,  in  the  desired  proportions.  Eight 
liters  of  such  a  gaseous  mixture  were  collected  in  a  large  bottle, 
over  water.  One  tube  (B)  was  then  brought  into  equilibrium 
with  this  mixture,  the  gas  being  bubbled  through  the  suspension 
for  at  least  five  minutes.  For  the  same  period  the  second  tube  (A} 
was  equilibrated  with  outside  air.  In  every  case  a  sample  of  gas 
was  collected  from  the  low  pressure  tube  toward  the  end  of  the 
equilibration,  and  its  later  analysis  accepted  as  giving  the  value 
of  the  initial  oxygen  and  carbon  dioxide  percentages.  The  air 
which  had  passed  through  tube  A  was  analyzed  in  several  experi- 
ments and  this  value  accepted  for  the  rest  as  giving  the  initial  oxy- 
gen and  carbon  dioxide  percentages  in  the  high  pressure  tube.  It 
showed,  after  passing  through  the  egg  suspension,  a  slight  diminu- 
tion in  oxygen  and  a  slight  increase  in  carbon  dioxide. 

At  the  conclusion  of  the  equilibration  the  two  tubes  were  closed 


INFLUENCE   OF    OXYGEN    TENSION    UPON    RESPIRATION.          85 

in  such  a  manner  that  the  contained  gas  was  left  at  atmospheric 
pressure  and  at  approximately  20°  C.  They  were  then  placed 
side  by  side  within  a  water  bath,  and  rotated  continually  throughout 
the  experiment,  turning  at  the  rate  of  about  thirty  times  a  minute. 
Under  these  conditions  the  eggs  were  always  evenly  distributed 
throughout  the  suspension,  and  kept  in  constant  motion,  the  water 
was  always  nearly  in  equilibrium  with  the  gas,  and  cleavage  pro- 
ceeded in  a  perfectly  normal  manner. 

Running  sea  water  was  used  in  the  water  bath.  Its  tempera- 
ture varied  slightly  from  day  to  day.  The  lowest  temperature  re- 
corded in  any  experiment  was  18.2°  C.,  the  highest  20.2°  C.  The 
experiments  continued  in  most  cases  for  two  hours ;  in  a  few  cases 
for  three  hours.  The  first  division  occurs  about  one  hour  after 
fertilization  at  this  temperature ;  subsequent  divisions  follow  about 
every  thirty  minutes.  At  the  end  of  the  two-hour  experiments 
the  eggs  were  in  the  four  and  eight  cell  stage;  at  the  end  of  the 
three-hour  experiments  they  were  in  the  sixteen  and  thirty-two 
cell  stage.  The  material  is  not,  therefore,  unicellular  throughout 
the  whole  experiment.  The  individual  cells,  however,  in  all  of 
these  early  stages  are  all  at  the  surface  of  the  dividing  egg  in 
intimate  relation  with  the  oxygen  supply  in  the  water ;  there  seems 
every  reason  to  believe  that  the  relationship  under  investigation 
will  not  be  materially  modified  by  this  increase  in  number  of  cells, 
unaccompanied  by  any  change  in  the  mass  of  respiring  tissue.  We 
have  reason  to  believe  from  the  work  of  Gray  (1925),  that 
cleavage  itself  does  not  affect  the  rate  of  oxygen  consumption,  and 
that,  after  the  first  sharp  rise  following  fertilization  the  consump- 
tion is  practically  constant  during  the  first  three  hours  of  develop- 
ment. The  unfertilized  egg  has  so  low  a  gaseous  exchange  that  it 
has  not  proven  practicable  to  follow  its  respiration  by  the  present 
method. 

At  the  end  of  the  experiment  samples  of  gas  were  secured  from 
both  tubes  and  analysed.  The  oxygen  and  carbon  dioxide  in  the 
gas  and  in  the  sea  water  were  then  calculated  for  the  beginning 
and  for  the  end  of  the  experiment.  For  this  calculation  the  ab- 
sorption coefficients  for  oxygen  and  carbon  dioxide  in  sea  water 
given  in  Tabulae  Biologicae  (Vol.  4,  pp.  571-578)  were  used.  The 
results  of  a  typical  experiment  are  as  follows : 


86  WILLIAM    R.    AMBERSON. 

Tube  A.  Tube  B. 

Oxygen  tensions  during  experiment .  155  to  142  mm.  Hg.       61  to  49  mm.  Hg. 

Volume  of  tube    106.15  c.c.  105.39  c.c. 

Volume  of  suspension   60       c.c.  60       c.c. 

Gas  Analysis  at  beginning: 

O0   20.87%  8.22% 

CO.,    05%  .02% 

N0_~ 79-08%  91-76% 

Gas  analysis  at  end   (corrected  for  volume  change)  : 

O,  19.16%  6.59% 

c60  62%  .58% 

N,  " 79.08%  91-76% 

Oxygen  in  air  and  water : 

At  beginning 9-96i  c.c.  3.863.  c.c. 

At  end   9.147  c.c.  3-095  c.c. 

Oxygen  Consumption    814  c.c.  .768  c.c. 

Carbon  dioxide  in  air  and  water : 

At  beginning    045  c.c.  .018  c.c. 

At  end 55§  c.c.  .517  c.c. 

Carbon  dioxide  production 513  c.c.  .499  c.c. 

Volumes  corrected  to  dry  values  at  O°  C.  and  760  mm.  Hg. 

Oxygen  consumption   .741  c.c.  .699  c.c. 

Carbon  dioxide  production 467  c.c.  .454  c.c. 

Respiratory   quotient    .630  .649 

Oxygen  consumption  at  low  pressure  =  94.4%  of  that  at  atmospheric  pres- 
sure. 

Carbon  dioxide  production  atJow  pressure  =  97.3%  of  that  at  atmospheric 
pressure. 

The  results  obtained  in  twenty  experiments,  carried  out  after 
the  preliminary  tests,  are  given  in  Table  2,  and  shown  graphically 
in  Fig.  i.  The  oxygen  consumption  is  seen  to  be  practically  con- 
stant from  an  oxygen  pressure  of  228  mm.  Hg.  down  to  about 
20  mm.  Hg.  Between  80  and  20  mm.  there  is  a  definite  downward 
trend  in  the  values,  but  at  20  mm.  the  consumption  is  still  about 
90  per  cent,  of  that  at  atmospheric  pressures.  Below  this  point 
the  consumption  falls  off  sharply. 

In  Fig.  i  the  experimental  values  are  shown  as  rectangles.  The 
height  of  this  rectangle  corresponds  to  i  per  cent,  on  the  oxygen 
consumption  scale;  the  length  indicates  the  oxygen  tension  range 
in  tube  B  during  the  course  of  the  experiment.  Each  rectangle 
shows  that  over  this  range  the  oxygen  consumption  of  the  egg 
suspension  in  tube  B  was  the  indicated  percentage  of  the  con- 
sumption in  tube  A,  run  at  atmospheric  pressure.  The  absolute 


INFLUENCE   OF    OXYGEN    TENSION    UPON    RESPIRATION. 


TABLE  II. 
RESPIRATION  OF  FERTILIZED  Arbacla  EGGS  AT  DIFFERENT  OXYGEN  TENSIONS. 


Respiration  in 

Respiration  in 

Ratio  be- 

Ex- 
peri- 

Oxygen 
Pressure  in 

Tube  A. 

Tube  B. 

tween  Ot 
Consumption 
in  Tube  B 

ment. 

Tube  B. 

02 

CO2 

O2 

C02 

and  that  in 

Cons. 

Prod. 

R.  Q. 

Cons. 

Prod. 

R.Q. 

Tube  A. 

mm.    Hg. 

*c.c. 

c.c. 

c.c. 

c.c. 

I 

228.8-220.0 

•423 

•394 

•931 

•433 

.294 

.679 

1.024 

2 

155.2-147.2 

•473 

•430 

.909 

.470 

•386 

.821 

•994 

3 

152.2-144.6 

•443 

•329 

•742 

•447 

•350 

•783 

1.009 

4 

142.0-135-7 

•309 

•257 

•832 

-315 

•  237 

•  753 

1.019 

5 

123.2-112.4 

•524 

•436 

•832 

•  533 

.326 

.611 

1.017 

6 

116.8-104.3 

.691 

•443 

.641 

•  733 

.448 

.611 

1.061 

7 

85.5-  76.0 

.650 

.496 

•763 

.621 

•403 

.648 

•955 

8 

70.6-  61.7 

-572 

.417 

.729 

.520 

.412 

.792 

-909 

9 

66.6-  55.4 

-665 

.528 

•794 

•653 

•493 

•754 

.982 

10 

61.2—  49.0 

.741 

.467 

•630 

.699 

•454 

.649 

-944 

ii 

44.6-  38.3 

-390 

•334 

-856 

•370 

-339 

•915 

-949 

12 

36.8-  24.2 

.818 

.611 

.768 

-735 

•  535 

.727 

•899 

13 

30.0-  24.2 

.406 

.279 

.688 

•345 

.217 

.628 

.850 

14 

23.9-  14.9 

•  592 

•524 

•  885 

15 

23-7-     8.7 

.856 

•  593 

.697 

.862 

•  558 

.648 

1.007 

16 

17.3-  10.1 

.674 

•444 

•658 

.419 

.360 

•  859 

.622 

17 

ii-S-     6-3 

.636 

•  543 

•  854 

•367 

•427 

1.160 

•  577 

18 

7-9-     3-3 

-565 

.667 

1.181 

.268 

.269 

1.004 

•457 

19 

7.1-        .8 

.746 

•  527 

.706 

•309 

.488 

1.582 

.414 

20 

4-3-     1-7 

.665 

•445 

.669 

•151 

.222 

1-473 

.227 

Average  R.  Q.      .783     Average  R.  Q.  (1-16)  .725 

*  Volume  measured  at  760  mm.  Hg.  and  O°  C. 


100;? 


120 


340 


^r 


I  TJKSIOJ.  1QL    Hg.  /£ 

FIG.    I.     Oxygen    consumption    of    fertilized    Arbacia   eggs    at    different  Lj  I  j_  |  B  F 
oxygen  tensions.     The   range   of   tensions   within   which   the   division   rate  \, 
is  affected  is  also  graphically  shown. 


88 


\VILLIAM    R.    AMBERSON. 


values  vary  considerably  from  experiment  to  experiment,  but  the 
graph  of  these  percentages  assumes  a  fairly  regular  and  consistent 
form. 

Correlated  with  the  diminished  oxygen  intake  at  very  low  oxy- 
gen tensions  retardation  in  development  was  observed  in  experi- 
ments 17-20.  In  all  other  experiments  the  eggs  in  the  low  pres- 
sure tube  had  developed  as  far  as  had  those  at  atmospheric  pres- 
sure. In  every  case  95-100  per  cent,  of  the  eggs  developed.  In 
experiment  i/,  continuing  for  two  hours,  a  slight  retardation  in 
division  rate  was  evident.  Counts  on  100  eggs  from  each  suspen- 
sion gave  the  following  values : 


i-cell. 

2-cell. 

4-cell. 

8-cell. 

Tube  ,4  (HighO2)  

4 

6 

^7 

-1-1 

Tube  B  (Low  Oz)  

c; 

14 

74 

7 

In  experiment  18  (2  hours)  a  more  marked  effect  was  observed. 
Counts  on  100  eggs  gave  the  following  values: 


i  -cell. 

2-cell. 

4-cell. 

8-cell. 

i6-cell. 

Tube  A  (High  O>)  .  .  . 

2 

o 

C2 

4O 

6 

Tube  B  (Low  O) 

"?O 

7 

o 

o 

In  experiment  19  (3  hours)  the  eggs  at  atmospheric  pressure  were 
in  the  sixteen  and  thirty-two  cell  stage.  In  tube  B  about  80  per 
cent,  had  reached  the  four-cell  stage,  but  none  were  found  in  later 
stages.  In  experiment  20  (2  hours)  the  eggs  at  atmospheric 
pressure  were  in  the  four  and  eight-cell  stage.  In  tube  B  a  care- 
ful search  failed  to  reveal  any  cleavage  whatsoever.  It  has  long 
been  known  that  in  the  complete  absence  of  oxygen  cleavage  in 
these  eggs  is  prevented.  (See  E.  B.  Harvey,  1926.)  My  own 
observations  would  suggest  that  a  certain  minimal  concentration 
of  oxygen  is  necessary  for  division,  but  the  matter  has  not  received 
a  thorough  study.  The  range  of  oxygen  tensions  within  which 
development  is  either  retarded  or  prevented  is  indicated  graphically 
in  Fig.  i.  The  values,  taken  from  four  experiments,  are  to  be 
considered  as  approximations  only.  Taken  in  conjunction  with 
the  curve  of  oxygen  consumption  they  show  the  great  ability  of 


INFLUENCE   OF    OXYGEN    TENSION    UPON    RESPIRATION.  89 

these  eggs  to  carry  out  a  normal  development  clown  to  very  low 
oxygen  tensions. 

It  is  of  interest  to  note  that  in  all  four  of  these  experiments  in 
which  retardation  or  inhibition  of  development  occurred  the  res- 
piratory quotient  rose  ahove  unity;  in  experiments  19  and  20  the 
quotient  reached  the  high  values  of  1.58  and  1.47.  These  figures 
suggest  the  presence  of  anaerobic  respiratory  processes  at  these 
low  oxygen  tensions.  It  is  not  possible  to  be  certain  concerning 
the  matter,  since,  under  these  conditions  of  oxygen  lack,  acid 
metabolites  may  collect  in  the  suspension  and  liberate  carbon 
dioxide  from  the  carbonates  of  the  sea  water. 

In  none  of  these  experiments  has  the  tension  of  carbon  dioxide 
risen  to  such  a  point  that  it  can  have  materially  affected  develop- 
mental rate.  Haywood  (1927)  has  shown  that,  in  high  concen- 
tration, carbon  dioxide  behaves  as  a  narcotic  and  completely  pre- 
vents cleavage  when  its  tension  rises  above  230  mm.  Hg.  Below 
this  value  cleavage  occurs  at  a  rate  slower  than  normal.  The 
threshold  tension  for  this  carbon  dioxide  effect  to  appear  was  not 
determined,  but  it  seems  evident  that  at  very  much  lower  con- 
centrations the  retardation  of  development  must  become  negli- 
gible. The  highest  carbon  dioxide  value  observed  in  the  present 
study  was  at  the  end  of  experiment  18,  when  the  partial  pressure 
reached  7  mm.  Hg  in  tube  B.  The  retardation  of  development 
observed  at  low  oxygen  tensions  must  therefore  be  caused  by 
oxygen  lack  rather  than  by  a  narcotic  effect  of  the  carbon  dioxide 
produced.  Haywood  also  reports  experiments  on  the  influence  of 
low  oxygen  tension  upon  developmental  rate  which  agree  with 
my  own  findings  in  showing  practically  no  influence  down  to  quite 
low  values. 

In  most  experiments  carried  out  below  an  oxygen  tension  of 
50  mm.  Hg  there  was  observed,  at  the  end  of  the  experiment,  a 
liberation  of  pigment  in  the  suspension  in  the  low  pressure  tube 
which  became  more  and  more  marked  as  the  oxygen  tension  was 
lowered.  This  liberation  of  pigment  apparently  arose  from  the 
cytolysis  of  a  certain  number  of  cells.  The  actual  percentage  of 
eggs  thus  destroyed  was  not  determined,  but  must  have  been  small, 
since  at  the  end  of  the  experiment  the  volume  of  the  eggs  after 
sedimentation  was  not  appreciably  diminished.  The  downward 


GO  WILLIAM    R.    AMBERSON. 

trend  in  the  oxygen  consumption  values  below  80  mm.  Hg  may 
be  in  part  due  to  this  destruction  of  a  small  number  of  the  eggs, 
although  we  know,  from  the  work  of  Warburg  (1914)  that 
respiratory  exchanges  may  continue  for  some  hours  even  in  com- 
pletely fragmented  sea-urchin  eggs,  at  a  level  not  far  below  that 
fovind  when  the  cells  are  intact. 

The  ability  of  both  protozoa  and  sea-urchin  eggs  to  carry  on  a 
normal  respiratory  exchange  down  to  very  low  oxygen  tensions 
points  very  definitely  to  the  normal  presence,  within  the  cells,  of 
a  considerable  oxygen  tension.  Oxygen  is  present  in  such  amount 
that  it  does  not  limit  the  metabolism,  whose  rate  is  determined 
by  other  than  oxidative  reactions. 

SUMMARY. 

By  standard  methods  of  gas  analysis  the  respiratory  exchanges 
of  Paramecium  and  of  fertilized  Arbacia  eggs  have  been  studied. 
The  respiratory  rate  in  both  materials  is  found  to  be  practically 
constant  over  a  wide  range  of  oxygen  tensions,  thus  confirming 
older  work  done  by  other  methods. 

In  the  fertilized  Arbacia  egg  the  oxygen  consumption  is  prac- 
tically constant  between  228  and  20  mm.  Hg  partial  pressure  of 
oxygen.  Between  80  and  20  mm.  Hg  there  appears  to  be  a  slight 
diminution  in  oxygen  intake,  but  at  20  mm.  Hg  the  consumption 
is  still  about  90  per  cent,  of  that  at  atmospheric  pressure.  Below 
20  mm.  Hg  the  consumption  is  sharply  reduced. 

The  cleavage  of  Arbacia  eggs  proceeds  at  a  normal  rate  down 
to  very  low  oxygen  tensions.  No  retardation  in  development  has 
been  observed  above  n  mm.  Hg.  Below  this  value  the  rate  be- 
comes slower  and  cleavage  ceases  entirely  below  4  mm.  Hg. 

BIBLIOGRAPHY. 
Alsterberg,  G. 

'26    Die    Winklersche    Bestimmungsmethode    fur    in    Wasser    gelosten 

elementaren    Sauerstoff.     Biochem.   Zeits.,    170,   30-75. 
Amberson,  W.  R.,  Mayerson,  H.  S.,  and  Scott,  W.  J. 
'24     The  Influence  of  Oxygen  Tension  upon  Metabolic  Rate  in  In- 
vertebrates.    Journ.  Gen.   Physiol.,  7:  171-176. 
Burrows,  M.  T. 

'24     Relation    of   Oxygen    to    the    Growth    of   Tissue    Cells.     Amer. 
Jour.  Physiol.,  68:  no. 


INFLUENCE   OF    OXYGEN    TENSION    UPON    RESPIRATION.          91 

Gray,  J. 

'25     The  Mechanism  of  Cell  Division.     Oxygen  Consumption  during 

Cleavage.     Proc.  Camb.  Phil.  Soc.,  i:  225-236. 
Harvey,  E.  B. 
'27     The    Effe,gt   of   Lack   of   Oxygen    on    Sea    Urchin    Eggs.     BIOL. 

BULL.,  ^53:  147-160. 
Harvey,  E.  N. 
'25     The  Total   Luminous   Efficiency  of   Luminous   Bacteria.     Jour. 

Gen.  Physiol.,  8:  89-108. 
Haywood,  C. 

'27     Carbon  Dioxide  as  a  Narcotic  Agent.     BIOL.  BULL.,  53:450-464. 
Heilbrunn,  L.  V. 

'15     The  Measurement  of  Oxidation  in  the   Sea-Urchin   Egg.     Sci- 
ence, N.  S.,  42:  615-616. 
Henze,  M. 
'10     Uber     den     Einfluss     des     Sauerstoffdrucks     auf     den     Gaswechsel 

einiger  Meerestiere.     Biochem.  Zeits.,  26:  255-278. 
Lund,  E.  J. 

'18     Relation   of   Oxygen    Concentration   and   the    Rate   of    Intracel- 
lular    Oxidation    in    Paramecium    Cau.datitni.  •  BIOL.    BULL.,    45: 

351-364- 
Novy,  F.  G.,  and  Soule,  M.  H. 

'25     Respiration  of  the  Tubercle  Bacillus.     Jour.  Inf.  Dis.,  36:  168- 

232. 
Putter,  A. 

'24     Die  Atmung  der  Planktonbakterien.     Arch.  ges.   Physiol.,  204, 

94-126. 
Stephenson,  M.,  and  Whetham,  M. 

'24     The   Effect   of   Oxygen   Supply   on  the   Metabolism   of   Bacillus 

Coli    Communis.     Biochem.   Jour.,    18:   498-506. 
Warburg,  O. 
'08     Beobactungen    fiber    die    Oxydationsprozesse    in    Seeigelei.      Zeits. 

Physiol.  Chem.,  57:  1-16. 
'14     Zellstruktur    und    Oxydationsgeschwindigkeit    nach    Versuchen 

am  Seeigelei.     Arch.  ges.  Physiol.,  158:  189-208. 

'14    liber  die   Rolle  des   Eisens   in  der  Atmung  des   Seeigeleis.     Zeit. 
Physiol.  Chem.,  92:  231-256. 


A  COMPARISON  OF  THE  OXYGEN  CONSUMPTION  OF 

UNFERTILIZED  AND  FERTILIZED  EGGS  OF 

FUNDULUS  HETEROCLITUS. 

MARJORIE  BOYD. 
(From  the  Marine  Biological  Laboratory,  Woods  Hole.) 

Since  Warburg  (i)  in  1908  measured  the  oxygen  consump- 
tion of  Arbacia  eggs  and  observed  the  marked  increase  follow- 
ing fertilization,  the  oxidation  processes  in  marine  eggs  and  em- 
bryos have  been  carefully  investigated.  The  rate  at  which  the 
oxygen  is  removed  from  the  surrounding  air  or  sea  water  has 
been  correlated  with  the  stages  in  development.  Thus  Shearer  (2) 
measured  the  oxygen  consumption  during  fertilization  of  Echino- 
derm  eggs,  and  found  a  decided  increase  upon  the  addition  of 
the  sperm ;  "  more  oxygen  is  taken  up  in  the  first  minute  of  the 
process  than  at  any  subsequent  interval  of  the  same  time."  In 
another  article  Shearer  (3)  states  that,  in  the  first  hour  of  devel- 
opment, the  fertilized  egg  consumes  six  to  seven  times  as  much 
oxygen  as  the  unfertilized  egg.  In  the  star  fish  egg,  however,  ac- 
cording to  Loeb  and  Wastenys  (4)  there  is  no  increase  in  the 
oxidation  rate  after  fertilization. 

The  rate  of  oxygen  consumption  is  also  correlated  closely  with 
heat  production.  Rogers  and  Cole  (5)  in  their  work  on  Arbacia 
eggs  have  shown  how  the  heat  production  varies  before,  during, 
and  after  fertilization ;  according  to  them  "  the  rate  of  heat  pro- 
duction at  the  instant  of  fertilization  is  ten  to  twelve  times  that 
of  the  unfertilized  egg." 

The  literature  upon  this  subject  reports  work  done  almost  ex- 
clusively upon  invertebrate  eggs.  Apparently  no  previous  study 
of  the  influence  of  fertilization  upon  respiratory  rate  has  been 
made  on  any  vertebrate  egg.  Scott  and  Kellicott  (6)  and  Hyman 
(/),  who  have  measured  the  oxygen  consumption  of  Fimdnlus 
embryos  at  various  stages  of  development,  made  no  observations 
on  the  respiration  during  the  first  two  hours  after  fertilization, 
and  secured  no  information  as  to  the  influence  of  fertilization 

92 


OXYGEN    CONSUMPTION    OF    EGGS    OF    FUNDULUS. 


93 


itself.  The  present  study  represents  an  attempt  to  secure  such 
information.  It  has  been  possible  to  show  by  several  methods 
that  fertilization  markedly  increases  the  oxygen  consumption  of 
the  eggs  of  Fiuidiilns  hctcroclitus.  The  time  relations  of  this 
increase  are  of  some  interest. 


i.  WINKLER  METHOD  FOR  DETERMINATION  OF  DISSOLVED 

OXYGEN. 

The  Winkler  method  as  applied  to  this  problem  was  employed 
in  the  manner  described  by  Amberson,  Mayerson  and  Scott  (8). 
600  eggs  were  placed  in  500  cc.  of  sea  water  in  each  of  two  small 
Erlenmeyer  flasks.  Samples  for  analysis  were  withdrawn  through 
siphons.  The  water  surface  was  covered  with  paraffin  oil  to 
minimize  the  diffusion  of  new  oxygen  from  the  air  into  the  water. 
The  sea  water  was  analyzed  for  dissolved  oxygen  previous  to 
experimentation ;  the  initial  sample  was  withdrawn  after  twenty 
to  forty  minutes.  In  order  to  secure  successive  determinations  of 
the  dissolved  oxygen  during  an  extended  time,  it  was  necessary  to 
adopt  a  micro-Winkler  method  as  suggested  by  Lund  (9).  For 
these  analyses  small  vials  of  6.5  cc.  capacity  were  used.  Fig.  i 
represents  the  graph  resulting  from  plotting  the  data  shown  in 
Table  I.  below.  The  values  for  the  amount  of  oxygen  consumed 
during  a  given  period  are  obtained  by  subtracting  the  amount 
remaining  in  the  sea  water  at  the  end  of  that  period  from  the 
amount  originally  present  in  the  sea  water  used  for  the  experiment. 

TABLE  I. 


Time. 

Sea  Water  Originally  Contains  5.1  cc.  Oxygen  per  Liter. 

Unfertilized  Eggs. 

Fertilized  Eggs. 

02 

Remaining. 

02 
Consumed. 

O2 
Remaining. 

Ot 

Consumed. 

20  min  

5-0 
4.9 

4.6 
3-8 
3-i 

O.I 

0.2 

0.5 

1-3 

2.O 

4-7 
4-5 
3-7 
3-4 

2.8 

1.8 

0.4 
0.6 
1.4 
i-7 
2-3 
3-3 

45  min 

7  hrs  

10  hrs   and  35  min 

25  hrs.  and  25  min  
31  hrs.  and  25  min  

94 


MARJORIE   BOYD. 


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OXYGEN    CONSUMPTION    OF    EGGS    OF    FUNDULUS. 


95 


It  is  to  be  noticed  that  the  fertilized  eggs  had  at  the  time  of  each 
determination  consumed  more  oxygen  than  the  unfertilized  eggs. 
Data  from  similar  experiments,  as  shown  in  Table  II.,  show  that 
the  rate  of  oxygen  consumption  is  most  rapid  during  the  first  two 
hours  following  fertilization. 

TABLE  II. 


Time. 

Sea  Water  Originally  Contains  5.2  cc.  Oxygen  per  Liter. 

Unfertilized  Eggs. 

Fertilized  Eggs. 

Oz  Remaining. 

Oz  Consumed. 

Oz  Remaining. 

Oz  Consumed. 

20  min.  .  .  . 
i  hr.  .  .    . 

5-i 
5-0 
4.9 
4.8 

O.I 
0.2 

0.3 
0.4 

4-7 
4-4 
4.0 

3-9 

0.4 
0.8 

1.2 

1-3 

2  hrs. 

4  hrs. 

2.  MICRO-RESPIROMETER  METHOD  FOR  DETERMINATION  OF 

OXYGEN. 

The  type  of  micro-respirometer  that  was  used  for  the  study  of 
oxygen  consumption  by  the  Fnudulus  eggs  is  one  that  has  been 
devised  by  W.  O.  Fenn  for  similar  studies  of  Arbacia  eggs.  A 
small  glass  bottle  with  a  ground  glass  neck  is  fitted  with  a  ground 
glass  stopper  which  is  connected  with  a  horizontal  fine-bore  man- 
ometer provided  with  a  centimeter  scale.  In  the  center  of  the 
bottom  of  the  bottle  is  a  small  compartment  into  which  0.5  cc.  of 
15  per  cent.  NaOH  is  introduced;  the  eggs  to  be  studied  are 
placed  in  the  space  surrounding  the  compartment.  The  NaOH 
serves  to  absorb  the  CO2  produced  by  the  eggs.  A  small  drop 
of  kerosene  is  introduced  into  the  manometer  and  its  movement 
across  the  tube  from  the  outer  to  the  inner  end  indicates  both  the 
amount  of  oxygen  consumed  and  the  rate  of  the  process. 

Five  cc.  of  sea  water,  containing  fifty  Fund  id  us  eggs,  were 
pipetted  into  the  micro-respirometer.  Two  micro-respirometers 
were  used  so  that  experiments  on  unfertilized  and  fertilized  eggs 
could  be  carried  out  at  the  same  time  under  identical  conditions. 
The  constants  of  each  apparatus  were  found  by  calibration  of  the 
respective  manometers.  The  micro-respirometers  were  immersed 
in  a  bath  of  running  sea  water;  the  temperature  for  all  of  the 


LIBRARY:; 
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96 


MARJORIE   BOYD. 


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OXYGEN    CONSUMPTION    OF    EGGS    OF    FUNDULUS.  97 

experiments  proved  to  be  20.2  ±  .6°  C.  The  readings  of  the 
meniscus  of  the  kerosene  drop  were  taken  at  intervals  of  fifteen 
minutes.  Over  twenty  series  were  run,  hut  in  only  the  last  five 
experiments  were  the  temperature  variations  observed  with  a  Beck- 
man  differential  thermometer. 

Figure  2  shows  typical  curves  for  the  results  obtained.  In 
Experiment  I.  the  number  of  cubic  millimeters  of  oxygen  con- 
sumed by  the  fertilized  eggs  is  a  little  less  than  twice  the  corre- 
sponding amount  in  the  case  of  the  fertilized  eggs  in  Experiment 
II.  Nevertheless  both  curves  show  the  same  marked  increase  in 
oxygen  consumption  45  minutes  after  fertilization.  This  in- 
creased oxygen  utilization  reaches  a  maximum  during  the  period 
from  60  to  90  minutes  following  fertilization.  From  this  time 
on,  the  amount  of  oxygen  consumed  per  unit  time  falls  so  that  the 
rate  of  utilization  approximates  that  for  the  unfertilized  eggs. 
It  would  appear,  therefore,  that  the  oxygen  requirements  of  the 
unfertilized  Fundulus  eggs  are  increased  by  fertilization.  The 
time  relations  of  this  increase  are  of  theoretical  interest ;  they  are 
indicative  of  some  oxidation  process  occurring  within  the  egg  for 
which  an  increased  oxygen  intake  is  a  necessity.  To  follow  the 
development  in  relation  to  the  time,  two  control  sets  of  50  Fundulus 
eggs  were  placed  in  sea  water  and  the  stages  of  development  were 
traced  by  microscopic  inspection.  It  was  found  that  the  increased 
rate  of  oxidation  occurs  at  a  time  before  and  during  the  appearance 
of  the  groove  in  the  surface  of  the  blastodisc  which  initiates  the 
first  cleavage.  The  subsequent  cleavages  evidently  do  not  require 
such  a  marked  rate  of  oxygen  intake.  A  single  run  with  twenty 
9-day  old  Fundulus  embryos  revealed  a  later  rise  in  the  oxygen 
consumption  which  can  be  correlated  with  the  marked  rise  in  the 
rate  which  Scott  and  Kellicott  found  to  occur  at  the  time  circula- 
tion is  established. 

To  show  still  further  the  peculiarity  of  the  time  relations,  the 
average  amount  of  oxygen  consumed  per  10  minutes  was  calcu- 
lated from  the  data  of  Experiment  I.  and  is  shown  in  Fig.  3.  The 
difference  in  the  rates  of  the  fertilized  and  unfertilized  eggs  is 
markedly  contrasted. 

A  few  experiments  were  carried  out  by  a  third  method  and 
gave  results  that  checked  qualitatively  with  the  two  mentioned 


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OXYGEN    CONSUMPTION    OF    EGGS    OF    FUNDULUSi  99 

above.  This  method  involved  the  analysis  of  air  above  25  cc.  of 
water  in  a  100  cc.  Haldane  gas  collector.  At  the  beginning  of  the 
experiment  200  Fundulus  eggs  were  pipetted  into  each  of  two 
collectors  with  the  sea  water,  and  the  water  was  equilibrated  with 
atmospheric  air  which  filled  the  vessel.  The  collectors  were  then 
sealed,  immersed,  and  rotated  in  a  bath  of  running  sea  water  for 
two  hours.  More  oxygen  was  found  to  have  been  lost  from  the 
sample  of  air  taken  from  the  collector  containing  the  fertilized 
eggs  than  from  that  containing  the  unfertilized.  This  method 
proved  to  be  only  approximate  as  the  rotation  caused  the  eggs  to 
stick  together  in  a  clump  and 'normal  development  did  not  take 
place.  The  data  secured  gave  a  qualitative  confirmation  of  the 
more  accurate  results  obtained  by  the  two  other  methods. 

SUMMARY. 

By  three  methods  it  has  been  shown  that  the  oxygen  consump- 
tion of  the  eggs  of  Fundulus  heteroclitus  is  greatly  increased  after 
fertilization.  This  increased  rate  of  oxygen  consumption  is  at 
its  maximum  from  60  to  90  minutes  after  the  addition  of  the 
sperm,  in  a  period  immediately  preceding  the  first  cleavage.  The 
oxygen  consumption  then  falls  to  a  level  practically  identical  with 
that  of  the  unfertilized  eggs. 

The  writer  wishes  to  express  her  appreciation  to  Dr.  \Y.  R. 
Amberson  and  Dr.  W.  O.  Fenn  for  their  suggestions,  and  to  Mr. 
J.  O.  Pinkston  for  his  assistance  in  the  oxygen  determinations. 

BIBLIOGRAPHY. 

1.  Warburg,  O. 

'08  Beobactungen  iiber  die  Oxydationsprozesse  in  Seeigelei.  Zeits. 
Physiol.  Chem.,  57:  1-16. 

2.  Shearer,  C. 

'22  On  the  Oxidation  Processes  of  the  Echinoderm  Egg  During 
Fertilisation.  Proc.  Roy.  Soc.,  B,  93:  213-229. 

3.  Shearer,  C. 

'22  On  the  Heat  Production  and  Oxidation  Processes  of  the  Echi- 
noderm Egg  during  Fertilisation  and  Early  Development. 
Proc.  Roy.  Soc.,  B,  93:  410-425. 

4.  Loeb,  J.,  and  Wasteneys,  H. 

'12  Die  Oxydationsvorgange  in  befruchteten  und  unbefruchteten 
Seesternei.  Arch.  f.  Entwick-Mechanik.,  35:  555-557- 


IOO  MARJORIE   BOYD. 

5.  Rogers,  C.  G.,  and  Cole,  K.  S. 

'25  Heat  Production  by  the  Eggs  of  Arbacia  punctulata  during  Fer- 
tilization and  Early  Cleavage.  BIOL.  BULL.,  49:  338-353. 

6.  Scott,  C.  G.,  and  Kellicott,  W.  E. 

'17      The  Consumption  of  Oxygen  during  the  Development  of  Fim- 

dulus  heteroclitusi.     Anat.  Record,  n:  531-533. 
/.  Hyman,  L.  H. 

'21  The  Metabolic  Gradients  of  Vertebrate  Embryos.  I.  Teleost 
Embryos.  BIOL.  BULL.,  40:  32-72. 

8.  Amberson,  W.  R.,  Mayerson,  H.  S.,  and  Scott,  W.  J. 

'24  The  Influence  of  Oxygen  Tension  upon  Metabolic  Rate  in  In- 
vertebrates. Jour.  Gen.  Physiol.,  7:  171-176. 

9.  Lund,  E.  J. 

'21  A  Micro-Winkler  Method  for  the  Quantitative  Determination 
of  Dissolved  Oxygen.  Proc.  Soc.  Exp.  Biol.  &  Med.,  19:  63- 
64- 


STUDIES  ON  DALLASIA  FRONTATA  STOKES. 
I.    POLYMORPHISM. 

GARY  N.  CALKINS  AND  RACHEL  BOWLING. 

Dallasia  frontata  is  a  common  fresh-water  ciliate  classified  in 
the  family  Chiliferidse,  sub-order  Trichostomina,  order  Holo- 
trichida.  It  was  originally  described  in  1886  by  Stokes  and  char- 
acterized by  him  in  1888  as  follows :  "  Body  elongate-obvate,  sub- 
cylindrical,  transparent,  longitudinally  striate,  and  finely 
reticulated,  five  times  as  long  as  broad,  the  lower  or  ventral  sur- 
face convex,  the  dorsal  slightly  concave,  tapering  posteriorly  to 
a  somewhat  retractile  tail-like  prolongation  forming  about  one- 
fifth  of  the  entire  body ;  anterior  extremity  narrowed,  obtusely 
pointed ;  oral  aperture  narrow,  ovate,  obliquely  placed  on  the 
ventral  or  convex  surface  at  some  distance  from  the  anterior  ex- 
tremity, enclosing  two  small  vibratile  membranes ;  contractile 
vacuole  single,  spherical,  near  the  center  of  the  dorsal  or  concave 
border ;  nucleus  presumably  represented  by  large,  ovate,  sub-cen- 
tral clear  space.  Length  of  body  1/180  of  an  inch.  Habitat.— 
Still  water,  with  Myriophyllum"  (1888,  p.  17 1). 

This  characterization  is  not  adequate  to  describe  the  many  form 
changes  which  this  remarkable  organism  passes  through  in  its  life 
history,  changes  which  we  are  not  yet  ready  to  interpret  as  to  cause 
or  full  significance,  but  which  we  will  describe  in  the  following 
pages. 

The  organism  may  be  found  without  much  difficulty  in  the  water 
of  Van  Cortlandt  pond  in  the  environs  of  New  York.  Unlike  Uro- 
leptits  inobilis  it  cannot  be  regarded  as  a  rare  form  and  is  probably 
widely  distributed  in  fresh-water  ponds  throughout  the  country. 
Many  individuals  were  encountered  in  the  autumn  of  1927  and 
individuals  were  isolated  in  different  types  of  media  in  isolation 
culture  dishes  usually  employed  for  this  work.  Initial  experi- 
ments with  culture  media  including  pond  water,  hay  infusion,  and 
the  combination  of  hay  and  flour  soon  showed  that  the  latter,  as 

101 


IO2  GARY    N.    CALKINS    AND    RACHEL    BOWLING. 

in  the  case  of  Uroleptus  mobilis,  was  the  most  favorable.  This 
medium,  made  up  in  the  same  way  as  for  Uroleptus  mobilis  during 
tight  years  of  culture,  has  been  consistently  used  for  some  of  our 
material  since  October  6,  1927.  Later,  media  made  up  with  rice 
and  with  cracked  wheat  were  tried  and  some  of  our  material  is 
now  successfully  running  on  the  latter.  In  this,  as  in  the  hay- 
flour  medium,  individuals  in  the  period  of  maximum  vitality  di- 
vide from  four  to  six  times  in  twenty-four  hours. 

The  material  of  the  isolation  cultures  is  run  in  "  series  "  and 
"  lines."  A  series  is  made  up  of  all  the  progeny  of  a  single  indi- 
vidual isolated  as  an  ex-con jugant;  lines,  usually  five  in  number, 
are  isolation  cultures  made  from  the  first  five  individuals  formed 
by  division  of  the  ex-conjugant.  Individuals  from  each  line  are 
picked  up  with  a  capillary  pipette  and  transferred  daily  to  another 
culture  dish  of  fresh  medium.  After  such  isolations  are  made  the 
unused  individuals  of  a  series  are  transferred  to  a  Syracuse  dish 
containing  about  10  cc.  of  fresh  medium.  Such  reserve  material 
is  allowed  to  multiply  with  no  change  of  the  medium  for  from  six 
to  ten  days.  It  constitutes  a  "  conjugation  test  "  such  as  proved 
successful  with  Uroleptus  mobilis.  In  this  way  abundance  of 
material  is  available  for  study.  With  Dallasia  after  a  few  weeks, 
epidemics  of  conjugation  occurred  in  the  Syracuse  dishes  and  pedi- 
greed series  were  started. 

Material  for  preparations  has  been  fixed  in  osmic  fumes,  Flem- 
ming's,  Hermann's  and  Schaudinn's  fluids.  The  latter,  made  up 
as  a  saturated  solution  in  95  per  cent,  alcohol  is  most  satisfactory 
for  general  staining.  Iron  haematoxylin  is  good  for  general 
topography  but  inner  cellular  structures  are  obscured  by  the  dense 
cortical  zone  of  deeply  staining  granules.  This  however,  may 
be  avoided  by  prolonged  treatment  with  turpentine.  Auerbach's 
combination  of  methyl  green  and  acid  fuchsine  (without  orange 
G)  is  excellent  for  cortical  structures  and  for  the  mouth  parts. 
Vital  stains  are  useful  for  demonstrating  some  structures  par- 
ticularly the  capsules  about  the  "  couples." 

The  derived  organization  of  Dallasia  is  so  delicately  adjusted  to 
its  environment  that  small  changes  in  the  latter  cause  remarkable 
changes  in  form.  This  leads  to  polymorphism  which,  more  than 
with  any  other  free-living  protozoon  known  to  us,  is  character- 


STUDIES   ON    DALLAS1A    FROXTATA   STOKES. 


103 


8 


104 


GARY    N.    CALKINS    AND    RACHEL    BOWLING. 


istic  of  this  ciliate.  Certain  well-marked  types  of  organization 
follow  in  the  same  order.  To  these  we  have  applied  purely  col- 
loquial names  which  have  no  resounding  classical  roots  indeed, 
but  which  enable  us  to  distinguish  clearly  between  the  forms  indi- 
cated by  them.  These  forms  are  (i)  tails;  (2)  boats;  (3)  couples 
(gametes)  ;  and  (4)  pairs,  and  they  will  be  described  in  this  order. 

i.  Tails. — This  term  is  an  abbreviation  for  "  tail-bearing  forms  " 
such  as  indicated  by  the  original  description  of  Stokes.  They  are 
relatively  large  (105^  to  140/^X22^  to  36^)  and,  owing  to  the 
remarkable  mouth  have  a  curious  resemblance  to  a  microscopic 
shark  a  resemblance  to  which  Stokes  called  attention.  The  an- 
terior end  is  rounded  and  in  most  cases  this  is  the  broadest  part 
of  the  organism  which  tapers  gradually  to  the  posterior  end  where 
it  narrows  into  a  well-marked  tail  (Fig.  I.,  4  and  Fig.  III.,  i). 
The  tail  is  quite  variable  in  length  and  shape.  Sometimes  it  is 
long,  resembling  the  handle  of  a  skillet;  again  it  is  reduced  until 
it  is  little  more  than  the  sharply-pointed  posterior  end  of  the  cell. 
In  other  cases  the  tail  disappears  entirely.  These  forms  are 
lairly  sluggish,  richly  stored  with  food,  and  are  usually  attached 
to  the  substratum  by  the  tip  of  the  tail  where  they  swing  about 
in  circles  with  the  tips  of  the  tails  as  centers.  The  cilia  are  long 
and  closely  set  in  longitudinal  rows  of  which  there  are  about 
forty. 

Another  type  of  tailed  form  is  much  longer  and  somewhat  more 
slender  but  unlike  the  fat  form  is  very  active  and  rarely  becomes 
attached. 

So  far  as  the  visible  structures  are  concerned  the  most  com- 
plex part  of  the  organism  is  the  mouth.  (Fig.  II.,  i).  This  is 
relatively  large  and  lies  in  the  anterior  quarter  of  the  cell.  The 
external  aperture  varies  in  shape  from  an  elongated  slit  to  a  cir- 
cular opening.  It  leads  into  a  spacious  buccal  pouch  (B.  p.)  which 
extends  inwards  and  diagonally  from  a  region  slightly  anterior  to 
the  mouth,  to  the  gullet  which  is  posterior  to  the  mouth.  The 
entire  apparatus  is  about  27/4,  long  and  15^  wide,  thus  taking  up 
about  one-fifth  of  the  organism.  On  the  floor  of  the  buccal  pouch 
is  a  long  tongue  (T},  triangular  in  cross  section,  which  runs  al- 
most the  full  length  of  the  pouch  (Fig.  II.).  On  one  side  of  this 
and  at  the  anterior  end  is  a  broad  endoral  membrane  which  fre- 


STUDIES   ON   DALLASIA   FRONTATA    STOKES. 


105 


quently  protrudes  from  the  mouth  (Fig.  II.,  E.m.}.  At  the  re- 
gion of  the  gullet  and  on  the  opposite  side  of  the  tongue  is  a 
long,  narrow  undulating  membrane,  the  adoral  membrane  (Fig.  II. 
A.m.}.  From  the  base  of  this  membrane  to  the  gullet  is  a  long 
ladder-like  structure  recalling  the  "  railroad  track  "  of  Chlamy- 
dodon  (A.c).  There  is  evidence  of  a  complicated  neuro-motor 
system  which  will  be  described  in  a  later  paper  on  the  cytology 
of  these  forms. 

The  macronucleus,  like  the  organism,  is  polymorphic.     In  many 
individuals  it  appears  to  be  emarginate,  frayed  out  and  of  a  de- 


E.m 


A.  m. 


B 


FIG.  II.     Mouth  and  buccal  pouch  of  Dallasia  frontata. 

A.  Total  preparation  of  tailed  form ;  mouth  and  buccal  pouch  only. 

B.  Transverse  section  of  tailed  form. 

A.c.,  ladder;  A.m.,  adoral  membrane;  £./>.,  buccal  pouch;  E.m.,  endoral 
membrane ;  M.t  mouth  opening ;   T.,  tongue  and  supporting  bars. 

cidedly  unhealthy  appearance.  It  is  often  splinter-like  and  irregu- 
lar, but  at  the  approach  of  division  it  becomes  more  condensed  and 
homogeneous  and  ellipsoidal  in  form.  It  divides  without  mitosis 
and  in  the  characteristic  manner  of  most  macronuclei. 

The  micronucleus  is  usually  single,  spherical,  and  homogeneous, 
and  is  closely  applied  to  the  macronucleus.  It  divides  by  mitosis. 

The  contractile  vacuole  is  a  single  vesicle,  in  the  middle  of  the 
ventral  surface;  feeding  canals  are  absent  but  a  ring  of  feeding 
vesicles,  clearly  visible  after  contraction  of  the  vacuole,  are  present. 

The  cytoplasm  is  filled  with  great  vacuoles  which  are  frequently 
so  abundant  as  to  distort  the  organism.  They  are  gastric  vacuoles 
for  the  most  part  but  are  frequently  merely  fluid-filled  vesicles. 


IO6  GARY    N.    CALKINS    AND    RACHEL    BOWLING. 

Granules  of  large  size  and  great  number  are  present  in  all  stages 
of  these  tailed  forms  and  are  a  nuisance  in  preparations  stained 
with  iron  haematoxylin.  The  majority  of  them  stain  well  with 
the  vital  dyes  neutral  red,  brilliant  cresyl  blue  and  methylene  blue. 
All  in  all  these  tailed  forms  are  remarkably  variable  in  size  and 
shape.  They  appear  to  be  highly  sensitive  to  environmental 
stimuli  readily  becoming  amorphous  and  variously  distorted.  If 
the  medium  is  too  rich  such  distortions  are  more  numerous.  For 
some  unaccountable  reason,  possibly  because  of  incomplete  reor- 
ganization after  division,  minute  dwarf  forms  with  grotesquely 
large  mouths  are  frequently  seen  (Fig.  III.,  4).  Such  types  are 
prone  to  change  into  distinctly  amoeboid  forms  with  protoplasmic 
processes  which  cannot  be  distinguished  from  pseudopodia 

(Fig.  III.,  4a). 

2.  Boats. — In  form  and  character  boats  are  quite  different  from 
the  tailed  forms.  They  are  considerably  smaller  (68/x  to  83^) 
and  are  derived  from  the  tailed  forms  by  gradual  absorption  of 
the  attenuated  caudal  extremity.  Both  anterior  and  posterior  ends 
become  rounded  and  the  organism  becomes  navicular  and  sym- 
metrical (Fig.  I.,  8;  Fig.  III.,  10).  The  environmental  condi- 
tions under  which  the  transition  occurs  have  not  yet  been  fully 
determined  but  the  period  required  for  it  varies  according  to  the 
age  of  the  series.  It  is  a  striking  phenomenon  to  see  a  rich  stock 
culture  in  fresh  medium  yield  nothing  but  boats  twenty-four  hours 
later.  Such  boats  are  not  attached  but  shoot  about  the  culture  dish 
with  amazing  speed.  After  another  24  hours  the  majority  of 
them  have  divided  four  times,  giving  rise  to  sixteen  minute  cells 
which  separate  off  in  pairs  to  form  the  couples.  After  the  first 
division  of  the  boats  the  daughter  cells  (gamonts  number  i)  are 
more  quiet  than  the  original  boat  and  have  a  tendency  to  rest  on 
the  bottom  where  the  second  division  takes  place.  The  daughter 
cells  of  this  second  division  (gamonts  number  2)  still  have  the 
ability  to  move  but  their  movement  is  more  or  less  spasmodic  and 
irregular  and  their  daughter  cells  (gametocytes)  derived  from 
a  third  division,  are  now  quiescent  (Figs.  I.,  10,  and  III.,  13). 
These  forms,  however,  are  rarely  found  on  the  bottom  but,  to- 
gether with  the  couples,  are  suspended  in  the  medium. 

The  early  stages  of  the  boats  show  mouth  parts  only  slightly 


STUDIES   ON   DALLASIA    FRONTATA   STOKES. 


107 


FIG.  III.     Dallasia  frontata  Stokes.     Life  cycle.     Camera  lucida  sketches 
from  preparations.     Same  magnification  throughout. 

1.  Vegetative  individual  from  isolation  culture. 

2.  Boat-shaped  individual  before  tail  is  formed  which  may  originate  at  any 

time  from  the  anterior  end  of  dividing  tailed  form. 

3.  Early  stage  of  division  of  tailed  form. 

4.  Degeneration  type  of   tailed   form  which   may  give  rise  to  an  amoeboid 

form  40. 

5.  6.  Later  stages  of  division  of  tailed  form. 

7.  Conjugation. 

8,  9.  Ex-conjugants  which  reorganize  into  tailed  forms. 

10.  Typical  boat-shaped  form  characteristic  of  mass  cultures  after  two  to 

three  days. 

11.  First  binary  division  of  boat. 

12.  Second  division  of  boat. 

13.  Third  division  of  boat  giving  eight  products. 

14.  15.  Fourth  division  of  boat  and  fusion  of  psedogamous  gametes  in  cap- 

sules to  form  eight  zygotes. 

16.  Development  of   zygote   into  young  individual. 

17.  Intermediate  young  individuals  from  couples. 


IO8  GARY    N.    CALKINS    AND    RACHEL    BOWLING. 

different  from  'those  of  the  tailed  forms  but  these  parts  are  slowly 
absorbed  and  in  the  second  generation  of  the  boats  they  have  en- 
tirely disappeared.  The  macronucleus  of  the  boat  form  is  more 
condensed,  more  definite  in  form,  and  stains  more  readily  than  in 
the  tailed  form,  and  this  intensity  of  staining  is  retained 
throughout  all  of  the  later  stages.  The  protoplasm  likewise  is 
denser  and  has  lost  its  included  vacuoles  but  there  are  still  many 
granules  which  are  partitioned  out  at  each  division  with  apparently 
no  increase  in  their  total  number.  The  contractile  vacuole  varies 
considerably  in  position,  sometimes  on  the  ventral  surface,  some- 
times on  the  dorsal  and  frequently  nearer  one  pole  than  the  other. 
In  the  third  and  fourth  divisions  of  the  boats  the  onset  of  di- 
vision is  always  indicated  by  the  presence  of  two  vacuoles  sym- 
metrically placed  in  the  cell. 

The  first  two  divisions  are  fairly  slow,  requiring  several  hours 
but  the  last  two  divisions  follow  one  another  in  quick  succession. 

Boat-shaped  forms  may  appear  at  any  stage  and  appear  to  be  a 
palingenetic  phase  of  the  organism.  Thus  in  ordinary  division 
of  the  tailed  form  the  anterior  half  is  navicular  until  the  tail  is 
regenerated.  Also  just  as  pathological  tailed  forms  turn  into 
amoeboid  cells  so  the  boat-like  individuals  may  undergo  a  similar 
pathological  change.  In  some  cultures  the  entire  population  ap- 
parently becomes  thus  transformed  into  amoebae. 

3.  Couples. — The  first  two  divisions  of  a  "  boat"  lead  to  small 
individuals  (2 I/A  to  25^)  of  broadly  ellipsoidal  form  and  with 
relatively  large  nuclei  (Fig.  III.,  12).  The  daughter  cells  formed 
by  the  second  division  still  have  the  power  to  move  and  usually 
become  widely  separated.  Each  divides  into  two  and  these  two 
quickly  give  rise  to  four.  Chains  of  four  cells  are  characteristic 
and  as  there  is  a  tendency  at  this  period  for  the  boats  and  their 
products  to  agglomerate,  great  masses  of  these  chains  are  fre- 
quently found  in  the  Syracuse  dishes.  The  four  cells  of  a  chain 
soon  become  associated  as  two  pairs  and  these  are  the  "  couples  " 
(functionally  gametes)  of  our  terminology.  These  pairs  measure 
from  22/x,  to  26/A,  each  individual,  from  n/*  to  13/x.  About  each 
couple  is  a  delicate  capsular  membrane  resembling  a  fertilization 
membrane,  but  there  is  as  yet  no  fertilization,  hence  the  resemb- 
lance is  closer  to  a  sporocyst  membrane  of  two  gregarines  in 
pseudo-conjugation  (Fig.  I.,  13;  Fig.  III.,  14). 


STUDIES   ON   DALLASIA   FRONTATA    STOKES.  109 

We  have  repeatedly  watched  the  process  of  couple  formation  in 
the  living  cells  and  the  further  changes  which  take  place  within 
the  capsule.  An  instructive  picture  is  obtained  by  use  of  neutral 
red  which  stains  some  of  the  endoplasmic  granules  and  these  fur- 
nish points  of  orientation.  The  two  cells  of  a  couple  fuse  to  form 
a  zygote  (Figs.  I.,  13,  and  III.,  15).  The  nuclei  also  fuse.  We 
have  watched  this  fusion  in  living  couples  under  an  immersion 
lens  and  have  noted  a  center  in  each  gamete  where  brownian  move- 
ment of  granules  is  evident.  Stained  preparations  show  that 
these  centers  are  nuclei.  After  fusion  of  the  cell  bodies  these 
centers  approach  and  melt  into  one  immediately  after  which  there 
is  a  more  violent  brownian  movement  of  the  granules. 

These  activities  show  that  the  boats  are  gamonts  which  give 
rise  to  gametocytes  and  the  latter  to  gametes  of  which  there  are 
sixteen  from  each  gamont.  Fertilization  is  strictly  paedogamous 
and  nothing  like  it  has  been  described  for  any  type  of  ciliate. 
The  nearest  approach  to  it  is  Brumpt's  account  of  encystment  and 
fusion  in  Balantidium  coli,  but  here  two  gamonts  come  together, 
no  gametes  are  formed  and  the  two  individuals,  as  hologametes, 
fuse  within  a  membrane  analogous  to  the  sporocyst  membrane  of 
gregarines. 

This  period  of  copulation  is  a  critical  one  in  the  history  of 
DaUasia.  Up  to  the  present  time  we  have  not  succeeded  in  rear- 
ing a  single  zygote  in  isolation  culture.  Many  young  forms  are 
found  in  the  Syracuse  dishes  in  which  an  epidemic  of  copulations 
has  occurred  (Fig.  I.,  16)  ;  some  of  these  are  not  yet  provided 
with  mouths  and  their  development  into  mouth-bearing  forms 
has  been  repeatedly  observed  (Fig.  I.,  15).  The  origin  of  these 
young  forms  from  the  stage  of  the  encapsulated  zygote  has  also 
been  observed  but  we  have  not  yet  succeeded  in  providing  a  suit- 
able environment  for  their  continued  life  in  isolation  culture.  In 
many  cases,  but  not  in  all,  the  zygote  apparently  encysts  within 
the  capsule  (Fig.  III.,  I5(/)  and  such  cysts  are  liberated  by  the 
dissolution  of  the  capsular  membrane.  The  further  fate  of  these 
cysts  is  unknown. 

4.  Pairs  or  Conjugants. — We  have  cultivated  Dallasia  in  isola- 
tion cultures  for  four  months  and  now  have  eight  series  of  dif- 
ferent ages  under  observation  each  series  derived  from  an  indi- 


HO  GARY    X.    CALKINS    AND    RACHEL    BOWLING. 

viclual  ex-con jugant.  Paedogamous  copulation,  described  above, 
occurs  in  conjugation  tests  made  within  a  week  of  the  first  divis- 
ion of  an  ex-conjugant  and  epidemics  of  such  unions  still  occur 
at  intervals  in  our  oldest  series.  They  occur  less  frequently  and 
in  much  milder  form  when  the  individuals  of  a  series  are  mature 
for  conjugation. 

Conjugation  epidemics  are  rare.  Tests  have  been  made  daily  by 
placing  the  reserve  individuals  left  over  after  the  usual  isolations 
are  made,  in  a  Syracuse  dish  with  about  10  cc.  of  fresh  medium. 
These  dishes  are  set  aside  in  a  moist  chamber  and  left  for  at  least 
one  week  and  usually  without  the  addition  of  fresh  medium. 
They  are  examined  daily  and  the  observations  recorded.  In  the 
early  life  of  a  series  boats  usually  appear  within  two  or  three  days 
and  the  boats  usually  give  rise  to  couples.  If,  however,  such 
boats  are  transferred  to  fresh  medium  they  change  again  into  tails. 
Sometimes  fully  100  per  cent,  of  the  original  tailed  forms  change 
into  boats  and  these  into  couples  and  zygotes  but  as  a  series  grows 
older  there  is  an  increasing  percentage  of  tailed  forms  which  do 
not  become  transformed  into  boats  and  an  increasingly  diminish- 
ing number  of  couples.  In  Syracuse  dishes  with  material  from 
older  series  there  is  thus  a  predominance  of  tailed  forms  at  all 
stages.  These  are  somewhat  smaller  (Fig.  I.,  6)  than  are  the 
individuals  of  the  isolation  cultures  and  they  show  the  same  type 
of  agglomeration  as  does  Uroleptus  mob  His  in  similar  conjugation 
tests.  As  with  Uroleptus  such  agglomerations  are  usually  although 
not  invariably,  followed  by  conjugation  of  the  individuals. 

The  first  epidemic  of  conjugations  occurred  after  thirty-five 
days  of  culture  of  a  wild  individual  and  gave  us  material  for 
Series  2  and  2a  of  our  pedigreed  races.  In  one  of  these  (2a)  a 
mild  epidemic  occurred  in  the  96th  generation  or  25  days  after  the 
first  division  of  the  original  ex-conjugant,  and  Series  3  and  4 
were  derived  from  it.  A  second  epidemic  occurred  in  the  i6oth 
generation  or  47  days  after  the  first  division  of  the  ex-conjugant 
and  from  this  epidemic  Series  5  and  6  were  started.  Three  other 
epidemics  have  appeared  in  Series  3  and  4  and  have  furnished  ma- 
terial for  Series  7  and  8. 

The  conjugating  individuals  are  relatively  small  (77/x.  to  iO2ju.) 
and  are  always  tailed  forms.  Union  occurs  as  in  Uroleptus  or 


STUDIES   ON   DALLASIA   FRONTATA    STOKES.  Ill 

Spathidhiui,  etc.,  at  the  anterior  ends  and,  again  as  in  Uroleptus 
the  mouth  parts  are  not  involved.  The  mouths,  however,  are 
greatly  reduced  and  apparently  are  absorbed,  new  ones  being 
formed  by  the  ex-conjugants.  The  period  of  actual  fusion  varies 
from  twelve  to  twenty-four  hours  and  the  period  of  reorganiza- 
tion of  the  ex-conjugant  varies  from  one  to  four  days.  The  cyto- 
logical  details  have  not  yet  been  worked  out  but  meiotic  divisions, 
interchange  and  fusion  of  nuclei  appear  to  follow  the  customary 
history. 

The  average  division  rate  for  the  initial  lo-day  period  is  high 
and  is  higher  in  most  cases  than  the  division  rate  for  the  same 
calendar  period  of  the  parent  series.  As  with  rrolcptus,  however, 
this  is  not  invariable  as  the  following  table  shows : 

Series  20  division  rate  ist.  10  days,  38.6.     Parent  ser  es  same  per  od  19.2 


Series  3  36.8. 

Series  4  40.8. 

Series  5  29.6. 

Series  6  "       "      "       "      33.4. 


40.2 
40.2 
24.4 
25.2 


It  is  too  early  to  draw  any  conclusions  as  regards  vitality  before 
and  after  conjugation,  this  subject  will  be  discussed  in  a  later 
study. 

DISCUSSION. 

So  far  as  we  are  aware  Dallasia  frontata  presents  a  unique  phe-. 
nomenon  hitherto  undescribed  for  the  ciliated  protozoa.  This  is 
the  interpolation  of  a  paedogamous  fertilization  stage  in  the  other- 
wise ordinary  cycle  from  ex-conjugant  to  conjugant.  Two  dis- 
tinct and  entirely  different  fertilization  phenomena  in  the  same 
life  cycle  certainly  furnish  food  for  reflection,  particularly  as 
regards  the  significance  of  fertilization  in  general.  The  nearest 
parallel  case  that  we  know  is  Cryptochilum  echini,  as  described  by 
Russo.  The  high  death  rate,  in  cultures,  after  copulation  may  be 
significant.  It  may  mean  that  the  culture  medium  is  not  suitable 
for  this  stage  of  the  organism  or  it  may  mean  that  the  encapsulated 
stage  is  taken  into  some  other  organism  where  part  of  the  life  his- 
tory is  spent  as  a  parasite  or  as  a  commensal.  Further  study  of 
the  organisms  in  culture  with  experiments  to  test  the  effect  of  dif- 
ferent media,  which  are  now  under  way,  may  throw  more  light  on 
this  problem. 

The  novelty  of  Dallasia  does  not  lie  in  the  copulation  of  micro- 


H2  GARY    N.    CALKINS    AND    RACHEL    BOWLING. 

gametes;  this  phenomenon  is  known  in  the  Opalinidae.  Nor  does 
it  lie  in  the  union  of  paedogamous  gametes  as  this  phenomenon 
is  well  established  in  the  case  of  Actinophrys  sol  and  in  the  case 
of  Actinosphaerium  eichhornii.  There  is  certainly  no  novelty  in 
the  phenomenon  of  conjugation  of  Dattasia  for  in  this  it  agrees 
with  the  vast  majority  of  ciliates.  The  novelty  lies  in  the  combi- 
nation of  fertilization  by  copulation  and  fertilization  by  conjuga- 
tion. 

It  is  well  known  through  isolation  culture  work  with  infusoria 
that  a  reorganization  process  without  union  of  individuals  occurs 
and  has  the  same  effect  on  vitality  as  does  conjugation,  it  is  a 
process  of  parthenogenesis  termed  endomixis  by  Woodruff  and 
Erdmann  (1914)  ;  and  in  some  form  or  other  it  occurs  in  prac- 
tically every  ciliate  that  has  been  studied.  It  takes  place  prior  to 
and  during  the  early  phases  of  encystment  in  the  Hypotrichida, 
without  encystment  in  various  species  of  Paramecium.  In  U ro- 
le ptus  mobilis  endomixis  with  encystment  is  a  characteristic  phe- 
nomenon of  the  early  stages  of  the  life  cycle  (Calkins,  1926)  ;  it 
becomes  infrequent  with  maturity  of  the  protoplasm  and  is  absent 
altogether  in  the  later  stages.  In  Dallasia  front  at  a  the  incidence 
of  couple  formation  in  the  early  stages  of  the  life  cycle,  the  for- 
mation of  capsules,  together  with  the  absence  of  any  evidence  up 
to  the  present,  of  the  ordinary  forms  of  endomixis,  lead  us  to  the 
conclusion  that  we  have  here  a  very  unusual,  perhaps  primitive, 
type  of  endomixis.  If  this  conclusion  is  correct  the  further  hypoth- 
esis is  permissible  that  endomixis  as  ordinarily  observed  in  ciliates 
is  a  reminiscence  of  ancestral  gamete-brood  formation. 

REFERENCES. 
Calkins,  G.  N. 

'26    The  Biology  of  the  Protozoa. 
Russo,  A. 

'26    Gli  exconiuganti,  derivati  dalla  ia  coniugatione  accessoria  fra  Gameti 
impuri    in    "  Cryptochilum    echini,"    producona    Gametogeni    puri    e 
Gameti  puri,  che  rinnovanno  il  ciclo  principale.     Rend.  Accad.  dei 
Lincei.,  Ser.  6,  Vol.  3. 
Stokes,  A.  C. 

'88     A   preliminary    Contribution   toward   a   History   of   the   Fresh-water 
Infusoria  of   the   United   States.     Jour.    Trenton   Natural   History 
Society,  Vol.  i,  No.  3,  Jan.,  1888. 
Woodruff,  L.  L.  and  Erdmann,  R. 

'14     A   Normal   Periodic   Reorganization   Process   without   Cell-fusion   in 
Paramecium.    Jour.  Exp.  Zool.,  XVII. 


THE  BACTERIOLOGICAL  STERILIZATION  OF 

PARAMECIUM. 

ARTHUR  K.  PARPART. 

(From  the  Biological  Laboratory,  Amherst  College,  Amherst,  Mass.) 

I. 

If  for  one  reason  or  another  it  is  necessary  to  control  the  bac- 
terial content  of  the  medium  in  which  Paramecium  is  living,  the 
first  step  is  a  reliable  method  for  the  bacteriological  sterilization 
of  the  animals. 

Hargitt  and  Fray  ('17)  devised  a  method  which  they  believed 
accomplished  this  end.  Their  procedure,  in  brief,  consisted  in 
transferring  a  single  animal,  by  means  of  sterile  pipettes,  through 
five  successive  washings  of  sterile  water  contained  in  sterile  de- 
pression slides,  the  latter  being  enclosed  in  Petri  plates.  There  is 
no  evidence  in  their  paper  as  to  how  many  animals  were  treated 
in  this  way  to  determine  the  efficiency  of  the  method.  According 
to  Philipps  ('22)  the  technique  of  Hargitt  and  Fray  "  is  undoubt- 
edly reliable."  However,  she  used  a  procedure  in  her  experiments 
which  "  made  it  necessary  to  wash  each  animal  seven  times  instead 
of  five." 

II. 

In  his  first  attempts  to  sterilize  Paramccia  the  present  writer 
increased  the  number  of  washings  to  ten.  The  animals  were  ob- 
tained from  a  pedigreed  culture  of  Paramecium  caudal  urn,  grow- 
ing on  a  0.7  per  cent,  infusion  of  pure  timothy  hay  in  tap  water. 
This  same  solution  was  used  for  washing.  The  solution  was  ster- 
ilized in  an  autoclave  at  12  to  15  pounds  pressure  for  45  minutes.1 

As  a  first  step  eight  animals  were  washed  ten  times,  with  the 
purpose  of  determining,  first,  the  diminution  in  the  number  of 

1  The  difficulty,  experienced  by  Hargitt  and  Fray,  of  getting  Pannnccia 
to  live  on  media  sterilized  in  an  autoclave  under  high  pressure,  has  never 
been  encountered  in  these  experiments  though  pure  lines  of  Paramccia  have 
been  carried  for  a  number  of  months  on  hay  infusions  and  beef  extracts 
treated  in  this  way. 


ARTHUR    K.    PARPART. 


bacteria  that  occurred  during  the  ist,  3d,  5th,  and  7th  washes; 
second,  the  number  of  animals  sterile  in  the  loth  wash.  To  ac- 
complish the  first  purpose,  the  ist,  3d,  5th,  and  7th  wash  fluids 
were  plated,  these  plates  incubated  at  37.5  degrees  C.  for  72  hours 
and  examined.  For  the  second  purpose  the  loth  wash  fluid  to- 
gether with  the  animal  was  broth  cultured,  and  the  cultures  treated 
in  the  same  way  as  the  plates.  The  results  are  recorded  in  Table  I. 

TABLE  I. 

REDUCTION  IN  NUMBER  OF  BACTERIA  IN  WASHES,   i,  3,  5,  AND  7. 
EFFICACY  OF  10  SUCCESSIVE  WASHINGS. 


Animal 

Number  of  Colonies  on  Plates  of  wash  Fluid  No. 

Broth  Culture  of 
Wash  No. 

No. 

10  +  Animal. 

i 

3 

5 

7 

i 

9,000 

I 

0 

o 

Infected 

2 

6,000 

3 

o 

o 

Infected 

3 

10,000 

o 

o 

o 

Sterile 

4 

9,000 

2 

o 

o 

Infected 

5 

13,000 

0 

o 

0 

Sterile 

6 

9,000 

O 

o 

0 

Infected 

7 

16,000 

2 

o 

o 

Infected 

8 

16,000 

O 

o 

o 

Infected 

The  diminution  of  the  number  of  bacteria  in  successive  washes 
up  through  the  5th,  as  brought  out  in  the  above  table,  concurs 
very  well  with  the  results  obtained  by  Hargitt  and  Fray.  The 
discrepancy  between  the  number  of  bacteria  present  in  their  first 
wash  and  the  infection  I  found  may  be  accounted  for  by  the  fact 
that  they  transferred  only  a  small  portion  of  these  washes  to  agar 
plates,  while  I  transferred  the  entire  amount. 

Superficially  the  fact  that  no  animals  contaminated  the  5th  wash 
might  be  taken  to  indicate  their  sterility.  However,  80  per  cent, 
do  contaminate  the  loth  wash.  These,  of  course,  could  not  have 
been  sterile  at  the  time  of  the  5th  washing. 

To  further  test  this  particular  point  18  animals  were  washed 
10  times,  and  the  loth  wash  fluid  together  with  the  animal  broth 
cultured.  The  latter  was  incubated  at  37.5  degrees  C.  for  72  hours 
and  examined.  The  results  are  tabulated  in  Table  II. 

Of  the  26  animals  included  in  these  tables  only  5  were  sterile 
in  the  loth  wash. 


BACTERIOLOGICAL    STERILIZATION    OF    I'AKA  M  ECIUM . 

TABLE  II. 
EFFICACY  OF  10  SUCCESSIVE  WASHINGS. 


Total  Number  of 
Animals  Tested. 

Broth  Cultures  of  the  lotli  Wash  -Fluids  +  the  animals. 

Sterile. 

Infected. 

18 

3 

16 

III. 

The  Hargitt  and  Fray  sterilization  method  differs  from  the 
above  method  in  that  they  employed  a  sterile  tap  water  solution 
for  the  washing  and  passed  the  animals  through  only  5  wash 
fluids.  Conceivably  sterile  tap  water  might  be  a  better  sterilizing 
agent.  At  any  rate,  it  speeds  up  animals  put  into  it  and  leads  to 
rapid  reversals  which  possibly  enable  the  Paramccia  to  throw  off 
more  readily  the  bacteria  lodged  between  their  cilia. 

Accordingly,  30  animals  were  washed  5  times  in  sterile  tap 
water.  Broth  cultures  of  the  3d  wash  fluid  and  the  5th  together 
with  the  animal  were  incubated  at  37.5  degrees  C.  for  72  hours. 
The  results  are  tabulated  in  Table  III. 


TABLE  III. 
EFFICACY  OF  5  SUCCESSIVE  WASHINGS. 


Total  Number 
of 
Animals  Tested. 

Broth  Cultures  of  the 
3d  Wash  Fluids. 

Broth  Cultures  of  the  sth  \Yasii 
Fluids  +  the  Animals. 

Sterile. 

Infected. 

Sterile. 

Infected. 

30 

28 

2 

3 

27 

Only  one  conclusion  is  possible.  In  the  majority  of  cases  5  and 
even  10  washings  in  sterile  media  cannot  be  relied  .upon  to  sterili/e 
a  Paramccinui. 

IV. 

As  washing  is  the  only  practical  method  for  ridding  Parameda 
of  bacteria,  the  following  technique  was  devised. 

i.  The  washings  were  performed  in  depression  slides,  each 
slide  being  enclosed  in  a  Petri  plate.  Those  plates  in  which 
Paramccia  were  cultured  had  a  thin  glass  slide  under  the  de- 


Il6  ARTHUR    K.    PARPART. 

pression  slide,  so  that  the  sterile  water  poured  into  the  plates  to 
make  them  serve  as  moist  chambers  could  not  get  into  the  de- 
pression. 

2.  The  pipettes  for  transferring  the  animals  through  successive 
washes  were  made  from  soft  glass  tubing  having  an  inner  diam- 
eter of  2  mm.  and  a  wall  of  I  mm.  thickness,  by  drawing  this  out 
to  capillary  fineness.     Of  the  60  pipettes  made  in  this  manner, 
10  were  chosen  at  random  and  the  inner  diameter  measured  at 
the  tip  of  the  capillary  portion.     The  average  inner  diameter  was 
213  micra;  none  varied  more  than  30  micra  from  this  average. 
The  large  end  was  plugged  with  cotton,  and  each  pipette  plugged 
into  a  separate  test  tube. 

3.  The  sterilization  of  the  pipettes  and  of  the  depression  slides 
in  Petri  plates  was  carried  out  in  a  dry  oven  at  between  160  and 
170  degrees  C.  for  45  minutes. 

4.  The  actual  washing  of  the  Paramecia  was  performed  under 
a  hood  which  was  placed  at  one  end  of  a  large  table,  and  con- 
sisted of  a  wooden  frame  (3  ft.  by  15  in.  by  n  in.),  with  a  glass 
top  and  cloth  sides.     The  front  cloth,  which  served  as  entrance, 
was  loose  at  the  bottom.     Toward  one  end  there  was  a  binocular 
microscope  with  sufficient  focal  length  so  that  its  oculars  extended 
through  and  above  the  top.     Cloth,  with  slits  in  it  for  the  oculars, 
was  glued  to  the  edges  of  the  glass  surrounding  the  oculars. 

In  handling  the  animals  the  transfer  pipettes  were  attached  to 
a  rubber  tube  plugged  with  cotton  and  operated  by  means  of 
mouth  suction  while  the  operator  was  looking  through  the  oculars. 

5.  All  of  the  various  types  of  culture  and  wash  media  used 
were  put  into  separate,  one-liter  flasks  fitted  with  glass   siphon 
tubes.    Rubber  tubing  with  a  glass  pipette  at  one  end  led  off  from 
each  siphon  tube.     After  the  flasks,  tubing  and  pipettes   (the  lat- 
ter plugged  into  small  test  tubes)  had  been  sterilized  in  an  auto- 
clave at  12  to  15  pounds  pressure  for  45  minutes  and  the  corks 
surrounding  the  siphon  tubes  sealed  with  paraffin,  they  were  ar- 
ranged outside  of  the  hood  and  the  rubber  tubing  and  pipette  led 
through  the  back  into  the  hood.     The  pipettes  were  suspended 
at  the  back  of  the  hood  in  such  a  way  that  their  tips,  after  the 
test  tubes  had  been  removed,  did  not  touch  anything.     By  use  of 
carefully  adjusted  pinch  clamps  the  size  of  the  drops  flowing  from 


BACTERIOLOGICAL   STERILIZATION    OF    PARAMECIUM.  117 

these  pipettes  was  regulated,  and  hence  the  volume  of  media 
could  be  determined.  The  fact  that  none  of  the  culture  media 
thus  treated  became  infected,  although  the  pipettes  were  exposed 
continually  for  a  number  of  months  to  the  air  of  the  hood,  is  very 
good  evidence  of  the  efficiency  of  the  hood. 

6.  The  wash  fluid  was  prepared  by  placing  250  ing.  of  Liebig's 
beef  into  200  cc.  of  sterile  tap  water.     This  solution  was  bacter- 
ized  from  the  cultures  of  the  pedigreed  series  of  P.  caudatnin 
being  cultured  on  0.25  per  cent,  beef  extract,  incubated  at  37.5 
degrees  for  2  days  and  diluted  up  to  I  liter  with  tap  water.     It 
was  placed  in  one  of  the  liter  flasks,  sterilized  and  arranged  for 
use. 

7.  The  actual  steps  in  the  washing  of  an  animal  were : 

(a)  Three  piles  of  5  Petri  plates  each  were  placed  under  the 
hood,  and  6  drops  (about  ^  cc.)  of  wash  fluid  was  put  into  each 
of  the  enclosed  depression   slides.     The  lowermost   Petri   plates 
served  as  moist  chambers  for  the  5th  wash  and  hence  contained 
slides  under  the  depression  slides. 

(b)  The   15  pipettes  necessary  for  the  transfers  were  placed 
under  the  hood. 

(c)  The  culture  containing  the  Paramecia  was  placed  on  the 
microscope  stand  and  a  single  individual  transferred  to  the  upper- 
most slide  in  each*  stack  of  plates.     Each  animal  was  transferred 
successively  to  the  depression  slide  in  the  Petri  plate  immediately 
beneath.     By  working  in  rotation  from  stack  to  stack,  the  ani- 
mals remained  in  each  wash  about  one  minute. 

(d)  When  all  three  animals  were  in  the  5th  wash,  from  3  to 
4  cc.  of  sterile  distilled  water  was  added  to  the  lowermost  plates. 
This  prevented  excess  evaporation  from  the  depression  slide  while 
the  5th  wash  fluid  and  the  animals  were  being  incubated  for  5 
hours  at  25  degrees  C. 

(0)  At  the  end  of  5  hours,  each  animal  was  again  transferred 
through  4  washes. 

(/)  From  the  last  of  these,  the  Qth,  the  animal  was  trans- 
ferred to  the  desired  culture  media.  The  Petri  plate  of  this,  the 
loth  wash,  was  converted  into  a  moist  chamber  as  above  (d). 


ARTHUR    K.    PARPART. 


V. 

The  data  demonstrating  the  efficiency  of  this  method  are  sum- 
marized in  Table  IV.  In  this  summary  are  included  the  data  on 
those  animals  which  were  placed  on  some  type  of  sterile  medium 
after  the  9th  wash.  The  data  were  obtained  by  broth  culturing 
the  5th  wash  after  the  animal  had  been  in  it  5  hours,  and  the  loth 
wash  together  with  the  animal  after  the  latter  had  died  in  it.  The 
death,  in  some  cases,  came  only  after  a  number  of  days,  during 
which  time  the  loth  wash  and  the  animal  were  incubated  at  25 
degrees  C.  and  examined  every  24  hours  until  the  death  of  the 
animal.  The  broth  cultures  were  incubated  at  37.5  degrees  C. 
for  72  hours  before  being  examined. 

TABLE  IV. 

SUCCESS  OF  10  WASHINGS;    THE  ANIMAL  REMAINING  IN  THE  sth  WASH 

FIVE  HOURS. 


Total  Number 
of 
Animals  Tested. 

Broth  Cultures  of  the 
5th  Wash  Fluids. 

Broth  Cultures  of  the  loth  Wash 
Fluids  +  the  Animals. 

Sterile. 

Infected. 

Sterile. 

Infected. 

50 

17 

33 

50 

o 

The  number  of  animals  tested  and  the  fact  that  all  were  sterile 
in  the  loth  wash  shows  conclusively  that  the  method  adopted  will 
rid  Paramccia  of  bacteria. 

The  length  of  time  that  an  animal  is  allowed  to  remain  in  the 
5th  wash  fluid  is  a  significant  factor  in  accomplishing  the  steri- 
lization. It  raises  the  question  as  to  why  many  animals  shed 
bacteria  into  the  5th  wash  fluid,  but  a  few  do  not. 

VI. 

Tables  III.  and  IV.  appear  to  demonstrate  that  Paramecium 
caudatuui  defecates  bacterial  spores.  According  to  Table  III., 
over  93  per  cent,  of  the  Paramccia  were  no  longer  shedding 
bacteria  into  the  3d  wash  fluid,  yet  over  90  per  cent,  of  these 
later  proved  infected.  Table  IV.  shows  that  66  per  cent,  of  the 
animals  left  in  the  5th  wash  for  5  hours  shed  bacteria,  yet  when 
these  animals  were  washed  four  times  more  they  proved  sterile. 


BACTERIOLOGICAL   STERILIZATION    OF   PARAMECIUM. 


119 


If  the  majority  of  Paramecia  can  be  passed  through  a  3d  wash 
fluid  without  shedding  any  more  bacteria,  and  then  later  do  so,  it 
seems  highly  improbable  that  the  bacteria  are  on  the  outside  of 
the  animals. 

To  test  this  point  the  following  experiments  were  performed. 
Seventeen  bacteriologically  sterile  Paramecia  were  left  for  24 
hours  in  a  pure  culture  of  Bacillus  prodigiosus  in  beef  extract, 
while  21  sterile  animals  were  left  for  the  same  length  of  time  in 
a  pure  culture  of  Bacillus  subtilis  in  beef  extract.  At  the  end  of 
this  time  the  animals  were  washed,  individually,  ten  times ;  the 
time  of  the  5th  wash  fluid  being  varied.  Those  fed  on  B.  pro- 
digiosus were  allowed  to  remain  in  the  5th  wash  for  ^2  hour. 
Those  fed  on  B.  subtilis  were  left  in  the  5th  wash  from  2  to  5 
hours.  About  24  hours  after  the  animals  had  been  put  into  the 
loth  wash,  this  together  with  the  animal  was  transferred  to  a 
broth  culture,  incubated  at  37.5  degrees  C.  for  72  hours  and  ex- 
amined. The  results  are  tabulated  in  Table  V. 


TABLE  V. 
EXCRETION  OF  SPORES  BY  Paramecia  FED  ON  B.  subtilis. 


Brotli  Cultures  of  the  loth 

Total  Number 

Bacterial  Culture 

Time  Each 

Wash  Fluids  +  the  Animals. 

of 

on  Which  Animal 

Animal  Spent 

Animals  Tested. 

Was  Placed. 

in  the  5th  Wash. 

Sterile. 

Infected. 

17 

B.  prodigiosus 

}/2  hour 

17 

O 

4 

B,  subtilis 

i  hour 

i 

3 

7 

B.  subtilis 

2  hours 

I 

6 

4 

B.  subtilis 

3  hours 

I 

3 

6 

B.  subtilis 

5  hours 

6 

o 

Bacillus  prodigiosus  has  never  been  known  to  produce  endo- 
spores,  while  Bacillus  subtilis  produces  endospores  very  readily. 

In  every  case  the  Paramecia  that  had  been  cultured  in  B.  pro- 
digiosus were  able  to  throw  off  all  bacteria,  although  the  animals 
remained  in  the  5th  wash  fluid  only  l/2  hour.  Eighty  per  cent,  of 
the  Paramecia  cultured  on  B.  subtilis  and  then  left  in  the  5th 
wash  fluid  up  to  3  hours  were  not  sterile  when  washed  five  times 
more. 

Since  both  of  these  species  of  bacteria  have  the  same  type  of 

9 


I2O  ARTHUR    K.    PARPART. 

flagellae,  it  seems  very  unlikely  that  B.  sub  tills  could  remain 
lodged  between  the  cilia  of  the  Paramecia  for  a  longer  period  of 
time  than  B.  prodigiosus. 

The  above  data  seem  to  clearly  indicate  that  Paramecium  def- 
ecates solid  material,  in  this  case  bacterial  spores. 

The  efficiency  of  the  sterilization  technique  adopted  is  further 
attested  by  the  data  in  Table  V.  Those  animals  washed  after 
having  been  cultured  in  B.  subtilis,  and  left  in  the  5th  wash  for 
5  hours,  were  all  sterile  in  the  loth  wash. 

The  author  wishes  to  thank  Professor  Otto  C.  Glaser  for  aid 
rendered  in  the  preparation  of  this  manuscript. 

LITERATURE  CITED. 

Hargitt,  G.  T.,  and  Fray,  W.  W. 
'17     Paramecium  in   Pure  Cultures  of   Bacteria.     Jour.   Exp.  Zool.,  Vol. 

22,  pp.  421-454. 
Phillips,  R.  L. 

'22     The  Growth  of  Paramecium  in  Infusions  of  Known  Bacterial  Con- 
tent.   Jour.  Exp.  Zool.,  Vol.  36,  pp.  135-183. 


THE  EFFECT  OF  MATERNAL  AGE  AND  OF  TEMPERA- 
TURE CHANGE  IN  SECONDARY  NON- 
DISJUNCTION. 

R.  R.  HUESTIS, 
UNIVERSITY  OF  OREGON. 

INTRODUCTION. 

The  following  figures  present  the  results  of  the  raising  of  XXY 
Drosophila  females  in  an  effort  to  ascertain,  particularly,  how  in- 
creasing maternal  age  and  how  differences  in  the  temperature,  at 
which  mother  flies  are  maintained,  affect  the  percentage  of  excep- 
tions to  sex  linkage. 

The  first  part  of  the  paper  deals  with  the  effect  of  maternal  age 
and  here,  because  of  some  lack  of  conformity  in  the  results  ob- 
tained at  different  times,  the  problem  has  not  been  solved.  How- 
ever, I  feel  that  the  figures  should  be  published  for  they  have  been 
accumulating  for  three  years  and  I  am  not,  at  present,  continuing 
the  investigation.  The  second  part  of  the  paper  deals  with  the 
effect  of  differences  in  temperature.  Here  the  different  tests 
which  were  made  check  in  a  fairly  satisfactory  manner.  I  am 
also  able  to  give  some  figures,  in  the  last  part  of  the  paper,  which 
have  a  bearing  upon  the  genetic  variation  in  exception-producing 
ability. 

EFFECT  OF  MATERNAL  AGE. 

The  first  experiment  was  carried  out  during  the  time  I  was  a 
graduate  student  in  Genetics  at  the  University  of  California,  the 
use  of  successive  subcultures  being  a  routine  method  of  rearing 
flies  there  and  fermented  banana  the  food  medium.  I  undertook, 
under  the  direction  of  the  genetic  staff,  a  rather  complete  repeti- 
tion of  Bridges'  1916  experiments  and  obtained  comparable  re- 
sults in  most  particulars.  However,  in  a  group  of  females  con- 
tinuously outcrossed  and  presumably  producing  a  "  normal " 
percentage  of  exceptions,  I  obtained  3423  (5.90  per  cent.)  of 

121 


122 


R.    R.    HUESTIS. 


these  in  58037  flies ;  this  being  somewhat  higher  than  the  4.3  per 
cent,  reported  by  Bridges. 

An  inspection  of  my  material,  made  after  most  of  the  data  were 
in,  showed  that  a  given  female's  later  subcultures  produced,  dur- 


16 


14 


12 


10 


30 


FIG.  i.  Curves  of  smoothed  percentages  of  exceptional  young  obtained 
from  groups  of  females  kept  at  different  temperatures.  Abscissas  ap- 
proximate the  age  in  days  of  mother  females  when  eggs  were  ripened. 
Ordinates  are  percentages  of  exceptional  young.  Curves  end  with  the 
group  number.  Under  each  curve  is  the  temperature  in  degrees  C.  Broken 
lines  denote  inbred  groups.  Thick-lined  curves  are  based  on  more  flies. 

ing  most  of  her  lifetime,  almost  one  per  cent,  more  exceptions 
per  week  than  her  earlier  subcultures.  The  total  production  of 
offspring  and  of  exceptions  to  sex  linkage  (exc.)  of  109  white- 
eyed  females  mated  with  red-eyed  males,  and  changed  each  week 
to  a  new  subculture,  is  given  in  the  totals  column  of  Table  I.  The 
curve  of  these  percentages  is  that  of  group  i,  Fig.  i. 

In  order  to  eliminate  the  possibility  of  the  weighting  of  the  later 
subcultures  by  reason  of  the  longer  life,  or  the  greater  production 
of  exceptions  in  certain  subcultures  of  a  few  high-producing  fe- 
males, I  excluded,  in  the  figures  presented  in  Table  I.,  the  data 
from  all  females  which  produced  over  10  per  cent,  of  exceptions 
or  which  had  any  sub-culture  failure  prior  to  the  one  which  ended 
the  females  production.  When  the  material  is  divided  into  groups 
of  females  which  produced  young  for  the  same  length  of  time, 


EFFECT   OF   AGE   IN    SECONDARY    NON-DISJUNCTION.      ,       123 


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R.    R.    HUESTIS. 


the  same  sort  of  "  curve  of  age  "  is  obtained.  The  production  of 
groups  of  females,  so  arranged,  being  also  presented  in  Table  I. 

All  the  females  used,  in  obtaining  the  above  figures,  descended 
from  the  one  original  white-eyed  female  which  started  the  XXY 
line,  so  the  X  chromosomes  were  presumably  constant  throughout. 
Male  flies,  however,  were  taken  from  a  number  of  stocks  of  flies 
trapped  around  Berkeley,  or  present  in  the  laboratory,  so  the  Y 
introduced  into  each  generation  was  not  constant.  The  flies  were 
kept  in  a  large  cabinet  incubator  at  25  degrees  C.  and  counted 
daily.  It  is  perhaps  worth  recording  that  the  mother  female,  of 
each  group  of  subcultures,  was  removed  from  the  incubator  in 
the  subculture  tray  during  the  counting  period.  This  was  long 
enough,  at  times,  to  cool  the  vials  down  to  room  temperature. 

Before  leaving  an  account  of  this  part  of  the  work  I  should 
like  to  report  briefly  upon  a  test  of  the  constitution  of  60  reg- 
ular daughters  of  XXY  females.  Twenty-seven  of  these  daugh- 
ters, when  mated  with  Bar  males,  produced  no  exceptions. 
Thirty-three  produced  exceptions,  but  of  this  number  8  daughters 
produced  just  one  or  two  exceptions  in  large  counts  of  flies,  the 
percentage  being,  in  these  cases,  consistently  in  the  neighborhood 
of  one  third  of  one  per  cent. ;  the  total  production  being  10  ex- 
ceptions (95  i  $  )  in  3,148  flies.  If  these  8  regular  daughters 
were  XX  females  which  produced  primary  exceptions  these  latter 
are  not  predominantly  male  as  in  Safir's  results  (1920). 

LATER  EXPERIMENTS. 

After  some  lapse  of  time  during  which  I  was  engaged  in  breed- 
ing Peromyscus  I  returned  to  the  problem  of  non-disjunction  with 
the  idea  of  checking  my  results  prior  to  publication,  and  also 
of  trying  out  the  effect  of  temperature  differences  upon  the 
percentage  of  exceptional  offspring.  I  obtained  a  stock  of  flies, 
through  the  courtesy  of  Dr.  R.  E.  Clausen,  and  after  inspecting 
the  progeny  of  white-eyed  females,  mated  with  normal  males, 
picked  up  an  exception-producing  strain.  The  flies  used  in  these 
latter  experiments  consisted  of  a  number  of  white-eyed  females 
from  this  stock,  a  number  of  white-apricot  compound  females, 
obtained  from  a  mating  of  white  and  apricot,  and  finally  of  a 


EFFECT   OF  AGE   IN    SECONDARY    NON-DISJUNCTION.  125 

number  of  white  and  apricot  females  obtained  by  equational  non- 
disjunction  from  the  white-apricot  stock,  females  of  which  pro- 
duced XXY  daughters  pure  both  for  white  and  for  apricot.  I 
could  not  observe  that  these  allelomorphs,  white  and  apricot,  dif- 
fered from  one  another  in  the  capacity  for  exceptional  production, 
in  comparable  experiments. 

The  culture  methods  in  this  latter  part  of  the  work  were  modi- 
fied somewhat.  Yeast-seeded  banana  agar  was  used  for  food. 
Females  which  were  producing  young  were  left  continuously  in 
the  incubator  except  during  the  interval  when  they  were  changed 
to  a  new  culture.  Subcultures  were  made  up  every  six  days,  at 
20  or  21  degrees  C.,  and  every  three  days  (in  group  6,  Table 
III.,  every  four  days)  at  temperatures  higher  than  this.  Two 
Freis  electric  incubators  and  one  electrically  controlled  cabinet 
incubator  was  used  and  the  temperature  checked  daily.  These 
machines  will  fluctuate  in  temperature,  within  a  degree  up  or 
down,  but  since  the'  routine  involved  the  growth  of  flies,  at  each 
different  temperature,  over  a  considerable  period  of  time  such 
fluctuations  should  cancel  out. 

Table  II.  summarizes  and  Fig.  I  depicts  graphically  the  results 
obtained  when  groups  of  females  were  kept  continuously  at  cer- 
tain temperatures  each  female  being  transferred  to  new  subcul- 
ture vials  as  long  as  she  remained  fertile.  Except  in  groups  6 
and  7,  in  which  inbreeding  was  the  rule,  male  parents  came  from 
several  cultures.  In  group  6  one  inbred  stock  of  wild  males  was 
used,  in  group  7  exceptional  brothers. 

A  comparison  of  Tables  I.  and  II.  and  of  the  curves  in  Fig.  I 
brings  to  light  some  very  obvious  differences  in  the  characteris- 
tics of  the  females  in  comparable  groups.  In  group  I  the  average 
fertile  lifetime,  in  round  numbers,  was  28  days.  In  groups  4 
and  5,  kept  at  comparable  temperatures,  the  average  fertile  life- 
time was  16  days.  It  appears  improbable  that  this  difference  is 
an  environmental  one  for  banana  agar  is  generally  conceded  to  be 
a  better  food  medium  than  fermented  banana. 

The  percentage   of   exceptional  progeny  produced   by  the    fe- 
males in  groups  4  and  5  (3.14  and  3.88  per  cent.)  is  considerably 
below  the  5.49  per  cent,  of  exceptions  obtained  in  group   I,  and  /c 
this   is  not  due  to  the  difference  in  longevity   for  the  group 
percentage  is  higher  at  any  comparable  age. 


126 


R.    R.    HUESTIS. 


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EFFECT   OF  AGE  IN    SECONDARY    NON-DISJUNCTION. 


127 


The  curve  of  age,  so  obvious  in  group  i,  is  not  exactly  repro- 
duced in  any  of  the  later  groups  although  a  curve  of  age,  as  may 
be  noted  in  Fig.  I,  is  usually  apparent  instead  of  a  randum 
fluctuation  from  a  model  percentage.  This  curve  of  age  differs 
at  different  temperatures,  in  the  figures  presented,  but  the  possi- 
bility that  genetic  differences  in  the  females  used  may  be  respon- 
sible for  this  cannot  be  excluded. 

A  noticeable  thing  in  Table  II.  is  the  increase  in  exception 
production  at  higher  temperatures.  Based  on  the  totals  in  each 
group  the  increase  was  slow,  for  a  given  temperature  increment, 
up  to  25  degrees,  then  more  rapid  between  25  and  28  degrees  and 
most  rapid  between  28  and  30  degrees.  For  these  temperatures 
the  curve  of  increased  exception  production  is  comparable  to  the 
curve  of  increased  crossing-over  of  second  chromosome  genes  in 
Plough's  results. 

EFFECT  OF  TEMPERATURE  CHANGE  DURING  THE  LIFETIME  OF 

XXY  FEMALES. 

In  order  to  make  tests  of  this  kind  females  were  hatched,  and 
their  first  eggs  ripened  at  a  high  temperature  and  then  kept  for 
the  remainder  of  their  lifetime  at  a  temperature  ten  degrees  lower. 
In  addition,  females  were  hatched  at  a  relatively  high  tempera- 
ture and,  after  a  period  at  a  temperature  eight  degrees  lower, 
they  were  returned  to  the  high  temperature  again.  A  difference 
of  eight  degrees  was  substituted  for  ten  because  too  many  females 
died  when  transferred  from  a  20  to  a  30  degree  incubator. 

It  was  found  by  Plough  and  Metz  that  females  about  twelve 
hours  old  have  approximately  140  oocytes  in  their  ovaries.  The 
practice  in  the  experiments  here  recorded  being  to  empty  cultures 
every  24  hours  it  can  be  assumed  that  the  average  age  at  mating, 
of  mother  females,  was  not  greater  than  twelve  hours,  for  younger- 
appearing  females  were  always  chosen  for  mothers  since  they 
are  most  likely  to  be  virgins.  It  takes  such  females  six  to  eight 
days  (this  period  must  differ  at  different  temperatures)  to  lay 
the  eggs  ripened  at  the  temperature  at  which  they  were  hatched, 
the  lag  of  layed  after  ripened  eggs  continuing  all  through  a  fe- 
male's lifetime. 


12 


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FIG.  2.  Curves  of  three  tests  of  temperature  change.  Abscissas  are 
days  of  life  of  mother  females,  ordinates  the  percentages  of  exceptional 
young.  Figures  at  intervals,  under  the  curves,  are  temperatures  at  which 
the  exception-producing  eggs  in  mother  females  presumably  ripened,  inter- 
mediate temperature  plateaus  being  left  blank. 


EFFECT   OF   AGE   IN    SECONDARY    NON-DISJUNCTION. 


129 


Table  III.  and  curve  N,  Fig.  2,  presents  the  results  obtained 
when  white-apricot  compound  females,  born  at  30  degrees  C., 
were  mated  with  normal  males  and  kept  at  20  degrees  C.  Sub- 
cultures are  six  days  apart. 

TABLE  III. 

THE  NUMBER  OF  FLIES  AND  THE  NUMBER  AND  PERCENTAGES  OF  EXCEPTIONS 

PRODUCED  BY  XXY  FEMALES  BORN  AT  30°  C.  AND 

THEN  MATED  AND  KEPT  AT  20°  C. 

Temperatures  are  those  at  which  the  eggs,  producing  the  exceptions,  are  pre- 
sumed to  have  ripened. 


Temperature. 

30 

30-20 

20 

20 

20 

C     K        It 

A 

B 

C 

D 

E,  F 

Flies 

Exc. 

Flies. 

Exc. 

Flies. 

Exc. 

Flies. 

Exc. 

Flies. 

Exc. 

1,751 

197 

2,005 

146 

I.93I 

40 

1,023 

28 

482 

5 

C7 

II.  2 

7-3 

2.1 

2.7 

I.O 

It  would  be  expected  that  the  eggs  ripened  at  30  degrees  would 
be  laid  in  the  A  and  in  the  first  part  of  the  B  subcultures,  being 
followed  by  eggs  ripened  at  20  degrees,  which  would  be  laid  in 
the  latter  half  of  the  B  and  in  subsequent  subcultures.  If  the 
percentage  of  exceptions  varies  with  the  temperature  at  which 
the  eggs  are  ripened  then  the  A  subculture  should  contain  the 
highest,  B  an  intermediate  and  all  subsequent  subcultures  a  low 
percentage  of  exceptions.  It  may  be  seen  that  this  expectation  is 
realized  completely. 

Table  IV.  and  curve  R,  Fig.  2,  presents  the  results  obtained 
when  white-apricot  females  were  born  at  28  degrees  C.,  mated 
with  normal  males  and  held  for  two  subcultures  (12  days)  at  20 
degrees  C.,  then  returned  to  28  degrees  C.  again. 

The  expectation  of  a  high  A,  intermediate  B,  low  C,  intermedi- 
ate D  and  of  high  subcultures  subsequent  to  D,  is  only  partly 
met.  The  poor  fit  in  the  A  and  B  subcultures  may  very  well  be 
due  to  the  chance  selection  of  very  young  mother  females.  The 
final  cultures  are  higher  than  expected. 


130 


R.    R.    HUESTIS. 


TABLE  IV. 

THE  NUMBER  OF  YOUNG  AND  THE  NUMBER  AND  PERCENTAGES  OF  EXCEPTIONS 
PRODUCED  BY  XXY  FEMALES  BORN  AT  28°  C.  MATED  AND  . 
HELD  FOR  Two  SUBCULTURES  (12  DAYS)  AT  20°  C. 

AND    THEN    RETURNED    TO    28°    C. 

Temperatures  are  those  at  which  eggs  are  presumed  to  have  ripened. 


Temperature. 

28 

28-20 

2O 

20-28 

28 

. 

A 

B 

C 

D 

E,  F 

Flies. 

Exc. 

Flies. 

Exc. 

Flies. 

Exc. 

Flies. 

Exc. 

Flies. 

Exc. 

1,084 

42 

1,146 

23 

834 

IS 

562 

18 

434 

37 

'. 

3-9 

2.0 

1.8 

3-2 

8.5 

Table  V.  and  curve  T,  Fig.  2,  presents  the  results  obtained  when 
apricot  females  were  born,  mated  with  one  inbred  stock  of  wild- 
type  males  and  held  for  one  subculture  (4  days)  at  28  degrees  C., 
then  kept  in  a  subculture  (6  days)  at  20  degrees  C.  and  finally 
returned  to  28  degrees  C.  for  all  subsequent  four-day  subcultures. 

TABLE  V. 

THE  NUMBER  AND  PERCENTAGE  OF  EXCEPTIONAL  YOUNG  PRODUCED  BY  XXY 

FEMALES  BORN,  MATED,  AND  KEPT  4  DAYS  AT  28°  C.,  THEN 

KEPT  Six  DAYS  AT  20°  C.  AND  THEN  RETURNED  TO  28°  C. 

Temperatures  are  those  at  which  eggs  are  presumed  to  have  matured. 


Temperature. 


28 

28 

20 

20  28 

28 

Subcult. 

A 

B 

C 

D 

E.  F 

Flies. 

Exc. 

Flies. 

Exc. 

Flies. 

Exc. 

Flies. 

Exc. 

Flies. 

Exc. 

2,342 

175 

3.825 

297 

2,108 

67 

1,033 

49 

485 

45 

' 

7-5 

7-8 

3-2 

4-7 

9-3 

This  was  the  most  adequate  test  of  the  effect  of  temperature 
for  a  fair  number  of  flies  was  raised,  the  males  used  as  parents 
were  all  of  one  type,  and  other  females  (group  6,  Table  III.) 
were  held  continuously  at  28  degrees.  The  C  subculture  should 


EFFECT   OF  AGE  IN    SECONDARY    NON-DISJUNCTION.  131 

be  the  low  one  but  should  not  go  down  to  2  per  cent,  for  the  6 
days  at  20  degrees  would  hardly  complete  the  oviposition  of  all 
the  eggs  brought  into  the  culture  which  were  ripened  at  28  de- 
grees. The  final  cultures  are  again  too  high  and  the  same  cause 
may  be  operative  here  as  in  Table  IV.  I  suggest  an  over-reaction 
to  heat  after  a  transfer  from  a  low  temperature. 

When  it  is  considered  that  the  curve  produced  by  the  percent- 
age of  exceptional  young  in  different  subcultures,  which  received 
eggs  ripened  at  different  temperatures,  is  superimposed  upon  a  curve 
of  age  which  has  been  shown  to  be  somewhat  variable,  it  may  be 
concluded  that  the  above  figures  are  as  close  to  the  requirements 
for  a  demonstration  of  the  effect  of  temperature  as  could  be  ex- 
pected. I  think  the  statement  that  the  percentage  of  exceptional 
progeny,  produced  by  XXY  females,  varies  in  being  higher  at 
high  temperatures  and  lower  at  low  temperatures  between  20  and 
30  degrees  C.  is  thus  quite  warranted  by  the  results. 

GENETIC  DIFFERENCES  IN  EXCEPTION  PRODUCTION. 

By  breeding  to  reintroduce  the  maternal  Y  chromosome, 
Bridges  obtained  a  "  high  "  line  of  exception  producing  females. 
He  suggested  that  this  high  production  was  due  to  increased 
heterosynapsis  resulting  from  the  peculiar  constitution  of  the 
introduced  Y.  Bonnier,  using  Bridges'  high  line,  showed  that  the 
introduction  of  a  new  Y  is  not  followed  by  a  return  to  the  "  low  " 
percentage  of  exceptions,  and  presented  evidences  to  show  that  a 
high  percentage  of  exceptions  is  a  matter  of  the  constitution  (gene 
basis)  of  the  two  X's. 

The  following  data  suggest  that  neither  of  these  theories  will 
account  for  all  the  inherent  differences  in  exception  producing 
ability.  I  mated  24  matroclinous  daughters  of  a  white-eyed  XXY 
female  (9  No.  116),  mated  with  one  male,  which  had  herself 
produced  14.5  per  cent,  of  exceptions  in  447  young.  These 
daughters  (they  are  included  in  Table  I.  above)  produced  an  aver- 
age of  6.2  per  cent,  of  exceptions  in  11,692  young,  the  percentage 
of  individual  cultures  being  from  2.9  per  cent,  in  one  culture  of 
820  young  to  10  per  cent,  in  another  culture  of  600  young.  This 
difference  between  2.9  and  10  per  cent.,  is  five  times  its  probable 


132 


R.    R.    HUESTIS. 


error 1  although  these  daughters  all  had,  theoretically  at  least, 
equivalent  sex  chromosomes.  Here  an  apparent  segregation  of 
exception-producing  ability  is  manifest. 

It  can  readily  be  demonstrated  in  another  way  that  this  varia- 
bility is  not  just  a  matter  of  random  sampling,  for  if  the  percent- 
age of  exceptions  produced  during  the  first  part  of  a  female's  life- 
time is  compared  with  that  produced  during  a  later  part  of  her 
lifetime,  positive  correlation  may  be  observed.  I  made  such  cal- 
culations within  groups  of  females  which  had  produced  a  se- 
quence of  complete  subcultures  at  23,  26  and  30  degrees  (groups 
3,  i,  and  5  already  tabulated)  and  obtained  the  following  corre- 
lation coefficients :  -|-  .56  ±  .12,  +  .39  ±  .07  and  -f-  .70  ±  .11.  This 
suggests  that  the  ability  to  produce  a  certain  percentage  of  excep- 
tions, tends,  like  any  other  measurable  character,  to  stay  within 
the  limits  proscribed  by  genetic  constitution  of  the  individual  in 
question.  That  being  the  case  one  would  expect  parent-offspring 
correlation,  in  exception-producing  ability,  of  about  the  same 
magnitude  as  that  which  has  been  found  for  other  characters.  I 
had  one  series  in  which  I  could  test  this  point,  for  in  group  I 
there  were  60  daughters  which  had  produced  300  or  more  young 
which  had  mothers  with  an  equal  productivity.  The  mother- 
daughter  correlation  in  exception  production  was  here  -f-  .37  ±  .07. 

I  selected  a  few  generations  of  flies  for  increased  exception- 
production  by  choosing  the  daughters  of  my  higher  producing  fe- 
males and  mating  these  daughters  with  their  exceptional  brothers. 
In  the  first  selected  generation  I  obtained  6.5  per  cent,  of  excep- 
tions in  4,493  flies,  in  the  second  generation  8.4  per  cent,  of  ex- 
ceptions in  4,414  flies,  and  in  the  third  generation  14.6  per  cent, 
of  exceptions  in  1,772  flies.  Exceptional  females  from  average 
exception-producing  mothers  which  had  been  similarly  mated 
with  their  exceptional  brothers  produced  5.6  per  cent,  of  ex- 
ceptions in  5,674  flies,  so  inbreeding  alone  did  not  increase  the 
per  cent,  of  exceptions.  An  inspection  of  the  pedigrees  showed 
the  selected  high  line  to  altogether  come  from  one  high-producing 

1  When  the  probable  errors  are  obtained  by  the  formula  ±\l— -,  where 

/>  =  the  percentage,  q  =  ioo-p  and  Af  =  the  number  of   observations  upon 
which  p  is  based. 


EFFECT   OF   AGE   IN    SECONDARY    NON-DISJUNCTION.  133 

female.  After  one  generation  produced  by  outcrossing  to  non- 
related  stock,  two  females  from  this  line  still  produced  14.1  per 
cent,  of  exceptions  in  546  flies. 

I  also  repeated  Bridges'  scheme  of  mating  to  reintroduce  the 
maternal  Y  chromosome  from  a  high-producing  female  by  mating 
her  daughters  with  their  exceptional  uncles.  In  two  generations 
of  this  line  I  obtained  15.5  per  cent,  of  exceptions  in  2,072 
flies,  a  figure  comparable,  since  sister  females  were  used  in  the 
two  cases,  to  the  third  and  one  later  generation  of  the  high  se- 
lection tabulated  above.  Either  of  these  methods  of  inbreeding 
would  tend  to  concentrate  factors  favorable  to  exception  produc- 
tion in  a  given  line  of  flies  and  these  need  not  be  intrinsic  char- 
acteristics of  the  sex  chromosomes. 

CONCLUSIONS. 

The  data  presented  above  together  with  those  of  previous  in- 
vestigations show  that  the  percentage  of  exceptions  to  sex  link- 
age may  be  affected  by  a  number  of  variables  which,  in  order  of 
importance,  are :  the  genetic  constitution  of  the  female,  the  tem- 
perature at  which  eggs  are  ripened,  and  maternal  age. 

With  regard  to  genetic  constitution,  XX  females  produce  well 
under  i  per  cent,  of  primary  exceptions.  XXY  females  may 
produce  from  i  to  over  20  per  cent.  Our  knowledge  of  the  causes 
of  this  latter  variability  does  not  appear  to  be  complete.  Bon- 
nier's  outcrossing  experiments  appear  to  remove  the  probability 
that  the  constitution  of  the  Y  chromosome  is  responsible  for 
Bridges'  high  eosin  line  and  although  Bonnier  was  not  able  to 
exclude  the  possibility  that  autosomal  genes  were  implicated,  his 
experiments  pointed  to  an  exclusively  X  chromosomal  effect  in 
the  production  of  different  percentages  of  exceptions.  It  is  the 
rule  to  have  a  constant  pair  of  X  chromosomes  in  all  lines  ot 
secondary  non-disjunction,  except  as  the  X's  may  interchange 
material  with  each  other  or  with  the  Y,  and  yet  genetic  variability 
is  still  present.  The  only  inference  that  is  possible  is  that  the 
percentage  of  exceptions  may  be  affected  directly  by  interaction 
of  the  sex  chromosomes  themselves  and  indirectly  by  autosomal 


134 


R.    R.    HUESTIS. 


genes.2  This  is  what  has  been  found  to  be  true  in  cross-over  per- 
centages. 

The  direct  effect  of  temperature  upon  the  percentage  of  ex- 
ceptions appeared  in  all  the  tests  I  made.  The  results  of  tem- 
perature differences  upon  protoplasm  in  modifying  physiological 
activity  are  so  well  known  that  some  temperature  effect  would  be 
an  a  priori  expectation  in  secondary  non-disjunction.  Although 
my  data  suggest  that  the  temperature  effect  increased  as  30  de- 
grees C.  was  approached,  I  cannot  exclude  the  possibility  that  this 
was  due  to  genetic  differences  in  the  groups  of  females  kept  at 
these  different  temperatures. 

Maternal  age  appears  to  affect  the  percentage  of  exceptional 
young  to  some  degree  but  apparently  by  interacting  with  other 
variables  for  different  age  curves  were  found  in  different  groups 
and  at  different  temperatures. 

The  inference  that  autosomal  genes,  temperature  and  ma- 
ternal age  all  affect  the  allocation  of  the  sex  chromosomes,  in 
XXY  females,  to  gamete-forming  cells,  follows  the  conclusions 
reached  above. 

LITERATURE  CITED. 

Bonnier,  Gert. 

'23     Studies   in    High   and   Low    Non-disjunction   in   Drosophila   mclano- 

gaster.     Hereditas,  IV.,  81-110. 
Bridges,  C.  B. 
'16     Non-disjunction  as  Proof  of  the  Chromosome  Theory  of  Heredity. 

Genetics,  I.,  1-52,  107-163. 
Plough,  H.  H. 
'17     The  Effect  of  Temperature  in  Crossing  Over  in  Drosophila.     Jour. 

Exp.  Zool.,  24,  147-208. 
Safir,  S.  R. 

'20     Genetic  and  Cytological  Examination  of  the  Phenomena  of  Primary 
Non-disjunction  in  Drosophila  melanogastcr.     Genetics,  5,  459-487. 

1  This  would  explain  why  a  high-producing  female  may  or  may  not  es- 
tablish a  high  exception-producing  line. 


OXYGEN  CONSUMPTION  OF  INSECT  EGGS.1 

ROY    MELVIN, 
IOWA  STATE  COLLEGE. 

Although  insect  eggs  present  unique  material  for  studies  in 
metabolism  the  literature  contains  comparatively  few  reports  in 
this  interesting  field  of  insect  physiology.  Bodine  (i)  has  pre- 
sented data  from  a  study  of  eggs  of  Orthoptera  which  show  the 
velocity  of  development  to  increase  in  direct  proportion  to  in- 
crease in  temperature  within  the  normal  limits  of  development. 
This  is  in  accordance  with  other  findings  on  the  effects  of  tem- 
perature on  poikilothermos  species.  This  author  shows  that  it 
is  possible  to  calculate  the  time  of  hatching  of  eggs  if  previous 
temperature  history  is  known.  Such  knowledge  of  insect  pests 
may  lend  itself  to  practical  application. 

Fink  (2)  has  conducted  studies  which  lead  him  to  conclude 
that  the  formative  period  in  the  development  of  eggs  of  certain 
insects  is  dependent  upon  whether  they  are  deposited  upon  foliage 
or  in  the  soil.  Data  to  be  presented  in  this  paper  tend  to  dis- 
prove the  above  explanation.  For  further  references  to  literature 
in  this  field  see  the  papers  of  Bodine  and  Fink  cited  above. 

Thanks  are  due  Doctor  Erma  Smith,  Professor  of  Physiology, 
and  other  members  of  the  Zoology  and  Entomology  staff  at  Iowa 
State  College,  for  many  helpful  suggestions  and  encouragement 
throughout  the  course  of  this  work. 

PURPOSE. 

The  purpose  of  this  paper  is  to  present  briefly  a  preliminary 
report  of  a  study  of  the  oxygen  consumption  during  embryonic 
development  of  certain  insects. 

1  Contribution  from  the  Department  of  Zoology  and  Entomology,  Iowa 
State  College,  Ames,  Iowa. 

10  135 


136 


ROY    MELVIN. 


METHODS  AND  MATERIAL. 

Bodine's  modification  of  Krogh's  manometer  was  used  for 
determining  the  oxygen  intake.  Constant  temperatures  were 
maintained  by  use  of  a  Freas  electric  water  bath.  The  data  pre- 
sented were  determined  from  the  eggs  of  the  following  insects: 
Squash  bug.  Anas  a  tristis  De  G. ;  Luna  moth,  Tropcca  luna  L. ; 
Cecropia  moth,  Samia  cccropia  L. ;  and  Smartweed  borer, 
Pyransta  dinslici  Hein. 

The  O2  consumption  is  expressed  in  millograms  of  O2  per  gram 
live  weight  (exclusive  of  shell)  per  hour.  Apparently  previous 
workers  have  not  taken  into  consideration  the  weight  of  the  shell. 
If  the  weight  of  the  shell  be  deducted,  as  it  evidently  should  be, 
the  O2  consumption  curve  will  be  raised  from  10  to  30  per  cent. 

The  per  cent,  of  shell  at  the  beginning  of  incubation  for  sev- 
eral species  of  insects  was  found  to  be  as  follows:  A.  tristis,  29.2; 
S.  cccropia,  22;  T.  luna,  23.3;  and  P.  ainslici,  31. 

Assuming  the  weight  of  the  egg  shell  to  remain  constant 
throughout  the  incubation  period,  the  percentage  of  shell  varies 
directly  with  changes  in  the  weight  of  the  egg.  It  is  thus  evident 
that  changes  in  the  weight  of  the  egg  will  alter  the  type  of  curve 
representing  O2  consumption.  For  this  reason  the  weight  was 
determined  just  prior  to  each  gas  determination  and  calculations 
made  accordingly. 

Determinations  were  made  on  egg  masses  as  soon  as  they  were 
deposited  and  every  12  or  24  hours  thereafter,  depending  on  the 
length  of  the  incubation  period,  until  hatched.  Calculations  were 
made  according  to  the  formula  of  Krogh  (3). 

TEMPERATURE. 

The  effects  of  temperature  upon  biological  processes  are  too 
numerous  and  too  well  known  to  warrant  detailed  discussion. 
Numerous  investigators  have  studied  the  effect  of  temperature 
upon  the  length  of  the  incubation  period,  but  few  reports  have 
been  found  dealing  with  the  effect  of  temperature  upon  the  rate 
of  metabolism  as  determined  by  the  oxygen  consumption.  With 
this  in  mind  experiments  were  undertaken  to  determine  the  ef - 
fects  of  temperature  upon  embryonic  development  of  insects. 


OXYGEN    CONSUMPTION    OF   INSECT    EGGS. 


137 


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OXYGEN    CONSUMPTION    OF   INSECT    EGGS. 


139 


The  same  eggs  were  used  to  make  the  determinations  at  both 
temperatures.  After  sufficient  acclimatization,  two  to  five  hours, 
depending  on  the  size  of  the  animal  chamber,  the  manometer  was 
closed  and  at  the  end  of  two  hours  the  reading  was  made.  In  like 
manner  the  reading  for  the  next  temperature  was  made  and  the 
eggs  returned  to  the  incubator  at  28  degrees  until  the  next  day. 


25 
2.0 
1.5 
1.0 
0.5 
0.0 


02MG  M 


TEMP.  C 


15 


20 


25 


30 


35 


FIG.  I.  Showing  the  effects  of  temperature  on  the  O>  consumption  of 
T.  lima  eggs.  (A)  last  day  of  incubation;  (B)  first  day  of  incubation. 

The  results  of  these  experiments  are  shown  graphically  in  Fig.  I. 

It  is  observed  from  Fig.  i  that  the  effects  of  temperature  on 
O2  consumption  are  very  slight  during  the  first  day  of  incu- 
bation and  very  pronounced  during  the  last  day.  In  order  to 
determine  the  effect  of  temperature  on  O,  consumption  during 
the  entire  incubation  period  daily  determinations  were  made  on 
two  series  of  S.  cecropia  eggs  at  28  and  34  degrees  C.  respectively. 
These  results  are  summarized  in  Table  I.  and  shown  graphically  in 
Fig.  2. 

For  comparative  purposes  the  eggs  of  four  species  of  insects 
were  run  at  28  degrees  C.  and  the  rate  of  O2  consumption  thus 
determined  is  shown  graphically  in  Fig.  3  and  summarized  in 
Table  II. 


140 


ROY    MELVIN. 


FORMATIVE  PERIOD. 

Upon  examination  of  Figs.  2  and  3  we  note  that  during  the 
early  part  of  the  incubation  period  temperature  has  very  little 
stimulation  on  the  O2  consumption  but  as  the  incubation  period 


i; 


QJ 


7 


/    a 


B/ 


0 
DAYS1       23       +        5G781 

FIG.  2.     Shows  the  effects  of  temperature  on  O2  consumption  during  the 
entire  incubation   period  of  S.   cecropia.      (A}    exclusive  of   shell  34°    C., 

(a)  same  as   above   but   including   shell;    (B)    exclusive   of    shell   28°    C., 

(b)  same  as  above,  but  including  shell. 

progresses  its  effects  become  pronounced.  This  is  in  accord  with 
and  substantiates  the  existing  theory  which  states  that  during 
early  embryonic  development  there  is  a  formative  period  during 
zvhich  metabolic  activity  is  comparatively  low  and  only  influenced 
slightly  by  environmental  changes.  Mention  has  been  made, 
above,  of  the  explanation  offered  by  Fink  for  the  variation  in  the 
length  of  this  formative  period  among  different  species  of  in- 
sects. In  the  case  of  S.  cecropia  and  T.  luna,  both  species  laying 
eggs  on  foliage,  the  formative  period  is  somewhat  lengthened. 
This  is  contrary  to  Fink's  explanation.  Data  presented  in  this 
paper  shows  the  length  of  the  incubation  period  to  be  a  greater 


OXYGEN    CONSUMPTION    OF   INSECT    EGGS. 


141 


factor  in  determining  the  length  of   the   formative  period  'than 
the  type  of  place  where  the  eggs  chance  to  be  deposited.     Fig.  3 


cu  3 

0. 


cu 


0 
OAY51        23       +       5Z1Z1 

FIG.  3.     O:;  consumption  of  eggs  of   (A)  P.  ainslici,  (B)  A.  tristis,  (C) 
T.  lima,  and   (D)   S.  cccropia. 

bears  out  this  explanation.     A  study  of  Fink's  curves  will  show 
that  they  too  substantiate  the  explanation  here  offered. 

SUMMARY. 


From  a  preliminary  study  of  the  factors  accompanying  and 
influencing  metabolism  as  determined  by  the  O2  consumption 
during  embryonic  development  made  on  four  species  of  insects 
the  following  conclusions  are  drawn : 

I.  The   weight   of   the   egg  shell   is   an   important    factor   and 
should  be  taken  into  consideration. 

II.  The  effects  of  temperature  are  not  as  pronounced  during 
the  formative  period  as  during  the  period  of  late  incubation. 

III.  The  explanation  offered  for  the  variation  in  the  length  of 
the  formative  period  is  the  length  of  the  incubation  period  and 
not  the  place  where  the  eggs  chance  to  be  laid  as  has  been  sug- 
gested. 


MELVIN. 

LITERATURE  CITED. 

1.  Bodine,  J.  H. 

'25     Effect   of   Temperature   on   Rate   of    Embryonic    Development   of 
Certain  Orthoptera.     Jour.   Exp.  Zool.,  42:   91-109. 

2.  Fink,  D.  E. 

'25     Metabolism    during    Embryonic    and    Metamorphic    Development    of 
Insects.     Jour.  General   Physiol.,  7:    527-543. 

3.  Krogh,  A. 

'15     Microrespirometrie.     In  Ahderhalden,    E.,   Handbuch   der   Biochem- 
ischen  Arbeitsmethoden,   Berlin,   8:    519-528. 


Vol.  LV.  September,  IQ28  No.  3. 


BIOLOGICAL  BULLETIN 


THE  INFLUENCE  OF  MOLDS  ON  THE  GROWTH   OF 

LUMINOUS   BACTERIA  IN   RELATION   TO   THE 

HYDROGEN     ION     CONCENTRATION,     TO - 

.GETHER  WITH  THE  DEVELOPMENT 

( )F  A  SATISFACTORY  CULTURE 

METHOD. 

SAMUEL  E.  HILL, 

PHYSIOLOGICAL  LABORATORY, 
PRINCETON  UNIVERSITY. 

At  the  Marine  Biological  Laboratory,  Woods  Hole,  during 
August  and  September,  1927,  it  was  observed  that  cultures  of 
luminous  bacteria  (Bacillus  Fischcri,  Rcijerinck,  Migula)  tended 
to  deteriorate  rapidly,  the  deterioration  being  progressive,  so  that 
finally  subcultures  were  made  daily,  the  luminescence  becoming 
steadily  less,  and  the  culture  was  finally  lost.  A  fresh  culture  was 
obtained  by  plating  out  luminous  material  of  the  same  stock, 
recovered  from  an  old  Petri  dish  culture  contaminated  with  mold. 
This  culture  grew  vigorously  for  a  time,  and  then  degenerated. 
Since  the  bacteria  in  contact  with  the  mold  continued  to  grow  and 
glow  for  some  time,  new  cultures  were  isolated  when  necessary. 

These  bacteria  were  being  used  for  physiological  experimental 
material,  and  it  was  considered  necessary  to  learn  the  reason  for 
the  deterioration  of  the  cultures  and  devise  a  cultural  method 
by  which  bacteria  of  the  same  strain  could  be  maintained  in 
vigorous  condition  throughout  any  given  series  of  experiments. 
Luminous  bacteria  live  normally  in  sea  water,  which  is  maintained 
constantly  in  a  fairly  definite  alkaline  pH  range.  They  are  con- 
sidered to  grow  best  on  culture  media  of  about  the  same  pi  I 
value  as  the  sea  water.  All  of  these  cultures  were  grown  on  the 
same  medium,  supposedly  of  the  proper  pH,  and  it  was  sug- 
10  143 


!44  SAMUEL  E.    HILL. 

gc.stt-d  bv  Professor  Harvey  that  the  trouble  might  be  due  to 
insufficient  alkali  reserve,  the  acid  produced  by  the  bacteria 
rapidly  lowering  the  pH  of  the  medium  to  a  value  unfavorable  to 
their  growth.  The  influence  of  the  mold  in  causing  continued 
light  and  growth  might  be  due  to  alkali  production.  This  expla- 
nation was  favored  by  the  result  of  pouring  a  solution  of  M/2 
NaCl  to  which  Clark's  phosphate  buffer,  pH  8.0,  had  been  added, 
over  the  surface  of  several  Petri  dish  cultures  which  had  ceased 
to  glow.  One,  in  which  the  light  had  been  out  only  a  few  hours, 
again  began  to  glow,  and  the  luminesceice  lasted  for  over  eight 
hours.  Others,  in  which  the  light  had  been  extinct  for  longer 
periods,  were  not  revived. 

Friedberger  and  Doepner  (1907)  had  studied  the  influence  of 
various  molds  on  the  light  intensity  of  cultures  of  luminous  bac- 
teria. They  grew  molds  in  bouillon,  filtered  the  bouillon,  and  used 
this  material  in  making  up  culture  media.  They  found  a  greater 
intensity  of  light  in  cultures  grown  on  these  media  than  on  con- 
trols prepared  with  ordinary  bouillon.  The  one  difference  which 
they  could  establish  between  ordinary  bouillon  and  bouillon  in 
which  mold  had  been  grown  was  an  increased  alkalinity  in  the 
latter.  Their  figures  show  that  10  cc.  normal  bouillon  neutral- 
ized .4  cc.  n/io  NaOH  to  phenolphthalein,  while  10  cc.  of  their 
"  mold  bouillon  "  neutralized  .2  to  .4  cc.  n/io  H.,SO4  to  phenol- 
phthalein. They  arrived  at  the  conclusion  that  the  greater  inten- 
sity of  the  light  of  cultures  grown  on  "  mold  bouillon  "  was  due 
in  part  to  the  increased  alkalinity,  and  in  part  to  "  other  proper- 
ties "  of  the  mold. 

Molisch  (1912)  had  shown  that  in  general  the  intensity  of 
light  of  cultures  of  luminous  bacteria  depended  on  the  rate  of 
growth.  It  is  the  opinion  of  the  writer,  for  reasons  given  below, 
that  the  only  cause  for  the  increase  in  intensity  of  light  and 
length  of  life  of  cultures  of  luminous  bacteria  grown  in  contact 
with  mold  is  that  of  alkali  production  by  the  mold,  which  thus 
acts  as  an  alkali  reserve. 

A  series  of  experiments  using  solutions  of  M/2  NaCl  plus 
Clark's  phthalate,  phosphate,  and  borate  buffers,  found  to  be  non- 
toxic,  showed  that  these  bacteria  glowed  brightly  in  the  pH 
range  5.7  to  8.7,  the  luminescence  lasting  for  over  an  hour. 


INFLUENCE    OF    MOLDS    ON    BACTERIA.  145 

(Observations  were  not  made  after  more  than  an  hour  had 
elapsed.)  Below  pi  I  5.2  the  light  lasted  only  a  few  seconds, 
above  pH  9.0  for  three  minutes  or  less.  The  pH  range  in  which 
growth  can  be  expected  lies  then  between  5.7  and  8.7,  pH  values 
outside  this  range  being  productive  of  rapid  injury. 

The  culture  medium  in  use  was  a  peptone,  beef-extract,  glycer- 
ine agar,  made  up  in  sea  water,  the  pH  being  adjusted  to  8.2  with 
NaOH.  As  these  bacteria  live  normally  in  an  environment  con- 
taining NaCl  in  about  one  half  molecular  concentration,  favor- 
able conditions  are  provided  for  the  use  of  buffer  mixtures. 
Molisch  (1912)  had  shown  that  a  number  of  salts  other  than 
NaCl  might  be  used  in  culture  media  for  luminus  bacteria.  A 
culture  medium  was  made  up  in  which  one  fifth  mol  of  sec- 
ondary potassium  phosphate  in  500  cc.  distilled  water  was  sub- 
stituted for  one  half  of  the  sea  water.  After  sterilization  the  pH 
was  adjusted  with  NaOH  to  8.2.  Separate  lots  of  the  same  batch 
were  colored  with  the  Clark  and  Lubs  selection  of  indicator  dyes, 
covering  the  pH  range  from  1.2  to  9.8.  Cultures  were  started  on 
slants  prepared  from  these  media,  six  tubes  of  each  being  inoculated 
with  luminous  bacteria  and  three  of  each  six  being  inoculated 
also  with  a  common  mold  at  one  end  of  the  slant.  (The  mold 
used  was  kindly  identified  for  me  by  Dr.  Charles  Thorn,  as 
Penicillinin  sp..  in  the  same  section  with  P.  commune  (Thorn) 
and  P.  soHtitin  (Westling).)  These  were  all  allowed  to  develop 
somewhat  below  room  temperature  for  two  weeks. 

Some  of  the  indicators  used  were  accumulated  by  the  bacteria. 
These  are  being  studied  further  to  determine  whether  they  pene- 
trated the  cell,  or  were  merely  adsorbed  on  the  surface.  They 
were  of  little  value  for  this  study,  since  not  enough  dye  was  left 
in  the  medium  to  indicate  its  pH  value.  However,  in  the  case  of 
several  of  these,  the  pH  was  indicated  roughly  by  the  color  of  the 
dead  bacteria,  which  was  not  markedly  different  from  the  medium. 
With  brom  cresol  green  (yellow  at  3.8,  blue  at  5.8),  the  dead 
bacteria  near  the  mold  were  a  more  intense  blue  than  elsewhere, 
and  the  acid  range  of  the  indicator  had  not  been  reached  any- 
where in  the  slant. 

On  the  chlor-phenol  red  slants  (yellow  at  5.2,  red  at  6.8)  the 
color  of  the  medium  indicated  that  the  pH  had  been  reduced  to 


146 


SAMUEL   E.    HILL. 


5.4  : :  .2.     The  pH  of  the  medium  near  the  mold  was  well  above 
the  alkaline  range  of  the  indicator. 

On  the  cresol  red  slants  (yellow  at  7.2,  red  at  8.8)  the  color 
of  the  medium  indicated  pH  below  the  range  of  the  indicator  ex- 
cept near  the  mold,  where  a  pH  of  8.6  =p  .2  was  indicated.  The 
results  with  meta  cresol  purple  were  about  the  same.  With  thymol 
blue,  the  color  of  the  indicator  was  masked  by  the  color  of  the 
medium  at  the  critical  value,  and  it  was  of  no  value. 

On  the  same  date  six  cultures  were  started  on  medium  of  the 
same  batch  without  addition  of  indicator.  At  the  end  of  two  weeks 
all  were  alive  and  glowing  brightly.  These  cultures  decreased 
slowly  in  brilliance  during  the  next  month,  but  were  still  glow- 
ing faintly  at  the  end  of  six  weeks,  and  viable  transfers  were 
made  at  the  end  of  the  seventh  week.  The  final  death  of  these 
cultures  appeared  to  be  caused  by  the  drying  up  of  the  medium. 

As  a  further  check  on  the  alkali  influence,  several  cc.  of 
M/NaOH  was  introduced  at  the  bottom  of  each  of  six  slants  of 
unbuffered  medium  colored  with  brom  thymol  blue,  and  an  equal 
number  without  indicator.  Streaks  made  on  these  slants  devel- 
oped rapidly  on  the  upper  half  of  the  slant,  away  from  the  alkali, 
and  grew  well,  the  cultures  on  the  uncolored  medium  lasting  for 
several  weeks  (average  of  six,  22.2  days),  until  the  alkali  was 
exhausted.  On  one  of  these,  more  alkali  was  added  and  a  fresn 
inoculation  made,  the  growth  lasting  this  time  for  less  than  a  week. 
It  was  observed  that  no  growth  took  place  below  the  line  which 
marked  the  limit  of  diffusion  of  strongly  alkaline  NaOH.  This 
limit  was  well  marked  on  the  uncolored  medium  by  the  precipi- 
tation of  magnesium  hydroxide. 

The  most  characteristic  activity  of  luminous  bacteria  seems 
to  be  that  of  acid  production.  They  are  killed  in  a  few  days  in 
their  own  acid  if  some  method  of  neutralization  or  removal  is  not 
employed.  In  their  natural  environment  the  excess  acid  would 
simply  diffuse  into  the  surrounding  sea  water,  but  within  the 
limits  of  the  test  tube  this  cannot  occur.  The  base  used  in  the 
culture  medium  was  NaOH,  which  in  contact  with  carbon  diox- 
ide becomes  NaHCO3,  and  since  NaHCO,  in  the  concentration 
used  (.01  M )  is  not  particularly  acid  when  saturated  with  carbon 
dioxide,  it  is  not  likely  that  the  acid  limiting  their  growth  is  car- 


IM'l.rF.NCE    OF    MOLDS    ON    BACTERIA.  147 

bon  dioxide.  That  it  is  a  non-volatile  acid  is  shown  by  the  fol- 
lowing experiment : 

A  constant  stream  of  sterile  air  was  drawn  in  series  through 
three  bottles  of  slightly  buffered  culture  medium  colored  with 
cresol  red.  The  first  of  these  was  the  control,  without  bacteria. 
The  other  two  were  inoculated  with  luminous  bacteria.  At  the 
end  of  24  hours  the  control  was  red,  as  at  the  start,  and  un- 
contaminated  as  shown  by  the  absence  of  turbidity,  and  this  con- 
dition lasted  until  the  close  of  the  experiment.  The  two  inocu- 
lated bottles  at  the  end  of  24  hours  were  down  to  about  pH  7.4. 
Enough  NaOH  was  added  to  the  third  bottle  to  restore  the  orig- 
inal pH  of  approximately  8.2.  and  this  was  repeated  every  two 
hours  until  the  close  of  the  experiment.  At  the  end  of  36  hours, 
the  PH  in  bottle  No.  2  was  down  to  about  5.5  (determined  by 
withdrawing  some  of  the  material  and  testing  with  other  indi- 
cators) and  the  light  was  extinct.  In  bottle  No.  3,  in  which 
pH  8.2  was  maintained,  the  bacteria  continued  to  glow  for  an- 
other 24  hours,  when  the  light  failed,  due  presumably  to  failure 
of  the  food  supply.  Carbon  dioxide  and  any  other  acids  volatile 
at  room  temperature  (if  any  were  formed)  would  have  been 
swept  out  by  the  stream  of  air,  leaving  behind  the  non-volatile 
acid.  This  is  probably  lactic  acid. 

Other  culture  media  were  tried  in  which  calcium  and  barium 
carbonates  were  employed  as  buffers,  and  also  higher  concen- 
trations of  K2HPO4  and  sea  water,  and  lower  concentrations. 
Luminous  bacteria  can  tolerate  a  considerable  range  of  salt  con- 
centration. It  was  found  that  on  phosphate  buffered  media 
where  the  total  salt  concentration  was  greater  than  in  sea  water,  but 
not  in  excess  of  molar  concentration,  growth  was  slower  than  on 
media  of  the  proper  concentration,  and  the  tendency  to  diffuse 
growth  was  absent.  The  resultant  crowding  gave  the  streaks  a 
fictitious  brilliance  for  a  few  days,  after  which  the  light  intensity 
decreased  to  a  low  value.  These  cultures  were  viable  for  fairly 
long  periods  of  time,  average  21  days,  but  not  as  long  as  cultures 
on  media  of  the  proper  salt  concentration.  When  media  of  lower 
total  salt  concentration  (as  about  *4  molar)  were  used,  there  was 
an  initial  rapid  growth,  accompanied  by  flowing  over  the  surface 
of  the  medium,  and  a  rapid  decay,  so  that  such  cultures  were 


148 


SAMUEL  E.   HILL. 


viable  for  only  a  few  days,  'the  average  of  six  cultures  being  five 
days.  Since  a  heavy  precipitate  of  calcium  and  magnesium  phos- 
phates was  formed  when  the  phosphate  buffer  was  added  to  sea 
water,  media  were  prepared  containing  various  concentrations  of 
NaCl,  from  .25  M  to  .75  M,  as  substitutes  for  sea  water,  but 
these  were  unsatisfactory,  the  best  of  them  lasting  for  only 
14  days. 

On  medium  buffered  with  calcium  carbonate,  growth  was  vig- 
orous, but  the  life  of  the  cultures  was  less  than  with  the  best  of  the 
phosphate  buffer  mixtures.  The  average  length  of  life  of  eleven 
cultures  without  indicator  was  17.8  days.  Curiously  enough,  the 
death  of  these  cultures  was  due  to  excess  alkalinity.  The  initial 
growth  was  rapid,  but  on  the  third  or  fourth  day  there  was  a 
decrease  in  brilliance  of  light  and  a  slowing  down  of  growth, 
caused  by  the  rapid  diffusion  of  the  acid  through  the  agar,  using 
up  the  small  amount  of  calcium  hydroxide  in  solution.  This  was 
followed  by  an  increased  brilliance  and  renewed  growth  as  the 
pH  rose  again,  due  to  the  solution  of  more  calcium  hydroxide 
(produced  by  hydrolysis  from  the  calcium  carbonate),  and  its 
diffusion  through  the  medium.  The  calcium  salt  of  the  acid  pro- 
duced by  the  bacteria  is  much  more  soluble  than  calcium  car- 
bonate, and  is  evidently  hydrolyzed  in  solution,  for  the  medium 
becomes  steadily  more  alkaline  until  the  alkaline  range  of  the 
available  indicators  is  passed.  Since  the  bacteria  are  soon  killed 
by  alkali  above  pH  9.0,  the  limiting  value  is  passed,  and  lumines- 
cence ceases.  This  can  happen  only  when  the  calcium  carbonate  is 
in  excess.  When  the  pH  of  the  medium  was  adjusted  with  calcium 
carbonate,  and  the  excess  carbonate  filtered  off,  initial  growth 
was  rapid,  but  the  decline  following  it  continued  until  the  death 
of  the  culture  occurred  on  the  sixth  day  (average  of  six  cul- 
tures), caused  by  acidity  as  shown  by  the  use  of  a  suitable 
indicator. 

On  the  medium  prepared  with  barium  carbonate  from  which 
the  excess  carbonate  was  filtered  off,  the  initial  fair  growth  was 
followed  by  a  rapid  decline,  the  average  length  of  life  of  14  such 
cultures  being  6.5  days.  When  an  excess  of  barium  carbonate 
was  present,  the  initial  growth  was  fair,  and  slowly  decreased, 
the  cultures  growing  steadily  more  alkaline,  the  average  length 


IM'I.rKNCE    OF    MOLDS    OX     BACTERIA.  149 

of  life  of  14  cultures  being  17.5  days.  Although  theoretically 
about  the  same  pH  value  should  be  produced  1>y  barium  and  cal- 
cium carbonates,  in  practice  the  medium  prepared  with  barium 
carbonate  was  always  the  more  alkaline,  and  was  too  alkaline  for 
good  growth  of  the  bacteria.  The  vigorous  growth  obtained  on 
calcium  carbonate  was  never  obtained  on  media  prepared  with 
barium  carbonate. 

The  medium  prepared  with  calcium  carbonate  has  the  advan- 
tage that  no  adjustment  of  pH  is  required,  the  hydrolysis  of 
the  carbonate  giving  approximately  the  right  value.  It  is  by  far 
the  best  buffer  substance  to  use,  both  for  slants  and  for  Petri 
dish  cultures.  The  medium  should  contain  20  grams  "  Bacto  " 
nutrient  agar,  10  cc.  glycerine,  and  5  grams  calcium  carbonate 
per  liter,  made  up  in  sea  water.  If  a  transparent  medium  is  de- 
sired, the  phosphate  buffer  mixture  with  the  same  amount  of 
nutrient  substance,  made  up  in  sea  water  and  filtered,  may  be 
used.  The  optimum  pH  value  for  this  medium,  probably  about 
8.6,  may  be  secured  by  titrating  the  hot  medium  by  the  drop 
method  until  a  good  red  is  secured  with  cresol  red,  and  a  barely 
perceptible  color  with  thymol  blue.  When  one  fifth  mol  of 
buffer  is  added  to  sea  water,  the  average  life  of  cultures  emitting 
strong  light  is  18  days.  After  this  time,  very  little  light  is 
emitted,  but  viable  transfers  may  be  made  for  several  weeks. 

Of  the  indicators  used,  several  appeared  to  be  slightly  toxic 
to  the  bacteria,  but  the  evidence  on  this  point  is  inconclusive. 

SUMMARY. 

The  influence  of  molds  on  the  length  of  life  of  cultures  of 
luminous  bacteria  may  be  simulated  by  the  use  of  buffer  mix- 
tures, or  by  supplying  fresh  alkali  continually.  The  maximum 
alkalinity  produced  in  these  experiments  by  the  influence  of 
Penicilliitin  sp.  was  pH  8.6  q=  .2.  Degeneration  of  cultures  of 
luminous  bacteria  may  be  caused  by  growth  on  media  insufiiciently 
alkaline,  or  so  slightly  buffered  that  it  soon  becomes  acid.  Dif- 
fuse growth  and  spreading  over  the  surface  of  the  slant  is  caused 
by  too  low  salt  concentration.  Long  life  of  cultures  may  be  se- 
cured by  growing  on  media  sufficiently  alkaline,  and  sufficiently 
buffered  to  resist  rapid  change  by  the  acid  production  of  the  bac- 
teria, which  are  killed  by  their  own  acid  at  about  pH  5.6. 


,  -0  SAM  CEL   E.    HILL. 

BIBLIOGRAPHY. 

Clark,  W.  M. 

'27     The  Determination  of  Hydrogen  Ions.     Baltimore. 
Fischer,  B. 

'88     Ueber   einen   neuen   lichtentwickelnden   Bacillus,    Centralbl.    f.    Bakt., 

etc.,  3,  Nos.  4  and  5. 
Friedberger,  E.,  and  Doepner,  H. 

'07     Ueber   den   Einfluss   von    Schimmelpilzen   auf    die    Lichtintensitat    in 
Leticbtbacterien-culturen,    etc.,    Centralbl.    f.    Bakt.,    etc.,    ist    Abt., 

43,   i. 
Migula. 

'00     System  der  Bakterien  Zweiter  Band,  Jena. 
Molisch,  Hans. 

'12     Leuchtende    Pflanzen,    eine    physiologiscbe    Studie,    Zweite    Auflage, 
Jena,   1912. 


THE  SEX  RATIO  IX  PEROMYSCUS. 
JOHN  J.  KAROI.. 

The  data  herein  presented  are  based  on  the  records  of  breeding 
experiments  with  Pcrontyscits,  conducted  by  Dr.  F.  B.  Sumner. 
In  an  earlier  paper  l  the  sex  ratio  in  Pcroinyscns  was  discussed  at 
considerable  length  and  data  covering  the  years  1915—1921  in- 
clusive were  presented.  The  present  report  is  based  on  the  rec- 
ords of  births  from  1922-1926  inclusive.  The  material  is  made 
up  partly  of  the  various  mutant  strains  of  the  niaiiicitlatus  series, 
variously  hybridized  and  partly  of  the  three  subspecies  of  Pero- 
inyscus  polionotus,  P.  p.  polionotus,  P.  p.  leucocephalus,  and  P.  p. 
albifrons,  both  pure  and  hybrid.  No  attempt  will  be  made  to  give 
comprehensive  interpretation  of  the  findings  but  reference  may  be 
made  to  the  paper  cited  above  for  more  detailed  discussion. 

I  take  this  opportunity  of  acknowledging  my  indebtedness  and 
sincere  thanks  to  Dr.  F.  B.  Sumner  who  suggested  the  subject 
and  under  whose  general  guidance  the  work  was  carried  out. 

The  influences  which  might  affect  the  sex  ratio  in  Peromyscut 
were  considered  in  this  treatment  of  the  data  to  be  (i)  season, 
(2)  size  of  litter,  (3)  race,  (4)  hybridization. 

The  total  number  of  broods  recorded  in  the  records  from 
1922  to  1926  is  760,  comprising  2,522  young,  or  an  average  of 
3.32  mice  per  brood.  According  to  sex  these  were  distributed  as 
follows : 

Males    1,316 

Females   1,1 14 

Sex  undetermined  (dead  or  escaped)    61 

.The  sex  ratio  (number  of  males  per  hundred  females)  for  those 
of  known  sex  is  114.93  ±  3- 19-~  lt  is  interesting  to  note  here 
that  the  sex  ratio  for  the  data  from  1915  to  [922  was  97.37  ±  1.93. 

1  Sumner,   McDaniel  and  Huestis,  BIOL.   BULL.,  No.  2,   1922. 

D 

-The  probable  error  here  employed  is   ±67.45    (i  +  R)  <J—,  in  whicn 
R  =  sex  ratio. 


JOHN    J.    KAROL. 

Since  the  number  of  individuals  considered  here  is  about  half 
as  great  as  that  in  the  previous  paper  on  Peromyscus,  we  shall 
present  the  data  of  this  later  period  only  for  what  they  may  be 
worth.  At  the  suggestion  of  Dr.  Sumner  it  was  considered  per- 
missable  to  combine  these  additional  data  with  the  earlier  records 
and  thus,  in  a  sense,  bring  some  of  the  results  on  the  sex  ratio  of 
Pcromyscus  up  to  date. 

The  total  number  of  broods  in  the  combined  data  from  1915 
to  1926  is  2,321,  comprising  7,547  young,  or  an  average  of  3.25 
mice  per  brood.  According  to  sex  these  were  distributed  as  fol- 
lows : 

Males    3-597 

Females     3,49^ 

Sex  undetermined   (dead  or  escaped)    458 

The  sex  ratio  for  the  combined  data  is  thus  103.01  ±  1.64. 

SEASON. 

The  following  table  gives  the  sex  ratio  for  each  month  of  the 
year  and  also  the  number  of  individuals  upon  which  this  ratio  is 
based.  The  table  contains  the  total  data  for  the  years  1922-1926. 

k 

January    ( 162)    123.61  +  13.29 

February    (154)     120.59  i  !3-21 

March    (360)     106.43  +    7.64 

April    (290)     H4-39  i    8.94 

May    (390)     1 10.50  +    7.65 

June    (277)     123.77  +  10.12 

July     (184)     111.90+11.30 

August    (220)     143.68  +  13.50 

September    ( 140)    140.35  +  19.42 

October    (143)     92.96  +  10.67 

November    (109)     76.67  +  10.15 

December    (68)     120.00  +  19.30 

As  it  is  obvious  from  the  graph  that  the  differences  between  the 
consecutive  months  are  of  little  significance  we  may  combine  our 
monthly  birth  records  into  four  seasons  of  three  months  each. 
In  both  the  earlier  data  alone  and  in  the  combined  data  we  may 
distinguish  two  high  periods  and  two  low  periods  annually.  The 
sex  ratios  for  these  four  periods  applied  to  the  later  data  are  as 
follows : 


THE    SEX    RATIO    IN    PEROMYSCUS. 


153 


1 I )  February-April     1 1 1.86  +  5.43 

(2)  May-July    1 14.99  ±  5-36 

(3)  August-October    126.55  ±  7-77 

(4)  November-January    105.56  +  7.92 

The  greatest  difference  between  two  of  these  ratios  is  that  be- 
tween the  third  and  fourth  periods  This  difference  is 
20.99  —  1 1-09. 


145 

140 

135 

130 

125 

120 

115 

110 

105 

100 

95 

90 

B5 

80 

75 


137 


553 


JAN    FEE    MAR    APR   MAY   JUN    JUL    AUG   SEP    OCT   NOV    DEC 


FIG.  I.  The  sex  ratio  of  Pcrotiiyscits  for  each  month  of  the  year  com- 
puted for  the  data  from  1922  to  1926.  The  figures  along  the  graphs  denote 
the  number  of  individuals  born  during  each  month  of  the  year. 

Grouping  the  same  data  according  to  the  seasons  of  the  year,  as 
employed  by  King  and  some  others,  we  get  the  following  sex 
ratios : 

Spring     1 10.12  +  4.90 

(March-May) 
Summer  126.28  +  6.71 

(June-August) 
Autumn    102.13  +  3.08 

(  September-November ) 
Winter   121.76  +  8.53 

(December-February) 

Here  the  greatest  difference  is  between  summer  and  autumn, 
being  in  this  case  24.15  ±  7.38.  Inasmuch  as  our  figures  are 
small  we  make  no  attempt  to  attach  any  particular  significance 


JOHN    J.    KAROL. 

to  these  values  but  we  may  say  in  passing  that  they  are  of  the 
same  order  of  magnitude  as  the  findings  of  King  1  in  the  Norway 
rat.  In  both  we  find  a  maximum  in  summer  followed  by  a  mini- 
mum in  autumn. 

Combining  the  earlier  data   (1915-1921)   with  these  additional 
data  we  get  the  following  monthly  sex  ratios : 

January   (395)    103.53  +  7.41 

February    (469) 99-54  ±  6.17 

March    (1,129)     106.26  +  4.30 

April    (660)     1 13-65  +  6.20 

May    (967)     101.13  +  4-61 

June    (707)     100.59  ±  5-29 

July    (592)    98.11  +  5.74 

August    (818)    113.46  +  5.46 

September    (617)    108.45  +  6.03 

October    (564)     103.3?  ±  6.02 

November    (367)     78.12  +  5.76 

December    (279)     96.38  +  7.93 


145 

140 

135 

130 

125 

120 

115 

110 

105 

100 

95 

90 

85 

80 

75 


6Z6 


346 


342 


JAN   FEB   MAR    APR    MAY    JUN    JUL    AUG    SEP    OCT    NOV    DEC 

FIG.  2.  The  sex  ratio  of  Pcromyscus  for  each  month  of  the  year  com- 
puted for  the  combined  material  from  1915  to  1926.  Numbers  along  graphs 
indicate  numbers  born  in  each  month. 

•  Here,  as  in  the  earlier  data  alone,  we  find  two  annual  maxima, 
one  occurring  in  March  and  April;  the  other  from  August  to  Oc- 

1  Arc hir  fiir  Entwickungsmeckanik,  1927,  61. 


'I  UK    SEX    RATIO    IN    PEROMYSCUS.  155 

tober.      In   the   graph   we  have  the   appearance   of    a    fairly   well 
marked  biennial  rhythm. 

Now  grouping  the  combined  data  according  to  3-month  periods 
we  find  the  following  sex  ratios : 

1 i )  February-April     106.96  +  3.07 

(2)  May-July   100.19  +  2.96 

(3)  August-October    107.87  +  3.36 

(4)  November-January    91.80  +  4.01 

The  difference  between  the  third  and  fourth  periods  is 
16.07  —  5-23  ar>d  may  be  considered  of  probable  significance 
according  to  the  conventional  statistical  standard.  These  figures 
still  show  a  rather  marked  biennial  rhythm  despite  the  fact  that 
the  later  data  showed  reversed  relations  for  the  February-April 
period. 

Again,  if  we  regroup  the  combined  data  by  the  ordinarily  rec- 
ognized seasons  the  figures  become : 

Spring    106.22  +  .2.78 

(March-May) 
Summer  104.76  +  3.18 

(June-August) 
Autumn   97.43  +  3.45 

(  September-November ) 
Winter 100.00  +  4.18 

( December-February) 

Here  the  greatest  difference,  between  spring  and  autumn,  is 
8.79  ±  4.43  and  of  no  probable  significance.  Likewise  the  bi- 
ennial rhythm,  apparent  in  the  case  of  the  later  data  seems  to 
have  been  eliminated  by  the  addition  of  the  earlier  data.  This, 
we  may  say,  is  typical  of  the  conflicting  results  pervading  the  entire 
literature  on  the  sex  ratio. 

In  the  previous  paper  on  the  sex  ratio  in  Peromyscus  it  was 
stated  that  the  records  were  "  unfortunately  not  adapted  to  re- 
vealing definite  periods  of  increased  or  diminished  reproductive 
activity,  since  the  matings  were  to  a  large  extent  controlled  in 
accordance  with  the  demands  of  the  breeding  experiments." 
Since  this  statement  is  equally  applicable  to  the  later  data,  we 
wish  to  stress  the  point  that  only  the  number  of  matings  was 
controlled  and  we  cannot  understand  how  this  could  possibly 
affect  the  normal  seasonal  trend  of  the  sex  ratio1 

1  Cf.  King,  1927. 


156 


JOHN  J.  KAROL. 
SIZE  OF  THE  BROODS. 


The  mean  size  of  the  760  broods  considered  in  the  later  data 
i-  3.32.  The  following  table  gives  the  sex  ratios  for  mice  be- 
longing to  broods  containing  from  one  to  seven  individuals  re- 
spectively. Double  broods  or  broods  in  which  individuals  of 
unknown  sex  are  known  to  have  died  have  been  excluded. 


No.  in  Brood. 

Males. 

Females. 

Ratio. 

i 

17 

12 

141.67  ±  36.07 

2 

119 

103 

iiS-53  ±  10.44 

3 

423 

336 

125.89  ±    6.25 

4 

352 

3i6 

111.39  ±    5.83 

5 

141 

129 

109.30  ±    9.02 

6 

59 

3i 

190.32  ±  28.36 

7 

18 

17 

105.88  ±  24.17 

Summarizing  the  combined  data  we  get  the  following  table  for 
the  sex  ratios  according  to  the  size  of  the  brood : 


Xo.  in  Brood. 

Males. 

Females. 

Ratio. 

i 

81 

73 

110.96  ±  12.09 

2 

35i 

355 

98.87  ±    4.96 

3 

1,047 

993 

105.44  ±    3-18 

4 

1,029 

983 

104.68  ±    3.18 

5 

405 

385 

105.19  ±    4.97 

6 

159 

in 

143.24  ±  11.96 

Considering  either  the  single  or  combined  data  we  can  find  no 
significant  differences  in  the  sex  ratios  of  various  sized  litters  and 
we  can  only  conclude  that  the  size  of  the  brood  does  not  seem  to 
have  any  well-defined  relation  with  the  sex  ratio  in  Pcromyscus. 

Separate  calculations  were  made  for  the  litters  in  which  no 
deaths  were  recorded  and  for  the  litters  in  which  deaths  are 
known  to  have  occurred.  In  the  later  data  we  find  the  sex  ratio 
for  incomplete  broods,  comprising  43  broods,  to  be  83.64  ±  11.26. 
For  the  673  complete  broods  the  sex  ratio  is  118.09  +  3.38— 
the  difference  between  incomplete  and  complete  broods  being 
3445  —  H-7^-  \Yhile  this  difference  is  large  enough  to  be  of 
interest  we  cannot  attach  any  great  significance  to  it  inasmuch  as 
only  43  incomplete  broods  were  considered.  In  the  combined 
data  we  find  sex  ratios  of  91.45  ±  4.76  and  104.65  ±  1.79  based 


THE    SEX    RATIO    IN    PEROMYSCUS. 


157 


on  309  and  1,974  broods  for  the  incomplete  and  complete  broods 
respectively.  Thus  we  do  find  a  difference  between  the  sex  ratios 
of  complete  and  incomplete  broods  but  we  do  not  feel  justified 
in  regarding  it  as  significant  in  view  of  the  meagre  record  of 
identified  dead. 


145 
140 
135 
130 
125 
120 
115 
110 
105 
100 
95 


270 


790 


2012 


706 


1 

FIG.  3.  Variations  in  the  mean  sex  ratio,  according  to  the  size  of  the 
broods.  Numbers  along  graphs  indicate  numbers  of  individuals. 

Combinations  of  the  Sc.vcs  in  Individual  Hroods. 

It  is  interesting  to  consider  the  possible  tendency  of  members 
of  a  litter  to  agree  with  one  another  in  respect  to  sex,  that  is, 
whether  or  not  we  encounter  broods  consisting  entirely  of  the 
same  sex  more  frequently  than  would  result  from  chance.  In  the 
following  table,  using  the  combined  data,  we  have  arranged 
broods  of  each  size  in  groups  according  to  the  number  of  each 
sex  present.  For  example,  broods  of  three  present  four  possible 
combinations :  3  <5  ,  2  £  +  i  9  ,  r  <J  -j-  2  9,39.  The  actual  num- 
ber of  complete  broods  containing  a  given  combination  of  males 


158 


JOHN    J.    KAROL. 


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TIIK    SKX     RATIO    IN    PEROMYSCrS. 


ami  females  and  the  "  expected  "  number  to  the  nearest  integer 
are  computed.  Since  the  percentage  of  males  in  the  combined 
data  is  50.74,  I  have  computed  these  last  figures  by  expanding 
the  binomial  (1.015  -)-  .985)".  In  the  case  of  an  equality  ratio 
we  should  use  the  ordinary  formula  for  probability,  e.g.,  (i  -f-  i)n. 
Considering  the  comparatively  small  number  of  broods  present 
in  most  of  the  groups  we  find  a  rather  close  agreement  between 
the  actual  and  the  expected  figures  for  all  of  the  broods  in  which 
all  members  were  of  the  same  sex.  The  actual  number  of  such 
homosexual  litters,  among  broods  containing  from  2  to  6  indi- 
viduals inclusive,  was  409;  while  the  most  probable  number  on 
the  assumption  of  purely  random  sex-production,  was  422.  If 
we  consider  fractions  (a  more  exact  procedure)  this  last  figure 
becomes  420.  In  the  earlier  data  alone  a  closer  agreement  than 
this  was  found,  the  figures  being  276  and  274  for  the  actual  and 
expected  number  of  broods  respectively.  It  would  appear  that 
the  distribution  of  the  sexes  in  single  broods  follows  the  laws  of 
chance  and  there  seems  to  be  no  tendency  for  fetuses  (or  germ 
cells)  developing  in  the  same  parents  at  the  same  time  to  give 
rise  to  organisms  of  the  same  sex.  We  may  likewise  reiterate  the 
conclusion  of  Sumner,  McDaniel  and  Huestis,  namely ;  ;'  the 
non-occurrence  of  polyembrony  or  true  twinning,  at  least  with 
sufficient  frequency  to  afreet  the  results." 

RACE. 

For  the  later  data  we  have  computed  the  sex  ratio  separately 
for  the  "pure"  (non-hybrid)  polionotus  series  and  in  the  fol- 
lowing table  we  have  listed  in  addition  the  sex  ratios  for  some  of 
the  other  geographic  races  (subspecies)  as  computed  by  Sumner, 
McDaniel  and  Huestis. 


Subspecies. 

Males. 

Females. 

Ratio. 

polionolus 

1  20 

89 

i  *4.8  }  ±  I  3.  IS 

e,ambeli  (La  Jolla)                 

77° 

840 

91.67   dh     3.07 

sotioriensts                                    .           .... 

SSo 

373 

93.83  ±     4.70 

rubidus 

ISO 

124 

120.97  ±    9-91 

The     difference    between     "  polionotus ':     and     "  gambeli "     is 
43.16  ±  13.50  and  may  possibly  be  regarded  as  significant.     But 
11 


JOHN    J.    KAROL. 

-f  cannot  say  definitely  that  these  figures  imply  the  existence  of 
any  actual  racial  differences  with  regard  to  the  sex  ratio. 

It  is  interesting  to  also  observe  here  that  in  the  subspecific  hy- 
brids of  Pcromyscus  pblionotus  we  find  a  sex  ratio  of 
114.61  ±  5.79  while  the  subspecific  hybrids  considered  in  the 
earlier  report  (mainly  P.  maniculatus)  give  a  mean  sex  ratio  of 
only  104.76  ±  3.41. 

Parkes  '  briefly  summarizes  the  data  of  many  workers  on 
specific  variations  in  the  sex  ratio  in  man  and  other  mammals. 

HYBRIDIZATION. 

In  235  broods  comprising  735  individuals  of  Fx  hybrids  in 
the  later  series  we  find  a  sex  ratio  of  114.61  ±  5.79.  While  this 
is  lower  than  the  ratio  for  the  pure  "  polionotus  "  stock  (see  p. 
159),  we  cannot  attach  any  significance  to  the  latter  figures  since 
they  are  so  small.  For  the  same  reason  we  do  not  feel  justified 
in  combining  the  later  group  with  the  earlier,  in  a  comparison  of 
pure  and  hybrid  ratios.  We  may  say,  however,  that  in  the  earlier 
series  alone  the  difference  between  the  ratios  for  pure  and  hybrid 
stock  was  found  to  be  11.49  ±4.1,  tne  hybrid  series  giving  the 
higher  ratio.  These  results  are  in  agreement  with  the  conclu- 
sions reached  by  other  workers,  e.g.,  Pearl  (1908),  King  (1911), 
and  Little  (1919),  that  hybridization  "per  se  "  may  result  in 
raising  the  sex  ratio. 

THE  YEAR. 

The  sex  ratios  and  the  number  of  individuals  upon  which  they 
are  based  for  the  year  1922-1926  are  as  follows : 

1922  (296)  106.34  +  8.33 

1923  (355)   106.43  ±  7.64 

1924  (519)  120.80  +  7.27 

1925  (966)  II3-93  +  5-03 

1926  (386)  125.60  +  8.80 

Although  it  is  quite  evident  that  there  are  no  significant  differ- 
ences here  it  was  thought  worth  while  to  present  the  figures  in 
view  of  the  fact  that  the  earlier  data  on  Peromyscus  (1915-1921) 
showed  such  marked  yearly  variations.  While  these  results  were 

]  A.   S.  Parkes,  "  The  Mammalian  Sex  Ratio,"  Biol.  Review,  Vol.   II., 
No.  i,  Nov.,  1926. 


THE    SEX    RATIO    IN    PEROMYSCUS.  l6l 

inexplicable,  they  were  statistically  speaking,  the  most  significant 
of  all  and  the  likelihood  of  obtaining  one  of  the  differences  by 
"  accident  "  was  less  than  one  in  40,000.  It  was  further  proven 
that  these  differences  were  "  not  due  either  to  the  seasonal  dis 
trilmtion  of  births,  to  the  preponderance  of  hybrid  births  in  one 
year  as  compared  with  another,  or  to  the  operation  of  any  of  the 
other  factors  previously  considered." 

Inasmuch  as  it  is  evidently  exceedingly  difficult  to  correlate  the 
annual  variation  in  the  sex  ratio  with  any  known  influences, 
accurate  data  on  the  subject  are  generally  lacking.  Of  course  it 
is  not  impossible  that  the  most  "  significant  "  figures  may  result 
from  chance. 

SUMMARY. 

Data  have  been  presented  based  upon  2,522  deer  mice  as  re- 
corded during  the  breeding  experiments  of  Dr.  Sumner,  from 
1922  to  1926.  Earlier  records  (1915-1921)  were  added  to  the 
above  and  the  combined  data  have  also  been  presented. 

The  following  results  seem  to  be  of  most  importance. 

1.  The  mean  size  of  760  broods  in  the  later  records  is  3.32. 
For  the  combined  data  comprising  2,321  broods  the  mean  size  is 
3.25  mice  per  brood. 

2.  The   sex   ratio    for   the   later   data   is    114.93  —  3-I9>   while 
that  for  the  entire  lot  is  103.01  ±  1.64. 

3.  Considering  the  possibility  of  a  seasonal  cycle  in  the  pro- 
portion of  males  and  females  born,  we  can  only  say  that  we  find 
in  the  later  data  a  maximum  sex  ratio  in  the  August-September 
period  followed  by  a  minimum  during  October  and  November. 
In  the  combined  data  we  find  two  annual  maxima,  one  occurring 
in  March  and  April,  the  other  from  August  to  October,  and  hence 
a   fairly  well  marked  biennial  rhythm.     Grouping  the  combined 
material  according  to  3-month  periods  we  find  in  one  arrange- 
ment that  the  biennial  rhythm  is  practically  eliminated  while  in 
another  it  is  rather  well  marked.     The  existence  of  a  seasonal 
cycle  in  the  sex  ratio  of  Peromyscus  is  not  definitely  proved. 

4.  The  size  of   the  brood  in  the  combined  material  does  not 
seem   to   have   any   well   defined   relation   with   the   sex   ratio   in 
Peromyscus. 

Although  we  find  a  difference  between  the  sex  ratio  of  complete 


JOHN    J.    KAROL. 

and  incomplete  broods  we  cannot  regard  it  as  significant  in  view 
of  the  meagre  records  of  identified  dead. 

5.  When  the  number  of  each  possible  combination  of  males  and 
females,   in  broods  of   each  size,  is  compared  with  the  number 
expected  according  to  chance,  the  conformity  is  found  to  be,  on 
the  whole,  very  close.     For  example,  if  we  compare  the  actual  and 
expected  totals  for  all  of  the  broods  in  which  all  members  were 
of  the  same  sex  we  find  409  as  the  actual  number  and  420,  the 
"  expected  "'   number.     Thus  there  is  no  preponderant  tendency 
toward  the  production  of  homosexual  litters  and  thus  the  non- 
occurrence  of  polyembrony  or  true  twinning  to  any  great  extent. 

6.  While  the  sex  ratio  for  the  three  subspecies  of  polionotus  is 
"  significantly  '    higher  than  that  for  other  pure  races  of  Pcro- 
myscus  we  cannot  say  definitely  that  these  figures  imply  the  ex- 
istence of  any  actual  racial  differences  with  regard  to  the  sex  ratio 
in  Peromyscus.     The  sex  ratio  of  polionotus  hybrids  is  likewise 
considerably  higher  than  that  of  other  Peromyscus  hybrids  which 
have  been  studied.  4 

7.  No  significant  yearly  variations  were  found  in  the  sex  ratio 
of  Peromyscus  from  1922  to  1926. 


COLD  HARDINESS  IN  THE  JAPANESE  BEETLE, 
POPILLIA  JAPONICA  NEWMAN. 

NELLIE  M.  PAYNE, 
NATIONAL  RESEARCH  FELLOW  IN  THE  BIOLOGICAL  SCIENCES. 

Cold  hardiness,  or  the  ability  of  an  organism  to  withstand  low 
temperature  may  be  considered  from  two  points  of  view,  (i) 
cold  hardiness  to  the  intensity  factor  or  the  ability  to  survive  ex- 
treme low  temperatures,  and  (2)  cold  hardiness  to  the  quantity 
factor  or  ability  to  withstand  long  periods  of  low  temperature. 
By  low  temperature  is  meant,  temperature  below  that  required  for 
normal  development. 

The  Japanese  beetle,  which  was  introduced  into  the  United 
States  about  1916,  can  be  secured  in  large  numbers,  thus  making 
intensive  study  possible.  This  insect  represents  a  type  of  ecolog- 
ical group,  the  soil  dwelling  insects.  It  passes  the  winter  in  the 
larval  stage;  about  97  per  cent,  in  the  third  instar;  about  3  per 
cent,  in  the  second.  Cold  hardiness  to  both  the  quantity  and  in- 
tensity factor  of  low  temperature  was  studied.  Both  external 
and  internal  factors  are  involved  in  cold  hardiness.  These  in- 
clude such  environmental  factors  as  relative  humidity  and  tem- 
perature, and  such  physiological  conditions  as  nutritional  state, 
health,  blood  conductivity  and  metabolic  rate.  Most  of  the  work 
was  done  on  larvae.  Some  studies  were  made  on  adults  and  a  few 
observations  were  made  on  cold  hardiness  in  pupae. 

METHODS  AND  APPARATUS. 

Respiratory  rate  and  quotient  were  determined  by  the  modified 
Krogh  manometer  of  Bodine  and  Orr  (1925).  Conductivity  of 
blood  and  body  fluids  was  determined  by  the  ionometer,  described 
by  Gram  and  Cullen  (1923).  pH  was  determined  with  the  type 
K  potentiometer,  using  a  small  vessel  capable  of  testing  the  pH  of 
a  drop.  By  this  method  several  readings  could  be  taken  on  the 
same  larva.  This  method  was  described  by  Bodine  and  Fink 

163 


NELLIE    M.    PAYNE. 

(1925).  Occasionally  a  larva  was  found  that  would  not  bleed 
freely  enough  to  give  sufficient  blood  for  a  reading.  Blood  was 
usually  taken  from  one  of  the  feet.  Relative  humidity  was  main- 
tained by  pulling  air  over  different  concentrations  of  sulfuric  acid 
by  means  of  a  suction  pump. 

COLD  HARDINESS  TO  THE  INTENSITY  FACTOR  OF  Low  TEMPERA- 
TURE. 

In  comparison  with  the  oak  borers  previously  studied  by  the 
author,  Payne  (1926),  the  Japanese  beetles  are  less  cold  hardy  and 
also  exhibit  less  variation  to  low  temperature.  In  Pennsylvania 
the  most  cold  hardy  Japanese  beetle  withstood  -  -  28°  C. :  the  most 
cold  hardy  oak  borer  -  -  47°  C.  The  most  cold  hardy  Japanese 
beetle  collected  in  the  field  thus  far  withstood  -  -  15°  C. 

Periodicity  in  cold  hardiness  to  the  intensity  factor  of  cold  is 
not  as  marked  in  the  Japanese  beetle  as  in  the  oak  borers  Synchroa 
punctata  and  Dendroidcs  canadcnsis.  Comparison  of  the  three 
species  in  question  tested  at  the  same  dates  is  shown  in  Table  I. 

Conditions  other  than  seasonal  which  modify  the  cold  hardiness 
of  the  Japanese  beetle  to  the  intensity  factor  of  low  temperature 
are  (i)  degree  of  dehydration,  (2)  disease  incidence,  (3)  nu- 
tritional state,  and  (4)  temperature  at  which  the  larvae  were 
kept.  Although  these  larvae  are  seldom  collected  in  dry  places 
normally,  they  are  able  to  withstand  a  high  degree  of  dehydra- 
tion. Larvae  dried  down  to  a  pulpy  condition  in  which  the  free 
water  is  reduced  to  a  minimum  are  cold  hardy  to  both  intensity 
and  quantity  factors  of  low  temperature.  Severe  dehydration  is 
accompanied  by  a  high  death  rate.  Larvae  can  be  dried  down  to 
one  third  of  their  body  weight.  In  the  dehydrated  condition  the 
Japanese  beetle  larvae  reach  their  greatest  cold  hardiness.  Since 
eighty  per  cent,  of  dehydrated  larvae  die  the  effect  of  dehydration 
may  be  considered  highly  selective,  killing  off  those  larvae  unable 
to'hold  water.  Those  larvae  capable  of  resisting  dehydration  are 
cold  hardy.  Relative  humidity  affects  cold  hardiness  in  a  de- 
cided manner.  The  results  of  a  series  of  different  experiments 
with  varying  relative  humidities  is  shown  in  Table 


COLD    HARDINESS    IN    THE    JAPANESE    BEETLE. 


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H 

-^. 

OO 
O    ro   1-1 

f 

U 

O    ro  O 

O\ 

IO     *t     HI 

. 

M     O      I- 

Ov 

b 

01 

-H^  -H 

1 

+J 

O, 

i    M 

10   10 

OJ 

c/3 

oj   a-1   g 

r-  -^  cs 
<s  od   ri 

S  !D  o 

1     1 

^  u 

BJ 

OJ 

s 

y 

•2  "w    e 

o 

a 

^    Q  .M 

s. 

R    3    § 

§35 

i  J3 

vj    >3  '** 

1 66 


NELLIE    M.    PAYNE. 


TABLE  II. 

1  '.\DucrrviTY  OK  BLOOD  OF  JAPANESE  BEETLE  LARVAE  KEPT  AT  DIFFERENT 

TEMPERATURES. 

(Conductivity  shown  in  %  NaCl  equivalent  uncorrected  for  protein.) 


o°C. 

10°  C. 

20-22°  C. 

25°  C. 

•65 

.6 

.38 

•33 

.68 

.61 

-45 

•35 

.72 

.604 

.42 

•38 

.64 

•58 

.41 

•39 

.70 

.604 

.42 

.40 

.69 

•55 

.41 

.41 

.67 

•58 

-45 

-375 

.66 

.6 

•44 

•39 

.68 

.6 

•435 

.40 

•  71 

.46 

.42 

•45 

•39 

-43 

.41 

•445 

Starvation  at  high  temperatures,  20°  C.  or  above,  is  fatal 
to  the  larvae  unless  the  relative  humidity  is  kept  high.  When 
kept  at  high  humidity,  larvae  are  able  to  withstand  comparatively 
long  periods  of  starvation.  One  hundred  larvae  were  kept  with- 
out food  for  the  month  of  May,  1927,  but  under  conditions  of 
100  per  cent,  relative  humidity  or  saturation.  Each  larva  was 
placed  in  an  individual  vial  and  weighed  before  and  after  the 
starvation  period.  During  the  process  they  lost  about  one  half 
of  their  body  weight.  None  of  them  survived  freezing,  the  low- 
est freezing  point  was  -  -  1.7  'C. ;  the  highest  -  -.65°  C.  Larvae 
kept  at  -|-  io°C.,  or  below  their  developmental  temperature,  lost 
one  half  of  their  body  weight.  Starvation  conditions  were  as- 
sured by  keeping  the  larvae  in  sterile  white  sand  kept  moist 
with  distilled  water.  About  one  fourth  of  these  larvae  survived 
freezing.  Changes  in  body  weight  under  different  conditions  of 
starvation  and  dehydration  are  shown  in  Fig.  i.  The  effect  of 
prolonged  exposure  to  low  temperature  as  well  as  starvation  was 
involved  in  the  experiment  described  above.  The  effect  of  differ- 
ent temperatures  on  cold  hardiness  as  measured  by  blood  con- 
ductivity is  shown  in  Table  "Hrl.  Larvae  starved  for  one  week  at 
-f  20°  C.  increased  in  cold  hardiness.  In  general  early  stages 
of  starvation  are  marked  by  (an  increase  in  cold  hardiness,  later 


COLD    HARDINESS    IN    THE    JAPANESE    BEETLE. 


I67 


BODY   WEI 

(0(o 

rtu 

WEIGHT 

2  MONT 
PERATUR 

POPlLLI/> 

I 

J 

or  LJ 

$ 

(T 

u  h 
h- 

LJ 

< 

<0 

O(- 

1  —  —  1 

rr5(0 

NORMAL 

ARVAL  WEIGH 
TARVATION  AT 

WEIGHT  AFTEI 
H'S  STARVATION 
TEMPERATURE 

i 

b  -i 

-JCO 

<t;i: 

L_    Q 

t  LJ 

cz^ 

CD  <£ 

LJ  cr 

j 

>  > 

n 

LiJ 

Q 

IN 


FIG.  i. 


158  NELLIE    M.    PAYNE. 

TABLE  III. 

EFFECT  OF  DIFFERENT  RELATIVE  HUMIDITIES  ON  CONDUCTIVITY  AT 

TEMPERATURE  OF  22°  C. 
(Conductivity  shown  in  %  NaCl  equivalent  uncorrected  for  protein.) 


Saturation. 

80%. 

50%. 

•33      

46 

72 

.38      

•  .45     -  • 

75 

•35       

•47    

76 

•39     

465   

78 

•40     

44     

73 

.41 

455   

77 

•375   

..   -46     

74 

•39     

44     

76 

40     

455   

77 

•375  

46     

76 

•38     

45     

82 

.32     

46     

77 

•445   

78 

•43 

stages  by  a  decrease.  The  point  of  decrease  in  cold  hardiness 
from  starvation  comes  when  the  digestive  tract  clears.  In  con- 
nection with  this  observation  it  is  interesting  to  note  that  freshly 
molted  larvae  are  unable  to  withstand  freezing  until  they  have 
eaten.  Pre-pupae  with  clear  digestive  tracts  are  not  cold  hardy. 

The  occurrence  of  wilt  disease  in  many  of  the  specimens  col- 
lected in  the  field  offered  an  opportunity  for  the  study  of  the 
effect  of  this  disease  on  cold  hardiness.  Larvae  were  collected  at 
the  same  date  and  subjected  to  the  same  conditions  of  temperature 
and  relative  humidity,  only  healthy  larvae  were  studied.  No 
larva  showing  typical  symptoms  of  wilt  disease  or  polyhedral- 
skrankheit  was  able  to  survive  freezing.  Since  thermocouples 
used  in  diseased  larvae  were  difficult  to  sterilize  and  might  infect 
healthy  larvae,  cold  hardiness  was  studied  by  measuring  blood 
conductivity  rather  than  freezing  point  depression.  Conductiv- 
ity decreases  as  the  disease  progresses.  On  the  first  day  of 
apparent  infection,  conductivities  of  blood  of  diseased  larvae  were 
below  that  of  healthy  larvae.  To  produce  such  a  marked  change 
on  the  first  day  of  infection,  the  causative  organism  must  affect 
the  blood  very  profoundly  and  very  rapidly.  On  the  other  hand 
the  change  in  conductivity  may  not  be  as  rapid  as  it  appears.  The 
disease  may  be  present  in  larvae  before  it  is  detected  by  discol- 


COLD    HARDINESS    IN    THE    JAPANESE    BEETLE. 


169 


\35 

\ 
\ 
\ 


RELATION   BETWEEN  WILT 
DISEASE  AND  CONDUCTIVITY 
FIG.  £ 


OD 
IT 

-§      20 
o 
(O 

c 


15 


o 
O 


10 


Period  of  Apparent  Infection  in  Days 


7 


8 


FIG.  2. 


10 


oration  or  wilting,  and  may  be  producing  conductivity  changes  in 
the  blood  before  other  symptoms  can  be  observed.  A  graph 
showing  the  relationship  between  day  of  apparent  infection  and 
blood  conductivity  is  shown  in  Fig.  2..  Table  IV.  shows  the  re- 


-Q  NELLIE    M.    PAYNE. 

TABLE  IV. 

CONDUCTIVITY  OF   HEALTHY   AND  DISEASED  JAPANESE  BEETLE 
(Conductivity  shown  in  %  NaCl  equivalent  uncorrected  for  protein.) 
Wilt  Disease.  Healthy.  Blackened  by  Freezing. 


OT 

.38     

•  6 

T7 

.4 

6 

•l/      

TO 

.41 

604 

T  T 

.4                 ... 

604 

58 

375 

575 

(17 

43 

61 

•w      

18 

.425 

63 

12 

.41 

62 

06 

."?Q 

625 

.4. 

64 

-25 
.27 

•15 

suits   of   conductivity   readings   made   on   the   blood   of    diseased 
larvae  in  comparison  with  healthy  ones. 

Wilt  disease  is  characterized  by  a  pronounced  blackening  that 
precedes  the  final  softening  that  occurs  just  before  death.  Black- 
ening also  has  been  observed  when  larvae  are  frozen  and  thawed 
quickly.  Blood  from  larvae  blackened  after  thawing  always 
showed  high  conductivity.  In  these  cases  discoloration  was  be- 
lieved to  be  due  to  changes  in  cell  permeability  releasing  certain 
oxidative  enzymes,  which  on  escaping  blackened  the  cells.  The 
prothoracic  segment  is  the  first  portion  of  the  larvae  to  discolor 
.after  freezing,  both  in  the  Japanese  beetle  and  in  the  oak  borers 
studied.  Changes  in  permeability  could  be  observed  during  the 
thawing  process.  Water  apparently  passes  through  the  body  wall 
where  the  chitin  is  thinnest.  This  water  was  frequently  reab- 
sorbed  when  the  larva?  were  kept  under  small  bell  jars.  Larvae 
losing  water  alone  were  generally  able  to  survive  freezing.  When 
the  fluid  exhuding  from  the  larva  gave  tests  for  amino-acids  or 
proteins  the  larvae  always  died.  The  exudate  remained  colorless 
for  several  days  unless  hydrogen  peroxide  was  added,  in  which 
case  it  blackened  quickly.  Larvae  which  showed  the  exudate 
after  thawing  were  fixed  and  sectioned,  but  in  these  sections  no 
gross  differences  from  normal  tissue  could  be  detected.  Broken 


COLD    HARDINESS    IN    THE    JAPANESE    BEETLE.  1JI 

cell  walls  were  not  in  evidence.  The  direct  cause  of  death  from 
extreme  low  temperature  has  been  interpreted  as  due  to  an  irre- 
versible change  in  permeability  rather  than  to  a  breaking  of  the 
cell  walls. 

If  larvae  capable  of  surviving  low  temperature  are  ground  up 
and  filtered  and  the  filtrate  precipitated  with  lead  acetate,  there 
occurs  in  the  filtrate  an  enzyme  capable  of  breaking  proteins  down 
to  amino  acids  at  low  temperatures  and  of  building  up  proteins 
from  amino  acids  at  high  temperatures.  A  similar  enzyme  has 
also  been  found  in  tussock  moth  eggs.  Reversible  reactions  with 
proteases  have  been  reported  by  Abderhalden  (1914)  from  auto- 
lyzing  tissues.  Taylor  (1909),  found  that  a  protein—  ;' plastein  " 
—could  be  formed  from  albuminose  and  proteolytic  enzymes. 
The  reversible  reaction  of  starch  to  sugar  at  low  temperatures 
and  sugar  to  starch  at  high  temperatures  is  a  well  known  reaction 
that  takes  place  in  potato  storage.  The  cold  hardy  mechanism 
of  these  larvae  studied  may.  in  part,  be  due  to  enzyme  action  which 
transforms  large  protein  molecules  into  smaller  amino-acids.  The 
larger  number  of  osmotically  active  units  thus  formed  would  lower 
the  freezing  point. 

Periodicity  to  cold  hardiness  is  not  as  marked  in  the  Japanese 
beetle  as  it  is  in  some  of  the  insects  that  are  exposed  to  extremes 
of  low  temperature.  Larvae  of  the  Japanese  beetle  live  close 
enough  to  the  surface  of  the  ground  to  experience  some  seasonal 
change.  During  the  spring  and  fall  they  are  in  addition  subjected 
to  diurnal  temperature  change.  Cold  hardiness  in  the  larvae  ap- 
pears to  be  closely  related  to  their  environment.  These  or- 
ganisms are  somewhat  seasonal  in  their  resistance  to  low  tem- 
peratures. This  periodic  cold  hardiness  is  shown  in  Table  I. 
Comparison  with  oak  borers  and  aquatic  insects  is  shown  more 
fully  in  a  previous  article  by  the  author  (Payne,  1926).  Al- 
though the  larva  stage  is  the  only  one  which  overwinters  in  this 
climate,  it  was  thought  that  studies  on  the  cold  hardiness  of  the 
adults  would  yield  valuable  material  for  the  comparison  of  a  stage 
exposed  to  winter  conditions  and  a  stage  not  normally  exposed. 
Adults  captured  in  summer  and  frozen  without  previous  condi- 
tioning were  able  to  survive  ice  formation  within  their  tissues 
and  to  survive  temperatures  as  low  as  -  -  20°  C.  Since  it  was  im- 


172 


NELLIE    M.    PAYNE. 


possible  to  obtain  enough  blood  from  the  adults  to  make  a  con- 
ductivity reading  none  were  made. 

A  beginning  was  made  on  the  study  of  cold  hardiness  of  the 
Japanese  beetle  pupae.  From  present  observations  the  age  of  the 
pupae  and  consequently  the  degree  of  hydrolysis  they  are  under- 
going determines  cold  hardiness. 

No  changes  in  blood  pH  were  found  to  be  associated  with  cold 
hardiness  in  healthy  larvae.  The  pH  obtained  from  a  series  of 
blood  samples  is  shown  in  Table  V.  In  the  early  stages  of  wilt 

TABLE  V. 
PH  OF  JAPANESE  BEETLE  LARWE  BLOOD  THIRD  INSTAR. 

Each  reading  is  an  average  of  3. 

Healthy.  With  Wilt  Disease. 

6.5 5-8 

6.78  5-7 

6.92  5.56 

6.94  6. 

6.5     : 5-Qi 

7.16   5-82 

7-i8   5-83 

6.66    5-97 

6.77    6.1 

7-1      5.84 

7-17    5-92 

7 5-96 

6-54    5.98 

6.66   5.96 

6.82   5.94 

7 5-9 

6-35   •  5-84 

6.51    5-97 

7-i      5-95 

7-i 

disease  the  pH  was  lower  than  in  healthy  larvae.  In  the  late 
stages  of  the  disease  the  larvae  were  in  such  condition  that  it  was 
difficult  to  obtain  blood  by  cutting  off  the  feet. 

The  respiratory  quotient  tends  to  be  high  in  both  cold  hardy 
and  non-cold  hardy  specimens,  ranging  from  .67  to  .72.  The 
respiratory  quotient  of  starving  larvae  tended  to  be  higher  than 
well  fed  larvae  regardless  of  the  temperature  at  which  they  were 
kept.  The  respiratory  rate  in  larvae  in  which  cold  hardiness  had 


COLD    HARDINESS    IN    THE    JAPANESE    BEKTI.K. 


173 


been  induced  was  much  lower  than  in  the  non-cold  hardy  indi- 
viduals. Associated  with  the  low  respiratory  rate  of  hibernating 
forms  was  the  slight  change  in  body  weight  occurring  over  a 
period  of  several  months,  as  shown  in  Fig.  i. 

COLD  HARDINESS  TO  THE  QUANTITY  FACTOR  OF  Low 

TEMPERATURE. 

Both  the  second  and  the  third  instars  of  the  Japanese  beetle 
larva  are  cold  hardy  to  the  quantity  factor  of  low  temperature 
except  directly  after  molting  or  when  the  digestive  tract  is  clear. 
Larvae  are  markedly  adapted  to  withstand  long  periods  of  low  tem- 
perature. At  the  present  writing  there  are  still  ten  larvae  alive 
of  one  hundred  which  were  placed  at  -|-  10°  C.  on  December  6, 
1925.  These  larvae  have  now  been  kept  over  two  years  below 
their  developmental  temperature.  Similar  lots  have  been  kept 
from  six  to  twelve  months  at  -f-  10°  C.  Graphs  showing  the 
number  of  larvae  surviving  plotted  against  time  in  months  in  these 
experiments  are  shown  in  Fig.  3. 

The  relationship  between  survival  for  long  periods  at  low 
temperatures  and  cold  hardiness  to  the  intensity  factor  of  low 
temperature  is  shown  in  Table  VI.  The  two  types  of  cold  hardi- 

TABLE  VI. 

SURVIVAL  AFTER  FREEZING  OF  JAPANESE  BEETLE  LARV.TI. 
Kept  at  constant  temperature  of  +  10°  C.  for  varying  periods  of  time. 


Length  of  Time  Kept 
at  +  10°  C. 

Number 
Frozen. 

Number 
Survived. 

%  Survived. 

2  weeks  

I  J.sO 

1,426 

08.^4 

4  weeks.  .  .  . 

I   4OO 

1,078 

77 

8  weeks  

I  ,OOO 

64=5 

64.  S 

3  months  

5OO 

2QO 

58 

6  months  

2OO 

48 

24 

ness  appear  to  be  inversely  related  after  a  certain  point  has  been 
reached.  This  decrease  in  cold  hardiness  to  the  intensity  factor 
of  low  temperature  cannot  be  interpreted  as  a  simple  loss  in  vital- 
ity since  larvae  kept  at  low  temperatures  are  able  to  complete 
their  development  when  placed  at  room  temperature  with  no 
higher  death  rate  than  larva-  maintained  at  room  temperature. 


174 


NELLIE    M.    PAYNE. 


SURVIVAL  OF  POPILLIA 
AT  10°  C. 


^o 


10 


45678 
TIME  IN  MONTHS 

FIG.  3. 


9        10       II         \& 


Col. I)    HARDINESS    IN    THE    JAPANESE    BEETI.K.  [75 

Long  periods  of  dormancy  accelerate  development  when  the  larva- 
kept  at  low  temperatures  are  raised  to  developmental  tempera- 
tures. Blood  conductivity  at  first  rises,  then  falls  after  two  or 
more  months  when  larvae  are  placed  at  or  below  -)-  10'  C. 

The  effect  of  rapid  alternation  between  high  and  low  tempera- 
tures on  cold  hardiness  was  tried  with  one  hundred  third  instar 
larvae.  Temperatures  of  o°  C.  and  -j-  30°  C.  were  alternated 
every  twenty- four  hours  for  one  month.  Neither  of  these  tem- 
peratures is  fatal.  As  controls  one  hundred  larva-  were  kept  at 
o°  C.  and  one  hundred  at  -j-  30°  C.  None  died  at  -f-  30°  C. 
Those  alternated  between  -|-  30°  C.  and  o°  C.  died  more  rapidly 
than  those  kept  at  o°  C.  Results  of  these  experiments  are  shown 
in  Fig.  4.  In  larva?  which  had  been  exposed  to  wilt  disease  alter- 
nating temperature  had  no  effect  ,on  length  of  life.  None  of 
these  larva;  lived  longer  than  ten  days  except  when  they  were 
kept  at  or  below  o°  C.  Healthy  larvae  were  considered  exposed 
when  they  had  been  bitten  by  larvae  having  wilt  disease. 

The  respiratory  quotient  of  larvae  cold  hardy  to  the  quantity 
factor  of  cold  was  somewhat  variable  but  not  connected  to  length 
of  survival  at  low  temperatures.  In  larvae  with  clear  digestive 
tracts  it  tended  to  become  lower.  In  larvae  kept  at  -(-  10°  C.  it 
ranged  from  .69  to  .73,  or  slightly  higher  than  in  larva-  cold  hardy 
to  the  intensity  factor  of  low  temperature.  In  larva-  with  clear 
digestive  tracts  low  respiratory  quotients  were  associated  with 
lack  of  cold  hardiness. 

Low  respiratory  rate  is  associated  with  cold  hardiness  to  the 
quantity  factor  of  low  temperature.  Changes  in  body  weight,  as 
has  been  stated  before,  were  very  small  with  larva-  kept  for  IOHL; 
periods  of  time  at  +  10°  C.  These  changes  occurring  in  differ- 
ent states  of  nutrition  and  under  varying  temperature  and  hu- 
midity conditions  are  shown  in  Fig.  I. 

Dehydration  of  larvae  is  associated  with  cold  hardiness  to  the 
quantity  factor  of  low  temperature  as  well  as  to  the  intensity  fac- 
tor of  low  temperature.  Dehydration  beyond  two  thirds  of  the 
body  weight  decreases  cold  hardiness  to  the  quantity  factor  of 
low  temperature.  Over  dehydrated  larva-  lived  but  one  day  at 
20°  C.  and  not  more  than  three  days  at  -f  10°  C.  or  not  move 
than  four  days  at  o°  C.  Dehydrated  larvae  have  been  kept  for 
12 


NELLIE    M.    PAYNE. 


\ 


SURVIVAL  OF  POPILLIA 


\ 


\ 


\ 


\ 


\ 


80 


70 


60 


o: 

50        JLJ 

T 

z 


40 


10 


10 


•At   0°  C 

'At  temperatures  alternating 
between  0°and  30°  every ^4  hrs. 


30  40 

TIME   IN  DAYS 

FIG.  4. 


5O  60 


COLD    HARDINESS    IN    THE    JAPANESE    BEETLE.  177 

one  year  at  -f-  10°  C.  The  experiment  has  not  been  continued 
long  enough  to  determine  whether  or  not  dehydration  increases 
the  cold  hardiness  to  the  quantity  factor  of  low  temperature. 
Untreated  larva  are  able  to  live  two  years  or  more  below  their 
developmental  temperature.  Dehydrated  larvae  show  very  nearly 
the  same  death  rate  as  undehydrated  ones. 

LITERATURE. 

Since  the  literature  pertaining  to  cold  hardiness  has  been  re- 
cently brought  together  it  seems  hardly  necessary  to  make  a  de- 
tailed list  and  discussion  of  it.  Robinson  (1927)  has  discussed 
and  given  experimental  data  on  water  binding  capacity  as  a  factor 
in  cold  hardiness.  Robinson  (1926),  and  the  author  (1926)  have 
summarized  the  literature.  Hibernation  in  regard  to  both  its 
ecology  and  physiology  has  been  recently  treated  by  Fink  (1925), 
Townsend  (1926),  and  Holmquist  (1926). 

From  a  survey  of  the  literature  it  would  appear  that  no  one 
factor  is  an  adequate  measure  of  cold  hardiness.  The  develop- 
ment of  a  cold  hardy  from  a  non-cold  hardy  insect  is  a  deep- 
seated  physiological  process  which  affects  blood,  and  body  fluids, 
respiratory  rate  and  permeability.  Nutritional  state  and  environ- 
mental conditions  also  influence  cold  hardiness. 

ACKNOWLEDGMENTS. 

The  facilities  for  this  study  of  cold  hardiness  were  furnished 
by  the  Zoological  Laboratory  of  the  University  of  Pennsylvania. 
To  Dr.  J.  H.  Bodine,  of  the  Department  of  Zoology,  I  owe  thanks 
for  many  helpful  suggestions,  especially  as  regards  apparatus  and 
methods.  To  Dr.  Henry  Fox,  of  the  Japanese  Beetle  Labora- 
tory at  Moorestown,  New  Jersey,  I  am  indebted  for  most  of  the 
material  used  and  for  suggestions  that  have  proved  of  service 
during  the  progress  of  the  work. 

SUMMARY. 

i.  Cold  hardiness,  both  to  the  intensity  factor  and  to  the  quan- 
tity factor  of  low  temperature,  were  studied  in  the  second  and 
third  instars  of  the  Japanese  beetle.  Brief  observations  were 
made  on  pupse  and  adults  with  regard  to  cold  hardiness. 


NELLIE    M.    PAYNE. 

2.  Japanese  beetle  larvae  are  somewhat  periodic  in  their  cold 
hardiness  to  the  intensity  factor  of  low  temperature,  less  so  than 
the  oak  borers  previously  studied  and  more  so  than  the  aquatic 
insects. 

3.  Disease  incidence,  nutritional  state,  and  degree  of  dehydra- 
tion are  associated  with  cold  hardiness  to  the  intensity  factor  of 
low  temperature. 

4.  Development  of  cold  hardiness  to  the  quantity  factor  of  low 
temperature  is  associated  with  loss  of  cold  hardiness  to  the  in- 
tensity factor  except  in  extremely  dehydrated  individuals. 

5.  Marked  permeability  changes  associated  with  enzyme  action 
occur  at  the  vital  temperature  minimum. 

LITERATURE  CITED. 

1.  Abderhalden,  Emil. 

'14  Versiiche  iiber  die  Synthese  von  Polypeptiden,  Peptonen,  und  Pro- 
teinen  mittels  Fermenten.  Fermentforschung  1  :  47-57. 

2.  Bodine,  Joseph  Hall,  and  David  E.  Fink. 

'25  A  Simple  Micro-vessel  with  Electrode  for  Determining  the  Hy- 
drogen Ion  Concentration  of  Small  Amounts  of  Fluid.  Jour. 
Gen.  Physiol.,  7  :  735-740. 

3.  Bodine,  Joseph  Hall,  and  Paul  Rudbert  Orr. 

'25  Respiratory  Metabolism.  Physiological  Studies  on  Respiratory 
Metabolism.  BIOL.  BULL.,  48 :  1-14. 

4.  Fink,  David  E. 

'25  Physiological  Studies  on  Hibernation  in  the  Potato  Beetle,  Lcpti- 
notarsa  decemlineata  Say.  BIOL.  BULL.,  49  :38i-4O5. 

5.  Gram,  H.  C.,  and  Glenn.  E.  Cullen. 

'23  The  Accuracy  of  the  "  lonometric "  Method  and  of  the  Protein 
Correction  in  Measuring  Conductivity.  Jour.  Biol.  Chem.,  67 : 
477-491. 

6.  Holmquist,  A.  M. 

Studies  in  Arthropod  Hibernation.  I.  Ecological  Survey  01 
Hibernating  Species  from  Forest  Environments  of  the  Chicago 
Region.  Ann.  Ent.  Soc.  Amer.,  19  :  395-428. 

7.  Payne,  Nellie  M. 

'26  Freezing  and  Survival  of  Insects  at  Low  Temperatures.  Quart. 
Rev.  Biol.,  1 :  270-286. 

8.  Payne,  Nellie  M. 

'27     Measures  of   Insect  Cold  Hardiness.     BIOL.  BULL.,  52 :  449-457. 

9.  Robinson,  William. 

'26  Low  Temperature  and  Moisture  as  Factors  in  the  Ecology  of  the 
Rice  Weevil,  Sitophilus  orysa  L.  and  the  Granary  Weevil, 
Sitophilus  (/ranarins  L.  Minn.  Agri.  Expt.  Stat.  Tech.  Bull., 
41 :  43  p. 


COLD    HARDINESS    IN    THE    JAPANESE    BEETLE.  179 

10.  Robinson,  William. 

'27     Water  Binding  Capacity  of  Coloids  a  Definite  Factor  in  the  Win- 
ter Hardiness  of  Insects.     Jour.  Econ.  Ent.,  20:  80-88. 

11.  Taylor,  Alonzo  Englebert. 

'09     On   the    Synthesis   of    Protamin   through    Ferment    Action.      Jour. 
Biol.  Chem.,  5:  381-387. 

12.  Townsend,  M.  T. 

'26     The  Breaking-up  of  Hibernation  in  the  Codling  Moth  Larva.    Ann. 
Ent.  Soc.  Amer.,  19:  429-439. 


PELAGIC  DISSOCONCHS  OF  THE  COMMON  MUSSEL, 

MYTILUS  EDULIS,  WITH  OBSERVATIONS  ON 

THE  BEHAVIOR  OF  THE  LARV.E  OF 

ALLIED  GENERA.1 

THURLOW  C.  NELSON. 

The  larvae  of  the  common  black  mussel,  Mytilus  eduJis,  are 
abundant  in  plankton  samples  taken  throughout  most  of  the 
summer  in  all  regions  where  this  mollusc  occurs,  Stafford,  '12. 
Recognition  of  the  larva,  as  Stafford  points  out,  is  rendered  easy 
owing  to  its  horn  yellow  color,  its  relatively  small  umbones  and 
its  small  depth.  To  these  characteristics  may  be  added  the  dis- 
tinctive shape  of  the  shell,  being  more  pointed  and  of  shorter 
height  at  the  anterior  end.  Fig.  i.  The  size  of  the  mature  pro- 
dissoconch  when  ready  to  attach  varies  considerably  as  judged 
from  measurements  of  the  largest  larvae  obtained  from  the  plank- 
ton, and  from  measurements  of  the  prodissoconch  shell  of  newly 
attached  dissoconchs.  Measurements  of -ten  of  the  largest  larvae 
found  in  the  plankton  in  Maine  waters  are  as  follows,  antero- 
posterior  axis  being  given  first. 

360  X  338  n,  368  X  320  P> 

360  )(  320  M,  336  X  304  p, 

376  >(  344  /*,  360X312^, 

35°  X  312  /A,  360  X  320  ju,,  exclusive  of  dissoconch  rim, 

336  X  304  ju,  304  X  280  p,  exclusive  of  dissoconch  rim. 

The  last  two  larvae,  although  caught  in  the  plankton,  each  bore 
a  narrow  rim.  of  purple  dissoconch  shell,  Jackson,  '88.  From 
these  and  from  other  measurements  made  upon  Mytilus  larvae  it 
appears  that  dissoconch  shell  may  be  secreted  at  any  time  after 
the  larvae  attain  a  length  between  approximately  300  and  360  u. 
Stafford,  I.e.,  gives  the  measurements  of  two  mature  prodisso- 
conchs  as  345  X  310/4  and  400  X  331/1. 

1  From  the   Zoological   Laboratory   of   Rutgers   University.     Paper   No. 
14,  New  Jersey  Oyster  Investigation  Laboratory. 

1 80 


PELAGIC    DISSOCONCHS    OF    THE    COMMON    MUSSEL.  I  X  I 

4 

The  observations  here  reported  were  made  during  August, 
1924  and  August,  1927,  in  Frenchman  Bay,  Mt.  Desert  Island, 
Maine.1  A  collecting  station  some  100  meters  from  the  labora- 


FIG.  i.  Stages  in  the  development  of  the  prodissoconch  larva  of  My  til  us 
cdulis:  U,  umbones ;  A,  anterior  end. 

tory  point  was  marked  with  a  buoy.  With  approximately  12 
meters  depth  at  mean  low  water  this  station  lay  in  the  full  sweep 
of  the  tide  through  Frenchman  Bay.  Plankton  samples  of  25 
liters  were  pumped  here  from  various  depths  using  a  hose  and 
oscillating  clock  pump,  the  majority  of  the  samples  being  taken 
at  the  surface  and  at  7  meters  depth.  The  Mytilits  larvie  were 
collected  by  passing  the  water  through  a  No.  18  treble  extra 
heavy  bolting  cloth  net,  adding  two  or  three  drops  of  formalin  to 
the  catch  and  then  drawing  off  the  supernatant  water  bearing  great 
quantities  of  the  diatoms  Chcetoceros  and  Rhisosolenia.  Table  I. 
gives  the  numbers  and  stages  of  development  of  the  mussel 
larva;  taken  at  the  station,  together  with  other  data. 

1  It  is  a  pleasure  to  acknowledge  my  indebtedness  to  the  former  Director, 
Professor  Ulric  Dahlgren,  for  the  facilities  given  me  at  the  Mount  Desert 
Island  Marine  Biological  Laboratory  at  Salisbury  Cove,  and  for  making 
early  summer  plankton  catches  for  me. 


1 82 


THURLOW    C.    NELSON. 


TABLE  I. 

\VATKK    CONDITIONS    AND    NUMBERS    OF   My  til  us    LAKV.I;    AT    STATION    OFF 
LAHOKATORY  POINT,  FRENCHMAN  BAY,  MT.  DESERT  ISLAND  IN  1924. 


Uatr. 

Time. 

Tide. 

Depth. 

Temper- 
ature 
0  C. 

Mytilus  Larvae  in 
25  Liters. 

Prodisso- 
conch. 

Disso- 
conch. 

Aug.      i 

— 

— 

Towing 

.  —  . 

—  . 

i 

5- 

11:30  A.M. 

2/3  flood 

2  m. 

10.6 

Many 

i 

7  • 

3:00  P.M. 

High 

7  m. 

II.  0 

262 

o 

13-3 

753 

8. 

3:45  P.M. 

High 

7  m. 

10.9 

336 

o 

17.1 

7i 

9- 

n  :3o  A.M. 

Low 

7  m. 

11.9 

278 

2 

o 

15-6 

5 

1  1  . 

10:15  A.M. 

1/2  ebb 

7  m. 

13-7 

63 

o 

14-3 

6 

n  . 

10:30  A.M. 

1/2  ebb 

Towing 

14-3 

Many 

8 

12. 

10:10  A.M. 

1/3  ebb 

7  m. 

ii.  i 

1,500 

o 

13-9 

4 

1.3- 

ii  :oo  A.M. 

i/6  ebb 

7  m. 

11.7 

650 

o 

13-8 

6 

13- 

2:50  P.M. 

3/4  ebb 

7  m. 

ii.  7 

213 

o 

iS-3 

20 

14- 

3:30  P.M. 

1/6  flood 

7  m. 

12.3 

240 

i 

o 

12.9 

320 

IS- 

10:45  A.M. 

High 

7  m. 

II.O 

390 

7 

o 

12.9 

i 

16. 

11:20  A.M. 

5/6  flood 

7  m. 

II.O 

i 

2 

0 

12.9 

177 

18. 

10:50  A.M. 

2/3  flood 

7  m. 

10.8 

65 

o 

iS-9 

i 

19. 

3:10  P.M. 

High 

7  m. 

12.2 

152 

o 

13-2 

2 

20. 

II  :30  A.M. 

1/3  flood 

7  m. 

II.9 

56 

o 

12.7 

2 

21  . 

2:50  P.M. 

5/6  flood 

7  m. 

1  1.  6 

30 

o 

12.0 

4 

22  . 

5:00  P.M. 

High 

7  m. 

II.4 

24 

I 

o 

14.7 

5 

23- 

3:10  P.M. 

2/3  flood 

7  m. 

II.O 

17 

0 

13.7 

7 

25- 

3:00  P.M. 

1/3  flood 

7  m. 

ii.  7 

40 

I 

o 

i3-i 

10 

27. 

3:45  P.M. 

1/6  flood 

7  m. 

12.3 

19 

o 

13-9 

7 

PELAGIC  DISSOCONCHS  OF  Mytilus. 
(a)    Buoyancy  through  Gas  Secretion. 

In  the  tow  sample  taken  by  Professor  Dahlgren  August  i  was 
found  one  Mytilus  larvae  which  bore  the  distinct  rim  of  purple 
shell  which  marks  the  commencement  of  the  dissoconch  stage. 


PELAGIC'    DISSOCOXC'IIS    OF    TIIK    COMMON     M  L'SSFL.  183 

Since  only  one  such  mussel  was  found  it  was  believed  to  have 
been  accidentally  introduced  through  the  townet  striking  some 
object  bearing  attached  mussels.  Subsequent  collections,  how- 
ever, revealed  numerous  dissoconchs  up  to  941  ^  in  length  freely 
floating  about  at  various  depths  up  to  /  meters.  A  25  liter  sample 
pumped  August  13,  from  the  surface,  20  cm.  from  a  Mytilits- 
covered  pile,  yielded  200  Mytilus  larva-  from  mature  prodis- 


FIG.  2.  Pelagic  dissoconch  of  Mytilus  cditlis  approximately  .8  mm.  in 
length,  bearing  a  large  bubble  of  secreted  gas  within  the  branchial  cham- 
ber. This  specimen  came  from  a  depth  of  7  meters. 

soconchs  to  advanced  dissoconchs  over  900  ^  in  length.  A  similar 
sample  pumped  August  15  from  a  depth  of  7  meters  at  the  col- 
lecting station,  more  than  100  meters  from  the  nearest  mussel 
beds,  gave  390  prodissoconch  Mytilus  and  7  dissoconchs  which 
ranged  in  length  from  445  to  784  p. 

The  presence  of  well-developed  dissoconchs  floating  freely  in 
the  water  at  once  raises  the  question  of  the  means  by  which  this 
is  effected  in  the  absence  of  the  swimming  organ  or  velum  of  the 
prodissoconch.  When  brought  to  the  laboratory  for  examina- 
tion these  dissoconchs  were  found  to  be  identical  with  others 
removed  from  sea  weeds,  save  for  the  presence  of  a  large  clear 
space  in  the  posterior  portion  of  the  pallial  cavity.  Believing 
that  some  change  might  have  occurred  in  the  molluscs  even  dur- 
ing the  fifteen  minutes  to  half  an  hour  which  elapsed  between 
their  capture  and  subsequent  examination  in  the  laboratory,  a 


1 84 


THURLOW    C.    NELSON. 


binoular  was  taken  in  the  boat  and  the  young  mussels  were  ex- 
amined immediately  after  their  capture.  The  result  is  shown  in 
Fig.  2.  A  large  bubble  was  found  to  occupy  the  posterior  part 
of  the  pallial  cavity,  its  buoyancy  causing  the  young  bivalve  to 
bang  suspended  in  the  water  umbone  downward,  with  the  postero- 
ventral  margin  of  the  valves  turned  upward.  On  one  occasion 
the  bubble  was  seen  to  form  through  the  coalescing  of  many 
minute  bubbles,  which,  passing  slowly  down  between  the  gill 
filaments,  united  to  form  a  single  large  bubble.  In  several  in- 
dividuals two  or  three  smaller  bubbles  were  found.  Where  a 
single  bubble  was  present  its  size  was  such  as  to  cause  a  thin- 
ning of  the  mantle  on  either  side  and  a  forward  displacement  of 
the  posterior  gill  filaments,  thus  accounting  for  the  large  clear 
space  already  noted  in  the  posterior  pallial  cavity  of  the  young 
Mytilus  dissoconchs  first  taken. 

Failure  to  observe  the  bubble  in  larvae  first  brought  to  the 
laboratory  was  due  to  the  fact  that  as  soon  as  a  Mytilus  dissoconch 
comes  in  contact  with  any  object  the  foot  is  rapidly  extruded 
from  between  the  valves  and  brought  into  contact  with  the  sur- 
face. The  extrusion  of  the  foot,  accompanied  as  it  is  by  a 
separation  of  the  valves  and  of  the  applied  lobes  of  the  mantle, 
results  in  the  immediate  escape  of  the  bubble  in  nearly  every 
instance. 

The  composition  of  the  gas  in  the  bubble  was  not  determined 
owing  to  its  small  size  and  lack  of  adequate  facilities  for  a  micro - 
chemical  test.  The  fact  that  it  forms  within  the  gills  would  indi- 
cate that  it  is  mainly  oxygen.  The  composition  of  gas  secreted 
into  the  swim  bladders  of  fishes  renders  this  still  more  probable. 

The  possibility  that  the  bubbles  within  the  branchial  chamber 
of  these  young  Mytilus  might  have  been  air  introduced  acci- 
dentally during  passage  through  the  pump  or  while  in  the  net, 
was  tested  in  the  following  manner.  The  hose  was  disconnected 
from  the  pump  and  allowed  to  siphon  water  from  a  depth  of  7 
meters  into  the  net  held  in  the  bottom  of  the  boat.  The  stream 
entered  the  net  under  water  and  great  care  was  taken  not  to 
agitate  the  net  or  to  break  the  water  surface.  Dissoconchs  of 
Mytilus  collected  in  this  way  revealed  the  same  large  bubbles  as 
before.  Dissoconchs  of  Mytilus  collected  from  sea  weeds  and 


PELAGIC   DISSOCONCHS    OF    THE    COMMON     MUSSEL.  185 

violently  shaken  with  a  little  sea  water  in  a  bottle  failed  to  ac- 
quire any  bubbles  of  air  in  the  process :  thus,  with  the  above 
experiment,  proving  that  the  bubbles  of  gas  were  not  accidentally 
introduced. 

To  determine  the  possible  effects  of  pressure  in  bringing  about 
gas  secretion  pieces  of  glass  tubing  2  cm.  long  were  cut  and  an 
early  Mytilus  dissoconch  obtained  from  sea  weeds  was  intro- 
duced into  each.  A  piece  of  coarse  bolting  cloth  was  tied  over 
each  end  of  the  tubes  which  were  then  fastened  to  a  line  at  one 
meter  intervals  and  suspended  from  a  float  at  the  collecting  sta- 
tion. One  string  bore  seven  tubes  which  hung  at  depths  of  from 
two  to  eight  meters.  A  second  string  was  attached  to  a  weight 
on  the  bottom  with  a  float  of  sufficient  size  to  hold  the  string 
vertically  in  the  water,  the  lowest  tube  being  at  u  meters  depth 
at  low  water  and  approximately  15-16  meters  at  high  water. 
When  removed  48  hours  later  all  of  the  bivalves  were  found  to 
have  attached  by  the  byssus  to  the  inside  of  the  tubes  or  to  the 
bolting  cloth  ends.  When  removed  to  a  dish  of  sea  water  they 
crawled  actively  about  with  the  foot.  In  no  case  was  a  bubble 
present.  Either  the  stimulus  to  gas  secretion  is  absent  when  the 
mussels  are  attached,  or  the  frequent  extrusion  of  the  foot  which 
occurs  while  the  mussel  is  attached  permits  the  escape  of  such 
gas  as  rapidly  as  it  is  formed. 

(b)    Attachment  to  the  Surface  Film. 

If  the  surface  of  the  water  near  a  mussel  bed  or  near  a  mus- 
sel covered  piling  be  skimmed  with  a  plankton  net  during  the 
latter  part  of  the  breeding  season,  numerous  dissoconchs  will  be 
found.  They  are  most  numerous  as  the  rising  tide  first  sweeps 
over  the  mussels  and  attached  sea  weeds.  A  microscopic  exami- 
nation of  these  dissoconchs  shows  that  none  contains  a  bubble, 
hence  it  is  obvious  that  these  young  mussels  must  maintain  them- 
selves at  the  surface  through  means  other  than  the  gas  secretion 
employed  by  larvae  at  a  depth.  When  placed  in  a  dish  of  sea 
water  such  larval  mussels  exhibit  great  activity,  gliding  about  upon 
the  long,  highly  adhesive,  ciliated  foot  as  rapidly  as  a  snail.  Ob- 
servations were  made  upon  these  young  molluscs  using  a  cham- 
ber 0.5  cm.  wide  made  of  two  microscopic  slides,  filled  with  sea 
water  and  viewed  horizontally  through  the  binocular. 


1 86 


THURLOW    C.    NELSON. 


Once  in  contact  with  a  solid  object,  such  as  the  wall  of  the 
chamber,  a  rock,  or  a  fragment  of  sea  weed,  the  mussels  exhibited 
a  marked  negative  geotropism  and  climbed  straight  upward  until 
the  surface  was  reached.  Here  the  distal  one  third  to  one  tenth 
of  the  foot  was  extended  in  the  surface  film.  Fig.  3,  and  with 
a  quick  contraction  of  the  foot,  aided  apparently  by  contrac- 
tion also  of  the  pedal  retractor  muscles,  the  ventral  margins  of 


FIG.  3.  Ventral  view  of  My  til  us  dissoconch,  4  mm.  long,  hanging  from 
the  foot  in  the  surface  film,  as  seen  from  the  side  and  partly  from  above. 

FIG.  4.  Lateral  view  of  4  mm.  Mytilus  dissoconch  hanging  from  the 
surface  film.  The  siphons  are  fully  extended. 

FIG.  5.  Lateral  view  of  3  mm.  Mytilus  dissoconch  hanging  from  byssus 
thread  attached  to  holdfast  secreted  in  the  surface  film.  The  foot,  which 
was  fully  extended  in  the  surface  film  while  secretion  of  the  holdfast  was 
effected,  has  been  wholly  withdrawn  between  the  valves. 

FIG.  6.  Lateral  view  of  3  mm.  Mytilus  dissoconch  holding  to  the  sur- 
face film  with  the  aid  of  the  tentacles  of  the  incurrent  siphon.  The  foot 
which  serves  to  hold  the  mussel  close  to  the  film  until  the  siphon  is  in- 
serted therein,  has  been  withdrawn  between  the  valves. 


PELAGIC    DISSOCONCHS    OF    THE    COMMON     Ml'SSKL.  187 

the  mantle  were  brought  into  contact  with  the  surface  film. 
While  lying  with  the  entire  ventral  margin  of  the  body  in  con- 
tact with  the  surface  film  the  byssus  gland  in  a  few  seconds  se- 
creted onto  the  surface  film  a  small  holdfast  similar  in  appearance 
to  that  which  is  laid  down  on  rock  or  piling  for  the  attachment  of 
each  byssus  thread.  A  thread  1-2  mm.  long  serves  to  support 
the  young  mussel  from  this  float  and  with  foot  withdrawn  it 
may  hang  suspended  indefinitely,  Fig.  5.  At  times  it  thrashes 
about  with  the  foot  fully  extended  as  though  in  search  of  some 
solid  surface  for  attachment.  When  the  foot  strikes  such  an 
object  the  mussel  glides  quickly  upon  it,  trailing  the  byssus  thread 
behind  or  breaking  it  off.  The  "float"  is  not  a  buoyant  struc- 
ture, since  when  pushed  beneath  the  surface  film  it  rapidly  sinks. 
It  maintains  its  position  in  the  surface  film,  supporting  mussels 
up  to  4  mm.  in  length,  solely  through  surface  tension. 

A  float  and  connecting  thread  are  not  always  secreted  when  the 
young  mussel  reaches  the  surface.  At  times  it  supports  itself 
solely  by  the  distal  end  of  the  foot  in  the  surface  film,  Fig.  4, 
after  the  manner  described  for  the  prodissoconch  oyster  larva, 
Nelson,  '243.  With  the  aid  of  the  numerous  short  cilia  covering 
the  foot  the  animal  glides  slowly  along  the  surface  film,  rocking 
the  body  slowly  from  side  to  side  and  occasionally  through  a 
quick  contraction  of  the  proximal  portion  of  the  foot,  bringing 
the  entire  ventral  margin  of  the  shell  in  contact  with  the  surface 
film.  This  behavior  will  recall  the  familiar  habit  of  pond  snails 
of  hanging  from  the  entire  foot  spread  out  in  the  surface  film. 

A  third  mode  of  suspension  from  the  surface  consists  in  ex- 
tending the  tips  of  the  tentacles  of  the  incurrent  siphon  into  the 
film  and  hanging  from  these,  Fig.  6.  This  behavior,  though  sel- 
dom observed,  serves  to  support  the  mussel  quite  as  effectively 
as  does  the  foot. 

Such  floating  dissoconchs  have  never  been  found  further  than 
approximately  25  meters  distance  from  mussel  beds  or  mussel 
covered  piling.  Their  abundance,  5  to  100  per  25  liters  of  water, 
close  to  such  habitats  indicates  that  young  mussels  frequently 
make  use  of  this  mode  of  transportation  for  covering  short  dis- 
tances. 

Examination   with  the   low   power   binocular   of    several   small 


j88  THURLOW    C.    NELSON. 

tide  pools  close  to  the  laboratory  revealed  numbers  of  Mytilus 
3-4  mm.  long  moving  actively  over  the  rocks  and  barnacles  while 
others  were  at  the  surface.  With  the  incoming  tide  the  latter  are 
carried  away  and  may  eventually  reach  a  place  of  attachment  at 
a  considerable  distance. 

METAMORPHOSIS  IN  ALLIED  LAMELLIBRANCHS. 

The  water  samples  taken  in  Frenchman  Bay  contained  in  ad- 
dition to  the  larvae  of  Mytilus  edulis,  great  numbers  of  the  young 
of  the  soft  clam,  Mya  arenaria,  together  with  occasional  speci- 
mens of  the  larvse  of  V enericardium,  of  Anemia  and  possibly  also 
of  Astarte.  During  more  than  ten  years  study  of  the  oyster 
larvse  of  the  New  Jersey  Coast  I  have  become  familiar  also  with 
the  larvse  of  Mytilus  recurvus  (the  southern  oyster  mussel), 
/  'cnus  mcrcenaria,  and  Teredo  navalis.  In  no  instance  have  I 
ever  observed  gas  secretion  in  any  of  these  forms  nor  have  I 
found  pelagic  dissoconchs.  When  the  time  for  setting  arrives 
the  mature  larvse  of  all  of  the  above  species  disappear  from  the 
water  within  24  to  36  hours. 

Reproduction  and  dispersal  of  marine  pelecypod  molluscs  occur 
through  the  medium  of  pelagic  veligers  which  are  free-swimming 
for  periods  ranging  from  a  few  days  in  such  incubatory  forms  as 
Ostrea  edulis  and  Teredo  bartschi,  to  approximately  three  weeks 
in  Mytilus  edulis,  Mya  arenaria,  and  in  probably  most  of  the 
marine  bivalve  molluscs  which  reproduce  at  temperatures  below 
20°  C.,  Nelson,  '28.  Owing  to  the  sessile  or  sedentary  habits  of 
the  adult  molluscs,  the  activities  of  the  larvse  become  of  first 
importance  in  the  dissemination  and  preservation  of  the  species. 
Through  the  aid  of  the  velum  the  larval  bivalve,  while  unable  to 
make  progress  against  a  current,  can  control  its  vertical  distri- 
bution and  thus  secondarily  may  determine  to  a  marked  degree  its 
horizontal  distribution  by  tides  and  currents,  Nelson,  '22. 

The  rate  of  growth  and  of  development  during  larval  life  is 
determined  chiefly  by  the  temperature.  The  long  series  of  ob- 
servations on  the  life  history  of  the  American  oyster,  Ostrea 
•rinjinica  Gmelin  (J.  Nelson,  Stafford,  Churchill,  T.  Nelson  and 
others),  indicates  that  at  a  given  temperature  the  duration  of  the 
pelagic  period  is  remarkably  constant.  With  an  average  tern- 


PELAGIC    DISSOCONCIIS    OF    THE    COMMON    MUSSEL.  189 

perature  of  23-24°  C.  the  period  from  spawning  to  the  attach- 
ment of  the  spat  in  New  Jersey  waters  is  13  days.  In  Richmond 
Bay,  Canada,  J.  Nelson,  '17,  found  that  at  temperatures  approxi- 
mating 20°  C.  the  minimum  time  required  for  oyster  larvae  to 
mature  was  17  days.  Stafford,  '13,  considers  three  weeks  to  be 
the  average  time  required  to  reach  maturity  in  Canadian  waters. 

The  close  of  the  free-swimming  period  of  pelecypod  larva:  is 
determined  apparently  by  internal  developmental  factors :  when 
the  time  for  "  setting "  arrives  the  larva:  must  attach  or  die. 
Since  the  veligers  during  their  pelagic  existence  have  been  dis- 
tributed widely  by  currents  it  follows  that  for  those  which  through 
chance  happen  to  "  fall  upon  good  ground  "  there  will  be  many 
more  which  through  this  same  chance  will  "  fall  by  the  wayside  " 
and  be  destroyed. 

Observations  of  the  oyster  larva,  T.  Nelson,  '22,  '24,  show  that 
approximately  24  hours  prior  to  attachment  the  young  bivalve 
becomes  positively  stereotropic  and  that  it  may  explore  numerous 
surfaces  with  the  aid  of  the  foot  before  it  finally  attaches.  Such 
behavior,  while  of  the  utmost  importance  in  securing  a  favorable 
spot  for  attachment,  is  without  avail  if  no  substrata  suitable  for 
attachment  are  present.  Little  is  known  of  the  factors  necessary 
to  provide  a  favorable  bedding  ground  for  such  burrowing  spe- 
cies as  Mya  and  J^ciius.  Although  attachment  of  young  Mya 
by  the  byssus  to  sea  weeds  or  other  objects  may  occur,  as  shown 
by  Ryder,  '89,  and  by  Kellogg,  '99,  it  is  pointed  out  by  fielding, 
'12,  that  survival  of  both  Mya  and  Venus  depends  largely  upon 
the  character  of  the  mud  and  sand  forming  the  surface  layers 
of  the  bottom.  All  who  have  studied  the  habits  of  larval  bi- 
valves agree  that  the  vicissitudes  of  larval  life  and  subsequent 
attachment  form  one  of  the  chief  barriers  to  wide  dispersal  of 
the  species.1 

1  A  survey  of  our  present  knowledge  of  the  habits  and  life  histories  of 
both  fresh-water  and  marine  pelecypods  shows  that  of  all  environmental 
influences  the  presence  of  a  suitable  substratum  is  the  most  important 
single  factor  limiting  distribution.  The  following  papers  may  be  cited  in 
this  connection:  fresh  water  mussels,  Coker  et  al.,  '21;  Mya,  Belding,  '09; 
Pcctcn,  Belding,  '10;  Venus,  Belding,  '12;  Ostrca  cucitllata,  O.  angasi, 
Roughley.  '25;  Mytilns,  Card  him,  Sa.ridomus,  Siliqua,  Paphia,  and  other 
genera  of  the  Pacific  Coast,  Thompson,  '13,  and  Weymouth,  '20;  Ostrc<i 


190 


TIM'RLOW    C.    NELSON. 


Of  the  known  genera  of  marine  pelecypods,  Alytilus  cdnlis  and 
Teredo  navalis  alone  are  circumpolar  in  their  distribution  over 
the  shores  of  the  northern  hemisphere.  General  adaptability  to 
changing  conditions  and  the  power  to  resist  adverse  surround- 
ings together  with  relatively  low  spawning  temperatures,  Nelson, 
'28,  have  aided  these  two  forms  in  attaining  their  present  wide 
distribution.  Transportation  through  attachment  to  vessels  or 
to  other  floating  wood  has  likewise  aided  in  their  dispersal,  being 
for  Teredo  the  only  means  by  which  any  great  distance  could 
be  covered.  In  the  case  of  Mytilus  edulis,  however,  the  ability 
to  bridge  the  period  of  metamorphosis  while  remaining  pelagic 
must  have  been  an  important  factor  in  securing  the  wide  disper- 
sal which  this  mollusc  now  enjoys;  as  well  as  a  great  aid  in 
bringing  to  a  suitable  place  of  attachment  a  fair  proportion  of 
the  larvae  produced  each  season.  The  largest  of  the  pelagic  dis- 
soconchs  found  in  Frenchman  Bay  was  fully  a  month  old,  dur- 
ing which  time  it  must  have  been  transported  over  long  distances 
by  the  tide.  If  during  this  period  it  had  come  in  contact  at  any 
time  with  a  solid  object  attachment  could  easily  have  been  effected. 

SUMMARY. 

At  the  close  of  the  larval  or  prodissoconch  stage  the  young  of 
Mytilus  edulis  which  fail  to  secure  immediate  attachment  may 
remain  pelagic  through  the  secretion  of  gas  into  the  mantle 
chamber. 

Short  distances  may  be  covered  through  the  aid  of  a  holdfast 
secreted  on  the  surface  film  or  through  holding  the  foot  or  the 
tentacles  of  the  incurrent  siphon  in  the  surface  film. 

The  ability  to  bridge  over  the  critical  period  of  metamorphosis 
while  remaining  pelagic  has  been  an  important  factor  in  securing 
the  present  wide  distribution  of  the  black  mussel. 

Absence  of  a  similar  adaptation  in  the  larvae  of  other  bivalves 
renders  them  still  dependent  largely  upon  chance  in  securing  at 
the  close  of  the  pelagic  period  a  proper  substratum  for  attach- 
ment. This  dependence  upon  the  substratum  is  one  of  the  chief 
barriers  to  the  wide  dispersal  of  the  species. 

I'irginica,  Moore,  '97,  Grave,  '01,  Stafford,  '13,  J.  Nelson,  '17,  Churchill,  '20,. 
T.  C.  Nelson,  '22. 


PELAGIC    DISSOCONCHS    OF    THE    COMMON    MUSS1.I-. 


191 


CITATIONS. 
Belding,   D.   L. 

'09     A    Report   upon   the   Mollusk   Fisheries   of    Massachusetts.      Boston, 

243  pages. 
Belding,   D.   L. 
'10    A  Report  upon  the  Scallop  Fishery  of  Massachusetts.     Boston,   150 

pages. 

Belding,  D.  L. 
'12     A  Report  upon  the  Quahog  and  Oyster  Fisheries  of   Massachusetts. 

Boston,  134  pages. 
Churchill,  E.  P. 

'20     The   Oyster  and   Oyster   Industry  of   the  Atlantic  and   (iulf   (Toasts. 

Rept.  U.  S.  C.  F.     Appendix  8-51  pages. 
Coker,  R.  E.,  Shira,  A.  F.,  Clark,  H.  W.,  and  Howard,  A.  D. 

'21     Natural    History    and    Propagation   of    Fresh-water    Mussels.      Bull. 

U.  S.  B.  F.,  37:  77-i8i. 
Grave,  C. 
'10     The  Oyster  Reefs  of  North  Carolina.     J.  H.  U.  Circulars,  No.   151. 

1-9- 
Jackson,  R.  T. 

'88     The   development   of    the   Oyster   with   Remarks   on    Allied   Genera. 

Proc.  Bost.  Soc.  Nat.  Hist.,  23 :  531-556. 
Kellogg,  J.  L. 

'99     Observation   on  the   Life   History  of   the   Common   Clam,   Mya  arc- 

naria.     Bull.  U.  S.  F.  C.,  1899. 
Moore,  H.  F. 

'97     Oysters   and   Methods  of   Oyster   Culture.     Rpt.   U.    S.   C.   F.,    1897, 

263-340. 
Nelson,  J. 

'17     Oyster    Propagation    in    Prince   Edwards    Island.      Contr.    to    Canad. 
Biol.   Supplement  to  6th  Ann.  Rpt.  Dept.   Naval   Service.     Ottawa, 
53-76. 
Nelson,  T.  C. 

'22     Aids   to    Successful   Oyster   Culture,      i.  Procuring   the    Scvd.      Bull. 

351,  N.  J.  Expt.  Sta.,  New  Brunswick. 

'24a     The  Attachment  of  Oyster  Larvse.     BIOL.  BULL.,  46:   143-151. 
'24b     Metamorphosis    to    the    Dissoconch    Stage    without    Attachment    in 
the   Veligers   of    Ostrea   and   of   Mytihts    (Abstract.)      Anat.    Rec., 
29:  97. 
'28     On  the  Distribution  of  Critical  Temperatures  for  Spawning  and  for 

Ciliary   Activity   in   Bivalve    Molluscs.      Science,   67:    220-221. 
Roughley,  T.  C. 
'25     The   Story   of   the   Oyster.     Australian   Museum   Magazine,   2,   Nos. 

5,  6,  7 

Ryder,  J.  A. 

'89     The  Byssus  of   the  Young  of  the  Common   Clam,  Mya  art'iutria    L. 
Am.  Nat.,  Jan.,  1889. 

13 


[92 


THURLOW    C.    NELSON. 


Stafford,  J. 

'12     On    the    Recognition    of    Bivalve    Larvae    in    Plankton    Collections. 

Contr.  to  Canadian   Biol.,   1906-10,  221-242. 
Thompson,  W.  F. 
'13     Report  on  the  Shellfish  of  British  Columbia    Report  of  B.  C.  Comni. 

Fisheries,  1913. 
Weymouth,  F.  W. 

'20     The  Edible  Clams,  Mussels  and  Scallops  of   California.     Calif.  Fish 
and  Game  Comm.     Fish  Bulletin  Xo.  4. 


STUDIES    ON    THE    SECONDARY    SEXUAL   CHARAC- 
TERS OF  CRAYFISHES. 

VI.    A  FEMALE  OF  CAMBARUS  IMMI'MS  WITH  OVI- 
DUCTS ATTACHED  TO  OPENINGS  OF 
SPERM  DUCTS. 

C.  L.  TURNER. 
ZOOLOGICAL  LABORATORY,   NORTHWESTERN   UNIVERSITY. 

The  specimen  to  be  described  was  taken  from  Root  River  near 
Racine,  Wisconsin,  on  July  10,  1924. 

The  presence  of  a  normal  annulus  ventralis  and  of  the  usual 
rudimentary  appendages  upon  the  first  abdominal  segments  mark 


FIG.  I.  Diagram  illustrating  external  secondary  sexual  characters.  S, 
openings  of  oviducts  at  base  of  fifth  walking  leg.  A.V.,  annulus  ventralis. 
R.A.,  rudimentary  first  abdominal  appendages.  O,  position  at  base  of  third 
walking  leg  of  oviducal  pore  in  normal  female. 

it  as  a  female  but  there  are  no  oviducal  pores  at  the  bases  of  the 
third  walking  legs.  On  the  other  hand,  a  pair  of  openings  ap- 
pears at  the  bases  of  the  fifth  walking  legs  at  the  position  ordi- 


194 


C.    L.    TURNER. 


narily  occupied  by  the  openings  of  the  sperm  ducts.     No  other 
male  characters  are  found,  however  (Fig.  i). 

Upon  dissection  the  specimen  was  found  to  have  a  well-devel- 
oped ovary  and  oviducts  which  were  attached  to  the  ovary  at  the 
usual  point.  In  normal  specimens  the  oviducts  make  their  way 
laterally  and  ventrally  and  open  through  pores  located  at  the  bases 
of  the  third  legs.  In  this  case,  however,  the  oviducts  slope  pos- 
teriorly as  well  and  finally  attach  themselves  to  the  openings  at 
the  bases  of  the  fifth  walking  legs.  A  closer  examination  of  the 
pores  themselves  shows  that  they  are  essentially  the  openings  of 
sperm  ducts  in  structure  although  they  are  considerably  modi- 
fied (Fig.  2). 


FIG.  2.  Diagram  illustrating  internal  relations  of  ovary,  oviduct  and 
walking  legs.  Position  of  oviduct  in  this  specimen  shown  in  solid  black. 
Position  of  oviducts  in  normal  female  shown  by  lines.  Positions  of  walk- 
ing legs  indicated  by  numerals. 

The  normal  oviducal  pore  is  oval  in  outline  and  has  slightly 
raised  and  thickened  rims,  but  the  structure,  as  a  whole,  is  not 
raised  conspicuously  above  the  surface  of  the  shell.  The  greatest 
width  of  the  oviducal  pore  in  a  specimen  of  this  size  is  about  one 
mm.  with  the  length  a  little  more.  The  plane  of  each  pore  is 
tilted  a  little  toward  the  median  line  and  a  little  toward  the  rear. 

The  normal  openings  of  sperm  ducts  are  much  smaller  and  are 
extended  by  means  of  membranous  projections.  They  open  toward 
the  median  line  and  there  is  practically  no  deflection  ventrally  or 
posteriorly. 


SECONDARY    SEXUAL    CHARACTERS    OF    CRAYFISHES.  195 

The  openings  of  the  specimen  described  here  resemble  the  pores 
of  sperm  ducts  in  their  general  position,  in  the  direction  in  which 
they  open  and  in  being  projected  somewhat  by  membranes.  On 
the  other  hand,  they  are  much  larger  than  the  openings  of  sperm 
ducts  but  smaller  than  normal  oviducal  pores  and  in  shape  re- 
semble oviducal  pores. 

DISCUSSION. 

Several  questions  of  interest  arise  in  connection  with  this  case 
but  most  of  them  can  be  treated  only  as  speculations,  (i)  How 
does  it  happen  that  the  oviducal  pores  are  absent  in  a  female  ani- 
mal? (2)  How  does  it  happen  that  the  openings  of  sperm  ducts 
are  present  in  a  female  animal?  (3)  Is  the  duct  to  be  interpreted 
as  an  oviduct  which  has  become  attached  at  its  distal  end  to  the 
opening  of  a  sperm  duct  or  as  a  vas  deferens  which  has  become 
attached  at  its  proximal  end  to  an  ovary?  (4)  To  what  extent 
has  there  been  a  modification  during  its  development  of  the  pore 
of  the  sperm  duct  by  virtue  of  its  attachment  to  the  oviduct? 
(5)  To  what  extent  does  the  oviduct  possess  the  potentiality  of 
shaping  a  structure  to  which  it  is  not  ordinarily  attached  (the 
opening  of  the  sperm  duct)  in  the  direction  of  an  organ  to  which 
it  is  usually  attached  (the  oviducal  pore)  ? 

As  regards  the  first  and  second  questions,  it  is  being  repeatedly 
shown  in  crayfishes  that  the  contemporary  occurrence  in  an  ani- 
mal of  the  ovary  or  spermary  and  a  fixed  set  of  secondary  char- 
acters is  by  no  means  necessary  for  normal  functioning.  Indeed 
it  has  been  shown  that  in  some  localities  the  occurrence  of  a  char- 
ter supposed  to  be  fixed  upon  one  sex  occurs  upon  the  other  sex 
considerably  more  than  half  of  the  time.  The  lack  of  one  or  both 
oviducal  pores  in  otherwise  normal  females  has  also  been  re- 
corded. The  absence  of  oviducal  pores  is  not  surprising,  there- 
fore, nor  the  presence  of  the  openings  of  sperm  ducts.  The 
presence  of  the  openings  of  sperm  ducts  in  the  absence  of  ovi- 
ducal pores  does  not  mean  that  these  are  mutually  exclusive  struc- 
tures, for  oviducal  pores  are  sometimes  found  upon  males  normal 
with  respect  to  the  sperm  duct  openings  as  well  as  otherwise 
normal. 

The  tube  connecting  the  pore  at  the  base  of  the  fifth  walking 
legs  and  the  ovary  may  be  considered  an  oviduct  for  the  follow- 


10,6  C.    L.    TURNER. 

ing  reasons.  First,  it  is  straight  and  shows  none  of  the  coils  of 
the  vas  deferens.  Second,  it  is  thin  walled  and  wide  while  the 
vas  deferens  is  rather  narrow  and  dense  in  texture.  Third,  it 
exhibits  the  same  types  and  arrangement  of  tissues  as  a  normal 
oviduct. 

In  Cainbarus  the  only  misplaced  oviducal  pores  discovered  have 
occurred  on  the  second  or  the  fourth  walking  legs  of  females  and 
then  only  as  supernumerary  pores,  the  normal  oviducal  pores 
being  present  on  the  third  walking  leg  as  usual.  It  is  assumed, 
therefore,  that  the  pores  of  the  fifth  walking  legs  are  the  open- 
ings of  sperm  ducts  and  not  of  oviducts.  The  extent  to  which 
the  pores  depart  from  the  features  of  the  normal  sperm  duct  is 
taken  to  represent  the  extent  of  the  influence  of  the  attachment  of 
the  pores  to  oviduct  instead  of  sperm  duct. 

There  is  no  evidence  to  show  that  the  influence  of  the  oviduct 
is  required  in  embryology  for  the  proper  shaping  of  the  oviducal 
pore.  Rather  to  the  contrary  the  oviducal  pores  are  sometimes 
present  and  perfectly  developed  in  the  absence  of  oviducts.  At 
the  same  time  the  resemblance  of  the  pores  of  this  specimen  to 
oviducal  pores  must  have  been  due  to  the  influence  of  the  oviduct 
to  which  they  were  attached.  The  case  is  roughly  parallel  to  the 
results  of  the  experiment  in  which  an  optic  cup  in  a  vertebrate 
embryo  was  transplanted  under  ectoderm  in  another  part  of  the 
body  and  under  the  influence  of  the  optic  cup  the  ectoderm  de- 
veloped a  lens  although  normally  it  would  not  have  done  so. 

The  condition  described  here  is  not  to  be  confused  with  the 
normal  condition  found  in  some  South  American  species  belong- 
ing to  the  genus  Parastacus.  In  these  there  are  regularly  both 
oviducts  and  sperm  ducts  but  only  one  set  is  functional,  that  one 
being  appropriate  to  the  sex  in  which  it  occurs.  The  case  de- 
scribed in  this  paper  cannot  be  considered  as  a  parallel,  for  here 
only  one  set  of  tubes  is  present  and  only  one  set  of  external  pores. 


STUDIES  ON  THE  SECONDARY  SFXTAL  CHARAC- 
TERS OF  CRAYFISHES. 

VII.  REGENERATION  OF  ABERRANT  SKCONDAKY 

SEXUAL  CHARACTERS. 

C.  L.  TURNER. 
ZOOLOGICAL   LABORATORY,   NORTHWESTERN    UXIVKRSITY. 

It  is  rather  generally  conceded  that  intersexuality  in  insects  has 
as  a  background  either  genetic  disturbances  or  metabolic  inter- 
ferences due  to  parasitism.  The  evidence  for  the  causes  of  inter- 
sexuality  in  the  Crustacea,  however,  is  by  no  means  as  distinct, 
due  to  the  fact  that  it  has  not  been  possible  to  separate  the  ques- 
tion of  the  development  of  secondary  sex  characters  from  that 
of  their  control  by  hormones  located  in  the  gonads.  It  has  been 
rather  concisely  demonstrated  in  the  insects  that  secondary  sex 
characters  are  independent  of  spermatic  or  ovarian  hormones, 
whereas  some  of  the  evidence  obtained  in  the  Crustacea  seems  to 
indicate  the  possibility  of  such  hormone  control. 

Among  some  six  or  seven  causes  which  might  be  able  to  ac- 
count for  intersexuality  among  the  decapods  there  is  the  repeated 
suggestion  that  some  ambiguous  or  accidental  event  in  embryol- 
ogy has  been  responsible.  When  in  one  locality  eighty-six  per 
cent,  of  all  the  females  of  Cambarus  virilis  and  in  another  lo- 
cality thirty-seven  per  cent.  of*Cambarus  propinquus  possess  well- 
defined  male  characters,  it  would  seem  that  no  accidental  event 
in  embryology  could  account  for  this  unusual  occurrence.  Rather, 
it  would  seem  necessary  to  seek  for  some  orderly  and  fixed  in- 
fluence. It  occurred  to  the  writer  that  the  tendency  to  form 
aberrant  structures  might  be  tested  for  its  duration  beyond  the 
embryonic  stage  by  detaching  the  appendages  bearing  these  struc- 
tures and  determining  whether  there  would  then  be  developed 
normal  or  aberrant  structures.  The  accumulation  of  this  body  of 
data  and  the  interpretation  of  it  has  been  carried  out  with  this 
inquiry  in  mind. 

197 


198 


C.    L.    TURNER. 


RKGKNKKATION  OF  NORMAL  SECONDARY  SEX  CHARACTERS. 

When  any  appendage  is  lost  in  the  crayfish,  unless  the  crayfish 
is  too  old,  a  new  appendage  will  grow  in  place  of  the  old  one  and 
if  the  crayfish  is  young  when  the  accident  occurs  the  regenerated 
appendage  will  come  to  have  almost  completely  the  size  and  char- 
acteristics which  the  original  appendage  would  have  had.  In  an 
i  >ld  specimen,  however,  there  will  not  be  sufficient  time  for  the 
complete  regeneration  of  the  appendage  before  the  crayfish  dies. 

Some  of  the  external  secondary  structures  which  have  to  do 
with  sex  are  either  modified  appendages  or  structures  located 
upon  the  appendages.  Such  are  the  first  and  the  second  abdom- 
inal appendages  which  are  modified  for  copulation  in  the  male 
but  are  rudimentary  in  the  female,  the  hooks  located  upon  the 
third  walking  leg  of  the  male,  and  the  openings  of  the  oviducts 
and  sperm  ducts  upon  the  third  and  the  fifth  walking  legs  re- 
spectively. When  one  of  these  appendages  is  broken  off  so  as 
to  include  one  of  the  modified  structures,  the  appendage  will  be- 
gin to  regenerate,  beginning  with  the  first  moult.  At  first  it  is 
juvenile  in  character  and  unmodified  but  eventually  it  becomes 
completely  differentiated  and  contains  the  modified  structure.  The 
regenerated  secondary  sex  characters  never  completely  resemble 
the  normal  secondary  sex  characters.  In  the  case  of  the  hooks 
which  are  found  upon  the  third  walking  legs,  the  regenerated  ones 
are  blunt  and  flatter  than  the  original  ones,  but  occurring  as  they  do 
in  a  definite  position  they  are  easily  recognizable. 

OBSERVATIONS  ON  THE  REGENERATION  OF  ABERRANT  SECONDARY 

SEX  CHARACTERS. 

Not  more  than  thirty  cases  have  been  recorded  in  Cambarus  of 
male-like  modifications  of  the  abdominal  appendages  in  females 
and  the  possibility  of  finding  cases  in  which  the  aberrant  appen- 
dages have  been  injured  and  regenerated  would  be  very  remote. 
The  occurrence  of  female  structures  upon  males  is  also  too  rare  to 
give  any  expectation  of  finding  regenerated  aberrant  structures. 
The  copulatory  hooks  upon  the  third  legs  have  been  selected, 
therefore,  as  the  most  likely  structures  for  observation  because 
their  occurrence  upon  females  furnishes  the  most  common  aberra- 
tion. 


SECONDARY    SEXUAL    CHARACTERS    HE    CRAVEISHES.  199 

In  Lake  Delavan  (Wisconsin)  eighty-six  per  cent,  of  the  fe- 
males of  Cambarus  virilis  carry  the  copulatory  hooks  like  those 
which  occur  upon  the  third  legs  of  the  males.  It  is  as  fully  de- 
veloped in  the  female  as  it  is  in  the  normal  male  and  developes 
in  ontogeny  at  the  same  stage.  In  the  Menomonee  River  (Wis- 
consin) thirty-seven  per  cent,  of  the  females  of  Cambarus  propin- 
quus  also  bear  these  hooks.  These  two  localities  were  chosen  as 
most  likely  to  produce  the  desired  specimens. 

In  the  course  of  three  summers  collecting  after  several  thou- 
sands of  specimens  had  been  examined,  seven  males  and  three 
females  of  Cambarus  virilis  from  Lake  Delavan  were  found,  each 
of  which  had  lost  and  regenerated  one  of  the  third  legs.  In  each 
specimen  the  third  leg  had  regenerated  to  a  point  where  it  was 
possible  to  determine  whether  or  not  a  normal  copulatory  hook  was 
being  formed.  Males,  ranging  from  56  to  88  mm.  in  length,  had 
all  somewhat  imperfectly  developed  new  hooks  upon  the  regener- 
ated legs  (Fig.  i).  These  normal  males  with  regenerated  hooks 


FIG.  i.  Diagram  illustrating  three  basal  segments  of  third  walking  legs 
and  copulatory  hooks  in  male  crayfish  which  has  lost  and  regenerated  a 
part  of  left  leg  containing  copulatory  hook.  Regenerated  copulatory  hook 
is  short  and  blunt. 

were  used  as  controls  with  which  to  compare  the  females  which 
had  likewise  lost  their  third  walking  legs  and  had  regenerated 
the  third  legs  together  with  the  hooks  upon  them.  Of  the  fe- 
males, two  had  well  developed  hooks  upon  the  uninjured  third 
legs  and  upon  the  regenerated  third  legs  the  hooks  had  reformed 
as  in  the  males  (Fig.  2).  In  the  third  female  the  uninjured  third 
leg  carried  no  hook  and  upon  the  regenerated  third  leg  no  hook 
had  formed. 

In  the  Menomonee  River,  two  specimens  of  Cambarus  propin- 
quus  were  found  which  could  be  used  for  this  study.  The  first 
was  a  male  in  which  the  uninjured  third  leg  was  entirely  normal 
and  carried  the  usual  hooks.  The  left  third  leg  had  been  lost  at 


2QO  C.    L.    TURNER. 

an  early  stage  and  had  regenerated.  Upon  it  was  the  blunt  type 
of  hook  usually  found  upon  the  regenerated  third  leg.  The  second 
was  a  female  which  also  carried  a  hook  upon  the  uninjured  third 


FIG.  2.  Diagram  illustrating  three  basal  segments  of  third  walking  legs 
in  an  aberrant  female  which  had  lost  and  regenerated  a  part  of  the  third 
walking  leg.  The  copulatory  hooks  are  aberrant  and  the  left  one  has  been 
regenerated. 

leg  and  had  also  developed  a  blunt  hook  upon  the  regenerated 
third  leg. 

EXPERIMENTS. 

Young  specimens  of  Cambarus  virilis,  about  thirty-six  mm.  in 
length,  were  selected  for  experiment.  They  were  taken  from 
Lake  Delavan  on  July  17.  Eighty-two  males  in  which  the  copu- 
latory hooks  on  the  third  legs  were  visible  were  divided  into  two 
equal  lots.  One  lot  was  used  as  a  control  and  in  the  other,  one 
of  the  third  legs  was  detached  in  each  specimen.  Fifty-five  fe- 
males in  which  a  copulatory  hook  was  visible  upon  the  third  leg" 
were  divided  into  two  lots  and  similarly  operated  upon  or  used  as 
controls.  All  were  kept  in  the  laboratory  under  conditions  as 
nearly  natural  as  possible  for  ten  months  and  about  one  fourth  of 
the  specimens  survived.  Six  of  these  were  males  in  which  one 
third  leg  had  been  removed  and  upon  the  regenerated  leg  there 
had  developed  the  copulatory  hook.  Seven  were  females  from 
which  one  third  leg  had  been  removed.  All  such  females  had  re- 
generated the  third  legs  together  with  the  copulatory  hooks  upon 
them.  The  hooks  compared  favorably  with  those  regenerated  by 
the  males  of  approximately  the  same  age. 

CONCLUSIONS. 

In  all  the  cases  cited  above,  whether  observed  in  nature  or  ex- 
perimentally produced,  females  bearing  aberrant  male  hooks  upon 
their  third  walking  legs  regenerated  hooks  whenever  an  injured 
leg  had  sufficiently  developed.  Some  were  one  year  of  age  and 


SECONDARY    SEXUAL    CHARACTERS    OF    CRAYFISHES.  2OI 

others  were  older.  It  is  reasonable  to  state,  therefore,  that  all  the 
evidence,  though  meager,  tends  to  show  that  whatever  influence 
was  present  in  the  first  place  to  produce  this  aberrant  secondary 
sex  character  was  also  present  and  operative  in  the  animal  later 
during  any  regeneration  period.  The  permanency  of  this  influ- 
ence during  the  life  of  an  animal  would  seem  to  take  it  out  of 
the  classification  of  accidental  or  temporary  embryonic  agencies. 
When  it  is  considered  together  with  the  fact  that  the  same  aber- 
rancy is  repeated  in  this  crayfish  population  (observed  for  six 
years)  it  seems  logical  to  give  the  influence  a  genetic  status  and 
to  postulate  that  there  has  been  a  definite  change  within  the  germ 
cell. 


REGENERATION    OF    LUMBRICULUS    IN    VARIOUS 

RINGER   FLUIDS. 

LEONARD  P.  SAYLES, 
TUFTS  COLLEGE. 

INTRODUCTION. 

In  the  course  of  work  with  Ringer  solution  on  Planaria,  J.  W. 
Wilson  ('26)  has  noticed  that  wound  closure  may  be  more  or 
less  completely  suppressed  in  an  isotonic  solution.  With  the  in- 
tention of  making  use  of  this  peculiarity  if  it  held  true  for  Lum- 
briculus,  I  have  experimented  with  various  strengths  of  Ringer 
solution  on  this  form.  Finding  various  modifications  of  the  usual 
method  of  wound  closure  and  regeneration,  I  have  made  studies 
on  the  effects  of  various  strengths  of  Ringer  fluid  on  regeneration 
in  this  form.  It  is  my  purpose  to  report  these  at  this  time. 

As  a  preliminary,  the  approximate  osmotic  pressure  of  the  body 
fluids  of  this  worm  were  determined  in  order  that  it  might  be 
possible  to  know  something  concerning  the  relative  strengths  of 
the  internal  fluids  and  the  external  solutions  used.  Adolph  ('25, 
p.  332)  concludes  that  we  can  "  probably  regard  the  maximum 
survival  concentration  for  freshwater  animals  as  a  measure  of 
the  osmotic  pressure  of  their  body  fluids."  The  maximum  sur- 
vival concentration  of  Ringer  solution  for  Lumbriculus  at  the  end 
of  24  hours  (the  arbitrary  time  adopted  by  Adolph,  '25,  for 
Phagocata)  was  found  to  be  O.I47M.  When  corrected  for  ioni- 
zation  this  gives  a  figure  of  O.257M  as  compared  with  O.2I5M 
for  Lumbricus,  as  assumed  by  Adolph  and  Adolph  ('25).  Ap- 
parently a  Ringer  solution  of  between  O.14M  and  O.I5M  concen- 
tration is  approximately  isosmotic  with  the  body  fluids  of  Lum- 
briculus. 

WOUND    CLOSURE    IN    VARIOUS    CONCENTRATIONS    OF    RINGER 

SOLUTION. 

The  usual  method  of  wound  closure  in  microdrilous  annelids 
has  been  quite  completely  described  (von  Wagner,  'oo  and  '06, 

202 


REGENERATION    OF    LUMBRICULUS.  2O3 

Iwanow,  '03  and  Krecker,  '10,  among  others),  both  from  the 
gross  and  from  the  microscopical  points  of  view.  Briefly  it 
takes  place  in  somewhat  the  following  manner:  Immediately  after 
a  worm  is  cut  the  muscles  of  the  body  wall  begin  to  contract. 
They  continue  to  draw  the  cut  edges  of  the  hypodermis  together 
until  only  a  small  opening  is  left.  This  aperture  is  then  clogged 
by  a  plug  of  cells,  many  of  which  have  been  torn  free  by  the 
cut.  The  wound  is  thus  completely  closed  and  the  body  fluids 
once  more  prevented  from  freely  mixing  with  the  solution  in 
which  the  worm  is  cut.  At  the  same  time  the  intestine  also  con- 
tracts somewhat,  closing  over  at  the  end  and  withdrawing  slightly 
from  the  contracting  body  wall.  This  preliminary  wound  closure 
is  completed  in  from  10  to  15  minutes  after  the  cut  is  made. 

The  behavior  in  an  isotonic  solution  is  in  distinct  contrast  to 
this  usual  behavior.  The  following  description  of  what  occurred 
in  one  series  of  observations  might  well  apply  to  many  cases  which 
have  been  followed  for  considerable  periods. 

An  individual  is  cut  in  a  O.I4M  Ringer  solution,  without  anaes- 
thetization,  at  2  154  P.M.  Both  pieces  move  about  quite  rapidly 
at  first  but  in  3  or  4  minutes  they  have  quieted  down  to  ordinary 
"  crawling "  movements,  such  as  are  commonly  found  in  unin- 
jured individuals.  During  this  time  there  is  a  loss  of  some  blood 
and  a  number  of  cells  due  to  the  fact  that  there  is  no  semblance 
of  contraction  of  the  body  wall.  At  3:03  there  is  evidence  of  a 
protrusion  of  the  gut  beyond  the  end  of  the  body  wall.  This 
protrusion  of  the  gut  gradually  becomes  more  pronounced  until 
a  portion,  perhaps  a  segment  in  length,  extends  beyond  the  plane 
of  the  cut  at  3:10.  At  this  time  the  cut  end  of  the  gut  begins  to 
show  evidence  of  a  rolling  back  upon  itself.  This  process  con- 
tinues until  at  the  end  of  an  hour  there  is  a  well  formed  bulb  of 
everted  gut  present  at  the  cut  end  (Fig.  i).  During  this  time 
there  has  been  a  gradual  contraction  of  the  body  wall  until  it  has 
reduced  the  diameter  of  the  opening  resulting  from  the  cut  to 
about  two  thirds  of  its  original  size.  The  gut  in  the  course  of  its 
eversion  has  now  come  in  contact  with  the  body  wall  so  that 
there  is  very  little  opportunity  for  interchange  of  materials  be- 
tween the  body  fluids  and  the  external  solution.  This  is  the  end 
of  wound  closure  from  the  macroscopical  point  of  view. 


204 


LEONARD    P.    SAYLES. 


As  a  result  of  this  process  of  "  wound  closure  "  there  is  present 
at  the  cut  surface,  at  the  end  of  i  or  2  hours,  a  bulb  of  everted 
gut  usually  of  almost  as  great  diameter  as  that  of  the  body.  The 
ciliated  portion  of  the  gut  cells  are  thus  exposed  to  the  outside 
solution,  in  which  they  continue  to  beat  with  apparently  the  usual 
rapidity. 


s 


^ 

FIGS,  i  AND  2.  Bulbs  of  everted  gut  at  the  posterior  end  of  pieces  re- 
generating in  o.ogiM  Ringer  solution.  Fig.  I  after  12  hours;  Fig.  2  after 
6  days. 

In  the  case  of  slightly  hypotonic  solutions  the  resulting  bulb  of 
gut  is  usually  smaller,  in  some  instances  not  more  than  one  fourth 
the  diameter  of  the  worm.  The  presence  of  these  smaller  bulbs 
is  due,  in  part  at  least,  to  the  fact  that  in  these  cases  the  gut  does 
not  protrude  as  far  at  first,  so  that  when  the  eversion  occurs 
there  is  only  a  short  piece  involved.  A  contributing  factor  is  the 
slight  pulling  together  of  the  body  wall ;  this  probably  tends  to 
hinder  the  protrusion  of  the  gut  and  in  addition  reduces  some- 
what the  diameter  of  the  portion  which  does  pass  through  the 
aperture. 

POSTERIOR  REGENERATION  IN  VARIOUS  RINGER  SOLUTIONS. 

If  we  assume  that  the  maximum  survival  concentration  at  the 
end  of  a  twenty-four-hour  period  is  isotonic  with  the  body  fluids 
of  an  animal,  it  is  difficult  to  keep  individuals  in  an  isotonic  fluid 
for  a  very  long  period.  One  experiment  may  be  cited  to  show 
what  occurs  when  individuals  are  left  for  a  long  period  in  O.I4/M 
Ringer,  the  solution  being  changed  each  day  at  the  time  of  ob- 
servation. Fifteen  worms  were  put  into  such  a  solution  and  on 
the  following  day  all  were  alive,  with  no  ill  effects  apparent.  On 
the  second  day,  2  had  died  and  3  others  were  clearly  not  far  short 
of  death.  On  the  third  day,  4  more  were  dead  and  4  others  were 
beginning  to  disintegrate  at  the  posterior  end.  On  the  fifth  day, 
only  5  were  alive  and  I  of  these  was  beginning  to  disintegrate 


REGENERATION    OF    LUMBRICULUS. 


205 


<it  the  posterior  end.  Because  of  this  high  mortality  in  Ringer 
stronger  than  o.i_|.M,  it  has  been  found  advisable  to  observe  the 
regenerative  processes  in  the  latter  concentration  in  which  the 
majority  of  the  regenerating  head  ends  will  live  for  a  considerable 
period.  Even  in  this  strength  many  tails  die  in  the  course  of  } 
or  4  days. 

In  the  case  of  heads,  regenerating  new  tails,  which  have  been 
cut  and  left  in  O.I4M  Ringer,  many  individuals  have  bulbs  very 
similar  to  those  found  after  2  or  3  hours,  even  at  the  end  of  a 
week.  For  example,  of  12  heads  put  into  such  a  solution,  9  had 
bulbs  of  everted  gut,  i  had  a  short  and  very  slender  bud  of  re- 
generating tissue  and  2  had  died  at  the  end  of  a  week.  This  pro- 
portion of  individuals  with  bulbs  of  gut  holds  true  within  fairly 
narrow  limits  in  all  experiments  using  O.I4M  Ringer. 

In  light  of  these  results  obtained  with  a  solution  almost  iso- 
tonic  with  the  body  fluids,  it  is  of  interest  to  determine  the  effect 
of  slightly  greater  dilutions  of  Ringer  solution.  The  number  of 
bulbs  of  gut  present  on  individuals  '"  regenerating "  in  various 
strengths  of  Ringer  solution  for  /  days  is  given  in  Table  I. 

TABLE  I. 

NUMBER  OF  INDIVIDUALS  WITH  BULBS  OF  GUT  ix  DIFFERENT  CONCENTRA- 
TIONS OF  RINGER  SOLUTION. 

10  worms  were  cut  in  each  concentration. 
Figures  in  parenthesis  indicate  number   which  also  show  new  tissue. 


Molar  cone, 
of  Ringer. 

Days  of  Regeneration. 

i 

2 

3 

4 

5 

6 

7 

O.OII              .... 

o 
o 
o 

3d) 

2(2) 

3d) 

8(2) 

7(3) 
8d) 
10(0) 
9(o) 
10(0) 

O 
O 
O 

id) 

o 

3(2) 

8(4) 
7(4) 
8(5) 
1  0(0) 

9(o) 
10(0) 

o 
o 

0 

id) 

o 

1(1) 

8(7) 
7(7) 
8(8) 

10(1) 
9(o) 
10(0) 

o 
o 
o 

id) 

o 

id) 

8(8) 

7(7) 
8(8) 

10(4) 
9(3) 
1  0(0) 

o 
o 
o 

id) 

0 

id) 

8(8) 

7(7) 
8(8) 
10(10) 

9(5) 
10(0) 

0 

o 
o 

id) 

o 

id) 

8(8) 

7(7) 
8(8) 
10(10) 
9(6) 
9(0)1 

0 

o 
o 

Id) 

o 

id) 

8(8) 

7(7) 
8(8) 
10(10) 
9(6) 
9(o) 

0.023 

0.034  

o.oj^ 

O.O^l 

o-.o68 

O.O7O 

0.091  

O.IO2  

O.  I  I  } 

0.125  

0.136 

1  I  dead  on  the  sixth  day. 


206  LEONARD    P.    SAYLES. 

After  one  day  of  regeneration  all  the  bulbs  of  gut  present  are 
practically  in  line  with  the  central  longitudinal  axis  of  the  body 
(Fig.  i).  The  anal  opening  (not  a  true  anal  opening,  for  it  is 
completely  surrounded  by  gut  which  forms  part  of  the  outside 
wall  in  these  cases)  is,  therefore,  terminal.  On  the  second  day, 
however,  those  pieces  in  O.IO2M  and  weaker  solutions  have  bulbs 
tipped  at  an  angle  so  that  the  opening  is  not  exactly  terminal 
(Fig.  2).  This  change  in  the  position  of  the  bulbs  does  not  occur 
as  soon  in  o.ii3M  and  O.I25M;  it  does  appear,  however,  usually 
in  all  individuals  by  the  fifth  or  sixth  day.  But  few  individuals 
show  any  evidence  of  this  tipping  in  O.I4M.  In  this  respect  the 
individuals  cited  in  Table  I.  under  O.I36M  were  exceptional, 
since  none  happened  to  show  evidence  of  new  tissue.  The  under- 
lying cause  of  this  slight  shifting  of  the  position  of  the  bulb  is 
apparently  the  growth  of  tissue  on  one  side  of  the  wound  region 
and  not  on  the  other.  The  region  in  which  growth  occurs  is 
found,  on  microscopical  examination,  to  be  always  ventral. 

There  is,  then,  from  the  macroscopical  point  of  view,  a  graded 
effect  on  regeneration  in  Lumbriculus  produced  by  a  series  of 
various  concentrations  of  Ringer  solution.  In  an  approximately 
isotonic  solution  the  majority  of  individuals  show  little  evidence 
of  the  production  of  new  tissue ;  except  in  rare  cases  those  indi- 
viduals which  do  produce  sufficient  new  tissue  to  make  it  apparent 
do  so  only  on  the  ventral  side.  In  weaker  solutions  new  tissue 
is  produced  in  practicaly  all  cases.  In  dilutions  but  slightly  hypo- 
tonic  (o.oSM  to  O.I3M)  this  process  is  confined  to  the  ventral 
region  in  most  instances.  In  most  cases  where  new  tissue  is 
produced  throughout  the  wound  region,  no  bulb  of  gut  is  present. 
Occasionally,  however,  a  very  small  bulb  may  be  found  at  the 
end  of  a  regenerating  bud  which  is  i  or  2  mm.  long.  In  solu- 
tions below  o.oSM,  on  the  other  hand,  practically  every  individ- 
ual produces  new  tissue  throughout  the  wound  region  and  bulbs 
of  gut  are  rare. 

SUMMARY  AND  DISCUSSION. 

In  Lumbriculus,  therefore,  it  would  appear  that  an  important 
factor  in  arousing  the  cells  to  regenerative  activity  is  the  dilution 
of  the  body  fluids  with  water.  When  an  individual  is  cut  or 


REGENERATION    OF    LUMBRICULUS.  2(>7 

broken  into  two  or  more  pieces,  there  are  open  wounds  through 
which  the  body  fluids  may  flow  out  and  water  may  enter.  This 
entrance  of  water  is  perhaps  increased  by  the  "  writhing  "  and 
"  crawling  "  movements  of  pieces  immediately  after  they  are  cut. 
These  movements  would  also  extend  the  region  of  dilution  a 
slight  distance  from  the  wound.  The  author  (Sayles,  '27)  has 
reported  that  for  mesoderm  and  intestine  regenerative  activity 
involves  between  10  and  12  segments  from  the  wound  region. 
In  the  case  of  the  hypodermis,  which  is  commonly  bathed  on  the 
outside  by  a  hypotonic  solution,  activation  occurs  only  in  a  re- 
stricted region  near  the  wound.  This  limited  activity  of  the  hypo- 
dermis is  due  perhaps  to  the  fact  that  the  diluted  fluids  come  in 
contract  with  its  inner  surface  underneath  the  muscles  which  are 
pulled  away  near  the  wound.  Farther  away  than  that,  however, 
the  diluted  body  fluids  probably  never  reach  the  hypodermal  cells 
through  the  relatively  thick  layer  of  muscle  cells. 

While  the  hypotonicity  of  the  water  to  the  body  fluid  seems  to 
be  an  activating  factor  in  Lumbriculus,  too  general  conclusions 
cannot  be  drawn  from  such  results.  In  other  fresh  water  forms 
this  factor  may  be  found  to  be  of  importance  but  in  marine  ani- 
mals regeneration  can  certainly  take  place  in  a  medium  which  is 
presumably  of  the  same  osmotic  pressure  as  their  body  fluids. 
In  these  animals,  however,  greater  regenerative  activity  may  occur 
in  slightly  diluted  rather  than  in  normal  sea-water.  This  has  been 
reported  by  Goldfarb  ('07,  p.  353)  in  the  hydroid,  Eudcndrimn, 
in  which  "  the  maximum  number  of  polyps  regenerated  does  not 
occur  in  normal  sea-water  but  in  solutions  diluted  with  about  20 
per  cent,  of  tap- water." 

REFERENCES. 
Adolph,  E.  F. 

'25     Some    Physiological    Distinctions    Between    Freshwater    and    Marine 

Organisms.     BIOL.  BULL.,  48. 
Adolph,  E.  F.,  and  P.  E. 

'25     The  Regulation  of  Body  Volume  in  Fresh-water  Organisms.     Jour. 

Exp.  Zool.,  p.  43. 
Goldfarb,  A.  J. 

'07     Factors  in   the  Regeneration  of   a   Compound   Hydroid,   Eudendrium 

Ramosnin.    Jour.  Exp.  Zool.,  4. 
Iwanow,  P. 

'03     Die  Regeneration  von  Rumpf-  und  Kopfsegmenten  bei  Lumbriculus 

variegatus.     Zeit.  f.  wiss.  Zool.,  75. 
14 


2O8  LEONARD    P.    SAYLES. 

Krecker,  F.  H. 

'10     Some  Phenomena  of  Regeneration  in  Limnodrilus  and  Related  Forms. 

Zeit.  f.  wiss.  Zool.,  95. 
Sayles,  L.  P. 

'27     Origin  of  the  Mesoderm  and  Behavior  of  the  Nucleolus  in  Regener- 
ation in  Lumbriculus.    BIOL.  BULL.,  52. 
von  Wagner,  F. 
'00    Beitrage    zur    Kenntnis    der    Reparationsprozesse    bei    Lumbriculus 

variegatus.     I.  Teil.     Zool.  Jahrb.  Anat,  13. 
von  Wagner,  F. 

'06    Beitrage   zur   Kenntnis   der   Regenerationsprozesse   bei   Lumbriculus 

variegatus.     II.  Teil.     Zool.  Jahrb.  Anat.,  22. 
Wilson,  J.  W. 

'26    Regeneration  of  Planaria  maculata  in  Isotonic  Ringer's  Fluid.    Anat. 
Rec.,  34. 


VARIATION  OF  HOOKS  ON  THE  HIND  WING  OF  THE 
HONEY  BEE  (APIS  MELLIFERA  L.).1 


W.  W.  ALPATOV, 
RESEARCH  FELLOW  OF  THE  INTERNATIONAL  EDUCATION  BOARD. 

The  present  paper  represents  partial  results  of  a  series  of  in- 
vestigations carried  on  by  4he  author  since  1924  in  the  field  of 
biometry  of  the  honey  bee  (See  Alpatov,  i-io).  The  material 
for  this  work  has  been  collected  partly  during  the  author's  work 
in  the  Zoological  Museum  of  the  Moscow  University,  and  partly 
(material  on  American  bees)  during  the  summer  of  1927  in  the 
apicultural  laboratory  of  the  Agricultural  College,  Cornell  Uni- 
versity. The  definite  calculations  and  the  preparation  of  the 
manuscript  have  been  completed  during  the  winter  1927-28  in 
the  Institute  for  Biological  Research.  The  author  is  glad  to  ex- 
press his  deep  indebtedness  to  Professor  Koshewnikov  (Moscow), 
Professor  E.  F.  Phillips  (Cornell  University),  and  Professor  Ray- 
mond Pearl  for  their  interest  and  help.  The  author  also  appreci- 
ated very  much  the  help  given  by  beekeepers  of  Russia  and  U.  S.A. 
in  collecting  bees  from  different  parts  of  both  countries.  Profeessor 
E.  F.  Phillips  has  also  been  so  kind  as  to  show  the  author  the 
manuscript  of  his  unpublished  paper. 

In  spite  of  the  fact  that  the  beekeeper's  literature  contains  a 
tremendous  number  of  observations  on  differences  in  bee  races, 
a  scientific  basis  of  racial  studies  in  bees  is  practically  absent, 
especially  in  comparison  with  racial  and  genetical  studies  on  other 
domestic  animals.  The  cause  of  this  lies  chiefly  in  certain  pe- 
culiarities which  characterize  the  honey  bee.  Firstly,  the  bees 
being  fecundated  in  air  do  not  allow  us  to  control  the  mating  and 
therefore  to  conduct  exact  genetical  experiments.  Secondly,  it 
is  more  difficult  to  study  the  characteristics  of  such  small  animals 
as  the  honey  bee  than  those  of  domestic  mammals  and  birds. 
Only  quite  recent  progress  in  artificial  insemination  of  the  queen 

1  From  the  Institute  for  Biological  Research,  Johns  Hopkins  University. 

209 


2IQ  W.    W.    ALPATOV. 

(Watson,  '20)  gives  us  the  hope  of  being  able  to  overcome  the 
first  of  these  obstacles. 

The  present  author  believes  that  a  careful  investigation  of  varia- 
tion should  be  made  before  any  attempts  to  study  the  heredity  of 
the  honey  bee.  In  this  direction  the  present  paper  brings  evi- 
dence of  the  importance  of  a  certain  characteristic,  namely,  the 
number  of  the  hooks,  characterizing  different  biological  groups  in 
the  limits  of  the  species  Apis  incllifcra  L.  Thanks  to  the  mod- 
ern investigations  mostly  of  Russian  scientists  (Koschewnikov, 
Chochlov,  Michailov,  Alpatov,  Alpatov  and  Tjunin)  two  very 
important  facts  in  the  field  of  variation  of  the  honey  bee  have 
been  discovered. 

The  first  of  them  is  the  geographical  regularity  in  the  varia- 
tion. The  changes  in  tongue  length  of  the  worker  bee  is  the 
most  striking  fact  in  the  geographical  variation  of  the  honey  bee. 
We  are  able  to  say  that  for  countries  with  native  bee  population 
each  locality  is  characterized  by  a  definite  tongue-length  of  bees 
inhabiting  the  given  locality.  Moreover,  the  change  from  one 
locality  to  another  is  regular  and  gradual.  A  general  rule  can 
be  established ;  the  more  to  the  south,  the  longer  the  tongue  length. 
Other  body  characteristics  also  show  some  regularities  in  geo- 
graphical variation  (Alpatov,  8).  The  author  of  the  present 
paper  believes  that  it  is  perfectly  justifiable  to  compare  the  dif- 
ferent r>  races  "  of  the  honey  bee  with  geographical  subspecies 
of  wild  animals. 

Family  variation  is  the  second  important  fact  which  every  in- 
vestigator in  the  field  of  variability  of  social  insects  has  to  bear 
in  mind.  It  was  shown  by  several  investigators,  Thomson,  Bell 
and  Pearson  (23,  24),  Warren  (25),  Arnddi  (12),  Z.  G. 
Palenitschko  (20),  Alpatov  and  Tjunin  (i)  and  Alpatov  (3,  4, 
6,  9,  10),  that  the  variation  of  single  families  is  smaller  than 
the  variation  of  the  whole  population.  Therefore,  in  establishing 
racial  characteristics  we  have  to  collect  our  material  from  as  many 
families  as  possible. 

Turning  our  attention  to  the  literature  devoted  to  the  special 
question  of  hook  variation  we  find  only  a  small  number  of  papers 
dealing  with  that  particular  subject.  Professor  Koschewnikov 
(19)  was  the  first  who  introduced  the  number  of  hooks  in  the 


HOOKS    ON    HIND    WING    OF    IIONKY    BEE.  211 

taxonomy  of  the  honey  bee.  E.  B.  Casteel  and  K.  F.  Phillips 
(14)  without  using  biometrical  methods,  tried  to  solve  the  prob- 
lem of  comparative  variability  of  drones  and  worker  bees.  Kel- 
log's  (i/)  data  have  also  a  very  restricted  value  from  the  point 
of  view  of  modern  biometry.  Wright,  Lee  and  Pearson  (27) 
then  attempted,  by  recalculating  Casteel's  and  Phillips'  data,  to 
draw  some  more  definite  conclusions.  The  most  extensive  work 
has  been  done  by  Bachmetjew  (13).  The  conclusions  of  this 
author  found  just  criticism  in  Koschewnokov's  (18)  and  Ray- 


20 


10 


/3    H    15    16    17    /g   19    20  21    22  23  24  25  26  27  28  29 

FIG.  i.     Hook  variation  of  3  colonies  of  the  Bulgarian  drones. 

mond  Pearl's  (21)  papers  and  need  not  be  mentioned  further. 
Fortunately,  Bachmetjew  published  in  his  paper  all  his  numerous 
countings  (about  2,500  bees  were  examined).  His  data  have 
been  worked  out  biometrically  by  the  author  of  the  present  paper, 
and  published  in  Russian  (4).  Professor  Phillips  did  the  same 
in  the  paper  which  is  now  in  press.  In  this  paper  Professor 
Phillips  turns  his  attention  mostly  to  the  individual  variation  in 


212 


W.    W.    ALPATOV. 


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HOOKS    ON    HIND    WING    OF    HONEY    BEE. 


213 


the  honey  bee,  and  on  that  account  his  conclusions  do  not  parallel 
those  of  the  present  paper. 

The  number  of  bees  examined  by  the  author  of  the  present 
paper  exceed  three  thousand — a  number  which  has  never  been 
reached  by  previous  investigators. 

Table  I.  shows  us  the  variation  of  Bulgarian  drones  belonging 
to  different  colonies.  It  is  evident  that  the  difference  between 
the  averages  are  in  many  cases  more  than  five  times  larger  than 
their  probable  errors.  Fig.  I  represents  3  variation  curves  of  the 
4th,  Qth  and  loth  colonies,  proving  the  conclusion  just  made. 
Table  II.  shows  the  same  for  worker  bees.  It  can  be  seen  that  in 

TABLE  II. 

CONSTANTS  FOR  WORKERS  OF  5  COLONIES  OF  BULGARIAN  BEES 
(DATA  FROM  BACHMETJEW). 


Number  of  the  Colony. 

i 

2 

3 

4 

5 

M. 

21.60  ±  .10 
1.487 
6.88  ±  .33 
99 

21.01    ±  .OQ 
1.367 
6.51   ±-3I 

99 

21.76  ±  .10 
1.566 
7.20  ±  .33 
no 

21.91  dh  .12 
1.867 
8.52  ±  .41 
IOO 

21.  II   ±  ,IO 

1.467 

6-95  ±  -33 
98 

<7 

COT 

Number  of  cases 

the  last  case  the  differences  are  not  so  pronounced  as  in  the  case 
of  the  drones.  Fig.  2  compared  with  Fig.  i  gives  the  same  im- 
pression. If  we  consider  the  coefficients  of  variation  we  find  that 
for  the  drones  they  vary  in  the  limits  7.52-13.02  per  cent.;  for 
the  worker  bees  6.88-8.52  per  cent.  It  is  obvious  that  the  aver- 
age variation  of  worker  bees  of  the  colony  is  smaller  than  the 
variation  of  the  drones. 

Are  we  justified  in  concluding  that  the  drones  are  more  vari- 
able than  the  worker  bees  ?  There  is  a  certain  weak  point  in  such 
conclusions.  We  are  not  convinced  that  the  method  of  collecting 
the  material  was  safe  enough  to  provide  us  with  bees  really  rep- 
resenting the  progeny  of  single  queens — i.e.,  members  of  one 
family.  The  proper  way  to  get  such  a  material  would  be  to  put 
a  sealed  brood  in  an  incubator  and  collect  the  emerging  bees.  In 
collecting  bees  directly  from  the  hive  there  is  a  danger  of  pick- 
ing up  bees  belonging  to  the  population  of  a  neighbour  hive.  It 


214 


W.    W.    ALPATOV. 


is  known  that  the  bees  and  especially  the  drones  sometimes  pene- 
trate into  neighboring  hives.     The  only  way  to  avoid  this  diffi- 


30 


20 


10 


17    /Z   19    20  21    22   23  24  25  26 
FIG.  2.     Hook  variation  of  3  colonies  of  the  Bulgarian  worker  bees. 

culty  is  to  calculate  the  coefficient  of  variation  for  the  whole  mass 
of  bees.  The  results  of  such  processes  are  shown  in  Table  III. 
It  can  be  seen  that  the  variation  of  the  worker  bees  belonging  to 

* 

TABLE  III. 

BULGARIAN  BEES  IN  DIFFERENT  GROUPINGS. 


Queens 

All  Drones 

Workers  from 

from  10 

Drones  from 

Drones  from 

i-io  Colonies. 

5  Colonies. 

localities 

1-5  Colonies. 

5-10  Colonies. 

in  Bulgaria. 

21.39  ±  -05 

21.49  ±  -05 

18.46  ±  .11 

21.82  ±  .07 

20.98  ±  .07 

2.352 

1.586 

1.892 

2.157 

2.438 

11.00   ±  -I? 

7.38  =fc  .16 

10.25  ±  .42 

9.89  ±  .42 

11.62  ±  .25 

997 

507 

139 

490 

507 

HOOKS    OX    HIND    WING    OF    HONEY    P.M.. 


215 


the  5  colonies  is  lower  than  the  variation  of  the  two  groups  of 
drones  each  representing  members  of  5  colonies.  The  coeffi- 
cients of  variation  calculated  from  our  original  material  on  worker 
bees  are  also  in  general  lower  than  8  per  cent.  Even  for  1000 
worker  bees  from  Middle  Russia  taken  from  106  colonies  the 
coefficient  of  variation  is  only  8.539  —  -I29>  as  can  be  seen  from 
Table  X.  We  believe  that  the  present  material  permits  us  the 
definite  conclusion  of  a  larger  variability  of  drones  in  respect  to 
the  number  of  hooks. 

Table  III.  contains  also  data  on  variation  of  hooks  in  queens. 
Firstly,  it  is  evident  that  the  average  number  of  hooks  is  far  lower 
than  in  the  drones  and  workers,  which  have  practically  the  same 
averages.  This  conclusion  is  given  here  in  statistical  form  for 
the  first  time,  although  G.  A.  Koschevnikov  has  already  given 
a  few  analogous  data.  In  respect  of  the  coefficient  of  variation 
the  queens  are  nearer  to  the  drones  than  to  the  workers.  A  very 
incomplete  material  collected  in  Table  IV.  shows  that  Middle  Rus- 
sian, German  and  American  black  and  yellow  queens  have  also  a 
much  lower  average  number  of  hooks  than  the  worker  bees  of 
the  corresponding  races. 

TABLE  IV. 

NUMBER  OF  HOOKS  OF  DRONES  AND  QUEENS  FROM  DIFFERENT  LOCALITIES. 


Drones. 

Queens 

Mos- 
cow. 

Kaluga 
(M.  Russia). 

N.  Wodolaga 
(S.  Russia). 

Black- 
Ontario. 

Italians. 

Moscow  and 
Darmstadt. 

M  .       .    . 

20  72 

2O  22   ±    2O 

20  83  ±    26 

16  2? 

1  8  oo 

18.67  ±  .22 

c%  

0.76  ±  .60 

12  oo  ±    87 

9.06  ±  .83 

No.  of  cases 

2  ^ 

AS 

48 

8 

10 

27 

It  is  interesting  to  ,note  that  among  the  bees  the  relations  of 
castes  in  respect  of  variation  differ  from  those  found  in  other 
social  insects.  It  was  shown  (Palenitschenko,  20)  that  among 
wasps,  termites  and  ants,  the  workers  are  more  variable  than  the 
sexual  forms — males  and  females.  The  worker  caste  among 
bees  is  therefore  an  exceptionally  constant  and  standardized  group 
of  individuals. 

Already  in  an  earlier  paper  (4)  some  evidence  has  been  brought 


2l6 


W.    W.    ALPATOV. 


together  to  show  that  the  bees  of  southern  localities  have  a  greater 
average  number  of  hooks  than  the  northern  ones.  In  order  to 
check  that  statement  on  a  more  solid  basis,  a  special  material  has 
been  collected  from  different  parts  of  European  Russia  and  the 
Caucasus.  The  map  in  Fig.  3  shows  the  localities  which  supplied  a 


FIG.  3.  Map  of  European  Russia  and  Caucasus.     The  figures  correspond 
to  localities  where  the  material  has  been  collected. 

corresponding  material.  The  plain  of  European  Russia  is  pop- 
ulated by  the  black  variety  of  Apis  mellifera  L — A.  iiiellifcra. 
mcHifcra  L.  An  introduction  of  foreign  blood,  mostly  of  Italian 
queens,  was  according  to  certain  statistical  studies  a  compara- 


HOOKS    ON    HIND    WING    OF    HONEY    BEE. 


217 


tively  rare  phenomenon  and  could  probably  not  produce  any 
significant  influence  on  the  whole  mass  of  the  bee  population  of 
Russia  (the  number  of  hives  in  Russia  according  to  certain  esti- 
mations runs  over  5,000,000).  Tables  V.  and  VI  show  the  fre- 

TABLE  V. 

FREQUENCY  DISTRIBUTIONS  AND  CONSTANTS  OF  THE  NUMBER  OF  HOOKS  OF 

BEES  FROM  MIDDLE  RUSSIA. 


No.  of  Hooks. 

Localities. 

9 

10 

II 

12 

13 

14 

15 

1  6 

17-18 

2O 

19 

2oa 

21 

22 

20 

i 

2 

i 
6 
3 

20 
21 
24 
13 

8 
I 

4 
14 
19 

22 

25 
14 
I 
I 

I 

I 

4 

12 

14 
13 

8 

2 
2 
2 

I 

3 
9 
8 
6 

13 

8 

5 
i 

i 

I 

0 

I 
6 

13 
16 

13 
II 
I 

2 
2 
8 
IO 
12 

18 

4 
4 

i 

I 

5 
II 
18 
13 
4 
7 
i 
i 

I 
I 
4 
9 
16 

12 

IO 

5 

2 

2 

3 

13 
8 

25 

22 

18 

4 

2 

3 

I 
O 

3 

0 

7 
13 

10 
12 

3 
I 

4 

4 

IO 

ii 
5 
4 

2 

i 

2 
10 
10 

17 

18 
17 
13 
9 

2 

I 

I 
2 

4 
ii 

21 
26 
22 
9 

3 

3 

9 

10 

8 
9 

10 

3 

28    

27 

26  

2"C     , 

24 

2"?  .  . 

22     

21 

2O     

10 

18  

17    . 

16 

No.  of  cases 

IOO 

IOO 

59 

55 

62 

61 

61 

60 

IOO 

49 

40 

99 

IOO 

52 

No.  of  colonies  

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quency  distribution  for  different  localities,  number  of  colonies 
and  corresponding  biometrical  characteristics.  Table  X.  gives 
summarized  frequencies  for  the  bees  distributed  in  Middle  Rus- 
sia at  the  level  of  55°  of  N.  latitude  and  in  South  Russia,  50°  N. 
latitude.  The  difference  between  the  averages  is  8.85  times 
larger  than  the  probable  error.  We  may  conclude,  therefore,  with 
.a  high  degree  of  certainty,  that  there  is  an  increase  in  the  average 


218 


W.    W.    ALPATOV. 


TABLE  VI. 

• 

FREQUENCY  DISTRIBUTION  AND  CONSTANTS  OF  THE  NUMBER  OF  HOOKS  OF 

BEES  FROM  SOUTH  RUSSIA. 


Xo.  of  Hooks. 

Localities. 

i 

2-3 

4 

5 

6 

7 

8 

26  

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3 
4 
10 

i? 

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10 

5 
3 
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7 

12 

18 
19 

22 
15 
3 

3 

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9 
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10 

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6 

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24 
21 
15 

7 

3 

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4 
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8 

4 
i 

i 

2 

7 

10 
20 

14 

4 
3 

2? 

24 

2^ 

22 

21 

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10 

18                          

17 

No   of  cases 

64 

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48 

60 

92 

50 

60 

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number  of  hooks  in  the  southern  direction  even  between  groups 
of  bees  in  comparatively  closely  situated  localities.  Turning  our 
attention  to  the  Caucasus  (Fig.  5)  we  must  say  that  the  situa- 
tion here  is  more  complicated  than  in  the  plain  of  European 
Russia.  Zoogeographically,  the  Caucasus  is  divided  into  several 
sharply  limited  provinces,  each  of  them  with  peculiarities  in  the 
composition  and  the  origin  of  the  organic  life.  The  Caucasus 
bees  are  also  not  homogenous.  The  best  characterized  is  the  gray 
Caucasian  mountain  bee  Apis  mellifera  caucasica  Gorbatschev 
and  the  yellow  Transcaucasian  so-called  Persian  bee.  This  bee 
was  first  recognized  as  an  independent  species  by  Pallas ;  although 
he  did  describe  the  Caucasian  bee  he  has  never  published  his 
manuscript.  The  specimen  with  the  original  label  is  preserved 
in  the  Berlin  Zoological  Museum  and  was  briefly  described  by 


HOOKS    OX     HIM)    \\TXG    OF     IIONKY     1!KH. 


219 


20%- 


16    17    IS    19    20  21    22  23  24  25  26  27  2S  29 

FIG.  4.  The  continuous  curve  represents  the  variation  of  hooks  of  bees 
from  Middle  Russia.  The  dotted,  is  based  on  material  from  South  Russia. 
The  frequencies  are  expressed  in  percents. 


Black 
Sea 


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oooooo 
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Yellow  trans- 
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FIG.  5.     Map  of  Caucasus  showing  the  distribution  of  fifUvn  variations 
of  bees  (after  A.  Gorbatschev). 
15 


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222  w-    w-    ALPATOV. 

Gerstacker  (15).  The  author  of  the  present  article  was  able, 
thanks  to  the  courtesy  of  the  curator  of  the  collection  of  Hy- 
nienoptera  of  the  Berlin  Zoological  Museum,  Professor  Dr.  H. 
Bischoff,  to  examine  Pallas's  specimen  as  well  as  his  manuscripts. 
Pallas  gives  in  his  manuscript  the  following  indication  about  the 
origin  of  his  Caucasian  yellow  bee:  "  Ad  Caucasum  lecta,  itemque 
ex  Hyrcania  transmissa  fuit."  The  small  size  and  pronounced 
yellow  coloration  of  the  specimens  preserved  in  the  Berlin  Mu- 
seum permit  us  to  conclude  that  Pallas  and  Gerstacker  described 
under  the  name  Apis  remipcs,  the  Transcaucasian  Persian  bee, 
but  not  the  north  Caucasian  darker  bees. 

Some  peculiarities — for   instance   a  much   longer  tongue — dis- 
tinguish Apis   mcllifcra   remipcs   Gerstacker    (not    Pallas)    from 
the  Italian  bee  Apis  mcllifcra  Hgiistica  Sp.     It  is  therefore  not 
correct  to  identify  the  A.  m.  remipcs  with  the  Italian  bees  (Apis 
lii/itstica)  as  it  has  been  done  by  G.  A.  Koschewnikov.     Accord- 
ing to  Gorbatschev  (see  the  map  in  Fig.  5  taken  from  his  article 
(16)  )   the  prairies  and  hills  of  the  northern  Caucasus  and  the 
valleys  of  Transcaucasia  are  populated  by  a  bee  of  intermediate 
type — hybrids  in  his  interpretation.     We  united  our  material  into 
four  groups:  (a)   N.  Caucasus  bees,   (ft)  bees  from  four  apiaries 
near  the  coast  of  the  Black  Sea — Abchasian,   (r)  gray  Caucasian 
mountain  bees    (A.   mcllifcra   cancasica  Gorb.)    and    (of)    yellow 
Transcaucasian    bees    (Apis    mcllifcra    remipcs    Gerst).      Table 
VII.   shows   the   frequency   distributions   and   Table   VIII.   gives 
us  material  for  estimating  the  importance  of  our  differences.    The 
Apis  m.  cancasica  and  remipcs  show  a  pronounced  higher  number 
of  hooks  than  bees  of  South  Russia.     Of  course  such  a  compara- 
tively  limited   number   of   colonies    from   N.    Caucasus   does   not 
permit  us  to  draw  a  perfectly  definite  conclusion.     It  is  interest- 
ing to  note  that  the  gray  Caucasian  bees  imported  to  the  United 
States   (see  Table  IX.)  gave  also  a  high  average  of  the  number 
of  hooks. 

Table'  IX.  n'ives  us  some  data  on  other  European  races  of  bees. 
The  Italian  bees  from  Italy  are  characterized  by  a  high  number 
of  hooks.  It  can  be  seen  that  the  progeny  of  Italian  queens  im- 
ported from  Italy  to  X.  Caucasus  shows  also  a  high  number  of 
hooks.  The  German  black  bees,  according  to  our  recalculations 


HOOKS    ON    HIXU    WING    OF    HONEY    BEE. 


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226 


W.    W.    ALPATOV. 


of   Annbruster's  data,  give  a  number  which  corresponds  to  that 
<>f  the  Middle  Russian  ones. 

Sun  lining  up  now  our  whole  material  on  European  races  we 
max-  say  that  there  is  much  evidence  for  an  assumption  of  a  high 
number  of  books  in  southern  races  in  comparison  with  northern 
ones.  The  Bulgarian  group  of  bees  also  supports  this  conclusion. 
It  would  be  interesting  to  test  this  rule  on  other  castes  of  bee  col- 
onies. Unfortunately  our  material  on  drones  from  Russia  is  very 
small  (see  Table  IV.),  although  it  can  be  seen  that  the  Middle 


20% 


10% 


>     — Black  bees  (USA) 
•\    —Italian  bees  (USA.) 
I  \\  Italian  bees  (Italy) 

\\ 
\\ 


16    17    18    19    20  21    22  23  24  25 

IMC.  (>.     Curves  of  variation  of  hooks.     Continuous  line — black  bees   in 
1.  S.  A. ;  dotted  line — Italian  bees  from  Italy;  dash  line — Italians  in  U.  S.  A. 

Russian  and  even  South  Russian  drones  have  a  smaller  average 
number  of  hooks  than  the  Bulgarian  ones.  It  would  not  be  wise 
to  draw  any  conclusions  about  the  geographical  differences  in 
queens  based  on  such  a  small  number  of  cases.  We  have  to  add 
that  Middle  Russian  and  South  Russian  drones  give  the  high  de- 
gree of  variation  (C'<  )  usual  for  drones. 


HOOKS    ().\     HIM)    WING    OF     HONKY    BEE. 


227 


It  is  well  known  that  at  the  time  of  the  discovery  of  the  New 
World,  America  had  no  native  bees.  The  first  bees  imported  to 
this  country  came,  according  to  historical  data,  from  Holland  and 
England  and  belonged  to  the  common  black  bees  A.  nicllifcra 
nicllifcra  L.  About  the  middle  of  the  last  century  the  American 
beekeepers  began  to  prefer  for  cultivation  the  yellow  Italian  bee, 
which  is  now  the  dominant  race  in  this  country.  Thanks  to  the 
help  of  many  beekeepers  I  have  succeeded  in  examining,  from  a 
considerable  number  of  apiaries,  Italian  bees  of  different  degrees 


200 


100 


7 


\ 


13 


15     16      17     18     19     20    21     22     23     2*     25     26     27    28    29 


FIG.  7.     Frequency  polygon  and  fitted  curve  of  the  variation  of  the  hooks 
of  the  Bulgarian  drones. 

of  development  of  yellow  color  as  well  as  pure  black  bees. 
Tables  IX.  and  X.  show  us  the  variation  of  bees  acclimatized  to 
the  United  States.  Firstly,  we  have  to  note  the  great  difference 
in  the  number  of  hooks  of  black  and  yellow  American  bees,  sec- 
ondly, a  little  lower  number  of  hooks  of  Americanized  Italian 
bees  than  that  of  true  Italians  reared  either  in  Italy  or  from 
Italian  queens  imported  directly  from  that  country.  This  is  il- 
lustrated by  curves  on  Fig.  6.  The  very  low  average  number  of 
American  black  bees  as  compared  with  our  material  discussed 


228 


\Y.    W.    ALPATOV. 


above  give  us  the  right  to  suppose  a  general  decrease  of  the  num- 
ber of  hooks  in  the  United  States  as  compared  with  Europe,  both 
in  black  and  yellow  bees.  Further  investigations  need  to  be  made 
with  special  attention  to  the  problem  of  influence  of  acclimati- 
zation upon  physical  characteristics  in  the  honey  bee. 


200 


100 


16     17     /g     19     20    21    22    23     24    25     26    27     28    29 
FIG.  8.     Frequency  polygon  and  fitted  curve  of  variation  of  the  worker 
bees  from  Middle  Russia. 

Our  comparatively  large  material  gave  us  the  possibility  of  de- 
termining the  character  of  the  frequency  distributions.  The  re- 
sults are  given  in  Table  X.  All  distributions  are  symmetrical 
and  only  one  shows  a  deviation  from  the  normal  distribution. 
A  curve  of  type  VII.  was  chosen  to  fit  this  distribution.  The 
distribution  for  the  Bulgarian  worker  bees  being  symmetrical  and 
normal  in  regard  to  the  /?,  gave  a  very  high  value  of  the  criterion, 


HOOKS    ON    HIND    WING    OF    HONEY    BEE. 


229 


which  leads  us  to  the  curve  of  type  IV.  It  was  not  possible  to 
calculate  the  probable  errors  of  the  criterion.  /?,  and  /?.,  being  too 
close  to  those  characterizing  the  normal  curve  of  error.  There- 
fore a  normal  curve  was  used  for  fitting.  We  used  for  calcu- 
lating the  ordinates  of  the  normal  curve  from  Pearson'^  "  Tables 
for  Statisticians  and  Biometricians."  Figs.  /  and  8  show  two  of 
our  curve  fittings. 


25 


FIG.  9.  Measurements  on  the  wing.  The  wing  shows  the  intercubitus 
vein  not  developed.  (Microphotograph  taken  in  the  Art  Department  of  the 
Institute  for  Biological  Research,  by  Mr.  Johansen.) 

During  the  author's  residence  at  Cornell  University  an  attempt 
was  made  to  study  the  influence  of  undernourishment  of  larvae 
upon  the  characteristics  of  adult  bees.  The  experiment  consisted 
in  putting  the  unsealed  brood  in  an  incubator  running  at  34.5°  C. 
The  brood  was  taken  from  a  comb  approximately  one  day  before 
normal  sealing.  On  the  following  day  the  cells  situated  in  the 
neighborhood  of  the  place  from  which  a  piece  of  comb  had  been 
taken  the  day  before  were  already  sealed  by  bees  and  also  put 
in  the  incubator  in  order  to  provide  us  with  control  insects.  Bees 
normally  developed  in  hives  were  also  collected  from  the  frame  of 
the  hive  which  gave  us  material  for  the  experiment.  The  pieces 
of  comb  with  unsealed  larvse  put  in  the  incubator  were  covered 
with  pieces  of  artificial  comb  foundation  in  substitution  for  the 
natural  capping  bees.  The  larvse  wove  cocoons  as  usual  and  the 
emerging  bees  were  collected  in  alcohol.  The  bees  emerging  from 
the  unsealed  brood  evidently  suffered  from  a  certain  underfeed- 
ing in  comparison  with  control  bees.  Table  XL  shows  that  the 


230 


\Y.    \V.    ALPATOV. 


TABLE  XI. 

('(INSTANTS     OK     WlXG     MlCASUREM  EXTS     OF     CONTROL     AND     UNDERFED      (iN 

LARVAL  STAGE)  BEES  IN  MM. 


Proximal  Length 
of  Wing 
(Meas.  N  24). 

Distal  Length 
of  Wing 
(Meas.  N  25). 

No. 
of 
Cases. 

Experimental  (underfed)  bees 

4.525   ±  -021 

4.192  ±  .023 

46 

(  Control  bee*>        

4.696  ±  .010 

4.353  ±  .012 

31 

experimental  bees  have  a  smaller  size  of  wings  than  the  controls. 
The  characteristics  have  been  measured,  as  is  shown  in  Fig.  9. 
Table  XII.  shows  the  average  number  of  hooks  in  three  groups. 

TABLE  XII. 

INFLUENCE  OF  UNDERFEEDING  ON   THE   NUMBER  OF   HOOKS   AND  THE  AB- 
NORMAL VENATION. 


Character. 

M  ±  P.E. 

C%±P.E. 

Percentage 
of  Wing 
with  Abnor- 
mal Vein. 

Number 
of  Speci- 
mens. 

I. 

Bees  taken  from  the  hive  .  .  . 

20.77  ±  -16 

7-34  ±  -56 

o.oo  — 

39 

2. 

Bees  reared  in  the  incubator 
from  brood  normally  fed.  . 

20.60  ±  .15 

7-33  ±  -52 

4.44  ±i.37 

45 

3- 

Bees  reared  in  the  incubator 
from  underfed  brood 

TO.  71    -t  .OO 

7.30  ±  .33 

19.82  ±  2.50 

116 

n 

iff.  1-3  

1.  06  ±  .18 

19.82  ±  2.50 

R  =  5-89 

R  =  7-93 

n 

iff.  2-3  

O.8o  ±  .17 

15.38  ±  2.85 

R  =  5.24 

R  =  5-40 

It  can  be  seen  that  the  underfed  bees  have  a  smaller  number  of 
hooks  than  the  control  bees  reared  from  the  sealed  brood  and 
taken  directly  from  the  hive.  The  same  is  expressed  in  graphical 
torin  on  curves  of  the  Fig.  10.  The  experimental  bees  showed  a 
quite  peculiar  type  of  abnormality  in  the  venation  of  the  first  pair 
of  wings.  The  abnormality  consists  in  the  incomplete  develop- 
ment of  the  second  intercubitus  vein.  The  percentage  expres- 
of  this  abnormality  in  our  three  groups  shows  that  the  ab- 


HOOKS    ON    1IIXU    \Y!.\<i    OF    HONEY    BEE.  23! 

normality  occurs  also  in  bees  reared  in  the  incubator  from 
normally  sealed  broods  but  reaches  a  very  high  tirade  of  develop- 
ment in  undernourished  bees.  The  abnormalities  in  insect  wings 
have  been  many  times  the  subject  of  careful  morphological  studies. 
Our  experiment  opens  a  way  for  studying  this  problem  by  means 
of  the  experimental  method.  It  is  worth  while  to  note  that  in 
geographical  races  the  southern  bees,  being  usually  smaller  than 


30% 


20% 


10% 


16       17       /$       19       20      21       22      23      24- 

FIG.  10.  Continuous  line — bees  from  the  hives  which  gave  larvae  for 
the  experiment  with  underfeeding.  Dotted  line — variation  of  hooks  in  the 
group  of  bees  emerged  from  underfed  larvae.  Dashed  line — control  bees 
developed  from  normally  fe_d  larvae  and  emerged  in  the  incubator  together 
with  underfed  bees. 

the  northern  one  (AJpatov,  8),  at  the  same  time  show  an  in- 
crease in  the  number  of  hooks.  Our  experimental  bees  gave 
the  contrary  relation.  Therefore  it  is  not  possible  to  explain  the 
smaller  number  of  hooks  of  the  northern  bees  by  the  assumption 
of  an  underfeeding  of  larvae. 


232  W.  W.  ALPATOV. 

SUMMARY. 

The  data  presented  in  this  paper  show  that  the  average  number 
of  hooks  in  the  honey  bee  is  a  characteristic  which  is  differently 
developed  among  single  colonies,  sexes,  castes,  and  races.  As  a 
general  rule  the  southern  races  have  a  large  number  of  hooks  in 
worker  bees  and  probably  in  drones.  The  queens  and  drones  are 
more  variable  in  regard  to  this  character  than  the  worker  bees. 
In  this  respect  the  relations  differ  from  those  in  other  social 
insects  (ants,  wasps  and  termites),  where  the  asexual  caste  is 
the  most  variable.  The  experiment  with  underfeeding  of  larvae 
showed  a  decrease  of  the  average  number  of  hooks  and  the 
producing  of  specimens  with  defective  venation — incomplete 
second  intercubitus  vein. 

LITERATURE  CITED. 

i.  Alpatov,  W.  W.,  and  Tjunin,  F.  A. 

'25     Beitrage  zur   Kenntniss   der   Variabilitat   der    Riissellange  bei   der 
Honigbiene    (in   German,    with    Russian    summary).      Rev.    Zool. 
Russ.,  Vol.  5,  pp.  79-108. 
2.  Alpatov,  W.  W. 

'25  liber  die  Verkleinerung  der  Riissellange  der  Honigbiene  vom  Suden 
nach  Norden  bin.  Zool.  Anz.,  Bd.  65,  pp.  103-111. 

3.  Alpatov,  W.  W. 

'26  Der  Riissel  der  Kasaner  Arbeitsbiene  in  variationsstatistisher 
Bearbeitung  (in  Russian  with  German  summary).  Bull,  of 
Kasan  Agric.  Exp.  Sta.,  pp.  23-33. 

4.  Alpatov,  W.  W. 

'27  On  the  Variability  of  the  Hooks  on  the  Hind  Wings  of  the  Honey 
Bee  (in  Russian).  Ptschelowodnoje  djelo,  January,  1927. 

5.  Alpatov,  W.  W. 

'27  The  Cardinal  Problems  in  the  Study  of  the  Caucasian  Bee  Races 
(in  Russian).  Ptschelowoni  Mir,  Vol.  i,  p.  3-8. 

6.  Alpatov,  W.  W. 

'27  On  the  Amelioration  of  the  Bee  Races  (in  Russian).  Ptschelo- 
wodnoje djelo,  8-9,  pp.  372-377. 

7.  Alpatov,  W.  W. 

'28  On  the  Variability  of  the  Tongue  Length  of  Caucasian  Gray  Bees 
and  Its  Inheritance  (in  Russian  with  English  summary). 
Ptschelowodni  Mir.  N  i,  2,  3. 

8.  Alpatov,  W.  W. 

'27  Biometrical  Study  on  Bees  of  Middle  and  Southern  Russia.  Rev. 
Zool.  Russ.,  Vol.  7,  livr.  4,  pp.  31-74  (in  Russian  with  English 
summary  ) . 


HOOKS    ON    HIND    WING    OF    HONEY    REE.  233 

9.  Alpatov,  W.  W. 

Variability  in  the  Honey  Bee  Tongue  Biometrically  Investigated  and 
Practical  Questions  Connected  with  the  Problem  of  the  Selec- 
tion of  the  Honey  Bee.  (In  press,  Jour,  of  Econ.  Entom.) 

10.  Alpatov,  W.  W. 

On  the  Improvement  of  Bee  Races.  (In  press,  Reports  of  Maryland 
Agricultural  Society.) 

11.  Armbruster,  T. 

'23  Wie  untersucht  man  Bienenstamme  und  Bienenkreuzungen  auf 
ihre  Farbe?  Archiv  fur  Bienenkunde,  Vol.  5. 

12.  Arnoldi,  K. 

'26  Studien  iiber  die  Variabilitat  der  Ameisen.  Z.  f.  Morphologic  und 
Okologie  der  Tiere,  Vol.  7. 

13.  Bachmetjew,  P. 

'10  Analytisch-statistische  Untersuchungen  iiber  die  Anzahl  der 
Fliigelhaken  bei  Bienen  und  die  daraus  hezvorgehende  Konse- 
quenzen.  Z.  f.  Wiss.  Zoologie,  Vol.  94. 

14.  Casteel,  D.  B.,  and  Phillips,  E.  F. 

'03  Comparative  Variability  of  Drones  and  Workers  of  the  Honey 
Bee.  BIOL.  BULL.,  5,  pp.  18-37. 

15.  Gerstacker,  — . 

'69  On  the  Geographical  Distribution  and  Varieties  of  the  Honey  Bee. 
Ann.  and  Mag.  of  N.  History.  III.  ser.,  Vol.  n. 

16.  Gorbatschev,  — . 

'16  The  Gray  Mountain  Caucasian  Bee  (Apis  mcliifcra  var.  Caucasica} 
and  its  Place  Among  Other  Bees.  Tiflis.  (In  Russian  with  an 
English  summary.) 

17.  Kellogg,  V.,  and  Bell,  G. 

'04     Studies  on  Variation  in  Insects.     Proc.  Wash.  Acad.   Sci.,  Vol.  6. 

18.  Kellogg,  V. 

'06     Variation  in  Parthenogenetic  Insects.     Science,  Vol.  24,  no.  622. 

19.  Koshewnikov,  G.  A. 

'00-'05  Materials  to  the  Natural  History  of  the  Honey  Bee,  part  i 
and  2.  Moscow,  Nachrichten  der  K.  Ges.  d.  Freunde  v.  Naturw., 
Anthrop.  und  Ethnographic,  Abt.  f.  Zool.  (In  Russian.) 

20.  Palenitschko,  Z.  G. 

'27  Zur  vergleichenden  Variabilitat  der  Arten  und  Kasten  bei  den 
Ameisen.  Z.  f.  Morphologic  und  Okologie,  Vol.  9. 

21.  Pearl,  R. 

'10  Recent  Quantitative  Studies  in  Variation  in  Social  Insects.  Am. 
Nat.,  Vol.  44,  521. 

22.  Phillips,  E.  F. 

Variation   and   Correlation   in   the  Appendages  of   the   Honey   Bee. 
(In  press.) 

23.  Thomson,  E.  Y.,  Bell,  J.,  and  Pearson,  K. 

'09  A  Second  Cooperative  Study  of  Vespa  vulgaris.  Biometrika,  Vol. 
7,  48-63. 

24.  Thomson,  E.  Y.,  Bell,  J.,  and  Pearson,  K. 

'11     A  Third  Cooperative  Study  of  V.  vulyaris.     Vol.  8,   1-12. 


234 


\\  .    \V.    ALl'ATOV. 


_>;.  Warren. 

'08     Some    Statistical   Observations   on   Termites.      Biometrika,   Vol.   6. 

26.  Watson,  Lloyd  R. 

'27     Controlled  Mating  of  Queen  Bees.     Hamilton,  Illinois. 

27.  Wright,  A.,  Lee,  A.,  and  Pearson,  R. 

"07     A   Cooperative   Study   on   Queens,   Drones   and  Workers   in   Vespa 
Biometrika,  Vol.  5,  pp.  407-422. 


Vol.LV. 


October,  1928. 


No.  4 


BIOLOGICAL  BULLETIN 


THE    DEVELOPMENT    OF   THE    SPERMATOZOON    IN 

CAVIA  COB  AY  A  i 

MARY  T.  HARMAN  AND  FRANK  P.  ROOT. 

INTRODUCTION     235 

MATERIAL  AND  METHODS   237 

(a)   Description  of  material   237 

1.  Period  with  little  change  in  the  shape  of  the  cell 237 

2.  Period  of  elongation  239 

3.  Histogenesis  of  the  elongate  cell  240 

DISCUSSION 241 

SUMMARY   244 

LITERATURE  CITED    244 

DESCRIPTION  OF  PLATES   248 

INTRODUCTION. 

The  development  of  the  spermatozoon  in  the  Mammalia  has 
been  observed  in  a  number  of  forms  but  a  detailed  study  has  been 
made  in  only  a  few  instances.  Among  the  workers  who  have 
published  observations  on  the  development  of  the  mammalian 
spermatozoon  are:  Lenhossek  (1898),  Meves  (1898),  Benda 
(1897,  1906),  Korff  (1902),  Duesberg  (1908,  1920),  Jordan 
(1911),  Oliver  (1913),  Stockard  and  Papanicolaou  (1918),  Gat- 
enby  and  Woodger  (1921).  There  is  a  general  agreement  in  the 
plan  of  the  development  but  many  differences  of  opinion  exist  with 
reference  to  the  detail.  Many  of  these  differences  are  significant 
not  only  from  the  development  of  the  spermatozoon  itself  but  also 
from  their  bearing  upon  other  biological  problems.  Since  mam- 
mals are  bisexual  and  have  not  been  known  to  reproduce  partheno- 
genetically,  the  continuity  of  the  different  parts  of  the  male  germ 

1  Contribution  from  the  Zoological  Laboratory,  Kansas  State  Agricultural 
College,  No.  100. 

16  235 


236  MARY   T.    HARM  AN   AND   FRANK    P.    ROOT. 

cell  is  of  as  much  significance  as  that  of  the  female  germ  cell. 
The  loss  of  a  part  of  the  nucleus  or  even  a  part  of  the  cytoplasm 
in  the  process  of  transformation  of  the  spermatid  into  a  sperma- 
tozoon may  affect  the  theory  of  the  vehicle  of  the  bearers  of  the 
hereditary  characteristics. 

Cavia  cobaya  has  been  used  as  a  subject  of  investigation  for 
the  development  of  the  spermatozoon  as  often  as  any  other  mam- 
mal and  the  work  has  been  done  in  as  much  detail  and  yet  there 
is  a  lack  of  agreement  upon  a  number  of  points.  All  authors 
are  agreed  that  the  spermatid  is  a  typical  one,  similar  to  that  de- 
scribed for  insects  and  other  animals  and  that  the  mature  sper- 
matozoon is  composed  of  at  least  three  parts  or  regions,  the  head, 
the  mid  piece  and  the  tail.  Also  a  fourth  region,  the  neck,  has 
been  described  by  ntany  workers.  What  parts  of  fhe  spermatid 
contribute  to  the  formation  of  each  of  these  regions,  of  what  each 
region  is  composed  and  whether  or  not  the  entire  cell  is  used  in 
the  formation  of  the  spermatozoon  are  questions  upon  which 
there  are  significant  differences  of  opinion. 

In  our  study  of  the  development  of  the  spermatozoon  of  Cavia 
cobaya  certain  things  have  been  impressed  upon  us  as  being  de- 
cidedly different  from  the  observations  of  other  authors.  Of 
these  we  shall  mention  five  as  the  most  outstanding :  ( i )  Follow- 
ing the  last  maturation  divisions  the  chromatin  material  goes 
through  an  abortive  preparation  for  division  before  there  is  much 
change  in  the  shape  of  the  cell.  (2)  We  have  found  no  loss  of 
cytoplasm  or  sloughing  off  as  has  been  described  by  many  authors. 
It  is  true  that  we  find  stages  when  the  entire  developing  sperma- 
tozoon is  smaller  than  in  previous  stages  but  this  seems  to  be  due 
to  a  condensation  of  the  material  rather  than  a  sloughing  off  of 
any  part  of  it.  This  will  be  discussed  in  some  detail  in  the  body 
of  the  paper.  (3)  We  have  not  found  in  any  stage  a  filament 
extending  out  from  the  cytoplasm.  We  have  diligently  looked  for 
it  because  we  were  very  anxious  to  see  the  nature  of  this  develop- 
ment and  at  what  particular  time  it  was  first  evident.  In  all  of  our 
observations  the  axial  filament  tapers  to  a  blunt  point  at  the  ter- 
minis.  There  is  no  naked  end  filament  even  in  the  fully  formed 
spermatozoon.  (4)  The  tail  is  made  up  of  three  segments  which 
are  not  only  shown  by  the  morphological  structure  but  also  by  the 


DEVELOPMENT   OF   THE   SPERMATOZOON.  237 

points  of  breaking  as  found  in  hundreds  of  broken  specimens. 
(5)  As  was  mentioned  in  our  previous  paper,  the  area  of  actively 
dividing  cells  are  elliptical  with  the  greatest  diameter  of  the  ellipse 
lengthwise  of  the  tubule.  Within  this  area  the  cells  are  generally 
in  the  same  stage  of  development  and  only  occasionally  a  stray 
cell  is  in  some  other  stage. 

It  has  not  been  our  purpose  to  describe  the  origin  of  the  cy'to- 
plasmic  structures  nor  to  say  much  about  the  confused  nomencla- 
ture of  the  same.  This  has  been  only  incidental  to  our  purpose 
and  we  have  discussed  them  only  in  so  far  as  they  contribute  to  the 
development  of  the  spermatozoon.  We  have  used  much  of  the 
nomenclature  of  Bowen  when  it  seemed  applicable  to  our  needs. 

MATERIAL  AND  METHODS. 

The  material  used  is  the  same  used  in  our  previous  paper  (Har- 
man  and  Root,  1926).  In  that  paper  will  be  found  a  detailed  de- 
scription of  the  fixing  and  staining  of  the  material.  All  drawings 
have  been  made  with  the  aid  of  a  camera  lucida  and  the  magnifi- 
cations are  given  in  the  description  of  the  plates.  With  one  ex- 
ception, our  drawings  could  be  duplicated  from  hundreds  of  cells 
in  our  material.  We  make  this  statement  to  emphasize  the  fact 
that  what  we  are  showing  is  universal  and  not  an  exception  which 
might  be  attributed  to  technique.  The  exception  is  the  bent  rod- 
shaped  cytoplasmic  inclusion  in  Fig.  7  which  we  have  called  a 
Golgi  body. 

(a)  Description  of  Material. — We  have  begun  with  the  changes 
which  take  place  in  the  cell  after  the  last  maturation  division  has 
been  completed.  This  is  where  we  stopped  in  our  last  paper. 
For  convenience  of  description  these  changes  may  be  divided  into 
three  periods  as  follows:  (i)  The  period  with  little  change  in  the 
shape  of  the  cell;  (2)  the  period  of  elongation  and  (3)  histogenesis 
of  the  elongated  cell. 

i.  The  Period  with  Little  Change  in  tJie  Shape  of  the  Cell. — 
Significant  changes  take  place  both  in  the  nucleus  and  the  cyto- 
plasm before  there  is  much  change  in  the  shape  of  the  cell.  At  the 
end  of  the  last  maturation  division  the  chromatin  passes  through 
a  typical  telophase.  It  becomes  finely  granular  and  a  definite  nu- 
clear membrane  is  formed.  Following  this  there  takes  place  what 


238  MARY   T.    HARMAN   AND   FRANK    P.    ROOT. 

we  have  chosen  to  call  an  abortive  attempt  to  divide  again.  The 
chromatin  forms  into  a  close  network  having  irregular  clumps 
and  the  nuclear  membrane  nearly  disappears,  Fig.  I.  Then  the 
nucleus  increases  in  size  and  the  chromatin  material  is  in  a  more 
nearly  continuous  spireme,  Fig..  2.  The  chromatin  clumps  be- 
come more  numerous  and  prominent.  These  changes  continue 
until  a  compact  unbroken  spireme  is  formed,  Fig.  3.  Then  there 
is  an  attempt  to  form  chromosomes,  Fig.  4.  The  chromatin  knots 
are  numerous  and  the  spireme  has  been  separated  into  irregular 
pieces  which  may  be  compared  to  chromosomes  but  which  lack  the 
smooth  contour  and  the  compact  appearance  of  chromosomes. 
We  have  called  these  masses  of  chromatin  material  "  chromatin 
knots."  There  remains  some  trace  of  the  spireme  but  it  is  little 
more  than  a  suggestion.  Following  this  the  chromatin  knots  be- 
come more  granular  and  there  is  no  further  indication  of  a  division 
of  the  cell,  Figs.  5  and  6.  Now  the  entire  cell  begins  to  contract 
and  to  become  compact.  At  first  this  is  more  evident  in  the  nu- 
cleus than  in  the  cell  body.  The  chromatin  material  becomes 
finely  granular  and  only  traces  of  the  spireme  are  discernible. 
The  entire  nuclues  occupies  much  less  space,  Figs.  7,  8,  and  9. 

While  these  changes  have  been  taking  place  in  the  nucleus, 
changes  have  been  occurring  in  the  cytoplasm.  A  number  of 
spherical  bodies  varying  in  size  appear  in  the  early  spermatid. 
These  are  the  Golgi  bodies.  There  is  a  lack  of  constancy  in  the 
number  and  the  size  of  these  Golgi  bodies.  They  are  found  in 
the  periphery  of  the  cell  as  well  as  near  the  nucleus.  Sometimes 
they  may  indent  the  nuclear  wall,  Fig.  2.  With  Heidenhain's 
haematoxylin  they  are  stained  like  chromatin  which  emphasizes 
their  spherical  form  and  distinguishes  them  from  the  surrounding 
cytoplasm  in  the  early  spermatids.  They  are  finely  granular  like 
the  surrounding  cytoplasm  but  the  granules  are  more  closely  com- 
pact than  in  the  other  parts  of  the  cytoplasm.  Each  Golgi  body 
has  the  appearance  of  a  sphere  surrounded  by  a  halo. 

An  idiosome  is  always  near  the  nucleus.  In  section  it  is  cres- 
centic  in  shape,  with  the  concave  side  toward  the  nucleus,  and 
stains  like  the  surrounding  cytoplasm  from  which  it  is  distin- 
guished by  its  more  homogeneous  structure.  In  older  stages  it 
comes  to  lie  in  contact  with  the  nucleus  then  there  is  a  more  den- 


DEVELOPMENT   OF   THE   SPERMATOZOON. 


239 


nite  orientation  in  its  position  than  that  of  the  Golgi  bodies  and 
the  nucleus.  At  least  one  Golgi  body  is  always  near  the  nucleus 
and  at  the  same  time  near  the  idiosome,  Figs.  I,  3,  4,  and  5.  Some 
of  the  Golgi  bodies  form  a  group  near  the  nucleus  on  the  side 
opposite  to  the  idiosome. 

The  idiosome  becomes  closely  applied  to  one  side  of  the  nu- 
cleus, Figs.  8  and  9.  The  idiosphere  is  in  the  concavity  of  the 
idiosome  between  it  and  the  nucleus,  NE  in  Figs.  8  and  9.  The 
entire  cell,  both  cytoplasm  and  nucleus,  has  become  smaller  and 
there  is  evidence  of  the  beginning  of  the  change  in  the  shape  of 
the  cell. 

2.  Period  of  Elongation. — With  the  diminution  of  the  volume 
of  the  cell  there  is  the  beginning  of  an  elongation  in  the  axis  de- 
termined by  the  idiosome,  on  the  one  side,  and  the  Golgi  remnant 
on  the  other.  This  elongation  occurs  in  the  entire  cell  affecting 
the  shape  of  both  the  cytoplasm  and  the  nucleus,  Figs.  10  to  22. 
During  this  time  the  cell  is  in  intimate  connection  with  the  Ser- 
toli  cell  which  is  at  first  small  but  later  increases  enormously  in 
size.  Fig.  20  illustrates  a  Sertoli  cell  with  some  of  the  associated 
spermatids  in  an  elongated  form.  The  part  of  the  spermatid 
destined  to  become  the  head  is  directed  toward  the  base  of  the 
Sertoli  cell  and  the  other  part  toward  the  lumen  of  the  tubule. 
This  is  true  regardless  of  the  stage  of  development.  When  the 
spermatozoa  are  freed  from  the  Sertoli  cell  they  are  not  in  a 
mixed  up  mass  but  are  in  bundles  lying  almost  parallel,  with  most 
of  the  heads  in  the  same  direction.  This  could  easily  be  accounted 
for  by  the  fact  that  they  have  a  definite  orientation  during  their 
development.  While  the  cell  is  elongating  the  idiosphere  becomes 
embedded  in  the  idiosome.  The  idiosphere  stains  more  densely 
than  the  idiosome.  Thus  the  idiosome  has  the  appearance  of  hav- 
ing a  core.  The  idiosome  and  the  idiosphere  form  an  elongate 
body  pointed  at  its  distal  end  and  truncate  at  its  proximal  end, 
Figs.  13  to  17.  Fig.  14  is  a  surface  view  while  the  other  figures 
show  the  idiosphere  surrounded  by  the  idiosome.  The  idiosome 
and  the  idiosphere  may  now  be  called  the  acroblast. 

The  chromatin  material  in  this  stage  has  become  finely  granu- 
lar and  can  scarcely  be  distinguished  from  the  cytoplasm  either 
by  its  staining  reaction  or  by  its  structure.  The  nucleus  elongates 


240 


MARY  T.    HARMAN   AND   FRANK    P.    ROOT. 


until  it  becomes  cylindrical,  Figs.  14  to  17.  A  thin  coating  of 
cytoplasm  surrounds  it  and  extends  in  the  direction  opposite  to 
the  acroblast.  Later  the  nuclear  material  takes  a  position  to  one 
side  of  the  cylindrical  mass  and  the  cytoplasm  forms  a  flattened 
area  to  the  other  side  extending  from  the  acroblast  to  the  other 
end  of  the  cell,  Figs.  18  and  19.  In  these  figures  the  acroblast  is 
becoming  rounded  and  is  beginning  to  take  a  position  to  the  side 
of  the  nucleus  instead  of  completely  anterior  to  it  as  in  the  earlier 
stages.  The  nuclear  material  is  beginning  to  become  more  con- 
densed and  is  spread  out  over  a  wider  surface.  Posterior  to  the 
nucleus  there  are  three  fine  thread-like  filaments  which  spread 
into  a  somewhat  fan-shaped  mass  in  the  surrounding  cytoplasm. 
Associated  with  these  filaments  are  two  areas  of  cytoplasmic  gran- 
ules. One  area  is  at  the  extremity  of  the  filaments  and  the  other 
area  is  near  the  base  of  the  nucleus,  Figs.  19  and  21.  Following 
this  stage,  the  cytoplasm  which  is  transforming  into  the  tail  of  the 
spermatozoon  condenses  rapidly  and  becomes  very  elongate. 

3.  Histogcnesis  of  the  Elongate  Cell. — In  the  histogenesis  of 
the  elongate  cell  the  three  regions  usually  recognized  in  a  mam- 
malian spermatozoon  begin  to  be  evident.  At  first  the  nucleus 
and  the  acrosome  which  make  up  the  head  are  much  longer  than 
they  are  wide  and  become  cylindrical  and  somewhat  enlarged  at 
the  free  end.  The  nucleus  is  now  at  one  side  of  the  cytoplasmic 
acrosome  and  it  does  not  extend  entirely  to  the  free  end  of  the 
developing  spermatozoon.  The  mid-piece  which  is  occupied 
largely  by  the  spiral  filament  in  the  adult  spermatozoon  becomes 
granular  in  regularly  arranged  clumps,  SF,  Fig.  23.  This  is  the 
region  which  was  occupied  by  the  three  thread-like  filaments  in 
Figs.  19,  21,  and  22.  One  of  the  most  noticeable  changes  is  in  the 
tail  region.  There  is  a  very  rapid  condensation  of  the  cytoplasm 
which  was  spread  out  in  a  fan-shaped  mass  to  a  tapering  whip- 
like  flagellum.  The  tail  is  composed  of  three  segments.  The  first 
one  is  about  as  long  as  the  mid-piece,  the  second  one  in  the  early 
stages  is  about  the  same  length  and  the  third  or  terminal  one  is  n. 
little  longer  than  the  combined  length  of  the  other  two.  It  gradu- 
ally tapers  to  a  blunt  point.  We  have  not  found  in  any  stage  of 
development  any  unsheathed  terminal  filament. 

As  differentiation  progresses  there  is  a  greater  difference  be- 


DEVELOPMENT   OF  THE   SPERMATOZOON.  24! 

tween  the  sizes  of  segments  one  and  two  of  the  tail.  The  second 
segment  elongates  more  than  the  first  and  tapers  more  as  it  in- 
creases in  length.  The  segments  are  recognized  by  distinct  mark- 
ings and  when  the  tails  of  the  spermatozoa  are  broken  off,  the 
break  is  always  at  the  union  of  two  of  these  segments.  Seldom 
is  the  tail  broken  from  the  head  at  the  anterior  part  of  the  mid- 
piece  and  practically  never  is  the  tail  broken  off  at  the  posterior 
part  of  the  mid-piece.  A  few  of  the  tails  are  broken  at  the  end 
of  the  first  segment.  Most  frequently  the  break  is  at  the  distal 
end  of  the  first  segment,  less  frequently  between  the  second  and 
the  third  segments.  We  never  find  the  tail  broken  within  any  seg- 
ment. 

Figures  24,  25,  and  26  are  illustrations  of  a  mature  spermatozoon 
viewed  from  different  positions.  The  acrosome  forms  a  hood- 
shaped  covering  to  one  side  and  anterior  to  the  nucleus.  The  head 
is  broad  from  side  to  side,  Figs.  24  and  25,  but  rather  thin  when 
seen  from  the  edge,  Fig.  26.  The  regularly  arranged  clumps  of 
cytoplasm  in  the  mid-piece,  mentioned  above,  develop  into  a  dis- 
tinct spiral,  with  the  coil  always  counter-clockwise  from  the  an- 
terior part  of  the  mid-piece.  The  last  two  coils  are  almost  rings 
and  might  be  termed,  annulus.  There  is  no  annulus  separate 
from  the  spiral  filament.  As  is  shown  in  the  drawings  the  coils 
are  not  always  regular.  They  remind  one  of  a  spring  that  has 
been  put  at  a  tension  and  the  rebound  has  not  been  the  same  in 
all  regions  of  the  spring.  The  first  four  coils  of  the  spiral  fila- 
ment are  inclosed  by  a  thin  bladder  of  cytoplasm. 

DISCUSSION. 

In  the  transformation  of  the  spermatid  into  the  spermatozoon 
little  attention  has  been  given  to  the  behavior  of  the  chromatin 
material  other  than  it  finally  becomes  condensed  into  a  more  or 
less  homogeneous  mass  which  appears  solid  and  is  stained  heav- 
ily with  nuclear  dyes.  Meves  (1899)  has  shown  the  nuclear 
material  formed  into  clumps  before  there  has  been  much  change 
in  the  shape  of  the  spermatid.  Ballowitz  (1891)  has  also  this 
clumping  of  the  chromatin  material  in  his  drawings.  Neither 
author  has  discussed  this  change  nor  has  mentioned  further 
changes  in  the  chromatin.  They  state  that  the  nucleus  forms  the 
greater  part  of  the  head  of  the  spermatozoon. 


242 


MARY   T.    HARMAN   AND   FRANK    P.    ROOT. 


In  one  of  the  Hemiptera,  Bowen  (1920)  says  that  "the  head 
undergoes  a  characteristic  change  resulting  in  what  appears  to  be 
a  complete  vacuolization  of  the  chromatin  lining.  Then  the 
chromatin  collapses  toward  the  axis  of  the  head,  etc." 

We  have  shown  that  after  the  last  maturation  division  the 
chromatin  material  passes  through  changes  which  are  similar  to 
those  in  a  cell  that  is  getting  ready  to  divide  until  there  is  the 
breaking  up  of  the  chromatin  material  into  clumps.  A  significant 
difference,  however,  between  these  changes  and  the  changes  previ- 
ous to  the  maturation  divisions  is  that  there  is  no  synezesis  and 
no  double  thread.  We  raise  the  question  whether  these  changes 
influence  the  behavior  of  the  cytoplasm  in  the  process  of  trans- 
formation and  thus  the  attempt  at  division  is  aborted  or  whether 
the  changes  in  the  cytoplasm  arrest  the  changes  taking  place  in  the 
nucleus. 

The  small  size  of  the  spermatozoon  in  comparison  with  the  early 
spermatid  is  recognized  by  many  authors.  Some  of  this  differ- 
ence in  size  has  been  accounted  for  by  a  loss  in  cytoplasm.  In  the 
formation  of  the  spermatozoon  of  vertebrates,  Kolliker  as  early 
as  1856  and  la  Vallette  St.  George  (1865)  described  the  "  slough- 
ing off"  of  the  cytoplasm.  Later  Biondi  (1885),  Benda  (1897), 
Hermann  (1889),  and  Neissing  (1889  and  1896)  agree  that  there 
is  a  loss  in  cytoplasm  by  a  sloughing  off.  Meves  and  Ballowitz 
have  shown  cytoplasm  loosely  connected  with  the  transforming 
tail  part. 

This  difference  in  the  size  of  the  spermatozoon  and  the  sperma- 
tid is  recognized  in  the  insects.  Montgomery  (1911)  states  that 
in  Euschistus  "  no  evidence  was  found  for  the  casting  off  of  any 
substance  by  the  sperm." 

In  the  formation  of  the  spermatozoon  in  Paratctiix,  Harman 
(1915)  did  not  find  any  loss  of  cytoplasm.  The  cytoplasm  con- 
densed around  the  axial  filament  but  there  was  no  indication  of  a 
sloughing  off  either  in  the  appearance  of  the  cell  or  the  remains 
in  the  follicle. 

In  our  material,  the  spermatozoon  is  greatly  reduced  in  size 
during  the  process  of  transformation,  but  we  have  found  no  evi- 
dence in  any  region  of  a  loss  of  material.  We  have  shown,  Figs. 
24,  25  and  26,  that  a  bladder-like  structure  of  cytoplasm  is  pres- 


DEVELOPMENT   OF  THE   SPERMATOZOON.  243 

ent  in  the  transformation  but  that  this  condenses  around  a  por- 
tion of  the  middle  piece  and  there  is  no  evidence  that  it  is  sloughed 
off. 

Most  authors  recognize  that  the  greater  part  of  the  head  of  the 
spermatozoon  is  formed  from  the  nucleus  of  the  spermatid  and 
furthermore,  they  recognize  that  this  head  is  much  smaller  than 
the  original  nucleus.  No  one  has  described  the  loss  of  nuclear 
material.  This  agrees  with  our  observations.  We  believe  that  this 
diminution  in  size  is  due  to  a  condensation  in  which  the  material 
appears  more  compact  than  in  earlier  stages. 

Meves  (1899),  Ballowitz  (1891),  and  Duesberg  (1910)  show 
a  thread-like  filament  extending  out  from  the  cytoplasm  in  the 
very  early  stages  of  development.  Meves  describes  this  filament 
as  arising  from  one  of  the  centrosomes  which  gives  rise  to  the 
posterior  nodule  and  this  filament  which  in  turn  becomes  the  axial 
filament.  He  represents  the  distal  end  of  this  filament  as  remain- 
ing unsheathed  and  forming  the  terminal  filament.  We  have 
found  no  unsheathed  filament  at  any  stage  of  development.  We 
have  shown,  Figs.  18,  19,  21  and  22,  three  filamentous  structures 
which  lie  deep  in  the  cytoplasm.  These  filaments  are  spread  out 
distally  into  a  fan-shape.  Associated  with  these  structures  are 
two  areas  of  granules.  We  have  not  traced  the  detailed  history 
of  these  granules  but  we  have  noted  that  they  finally  become  in- 
closed in  the  cytoplasm  which  rapidly  condenses  and  with  the 
associated  filaments  form  the  tail  of  the  spermatozoon.  There 
is  a  gradual  tapering  of  the  tail  to  a  blunt  point.  This  tapering 
takes  place  in  the  axial  filament  as  well  as  in  the  sheath  which 
encloses  it  entirely  to  the  distal  end. 

The  tail  is  made  up  of  three  segments  as  we  have  shown  in  Figs. 
23,  24,  25,  and  26.  Early  in  our  study  of  the  mature  spermato- 
zoon, among  mutilated  specimens  we  were  impressed  with  the  reg- 
ularity of  the  lengths  of  the  pieces  of  the  tails.  These  lengths 
were  quite  constant  whether  the  spermatozoa  were  in  bundles, 
merely  a  few  together  or  even  if  a  single  spermatozoon  was 
broken.  The  pieces  were  in  three  different  lengths  which  corre- 
sponded to  the  three  segments  of  the  tail.  Measurements  showed 
only  a  slight  variation.  It  would  seem  that  the  tail  is  weaker  at 
the  points  of  junctions  of  the  segments. 


244  MARY   T.    HARMAN   AND   FRANK    P.   ROOT. 

The  transformation  of  the  spermatic!  into  a  spermatozoon  takes 
]»lace  in  definite  areas  which  are  elliptical  in  shape.  The  greatest 
diameter  of  the  ellipse  is  always  lengthwise  of  the  seminiferous 
tubule  and  the  shortest  diameter  never  exceeds  two-thirds  of  the 
circumference  of  the  tubule. 

SUMMARY. 

1.  The  transformation  of  the  spermatid  into  a  spermatozoon 
takes  place  while  the  spermatid  is  closely  associated  with  a  Ser- 
toli  cell  and  it  does  not  become  free  in  the  lumen  of  the  seminif- 
erous tubule  until  the  spermatozoon  is  matured. 

2.  In  the  e*arly  stages  of  transformation  the  cell  goes  through 
a   growth   period   in   which   the   entire   cell   gets   larger   and   the 
chromatin  material  goes  through  an  abortive  preparation  as  if  for 
division. 

3.  During  the  period  of  elongation  there  is  a  reduction  in  the 
volume  of  the  cell  and  a  rearrangement  of  its  parts. 

4.  No  "  sloughing  off"  or  loss  of  cytoplasm  has  been  observed. 

5.  The  head  of  the  spermatozoon  is  composed  of  two  parts,  the 
head  proper  which  arises  from  the  nucleus  and  the  head  cap  or 
acrosome  which  arises  from  the  idiosome  and  the  idiosphere. 

6.  There  is  a  cytoplasmic  bladder-like  structure  around  the  an- 
terior part  of  the  mid-piece. 

7.  The  tail  is  composed  of  three  segments  terminating  in  defi- 
nite nodes. 

8.  We  find  no  indication  of  an  unsheathed  terminal  filament 
either  during  the  transformation  or  in  the  mature  spermatozoon. 

9.  The  tails  of  the  spermatozoa  are  always  toward  the  lumen  of 
the  seminiferous  tubule. 

10.  The  areas  of  transformation  are  elliptical  in  shape  with  the 
long  axis  of  the  ellipse  corresponding  to  the  length  of  the  semi- 
niferous tubule  and  the  short  diameter  of  the  ellipse  never  exceeds 
two  thirds  the  circumference  of  the  tubule. 


LITERATURE  CITED. 
Ballowitz,  E. 
'86     Zur    Lehere    von    dcr    Struktur    der    Spermatozoen.      Anat.    Anz., 

Jahrg.  i. 

'91     \VrikTe  Beobachtungen  iiber  den  feineren  Bau  der  Saugethier-sperma- 
tozoen.     Zeitschr.  f.  wiss.    Zool.,  Bd.  52. 


DEVELOPMENT   OF   THE   SPERMATOZOON.  245 

Benda,  C. 
'87     Untersuchungen  iiber  den  Bau  des  funktionerenden  Samenkanalchens 

einiger  Saugethiere  und  Folgerungen  fiir  die  Spermatogenese  dieser 

Wirbelthiere.     Archiv  f.  mikr.     Anal:.,  Bd.  30. 
'97     Neuere   Mitheilungen   iiber   die    Histogenese   der    Saugethiersperma- 

tozoen.    Verb.  d.  Physiol.  Ges.  zu  Berlin. 
'06     Die  Spermiogenese  der  Marsupialier.     Semons  Zoologische  Forsch- 

ungsreisen  in  Australian.     Jena,  Fischer. 
Biondi,  D. 
'85     Die  Entwicklung  der  Spermatozoiden.     Archiv  f.  mikr.     Anat.,  Bd. 

25- 
Bowen,  Robert  H. 

'20     Studies   on    Insect    Spermatogenesis.      I.  The   History   of   the    Cyto- 

plasmic    Components    of    the    Sperm    of    Hemiptera.      BIOL.    BULL., 

Vol.  39- 
'22     Studies    on    Insect    Spermatogenesis.      II.  The    Components    of    the 

Spermatid  and  the  Role  in  the  Formation  of  the  Sperm  in  Hemip- 
tera.   Jour.  Morph.,  Vol.  37. 
'22     Studies  on   Insect   Spermatogenesis.     III.  On  the   Structure   of   the 

Nebenkern   in   the   Insect   Spermatid   and  the   Origin   of    Nebenkern 

Patterns.     BIOL.  BULL.,  Vol.  42. 
'24     Studies  on  Insect  Spermatogenesis.     VI.  Notes  on  the  Formation  of 

the  Sperm  in  Coleoptera  and  Aptera,  with  a  General  Discussion  of 

Flagellate  Sperms.    Jour.  Morph.,  Vol.  39. 
'27     Golgi  Apparatus  and  Vacuome.    Anat.  Rec.,  Vol.  35. 
Duesberg,  J. 

'08     La  spermiogenese  chez  le  rat.    Archiv  f.  Zellforsch.,  Bd.  2. 
'20     Cytoplasmic  Structures  in  the  Seminal  Epithelium  of  the  Opossum. 

Cam.  Inst.,  Washington,  Contrib.  to  Emb.  No.  28. 
Gatenby,  J.  B.,  and  Woodger,  J.  H. 

'21     The   Cytoplasmic   Inclusions  of   the  Germ-cells.     Part   IX.     On  the 

Origin   of   the   Golgi   Apparatus    on   the   Middle-piece   of    the   Ripe 

Sperm  of    Cavia,   and  the  Development  of   the  Acrosome.     Quart. 

Jour.  Micro.  Sci.,  Vol.  65. 
Harman,  Mary  T. 

'15     Spermatogenesis  in  Paratettix.     BIOL.  BULL.,  Vol.  29. 
Harman,  Mary  T.,  and  Root,  Frank  P. 
'26     Number  and  Behavior  of  the  Chromosomes  in   Cavia  cobaya    (the 

Common  Guinea  Pig).     BIOL.  BULL.,  Vol.  Si- 
Hermann,  F. 
'89     Beitrage  zur  Histologie  des  Hodens.    Archiv  f.  mikr.    Anat.,  Bd.  34. 

Jordan,  H.  E. 

'n     The  Spermatogenesis  of  the  Opossum   (Didclfltys  firi/iiiuma)    with 
Special  Reference  to  the  Accessory  Chromosome  and  the  Chondrio- 
somes.    Archiv  f.  Zellforsch.,  Bd.  7. 
v.  Kolliker,  A. 

'56     Physiologische   iiber   die    Samenfliisigkeit.      Zeitschr.    f.    wiss.    Zool., 

Bd.  7. 


246  MARY  T.    HARM  AN   AND  FRANK   P.   ROOT. 

Korff,  K.  V. 
'02     Weitere  Beobachtungen  iiber  das  Vorkommen  V-formiger  Central- 

Korper.    Anat.  Anz.,  Bd.  19. 
Lenhossek,  M.  V. 
'98     Untersuchungen    iiber    Spermatogenese.      Archiv     f.     mikr.    Anat., 

Bd.  51. 
Meves,  F. 
'97     tiber  Centralkorper  in  mannlichen  Geschlechtszellen  von  Schmetter- 

lingen.     Anat.  Anz.,  Bd.  14. 
'98    XJber    das    Verhalten    der    Centralkorper    bei    der    Histogenese    der 

Samenfaden  vom  Mensch  und  Ratte.     Verh.  Anat.  Ges.,  Bd.  14. 
'99    Ueber  Struktur  und  Histogenese  der  Samenfaden  des  Meerschweinch- 

ens.     Archiv  f.  mikr.  Anat.,  Bd.  54. 
Montgomery,  T.  H. 

'n     The  Spermatogenesis  of  an  Hemipteron,  Euschistus.    Jour.  Morph., 

Vol.  22. 
Niessing,  G. 

'89    Untersuchungen   iiber   die   Entwicklung   und    den    feinsten    Bau   der 
Samenfaden    einiger    Saugethiere.      Verh.    d.    phys.    medic.    Ges.    in 
Warzbur  N.  F.,  Bd.  52. 
'97     Die   Betheiligung   von    Centralkorper   und    Sphare   am   Aufbau    des 

Samenfadens  bei   Saugethieren.     Archiv  f.  mikr.  Anat.,  Bd.  48. 
Oliver,  J.  R. 
'13     The  Spermatogenesis  of  the  Pribilof  Fur   Seal.     Am.  Jour.  Anat., 

Vol.  14. 

Papanicolaou,  George  N.,  and  Stockard,  Charles  R. 
'18     The  Development  of  the  Idiosome  in  the  Male  Germ-cells  of  the 

Guinea  Pig.    Am.  Jour.  Anat.,  Vol.  24. 
v.  la  Valette,  St.  George. 
'65    Ueber  die  Genese  der  Samenkorper.  Erste  Mittheilung.     Archiv  f. 

mikr.  Anat.,  Bd.  I. 
Weigl,  R. 

'12     Vergleichend-zytologesche  Untersuchungen  iiber  den  Golgi-Kopschen 
Apparat.    Bull,  de  1'Acad.  Scient.  Cracovie. 


248  MARY  T.    HARMAN  AND  FRANK    P.   ROOT. 


EXPLANATION  OF  PLATES. 

All  the  drawings  were  made  with  the  aid  of  a  camera  lucida,  a  1.9  oil-im- 
mersion objective  and  a  number  6  compensating  ocular  at  table  level.  Figure 
20  was  enlarged  two  diameters.  All  other  drawings  were  enlarged  four  and 
one  half  diameters.  The  reproductions  were  reduced  one  half  from  the 
original. 

PLATE  I. 

FIG.  i.  Spermatid  soon  after  the  last  maturation  division.  G,  Golgi 
bodies ;  N,  nucleus ;  /,  idiosome. 

FIG.  2.     Spermatid  showing  increased  size.     G,  Golgi  body. 

FIG.  3.  Spermatid  with  the  chromatin  in  the  form  of  a  spireme.  /,  idio- 
some ;  G,  Golgi  body. 

FIGS.  4,  5,  AND  6.  Spermatid  showing  an  abortive  attempt  to  form 
chromosomes.  I,  idiosome ;  G,  Golgi  body. 


BIOLOGICAL   BULLETIN,    VOL.    LV. 


PLATE  I. 


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PLATE  II. 

FIG.  7.  Spermatid  showing  the  chromatin  finely  granular  and  the  begin- 
ning of  the  contracting  of  the  entire  cell. 

FIG.  8.  Spermatid  showing  the  idiosome  closely  applied  to  the  nucleus 
and  the  appearance  of  the  idiosphere.  /,  idiosome ;  NE,  idiosphere ;  G,  Golgi 
body. 

FIGS.  9  AND  10.  Spermatids  showing  a  great  reduction  in  size.  I,  idio- 
some; NE,  idiosphere. 

FIGS,  ii  AND  12.  Spermatid  showing  the  ovoid  shape  which  is  the  be- 
ginning of  the  elongation.  /,  idiosome;  NE,  idiosphere;  G,  Golgi  body;  C, 
cytoplasm. 

FIGS.  13  AND  14.  Spermatid  showing  the  beginning  of  the  elongation  of 
the  idiosome  and  the  idiosphere.  /,  idiosome ;  NE,  idiosphere ;  N,  nucleus ; 
C,  cytoplasm;  G,  Golgi  body ;  A,  acroblast. 

FIGS.  15,  16  AND  17.  Spermatids  showing  the  elongation  of  the  nucleus 
and  the  spreading  out  of  the  cytoplasm  in  a  fan-shape.  A,  acroblast,  N, 
nucleus ;  C,  cytoplasm. 


BIOLOGICAL   BULLETIN,     VOL.    LV. 


PLATE  II. 


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MARY  T.    HARMAN   AND   FRANK   P.   ROOT. 


PLATE  III. 

FIGS.  -18  AND  ig.  Spermatids  showing  appearance  of  filaments  from  the 
nucleus  and  the  extension  of  the  acroblast  to  the  side  of  the  elongated  nu- 
cleus. A,  acroblast ;  N,  nucleus ;  C,  cytoplasm ;  F,  filaments  ;  G,  Golgi  body ; 
Gr,  granules. 

FIG.  20.  Sertoli  cell  with  some  of  the  associated  spermatids  in  an  elon- 
gated form.  S,  spermatids;  N,  nucleus;  AC,  spermatogonial  cell. 

FIGS.  21  AND  22.  Spermatids,  a  continuation  of  the  development  shown 
in  Figs.  18  and  19.  A,  acroblast;  N,  nucleus;  F,  filaments;  C,  cytoplasm; 
Gr.,  granules. 


BIOLOGICAL  BULLETIN,    VOL.  LV. 


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254  MARY   T.    HARMAN   AND   FRANK    P.    ROOT. 


PLATE  IV. 

FIG.  23.  Spermatid  almost  transformed,  viewed  from  one  edge.  N, 
nucleus;  SF,  spiral  filament  in  formation;  A,  acrosome;  LT,  tail  segment; 
NO,  node. 

FIGS.  24,  25,  AND  26.  Mature  spermatozoa.  Fig.  24  viewed  from  con- 
vex surface,  Fig.  25  from  side  angle  and  Fig.  26  from  edge  of  head.  A, 
acrosome ;  N,  nucleus ;  R,  residual  cytoplasm ;  SF,  spiral  filament ;  T,  tail ; 
NO,  node. 


BIOLOGICAL   BULLETIN,   VOL.    LV. 


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MARY  T.    HARMAN  AND   FRANK   P.    ROOT. 


STUDIES    ON   THE    SECONDARY   SEX    CHARACTERS 

OF  CRAYFISHES.     VIII.    MODIFIED  THIRD 

ABDOMINAL  APPENDAGES  IN  MALES 

OF  CAMBARUS  VIRILIS. 

C.  L.  TURNER, 
ZOOLOGICAL  LABORATORY,  NORTHWESTERN  UNIVERSITY. 

The  first  and  second  abdominal  appendages  of  males  are  ha- 
bitually modified  in  Cambarus  for  the  purpose  of  copulation. 
The  individual  parts  of  the  first  pair  of  appendages  are  fused  and 
twisted  and  lie  compactly  in  a  groove  on  the  ventral  side  of  the 
thorax.  The  appendages  of  the  second  abdominal  segment  re- 
semble the  typical  swimmeret  in  general  plan  (Figs.  I  and  6). 
However,  the  protopodite  is  elongated  and  heavier,  the  basal  un- 
segmented  portion  of  the  endopodite  is  likewise  reinforced,  bearing 
a  conspicuous  triangular  shoulder.  The  terminal  segmented  por- 
tion of  the  endopodite  is  much  reduced.  The  remaining  swimmer  - 
ets  are  unusually  quite  typical. 

There  is  apparently  only  one  published  record  of  a  modified 
third  abdominal  appendage.  Moenkhaus,  Proceedings  of  the 
Indiana  Academy  of  Science,  1903,  pp.  in  and  112,  describes  a 
specimen  of  Cambarus  virilis  bearing  such  a  modification  as  fol- 
lows :  "  The  first  and  second  pairs  of  appendages  were  modified 
in  the  usual  way  and  in  no  way  differed  from  corresponding 
appendages  in  the  normal  male  of  the  same  species.  The  addi- 
tionally modified  third  pair  resemble  in  plan  almost  exactly  the 
second  pair.  The  exopod  and  the  segmented  flabellum  of  the 
endopod  are  much  less  reduced  and  much  more  extensively  pro- 
vided with  feathered  setae  than  the  second  pair.  They  are  about 
the  same  size  and  in  position  converge  and  fit  against  the  sec- 
ond pair  of  appendages  much  in  the  same  manner  that  these  do 
against  the  first.  Whether  they  were  in  any  way  functional  I 
am,  of  course,  unable  to  say."  Another  specimen  with  a  modi- 
fication similar  to  but  not  so  fully  developed  as  the  one  described 
by  Moenkhaus  was  collected  by  Dr.  H.  J.  Van  Cleave  of  the 
University  of  Illinois  and  appears  in  his  collection. 

255 


256  C.    L.    TURNER. 

Since  crayfishes  are  in  such  common  use  as  laboratory  subjects 
it  seems  likely  that  any  considerable  occurrence  of  this  aberrancy 
would  have  been  noted  and  described.  The  writer  has  examined 
thousands  of  crayfishes  during  the  past  seven  years,  always  with 
the  object  of  finding  peculiarities  in  the  secondary  sex  characters 
and  while  large  numbers  of  specimens  have  been  found  in  which 
other  aberrancies  occurred,  not  one  was  found  with  this  type  of 
peculiarity  until  the  lot  described  came  to  light. 

A  collection  of  several  hundred  specimens  which  had  been  taken 
from  the  Fox  River  between  Green  Bay  and  DePere,  Wisconsin, 
during  the  summer  of  1927,  was  being  used  in  the  Zoology  Lab- 
oratory at  Northwestern  University.  A  specimen  having  pecu- 
liar appendages  was  discovered  by  chance  and  the  writer  then  ex- 
amined the  entire  lot.  Forty-six  of  a  total  of  three  hundred  and 
forty-two  males  were  found  which  had  third  abdominal  appendages 
modified  somewhat  like  those  of  the  second  abdominal  appendages. 
No  other  peculiarities  were  noted  among  the  males,  but  one  fe- 
male in  seventy  possessed  a  pair  of  copulatory  hooks  on  the  third 
walking  legs  like  those  of  the  male.  The  latter  type  of  aber- 
rancy is  the  most  common  and  it  is  surprising  to  find  a  type  that 
is  apparently  rare  in  greatly  superior  numbers. 

DESCRIPTION  OF  SPECIMENS. 

The  male  specimens  with  the  modified  third  abdominal  ap- 
pendages are  about  thirteen  and  a  half  per  cent,  of  the  total  num- 
ber examined.  They  range  in  length  from  79  to  107  mm. 
Twenty-eight  are  second  form  and  eighteen  are  first  form  males. 
A  fairly  complete  series  is  represented  in  the  aberrant  appendages. 
In  some,  the  third  abdominal  appendages  varies  only  in  the 
presence  of  a  slight  projection  upon  the  inner  border  of  the 
endopodite  between  the  basal  unsegmented  and  the  terminal  seg- 
mented portions  (Figs.  2  and  3),  while  at  the  other  end  of  the 
series  the  modifications  are  practically  like  those  of  the  second 
abdominal  appendages  (Figs.  4  and  5).  There  is  apparently  no 
relation  between  the  extent  of  modifications  of  the  appendages 
and  the  size  of  the  animals.  In  form  I.  specimens  the  angles 
upon  the  shoulder  of  the  aberrant  appendages  are  sharper  and 
stronger  than  those  of  form  II.,  but  this  might  have  been  expected 


STUDIES  ON   SECONDARY  SEX    CHARACTERS   OF   CRAYFISHES.    257 

since  the  same  is  true  of  the  usual  modified  appendages  in  normal 
form  I.  and  form  II.  males. 

The  first  and  second  abdominal  appendages  are  normal  in  every 
respect  in  all  the  specimens. 

DISCUSSION. 

In  attempting  to  find  an  explanation  for  the  large  occurrence 
of  a  rare  aberrancy,  age,  accidental-  embryonic  development,  effect 
of  environment  or  peculiar  genetic  constitution  might  be  sug- 
gested at  first  thought  as  causal  factors.  The  fact  already  noted  in 
this  description  that  size,  and  therefore  age,  and  degree  of  develop- 
ment of  the  peculiarities  in  the  appendages  are  independent  would 
seem  to  eliminate  age  as  a  factor.  Accident  might  be  called  upon 
to  account  for  a  specimen  or  two  but  scarcely  for  so  large  a  num- 
ber as  is  represented  here.  It  has  yet  to  be  shown  that  environ- 
ment has  played  any  part  in  the  development  of  the  secondary 
sex  characters  of  crayfishes,  nor  indeed,  in  modifying  them. 

Peculiar  genetic  constitution  seems  to  be  the  logical  factor  here. 
It  has  already  been  shown  for  other  'aberrant  conditions  in  sex 
characters  of  crayfishes  that  there  is  a  strong  tendency  for  the  de- 
velopment of  one  type  of  peculiarity  in  one  locality  and  the  pres- 
ent case  is  another  instance  of  the  same  tendency.  It  has  been 
argued  in  these  other  instances  that  the  peculiarity  might  easily 
arise  and  perpetuate  itself  as  a  mutation  and  the  explanation  is 
again  offered  for  the  case  in  hand. 

It  does  not  seem  likely  that  this  modification  has  any  functional 
significance.  Specimens  more  radically  modified  in  other  sex 
characters  have  been  functioning  normally  and  there  is  no  reason 
to  believe  that  this  slight  peculiarity  would  make  males  any  more 
efficient  nor  that  it  would  interfere  with  copulation. 

The  series  offered  in  the  specimens  here,  from  the  slightly  modi- 
fied to  the  most  completely  modified  may  give  a  clue  as  to  the 
evolutionary  changes  through  which  the  normal  second  abdominal 
appendages  came  in  the  course  of  their  development.  This  is 
speculative,  of  course,  but  we  have  here  an  actual  series  ranging 
from  a  practically  unmodified  third  abdominal  appendage  to  one 
which  almost  exactly  duplicates  the  normal  second.  Unless  the 
highly  peculiar  second  abdominal  appendages  arose  with  all  their 


258 


C.    L.    TURNER. 


STUDIES  ON   SECONDARY  SEX   CHARACTERS  OF   CRAYFISHES.    259 

pecularities  fully  formed  in  one  stage  it  is  easy  to  believe  'that 
they  arose  through  a  series  of  changes  such  as  is  represented 
here.  The  first  stage  would  be  represented  by  the  development  of 
a  low  projection  on  the  inner  surface  of  the  endopodite  between 
the  unsegmented  basal  portion  and  the  segmented  terminal  por- 
tion. Subsequent  changes  would  involve  an  enlargement  of  this 
spur  and  a  molding  of  it  until  it  had  assumed  the  shape  found  in 
the  normal  second  appendage  of  the  male.  Other  changes  would 
involve  an  elongation  and  an  enlargement  of  the  propodite,  and  an 
enlargement  and  a  strengthening  of  the  basal  portion  of  the  endo- 
podite together  with  a  reduction  of  the  terminal  segmented  portion 
of  the  endopodite. 

In  aberrant  females  having  first  abdominal  appendages  modified 
like  those  of  males  the  second  abdominal  appendages  are  also 
sometimes  modified.  Such  aberrant  females  are  rare  but  even 
in  a  small  number  various  degrees  of  modification  are  shown  in 
the  second  appendages.  These  second  abdominal  appendages  are 
identical  in  their  structural  peculiarities  with  the  third  abdominal 
appendages  described  here  and  are  similar  also  in  that  they  show 
various  stages  of  development. 

EXPLANATION  OF  FIGURES. 

Note:  All  figures  are  drawn  to  the  same  scale. 

FIG.  i.     Unmodified  left  third  abdominal  appendage  of  normal  male. 

FIG.  2.  Left  third  abdominal  appendage  of  aberrant  male  measuring 
88  mm.  Callosity  on  endopodite  is  showing  first  stage  of  development. 

FIG.  3.  Left  third  abdominal  appendage  of  aberrant  male  measuring 
82  mm.  Callosity  on  endopodite  much  larger. 

FIG.  4.  Right  third  abdominal  appendage  of  aberrant  male  measuring 
80  mm.  The  protopodite  is  longer,  the  basal  portion  of  the  endopodite 
elongated  and  the  shoulder  upon  the  endopodite  is  more  prominent. 

FIG.  5.  Left  third  abdominal  appendage  of  aberrant  male  103  mm.  in 
length.  Modifications  almost  equal  to  those  of  the  normal  second  abdom- 
inal appendage. 

FIG.  6.  Left  second  abdominal  appendage  of  normal  male  measuring 
98  mm. 


18 


NATURAL  HISTORY  OF  SHIPWORM,  TEREDO  NAVA- 
LIS,  AT  WOODS  HOLE,  MASSACHUSETTS. 

B.  H.  GRAVE, 
WABASH  COLLEGE. 

From   the  Marine  Biological  Laboratory,  Woods  Hole,  Mass. 

SECTION  I.     OCCURRENCE. 

The  common  species  of  shipworm  at  Woods  Hole,  as  identi- 
fied by  Kofoid  and  Clapp,  is  Teredo  navalis.  The  date  of  its 
first  appearance  in  this  region  is  not  known.  Verrill  lists  it  in 
his  '  Invertebrate  Animals  of  Vineyard  Sound  and  Adjacent 
Waters  "  (1871).  Whatever  its  history  in  American  waters  may 
have  been,  it  is  now  known  to  occur  throughout  the  entire  North 
American  coast  from  Alaska  to  Labrador.1  The  present  study 
has  been  carried  on  during  the  past  four  years  and  in  that  time  no 
other  species  has  been  collected.  It  is  known,  however,  that 
Utinkia  fiinbriata  occurs  in  this  region,  although  in  comparatively 
small  numbers.  During  the  year  this  work  was  first  undertaken 
it  was  difficult  to  obtain  Teredo  in  sufficient  numbers  for  satis- 
factory study,  but  this  is  not  an  indication  that  the  species  is  not 
abundant  in  New  England  waters.  The  reason  for  an  apparent 
scarcity  is  that  shipworms  are  inaccessible,  being,  for  the  most 
part  imbedded  in  piles  and  permanent  structures.  Subsequently, 
by  putting  out  suitable  timbers  during  one  summer  to  be  studied 
the  next,  it  has  been  an  easy  matter  to  obtain  Teredo  in  abundance. 
Lobster  pots2  and  2X4  stakes  have  been  found  to  be  the  most 
convenient.  If  these  timbers  are  exposed  to  the  water  during 
the  latter  part  of  the  summer  they  are  found  to  contain  sex- 
ually mature  worms  by  the  beginning  of  the  breeding  season  the 
following  June.  The  2X4  stakes  give  best  results  if  exposed 

Nelson,  '22,  speaks  of  an  infestation  of  Teredo  navalis  in  Barnagat 
Bay,  New  Jersey,  as  a  sudden  outbreak.  He  is  probably  in  error  in  think- 
ing that  this  species  arrived  so  recently  on  the  New  England  coast. 

2  Lobster  pots  are  constructed  of  small  slats  about  the  size  of  ordinary 
plasterer's  lath,  2  in.  broad  and  y2  in.  in  thickness. 

260 


NATURAL    HISTORY   OF    SHIPWORM.  26! 

during  July  or  early  August,  but  the  smaller  timbers  are  liable 
to  complete  destruction  before  winter  if  put  out  early  in  the 
summer. 

Teredo  do  not  grow  large  in  small  timbers  such  as  are  used 
in  the  construction  of  lobster  pots,  but  are  easily  removed  from 
such  small  strips  of  wood,  thereby  facilitating  study.  The  size 
attained  depends  upon  the  degree  of  crowding.  To  ascertain  the 
size  to  which  Teredo  will  grow,  it  is  necessary  to  supply  larger 
pieces  of  wood  and  2X4  stakes  are  excellent  for  the  purpose. 
With  a  drawing  knife  it  is  possible  to  expose  the  entire  burrow 
in  a  few  minutes  because  Teredo  tunnels  with  the  grain  of  the 
wood,  usually  within  half  an  inch  of  the  surface.  A  study  of  such 
stakes  has  shown  that  Teredo  larvae  attack  the  wood  in  great 
numbers  at  the  mud  line  but  less  and  less  abundantly  from  the 
bottom  to  the  surface  of  the  water.  Three  fourths  of  the  Teredo 
burrows  in  an  exposed  timber  occur  within  two  or  three  feet  of 
the  mud  line.  Very  few  are  found  more  than  four  feet  above 
the  bottom. 

SECTION   II.     ANATOMY,   PHYSIOLOGY  AND  BEHAVIOR. 

The  anatomy  of  Teredo  has  been  accurately  described  by  sev- 
eral early  investigators  and  more  recently  the  shell  and  digestive 
tract  have  received  attention  by  Miller  and  Lazier,  whose  ad- 
mirable work  is  published  in  four  papers.  It  is  sufficient  here  to 
say  that  the  shipworm  has  the  structure  of  an  ordinary  lamelli- 
branch  in  which  the  body  is  much  elongated  and  in  which  the 
bivalve  shell  is  highly  modified  in  adaptation  to  the  burrowing 
habit.  In  one  particular  my  observations  are  not  in  agreement 
with  those  of  Miller.  He  attributes  the  formation  of  the  rings 
of  growth,  the  rasping  ridges,  and  denticles  of  the  shell  to  altera- 
tion or  fluctuation  in  the  food  supply  which,  according  to  his 
conception,  results  in  corresponding  periods  of  slow  and  rapid 
growth.  This  may  account  for  the  annual  rings  of  growth  of 
certain  mollusks  and  has  been  so  interpreted,  but  it  could  hardly 
account  for  the  rings  and  ridges  on  the  shell  of  this  young  ani- 
mal which  adds  two  rings  per  week  in  the  early  stages  of  its  de- 
velopment. These  sculpturings  of  the  shell  which  adapt  it  to 
burrowing  are  undoubtedly  due  to  the  action  of  little  tongues 


262  B.    H.    GRAVE. 

of  mantle  tissue  which  are  pushed  up  over  the  edge  of  the  shell 
during  deposition  of  the  shell  material.  This  process  of  shell 
sculpturing  was  observed  in  the  large  lamellibranch  Atrina 
rigida  (Grave,  '09).  The  peculiar  form  and  pattern  of  the  shell 
is  specific  and  is  a  matter  of  inheritance,  but  the  building  process 
is  due  to  the  peculiar  manipulation  of  the  mantle  and  not  to 
alternate  periods  of  starvation  and  plenty. 

The  physiology  of  digestion  has  been  studied  particularly  in 
recent  years  by  Dore,  Miller  and  Potts. 

Potts  ('24)  corroborates  the  work  of  Dore  and  Miller  ('22)  in 
showing  that  as  the  shipworm  burrows  through  the  wood  it  swal- 
lows the  chips  and  derives  some  nourishment  from  them.  A 
large  section  of  the  digestive  tract  seems  to  be  devoted  entirely 
to  the  digestion  of  wood  (the  caecum  and  liver).  Potts  believes 
that  wood  is  the  only  food  of  Teredo  but  Miller  shows  that  the 
digestive  tract  contains  diatoms  as  well  as  wood.  The  burrow 
mainly  serves  as  a  means  of  protection. 

As  the  Teredo  grows  it  enlarges  its  burrow  proportionately 
until  at  maturity  it  may  be  16  inches  in  length  and  have  a  diam- 
eter of  Y%  of  an  inch  (40  X  i  cm.).  A  pile  or  other  exposed 
piece  of  timber  may  be  honeycombed  with  Teredo  tunnels  with- 
out showing  on  the  surface  that  it  is  infested.  The  only  open- 
ing of  the  burrow  leading  to  the  outside  is  the  minute  pore 
through  which  the  young  Teredo  entered  the  wood  as  a  meta- 
morphosing veliger.  Although  less  than  .35  mm.  in  diameter 
and  therefore  too  small  to  be  seen  readily  by  the  unaided  eye,  it  is 
through  this  passage  that  the  siphons  are  protruded  to  obtain 
respiratory  currents  and  food  other  than  wood.  The  shipworm 
feeds  upon  minute  organisms  derived  from  water  currents  that 
pass  over  its  gills  for  respiration,  just  as  in  ordinary  lamelli- 
branchs.  It  is  in  fact  an  elongated  lamellibranch,  whose  bur- 
rowing shell  covers  only  its  anterior  tip,  leaving  most  of  the  body 
and  the  siphons  unprotected  except  for  the  wooden  shell-lined 
burrow. 

CHARACTER  OF  THE  BURROW. 

The  burrows  are  always  lined  by  a  calcareous  substance,  except 
at  the   anterior   end,   where   further   excavation   is   taking   place. 


NATURAL    HISTORY   OF    SHIPWORM. 


263 


This  shell-like  material  is  secreted  by  the  general  surface  of  the 
body  or  mantle.  It  has  been  suggested  that  this  lining  of  the 
burow  not  only  makes  a  smooth  surface,  but  shuts  out  wood 
acids  as  well  as  external  enemies  which  might  otherwise  injure 
the  soft  body  of  the  animal.  Even  the  outer  pore-like  opening 
is  lined  with  this  secretion  and  is  divided  transversely  by  a  par- 
tition, so  that  the  siphons  protrude  through  two  minute  pores  just 
large  enough  to  transmit  them.  While  the  shipworm  is  not  feed- 
ing, or  when  it  is  disturbed,  the  siphons  are  withdrawn  and  the 
external  openings  are  plugged  by  two  curious  horny  pallets,  as 
they  are  called,  situated  one  on  each  side  of  the  siphonal  region. 
See  Figure  I. 


s   s 


FIG.  i.  Young  Teredo,  length  2  cm.,  age  five  weeks  from  metamorphosis; 
drawn  by  camera  lucida.  S  shell,  F  foot,  i.  s.  incurrent  siphon,  e.  s.  ex- 
current  siphon,  p.  pallet. 

Effect  of  Adverse  Conditions.  (Repairing  the  burrow,  etc.) 
In  case  the  tunnel  is  broken  by  accident,  or  by  the  wearing 
away  of  the  surface  of  the  wood  from  any  cause,  the  adjacent 
glands  secrete  shell  substance  in  greater  abundance  and  mend  the 
breach.  The  integrity  of  the  burrow  is  carefully  preserved.  In 
case  adverse  conditions  arise  which  make  the  environment  diffi- 
cult either  from  enemies  or  poisons  in  the  water,  or  from  over- 
population by  its  fellows,  this  shell  substance  is  secreted  in  the 
form  of  a  heavy  casing,  not  only  on  the  sides,  but  over  the  an- 
terior burrowing  end  as  well.  This  is  the  invariable  reaction  of 


264  B.    H.    GRAVE. 

Teredo  to  adverse  external  conditions,  the  most  common  cause 
of  which  is  the  crowding  of  individuals  in  small  timbers.  As  a 
consequence,  the  wood  becomes  extremely  fragile,  a  mere  shell, 
so  porous  that  enemies,  such  as  bacteria  and  parasitic  protozoa, 
find  entrance  and  menace  the  life  of  the  community.  Under 
these  conditions  the  worms  die  within  the  first  year.  It  may  be, 
too,  that  wood  is  an  essential  part  of  their  diet,  but  it  is  more 
probable  that  the  trouble  is  a  lack  of  adequate  protection  against 
adverse  conditions  and  dangers  from  without. 

No  Teredo  ever  molests  the  burrow  of  another.  When  two 
come  close  together  they  face  about  and  proceed  in  another  di- 
rection, thus  avoiding  each  other.  When  they  become  so  closely 
crowded  that  further  burrowing  would  infringe  upon  a  neighbor, 
growth  seems  to  stop.  The  size  attained  depends  upon  the 
amount  of  crowding.  As  stated  above,  the  Teredo  responds  'to 
these  conditions  by  greatly  thickening  the  shell  lining  of  its  bur- 
row on  the  front  as  well  as  on  the  sides  so  that  the  whole  is 
strongly  encased.  However,  it  is  at  best  a  brittle  affair  and  para- 
sitic protozoa  and  bacteria  are  admitted  which  soon  destroy  the 
occupant.  The  protozoan  Architophrya  (a  holotrich)  is  always 
abundant  in  such  situations. 

It  is  difficult  to  see  how  growth  may  cease  and  the  animal  sur- 
vive, but  it  is  perfectly  clear  that  Teredo  three  months  old  living 
in  crowded  situations  are  often  less  than  one  fifth  as  large  as 
others  of  the  same  age  growing  under  better  conditions.  The 
stunted  worms,  though  packed  closely  together  are  frequently 
all  alive  and  reproducing.  As  many  as  seven  young  Teredo  per 
square  inch  have  been  observed  in  test  blocks  although  the  aver- 
age is  by  no  means  so  high.  When  these  worms  all  become  two 
or  three  inches  long,  a  crowded  group  results  unless  they  hap- 
pened to  have  entered  a  large  timber  which  permits  of  unlimited 
expansion. 

Shipworms  rarely  go  from  one  board  to  another,  no  matter  how 
closely  the  boards  are  applied  to  each  other.  Only  two  exceptions 
to  this  rule  have  been  observed  among  the  thousands  of  burrows 
studied.  They  seem  to  avoid  anything  that  threatens  to  interrupt 
the  continuity  of  their  tunnels. 

Teredo  seems  not  to  orient  to  gravity  since  it  burrows  down- 


NATURAL    HISTORY   OF    SHIPWORM.  265 

ward  about  as  frequently  as  upward.  The  burrow  of  a  single 
individual  often  shows  that  there  is  no  tropistic  response  of  this 
kind.  If  in  tunneling  downward  a  Teredo  approaches  the  end  of 
the  timber,  another  Teredo  burrow  or  a  knot,  it  may  turn  directly 
about  and  proceed  in  the  opposite  direction,  paralleling  the  first 
part  of  its  burrow.  By  some  means  it  is  able  to  detect  any  nearby 
surface  of  the  wood  and  avoid  it.  Two  Teredo  tunnels  may  ap- 
proach within  an  eighth  of  an  inch  of  each  other,  but  they  remain 
quite  separate.  They  have  some  sense  also  which  warns  them, 
when  approaching  the  end  of  a  timber,  to  face  about  before  reach- 
ing the  end,  retreating  usually  at  a  point  5  to  10  mm.  from  the 
tip. 

SECTION  III.     THE  BREEDING  SEASON. 

My  interest  in  Teredo  dates  from  1922  when  the  National  Re- 
search Council  suggested  the  study  of  the  breeding  season  of  this 
species  and  appropriated  funds  to  meet  preliminary  expenses. 
The  results  of  this  study  were  reported  at  the  Washington  meet- 
ing of  the  American  Association  for  the  Advancement  of  Science 
in  1924,  and  an  abstract  was  printed  at  that  time.  The  publication 
of  the  paper  as  a  whole  was  deferred  until  the  study  of  various 
details  could  be  completed. 

The  fact  that  the  female  carries  the  young  embryos  in  the  gill 
chamber  for  a  short  time  makes  an  accurate  study  of  the  breed- 
ing habits  a  comparatively  easy  matter.  It  may  be  ascertained  at 
a  glance  whether  a  female  is  carrying  embryos  or  not  and  the 
presence  of  eggs  or  embryos  in  the  suprabranchial  chamber  is 
conclusive  evidence  of  recent  spawning.  A  further  useful  indi- 
cator is  that  of  color.  The  eggs  and  young  embryos  are  pure 
white,  but  they  gradually  take  on  a  dark  gray  color  with  age. 

The  first  spawning  at  Woods  Hole  occurs  from  the  first  to  the 
middle  of  May,  and  the  last  about  the  middle  of  October.  Dur- 
ing 1925  eggs  were  first  obtained  on  May  15  and  these  were  in 
a  late  cleavage  stage  when  discovered.  Two  of  twenty  females 
examined  had  spawned  at  this  date.  In  1926  eggs  were  first  ob- 
tained on  May  16.  Two  of  the  twelve  females  examined  had 
spawned,  and  the  embryos  were  in  the  gastrula  stage  of  develop- 
ment. Frequent  previous  examinations  in  April  and  May  had 
shown  no  spawning  individuals. 


266  B.    H.    GRAVE. 

During  the  fall  of  1925  and  1926  special  trips  were  made  to 
Woods  Hole  in  order  to  determine  the  extreme  limits  of  the 
breeding  season.  At  this  time  an  effort  was  also  made  to  learn 
how  late  in  the  fall  veligers  were  metamorphosing  and  entering 
wood.  On  September  22,  1926,  numerous  females,  both  in  Eel 
Pond  and  at  the  Cayadetta  Wharf  in  Vineyard  Sound,  were  carry- 
ing embryos  in  various  stages  of  development.  On  October  10, 
of  sixty  Teredos  taken  from  Eel  Pond,  none  were  carrying  em- 
bryos, while  five  of  twenty  five  taken  from  the  Sound  had 
quantities  of  veligers  in  their  gills.  The  embryos  of  one  of  these 
were  late  trochophores  or  early  veligers  and  repeated  observa- 
tion on  the  rate  of  development  in  Teredo  has  shown  that  these 
would  normally  be  carried  from  ten  days  to  two  weeks  longer. 
None  were  found  carrying  embryos  on  November  4.  These  and 
other  data  show  that  the  breeding  season  in  Eel  Pond  ended  two 
weeks  earlier  than  in  Vineyard  Sound.  The  difference  in  tempera- 
ture is  apparently  the  cause  of  this  diversity  in  'the  duration  of 
the  breeding  season,  Eel  Pond  being  approximately  two  degrees 
colder  during  the  fall  than  the  deeper  water  of  the  Sound. 
Kofoid  noted  a  similar  difference  in  the  breeding  season  in  vari- 
ous parts  of  San  Francisco  Bay  where  wide  stretches  of  shallow 
water  become  several  degrees  warmer  in  early  spring  and  cooler 
in  the  fall  than  the  deeper  portions  of  the  same  body  of  water. 
His  estimate  of  two  weeks  difference  is  no  doubt  conservative. 
Observations  just  completed  at  this  writing  show  that  the  first 
spawning  by  Teredo  in  Eel  Pond  in  1927  occurred  on  May  I  and 
in  Vineyard  Sound  on  May  12.  Spawning  occurred  in  each  case 
when  the  water  had  reached  a  temperature  of  approximately  11° 
C.  (between  11°  and  12°  C.).  Since  spawning  ceased  in  Ed 
Pond  on  October  i  and  in  Vineyard  Sound  about  October  15  we 
have  the  same  variation  due  to  temperature  difference  and  the 
total  spawning  season  for  Teredo  at  Woods  Hole  is  shown  to 
be  nearly  or  quite  five  months  in  duration. 

It  should  be  explained  that  the  larva  has  a  free  swimming 
period  of  approximately  two  weeks  after  leaving  the  supra- 
branchial  chamber  of  the  mother  before  it  is  ready  to  enter  wood. 
In  accordance  with  the  fact  that  veligers  are  carried  by  the  mother 
as  late  as  October  20  in  Vineyard  Sound,  one  would  expect  to 


NATURAL    HISTORY   OF   SHIPWORM.  267 

find  that  wooden  structures  are  being  entered  by  the  metamorphos- 
ing veligers  until  the  first  of  November.  The  facts,  however, 
do  not  bear  out  this  expectation.  The  last  date  on  which  veligers 
successfully  metamorphosed  and  attacked  wood  in  Eel  Pond  was 
September  23,  whereas  larvae  were  no  doubt  present  until  about 
October  5.  Lobster  pots  placed  in  Vineyard  Sound  on  October 
10  were  entered  by  metamorphosing  veligers.  It  is  certain  that 
larva?  are  present  in  the  water  in  Vineyard  Sound  until  No- 
vember i  or  the  last  week  in  October.  In  other  words,  larva?  are 
present  in  the  water  at  least  two  weeks  after  the  last  ones  suc- 
cessfully attack  wood.  The  reason  for  this  is  not  evident.  The 
cilia  of  the  swimming  mechanism  of  the  larva  possibly  become 
less  and  less  active  as  the  water  cools,  with  the  result  that  mor- 
tality among  the  last  generation  of  larvae  of  the  season  is  high. 
In  Bugula  also  the  last  larva?  of  the  season  fail  to  metamorphose, 
but  not  to  so  great  an  extent  as  is  the  case  with  Teredo. 

An  examination  of  the  gills  of  a  large  number  of  Teredo  on 
November  4  showed  a  spotting  of  these  organs  as  if  the  last  em- 
bryos contained  had  been  resorbed.  It  is  quite  likely  that  the 
belated  ones  lose  ability  to  swim  and  therefore  remain  inactive 
and  disintegrate  in  the  gill  chamber.  (This  may  not  be  the  correct 
explanation  of  the  cause  of  the  failure  of  the  last  embryos  of  the 
season  to  metamorphose.)  The  larva?  of  Bugula  and  those  of  cer- 
tain hydroids  continue  to  metamorphose  successfully  into  No- 
vember and  the  latter  into  December  although  dependent  upon 
cilia  for  locomotion. 

The  data  in  hand  indicate  that  the  breeding  season  of  Teredo 
at  Woods  Hole  extends  from  about  May  10  to  October  10  or 
possibly  to  October  15,  a  period  of  five  months. 

Fecundity. 

Teredo  is  tremendously  prolific.  Each  female  spawns  three  or 
four  times  in  a  season.  The  number  of  eggs  produced  varies 
with  the  size  of  the  individual  and  is  estimated  to  be  from  one  to 
five  millions.  At  the  end  of  the  season  the  female  seems  to  be 
exhausted.  Many  molluscs  survive  for  several  years  but  Teredo 
dies  during  the  second  year  as  test  blocks  have  shown  repeatedly. 
This  unusual  fecundity  may  explain  the  early  loss  of  vitality. 


268  B.    H.    GRAVE. 

As  evidence  that  the  female  Teredo  spawns  every  four  or  five 
weeks,  the  following  data  are  offered.  Several  cases  of  this  kind 
were  observed. 

June  20,  1925.  Two  large  females  which  were  carrying  gray 
vi-ligcrs,  were  ready  to  spawn  a  second  time.  The  ovaries  were 
large  and  distended  with  eggs  which  were  full  size  and  fertilizable. 

June  24,  1925.  Two  among  several  females  examined  had 
spawned  a  second  time  this  season,  numerous  late  veligers  mixed 
with  cleaving  eggs  were  found  in  the  suprabranchial  chamber. 

Periodicity. 

One  of  the  specific  objects  of  this  study  was  to  ascertain  the 
characteristics  of  the  breeding  season,  whether  or  not  there  is  a 
lunar  or  other  periodicity  in  the  production  or  shedding  of  the 
gametes.  It  was  made  apparent  during  the  first  year's  study  that 
no  lunar  periodicity  occurs  in  the  spawning  of  Teredo.  From  the 
beginning  to  the  end  of  the  breeding  season,  the  water  contains 
abundant  larva?  in  all  stages  of  development.  The  records  of 
examinations  of  hundreds  of  stakes  and  lobster  pots  indicate  that 
larvae  are  abundant  in  the  water  ready  to  attack  any  exposed 
timber  each  day  of  the  summer.  The  evidence  bearing  on  this 
point  is  derived  from  two  types  of  experiments  which  are  here 
described  in  some  detail  because  other  workers  have  stated  that 
the  spawning  of  Teredo  is  periodic  and  that  definite  broods  ma- 
ture at  definite  times. 

ist.  The  following  tables  show  that  no  periodicity  in  the 
spawning  by  this  species  occurs.  Of  a  large  number  of  ship 
worms  that  may  be  examined  at  any  time  during  the  summer, 
some  will  be  found  to  carry  cleaving  eggs,  some  gastrulae,  and 
some  trochophores,  some  young  veligers  and  some  typical  veligers, 
thus  showing  that  spawning  is  continuous  and  not  synchronous. 

TABLE  i. 

Teredo  EXAMINATIONS   1925,  JULY   i. 
Material  frnin   Lobster  I'ot  I'laeed  in   U'aler  .-]»</.  16,  1924.     Eel  Pond. 

!•  i-males  carrying  unspawnecl  eggs  4 

Females  carrying  cleaving  eggs  in  gill  chamber   i 

Females  carrying  young  veligers  in  gill  chamber   i 

I  •(•males  carrying  typical  veligers  in  gill  chamber   4 

Mature   males    with   active   sperm    7 

Total    ! 


NATURAL  HISTORY  OF  SHIPNVOKM.  269 

TABLE  2. 

Teredo  EXAMINATIONS  1924,  JULY  5. 
Material  from  Lobster  Pot  Placed  in  Water  Aug.  20,  1923.    Cayadctta  Dock. 

Females  carrying  mature  eggs   5 

Females  carrying  immature  eggs   I 

Females  carrying  cleaving  eggs  in  gill  chamber    7 

Females  carrying  gastrulae  in  gill  chamber   5 

Females  carrying  young  veligers  in  gill  chamber  4 

Females  carrying  typical  veligers  in  gill  chamber    5 

Mature  males  having  motile  sperm   7 

Immature    males     3 

Total    37 

TABLE  3. 

Teredo  EXAMINATIONS  1924,  JULY  19. 
Material  from  Lobster  Pot  Placed  in  Water  Aug.  20,  1923.    Cayadctta  Dock. 

Females  carrying  eggs    8 

Females  carrying  cleaving  eggs  in  the  gill  chamber  2 

Females  carrying  gastrulae  in  the  gill  chamber   n 

Females  carrying  young  veligers  in  the  gill  chamber   3 

Females  carrying  typical  veligers  in  the  gill  chamber   5 

Mature  males  with  active  sperm    5 

Total     34 

TABLE  4. 

Teredo  EXAMINATIONS  1924,  AUG.   10. 
Material  from  Lobster  Pot  Placed  in  Water  Aug.  20,  1923.     Eel  Pond. 

Females  carrying  eggs   I 

Females  carrying  cleaving  eggs  in  the  gill  chambers   i 

Females  carrying  blastulaa  or   gastrulae    4 

Females  carrying  early  veligers   2 

Females    carrying    typical    veligers     5 

Females  carrying  a  few  veligers  in  the  gill  chambers,  apparently 

spent  i 

Mature  males  with  abundant  active  sperm    12 

Total     26 

These  four  tables  show  that  spawning  takes  place  at  all  times 
during  the  month  and  not  synchronously.  They  show  conclu- 
sively that  there  is  no  lunar  or  other  periodicity  such  as  that 
sometimes  caused  by  variations  of  temperature.  Attention  is 


27O  B.    H.    GRAVE. 

called  to  the  fact  that  the  spawning  of  these  animals  took  place 
not  in  the  laboratory,  but  normally  in  their  natural  habitat. 

It  is  also  apparent  from  these  tables  that  there  are  no  "  broods  " 
or  special  times  of  infestation  of  exposed  timber.  As  further 
evidence  on  this  point  the  test  blocks  (lobster  pots)  were  put  out 
every  ten  days  during  the  summer  and  all  became  infested  with 
metamorphosing  Teredo  larvae  almost  at  once,  certainly  within  a 
day  or  two  after  exposure,  as  numerous  experiments  on  rate  of 
growth  show.  At  Woods  Hole  the  first  larvae  settle  and  begin  to 
burrow  toward  the  end  of  June  (June  20).  From  that  time  on 
until  early  fall  the  water  contains  a  copious  supply  of  swimming 
larvae  ready  to  burrow  into  any  exposed  wooden  structure. 

T.  C.  Nelson  in  his  report  for  the  year  1923,  Table  5,  page 
208,  concludes  on  very  meager  and  insufficient  data  that  one  brood 
of  larvae  settled  in  Barnegat  Bay  in  June  and  that  a  second  brood 
matured  some  time  between  July  26  and  September  4.  The  evi- 
dence derived  from  my  experiments  covering  four  years  show 
that  there  are  no  broods  but  rather  a  continuous  entrance  of 
timbers  by  larvae  maturing  throughout  the  breeding  season.  The 
evidence  of  many  experiments  shows  that  one  can  not  depend 
upon  green  timber  or  even  seasoned  2X4  stakes  for  such  ex- 
periments, as  they  may  remain  uninfested  for  weeks  for  no  ap- 
parent reason.  Seasoned  lobster  pots,  however,  regularly  became 
infested  either  the  day  they  were  exposed  to  the  water  or  very 
soon  thereafter.  This  is  possibly  due  to  the  horizontal  position 
of  the  timbers  in  the  water,  as  contrasted  with  stakes  standing 
vertically.  The  answer  to  the  question  whether  Teredo  larvae 
enter  wooden  structures  in  broods  at  special  times  or  continu- 
ously has  important  practical  bearings  as  well  as  scientific  interest. 

It  is  also  apparent  from  the  data  of  these  tables  that  Nelson's 
statement  that  there  are  five  hundred  females  to  one  male,  does 
not  hold  for  the  Woods  Hole  region.  Females  outnumber  males 
but  by  no  means  to  so  great  an  extent. 

Kofoid  has  shown  that  the  number  of  larvae  in  any  particular 
region  depends  upon  the  extent  to  which  infested  timber  is  pres- 
ent. Regions  far  from  wooden  warves  have  relatively  few 
larvae  in  the  water.  I  was  able  to  show  that  Teredo  is  much 
more  abundant  at  the  Cayadetta  Wharf  than  in  Eel  Pond,  the 


NATURAL    HISTORY   OF    SHIPWORM.  271 

ratio  being  approximately  2:1.  The  distance  between  these  lo- 
cations is  less  than  one  hundred  yards  and  the  difference  in 
numbers  in  this  case  is  not  due  to  a  difference  in  the  amount  of 
wood  present.  The  biological  conditions  in  the  more  or  less  iso- 
lated Eel  Pond  are  clearly  different  from  those  of  the  open  waters 
of  Vineyard  Sound  because  species  inhabiting  them  are  differ- 
ent to  some  extent,  as  shown  in  another  paper  (See  Bugula).1  A 
study  of  these  conditions  is  contemplated  but  at  the  present  no 
adequate  explanation  is  suggested  unless  the  large  amounts  of 
formalin  and  other  poisons  and  oils  from  the  supply  station  seri- 
ously affect  the  Eel  Pond  water  at  times.  There  are,  however, 
differences  in  natural  conditions.  The  tidal  currents  outside,  at 
any  rate,  are  much  stronger  than  those  in  Eel  Pond. 

SECTION   IV.     EMBRYOLOGY,  AND  RATE  OF  DEVELOPMENT. 

The  extensive  contributions  of  Sigerfoos  and  Hatschek  give 
satisfactory  descriptions  of  embryological  development  so  that  I 
shall  avoid  duplication  and  emphasize  only  facts  that  are  new. 

The  egg  of  Teredo  is  comparatively  small  and  white  in  color. 
It  measures  in  extreme  limits  from  .050  mm.  to  .061  mm.  with 
an  average  diameter  midway  between  these  figures.  The  oviducts 
open  into  the  suprabranchial  chambers  which  are  extensive  and 
serve  as  brood  pouches.  When  the  eggs  are  extruded  they  are 
retained  in  the  suprabranchial  chambers  for  a  period  of  two  or 
three  weeks,  during  which  time  they  pass  through  the  early 
stages  of  development.  When  liberated  into  the  sea  water  they 
are  typical  lamellibranch  veligers,  vigorous  and  hardy.  A  large  fe- 
male may  liberate  from  500,000  to  1,000,000  eggs  at  a  single  spawn- 
ing, so  that  the  gill  chambers  are  tightly  packed  with  embryos 
distributed  in  two  parallel  rows  along  the  sides  of  the  slender 
elongated  body.  The  approximate  age  of  embryos  can  be  esti- 
mated by  their  color  since  they  gradually  change  from  white  to 
a  dark  muddy  gray  during  development. 

The  embryo  is  not  parasitic  upon  the  mother,  but  the  egg  will 
not  develop  outside  the  gill  chamber.  Ripe  eggs  were  several 
times  removed  from  the  gonads  and  artificially  fertilized  in  an 

1  Bugula  flabcllcta  lives  readily  in  Eel  Pond  but  will  not  thrive  in  the 
adjacent  waters  of  Vineyard  Sound,  while  the  reverse  is  the  case  with  B. 
turrita. 


272 


B.    H.    GRAVE. 


attempt  to  observe  them  in  development.  Development  was 
initiated  but  no  egg  cleaved  beyond  the  sixteen  cell  stage,  and 
many  stopped  at  the  two,  four,  and  eight  cell  stages.  Develop- 
ment in  these  cases  was  extremely  slow  and  cleavage  was  ir- 
regular and  abnormal.  Eggs  fertilized  at  six  P.M.  had  reached 
the  eight  cell  stage  at  9  P.M.  It  is  probable  that  development  in 
this  species  is  normally  slow,  but  this  rate  can  hardly  be  con- 
sidered normal.  Very  young  embryos  in  the  two  and  four  cell 
stages  were  several  times  found  in  the  suprabranchial  chambers 
and  these  when  removed  developed  no  better  than  the  artificially 
fertilized  eggs.  In  common  with  artificially  fertilized  eggs,  they 
finally  became  viscid  and  adhered  to  the  containing  dish.  It  was 
found  also  that  blastulae  and  gastrulse  would  fare  no  better. 
They  failed  to  develop  into  swimming  larvae.  Late  trochophores 
and  early  veligers  on  the  other  hand  continued  to  develop  nor- 
mally when  removed  from  the  gill-  chamber  to  sea  water.  Veligers 
removed  prematurely  showed  great  vigor  and  swimming  ability, 
and  were  several  times  kept  for  two  weeks  in  sea  water.  Velig- 
ers withstand  much  rough  treatment  and  survive  in  poorly  aerated 
and  even  foul  water.  Some  were  kept  in  glass  aquaria  and  fed 
on  diatoms  for  three  weeks,  but  to  what  extent  they  meta- 
morphosed and  entered  the  wood  that  was  provided  was  not 
learned. 

The  gastrula  is  invaginate,  similar  to  that  of  many  other  mol- 
luscs and  annelids  that  produce  small  eggs  with  little  yolk.  The 
trochophore  is  especially  interesting  because  in  adaptation  to  its 
parasitic  mode  of  life.it  fails  to  develop  a  strong  protoroch.  The 
cells  which  normally  develop  this  larval  swimming  organ  are 
undoubtedly  present  and  distributed  in  a  broad  equatorial  band 
similar  to  that  of  many  molluscs,  and  they  are  more  extensive 
than  in  most  annelids.  The  protroeh  is  apparently  present  and 
was  described  by  Hatscheck.  I  found  it  either  absent  or  so  feebly 
developed  as  to  be  easily  overlooked.  The  trochophore  is  pear 
shaped  or  slightly  elongated  and  on  the  average  measures 
.059  X  -060  mm.  in  length.  As  it  begins  to  transform  into  the 
veliger,  strong  cilia  develop  on  the  velum,  and  the  embryo  be- 
comes motile  long  before  it  is  ready  to  be  expelled  into  the  sea 
water  to  shift  for  itself. 


NATURAL    HISTORY   OF    SHIPWOR.M.  2J3 

Duration  of  Larval  Period. 

Sigerfoos  failed  to  find  free  swimming  vdigers  in  the  water 
and  both  he  and  Nelson  speak  of  the  habits  and  duration  of  the 
larva  as  being  unknown.  The  larval  period  from  fertilization  to 
metamorphosis  has  usually  been  estimated  at  about  one  month. 
It  is  evident,  however,  that  it  varies  somewhat  with  temperature 
being  shorter  in  tropical  and  sub-tropical  regions  than  at  \Yoods 
Hole. 

I  have  frequently  found  Teredo  veligers,  in  various  stages  of 
development,  settling  upon  horizontally  placed  boards  and  Nelson 
has  more  recently  taken  them  in  "  tow,"  as  well  as  hovering  about 
piles  ready  to  settle  permanently.  In  fact,  he  corroborates  the 
observations  of  Harrington  that  the  mature  veligers  of  Teredo 
are  attracted  to  wood  and  wood  extracts.  The  duration  of  the 
free  swimming  period  has  never  been  accurately  determined.  To 
give  attention  to  this  phase  of  the  life  history  publication  of  this 
paper  has  been  delayed  until  its  study  could  be  completed  and 
verified.  The  evidence  now  at  hand  indicates  that  the  entire 
developmental  period  from  egg  to  metamorphosing  larva,  is 
about  five  weeks.  At  least  half  of  this  time  is  required  for  de- 
velopment in  the  gill  of  the  mother,  leaving  for  the  free-swim- 
ming period  not  to  exceed  two  or  three  weeks.  The  evidence  on 
which  this  conclusion  is  based  is  derived  in  various  ways  but  is 
indirect.  Since  the  method  and  conclusion  may  be  questioned, 
the  data  are  explained  in  considerable  detail  in  the  following  pages. 

In  1925  the  first  eggs  were  laid  between  May  12  and  May  15, 
while  the  first  young  metamorphosed  Teredo  were  found  in  test 
blocks  on  July  2  and  July  5.  These  young,  metamorphosed  ship- 
worms  measured  .35  mm.  to  .5  mm.  Evidence  collected  from 
many  experiments  carried  out  during  the  past  two  years  shows 
that  young  Teredo  of  this  size  have  spent  from  15  to  18  days  in 
the  wood,  or  rather,  that  they  settled  and  began  to  metamorphose 
and  burrow  15  to  18  days  previously.  If  we  subtract  15  days 
from  July  2  or  18  days  from  July  5,  June  17  is  the  approximate 
date  when  these  Teredo  ended  their  careers  as  free  swimming 
larvae.  Other  young  Teredo  collected  on  July  7  measured  I  mm. 
and  these  are  known  to  be  three  weeks  old,  or  that  three  weeks 


274 


B.    H.    GRAVE. 


had  elapsed  since  they  settled  upon  wood.  Subtracting  twenty- 
one  days  from  July  7  gives  the  date  June  17  when  metamorphosis 
began.  The  total  larval  period  is  therefore  between  four  and  five 
weeks.  Other  data  collected  during  1925  lead  to  the  belief  that 
the  time  is  more  nearly  five  weeks  than  four.  This  method, 
though  indirect,  is  accurate,  and  was  repeated  many  times  at  the 
opening  of  the  breeding  seasons  of  1925  and  1926.  The  experi- 
ments made  to  determine  the  rate  of  the  metamorphosis  and  rate 
of  early  growth  were  also  repeated  many  times  during  the  past 
two  years  and  are  also  reliable.  They  show  conclusively  that 
young  Teredo  which  measure  one  millimeter  are  approximately 
three  weeks  old.  The  spring  of  1925  opened  unusually  warm, 
although  the  winter  was  severe,  so  that  animals  came  out  of  win- 
ter hibernation  a  few  days  earlier  than  usual.  The  effect  of 
this  was  shown  most  markedly  in  the  rapid  somatic  growth  of 
many  animals,  but  it  also  affected  to  a  slight  extent  the  breeding 
seasons  of  most  animals.  The  date  of  first  settling  of  Teredo 
iarvse  at  Woods  Hole  is  usually  about  June  20,  and  the  first 
spawning  about  May  10.  The  variation  in  the  spawning  season 
from  year  to  year  does  not  usually  exceed  two  or  three  days  but 
it  may  vary  more  than  a  week.  There  is  evidence  that  some  ani- 
mals begin  to  breed  only  when  the  water  rises  to  a  certain  tem- 
perature. This,  however,  is  by  no  means  a  universal  rule. 

Rate  of  Groivth. 

The  veliger  of  Teredo  has  the  typical  form  common  to  lamilli- 
branch  larvae,  but  is  not  so  thick  or  nearly  spherical  as  sometimes 
described.  Young  veligers  taken  from  the  gills  in  an  early  stage 
of  development  measure  on  the  average  .065  x  .080  mm.  Five 
specimens  taken  from  two  individuals  measured  as  follows : 
.060  x  .080  mm. ;  .065  x  .080  mm. ;  .065  x  .082  mm. ;  .070  x  .083 
mm. ;  .070  x  .085  mm.  These  measurements  represent  the  range 
of  variation  in  length  and  breadth.  One  of  these  seen  in  edge 
view  measured  .082  x  .05  mm.,  and  an  older  one  .09  x  .05  mm. 
Veligers  ready  to  begin  their  free  swimming  life,  after  spending 
two  or  three  weeks  in  the  gill  of  the  mother,  measure  some- 
what larger,  as  the  following  examples  show :  .070  x  .090  mm. ; 
.072  x  .090  mm. ;  .075  x  .085  mm. ;  .075  x  .088.  These  measure- 


NATURAL    HISTORY   OF    SHIPWORM.  275 

ments  not  only  indicate  some  growth  but  also  that  a  considerable 
variation  exists  in  the  relative  measurements.  The  range  of 
variation  in  ratio  of  length  to  breadth  and  also  in  length  of  hinge 
line  is  great.  Veligers  fed  upon  diatoms  for  one  week  meas- 
ured from  .077  x  .090  mm.  to  .081  x  .093  mm.  The  size  at-  (  ^  I  B  ft  A  R  Via 
tained  at  the  time  of  metamorphosis  was  not  learned,  but  Nelson 
('23)  gives  it  as  .25  mm.  in  length.  I  have  collected  several 
hundred  young  metamorphosing  Teredo,  which  had  burrowed 
into  wood,  varying  in  age  from  two  to  three  weeks  after  settling. 
These  range  in  size  from  .35  mm.  to  I  mm.  The  smaller  ones 
in  two  weeks  have  almost  completed  metamorphosis  and  have 
from  two  to  three  rasping  ridges  or  rings  of  growth  on  the  shell. 
Individuals  three  weeks  old  have  four  rings  of  growth  and  a 
typical  Teredo  shell.  At  three  weeks  of  age  the  Teredo  is  prac- 
tically spherical  and  its  burrow,  when  exposed  by  cutting  away 
the  surface  of  the  wood,  is  a  hemispherical  pit.  The  young  worm 
now  begins  to  elongate  rapidly  and  at  the  end  of  one  month  its 
burrow  measures  from  5  to  7  mm.  in  length,  and  has  a  diam- 
eter of  2  to  2.5  mm.  The  shipworm  when  expanded  fills  its  bur- 
row so  that,  in  measuring  the  rate  of  growth,  the  size  of  the 
burow  may  be  taken  as  the  correct  measure  of  the  enclosed  worm. 
When  the  shipworm  is  removed  from  its  burrow,  it  contracts  to 
one  half  or  two  thirds  of  its  expanded  measure.  Tables  5  and  6 
show  the  rate  of  growth  from  the  egg  to  adult  size,  and  need 
not  be  described  in  detail.  Measurements  were  taken  every  three 
or  four  days,  and  the  rate  of  increase  in  size  from  day  .to  day 
was  found  to  be  surprisingly  rapid. 

It  should  be  noted  that  the  ages  given  in  the  tables  include  only 
the  time  that  elapsed  from  the  time  of  settling.  If  the  age  from 
the  egg  is  desired,  about  thirty-five  days  should  be  added  to  these 
figures  to  include  the  time  from  fertilization  to  the  end  of  the 
free  swimming  period.  The  larval  period  is  excluded  in  the  fol- 
lowing description  and  from  the  tables. 

Growth  during  the  first  twenty-five  days  seems  small  but  when 
the  minute  size  of  the  animal  at  the  beginning  is  taken  into  ac- 
count, the  growth  is  not  slow.  From  one  month  to  five  months 
the  increases  shown  during  the  intervals  of  three  or  four  days, 
between  measurements,  are  seen  to  be  remarkably  great.  For 
19 


276 


B.    H.    GRAVE. 


TABLE  5. 

RATE  OF  GROWTH  OF  Teredo  navalis   (SUMMER). 
Measurements  of  the  Largest  Burrows  of  Specified  Ages. 


Date  and 
Period  of 
Growth. 


July  16  to  July  26. .  .  . 
July  16  to  Aug.  3.  .  . 
July  26  to  Aug.  13  .  .  . 
July  16  to  Aug.  8.  .  .  . 
July  16  to  Aug.  ii.. 
July  26  to  Aug.  20.  .. 
July  16  to  Aug.  13.  .. 
July  16  to  Aug.  16.  .  . 
July  16  to  Aug.  18.  .  . 
July  16  to  Aug.  23.  .. 
July  16  to  Aug.  28.  .. 
July  16  to  Sept.  i.  .  .  . 
June  20  to  Aug.  20.  .  . 

July  3  to  Sept.  6 

June  22  to  Sept.  3. . . . 
June  22  to  Oct.  20.  .. 
June  22  to  Dec.  i .  .  .  . 
July  1923  to  July  1924 


Age. 


10  days 

18  " 

18  " 

23  " 

25  " 

25  " 

28  " 

30  " 
33 

38  " 
43 

46  " 

60  " 

65  " 

72  " 

130  " 

160 

One  year 


Size  of  Burrows  in 
Length  and  Widest 
Diameter  (Metric). 


No  visible  Teredo  burrows. 
No  visible  Teredo  burrows. 
.35  to  .5  ram.  x  .35  to  .5  mm. 
.35  to  .5  mm.  x  .35  to  .5  mm. 
.5  to  .1  mm.  x  .5  to  i  mm. 

1  to  1.5  mm.  x  i  to  1.5  mm. 

2  to  3  mm.  x  i  to  2  mm. 

5  to  7  mm.  x  2  to  2.5  mm. 
8  to  10  mm.  x  2  to  3  mm. 
14  to  17  mm.  x  3  mm. 
35  to  45  mm.  x  4  mm. 
50  to  57  mm.  x  4  to  4.5  mm. 
70  to  75  mm.  x  4  to  4.5  mm. 
80  to  90  mm.  x  4  to  4.5  mm. 
100  to  120  mm.  x  4.4  to  4.8  mm. 
140  to  170  mm.  x  6.5  to  7  mm. 
175  to  200  mm.  x  7  to  7.5  mm. 
250  to  400  mm.  x  7.8  to  9.4  mm. 


Approximate 

Length  in 

Inches. 


.014  to  .02  in. 
.014  to  .02  in. 
.02  to  .04  in. 
.04  to  .06  in. 
.08  to  .12  in. 
.2  to  .28  in. 
.32  to  .4  in. 
.56  to  .7  in. 
1.4  to  1.8  in. 
2  to  2.3  in. 
2.8  to  3  in. 
3.2  to  3.6  in. 
4  to  4.8  in. 
5.6  to  6.8  in. 
7  to  8  in. 
10  to  16  in.  x 
to  f  in. 


Note: — The  left  hand  columns  of  Tables  5  and  6  represent  the  time  of 
exposure  of  timber  to  the  sea  water.  Thus,  if  we  consider  the  first  item 
of  Table  5,  timbers  were  exposed  to  sea  water  on  July  16  and  examined 
for  Teredo  on  July  26,  making  ten  days  as  the  maximum  age  of  the  infest- 
ing Teredo  as  indicated  in  the  second  column  of  the  table.  Column  three 
gives  the  measurements  of  the  infesting  Teredo  if  any. 

The  measurements  given  in  these  tables  are  actual  cases  and  not  aver- 
ages. Many  more  were  measured  than  are  given  here  but  the  data  given 
are  considered  typical. 

In  all  cases  the  larval  period  is  omitted.  The  age  from  fertilization 
may  be  approximated  by  adding  thirty-five  days  to  the  age  as  given  here. 

Timbers  exposed  forty-three  days,  July  16  to  August  28  contained  sex- 
ually mature  worms.  Other  similar  data  show  that  Teredo  under  favor- 
able conditions  becomes  sexually  mature  in  six  weeks  at  Woods  Hole. 
The  item  second  from  the  bottom  of  Table  5  shows  that  Teredo  does  not 
reach  adult  size  during  the  first  season  (June  23  to  December  i).  No 
growth  takes  place  after  December  i.  (See  also  Table  6.)  The  last  item 
of  the  table  shows  that  adult  size  is  attained  in  one  year  (July  1923  to 
July  1924).  The  largest  specimen  found  at  Woods  Hole  measured  forty 
centimeters  in  length.  It  was  precisely  one  year  old. 

example,  the  length  of  the  largest  burrows  at  twenty-five  days  is 
1.5  mm.,  at  thirty  days  5  to  7  mm.,  at  thirty-eight  days  14  to  17 
mm.,  at  forty-three  days  35  to  45  mm.  Davenport  claims  thai 


NATURAL    HISTORY   OF    SHIPWORM.  277 

growth  in  size  is  partly  due  to  swelling  by  the  absorption  of  water. 
It  is  easier  to  account  for  this  phenomenal  growth  in  this  way  than 
to  suppose  that  the  change  in  size  represents  only  protoplasmic 
growth  and  actual  cell  multiplication. 

It  has  been  repeatedly  found  that  Teredo  navalis  at  Woods  Hole 
leaches  sexual  maturity  and  spawns  from  six  to  eight  weeks  after 
entering  the  wood  as  a  metamorphosing  larva.  The  youngest 
to  spawn  were  six  weeks  old,  and  their  burrow  measured  one 
and  one  half  inches  (38  mm.)  in  length.  They  spawned  in 
abundance  at  the  age  of  two  months  when  they  measured  2-2^/2 
inches  (50  to  63  mm.)  in  length.  Sexual  maturity  is  reached 
long  before  adult  size  is  attained,  since  a  fully  developed  ship- 
worm  measures  from  12  to  16  inches  (30  to  40  cm.)  in  length. 

The  larvse  that  metamorphose  first  in  a  season  almost  reach 
adult  size  by  December  I,  the  largest  ones,  measuring  from  seven 
to  nine  inches  (17.5  to  22.5  cm.)  in  length.  During  December, 
January,  February  and  early  March  ship  worms  are  practically 
dormant  and  do  not  grow  perceptibly.  Then  they  may  be  said 
to  awaken  and  by  the  first  of  July  the  oldest  have  reached  maxi- 
mum size.  The  largest  specimen  found  at  Woods  Hole  meas- 
ured 16  inches  in  length  and  %  inch  in  widest  diameter 
(40x1  cm.).  It  grew  in  a  2x4  test  take  which  was  in  the 
water  from  July  i,  1923  to  July  i,  1924.  Others  measuring  from 
12-15  inches  are  common.  Larvse  which  enter  the  wood  later 
in  the  summer,  even  to  October  i,  lie  dormant  over  winter  and 
resume  growth  the  following  spring.  It  was  shown  that  growth 
is  greatly  retarded  after  the  first  of  November,  especially  on  the 
part  of  the  youngest  Teredo.  Table  6  shows  the  rate  of  growth 
for  the  entire  year  including  the  winter.  It  has  already  been 
stated  that  Teredo  reaches  adult  size  in  one  year  and  dies  during 
the  second  year. 

Teredo  navalis  is  said  to  grow  to  a  slightly  larger  size  in  sub- 
tropical climates  than  at  Woods  Hole.  As  stated  above  the  largest 
specimen  found  in  this  northern  locality  in  four  years'  study 
measured  forty  centimeters  in  length  and  one  centimeter  in  great- 
est diameter. 


278 


B.    H.    GRAVE. 


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NATURAL    HISTORY   OF    SHIPWORM.  279 

Note: — Table  6  shows,  primarily,  the  amount  of  growth  attained  by 
Teredo  during  the  first  season  up  to  the  beginning  of  the  hibernation  period 
(Dec.  i).  It  shows  not  only  that  the  earliest  larvae  of  the  season  fail  to 
reach  maturity  (adult  size)  the  first  season,  but  also  gives  the  amount  of 
growth  attained  by  the  later  larvae  of  the  season  as  well. 

The  first  five  items  of  this  table  show  that  Teredo  larvae  which  enter  the 
wood  after  September  i  will  not  attain  sufficient  size  to  be  detected  in  the 
wood  up  to  Decvember  i.  Timbers  exposed  to  the  water  as  late  as  Au- 
gust 20  on  the  other  hand  are  likely  to  contain  Teredo  measuring  35  to  45 
mm.  in  length  by  the  end  of  the  growing  season  (Dec.  i). 

The  last  two  items  of  the  table  show  that  timbers  exposed  to  the  water 
between  Sept.  12  and  Sept.  23  become  infested  by  Teredo  and  that  they 
appear  in  the  wood  the  following  July,  although  they  remain  too  small 
during  the  winter  to  be  detected. 

The  latest  infestation  observed  at  Woods  Hole  occurring  in  Eel  Pond 
was  Sept.  23,  and  in  Vineyard  Sound  Oct.  10.  It  may  sometimes  occur 
somewhat  later  than  this,  since  the  point  was  not  sufficiently  investigated. 
Table  6  shows  that  growth  in  the  late  fall  is  very  slow  compared  with 
summer  growth  as  given  in  Table  5  and  data  not  tabulated  show  that 
practically  no  growth  takes  place  in  Teredo  at  Woods  Hole  between  Dec- 
i  and  March  I.  Some  observations  indicate  that  the  gonads  begin  to 
proliferate  extensively  before  there  is  detectable  body  growth  in  the  spring. 

SUMMARY. 

Teredo  navalis  occurs  in  abundance  at  Woods  Hole  and  vicinity 
and  has  been  known  there  for  many  years. 

The  breeding  season  extends  from  about  May  10  to  October 
10.  Spawning  begins  in  the  spring  when  the  water  reaches  a  tem- 
perature between  11°  and  12°  C.  Spawning  by  each  female 
occurs  several  times  during  the  season.  No  lunar  periodicity  in 
spawning  occurs  and  there  are  no  broods  caused  by  synchronous 
spawning. 

The  eggs  are  retained  in  the  gills  of  the  mother  during  cleav- 
age and  early  larval  development. 

The  time  required  for  the  fertilized  egg  to  complete  larval  de- 
velopment to  metamorphosis  is  approximately  five  weeks  at  Woods 
Hole.  About  half  of  this  time  is  passed  in  the  brood  pouch  and 
half  as  a  free  swimming  veliger. 

When  eggs  and  early  embryos  are  removed  from  the  gills  they 
do  not  develop  normally. 

The  trochophore  of  Teredo  is  non-motile,  having  either  a  feeblv 
developed  protroch  or  none. 

Teredo  navalis  reaches   sexual  maturity  in  six  weeks  or  two 


28O  B.    H.    GRAVE. 

months  after  metamorphosis  when  it  measures  four  or  five  centi- 
meters in  length.  It  reaches  adult  size  in  on  year,  and  dies  dur- 
ing the  second  year.  The  largest  specimen  collected  in  four  years 
measured  forty  centimeters  in  length  and  one  centimeter  in  great- 
est diameter. 

The  rate  of  growth  during  the  summer  months  and  also  during 
the  winter  was  determined  and  tabulated.  Certain  habits  of  ship- 
worms  were  also  observed  and  recorded. 

APPENDIX. 
Practical  Measures. 

Because  of  numerous  inquiries  by  lobstermen  and  owners  of 
small  boats  concerning  methods  of  preventing  damage  by  ship- 
worms,  a  series  of  experiments  was  made  on  the  effect  of  drying 
upon  Teredo.  These  experiments  were  not  extensive  but  suffi- 
cient to  show  that  simple  but  effective  precautions  may  be  taken. 

Teredo  larvae  first  begin  to  enter  wood  between  June  20  and 
June  25  and  stop  about  October  10.  Shipworms  do  not  enter 
wood  at  any  other  time  during  the  year.  Little  or  no  damage  is 
done  to  wood  until  it  has  been  in  the  water  one  full  month.  The 
largest  of  the  young  shipworms  are  only  one  fourth  of  an  inch 
long  at  the  end  of  one  month  but  they  attain  a  length  of  one  inch 
in  six  weeks.  It  is,  therefore,  advisable  to  dry  lobster-pots  and 
boats  once  per  month  and  leave  them  out  of  the  water  exposed 
to  the  sun  for  one  week.  This  is  especially  true  in  July  and 
August  when  most  of  the  damage  is  done.  Shipworms  in  small 
timbers  are  killed  by  five  days'  exposure  to  sunlight,  but  2x4 
stakes  and  larger  timbers  require  from  a  week  to  ten  days  for 
drying  sufficiently  to  kill  all  of  the  worms. 

EXPERIMENTS. 

(i)   Infested  lobster-pot  lath: 

(a)  After  exposure  in  air  to  bright  sunlight  for  I  day, 
seemed  to  be  dry  but  some  of  the  infesting  shipworms 
were  still  alive. 

After  exposure  in  air  to  bright  sunlight  for  2  days,  some 
shipworms  alive. 


NATURAL    HISTORY    OF    SHIPWORM.  28l 

(c)   After  exposure  in  bright  sunlight  for  3  days,  all  ship- 
worms  dead. 

(2)  Infested  lobster-pot  lath: 

(a)  After  7  days  on  shelf  in  laboratory,  all  worms  contracted 

and  shrunken,  some  of  which  regained  plumpness  and 
normal  activity  when  placed  in  sea  water.  Sperma- 
tozoa and  larvae  taken  from  these  shrunken  worms 
showed  activity. 

(b)  After  10  days  on  shelf  in  laboratory — all  worms,  sperm 

and  larvae  dead. 

(3)  Infested  2x4  stakes  : 

(a)  After   exposure   in   air   to   sunlight   for   5   days;   many 

shipworms  dead,  some  living. 

(b)  After  exposure  in  air  to  sunlight  for  7  days;  all  ship- 

worms  dead. 

(4)  Infested  2x4  stakes : 

(a)  After  exposure  in  air  in  shade  for  7  days;  many  worms 

dead  but  some  living. 

(b)  After  exposure  in  air  in  shade  for  10  days;  none  living. 

Note:—li  infested  2x4  stakes  are  exposed  in  air  in  the  shade  but  kept 
wet  some  worms  may  live  for  several  weeks. 

BIBLIOGRAPHY. 

1.  Barrows,  A.  L. 

'17     An    Unusual    Extension   of    the    Shipworm   in    San    Francisco   Bay. 
Univ.  of  Calif.  Pub.  in  Zool.,  Vol.  18,  p.  27. 

2.  Blum,  H.  F. 

'22     On  the  Effect  of  Low  Salinities  on  Tcrcgo  navalis.    Univ.  of  Calif., 
Pub.  in  Zool.,  Vol.  22,  p.  349. 

3.  Davenport,  C.  B. 

'97     The  Role  of  Water  in  Growth.     Proc.  Boston  Soc.  Nat.  Hist.,  Vol. 
28,  p.  73. 

4.  Core,  W.  H.,  and  Miller,  R.  C. 

'22     The  Digestion  of  Wood  by  Teredo  navalis.    Univ.  of  Calif.  Pub.  in 
Zool.,  Vol.  22,  p.  383. 

5.  Grave,  B.  H. 

'09     Anatomy   and    Physiol.   of   Atrina   rigida.     Bull.   U.    S.    Bur.   Fish., 
1909. 

6.  Harrington,  C.  H. 

'21     A  Note  on  the  Physiology  of  the  Shipworm,  Torcdo  norz'cgica.    Bio. 
Chem.  Jour.,  Vol.  15,  p.  736. 

7.  Hatschek,  B. 

Ueber    Entwicklungsgeschichte   von    Teredo.     Arbeit.    Zool.    Inst.    Wien, 
Vol.  3,  1881,  p.  i. 


282  B.    H.    GRAVE. 

8.  Kofoid,  C.  A.,  and  Miller,  R.  C. 

'22  The  Specific  Status  of  the  Teredo  of  San  Francisco  Bay.  Report 
No.  2  on  the  San  Francisco  Bay  Marine  Pilings  Survey. 

9.  Kofoid,  C.  A. 

Reports  Nos.  i,  2  and  3  on  the  San  Francisco  Marine  Pilings  Survey, 
'21,  '22,  and  '23. 

10.  Lazier,  E.  L. 

'24  Morphology  of  the  Digestive  Tract  of  Teredo  navalis.  Univ.  of 
Calif.  Pub.  in  Zool.,  Vol.  26,  p.  455. 

11.  Miller,  R.  C. 

'22     Variations  in  the  Shell  of  Teredo  navalis.     Univ.  of  Calif.  Pub.  in 

Zo61.,   Vol.    22,    p.   293. 

12.  Miller,  R.  C. 

'24  The  Boring  Mechanism  of  Teredo.  Univ.  of  Calif.  Pub.  in  Zool., 
Vol.  26,  p.  41. 

13.  Nelson,  T.  C. 

'22     The  European  Pile  Worm.     Circular  139,  N.  J.  Ag.  Exp.  Sta. 

14.  Nelson,  T.  C. 

'23  Marine  Borers.  Report  Dept.  Biol.  N.  J.  Ag.  Col.  Exp.  Sta.  year 
ending  June  30,  1923,  p.  204. 

15.  Potts,  F.  A. 

'20  A  Note  on  the  Growth  of  Teredo  navalis.  Rep.  Dept.  Mar.  Biol. 
Carnegie  Inst.  Wash,  year  book  No.  19. 

16.  Potts,  F.  A. 

'24  The  Structure  and  Function  of  the  Liver  of  Teredo.  Proc.  Cam- 
bridge Phil.  Soc.  (Biol.),  Vol.  I.,  p.  i,  and  Jour.  Mar.  Biol.  Assoc., 
Vol.  13,  p.  511. 

17.  Sigerfoos,  C.  P. 

'08  Natural  History,  Organization  and  Late  Development  of  the  Teri- 
dinida?  of  Shipworms.  U.  S.  Bur.  of  Fish.,  Vol.  27,  p.  191. 

18.  Verrill,  A.  E.,  and  Smith,  S.  I. 

'71  Report  upon  the  Invertebrate  Animals  of  Vineyard  Sound  and  Ad- 
jacent Waters.  Report  of  the  U.  S.  Commissioner  of  Fish  and 
Fisheries. 


STUDIES  OF  HUMAN  TWINS. 

I.     METHODS    OF    DIAGNOSING    MONOZYGOTIC    AND    DIZYGOTIC 

TWINS. 

H.  H.  NEWMAN, 
THE  HULL  ZOOLOGICAL  LABORATORY,  UNIVERSITY  OF  CHICAGO. 

INTRODUCTION. 

This  is  the  first  of  a  series  of  studies  of  human  twins  based 
upon  a  considerable  collection  of  pairs  taken  from  the  environs  of 
Chicago.  These  studies  have  been  carried  on  in  collaboration 
with  Professors  F.  N.  Freeman,  K.  J.  Holzinger,  and  Mrs.  Blythe 
Mitchell.  The  original  objective  of  this  research  project  was 
to  secure  an  adequate  collection  of  monozygotic  and  same-sexed 
dizygotic  twins  about  whose  diagnosis  we  could  be  certain.  With 
this  objective  attained,  it  was  proposed  to  make  an  intensive 
comparative  psychological  study  of  the  two  types  of  twins  to 
determine,  if  possible,  the  influence  of  heredity  and  environ- 
ment upon  the  various  mental  traits.  This  is  an  old  and  some- 
what hackneyed  problem,  but  one  that  has  never  been  at  all  sat- 
isfactorily solved.  It  seemed  to  us,  however,  that  all  previous 
studies  had  been  inadequate  because  methods  of  diagnosing  the 
two  types  of  twins  were  unsatisfactory.  The  one  crying  need 
then  was  for  a  satisfactory  method  of  diagnosing  monozygotic 
twins,  and  the  working  out  of  such  a  method  was  assigned  to  the 
present  writer. 

COLLECTION  AND  CLASSIFICATION  OF  MATERIAL. 

The  objective  set  by  the  collaborators  in  this  study  was  the  col- 
lection of  fifty  pairs  of  identical  twins  and  fifty  pairs  of  fra- 
ternal twins.  In  order  to  simplify  our  task,  we  decided  to  elimi- 
nate the  disturbing  factor  of  sex  dimorphism,  and  therefore 
confined  our  study  to  twins  of  the  same  sex,  pairs  in  which  the 
twins  were  both  boys  or  both  girls. 

At  first  no  selection  was  practised  among  same-sexed  twins, 

283 


284 


H.    H.    NEWMAN. 


but  all  cases  were  taken  as  they  came.  As  each  case  was  com- 
pleted an  informal  vote  of  the  three  or  four  workers  present  was 
taken  as  to  the  category  (identical  or  fraternal)  to  which  the 
pair  belonged.  Rarely,  if  ever,  was  there  any  difference  of  opin- 
ion, but  in  about  one  tenth  of  the  cases  there  was  some  uncertainty 
and  these  cases  had  to  be  studied  more  intensively. 

It  soon  appeared  that  the  collection  of  identicals  and  fraternals 
was  not  going  evenly,  the  fraternals  being  more  numerous.  If 
our  preliminary  judgments  as  to  their  classification  were  accu- 
rate we  would  need  to  stop  the  collection  of  fraternals  and  collect 
only  identicals  during  the  last  stages  of  the  period  of  study. 

When  the  adjudged  "  fraternals  "  mounted  to  fifty-two  cases 
(consisting  of  twenty-four  male  pairs  and  twenty-eight  female 
pairs)  there  were  only  forty-three  :<  identicals  "  (consisting  of 
twenty-five  male  pairs  and  eighteen  female  pairs).  The  sex 
ratio  at  that  time  was  very  close  to  normal  expectancy :  forty-nine 
male  pairs  to  forty-six  female  pairs.  The  question  arose  as  to 
whether  the  proportion  of  identicals  to  fraternals  was  running 
according  to  theoretical  expectancy. 

Various  methods  have  been  used  to  determine  the  proportion 
of  monozygotic  to  dizygotic  twins.  One  method  involved  the 
examination  of  the  fetal  membranes  of  considerable  numbers  of 
twin  births  in  institutions  where  competent  observers  were  able 
to  secure  these  important  diagnostic  data.  Spat  in  1860  reported 
that,  in  a  total  of  one  hundred  eighty-four  cases  of  twins  ex- 
amined as  to  the  membranes,  24.6  per  cent,  were  monozygotic. 
Brem  in  1891  reported  22.7  per  cent,  of  monozygotic  twins  out 
of  one  hundred  twenty-six  twin  births.  Krahn  in  1891  reports 
19  per  cent,  of  monozygotic  twins  among  one  hundred  twenty- 
seven  twin  births,  but  includes  as  monozygotic  two  opposite- 
sexed  pairs.  Tigges  found  in  1896,  21  per  cent,  of  monozygotic 
twins  among  fifty-two  twin  births,  and  Quenzel  in  1894  reported 
20.4  per  cent,  of  monozygotics  among  one  hundred  eighty-one 
pairs  of  twins.  These  percentages  range  from  19  per  cent,  to 
24.6  per  cent. 

A  second  method  used  by  several  investigators  for  computing 
the  proportion  of  identical  twins  is  statistical  in  character.  The 
best  known  of  these  methods  is  Weinberg's  "  differential  method." 


STUDIES    OF    HUMAN    TWINS.  285 

In  1902  Weinberg  described  his  method  as  follows :  "  Assuming 
that  sex  is  determined  at  the  time  of  fertilization  and  that  about 
half  of  all  zygotes  will  produce  males  and  the  other  half  fe- 
males, it  follows  that  there  will  be  equal  numbers  of  same-sexed 
as  opposite-sexed  fraternal  twins.  If,  therefore,  we  double  the 
number  of  opposite-sexed  twins  and  subtract  the  product  from 
the  total  of  all  twins,  the  remainder  will  represent  the  number  of 
monozygotic  twins." 

Applying  this  method  to  large  masses  of  twin  data  he  found 
that  the  percentage  of  monozygotic  twins  varies  from  23.4  per 
cent,  to  31  per  cent.,  the  percentage  differing  in  different  coun- 
tries. This  agrees  rather  closely  with  the  percentages  determined 
on  the  basis  of  fetal  membranes. 

Recently  Knibbs   (1926)   has  worked  out  a  formula  for  com- 
puting the  number  of  monozygotic  twins  in  the  twin  population, 
using  data  taken  from  the  census  of  Germany.     His  formula  is 
as    follows :    The    ratio    of    monozygotic    twins    to    all    twins    is 
(M  +  F-  -  P)  -f-  (M  -f-  F  +  P),  where  M  is  the  number  of   $  $ 
pairs,  F  the  number  of    9  5    pairs,  and  P  the  number  of    $  9 
pairs.     This  method  gives  24.4  per  cent,  of  monozygotic  twins  in 
Germany  from  1906  to  1911. 

Applying  Knibbs'  method  to  the  extensive  twin  data  for  the 
United  States  that  is  presented  by  Nichols  (234,497  $  $  ; 
264,098  $  9  ;  219,312  $  $  ),  we  discover  that  26.42  per  cent,  of 
this  large  group  are  monozygotic  and  that  nearly  42  per  cent,  of  all 
same-sexed  twins  are  monozygotic. 

The  question  now  arises  as  to  whether  our  small  random  col- 
lection of  ninety-five  pairs  of  same-sexed  twins  was  composed 
of  the  expected  number  of  identical  and  fraternal  pairs.  Ac- 
cording to  our  diagnosis  there  were  forty-three  pairs  of  identicals 
and  fifty-two  pairs  of  fraternals — i.e.,  45  per  cent,  identicals  in- 
stead of  the  expected  42  per  cent.  This  is  but  a  small  discrep- 
ancy and  may  have  two  meanings :  Either  the  random  selection  of 
twins  has  brought  in  two  or  three  too  many  pairs  of  identicals 
or  else  some  two  or  three  of  the  pairs  diagnosed  as  "  identicals  " 
should  be  classed  as  "  fraternals."  It  is  probable  that  the  former 
explanation  is  correct,  for  it  is  very  unlikely  that  the  ideal  ratio 
as  determined  on  the  basis  of  717,907  pairs  of  twins  would  be 


286  H-    H-    NEWMAN. 

realized  exactly  in  the  first  ninety-five  cases  selected  a't  random. 
In  fact,  the  close  approach  to  theoretical  expectation  actually 
realized  is  almost  too  close.  The  conclusion  may  then  be  drawn 
from  this  that  our  methods  of  diagnosing  identical  and  fraternal 
pairs  cannot  be  far  astray. 

In  order  to  complete  the  proposed  collection  of  fifty  pairs  of 
identicals  and  fifty  pairs  of  fraternals,  it  was  then  necessary  to 
select  seven  cases  of  certain  identical  twins.  Two  cases  of  fra- 
ternals were  eliminated  from  the  fifty-two  cases  of  fraternals 
in  order  to  get  down  to  fifty  cases.  The  two  cases  eliminated 
were  chosen  for  the  following  reasons :  In  one  case  one  twin  had 
lost  three  fingers  and  his  palm  was  so  scarred  that  no  adequate 
palm  print  could  be  taken;  in  the  other  case  one  of  the  twins 
showed  up  with  an  infected  hand  and  no  palm  print  could  be  ob- 
tained. Since,  in  our  diagnosis  of  monozygosity,  the  palm  prints 
were  used  as  highly  important  criteria,  it  seems  well  to  eliminate 
these  two  pairs  in  which  the  palm  print  evidence  was  incomplete. 
The  two  pairs  eliminated  were  unequivocal  cases  of  unlike  fra- 
ternal twins. 

We  have  now  complete  data  on  one  hundred  pairs  of  same- 
sexed  twins,  fifty  of  which  have  been  classed  as  identicals  and 
fifty  as  fraternals.  No  doubt  some  of  our  readers  are  wondering 
how  we  can  speak  so  confidently  about  our  ability  to  classify  all  of 
our  cases  as  either  identicals  or  fraternals.  It  may  be  said  that  the 
method  was  slow  in  taking  shape  and  was  arrived  at  only  after 
intensive  study  of  the  materials. 

DIAGNOSIS  OF  MONOZYGOTIC  TWINS. 

The  majority  of  workers  on  human  twins  seem  to  have  despaired 
of  arriving  at  an  adequate  classification  of  twins  into  clean-cut 
categories :  monozygotic  and  dizygotic.  Years  ago  Thorndike 
found  so  much  difficulty  with  his  cases  that  he  came  to  the  con- 
clusion that  all  twins  belong  to  a  single  series  and  have  a  similar 
origin.  Lauterbach,  1925,  after  the  study  of  nearly  two  hundred 
pairs  of  twins,  found  himself  unable  to  separate  the  same-sexed 
pairs  with  any  assurance.  He  tentatively  classified  59  per  cent, 
of  the  same-sexed  twins  as  monozygotic,  a  percentage  much  too 


STUDIES    OF    HUMAN    TWINS.  287 

high,  suggesting  that  he  has  included  a  good  many  cases  of  similar 
fraternal  twins  in  his  "  identical  "  group. 

The  most  recent  study  of  twins  is  that  of  A.  H.  Wingfield 
(1928)  who  studied  one  hundred  two  pairs  of  twins  selected  at 
random  from  the  public  schools  of  Toronto  and  Hamilton,  On- 
tario. Taking  all  pairs  of  twins  as  they  came  there  were  accumu- 
lated seventy-six  like-sexed  pairs  and  twenty-six  unlike-sexed 
pairs.  The  expectation  would  be  about  65  per  cent,  of  like-sexed 
twins  instead  of  about  74  per  cent.,  the  number  found  in  this  col- 
lection. It  seems  probable,  therefore,  that  some  unlike-sexed  twins 
were  overlooked.  Wingfield  made  an  attempt  to  separate  the 
seventy-six  like-sexed  pairs  into  two  groups,  identicals  and  fra- 
ternals.  His  method  was  somewhat  precarious.  He  classed  as 
"  identical  "  all  those  which  seemed  to  himself  and  the  teacher  to 
have  a  higher  degree  of  physical  identity  than  siblings  are  likely  to 
exhibit.  "  Only  those  pairs  of  twins  showing  practically  indis- 
tinguishable physical  traits,  as  judged  by  the  teachers  in  the  school 
and  myself,  were  included  in  the  identical  group.  While  it  is 
not  absolutely  certain  that  all  pairs  included  in  the  identical  group 
had  identical  heredity,  the  chances  in  favor  of  this  being  the 
case  are  very  great."  The  fact  that  he  classed  as  identical  over 
44  per  cent,  of  all  the  twins  in  his  group  is  surprising  in  view  of 
the  fact  that  the  statistical  expectation  is  only  about  26  per  cent. 
It  seems  probable  then  that  Wingfield  has  included  among  the 
"  identicals "  several  cases  of  similar  fraternal  twins.  This  is 
further  suggested  by  the  fact  that  he  found  a  coefficient  of  corre- 
lation of  only  about  -j-  0.90  for  this  group  as  compared  with 
-)-  0.95  obtained  for  our  identicals. 

That  it  is  possible  to  develop  a  method  of  distinguishing  be- 
tween identical  and  fraternal  twins  is  strongly  suggested  by  the 
fact  that  two  European  twin  specialists  claim  to  be  able  to  make 
such  a  distinction  with  a  high  degree  of  infallibility. 

Dahlberg  (1926),  in  his  monograph  on  ;  Twin  Births  and 
Twins  from  a  Hereditary  Point  of  View,"  makes  this  statement: 
'  The  following  demands  should  be  satisfied  for  a  diagnosis  of 
monozygotism  for  a  grown-up  pair  of  twins : 

"  i.  That  the  appearance  of  the  twins  give  an  impression  of 
very  great  resemblance  or  identity. 


288  H-    H.    NEWMAN. 

"  2.  That  during  childhood,  neighbors,  school-fellows,  etc., 
have  had  difficulties  in  distinguishing  them  and  have  sometimes 
confused  them. 

"  3.  That  the  configuration  of  the  ears  does  not  show  great 
dissimilarity. 

"  4.  That  the  finger  prints  show  a  certain  high  degree  of  simi- 
larity. 

'  5.  That  the  anthropological  measurements  do  not  show  too 
considerable  differences." 

Siemen's  method  (1927)  is  somewhat  more  detailed  and  exact- 
ing. He  takes  the  very  sensible  view  that  no  single  criterion  of 
monozygotic  origin  is  reliable,  but  that  judgment  in  doubtful  cases 
should  be  based  upon  identity  in  as  many  traits  as  possible.  He 
emphasizes  the  rarity  of  really  questionable  cases.  Many  years 
of  experience  in  the  study  of  twins  has  developed  in  him  such  a 
degree  of  confidence  in  his  method  of  diagnosis  that  he  consid- 
ers that  he  has  been  able  to  reach  "  a  certain  diagnosis  in  virtually 
every  case  of  twinning." 

He  finds,  as  others  have  found  before  and  since,  that  the  great 
majority  of  all  twins  are  either  so  completely  alike  or  so  markedly 
different  that  there  is  no  question  about  their  diagnosis.  A  care- 
ful study  of  the  certain  cases  should  furnish  criteria  for  diag- 
nosing the  few  doubtful  cases.  Thus  a  study  of  over  a  hundred 
pairs  of  unquestionable  identical  twins  has  resulted  in  the  fol- 
lowing "  scheme  "  for  diagnosing  monozygosity : 

A.  Traits  in  which  one-egg  twins  practically  always  agree  and  in 
which  two-egg  twins  agree  only  very  rarely: 

1.  Hair  color  and  form. 

2.  Eye  color. 

3.  Skin  color. 

4.  Downy  hair  of  the  body. 

B  Traits  in  which  one-egg  twins  differ  only  within  narrow  limits 
and  in  which  two-egg  twins  usually  differ  more 
widely. 

5.  Freckles. 

6.  Appearance  of  blood  in  the  skin. 

7.  Follicular  processes. 

8.  Tongue  (furrowed  or  not)  and  teeth. 


STUDIES   OF    HUMAN    TWINS.  289 

C.  Traits    in   which   one-egg  twins    usually,    and   two-egg   twins 

rarely  show  strong  resemblance : 
9.  Form  of  face. 

10.  Form  of  ears. 

11.  Form  of  hands. 

12.  Body  build. 

13.  Mentality. 

14.  Illness  and  abnormality. 

15.  Traits  studied  by  special  methods — finger  prints,  etc. 

Our  own  method  of  diagnosis  has  been  considerably  influenced 
by  the  methods  of  Dahlberg  and  of  Siemens,  especially  by  the 
latter,  but  is  somewhat  different  from  any  previously  used.  Our 
effort  has  been  to  combine  the  best  features  of  all  known  methods. 

After  our  own  method  was  developed  and  while  reading 
Wingfield's  monograph,  the  writer  noted  a  reference  to  a  short 
note  in  Science  by  Taku  Komai  (1927)  entitled  "A  Criterion 
for  Distinguishing  Identical  Twins  from  Fraternal  Twins."  The 
criterion  described  has  to  do  with  finger  prints  and  palm  and 
sole  prints  of  twins.  "  Generally  speaking,"  he  says,  "  the  same 
hands  or  feet  of  the  identical  twins  resemble  each  other  more 
closely  in  their  patterns  than  the  two  hands  or  feet  of  the  same 
individual."  This  I  have  found  to  be  very  frequently  true,  but 
the  formula  needs  modification,  as  will  be  shown  below. 

OUR  OWN  METHOD  OF  DIAGNOSIS. 

The  method  of  identifying  monozygotic  twins  used  in  the  pres- 
ent work  may  now  be  described  in  detail.  A  great  deal  of  atten- 
rion  has  been  given  to  this  matter,  for  we  realize  that  the  sound- 
ness of  our  conclusions  as  to  heredity  and  environment  depend 
upon  the  correctness  of  this  diagnosis. 

At  the  beginning,  it  may  be  said  that  in  over  90  per  cent,  of 
our  cases  there  was  at  no  time  any  doubt  as  to  their  classifica- 
tion. The  great  majority  of  one  type  of  twins  are  so  strikingly 
similar  that  their  monozygotic  origin  is  obvious.  Their  resemb- 
lance is  not  confined  to  gross  physical  correspondence,  but 
extends  to  tones  of  voice,  gestures,  and  peculiar  mannerisms. 
One  soon  becomes  sensitized  to  the  intangible  correspondences  of 


290 


H.    H.    NEWMAN. 


identical  twins  and  diagnoses  them  almost  at  a  glance.  The  great 
majority  of  the  other  type  of  twins  strike  one  at  once  as  en- 
tirely unlike,  often  being  more  different  than  average  brothers 
or  sisters.  About  these  there  is  no  question  after  the  first  glance. 
Our  ability  to  diagnose  cases  improved  during  the  course  of  our 
study  and  we  found  that  there  was  no  difficulty  at  all  in  diag- 
nosing the  last  half  of  the  pairs  that  presented  themselves.  Two 
of  the  very  early  pairs  were  diagnosed  doubtfully  that,  when 
reexamined  after  a  year  of  experience,  offered  no  difficulty  at 
all.  Two  other  cases  were  left  uncertain  because  we  allowed 
ourselves  to  be  influenced  by  statements  of  the  mother.  About 
these  cases  there  should  never  have  been  any  question  had  the 
mother  not  been  loquacious. 

Out  of  one  hundred  two  pairs  of  twins  there  was  justifiable 
doubt  about  only  six  cases.  These  cases  have  all  been  diagnosed 
satisfactorily  with  the  possible  exception  of  No.  61,  which  still 
remains  slightly  uncertain. 

The  following  are  our  criteria  for  diagnosing  identical  (mono- 
zygotic)  twins. 

1.  They  must  be  strikingly  similar  in  general  appearance  in- 
cluding various  intangible  resemblances. 

2.  They  must  be  essentially  identical  in  hair  color,  texture  and 
form. 

3.  They  must  have  the  same  shade  of  eye  color  and  form  of 
iris. 

4.  They   must   have   the   same   skin   color   and   texture    (com- 
plexion) except  when  one  is  more  tanned  than  the  other. 

5.  They  must  have  no  marked  differences  in   features ;  shape 
of  ears ;  shape,  size  and  arrangement  of  teeth. 

6.  They  must  have  hands  of  the  same  type  and  nearly  equal 
in  size. 

7.  The  general  microscopic  character   of   the   papillary   ridges 
in  fingers  and  palms  must  be  essentially  the  same. 

8.  There  must  be  stronger  cross  resemblance  than  internal  re- 
semblance  in  one  or  more  of  the  details  of  finger  and  palm  pat- 
terns. 

9.  The  presence  of  reversed  asymmetry  in  handedness  or  hair 
whorl  in  one  twin  is  confirmatory  evidence  of  monozygosity,  but 


STUDIES   OF    HUMAN    TWINS. 

its  occasional  presence  in  unlike  twins  is  not  to  be  'taken  as  an 
indication  of  monozygosity. 

A  great  deal  of  stress  has  been  laid  upon  the  diagnostic  value 
of  the  palm  and  ringer  patterns.  While  this  criterion  alone  is 
inadequate  for  certain  diagnosis,  it  is  surprising  how  few  mis- 
takes were  made  in  our  effort  to  diagnose  monozygosity  on  this 
basis  alone.  In  the  first  forty-two  cases  in  which  a  judgment 
was  attempted  on  the  basis  of  palm  and  finger  prints  alone,  there 
was  disagreement  in  only  two  cases  with  the  judgment  based  on 
general  resemblance.  Our  method  has  been  to  classify  all  cases 
on  the  basis  of  the  first  six  criteria  and  then  to  check  this  classifi- 
cation by  criteria  7  and  8. 

PALM  AND  FINGER  PRINTS  AS  CRITERIA. 

The  intensive  study  of  palm  and  finger  patterns  is  perhaps  the 
best  single  diagnostic  aid.  After  a  scrutiny  of  the  first  thirty  or 
forty  sets  of  palm  prints  the  writer  began  to  notice  an  important 
fact  about  the  palm  and  finger  patterns  of  strikingly  identical 
twins :  namely,  that,  instead  of  showing  mirror-imaging  of  pat- 
terns (involving  the  resemblance  of  the  right  hand  of  one  to  the 
left  hand  of  the  other)  the  two  hands  of  one  of  the  twins  were 
direct  duplicates  in  major  features  of  the  two  hands  of  the  other. 
Specifically,  the  right  hand  of  one  twin  is  more  like  the  right 
hand  of  the  other  than  like  own  left  hand,  and  the  left  hand  of 
one  twin  is  more  like  left  hand  of  other  than  like  own  right  hand. 
Thus  cross  resemblance  between  the  two  twin  individuals  is 
stronger  than  resemblance  between  the  two  hands  of  the  same 
individual. 

Among  twins  that  are  somewhat  less  alike  the  same  rule  holds 
in  a  somewhat  modified  form.  Thus  right  hand  of  one  twin  may 
be  like  right  of  the  other,  or  left  of  one  like  left  of  the  other, 
but  the  close  resemblance  does  not  extend  to  both  sides.  In  still 
other  pairs  of  twins  in  which  one  is  distinctly  left-handed,  there 
is  a  reversal  of  asymmetry,  so  that  the  right  hand  of  each  twin 
is  like  the  left  hand  of  the  other.  In  every  pair  of  obviously 
monozygotic  twins  the  rule  holds  that  there  is  stronger  cross  re- 
semblance betzuecn  the  hands  of  one  twin  and  those  of  the  other 
than  between  the  two  hands  of  the  same  individual.  The  same 
20 


292 


H.    H.    NEWMAN. 


is  true  of  ears,  teeth,  and  other  structures  that  show  more  or 
Jess  asymmetry,  but  there  is  more  detail  in  palm  and  finger  prints 
and  a  more  objective  method  of  comparing  them.  In  the  case 
of  the  fingers  the  types  of  patterns  have  been  formulated  in  all 
cases  in  order  to  obtain  a  qualitative  basis  of  comparison,  and  the 
friction  ridges  in  all  patterns  (following  the  method  of  Bon- 
navie,  somewhat  modified)  were  counted  under  binocular  so  that 
a  quantitative  comparison  between  the  fingers  of  one  hand  and 
those  of  another  is  possible.  In  both  qualitative  and  quantita- 
tive respects  the  rule  that  cross  resemblance  is  stronger  than  in- 
ternal resemblance  holds,  for  identical  twins. 

The  studies  of  palm  main  line  formulae  and  of  the  occurrence 
and  varied  expression  of  the  six  fundamental  primitive  patterns 
have  been  greatly  facilitated  by  the  study  of  a  paper  now  in  • 
manuscript,  the  work  of  a  considerable  group  of  experts,  entitled 
"  A  Study  of  Error  in  the  Interpretation  and  Formulation  of 
Palmar  Dermatoglyphies,"  by  Cummings,  Keith,  Midlo,  Mont- 
gomery, H.  H.  Wilder  and  I.  W.  Wilder.  Professor  Cummings, 
evidently  the  guiding  spirit  of  the  group  in  this  collaborative 
inquiry,  has  very  kindly  furnished  me  with  a  copy  of  the  manu- 
script and  has  thus  made  it  possible  for  me  to  study  the  palms  of 
our  twins  with  far  greater  efficiency  than  would  have  been  pos- 
sible without  this  assistance. 

With  few  exceptions  the  same  rules  of  cross  resemblance  apply 
to  the  palmar  main  lines  and  patterns  that  apply  to  finger  prints. 
Most  frequently  the  cross  resemblance  runs  similarly  in  all  four 
respects:  in  qualitative  characters  of  finger  patterns,  in  quanti- 
tative values  of  finger  patterns,  in  palmar  main  line  formulae,  and 
in  the  occurrence  of  palmar  patterns.  Sometimes  the  cross  re- 
semblance is  obvious  in  only  three  of  four  respects,  sometimes  in 
two,  or  only  one ;  but  if  it  is  greater  between  one  hand  of  one 
twin  and  either  the  same  or  opposite  hand  of  the  other  twin  than  in 
own  hands,  the  rule  is  considered  to  hold  good. 

While  it  is  of  importance  that  the  detailed  analysis  of  the 
finger  and  palm  characters  of  this  collection  of  twins  should  be 
published,  this  is  hardly  the  appropriate  place  for  it.  One  or 
iwo  separate  papers  devoted  to  a  special  presentation  and  analysis 
of  these  data  are  planned  for  subsequent  publication. 


STUDIES    OF    HUMAN    TWINS.  2Q3 

At  this  time  we  must  ask  the  indulgent  reader  to  accept  ten- 
tatively our  criteria  for  diagnosing  twins.  With  the  publication 
of  the  complete  data  used  in  this  diagnosis  the  methods  used  may 
be  put  to  any  test  that  seems  necessary. 

Applying  the  criteria  of  diagnosis  above  described  to  the  six 
pairs  of  twins  about  which  there  was  some  doubt,  three  of  them 
fell  readily  into  the  category  of  identicals  and  three  were  classi- 
fied as  similar  fraternals.  At  the  present  time  the  writer  feels 
quite  confident  as  to  the  correctness  of  diagnosis  of  the  whole 
collection.  The  cases  'that  might  be  questioned  by  some  are  the 
three  cases  of  similar  fraternals  just  referred  to.  Before  dis- 
cussing the  problems  arising  out  of  a  study  of  identical  twins, 
it  seems  advisable  to  devote  a  few  paragraphs  to  the  fraternal 
twins,  especially  to  the  three  cases  most  difficult  to  diagnose. 

THE  DIAGNOSIS  OF  FRATERNAL  TWINS. 

Of  the  fifty-two  pairs  of  fraternal  twins  in  our  collection,  three 
may  be  classed  as  "  similar  fraternals,"  and  twenty  as  "  slightly 
similar  fraternals,"  and  twenty-nine  as  ;'  unlike  fraternals." 
None  of  the  pairs  show  as  much  resemblance  as  the  least  similar 
of  the  identical  twins.  The  only  cases  that  could  possibly  be  at  all 
in  question  as  to  their  classification  are  the  three  "  similar  "  pairs, 
numbered  61,  15,  and  74.  Let  us  carefully  scrutinize  these  rather 
crucial  cases  as  to  the  possibility  that  they  might  be  monozygotic 
twins  of  the  less  nearly  identical  sort. 

Pair  61. — These  girls  at  first  impressed  us  with  their  similar- 
ity. They  were  dressed  exactly  alike,  arranged  their  hair  alike 
and  had  very  similar  coloring.  In  height  there  was  but  three 
eights  of  an  inch  difference;  there  were  two  and  three  fourths 
pounds  difference  in  weight.  Head  length  of  A  was  13.95  mm., 
of  B  14.35  mm.;  head  width  of  A  was  17.7  mm.,  that  of  B  was 
17.9  mm.  The  hair  of  both  was  in  general  rather  similar,  but 
that  of  B  was  a  shade  darker,  softer,  finer  and  not  so  heavy. 
Eye  color  was  the  same  in  both,  a  type  of  hazel.  There  was  no 
difference  in  skin  color.  Ears  of  A  were  higher  and  narrower 
than  those  of  B,  and  had  a  shorter  lower  lobe.  A  has  fuller  lips ; 
B  has  the  longer,  more  prominent  chin.  A  holds  eyes  wide  open ; 
B  has  them  nearly  half  closed.  Bridge  of  A's  nose  more  bowed 


294 


H.    H.    NEWMAN. 


than  that  of  B.  The  teeth  of  the  two  differ  rather  sharply,  the 
upper  arch  of  B  being  narrower  and  the  teeth  crowded  and  ir- 
regular, while  those  of  A  are  regular. 

The  finger  print  formulae  are  decidedly  different : 

Left  Hands.  Right  Hands. 

I,  2,  3,  4,  5  i.  2-  3,  4,  5 

A— U,  R,  A,  W,  W  A— U,  U,  U,  U,  U 

B-W,  U,  R,  U,  U  B— W,  R,  U,  U,  U 

The  quantitative  values  of  the  finger  prints  are : 

A — right  hand  24  A — left    hand    28 

B— right  hand  38  B— left    hand    25 

All  four  palm  main  line  formulae  are  different  and  the  patterns  are 
also  different. 

Left  Hands.  Right  Hands. 

A— (9.8.5".5')  B.O.O.O.O.  A— (11.9.7  -5.')  B.O.O.L.O. 

B— (9.8.s".3  )  A.0.0.0.0.  B— (  9.7.5" -3  )  C.O.O.O.O. 

Both  are  equally  right-handed  and  both  have  clockwise  hair- whorl. 

In  spite  of  a  superficial  rather  close  resemblance,  then,  there  is 
no  indication  that  these  twins  have  had  a  monozygotic  origin.  This 
was  the  most  difficult  case  to  diagnose,  but  there  seems  now  no 
doubt  that  these  twins  are  dizygotic  in  origin. 

Pair  65. — This  case  was  somewhat  puzzling  because  the  two 
girls  are  both  rather  peculiar  in  appearance  and  are  similar  in 
many  peculiarities. 

In  height  A  is  57^2  inches,  B  56%  inches.  In  weight,  A  is 
II31/4  pounds,  B  is  m1^  pounds.  Head  length  of  A  is  14.5  mm.; 
that  of  B  is  14.4  mm.;  head  width  of  A  is  17.7  mm.;  that  of  B  is 
17.1  mm.  Hair  of  both  is  the  same  in  color  and  texture;  eye 
color  of  both  is  of  the  same  shade  of  blue;  B  has  a  lower  brow 
and  a  sullen  expression  about  the  eyes,  while  A  has  a  contented 
expression.  The  skin  is  somewhat  more  florid  in  B.  The  ears  of 
the  two  differ  greatly,  B  having  much  longer  lower  lobe.  The 
hands  differ  in  shape,  those  of  A  being  broader  and  thicker.  B 
has  shorter,  more  turned-up  nose,  a  distinctly  wider  mouth,  fuller 
lips  and  fatter  face.  The  teeth  differ  radically,  the  upper  arch  of 
B  being  wider  and  straighter  across  the  front  and  with  wider 
teeth. 

The  finger  print  formulae  read  as  follows : 


STUDIES   OF    HUMAN    TWINS. 


295 


Left  Hands. 

i,     2,      3,    4,     5 
A— W,  R,    W,  W,  U 
B— W,W,    U,  W,  U 


Right  Hands. 
I,      2,      3,     4,     5 
A— W,  W,  W,  W,  U 
B— W,  W,  W,  W,  U 


The  quantitative  values  of  the  finger  prints  are : 


A — right  hand  53 
B — right  hand  52 

The  palm  formulae  are  as  follows : 

Left  Hands. 

A— (11.7.7.  3  )        A.O.O.O.D. 
B— (  g.8.5".5')  A/B.O.O.O.D. 


A — left    hand    54 
B— left    hand    60 


Right  Hands. 
-(11.9.75')  A.O.M.O.D. 


B— (11.8.7.5')  O.O.M.L.O. 

In  several  respects  there  is  a  little  more  resemblance  between  right 
palm  and  fingers  of  the  two  than  to  their  respective  lefts,  but  this 
does  not  extend  to  details.  On  the  whole  these  'two  girls  make  an 
entirely  different  impression.  One  has  a  rather  pleasing,  happy 
expression,  the  other  a  sullen,  lowering  expression.  The  fact  that 
B  is  ambidextrous  in  both  finger  and  wrist  tapping  suggests  that 
she  might  be  the  left-hand  component  of  a  monozygotic  twin  pair, 
but  there  are  too  many  differences  between  them  to  permit  such  a 
diagnosis. 

Case  24- — These  girls  have  many  traits  in  common,  but  show 
also  some  extreme  differences.  A's  height  is  59  inches ;  B's  is 
53^4  inches.  A's  weight  was  70^2  pounds;  B's  66^/2  pounds.  A's 
head  width  is  14.1  mm.;  B's  13.5  mm.  A's  head  length  is  17.6 
mm.;  B's  is  17.1  m.m  Hair  color,  texture  and  crown  whorl  same 
in  both.  Eye  color  of  both  a  gray  brown,  but  A's  eyes  are  dis- 
tinctly grayer  and  B's  browner.  B's  ears  are  distinctly  larger  and 
wider  although  her  head  is  considerably  smaller.  A's  eyes  are 
wider  spaced  than  B's.  A's  nose  is  larger,  longer  and  different 
in  shape.  B's  teeth  are  crowded  and  overlap  in  front,  while  A's 
are  straight. 

Finger  print  formulae : 


Left  Hands. 

i,     2,    3,    4,    5 

A— W,  R,  R,  U,  U  : 

B— U,  R,  U,  U,  U 

Quantitative  values  of  finger  patterns 

A — right    hand    44 
B — right    hand    27 


Right  Hands. 

A— W,  R,  U,  U,  U 
B—  U,  A,  A,  U,  U 


A — left    hand    30 
B— left    hand    26 


296  n-    n-    NEWMAN. 

Palm  formulae: 

Left  Hands  Right  Hands. 

A— (n.77-3)  O.O.O.O.D.  A— (11.9.7-3)  O.O.O.L.D. 

B— (11.7.7.3)  O.O.O.O.O.  B— (11.7.7.3)  A.O.O.O.O. 

Here  again  the  palm  formulae  suggests  a  closer  resemblance  than 
actually  exists,  in  that  we  have  the  same  pattern  for  the  two  left 
hands,  but  the  two  palms  of  B  also  have  the  same  pattern  and 
are  far  more  similar  in  detail.  Nowhere  is  there  stronger  cross 
resemblance  than  internal  resemblance.  On  the  whole  there  can 
be  no  doubt  that  these  are  fraternal  twins. 

Apart  from  these  three  cases  there  are  no  decidedly  similar 
twins  among  the  fifty-two  pairs  in  our  collection.  Twenty  pairs 
are  designated  as  "  slightly  similar  "'  fraternal  twins  and  the  re- 
maining twenty-nine  cases  are  designated  as  "  unlike  "  fraternal 
twins.  The  slightly  similar  fraternals  show  merely  the  degree 
of  resemblance  common  among  siblings,  while  the  unlike  frater- 
nals seem  to  show  hardly  as  much  resemblance  as  do  average 
siblings.  Even  the  three  cases  of  decidedly  similar  fraternals, 
except  for  their  identity  in  age,  are  no  more  alike  than  are  occa- 
sional siblings.  On  the  whole  then,  there  seems  to  be  nothing 
about  these  fifty-two  cases  out  of  accord  with  their  classification 
as  fraternal,  or  dizygotic,  twins.  Hence  there  is  now  no  ground 
for  doubting  the  validity  of  our  classification  of  the  one  hundred 
two  pairs  of  twins  used  in  this  study,  into  the  two  categories, 
monozygotic  and  dizygotic. 

SUMMARY. 

1.  The  original  objective  of  these  studies  was  the  study  of  the 
roles  of  heredity  and  environment  in  determining  mental  capaci- 
ties of  various  sorts. 

2.  The  first  essential  was  to  learn  how  to  diagnose  with  cer- 
tainty the  two  types  of  twins,  monozygotic  and  dizygotic. 

3.  Only  about  25  per  cent,  of  all  twins  are  monozygotic.     Col- 
lections that  depart  widely  from  this  figure  have  probably  been 
incorrectly  diagnosed. 

4.  Only  about  42  per  cent,  of  same-sexed  twins  are  monozy- 
gotic. 

5.  The  method  of  diagnosis  used  in  this  study  combines  the  best 


STUDIES   OF    HUMAN    TWINS.  2Q7 

features  of  the  methods  of  Dahlberg,  Siemens,  and  Komai.  Cer- 
tain refinements  of  technique  are  added,  the  details  of  which  are 
explained  in  the  text. 

6.  Out  of  a  collection  of  one  hundred  two  pairs  of  same-sexed 
twins,  only  six  pairs  caused  any  difficulty,  three  of  which  are  now 
classified  as  monozygotic  and  three  as  dizygotic. 

7.  The  details  concerning  the  three  "  similar   f  raternals  "   are 
presented  and  the  reasons  for  their  diagnosis  as  dizygotic  twins 
are  given. 

8.  The  result  is  that  we  have  now  a  collection  of  fifty  pairs  of 
monozygotic  and  fifty-two  pairs   of   dizygotic  same-sexed  twins 
accurately  diagnosed.    These  are  to  be  used  for  further  biological 
and  psychological  study. 

BIBLIOGRAPHY. 

1.  Bonnevie,  K. 

'24     Studies  of  Papillary  Patterns  of  Human  Fingers.     Jour.  Genetics, 
Vol.  XV.,  No.  i. 

2.  Dahlberg,  G. 

'26     Twin  Births  and  Twins  from  a  Hereditary  Point  of  View.     Stock- 
holm. 

3.  Knibbs,  G. 

'28     Multiple   Births,   their    Characteristics    and    Laws    Mathematically 
Considered.    Jour,  and  Proc.  Roy.  Soc.,  N.  S.  Wales,  Vol.  59. 

4.  Komai,  T. 

'27     A    Criterion    for    Distinguishing    Identical    Twins.      Science,    Vol. 
LXV.,  No.  1681. 

5.  Lauterbach,  C.  E. 

'25     Studies  in  Twin  Resemblance.     Genetics,  Vol.  X.,  No.  6. 

6.  Newman,  H.  H. 

'15     The  Biology  of  Twins,  Chicago,  1915.     The  Physiology  of  Twin- 
ning.    Chicago,  1923. 

7.  Siemens,  H.  W. 

'27     The  Diagnosis  of  Identity  in  Twins.     Jour,  of  Heredity,  Vol.   18, 
No.  5. 

8.  Thorndyke,  E.  L. 

'05     Measurement  of  Twins,  Archives  of  Philos.,  Psychol.  and  Scientific 
Methods,  No.  I. 

9.  Weinberg,  W. 

'02     Probleme    der    Mehrlingsgehurtenstatistik.      Zeitschr.    f.    (it-nurtsh. 

u.  Gynak.,  Bd.  47. 
10.  Wingfield,  A.  H. 

'28     Twins  and  Orphans.    The  Inheritance  of  Intelligence.    London  and 

Toronto. 


STUDIES  OF  HUMAN  TWINS. 

II.    ASYMMETRY  REVERSAL,  OF  MIRROR  IMAGING  IN  IDENTICAL 

TWINS. 

H.  H.  NEWMAN, 

INTRODUCTION. 

One  of  the  most  striking  phenomena  observed  among  monozy- 
gotic  'twins  is  that  of  the  reversal  of  asymmetry  between  the  in- 
dividuals of  a  pair.  Among  armadillo  quadruplets  the  present 
writer  (Newman,  1916)  found  numerous  instances  in  which  a 
band  or  scute  doubling  occurred  on  the  left  side  of  one  twin  and 
on  the  right  side  of  the  other.  Such  cases  call  to  mind  the  fact 
'that  in  human  double  monsters  (Siamese  twins)  situs  inversus 
viscerum  occurs  in  many  cases.  The  same  type  of  asymmetry 
reversal  was  noted  by  Swett  and  by  Morrill  in  double-headed 
fish  embryos.  In  separate  identical  twins  in  man  it  has  been 
noted  that  the  incidence  of  left-handedness  in  one  twin  of  a  pair 
is  very  much  greater  than  among  fraternal  twins  or  in  the  gen- 
eral population  of  single  individuals.  Asymmetry  reversal  in  the 
direction  of  crown  whorl  of  the  head  hair  seems  to  have  about 
the  same  incidence  in  monozygotic  twins,  dizygotic  twins,  and 
single  individuals  as  has  left-handedness.  These  two  expressions 
of  asymmetry  have  been  studied  intensively  in  the  present  in- 
vestigation and  their  significance  will  be  discussed  in  some  detail 
later. 

HANDEDNESS  AN  EXPRESSION  OF  ASYMMETRY. 

As  an  introduction  to  this  study  it  seems  well  to  examine  the 
phenomena  of  handedness  as  it  is  found  among  human  beings. 
In  the  first  place,  there  are  two  distinct  kinds  of  handedness: 
that  which  is  genetically  determined  and  that  which  is  the  result 
of  twinning  and  therefore  epigenetically  determined. 

Genetic  handedness  is  evidently  transmitted  in  such  a  way  that 
any  given  zygote  will  give  rise,  when  no  twinning  occurs,  to 
a  right-handed  or  left-handed  single  individual.  There  seems, 
however,  to  be  varying  degrees  of  right-  or  left-handedness. 
The  majority  of  individuals,  apparently  about  eighty  per  cent. 

298 


STUDIES   OF    HUMAN    TWINS.  299 

of  single  individuals,  are  definitely  righ-handed ;  about  four  per 
cent,  definitely  left-handed,  and  the  remaining  sixteen  per  cent. 
partially  left-handed  or  ambidextrous.  The  incidence  of  right- 
and  left-handedness  is  about  what  one  would  expect  if  right- 
handedness  is  a  dominant  Mendelian  unit  character  and  left- 
handedness  recessive.  The  ambidextrous  individuals  and  those 
showing  lesser  degrees  of  left-handedness  may  be  heterozygous 
individuals  in  which  the  dominance  of  right-handedness  is  in- 
complete. 

The  other  type  of  left-handedness,  quite  different  in  origin  and 
heritability,  is  that  which  results  epigenetically  as  the  result  of 
the  twinning.  Such  left-handedness,  being  a  somatic  modifica- 
tion would  not  be  hereditary:  it  would  be  merely  an  expression 
of  asymmetry  reversal  due  to  the  development  of  a  whole  in- 
dividual from  a  half  embryo  which  had  already  become  more  or 
less  differentiated  in  a  left-handed  direction  before  the  separation 
into  twins  has  taken  place. 

Thus  in  genetic  right-handed  embryos  which  undergo  twinning 
after  some  asymmetry  has  been  established,  the  left-hand  half 
embryo  would  be  the  superior  one  and  would  give  rise  to  a  right- 
handed  individual,  since  right-handed  superiority  is  due  to  left- 
sided  superiority  in  the  brain.  Conversely,  in  a  genetic  left- 
handed  embryo,  the  right  side  would  be  superior  and  the  left 
side  the  inferior  side,  in  which  case  the  left-handed  individual 
would  retain  the  genetic  asymmetry  and  the  right-handed  individ- 
ual would  exhibit  asymmetry  reversal. 

In  embryos  genetically  ambidextrous  the  right  and  left  sides 
would  be  equal  and  would  produce  twins  both  of  whom  would 
be  ambidextrous. 

PREVIOUS  DATA  ON  HANDEDNESS  IN  TWINS. 

A  good  deal  of  attention  has  been  paid  by  various  authors  to 
the  peculiar  incidence  of  left-handedness  in  twins.  Siemens 
(1924)  found  in  thirty-seven  pairs  of  identical  twins  twenty-six 
cases  both  right-handed,  ten  cases  in  which  one  was  right-handed 
snd  the  other  left-handed,  and  one  case  where  both  were  left- 
handed.  In  a  later  paper  the  same  writer  reported  on  a  larger 
number  of  identical  twins  (the  total  number  not  given)  in  which 
21 


3QO  H-    H-    NEWMAN. 

there  were  twenty-one  cases  where  one  was  right-  and  the  other 
left-handed  and  three  cases  where  both  were  left-handed. 

Weitz  (1924)  found  among  eighteen  pairs  of  identical  twins, 
seven  pairs  composed  of  a  right-  and  a  left-hander,  ten  pairs  both 
right-handed,  and  one  pair  both  left-handed. 

Dahlberg  (1926)  reports  for  sixty-nine  pairs  of  identicals 
fifty-three  pairs  both  right-handed,  twelve  pairs  one  left-handed, 
and  four  pairs  both  left-handed.  Adding  the  three  sets  of  cases 
together,  -we  have  one  hundred  and  twenty-four  cases  of  iden- 
tical twins  divided  as  follows : 

89  pairs,  biilh  right-handed,   7  I  .X  per  cent. 

29  pairs,  one  left-handed,      23.4  per  cent. 

6  pairs,  both  left-handed,       4.8  per  cent. 

Dahlberg  has  also  studied  the  incidence  of  left-handedness  in  one 
hundred  and  twenty-eight  pairs  of  dizygotic  twins.  The  follow- 
ing figures  indicate  his  results  : 

in   pairs,  both  right-handed,  86.7  per  cent. 

16  pairs,  one  left-handed         12.5  per  cent. 

i  pair,  both  left-handed,          0.8  per  cent. 

It  will  be  seen  that  the  incidence  of  left-handedness  among 
identical  twins  is  over  twice  as  great  as  among  fraternal,  or  four 
times  as  great  in  proportion  to  the  number  of  zygotes  involved, 
for  a  pair  of  identical  twins  involves  only  one  zygote.  Even 
among  fraternal  twins,  the  incidence  of  left-handedness  is  rela- 
tively high  as  compared  with  the  general  population,  which  is 
reported  by  Jones  (1918)  to  be  about  four  per  cent.  Jones'  esti- 
mate, however,  is  probably  much  too  low  and  takes  account  of  only 
the  most  complete  cases  of  left-handedness. 

Lauterbach  (1925)  reports  among  fifty-seven  same-sexed  twins 
(not  distinguished  as  to  monozygotic  or  dizygotic  origin)  twenty 
pairs  in  which  one  was  left-handed,  about  35  per  cent,  of  all  cases. 
This  is  a  higher  incidence  of  left-handedness  than  any  previously 
reported,  especially  when  it  is  taken  into  consideration  that  the 
group  examined  consists  of  both  identical  and  fraternal  twins. 

The  most  recent  data  on  handedness  in  twins  is  furnished  by 
Verschuer  (1927).  He  found  one  or  more  left-handed  individ- 
uals in  26.8  per  cent,  of  seventy-nine  pairs  of  identical  twins  and 


STUDIES    OF    HUMAN    TWINS. 


301 


in  26.3  per  cent,  of  the  thirty-eight  pairs  of  fraternal  twins.    They 
were  distributed  as  follows : 

Identical  Twins.  Fraternal  Twins. 

58  both  right-handed. 


28  both  right-handed. 


one    uSi 


10    one    right-    the    other    left- 
handed. 
o  both  left-handed. 


right-  the  other  left- 
handed. 

5  both  left-handed. 

i  one  right-handed,  the  other 
ambidextrous. 

The  percentage  is  rather  low  as  compared  with  those  of  others, 
particularly  those  of  Lauterbach  and  the  present  writer,  but  the 
difference  is  probably  due  to  the  inclusion  of  only  the  cases  of 
complete  left-handedness.  The  percentage  of  pairs  showing  left- 
handedness  among  fraternal  twins  is  exceptionally  high  and  not 
in  accord  with  the  findings  of  others.  Possibly  the  relatively 
small  number  of  cases  may  be  the  cause  of  this  discrepancy. 
Even  more  probable,  it  seems  to  me,  is  the  inclusion  among 
fraternal  twins  of  a  few  of  the  least  similar  identical  twins  among 
whom  left-handedness  is  common. 

CRITERIA  OF  HANDEDNESS. 

It  is  by  no  means  a  simple  matter  to  diagnose  left-handedness. 
There  are  many  cases,  of  course,  where  the  twins  are  (or  were  at 
an  earlier  period)  obvious  left-handers,  but  there  are  also  many 
cases  where  congenital  left-handedness  is  obscured  by  training 
the  right  hand  and  suppressing  the  left.  Such  cases  often  result 
in  a  sort  of  ambidextrality  in  ordinary  manipulations.  In  our 
work  we  have  used  as  a  test  of  handedness  speed  in  tapping  with 
wrist  and  fingers.  In  all  cases  of  complete  left-handedness  the 
tapping  tests  confirm  the  left-handed  diagnosis.  It  appears  to  be 
safe  then  to  use  the  tapping  tests  to  reveal  native  left-handedness 
obscured  by  right-hand  training  or  various  degrees  of  partial  left- 
handedness. 

A  good  many  cases  of  partial  left-handedness  were  revealed  by 
tapping  tests.  Among  identical  twins,  in  addition  to  the  eleven 
pairs  showing  complete  left-handedness,  there  were  thirteen  pairs 
(both  of  whom  considered  themselves  right-handers)  in  which 
some  degree  of  left-handed  superiority  was  revealed  in  one  or 
both  members  of  the  pair.  In  three  of  these  pairs  both  members 


-Y 

<*{* 
L  I  B  R  A  R  Y  ' C 


-2Q2  H-    H-    NEWMAN. 

were  shown  to  be  partially  left-handed,  and  in  two  pairs  both 
members  were  definitely  ambidextrous. 

Among  fraternal  twins,  in  addition  to  six  pairs  in  which  one 
individual  was  completely  left-handed,  there  were  five  pairs  in 
which  one  individual  was  partially  left-handed,  two  pairs  in 
which  both  were  partially  left-handed,  and  two  pairs  in  which  one 
individual  was  right-handed  and  the  other  ambidextrous. 

Assuming  that  all  these  cases  represent  grades  of  left-handed- 
ness  we  have  added  to  the  seventeen  pairs  showing  complete  left- 
handedness  twenty-two  pairs  showing  partial  left-handedness,  a 
total  of  thirty-nine  out  of  one  hundred  pairs  in  which  some  degree 
of  left-handedness  appears  in  one  or  both  members  of  a  pair-. 
This  high  percentage  would  be  much  like  that  found  by  Lauter- 
bach  (35  per  cent.)  if  we  omitted  the  cases  of  ambidextrality. 

CROWN  WHORL  AN  EXPRESSION  OF  ASYMMETRY. 

As  is  well  known,  the  head  hair  at  the  crown  twists  or  whorls 
in  either  a  clockwise  or  a  counter-clockwise  direction.  The  great 
majority  of  individuals  show  clockwise  hair-whorl,  and  therefore 
clockwise  asymmetry  may  be  considered  as  the  normal  and  coun- 
ter-clockwise asymmetry  as  the  reversed  asymmetry.  Various 
writers  have  called  attention  to  sporadic  instances  of  reversed 
crown  whorl,  and  a  few  cases  have  been  described  for  identical 
twins. 

Only  one  writer,  however,  has  thus  far  made  a  systematic  study 
of  crown  whorl  in  twins.  Lauterbach  (1925)  in  a  study  of  re- 
semblances and  differences  in  twins  has  presented  some  very  in- 
teresting data.  Out  of  fifty-seven  pairs  of  same-sexed  twins 
there  occurred  fifteen  pairs  in  which  one  or  both  twins  showed 
counter-clockwise  hair-whorl.  In  one  of  these  cases  both  twins 
were  counter-clockwise.  This  means  that  about  twenty-six  per 
cent,  of  the  pairs  of  same-sexed  twins  showed  asymmetry  re- 
versal in  hair-whorl.  In  addition  to  these,  there  were  six  cases 
showing  double  crown  in  which  one  half  of  the  whorl  has  a  clock- 
wise and  the  other  a  counter-clockwise  direction.  These  cases 
are  possibly  comparable  to  ambidextrality  in  handedness  and 
should  probably  be  listed  as  a  form  of  asymmetry  reversal.  Add- 
ing these  six  cases,  the  percentage  of  pairs  showing  more  or 
less  reversed  hair-whorl  among  same-sexed  twins  comes  to  nearly 


STUDIES   OF    HUMAN    TWINS.  303 

37  per  cent.,  not  unlike  the  percentage  of  left-handedness  in  the 
same  set  of  twins,  which  was  35  per  cent. 

In  our  own  collection  of  one  hundred  pairs  of  same-sexed 
twins  there  are  in  all  ninety-five  pairs  in  which  it  was  possible 
'to  determine  the  hair-whorl.  In  five  pairs  (three  identicals  and 
two  fraternals)  the  kinky  or  closely  matted  character  of  the  hair 
rendered  diagnosis  of  hair-whorl  extremely  difficult  or  impos- 
sible. Among  the  identicals  there  were  twenty  pairs  showing 
some  form  of  asymmetry  reversal  in  crown-whorl.  In  fifteen 
pairs  one  twin  showed  clockwise  and  the  other  counter-clockwise 
whorl,  in  three  pairs  both  twins  were  counter-clockwise,  and  in 
one  pair  one  twin  had  a  double  crown  and  the  other  a  clockwise 
whorl.  The  remaining  twenty-seven  diagnosed  pairs  showed  clock  •- 
wise  hair  whorl  in  both  twins. 

Among  fraternal  twins  there  were  but  five  pairs  having  any 
form  of  asymmetry  reversal  in  hair-whorl.  In  four  of  these  pairs, 
one  twin  was  counter-clockwise,  and  in  one  pair  one  twin  had  a 
double  crown  and  the  other  a  clockwise  whorl. 

As  in  the  case  of  handedness,  there  are  doubtless  instances  of 
incomplete  asymmetry  reversal  that  are  not  recognizable.  Prob- 
ably some  of  the  crowns  diagnosed  as  slightly  clockwise  or  in- 
definite may  be  cases  of  partial  asymmetry  reversal. 

Crown-whorl  has  one  advantage  over  handedness  as  a  criterion 
of  reversal  of  asymmetry  in  that  it  is  not  subject  to  modification 
by  training  and  is  therefore  a  somewhat  surer  sign  of  asymmetry 
reversal  than  is  left-handedness. 

THE  RELATION  BETWEEN  HANDEDNESS  AND  CROWN  WHORL. 

In  only  ten  pairs  of  our  identical  twins  do  we  find  reversed 
asymmetry  of  any  sort  (either  left-handedness,  counter-clockwise 
hair-whorl,  or  both)  in  both  twins  of  a  pair.  In  eight  of  these 
cases  (73,  25,  23,  87,  43,  38,  7,  27)  both  twins  of  a  pair  are  left- 
handed  or  both  have  counter-clockwise  hair  whorl.  It  would 
seem  natural  to  assume  that  all  such  pairs  have  been  derived 
from  zygotes,  genetically  left-handed.  But  what  shall  we  do 
with  the  other  two  cases  (13  and  72)  in  which  one  twin  of  each 
pair  is  plainly  left-handed  and  the  other  clearly  counter-clock- 
wise in  hair  whorl?  Since  both  of  these  indications  are  valid 


304  H-    H-    NEWMAN. 

criteria  of  reversed  asymmetry  there  seems  no  escape  from  the  con- 
clusion that  these  two  pairs  also  are  derived  from  genetically  left- 
handed  zygotes. 

THE  INCIDENCE  OF  ASYMMETRY  REVERSAL  IN  OUR  OWN  COL- 
LECTION OF  TWINS. 

When  the  present  study  began,  the  writer  was  keenly  on  the 
lookout  for  evidences  of  asymmetry  reversal  in  identical  twins. 
The  expectation  was  that  the  more  strikingly  identical  the  twins 
were,  the  more  evidence  of  asymmetry  reversal  would  be  present. 
Before  the  study  was  half  over  it  seemed  certain  that  this  expecta- 
tion was  not  to  be  realized.  In  fact,  the  very  opposite  of  this 
appeared  to  be  true,  namely,  that  the  least  evidence  of  asym- 
metry reversal  appears  among  those  twins  that  are  practically 
indistinguishable,  while  the  twins  that  are  less  nearly  identical 
show  the  most  evidence  of  reversal  of  asymmetry. 

In  order  to  test  out  this  conception,  the  writer  tried  to  arrange 
the  fifty  pairs  of  identical  twins  in  the  order  of  their  closeness  of 
resemblance,  including  resemblances  in  features,  height,  weight, 
headsize,  finger  prints  and  palm  prints.  After  this  was  done, 
Mrs.  Blythe  Mitchell,  the  one  who  has  had  the  most  intimate  and 
prolonged  acquaintance  with  the  twins,  was  asked  to  rearrange 
the  cases  according  to  her  impression  as  to  the  degrees  of  identity. 
On  the  whole,  there  was  a  very  close  agreement,  no  case  being 
changed  more  than  a  few  places  up  or  down  in  the  series.  Using 
the  photographs,  we  worked  over  all  cases  together  and  arrived 
at  the  arrangement  shown  in  Table  I.,  which  is  not  intended  to 
be  exact,  place  for  place,  but  certainly  represents  a  real  grouping 
in  the  sense  that  the  first  five  pairs  are  more  similar  than  the 
second  five,  the  second  five  than  the  third,  the  ninth  five  than  the 
tenth.  Within  groups  of  five  the  order  might  be  more  or  less 
shifted  as  the  criteria  of  resemblance  are  not  entirely  objective, 
but  depend  to  a  large  extent  upon  one's  judgment  of  degrees  of 
facial  resemblance.  In  the  following  table  asymmetries  in  hand- 
edness,  crown-whorl,  and  head  dimensions  are  given  for  the 
fifty  pairs  arranged  in  fives,  beginning  with  the  most  alike  and 
ending  with  the  least  alike.  In  this  table  R  and  L  indicate  defi- 
nite right-  and  left-handedness,  /  indicates  partial  left-handedness, 


STUDIES    OF    HUMAN    TWINS. 


305 


A  indicates  ambidextrality ;  -(-  indicates  clockwise,  or  the  com- 
mon type  of  hair-whorl;  and  -  -  indicates  counter-clockwise  hair- 
whorl.  Double  hair-whorls  are  indicated  by  (-f-  -). 

TABLE  I. 


t 

, 

T) 

£        • 

r* 

^2 

.2  d 

_d 

1 

Hande 
ness. 

1  o 
££ 

u£ 

J 

0  •£ 
'<s>  S 
pq 

Remarks. 

62 

A 

cf 

R 

+ 

17.4 

15-5 

B 

cf 

R 

+ 

17.2 

15.2 

98 

A 

9 

R 

+ 

17.7 

i3-9 

B 

9 

R 

+ 

17-5 

14.1 

63 

A 

j 

R 

+ 

lS.2 

14-3 

B 

cf 

R 

— 

18.1 

i3-9 

40 

A 

cf 

R 

(+-) 

17.85 

15.0 

B 

cf 

R 

+ 

18.1 

15-5 

3 

A 

cf 

R 

? 

20.0 

14-75 

Negroes.       Crown-whorl 

B 

cf 

R 

? 

19.7 

i4-3 

could  not  be  made  out. 

9 

A 

9 

R 

_ 

17.0 

13.6 

A  shows  partial  asymmetry 

B 

9 

R 

+ 

17.7 

13-7 

reversal  in  crown. 

8o 

A 

9 

R 

+ 

I8.7 

13-5 

B 

9 

R 

+ 

18.1 

13-2 

67 

A 

j 

R 

+ 

18.9 

14.6 

B 

cf 

1 

+ 

18.7 

14.1 

B  left-handed  in  wrist  tap- 

ping. 

55 

A 

cf 

R 

+ 

ig.2 

14.8 

B 

cf 

R 

+ 

19.1 

14.4 

35 

A 

tf 

R 

— 

18.55 

15.0 

A  shows  partial  asymmetry 

B 

cf 

R 

+ 

18.55 

iS-i 

reversal  in  crown. 

96 

A 

cf 

R 

— 

18.10 

13-9 

B 

o* 

R 

+ 

18.15 

13-9 

*73 

A 

9 

1 

— 

17.1 

13-35 

A,  incompletely  reversed 

B 

9 

A 

— 

17-5 

13-5 

crown;     left-handed    in 

finger    tapping. 

B,   completely  reversed  in 

crown;   nearly  ambidex- 

102 

A 

9 

R 

— 

17.8 

15.0 

trous. 

B 

9 

R 

+ 

18.1 

15.0 

*25 

A 

cf 

R 

— 

18.3 

14.2 

B 

cf 

R 

— 

17.9 

13-9 

30 

A 

9 

R 

+ 

18.8 

13-7 

B 

9 

R 

+ 

18.7 

13-55 

306 


H.    H.    NEWMAN. 


TABLE  I.     (Continued.) 


^ 

_g 

T-J 

•s  g 

>    *r 

O  _r* 

•o  -S 

ctf    b/3 

N  "2 

Remarks. 

dj  *Z, 

^ 

f/1 

s  a 

1-1  ^ 

VjH      " 

W    <U 

rt  C 

c 

a 

m 

*23 

A 

9 

A 

+ 

16.2 

14-25 

A  more  left-handed  than  B. 

B 

9 

A 

+ 

16.1 

14.0 

94 

A 

9 

R 

+ 

18.4 

13-5 

B 

9 

L 

+ 

18.15 

13-2 

68 

A 

9 

R 

— 

17-45 

14-25 

B 

9 

R 

+ 

17-5 

14.4 

49 

A 

9 

1 

— 

18.0 

14.6 

. 

B 

9 

R 

+ 

18.2 

14.1 

*I3 

A 

9 

R 

— 

17.0 

14-5 

B 

9 

L 

+ 

17.0 

14.6 

78 

A 

d1 

L 

+ 

18.45 

14.7 

B 

d1 

R 

+ 

18.40 

14.7 

*87 

A 

d1 

A 

— 

19-95 

13-9 

A    more    decidedly    left- 

B 

d1 

A 

+ 

19.7 

14.0 

handed. 

*43 

A 

d1 

1 



19.6 

15-7 

Bats  left-handed  naturally. 

B 

cT 

1 

+ 

19.1 

15-3 

Left-handed  in  finger  tap- 

ping. 

*38 

A 

9 

1 

— 

19.4 

15-5 

Left    in    both    wrist    and 

finger  tapping. 

B 

9 

1 

— 

19.1 

15-5 

Left  in  finger  tapping  only. 

79 

A 

d1 

R 

+ 

17-35 

13-9 

B 

d1 

L 

+ 

18.1 

13-3 

*  72 

A 

d1 

L 

+ 

19-15 

14-5 

B 

d1 

R 

— 

19-25 

15.0 

99 

A 

d1 

R 

+ 

18.2 

14-5 

B 

d1 

R 

+ 

18.25 

14-5 

33 

A 

d1 

L 

+ 

18.05 

14.9 

B 

d1 

R 

+ 

18.05 

15-25 

53 

A 

d1 

R 

+ 

19.0 

13-9 

B 

d1 

L 

+ 

18.8 

13-4 

44 

A 

d1 

L 

+ 

18.5 

14-3 

B 

d1 

R 

+ 

19-5 

13-9 

2 

A 

9 

L 

? 

17-45 

14-45 

B 

9 

R 

? 

17-95 

14-35 

91 

A 

9 

R 

+ 

18.0 

13-8 

B 

9 

R 

+ 

17.9 

13-9 

IOO 

A 

d1 

R 

+ 

17-65 

13-6 

B 

d" 

R 

+ 

17.2 

14.0 

STUDIES   OF    HUMAN    TWINS. 


307 


TABLE  I.      (Continued.} 


-a 

. 

'C  ° 

'^ 

* 

4i  °5 

TJ    to 

c   u 

H 

v  c 

N  ii 

Remarks. 

* 

H 

rt    C 

£ 

o£ 

a  5 
^ 

55  a 

pq 

101 

A 

d1 

R 

+ 

18.6 

15-5 

B 

d1 

L 

+ 

18.9 

iS-5 

70 

A 

d1 

R 

+ 

18.5 

15-6 

B 

d1 

L 

+ 

18.1 

15-3 

37 

A 

d1 

R 

+ 

19-15 

14-75 

B 

d1 

1 

+ 

19-35 

14.65 

Slightly    left-handed    in 

wrist  tapping. 

34 

A 

d1 

R 

+ 

iS.8 

14.8 

Inclined    to    be    ambidex- 

trous. 

B 

d1 

1 

+ 

19.0 

14.8 

Slightly    left-handed    in 

finger  tapping. 

28 

A 

9 

R 

— 

16.8 

13-8 

B 

9 

R 

+ 

17.7 

13-9 

*7 

A 

d1 

1 

_ 

17-3 

iS-7 

Strongly  left-handed  in 

finger  tapping. 

B 

d1 

1 

+ 

17.6 

iS-5 

Strongly   left-handed  in 

finger  tapping. 

6 

A 

9 

R 

+ 

17.5 

13-9 

B 

9 

R 

+ 

17.2 

13-7 

97 

A 

9 

R 

+ 

17-85 

16.0 

B 

9 

R 

+ 

18.0 

16.0 

17 

A 

9 

R 

+ 

17-85 

14-45 

B 

9 

R 

+ 

17-75 

14.65 

14 

A 

9 

R 

— 

17.2 

14-45 

B 

9 

R 

+ 

17-45 

14-45 

15 

A 

d1 

R 

+ 

19.65 

14-6 

Both  ambidextrous  as 

B 

d1 

R 

— 

19-45 

14.9 

babies. 

69 

A 

d1 

R 

+ 

18-75 

14.9 

B 

d1 

R 

+ 

18.9 

14-3 

24 

A 

d1 

R 

4- 

17-45 

14.65 

B 

d1 

1 

+ 

17.4 

14.65 

Slightly    left-handed    in 

playing  marbles. 

18 

A 

d1 

R 

+ 

18.6 

13-8 

B 

cT 

R 

+ 

18.8 

13-8 

*27 

A 

d1 

R 

— 

19-25 

13-5 

B 

d1 

1 

— 

19-3 

13-7 

Slightly    left-handed    in 

wrist  and  finger  tapping. 

4i 

A 

9 

L 

p 

17.26 

15.2 

Hair  whorl   could   not   be 

B 

9 

R 

p 

17.6 

15-6 

determined. 

60 

A 

9 

R 

+ 

17-6 

14.1 

B 

9 

1 

+ 

17-5 

14-15 

Slightly   left-handed    in 

finger  tapping. 

3o8 


H.    H.    NEWMAN. 


HANDEDNESS  IN  RELATION  TO  DEGREES  OF  RESEMBLANCE. 

In  this  table  there  are  listed  twelve  pairs  of  twins  one  member 
of  which  is  fully  left-handed  and,  in  addition  to  these,  there  are 
eleven  cases  that  show  partial  left-hancledness  in  one  or  both  in- 
dividuals of  the  pair.  Besides  the  twenty -three  cases  showing 
some  degree  of  left-handedness,  there  are  two  cases  in  which 
both  members  of  the  pair  are  classed  as  ambidextrous.  Thus  in 
exactly  fifty  per  cent,  of  our  pairs  of  identical  twins  there  is  some 
degree  of  left-handedness. 

It  is  significant  that  the  first  case  in  the  series  to  show  complete 
left-handedness  is  seventeenth  out  of  fifty.  There  are  only  two 
cases  of  partial  left-handedness  among  the  fifteen  most  strikingly 
similar  set  of  twins,  while  some  degree  of  left-handedness  be- 
comes the  rule  rather  than  the  exception  from  the  sixteenth  to 
the  end  of  the  list. 

CROWN  WHORL  IN  RELATION  TO  DEGREES  OF  RESEMBLANCE. 

The  incidence  of  asymmetry  reversal  in  crown  hair  whorl  fol- 
lows the  same  general  lines  as  does  left-handedness.  In  the  first 
ten  pairs  there  is  but  one  case  (No.  63,  in  third  place)  that  shows 
true  counter-clockwise  hair-whorl.  Two  other  cases  (No.  9, 
in  sixth  place,  and  No.  35,  in  tenth  place)  show  a  mixed  hair- 
whorl  partly  clockwise  and  partly  counter-clockwise.  There  is 
also  one  case  of  a  double  hair-whorl,  one  whorl  being  clockwise, 
the  other  counter-clockwise  (No.  40,  in  fourth  place).  The  most 
frequent  incidence  of  counter-clockwise  hair-whorl  in  one  twin 
occurs  among  the  middle  grade  twins,  neither  the  most  alike  or  the 
most  different.  This  is  true  also  of  left-handedness,  and  such  :\ 
correspondence  in  the  incidence  of  two  forms  of  asymmetry  re- 
versal must  have  some  real  significance. 

THE  RELATION  BETWEEN  HANDEDNESS  AND  HEAD  SIZE. 

In  the  following  study  both  left-handedness  and  counter-clock- 
wise hair-whorl  are  taken  to  be  equivalent  criteria  of  either  genetic 
or  epigenetic  reversal  of  asymmetry.  For  the  present  we  shall 
omit  from  consideration  the  ten  pairs  of  twins  (Nos.  73,  25,  23, 
13,  87,  43,  38,  72,  7  and  27)  that  were  diagnosed  as  derived  from 
zygotes  genetically  left-handed.  These  are  starred  in  the  list. 


STUDIES    OF    HUMAN    TWINS.  309 

Before  discussing  the  relation  of  head-size  to  handedness  it 
should  be  said  that  there  is  undoubtedly  some  inaccuracy  in  the 
figures  for  head  dimensions.  Repeated  measurements  of  the  same 
head  rarely  give  exactly  the  same  result.  Dahlberg  has  calculated 
that  the  average  error  in  head  measurements  is  about  0.5  mm. 
It  seems  probable  that  our  own  errors  were  at  least  as  great  as 
this,  and  probably  greater.  Hence  differences  of  no  more  than  i 
mm.  may  be  ignored  or  considered  as  without  significance. 

Glance  with  me  down  the  list  of  forty  pairs  of  identical  twins 
not  previously  diagnosed  as  derived  from  genetically  left-handed 
zygotes.  In  all,  there  are  seventeen  pairs  in  which  one  twin  may 
be  classed  as  right-handed,  the  other  left-handed,  and  in  which 
there  is  a  significant  difference  in  head  size.  In  thirteen  of  these 
pairs  (63,  9,  67,  102,  68,  33,  53,  44,  2,  70,  28,  14  and  41)  the  right- 
handed  individual,  derived  from  the  superior  side  of  the  embryo, 
has  a  distinctly  larger  head. 

The  four  other  cases  (49,  79,  101  and  34)  reverse  this  condition, 
the  left-hander  has  the  larger  head,  though  case  34  is  ambiguous 
in  that  one  twin  is  slightly  left-handed  in  tapping  and  the  other 
nearly  ambidextrous  and  may  therefore  belong  with  the  list  of 
ten  diagnosed  as  derived  from  a  left-handed  zygote.  The  other 
three  cases  (49,  79  and  101)  are  valid  exceptions.  Let  us  con- 
sider these  cases  carefully.  What  would  happen  in  the  case  of  a 
genetically  left-handed  zygote  if  one  of  the  twins  underwent 
asymmetry  reversal?  Obviously  the  reversed  twin  would  be  a 
right-hander,  and  should  have  the  smaller  head.  This  interpre- 
tation appears  to  fit  cases  49,  79  and  101.  It  would  be  strange  if 
some  cases  such  as  these  did  not  occur  in  view  of  the  existence  of 
genetically  left-handed  zygotes. 

This  hypothesis,  that  head  size  is  correlated  with  handedness, 
may  be  checked  still  further  by  examining  the  ten  pairs  of  twins 
diagnosed  as  derived  from  genetically  left-handed  zygotes.  Of 
these,  eight  show  a  significant  difference  in  head  size.  These  eight 
cases  deserve  individual  attention : 

Pair  No.  73. — This  is  a  confusing  case.  Twin  A  shows  left- 
handedness  in  finger  tapping  and  has  a  partially  reversed  hair- 
whorl  ;  twin  B  is  practically  ambidextrous  in  tapping  and  has  a 
well-defined  counter-clockwise  hair-whorl,  the  only  really  posi- 


-2IO  H.    H.    NEWMAN. 

tive  indication  of  left-handedness  present  in  the  pair.  This  twin 
(B)  has  the  larger  head. 

Pair  No.  25. — In  this  pair  both  twins  are  right-handed  and 
both  have  counter-clockwise  hair-whorl.  It  is  impossible  to  decide 
which  of  these  has  been  derived  from  the  superior  side  or  whether 
they  are  derived  from  a  right-handed  or  left-handed  zygote,  for 
the  handedness  and  hair-whorl  completely  contradict  each  other. 

Pair  No.  23. — Both  twins  are  ambidextrous,  and  both  have 
clockwise  hair-whorl.  Twin  A,  with  the  larger  head,  is  more 
nearly  left-handed  than  B. 

Pair  No.  87. — Twin  A,  while  ambidextrous,  tends  to  be  more 
left-handed  than  B,  and  has  counter-clockwise  hair-whorl;  twin 
B  is  ambidextrous  and  has  clockwise  hair-whorl.  Evidently  A  is 
the  left-handed  (superior)  individual,  and  he  has  the  larger  head. 

Pair  No.  43. — Both  twins  are  partly  (probably  natively)  left- 
handed.  Twin  A  has  counter-clockwise  hair-whorl,  twin  B  clock 
wise.  A,  the  more  distinctly  left-handed  twin,  has  the  larger  head. 

Pair  No.  38. — Both  twins  are  partially  left-handed  and  both 
have  counter-clockwise  hair-whorl.  A  is  left-handed  in  both  wrist 
and  finger  tapping;  B,  only  in  finger  tapping.  A,  the  more  left- 
handed,  has  the  larger  head. 

Pair  No.  J2. — Twin  A  is  strongly  left-handed  but  has  clockwise 
hair-whorl ;  twin  B  is  right-handed  but  has  counter-clockwise  hair- 
whorl.  It  is  impossible  to  say  which  individual  should  be  diag- 
nosed as  from  the  superior  side,  since  the  two  criteria  seem  to  be 
of  equal  value.  Of  the  two  the  reversed  hair-whorl  is  somewhat 
safer  as  a  criterion,  and  it  happens  that  the  twin  (B)  with  the 
counter-clockwise  hair-whorl  has  the  larger  head. 

Pair  No.  27. — Twin  A  is  right-handed ;  B,  slightly  left-handed 
in  wrist  and  finger  tapping.  Both  have  counter-clockwise  hair- 
whorl.  Twin  B,  the  partially  left-handed  member  of  the  pair, 
has  the  larger  head. 

All  of  these  eight  cases  except  pair  25,  which  is  neutral,  support 
the  conclusion  that  the  twin  derived  from  the  genetically  superior 
side  (the  right  side  in  these  cases)  of  the  embryo  has  the  larger 
head. 

One  other  class  of  cases  remains  to  be  dealt  with,  those  in  which 
a  significant  difference  in  head  size  exists  without  any  complete 


STUDIES    OF    HUMAN    TWINS.  31! 

asymmetry  reversal  in  handedness  or  hair-whorl.  There  are  nine 
such  pairs  (62,  3,  80,  55,  30,  6,  97,  69,  18).  In  all  but  two  of 

» 

these  cases  (3,  55,  30,  6,  97,  69,  18)  one  twin  was  definitely 
more  right-handed  than  the  other  and  the  more  right-handed  in- 
dividual has  the  larger  head  in  all  pairs.  In  pairs  62  and  80, 
both  twins  are  equally  strongly  right-handed  and  offer  neutral 
evidence.  Instead  of  weakening  the  general  theory,  then,  all  of 
these  cases,  where  varying  degrees  of  difference  in  right-handed- 
ness but  no  true  left-handedness  occur,  tend  strongly  to  support 
it.  There  is  beyond  question  a  strong  correlation  between  hand- 
edness and  head  size.  With  very  few  exceptions  indeed,  the  twin 
having  the  larger  head  shows  evidence  of  having  been  derived 
from  the  genetically  superior  side  of  the  embryo;  from  the  left- 
hand  side  in  twins  derived  from  zygotes  genetically  destined  to 
form  right-handers,  and  from  the  right  side  of  zygotes  destined 
to  form  left-handers. 

TWINNING  AND  THE  ASYMMETRY  MECHANISM. 

The  data  just  presented  have  given  rise  to  a  theory  that  seems 
to  rationalize  for  the  first  time  the  peculiar  incidence  of  reversal 
of  asymmetry  in  twins.  It  is  well  known  that  in  some  groups 
of  animals,  notably  those  characterized  by  a  striking  degree  of 
determinate  cleavage,  bilateral  symmetry  and  asymmetry  are  es- 
tablished in  the  undivided  zygote  before  or  at  the  time  of  the 
first  cleavage.  In  those  forms,  on  the  other  hand,  that  show  a 
strong  tendency  toward  indeterminate  cleavage,  notably  the  ver- 
tebrates and  echinoderms,  symmetry  and  asymmetry  are  not 
definitely  fixed  until  considerably  later  in  development.  The 
writer's  work  (Newman,  1924)  on  asymmetry  reversal  in  the 
starfish  indicates  clearly  that  asymmetry  is  fixed  before  the  time 
of  gastrulation,  for  no  reversal  of  asymmtry  could  be  induced  in 
embryos  older  than  late  blastulse. 

There  are  also  indications  among  the  vertebrates  that  asym- 
metry is  established  prior  to  or  during  gastrulation.  Thus  in  the 
nine-banded  armadillo,  the  only  case  of  twinning  among  mammals 
where  the  stage  at  which  twinning  occurs  is  definitely  known,  it 
has  been  found  that  the  first  step  in  the  twinning  process  usually 
precedes  the  period  at  which  symmetry  and  asymmetry  are  es- 


312 


H.    H.    NEWMAN. 


tablished  and  that  the  second  step  in  twinning  takes  place  during 
the  process  of  the  establishment  of  the  axis  of  symmetry.  By 
analogy,  we  may  infer  that  twinning  in  man  takes  place  in  close 
association  with,  and  possibly  as  an  aberration  of,  the  process  of 
establishing  and  fixing  the  relations  of  symmetry  and  asymmetry 
in  the  embryo. 

Now,  since  no  biologic  processes  takes  place  with  the  same 
clock-like  precision  in  all  specimens,  we  may  suppose  that  the 
twinning  act  in  some  cases  is  consummated  during  relatively  early 
stages  of  the  establishment  of  symmetry  and  asymmetry,  and  that 
in  other  cases  it  is  established  later.  In  the  cases  in  which  twin- 
ning occurs  relatively  late,  the  establishment  of  a  single  bilateral 
individual  may  have  gone  so  far  that  complete  twinning  is  im- 
possible. This  is  probably  the  case  in  all  partial  twinning,  re- 
sulting in  conjoined  twins  and  double  monsters.  In  such  twins 
one  of  the  most  striking  features  is  the  occurrence  of  profound 
reversal  of  asymmetry,  as  expressed  in  more  or  less  complete 
situs  inversus  mscerum. 

If  then,  we  may  assume  that  conjoined  twins  with  the  most 
extreme  reversal  of  asymmetry  in  the  inferior  component,  repre- 
sents one  end  of  the  series  of  twins,  it  is  natural  to  assume  that 
the  opposite  end  of  the  series  is  represented  by  cases  in  which 
twinning  is  consummated  before  any  asymmetry  is  fixed.  In  such 
cases  the  twins  would  be  derived  from  two  equivalent  primordia 
which  had  not  yet  been  differentiated  into  right-  and  left-hand 
sides.  When,  later,  asymmetry  comes  to  be  established  in  these 
two  genetically  equivalent  and  still  undifferentiated  embryos,  it 
should  follow  the  same  course  in  both  and  each  should  develop 
the  same  asymmetry  as  the  embryo  would  have  done  had  it  not 
undergone  twinning.  Thus,  if  the  original  embryo  was  genet- 
ically a  right-hander,  two  right-handed  twins  should  result;  simi- 
larly, if  the  original  embryo  was  genetically  a  left-hander,  two 
left-handed  twins  should  result — a  condition  not  uncommon 
among  twins,  but  hitherto  unexplained.  In  such  twins  we  would 
expect  a  high  degree  of  same-sided  asymmetry  in  such  details  as 
palm  and  finger  prints,  ear  shape,  dentition,  handedness,  hair- 
whorl,  etc.  Moreover,  since  the  two  twins  are  derived  from  two 
primordia  that  have  not  yet  become  differentiated  as  right-  and 


STUDIES    OF    HUMAN    TWINS.  313 

left-hand  components,  the  two  resulting  twins  would  be  expected 
to  be  very  strikingly  similar,  more  similar  than  would  be  twins 
separated  after  asymmetry  had  been  more  or  less  fixed  in  the 
embryo  from  which  they  are  derived. 

Thus  the  earlier  twinning  occurs  with  respect  to  the  establish- 
ment of  asymmetry,  the  more  similar  should  he  the  resultant  twins 
and  the  less  should  they  show  such  evidences  of  reversal  of  asym- 
metry as  left-handedness  and  counter-clockwise  hair  whorl.  This 
explains  why  these  criteria  of  asymmetry  reversal  are  rarely  pres- 
ent in  the  most  strikingly  similar  twins  and  are  increasingly  com- 
mon among  identical  twins  that  are  less  similar. 

If  this  theory  be  sound,  and  there  is  much  evidence  in  its  favor, 
we  have  discovered  another  mechanism,  not  classifiable  as  envir- 
onmental, that  operates  to  make  identical  twins  different.  This 
factor,  the  asymmetry  mechanism,  may  be  the  main,  if  not  the 
only,  factor  responsible  for  observed  differences  between  identical 
twins  reared  together.  Consequently  it  would  be  quite  unsafe  to 
infer  that  any  differences  between  such  twins  are  due  to  differ- 
ences in  environment  or  in  training.  On  the  other  hand,  once 
we  have  established  the  average  degree  of  difference  between 
identical  twins  reared  together,  we  should  be  able  to  use  this  as 
a  base  line  in  determining  to  what  extent,  in  cases  of  identical 
twins  reared  apart,  the  differences  in  environment  have  operated 
to  increase  the  physical  or  mental  difference. 

This  theory  goes  far  to  explain  why  some,  but  not  all,  pairs  of 
twins  show  left-handedness  and  counter-clockwise  hair-whorl  in 
one  twin  of  a  pair ;  why  there  should  be  occasional  cases  in  which 
both  twins  of  a  pair  are  left-handed  or  have  counter-clockwise 
hair-whorl ;  why  there  should  be  various  degrees  of  incomplete 
asymmetry  reversal  as  the  result  of  separation  of  twins  prior  to 
complete  establishment  of  asymmetry.  The  establishment  of 
asymmetry  is  a  progressive  process  and  takes  some  time  to  be- 
come fully  fixed.  Hence  we  may  expect  to  find  that  twinning 
early  in  the  process  will  result  in  little  if  any  signs  of  asymmetry 
reversal  in  one  of  the  twins,  and  that  twinning  occurring  late  in 
the  process  will  result  in  extensive  reversal  of  asymmetry  in  one 
of  the  components. 

In  brief,   this   theory  seems  to   clear  up  many   if   not  all  the 


314 


H.    H.    NEWMAN. 


formerly  baffling  asymmetry  situations  found  in  twins.  It  lacks 
experimental  confirmation,  but  this  must  be  so  from  the  nature  of 
the  material.  Yet  the  data  themselves  almost  speak  out  the  theory 
of  their  own  accord. 

SUMMARY. 

1.  Reversal  of  asymmetry  in  monozygotic  twins  expresses  itself 
in  varying  degrees,  ranging  from  complete  situs  inversus  viscerum 
in  conjoined  twins  to  left-handedness  or  counter-clockwise  hair- 
whorl  in  separate  twjns. 

2.  There  are  two  kinds  of  handedness :  genetic  and  epigenetic. 
Genetic  right-  and  left-handedness  have  about  the  incidence,  re- 
spectively,  of   dominant  and  recessive  allelomorphs.     Epigenetic 
left-handedness    (or    in    genetic    left-handers,    right-handedness) 
results    from   twinning,   the   inferior   side   having   an   asymmetry 
opposite  to  that  of  the  superior  side. 

3.  Arranging  fifty  pairs  of  identical  twins  in  the  order  of  their 
closeness  of  physical  resemblance,  it  is  found  that  there  is  very 
little   evidence   of   asymmetry   reversal   among   the   most   similar 
twins,  while  the  less  similar  twins  show  a  high  degree  of  it. 

4.  Clockwise  hair-whorl  has  about  the  same  incidence  as  right- 
handedness,  and  counter-clockwise  hair-whorl  that  of  left-hand- 
edness. 

5.  Varying  degrees  of  partial  left-handedness  and  of  ambidex- 
trality  are  revealed  by  tapping  tests. 

6.  Ten  pairs   of    identical   twins   show  asymmetry   reversal   in 
both  members  of  a  pair  and  are  therefore  diagnosed  as  derived 
from    genetically    "  left-handed "    zygotes ;    three    pairs    showing 
asymmetry  reversal  in  but  one  twin  should  probably  be  classed 
as  "  left-handers  " ;  the  remaining  thirty-seven  pairs  are  believed 
to  be  derived  from  right-handed  zygotes. 

7.  There   is   a   very   close   correlation   between   head   size   and 
handedness.    The  twin  derived  from  the  superior  side  of  the  em- 
bryo nearly  always  has  a  significantly  larger  head. 

8.  The  reason  why  many  but  not  all  identical  twins  show  asym- 
metry reversal  in  one  twin  is  that  the  epigenetic  establishment  of 
asymmetry   takes    place    sometimes   before   and    sometimes   after 
twinning.     If  it  takes  place  before  twinning  the  twins  will  show 


STUDIES    OF    III  "MAX    TWINS.  315 

a  high  degree  of  asymmetry  reversal:  if  it  takes  place  after  the 
twinning  the  twins  will  both  show  the  same  asymmetry  and  be  in 
other  respects  more  alike  than  when  the  establishment  of  asym- 
metry precedes  twinning;  if  it  takes  place  during  the  twinning 
process  the  twins  will  show  varying  degrees  of  asvmmetry  re- 
versal in  one  individual  and  varying  degrees  of  close  resemblance 
in  physical  and  mental  characters. 

REFERENCES. 
Dahlberg,  G. 

'26     Twin    Births    and    Twins    from    an     1  Invditary     Point    of    View. 

Stockholm. 
Jones,  W.  F. 

'18     A  Study  of  Handedness.     Vermilion,  S.  D. 
Jordan,  H.  E. 

'ii     The    Inheritance    of    Left-handedness.      Anicr.    Breeders'    Magazine, 

2:  19-29. 
Lauterbach,  C.  E. 

'25     Studies  in  Twin  Resemblance.     Genetics,  Vol.  10. 
Meirowsky,  E. 
'27     Zwillingsbiologische   Untersuchungen.     Arch.    f.    Rasscn   und   <  u -scll- 

schaftsbiol.,  Bd.  18. 
Verschuer,  O.  von. 
'27     Der     Vererbungsbiologische     Zwillingsforschung.       Ergeh.     Innerui. 

Med.  u.  Kinderheit.,  Bd.  31. 
Newman,  H.  H. 
'16     Heredity  and   Organic   Symmetry   in   Armadillo   Quadruplets.     Bun, 

BULL.,  XXX. 

'17     The  Biology  of  Twins.     Chicago. 
'21     The  Experimental  Production  of  Twins  and  Double  Monsters  in  tin- 

Larvre  of   the   Starfish   Pateria.     Jour.   Exper.   Z<>"1..   Vol.  33. 
'23     The  Physiology  of  Twinning.     Chicago. 
Siemens,  H.  W. 

'24     Die     Bedeutungs     der     Zwillingspathologie      fur     die     aetiologischo 
Forschung  elautert  an   Beispeil  der   Linkhandigkeit.     Setzungsber.   •!. 
Ges.  f.  Morph.  u.  Physiol.  in  Munich,  Jhg.  35. 
Weitz,  W. 

'24     Studien  an  eineiigen  Zwillingen.  Zeitsch.  f.  Klin.   Med.,  Bd.   101. 


Vol.LV. 


November,  1928. 


No.  5 


BIOLOGICAL  BULLETIN 


SEX   DIFFERENTIATION   IN   GONADS   DEVELOPED 

FROM  TRANSPLANTS  OF  THE  INTERMEDIA'!!; 

MESODERM   OF  AMBLYSTOMA. 

R.    R.    HUMPHREY, 

DEPARTMENT  OF  ANATOMY,  SCHOOL  OF  MEDICINE, 
UNIVERSITY  OF  BUFFALO. 

INTRODUCTORY  AND  HISTORICAL. 

From  his  studies  on  parabiotic  twins  in  A  mblystoma  punctatum 
Burns  ('25)  is  led  to  the  conclusion  that  in  this  species  there  may 
occur  a  complete  reversal  of  sex  previous  to  the  period  of  sex- 
differentiation.  Embryos  joined  in  pairs  in  early  stages  should, 
by  the  laws  of  chance,  be  combined  in  the  proportion  of 
i  cf  cf  :  i  cf  9  :i  9  cf  :  i  99.  Instead  of  this  expected  ratio, 
Burns  obtains  exclusively  one-sexed  pairs,  in  the  proportion  of 
44  cf  cf  to  36  99.  This  result  he  is  inclined  to  interpret  as  a 
i  :  i  ratio.  Having  no  evidence  that  the  two-sexed  pairs  had 
been  eliminated  through  selective  mortality,  Burns  postulates 
that  half  the  pairs  reared  must  have  been,  originally,  9  cf 
combinations;  in  these  pairs,  from  a  condition  of  near-equilibrium 
as  regards  sex,  one  or  the  other  sex,  he  assumes,  had  eventually 
gained  the  ascendency,  so  that  at  sex-differentiation  the  gonads 
of  the  two  members  of  the  pair  were  identical.  Since  the  sex- 
ratio  found  was  approximately  i  cf  cf  :  i  99,  Burns  infers  that 
there  can  be  no  prepotency  constantly  favoring  either  male  or 
female,  since  in  this  event  a  3  :  I  ratio  favoring  the  prepotent 
sex  would  be  expected. 

The  more  recent  studies  of  Witschi  ('27)  on  frog  embryos 
joined  in  parabiosis  show  that  in  these  amphibia  the  early  sex 
reversal  assumed  by  Burns  does  not  occur.  Witschi  finds  in  56 


21 


R.    R.    HUMPHREY. 

pairs  the  following  combinations:  cf  cf ,  16  pairs;  cf  9  ,  17  pairs, 
with  7  of  the  females  undergoing  sex-reversal ;  9  cf ,  10  pairs,  with 
4  of  the  females  undergoing  sex-reversal;  9  9  ,  13  pairs.  This 
approximates  very  closely  the  expected  ratio  of  i  cf  cf  :  i  cf  9  : 
i  9  cf  :  i  99,  and  shows  conclusively  that  there  could  have 
been  no  sex-reversal  previous  to  the  time  of  sex-differentiation. 
From  the  fact  that  in  many  of  the  two-sexed  pairs  the  females 
were  found  undergoing  sex-reversal,  while  a  female  united  with  a 
male  undergoing  reversal  was  never  found,  Witschi  concludes 
that  the  male  is  always  dominant  in  the  sex-reversal  which 
finally  occurs.  Though  he  believes  that  the  independent  sex- 
differentiation  in  the  individuals  of  genetically  two-sexed  pairs 
favors  the  theory  of  localized  sex-differentiators  ("lokalisierte 
Innenfaktoren,"  probably  comparable  to  Spemann's  "Organisa- 
toren"),  he  states  that  in  the  later  sex-reversal  of  the  female  of 
the  pair,  "the  cooperation  of  hormones  is  not  improbable." 

The  method  of  parabiosis  used  by  Burns  and  Witschi  has 
certain  obvious  disadvantages.  If  used  with  a  species  in  which 
the  zygotic  sex-determination  can  be  completely  reversed  previous 
to  sex-differentiation,  as  is  possibly  the  case  in  Amblystoma,  there 
can  be  no  certainty  regarding  the  original  state  of  any  one- 
sexed  pair  examined  after  sex-differentiation  has  taken  place. 
In  drawing  conclusions  as  to  the  occurrence  of  sex-reversal  in 
these  one-sexed  pairs,  one  must  depend  entirely  upon  the  sex- 
ratio  obtained.  Further,  if  the  death  rate  among  pairs  joined  in 
parabiosis  is  high,  the  possibility  of  a  selective  mortality  cannot 
be  entirely  eliminated,  even  though  evidence  in  favor  of  it  may 
be  scanty  or  lacking.  Hence  there  is  no  absolute  proof  that  the 
one-sexed  pairs  found  at  sex-differentiation  were  not  all  of  this 
character  genetically  at  the  time  they  were  joined;  the  proof  of 
sex-reversal,  therefore,  remains  inconclusive. 

The  method  of  parabiosis  is  relatively  advantageous  if  used 
with  a  species  in  which  an  early  reversal  of  sex  does  not  occur 
(as  Rana  sylvatica;  Witschi,  '27).  In  such  a  species,  pairs  pre- 
served at  a  suitable  period  in  development  would  show  the 
actual  progress  of  sex-reversal  in  one  member  of  the  pair.  If, 
however,  the  reversal  becomes  complete,  all  pairs  killed  at  later 
periods  would  be  found  to  be  one-sexed.  Although  sex-reversal 
could  be  confidently  asserted  for  a  species  of  this  type  as  a  result 


SEX    DIFFERENTIATION    IN    GONADS.  319 

of  the  disappearance  of  the  two-sexed  condition  observable  in 
younger  pairs,  the  identity  of  any  of  the  originally  mixed  pairs 
could  be  established  in  adult  animals  only  with  great  difficulty 
if  at  all. 

In  the  spring  of  1926  the  writer  undertook  to  transplant  the 
intermediate  mesoderm  of  Amblystoma  from  one  embryo  to  a 
latero-ventral  site  in  another  in  order  to  determine  the  fate  of 
the  primordial  germ  cells  included  in  such  grafts.  Among  the 
seven  embryos  surviving  the  implantation  was  one  in  which  at 
forty-four  days  after  operation  the  germ  cells  of  the  graft  were 
found  to  have  given  rise  to  a  gonad  of  considerable  size 
(Humphrey,  '27).  This  suggested  the  possibility  that  such 
grafts,  if  allowed  to  develop  until  after  the  period  of  sex- 
differentiation  of  the  host,  might  be  found  to  contain  gonads 
which  had  likewise  undergone  sex-differentiation.  The  donor 
serving  as  the  source  of  the  transplant,  and  the  host  into  which 
it  was  engrafted,  though  selected  at  random  long  before  sex- 
differentiation  had  occurred,  must  in  many  cases  be  unlike  in  sex. 
Since  the  donor  could  be  reared,  its  sex  could  be  determined 
from  the  gonad  it  possessed,  and  since  donor  and  host  were  not 
joined,  the  sex-differentiation  in  either  could  not  be  influenced  by 
the  other,  except  in  so  far  as  the  graft  might  be  able  to  modify 
the  sex-differentiation  of  the  host.  If,  then,  after  sexual  differ 
entiation  the  gonad  of  the  graft  were  found  to  agree  in  type 
with  that  of  the  host  regardless  of  the  sex  of  the  donor,  the  fact 
of  an  early  sex-reversal  would  be  established  beyond  question. 
If,  on  the  other,  hand,  the  gonad  of  any  graft  differed  in  type 
from  that  of  the  host,  agreeing  with  that  of  the  donor,  it  would 
show  conclusively  that  sex-reversal  previous  to  sex-differentiation 
had  not  occurred.  By  the  method  of  grafting,  therefore,  it 
seemed  possible  to  obviate  certain  difficulties  inherent  in  the 
method  of  parabiosis.  The  donor  furnishing  the  graft  would 
undergo  sex-differentiation  according  to  the  factors  present  in 
the  egg  at  fertilization;  its  gonad  could  be  compared  directly 
with  the  gonad  developed  in  a  transplant  removed  during  the 
germ-layer  stage  and  grown  in  a  host  of  the  opposite  sex.  Con- 
clusions as  to  the  occurrence  of  sex-reversal,  therefore,  could  be 
drawn  from  comparison  of  structures  rather  than  by  reasoning 


320 


R.    R.    HUMPHREY. 


from  sex  ratios  in  which  the  factor  of  selective  mortality  might 
possibly  be  involved. 

MATERIAL  AND  METHODS. 

The  removal  of  the  intermediate  mesoderm  (preprimordia  of 
gonad  and  mesonephros)  of  Amblystoma  and  its  implantation  into 
another  embryo  is  a  relatively  simple  operation,  the  technique 
for  which  has  been  outlined  elsewhere  (Humphrey,  '27).  During 
the  operating  season  of  1927,  180  such  implantations  were 
carried  out.  The  graft  always  included  a  large  part  of  that 
region  of  the  intermediate  mesoderm  in  which  it  had  been  found 
that  primordial  germ  cells  develop  (i.e.,  the  territory  of  the 
seventh  to  the  seventeenth  somites,  approximately) ;  in  addition 
it  included  parts  of  the  adjacent  axial  and  lateral  mesoderm, 
together  with  the  overlying  ectoderm. 

Following  operation,  the  host  receiving  the  transplant  and  the 
donor  furnishing  it  were  reared  to  the  age  of  fifty  days  or  over— 
i.e.,  until  after  the  beginning  of  morphological  sex-differentiation. 
At  autopsy  of  the  host  the  graft  derivatives  were  found,  as  a 
rule,  attached  to  the  inside  of  the  ventral  or  lateral  body  wall. 
In  the  donor,  at  autopsy,  the  gonad  was  always  very  small  or 
entirely  lacking  on  the  right,  the  side  from  which  the  transplant 
was  invariably  taken  in  the  embryo. 

RESULTS. 

Of  180  pairs  (donor  and  host)  only  49  or  27  per  cent,  of  the 
.total,  were  reared  to  the  age  of  50  days  or  over.  This,  however, 
does  not  indicate  an  actual  mortality  of  73  per  cent,  in  the 
grafted  animals,  since  25  additional  hosts  were  reared  to  the  age 
of  50  days  or  more,  although  the  donors  which  had  furnished 
them  transplants  had  died  in  early  stages  of  development. 
Several  hosts  were  also  killed  before  reaching  the  age  of  50  days, 
in  order  to  study  the  development  of  the  gonad  and  other 
structures  in  the  graft;  these  hosts  were  always  those  of  pairs 
from  which  the  donor  had  already  died  from  operative  injury 
or  other  causes.  In  all,  74  grafts  were  recovered  after  sex  of 
the  host  had  become  distinguishable.  Of  these  grafts,  40  con- 
tained a  gonad,  the  sex  of  which  was  determinable  with  a  fair 
degree  of  certainty  in  33  cases.  In  the  remaining  grafts  the 


SEX    DIFFERENTIATION    IN    GONADS.  32! 

gonad  was  small  with  few  germ  cells  and  no  features  permitting 
it  to  be  classified  as  either  ovary  or  testis. 

Unfortunately  for  this  study,  the  majority  of  the  gonads  which 
developed  were  in  homoplastic  transplants  in  Amblystoma  jeffer- 
sonianum.  In  this  species,  instead  of  the  expected  i  :  I  sex- 
ratio,  the  animals  reared  in  the  laboratory  in  1927  were  in  the 
proportion  of  56  females  to  19  males,  essentially  a  ratio  of  3  :  I. 
As  a  result  of  the  predominance  of  females,  donor  and  host  were 
both  of  this  sex  in  an  excessive  number  of  cases.  In  only  two 
instances  were  donor  and  host  unlike  in  sex  and  in  these,  un- 
fortunately, the  gonad  of  the  graft  was  in  each  case  of  somewhat 
atypical  structure  due  to  unfavorable  environmental  factors. 

To  the  writer's  knowledge  a  sex-ratio  such  as  the  one  here 
reported  for  Amblystoma  jeffersonianum  has  not  been  previously 
recorded  for  this  species.  Whether  it  is  to  be  explained  on  the 
basis  of  a  selective  mortality  among  operated  animals,  or  whether 
it  is  due  to  an  induced  reversal  of  sex  in  certain  males  resulting 
from  nutritive  disturbance  or  other  alteration  of  environmental 
factors,  or  whether  an  excess  of  females  is  a  normal  condition  in 
this  species  or  at  least  in  its  local  strain,  cannot  be  positively 
stated.  It  is  worthy  of  note  that  in  Amblystoma  maculatum 
(punctatum)  reared  in  the  laboratory  under  identical  conditions 
and  after  similar  operative  procedure,  the  sex-ratio  is  apparently 
quite  normal.  The  collection  of  large  numbers  of  A.  jeffer- 
sonianum larvae  from  local  ponds  and  a  study  of  their  sex- 
ratio  has  not  been  possible.  The  few  specimens  picked  up  near 
ponds  after  metamorphosis  have  been  found  to  be  females  in 
the  great  majority  of  cases. 

A  second  feature  of  interest  noted  particularly  in  this  species 
is  the  occurrence  of  spermatocyte  stages  in  the  testes  of  males 
60  to  80  days  of  age.  This  cannot  be  due  to  the  presence  of  a 
graft  furnished  by  a  female,  since  spermatocytes  are  no  more 
frequent  in  hosts  than  in  donors.  Though  Burns  ('25)  makes  no 
mention  of  spermatocytes  in  A.  maculatum  of  similar  age,  the 
writer  has  encountered  such  stages  occasionally  in  this  species 
as  well  as  in  A.  jeffersonianum.  In  the  latter,  however,  they 
occur  in  a  higher  percentage  of  the  males  examined,  and  usually 
in  greater  numbers  than  in  A.  maculatum.  In  neither  species, 
were  the  spermatocytes  found  in  stages  later  than  the  pachytene 


322 


R.    R.    HUMPHREY. 


condition  of  the  heterotypic  prophase.  Though  the  presence  of 
heterotypic  prophases  in  males  renders  these  stages  of  little 
value  as  a  criterion  of  sex  when  considered  alone,  it  may  be  noted 
that  their  number  in  the  male  is  small  as  compared  with  the 
number  of  other  germ  cells,  and  that  they  were  not  found  in  the 
diplotene  or  later  stages  characteristic  of  the  oocytes  of  amphibian 
females. 

Of  56  Amblystoma  maculatum  reared  in  the  laboratory  in  1927 
30  were  females  and  26  males.  These  numbers  give  an  approxi- 
mation of  the  expected  I  :  I  ratio.  In  this  species,  however, 
the  majority  of  the  transplants  used  were  furnished  by  very 
young  donors  (stages  21  to  25  *)  and  but  few  gonads  developed. 
In  only  two  cases  in  which  the  sex  of  the  donor  was  known  to 
differ  from  that  of  the  host  was  a  gonad  present  in  the  graft. 
In  one  of  these  two  the  gonad  was  small  and  of  the  indifferent 
type,  while  in  the  second  it  was  of  a  type  combining  features  of 
both  ovary  and  testis. 

From  the  above  it  may  be  seen  that  relatively  little  evidence 
bearing  upon  the  problem  of  sex-reversal  was  obtainable  from 
grafts  the  donors  of  which  had  survived  to  sex-differentiation. 
But  in  several  cases  in  which  the  donor  had  died  before  reaching 
this  period,  the  transplant  furnished  by  it  was  found  to  contain 
a  gonad  differing  in  sex  type  from  that  of  the  host  in  which  the 
graft  had  developed.  In  these  cases  it  would  appear  that  donor 
and  host  must  have  been  unlike  in  sex,  but  that  the  gonad  of  the 
graft  had  differentiated  in  a  fashion  determined  by  the  organiza- 
tion of  the  transplant  previous  to  its  isolation  from  the  donor. 
These  cases  may  now  be  described  in  some  detail. 

No.  211. — Transplant  from  A.  jeffersonianum  of  stage  29 
implanted  in  A.  maculatum  of  stage  25.  The  donor  died  18  days 
after  operation.  The  host,  killed  61  days  after  operation,  proved 
to  be  a  female.  A  section  of  the  ovary  is  shown  in  Fig.  i.  The 
central  ovarian  cavity  is  well  developed,  and  the  germ  cells  are 
peripheral  in  position.  Their  nuclei  are  largely  in  the  heterotypic 
prophase  stages  characteristic  of  the  early  urodele  ovary,  although 
few  in  number  or  lacking  in  the  testis,  as  a  rule,  until  a  much 
later  period  of  development.  The  graft  removed  from  this  host 

1  The  stages  referred  to  throughout  this  paper  are  those  of  Harrison's  series  of 
standard  stages. 


SEX    DIFFERENTIATION    IN    GONADS.  323 

included  a  fairly  large  gonad  of  testicular  type  (see  Fig.  2).  No 
central  cavity  is  present.  The  germ  cells  are  somewhat  uniformly 
scattered  through  the  organ,  intermingled  with  numerous  smaller 
cells  which  constitute  the  'sex  cords'  (anlagen  of  duct  system), 
and  the  stromal  and  sustentacular  elements  of  the  testis.  The 
germ  cells  are  all  in  spermatogonial  stages;  heterotypic  prophases 
are  entirely  lacking. 

No.  284. — Transplant  from  A .  maculatum  of  stage  30  implanted 
in  host  of  same  species  and  stage.  The  donor  was  killed  by  the 
host  37  days  after  operation.  The  host,  autopsied  58  days  after 
implantation  of  the  graft,  is  a  female.  Although  sex-differ- 
entiation had  but  recently  occurred,  the  ovary  has  the  charac- 
teristic central  cavity  and  peripheral  oocytes  with  nuclei  in 
heterotypic  prophase  (see  Fig.  3).  The  gonad  found  in  the 
transplant  is  a  pear-shaped  testis  attached  by  a  stalk  to  the 
surface  of  the  graft  mesonephros.  It  lacks  the  central  cavity 
characteristic  of  the  ovary,  and  shows  the  more  uniform  distri- 
bution of  germ  cells  typical  of  the  young  testis  (see  Fig.  4).  No 
heterotypic  prophases  are  present,  all  germ  cells  being  in  sper- 
matogonial stages. 

No.  244. — Transplant  from  A.  jeffersonianum  of  stage  31, 
implanted  in  host  of  same  species  and  stage.  Donor  presumably 
devoured  by  host  at  about  31  days  after  operation.  The  host, 
autopsied  61  days  after  operation,  is  a  male;  a  section  of  one 
testis  is  shown  in  Fig.  5.  As  is  frequently  the  case  in  males  at 
this  stage  of  development,  the  testes  of  this  animal  show  a  few 
cells  in  the  spermatocyte  stage,  but  the  germ  cells  are  distributed 
in  the  fashion  characteristic  of  the  testis,  and  no  central  cavity  is 
present.  For  comparison  with  the  testis  of  the  host  a  section 
of  the  gonad  of  the  graft  is  shown  in  Fig.  6.  This  gonad  must  be 
interpreted  as  an  ovary  in  an  early  stage  of  sex-differentiation. 
Although  no  central  cavity  is  yet  present,  the  germ  cells  are 
arranged  in  a  layer  around  the  periphery  of  the  gonad  and  are 
for  the  most  part  oocytes  in  earlier  stages  of  the  heterotypic 
prophase.  By  comparison  of  Fig.  6  with  Figs.  I  and  3  (ovaries 
of  fairly  early  stages  of  differentiation)  it  will  be  readily  appreci- 
ated that  this  graft  gonad  is  ovarian  in  nature.  The  differences 
between  it  and  the  graft  gonads  of  Figs.  2  and  4  (testes)  are 


324 


R.    R.    HUMPHREY. 


clearly   evident   from    the    photographs,    and    need    no    further 
comment. 

The  three  cases  above  described  show  clearly  that  a  gonad 
developing  in  a  graft  need  not  agree  in  sex  type  with  the  gonad  of 
the  host.  It  may  be  logically  inferred  that  in  these  three  cases 
the  sex-differentiation  of  the  graft  gonad  was  determined  by  the 
organization  in  the  implanted  mesoderm  previous  to  its  removal 
from  the  donor  embryo. 

In  a  few  cases  in  which  both  donor  and  host  lived  until  after 
sex-differentiation  and  were  found  to  be  of  unlike  sex,  a  gonad 
was  present  in  the  graft.  These  cases,  however,  are  less  satis- 
factory than  the  preceding,  since  the  gonad  of  the  graft  is  either 
in  an  early  stage  of  sex-differentiation  or  is  of  atypical  structure. 
Three  such  cases  will  now  be  described. 

No.  207. — Transplant  from  A.  jeffersonianum  of  stage  29 
implanted  in  A.  maculatum  of  stage  25.  The  host,  killed  61 
days  after  operation,  is  unquestionably  a  male,  although  a  few 
germ  cells  in  heterotypic  prophase  are  found  in  one  of  the  testes. 
A  section  of  the  testis  is  shown  in  Fig.  7.  The  donor,  a  female, 
was  not  killed  until  78  days  after  operation.  The  gonad  shown 
in  Fig.  8  is  therefore  more  advanced  in  development  than  the 
testis  of  Fig.  7.  The  gonad  found  in  the  graft  is  small  and  in 
an  early  stage  of  sex-differentiation.  Although  no  central  cavity 
is  present,  the  germ  cells  tend  to  take  a  peripheral  position. 
Of  the  95  germ  cells  present,  38  are  in  early  stages  of  the 
heterotypic  prophase.  Considering  all  its  structural  features, 
this  gonad  should  be  classed  as  an  ovary.  In  the  peripheral 
arrangement  of  its  germ  cells,  and  in  the  high  proportion  of 
these  cells  found  in  heterotypic  prophase,  it  is  clearly  similar 
to  the  gonad  of  the  donor  rather  than  to  that  of  the  host. 

No.  igo. — Transplant  from  A.  jeffersonianum  of  stage  33 
implanted  in  host  embryo  of  same  age  and  species.  The  host, 
killed  64  days  after  operation,  is  a  female  (see  ovary  in  Fig.  10). 
The  donor,  killed  at  the  same  age  as  the  host,  is  a  male  (see 
Fig.  n).  The  gonad  of  the  graft  is  atypical  in  structure  in 
that  an  unusual  amount  of  stroma  is  present,  in  the  form  of  a 
mucous  type  of  connective  tissue  (Fig.  12).  It  may  nevertheless 
be  classed  as  testis  rather  than  ovary.  The  germ  cells,  though 
frequently  included  in  the  covering  epithelium,  are  predominantly 


SEX    DIFFERENTIATION    IN    GONADS.  325 

scattered  through  the  central  part  of  the  organ.  No  central 
cavity  is  present.  Sex  cords  (anlagen  of  duct  system  of  testis) 
are  recognizable  as  groups  or  strands  of  smaller  cells,  in  some 
sections  extending  a  third  or  more  of  the  length  of  the  gonad. 
The  germ  cells  are  for  the  most  part  spermatogonial  in  type, 
only  three  or  four  of  the  several  dozen  present  being  in  heterotypic 
prophase,  and  none  of  these  having  the  characteristics  of  growing 
oocytes.  Though  of  atypical  structure,  this  gonad  cannot  be 
considered  as  undergoing  transformation  from  testis  into  ovary. 
Aside  from  the  abundance  of  mucous  connective  tissue,  its 
structural  features  are  clearly  similar  to  those  of  the  testis  in 
the  donor.  Atypical  gonads  of  the  same  general  appearance  may 
develop  in  grafts  from  a  male  donor  implanted  in  a  male  host. 
The  peculiarities  of  structure  exhibited  are  therefore  due,  prob- 
ably, to  the  action  of  local  environmental  factors  rather  than  to 
the  activity  of  sex  hormones  secreted  by  the  gonads  of  the 
host. 

No.  188. — Transplant  from  A.  jeffersonianum  of  stage  33 
implanted  in  host  of  same  age  and  species.  Both  donor  and  host 
were  killed  64  days  after  operation.  The  host  is  a  female,  the 
donor  a  male  (see  Figs.  13  and  14).  The  gonad  of  the  graft  is  an 
atypical  structure  difficult  to  classify  (see  Fig.  15).  Neither 
typical  ovarian  cavity  nor  testicular  duct  system  is  recognizable. 
The  germ  cells  are  predominantly  peripheral  in  location,  although 
frequently  scattered  or  in  masses  deeper  within  the  stroma. 
In  one  instance  a  mass  of  germ  cells  lies  in  a  cavity,  with  no 
apparent  attachment  to  other  tissues  of  the  gonad;  these  cells 
show  marked  degenerative  changes.  The  cells  at  the  periphery 
of  the  gonad  frequently  exhibit  a  grouping  or  'nesting'  com- 
parable to  that  of  young  oocytes  in  a  normal  ovary.  Though 
for  the  most  part  in  heterotypic  prophase  (several  hundred  such 
cells  must  be  present)  these  germ  cells  seem  never  to  progress 
beyond  the  pachytene  stage.  If  the  gonad  were  actually  ovarian, 
some  few  at  least  of  these  cells  might  be  expected  to  pass  through 
the  diplotene  stage  and  then  enlarge  as  growing  oocytes.  This 
has  been  found  to  occur  in  those  atypical  gonads  which  have 
developed  in  grafts  from  female  donors.  In  this  gonad,  how- 
ever, no  growing  oocytes  are  present,  numerous  pyknotic  and 
fragmenting  nuclei  indicating  the  degeneration  of  the  germ  cells 


326  R.    R.    HUMPHREY. 

during    the   pachytene   stage   rather   than    their   continued   de- 
velopment. 

While  it  might  appear  at  first  glance  that  the  features  exhibited 
by  this  gonad  have  resulted  from  the  action  of  the  hormones  of 
the  host,  it  is  highly  probable  that  many  of  its  peculiarities  are 
referable  to  the  growth  potentialities  of  the  implanted  tissue  as 
modified  through  local  environmental  influences.  The  donor 
furnishing  this  particular  transplant  exhibits  an  unusual  number 
of  spermatocytes  in  its  one  (left)  gonad.  Four  such  cells  may 
be  recognized  in  the  section  shown  in  Fig.  14  (at  left).  Pre- 
sumably the  tissue  implanted  possessed  the  potentiality  for 
developing  a  gonad  in  which  unusual  numbers  of  heterotypic 
prophases  would  have  appeared  precociously,  even  without  an 
endocrine  stimulus  from  a  female  host.  As  to  local  environ- 
mental conditions,  it  may  be  noted  that  the  gonad  was  attached 
by  a  very  delicate  fold  of  tissue,  and  was  apparently  poorly 
vascularized.  The  latter  condition  alone  would  be  unfavorable 
to  the  development  of  a  gonad  of  normal  histological  structure. 

In  addition  to  the  graft  gonad  above  described  (No.  188)  two 
other  specimens  exhibit  features  which  might  possibly  be  in- 
terpreted as  modifications  due  to  the  action  of  sex-differentiating 
hormones.  In  one  of  these  the  graft  gonad  consists  of  a  central 
core  of  testicular  character  overlaid  by  a  cortex  ovarian  in  type. 
This  structure  resembles  the  modified  testes  described  by  Burns 
('28)  as  resulting  from  the  action  of  ovarian  hormones.  The 
position  of  this  particular  graft  in  the  body  of  the  host,  however, 
is  such  that  some  of  the  primordial  germ  cells  of  the  host  may 
actually  have  entered  into  the  make-up  of  the  graft  gonad.  If 
this  be  the  case,  this  structure  must  be  regarded  as  a  'mosaic' 
gonad  derived  from  two  preprimordia  of  unlike  sex-potentialities 
rather  than  as  a  testis  undergoing  sex-reversal  due  to  the 
endocrine  influence  of  a  female  host.  It  is  significant  that  graft 
gonads  developing  in  sites  sufficiently  far  ventral  to  exclude  the 
possibility  of  actual  contribution  of  host  germ  cells  generally 
show  no  indication  of  sex-reversal  (see  Figs.  2,  4,  and  6). 

Among  those  cases  in  which  only  the  host  survived  until  the 
period  of  sex-differentiation  are  seven  in  which  the  gonad  of 
the  graft  agrees  in  type  with  those  of  the  host.  While  a  reversal 
of  sex  in  these  few  cases  cannot  be  positively  excluded,  it  is 


SEX    DIFFERENTIATION    IN    GONADS.  327 

rendered  exceedingly  improbable  by  the  fact  that  in  five  other 
cases  the  gonad  of  the  graft  is  of  opposite  sex  from  those  of  the 
host.  Examples  of  this  latter  group  have  already  been  described 
(Nos.  211,  284,  and  244;  Figs,  i  to  6). 

DISCUSSION. 

The  outstanding  feature  of  the  results  described  in  the  pre- 
ceding pages  is  the  apparently  independent  sex-differentiation  of 
the  gonads  which  develop  in  grafts.  Although  in  one  or  two 
cases  such  a  gonad  has  been  modified  in  a  fashion  suggesting  an 
influence  from  sex  hormones  of  the  host,  in  no  case  is  a  complete 
early  reversal  of  sex  clearly  indicated.  So  far  as  can  be  deter- 
mined from  cases  in  which  the  sex  of  the  donor  is  known,  the 
primary  sex-differentiation  in  the  gonad  of  the  graft  always 
proceeds  in  a  fashion  determined  by  the  sex  of  the  donor.  In 
five  cases  in  which  the  sex  of  the  donor  is  not  known,  the  gonad 
is  of  opposite  sex  from  that  of  the  host.  In  four  of  these. cases, 
gonads  with  the  features  characteristic  of  a  testis  have  differ- 
entiated in  grafts  implanted  in  female  hosts,  while  in  the  fifth 
an  ovary  has  developed  in  a  graft  implanted  in  a  male. 

It  is  difficult  to  reconcile  these  findings  with  the  conclusions 
reached  by  Burns  ('25)  from  his  studies  on  the  sex  of  parabiotic 
twins  in  Amblystoma.  Burns  finds  that  the  sex  of  the  two 
members  of  any  pair  is  always  the  same.  From  this  he  is  led  to 
infer  that  complete  reversal  of  sex  has  occurred  in  one  member 
of  all  two-sexed  pairs,  such  reversal  being  accomplished  before 
sex  differences  in  the  gonads  become  morphologically  distin- 
guishable. He  assumes  that  when  embryos  of  unlike  sex  are 
joined  in  parabiosis  there  results  a  condition  of  close  balance  or 
unstable  equilibrium,  which  is  broken  if  one  animal  of  the  pair 
gains  a  slight  advantage,  presumably  through  earlier  or  more 
abundant  output  of  sex-differentiating  hormones.  All  hormones 
being  mingled  in  the  blood  stream,  and  neither  sex  being  con- 
stantly prepotent,  either  the  male  or  the  female  hormone  may 
become  dominant.  Such  domination  being  established  before 
the  onset  of  morphological  sex-differentiation,  the  phenomena  of 
this  period  will  be  identical  in  the  two  members  of  any  parabiotic 
combination,  or  essentially  so.  The  twin  which  has  undergone 
reversal  thus  differentiates  directly  without  first  exhibiting  the 


328 


R.    R.    HUMPHREY. 


sexual  characters  to  be  expected  from  its  genetic  constitution. 
Under  these  conditions,  a  reversal  cannot  be  detected  by  study  of 
developmental  stages  of  the  gonads  but  must  be  inferred  from  the 
absence  of  two-sexed  pairs  after  morphological  differentiation  has 
been  completed,  unless  it  be  assumed  that  all  such  pairs  have 
been  eliminated  through  a  selective  mortality. 

In  discussing  his  results,  Burns  considers  the  possibility  that  a 
'selective'  mortality  has  operated  to  eliminate  all  heterogeneous 
(male-female)  pairs,  permitting  only  homogeneous  pairs  to  sur- 
vive. While  this  explanation  cannot  be  positively  rejected, 
Burns  regards  the  occurrence  of  a  selective  mortality  as  highly 
improbable.  Although  the  death  rate  among  his  operated 
animals  is  very  high  (about  77  per  cent.),  he  believes  that  it  is 
possible  to  explain  it  without  postulating  a  physiological  incom- 
patibility of  the  sexes  so  profound  as  to  induce  the  death  of  all 
two-sexed  pairs.  Witschi  ('27)  has  demonstrated  that  no  such 
incompatibility  exists  in  the  frog,  since  he  finds  the  expected 
number  of  mixed  pairs  at  metamorphosis  of  his  parabiotic 
animals.  If  we  assume  that  among  Burns's  experimental  animals 
there  was  likewise  no  selective  mortality  eliminating  mixed  pairs, 
we  are  forced  to  conclude  that  parabiosis  induces  an  early  sex- 
reversal  in  one  member  of  every  two-sexed  pairs. 

If  sex-reversal  in  parabiotic  twins  in  Amblystoma  be  assumed  to 
have  occurred  in  the  manner  postulated  by  Burns,  it  would  be 
logical  to  expect  a  reversal  of  sex  in  the  gonad  of  a  graft  implanted 
in  a  host  of  opposite  sex  from  that  of  the  donor.  The  bulk  of 
the  transplant  is  small  compared  with  the  entire  body  of  the  host, 
and  the  gonad  to  which  the  graft  gives  rise  is  but  a  fraction  of 
the  size  of  the  host's  own  gonads.  Under  these  conditions  there 
should  exist  no  state  of  near-equilibrium  as  regards  sex.  If  sex- 
differentiating  hormones  are  produced  previous  to  morphological 
sex-differentiation,  those  of  the  host  should  always,  from  their 
greater  abundance,  be  able  to  dominate  the  differentiation  of 
the  gonad  in  the  graft;2  the  latter,  therefore,  should  always 

2  The  gonad  of  the  graft  is  often  somewhat  retarded  in  development  as  com- 
pared with  those  of  the  host,  possibly,  in  some  cases,  because  of  inadequate  nutrition. 
Such  retardation  of  its  development  should  favor  modification  of. the  graft  gonad 
by  the  gonads  of  the  host,  assuming  that  sex  differentiating  hormones  are  poured 
into  the  circulation  when  the  gonads  reach  a  certain  stage  in  their  differentiation. 


SEX    DIFFERENTIATION    IX    GONADS.  329 

agree  in  type  with  the  gonads  of  the  host.  Yet  the  gonad  of  a 
graft  is  clearly  able  to  develop  as  ovary  in  a  male  host,  or  as 
testis  in  a  female.  In  none  of  my  animals  could  sex-reversal  be 
demonstrated  as  having  preceded  the  primary  sex-differentiation. 

Since  the  extent  to  which  a  hormone  may  modify  an  embryonic 
structure  probably  depends  in  part  upon  the  period  of  develop- 
ment at  which  it  is  introduced  and  the  time  during  which  it  is 
allowed  to  act,  these  conditioning  factors  may  well  be  compared 
for  parabiotic  twin  and  graft. 

In  Burns's  experiments,  Amblystoma  embryos  were  joined  in 
parabiosis  at  about  stage  28  of  Harrison's  series.  In  my  own 
experiments  many  of  the  grafts  were  implanted  at  this  or  even 
earlier  stages.  In  none  of  the  cases  considered  in  this  paper 
was  either  donor  or  host  more  advanced  in  development  than 
stage  34  at  the  time  of  operation.  In  neither  the  parabiotic 
twins  at  the  time  of  union  nor  in  the  host  receiving  an  implant 
has  the  blood  yet  begun  circulation.  While  it  is  probable  that 
the  blood  streams  of  embryos  joined  in  parabiosis  are  in  com- 
munication from  the  time  the  circulations  of  the  two  first  become 
established,  my  observations  indicate  that  the  graft  becomes 
vascularized  at  a  correspondingly  early  period  in  its  development. 
In  short,  the  sex-modifying  influence  of  the  host  upon  the  graft 
should  be  exerted  fully  as  early  as  the  influence  of  an  embryo 
upon  its  parabiotic  twin,  assuming  that  this  influence  is  mediated 
through  the  activity  of  substances  transported  by  the  blood. 

As  regards  the  actual  time  elapsing  between  operation  and 
autopsy,  the  advantage  appears  to  lie  with  the  parabiotic  twins. 
Burns  states  that  among  the  pairs  of  his  series  even  the  best  did 
not  show  sex-differentiation  until  seventy  days,  while  the  general 
average  required  considerably  longer  (eighty  to  ninety  days) 
for  sex  to  become  clearly  distinguishable.  In  my  own  animals 
sex  was  usually  determinable  without  difficulty  at  fifty  days 
after  operation.  The  longer  indifferent  period  in  the  parabiotic 
twins  doubtless  results  chiefly  from  growth  retardation  due  to 
difficulties  in  feeding.  In  any  event,  it  greatly  increases  the 
period  over  which  one  animal  is  subjected  to  the  influence  of  the 
other  before  morphological  sex-differentiation  occurs.  Possibly 
in  this  prolonged  indifferent  period  the  physiological  state  of 
the  gonads  in  one  animal  may  be  so  altered  through  the  influence 


330 


R.    R.    HUMPHREY. 


of  its  opposite-sexed  twin  that  at  the  time  morphological  sex- 
differentiation  finally  occurs  the  gonads  of  the  two  animals 
differentiate  in  identical  fashion.  In  my  own  experimental 
animals  the  shorter  indifferent  period  may  be  insufficient  to 
effect  such  a  physiological  reversal  in  the  gonad  of  the  graft, 
which  in  consequence  differentiates  as  determined  by  the  genetic 
constitution  of  the  donor.  In  the  parabiosis  experiments  of 
\Vitschi  the  indifferent  period  (in  Rana  sylvatica)  is  likewise 
short,  which  may  possibly  explain  the  fact  that  sex-reversal  of 
the  female  follows  rather  than  precedes  the  primary  morpho- 
logical differentiation  of  the  gonad. 

It  is  also  possible  that  conditions  attendant  upon  development 
of  the  graft  may  render  it  less  subject  to  hormone  influence 
from  the  host  than  is  a  parabiotic  twin  to  the  influence  of  its 
mate.  Since  the  graft  usually  becomes  well  vascularized,  how- 
ever, it  would  seem  that  the  nutritive  materials  and  hormones  of 
the  host's  blood  stream  should  be  as  readily  available  for  the 
gonad  of  the  graft  as  for  the  host's  own  gonads.  As  has  been 
stated  before,  sex-differentiating  hormones  of  the  host  should  be 
but  little  diluted  by  antagonistic  hormones  secreted  in  the  graft. 
Moreover,  the  removal  of  the  graft  from  its  natural  environment 
in  the  donor  while  in  a  germ-layer  stage  and  its  implantation 
into  an  essentially  foreign  situation  should,  if  anything,  disturb 
the  action  of  local  factors  affecting  sex-differentiation,  and 
facilitate  the  modification  of  this  process  through  hormones 
produced  by  the  host.  It  would  seem  that  in  a  graft  the  de- 
veloping gonad  has  been  removed  from  both  the  endocrine  and 
environmental  influence  of  the  donor  and  subjected  to  the 
influence  of  the  host  in  a  far  more  complete  fashion  than  the 
gonads  of  one  parabiotic  twin  can  be  brought  under  the  influence 
of  the  other  embryo  of  the  pair. 

From  comparison  of  the  conditions  acting  upon  parabiotic 
twin  and  graft,  we  may  conclude  that  two,  at  least,  possibly 
have  significance  in  determining  the  difference  in  the  results 
obtained.  First,  the  greater  time  required  for  morphological  sex- 
differentiation  in  parabiotic  twins  may  permit  an  influence  of 
one  animal  upon  the  other  such  as  would  not  be  possible  in  the 
case  of  a  graft  gonad  differentiating  in  from  half  to  two-thirds  of 
the  same  period.  Secondly,  the  fact  that  in  one  case  (parabiosis) 


SEX    DIFFERENTIATION    IN    GONADS.  33! 

the  gonad  has  remained  undisturbed  in  the  organism,  while  in  the 
other  its  preprimordium  has  been  implanted  in  an  ectopic 
situation  in  another  individual,  may  possibly  explain  the  different 
way  in  which  it  reacts  preceding  or  during  sex-differentiation. 

The  results  obtained  by  the  writer  in  Amblystoma  are  not  with- 
out parallel  from  experimental  work  on  other  vertebrates. 
Willier  ('27),  from  his  study  of  the  differentiation  of  chick  gonads 
implanted  in  the  chorio-allantoic  membranes  of  either  male  or 
female  hosts  is  led  to  the  conclusion  that  "the  course  of  sex- 
differentiation  in  the  chick  embryo  is  apparently  not  determined 
by  the  action  of  sexual  hormones  circulating  in  the  blood  stream." 
He  believes  that  "hormonic  sex-differentiating  factors  of  the  host 
embryo  are  either  absent,  or  if  present,  they  are  ineffective  in 
the  modification  of  the  engrafted  sexual  glands."  Witschi  ('276) 
reaches  similar  conclusions  from  one  of  his  latest  studies  on  sex- 
differentiation  in  Rana  temporaries.  He  finds  that  the  implanta- 
tion of  a  large  graft  of  adult  frog  testis  in  tadpoles  of  this  species 
does  not  "exert  the  least  influence  upon  the  larval  and  early  post- 
larval  development  of  the  gonads."  In  both  frog  and  chick, 
therefore,  the  indifferent  gonads  are  found  to  undergo  their 
primary  sex-differentiation  apparently  unmodified  by  sex  hor- 
mones from  outside  sources.  In  cattle,  too,  recent  studies  may 
be  interpreted  as  showing  that  even  when  the  chorions  of  two- 
sexed  twins  are  fused  at  a  very  early  period,  the  gonad  of  the 
female  co-twin  first  begins  to  differentiate  as  an  ovary,  and  only 
later  undergoes  modifications  leading  to  the  production  of  the 
characteristic  free-martin  gonad  (Lillie,  '23;  Bissonnette,  '28). 

That  the  vertebrate  ovary  in  situ  may  be  modified  in  its 
development  subsequent  to  its  primary  sex-differentiation  is 
apparent  from  the  studies  of  Lillie  ('17)  and  others  on  the  free- 
martin,  or  from  the  cases  of  sex-reversal  in  parabiotic  frogs 
reported  by  Witschi  ('27a).  That  these  same  gonads  would 
have  undergone  a  comparable  modification  if  implanted  as  grafts 
in  a  host  of  the  opposite  sex  has  not  been  actually  demonstrated. 
According  to  Willier,  no  modification  of  engrafted  gonads  of  the 
chick  is  demonstrable  after  a  period  of  nine  days  on  the  host 
embryo.  It  is  conceivable,  however,  as  W'illier  states,  that  the 
transplantation  of  the  embryonic  sexual  glands  into  chicks  after 
hatching  might  yield  different  results  than  when  these  same 


332  R-    R-    HUMPHREY. 

glands  are  implanted  on  the  membranes  of  embryonic  hosts. 
Greenwood  ('25)  has  reported  the  development  of  spermatic 
tubules  in  grafts  of  the  left  ovary  taken  from  chicks  two  to  four 
days  after  hatching  and  implanted  in  young  chicks  of  the  same 
age.  It  would  appear  probable,  therefore,  that  isolation  and 
implantation  of  a  gonad  (or  its  preprimordium)  do  not  necessarily 
prevent  the  modification  of  that  gonad  through  the  action  of 
sex-hormones  of  the  host:  i.e.,  there  remains  possible  an  inhibition 
of  growth,  or  an  induction  of  growth,  in  those  parts  (as  for 
example  the  medullary  cords  of  the  bird's  ovary)  which  have 
retained  their  embryonic  capacity  to  react  in  a  specific  fashion  to 
growth  stimuli. 

The  grafts  described  in  this  paper  were  in  no  case  left  im- 
planted in  the  host  for  a  period  longer  than  seventy  days. 
Although  in  none  of  the  grafts  recovered  had  the  gonad  under- 
gone a  complete  reversal  of  sex  previous  to  its  primary  differ- 
entiation, it  is  possible  that  in  one  or  two  cases  it  had  undergone 
some  slight  modification  which  might  be  ascribed  to  the  action 
of  sex  hormones  of  the  host.  Whether  a  complete  reversal  of 
sex  might  have  occurred  had  the  graft  remained  implanted  for  a 
longer  period  is  problematic.  From  grafting  experiments  recently 
reported  by  Burns  ('27)  it  is  evident  that  sex-reversal  in  the 
gonads  of  Amblystoma  is  not  complete  even  after  periods  of  from 
fifty  to  seventy-six  days  in  a  host  of  the  opposite  sex.  Burns 
transplanted  gonads  from  larval  stages,  just  before  and  just 
after  the  beginning  of  morphological  sex-differentiation,  into 
older  larvae  in  which  sex-differentiation  was  more  advanced. 
Since  several  of  the  grafts  showed  an  admixture  of  the  charac- 
teristics of  the  two  sexes,  it  is  possible  that  a  complete  reversal  of 
sex  might  eventually  have  been  effected. 

Whether  or  not  complete  reversal  of  sex  in  Amblystoma  may 
occur  subsequent  to  morphological  sex-differentiation,  a  reversal 
of  sex  preceding  this  period  does  not  appear  to  be  effected  in 
gonads  developed  in  grafts,  when  such  grafts  are  implanted  in  an 
ectopic  situation,  such  as  the  latero-ventral  body  wall.  Whether 
implantation  of  the  graft  into  its  normal  site  would  insure  reversal 
of  the  gonad  as  postulated  for  animals  joined  in  parabiosis  still 
remains  a  question.  The  writer  now  has  in  progress  an  extensive 
series  of  experiments  to  test  this  point. 


SEX  DIFFERENTIATION  IN  GONADS.  333 

SUMMARY  AND  CONCLUSIONS. 

1.  An  area  of  mesoderm  which  included  the  preprimordium  of 
the  gonad  was  transplanted   from  one  Amblystoma  embryo   to 
another  at  stages  21  to  34.     Such  transplants,  when  taken  from 
donors  older  than  stage  25,  gave  rise  to  a  gonad  in  a  high  per- 
centage of  cases.     This  gonad  was  ectopic   in  position,   being 
attached  to  the  inside  of  the  lateral  or  ventral  body  wall,  and 
was  always  far  smaller  than  the  normal  gonads  of  the  host. 

2.  Morphological  sex-differentiation  .occurred  in  the  grafts  at 
from   fifty   to  sixty  days   after  implantation.     All   grafts  were 
removed  and  fixed  within  seventy  days.     In  several  cases,  gonad^ 
of  testicular  type  were  recovered  from  female  hosts.     In  two 
cases  gonads  of  ovarian  type  were  found  in  grafts  implanted  in 
males. 

3.  In  two  cases  in  which  donor  and  host  were  of  opposite  sex 
the  gonad  of  the  graft  was  modified  in  such  fashion  as  to  suggest 
an  influence  from  sex  hormones  of  the  host.     In  no  case,  however, 
was  the  sex  of  the  graft  gonad  completely  reversed  previous  to 
the  period  of  morphological  sex-differentiation. 

4.  It  may  be  concluded  that  gonads  developed  in  ectopic  grafts 
of   the   gonadal   preprimordia   undergo   their  primary   morpho- 
logical sex-differentiation  according  to  the  organization  of  the 
graft  at  the  time  of  its  removal  from  the  donor. 

5.  If  sex-differentiating  hormones  are  produced  by  the  host 
previous  to  morphological  sex-differentiation,  they  are  apparently 
incapable  of  bringing  about  reversal  in  the  gonad  of  the  graft. 
The  possibility  of  reversal  at  a  later  stage  of  development  is  not 
excluded,  since  no  grafts  were  allowed  to  develop  for  periods 
longer  than  seventy  days. 

6.  The  failure  of  the  gonad  in  a  graft  to  undergo  sex-reversal 
previous  to  its  morphological  differentiation  is  in  marked  con- 
trast to  the  complete  reversal  which  appears  to  occur  in  parabion- 
(cf.  Burns  '25). 3 

3  Studies  completed  while  this  paper  was  in  press  indicate  that  the  graft  ovui  i<-- 
of  Figs.  6  and  9  possibly  owe  certain  features  of  their  structure  to  the  action  of  the 
testicular  hormones  of  the  host.  These  studies  show  that  the  developing  ovary  is 
readily  modified  if  subjected  to  the  continued  influence  of  a  testis  resident  in  th«- 
same  host,  and  that  one  of  the  first  perceptible  indications  of  this  modification  is  tin- 
absence  of  the  characteristic  central  ovarian  cavity.  These  studies  will  be  repoi  t<  >1 

in  a  separate  paper. 

22 


•534  R-    R-    HUMPHREY. 

BIBLIOGRAPHY. 
Bissonnette,  T.  H. 

'28     Notes  on  a  32  Millimeter  Freemartin.     BIOL.  BULL.,  Vol.  54,  pp.  238-253. 
Burns,  R.  K. 

'25     The  Sex  of   Parabiotic  Twins  in  Amphibia.     Jour.  Exp.  Zool.,  Vol.  42, 

pp.  31-89- 

'27     -Some  Results  of  the  Transplantation  of  Larval  Gonads  in  Urodele  Am- 
phibians.    Anat.  Rec.,  Vol.  37,  p.  163. 
Greenwood,  A.  W. 

'25     Gonad  Grafts  in  the  Fowl.     Brit.  Jour.  Exp.  Biol.,  Vol.  2,  pp.  469-492. 
Humphrey,  R.  R. 

'27     The  Fate  of  the  Primordial  Germ  Cells  of  Amblystoma  in  Grafts  Implanted 
in  the  Somatopleure  of  Other  Embryos.     Anat.  Rec.,  Vol.  35,  pp.  40-41. 
Lillie,  F.  R. 

'17     The  Free-martin;  a  Study  of  the  Action  of  Sex-hormones  in  the  Foetal 

Life  of  Cattle.     Jour.  Exp.  Zool.,  Vol.  23,  pp.  371-452. 
'23     Supplementary   Notes  on   Twins  in   Cattle.     BIOL.    BULL.,   Vol.  44,   pp. 

47-77- 
Willier,  B.  H. 

'27     The  Specificity  of  Sex,  of  Organization,  and  of  Differentiation  of  Embryonic 

Chick  Gonads  as  Shown  by  Grafting  Experiments.     Jour.  Exp.  Zool., 

Vol.  46,  pp.  409-465. 
Witschi,  Emil. 

'270     Sex-Reversal  in   Parabiotic  Twins  of   the  American  Wood-Frog.     BIOL. 

BULL.,  Vol.  52,  pp.  137-146. 
'27^     Testis  Grafting  in  Tadpoles  of  Rana  temporaria  L.  and  its  Bearing  on  the 

Hormone   Theory   of   Sex   Determination.     Jour.   Exp.   Zool.,   Vol.   47, 

pp.  269-294. 


336  R-  R-  HUMPHREY. 


PLATE  I.     EXPLANATION  OF  FIGURES. 

All  figures  on  this  plate  are  photomicrographs.     Magnification  145  X. 

FIG.  i.  Ovary  of  host  No.  211,  Amblystoma  maculatum,  killed  61  days  after 
implantation  of  graft  at  stage  29.  The  central  ovarian  cavity  is  well  developed, 
and  the  germ  cells  peripheral  to  it  are  chiefly  oocytes  in  heterotypic  prophase. 
Compare  with  Fig.  2. 

FIG.  2.  Testis  of  graft  recovered  from  host  No.  211.  Note  the  absence  of  a 
central  cavity.  The  germ  cells  are  uniformly  distributed,  and  none  are  in 
heterotypic  prophase.  Compare  with  the  ovary  of  the  host  (Fig.  i).  This  testis 
was  attached  to  the  body  wall  by  a  slender  stalk  not  included  in  this  section. 

FIG.  3.  Ovary  of  host  No.  284,  Amblystoma  maculatum,  killed  50  days  after 
implantation  of  graft  at  stage  30.  Ovarian  cavity,  peripheral  arrangement  of 
germ  cells,  and  abundance  of  heterotypic  prophase  stages,  as  in  Fig.  i.  Compare 
with  graft  gonad  of  Fig.  4. 

FIG.  4.  Testis  of  graft  recovered  from  host  No.  284.  Note  absence  of  central 
cavity  and  heterotypic  prophases,  and  the  uniform  distribution  of  the  germ  cells. 
Compare  with  the  ovary  of  the  host  in  which  this  testis  developed  (Fig.  3).  The 
slender  stalk  attaching  the  testis  to  the  mesonephros  of  the  graft  is  not  included  in 
this  section. 

FIG.  5.  Testis  of  host  No.  244,  Amblystoma  jeffersonianum,  autopsied  61  days 
after  implantation  of  graft  at  stage  31.  This  gonad  exhibits  the  scattered  arrange- 
ment of  germ  cells  and  the  absence  of  a  central  cavity  noted  in  the  testes  of  Figs. 
2  and  4.  Compare  with  graft  gonad  shown  in  Fig.  6. 

FIG.  6.  Ovary  of  graft  recovered  from  host  No.  244.  Although  the  central 
cavity  is  not  yet  developed,  the  germ  cells  are  peripheral  in  position  and  are  for 
the  most  part  in  heterotypic  prophase.  This  gonad  thus  resembles  an  ovary 
(see  Figs,  i  and  3)  rather  than  the  testes  of  the  host  from  which  it  was  recovered 
(see  Fig.  5). 


BIOLOGICAL  BULLETIN,    VOL.    LV. 


PLATE  I. 


R.    R.    HUMPHREY. 


338  R-    R-    HUMPHREY. 


PLATE  II.     EXPLANATION  OF  FIGURES. 

All  figures  on  this  plate  are  photomicrographs.  The  magnification  is  121  X 
except  for  Figs.  8  and  9,  in  which  it  is  162  X. 

FIG.  7.  Testis  of  host  No.  207,  Amblystoma  maculatum,  killed  61  days  after 
implantation  of  graft  at  stage  25. 

FIG.  8.  Left  ovary  of  donor  No.  207,  Amblystoma  jeffersonianum,  killed  78 
days  after  furnishing  graft  (at  stage  29)  for  implantation  in  host  No.  207.  Due 
to  the  age  at  which  this  animal  was  killed,  the  ovary  is  advanced  in  development  as 
compared  with  those  of  Figs,  i  and  3. 

FIG.  9.  Gonad  of  graft  recovered  from  host  No.  207.  Though  retarded  in  its 
differentiation,  this  gonad  is  apparently  an  ovary,  since  its  germ  cells  are  peripheral 
in  arrangement,  and  a  large  proportion  of  them  are  in  heterotypic  prophase  stages. 
Compare  with  Figs.  7  and  8. 

FIG.  10.  Ovary  of  host  No.  190,  Amblystoma  jeffersonianum,  killed  64  days 
after  implantation  of  graft  at  stage  33. 

FIG.  ii.  Left  testis  of  donor  No.  190,  Amblystoma  jeffersonianum,  killed  64 
days  after  furnishing  graft  for  implantation  in  host  No.  190. 

FIG.  12.  Gonad  of  graft  recovered  from  host  No.  190.  Though  atypical  in 
structure,  due  to  the  presence  of  mucous  connective  tissue,  this  gonad  is  apparently 
a  testis.  No  central  cavity  is  present,  the  germ  cells  are  scattered,  and  but  very 
few  of  them  are  in  heterotypic  prophase.  Compare  with  gonad  of  donor  (Fig.  n). 

FIG.  13.  Ovary  of  host  No.  188,  Amblystoma  jeffersonianum,  killed  64  days 
after  implantation  of  graft  at  stage  33. 

FIG.  14.  Left  testis  of  donor  No.  188,  Amblystoma  jeffersonianum,  killed  64 
days  after  furnishing  graft  for  implantation  in  host  No.  188. 

FIG.  15.  Gonad  of  graft  recovered  from  host  No.  188.  It  lacks  a  central 
cavity,  but  has  its  germ  cells  predominantly  peripheral  in  position,  and  frequently 
in  groups  or  "nests"  as  in  the  ovary.  Although  many  of  its  germ  cells  are  in 
heterotypic  prophase,  this  is  true  also  of  the  testis  of  the  donor.  This  gonad  is 
possibly  a  testis  modified  by  reason  of  its  development  in  a  graft  in  a  female  host. 


BIOLOGICAL   BULLETIN,    VOL.    LV. 


PLATE   II. 


Wfc 


i ! 


13 

R.    R.    HUMPHREY. 


I14 


15 


ON    THE    PROPERTIES    OF    THE    GONADS    AS    CON- 
TROLLERS OF  SOMATIC   AND    PSYCHICAL 
CHARACTERISTICS. 

XI.    HORMONE   PRODUCTION  IN  THE  NORMAL  TESTES,   CRYP- 

TORCHID  TESTES  AND  NON-LIVING  TESTIS  GRAFTS 

AS  INDICATED  BY  THE  SPERMATOZOON 

MOTILTTY  TEST.1 

CARL  R.  MOORE, 

HULL  ZOOLOGICAL  LABORATORY,  THE  UNIVERSITY  OF  CHICAGO. 

I.   INTRODUCTION. 

Advances  in  the  study  of  the  internal  secretions  are  very  often 
largely  dependent  upon  the  development  of  successful  indicators 
for  the  substances  concerned.  Since  the  studies  of  Brown- 
Sequard,  innumerable  attempts  have  been  made  to  increase  our 
knowledge  of  the  internal  secretions  of  the  sex  glands,  and 
indeed  vast  stores  of  information  have  been  accumulated  through 
these  investigations.  The  chief  difficulty  in  many  of  these 
attempts  and  especially  in  attempts  to  obtain  the  hormone 
principle  in  extractions,  has  been  the  lack  of  an  applicable 
indicator  of  the  substances  sought  for  isolation. 

A  tremendous  step  forward  in  the  study  of  the  female  hormone 
was  the  demonstration  of  the  details  of  the  oestrous  cycle  as 
indicated  by  vaginal  smears  first  by  Stockard  and  Papanicolau 
('17)  in  the  guinea  pig  and  later  by  Long  and  Evans  ('22)  in  the 
rat.  By  the  vaginal  smear  method,  one  is  enabled  to  determine 
the  presence  or  absence  of  substances  concerned  with  the  regula- 
tion of  the  oestrous  cycle.  With  such  a  useful  indicator,  the 
advances  made  in  the  study  of  the  internal  secretions  of  the 
ovary  have  been  indeed  marked. 

On  the  male  side,  however,  the  situation  has  been  a  less  happy 
one  from  the  standpoint  of  real  advancement.  In  some  species 

1  This  investigation  has  been  aided  by  a  grant  from  the  Committee  for  research 
in  problems  of  sex  of  the  National  Research  Council;  grant  administered  by  Prof. 
F.  R.  Lillie. 

339 


340 


CARL   R.    MOORE. 


of  the  bird,  notably  certain  breeds  of  the  domestic  fowl,  the 
male  feather  pattern,  behavior,  and  head  furnishings  have 
afforded  a  good  criterion  of  testicular  presence  and  activity 
though  many  details  were  insufficiently  known  until  of  late  to 
make  conditions  as  well  understood  as  was  needed.  The  ex- 
tensive work  of  Domm  ('27)  on  the  brown  leghorn  breed  has 
given  a  much  greater  appreciation  of  the  many  pitfalls  that 
present  themselves  in  this  field  (for  a  review  of  the  extensive 
literature  on  this  subject,  see  Domm's  paper). 

\Yhen  we  approach  the  study  of  the  internal  secretions  of  the 
gonads  in  the  male  mammal,  however,  a  careful  analysis  will 
show  the  marked  absence  of  useful  criteria  to  indicate  the 
activity  of  the  testis  hormone,  operating  over  limited  periods  of 
time.  It  is  true  that  there  is  the  sex  impulse,  supposedly 
entirely  under  the  control  of  the  internal  secretions  of  the  testicles, 
but  many  things  lead  us  to  believe  this  supposition  to  be 
erroneous.  A  castrated  male  theoretically  should  lose  its  attrac- 
tion for  the  female,  but  I  have  repeatedly  utilized  guinea  pigs 
castrated  at  30  days  of  age  as  testers  for  the  period  of  female 
acceptance  for  some  months  after  castration.  Stone  ('27)  has 
recently  reported  that  young  male  rats  castrated  at  the  age  of 
three  months  will  continue  to  copulate  with  females  for  periods 
of  four,  five  and  even  eight  months.  And  it  is  reported  that  the 
eunuch,  though  castrated  early  in  life,  will  years  afterward  have 
not  only  an  attraction  toward  the  female  but  experiences  a 
degree  of  satisfaction  in  this  association. 

It  is  likewise  true  that  the  growth  of  the  penis,  seminal  vesicles 
(when  present),  prostate,  etc.,  are  to  a  large  extent  dependent 
upon  the  internal  secretions  of  the  testis  but  not  only  have  the 
variations  in  such  structures  proven  so  great  as  to  make  an 
assay  of  a  given  experimental  procedure  difficult  and  often 
impossible,  but  also,  if  castrations  are  made  on  adult  animals, 
to  be  followed  by  such  procedures  as  testis  transplantations, 
injections  or  other  possible  approaches,  the  question  of  the 
condition  of  these  structures  as  representing  a  balance  between 
postoperative  regression  or  possible  stimulation  from  the  materials 
or  conditions  utilized  often  presents  insurmountable  barriers. 

Such  other  indicators  for  testicular  internal  secretions  as 
individual  body  weight,  body  length,  fat  deposition,  hair  coat, 


ON    THE    PROPERTIES    OF    THE    GONADS.  34! 

and  pugnacity  as  have  been  utilized  by  other  workers  serve 
often  to  mislead  the  investigator  due  to  the  lack  of  specificity 
of  the  indicator  (for  further  criticisms  of  this  phase  see  my 
papers,  '21  and  '22). 

During  the  course  of  a  study  of  the  physiology  of  the  scrotum 
or  its  heat  regulating  effects  on  the  generative  tissues  of  the  te^ti- 
(Moore,  '240,  6;  '26,  '27,  and  '28;  Moore  and  Quick,  '24)  a 
possible,  fairly  satisfactory  indicator  for  the  internal  secretions 
of  the  testis  in  the  differential  survival  of  the  capacity  for 
motility  of  epididymal  spermatozoa  was  discovered  by  accident. 
The  same  conditions  were  also  discovered  by  Benoit  ('26)  a 
little  earlier,  in  the  course  of  his  beautiful  work  on  the  histology 
and  cytology  of  the  epididymis.  The  application  of  this  "sper- 
matozoon-motility"  test  for  the  testis  hormone  has  been  under 
investigation  in  this  laboratory  for  longer  than  three  years. 
The  principle  of  the  test  may  be  expressed  in  details  for  the 
guinea  pig. 

When  both  testes  of  an  adult  guinea  pig  are  removed  from  the 
animal,  leaving  the  inferior  portion  of  the  epididymides,  con- 
taining their  millions  of  spermatozoa,  in  the  normal  scrotal 
position,  one  finds  that  the  spermatozoa  gradually  lose  their 
capacity  for  motility  when  these  are  suspended  in  physiological 
saline  solution.  A  lessened  capacity  for  motility  is  evident 
within  a  few  days  after  testis  removal  and  seldom  can  one  see 
any  degree  of  motility  in  the  spermatozoa  after  a  period  of 
twenty-three  days  following  the  operation.  However,  if  instead 
of  removing  both  testes,  one  is  allowed  to  remain  normal,  the 
single,  opposite,  epididymis  will  contain  spermatozoa  that  show 
motility  when  suspended  in  saline  solution  for  a  period  of  sixty- 
five  to  seventy  days  (Moore,  '28).  The  difference  between  the 
23  days  retention  of  the  capacity  for  motility  when  both  testes 
have  been  removed  and  that  of  65  days  when  one  testis  has 
remained,  has  been  proven  to  be  an  expression  of  the  internal 
secretion  of  the  testis  (for  further  details  of  this  reaction  see 
Moore,  '28). 

While  it  is  freely  admitted  that  the  spermatozoon  motility 
reaction  has  many  limitations  we  have  found  it  very  useful  and  it 
will  continue  to  be  useful  until  a  better  hormone  indicator  has 


342 


CARL    R.    MOORE. 


been  discovered.     In   the   following  pages  a   few  items  of  in- 
formation with  respect  to  its  usefulness  will  be  presented. 

II.   HORMONE  PRODUCTION  BY  NORMAL  TESTES. 

Utilizing  the  spermatozoon  motility  reaction  in  the  guinea  pig 
as  described  above,  I  have  attempted  to  study  hormone  pro- 
duction in  the  normal  testis  to  learn  more  concerning  its  action 
upon  the  life  of  spermatozoa  when  it  is  removed  from  the  animal 
through  castration  at  varying  periods  during  the  possible  life  of 
the  mature  germ  cell.  It  has  been  indicated,  for  example,  that 
under  the  influence  of  the  full  hormone  compliment  of  one 
testis,  the  spermatozoon  life,  as  shown  by  its  capacity  to  exhibit 
motion  in  physiological  saline  solution,  gradually  wanes  until 
after  approximately  65  to  70  days  it  no  longer  responds  to  this 
stimulus.  Should  we,  for  example,  wish  to  supply  the  hormone 
by  testis  transplantation  or  by  injection  of  material  supposed 
to  contain  it,  we  should  be  able,  if  possible,  to  test  the  effect  in 
the  shortest  time  within  which  the  reaction  will  indicate  any 
effect.  What  influence,  therefore,  does  hormone  supplied  by  a 
testis  in  situ  exert  when  it  acts  for  ten,  fifteen  or  twenty  days? 
The  following  procedure  will  present  the  method  employed. 

Young  adult  guinea  pigs  are  operated  under  ether  anaesthesia 
through  a  low  mid-abdominal  incision  and  one  testis  withdrawn 
into  the  field  of  operation.  The  testis  is  carefully  separated 
from  the  inferior  pole  of  the  epididymis  (tail  portion),  the 
internal  spermatic  vessels  are  ligated  and  the  testis,  along  with 
the  head  and  body  of  the  epididymis  removed.  The  remaining 
(inferior)  portion  of  the  epididymis,  connected  with  its  vas 
deferens,  is  then  carefully  replaced  in  the  scrotum — a  necessary 
precaution  (see  Heller,  '29).  Ten  or  fifteen  days  later  the 
opposite  normal  testis  is  removed  entire  through  a  scrotal 
incision.  At  selected  intervals  after  the  second  operation,  the 
animals  are  sacrificed,  the  isolated  epididymis  finely  hashed  with 
scissors  in  a  small  quantity  of  physiological  saline  and  examined 
immediately  with  the  microscope  for  spermatozoon  motility. 
To  properly  express  gradations  in  motility  *  signs  have  been 
employed  in  which  the  normal  movement  is  expressed  by  ****; 
the  barest  vibratile  movement  on  the  part  of  a  few  spermatozoa 
(perhaps  I  in  10,000  will  contract  weakly  with  little  or  no  trans- 


ON    THE    PROPERTIES    OF    THE    GONADS. 


343 


lation)  is  rated  *.     Where  no  movement  can  be  detected  the 
observation  is  designated  o. 

Table  I.  will  serve  to  illustrate  the  observations  on  the  motility 
of  spermatozoa  obtained  from  the  isolated  epididymis  of  animals 
whose  normal  testis  was  allowed  to  remain  for  10,  15,  20,  30, 
and  40  days  after  the  epididymis  to  be  tested  had  been  isolated. 

TABLE  I. 

UNILATERAL  EPIDIDYMAL  ISOLATION;  OPPOSITE  TESTIS  REMOVED  SUBSEQUENT  TO 
ISOLATION  AS  INDICATED  IN  DIFFERENT  GROUPS  (GUINEA  PIG). 


Excess  Life 

beyond  23 

Animal. 

Epididymal 

Isolation. 

Killed. 

Days  after 
Isolation. 

Motility. 

Days  At- 
tributed to 

Hormone 

Effect. 

(delayed  removal  i< 

)  days) 

5i7 

i-3i 

3-i 

30 

* 

7  days 

5i8 

i-3i 

3-3 

32 

* 

9  days 

520 

i-3i 

3-5 

34 

o 

521 

i-3i 

3-5 

34 

o 

- 

(delayed  removal  15  days) 

406 

6-10 

7-1  1 

3i 

** 

8  days 

407 

6-10 

7-14 

34 

*** 

ii  days 

409 

6-10 

7-20 

40 

0 

409 

6-10 

7-20 

40 

** 

17  days 

410 

6-1 

7-24 

44 

** 

20  days 

411 

6-1  1 

7-29 

48 

o 

412 

6-1  1 

7-29 

48 

o 

4i3 

6-1  1 

7-29 

48 

o 

414 

6-1  1 

7-29 

48 

o 

415 

6-1  1 

7-29 

48 

* 

25  days 

(delayed  removal  20  days) 

441 

IO-2I 

11-29 

39 

* 

1  6  days 

442 

10-21 

11-29 

39 

* 

1  6  days 

443 

IO-2I 

12-3 

43 

* 

20  days 

444 

IO-2I 

12-3 

43 

* 

20  days 

445 

10-21 

12-6 

46 

o 

446 

10-21 

12-6 

46 

o 

(delayed  removal  30  days) 

450 

10-25 

12-8 

44 

** 

21  days 

447 

10-25 

12-17 

5 

* 

30  days 

451 

10-25 

I2-2O 

56 

* 

33  days 

452 

10-25 

12-24 

60 

0 

(delayed  removal  40  day~ 

469 

12-3 

1-25 

53 

* 

30  days 

472 

12-3 

1-25 

53 

o 

470 

12-3 

1-27 

55 

0 

473 

12-3 

1-27 

55 

* 

32  days 

474 

12-3 

I-3I 

59 

o 

344 


CARL    R.    MOORE. 


To  understand  the  observations  recorded  it  must  be  re- 
membered that  when  an  epididymis  is  isolated  from  its  testis 
and  both  testes  are  removed  from  the  animal,  the  spermatozoa 
contained  within  an  epididymis  retain  their  capacity  to  show 
motility  for  a  period  of  23  days;  this  we  may  call  the  basic  life 
period  and  realize  that  they  will  live  for  this  period  without  any 
hormone  being  produced  by  the  testicle. 

From  the  table  it  can  be  seen  that  under  "delayed  removal  10 
days"  the  spermatozoa  were  observed  to  show  motility  for  32 
days  or  nine  days  longer  than  expected,  had  both  testes  been 
removed  at  the  time  of  epididymal  isolation.     We  see,  therefore, 
that  the  hormone  supplied  by  the  normal  testicle  for  a  period 
of  ten  days  before  its  removal,  actually  extended  the  life  of  the 
spermatozoon  nine  days.     Similarly,  hormone  supplied  for  fifteen 
days  extended  the  retention  of  the  capacity  for  motility  for  a 
similar  length  of  time  (actually  slightly  longer  since  in  animal 
415  a  few  sperm  were  seen  to  move  slightly  on  the  48th  day  after 
isolation  or  an   increase   of  25   days  above   the  basic  23   days 
expected).     When  the  normal  testis  was  allowed  to  remain  20 
days  after  epididymal  isolation,  motile  capacity  was  increased  20 
days  beyond  what  it  would  have  been  had  both  testes  been 
removed    at    the    first    operation.     Hormone    supplied    by    the 
normal  testis  for  30  days  permitted  retention  of  spermatozoon 
motility  up  to  56  days  or  33  days  longer  than  the  natural  life 
without  hormone  being  supplied.     A  hormone  supply  from  the 
normal  testis  for  forty  days  increased  the  sperm  life  by  little 
more  than  thirty  days.     But  it  must  be  remembered  that  as  we 
add  to  the  length  of  time  after  operation  we  gradually  approach 
the  natural  limits  of  spermatozoon  life  even  with  a  full  compli- 
ment of  hormone;  this  limit  is  65  to  70  days.     We  could  not 
therefore  expect  the  relative  progressive  effectiveness  to  continue 
much  beyond  a  4O-day  normal  testis  retention  because  of  the 
approach  to  the  maximum  period  of  persistence  of  sperm  under 
a  continuous  hormone   influence.     In  an  earlier  paper   I   have 
emphasized  that  even  utilizing  the  greatest  possible  care  in  the 
selection  of  standard  animals  for  operation  and  in  doing  the 
operation  itself,  there  is  an  individual  animal  variability  that 
cannot  be  eliminated;  at  best  we  can  only  make  an  approach 
toward  quantitative  relationships. 


ON    THE   PROPERTIES    OF   THE   GONADS.  345 

Utilizing  the  basic  23  days  as  the  approximate  maximum  of 
retention  of  the  capacity  for  motility  on  the  part  of  spermatozoa 
when  no  hormone  is  being  supplied  (and  in  scores  of  observations 
I  have  never  observed  motility  for  periods  above  23  days)  we 
see  that  one  can  actually  detect  the  influence  of  the  testis 
hormone  when  it  is  supplied  for  only  ten  days.  Due  to  the 
individual  animal  variability,  I  would  consider  attempts  to  read 
reactions  more  finely  as  decidedly  unprofitable.  In  fact,  to  err 
on  the  safe  side,  I  have  arbitrarily  chosen  to  regard  any  tested 
substance  or  condition  involving  gonads  as  negative  unless  the 
capacity  for  motility  is  retained  for  thirty  days  or  longer. 

It  is  of  interest  to  examine  the  data  of  the  above  table  with 
reference  to  what  they  may  tell  us  of  hormone  production  and 
storage.  When  a  ten  day  hormone  supply  by  the  normal  testis 
shows  an  effect  of  ten  days  in  the  reaction,  and  likewise  when 
spermatozoon  motility  is  extended  15,  20,  and  30  days  beyond 
the  basic  expectations  in  conditions  wherein  the  testis  was 
present  for  15,  20  and  30  days  after  epididymal  isolation,  one 
must  conclude,  I  believe — (i)  that  hormone  secretion  is  a 
continuous  process  and  (2)  that  the  hormone  is  not  stored  within 
the  body.  When  hormone  is  supplied  by  the  normal  testis,  for 
10  days,  the  reaction  indicates  an  effect  for  the  same  length  of 
time. 

III.   HORMONE  PRODUCTION  IN  CRYPTORCHID  TKSTES. 

It  has  long  been  known  that  man  or  the  domestic  mammals 
may  experience  a  failure  of  testicular  descent  into  the  scrotum. 
Such  animals,  although  always  sterile,  nevertheless  possess  their 
full  compliment  of  secondary  sex  characters;  they  are  spoken  of 
as  Cryptorchid  individuals.  The  undescended  testes  of  such 
animals  have  long  been  known  to  lack  a  germinal  epithelium; 
the  gametogenetic  function  of  the  testis  is  deficient  but  its 
internal  secretory  effects  are  not  visibly  diminished.  It  is  now 
known  that  a  normal  testis  removed  from  the  scrotum  and 
confined  within  the  abdomen  very  rapidly  loses  its  germinal 
epithelium  and  assumes  within  a  month  or  two,  almost  identical 
characteristics  to  those  testes  that  have  never  descended.  The 
cause  of  the  degeneration  of  testes  confined  within  the  abdomen 

has  been  found  to  be  the  warmer  environment  of  the  abdomen 
23 


346 


CARL    R.    MOORE. 


and  the  function  of  the  scrotum  has  thus  been  seen  to  be  that  of 
a  local  thermoregulator  (for  details  of  this  work  see  Moore, 
I924&,  19246,  and  1926;  Moore  and  Quick,  '24). 

Regarding  the  amount  of  hormone  produced  by  such  a  cryp- 
torchid  testis,  occurring  normally  or  artificially  made,  little 
is  known.  It  could  be  assumed  perhaps  that  a  smaller 
quantity  of  hormone  might  be  required  to  produce  or  to  maintain 
the  secondary  sex  characteristics  than  would  be  required  to 
maintain  completely  normal  male  conditions.  Lipschutz  and 
his  co-workers  have  maintained  that  in  the  rabbit  I  per  cent, 
of  the  normal  amount  of  testicular  tissue  is  sufficient  to  maintain 
all  the  secondary  sex  characters  ('22).  On  the  other  hand,  it 
has  been  assumed  by  some  investigators  that  any  condition 
leading  to  an  "Apparent  increase  in  interstitial  cells"  whether 
by  testis  transplantation,  X-rays,  vasoligation  (this  latter  is  the 
basis  of  the  contentions  underlying  the  ideas  of  the  Steinach 
rejuvenation  hypothesis)  or  any  other  means,  presages  an  in- 
creased production  of  hormone.  The  argument  proceeds  from 
the  assumption — First,  that  the  apparent  compensatory  hyper- 
trophy of  interstitial  cells  is  real  (see  discussion  Moore,  '240; 
Bascom,  '25) ;  and  second,  that  the  hormone  is  produced  ex- 
clusively by  the  Leydig  cells.  Some  authors  have  gone  so  far 
as  to  speak  of  castrated  males,  bearing  testis  grafts  containing 
appreciable  amounts  of  interstitial  tissue,  as  "supermales,"  an 
implication  that  I  consider  without  any  basis  of  fact. 

In  order  to  gain  any  new  information  possible  regarding  the 
quantitative  aspects  of  hormone  production  by  cryptorchid 
testes,  I  have  utilized  the  guinea  pig  in  the  following  manner: 
Young  adult  guinea  pigs  have  been  operated  so  that  one  testicle 
was  removed  from  the  scrotum  into  the  abdomen  and  the 
inguinal  canal  closed  to  prevent  scrotal  redescent.  Four  months, 
and  five  months,  later  a  second  operation  was  made  to  isolate 
the  normal  epididymis  from,  and  to  remove,  the  normal 
testicle.  We  thus  have  an  isolated  epididymis  with  its  sper- 
matozoon content  to  use  as  a  test  for  the  hormone  produced  by 
the  opposite  degenerate  four  or  five  months  experimental  cryp- 
torchid testis. 

Table  II.  is  a  record  of  observations  made  on  eleven  animals 
in  which  one  testis  was  confined  to  the  abdomen  for  four  months 


ON    THE    PROPERTIES    OF    THE    GONADS. 


347 


and  upon  six  animals  where  hormone  supply  came  from  a  testis 
confined  in  the  abdomen  for  five  months. 

TABLE  II. 

UNILATERAL   CRYPORCHIDISM  FOUR   MONTHS;   NORMAL  TESTIS   REMOVED  FROM 

EPIDIDYMIS,  AND  EFFECT  OF  FOUR  MONTHS  CRYPTORCHID  TESTIS 

ON  SPERMATOZOON  MOTILITY  DETERMINED. 


Isolation  of 

Days 

Wt.  of 

Animal. 

Normal 

Killed. 

since 

Motility. 

Cryptorchid 

Epididymis. 

Isolation. 

Testes.* 

393 

Oct.  13 

Nov.  25 

43 

*** 

Not  recorded 

394 

Oct.  13 

Dec.     5 

53 

** 

0.26    gms. 

402 

Oct.  17 

Dec.     8 

52 

*** 

0.424 

396 

Oct.  13 

Dec.    12 

60 

* 

O.2I 

397 

Oct.  13 

Dec.    12 

60 

o 

0.175 

398 

Oct.  13 

Dec.    17 

65 

* 

0.158 

400 

Oct.  13 

Dec.    1  7 

65 

o 

0.130 

403 

Oct.  17 

Dec.   21 

65 

# 

Not  recorded 

404 

Oct.  17 

Dec.   26 

70 

* 

0.170  gms. 

416 

Oct.  17 

Dec.  31 

75 

* 

0.255     " 

4i" 

Oct.  17 

Jan.      7 

82 

0 

0.205 

UNILATERAL  CRYPTORCHID  FIVE  MONTHS;  SUBSEQUENT 
TREATMENT  SIMILAR  TO  ABOVE. 


475 

Apr.  28 

June  26 

59 

** 

0.153  gms. 

476 

Apr.  28 

June  30 

63 

* 

0.13       " 

477 

Apr.  28 

July     6 

69 

o 

Not  recorded 

478 

Apr.  28 

July     6 

69 

* 

Not  recorded 

479 

Apr.  30 

July    12 

73 

o 

Not  recorded 

481 

Apr.  30 

July    12 

73 

* 

0.095  gms. 

*  Testis  weight,  without  epididymis  or  fat  body. 

It  will  be  seen  from  Table  II.  that  spermatozoa  within  the 
isolated  epididymis  have  been  observed  to  retain  their  capacity 
to  show  movement  on  proper  stimulation  for  seventy  to  seventy- 
five  days.  Since  the  normal  testicle,  actively  carrying  on  its 
spermatogenetic  activity  supplies  only  sufficient  hormone  to 
permit  the  sperm  to  live  for  the  same  length  of  time,  we  must 
conclude  that  the  hormone  producing  capacity  of  a  non-gameto- 
genetic,  degenerative,  or  cryptorchid  testicle  as  measured  by  the 
spermatozoon  motility  test  is  the  equivalent  of  the  normal 
testis. 

Figure  i  is  introduced  to  show  the  histological  character  of 
the  testicle  after  abdominal  confinement  (experimental  cryp- 


348 


CARL    R.    MOORE. 


torchidism)  for  a  period  of  seven  months;  the  microphotograph 
shows  a  portion  of  a  section  from  the  testicle  of  animal  No.  481. 
This  testicle  had  been  confined  within  the  abdomen  for  a  period 
of  five  months,  when  the  epididymis  of  the  opposite  testicle  was 
isolated  for  the  subsequent  spermatozoon-motility  test  and  the 
normal  testicle  removed  from  the  animal.  Reference  to  Table  II. 


FIG.   I.     Photomicrograph  of  portion  of  7  months  cryptorchid  testicle  (animal 
no.  481)  showing  shrunken  seminiferous  tubules  separated  by  interstitial  tissue. 

will  serve  to  recall  that  the  test  epididymis  contained  a  few  living 
spermatozoa  73  days  after  epididymal  isolation  and  these  few 
exhibited  very  weak  motility  on  suspension  in  saline  solution. 
When  the  animal  was  sacrificed  on  the  73d  day  after  epididymal 
isolation  the  testicle  had  been  confined  in  the  abdomen  for  a 
period  slightly  longer  than  seven  months.  The  weight  of  the 
organ,  after  removal  of  its  attached  epididymis,  was  0.095 
grams.  Since  the  average  weight  of  eight  normal  testicles, 
without  the  epididymis,  removed  from  similar  sized  animals  and 
at  the  same  time  of  the  year,  was  1.7  grams  (1.34  minimum  wt.— 


OX    THE    PROPERTIES    OF    THE    GONADS. 


349 


2.06  maximum)  it  will  be  appreciated  that  the  weight  of  this 
cryptorchid  testis  represents  2.8  per  cent,  of  the  total  testicular 
weight  of  the  normal  animal.  Had  the  epididymides  been 
included  in  this  weight  the  percentage  of  the  normal  testicular 
weight  represented  by  this  cryptorchid  testis  would  have  been 
considerably  less;  the  spermatozoon  and  secretion  mass  within 
the  normal  epididymis  being  very  much  greater  than  the  slight 
fluid  content  of  the  cryptorchid  epididymis.  It  can  be  concluded, 
therefore,  that  the  cryptorchid  testis  representing  2.8  per  cent. 
of  the  normal  testicular  mass  was  producing  sufficient  hormone 
to  maintain  the  life  of  spermatozoa  in  the  isolated  epididymis  for 
the  same  period  as  would  the  hormone  produced  by  two  normal 
testes. 

Figure  I  shows  that  the  seminiferous  tubules  of  the  seven 
month  cryptorchid  testis  were  very  much  reduced  in  caliber  and 
consisted  of  a  basement  membrane,  somewhat  thickened,  and  a 
few  Sertoli  nuclei  and  reticulum;  the  tubules  were  rather  widely 
separated  by  interstitial  tissue.  Fig.  2  is  a  drawing  of  the 


.-1C 


FIG.  2.     Drawing  of  tubule  marked  off  by  white  lines  in  Fig.  2.     bm,  basement 
membrane;  ic,  interstitial  cell;  S,  Sertoli  nucleus. 

tubule  marked  off  by  white  lines  at  the  upper  part  of  Fig.  i. 
The  thickened  basement  membrane  is  more  clearly  shown  and 
the  character  of  the  contents  of  the  tubule  indicates  an  absence 
of  any  germ  cells;  the  nuclei  that  are  visible  are  believed  to  be 
Sertoli  nuclei.  Careful  microscopic  study  has  failed  to  bring  to 
my  attention  any  cell  that  appeared  different  from  those  repre- 
sented in  this  figure  and  it  is  for  that  reason  that  I  believe  no 


350 


CARL    R.    MOORE. 


germinal   cells  were  present  in   this   testis,   at   the   time   of  its 
removal. 

IV.   HORMONE  PRODUCTION  BY  TESTIS  GRAFTS. 

The  question  of  the  function  of  testis  transplants  must  of 
necessity  be  considered  under  at  least  two  categories:  (i)  The 
function  of  grafts  which  have  become  successfully  incorporated 
into  the  host  organism  and  remain  as  living  masses  of  testicular 
tissue,  and  (2)  the  function  of  such  masses  of  testis  tissue  trans- 
planted into  various  parts  of  the  host  organism,  which  by  reason 
of  host  resistance  to  the  transplant  or  because  of  too  great  a 
mass  of  tissue  for  vascularization,  dies  and  is  resorbed  or  sloughed 
out  of  the  incorporation  bed  often  with  pronounced  suppuration. 
It  is  known  that  living  testis  grafts  can  be  obtained  and  that 
they  will  function.  This  question,  along  with  the  presentation 
of  personal  observations,  has  been  reviewed  by  me  at  some 
length  (Moore,  '26).  The  question  of  the  function  of  testis 
tissue  transplanted  into  a  host  organism  under  conditions  that 
have  been  so  unfavorable  as  to  prevent  its  retention  and  growth 
has  been  dealt  with  most  usually  by  the  clinician.  For  various 
reasons  many  cases  of  transplantation  in  man  of  human  testis 
tissue  or  testicular  tissue  from  another  mammal  such  as  the  ram, 
boar,  monkey  or  deer,  have  been  done.  The  effects  reported 
are  so  all-embracing  that  discredit  of  all  effect  is  engendered 
(for  discussion  of  this  work  see  Moore,  '26).  In  general  it  may 
be  repeated  that  the  effects  reported  have  been  expressed  in 
terms  of  the  subjective  feelings  of  the  patient — whether  he  may 
feel  better  after  remaining  quietly  in  bed  for  a  week  or  longer 
after  the  operation;  or  whether  after  the  suggestions  and  dis- 
cussion of  the  question  and  the  anticipations  of  the  operation 
and  its  outcome,  he  has  a  greater  desire  for  coitus;  or  whether 
the  patient  feels  that  he  can  walk  more  sprightly  or  feels  that  he 
can  climb  a  stairs  two  steps  at  a  time  instead  of  the  customary  pre- 
operation  'One  step.  In  short,  such  evidence  is  worthless  from 
the  scientific  point  of  view. 

In  order  to  study  by  objective  means  the  question  of  the 
function  of  such  non-living  testis  transplants,  I  have  utilized  the 
guinea  pig  as  the  experimental  animal  and  the  spermatozoon 
motility  test  as  an  indicator  of  effectiveness.  In  an  earlier  paper 


OX    THE    PROPERTIES    OF    THE    GONADS.  351 

(Moore,  '28)  I  included  a  few  observations  then  at  hand  and 
have  since  given  additional  attention  to  the  problem.  The 
method  employed,  in  brief,  is  the  bilateral  isolation  of  the 
epididymides  along  with  removal  of  the  testes  from  the  animal. 
The  two  testes  removed  were  replaced  immediately  in  an  es- 
pecially prepared  subcutaneous  incorporation  bed  made  by 
tunneling  under  the  skin,  with  some  destruction  of  skin  muscula- 
ture and  a  general  scarification  of  the  particular  region.  Each 
testicle,  cut  into  two  parts,  was  placed  in  a  separate  implantation 
bed,  one  on  each  side  of  the  mid-ventral  line  of  incision. 

In  addition  to  the  subcutaneous  transplantation  of  the  animal's 
own  two  testes  at  the  time  of  epididymal  isolation,  a  few  cases  of 
multiple  transplantations  were  studied  with  the  idea  that  perhaps 
a  small  amount  of  hormone  might  be  liberated  from  the  intro- 
duced tissue  which  if  introduced  more  than  once  would  con- 
ceivably show  a  greater  effectiveness.  Accordingly  at  the  time 
of  bilateral  epididymal  isolation,  two,  one-fourth  testes,  were 
introduced  subcutaneously  at  the  time  of  the  first  operation  as 
well  as  on  the  3d  and  5th  day  following.  In  each  animal, 
therefore,  six  transplantations  were  made,  the  aggregate  amount 
of  tissue  transplanted  being  one  and  one  half  testes.  The 
observations  on  four  such  experiments  are  recorded  in  Table 
lllB. 

Since  an  arbitrary  choice  of  thirty  days  after  operation  for 
effectiveness  to  be  registered  was  made,  animals  were  sacrificed 
close  to  this  period  for  the  study  of  the  spermatozoon  content 
of  the  isolated  epididymides.  From  section  II.  it  will  be  seen 
that  motility  of  spermatozoa  at  this  time  would  indicate  the 
equivalent  of  effectiveness  of  the  normal  testis  present  for  seven 
to  ten  days.  Too  much  uncertainty  surrounds  the  application 
of  this  test  to  make  it  profitable  to  attempt  readings  at  an  earlier 
date. 

Table  III.  presents  some  of  the  observations  recorded. 

Among  the  nineteen  animals  whose  isolated  epididymides  were 
studied  for  spermatozoon  movement  between  the  25th  and  36th 
day  after  autoplastic  transplantation  of  two  testes,  only  two 
animals  have  shown  any  movement  of  spermatozoa  and  in  each 
case  (animals  No.  320,  No.  456)  observed  on  the  3ist  day  the 
motility  was  the  weakest  possible  for  a  positive  reading.  Amid 


352 


CARL   R.    MOORE. 


TABLE  III. 

A.   THE  EFFECT  OF  NON-LIVING  TESTIS  GRAFTS  ON  SPERMATOZOON 
MOTILITY  (GUINEA  PIG). 


Animal. 

Date- 
Operation. 

Date- 
Killed. 

Days 
after 
Opera- 
tion. 

Motility. 

3<>4 

4-  6-27 

5-12-27 

36 

o 

many  non-mot. 

sperm 

365 

4-  6-27 

5-12-27 

36 

0 

370 

4-  8-27 

5-12-27 

34 

0 

«                          «                     (I 

11 

371 

4-  8-27 

5-12-27 

34 

o 

1                          t(                     <  t 

it 

372 

4-  8-27 

5-12-27 

34 

o 

I                          It                    tt 

n 

373 

4-  8-27 

5-12-27 

34 

o 

<                          It                     « 

it 

320 

11-22-26 

12-23-26 

3i 

* 

(i    n  1000  weak 

mot.) 

436 

10-15-27 

11-15-27 

3i 

* 

(i  in  1000  weak 

mot.) 

435 

10-15-27 

11-15-27 

3i 

0 

347 

3-  8-27 

4-  7-27 

30 

o 

many  non-mot. 

sperm 

376 

4-15-27 

5-15-27 

30 

o 

377 

4-15-27 

5-15-27 

30 

0 

«  t                    i                ti 

* 

439 

10-20—27 

11-19-27 

30 

o 

440 

10-20-27 

11-19-27 

30 

0 

it                    i                4  . 

1 

437 

10-20-27 

11-19-27 

30 

0 

455 

10-26-27 

11-25-27 

30 

o 

it                     t                it 

* 

457 

10-27-27 

11-25-27 

29 

o 

(  t                     t                t( 

* 

458 

1  0-2  7-2  7 

11-25-27 

29 

o 

"                     '                ** 

* 

453 

10-25-27 

11-19-27 

25 

o 

t  (                    t                i  « 

' 

B.   MULTIPLE  GRAFTS  AFTER  BILATERAL  EPIDIDYMAL  ISOLATION. 


Animal. 

Transplantation  Days  after 
Epididymal  Isolation. 

Killed,  Days  after 
Testis  Removal. 

Motility. 

553 

ist,         3d,         5th 

30 

0 

554 

ist,         3d,          5th 

30 

0 

555 

ist,          3d,          5th 

30 

* 

549 

ist,          3d,          sth 

32 

o 

the  field  of  millions  of  spermatozoa,  here  and  there,  an  individual 
cell  could  be  seen  to  show  a  weak  contraction,  perhaps  a  weak 
vibratile  movement  every  thirty  seconds;  a  rough  estimate  of 
i  in  1000  was  made  to  give  a  relative  notion  of  the  quantity 
of  spermatozoa  capable  of  movement.  In  contrast  to  this, 
eleven  animals  observed  a  shorter  period  of  time  after  operation 
(25  to  30  days)  were  all  negative;  no  spermatozoon  movement 
could  be  seen.  One  animal  (No.  555)  receiving  six  transplanta- 
tions of  one  fourth  of  one  testis  at  three  different  operations 
subsequent  to  testis  removal,  showed  a  few  spermatozoa  capable 
of  weak  movement  on  the  3Oth  day  after  operation,  whereas 


ON    THE   PROPERTIES    OF   THE   GONADS.  353 

two  others  on  the  same  day  and  one  on  the  32d  day  after  operation 
failed  to  show  any  spermatozoa  capable  of  exhibiting  movement 
despite  the  fact  that  quantities  of  normal  looking  sperm  were 
present. 

These  results  show,  therefore,  that  subcutaneous  transplanta- 
tion of  testes  provided  such  a  small  amount  of  hormone  (if  any 
at  all)  that  its  effect  was  less  than  the  effect  of  a  normal  testis 
remaining  in  situ  for  seven  to  ten  days  after  operation. 

It  is  difficult  or  impossible,  as  pointed  out  above,  to  prevent 
individual  animal  variation.  Whether  the  two  positive  readings 
on  the  3 ist  day  are  to  be  explained  on  this  basis  of  more  virile 
spermatozoa  or  as  an  indicator  of  some  hormone  effect  cannot  be 
stated.  But  since  all  operations  were  done  alike,  and  equivalent 
masses  of  tissues  transplanted,  it  would  seem  as  if  animals 
sacrificed  earlier  (between  25  and  30  days)  would  have  given  as 
strong  or  a  stronger  reaction  than  these  three.  In  any  event, 
should  we  attribute  the  results  to  hormone  production  and 
express  it  as  a  positive  effect  of  the  transplantations,  the  mildness 
of  the  reaction  would  still  be  evident.  At  best  it  is  a  questionable 
indication  of  hormone  production. 

The  transplanted  tissue  reactions  have  been  characteristic  in 
all  cases.  A  few  days  after  transplantation  the  graft  site,  con- 
siderably removed  from  the  line  of  skin  incision,  is  swollen  and 
decidedly  reddened;  the  elevation  caused  by  the  transplanted 
tissue,  at  first  scarcely  visible,  becomes  approximately  the  size 
of  a  pigeon's  egg  or  larger.  It  is  typically  an  inflammatory 
reaction.  Ten  days  after  operation  the  swelling  may  be  almost 
as  large  as  three  days  after  transplantation  and  an  active  pus 
discharge  may  be  noted.  In  many  cases,  the  pus  spreads  toward 
and  escapes  through  the  healing  incision  but  in  many  cases 
erosion  of  the  skin  over  the  site  of  transplantation  may  provide 
an  escape  for  the  discharge  through  the  new  opening.  Pus  is 
often  seen  exuding  from  such  areas  up  to  the  termination  of 
the  experiment  (30  days).  In  some  cases  the  transplanted  mass 
has  so  completely  sloughed  by  the  termination  of  the  experiment 
as  to  be  invisible  excepting  as  the  site  of  transplantation  may  be 
marked  by  scar  tissue.  In  other  cases  small  encapsulated 
masses  of  pus  are  present. 

It  is  evident  from  these  observations  and  considerations  that  a 


354 


CARL    R.    MOORE. 


mass  of  testicular  tissues  undergoing  autolysis  after  transplanta- 
tion gives  little  or  no  evidence  of  having  liberated  hormones  into 

the  organism. 

V.    DISCUSSION. 

Our  three  years  experience  with  the  spermatozoon-mo  til  ity 
reaction  as  an  indicator  for  the  testis  hormone  have  increased  our 
confidence  in  the  test  as  a  dependable  objective  test  for  hormone 
production.  Readily  admitting  that  it  lacks  several  desirable 
qualities  to  make  it  entirely  adequate  for  many  different  ap- 
proaches to  the  subject  it  must  still  be  recognized  as  a  valuable 
means  of  studying  hormone  production. 

Relative  to  our  interests  here  under  discussion,  we  realize  for 
the  first  time  that  the  hormone  produced  by  mammal  testes  is 
not  stored  within  the  body  of  the  organism  and  the  internal 
secretions  of  this  organ  are  thus  brought  into  line  with  such 
other  organs  producing  internal  secretions  as  parathyroids, 
pituitary,  ovary,  etc.  In  the  ovarian  follicle  it  has  been  realized 
that  a  temporary  storage,  perhaps  at  the  site  of  production, 
does  occur,  but  that  the  body  does  not  ordinarily  store  it  up  for 
future  release  is  shown  by  the  failure  of  recurrence  of  cestrous 
in  spayed  females.  Removal  of  the  testis  eliminates  the  hor- 
mone source  and  there  is  no  evidence  that  any  appreciable 
quantity  is  retained  within  the  organism.  This  is  especially 
emphasized  when  one  realizes  that  a  ten  day  hormone  output 
by  a  normal  testicle  expresses  itself  with  an  increase  of  ten  days 
in  the  length  of  sperm  life  (as  indicated  by  their  motility) ; 
similar  additional  periods  can  in  general  be  detected  by  the 
reaction. 

When  the  aspermatogenetic  or  cryptorchid  testicle  is  studied 
it  is  indeed  interesting  to  learn  that  a  testicle  reduced  in  weight 
to  0.095  grams  produces  as  much  hormone  as  two  normal  testicles 
carrying  on  spermatogenetic  activity  and  weighing  approxi- 
mately 3.4  grams.  These  cryptorchid  testes  had  originally 
produced  sperm,  but  due  to  experimental  elevation  into  the 
abdomen  their  germinal  epithelium  had  undergone  dissolution 
and  removal.  Sections  show  the  typical  picture  of  natural 
cryptorchid  testes  in  that  the  seminiferous  tubule  outlines  are 
reduced  to  small  cavities  with  the  characteristic  single-celled 
layer  of  Sertoli  elements;  between  the  tubules  the  interstitial 


ON    THE    PROPERTIES    OF    THE    GONADS.  355 

cell  masses  present  the  typical  picture  of  "apparent  interstitial 
cell  hypertrophy."  Such  anatomical  characteristics  have  been 
sufficient  stimulus  for  some  writers  to  designate  such  an  animal 
as  a  "supermale"  but  I  have  never  been  able  to  see  adequate 
justification  for  the  introduction  of  such  a  term. 

The  fact  that  such  cryptorchid  testes,  having  a  fraction  of  the 
weight  of  the  testicular  mass  in  normal  male  animals,  generated 
the  same  quantity  of  hormone,  as  do  two  normal  testes  (judging 
from  the  spermatozoon  motility  test)  suggests  again  speculation 
as  to  the  actual  elements  producing  the  hormone. 

It  must  be  admitted  that  of  all  possible  elements  within  the 
testis,  the  Leydig  cells  appear  to  have  the  weight  of  evidence  in 
their  favor  as  being  the  source  of  origin  of  the  internal  secretions. 
However,  since  no  one  has  ever  satisfactorily  eliminated  all  other 
elements  such  as  the  general  connective  tissue,  but  more  es- 
pecially the  cells  of  Sertoli,  there  remains  the  same  debatable 
conditions  regarding  the  actual  source  of  origin.  \Yhen  one 
views  the  structure  of  such  degenerate  testes  as  these  six  and 
seven  month  cryptorchid  testes  (cryptorchid  four  and  five 
months  before  epididymal  isolation  and  two  months  of  the 
experiment)  and  realizes  that  each  is  functioning  in  producing  a 
full  hormone  quotient  (again  judging  by  the  test  employed)  one 
is  certainly  inclined  to  favor  the  Leydig  cells  as  the  source  of 
origin  and  to  minimize  the  apparently  inactive  cells  of  Sertoli. 
The  question  however  is  not  yet  settled  despite  the  suggestiveness 
associated  with  the  Leydig  cells. 

The  chief  interest  connected  with  the  transplanted  testis 
materials  was  to  see  if  the  spermatozoon  motility  test  would 
reveal  the  liberation  of  any  hormone  during  the  process  of 
autolysis  of  the  tissue.  Since  certain  writers  had  reported  such 
imaginary  effects  from  testis  transplantation,  it  was  thought 
possible  that  during  the  breakdown  of  the  incorporated  tissues 
some  action  might  be  detected.  The  results  of  my  investigations, 
however,  have  failed  to  show  the  liberation  of  sufficient  hormone 
to  be  detectable.  Despite  the  capability  of  the  test  to  reveal 
hormone  action  for  a  period  of  ten  days  by  a  normal  testicle  it 
becomes  evident  that  should  any  hormone  effect  be  derived  from 
transplantation  of  two  entire  guinea  pig  testicles,  its  effect  must 
be  less  than  that  of  the  normal  ten-day  production  period. 


356 


CARL    R.    MOORE. 


As  a  further  defense  of  the  capability  of  the  spermatozoon 
motility  test  to  indicate  the  presence  of  substances  produced  by 
the  testicle  I  may  mention  that  subcutaneous  injections  of  lipoid 
extracts  of  the  bull  testicle,  prepared  by  McGee,  and  injected 
by  me  into  guinea  pigs  whose  testes  had  been  removed  from 
their  epididymides,  resulted  in  prolonging  the  life  of  epididymal 
sperm  to  the  54th  day  after  operation  (Moore  and  McGee,  '28). 
Therefore,  had  the  transplanted  testis  masses  been  liberating 
hormone  into  the  host  organism,  we  should  have  been  able  to 
detect  it  by  the  test  under  discussion.  Such  a  result  certainly 
lends  no  credence  to  the  idea  expressed  by  others  that  similar  non- 
viable  testis  grafts  in  man  are  sufficiently  effective  to  be  noticeable 
for  a  period  of  approximately  two  years,  or  again  that  such 
transplantations  are  able  to  aid  in  the  cure  of  asthma,  tubercu- 
losis, myopia,  or  the  host  of  other  debilities  attributed  to  its  effect. 

The  evidence  is  very  direct  that  as  soon  as  the  hormone 
producing  tissues  are  removed  from  the  organism  the  lack  of  the 
substance  produced  begins  to  be  detectable  in  a  very  short  time. 
No  storage  within  the  body  for  future  utilization  is  evident. 

SUMMARY  AND  CONCLUSION. 

The  spermatozoon  motility  test  has  been  found  capable  of 
detecting  the  production  of  testis  hormone  for  a  period  as  short 
as  ten  days. 

The  hormone  produced  by  the  testicles  is  not  stored  within  the 
animal  body. 

An  experimental  cryptorchid  testicle  of  five  months  duration, 
having  a  weight  of  approximately  one-tenth  of  a  gram,  produces 
as  much  hormone  (indicated  by  the  spermatozoon-motility  test) 
as  do  two  normal  testicles  at  the  height  of  their  spermatogenetic 
activity.  The  experimental  cryptorchid  testis  is  by  weight 
approximately  2.8  to  3.5  per  cent,  that  of  the  normal  testicular 
mass. 

Autoplastic  subcutaneous  transplantation  of  two  testes  results 
In  the  liberation  of  hormone  in  such  small  amounts  (if  at  all)  as 
to  be  scarcely  detectable :  if  any  hormone  is  liberated  by  these  non- 
viable  testis  transplants,  the  effect  upon  the  animal  is  no  greater 
than  the  effect  of  a  ten  day  normal  hormone  production.  So  far 
as  present  means  will  indicate,  there  is  no  storage  or  cumulative 
effect  of  the  hormone. 


ON    THE   PROPERTIES    OF   THE   GONADS.  357 

BIBLIOGRAPHY. 
Benoit,  J. 

'26     Recherches  anatomique,  cytologique,  et  histo-physiologiques  sur  les  voies 
excretrices  du  testicule  chez  les  mammiferes.     Arch,  d'anat.  D'physiol. 
et  D'embryol.,  5:  173-412. 
Bascom,  K.  F. 

'25     Quantitative  Studies  of  the  Testis.     Some  Observations  on  the  Cryptorchid 

Testes  of  Sheep  and  Swine.     Anat.  Rec.,  30:  225-241. 
Domm,  L.  V. 

'27     Ne\v  Experiments  on  Ovariotomy  and  the  Problem  of  Sex  Inversion  in 

the  Fowl.     Jour.  Exp.  Zool.,  48: 
Heller,  R.  E. 

'29     New  Evidence  for  the  Function  of  the  Scrotum.     Physiological  Zoology 

(in  press). 
Lipschutz,  Ottow  and  Wagner. 

'22     On  the  Hypertrophy  of  the  Interstitial  Cells  of  the  Testicle  of  the  Guinea 
Pig   under   Different   Experimental   Conditions.     Proc.    Roy.   Soc.,   93: 
132-142. 
Long,  J.  A.  and  Evans,  H.  E. 

'22     The  OZstrous  Cycle  in  the  Rat  and  it?  Associated  Phenomena.     Memoirs 

Univ.  of  California,  Vol.  6. 
Moore,  Carl  R. 

'21  On  the  Physiological  Properties  of  the  Gonads  as  Controllers  of  Somatic 
and  Psychical  Characteristics.  IV.  Gonad  Transplantation  in  the 
Guinea  Pig.  Jour.  Exp.  Zool.,  33:  365-389. 

'22     V.   The  Effects  of   Gonadectomy  in  the   Guinea   Pig  on   Growth,    Bone 
Lengths,  and  Weight  of  Organs  of  Internal  Secretion.     BIOL.  BULL.,  43: 
285-312. 
'240     VI.  Testicular  Reactions  in  Experimental  Cryptorchidism.     Amer.  Jour. 

Anat.,  34:  269-316. 
'24^     VIII.   Heat  Application  and   Testicular  Degeneration,   the  Function  of 

the  Scrotum.     Amer.  Jour.  Anat.,  34:  337-358. 
'26     IX.    Testis-graft    Reactions    in    Different    Environments    (Rat).     Amer. 

Jour.  Anat.,  37:  351-416. 
'27     A  Qualitative  Test  for  the  Testis  Hormone.     Proc.  Soc.  Exp.  Biol.  and 

Med.,  24:  847. 
'28     X.  Spermatozoon  Activity  and  the  Testis  Hormone.     Jour.  Exp.  Zool., 

50:  455-494. 
Moore,  Carl  R.,  and  Lemuel  C.  McGee. 

'28     On  the  Effects  of  Injecting  Lipoid  Extracts  of  Bull  Testes  into  Castrated 

Guinea  Pigs.     Amer.  Jour.  Physiol.,  87  (in  press). 
Moore,  Carl  R.,  and  Wm.  J.  Quick. 

'24     The  Scrotum  as  a  Temperature  Regulator  for  the  Testes.     Amer.  Jour. 

Physiol.,  78:  70-79. 
Stockard,  C.  R.,  and  G.  N.  Papanicolau. 

'17     The  Existence  of  a  Typical  CEstrous  Cycle  in  the  Guinea  Pig  with  a  Study 
of  its  Histological  and  Physiological  Changes.     Amer.  Jour.  Anat.,  22: 
225-264. 
Stone,  Calvin  P. 

'27  The  Retention  of  Copulatory  Ability  in  Male  Rats  following  Castration. 
Jour.  Comp.  Psychology,  7:  369-387. 


INITIATION    OF    DEVELOPMENT    IN    ARBACIA.     V 

THE  EFFECT  OF  SLOWLY  EVAPORATING  SEA-WATER 

AND  ITS  SIGNIFICANCE  FOR  THE  THEORY 

OF   AUTO-PARTHENOGENESIS.* 

E.  E.  JUST, 

ROSENWALD  FELLOW  IN  BIOLOGY,  NATIONAL  RESEARCH  COUNCIL. 

In  1901  Hunter  published  results  of  experiments  which  showed 
that  uninseminated  eggs  of  Arbacia  exposed  to  sea-water  con- 
centrated by  evaporation  develop  on  return  to  normal  sea-water. 
The  present  writer  has  been  able  to  confirm  these  results  though 
his  method  differs  from  Hunter's.  The  sole  reason  for  reporting 
the  findings  here  presented  is  that  they  lead  to  some  interpreta- 
tions of  significance  for  Lillie's  fertilizin  theory  of  fertilization. 
The  work  was  done  during  several  summers  spent  at  the  Marine 
Biological  Laboratory,  Woods  Hole,  Mass. 

THE  EXPERIMENTS. 

Normal  uninseminated  eggs  of  Arbacia — free  of  perivisceral 
fluid,  of  high  fertilizin  content,  and  capable  of  giving  one  hundred 
per  cent,  fertilization  and  cleavage — show  a  small  per  cent,  of 
cleavage  and  of  abnormal  blastulse  that  do  not  rise  to  the  surface, 
if  after  having  lain  in  a  small  volume  of  normal  sea-water  for 
one  or  more  hours,  they  are  removed  to  a  larger  volume  of 
normal  sea-water.  Two  conditions  are  important  for  this  method 
of  initiating  development  in  the  egg  of  Arbacia.  First,  it  is 
best  to  use  fairly  dense  egg  suspensions.  The  less  dense  sus- 
pensions prolong  the  time  of  exposure  necessary  to  initiate 
development.  Secondly,  it  is  indispensable  for  the  experiment 
that  the  dish  containing  the  eggs  be  left  uncovered  to  insure  evapo- 
ration. A  concentration  of  I  cc.  of  "dry"  eggs  plus  99  cc.  of 
normal  sea-water  was  the  least  dense  suspension  successfully 
used.  In  some  cases  it  was  necessary  to  leave  such  a  suspension 
uncovered  for  twenty-four  hours  before  transfer  to  the  larger 
volume  of  sea-water;  but  it  was  clear  here  that  evaporation  was 

*  From  the  Marine  Biological  Laboratory,  Woods  Hole,  Massachusetts,  and 
the  Department  of  Zoology,  Howard  University,  Washington,  D.  C. 

358 


INITIATION    OF    DEVELOP  M  K. XT    IX    ARBACIA.  359 

responsible  since  suspensions  of  this  kind  always  gave  better 
results  when  placed  in  larger  dishes  with  greater  surface  for 
evaporation.  And  if,  moreover,  a  I  cc.  suspension  of  eggs  in 
100  cc.  of  sea-water  be  poured  on  a  glass  plate  thereby  insuring 
greater  evaporation,  the  results  were  indeed  striking.  How- 
ever, I  am  here  interested  mainly  in  the  results  obtained  with 
smaller  volumes  of  eggs  and  of  sea-water. 

In  all  the  experiments  it  was  first  ascertained  that  the  eggs  to 
be  used  were  in  optimum  fertilizable  condition  by  trial  insemina- 
tions for  the  estimation  of  their  capacity  to  separate  normal 
membranes.  First,  the  eggs  were  carefully  collected  uncon- 
taminated  by  perivisceral  fluid,  washed  in  four  changes  of  200  cc. 
of  sea-water,  and  allowed  to  settle.  The  supernatant  sea-water 
was  decanted  and  a  highly  concentrated  bulk  of  eggs  thus 
obtained.  For  each  experiment  the  eggs  were  from  one  female. 
These  were  divided  into  two  equal  lots  whenever  their  bulk 
permitted;  one  lot  was  placed  in  an  open  dish,  the  other  in  a 
glass  vial  closed  with  a  ground  glass  stopper.  At  intervals  of  30 
minutes  a  drop  of  eggs  from  each  lot  was  removed  to  250  cc.  of 
normal  sea-water  and  their  development  observed.  The  per 
cent,  of  cleavage  was  as  carefully  counted  as  possible,  though 
the  count  is  often  made  difficult  because  of  the  number  of 
cytolyzed  eggs.  With  further  development  complete  cytolysis  of 
eggs  makes  the  counting  of  "swimmers"  more  difficult  and  of 
doubtful  value  since  these  counts  cannot  take  into  consideration 
eggs  that  have  disappeared  through  complete  disintegration. 
Moreover,  many  of  the  eggs  exposed  to  evaporation  develop  with 
their  blastomeres  separated  because  the  eggs  do  not  possess 
membranes.  In  such  cases,  two  "  micro-blastula? "  counted  may 
have  developed  from  one  egg  or  each  from  a  different  egg,  its 
fellow  mass  of  blastomeres  having  disappeared  completely 
through  disintegration.  I  therefore  early  abandoned  attempts 
at  making  accurate  counts  and  merely  noted  the  presence  or 
absence  of  "swimmers." 

I  wish  to  emphasize  that  in  not  one  single  experiment  did  I 
ever  find  an  egg  in  the  stoppered  vial  that  showed  the  least  sign 
of  development.  That  these  eggs  were  not  impaired  I  deter- 
mined by  inseminating  them — in  normal  sea-water  in  the  case 


360 


E.    E.    JUST. 


of  highly  concentrated  suspensions,  or  in  the  vials  in  the  case  of 
the  less  concentrated  suspensions. 

One  other  point  before  we  consider  the  experiments  in  detail. 
The  reader  appreciates  the  fact  that  the  rate  of  evaporation 
varied  from  day  to  day.  I  made  no  attempt  to  control  this 
variation.  It  is  also  obvious  that  the  rate  of  evaporation  depends 
upon  the  volume  of  solution  employed — smaller  volumes  evapo- 
rating more  rapidly  than  larger.  Finally,  the  vessels  used  make 
a  difference;  in  my  experiments  I  used  either  shallow  dishes, 
with  a  large  surface  for  evaporation,  or  for  the  greatest  volumes 
of  solutions  employed  glass  plates,  30  x  30  cm.  For  volumes  up 
to  4  cc.  Syracuse  watch  glasses  served  admirably. 

There  now  follow  a  summary  (Table  I.)  of  the  first  type  of 
experiment  and  a  brief  comment  for  the  purpose  of  elucidation. 

TABLE  I. 

THE  EFFECT  OF  SLOWLY  EVAPORATING  SEA-WATER  ON  THE  UNINSEMINATED  EGGS 

OF  Arbacia  AS  SHOWN  BY  THE  PER  CENT.  OF  THEIR  DEVELOPMENT 

ON  RETURN  TO  NORMAL  SEA-WATER.     EXPERIMENTS 

ON  EGGS  OF  45  FEMALES 


Per  Cent,  of 

Per  Cent,  of 

No. 

Bulk  of  Con- 
centrated 

Volume  of 
Sea-water 

Cleavage. 

"Swimmers." 

Eggs  (in  cc.). 

(in  cc.). 

Exp.  No. 

i 

2 

3 

4 

5 

Exp.  No.  i. 

i 

O.I 

0.9 

8 

II 

7 

14 

20 

9 

2 

0-5 

o-5 

7 

O 

ii 

14 

3 

5 

3 

0.6 

1.2 

6 

12 

4 

9 

o 

4 

4 

i 

I 

0 

6 

9 

7 

2 

o 

5 

i 

I 

10 

7 

8 

4 

14 

13 

6 

i-5 

3 

21 

i? 

23 

27 

18 

23 

7 

2 

2 

0 

3 

4 

o 

5 

o 

8 

2-5 

5 

13 

6 

18 

21 

19 

15 

9 

3 

3 

14 

24 

S 

O 

6 

IO 

Eggs  from  the  same  females  in  stoppered  vials:  No  cleavage,  no  "swimmers 
Same  volumes  of  eggs  and  sea  water  in  each  case  except  as  follows:  No.  7,  0.5  cc. 
of  eggs  +  0.5  cc.  sea-water;  No.  8,  0.5  cc.  of  eggs  +  0.5  cc.  of  sea-water;  No.  9, 
0.5  cc.  of  eggs  +  i  cc.  of  sea-water. 

The  data  given  in  Table  I.  are  for  eggs  exposed  to  slowly 
evaporating  sea-water  for  two  hours.  This  one  length  of  exposure 
is  arbitrarily  taken  for  the  purpose  of  simplicity,  instead  of 
presenting  the  results  of  each  3O-minute  exposure.  In  some 


INITIATION    OF   DEVELOPMENT    IN    ARBACIA.  361 

instances  the  per  cent,  of  development  was  greater  after  a  longer 
or  a  shorter  exposure;  the  results  of  the  two-hour  exposure  is 
very  nearly  the  average  of  all  exposures  made.  Though  the 
per  cent,  of  development  in  no  experiment  is  high,  yet  it  shows 
that  the  evaporating  sea-water  does  initiate  development.  I  do 
not  regard  this  as  an  efficient  method  for  experimental  partheno- 
genesis: it  has  been  very  suggestive,  however,  for  other  lines  of 
my  work. 

Eggs  in  sea-water  protected  against  evaporation  never  show 
indication  that  development  is  initiated.  This  statement  is 
certainly  superfluous  for  suspensions  of  uninseminated  eggs  of 
Arbacia  that  are  ordinarily  employed  as  controls,  as  all  worker- 
know.  Of  the  more  dense  egg  suspensions  it  might  be  that 
lack  of  oxygen  or  CO2  concentration  makes  initiation  of  develop- 
ment impossible.  The  fact  that  such  eggs  from  such  suspension 
fertilize  on  return  to  larger  volumes  of  sea-water  does  not  meet 
this  possible  objection.  However,  I  might  repeat  that  some 
suspensions  made  of  I  cc.  of  eggs  plus  99  cc.  of  sea-water  exposed 
to  slowly  evaporating  sea-water  showed  initiation  of  development 
whereas  similar  suspensions  in  stoppered  vials  never  did. 

Eggs  that  show  initiation  of  development  as  the  result  of 
exposure  to  evaporating  sea-water  never  separate  membranes, 
their  cleavage  is  irregular,  and  the  blastomeres  tend  to  fall  apart. 
Many  eggs  do  not  cleave  and  of  these  some  reach  the  monaster 
stage  with  rhythmical  dissolution  and  re-formation  of  the  nucleus. 
All  uncleaved  eggs  on  insemination  separate  membranes,  cleave, 
and  reach  the  pluteus  stage. 

The  abnormal  swimming  forms  developing  from  these  eggs 
subjected  to  treatment  with  slowly  evaporating  sea-water  never 
swim  at  the  surface,  but  merely  rotate  on  the  bottom  of  the 
dishes;  among  them  are  micro-  and  mega-"blastuke"  —i.e., 
swimming  forms  developed  from  blastomeres  that  have  fallen 
apart  and  those  developed  from  two  or  more  cleaving  eggs. 
It  is  this  fact  that  makes  difficult  the  counting  of  swimmers; 
hence,  the  reader  will  note  that  except  for  the  first  experiment 
(Table  I.)  and  for  one  experiment  described  below  (Table  II.), 
I  give  no  counts,  but  simply  note  with  a  +  or  o  sign  their  presence 
or  absence. 

I    interpret    these   experiments   to   mean    that    these   eggs    in 

24 


362 


E.    E.    JUST. 


evaporating  sea-water  are  by  such  evaporation  exposed  to 
hypertonic  sea-water.  It  is  the  hypertonicity  that  is  responsible 
for  the  initiation  of  development  and  not  the  mere  crowding  of 
the  eggs  since  equivalent  volumes  of  eggs  from  the  same  females 
and  of  sea-water  protected  against  evaporation  do  not  give  any 
evidence  of  initiation  of  development  after  transfer  to  larger 
volumes  of  sea-water.  These  eggs  as  noted  above  had  been 
thoroughly  washed  before  exposure  to  evaporation;  they  would 
nevertheless  continue  to  produce  fertilizin — but  so  would  the 
eggs  protected  against  evaporation.  If  fertilizin  production, 
therefore,  were  responsible  for  the  initiation  of  development  we 
might  expect  that  at  least  the  highly  concentrated  eggs  in 
stoppered  vials  would  show  some  signs  of  cleavage  and  farther 
development.  And,  what  is  more,  the  use  of  "egg  water" 
instead  of  normal  sea-water  does  not  increase  the  per  cent,  or 
improve  the  development.  Table  II.  gives  the  results  of  a 
typical  experiment  on  eggs  exposed  to  evaporating  "egg  water." 
Drops  of  eggs  from  both  the  uncovered  and  the  stoppered  lots 
were  returned  at  half  hour  intervals  to  200  cc.  of  normal  sea- 
water.  The  percentages  given  are  those  of  eggs  having  had  a 
two-hour  exposure  to  the  evaporating  "egg  water."  This  experi- 
ment was  made  five  times. 

TABLE  II. 

THE  EFFECT  OF  SLOWLY  EVAPORATING  EGG  WATER  ON  THE  UNINSEMINATED  EGGS 

OF  Arbacia  AS  SHOWN  BY  THE  PER  CENT.  OF  THEIR  DEVELOPMENT 

ON  RETURN  TO  NORMAL  SEA-WATER.     EXPERIMENTS 

ON  THE  EGGS  OF  9  FEMALES. 


No. 

Bulk  of  Con- 
centrated Eggs 
(in  cc.). 

Volume  of  Egg 
Water  (in  cc.). 

Per  Cent,  of 
Cleavage. 

Per  Cent,  of 
"Swimmers." 

i 

0-5 

i-5 

9 

7 

2 

0-5 

2-5 

13 

ii 

3 

i 

i 

7 

o 

4 

i 

i 

18 

14 

5 

i 

2 

n 

5 

6 

2 

I 

3 

5 

7 

2 

2 

IS 

12 

8 

2 

3 

12 

10 

9 

2-5 

5 

9 

6 

Equivalent  volumes  of  eggs  from  the  same  females  and  of  "egg-water,"  except  for 
No.  9  where  0.5  cc.  of  eggs  and  i  cc.  of  "egg  water"  were  used,  in  stoppered  vials 
gave  no  trace  of  development  after  return  to  normal  sea-water. 


INITIATION    OF    DEVELOPMENT    IN    ARBACIA.  363 

It  would  appear  from  a  study  of  Table  II.  that  there  is  no 
advantage  in  substituting  "egg  water"  for  sea-water.  As  a 
matter  of  fact,  other  experiments  with  "egg  water"  gave  inferior 
results.  In  addition,  one  gains  the  impression  that  exposure  to 
evaporating  "egg  water"  causes  more  eggs  to  separate  blasto- 
meres,  and  that  there  are  more  micro-  and  mega-"blastulae." 
This  I  did  not  properly  investigate,  i.e.,  by  running  experiments 
on  lots  of  concentrated  eggs  from  the  same  females,  one  lot 
exposed  to  evaporating  sea-water,  one  to  stoppered  sea-water, 
one  to  evaporating  "egg  water,"  and  one  to  stoppered  "egg 
water"  counting  both  the  eggs  that  showed  blasto meres  falling 
apart  and  the  micro-"  blastuke."  However,  some  older  un- 
published observations  made  independently  by  Lillie  and  by 
the  writer  may  be  cited.  These  showed  that  "egg  water' 
actually  possesses  a  deleterious  effect  on  development.  Thus,  I 
found  that  if  eggs  from  the  same  female  be  divided  into  two  lots, 
one  suspended  in  sea-water  and  the  other  in  strong  "egg  water' 
before  or  after  insemination,  the  development  of  the  eggs  in 
"egg  water"  are  markedly  inferior  to  that  of  the  eggs  in  normal 
sea-water  as  measured  by  the  per  cent,  and  normality  both  of 
cleavage  and  of  plutei.  Lillie  also  has  commented  on  the  adverse 
effect  of  "egg  water"  in  other  ways  on  eggs.  There  is  indeed  no 
reason  why  this  should  not  be  true  and  several  reasons  why  it 
should.  "Egg  water"  is  not  simply  sea-water  charged  with 
fertilizin — it  contains  products  of  metabolism  of  the  uninsemi- 
nated  eggs,  even  though  metabolism  is  at  a  low  level;  this  would 
be  especially  true  of  eggs  highly  concentrated  in  strong  "egg 
water,"  which  perhaps  also  contains  more  bacteria  than  normal 
sea-water. 

Glaser  likewise  notes  that  "addition  of  the  extracts  ["egg 
water"]  in  certain  concentrations  to  normally  fertilized  eggs, 
resulted  in  a  retardation  of  development;  normal  blastulse 
instantly  slowed  their  movements,  and  underwent  a  noticeable 
increase  in  volume  when  subjected  to  the  extracts.  Similar 
observations  were  made  on  the  larvae  of  Arenicola  whose  rate 
of  movement  was  also  slowed  down,  to  be  followed  instantly  by 
an  outflow  of  their  yellow  pigment  and  a  slight  reversible  aggluti- 
nation." Unfortunately,  however,  Glaser's  method  of  preparing 
his  egg  extracts — by  removing  the  eggs  directly  from  the  ovaries 


364  E-   E-   JUST. 

into  twice  their  volume  of  sea-water — is  open  to  objection  since 
he  must  have  carried  over  some  peri  visceral  fluid.  The  peri- 
visceral  fluid  alone  may  have  been  responsible  for  his  results. 

The  following  experiment  was  also  made  ten  times:  eggs  from 
one  female  were  placed  (i)  in  sea-water  exposed  to  evaporation, 
(2)  in  sea-water  in  a  stoppered  vial,  (3)  in  "egg  water"  exposed 
to  evaporation  and  (4)  in  "egg  water"  in  a  stoppered  vial;  at 
30  minute  intervals  drops  of  eggs  were  removed  from  each  of 
the  four  lots  to  dishes  each  containing  200  cc.  of  normal  sea- 
water.  I  give  now  the  summary  of  one  long  experiment  because 
it  shows  the  results  with  varying  concentration  of  eggs  from  one 
female : 

No.     i.     10  drops  of  eggs   +  90  drops  of  uncovered  sea-water  gave  18  per  cent. 

cleavage,  +  +  "swimmers." 
No.    2.     10  drops  of  eggs  +  90  drops  of  sea-water  in  a  stoppered  vial  gave  o  per 

cent,  cleavage,  o  "swimmers." 
No.    3.     10  drops  of  eggs   +  90  drops  of  uncovered  egg  water  gave  6  per  cent. 

cleavage,  +  "swimmers." 
No.    4.     10  drops  of  eggs  +  90  drops  of  egg  water  in  a  stoppered  vial  gave  o  per 

cent,  cleavage,  o  "swimmers." 
No.    5.     20  drops  of  eggs  +  80  drops  of  uncovered  sea-water  gave  27  per  cent. 

cleavage,  +  +  "swimmers." 
No.    6.     20  drops  of  eggs  +  80  drops  of  sea-water  in  a  stoppered  vial  gave  o  per 

cent,  cleavage,  o  "swimmers." 
No.    7.     20  drops  of  eggs   +  80  drops  of  uncovered  egg-water  gave  8  per  cent. 

cleavage,  +  "swimmers." 
No.    8.     20  drops  of  eggs  +  80  drops  of  egg  water  in  a  stoppered  vial  gave  o  per 

cent,  cleavage,  o  "swimmers." 
No.    9.     30  drops  of  eggs  +  70  drops  of  uncovered  sea-water  gave  31  per  cent. 

cleavage,  +  +  "swimmers." 
No.  10.     30  drops  of  eggs  +  70  drops  of  sea-water  in  a  stoppered  vial  gave  o  per 

cent,  cleavage,  o  "swimmers." 
No.  ii.     30  drops  of  eggs  +  70  drops  of  uncovered  egg  water  gave  n  per  cent. 

cleavage,  +  "swimmers." 
No.  12.     30  drops  of  eggs  +  70  drops  of  egg  water  in  a  stoppered  vial  gave  o  per 

cent,  cleavage,  o  "swimmers." 
No.  13.     40  drops  of  eggs  +  60  drops  of  uncovered  sea-water  gave  21  per  cent. 

cleavage,  +  +  "swimmers." 
No.  14.     40  drops  of  eggs  +  60  drops  of  sea-water  in  a  stoppered  vial  gave  o  per 

cent,  cleavage,  o  "swimmers." 
No.  15.     40  drops  of  eggs  +  60  drops  of  uncovered  egg  water  gave  17  per  cent. 

cleavage,  o  "swimmers." 
No.  16.     40  drops  of  eggs  +  60  drops  of  egg  water  in  a  stoppered  vial  gave  o  per 

cent,  cleavage,  o  "swimmers." 


INITIATION    OF   DEVELOPMENT    IN    ARBACIA.  365 

In  this  experiment  because  of  the  rapidity  of  evaporation  on 
this  particular  day  the  eggs  were  removed  to  normal  sea-water 
after  one  hour.  The  experiment  reveals  that  the  effect  of 
evaporating  "egg  water'  is  certainly  not  superior  to  that  of 
evaporating  sea-water  in  causing  initiation  of  development.  It 
shows  also  as  other  experiments  cited  show  that  more  concen- 
trated suspensions  do  not  yield  markedly  higher  percentages  of 
development  than  less  concentrated  ones. 

On  the  whole  I  think  that  the  evidence  which  I  have  submitted 
indicates  that  eggs  exposed  in  uncovered  dishes  develop  because 
of  an  increasing  hypertonicity  due  to  evaporation.  Further, 
the  evidence  indicates  that  "egg  water"  is  not  necessary  for 
this  effect;  indeed,  "egg  water"  appears  to  be  less  efficacious  if 
not  actually  more  harmful  in  some  small  degree  than  normal 
sea-water.  If  this  evidence  be  accepted,  Glaser's  work  on  auto- 
parthenogenesis  must  be  questioned.  A  brief  discussion  of 
Glaser's  work  and  its  significance  for  the  fertilizin  theory  in 
the  light  of  the  work  which  I  herein  report  now  follows. 

DISCUSSION. 

In  1914  Glaser  reported  for  eggs  of  Arbacia  and  Asterias  a 
type  of  initiation  of  development  due  to  exposure  to  "egg  water" 
for  which  he  chose  the  name,  auto-parthenogenesis.  Glaser's 
procedure  was  as  follows:  "Standard  secretion  ["egg  water"] 
was  prepared  by  adding  to  a  certain  number  of  "dry'  ripe 
ovarian  eggs,  double  their  volume  of  sea-water.  At  the  end  of 
ten  minutes,  during  which  the  eggs  were  slightly  agitated  at 
intervals,  the  suspension  was  centrifuged,  and  the  eggs  cast  down. 
After  100  revolutions  the  supernatant  fluid  was  carefully  decanted 
and  set  aside  for  use. 

"Ripe  eggs  were  then  shaken,  usually  from  the  ovaries  of  a 
single  individual,  into  a  small  quantity  of  fresh  sea- water,  and 
to  i  cc.  of  a  concentrated  suspension  of  these  was  added  I  cc. 
of  the  secretion.  In  this  mixture  the  eggs  were  allowed  to  stand 
2  hours,  when  cleavages  were  usually  found  in  all  the  dishes." 

And  further:  "Many  experiments  were  tried  varying  the  con- 
centration of  the  secretion  as  well  as  the  time  of  exposure.  My 
records  indicate  cleavages  at  higher  concentrations  as  well  as 
lower,  and  also  in  less  than  two  hours,  but  the  greatest  number 


366  E.    E.    JUST. 

was  always  obtained  when  I  volume  of  the  concentrated  egg 
suspension  was  exposed  for  2  hours  to  I  volume  of  the  standard 
secretion.  If  at  the  end  of  this  time  the  supernatant  fluid  is 
poured  off  and  replaced  by  fresh  sea-water,  free  swimming 
blastulse  will  be  found  within  24  hours.  In  one  case  only  did 
development  proceed  to  the  pluteus  stage." 

As  I  have  stated  above,  Lillie  was  never  able  to  repeat  this 
observation  made  by  Glaser.  Nor  was  I  until  by  chance  I 
observed  the  extent  of  evaporation  that  had  taken  place  in  two 
cc.  of  egg  water  put  in  a  Syracuse  watch  glass  one  hour  before. 
Deliberately  repeating  this  chance  observation  on  eggs  suspended 
in  either  "egg  water"  or  sea-water  through  several  seasons  I  have 
obtained  initiation  of  development  in  Arbacia  eggs  provided  the 
"egg  water"  or  sea-water  be  allowed  to  evaporate.  I  am  there- 
fore constrained  to  believe  that  Glaser's  auto-parthenogenesis  is 
a  hypertonic  effect  due  to  evaporation. 

Glaser  has  also  reported  what  he  calls  an  improved  method  of 
auto-parthenogenesis.  Says  Glaser:  "Loeb's  improved  method 
of  artificial  parthenogenesis  consists  in  following  the  treatment 
with  parthenogenetic  agents,  by  an  after  treatment  with  hyper- 
tonic  sea-water,  8  cc.  of  2.5  M  NaCl  plus  50  cc.  of  sea-water. 
It  seemed  likely,  therefore,  that  a  better  yield  of  larvae  could 
be  secured  if  eggs,  after  having  been  subjected  to  the  action  of 
the  secretion  for  two  hours,  were  afterwards  treated  with  the 
hypertonic  solution  for  forty  minutes.  This  surmise  proved 
correct."  The  proof  offered  is  the  outline  of  a  typical  experi- 
ment showing  the  development  in  two  sets  of  eggs  both  of  which 
were  exposed  to  hypertonic  sea-water  after  treatment  with  the 
egg  secretion.  There  are,  it  seems  to  me,  two  objections  to  this 
experiment. 

In  the  first  place,  in  the  improved  method  of  artificial  partheno- 
genesis Loeb  typically  used  butyric  acid  which  alone  is  not 
capable  of  causing  development  of  the  egg  beyond  the  separation 
of  the  vitelline  membrane  and  formation  of  a  monaster  around 
the  egg  nucleus;  according  to  Glaser,  the  egg  secretion  which  he 
used  causes  development  at  least  to  the  blastula  stage  without 
separation  of  membranes.  Moreover,  when  one  uses  butyric 
acid  one  must  replace  the  acid  sea-water  with  normal  sea-water 
and  allow  a  certain  time  to  elapse  before  beginning  the  treatment 


INITIATION    OF    DEVELOPMENT    IN    ARBACIA.  367 

with  the  hypertonic  sea-water;  Glaser  exposed  his  eggs  to  the 
egg  secretion  and  at  once  transferred  them  to  the  hypertonic  sea- 
water.  There  is  here,  therefore,  no  similarity  between  the 
improved  method  of  Loeb  and  that  of  Glaser. 

Secondly,  and  this  is  far  more  serious,  Glaser  does  not  tell  us 
to  what  extent  there  is  an  improvement  through  the  after  treat- 
ment with  the  hypertonic  sea-water;  he  gives  no  information 
concerning  the  development  of  two  lots  of  eggs  from  the  same 
female,  one  with  and  one  without  hypertonic  sea-water  after  the 
exposure  to  the  egg  sea-water.  Obviously,  Glaser  should  have 
set  up  an  experiment  on  four  lots  of  eggs  from  the  same  female, 
one  an  uninseminated  control  in  normal  sea-water,  one  exposed 
to  hypertonic  sea-water  alone,  one  to  egg  water  alone,  and  our 
to  hypertonic  solution  after  a  treatment  with  "egg  water." 

In  the  same  communication  Glaser  also  described  auto- 
parthenogenesis  in  eggs  of  Asterias.  For  this  he  used  either  I 
or  2  volumes  of  maturing  Asterias  eggs  plus  one  of  "egg  water' 
and  obtained  fertilization  membranes,  cleavage,  and  "much 
gastrulation."  I  would  suggest  that  this  result  was  due  in  part 
to  CO2,  which  in  Asterias  eggs  initiates  development,  and  to 
hypertonicity. 

Glaser's  "hetero-parthenogenesis"  is  the  effect  of  Arbacia 
"egg  water"  on  Asterias  eggs.  Here  again  he  used  I  volume  of 
"egg  water"  (from  Arbacia  eggs)  to  i  volume  of  Asterias  eggs. 
The  foreign  "egg  water"  gave  fertilization  membranes  and 
numerous  cleavages.  I  venture  the  opinion  that  the  initiation 
of  development  was  due  to  one,  two  or  a  combination  of  three 
factors:  CO2,  hypertonicity,  and  the  foreign  perivisceral  fluid 
which  from  Glaser's  method  of  procuring  the  Arbacia  "egg 
water"  must  have  been  present. 

On  the  basis  of  my  findings  and  the  possibility  that  these 
adverse  criticisms  of  Glaser's  work  be  correct,  I  suggest  that  auto- 
parthenogenesis  is  an  initiation  of  development  due  to  hyper- 
tonicity of  either  "egg  water"  or  sea-water.  If  this  be  true 
Glaser's  criticisms  of  Lillie's  fertilizin  theory  based  on  his  findings 
are  without  foundation. 


368  E.    E.    JUST. 

LITERATURE    CITED. 

Glaser,  Otto. 

'14     On  Auto-parthenogenesis  in  Arbacia  and  Aslerias.     BIOL.  BULL.,  26,  pp. 

387-409. 
Hunter,  S.  J. 

'01     On  the  Production  of  Artificial  Parthenogenesis  in  Arbacia  by  the  Use  of 
Sea-water  Concentrated  by  Evaporation.     Amer.  Jour.  Physiol.,  6,  pp. 
177-180. 
Lillie,  Frank  R. 

'14     Studies  of  Fertilization.     VI.  The  Mechanism  of  Fertilization  in  Arbacia. 
Jour.  Exp.  Zool.,  16,  pp.  523-590. 


INTRACELLULAR  HYDRION   CONCENTRATION 

STUDIES. 

I.   THE  RELATION  OF  THE  ENVIRONMENT  TO  THE  pH  OF  PROTO- 
PLASM AND  OF  ITS  INCLUSION  BODIES. 
ROBERT  CHAMBERS, 

LABORATORY  OF  CELLULAR  BIOLOGY,  DEPARTMENT  OF  ANATOMY,  CORNELL  I 
VERSITY  MEDICAL  COLLEGE,  NEW  YORK  CITY,  AND  THE  ELI 
RESEARCH  DIVISION,  MARINE  BIOLOGICAL  LABORA- 
TORY, WOODS  HOLE,  MASSACHUSETTS. 

Recent  micrurgical  investigations  (i,  2,  3)  on  the  colorimetric 
determination  of  the  protoplasmic  pH  have  emphasized  the  need 
of  studying  the  relation  between  the  pH  of  the  protoplasm  of  a 
living  cell  and  that  of  its  environment.  Of  the  acids  and  bases 
which  affect  the  pH  of  the  environment  some  penetrate  living 
cells  while  others  apparently  do  not.  This  has  been  demon- 
strated by  the  change  in  color  of  cells  stained  with  indicators. 
For  example,  with  the  use  of  neutral  red  it  has  been  shown  by 
previous  investigators  (4,  5)  that  living  cells  are  readily  per- 
meable to  CO2  and  NH3  but  not  to  HC1  nor  NaOH.  This  fact 
that  the  color  of  the  intracellular  stain  can  be  readily  shifted  to 
the  acid  or  the  alkaline  side  suggests  that  the  intraprotoplasmic 
pH  can  be  changed  very  easily  by  environmental  conditions,  a 
conclusion  which  is  at  variance  with  experiments  which  indicate 
that  protoplasm  has  a  marked  buffering  power.  Thus,  when 
solutions  of  indicators,  both  in  the  acid  and  the  alkaline  states 
of  their  color  ranges,  are  injected  into  living  cells  the  colors 
quickly  shift  to  those  characteristic  of  a  constant  pH  (6.9±o.i). 
This  has  been  found  true  for  such  varied  types  of  cells  as  the 
ameba  (i,  6),  marine  ova  (2,  3),  and  various  tissue  cells  of  the 
frog  and  the  mammal  (6).  In  addition,  there  is  the  significant 
result  that  the  localized  increase  in  intraprotoplasmic  acidity, 
caused  by  mechanical  injury  is  almost  immediately  neutralized 
as  long  as  no  cytolysis  results  (i,  2,  3,  6). 

In  view  of  these  facts  it  was  considered  advisable  to  test 
further  the  constancy  of  the  intraprotoplasmic  pH,  to  discover 

369 


370 


ROBERT    CHAMBERS. 


whether  this  pH  can  be  shifted  appreciably  without  detriment 
to  the  cell  and  to  obtain  evidence,  if  any,  of  localized  variations 
in  the  intracellular  pH. 

The  purpose  of  the  experiments  described  in  this  paper  is  to 
determine  whether  the  intraprotoplasmic  pH  can  be  shifted  by 
exposure  to  COa  or  to  NH3  and  whether  the  reaction  to  indicators 
of  such  intracellular  structures  as  granules  and  vacuoles  are 
comparable  to  those  of  the  optically  homogeneous  protoplasmic 
matrix. 

Before  dealing  with  the  actual  experiments  performed  it  is 
necessary  to  describe  the  manner  in  which  the  protoplasm 
becomes  colored  with  neutral  red  and  with  the  other  dyes  used. 
When  cells  are  stained  with  neutral  red  or  certain  other  basic 
dyes,  the  dye  accumulates  in  or  on  the  intracellular  granules  and 
vacuoles  while  the  hyaline  protoplasmic  matrix  remains  colorless. 
This  occurs  not  only  when  cells  are  stained  by  immersion  in  a 
solution  of  the  dye  but  also  when  the  dye  is  injected  directly 
into  the  cell.  In  the  latter  case  the  color  appears  at  first  diffuse 
but  gradually  the  granules  and  vacuoles  take  up  more  and 
more  of  the  color  until  none  of  it  can  be  detected  in  the  hyaline 
cytoplasmic  matrix.  On  the  other  hand  the  acid  dyes  used, 
e.g.,  brom  cresol  purple,  phenol  red  and  cresol  red,  do  not  pene- 
trate from  the  environment  into  the  cells.  When  injected, 
however,  they  quickly  spread  through  the  cytoplasm  giving  to 
its  hyaline  matrix  a  more  or  less  permanent  and  diffuse  coloration 
(i,  2,  3,  6). 

The  fresh  water  Amceba  dubia  and  the  unfertilized  eggs  of  the 
starfish,  Asterias  forbesii,  and  sanddollar,  Echinarachnius  parma, 
were  used  in  these  experiments.  The  amceba  and  the  eggs  were 
colored  with  the  dyes  either  by  the  immersion  method  or  by  the 
microinjection  method.  Both  methods  were  also  used  simul- 
taneously on  the  same  cell.  The  cells  were  then  immersed  in 
various  acid  and  alkaline  solutions  and  the  color  changes  noted. 
For  a  study  of  the  effect  of  NH3  and  CO^  the  cells  were  suspended 
in  hanging  drops  of  water  from  the  roof  of  a  special  form  of 

moist  chamber  which  was  closed  except  for  narrow  inlet 
and  outlet  tubes  (7).  The  hanging  drops  were  then  charged 

with  either  CO2  or  with  NH3  by  passing  the  moist  gas  through 
the  chamber. 


INTRACELLULAR    HYDRION    CONCENTRATION    STUDIES.          371 

i.    EFFECT  OF  ACIDS  AND  BASES  ON  AMEB.E  COLORED  BY  TIII; 
INJECTION  OF  ACID   INDICATORS  ONLY. 

Amebae  were  injected  with  0.4  per  cent  solution  of  brom 
cresol  purple,  phenol  red  and  cresol  red  (8).  These  indicators 
were  selected  because  they  change  color  within  the  pH  ranges 
tested  (i,  3).  Amebae,  injected  with  brom  cresol  purple,  are 
uniformly  blue  (the  alkaline  range),  with  phenol  red,  a  pale 
orange  yellow  (approaching  the  acid  range).  These  findings 
accord  with  those  already  published  (6)  from  which  the  pH  of 
the  freshwater  ameba  was  placed  at  6.9  ±  o.i. 

Amebae,  colored  by  the  injection  of  the  above-mentioned  dyes, 
were  immersed  in  solutions  of  HC1  (pH  5.5),  NH4C1  (pH  5.5), 
CO2  charged  water  (pH  5.5),  NaHCO3  (pH  8),  NH4OH  (pH  8) 
and  NaOH  (pH  8).  The  acidity  of  the  first  three  solutions  is 
sufficient  to  cause  the  indicators  to  take  on  the  yellow  color  of 
their  acid  ranges,  while  the  alkalinity  of  the  last  three  solutions 
is  sufficient  to  give  to  brom  cresol  purple  the  purple  blue,  and  to 
phenol  red  and  cresol  red  the  bright  red  color  of  their  alkaline 
ranges.  It  was  found  that  the  immersed  amebae  all  maintained 
their  original  colors  as  long  as  they  remained  alive.  The  color 
of  those  which  rounded  up  and  died  changed  to  that  characteristic 
for  the  pH  of  the  environing  medium. 

These  results  indicate,  either  that  there  is  no  penetration  of 
the  acid  or  of  the  alkali  from  the  solutions  used,  or  that  the  proto- 
plasm is  sufficiently  buffered  to  neutralize  the  acid  or  the  alkali 
which  does  penetrate. 

2.    EFFECT  OF  ACIDS  AND  BASES  ON  CELLS  STAINED  WITH 

NEUTRAL  RED  AND  INJECTED  WITH  ACID 

INDICATORS. 

a.  Amoeba  dubia. 

Since  the  permeability  of  cells  to  certain  acids  and  bases  can  be 
demonstrated  by  the  change  in  color  of  neutral  red,  amebae  were 
immersed  in  a  solution  of  neutral  red  until  various  intracellular 
inclusions  took  on  a  red  color.  These  amebae  were  then  injected 
with  solutions  of  the  indicators  which  color  the  cytoplasm  dif- 
fusely. On  immersing  these  doubly  colored  amebae  into  the 
various  acid  and  alkaline  solutions  the  following  results  were 
obtained : 


372 


ROBERT    CHAMBERS. 


In  accordance  with  the  previous  experiment  it  was  found  that 
immersion  produced  no  change  whatever  in  the  diffuse  coloration 
of  the  hyaline  cytoplasmic  matrix.  On  the  other  hand,  the 
inclusion  bodies  which  were  stained  with  neutral  red  quickly 
became  yellow  in  the  solutions  containing  the  NH3  (NH4OH 
and  NH4C1)  and  bright  red  in  those  containing  CO2  (NaHCOs 
and  CO2  charged  water). 

These  results  imply  that  the  pH  of  the  hyaline  cytoplasm  does 
not  change  even  when  sufficient  NH3  or  CO2  penetrates  to  change 
the  color  of  the  intracellular  inclusions.  In  other  words,  the 
pH  of  the  intracellular  inclusions  can  be  shifted  readily  by  the 
presence  of  CO2  or  of  NH3  in  the  environment  while  that  of  the 
protoplasmic  matrix  remains  constant. 

b.  Unfertilized  Eggs  of  the  Sanddollar  (Echinarachnius  parma} 
and  the  Starfish  (Asterias  forbesii). 

The  protoplasm  of  these  eggs  is  uniformly  crowded  with 
granules  or  macrosomes  practically  all  of  which  ultimately  stain 
a  deep  rose  red  with  neutral  red.  The  eggs  were  allowed  to 
remain  in  sea-water  containing  neutral  red  only  long  enough  to 
stain  a  small  percentage  of  the  granules.  The  eggs  were  then 
washed,  transferred  to  hanging  drops  of  sea-water  in  the  moist 
chamber  and  injected  with  the  indicator  solutions.  In  the  same 
chamber  were  placed,  as  controls,  other  hanging  drops  of  sea- 
water  colored  with  the  same  indicators.  Ammonia  gas  was 
then  passed  through  the  chamber  until  the  hanging  drops  became 
sufficiently  saturated  with  ammonia  to  change  the  color  of  the 
control  drops. 

The  color  of  the  eggs  was  noted  when  the  dyes  in  the  control 
drops  had  assumed  colors  indicating  a  pH  more  alkaline  than  8.4. 
In  every  case  the  color  of  the  granules,  stained  with  neutral  red, 
changed  from  red  (acid)  to  yellow  (alkaline)  while  the  diffuse 
coloration  of  the  indicators  in  the  hyaloplasm  of  the  eggs  per- 
sisted in  registering  the  originally  recorded  pH  of  6.8  ±  o.i  (3). 

An  experiment  giving  striking  color  contrasts  is  one  in  which 
three  dyes,  neutral  red,  phenol  red  and  cresol  red,  were  used  for 
the  purpose  of  detecting  simultaneously  the  pH  changes  in  the 
cytoplasm,  the  cytoplasmic  granules,  and  the  sea-water  sur- 
rounding the  eggs.  It  is  to  be  remembered  that  neutral  red 


INTRACELLULAR    HYDRION    CONCENTRATION    STUDIES.          373 

which  stains  the  granules  is  red  at  a  pH  more  acid  than  6.8  and 
yellow  at  a  pH  more  alkaline  than  7.4.  Phenol  red  which  colors 
the  hyaloplasm  is  yellow  at  a  pH  more  acid  than  6.8  and  red  at  a 
pH  more  alkaline  than  7.4,  and  cresol  red  which  was  used  for 
the  environing  sea-water  is  yellow  at  a  pH  more  acid  than  7.8 
and  red  at  a  pH  more  alkaline  than  8.0.  The  experiment  was 
the  following:  Eggs,  stained  with  neutral  red,  were  immersed  in 
a  hanging  drop  of  sea-water  colored  with  cresol  red  and  were 
then  injected  with  phenol  red.  The  result  was  a  striking  picture 
of  yellow  eggs  containing  scattered  red  granules  and  surrounded 
by  a  medium  of  yellow  sea-water.  Ammonia  gas  was  then 
passed  through  the  chamber  until  the  cresol  red  in  the  sea-water 
changed  from  yellow  (acid)  to  red  (alkaline).  As  soon  as  this 
occurred  the  cytoplasmic  granules,  stained  with  the  neutral  red 
turned  yellow  (alkaline)  while  the  hyaloplasm  maintained  the 
original  yellow  (acid)  color  of  the  phenol  red.  The  result  was 
now  a  picture  of  uniformly  yellow  eggs  standing  out  against  a 
background  of  red  sea-water.  Carbonic  acid  gas  was  then 
passed  through  the  chamber  until  it  displaced  the  NH3  in  the 
hanging  drops.  As  a  result  the  original  colors  returned,  viz., 
the  sea-water  again  became  yellow,  the  cytoplasmic  granules 
turned  from  yellow  to  red  but  the  cytoplasm  itself  remained 
yellow. 

Since  the  cytoplasm  has  a  pH  of  6.8  ±  o.i  (3)  which  is  in  the 
acid  range  of  phenol  red  the  above  experiment  is  not  suited  for 
detecting  a  possible  effect  of  the  CO2  on  the  cytoplasmic  pH. 
For  this  purpose  it  is  necessary  to  use  brom  cresol  purple  (yellow 
at  a  pH  more  acid  than  6.0  and  blue  at  a  pH  more  alkaline  than 
6.2)  which,  upon  injection,  colors  the  hyaloplasm  blue.  These 
eggs  were  immersed  in  a  hanging  drop  of  sea-water  colored  blue 
with  the  same  dye.  The  hanging  drop  was  suspended  in  the 
hermetic  chamber  through  which  moist  CO2  gas  was  made  to 
stream  until  the  sea-water  became  charged  with  CO2  sufficiently 
to  change  its  color  from  blue  to  yellow.  The  eggs  in  the  yellow 
water  kept  their  original  blue  color. 

These  experiments  indicate  that  NH3  and  CO2,  both  of  which 
penetrate  the  protoplasm  and  affect  the  pH  of  the  intracellular 
granules,  do  not  shift  the  pH  of  the  hyaloplasm  as  measured 
by  the  indicators. 


374 


ROBERT    CHAMBERS. 


3.    EFFECT  OF  CO2  AND  OF  NH3  ON  AMEB/E  WHOSE  CYTOPLASM 

AND  INCLUSION  BODIES  ARE  COLORED 

WITH  THE  SAME  INDICATOR. 

A  possible  error  in  the  previous  experiments  lies  in  the  fact 
that  the  coloration  of  the  cytoplasmic  inclusions  and  of  the 
hyaline  cytoplasm  were  not  made  with  the  same  dye.  For 
example,  neutral  red,  which  colors  the  cellular  inclusions,  is  a 
basic  dye,  while  the  dyes  used  for  producing  a  diffuse  coloration 
are  acidic.  It  is  conceivable  that  this  may  be  responsible  for 
their  difference  in  reaction  to  the  penetrating  CC>2  or  NH3. 

To  meet  this  objection  it  was  found  that  methyl  red  could  be 
used.  Methyl  red  has  already  been  used  as  a  vital  stain  for 
plant  protoplasm  (9)  and  is  a  pH  indicator,  being  red  at  a  pH 
more  acid  than  5.0  and  yellow  at  a  pH  more  alkaline  than  5.4. 
Immersion  of  amebae  in  an  aqueous  solution  of  this  dye  stains 
the  hyaline  cytoplasm,  its  various  inclusions  and  the  nucleus 
an  intense  yellow.  Amebae  colored  in  this  way  were  placed  in  a 
moist  chamber  in  hanging  drops  of  the  yellow  aqueous  solution 
of  methyl  red.  Moist  CO2  gas  was  then  passed  through  the 
chamber  until  the  hanging  drops  turned  from  yellow  to  red. 
When  this  occurred  it  was  found  that  the  yellow  stained  inclusions 
of  the  ameba  had  also  become  red  while  the  cytoplasm  and 
nucleus  remained  yellow.  Ammonia  vapor  was  now  passed 
through  the  chamber  whereupon  the  color  of  the  hanging  drops 
and  of  the  intracellular  inclusions  quickly  changed  back  to 
yellow. 

These  experiments  with  methyl  red  clearly  demonstrate  the 
penetration  of  CO2  into  the  living  ameba  l  as  registered  by  the 
change  in  color  of  the  intracellular  inclusions.  The  hyaline 
cytoplasm  and  the  nucleus,  however,  maintain  their  original 
color  and  give  no  evidence  of  a  change  in  pH. 

1  The  neutral  red  method  is  not  very  favorable  for  detecting  the  penetration  of 
CO»  into  cells  since  the  granules  stained  with  neutral  red  under  normal  conditions 
already  have  the  rose  red  color  characteristic  for  the  acid  range  of  the  dye.  On 
the  other  hand,  methyl  red  under  normal  conditions  stains  the  intracellular  granules 
the  yellow  color  of  its  alkaline  range.  Upon  exposure  to  CO2  the  color  of  the 
granules  changes  to  red,  which  is  as  decided  an  evidence  for  the  penetration  of 
the  CO2  as  is  the  neutral  red  method  for  the  penetration  of  NH3. 


INTRACELLULAR  HYDRION  CONCENTRATION  STUDIES.       375 

4.   THE  EFFECT  OF  PENETRATING  ACIDS  AND  BASES 
ON  THE  NUCLEAR  pH. 

The  nuclei  of  immature  starfish  eggs  were  used  in  these  experi- 
ments. The  nuclei  of  different  eggs  were  colored  with  cresol 
red,  neutral  red  and  phenol  red  by  the  microinjection  method 
after  which  the  eggs  were  exposed  to  CO2  and  to  NH3.  In  every 
case  the  color  within  the  nuclei  of  living  eggs  remained  constant 
irrespective  of  the  color  changes  of  the  granules  in  the  sur- 
rounding cytoplasm.  In  other  words,  the  nucleus  was  found  to 
be  sufficiently  buffered  so  that  the  intranuclear  pH  of  7.6-7.8 
(3)  remains  unchanged.  When  the  egg  disintegrates  by  crushing 
or  tearing,  the  nucleus  undergoes  changes  (3)  and  loses  all 
buffering  action.  The  persisting  spherical  nuclear  remnant  is 
then  immediately  susceptible  to  acid  and  alkali  changes  in  its 
environment. 

SUMMARY. 

The  presence  of  CO2  or  of  NH3  in  the  aqueous  medium  sur- 
rounding living  cells  readily  changes  the  pH  of  the  intracellular 
inclusions  which  stain  with  neutral  red  but  does  not  change  the 
pH  of  the  protoplasmic  matrix  nor  of  the  nucleus  as  long  as  the 
cell  is  alive. 

BIBLIOGRAPHY. 

1.  Needham,  J.,  and  Needham,  D.  M. 

'25  The  Hydrogen-ion  Concentration  and  the  Oxidation-reduction  Potential 
of  the  Cell-interior:  A  Micro-injection  Study.  Proc.  Roy.  Soc.,  B. 
98,  259. 

2.  Needham,  J.,  and  Needham,  D.  M. 

'26  The  Hydrogen-ion  Concentration  and  Oxidation-reduction  Potential  of 
the  Cell-interior  before  and  after  Cleavage:  A  Micro-injection  Study 
on  Marine  Eggs.  Proc.  Roy.  Soc.,  B,  99,  173-199- 

3.  Chambers,  R.,  and  Pollack,  H. 

'27  Micrurgical  Studies  in  Cell  Physiology.  IV.  Colorimetric  Determination 
of  the  Nuclear  and  Cytoplasmic  pH  in  the  Starfish  Egg.  Jour.  Gen. 
Physiol.,  10,  739-755- 

4.  Harvey,  E.  N. 

'14  The  Relation  between  the  Rate  of  Penetration  of  Marine  Tissues  by 
Alkali  and  the  Change  in  Functional  Activity  Induced  by  the  Alkali. 
Publ.  Carneg.  Instit.,  Wash.,  No.  183,  131. 

5.  Jacobs,  M.  H. 

'20  The  Production  of  Intracellular  Acidity  by  Neutral  and  Alkaline  Solutions 
Containing  Carbon  Dioxide.  Amer.  J.  Physiol.,  53,  457. 

'22  The  Influence  of  Ammonium  Salts  on  Cell  Reaction.  J.  Gen.  Physiol., 
5,  181. 


376 


ROBERT    CHAMBERS. 


6.  Chambers,  R.,  Pollack,  H.,  and  Hiller,  S. 

'27     The  Protoplasmic  pH  of  Living  Cells.     Proc.  Soc.  Exp.  Biol.  and  Med., 
24,  760-761. 

7.  Cohen,  B.,  Chambers,  R.,  and  Reznikoff,  P. 

'28     Intracellular  Oxidation-reduction  Studies,  I.     J.  Gen.  Physiol,  II,  585. 

8.  Clark,  W.  M. 

'25     The    Determination    of    Hydrogen    Ions.     Williams    and    Wilkins    Co.. 
Baltimore,  2d  ed. 

9.  Schaede,  R. 

'24     Uber  die  Reaktion  des  lebenden  plasmas.     Ber.  d.  bot.  Ges.,  42,  219. 


INTRACELLULAR   HYDRION   CONCENTRATION 

STUDIES. 

II.    THE  EFFECT  OF  INJECTION  OF  ACIDS  AND  SALTS  ox  THE 
CYTOPLASMIC  pH  OF  Ama>ba  dubia.1 

PAUL    REZNIKOFF   AXD    HERBERT    POLLACK 

In  a  previous  communication  (i)  from  this  laboratory  the 
pH  of  the  cytoplasm  of  Amceba  dubia  was  reported  to  have  a 
value  of  6.9  ±0.1.  To  determine  whether  any  permanent 
variations  in  the  intracellular  pH  could  be  artificially  produced, 
solutions  of  acids  salts  and  simple  salts  having  toxic  actions 
were  injected  by  the  micrurgical  technique  (2)  into  amebre 
previously  colored  with  indicators. 

EXPERIMENTAL. 

The  ameba  and  methods  used  in  these  experiments  were  the 
same  as  those  described  previously  (2).  The  hydrion  indicators 
(3)  employed  were  thymol  blue,  brom  phenol  blue,  brom  cresol 
green,  methyl  red,  chlor  phenol  red,  brom  cresol  purple,  phenol 
red,  and  orange  III.  Of  these  phenol  red  was  the  most  exten- 
sively used.  The  advantages  of  this  dye  are  twofold.  It  is  the 
least  toxic  of  all  the  indicators  and  is  the  most  valuable  one  in 
experiments  of  this  type  since  its  useful  range  covers  the  normal 
cytoplasmic  pH.  The  other  indicators  were  used  in  extreme 
changes  of  pH. 

In  the  case  of  each  solution,  at  least  10  ameba?  were  used,  and 
for  critical  concentrations  from  25  to  50.  Small  quartz  cover 
slips  were  employed  in  these  experiments.  They  were  attached 
by  means  of  water  films  to  the  ordinary  long  glass  cover  slips. 
On  the  quartz  slips  were  placed  amebae  in  a  drop  of  their  medium, 
varying  in  reaction  from  pH  5.8  to  7.5,  a  drop  of  indicator,  and 

1  From  the  Laboratory  of  Cellular  Biology,  Department  of  Anatomy,  Cornell 
University  Medical  College,  New  York  City,  and  the  Marine  Biological  Laboratory. 
Woods  Hole,  Massachusetts. 

2  Expenses   connected   with    this   investigation   were   in   part   drtrayril    from    a 
grant  by  the  Ella  Sachs  Plotz  Foundation. 

377 


PAUL    REZNIKOFF    AND    HERBERT    POLLACK. 

a  drop  of  the  solution  the  effect  of  which  was  to  be  tested.  The 
pipettes  used  were  made  of  pyrex  glass  and  were  rinsed  several 
times  in  distilled  water  and  then  in  solutions  of  the  substances 
to  be  injected.  The  dyes  were  injected  into  the  amebae  which 
were  permitted  to  recover.  After  recovery  the  next  solutions 
were  introduced. 

As  a  control,  the  degree  of  injury  caused  by  the  insertion  of 
the  pipette  was  determined.  The  method  employed  was  to 
note  any  change  in  color  of  the  previously  injected  dye  from 
the  possible  formation  of  acid  associated  with  injury  (4).  The 
simple  introduction  of  a  pipette  into  an  ameba  was  found  to 
give  no  indication  of  acid  production.  If  the  pipette,  however, 
stirred  up  the  cytoplasm  so  vigorously  that  the  injured  area  was 
ultimately  discarded,  a  distinct  acidity  was  produced.  When 
death  occurred  in  the  presence  of  those  dyes  covering  the  range, 
a  pH  of  about  5.5  was  indicated,  unless  the  pellicle  surrounding 
the  mass  was  broken  in  which  case  the  color  was  rapidly 
washed  out. 

Solutions  of  HC1  (pH  2)  when  introduced  into  an  ameba, 
which  is  colored  an  orange-yellow  with  previously  injected 
phenol  red,  cause  an  immediate  and  intense  yellow  coloration  of 
the  injected  area.  If  the  injected  region  is  not  irreparably 
injured  by  the  acid  the  pH  of  the  area  reverts  within  a  few 
seconds  to  that  of  the  normal  cytoplasm.  Usually,  however, 
the  injected  portion  is  injured  to  such  an  extent  that  it  is  pinched 
off  in  a  manner  previously  described  (2)  after  which  the  yellow 
color  (acid)  of  the  discarded  sphere  gradually  changes  to  that 
indicative  of  the  pH  of  the  environment.  In  time  the  color 
entirely  washes  out.  The  unaffected  remnant  of  the  ameba 
retains  its  orange-yellow  color. 

When  CaCl2  is  injected  in  concentrations  stronger  than  M/2OO 
the  phenomenon  of  solidification  and  pinching  off  is  accompanied 
by  distinct  evidence  of  acid  production.  If  a  solution  of  M/2OO 
CaCl2  is  injected  into  amebae  colored  with  phenol  red,  the  flash 
of  yellow  color  indicating  acid  production  rapidly  returns  to 
that  of  the  normal  pH,  provided  the  injected  area  is  not  dis- 
carded. If  the  color  does  not  revert  within  a  few  seconds  the 
affected  portion  is  pinched  off. 


1NTRACELLULAR    HYDRION    CONCENTRATION    STUDIIs  379 

In  only  three  cases  out  of  several  hundred  did  the  maintenance 
of  a  localized  acid  reaction  persist  for  as  long  as  a  minute  after 
HC1  or  CaCl2  had  been  introduced  without  subsequent  pinching 
off.  To  investigate  further  this  condition  in  which  a  localixed 
acid  reaction  is  maintained  for  an  appreciable  time  with  subse- 
quent complete  recovery  solutions  of  M/^2  Aids  were  introduced 
into  amebae  colored  with  phenol  red.  Such  a  concentration  of 
A1C13  causes  a  solidification  of  the  injected  portion  but  this 
region  is  not  infrequently  reincorporated  after  being  almost  dis- 
carded (5).  Of  at  least  50  cases  only  one  showed  a  delay  of  two 
to  three  minutes  in  the  return  of  color  from  yellow  (acid)  to  the 
original  orange-yellow  after  the  affected  area  had  been  re- 
incorporated.  In  every  other  test  the  reversal  of  color  was 
immediate  if  reincorporation  occurred  or,  if  the  area  was  dis- 
carded, its  color  remained  yellow. 

The  introduction  of  solutions  of  MgClo  of  pH  6.5  in  con- 
centrations of  M/3O  and  stronger  into  amebae  previously  injected 
with  phenol  red  causes  an  immediate  shift  to  yellow,  indicating 
acid  production.  The  cell  breaks  and  the  color  diffuses  out. 
When  an  M[6o  solution  of  MgClo  is  injected  the  yellow  color 
reverts  rapidly  to  that  normal  for  healthy  cytoplasm  and  the 
ameba  recovers. 

In  order  to  determine  the  degree  of  .acid  production  by  the 
injection  of  CaCl2  and  MgCl  (pH  6.6)  amebae  were  injected 
with  this  salt  after  having  been  colored  with  thymol  blue,  orange 
III,  methyl  red,  brom  phenol  blue  and  brom  cresol  green.  All 
these  dyes  were  injected  with  the  exception  of  methyl  red  (6). 
Amebae  were  stained  with  methyl  red  by  immersing  them  into 
5  cc.  of  distilled  water  into  which  a  few  drops  of  a  0.4  per  cent, 
aqueous  solution  of  methyl  red  were  placed.  With  methyl  red 
a  distinct  red  is  produced  when  either  CaCl2  or  MgClo  is  injected 
into  amebae.  With  orange  III  the  yellow  color  persists.  This 
places  the  reaction  of  the  acidified  portion  of  the  cell  between 
pH  4.0  and  4.6.  It  is  difficult  to  determine  a  more  exact  pH 
value  because  the  color  changes  with  brom  phenol  blue  are  not 
sufficiently  distinctive  within  the  critical  range.  These  results 
show  that  the  acid  produced  by  injection  of  CaCl2  or  MgCl2  is 
more  marked  than  the  acid  of  injury  which  was  found  to  be 

about  pH  5.5. 

25 


380  PAUL    REZNIKOFF    AND    HERBERT    POLLACK. 

When  NaCl  and  KC1  (pH  7)  are  injected  into  amebse  colored 
with  phenol  red,  no  immediate  change  in  color  occurs.  If  the 
concentration  of  these  salts  is  lethal  (2)  the  rounded  amebae 
gradually  take  on  the  color  indicative  of  the  pH  of  the  environ- 
ment. Injection  of  non-lethal  concentrations  of  these  salts 
results  during  the  quiescent  period  in  a  slight  shift  in  color 
toward  that  suggestive  of  the  pH  of  the  surrounding  medium 
whether  this  be  acid  or  alkaline.  But  as  soon  as  the  ameba 
recovers  the  color  reverts  to  the  normal  orange-yellow. 

The  change  in  color  of  the  discarded  spheres  after  HC1  and 
CaCl2  had  been  injected  or  of  the  dead  ameba  when  lethal 
amounts  of  NaCl  or  KC1  were  introduced  is  due  to  the  penetration 
from  the  environment.  It  is  quite  obvious  that  any  uncontrolled 
changes  in  the  environmental  pH  would  be  confusing.  Therefore 
it  was  necessary  to  take  precautions  to  obviate  this  factor. 
In  preliminary  experiments,  when  ordinary  cover  slips  were 
used  the  medium  increased  in  alkalinity  markedly  during  its 
contact  with  the  coverslip.  The  use  of  pyrex  glass  or  coverslips 
coated  with  balsam  or  collodion  did  not  prevent  this  change  in 
hydrogen  ion  concentration.  To  maintain  a  constant  pH  of  the 
environment  a  buffer  calcium  acetate  solution  l  of  pH  6  was  used. 
In  this  ameba?  were  immersed  and  the  various  salts  and  acids 
injected.  In  this  case  the  dead  spheres  and  the  amebae  killed 
with  NaCl  or  KC1  took  on  the  color  representing  the  reaction 
of  the  surrounding  medium,  viz.,  pH  6.  With  quartz  cover 
slips,  which  do  not  affect  the  pH  of  solutions  coming  into  contact 
with  them,  the  medium  remained  constant  and  the  discarded 
spheres  and  dead  amebse  assumed  the  reaction  of  any  environing 
medium  into  which  they  were  placed.  These  results  show  that 
the  injection  of  the  individual  chlorides  are  ineffective  in  changing 
the  intracellular  pH  except  when  toxic  concentrations  were  used. 

DISCUSSION. 

In  the  marine  egg  the  production  of  acid  due  to  injury  is 
much  more  easily  manifested  than  in  the  ameba.  As  previously 
shown  (4)  a  localized  flash  of  color  indicating  acid  production  is 
apparent  in  the  starfish  egg  if  the  needle  is  introduced  abruptly 

1  We  wish  to  thank  Dr.  William  Perlzweig  for  the  preparation  of  this  buffer 
solution. 


INTRACELLULAR    HYDRION    CONCEN  TK.VI  H  ).\    STUDIES.          38! 

into  the  interior.  This  change  is  not  evident  in  the  ameba  unless 
the  mechanical  trauma  is  vigorous  enough  to  cause  death  of  the 
disturbed  part.  This  difference  points  to  a  greater  susceptibility 
to  injury  of  the  egg  or  an  increased  buffering  power  of.  the  ameba 
which  may  in  turn  be  due  either  to  an  increase  in  the  amount  of 
buffer  present  or  to  a  greater  ease  in  diffusion  of  buffers  through 
the  cell.  This  faster  rate  of  mobilization  of  buffers  in  the  ameba 
as  compared  to  the  egg  is  suggested  by  the  constant  flow  of  cyto- 
plasm of  the  ameba  in  contrast  to  the  relatively  '  quiescent 
cytoplasm  of  the  egg. 

The  production  of  acid  when  CaCl2  or  MgCl2  is  introduced 
into  the  ameba  may  be  due  to  the  production  of  insoluble  Ca 
or  Mg  salts  wTith  the  liberation  of  free  acid.  Aub  and  Reznikoff 
(7)  have  suggested  such  an  explanation  for  the  effect  of  Pb  salts 
on  red  blood  cells.  Ca  may  also  unite  with  the  carbonate  and 
phosphate  to  form  insoluble  salts  with  the  production  of  free 
acid.'  This  acid  formation  is  evident  until  some  alkali  diffuses 
into  the  solidified  mass  and  neutralizes  the  acids  present.  Such 
an  explanation  does  not  preclude  the  possibility  also  of  the  for- 
mation of  a  Ca  or  Mg  organic  compound. 

CONCLUSIONS. 

1 .  The  cytoplasm  of  the  living  Amoeba  dubia  shows  considerable 
buffering  power  to  pH  changes  induced  by  the  injection  of  salts 
and  buffers. 

2.  If  HC1,  injected  into  the  ameba,  is  immediately  buffered  by 
the  cytoplasm  no  toxic  effect  results.     If  the  quantity  injected 
is  too  great  to  be  buffered,  the  affected  portion  of  the  cell  dies 
and  is  discarded. 

3.  CaClo,  MgCl2  and  A1C13,  injected  into  amebac  colored  with 
indicators,  give  colorimetric  evidence  of  the  production  of  acid 
greater  in  amount  than  can  be  explained  by  acid  produced  by 
mechanical    injury.     Unless   this   color   reverts   immediately   to 
that   indicative   of   normal   cytoplasm,    the   affected   portion   is 
discarded  in   the  case  of  CaClo  and  A1C13  and  the  entire  cell 
dies  in  the  case  of  MgCl2. 

4.  Upon  death  permeability  changes  occur  so  that  the  dead 
mass  of  the  ameba  quickly  assumes  the  hydrogen  ion  concen- 
tration of  the  environment. 


382  PAUL   REZNIKOFF   AND    HERBERT    POLLACK. 

BIBLIOGRAPHY. 

* 

1.  Chambers,  R.,  Pollack,  H.,  and  Hiller,  S. 

'27  The  Protoplasmic  pH  of  Living  Cells.  Proc.  Soc.  Exp.  Biol.  and  Med., 
xxiv,  760. 

2.  Chambers,  R.,  and  Reznikoff,  P. 

'26  Micrurgical  Studies  in  Cell  Physiology.  I.  The  Action  of  the  Chlorides 
of  Na,  K,  Ca,  and  Mg  on  the  Protoplasm  of  Amoeba  proteus.  J.  Gen, 
Physiol.,  viii,  369. 

3.  Clark,  W.  M. 

'25  The  Determination  of  Hydrogen  Ions.  Williams  and  Wilkins  Co.. 
Baltimore,  2d  ed.,  81. 

4.  Chambers,  R.,  and  Pollack,  H. 

'27  Micrurgical  Studies  in  Cell  Physiology.  IV.  Colorimetric  Determina- 
tion of  the  Nuclear  and  Cytoplasmic  pH  in  the  Starfish  Egg.  J.  Gen. 
Physiol.,  x,  739. 

5.  Reznikoff,  P. 

'26  Micrurgical  Studies  in  Cell  Physiology.  II.  The  Action  of  the  Chlorides 
of  Lead,  Mercury,  Copper,  Iron,  and  Aluminum  on  the  Protoplasm  of 
Amoeba  proteus.  J.  Gen.  Physiol.,  x,  9. 

6.  Chambers,  R. 

'29  Intracellular  Hydrion  Concentration  Studies,  I.  The  Environment  and 
the  pH  of  Cytoplasm  and  of  Inclusion  Bodies.  Biol.  Bull.,  LVI,  3£9. 

7.  Aub,  J.  C.,  and  Reznikoff,  P. 

'24  Lead  Studies  III.  The  Effects  of  Lead  on  Red  Blood  Cells.  Part  3. 
A  Chemical  Explanation  of  the  Reaction  of  Lead  with  Red  Blood 
Cells.  J.  Exper.  Med.,  xl,  189. 


INTRACELLULAR   HYDRION   CONCENTRATION 

STUDIES. 

III.   THE  BUFFER  ACTION  OF  THE  CYTOPLASM  OF  Amoeba  dubia 
AND  ITS  USE  IN  MEASURING  THE  pH. 

HERBERT    POLLACK, 

LABORATORY  OF  CELLULAR  BIOLOGY,  DEPARTMENT  OF  ANATOMY,  CORNELL  UNI- 
VERSITY MEDICAL  COLLEGE,  NEW  YORK  CITY. 

Recent  determinations  of  intracellular  pH  have  been  made  by 
noting  the  color  of  indicator  dyes  injected  directly  into  the 
protoplasm  (i,  2,  3,  4,  5).  The  recorded  value  was  found  by 
comparing  the  results  of  injecting  a  series  of  overlapping  dyes. 
The  color  of  the  dye,  whose  range  was  found  to  include  the  pH 
of  the  cytoplasm,  was  compared  with  known  standards  projected 
optically  into  the  field  of  the  microscope. 

While  attempting  to  determine  the  buffer  action  of  the  cyto- 
plasm it  was  found  that  an  indirect  method  could  be  used  to 
check  the  results  obtained  from  the  direct  color  comparisons. 

It  is  known  that  a  drop  of  a  solution  at  a  certain  pH  added 
to  another  buffer  solution  containing  an  indicator  dye  will 
cause  a  momentary  localized  change  in  the  color  providing  the 
reactions  of  the  two  solutions  are  different.  The  closer  the  pH 
values  of  the  two  solutions  are  to  one  another,  the  less  marked 
will  be  the  color  change.  When  they  have  the  same  pH  there  will 
be  no  change  in  color.  It  is  possible  to  take  advantage  of  this 
fact  in  measuring  the  intraprotoplasmic  pH  by  injecting  a  series 
of  solutions  of  known  pH  into  cells  colored  by  the  previous 
injection  of  indicator  dyes.  As  will  be  brought  out  later,  this 
technique  is  only  approximate  but  serves  to  check  wide  deviations 
from  direct  tint  comparisons. 

It  has  been  shown  that  M/4  solutions  of  mono-sodium  phos- 
phate may  be  injected  with  no  toxic  effect  (6),  and  that  the 
potassium  ion  has  about  the  same  toxicity  as  the  sodium  ion  on 
injection  (7).  Hence  the  Clark  buffer  solutions  (8)  whose 
KH2PO4  concentration  is  M/2O  should  be  non-toxic  from  the 
point  of  view  of  salt  concentrations,  and  any  toxic  effect  must 
be  due  to  the  buffered  hydrogen  ion  concentration. 

383 


384  HERBERT    POLLACK. 

Amoeba  proteus  and  Amoeba  dubia  were  used  in  this  study 
since  their  pH  has  been  determined  by  direct  tint  readings. 

The  amebae  were  injected  with  brom  cresol  purple  and  phenol 
red  which  were  the  indicators  whose  ranges  cover  the  pH  as  found 
by  previous  work.  The  colored  amebae  were  then  injected  with 
the  phosphate  buffers  from  pH  5.6-8.0  and  observations  made 
on  the  changes  in  color. 

When  buffer  solutions  of  5.6,  5.8,  6.0  were  injected  into 
amebae  colored  with  brom  cresol  purple,  a  temporary  but  distinct 
yellow  flash  was  produced.  Those  above  6.2  produced  no  color 
change  with  this  indicator  which  is  already  blue  in  the  cell. 
Buffer  solutions  of  pH  6.2  and  6.4,  when  injected  into  amebae 
colored  orange  yellow  with  phenol  red,  gave  temporary  yellow 
flashes.  With  the  same  indicator,  solutions  having  a  pH  of  6.6, 
6.8  or  7.0  showed  no  color  change.  Those  whose  pH  was  7.2 
and  above  showed  reddish  flashes  in  the  orange  yellow  colored 
cytoplasm. 

This  shows  that  the  pH  of  the  amoeba  is  not  less  than  6.6 
and  not  greater  than  7.2.  This  is  in  accord  with  the  results 
obtained  in  this  laboratory  in  previous  investigations  and  not 
with  those  obtained  by  the  Needhams  (2).  They  also  used  the 
microinjection  technique  with  direct  color  comparison  for  reading 
the  pH  values.  Their  value  for  the  cytoplasmic  pH  of  the 
amoebae  was  7.6,  as  was  Pantin's,  who  used  the  neutral  red  vital 
staining  technique  (9). 

As  for  the  Needhams'  results  it  must  be  remembered  that  they 
were  using  a  European  species  and  also  that  they  report  the 
amebae  died  within  five  minutes  after  injection.  In  the  investi- 
gation reported  in  this  paper  the  amebae  were  allowed  to  recover 
fully  after  the  injection  before  treatment  with  the  buffer  solutions. 
With  a  proper  injection  of  phenol  red  and  brom  cresol  purple, 
amebae  can  be  kept  alive  and  apparently  normal  for  at  least  two 
days  (4).  Frequent  checks  on  the  color  by  direct  comparison 
with  standard  buffers  showed  no  change  during  that  time.  The 
amebae  colored  with  phenol  red  maintained  the  same  orange 
yellow  tint  (pH  6.9  ±  o.i)  as  long  as  they  were  kept  under 
observation.  On  the  other  hand  moribund  and  dead  amebae 
take  on  the  pH  of  the  environment  which  is  usually  alkaline 
when  the  ordinary  glass  coverslips  are  employed  without  proper 


INTRACELLTLAR    IIVDRION    CONCENTRATION    Sl(  DIES.         385 

precautions  (5).     As  for  the  value  obtained  by  Pantin   (8)  tin- 
inefficacy  of  neutral  red  staining  has  been  shown  (3,  10). 

The  interesting  fact  is  that  regardless  of  the  pH  value  of 
the  buffer  solution  injected  the  return  of  color  of  the  indicator 
present  to  its  usual  one  is  quite  rapid  and  constant.  If,  how- 
ever, sufficient  buffer  was  put  in  to  change  the  pH  of  the  cell, 
the  cell  died.  These  facts  emphasize  two  important  point- 
relating  to  intracellular  hydrogen  ion  concentration.  One,  that 
the  cytoplasm  has  a  considerable  buffering  power,  and  t\v<> 
that  when  the  pH  of  the  cytoplasm  is  changed,  t,he  cell  dies. 

BIBLIOGRAPHY. 

1.  Needham,  J.,  and  Needham,  D.  M. 

'25-'26  The  Hydrogen  Ion  Concentration  and  Oxidation-reduction  Potential 
of  the  Cell  Interior  before  and  after  Fertilization  and  Cleava  •<:•:  A 
Micro-injection  Study  of  Marine  Eggs.  Proc.  Roy.  Soc.  London, 
Series  B,  XCIX,  173. 

2.  Needham,  J.,  and  Needham,  D.  M. 

'26  Further  Micro-injection  Studies  on  the  Oxidation-reduction  Potential  of 
the  Cell  Interior.  Proc.  Roy.-Soc.  London,  Series  B,  XCIX,  383. 

3.  Chambers,  R.,  and  Pollack,  H. 

'27  Micrurgical  Studies  in  Cell  Physiology,  IV.  Colorimetric  Determination 
of  the  Nuclear  and  Cytoplasmic  pH  in  the  Starfish  Egg.  J.  Gen. 
Phys.,  X,  739- 

4.  Chambers,  R.,  Pollack,  H.,  and  Killer,  S. 

'27  Protoplasmic  pH  of  Living  Cells.  Proc.  Soc.  Exp.  Biol.  Med.,  XXIV, 
760. 

5.  Reznikoff,  Paul,  and  Pollack,  H. 

'29  Intracellular  Hydrion  Concentration  Studies.  II.  The  Effect  of  In- 
jection of  Acids  and  Salts  on  the  Cytoplasmic  pH  of  Amoeba  dubi,i. 
Biol.  Bull.,  LVI,  377- 

6.  Reznikoff,  Paul,  and  Chambers,  R. 

'27  Micrurgical  Studies  in  Cell  Physiology.  III.  The  Action  of  CO2,  and 
Some  Salts  of  Xa,  Ca,  and  K  on  the  Protoplasm  of  Amoeba  dnbia.  J. 
Gen.  Physiol.,  X,  731- 

7.  Chambers,  R.,  and  Reznikoff,  P. 

'26  Micrurgical  Studies  in  Cell  Physiology.  I.  Action  of  the  Chlorides 
of  Na,  K,  Ca,  and  Mg  on  the  Protoplasm  of  Amoeba  proteiis.  J.  Gen. 
Phys.,  VIII,  369- 

8.  Clark,  Wm.  M. 

'22  The  Determination  of  Hydrogen  Ions.  Williams  and  \Vilkins  Co., 
Baltimore,  Md. 

9.  Pantin,  C.  F.  A. 

'23     On    the    Physiology    of   Ameboid    Movement.     J.    Marine    Biol.    A 

XIII,  i,  24. 
10.  Chambers,  Robert. 

'29     Intracellular  Hydrion  Concentration  Studies.     I.  The  Relation  of  ilu- 

Environment    to    the    pH    of    Protoplasm    and    its    Inclusion    Bodir-. 

Biol.  Bull.,  LVI,  369- 


THE    EFFECTS   OF   CHANGES    IN    MEDIUM    DURING 

DIFFERENT   PERIODS   IN   THE   LIFE   HISTORY 

OF    UROLEPTUS  MOBILIS  AND 

OTHER   PROTOZOA. 

LOUISE  H.  GREGORY. 
3.   THE  EFFECTS  OF  YEAST  EXTRACTS. 

The  effect  of  vitamines  on  the  vitality  of  protozoa  has  been  a 
subject  of  but  little  investigation.  In  1917  Calkins  and  Eddy  (i) 
reported  no  effect  of  treating  paramecia  with  pancreatic  vitamine 
extracted  with  Fuller's  earth.  In  1918  Lund  (2),  working  with 
yeast  extracts,  found  that  if  Paramecia  had  been  starved  before 
being  fed  with  boiled  yeast  their  size  and  speed  of  oxidation  were 
increased  but  not  the  cell  division.  In  1919  Chambers  (3) 
reported  a  slight  increase  when  the  animals  were  fed  yeast, 
especially  ground  yeast,  and  in  the  same  year  Flather  (4)  obtained 
similar  results  with  the  unpolished  rice.  All  of  these  experiments 
were  upon  Paramecium,  which  is  not  a  favorable  subject  for 
investigation  unless  pure  lines  are  established  and  endomixis 
watched,  for  a  change  in  the  vitality  may  be  due  to  a  reorganiza- 
tion of  the  protoplasm  rather  than  to  a  change  in  the  environ- 
ment. Abderhalden  and  Kohler  in  1919  (5)  reported  a  slight 
stimulation  of  Colpoda  cucullus  when  treated  with  yeast  extracts 
but  the  evidence  is  not  decisive. 

Through  the  courtesy  of  Professor  W.  H.  Eddy  and  Dr.  Ralph 
Kerr,  of  Teachers  College,  I  have  had  placed  at  my  disposal  the 
following  yeast  extracts,  (i)  Alpha  bios  No.  223  extracted  by 
Professor  Eddy  in  1924  (6).  (2)  Beta  bios  isolated  in  1928  by 
Dr.  Kerr  (7).  (3)  Gamma  bios  a  residue  substance  similar  to 
bios  II  reported  by  Lucas  and  Miller  (8)  in  1924.  These  three 
substances  were  prepared  as  indicated  in  Table  I.,  which  has 
been  compiled  by  Dr.  Kerr. 

TABLE  I. 

THE  SEPARATION  OF  YEAST  AUTOLYZATE  INTO  VARIOUS  Bios  FACTORS. 
I.  Preliminary  Fractionation. 

1.  Make  autolyzed  yeast  66  per  cent,  alcoholic  by  volume  to  precipitate  proteins. 

Filter. 

2.  Filtrate  from  i.     Add  hot  saturated  baryta  so  long  as  an  immediate  precipitate 

386 


EFFECTS    OF    CHANGES    IN    MEDIUM. 


387 


forms.     Add  alcohol  as  necessary  to  maintain  66  per  cent,  strength.     Filter. 

Save  ppt.  for  8. 
3.  Filtrate  from   2.     Contains  alpha  bios  and  some  gamma  bios.     Neutralize 

immediately  with  sulfuric.     Adjust  to  pH  4.7.     Precipitate  with  iron  sol. 

and  discard  pptate. 
4    Filtrate  from  3.     Adjust  to  pH  5.3      Precipitate  with  iron  sol.     Filter.     Save 

nitrate  for  7.     Precipitate  contains  all  the  alpha  bios. 


II.  Isolation  of  Alpha  Bios. 

5  Ppt.  from  4.  Work  up  with  water 
and  refilter  to  remove  water 
washings.  Dissolve  ppt.  in  30 
per  cent,  sulfuric.  When  solu- 
tion is  complete  dilute  with  water 
and  neutralize  with  baryta.  (Fe, 
SO4,  ions  removed  as  Fe(OH)s 
and  BaSO*).  Filter  by  suction 
and  discard  ppt.  With  baryta 
and  sulfuric  remove  quantita- 
tively all  Ba,  Fe  and  SC>4  ions. 

6.  Filtrate  from  5.  Evaporate  to  dry- 
ness  and  recrystallize  from  hot 
95  per  cent,  ethyl  alcohol.  Puri- 
fied product  has  melting  point 
223°  C. 


III.  Concentration  of  Gamma  Bios. 
7.  Start  with  nitrate  from  4  Evapo- 
rate to  small  volume.  Add  sul- 
furic to  make  5  per  cent,  by 
weight.  Filter  if  necessary  and 
discard  ppt.  Now  add  phospho- 
tungstic  dissolved  in  5  per  cent, 
sulfuric  so  long  as  any  ppt.  forms. 
Filter  and  discard  filtrates.  Ppt. 
contains  gamma  bios. 
15.  Start  with  phosphotungstates  from 
7  and  12.  Decompose  with  ba- 
ryta in  the  usual  way.  Filter. 
Make  filtrate  decidedly  alkaline 
to  litmus.  Add  alcohol  to  80 
per  cent,  by  volume.  Filter  and 
discard  ppt.  if  any.  Free  filtrate 
of  Ba  and  SO4  quantitatively. 
Filtrate  now  contains  a  product 
not  yet  purified  but  which  sug  - 
gests  Miller  and  Lucas*  bios  II. 
We  designate  it  here  as  gamma 
bios. 


IV.  Isolation  of  Beta  Bios. 

8.  Start  with  ppt.  from  2.     Wash  with  alcohol.     Then  stir  washed  ppt.  repeatedly 

with  water  and  filtrate  by  suction  so  long  as  the  water  is  colored.  Neutralize 
the  filtrates  immediately  with  sulfuric.  Refilter  and  discard  pptates  and 
residue. 

9.  Filtrate  from  8.     Treat  with  hot  saturated  Ag2SC>4.     Filter  and  discard  ppt. 
10.  Filtrate  from   9.     Treat  with  hot  saturated  acid   mercuric  sulfate.     Filter. 

Discard  ppt. 

n.  Filtrate  from  10.     Free  from  Ag  and  Hg  ions  with  HjS.     Remove  excess  SO« 
with  baryta.     Save  clear  filtrates.     Evaporate  at  40°  C.  to  small  volume. 

12.  Filtrate  from  n.     Make  5  per  cent,  sulfuric  by  weight  and  extract  five  times 

with  ether  of  equal  volume  discarding  ether  extract.  Add  phosphotungstic 
acid  in  5  per  cent,  sulfuric  until  no  further  ppt.  forms.  Filter.  Add  the 
ppt.  to  7.  (See  III,  7  above.)  Make  filtrate  slightly  alkaline  with  baryta 
and  refilter.  Discard  this  ppt. 

13.  Filtrate  from  12.     Neutralize  with  sulfuric  and  evaporate  to  small  volume. 

Make  80  per  cent,  alcoholic  by  volume  and  again  ppt.  with  baryta.  Filter 
and  discard  filtrate. 


388  LOUISE    H.    GREGORY. 

14.  Precipitate  from  13.  Free  of  Ba  with  sulfuric.  Evaporate  to  a  thick  sirup 
at  40°  C.  Dehydrate  by  stirring  and  grinding  with  dry  acetone  to  a  fine 
white  powder.  Filter  nearly  to  dryness  on  suction  filter  but  leave  enough 
acetone  to  make  a  moist  mass.  Transfer  acetone-wet  product  to  vacuum 
desiccator  and  here  free  of  acetone  by  suction.  Product  is  Beta  bios. 

I  wish  to  express  my  appreciation  to  Professor  Eddy  and  Dr. 
Kerr  for  their  interest  and  helpful  suggestions. 

The  work  with  the  bioses  was  begun  in  1926-27  at  the  same 
time  when  experiments  with  di-sodium  phosphate  were  being 
conducted  on  Uroleptus  mobilis  in  order  to  determine  any  vari- 
ations in  response  according  to  the  age  of  the  protoplasm. 
Since  then  Dallasia  from  pure  lines  of  Professor  Calkins, 
Stylonychia,  and  Pleurotricha  have  been  used  in  addition  to 
Uroleptus  mobilis.  Whenever  possible  the  material  was  taken 
from  pure  lines  started  from  an  exconjugant  or  cyst  so  that  the 
age  of  the  protoplasm  was  known.  In  the  case  of  Stylonychia 
conjugation  did  not  occur  and  the  material  was  obtained  from 
a  single  individual  isolated  from  the  wild  culture.  The  methods 
used  in  all  experiments  were  the  same  as  those  of  earlier  papers 
and  as  usual  the  rate  of  division  is  considered  an  indication  of 
the  vitality  of  the  protoplasm. 

In  earlier  papers  (9,  10),  results  of  experiments  have  been 
reported  which  indicate  that  the  protoplasm  of  Uroleptus  mobilis 
varies  in  its  response  to  treatment.  Beef  extracts  and  di- 
potassium  phosphate  cause  an  increase  in  the  division  rate 
only  when  the  protoplasm  is  mature  while  di-sodium  phosphate 
causes  an  increase  in  the  division  rate  of  cells  of  all  ages  but 
the  greatest  increase  occurs  in  the  mature  cells.  Experiments 
with  di-sodium  phosphate  have  been  continued  and  will  serve  as 
an  additional  control  in  the  majority  of  the  experiments  with 
the  bioses. 

I.  Experiments  with  Alpha  Bios. 

Three  series  of  Uroleptus  mobilis  were  used  in  these  experi- 
ments. Various  concentrations  of  alpha  bios  were  tried  and 
finally  a  concentration  of  .05  mg.  per  cc.  was  fixed  as  the  best. 
The  bios  solution  was  added  to  the  normal  hay  flour  medium 
daily  and  controls  were  carried  on  in  the  normal  medium  and 
also  parallel  experiments  were  conducted  at  the  same  time  with  a 
medium  containing  di-sodium  phosphate  in  the  Packard  (n) 
concentration  of  .M/yooo.  The  results  are  shown  in  Table  II. 


KFKKCTS    OF    CHANGES    IN     MEDIUM. 


389 


TABLE  II. 

EFFECTS  OF  DI-SODIUM  PHOSPHATE  AND  ALPHA  Bios  ON  THE  DIVISION 
RATE  OF   Uroleplus  mobilix. 


Amount  of  Variation  from  Control  in 

Division  Rate  per  Line  in 

Series 

Age  in 

ro-day  Periods. 

No. 

Gen. 

* 

Di-sodium  Phosphate 

Alpha  Bios 

Series. 

Series. 

139 

19 

+  3-8 

— 

64 

+  3-4 

— 

125 

+    .6 

-  3-o 

175 

+    .6 

-  2.4 

225 

+  4-4 

+  3-0 

240 

+  1.6 

-  2.6 

275 

+  3-4 

+  3-0 

140 

19 

+    -4 

— 

60 

+  2.4 

+  1.8 

117 

-  3-0 

-  3-2 

157 

—  i.o 

-4-8 

218 

+  3-4 

+  2.O 

227 

+  4.2 

+    .6 

141 

6 

+   1.2 

-  i.S 

36 

-   I.O 

—   2.2 

66 

+  1.8 

—  2.2 

125 

+  1.6 

-  2.4 

180 

+  4-2 

+  1.9 

192 

+  4-4 

+  2.4 

234 

+  3-2 

+  4-0 

243 

-     .6 

-  4.0 

As  in  former  experiments  the  sodium  phosphate  caused  a 
stimulation  of  the  vitality  throughout  the  life  history  of  Series 

139  and  practically  throughout  the  life  of   Series    141.     Series 

140  was  the  least  vigorous  and  died  out  in  the  227th  genera- 
tion after  showing  instability  throughout  its  life.     The  greatest 
stimulation  however  in  all  three  series  appeared  during  maturity. 

Alpha  bios  failed  to  act  as  a  stimulant  save  in  the  225th  and 
275th  generation  of  Series  139,  three  times  at  slightly  irregular 
intervals  in  Series  140  and  only  slightly  after  the  iSoth  generation 
in  Series  141  save  in  the  234th  generation  when  there  was  a 
slightly  higher  division  rate  than  that  of  the  control  or  sodium 
series.  Thus  alpha  bios  with  few  exceptions  has  a  depressing 
effect  on  the  vitality  of  these  three  series  of  Uroleplus. 

These  results  may  be  due  to  at  least  two  factors:  (i)  too  acid  a 


390 


LOUISE    H.    GREGORY. 


condition  of  the  medium,  (2)  a  general  lowering  of  the  vitality 
of  Uroleptus  mobilis.  Undoubtedly  the  protoplasm  was  weaken- 
ing as  it  did  not  respond  as  vigorously  to  sodium  stimulation  as  it 
did  in  the  experiments  of  1926.  On  the  other  hand  the  H  ion 
concentration  of  the  alpha  bios  medium  was  slightly  lower  than 
that  of  the  normal  medium  (7.2).  Since  trial  experiments  with 
beta  bios  known  to  be  more  acid,  resulted  in  a  decided  lowering 
of  the  vitality  and  since  it  seemed  uncertain  whether  any  bios 
would  cause  a  definite  stimulation  of  an  animal  cell,  experiments 
were  conducted  in  which  a  yeast  extract  containing  all  the  bioses 
was  used  and  in  one  series  di-sodium  phosphate  was  added  to 
the  yeast  extract  medium  to  increase  the  alkalinity. 

II.  Experiments  with  Harris  Yeast  Extract. 

Four  experiments  with  Uroleptus  at  varying  ages  and  two  with 
Dallasia  were  carried  on  in  which  the  animals  were  kept  (i)  in  a 
normal  medium  to  which  was  added  daily  yeast  extract  of  a 
concentration  of  .01  mg.  per  cc.,  (2)  in  a  normal  medium  to 
which  was  added  di-sodium  phosphate,  (3)  in  the  same  medium 
as  in  (2)  with  the  addition  of  the  yeast  extract  and  (4)  in  normal 
hay  flour  medium  as  the  control  series.  These  results  are  shown 
in  Table  III. 

TABLE  III. 

EFFECTS  OF  YEAST  EXTRACT  ON  THE  DIVISION  RATE  OF 

Uroleptus  mobilis  AND  Dallasia. 


Series 
No. 

Age  in 
Gen. 

Amount  of  Variation  from  Control  in  Division 
Rate  per  Line  in  lo-day  Periods. 

Sodium 
Phosphate 
Series. 

Yeast 
Extract 
Series. 

Sodium  Phos- 
phate Yeast 
Extract  Series. 

Uroleptus 
146.  .  .  . 

143  
142  
141  

15 
30 
84 
250 

died 
—  2.0 

-  3-4 
o.o 

—      .2 

-3-6 

-  2.8 

-  5-2 

died 

+      -4 

-      2.6 
-      2.8 

Dallasia 
I  

75 

75 

-  3-4 

+  3-2 

+  5-0 

+  7.2 

+    9-0 
+  10.4 

2  

EFFECTS    OF    CHANGES    IN    MEDIUM. 


391 


The  experiments  with  Uroleptus  show  practically  no  stimu- 
lating effect  of  the  yeast  extracts.  The  protoplasm  was  too 
weak  to  respond  and  the  entire  race  died  out  shortly  afterwards. 
The  two  experiments  with  two  different  series  of  Dallasia  both 
in  the  75th  generation,  showed  a  definite  increase  in  division 
rate  in  all  the  experimental  series.  It  was  especially  marked  in 
the  yeast  sodium  hay-flour  medium  when  the  rate  was  10.4 
divisions  higher  than  that  of  the  normal  control  series  and  12.4 
higher  than  the  sodium  hay-flour  series  for  the  same  period. 
Since  the  yeast  extracts  caused  a  marked  stimulation  of  the 
vitality  of  Dallasia  both  with  and  without  the  addition  of  sodium 
to  the  normal  medium  the  fractional  extracts  of  the  yeast  were 
then  used. 

III.  Experiments  with  a  Neutral  Salt  of  Beta  Bios  and 

with  Gamma  Bios. 

Dallasia,  Pleurotricha  sp.  and  Stylonychia  sp.  were  treated 
with  the  two  bioses  using  the  same  methods  as  above.  These 
three  animals  differ  in  their  normal  rate  of  cell  division.  Dallasia 
when  young  undergoes  from  3-5  divisions  daily,  Pleurotricha  like 
Uroleptus  not  more  than  1-2  divisions  and  Stylonychia  divided 
every  other  day.  Stylonychia  may  have  been  more  mature  as  in 
this  series  no  conjugations  occurred  and  the  age  is  unknown. 
The  results  of  the  experiments  are  shown  in  Table  IV. 

TABLE  IV. 

THE  EFFECTS  OF  BETA  AND  GAMMA  Bios  ON  THE  DIVISION  RATE. 


Series 

XT/-* 

Age  in 

C^f^rt 

Amount  of  Variation 
Division  Rat 
lo-day 

from  the  Control  in 
2  per  Line  in 
Periods. 

Beta  Bios  Series. 

Gamma  Bios  Series. 

Dallasia 

2               .... 

115 

-     .8 

+    4-8 

130 

+      -2 

+    4-8 

Pleurotricha 

90 
110 

+  5.0  (ist  10  da' 
died  (2d                 ) 

+  10.2  (ist  10  days) 
+    1.6  (2d    "      "    ) 
+    4-8 

Stylonychia 

— 

+    3.8  (ist  10  days) 
+    4-8  (2d                 ) 

392 


LOUISE    H.    GREGORY. 


Beta  bios  apparently  had  no  effect  on  Dallasia  when  older 
gamma  bios,  however  acted  as  a  definite  stimulant  increasing  the 
division  rate,  4.8  divisions  per  line  in  10  days.  Pleurotricha  was 
stimulated  in  the  goth  and  noth  generation  and  showed  a 
marked  response  to  gamma  bios.  Stylonychia  had  its  division 
rate  almost  doubled  in  the  gamma  bios  solution  and  this  effect 
continued  for  twenty  days.  When  Pleurotricha  was  stimulated 
for  twenty  days  the  effect  died  out  during  the  second  ten-day 
period.  This  may  be  correlated  with  the  variation  in  norma1 
vitality  of  the  two  species. 

IV.  Experiments  with  Alpha,  Beta  and  Gamma  Bios. 

Finally  experiments  were  conducted  to  compare  the  effects  of 
the  three  bioses  on  Pleurotricha  and  Stylonychia,  when  added  to 
the  normal  medium  and  in  a  few  experiments  to  the  sodium 
medium.  The  results  are  shown  in  Table  V. 

TABLE  V. 

EFFECTS  OF  ALPHA,  BETA,  AND  GAMMA  Bios  ON  THE  DIVISION  RATE 
OF  Stylonychia  AND  Pleurotricha. 

Amount  of  Variation  from  the  Control  in  the  Division  Rate 
per  Line  in  lo-day  Periods. 


Alpha 
Bios 
Series. 

Beta 
Bios 
Series. 

Gamma 
Bios 
Series. 

Na2HPo4 
Series. 

NajHPo4 
Alpha 
Bios. 

Na2HPo4 
Beta 
Bios. 

Na2HPo4 
Gamma 
Bios. 

Stylonychia 

+  1-4 

+     4-2 

o.o 

•  +  4-4 

+    3-2 

Pleurotricha 
115  gen  +10.2 
130  gen.  died.  . 

+  II-  4 
+     4-8 

+   IO.2 

+     8.0 

-I-  8.2 
-     .8 

died 

9.0 

+  16.0 

+   12.8 

In  these  experiments,  Stylonychia  quickened  its  division  rate 
in  all  media  save  that  of  the  Gamma  Bios,  where  the  division 
rate  equalled  that  of  the  control.  Pleurotricha  in  the  H5th 
generation  showed  a  definite  stimulation  in  all  media,  especially 
in  that  with  the  HNa2PO4  and  gamma  bios.  In  the  second 
experiment  the  division  rate  of  the  control  dropped  to  5.4 
divisions  per  line  in  10  days  while  the  experimental  series  kept 
a  much  higher  level,  the  climax  being  reached  with  18.2  divisions 
per  line  for  the  same  lo-day  period  in  the  series  kept  in  normal 


EFFECTS   OF    CHANGES    IN    MEDIUM.  393 

medium  to  which  HNa2PO4  and  Gamma  Bios  solution  had  been 
added. 

SUMMARY  AND  CONCLUSION-,. 

These  preliminary  experiments  indicate  that  while  the  proto- 
plasm of  Uroleptus  mobilis  was  usually  depressed  when  treated 
with  yeast  extracts  due  probably  to  its  weakened  condition,  that 
of  Dallasia,  Pleurotricha  and  Styloiiychia  were  definitely  stimu- 
lated by  the  addition  of  fractional  extracts  of  yeast  to  the  normal 
medium.  Alpha  bios  in  general  causes  the  least  effect  and  gamma 
bios  the  greatest  increase  in  division  rate.  \Yhile  there  is  usually 
an  increase  in  the  protoplasmic  activity  when  sodium  phosphate 
is  added  to  the  medium  already  containing  the  bios  solution, 
this  may  not  be  -due  to  an  increased  alkalinity  as  the  variation 
in  Hydrogen  ion  concentration  were  not  more  than  .I-.2  of  a 
point.  The  explanation  may  lie  in  an  increase  in  the  per- 
meability of  the  cell  allowing  a  far  more  reaching  effect  of  the 
bios  solution.  The  age  and  general  characteristics  of  the  proto- 
plasm must  also  be  taken  into  consideration  and  further  experi- 
ments are  planned  with  pure  lines  of  varying  ages. 

The  fact  of  a  sudden  marked  increase  in  the  division  rate  of  a 
protozoan  cell  when  treated  with  these  yeast  extracts  brings  to 
mind  the  theory  of  Burrows  in  which  the  rapid  growth  of  cells 
and  formation  of  tumors  is  assumed  to  be  due  to  a  lack  of  balance 
between  vitamines  in  the  cells.  The  relation  of  these  extracts 
to  vitamines  has  yet  to  be  proved.  They  are  however  stimu- 
lating substances  to  yeast  cells  and  to  certain  animal  cells,  their 
effects  varying  according  to  the  age  and  general  conditions  of 
the  protoplasm. 

BARNARD  COLLEGE, 
June,  1928. 

REFERENCES. 

1.  Calkins,  G.  N.,  and  Eddy,  W.  H. 

'17     Soc.  Exp.  Biol.  and  Med.,  Vol.  XIV.,  162. 

2.  Lund. 

'18     Am.  Jour.  Phys.,  Vol.  XLVIL,  p.  167. 

3.  Chambers,  B. 

'19     BIOL.  BULL.,  Vol.  XXXVI.,  p.  82. 

4.  Flather. 

'19     BIOL.  BULL.,  Vol.  XXXVI.,  p.  54- 

5.  Abderhalden  and  Kohler. 

'19     Arch.  f.  ges.  Physiologic,  Vol.  CLXXVI.,  p.  209. 


394  LOUISE    H.    GREGORY. 

6.  Eddy,  W.  H.,  Kerr,  and  Williams. 

'24     Jour.  Am.  Chem.  Soc.,  Vol.  XLVI.,  2846. 

7.  Kerr,  R.  W. 

'28     Soc.  Exp.  Biol.  and  Med.,  Vol.  XXV.,  3847. 

8.  Lucas  and  Miller. 

'24     Jour.  Phys.  Chem.,  Vol.  XXVIII.,  1180. 

9.  Gregory,  L.  H. 

'25     BIOL.  BULL.,  Vol.  XLVIII.,  No.  3. 

10.  Gregory,  L.  H. 

'26     BIOL.  BULL.,  Vol.  LI.,  No.  3. 

11.  Packard,  C. 

'26     Jour.  Cancer  Research,  May. 

12.  Burrows. 

'26     Soc.  Exp.  Biol.  and  Med.,  Vol.  XXIV.,  3240. 


Vol  LV  December,  1928  No.  6 


BIOLOGICAL  BULLETIN 


INSECT   METABOLISM. 

THE  ANAEROBIC   METABOLISM  OF  AN    INSECT    (ORTIIOPTKKA). 

JOSEPH  HALL  BODINE, 

ZOOLOGICAL  LABORATORY,  UNIVERSITY  OF  PENNSYLVANIA. 

That  insects  can  live  anaerobically  for  varying  periods  of  time 
has  been  repeatedly  pointed  out  (Winterstein,  1921;  Lee,  1924, 
1925;  Willis,  1925;  Davis  and  Slater,  1926,  etc.).  When  de- 
prived of  oxygen  they  enter  into  a  state  closely  resembling 
anesthesia.  Various  methods  for  the  withdrawal  of  oxygen  have 
been  experimentally  employed,  such  as  replacing  the  air  by  the 
gases  hydrogen,  carbon  dioxide,  nitrogen,  etc.;  by  evacuating 
the  vessel  in  which  the  insects  are  contained  and  by  immersing 
the  insects  in  water.  The  results  produced  by  all  of  these 
methods  closely  resemble  each  other  and  if  the  deprivation  of 
oxygen  has  not  been  too  long  the  insects  recover  and  appear  quite 
normal.  A  state  of  anaerobiosis  thus  produced  in  insects  offers 
rather  unique  conditions  for  studying  the  gaseous  exchange 
of  an  organism  during  oxygen  lack. 

The  present  work  deals  with  the  rates  of  oxygen  consumption 
and  the  blood  pH  changes  in  grasshoppers  under  normal  as  well 
as  anaerobic  conditions. 

MATERIAL  AND  METHODS. 

The  grasshoppers,  including  individuals  of  the  following 
species,  M elano plus  differ entialis,  Melanoplus  fenuif-nibnini,  and 
Chortophaga  viridifasciata,  were  hatched  and  raised  under 
laboratory  conditions  and  fed  lettuce.  Organisms  of  known  and 
varied  ages  were  used  in  the  experiments.  Oxygen  determina- 
tions were  made  by  means  of  the  modified  Krogh  Manometer 

395 


396 


JOSEPH    HALL    BODINE. 


(Bodine  and  Orr,  1925),  immersed  in  a  Freas  constant  temperature 
water  bath  maintained  at  25°  ±0.1°  C.  Animals  were  subjected 
to  anaerobic  conditions  largely  by  immersion  in  water  at  25°  C. 
for  varying  periods  of  time.  Immersion  in  water  was  found  to 
give  results  identical  with  those  produced  by  the  gases  hydrogen, 
carbon  dioxide  or  nitrogen. 

Animals  were  first  put  in  the  manometers  and  their  normal 
rates  of  oxygen  consumption  determined.  After  removal  from 
immersion  in  water  they  were  again  quickly  put  in  the  same 
manometer  and  their  rates  of  oxygen  consumption  during  re- 
covery noted.  By  such  a  procedure  a  continuous  record  of  the 
oxygen  consumption  of  the  organism  was  obtained  except  for 
the  actual  period  of  immersion  in  water. 

Blood  pH  determinations  were  made  by  micro-colorimetric 
methods  (Bodine,  1925).  By  means  of  fine  capillary  pipettes 
blood  was  easily  obtained  from  minute  punctures  made  by  fine 
needles  in  the  lateral  abdominal  wall  of  the  animal. 


f  6  7  ft  9  /» 

TIME    //y  HOURS 

FIG.  i.  Curve  showing  the  effect  of  immersion  in  water  for  75  minutes  on  the 
rate  of  oxygen  consumption  of  a  male,  nymph,  Melanoplus  differ  entialis.  Space 
within  arrows  indicates  the  period  of  immersion  of  the  animal;  points  on  curve,  the 
rates  of  oxygen  consumption  before  (which  is  taken  as  100  per  cent.)  and  after  im- 
mersion. Abscissa,  time  in  hours. 


JNSI-XT    MKTABOLISM. 


397 


RESULTS. 

Oxygen  Consumption. 

The  normal  rate  of  oxygen  consumption  for  each  organism 
was  determined  over  a  period  of  an  hour  or  more  until  a  constant 
rate  was  obtained.  The  animal  was  then  removed  from  the 
manometer,  placed  in  a  glass  tube,  the  open  ends  of  which  wen- 
CD  vered  with  wire  gauze,  and  immersed  in  water  at  25°  ('.  to  a 


TIME  iK  HOURS 

FIG.  2.  Curve  showing  the  effect  of  immersion  in  water  for  i5<j  minutes  <ni  t he- 
rate  of  oxygen  consumption  of  a  male,  nymph,  Mdatwplus  diffcn-nliuli^.  Space 
within  arrows  indicates  the  period  of  immersion  of  the  animal;  points  mi  <  urve, 
the  rates  of  oxygen  consumption  before  (which  is  taken  as  100  per  cent.)  ami  alter 
immersion.  Abscissa,  time  in  hours. 

depth  of  1 80  mm.  All  air  bubbles  were  removed  from  tin- 
surface  of  the  animal  and  the  ends  of  the  tube  by  gentle  shaking. 
The  animal  becomes  motionless  within  a  very  short  time  after 
immersion  and  remains  so  throughout  the  immersion  period. 
After  immersion,  the  organism  is  quickly  removed  from  the  glass 
tube,  dried  on  filter  paper  and  returned  to  the  same  manometer 
originally  used  to  determine  its  normal  rate  of  oxygen  consump- 
tion. The  recovering  animal  is  left  in  the  manometer  and  its 

• 

rate  of  oxygen  consumption   followed   until   complete   recovery 

26 


398 


JOSEPH    HALL    BODINE. 


occurs.     Since  the  general  response  of  all  organisms  is  essentially 
the  same,  only  typical  experiments  will  be  presented. 

Figures  I,  2,  3  and  4,  in  which  the  rates  of  oxygen  consumption 
are  expressed  in  terms  of  the  normal  rates  (100  per  cent.),  show 
graphically  the  changes  in  the  rates  of  oxygen  consumption  pro- 
duced by  exposures  to  lack  of  oxygen.  An  examination  of  these 


100 


kl 

3 


" 


CHOR.  V/RID 

(,0  Min. 


•«  -*.-*-«-  tf-  -a-  -A  . 


/  2  *  t  e  9  "» 

T/rtE  Ifi  HOURS' 

P"IG.  3.  Curve  showing  the  effect  of  immersion  in  water  for  60  minutes  oji  the 
rate  of  oxygen  consumption  of  a  male,  numph,  Chorlophaga  viridifaciata.  Space 
within  arrows  indicates  the  period  of  immersion  of  the  animal;  points  on  curve,  the 
rates  of  oxygen  consumption  before  (which  is  taken  as  100  per  cent.)  and  after  im- 
mersion. Abscissa,  time  in  hours. 

figures  further  shows  that  when  the  animal  is  readmitted  to 
oxygen  after  immersion  its  rate  of  oxygen  consumption  increases 
considerably  over  the  normal  rate  or  that  characteristic  for  the 
animal  before  deprived  of  oxygen.  This  excess  oxygen  taken  up 
by  the  organism  can  be  shown,  in  carefully  controlled  experiments, 
to  be  approximately  equal  to  the  amount  the  organism  would 
have  taken  up  normally  during  the  period  it  was  deprived  of 
oxygen.  In  other  words,  it  seems  that  the  grasshopper  when 
deprived  of  oxygen  or  existing  anaerobically,  goes  into  debt  for 
oxygen  in  a  manner  quite  similar  to  that  pointed  out  for  the 


INSECT   METABOLISM. 


399 


cockroach  (Davis  and  Slater,  1926)  and  for  heavy  physical  work 
in  man  or  for  isolated  muscle  (Hill,  1922). 

The  length  of  exposure  to  lack  of  oxygen  that  can  be  with- 
stood by  different  species  of  grasshoppers  varies  as  pointed  out 
below.  Some  species  have  been  found  to  successfully  withstand 
as  high  as  7  hours  immersion  in  water.  The  rates  of  oxygen  con- 
sumption during  recovery,  as  indicated  in  Figs,  i,  2,  3  and  4, 


CHOR 


no  ft,n 


. 


9  6 

TIME  //Y  HOURS 


e 


/o 


FIG.  4.  Curve  showing  the  effect  of  immersion  in  water  for  120  minutes  on  the 
rate  of  oxygen  consumption  of  a  male,  nymph,  Chortophaga  viridifasciata.  Space 
within  arrows  indicates  the  period  of  immersion  of  the  animal;  points  on  curve,  the 
rates  of  oxygen  consumption  before  (which  is  taken  as  100  per  cent.)  and  after  im- 
mersion. Abscissa,  time  in  hours. 

seem  to  be  greatly  influenced  by  the  length  of  the  immersion 
period.  Animals  immersed  for  60  to  120  minutes  usually  recover 
in  a  typical  manner  as  shown  in  these  figures.  When  the  im- 
mersion period  is  lengthened,  however,  there  is  a  strong  tendency 
for  the  rates  of  oxygen  consumption  to  return  to  normal  in  an 
extremely  slow  fashion  as  shown  in  Figs.  2  and  4.  This  slowness 
in  recovery  to  a  normal  rate  of  oxygen  consumption  is  probably 
correlated  with  the  physiological  condition  of  the  organism  as  well 
as  with  the  fact  that  the  exposure  might  be  just  a  sub-lethal  one 
for  the  animal. 


400 


JOSEPH    HALL    BODINE. 


There  also  appears  to  be  a  marked  difference  in  the  rates  of 
recovery  in  the  different  species.  Melanoplus  differ entialis 
seems  better  able  to  reversibly  withstand  long  immersion  than 
Chortophaga  viridifasciata,  as  indicated  in  Figs.  2  and  4.  Age  is 
also  an  important  factor,  since  younger  individuals  withstand 
and  recover  from  long  immersions  better  than  older  ones. 

The  relations  between  length  of  immersion  in  water  and  re- 
covery time  for  the  different  species  of  grasshoppers  examined  are 


K 

f 

I 


CHOR.  v/x/a 
VEL.  01  rr. 


tOO  IfO  20O  25O  3OO 

LEVGTH   Or  EXFDSURE- 


35O 


fD 


FIG.  5.  Curves  showing  relation  between  average  mean  recovery  time  and 
length  of  immersion  in  water  in  young  adult  grasshoppers  of  three  species,  Melano- 
plus femur  rubrum,  Melanoplus  differentialis  and  Chortophaga  viridifasciata.  Each 
curve  based  on  several  hundred  observations.  Recovery  time  indicates  return  of 
"turn-over  or  righting"  reflex. 

graphically  shown  in  Fig.  5.  From  a  study  of  this  figure  it  is 
evident  that  a  linear  relationship  exists  between  length  of  im- 
mersion and  recovery  time.  That  the  causes  of  the  anaesthetic 
condition  produced  by  lack  of  oxygen  are  doubtless  due  to  the 
carbon  dioxide  and  lactic  acid  produced  within  the  organism 
seems  reasonable  when  a  comparison  is  made  between  the  results 
obtained  by  immersion  in  water  and  those  obtained  by  subjecting 
the  organism  to  carbon  dioxide  (Willis,  1925),  as  indicated  in 
Fig.  6.  These  curves  are  qualitatively  similar.  In  view  of 
similar  evidence  gained  from  studies  on  anaerobiosis  of  mam- 
malian muscle  (Hill,  1922),  such  an  hypothesis  seems  reasonable 
in  explaining  these  phenomena  in  grasshoppers.  As  a  matter  of 


I.SMCCT    METABOLISM. 


401 


fact,  Lee  (1924)  has  shown  that  injection  of  carbonic  acid  or 
lactic  acid  into  a  grasshopper  produces  results  quite  similar  to 
the  present  ones  on  immersion  of  the  animals. 


FIG.  6.  Curves  showing  comparisions  between  average  mean  recovery  times 
and  rates  and  length  of  immersion  in  water  and  exposure  to  carbon  dioxide  in 
Melanoplus  differ entialis.  Carbon  dioxide  data  from  Willis  (1925). 

BLOOD  pH. 

As  previously  pointed  out  the  normal  pH  of  the  blood  of  the 
grasshopper,  M.  differ  entialis,  seems  to  be  6.8.  (Bodine,  1926.) 
A  careful  study  has  been  made  of  the  blood  of  individuals  through- 
out the  present  immersion  and  recovery  experiments.  Fig.  7,  in 
which  are  plotted  together  results  of  experiments  carried  out  for 
different  periods  of  time,  shows  graphically  the  pH  changes  oc- 
curring in  the  blood  during  the  anaerobic  and  recovery  periods. 
A  progressive  lowering  in  pH  values  with  length  of  immersion 
occurs  and  seems  to  reach  a  minimum  at  about  5.8.  Below  this 
minimum  the  animal  seems  irreversibly  affected.  Upon  re- 
covery, a  slow  blowing  off  of  acids  (HzCOs),  probably  occurs  and 
the  blood  then  gradually  returns  to  its  normal  pH  value.  An 
interesting  fact,  indicated  in  Fig.  7,  is  that  no  marked  changes  in 
blood  pH  occur  in  immersed  animals  when  removed  from  the 
water  until  after  respiratory  movements  have  become  established. 
Upon  careful  examination  of  the  animal  it  is  found  that  initial 
respiratory  movements  upon  recovery  are  extremely  slow  and  of 
great  depth.  As  recovery  progresses  the  movements  become 
more  regular  and  normal.  These  changes  in  blood  pH  strongly 


4O2 


JOSEPH    HALL    BODINE. 


suggest  that  during  anaerobiosis  large  quantities  of  acids, 
carbonic  and  lactic,  are  produced  and  that  recovery  consists 
largely  in  their  elimination  by  the  organism. 

As  pointed  out  by  Davis  and  Slater  (1926),  who  have  found 
similar  results  for  the  anaerobic  metabolism  of  the  cockroach, 
insects  seem  to  be  extremely  favorable  material  with  which  to 
further  elucidate  the  changes  occurring  during  the  anaerobic 
existence  of  organisms  and  the  question  of  energy  formation  under 
such  conditions. 


7.0 
(,.8 


I 


f-7 


VELDIFF; 


/oo  /fo 

Tint  in 


zoo 


FIG.  7.  Curve  showing  the  effect  of  immersion  in  water  for  120  minutes  on  the 
pH  of  the  blood  of  a  male,  nymph,  Melanoplus  differ entialis.  Space  within  arrows 
indicates  period  of  immersion  of  the  animal;  points  on  curve,  actual  pH  determina- 
tions of  blood  before  and  after  immersion.  Broken  portion  of  curve  is  composite, 
being  made  up  from  pH  determinations  of  the  blood  of  individuals  immersed  for 
periods  of  time  ranging  from  50  to  120  minutes. 

SUMMARY  AND  CONCLUSION. 

1.  Rates  of  oxygen  consumption  in  grasshoppers  before  and 
after  immersion  in  water  (lack  of  oxygen)  have  been  determined. 

2.  During  oxygen  lack,  grasshoppers  build  up  an  oxygen  debt. 
When  readmitted  to  oxygen  an  increased  rate  of  oxygen  consump- 
tion occurs  and  an  excess  of  oxygen,  approximately  equal   in 
amount  to  that  which  the  organism  would  have  taken  up  normally 
during  the  period  it  was  deprived  of  oxygen,  is  consumed. 


IN  si  (    I     MKTABOLISM.  403 

3.  During  anaerobiosis  Mood  pH  falls.     I'pon    recovery  pll 
values  slowly  return  to  normal. 

4.  It  is  suggested  that  the  chemical  change  responsible  for  the 
anesthetic  condition  accompanying  anarrobiosis  is  the  production 
of  an  excess  of  acid,  carbonic  and  lactic,  and  that  recovery  con- 
sists in  their  elimination. 

LITERATURE    CITED. 
Bodine,  J.  H. 

'25     Physiology  of  the  Orthoptera.     Hydrogen  Ion  Concentration  of  the  Blood 
and    Alimentary    Tract    of    Certain    Orthoptera    (Grasshoppers).     BIOL. 
BULL.,  Vol.  XLVIII.,  pp.  79. 
Bodine,  J.  H. 

'26     Hydrogen  Ion  Concentration  in  the  Blood  of  Certain  Insects  (Orthoptera). 

BIOL.  BULL.,  Vol.  LI.,  pp.  363. 
Bodine,  J.  H.,  and  Orr,  P.  R. 

'25     Respiratory    Metabolism.     Physiological    Studies   on  Respiratory  Metab- 
olism.    BIOL.  BULL.,  Vol.  XLVIII.,  p.  i. 
Davis,  J.  H.,  and  Slater,  W.  R. 

'26     The  Aerobic  and  Anaerobic  Metabolism  of  the  Common  Cockroach   (P. 

Orientalis).  Biochemical  Jour.,  Vol.  20,  pp.  1167. 
Hill,  A.  V. 

'22     Mechanism  of  Muscular  Contraction.     Physiol.  Reviews,  Vol.  2,  pp.  310. 
Lee,  M.  O. 

'24     Respiration  in  Orthoptera.     Amer.  Jour.  Physiol.,  Vol.  68,  pp.  135. 
Lee,  M.  O. 

'25     On   the    Mechanism   of    Respiration   in   Certain   Orthoptera.     Jour.    Exp. 

Zool.,  Vol.  41,  pp.  125. 
Willis,  J. 

'25     Effects  of  Carbon  Dioxide.     Effects  of  Different  Tensions  of  Carbon  Di- 
oxide on  Certain  Orthoptera  (Grasshoppers).     BIOL.  BULL.,  Vol.  XLVIII.. 
pp.  209. 
Winterstein,  H. 

'21     Handbuch  d.  vergleichenden  Physiologic,  Jena. 


THE   PULSATORY   RHYTHM   OF  THE   CONTRACTILE 
VESICLE   IN   PARAMECIUM. 

FRANCIS   E.   LLOYD  AND  J.  BEATTIE. 
I. 

It  became  apparent  to  the  senior  author  a  year  ago  that  there 
are  discrepancies  between  the  more  recent,  and  therefore  pre- 
sumably the  most  correct,  accounts  of  the  behavior  of  the  con- 
tractile vesicle  in  Paramecium  and  the  objective  facts  as  ap- 
prehended by  him.     As  to  these  facts  both  the  present  authors 
have    found    themselves    in    agreement,    and    it   was   therefore 
determined  to  make  an  investigation  of  said  behavior  by  such 
refined  means,  by  way  of  control  of  direct  and  unaided  vision, 
as  are  available.     Two  methods  were  used,  that  of  recording 
visual  observations  of  critical  points  in  the  contractile  cycle  on  a 
rotating  drum  and,  still  better,  that  of  making  motion  pictures  at 
normal  speed,  viz:    16  per  second,  without  any  lapse.     It  is  not 
easy  to  get  a  Paramecium  to  remain  within  the  field  of  vision  long 
enough  to  take  a  motion  picture  so  as  to  get  a  continuous  view  of 
the  contractile  vesicle  for  two  or  three  cycles  of  movement.     We 
have  however  succeeded  by  making  use  of  very  slight  compression 
between  cover  slip  and  slide — a  method  to  which  there  is  some, 
but  we  believe  not  wholly  justified  objection — and  by  surrounding 
the  animals  with  a  suspension  of  Chinese  ink,  a  time  honored 
method  for  demonstrating  the  expulsion  of  fluid  from  the  con- 
tractile vesicle,  as  used  by  Carter  (1861)  and  by  Jennings  (1904) 
and  by  many  others  before  and  since.     It  happens  that  a  rather 
thick  suspension  of  this  insoluble  pigment  impedes  the  move- 
ments of  the  animal,  so  that  the  chances  for  observing  a  relatively 
quiet  one,  with  the  contractile  vesicles  in  full  view,  is  much  in- 
creased without,  we  think,  in  the  least  affecting  the  behavior  of  the 
vesicles  themselves.     We  suspect  that  in  Chinese  ink  there  is  an 
admixture  of  some  aromatic  substance  which  may  act  as  a  mild 
depressant,  narcotic  or  otherwise,  but  of  this  we  have  no  sure 
knowledge.     However,  the  animals  can  live  for  several  hours 

404 


CONTRACTILE    VESICLE    IN    I'ARAMI.i  HM. 


405 


under  a  cover-glass  in  a  thick  suspension  of  Chinese  ink  and  ap- 
pear quite  undamaged.  We  have  no  doubt  at  all  events  that  the 
behavior  of  the  vesicles  is  normal.  The  motion  pictures  produced 
by  the  senior  author  have  been  shown  l  in  illustration  of  this 
paper;  in  the  accompanying  plate  we  present  a  few  excerpts  from 
one  of  the  films  to  serve  present  purposes. 

The  point  of  departure  of  this  study  may  be  better  appreciated 
by  considering  in  the  first  place  the  latest  pronouncement  on  the 
subject,  that  of  Nassonow  (1924).  In  the  text  which  follows  we 
shall  speak  simply  of  the  vesicle  2  (--  contractile  vacuole)  and  the 
canals  (=  rays,  canaliculi,  radial  canals).  The  general  topog- 
raphy of  the  apparatus  is  so  wrell  known  that  we  may  take 
familiarity  with  it  for  granted.  Nassonow  says : 

"After  the  emptying  and  the  complete  disappearance  of  the 
vesicle  there  become  visible  in  its  immediate  vicinity  the  5-7 
canals.  The  ends  directed  toward  the  center  of  the  vesicle  are 
strongly  swollen  and  no  continuity  between  them  is  to  be  seen. 
The  swollen  ends  approach  each  other,  flow  into  each  other  and 
form  a  new  vesicle,  into  which  the  fluid  of  the  canals  now  flows. 
Thereupon  the  canals  quite  disappear  and  only  later  do  new 
canals  begin  to  be  formed  in  their  place  the  ends  of  which  after 
the  emptying  and  disappearance  of  the  vesicle  suffers  enlarge- 
ment and  in  this  manner  complete  the  cycle  "  (Nassonow,  IQ24,3 
p.  454).  Nassonow  then  goes  on  to  recall  the  views  of  others, 
including  that  of  Stempell  (1914)  in  regard  to  the  existence  of  a 
membrane,  with  which  Nassonow  was  particularly  concerned,  ap- 
parently acquiescing  completely  with  this  author  in  respect  of  the 
progress  of  the  cycle  of  behavior.  We  therefore  quote  Stempell 
also  as  follows: 

1  Winnepeg  meeting  of  the  Royal  Society  of  Canada,  May,  1928. 

2  It  seems  to  us  that  Claparede  and  Lachmann  chose  the  better  terminology, 
and  we  follow  them,  with  Pritchard. 

3  Among  the  figures  illustrating  the  paper  by  Nassonow  occur  two  which  wo 
may  remark  in  passing  to  be  capable  of  precisely  the  opposite  interpretation  to 
that  given  by  him.     His  Fig.  40  is  labelled  "Diastole  of  the  excretion  apparatus" 
while  Fig.  42  is  labelled  "Systole  of  the  excretion  apparatus."     If  by  excretion  ap- 
paratus he  means  the  vesicle  then  figure  40  represents  early  systole  and  figure  42 
early  diastole.     If  however  he  means  the  canals  then  his  labelling  is  correct.     It  is 
not  easy  to  understand  his  exact  meaning.     It  is  certain  that  diastole  and  systole 
of  the  vesicle  ar,e  not  synchronous  with  those  of  the  canals. 


406  FRANCIS    E.    LLOYD   AND    J.    BEATTIE. 

'The  end-products  of  metabolism  collect  in  dissolved  condition 
in  certain  places  in  the  protoplasm,  namely,  in  a  canal-  or  space 
system  (probably  a  branched  one)  the  exits  of  which  run  towards 
the  two  pulsating  vesicles  as  afferent  canals.  As  soon  as  the 
vesicle  is  emptied  ('Sobald  die  Vacuole  sich  entleert')  the  ends  of 
these  afferent  canals  swell  up  to  form  '  Bildungsvacuole'  since 
the  fluid  flows  hereinto  as  to  the  place  of  minimal  pressure,  and 
is  here  dammed  up.  As  a  result  of  this  pressure  delicate  proto- 
plasmic valves  open  and  permit  the  volumes  of  fluid  which  have 
collected  in  the  canal-ends  to  flow  together  in  the  vesicular  space, 
on  which,  after  this  is  filled,  the  valves  promptly  close.  Since 
the  fluid  now  collected  in  the  vesicle  has  a  high  osmotic  pressure, 
there  results  a  lasting  addition  of  water  to  the  fluid  already  held 
in  the  vesicle  by  diffusion  through  the  semipermeable  membrane 
formed  ad  hoc.  As  soon  as  the  pressure  of  the  fluid  in  the  vesicle 
has  reached  a  certain  height,  that  is,  has  become  higher  than  that 
of  the  external  water  pressure,  a  second  valve  at  the  apex  of  the 
papilla-like  dome  of  the  pellicula  opens  and  there  results  from  the 
pressure  of  the  protoplasm  and  of  the  surface  tension  of  the 
vesicular  drop  a  complete  emptying  of  the  vesicle,  whereupon  the 
process  is  repeated  in  the  same  way"  (Stempell,  1924,  p.  460). 
Here  it  may  be  remarked  that  Fortner  (19260  and  b}  and  v.  Gelei 
(1926)  without  inquiring  into  the  validity  of  the  above  view, 
proceeded  on  the  assumption  of  its  truth. 

With  regard  to  Stempell's  ideas,  as  above  expressed,  there 
can  be  no  doubt  since  he  has  furnished  us  with  a  diagram. 
From  both  description  and  diagram  we  learn  that  Stempell  does 
not  entertain  at  all  the  idea  that  there  is  any  flow  of  fluid  from  the 
vesicle  into  the  canals.  Nassonow's  idea  is  identical,  we  believe 
we  are  right  in  saying,  but  his  diagram,  taken  from  Putter  (1903), 
might  be  interpreted  otherwise,  as  witness  Figs.  5,  6  and  7,  which 
show  the  canals  enlarging  before  the  contraction  of  the  vesicle. 
Whether  this  enlargement  results  from  backflow  from  the  vesicle, 
or  from  the  collection  ("ponding  back"  as  Carter  expressed  it) 
of  fluid  derived  from  the  surrounding  protoplasm,  is  the  question 
with  which  we  are  concerned.  We  are  now  in  a  position  in  the 
second  place  to  examine  the  view  of  earlier  observers  of  the  same 
phenomenon. 

Felix  Dujardin  (1841)  made  a  drawing,  reproduced  in  his  Plate 


COXTRACTII.K    VKSICI.K    IN     I'\R\M|.(  HM.  407 

8,  Fig.  6a  and  6b  in  which  the  radiating  vesicles  ("taken  by  Khren- 
berg  for  seminal  vesicles")  are  seen  in  the  condition  just  before 
the  systole  of  the  vesicle,  the  canals  being  expanded.  Dujunlin 
does  not,  of  course,  have  anything  to  say  about  the  matter,  but 
his  drawing  could  not  be  correct  if  the  canals  do  not  fill  before  the 
systole  of  the  vesicle.  The  question  above  indicated  therefore 
recurs,  whence  the  fluid  which  fills  the  canals. 

As  to  this  there  was  no  doubt  in  the  minds  of  Claparede  and 
Lachmann  (1858)  (Lachmann  1857  for  1856).  It  was  these 
who  held  the  view  that  the  apparatus  is  the  homologue  of 
the  circulatory  apparatus  in  the  more  differentiated  animals  and 


<5tC(jndj.  f          2s          3          tj.          &•         6  &         y         /a        // 


it  was  consistent  with  their  view  that  there  was  no  opening  afford- 
ing an  exit  for  the  fluid  to  the  outside.  Their  unfortunate  error 
in  this  seems  to  have  led  to  a  general  condemnation  of  their  win  >lr 
conception  and  thereby  their  critics,  in  overlooking  what  they 
did  see,  fell  into  an  error  as  grievous,  namely,  in  failing  to  see 
that  the  canals  are  in  the  first  instance  filled  at  the  expense  of  the 
vesicle,  as  we  hold.  Lachmann 's  description  (1857)  will  serve 
our  turn  at  the  moment. 

He  says  (1857,  p.  224),  maintaining  that  the  thin  area  of  the 
body  wall  over  the  vesicle  is  only  a  thin  place  fit  for  diffusion  and 
with  no  opening,  that  when  the  vesicle  is  fully  expanded  the 
canals  are  fine  lines.  By  the  sudden  contraction  of  the  vesicle, 
however,  the  canals  instantly  swell  into  pyriform  spaces  close  to 
the  contractile  vesicle,  which  has  disappeared.  During  the  slow 
reappearance  of  the  vesicle,  the  canals  gradually  decrease  and 
they  have  again  been  reduced  to  fine  lines  by  the  time  the 
vesicle  has  become  fully  inflated. 

It  must  be  clear  that  Lachmann  believed  that  the  swelling  of 
the  canals  is  synchronous  with  the  early  period  of  systole  of  the 


4o8  FRANCIS    E.    LLOYD   AND   J.    BEATTIE. 

vesicle.  Carter  (1861)  does  not,  we  think,  correctly  take  his 
meaning  when  he  says:  "Claparede  and  Lachmann  have  said 
that  the  fluid  of  the  vesicle  is  returned  into  the  vessels  on  the 
systole  or  contraction  of  the  vesicula  because  the  sinuses  and 
vessels  become  filled  immediately  afterward"  (italics  ours)  as  this 
is  not  what  Lachmann  said.1 

It  is  in  this  connection  that  Carter  suggests  that  the  swelling  of 
the  canals  into  the  characteristic  pyriform  is  due  to  the  "ponding 
back"  of  the  fluid  which  flows  through  the  canals  into  the  vesicle 
for  the  short  time  that  the  latter  empties  itself,  like  the  ventricles 
of  the  heart  but  in  the  other  direction  (1861,  p.  282).  We  may 
here  remark,  what  we  shall  endeavor  to  show  to  be  true,  that  the 
rate  of  swelling  of  the  canals  does  not  consist  with  the  idea  that 
the  fluid  reaches  the  lacunae  by  diffusion  through  the  walls,  the 
rhythm  of  diastole  and  systole  in  these  being  of  the  same  character 
as  in  the  vesicle ;  and,  if  the  rhythm  of  the  vesicle  can  be  under- 
stood only  when  it  is  admitted  that  the  fluid  of  the  canals  gushes 
into  it,  the  same  must  be  admitted  for  the  canals,  but  in  the 
opposite  sense. 

Somewhat  earlier,  and  in  contrast  to  Lachmann  and  Claparede 
Lieberkiihn  (1856),  while  agreeing  with  them  as  to  time  relations 
of  vesicular  systole  and  canalar  diastole,  saying  that  "a  little 
before  we  observe  the  commencement  of  the  systole,  the  vessels 
begin  to  expand  slowly,  etc.,"  simply  denied  that  there  is  any 
backflow.  The  interest  here  is  obviously  the  correct  observation 
in  regard  to  time  relations  in  question.  Spallanzani  also  believed 
that  the  canals  become  empty  as  the  vesicle  fills,  and  do  not  re- 
appear until  some  time  after  it  has  contracted  and  that  therefore 
"The  fluid  with  which  the  vesicula  is  distended  comes  through 
the  sinuses,  but  is  not  returned  by  them  to  the  body"  (through 
Pritchard,  1861). 

J.  Miiller  (1856)  appears,  according  to  Claparede  and  Lach- 
mann (1858,  p.  51),  to  have  taken  the  same  view  of  the  time  rela- 
tions. We  transcribe  their  summary  of  his  views,  since  we  have 
been  unable  yet  to  see  Miiller's  original  paper.  This  author 
distinguishes  in  the  behavior  of  "central  circulatory  apparatus" 
of  Paramecium  two  partial  systoles  which  alternate  with  each 

1  We  have  not  been  able  to  see  Claparede's  paper,  but  it  appears  that  these  two 
observers,  Claparede  and  Lachmann  worked  in  harmony,  sharing  each  other's  views. 


CONTRACTILE    VESICLE    IN    I'AKAM  KCIfM.  409 

other — the  systole  of  the  vesicle,  then  the  systole  of  the  fusiform 
or  pyriform  swellings.  The  latter  coincides  with  the  diastole  of 
the  vesicle.  Lieberkiihn  had  already  observed  that  "un  instant 
avant  le  systole  des  vesicules  les  rayons  se  ren fluent  considerable- 
ment."  Miiller  explains  the  phenomenon  by  showing  that  the 
vesicle  contracts,  diminishing  insensibly  in  volume  in  the  instant 
which  precedes  systole  and  forces  at  once  a  part  of  its  content- 
into  the  "rays  of  the  star."  Then  the  systole  of  the  vesicle  takes 
place,  which  produces  a  further  swelling  of  these  rays. 

We  cannot  refrain  from  mentioning,  in  passing,  the  work  of 
Wrzesniowski  (1869),  who  studied  Enchelyodon,  Trachelopliyllnni 
and  Loxophyllum,  (but  was  however  chiefly  concerned  with  the 
question  of  the  absence  or  presence  of  a  contractile  membrane), 
because  there  is  some  evidence  in  his  results  which  point  to  the 
presence  of  a  contractile  vesicular  apparatus  similar  to  that  of 
Paramecium,  though  the  author  himself,  if  he  adhered  to  the 
original  account,  would  deny  this.  One  point  may  be  mentioned, 
however,  namely,  that  a  series  of  small  vesicles  is  formed  on  the 
surface  of,  and  from  the  contractile  vesicle  during  early  systole, 
and  these,  upon  growth,  run  together  later  to  form  a  new  con- 
tractile vesicle  (not  the  old  one  reextended).  This  view  of 
Wrzesniowski's  seems  to  be  strongly  linked  with  his  conviction 
that  the  vesicle  is  formed  de  novo  and  totally  lacks  a  membrane 
in  any  but  the  sense  of  molecular  physics  as  Khainski  (1911) 
would  express  it.1 

We  pass  to  the  year  1883  when  Maupas  attacked  the  subject. 
According  to  him  the  systolic  movement  of  the  vesicle  is  sudden 
and  rapid.  A  little  before  it  happens  the  canals  commence  to  till 
in  the  form  of  elongated  pears  at  a  little  distance  from  the  point 
where  they  open  into  the  vesicle.  Maupas'  account  indicates  a 
high  degree  of  meticulous  care  in  observation  He  goes  on  to 
remark  for  example  that  the  systole  of  the  vesicle  takes  place 
more  often  before  the  pyriform  swellings  (of  the  canals)  have  at- 
tained their  full  size.  In  spite  of  the  fact  that  he  correctly  ap- 
prehended the  time  relations  involved  he  pronounces  for  the  VH-NV 
that  the  canals  are  simple  afferent  conduits  and  sententiously 

1Samuelson  observed  in  1857  that  the  single  globular  vesicle  in  Glaucoma 
scintillans  when,  it  contracts  forces  the  fluid  into  others  which  appear  temporarily 
around  it. 


410  FRANCIS    E.    LLOYD   AND   J.    BEATTIE. 

remarks  "I  have  never  seen  the  liquid  of  the  vacuole  reenter 
them."  This  would  indeed  be  difficult  and  his  failure  cannot  be 
charged  to  his  discredit,  for  at  all  events  he  very  correctly  de- 
scribes the  at  first  irregular  contours  of  the  vesicle  during  the 
early  stages  of  its  diastole  when,  under  systole  of  the  canals,  these 
empty  themselves  into  the  vesicle.  Maupas  was  on  the  side  of 
the  non-membranists. 

It  will  be  seen  that  these  earlier  observers,  while  disagreeing  in 
regard  to  the  afferent-efferent  nature  of  the  canals,  support  a 
majority  view  which,  as  we  believe,  correctly  describes  the  time 
relations  between  the  behaviours  of  the  vesicle  and  the  contrib- 
utory canals.  It  is  therefore  a  curious  fact  that  later  observers, 
as  we  have  already  shown  at  the  outset  of  the  paper,  siding  with 
the  view  of  the  solely  afferent  nature  of  the  canals  have  in  some 
way  been  led  to  overlook  the  true  time  relations. 

Closely  connected  with  the  general  trend  of  inquiry  above  out- 
lined is  the  parallel  inquiry  into  the  nature  of  the  membrane  lining 
the  vesicular  cavity  It  will  be  easily  apprehended  that  very 
convincing  evidence  has  been  so  difficult  to  obtain  that  only 
recently  has  Miss  Howland  (1924)  favored  the  view  that  a  proper 
membrane  in  the  morphological  sense  is  present  constituting  the 
branching  cavity  composed  of  the  central  vesicle  and  its  con- 
tributory canals.  She  succeeded  in  isolating  the  membrane  with 
little  distortion  by  micro-dissection  from  an  animal  (Paramecium 
catidatum)  treated  with  a  strong  solution  of  alizarin  blue.  This 
author  expressed  some  doubt  of  her  interpretation  based  on  the 
possibility  that  the  dye  had  coagulated  the  surface  material  of  the 
vesicle  and  so  produced  an  artefact.  In  the  same  year  Nassonow 
presented  evidence  based  on  the  method  of  osmication  which 
would  convince  even  the  elect  were  it  not  for  a  doubt  similar  to 
that  expressed  by  the  former  author.  We  venture  to  think  that  a 
weak  link  exists  in  the  chain  of  his  argument.  We  are  not  here 
concerned  with  this  author's  views  of  the  homology  of  the  con- 
tractile vesicle  with  the  Golgi  apparatus  although  we  subscribe  to 
the  general  view  supported  by  Nassonow  that  the  pulsating 
vesicle  is  a  true  organelle  of  morphological  value,  as  Lachmann  so 
long  ago  held.  With  regard  to  earlier  observers  it  will  boot  us 
little  to  bring  forward  the  details  of  their  views,  a  summary  of 


CONTRA*    III. !•;    VESICLE    IN     I'AKAMKM  I'M  .  4!  I 

which  will  he  found  in  a  paper  by  Taylor  (1923).  Portlier,  by 
compressing  animals  in  a  hypertonic  solution  of  cant-  sugar.  \va> 
able  to  set  free  the  apparatus  surrounded  by  protoplasm  and  in  a 
state  of  approximate  diastole.  Their  behavior  he  argues  un- 
qualifiedly postulates  the  impermeability  of  the  membranes;  but 
these  membranes  he  believes  arise  ad  hoc,  that  of  the  vesicle  at 
the  completion  of  each  systole  affording  the  new  membrane  for 
the  papilla  pulsatoria.  Without  further  discussion  of  this  matter 
from  the  historical  point  of  view  we  may  be  permitted  to  remark 
that  had  the  true  time  relations  in  the  cycle  of  events  not  been 
lost  sight  of,  the  protagonists  of  the  "non-membranous"  view 
would  have  suffered  pause. 

II. 

No  special  technique  is  required  to  demonstrate  the  phasic 
activity  of  the  contractile  vacuole  and  canaliculi  in  Paramecinm. 
Care  must  be  taken  that  the  cover  slip  over  the  preparation  does 
not  press  untowardly  on  the  animal,  otherwise  the  pore  to  the 
exterior  may  be  blocked  and  the  contractile  vesicle  fail  to  dis- 
charge in  the  normal  manner,  and  at  normal  rate. 

After  the  preparation  has  been  made  it  is  well  to  allow  some 
minutes  to  elapse  before  the  preparation  is  examined  as  it  is  a 
hopeless  task  to  attempt  to  observe  the  contractile  vesicle  in  one 
single  animal,  while  the  animals  are  in  rapid  motion  immediately 
after  they  have  been  placed  on  the  microscopic  slide.  In  a  short 
time  the  animals  settle  down  to  feed,  and  it  is  then  possible  to 
watch  a  whole  group  and  to  pick  out  one  animal  for  observation. 
It  is  possible  also  to  trap  the  animals  in  a  very  fine  capillary  tube 
and  so  limit  their  movements  except  round  a  longitudinal  axis. 
A  better  method,  but  open  to  the  objection  of  an  abnormal  en- 
vironment, is  to  mix  finely  ground  China  ink  with  the  mounting 
medium.  This  appears  to  impede  the  movements  and  so  far  a> 
one  can  see  there  is  no  interference  with  the  normal  cycle  of 
events  within  the  contractile  vacuole  system. 

After  close  observation  for  a  few  minutes  the  following  series  of 
changes  can  be  seen  The  contractile  vesicle  will  be  observed  as 
a  highly  refractile  almost  spherical  droplet  lying  in  the  m<»t 
superficial  part  of  the  cytoplasm.  When  the  animal  rolls  over  on 
its  side  it  will  appear  that  at  one  point  there  is  a  close  attachment 


412  FRANCIS    E.    LLOYD   AND   J.    BEATTIE. 

of  the  vacuole  to  the  pellicle.  At  this  time  the  vacuole  when 
viewed  from  the  side  will  appear  as  three  quarters  of  a  sphere  with 
a  conical  apex  attached  to  the  pellicle.  When  the  animal  rolls 
so  that  the  vacuole  is  observed  from  above  with  careful  focusing  a 
bright  minute  ring  will  be  seen  in  the  center  of  a  small  clear  area 
in  the  pellicle.  This  is  the  pore  through  which  the  vesicle  expels 
fluid  to  the  exterior.  The  vesicle  gradually  enlarges  and  in  doing 
so  changes  its  shape  from  the  conico-spherical  form  to  a  perfect 
sphere.  Enlargement  after  the  spherical  shape  has  been  attained 
is  slow  and  very  small  in  amount  as  to  linear  dimensions.  Sud- 
denly at  the  end  of  diastole  the  vesicle  appears  to  get  smaller 
(Plate  I,  Figs.  3  to  4)  and  at  the  same  moment,  not  afterwards, 
radiating  canals  appear  surrounding  the  vacuole  (Plate  I,  Fig.  3). 
Seen  from  above  the  inner  ends  of  these  structures  are  separated 
from  the  vacuole  by  a  distinct  area  of  protoplasm.  Seen  from  the 
side  the  bulbous  or  pear-shaped  ends  of  the  canals  are  observed 
to  lie  in  the  most  superficial  layer  of  cytoplasm  and  to  be  con- 
tinued more  distally  into  the  deeper  parts  of  the  cytoplasm  as 
fine  canals. 

After  this  phase,  which  can  only  be  interpreted  as  a  distinct 
diastole  of  the  canaliculi  caused  by  systole  of  the  vacuole  and  not 
merely  as  a  damming  back  of  liquid  attempting  to  flow  into  the 
vesicle,  the  vacuole  suddenly  contracts  (Plate  I,  between  Figs.  4 
and  5)  and  expels  the  remaining  contents  to  the  exterior.  There- 
fore systole  of  the  contractile  vesicle  consists  of  two  distinct 
phases : 

(a)  First,  an  early  systolic  phase  during  which  the  contractile 

movement  of  the  vacuole  is  slow  and  diastole  of  the  canals 
rapid  (Plate  I,  Fig.  3,  4). 

(b)  Second,  a  later  period  during  which  the  vacuole  expels  the 

remainder  of  its  contents  to  the  exterior  (Plate  I,  Fig.  5). 
The  behavior  as  thus  set  forth  has  been  displayed  graphically 
in  the  accompanying  diagram,  in  which,  to  some  extent  provi- 
sionally, we  have  attempted  to  express  the  time  relations  seen  in 
the  rhythm  of  the  contractions  and  expansions  of  vesicle  and 
canals,  while  the  volume  relations  are  avowedly  inexact,  but  ap- 
proximate. Time  is  plotted  on  the  abscissa,  and  the  volume  of 
the  canals  and  of  the  vesicle  on  the  ordinates,  the  total  volume  of 
the  vesicle  being  taken  as  one.  The  hatched  areas  are  bounded 


CONTRACTILE    VESICLE    IN    PAKAMKCIUM.  413 

by  the  curve  of  diastole  and  systole  of  the  canals;    the  areas 
bounded  by  the  curves  for  the  vesicle  are  left  blank. 

There  is  no  doubt  that  there  is  a  discharge  of  vesicular  con  ten  t  - 
to  the  exterior.  Jennings  showed  this  first  convincingly  as  has 
been  stated  above  and  we  have  been  able  to  confirm  his  observa- 
tions and  to  make  a  motion  picture  of  the  process. 

There  has  been  some  doubt  expressed  as  to  whether  or  not  the 
pore  through  which  the  vacuole  discharges  can  admit  fluid  from 
the  surrounding  medium.  We  have  found  no  evidence  to  support 
this  theory.  All  our  observations  go  to  show  that  after  the 
vesicle  has  discharged  its  contents  reconstitution  of  the  vacuolar 
space  takes  place  by  the  discharge  into  the  collapsed  cavity  of 
the  fluid  contained  in  the  canals  (Plate  I,  Figs.  1-2;  6-8). 
Discharge  of  the  contents  of  the  canals  into  the  vesicular  space 
takes  place  within  one  second  after  the  completion  of  systole  of 
the  vacuole.  The  canals  do  not  however  discharge  simultane- 
ously but  by  careful  observation  one  is  able  to  make  out  that 
first  one  canal  may  discharge  into  the  collapsed  vesicular  region 
which  then  forms  an  irregular  angular  cavity  1  soon  followed  by 
another  and  then  by  the  remainder.  When  the  last  canal  has 
discharged  the  space  is  seen  to  be  conico-spherical  as  described 
above.  It  is  possible  to  analyse  the  discharge  of  the  canaliculi 
into  the  vacuolar  space  only  by  study  of  the  motion  picture  film. 

After  the  reconstitution  of  the  vesicle  enlargement  takes  place 
and  this  phase  of  diastole  of  the  vacuole  occupies  the  longest 
period  of  the  cycle  of  events.  One  notices  that  the  conico- 
spherical  form  persists  for  quite  a  time  (almost  three  quarters  of 
diastole)  before  the  spherical  form  is  assumed.  Once  the  vesicle 
becomes  spherical  systole  of  the  structure  takes  place  within  a 
second  or  two. 

The  cycle  of  events  occupies  normally  about  eight  seconds. 
Records  which  we  have  made  show  that  in  fresh  specimens  cycles 
of  seven  and  one  fifth  seconds  were  common.  \Ye  have  observed 
cycles  which  required  ten  seconds  for  completion.  When  the 
cycle  lengthens  it  is  the  diastolic  period  which  is  chiefly  prolonged. 
When  the  animal  is  compressed  gently  it  is  possible  to  occlude 
the  pore  and  so  prevent  the  second  phase  of  systole  taking  place. 
The  first  phase,  i.e.  diastole  of  the  canals  takes  place  but  then  is 

1  Beautifully  recorded  by  Nassonow,  Fig.  42. 
27 


* 


414  FRANCIS  E.  LLOYD  AND  J.  BEATTIE. 

no  discharge  to  the  exterior.  In  a  short  time  the  canals  reappear 
and  so  the  cycle  goes  on.  The  vacuole  continues  to  enlarge  and 
before  very  many  minutes  the  pellicle  ruptures  and  the  proto- 
phism  is  extruded  carrying  with  it  in  some  cases  the  entire  con- 
tractile vesicle.  The  vacuole  may  be  seen  lying  as  a  spherical 
body  in  the  surrounding  fluid.  We  have  not  observed  any  sign 
of  a  canal  when  the  protoplasm  is  examined  after  bursting. 

When  neutral  red  is  used  in  solutions  of  one  part  to  four  hundred 
or  higher  concentrations  it  is  frequent  to  observe  the  gradual  con- 
traction of  the  cytoplasm  from  the  pellicle  and  the  formation  of  a 
peri-cytoplasmic  space  rilled  with  fluid.  In  one  specimen  of 
which  a  photograph  is  shown  (Plate  I,  See  Fig.  9)  the  cytoplasm 
in  contracting  pulled  a  fine  cone  of  pellicle  downwards.  At  the 
apex  of  this  was  attached  the  pulled-out  contractile  vesicle 
which  extended  as  a  conical  cavity  through  the  peri-cytoplasmic 
space  to  the  dimple  in  the  pellicle.  The  actual  interface  between 
the  vesicle  and  the  surrounding  fluid  could  be  seen.  The  apex  of 
the  dimple  was  the  pore  through  which  the  vacuole  discharges. 
It  also  would  appear  to  show  that  the  vacuole  when  it  discharges 
to  the  exterior  is  not  reconstituted  de  novo  in  the  old  site  but  rather 
that  there  is  something  of  a  permanent  nature — a  vesicular 
membrane  into  which  is  discharged  the  contents  of  the  canaliculi 
when  the  vesicle  is  reformed. 

Miss  Rowland,  as  we  have  already  said,  has  been  able  to  isolate 
the  vesicular  membrane  from  preparations  treated  with  alizarin 
blue. 

By  way  of  summary  of  the  above  we  draw  attention  to  the 
following  important  facts. 

Diastole  of  the  vesicle  falls  into  two  phases — an  early  rapid  and 
a  later  slow  one. 

The  early  rapid  phase  is  due  to  the  systole  of  the  canals  during 
which  their  fluid  content  is  forced  into  the  vesicle. 

i 

The  later  slow  phase  of  diastole  of  the  vesicle  is  due  to  further 
distention  by  diffusion  of  water  into  the  vesicle. 

Systole  of  the  vesicle  falls  similarly  into  two  phases,  an  early 
slow  phase  during  which  the  fluid  is  forced  into  th'e  canals 
(diastole  of  these)  and  a  later  rapid  phase  during  which  the  re- 
mainder of  the  vesicular  fluid  is  forced  through  the  spore  into  the 
surrounding  medium. 


CONTRACTILE    VESICLE   IN    PARAMECIUM.  415 

It  appears  that  early  diastole  of  the  vesicle  is  synchronous  with 
the  systole  of  the  canals ;  and  that  early  systole  of  the  vesicle  is 
synchronous  with  diastole  of  the  canals.  During  early  diastole 
of  the  vesicle,  this  is  partly  filled  with  fluid  from  the  canals, 
This  is  the  residual  fluid  plus  that  which  has  in  the  interim  en- 
tered by  diffusion  into  them.  During  early  systole  of  the  vesicle 
the  canals  are  partly  filled  with  fluid  from  it — this  we  may  speak 
of  as  the  residual  volume.  That  volume  which  is  discharged  l>y 
the  vesicle  is  the  overplus  accumulated  by  diffusion  into  the 
vesicle  and  canals  during  their  diastolic  periods. 

Viewed  thus,  the  mechanism  is  one  in  which  a  certain  quantity 
of  fluid  of  relatively  high  osmotic  pressure  is  retained  in  the 
canals,  derived  by  them  from  the  central  vesicle,  and  which  is  at 
once  put  into  service  to  withdraw  water  from  the  body  into  the 
pulsatory  apparatus.  Thus  an  important  feature  of  Stempell's 
view  receives  support,  even  though  his  conception  of  the  methods 
of  working  of  the  apparatus  is  incomplete.  If  it  depended  solely 
upon  diffusion  for  filling,  from  the  completely  collapsed  state  to 
the  completely  replete,  it  could,  in  our  opinion  not  work  so 
rapidly  and  efficiently. 

REFERENCES. 
Carter,  H.  J. 

'61     Notes  and  Corrections  on  the  Organization  of  Infusoria,  etc.     Annals  & 

Mag.  Nat.  History,  ser.  3,  8:  281-290. 
Claparede,  Ed.,  and  Lachmann,  Johannes. 

'57-'6i     Etudes  sur   les   infusoires  et   les  rhizopodes.     Mem.   de   1'inst.    Nat. 
Genevois  5:  (for  1857)  1-260,  pi.  1-13,  1858;  6:  (for  1858)  261-482,  pi.  1-24. 
1859;    7:  (for  1859-60)  5-291,  pi.  1-13,  1861.     Vol.  5  contains  the  general 
discussions. 
Dujardin,  Felix. 

'41     Infusoires,  etc.,  Histoire  naturelle  des  zoophytes.     Suites  a  Buffon,  Paris. 
Fortner,  H. 

'263     Ueber  die  Gesetzmaessigkeit  der  Wirkungen  des  Osmotischen  Druckes 
physiologisch  indifferenter  Loesungen  auf  einzellige,  tierische  Organismen, 
Biol.  Centralbl.  45:  417-446. 
'26b     Zur  Frage  der  discontinuirlichen  Excretion  bei  Protisten,  Arch.  f.  Protist- 

enk.  56:  295-320. 
v.  Gelei,  J. 

Nephridialapparat  bei  den  Protozoen.     Biol.  Centralbl.,  45:  676-683. 
Rowland,  Ruth  B. 

'24     Dissection  of  the  Pellicle  of  Amoeba  verrucosa.     Journ.   Exp.  Zool.,   40: 

263-270. 

'24     On   Excretion  of   Nitrogenous  Waste  as  a  Function  of   the   Contractile 
Vacuole.     Ibid.,  40:  251-250. 


41 6  FRANCIS   E.    LLOYD   AND   J.    BEATTIE. 

'24     Experiments  on  the  Contractile  Vacuole  of  Amoeba  verrucosa  and  Para- 

mecium  caudatum.     Ibid.,  40:  251-262. 
Jennings,  H.  S. 

'04     A  Method  of  Demonstrating  the  External  Discharge  of  the  Contractile 

Vacuole.     Zool.  Anz.,  27:  656-658. 
Lachmann,  C.  F.  J. 

'57     On  the  Organization  of  the  Infusoria,  especially  the  Vorticellae.     A.  &  M. 
N.  H.  19:  ser.  2,  113-128;   226-241.     (Translated  from  Miiller's  Archiv.  p. 
240,  1856.) 
Lieberkuhn,  N. 

'56     Contributions  to  the  anatomy  of  the  Infusoria.     A.  &  M.  N.  H.  18:  ser.  2, 

319.     (Translated  from  Miiller's  Archiv.  Jan.  1856.) 
Maupas,  £. 

'83     Etude  des  infusoires  cilies.     Archives  de  Zool.  Exp.  et  Gen.,  i:  634. 
Miiller,  J. 

'56     Beobachtungen  an  Infusorien.     Monatsbericht  der  Berliner  Akad.,  p.  393. 
Nassonow,  D. 

'24     Der  Exkretionsapparat  (Kontraktile  Vakuole)  der  Protozoa als  Homolog  des 
Golgischen  apparats  der  Metazoazellen.     Arch.  f.  mikr.  Anat.  u.  Entwick. 
mech.,  103:  437-482. 
Putter,  A. 

'03     Die  Reizbeantwortung  der  ciliaten  Infusorien.     Zeitschr.  f .  allgem.  Physiol., 

3:  (Heft  i). 
Pritchard,  A. 

'61     A  History  of  Infusoria.     4  Ed.     London. 
Schmidt,  O. 

'53     Lehrbuch  der  Vergleichenden  Anatomic,    1853   Froriep's  Notiz.,  Vol.   9: 

(through  Pritchard). 
Stempell,  W. 

'14     Die  Funktion  der  pulsierenden  Vacuole.     Zool.  Jahrb.,  34:  437-478. 
Taylor,  C.  V. 

'23     The  Contractile  Vacuole  of  Euplotes,  an  Example  of  Sol-gel  Reversibility  of 

Cytoplasm.     Journ.  Exp.  Zool.,  37:  259-290. 
Wrzesniowski,  A. 

'69     Ein  Beitrag  zur  Anatomie  der  Infusorien.     Arch.  f.  mikr.  Anat.,  5:  25-48. 


FRANCIS    E.    LLOYD   AND    J.    BEATTIE. 


EXPLANATION   OF    PLATE. 

FIGS.  1-8.  Eight  episodes  from  motion  picture  (photomicrographic)  of  Para- 
mecium  caudatum.  The  animal  was  slightly  compressed  between  slip  and  cover; 
the  periodicity  was  slightly  slower  then  normal  therefor.  The  position  in  the  film 
is  indicated  for  each.  Exposure  16  per  second.  Enlargements  at  constant  distance. 

FIG.  i.     Foot  887  frame  16.     Mid-diastole.     Canals  are  emptying  into  vesicle. 

FIG.  2.     883-1.     Late  diastole.     Traces  of  canals  visible. 

FIG.  3.     880-16.     Early  systole  of  vesicle;   canals  beginning  to  fill. 

FIG.  4.  876-10.  Mid  systole  of  vesicle  which  is  now  smaller;  canals  nearly 
filled. 

FIG.  5.     873-15.     Systole  of  vesicle  complete,  canals  full. 

FIG.  6.  871-10.  Mid-diastole  (somewhat  later  than  Fig.  i);  canals  emptying 
into  vesicle. 

FIG.  7.     870-3.     Later  diastole;   canals  nearly  disappeared. 

FIG.  8.  868-9.  Diastole  complete;  canals  empty.  One  canal  persists  longer 
than  the  others:  note  that  it  occurs  in  Figs,  i,  2,  5—8. 

FIG.  9.  An  animal  treated  with  neutral  red  (see  text),  showing  the  vesicle 
pulled  away  from  the  pellicle,  and  dimpling  it  by  pulling  on  the  pore  rim.  The  result 
follows  from  the  shrinkage  of  the  cytoplasm. 


BIOLOGICAL  BULLETIN.    VOL.    LV. 


PLATE  I. 


FRANCIS  E.  LLOYD  AND  J.  BEATTIE. 


OBSERVATIONS    ON    HYDRA    AND    PELMATOIIYDRA 

UNDER   DETERMINED   HYDROGEN    ION 

CONCENTRATION. 

W.    L.    THRELKELD   AND   S.    R.    HALL, 
UNIVERSITY  OF  VIRGINIA.' 

Much  has  been  written  recently  concerning  reduction,  de- 
differentiation  and  resorption  in  Hydra.  It  is  generally  conceded 
that  reduction  in  hydra  is  accompanied  by  a  loss  of  tentacles. 
The  literature  enumerates  the  following  causes  by  which  hydras 
lose  their  tentacles.  N.  Annandale  ('07)  observed,  in  studying 
Hydra  orientalis,  that  during  the  hot  season  of  the  year  this 
species  has  but  four  tentacles  while  during  the  cold  season  it  has 
six  tentacles.  G.  Entz  ('12)  observed  that  an  infection  with 
Amoeba  hydroxena  may  lead  to  a  degeneration  of  tentacles. 
Reynolds  and  Looper  ('28)  have  come  to  the  conclusion  that  this 
parasite  is  responsible  for  the  degeneration  of  the  tentacles. 
Certain  ciliates  recorded  by  E.  Reukauf  ('12)  and  P.  Shultze  ('13) 
also  caused  the  loss  of  tentacles.  E.  Shultz  ('06)  observed  that 
hunger  set  up  a  process  of  dedifferentiation  within  the  tentacles. 
Huxley  and  DeBeer  ('23)  observed  that  adverse  environmental 
conditions  accelerate  dedifferentiation  and  resorption  of  the 
tentacles  of  Obelia  and  Campanularia .  They  also  found  that 
this  process  of  dedifferentiation  and  resorption  might  involve  not 
only  the  tentacles  but  also  part  of  the  zooid.  Berninger  ('10) 
found  that,  in  response  to  inanition,  hydra  lost  its  tentacles. 
Finally  Kepner  and  Jester  ('27)  also  observed  that  the  loss  of 
tentacles  was  brought  about  in  response  to  inanition.  This  loss, 
according  to  them,  was  accomplished  by  ingestion  of  the  tips  of 
the  tentacles  through  the  mouth.  This  may  occur,  but  undoubt- 
edly is  not  the  usual  method,  as  Hyman  ('28)  indicated. 

It  is  a  well  known  fact  that  the  concentration  of  the  hydrogen 
ion  medium  that  bathes  the  protoplasm  or  protoplasmic  tissue 

1  These  investigations  were  carried  on  under  the  direction  of  Professor  W.  A. 
Kepner.  Acknowledgments  are  due  Mr.  Carl  H.  McConnell  of  this  laboratory,  for 
the  preparations  of  the  photomicrographs. 

419 


42O  \V.    L.    THRELKELD   AND    S.    R.    HALL. 

has  a  profound  effect  upon  it,  therefore  it  seems  strange  that  no 
attempts  have  been  made  to  account  for  reduction,  dedifferentia- 
tion  and  resorption  on  the  basis  of  such  environmental  conditions. 
The  following  observations  and  results  have  been  obtained 
through  an  effort  to  determine  whether  or  not  the  concentration 
of  the  hydrogen  ion  is  an  important  factor  with  reference  to  the 
three  above  mentioned  phenomena. 

METHODS  AND  MATERIALS. 

Filtered  spring  water  in  300  cc.  portions  kept  in  thoroughly 
cleansed  glass  dishes  was  used  as  a  culture  medium. 

Very  dilute  solutions  of  N/2O  sodium  hydroxide  and  of  hydro- 
chloric acid  were  used  in  quantities  to  adjust  the  pH  of  the  solu- 
tions. The  colorimeter  method  was  used  for  the  pH  determina- 
tion of  the  solutions  and  LaMotte  color  standards  were  employed 
for  color  matching.  Tests,  adjustments  and  observations  were 
made  every  twenty-four  hours  except  where  otherwise  indicated. 
The  temperature  was  maintained  between  18  and  22°  C.  During 
these  investigations  frequent  examinations  were  made  of  both  the 
culture  and  of  the  animals  for  protozoa  which  might  have  been 
responsible  for  reduction.  None  were  found  except  where 
stated.  Observations  were  made  with  a  dissecting  binocular  of  a 
magnification  of  twenty  diameters.  These  observations  were 
supplemented  by  histological  preparations. 

At  first  distilled  water  was  tried  as  a  culture  medium  with  the 
idea  that  a  more  accurate  determination  could  be  made  of  the 
hydrogen  ion  concentration.  Various  deleterious  factors  enter 
into  the  use  of  such  a  medium  so  it  was  discarded.  In  the  sub- 
sequent experiments,  filtered  spring  water  was  used. 

The  terms  reduction,  dedifferentiation  and  resorption,  as  used 
by  other  authors  and  us,  may  be  defined  as  follows:  Reduction 
is  a  uniform  decrease  in  surface  area  in  which  process  the  ecto- 
derm, mesoglea  and  endoderm  remain  intact  and  maintain  a 
normal  position  in  relation  to  each  other.  Dedifferentiation  and 
resorption  represent  a  dual  phenomenon  which  involves  a  local 
reduction  of  surface.  The  presence  of  this  dual  phenomenon  in 
the  tentacles  is  indicated  by  a  thickening  and  knobbed  appearance 
at  the  tips  of  the  tentacles. 


OBSERVATIONS  ON  HYDRA  AND  PELMATOHYDRA.          421 

EXPERIMENTAL. 

Culture  i. — Four  Pelmatohydra  oligactis  (Pallas),  were  placed 
in  a  culture  medium  consisting  of  distilled  water  and  NaOH  was 
added  to  maintain  a  constant  pH  of  7.8.  At  the  end  of  a  period 
of  six  days  there  was  much  apparent  reduction  and  resorption 
of  the  tentacles  in  all  specimens.  One  polyp  was  fed  on  the  sixth 
day  and  one  on  the  seventh.  At  this  point  the  experiment  was 
terminated  through  an  accident. 

Culture  2. — Four  Pelmatohydra  oligactis  were  placed  in  a 
culture  medium  of  distilled  water.  The  culture  maintained  a 
pH  of  6.8  without  the  addition  of  either  hydrogen  or  hydroxyl 
ions.  These  polyps  disintegrated  in  five  days. 

Culture  j. — Four  Pelmatohydra  oligactis  were  placed  in  a 
culture  medium  consisting  of  distilled  water.  This  culture 
maintained  a  pH  of  7.0  which  was  fatal  to  the  polyps  in  five  days. 
At  this  phase  of  our  observations  we  came  to  the  conclusion  that 
we  were  imposing  other  factors  than  the  controlled  pH  repre- 
sented, upon  the  hydras  in  using  distilled  water.  A  change  in 
osmotic  pressure  was  undoubtedly  involved  when  distilled  water 
was  used  instead  of  spring  water.  So,  from  this  point  on,  spring 
water  was  employed  as  the  medium  in  which  to  keep  the  observed 
polyps. 

Culture  4. — Four  Chlorohydra  viridissima  (Pallas)  were  taken 
from  spring  water  which  tested  pH  7.6.  They  were  normal  in 
every  respect.  The  pH  of  the  second  lot  of  spring  water  was  now 
maintained  at  6.6.  The  only  change  being  made  here  was  using 
a  second  glass  dish  similar  to  the  one  in  which  the  pH  tested  7.6 
and  in  the  pH  now  being  6.6.  In  five  days,  six  of  the  polyps  had 
disintegrated  and  the  remaining  one  had  undergone  advanced 
dedifferentiation  and  resorption.  It  was  placed  in  filtered  spring 
water  of  pH  8.6  in  an  effort  to  bring  about  regeneration  but  it 
disintegrated  in  a  few  hours.  This  result,  together  with  general 
observations  made  on  various  cultures,  in  the  laboratory,  in 
which  the  polyps  displayed  marked  dedifferentiation  and  re- 
sorption, indicates  that  the  acid  condition  of  the  medium  in- 
duces dedifferentiation  and  resorption.  Our  observation  upon  a 
lower  hydrogen  ion  concentration  (higher  pH)  proved  to  be  little 
more  instructive  as  seen  by  the  following  culture. 


422  W.    L.    THRELKELD    AND    S.    R.    HALL. 

Culture  5-  Six  Pelmatogydra  oligactis  were  isolated  in  filtered 
spring  water  the  pH  of  which  was  maintained  between  7.6  and 
8.2.  On  the  8th  day  all  of  the  hydras  appeared  perfectly  normal; 
however,  on  the  9th  day,  all  except  one  had  disintegrated.  The 
one  remaining  hydra  showed  no  apparent  reduction  or  de- 
differentiation  and  resorption  of  the  tentacles.  This  hydra  was 
sectioned  and  its  histology  appears  later  in  the  paper. 

On  several  occasions  similar  results  were  obtained  when  the 
pH  was  held  within  the  range  from  pH  7.8-8.0.  It  appears  that 
the  first  ten  days  represent  a  critical  period  when  the  polyps  are 
exposed  to  inanition.  After  the  loth  day  has  passed  we  have 
had  uniform  results  as  the  following  observations  indicate. 

Culture  6. — Four  Chlorohydra  viridissima,  in  which  some  re- 
sorption was  displayed,  were  isolated  in  filtered  spring  water 
pH  6.6.     This  water  was  over  Elodea  which  had  been  previously 
boiled.     The  Elodea  was  separated  from  the  polyps  by  a  double 
thickness  of  cheese-cloth  spread  over  the  bottom  of  the  container. 
The  Elodea  was  removed  after  six  days  and  spring  water  alone  was 
used.     As  indicated  above,  these  hydras  were  in  a  somewhat  re- 
sorbed  condition.     The  pH  of  this  culture  was  varied,  first  de- 
creasing the  concentration  of  the  hydrogen  ions  after  the  first 
two  days  up  to  7.6,  then  increasing  to  7.0,  then  again  decreasing 
to  7.8.     A  pH  of  7.8  was  maintained  for  the  last  thirteen  days. 
Immediately  following  these  changes  in  pH,  we  observed  the 
physiological  aspect  of  the  polyps.     It  was  seen  that  the  greater 
the  concentration  of  the  hydrogen  ions  the  greater  was  the  degree 
of  dedifferentiation  and  resorption  in  the  polyps.     If  the  concen- 
tration of  the  hydrogen  ions  was  lessened  the  hydras  returned  to 
normal.     Two  of  the  four  hydras  survived  for  a  period  of  twenty- 
three  days.     One  of  these  was  sectioned  (its  histology  is  referred 
to  later  in  the  paper)  and  the  other  was  lost  during  a  transfer  for 
examination.     On  the  nineteenth  day  a  green  hydra,  with  much 
resorbed  tentacles  and  bearing  gonads,  was  introduced  into  this 
culture.     In  two  days  this  hydra  had  gained  its  normal  appear- 
ance but  its  gonads  had  partially  disappeared.     It  was  fed  and 
placed  in  an  aquarium  containing  food  where  it  developed  into  a 
fine    vegetative    specimen    apparently    normal.      In    this    last 
specimen  the  change  from  laboratory  culture  water  to  filtered 
spring  water  must  have  been  a  factor  as  well  as  the  change  in  pH. 


OBSERVATIONS    ON    HYDRA    AND    I'KI.M  A  I  ( )ll  VDK  A  .  423 

This  does  not  however  lessen  the  significance  of  the  reaction  of  the 
other  individuals  of  culture  6,  wherein  only  the  pH  concentration 
has  been  the  factor  involved. 

Culture  7. — Six  Chlorohydra  viridissima  in  a  slightly  resorbed 
condition  were  placed  in  filtered  spring  water  without  Elodea 
the  pH  of  which  tested  8.6.  After  the  first  two  days  the  pH  was 
maintained  at  7.8  until  this  experiment  was  terminated.  On  the 
fourteenth  day  one  hydra  was  sectioned.  At  the  end  of  a  period 
of  twenty-four  days  three  hydras  remained.  They  were  much 
reduced  in  size  but  their  tentacles  were  apparently  normal. 
On  the  twenty  fifth  day  they  were  placed  in  an  aquarium  con- 
taining food  where  they  lived  for  several  days  and  attained  nearly 
normal  size.  At  this  point  our  observations  on  these  animals 
ceased. 

These  most  interesting  cases  (cultures  6  and  7),  in  which  the 
polyps  that  had  been  reduced  and  in  which  apparent  dedifferentia- 
tion  and  resorption  had  taken  place  at  a  hydrogen  ion  concentra- 
tion above  the  optimum,  were  restored  to  a  completely  normal 
condition  when  subjected  to  hydrogen  ion  concentration  at  or 
near  the  optimum.  This  undoubtedly  indicates  that  food  is  not 
necessary  for  the  regeneration  of  hydra,  but  regeneration  depends 
rather  upon  the  hydrogen  ion  concentration  of  the  culture  water. 
Kepner  and  Jester  ('27)  record  one  hydra  which  had  lost  all  <>t 
its  tentacles  and  without  the  presence  of  food  the  lost  tentacles 
were  replaced  by  regenerated  ones  in  eight  days.  As  the  culture 
medium  was  frequently  changed  it  is  probable  that  a  favorable 
pH  was  accidentally  maintained.  Hyman  ('28)  records  the 
same  phenomena  when  she  says:  "Depressed  specimens  may  be 
caused  to  regenerate  if  the  water  is  replaced  by  culture  water' 
(page  78).  Huxley  and  DeBeer  in  working  with  Obelia  and 
Campanularia  were  unable  to  cause  the  regeneration  of  dedifferen- 
tiated  and  resorbed  tissue. 

Culture  8. — Eight  Pelmatohydra  oligactis  were  isolated  in 
filtered  spring  water  the  pH  of  which  was  maintained  for  the 
first  two  days  at  8.4  and  for  the  remainder  of  the  period  it  \\.i- 
kept  at  pH  7.8.  On  the  tenth  day  three  hydras  had  completely 
disintegrated  without  displaying  reduction,  dedifferentiation 
and  resorption.  On  the  i/th  day,  Halteria  appeared  in  the 
culture.  These  were  not  abundant,  about  ten  being  found  in  the 


424  W.    L.    THRELKELD   AND    S.    R.    HALL. 

field  of  the  binocular  dissecting  microscope.  As  all  the  hydras 
appeared  in  the  same  condition  one  was  sectioned.  These  sec- 
tions showed  no  Halteria  present  within  coelenteron  or  the  food 
vacuoles.  But  menatocysts  were  present  in  the  epithelio- 
muscular  cells  of  the  endoderm  and  within  the  coelenteron,  hence 
the  histology  indicates  that  resorption  had  taken  place.  This 
resorption  was  so  slight  that  it  is  overlooked  by  examination  of 
the  living  polyps  under  a  dissecting  microscope.  The  culture 
medium  was  changed,  so  as  to  have  water  free  of  protozoa,  and 
the  observations  continued.  On  the  twenty  third  day  one  hydra 
was  sectioned  (its  histology  is  referred  to  later).  On  the  twenty 
fifth  day  the  remaining  three  hydras  were  given  bits  of  liver 
which  they  readily  accepted.  Thus  indicating  that  they  were 
not  in  a  "depressed"  condition  as  described  by  Hyman  ('28). 
They  were  placed  in  an  aquarium  containing  food  where  they  were 
observed  for  several  days.  No  indication  of  "depression" 
became  evident  during  these  observations  nor  was  there  any 
evidence  of  it  at  the  time  the  observations  ceased. 

In  order  to  determine  wherein  the  optimum  range  of  hydrogen 
ion  concentration  for  the  medium  lay,  both  green  and  brown 
hydras  were  exposed  to  varying  degree  of  hydrogen  ion  concentra- 
tion ranging  from  pH  5.2-8.0  and  the  time  recorded  when  all 
hydras  had  disappeared  in  each  culture.  The  result  of  this 
experiment  is  given  in  the  following  table. 

Four  more  cultures  were  run,  with  both  green  and  brown 
hydras,  one  with  a  pH  of  7.8,  the  other  at  pH  8.0.  All  the 
polyps  in  these  cultures  were  alive  at  the  end  of  a  period  of 
twenty  four  days. 

This  indicates  that  the  optimum  hydrogen  ion  concentration 
lies  near  pH  7.8.  And  further  hydrogen  ion  concentration  is  an 
important  factor  in  the  determination  of  dedifferentiation  and 
resorption;  for,  in  the  same  medium  (filtered  spring  water)  with 
only  the  concentration  of  hydrogen  and  hydroxyl  ions  altered,  we 
have  been  able  to  either  induce  or  inhibit  dedifferentiation  and 
resorption.  This  does  not  support  the  later  part  of  Hyman  ('28) 
page  93,  paragraph  2,  Biological  Bulletin  volume  LIV,  January 
1928,  number  I  in  her  explanation  of  the  phenomenon  of  depres- 
sion when  she  says  that  "it  is  induced  by  transfer  to  clean  fresh 
water."  It  is  quite  evident  that,  if  two  different  lots  of  hydra 


OBSERVATIONS    ON    HYDRA    AND    I'KLMATOHYDRA. 


425 


TABLE  I. 

THE  x  MARK  INDICATES  THE  DAY  OF  THE  DEATH  OF  THE  LAST  HYDRA  IN  THE 

CULTURE. 


Existence  in  D;iys. 

2 

3 

4 

5 

6 

7 

8 

0 

IO 

1  1 

12 

13 

X 

Brown  hydra  in  pH  5.2  ... 

X 

X 

X 

X 
X 
X 
X 
X 

X 
X 
X 
X 

X 
X 
X 
X 

X 
X 
X 
X 

X 

X 

X 

X 

X 

Green        "             "    5.2  ... 

Brown       "             "     5.4 

Green                           5.4.  .  .     . 

Brown       "              "    5.6.  .. 

Green                           5.6.  .  . 

Brown       "              "    5.8.  . 

Green                       "    5.8  .  . 

Brown                     "    6.0.  . 

Green         '             "    6.0  

Brown        '              "6.2 

Green         '             "6.2  

Brown                           6.4  

Green                           6.4  

Brown        '               '    6.6  

Green         '               '    6.6  

Brown        '               '    6.8.  .  .  . 

Green         '              '    6.8  

Brown                           7.0  .  . 

Green                           7.0  

Brown                           7.2  

Green         '               '     7.2  

Brown        '         '      '     7.4. 

Green         '         '      '    7.4. 

Brown                           7.6  

Green                            7.6  .  . 

taken  from  the  same  culture  or  aquarium  are  placed  in  identical 
spring  water  cultures  save  for  the  concentration  of  the  hydrogen 
ions  and  favorable  reactions  are  repeatedly  to  be  noted  in  the 
culture  of  low  hydrogen  ion  concentration  while  unfavorable 
reactions  are  always  to  be  noted  in  the  culture  of  high  hydrogen 
ion  concentration,  undoubtedly  the  pH  of  the  culture  must  be  a 
strong  factor  in  determining  this  difference  in  the  reaction- 

HISTOLOGY. 

The  histology  of  reduction,  dedifferentiation  and  resorption  in 
Hydra  has  been  observed  by  E.  Shultz  ('06)  and  \Y.  Rehm  ('25). 
Huxley  and  DeBeer  ('23)  also  described  histologically  dediffer- 
entiation and  resorption  in  Obelia  and  Campanularia.  Our  obser- 
vations are  almost  in  exact  agreement  with  those  of  the  above. 

Studies  on  reduction,  dedifferentiation  and  resorption  always 


426  W.    L.    THRELKELD   AND    S.    R.    HALL. 

involve  the  histology  of  the  animal.  Hydra  is  a  diploblastic 
animal  having  only  an  ectoderm  and  endoderm.  The  ectoderm 
presents  in  its  vegetative  condition,  epithelio-muscular,  inter- 
stitial, cnidoblastic  and  nerve  cells.  The  endoderm,  on  the  other 
hand,  is  made  up  of  epithelio-muscular,  glandular,  interstitial 
and  scattered  or  isolated  nerve  cells.  In  the  ectoderm  there  is 
no  great  local  specialization  or  differentiation  into  regions. 
The  ectoderm,  however,  shows  three  distinct  regions:  (i)  the 
oral  two-thirds  in  which  there  are  scattered  gland  cells  and  a 
general  covering  of  epithelio-muscular  cells  that  are  heavily 
charged  with  absorbed  alimentary  products;  (2)  a  basal  third 
that  has  few  if  any  gland  cells  and  in  which  the  epithelio-muscular 
cells  are  usually  highly  vacuolated,  except  for  those  at  the  basal 
disc  and  (3)  the  endoderm  of  the  tentacles.  In  this  third  region 
there  are  no  gland  cells  and  the  epithelio-muscular  cells  are 
highly  vacuolated.  Thus  it  appears  that  the  endoderm  of  the 
highly  active  or  moving  tentacles  resembles  that  of  the  relatively 
quiet  basal  third  of  the  body  proper. 

Dedifferentiation  and  resorption  have  been  referred  to  fre- 
quently above.  This  has  been  denned  as  a  dual  phenomenon 
which  involves  a  local  reduction  of  surface.  We  take  the  presence 
of  ectodermal  elements  (nematocysts  being  the  most  easily 
recognized)  within  the  coelenteron  or  endoderm  as  evidence  that 
dedifferentiation  and  resorption  have  taken  place. 

The  question  now  remains:  How  is  the  surface  reduced  locally, 
and  how  do  ectodermal  elements  gain  their  entrance  into  the 
coelenteron?  As  this  phenomenon  is  most  often  seen  in  the 
tentacles,  we  have  studied  it  there.  In  response  to  adverse  en- 
vironmental conditions,  the  cells  at  the  tips  of  the  polyp's 
tentacles  coalesce  or  become  dedifferentiated.  The  ectoderm  is 
apparently  affected  first.  Here  the  dedifferentiated  cells,  pre- 
paratory to  resorption,  group  themselves  into  rounded  or 
spheroidal  masses.  (Fig.  i-A.}  Nematocysts  as  well  as  numer- 
ous cell-fragments  may  be  seen  within  these  aggregates.  Ob- 
viously there  must  be  some  change  in  the  non-living  mesoglea  as 
well  as  the  living  endoderm  before  resorption  of  the  modified 
ectoderm  can  proceed.  Dedifferentiation,  therefore,  starts  in  the 
endoderm.  These  cells,  apparently,  break  away  from  the  walls 
of  the  tentacles  and  soon  assume  a  globular  form  (Fig.  I,  B). 


OBSERVATIONS    ON    HYDRA    AND    PELMATOH YDKA.  427 

They  migrate  down  the  lumen  of  the  tentacle  (Fig.  i,  5). 
Now  the  mesoglea  breaks  or  is  resorbed  (Fig.  i,  C)  and  the  endo- 
dermal  elements  apparently  have  little  trouble  in  finding  their 
way  to  the  coelenteron.  The  cellular  masses  of  ectoderm, 
spheroidal  in  shape  and  often  with  contained  nematocysts, 
together  with  the  above  mentioned  dedifferentiated  endo-epi- 
thelial  masses,  may  be  found  in  the  coelenteron  as  far  down  as 
the  basal  disc.  Thus  the  surface  of  the  tentacle  is  decreased. 
To  use  the  language  of  Huxley  and  DeBeer  ('23)  in  describing  a 
similar  phenomenon  in  Obelia  and  Campanularia,  "The  ecto- 
dermal  cells  may  be  compared  with  that  of  a  rear  guard,  retreat- 
ing yet  always  maintaining  an  unbroken  front."  These  histo- 
logical  details  serve  as  a  final  criterion  for  determining  whether 
dedifferentiation  and  resorption  have  taken  place.  But  with  the 
aid  of  low  magnification,  one  can  see  that,  as  resorption  proceeds, 
the  tips  of  the  tentacles  increase  in  diameter,  and  finally  appear 
knobbed  and  the  involved  area  becomes  darker  and  darker, 
The  endodermal  cells  lining  the  tentacles  are  normally  highly 
vacuolated.  These  cells,  however,  appropriate  relatively  much 
food  during  the  later  stages  of  resorption. 

It  is  certain  that  this  dedifferentiated  and  resorbed  tissue  is 
used  as  food  by  the  animal  because  nematocysts  in  various  stages 
of  digestion  may  be  found  in  the  epithelio-muscular  cells  in  all 
parts  of  the  endoderm.  This  confirms  Kepner  and  Jester  ('23) 
in  their  minor  claim  that  the  ingested  parts  were  used  as  food; 
but  Kepner  and  Jester  were  misled  by  the  occasional  biting  off  of 
the  tentacles.  Dedifferentiation  and  resorption  are  the  usual 
reaction. 

Since  it  was  seen  that  both  the  cells  of  the  ectoderm  and  the 
endoderm  of  the  tentacles  were  almost  exactly  like  those  of  the 
lateral  walls  of  the  basal  one  third  of  hydra,  dedifferentiation  and 
resorption  was  looked  for  in  this  basal  region.  It  was  found  to 
occur  in  the  case  of  the  sectioned  hydra  recorded  in  culture 
numbers  (Fig.  2,  A).  No  explanation  is  offered  for  deditfrivntia- 
tion  and  resorption  being  found  in  the  basal  disc  in  this  and  no 
other  case.  It  was  noticed,  however,  that  in  this  case  resorption 
was  not  found  in  the  tentacles.  Resorption  has  not  been  re- 
ported before  as  occurring  in  the  basal  region  prior  to  its  inception 

in  the  tentacles  and  peristome.     All  other  writers  state  that  it 

28 


428  W.    L.    THRELKELD    AND    S.    R.    HALL. 

starts  at  the  tentacles  and  proceeds  towards  the  base.  The 
peristome  is  affected,  according  to  them,  after  the  tentacles  have 
been  removed.  But  this  specimen  showed  dedifferentiation  only 
in  the  basal  region. 

Green  hydra  reported  in  culture  number  6  which  was  carried 
twenty-three  days  without  food,  showed  histologically  only  slight 
resorption. 

Rehm  ('25)  says  that  at  the  end  of  twenty  one  days  the  body  of 
hydra  subjected  to  inanition  was  reduced  to  a  mere  rounded 
form,  which  he  calls,  following  Will  and  other  investigators, 
" Reductionskorper "  (§371).  At  other  places  he  refers  to  these 
rounded  hydras  as  presenting  planula-like  pictures  ("planula- 
ahnliches  Gebilde,  der  Reductionskorper")  (§382).  We  have 
carried  brown  hydra  for  twenty  three  days  within  the  optimum 
hydrogen  ion  concentration.  This  polyp  showed  so  little  de- 
differentiation  and  resorption  that  they  could  only  be  detected 
histologically.  Under  low  magnification  the  living  polyp,  though 
reduced  in  size,  appeared  to  be  complete  and  have  no  broken 
surface.  The  brown  hydra,  as  recorded  in  culture  number  8, 
which  was  sectioned  after  sixteen  days  of  inanition  within  the 
optimum  hydrogen  ion  concentration,  presented,  while  living, 
no  evidence  of  dedifferentiation  and  resorption  under  low  magni- 
fication. However,  the  histology  of  this  animal  shows  frequent 
nematocysts  in  the  coelenteron  hence  slight  dedifferentiation  and 
resorption  must  have  taken  place  during  the  seventeen  days  of 
inanition.  Examination  on  this  day  under  the  dissecting  micro- 
scope disclosed  no  difference  in  appearance  between  the  remaining 
hydras  and  the  one  sectioned.  On  the  twenty  third  day  another 
hydra  from  this  culture  was  sectioned.  From  the  histology  of 
this  polyp,  it  is  seen  that  dedifferentiation  and  resorption  which 
were  shown  in  the  histological  examination  of  the  hydra  sectioned 
on  the  iyth  day  not  only  has  ceased  but  the  resorbed  tissue  has 
been  digested  by  the  polyp  sectioned  after  twenty  three  days  of 
inanition  within  the  optimumrange  of  hydrogen  ion  concentration. 
Similar  phenomena  have  been  observed  for  green  hydras.  For  a 
green  hydra,  which  had  suffered  14  days  of  inanition  at  optimum 
hydrogen  ion  concentration  showed  slight  dedifferentiation  and 
resorption;  while  a  second  green  polyp,  from  the  same  cultuie 
sectioned  after  twenty  three  days  of  inanition  at  optimum  hydro- 


OBSERVATIONS    OX    HYDRA    AND    I'KI.MATOH YDKA.  429 

gen  ion  concentration,  showed  no  evidence  of  dedifferentiation 
and  resorption. 

Thus  it  appears  that  during  inanition  at  optimum  hydrogen  ion 
concentration  a  crisis  is  reached  after  about  two  weeks.  During 
this  crisis  slight  dedifferentiation  and  resorption  make  their 
appearance.  The  resorbed  material  may  supply  sufficient 
nourishment  to  tide  the  polyp,  now  reduced  in  size,  through  a 
long  period  before  a  second  crisis  develops  and  compells  the  de- 
differentiation  and  resorption  of  more  tissue. 

SUMMARY. 

1.  The  optimum  range  of  hydrogen  ion  concentration  for  both 
Hydra  viridissima  and  Pelmatohydra  oligactis  lies  within  the  range 
pH  7.8  and  8.0. 

2.  Polyps  allowed  to  develop  pronounced  dedifferentiation  and 
resorption  in  a  high  hydrogen  ion  concentration  (low  pH)  were 
induced  to  completely  restore  their  lost  parts  when  the  medium 
was  altered  to  be  within  the  optimum  range  of  pH. 

3.  Hydras  carried  within  the  optimum  range  of  pH  were  sub- 
jected to  periods  of  inanition  as  great  as  twenty  five  days  without 
showing  any  external  evidence  of  dedifferentiation  and  resorption 
at  the  end  of  this  period. 

4.  Histological  preparation  of  polyps,  kept  for  long  periods 
without    food    at    the    optimum    hydrogen    ion    concentration, 
show  slight  evidence  histologically  of  dedifferentiation  and  re- 
sorption at  a  critical  period.     This  critical  period  appears  some- 
where between  ten  and  seventeen  days  after  inanition  within  the 
optimum  range  of  pH.     Such  microscopic  dedifferentiation  and 
resorption  are  not  progressive;    for  after  this  critical  period  has 
passed  no  further  histological  evidence  of  dedifferentiation  and 
resorption  has  been  observed. 

(b)  This  microscopic  dedifferentiation  and  resorption  usually 
appear  at  the  tips  of  the  tentacles;  but  in  one  case  we  have  seen 
it  involve  the  basal  third  of  the  polyp  and  not  the  tentacles. 

5.  Hydras  subjected  to  long  periods  of  inanition  within  the 
optimum  range  of  pH  accept  food  readily.     There  is,  therefore, 
no  evidence  of  depression  given  by  these  polyps. 

6.  Dedifferentiation  and  resorption  are  induced  rather  by  un- 
favorable hydrogen  ion  concentration  than  by  inanition. 


430  W.    L.    THRELKELD   AND    S.    R.    HALL. 

LITERATURE. 
Annandale,  N. 

'07     Seasonal  Variations  in    Hydra  orientalis.     Jour,   and   Proc.   Asiatic    Soc. 

Bengal,  N.  S.,  111. 
Berninger,  von  Julius. 

'10     Uber  Einwirkung  des  Hungers  auf  Hydra.     Zool.  Anz.,  Bd.  36. 
Entz,  G. 

'12     Uber  eine  neue  Amobe  auf  Susswasser — Polpen    (Hydra  oligactis  Poll). 

Arch.  Protistenk.,  Bd.  27. 
Huxley,  J.  S.  and  G.  R.  DeBeer. 

'23     Studies  in  Dedifferentiation.     IV.  Resorption  and  Differential  Inhibition  in 

Obelia  and  Campanularia.     Quart.  J.  Mic.  Soc.,  Vol.  67. 
Hyman,  Libbie  H. 

'28     Miscellaneous  Observations  on   Hydra,  with  Special  Reference  to  Repro- 
duction.    BIOL.  BULL.,  Vol.  LIV. 
Kepner,  W.  A.  and  Jester,  P.  N. 

'27     The  Reaction  of  Hydra  to  Inanition.     BIOL.  BULL.,  Vol.  52,  pp.  173-84. 
Kepner,  W.  A.,  and  Miller. 

'28    A  New  Histological  Region  in  Hydra  oligactis.     BIOL.  BULL.,  Vol.  54. 
Marshall,  Shema. 
'23     Observations  on  the  Behavior  and  Structure  of  Hydra.     Quart.  I.  Mic.  Soc., 

Vol.  67. 
Rehm,  W. 

'25     Uber  Depression  and  Reduktion  bei  Hydra.     Zeitsch.  f.  Morphol.  u.  Okol. 

d.  Thiere,  Vol.  3,  pp.  358-88. 
Reukauf,  E. 

'12     Selbstumstulpung  and  Armanputation  durch  ein  Wimperinfusor  (Prorodon 

teres)  bei  Hydra  Fusca.     Zool.  Anz.,  Bd.  39. 
Reynolds,  B.  D.,  and  Looper. 

'28     Infection  Experiments   with  Hydramaeba  hydroxena  (nov.  gen.)     Jour,  of 

Parasitology.     Vol.  XV  pp.  23-31. 
Schutz,  E. 

'06     Ueber  Reductionen.     II.   Ueber  Hungerersheinumgen  bei    Hydra   Fusca. 

Arch.  Entw.  Mech.,  Bd.  21. 
Schutze,  Paul. 

'13     Hypertrophic  der  Tentakeln  von  Hydra  oligactis  Poll,  infolge  massenhaften 
Befalls  mit  Kerona  Pediculus  O.  F.  M.     Zool.  Anz.,  Bd.  42. 


432  W.    L.    THRELKELD   AND    S.    R.    HALL. 


PLATE  I. 
Explanation  of  Figures. 

FIG.  i.  Longitudinal  section  of  the  free  end  of  a  tentacle  of  Pelmatohydra 
oligactis  which  had  been  starved  twenty-four  hours  in  spring  water  at  pH  6.8.  This 
shows  the  inception  of  dedifferentiation  and  resorption.  The  mesoglea  has  broken 
down  at  end  of  tentacle.  Rounded  masses  of  coalesced  ectodermal  cells  are  forming 
(A).  Similar  rounded  masses  of  coalesced  endodermal  cells  are  forming  (B); 
at  B'  we  see  a  mass  of  coalesced  endodermal  cells  having  migrated  towards  the 
lumen  of  the  tentacle;  at  C  a  mass  of  coalesced  ectodermal  cells  is  passing  through 
the  region  of  the  broken  down  mesoglea.  X70O. 

FIG.  2.  A  longitudinal  section  involving  a  part  of  the  basal  disc  of  Pelmato- 
hydra oligactis.  (Culture  number  5.)  This  specimen  had  been  starved  nine  days 
within  optimum  hydrogen  ion  concentration.  The  inception  of  dedifferentiation 
and  resorption  is  shown  at  A;  BGC,  basal  disc  glands  cells;  E,  endodermal  cells; 
L,  lateral  ectodermal  cells.  X70O. 


BIOLOGICAL  BULLETIN,    VOL.    LV 


PLATE    I. 


W.    L.    THRELKELD  AND  S.    R.    HALL. 


THE   OCCURRENCE   OF    NUCLEAR    VARIATIONS    IN 
PLEUROTRICHA   LANCEOLATA    (STEIN). 

REGINALD    D.    MANWELL.i 
SCHOOL  OF  HYGIENE  AND  PUBLIC  HEALTH,  JOHNS  HOPKINS  UNIVERSITY. 

The  occurrence  of  variations  from  the  accepted  type  among  the 
protozoa  has  received  much  attention  in  recent  years,  and  a 
number  of  such  cases  have  been  reported,  both  of  the  artificially 
induced  and  spontaneously  appearing  sort.  Most  of  the  former 
have  been  of  the  "enduring  modification"  type,  that  is  they  per- 
sist throughout  a  longer  or  shorter  period  of  vegetative  division, 
but  are  eventually  lost  when  conjugation  or  endomixis  takes 
place.  The  latter  may  be  divided  into  two  classes.  The  first 
group  would  include  the  true  mutations,  of  which  the  tetraploid 
Chilodon  described  by  MacDougall  (1925)  is  probably  one  of  the 
best  authenticated  examples.  In  this  case  the  mutation,  which 
consisted  in  the  possession  of  twice  the  usual  number  of  chromo- 
somes, combined  with  unusual  size  and  certain  other  minor  char- 
acteristics, persisted  through  both  conjugation  and  division. 
To  the  second  group  would  belong  all  other  departures  from 
normal,  such  as  the  production  of  monsters,  the  amicronucleate 
.condition  in  infusoria,  and  various  other  unusual  physiological 
and  morphological  characters  which  persist  through  division 
but  tend  to  revert  to  normality  eventually.  Examples  of  this 
kind  of  variation  are  quite  numerous.  Among  them  may  be 
mentioned  the  amicronucleate  Oxytricha  studied  by  Dawson 
(1919),  the  race  of  Paramecium  which  possessed  extra  contractile 
vacuoles  (Hance,  1917),  the  rapidly-dividing  race  of  Didinium 
reported  by  Mast  (1917),  and  the  sudden  appearance  of  an 
Arcella  having  double  characteristics  described  by  Reynolds 
(1923).  Since  the  latter  investigator  found  that  these  abnormal 
characteristics  could  be  diminished  until  a  completely  normal 
condition  was  reestablished,  or  increased  by  selection  of  suitable 

1  From  the  Department  of  Protozoology,  School  of  Hygiene  and  Public  Health, 
Johns  Hopkins  University,  and  the  Marine  Biological  Laboratory,  Woods  Hole, 
Mass. 

433 


434  REGINALD   D.    MANWELL. 

individuals  this  last  variation  evidently  belongs  with  those  found 
by  Jennings  (1920)  and  Root  (1918)  to  exist  in  Difflugia  and 
Centropyxis,  with  this  difference,  however — the  former  occurred 
suddenly,  while  the  latter  were  of  lesser  degree  and  appeared  more 
gradually.  More  recently  Dawson  (1924)  has  reported  the  oc- 
currence of  a  peculiar  form  of  Paramecium  aurelia  which  has  been 
carried  in  culture  for  several  years  since.  The  abnormal  char- 
acter in  this  case  consists  of  a  "notched"  condition  which  is 
definitely  heritable,  at  least  in  ordinary  asexual  division. 

The  present  paper  deals  with  variations  in  the  number  of  both 
micro-  and  macronuclei  in  Plcurotricha  lanceolata.  Pedigreed 
cultures  of  this  ciliate,  which  is  a  hypotrich  belonging  to  the 
family  Oxytrichidae,  were  maintained  for  18  months  and  studied 
mainly  from  the  standpoint  of  the  cytological  changes  occurring 
during  conjugation  and  division,  as  described  in  a  previous  paper 
(Manwell,  1928). 

The  normal  animal  is  shown  in  Fig.  I.  It  will  be  noted  that 
it  possesses  two  nuclei  of  each  sort,  and  according  to  Stein  (1858) 
who  first  described  both  the  species  and  genus,  the  presence  of 
two  macro-  and  two  micronuclei  is  a  generic  character.  About 
two  months  before  the  culture  was  discontinued  however,  and 
while  to  all  appearances  it  was  in  a  very  vigorous  condition  with 
division  taking  place  very  actively,  individuals  possessing  only  one 
macronucleus  were  noticed  in  some  of  the  stained  preparations. 
The  micronuclear  condition  varied ;  in  some  cases  there  was  only 
one  and  in  others  there  were  two  as  in  normal  individuals. 
Animals  possessing  the  normal  macronuclear  complex  but  with 
three  micronuclei  have  also  been  observed,  and  such  changes  are 
indeed  not  very  uncommon,  not  only  in  Pleurotricha  but  in 
Oxytricha  and  other  ciliates  containing  more  than  one  micro- 
nucleus.  But  no  individuals  have  been  observed  with  only  one 
macronucleus  and  more  than  two  micronuclei.  Fig.  2  shows  an 
individual  possessing  but  one  nucleus  of  each  sort  in  division,  and 
in  Fig.  3  a  similar  individual,  differing  only  in  having  two  micro- 
nuclei,  may  also  be  seen  dividing.  The  next  two  figures  show 
later  stages  in  the  division  of  such  individuals,  and  in  Fig.  6  a 
unimacro-  and  micronucleate  animal  is  shown  just  after  division. 

From  these  figures  it  can  be  seen  that  division  takes  place  in 
exactly  the  same  way  as  it  does  in  individuals  having  the  normal 


NUCLEAR    VARIATIONS    IX    PLKTROTRKTIA    I.AN< 'KOI  .ATA .       435 

nuclear  complex,  and  that  the  variations  are  heritable,  at  least  in 
ordinary  vegetative  fission.  To  settle  this  point  still  more 
definitely  several  lines  were  started  from  individuals  possessing 
hut  one  nucleus  of  each  sort  and  followed  for  10  days.  At  the 
end  of  that  time  these  subcultures  were  lost  by  accident  and  other 
circumstances  made  it  necessary  to  conclude  the  experiment,  but 
stained  preparations  made  from  each  generation  showed  clearly 
that  the  reduced  number  of  nuclei  was  being  passed  from  one 
generation  to  the  next. 

A  careful  examination  of  stained  preparations  has  been  made 
in  an  effort  to  discover  whether  the  abnormal  nuclear  complex 
was  accompanied  by  any  other  morphological  changes,  but  ap- 
parently there  were  none.  During  the  early  stages  of  division 
however  (about  the  stage  shown  in  Fig.  2)  it  was  frequently  pos- 
sible to  distinguish  animals  possessing  but  one  macronucleus  from 
normal  individuals  in  the  same  culture  in  a  similar  stage,  for  the 
bodies  of  the  former  were  definitely  broader  about  1/3  of  the  way 
back  from  the  anterior  end  and  then  tended  to  become  narrower, 
while  in  the  normal  animals  the  entire  middle  third  of  the  body 
was  of  a  fairly  uniform  width.  If  there  were  any  differences  in 
size  they  were  in  favor  of  those  individuals  possessing  but  one 
nucleus  of  each  sort. 

No  evidences  of  conjugation  among  these  abnormal  individuals 
was  ever  observed,  but  since  as  previously  reported,  conjugation 
occurred  but  rarely  in  all  the  cultures  from  start  to  finish  of  the 
experiment,  not  much  stress  can  be  laid  on  this  point.  Encyst- 
ment  was  also  not  observed.  Consequently  it  cannot  be  said 
whether  such  a  variation  as  this  would  survive  endomixis  and 
conjugation,  although  it  seems  probable  that  in  some  cases  at 
least,  unimicro-  and  macronucleate  conjugants  might  produce 
similar  individuals. 

In  view  of  the  work  of  Baitsell  (1914),  and  the  fact  that  conju- 
gation in  this  species  has  been  shown  to  result,  at  least  when  it  oc- 
curs under  cultural  conditions  favorable  to  vegetative  division,  in 
almost  100  per  cent,  mortality  (Manwell,  1928)  the  question  of 
the  occurrence  of  such  morphological  variations  as  herein  described 
becomes  of  some  practical  importance.  For  obviously,  if  under 
favorable  conditions  multiplication  by  fission  can  continue  in- 
definitely, then  such  changes  might  be  perpetuated  for  a  very 


436  REGINALD   D.    MANWELL. 

long  time  in  nature,  as  well  as  in  artificial  cultures.  And  if  this 
is  so  account  should  be  taken  of  the  fact  in  the  description  of 
genus  and  species,  since  the  number  of  nuclei,  especially  of  the 
macronuclei,  is  a  conspicuous  character.  If  asexual  reproduction 
can  continue  indefinitely  then  the  sudden  appearance  of  changes 
of  the  kind  described  would,  for  practical  purposes,  have  the 
value  of  a  mutation. 

The  occurrence  of  abnormal  micronuclear  conditions  has  been 
reported  a  number  of  times  before,  particularly  with  respect  to 
the  total  absence  of  a  micronucleus,  and  the  presence  of  one  or 
two  supernumerary  micronuclei  is  not  very  uncommon  in 
species  ordinarily  possessing  two  or  more,  as  already  noted,  but 
apparently  the  number  of  macronuclei  is  a  much  more  constant 
character.  The  only  instance  in  which  a  variation  in  the  latter 
has  been  reported,  to  the  author's  knowledge,  at  least,  is  that 
given  by  Calkins  (1926).  Here  he  states  (p.  579)  that  in  early 
cultures  of  Uroleptus  mobilis  the  number  of  macronuclei  was  al- 
most uniformly  8,  but  as  the  age  of  the  cultures  increased  indi- 
viduals with  a  greater  number  of  nuclei  became  common,  until 
finally  the  number  was  nearly  always  14  or  15. 

SUMMARY  AND  CONCLUSIONS. 

In  a  pedigreed  culture  of  Pleurotricha  lanceolata,  a  species  o, 
hypotrich  normally  possessing  two  macro-  and  two  micronuclei 
individuals  with  only  one  macronucleus  and  one  or  two  micro- 
nuclei  suddenly  appeared,  at  a  time  when  division  was  rapid  and 
the  culture  apparently  very  vigorous. 

That  the  difference  in  nuclear  number  was  heritable,  at  least  in 
asexual  multiplication,  was  shown  from  stained  preparations  and 
pedigreed  lines,  and  the  fact  that  it  has  been  shown  that  this 
species  will  live  and  divide  normally  apparently  indefinitely  under 
favorable  conditions,  without  conjugation,  makes  it  probable 
that  such  variations  as  have  been  described  would  continue  for  a 
very  long  time,  and  that  animals  with  such  peculiarities  may  be 
common  in  nature  as  distinct  varieties. 

BIBLIOGRAPHY. 
Baitsell,  G.  A. 

'14  Experiments  on  the  Reproduction  of  the  Hypotrichous  Infusoria.  II.  A 
Study  of  the  So-called  Life  Cycle  of  Oxytricha  fallax  and  Pleurotricha 
lanceolata.  Jour.  Exp.  Zool.,  Vol.  13,  pp.  211-234. 


NUCLEAR    VARIATIONS    IN    PLKUROTRICII A    I.ANCKOLATA.      437 

Calkins,  G.  N. 

'26     Biology  of  the  Protozoa.     Philadelphia.     623  pp. 
Dawson,  J.  A. 

'19     An  Experimental  Study  of  an  Amicronucleate  Oxytricha.     I.  Study  of  the 
Normal  Animal  with  an  Account  of  Cannibalism.     Jour.  Exp.  Zool.,  Vol.  29, 

No.  3.  PP-  473-513- 
'24     Inheritance  of  Abnormality  of  Form  in  Paramecium  aurelia.     Proc.  Soc. 

Exp.  Biol.  and  Med.,  Vol.  22,  pp.  104-106. 
Hance,  R.  T. 

'17     Studies  on  a  Race  of  Paramecium  Possessing  Extra  Contractile  Vacuoles. 

Jour.  Exp.  Zool.,  Vol.  23,  No.  2,  pp.  287-327. 
Jennings,  H.  S. 

'20     Life,  Death,  Heredity  and  Evolution  in  the  Protozoa.     Boston,  233  pp. 
MacDougall,  M.  S. 

'25     Cytological  Observarions  on  Gymnostomatous  Ciliata  with  a  Description  of 
the  Maturation  Phenomena  in  Diploid  and  Tetraploid  forms  of   Chilodon 
uncinatus.     Quart.  Jour.  Mic.  Sci.,  Vol.  69  (new  series),  Pt.  3.  PP-  361-384- 
Manwell,  R.  D. 

'28     Conjugation,  Division  and  Encystment  in  Pleurolricha  lanceolata.     BIOL. 

BULL.,  Vol.  54,  No.  5,  May,  pp.  417-463. 
Mast,  S.  O. 

'17     Mutation  in  Didinium  nasutum.     Amer.  Natur.,  Vol.  51,  pp.  35i-3°o. 
Reynolds,  B.  D. 

'23     Inheritance  of  Double  Characteristics  in  Arcella  polypora.     Genet.,  Vol.  8, 

pp.  477-493- 
Root,  F.  M. 

'18     Inheritance   in   Asexual    Reproduction   of    Centropyxis   aculeata.     Genet., 

Vol.  3,  pp.  173-199- 
Stein,  F.  R. 

'59     Der  Organismus  der  Infusionsthiere.     Leipzig.     Abdruck  i,  206  pp. 


438  REGINALD   D.    MANWKLL. 


EXPLANATION    OF   THE    FIGURES. 

Magnification  X  550;  all  drawings  made  with  camera  lucida 

PLATE  I. 

FIG.   i.     A  typical  vegetative  individual. 

FIG.  2.  An  individual  with  one  macronucleus  and  one  micronucleus  in  a 
moderately  early  stage  of  division. 

FIG.  3.  A  division  stage  similar  to  the  above  in  an  animal  having  two  micro- 
nuclei,  but  only  one  macronucleus. 

FIG.  4.     A  more  advanced  stage  in  an  individual  similar  to  the  above. 

FIG.  5.     The  final  stage  of  division  in  a  unimicro-  and  macro-nucleate  individual. 

FIG.  6.     A  daughter  individual  just  after  fission. 


BIOLOGICAL   BULLETIN,    VOL.    LV. 


PLATE  I. 


•£5  // 

_;    [    K  "''v'    .— ' 
•-?'•>   -^- 


Op-<      i- 

^ 


REGINALD  D.    MAMWELL 


OBSERVATIONS   ON    THE    LIEE    HISTORY   AND 

PHYSIOLOGICAL   CONDITION    OF   THE 

PACIFIC    DOG    FISH 

(SQUALUS  SUCKLII). 

J.    P.    QflGLEY.1 

Incidental  to  an  investigation  of  the  reactions  oi  .SV/ 
sucklii  to  variations  in  the  salinity  of  the  surrounding  medium  (i) 
observations  were  made  regarding  the  life  history  and  physio- 
logical condition  of  this  fish. 

The  fish  were  captured  during  the  months  of  June,  July  and 
August  of  1926  from  the  Straits  of  Georgia  in  the  vicinity  of 
Departure  Bay,  Vancouver  Island,  B.  C.  They  were  taken  on  a 
set  line,  the  hooks  of  which  were  baited  with  pieces  of  salted 
herring.  Most  of  the  fish  were  obtained  at  a  depth  of  about  30 
meters,  and  they  were  generally  caught  near  kelp  beds.  A 
sample  of  water  taken  at  a  depth  of  30  meters  in  the  region  where 
many  of  the  fish  were  taken  was  found  by  Lucas  (2)  to  have  tin- 
following  characteristics;  pH  8.4,  temperature  10.3°  C.,  density 
1. 0218,  oxygen  content  4.41  cc.  per  liter,  sodium  chloride  content 
27.37  gm-  Per  liter. 

Weight  of  Fish. — It  was  found  that  many  of  the  factor^  as- 
sociated with  the  weight  of  the  fish  could  be  emphasized  by 
grouping  the  fish  according  to  weight  as  has  been  done  in  Table  I. 
Examination  of  this  table  shows  that  with  the  fish  of  lighter 
weight  the  two  sexes  are  nearly  equally  represented,  the  number 
of  males  being  slightly  greater.  As  heavier  fish  are  considered, 
the  relative  number  of  males  shows  a  marked  iiiriva-r,  then  a 
sudden  decrease  so  that  in  the  weight  divisions  above  4,000  gram> 
the  males  are  entirely  absent. 

These  results  probably  indicate  that  male  fish  with  body  weight 
over  4,000  grams  do  not  exist  in  this  locality  during  the  Bummer. 
It  cannot  be  definitely  stated  that  the  figures  obtained  with  li^h 
of  lighter  weight  indicate  the  relative  proportion  in  which  tin- 

1  From  The  Pacific  Biological  Station,  Nanaimo,  H.  C.,  and  Tin-  Department  »i 
Physiology  and  Pharmacology,  Fnivcrsity  of  Alberta,  Kdmonton,  Alberta. 

439 
29 


44<> 


J.    P.    QUIGLEY. 

TABLE  I. 


\\Vinht 

Limit  - 

Number 

<it   Fish 
i  il.l.iiunl. 

Number  of 

Percentage. 

Average 
Length 
(Cm.). 

Average 
Increase 
in  Length. 

M.il'  3. 

Females. 

Males. 

Females. 

300  ,i<;9-  .  . 

12 

7 

5 

58 

42 

39-9 

joo    [99 

15 

9 

6 

60 

40 

43-6 

3-8 

500  500 

[6 

ii 

5 

69 

31 

45<7 

2.1 

ooo   009.  .  . 

13 

8 

5 

62 

38 

48.4 

2.7 

0    709-  -  • 

5 

5 

o 

100 

0 

52.5 

4.1 

,Xoo    S<;o.  . 

7 

4 

3 

57 

43 

53-8 

i-3 

000-999.  .  . 

5 

5 

0 

IOO 

0 

54-7 

0.9 

i  .DOM    i  ,499  . 

22 

20 

2 

91 

9 

60.3 

5-6 

1.500    i.  <><><) 

I  I 

9 

2 

82 

18 

69.2 

8.'9 

2,  OOO-2,  <)<i<j 

30 

26 

4 

87 

13 

74-9 

5-7 

3,000-3,999 

13 

5 

8 

38 

62 

83.3 

8.4 

4.OOO     \.<)<>'i 

10 

o 

16 

o 

IOO 

90.5 

6.2 

5,000  s,999 

c6 

o 

16 

0 

IOO 

91.6 

i.i 

6,000-6,999  . 

4 

o 

4 

0 

IOO 

95-5 

3-9 

7.000-7,999  . 

i 

o 

I 

o 

IOO 

99.0 

3-5 

i\v<>  sexes  occur,  although  such  probably  is  the  case.  Since  the 
lish  were  taken  on  a  set  line  hunger  or  greed  might  conceivably  be 
a  factor  in  determining  whether  or  not  fish  would  take  the  bait. 
The  stomach  of  fish  captured  usually  contained  much  food,  a 
l.ict  which  indicates  that  feeding  for  this  fish  is  determined  more 
by  the  availability  of  food  than  by  hunger. 

Out  of  219  fish  captured,  128  (58  per  cent.)  were  males.  Craigie 
(3)  examined  the  fish  obtained  in  the  same  region  during  July 
and  August,  1925,  and  found  that  among  76  specimens  44  (60 
per  cent.)  were  males,  while  during  December  of  1925  by  examin- 
ing 1 17  specimens  he  found  47  (40  per  cent.)  males. 

As  was  to  have  been  expected,  there  is  a  comparatively  definite 
relationship  between  weight  and  length  of  fish.  The  increase  in 
length  is  rather  steady  though  not  entirely  uniform  as  heavier  fish 
are  compared  with  those  of  lighter  weight.  It  could  not  be  shown 
thai  sex  altered  the  relation  of  weight  and  length.  There  was  a 
slight  though  inconstant  indication  that  nonpregnant  females 
were  longer  than  pregnant  females  of  the  same  weight.  The 
longest  fish  captured  measured  99  cm.,  the  shortest  35.5.  The 
lu-a\ -icst  Ii-h  weighed  7,550  grams  and  the  lightest  300  grams. 
When  increasing  their  weight  100  grams  the  smaller  fish  made  an 
increase  in  length  of  approximately  the  same  magnitude  as  did 
the  larger  fish  when  making  a  weight  increase  of  1,000  grams. 


LIFE  HISTORY  OF  PACIFIC  DOG  FISH.  441 

Pregnancy  and  Embryos. — Of  the  females  captured,  43  per  cent, 
carried  embryos  large  enough  to  be  readily  noted  in  a  cursory 
inspection.  The  lightest  fish  having  embryos  weighed  3,440 
grams  and  was  85  cm.  in  length.  These  figures  give  an  approx- 
imate minimum  limit  of  the  size  of  the  mature  female.  Among 
the  50  females  captured  with  a  weight  equal  to  or  above  3,440 
grams,  39  (78  per  cent.)  carried  embryos. 

Ford  (4)  quotes  the  conclusion  of  several  investigators  that 
Squalns  acanthias  breeds  throughout  the  year  and  of  other 
investigators  that  this  species  breeds  only  during  certain  periods. 
The  results  of  his  own  investigations  support  the  latter  conclusion 
and  tend  to  show  that  near  Plymouth,  England,  specimens  ready 
for  birth  would  not  be  found  earlier  than  the  end  of  August. 
I  found  specimens  of  Squalus  sucklii  embryos  at  all  times  during 
the  summer  which  ranged  through  all  the  sizes  from  the  smallest 
to  those  with  the  umbilical  scar  healed  completely  and  apparently 
ready  for  birth.  This  observation  naturally  suggests  that  in  the 
vicinity  of  Nanaimo,  Squalus  sucklii  breeds  at  all  times  of  the 
year. 

In  any  one  parent,  the  embryos  were  of  the  same  general  size. 
A  set  of  developing  eggs  was  always  found  in  females  carrying 
embryos.  The  number  of  embryos  obtained  from  16  fish  varied 
between  3  and  II  with  an  average  number  of  6.87.  Although  it 
could  not  be  definitely  stated  that  none  of  the  embryos  had  been 
lost  from  the  mother  in  the  course  of  capture  it  is  believed  that  this 
was  a  rare  occurrence.  No  embryos  were  lost  after  the  mother 
was  taken  from  the  set  line  and  in  most  cases  egg  capsules  still 
unruptured  wrere  obtained.  In  an  examination  of  Squalus  acan- 
thias Ford  (4)  found  that  females  of  this  species  could  carry  as 
many  as  n  embryos  but  the  greatest  number  of  pregnant  fish 
carried  only  3.  In  Squalus  sucklii  I  found  that  embryos  of  both 
sexes  usually  occurred  in  the  same  uterus  but  there  was  no 
relation  between  the  number  of  either  sex,  e.g.  in  one  fish  I  found 
6  females  and  I  male,  in  another  3  males  and  no  females.  Of  the 
embryos  obtained  50  per  cent,  were  males.  This  figure  is  to  be 
contrasted  with  that  previously  noted  for  the  fish  of  small  size 
taken  on  the  set  line  where  a  preponderence  of  males  existed. 
A  blue  shark,  Prionace  glance,  (identified  by  Professor  J.  R. 
Dymond)  received  at  the  Pacific  Biological  Station,  August  19, 


442  J-    P-    QUIGLEY. 

1926,  was  found  to  have  11  females  and  8  male  embryos  all  the 
same  size  nearly  ready  for  birth. 

Constitution  of  Shoals. — Throughout  the  period  fish  were  being 
taken,  the  specimens  obtained  on  any  set  line  usually  consisted  of 
both  sexes  in  approximately  equal  numbers  and  of  all  sizes. 
The  conclusion  was  reached  that  the  shoals  consisted  of  both  sexes 
and  all  sizes  of  fish  or  else  the  line  had  been  visited  within  a  few 
hours  by  several  different  shoals.  It  was  also  noted  that  the 
largest  fish  were  usually  taken  at  a  greater  depth  (very  near  or 
actually  on  the  sea  bottom)  than  the  smallest  and  it  may  be  that 
the  composition  of  shoals  is  in  part  determined  by  size.  From 
his  study  of  Squalus  acanthias,  Ford  (4)  concluded  that  for  this 
species  the  mature  males  and  females  each  form  separate  shoals 
while  these  shoals  in  turn  are  distinct  from  those  composed  of 
immature  males  and  females  together.  I  obtained  fish  in  the 
same  region  throughout  the  summer.  It  is  therefore  likely  that 
certain  shoals  inhabit  this  region  during  the  entire  season. 

SUMMARY. 

1 .  Among  the  smaller  fish  males  were  slightly  more  prevalent 
than  females.     Males  weighing  more  than  4,000  grams  were  not 
obtained.     Females  attain  a  much  greater  length  and  weight  than 
males.     The  greater  weight  of  the  females  was  not  always  due  to 
the  presence  of  eggs  or  embryos. 

2.  A  comparatively  definite  relationship  exists  between  weight 
and  length  of  fish.     The  relationship  of  length  increase  to  weight 
increase  for  small  fish  is  approximately  ten  times  as  great  as  for 
large  specimens. 

3.  Of  the  mature  females  captured  78  per  cent,  carried  embryos. 
This  species  apparently  breeds  throughout  the  year.     The  average 
number  of  embryos  carried  by  the  females  is  greater  than  six. 

4.  The  shoals  apparently  consist  of  fish  of  all  sizes  and  of  both 
sexes.     The  shoals  probably  remain  in  the  same  region  throughout 
the  summer. 

REFERENCES. 

1.  Quigley,  J.  P. 

'28     BIOL.  BULL.   LIV.,  165. 

2.  Lucas,  C.  C. 

Personal  communication. 

3.  Craigie,  E.  H. 

'27     Contrib.  to  Canadian  Biol.  and  Fisheries,  N.  S.,  Ill,  No.  22,  491. 

4.  Ford,  E. 

'21     Jour.  Marine  Biol.  Assoc.  of  the  United  Kingdom,  N.  S.,  XXII. ,  468. 


ALG^E  OF   PONDS  AS   DETERMINED    BY   AN 

EXAMINATION   OF  THE   INTESTINAL 

CONTENTS  OF  TADPOLES. 

VIVIAN    FARLOWE, 
UNIVERSITY  OF  VIRGINIA. 

INTRODUCTION. 

During  the  last  few  years  a  considerable  amount  of  research 
has  centered  around  the  food  taking  of  small  fresh-water  fish. 
This  work  has  emphasized  the  dependence  of  small  fish  on  algae 
and  in  turn  these  fish  as  a  source  of  food  for  the  game  fish.  In 
reviewing  literature  the  writer  has  found  comparatively  little 
scientific  work  on  the  feeding  habits  of  the  tadpole  and  frog. 

The  tadpole  as  well  as  the  small  fish  is  an  indirect  source  of 
food  for  the  human  race.  Tiffany  ('22)  states:  "For  most  of  the 
young  fishes  examined  the  complete  story  reads:  'no  phyto- 
plankton,  no  gizzard  shad.' '  It  may  also  be  said,  no  algae,  no 
tadpole. 

The  writer  wishes  to  express  her  gratitude  to  Dr.  Bruce  D. 
Reynolds,  who  suggested  this  problem  and  who  has  greatly  as- 
sisted by  his  advice  and  criticism  in  the  preparation  of  this  paper; 
also  to  Professor  I.  F.  Lewis  and  Dr.  E.  M.  Betts  for  helpful 

criticisms. 

METHODS. 

During  the  summers  of  1927  and  1928  one  hundred  tadpoles 
and  one  hundred  pond  collections  were  taken  from  five  ponds  on 
the  campus  of  the  University  of  Virginia  and  in  the  surrounding 
vicinity.  Two  of  the  ponds  measured  approximately  250  ft.  x  100 
ft.,  one  150  ft.  x  50  ft.,  one  100  ft.  x  30  ft.,  and  one  50  ft.  x  20  ft. 
The  ponds  which  were  studied  did  not  have  active  outlets. 

Two  examinations  of  each  of  these  ponds  were  made  during  the 
summer  of  1927  from  July  15  to  August  28,  and  two  were  made 
during  the  summer  of  1928  from  June  20  to  July  5.  Each  collec- 
tion from  a  pond  consisted  of  five  tadpoles  1  which  measured  from 

1  Of  the  100  tadpoles  used  in  these  experiments,  94  were  Rana  clamilans  and  6  R. 
catesbeiana. 

443 


444  VIVIAN    FARLOWE. 

one  and  three-fourths  inches  to  five  inches  long  and  five  collections 
of  sediment  taken  from  the  edges  of  the  ponds.  The  tadpoles 
and  pond  collections  were  put  in  separate  containers.  Im- 
mediately after  returning  to  the  laboratory  the  tadpoles  were 
killed  and  the  intestines  removed.  Three  slides  were  made  of 
material  taken  from  each  digestive  tract,  one  from  the  anterior 
and  one  from  the  middle  regions  of  the  small  intestine,  the  third 
from  the  anterior  region  of  the  large  intestine.  A  study  of  each 
of  the  slides  was  made  under  the  high  power  of  the  microscope. 
The  algae  from  each  region  were  identified  and  recorded.  The 
pond  collections  were  studied  in  a  similar  way.  Three  slides 
were  made  from  each  of  the  pond  collections.  The  algae  from 
each  slide  were  identified  and  recorded. 

During  the  summer  of  '27  the  tadpoles  were  collected  from  the 
pond,  and  then  the  pond  collections  were  made  without  any 
effort  to  correlate  the  position  of  the  tadpole  and  the  pond  collec- 
tion, but  in  the  collections  made  during  the  summer  of  '28  a  tad- 
pole was  caught  and  from  the  same  place  a  pond  collection  was 

made. 

THE  PROBLEM. 

The  experiments  presented  in  this  paper  were  not  undertaken 
primarily  for  the  purpose  of  studying  the  food  of  tadpoles,  but 
rather  in  order  to  ascertain  if  the  algae  found  in  the  alimentary 
tract  of  tadpoles  can  be  relied  upon  as  an  index  to  the  micro- 
scopic flora  of  the  ponds  in  which  the  tadpoles  are  living.  In 
other  words,  does  the  tadpole  feed  on  different  kinds  of  algae  or  is 
it  selective  in  its  feeding  habits?  If  not  selective,  is  it  as  good  a 
collector  of  algae  as  the  investigator  interested  in  studying  them? 

EXPERIMENTAL. 

In  following  up  this  problem  observations  were  made  on  four 
collections,  made  at  different  times,  from  each  of  five  ponds. 
The  results  obtained  are  shown  in  tabular  form. 

By  referring  to  Table  I.  it  will  be  seen  that  the  number  of 
species  of  algae  obtained  from  the  intestine  of  the  tadpoles  ex- 
ceeded the  number  obtained  from  the  pond  collections  in  every 
case  except  two,  and  in  these  instances  they  were  the  same — -the 
pond  collections  being  made  where  the  tadpoles  were  caught. 

Attention  is  also  called  to  the  relative  number  of  algae  found  in 


ALGJE   OF    PONDS. 


445 


the  intestines  of  tadpoles  and  the  ponds  from  which  they  were 
taken,  in  large  and  small  ponds  (Table  I.).  It  is  evident  that, 
when  making  collections  from  small  ponds,  the  investigator  is 
able  to  find  most  of  the  algae  present;  whereas  if  the  pond  is  a 
large  one  there  is  an  appreciable  difference  between  the  number 
of  species  of  algae  obtained  by  the  two  methods — the  ratio  being 
approximately  4 :  3  in  favor  of  the  tadpole. 

TABLE  I. 

SHOWING  THE  TOTAL  NUMBER  OF  SPECIES  OF  ALGAE  TAKEN  FROM  THE  INTESTINAL 

TRACT  OF  FIVE  TADPOLES  AS  COMPARED  WITH  THE  TOTAL  NUMBER 

FOUND  IN  FIVE  COLLECTIONS  MADE  FROM  THE  SAME  PONDS. 


Size  of  Pond. 
250  x  IOO  ft. 

Collections  Made  during 
Summer  of  1927. 

Collections  Made  during 
Summer  of  1928. 

Jun.  is-Aug.n. 

Aug.  n-Aug.  28. 

Jun.  2i-Jun.  27. 

Jun.  27-July  5. 

Tadpole. 

Pond. 

Tadpole. 

Pond. 

Tadpole. 

Pond. 

Tadpole. 

Pond. 

50 

54 
52 
35 
35 

32 

42 
46 
30 
30 

59 

45 
47 
63 
46 

39 

37 
44 
50 

44 

63 

44 
65 
56 
47 

49 

44 
56 
47 
39 

58 
56 
59 
47 

44 

48 

49 

46 

41 
44 

250  x  IOO  ft  

150  x     50  ft.  ... 

IOO  x     50  ft.    .  .  . 

50  x    20  ft. 

As  stated  in  a  paragraph  under  Methods,  three  examinations 
were  made  of  each  pond  collection  and  of  each  tadpole — one  from 
the  anterior  region  of  the  small  intestine,  one  from  the  middle 
region  of  the  small  intestine,  and  one  from  the  large  intestine. 
Table  II.  shows  the  distribution  of  the  species  in  different  regions 
of  the  intestinal  tract  as  compared  with  the  total  number  found 
in  the  tadpole  and  the  total  number  found  in  the  pond  collections. 
Usually  more  species  of  algae  were  found  in  the  anterior  end  of 
the  small  intestine,  but  there  is  not  a  great  variation  in  numbers 
in  the  three  regions.  Most  of  the  algae  found  in  the  large  in- 
testine show  slight  evidence  of  having  been  acted  upon  by  the 
digestive  juices. 

Even  though  the  species  of  algae  found  in  the  tadpoles  out- 
numbered those  in  the  pond  collections,  algae  which  did  not  occur 
in  the  tadpoles'  intestines  were  found  in  collections  made  from 
the  pond.  There  was  one  exception,  and  in  this  case  the  tadpole 
and  pond  collection  were  taken  from  the  same  place.  In  this 
entire  work  only  five  species  of  algae  were  found  in  pond  collections 


446 


VIVIAN    FARLOWE. 


TABLE  II. 

THE  TOTAL  NUMBER  OF  SPECIES  OF  ALG.E  FOUND  IN  DIFFERENT  PONDS, 
TIIK  NUMBER  FOUND  IN  TADPOLES  AND  THE  NUMBER  FOUND  IN 

DIFFERENT  REGIONS  OF  THE  INTESTINE. 

A.  S.  Int.,  anterior  end  of  small  intestine;    M.  S.   Int.,  middle  region  of  small 
intestine;   A.  L.  Int.,  anterior  end  of  large  intestine. 


Pond. 

Tadpole. 

A.  S.  Int. 

M.  S.  Int. 

A.  L.  Int. 

29 

36 

23 

22 

20 

30 

34 

22 

16 

19 

35 

44 

23 

24 

19 

33 

33 

24 

19 

19 

30 

45 

21 

24 

31 

28 

33 

28 

26 

21 

3i 

40 

26 

20 

24 

32 

3i 

19 

M 

14 

27 

50 

26 

21 

36 

16 

34 

18 

13 

16 

25 

35 

29 

12 

16 

18 

35 

26 

14 

22 

35 

34 

21 

26 

16 

24 

36 

21 

21 

27 

32 

38 

21 

16 

,  21 

27 

36 

2O 

22 

22 

32 

34 

31 

15 

18 

25 

38 

22 

21 

25 

24 

32 

18 

18 

19 

36 

49 

32 

26 

30 

TABLE  III. 

COLLECTIONS  MADE  DURING  SUMMER  OF  1927. 


Total 

Total 

Number 
Species 
from  Both 

Percentage  of 
Those  Found 
in  Tadpoles. 

Percentage  of 
Those  Found 
in  Pond. 

Number 
Species 
from  Both 

Percentage  of 
Those  Found 
in  Tadpoles. 

Percentage  of 
Those  Found 
in  Pond. 

Sources. 

Sources. 

50 

86.20 

55-17 

70 

82.85 

55-71 

68 

79-32 

61.76 

58 

83.10 

63.79 

70 

74.28 

65-71 

57 

82.62 

77.19 

45 

77-77 

66.66 

68 

93.64 

73-23 

37 

94-59 

81.08 

53 

86.79 

75.28 

COLLECTIONS  MADE  DURING  SUMMER  OF  1928. 


67 

94-03 

73-13 

64 

95-31 

77-50 

50 

88. 

88. 

58 

96.55 

84.48 

66 

98.48 

84.84 

62 

95-17 

74.19 

60 

94-33 

78.33 

56 

IOO. 

83.91 

48 

97.91 

81.25 

47 

93.61 

93.61 

Showing  total  number  of  species  of  algae  taken  from  each  pond,  including  the 
percentage  of  those  obtained  from  tadpoles  and  from  pond  collections. 


OF    PONDS.  447 

which  were  not  also  observed  in  the  tadpoles.  Evidently  these 
species  were  very  rare,  for  only  one  was  encountered  the  second 
time.  The  fact  that  these  algae  were  not  found  in  the  tadpoles 
does  not  indicate,  therefore,  that  the  tadpoles  refuse  to  eat  them. 
The  variation  in  percentage  of  algae  from  the  two  sources  is 
less  when  pond  collections  and  tadpoles  are  taken  from  the  same 
place.  This  may  be  seen  by  referring  to  Table  III.  The  pond 
collections  made  during  the  summer  of  1928  were  taken  from  the 
immediate  vicinity  in  which  the  tadpoles  were  caught,  while 
those  made  during  the  summer  of  1927  were  taken  without  regard 
to  this  matter. 

SUMMARY. 

It  is  a  well  known  fact  that  tadpoles  feed  on  microscopic 
plants.  The  importance  of  this  animal  as  a  collector  of  algae  is 
clearly  demonstrated.  In  comparing  the  intestinal  contents  of 
one  hundred  tadpoles  with  pond  collections  made  from  the  same 
ponds,  the  number  of  species  of  algae  obtained  from  the  tadpoles 
exceeded  the  number  obtained  from  the  collections  in  every  case 
except  two;  and  in  these  instances,  they  were  the  same.  It  may 
be  stated,  therefore,  that  an  examination  of  the  intestinal  con- 
tents of  tadpoles  affords  one  of  the  best  and  easiest  methods  of 
determining  the  species  of  algse  present  in  ponds.  This  is  es- 
pecially true  in  large  ponds,  and  applies  particularly  to  the  phyto- 
plankton. 

In  this  examination  one  hundred  and  seventy  species  and 
varieties  of  phytoplankton  were  found.  Of  this  number,  one 
hundred  and  sixty-five  were  encountered  in  the  intestines  of 
tadpoles. 

CONCLUSION. 

1.  The  food  of  green-frog  tadpoles  consists  chiefly  of  algae. 

2.  The   algae  from  pond  collections  and  from  the  intestinal 
contents  of  tadpoles  taken  from  the  same  ponds  do  not  differ  as 
much  in  small  ponds  as  they  do  in  the  larger  ones. 

3.  The  anterior  region  of  the  small  intestine  is  considered  to  be 
the  best  region  for  making  examinations  for  algae. 

4.  The  species  of  algae  taken  from  the  intestines  of  tadpoles 
constituted,  on  the  average,  89.73  +  per  cent,  of  the  total  found. 


448  VIVIAN    FARLOWE. 

An  examination  of  the  intestinal  contents  of  tadpoles  affords 
one  of  the  best  and  easiest  methods  of  obtaining  a  collection  of 
alga;  from  ponds. 

BIBLIOGRAPHY. 

Cahn,  A.  R. 

'27     An    Ecological    Study    of    Southern    Wisconsin  'Fish.     Illinois    Biological 

Monographs. 
Coker,  R.  E. 

'18     Principles  and  Problems  of  Fish  Culture  in  Ponds.     The  Scientific  Monthly. 
Forbes,  S.  A. 

'14     Fresh  Water  Fish  and  their  Ecology.     Illinois  State  Laboratory  of  Natural 

History. 
Mann,  A. 

'21     The  Dependence  of  Fishes  on  the  Diatoms.     Ecology,  2:  79-83. 
Tiffany,  L.  H. 

'20     Algal  Food  of  the  Young  Gizzard  Shad.     Ohio  Journal  of  Science,  21:  113- 

122. 
Tiffany,  L.  H. 

'22     Some  Algal  Statistics  Gleaned  from  the  Gizzard  Shad.     Science,  56:  285- 

286. 
Tiffany,  L.  H. 

'26     Algal  Collection  of  a  Single  Fish.     Michigan  Academy  of  Science,  Arts  and 
Letters,  Vol.  VI. 


FURTHER    OBSERVATIONS    ON    THK    CHEMICAL 

COMPOSITION   OF  WOODS    HOLE   SEA 

WATER— THE  CHLORINE 

CONTENT  AND   SALT 

ANALYSIS. 

IRVINE    H.    PAGE, 

ELI  LILLY  RESEARCH  LABORATORY,  MARINE  BIOLOGICAL  LABORATORY,  WOODS 

HOLE,  MASS. 

From  time  to  time  we  have  had  occasion  to  make  further  ob- 
servations on  the  sea  water  at  Woods  Hole  since  the  publication  of 
the  original  analysis  (i).  Though  not  in  any  sense  complete  it  is 
believed  that  the  following  data  may  prove  useful  and  therefore 
they  are  presented. 

It  should  be  pointed  out  that  our  aim  has  been  always  to  select 
methods  of  analysis  which  would  adapt  themselves  to  the  use  of 
relatively  small  fluid  volumes,  as  only  in  this  way  can  they  be- 
come applicable  to  the  investigation  of  physiological  and  biolog- 
ical problems.  From  the  large  number  of  analyses  of  sea  water 
tabulated  by  the  Hydrographic  Laboratory  of  Copenhagen, 
Knudsen,  Dittmar  (2)  etc.,  further  data  of  this  kind  have  oceano- 
graphic  interest  but  little  more.  There  has,  therefore,  been  made 
a  conscious  attempt  to  utilize  more  sensitive  methods  which 
require  small  samples  for  analysis,  albeit  the  absolute  values 
may  not  be  quite  as  accurate. 

DETERMINATION  OF  CHLORINE. 

Since  many  physiological  activities  are  sensitive  to  slight 
changes  in  the  tonicity  of  the  surrounding  medium  it  seemed  of 
interest  to  determine  whether  the  chlorine  content  of  the  Woods 
Hole  sea  water  varied  to  a  significant  degree  from  day  to  day. 
The  method  employed  "was  as  follows:  Standard  AgNO.3  was 
made  such  that  I  cc.  was  equivalent  to  10  mg.  chlorine.  This  was 
standardized  against  pure  NaCl  since  it  has  been  shown  by 
Thompson  (3)  that  this  salt  may  be  substituted  for  standard 
water  from  the  Hydrographic  Laboratory.  The  AgNO3  was 

449 


450 


IRVINE    H.    PAGE. 


kept  in  the  dark  in  a  glass  stoppered  brown  bottle  and  the 
standardization  repeated  at  the  end  of  the  series  of  determina- 
tions. The  method,  thereafter,  followed  in  detail  that  presented 
by  the  Association  of  Official  Agricultural  Chemists  (4).  The 
burette  used  was  of  50  cc.  capacity,  standardized  by  the  Bureau 
of  Standards,  Washington.  15  cc.  samples  of  sea  water  were 
measured  with  a  standardized  pipette  and  diluted  with  distilled 
water  to  35  cc.  before  titration. 

Samples  were  taken  from  the  laboratory  tank.  This  tank  is 
fed  by  water  taken  about  125  feet  from  shore.  The  other  samples 
were  taken  from  surface  water  as  follows:  (i)  Buzzards  Bay  one 
half  mile  North  of  Robinson's  Hole.  (2)  Cuttyhunk  300  feet 
from  shore  on  the  "Sound"  side.  (3)  Tarpaulin  cove  one  half 
mile  out  in  the  Sound;  water  80  feet  deep.  (4)  East  of  Nobska; 
water  28  feet  deep. 

Duplicate  titrations  were  made  and  it  may  be  said  that  these 
determinations  but  rarely  disagreed. 

The  temperature  was  taken  with  not  great  accuracy,  employing 
a  standard  50  degree  laboratory  thermometer.  Such  slight 
changes  as  observed  during  these  observations  were  not  consid- 
ered significant. 

Grams  of  chlorine  per  kilogram  were  calculated  from  Thomp- 
son's empirical  formula— 

Clw  =  =  0.008  -f  0.99980  CU  --  0.001228  C\v2 

where  C\w  •-  =  grams  of  Cl  per  kilogram  and  Clt,  =  grams  Cl  per 
liter  at  20°  C.  A  graph  prepared  by  using  the  more  common 
range  of  Cl  contents  was  found  useful. 

The  salinity — defined  as  the  weight  in  grams  of  all  the  salts 
dissolved  in  a  kilogram  of  sea  water,  after  the  carbonates  have 
been  converted  to  oxides,  the  Br  and  I  have  been  replaced  by  Cl 
and  the  organic  matter  has  been  completely  oxidized — was 
calculated  from  the  relation  derived  by  Knudson— 

So/oo  ==  0.030  +  1.8050  Clw 

Of  course  it  must  be  recognized  that  this  is  only  an  approxima- 
tion, as  Giral  (5)  has  emphasized. 

During  these  observations  it  should  be  stated  that  the  weather 
was  in  general  extremely  bad,  rain  alternating  with  fog  for  dis- 


CHEMICAL   COMPOSITION    OF    WOODS    HOLE    SEA    \\ATER.      45! 


TABLE  I. 

CHLORINE  CONTENT  OF  \\~OODS  HOLE  SEA  WATER  I)i  RIM,  TIIK  Si  M.MER  OF  ig28. 


Date. 

Source. 

Tcinprraturf. 

Grams  Cl 
per  Liter. 

1  .rams  Cl 
IKT  Ki'.oKr.im. 

So'oo. 

July  16.  .  . 

Laboratory  tank 

2  1      degrees 

17.80 

17.42 

.Si.  17 

18.  .  . 

* 

22 

17.80 

17.42 

31-47 

21  ... 

' 

21 

17.86 

17.48 

31.58 

23.   •• 

' 

21 

17.77 

17-39 

31.42 

26.  .. 

' 

21.8 

17-77 

17-39 

31.42 

28.  .  . 

* 

21 

17.86 

17.48 

3I.S8 

August  i   . 

* 

20-5 

17.80 

17.42 

31-47 

July  17.  .. 

Buzzards  Bay.  . 

2O 

17-93 

17-54 

31.69 

* 

17.  .  . 

Cuttyhunk 

20 

18.00 

17.60 

.51.7'; 

* 

'        21  ... 

Off  Tarpaulin  Cove 

20 

17-93 

17-54 

31.69 

t 

'        21  ... 

East  Nobska 

20 

17-70 

17.32 

3I-27 

agreeably  long  intervals.  The  results,  do  not  show  any  very 
marked  changes  in  the  Cl  content  of  the  water  but  it  is  altogether 
possible  that  a  dry  summer  may  increase  the  Cl  content.  Sam- 
ples taken  from  other  points  along  the  uneven  coast  of  Woods 
Hole  show  more  evident  variations,  as  was  to  be  expected. 

SEA  SALT  ANALYSIS. 

Samples  of  the  dried  sea  salt  taken  from  the  laboratory  tank 
during  the  summer  of  1926  have  been  analysed,  employing  the 
classical  methods  as  given  in  the  Bulletin  of  the  Official  Agricul- 
tural Chemists  (4)  and  by  Scott  (6).  Though  not  complete, 
these  data  are  presented,  as  they  may  be  found  useful. 


SEA  SALT  OF  WOODS  HOLE. 


Xo.  i 


PerciMitam-. 


Sodium 30.68 

Magnesium 3. 3  i 

Calcium 1.27 

Silica 0.014 

Phosphate Trace 

Nitrate.  .  .  Trace 


No.  2. 

30.49 
3.48 

1. 12 
0.018 

Trace 
Trace 


The  above  analyses  would  tend  to  confirm  the  suggestion  made 
in  our  former  paper  that  the  Kramer-Gittleman  direct  method 
for  the  determination  of  sodium,  while  very  convenient  for  rela- 
tive data,  may  give  an  absolute  value  which  is  low.  One  must 
remember,  however,  that  using  the  Haywood  and  Smith  Method 


IRVINE    H.    PAGE. 

or  that  of  Dittmar  the  sodium  determination  comes  out  low, 
as  has  been  the  universal  experience  of  analysts.  The  values  are 
then  corrected  by  employing  Dittmar's  method  (2)  of  "total 
Milphates."  The  older  methods  for  sodium  determinations  are  so 
nbersome  (as  reference  to  Dittmar's  article  will  show)  that 
there  is  still  some  doubt  as  to  the  accuracy  of  the  results. 

During  the  Summer  of  1928  we  have  again  confirmed  Atkins' 
(8)  and  Harvey's  (9)  work  on  the  nitrates  and  phosphates. 
Samples  of  the  Woods  Hole  water  showed  only  the  smallest  trace 
of  NO3  and  PO4  during  July  1928,  the  time  at  which  our  analyses 
were  made  this  year.  This  change  is,  as  they  have  shown,  due  to 
seasonal  variations  in  the  plankton. 

SUMMARY. 

1.  The  chlorine  content  of  Woods  Hole  sea  water  has  been 
examined  over  a  three-week  period  and  shown  not  to  vary  within 
any  large  range. 

2.  Analyses  of  the  sea  salt  are  presented. 

REFERENCES. 

1.  Page.     BIOL.  BULL.,  52,  161  (1927). 

2.  Dittmar.     Challenger  Report  I.      (Phys.  and  Chem.)  i,  (1884). 

3.  Thompson.     Jour.  Am.  Chem.  Soc.,  50,  681  (1928). 

4.  Methods   of   Analysis    of    the    Association    of    Official    Agricultural    Chemists, 

Washington,  D.  C.,  1925. 

5.  Giral.     Publications  de  Circonstaiice  No.  90  (1926). 

<>.  Scott.     Standard  Methods  of  Chemical  Analysis.     3d  Ed.,  D.  van  Nostrand 

Company,  New  York,  N.  Y. 

7.  Hay  wood  and  Smith.     Bull.  91,  Bureau  of  Chemistry. 
vS.  Atkins.     Jour.  Marine  Biol.  Assn.,  15,  191  (1928). 
9.  Harvey.     Jour.  Marine  Biol.  Assn.,  15,  183  (1928). 


THE  PRECIPITATION  OF  CALCIUM  AND  MAGNESIUM 
FROM    SEA   WATER    BY   SODIUM    HYDROXIDE. 

ELEANOR    M.    KAPP.1 

In  the  course  of  an  investigation  into  the  modification  of  sea 
water  for  use  as  a  perfusion  medium  (Kapp,  '28),  it  became 
necessary  to  know  something  of  the  relative  amounts  of  calcium 
and  magnesium  precipitated  by  sodium  hydroxide.  Haas  ('16) 
suggested  that  the  first  flat  portion  of  his  titration  curve  for  sea 
water  was  coincident  with  the  precipitation  of  Mg  as  hydroxide, 
the  second  with  that  of  Ca.  That  this  was  a  reasonable  assump- 
tion is  further  suggested  by  the  solubility  product  constants  for 
the  hydroxides  of  Mg  and  Ca,  which  are  1.2  X  io~n  and  4.1  X 
io~6,  respectively  (Johnston,  '15).  To  obtain  more  exact  in- 
formation concerning  this  behavior  of  Mg  and  Ca,  the  following 
experiments  were  run  on  sea  water  taken  from  the  English  Chan- 
nel outside  the  Plymouth  breakwater,  and  from  Great  Harbor, 
Woods  Hole,  Mass. 

Graded  amounts  of  10  normal  NaOH  (practically  carbonate- 
free  2)  were  added  to  100  c.c  portions  of  sea  water.  The  flasks 
were  stoppered  and  the  contents  thoroughly  mixed.  The 
supernatant  fluid  was  filtered  off  as  soon  as  the  precipitate  had 
settled  somewhat  (within  four  hours  in  all  cases),  and  Ca  and  Mg 
were  determined  in  separate  samples  of  the  filtrate.  Ca  was 
precipitated  as  oxalate  from  25  cc.  samples  according  to  McCrud- 
den's  ('09)  method,  and  allowed  to  stand  in  the  refrigerator  for 
at  least  18  hours.  The  oxalate,  after  washing,  was  determined 
with  permanganate.  The  Mg  determinations  were  carried  out 
according  to  the  method  of  Willstatter  and  Waldschmidt-Leitz 
('23)  on  duplicate  5  cc.  samples  from  each  filtrate.  Values  for 
total  Ca  and  Mg  were  obtained  by  the  same  techniques  from 
samples  of  untreated  sea  water,  and  show  good  agreement  with 
the  figures  compiled  by  Clarke  ('24)  for  sea  water  from  a  wide 
range  of  sources. 

1  From  the  Laboratory  of  the  Marine  Biological  Association,  Plymouth. 
-  Made  up  from  the  filtrate  of  a  50  per  cent,  solution  in  which  the  carbonate  had 
been  allowed  to  settle. 

453 


454 


ELEANOR    M.    KAPP. 


The  behavior  of  Mg  and  Ca  was  investigated  by  Irving1 
('26),  but  major  emphasis  was  placed  by  him  on  equilibria  within 
the  biological  limits  of  alkalinity.  An  extension  of  these  in- 
vestigations and  an  explanation  of  certain  discrepancies  which 
were  encountered  follow. 

The  data  for  Mg  for  Plymouth  sea  water  are  given  in  Fig.  i, 
and  roughly  agree  with  my  results  obtained  on  Woods  Hole  sea 
water  by  a  less  reliable  technique.  The  curve  for  the  precipita- 
tion of  Mg  as  drawn  by  Irving  is  inaccurate,  as  owing  to  the 
scarcity  of  his  points  he  completely  missed  the  plateau.  Fig.  I, 
however,  substantiates  the  points  he  did  determine. 


.050 


h 

0) 

•*»  .040 


0) 
Pi 


43 

«H 

P. 


030 


O 
g 

a.020 


.010 


,000 


Total  Magnesium 


o  -  determined  immediately 
•  -  at  equilibrium 
+  -  C08- saturated 


j_ 


.100  .200 

Mols  NaOH  added  per  liter 

FIG.   i.     The  precipitation  of  Mg  from  Plymouth  sea  water  in  relation  to  the 

amount  of  NaOH  added. 

1  Unknown  to  me  when  this  work  was  undertaken. 


PRECIPITATION    OF   CALCIUM    AM)    M.\(.NI-;sirM. 


455 


The  data  for  Ca  show  that  the  results  may  be  considerably 
modified  by  a  slight  variation  in  procedure.  The  Ca  curve  - 
plotted  as  hollow  circles  in  Figs.  2  and  3  both  differ  markedly 
from  the  one  obtained  by  Irving.  His  technique  was  substanti- 
ally the  same  as  mine,  with  the  exception  that  his  original  sample- 
of  sea  water,  after  the  NaOH  had  been  added,  were  shaken  for  24 
hours  instead  of  being  filtered  at  once,  so  that  equilibrium  was 
insured.  Since  CaCO;!  tends  to  remain  supersaturated,  it  was 
suspected  of  being  the  cause  of  the  discrepancy.  A  control 
experiment  was  therefore  set  up,  in  which  the  NaOH  was  added 
very  slowly  as  a  normal  (instead  of  10  normal)  solution,  in  order 
to  avoid  local  high  concentrations  of  hydroxide,  and  the  stoppered 
mixtures  were  allowed  to  stand  with  occasional  shaking  for  one 
week.  At  the  end  of  this  time  they  were  filtered  and  analyzed. 


$-. 

<D 


5.010  - 


-d 
a> 
•p 

05 
-P 

TH 

p< 

•H 
O 

I 


«J 
O 


.005  - 


determined  immediately 
at  equilibrium 
C0a- saturated 


.  100  . 200 

Mols  NaOH  added  per  liter 

FIG.   2.     The   precipitation   of   Ca   from   Plymouth  sea  water  in  relation   to  the 


amount  of  NaOH  added . 


30 


456 


KLKANOR    M.    KAPP. 


The  ('a  curve  thus  obtained  differs  from  the  first  ones,  this  time 
confirming  the  results  of  Irving.  Its  points  are  shown  in  Fig.  2 
as  black  circles.  The  difference  between  the  two  curves  is 
therefore  due  only  to  the  slowness  with  which  CaCOs  is  precipi- 
tated, and  can  be  controlled  by  taking  the  time  factor  into  account. 
The  same  situation  does  not  exist  in  the  case  of  Mg,  as  can  be 
seen  from  the  black  circles  plotted  in  Fig.  i,  which  coincide  with 
the  original  curve. 

The  effect  of  increasing  the  amount  of  carbonate  was  obtained 
by  saturating  several  samples  of  sea  water  with  CO2  before  the 
addition  of  the  alkali.  Increasing  quantities  of  normal  NaOH 
were  then  added  very  slowly,  to  allow  the  gelatinous  precipitate 
which  formed  to  redissolve,  until  the  third  sample,  to  which  11.5 
cc.  had  been  added,  remained  cloudy.  The  mixtures  were  aerated 
to  drive  off  excess  CO2,  and  allowed  to  stand  in  contact  with  the 
atmosphere  for  one  week.  During  this  time  a  crystalline  precipi- 


.010 


o 
O, 

•tf 

<u 
•p 

efl 

•p 

*H 
ft 
^ 

o 

8 

p. 

05 
O 

VI 


.005 


Total  Calcium 


•100  .200 

Mols  NaOH  added  per  liter 

FlG.  3.     The  precipitation  of  Ca  from  Woods  Hole  sea  water  in  relation  to  the 

amount  of  NaOH  added. 


PRECIPITATION    OF   CALCIUM   AND    MAGNESIUM. 


457 


tate  had  formed,  and  the  solutions  were  filtered  and  analyzed  as 
before.     The  results  are  shown  by  the  crosses  in  Figs.  I  and  2, 
and  are  strikingly  different  from  the  other  precipitations.     In. 
this  case  the  addition  of  a  small  amount  of  alkali  precipitates 
only  the  Ca,  while  the  Mg  is  affected  by  larger  amounts. 


PH 
12 


11 


10 


3 


.05      .10      .15 
Mols  NaOH  added  per  liter 

FIG.  4.     The  effect  of  NaOH  on  the  pH  of  sea  water  (after  Haas). 

With  reference  to  the  reason  for  the  shape  of  the  Haas  titration 
curve  (Fig.  4),  it  is  clear  that  Haas'  own  statement,  mentioned 
previously,  must  be  modified  somewhat.  As  he  suggested,  Mg 
is  precipitated  rapidly  by  NaOH  over  the  range  where  his  titra- 
tion curve  shows  a  plateau.  At  a  region  corresponding  to  the 
addition  of  o.i  mols  of  NaOH  per  liter  of  sea  water,  the  titration 
curve  begins  its  second  rise,  and  the  Mg  curve  flattens  out.  A 
small  amount  of  Ca,  however,  is  precipitated  throughout,  owing 
its  first  precipitation  to  the  insolubility  of  the  carbonate,1  which 
is  intermediate  in  this  respect  between  Mg  and  Ca  hydroxides. 

I  am  deeply  indebted  to  Dr.  E.  J.  Allen,  F.  R.  S.,  of  the  Marine 
Biological  Association,  Plymouth,  for  facilities  extended  to  me 

1  Ks.p.  =  .98  X  io-»  (Johnston,  '15). 


458  ELEANOR    M.    KAPP. 

during  this  investigation.     1   also  wish   to  thank  Prof.   M.   H. 
Jacobs  and  Mr.  H.  W.  Harvey  for  their  helpful  interest. 

REFERENCES. 
Clarke,  F.  W. 

'24     U.  S.  Geol.  Survey,  Bull.  770,  p.  127. 
Haas,  A.  R. 

'16     Jour.  Biol.  Chem.,  Vol.  26,  515. 
Irving,  L. 

'26     Jour.  Mar.  Biol.  Ass.,  Vol.  14,  441. 
Johnston,  J. 

'15     Jour.  Am.  Chem.  Soc.,  Vol.  37,  2001. 
Kapp,  E.  M. 

'28     Science,  Vol.  67,  513. 
McCrudden,  F.  H. 

'09     Jour.  Biol.  Chem.,  Vol.  7,  83. 
Willstatter,  R.,  and  Waldschmidt-Leitz,  E. 

'23     Ber.  v.  d.  deut.  chem.  Ges.,  Vol.  56,  488. 


FURTHER  OBSERVATIONS  ON   THE   EFFECT    OF 

HIGH    FREQUENCY  SOUND   WAVES  ON 

LIVING   MATTER. 

E.    NEWTON    HARVEY,    ETHEL    BROWNE    HARVEY   AN-D 
ALFRED    L.    LOOMIS.i 

» 

Interest  in  the  biological  effects  of  very  high  frequency  sound 
waves  started  with  the  investigations  of  Wood  and  Loomis  (i) 
who  devised  methods  for  producing  intense  "supersonic"  vibra- 
tions and  described  many  of  the  phenomena  connected  with  them. 
The  reader  is  referred  to  this  paper  for  a  description  of  the  two 
kilowatt  generator  and  methods  of  working  with  the  waves.  The 
apparatus  was  of  such  high  power  and  the  sound  waves  of  such 
great  intensity  as  to  produce  considerable  heating.  It  seemed 
highly  desirable  in  working  with  cells  to  reduce  the  heating 
effects  of  the  vibrations,  and  to  observe  the  cell  with  the  micro- 
scope while  being  radiated.  After  many  attempts  to  use  the 
high  power  oscillator  as  the  source  of  the  waves  and  to  lead  them 
to  the  material  on  the  stage  of  a  microscope  along  capillary  rods 
and  tubes,  a  low-powered  apparatus  was  decided  upon  as  the 
most  convenient  for  the  purpose.  This  has  previously  been 
described  by  Harvey  and  Loomis  (2)  together  with  some  of  the 
effects  of  these  supersonic  waves  on  living  organisms,  cells  and 
tissues.  The  outfit  consists  of  a  75  watt  high  frequency  oscillator 
and  a  quartz  crystal  whose  vibrations,  produced  in  the  electric 
field  by  reversal  of  the  piezo-electric  effect,  travel  through  any 
medium  in  contact  with  the  crystal.  A  frequency  of  400,000  per 
second  was  used  and  the  material  mounted  directly  on  the  crystal 
which  served  as  a  microscopic  slide.  Schmitt,  Olson  and 
Johnson  (3)  have  also  described  various  biological  effects  using  a 
250  watt  generator  with  crystal  immersed  in  xylene.  They  lead 
the  sound  waves  along  a  rod  of  small  diameter  ending  in  a  micro- 
needle,  which  could  be  inserted  into  the  material  to  be  studied. 

Some  additional  effects  have  been  recently  observed  with  our 

1  From   the   Marine   Biological  Laboratory,   Woods   Hole,   the   Physiological 
Laboratory,  Princeton  University,  and  the  Loomis  Laboratory,  Tuxedo  Park,  N.  Y. 

459 


460     E.  N.  HARVEY,  E.  B.  HARVEY  AND  A.  L.  LOOMIS. 

75  watt  outfit  in  its  original  form  and  also  modified  to  use  higher 
frequencies  by  changing  the  capacity,  inductance  and  crystal. 
The  new  quartz  crystal  was  a  spectacle  lens  which  happened  to  be 
cut  in  the  proper  direction,  kindly  loaned  by  Dr.  Kenneth  Cole. 
The  natural  frequency  of  this  crystal  was  approximately  one  and 
one  quarter  million  per  second.  Its  thickness  varied  from  I  to 
1.8  mm.  and  consequently  the  distance  between  the  tin  foil 
electrodes,  was  much  less  than  in  the  original  7  mm.  crystal,  giving 
a  far  more  intense  electrical  field  and  greater  effects.  A  few 
experiments  have  been  made  with  a  2.25  million  crystal  which 
vibrates  well  and  gives  the  same  effects  with  Elodea  as  the  1.25 
million.  A  6  million  crystal,  0.45  mm.  thick,  does  not  vibrate 
strongly.  We  are  at  present  engaged  in  increasing  the  frequency 
to  the  highest  point  possible  to  see  how  biological  effects  will  vary 
with  the  frequency. 

A  convenient  means  of  finding  the  resonant  frequency  of  the 
crystal  is  to  set  it  up  between  the  two  tin  foil  electrodes  with 
holes  in  their  centers  (to  allow  light  to  pass  for  microscopic  ob- 
servation) and  then  place  a  drop  of  water  on  the  crystal.  At 
various  settings  of  the  condenser  the  water  will  be  violently 
agitated  and  broken  up  into  fine  droplets  like  steam.  Low 
melting  point  crystals  placed  in  the  water  show  that  the  tempera- 
ture does  not  rise  but  that  the  "steam"  is  mechanically  formed, 
as  observed  in  various  ways  by  Wood  and  Loomis  (i),  and  not  a 
condensation  from  vapor.  The  exact  specifications  for  an  oscil- 
lator giving  various  frequencies  will  be  found  as  an  appendix  to 
this  paper. 

If  an  Elodea  leaf  covered  with  a  cover  slip  is  mounted  on  a 
crystal  whose  resonant  frequency  is  400  kilocycles,  and  relatively 
weak  (by  reducing  filament  current)  sound  waves  sent  through 
the  leaf,  it  can  be  observed  under  the  microscope  that  only  certain 
areas  in  the  leaf  show  the  characteristic  whirling  of  the  chloro- 
plasts  described  in  our  previous  paper  (2).  The  areas  do  not 
correspond  to  any  position  on  the  crystal  but  to  some  peculiarity 
in  the  leaf,  as  moving  a  leaf  to  a  new  position  over  the  crystal 
does  not  necessarily  change  the  areas  of  marked  whirling.  These 
areas  of  whirling  are  most  marked  where  air  bubbles,  which  vi- 
brate strongly,  are  caught  under  the  leaf  and  where  the  cells  are 
several  layers  in  thickness,  near  the  midrib  (which  also  contains 


EFFECT    OF   SOUND    WAVES    OX    LIVING    MATTER.  461 

air  in  intercellular  spaces).  Part  at  least  of  the  condition  for  rapid 
whirling  is  the  distance  of  the  leaf  from  the  crystal.  By  attaching 
the  coverslip  to  a  mechanical  device  for  adjusting  its  distance 
from  the  crystal,  the  amount  of  water  between  coverslip  and 
crystal  can  be  varied  and  a  slight  change  in  this  layer  of  water 
will  cause  whirling  in  a  given  area  to  start  or  to  stop.  These 
effects  are  no  doubt  due  to  interference  of  two  sets  of  sound  waves 
resulting  in  complicated  interference  patterns  with  nodes  and 
internodes.  Fine  particles  like  red  blood  corpuscles  suspended 
between  crystal  and  coverslip  can  be  observed  to  collect  in  nodes 
forming  such  a  pattern.  The  chloroplasts  in  Elodea  cells  cannot 
do  so  since  they  are  restricted  in  movement  by  the  cell  walls  but 
in  a  region  which  happens  to  be  an  internode,  they  will  undergo 
rapid  whirling  movements.  The  part  played  by  an  air  bubble  in 
causing  rapid  whirling  is  no  doubt  to  offer  a  reflecting  surface 
around  which  interference  pattern  and  nodes  appear.  The 
whirling  itself  is  probably  due  to  the  radiation  pressure  of  the 
sound  waves  as  they  pass  through  the  cells. 

Another  phenomenon  regularly  observed  is  a  variation  in  the 
rate  and  character  of  the  whirling  as  the  variable  condenser  is 
changed  to  vary  the  frequency.  For  instance,  over  a  range  of 
10  kilocycles,  there  appeared  maximum  whirling  in  a  given  area 
of  the  leaf  at  407,  409,  410.4,  412.5,  415,  and  417  kilocycles,  i.e. 
a  maximum  approximately  every  2  kilocycles,  with  no  whirling  or 
very  slow  whirling  between. 

In  order  to  understand  the  changes  in  whirling  motion  imparted 
to  the  biological  material  placed  upon  the  quartz  as  the  frequency 
is  varied,  it  is  necessary  to  digress  a  moment  and  consider  the 
forces  acting  upon  an  oscillating  quartz  disk.  As  is  well  known, 
a  natural  quartz  crystal  has  three  electric  axes  perpendicular  to 
the  optic  axis.  (See  Fig.  i.) 

The  disk  is  cut  as  indicated  by  the  shaded  portion,  i.e.  so  that 
one  of  the  electric  axes  shall  be  perpendicular  to  the  plane  of  the 
disk.  If  pressure  is  applied  to  the  side  of  the  disk  corresponding 
to  A  -  a  negative  charge  will  accumulate  there,  while  corre- 
spondingly if  a  negative  charge  is  applied  there  without  pressure 
the  disk  will  contract  as  if  the  equivalent  pressure  had  been  ap- 
plied. The  same  holds  true  with  positive  charges  on  the  A  +  sidr. 
On  the  other  hand,  when  a  positive  charge  is  placed  on  the  nega- 


462  I   .    N-    HARVEY,    E.    B.    HARVEY   AND   A.    L.    LOOMIS. 

tivc  side  and  a  negative  charge  on  the  positive  side,  the  crystal 
will  expand.  A  rapid  alternation  of  charges  causes  the  crystal 
to  oscillate  and  as  a  first  approximation  the  crystal  can  be  con- 
sidered to  be  an  oscillating  rigid  piston.  This  would  be  rigorously 
correct  if  the  disk  were  perfect  and  infinitely  large  but  with  a 
finite  disk  the  forces  are  not  symmetrical  near  the  edges  and  a 
complex  wave  pattern  is  formed  in  the  crystal.  This  can  easily 
be  seen  by  first  considering  a  point  0  on  the  surface  of  the  crystal 
near  the  center  (Fig.  2).  If  a  unit  negative  charge  is  placed  on 
the  under  surface  with  the  corresponding  positive  charge  on  the 


A- 


FIG.   i.     Quartz  crystal   (shaded)    cut   perpendicular  to  optic  axis.     Electric 
axes  indicated  by  A  A,  BB,  CC. 

FIG.  2.     Vectors  showing  forces  in  point  O  in  crystal. 

upper,  the  crystal  at  "0"  will  tend  to  contract  along  the  axis 
OA  +  and  expand  along  the  axes  OB  —  and  OC— .  The  intensities 
of  these  forces  are  directly  proportional  to  the  potential  gradiants 
along  the  respective  axis.  The  forces  along  OB—  and  OC--  are 
therefore  only  half  as  great  as  along  OA  -  since  the  distances 
through  the  crystal  along  these  axis  are  twice  as  great  as  along 
the  axis  OA-  (the  angles  between  the  axis  being  60°).  The 
vector  resolution  of  the  forces  OC—  and  OB—  along  OA  —  shows 
that  they  are  equivalent  to  a  force  opposed  to  the  force  along 
OA  -  •  and  of  magnitude  equal  to  one  half  that  of  OA  — .  The 
vector  equivalent  of  all  three  forces  is  therefore  a  single  force  along 
OA  —  equal  to  half  of  what  that  force  would  be  if  the  forces  along 
the  axes  OC-  -  and  OB—  were  not  present. 

This  symmetry  does  not  maintain  however  near  the  edges  of 
the  disk.     Consider  the  point  Q,  Fig.  3.     The  axis  QB  is  not  in 


EFFECT    OF   SOUND   WAVES    ON    LIVING    MATTER.  463 

the  crystal  at  all.  The  resolution  of  the  forces  along  QA  --  and 
QC-  -  gives  ajbrce  along  QX  equal  to  the  force  along  QA  +  multi- 
plied by  IA/S.  It  is  clear,  therefore,  that  the  forces  near  the 
edges  are  not  symmetrical  and  tend  to  produce  distortions  which 
travel  in  waves  across  the  disk. 

A  second  system  of  forces  are  also  acting  on  the  disk.  As  the 
quartz  contracts  normally  to  the  surface  it  expands  parallel  to  the 
surface  (this  effect  is  best  seen  in  a  rectangular  plate).  Thus  the 
series  of  longitudinal  waves  create  interference  patterns  with  the 
traverse  waves. 


A- 

FIG.  3.     Vectors  showing  forces  in  point  Q  in  crystal. 

Thirdly,  it  has  been  shown  that  even  with  a  perfect  quartz 
crystal  the  intensity  of  the  piezo  electric  effect  varies  in  different 
parts  of  the  crystal.  Dye  has  photographed  the  distortions 
produced  in  the  interference  fringes  of  an  interferometer  when 
one  of  the  plates  is  an  oscillating  quartz  disk.  These  photo- 
graphs show  most  beautiful  and  complex  patterns  which  showly 
drift  across  the  plate  when  the  frequency  is  slowly  changed. 

Fortunately  in  biological  investigations  under  the  microscope 
good  use  can  be  made  of  these  complex  patterns.  Thus,  without 
changing  the  position  of  the  specimen  on  the  crystal,  one  can  by 
merely  changing  slightly  the  frequency,  cause  these  patterns  to 
shift  so  that  any  particular  part  of  the  specimen  can  be  made  to 
experience  forces  of  varying  magnitude  and  direction.  Thus  in  a 
particular  cell  of  Elodea  the  chloroplasts  can,  at  will,  be  made  to 
rotate  slowly  or  rapidly,  clockwise,  or  counter  clockwise,  in  one 
vortex  or  in  a  series  of  vortices,  while  merely  watching  the 
specimens  under  the  microscope  and  observing  the  effects  pro- 
duced as  the  frequency  is  slowly  varied. 

By  increasing  the  intensity,  the  leaf  of  Elodea  can  be  agitated 
so  violently  that  the  chloroplasts  themselves  are  broken  up  into  a 


464  E.    X.    HARVEY,    E.    B.    HARVEY   AND   A.    L.    LOOMIS. 

fine  green  emulsion  which  completely  fills  the  cell.  This  effect  is 
not  due  to  heating,  since  crystals  of  ethyl  stearate,  melting  at 
30—31°  C.  and  placed  on  the  Elodea  leaf,  are  not  melted  even  after 
1 5  minutes,  nor  does  slowly  heating  Elodea  leaves  bring  about  this 
effect.  Neither  is  it  due  to  possible  mechanical  rupture  of  the 
cellulose  wall  or  mixing  of  the  vacular  sap  with  the  chloroplasts, 
since  unrayed  cells  can  be  crushed  with  a  needle  and  their  chloro- 
plasts do  not  break  up  in  this  characteristic  manner.  The  emulsi- 
fication  is  caused  by  the  tearing  action  of  the  sound  waves. 

Perhaps  it  should  be  emphasized  at  this  point  that  these  effects 
are  all  due  to  high  frequency  sound  waves  and  not  to  any  influence 
of  the  oscillating  electrical  field,  as  control  experiments  using 
glass  plates  of  a  size  similar  to  and  replacing  that  of  the  quartz 
crystal  have  shown. 

Some  of  the  more  interesting  of  the  effects  observed  with  the 
spectacle  lens  crystal  and  frequencies  of  1,250  kilocycles  are  as 
follows : 

AmcebcB  proteus  or  dubia,1  moving  along  the  surface  of  the 
crystal  are  not  particularly  affected  by  an  intensity  that  causes  the 
inclusions  in  small  vacuoles  of  the  Amoeba  to  rotate  on  their  axes. 
Higher  intensities  cause  a  mild  whirling  of  the  more  liquid  regions 
of  the  Amoeba  followed  by  rupture  of  the  pellicle  on  one  side  and 
extrusion  of  the  contents  which  join  the  general  whirl  of  fluid  in 
the  medium.  There  is  a  tendency  for  the  Amceba  to  move  more 
rapidly  during  the  raying  as  if  the  endoplasm  became  more 
liquid.  After  this  there  is  a  sudden  change  in  direction  of  move- 
ment. 

Both  unfertilized  and  fertilized  sea  urchin  (Arbacia)  and  star- 
fish egg  are  violently  agitated  and  may  spin  around.  The  jelly 
is  torn  off  and  the  fertilization  membrane  may  be  broken.  The 
eggs  are  thrown  into  rows  or  clumps  and  eventually  cytolyze 
either  partially  or  completely,  the  cytolysis  taking  place  on  one 
side  and  sometimes  within  the  fertilization  membrane.  There  is 
no  movement  of  materials  inside  the  egg  caused  by  raying,  as  can 
be  determined  with  certainty  by  using  centrifuged  eggs,  the 
stratified  layers  remaining  intact  until  cytolysis  takes  place. 
Cytolysis  may  take  place  from  any  of  the  stratified  layers.  How- 
ever, if  the  unfertilized  centrigufed  eggs  are  placed  in  diluted  sea 
water  (40  distilled  water  to  60  sea  water)  and  thus  made  less 


!.!•  KELT    OF    SOUND   WAVES    ON    LI VI. Ml    MATTER.  465 

viscous,  the  inside  may  be  made  to  whirl.  The  whirling  takes 
place  in  the  lighter  layers,  including  the  oil,  clear  and  granular 
layers,  but  only  along  the  edge  of  the  pigment  layer,  most  of 
which  remains  intact.  The  oil  drops  tend  to  remain  together, 
but  the  clear  and  granular  layers  become  mixed  and  after  ten  or 
fifteen  minutes  of  whirling  a  clear  zone  can  not  be  distinguished. 
The  direction  of  rotation  may  be  reversed  instantly  by  a  slight 
change  in  frequency.  Sometimes  instead  of  a  whirling  of  the 
protoplasm,  there  is  a  streaming  of  granules  similar  to  that  of  an 
Amoeba.  No  whirling  of  protoplasm  nor  movement  of  granules 
has  been  observed  in  eggs  put  in  dilute  sea  water  and  not  centri- 
fuged.  This  may  be  due  to  the  fact  that  the  less  dense  material, 
where  the  whirling  takes  place  is  not  separated  out  from  the 
heavier  pigment  granules. 

The  asters  are  quite  unaffected  by  raying.  Cleavage  furrows 
will  come  in  normally  during  raying,  even  when  the  egg  is  vio- 
lently agitated.  When  an  egg  has  been  slightly  cytolyzed  by 
raying  we  have  observed  that  the  furrow  may  come  in  at  the 
proper  place.  Eggs  in  the  two  or  four  cell  stage  may  have  one  or 
two  blastomeres  cytolyzed  and  the  others  unaffected. 

Arbacia  plutei  swimming  slowly  are  paralyzed  by  a  momentary 
raying,  presumably  because  the  cilia  are  torn  off.  Otherwise  they 
look  uninjured  but  more  prolonged  treatment  or  greater  intensity 
will  tear  them  to  pieces,  leaving  only  fragments  of  the  skeleton 
behind. 

The  gill  cilia  of  Mytilius  do  not  seem  to  be  affected  by  violent 
agitation  of  the  sea  water  about  them,  until  the  cilia  and  gill 
filaments  are  actually  torn  to  pieces. 

Pigment  cells  well-expanded  in  the  scales  of  Fundulus  are  not 
affected,  although  the  scales  are  rapidly  agitated  as  the  waves 
impinge  upon  them. 

Frog  abdominal  muscles  mounted  on  the  crystal  show  no  con- 
traction or  movement  although  air  bubbles  and  blood  corpuscles 
on  top  of  the  muscle  tissue  whirl  rapidly.  The  waves  must  have 
passed  through  the  muscle  tissue  to  reach  the  air  bubbles  and 
corpuscles. 

Fragments  of  the  rays  of  the  ctenophore,  Mnemiopsis,  con- 
taining luminous  material,  mounted  on  the  crystal  in  the  dark 
and  waves  passed  through,  are  agitated  and  occasionally 


466  I'..    N.    HARYKY,    K.    B.    HARVEY   AND   A.    L.    LOOMIS. 

luminesce.  There  is  no  continual  luminescence  which  can  be 
attributed  to  the  waves  but  only  the  sporadic  luminescence  con- 
nected with  sudden  movement  of  the  fragment  such  as  can  be 
( ibtuined  on  jarring  the  table  containing  fragments  of  Mnemiopsis, 
even  when  not  exposed  to  high  frequency  sound  waves. 

Fundulus  embryos  within  the  egg,  with  beating  hearts,  sub- 
jected to  waves  of  an  intensity  to  agitate  the  eggs  but  not  so 
great  an  intensity  as  to  interfere  with  observation  of  the  heart 
beat  show  no  marked  effect  upon  the  character  of  the  beat  or 
circulation.  In  fact  only  the  effect  observed  was  a  slight  increase 
in  rate  during  raying  which  can  be  accounted  for  by  a  slight  in- 
crease in  temperature,  that  undoubtedly  occurs  when  these  high 
frequency  waves  carrying  considerable  energy,  are  absorbed  by 
the  medium.  The  embryos  were  rayed  I  minute  and  then  not 
rayed  for  one  minute  while  the  heart  beats  were  counted.  In 
four  experiments  the  rates  were:  Rayed — 148,  157,  140,  132; 
unrayed  --140,  148,  122,  122,  respectively.  The  average  in- 
crease in  rate  was  about  8  per  cent.,  which  can  be  accounted  for 
from  the  known  effect  of  temperature  on  the  heart  beat  of 
Fundulus  heteroditus ,2  by  a  rise  of  temperature  from  22°  C.  to 
about  23°  C. 

Perhaps  it  should  be  emphasized  again  from  the  experiments  on 
muscle,  heart,  luminous  cells  and  chromatophores  that  there  is  no 
stimulating  effect  of  these  waves  similar  to  the  stimulation  by 
electrical  or  sudden  mechanical  disturbance. 

Fertilized  Fundulus  eggs  mounted  on  the  crystal  can  be  very 
violently  agitated  and  the  oil  drops  and  granules  within  made  to 
dance.  The  yolk  can  be  thoroughly  stirred  and  the  surface  of  the 
protoplasm  can  be  observed  to  move  and  bend.  Dr.  Elmer 
Butler  has  carried  these  eggs  to  the  point  of  hatching  and  finds 
the  development  and  the  embryos  normal.  If  the  agitation  has 
continued  so  long  as  to  burst  the  protoplasmic  surface  develop- 
ment does  not  proceed.  An  intensity  of  raying  which  does  not 
destroy  the  surface  has  no  effect  on  development  while  a  slightly 
greater  intensity  results  in  dissolution  and  cytolysis. 

Study  of  a  large  number  of  cells  and  tissues,  some  of  which  are 
recorded  above,  has  led  us  to  the  conclusion  that  the  effects  of 
these  waves,  apart  from  slight  heating,  are  purely  mechanical. 
If  intense  enough,  practically  all  cells  can  be  cytolyzed.  It  is  as 


KFFECT    OF    SOUND    \\AYHS    ON    I.I\I\(-    MATTER.  467 

if  one  could  grasp  a  cell  in  both  hands  and  bend  it  violently  back 
and  forth  at  a  very  rapid  rate.  Delicate  structures  on  the  out- 
side of  a  cell  are  torn  off.  If  the  cell  is  very  small  it  is  thrown  into 
nodes  so  quickly  as  to  escape  injury.  If  the  cell  can  be  held  fixed 
and  is  not  too  viscous,  its  contents  can  often  be  made  to  whirl 
before  it  breaks  down. 

From  the  whirling  one  can  gain  an  idea  of  the  viscosity  of  the 
cell  contents.  Perhaps  the  chief  value  of  the  waves  for  biological 
investigation  lies  in  the  evidence  obtained  from  their  action 
regarding  the  viscosity  of  cells.  It  should  be  emphasized,  how- 
ever, that  comparative  studies  of  viscosity  are  difficult  because  of 
the  great  complexity  of  the  sound  wave  patterns  under  the  cover 
slip,  both  horizontally  and  vertically.  Two  cells  in  different 
portions  of  the  same  microscopic  field  are  not  necessarily  exposed 
to  the  same  radiational  forces  and  great  caution  must  be  used  in 
drawing  conclusions  regarding  viscosity  or  resistance  to  tearing 
by  difference  in  behavior  of  cells. 

High  frequency  sound  waves  offer  a  new  means  of  affecting 
the  interior  of  cells  without  necessarily  breaking  down  the -cell 
wall.  They  will  be  of  most  value  when  a  beam  of  given  fre- 
quency and  controlled  intensity  can  be  sent  through  a  cell  or 
tissue  in  a  particular  direction. 

APPENDIX. 

For  those  biologists  who  desire  to  construct  a  low-powered 
oscillator,  the  following  constructional  details  ought  to  suffice. 

The  following  apparatus  is  recommended. 
One  No.  852  Radiotron  75  watt  tube, 
One  tube  holder 

One  filament  transformer  to  give  10  volts 
One  plate  transformer  to  give  2,000  volts 
One  5,000  ohm  resistance 

Several   transmitting  condensers   (designed  to  withstand   5,000 
volts)  with  an  aggregate  capacity  of  about  o.i  microfarad 
One  rheostat 

Some  heavy  copper  strip  to  wind  the  inductance 
Some  fine  wire  to  make  the  secondary 

All  of  the  above  can  be  bought  from  any  radio  store  carrying 


468     E.  X.  HARVEY,  E.  B.  HARVEY  AND  A.  L.  LOOMIS. 

parts  for  transmitting  sets,  and  should  not  cost  more  than  $100 
in  the  aggregate. 

Fig.  4  shows  the  wiring  diagram  and  a  suggested  arrangement 
of  the  parts.  The  iron  of  the  transformers  should  be  on  the 
side  of  the  tube  away  from  the  oscillating  parts  and  should  be  at 
least  a  foot  from  the  tube.  All  the  parts  can  conveniently  be 
mounted  on  a  board  30  x  10  inches. 


IIOA.C. 


FIG.  4.  Constructional  diagram  for  a  75  watt  oscillator.  A,  plate  transformer; 
B,  filament  transformer;  C,  rheostat;  D,  choke  coil;  E,  Radiotron  No.  852; 
F,  Blocking  condenser.  G,  Grid  leak  condenser.  H,  Grid  lead.  I,  Inductance  coil; 
J,  tuning  condenser;  K,  secondary  coil;  L,  Variable  condenser;  Q,  Quartz  plate 
between  electrodes. 

The  rheostat  should  be  mounted  in  the  lead  from  the  no-volt 
A.  C.  house  circuit  and  can  be  used  to  regulate  the  voltage.  The 
primaries  of  the  transformers  should  be  connected  in  parallel 
across  the  house  circuit.  One  side  of  the  secondary  of  the  plate 
transformer  should  be  connected  to  the  center  tap  of  the  filament 
transformer  which  point  should  also  be  grounded.  The  other 
side  of  the  secondary  should  go  through  a  choke  coil  to  the  plate. 
The  choke  coil  can  be  made  by  winding  about  100  turns  of  fine 
wire  on  a  bakelite  tube  one  or  two  inches  in  diameter. 

The  inductance  can  be  made  by  winding  fifteen  or  twenty  turns 
of  heavy  copper  wire  on  a  bakelite  tube  six  or  eight  inches  in 
diameter.  The  plate  should  be  connected  to  one  tap  on  the 
inductance  through  a  blocking  condenser  of  about  .002  microfarad 
capacity.  The  grid  should  be  connected  to  the  other  tap  on  the 
inductance  through  a  by-pass  condenser  of  about  the  same 
capacity  and  a  grid  leak  of  above  5,000  ohms  resistance.  The 
center  tap  of  the  inductance  should  be  grounded.  The  secondary 
can  be  made  by  winding  100  turns  of  fine  wire  on  a  bakelite  tube 


EFFECT    OF    SOUND    \\AVKS    ON    I.lVI.Mi    MATTER.  469 

which  can  be  slipped  inside  the  primary  inductance.  One  end  of 
the  secondary  should  go  to  one  plate  of  the  crystal  holder  (the 
other  plate  of  the  holder  being  grounded).  The  other  end  of  the 
secondary  should  be  connected  to  ground  through  a  variable 
condenser  or  to  a  rod  of  metal  perhaps  i  inch  diameter  and  ten 
inches  long,  which  is  not  grounded. 

The  quartz  crystal  need  not  be  larger  than  one  square  inch. 
It  should  be  cut  perpendicular  to  an  electric  axis.  Its  natural 
frequency  of  oscillation  will  depend  on  its  thickness. 

Mm.  Thick.  Frequency  (Approx.). 

i ....  2,900,000  cycles  per  sec. 

2.  .  1,450,000 

3 966,000 

4-  725,000 

5 580,000 

etc etc. 

The  oscillating  circuit  should  be  tuned  to  approximately  the 
frequency  of  the  crystal. 

The  crystal  holder  can  conveniently  be  made  out  of  two  micro- 
scope slides  and  two  thin  brass  strips  with  holes  cut  in  them  for 
use  with  the  microscope.  The  microscope  should  be  at  least  three 
feet  from  the  oscillator  so  that  movements  of  the  operators  body 
shall  not  change  the  frequency.  The  high  tension  lead  to  the 
microscope  should  be  shielded  by  surrounding  it  with  a  grounded 
metal  tube  and  the  microscope  itself  should  be  grounded  to  pre- 
vent small  spark  discharges  to  the  observer. 

REFERENCES. 

1.  Wood,  R.,  and  Loomis,  A.  L. 

'27     Phil.  Mag.,  4,  417. 

2.  Harvey,  E.  N.,  and  Loomis,  A.  L. 
'28     Nature,  121,  622. 

3.  Schmitt,  F.  O.,  Olson,  A.  R.,  and  Johnson,  C.  H. 

'28     Proc.  Soc.  Exp.  Biol.  Med.,  25,  718. 

1  Kindly  supplied  by  Dr.  J.  A.  Dawson  of  Harvard  University. 

2  Unpublished  data  of  Dr.  Otto  Glaser  of  Amherst  College. 


BIOLOGICAL    BULLETIN 


OF  THE 


flDarine  Biological  Xaboratorj) 


WOODS  HOLE,   MASS. 


VOL.  LV 


JULY,   1928 


No.   i 


CONTENTS 

Thirtieth  Annual  Report  of  the  Marine  Biological  Laboratory 


PUBLISHED  MONTHLY  BY  THE 

MARINE  BIOLOGICAL  LABORATORY 

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EOttortal  Statf 

GARY  N.  CALKINS  —  Columbia  University. 

E.  G.  CONKLIN  —  Princeton  University. 

M.  H.  JACOBS  —  University  of  Pennsylvania. 

FRANK  R.  LILLIE  —  University  of  Chicago. 

GEORGE  T.  MOORE  —  The  Missouri  Botanic  Garden. 

T.  H.  MORGAN  —  Columbia  University. 

W.  M.  WHEELER  —  Harvard  University. 

E.  B.  WILSON  —  Columbia  University. 


Bbitor 

C    R.  MOORE  —  The  University  of  Chicago. 


All  communications  and  manuscripts  should  be  sent  to  the  Man- 
aging Editor,  the  University  of  Chicago.  Subscriptions  and  other 
matter  should  be  addressed  to  the  Biological  Bulletin,  Prince  and 
Lemon  Streets,  Lancaster,  Pa. 


Notice  to  contributors.  Every  paper  to  appear  in  the  Biological 
Bulletin  should  be  accompanied  by  an  author's  abstract  presenting 
the  chief  results  of  the  investigation.  The  abstract  should  not  exceed 
225  words  in  length. 

For  indexing  purposes  there  should  be,  in  addition  to  the  title, 
one  or  more  subject  headings  indicating  in  a  word  or  two  the  divi- 
sions of  the  subject  discussed  in  the  paper.  The  entire  name  of  the 
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desired. 


BIOLOGICAL    BULLETIN 


OF  THE 


fiDarine  Biological  laboratory 


VOL.  LV 


WOODS  HOLE,  MASS. 


AUGUST,  1928 


No.  2 


KING,  ROBERT  L. 


MAIN,   ROLLAND  J. 


AMBERSON,  WILLIAM  R. 


BOYD,   MARJORIE 


CALKINS,  GARY  N.,  AND 
BOWLING,  RACHEL 

PARPART,  ARTHUR  K. 


HUESTIS,  R.  R. 


MELVIN,   ROY 


CONTENTS 

The  Contract-He  Vacuole  in  Paramecium 
trichium  .......................... 

Observations  of  the  Feeding  Mechanism  of 
a  Ctenophore,  Mnemiopsis  leidyi  ......  ''',' 

The  Influence  of  Oxygen  Tension  upon  the 
Respiration  of  Unicellular  Organisms  .  .  ~n 

A  Comparison  of  the  Oxygen  Consumption 
of  Unfertilized  and  Fertilized  liggs  of 
Fund  nl  us  heteroclitus  ...............  (>- 

Sti/dics  on  Dallasia  frontata  Stokes  .......     i«'i 

The  Bacteriological  Sterilization  of  Parame- 


i i  .1 


The  Effect  of  Maternal  Age  and  of  Temper- 
ature Change  in  Secondary  Xou-Disj  unc- 
tion .............................  TJI 

O\v«en  Consumption  of  Insect  I''.ggs  ......    i;-,5 


PUBLISHED  MONTHLY  BY  THE 

MARINE  BIOLOGICAL  LABORATORY 

PRINTED    AND    ISSUED    BY 

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AGENT  FOR  GREAT  BRITAIN 

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Entered  October  10,1902.  at  Lancaster,  Pa.,  as  second-class  matter  under  Act  of  Congress  of  July  16,  1894 


Eotforial  Staff 

GARY  N.  CALKINS  —  Columbia  University. 

E.  G.  CONKLIN  —  Princeton  University. 
M.  H.  JACOBS  —  University  of  Pennsylvania. 

FRANK  R.  LILLIE  —  University  of  Chicago. 

GEORGE  T.  MOORE  —  The  Missouri  Botanic  Garden. 

T.  H.  MORGAN  —  Columbia  University. 

W.  M.  WHEELER  —  Harvard  University. 

E.  B.  WILSON  —  Columbia  University. 


lEMtor 

C.  R.  MOORE  —  The  University  of  Chicago. 


All  communications  and  manuscripts  should  be  sent  to  the  Man- 
aging Editor,  the  University  of  Chicago.  Subscriptions  and  other 
matter  should  be  addressed  to  the  Biological  Bulletin,  Prince  and 
Lemon  Streets,  Lancaster,  Pa. 


Notice  to  contributors.  Every  paper  to  appear  in  the  Biological 
Bulletin  should  be  accompanied  by  an  author's  abstract  presenting 
the  chief  results  of  the  investigation.  The  abstract  should  not  exceed 
225  words  in  length. 

For  indexing  purposes  there  should  be,  in  addition  to  the  title, 
one  or  more  subject  headings  indicating  in  a  word  or  two  the  divi- 
sions of  the  subject  discussed  in  the  paper.  The  entire  name  of  the 
author  (of  each  author  if  a  joint  paper)  and  the  year  of  birth  is  also 
desired. 


BIOLOGICAL    BULLETIN 

OF  THE 

flDarine  Biological  laboratory 


WOODS  HOLE,   MASS. 


VOL.  LV  SEPTEMBER,  1928  No.  3 

CONTENTS 

HILL,  SAMUEL  K.  The  Influence  of  Molds  on  the  Growth  of  Lumi- 

nous Bacteria  in  Relation  to  the  Hydrogen 
Ion  Concentration,  Together  with  the  Devel- 
opment of  a  Satisfactory  Culture  Method. .  143 

KAROL,  JOHN  J.  The  Sex  Ratio  in  Peromyscus 151 

PAYNE,  NELLIE  M.  Cold  Hardiness  in  the  Japanese  Beetle,  Po- 

pillia  japonica  Newman 163 

NELSON,  THURLOW   C.     Pelagic  Dissoconchs  of  the  Common  Mussel, 

Mytilus  edulis,  with  Observations  on  the 
Behavior  of  the  Larva  of  Allied  Genera  ....  1 80 

TURNER,   C.   L.  Studies  on  the  Secondary  Sexual  Characters  of 

Crayfishes. —  VI.  A  Female  of  Cambarus 
immunis  with  Oviducts  Attached  to  Openings 
of  Sperm  Ducts 193 

TURNER,   C.   L.  Studies  on   the  Secondary  Sexual  Characters 

of  Crayfishes. —  VII.  Regeneration  of  Aber- 
rant Secondary  Sexual  Characters 197 

SAYLES,   LEONARD   P.         Regeneration  of  Lumbricul 'us  in  Various  Ringer 

Fluids 202 

ALPATOV,  W.  W.  Variation  of  Hooks  on  the  Hind  Wing  of  the 

Honey  Bee  (Apis  meUifera  L.} 209 

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MARINE  BIOLOGICAL  LABORATORY 

PRINTED    AND    ISSUED    BY 

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Entered  October  10.1902,  at  Lancaster,  Pa.,  as  second-class  matter  under  Act  of  Congress  of  July  16,  1894 


jEDttorial  Statf 

GARY  N.  CALKINS — Columbia  University. 

E.  G.  CONKLIN — Princeton  University. 

M.  H.  JACOBS — University  of  Pennsylvania. 

FRANK  R.  LILLIE — University  of  Chicago. 

GEORGE  T.  MOORE — The  Missouri  Botanic  Garden. 

T.  H.  MORGAN — Columbia  University. 

W.  M.  WHEELER — Harvard  University. 

E.  B.  WILSON — Columbia  University. 


C.  R.  MOORE — The  University  of  Chicago. 


All  communications  and  manuscripts  should  be  sent  to  the  Man- 
aging Editor,  the  University  of  Chicago.  Subscriptions  and  other 
matter  should  be  addressed  to  the  Biological  Bulletin,  Prince  and 
Lemon  Streets,  Lancaster,  Pa. 


Notice  to  contributors.  Every  paper  to  appear  in  the  Biological 
Bulletin  should  be  accompanied  by  an  author's  abstract  presenting 
the  chief  results  of  the  investigation.  The  abstract  should  not  exceed 
225  words  in  length. 

For  indexing  purposes  there  should  be,  in  addition  to  the  title, 
one  or  more  subject  headings  indicating  in  a  word  or  two  the  divi- 
sions of  the  subject  discussed  in  the  paper.  The  entire  name  of  the 
author  (of  each  author  if  a  joint  paper)  and  the  year  of  birth  is  also 
desired. 


BIOLOGICAL    BULLETIN 

OF  THE 

flDarine  Biological  laboratory 


WOODS  HOLE,  MASS. 


VOL.  LV  OCTOBER,  1928  No.  4 


CONTENTS 

HARMAN,  MARYT.,  AND 

ROOT,  FRANK  P.  The  Development  of  the  Spermatozoon  in  Cavia 

cobaya 235 

TURNER,  C.  L.  Studies  on  the  Secondary  Sex  Characters  of 

Cray  fishes,  VIII.  Modified  Third  Abdom- 
inal Appendages  in  Males  of  Cambarus 
virilis 255 

GRAVE,  B.  H.  Natural  History  of  Shipu'orm,  Teredo  n aval-is, 

at  Woods  Hole,  Massachusetts 260 

NEWMAN,  H.  H.  Studies  of  Human  Twins,  I.     Methods  of  Di- 

agnosing Monozygotic  and  Dizygotic  Tuins  283 

NEWMAN,  H.  H.  Studies  of  Human   Twins,   II.     Asymmetry 

Reversal,  of  Mirror  Imaging  in  Identical 
Tirins 298 


PUBLISHED  MONTHLY  BY  THE 

MARINE  BIOLOGICAL  LABORATORY 

PRINTED    AND    ISSUED    BY 

LANCASTER  PRESS,  INC. 
LANCASTER,  PA. 

AGENT  FOR  GREAT  BRITAIN 

WHELDON  &  WESLEY,  LIMITED 

2,  3  and  4  Arthur  Street,  New  Oxford  Street,  London,  W.  C.  2 

Single    Numbers,  $1.00.     Per  Volume    (6  numbers),  S4.50 
Entered  October  ro,  1902,  at  Lancaster,  Pa.,  as  second-class  matter  under  Act  of  Congress  of  July  16,  1804 


Staff 

GARY  N.  CALKINS  —  Columbia  University. 

E.  G.  CONKLIN  —  Princeton  University. 

M.  H.  JACOBS  —  University  of  Pennsylvania. 

FRANK  R.  LILLIE  —  University  of  Chicago. 

GEORGE  T.  MOORE  —  The~Missouri  Botanic  Garden. 

T.  H.  MORGAN  —  Columbia  University. 

W.  M.  WHEELER  —  Harvard  University. 

E.  B.  WILSON  —  Columbia  University. 


BDitor 

C.   R.  MOORE  —  The  University  of  Chicago. 


All  communications  and  manuscripts  should  be  sent  to  the  Man- 
aging Editor,  the  University  of  Chicago.  Subscriptions  and  other 
matter  should  be  addressed  to  the  Biological  Bulletin,  Prince  and 
Lemon  Streets,  Lancaster,  Pa. 


Notice  to  contributors.  Every  paper  to  appear  in  the  Biological 
Bulletin  should  be  accompanied  by  an  author's  abstract  presenting 
the  chief  results  of  the  investigation.  The  abstract  should  not  exceed 
225  words  in  length. 

For  indexing  purposes  there  should  be,  in  addition  to  the  title, 
one  or  more  subject  headings  indicating  in  a  word  or  two  the  divi- 
sions of  the  subject  discussed  in  the  paper.  The  entire  name  of  the 
author  (of  each  author  if  a  joint  paper)  and  the  year  of  birth  is  also 
desired. 


BIOLOGICAL    BULLETIN 

OF  THE 

flDarine  Biological  laboratory 


WOODS   HOLE,   MASS. 


VOL.  LV 


NOVEMBER,   1928 


No.  5 


CONTENTS 

HUMPHREY,  R.  R.  Sex  Differentiation  in  Gonads  Developed  from 

Transplants  of  the  Intermediate  Mesoderm 
of  Amblystoma 317 

MOORE,  CARL  R.  On  the  Properties  of  the  Gonads  as  Controllers 

of  Somatic  and  Psychical  Characteristics,  XI.  3  39 

\ 

JUST,  E.  E.  Initiation   of  Development    in    Arbacia,    VI. 

The  Effect  of  Slowly  Evaporating  Sea-  Water 
and  its  Significance  for  the  Theory  of  Auto- 
Parthenogenesis  358 

CHAMBERS,  ROBERT.          Intracellular  Hydrion  Concentration  Studies, 

I.  The  Relation  of  the  Environment  to  the 
pH  of  Protoplasm  and  of  Its  Inclusion  Bodies.  369 

REZNIKOFF,  PAUL,  AND 

POLLACK,  HERBERT.      Intracellular  Hydrion   Concentration  Studies, 

II.  The  Effect  of  Injection  of  Acids  and 
Salts  on  the   Cytoplasmic   pH   of   Amoeba 
dubia 377 

POLLACK,  HERBERT.  Intracellular  Hydrion  Concentration  Studies, 

III.  The  Buffer  Action  of  the  Cytoplasm 
of  Amceba  dubia  and  Its  Use  in  Measuring 
thepH 383 

GREGORY,  LOUISE  H.          Th?  Effects  of  Changes  in   Medium  during 

Different  Periods  in  the  Life  History  of 
Uroleptus  mobilis  and  Other  Protozoa 386 

PUBLISHED  MONTHLY  BY  THE 

MARINE  BIOLOGICAL  LABORATORY 

PRINTED   AND    ISSUED    BY 

LANCASTER  PRESS,  INC. 
LANCASTER,  PA. 

AGENT  FOR  GREAT  BRITAIN 

WHELDON  &  WESLEY,  LIMITED 

2, 3  and  4  Arthur  Street,  New  Oxford  Street,  London,  W.  C.  2 

Single   Numbers,  $1.00.    Per  Volume   (6  numbers),  S4.5O 


Entered  October  10,  1902,  at  Lancaster,  Pa.,  as  second-class  matter  under  Act  of  Congress  of  July  16,  1894 


EDttorial  Staft 

GARY  N.  CALKINS — Columbia  University. 

E.  G.  CONKLIN — Princeton  University. 

M.  H.  JACOBS — University  of  Pennsylvania. 

FRANK  R.  LILLIE — University  of  Chicago. 

GEORGE  T.  MOORE — The  Missouri  Botanic  Garden. 

T.  H.  MORGAN — Columbia  University. 

W.  M.  WHEELER — Harvard  University. 

E.  B.  WILSON — Columbia  University. 

Managing  BMtor 

C.  R.  MOORE — The  University  of  Chicago. 


All  communications  and  manuscripts  should  be  sent  to  the  Man- 
aging Editor,  the  University  of  Chicago.  Subscriptions  and  other 
matter  should  be  addressed  to  the  Biological  Bulletin,  Prince  and 
Lemon  Streets,  Lancaster,  Pa. 


Notice  to  contributors.  Every  paper  to  appear  in  the  Biological 
Bulletin  should  be  accompanied  by  an  author's  abstract  presenting 
the  chief  results  of  the  investigation.  The  abstract  should  not  exceed 
225  words  in  length. 

For  indexing  purposes  there  should  be,  in  addition  to  the  title, 
one  or  more  subject  headings  indicating  in  a  word  or  two  the  divi- 
sions of  the  subject  discussed  in  the  paper.  The  entire  name  of  the 
author  (of  each  author  if  a  joint  paper)  and  the  year  of  birth  is  also 
desired. 


\ 


BIOLOGICAL    BULLETIN 

OF  THE 

fiDarine  Biological  Xaboratorp 


WOODS   HOLE,   MASS. 


VOL.  LV 


DECEMBER,   1928 


No.  6 


CONTENTS 

BODINE,  JOSEPH  HALL.  Insect  Metabolism 395 

LLOYD,  FRANCIS  E.,  AND  The  Pulsatory  Rhythm  of  the  Contractile 
BEATTIE,  J.  Vesicle  in  Paramecium 404 

THRELKELD,  W.  L.,  AND          Observations  on  Hydra  and  Pelmatohydra 
HALL,  S.   R.                                  under  Determined  Hydrogen   Ion   Con- 
centration      419 

MANWELL,  REGINALD  D.         The  Occurrence  of  Nuclear  Variations  in 

Pleurotricha  lanceolata  (Stein) 433 

QUIGLEY,  J.   P.  Observations    on    the    Life    History    and 

Physiological   Condition  of  the  Pacific 
Dog  Fish  (Squahis  sucklii] 439 

FARLOWE,  VIVIAN.  Algae  of  Ponds  as  Determined  by  an  Ex- 
amination of  the  Intestinal  Contents  of 
Tadpoles 443 

PAGE,  IRVINE  H.  Further    Observations    on    the    Chemical 

Composition  of  Woods  Hole  Sea  Water 
—  The  Chlorine  Content  and  Salt  Analy- 
sis      449 

KAPP,  ELEANOR  M.  The  Precipitation  of  Calcium  and  Mag- 
nesium from  Sea  Water  by  Sodium 
Hydroxide 453 

HARVEY,  E.  NEWTON,  Further  Observations  on  the  Effect  of  High 
HARVEY,  ETHEL  B.,  AND  Frequency  Sound  Waves  on  Living 
LOOMIS,  ALFRED  L.  Matter 459 

PUBLISHED  MONTHLY  BY  THE 

MARINE  BIOLOGICAL  LABORATORY 

PRINTED    AND    ISSUED    BY 

LANCASTER  PRESS,  INC. 
LANCASTER,  PA. 

AGENT  FOR  GREAT  BRITAIN 

WHELDON  &  WESLEY,  LIMITED 

2,  j  and  4  Arthur  Street,  New  Oxford  Street,  London,  W.  C.  2 

Single    Numbers,  S1-OO.    Per  Volume    (6  numbers),  S4.50 
Entered  October  10,  1902,  at  Lancaster,  Pa.,  as  second-class  matter  under  Act  of  Congress  of  July  16,  1894 


EDttorial  Staff 

GARY  N.  CALKINS — Columbia  University. 

E.  G.  CONKLIN — Princeton  University. 

M.  H.  JACOBS — University  of  Pennsylvania. 

FRANK  R.  LILLIE — University  of  Chicago. 

GEORGE  T.  MOORE — The  Missouri  Botanic  Garden. 

T.  H.  MORGAN — Columbia  University. 

W.  M.  WHEELER — Harvard  University. 

E.  B.  WILSON — Columbia  University. 


C.   R.  MOORE — The  University  of  Chicago. 


All  communications  and  manuscripts  should  be  sent  to  the  Man- 
aging Editor,  the  University  of  Chicago.  Subscriptions  and  other 
matter  should  be  addressed  to  the  Biological  Bulletin,  Prince  and 
Lemon  Streets,  Lancaster,  Pa. 


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