, 
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Embryology Course Materials 
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EMBRY IJ)GY COURSE 1947 
Staf'f 
Donald P. Costello, Professor of Zo~logy, University of North Carolina, 
in charge of the co~rse. 
Jean Brachet, Professor of Experimental ~rphology, ~niversity of 
Brussels. 
Howard L. Hamilton, Assistant Professor of Zo~logy, Iowa State College. 
John A. r~oore, Assistant Professor of Zoology, Barnard College. 
Ray Watterson, Assistant Professor of Zoology, The University of Chicago. 
Assistants 
Catherine Henley, The University of North Carolina. 
Alice Ferguson, Louisiana State University. 
a:-', 
Charles 
• 
< r • 
. ' c. 
Dickason , ur r lilizabet.u 
Edv.ards , ··ohn Farmf'n 
Go, bcr , Gharles ~•~ence 
Greg'" , anes •• 
Hill , Henrietta J o 
inchey, • Catherine 
Holtze , How 
Fo ki s, . os wrence 
Isaac , Isaac B b 
l~u, Helene ope 
:;-.Pone, Cha los A. 
~cleb'JT'gPr, l 'co lv · r 
axon , 'arion Gray 
rolson , Bot .. Geneva 
<J Odum, ~ o ·;ard '.1 h as 
Pany'cula , He len Jl.nn 
1U s , " usten ox II 
Robbins , .aril.-n. lizabeth 
Rosenbloom, ~ibby 
Russo, .... 'velyn ~~sther 
Shrader , (uth ~lizabeth 
Stolac~ , .ichard B. 
.einstein, , ' . G. 
le~er , D;rron ·3 . 
Zuckerkandl, ~1 
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ni ersi ty (A. • ) 
TTni versit-:r of ~ <'.higa ( '. . • Hro') 1' ·n) 
o sernont (.A . • ) 
.."ale :Jni versi t ~· ed i.<'al cho l ( .A •. ~linnesot.r \ 
Universlt. of or1h Car ljn (A.B. Hi , oin· ;oJl€! o) 
Univer sity of 1linois (I, . <i . Worcector olyt.och . I c . ) 
lashington University (B . S., ' · omin1 ·) 
Unive s i t y of Tll inois (SorbonnP) 
1 
31 air 
32 
33 Odu:ra 34 Io ins <')9 c 
36 Bolton 
3? Leone 
40 ·e •er 
.11 c' ader 
4~ 'axon 
45 Hinche,· 
48 Cloud 
49 Robbins 
52 Neloon 
53 Calvert ~\"j+c.\~ 
56 Dic'<ason 
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1 deb r 
1 Ri. --s 
1!1 1 1tzer 
14 Russo 
1 I i 1 
1 C'.o7llberg 
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6 au 
? Chmielowski 
1 7uc erk nell 
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• creet in the mouse embryo • 
Origin of pigment cells from the neural • 
Factors involved in gastrulation with special reference to the 
work of Holtfreter. Dr. Hamilton. 
.c. 
E.,ffiRYOLOGY COURSE 1947 
Monday, June 16, 9:00 A. M. 
Tuesday, June 17, 9:00 A. M. 
Lectures 
Introductory Remarks Dr. Costello 
!ertilization I· Dr. Costello 
Some Experi ents on the att inment of 
the fertilizable condition by the ovum. 
Dr . Costello. 
re nesli.;'1. ·, :r· fl 1 
~hursd'1 ·, :run a l 
.crid -w:o "' y , 
urdo.' , Juno "1 
onda , .Tune ~3 
Tuesday , .June 24 
9 : 
9 : 
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9 : J • 
9 : 0 1\ii 
1 CO GE , 1 ? 
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f 
:1r:1 1 n e. or 
ethods o 
of the s er 
s cr"7l to ore t nsfcr 
to 1} ro in t o squid , 
Dr. runilt.on 
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tt e s ,. d . 
... t ctm·o 
__ oo_lii_. 
(To be held in the E~br~olo Class Roo ) 
.:ednesC.ay, Ju~e 25, 9:.00 A. 1 .. • 
.. eC.~esC.ay, June 25, 8 : 00 P . , • 
Thursday, Jur..e 26, 
Friday, June 27, 
Saturday , June 28 , 
r.~onday , June 30 , 
rt.onday , June 30' 
Tuesday, July 1 , 
''ednesday, Ju l y 2 
9:00 A. M. 
9 : 00 A. M. 
9 : 00 A. M. 
8 : 00 P . M. 
9 : 00 A. M. 
9 : 00 A. M. 
Cell-lineaee an Silral leavage 
r. CosteJlo 
Genes and De'\i elol ' ent - th 
s ec:~l re erence to tle deve op-
ment of color patternn o.n u'e elia.n 
characters n fish 
Dr. H. t. uoodr ch 
No lecture Laboratory \~ork 
Normal development of starf sh 
and sea urchins prior to ons t 
of metamor1hosis . 
Dr . !a. t ters on 
Experimental analysis of sea 
urchin development 1 . 
Dr . Vatterson 
Experimental analys s of sea 
urchin development 11 . 
I:'r . vratterson 
Moving picture in audit orium in 
brick buildine . "Devel opment 
of the sea. urch:n through 
]lffetamorphos s " 
Dr . \"atters on 
No l ecture Ent re period 
9 : 00 a . m. on used for exper -
mental work by class 
E~ RYOLOGY LECTURES 1947 
(To be held in the Embryology Laboratory) 
Wednesday, July 2 8:00 P. 
Thursd e;J, July ~ 9:00 A. M. 
Friday, July 4 9:00 A. M. 
Saturday, July 5 9:00 A. M. 
Monday, July 7 9:00 A. M. 
Tuesday, July 8 9:00 A. M. 
Thursday, July 10 9:00 A. M. 
Friday, July 11 9:00 A. M. 
Saturday, July 12 9t00 A. M. 
Seminar on Theories of Astral Cytokinesis. 
Dr. Jean Clark Dan. 
Embryology of Annelida. 
Dr. Costello. 
Fertilization Studies on Paramoecium. 
Dr. Charles Metz, Jr. 
Embryology of Mollusca 
Dr. Watterson. 
Experimental Embryology of Annelida and 
Molluscs. Dr. Costello. 
~~-1 ana lilltpeJ'i.MIIdial Bmbl) elegy of &he 
Regeneration in Hydroids. 
Dr. Moore. 
Development of Coelenterates. 
Dr. Mooro. 
Development of Tunioates. 
Dr. Moore. 
Asexual Reproduction in Tunioates. 
Dr. Moore. 
EMBRYOLOGY COURSE 1947 
Thursday, July 10 8:00 P. M. 
Monday, July 14. 9:00 A. 
r.uasda.y, July 15. 9:00 A. M. 
Wednesday, July 16. 9:00 A. M. 
Wednesday, July 16 8:00 P. M. 
Embryology Seminar: Origin of Pigment 
Cells from the Neural Crest in the Mouse 
Embryo. Dr. Mary Rawles. 
The Biochemistry of Fertilization. 
Dr. Brachet. 
The metabolism during cleavage of the egg. 
Dr. Brachet. 
The biochemistry of mosaic and regulatcry gge. 
Dr. Brachet. 
Some factors involved in gastrulation with 
special reference to the work of Holtfreter. 
Dt. Hamilton. 
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VIII: 
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· tinati n 
2) Spocifici t • of fertilizin d fert'lizn ion t 
3) "'recipit tion in the fort'l'zi - tifo tn·~in r ct.· 
Experiments on Il.' ss and t c ~ u'd . iG'" C lY t 
5) Iaolation of blasto 1orcs of r·e.rcia . : · ss No o 
6) Ccntrifu ~inc the eg ~s of · ractr • li ss ad ln 
7) Isolation of bl stomcres of H. dra.ct niA. . Ii ss Iinche. r 
9) Constriction of unclca.ved · bac ·a o r. ·ron ,or 
9) 1 othods for injecting teleost ombr.ros . r . II nkins 
. 
. 
r. 1 0 ~ 
lto 
10) Hctcronlastic erafts of Fundulus to "Ol" sac of t adfin, on th 
roblcm of uigment cell differcntiat.ion. ,.. . Od11ln 
11) a . 
b . 
c . 
:Tertilization of Fundulus ee;r;s in 30' so 
Develop."llent after treatnent v:i th alcohol 
}1rncturins blastomeres 
I.:iss Shrader 
·mt.or 
and ·bCl.., 
.... 
12) Axial gradient and inhibitor substance in 'fubularia. ilr . Chm.ioloHs';;i 
13) Chemical embryology of the unfertilized Chaotopterus e, .• rr. Gtolack 
Papers not presented by 1~ : 00 noon will be deferrect until 1:30 nr. 
I 
Developnent of 
iron- Pelagic (Demersal) Eggs 
Type - Fundulus ~. 
Breeding Season: :aterial is best and most abundant during tlw first 
- three weeks of June but small nu bers of fertilizable ee:,rs can 
b Q procured through July 15. 
Equipment: 
-= Living fl•aterial-Fundulus heteroclitus and/or Fundulus majalis 
Glassware etc. -
General 6lass'Equipment- 3 laree aquaria 
Individuc:.l Equipment - 3 clean finrer -bowls (4 by 2 11 ) 
3 glass plates to cover finger-1:: owls 
\ 2 syracuse dishes 
3 ordinary pipettes and bulbs 
1 fine-tipped pipette and bulb 
paper to ·1el inr; or filter paper 
lens paper 
2 syracuse dishes 
hair loop 
culture slide - 1.7-1.8 mm. depression 
plain g lass slides 
thin sheet of mica 
I' cover slips 
I! 
Solutions - stock 0.5% Neutral Ped solution 
Dilute sea H20 (70% sea H20, 30% fresh water) 
Additional Reagents needed if eggs are to be fixed for sectioning or 
total preparations: 
1- Total preparations: 
Stockard's solution: formalin- 5 parts 
2- For Sectioning: 
glacial acetic - 4 parts 
glycerine - 6 parts 
distilled water - 85 parts 
Bouin's or Zenker's Fluids 
Graded series of Alcohols 
amyl- acetate 
56-58 degree paraffin 
Technique of Preparing and H andling Material: 
A. Care of Adults 
Although fish are usually brought to tho laboratory in mixed 
lots of males and females it is advisable to serrcgate tho sexes to 
prevent spawning. Males e.nd females should be placed in separate 
tanks until needed and after stripping removed to a discard tank. 
The sexes of both species of Fundulus arc easily identified. The· 
mature female F. heteroclitus is pale olive in color and usually 
possesses no distinct ·bars or spots, although the young females 
have indistinct, dark, transverse bars on tho sides. The dorsal 
fin is non~pigmontod . The adult male of this species is a dull , 
dark-green, the sides bearing narrow , ill-defined transverse bars 
B. 
c. 
D. 
E. 
-2-
composed of silvery spots. 1h0 dorsal fin possesses black 
pibment arranged in a mottled pattern. The ody markin~s of 
F. majalis are more conspicuous. The palo olive female has a 
pattern of heavy black longitudinal stripes on the sidos and a 
non-pigmented dorsal fin. The sides of th; ~li ,htly d~rkor male 
bear a dozen broad, dark, transverse b~rs. Tho black patch on 
the dorsal fin is striking. 
Procuring Gametes 
Both e gs and sperm are procured by stripping. The fish is 
held firmly with the left hand while gentlo pressur i applied 
to the abdomen using tho thumb and forofin er of the right 
hand. As these fingers are drawn towards the anus the pr sure 
forces out the gametes . If the fish is held against the li ~ht 
while stripping the eggs may be seen passing through tho ovi-
duct v1hich runs along the anal fin . 
Preparation of Cultures 
Strip eggs into a clean fin~er bowl which has been moisten d 
with sea water. Strip milt into a sma~l r,mount of s a water, 
mix with eggs , and allow to stand in ~~ of salt water . N ithor 
e~gs nor sperm should be allowed to stand eforv fertilizin, . 
After 30-45 minutes chango the soa water nnd leave tho eggs in i -i". Keep bowl covered with a glass plate. Do not allow eggs 
to clump or accumulate in one spot. Label oach lot with tho 
exact time of fertilization. Chango the water at least twice 
a day. 
Methods of s tudying eggs 
The eggs should first be studied in the condition in which 
they are spawned, but for many purposes it is desirable to 
prepare them for microscopic study as foll ows: roll tho eggs on 
a piece of filter paper or paper towel until the jolly t ~ nd the 
outer fibres are removed leaving the surface of tho outer mem-
brane smooth and clean. The same procedure should be followed 
for day-old stock cultures in order to prevent clumping of tho 
eggs. 
For experimental work where absolutely normal development is 
essential, eegs are usually examined uncovered in s~~llow de-
pression slides and manipulated with h~ir loops. For laboratory 
study where eggs are to be observed · over long periods of time 
and specific orientati m is des ired, either of the following 
methods is suggested: Place the eggs in sea water in culture 
slides having a 1.7-1 .• 8 mm. depression (slic;htly less than the 
diameter of tho egg). The egg may now be rotated by moving 
the cover slip. If these special slides are not available 
eggs may be placed in a drop of sea water on an ordinary g lass 
slide and covered with a very thin, flexible sheet of mica. 
Water is then withdrawn with lens paper until capillary attrac-
tion causes a pressure on the egg and it may be rotated as in 
the previous method. 
Permanent Total Preparations 
Fix the eggs in 'Stockard's solution. 'rhis 
plasm white but leaves the yolk transparent. 
be used as ['. preservntivu or the material may 
to 10% formalin after 2 days. 
turns the proto-
The fixative mny 
bo transferred 
Preparation of Eggs for Sectioning 
Eggs must be dechorionated before fixation to nllow fluids to 
penetrate the interior. (For details of this process soc 
Nicholas, '27). The following method of ombeddin~ is that of 
-3-
Barron '34, with some modifications of tiui sug e ted by J. 
OppenheiMer . 
1- Fix in Bouin 1 s or Zenker's solution 12-24 hours . 
2- Run up in ordinary manner through the alcohols leavinr, o fjS 
in each through 95% for o e hour . 
3- Place in absolute for 2 hours - runninr.: throu h se •ernl changes . 
4- Place in equal parts absolute and ar1yl -ac~tate for 2 hour .• 
5- Place in amyl - acetate for 24 - 48 hours . 
6- Place in equal parts of amyl-acetat . an para fin and in ubat 
at about 30 degrees for about 12 ho rs. 
7- Transfer through three changes of paraffin (15 minuteq in 
each) and embed in 5G - 58 degree paraffin . 
Observations of Normal Development 
1- The Unfertilized Eg~: Strip the eggs from a femRle intC' di-
luted sea water ( 70 fresh water, 30% sea water) • K e p them 
in this solution to retain the morpholorica1 characteristics 
observable at time of extrusion . Note the details of struc-
tures of the unfertiliz ed ripe ovum . These include plate-
lets, oil drops, protoplasm , 11 embranes , microtylo, o tc . 
(The micro}Jyle must be observed before remova of chorioni 
jelly). If young ova are present compare with ripe ova. 
2- The Sperm: Sperm may be stripped into sea water and a drop 
of the suspension examined under a cover slip under high 
power. Note the g eneral structure and the enormous size 
difference betv;een egr,s and sperm. 
3- Fertilization: Prepare a culture of fertilized e ggs according 
to the method outlined in part C of the section on technique. 
~o sure to record exact time of insemination . Bo prepared 
to transfer eggs immediately to a slide for observation. 
Record time of fading of pJ.atolets, of tho formation of the 
the heri vi to lline space. What are your conclusions in r o gard 
tot e rapidity of activation of the egg? If practicable, 
place a number of newly-extruded unfertilized eggs in a do-
pression slide and partially cover the depression with a 
cover slip l eaving uncovered a space large enough to permit 
the introduction of a fino pipette. Rotate eggs until the 
micropyle of one comes into view. Intruduce a drop of sperm 
suspension into tho depression without disturbing the cover 
slip and watch the entrance of the sperm and tho sproad of 
the fertilization reaction from the lucus of the micropyle. 
Since polar bodies have not been soon in Fundulus it has not 
b een determined at what stage of maturation tho sperm enters 
the egg. 
4- ~ormat ion of tho Blastodisc: Note the gradual accumulation 
of the protoplasmic cc,p. This is tho blas tod is c or gorm disc . 
Compare polar and lateral views. \.hat is tho relation of~ 
polo of the egp; tc grnwity? How does this compare with tho 
condition in the frog ogg; with the chick egg? Do any pro-
cesses take place in the unfertilized egg similar to those in 
the fertilized egg? 
5- Cleavo.ge: Watch for the appearance of a groove on tho sur-
face of tho blastodisc- tho indication of the first cleavage 
- 4-
plane . This usually occurs vlithin 2- 3 hours aft r furtilizntion . 
:' : to the eometric relation, and ti e sequ nc of th subsequent 
cleavages . Do the clcava e planes di ide the e tire ovum? The 
entire blastodisc? Durine; interkinesis tLc nuclei ar sometimes 
visible . Dis tine;uish betv1een central and margin 1 cells . 
Follow cleavage carofully to tho 32 cell staro . Not0 irro u -
laritios . ·men do horizontal clea aET,e plan~.- s fi ·st occur; Doc 
the blastodisc increase in size or altvr in form. to that 
cleavages continue for a considerable period •Jithout r:1U h chnnr: 
in form from that of thu original blastodisc . Tlli is ,alled 
the period of tho ¥if)h blastula . ·v'hon docs the <'hnne- of form 
to tho flat blastu a occur? {Soc Oppenheim r 137 for chrono-
logical terms) . 
s .- Time TablD of Development: T1.1e rate of dovolopm nt vnr:i.t-S with 
temperature and other external environmental conditi : ns . Tho 
approxima to developmental stages which may b . oxpoc ted under 
ve.rious conditions of temperature , nd salinity uay be soon in 
chart 1 . The stages arc numbered accordinG to tho chronoloe;y 
established by Opponheinvr 1 37 . 
7 - The Periblast : (Appears 16- 24 hourD after fertilization) 1ho 
uncleaved protoplasm around the margin of the croup of blnsto-
meres is co.llod the marginal J20riblast; that bonoat1 the 
blastodis c (only visible in sections ) the central periblast . 
In the l ate blastodisc (18- 20 hours) observe particularly the 
behavior of tho marginal cells and distinET,uish between circular 
and radia l cleavages . The larGe pinkish nuclei of tho periblast 
are easily vis iblo . r oto how the nucla i of tho marginnl row of 
cells become free of cell outlines , continue their divisions 
and migrate into the margj_nr,l peribL.st , convertinG it into a 
nucleated but non- cellular structure . Foll0w tho periblast 
struc t u r e in l ater stages . 
8- Th o Germ Ring and tho Extension of t ho Blastodis c (18- 48 hours ) 
Subsequent to the nucleat i on of the per iblast note the chnngo 
in form and size of the blastoderm. Tho embryo is now referred 
to ~s a bastula . Soon tho margin of the disc appears relative -
ly thicker . This thickeninET, is t 0rmcd the germ- ling and is 
du e both to an actua l periphor&l increase in col s and to a 
t h i nning of the centr al part of tho disc . This germ ring can 
bes t be obs erved i n F .• m8. j c-.1 i s . During tho noxt few hours the 
germ ring grows completely over the surface of tho yolk mass . 
The un c overed por tion cf tho yol k is tho blastopore . The 
fina l covering of the yol k or tho closing of tho blastopore 
occurs af t er t h o f i rst stages of th8 formation of tho ombryonic 
axis . Under favor able conditiuns the boeinnine of ~astrulation 
may be observed in the appoar,ncc of n slirpt inden ntion nt the 
edge of the germ ring at tho time whon the yolk :ts about ~~­
covered . Stcining v1i th neutral red will aid in idontific< tion 
of the germ ring and periblast . (Add 1 or 2 drops of stock 
solutlon to a syracuse dish of son water) . 
9- The Formation of the Embryo (Begins in 24- 36 hours ) 
While the germ rinG is exte!1ding around tho yolk the embryonic 
axis is being established . Its first indication is a cellular 
-5-
thickening known as the embryonic shield caused by a ore activo 
movement of cells in one part of the germ ring. This formation 
is initiated when tho blastoderm has covered fr om one-quarter t o 
one -third of the surface of the yolk. By the time that the 
blastoderm has covered about one-half of the yolk the embryonic 
shield has become a blu nt l y trian ular area extending fro the 
margin of one portion of the blast oderm to ncar tho center of 
the blastoderm. The shield can best be idontifi~d in profil 
viev1 . As the blastoderm spreads ov r the surface of th yolk 
the embryo grows r apidly in length. 
Observe the wh ole egg in profile view , so that the embryo is 
seen in sagittal optical section, when the y olk is 1/3, 1/2, 2/3, 
and 3/4 cove r ed . ~~at proportion d oos the length of th ~mbryo 
bear to the diameter of tho blast ode rm and to tho longth of tho 
germ ring i n each of these successive stages? 
After the yolk is 7/8 covered, look for a large clear vesicle 
near the hind end of the embryo . (Do not conf use this with A 
cluste r of small oil drops frequently f ound in a similar p osition) . 
This is Kupffor's vesicle. 
During this period the embryo becomes segmented . This seg-
mentation is confined to the mesoderm which lies on each sid of 
the axis of the embryo formin8 mesoblastic s omitcs . How many 
somites arc present at tho time o f the closure o f tho blastopore. 
Look for tho notochord • . Study it in longitudinal and trans-
vers o optical s e ctions. vVhero docs it t erminate anteriorly and 
post eriorly? 
10. Later De v e lopment Obtain series o f embryos of 2 , 3 ,4, 5 , 6 days and 
make a detailed c omparative study. It is suggested that drawings 
be made at 24 hr. intervals and that a chart b e made showing 
the first appearance and later development of tho organ systems. 
The following method of chart construction may bo used: 
Days after fertiliz a tion 
2 I 4 
Somit e Numbur 
~ ' 
Nervous Sys tom I II I Erain Divisions I L Eye l I 
Otocyst i • • 
Olfactory Pit ' 
Circulatory 0ystom I ' ' . I . J . . 
. 
I 
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I e tc 
If you a r o unfamilia r wi th tho form of chronological charts good 
examples may bo found in tho following t exts: 
Lillie, F • . R. 1940 
Arey, L. B. 1940. 
Tho development of tho chick. !lew 
Holt . (opposite page 68) 
De v elopment a l ana tomy, Philadelphia 
Saunders. (opposite page 148) 
York: 
and London : 
--6-
It is suggested that embryos be rer1o ed fro ', th" r cl or ons in 
the later stages, for better observatio of structural detail~ . Al-
though this is difficult to perform in early stages and req ires 
special instrur.ents (See experiMental section, pa e ••• ) n later stages 
the loose chorion may be torn off with sharpened forceps, or with the 
beading needles. Be careful to avoid injury to the yolk sac . 
The following developmental features should be obser ed and 
incorporated into drawings and chart; 
a. Semites. Note first a pearance and determine numbers formed 
on successive days. 
b. Nervous sytem Find in early stages the optic vesicles, 1 ns 
nouromeros, mid -, fore -, and hi~d- rain regions and trace tho 
do·.:elopr;ent into cerebrum, optic lobes, cercbollur1, m__Qdulla, tc. 
Study the development of the ~ olfactory ~ and otocyst. 
How is the lons of tho eye formed? 
c. Circulatory system. ~ ote the extra-embryonic body cavity ; form-
ation of the pericardium; first blood vessels (and especially 
their mode of formation from wandering mesenchyme cells); the 
first action of the heart; form and position of tho heart. Com-
pare the course of circulation on the 4th and 6th days . Consult 
chart No .2 for an outline of the devolop 1ont of the circulatory 
system. 
d. Mesenchymal cells. Note wandering mesenchymal cells, especially 
abundant beneath the posterior part of the embryo on tho 2nd , ' 
I· and 3rd days. Can you distinguish various typos? By successive 
observations at brief intervals, tho change in form , migration , 
and division of those cells may be noted. 
e. Note first appearance of fins; of the urinary vesicle or bladder 
(a bi-lobed outgrowth of the hind-gut); the liver posterior to 
the left fin bud. 
11. After Hatching. Tho young fish may be studied just after hatch-
ing by anaesthetizing with chlorctono . Consult paper by 
Oppenheim '37 for further details of dovolopmentul stages. 
I 
--7-
Bibliogrr.phy 
DEVELOP :ENT OF THE TELEOST 
ThiS bibliography contains only a few of tho older papers . (for more 
complete references see general works and bibliogrcphie in papers 
here lis ted) 
1. General Reference Works 
Brachet, A. · 1935 Trnite d'embryolorio des vertobros . 2nd d. 
Oppenheimer , J . 1. 1936 Historical introduction to tho study of 
t ele ostean development . Osiris , 2 , 1936. 
11 •. Special References on Descriptive Embryolor;y 
Armstrong, P. B. 1936 1ochanism of hatching in Fundulus h toroclitus 
Bio. Bull . 71 . 
s~ner , F . B. 1900 Kupffor 's Vesicle ann its r olr.tion · to gastrulation 
and concrescence . :em. N. Y. Acad. Sci ., 2 . 
-------- 1904 A study of early fish dovc lopmont , experimental and 
morphological. Arch. £ntw. 17 92-J l9 . 
Stockard, c. R. 1905 A study of wandering mesenchymal cells on tho 
livin8 yolk sac. Am . Jour. Anat. 18. 
Wilson, H. v. 1891 Tho embryology of tho Sea bass (Serranus atrarius) 
Bull • U • 8 •. F is h C om. , 9 • 
111~ Stage Series 
Oppenheimer , J. M. 1937 The normal stagvs of Fundulus hctoroclitus 
Anat. Ro c •. 68 
Solberg, A. N. 1938 Tho development of a bony fish (Fundulus) Progr . 
Fish. Culturist, and Do ~. of Comm., Bureau of Fish. 
Was hington, D. c. 
1V •. Reproductive Cycle and Breeding Hnbits 
Craig-Bennett , A. 1931 The r eproductive cycle of tho throe-spined 
stickle-back, Gastorostcus acul oatus, Linn. Phil . Trans . 
Roy. Soc. Lond., B 219, pp 197-279 
Newman, H. H. 1907 Spawning behavior ond sexual dimorphism of 
Fundulus hctcroclitus and fillicd fish . Biol Bull. Vol 12 
v. Girculatory System 
Armstrong, P . B. 1931 Functional reactions in tho embryonic heart 
a ccompanying tho ingrowth and development of tho vagus 
inncrvr.tion . J. E . z., 58 . 
Stockard , c. R. 1915 An experimental analysis of tho oriein of blood 
and vascular endothelium. Mom. Wis t a r Inst . Anat. 
Biol . No . 7 Also Am. Jour. Annt . 18 . 
V1 Germ-c ells 
Okkelberg , Peter 1921 The early history of tho germ cells in tho 
brook lrumprey, Entosphenus wildori (Gnr,o ) up to 
and includine tho period of sox differentiation. 
Jour. Morph., vol 35 (This paper has a complete 
bib1iograph of work on germ cells in other groups) 
1·1 
Richards, A. and 
J. I. Thompson 1 
Child, c. .. 1943 
Eakin, R. T 1939 .. 
Kagan, B. ~ 1935 ...... . 
Luther, W. 1936 
1936 
- 8 -
1921 .igration of primary sex cells of Fundulus 
Biol . Bull . vol . 40 
Vll Experimental ~ork 
Oxidation-reduction pattern in development of a 
Teleost . Physiol. Zool., vol. 16 
Regional determination in the development of the 
trout. Arch. Ent . -Mech. 139 
The fertilizable period of the e gs of Fundulus 
heteroclitus and some associated phenom na, Biol . 
Bull . vol. 69 . 
Potensprufunger an isolierten Teilstucken der 
Forellenkeimscheibe . Zool . Anz ., Suppl . 9 
Austausch von prasumptivcr Epidermis und 
1edullarplatte bein Forellenkeim. Arch. ~nt . 
ech. 135 
Nicholas, J . ~. 1927 The application of experim ntal methods to tho 
study of developing Fundulus eMbryos . Prof. Nat . 
Acad. Sci 43: 695 - 698 
Nicholas, J. s . 1942 Regulation and rec onst itution in Fundulus J. • 
z~, vol 90 , 127 
Oppenheimer, J. M. l936a ~J:~hc development of isolated blastoderms of 
Fundulus heteroclitus . J. E . z ., vol. 72 
l 936b Transpl antation of experiments on developing 
teleosts (Fmndulus and Perea) J.E.z., vol. 72. 
l936c P rocesses of localization in developing 
Fundulus J . E . z . vol. 73. 
1938 Potencies for differtiation in the Tcleostean 
germ ring J. E . z., vol. 79 
1939 Tho capacity for differentiation of fish 
embryonic tissues implanted into amphibian 
embryos J. E . z. vol .• 80 
Pasteels, Jean 1936 Etudes sur la gastrulation des Vertobres mere-
blastiquos 1. Te leostoens , Arch.Biol. vol. 47. 
1940 Un apercu comparatif · de la gastrulation chez les 
Chordes . Biol . Rev,, vol. 15 . 
Spek, J. 1935 Die biplare Differenzierug des Protoplasmas des 
Tcleosteoneies und ihre Entstehung. Protoplasma , 
vol. 18. 
Stockard, c. R. 1910 Tho effects of alcohol and other anesthetics on 
embryonic development. Am. Jour .• Anat. 10. 
Vandebrock, G. 1936 Los Mouvements morphogenetiques au cours de la 
gastrulation chez Scyllium eanicula Cuv. Arch. 
de Biol., 47, pp 499-584 -
Weissenberg , v. R. 1934 Untersuchungen ueber den Anlagephlan beim 
Naunaugonkeim; Mes oderm, Rumpfdarmbildung und 
Ubersicht der centralen Anlagezonen. Anat. Ans., 
79. 
.Y:;L.J-1 H_ybridizat1:sm __ 
Bancroft, F .• 'vV. 1912 Heredity of pigmentation in Fundulus hybrids. 
J. Exp. Zool ., vol. 12 
Morris, Margaret 1914 The behavior of chromatin in hybrids between 
Fundulus and Ctonolabrus . J. Exp. Zool ., vol.l6 
Newman, H. H. 1918 Hybrids between Fundulus and mackerel. J. Exp . 
Zool ., vol 26 (See this for reference to other 
paper by samo author) 
- 9-
lX Technique 
Barron, Donald H. 1934 Amyl Acetate : a useful s olvent for embedding 
masses . Anat . Roc.,~ (1), nnd Sup . : 1-3. 
-10-
Te chnique of HandlinG ola~ic E~gs 
Type - Tautogolabrus adap rsus, the 
American cunner or Chogs ot 
~/henever possible, obs ervations should be made on pola ic egGs 
e.s well as on the deme rs al eggs of Fundulus. r.1any structures such o.s 
he ge r m-ring , c:r.1bryonic shield, and Kupffcr 's vesicle will b easi r 
to see in t he former because they have fewer oil Globules and a loss-
ramllar protoplasm. The formation of polar bodies may also bo so n 
in this typo of egg. Po lagic oegs arc far more sons i ti vo, how vor , 
in the ir oxygen requirements, so require careful hRndling . 
Pelagic eggs may be obtained from the scup (Stonotomu chrysops, 
Linn.) and the macke r e l (Sc omber scombrus, Linn.), but must b"' stripped 
and fertilize d o.s tho fish arc t~ken fresh from tho live car. Tho 
cunner· will prove far more us eful, particularly for tl,o study of oo.rly 
stages, for it may be brought to tho Lr,boratory aquo.rio. and strlppod 
as needed. 
Cunners should be caught the same day o.s needed; females nrc ordi-
nnrily obtained only o.fter 12 T. The ma l o has a somewhat brighter grt.on 
color and can also be distinguished by its bright rod cloo.cal lining 
epithelium. Milt is stripped intu a large finger bowl which contains 
sufficient s ea water to cover the bottom. ~ggs arc stripped into a 
separate bowl containinG c. smo.ll amount of sea water . It will provo 
helpful to us c c. cloth tov1cl for h olding tho fish VJhilo they arc boin 
stripped , be cause they a rc extreme ly slimy and have sho.rp spines in 
tho dorsal fin. 
As soon as possible o.ft er stripping , tho sperm-suspension should 
bo poured into tho egg dish and tho time recorded. Lot tho mixture 
stand for h a lf a minute; then dilute vlith sea water a nd decant into 
smaller finger bowls, or pour into a cylinder or Erlenmeye r fl ask and 
add sufficient sea water to fill. Good eggs will float t o tho top and 
collect princip2.lly at tho edge of tho meniscus . They should be 
pipet ted off end plo.cod in covered finger-bowls containing i-11 of clean 
sea water, and s ot in the sea water t able whore they will keep cool. 
Only glo.ss-cloar e ggs are suitabl ~ for study; i f the eggs show tho 
slightes t opacity they arc e ither immature or dead. If b its of tissue 
are clinging t c the egg, it is immature and should bo discarded. Tho 
porivetelline space appears immedia t e ly after fertilization; time 
should not be watcd in observing eggs which do not develop such o. 
space within a·few minutos . For observing tho forma tion of polar bodies, 
tho blastodisc, and early cleavage , it is advanta geous to place the 
mis croscope in a horizont a l position so t hat tho blastodisc may be 
observed in profile. It is difficult t o sao tho polar bodies by any 
other method. They appear 5 to 10 minutes after f ertilzation as smnll 
clear beads on the surface of the blastodisc . Cle vagc is rapid, occur-
ing approximately once every 20 minutes at a t cmpo rnturo of l 6 -l8oc . 
The cleaving eggs a rc crystal-clear, thoro arc no obscuring oil drop-
lets, and the nuclei appear a s pinkish objects for n short time 
between divisions . If neutral red i s us ed for outlining tho clea vage 
spindles , it should be extremely d ilute . Too much stain will stop 
cleavage or make the pattern irregul a r . 
Cunner embryos may be kept alive for several days if care is 
-11-
aken not to cro rvd them and the v1a ter is changed frequently. It is 
uggested that not more than 3 to 6 e s be pla ed in each finger- · 
owl, that they be transferred to clean sea water mornin[" and night , 
and that the bo 1ls be covered and kept in the sea '/8.ter ta le. 
Opaque (dead) eggs should always be removed. The em ryos usually 
hatch vlithin 4 or 5 days after fertilization. 
~----------------------------~~ --
- - .. ·· -··- - -- ··- ·---· . 
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------ --·----
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Lis t . v.rn t cr ~-- -- --- --·-- -- ---- .... ·-·· . . 
-·- ... - ·-·. l _ _ __ ..._ ________ . -~---·· ... ____ .. __ _ 
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Soc n&tor 1 , 2 
--7-------- ------- ···--·- - ·-·- .. . ---·--· ---- --· ·- ·-----.. ,- . --- -- -----·- . - ---. --- ·----· --- . -..... -
1 2 sua · , ! 
li.? _ __g_iE_t il. ·- - - . -· -- --- --- . - -·----- ____ ... -· . -- -··· - ..... 1-r- . . -· . - ---- . ·- --- _g_ __ __ . ·--- .. 
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f'E.E_V!_D._t_9F._ ·-- ... ____ _ ..... · .. 
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Dist . Tirtsr l' 2 
- ·--- .. -------- .. ·-. -- ----- ----- .. ---· .... ------ -- .. - ------- --- -·· ---.. ------- ----------- ·-- . 
t. t8.b:;_u to s 1~. 0"I the ;J.umber of days (24 hrs) r c quir•- d by F'u nduJ.us J:~cter-
oclitus embryos to ret. cl":' d,,ffcront stc. '·c s of dovc_l opmcntfron1Erie _ ___ _ 
blrsti'ia to th~_; f orr~·"'t5on of th-- s··•i. b1ctdd ... r just befor e hntc~~i_nG 
ndor v~r-)inc c::mditions of tcmyorcturc , s'~1init;·;, e tc . 
Drmiol ·Grrim~ n, Osborn Zoo1 ogl C<. 1 Lnborn tor-:i nnd ;_,ing "'l_r.m Ococ..noe;rc ph 5 
Laborntory , Yale liniv ..... rf.'it y . 
~2:£12 5 t-2~-?~?~ :~~T2·9· ;0~.;+~1~ =.-_ =-~ _- -· · - · 
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I 
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I t 
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4 I 5 7 1 8 :1e :11 
----·-·- --j··· .. ·-· ·-1- ··-r- !· .. - .. 
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5 ' 6' 7: 9 :11 ' 14 
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---·---· _: __ .;..- ---- ·--~ ----.-- -··. -··-
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i . 
;3: 1 4'516'7 --~---· :...J - ---· - - ----· - ---· 
·se o!'ll:- the pi,r:ettes at the stocl· dis.1e to o ta1 ~ ametes . Car~ ­
lessness will result ir contaninati. t' e s • c1• f unf r-:.ili'7'e 
e gs Jit:. s ermate z;)a . :~1e stoc1• c-'..sl of e ?:S .,·:ill ,_ la }.:t at V,' 
front of -r.he roo~ t':.e stocl dis: of s 'en .. atoz • a . at t'1e r ar . vnre 
:'".ust be taken t e avoid inseminatincr heavil · • roo mar:y s en~atozoa 
eft en cause oJ yspermy \"hie.·. resul·cs in a .. r .al lea ra · and d ~ . eJ p -
men t . Polys perr1ir e? c;s of s one foiT"s "' e el e n:e re r pidly t en r-
:·lal ly fertilized eggs . T e pol-:;spem.~ c e · S e f '.ere is, hm e\· r, 
usually feil to cleave . 
Use th<> ordinarJ low power ')f the ror pound micresc p (ap1-r xinAto y 
100 d5_a~"'eters n·at_-nifica t ion, i . e . , lJX e~·e -piece and 1 X obi c-1.:. lve) 
f;)r observation and study . lhis _r:er its mai-tainin~ the eg's in a 
considerable:~ volur1e of water in a Syracuse v·atch g ass so that n-
centration of sea water by Qva1-oration is not ra1-id. ~.ost phenomena 
can be seen readily with this ma,_,nification, whic'!J. nff erds ceJ.l nt 
definition . 3:ig..h.er powers may be used if ne desires to • b erve 
spermatozoa in detail under a coverslip . After sperm penetration L1 
:~ereis has been f e llowed by observin t?>e er;gs in t :. e wat • f,L s~ fo,· 
at least 95 minutes , it will be instru.ctive to i'1sem1.nate a SP 1. o d 
batch , and periocHcaJly mount s eFe o'· the"e under roverrla "' to 
observe under higher po\'ler . Herurunber that t~e:r r or1a in normal 11 l t 
a short tir.'e under these c enditions . 
Obtain a few unfertilized e rgs in Sfla v'.'ater in a Syracuse d:i.sh . 
'l':1ey are approximately 140 mir.rons in dian~eter as seen fr011 abo,·e , 
and 100 :r.icrons high in side v. e ·1 . L·ecaus e of their s 1•ape, tho~r 
tend t o orient on a flat surface with the animal p e le either above or 
below , rarely t • the side . 0')s erve the large in;r:e. + ure nucleus 
(germinal vesicle or nucleus e f the pr~nary oocyte), and the oil 
droplets and yollc spheres in the cytoplasm surreundinry the nucJeus . 
Note also the t hick cortex ef tl:..e egg . 
After beceming familiar with the unfertilized egg , inseminate by 
adding a drop ef freshly prepared s_ erm suspension . Stire t 11.e egc'"' 
at once by a circular movement of the dis't' and ebserve chanr.es . 'Jll""~orc 
first changes will begin a few seconds after insemination . At 21°C . , 
the fo l lowing schedule applies to·-scY~'~ ·of a population ) f eggs ( t :i 'N' 
after f ertilization) . 1st . polar body, 45 minutes; 2nd polar body, 
6tl minutes; lst cleavage 9~ minutes (ref . /'22) . 'Ihe laboratory wi1l 
pro')ably be V'!armer than 21 • c ., so that devolopmr;nt v1ill J roceed moro 
rapidly . There are many change s in the egg to observe before the 
first polar body forms . 01""~serve and r c c erd the foJ lowing : laboratory 
temperature, time of breakdown of the c;e rminaJ. ves i cl c , _time ~f 
final penetration of sperm head thrm.J.fZ~'}. membrane , time .., f first 
polar body formation, time. of s ecend :.)ola.r body formntion, t imG of 
cleavage . 'I'he time schedule in the dcscriptivo t ex t l oJ. ew s11ould 
hold approxirnatoly if t he air te:1peraturc of tho laboratory is 
about 24°c . 
If possible , arrange t e o~serve two or mor ~ o~ps Nhlch are touching 
imrr.ediately after fertilization . 2 - 3 minutes after fortil zation 
they will becin to be pushed apart by transpar(:nt j olly secreted by 
2-
the eggs external to the vitel line mem rane. By 2 minttcs, tho zo1o 
of j elly around each egg v1ill ' e as wide as tho c.:, diameter. 1he 
mar gin of th e jGlly can ef ten be maoo out y observin0 super-~umc ' -
ary sperma tozoa and othe r particles at the edge of th jolly . 5 vr 
6 minutes after fertilization thEJ vi t oll ine (fer til iza t i n )'"~r::.· rano 
~ill b e noticeable due to the fennati en of the narr ~ perivitelline 
space upon jelly extrusion . At 7 or 8 minutes, the entrance cone 
begins visibly te form . r ind an egg shov1ing a 1refile vic\ of tho 
entrance cone and the sperm which is to ent er. Fer 8 te 12 minut 
or longer, the sperm is clearly visibl e outside the vitelline 
(fertilization) mcmbra.ne at th e tip ef the censpicueus entrance cone. 
In the course of the next 8 te 11 minut~s , the vitell in membrane is 
i!"ldent ed sligt tly at its peint e f centact ith the entrcnce ~:tn e . 
lhis tends to ebscure the sperm fr em vic to some ext ent, and at 
about 21 minutes after fertilization tho e gg wrinkl -s, becomin 
dis torted and qui t o irregular in somcvJhat amo~.:.boid fe hi on. Th 
ent rance cone ra s already flatt ened censiderahly, but is still 
pre s ent, and although the sperm is pa rtially ebscurcd from vi ew , tl:~.:. 
entrance of its he nd into tho egg is not c ompleted until semc ti ·llt, 
later (ref. #12 ,1 5 ,16). The final enotr r.ti on e f tho s e rm henn 
thru tho membrane, leaving the middlE> piece and t nil outside , nay 
2. lse be observe d. At about 30 minutc..s , the egg reunds up ag in , 
but as the time appreo.chEJs for lst polar bedy formo.ti en, the ogr 
e l~ngatos in a direction perpendicular to ~he pelar axis. Ir no 
eggs lie s e that tho fanning p•lar body is on tho h eri zon, tho di~! 
should be shaken. The 1st polar body may form at about 36-40 minu t 
and it li es in t he space betwe en the egg and the vitelline mombrono. 
This spac e is ~ ·· id er in the re g ion of tl1 c animal polo than elsewhen . 
Tho s ec ond po1a.r body ttft en forms at about 51 minut es and commonly 
d~cs so immediately under tho first polar body, whic:: is thus lit ted 
into perivitelline space. At per haps 80 minut es tho eggs will begin 
t..., d ivide into tw• unequal blastome r os. Observe 2nd 8nd 3rd 
cleavages also, if time permits . The 3rd division, fr~n 4 too ce lls , 
produce s 4 micromeres by spiral clea,·age (ref . 7/24 .). 
Place some very recently fertilized eggs ~f re r c is in a drop of 
frosh , thick chinese ink suspension (mad o up b y rubbing a pioco of 
ink en a Syracus e dish moist ened with sea water) i n the center o~ u 
Syracuse dish. As the jelly is s ecreted , t ho attcchod sperm cauS Lf 
a canal to form in the s ecret ed ,jelly into which particl es of inl-:: 
will penetrate . This is due to inhibi tio n ef jelly outflow at tho 
peint of sperm attachmen t . Tl'tc inl-:" t·hus marks th e.. entrance po) 11t 
of the sperm . Arte r th(, cannl he.: s fiJ l od with inJ - , add s ua wa tc..r and 
if time permits, o~ s ervs end r e cord for a number ef eggs the r ela -
tion of the first cleava ge pJ.a:-:o to the polar bodi es and t }le entrance 
point of the sporm as marked by tho ink (ref . 11 12,16 ,1 9) . (Caut ion. 
d o not leave the pioc0 of ch i nese ink in a disb of s ea water; it 
wi l l disintegra te) . 
NERE IS! Exagg~:_r_nt c q E~tranc~~on~s :_ 
Place some Noreis eggs inseminated 5 to 8 minutes enrlior in a 
Syr acuse wat ch glass containing alkaline NaCl (pi 1 11.3-10.5. Ob-
E._e_Tve innncdiato_J.:L~. The vit elline memb r an es will c l c va t duo te n 
sudden inhibition of jelly r e lease through the mcmb r ono and a sub-
sequent accumulati on of the j elly in the perivitelline space(ref . // 1 ) . 
3 -
7 _e vit e lline membr ane re; aL.s err:er' J t o v:a er 
p-,rivit.elline s ac0 as the jelly S'"ells . 'lhe el ratio of t!1e 1:'0111 --
,_rP.ne str8tc~!es out the s er~ entrance co e '.:>etv: "'en L J.lorane and 
s·.1rface , forr.ing a lor..g file--.e·1t w'1,_ch . rec_u.e'!t} . a· e .i r-r' ed ;~1 · · -
cat5 or.. <i f the 1;1em' rane . If t'•1e e ct·s '.ave >e n 1ce t in en i ;e bo · t.~ 'j 
J.:3.> beco:::e polyspermic upon inse::-r,ir:.a tiOtl anc. shov :1t1.1:1er US exar:zer, t < 
o~ ;:;ro.nce co"les _on treatment wi t1·1. allcal i ·.e :i'-laCl . l\.1-.ou t ten min . t. "': 
a: ;cr treatment the sperm head may be cen ~ vin~ aciOSR the eri-
'..5.JeJlinc space to fllse with the e:-g surface; at v:.ic· tL·e t1.. 
:.1"r. rane indentation is relaxed . · f t cse e rgs ere en:: ft4ll;t ren1ovc>c: 
~:-o:· t~1e all--aline •. aCl to sea water , Rnc. n.\S11ed , so:--e ·il d vE=>lOl' 
r:ormnlJ.y ·:i t h.in the raised meJ ranes . .1.f 1 eft in P 1 ' \aline ·· n J t''..e 
optin·ur: lengtb of tir-e uefore v.re.r,hi"l- , and if the ul,·aJjne :'::>.Cl l.c ... 
'een chc:nt:ed once or twice to renov e nos t of the se ~a ter , L1e e? :s 
r1ay be cor.·pletely freed of tl:eir ner.·')ranos . ·_ hese "no.:,ed 11 e~ rs "\a'. 
been used for experi•-:1ents on the develo· .. ent Qf isola ted ')}DS o · .r•" 
(ref . i,[) . 
If time ·er~~::.ts , ccntrifuse sor:e unfertili :·ed t(: reis E'!£!" ·· i"l t'.\o 
Enerson electrir centrifuge ( cove1· off) for 60 111inu~ cs . A lryor o~' 
0 . 95 molar sucrose (j_ndistilled water) at t 1:e bottem • f tl-c en r·Li,l 
tubes prevents injury to the eggs . nt ·\~:· is S4110'.':lat 11y]:.'c .. ·tonic, ........ L 
provides an adequat e sup1 Qrt for t~e ~crc1s e~cs witheut injuri~f 
t~lem in any way . This ar~;ouDl: of ..:ontrifu~ins separa tos the variO''D 
fo rmed cor::ponents of tlle er-r- into s e ·ct'a] strai.a (ref . #7 . ). Tnsen;' -
nate t e centri:f'l.ged ep-gs after \'Jash:tng off t).,e surrose •1it'. s en 
~ater , and observe as~ meterical jel y - extrusion . Is more jelly o' · 
truded at the centripetal or co~trifupal p ole? 
If ergs stratif :i.ed in an ultr~centrif:t.ge a'·e availabl t"· , c. mpare thc"'o 
wit~l t he joreis ergs c cntrifur:e d in t he ..... merson electric centrifu c 
at about 1• , 000 X gravity (ref . 7fl 5 ) . 
Yereis swarm in Eel Fond ar.1out an !leur after dart at ccri·ain pl:lasr,s o"' 
the lunar cycle . (S ee :Lillie and Just , 1913) . On e c'l of tw0 appro-
priate evenings , about Dn hou~ aftor suns e t, half of the class will 
gather en the floati~1g stage behind t .1c :::.u~ ply De},artmont to obscr·vc 
this inte r esting phenomenon . 
The chactopterus e:g i s rather dar~ and granular . It is sliphtlv roro 
than 10~ microns in diamtor , but before fcrt:i.lizr·tio.1 t r'c Off:S arc 
oftvn not quite spherical . t..l-Jc polar bodies arc larc:or t•·w.n i.n 1\Jc J'c·'J s, 
and the egg divides to form t wo unequal cells by m~:;ansc • f a polar 
1 o be at t :1 e v e get a 1 p o J. e ( r ef • · 14 , 1 8 ) • 
When the ege; is taken frcm t~-,_c fcmaJ.o lt contcins a larn;e itturatur, 
nucleus (germinal vcs ~clc) , as docs V1c :ere is e, f. , but unliko tho ogr: 
of ·c reis , it spontaneously undergoes pa~tial maturution when ~laced 
in sea v1ater , even if not f'crtilizcn. . 2. num')cr of spreies of CP:CS 
partly ma turc v1hen they ontcr s ca. 1e tor nnd Fas to cls ( rof . #21) has 
shown that this is dependent upon t~e pr8sr.nc~, e f Calchun in the sea 
''ator . 
~ -
J_1aetopte!"'..lS eggs deve]. e p quitE' ra_"id . . r . ::.f e,·gs are fc, ilized 
.i -<:' t after t:J.e art ial r1a tura tio:-- in s ca \ a ter :"las been co: '.t:l etect 
they develop as ra idly as aggs insm inated 12 - 15 min tes earl:er 
when first laced in sea "Jate:;., (ref. , 23) . (timE' co nted fror. fer-
~ ilization) : lst pelar body 14 . 5 nin tes; 2nd polar b ody, 2 - ~in te~; 
n~earn shaped stage , 4~ minutes; polar 1 e bulge , 52 ni.nutvs; 
c :eavage wi~ polar lo~e attached , 58 minutes; co pleted cava -
rvi th polar 1 obe resorbed i~to one blastomere, 62 r.inut s; _ 
4 c8ll stage , 82 minutes . If the laboratory eir t m erat tr is a ott 
24°C . the development will be . ore rapid, and a 't out at t .e re.t v 
indica ted in the des cri t i ve text 1 elm; . 
'ivnen the eggs are taken from the f e ~a:t l ' , t e lare;c immature mu.ol 
is in the center of the egg . After about 15 m1nutcs in sea ·n ccr, 
matura .1. ; on wi 11 have proeeed ed to t'-,e metapl-J a"' e of t ' 1e f i rr- t .1: l o.r 
spind~e at ~hie~ stage devel ~p ent is arre~ted unless ~1e e ·r ' S 
inseminated or artificially activat "'d . 'l" e spinC:lc canl10t b d is-
tinguished as much in the livinf ecP, but it will e observed tha t 
the relatively clear region of the rr.ucleus and spindle is now lo . rt c> 
quite eec entri C81 J y . It reac.hes tr e surface of t-.... E: egg in the po) ., l 
region , where t he p elar bodies wiJl be given off . 
If eggs are now lightly insemi nated and stirred, a few spe rm aa· be 
seen adhering to the e ggs almost irmoed:l ately . With in 5 ' r 6 nd Tn t e s, 
the vitelline membrane ma y be seen to s epara te from the e r,g s u r 1'8 e 1 
after whic time it is called the f e Pt i lization mem·hrane . It is n ot 
conspicuous nnd does not elevat e much above the rr surf ace. By 10 
or 12 minu t e s , t h e e ggs, whic"IJ had become eJ.mos t s · 'he ri al afte r 
fertilization , are se en to elongate in an axis perpendicular 1o ~~e 
p~lar axis . This is p~eparatory to f er~atio~ of the 1st polar body 
at about 12 minutes . In this division the egr; thus assumes a ppr oi -
i ma t ely the shape of a bl a3 tomere , al t l, ough t~ ~e polar b ody is a 
vestigial cell . After t he 1st polar b ody f e rrns , the egg agP in r ou nd s 
up ( 16 -1 9 mi"rmtes) 1 but it elonr:ates ae;ain in the same manner to f orm 
the 2nd polar body at perhaps 23 minutes . The 2nd polar body often 
forms under tbe first '- whicb is t l·rus pushed a ".fray from the surface of 
the egg . The egg 2gain round s up (26 - 30 minutes); The e gg pr ~ ­
nucl eus may nov1 sometimes be se en mi g r a ting toward t he c enter of tl l ~ 
e[!;g , and occasionally the sperm pronucl eus may also b e det e ct ed . rv 
35 minutes, the clear zone has extendGd from the polar re ; ion toward 
the equator of the egg , 2nd a t 3 ·1 - 40 minutes a typricel 11 pear s ha.l-;c1 11 
stag e is r eached . ~h e p olar bodies lie at a position rorrespond in; i;o 
whe r e the stem attaches to a pear , ar• d the bulge whicl forms th e po·:.., J 
lobe b er; ins quit e sudd ently at tl;.e 8nti - polar end of the er g (40 - 43 
minut es) . When the polar l ~b e is fuJ l y develo1 ·ed , h ow ev e r , it 
corresponds to the st em e nd of t h e 11 pe ar 11 , 2nd th e pola r bodies ar c 
opposite . 
Obs e rve the extrusion of the pol a r lo'::lo wt. i cr contol ns coars u r,l obu -
l a r mat e rial . By 47 - 48 minut e s t he cleava r e furrow bvrr;ins at the 
anima l pole an d pa ss os to one s ide of t :-1.0 polar 1 obo , 11- ich t h u s 
comes to be a.ttac'-1.8 d to one of t r1e t wo b l8.stomcr c s . 'l'ho r r s orp t ion 
of t he polar lohc into thi s b lastome r e c aus e s it to b e comu l arrr r 
t~1an its mate , and a t about 51 mi utos two St"!"ootr u nequ a l b l ::r. :Jt omc r us 
li e a gainst each ot:"!_er . l o:l ys rJe r mic cr~s 'lll i l 1 novr often he i n on 
abnormal 3 cell stag e . By 60 minut s s t he tvJO IJJ.a ~ tor e r cs ar c qui Lc 
f us ed toge the r . At 67 - 'i O mi nut es t >,e 2nd c J. oavapc '-a}'GS pJn c ( . 'l~o 
l a r ge blastomer e again f nrrns a poJ nr 1 obe , a nd r 4 cel l stag, res uJ t s 
with one blast ome r e larr:c r ti:".an t bo othe r thr·~c . By 91 mi nu t r s , or 
5 -
carliE-r, the clear n c -.:i in tb · 4 co~ls m2•~ r e.di v be uadl out . 
At 93 - 9 rr.inutcs the 3rd divisi n tcl;:L.s :'la~c, forr.inr: ;~0lt.t v·lv 
large n:icron:ercs . - profile view · i 1 rE.v .s.l tl"~~:: macrom .r('s , ,.licro -
mer(;s , and e lar bodjcs . A olar vic" ·; i}l S.lO': t.o rotat0d dis-
placem.::nt of the m; cromeres, r es 11 tin[ from spira lL ava(""c, r.l t ' un::h 
t'.,e dis laccment is not great or onsyicllous in c~)e~-~-o~:t.~~tus. 
cc·;:;E1!'l2. TIC! A1~D .~CTI\'I'l.:' 0? Sr'E .:1~~- Z lJ',: 
Place E -~-m_~l} ___ s_~._!>_l_l_o~ dro of 11 dry 1' S l err!a t zoa directly fren: th 
testis en a slide . lhe sperm must not be diluted \'lit• o..ny sea ,.,..,tvr 
Vlhc:, tsoGvcr . Sec urchin spor .. atozoa ar · ·ood f01 this bcc?u t.;; i.t ·s 
espec~ ally easy to obtain t e111 11 dry" dirt-ctly from the tt·sti.s . J.. ac 
a t rop of s ea wa.ter ncr.rby on the slide so that it doos not touC'h 
the drop of spermatozoa . "v.: th ord i.no..r. · low powt-r ( r.pp'ox . 100: ) 
e xamine t~e spermatozoa and note t. c degree of activity e r0 in ly at 
the ed[O of the drop ''!here they may be seen more rcacll.ly. ;1 --,v; tc. 1,e 
a small g lass rod, or a match stick, or o~.er objlct and drarr it 
tip from the d2 op of se ~ water into tho dro of spOI'l1'f'toxoa so I hr..t 
n. connecting 1: ridge is e stablished . As tho sperm diffuses int~ t'1. 
sea v1ater a grs.dient of concentration is e sto.blisl ed . ."ote swb·mLlG 
activity in relation to concentration . 
If r:~n~pgia err_:s arE. a~ai1able , observe the migre:.t5.on o.nd fusion of 
the pr cnucl c i. '. ·h:l.lo :?_y.mingia oe:ss or·"' small (about 60 nicrons in 
diamter), tho:r aro cl e ar and shmv ·chc pronuclei in the llvJnC"" "'to.tc 
especialJ.y v~ell . lhoy cyt eplo.smic corstitucnts of thcsC' cre;s arc 
very readily stratified into four zones in tho ccntrifuco . 
S~ECIAL ~ROJ·CTS: 
DJ.ring spar8 timo stude nts may undo r tnkc spuciRJ projects rclutinG 
to fertilazation or coll - linoafe . 1~e follawinrr ~roblems ere sug -
gested , but students may formulate their OV'n project"' sub j ect to tho 
appreval of tho instructor . 
1 . B'ertil ization of Pla t_yncrcis . Collect 1latynorcis fr em the 
Cayadc-tTr,- ~-1arf fioatin ; sta-~e D't the richt phase of tho moon . r;on -
sul t ,.: • E . Just 1 s papers on breeding ha ;;its c. nd fort il izu tion. -~on·­
pare your observ2tions with those on Norcis l ibata . 
2 . Devel opment of lsolc:tcd Ll 8.stm1crns of r~ercis . h e ovc mur.brcn .J 
o(·-:-ins-emi·n·a-~C"cC1Tcrc-is .. ·c-f.:gs .. hy.-th-c .2j .l;;:.(.:.fi,;l·e-·1-;-a-c·J-method previous 1 y 
described . Dso s cmi - stcriJ.c tech.."'liqu<.J viith all dishes und instrumcnt::-o. 
Hal~c Spcmann c lass needles r..ftor di.rcctions of l·1orst r..dius in ~:cSJunri~ 
~·icroscopicc:l 'l.cchniquv . Usc sm~'1 1 mtch clrsscs (prof orubly ColU!ll-
bia) v1i tb E thin layer of fil to rod cr:.ar made up in son '\olatcr , as 
operating and isolatio~ dist~s . SvpnrLto tho blastomorcs wit~ tho 
fino tips of the gle.ss nc cdJ.os ir.1mcdi.:..t c J.y after the cleavar:c fui'row 
is complete . Transfer to sop:.ro.tc isolrtion dls:·vs wit11 mouth 
pipette and pcrrrdt isol2tcd 1' D.storr,crcs to dovcl01J in cool noist 
chambers . Observe c.t .frequent intcrvnls . 
3 . ~~-~c_l_C?_Em_c_r:-_~ of LE.;!lt.~_if~~~l .. Nc:r:_<.._i_~ Lo·s . c~...~trifurc ((,roia err n 
for 60 to 90 minutcf:! in I~morson eluctric ccntr1 f1.wc wi tl" covur off 
(or in n.ir turbin·-·, if a v o..ilo.blt;) , \•itl sucrose, a:J r ev 01..l.Bly 
des cribcd . :.c::.sh off sur eros c in .... :-. . ·c tcr , in v .inc t c:nd s t tdy 
clvC.V£c ~,., . Stc.tistics o.s to tl'l~.- numb e r of:-:: c:n<l cr, lcstOL1Crcs 
i'orr.-.in0 fron~ c c:ntri •c tell e r ccntrifu 1 ds of tl:~.: c ·ntrifug\.;d C""GS 
~auld be of inter ~ st . Positio~ of micr m res ncy clso b not L in 
r t.. lction to stratificr..tion e.nd i'1 relati ~'1 t e vgg lJOL. ity . 
7 -
General ='c fcrencc : 
2 . 
1 . Gray, J ., 1931 1~ t .... xt ook of ox1,.-~..ri:'10ntal cytolOGY · Cn. l·Ln-t.' 
:ni vers i t;r 1 rec f:. 
3 . 
4 . 
5 . 
Just, :. • .C. . 193!) The biolog..,r e f t''lt: cell surfacv. Blcl::iston 
Lilli e , F. 
. :or[' an , 'T' 
- . 
• l E • . l_S0'1, 
-
H • 
B. 
F~J.ila . 
C \''dr·u 
,J , 
" _;en o -res 
and L • L. JUSt 1924 C_.fc tcr 8 i 
G~noral ytolo~y , Lniv . f 
192'7 J..JX'Crl":\-ntv.l ~mbr olOiTY. 
1925 T:'"le c o 11 in d C; 'Cl or·:· en t 
r:ac1'iJ len, 1 c., ·: York 
Col :un :r. r-niv . 
an1 lOr~.-dity . 
Further references relatine; to lr.~, orrtor~r \'!Or~ : 
6 . Costello, I . 1- . 1~39 lhe c e lurr.cD occupied y the formed 
cytoplasmic crn~JOntnts in marino ug~ • 
P~ysiol . Zool . 12, 13 
7 . Costello , D. • 194• The cell ericin 
Nereis limbnta, • i e l . BuJl ~ 369 
8 . Coste l lo, ~ . p . and R. ~ . YounG 1!39 ~he moc~n ism Qf mombr~nc 
elevation in the er,r of ere:ls limbntn 
Collectinr Net , 14, 211 
~. CostcJlo, D . P . 1940 'l· ·o fcrtilizvbilit'T , f nucleated [lnd non-
nucl eated frE' gmcn ts of ccntrifuc;cd hJro is ct.:: s 
J . i·.·: orph. , 66 , 99 
10. Hoadl ey , L . 1934 ?ulsations i n the Ncr cis ogg . biol . }".ull 
vel . 67 1 pp 484 - 493 
11. Just , 1 . L . 1 912 The r...:lation or tho.: first clcnvo go pln.no to 
tho entranc e point of tho "'perm . :: iol. l. ull . 
22 , 239 
12. Just . ~ . £ . 1930 The present status of th~ fertilizin theory 
of fertilization . }rotoplasmn, 10) 301 
13. Lilli e , F . h . 1916 Obscrv8.tions and cxperimontsconcorning tho 
elementary p"lonemona of embryonic dcvel e pmcnt 
in C11a otoptc~'US . J . Exp . Zool. , 3, 153 
14. Lillie, F . R . 1911 Studies of f(.;rtil.iza.tion in Ncrcis 
J . ~orph . 22 , 361 
15. Lillie , F . 11. . 1912 Studie s of fortilizo.tion of No r cis 
111 , l V, J . Exp . Zool . 12, 413 
16. Lillie , F . h . 1 914 Studios o.t Fcrtilizntion Vl J . Exp . Zool . 
-~ 523 
1 7 . Mead, A. D . 189r/ 'lhe eo.rly development of marino o.nn clids . 
J. Mor ph ., 13, 271 
18. Lorgan , T . h . cmd Albert 'I'yJ.Gr 193t 'Tho point of cntrc..nco of 
the spermatozoon in r clo.tion to the ori e ntation 
of tho embryo in eggs ·. ·• ith S}Jiro.l c1o avago . 
Biol . Bul l. 58 , 59 
19. Novikoff, P • • B. 1939 Cho.ngosn t. tho Surfac e of Nercis limb oto. 
eggs af t e r insem~nation . Jour . Rxp . Biol . 
v 01 • 16 , 4 0 3 - 8 
20 . l'ast e;oJ. s , J . l~o5 Re che rche s sur lo detcrminismo d 0 1 1 cntr( c 
on maturation d (.; 1 1 o(.;uf chez divers invurte -
brcs mFrinc . J:.rch . Diol. 46 , 229 . 
22 . Vihito.ko r, v . J-., . 1 931 On the rntc of oxygen c ttnsumption by 
fertiliz C;d r.nd unfertiliz ed C;r;gs . 111 No.rcls , 
J . Gen . thysiol ., 15 , 191 
8 -
2:3 . ···hitc,ker , r . ·.: . 1933 Ibid 1V Cho. ..... to .... rus, J . Gen . hy""ioJ Of='J . 
15 , 191 
24. :Ii15on , ~. B. 1892-,-C~v1l line r:.c of .'rv re;is, J •. rp· · . 6 , ··:-.1 
Lunar terioC::.city 
- --- ----- --. ~. 
1 . Clc:rl~ , L . B . r,nd \ . ! • 1-icss , 194.0 (.htluntic rr:.1ol o) 
:tublicat1on No. 524 , . 21, C8rnc:~ i\_, In~·t . • of 
Was: .ine;ton 
2 . Fox 1924 !roc . Roy . Soc . London . L . 
3 . r:cmp c1l"1£';nn, Fr .' 1911 (/ercis dumcrilii) LJOOl gicn , BeL r\5 
4 . Izuka , A., 19•3 (Japcnse l:alolo) J . Col1 • .:-ci. Imp . T. okyo 17 
5 . Just , E . ~ · 1914 (ilatyncreis mogo.lops) Biol . Bull . 27 , 2 1 
6. Lillie, F . li: . ::mr . .l:. . E . Just, 1913 (.!crois limbr.ta) Biol. tull . 
24 , 147 
7 . Mayer , A. G . 1908 (J:.tJ.antic .h?.lolo v;orm) 1-u'~-•lication 102 
Carncf!io Institution of "a"'~ inrton 
8 . Scott, J . ''i. 1909 (Aml..~l1 itrit c ) Eiol. Bull . 17 
9 . ·,·foodv/Orth , \, . 1,-c 1907 (tE>d.fic lclol e ) Eulr.·:·u . Comp . Sool . r::1 
Fertilization 
Gametes of Arbacia are best obtaine b·.~ cuttin > around the 
pe istome ( on oral surface) arrl re wvin Aristotl ' l llntc ., . , t o. · in , 
care not to injure the t;onads . The pe i visceral fl1id i then .T''. ~ ·'e.: n 
f ro. the body and the animal , aboral surface do ''Il, plncPd to h "" d n 
a Syra cuse ·ratch glass slip:htly r.10ist.ened vi th s t:~ ·.·a 0 • 'lash th :-
ha nds a nd scissors vri th running fresh •rater aftc · cnr.1 nima l is 
o_;::> cned to avoid contamination of one sex 1f'ith g nno t(S orb dy fluid 
of the other. There is no convenient r.! e thod of ':< .-> Cortninin the 
sex of sea urchins v:i thout cutting into their ne rivisceral cavi ti er, , 
but v~en this is done, the ripA ovarils will bEn serv d to be dEco 
reddish brown in color, and th ·.:> ri_:)e t e stes a r( ~- -~ sh v1hi te, \"i th 
flecks of bro~ 'n . If the eg[,s an , s hed through the gonopore s of th 
fenale into the Syracuse disb. , the~r should be removed to 200 cc . of 
sea water in £:t finger bowl within ten minutes , to nvoid the injn ·-
ious effe cts of crovTding. The spc. rm ar tJ best kept "dry" ju t as 
they exude from the testes. Ad ·op of "dry" sperm mny be dilut(d 
VJi th 10 cc. sea 1·:ater in a v:atch glass just before in~ E'mination. 
Do not use sperm that have been diluted more than 20 linnt t' a nd 
avoid too high a sperm concentration. The latte r leads to poly~ po r·1~ ' 
and abnormal cleavage. 'l\ ra drops of dilut~d sporr, should bo suf:f'j · 
ciont for a fingEJr bov:l of c·p.-,gs . Stir the egg dish imME'dia tely :t f ·t ··' 
adding sperm for insemination. 
If shedding of tho s.nimals does not tnkc place v·ithin t e n 
minut e s afte r rc r~wval of the lantt:!rn, make a second cut around the 
shell near the equa tor, just above the oral endu of the ovaries or 
testes. If, after this second cut , gamete-1 do not stream fror1 tho 
gonopores , ovaries mRy be r r:r.toved v·ith curve¢!. for0eps to 200 cc . of 
sea watsr in a fin,q;er bow~ and the riQe er,gs v·ill be. ext::;u~eG 
through the ruptured ov:-1r1Pn v-ralls . The ee,g suspens1on lS then 
filt e r~d through chees~ cloth ( ~hich h a s been soaked in s ea wa t e r) 
to :i:'PP.H)v·e ovnrian debri3 , and after the eggs ·scttJ.e. the sea "at c r 
is -t v:ice (at least) de canted and replaced ,:.·i th fresll sea vm t e r to 
remove tissue r:Y.tn cts. V!nshing o'f the er.,gs shed after cuttinp. ... 
around the peristome may c.tlso be necessary if nn ovary has boon · 
injured or th.:; ege:> otherwise contaminated v.rith perivisceral fluid. 
Testes rr,ay be similarly removed to a dry watch glass v•i th forcop , 
aHd exuded " dry" sperm used as indicated above . 
A good batch of eggs from a rip e female should show unifomity 
of size, perfect l ~r spherical form, and complet e aos e nce of immature 
eggs ( ir1 the e;eminal vesicle stae;e) . The egg of Arbacia i s shed 
in the fully mature state, both pola r bodies having been give n off 
in the ovary. Very rarely, the po l ar bodies remain on the egg 
surface . Usually-they are not present, and the only good index of 
polari tr of the e [S ~ is the fu.rm e l - sh aped space in the jelly v1hich 
marks the previous point of att a chment of the egg to t he ovnri a n 
VIall . 
The unfertilized e ge,s should be examined in a drop of s ea 
water under a cover ~las s a n d the follovrinf.; parts noted: jelly 
layer ( rendered readily visible with a l .ttle J a nus ~reen solut-
iorl or by a suspension of Chinese L ·: ); vi tel line membrane (very 
Ar acin - ...,-
thin); r.:ature egr. nucleus (a sr.:all, clear sphe 'C); , rotopla~mic 
granules of t ·:o klnds, ( l) re pigment granules ofjnt•e:timin echinochrone 
(2 smaller, colorless yolk grar. les. vo othe types of granules, i .. 
the ~itochondria and the oil spheres are present but usually di ti 1 -
guishab le only after stratification b• centrifugin~ . 
Inser.inate some of the eggs as indicated abov nn e.·runine iruned-
i '1tely under low povrer . Spern penetration occurs too ri.1Iidlv to b · 
easilY detected . 'li thin a fev • seconds after insemination the cortica 
responses of the egg begin. The vitelline membrane elevates ru idly 
from the egg surface , leavihg a peri vi tell inc space. Thi c. mer.1 rn n ·: 
thickens and hardens during the next five minutes and after alt~rnti~n 
is called the fertilization membrane . At the proto~ Iesmic ee..g surf: c · 
(which is at first slightly disru~d by the elevation of the v:i tellil1C 
-·"embrane) a new, clear layer is se cret od - the hyaline pl<- no. m r1brr, •~" 
·nich is presumably a calcium-prot einate ac ti ng as o ce 1cnt to ho) d T 1 (• 
blas tomercs together after clcavaeo . It disappears in cnlcil~-freo 
sea water . After insemination, the jelly layer can often be clearly 
r..ade out b:V the :bing of sperm entrapped near its surface. :r-~oser ( 1 0~~~) 
has corre lated the elevation of the vitelline membrane with the br l'n..· -
do,·n of certain cortical granules . These granules arc embedded in trw 
cortex and are not easily displaced by centrifuging us a re thC' grann·t s 
of the underlying fluid endoplasm . In the centrifuged eggs these 
cortical granule s show up clearly at the hyaline zone . 
Examine eg~s centrifuged in an electric centrifuge at 6,000 to 
10,000 times gravity and identify the follovTint$ layers in order fro ,l 
centripetal to centrifugal pole : ( l) oil cap, ( 2) hyaline zone cont·d , . 
ing nucleus, with cortical granules visible at periphery in optica l 
section , (3) "Fifth layer", or so-called "mitochondria, (4) zon of 
yolk spheres , (5) zone of pigme nt granules . 
1;iount c entrifuged unfe rtilized eggs under a covor r,lass on a slide: 
and focus on cortical granul es c.t hyaline zone under high power . Then 
carefully add a drop of dilute s:;?erm at tho edge of the covergl a"'s . 
As tho spermatozoon penetra t es tho ege; there is a mvo of breakdovm of 
the cortical granules , which is followed by r ap id membrane elevation . 
Thc cortical respons e s of the norma l and ce ntrifuged eggs to in-
semination may be slowed dovm by dGcrcasing tho t f_,mp~Jrature . This cnn 
be done by placing small dishes of eggs and r.po rm in an ico bath tnd 
cxami ng thoro on a chille d slide aft , r insemination. 
It is also of interest to ox£lr.1ino f e rtilize d ~~nd unfe rtilize d er,ss 
by ds rk-fi e: ld illumination (oven n dark-field stop elise plncod in nn 
ordinary C'ondenser is ofte n off~~ cti v e ) and not• thA cho.ng . in the cr.,r 
cortex upon inseminr.tion. This dise.ppot...r 'l.ncc at f~ rtilizntion of n 
cortice.l laye: r which is brirht unde r dnrk-fiold illumination was 
first described by Runnstrr~rt (1928 ). 
Make sketches of all distinctive f eature s of the Ar'bncia cgf 
mentioned abov 8 . 
Arbacia -3-
Inauguraion of Cleav_~G£ 
The tine betwe en f e rtilization and fi st clC''\V,, ..,c vL ie 
s ightly for different lots of eggs at a given te .p ·a ture. o~e 
details of the mitotic pr cess ( includin.g s OI"!T'. a~ ter, din t e r f 1 ~ 
spindle) can be observed in theliving e ~~s, partie l a rly if thev are 
s lig_h tly flattened by compress..:. on under a covf rgla s . Re e1:be r that 
egos under a coverglass develop nor!"lally for only a short ti"e and 
under compression this p{riod may be markedly decrea s e d . 
One of the fir~t internal phenomena clearly visible i~ the 
formation of the spenn aster, which appears o.q a round, om' ha t cle nr 
reBion at the time the pronuclei cor-e into contact. The spern a tor 
then elongates, to give the "streak'' stage, ···itb u s omevrhat cl ar 
crescentric streak extending < tcros::.~ most of the di c:.meter of the ese; . 
The streak then fades, ar. c~ is re')laced by tv·o clenr regions, ma 'kinr. 
the two asters of the cler~vage spindle . The follov.•ing ti . e C'lchc dul 
applie s to 50 per ce nt of the ef!.gs at 20° C. (Fry, 19 :S6): Round "'p orr. 
aster, 11 min.; " streak" stage, 30 min .; full~r forn c d dies t e r, 45 - 50 
.. in.; first cleavage ,_::::----::_-,- 65 min., second cleav<lfe , 106 min.; 
third cleavage, 145 min . It is i mportant to note thBt fi s t cleavac ' 
of s ome eggs will occur as early as 56 minutes at 20 c., a nd a t 4 5 
to 47 minutes at 22° C. ,Obs1'1"Ve and sketch cleavages throug h the 
cell stage , keeping D time -schedule of oven ts at the tempe r a ture o f 
the laboratory. Note t he behavior of the hyaline plasma membrane 
during the cle avage proce s s, the r 8lation of the first three cleavc: ""e 
planes to each othe r and to the egg polarity, an:l from time to U no , 
cxruninc a fresh sample of e ggs under compression to obse rve such 
internal phenomena of mitos is as are visible in these living eggs . 
References 
Boveri , Th., 1907. Die Entv:icklung disper7;1er Seeigel-Eies. Ein 
Be,]trag zur Befruchtungslehre und zur Theorie des Kerns. Jenaische 
Zeitschr ., 43. 
Fry, H. 1936. Studies of the mitotic figure. V. The time schedule 
of mitotic changes in developing Arbacia eggs. Biol. Bull., ~ 89 . 
Ha rvey·, E .· B. , 1940. A comparison of the development of nucleate 
and non-nucleate eggs of Arbacia punctula t a . Biol. Bull., 79. 
Just, E. E ., 1939. Busic methods for expe riments on eggs of marino 
animals . Blakiston, Philadelphia . 
Horgan , T. H. , and Lyon, E. P. 1907. The relation of the substo.nc os 
of the egg, separated by a strong centrifur,al for-ee, to the locati on 
of the embryo . Arch. f . Entw., 241 
loser, F. , 1939. Studies on a cortica l l a yer r e s ponse to s timula t-
ing agents in the Arbaci a e gg . I, II. J our. Exptl . Zool., 80: 423, 
447. III, IV, 1910, Biol . Bull., 78. 
Runnstrom, J., 1928. Diu Ve r a ndr rung de r Plasnu.kolloide bc i dor 
Entwicklungserregung de s Soo i ge l c ts. Protop~asma , ~ 388 . 
J . SpE-k 
s . V/ataso 
- 2-
1834 Ober die bipolare uifferenzierung des Cephalopoder. 
und ~rosobranchier - Ei~s . Roux Archiv . vol. 131 
1891 Studies on Cephalopods . 1. Cleavage . J . vf 
~orphology vol . 4 . (See figurEJs) 
L. VI . 'lilliams The Anatomy of the Connon Squid . (Excellent 
Figures of the structures of the adult squid) . 
·-
References on Develo~ent of Cephalopods 
YJ. K. Brooks 
G. A. Drew 
L. Hoadley 
E. Korschelt 
E. Kerscholt 
E . VJ . Jv acBride 
A. Naef 
A. P ortmann 
1880 Development of the Squid • . :er:;.oirs Boston Soc . 
of Nat . Hist . (Good fi ·res of all stages) . 
1911 Sexual activities of the Squid. (Loligo Pealii) 
1 . Copulation, egg laying and fertilization . 
J . of f.orphology . vol . 22 . 
1919 11 . The spermatophore; its structure, ejacula -
tion and formation . J . of Morr;hol ogy , vol. 32 . 
(Both papers are recommended for col lateral 
reading; consult figures in #11 f or details 
of the structure of the spermatophore). 
1930 Pol ocyte formation and the cleavage of the 
Polar Body in Loligo and Chaet opterus . Biol. 
Bull . vol. 58 
(data on artificial insemination) . 
1892 Entwicklung des DarF..kanals und l'Tervensystems 
do r Cephalopodon . 
Festschrift fur Leuckart 
(good fieures of deve lopment of intestine) 
1936 Vergleichende Entwicklungsges chicht e der Tie r e . 
vol. 2 pp 968 -1009 . (Contains Bibliography) 
1914 Textb ook of Embryology. 1. Invertebrata. 
1928 Die Cephalopoden, vol. 2 ; Embryol ogic . Fauna 
o Flora del Golfo di Napoli. 
(C omplete series of figures of development of 
Loligo on plat es 1-Vll ; seriation of stages. 
Consult particularly plate 7, figs, 4 and 4a 
for newly hatched squid . Note that Naef de-
scribes the Mediterranean species, L, vulgaris, 
whi ch devel ops more $lowly than L. P. ) 
1926 Der embryonale Kreislauf und die Dotterresorp-
tion bei Loligo. 
Zo itschrift f, Morph ol. u, Ok ol. vol. 5. 
(good figs. of yolk sac circulation). 
A. P ortmann and Bidder 1928 Yolk absorption in Loligo and the .func-
tion of tho embryonic liver. Quart. Journ. 
Micro. Sci . vol . 72 
s . Hanzi 1931 Sviluppo di parti is olate di cmbrioni di 
Cofalopodi . Pubbl . della Staziono Zool, di 
Napoli. vol , 11 
1931 Duplicitas oruciata in embrioni di Cefal opodi . 
ibid . vol. 11. 
--·~· ... - .. ~- ' -··":"· .:.· -· ~ ~ ~~---
-5 -
1) Obacrvc r:;rov1th of r.wntlr; 8.nd fins. {The shell gland is 
muanwhilc invag nn.tcd and not Yisibl~:; . ) 
2) Tho e~re-ctE· lks arc prominent. 
On the posterior s de 
3) ':he formation of the siphon by concrescence of the 
antorior siphonal folds . The posterior siphonal folds 
continue as ridges to th~ anterior side . 
4) The anus, between the gill primordia . 
Note the further grov1th of other primordi2. ; contractions of 
tho yolk s ... c, etc. 
c. Old Stagu3 . {Before hatching - Chart figs . 5 and 6) 
1) Eye-stulks arc very prominent. They contain tho primordia 
of tho optic and cerebral ganglia, the so - called 11 v1hi te 
bodics 11 , also D. separate mass of yolk . 
2) 'I'hc l0nu. Tht.., inner sector which is forned by the outer 
pu.rt of the optic vesicle (not like Vertebrates ( is 
clearly visible as a club-shaped rod extending into the 
u~ru vesicle . 
3) The mantle has overgrown the anus and gills • It is c on -
tractile . In the older stages it is beset with 
4) Cn.romatophorus . Nutc different types and culors: obse r ve 
their contraction and expansion . They arc equipped wi t h 
muscle and arc innervated . 
5) Tho statocysts lie close together . 
6) The fcathcr - liku gills can be observed through the mantle . 
7) Tho bronchial hearts wil l be found at the bases of the 
e;iJ.ls and the systumic heart between them . All three 
pulsnto . 
8) Thu roctum ~nd the ink- bag 
9) 'rraco the outl ino of tho internal yolk mo.s s and note the 
grr.cua1 dccr~Hlse in size of the extornnl yolk sac . 
10) Observe tho locomotion of ~ old embryo after it has 
hatched . 
Time after Fertilization 
20 Min. 
1 hour 
3 hours 
12 hours 
24 hours 
2 days 
3 days 
3 1/2 days 
5 1/2 days 
6 1/2 days 
11-12 days 
Stage 
1st polar body 
2nd polar body 
1st cleavage 
Blastoderm over top of egg 
11 Gastrula 11 , thickened peripheral 
ring 
Blastoderm half way over the egg 
Blastoderm nearly covering the egg 
Appearance of shell gland and eye 
stalks 
Siphonal folds and arms appear, eyes 
prvject 
Siphonal folds fused into a tube , 
eye stalks prominent 
Hatching 
6. Spreadin~ of the Blastoderm. Study eggs about 24 hours after 
insomina ion. Lacer blastodermal stages may he obtained from 
egg strings. Note the gradual extension of the blastoderm about 
the yolk . Tho "blastocones 11 which are supposed to give rise to 
the yolk epitholirun are not very distinct in Loligo. Note tho 
thickening of the m~rgin of tho blastoderm - the formation of 
the antomosoderm (gastrulation). 
7. Organ Formation. It is convenient to call the pole where the 
shell gland and mantle appear 11 dorsal 11 and the 7,ok pole 11 vontral 11 
'l'rEJ siphon side 11 posterior 11 and the mouth side 'anterior , 
although those designations aro not correct from a comparative 
anatomical point of view. Study a sequence of at least 6 stages 
as ro pros en ted. on chart ••• Study c mbryos from all sides. Make 
drawings of different stages. Note : 
A) Early Stages (Cl>.a rt figs. 1 and 2). cf. also text books of 
MacBride and Korschelt . 
l)Shnll Gland at dorsal pole 
2) Mantle Primordi~m, an ectodermal concentric fold beneath 
tho sroll gland . {fig. 2) 
On tho anterior side: 
3) Mouth 
4) Eye primordia - ectodermal invaginations. 
On the posterior side 
5) Anterior and posterior siphonal folds. The former are 
tho primordia of tho siphon; the latter will form its 
retractor muscles . 
6) Statocysts . 
7) Gill primordia 
At the bounda of blastoderm and olk 
8 Primordium of tho anus 
9) Note tho rhythmical contractions of the yolk epithelirun. 
They servo the purpose of circulcting the liqu fiod yolk 
111atcrio.l in the yok- sac vessels. The material is 
carried into the embryonic tissues in this way. (See 
Portm..'lnn, 192 6). 
B) Mod m 1 Stag s (Ch'lrt figs. 3 and 4 l. Note the gradual 
constriction of the yolk sac. Tho letter continues into 
tho embryo \'lhich is thus formed around a core of yolk- mass . 
3 . 
4 . 
o. 
5 . The ~ and cap thread at the smaller tip end . 
£, • 'i'he r:iddle tunic ':Jhich r:1ay be sli[;htly S 'lOllen . 
7. A liquid apace aro'-lnd the s er:-.1 ::Lass , lined by r::iddle 
and inner tunics . 
8 . Of the three 11ne;:'lbranes 11 vhi~h are forr.1ed arotald the 
ejaC'llatory arJpare.tus and ins ide the 11 tu.Ylicsn , the niddle 
neml:lrane can be nost easily identifiec~. . It is relative ly 
thick and extends fron the cenent body to the cap . It is 
fastened to the outer tunic at the cap end . This fusion 
vJill never break curing the process of explosion; but the 
entirE; con tents of the capsule ;ill e va~ina te 8.t this 
point . ( cf . dia ~rarr,s of Drew , 1919) • 
~ . The outer meMhrane begins also at the cement body . It is 
so closely applied to the inner tunic that it is 
difficult to dis tine;uish betw0en them . The oral end of 
tho inner tunic and outer menbrane can bo easily identi-
fied as a thickened ring around the niddle membrane , at 
a short distance from the cap . 
The cvae;ina tnd inner t U.Ylic and outer membrane will form the 
sterm reservoir after tho explosion . The sperm reservoir is 
c os ··c1 at onu end by cement fror'1 the cement body and open at the 
othc1· end (sec under 9) . The sperm , mixed with a ge latinous 
mass , v1ill ooz c out s lov1ly in " clouc'i; this v1ill cont invle for 
hours or days . All other structures are left behind after 
explosion. 
Fertilization and J, aturation . Artificially fertilized egrs are 
more favorable for the stud~r of the first phases of developne nt 
than n.re those laid by tho feme.le because they lack the jelly 
onvclopos . Fertilization and clcava[·o can be readily observed 
in this wv.y . Howovor , these C[.f!S 2.rc very s ensitive and must be 
kol,t in a large volume of w at0r . Inserlinate a watch glass of 
eggs and transfer a few ocgs i~nediately into a depression slide 
(sec sc ct ions C, D, and F of '.L'o chniquo) • Observe the fort il iza-
tion under hie;h power . Note tho penetration of a sporm throuc;h 
the micr opyle . !l.ftcr a short time , tho cytoplasmic cap vlill 
withdr<lV/ from the chorion , and a clear perivitelline space wil l 
appear , 1hich indicates thc.'l.t fertilizati on is to.king place . 
Tho first p olar body appears o.bout 20 minutes after fortili-
~ uti on . Observe and time tho appearance of tho se c ond polar 
~~c'l)' and the further divisions of tho polar b odies . (Soc Hoadley , 
. 
Clcnva~e . Mount 6 - 12 eggs in an upright p osition in a depression 
slide Sec section F of Te chnique) . Usc eggs which have b o th 
polar "b odj.cs f ormed, and obs e rve t horn a t shor t int erva ls. 
Observe the firs t cleavages and note t heir re l ati on to the 
axes of symmetry of the egg . Tho first cleavage plano coincides 
with tho median pl)ne of tho future embryo . (Consult tho figs . 
of 'ria tns u , 1 , 1891 • 'rho clon. v::> ce is mcroblas tic , and not spiral 
in contrast to other molluscan eggs . 
Time TnlJlc of Dovolopmcnt . Thoro is considerob1e vnriation duo 
to tompor,tur<J difforonc<'s , and the followinc; table givus only 
n rough approximation of tho times at v1hich cort8.in s tagos are 
roached . 
- 3 -
2) Rinse vii th distil led •;Jater several ; inutes . The tra!'lsfer from 
formaldehyde to distilleC. 'later nm~t be gradual . . . 
3) Stain v1ith .!.~hrlich 1 fl ':'riacid for 5 - 10 :-:inutes . The stP.ln lS 
made by cilutin;-: the steel( solu.~~iG~: 6 drops to 8 cc of dis -
ttllcd v1ate:' (8 c:,., fills a sy;."a~-_;_se dish about half full) 
4) ::1inse stc..iY' ')ff •,-,1~ -- distilled -::o.ter and put the sper~.12tophores 
m L s J i d e u rt rl r-: r c; o--o r s l i p • 
H .. Prop~:ra.tio"" o•· -.. 'iclu ,:c,,nt-.s t.r,d S9~tions 
---"'-:-c~c>~:lf:- o~ '];r]lr :=-·{;(~---f.!~.-c,u!-if :::-f ;olk '.I hi ch they contai::1 , squid 
cm'J ·JO[~ have 11. 1 c.n-J·.:n-:('7 tv be friDble anrl dif'ficul t to section 
cs ?;)O j .;..n:.L? in yr :n ~cr .:: tn. GGS . r:'rlo ru1.yl acetate technique nay be 
used (serj r vl':Y1 ~:t8) cr ti:G 't::_o,~an t~chnique as outline d below: 
-;, Fi:r.. L•.o enbryo~, ir- "jou-tn 's solutiono (If the embryo is 
hir~hly r.-wtilo it 3houlc1 firClt be anesthetized in sea water 
containinG chl.oretone before ;..,einr; dro: }Jed in the fixative) . 
~ . Tronsf~r the: et:t1Jryos fror>l th8 fixative i""to pure dioxan . 
C1 n ngo to frvsh cJio;:an at hourly intervals (2 chances) 
until thC;J have been in dioxan for 3 hours . 
3 . Transfer to pure varaffin for 1 hour; chanre to a fresh 
paraffin bath for a second h<;ur , and then to a mixture of 
paraffin c on ·i.:.aj_ninc; 8 to 10% bayberry v1as for a third hour . 
4 . Embed in paraffin- bayberry v1ax.: 
tJ . Section u.t 5 or 6 microns and stain with Hcidenhain ' s 
haomatoxylin or with Prenant ' s triple stain . 
Observations of Hormal Development 
1 . Tho Unfertilized ~gr; . Study mature eggs taken fr om the ovary of 
tho squid . Tho mature egg is surrounded by a transparent 
chorion v1hich is cl osely applied t o it . At the po inted end , 
f i nd a depression and a minute canal extendinr, entirely through 
it- . This is the micropyle . lToto th0 p olari t y (blunt and 
pointed p oles) and tho bilator8.lity of tho egg by turning i t 
over . Tho more convex sido of the 0gg is the f u t ure 11 anterior 11 
or mouth side of tho embryo. !Jote tho thin cytoplasmic .£EJ2 
covering the yolk at the pointed polo . S t udy t ho ext ent of t he 
cytoplasnic cap by rolling tho egg . This cyt op l asm wil l give 
rise t o tho embryonic s t ructures . 
Uake a drawing of the ogc; and tho cl1.ori on . 
Study a normnlly laid e~g strin~ . The eggs a r e embedded in a 
ge l atinous matrix which i s produced by glands of tho ov idu ct , 
nnd covorod by a jolly momb r nne produ ced by tho nidnmen t al ~:;lands . 
Arc tho eggs vv ound spiral l y around a central c ore? Compare w:i. t h 
tho structure of the sperm mass be low (2 ). 
2 . T; to S porr.1a tophoro . 'l'ho exccll nt pr,_pers of Drev1 ( 1911 nnd 1919) 
and th0ir illustrations should bo consult.d for u.ll details . 
0 t'tin spormatophoros (see section C of Tochniquo ) and watch 
tlloir cxplos ion and thL) o jaculation of sperm . 
Prepare some j_ntact syermatophorr1s for study (Se c tion G of 
Tochniquo) . Observe the followtng structures: 
1 . 'l'hc opaque spem mass in tho center , surrounded by the 
inner tunic . 
2 . Tho flnslc- shnp d cement body in front of it . 
3 . 'lho "'pria 1 filnmer.t in front of th' ' cvmont body . 
4 . The outer tunic is the outer,. ost layer of t~1.c entire 
onvolopo . -
C., 
D. 
E . 
F . 
G. 
- 2 -
collected at lov! tide along the sandy beaches of ::onamessett 
Isla~o. The clumps of strings are found attached to submerged 
objects in the shallo':J ·:;ater . T."ley can be kept in aquaria 
with runninc sea-·latf;}r.., The eg[ strings containing the older 
stace~ are usually darker and more v;eathered in appearance . 
Procuring GaMetes 
OI,en r.wl6 rna female by making a lo:1pitudin2l section (use 
larr,e scissors) throu;): the nantlo fro:·.1 the si}-hon to the tip, 
cuttlnc f'long the IJOSterior (funnel) side . -~er-.ove the in1: sac . 
In ~~ ,c female - tear the thin wall of every with forceps and 
3"h.ai-::r:: all transparent ep:~s into a fin;er bowl of v;ater . If 
og~s c:.ro fully mature t:1ey separate readily fror1 the ovary 
and a 11pear as beautifully transparent as gla.ss . Inuaature 
eggs nre not transparent and will not develop . 
In tho malo - piek up the t·unrJ.lcs of s r)erna tophores at the 
opening of tho sr,erm duct, transfer them to a vmtch glass . 
The spormatophores vlill explode when placed into sea vu?.ter; a 
concentrated sperm suspcn8ion v1ill thus be obtained . 
Note - If na Jos arc not avcilable the sperm in the sperm 
receptacle of the fomr.lc may be used . 
PrLpnrrti .n of Cultures 
--obtaTn-gametos as directed in section abore . Place eggs in a 
finger bov1l and add several drops of sperm suspension. After 
20-30 minutes transfer to a largC; dis:t filled with clean sea 
water and do not disturb f or 2 1/2 - 3 hours . Keep in sea 
water table a nd (,hange ·water at least twice a day . 
Hemoval of Embryos from strings 
Remove an ogg strinr, t o a syracuse dish . Using tho beading 
needles in the manner of knives cuttinc aGainst each other , cut 
it in half . Place left hand noodle so that the pressu're forces 
several embryos clear of the jelly at the open end of one of 
the halv0s . Keeping this neople in place , puncture tho chorion 
of one of the eggs with the tip of tho rir;ht hand needle . Tear 
the chorion with a sharp jerk. The pressure of the enclosed 
fluid will pop the embryo from the membrane . \lfhcn the exposed 
row of embryos has been removed , cut off tho empty jelly and 
ropoat the process . If tho eggs arc not first forc e d clear 
of tho jelly they arc difficult to remove without injury . This 
method can be used on all dmbryos , t!1.0ue;h the younger stages 
are more difficult t o remove . 
Methods of Studyinf,: Artificially Fertilized Ep;gs 
For short observationa l periods ages rnay bo studied in de -
pression slides LA.nd y1nnipulatod with tho hair-loop . To obtain 
n polar viow of tho c;rtoplnsmic cap which a lone will unde rgo 
clonvap:c it is necussury to mount the em~s in an upright position , 
PJnc n mall amount of vaso1ino in a depression slide , fill 
tho latter with water nnr~ 1'1Jount tho eggs v1ith a hair- loop so 
that they stand up . 
reparation of Intact Sperm::ttophores for ~tudy 
l)Trflnsfor some unexplod "d ~permatophores quickly into cone . 
(40?~) formaldehyde; fix for 10 Minutes . (They •Jill explode in 
a w.nkcr solution) . 
1947 
Development of the Cephalop~d E~g 
Type - Lolico pealii, the 
common squid 
Breedine Seacon: Females with ;r.;ature ege;s are available at v:ooC.s Hole 
fror1 June throut;h se· tember , althouGh t!1.e majority are spent by 
September. 
L1ving Naterial - Loligo yealii, mature r.1ales and females. 
Er~g s trin~n of Lol iGo !.;Gal ii c onta i:1ing ea.ll the developmental stages 
Der1orstration Specir1ens- Dissected specinens of' adult male and female 
squjcl v1hich shOV/ the adult structures . 
Equipment: 
Goner·, 1 Glass Lquipmont 
Individual Equipment -
Solutions, etc . -
- Aquarta in 1hich to keep adults and 
egg strings . 
clean finror bov1l 
2 watch r;la13ses 
1 large glass dish or fin8er bowl 
several syracuse dishes 
pair of coarse scissors 
2 pair forceps 
hair loop 
2 fine beading needle8 annealed into 
class handles 
pipettes and bulbs , fine - tipped and 
wide -mouthed 
several depression slides 
ordinary microscqpic slides and coverslips 
c oncentrated (40%) formaldehyde 
Ehrlich ' s Triacid Stain 
distilled water 
vase line 
Technique of Preparing and Handling Material: 
A. Care of Adults 
If adults are transported without undue dis turbance fr om 
fish traps in live cars with largo amounts of v1ater and trans-
ferrod to larse aquaria with a good supply of running water 
they wiJ.l live for a few days in tho laboratory . 
The sexes are distinguishable . The testis vf the male shows 
white through the mc..ntle at tho posterior end . The femal e s 
<ro usually smaller than the mal es , and arc shorter and 
bro dcr . Tho fcmal s often have a small pocket of white sperm 
in tho collar het~uon tho head and the free Bd~o of tho mantle. 
Tho accorsory nidamontal g lands of tho female are red during 
the broocUne season and can usually be seen through the mantle. 
Aclult squlc'l. aro used for obtaining gametes for studies of 
artificir,J. fertilization, th formntion of polai' bodies , and 
cloo.vago . rrhe male o.lso provides spornatophorcs for subse -
qucm ty study . 
B. Procuring oge strings 
S tngos showing or8A.n format ion are more rene :i.ly obta incd 
from naturally J.aid e Stj strings . Tho so s trin8s G<'ln bo 
0 
i 
e 
f , 
I 
t 
t 
IIi 
], , 
t 
s 
s 
r 
t ' 
a~ 
(~ 
c 
t 
i 
s 
T 
t 
E 
a 
s , 
ir 
S} 
es 
s 
Arb a c i..!...__E_'ll£'_~-~ u 1 at a 
-biS species is usually ripe from mid - June to mid-Septe --be r in th 
:ioods H0le re gion. When stored in labor8 tory aquaria tr_ey may 
,s,intain t_ eir ripe condition even beyord the hreec i ng: s as on and 
~upply apr:arently normal e ggs and sperm. In general, howev r, it s 
safest to use anima ls within a f ev1 days after they are colle rted . 
ile in sor1e s pecies of echinoids the sexe s me.y be distinp-ui hod 
externally (see karx, 1 930..), no differentiat inp: haro.ctoristics ha o , 
as yet, been des cribe,d f or A. punct.ula ta . The sexes o.ro readily 
identified afte r the animals are open ed by tho deep-red or purpl 
ovaries and the yellowish- gray testes, or, if unopened animals shod 
spontaneously, by tho red eggs and the white sperm. 
Obtaining the _ _gametes :- A..Yly of tho follvwing methods may be us d . 
eT Cut around the pcris t omo and r vmove the .hristotlo 1 s lantern. Pour 
out the body fluid and place tho animal, aboral side dorm in a dish 
containing a small amount of sea v1ater . 'l'he animals then frequently 
sbed thru the gonopores . After 10 m1nutes r emove any eg s that have 
been shed to a finc;er b ov1l (or othur large flat di h) col ta ining en. . 
200 ml of sea water. Sper:n should be kept in concuntratcd susp0nsion 
or 11 dry11 (i.e. a s ::...t exud ~ s from tho testes) . b) Cut around tho tust 
about half vvay botwc0n t ho mouth and the oqu 2tor and proceed as in a . 
Shedding is more frequently obtain d by this mothod, but t hor o is a lso 
more liklihood of cutting the gon~.ds . c) Cut r,s in b , pour out body 
fluid '-n ~: r vmovc _onads (at gunoduct end ) with blunt forcep5 , spatula 
or spoon. The ovnrios should. be placed in about 200 ml of son wat r· r 
in a finger bowl r.nd allowed to shod . If undisturbed the eggs arc 
extruded in compact clumps or strings anc1 may be r eadily removed to a 
fresh dish without ovarian tissue by means of a wide -mouth pipette . 
If larc o quanti tics of o ggs 8. re d c s ired the ovaries s hot.ld be a llowed 
to shed for about ~ hou.r W·ith occo.sional stirring , then pour•;d gently 
thru washed (Pnd sua vater s oaked) chcescloth or bolt :tng silk . d ) 
Inject about 0.2 ml of 0 .5M KC l into tho poristomia.l cavity. Ripo 
aTlimals wil l beg i n to s h ed 'Nirithin 2 mi nut..::s . 'lno eggs can be col-
l ected by i:1vorting t h o anima.1. in a dish of sea vwt..:.r or by v1ashing 
the eggs gontly from the surfc.ce of the animc.l w:l.th a. pip.otto . Tho 
sperm should be removed 11 dry 11 or i n concontrntod suspension . 
Spermatozoa~- Upon dilution with sea v1o.. t c r tho sperm bocomo tompor -
rnrily intensely ~ctiva . They lose their motility sooner in concen-
trated than in dilute suspension , duo presumably to the moro r c.pid 
accumulation of C02. On tho oth'l r hand the ir ability to fertiliz e 
eggs is lost mor o r apidly in dilute thr.n in concentr[t t od suspension . 
(See F . R. LiJ.liu, 1 91 5 ; C otm 1918; Ha.yr> shi 1945) • 1fiihon k opt in tho 
cold(2°C.) udry" sperm mo..y r ema in good for so vc.. r n l drys. At room 
temperature dilute sperm suspunsions mo.y lost their f urt:tl izing power 
in an hour or l ess . It is adviscble , thLn , tc us c freshly dilut~d 
sperm for fertilization . 
Tho head of t ho sperm is compris :~d of ~'. cr osomc , nucl ;:;us and midp ceo 
t ho.t arc rouc;hl y 0 . 3~4:tnd 1 mic r ons r 03pcctivcly in l vnp;th and 0 . 3 , 1 . 3 
nnd 1.2 micrcns in groo..tcst width. Th3 tcil is about 45 microns lon 
and 0.1 rricron in grc~tost width . Its axio..l filo.mcnt protrud0s a 
short distanr.c, b eyond tho end cf tho shor.th. Exc-min~J under oil 
immcrs i·:)'l a nd sketch n. _spermo..tozoC:n . Ex mine moderately activo 
spermatozoa under 'high-dry( and descr ibe their mode of s ~imming . 
Ar c.c i['. • 2 
:nf'-rtilizoc .Scgs :- Arbrcic.. ergs co::.:r . .:to bvt r.1:ioti d vision 
::;hile still in the ovnr:r ar.d tl;e olc.r ' ociics vury s ld~n r ',.c. in 
o.ttr.ched :;:b_.:m the eggs nrc shed . Occc.si~:n::-ll:r, cspcci,..~Jlv from 
rJlctivel~ unripo ~nimals or ~ft~r nac ~r~ting ~vari~s, og s na! o 
round thr~t c.rc in the orr.:ino..l vesicle (ci".:<incsis of pr mc.r: cdcj.·tc) 
str.ge reccgn zr.blc by t:1 .. 1~rcc. cloc.,r n'1Clvus (c. ~ut · n ' -h"lf erg 
dir.mcter) c.nd nuclc·lus. Such eggs may cx...~ibit <"'Qmu "ur!cco r's u1so 
to spcr:'1 but they c'"' not devolo upon inssminc.tion . 'lb0 ripe cgr-r 
(75 micr:-.ms dic..,'"!1ctor )has c. smc.ll clcc.r n 1..cleus. It cont".ins unifor·:1ly 
cispcrscd pale yolk grr.nulos r.nd slightly lo..rgcr red grr'.nul0s c n-
.· to.ining c. pignont cnllod <..ch incchroPlf..'.':b.ich is a substitut~...d nnphthc-
-.. quincne rclotcd tc the K. vitnmins . (3all , 1936, Hhrtmo.nn ~C~t nl . 1939, 
Tyler l r 39). Upcn centrifugaticn~ mitoch~ndric. and oil sphcrul s nrc 
also distinguishc.bl c. 'l!1c r:uclctfs is gcmorc..lly locc.tod excontric~lly . 
Since tho pclc.r bodies :-.re n·::>t usur.lly pr.:.sont tho p0siti::'n of the 
nucleus vii th ros pc·c t to tho polc.r r.::~is i <"' not rend 1~- · termim d. 
Occc.s ionally, h011ev.:.r , bntchos · of ogrs r.ro obtr noc in :h ich the pol.nr 
bodies nrc c..tta ch~d. In th&sc , obsorvntions (Ho8dl~y , 1934) have sh ~ 
th2-t tho nucleus may lie in o.ny 1'C'.rt cf the cytopln.sm bot Pen tho 
ccrtcx c.nd tho center. In the tronopc.ront gclr.tj_n•)US C\_,".t (about 30 
micrcns v1ido) cf the egg thoro is a funnel-she-pod sprco which ccnor-
ally lies in the polcr r.xis . The funnel is rendered visible by 
str.ining the jelly ''.'i th Jf'nus g r een ur by plc.cint; tho eggs in 
suspension of Chinese ink . For th is purpose tr.c ogc-s should be tnkon 
i11Jr.1CC.io.tely 2.ftc r shedding since the micropyle (fun::-J.cl) mn. · disr~ppenr 
c.s the jelly swells. E.xc.1~ine and skc tch some unfortiliz · d eggs ndor 
high pov;er noting fc['_turus described c-b·:.>vc . ~~ ~-ooc 0-.LL~ ~~-
Centrifuge c. nc.mplc c-f unfertilized ergs o.t ... o~u-t 10,000 g for~ hou.r 
and sketch one in "side vicwn ncting the following five lo.yors of 
stratified material;- oil cnp (centripetal end) , hyc.linu zcno , . 
mi tochondric., yolk zone nnd pig:"'l~c nt laye r ( contrifuge l end) • \1horo 
is the nucleus locntod? Rc.vo tho granules in the cortex of tho ogg 
boon displaced? 
Fertilization: - Inscmi:::1nto c. smn.plc c: eggs , using one drop of 
freshly diluted 1% sperm (0no drop of "dry" sperm in 5 ml of seo. 
water) fer eo.cr:. · 10 r.1l of fr os~1.ly wnshed dilute egg sus pens i:Jp (con-
taining nbout 5 1 000 egg~ per ml) . Stir/ ~pc dish i m.mEldio.tely c.ftur 
adding the sperm and <..•bserve thu process vpmcmbrmtc elevation . How 
soon docs it begin? whon is it complc tol~r s ~po.rntcd from th.:. sur-
face of the egg? Y·11on dous the perivitelline spo.co r•.ttnin its 
ma.ximw:1 vJidth? .Leasure the diameters of c.n unfertiliz ~Jd egg and o.n 
egg nt 10 to 15 minutes after fertiliz ation . Is thoro any npprcciablo 
difference in volume apart frvm tho.t of tho pori vi toll inc spnco? 
The sperr.wtozoch ent e rs tho ogg within a few second rfter o.ttnchmcnt . 
To obse rve th~ process place o.. drop 0f oggs in t~u c~ntcr of n 
vaseline -ringud slide and add a drop )f sperM of just sufficient 
concentration tc fertilize all of the uggs . Acd n coverslip '\nd lo-
cate as quickly o..s possible n.n egg tho.t shov;s only ono spermrt - zoon 
on its surface . Nvtv t J:1c chnngos th,,t occur upon p0nctrc.ticn of tho 
sperm. Whore docs !'1Cmbrcno olovnticn first bor,in? In the cortex of 
t he unfertiliz ed ege; thoro is a single 1P.7fvr vf rr;~nulos v1hich dis-
appear (!;Ioser , 1939) upon fcrtiliz o.t ion o.nd cc:.·ntributc (according to 
Runnstrom , 1944) to the fcrrK.ticn uf tho fortiliz--·.ti 1n mGMb r ano . 
Those arc best seen in the cyr.lino zono of tho centrifuged cggc . In-
seminate n sar.1plo of centrifuged eggs vn a s lido 1 c..s do ncr ibr;d c.b .Jvc 
and observe the behc.viour of tho cortica l g r anulos . 
Arbacia p. 3 
--a.rk - f ield illlli:lina tio ~ shows a ri ~ t reddish 11 luninous 11 layer on 
;he surface of the ur.fert .:.1.:.ze eg • 'he lumit.os i t:v .11 ish s and 
becomes paler u ... o~. fertil_za ~o (:~ r.'"'S ron, 1.::.. ; 'hl n.n, 194.5) 
Using the dark-field stop disc for the condenser of your microscope 
exaMine a sample of unfertili~ed and fertilized eggs. 
p relininaries to Cleavage:- At 10 min~tes after fertilization a hyn-
:Tne layer (about I micron wi 'e) forms on the urface of the eg o 
1hiS layer later follov1s the clea vo.ge furrows and is the m..<t terial y 
1hich the blastomeres are held together . In calcium-fro sea water 
the hyaline layer disappears. 
r;he sperm can..'1ot be distinguished in the livine; egg. At about 15 
minutes after fertilization (at 20°C) a sperm aster is visible as n 
spherical region containing clear rays extendinc; from a clear c nto1 r 
ThiS attains its maximum devel opment at 20 to 30 minutes . Th n a 
clear streak appears in tho egg slig1tly above the equator and at 45· 
50 minutes this is replaced by two clear areas, th · as tors of th fir· . 
cleavage spindle . 
Cleavage: - The following figures·give 
three cleavages (after Fry, 1936). 
the average time for tho fir~t 
Cleavage :- ~F~i~r~s~t~~~~~~~-S~e~c~o~n~d~--~~~th~i~r~d~ 
Minutes after fertilization:- 42(250); 113(150) 107(20°) 145 (200) 
Different batches of eggs vary slightly (1-2%.;<-in averap,e cleavage 
time and; while within a batch of 0e;gs most will dEJvelop at the avo:e-
ago rate, sor.1e nay vary by about 10%. For any termperaturo botvroon 1'-:) 
and 25° the average time of development-can be calculated fron thn.t 
at 20° by means of the following formula:-
log (time at tE>mp.t): log (time at 20°) - (tv20)/lO.log 2.6 
/ Temperatures above 30-32° are lethal for Arbacia eggs. ~e first 
cleavages divide the egg into eight equal sized blastomeros. Tho 
planes of the first two cleavages arc meridional (in the polar axis), 
that of tb.e.third is equatorial or horizontal (at right angles to the 
polar axis). Follow the progress of the cleavage furrow in divirlin~ 
eggs. Note that the hyaline layer forms tho surface of the furrow, 
and later, when the cells flatten against one another , that it forms 
the boun~ary between thorn. 
At the fourth cleavage the upper four cells divir1 c moridionally form-
ing eight equal colls called mcsomeres, VJhil c thG lower four cells 
divide unequally and horizontolly for inrr 1 our lar t;e c0lls cnllod 
macromorcs and below tncm, at the V(' [;Ct~: 1 pole 1 four small clcnr cells 
called micro:meres . At the fifth divi::don th~;.; clght r•Hsomoros divide 
equally and hvrizontally forninr: t\·10 tiers of cella tormod an1 (at the 
animal polo) and an"" (seo H{jrs tad ius, 1939) wh:i.lc the rno.crm:1c:rcs o.nd 
nicromercs divide meridionally . At the sixt:1 clcJavar;o tho an nnd. o.n2 
cells divide in more or less radial direction while the mncromoros 
divide horizontally forning two tiers torned vcgl and vog2• o g2 is 
next to tho micromeres v1hich have r~lso divided at this tiyne but v1hich 
do not form distinct layers. I..(),yers of cells arc not rv cHly d is-
tinguished in later cleavage stages and no spacial clcnignntion is 
ap 1 iod to t~1u cells aftor the 64-cell stage. It has boon shown (soc 
Horstadius 1939) _than the an1 and an and veg1 cells form tho ao~o-~,.. l.li/.-j.S. Fdlfi!J V"h£ ?11J,1JP>~If'ftJ 2 d.'erm 11 ~ gu1; J ana par1; of tho mes oderm (cool om) nnd tho micromeros form 
mesoderm (skeleton). Sketch the various sta8os up to the sixth 
cleavage . 
Arbncin p . 4 
-::1astula : - At the eight cell stage there is a very sr.mll entra.l 
~ic.h enlarges , as cleava e continues , to for the blastocoel . 
, abo t 6 hours afte!' fertilization a smooth- s rfaced s horicnl 
~onng blastula is forned , the wall of w..ich is one~·cell thick . Cilia 
500n develop on the surface and the blastu a is rotate by thei~ 
6ction ~ithin t.e fertilization me 1 rune . At about 10 hours th 
- -astula hatches out of the fertilization nerr:brm e .. It has b n rhown 
·::opac , 1941) that the blastula releases a 11 h['.tchL ~ enzyre 11 at this 
"cir.JC that 'JCalcens and cis solves the .. 1en!brane suffic io1 tly for tho 
blastula to break thru . A small tuft of lOYlf, cilia d velo s at the 
·nir1al pole of the bla" tula •:hich is the ::'orv;ard end whon it is 
~~·;ir11Tiing . At the base of this 8. ical tuft the blastula wall is 
-~hickened , forminP:: tho e.pical plate . At the vegetal ole th blart.nla 
Ylall becomes flatt ened and the micromeres migrate into the blastocoel , 
rorming the primary mesenchyme v;hich gives rise to th skeleton. 
sketch early and late blastulae . 
Gastrula :- At about 20 hours after fertilization tho c lls at the 
vegetal pole invaginate to f orm a blind tube , the archenteron ~ This 
roaches tho opposite end of the blas to c oo 1 in about five hours. 'l'he 
gastrula contains a out 1000 cells and its outer wall as w ·11 as the 
... rll of the archenter on has a single layer of cel ls . Tho primary 
:.oscnch:r-.. c cells fOTiil e. ring ar ound the blastoporal end of th archun ... 
teron . secondary mesenchyrw and , later , coel om arc budded off from 
the tip o f the archenteron . Draw bor;inning and conplcted gastrulae . 
Prism: - At the; cor.1plotion o f gastrulation the tip of tho archenteron 
turns to one side of the gastrula which becomes flattened over an ar(~ o. 
cxtendine; fror.~ the animal pole nearly to tho blns toporo . Tllis is the 
first sign of bilateral S".)rmme t ry , tho flattened area roprcsontinf, th 
ventral side of t ho umbryo. The primary mos cnchymo cells aggroga to in 
tVJ o groups , one on each postero-vontral side , and each group secretes 
a triradiate spicule , tho boginnine;s of tho skeleton . Vlher the tip 
of the archenteron t ouche s tho ectoderm thoro is forned a depress ion 
which later acquires an openin0 into tho archenteron to ~oc omo tho 
stomadeum . Tho archGntcron becomes div ided by two constrictions into 
oes ophagus , stomach and intestine . The apical tuft disa· pears , a cil-
iated be.nd surrounds tho oral field , tho dmbryo bee;ins to e lonr,atu in 
the dorso-ventral axis and t ho direction of swim.'1'lins che.ngos so that 
the ventral side is forward . Draw a prism larv& . 
Plutuus :- After about 48 h ours the embryo enters the pluteus stage 
which is fully devel oped at thu end of tho third day . Tho orir;inal 
apica l plato r;rows out in a ventral d irection to form tho oral J. obe 
which includes tho stomadou.-rn am1 anterior part of the o us ophar-us:-'I'wo 
short outgrowths , tho ora l kntero- latoral) arms ar formt·d on tht.J ornl 
_obo , and, at tho anal s ido , two lo!lp;o r anal--ro:boraJ. or lJ OS toro.l) nrms 
grow out in the so.mc gonerul direct ion . --rrFie ori~in[,l trirfldi to 
spicules form skolE.ltal rods which extend intc tho oral armn (ore l rods) 
the anal arms (anal rodsr;-dorsc..ll~r through the boc1y (boc'ty l~ od'"') c ... nd 
laterally (ventr['.l trt>nsvor3o rods J . Tho roc'ls e re A.\ch P1uc1o np of 
three or ::·our paralloJ. parts j oinud by cross bars . Diffel'ont 0 yoc ios 
of sea - urchins differ i 11 this regard , no tho structure of tho 
r,{elo tal rods is a usc ful d1ar8.cteris tic in hybridizu t ion s tudics . 
The embryo continuJs to c· longr:.e;o in the dorso-ventrnl direction and 
ccomes pointed at tho pos tcro- dorsal end whore tho bor1 y rod"' moe t . 
Arbacia p . 5 
r~e axis runnin thru oeso 4 harus , stom.ach and int stines be .o..,:es J -
sLaped . The stomach expands to form a spherical structure t~1nt fills a 
large part of the body of the pluteus and s h 1 cter •--us les co ne t it 
with oesophagus and intestinE" . T!1e two coelomic sacs extend 'lOStero-
laterally fr an the oeso hagus . That on the left side becones lar cr 
;nd later acquires a dorsal opening celle~ the pore canal . r1e r ht 
coelom buds off cells to form the madreporic vesicle but otherw ~e re~ 
r-ains rudimentary . The left coelon undergo3s extensive later cv0lop-
rnent in the fo r ;Tlation of t.."l-13 structur;.;s of tl e adult sea - urchin . '111 so 
chc.n ge s d o n o t o ccur until the suconc vwek when metamorphosis cgin 
in properly fed l arvae end will not be studied her • It shoul bo ~cn­
tioned , h owever, that the adult organs are built up in and around a 
structure torm ..... d the Echinus rudim8nt which is fon C. by tho fusiOl~ of 
an invagination (amniotic inve.g ination) of the ectoderl!l on the loft 
side 'N it' the mid- portion (hydr oco<0l ) of the left coelum . 1 l1e left 
side of thu pluteus becomes , then , th~ futur oral face of the adult . 
Jra ·: a 3 - day old l·lutJus in pos tcro-von tro.l and side view 
Refe r ences :-
Fall , ..t:. . G. 1936 Eehinochromo , · its isolation and composition 
J our. oi ool. Chom., 114 , Pt. Vl 
Cohn , E . J . 1 918 . Studius in the phys~iology of spermatozoa . Biol . 
Bull ., 34 : 167 - 218 
Fry , J ., 1 936 . Studies of tho mitotic figurt:: . v. The t;jmo schedule 
of mitotic chan_zc s in C.o7oloping Arbacia cr,gs . Biol . Bull, 
7 9: 89- 99 
Hart mann , ~:.~charto.u , o., Kuhn , R. and iiiallcnfcl , K. 1939 . Ubi:Jr di, 
Scxual st offc der Seeic;c l Naturviiss . 27 : 433 
Hayashi , 'I' . 1 945 . Dilution modhun and-su:rvivcl of t'·1c spermatozoa 
of Abo.cia . 1. Effect of th .:.; rrodium on fertilizing powE:r . 
·Biol. Bull ., 89: 162- 179 
Hoadl ey , L., 1 934 . The rt"l l ation between tho position r_,f the fomo.lo 
pronucleus and tho polar bodies in tho unfertilized OG~ of 
Arbacia punctulata . b iol . Pull ., 67 : 220- 222 
Horstadiu s , Svon, 1939 . The mechanics of so~ urchin devu lo~·ont , 
s tudied by opc r ntivo mothoCs . Blol. Rev ., 14:132- 179 
Kopac , M. J ., 1941. Disintegration of th0 fortilizationmomhr8.ne of 
Arbacia by the notion of o.n 11 enzymo 11 • Jour . Call . Comp . 
Phys i ol., 18 : 21 5 - 220 . 
Kuhn , R . and N<ll l enfel"S; K. 1 940 . Echlnochromc nl s prrsthotischo 
Gr upp en hochmolokulo.rcr Symploxc in den Eh:rn v on Arbac io. 
pas tul osG . Ber . Deutsch. Chem. Gesell ., 73 : 458 - 464 
Lillie , F . R. 1915 . Studios of fertilization . Vll . Analysis of 
variations in the fertilizing power of sperm susponsions 
of Arbac io. . Biol . Bull ,, 28 : 2~-251, ,. 
,:~arx , Wilhelm 1031. Zv.m f· robl om dcr Dutorminlltion c:c r Bilat\ r ... litat 
im Seeigc lkoim (Nebst e inom Bcitro.g zur AGnntnls des 
Goschlochts - dir11crphismus c inigcr Socicol) . Arch . •.ntwick-
lungsmc ch . Org ,, 125:~6 -147 
, oser , F ~ 1939 . Studies on [' c orticnl l a y e r response to stimulating 
agunts in thG Arbacia ege . 1 . 11 . Jour . Exp . Zool ., 
80 : 423 , 447 . 111, IV . 1940 Biol . Bull ., 78 
Obman , L <! 1945 . On tho lipids of tho sea -urchin ogg . Ark . F . 
Zoologi , 36A . N: 07 , 1 - 95 · 
Runns trom , J .' Monnt- , T r.r.d Wicklund I E . 1944 ~ rr.c cho.nism of 
formation uf the fortilizrtivn membrane in the sea u1·chtn 
egg . Nature , 153 : 313- 314 
'Iylor , A. 
Tyler , A. 
I 
I . 
p . 5 A 
Ricci, :tr . c.nd. oro-Nitz , :· .• E., 1938 . Tho r-s irr.ti n 11."1d 
fertilizable life cf t.r~~cin egrs undvr st~ril0 end on-
sterile conditions . Jour . Fxpcr. Zeal ., 79 :1~0-143 
1939. Crys tc.ll inc cchincchrom0 ['.nd s pin0chrcmo: 'lbc r 
failure to sthm1Qt0 the r csp iro.tL .. ·n cf , £S c d f sperm 
of Strongylocentrotus . Yroc. nt , Acnd. Sci ., ~:523-528 
• 
r 
,, 
I ' 
I 
I 
I 
Astorias f rbosii (or A. ~1 ~ris) 
A) Obtc ining go.metos , nc.turatic;n rend f0rtilizntion: 
The sexes a.re sepc,rate in Asterins , b'.lt it is not possible 
to distinguish them on the bo.sis of exttJrnnl cha.rnctoris-
tics . Only animnls vd. th soft , bul f ing nrms ere· fully 
ripe , nnd it is n waste cf mrtcrirl to open smnll, hnrd-
skinned storfish in ~n attempt to obtain gametes . · Fill 
t?o 1750 cc . fin~er bowls wit h clean sea 1a.t0r from n 
2- litor fl'"tsk in 'lhich the edimunt has boon parmi ttod 
to settle by nbout fifteen minut 0s of standin~ nftor 
vJithdr::mnl of the soo.. v1ator from the tl:'.p . t ith largo 
scissors , make a small puncture in one arm close to tho 
disc , r.nd pipette 8. few drops of cells fro!TI the GOnad 
to ascertain the sox of tho r,nirnc,l . If tho animal i 
a femr-lc , renovo this arm ccr::pletely , ::md slit it c.long 
the mid- dorsal line to expose tho bulging pnir of 
ovaries , of a typicc.l pl:'.le SE'.li'lon color . Then v i th o. 
pair of forceps co.refully detach oach plume - like ovo..ry 
by grasping it ncar its point of cttcch~ent nt the disc 
end , closin8 the gonc_duct , ond rinse it •.;ith l:',s little 
injury o.s possibl e in the first bowl of soa. - w"ctor , then 
trr.nsfor it to tre second bowl. Tho nnimc.l from \':hich 
the o.rm has boon sepnrc.tod mc..y be returned to a separate 
aquariu.."TT of running son v1nter, o..nd r thor C'.rns may be 
used for ga.netes later in tho da.y . Such· o.n injure d 
f0mr lo will net keep indefinitely , however~ end gametes 
ere rc.ro ly us a.b lo at the tir.lC the c ninc.l b v p;ins to show 
autotomy . · Do not cut up to ovaries in the b c,wl of 
sea water , merely r.llovJ the oggs to exud e; fr or.1 tho blunt 
t nd of the ovo.ric s for C'. poriod of fi vc minute a . 
At the end of this ti~e, remove 
.... 
.. be ovo.rios tv r.nothor C0!1tr.iner, or discnr the.~ . 'Iho o.r€) 
;hcsc first shed . Gently stir the ··a. ter in the l~r -"e fi g r b 'il nnd 
;-loW the eggs to settle. Settling occurs vcrr slc~ly . Then r cff 
tbC sup€;rnntnnt sec. wo..ter c.nd cnrefull7 ro_lnce \'.'ith o.n oqunl vo 
fron a 2-l iter flask. r:l'hen leave tho eGgs LL'J.disttrbod, ':ithout s "..king 
~r stirring, for r,b:.:.ut 20 to 30 rr. inut c.s . Dt ring this tir~c s .. nll sr .. 1plo'"' 
--:c.y bo rerr:c.vod \'lith c. pipette for exm"lin:-.tiun under tho m:l.croscop , 
:nd the steps in germinal vesicle br~akdJ~n obs crv d. N0to the jelly-
hull about the eggs. This r..o..y bo der:wnstrr.tcd more rondily in dir 
illUinina t ion or by o..dding r. trace of Janus Green t ~~ one slid<' prcp".rn-
tion. Eggs fror.1 o. ripe fer.:c.le which v:o.s ·kept under proper conditions 
of coolness o.nd adcquc.to oxygen supply fror:1 the nomcnt of ..... ollocti~...n, 
and properly manipulated in obtaining gnmotcs 1 should shov,r 8 to 90% 
gcminc.l vesicle broo.kd oVll.1 c..t o.pproxL:-u:.tely the sa 10 time. Reto.in . 
~ood sample of eggs in n snc.ll finger bowl t c follow tho naturation 
~tc.ges t11rough the second poln:r division in tho uninsoninntcd 0 1_, s. 
If the anir1o.l opened is a mo.lo, the tos tos ';Vill be hi tc ~)r ivory. 
Since it is i mp ~rtnnt to usc o. fresh sperM suspensi• .. m , this e.nil.nl no.y 
be placed in a dry fir...garbowl until tho eggs r..re re nd-r for fortiliz['.-
tion. Then a single testis is removed, rins ed in clean sen wrter, ond 
u small piece from tho blunt end cut off o.nd placed in 200 ec oon VJntor 
To or three pipettes of this suspension should be added to n 1750 cc. 
finger bowl of eggs ~ with nn irnr.odiat ~., but net violent rotationo..l l710vc -
nent to ensure conplete :nixing . The aptinun pori, ·d f ·r fertilization 
is after tho brcf'.kdown cfl tho gornino.l v0s icle ·and of oro the 1st 
polar body ho.s be on extruded . It is, there fore, c ('nvoniont to insemi-
nate when tho distal end of the first neturc.tion spindle bec;ins to pro-
trude o.bove the previously sncoth surface of tho cO'c~rto, in n f"..ir por-
centngo of the eggs shovling go rmino.l v cs reclo bronkdown . ggs inser:i -
nuted in tho .stage of the intc..ct germinal vesicle are non- fortiliznble . 
Even tho they may elevnto o. fortiliz f'.t ion !!l0l7lbr[~no they do not develop 
further . Tho details of s porr.1 penotrc.tion r.~ny be rof'cdily studied , if 
the obse rve r oxnmines the o c;gs vJi thou t dolny . It VIas in tho egg of tho 
strrrfish thnt Fol (1879) first .;bserved the nctunl penetration of an 
egg by n sperm. Chambers (193 -:- o) hc.s confirmed these early · observntions. 
A microsc ape with clonr objectives, clean slides nnd covers , and good 
illum.ino.tion o.re prerequi sites for observing tho finer details of this 
process in the l c..bcratory . 
It nru.st be remonberod thnt tho egg of Astorias is very delicnte o.s 
compared with most eggs used for r outine lr.bcro.tory work . Sn.tisfnct.Jry 
results nrc not obtninod without tnking ndoqunte precautions. Ir.1port::mt 
preco.ustions are: (1) to o.•J;oid ccnto.minntinc; either t ype of c;o.moto with 
~eriv is cero.l fluid-it is becnuse :.:.f this tho. t tho t;cmncl.s arc rins o d; 
(2) ·do not overinsomino.te; (3) do n ·:.t crowd the eggs: therE; should be 
no norc than one lo.yor of well-spaced eggs on the bottom of tho dish] 
(4) use only fresh, r:iOt ilo s porn. 
Cleavage : 
The blustoneres of Asterins nrc ruther loose ly connected , because tho 
perivitelline space is ·wider and tho hyaline plr .. sma membrnne thinner 
and weake r than in tho Arbacir, ecg . N')to the r elc.tion betvwen tho fir3i 
and second polar bod ies ~2nd tho fertilization rnembrr.no . Cho.nbors ho.s 
pointed out thnt in the absence of fertilizo.tion mombro.ne , the blnsto-
no res tend to sopnro.tc crnnplotoly. Because of tho rolntivo trans- · 
purency of tho yolk of this e gg , details of living aste rs mny bo soon . 
A dctcilcd study of the cl ~~ngc of this fo~: is 
undcrtc.k::n b-r s tud~nts of t~~c our" c unlo'"' s "'"hv' 
spccir.l intci--cst in this ~ ·c.tcri~l, L 1. t. t:1 ~ lc.t.~.-r 
of considcrcblc i0nificc~co . 
Lc. tor Str. '":'OS : 
----.- ~--· 
suclly not 
h"'· [" 
s tt". , s ~ .. r..., 
To rc.is c Ar.tc.rir" to lntcc:r.~br;>~o ic s'·c.g<: t i~ n ~.f' c-.· 
to chc.ng.,:; the s · c. ,_..,ntcr in the cl.!lt 1r ... disrc.s "bout h:-tlf -
hour intcr\·c.ls curi·1c cc.rly clccH'.gv to 1 h:'i~1c. c the ~ .· IJ • 
sparr: V' 1lic~· V;Ov1lc ot:lCr'::h ·c f oul the cult rc· , Tl <'1, l"· cn tbc 
first SDi:rin~ stc.gc s (blcstulc.c ) cpp~cr , lhc u lr hnlf of t c 
cu~.ture , eontf'.ining tl: c :1.or0 non;:c.l toi:- - S'.'il''.Jli'11 ' bl~~tuln, is 
poured off into~ s e ries of t:ll bc.ttc ry j. r" nhich nrc u-
sequc ntly fill(,d to t:'c top v:ith fr , sb s•J::l v . .:-tvr . C;,l'C r11c·t 
be tckcn to f'.vo:o c :-~ rrying over d c.d ~r.bryos of unf ~rt lin; ud 
eggs . 7[).11 jc.rs nrc superior to "hf'.llov; dishc·s , s i 1 L ev['_ ort.-
tion in considcrcbly r educ ed . It i" assent ~ l th~t r c lntivoly 
f C1tl larvae be pJ.[>.ccd in c j c.r . .a...nrl~I bipi nc.r:t: L:~:.y bo obtci n<1c1 
v1ithout spcc:ir..l fc\.din[: but the cultures of h.stlrio.s lnrvC\o 
nus t 1-.:e fed di~ . tor.·c ( prcpnrc.d by Jus t 1 s L<.;thod«) to obtr. n 
brachiolc.ric. or lwtcr ntc.gc.s . 
Ref erences~ 
Chnnb c rs, k . 1 930 The r.u.mnc r of s pcr"l en try ::.n tho s tr.rfisl1 os(T . 
Biol. Bull 58 . 
Costello , D. l . 1935-portilizntion l~l'.n,rrD.nc~s of contrifug<.d 
As tc.... rir.s Egr.;s . Ph:;s iol . ZooJ • 8 65 - 72 
Dolngo , Y 1904 El cvC\~~ de~ lC\rvGs p~rth-no ~~n~tiques 
d 1 Astor io.s g l c.ctnlis . Arch . cc Zool . ~xper. 43 ~0r . 2 
Fol , H. l879 . Rechcrchus sur la focond~tion o t lc conwonc oiTent -
d e 1 1 hcro_on~.c . :.em . Soc . Phy . ct 1-Tist . Nat . Genove__g§_ 
Fry , H. J . 1937. Article (p . 547) in Culture Methods for 
Inv crt cbrc.t " Ani;·;c.ls , 0ritod by G['ltsoff, Lutz , etc . 
Consto~k u ~ l . Co . 
Gom.'11ill, J . F . 1 914 . 'lhe- d e volop1 ··cn t 1'\l1d ccr t 2.in point'1 in the 
ndul t structure of tho stc.rfish , Astorio.s rub,·ns . 
Philos . ~:rnns . Ro:'t • Soc . Lo,Jdon B . vol 205 . 
Just, E . E . 1939. Bc.sic nct'10ds for cxpcrminc.nts on egrs of 
of mnrinc c.ni!m:-.ls . Blc.kiston, 1-'l·i il. 
Lr rs on , L . J . Article (p 550) in Culture kethods for Invcrtc -
bro.to AniDc.ls edited by G2.ltsoff , Iutz . e tc . 
Cw1bs tock fub . Co . 
Astcrio.s Forbcsii 
B . DevclopQc.nt 
Prcpnrc. your ovm cultur:..s for C.[.rly stcgc.s ".nd u3o thos e 
pre pared by the c.ss is tr..nt for s t o..[;r· s frm blns tulo. on . J;c-
vclOJ-:r:'Cn t Ul-· to t~1c cr. rly Bipinnnrio. (J.:l ~ j,ilcurulo.) ern bo 
follovvod on l i ing Lo. t c rir.l . Older Eipinnr·. r :J c· , Brnchiolo.rin 
lo.rvc.e and mctc.r.·o):phos is co t c.gos vlill be stud i r.;d on 3 t[' incd v·lholo 
mounts . ·:~holE.. r.10unts of cr.rly Bipinnc.ric. ' r\.J c.ls o t. vr•.:ilr•blc 
for conp~rison . It is c.dviso.ble to begin ~ith early go.strul 0 
sto.gos end fol2 ow the dcvolopnc.nt through to tl•c Diplcurul£' . 
4 
Study f c. rt il iz c:. tion, clc!'.vr-.ge r-.. ' bl~.s tul8. 1 , t .r on us in T 
your m ·n culture s • . Consult the illustr".t .... o s i J. r.cBridt."' , 
Agc.ssiz (1877) c.nd Ge:.1mill (1914) uc ti:".e t ", Jc in ~-P nd~' 
1. Fertilizo.tion (s co lo.'c direc.tions for "I-ortiliz, tion") 
2 . _9_1C?_'::_~_:_ _Tnc first two cl c rv'"', 3 L." c.r o n r : dionf'.l, th .. t 
is, they go through o.nlr.:o. l r.nd vee;ctc.l pol e <· r.nd c..r p rpundi -
ulc.r to ec.ch oth(.,r . 'iho third clec.v".[C is '"~orri" on tf' 1, the 
ei5:'lt c ello of this stc.gc r:rc .:'.pproxir.; f'. t ~ l.r equr.l in size . 
In th~; 16-c cll stc.ge , no d efinite o.rrc.ngcr-:c nt of cells in rov1s 
to.kcs plc.c c , and clu~ vc.gc fro:~ nov; 0'1 is irrcgul :--.r. 'lhrouc:hou t 
these early cl cr~ vc.gc sta.gcs the blo.sto::cr c s hc.v o i tond t:. ~1c.y to 
a.ssui1C sp" ericc. l sh:-.p <.- , r esulting in c. r G thc:.r loon c nrr".n£jd1Cnt 
of cells . 
3. Blastula . ~vun~cuo.lly the cell" crrnn,.,.c ther:~.clv ' s in r.n 
cpi thcl ial v1r.1 l cnclos ing the blnstoco lo . 'lhc: s rf .. cC' c "lls 
c.cquirc cilic. , c.nd the blrstulo. rotate<"! witl':in t1e v it c llLlt..; 
membrc.ncc . The two pola.r bodies c.rc still visibl , e ither 
a.tt ·. ched to the c.ntnr.l pole, or dcto.ched fror~ the onbryo. The 
embryo ha. t chcs in tho lc. t o blo.s tulf'. s tc.ge. 
4. Ec.rly go.str~1.ln . 'Iho vcgcto.l pole. nr oc. thich ns r.nd 
flattens and i n vc.cination begins . 1hc blastopore is destined 
to become tr~ c c.nus . The l nrvc.. clon6 c.tcs o.lonc tllc o.nirr.c.l-
vogetc.l axis . 
5. _}g_d_dl_o_ L_~d __ l_~t E? gc.str:!:lln. The r;c,strulo. b oconos pcc.r-shoped . 
Tho blind inner end of tho nrchonteron b e come s thin-wnllod r.nd 
cxpo.nds . Frorr. this end mcs enchjl11C cells wnndicr out into tho 
blns tococle. In o. sl igr1 tly lt\ ter s tq:; c , two out pockctings of 
the dj_stctl end becor::c distinct , the prin:ordin of the. c oclor1 c 
so.ca. At the sr.rrJ.e tirr·G , the c.rchentcron b c. nds towards onu s· d e 
v;hich j_s tJ-~ e futur e vcnt r c.l side • . This is tr1c first s i c n of 
the chnngc of r<:.dial into bilo.torc.l sy:rr::etry . Note tho 
cil i~tion in the a. rchontoron . 
6 . Tr~~~~tio_~_t_~ __ Dil2_l_c:_~~-tQ£:~-~_r_va . Tho blind end of tho 
e.rchentoron bonds shc.rply towc.rds the vcntrc.l body wnll , no.Y.:c s 
contc.ct with a.n eotodormc.l depr ession , tho stomodo. oun, o.ncl 
tho r:10u th brco.ks throue:;h. Tho tv1o co cl or.1ic vcs ic.l0s hrv o b ee n 
cons tricted off fror,1 the etl"'che ntcron.--T-hclcft. orie is 
l £>, r gor from onrly s t nce s on . It fon"'l s o. tubular out(Srowth 
to the dorsc.l body wnll vihich op ns to tho outs id e. . This is 
the .P_Qro_-:_c c. n_€-..~ .• 
7. f.uJ:.l.:~ _f o~'l_ e;_d_ p_~p_l_(Y:~li: __ (_ O;"·_rl y Bip_i .!.nc.rJ..n )_ ~arv~": . Th j s 
lnrvc. represents r..n oc.rly 1o.rv o.l type C01!.U:10n to Asteroid in , 
Echj ·1.oidec. , Op!:1iuroid or. ·"''1d I:olothurc c, (3o o Korscht lt , vol . 1 
p . 49 9 ). Study carefully c. vontrr l, dorsrl o.nd lnt ro.l 
( profc ra.bl :· loft) vi <:vv 
Observe tJ:"le follo ·Jing : 
Shape of l~rvc.. Notic e conv exity of vcntr 1 side c.nd 
mouth oponing--uridorriec.th the ovcrhr n g inc ornl lob o . 
r 
I' 5-
Locomotion 
Ciliation . 5mall cilia co e r the e~ti e surface . The ciliary 
band is at first continuous, a lone-itud !1al band with two cross ars. 
:he longitudinal band above the upper cross bar loo s to\·ards t1e 
rr.idline . Eventually the loops meet in the midline, and a frontal 
field , the pre - oral ciliary band is separated in the upper ventral 
parr-or the larva, overhanging the oral field . 1bis separate 
frontal field is characteristic of Asteroid larvae . bs rve care-
fully the course of the entire longitudinal band . Observe the beat 
of the cilia in dark field illumination , if available . 
Alimentar~raet . The three parts, characteristic of Echinod rm 
larvae : oesophacus -rwi th constrictioa ncar entrance into stomach,) 
stomach and intestine . Observe in lateral view the bend of th 
intestine . Study ciliation in oral field and different part of tho 
tract . 
Coelom. Study the two coelomic vesicles fron all sidvs. Thoy 
are clearly visi le at the lower end of the oesophagus near its 
entrance into the stomach . A subdivis ion of the vesicles is not yet 
clearly denarcated , but the narrow tubE; connoctinr tho larp(-'1" left 
coelomic vesicle with thEJ dorsal body wall , the .J2.£!:£ canal, and its 
opening . the madreporic pore , can be readily s eon . Loose mesenchyme 
cells are scattered in tb0 body cavity which is tht.. persistinB 
blastocoele . 
Vits.l staining wit! neutral red is helpful; but study unstained 
specimens firs t . Study a lso stc.ined whole mounts of those stages . 
The following stacus are r~'.ro ly found in dredges of pl ankt on and 
difficult to raise fr'Om 0gf:s in tho laboratory . Prepo.red and stained 
whole mounts will be provided . 
8. Fully formed Bipinno.ria . Consult figures in Gemmill , kacBr c1c , 
and Agassiz . This l e.rvc is characterize S. by a number of pn.irs of 
lobes or arms which g rov1 out from tht- margin of the cctodurm and 
which carry tho ciliary band a long . They arc not supported by 
skeleton . Pairs of arms foll~' each other in succession . Youn ~ 
stages may not havu a ll arms developed . 
Arms Identify mp2 ircd mudinn d orsal , po.irod r,ntcrior dorsal , pos-
t erior d~~nl , ~torior l c.tera l, pos~ora.l and pre oral arms . se ; 
Gemmill , late 18 , fig . 7 , and Mo.cBrido , p. 465. 
Intestinal Tract 
Coelom . The coslorr.ic vesicles hCJve gro··m out into long tubes and 
have fused in the ant-erior part of tho l arva . ·o further subdivisionc 
have yet occurred . 
9 . Brachiolaria and met_~plorph~~i-~_sta._ges . Study ventral o.nd latcre.l 
vie'!JS . Consult the exc0llent figures in Ge:··1r.lill, },llates 19 and 20 . 
The Bipinnaria arms are lonrr , hollow tubes. 
The three Brachiolaria arms (brachia) are s:1ort . 
diverticula of the c oelom. 'I'hey arE; not ciliated 
discs differentiate smnll papilla e and can adhere 
They contain 
but their end 
to the substrate 
r 
6-
Asucker, the gl anC. cells of ::hich s"'crcte [. sti ry s:t st.o.1 e , is 
"ormed bet·,;een the brachi.s. . Bra "lia anc. sucker serv f or nttnchmert 
;: the larva to the substrate in lc. ter ctages Of ;:leta 10r OS is • 
1vernmi ll , p . 250) . 
~~-tes tine 
:;:.:.. . 
coel om, in diff Jrcnt staga s of S'J. division 
-
21sk~ or developinG starfish, on left side. 
In late ~tages of metamorphosis, the r.nte r i or part of he larvn in 
front of disk shrinks to form tho stalk v·hic. is nttc chod to the 
substrate by sucker and branchia ar:.d which C['..rr:ttJs t o Asteries etnla c 
at i t s dis t a l end . For details of e tru;10rphos is consult Ge::rr ill , 
~acBride , and Korschelt. 
Echinaro.c_hnius parma. 
Ob t e in ing gru11etes 
T'he sexes are separate i n Echinarachinus (th.e s cmd d ollar) t~ut it is 
imposs i b l o to distinguish tho ma l e fro:n th\..' f,)malo by "upcrficinl 
examino.t ion. A cut is made about one - quart e r inch .f'ror. tho mar g in 
around tho .::: ntj r e s.nima. l thront h o ath orfl l c.no aborc.l c lear ~ ous 
ske leta l parts . Then D. scalpel is c~refully insort 'd , just boneo.th 
the ora l s kclutcm , scpnrc.ting the or8 l an d a oral portions . Tho orn. l 
portion is l ifted a'.Jay n.nd d iscard0d , taking c ro not to destroy th 
gonads, which adhere to the abora l portion . ~he uboral portion is 
then . placed (outs i de surfc. cc dovm) on a c l ean , dry Syracuse VJatch-
glass. If the anime.l i s ripe , gc.mo t o" \Ji ll ooz e from the g on .. ds . 
Allow the opened !nal c to ronw. in undisturbed unt il the or__~ gs arc to be 
inseminated. Tho ovorh.s of the; f (~mr.lo ara o. reddish purple color , 
and the e ggs o. r e usuc.lly mixod with an opa l es c ent or millcy pcri-
visc c r n l fluid~ From the f omnlo c a r efully pipette tho oggs · to a small 
fin ge r buv'.'l of soo. wat e r . After n1 lcwing thrJ eggs to settle, carefully 
pour aff the suporn:::tn:qt fluid o.nd re pl a c e ·. ith fresh sen wat e r . If 
tho f ema l es nrc n o t in cr, ood condi tion (if t ho o ggs do not ro o.di ly 
stream fr om the ova ries) , the c on,__ ds may be r <J movod with a. forc eps to 
a finger bowl of sea wntor, o. nd tho e gg s uspens ion stra.:i.nod through 
clean , washed choosocloth prev i ously so~kod in s en wctcr . 
Fertilizati on 
Tho eggs of Echino.rc. chinus nrc large r than thos c of Arbnc i n ( 135 
mic r - ns o.s compared 1.~1i t~ 75) o.nd sur~2_qu~ by a much thicke r j olly-
hul l i n vJhich beRutiful r ed p i gment . nUtes- nr c suspcndr.::d . 'fhu e gg 
itself , fr ee of t ho jolly , i s pal e: yellow . Bxrmino the unfertilized 
eggs under lm•r o.nd h ie;h magnifico. ti un . Then ins e;minc. t e thu o es o.n 
was dono in tho cas e of Arbncin , o.ncl ox~mino thu ogt":-.s i mnodia.tuly nf'tor 
adding tho d iluted s perm susp0nsicn . Bo co.use of tl1G rolntivC; ly l a r go 
size of tho egg , tho fortiliza.tion rt o.ction nr'.y b e reno·ly follovrf")d . 
r.·:omb r nno e l ev a ti on proc ..:ods f r ·1m t~ 'J cntro.r. c c point vf the t,p<; rm 
around tho egg cortex in o. vrc.vo . The membrane be c;ins to clove tv in 
from seven to t\•; cnty-two s o c unds o.f t c r sper m p<::nctrr. t i on , o.nd is com-
p l o t c d in from ninr.; to thirty o oc :>nds c.ftGr it tc~ins . Sinco perm 
7-
cc!'lctration occurs from fourtnon to fcrty- fi ve ~ ..:..cc'1 .... ft.cr in" o ni -
f.ation , both processes (i . e ., sperm pen·--tr~.ti(>n r.r.c: ''1m r~n e lL 70. -
t.icn) m['..y be co"':plct0d v:ithin nb::mt 40 sccc· .ds nftcr in 0! i .:->t on 
' Just , 1 919) . 
cleavage of the· egg of Ec~1.inarnc!1nius is not nnrk~Jly d ff~r~.,nt ''Om 
tho.t of Arbcci a , nnd unless this forn: is of spt.Jcinl ir.tcro~ t to th. 
student , dotnilcd drm··ings of the clcnvag' need 0t c 1~1ndu . On 
culture should bG prepared end kept t prv\·ido plut i for ccmpnri n 
dth these of Arbo.cir. . 
References : 
Just , E • .c . 1919 Tho fertilization reaction in l:.chinurnchinns .. nrm~ . 
1. Eiol . Bull . 36 : 1 - 10 
Just , E . E . 1923 Vl . Eiol . Bull . 44: 1 - 9 
,... 
r, 
8-
Bibli~aphy o~ Echinoderm Develo~er.t 
_, :Jorrr.al devel opr:ent and metamorphosis 
Aga ssiz ; A. 18'77 . Embryolog of the tarfish . •-em . ·:us . Com • Zool 
Harvard , vol 5 (first publ . ir. 1864) . 
Bury , H . 1895 . ;!e tamorphosis in Echinoderms . ~Juart . J . ,:ic r . Sci . 
llOl. 38 , 
Chadwic k , H. C. 1 1 914 . Echinoderm larvae . Liverpool I1 arine i oih . 
Committee 1emoirs , No . 22 . 
Gemmill , J . F ., 1914 . 'l'he development and certain point in the 
adult strucb.u·e of the starfish, Asterias rubens . Fhilos . Tran • 
R. oy . Soc . Lond on ( Ser . B., vol . 205 (excellent figs . of Bipinnar n 
and Brac hiol ariaJ . 
Got o, s . 1 898 . The metamorphosis of AstDrias pa .. lida . Jour . Coll . 
Sc., Imp . Univ. Tokyo , vol. 10 . 
Grave , Caswell, 190Q, EmbryolOGY of Ophiocoma ec\linata . 1'1em . Biol . 
J ohns Hopkins Univ ., vol. 12 . 
Harvey , Ethe l Brovme , 1939 . Arbacia . Collecting Ne t , ol . 14 
-~---------, 1 941 . The cyt ol ogy · of fertilization and cleavage of 
Arbac ia punctulate .• Turtox News , vo1 . 19 1 n o. 7 . 
Hoe r s t adius , Sven , 1 939 . Ueber die £n twickl ung von Astropect cn 
auranc i acus. Pub~ . Staz . Zool . Napoli , v ol. 17 . 
MacBride , E • . i . 1903 , Development of Echinus esculentus . l hi1os . 
Tr ans. Roy . Soc . London , Ser . B., vol. 195 (good figs . ) 
Mor t ensen , Th., 1 931-1938 . Contributions t o the study of the 
development and larv a l forms of Echinode r ms . l - 1 V. r.':em do 1 1 Acad . 
de Sci. Copenhagen . Se ct . de Sc i., Ser . 9 , vol . 4 nnd '7 , 
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Echinodermen . Abh. Kb'n i gl . 1-.kad . 'l'liss • .[jerlin 
S chmidt , H. 1 904 . Zur Kcnntnis der Lnrvenentwicklun v on Echinus 
microtuber cu1a tus·. V Grh, f'hy . - me d . Gesc1ls ch . '-Jurzburg , vol , 36 
v on Ubis ch , L. 1937 . Dio norma1e Sko l o ttbildung boi Echinocymus 
pu sillus und Paammcchin'.ls miliaris . Zuitschr . ·,iis s , Zool ., vol . 
149 (good fi gs . of origin of l &rval s ke l e ton) . · 
11. Experimenta l: gono r a1 r eviews: 
Brachct , J • 1 944 Embr y·clog i c Chimique . rliass on Z:. C ic ., l' aris 
Chi l d , c. Ivi , 1941. Patterns ond problems of dovolopmont . Univ . of 
Chicago Pross . 
Jtnlcq ., A, 1928· .Los BasEJs 1·hysiologiquos de lo. Fc condation . Pr essu8 
Univ. of Fr an c o , P ~ris . 
9 
alcq ., A. 1938 . Form and Csusality 
·' 
Fauro -Fru~ict , E. 1925 . La Ci~otiquc 
Univcrsit~rios de France , Paris 
nEarly De·~l o mcnt . 
· ,. Cr..n. rid;re Tniv . fr"'"' . 
cv lo:pr :ent , ro"' s 
Horstcdius , s ., 1939 . 0 ~ochanics of sea urchin dcvclop1ent, 
studied by oporr. ti ·rc methods . Biol. Rev . 1 14 : 1 - 17 
Huxley , J . and DeBGer , R. 1934 . Elenonts of ExporiLcntnl Embry~l . 
Lindahl , P. E . 1 942 . Ccntri utic-ns to th0 physiology of form 
generation i n the dc\·c. lop .. c,nt 0.f ti1o soa urchin. "'-U·::trt . Ho·-. Jic l . 
17 
Lindahl , · • E . 1 941. ?hysiologisch- chc.r.1ischo Pro lemv dor 
~mbrvonalcntwicklunR: . Fortschr. dor Zool. : 187 - 359 
~ ~· -
Tv'Iorgan , ·r . E . 1027 . r..xpor imento.l Embryology . Columbio. Univcrsi ty 
Prd.:s , Ne·: Yor .{ 
Koodham , J ., 1 942 . Biocho1~1 istr"'· , nd I1:orphogcn~}sis , pp . 476 - 502 . 
Cambridge Dniv . Iross , Londvn 
Schlo ip , \'i ., 192 9 Dc t crmina tiun dcr Pr i ni ti v - .Snt wicklung . 
542 pp . Akc.d . Vcrlagus . Leipzig . 
Tyler , A. 1 941 Artificia l 1-e..rthenogc;nos is . biol. Ruv . 16 ~ 291-336 
Ty l e r, A. 1942 Dovolopmontcl proc ossc s and cnor~cctics . Qunr t . 
Re v. Biol. 17: 1 97 - 212 & 339- 353 
Weiss , P . 1939 Principl es of Dovo loptc.nt . E~lt ~ Co . New York 
Artificic.l ~crthe .ogcnc~sis in h.rbr. ·r. r:-1d lchinDrachAJus 
In l896 -189G :·or bc.n , .~eed, Hcrt·.~ig- r.nc4 lo' dv:n nstr ted thD.t 
devol o ~~c-;.1t c euJ.d be in it i<.t E.d c.nC:. • or! .r 1 lrr ·nc bt~ i •d b;r 
trcC'.tr.!0nt of unf0rtilizoc en;s \:itL S [ lt solutions . Sin e ti r>.t 
time !'.rti~icicl rctivr,tion cf em;s :u'." C.:)el the su-....jcct of a 
great many invsstig~tions . (Sec r~vi by Tyler, 1941 end your 
cxp6rimcntr.l embryolOGY te;:ts for di~ c lS'"' ion nnd ref renee"') . 
The present exercise is cs scntic ly ~ r 0pct ition of Lol 1 s 
classical double tr~a~mcnt rncthod (~odifi d after Just, 1930). 
EquiJ2~~-nt: - Sci··sors , blunt force f~ , 6 11 squcrc of hoc"'c loth, 
10 finrer bo~ls , 6 syrccure dish0S 1 4 stond·r dishes uit~ lid 1 
3 slidE's r.nd covers lips, 1 grrduc.ted yi1-cttc ( 5 ml , ''ide tip), 
1 grc,duo.to (lOll nl) , 3 ordincry pipettes , 1 n£~.rrm•J long-tipp~d 
pipctt(; . 
Solutions: - 2 liters filtered scn - unt0r 
20 ml butyric o..cid sen vmtor (8 ml ·;10 butyric 
ncid ·t 1 00 ml sen Jat0r) 
200 ml hypertonic sec. wr. tcr ( 32 !Yll 2B 1l Nr Cl \- 100 ml 
sec wrtc r). 
Troo.tment: - Obto..in eggs of Arbo..ci o or of ·~ch innrrchnius ns pre -
viously dco cribod . Guo.rd r.gc. ins t ace idon tt~l intrn.duc tion of 
sperm into the dishes of eggs . To 3 r:1l of r. dC'1SI susyen"' ion of 
tho eggs o.dd 3 ml of the butyri c o.cid sen \'lc.tcr • .After 90 , 105, 
120 end 135 seconds ro~ovo samples '"i th 11.ot mor '~ thnn 1 ml of thC' 
solution to finger bowls contcining 200 :r1l of n~c. wr.tor . Dctermin( 
in YJhich of tho fou r bo-iJls t ho cggo show tho hi£)!0st porc~Jnt<tge of 
mcmbrnne ~' levo.tion . }'our off n.b out hclf of the VJrter from tho 
bowl to facilitate concontro..tine t he eggs , then trcns fer ~ o eggs , 
with not core t~on 3 ml of tho solut ion , to n s rrduntc and m ke 
tho tote'.l volume up to 50 mJ. with s en \Wtcr . At 10 minutes rfter 
t he sta rt of tho cut:·ric ,. cid t rcotment p our tho og8S gently into 
o. finger bowl cont:i~ing 66 ~1 of th0 hypert on ic solution . After 
20 , 25, 30 c.ncl 35 mi11.u tcs in this s ol1.1. Lion trc.ns fer scmple s , with 
not more thnn 1 ml of solution , to 100 ml of sec ~oter, wn~h 
once nnd cul ture in covcrGd finGer bo~ls or stander disMas . 
Devc lopn-, cnt: - .:.~~nminc.: , r.. t intorvrls of about -~hour for 3 hours , 
some o-.:Ctht eggs t h<:.t hnvo r ::: c c,ivcd only t}lO butyric l'Cid trent -
mont nnd note the r epeo..to d formation nnd disopponrnnce of r 
mono.s t e r. In tl"o doub l y trentcd cggo loolc for r~ccc:...sory cytrotcrs 
and ~ or amphinstcrs . Note tho time rnd po.ttcrn of clcrv~~o rnd 
s kctc~ some of the enr l y cl cnvr•.gc ntnccs • .t.t r.bout 3 hours osti -
mo. tcthe pcrcc·1trgc clCCVC[.C rYld tllc 10llO'.'i·~.nr rl[•r th pcrc<ntr~'rO 
of <~t o i; svlim:.-c r~}1 in the four dishes • .-{CJ.10\ '~ t'b c to sv1immors t o 
c.nothcr bov11 , 8Ul turo f .>r two or tl-).r0e mor e d ~_::; s :-..nd es timr. t.;c 
the perc~.mtc.go of nornnl plutci obt r..in( d . 
Refcrcnco~: -
E crt\·IIg ~ · TC 1 '396 , F 0::: ts chrift f{ir C".rl Gr,gc n1":'ur , 2:23 - 86 
Just , E . ~ . 1939, Basi c ·c t hods for ~xpcrincnts on Lrpc 
Lo eb , J . 1899, Amcr . J . J·h.ysi ol ., 3::135 - 138 
Ii:eo.d , A. D. 18q8 , Bio: . Lecturer-. , t . 1' . I . Gth Lr ct ., 1-'P 203 - 218 
Morgen, 'l' . H. 1896, Al1 Ch . J..,ntvJ ,-n,o ch ., 3:339 - 3Cl 
Tyl er , A. 1941. Liol . 2-c v ., 16:291 - 336 
Specific Ineracting Substances of Eggs and Sperm 
p. R. Lillie (1912) demonstrated that ripe egg!'l of Arbacia and of 
1·greis give off a substance, called fertilizin, 'hi .h activates 
and agglutinat es the species sperm. SDnilar fertilizins have 
1 since been reported in many species ~f invertebrat s and they, to~ 
g:ether with the antifertilizins from sperm with ,.. hich they react, 
bave been the subject of many investigations, of which most of the 
more recent are listed below along ~ith some of the older ones. 
The present exercise includes more tests with this material than 
can be completed in the time allotted. Only the s~1pler tests in 
the first part of each of the following sections should be under-
taken during the class period assigned to the work. The additional 
material is presented as a guide t•' further w0rk for those who 
may elect such invest igations at the end of the course. 
Eq)i:emen.!:- 4 finger bowls, 50 syracuse dishes, 1 graduate (100 
, 1 graduated pipette (5 ml), 4 ordinary pipettes (droppers), 
1 large pipette ( 25-50 ml cap. + 25-50 ml rubber bulb), 2 
centrifuge tubes (15 ml). 
Solutions:- 1 liber filtered, sea water; 10 ml l N HCl; 10 ml In NaOH 
Fertilizin:- Obtain eggs and sperm of Arbacia by one of the usual 
methocfs:-.Vash the eggs once and concentrate the suspension to about 
25% by volume. After about 15 minutes mix 2 dr•ps of the supernatant 
egg water with 2 drops of a 1% sperm suspension (one drep of "dry" 
sperm in 5 ml of sc::a water) and examlne v;ith the microscope. Note 
the agglutination of tho sperm and, a fcv1 minutes later, the r e -
versal of th8 clumping. Are the sperm still motile after r eversal? 
To 2 drops of a strong egg-water (in which eggs have stood several 
hours, or obtained by acidification-see below) and to 2 dreps of 
sea water in a control dish add 2 drops of 1% sperm. After rover -
sal of t~e agglutination add 2 dreps of egg-water to each dish. Do 
the reversed sperm ro-agglutinate? 
To 5 ml of a strong egg-water and to 5 ml of a c~ntrol dish of 
sea wa tor add 1 drop of 1 dry 1 sperm. Shako the dishes . V·ihat differ-
enc e in behaviour of the dreps of sperm de you ebs orve and how do 
you account for it? 
To 1 ml of a strong egg-water and to 1 ml of a control dish of 
sea water add 2 drops of 1% sperm. Aftor agglutination has r evers ed 
add 1, 2,4 and 8 drops from each to dishes containing 5 ml of a 
dilute suspension of eggs (about 101 eggs per ml). Determine tho 
percentage fertilization in each of tho 8 dishes. Ilas the egg-
water treatmen t had any effect on the fertilizing povwr of the sr,crm 
Titration of fertilizin solutions muy be done by testing SGrlal 
dilutions of the solution with a standard sperm suspGnsion. 'l'he 
dilutions may be prepared with c.n ord inc.r y pipe tte (dropper) u~ 
follows:-Placo 2 drops of sou water in each of a set of dry dishes . 
Add 2 drops of cgg-vmtcr to tho first dish, rinse tho pipette 
with sea water, mix tho drops, drav1 up most of tho mixture, expel 
2 drops into t he s e cond dish and r eturn r emainde r to tho first dish. 
Re peat this procedure with tho succeeding dishes. Then add 2 drops 
of 1~; sperm to o 8 ch dish and oxnmine at on co. 'lhe first dish con-
tains a four-fold dilution of the egg water, the s econd ~i~pt fold, 
etc. The fertil:i.zin titer can be expressed as the r r nt ·Jst dilu-
ti en of ogg-wa tor thnt gives & microsco}Jico.lly porcc..ptilJlo 
agglutination rea ction . 'l' itratc your erg water usjng e ight 2 -fold 
dilutions. 
I 
I 
.. -
S cific Substances • 2 
~vidence c oncerning the sourc of the fertilizin may e obtained 
rror.1 the follm·:ing tests . Divide about 200 ml of u freshly pre .. rod 
10~ eg~ sus~e~sion i n t~o equal pars and aci.ify on· part to about 
vH 3 t.o 3 . 5 (recf: .. lires ab out 1 ml of • HCl r 100 nl) . A f~.,w 
;.inutos later dre.w off '51 n:l of sup rno.te.nt fr n;. each dish, 
nclltralizc tho acid t::gg- :w.ter 'li th J. 5 ·:11 of L' 1'n..OH end de term n 
the fcrtil1.zin titers. Excr.1ino the acid-trE:.ntod ~g )s w th the 
microscope and note tho abs en ce of the gcletinous co"t. N-utrnlizo 
r.nd IE.sh the- ucid-trE;a.t od eggs. After scvc.~al hours deter inc 
fGrtilizin-titer along with that of the sb·ila.rly agud con rol. 
Acidify tho contr0l to pH 3 to 3 . 5, dro'i off th . u crnatant , 
neutralize cnd compare its fertilizin titer with that of the first 
acid - egg- we.ter. Is thor o evidence ef secretion of fertilizin by 
the eggs? What is tho apparent source of the fertilizin? 
To test for activating action of f ertilizin alln~ n dilut· (1~) 
sperm suspension to stand for 1 hour or until motility hns d crcc.sod 
considerably ; then add 2 drops to 2 drops of a. strong eg~ \'/Cltcr 
and to 2 drops of sen wntor, Examine th~.. two dis GS microscopic~lly 
and n ote roughly the activity exhi bit ed by the pormr.tozo£1 . • 
Absorption t~.Jsts may be mr.de by o.dding r. concentrntud sp~;rm sus-
pension (10%) or greater) to an equo.l volume of moderately strong 
egg -water, centrifuging aftor 1 hour nnd testing the supernntnnt us 
well as a similarly dlh~tcd s ar1plo of the egg-vmtor for o.gglutinat -
ing action on dilute (l7c) sperm. Docs c.bscnce of aggl utinating 
action nec ess~rily moan binding of fertilizin by tho sperm? Vfuat 
other ~es ts would be nccc.,sso.ry? 
Specifycity mo.y be examined by t esting Arbncin f ertiliz in on sperm 
of closely r oL..t od o.nd distc.ntly r el nt ed c.nimo.l s, that nro 
availabl e in tho l eboratory. Whe r e r eactions c..re obtc. inod absorb 
the Arbncio. egg -vw.ter with tho foreic;n sperm, r.s described c..bovo , 
and t est the supernr.b :mt on both s oc i es c.nd f oreign sperm . 
Antifortilizin:- This mr. t cria l m£'..y be prepared from a concentrctc d 
Tl0% or -greater) sperm suspension by (a.) freezing c.nd thuvling tho 
suspension, (b) he a tinG to 1oooc . for one minute or (c) acidifying 
to pH3 . The tr~E t o d suspension is t ncn ccntrifucod or filt er ed 
and the supcrnc. t o.n t or filtrc.to will be f .ound to c ont r~ in the nctivo 
mo.terinl . 
To demonstr ate tho agglutinating action of nntifcrtilizin add 2 
drops of t ho solution prepared by me thods , o. , b or c to 2 dreps of 
a 1% sus pens ion of fr eshly wc.shcd eggs . S!:-;:-kL. -tho dish several 
times and cxc..mine macrosc opicclly c.nd microscopic8lly c.fter 1 to 
5 minutes . Note the formo.tion of a precipitati on mombronce on tho 
surface of tho ge lntinous coo. t of the egg . 
Titration of the ant ifertil iz in is perf armed in t:.,o m:1nner def>-
cribed ab ove f er fortil iz in. Pr epnr c n s ot of 8 tv:o-fold s or inl 
dilutions of 2 drops of the antifort i l izin solut ion c.nd add t o ce.oh 
2 drops of c. dilute (en . 100 eggs pe r drop) fr Gshly wrshod, egg 
suspension . Examine at once and c. ~Pin afte r ~ hour . ~oto differ -
ences i n widt~ of tho gc luti~ous c ent nnd in tho pr ~ cipitatlon 
mcmbrancc in tho different dilutions and a t different times . T~ 
end point mc.y be tc,kcn o.s the. t di l ution beyond which r. pre cipi t o. -
tion membrane is no lon ge r visible . Dctcrmino tho titer of your 
preparation . Docs tho ogg - agglutinntio1 r oc.ction r everse 
spontane ously? 
To demonstrate neutralization ~f the fertilizin add 2 drops of 
strong nntifert i liz in solution to 2 drops of a modo r o.toly s trong 
egg-water . Pr opo.r0 a control of 2 dro:r-s &f egg wa t er plus 2 drops 
of s en wn t or. After i hour add 2 drops of c 1% spor m suspension 
Specific S bstanccs p. 3 
to both dishes. !!oto the degree and duration of the agglutination 
rer.ction. Titrntions may be performed :ith d plic~.te seri~l 
dilutions of the egg water to one set of vhich is cdded censtcmt 
emount of the o.ntifertilizin solution v1hile tht; other gets o.n 
equal volume of sea water then sperm r.ddc-d after f1 hour; or \ ith 
dupliccte surinl dilutions of the cntifcrtilizin lus const~nt cgg-
wc.ter to one set , nd sea mter to the othe:', th~n eggs nddcd "ftt-r 
~hour . 
Rcferences:-
Ccrter, P . s., 1932 Brit . Journ. Exp. Biol ., 9:253-263 
Cornman, I ., 1941. Biol . Eull ., 80:212-217 
Frank , J. A., 1939, Biol. Bull.,~6:190 -216 
Glaser, o., 1914. Biol . Bull ., 26:367-386 
Glaser , o., 1921. Biol . Bull ., 41:63-'72 
Hr.rtman, M., 1940. No.turwissenschcftcn 51:807 - 213 
Ho..rtman, M., R . Kuhn , o. Sche:.rtnu c.nd r.-vJnllenfc.ls, 1939. Jcturwis 
27:433 
Hurtmo.n, F., R. Kuhn, 0. Schrrto.u "nd K, Wr..llenfcl , 1941-:--rr..tur\Jis 
28:144 
Hurtmo.n, M. o.nd o. Scho.rtnu, 1939. Biel. Zentro1bl . 59:571-587 
Ho.rtmnn, M., o. Schortnu ond K. ~~llenfols, 1940.Bioi: Zbl 60:398-
-423 
Just1 E. L., 1915. Biol. Bull ., 28: 93-114 
·Just, ~.E ., 1919. Biol . Bul l., 36:11-38 
Just, E . ~., 1930. Protoplf'.Sm2., 10 :300-342 (n rC;vicw) 
Kuhn, R. and K. \'irllenfels , 1939-.-Bcr. dtsch Chom Ges 72:1407-1413 
Kuhn , R. o.nd K. 1iinllc'1fels , 1940. Ber dtsch Chem Ges . 73:458-464 
Lillie, F . R., 1912. Science, 36:527-531 ---
Lillie, F. R., 1913. Jour. Exp. Zool., 14:515-574 
Lillie, R. R. 1914. Jour. Exp~ Zool., 16:523 -589 
Lillie, F . R. 1915. Biol. Bull., 28: 18-33 
Lillie, F . R. 1 919 . Problems of Fertizo.tion. Univ . Chicago Pross 
Lillie, F. R., Eiol. Bul l., 40:1-23 
Ll~:! . i.e, F. R., cmd E . E. Jus-:r-;---1924. Fortili?ntion . In Cowdry's Gen. 
Loeb , J. 1914, Jour. Exp . Zool. 1~:123-140 Cytol 
Loeb, J. 1915, f.mer. No.turc.list,-49:257-285 
Medem , F. V. 1942. Biol. Zentralb~ 62:431-446 
Metz, c. B·. 1 942 . Biol. Eull., 82:446-454 
Me tz, c. D. 1915. Eiol. Bull ., 89:84-94 
ront r. l cnti , G. rnd O. Schr.rtnu-:-I941 Pubbliccz. Stnz. Zool . Nr.poli 
Moore, c., 1916, Biol. Bull .• 31:137-180 19:48-55 
Moore , C. 191?, Blol . Bull. 33:285-295 
Nomurc., s. 1924 Sci .• _ Rc.P • Tohohu Imp. Univ . 4th :::,er. Biol 1.:83.-87 
Richc.rds, A. o.nd A • .w .. .. oodwr.rd 1915. Biol . Bull.. 28:140-148 
Runnstrom, J .• , A. Tisolius o.nd i. Vo..ss cur 1942, Ark-:-f.I\cmi l5A: i'f: 
Runnst rom, J , A. Tisclius rnd ~ . Lindvo.ll ,l944 Nctur c 153 016,1-18 
Runnstrom, J.:, 1-. • Tis c liur. end s. Lindv~ll, 1945, Ark. 285 
f. Zool . 36c:N:o 22,1-25 
Sampson, M. ~., 1922, Biol . Bull. , 43:267-284 
Southwick, 1Y. E. 1939 , :Oiol. buJ.l ,--n- :147-156 
Torno, A. 1926, Sci he ~. 'l'ohol:u Imp-.-Univ . 4th ser, Biol. 2:127-132 
Tyler, A. 1939, Proc . ~ct . Aco.d. Sci ., 25:317-323 
Tyler, A. 1939, Proc . 1 r. t. Aco.d .• Sci ., 25:523-528 
Tylor , A. 1940, Biol. Bull ., 78:159-178--
TJl or, A., 1940. rroc . "Jc.t . Acnd. Sci., 26:249-256 
Tyler, A., 1941 Siol. Bull ., 81:190-204--
'Tyler, A. 1942, 'f. est . Journ. Surg. Obstet, & Gyncc . 50:126 -138 
S · cific CJ -;.~· t::-., c ,< 1 . 4. 
Cy1c::: , A., 19~2 , l-roc , -''1t . A'2::.d . c ... . , 2~~~91 -:rt 
':.'y1Gr, ~ . r-nd s . · .. . Fox , 1339, Scic Cl oo:51 - ,17 
'lylcr· , P .- c.nd :.., . ';· . Fox, 19£20, B5.o1 . f'..J.-~ ., "f(2 :1 ~'7, -1)r: 
Cy1cr , r~r..d Y . C1i'clvcny:; 1 9·H . "iol . 3u.~l-.-8 ::::6·~ -~74 
i·;oodvc.rd, 1 .. -- ., 1918, J o r. K"P · Zool ., g_:459 - "'C0 
c., .... 
Curt Ecrbs t in 1892 dis co or d trH' t tree- '·1 •• -nt o.f t v lo. i. · sco;-
urchir:. eggs .... ith sec. \''nter contni':.in[ lit! iam sc.lts r:),'llts in 
th '- forr:-tion o exogc.strulc.c and ot.1cr r t. l['.t .d t.-. s of . bnor. 'l.l 
cn-...ryos . <is hc.s been the subject of n-w·1crc l." iuv sti5Ltions 
since tr..r,t t:Lr.:.c (Sec Child , 1940, 1941; Lind~.l, 1 .tJ ~nd .~our 
cxporir:1cntcl ~brrology texts for co .p o rt.: fc r n s) . The x-
ogr.strulo.ting c.ction of the lithiu· is i-:J.tvr rctcc1 r: "' r "'Ult 
of r ~cnerr.l cgctr.liz~tion of tht. eGg in vh. ·h t1c v 0r t~l, 
endod~- rsr-. 1 c.nd w.cs '-'n h.{t: r 1, nc tar ir 1 :in rcrts " · t tl o o.- o~ r-. 
of the nnino.l , cctodornr-.1 , natcricl . ther t ·.nts ;1"'· Y brin:" 
cbout this effect but none c.rt- , C." yet, kno\'il to give .. "' 
tent results "'.s lith iur:1 . 
f_gui :r.wnt: - Scissors, blunt forcep", 6 11 squcr 
3 finger bO~lS 1 12 ayrCCUSO dinhe• , 4 ~t ndLr 
3 slides o.nd covorslip8, 1 gr~dunto (100 rl) , 
1 no.rron long - ti pod pipette, 1 fin o pipette . 
of lt'un t.c~ ot.1 , · 
dis}L, ·;it.h licl.>, 
3 ord:in .. ry pi ott vs , 
Solut ions: - 1 litor filter ed sec. - water 
--- -- 100 ml Li - scc. - vJetcr (20 nl of m/2 Li.CJ.·~ 80 nl so~. 
v1o. t or) • 
Treatment~ - Obtcin eggs o.nd sperm of Arbncia or of 1 hinnrn chniu8 
intheusur.l vmy . Inse1~1jnc.tc a fr.irly L .r rc sc.mplc of the .r:Gs 
in o. f ine·cr bow . At the tine of first clo8. V~\c;v trr'ns fer n spmp1 o 
with not more then 1 ml of s en wo.tor , tor. f:i.lJ.f'Gr bowl contc· ln~'\g 
e.bout 50 ml of th o Li - s oo. - vmtor o.nd , o.t t:1o snr.10 tir:c, another 
so.mple to c. control dish of s en \!'Inter . J~ftor 2 , 4.,8 ond 12 ho11rr> 
trans f e r s crn}-les (co. . 0 . 05 ml) throurr,h thr uo dl }•~.,,.-. of 10 ml of 
s en wo.tor and culture in ho.lf-fillcd , covor 0d , sttndcr dishes . 
Development:- Observe the eggs o.t vr:rio1s tir·t s during tho lit''hll 
t~~(; o.tmont and compo.rc their r ate r.nd form of cleavr•gc w:i.th t1! 
controls. Ex<'mine tho cul turcs tvlico n dry durins the n uxt thrc v 
drys , r.nd sketch vo.rious types of cxocnstrulac , notinr inhihition 
of development of o. r ms o.nd octodorno.l s tructuros, triparti t o 
structure of a rchenteron, etc . DctormlnO th o rp~roximo.tc propor -
tion of normal to z.bnormr-.1 embryos in the four cul turc" . 
Li-tro o. tmcnt of Is ole. ted Animnl Hal v c s . 1-..n i mc 1 he 1 vcJs of s or -
urchin e-ggs isolatc~d-·:fi-1 '-the 8=-or 1 6-:.-ccil str gc fo.:n to r;nstrulatc 
g cnoro.lly forming 11 Douorblc.s tulno it . Von Ubin ch (1 929) ma.do tho 
interesting discovery that lithiur" tror,tr:1 cnt would cn ... blo some 
of tho o.nimc. l helves to dcv t.. lopc in t o normel plutci. Students 
who a r c skillful in micro - dissection mny substitute this ex -
periment in pl o. c e of the above , eSter discussing d e tr'ils of 
procedure with the instructor . 
Rof orcnccs: -
CLild -, C . !" ., 1940 l)hys iol . Zool . 13:4-42 
Cl'ild , c. f{ . 1 941. 1-'nttcrns nnd lroblL:n8 o " Dovelop,ment . Un:tv . 
Herbs t,C. 1892,Zoitschr. viiss . Zool 55: !l.46 - 18 Cl, icr.ro Pre::;::: 
Lindahl, P . E . 1940, Arch . 1ntw .-mcch ., 1<10:168-19'1 
MncArthur, J •.. J., 1 924 , Biol. Bull , 46 - 60 - 87 
Von Ubisch , L . 1 929 , Arch . Entw .-!':lC ch ., 117 : 80 -122 
Tamini, E ., 1943, Reale Institute Loml o.rd o (H e; d . Sci . ) , 76 :36?>-:)92 
Developnent of Is~lnted n)-".s tomc r es of t r r.ci"l. 
and 2chin~r~ch1ni s 
Hans Dries ch in 1891 denons trc. ted thr. t 1hol e c :1 ryos mo.y be 
obta.ined fr01:1 is ole. ted blr.s toE1eres of the soc. -urchin e gr , The 
present exercise is essenticlly n repetition of th".t clcssie~l 
experiment wi t h s o-::1e addi t ionrl fuc b1rvs provided by l~ter v1ork. 
Equipnent:- scissors, blunt forccpts, 6 11 squer<.. of che sccloth , 
3 finge r bowls, 12 syr~cuse dishes , 3 stondor dish s with lids, 
6 onbr yol og icc. l 1o.tch gl asses , 1 t es t tub e (en . 5/8 11 x6 11 ), 2 fin~.. 
glass needles, eyepiece micrometer , 3 vusoline -rincod s lid s r.nd 
coverslips, 3 ordinc.ry pipettes, 1 narrow long -tipped pipette, 
1 fine pipette. 
Solutions:- 1 liter filt e r ed soa-wcter 
100 ml hypertonic seo.-wGtcr (30 ~r~ms !a.C l in 1 
lite r of set:t-W['ter) "/'taJ.cQ_ 
100 ml of Cc -free s c~ -VJf'. tor ( 1000 m1 t/2 r 22 m1 
M/2 I\Cl ·; ·195 11 m/3 1gC l2 . 61 20 ~· 1G3 1"11 V3 Na2E>o4 t-
6 ml :/2 NuHC03 , adjusted to pH 7 . 9 - 8 .3 (based on 
Lymc.n end F l eming 194~. ). 
5~ ml of 5% formnlin in soa-wnt r 
Remova l of Fortilizo.tion Membrane . For the purpos e of isolnting 
bln-stomeres--the--fe-rtiliza.tion monbr:"'.ne must first be remov ed . 
This is Dccomplished by shr.king c suspension of freshly fertilized 
Gggs. Obtc.in eggs nnd sperm of Arbo.ein or ef Echinnrach@nus in thG 
usual way. Inscmino.te (noting tine) a. s nmpl o to t est fo'r4--'fortil-
izabil i ty a.nd to de t er mine first cle nvcge tine . Te n minutes lntor 
fill a test tube a. bout four -fifths f ull with c fre shly wr shod 
sample of eggs a.nd insc~innt e , mixing by inv e rting the t es t tube 
once. About ono-hnlf minute lnter pour about ~ of tho eggs from 
the t est tube gently into r. syrr.cuso dish end o:;remine for momb r une 
c l evc.tion . Vvhen t he mcr:lbr r.nca have scpuro.tod from the surff'.ce of 
pro. ctic o.lly nll of t he eggs (o.bout 1 to 2 minutes o.ftor ins eminn -
tion), sho.ko the t es t tub e t en times r c.p idly up and down using o 
full foro-o.rm swin g end holding long 2x is of tube in direction of 
swing with thumb ov e r open ond . Pour r.bout ~- of t ho eggs into a 
syro.cuse dish, i m.'Tio dir.t oly shc.ke tube o.gr.in ton times , r emov e 
nnother ± of the eggs , r op en t n third time c.nd r em ov e the r e -
mo.ining -;} . Exo.mino the thr ee dishes of shnkon egg·s nnd s ole ct the 
one contnining tho highest p orc cnt f'.go of nnked cr,gs . Vvo.sh t wic 
with filter ed scn - wc.tor . 
An alternc.tivo me thod of momb rc.ne r omov:1 l us es n p ipe tte v1ith a 
nnrro~ opening . Tho opening c~n bo scpe r o. l tiMo s the ogg d i ame t er. 
f.. concontrr-,t od suspens ion of eggs c.t the time of ncnbrono elcvn -
tion is r o.pid l y dr o.vm into t ho pipette , ".nd oxpcllcd iYJ.to vn 
empty dish r.nd oxo.nined . If one trcc. t mon t is inadequate it may 
b e r opoo. t od . 
S cpo rn t~~n of Blns~~m~r c~. · For this purpose the s tudent m~y usc 
ei thor the Co.-fr ee sor. -v10.tc r (Herbst , 1900) or t ho byportonjc 
sco. -wnter (E . B. Hr.rvoy , 1940) li s t ed above . 'l.he time of fir t 
cl onvngc is de t e r mined on tho control s ompl o thr. t vJns f ort il izod 
10 minutes b e fore the cxpo rimcnt o.l set . 
Isolat d lasto~~r s p . 2 
-
2. C.Ufre c s ec. - w&ter . At ubout five :.:in tcs befor e t'l-}e tine of 
first division ,--c onccntrn te the mer1br ano -les s c g s in the c ent e r of 
the dish by gentl e revolution of the dish (tho c~ntor of the dish 
should follo1 the circ~~ferencc of~ circle n ott l to 2 em . in 
dinnetcr) nnd transfer, with the narrow long - tip od pipottc , o 
s <1mple of the eggs with less thn.n 0 . 1 ml of seo.-woter through throe 
dishes of 10 r::l of Cn - frcc s co. - we tcr. Ex"ninc, un cr high power, 
so.mple of tho eggs in the Ca - frco soc. v ntor end co1 pu r o their 
ccto l"snic l c.ye r with tha t of the control ggs ! ~ftcr 1 0 to 20 
r:~inut c s renovo c. s~nple to r. dry finger bov•l . lf tho blnston~...r0 
hr.vc not sepe.rc.tcd dr r.w the so. .l le rapid l y in <..nd out of the 
pipette severnJ. time::> . Fill the bo\·!l 'I:Ji th son WDtcr r.nd trr.nsf ·r 
onc e to o. s t ende r dish hc.lf fill ed with fresh sec. V!O. tor r.nd ovcr. 
This will s e rvc c. s a r::as s culture of is ole. t ed blc.s torlCres nlon[~ w th 
so:ne whole eggs . To :::>tudy pr.irs of blr.stOl:lCrcs from the sc.J;c Cf,[j 
pick out of the Cu. - free s co. we.te r dish, under th(, dissecting micro-
scope, eGgs i n which the bl~st omer oo r.re still toccthor or r.ro 
close enough t oge thcr to -be r e co[;nized ns sis tor . 'Irc.nsfor eo. ell 
pair nlone with o. whole esg to ~ s opo. rr.t e crbr yolo(icc l watch g l nss 
containing s ea water . If the blc.stomcros of tho rair v10rc not 
compl e t el y sepc.rr.ted r. t the tine of selection bounce the egg in 
the dish o. fc'.'' t i rr.es or s opc. r c. t o the b l ns t omo r os by manns of n 
glass needle be for e trnnsfe rring to tho soc. wo.tor . •• ftc r one or tv1o 
cl ec. vages n oun t r. pnir of is ole. t ed blu.s t omo r cs c. long with n whole 
egg on each of tv1o or thr oe of the vc.s ol i nc -rin[.;cd s lidos . To do 
this plo.c c n smc.ll d rop conto.ining t he O[!,gs in the center of the 
ring , c.dd a coverslip nnd pr <:.ss it dovm so thc.t it touches tho drop 
c.nd c. continuous s oo. l is nude v1i th th e v<1se:line , but c.void having 
the drop touch the vc.seline . 
b . Bl:E erton_~~_2_oo.._:-v!_~.._t_~-~· - With this ne thod, t he e ggs (on e drop) o.ro 
plf\.cod in the solution ( ::>.bout 10 r.:l) wh en most of t hcr•l hnvo jus t 
cor.1pleted the first cler.vc.go but before maximc. l s epnr a t ion of the 
blc.stome r os h.:'..s occurred . Ex<.:minc , under high pov1er, o.. sample of 
th0 cc.gs in tho hypertonic solution o.nd n ote the e ffect on tho 
ectoplns mic lr.yc r. rl'on mi nutes lat er, trr.nsfor th e eggs with f\. 
minimum amount of solution through thr ee d ishes of n ormo. l scn-wntor . 
The trontmont, if succ essful, caus es th e r..: ctoplnsmi c l o.ye r, by which 
the two blns tor:1o r e s nrc joined, to b c cor:1e t h in o.nC1 ge lntinous, end 
tho tv10 blc.s tomer c s nrc oft en wid ely s epnr o. t ed vii th only c. thin 
film b e t wee n them . Such po.irs may be cultured r long wi th control 
whole eggs , in ombryologicr l wr.tch gl csscs o.nd ~vnscline-rinr:cd 
slides o.s described above , They may be picked out ~t c. l rtcr 
stage of dovo lopr:ccnt sinc e the pc.irs gone ro.lly r emain o. t t:~ chcd by 
8. thin hyc.line strnnd until t hey nrc swin.1j.ng blcstulr.c . Only n 
few twin blastulae ere ordinc. rily obtained by t his method since 
the .gr ec. t mo. j ori ty fus c toge ther during CL'.. rl y devo l opmcn t. 
Development. Obs erve c.nd sketch t ho isoln t cc. blastor:ercs in thdr 
4 -, 8 -, and l 6 ,..col l stc.ges , How r.w.ny 1nicror.'crcs o.ro formed c.t vthttt 
sto.go? Do es the isol nt ed b l f'.s tomc r o cloo.vG ns though it wore still 
part of a VIhol e or,g? J,t tho beginning of go.strulc.tion fix some 
11 h nlf" - embr yos r.nd whole onbr yos i n 5~b forr.w.lln o.nd measure tho ir 
r e s p oet i ve d i r.ne t0rs • V.'hu t r.ppr oxima. t c r o.t ios c, re obtained for 
their r ospo c t ive~olunes , surfu. cc nr ocs and wall -thickness? 
Isolcted l~sto~crcs p . 3 
Excmine r.nd sketch the e:nbryos in the CO'": ' lcte: -::rstrul'"'- , risn "nd 
pluteus st<'.t;es . Do the 11 h~lf 11 -enbryo develop ~~t the sr.r r nte r.s 
the controls? ··-re the 11 hnlf 11 - plutei cor lete in r cgr.rd to ::-.11 
structures s oon i n the whole plutei? Dotorr.1ine ',Jhether or not oth 
nembers of the nirsj'Of is ole. te d blesto:r.a r ,s for!" norr:c.l plutoi . 
(Sec Horstr-,dius , 194'6; Tyler, 1942 r.nd experi..rc1tn.l cmbryolo y 
texts for further nnr. l ysis) . 
References: -
Driosch , H., 1891. Entwicklungsncch~nischc St~dicn . 1. Dar ~crth 
dcr b eiC.en c rst cr FUrchungszcllcn in dor Echinodcrr:cnhJickl 1; . 
Experinentel l e Erzuu(;UnC vo!'l '1'heil - und Doppl- l bil c"ug .... n . 
Zeitschr . f . Wiss .• Zool. 53: 160- 184 
Harvey , E . B., 1940 . " nev1 methoc't of producing twins, triplets nnd 
quadrupl e ts in Arb8.ci~I?-.ctuln.t~ , o.nd their develop,'lCnt. B ol . 
Bull ., 78: 202 - 216 . 
Herbst, C ., 1900 . trber dn.s ·us c inc.ndorrchcn von Furchungs - cmd 
Gc-,.-h .. b e zollon in knlkfrcior.1 I:l ediur.l . Arch . f. En tY!. -1'1e ch . , 
9: 424 - 463 
Ho~str.dius , s ., 1939 . T' e mcchc.nics of se n - urchin development 
studi ed by oper ~. tivc methods . t- iol . Roy ., 14: 132-179. 
Lymnn , J . [.'.n d R . H. Fleming , 1940 . Cor:1positiou of sen wc. t 0r . Jour . 
Marine Resoc.rch , 3 : 134 - 146 
Tyl er , A ., 1942 . Devc l opmentc.l process cs [\DC~ cnor r;e tics . Qunrt . 
Rev . Biol . 17: 197 - 212 , 339 - 353 
The follo· ing is a brief account of n rapid s lide-1:1alcin ~ that 
, 5 use f·..:.:.. for such pur cses r-s r.P..l:ing chron SOI'1G c ou t~ , d tort inin 
~ages of mitosis, fertilizn~ion , etc . , on devolot5,~ ~ g ~nd o her 
-:e.terial . It involves flattening the ec;gs or umhryos betw n t\"o 
coversl ips and is, t':lerc f0re , unsuitable f or any ·:ork in w1 ich it i 
~csired to retain the n0rmal shape of the cells . 
~ipment: 6 Syracuse dishes , 2 or more cloa~ 
c volur.lbia stai!'lint; dichcs (coverslip- size) , 1 f 
::cedle , 2 or r..ore- sliJ.cs e 
? o . 1 cov rsliJ:S , 
caps , 1 disacetinE 
solutions:- Bouin 1 s fixin~~ f luid , Dt. l "..fie l d 1 s h. • ,ct o.-ylin, Alco lOl 
~cries (3o;; , 50% . 7oc;; J. 85/;, 95~; , and lOO%) _, Adc'l·nlc.o.lOl (1~ TICl in 
30% a lcohol), Xylol , c;anada Balsar or EupnraJ. . 
~oceduro :- The ~ethod consists simply in joini .g ~nd later S(parnt~ 
-iv~~--t;·uo coverslips, ono of \'Jhich conto.ins a dr~~) of tho fixing fluid , 
ue other a drop of ·:'1.0 egr; suspension . Tho covorslips VJith adlwrint: 
f]nttenod eo~s a:rc SU1)Sequently _ D.ndlod in tho S['me l'Jnnner as slides 
of sectioned matcri8..i. To fc:cilitate later se 1 aret.icn the cover lips 
should be joined crcscnise . Tho f ollowing illustrates the procedure . 
support one coverslip on tho edges of tv1o Syracust. dishes p1acod next 
~o each other (or so.:1e other convenient s u pport) and add a small drop 
of fixing flu id (Bouin's) . Hold tho other coverslip in ono hand rnd 
add a small drop of ~iiD egg ousponsion at tho de~ ired sta~e . Invert 
the second coverslip o•rcr tho first i n crossvliso posj_tion (so thn.t the 
corners do not coi'1Ci.do ) nne' rele r~se it as s o un as the drops touch e 
Tho drops should noc be 2.ll cw~:::d to u.ndcrgo any n.pprecinble evaporation 
before J Olnlng . The size of the cl.rops shou.ld bl.i s uch that when 
joined the fluid docs not qu i to fill the space between tho tv•o c oYor--
s1 ips . The eggs [',ro thus fl2..ttunod and the dogreo of this flattonin;~ 
can be r 0Gula t ed to s orne exton t by the size of t 1c drops . Tho f ixint; 
fluid acts r apidly on cells of s mnll clit'.mete-r , cspoc ic.lly when they 
are f lattened . 'ii5_th most marino e ggs of 0 . 05 to 0,.2 nnn ., two t o ton 
ninutos usua lly suffices for good fixr.ti:m in I3ou.in 1 s fluid . The 
jci:1ed covcrslips arc then co..refully pJ.acod in 8. Syro.r:uso dish and 70/.~ 
aJ cohol is added whereupon tho cuve rs1 i11s tend to s ~: po.r['. to . Lift th0 
upper covcrsl ip off by moc.ns of o.. fino fore< :ps using a neefl lo placed 
nt tho opposite edge of tho upper cove rslip to .revon t it from slidin~ 
·:l, il8 being removed , and plPce it egg- side up in r.nothor dish of 70% 
flcohol. Slidine of one coverslip over tho othe r wil l cnuso d i storti-:m. 
r.nd loss of egg s or frngments thereof . 0rdinarll7l nbout hnlf of tho 
eegs v1ill adhere to o c..ch c overslip . If the' fixing fluid had b een 
ullov1od to ret for t c,r, long u timo before tho o g:·s wo re actur_lly 
I'l'1ttoned , many of tho egrs v!Uuld fail to ['dhcro to tho covcrslips when 
they are separated . Allow tho coverslip to rem' in in two or three 
changes of 70% o.lcoh•)l for a sufficient l ength of timo (usurlly rt 
lea st an h -.ur) to remove tho yellow color <;f picric ucid ( of th I3oujr • .. 
fluid) fror.1 the ec:-r;s . 
Transfer the ccvorslips through 50~S and 30~; a lcoh0l (about 
one minute in each) to Delafi e l d 1 s hem.atu;', lin fur ten r.~j nu tos or 
- 2-
1 nger . The Colu; bia st[>..i. inc ci"'hes .re conye ic·nt cr t ... is nn 
;:t'bscquent • andl inc.. Thtm ':Jnsh ;:mce in t a plr,c the 
: 0verslips in t.1c o.cid alcohol . 71 L latter shoul in n S;.rrncuso 
~ .... s:h and the ... rosros s of the des n · n:_nr folluwG un .or the mi r s o e . 
·,·_e:t the str. in n~ 1 gcr corr:cs out cf the ege.;s in 'is iblc cl d 
rbvUt . :'10 - half to one Li'1Utl' f ftcr 10 - 15 ninut S S tR 111. • ) il. 
~~ cov0rslip in tr ~ntcr . After at lccst throe hn g "' of tn 
·atcr durinf 5 tc 10 minutL.s run the cover 1 ips ll thr:..>ugh th~.. 
-lcoht-ls c.nc. Xylol c.nd nount on a slide vlith bn. l'' or ''lli.t the .·ylvl ;;d momt in oupnr['l . Count rstDini'1g v•ith eosin o other dye 1.n.: 
~used in the usu~l vPy if desired . 
aoferences : - ': ylcr , A ., 1946 . c.pid slidc -n~ ··in~ r10tl od for 
Drcparctions of eggs , protoz.oa , ote . The Cullcctinr- Nl't , ol 10 , 
PP • 40 - 41 . 
CULTUR r;;G E ,.:BRYOS 0. VASELI:ffi EALED SLIDES 
It is often desirable to cult1re sinGle e brros or s nll nw-
bers of then in a sr.1all ·-f'lU:.C' of :.ocUUl. so that thy can be readily 
observed fro:"'l time to ti_me ancl so t .at- t1,eir ~·;·il.l: ·~pr: activities do 
... 0t r1ake searching for· ·:.; · lc '· •· J(• t Llle co~sur .i.n ~. !.'o:;." thi rurpose t.1ey r.:ay be kept in ::. s. ':1.:.:_~, ~.,...op i"'l n cho.!:"cr fo ": e 1 by a Rlicl a.n 
":n·erslip sealed toce-t~._,· .. · \'lit~ vaseli: eo It is nls oft 1 de ira le 
to c.1l t·.tre l3.re;e nnr.Jer'3 0::' e~1bryos in ra "ther concentrn ted sus on ion . 
?!1e use of ~bllcpha _e tubing helps overco~1e t.1e difS"iculty of o.tte•·1 t-
;,.,r: to transfc:c ls.~'ge ·.ll'1ters ( f swir11r.1incr er ::."vos to frnsh sea 'later 
o~-•"'0 • .. 
e.n1 1-ernits satisfa·.;tc!'y develop ~ent of ratlv3r col'lcentrntod su"'pen-
sir:>ns. 
£~[!_5 pment : Vaseli'tc ~:>:-rly pure petrc le1.1n!. slide", ~over 
cr.i ~nary dropper'"', ~ ·.:.-.E- ·-'· :q 1:--ed qroppers) cello 'h:mo tu lTI[; ( 
ce..'..!.ulos0 sau8£J [··' c .. i' ir:?;S ca., 1 1' diar.to) .• fl:"'..12~. (6 11 ) > Syracu 
fL1ger b8VIls: R:li s e:.ss ors. 
S0- ·.tions: 
- --·-----
F:U ·. ·:-od sea water, Diaton cul tn~·e . 
~-·=J.!.>c:ine -Slidc .'ct,l:--r: \'!arm a small amount of vaselino in a ben.ker 
'f5 -J~s ~above- i}i'A - -1 ~~-t ing point and with a warm dropper nake a hollow 
squn.re of vas c:1 i l'C .-,n a. clean, dry s lido . A bent metal rod can also 
be u1: ed for thj.s purpc.'3o in place of tho dropper. The outs ido dimen-
sions of tho square of vaseline should be rou[;hly the same as that 
of the coverslip that is to be used and tho height should be about 
1 nm. P lace a small drop containing the eggs or embryos within tho 
s(j_uare on the slide . This drop should be of such sj.ze that when 
the coverslip is added, completely sealed to the slide and contact 
made with the drop, the d iamcter of the drop will bo about 2 to 3 runo 
The coverslip should be pressed down sufficiently so that o. completo1y 
sonled chamber is formed , with no air chfl.rl no ls running throur,h tho 
vasoline . Avoid having the drop como in contact vlith tho ,rasolinoo 
The slide should bo kept in a cool place and the emlJryos transfcrrctl. 
to a fresh preparation at invervals of one to four days depending 
upon the amount of living materlal present . 
f_ellophane Tubing Method : The seamless cellophane tubing is usually 
supplied in flat roll .s of about 100 feet. Cut off a piece about 
0110 foot in length and tie a knot in one end of it . Soak tho piece 
in sea water for a fow minutes , then open the other end by sliding 
':;he two sides of the flattened tube against one another . Introduce 
1 funnel into the open end of tho tube nnd pour in about 100 cc. of 
~i1e sus pens ion of eggs or cmhryos. Tie off the tube in such a way 
t"lan an air pocket of about one-fifth of the volume of fluid is loft. 
·r':len place the tube in a dish or aquarium of runnine; sea water in 
such a way that tho water tonds to flow past it . \'1ith small em1 ryos 
of annelids, mollusks, or echinoderms , several thousand embryos por 
lJO cc . can be kept alive in this manner providod they arc chan~ed 
to fresh tubes once or t wice a vJcelc . To obtain later devolopmental 
stages and growth food matcrin.ls must be added . For this purpose 
a small amount of a dense suspension of diatoms (Nitschia) should bo 
added at each transfer . 
1947 
CHE.:ICAL E1.BRYOLOGY OF 1.ARIHE D VERTE RATE EGGS 
.~. cytochemical methods: 
These methods allow the detection of sor..e of the chenical consti-
;;uents of the cell (proteins, lipids, glyco en, ucleic aci s, en-
~pes, etc.). They have· not been used on marine egg very extensiv-
lY so far, \'lith the exception of Ries ' papers on r s ira tory en~ymes 
in nosaic and regulatory eggs. The results obtained Yith the e 
r:ethods should be examined critically. For such a critical discuss ion 
of cytochemical methods see Lisen's and Brachotrs books. 
The experiments essentially consist in the application of a 
series of these cytochemical tests to unfertilized and fertilized 
eggs of both the regulatory (Arbacia, Asterias, Echinarachnius) and 
the mosaic (Chaetopterus, Here is , Ciona) types, as well as cl a vag 
stages and gastrules . Arbacia is not a very good material for this 
sort of work because of tho presence of the rod pigment echinochrome. 
~e same tests should also be made on unfertilized and fertilized 
eggs which have been centrifuged for 10 minute" . Such egGs should o 
fixed inmediately after centrifugation. 
Do all tests on a large number of eggs (several hundred) . Obsorv 
the eggs carefully and make accura to drawings . 
1 . Detection of Amino Acids present in tho protein: (cf . Sorra: 
Stain Technol . 21: 5 . 1946) 
a. Test for sulfhydril group: 
(Method of Giroud and Bulliard - Lison, L. Histo Chimi 
Animale 1936) 
a) Fix with 10% trichloracetic acid for 10 minutes. 
b) Immerse in 5% aqueous solution of zinc acetate for some 
seconds (excess docs no harm) . 
c) Then immediately treat with a 10% aqueous solution of 
s odium nitroprusside , containing about 2j& concentrated 
ammonia . The pieces (eggs) acquire a bright rod colora-
tion, which attains its maximum in 3 - 5 minutes . 
d) ~.1ount in pure glycerine for mi eros copic obsorva t ions , 
if ne cessary , with a preliminary washing in distilled water. 
b. Test for tyrosine (Millon's modified) 
(Method of Serra, J . A., and Queiroz Lopes , A., Nature 155 
page 792., 1945) 
a) Immerse tho ege for 30 minutes in a few cc of mercuric 
solution (composition : Hgso4 7.5 g; HgC12 5 . 5 g; Na 2so4 
7.0 g; - dissolved in 85 ml. of distilled water to which 
12.5 g . of concentrated H2S04 is added; after dissolving 
diluted to 100 ml • .. with distilled water). Perform tho 
treatment in a small gl ass st oppered bottle placed in a 
water bath whi ch is maintained nt 600 c. 
b) After the 30- minuto tro~tment , cool the bottle in 
running water and allow to stand at room temperature for 
10 minutes . 
c) Dilute the mercuric solution in a bottle by addition of 
an equal volume of distilled wRter . 
d) Develop the color , adding now some drops of a freshly 
prepared 1 M solution of sodium nitrite (6 . 9 g NaN02 in 
100 ml . of water} . The coloration attains its maximum 
in three minutes , and lasts for some months. The eggs arc 
mounted and observed in pure glycerine whore they can be 
squeezed or squashed if necessary . 
,... 
c. Test for Arginine: 
(Thomas, Lloyd E., J. c. c. P. 28 1 #1, p. 145, 1946) 
n) Fix in Bouin 1 s fixative for five minutes. 
b) V'ash in running wa tcr 
c) Add alpha naphthol for at least throe minutes, at room 
tempera turo. 
d) Sodium hypochloride - 20 seconds 
c) Urea - 5 seconds 
f) Mount and observe, in glycerine 
g) Red color will appear if arGinine is resent 
Reagents: lo% alpha naphthol in 95% alcohol. 0.15 so ium hypo-
chlorite in 0.05 N NaOH. 20% urea in 0.05 N eOH. 
2. Detection of lipids (plasmalogen) 
TecP21ique: Fix (eggs) in seturnted corrosive sublimate (hgClz) 
(poisonl ) for 5 minutes. Then wash with distilled w tor and put 
into Foulgen rev gent for 15 minutes. Hash throe times in wo. tor 
saturated \'lith so2 , then rinse in water. Observe under the 
binocular. Then make a slide vJi th glycerine and observe under the 
1 scope . 
Controls: 
1. Same as above, but omit tho sublim~to. 
2 • Fix the eggs in Carnoy' s fluid for 30 m:l.nutcs, and wnsh in 
alcohol twice for 15 minutes . (This extracts most of tho 
lioids). Then s~me as abovo . 
(Carney 's fluid is: alcohol 6 p.; chloroform 3 p.; acetic acid lp) 
3. Detection of glycogen (Bauer) 
Place a few of the eggs which were fi:v:ed in Carney and tr ated 
with alcohol into 4% chromic acid for one hour . (Tr.is treatment , 
liberates aldeyhyde groups in polysaccharides) . lash in distilled 
water, then stain with Feulgon ' s roagvnt as above . 
4. Detection of r espiratory enzymes . 
a ·. Peroxidase (Pre punt 1 s mothod) 
Fix one group of eggs in 10% formol for 10 minutes (this treat-
mont destroys tho catalase) and wo.sh thoroughly in distilled 
water . Immerse tho e ggs in a saturated solution of benzidine 
in water contai n inG one drop of acetic acid per 10 c ~~· 
Then immerse in HzOz (perhydrol diluted 1%). Note the oxygen 
bubbles (ca taluse) and tho blue , then brown color·. Repent tho 
test on frush eggs. · 
b• Indophenoloxidase (Child's method in ~hysiological Zoology 17. 
129 1944). 
a i 1 drop of dimethylparaphonylonodiamino in 10 cc HzO 
is 1/40 of a cc) 
b) 1 mgm alpha naphthol in 10 cc Hz) • Add a drop ~f each 
to 1 cc of tho solution containing tho fr esh eggs to be tested . 
Do you observe tho gradient pattern described by Child? 
5. Detection of nucleic acids. This is best dono on sections of 
eggs v1hich wore fixed and embedded in parrq.fin . 
Material: Arbacia: fertilized eggs and plutci (or gastrulae) 
Chaetopterus: unfertilized , fertilized eggs and 
trochophores 
Fixation: Fix for·l5 - 30 minutes in Ccrnoy or Serra (9 parts 
alcohol, 1 part formol , 0 . 5 part acetic acid) 
Embed as usual. 
a . Thymonuclcic acid (F'oulgon reaction) : Paraffin is removed 
as usual and sections are rehydrated . Tho slides arc hydrolyzed 
for six minutes in N HCl at 60° c., then rinsed in H20 . They 
:; 
I 
, 
' 
are left for one hour in Feulgen' s fuchs in sulphurous rea ent 1 the 
washed three times in so2 water (1 gm NaHS03 i 100 cc H2o plus 
1 cc concentrated HC 1~. Vash in water, dehydrate in alcohols as 
usual a nd mount in Canada balsar.t . 
As a control, treat another section the same way, but or.ittin 
the HCl hydrolysis. 
b. Ribonucleic acid: Stain the sections in: 
1. A saturated aqueous solution of toluidine lue for twenty 
minutes. Wash in running v1a ter. Leave thf' s lidos in 95f, alcohol 
for five ~inutes. Mount as usual. 
2. Unna 's methyl green - pyronin mixture 0 .15 gm . methyl r,rcen; 
0.25 gm . pyronin P ; 2.5 cc. 95% alcohol; 20 cc. glycerine; 0 . 5 
gm. carbolic acid; 100 cc. H20). Stain for twenty inutes, 
rinse in water, differentiate in 95% alcohol, then into xylol 
and balsam. 
Both nucleic acids stain with all basic dyes. However, wlon 
the Unna mixture is used there is a competition between tho 
two dyes and the nucleic acids, so that ribonnucloic acid 
stains red and th~nonucl cic acid stains green . Dut tho reaction 
is s pecific for ribonucleic only when it is shovm that thoro is 
no staining v1hon this acid has been previously digested away 
with ribonuclease as f ollov1s : 
Incubate one slide for one h our at 56 ° in a :Mcilwain citric 
acid buffer at pH 6.75. This is usod as a control. Treat a 
second slide the same way but add 0.1 mgm of ribonuclease per 
cc. of buffer . Then stain both slidos with methyl green- pyronin . 
B. Properties of the nucleic acids and nuclcoprotoins. 
Ribonucleic acid is mostly present in the form of cytoplasmic 
ultramicroscopic granules (microsomes) which als o contain lipids (in-
cluding plasmalogen) proteins and r espiratory enzymes . Tho thyr10nu-
cleic acid is bound to histone in the nucleus. This thymonucleohistono 
forms very long , thr ead-like macromolecules which have considerable 
viscosity (11· irsky and PolD::istcr: J. Gen. Physiol 194G) 
a. Isolation of nicrosomos from c entrifuged Arbncia eggs: 
Collect and wash the eggs from two ripe females. Remove tho sea-
water after centrifugation. Then, crush tho eggs in a mortar (in 
an ice-bath) with sand and add 10 volumes of cold phos phate 
buffer (M/200, pH7). Mix well and centrifuge for 10 minutus at 
3 1 000 r.p.m. Observe the different lay0rs under tho microscope-
yolk platelets, pigment, fats. The microscope l ayer can bo soon 
as a cloudy layer between the fats and tho pigment ·, 
Remove this layer with a thin medicine dropper, and centrifuge 
for 20 minutes at 12.,500 r.p.m. in tho ultracentrifuge. Observe · 
the supernatent fluids and tho pollots. Do tho indophcnoloxidaso, 
peroxidase, - SH and plasraal t ests on those pellets. 
Add 0.1 cc. of 10% trichloracetic acid for each pellet , and 
mix well . Precipitate tho protoin fro~ the supornat8nt fluids 
(soc abovo) with o.l cc of 20% trichloracvtic acid for oc ch o.l cc 
of fluid . Then c0ntrifuge both mixtures and repeat tho extrac-
tion with 0.1 cc trichloracetic on the supernatant fluids . Add 
0 .1 cc of water and 0 .1 cc of Bi al ' s pontoso reagent to o~ch. 
Innnorso in a boiling v1ator bath for 10 minutes. (Bial's ro gent: 
Dissolve 10 mgs . orcinol pcJr cc of a solution of concentrated HCl 
containing 0.1% ferric chloride .) 
b. Isolation of thymonucloohistono from Arbacia t estis : 
Crush and extract a sea-urchin t estis ·1i th ar1 NaCl . Observe tho 
formation of a viscous solution of thymonucloohistoncs . Centrifuge 
4 . 
at ordinary speeds for 10 minutes . Dilute tho su1ernatant vith 10 
·olumes of distilled water. A fibro s . recipitnt of thymonu leo-
"'istone settles out . 
·· De~onstrate the presence of large amounts of argini e in the 
~ecipitate ~ith the cytochemical reaction used a ove . 
· Demonstrate the presence of thymonucleic acid in the prec pitnte 
?lith the Dische diphenylamine test . Try he same reaction on the 
... icros ones \'/hich have been isola ted in the previous exporir1cnt . 
-ische 1 s test: ... ~eat on the water bath durin r, 10 minut s 1 vol. of the 
solution containinG nucleic acid A.nd 2 volumes of the rea ent: 2~ 
diphenylamine in a mixture of 39 parts acetic acid o.nd 1 part of sul -
furic acid. 
Tho presence of thymonucleohistone in the sperm heads (v siclos) 
can be demonstrated by doing the arginine and Feulgen cyto h micnl 
tests on smears of sea -urchin testis . (for tho Feulgon test it i 
necessary t o get rid of the intcrferinc; plasmalogen by leaving tho 
slides in 95% alcohol for 20 minutes . ) 
Repeat the arginine and Feul en tests on sm ars which hav . bo n 
treated for 10 minutes in 2 M NaCl and then wash d with water . 
See Sc hneider ' s papers f or a auantitative method of csti~ntion 
of both types of nucleic acids with tho Bial and Dische t sts , in 
~ · Biol. Chen . 161 : 293 1 1945, and 164: 747, 1946 . 
-2-
:te :ovc the fixc.tive usin[, a pi~ +te of 
t"'i th a syringe bulb, c::.nd fill the vial ··it 
70; until the e ,._:-s are 'Jhi te . It i adyj s bl to ·void w inc: to 
10~~ in 701, since the stain c~ployed is bes~ ·to, it do-s not pon~­
trs. tc the acromeres . ThE sc 12 tt r should therefore bP left 1 ig:.:. t- ~ 
, cid . Thus tht> cgss arc r e1.ovcd for: ?O· ~ ir..::cdi o.t~...l : o.ft r the l ~st 
-~sh ·~ich rcnoves no picric fro then, h•dratod in 50~, ~51 ad 
r·ashed thoroughly in .:._- 3 changes of r:a tcr . 
II. Stuining : 
After Hashing \'ti th v:a tt,.;r, fill the vinl ·:i th undiluted To.:l'l" ,' 
haccalum , and stain for 5 - 10 minutes . For the pola b~dy tn('\'S , 
5- ? r:1inutss is ua~Qlly sufficient . Aft r stnininc; , nash tho ·our Jy i 1 
~ator , dehydrate, and clenr in xylol . Renov th ·rlol usod i1 
cJ:caring and r epl ace it vvi th a sr.wll amount of thin dnr.w.r. 
III . :ountine : 
Cover gl asses 1:1us t be s upporto d . For this nrpo · it hns be u 
convenient to usc paper squnrc:s the size of 7/811 cover glnssf' . A 
hole is punche d in the cC'ntor of each souarc "ith c p po punch . 
In mounting the square s arc cleared in xylol , and fixed to th e co -
tcrs of slides b~• &ddine.; throe or four<ihrops of thin clnr11ar b c fo ,, tLr 
cvaporo.tion of the xylol . ~'lhcn the paper mounts hnvo dried, th .., 
c~gs arc r emove d fro.l the ,vial in "'hich the~ arc stored by tho usc ,·, 
a pipette draV''n out to a long tape r and having a sr.,nll diamctor [ t 
its tip . The CCGS nrc allowed to se ttlc tm"~rd the tip of the 
pipette , e.nd one drop of the egg- damar susp ~:nsion is pl8ce d in tho 
center d epr ession of cc.ch paper mount . The dnr.1ar in nllowed to dr:y 
to tho point of formation of a thin film in order thnt the cc,r;,s r.ay 
renain dispersed and ~th thG .acrornc r c quar~otte adjacent to tho 
slide when mounted . A~ply thick d.:~mar to the edcc of tho pape-r 
mount , ii!".ncrse o. #(J'J cover gloss in xylol and a 'rJPl? it to the slide 
over tho pnpcr mount. 
Alternative Yc thod . 
The above technique Y'e,s used .succosffullv by ~:r . John Yilford 
in 1939 . An ol der mc.:ihod. sor'1otimos ~ric:ldinc, r~ooc1 r sul ts i ~; <:.s 
follqws : 
1. Fix for 30-120 minutes in ~~yc r ' ~ Picro- s ulphuric. 
2 . Wash in 35 , 50 , 70 .~ a lcohol . Lcnvo in l n ti;. c: r until yellow 
color ceases to come out . 
3 . 50 , 35~& a lcohol , to v·r t c r ( 5 f' in.. c ach ) . 
4 . Sto.in in Conklin ' :;. h':~omn toxylin ( l pr rt Dc l o.fi old ' s to 4-5 
volumc·s dis t . ~.·o.tcr , to •'hi ch 1 CJ.rop of picro-sulphtuic 
fixative is nddod for oo.ch 10 cc . oft~ dilut d stnin,) 
for 5-10 Llint1 t es . 
5 . Wash in '~trr , de hydra t e 5 min . in e~ch alcohol; 10 ~in . in 
95~; 2 chnn gos of absolute alcohol; Y~rlol . 
6 . 1:ount in thick: b nlsru"l 1Tith supportr..)d covC'r r, l n.s0 . 
1 . Ge:1eral : 
,.,. 
- .. -
Euxle~- and DeBeer, 1934 . ::::ae ,ents .1. EX) tnl En ~·olo!l.r 
Chap . 5 . Ca~brid6e t iv . Pr~c . 
,:cEride B. .! • 1914, Textbook of Er1brrol c,: , V 1 . 1 . 
~.:organ , 1'. E . 1934: Er.1brw olOG'l and Genetics, Chap . 4. , C lw·bia 
Univ . Press . Nev York. 
Richards, A. 1931 . P1tline of CorlpE>ra•.iv D·,bry l')gy, p . r::o 
Yviley e:. Sons, Ner Ycrk 
8 . Special: 
Conklin, E . G. 
Child, C. ? : . 
Lillie, F. R. 
Lillie, F. R. 
J.;iead , ;~.D. 
Newby, y,r . \'[. 
Heath , H. 
1897 
1900 
1895 
1899 
1897 
Cre · idula . J. ~ornh . 13. 
. Arenicola, Sternapsi"":-Arch, f. Entwic :1 . ) 
Unio. J. ' 0 rph . 10. 
Adaptation ' . P . Lectt ~s, lBSO . 
Eerl~' D0 velop .ent of r,:nrine Annelids . 
J. r-:orph . 13. 
1932 Eorly Deve.ll:opr.ent of Echiuroid Urcchj s . 
Biol. Bull . ~ 387 . 
1899 Development of I schnochi ton. G. F' sch r, 
Jena. 
l\r. eisenheirner , J. 1901 Dreissensia (} 'ollusc). 7,eit. f . ~'i G . 
Treadvre 11 , 1~. 
Ylierzejski 
Wilson , E . B. 
Vlhi tman , C . 0 • 
\.'Joltereck, 
Wilson , Y.: . B. 
Zool. ~ 1. 
L. 1901 Podarke. J. }.~or ph . , 11._ 
1905 Physa . Z . F. wiss . Zool ., 83 
1892 Ner eis. J. r 0rph . 6 . 
1878 Clepsine. Q. Jour. -Pier . Sci ., 18 . 
1903 Polyr,ordius. Arch . f. Bntv ick . ~ 
1898 Cell-lineage . ~'!. H. Lc>ctures , 1898 . 
J.,nn . N. Y. A cad. Sci . ..ld..!_ 
En:bryol ogy of_ A~ _e_l j._d_!3: 
:·os. 1 , 2 a .d 3 are required, ~ 4 ho ld e done if ti 1 .. ermits . 
T''1e three forms to be studied are ~·y_dr:_oides (I:u omat s) ':c:.:.~e;onu. , 
Jereis and e>abellaria . 
---- -------
Cultures of advanced stages v:ill be _..,re a red . If y 'l. vi 1 to l"'r parfl 
your o·.n cult J.res r:-oce8d as follows: _:Y.0_l::_o_i_de~ both 1r'1l an fpu•nles 
will spawn in:...:1ediat(_ly after being removed fror th ir cnl 
tubes . ?emove several and place them in fi 2:er bO\:ls l on r rn 
dish to keep t _e sexes separate) . Remove the anin1als aft ... r tl 
r .. -av.rned . Let s .~:-· erm stand for about ~ h01.1r . Add n few dr o1 ~ pe •m 
suspension to a dis'l-1 of ergs . (Extrustion of olar odi t cmd 
mr...y Le 8asily studied) . 'lhe blastula stage is r rc1 ed nft r f-' L ·s, 
cas·:·r·.t=· e.tion after 8 -12 hrs; the troc'!:lophore stage lest" fpom :~o U'8. 
to 2 VJPPks . _he trocho _,orcs are best fore study when dny s ld . 
Sabellaria may be treated in the same way . Fertilization of N r is 
b.as __ b_e-en ·sludied in a previous lab . period 
1 . 
The ,. rochopr' ore of Hydrolde • 
The trochophore is a typical Annelid trochophorc. Consult the ex-
cellent figs . in Hatschek ( 1 86) and ~hPvrer ( 1 11) . 'l~e larvae show 
positive phototaxis and gather at the window s do of the dirJh . ~·m nt 
trochophores, 3-5 days old, on a s1ide on which a fev: s 1rcds of 1 ns 
aper have been placed to entangJ e t'l-lcm and £10ld them quiet~ }Tare tics, 
e . g . a few drops of chloretone or of ¥gS04, are not very effective 
but may be tried . Vital staining obscures rather than clarifi Ps the 
structures . The larvae are trans:t-·arent, 2nd proper adjustment of tho 
illumination by moving the mirror and t~1e Abbe condenser will bring 
out all structures . Study animals in lateral and in polar views (both 
from animal and fron• vegetal pole) . 'l'l1e apical tuft and the anal 
vesicle are landmarks for the poles , the mouth is on the ventral side; 
the eye is on t he ri ght side. Observe the locomotion firs t. 
Observe : 
1) _?hap_e._ of the trochophore 
2) J.pical tuft (several long cilia, probably functioning as 
a sense organ). 
3)_!pical _2Egan , a thickening of eetoderm at the animal pole ; 
a nerve c e tlter and probably the }:.- r imordium of the cerebral 
ganglia . 
4) The Frototroch, an equatorial 1:and of large cilia . In oJder 
troch op1.'loros-,-two rows of cil ia will be found ; a row of short 
cilia nnterior to the large cilia . The prototroch is the most 
characteristic structure of the larva , and rave it its nam( . 
It is always anterior to th0 mouth (prooral) . It consists of a 
few large prototroch cells v•hicl:J become pigm8ntcd in older 
stages . 
2-
5) The metatroch (paratroch), a ire 1'1r and of cilia in the 
middle of the posttrochal emisf'her - . 
6) A .2_iliated groove on the midventral line connectin the mouth 
and anus. This groove is interestiYlg in that it narks the 
line of closure of the blastopore. The mouth is the remn nt 
of the blastopore; the anus is a secondary opening at the 
lower end of the origina l blastopore slit. 
7) One~ on the right side of the retrochal homis here. Not 
the red eye pigment. 
8) ~No s t~toc~sts on the vent r al side . 
9) The digestive tract, consisting of: mouth opening, stomadaoum 
r-- oesophagus; ectodermal)' enlarged stomach ( ontodormal ) ' 
narrow intestine (entodermal except f or the end por"-ion which 
is invaginated ectoderm :: proctodaeum), and t1c anus, an 
opening behind tho vegetal pole. All parts arc besot with 
cilia. Feed i nd ia ink and study the mechanism of food intake . 
10) The anal vesicle, a large vacuolated cell at tho posterior end , 
not found in other trochophoros . 
11) The cavity between intestine and outer body wall is not a truo 
coelom but a Rrimary body cavity, the persis ting blastocoele . 
12) The larval kidneys (paired) are typical protonophridia with 
flame cells; they open near tho anus. They appear as slende r 
cords near the statocysts, extending between oesophagus and 
anus. 'rboy are best identified in animals with ve getal polo 
up (consult figs. in Bctschek and Schearer 
13) Mus cles. Two fino strands will be seen bifurcating at tho 
upper end of the larval kidney. One of them can be traced to 
its insertion at the apical plate, the other inserts at the 
oesophagus. These are longitudinal muscles. Other longi tud-
inal muscles extend from the stomach to points of the upper 
hemisphere. A strong circular muscle is near the metatroch: 
the constriction of the larva caused by its contraction will 
be frequently obs_erved. Note also circular (sphincter) muscles 
in the digestive tract. 
14) Undifferontia.ted octomosodorm cells, · single or in small groups, 
will be soon attached to the stomach, to tho inner body wall, 
near the apical orga n, etc. 
15) The i mportant ontomesodormal cells (derivatives of 4d e loblna t.::~ J 
which will give rise to the mesodermal otructuros of tho worm 
body &re difficult to distinguish. They o.ro srraJ 1 g1·oupe of 
cells ncar tho lower end of the huau kidney . 
Draw lateral and polar vi ews. 
3-
11. 
rte t a.n:orphos is of t}?._9_~ero ~::; __ L~ 
The metamor.hosis of an Annelid larv~ into n sogm ntod ~ orm c~n be 
studied best in rereis , 1 to 7 days old. No r ois hn~ no typic 1 
trochophore but an abbreviated , teloscopud larvnl dovolopmont . Tho 
first signs of the adult, segmental orgtnization appear very curly. 
Prepare slides as under 1. Study larvae from all sides. Consult 
tho figs . in E. B. Wilson ' '92). 
A. Trochophore-~ stage 24 hrs. (Wil~on, fig . 84 ) 
Observe: 
1) The prototroch, composed of 12 ery largo ciliat,d cells . 
2) The paratroch, near tho vegeta l p ~ lo. 
3) A pigmented aron at the anal p0l o : the anal pi@nont. 
4) The mouth and stomodaoun1; the latter is a short oc t odormal 
invagination. 
5) The l ar ge mncromores have not yet differentiated into tho 
entodermal parts of th~ intcst no; their cell boundaries 
may bo s een . No cnus i s formed r.r· yet . 
6) Several 11 fr0nt a l bodios 11 neo. r tbu upper end . Circular 
disc-like structures of unknown function . 
7) Two pairs of seta sacs, spherical structures in tho postrochr 
hemisphere. ~sotUO (~chaetae) will be differentiated 
inside of them. Those sacs are the first indication of tho 
first two s egments of the worm. 
8) Observe the trochophcro in l ocomotion . 
B • Advanced trochophoro, .£days old (Wilson, figs . 
85-89 
Identify a ll structures found in A. In addition observe : 
1) Eye s ;pots 
2) A third pair of ~~a ~ hns appeared behind the first tw o . 
All three nre lined up in a rov1 indicr.ting the firnt 3 nog-
m8nts . Soto.s e can be sean in tho pr Jcoss of formnti un , 
inside cf these so.cs; in slightly vl dor sto.gos , they vii) 1 
be soon projecting from the sacs. 
3) Pigment appeQrs in the prototroeh cell . 
c •.. :ot!:lmorphcnis . 21?--3 dr.ys Uilson, figs . 90-91 
Notice the clwngo of shape and tho gro.dun l demarcation of tho 
first three segment s . 
4-
Observe tho follcvling nov: features: 
1) Sense heirs ct the apical pole . 
2) Additi nal E..J£2.. spots; number vnrin lc 
3) ~1 es otrochc.l c ilio.ry bnnd ... at tho ound~rios of th segments • 
4) Pcrc.podic.. \'iith pnrcpodic..l cirrhi npponring (In segment • 2 ~md 
3. 
5) Tentnculo.r cirrhi on tho hond. 
6) Stomndacum, stomnch, intestine bocom distinct . 
This stage combines lnrva.l (tr<..chophoro) nnd :"'dult structures in n 
peculia.r wo.y . 
D. Lc.to Stages of Mcto.morrb.os is. 4 d~:rs nnd 
(Wilson, fig . 92 
ldor. 
The heo.d ho.s lost its bnll8on sho.pe Qnd develop the o.ppond~~os 
chnro.cteris tic for the o.dul t. Locomotirm is still by moons of 
cilia. The end of metamorphosis is marked by tho shedding of tho 
prototroch nnd tho mesotrochs 1 and tho chnngc from s-..:1ir:uninr to 
crawling. 
Observe: 
1) Frontal o.n tonnno, tentc.culo.r cirrhi, pnlpi, nll on tho hand. 
2) Anal cirrhi. 
3) '1\vo large and s ovor c. l smo.ll eyes . 
4) JavJS inside ·:Jf the oec·spho.gus; they co.n bo protruded . Tho 
an1mo.l is carnivorous o.nd r apacious . 
5) Study tho finer structure of tho scto.c , and their motility. 
(muscles o.t their bases) . 
111. 
Tho Trochophoro of So.bol1nrio. . 
This lo.rvo. shov1s very long bristles ;hie h h::tvo probably both n 
suspensory and a protective function . Study trcchophoros , 2 dnys 
o.nd older (consult D. P. ~ilson ' 29, ~nd Novikcff '38). 
Observe: 
1) Stiff o.p i cc.1 ci1ic. in the p1c.cc of the nptcr 1 tuft. 
2) Th~ ..E.E...Ototroch consists of 3 rows of 0ilic., rnd shows t~ gnp 
on tho dorscl side . 
3)The nourctrvch in tho midvon trnl line. 
4) One ~on tho left side . toro oyo spots dovo lop lo.tor . 
5-
5) Tho hcod , o. fold oYorhnnging the 1:10 th 
6) Very l ong bristl0s de·ro l opo in sotn sr..cs . Study their 
fine structure . ~hoy c..ppenr one pc. ir nft r nnct·.hor , ovcr.tur:l-
l y 10 pairs . They ·Jill be replaced ..,_ t n et!".P,orpho" s by 
ordino.ry sotne . 
l V. 
Gns trulo.t i on nnd ForrJr, tion of the 
Trochophoro in Hydrcides--
Prcpare your ovm cultur .. ,s (sec p . 1). Gastrulation by in n.ginc~tion 
occurs uppr c,xinetely 7-10 hrs . c.ftcr fertilizc.tion . C· nsult tho 
figs . in Shco.rcr ( 111) o.nd Hntschol{ ('8o) . 
Child 1 C. -... 
Frnipont , J . 
H.a. ts chc k, B. 
Ivmnoff , c. P. 
Just, E. E. 
Lillie 1 F . R • 
M cad , A. D. 
Newby, W. w. 
Novikoff , A. B. 
Shenrer , c. 
Wilson, D. P. 
Wilson , E . B. 
Wilson , E. B. 
Wistinghaus on , 
c. A. von 
Woltereck , R. 
1900 
1887 
1886 
1928 
1922 
1902 
6-
Enrly dovelopm""'nt of Arcnicoln . Roux' 
Archiv . Bd. 9 
LeGenrc Pol- ordius . Fa.unn u . Flor n des Golf's 
·. 0n o o. pc 1 , V 1 • Xl V • 
Ent~ickl~~g der Trochophoro von Eup mo.tus. 
Arbeit . n .d . Zool . Inst . \"den, Vl . (Hydr ides) 
Entvlicklung der Lctrvo.lsogmento boi don 
A.nncliden. Zeitschr . hlorph. Ouk . Bd . 10 
On the r ccring of scxuclly mature Norois from 
eggs . A 1• 1 o. t . vol . 56 . 
Differentiation without cloo.vo.go in tho ogg 
of the c.nne lid Chnotopterus . loux Archiv' f. 
Ent\'' • Bd . 14. 
1906 Observations o.nd ex,. cri nents cc,nccrning tho 
•Jlemento.ry phenom. of embryonic dovolopmont 
in Chaotoptcrus (F0rmnt ivo stuff) Jour. Exp. 
Zool . 111 . 
1909 P clarity nnd bi1nternlity of tho Annolid egg. 
Bio1. Bull . XVl. 
1897 The early dovo l opmont ~r mc.rino annelid • 
J . Morph. vol . 13 . 
1940 The embryology of tho Echiuroid worm . Urcchi 
cnupo . Mom. Amer . Philos . Sc . vol . 16 1- 219 
1938 
1911 
1929 
1892 
1898 
1891 
1904 
1904 
190 5 
Embryonic de t ermination in tho Annelid , 
Snbellario. vulgaris , Biol . Bull . vol . 74 
Deve lopment and structure of tho trochophore 
of Hydroides , Quart . J . 1 icro . Sci . vol . 56 . 
Lo.rvno of British Snbcllo.rio.ns . J . IYo.rine 
Biol . Assoz . N. 5 . 16 . 
Cell linoo.ge of Nere is , J ourn . of Morph v . 6 
Considerntions of cell lineage and nnccstr nl 
reminiscences . Ann N. Y. Acad . Sci . vol . 11 
Utorsuchungen Ubor die Entv!ickl . von Nero is 
dumerilii . Mitt . n . d . zool . Stat . Nenpo l . 
Bd . 10 . 
Wurrnkopf , 1!/urmrumpf und Trochoph:.:>rr. . z . .~ol . 
Anz . Bd . 28 . 
Boi trnogo zur pro.kt ischen /'.nclyso dor Poly-
gordius -EntvJicklun(i. , o to . Roux Arc hi v . Bd . 18 . 
Zur Kopffrnge dar tnnolidcn . Verb. d . Doutochon 
Zool . Gas . (His figs . nrc roproduc od in McBride ) 
Gastropoda. 
1. Tne Veliger Larva. 
Stud~'" the typical Veliser larva of Cr 1-i la fornicata . Ob· 
tain naterial by breaking t.e animal fr the su~strate t Y'hich 
it is attached. Yo ~ill find the yello?ish eggs tencl sed il 
transparent <- c.ps'..lles) attac ed to the substrate or in th s~ .. ell •!' 
the mother . Tease t::e embryos cut ~f the capsules. Obt~in swim-
ming larvae in different stages, particularly o d ones ~ t~ t~~ 
yol resorbed. They are trans · arent and sh0w the inner rsa • 
Consult Conklin ( 1 97) figs. 80 -82 and the tt:xtbooks f Pe.rl=or - ~­
,.!ell vol . 1 (1928) fig. 65'"1 on p . 734, r ,.'acBr de figs. 263 ff . 
on pe.t;e s 30!_ ff, or Korschclt , 1936 , vol. 2 , ,.p . 881 -891 . Study 
dorsal, ventrnl and lateral vie'<·:s . ?:outh and foot are on t'.., vcn .. 
tral side, the anus is on t~e right side. 
Note: 1. 
-
2~ 
3. 
4 . 
5 . 
6 . 
7 . 
Velum with powerful cilia. 
Head vesicle (dorsal) 
Eyes with lens (dorsal) 
F~ot (ventral), st1dy it in lateral view. 
Statocysts (ventral , on basis of foot). 
:outh v!ith powerful cilia (ventral, a.bo7e the foot . 
Oesop1~gus (dorsal), stomach, liver . Tne different parts 
of the intestine c c-.n be distinguished onJ.v in older 
embryos which i.1a\Te resorbed the yolk. 
8 . Anu.s (on right; side) • 
9 . External kidneys (lat eral to foot.) 
10. Heart (dorsal). Observe the heart beat in older embryos. 
11 . Transparent shell. 
II. EARLY S'IJ-.GLS OF THI Vt.LIG!:R LJ.HVA . 
There is no typical trochophore stage in CrepiduJe. Study 
stages of direGt transformation of the gnstrula into t ... e VeJiger 
larva. (Conklin, '92, figs. 77 -~'9 ,) Observe the gradual d~velop­
ment of shell gland, shell , velum and foot . 
Literature : 
Conklin, E. G., 189~. Embryology of Crepidula. J. 
Morphol. vol. 13 . 
Moritz, c. E., 1938. The Anatomy of the Gactrop()d, Crepill-
ula adunca. Univ. Calif . Publ . Zool., vol. ~3 , 83-92 . 
----~~--~-1939 . Organogenesis in the Gautropod Crevid-
ula adunca. Ibid. vol. 43, 217-240. 
I 
- 2 -
Lamcllibrsnc~ ia ( ~ lccy od n ) 
IJ I. T e 'el iger Stag ~ 
Stu dy the Veligt.;r larva of C ninc-ie. . u tu ,? J::•r •ec :.. , ~, f 1 
c.nd 8 Cl.nrs old; mount ther.1 ·:i th a few shreds of h s ~~er to cn -
tc"gle ~~e~ or use Chloralhydrate as an nncsthbti • It vi1l bo 
neceS[:'E:.ry ~o f i nd r..ort'l or less qnie individuals \:ith v l:tm e xtended. 
Controeted individuals wi ll not do. 
T~·10s e w~. ich arc ex. c.nd in __ and contro c t i nr· tl.0 v 11 m nr 
best for study since t'1is move" the digeotivo trrct . Us" tl1c loiV 
:tc·•;cr fi rst and r>.f t c r a good S}ccimon is found turn on tl o ~ i h 
; ov'o:' . r·r.- 1:o out t'!.E.- follm··inc: 
1 . The gcncr2.l shE:cpo of t.10 body' ith strc.i"·ht hinc;c lin~ t:nd 
sc~i~~rcll~r s~cll . 
~ . The clli8.tod vclun1 E.l-.:l .. [ _ ical ttlJ't . 
3 . 'lhe d'lgosti vo trc;ct con1..istiner of oc so.t-hn~s , stcmt ch 1nd 
inrcstj.ne . Is the lc.t~ ur c c ilc d or stro.ight~' 
·:1: . }osition of :)1outhv•ith refe r ence to tlo ve lum. 
tion of r,n l.S . 
1 0 j,.OS i-
s . Ci1 i a t ion of tlx; d:i.gcs tive trnct. How much o f it is 
c :lio.ted? 'i•hr.t is the !'Ode of feeding? Pdd ehin sc ink to the wn t r . 
6 , 'lhe J! r l1-:1e. r y ;)ody C["vity is a rc"t'1er lf,r[:'-.; sr:ncc soon C.)OVO 
and '"E: low the diges tive trc.c.t . It is not a truo coolom but j,.·( r hnp3 
8. 1-c.r s i sten t c leo.vf.!.ge cavity . 
7 . Extending from t h<:. 1-J.ine:c l i ne to the velum o.re scv(.,rul 
strm1.Cl.s of muscle fi1:.H'us . 
IV . Tho 'Irochophoro _ _9_~-~gs_._ 
Study the Trochof;hore stc.g_e of Cum ·i ngio. (12 -17 hrs . rf tor 
f ur tilizo. tion) and its t.rnnsforr.12tion into tho VCl ie;e r l a rva . (18-24 
hr s • a fter f c r t iJ i z at ion ) • 
The t rocho hore is · of s hort dura tion , (loss than ten h ours .) 
It is best studie d 12 to '. 7 hrs . 8fte r inseminr,tion . JVI.ount some 
trocho};.l)orcs i.n a 1:i.ght sus1.0nsion of india ink , o.nd en tanr:le thorn 
in s~rects of lens ~aLer . 
1. Obs erv0 the gon C;rr.1 s:<.a}:.e in side nnd l' olc r vic ws . 
2 . J> os :l. tion of the band of c 3lia . 
3 . Ap ica l tu f t? 
None of th tJ internal organs c o.n bo so un bc:causo thvy aro 
obscured by yolk . 
Dl'P..\'11 ejf\.o rmcl poll.',r views jn ontJ i n - , P!1o·dnr eLl h:.tion • . 
- 3 -
.'Literature: Grave , B. H. 1? 27 . lfaturo.l !,i tor• .r Cwi.ingia 
t ellinoides . iol . Bull . vol. ~3 . 
;\·organ , T. :r: . 
fuged eggs . 
1 10 . Cytolocical St 
J . ::.::xp . Zool . ~ol . 9 . 
i e s of Centr -
BIBLIOGRAPHY 
EXPERIJAEHI'AL EHB:tr· OL GY OF ANNELIDS MID fOLLU {u 
Huxley and de Beer 
Horgan, T. H. 
_leedham, J, 
Schleip, w. 
\"Iei ss, P, 
Conklin, E. G. 
Conklin, E. G, 
Costello, D, P . 
Costello, D. P. 
Costello, D. P. 
Crampton, H. F.,. 
Hatt , P. 
Horstadius, s. 
Lillie, F, R. 
Lillie , F. R. 
Horgan, T. H. 
Novikoff, A.:e. 
'I'"'OO'BOOK 
1934 Blements of Experimental Embryolo~'· Cambridge . 
Crac fi' len) 
192? Experimental Embryology. Col unbia TTniv. ess. 
1942 Biochemistry and orohop:enesis. CE~mbrid~e ( · aolfillan) 
19?9 Die Determinat·on oer Primi iv-Pntwicklun • Ale-ad . 
Verl. Lei. wi.g 
1939 P rinciples of Develo~ment !To rr H 1 '~ 
SPECIAL RJ ~ F.ERENCJi'S 
191? Effects of centrifugal f rce on the structur and 
development of the e f"S of Crepid 11a, J. Exp. Zool .2~) 
1938 Disorientation of development in Cr pidula plana pr · 
duced by low temperatures. Proc. J\mer. Phil. '~oc. r;c, 
1938 Studies on fragments of centrtfugod Nereis egr;s . 
Biol. Dull. v. ?5 
1945 Experimental studies of germinal localization in 
Nereis. I . The development of isolaton blastomoror,, 
J. Exp. Zool. 100_ 
1945 Segre(~ation of oo"Olasmic const.i tuents. Jour. El i.sh:1 
Mitchell Sci. Soc., 61 
1896 Experimental si uc'li.es on Gestrouon develonment. it rch. 
f. ~ntw . 3. 
193? Essais exneri.mentAux sur 1es localisations ~erminales 
dans l'oeuf d'nn A n11eli.de (Sebellaria alveolate L.) 
Arch. d'Anat. Mi.cr. r; . ?8 
1937 Experiments on determination i.n the early development 
of Cercbratulus lacteus . Biol . Bull . 73 
1902 Differentiation without cleavage in the egg of the 
annelid Chaetoptcrus. .1\rch. f. Entw. 14 
1906 Observat i ons and expGriments conccrnil\~ tho elemen-
tary phenomena of embryonic developm,nt in Chaetopter-
us. .r. Exp. Zool. 3 
1910 Cytological studies of centrifu~ed eggs. J. I~ . 7.ool9 
1938 Embryonic determination in tho annelid, Sabellaria 
vulgaris. 1. The differentiation of ectoderm when 
separaterl throu,.h i nd1 ced exo~a strulat ion. ?-. ' '"1rans-
p1.antation of polar lobes and blAsi omeres. Bi ol. null. 
74 
ovikoff, A. B. 
Penners , A. 
Schloip , H. 
Tyler , A. 
1 ilson, "' B. 
· "i lson , E. B. 
Pilson , J~ . B. 
1
.lilson, E . B. 
Yatsu , N. 
Yatsu , N. 
Zeleny, c. 
2. 
1940 aornhogenctic substances r or anizers in annelid 
developncnt. J . Exp. Z ol . 85 
1938 Abhangigkoit der Formbildune v m ·~csodern im Tubifcx-
fu' ryo. Zoi t. f. wiss. Zool. 150 
1925 Die Furchung dispermor Dontalium-Eier. Arch . f . Entw. 
106 
1930 Experimenta l production f double cmbry s in annelids 
and mollusks . J. r~. Zo l. 57 
1903 Exner imcnts n clcavBge an<'l local i.7.ati n i.n tho 
nemertine e gr . Arch . f. Entw. 16 
1904 Exueri rnentel st 1dies n Germi.nal. 1ocal·7.ation. 1. 'J'h . 
~erm-regions in the er: ' of Dent al.itU'l. ::> . EXD rirncnts 
on the cleavape m sai.c i.n Patel 1~ E~nd n nt alium. 
J . Fxn. z ""1 . l 
1904 11:osE-ic dcvelonmont in 4 ho an11el i<'l ow. f'cicncc ::>O 
1929 ~he development of eg~ frep.menis in en·clids. Arc h . 
f. Entw. 11? 
1904 Exper iment s on tho development of egg fragments of 
Cer obratulus. Biol. Bull. 6 
1910 Experiment s on germinal locali7etion in tho ogr, of 
Cerebr atu1us . J . Coll. Sci. ~ okyo. 2? 
1904 Experiments on the 1 o.!alization of developmental 
fac t ors in the Nemert in, ogc. J . Exp. Zoo1 . 1 
I~troduc ior. to Eydrozoa 
----------- _.,___ __ _ 
n) ~ensitivencss of the mntcrinl 
·:1':1ile; working with coel~nterct < s in the 1~ or:}tor· it is ss cntietl 
to remember that tho hydroids nrc very scms:l.ti c t cn\'jron:· ntcl 
conditions . They do not survive well in th -. lcborc, or.' vn1 in 
aau&riet of running water . Do not crowd oit~er the ~d1lt st ~~ or 
u;:c eggs cnd onbryc s . In gcnerc.l, your pr c .. : dur will c to look 
over r, good - s iz d colony of hydroids und~.-r th(.. lov:c t pov r of 
magnifica tion, nnd to eli~? off r. fow piec~..'s cont". ining tl. ~..st 
embryologic&l mctcrinl . J.hosc ccn be rins ..... d in run1in '"' et w:-- i:rr~ 
and scgrc_a t cd in plenty of S\'c, wct e r in n disl' for 1:10rc. dct-: · J. d 
study . 
There arc two phr.s c s of c.., . r : 'oJ o ::_ y i n the. Eydrozo[;. '•nrnct t ri ... t :l -
co.lly , a lon g scri .... s of a · <.., xufl l rcprodu ~tions (by budding ::nd 
other methods) is int c rru~-t" cd-··c"f -frrc gul."i,_-r.-- in tc rve-ls by i~: olnt\ d 
exampl e s of, scxu[.~]_ _r o_.eroc'uct_~ o__::_:.~ ri'_1 is [\ltcrnation of so 'Uftl , 1c: 
asexua l gcncro.tiono is clos · .. l~- 'arr.ll c l vd by f'n ['. lt0!'11t... ~ ion of 
s true tur c , s inc <..: zygot e production is usuc.J ly ::.ccompl isht,d by 
mcdusc.c nnd the production of bu<'l.s by pol_yj)_E_ . 
In o omc hydrozoo. no medusa - f orm is l·now, in othGrs no pol yp -fon: 
is known , and tl~erc e r e nl l stc.gcs b c tw <Jcn. 'l'hc hydrozo . et v.~i.Jc 1J"' 
at ~\'oods h ol e i1lustrc.te well th e struc.turc.l veri C'. "lilit:r of tho 
medusa - stat E:; . j_l:.c bcst - kr..o m ox rr:vlo of c compJ f'tG modusn vil t 11 '"' 
de genera to polyp s t ngc is the td c r·l izod j clly -f ic:h Gon jon emus , but: 
it is unfortunn toly now n uc.rly extinct at .voods Hole . 11 ht. gon tJ r [' 
picke d for study ~rc firrnng cd in tlL order of dlmin i nhing com-
pleteness of the modusr. form, th e f5.rst h ::: vj_n e- fr oc -swirnminr; 
medusae o.nd t~o laot more sporosacs . ~~c utt e r d <..,gc noration of 
tho mcdusa -fonn is illustrfit :; d by the f['.T:l i1i::1.r :-;ydrc . 
c) Orde_E...__of Study 
Because of sc~sonvl variations end unpr Gdict=~ l G fluctuntjo ns 1n tl. 
Coc lentcrc.tc faunc., so1:' c of th e T"lntcri r' l dGscrib c., d mc.y not b e 
availn~le , and t~c order of study will hnv o to br announced . 
Lnborc..torv ~roccdurc 
- . -·-------~ ..... _ -· -- - - - .. ·--
A. Stu_dy of_}E_!!~s_ -~'j._t}\_!'_<:_r..fc et .. ::c. ~:u_~-~-
Exampl os ; Bougc. invD 1 io , Ob ol i::- , .t'or:ocoryne . 
Chnr"]~(fto~~s_t):_ c _ _l iJ> _0_is t ?.rz! Zyc·ct,_ sh.;d from mcdus c.; Dovel op-
mcnt to plc.nulf. lnrvc ; I.:ctc:.wrp1lo~i c; t o 1clyp; .Ancxuc.l ~.ulti ..­
plicatiorl of polyps b".J- 1 u c'dinr_· , '-'lhic' l;rod,..lc ,r c. coJ.on;r; .. 1 du:J" O 
formed by s p c c i.n 1 buc1n , in :·. ··onD[J one ( 01 <.. 1 ir ) or s < p o.. r;:d: .:.1 y 
(Bougc.invillic~); Shcdd:1.nr: ofm.:Ci11s [~- , --· .. ,1·i ch ''h. t ,' r 0 slowly us 
scpo.ratc individuols reforc forr1,nr cr.e· s or :::perm . 
BOUG!~INV ILLIJ"~ . (Juno , J·uly, .Lur·-u~ t; not , lvJf J G c. vniln oJ o in Juno) 
2-
'The gonophores c..r<; orne singly or in clust .., rs n the mnin. stem 
and brc.nch~...s, and in this genus devvlop into cor: lo : rK duso • Th 
ncd'.lsn.-buds nrc ncottcrcd irregulrrl:r thr out t1 <.. colony, th ro 
bci:!G no orderly c..rrc.ngcmcn t r.c ord · ng to nge. ~.J oct u .. th ·t 
sho ; vc.r ious s tr.gcs of mcdusn do vel op· ent c. d rount them under 
cover lips, c.nd study their Qnfoldi .g structur~. ru· off vote 
from under th 8 cover slip with r.bsorbont .. c.p(sr to pro uc n slight 
pressure on tho buds . Sketch three " tcgos in ~~ u <:'. dovclo \'10 t. 
·:·hen c.ll of its pnrts except the gonads arc fullv formed, the 
r-:odusr. bronks loose "..nc swins awcy. It lin;s ind"'p~ndontl:- one or 
t·1o nonths, the gono.ds gro.dunlly nnturin;:. ·' in(1 ;_ v~cll- uvclo' ",l 
spccin~en that is swir ning c.ctivGly nnd s~:ctc lts dingrcrm "..t c 
structuro. Ind~ntify r.mnubriun, rc.die l .nd circulrr cru1r.ls, vd. ··1, 
oral tentc.clcs , 4 groups of mnrginnl t entncl s. 
Illustrations of medusn in Ho..rgitt, C. 11. '01, '04; .1uttinc: ' ol , oi 
mcduso. development in Goctto 1 07 . t.lso HY1:10.n 1 40. 
Obcl in (June , Jul:,-, Iugust) 
The gi'ei:siftes .B.._<?_!lOS_9!·:.~~ t-.r<-- s )V1...r2.l times c.s lr.rgo ns the hydrcnths 
Exo.mino specimens o.nd "'oc if the.' c.rc lac .:.. ted nt rnndom nl ong th '"' 
stalks, or in r egular plnc~s . ' 
J';,n Obcl ie'. gonos omc hns em cnlnrg~d trcmspo.r ent covering, tho 
_g_onothe cn, ·ni tb c. blc.s tos txl o extending thru it from bus o to t i • 
Tho outer end , or tip , of the blastostylo expands to make o. l ooo~... 
pluc for tho gonothcca when nuturc·. 
A clus tor of, _gQ_nopl'1orcs is borne on tho. blas tos t:·lo ins ide tho 
gonotho co. . l'l1C' gonophorcs mo. turc cs rncdusnc, c.nd brenk loos l , (' 
co.ping to the outside po.st the blnstostylc plug. They uro connon:. 
cr.ught in tow nets . ThE: ir free -s"'''in.min0 1 lfc lns ts two months 
or so , the gonads maturing slowly . 
Tho older gonophoros shoul d show developing tento.clos, when 
pressed slightly under a cover s 1 ip. v•n·1icl·· gonophor cs on a bln 'l to--
style arc oldest? 
~N ith noodles , pr oss on the gonothcco. of r. wc1 1-rr.etur cd gonosomc 
o.nd exo.mino under hi[h magnification the p·onophorcs tho t nrc 
r ole as c d . 'I'hos o the. t r..rc old os t may show swimP1ing r1ovor.1onts. 
Younger stc.gcs of develop:nent mc.y bu tt;c.s vd out froFl tho gonosouc 
und studied. Sketch several stc.gos. 
If ripe Obelia coloni es arc ~opt for ~n hour or two in n dish 
of sea water on the desk (rene .wcr net to crowd them) , sv1imming 
medusae can usually be do t oe ted in r. cood 1 ight tJj. th tho nD.kcd (;yo . 
Exr..minc some of thcr:1 \'hon c.v.:·.ilc.blo, ~ nci note thct their utructur0 
is slightly imperfect conpf'rcc1 rJit1 tl1et of bougr.in,~illin . 
'Ibe v e lum is reduced to r no.rr0\1 c.nd nomcwhn.t lobed mombrr-nd nocr 
tho bases of tho tonto.cl ~s . 'l'his :nck.s po"siblo nn aver~:· ion of tho 
bell when tho modust> comes to rest, so thnt tho mc..nuhrium ntlckn 
out from tho c enter of tho convex sic'lt- , like the ho.ndlo of o. pGst-
hurrlcanc umbr cllo . Wo.tch thu svli'''J'1 ng movor%nts , o.nd n r,o how thlo 
3-
happens. n t.1e everted condition, the , anll"l-ri · 
rr:or hological~y sut -1..m:brellar , tho 1-:h this ten, ha 
priateness. 
is still 
lost t 
The newly shed medusa of O.elia genic lat hcs 2x tentacles, ~hil~ 
that of Obelia co rrnissuraliz has 16. Both forr·s nay e a ailuble 
in the laboratory . 1Jeither has gonads develo ed ut this stnge. 
Illustrations of Obelia embryology in HJ11~lan 1 40 , ~ ctte ' 07 
PODOCOLYJTE (June, July. 1 or 2 colonies will b collected with 
each 100 Eydractinia colonie from ::,bee ... - l-en .ar· Ol' , 
1 r one fror.J Pas que . ) 
The highly specialized colony grovs i1 a ncrustin[; r1at on nn L. 
she}Js, etc., and is almost exrctly like hydractini • Bot•1 DV 
three type ~ of individuals: feeder~, goyos~!esJ_ stin rs (~f. 
descriptions of Hydrnctinia below, p . 7 lodocryne is includ ~d 
here because of its startling meta e~e tic contrast to Hydra·ti. l~ . 
·'tedusae of Podocoryne are ne nrJ. y perfect , and may prodn e s e \~ · r 1 
gene rations of new medusae by asexual buddinG b~fore ge ting 
around to tl.eir main busin6ss of gamete produ tion. Cf. Goott~;., 1 1 
(The sporosacs of Lydra.ctinia boar very litt1e rescmbhmCL to 
medusae, being highly degenerate). 
Sketch the three types of polyps and show s cvt..ral stages in n och El 1. 
develop:-:tcnt. Gonads can be made out nl ong the radial cana1s of \11 
swiil'lr..:i.ng medusae , very innnature but sexually di"'tinrutsha~· lo. 
The asexual colony gives off ci tiler male or female mod 1sao, hot · o·:· 
B . Study ef Forms '.'lith Imncrfoct hcdus ao . 
--------:.E-.:.----·--... --·--~----
Examples: l·ennaria, Tubulrr:i.f.' . • 
Life Histori es~ Zysote shed from short-11. vcd im1-.erfec t modus[) 
TPcnna-riaT--or "r"(."tainod in r educed s cs ilo med1"'.S£l form. 
(Tu~ ularia) : Devol oprren t to .El:_n_n~~:.'.'\ l~r._y~ and n·c tnmorpho8 is to 
polyp (Pennnri~) or devele~~nt to Actinula lnrv~ ond gro uth 
to polyp ( 'Lubularia) : As oxual mul ti'pl ic-6-ffon -6-.fpclyps by 
budding to produce colony; GonoplJorcs formed by special bud:.: 
on hydro.nths; Maturation e f gonop1·· orcs (imperfect medusaG) ox1c. 
f0rtilization either i n 8itu (Tubulrria) or within tho limitn 
of the colony durinr·:. tho ir detachm,.nt (:t cnne rin) • 
PSJ'T_?iA~\-~ .h (J"uly , !m.:_,ust , Scptcmb,,r; bcp_;l.ns ·co ripen mj_ddle of Jul1.r) 
Gonoyhorcs bud off sin~ly around the low ~r portion of tho hydrcnt1 • 
They form slightly rcduc•.;d medusae with rudimcntcr·r tuftli]~f: tont~­
cl es. Before open inc out c.s tr:::.nsparant b Gll - shr.pocl forms they 
suggcs t coconnu ts. A s ingl ~,, col oYJy boars r;onopr orcs of one sex 
only, but in the living indi,iduaJ.c, s ox can be diagnoGod onlr 
With difficulty until they HJC.turo , '~1hCn th•. lJinknf;;f.:S Of C["'WJ 11nd 
the whiteness of sperm o.ppco.r . (S:•.nllvJOod 1 89) . 11 1':1a lc 11 E~nd fcr.wJc" 
colonie s aro actu~Jly asexual , boarinr molo rnd fcmrJc ~onophorcs 
respectively . 
Maturo PGnncria colonico festooned witb nhcddinr medusae provide 
4-
an astonishing end bc~utif 1 spcctacl t thct very s ud~ t should 
ccrta inly sec . The we. t ·ric 1 ri ens in th~.; s cc.s on of \ nrm t \ f' tor, 
nnd a demonstration of the sheddinr ~ill b. mqd \hOI pocsi lc . 
-t starts early in the evening c.nd continues throu h midnir::ht . 
It is usually best s een in mc.torir-,1 brought into the lnborr.tory 
the preccdine dey (i . e . tho second nicht) . 
The ripe mcdusc,o gro.dually start ~ rhythmic tvitching . Those \':hi l 
2.ro mc.les Gr.li t puffs of v1hi tisl-l sperm, .nd tllos which r.rc f m".l . 
eject v1ith greeter trcvc.il tho t':r~.c t.o six opc.qu pink ggs . In 
the south, Fenn~rio. r.wdusac generally br ale loose from the colony 
and swim about during this discho.rcc , but o.t i1oods Hole they gc n-
erally remain attached , c.nd tho eggs may not b0 cjoct~d until lmf 
after fertilization . Tho medusae fin lly drop ff, swim very fu~l~ 
if ut c.ll , shrivel rnpidly c.nd die in n fow hours . 
Put small so lee ted sterns from ripe "male" and 11 f m;.l~_, 11 coloniL-S 
together in a fingorbowl after careful ri11sing, nt 3-4 p. m. 0.1 d 
leave them overnight . 'l'hoy o.r '· oxtrc.moly s ens it i v to ovo rcrowd-
ing t Next day, roll'ovc., tho stems and look witr nc.k< .. d ,__yo for frl"' 
modusnc as evidence of shedding . lf they arc found, lool for 
developing eggs . 
The eggs nrc very s impl c :.~nd sl 5.£1"~. tly ameboid, with no nppr-,rc nt 
mcmbrnn.c=' . FullovJ the ir dcvclopn·cnt r.s fnr os possihl(· . lf t :lt 
wo.tor is cho.nged seve rc.l times , tl c plnnul a s tc-.c; '' should bo ru' ~l·~d 
in 24 hours, and st8.gcs in tho very simpJ.o mctr,morpl!Osis to t1.c 
polyp form may bo observed . (Hnreitt, G. T . 1900, 1909) . 
tease apart mnlo nnd f cmnlc c;onophorcs rnd exc-.mine the oc;gs nnd 
sperm. Ske tch scverr.l ::1 tc\gcs i n dovclopn·cnt of the mcduso.; sketch 
the matur~ male end female mcdus rc; sketch sovernl clcnvngo stages 
if found . 
Illustrations of medu~a development i~ Goette 1 07; of cloavag 
in Hargitt, c . V'J ., 1 00 . Also Ll:t'111C'n 1 40 
I'U ~:·u LA1UA 
------
(June, July) 
'1'1_, c Gonos omos of n wcll -mc.turcd s po cimcn form long r ncumcs or 
clusters of gonOi-·horcs croHding and !droopin;:· from the re gion 
between tho cira.cs of t entacles on r. hydrrnth. '1 he gonophor 0s 
qui to seve rely reduced modus r-.e v1hj_ch never 'bee omc free -swimming , 
usurlly have no evident radial or circul~r cnnclo ond dcvcl epc 
nothing but buds for t cntrclcs . ~rlc and female gonophoros occur r 
scparcto colonies . Diagnosis of sex by sight is impossible in the 
imnw.turc , but gonophoros tbGt contain embryos nrc easy to t ell fro 
those that o.ro fiJ1cd ',iith G cloudy mnss of npcrm . Early ntngcs of 
developing embryos nrc found b:r t cr.3 :l.ng v1i th noodles , thoso ncnr 
tho hatching stngc nrc visible in situ . 
Exuminc n ripe mnlc gonopr ore , c OlllS idorinr it c- s a. vor:r dcgonornt o 
moduso. . Notice ['nd "' ketch its mode of attcchrr'-nt, its shnpc, the 
structure of ito free ond r.nd the position of thG sperm surround-
ing the dc.rk r ed mc.nubrium. Crush it on c. s lidc and inspect the 
motile sperm under high power . 
5-
Examine and sketch a ripe female gono hore . Ts ally the tentacl' 
at its distal end appear only as four short ~1 nt knobs, but one 
or r:ore of them are sometimes slightly elongated . l e eggs om 
from favored oocytes that progressively swallm up their neig: b 1s . 
lying in the space around the spadix (manubrium) . 
!Vhen ripe the egg is very large and somewhat irregular in shape 
(Allen '00, Lowe '26) . After fertilization, cleava[ is often 
chaotic, apparently either a coeloblastula or a morula may be 
formed (Love 1 26) , and gastrulation of the formAr has been de-
scribed (Benoit '25), as a mixture of delamination and multift lnt' 
proliferation. The emtrvos are developed up to the 11 Actinula 1 s+.:, e 
within the gonophore. 1be Actinula larva is to b considered ~s 
a precociously metamorphosing form, ·art planula and part polyp . 
By teasing some female gonophores open wit _ needles, col1oct nd 
sketch eggs and larvae in various stages . The larvae just tal·i 1£" 
shape are flattened tith blunt marginal processes. At first tho~ 
are not symmetrical in outline but radial symnotry is attn ned 
later. When the larva has reached t!'1e Actinula stage it as o. 
mouth and aboral tentacl es , and a rounded r.boral body that l~tor 
becomes attached at its tip . 
Study and sketch the structure oJ' a fully formed Actinulo. larvn , 
Look over some ripe 11 fomale 11 colon j c.s for Actinula beginninG t .. 
escape from the gonophores . .·.ctinulae will not develop furtho1· 
wi tl.1out feeding . 
1ubularia anatomy and development is illustrated in the texts of 
MacBride '14 , Korschelt 1 36 and E~1an 1 40 . }or cleavage cf . All en 
'00, Hargitt , G. T. 1 09 . For e: onopJ.1oro development , cf.Goettu 1 0? . 
C. _ . .S.~~_dy: ·-<if~-<?.!!~~ _v1 i th J:2~ger~~_r_a t_e __ } :Io_d_us 0.~ 
~~~.!'0J?_l_e_E_ __ Ii t~ _9].. o.s.~o.s_t_yJ.~- --iT?:S 2:~9. _E_O_!?._otl!:_G_C?_~-: Campnnulario. , 
Gonothyrea . 
Life Histories : 
--a-r-compa!1ufnria , Gonothyreo.; Zygot 0 devel ops into planula 
larva inside sessile degenerate modusn; .t1anula escapes , livv 
fr ee awhile, metDmorphoses into n polyp ; Asexual multiplicatir .... 
by buds ; Colony form~tion; Degenerate mc.dusno (gonop!loros) 
formed on o. blas tos tylc; Gonop'- orcs m::l t u r c. in situ; Sperm ar 
shed, eggs fertilized in situ . 
b) Hydro.c t inia, Ludcndriul11: Zygot e dov.:.:lo~s into pl nulo. lo.rva 
ei thcr ins ide gon::;s orne (Eud ondrium) or c fter b e inr' shod from 
gonos omc (Hydrc.ctinic.) ; Plr.nul<c motor:orp} os en in to polylJ ; 
Asexual multiplicn tion by buds; Colony formntion : Gonosomus 
formE-d fror: Eydror1~ izc (~·Iydc.c ti n a ) or by trans forme, tion of 
hydranths (Eudcndrium) : -.7onOlJl- orGs (h1 chly r cducGd medusa e or 
sporosacs) I3orne on gonosorr.._s ; ·Jgp.:.s r··1d S}:)Grm f ormed in tho 
sporosncs; Eggs f ertilized in situ ( :i~udondrium) or during shod-
ding ( Hydro.ctini~) . 
6 -
(June, July) 
It is not safe to try to distinguis .. this g nus fr r. Ob ~lin by 
the anatomy of the feed in indi viclar.ls . ·v ...,n ::C" co 10s on cs arc 
similar in appoarRnce in tho two e;0nern , en ~- o 1s is til of n 
trans pnren t gonot!Lecn .lit 1 the blns tos t?1e ext ; ding from bas o to 
tip end gonop~ores budding from it . 
The strE:ing difference is that Obelin 'rod cos n arly perfect 
free -sv1brr:.ing medusae, who reas CDm~nnulnrir. roduc~..s conophor~ s · o 
utterly dogcncrc.tc that ttcir modusc. - lilcc structur ct:m only be 
madcout in sections. Each go~ophorc o tho lastostylo of a 
"F cmc.lc 11 colony conto.ins c. vo..-ry largt-: irrer:ulo.rl:; shr..pud og. v:' 
is fertilized in situ, cler'.vos, forms fl. moruln, gns:trulo.tus l:y 
delamination <:'.nd rec.chcs the frcc - s•1i ... ing plo.nula star;, sti]l ia 
si.tu. Campanulr.rin , therefore , rcl or s os from its gonothcc , not 
meduso.e but planulae . 
Because the gonophores arc so inconspicuous and tho embryo o 
obvious, tho celonios whicl' produce fcm·;lc gonophoros nnd lat r 
contain embryos c..re loos e ly spoken o.f o.s 11 fcm::.lc" colonic , nl-
though they r.rc c-.sexuc.l . 
SoJ.c~t fror.1 c.. ''fcmc.le" colon.r o. sonosomo show.i.'!g eggs in tho 
bo.sc.l gonop~"lOrc s . ~'- ount it 0 1 r. slide, studJ with v rious m, . 1· · 
fico.tions under the microscope, end sketch it . 
Select nnd akctch r.nothor e;onosomo \'lith plrnuJ.no shovrinc; noo.r t~· 
tip. Squeeze or open the gonotheca with noodles f.'.nd libcrcto th ~.. 
plnnulc,o . r oticc their ciliatE:;d ectoderm nnd \'JO.tch their move -
ments . If tho planulae arc well matured , they arc two or three 
times l onger than broad . They show maggot -l ike movements even 
v1hil o within the gonothc ca • 
Cc.mpnnulo.rio. is very favorr-.bl o f .. r the studl:r of planuln mcta.rror-
phosis . Put o. few mc.turo plnnulr.e us ide in st.r v·c.tc·r (not more 
than 2 or 3 to c. vmtch glc.ss ,) and cover t ;-'om. rrhoy should attP.ch 
to the glass in 4 to 10 h ours. Er.ch should then opon a mouth , 
bud out tentacles, secrete hydrothcc~ nnd porisD.rc , and become n 
full formed indi viduc.l polyp in two or thrco dr-ys . ~.~hon the 
plo.nulo.c have attr~chcd , the vJc,tor should be: chf ngcd in tho dis r,t 
leo.st twice a dD.y . Sketc::t soverr.l st[.tgcc of attflchmcnt ['nd 
metamorphosis . 
The g~osorr.os of "mo.le " colonios ::'rc· similar in form to tho 
fcme-l e . The gonop!:.or c s , v1hcn mo. tur c , n r o r eund od c.nd ho. v c o. thln 
milky- gr ay c·•lor. 'The sperm be come t:.ctivo ·when they nrc dis-
cho.rged into contc,ct witr' so< v1~tor , ns i1'ay be seen by crushing 
r.. mule gonophore under c co ve r slide v1 1ilc watchinrr it under the 
microscope . 
F'or iJ.lustre tio:J.s of Cr.mpr-.nuJ.r.ric g ono:,/ ·orc dcvolopmr;nt c . f •. 
Goct te ' 07 . 
G0?-10THJT~A ( Jul:t , August) . 
As in Abcli[', the medusc.o dov vloy ·.d.t ·1j_n tho fOnothccn . t'lhon mr'-
ture , instc8d of swinmjng n 1ey thoy remain nttrchcd to tho end 
of the blnstostylc , projecting outside the gonotheev in group3 of 
7-
three er four, like tey baloons. \.it in the balls of th med sa 
the eggs of "female" colonies are fertilized ~ • the shed spf'rm 
from "male" colonies. The zygotes develo to thE' planula stage 
before being set free, after ~hi~- the med·sae r p of • 
Sketch plan lae in various stages f development in the projE>ct-
ing attached medusae of a ripe 11 female 11 colony. c.·.ulfert 1 02 • 
Gonoth_.rrea cleavage illustrated in Vlulfert 1 02; ... edusa develo1.-
ment in Goette 1 07 . Cf. also texts of Hm~n 140 and KorschE>lt '36 . 
HYDRACTI1r iA. (June, July, Aug..1st) 
Colonies of this form are fairly conlr"On or· Littorina snail ... lls 
inhabited by the small hermit crab, 1-agurus . T ere are thr 
of individuals in the full developed colony: ord inary poly s 
(feeders), threadlike coilinr forms with no mouth and an apical 
knob of nema tocys ts (stingers, co1 mones t around t 10 1 ip of th 
shell), and go nos omes. 'l'he three types all arise f rom a hydror 1izc 
network covering a rust-red spine-studdied crust. 
a) Gonosomes and Gon onbores: 
- ·--· -- --- · - -·~- - ·----
The gonosomc s or r e productive individuals ar v usualJy withr,ut 
t entacl es and have a large knob of nematocysts on the p .. : ol. .. :Jc. 
each boars a number of gonophoros 0 vlhic1'"' art' )1'e dusa-buds r c flu cc. .. : 
to the status of s 0rosacs . Ripe 'male " and " female" colonj os cr~n 
be told apart with the naked e y e since the eggs within the 
sporosacs are dull green against the red hydrorrizc, and tho pur 
when mature are a whit e mass. 
Remove several 11 male 11 reproductiv e ind l viduals show ing ripe 
sporosacs , and crush them slightly und e r n coverslip on a glass 
slide . This may be done by drawing off some o f the water with 
9lotting paper. The sporosacs fall far short of b eing pcrfoct 
medusa e . Note stages iP tho produ c tion of spc rm within t11 em . 
Burst 2. mature sporos.ac and study tho r: por m u n der high powor . 
Simularly, crush severa l 11 f emale 11 r eproductiv e individuals , and 
observ e the eggs wi t h their largo gcr·mina l vo c· icl cs , in variou.s 
stages of deve lopmt..nt . Sk c tc~~ tllo 11 f omnlc 11 g onosomc with its 
femal e g onophor e s snd containe d eggs. 
}2j __ Q}ee.vag£_ an<L~~Y..~l?_PI:_lent. 
If a numh.cr of 11 mo.l c 11 ond 11 f emo.l o 11 celontc s of Hydractinia arc 
put together in c. large d:1.s:r of s ea wat e r ( er a pa ir of prime 
coloni e: s in a fingorbowl) and l of t ovc rn J.vl1 t, egfs shou1d b e shud 
and fertilized. b otw oe:n 7 and 9 e . m. \f1S'l . '1'11.0 shcddinrr con be 
controll ed by ligr.t, If fcrti1izr·. tion end. cl\.cvap:o stac;(Jo nrc 
n eede d lat e r in the or.y or in th e.. ovcninf , t:'lc c olon c· mry bo 
kept illuminntod during the prt;C.din~"' ni ~ 1-~t , put in tho do.rr fo r 
a couple of hours r.nd r o i J.lu minat ccl one hour 'H.Joro tho tim0 '<Jhcn 
shedding is d ., sired. Ef!gs c.rc sh~;d in b5 mirutc~· , sperm in 50 
minutes . Cf . La1lurd , 1 42 . 
8-
.:atorials for the study o th entire de' l o • nt of Hydra +-inin 
from egg to polyp will be made uvGilc.ble. If possi lo, obsor•vo 
tho shedding of eggs E'.nd sperm. Sl·ct"'h (:;r:.f:s l.l.nd~..:rgoinK first 
three cloavc.ges, elonc-atod gr.s truln, 3wiru. ing r..nd o.ttnchod 
planulae, mctruLorphosine form and oung ply~. 
Eggs arc Gavily yolky and suaJly ~rccn, ut occcsionally gray, 
orange or pinlc. ~~ature. tion take p :.'. durin···· the hr.lf h ur pr 
ceding shedding and polrr bodies aro lost soon after, a loose 
jelly being the only covering of the eg.:. 
Cleavage may b e irregular or even ,haotic, but usually th 
slightly ameboid egg undergoes throe. equal total clonv\gus, 1..f'<'h 
at richt angles to tue procuodinc . Tho so arnting pairs of 
blastomeres tend t o retain broad protoplnsn.ic connoctions \'itl• 
each other on the side opposite to the clcav~ c furrow, until 
just before the succeeding cleavages b0gin . It i~ soon o.p nr nt 
that there is much variation in tre time and derrev of shiftin 
of positions of the blastomcrcs, but the extro.ordinc.ry and 
choa tiu cl ca vago patterns corr.Jnonly seen in the cl~ room o.r oftc~n 
the rosul t of drying up c.nd c onccntro.tion of s0o wnter, or otl"tJl' 
unfavorable circumstances . 
r.~itotic sychronism quickly disappears . GastruL .. tion is s id to 
start even as early as the 16 C(l l sto~e, y ) i .od del~min<tion 
and multipolar proliferation. rl"hc gastrula loses its sphcr1c~>.l 
for m a.nd spends n fcvJ hours 8.S an i .. rcgulerly bumpy obl0 1£: H [l • 
then returns to the sphericr~ form and -:radua. 1 v lcngthuns :l.nto 
the p1c:nulc. form. 
At th0 end of 24 hours the omhryo is a 11 prcplanulc. 1i (T eissic r 1 2' 
with an elongo. ted ovoid form , r <:. c • rrnizt.blo polnri ty and c 11 in t -~on 
which enables it to swim heavily . B.1rinr::: several days it lc ·n r -
thcns , one end becomine; slirmnE.lr cmd slirr .. mer, whilo it r olls nnr1 
and cra::ls along the b ottom like a planc rlen . The bie; end whic. • 
goes first in this movement is the one V'hich lntor produces t 1e 
adhesive disc by which it attt:'.Cl1 ,..; S fo r mc t0.n or_.,)1osis . It bec011c s 
tho aboral end of tho polyp . 
}'ollowing attachrr0nt of tho ntte;nunt cd plcnula, there is a 
dels.y of a fow h ours t• s everal days , c.nd then tho tr.poring f''C c 
end shrinks down almost t o the substrate , ¥he r o it shortly pro -
duces a mouth nnd a succession of tentacles . The nc:w polyp c. lon~ ­
atcs , its attached and meanwhile actively ~ending wt n numt c r 
of anastomosing and encrus tine: !.1.ydrorl"dzo procossos from v..bic~ 
sprout nc ~, polyps . 
Illustrations of cloavo.f:/ in Bunting 1 94 , F.>cckwith 114; of lat v:-
planulc~ devel opment in .t c issi <:. r 1 37 ; of dov0loping gonosomc) in 
Goette 1 07 and 1 16 . 
EUDEJDR-m (July, .t- ugust) 
Tho gonos omc s f thi s gonus arc dc rnmc :r·a t c s c·s s j lc mudus .. - forma 
or gonop1'1.orcs , s tr :i.kinp.: l 'T diff c. r ~,nt :i.n t "IJ.c t· 10 scxc s , borne a.t 
the bas e s of s pocj_aJ :1.ydrr.nt hs ',vh:i c: los e thei r t cntnclr.s c nd 
d cgene re. te v1hil u the gonophore a arc r ipen . n~ . 
g _ 
'' :?cmnle;1 colonies b cr loose irrc b~lla.' ~ ftc:- f sporosn s 
r.tt~chcd t the stvn:s, C!"l. uric S'Cl'OS['C "" '-' · n brie:'-lt orcv"c in 
color . ;;: .. rlc 11 color:i ... s ben. ... lL_:ht ph~l· s ... orosr. s nrr"'.l1Nld in 
groups of t"!O tn four r norc L e li:1, th~ lin r<>.d'~ting fro' 
c comr.:on point on th.: br.s c of t:J0 dog n rn t d ydr~,.1tl . 'lh 
ripest mule sporosncs ~cc r at t~c ri ·Lrv f the clu t r a.nd 
r.r: •:rh 'te v:ith sperm. l~c.t h bbth n.r.lc ~·nd fcmr.l S}orosr~cco . 
Eggs r.rc fc.rtilizc.d \"it>i.:: the f~·malc £'Onop' or or _orosr-.c, n1 
de rclop to tho pla.nuln s tns;e be for~ ·c ing l' ore tvd . (Herr-itt, 
C . · .. . , 1 04 ) . rfcn3o out c·~r:r s fro:l~diffcrf· t coJonl.,s end •·tudy 
nJ l s tagcs f eu no . The cr.:gs c.re so ric.1 i1 yolk the t the~r cl c c.v~.. 
li':e insect eggs . The gastrulation is by nn xtro.ordin~.ry svn-
cytinl delnminatien • . .'etnmerphos is s sir.pl; . 
Eudendriurr cleavage i1lustrntcd ir~ Hargitt 1 04 (::.ool . lTnhrb . ); 
Gonosomr.:: dcvclop11'-..nt in Go~ttt 1 0~ . Cf . ~.lso texts b_r "or c~1l'lt 
1 36 and nyrnan 1 4 t. 
_De~cls>_p~c.~ t _ _?_f_ S~_Y.P..h •~o.a. 
AHE{ELIP. O~ __ q_Y_j~TI.t: (April - Jun L. ) 
Both these j ellyfish c~ hav J oral lobes uYt~ d,~~ downwc.r ~ . In 
maturE- specimens grnnu1nr material v;ill be fomd ontc..nglccl n t1ll· 
lobes or contained in small brood ss.cs in th lol'hs . 'lt;e t off 
some of t:'lj.s materia l into a drep ef s,A1 vc.t01' on n slide, n.nd 
exnmine under the microsc ope . Embryos of diff~,.,rcnt stag, s ccr. !1t 
f•und, frO!" spherical cl eaving ecgs to oval r;nstrul['ting fori s 
and full,r formed stock~r, active plc.nu1r.o . (Ho.r itt, G. T . 1 00) 
(Hein 100). Is clcc.vago r0 [,ulnr? 0l~otch thecmbryonic sto.~es t.h<tt 
nrc available. 
S e l e ct a number of activo plc.nulo.e r.nd place them in clcc..n 
watch glasses of soo. ~atcr for further study on l3tor days . ~huir 
gradual chango in form, at: tac·r on t to ti1 l"' bottom, o.cqu is t ion of 
tentacles and elongation into the sessile scyphula stage can be 
easi l y fallowed . 'l~he rGsc1~1bla 'Ce of the scyphulll to o. si!: ple 
polyp is obvious . Attn.c'bJ •ont plus the formation of 2 t • 4 
t entacles occurs ,, on tho second dD 7" , a.s docs the d'JvOl !'pmcnt of 
an opon mouth. lhore arc 8 t entacles at 4 days, 16 at 2 wcGl 8 1 
24 c.t 1 month . Sketch the scyphula in side vievJ and in t ep vic'., . 
The s cyphuJ.o. or s cyphis toma s tagc lasts throucr10u t th o vlint cr . 
T'n.o animaJ.r.: increase in s izo r.nd undergo asexual r c preducti en 'r.)y 
transve rs e fissitn into Gphyr ae ( 11 Strob i1izo.U.on 1'), n.nd by oth .r 
methods (Percival 1 23) . The tiny ..:..pl"lrD.E:; (lnrvaJ j e llyfishes) o.r 
lib c ru.t cd c.nd grc.duG.lly transform into tl:.c adult form over a 
period of many months . 
Aurelic.. lif<.; h~Lstory illus trntcd in the texts of J acDrido 114, 
:<.:ors chcl t 1 36 and Hymnn 1 40. 
·-
Brooks, 
1886 
,~ • K . 
The life history of the Hydromedussac : A discu ion of 
the orig in of the medusae , o.nd of t~1o signifi nne{ of 
metagt-ncsis . :·cmoirs , Boston Soc . Tnt . Hist .,___1.:359-ttj0 
F oyn, B 
1928 
Goette , 
1907 
A 
Studicn ·u~er Geschlecht und Gcschluchtsz,llcn hc i 
Hydroidcn . Bcmcrkungen ubcr die Intst chung dcr K"imz l l e n 
und die I:,ntwicklung dcr Gonophorcn bci Clava. squmno.te . 
Houx 1 Archi v . ,...1:_~_4 : 501 - 510 
Verglcichcnde Lntv,·i cklungsgcsc., ichte dE.r c s hl cht"' -
individuen dvr Hydropoly en . Zeit . f . ~ is~ . Zool ., 
87: 1 - 336 
Hn r e; itt , C • y., • 
1901 Synopsis of i:~ orth American In v e rt ebra t o • XI\ . rl'he 
Hydromcdusae . Am . Nat ., 35 : 301-315, 379-395,57'- 95 
1903 XIV . The Scyphomedusno . Ibid ., 37_: 331-345 
1904 The medusr,o of the \ ioods Hole ro g ion . Bu11 . Bur . Fish ., 
24: 21 - 79 
Hargitt, G. T. 
1919 Germ c e lls of Coelenterat es. VI. General considerations, 
discussion and conclusions. Jour • .!v1orph . 33: l - 6 J 
Hyman , L . H. 
1940 The Invertebrates . l. Protozoa throuP,h Ct enophore . 
McGra-Hill 
Kings l ey , J . S. 
1910 A synopsis of t ho fixed hydro ids of Nev1 Englo.nd . Tufts 
College Studies,~: 1 3 - 38 
Mayer, A. G. 
1910 r:edusae of the VIOrld. I. and 11 . rl h e Hydromedusac . 
111. The Scyph omedusae . Carnegie Ins t . v'a shington 
109: 
Nutting , C . C. 
1901 They Hydr oids of the .1 oods Hole r egion . Bull . U . ~ . Fish 
Commission , 19: . 325-386 
Sumner , F . G. e t al 
1913 A biological survey of the wators of ,. oodc- Holo o.nd 
vicinity . Bull . Bur . F ish .~: 
A. GENEBAL H1_BRYOLOGL_ _ _P.~_SCR_IPTIVE 
Allen , Carrie M. 
1900 A contribution to the dev elopment of Pury hn (Tubulorin) 
croce a . Biol. Bull ., l: 291-315 
2 -
Beckv:ith, Cora J . 
1914 The g<.-ncs is of the plnsmo. s tr'..lcturc in the cg~ of ::1yd1 l'.o-
tini& cchinc.tn. Journ . :orph . ,__g_~: 189 - 2 1 
Benoit , P 
1925 L1 ovogenese et lcs rcn1icrs st['.dC'"' d..l dc\rt-1 rc nvn 
la :yriothele ct chez ln. Tu ula ire . ;~rch . du Zool . 
Exper ., 64: 85- 326 
hez 
B nting, 
1894 
>artha 
Do flo in, I<' 
1896 
Goette , A. 
1916 
'Il'c origin of sex cells in Hydractir~io. o.nd 1 odocoryno, 
and tho dcvcloprr:cnt of Hydr~ctinia . J urn • .. orph., 
9 : 203 - 236 
Die Eibildung bci Tubularin . Zeit. f . !!iss . ZooJ ., 62~ 
61 -73 
Die Gattungen Podogorync , Stylactis und Hydrcctini~ . 
Zool . Jahrb ., Abt . System . Gcor . ~~ol. Ti~rc 3~:443-510 
GGr6hberg , G. 
1898 Be i tra c qur Kcnntnis s der Gat tuns 'l'ubulo.ria . Ze ol. 
Jahrb ., A~th . f. hnat . 11 : 61-76 
Hargitt , 
1900 
c • 1"i . 
1904 
A contribution to th~ natural history nnd dcv~lopmcnt of 
~ennarin Tiarella . Am . lnt ., 34:387 - 415 
Tho early dev elopment of :Eudcndrium . Zool. Jahrb . , Abt. 
Anat . u . Ont ., 20: 257-277 
Hargit t, 
1909 
G. T . 
Ma turation, fer tilization and segmentation of lcnnaria 
tiarella nnd of Tubularia crociea . Bul l. il1us . Comp . Zool . 
Ha rvarc, 53 :1 59 - 212 
Lowe , E . The embryology of 'l'ubularia larnyx. Quart . Journ. r~icr . 
19 2 6 s c i • , 7 0 : 59 9 - 6 2 5 
May , A. J . A contribution to the morphology and development of 
1903 Cor ymorpha pcnduln Ag . Am . Hat ., 3?: 579-599 
Smallwood, 
1899 
·,.; • M • 
A con tribution to the morphology of .t'(;nnaria tinrella 
l~.m . liat., 33: 86l - 87 G 
Smallwood , w. M. 
1909 A re - examination of the cytoloe:y of HydractinL and 
Pennaria . Biol. Bull . 17:209-240 
Teissicr , G. 
1926 Not on critiques sur la morpholoflie des gonophoros chf'Z 
los hydraires . ~rch . de Zool. Lxper . et Gen~ :7 5 -86 
1931 Etude Expcrimentalc du dcvcloplomcnt do quelques hydrairc 
Ann . des Sci . Nn t. Zool . 14: 5 -60 
3-
T issicr, L. ~nc G. 
1927 Lcs rinci r.los tnpcs d d~Vt lo.r,. n~1ent d q·, dr".ctinin 
ochinatu. ~ull . Sbc . Zool. Fr . , 52: 537·-47 
,-:ulfort, J 
1902 Di u -'--'mbr;onc.lc Entwicklu~1g von Gonotlyra ~ lov i. Zit . 
f . ':iiss. Zool., 71: 296-327 
B. S1ECikL TOl-ICS -HER: Al-H~ODITE G01! r:· 
Bunting , .:artha (listed o.bovo) 
foyn , B 
1924 
Kuhstlic.1 hcrgcstclltor Hermcphroditsmus boi 
Zool . tnz . , 61: 105- 110 
r~X1 :.RI 
ydroid(..n. 
1927 Studien ub cr Gcschlccht und Gv'c:blochtszcJl~__;n oi 
Hydroidcn. 1 . lst Clava squruno.ta (I/ull r) 'inl' ~onochor ­
istischc oder hemmnphrodite Art Roux' Archiv 109: 13-)34 
1927 
Goto, s . 
1910 
ltll: . Ausprossungsversuchc e.n C:lr,va squ01no.ta ( Hler) 1it 
misch~ng von Zellcn aus Polypcn dos~Glbon oder v r~c io-
donen Geschlechts , Ibid . , 110: 89-148 
On two species of Rydractinlc living in sym iosis ·.ith 
h ermit crab . Jour . ~xp . Zool ., 9:469-496 
Hs.r r;i tt , C . ~,.- . 
1906 Tho organization Pnd early devGlopmlnt of the egg of 
Clava lcptostyla. Biol . Bull . , 10 : 207-232 
C. Sl-ECIAL TOPICS - SYNCHRONOUS SI A!n!lNG ArlD ITS :ELA'l'I - TO LIGr~rr 
·----- --- - -
Baker, E . G. S , 
1936 Photoperiodicity in tho spnvming r eaction of l ·nnarin. 
tiarolla. l-roc . Indiana Acad . Sci . 45 : 251-252 
Ballard , \v . W. 
1942 The mechanism for synchronous s pawninr in Ilydractinia 
and Ponnaria . Biol . Eull . , Juno 
Brooks , ';! . K . (Listed above) 
Hargitt , C. W. (Listed abov e , 1900 and 1904) 
Hnrgitt , G. T . (List ed above , 1909) 
Mayer , A. G. (Listed cbovo , 1910) 
Murbach, L 
1895 Pr eliminary notG on th · lif e '-listorv of Gonioncmus . Jour. 
Morph . ,__!!: 493 - 496 
Ri ttenhous e , S . 
1919 T'c embryology of Stomotoca c.p ic o. to , Jour. kp. Zool . , 
9: 333 - 346 
Sigcrfoos , c: l-' . 
1899 A nov1 hydroid from Long Islo.nd Sound. Am . 1~a t . , 33 : 801-807 
4-
Smallwood, ' • 1 •• • (Listed abov 1909) 
Torrey, H. B. 
19 7 Biological studies n Corporpha 11 . 1he devclo ment 
of C. alna from the e~~ . cniv . Cal . u 1 . 3:2 3-2,8 
Beckwith, C. 'J. (Lis ted above) 
Hargi t t, C • r .• (Listed above 1900) 
1911 Some pro lems of Coelenterate o. togcny. Jotr. to •pl . 
22: 493 - 550 
r-:aas 1895 Experimentelle Be i tra,.e ZUl' fntv·icl·l U.1gs ,es c'1.:l Cl"'t 
der Medusen . Zeit . f . wiss . Zool . , 82 : 601- 10 
Teissier, G. (Listed above) 
Torrey, II . B. (Listed above , 1907) 
Zoja , R . Sullo sviluppo dei blastomeri isolate dalle unova di 
1895 alcune meduse . Arch . f . ~ntwicklungsmechenik 
1:578 - 595 and 2: 1 - 37 
Beadle , L. G. and Booth, F . A. 
1938 The reorganization of tissue masses of Cordylo~1ora 
lacustris and the effect of oral cone grafts, with 
supplementary observations on Obelia gelatinosa . 
Jour. exp . Biol ., 15 : 303 - 326 
Child , C. M. 
1 928 Axi al development in aggregations of dissociated c,lls 
from Corymorpha . 1-'hysiol . Zool ., 1: 419-461 
DeMor gan , 
1 914 
Foyn , B 
W. and Drew , G. ~- . 
A s t udy of t he restitution masses formed by dissociat-
ed cells of the hydroids Ant ennularia ramos a o.nd A. 
antennina . Jour . Mar ine Di ol . Assoc . of l lymouth 
10! ' 
(Listed above ) 
Ha r c; itt , C • \1 • 
1915 Regenerative potenci es of dissociated cells of lydro-
medusae . Biol . Bul l . 28: 370-384 
Wil son , H. V. 
1911 On the behavior of the dissociated cells in hydroids , 
Alcyonaria and Asterias . Jour. Lxp. Zool . 1:J::281-338 
REGENER1'.'l'IO: FROt NOJ - DISSOCJAr1'LD CI:Li S 
Barth , L. G. 
1940 The process of regeneration i n hydroids . U:lol 
5-
15:405- 420 
Chid, C • .. • 
1941 
rattern ar..d .t-roblems of e ·el PP""'ni . • 
~ress (Con cins references t all Child' 
f hi a~ o 
pnp rs) -
Gilchrist, F . G. 
1937 The h.ydroid polyp Corymo:r ~': a t-alma as sost . t a d as 
h istory Am . "rat . , 71:382 - 403 
Hazen, Annah 
1902 Regeneration in Aydractinia and todoc ryne . A~ . tat . , 
36: 193 - 200 
:-:argitt, 
1899 
c . -.. . 
Lund, =-. . 
1921 
1923 
1924 
J . 
Experimental st dies upon udromed'...l.sae. Bioi . [ulll:3~ -l 
Ex1erimental control of organic polarity by the electrjc 
current. 1 . Effects of the electric current on rq;cn rntlnr; 
internodes of Obelia corr1r1iS sural is . Jour . :E.x}..;. Zool. 3 ~ 4, l -
493 - -
111 . Normal and experimenta+ delay i1. the ini tiat 011 of 
polyp formation in Obelia internodes . Ibid . , 37:69-80 
lV . The quantitative relations between current density; 
orientation and inhibit ion of regene ra tion . lbicl~:~f:7-380 
Morgan , T. H. 
1906 Hydranth formation and polarity in 'lubularia . Ibid 
3:501 - 516 . 
~.T T 
1 ... organ , • 
1908 
1 909 
,1ors e, M 
1909 
M~ller , H. 
1913 
H. 
Some further records concer ning the physiology of regen -
eration in Tubularia . Bioi . bull ., 14 :149 -162 
The dynamic fact or in regeneration .-rbid ., 16 : 265 - 276 
The autot-omy of the hydran th of 'l'ubularia Ibid 16:172-182 
c . 
Regenera tion der Gonophoren bei den .iydroiden und 
ans ch l iessende bi ologische Beobachtungen . 1 . Athccates . 
Ar chiv . f . Entwickl ungsmechanik , 37:319 - 419 11. Thecates. 
Ibid ., 38 :388 - 331 -
~cables , Fl orence 
1 900 Lxperiments in regeneration and in grafting of Hydrozoa 
Ibid ., 10:435 - 488 
Torrey , H. B. 
1910 Biological studj_es on Corymorpha . lV • .buddin£! and fission 
in heteromorphic pieces and the control of polarity. 
Biol . Bull . 19:280- 301 
Zwilling , E . 
1939 The effect of removal of perisarc on regeneration in 
Tubularia crocea~ rliol . Bull . 76 : 90-103 
6-
~ArERS 0?~ 'CYi-"OZ A 
Friedemann , 0 . .. 
190 2 Untersuchungen uber die est r.: , r7·onnle ·nt viclc un ~ von 
Aurelia aurita . zeit . f . wiss . ::ool .,_2~:227 -2 7 
Hargitt, c . . , and 2argitt , G . T . 
1910 Studies in the develo ment of Scy hoMed sa • J 1r . h 0r1 . 
21: 217 - 262 
H8 in , i 
1900 Untersuchungen \ibcr die Entv:icklu, g von A r lia nuritn 
Zeit . f . wisQ . Zool ., 6? : 401 - 438 
-~or :-.('h e l t , E . 
1936 Vergleichcnde Entwicklungsgeschichte der r iore . ,T .nn 
r:ac:-' r-1. °C ' 
l Gl4 
Percival =: . 
Text - book of Embryology . 1 . Invertebrata . 1 11 t• 1 o.n 
1923 On the st r obilization of ~urelia . Quart . Journ . I icr . 
Sci ., 67: 85 - 100 . 
I 
In Tubularia we have a system i~ whicr .. eac ar i8 abl t· ref r1 
er rec enstitute int • a differentiated l ~dranth s.u.:-·. " d \el• 
n·omally from the egg . '"hen tl1t=> sten is cut t. r'..l, the • e .ing 
closes a nd the sten cel l s are c enverted directlv 'nt a hvdranth 
witho·..lt furt e>r cell divisi:m. 'There are, ther~ f re, 1 especial 
reserve cc1ls whic. supply t,1e cells for the h_rclranth 10r any o t-
growtb fr en the cut surface. Thus, a _ydranth can form in tvo en-
tirely different vays: 1) ycell di ·isi • n of th egg \'Jith the e rc'er 'r 
formation of a larval stat;c (tle actinula) and:.;,) by ad rcct tran-
formatio::1 of adult stem cells into a hydrenth. B studying the 
second precess we con separate the verio s em~ryoni pr ce~ses and 
deal vith differentiati n al ~ne . 
The process of differentiati en by definiti n ·~c:t"' - the pr e ces"' 4L 
becoming different 'le must either start wit'" a syst"'m in wh ch tl1er•o 
are differences ~r create differences in a ho~ogen us system. 
'I'ubularia stern s! ows s C'\n e d ifferencc"' ut t s d iffercnccs are S 11c:1 
that the chimical envir enment can everride the . es ta bl is, ing a new 
polarity . If the s tom be cut int •. ·a numr er of small , icc es each 
piece will form a hydranth at the ·distal end but n~n8 at tho proximal 
end , thus exibi ting a polarity . ::.owever 1 if each dis tal end c 
placed in a glass tube t1is r elarit7 can be reversed nnd a hydrnnth 
placed in a glass tube this pblarity can he rev e rs ~ d and n hydrnnth 
will develop at the , pposite end, the pr ()4imal. Obviously then 
both cut ends tend to· forrn a hydranth, but the t end:'nc-y is grc8ter 
at the distal end and this i n s'me Nay represses the forntation of 
the preximal hvdranth (DominanceJ . r ~ 
Dominance is i rrportant embr~roJogical.ly as it allows f • r the f ormat ion 
of u single structure from a mass of' t is sue w'l.:i.c is capabJ c of 
forming more tl'"'.an one structure . '£:1e ree;io'1 which has the greater 
tendency to form a structure represses adjacent tissue fror.1. f e rming 
the structure . '11his might be explained on a nutritional bas is wi t~1 
the r egi on of greatest activity drawning materials away from adjacent 
regions . In a crowded mass of c ells as o'otaJ.nGd in the g[1st rula 
sts.r:c there must be a keen cempetition of the vG.r·ious ceJJ s for 
nutrime '_1ts :;;.nd a mutual inhibition by c el ls caused by tboir cxocrct or~r 
products . In both of those phenomena t t.e more rapidly metabolizing 
cell has the advantage and is most likely to differentiate . 
The above situation can be br,.ught about o~perimonta l ly by taking a 
mass of cells and placing a barrier to free diffusion on one side of 
the mass . This has the dual effect of an incr ea se in execrctory 
products and a decr ease in e xygcn t cns lon and the r esul t is that 
difforc:mtlation is inhi' ' ited on this side v;hjlc the hydranth forms 
from the cells of the opvosit c side . 
In the naturnl environment with the pcrisarc ar eupd the coJls it ls 
highly probable that thj_s perisarc acts as a n £· turnl barrier to 
free diffusion o.nd that the hyclranth forrcs at t1'"'.o cut E' nd bocaus , 
these ce1ls are· r clcas0d from an inhiu_ tio 1 caused by both oxocretOl'l 
products and low oxygen t ension . 
COlii.C'IlOi: JJ.l·ffi CL -._: OF 'l'iJ~· lJL.f.. ... I A 
Tho ereticaJ.ly Tubularia is a so] itr,ry forrr 1:'ut pro.ctically so many 
2-
indiv:dwLs ;ro~: · t cct1.c r t .... a ... £. dense tr.. ,· ·d 
in t"IJ.e ole <:-"' for: ~s . ~'! o'...l. c s .ort stc: ·s ar . t:1 
··ork .nd c,.n · c ob tcined f ror.: flouts rnd r -s I:1. 
[t.ncra_ ;_ t is' ust to CO~-LCt <To• r 0'.'."1 st 1''8. uinc tums no"d 
r ur..nin £ \'lat er and a lo" b::;"crt.turc 1.::,"' t _ · ~.-o :1 • t c 1 l in th 
lc.bora tory . In nr. t rc the _ ydrunths drop eff L 1 0' t d o" July 
~.nd t he stens r emain d err.w.n t mtil th ~ r::-.·c.r cool~ i1 th f.ll. 
At JOods Hole t'1.e s tens t""\ 'Cecr in 1i d JT1C' ~ n ,_ cd "t. 1 
Aucust . Hove cr , s:ncc t e. ·:aters of 'jape '-'od 1 .~ n t1{ :·ortl . s· or 
arc "!':uch colccr , J.'ub'.llario. r:a~- be o, tr.incd f .'cr· t _c 1 ort _ nd .. t'~ . 
c anal t~r o-"',·['-lOu t ... L.ugus t. 
The stems 8.S collected vary in l l n ...: th, t"l1icl:ne~s and in rcnt.ra 
physiologicrl condit ion (somt,; are:, n .'S\"'cd, sor:.: stnrv~..d, ot ."'r v c.ry 
old ·1i t·~ large gonophor c s . Ther e f ro , \'H cut t c sterns off nd 
sort th ":~l ·out i n 2. l t-rge f i .. ger ov1l · i nc cart.ful ~1ot to er s' 
F or most ·:ork the s h ort sterns aru aho 1t 10 mm , 1 nr~th an<1 or 
suitable . Tl ese a: c s e lected for unifor·; dirr et" r and a 1 o. r anc• 'lnd 
the hydrant l is cut off a fcv· mm from its bnse . It is no .lSS'·,r,· to 
cu t of f f: omc of t£1e stem \'Jith the hvdrantr~ as t is p .... rt of t1:.o "tel 
dovs not rcgsnorate cons i stently, os prc.ialJ.y in older stLn' • 
Of cours e , even aftt·r s elect i on the stems s how so1· e voYic:b ility in 
regcnerati~n and so it is bes t t e pool all th~ st~res for one 1xp rl -
mcnt and se1cct at r and ern fo r the: various parts of the cvpt rir·C''1t. 
TJ"'.U.S, if you are treat ing th e stems in 4 differ ( nt vr.~rs you "houJ d 
s eparate tt.e stems into 5 lots at rc:-.ndo-,, usinc; one lot os o. control . 
The number in oacr lot do ·0nds on th e. :w t urc of t' 'v t. pc r imcnt . 
t1~any expe riments art: of the o.l. l or none natu rc o.nd 1 stems il'l t c c.) 
lot are sufficient . Ii ox pGrimunts , .. h0rc r 8tc of rcc;cncration is 
compared und~r different cond i tions tt is bcr;t to us E:. about 25 stv.~s 
in 0a cl1 lot . 'l11.is gives a v e ry h igh o.c.curDcy in av ra[7ing rate • 
The st erns are kept in run.r:. inr, r-o. tor nnd cool by p1o.c in~ them in 
syracus e watch glassus vhich arc first r.;ubmcrcod i n lorc;c finrcr >O'ils 
throught which s c&~. '~·a tcr i s runnin. r-; . S orr.c c2.r" must be. tr ken tho. t 
t~e stems arc not ~cshod nwey . 
'l'he instrur.:cnts u s ed for cuttin[ and )'1rnc1 lin~ arc 2 pcir of shan~ 
scissors , a pair of forc eps and o medicin~ dr opper . In usinf th~ 
f ll reeps cnro must be talre n tho. t only t'::t: pn rts which 2. re fina11 y 
cut off and discarded arc handl ed . 1-of t . r the ston1s h a v e l)ccn cut 
to s :Lz e they nrc trans f e~rcd wi t1~ c pipe tte , Gnd must not be han01 c d 
'Ji t h forceps . 
When the tan perature of running s ea. ,,mtor ro ts c·.bovc· 25 c it is 
neces sary to keep tl"e st ems in t.. rofrircpntGd br-th or regeneration 
will net be consistent . 
r.!.'he "!arbur gs appear to lJG 8.n GXCCllc-nt plCCL f e r 'lubu}o.rja D.S tl 
cons t rnt shakinr: kc;,..ps them •·;ell o.·.rutod end bo.th ~,;d ,.,i t'1 no utjon 
with the r csul t tb.o. t t:'lo'r r cg0nc r o to rnpidJ. y rmd form la rgc hydrc.n t'1[.:. 
F our lots of stem~ v-1ith 10 stems i::-! e<c· Jot \Jill rc used . 1) long 
stems from 10 to 15 mm in l ent;t1'1 aft.:::r c1.1 ttinr:; 2) 'hort stems ubout 
6 nun long 3 ) short stt:.ms 'nith a lie;atu r·o tjvd in the. middl ( of t h 
stem; 4/ v r~ r-i short stems cbout 1 or 2 mm long . S• lcct stems t.bout 
20 to 25 mm l e.ng for 1tl c:-ncJ #2 , 3 o.nd 4 sttms up to 10 mm arc 
suito.ble . These can th0n be trimmed to sizo be in;. v c. r7 co.r~,ful t(') 
3 -
< 
rcme ve some e f the stem ~-:hen cutti.:: off tho -.~ r:.'.nth. 
This ex;crine,t is ef the Gll or none n~tur~; th~t i , th~ proxi~nl 
h~rdro.ntl '"'ill usur.ll~r either du c lo in •11] tt..ms :>r be nbs 1t in 
o.ll st..;ms bccr,Qse of d:>min::mct. of th"' istc.l h:·drc.nt11 . 
Tor this cxperi ent it v:ill be necessary 8 _ly to r'" cord th'- UI: cr of 
hydranths dev·.::l.., inc i:1 each of the four lots . lt I.'lt:Y b' s .f ly 
assumed th8-t t..ne distal hydranth alwcys dt'\' lops so thr. if on~.; e d 
fc:::.ils to forr.1 a hydrc.nth it is alr:os t ccrto inl y th\.., roximc.l nd. 
After the stems hcvc been cut to si~c they arc 
glc:>.s ses in t, e circulating s en wc.ter. Ca:.,e must 
incoming sec. water d e os not wr.sh the stems "\'Ja"~. 
enter at t~e bottom of the lr.rge finger bowl and 
water run too fast . 
lactd in thu w tch 
be t: lct.n tho. t tl o 
-~rvc th sea ,·;at .r 
d 1 ot h v o tho 
At 48 hrs . tho hydrv..nths should be fully f e 1 .ed ··ithin t.hEJ cri n 'C 
and s emc should be emergine; . hecord both the numbers . D1scard st m 
v1hich do not shov1 re generatlon at eitJ.1er end . 
This again ls an alJ or none typl.' of cxrermont i nd only l t s turns nr 
needed in E::a ch lot . Dr[,'tl out glass tubes about 1 mm in inside 
diameter so that tho stems v:ill fit into these tubes loosel y . 'l'r;l{ 
l~ stems about 6 rrrrn long tnd :i.nsort the disto.l end of tho stCI int 
tho tub e leaving the pr e>ximal end fr ee . Usc 10 controls to doto1 11inL~ 
tho behavior of the pr exinw l and disto.l ·nds under normal conditjon:.,, 
GRADIL:'l'S p.; 'F_TBULP.TilA 
Gradivnts arc quantitative diffcronc0s which can be dotocted in a 
variety of "~aays but perhaps the most significant difference is a 
diffcronc 0 in tho rate at nhich the ston forms a hydrnnth at diffcr-
e.nt levels of the st em . (Ac~ompanio d v:it'1 this is a difference in size 
e f the r egenerating hydrnnth . ) We ~ill cttonpt to measure the time 
from cutting of tho st.em to the fully formed hydrcJnth . Sinco th<.; 
first hydranths dcvol ep 8.t r,bout 24 honrs and others contj.nuc to 
develop at later times it is best to stc.rt the; experiment as early jn 
tho morning ns possible so as to hnv c nll the next day in v1hich to 
make observntions . 'Iho stems should be examined every 2 hrs . after tho 
first hydranths have formed for further r ec:cn<.; r r. tion . 'l'he cri t ori on 
o.sod for a fully formed hydranth is tho presence of tho t ontaclco and 
especially a sharp constriction at t he base of the hydranth while 
still within tl•e pcrisarc . Simply 1 oolr at the ends of the sterns under 
low power and record tho time ~han this censtriction is first noted . 
Sole;ct stems about 25 rnm ol' the stem ns before . 'i'hcn cut tbe rom··,lndc r 
into pieces about §G 5 to 6 mm . long discarding which is ltft . As 
t~cse pieces are cut place them in scpo.rrtL dishes labelled distul, 
middle and proximrrl thirds . CoYt ti uc 2 hr . obscrvr1tionc- as lontr ~ t> 
possible o.nd take n finr l observation tLc second doy at 4f3 hours ond 
a vera go the t imt;s, by totr.J.l in[:.. tn c tot" 1 numbe;r of hours for cnch 
lot and dividing by the tott .. l numt:,cr of stems . If some stc,ms havo 
not yet r egenerated inclurlc th_so in thl- total numb0r as they would 
regenerate if given time . 
4. -
Chi..d - 1941 Patte rns an d rot' cn:s o f )(;; lop cnt 
Pagts 313 , 333, 345, 359, 372 , 378 , 41 3 
Also Cb£~.ptcr 8 v:hich is th c orctico.l . 
3arth - 1940 
PL1:rs iol . 
Rose und h os e 
Biol e gical • v i o\'IS 15. !.lso Biol Eull . 74; 
Zoel 11; Biol . 0ull . 78 
1941 rhysiol. zool . 14 
'/ o • r o - 1 9 3 9 B i o 1 • Bu 11 • 7 6 
!.:il l e r - 1937 Biol • .dul l. 73 
1o og and Spiogclmnn 1942 . l roc . Soc. ::..x1- . uiol. "cd . 49 
1oog - 1941 Biol. Bul l. 81 
Goldin - 1942 Biol . Eul l. 82 
Zvlilling - 1939 Bio1 . Bull . 7 4 
:::.:BRYOLOO Y OF THZ COZL3 IT ER:.TZS 
l. L IFZ CYCLZ 
C elentert:tes-1 
The "typict1.1 11 hydrozo·'n exhib1t 0 · n ·•lt rn<. ti n 
of gene r rt ions - the sucession.of t1.o morph lo lc~'lly different 
t ~ult types, each chcrecterized by 1 different m~nner of r6product-
ion. These gene r btions are-
1. The HYDROID G~.BR.",TIOr. The hydroi is u u"lly 
attached ?nd coloniP.l. 'I he ind iv idu<'l members of th 
colony !!BY be <' like, or the colony mPy exhi")it poly-
morphism. In the l<~tter cese some, the TROPHOZO IDS, 
are speciolized for securinr, food;others, the G~NO­
ZOOIDS, are speciclozed for reprodu tion. In some 
forms ~d it ion<'l ty e s e f soec ietlized individuals o r. 
;..szxu~·.L reproduction by budding, forms the medusu. In 
general u colony proAuces either male or femtle medusaG. 
2. The :.:3DUSA GENER:,TION. The medusa ori in<: tos as tl bud 
from the hydro id colony. This ind iv idu<-11 le<wes the 
colony and carries on P. free living existance. Gon<,:.ds 
develop en the r<.ld ial conC:lls or on the sides of the 
m<.lnubrium. Sperm or eg s are produced in these go n <rls, 
reproduction is therefore e f tho s:z..xu;.L typG. ThEJ 
gP-metos arG lib(r'Bted <md fortilizPtion occurs. The 
fertilized Ggg develops into a PL:..NUL.'> lPrvti. The 
plcmula attaches e,nd dcvGloos t~ hydr nnth. Ll~tcr<~ l 
budding occurs and in this rnenn<")r a colony is formed , 
~n altcrnution of gc:n0rutions, ~ ith nell dcvclo ad 
nydro id c,nd mcduse gcnE:rut1ons occurs only 1n the CL .'SJ HYDROZo !· .. 
In the CL;.Ss SCYPHOZo:. the mcd usa gc ncr(; tion is . ell dcv c: loped but. 
the: ):lydroid gencr<,t1on is reduced or w<!nting. The modusa gcncr<·t~on 
is entirely CJ.bscnt in the CL.ISS :.Nl'HOZO.'~. Zvcn within the Cl<·S 
HydrozoD the re is considcra.blc vHriotion in r c l vt iv c dcvc·lopmi· n:. 
of the two goncr<~tions . 
GEI'BRAL REFSRENCZS. 
Fraser, C.H. 1944. Hydroids of thr :,tlLintic Co vst ,f North 
:illlrr icc:. Univ . 4 f Toronto ru>rcs s. T f!J(ononry , .. nd 
distribution. 
Hymon, L. H. 1941. The: invc rtcbrctrs: Protozo<~. through 
Ctcnophorci·. ~.1 cGr BV7 -H ill. Good gc:nr:ral discussion 
of the coclcnt r r ~ tcs . 
Mc:~ycr, · .. C. 1910. Modus ec of the; werld. Cnrnc:g io Ins t. of 
'!1<~S hington. Publicvtion 109. T<.txonomy <:nd d1strib-· 
ut ion. 
DZSCRIPT IONS OF LOClJ.. FORMS. 
Hargitt, C. '.V. 1914 The mr.dusC:Ic of the 'Jiloodc Holr r eg ion. 
Bull. U.S. Burca.u of Fisher i es. 24: 21-79. 
Eurgi , C . . /. 1914. Th\. _·,nthozoP ot he tlo d lol-
rcgion. Bull . 5 r c< of ?Lh-rics . 32: 225-2 54 . 
... 'uttins, c.c. 1901. Tht hydr ids f ;- · '"' Hole r~..-ion. 
Bull . 0 .S. ~ish . Coo~. for 189~: 325-386. 
Sumner, ?.B., R.C. 
b iol g iN' l 
vicinity. 
Section 3, 
OSb rn and L.J. Col 1913 . 
survey f th,' ·;;< •t c r"' of ·'ioods Hole l·nd 
B ll. · Burc c:u f Fish. 31:. p··rt 2 , 
ptgrs 559- 578 covr rs t~ c ~ 1 · tL r ~t.s . 
DZVELOP~3tn: OF TH:Z HYDROZO:"-! 3GG ;liTH SP:ZCI '.L FBnlD C::!: TO 
P:: .. ~ ;Jn _·. TI~'..R:LL· . • 
Fcmulc mcdus<:.r <Ire produce on the hydr~•nths. 
1mmcdi<.~ tcly above the bBsvl 1horl of trnt<~clcs. ::<l. m;~ dusuc <r~: 
produced in<~ sim il c-r position, but on diffcr.· nt colonies. 
The mcdusc c of P nn< ·riE Pre: libe r<·trd from the 
pa r ent colonies in th0 c<.rly hours of tha : v ~ ning. They cont b in 
m<~ turc gHrn c. tc s Dt this t imc. _·.ccorci in . to Bcclc\'; ith mnt~r< tion 
occurs be tYJo r n 4 e:-nd 6 ... ·,. n~ . in the morning pre coding th•: shedd inc 
of the; cgcs . In som·· cc.scs the mc:dus<Jc rcmnin <.• tt<•ched <nd libc r< l t,; 
thc;ir g<.•mc;tc;s from th r pvrcnt colohy. 
Fertil iz<"tion oc curs in the occ< n. Clc<·v< -gt· is 
very irrq~ul<! r- both in loc<· tion of the clc<!V~f gc pl~ · nt s bnd in 
tempo. Nu clc <.lr division, Y.' ith dr:lryc:d cytool<:smic division, i s 
fll'cquv.nt. Irrrgulvr c le:cw<-gc is frequent in the hydrozot;n 13G · 
:, typicBl blcstulD riith ccntr<ll blrstococl, io 
form ed 1n some hydrozo<..' n eggs . Th is c<.iVit y subs :qw .ntly becomes 
f illc-d '.-;it h c r: lls ·:; h ich orig inetc by prDl ife r< tion or irn n i rr< t ion 
from the c r lls of th~ bl<stulu TI~ ll. In some sp. cic s thiS 
prolifcr<~ tion <- ppc;rs to occur Hll <:round the bl<~ stococl ;.hilc in 
othe rs <m immigr<, tion occurs from the rnd ':ihich ·.-~ill be posterior 
in th r l a rv<: < ~ nd the distal end of the hydroid . The s olid rmbryo 
formr:d by the se- movr:m cnts is known 1.: s '-' morulr. Thc: outer ccl1s c."~f' 
the moru l n Yi ill form the; e ctod er m und trr in tK r cells the endod e r m. 
Thl: morult: c longl~tco , br.com("G ciliL'tcd , und iu th,.J 
kno·.in <.·S 1 .~ pl v nul<~ . The pl<. .. nul<; 1s blunt <·t on,J e nd. This end ia 
for'.1<~rd ri hc n the <.·nimnl mov ' s throue;h the ·1;1.~ tc:r :' nd 1t is the pole 
of <~tttJ chmcnt. The pl<:nul<,. ca rries on u. free livine; exist<:nce for 
a, vari8ble pertod of time t:i.nd then becomes C?t.t<l ched vt the blunt 
unterior end. :. centr <• l cbv ity <;ppe<~rs in the endoderm . Tl1iS viiL!. 
become the gustro-v8scul~r cav ity of the ~dult. The body is no~ 
composed of t•.:o lc..;yers - <m outer ectoderm <~nd c. n inner endoderm. The 
free end differentiBtes into th t; first hydranth. Other hydrunths 
<lppear by l oter<:·l bu<ld ing. 
TUBUL.1RLi. 
In this genuo thero 1'1 c.• further dogener1~ tion of tll 
medusa (in compa rison ··: ith PGnnPtiu) . The medusv never b o comGs fro 
but romai ns Bttachod to the hydr<;nth from v1hich it vrosu 11s u bud. 
~gg production, fcrtilizHtion, <~nd (;H rly dGVl~ loom(;nt takr; pl<~co 
n 1thin the modified mc;dus tt . 
ThG -gg do·elops in the c -:vit bct11c -:n the modifi 1d 
manub rium cmd he in .c r surf ·: c.: f th -:- bell. :.s is freque nt in he: 
coclc ntcrbtcs the eggs g ro~ by e n ~lfing n-i~h orin c 'lls . Th( 
f lly formed gg is concave on the surf <~c~ c-dj~:tc? n to hr 
manubrium a nd convex en the surfc.co rd j P.ccn t the "11 . Th\; P 1 · r 
b dies crisu ncar the middle of the convex surf < e · . T, 1 '"i ill b~. 
the b<S~l cn:i •f the future: hydroid col ny . J clear division i 
mo r e r iloid than cell division in the o1•rly t <gos . ·, bl<~stocool 
m<-'y or may not b e formi:d. If it docs form multi l <~r ud "ing fills 
thG c t'v ity . The: e ctoderm diffcrcntie.·tcs first Pnd l<tcr ., c ·vity 
forms in the endoderm. Tt1o s ts f t cnt c•cl cs <p car : c. circle of 
long oboral t c nt'-' clcG at tho:; mc.rg in of the diSc- h<~pcd embryo; < rrl 
four srnull oral tcnt<·clcs on th· conv ex side. The l<..rv<· iS no\. 
kno'.in &s on .:::ctinul c.:. It l ceves the t' rcnt medusa Pnd ottuchcs . 
Th t: tcnt&clc;s oi th · first hydr<mth h<~v · tlrc<'dy formed .. : tolon 
grows out from thr- b ·s<: l end c.1nd nc r; 1ndividu <> l s gri r by budd in . 
GONarHYRZ~. 
In this genus the ncdus~c ~rc tlso dcgcnc r Fto . 
They nrc produced by cc rt<: in modified individuc.ls knor,n c.·s g nrnri 
The mcdus t:;. buds form on the st< lk of the bl<·stoutyl\-: . Within c· ch 
mcdusr. 2-3 eggs c.:rc formed. The fully formed m ·dusrc h r:vc tiny 
tentacles end tr~c rs of r1~iBl c~ n < ls. They pusk out from th 
gonethc:cn but rem<~ in t•ttc.:chr;d to the' too of thr blPStostyll-. Tb : 
emb ryos d c; v clop to the pl<.' nul<~ st<~g e w lth i n the mcdus r. The 1:rc 
libc:rC: t odas pl c.>nula<: c;nd 1Stcr a short frc:c living cxiGtl•ncc r:tt<;ch 
by th e: blunt e nd. The smt.ll end dcvc:lopG the first hydr<.nth. 
C ;~1P "1NUL ."1R I: . . 
Th0 d e ge ne r a tion of the medusa i G cLrr1cd onr Gtrgc 
furthe r in this ge nus. ··11 th<:t r r mu ins of th ( mcdw:w is < ~ sm<lJ 
bud on the bl <:'. stostylc . :!:ach of thr.s c produces < n egg nhich rcm<:·inG 
·,·i ith1n the gonothe c<~ until thc: pl t~ nul< st<::gc . 
The dcgc ncrPtion of thr: mcdunv s t <:.g G t ou ches it ::~ 
comple tion in some fresh He:tcr forms such c.:s Hyd r<· . 
2xpc r imr nt<n :2t~:BRYOLOGY. 
The egg of the co Gl cnt r. r <· t r , cs pc ci<·lly of the 
hydrozo<:<, i s thought to b e of the c:xtr::mr rc;gul (~ tiv c type . Zoj t~ 
i sol<· ted th r. bl<:. s tom er e s of Clyt i < .. bnd found-
1/2 blas tom r. r c s form ed pl<.mul<.,c and hydro id s 
1/4 bl Gstomr: r cs form ed plc.mulcc <md hydroids 
1/8 blestom r rcs form r: d pl<::nul<~c only ( M,?sa got hydroids ) 
l/16 blus tome r c s form ed plu.nulv c . 
In Liriopc c. 32 c c: ll cd bl<. stul<: i s fo rme s ri hich consists of < s ingl r 
1 1:ycr of cells. ;~t nc xt d iv i s ion 32 innc r c r lls <' rc formc:d. 
1/2 blc.'stomcmc r cs produce endoderm <'t 4 cl N ·v tgcs the n l<.• t c r 
form rncdus c.~c . 1/4 bl<·s tomc rc s foro t vio l<·y\ r c:d emb r yo . 1/8 
bl r stomr. ros t~m do not form c: otodc rm end r ndodcrm- m• rely <J ophc r c . 
In :,mph1sb c: t1 c the un utt < chcd pole of the rgrr, is 
ycllovr. :~t this pol r th.: mEtur<:tion divisions oc ur, lst. cl c<~ v1 · g 
b egins, ge.strulc:tion b,: g ins~ it 1£; the pos t \· rior pol e of th ( pl<. nul < 
E•nd the orr l pol e: of the hya.roid. Frrgrn c ntG o f t h r egg- do,,; n to 
1/10 the norm<l s iz e form pl<:nul <:- C-. but l l :;rys · . ith the origint.l 
pol vrity. 
.t... cor;prehensive survey of devel menta pro sse i n the tuni-
cates would incl de examination not only of eggs a d enbryos 
but also of metamorphosing larvae, various ty es of • getntive 
reproduction, and regenerating forms . Be ause t ese dev ~ l 111antP~ 
forms must be constantly related to th struct1re of larva and 
adults, it is necessary that the student should first of all re-
view in a standard text the anatomtt and life history of the 
co~on types of t~nicates. 
A. S n· r LS ASC IDIJ.J S 
DEVELOh ENT TO '.i' T TADP OlE S'lJ. _ . 
Styeln (Old name Cynthia) 
Conklin's classic description of t h e developr.ent of Styela e f!S 
(1905) provides the morphological baclq:; round for t 11e xperiments 
(Conklin 1 31, 'I'ung 1 34, Dalcq 1 38 , Rose '39, Tung , 1\u~1'ung 1 41 , 
etc.) which are actively shap in eur conception of the or ~ani­
zation of Tunicate eggs and emtryos . Conklin's fi gures should 
certainly be referred to during the following studies. 
a . Me thods 
Though trul hermaphroditic, btyela is ordinar i l self-st 0ril o 
like several ot 1er .hscidians (Morgan 138). It sheds t he eggs 
and sperm bebveen 4 and 7 p. m., and fertilization takes place 
when ri pe gamete s from two differ ent individuals got mixed. 1·'·, 
is easy to have eggs shed and f e rtilized on normal schedule in 
the laboratory, but t his entails t he disadvantage of havinr to 
study the migrations of yellow pigment within them b y artifi ci£!.1 
light. 
The classic method of obtetining eggs and embryos from Styola ho.s 
been to mince the gonads from R number of lar r~; i ndividuals 
together in a dish of s ea water . ::Jh is l il~erat c.s all stares in 
the maturation of eggs and sperm, and usually c f ew e , g s wiJ J lJe 
fertilized, whatever the time of day or nicb t, on• will couJmcmco 
normal d evel opment. 
s. ~.i . Rose 1 39 has developed a method of controlling the no.tur, J. 
spawning in the laboratory by illumination, and this ~s t ho boot 
way to get fertilization for experimental mat e rial. It we rks 
well except .f er a f ew weeks in mid-sur.1mor, whon the animals arr; 
spent . By such control, tho s rune bo. tch of animDl s can b e lnducod 
to shed a number of timos on succcssivt; days. ,'I'hoy arc kept 
darkened until e ll-v on or twclvo h ours boforo f e rtiliz a tion is 
des ired, and then an r rt ific io.l dai;" i s s tartod by turn in[" on en 
e lectric li g.h.t . A 40 wa tt bulb 18 1 from tho animals is suffic:i unt 
Eggs and sperm ar c dischar;;ed in clouds Ht tho des ired tlmo . 
b. Ma t1::.1r~2n_f_~_rt_~l :i.:_~_o? ~_gg_ . 
~ketch the mature unfertilized O[!g , which should show the followin 
(1) Chorion, o tough mumb ra.no vli t h pe rhaps n few follicle c c;lls 
adhe ring to its outer surfnc0 . 
2-
T nicc.tes 
( 2) Smc.ll s p .or icc 1 inner _ oll ic lc c lls ( 11 11. r" c cells") · .c tween 
chorion f'-nd ec;g itself . Th(.;y contr. in ycllo '' 8 rl1n 1 s. 
(3) Pcripl:crc.l layer of cg.c , r. cld~r l ny'"' r ont:-.ini . I:1 mt~... . 
yello '' <..· ranul ~ s. 
(4) c~ntrc.l pc.rt, consisting of grc.y yolk 1~~ lets. 
(5) Gorminc.l vesicle, a vcr large ccc(.;ntricnlly plc. d mn~ • 
.::. :r· ~s~rortili.z.C:.t_ion~~a:rrn.nJl~mcnt~_ 
~1is is c. difficult procc~s to observe c.s m st cg~s hn.v too 
littlc. piCJ:' cnt, o.nd f ew c.rc fertile in 11 min ~d 11 cultures. It is 
be tter to omit this section end to concentrate on clcav~gc n.nd 
gcstrulc.tion . Then, if time is r.vc..ilnblo c.nd if tho .... g"' h s 
yor.r contc.in sufficient pigment, these r ccrrr.ngc. ent mc.y be 
studied . 
Ccrofully wr.tch for the r cr.rro.ng~cnt of o g subst~nc0s st.rting 
vithin 2-8 minutes o.ftcr fortilizr.tion. The clc~r ycllow i h 
periphcrr.l mo.ttcr strcc.ms to the lower pol(.; over tht.. yo1lc, 
follov1cd by th0 clccr protoplcsn frorr. tbo rnimcl pol • If th '"' 
y el lov1 pigment is not v. !:i 1blc, try c. more brir-htly , olor~... d or-• , 
Us0 only dnylight for illuminc.tion, r..nd hrvc th dio.phr~· r,m on ·o.o 
microscope wide epen. Roll t~c er rround by mo in ~ tho cov~r 
slip with o. noodle. 1he yellow inner follicl t C(lls may nlso ~ 
migrating; wntch the egg cortex. 
Tho gro.y yolk ris cs to occupy th t: u per polo, c.ll e xcopt th~J 
space thc.t surrounds the maturation-"'pindle compl , x. Soon the 
y cl l'lw subs to. nee c. ccumuln t es on on u pnrt of tl. e lOv'J e r hem !'l pl · Vt'< , 
Ylhore it assumes c rescentic form . lmmediC>.tcJ.y rbovc tho broco 
part of the: ye llow crt;sccnt is a l nyor form '- d by tho clur.r 
cytoplo..sm . 
The most rcmc,rkablo ch['.ro.ctcrist ic of Styolo. is t'Lnt now rt t' ·-
conclusion of these movements tho posttion of t,c future lcrvn -~'1 
visibly marked on tl'c surfac e of thu uncl oC'.ved <..Jgr, . Th e bror,d• 'l 
po.rt of the yellow eros cent ia r t t1-- c. pos tcrior polo, r.nd the 
borns wrc.p nround t1c' ri cht 1:.md l eft sides. 'l'he cnimc.l ol e , 
wh e re the gcrmincl vesicl e l~y , becm·c s the vontrnl-rn~ crior side 
of tho lc.rva . Tho vegetr.l pole, v1hcre t l c spermc>.tozoon ent e r, 0 , 
is th9 futuro dorsr.l sioc. To o.void confusion, fix firmly in 
your mind the r ol n tionship l)e tv1cen ogg ori e nte.. tion nnd dofini t i vo 
axis of ombryo . 
D. Cleavo.gc 
The follmvinc; approximf,t c time sch(; dulc for the embryolo r.ry of 
Styol o. is from the g r ea t menogr~ph of Conklin ( 1 05c.) . Follo" ·:11..; 
events of cl eo..vc.gc in o.s much dcto.il c,s possibl ~,., . Obs erve 
gc.s trulc.tion c:nd vwtch tho trdpole ta1ro shc.pc . Sk c. tch ,_ succ <. ~.si on 
of cleo.v8gG stc.gc.:s , showing 'Jilr.terr.l symnetry r.nd locc.tton of 
yellow cr esc ent mntorir.l . Skc tc .. r.t l er.s t 2 sto.ges of cr.strulC'-
tion r:nd 2 of longctinr; pro-to.d pol rc . (Tris schedule, ic for 
norm8 lly shed eggs . I f eggs r.ro obtr.in '-d from "minced 11 cul tu rr o , 
cleo.vn3e is dolPyed, the opgc ~~~~ront ly maturinr ct va ricbl o 
intervals aft er strikinG tho s 0~ w~t~r .) 
F'irst cle<".vr.gc to 2 c ells rcft cr 48 minutLs 
2nd " 4 II " 30 II 
3rd ii " 8 ,) " 30 " 
4th " 
11 16 d 2. " 
5th j, 
,, 32 , j 20 
6th II II 64. " tt 20 !l 
( 1) 
7th clecvcge to 112 
8th II If 218 
3-
cclls 
II 
1ftcr 20 11in lt. ·s 
it 2 II 
o , 2 r10rc ho 1'8 • To neu r~l ·J.Lte st~ 
~ul ly ~armed ted olo 12 .. 0' rs r.ft .r fc ~ti io1 
First cl~...-~.vr.cc. hquc-1, s0 ['.rnti .. "l[~ tl11... t'.·o hornr 
yello '! crcs cent fro.-· co c 1 other, l i {C\' is b s c t i~1 
clccr protoplr..sM c.ntcrior to tho :r llow. 
f the 
th<.. 
(2) Second clcr.vr~g . !~er.rl..r ~.q rl, \"<'rtic~.l "t ri•:'Lt" glc<> 
to the first. rrhe two paste rior c~,.,lls ( 03 on the lt,;ft ~.nd 
B3 on t~c. right) ccntr.in little yol): e-nd prr-.cti ~Jly ~.11 
the yell o ~J eros ccn t G 1:-s tr.ncc. Tho two r.nt~rior C(' ls 
(A3 on the l eft r.nd A.3 on t c rirht) cont~·. in n cl ... yolk, 
end prCtcticr.J l·· none of the yollov· erose 'nt subst[lncc. 
The clcLr protoplrsn ~ocs CQUr. ly to the for c . ls. 
(3) Third cl.:.::r.. .:--::;c . Borizontrl, t .1c u Pl' r qu~.dr~ . nt (c..:-11'"' r'l, 
o.4, b4 f'.nd b4) some'nh .. t smr..J.lvr th~n the 10\''<..r qur.rdrrnt 
cells A4' .n4'; B4 nnd B 4 . ) 'l'hv yvll \1 Cr(;S \.-1"'1 t "Ubco t".n l 
is Qlmost entir ely confined to tiw two posterior dors~l 
cells (B4, B4) . 
(4) Fourth clervc..;o . The pl~ 'C S of clcr ·c.fi,C v~ry in diffcr on t 
quc.rdr r.nts , but the now cells do not ovcrl p tho s ::.fi tt '"' l 
pl ene of the embryo . Two of thu c.ntcro-dor"'~l ccJ.ln r.nd 
tv1o of th e postoro - vcntrr.l c0lls o.f t e 16-ccll em ryo :•r 
crovJdcd r.vir.·r from this s2.rittr.l plC'1C , but 11 other c el l" 
touch it . 1:l"'e dorsr.l r.nd vcntrr.l 1 omi t.p'herc"' at this st['g 
c.ro mirror imrr cs . The ~rollm1 pic;mc >1 t l ies 1n four posterior 
cel ls (B5 . 1, B5 . 2,_j?.5 1 1 o.nd P5 ._~h 
(5) Fifth clec.vago . Glc~v~gc in th ; dorarl (vo~otQl) hemisphere 
precedes thrct in the v cn 'cr~.l (c.nimnl) :-t vmi"'phcro, rnd 
cler:vr.ge in the r ntcrior part of c~ch hcmtsphcrc prccc.d<"' 
that in the posterior pr.rt . 
At the 32 cell stnc;o , tr o yGllon sub" t.".nco is c. l mo'"'t en tire l y 
confined to six dorsa - posterior c.:.ll3, three on each side of th 
midline (B6 . 2 , B6 . 3, B 6 . 4 , _B6.2 , £.6~ B~-·-~J .. 'l'hey give rif.c 
to rr .. es ode rm r.nd mos en chyme . 
Six yolk - filled c el ls at tho vcg o t~l (definitive dorso-postvrior) 
p ole ~ntcrior to t~e yellow mcsodor~ ctJls, will givo riso to 
endoderm: AG. l , A6 . 3 , B6 . 1 , A6 .)_ 1 .hE . 3, B6 . 1 . 
Four cells c..t the r.ntc;rior bord c: r of the cmbr;;·o just below the 
equctor (.A5 . 2, P.6 . 4 , AC-) . 2 o..nd A6 . 4) , end t 'JO just rb ovo tho 
cqur..tor ( r.: . 5 ~-.nd r.6 .5}·-:rir·· giv-e-rise to tho not o~ord rnd 
neurr.l plr,to . ---
All the r es t of the c ells o.ro octodC;rmd . . 
(6) L[1ter clcnvrge , grst rul r.tion, ~curuJ[ : ion. '}'he f,"'Gtrulr. 
passes throue:;h disf'-shc.p ~. d , sc.uccr-shCl)Ud r.nd cu - :1 rpcd nt2.c<.-s , 
sto.rting ~ith the 7th cleavage . ~ it ftnrlly becomes cg~ ­
chc.ped , t ho gr.strulc 1 s blo.stoporv, founc; r.t t) 'c ~m£11 hind end, 
4 - T'J.nicat~s 
becomes 1 -shaped ' the stem of the 'I bordered by t... rello v 
meso6erm - mesenchym cells. 
~he ce l ls ov e rhanging the crossbar of tho T -sha~o blastopor~ 
constitQte its dorsal lip. They overgrov it f nr1ly enb'lfin 
the yello cells which from then o_-:: are only seen diml ~ throu~rh 
the translucent ectoderm . 
'i.:atch the gradua l elongation of the em ryo and the app o.ran of 
definite tadpole form . 
:olg~l~ 
The eggs of 1iolgula follow almost e:xe.ctly the pattern of dcv 1 p -
ment seen in Stye la, but the ~: do not hav e pigment. 1'h an imals 
are s e lf-ferti l e . A few fertilized e~gs ~ay be obtaine d at any 
time by mincing the gonads in sea ·:·ator, although spawning 
normally occurs at daybreak . 
Rose's method for controlling the spa 1nin g of · Ol[ula or Ci na is 
to put a few individuals in a lar>e dish of sea wat e r and stor ~ 
them in the dark, for instance, i~ a des k drav1er, until OGgs o.r 
needed . Molgul r\ wiJ.l shed 15 minutes or so after b cin brought out 
into the light , Ciono. immed ia tcly . 
Start some ,·:olgula eggs devel oping , and follow tho cloava , o throur 1 
gastrulation . If developing eggs are isolat ed in a watch g l osr, 
tadpol e s and youn g stage s of metamorphosis aro obtainable . 
Tadpoles arc fully fonned in 8 hrs . (Conklin 1 05a) (Grave 1 2) . 
B . __Q_OLO~__J}-.J;: ASq_I.P._~~J~S -· 
1 . ALAROUCIUY: TADIOLLS, i E'£/>J:O-rHROSIS, ...:,}·lCfi.HDIAL BUDDT:-G . 
(Usually not available un t il July) 
a) Methods . 
Tholarvao of Styela and r;:olgula arc so small that study of thei r 
internal organiz a tion is difficult . 'l'he compound ascidian 
Amarouciu10 is vi vipo.rous, and the lar ge tadpol e s arc oas ily ob -
tained from parent colonies , p rovid e d the motericl is rip e and 
has boon quite r e c en t ly collect e d . 
Squeeze a mGss of fresh Amarouciurn ov er o. littJ e s oo. wo.ter i n c. 
fin gerbowl . r!J:any highly colored fragments of the n.dul t individuals 
will bC; e jected , togc thE>r with eggs D.nd embryos . !.• ill tho bov11l 
with water and pour off the coars e r particles whirl i n g n car t ho 
top. Tadpoles and eggs in a ll s t ages of d e v elepr.,ont may present l y 
be f ound at tho bottom . 
Tndpoles usua J l y l eave the parent cololly s h ortly ft or sunrise . 
'I:1cse undamaged and fully dovulopcd indivi u c.l s c an be col ectcd 
in thc lc.boratory , and they nrc tho bes t mo. t or i o. l for t ho stud] of 
swimming and of mo tc.morph os is . rlhey co~lc.ct nt the top of tbo 
wat er on th o s ide of the tank nGar cst the li O"h t . 
If colonies arc k ept in 8. shrouded nquar 1um thL- shcddin13 ern be 
pos tponcd until n mor e s cas ono.blc hour . S·,·c. rms of ne t i v o t dpolo s 
usually appear \'Ji thin fift een -,. inu t cs of b r j nr·inc:r, ripe co] onies 
eut of th€- dark . About a third of th e t adpoles •1111 cwcrgu wj U in 
h alf an hour , if iJ.lumino. t ed first o t nj_n c o'clock in tho morn 1'f ; 
5 - Tunicf'.tcs 
if thEi colony is kept dark until mid - c.f-'-crnoon ubo t throe quarters 
of them will cme rg Ei ~ int •. in h a lf ~n hour , ·onrly c,ll of th 'm will 
connnencc metamorphosis 'Ji thin un hour of being shed . 
b . Early s tali£E_ 
From the debris in the fingerbowl, s e l ect ends ·.tch n s rios of 
embryos and developing larvae that shovJ the grndunl cmcrgen e of 
tadpol c form. None of tho so squeezed tadpol\~s will swir.t immed a tcly 
and f 0v1 vlill attach normr.lly or dcvc.l ep further . TvJit hin mo a -
ments like thos e of vertebrate ombrvos or fetuses \':ill be otscrv d . 
v 
c. Tc.dpol c ~1:;_T.uctur c r.n_9_ b<_?ha v.1:o~ 
v·Jn tch Amr.roueium tadpoles sv1armin~ in a dish of sen vn+-cr . \',hat 
a r c tho r cactio ::J.s to light end e ravity? How do thc:r prop< 1 th· 1 -
s clvcs? Plac e n few tadpoles on n slide in ~ sma 1 drop of 
·water r.nd study their c. natomy under tho microsctopo . (Grnvc 1 20 , 2l . ) 
Not all structure s can be s een i n ~ living tadpole . otc the 
thick test with centainc d t es t-cells, the adhesive po.pillc.c , th0 
n tri2l siphon ( neo.r the t n il) , the orel siphon (usur.lly l arger) ; 
end the s onsory ves icl e bob'J ocn tbc tv· .. o siphons . The- mall pirnncnt 
cup with l ens is c. li ght-pe rceivin g orgo.n, end thor is o. small 
round blnck stntic ergc.n o.lso within the son"'ory vesicle . In th 
t a il, note tho mus cle c ells end notocor~ . 
Some Amo.roucium tc.dpolcs hnvc been fix ed in Louins and stained 
with borax ca r mine . In one of th ese sta i ned tadpol es , idcntLfy 
tho phnrynx v1i th its viscero.l c l oft s , tho ccntro.l yolk mo.s s, th o 
intestines o. nd tho ne rvous system (Gra v e '~). Skctch c. t ndpol c , 
showing structura l dcte ils s een . 
d. Mc ta~~~osis .. 
Isol a te a doz en or so tRdpolcs i n scpo.ro.tc drops of wutc r in wctch 
gl G.sscs , for study of behavior during metnmorphosis . ·,hem in tho 
cours e of the next hour tho tr.dpol os h c, v c fi r ml ;r at t ache d t o tho 
g l ass, add more so:::. wo.tcr to the dish . After ['.tti c11.me nt (nomotimcs 
b efore there is time to attach , the fl e s h of tho tail is wither ed 
and dravm in, tho t es t swe lls nn d metamorphosis hr.s sto.rtod . ·, i thin 
a coupl e of hours, mov emen ts of tho b ody mo.y be observ e d . T~c 
results of tho ext ensive o.nd r a pid intcrnc.l r e orgc.nizo.ti on thot is 
c;oing on cc.n be obs e rve d b es t t1flo dr.y s or c, ':·•cbk l ntcr . (Gr c':VO 135). 
Mo.kc s c v or nl time d ske tches of t he oxtern< 1 o.s po cts of mctc.mor p1-, os is 
o . Lnter Sto.gus 
Obs e rve o.nd s ke tch mctc.morphos cd Arnnroucium indi viduo.ls which 
hnvo boon g rovling for f our dR~rs or s o o.ftcr rt tnchmcnt. They c.ro 
f o.st cno d to vmtch g l o.sscs v1hj_cr.t ho.vc boon stored in fro.rnc s undGr 
v1ntcr. Gently f lush debris from t. om nt tho t o.p , c,nd c.void tipping 
off th c.. ir cover of .,1o.t c r . !-;.ftor mcking ~rou r r ecords you will re-
turn the specimens, still l ivinf; in the;ir 'v'iatch Gl t:'.33C3 , to tho 
fr o.mes they ccmc fron . 
Examine tho spc cimcn s unde r 1 01.'1 r)ocr firs , f or oricn to.t ion . If 
they nrc grovling upric;ht thoy r:lC",J bu f l c. tt (.nod out by gon tly 
lovJO ring n cover g l nss on them . (If g rovm on cover slip , turn 
it ov er). 
6 - T nicntt.:s 
At one end of this cnimcl ~re the ntrinl nnd ornl siphon, rt th , 
other is the post-abdomen, v•ith the htnrt ~t it ti • t".tch tho 
henrt for periodic revers 1 of beat. Identify th cpic~rdirn, ~ 
usue.lly pigmented strand of tissu~ running thruout tho po t-n \.o en 
from phc.rynx to heart region . It is th ~gent in n::>e cunl r poroduc 
tion and colony formction . 
Below the siphons is tho pharynx ;ith its three rows of numt;rou 
stigm~ta (visceral clefts). It opens into ~ short vSO hngu v:hich 
connects vlith c. round yellow stor:o. ch mo.rlced b r mus ulnr b nds. The 
intestine turns shc.rply after loo.ving tho stomach, c.nd ends nC't:.r 
tho atrial siphon . Tho endostyle is sharply indicnt don the v ~ll 
of the pharynx, delimiting tho two c.triLl pouches. 'l'hcsc pou h 
surround the pharynx nnd open to th(. exterior nt the atrin.l "ipllon. 
f . ~icnrdial Budding 
Asexu.al reproduction of ne ~ individuals m£>.y bo s<..cn in ln'..:>or tory 
cultures about 17 days after o.ttr.cbnent of the trdpolo. Or, swnrms 
of buds in all stages of growth c.nd migrr.tion con usually o found 
at the bases of tho tiniest trannparcnt fingorlikc lobes of a o.r c 
henl thy colony . Demonstro.tions vlill be mr.do of them, which should 
be sketched at low magnification . 
Asexual reproduction is accomplished by strobilization, i . c . 
s e gmentr, t ion of the post -abdomen whic:b contains the opicardinl 
strand . Tho buds consist nt first of inner vesicle (from epicardium) 
and outer covering (from parent epid<..l~is) . All internal orgnns 
of the ncv1 lndi viduals form from the cpicerdium tissue, v1hicl• was 
a pharyngeal derivativ e , io . endoderm . 
This method of asexual reproduction is distinguished from~hers 
in Tunica t es by being called Phcryngoal er Epicnrdio.l Budding . 
(Kowal evsky ' 74 , B<..rrill 1 35) . Tho opicaricl buds while diffcron -
tic.ting int o now zooids movo up c.nd tnk8 their l llo.co c.round tho 
parent . Dur ing tho strobilization of the parent ' s postabdomon, 
tho old heart is isolated ~nd dogcncro.t cs , nnd n now heart is re-
generated in the par~nt . 
2 . BOTRYLLUS : TADPOLES, i;! LTfJ,WHl:- :J OS IS, ATH I AL BUDDE~G 
Botryl l us is c.nothcr compound Ascidian, 'Nhich is found e-ncrusting 
on rocks , whc.rvos and fl oo.ts . Corte. in colonie s , brought into 
tho Laborctory, and placed in dishes of soc. water , will liber~tc 
many to.dpolo s . Tndpol cs and all stages of clco.vagc and devoloJ:.;rllont 
may c l so bo obtecined by mincing the colonies and hunting in tho 
debris . If the normn.lly shud t0dpol cs nrc plncud in littl e s cf' 
v1c.tor in a watch glr.ss, they soon c.ttrch to the dish and comr,cnco 
their rapid motc.norphosis . (Hcrmnn F . c. 1 24) . 
a) Structure of T. dpole 
Thc .. te. d·p-ofois no-f ·as -large as thr.t of Amr roue iur , but show em 
interesting nov1 fen turc . Just undE-r tho r.dhos i vc pc.pillac is r. 
ring of 8 nmpuJ.lno which nrc divc rt jculr. of the body wrll, dustin0d 
to be ports of tho r.s yet non-function~l 8nd inco~plotc circulotor; 
system (Grcvcs and \:oodbridgc 1 24) . In motcMorpr!Oois they unfold 
7- Tunica tes 
like petals of ~ flower, and s rend r •o n~ the ~ 
ing tunico.te. As the colony c-rows these incr r.s 
of tho dcvclop-
nun.b r. 
In addition, identify the stntolitn, ~densely bl~ck .up 1s~·n ed 
in the sensory vesicle by n slender stalk ~nd closely a sociLtod 
·1ith lie:ht-sensc,tive clements (Gr['.ve f'.nd Riley 1 "'5); the t\J 
sipho!1s; the phnrynx v1ith sever~l sti f'.tr.; the sto1 nch r.nd 
intestine. Sketch tho tndpolc. 
Tadpoles nttnch to n substrrto withir nn hour or ~·o after 
hatching. Tho mete-morphosis is extremely rcpid. (Grnvo 1 3r.: 
Grave and Ni chol 1 39). 'l'hcy ofto!1 nctamorphos o v:ithout ".ttrchinr; 
and stick down c. day or so lntcr. 
b. ~llo -D_gr.Old _ _E_~:r:m.. _Atrial Buddin0 
Tndpolcs have been c.llowed to c.ttch to watch glnsscs c.nd grm for 
tviO dc.ys. 'Ihey nrc now large r and clearer, nnd usurlly o ori entad 
thnt the observer looks dir octl·r d o\'m into the J.trif'l and orr_l 
s ipho:1s. The largo pharynx is in tho sho.p( of ~- trunc"'.tcd con~.. 
o.nd bec.rs up tho throe r ev1s of stigmC'.tr. (visccrrl clefts) wh ch 
lot water pnss out int• the atrlr.l cc.vities of the tvJO side~. Tho 
endos tyl e 1 ies on the under s ldl~ of t •o phnrynx c.nd thu Q pc nrs 
as a rod under the mouth . 
The stomach ordinnrily appcc.rs r.s o yel low body under t he f'.trinl 
opon int; . '111 c intestine, a fter turn in[ to one side from the 
stomach, r etu rns to empty noar t Cl ntrium . 'l'hc pulsating hLt. ·t 
nnd its v oss els nrc of pc.rticulr,r interest, os pee irJ ly the flov' of 
blood nround t he mouth end phnrynx . 
A new bud ( I<'irs t t l a stoz ooid) nypcr.rs from tr. ... s oxunll y de cl O} co 
animnl (tho oozoid) as r.n cvaglr.ation o f th e f'.trium nt onc ide . 
It is furnished with 2. blood supply, r.nd p r c::;ont l y the differenti-
ating parts c2.n b o distinzuisllcd. Com~n8 frou the, o.triur1, tho 
whole bud , and all parts of the n ew individual, a r c derived from 
ectode rm. This is the Atri~l typo of buddinG• (Berrill 1 41) 
Ske tch an oozooid showing buds . 
c Week - old form . 
In thes o nnimnJ. s , gene ral G true turc c.lrcndy stud iod -:nf'.y be OC\S iJ.y 
s een unde r low power . The ph~rynx of the oozooid has developed 
4 rows of stigmntc, c.nc: the first blc.stozooid mo.y c lso ho.vc 
thr ee or four ro'N f> . Blo.s toz ooid buds of tl1 o second c.nd third order 
mo.y be present . 
Tho first blnstozooid buo in Dotr:rllus is sin r·lc, o. ll the l ntcr 
ones c. r e in s srnmo tricr.l pr.irs. '1!1~ snro orgrn structures mny 
be seen in a ll t he individuo.ls , no tv1j thstending their divorsc 
embryology , with the minor exception thc.t the oozooid docs not 
develop gonc.ds . 'l'hru r cn.rrr n·cncn t of tho ind i v1c'tur.ls, the com-
pleted colony shov!s o. common c.trinl pit in the Cl-ntor, with 
sopnrc.tc phnryne;ccl openings c.. t the periphery. 
8- Tunic['..t 
For the cn~tomy of the zooids ~nd structure f vcrio steg s 
in bud devcloprcnt co~pnre the livincr preprr~ti 1 in h ~~tch 
glnsses with fixed-stcincd -1:10untcd propcrr\ i n thct ".re> :-.vr.ilc,blu . 
These 'iH;re mr.dc from o. colony tho.t \'/Ci.S ncnrly trc. " t'.rcnt \':h n 
living . Lost colonies develop so nuch pigrr.ent i the tuni thc.t 
·1holc mounts r.rc uscles~. Erch bud consists ~t fir t of r. dis , th 
n sphere . The sphere extrudes sex calls r.t one or oth sides end 
becomes pr.rtitioncd into three veJiclcs, tho lrt r~l ones fo~.in 
o.t renl chr.mbers, tho middle one the pharynx . Lr.tcr st~gc shovl 
differcntic.tion of the rest of the organs frm1 the rhr.rynx-vc"' .• lc. 
(Bcrrill , '41). 
3 . PEROFRORA: S..!.FTAL BUDD:: G 
Peropb.orn is o. little grcon - color c.d r.scidinn, which by 1.crns of 
stolons foms loose colonies on ,·:hr.rf pilin s, etc . Pico~.,;S of 
the colony mr.y be gently stuck to wntch glrss .. n •Jit vn"cl nl., 
and stored in running s c~ YJo.tcr. After o. dr.y or so , 3tolons ·•· 1 
be s cnt out over the surf~cc of tho glc.ss ::md now blnstozooids 
will be f ormed c t intcrvr.ls . Lx['Jninc n wo.tch - glnss culture of 
Po r ophor n v1h ich has been growing for two weeks . 
Notice the brnnching pnttcrn of the stolons . 'rh tips shov 
exploro.tory t cndcmcies like smc.ll pseudopod in . 'l'he outg oing r.nd 
incoming blood strcnms in the ntolons c,rc sopnrr.tod by n moscnchy e 
septum . All stcge;s in the fornction of now individu~ls will be 
found , o.rrc.ngcd 1 iko pumpkins on n vine , v1i th th e youngcs t n o .1' • t 
the t ips • 'l·hc youngest buds consist of nn outer vc s i clo dori v-...d 
from the epiderm of tho colony c~nd c..n inner vesicle formvd by t'1c 
spl it t ing of the moscnchynr.tous stolon septum . 
OrgnnoGenesis tnkes place ir. the inner vesicle , which is d c riv oo 
from mcs cnch~'1110 (Be r ril l 1 35) . 'I'his method of budding is 
dis t inguished r.s tho septa l t ypo . 
PRO'IOCHOHDAT ..... S 
Conklin, E . G. 
1932. The cnbryo1ogy of Anphioxu • Jour. ·.: rph. 54:- -14 • 
1933. Deve1op~cnt of iso1~ted ~nd p~rtinlly scpnrr.. d 
b1nstonorGs of Anphioxus. Jour. xp. Zool . ~4:303-37S 
Dc,1cq, A. 
1935 L 1 Orgo..nizntion de 1 1 Oeuf chez los Chordo"s . l'r.ri" . 
1938 Form end Cr.usc..1ity in .. -..n r1y Dcvolopit·cnt . C""r;~ rid. c . 
Dc,wydoff, C . ~ I I 
1928 Trc.. ito d 1 E1nbryo1 ogic co .1pu.reo des Invcrtcbrcs . 1:-nr. 
Hcrdmc.n, E . C. 
1924. Botryllus. Liverpool Mo..rino biologicr.l Committe 
Memoirs, XXVI: 
Hordmr.n, U. A. 
1899 As cidiu. Liverpool 1 r. rin c b iolorico.l Co1 •n:1 ittec Mcnoirn ...:._:r: · 
Korschc1t , E . 
1936 rc r g1oichcndc 1ntwick1ungsgoschichto dcr 'I'iorc . Jcn< . • 
Sco1igcr, 0 
1893 
- 1907 Tunic~ ton . Bronn 1 s C}c· .. c:-" en u . Ordnungcn do"' Ticrro ichs, 
III: Suppl . 
\'f illey, A. 
1893 Studi os on the protochordr.to . • (.ur.rt . Jour . r~icr . Sci ., 
35: 295 - 324 
TUl ICATE EIIffiliYOLOGY 
Bcrri11, N.J. 
1929 Studi e s in Tunica to Devol oprr.0nt . 1. Gt.n cro..1 Phys i.ol ogy 
of Dcvo1o~1ent of Simple Ascidirns . lhil . Tr . Roy . Soc . 
London, 21~( B ): 37-78 
1931 11. Abbrcvio.tion of Dcv o1o}:;r ant in th~.- : olgulidr.c . 
Ibid .,_§]J ( B) :281-346 
1935 111 . Differentic.1 Hc to.rdr.tion r.nd Accolerr,tion . Ibid ., 
225 ( E ) : 255- 326 
1932 • . roso. ic l.Jcveopr·cnt of Ascidic·.n .!.'..cgn. ~ iol. Bull~3:381-3G 
Gho.bry, L 
1887 ' Cont ributi or: r.. 1 1 c,nhryoli ['"i.G normu1c ot tcrnto1ogiquo 
des P.scidics si!YJp1os . Jour . Anot . ct li1.yaiol . , 23:167~319 
2 -
Cohen, A, & Berrill, J. 
1936 The dc.velopl""cnt of isolot •d blC' tor·.cr0" of the c. cidif'.n 
egg . Jour . ·xp . Zool .~91 -ll8 
Conklin, E . G. 
1905a Orgcniz~tion and ccll -linc~sc of tb As 
hCnd . Net . Sci . (~hiladcl hia), 13 (2r.d 
id i "'1 c p:_ . J 0 U'' . 
~,.; ri s):i - 21 
1905b t..osr,ic devol pncnt in nscidic.n en;t· . Jo lr . L·p . Zool . , 
2:146 - 223 
1905c Orgen - forning substt.nc::; int e E::.,[;s of!.Scidirns . Fi 1 . 
Bull . , _§...:_205 - 230 
1911 'I'hc orgC'.n izrtion of the C[g "'.nd the dC'VClO l'Ont Of ::;in 1 ... 
glcstomercs in Phcllusir. . ~. our . Exp . Zool . , 10:39'~-107 
1931 The dcvclo~ ent of ccntrifu~0d eg~~ of As idi~ns . bi( . , 
60:1-120 . 
Kings l ey , J . S . 
1883 Sor1e points in th e: c. ... vclopnGnt of LolljUlc. . 1.:-roc • .t. osto · 
Soc. . NC'.t . Eist ., 21:441 - 84e 451 
Kowalcvsky, A. 
1871 Wei t e r e Stud ion 'i..1bcr die Lntwicklung C:o r . infr.ehcn 
J",scidinn . J..rch . l'Hkr . 1~nrt . ,_'ZJ)Ol - 138 
fl orgo.n , rr· . n. 
1 938 The genetic nnd pl1ysiolozicr l probl cr's of sclf-stcril1ty 
in Cionc . • J our . Rxp . Zool ., .78_~271 -334 
1 942 
Rose , s . 
1 939 
Cross - nnd self - fort :i.l tzc-. t ion in tho r.s c idi[tn Sty~.; ln 
Biol. Bull ., 82: 161-171 
iv1 • 
Er.1b r yon i c InC:uction in the .hscidio. • .. ·io1 . 0ul1. , 77: 
21 6 - 232 . 
de S c1 y s - Longchc.mps o t Dc.mns , D. 
1 901 Recherches sur 1 c devc 1opP.'!E:.nt post - t r!lbryonnniro ct 
1 1 Anet tomic dcfini ti vu d-2 Folsul o.nyu11oidcjs . l~rch . 
do Bio1 ., ~3:451 -469 
Tung , Ti-Chow 
1 932 Expc'r icnc os de colorrtion vi t n. 1 c sur 1 1 ocuf c'l. 1 J.scidiellc. 
c.spcrsc . Ibid ., 13:451 - 460 
1934 Reeherch0s sur 1 cs potent ir.ll t0s des bJ.r.s tonC:ros chc z 
Ascidicllr. sccbrC'. . Zyp~ri0ncc~ de transloca tion , de 
cor.1binc.ison ot c1 1 isolcnc;nt do b lrstm.1c rcs. J..rch . 
d 1 .hnc.t . nicro . , 30:381 - 410 
Tung , T - Ku, S . c,nd 1 uny , Y. 
1 941 Tho dovr.;loprl.ont of th<., ,_s cid i,.n ogg c~,ntrifugcd before 
fcrtiliz ~ tion. Biol . Bul l .,~Q:l53-1G8 
3 -
TUNic::.rE ·.sEx.·•: .. L .:.:. RG ·1 .TIO"T 
i • J . Berrill, 
1935 Studic.s in Tunicctc cv-lo ,,.Cl1t . 1·;. :~so ... ru".l 1rod1 tin . 
?hil . Tr~ns . Rony . Soc . , London, 225 (B): ~27-379 
1941r:.. The de c1op;"cnt of the bud in Eotry11 f.; . Biol . Bull., 
80:169 - 3 ... 9 
194lb S izc r.nd norphogcncs is in the. bud of E otr~ 11 "". I id., 
80:185 -193 
Brien , f . 
1927 B1c.stogencsc des Tunicicrs . ;.rch . c:c .=-io1., 3?: 1-46 
Cc.ullcry, M 
1909 Contributions '' 1 1Etudo de<: :.scidi s conpos ~~s . 
Bull . Sci . F'r . Bclg . , 27: 1-158 
KovT0.1evsky, ;.._. 
1874 Ubcr dcr Knospung d r :.sc idicn . (Didcr.·niiD1 .:.:. l.mr rouciu ·) 
.trch . Hikr . l.nn t . , 10:441 - 47 I 
Pizo::-1, A. 
1905 L ' evolution des Diplosomos (.<'..scidios conposees) . 
Lrch . de Zool . Exp . o t Gcn . , IV: 1-68 
R it tor, \V . E. 
1897 Budding in co~pound nscidians , bws cd on studios on 
Goodsirir and Porophorr . Jour • • 1orph . , 12: 149 -~ 238 
de Se1ys - sur 1 e bourgconncr.1ent dor. Fo1ystydlinos . Bull . 'ci . I'r . 
192~ Bclg . , 50~ 170- 276 
1916 -
S pck, J . 
1927 Ubc r die ·;,intcrknospenonhlicldung, Rcgcncrr.tion und 
Reduktion bci C1r.veJ line. 1epc.difor nis , und die. Bodoutunr; 
bes onderor 11 omnipotcnter 11 Zc11e1or·.ont c fur dies o 
Vergchgo Roux ' Archiv . III: 119- 172 
TUNICKTE REGEr~ERl'.TI ON , FTC . 
Bc r rill , N . J . end Cohen 1 • • 
1 936 Regonerf' t ion in C1 r:.v olinr. 1e pnr1iforr.1is . Jour . Exp . Di ol 
5; 13 :352 - 362 
Brion , F 
1 933 Regeneration thor r.c iquc che z Lrchir.scidic . Bull . Diol . 
Fr . Bclg .~l: 100- 124 
Driesch , H 
19t 2 Die RestitutionC'n dcr Clr.vc1lino. lupcelifor. iG . :.reb . 
Huxley, 
1921 
Entvlicklungsrn.cl,r·nik, 1~: 24.7-287 
J . s . 
Studj c s in ~cdiffercntirtion 11 . Dcfiffcrcntiction 
resorption in .t'crophcrf' . • r.,:ucrt . Journo.l Lic r . Sci. 
65 : 643 - 697 
enc: 
4-
1926 VI . Red cti on p~eonomena i~ C avclline lepadifo .is 
Publ . Staz . Zool. ITapoli..L.-..!_.:_1-35 
TU IC/-.'1~ T h!h LES :.YD il:_.IR 1 ;t<TA~ : ORPtC 'IS 
Grave, C. 
1920 .Amarouciu1'1 constellattm: . I . Th . actiYities an r actions 
of the tadpole larvae. Jour . Exp . Zool . , 30:~ 239 -2 ~8 
1921 11. The structure and organization of the tad ole 1 r 
Jour • . ;orph., 36 : ? 1-101 
1926 i. olgula citrina. Activities r.nd structure of th fr t "-
swimming larva. Jour • . :orph . and rhysiol. 42:453-4',1 
1935 ~etamorphosis of Ascidian Larvae. Papers fron tho 
Tortugas Laboratory, 29: 209-292 
Grave, C . and r icol1, 1- . A. 
1939 Studies of larval lif e and mctamorr'1osis in _ .. sc Ldi 
nigra a nd species of Poly8n dro carpa . Ibid 32:1 - 46 
Grave, C . and Riley~ . 
1935 Development of t_:e s ens organ s of ttc lnrva of 
Botryllus sr-hloss eri. our . Iiorph .~?_ :l8 5 -211 
Gr ave, C . and ••oodbridge B. 
1924 Botryllus schlosseri; the behavior and morphology of 
the free-swimmi ng l a rva. Jour . ;,iorph. nnd Physiol., 
39:2-7-247 
Kowalevsky , A. 
1892 Beitrage zur Bildung des ... r.n t els der Ascidicn . Mcm . 
Mast, S. 0. 
1921 
Acad. ~t. ~c tersburg ., 38: 
Reactions to 1 i gh t in th.: lo.rvo. e of the as c j_d ians 
hm[roucium constellatum £.nd Anaroucium pel licidum 
with r efe r ence to photi c oricn t c t:i.on. Journ . Exp. Zool. . 
34:149-188 
van Bened en , ~ . and Julin c. 
188 4 Re cherches sur lc devel opment post cmbryonnai r o d'uno 
Phallusi e , }hallusio. scabroides. Arch . de Biol., 
5 !611-638 
1 887 Recherches sur 1a morpno1og i c d es tun icior s (the 
development of Clcvcl1ino.). lt.:ld . 6:237 - 1'76 
r-. 
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