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BIOLOGICAL BULLETIN

! HE

noannc Biological laboratory

\\i M >DS II' 'I i , M.\

stall

1". . (.. ( 'I\KII\ 1'rincft-n University,

I \. '<; i- I. «•!•»- '/'It,- l\ t'n-r In>titm< I/ Heal Research.

T. II. \Iom..\\ ('••/nmf>t\i ( 'm'

\\ . M. WHEELER ///':•/

^ . i » W ii 1 1 MAN I'ii,- I Diversity

II. II. \\'n-.i\ i lumbia f ''.'.'':•> f-.-'/v.

I I;\\K \< . I. ii i n '/'',,- University

V< 'i i Mr X I X

l< >l I , M \>-
II \l l< ' Ni 'VI Ml:|- U I ,i,,.

PRE-_.
T«l N'cw ERA PRINTING

LA-NCASTFB "»•

CONTENTS OF VOLUME XIX.

i . JUNE,

M(i\'[<.«'\irk\ . Tii'i- II.. IK

tirnnn-: \

I h -.si K KI 'i;i k I \V ..... i ^

LEFEVRE, G IND ( ' . Mumupim; ;i

IHN. I I. >. /

I i i\i \V I'. I ' '!nt y f>h \.

•

\\ \\. I'll"
I I I \ I II. I I MM I 7 >

\I< ikun i ( ii \I-M i •- \

timi ii>:< ;• i

I 'I IK I SM \ I I. M. \l 1 \ \\lil-K. I -'7

\\ mi i i K. \\ \i \l An A '• rranl .

\N. M \\

1910.

BRADLEY, H. ( i('i

\ n HI ii •,. \i . \ «( i-i .'
BENSLEY, R. R.

-I ' '7')

H EFFNER, BARBARA A

.ijiii. .

STURTEVANT, \ II.. JK /''.•< . l >• •

llnrn,^ I!
( >u i MASS. \. I The Mo - 17

GUYER, MICHAEL F. . !• '/ M<m n>>

MIK.KI \ K. <>i: tin- !•: : ;-

i ,i: \\ i . ( \-\\ ELL, \M' < -i \-ri;. OTTO < ;

;,•///; ///. - i"

iii

i\ CON rENTS Ol \ <>!.! Ml \l\.

•• K\KI> ("l(\KI.K> R. I'lh . \tvmniftr\- ill

Regenerating Cln-ln «/ Crt i **

No. 5. O( 1MHKK. li) I (i.

I i KNKK, C. II. Experiments on Color-i ision of the IInnry-t>ee ..... Js;
TORREY, HARRY BEAL. Biological Studies on Corynmrplia. 1 1'. Hud-
ding and Fission in Ilftcroniorfthii /';V(C.v and tlicCoiit/'nl of J'olurity. J

No. <>. NMYKMHKK.
M(|NDOO. NOKMAN !•". Biology of the Shawnee ('u;> .s'/^'i/rr.s ...... .v>.> N

POWERS, J. 11.. AND MirciiKi.i.. CLATDK. .1 Ne-u Species »f j \ini-
mecinm (P. multimicronucleata) Experimentally Determined. . . . ,\

I HII. i), C. M. The Central .\em>:i* System </.v a Factor in the Regen-
eration of Polyclad Titrfie'laria .............................

( \\<\ . I.K\\IS K. '/'//<- i'orniution m (!erm J.ayer.s in Actinia l/ermn-
;'.v \'crr. . ............

Vol. XIX. June, i9io. No. i

BIOLOGICAL BULLETIN

ARK PARTICULAR CHROMOSOMES SEX
DETERMIN \\ I -

TMi >S. H. M<).\ I i.« 'Ml- KV.
Tin- pa-t decade li.i- \\itii' -\\- renewed intere-t in the

em- "i' sex determination, <lin- \cr\ largely in ilu- -tud\ <>\

h\ liridi/atioii and tin- I'mad application of tin- iv-ult-. Con-
rurrentK tin- in\ i -i i'catiou <>f tin- cerm cell- h.i- iiuTc.i-i'd in
.iinmiiil in .1 •crMiiiciric.il IMUM; nc\cr lirtV.rr h.i\-r ilic-c ci-ll-
t'iill\ i! '. ihc iliMii-cln- "I" liinln'ci^i-. .ind MUI- knoulcd'cr of

ill.- coniplcx i lirMm<>-Min.il .t.tixitic- li.i- incn-.i-cd to .111 extent
unpredicted.

In ]>.ii tii ul.tr h.i\c multiplied im 'ii>n- of llic modified

chromosomes, no\\ de-cril>ed in .niim.iU for the -pcnn
in in-eci-, .ir.nic.iil-. m\ rioj | lod.i, S.i^ii l.i .ind hinU:

and for tli. tiesis in ri liinodrrm-, tin- c.u. and ]io--ilil\ -oinc

insects /' • l;olli>\viiu ilie tenninol .en

|i\ me iii i«)o(., tlu-r ni.i\ In- collccti\rl\ n. lined .illo-oii, ••rm

nion- conxciiicnt than in> earlier oin- of heterc .. hronio-oine-. in
contrast to the unmodified chrom<i-omi-- or autosomes I'liry
havereceived ; \ariet\- of nam« >r\ . -|ic< ial. lac^in.u,

hcleroiropic, -r\ cliromo-omr-; idiochronio-omr-. microchromo-
-ome-. tli|)lo-ome-. gonochromosomes, chromatin nnclcoli, i
.\n\ 1'Mtly within a nuclcu- that -tain- like chromatin -hotild not,
ho\\c\cr. lie con-idered .m allo-oiiK- until it- chromo-omal nature
lie a-ceriaillrd.

\\ilhin the pa-t cijjit years an h\-]»othi--i- ha- ari-cii a-criliini;
-cx-drterminin;c |iropertie- to the-e allo-ome-, and m\ ol.jcct U
to tre-at thi- lupoihe-i- fir-t hi-toricalK , and st-cond crit icall\'.

THOS. II. MoMi.oMKRV, JR.

A. Tin 1 h I'M] HKSIS.

The fir-t -tatemeiit of the hypothesis is due to McClung
in [902, after in\ estimation of the unpaired accessory chronio-

me in the spcrmatogein-si- of < )rthoptera llial kind of
modified chromosome named by me in 1906 the monosome.
Mi ('lung's conception of the function exerci-cd by llie accessory
chromosi mic is "that it is the bearer of those qualities which
pertain to the male organism, primary among which i- the faculty
of producing -ex cells that have the form of spermatozoa." He
i/cd al-o ih.it there must he selective fertilization, that
to the ovum "come the two forms of spermatozoa from which
-election i- made in response to environmental necessities." At
that time nothing was known of the maternal chromosomal num-
ber, so that it was natural for McClung to reason that the mono-
some \\as .1 pairnial chromosome not represented in the female.

In the same year Sutton (1902) described for Bracliystola that
"twenty-three is the number of chromosomes in the male cells,
\\hile i\\rnty-two is the number I have found in the female cells,
and thn- \\ e -ecm to find a confirmation of McClung's suggestion
that the accessory chromosome is in some way concerned in the
determination of sex." Subsequent studies have shown that
Sutton \\as wrong in his count of the oogonial chromosomes.

Then Stevens ! 11105) found in Tcnchn'n "that in both somatic
and germ cells of the two sexes there is a difference not in the
number of chromatin elements, hut in the size of one, which is
\vr\ -mall in the male and of the same size as the other nineteen
in the female. . . . The small chromosome itself may not he a
deierminant, but the conditions in Tcuchrio indicate that sex
may in some < as( - he determined by a difference in the amount
or (|ualil\ of i he chromatin in different spermato/oa." In K)O6
she wrote: "The scheme also assumes either select i\ e fertilization,
or, what amounts to tin- same ih ing, infertility of gametic unions
where like sex chromo-ome- are present"; and in H)o<)</ : "Tin-
only other alternative in these insects seems to be that sex \^
already determined in ih. egg helon- fertili/ation, either as a
matter of dominance, or as a re-ult of maturation, and that
zation is selective . . . but any -uch general application
i- premature until adequate evidence i- at hand to prove thai

ARE PARTICULAR CHROMOSOMES SEX DETERMINANTS? 3

the sex character i- repre-ented in the chromosorn< Net in

another paper (1909 that appeared simultaneously, Su-ven-
\vri' \- to tin.- fact that the- la^in- chromosome ol the aphid-

i- ,1 heterochnuiio-oim- intimately connected with the phenome-
non "I ••ti-rminatiim. 'hr pre-ent im • ion ot the- male
licnn ( r]l~. I think, leaves no doubt."

Independently i .f Mi » vens, .UK] simultaneously, \Yil-on
found ih.it \\herc tlicn- i- Je mono-onie in the- male, it i-

repre-i nt.-d 1,\ ,1 pair in ii •!.•; .uid \\hctv a lar^e and -mall

idiochromosome in the former, tl air of 1 nes in the

lattrr. In lr d him-elf

yjiardedly a- to Mich chrom, ilril with -r\ual

|.h. -110111. n. i. In hi- third -tud\ i much fulK-r

di-cn--ii>n. .unl it i- tin- trratnu-nt nmrr than an\ other, that ha-
.iroii-cd -, iirr.il in1 in th<- su ll<- mention- as one

,dtcrn.iti\e th.it nn-n-ly ((iiant hat i\ i- dilVerencc in amount of
ill.- < liroin.itin may 1 >e the determinir. >r. lull he crilici-c-

ihi- for the reason thai in ' iiochromo-..me- are o|

eipial -i/e in hoth \\hilr .1 .id.ttioii- are

ki'o\\n I •«•! \\ern -tich .1 .otiditioii and the one \\here I!ICM- . lc-
ments are di--imilar in -i/e in tin I he maintain-

(In- alternative \ ic\\ . th.u the all - ha\e <|iialit.iti\e dif-

1,1, n. i - ih.it an \\ilh Mendcli.m doinin.i'

and with selective fertilization. ral interpretation . . .

mii-t include tin- .1 — umpiion th.u ih- two kind- ot ,

(piv-umalil\ in ,i| i]'io\im.iicl\ cijiial numl'er- thai contain re-

-|Kcii\cl\ the mali- and the female-determinant, and that the
former are fertilized only 1>\ -pern. i that lack tin- hei.

tropic chnuiio-i .111. the n: -ninant i and

. . . Su.-h a selective leiiili/.uion i- then-fore a sine '/mi unit
of Hi. .1 — umptioii thai the h> ropic chroino-onie i- a -pe. itic

determinant." In tin- ar-nment \\'il-oii makes Use ot
( tie's 1903 hypothesis that sex follows the rule of Mendelian

ilion and dominance. To thi- \ie\\ he adhen- al-o in

hi- fourth and tilth studies i- Hut in hi- la-t paju-r

loo,,, i he oppo-e- tin- Mi lidelian interpretation, U-. ause -• I- -

the fertili/ation i- impn.liaMe. He !•• - anotlu-r objection

the case of tin- bee; luie ih. 'ter two maturation di\i-ions

TH"-. II. MoNTi, oMKKY, IK.

form- a inaK- il" n»t fertili/ed, a female if fertili/ed; under the
hypothesis the female tendency .-hoiild In- derived from the
spermato/oou- -"a rednctio ad absurdnm; for tin- male i- derixed
from an unfcrtili/ed e^g which has by tin- hypothesis eliminated
the female tendency."

\.i\\ lin (ig nd Boring (19071 anal} /rd tin- phenomena of

the allosomes in Hemiptera and Colcoptera respectively, and
J..rdan (1908) in an orthopteran. all comparing chromosomal
numbers in the- female and male cells, and all inclined to regard
the allosomes .,- -ex-determinants. Morrill (1909) found that
in Prole nor and other species all the cleavage cells of one indi-
vidual have either thirteen or fourteen chromosome^, /. e., all
either a Dingle allo-ome or a pair, cmiformalile with \\'il>on's
re-ults on the germ cells.1

l'a\-ne (1909) has accepted Wilson's views of a qualitative
-ex-determination by allosomes; while von Baehr (1909), in dis-
cussing the subject at length, is inclined towards the quantitative
explanation. \\"allace (1909) concluded that in Ayleua some
rmatozoa ha\-e two allosomes, others have none, and argued
that a male would result when an ovum is fertilixed by a sperm
with the>e two elements; it should be noted, however, that the
different describers of aranead spermatogc-nesis have reached
qnile eonllii ling results.2

l!ali/er i too*)) on echinoids, drew attention to the occurrence
of allo-ome- (idiochrosomes) in the female line, though he studied
the rhro-oinoine- only in the pronuelei and cleavage cells, inn
in the -ro\\ih period of the ooc\ie-. ' We distinguish accord-
ingly two types of eggs: some with and some without an
unpaired element. The chromosome number i> in both cases
ei-liieen. Therefore, \\ e miiM conclude that the unpaireil chromo-
some in l lie egg type \\ here il is \\ anting is repre-enied by a rod-
shaped element. The sperms al\\a\s \\ith eightei-n elemeni>
are all alike. According to the discoveries on insect-, it is not
improbable that the determination of sex, which \\ould lie with
the female, is connected with this difference of the egg nuclei."

'Sonic writers ha •• '. • >• Prolenor-lype," evidently

rant that the spi-m. ia 1.1 tlii- l»im u;i- Kitlicr lully dea nln-,1 l,y im-

(1901) some years before \Vil-mi |ml.li-ln-<| liis stmli

the papers m \\ Ben .nui IU\M-II.

ARE PARTICULAR CHRO . DETERMINANT - ' ;

Boveri II')<KJ</ refers to Baltzer's work as indicative of -•
determination by partieul.if ehmmo-oir.e- the -hort lu>ok-
-haped one-'. Hut in oppo-iiion to \Yil-on'- explanation. he
d'M- IK. i lielie\e ih, 'i "in- rhron maK- and the other

a I'rmalr tendency, luit that they differ only in activity: the
larger allosome would givi « II a larger power of assimilation,

and -m h ,t \ic\\ "\\ould perhap- In- qualified a Ki-i-

for a determination." Boring i<io.|< de-

lied il:. nee in th' f'lmld of a

fifth, -in. ill chroii: nt, hut \\.i> unal'le to deride

\\ hi-tlier tin- i- "a i hroino-onu- unit in it -el I. or a I raiment ot one
•In Ion- i hroin d con-idered it niereK |»o»ilile

that it mivjit : H"\«-:i i 'inld- \}\\>

i-i.'iial -tru. -ture i" 1-e undoulitedK -e\-«letcrniinin;^. a rhio-
nio-oiiu- unit that i- in n itta< ln-d to the end of one of

th. other-. At the -ame time he report- the oeeurremv of a
iiiono-oiiie in iln- -perm. i . and ei -in hide-.

in the sen Wilson: 1 « ' nli/ation of an em !•> a -pel in with

li\e ( hloiuo-oii:( - I' de\ elopinent ot a female. li\ a -perm

with four element- to prodiieiion of a male."1

|-in.il!\. in the la-t |>apti on thi- -ul.jeit. |.d\'
de-« ill" - allo omes in the -pern. 'tain indi\ idu.d-

ot .! >.nd dm-- not lu -itate t» call them sex-

determinants.

I1-. I'KI \ i it i-\t- MI mi 1 lvr..i in sis.

In tlu- : n:< lin- \ ored to state, in all

brevity, the nature ofthe arguments ad\amed to pr..\e that

paitii-ular allo-oiue- pr.'duee ..in r the other by their

presence or al--« -m e.\\ he t her liy (jlialitatix • or quantitative dif-
fereno N"\\ \\e ma\ (on-id. iin olijection- that ha\e

rai-ed to -ueh interpretation-.
I have remained -keptieal \\ith regard to the-e lupothe-

I ha\e de-rrilied main . - in a -ucre--ion

i p. i .i> !'• ry in iO"-j that "tli' <ii the

lii -i ..... \ tc ili\ i-ic Hi : • . i

ilarly (!• figured Ijy

IIM- • .i! In-i in th<; -.11111- \

6 THOS. II. MoM'.OMKKY, JK.

<>t" papers, and ha\e expre-- cd my-elf only once on tin- matter,
in H)o6, in considering \Yil-on'-, conu-ntion which 1 iv-anled
"a very plausible conclusion, but thriv are in particular two
phenomena which must be explained before it can be accepted.
One is, how an allo-ome becomes lost in the spermatogenesis;
and the other is, how the allosomes introduced by the spermato-
zoon inio the ovum behave during the ovogenetic cycle; on both
of the-e questions we know as yet practically nothing."

Gross (1904) objected to the hypothesis of McClung, (i) that
it i- not proven that accessory chromosomes arc ab-ent in female-,
and ( 2 ) the case of the bee, where males develop from unfertili/ed
fS. He also believed that the spcrmato/oa with mono-oiiies
may be incapable of fertilization; but 1'ailrd to note that such
supposition could not be applied to -perm with idiochromo-onie-.

Foot and Strobell (1909) urged that the theory of the indi\ id-
uality of the chromosomes is not proven — today, a decided minor-
it} view. They also held that the allosomes of Eiischistits are
not chromo-onies at all, and an- variable in number, which is in
direct opposition to the discoveries of Wilson and myself. "In
the case of J-'.nscliislits we are told that the larger of the two
chromatin nucleoli of the spermatocyte is the homologiu- of the
accessor) chromosome of other forms, and if this interpretation
i- correcl \\e may expect to find a large bixaleni or two unixalent
chromatin nucleoli in the growing oocytes." Hut they find no
such bodie- in oocytes, and therefore conclude that the chroiiialin
nucleoli of the male AVC never transmitted to the egg, an- not
chromosomes at all, and hence cannot lie ^ex-determining", this
objection lo the Wilson-Stevens theory i- inadmissible

An explanation suggests itself to me \\liy allo-onies, \\hich all
lence lead-- us to belie\c mu^t be transmitted to eggs by
fertilization, acl in a dilfereni \\a\ in the oogenetic cycle. That
is, in spermaii);jeiie-i- ilie -ingle mono-ome, or the pair of un-
C(|ual idiochromosonu-^. bi-ha\e dillereiith troni the oihi'r chro-
mosomes, remaining den-e and compact in the grout h |»eriod
of the SpermatOCytCS, probablx because lhe\ are there unpaired
(monosome) or of unequal size • idiochromo-ome!. \\hili' all the
other chromosome- are paired, and the tun of each pair seemingly
alike. In the growth period of the OOC} tes, on the contrar\-. ilie

ARE PARTICULAR CHROMOSOMES SEX DETERMINANTS? J

allo-onie- of the spermatocyte- seem to be repre-eiiied by a pair
• it' element- similar in all respects to each oilier; there i- no dis-
similarity of tin- pair, hence no > on-picuou- behavior different
from thai of tin- oih.-r chn>mo-ome- aiitosonies). In other
word-, it i- the -iivjene-- of tin- element- niono-ome->, or tlu-ir
di-parity in -i/<- and a« tivity (idiochromosomes), that may In- a
reason why the allo-omr- behave so peculiarly in -permato-

esis. For the-e \ ariou- con-id, -r.it ion- tin- argument- ot Foot
and Strobell a^ain-t ilu- hypotlu-i- .in- u«[ \alid, though
ihi--c in\ v-iii^ator- .irr quitr rii;ht in -crin^ the IKTI-— it\" ot
tin- odgenesis.

i KXJ has entered other arguments against the deter-
mination of sex b\ .illo-onic-, an h\pothr-i- thai lu- \\holly
II, refers to tlu-ir limited «.i « urrrm -i-. which -how- tlui\-

!(| imi In- imi\er-.il sex-determinants. Then to the oci-ur-
reno >ry i-hromo-miH1 in ilu nesis, paralleling

lh.it in i he -perm.itoi^-iir-i- <.f (,'rv///rv, he call- particular atten-
tion, in-i-tin^ ili.it it i- n- - for the \\'il-"ii-Ste\ en- ihn.ry
ill. n sperm-Cells alone -hoiild ha\e .illo-om« "In (!ryllns
ih. -I.- can bf no talk of .1 -i-x-di-terminini; function, and thereby
naluralK al-o not in tlu- other animal- \\ilh 3Ory chn>mo-

-ollli

Guthei i ias, however, combated Hnclmer'- opinion

that the chromatic bod\ in the o«.c\t' • Iryllus i- a chioino-

-oine. and -ho\\- that it diltri- from -in h in many detail-. I l«
also In id- that "the diploid chnniio-ome ^roiip of the male num-
ber- Ji. that of tin- female 22 chromo-nm. 'The doctrine

i ;

iiiiiidt h.. niiv;lit :

" ti-'|'!ii>- i lii.'in.niti." ai. liniiilt's

ity. A- a in. if- i: "Tli lit !"• th.it in th<- in-

nu-t.il".lh arlivity tli.in tin- otlu-r i lirinn

kin, I ot mrt.it..,li-m." I •

incut "I tlii- I li.-n on p. 415, B !""-

,,,,,.,,,11, mi unit 'iiin-'l ii|) in thi-<

tini--, I. in .1 lii\.il--iu wiili niin-i-,|iii\-.i],-nt cornp Hut in i';"i. a;

in 1905, 1 argued that the largei m !"ay \»: bival,-nt i-h-ni

tin- |«air that i- r-cp.ii.it'- in tl l'.i>r U1 tu-i-iii in -pcrma-

togent

Tllns. II. MONTGOMERY, JR.

dt" tin- connection between heterochromosomes and sr\-deier-
niin.ttiiin i- accordingly imt disturbed by this discovery."

Morgan [909, I'm"! classes the theories of sex determination

by chroiuo-oi qualitative ,ind (|ii.uit itat i\ e, and incline- to

the latter \ iew he bring the first to take this stand positively.
By a quantitative interpretation In- does "not mean that the
triii. dc is simply male plus something rl-e, a view recently ad-
\anced hy Castle, but that male and female are two alternate
of ilu' li\'ing material, which po»ihilit\- is realized
on quantitative factors. . . . The gametes are not,
therefore, male and female, but contain certain factors which,
when combined, -i\< rise, in an epigenetic fashion, to one or the
other altcrnath e." In the phylloxerans, the "loss of certain
chromosomes from the male egg appear- to follow, not to pre-
cede the -i/e relation. . . . But there i- nothing in these facts
that -how-- that- the effect^ are directly quantitative rather than
that observable quantitative differences accompany, or follow
in some cases, more profound changes." He considers as the
most -eiious objection to the qualitative interpretation "that
although the hypoihe-i- is ostensibly based on the presence of
certain chromosomes which are assumed to be male and temale
• •running respectively, yet to these chromosomes, which are
to all appearances identical, are ascribed exartly opposite func-
tion-." Morgan's whole attitude i> rather hostile to the view
that particular chromosomes are sex-determinant-, and his argu-
insl the \ iew are the- most cogent yet presented.

C. FURTHER CRITICISMS OF THE Hvmi in >is.

In a pivxioii- treatment (ic^oGa) of the phenomenon of sex-
uality, I \va- led to define it (p. *5 ) as "essentially t hi- condition
of ditteicnce obtaining between conjugating individuals...-
Because conjugation is a pn>< . — distinct from reproduction, sex-

Uality, being intimateK associated wit h conjugal i«>n, has no jiri-

mary connection with reprodut tion. . . . The genesis of sexuality
has been this: that out of a state where all individuals were equally
capable of reprodm lion aconditionof division of labor has ensued,
inducing morpholo-ji al and chemical differences, between individ-
uals capable of re|>rodui tion and conjugation and other individuals

ARE PARTICULAR CHROMOSOMES SEX DETERMINANT 9

capable- of reproduction and conjugation alone. Thi- hold- true
in tlu- Metaxoa, both for tin- i^erm cells and for the per-on-. and
tin- male is rhara< teri/ed by hi- power to conjugate or fertili/e,
tin- female \>y her power to reproduce. A micro^aniete in the
I'p.io/o.i. or a -permato/onn or male per-on in the Meta/oa. i-
an indi\ idual that ha- lo-t the power of reproduction in becoming
-pe< i,ili/ed for tli •!" conjugation. Sexuality i- then the state

of occurrence of di--imilar conjugating individual-, and the
-ential point in thi- di — imilarit y i- that only one kind of the-e
indi\ idual- ha- the power to reproduce. Thi- -imple interpreta-
tion w.i- entiri-ly overlooked b\ ' and Thom-on in their
theor\ of 'The b.\olution • 3ex.' ".

The germ cells are then not w it lion t & Morgan would ha\e
us believe, but have an actual ^x utility with re-pec t to each other,

an o\ um bein- lemale and a -permalo/, „ >n male; a- well a- a

\nality with j 1 to the kind of individual they

may engender. \\ f are here ( <ni< . -i m d with the i|iie-tion of the

determination b\ particular i lironm-oine- of |>i-o-pecti\i- sexu-
ality .

I urther, a hermaphiodite i- bi-e\ual. and ii therefore

pecti\el\ bi-e\nal. engendering both kind- i-t gametes. Thi-
iiulicate- that an egg nia\ i on lain potentialK the chara< t< rs of
both g< .r better -taled, that both >lat< max ari-e

I'l'-m the -anie egg. Thi- ma\ abo be true for -pecie- thai are
no] hei-maphiodile, for a female indi\ idual fre(|Ueinl\ sho\\ -
Certain male charai teri-tic-, and a ma! dn female <|ualili« -,

even it in a more <,r less latent condition. The-e londition-
indieale that ai not (ontain pro-| ie. i i\ ely one -e\ual

-tale to the e\i lu-ioll of the other, but father that malcnc-- and
femalene-- are closelj iateil phenomena that may inter-

change within the same individual; a po--ibility -u^>;e-t»-d by
M 'i -an 'I'n

Bearing th< se idea- of the \ a hie of sex in mind, the following
main objection- max be made to the h\pothe-i- that particular
allo-ome- act • ' minani- :

I. While the phenomena appear to admit ot a -impK1 explana-
tion in cases w hen- there are only a pair ot idiochromosomes, or a
.-in-le moiio-ome, iii the spermatogenesis, ohcn the condition-

IO TIK'S. II. MONTI iO.MKKV, JR.

ot the .illi >-< 'inc- are »i much morecomplex than this, allowing so
main different chromosomal combination- in tin- -permato/oa,
that tin- interpretation of \vhai -permato/oa are male-producing
and what are female-producing becomes very difficult. Atten-
tion may be drawn, for example, to a case in the Hemiptcra
dc-cribcd by me (1901, 1906^). In -permatocytes of Calocoris
rapidus there are: "twelve autosomes that divide in both mitoses,
l\\o diplo-i.me- that do likewi-e (therefore arc probably al-o bi-
valent |, a -mailer monosome that does not divide in the tir-t but
dot- divide in the second mitosis, and a larger monosome that
divides in the first but not in the second mitosi-." Other com-
plex associations of allosomes have been de-scribed by McClung
and Payne. Were tin- allosomes sex-determinant-, \\c would
have to conclude that in certain species a considerable number of
the chromosomes subserve this end, which would be allotting an
undue amount of the nuclear material to this purpose.

2. In all plants, with the exception of one (Salomon io) described
by Cardiff (1906), and in may animals, no allosomes are known,
yet ihe-e species have sexuality. It is probable that such struc-
ture- will be found in certain cases where they have m i\ been
o\ erlooked ; yet they are apparently absent in some cases win -re
-pecial >earch ha- been made for them; accordingly, at the most
they can IK- -ex-determinants in only a limited number ot cases.

.v In certain species there is the phenomenon ot two sizes of
i-ggs, some larger that produce females, others smaller and male-
produeing. This is known for the Ph\ lloxerans, Rotatoria and
/)ino/>liilns nfxilris; another < LS6 ha- bei'ii de-cribed for an acar-
ine b\ Renter (19071; and I have -hown il'io;) that there are
two -i/es of eggs in thearaiie.nl '/'In'riili iini , though I did not
raise these e--- to determine their prospective sex values. These
two kind- of e-g- may be produced by the -ame individual, or
(Rotatoria, Punnet t , hjodj b\ different indi\ idnal-. These eggs
become distinguishable in the gn>\\ ih pei-jod, and for the 1 Mix llox-
erans Morgan (I'jooi has shown that the e^g i- "-e\na!l\ deter-
mined" before the formation < ,\ the polar bodie-. Malsen (1906)
held lor DitlOphiluS apdtris that the "dilTerence betueen male and
female eggs apparently lie- chielK in the greater or less number
of fusing ovogonia"; but his brief description and le\\ figures do

ARE PARTICULAR CHROMOSOMES SEX DETERMINANTS? II

noi prove thi- point. But however the-c differences arise they
arc clearly pre-ent early in the growth period, which i- -trong
c\ id cm c that they cannot he pn >duccd l>y any Mining of allosomes
in I'erlili/ation. And it i- (|tiite pn— ihle.a- Beard ha- iva-oned.
lli.it a di-tinctioii "I" male and female eggs may l>e a general
phenomenon, though not u-uall\ .1— ociated with dinuxaly.

4. In partln-n"ueiif-:- rily determined without
fei i ili/ation ; from Mich eggs of I\ot.it< >ri.i. aphid-, l'h\ llo\er,m-
.ind d.iphnid- limh mali-- .md female- dexelnp. SiiU'e there i-
110 fertili/.ition tin- daughter indi\ idnal- -hoiild ha\e the -ame
chromosome compl. he parent, -Imuld all 1 >e it-male-, were

determined 1>\ particular dimmo-oiii. 1 Uc there -hoiild

l>i .intii i]i.iied -eparatioiis i.f particular chroin, .-< une- in definite
m. inner-, \\hich \\mild -eem to ini]p|\ nio-t coni]iK-\ mechanit .d

i no \ ci in-lit-; asyel \\»- kno\\ nothing definite of such mo> emem-.

5. In hermaphroditic -; ri-eto a l>i-e\ual
iiuli\ iilu.il, ne\t-r to ,i uni-e\ual. \\Vn- t!n-r«- -« \ determination
l>\ |>.u -iii ul. u coinliination- of allo-..me- in the lertili/cd egg, \\ e
\\ould nei c--.ii il\ i -\p. i-ioii.il uni-e\ual indixidual- to re-
Mill, [n Sagitta Stevens 1005 found an allosome in the sperma-

1'iit neither in odgenesis nor in the tir-t cleavage; and

>he. ' uliii ivho described one in the plant S ».'/</,

|ioint- out t hat -in h .in element can ha\«- no -c \ii.il \ a hie in 1 1

hermaphrodit

A- \\'il-oii and other- ha\ e n-ali/ed. to regard ]i.irtii nl.ir allo-
SOmi e\-deiei ininant- logically n. elective

li i i ili/ation. I mil a .e fert ili/at ion ha- ln-en

demoii-l rated. l)o\\,-\,-r. the di-cu— ion mi tlli- point had lietler
In- t.lMed.

7. M..i-an ha- ur-^ed th.it it m.i\ In- tin- ma— rather than the

<iualit\ oi the chromosome substance that ma\ be sex-determina-
tive, pio\id,-d that -uch -uli-tance i- d«-ti nninativc at .ill. Il i-
the general rule in in-ect- that the male ha- less chromosome

Mili-iance than the lem.ile. in ha\ : -in.Je mono-, ,nie, or a

-mall and l.ir^e idiochromosome in the place of tuol.ir^e one-.
It mi;Jit then he .tinned that -uch allo-. .nu--, 1 >\ tin- diflen in e
in ma— \\hich the\ occasion, e-taMi-h the proM>ecti\'e sex \alue.

!lt is net actually |n»\-i-n that i .111. il n.itun-.

12 THOs. H. MoMooMEKY, IK.

Tlii- .urn- with the fact that eggs \\hich h,i\c given off both
pol.tr bodic- and an- m>t Icrt ili/ed -i\c ri-e to males, as in the
Rotatoria i \\'hit nc\ i and -oinc Hymenoptera.1 Ho\\ever, this
does not nece--arily imply that particular chromosomes are sex-
detcrminative even quantitatively, hut that tin- mass of all chro-
mosomes collectively may lie determinate e.

8. The hypothesis neglects the part that other substances,
such as the cstopta-m and the mitochondria, may have in sex
determination.

9. The strongest objection to the hypothesis of particular
chromosomes being specially sex-de!erminative remains to be
discussed, and it max equally well lie made against certain current
explanations of heredity in general. There can belittle question,
at least in the present state of our understanding, that chromo-
somes are of great importance in cellular metabolism, and even
evidence that they are in part enzyme masses. But these chro-.
niosomes, while preserving their continuity from generation to
generation, \\hich I hold to be abundantly established, are in no
sense independent units, but parts of a larger whole, the "nuclear
element," composed of the sum of the chromatin and linin.
Further, this nuclear element is not an independent unit, but
only a part, even if it be the most important part, of the cell
whole. Thus the idea is erroneous to speak of the chromosome-
as automatic units, for they are but parts of the cell or cell com-
plex. The whole, as Whitman (1893) argued, cannot be the
single cells or parts of them, but the entire inclusive organization.
For the organism acts as a whole, not simply as the sum of main-
parts; it is the interrelation of the activities of the many parts,
added to these, that constitutes tin- behavior of this major unit.

Now to assume that particular chromosomes alone- are --ex-
determinant- is to disregard this complex inter-activity. At the

•••-.\ taiilv \\«-ll r-talilNhcd tli.u dronea "i tin- honey-bee, hornet, \\a-p

and am all ; the n-clinvd numl»-i "t i In .mio-oim^. and tlu'irii.ic mu<t have-

ted IK-HI unl'ci lili/rd i-.y.i;^ lli.it llild piodllcrd two piil.il liodir-. "lhr\Milk

Mcves (1907, IQOS), Mark and ( opi-l.md ">"<•, i<}"~). Lain- I Srlik-ip

(1908) is thoroughly corroborative »t tin- conclusion. But this docs not prove
that in th<- Ilyini noptcia all uniVi tili/rd r^- x\\-<- rise to nuilrs, ioi there seem to

ilished records oi icinal'-- i< -lilting from nnlfitilixi-d CKK^. \vhi'-h
• ve been coll. , i,-d t,,,m tin- lit.-iatuir l.v \\ lu-eler (1903) and Slmll M

ARE PARTICULAR CHROMOSOMES SEX DETERMINANT 13

mo-t \vc are justified in concluding only that the chromosomes
have a share in tin- establishment of sex. He would be ra-h who
\\-mld \i-nturc I- 1 claim that a particular chromosome determines

excretion, another determine- locomotion; yet these proces
are relatively -imple compared with that of -exuality, which
-(.me ha\e contended may 1 .e . ..m rolled by a particular chronio-
some. The hypothe-i- i- toonaive.il assumes too great -im-
lilicity of the cell, i> trong of rkid predetermination.

The ide.i of unit character-, promulgated mainly by the work ot
Mendel, I >eYrie- and their follower-, i- largely to blame for -uch
hypoth. < I ms to me that phy-iological -tudy ha- siilYi-

rienily demon-ir.ited thai there are no actual unit character-,
ami it i- but natural that phy-iologi-t- have refu-ed to accept
them. hi the analyst! --breeding, the in\e-tigator ha-

tn focus hi- attention upon one or but a few i haracteri-t ic- ot
the orgaiii-m: he ha- to close lr. to the great multitude ..(

Characteristics, for the\ ., numer«>u- for an\ one mind to

p ,ii once. The i ha- he m,i\ -elect lor examination

are hi- unit- of -tud\ , and he i- en ti rely jn- titled in -peaking ol
them as unit characters, provided he doe- not that they

are merelj .ubinai\ units of convenience, lint mo-i h\ bridi-t-

ha\e ^oiie further t han t hi- : t he\ have SOUght to directl) compare
SUCh arbitral • |.t- \\ith unit- of or-aiii/alion. scarcely |>an-

in- i.i coii-ider uhat i- a unit of oixani/ation. sun-ly it i- the
mi-m as a \\lmU- that i- the miU unit, and ju-t a- -uiel\ all
it- part- are mo-t coni].le\l\ interrelated. The lixin^ l"'d\ i- a
linitN . not a colon\ .

Modern Meiuleli.ui explanation- repre-eiit a determinant
theoi\ far mor< i and complex than that of \Vei-maiin,

lhoiii;h, -iran^eU enough. m«-t M-ideli-t- in the iniTption ot
their -tudie- \\etv im-\ ni|)athetic to \\ ei-mannian interpretation.
Thi- i- the mo-t curioii- in-tance of h«'\\ men ha\e come to
identif\ an arbiiiarx term of convenience \\ith a part of the

When Sutton i.,o.V |'ointe<l out that the paternal and mater-
nal chromosome series parallel in their pairing and -cparation
phenomena of alteruati\e inheritance, thu- -eeining to ptv-cnt a
cellular ba-i- for Mendcli-m. and Castle (IQO3) argued that sex

14 TIIOS. H. MONTGOMERY, JR.

follt>\\- such inheritance, the i In night originated of identifying
unit characters with chromosomes. It was made to appear that
unit characters are present in the germ, though just what relation
a rose coml> lias to a particular chromosome was not elucidated.
Mich a concatenation of ideas as this naturally led to the iden-
tification of "sex-units" with certain chromosomes.

The better founded idea that the organism behaves as a
whole, whether it be a germ cell or a multicellular body, should
make iis lu-.il ate to localize any particular function solely in one
particular structure, for that would mean to disregard the im-
portance of interrelations of parts. Thus when we find partic-
ular chromosomes in one sex and not in the other, it by no
means follows that these are the cause of the sex difference. All
we can say at the present time is that the two phenomena are
coincident. Thus I am inclined to agree with Morgan's (1909)
closing thoughts: "The accessory (chromosome) may follow sex
or be associated with other differences that determine sex,
rather than be its sole cause."

In all probability the activities of the chromosomes are in-
thiential in establishing sex, but not in the crude way in which the
process has been imagined.

One point is quite clear, that fertilization is not necessary
for the establishment of sex, for any unfertilized egg that de-
velops furnishes a sexual individual. At the same time sex may
be changed by fertilization ; thus Whitney (1909) has shown it to
be probable that the male eggs of Rotatoria furnish males it noi
fertilized, but females when fecundated. Sex is then established
before, but may bechanced by fertilization. This clearly im-
plies that malcness and feinaleness are not unchangeable unit
chancier-, as does al-o the fact that an individual of one sex
may develop some of the characteristics of the other sex, a phe-
nomenon ><> apparent in the human body. Malcness and fern. ile-
tiess would appear to be two modes of one process, the proce--
of germ cell production, not radically dill'erent conditions. In
oilier words, there i> no valid reason to interpret sex as an im-
mutable unit chancier resident in or presided over by particular
Chromosomes, and sorted oul and disiribuled by Mendelian
lion \\ilh all ihe complex mechanisms ol dominance and

ARE PARTICULAR CHROMOSOMES SEX DETERMINANTS? 15

determiners; but rather as a growth, the result of a labile pro<
which may be changed by a variety nf influences.

LITERATURE.
Baehr. W. B. v.

'09 I>i'- < togenese U-i einiyen viviparcn Aphididen. etc. Anh. Zclll'., 3.
Baltzer, F.

'09 !»:•• Chroi 18 Hvidti- und Ki/himi< mi

tub«-r< ukitu-. Ilti-i
Boring, A M

'07 A - the Membi

'09 A -in. ill Chi .1.4.

Boveri. T

O'i . •Minnuni; S

l..-r. I'l.
•Qo i Ar. h. /i-lli.. 4.

Buchncr, P.

'09 I '

< >i tin. | •
Castle. W. E.

'03 I li.- I!-
Cardiff. T D

'06 A Mil'. '

Edwards. C I

'10 11
Foot. K and Strobell. E C.

oy i

Gross. J
'04 I '
Guthcrz. S.

'06 /in Ki inn • iinki A:

'09 \\'iid di<- Ann.ihi ! '• "id

:MK elm

•'in.
Jordan. H E

'08 I In- >PI-I i
Lams, li

'08 I ' '"•" "''"

I \: /rill.. I.

McClung. C. E.

'02 I i • • I IlK'Il: 1.. 3-

Malsen. v.
'07 G .!>ililunn '•' - Dinophilus apatris.

An li. mikr . An.:

Mark. E. L . and Copeland. M.

'06 - 5 Hoc. Bull. Mus. Comp.

Zool. Il.iiv.nd.

1 6 THUS. II. MONTGOMERY, JR.

'07 M.ituiatiuii Stall's in the Spermatogenesis of Vespa maculata. Proc.

An. Vrts .aid Sci., 43.

Meves, F.

'07 Dii' Spermatozytenteilung bei dor Honigbicne. Ibid., 70.
Meves. F. and Duesberg. J.

'08 Die Spermatozytenteilungen bei der Hornisse. Ibid., 71.
Montgomery. T. H.

'01 A Study of the Chromosomes of the Germ Cells of Metazoa. Trans. Amer.

Phil. Soc., 20.
'04 Some Observations and Considerations upon tin Maturation Phenomena

of the Germ Cells. Biol. Bull., 6.
'05 The Spermatogenesis of Syrbula and Lycosa, etc. Proc. Acad. Xat. Sci.

Philadelphia.

'06a The Analysis of Racial Descent in Animals. New York.
'Q6b Chromosomes in the Spermatogenesis of the Hemiptera heteroptera. Trans.

Amer. Phil. Soc., 21.

'07 Probable Dimorphism of the Eggs of an Aranead. Biol. Bull., 12.
Morgan. T. H.

'07 Experimental Zoology. New York.

'09 A Biological and Cytological Study of Sex Determination in Phylloxerans

and Aphiils. Journ. Exp. Zool., 7.
Morrill, C. V.

'09 Preliminary Note on the Chromosomes in the Oogenesis, Fertilization and

Cleavage of certain Hemiptera. Science, N. S., 30.
Nowlin, W. A.

'06 A Study of the Spermatogenesis of Coptocycla aurichalcea and Coptocycla

guttata, etc. Jour. Exper. Zool., 3.
Payne, F.

'09 Some new Types of Chromosome Distribution and their Relation to Sex.

Biol. Bull., 16.
Punnett. R. C.

'06 Sex Determination in Hydatina with some Remark- on Parthenogenesis.

Proc. Roy. Soc., 78.
Reuter, E.

'07 IVher die Eibilduni,' bei der Milbe Pedicnlopsis graminion (E. R-'iit.).

}• es1 -< lir. f. Pahiii'-n. I lei
Schleip, W.

'08 Die Richtungskorperbilduii.u im Ei von Formica sanguinea. Zool. Jahrb.,

26.
Shull, A. F.

'10 Do parthenogenetic Eggs of Hymnioptera prod in <• only Male.-.' Amer.

Nat., i ).
Stevens, N. M.

'05 Stndie- in Spermad i«i -iu-i- with e-pecial referenrr to the ". Veossory

mOSOme." Plllil. < ai llrr.ie- lll-t.

'06 Studies in Spermat. I'.ut II. Ilii«l.

'08 The Chroino-oiii, in I tiai.i.n i, l( vittata, Diabrotica soror and

I2-punetata. Journ, I \|"T. Zool., 5.

'09a Fmtliet Sturlie- oil the Chromosome- o! the Coleoptera. lliid.. <>.
An unpaiii ••! Heterochromosome in the Aphids. Ibid.

ARE PARTICULAR CHROMOSOMES SEX DETERMINANTS? I~

Sutton, W. S.

'02 On the Morphology of the Chromosome Group in Brachystola magna.

Biol. Bull.. 4.

'03 The Chromosomes in Heredity. Ibid.
Wallace, L. B.

'09 The Spermatogenesis of Agalena naevia. Ibid., 17.
Wheeler, W. M.

'03 The Origin of Female and Worker Ants from the Eggs of parthenogenetic

\Y-.rk«-rs. Science.
Whitman, C. O.

'93 Tli«- 1: :y of Development. Woods Holl Biol.

I •
Whitney. D. D.

'09 < >'• >n Stages of the parthenogen i sexual

nta. Jour: /ool., 6.

Wilson, E. B.

'05.; ,2.

'05/i Stu-1 . II. II. id.

'06 .III. Il.i'l.. 3.

'00 IV. 1

'09b Stir' . V. II

'09 KI-I i-nt 1< aii'l i l'T'-<li-

N. S.. 29.

EXPERIMENTS WITH CHRYSOMELID BEETLES.1

ROBERT \V. HEGXER.

Results obtained by the writer within the last five years from
experiments upon Calligrapha bigsbyana and other chrysomelid
beetles have shown that the eggs, larvae, pupae and adults of these
insects are admirably suited for the study of many of the external
and internal factors of development. The eggs may be definitely
oriented with regard to the future position of the embryo quite
easily; they continue to develop when subjected to extremely
violent mechanical conditions; and various parts may be removed
without retarding their development(Hegner, 19080, 19086, 19090,
19096). The larvae are easily reared in the laboratory in an en-
vironment similar to that which they encounter in nature; they
usually thrive well under experimental conditions. The same
things may be said of the pupae and adults.

It is the writer's intention to present in this paper, and those
that are to follow, the data and conclusions derived from experi-
ments dealing with the growth and external and internal factors
which influence the development of the various stages in the life
history of certain chrysomelid beetles. Thus far the willow
beetles, Calligrapha bigsbyanaand C. miiltipunctata, have received
the largest share of attention, but other species have also been
employed.

I. THE NORMAL RATE OF GROWTH OF Calligrapha Bigsbyana.

i . The Weight of Developing Eggs.

Method and Data. — Two series of weighings were made to de-
termine the loss of weight of developing eggs of C. bigsbyana.
Table I. shows the results for twelve eggs laid by four different
beetles at practically the same time. Two batches of two eggs
each, one of three, and one of five were taken at I P.M. on June
29, placed in a watch glass, and covered by another watch glass.
The loss in weight is quite striking. The pigmentation of ilu-

'Contributions from the Zoblogical Laboratory of the I'nivcrsity of Michigan.
No. 128.

I.KIMI.MS WITH CHRYSOMELID BEETLES.

embryos became \i-ible through the chorion on the sixth day
(July 5). Ho\\e\er, the eg'^- (li'l not hatch, as is usually the
case, on the fifth or -ixth das i Hefner. 19080), nor on the seventh
or eighth clay. Loss of water no doubt prevented the larva-
from breaking through tin- i horion.

TABLE I.
M \M. "i i. .rapha > : LAID BY 4 BEETLES AT

I I' M . ' '•• J1 tn ... I.RATURE. 2.}

Juni

Jim-

Jui:

Ju!

jui:

Jul

5-1

Jul

3-8 1

I
.8

A\n age 1 M

KgK ill in^s.

.OS
.Of.

•5
.6

.0417

•3

•7

• 7

.0;

•05

I Mil I II.

WEIGHINGS M : LAID BY 5 Mi i n rs \r

1 '

Jul

JuK •;

Ju!\ i-i

l 1 i

Jul)

Av<

.2
.3

• 71
6

It ha- brt-n m\ < u-tom t<- kcr|i a -mall |)ii-ce <if tiller |iapi-r.
IlK.i-trlled \\ith di-lilled water, in the \\atrll glaSS \\ith the •
(o prc\ciit df-iccatioii. 'I'oo mufh mni-ture frei |iieiit 1\ enable^

a tnngii- to establish a growth upon the < 1 mi-ion, sometimes check-
ing the hati liin- i >\ tin - \>,,> little moisture' ma> al-o
|'ic\ciu hatehing. In nature the egg- are laid on the under -ur-
face ot lea\e- uliele tiles ale kept -lllt'u iellt 1\ HK'i-t b\ the COH-
deii-ation »\ \\ater \a|mr at night.

1 1 In- inui-i.-nr,! tih. -i I'.ip.T u.i- removed ••» July Qth and returned on July 10.

Phis account* r.irtl>- ti>i the comparatively great l< in weight during tins inu-rv.d.

-I\\int\ <'t tin- egga li.itih'-il i'ii Jul\ ij. The chorions from which tlu-

i|)i-il \\richrd 1.170 mi;., "i ."5>-.s m^. per chorion.

2O ROIJKKT \V. HKGNIiR.

The eggs whose egg weights are recorded in the second series
(Table II.) were placed in a covered watch glass along with a
piece of filter paper which was moistened with a drop or two of
water every day. The loss in weight of these twenty-two eggs
during embryonic development was not nearly so great as was
that of the first series (Table I.), and doubtless represents more
closely the state of affairs tinder normal conditions. Twenty of
the twenty-two eggs hatched on the fifth day, the usual time for
eggs of this beetle.

Discussion and Conclusions. — The belief has been held for many
years that eggs diminish in weight during the early embryonic
stages, and before extraneous food is consumed. That this belief
is well founded has been proved by careful experiments with the
eggs of several species of animals.

Pott and Preyer (1882) have shown that the hen's egg loses
weight during incubation. The amount of oxygen absorbed by
the eggs equaled the amount of CO2 excreted. This excretion,
produced in the physiological processes taking place during in-
cubation, does not, at least in this case, account for the loss in
weight, as is usually supposed, since the decrease is equalized by
the absorption of oxygen. The conclusion was reached that a
gradual evaporation of the albumen caused the loss in weight.

When hens' eggs are incubated in desiccators the rate of de-
velopment is accelerated during the first three days, but later is
retarded, and many of the embryos become abnormal or die
(F6re, 1894).

Eggs that develop in water have also been used to determine
the loss in weight of developing eggs during development ( Kit ter
and Bailey, 1908). Bailey used for his experiments the eggs of
the California mud-fish, Funduhis parvipinnis. Starting ten days
after ensemination, 93 eggs were weighed at intervals of about 20
hours, covering a period of 9 days. Of the 30 weighing:- made,
only 10 showed a gain, and this was accounted for l>> the pre-eiiee
of dirt upon the eggs. Bailey believes that the "lo>s in weight
must have been due to carbon dioxide (CO.,) and organic salts
representing the albuminoid loss, which had passed «m through
the egg-membrane and been washed away in the -e.i-\\.iter."

A loss of energy also takes place during segmentation, and, in

EXPERIMENTS \VJTH CHRYSOMELID BEETLES. 21

the case of the sea-urchin egg, has actually been measured, though
not enough experiments were performed to make the resultant
figures of much value (Spaulding, 1907).

The eggs of chrysomelid beetles differ in several respects from
any thu- t'.ir u-ed for \\eight experiments. In the first place they
an- < overed l>y a <hitinou- chorion which is comparatively im-
pervious to fluid-, and i- ially well adapted to with-tand

desiccation. The method of cleavage, •/. e., superficial, dii't'ers
from that of tli. 'Heretofore examined.

The rc-nlt- •.( the t\\o -eric- of \\eighings recorded in Tables
I . and II. prove conclusively that there is a loss in weight, and
thai this loss is largely due to evaporation. A comparison of the

data in Table- I. and II. -lm\\- thi- quite clearly, since the i
\\ei-h"d in Table 1 . \\ ci c allo\\ ed tO develop Without the addition
of moisture, and < on-equentK d. • .1 in weight more rapidly.

2. 7 'In • A'I/'I and Adults.

Method nml l>,it<i. The tuenty l.u\a that hatched from the
n-c(| in del. -i •mining the lo— in \\iijn of developing upated ; the pupa'
\\ere then \\eiijicd dail\ . and I'm. illy the adults. These wei'Jim^-
extended over the peri.-d from Juls i: to August 14, 1908.
I'-c. .HIM ..| (he i|ail\ di-turbaiice- made neee — ary by the \\ciijl-
. in.inx .'! the lai \ a- die<|. "I" hi- mortalit\ \\ as great e-t during
the lir-t lour da\-; ho\\e\er, under normal n mditions, main of
the lai \ a- die during thi- earl\

'fhe data obtained ha\ e been arraiued chronologically in Table

III. 1:^ i gives the curve showing the dail) increase in weight

and l-'ig. j i^i\c- the curve -ho\\iu- i he daily percentage iiu ie-
nu-nt- in \\ci-ht.

/'/x /<.v.v/o;; mid i'i>ni'lnsinns. The |)roblem of growth i- one of

t intere-t to /.Milo^i-t-. and its Study has been gixt'ii added

im|>etus b\ the \\oik <>t Minoi I^MI. 1907). Thi> in\ e-ti^ator

considered -ro\\th not as in increase in -i/c or \olnme, but as

an increa-e in ma— or \\cijn . The rate of growth was measured

b\ him b\ taking the incrca-e in \\eight during a definite period
and e\|u-i'— ing it as a percentage o| ilu- \\eight at the beginning
of that period. Any change in weight can thus be -hown by
successive percentages for equal period- of time.

KUUKKT \V. HKCXER.

I MtLE III.

THE RATE OF GROWTH OF LARV^, PUP.*: AND ADULTS OF Calligrapha bigsbyana.

i c ~

H -

j;
rt

°. >

~~+ =

.- y

i~-'

~" ~.^i L'

'r. 'f '•£. =

-
- -
-

'""

— ~ :_ —

July 12

"•3

•565

July 13

12. .6

•035

6.2

July 14' 2 20

12.6 .63

•03 5-

July I52 3 i?

19. 1.118

.488 77.4

July i63 4 9

15-6 1-733

•615 55-

July 17

5 9

1.889

.156 9-

July 18

34-2

3-8

1.911 101.1

July I9<

39-4

4-377

• 577

15-2

July 2O5

44-6

5-575

1.198

27-3

July 21

7-25

1-575

28.2

July 22

107

12.375

5-125

70.7

July 236

134

16.75

4-375

35-3

July 24

140.5

17.582

.832

4-9

July 25

193-5

24.187

6.605

37-5

July 26

287

35-875

11.688

48.3

July 277

294.8

42.114

6.239

17.4

July 28»

284

40-57

- 1-544

— 3-6

July 29'

249

41-5

•93

2.2

July 30

235

38.917

— 2.583

— 6.2

July 31

257

42.833

3.916

IO.

August i

257

42.833

August 2

259

43.166

•333

•77

August 3

250

41.666

— 1.5

- 3-4

August 4

236

39-33

— 2.333

— 5-6

August 5

239

39-833

• 5

1.2

August 6

242

40-33

-5 1-2

August 7

238

39-66

— -67

— 1.6

August cS

235

39-166

— .5

- 1.2

August 9

234

39-

— .166

.42

August 10

235

39-166

.166

.42

August II

234

39-

— .166

.42

August 12

231

38.5

— -5

— 1.25

August 13'°

194

38.8

-3

• 77

August 14

[go

38.

— .8

'Larvae began to feed.

'Three larva; died.

'Moulting began on the fourth day; eight larvae died.

••Second moult in progress.

6One larva died.

"Third moult in progress.

7()ne larva died.

•Fee-ding practically stopped and larvce prepared for pupation.

"One larva died.

10Onc larva died.

Minot's results from weighings made of ^iiiiu-.i -p'.^ ^H<»\v that
the growth rate increases almost immediately alter liirtli, the
decline being very rapid at first, but less rapid a-> the age of the

EXPERIMENTS WITH CHRVSOMEL1D BEETLES.

animals increases. That there is a corresponding prenatal de-
cline in the rate of growth was shown by means of rabbit embryos.
Curves representing the change in the rate of growth with age

A
i

/ Praparation

V^^^

Period of

I /

Pupation.

Pa p at i o n

1- '

t—>

. ••• ^X'

ll_

111 4 i

-o
o

. 3.

. I :

I- 1C. 1.

3 6 8 10

Age in days.

FIG. 2.

have been o»n-tnirtr«l for the embryos and young of many
aiiimaK, ami almost without exception the growth-rate declines
as development proceeds.

24 ROBERT \V. HKGNER.

Davenport (1897) has shown for the tadpoles of Rana, Bnfo
and Amblystoma, that, during the first two weeks of larval life,
growth is largely due to the absorption of water, which increased
from 56 to 96 per cent. During later development, however, the
storing up of formed substances is mainly accountable for their
growth. The curve of the growth-rate for tadpoles does not
agree with the general rule; it rises first, then declines, and
finally rises again. This result is probably due to the absorption
of water.

TABLE IV.
THE RATE OF GROWTH OF LARV^: OF Telea polyphemus (TROUVELOT, 1867).

W- iii Days.

\Vt-ijiht in t *ruins.

Increase in Weight
in Grains.

1'er Cent. Increase.

Just hatched

•05

-5

•45

9OO

20
30

3
31

2-5
28

5OO
933

40
56

207

59
117

190
130

Trouvelot (1867) has given a few weighings of the larva? of the
moth, Telea polyphemus. These have been arranged in Table IV.
so as to show the actual increase in weight, and also the percen-
tage increments for ten day intervals. The decline in the rate
of growth is not regular, probably because of the meager data,
but it is no doubt similar to that exhibited by the guinea-pig and
other animals.

Fig. I shows the weight of developing beetles of the species
C. bigsbyana from the time of hatching to the emergence of the
adults, a period of 33 days. The following data will make clear
certain irregularities in the curve. The larva? usually devour a
part or all of their cast-off egg-shells soon after hatching, but do
not begin to feed actively until the second day; this accounts for
the very slight increase in weight during the first two days. An
actual decrease in weight \\ould be expected at the moulting
periods, when food-taking ceases and tin- rhitinous covering is
shed, but all larva? do not moult at the same time (see Table V.).
and instead of a decrease in the average \\cight, there is a slight
increase. This is shown in all of tin- moults. The period of
most rapifl increase is that I >n \\rcn the fifth and the fifteenth

EXPERIMENTS WITH CHRYSOMELID BEETLES. 25

TABLE V.
THE WEIGHT OF INDIVIDUAL LARV.« OF Calligrapha bigsbyana WHEN 7 DAYS OLD;

J'-ST AFTER THE FlRST MOL'LT.
M'lulting. j -it in mgs.

July 16 5.8

Jul- 5.4

Jul> 5-4

July 17 4.8

July 17 4-6

Jul 4-

Jul 2.6

Jul- 2.
July P, .9

days. l-'roin tin- 1. ittcr tinn- onuard the larva? gradually cease

ling .nnl lie on their l>a<k- in the earth provided for them.

1 hiring thi- |>re|.,ir,it \< >n for |»ii|iat ion, and during the period of

|iii|i.iiioii. there i- a -tead\ dediiu- in weight until the adults

emerge.

I i.1. 2 -h<>\\- the daily pen vnta^e increments in the \\eiijit of
the de\elopin;c beetle-. The remarks made in explanation of
I . . I .iUo explain the irre^ularit ie- in this curve. The percent-
age increment- decline very rapidly during the moulting period-.
It all of the l.ii\.e moulted .it the -umc time, the rate \\mild he

iti\ e.

The dat.i obtained fri'in the-e uei^hiir^- conl'irin \\hat Miimt
[891, l '107 h.i- found to l.e true of miinea-piy;-, i. e., the rate of
Drouth decline- rapidl\ during the early ^-t.i^e- of dexelojunent
and more -lo\\l\ during the later stages. jYnkin-oii I')'") has
ohtained -imilar n-iilt- for many other animal- l.y u-ini; the
data alreadx a\ailaMe in literature.

II. Tin 1 'MM i- MI- LIMIT UPON Tin I >HVELOPMI-:N r OF

I. The Influence of Darkness.

Method and l\it<i. l-".\|K-riinent C.B. 42. Kight eggs of C.
hi^/'yaiui \\en- laid at \2 M., June 10. Four were allowed to
de\elop in an ordinal \ -lender di-h (7 cm. in diameter), and the
other four were placed in a >imilar receptacle which had been
covered externally \\ith a coat of opaque paint. The same
amount of moi-ture was -upplied to each dish, and the tempera-
ture did not vary a d< gr< e.

J-

ROBERT W. HEGNER.

Two eggs in the light and all of those- in darkness hatched on
June 19; the two remaining in the light hatched on June 20. On
June 23 two of the larva' in the darkness moulted. Three of
those in the light died on June 23; the other moulted on June 24,
as did the two remaining in the dark. All of the larva? were
accidentally destroyed on June 25.

Experiment C.B. 70. Four batches of eggs were laid by four
different beetles at approximtaely 10:30 A.M. June 26. One
half of each batch were allowed to develop in the light several
feet from a window; the other half were placed in darkness as in
experiment C.B. 42. The conditions of moisture and tempera-
t ure were similar in the two dishes. The data have been arranged
in Table VI.

TABLE VI.

DATA RECORDED IN EXPERIMENT C.B 70. SHOWING THE RATE OF DEVELOPMENT

OF EGGS, LARV.E AND PUP^E OF Calligrapha bigsbyana IN WHITE

LIGHT AND IN DARKNESS.

Date 1909. White Light.

June 26 15 fresh eggs from 4 batches

July i — 8 A.M. 5 hatched
July i— i P.M.
July 2
July 3

July 4

2 hatched
7 larvae
7 larvae

7 larvae

July 5
July 6

July 7

July 8

July 18
July 21
July 22
July 23
July 26
July 28
July 29
July 30

August i

1 moulted

4 moulted

:i second moult
i still in ist instar
• 2 second moult
i still in ist instar

5 ready to pupate

2 pupae

4 pupae

5 pupae
.=; pupae

2 adults

3 adults
5 adult -
5 adults

2 larva" did not pupate

Darkness.

1 6 fresh eggs from 4 batches

5 hatched
2 hatched

2 hatched
9 larvae

f 8 larvae alive
I. i larva dead

6 moulted
i dead

/ i second moult
t i dead

4 ready to pupate

i pupa

4 pupae

5 pupae

6 pupae
i adult

3 adults
5 adults

f 5 adults

I i adult did not emerge

Discussion and Conclusions. — The eggs of C. hi^shyana are
attached to the under surface of the leaves of the food plant of
the larvse, Salix longifolia, and are thus never exposed to the
direct rays of the sun except for exceedingly brief intervals when
the leaves twist in the wind. They develop therefore in light of

EXPERIMENTS WITH CHRYSOMELID BEETLES. 2/

moderate intensity. Eggs that develop within an opaque mother,
or thai possess an opaque envelope, pass through their embryonic
stages in darkness; but there can be no doubt that the chorion
of the beetle's egg allows the light to penetrate, since, as 1 shall
-how in a later paper, sunlight has a decided influence upon
embryonic de\ elopment .

In certain cases experiments have seemed to prove that dark-
lie-- dela\- the growth of the eggs or larvae, e. g., Yung (1878)
recorded not onl\ a retardation in the development of fro- larv.e,
but al-o ,i hi'Ji death rate. The same investigator noted a -liijit
irdation in tin- de\ el. .| .nieiit of the eggs of the snail, Lymmcd
ntlli'i. \\heil placed ill the dark.

\"'-ni< .11 [895 . < m the i .1 her hand, found that echin< .derm lar\ ae
-niter \er\ little, it any, i haiue from the normal when reared in

absolute darkness Loeb [896 also has brought forth evidence

pr«>\ in- that darkne-- doe- n,,i n-tard the embryonic de\ elopment
of the ti-h 1'nmlnlns, but <]<» ; a decrease in the number of

pigment cell- on the \ oik 3a< .

In other cases, darkne-- does not hinder the growth of the
emluAo or lar\a. but tail- to -timnlate the hatching pn>< •
I'r/ibrain i mid that the larva? of the praying manti-.

Sphodromanti mled if the cocoon is placed in

the dark.

In di-cu--in^ experiment- ( . H. ^2 and (\H. 70, the normal rate
of de\elopmeiil and it- \ariations must be noted. Records of

OVer 2, . >!<i an<l the clo-ely allied species C.

ninltipntitttitd ij\e 5 da\- and to hours as the average- hatching
period rlegner, [9080). This period varies according to condi-

lioii- of nioi-uire, teni|)eial m e and probabK" other external fac-
tor-. I nun 4 to 7 day-. Record- \\eie also made of o\ er I ,OOO
Kir\.e. The average larval life i- :M> da\'s; but, a- in tin- case
of the hat chini; time, tin- period may be shortened to 17 days or
extended o\ er 24 da> - b> ditfeiences in external conditions.
The ,i\ pupal period i-, u days, though adults frequently

emerge in a -horter time, and a few do not escape until 13 or 14
day- have elapsed. These variations in the duration of the dif-
ferent stages may occur in eggs, larvae or pupae from different
batches of eggs or from the same batch.

28 ROBERT \V. HKGNER.

The data from experiments C.B. 42 and C.B. 70 indicate that
darkness has no retarding nor accelerating influence upon the
embryonic development, upon the rate of larval growth, or upon
the period of pupation.

One other conclusion that may be arrived at from these experi-
ments is that darkness has no effect upon the coloration of the
eggs, larvae, pupae or adults of the species studied. Frequent
examinations were made during the growth of the beetles reared
in the dark, but no variations from the normal were discovered
that could be attributed to the absence of light. This confirms
Przibram's (1906) results for the praying mantis, the entire post-
embryonic development of which was carried out in the dark
without producing any effect upon the coloring.

2. The Influence of Colored Lights.

Method and Data. — Experiment C.B. 64. This experiment is
the only one attempted with a view to testing the effects of
colored lights upon the embryonic development of beetles' eggs;
but it indicates that color has no very striking influence upon the
rate of development.

Several eggs from a single batch of 15, which were laid at 10:30
A.M. on June 24, were placed in each of six cylindrical tubes.
These tubes were then closed with rubber corks through each of
which were inserted a thermometer and a tube for ventilation.
These cylindrical tubes were then immersed in different colored
liquids prepared according to Yung (1878). The colors used
were red, blue, yellow, green and violet, and a tube was kept in
pure water as a control. The temperature in the different tubes
was practically identical. The eggs in the white, yellow, green
and red lights hatched on June 29; those in the violet and blue
were ready to hatch on the same day, but were prevented by
fungus growths.

Discussion and Conclusions. — Many experiments have been
performed with eggs of a number of species of anim.ils to deter-
mine the influence of colored lights upon their dc\ dopment.
Yung (1878) used freshly laid eggs of the frog, Rana esculeiita and
R. temper aria. At the end of two months all of the tadpoles in
the green light were dead, thoM- in tin- white and yellow liglits

EXPERIMENTS WITH CHRVSOMELID BEETLES. 2Q

were greater in number, those in the red light were retarded and
finally died, and those in the violet light were larger, but less
advanced and had greater powers of resistance.

These results have not been confirmed for the frog and other
animal- by later inve-tigators. For example, Yernon (1895)
found that the larva- of < < hinoderms, in some cases, were not
killed by the green light, and that yellow light caused greater
injury than red. Dric-< h 1*92), moreover, claims that the eggs
of Riuiti. I-.chinns and riunorbis are not influenced by any of these
colors.

In e\|u -riment- on the p: j mantis, Przibram (1906) found
that the influence of green, red ami yellow gla--< •> \\ as unfavor-
able-, though t hi- may ha\ e br.-n due to differences in the tempera-
ture, \\ hii li \\ a- not controlled.

M riment \\ith ih« '. bigsbyana confirms for the

• thi- beetle the if-ult- obtained by Drie-cli for eggs of
Rumi, /•'.< l;i>i;^ and /'/<:

/••• L LABI IB > n -v\.

Mn HI-.
Ajuil i

1 I II R A I 1 RE.
Davenport (

or :,.xr. Boston Soc. Nat. Hist., \'..l

Driesch 1!

'/J : ..!.. B.|.

Fere. C

'i4 . ;it 1 intl; 'it 'I'- 1'i-inlii

.!<• C. R. S 10.

Hegner. R W.

'OS,: • ' Beetles, I al-

pha '•!. Miultipu: ' iiiatn. l'-\'l"'. V»L 15.

'08 .-iininants fr tin- l~.^

Soiin- ( b 16.

OQa [I,,. ] the Eggs of N-IIIC- C In \ -..iu.-li.|

|. .;ii n I \;> Z '• I. (>.

'00'' II;. < I:I-MI .in. I l-.iii\ Hi-:.. iv -if the Germ-Cells in S»un«- ("lirvMirnelid

Hi-«'tlt-.. Joiini. M-itph.. \'--l. JO.
Jenkinson. J. W

'09 K.\|><-iiiiiriu.il l-liii! •
Loeb. J.

•96 lYliri '.nhihlung bei Thi- H-n. PflUger's

i.. M-l.

3<D ROBERT \V. HEGNER.

Minot. C. S.

'91 Senescence and Rejuvenation. Journ. Phys., \'ol. 12.

'07 The Problem of Age, Growth, and Death. Pop. Sc. Month., Vol. 70-71.
Pott. R., und W. Preyer.

'82 Ueber den Gaswechsel und die chemischen Veranderungen des Hiihnereies

wahrend der Bebrutung. Pfliiger's Arch., Bd. 27.
Przibram, H.

'06 Aufzucht, Farbwechsel und Regeneration einer agyptischen Gottesan-

beterin (Sphodromantis bioculata Burm). Arch. Entwm., Bd. 22.
Ritter, W. E.. and S. E. Bailey.

'08 On the Weight of Developing Eggs. Univ. of Cal. Pub., Vol. 6.
Spaulding. E. G.

'07 The Energy of Segmentation. Journ. Exp. Zool., Vol. 4.
Trouvelot. L.

'67 The American Silkworm. Am. Nat., Vol. i.
Vernon, H. M.

'95 The Effect of Environment on the Development of Echinoderm Larvae:
an Experimental Inquiry into the Causes of Variation. Phil. Trans. Roy.
Soc., Vol. 186.
Yung. E.

'78 De 1'influence des milieux physiques sur les etres vivants. Arch. Zool.

Exp. et Gen., Tome 7.

'81 Des l'influence des lumieres colorees sur le developpement des animaux.
Mitt. Zool. Stat. Neapel., Bd. 2.

THE MAk^lTH M OF THE UXIOXID/E.

GEORGE LEFEVRE AXD \VINTERTON C. CURTIS.

In a reo-nt preliminary announcement of a new -y-tern of
< l.i— ifiration <.|" the I 'nionida- which is based upon a -tudy of the
anatomy of the fre-h-water mu— els of Pennsylvania, nrtinann1
suggest^ a divi-ii.n • •!" the tainii\ exclusive of the Hyriina-i into
four subfamilies, nameK. Margaritaninae, Unioninae, Anodontina-

and I.amp-ilii'.e. re-pect i\ ely. Hi- arrangement lav- especial
enipha-i- fii tin- ilia: of tin- marsupiuiu and involves

several important modifications of Simpson's classification, whirh
In m.iintain- nni-t be recast to a I "ii-iderable extent in ordi i
make it repre-ent tin- nalural attmitie- • .f the group. Although
it will not In- pn--ible in fiiMn an opinimi in regard to tin- validity
• it ihc priipo-cd change- before tin- appearance of his detailed
results, lie h. - undoubted I \ I alien intu a M-riotis error \\ i h res]
in ihe iiiar-upiinn "I "lie • it lii- >ul itamilies, and it is the object
"1 ilie present imie tn |i<iint out tin- mi-take.

In i • -I i net i imi \\ iih tin- tie-li- \\aier m n --i I in\ e-ti-ations \\ hit h
ha\ e been u l)< lei u a V in tlli- la 1 ii ua 1 1 'I • ime time and whieh

ha\e bi-en t amid ..n |..r the I nited ^lates Bureau of Fi^hei
I'limaiih Im" the purpn-e "I deifrminin- the feasibility' of arti-
ficial pnipa.uaiiiin of the I nimiid.!-. we have had occasion io
give attention to the anatomical and histological structure of the
marsupium in a lar^e number "I ^i nera, and, furthermore, \\ e
ha\e been paitit ulaib • in d \\ith the change- that oeeur in

the gills during the pt rind ••! -^ia\ itlity. Since a fundamental di —
i re|'.un > e\i-i- between < >itinann'- de-rri|>tion of the gravid i;ill
in hi- -ublamiK Am tlmnina' antl our o\\ n observations on at lea-t
three ul the i^eiieia \\liith lie include- in thi- ^r<Mi]i. nameK',
Alasmidonta, Anodmita and >\ inphynota, we ha\i- thought it
ad\ i-able t" call attention t.. the tact.

Ortmann tp. 117 makes the following rather a-toni-hing state-
ment concerning the -tnicture "I the marsupium of the Anodon-

'.\ \r\\ >\ -t.-in 1.1 tin- t Fnionidse," A. V.. ' 'rtmann. Xantilus, XXIII, February,
1910, pp. 1 14-120.

GEORGE LEFEVRK AND WINTKKTnX C. CL'RTIS.

tiiuc: "Water-tubes in the gravid female divided longitudinally
into three tubes, one lying toward each face of the gill, the third
in the middle; only the latter contains eggs or embryos, and is
much larger than the other tubes. This division into three parts
is not present in the sterile (sic] female." Although it is not
specifically stated, it is to be inferred from the above description
that the divisions of the water-tubes into three parts is due to

• T- .-;•

'• .'c;

FIG. i. Anodonta cataracla Say. — Hori-
zontal section of portion of gravid mar-
supium, showing a water-tube, undivided
and filled with embryos. O.L, outer
lamella of gill; I.L, inner lamella; I.J,
interlamellar junction; W.T, water tube;
E, embryos, X3I.5 Kline del.

FIG. 2. Alasmidonla truncata
Wright. — Horizontal section of por-
tion of gravid marsupiiim. showing
a water-tube, undivided and tilled
with embryos. The mass of em-
bryos is somewhat contracted into
the middle of the tube. X3I.5
Kline del.

the presence of longitudinal partitions running parallel \\ith the
lamella1, but no intimation is given as to how they arise, <>t how
they disappear after the marsupium has discharged its contents.
To any one familiar with the structure of the gills of the I 'nionidoj
the statement that the water-tubes exhibit a temporary division
into three parts is on its face improbable, for it would be difficult
to imagine how such a division could be brought about, and still
more difficult to understand why, when once established, it

THE MARSUPIUM OF THE UNIONID.E.

should di-appear after spawning. It is true that one occasionally
encounter- a partial fu-ion of two adjacent interlamellar junc-
tions, with a consequent division into two or more parts of the
water-tube lyiiiL; between them, but this is not constant in occur-
rence for the species and, when it is pre-ent at all, it involves
only a -iiiije tube here and there in the i;ill. \Ye have observed
such fu-ions in a few indi\idual-
bclon^inu to different genera in
both i;ra\ id and non-gravid gill>,
but it : ndition that mu-t be

rded nicrch a- an occa-ioiutl
variation and i- entirely different
from that \\hich i- -uppo-<-d by
( )rtmami t" e\i-i in the Anodon-
tin.e. Hi- de-i riiition. m
i- at total variance \\ ith our ob-

-er\ ationa in the th- -. re-

ferred t' >, a- -••< lion- of the ^ill-
in the-e form-, taken at \arioii-

\ id period,
-hov. :/</<•/ v no :

diri.^inn nf the tu't'f s. h i ;.

which are drawn from hori/ontal
section- of the v;ra\id mar-u|>ium
of Anoilontti cnttiniitii Sa\ . .
mitlnntti trun«iUi\\'nv.\}( and v
phynota compfanata Mann--.
ti\el\ , the water-tubes, containing

embr\o- and :<lochidia, are -een in
their u-ual form, undivided lon-
gitudinally and bounded by the witli nlocliidia. x 31.5 Kline del.
inner and outer lamella- « <i t In- ^ill

and by he interlamellar junctions. If such a divi-ion of the
tube- into three part-, a- Uninann describe-, were present, it
\\onld of cour-e be indicated in the-e sections.

\\ e are at a lo-- t,. under-tand what appearances observed by
Urtmann could ha\e i^i\en ri-e to his error. The only tiling
that Miji.ne>t> it-elf i- that the material which he used had been

• i complttnata

', . • \<>n of por-

ti.ii. ipitini,

a w.itrr-tulir. uiKlividciJ and

34 <;EORGE LEFEVRE AND \VINTERTON c. CURTIS.

badly preserved and the gills in consequence much shrunken.
In this event, it is quite possible that the embryo- might have
been contracted into a mass in the middle of the water-tube and
the mucus, by which they aie surrounded, coagulated in such a
way as to cause the appearance of septa stretching between the
interlamellar junctions when observed under a low magnification.
It is not uncommon to find the embryos contracted in this manner
to a greater or less degree as a result of fixation, as may be seen
in Fig. 2, in which the mass of embryos has been withdrawn
slightly from the inner surface of the lamellae. The fact that he
states that the divisions are only present in the gravid gills
would lend some degree of plausibility to this explanation.

ZOOLOGICAL LABORATORY,

IMVERSITY OF MISSOURI,
April 26, 1910.

THE TYI.nKir GLAND" OF THE ASCIDIAN

BOTKYI.I.l ^ AN ORGAN ( >F EXCRETION?

) LTON,

IM k« il.i i riON.

This contribution deals with tin- anatomy and physiolo^ of an

n found in mosl of tin- :croup- of the Tunicata. Thi- organ

i- u-uall\ compoxd ol -tern i'|" tine tul>e- which ramif\in^

over tin- \\all-ot th.- inie-iin. ^incd to -..me portion of the

Btoma< h l'\ "He i.j- in,, ic din ts. Although of general occurrence

in theAscidiacea andThaliacea.yel there ha- lucn n.> concurrence

"I o|.inion ,i~ to its function. In c. .n-.-i|ucncr of thi- f.ict hardly
luo author- ha\c n : to it \<\ Hi.- -am.- name. Hence \\ <•

lia\e ilii- organ loured a- I Claude- di- , i;;n\ , [8lO

and let. ii.-d to, il ue leave out literal t ran-lat i. «n- as, 2) lixer

Hancock, [866 Kn.lui. 1852 Milne-l-'.duar.U, 1841 . ;
glande annexe du tube digestif ( 'hamldon. 1875 v. Winiwarter,
1896), i 'Jandola epato-pancreatico I >ella \ all.-. 1881 . -
la. teal system Lister, [834 llu\le\. 1851 Kilter.

inlc-tiual inland llerdinan. [882 Maurice, lx"N . 7 la. line-

stomaco-intestinales l\..ule. 1884 .lande -t.miacale (van

Hellfdell et Jlllill. 1 W} . >, d.inillllll-| 'i IlIlelK 11' l>IH-e Seelij

[882 I 'ahl^riin. [QOl Isert, [903 . [0 j ne r.'trin^ent

Giard, Is;-1 l'i/..n. [893 and II Jandr m |orii|Ue l.na/e-

1 >uthiers ••« I telagi i 889 \\"ille\ , i -

\\iih -uch a .li»i.c .if name- \\hat -hall \\ e call the or^an in
(|iie-iion' >inci- tin- name p\ loric ^land i- -Imn and -ufliciently
non-coniniittal and ha- 1-eeii dignified li\ usage, \\ c \\ill use- it in
preference i. . the other-.

The follo\\in^ -tudv i- an outi;ro\\th of one that the writer
ha- lieeii at work on for the pa- 1 two years, and as it ha- developed
into -lii;htl> other line- than \\a- ori.uinalh planned, he ha- Liken
thi- e\cu-e to make a separate I'apcr of thi- |)ortion. The \\ork

• ( 'li;ni.|.-l,.iL
• < .I.U.I. [8720.

36 HAROLD SELLERS COLTi'V

.1- .i whole \\as be^un in the /< >< »1< >^ical laboratory of the Univer-
sity of Pennsylvania. It \\a- continued at the Zoological Station
at Naples, at the Fisheries Laboratories at Woods Hole, and
I'., an fort , and the following part of it completed in the Zoological
Laboratory of the University of Pennsylvania. At this point
the writer wishes to express his great thanks to the Carnegie
Institution for the use of one of their tallies at the Naples Labora-
tory, to the authorities of the station for their many kindnesses
and hospitality, to the United States Commissioner of Fisheries
for the use of a table for two weeks at Woods Hole, and one for
t\\o weeks at Beaufort, and also to the directors of those stations,
1 )v. I-'. B. Sumner and Mr. H. D. Aller in particular.

Although for this study most of the living material was pro-
cured in the salt-water tanks of the vivarium of the University
where Botryllus colonies have been established for many years,
\et the wealth of material preserved in Naples and Woods Hole
has often been called into requisition while living material of
other families of ascidians were studied at Beaufort.

According to Bancroft ('03) there is but a single species of
Botryllns found in the north Atlantic Ocean and its extensions.
Many have been described, but they are found to be based on
color variations and habit of growth depending partly on the
age and partly on the physiological state of the colony. The
writer having worked at both Naples and at Woods Hole sup-
port > tin- view of Bancroft and considers that Botryllus schlosseri
(Pallas) Savigny, is the form represented on both sides of the
ocean.

The material was fixed in Flemming's solution, in corrosi\e
sublimate, sublimate acetic, formol, etc. The best results were
procured with Flemming's solution. Sections were cut 6 ^ and
stained in Delaficld's ha'inatoxylene and eosin. However, most
of this study was made on t he living animals and sect ions were
Used only to check up the results.

MoKi'in >\.( ii. V.

The alimentary tract of Botryllns is of the typical ascidian
type and may be represented by the letter U of which one arm
will be the oesophagus and stomach while the other is represented

' 1'YLORIC GLAND OF THE ASCIDIAX BOTRVLLUS. 37

by the inte-tine and rectum (Fig. i). In the angle between the
two arm- lies a small blind sac, an out-pocket from the pyloric
end of the stomach. It- walls are thick and glandular, similar
to the walls of the stomach, but the cells that compose it do not
contain the -e. retion that gives the -tomach it- characteristic
yellow color.

Thi- ortMii \\a-< ailed by I.ahille '90 the pyloric coecum. It
i- into thi- sac, at the point \\here it niter- the -tomach, that the
dm t of the p\ l.iric ^l.ind empti-

1 1 \\ e follow the duct from the p( >int where it enters the pyloric
CGBCUm and trace it to the inte-tine we will find it divide iu-t
before reaching that or^an. -ending a branch both to the risjit
>ide and to the left -idc. At oner on reaching the walls of the
inte-iine both branch many time-, finally ending in blind bulbs
or ampulla-. Ilo\\c\cr. all the brain he- do not end thus, but a
leu not more than ti\e or -i\ pi. Meed half \\a\ to the aim-,
often \\ithoiit branehiiu again. The-e tube- do not end in
ampulla-. A- ti • h the region oi the rectum in -ome cases,

We U ill for ( oil \ el) iein e It tel to I hell 1 a- I e. tal t llbllle-.

It i- \ei to -tud\ the oi'van in the li\inv; Hntryllnx and

indeed it i- |)o--ible in that ua\ to -ee much more than can be
ob-ei \ed in the PM served material, either in -ei ti..n- or in -in :
\ ieu . When a <"imii- i- ivnio\ed tiom it- -ub-tratuni and
p'.u ed on a mil i '< -i oj ,\< -lide, tin- -toinach and inte-tine can be
tea-ed out \\itll a ]).iil o| needle- under a di— ectin.^ mi( lo-cope.
The coiniu- i- then relinked ir.'iii the -lide. a drop of sea \\ a lei-
added and the \\h..le d \vith a COVCr glass. The writer
foimd .irtilicial li^lit in the -ha|>e of ,( Wei-bach burner, a Xei--

apochromatic 2 mm. objective, and compensai 3 \ and

u • . necessities in the pre-.-nt -tud>.

In tin- li\ in;^ tissue the tube- and bulb- appear highly refractive.
In -ome cases it i- quite e.i-\ to see tin- nuclei and e\en the

chromatin in tin- nuclei. lnnian\ « ell boundaries are quite

clear and the pre-eiu e of cell granule- i- ea-\ to determine.

The ampulla- are bounded by a rather Mat epithelium, the cell
\\all- of which contain often refractive granule- ot a \ellow color.
The cell- bear Ion;; \\hip-like Maxell. i. but it i- difficult to deter-
mine if all are SO provided. The-e lla^-lla -oon lo-e tlu-ir mo\e-

SKI.l.KKS 0>| n.N.

inrnt after being placed on a slide, although the writer has ob-
served tin-in beating I'm- live hours after the alimentary tract was
it-moved from the organism. Fig. 2 represents the character of
the epithelium and llagella in two adjoining ampullae.

In Botryllns this organ was reported ciliated by Delia Yalle
(*8i) who wrote, p. 45*: "La struttura intima di questa glandola
e simplicis>ima trattadosi d'un semplice epitelio, che io ho
veduto sempre sfornito di cigli vibratili." He gives no figure.

Pizon ('93) particularly mentions that he is unable to verify
Delia Yalle and finds the lumen of the ampulla? unciliated. There
are three other cases in which the pyloric gland has been found
ciliated — Chandelon ('75) in Perophora, text Fig. I, Uljanin ('84)
in Doliolum. text Fig. 2 and Isert ('03) in Microcosm us. The

FIG. i.

FIG. 2.

After Chandelon. After Uljanin.

Ampulla? of the pyluric gland as seen in Doliolum and Perophora respectively.

latter found tin- ductsand believed tin- ampulla- were ciliated too,
although he could not see it. The cases in which cilia have been
,i in the ampulla- were all observed before the modern meth-
ods of mien.-! npic technique were evolved. Since then we have
been carried away by the use of dead material when in main
cases, perhap-, as much if not more could be -ecu in the living.

Although the protoplasm of the \\.ilU of tin- duct- and ampulla-
seems to U- clear and refractive in the living animal, yel there are
here and there yellowish granules in thecells. These are exceedingly
minute, .1 to .2 n in diameter, and an- found in that portion ot

I <

I >

PYLOKIC i, LAND OF THE ASCIDIAN BOTKVLLUS. 39

tin- cell neare-t to the lumen of the gland. One rarely sees more
than a single granule in a cell. Among these small granules are
larger ones, o to .8 /*. di-tingui-hed from them by being more
refractiv. In character they seem much like the brown con-
cretions found in certain blood cells (Fig. 18). However, these
concretion- < .mnot be found in the preserved material, so the
supposition i- th.it they are compiled of a material that is not
coagulate.! l,\ the killing lluid. this material being of the nature
nf ,1 secretion. The concretion- in the blood cell- are not ea-ily
di--olved b\ an\ ordinary reagents u-ed in micro-. -opic technique
therefore they .ire found in the piv-. r\rd material.

At IVatifort the \\riter had the good fortune to be able to
examine the .impull.e of tin- H\ in- . 1 n;nr<><; inni stcllntiini , P, -
f)lit>rln>ni -ome aminill.e \\ere like tho-r toimd in /v»/ry///<.s 1 ig. 22),
but other- had ex< cedin^U |e\\ » ilia \\hich \\ere direct, d io\\ard
the month of the tube and not av. in < 'handelou'- figure

l g. 21 . Since the writer was able to observe movement in all

the . ilia in thi- ampulla their direction can be determined to a

tainty. 1'he \\alU of the ampulla- ..I houed no ,•--

-riitial character dilleivnt from that of Hotryll;- \ :. [9 In

the cells of the gland oi I i (I •; » found the yellow secre-
tion iu-l as IM-I-I ile-ciibed it in M : and in the lumen of
the gland are tound globule- of it. In Mol^nln the organ is
t\pical I 5il • main' diver-e lamilie- ot ascidians
-h..\\ tlagella in the luinrii "1 the p> loric gland, the writer believes
that i! carelullx lo,,k»-d for tin- organ in all tnni.ate- \\ill be
found to bear llagella.

The \\all- of the tubule- are -imilar to those of the ampulla-
except that the cell- are more cuboid ! and the tlagella

shorter. N • granules are to be detected in the lumen of the
tube- of Hotryllus >uch as l-ert found in the ducts of the pyloric
gland of Microcosm us, and a- the writer has tound in the ducts
of the gland in Stycld.

When \\e compare the rectal tubule- with the other portion -
..I the organ in Hotryllit* several things may be noticed. Among

4O HAROLD SELLERS COLTON.

these things our attention is particularly called to the relative
fewness of flagella and nuclei in the terminal portions of the tu-
bules. Figs. 4-13 show flagella while Figs. 14 and 15 show nuclei.
Although it is not possible to distinguish cell boundaries either in
the preserved material or in the living tissue, yet the fewness
of thr nuclei might suggest the possibility that the cells composing
this part of the gland have intracellular lumens. Again the ter-
minal ends of these tubes exhibit two different types. We may
ha\ e those unsegmented with a very small lumen, 2-6 p (Figs.
8-12, 14 and 15), or we may have segmented tubes with a much
larger lumen, 4-10 fj.. In the first type of tube our attention is
at once attracted to the terminal end of the tube. In most cases
there seems to be a very thin place in the walls of the tubes.
This thin place may be formed in three ways: (i) by the lumen
of the tube approaching the exterior (Figs. 8, 14, 15), (2) by a
cup-like depression in the end of the tube (Figs. 9-12, 15), (3)
by a vacuole in the wall of the tube which does not communicate
either with the exterior or the interior. To these three cases
there is a fourth effect that the writer has observed. He thought
that he could see tubes less than a micron in diameter that formed
a direct communication between the interior of the tube and the
blood space. The structures found at the end of the rectal
tubules are so small that what we may interpret as a duct may be
nothing more than a division bet ween two cells — or a nucleus—
both of which look clearer in preserved material and in the living
tissue than the cytoplasm of the cell. The cup-like depression
at the end of the tube suggests the organ of Boveri as found in
Amphioxiis or perhaps a nephridial funnel. Tin- writer has
searched tin- neighborhood about the ends of the lubes to see if
he could find solenocytes as described by ('.oodrich ('09) in
Anif>liio\iis but without re>ult. Again he has watched particles
in the blood in the neighborhood of the possible opening, yet in
no case ha-; he been able to obser\ e Mich a panicle enter the tube.
In this connection the experiment of KuptTer ('72) is interesting.
He sa\ - (p. 381): "Mir ist es audi bei Asiitiin rnnina gelungen,
dieses Sy-tem wenigsten- partiell vom Herzen ails zu injiciren.
Die Injectionsmasse war in melm-tv der blinden Anhange einge-
drungen. Solche blinde kolliige Anhange sind auch niehts Neues

" PYLORIC GLAND" OF THE ASCIDIAN BOTRYLLUS. 41

im Gefasssystem der Ascidien. Man findet dasselbe an den
colonialen Gefassen in der gemeinsamen Tunica der Synascidien.
Ich hake <l.th<-r das Gan/e fiir cinen besonders enwickelten
Theil dt> Circulationsapparates di-m wohl neben der Resorption

des ("hymn- noch anden- Fum t imu-n zukommen."

It" Ascidin i'd H hid -hould have open communication between
tin- lunicn nf the-e tube- .ind the him id ca\ity it would easily
explain IHAV Kupt'fer found tin- injection ma-s in the lumen of
the idand. Since no communication ha- been demonstrated, it
\\ould U- ea-ier t.i c\|ilain tin- iv-ult of Kuptfer in the liijit of the
\\riter'- o\\ n «-\| leriment - with indie" c.irmin on Styelii (p. 43).
With tin- |in--cni i-\ idi-nc.- l.rfon- u- we caniioi a — nine that there
i~ .m\ i omnuiiiit ati-ui ln-i\\rrn the Mm id -pace-, of ttntryllns and
the lumen ol the p\ loric inland.

Tin cases in uhich cilia ha\e Keen -ecu in the ampulla- were
all oli~er\ed liefure ihe uioilem method- nf ni; pic iechni<|iie

\\ere i \o|\ed. ^iine then \\ e lia\e Leeii carried a\\a\ hy the
u-e of dead material \\heii in man -. perhap-. a- much, it

not ill' 'IV. i . >\\\(\ lie -eell ill tile li\ il

I'i/on '•) ^ In- -tudied tli' :i of thi- or-aii Imth in tin-

tadpole and Inn I i.l l^'lryllns. < n ilii- -tudy the \\ riter ha- veri-
fied the 11 -nit- and can Imt accept the conchi-ion of I'i/on ('
that in both the origin of the p\lori«- inland i- I nun the

endoderm b\ a -imple < li\ ert iculum of the i^iit. Thi- agl
]ie|-|ectly with \'an Helledeii and Julin '84 and '86) in I'lldllu^ id,

l.etc\ re '98 in /' na, Kit ter '96 in

2 in ('A/re//;/./, I'ljanin '84 in /><>ln>!iini, etc. In all cases
it arises as an out-pocket of the -toinach.

I \ci KIMI \ I-.

The -mallnc-- of the p> loric inland in Hntrylln* and the tmeiie--
of the tubes and ampulla- a- found in the larger a-cidian-, to-
gether with the clo-e ap|ilication of the ^laild to the walls of the
inte-tine, to the reproductiv ins or to the renal vesicles,

\\onld forbid, in an\ form that ha- yet been available to the writer,
direct phv-iol«»ijcal determination-. It is due to this that the
nature of tin- oi-an has been problematical. To be sure Henri
('03) claim- to ha\e i-olated the -land in Sdlfxi, but a- \et the

4- HAROLD SELLERS COLTON.

writer has been unable to procure the form in question. As no
direct experiments on the nature of the fluid contained in the
ducts and .mipull.i- have in this case been possible, the writer
has'resorted to indirect means — that of the use of certain in-
\ it am stains.

In the use of these stains the writer has not proceeded far and
hopes at another time to undertake a fuller discussion of their
-i-mtu -.mce. Suffice it to say that certain dyes, when introduced
into the blood of a living organism in solution, have affinities
for formed substances in the cells of certain tissues, as methylene
blue in nervous tissue. Others, such as neutral red, act as
indicators, telling us whether a given substance has an acid re-
action or not, while still other dyes are segregated out of the
blood as solids and deposited in cavities often connected with
the exterior.

Following the experiments of Chrzonszezewsky ('64), Heiden-
hain ('74)' by injecting certain dyes, principally indigo carmin
and ammonium carminate, into the veins of vertebrates came to
the conclusion that tin- former dye was excreted by the Malpig-
hian tubules of the kidney, while the glomeruli excreted the car-
minate. Kowalewsky ('89) carried this idea into his experiments
on invertebrates, concluding that renal cells show either acid or
alkaline reaction which determines the character of the secretion.
However, Schmidt ('cji)2 has shown this idea false, as both am-
monium carminate and indigo carmin may lie excreted by the
same organ. Nevertheless, it is a rather general characteristic
of renal organs that they excrete carmin in some form.

In this study of Botrylliis the writer has placed colonies in
neutral red, in Bismarck brown, in ammonium carminate and
in indigo carmin, studying the reaction of the pyloric gland to
these dyes. Neutral red in concentrations rendering the sea
\\ater a pale yellow, stain- i he secretion in the re IN of" the organ
an intense red and colors the liquid in the lumen of the <lnct- and
ampnll.r also. The probable -i-niln MIICC of this is that the secre-
tion has an acid reaction. Ui-marck brown coloring the water
much like that of the neutral red, stains the granules brown and

• I IKIMI I'.i nut /. '

'PVLORIC GLAND" OF THE ASCIDIAN BOTRYLLUS. 43

in contrast to the blood and to the sea water, the contents of the
lumes of the tubes and bulbs i- very bmxvn. There are two alter-
natives by which IM interpret tin--. t\\ . . experiments — either these
stains act as indicator- or th< actually concentrated in the

lumen of the organ brin- ted from the blood by the cells

of the \\ all- of the tube- and ampul I.e. Beyond certain leucocytes
located in the neighborhood of tin \\hich -eem to collect

tin- dye, the only other cell- that take up the ammonium car-
mi nate and indigo i a rm in are the vat ii"K-- in the inte-tinal cells.

A cro « . lion of the inte-tine ha- the -hape of a rectangle (Fig.

17 . the end- of \\hich are made up of an epithelium of Hat cells
bearing cilia, \\hile the -ide- are thick and are compo-nl of iwo
^orl- of i olumiiar cell-. «ilialed and glandular I u. 14). The
Inland i ell- muler ordinary condition contain a cK-ar lit|iiid. It
is the vacuolea ot tin se -Jand < ill- \\ hich -lain -lightly in ammon-
ium carminati- and im; rmin. The -ignilicance of this will
be ili-i USSed later on.

Both Mul'^iiln and . 1 M ?•/;</ \\eiv tn.ited \\ilh indigo carmin at
I'" mforl but llie p\ loric gland -hou.,1 no reaction to them.

In .S7\'(7</, ho\\e\er. indigo carmin in concentrations as shown

in the table ;^,i\e in e\ ei 'eri-tic reaction. The

malerial in < |iie-t ion \\eie rather -mall .S'/vc/.: 20 ^> mm. The
large ones i e< |nii i-d too 1., epiacl-

I- \|.i-iinn nt i . rater 4 d

dip' r.uinin in
2 I"" C.C. do

i" • .!•> 4

•I

The animal- in experiment- I, J and ^ li\ed, in experiment 4
the\ died.

\\lien the animal- \\ < ruined lar-e blue concretion- \\ere

found in the ampulla- and duct- of the p\ loric gland 1 igs. 25
and 26). These concretions gave that portion »l the inte-tine
Covered b\ tin- p\ loric -land a blue color. The writer con-
sider^ that the indigo carmin wa- excreted from the blood into
the canal- of the J md.

HAROLD SELLERS COLTOX.

DISCUSSION.

Turning now to the character of the pyloric gland in other
tunicates, we may as well begin with the Larvacse. Although
this organ is absent in most of the genera, it seems to be repre-
sented at least in a rudimentary form in two described by Chun
('81) from deep water of the Mediterranean Sea — Stegasoma and
Megalocerciis. Here we have a diverticulum of the gut which
may be a possible homologue of the pyloric gland or at least to-
the pyloric caecum. However, in no case is the organ developed
as it is in the other orders of tunicates.

Our present knowledge of the pyloric gland in the Thaliacea
and Ascidiacea can best be presented in tabular form. The table
in question does not pretend to be complete but gives in con-
densed form the observations of various investigators.

Family. ' • nus.

Authority.

No. oi

I >m Is.

Type.

Doliolidae
Salpidae
Pyrosomidae

Polyclinidae

Distomidae
Botryllidae

Polystyelidae
Clavelinidse

Perophorid.i
Phallusida-

< \ mhiadae
M nl-iili'l.i-

Doliolum
Salpa

Pyrosoma

Fragaroides
Amaroecium
Distaplia
Bolryllus

Goods! riu
Clave 1 inn

1'crophora

1'hullnsiti
scabra
•din sp.
Corellti
Mil rin<>\iiiH\
Pol year pa

\/vc/i; >-ii\lh<i
.Vvi7(j filicata
Styelina

Mnli'jila -p.

Uljanin ('84)
Chandelon ('75)
Huxley ('59)
Seeliger ('89)
Maurice ('88)
Author
Delia Valle ('82)
Delia Valle ('82)
Pizon ('93)
Author
Hitter ('96)
Seeliger ('82)
Author
Chandelon ('75)
Author
v. \\'ini\varter ('96)

All! In '!

v. \Viniwai ter ('96)
I sert ('03)
Lacaze-Duthiers
('89)
Wagm
Author
1 ..ii .i/f-l Mithii-i <

•S,,

Authoi

I
2

I
I

Dendritic
Reticular
"Ramifying
Dendritic
Reticular

Ciliated.

1 1

1 nciliated.
Ciliated.

I
I

I
2

5-i i

Reticular

1 Vmlritir
Dendritic
1 )ciidritic
Brandling

1 )cn«li it ir
1 >rmli itic
Dendritic
Dendritic

Ki't icular

( liliated.
I'nciliated.
C'iliated.

( liliate I.

Ciliatcil.
Ciliated.

( iliated.

Eteticular

ki i n uhir
1 )cinlt iti>-

Reticular

Ciliated.

I "iH-iliatrd .

Ciliated

1 >i-ii'li itir

Ciliated.

As far as thi*. di^.m h.i^ been particularly dr^i nbrd it is much
the same in all families. The usual number of the ducts is one,
but there may be more. The tubules are eilher dendriiir or

"PYLORIC GLAND" OF THE ASCIDIAN BOTRYLLTS. 45

form a network over the intestine. All the tubes, ducts and
ampulla that have been examined carefully have been found
to be lined with a ciliated epithelium. Moreover, it is worthy
of note, perhaps, that Huxley. '51 and Delia Yalle ('81) have
observed a bladder-like -welling of the duct in Didcnunim. To-
daro figure- the -ame for Salpa. A type of gland differing
-lijitly from the one- referred to above was described by Julin
('041 in Ari-lnnsfidia. The single dun was -hort, ami branched
into but ~i\ tubule- \\hich did not branch on the inte-tine but
ran par, il lei to one another almost -ill the \\a\ to the ami-. There
were no l\ pii al am pi 1 1 I.e. The-e tube- r.m be-t be compared to
(he ivetal tubule- de-i ribed above for Jititryllns.

\- llie \\riter mentioned in the int rodliclory paragraph great
ditli-n -nee o! opinion exists a- to the function of thi- "r.;an. To
be -me. few author- ha\e \eiitured to -tron^ly -upport OIK- idea

and Illo-l lla\e been i|llite te-er\ e< 1 in their Conclusion-. \ct it is

o| -uttn ient iniere-t t» \\arrant the \\riter'- re\ie\\ing their
opinion- biielly. n-iderin^ those \ieu- that \\ere ba-ed

< le.irly on mi-, on. vpt ion- ct. \ to the -tructureof

the or^aii. \\e \\ill take up a fe\\ of the other-. I\..ill\ \\hell
I lancoi k i lied (he or^aii in <|ue-ti«m a true liver much can

be -a ill to -uppoit the \ie\\. It- relation to the blood -upply
plainl\ rei all- that of the \ ertebraie li\ er. Indeed u ith \\ hat \\ e
kllou at pre-ent of the or^ail. il \\"llld be \er\ dilticillt to relute
(hi- idea, particularly a- the \ertebrate li\er ha- been known to
6» icte ( armin.

Chandeloii 7^ . |)ella X'alla '85 ami \--ii \\ini\\aitei ' 'c)6)
and l-ett '03 Consider, after thoroughly re\ iewing the -ubje.t,
that the function is digestive. Henri '03 ha- other than mor-
pholo-ic.il exidence. 1 |.- -i\-, \>. ~> •; : "Kn fai-ant ile- macei \i-
tion- de cette u'aiidi1 p\lori.|ue. on obtient un liijiiide riche en
am\ la-e, il ne digere ni ralbumine, ni la tibrine; cette maceration
.igit an coiitraire faiblement -m la gelatine. Cette glande con tient
done bien de- ferment- digestifs. Les macerations des autres
parties du corp- de la Sdlpt donneiit de- re-ultats n^gatifs."

Kupfter /.sj) and Koule '84 \>\ means of injections arrived
at the conclusion that the-e tube- \\ere part of the blood vascular
>y-tem. l.i>ter ('34), Huxley (,'511 and after them Pizon ('93)

46 HAROLD SEUERS COLTON.

and Lefevrc ('98) ha\e agreed that ii probably serves an ab-
sorbing function, something like tin gastrovascular canals of
C.elentcrata. Huxley ('51) asked: "Does this tubular system
represent a hepatic organ or is it not more probably a sort of
rudimentary lacieal -\ stem — a means of straining off the nutri-
tive juices from the stomach into the blood by which these tubes
are bathed ?" It is very probable that the organ has a digestive
function ; there seems strong evidence to support that idea. But
the direction of the cilia in the duct would forbid the conclusion
of Huxley, etc., that the function is that of absorption.

There is yet another function that lias been attributed to the
pyloric gland. Kowalewsky ('74) was inclined from what he
knew of the structure of the organ in Peropora to attribute to
it urinary functions.

Krunkenberg ('80) says: "Ich finde sie als constantes, durch
die Murexideprobe leicht und schon nachzuweisendes Product
der als Xieren angesprochnen driisigen Darmanhange bei Phal-
litsia mentnla." This statement is based on a misconception.
He did not distinguish that the pyloric gland and the renal
vesicles were not part of one system. What he analyzed were
the concretions which others have found to contain uric acid.
This interpretation is supported by the fact that he could not
find uric acid in the gland of Ciona and of Cynthia, neither of
which have renal vesicles covering the intestine.

In SaJpa, Todaro ('oi-'o2) described three pairs of diverticula
from the alimentary canal that had the power of taking up car-
min. The first pair was in the pharynx, the second pair in the
oesophagus and the third pair the pyloric gland.

Let us now turn and inquire as to what organs have been pre-
viously described as possessing the power of elimination of waste
products of metabolism from the body of tunicates. Roule ('84)
make- ihe di-t inction bet \\eeii a kidney of excretion and one of
accumulation. Through the investigations of Van Beneden ('46),
Kupffer ('72), Laca/e-Duthiers ('741, Ko\\alc>\ ky ('89) and Dahl-
griin ('oo), we have a knowledge of this latter type of organ at
least in a lew groups. The kidney accumulation may be said
to COM^JM of i wo t> pe-. In .^nl/Mi, ( 'i<»ia and Botryllns it consists
of blood cells containing brownish concretions. The second t\ pe

' PVLORIC GLAND"" OF THE ASCIDIAN BOTRYLLUS. 47

is composed of closed vi-ick-- lined by an nonciliated epithelium
which encloses a fluid in which are suspended one or more rela-
tively large concretion-. There may be many small vesicles as
in Ascidia and Ascidiella. In Cynthia we have a few larger
\e-icle- and in Mnl'^uln a -inje large one.

In a kidney of a< < umulati< .n. ihc waste matter of the organism

isston-d ii|) in the form of a -olid \\hich i- freed from theon;ani-m

only by death. Harmer '«ij has described such an organ in the

ectoprocta and the kidney of arcumnlation can In- found in -everal

I]'- of .uiimal-. In the Tunicata other i.ruan- have been

described as kidneys of i -ion. Julin '«)i -u-^c-ted that the

neural jjand had perhap- an .-\. function. Metealf t'oo),

On reviewing the sul if the neural -land. c.>n-ider- that there

is no evidence to support the \ie\\ ..t Julin. Koule Vj '85) on

the other h.nid. d«-< ribeil about tin- opening of the deferent ranal
in Cimiti a nia-- ol pigment cell- uhich according to thj- author
i- a kidne\ ii| t -\« M lion.

I" the \ie\\ that tlu- p\l<>ii< Jand i- a kidney, there i- one

serious objection. This is the fact that Hem found in ihe

p\ lorn vjand .,! Siil{»: a ilia-:.iiic ferment i ' abundaiicf.

'1 hi- \\tnild -i t-in in be a -n. ument in fa\-»r of a dii^e-tixe

function for the or^.m \\ere it not that -uch a fernii-nt i- found in
the kidnex- ol certain mammal-, -parin^K in the do^ it i-> true
but lit hl\ in the rabbit < Ippenheiniei , '< n

\\ e ha\ e ill t ertain I or m- of tunicate- a kidne\ of a< • umulat i- •!!,
but in nearK all i;roii|>- a |i\loii< eland. The Appendicularia-
\\hith are \\ithout it an minute that all their

ti--ue-. it not actualh bathetl b\ the sea uater, are in cl"-e
proximity to it. SO that the need of -pet ial or^an- of tAcretimi i-
not unite SO urgent. He that a- it may the i|ue-tion naturalh'
ari-e-, lia- thi- limit ale ot-an au\ charaiN-r- in common with
the e\cretoi\ or-aii- of other ^nuip- of animal.--'

The -hape and characier of the terminal bulb-, and the duct-
ate parallelled in part by the multit ellular ciliai) (lames found in
the Nemertinea. A -ection of the terminal end of a duct in
Linen* "by 1'imiiett (*u shows a condition verj -imilar to that

of the a-cidian. Mach > ell of the or-an beai> a single ilagellum
whit h i- direct eil a\\a\ from the blind end of the tube. That the

48 HAROLD SELLERS CO I. To N.

duct of the pyloric gland opens into the stomach and is derived
in development from the eiitoderm is a condition characterizing
no other excretory >ystem out side of the Arthopods. How-
ever, HIUV tin- vertebrate liver has been shown by C'hr/onsze-
/r\\-k\ '66) to excrete carmin, and has morphologically the
>.mu po-ition as has the pyloric gland of the ascidian and has a
Hinil.ir development, there is good reason to believe that the
t\\<> are homologous. \Yilley has homologized the organ with
the hepatic ca'cum of Amphioxus which Hammar ('93) about
the same time compared to the liver of the Craniota. Can we
not conceive that in the hypothetical ancestor of the vertebrate
the liver arose as an organ of excretion and in the tunicate it has
retained more of those characters ?

SUMMARY.

1. There are in Botryllns two sorts of terminations to the tubes
that compose the pyloric gland, bladder-like ampulla? and long
straight blind tubes — the latter we have called rectal tubules
because in many cases they extend to the region of the rectum.

2. The ducts and ampulla? of Botryllus as well as Ascidin.
Styela, Molgula, Perophora, Clavelina and Amaroecium are lined
by cells- bearing long wrhip-like flagella, the ends of which are
directed toward the mouth of the duct.

3. Many of the rectal tubules have a termination difficult to
interpret. This has the appearance, in most cases, of a cup-like
depression in the end of the tube which seems to form a communi-
cation between the blood cavity and the lumen of the tube. In
no case, however, could such a communication be demonstrated.

4. The direction in which the free ends of the flagella point
indicates that the contents of the lumen pass toward the stomach
and therefore the function of the organ is secretory rather than
that of absorption.

5. Part of this secretion is probably found in the minute yellow
globules found in the cells of the ducts and ampulhu. It these
yellow globules represent a secretion, this is soluble in water
and does not form masses in the lumen of the tube as in Micro-
cosmus and Styela.

6. Bismarck brown and neutral red are concentrated in the

'I'M OKIC GLAND" OF THE ASCIDIAN BOTKVLLIS. 49

lumen of the organ in the form of a liquid while the indigo curium
is found concentrated in solid form in the gland of Styela.

7. In the tunicates in general no special kidney of excretion has
been recognized. Although the gland in question may have
other function- al-o, \vt its structure and properties seem to
indicate that it i- the kidney of excretion of the tunicate-, and
i^ in turn homnlogmi-. to the vertebrate liver.

LITERATURE.

Bancroft

'06 Vaii.itii.r. and Fusion in Compound Ascidians. Proc. Gala.

A. ' ;;.

Van Beneden

'46 KI-I ln-i. li< •- !Ui 1'i-inl-: .mie ct la physiologic d ii<3

•
Van Beneden et Julin

'84 !<• nt postembrionnaire d'une Phallu-i'-. Ai<h.

<li- I'.i.il., \ '.. '•> i .

'86 l<. I uniders. Arch, de Biol.. VI.. -• ,7.

Chandelon

75 !<• i mix? digestif des Tuniciers. Hull. .V.i.l.

5d. Bel \XX1X . .,i i.

Chrzonszezewsky

'64 /m A: .'s Archiv. XXXI.. 153.

'•'• . ii Aii.iii'iiiir iiinl I'd1 • r. X'irchow's Archiv. X \ X \'.. 157.

Dahlgrun

'01 I ut. I-IK hunv;« n iil«-r ili-n i Excri-lionsorganc der Tunicaton. An h.

I Mlk! Ai.at . I \ III

Delle Vail.-

'81 Nin.vr o>mir .ituralc dcllc Ascidie composte del goli<.

• li Nai'i'li. Atti ilt-i I ii. X.. 431.

Giard

'72.i 1-inl- i.li<-. Ar» h. de Zool. Gen. et Exp.. I., 397.

'72>' \<< •< In -i> In •- -MI l< •- A- nil- -<jesou Synascidians. Arch. Zool. I-"x|»-r.

• •i < .1 n. i . I . 507.
Goodrich

'09 MMHtun- ni tin- • .ins of Ainpliioxus. Quart. Jour. Micr. Sci..

I l\ . ;-
Hancock

'68 J'lin . 1 inn. >. H , 1 X.
Hammar

'98 /in Kriiiitiii- il.-i 1 • 1" i- •!'.! -A :• K' lung bei Anpliioxus. Anal. An/.. XIV.,

602.
Harmer

'92 ( >n tin- Naiuit- ..i tli<- I Processes in Marine I'oly/na. Quar. Jour.

Micr. Sci a). XXXIII.. i:

5O HAROLD SELLERS COLTON.

Henri

'03 Etude des ferments digestifs chez quelques Invertebres. C. R. Acad. Sci.,

CXXXVII.. 763.
Herdman

'82 Challenger Reports. II. and XIV.
Huxley

'51 Observations upon the Anatomy and Physiology of Salpa and Pyrosoma.

Phil. Trans., Part 2.
Isert

'03 Untersuclnmg iiber den Bau der Driisenanhangen des Darm bei den Mona-

scidien. Arch. Naturg., LXIX., 237.
Julin

'81 Recherches sur 1'organization des Ascidies simples. Arch, de Biol.. II., 59.
'92 Les Ascidiens des C6tes du Boulonais. Styelopsis grossularia, Anatomic

et embryogenie. Bull. Sci. France et Belgium, XXIV., 208.
'04 Archiascidia neapolitana. Mitth. Zool. Stat. Neapel., XVI., 489.
Kowalewsky

'71 Entwicklung der cinfachen Ascidien. Arch. f. Mikr. Anat., VII.

'74 Sur le bourgeonnement de Perophora listeri. Rev. de Science Naturelle,

Sept., 213

'90 Ein Beitrag zur Kenntnis der Excretionsorgane. Biol. Centr., IX., 43' 57.
Krohn

'52 Ueber die Entwicklung der Ascidien. Arch. f. Anat. u. Phys. u. Wiss.

Med., XIX., 313.
Krukenberg

'80 Yergleichend Physologische Studien. Heidelberg, i Reihe, 2 Abt., B. 22.
Kupffer

'72 Zur Emvicklung der einfachen Ascidien. Arch. f. mikr. Anat., VIII.
Lacaze-Duthiers et Delage

'89 Etudes anatomiques et zoologiques sur les Cynthiadees. Arch, de Zool.

Exp. et Gen. (2), VII.
Lahille

'90 Recherches sur les Tuniciers des Cotes de France. Toulouse.
Lefevre

'98 On the Budding of Perophora. Jour, of Morph., XIV., 367.
Lister

'34 Some Observations on the Structure and the Functions of tubular and cel-
lular Polypi and of Ascidiae. Phil. Trans., 380.
Metcalf

'00 Notes on the Morphology of the Tunicata. Zool. Jalirb., XIII., 495.
Maurice

'88 Etude monographique d'une espece d'Ascidie composee. Fragunmlcs

auranliacum. Arch, de Biol., VIII., 367.
Montgomery

'08 On the Morphology of the Excretory Organs of the Metazoa. Proc. Am.

Phil. Soc., XI. VI I.
Oppenheimer

'09 Handbuch der Biochemie, II., 628.
Pizon

'93 Histoire de la Blastogenese chez les Botryllides. Ann. .!«•> s< i. \.it.,
XIV.. i.

GLAND" OF THE ASCIDIAN BOTRYLLUS. 51

Punnett

01 Lineus. L. M. B. C. Memoirs. Xo. VII.
Ritter

'96 Bii'MiriK in the Compound Ascidians based on Studies on Goodsiria and

• phora. Jour, of M'Tph., XII.. 149
Roule

'84 Rechf i ••* simples des Cdtes de Provence. Ann. Mus.

Mar--i!li-. II.. M«-m»ir I.
Seeliger

'82 / u I ntui< kl . • r Ascidieri. Sitzb. der K. Akad. der \Y

LXXXV .
Todaro

'02 Sur I' Salpides. Arch. Ital. de Biol., XXXVIII.

Uljanin

'84 DI-- Art. -n i|.-r <,.it(ui;. D urn im Golfe von Neapel. Flora u. Fauna

Vogt

54 !<••, li. urs de la Mediterranee.

Wagner

'85 Di- u -on Mecres.

Willy

03 Studiefl .iii il. I. Quart. Jour. Micr. Sci., XXXIV., ,U7-

Willey

'03 Ampin • !if Vertebrates. New York.

von Winiwartcr

'96 Note BUI digestif des Ascidics simples. Arch, de

52 HAROLD SELLERS COLTON.

DESCRIPTION OF PLATES.

The figures were all drawn with a Zeiss microscope and apocromatic lenses. In
making all the drawings a camera lucida and artificial light was used, in the case
of Botryllus a Welsbach burner, and in the case of the other forms an ordinary oil
lamp. With the exception of Fig. i, Fig. 16 and Fig. 17 all were drawn at a mag-
nification of 1,500 diameters, and have been reduced in reproduction to 1,000.
.4 = ampullae. PS == peribranchial sac.

= blood cell. R rectum.

C = concretions of indigo carmin. RT = rectal tubules.

= intestine. RO = pyloric gland.

Nu = nucleus. S = stomach.

0 = (Esophagus. V = vacuole in intestinal cell.

PC = pyloric ccecum.

EXPLANATION OF PLATE I.

FIG. i. The stomach and intestine of Bolryllus. X 52.

FIG. 2. Two ampulla? of the pyloric gland in Botryllus. This drawing shows
the long whip-like flagella and also the granules in the cells. X 1,000.

FIG. 3. Portion of one of the main ducts. X 1,000.

FIGS. 4-7. Optical section of a rectal tubule showing segmentation, granules,
and flagella. X 1,000.

FIG. 8. Optical section of a termination of a rectal tubule showing a thin place
in the wall of the tube. X 1,000.

FIG. 9. Optical section of a cup- like depression in the end of a tubule. < 1,000.

FIG. 10. Optical section of a tube with two cup-like depressions at the end.
X 1,000

FIGS. ii-i2. Termination with a single cup-like depression. X 1,000.

FIG. 13. Optical section of a segmented tube in the walls of which there are
no granules. X 1,000.

BIOLOGICAL BULLETIN.

PLATE I.

;-;- 1

// <f-\— '

H. 8. COLTON.

54 HAROLD SELLERS COLTON.

EXPLANATION OF PLATE II.

Fie. 14. Section 6/t thick of the termination of a rectal tubule of Bolryllus
which was fixed in Flemming's solution. This shows a thin place in the wall of
the tube and also a vacuole. In this figure is also shown the relation of the tube
to the walls of the intestine. The vacuoles in the intestinal cells are those which
stained red and blue in ammonium carminate and in indigo carmin respectively.
X 1,000.

FIG. 15. Drawing made from two adjoining sections of a rectal tubule, which
show terminations with both a thin place and a depression. X 1,000.

FIG. 16. A section of a bud of Botryllus showing the origin of the pyloric gland
as an out pocket to the stomach. X 320.

FIG. 17. Sections of the rectum of Bolryllus fixed in absolute alcohol, glacial
acetic acid and chloroform. This shows the relation of the tubes to the canal.
X 150.

FIG. 1 8. Two living blood cells, in one of which the nucleus is visible in the
other it is not. X i.ooo.

FIG. 19. Optical section of ampulla of Ascidia. X 1,000.

FIG. 20. Optical section of rectal tubule of Ascidia. X 1,000.

FIG. 21. Optical section of ampulla of Perophora. 'X 1,000.

FIG. 22. Optical section of ampulla of Perophora. X 1,000.

FIG. 23. Optical section of ampulla of Molgula. X 1,000.

FIG. 24. Optical section of ampulla of Styela. X 1,000.

FIG. 25. Optical section of ampulla of Styela, containing a concretion of indigo
carmin. X 1,000.

FIG. 26. Optical section of a tube of the refringent organ of Styela containing
a concretion of indigo carmin. X 1,000.

BIOLOGICAL BULLETIN.

PLATE II.

«. 8. COLTON.

THE SI'KKMATOGENESIS OF THE CADDIS FLY
I'l.ATYPHYLAX DESIGXATUS WALKER).

B. F. LUTMAX.

The numeroii- w< >rk- that have appeared in recent yours on
the -pern nesis <>f insects have covered practically every

order .ind in ~<mir order-, such as the Hemiptera, main of the
import. mi l.iinilic-. There still remain, however, a number of
the -in. ill« T order- \\liirh have not been investigated, and there
i- ,il\\ a> - the po^jl.jljty th.u these may contain new or especially
l"a\orable structures. The Trichoptera is one of tlu--e -mall
orders. The \eun>ptera and Lepidoptera to which it i- belie\ ed
to be nio-i iiearK related haxebeen investigated by Miss Mi < ".ill
ind l»\ 'l"o\ama i > .in. ! II' nking (2). The small size of the

lar\a- and the diltu ill' ;i--ecting out the very youni; tests

ha- ptobabh deterred in\ e-i uators from attacking thi- ^roup
\\hen larger, belter kiioun and more easily ol)tainable form-urn-
to be had iii the Hi inipieia and Orthoptera.

I -hall HOI gO ilU" all extensive discussion here of tile general

liieiature "I -perma t o^me-i-. Phityphylax has in the main the
-aim- de\ e|.i|unein that ha- been described for all insect lorm-.
There are certain points in which it is different and the literature
on them uill be di-cu--ed in connection \\iih my own ob-er\a-
t ii Hi-.

The onl\ p.iper iii \\hich the spermatogenesis of the Trichop-
tera i- di-cu— ed .ii .ill i- in that of Lubben (3). Lubbc-n \\a-
moie init re-ied in the external morphology and the development

of tin- te-le- than he \\a- ill the CVtological details, but heob-er\ed
a number of f.ut- \\ell \\orth noticini;. The sperm. i^onial cells
ari-e dit of the original genital cells by division. They Kr"w
into l.ir-e cell- \\ hich appear, united with two cy-t epitheli.il cell-.
The origin of the-e epiihelial cells of the cysi- \\as not clear to
him. In-ide the-e epithelial cells now ari~e- a mass of cells by
divi-ion. Then b\ two more division- the spermutids ure pro-
duced; the-e ha\e -harply bounded nuclei \\ith cli-ar pla-ma
around them. The -|ierm.itid- iiuTea-e in length and the whole

56 B. F. LUTMAN.

complex of spermatozoa takes on the form of a long-drawn out
cylinder. The nuclei are all arranged at one end, pointing one
way, while the body of the sperm fills the remaining space.
These he calls spermatocysts.

The formation of the free spermatozoids does not seem to
occur either in the free or the enclosed larvae. At the time of
pupation there are only spermatocysts. In the pupa there re-
sults along with growth and the further differentiation of the
spermatozoids the resorption of the walls of the spermatocysts.
In the mature pupa the spermatozoa are free and lie in thick
knots in each division of the testes.

The caddis-flies, as has long been known, pass practically their
entire life in the larval and pupal stage. Vorheis (17), who has
followed the life-history of Platyphylax designatus, has found that
the eggs are laid in April and that the larvae appear about two
\veeks later. During summer and fall the larvae grow, and from
November to January more and more larvae are found. The
period of pupation begins about the middle of February and is
indicated by larger irregular stones being attached to the anterior
end of the sand cases "while some are attached to the lower sur-
face of the large rocks by a mass of silk at the anterior end."
For a few days after the closing of the case the larvae remain
inactive but unchanged, before becoming pupae.

The caddis-fly larvae offer the advantage that while the life-
history just sketched is gone through with at about the time
indicated, larvae of almost any size may be still obtained up until
January and February. The material was obtained during three
years; the first year from about the first of December to the first
of February; the second year during the month of June, and the
third year in May. The spermagonial and reduction division
occur in the larval stage, so the half-grown larva- of a length of
6-lo mm. was the material in which it was found. The sperms
are apparently all formed at or soon after pupation.

In the older specimens the testes were dissected out, but in
order to get the very young spermagonia it was nere>.-ary to
section the entire abdomen.

Practically all the material was fixed in Fleming's weaker
solution although sublimate-acetic was used on some abdomens

-I T.KMATOGENESIS OF THE CADDIS-FLY. 5/

with very good results. The sections were stained in Heiden-
hain's iron alum ha-matoxylin or in Flemming's triple stain.

THE TESTES.

The testes, a- Yorln -i- (17) has already noted, occur in the
fourth and fifth -e-ment of the abdomen. They are small five
loltrd -trurtnre- n-tially -urrounded by a mass of fat.

Tin -.• ti\r lob.-- <ir fi.llicles are about equal size but of various
shape-. In general the\ are somewhat wedge-shaped, tin- thinner
part nl the \\< •(!-«• l.i in-, of course, toward the point of attach-
in" -in of the oriMn I U. i.}). In longitudinal section about fifty
ippear in cadi f"lli«le (Fig. 14), the larger one- at it-
-mailer end.

Karh folliele ha- it- ou n wall and the flattened nuclei of th.
\\all i ell- |ie.|ueiitly appear in the sections. In addition each
• i- pio\ided \\iih a \\all. The youngest cells are fart he- 1
Iti'in the point ol aiia< hmeni of the testes. In one -ertion. as
-hounin li^. i( ntaining spiremes for the first division,

tei rads, in -i and se< . md di\ i-ions, transformations of the speiina-
tiiU. and Inllx de\ duped -perms occur in the order named at ;
the -e( limi. The \ min^ i \ -IN show in section, 6-8 spermatot \ tes;
the older mie-. \\ here the eell- are smaller, due to the two di\ i-inn-
and to the growth of th( ~, show 12-18. There is no del in it r

/oiiation i.| -piienie-, lir-i divisions, second di\i-ioiis, etc., surh
a- \Vili <i\ i'i has loured in Caloptenius femur-rnbrntn, but the
m-m-ral lendeiK \ i- for the cysts containing the younger cell-
to be further a\\a\ limn the smaller end of the follicles. 'I In-
tact that there i- in. definite Donation of su< ve--i\ely older spcrm-
atocytes make- it at fir-t confusing in picking out the se(|in m e
• •I de\eloiMueiu. The difference in size and number ol them in
the different cysts ho\\ ever makes this possible after a little
observation. The -permatid- in developing in the cysts, as is
n-ual, ha\e their head- all pointing in one direction as they lie
-ide \>\ -ide.

Tin SIM KMAGONIAL CELLS.

As tin earl\ hi-t«>r\ of the -permagonial cells does not seem to
have been worked out on many species of insects a rather full
description of that process \\ill be given here. In this insect the

58 B. F. LUTMAN.

primary and secondary spermagonial cells are sharply distin-
guished in their division stages while the development of the
cysts with their surrounding epithelial cells is almost diagram-
matic in its clearness.

There seems to be a general agreement in the literature that
each of the cysts that lie in the follicles of the testes has its
origin from a single germ-cell. This conception seems to have
originated in the work of ValletteSt. George (12) on the testes of
Rana temporaries. He found that if he teased apart the paren-
chyma of the testes, groups of cells, that he called spermatocysts,
would drop out. These structures had walls of their own in
which were imbedded one or two nuclei. St. George believed
that each of these cysts arose from a single cell, one of his "Ur-
samenzellen." These would correspond to the last of what is
now known as the primary spermagonial cells. By the division
of these cells there was produced the spermacysts containing the
spermagonia; or as now called, the secondary spermagonia.

Montgomery (10) describes the cysts in Pentatoma but did not
work out their origin. The connective tissue network of the
young testes contains, he believes, in each mesh a single sperma-
gonium or at least only a few spermagonia. These cells divide
and the cells produced by their division are surrounded by a
cyst-membrane derived from an extension of the connective tissue
investment of the follicle. The germ-cells and the cells of the
cyst wralls have then a different origin.

Several others, among them Henking, Paulmier and McGregor,
are mentioned by Sutton (14) as having noted the arrangement
into cysts and the cyst walls but none of them seems to have
followed their development.

Sutton (14) in studying the spermagonial divisions in Brack ys-
tola magna also worked out fairly completely the development
of the cysts. A cyst membrane with nuclei in it is formed even
in the two-celled stage of the secondary spermagonia. The c> sts
assume a roughly pyramidal shape, the cells inside it largely
dividing tangential!} to the surface of the cyst. All the cells
in one cyst seem to divide simultaneously producing by means
of about eight divisions, 256 cells. The cysts are not attached
to the walls of the follicles.

-I'KKMATOoENESIS OF THE CADDIS-FLY. 59

In all of the species so far described, however, the cysts lie
so clo-i -ly appre--ed against each other and against the follicu-
lar wall that ii i- impossible to decide certainly as to the identity
of ill.- cyst \\.ills. In all of them too the number of cells seem-
to In- lar^e. In the-e n --peels Platyphylax offers a much better
opportunity for deriding the question of cyst wall and cell num-
l"-r in a cy-t .

Tlu t-.irli. of the reproductive organ that I wa- able

to 1,..- -UK- \\ ,i~ tin- testes \vas that shown in Fig. I . At this stage
then- ha- already appeared the lobing into five divi-ion- that i-
so characteristic of tin- mature organ. The cells compo-ing it
are apparently .ill ot "about the same size; there being at this time
no \ i-ible dill- nil t ia' ion into germ-cells and cyst epithelial rell-,
it -IK Ii d<- i. I In- nuclei of these cells are a little la:

than iho-e in other part- of the body. The distribution of
chromaiin in the-e nm lei i- characteristic of that in all parts of
the bod\ , the piece- l>ein;< of rather large size and ot a rather
regular number iii-tead o| having the form of tine granule-.
Sometime- these pii : chromatin are in the form of bodie-

uhich miijit \\ell be taken for split chromosomes, while other-
ha\e the diamond -hape characteristic of the tetrads with \\hich
the\ might leatliK be i . .niounded ( Fig. 3). Divisions are rather
ran at tin- linn- and ^louih is apparently rather slow. The
di\i-ion ti^iuv- ob-ei \ ed \\ere of typical initotic type. The
pei uliar p. irt that the nm leolus plays in thi- division \\ill be
di-cu— cd under the reduction divisions as in them it is much

ger and UK ni le.idiK followed. All that i- nece-sary to say
hen- i- that it -i em- to change its spherical -hape, bet . mie-
elon-.ii,-d and a|>]>an-nil\ lorni- a chromosome.

In the testes next adxanced in the stage of development the
|irimar\ -]n-i in.i-onial ci-lls occup>" onl\ about a third of the S] •
while the remainder of ii i- Tilled with the secondary -pernia-
gonial cells in various s >f di\ ision to form the mature c\ sts

call be lolloued .it e\ el \

The testes ha\e ;^roun cmi-iderably. The !i\e tli\ i-ions are
fully formed: the\ ha\e aci|iiired distinct cellular boundaries;
and the original -ecoiidar\ -permagonial cells, instead of being
rather C!OM-|\ packed iu the te-tes, are now lying with consider-

6O B. F. LUTMAN.

able free space between them. This free space is what makes
the task of following them, and their boundaries, so easy. About
two thirds of the primary cells, spermagonia, or "Ursamenzellen"
as they are variously called, have divided to form groups of 2, 4,
8, 1 6 or 32 cells while the other third is undivided.

A closer examination of these undivided cells will now show
that in addition to the larger germ-cell there is lying closely
appressed against it a smaller kidney-shaped nucleus (Fig. 4).
The chromatin in both nuclei is distributed in the form of little
flecks rather regular in size and number and united by strands.

The origin of this nucleus that lies in the epithelium of the
cyst is not clear. There is no such differentiation in the cells in
the testes at the preceding stage. If these epithelial cells were
in the testes at that time it was impossible to distinguish them.
It may be that it is an epithelial cell that has made its way in
some fashion into the interior of the testes and there surrounded
the germ-cell. It is a difficult question to decide and one on
which my material gives little evidence.

The further development of the cysts of the follicle can be
readily followed in the two- (Fig. 5), four- (Fig. 6), eight- (Fig. 7),
sixteen- and thirty-two-celled (Fig. 8) stages. The cell-walls are
difficult to distinguish at these stages as the plasma membrane
which is all that surrounds the cells is very thin and the cells
are pressed tightly against each other. The same difficulty was
noted by St. George (12) who found, however, that he could dis-
tinguish the walls in material fixed in "quick acting reagents."
At any of these stages the nucleus of the cyst epithelium will
show. In the older cysts there appear quite frequently to be t\v<>
such nuclei, but in the younger stages at least I have observrd
only one. These nuclei do not seem to divide.

The divisions in the secondary spermagonial cells are vrr\
regular, all the cells in one follicle dividing at one time (Fig. 9).
All these divisions are typically mitotic. Sutton (14) found th.ii
the division spindles were largely tangential to the walls ot ilu-
cyst, owing to pressure in those directions. This caused the c> -4
to grow in length. As the cysts of the caddis-fly are spherical, the
divisions occur in equal numbers in .ill planes. It can be readily
determined that there are regularity 32 cells in each cy>t ot the

SPERM ATOGENESIS OF THE CADDIS-FLY. 6 1

follicles, which indicates, of course, that five divisions have oc-
curred.

After the la-t division, the cells of these mature cysts grow
;md the nucleu- becomes much larger in proportion to the size
of the cell. Fig. 8 shows the size of the cyst immediately after
it ha- acquired it- full number of cells and Fig. 10 shows a cv-t
after tin- gnmth period in which the cells are in the -pireme for
the reduction di\ i-ion-. There are therefore two growth periods:
after the la-t primary spermagonial divi-ion and the >econd
i In- reduci ion di\ isions.

Tin REDUCTION DIVISIONS.

Alter the l.i-t -pel in.e^oiiial divisions the growth i- apparently
very rapid. The nucleus is very large, occupying almo-t the
entire cell and the chromatin is in the form of regular little pi,

Fij it. The remain- of the spindle of the last >permagonial
division lie- near tin- IUK leu> in the form of an <>\oid body, the

• ii-iikern.

Apparently the fn-t -tage in division shows the little pieces
ol i hioinatin draun out until they resemble ehroino-, ,nn-> while
the mi, leo lu- become- oval or spindle-shaped big. 12). The
pieces o| rhiomatin lu-comc united by a slender connection and
-eem to -pin out and lo-»- their identity. The niu leolu> in the
meantime becomes more and more elongated Fij 15).

The chiomatin n»\\ -e.-ms to go into a \»u^ -lendi-r >|)in-ni, ,
the ma-- ol the thread- King at one side <»t" the nucleus and oc-
eii|)\ini; about one half of it. This is apparently the stai^e of
>\ nap-i-. The -i rand- are very fine and delicate and as the nuclei
are not -, . \,-r\ lar^e. it is impossible to make out an\ pairing
ol the threads, if such occurs. The nudeolus lies as a long drawn
out l>od\ either in thi> tangle of the threads or on its surface.
' ', • a-ional loop- >tick up out of the denser clum]> but they are
too -mall to follou or to make much out of. After tlu-e -tages,
\\hich are a|)parentl\ -hort a- they are not nunierou-, the chro-
matin again till- the entire cell in the form of the spireme. This
spireme gradual!) become- -horter and thicker. This stage is a
\er\ Ion- one as i- -hown by the fact that in sections where any
di\i-ion- ,u all occur, about half of the cells are in this stage.

62 B. F. LUTMAN.

It may be that the stage of synapsis is much shortened in the
caddis-fly and that of the spireme is correspondingly lengthened.

The spireme now breaks up into chromosomes and these lie in
the nucleus as long slender paired bodies (Fig. 22). The chromo-
somes now come to have the peculiar shapes such as X's, Y's,
twisted figures, etc., characteristic of the stage of the reduction
division (Fig. 23). These soon shorten into the tetrads (Fig. 24).
The tetrads take on the typical lozenge form and sometimes show
an opening in their center. The manner in which these bodies
arrange themselves on the spindle could not be definitely deter-
mined as they are small in size and the four segments of the
lozenge are all of about the same length.

The metaphase shows a sharp pointed spindle with its extremi-
ties in a centrosome just inside the plasma membrane (Fig. 25).
The telophase of this division shows the chromosomes pulled
about two-thirds of the way back to the centrosome and the
nuclei still connected by the remains of the central spindle (Fig.
26). The centrosome at this time is still apparently divided and
the rays from it extend down to the cluster of chromosomes.

The second division, following almost immediately, has little
to distinguish it in the metaphase from the first except in its size
and in the size of its chromosomes (Fig. 29). In the telophase of
this division the centrosome was not to be found — just what had
become of it has not been ascertained. The remains of the cen-
tral spindle is the most conspicuous feature of this stage. The
chromosomes seem to spread out at once, as soon as a nuclear
membrane is formed, and make the ordinary reticulated network
of a resting nucleus (Fig. 34).

There is a period of growth after the reconstruction of the
nucleus, such as Paulmier (n) and other authors have described,
followed by a stage in which the nucleus shrinks. Even before
the chromosomes are entirely distributed and while they are still
present as little pieces of chromatin (Fig. 34), the cell begins to
lengthen and the axial filament to form. In fact the long drawn
out form of the cell which it has from the last division does not
seem to change but passes over at once to the young sperm.
The transition stage takes a long time for its completion and any
number of transition stages can be found. The chromatin col-

-l'I KMATOGENESIS OF Till-: CADDIS-FLY. 63

lects more or less to the outside of the nucleus, forming a hollow
sphere, just inside the nuclear wall. The remains of the spindle
lie near the nucleus as an oval body in a clear zone. This body
apparently divide-; as Baumgartner (i) has described and a part
of it -eem- to pa-- around the nucleus. Soon after these stages
-howii in 1 ii;. ;>4 the nuclei become very sensitive to the fixing

gents .iml .1- a <"n sequence practically all of them have col-
lap-ed. Tin- would probably be the stages when the chromatin
i- in the form of .1 tube with a very small lumen. The fully
developed -perm i- -hown in Fig. 36.

I In detail- v;i\ en .ibovc arc in the main the same as h,i\ e 1 >een
dc-i ribed many time- for animal spermatogenesis. In the action
oi (.mi- p nt- of the ( rll mechanism there is always quite a little
variation and it i- to the-e parts that particular attention \\ ill be
p.iid. The t\\o -tun tures to be especially noted are the centro-
.iinl tin- "chrontatin nuclcolus."

1 UK CENTROSOMK.

h i- impo— ibU- to locate the centrosome except \\hen it is
o. iiipxiii- a p.i-ititui at the end of the sj>iiulle. At any other
pl.iee it i- ini| >o--il •!«• to say wlii-ther a certain dark staining body
i- .i centrosome or not. HtKlies appear near the nucleus where
the ceiitro-. .me -hould be anil have all the characteristics of
Centrosomes, U-in^ darkly staining little granules surrounded b\-
.1 i lear space -ind trequeiitly with what ajjpear to be rays running
up to them, ilioii^h these latter are very faint and small, but the
miihiplii it\ of -urh bodies makes it impossible to trace the hi-tory
of the ( entrosome u ith any certainty for any distance or even to
be -tire tli.it -mil a centre is present at all times. At tin end-
ot the -pimlle. it- position is of course always easy to determine.
It u-u.ilh lir-t appears so as to be definitely recogni/ed at the
tetrad >ta^e a- a dark body on the plasma membrane. It ap-
peal - as .1 \ ei \ -mall blark -raiiiilc on which the fibres terminate
and lie- ju-i in-ide the pla-ma membrane and apparently at-
lai hed to it. hi- mo-t eon-|m-uous during the meta]iha-e \\ lien
the >harp ])ointed -pindle end- in this little grannie at the plasma
membrane. It ma\ be. of course, that here it i> not a bod\' at
all but onl> the i omnion point of att.ichment of the Iil)res of the

64 1?- !•"• I-l'TMAN.

central spindle and of the aster. Whatever the origin and nature
of the centrosomes may be at this time it is at least something
that will take a stain and that has a definite location. After
the second division, at which time it lies at the ends of the
spindle again, it seems to disappear until a dark staining granule
appears at one side of the nucleus from which the axial filament
seems to be growing out. I have not traced the centrosome
around to discover whether the two are identical or not but from
the results on other animals it undoubtedly is. From this stage
on then, it would form the middle piece of the sperm.

THE CHROMATIN NUCLEOLUS.

McClung (4) has described in the germ-cells of certain grass-
hoppers a body which he calls the accessory chromosome. Pre-
vious to this discovery of this body the "chromatin nucleolus"
had been described by Montgomery (10) in the Hemiptera.
More recently the discussion of heterotypic chromosomes has
been given special importance by the papers of Stevens (13) and
Wilson (20, 21) especially in connection with the theory of sex-
determinants.

The divisions in the nuclei of Platyphylax show a body which,
while it seems to have something in common with these described
structures, is in other respects quite different. Its behavior has
been reserved for this separate discussion.

The various changes undergone by this body have been fol-
lowed to some extent both in the spermagonial and reduction
divisions. As the cells, and consequently this chromatin nucleo-
lus, are larger in the reductions divisions it will be described there
first. The nucleus of the young spermatocytes contains a nu-
cleolus that stains typically both in the triple and the iron
haematoxylin. This body is either spherical or ovoid in shape.
In the preparatory stages of division it begins to lengthen and
become spindle shaped. It frequently lies twisted over on it -dl
or is spoon-shaped at this time (Fig. 15).

In a later stage when the chromatin has gone into the s\ naptic
condition this body seems as a rule to be somewhat smaller in
diameter as though it were spun out as the other chromatin has
been (Fig. 16). It does not lie among the chromatin strand-, but

MT.KMATOGENES1S OF THE CADDIS-FLV. 65

as a rule re-ts outside the chromatin mass which at this time
in i upics about half the nucleus. At this time the body seems
to IH- a part of the spireme. In the next stage when the chroma-
tin conic- out <»f -> napsis this body appears as a pan of the much
thickened -pin-me thread (Figs. 17-21). It is quite large at this
time and much resembles a nucleolus in its intense -taining
n MI i ion- lull it i- -pindle shaped and from each end run- out the
continuation ot tlie -piremc thread. In the triple stain at thi-
time it -till take- thesafranin color. A still closer examination
-how- that the threads leading up to this body are double and
the body itself is divided into two halves by a longitudinal furrow.
It lie- .n thi- time, when seen in side view, as a flat spindle-
-haped figure immediately pressed against the nuclear wall. It
now tv-embie- \t-ry much the accessory chromosome as drawn
l>> M<< li, in hi- Fig. 2, except that he at this period dis-

co\rrcd no l.n-.ik and the body that he drew was proportionally
coii-ider.ibly l.uxi r. He describes and figures a stage (Fig. 5)
\\here thi- liod\ goes into a spireme of its own but no split \\a-
ob-cr\ed in ihi- -eparati- thread. Considering the subse< jucnt
behavior of thi- body — the formation of a tetrad and of chronio-
somes, -mli .1 -plit i- to be expected. A split was to be observed
in thi- chromatin nm leolus of Platyph v/a.v; its spireme is a part
ot tin -pin me formed from the remainder of the chromatin.
The -plit in it becomes more marked (Fig. 19) and the body
finally open- out a- a lo/enge-shaped tetrad (Fig. 21). At thi-
time the other ihiomosomes have not yet formed, although the
lon-iiMilinal -plit has taken place. In some cases it look- as
though the tran-\er-e splits have already occurred, but the thread
-till remain- int.n i with this body as a part of it. This black
-taming tetrad is one of the most conspicuous parts of the nucleu-
at thi- time i Fig. 2l).

The other chromosomes are now formed and assume the pe-
culiar -h.ipe- characteristic of them at the time before they form
the tetrads. Thi- body is still recognizable at thi- time on
account ot it- regular lozenge shape while the other- are in the
form ot K's, Y's and various other twisted shapes (Fig. 23). At
the next -tage. however, when all the chromosomes have become
tetrad- thi- body is indistinguishable from them (Fig. 24).

B. F. LU I.MAN.

There is no evidence that it has disappeared as tin- nucleolus
usually does; it seems simply to have become a tetrad. The
elaborate formation and dividing of the tetrad would argue
against this disappearance also. This chromatin nucleolus can
be traced no further. During the equatorial plate stage of divi-
sion the chromosomes all lie in one plane and it is impossible to
identify any particular one as the transformed nucleolus. Nei-
ther does any one lag behind in divisions in the metaphase nor
in the movement toward the poles in anaphase (Figs. 26 and 32).
If this body forms a chromosome, as it undoubtedly does, that
chromosome behaves exactly like all the others.

The number of chromosomes is of great interest here if this is
a true accessory chromosome. According to cither the McClung
or the Wilson type of an accessory chromosome, or the Wilson
type of a hcterotypic one, there should appear an odd number of
chromosomes plus this additional one; or as McClung has found
in Orchesticus sixteen chromosomes and the accessory one.

In all the counts made in Platyphylax, however, the number of
chromosomes for both the reduction divisions \vas found to be
thirty. This is the result of repeated trials. These countings
are as easy to make as of dots on a piece of paper (Figs. 27, 28, 30)
as the polar plate views are numerous and the chromosomes are
short. There is some variation in size in the chromosomes in
polar view but it is impossible to pick out one of them as the
special structure that has been followed.1

It will be seen from this description that while this body re-
sembles the accessory chromosome of McClung in many respects
still it differs from it in one very essential one. It apparently
forms a tetrad that divides in both divisions and so each sperm
would receive one fourth of it. This wrould make it impossible
for it to serve as a sex-determinant, for all the sperms would
receive a part of it, and not half of them, as would happen if we
credit the observations of McClung or of Miss Wallace (18).
This body in that case could not be a sex-determinant.

'In cutting abdomens to get the development of the testes I have cut and stained
as many, or more, females than I have males. The divisions in the former can be
readily obser%rcd here as they are much larger than in the testes. The nucleolus
undergoes a similar lengthening out, and then forms a part of the pireme. M.n-
shall (9) in his paper on the development of the ovary also shows several figures
that strongly suggests this.

SPEkMATOi.KNESIS OF THE CADDIS-FLY. 6/

\oinov i 1 6; has described in the divisions preceding sperm
formation in a beetle (Cybister roeselii] a. body which very much
resemble- in it- appearance the one under discussion. He, how-
ever, <li<l not ob-erve it forming a chromosome tetrad, nor did he
follow it during the nuclear divisions except to figure a small
darkly -tainin^ body lying outside the nucleu- in the cytoplasm,
which he belit -\. •- to be the same. It may be that this body
niiuht t'»r:n a tetr.nl a- in Platyphylax.

Heidenhuin in "IM.i-ma und Zelle" approves Flemming's opin-
ion that the nu< leolu- is always surrounded by a thin Liver of
true chromatin. Some appearance such as those shown in Fig. 2
would seem to -u^e-t at least that there might be a ring of some
other material around the nuclcolus but the structure is so small
that it is impossible to say certainly. If this were true, however,
tin- ( haiue- de-eribed \\ould only mean that the nucleolus loses
ii- loim dining the di\i-ions and becomes pulled out and split,
bet \\een iln (hi.. matin -trands surrounding it.1

\i an\ rail \\hate\er this structure in Platyphylax may be.
wli«-ili "accessor} < luomo>ome" or "chromatin nucleolu-."
>oun- di-po-itioii o| it iiui-t be made in discussing these aberrant
ehn>nio-i mie- in t|lr ,lll( |rj <,f insects.

Thi- \\oi k u as d' me under the direction of Prof. \V. S. Marshall,
ot the 1 ni\er~ii\ oi Wisconsin, to whom my thanks are due imt
onl\ for assistance with methods and literature but also for quite
a portion o| the material from which the sections were made.

SUMMARY.

1. The de\elopment of the follicular cysts can be readily fol-
lo\\ed in thi- in-ect. Each cyst contains 32 cells derived by
5 di\i-ion- tiom a primary spermagonial cell and enclosed in a
membrane containing one or two nuclei.

2. The reduced chromosome number is always 30; the somatic
number i- probably 60 from a count in the oogonial divi-ions.

,v The centro-ome i- onl\ to be followed from the tetrad stage
to the anapha-e but probably forms the middle piece of the
sperms.

A CO nu i>t tin- si'iiKiiic nuinlx-r nf chromosomi.-< in the ix'.iKonial division gave
5> 60. 1 h«- <-\i'i-«-<linnly lar.m- number makes counting difficult and not very ac-

i male in tlu-M- ,livi-:

B. F. I.rTMAN.

4. The nucleolus of the spermatocyte seems to form a tetrad
which becomes one of the thirty of the reduced number.

BIBLIOGRAPHY.

1. Baumgartner. W. S.

'02 Spermatid Transformations. Kans. Univ. Soc. Bull.. Vol. I., 1902.

2. Henking. H.

'09 Das Ei von Picris brassicae neben Bemerkungen iiber samen u. Samen-
bildungen. Zeit. f. Wiss. Zoo!.. XLIX., 1890.

3. Lubben. H.

'07 t'ber die inncre Metamorphose der Trichopteren. Zool. Jahrb., Bd. 24,
1907.

4. McClung. C. E.

'99 A Peculiar New Element in the Male Reproductive Cells of Insects. Zool.
Bull., Vol. II.. 1899.

5. '00 The Spermatocyte Division of the Acrididae. Kans. Univ. Quart.. Vol.

1-X, 1900.

6. 02 The Spermatocyte Divisions of the Locustidae. Kans. Univ. Sc. Bull.,

Vol. I.. 1902.

7. 09 Spermatogenesis of Xiphidium fasciatum. Kans. Univ. Science Bull., IV.,

1909.

8. McGill. C.

'04 The Spermatogenesis of Anax junius. Univ. of Missouri Studies, II., 1904.

9. Marshall. W. S.

'07 The Early History of the Cellular Elements of the Ovary of a Phryganid,
Platyphylax designatus Walk. Zeits. f. Wiss. Zool., Bd. 86, 1907.

10. Montgomery. T. H.. Jr.

'98 The Spermatogenesis of Pentatoma up to the Formation of the Spermatid,
Zool. Jahrb., Bd. XII., 1898.

11. Paulmier. F. C.

'99 The Spermatogenesis of Anasa tristis. Journ. of Morph., Vol. XV., Supp.,
1899.

12. St. George. Valletta

'76 Die Spermatogenese bei den Amphibien. Arch. f. Mik. Anat., B<1. XII..
1876.

13. Stevens. N M

'05 Studies in Spermatogenesis with Special reference to the "Accessory Chro-
mosome." Carnegie Inst. Pub., 1905.

14. Sutton. W. S.

'00 S|K-rmatogonial Divisions in Brachyostola magna. Kans. Univ. Quart.,
Vol. IX., 1900.

15. Toyama. K

'94 On the Spermatogenesis of the Silk-worm. Bull. Coll. Ag. Imp. Univ. Japan,
1 1., 1894.

16. Voinov. D. N.

'03 La Spermatogenesis chez le Cybister Roeseli. Arch. Zool. Exp., 1903.

17. Vorheis, C. T.

'05 Habits and Anatomy of the Larva of the Caddisfly. Platyphylax desig-
natus Walker. Trans. Wis. Ac. Sc., Arts and Let., Vol. XV., 1905.

SPERMATOGENESIS OF THE CADDIS-FLV. 69

18. Wallace. L. B.

'05 On the Spermatogenesis of the Spider. Biol. Bull., VIII.. 1905.

19. Wilcox. E. V.

'95 Spermatogenesis of Caloptenus femur-rubrum and Cicada tibicen. Bull.
Mus. Comp. /.".I. Harvard, XXVII., 1895.

20. Wilson. E. B.

05 06 Miidi«-5. »n Chromosomes. Journ. Exp. Zool.. Vols. II. and III., 1905
and i',

21. '09 >n the Determination and Heredity of S< : nee,

X. S., XXIX., i

72 B. F. LUTMAN.

EXPLANATION OF PLATE II.

13. Longitudinal diagram of the entire mature testes.

14. Section of one of the follicles showing size, shape, and distribution of the
cysts.

15. Formation of the spireme in a spermatocyte.

1 6. Synapsis.

17. Nucleus showing nucleolus on spireme.

1 8. Nucleus showing nucleolus on spireme.

19. Entire cell showing same.

20. Enlarged views of the nucleolus at this same stage showing the split.

21. Chromosomes formed by the transverse split.

22. Chromosomes lying free in the nucleus; also the split nucleolus.

23. Chromosomes forming the tetrads; nucleolus at n.

24. Tetrads.

25. Metaphase, first division.

26. Anaphase, first division.

27-28. Polar view of the equatorial plate.

29. Metaphase, second division.

30. Polar view, equatorial plate.
31-32. Anaphase, second division.

33-35- Transformation stages of the spermatid.
36. Sperm.

BIOLOGICAL BULLETIN.

PLATE II.

Vol. XIX. July, igio. No. 2.

BIOLOGICAL BULLETIN

Illl ASSOCIATION OF A FISH WITH A HVDROID.

HAROLD HEATH.

Am- mi; the most interesting phases of animal existence are
tho-e examples of commensalism or "messmateism" known in
iii.niN in-tain • •- to exist among species of widely different plnla.
At time- tin a--ociation. while no doubt beneficial to both parties,
i- pnivK .11 ( -ill. -ntal, such as that occurring occasionally betucni
the (-..!• mil •- of various hydroids and crabs or mall uses ami on
two OG i -ions I have found flourishing colonies of Clava leptost via
• tti.u hed tn i In- spines of the sea urchin, Strongylocentrotns fnin-
Kinus. A^.iin what appears to be a communistic associ.ition
in.i\ in realit\ he a case of parasitism as, for example, the relation
nt certain h\ (In lids and the eggs of a number of fishes, or po--il>ly
"iir h\ Mi. -ni to another as noted by All man in his monograph mi
i;\ miii '1 il.i-i ic h\ droids, or the attachment, mentioned by Feu 1
nl the |]\«lnii<l. Ilydrichthys mints, to the fish, Seriola z/nuitn.
In undoubted cases the association is not only invariable, or
lairK cim-iant. lnn mutually beneficial and very intimate a^ i^
\\ it m --eil 1>\ tlie l.ict that the hermit crab, Eupagurns pridcauxii,
\\ln n rli.in;<iii^ its abode removes the commensal anemone, or
the case uieiuiiMied by Miss Rathbun2 of the Hawaiian Ishmd
cr.ib. I.ylna tcssclatn, that held "little sea anemones one in each
i l.i\\ .mil presented them in a boxing attitude whenever teamed
in .ippi-D.ifhed by another crab." And, on the other hand, it is
m'lier.dly belie\ed that the anemones enjoy a larger food supply
o«iiM'<|ueiit upmi the improved method of locomotion.

In i^'ij Almck added what he considered to be another of

'/'/ '. it. 11: t. SOC.. \"ol. XXIII.

1 S. F. C. Buli.-tin, 1903. p. 866.

3 Ann. M .. \ -. li .6 ser.. \rol. IO.

74 HAROLD HEATH.

these remarkable mutual benefit societies, describing the associa-
tion of the hydroid Slylactis minoi with the rock perch, Mhinns
inennis. Several specimens wen- taken at depths varying from
forty to seventy fathoms in the Hay of Bengal and the Laccadm-
or Malabar sea; and in every ca^e the hydroid was found at-
tached in large numbers about the gill opening, on the throat
and in the axilla. And not only were no fish of this species ever
discovered without being coated with the hydroid, but none of
these hydroids was ever found upon the multitudes of other
animals dredged in the same locality, though among these were
specimens of Minous coccineus. Accordingly it thus appears to
Alcock that, unusual though it is, this is a case of true com-
mensalism.

Several years later Doflein collected three more specimens of
M. inermis in Sagami Bay, Japan, and again all were coated,
especially between the pectoral and ventral fins, with this same
hydroid. In the first account the coelenterate was assigned by
Alcock, on characters associated with the reproductive sacs, to
the genus Podocoryne; but other specimens, seemingly more
highly developed, led the author later to place them in the genus
Stylactis. On the other hand, Stechow,1 who described the
Japanese specimens, finds no evidence of spordsacs, but young
medusa5 with tentacles and four radial canals and accordingly
this author places the species once more in the genus Podocoryne.

During the past summer my friend and colleague, Prof. E. C.
Starks, dredged upwards oi a hundred specimens of an agonoid
fish, Hypfdgonus quadricornis, in Puget Sound at a depth of
approximately forty fathoms. The area over which the dredging
extended was in the neighborhood of Friday Harbor and em-
braced an area of at least two hundred square miles where the
bottom varied from sand to mud. Of the 37 specimens preserved
in the Stanford University collection 10 of them are coated with a
new species of hydroid, Perigonimus pugetensis, whose descrip-
tion is given later. In every specimen the ccelenterate was more
abundant on the ventral surface of the body, especially in tin-
axilla, and a luxuriant growth was usually found on the pectoral,
ventral and, to a less extent, on the anal and caudal fins. With

'Zoo/. Am., Bd. 32, p. 752, 1908.

I HE ASSOCIATION OF A FISH WITH A HVDKOID.

one or t\\<> exceptions the polyps were much more sparsely dis-
triUitecl over the body and dorsal fins. In no case were they
found on the head.

.\]<"<k, referring to several species of h-hes of the family
:p.enid.e th.it "have the body and fin-- capriciously co\ ered
with Ion-, wavy often tufted cutaneous filament-," l>clic\ e-. w ith
a I. 'inpany of zoologists, that these structure- "assist in

i;ivin^ i In- li-h a deceitful resemblance to the incrusted rocks of
it- en\ iroiimeut . in order to allure, or at any rate not to scare,
prey. And it appears probable that Slylaclis nnnoi enables it-
( oinpaiiioii Minoits inerniis in the same way to assume the same
< «-ii\eiiiein .nid successful disguise." \\'hile the e\ idence i-

|-ii.. i. A id fish (Hypsagonus quadricornis) bearing a hydroid ii>l<my

/','.. •::••:: . Natural size.

-Hour, that these devices do enable their possessor to escape
detection and \\ai;e more successfully it> battle for exi>ten. e.
and \\hile the hydroid may enable the fish in question to more
( lo-el\ harmoni/e with its surroundings it does not follow even
then that this is a case of commensalism. Nevertheless, a-
I li. k-on p..int- out,1 the fact that "the fish is never found without
t hi- hylroid, nor the hydroid without this species of tis.h. suggests
\.i\ -trough that there is a mutual advantage in the associa-
t imi."

In the iu'eseiu case the evidence is not so cogent. About one
fourth of the fishes only were overgrown with the hydroid and

'"( .uul.. Nat. Ili-t.." Vol. I., p. 268.

76 HAROLD 1 1 1. A I'll.

other specimens taken by the U. S. F. C. Str. "Albatross" in the
open ocean off the Washington coast and in Bering Sea, are
totally without them. These last named specimens, coming from
the same' depth (40 fm.) occurred on a pebbly bottom or one of
broken shell and it is possible that the Puget Sound individuals,
without the coelenterate, occurred in a similar habitat. Be that
as it may, it is a suggestive fact that in the fishes under con-
sideration the hydroid was "attached in large numbers about the
gill opening, on the throat and in the axilla," in other words over
the ventral surface that is already the most concealed portion of
the body. Referring to Hypsagonus quadricornis Prof. C. H.
Gilbert writes in Jordan and Evermann's "Fishes of North and
Middle America" (p. 204): "In the aquarium the fish appears to
walk, resting alternately on the upper and lower pectoral rays
and on the front rays of the anal." Under such circumstances
the eddies produced in the bottom ooze would naturally bring
the greatest amount of organic material to animals ventrally
situated. The appearance strongly suggests that the advantage
lies rather with the- hydroid just as it does with the several species
of barnacles attached to the skin of the whale. Whether the
association is any more intimate in the case Alcock cites it is
impossible to state conclusively, but the evidence is certainly not
entirely convincing.1

Prof. C. C. Nutting, to whom I have submitted specimens, has
kindly identified them as a species of Perigonimus, its nearest
relative being apparently P. vestitus Allman. As in other mem-
bers of the genus the hydrorhixa forms a highly branched, fre-
quently anastomosing, system over the surface of the fish, but
so far as noticed this contact is purely superficial, there being no
evidence of parasitism. And furthermore the presence of sin. ill
cntomostracans and nondescript organic remains in the gastric

'Since this paper was sent to press I have examined upwards of two d<
specimens of this same species of rock perch (M. inermis) collected by my
colleague Prof. J. O. Snyclcr, at Onomichi, on the Inland Sea, in the Province of
Hingo. Japan. All of these are excellently preserved and in no instance has a
hydroid been found upon them. It thus becomes more certain that the associa-
tion described by Allcock is not an undoubted case of commensalUin. I'i>''<
Snyder has called my attention to the fact that according to K> -.MM \iin. and
Mag. N'at. Hist., 1905, Vol. XV., p. 20) Minous inermis should be Alhiotn m mo-
dactyhis (Bloch and Schneider).

HIE ASSOCIATION OF A FISH WITH A HYDRO ID.

cavity <>f the hydranths shows the feeding processes to be those
nt' a mm-par, i-iiic species.

At frequent intervals branches, 3 to 4 mm. in height when fully
<|r\rl"pr<l. spring from this root system and each i> terminated
by a -hvje hydranth. In no case does a hydranth arise as a
lateral bud l'n>m the hydrocaulus, as in P. vestitus, for example.
( )n i he (»i In -r hand, the medusa buds almost invariably appear as
. verj rarely closely associated pairs of out^n-wth- <li--

2. Portion of hydroid colony (P. pugetensis).

IK.-. -.1 at comparatively regular intervals along the stem. Thnr
order <>! appearance is seemingly not so definite, though this, in
part at least, is perhaps due to the escape of an unknown number
of mediae fn.in the older stems. On the shorter, younger
branchr- one bud appears usually in the vicinity of the ba-e. or
tin- hydranth, and about the time its development is half way
compli-trd a >econd one arises in the middle section of the stem,

78 HAROLD HEATH.

while a third frequently makes its appearance in the vicinity of
the hydrorhiza about the time the first medusa is liberati-d.
Beyond this point the order of development is not known, Inn
Fig. 2 illustrates a few of several different stages. The mode of
development of the medusa is typical, and results in a bi-tentacu-
late type.

With the exception of the distal portion of each hydranth,
including the tentacles, the entire colony is ensheathed in a cuticle
often coated with minute organisms and sediment. In the older
portions this investment is comparatively firm with the exception
of that surrounding the hydranths, which is less dense and more
flexible. The medusa buds are likewise covered, and for a time
prior to their detachment are bound to the stem by an irregular
cuticular bridge.

In the younger hydranths the line of demarcation between
them and the stem is not clearly defined, but as they become older
the boundary is more distinct, the hydranth growing more
globular owing, to some extent at least, to the greater height of
the endoderm. In the younger stages each hydranth bears four
tentacles, later four others appear, often with slight irregularities
in the time intervals, and finally with the appearance of four more
the number is complete.

The following diagnosis will distinguish the present species
from other known forms: Perigonimus pugetensis new species,
twelve tentacles. Hydranths arising invariably from the hydro-
rhiza, and bearing as many as four scattered bi-tentaculate
medusa?. Cuticle relatively thin. Occurs on the agonoid ii>h,
Hypsagonus qiiadricornis, in Puget Sound, Washington.

THE CHROMOSOMES IX THE OOGEXESIS,

FERTILIZATION AND CLEAVAGE OF

COREID HEMIPTERA.

CHARLES V. MORRILL.

I. INTRODUCTION.

Tin- n-ci-iit i onclu-ions regarding sex-production based on the
IHK lr.tr dimorphi-m of the spermatozoa in the trarheate- have
invoked certain assumptions regarding oogene-i- and cleavage
which, though inaile in conformity with main- well-determined
facts, are -till in need of more adequate support from direct
observations. Tin- principal of these assumption- i- that in the
formation of tin- polar bodies, the diploid chromosome-groups
oi i IK- female an- reduced to haploid groups that are alike in all
the matin s. Further, since the spermato/oa are «>t two

sorts, the embryos produced would be correspondingly different
.nid tin- diltctvnce should be apparent from a study of the em-
br\onic inn lei. At present, however, the complete chnnno-ome-
le ha- been uorked out in only two groups of tracheates, the
ph\ Hi ixer.m- and aphids. In the phylloxeran-. Mor-.m 'o>, '< " i
ha- tiait-d the lull history of the chromosomes through several
genera tion- and the combined observations of Stevens ('050, 'o6a,
oil \dii Baehr '08, 'oi>) and Morgan ('09) practically completes
the . \( le iii the aphid-. The very recent observations of Boveri
and C.nlii k hough as yet published only in brief outline,

-how that in ll<:>rak\s, a nematode, the chromosome-cycle i-
-iniilar to that a--umed for many insects and bears the same
relation to -ex-production, while Baltzer ('08) has found that in
-ea-un hin-. the conditions are the reverse of those in insects,
the ei;i;- Uiii- dimorphic and the spermatozoa all of one kind.

The pre-eiit \\oik1 was undertaken with the hope of demonstrat-

I !n~ 1. 1. .1.1. in \\a- begun in the zoological laboratory of Columbia t "iiivi-i>ity
.in.l . ..inpli -ti -.1 in th.- anatomical laboratory of the College of Medicine, Syracuse
t "nivi T-ity. A part ni" the material was collected while occupying a John D. Jones
N l...lai-hi|> I.M, m at tin.- liinlivuical laboratory, Cold Spring Harbor. N. V., during
t In- Mimiiu-r ol' n;"7. an. I a \Vi>tar Institute room at the Mann. Biological Labor

SO CHARLES V. MOKKIII

ing the chromosome groups in the oogenesis, fertilization and
cleavage in certain coreid hemiptera and of determining in this
way, if possible, whether the assumptions' made in regard to tin-
number and behavior of the chromosomes in these stages is in
accordance with the facts.

Four species of the Coreidae were examined: Archinierus altcr-
tiatus Say, Anasa tristis De Geer, Protenor beljragei Hagl., Chelini-
dea vittigera Uhler.2 In all of these forms the spermatogonia
have been found to contain an odd number of chromosomes, one
of which (the unpaired idiochromosome3) fails to divide in one of
the maturation divisions. One half the spermatozoa thus con-
tain this chromosome, the other half lack it, and a dimorphism of
the spermatozoa arises. The oogonia have been shown to have
an even number of chromosomes, the unpaired element of the
spermatogonia being represented here by a pair of equal size.
The maturation of the egg had not been fully worked out, but
it was assumed that every chromosome divides equally in both
polar divisions, giving to the mature egg a group of chromosomes
similar to that borne by a spermatozoon having the idiochromo-
some. The eggs were accordingly assumed to be all of one kind
with respect to their chromatin content, as direct observation
has shown to be true in phylloxerans (Morgan), aphids (Stevens,
von Baehr) and more recently in Heterakis (Boveri and Gulick.)

atory. Woods Hole, Mass., in 1909. To the directors of these laboratories I am
indebted for the facilities placed at my disposal. I wish also to express my grati-
tude to Professors E. B. Wilson and T. H. Morgan for the many helpful suggestions
they have made during the progress of this work.

:I am indebted to Mr. E. P. Van Duzee, of Buffalo, N. Y., for the identification
of my material. The species Archimerus altcrnatns is almost, if not quite, identical
with the A. calcarator of Professor Wilson's material (identified by Mr. P. R. Uhler)
and was collected from the same locality in Van Cortlandt Park, NV\\ York. Pro-
tenor was found at Woods Hole, Mass., and Anasa at Woods Hole, and Cold spring.
Harbor, N. Y. Chelinidea was taken by Professor Wilson at Southern Pines, N. C.
A part of the living specimens of Anasa were also furnished by Professor \Yilson

3For the sake of simplicity, the term "idiochromosomes" will be used in this
paper to designate those chromosomes which are associated \\itli sex-production,
irrespective of their detailed behavior in the growth periods or maturation division*.
It will thus include the "idiochromosomes" (in the more restricted sen-n, "acces-
sory," "odd" or "hcterotropic" chromosomes, "monosomes," "heterochromosomes,"
"X- and Y-clements," etc., of recent writers. Professor Wilson ha- u^-.l this term
in a similar sense in the fourth of his "Studies" ('ogb).

CHROMOSOMES IN COREID HEMIPTERA. 8 I

It was fun her -npposed that if an egg is fertilized by a spermato-
zoon bearing the idiochromosome an embryo will result whose
nuclei all have an even number of chromosomes, similar to the
oogonial group-, but if fertilized by a spermatozoon lacking that
chromosome, the embryonic nuclei will have an odd number of
chromo-ome- -imilar to the spermatogonial groups. Accordingly
i In- former will be- females, the latter males. It should be pos-
-ible. then, to distinguish the sex of an embryo by an inspection
of the embi \oni, -omatic) chromosome groups. 'Again, it" the
number of < hroni.^omes in the male and female pronuclei could
be accurately determined just before the first cleavage spindle
i- loniied. it would afford additional evidence of the dimorphi-m
ot the -perni.ito/oa and the relation this condition bears to sex-
p reduction.

II. MAIKKIAL AND METHODS.

The insects were brought into the laboratory or gnvnhon-e
and pi. u id in rages in which their food plants were growing.
1 1' M- the\ p. i ire. I readily and laid their eggs either on the plant-
or on the -ide- or bottom of the cages. The breeding period- of
the tcnii gener.i employed differ widely. Anasa may be found
pairing mi -i|ii.i-h plants in the vicinity of New York or YVood-
llolr i.uK in July, the eggs being laid in clusters on the under
-in1 ! i lie leaves, but specimens kept in a greenhouse o\er

t lie \\ inti-r laid early in May. Chelinidea, brought from the south
and kept in nhouse during the winter, began to la\ ii-

• hi-ter- ol in the latter part of March. Archimerns begin-

la\ ing on i he ^"Idenrod in the vicinity of Ne\\ York in the latter
I MI! "i M.i\ or tir-t of June. The eggs are laid singly, and it
\\a- found impo--ible to collect sufficient numbers in the field
-o tli.u .ill thi'-e n-ed were taken from caged individuals. Pro-
tenor al-o l.i\ - ii- eggs one at a time and, in the laboratory, rarely
make- an\ attempt at fastening them to any object, but drops
them to tin1 bottom of the cage where they were collected in small
i|iiantitie-. In addition to these, a number of egg;- were taken
from the oviduct- of Anasa and Protenor.

The eggs "I" the lour species differ markedly in size, Archimerus
ha\ ing tin large-t and Protenor the smallest, those of Anasa and

82 CHARLES V. MOKKII.L.

Chelinidea being intermediate; these size differences correspond
roughly with the difference in size of the several species. All the
eggs, whether in the oviduct or after laying, are enclosed in a
tough brown chorion.

Several different fixing fluids were tried. Flemming's strong
fluid, Gilson's mercuro-nitric and Bouin's picroformol wnv found
very uncertain in result, as they seldom penetrate the thick
chorion. All these can be used, however, if the eggs are pricked
with fine needles before placing them in the fixing fluid, but their
action is such as to render the yolk very brittle and difficult to
cut. By far the best results were obtained by placing the eggs
immediately in the Gilson-Carnoy acetic-alcohol-chloroform-sub-
limate mixture for fifteen to thirty minutes or in a mixture of
glacial acetic, one part, absolute alcohol saturated with sub-
limate, two parts, for five to ten minutes. After either fluid,
the eggs w'ere transferred to iodized 95 per cent, alcohol for twelve
hours and preserved in 80 per cent, alcohol. The acetic-alcohol-
sublimate mixture was found invaluable for the earliest stages
of maturation which occur while the eggs are still in the lower
part of the oviduct and directly after laying. For later stages,
the Gilson-Carnoy mixture gave excellent results. After immer-
sion in alcohol, the egg shrinks away from the chorion which can
then be removed with fine forceps and cutting needle. After
removing the chorion, the eggs were dehydrated, cleared in ced.n-
oil and immersed in melted paraffin for two hours. They WITI-
then oriented in a drop of paraffin and embedded. Serial sec-
tions were cut 6-8 n thick on a sliding microtome. Very good
series can be obtained in this way though the yolk sometimes
becomes brittle and troublesome. The stain most frequently
employed was iron-haematoxylin with or without a counter-stain.
In addition to the eggs, ovaries and testes were fixed in Flem-
ming's strong fluid and stained in iron-haematoxylin or >al"ranin.

About twelve hundred egg- in all were sectioned, but owing
to mechanical difficulties in technique, only about two hundred
of these were of any value for study. In the maturation and
fertilization stages particularly, one or two poor sections may
render an entire series worthless, though in later embryonic stages
this difficulty is not so serious. For this reason the rr-ulis an-

CHROMOSOMES IN COREII) HEMIPTEKA. 83

nece-- arily -onieNvhat meagre, but they are perfectly clear as far
as they go.

III. DESCRIPTIVE.

A. Oo gene sis.

Tin- n -uli- on ••'i^i.-nesis are confined to the chromosomes of
tin- oo-i.nial and o.">cyte divisions. No attempt ha- been made
lot race tin- full history of the growth period, but an * •xamiiuit it >n
ct a leu eggs tak»-n from the ovarian tubes seems to show that
no definite < hioinat in-nucleolus or persistent oogonial chromatin
eleineiit i- pre-em . as stated by Wilson ('06). The nucleus at
tin- time contains main- faintly staining chromat in threads and
-e\t ral -mall nuclei di whose nature was not determined. Foot
ami "Mr.il.ell ia\e found the same condition to In- true in

l:nsthisti(^, a pi -iiiatomid.4

i. Archimcrus alternatiis.

The spermatogonial groups1 have been figured by \\iUon in
the -t i ..ml ..I his "Studies ' ('056), but for the sake of comparison
i\\o more are -hown (Fig. i, c and d). Each group has 15 < hn>-
mosomes, tu.> <>i uhich, the m-chromosomes (following \\il-on'-
tei niino|oL;\ . <an aluays be identified by their very small -i/e.
< )| the icniainiir^ ihirtei-n no one ran be positive!) ideiilitied, by

its size or shape, as the idiochromosome. In tlu- spermatocyte

di\i-ion- thi- t In > Milosome passes undivided t<> our pole ol the-
^pindle in the 'tr*t mitosis and divides equally in the -econd
\\il-..n, '05 I 'hi* condition is peculiar to Archimcrus alone

«.| all the (".'ivid.i- >o far described,6 but a reexamination of the
>prrmaiiic\ ie stages in new material shows beyond doubt that
\\il-.. n'- account i> correct. The idiochromosome can be easily
identified in the- first mitosis by its peripheral position on the
>pindle ami by it- inn t mstricted contour when the other chromo-
somes are in eail\, and even late, anaphase. It passes to one
pole o|" ihe -pindle a little behind the others. Further, in the

•St. \, i ! In t.n .chromosomes" in certain stages of the growth

I of the -»n homopteran.

. i.,,,ni(.ii- j. .it li..it. .in ..t page 80.

l'i,. i, 3801 \\il-t.n li.i- .iN.. i..iind a similar condition in Pachylis gigas (unpub-
lishi

CI1AKI.KS V. MOKRILL.

cysts of second spermatocytes, one finds metaphase groups with
eight and seven chromosomes side by side.

The oogonial groups7 have not been figured previously, but
Wilson gives the number of chromosomes as 16 in the fourth of
his "Studies" (096). In Fig. I, a and b, two plates arc shown

^K.

FIG. i. Archimerus alternatus. a and b, oogonial, c and d, s
metaphase-groups. e-j, first oocyte- (polar) division; e, daughter groups from the
same anaphase-spindle, polar view; /and g, anaphase, side view, in two sections;
h and i, inner daughter groups, polar view, of two anaphases; j, outer
group, polar view, of an anaphase.

each with 16 chromosomes. A pair of ^/-chromosomes
appears in each group. The fourteen larger element^ pir-cnt a
somewhat graded series and cannot be paired off readily. T\\<>

7The unreduced female groups in all the species, were taken from tin ir^iun In
the ovary lying just below the nutritive chamber, where i>".Lr'ini.i. inllii-Ir-i-rll- .i
young oOcytes are found together. For convenience they will !«• c.illeil "oii

' IIROMOSOMES IX COREID HEMIPTEKA. 85

of the largest are probably the idiochromosomes, though they do
not differ sufficiently from the others, in size or shape, to admit
of exact identification.

The anapha-e of the first mitosis was the earliest oocyte stage
obtained ;md was found in eggs just after laying. Fig. I, e
i from t\\o -ection>i, shows a polar view of this stage in which
ei-ht chromo-onir- can be accurately counted in both outer and
inner group-. Tin-re are seven large dyads and one very small
OIK- in c.ith. corresponding in relative size to the fourteen large
ami tuo -m. ill chromosomes of the oogonia. Fig. i, / and »,
-ho\\ .1 -idi \ it-\\ of a late anaphase from two sections. The inner
- 1, >up c oi n,i in- right dyads; one of them has divided prematnn 1\ ,
i IK- t\\o part- appearing in neighboring sections. The chromo-
somes in i IK- outer group are too crowded to be counted. PI. I.,
</, -ho\\- aiioilit-r anaphase in which the inner group is complete
in one ->' lion and contains eight dyads. Fig. I, /; and /, -ln>\v
i IK inin-r giMup- of two more anaphases, polar view. The chn>-
mo-omr- in both are all dyads, eight in each, the component
parts "I \\lii< li -how all degrees of separation. This premainre
di\i-ioii oi i In dyads is very common in the final anapha-i-- of
tin In -t c]i\i~i<>n and might lead to mistakes in counting it it
were noi that one finds all stages of division up to the complete
-eparation -houn in Fig. I, /and g. Menking found this condi-
tion in tin ' . j of Pyrrhocoris ('92, PI. III.) and it has also been
l"iiinl in the lust spermatocytes of Aphrophora an homopteran
(Stevens, 'o6ft and A mix a dragon-fly (Lefevre and McGill. '08).
It i- probable that even in cases of extreme separation, the halves
oi tin <l\ad- remained connected by fine strands of chromatin or
linin \\hieh beeoine invisible after long extraction of the stain.
The -ei oml di\i-ion is thus foreshadowed, in the anaphase of the
first, and e\en before as will be shown in Anasa. Since there is
no pei i. id beiueen the first and second divisions when the chromo-
some- lo-<- their in<li\idual contour, in tact no telophase in the
>ti it t sense, the d\ ad- pass practicalh" unchanged into the second
maturation -pindle.

The ehromo-omes which enter the first polar body retain their

contour and grouping for some time, forming a flat plate when

n in Mirfatv view. Fig. i, j, shows such a plate with seven

86 CHARLES V. M( iRK ILL.

large chromosomes all more or less constricted and a small nodule
close to the central one, which probably is the w-chromosome.
Fig. 2, b, shows another in somewhat oblique view with MAID
large dyads, and the w-chromosome dyad, the latter faintly
stained. Fig. I, /, and PI. I., ti, show side views of two more
polar body groups; in Fig. i,/, the w-chromosome dyad is dis-

**«'

t t *

A* '

"•'•'

• •

FIG. 2. Archimerus allcrnatus. Second oocyte- (polar) division, a. inctaphase-
Rroup, oblique view, b, chromosome-group of the first polar body from the s;uiu-
egg as the last, c, metapbase-group, polar view, d and c, metaphase groups, >idi-
view. / and g, daughter groups from the same anaphase-spindlc, oblique vi<-\\.
h and i, anaphase. oblique view, in two sections.

tinctly seen. All the chromosomes show the typical con^i i id ion
as though ready for a second division. Il<>\\r\rr no spindK- i-
formed and no division takes place except that in indixidual c hro-
mosomes the halves of a dyad may separate of their own accord,

CHROMOSOMES IN COREID HEMIPTERA. S/

ju-t a- in those of the inner groups. At the close of the second
division tin- chromosomes of the first polar body are finally
merged together in a deeply-staining mass.

The -e. Miid division tollows closely upon the fir>t a- Mated
above. The chromosomes do not crowd together in the final
ana]iha-e < >\ the fir-t division as in the spermatocytes, but merely
rotate about lorty-five degrees and become disposed upon a new
-|>indle which lorins out of the enveloping cytoplasm. Fig. 2, a,
-ho\\- .1 metaph.e-e of the second division in slightly oblique
pol.ir \ iew. The -even large dyads and w-chromosome dyad are
-harpl\ < Mil-nit ted and ready for division. The first polar body
\\ith ii- eight d\ads taken from the same section is shown in
2,i'. Fij , shows another polar view of a second division
metaphase, and I ig. 2, d and c, are side views of the same, each
-houing eight chromosomes. All the dyads are more or less
dia\\n Mm iii preparation for division, and in anaphase, separate
into i UM -i MI 1 1 1- df monads. Fig. 2, /and », show outer and inner
group- re-pecti\ely taken from the same anaphase; each ha-
M iiion.id-. I ig. 2. h and i, show an oblique view of an ana-
phase in t\\o -ertions (the surface of the egg is indicated by a
doticd lineal iheriijn of each section). The outer group which
passes into tin- -e« ond polar body is shown above, the seven
Luge monads in //, the w-chromosome monad in /. The inner
ip \\hii h remains in the egg, is shown below; six of the large
moii. id- and i he w-chromosome monad in /, the remaining large
monad in /: The -t ippled object in /; is part of one of the chromo-
-oine- in ;•. Tim- the end result is, that the female pronucleu-
i- lormed I nun eight single elements or monads, comparable to
tho-e borne b\ ,i spermatozoon containing the idiochromosome
\ id. \\'il-on. 'i i-

There are no peculiar or "lagging" chromosomes in either
di\ i-i< >n. Which . -t t he polar divisions is reducing, i. e., -eparates
\\hole chidmo-ome- which have paired in synapsis, could noi
be determiiu'd, -ince the first oocyte pro])hases were not ob-
tained.

While it i- not possible to identify the idiochromosomes in the
i\\o di\i-ion- ju-t di-cribed, the w-chromosome appears as a
constant element dividing in both. In the >permator> les \\il-

CHARLES V. MORRILL.

son's ('056) figures show it occupying tin1 centre of spindle in
both divisions. However, in the oorytes its position is variable
in the first division (Fig. I, e, //, and /; PI. I., a) but always
peripheral in the second (Fig. 2, a, c, d,f and g, h and i).

* e

s * *

•-M

.-A"

FIG. 3. /Inaso tristis. a and 6, oogonial, c and d. spermatogoni.il nntaphase-
groups. e-j, first oocyte- (polar) division: e. /, g and /;. mctaphase-K""ij>>. in>lar
view. / and j, daughter groups from the same au.i|>lM-< spindle, IM.I.H view.

CHROMOSOMES IN COREID HEMIPTERA. SQ

2. Anas a tristis.

The spermatogonial groups have been figured by Wilson ('056
and '061, and the number of chromosomes stated as 21. Wilson's
count ha- been corroborated by Montgomery ('06) and by Le-
fevre and M<< ,ill ('08) though Paulmier ('99) and Montgomery
in .in earlier paper ('01) had given the number as 22. Foot and
Sirol-ell 07-.' .UK! 076) have disputed the twenty-one count and,
on i In- li.i-i- D| photographs of smear preparations, have concluded
thai tin- iminb'-r is 22, confirming Paulmier's original account.
A r<< \aininatioM of fresh material from two different localities
ga\e j i a- il:' correct number of spermatogonial chromosomes
in a-niineiit \\ith accounts of Wilson, Montgomery, and of
l.i !( \i. and M< ('.ill. Imperfect plates it is true may show less
i ban ilii- inimU-r, but none were found with more. In Chelinidca
I'itti'j.i-ni, \\lnn- the spermatogonial groups are aln ost identical
\\itli tho-r o| Anasa, a single exception may be cited, in which
22 > In were foil nil. Apart from this, all clear plates

. • j i .

A- poinieii out l>\ Wilson and by Lefevre and Mc(iill, the
-pi mi il groups of Anasa show two m-chromosoraes and

three ! chromosomes one of which must be the unpain-d

chromosome heterotropic" or "accessory" chromosome) though
ii cannot be precisely identified by its size or shape. Fig. 3, c
and •:'. -li"u- tuo spermatogonial groups illustrating these points.
• •o-^oiiial groups (Fig. 3, a and b) contain 22 chromosomes,
iiu lining two m-chromosomes and four largest chromosomes.
(>t the la-i named, two must be the idiochromosome pair, cor-
"ii.lin- to du- unpaired idiochromosome of the spermato^oui.i,
as poinied out b> Wilson in the third of his "Studies" ('06).

The nieiapliase of the first oocyte division was found in eggs

taken troiii i he lower part of the oviduct. Fig. 3, e,f, g and //,

-liou- toni Mich phases, /; being a composite figure from two

There are ii chromosomes in each, all undoubtedly

- i he in size between the chromosomes as a whole of Fig. 3, < ami /.

.in, I tha . and It. is probably due to a difference in the length of fixation.

In tin- formi ie eggs were left for ten minutes in the fixative (alcohol-acetic-

-iil.liiii.il. in i la latt.-r. for five minutes. The chromosomes of Fig. 3, g and /;,

-h,.u tin- -u riling action of the acetic unchecked by the sublimate which

pen<

* \

- -

FIG. 4. Anasa Iristis. a-f, first o6cyte- (polar) division; a, four chromosomes
in initial anaphase, side view, showing tetrad-character; b, final anaphase, side
view; c and d, daughter groups from the same anaphase-spindle, slightly oblique
polar view; e and /, the same from another egg; g, metaphase-group. side view,
second oocyte- (polar) division, h, the chromosomes of the last drawn at three
different focal levels. *, the chromosome-group of the first polar body from the
same egRS

CHROMOSOMES IN COREID HEMIPTERA. 9!

double, arranged in an irregular ring with one in the centre and
one or two outside. Unlike the first spermatocytes, the central
chromosome i- not the w-chromosome bivalent but one of the
larger ones, while the w-chromosome itself lies just within the
riii'.. I ig. 3, -•, /and h) or somewhat outside (Fig. 3, g). In these
lour meiapha-e-., the axis of the spindle is somewhat oblique to
tin- plane of section and the structure of the chromosomes can
thus bt- readily obsemxl. As might be expected many have the
-hape o| t \pic.il tetrads foreshadowing the two oocyte divisions.
Thi- i- r-prrially well seen in Fig. 3, e, where even the w;.-chromo-
some has the quadripartite form. Whether distinctly quadri-
partite, or -imply dumb-bell shaped, the plane of the first division
le.irly indicated in every chromosome. In Fig. 3, e and //,
tin-re are t\\o bivalents (M and AT) perceptibly larger than any
ot the- other-. In all probability these arise from a pairing of
i he | < ii ic 1 chromosomes of the oogonia. Accordingly one

o| i In in m.i\ lie- considered to be the idiochromosome bivalent.

The anapha-e of the first polar spindle was found in <
diceiiK .liter laving. y The- chromosomes divide in the plane
ahradv indie ated at metaphase. Fig. 4, a, shows a side view of
louc i hioiiio-tniie- in initial anaphase; the tetrad character of
e.u h i- ( le.uK indicated. Fig. 3, i and j, shows polar views of
i he outer and inner groups respectively from the same spindle;
ea( h ha- i i chromosomes. All, with the exception of the ni-
( hromosome in j, are obviously dyads showing all degrees of con-
striction, as in the first polar ana phase of Archimerus. The outer
group \\hirh passes into the first polar body shows two dyads
completely di\ided though the polar body itself neither divides
nor form- a -pirn lie. lu both groups two largest dyads (M and
N) can be distinguished. Fig. 4, c and <7, are outer and inner
group- iv-pert i\ely from another spindle, in slightly oblique pol.ir
\ ie\\ in (- the large chromosome, AT, was found in the same section
as tin inner group, d). Both groups contain II dyads of which
t\\o i M and .Y> are larger than any of the others. Fig. 4, e and
Me outer and inner groups respectively of a third anaphase in

•A Millie i-Mi-1'ti..n to this was found. One egg taken from the lowest part of

tin- .'Yicliut >li(«\scil tin- first polar spindle in initial anaphase It is possible that
n.uuli liaiKllinv; -tan.',! the maturation process, as has been found in other groups
of aniin

92 CHARM'S V. MOKKII.L.

somewhat oblique polar view (the entire outer group, e, and six
chromosomes of the inner group,/, appeared in one section, the
remaining five of the latter group, in the next section). Both
groups contain n dyads of which several in / show premature
separation of their parts.

The peripheral position assumed by the w-chromosomes in
metaphase (Fig. 3, e, f and g) is again seen in all the anaphase
groups (Fig. 3, i and j; Fig. 4, c, d, e and/). This is undoubtedly
their normal position in the first polar division. A side view of
the final anaphase (Fig. 4, b) shows no peculiar or "lagging"
chromosome on the spindle. All the chromosomes divide equally,
as the polar views of three final anaphases show, where every
chromosome is distinctly visible.

A single example of the second polar division was found (Fig. 4
g). It is a side view of a metaphase with II dyads. In //, the
dyads of this group are drawn at three different focal level-.
The first polar body (i) of the same egg also contains eleven dyads.
In both the second polar spindle and the first polar body, there
are two dyads (M and N) somewhat larger than any of the others.
These are undoubtedly the products of division of the two largest
tetrads of the first polar metaphase (Fig. 3, e and //). The m-
chromosome in the second division (Fig. 4, g and //) takes a
peripheral position as it did in the first. It appears also in the
first polar body (i}. In short there are three chromosomes dis-
tinguishable by their size, which can be identified in both polar
divisions. These in all probability arise I nun a pairing of the
four largest and two smallest oogonial chromosomes.

Though no anaphases of the second division were found, it U
almost certain from the results in Archimerus thai all the d\-. id-
divide equally, in a plane corresponding to the constriction shown
at metaphase. Accordingly the female pronucleus, as well as
the second polar body, will contain 1 1 monads and correspond t<>
a spermatozoon bearing the idiochromosome as assumed by-
Wilson ('06).'

3. Pro tenor belfragei.

The spermatogonial groups have been described and figured
by Montgomery ('01 and '<>(>) and by Wilson ('06). Tlir-e ob-
servers agree that there are 13 chromosomes, one of which i-^

CHROMOSOMES IN COREID HEMIPTERA. 93

more than twice as large as the next in size. Of the remaining
twelve, two are much larger than the others, and the rest form a
graded series of pairs. The smallest pair, the m-chromosome, is
relatively larg«-r in this species than in Archimerus and Anasa.
In Hg. 5, c and d. are shown two spermatogonial groups illus-

>
.» *

^ '" h

Ifragfi. a and t. oogonial. f and d. spermatogonial nx-tu-

' vte-(polar) division; <•, metaphase group, polar vi>-\v;

/. ini-t.i; .up. nlilii|iii- polar view; g. six chromosomes in metaph;i-<-,

a metaphase, side view.

the .il»>\f mentioned points. The idiochromosome does
not .i|)|>t .11 • oii-tricied as figured by Montgomery ('oi), Fig. 134.

Ili« odgonial groups were figured by Wilson in the third of
hi- "Studies" ('06). In them "there are two very large chromo-
SOIIH-, rqii.il in -i/c, in place of the single one that appears in the
m. ill', \\hili- tlir 1 1 inaining chromosomes show the same relations
a- in tin- in.il In Fig. 5, a and b, two of these groups are shown.

< M thr m. it ur.it ion stages, only four preparation- were obtained,

94 CHARLES V. MORRILL.

all of the first polar metaphase (Fig. 5, e, f, g and //).10 Two of
these U> and /) are complete, showing se\vn bivalents. The idio-
chromosome bivalent can be positively identified by its relatively
enormous size, having been formed, no doubt, by the synapsis
of the two largest chromosomes (idiochromosomes) of the oogonia •
It does not in any way resemble a nucleolus. The bivalent next
in size, corresponding to the two next largest chromosomes of the
oogonia, can also be identified. Indeed the bivalents as a whole
show the same relative size differences as the chromosome pairs
in the oogonia. The w-chromosome bivalent is the smallest but
only slightly smaller than the next in size. In Fig. 5, /, three
chromosomes of intermediate size are seen in face view; two of
these exhibit a quadripartite form, clearly indicating their bivalent
nature. Fig. 5, g, is a side view of an incomplete metaphase,
drawn from two sections, showing six of the seven chromosomes,
and Fig. 5, h, shows a metaphase, side view with only five chromo-
somes, again taken from two sections. In both of these figures
the idiochromosome bivalent can be readily identified by its n/e
and the plane of the second division is clearly indicated in all the
bivalents. While the further processes of maturation were not
followed, it may be inferred, on the analogy of Archimeriis, that
in the anaphase of the first division the seven tetrads divide into
two groups of dyads and the inner group of these separate in
the second division into two groups of monads; the inner group
of these last named, seven in number, enter into the formation of
the female pronucleus, which is thus similar in chromatin-content
to a spermatozoon bearing the idiochromosome.

4. Conclusions Regarding Oo genesis.

The results on maturation are somewhat meagre it is true but
perfectly clear as far as they go, and point to the conclusion that,
unlike the spermatozoa, all (he mature eggs are of OIK- kind with
respect to their chromatin-content, as has been assumed. Tin-
female pronucleus contains a reduced group of chromosomes
similar in size and number to that carried by a "female-pro-

l°Thc excessive size of the chromosomes in these figures, especially those of g
and h. is probably due to the peculiar action of the fixative (see footnote 8, on
page 89).

CHROMOSOMES IN COREID HEMIPTERA. 95

ducing" spermatozoon, i. e., one bearing the idiochromosome
(directly proved in Archimerus, but only in part in Anasa and
Protcnnr). The idiochromosome bivalent is not distinguishable
l>y its behavior from other chromosomes, and divides in both
mitoses, giving an equal portion to each ootid. It never assumes
a run -h-ohi— like form either in the oogonial or oocyte divisions.

B. Some Details of Polar Body Formation.
The pi. ire of polar body formation has been found to vary in
different ^n mp- of insects. It may be on the dorsal surface,
either ,ippro\im.itely midway between the poles as in Blatta
i Blo< hmann, XYheeler) and Pyrrhocoris (Henking), or a short
di-iance behind the anterior end as in Musca (Blochmann, Hen-
kin. . In ( hrysomelidae (Hegner) it is ventral, while in Pieris

II. nl in. ii is close to the anterior (micropylar) end. In 7/v-
drnplnlns > Heider) and Aphis (Stevens) it is lateral. In A r chi-
me r us the polar bodies are given off on one of the flat surfaces of
the approximately midway between ^the poles. In this

spei iea the two surfaces cannot be distinguished after the chorion
ha- been renio\ed. but in Protenor the dorsal surface is markedly
convex \\hile the ventral is flat or slightly concave, and it was
hen- drier-mined by proper orientation that the place of polar body
forrn.il ion i- on the dorsal surface. The first polar spindle lies
in a -in. ill thickening of cytoplasm with its axis at right angles
to the egg Miitace (Fig. i,/ and g; Fig. 4, a and b; PI. I., a).
The .ui.iph.iM- of the first polar division occurs just after laying
(in Artliinti-rus a single exception was found; see footnote 9,
page 'H \'o centrosomes or asters could be demonstrated
b\ the method of fixation used just as Henking ('92) found in
Pvrrhth-oris though he used a different method. In late anaphase
.i cell-plate i- formed by swelling of the spindle fibers and the
MII t.ne of the egg dips down and around the outer group of
chronio-onies, until finally a little mass of cytoplasm containing
the 1. itter comes to lie free in a depression of the egg surface
(J-'ij.. i. < and g; Fig. 4, b\ PI. I., a). In both the first and the
second polar divisions (Fig. 4, b, and PI. I., b) the constriction
probably does not involve the cell-plate but passes between it
and the outer group of chromosomes as Henking observed in

Pyrrhocoris.

96 CHARLES V. MOKKILL.

In Anasa, a rounded cytoplasmic body was found, in two cases,
in or near the first polar spindle (Fig. 3, i; Fig. 4, c-d}. This is
perhaps comparable to the "eigenthiimliches Korperchen" which
Henking described in Pyrrhocoris. It may be a plasmosome, but
it is difficult to decide, since one frequently finds a number of
cytoplasmic bodies in the neighborhood of the first polar spindle
(PI. I., a) which cannot be distinguished from yolk granules and
which are inconstant in appearance and number.

At the conclusion of the first polar division the spindle gradu-
ally fades away; there is no persistent cylindrical mass of spindle
fibers or "thelyid" as Henking ('90 and '92) found in Picris, a
lepidopteran, and Agelastica, a coleopteran. The chromosomes
left in the egg, as stated before, remain separate and there is no
telophase in the strict sense. After a short resting period, they
rotate about 45° and become disposed on a new spindle which
has formed out of the cytoplasm surrounding them. The axis
of the second polar spindle lies very obliquely to the surface of
the egg (Fig. 2, h and i; PI. I., b). As in the first division there
are no centrosomes or asters. In late anaphase a cell-plate is
formed by swellings of the spindle fibers. The second polar
body is constricted off in the same manner as the first and lies
alongside of it in the same depression. The first does not divide.
The two bodies finally become embedded in the surface cytoplasm
and can be distinguished as late as the third or fourth cleavage.

At the close of the second polar division, the chromosomes left
in the egg become massed together and are converted into the
female pronucleus (PI. I., b). Those which have entered the
polar bodies may remain separate for some time but eventually
fuse into one or two deeply staining masses.

C. Fertilization.

The spermatozoa enter the egg through the micropyles which
form a conspicuous ring at the anterior end. Polyspermy is
undoubtedly normal, for accessory sperm nuclei were found in
the egg as late as the copulation stage shown in PI. II., h. As
many as three of these nuclei appeared in some cases. At the
time when the first polar spindle is in late anaphase, the -.perm
head enveloped in a mass of cytoplasm has moved some

CHROMOSOMES IN COREID HEMIPTEK A. 97

into the egg among the yolk spheres leaving a train of cytoplasm
behind it. It appears as a compact deeply staining rod sur-
rounded by a clear area and preceded by an aster (PI. I., d).
Tin- ( lear area is probably the "arrhenoid" mentioned by Henking
t'<;2) iii hi- account of Pyrrhocoris. The sperm head often ap-
pears <"il<<! at the end which points away from the direction
of it- mi .\ i -ment. Later it loses its staining power and opens
out into .111 oval vesicle (PI. II., a). The clear area and aster
are Inn- \\<11 marked though no centrosome is visible in the
l>rc|i.ir.iiion. Still later, the vesicle becomes considerably larger
ami -in. ill irregular masses of chromatin can be seen in its interior.
li i- tin n n.idy for copulation.

In th» mi Miitime, the egg nucleus, formed from the inner group
of i litMiii'i-omes of the second polar spindle, has begun to move
into tl: -urrounded by a small mass of cytoplasm (PI. I., c).

Tin i \ topla-m frequently contains one or more yolk sphere-.
Tin inn leu- is at t\r>( round in outline and the chromatin i-
di-ti ibutt •<! in -mall nodules lying chiefly against the iiinli.n
membrane. Subsequently it loses its capacity for staining, and
apt" >mewhat like the sperm head, but more rounded. It

i In 11 lii-ins to increase in size, becoming at the same time ir-
il.u in -hape and the chromatin once more appears in irregular
mass*

\- tin two pronucki approach each other their cytoplasmic
areas lu-e and they come to lie side by side with an amphiaster
bet \\ern I'l. II., h; the aster on the upper side of the nuclei
was iliaun from the next section). In contact with each pro-
nueleu-. may often be seen a large clear vesicle. These probably
repie-eni the structures mentioned by Henking as the "descend-
ant- "I tin arrhenoids," ;. e., derived from the clear area sur-
rounding tin' male pronucleus. In PI. II., b, one of the aster-
contains a minute centrosome. The entire amphiaster is prob-
abl\ lornu'd under the influence of the male pronucleus, for, in
the same egg from which PI. 1 1. .6, was taken, an accessory -perm
nui-kii- was found with a very small amphiaster lying in contact
with it. The further history of the clear vesicles could not be
foll.)\\rd as very few first cleavage figures were found; at a later
stagi of (,.[, ulation (PI. II., c) they did not appear. Henking

98 CHARLES V. MOKRILL.

described them in Pyrrhocoris as forming the poles of tin- first
cleavage spindle ("Polkorperchen") and apparently considered
them to be archoplasmic masses. That he did not see an aster
in front of the male pronucleus nor an amphiaster at copulation,
in addition to these structures, is perhaps due to his methods of
technique.

During the approach of the pronuclei the chromatin in each
becomes more and more condensed until the compact somewhat
elongated chromosomes appear. PI. II., c, the single example of
this stage found, shows the pronuclei of Archimerus, still slightly
separated. An indistinct aster appears at the right. In the
lower nucleus seven chromosomes of different sizes can be dis-
tinctly seen. The w-chromosome is missing and because of its
small size could not be identified in the next section. There
are no nucleoli in either pronucleus.11 The chromosomes in the
upper nucleus are not yet fully condensed. The two pronuclei
are so nearly equal in volume that one cannot distinguish which
is male and which female even before copulation (PI. II., c).
It is apparent from a comparison of PI. II., b and c, that both
undergo a marked decrease in volume just before their nuclear
membranes fade out. PI. II., d, shows a late copulation stage or
prophase of the first cleavage spindle of Protenor in polar view.
The nuclear membranes have faded out but the chromosome
groups derived from each pronucleus are still separate. This
figure is obviously incomplete but it shows distinctly one reduced
group (at the right of the figure) in which all the chromosomes
appear, seven in number. Just as in the first oocyte division, the
idiochromosome is here recognizable by its relatively large size,
and does not in any way resemble a nucleolus. A next largest
chromosome and an w-chromosome also can be identified, the
remaining four being intermediate in size. The group at the left
of the figure shows the idiochromosome and three others, the
remaining chromosomes being too crowded in the next section
to identify. Since each group contains an idiochromosome it is
not possible to say which was derived from the egg nucleus and
which from a sperm of the class which bear this chromosome.

"Stevens ('060, PI. IV., Fig. 119) has figured this stage in the "Goumi aphid"
•where there are five chromosomes in each pronucleus, and, in the female, two
plasmosomes in addition.

CHROMOSOMES IN COREID HEMIPTERA. 99

\ l< iwever, the embryo arising from this union would have been a
female, for all the products of the first cleavage nucleus would
contain two i<li< (chromosomes as in the oogonia. A chromosome
uroup taken from a female embryo is shown in Fig. 12, c. Even
these meagre results make it probable that the chromosomes
coming "in <>f the male and female pronuclei at copulation are
of the -a n it- n umber and show the same relative size differences as
tho-e which previously entered into the formation of the gametic
nuclei.

1 ). The Cleavage and Blastoderm Nuclei.

'l"h«- < le.i\at;e nuclei are formed by successive division of the
t'i-i -iili/ati«.n nucleus. After each division the daughter nuclei
move apart , each surrounded by a star-shaped cytoplasmic island.
The\ \\aml. -r toward the periphery, continually dividing by mito-
-.!-, and ih. -re form the blastoderm. Xo instances of amitosis
\\en- ..I.-. -r\t -d in these stages such as Wheeler ('89) described in
/•?/<///<;. Ah hough but few first cleavage divisions were found,
the ( In OIMOM >mes in them do not differ from those of somewhat
later -ta^es described beyond. The cleavage mitoses all sho\\-
>pindle-, ( < ntrosomes and asters with diagrammatic clearness
and the chromosomes, though somewhat elongated, can be
c'.imii-d as readily as in the oogonial or spermatogonial divisions.
In metaphase each chromosome appearson the spindle split length-
\\ i-e and in anaphase the halves separate as in ordinary homotypic
division. In telophase the chromosomes at either pole become
\e~icular. fuse together and form a daughter nucleus. At first
tin- t ontt -in- of the resting nucleus entirely lacks staining power,
n<> mi. l.-oli of any kind appearing. As the time for the subse-
quent di\i-ion approaches, small Hakes of chromatin appear
\\ hi. h increase in number and gradually unite to form thechromo-
somes. In the cleavage stages, no definite chromatin nucleoli
or pla-moNonies could be seen with the methods of fixation em-
l)lo\i-d, nor was there any elimination of chromatin during the
earlier mitoses as described by Boveri in Ascaris.

i. Archimems alter natus.

A careful Minly of the eggs after fertilization revealed the fact
thai there are two sorts of embryos, one having 15 chromosomes

IOO

CHARLES V. MORRILL.

in all its cleavage — and blastoderm — nuclei and the other 16.
These chromosome numbers are the same as those found in the
spermatogonia and oogonia respectively. The size-relations al>o

FIG. 6. Archimerus alternatus. Chromosome-groups of embryonic cells, 15-
chromosome type, a-e, from the same embryo; /-/, from other embryos.

are in general the same as in the gonads. It seems fair then to
conclude that the is-chromosonu- embryos are males, the 16-
chromosome, females. In Fig. 6 are shown twelve 15-chromo-

CHROMOSOMES IN COREID HEMIPTERA. IOI

groups '.iken from embryos at different stages. Fig. 6,
a, b, c. (I and e, are from the same embryo early in the formation
of tin- hi. 1-1 1 .derm. Fig. 6, f and g, are from another embryo in
tlii-~.i; ,e. Fig. 6, h and i, are from an embryo in a slightly

I.i t IT stage of the blastoderm. Fig. 6, j, is from still another at
tin- -.inn- -t.ige as the last, and k from a late blastoderm. Fig. 6,

1 i,. 7 • :«s [allfrnatiis. Chromosome-groups of embryonic cells, 16-

.iin t\po. rf. e. and /. from the same embryo; others from

/ i- taken from a stage'in which the blastoderm is invaginatin;^

to loiin the embryonic fundament and membranes. The 16-

chronioMniH- Croups are shown in Fig. 7. Fig. 7, a, shows a

:i|. t.iki-n from tin- interior of an egg in the cleavage stage.

7, /> .UK! / . .ire from an 'embryo in the early blastoderm stage.

IO2 CHARLES V. MORRILL.

Fig. 7, d, e and /, are from another embryo in the same stage as
the last. Fig. 7, g and //, arc from a slightly later blastoderm
and i is from a completed blastoderm.

An inspection of Figs. 6 and 7 as a whole, shows that in the
earlier stages, the chromosomes are somewhat elongate (Fig. 6,
a— g; Fig. 7, a-/) and that as development proceeds, they become
shorter and thicker until at the time of invagination or just before
(Fig. 6, k-l; Fig. 7, i) they have about the same contour as those
of the spermatogonia and oogonia (compare with Fig. I, a-d~).
In every stage the tw-chromosomes, though very minute, are
constant elements. The unpaired idiochrosome in the male
groups and the paired idiochromosomes in the female cannot be
distinguished by their size or contour but are probably repre-
sented among the larger chromosomes. The remaining chromo-
somes cannot be readily paired off.

2. Anasa tristis.

The embryonic mitoses of Anasa, though having a larger num-
ber of chromosomes that those of Archimerus, are much more
favorable for making chromosome counts, especially in the early
(incomplete) blastodern stage, i. e., at a time when many of
the cleavage nuclei have reached the surface and are still rapidly
dividing. The embryos are of twro classes: one having 21 and
the other 22 chromosomes. Since these numbers correspond to
those in the spermatogonia and oogonia respectively, it may be
concluded that the 21 -chromosome class are males, the 22-chro-
mosome class, females. Fig. 8, a-h, show eight metaphase groups
from the 2 1 -chromosome class. Fig. 8, a, b, c, d, e and /, are
taken from an embryo in the early blastoderm stage. In this
embryo ten more perfectly clear groups were found each with 21
chromosomes, making sixteen in all from the same embryo. Fig.
8, g and h, are from another embryo in the same stage. In Fig.
9 six groups of the 22-chromosome class are shown, all from the
same embryo in the early blastoderm stage.

One exceptional group was found in an embryo of the 22-
chromosome class (Fig. 8, i). This group contains 23 chromo-
somes, of which three arc larger than the rest. It is difficult to
suggest an explanation for this condition. It may be due- to an

CHROMOSOMES IN COREID HEMIPTERA.

1 03

accident of technique, the microtome^knife tearing/a chromosome
in half as it passed through the block, or it may be the result of
an abnormality in a previous division. There were no other

Kn.. s. I i;.j d tri.\tis. Chromosome-groups of embryonic cells, a-h, 21-
Miii-.'iiu' t\ p< ,1. b, c. d, e and/, from the same embryo; g and h, from anothrr
<iiil>i\.. i. i \..ptional group with twenty-three chromosomes.

in tin- immediate vicinity from which an extra chromo-
i ould have been derived. The chromosome groups repre-
iii Fii^. 8 and 9 were selected from a large number of

lO-j- CHARLES V. MORRILL.

very clear preparations. Many more could have been shown, but
it seemed needless to multiply the number of figures.12

A comparison of the male groups (Fig. 8, a-h) with the female
groups (Fig. 9) shows clearly that in the- former there are three
chromosomes larger than the rest, while in the latter there are
four such elements. These size relations are the same as those
in the spermatogonia and oogonia respectively (vid. page 89).

$ £

V. r«» n^f£/

*~P e *QsS

FIG. 9. Anasa tristis. Chromosome-groups of embryonic cells, 22-chromo-
some type, all from the same embryo.

Accordingly, one of the large chromosomes of the male groups
must be the unpaired idiochromosome and two of the large chro-
mosomes of the female groups, the paired idiochromosomes. The
m-chromosomes appear as constant elements in both groups and
are usually more, elongated than in the germ-cells, a feature which
is common to all I he chromosomes. Apart from the largest and
smallest elements, this elongated condition makes it impossible to
pair off the remaining chromosomes with any degree of certain i \ .

12A11 the figures were drawn with camera lucida, Zeiss apochromat. 2 nun.,
compens. oc. 12. With the exception of Plates I. and II., they were again rnl.r
with the camera and subsequently reduced in reproduction one-half, giving a final
magnification of 2,650 diameters. The magnification of Plates I. anil II.. is i.sv.s
diameters. Achromatic structures, except those of Plates I. and II.. havr Inn.
represented semi-schematically.

CHROMOSOMES IN COREID HEMIPTERA.

IO5

3. Chelinidea vittigera.

Tin- i-mlir\onic groups of this species are very similar to those
Ml" A>i<i 'i and equally favorable for making chromosome counts.
The embryos are again of two sorts, one having 21 chromosomes,
i lit- other 22. In fact the number and size-relations are so much
like tlio-t.- in Anasa that the two forms cannot be distinguished
;h( ir i hroniosome complexes alone.

Pic UniJeo vittigera. Chromosome-groups of embryonic cells. o-/z.

8IH hromosome type; 6. c, d. e and /.'from the same embryo; o. from an early
onal group with twenty-two chromosomes.

IO6

CHARLES V. MORRILL.

In Fig. 10, a-//, are shown eight 21 -chromosome groups. Fig.
10, a, is from an early cleavage stage corresponding approxi-
mately to the fourth cleavage of holoblastic eggs.13 Fig. 10, b, c,
d, e, and / are from an early blastoderm stage. Seven more
perfectly clear counts were made in this embryo, all giving 21
chromosomes, making twelve in all. Fig. 10, g and h, are from
another embryo in the same stage. Fig. 1 1 shows six of the 22-
chromosome groups. Fig. n, a, is taken from an early cleavage

FIG. ii. Chelinidea villigera. Chromosome-groups of embryonic cells, 22-
e, b, c, d and e, from the same embryo; /, from another embryo;

chromosome type

a, from an early cleavage

stage, approximately the fourth. Fig. u, b, c, d and e, are from
an early blastoderm stage in which eleven perfectly clear groups
were found all with 22 chromosomes. Fig. u,/, is from another
early blastoderm.

As in Anasa, it seems fair to conclude that the embryos with
21 chromosomes are males, those with 22 chromosomes, females,

"After the "second cleavage," the nuclei apparently do not divide quite syn-
chronously so that one may find at times an odd number of nuclei, some resting
and some in division.

CHROMOSOMES IN COREID HEMIPTERA. I O/

for these groups correspond in number and size-relations with
those of the spermatogonia and oogonia respectively. Like
.1 nasa again, the male groups contain three chromosomes which
are distinctly larger than the others (Fig. 10, a-f and h), one of
ilu-m probably representing the unpaired idiochromosome. In
the ft -in. ilc Croups four largest chromosomes can frequently be
di-tinv:iii-hed (Fig. II, b, c and/), though they cannot be identi-
fied in all the figures, probably on account of fore-shortening.
Two of the-e may be considered as the paired idiochromosome-.
in plan- of the unpaired element of the male groups. The m-
( -hromo-oiMi •- are typically paired elements of both male and
fcin. tli ^nuipx. All the chromosomes as in Anasa are more i-lon-

<1 tli.in in the gonads but still preserve the same size-relations.
Tin groups -hown in Figs, 10 and 11 were selected from a large
number of clear preparations. Fig. 10, /, is a group of 22 chromo-
somes taken from an embryo in which all other counts gave 21.
I or t hi-, tin- single exception of its kind observed, it is difficult
ic. give .1 -aiisfaclory explanation, though the same possibilities
Mii^r-trd in connection with the exception found in Anasa (vi<l.
page i"-' mil i also apply here. There were no neighboring

ip- In. m which the extra chromosome could have been de-
rived.

4. rrotcnor belfragei.

The mil T\ • >nic mitoses of this species are not quite so favorable
for making chromosome counts as those of the two preceding
form-, on a.voimt of the elongation of the chromosomes in the
rail\ ami even late cleavage stages. In the blastoderm stage
tin rliromo-omes arc more compact, but very few embryos were
obtained at this time so that the results are very meagre. Fig.
u -ho\\- t\\o [^-chromosome groups (a and b) taken from an
Ma-todcrm stage, and one 14-chromosome group (c) from
i-mbryo in the same stage.

In tlu- i .^-chromosome groups (Fig. 12, a and b) a very large
chrom.i-.onu-. unquestionably the unpaired idiochromosome,
Man.l> out cli-arly, being more than twice as large as any other.
A . .nd laixr-t pair can also be readily identified and a smallest
pair, tin- rn-chromosomes.

IO8 CHARLES V. MOKRII.L.

In the i-j-chromosome group (Fig. 12, c) there are two very
large chromosomes equal in size, in place of the unpaired element
of the first two groups. These arc no doubt the paired idiochro-
mosomes. There is also a next largest pair but the w-chromo-
somes cannot be identified with certainty.

A comparison of these embryonic mitoses (Fig. 12) with those
of the gonads (Fig. 5, a-d) shows that the 13-chromosome groups
are similar in the number and size-relations of their chromosomes

FIG. 12. Protenar belfragei. Chromosome-groups of embryonic cells, a and b,
13-chromosome type, from the same embryo, c, i4-chromosome type, from another
embryo.

to those of the spermatogonia, the 14-chromosome group, to
those of the oogonia. Though the results are too few to justify
broad conclusions, it is most probable that the embryo with 13
chromosomes is a male, the one with 14 chromosomes a female,
thus bringing Protenor in line with Archimerus, Anasa and
Chelinidea.

IV. SUMMARY AND CONCLUSION.

Among the results described in this paper,14 those of particular
interest are as follows:

1. In Archimerus, Anasa and Protenor there is an odd or un-
paired chromosome in the spermatogonia which in Protenor is
distinguishable by its size. The oogonia contain in addition to
this chromosome, a second chromosome of the same size. These
observations are in agreement with those of Wilson, Montgomery
and of Lefevre and McGill for the forms mentioned.

2. The chromosomes in the reduced female groups (polar or
oocyte divisions) show the same relative size differences as the
corresponding pairs in the oogonia (particularly well shown in
Protenor).

3. All the chromosomes divide in both polar divisions (proof

J<A preliminary note giving the most important results was published in Science
for December 31, 1909.

CHROMOSOMES IN COREID HEMII'TKKA. IOC)

j-ive in Archimerus, less complete in Anasa and Protenor).
Then- are n<> peculiar or "lagging" chromosomes in either of these
divisions.

4. Tin- female pronucleus contains a group of chromosomes
similar in that borne 'by a spermatozoon having the "accessory1
or i<lio< hromosome (directly proved in Archimerus).

•tilization the reduced groups from each pronucleus
an- -<-p. irately distinguishable and the chromosomes show the
-.inn -i/e relations as those of the spermatocyte and oocyte di\ i-

I here are no nucleoli in either pronucleus.

In tin- i leavage and early blastoderm nuclei of Archimerus,

:elini(lfd and Protcnor, the chromosomes are pertectly

ili-tim t ,m<l can IK- counted as readily as those in the gon.uls.

T\\o i\pes of embryos are found, one having an odd and the

niher .in even number of chromosomes, these numbers bein-

re-pet ii\ely the same as occur in the spermatogonia and oogoni.i.

A. • ordingly it >eems fair to conclude that the former arc male-,

the l.i 1 1 er females, and it thus becomes possible to distinguish

tin sex "I an embryo by counting its chromosomes.

7 Tin i< li< >rhromosomcs behave exactly like the other chromo-
v, .in.-, in i he oocyte divisions, at fertilization and in the cleavage
ami e.uK blasUxlerm stages. They never show any rcsemblan.v
t«> iuitle.,li ami in Protcnor they can be identified in all sta
\\ iih al.-« ilute certainty.

li \\ill be >een that the results in general bear out the assump-
tion- ina.le by Stevens, Wilson and others regarding the number
.1111 1 bi-h.i\ii«r of the chromosomes in the maturation of the female
and in the somatic cells. They give additional morphological
support to theories of sex-production based upon the presence or
absence «>f certain chromosomes and to the hypothesis of chronio-
some -intlix iduality or "genetic continuity of chromosomes" as
\\ il-on 'iwx) more cautiously calls it.

\'. RKVIEW AND DISCUSSION.

The literature on the maturation and early development of
the of in-ects and allied forms is very extensi\e. onering

a period of over fifty years, but it is beyond the scope of the
pre-eiit paper to review it in detail except in so far as it concerns

IIO CHARLES V. MORRILL.

the history of the chromatin in the early stages. Considered
from this standpoint, the results briefly are as follows:

Dipt era. — Apart from the earlier works of \Yeismann and
Blochmann in which the chromosomes were not especially con-
sidered, there are no observations except those of Henking ('88
and '93) on Musca vomitoria. In the first paper Henking figured
the cleavage spindles but did not determine the number of chro-
mosomes. Moreover, the results are difficult to interpret because
of the standpoint taken in regard to "free-nuclei-formation."
In the later paper Henking summarizes his previous results but
gives no new observations.

Lepidoptera. — Platner ('88) described briefly the maturation
and early cleavage of the parthenogenetic and fertilized eggs of
Liparis dispar but gave no figures and no account of the chromo-
somes. . Henking ('90) described and figured the maturation,
fertilization and early cleavage of Pieris brassiccz. He found
the haploid number of chromosomes to be 14 in both polar
spindles and in the female pronucleus, but did not accurately
determine the diploid number, though in a later paper he gives
the probable number as 28. The same author ('92) gave a brief
account of the maturation and early cleavage of Bombyx mori
and Leucoma salicis in which the haploid chromosome-group was
stated to be "at least 12," in both species. The diploid number
was not determined.

Neuroptera. — As far as I am aware, there are no observations
on the maturation and cleavage of mitoses of the eggs of this
group. Miss McGill's ('06) observations on An ax junius and
Plathemis lydia were confined to the nuclear changes during the
growth period of the oocytes.

Coleoptera. — Wheeler ('89) observed the formation of the first
polar spindle of Leptinotarsa (Doryphora) decemlineata but did
not determine the number of chromosomes. Henking ('92) in
Agelastica alni found the haploid number of chromosomes to be
about 12 in both polar spindles and the diploid number, 24-30 in
the cleavage spindles. He also observed the approximate num-
ber in Lampyris splendidiila, Adlmonia tanaccti, and Doinuin
(sericea L.?). In none of these, however, did he observe the
diploid groups in the cleavage stages. The- nb-i-rvutions of

CHROMOSOMES IN COREIU HEMIPTERA. I I I

r.iardina 'oi) on Dytiscus marginalis and likewise those of
I » laisieux '09) on the same species were confined to the growth
period <>t" the oocytes. Both authors describe a large chromatic
ma— in th.- nucleus of the oocyte, distinct from the chromosomes,
which appears to be eliminated just before the maturation divi-

i- Tin- latter are not described.
Orlhoplcra. -The observations of Blochmann ('87) and Wheeler

, on lilatta germanica were not very extensive from our point
"1 \ie\v. 'hiT author determined the number of chromosomes

in tin- maturation spindles. Wheeler, however, gives a good
h^tin nt a cleavage spindle showing 10 chromosomes. Guther/
i a brief paper, described a chromosome-nucleolus in the
! Pyrrhocoris but found no such body in the somatic
mi ' Gryllus domestic us. He therefore questioned the occur-

rence ..i "heterochromoeomes," maintaining that there were
prob.il.K 20 chromosomes in the somatic cells of the last nanu •<!
-pecie- and no "heterochromosomes." However in his later papers
'08 ami 'o<><2) he abandoned this view, describing typical "hetero-
chromosomes" in the spermatogonia and oogonia of Gryllus as
in oilier Orthoptera, and stating further, that the somatic cells
ha\e the same number of chromosomes as the oogonia and
spermatogonia respectively though he gave no observations in
Mipport i -I tin's last statement. The observations of von Baehr
n the parthenogenetic egg of the phasmid, Bacillus rossii,
though dt tailed in some respects, are not quite conclusive in

.nl in the number of chromosomes. The egg nucleus, just
before the lust polar division, contains 18-20 chromosomes many
of \\hich are tetrads. In anaphase the double nature of the
daughter halves often becomes apparent. Moreover, there is < me
lar-e tetrad in the first division which again appears in the second.
Tin number of chromosomes in the latter division was not deter-
mined. In a recent paper Buchner ('09) has described in Gryllus
«inif>t-stris an irregular nucleolus-like structure, the "accessory
|po.l\ ." \\hich persists through the growth period of the oocytes.
It i- ileri\i-i| from a similar body in the oogonia which he ap-
parentU considers identical with the "accessory" chromosome
ol other torin-. The maturation mitoses were not observed.
( .inher/ '09 working on a nearly related species, Gryllus domes-

112 CHARLES V. MOKRILL.

ticus, finds a similar body in the growth period of the oocytes.
After tracing its history, he concludes however that it is com-
parable to a nucleolus and is not to be confused with a "hetero-
chromosome." Buchner's identification of the "accessory body"
as a chromosome thus appears very doubtful.

Hymenoptera. — Blochmann's ('89) observations on the matura-
tion of the fertilized and parthenogenetic egg of the bee were con-
cerned chiefly with the number of polar bodies formed in the two
sorts of eggs. Though the polar spindles were figured the exact
number of chromosomes was not determined. Henking ('92)
found the haploid number of chromosomes to be 10 in the polar
spindles of Lasius niger and the diploid number in the cleavage
spindles, 20. In the unfertilized egg of Rhodites rosa he found 9
chromosomes in the polar spindles. In the cleavage nuclei the
number was 18-20, i. e., the number of chromosomes in the female
pronucleus had been doubled. Petrunkewitsch ('01) working on
the fertilized and parthenogenetic eggs of the bee, found that
the first polar division was equational, the number of chromo-
somes being 16. In the second division there occurred in both
sorts of eggs, a reduction of the chromosome-number to about
half, i. e., from 16 to 8. In the parthenogenetic (drone-) eggi
the female pronucleus contained at first 8, but later 16 chromo-
somes, the latter being produced by a doubling of the haploid
group, so that in the equatorial plate of the first cleavage spindle
the diploid number, 16, again appeared. In later cleavages there
was a progressive doubling of the chromosome-number, producing
multiple groups of 32 and 64. Silvestri ('06 and '08) has de-
scribed in detail the maturation, fertilization and cleavage of
several species of parasitic hymenoptera (Litomastix, Encyrtus,
Oophthora, Ageniaspis}. However, since his results do not in-
clude the determination of the exact number of chromosomes in
the early stages, it will be unnecessary to review them here.
Doncaster's ('07) results on Nematns ribesii (Tenthredinidae) are
quite anomalous and difficult to interpret. He finds that there
are two types of maturation in tin- female. In some egg^ there
is no reduction of chromosomes, the female pronucleus receiving
the diploid number, 8. In others typical reduction occurs, the
egg nucleus receiving in all probability the haploid number, 4.

( HKOMOSOMES IX COREID HEMIPTERA. 113

The former type of egg develops parthenogenetically, the latter
only being capable of fertilization, it is supposed. In some
somatic tissues, such as the ovary sheath, there are more than
the diploid number of chromosomes, as in the bee and in Ascaris.
In a later brief communication, however, Doncaster ('ioa) states
that hi- observations on the polar mitoses may require revision
ami th.it the behavior of the chromosomes in Nematus ribesii is

•lithe -ult to follow that it is doubtful if a satisfactory interpret a-
ti"n can 1" obtained in this species. In a very recent paper, the
same author Ciofr) describes in detail the maturation, fertilization
.Hi'] i-.irly eleavagc of Neuroterus lenticularis (Cynipidae). Here

in -ome of the results are quite novel. The mitoses of the
primiti\r «.\ a found in young female larvae of the summer gi -in T,I-
t ion (oni.iin about 2o chromosomes, like those of the somatic
cells. In the maturation of the summer eggs apparently two
di\i-ion- ...cur, the female pronucleus probably containing 10
< In i niic.- is. The eggs are fertilized and in the cleavage spin-
die- about 20 chromosomes appear. The results on the matura-
tion ,,i tlu- spring (parthenogenetic) egg are so anomalous that it

ms l.«--t to quote from Doncaster's own summary (loc. tit., p.
[O2 "The maturation of the spring egg has not yet been sutli-
i it ntl\ -tuilied, but it appears that some eggs undergo at le.i-t
OIK in. it mat ion division, others probably none. In eggs in which
in.itnrai ion has occurred segmentation mitoses show 10 chromo-
BOmes; all the eggs laid by one individual female in which tlie
c htoiiio-oincs could be counted were of this type, and it is sii^-

ted that the-e develop into males. In the eggs laid by other
1'emale-. however. 2O chromosomes appear in the segmentation
di\ i-ion-; in these, polar chromosomes appear to be absent, and
it i- prob.ible that there has been no maturation division, and that
the \\ould develop into females." It will be seen that no

definite conclusions can be drawn without further confirmatory
observations. Schleip's ('08) observations on the polar body
formation in Formica sanguined were confined chiefly to the
part hen.. ^onetic egg. He found in the latter the haploid number
of chromo-omes to be about 24 in the maturation spindles and
female proiiiu lens. This number also appears in the first clea-

e nmleii-. In the fertilized egg, the number of chromosomes

114 CHARLES V. MOKRILL.

in the male and female pronuclei was not determined with cer-
tainty but is probably 24 in each. On the whole, the chromo-
somes were small and the size-differences not well marked.

Hemiptera-homoptera. — The older papers of Weismann, Wit-
laczil, Blochmann and Will on the early embryology of aphids
contain no detailed account of the chromosomes. Stschelkanov-
zew ('04) in a brief paper on the maturation and early cleavage
of the summer (parthenogenetic) egg of Aphis roses, gave 14 as
the number of chromosomes in the maturation spindle (only one
polar body is formed). In one first cleavage spindle there were
only 1 1 chromosomes but he considered that three of these might
be double elements, thus giving the diploid number, 14, in both
maturation and cleavage. Miss Stevens ('050) and Hewitt
('06), 15 however, found the diploid number in the parthenogenetic
egg of Aphis roses, to be 10 and this count has been confirmed by
von Baehr ('09) in the same species. Miss Stevens also found
that the winter (fertilized) egg gave off two polar bodies in which
the haploid number, 5, was present. Three of these authors
observed marked size differences in the chromosomes of the ma-
turation and early cleavage stages, both Miss Stevens and von
Baehr finding four smallest chromosomes constantly. Miss
Stevens ('o6fr) in a very extensive paper described the maturation
and cleavage in a large number of aphids, with especial reference
to the number and behavior of the chromosomes. Without
giving a detailed review of her results it may be said in general
that the number and size differences of the chromosomes was
found to be constant for the species and that this constancy
applies to the diploid groups whether in the maturation spindle
of a parthenogenetic egg or in its cleavage spindles. In many
cases also the haploid group was found to exhibit the same rela-
tive size differences as the diploid group of the same species.
In the fertilized egg of the "Goumi aphid," the number of chromo-
somes in the male and female pronuclei just before copulation
was shown to be 5 in each, i. e., the haploid number. Mi^
Stevens concluded from her observations on aphids up to this
time that there were no "heterochromosomes" in this group, but

"Hewitt's results are known to me only through the brief mention made l>\ vmi
Baehr ('09, p. 285).

CHROMOSOMES IX COREID HEMIPTERA. 115

more recently ' '09) she has abandoned this view and has reached
conclusion-, in agreement with those of Morgan and von Baehr,
mtntioned U-luw. The results of Tannreuther ('07) on the
maturation and cleavage of several species of aphids differ in
many important respects from those of other workers on the
same group. They have been questioned by Morgan ('09) and
'on Bat-In '09) and will not be considered here. Morgan ('08
• ind '09 has traced the full history of the chromosomes through
.«m rations of phylloxerans. He observed that the par-
-. -iii-tic eggs of the second generation, i. e., those which
. r the sexual individuals, are of two sorts both as to their
and tin- number of chromosomes in the embryos which they
produce. The male embryos have actually two less chromo-
somea than the female though this difference is not always ap-
parent o\\ing to fusions occurring between certain chromosomes
i tin- "ai • •••--ones.") The- embryonic chromosome-groups of fe-
male indis idtials contain four "accessory" chromosomes. Those
<il tin- malt •-, however, have but two "accessories" since although
malt- an- produced parthenogenetically from females, two of
tin •-<• chromosomes are given off to the polar body in the matura-
tion < -I t In- male-producing egg. The most important conclusions
to IK iliaun In mi these results for our purposes are, that idio-
rl Mom. • - are present throughout the entire life-cycle- and
that it i- |>o--ili|e to diagnose the sex of an embryo by counting
it- t hi 01 no -mnes, though here it is true, sex is also associated
\\ith t! of tin- egg. Yon Baehr ('09) described the matura-

tion and ( lea\agc of the parthenogenetic eggs of several species
ot aphid-. Hi- results were in general similar to those of Miss
Ste\cii-, the maturation and cleavage mitoses being similar in
tin- number and size relations of the chromosomes. He did not
obsen <• an\ elimination of "accessory" chromosomes in the polar
di\ i-ii-n i-t" male eggs as in the phylloxerans. However, in one
maturation -pindle of Aphis saliceti (loc. cit., PI. XIV., Fig. 42)
hr ligute- 5 chromosomes, the remaining figures showing 6, and
in a male -omaiic cell i.1'1. XV., Fig. 94), as well as in the sperma-
to-<,nia. 5 chromosomes again appear. Moreover his results on
the -permato-eiie-is of Aphis salicdi as well as those ot Mi--
('09) on the spermatogenesis of other aphids would seem

Il6 CHARLES V. MORRILL.

to indicate that the behavior of the chromosomes in the female
line is probably similar to that in phylloxerans.

Hemiptera-heteroptera. — The only observations on the matura-
tion and cleavage of the egg in this group are those of Henking
('92) on Pyrrhocoris. He has given a very extensive and detailed
account of the chromosome history in this form and in a previous
paper ('91) described the spermatogenesis. He found that in the
diploid groups of the oogonia, there were 24 chromosomes. The
follicle and connective tissue cells, both larval and adult also
showed this number. In the haploid group of the first polar
spindle 12 dumbbell-shaped chromosomes appeared. In one such
group ('92, PI. III., Fig. 83) one chromosome is much larger than
the rest and is probably the idiochromosome pair (cf. Wilson's
('O9</) figures of the oogonial groups). The second polar spindle
showed again 12 dumbbell-shaped chromosomes. The number
of chromosomes in the male and female pronuclei was not ac-
curately determined but in Henking's Fig. 90 (PI. III.) one such
nucleus shows 12 chromosomes. The early cleavage spindles
were figured but of them the author says (loc. cit., pp. 29-30):
"The number (of chromosomes) cannot be accurately determined
on account of the smallness of the spindle and the close grouping
of the chromosomes ... it should be 24." He thus did not
distinguish two classes of embryos with reference to the chromo-
some number. This, no doubt, was partly due to the fact that
he had not observed any difference in the number of chromosomes
in the spermatogonia and oogonia and did not appreciate the
significance of the idiochromosome ("accessory" chromosome)
which he himself was the first to describe. Foot and Strobell
('09) have described the growth period of the oocytes of Euschis-
tus variolarius. In accordance with the earlier account of Wilson
('06), they find no chromatin nucleolus in the young oocytes or
germinal vesicles of this species but in the older oocytes and in
the germinal vesicles there is a relatively large achromatic nucleo-
lus. The maturation divisions were not described.

Arachnida. — Montgomery's ('07) results on Theridium are not
very extensive from the chromosome-standpoint. He found in
the second polar spindle 12 chromosomes and in a fourth cleavage
spindle 24 chromosomes. No idiochromosomes were <>li-rrved.

CHI i.MES IX COREID HEMIPTERA. I I/

I .\chxling the early cleavage groups mentioned above, a num-
ber '•) authors have described the chromosomes of older somatir
< • 11-. II. nking ('92) found the number of chromosomes in the
fnllii It- and connective tissue cells, of Pyrrhocoris to be 24.
I' . it^ch ('oi) observed that young blastoderm-cells of

tlif l'f tin multiple groups. Miss Stevens ('05$ and '066)

df-cribed the somatic groups of several species of Coleoptera
.nxl l< 'inxl i hat the small idiochromosome which occurs only in
tlif malt n! ihf-e forms could be readily identified. Yon Bachr
'1 that the male somatic groups of Aphis saliceli
•aiii 5 • hromosomes, one less than the female. Gutherz ('»••
liidfl Mi.it in Gryllus domesticus the somatic cells have the
-aim- iniiiil.fr of chromosomes as the oogonia and spermatogonia
though his obser\-ations on this point were not very
nxl no figures of somatic mitoses were given. Don-
•bserved the somatic groups in the male and female
I nil if gall-fly. He found that in the male, some somatic

mil. I-M - -li«.\\ thr diploid number oi chromosomes while others
lx>\\ the haploid number. In the female, all somatic
mill i -i - lia\c the diploid number. The very anomalous condi-
tion- di -. liU-d for the male do not at present rest upon dcmon-
Btrative < \xlence as the chromosomes were found to be small
.nxl diitii ult to count. In addition to the above-mentioned ob-
servations, most recent papers on the spermatogenesis of insects
(i 'iiiain ai i < 'iints of the oogonial groups in which idiochromosomes
can I'tii- n he identified.

1 i. mi iln luregoing brief view of the literature on the chromo-
M.iiif- in .... genesis and cleavage, it is evident that with the'excep-
t inn "I Mi— Elevens and Morgan none of the authors have traced
tin iilii.t Im-mosomes into the cleavage and later somatic mitoses,
and ix 'in but Miss Stevens, Morgan and von Baehr have sho\\n
that tin embryonic or larval somatic cells of male indi\xluaU
tliltt i limn those of females in the number or size of their chromo-
somes. Morgan h.is also shown that idiochromosomes are pres-
ent in tin- ]>. -lar spindle where, in his material, they behave in .1
characteristic manner. The results on the whole show, 1 think,
that idii -chromosomes ("heterochromosomes") are constant chro-
nx-omc-flcmeiits and not merely temporary structures (nucleoli)
(luring maturation.

Il8 CHARLES V. MORR1LL.

Outside of the air-breathing arthropods, there are, as mentioned
before, two other groups in which idiochromosomes or similar
structures have been found in maturation and cleavage. Baltzer
('08) has found that in two species of sea-urchins there is a par-
ticular hook-shaped chromosome which occurs in only a part of
the mature eggs. The eggs are thus of two types with respect
to this element. (It is replaced by a chromosome of the ordinary
sort in the eggs which lack it.) The sperm nuclei on the contrary
are all alike. It is not improbable, Baltzer concludes, that the
determination of sex depends upon this dissimilarity of egg nuclei,
and therefore lies with the female (i. e., with the egg), as in the
male and female (parthenogenetic) eggs of aphids and phyllox-
erans. The peculiar hook-shaped elements might thus be called
"idiochromosomes." Eggs which contain this element would
develop into females, those without, into males. In a very
recent paper Boveri and Gulick ('09) have described briefly the
chromosome-cycle in Heterakis, a nematode. Its cycle corres-
ponds exactly with that of Protenor as given by Wilson ('06).
The diploid number in the male (spermatogonia) is 9. During
spermatogenesis the odd chromosome goes undivided to one pole
of the spindle in the first spermatocyte division but divides in
the second. The spermatozoa are thus of two classes, with 5
and 4 chromosomes respectively. The diploid number in the
female was not determined with certainty but the haploid number
in the germinal vesicles and polar spindles was found to be 5.
The eggs are thus all alike and, it is assumed, will develop into
males or females according as they arc fertilized by 4-chromosome
or 5-chromosome spermatozoa. The chromosomes of the cleav-
age nuclei were not described.

Since the results here described for coreid Hemiptera do not
give any further insight into the fundamental question of sex-
determination but only render the data more complete, it seems
needless to add a lengthy discussion on this point. In the recent
papers of Wilson, Bateson Castle, Boveri and Morgan, the cyto-
logical evidence relating to sex-determination has been thoroughly
analyzed. It may be pointed out, however, that apart from theo-
retical considerations this evidence has been questioned from tin-
standpoint of fact by several workers who have supported their

CHROMOSOMES IX COREID HEMIPTERA. 119

contentions l>y direct observations on insect spermatogenesis.
Arnold '08) from his observations on the spermatogenesis of
Ilydrnphilns piceiis concluded that there were no idiochromosomes
in that torin, although Miss Stevens has found these elements in
all tin Colcoptcra which she has examined (42 species). The
ol.jc. -lion "Mi-red by Foot and Strobell ('07) to the presence of an
odd mimlu-r of chromosomes in the spermatogonia of Anasa
Iritis, hence of an "accessory" chromosome and the replies to
tin- objr< ti« .n have already been considered. The present work,
particularly the section dealing with the cleavage and early bias-
toil, -mi miclri, gives further proof that in this species, as well as
in the other three examined, the number of somatic chromosomes
in the male is one less than in the female. Gross, from hi>
studic- on .^yromastes mar^inatus ('04) and Pyrrhocoris aptcrns
'<"• . ' oni hided that the "accessory" chromosome could have no
ell. it on sex-production in these two forms, for he believed that
the number of chromosomes is the same in both sexes — 22 in
S\> \ and 24 in Pyrrhocoris. In the last named form his

count- with the earlier ones of Henking ('91), though the

lattet \\.i- uncertain of the spermatogonial number. Recently
ho\\<\ci \\ ilson ('096, 'oox/) has reexamined both these forms
and linds that in Pyrrhocoris, the male has one less chromosome
than the li male, /. <-., 23 instead of 24, while in Sryomastes, the
male ha- 22 as described by Gross but the female has 24 instead
oi _v I'yrrhocoris may thus be placed in the same class with
Mich tomis as Archinterns, A nasa and Protenor. Syromastes, how-
-. !- unique among the Hemiptera hcteroptcra in having a
l.i\alent "accessory," though a similar condition has been de-
l.\ Morgan ('09) in the homopteran, Phylloxera caryce-
is, \\hile Payne ('09) describes several cases among the
K.duxiid.c /•';/<///(/. Rocconota, Conorhinns) in which the large
idiot htomo-ome (which represents the "accessory") is double.

In addition to the objections cited above, a number of authors
ha\c cither expressed their doubts of the presence of two sorts
ot -pci inato/oa. or, while admitting the existence of such a
ilimoi phi-m, ha\c questioned its sexual significance. In recent
years, however, «-\idence has been steadily accumulating in sup-
port ot the conclusion that a nuclear dimorphism of the sperm—

I2O CHARLES V. MORRILL.

or of the eggs — does occur, not only in insects hut in at least two
other groups of animals and that it bears a definite relation to
sex-production. Whether the sexual tendencies are carried by
specific chromosomes or whether certain combinations of chromo-
somes cause one sex or the other to arise, is still an open question,
but that it is possible to demonstrate a nuclear difference in the
gametes, and frequently indeed, in the somatic tissues of the
two sexes, is now, I think, placed beyond doubt by many decisive
observations.

April 27, 1910.

LITERATURE.
Arnold, G.

'08 The Nucleolus and Microchromosomes in the Spermatogenesis of Hydro-

philus piceus (Linn.)- Arch. f. Zellf., II., i.
Baehr. W. B. von

'07 Ueber die Zahl der Richtungskorper in parthenogenetisch sich entwickelnden

Eiern von Bacillus rossii. Zool. Jahrb., Anat. u. Ontog., XXIV.
'08 Ueber die Bildung der Sexualzellen bei Aphididae. Zool. Anz.. 33.
'09 Die Oogenese bei einigen viviparen Aphicliden und die Spermatogenese

von Aphis saliceti, u. s. w. Arch. f. Zellf., III., 2.
Baltzer. F.

'09 Die chromosomen von Strongylocentrotus lividus und Echinus microtuber-

culatus. Arch. f. Zellf., II.
Blochmann

'87 Ueber die Richtungskorper bei Insekteneiern. Morph. Jahrb., XII.
Boveri, Th. (and Gulick. A.)

'09 t'eber "Gcschlechtschromosomen" bei Nematoden. Arch. f. Zellf., IV., i.
Buchner. P.

'09 Das accessorische Chromosom in Spermatogenese und Ovogenese der Or-

thopteren, u. s. w., Arch. f. Zellf., III., 3.
Debaisieux, P.

'09 Les debuts de 1'ovogenese dans le Dytiscus marginalis. La Cellule, Tome

XXV.
Doncaster, L.

'07 Gametogenesis and Fertilization in Nematus ribesii. Quart. Jour. Micr.

Science, LI.
'10a Gametogenesis of the Sawfly, Nematus ribesii. A correction. Science,

N. S., XXXI., February.
'106 Gametogenesis of the Gall-fly, Neuroterus lenticularis (Spathegaster

baccarum). — Part. I. Proc. Roy. Soc., B, Vol. 82.
Foot, K., and Strobell, E. C.

'07<i The "Accessory Chromosome" of Anasa tristis. Biol. Bull., XII.

'076 A study of the Chromosomes in the Spermatogenesis of Anasa tristis.

Am. Jour. Anat., VII.. 2.

'09 The Nucleoli in the Spermatocytes and Germinal Vesicles of Euschistus
variolarius. Biol. Bull., XVI., 5.

CHROMOSOMES IN COREID HEMIPTERA. 121

Giardina. A.

'01 < irigine dell'oocite e delle cellule nutrici nel Dytiscus. Internal. Monatschr.

f. Anat. u. Phys., XVIII.. 10-12.
Gross. J

'04 \> latogenese von Syromastes marginatus. Zool. Jahrb., Anat. u.

. XX.

'06 1 1 .atogenese von Pyrrhocoris apterus. Ibid., XXIII.

Gutherz. S.

'07 Zur K'-nntnis der Heterochromosomen. Arch. f. micr. Anat.. LXIX.

'08 I r Beziehung zwischen Chromosomenzahl und Geschlecht. Zentralbl.

i. Phys., XXII.. Nr. 2.

'09. i V zur Geschichte des Heterochromosomes von Gryllus domesticus

I. Sitz. der Gesell. Naturfors. Freunde. No. 7., Jahrgang 1909.

Annahme eincr Beziehung zwischen Heterochromosomen und
• >bestimmung durch das Studium der Gryllus-Oogenese wider-
-itz. der Gesell. Naturfors. Freunde. Xo. 9. Jahrgang 1909.
Henking H

'88 :i Entwicklungsvorgange im Fliegenei und freie Kernbildung.

wiss. Zool.. XLVI.
'QO I 'in- tsuchungen ilber die ersten Entwicklungsvorgange in den Eiern der

kten. I. Ibid.. XL1X.
'91 Uebei Spermatogenese uncl deren Beziehung zur Eientwicklung bei Pyr-

aptvrus. Ibid.. LI
'92 i !;•• : ~uchunKi-n Ulxr die ersten Entwicklungsvorgange in den Eiern der

u.. III. Ibid.. LIV.. i.
Hewitt. C. G.

'06 "I In- < yt.iloKU-al Aspect of Parthenogenesis. Mem. Proc. Manchester Lit.

•c.. Vol. 50.
Lefcvt. Q and McGill C.

'08 Tin- Chromosomes of Anasa tristis and Anax junius. Am. Jour. Anat.,

VIII.. 4.
McGill. Caroline

06 I vior of the Nucleoli during the Oogenesis of the Dragonfly with

: Reference to Synapsis. Zool. Jahrb., Anat. u. Ontog.. XXIII., 2.
Montgomery, T. H.

'01 A ^tiiily of the Chromosomes of the Germ-cells of Metazoa. Trans. Am.

Plnl. Soc., XX.
'06 i !.i inostimes in the Spermatogenesis of the Hemiptera Heteroptera.

I i. in-*. Am. Phil. Soc.. XXI.. p. 97-

'07 M -i and Fertilization in Theridium. Zool. Jahrb., XXV., 2.

MorRan. T. H.

'08 Mi.- IV. iuction of Two Kinds of Spermatozoa in Phylloxerans. Functional
\.il.--Producing" and Rudimentary Spermatozoa. Proc. Soc. Exp.
1. md Med.. V.. 1908.
'09 A al and Cytological Study of Sex Determination in Phylloxerans

;ui'l .\i>lii'ls. Jour. Exp. Zool.. \"II.. 2.
Morrill. C. V.

'09 Pn-liminary Note on the Chromosomes in the Oogenesis, Fertilization and
Cleavage oi Certain Hemiptera. Science, N. S., XXX., December.

122 CHARLES V. MORRILL.

Paulmier. F. C.

'99 The Spermatogenesis of Anasa tristis. Jour. Morph. Supplement to

Vol. XV.
Payne. F.

'09 Some New Types of Chromosome Distribution and their Relation to Sex.

Biol. Bull., XVI., 3 and 4.
Petrunkewitsch, A.

'01 Die Richtungskorper und ihr Schicksal im befruchteten und unbefruchteten

Bienenei. Zool. Jahrb., Anat. u. Ontog., XIV.
Platner. G.

'88 Die erste Entwicklung befruchteter und parthenogenetischer Eier von

Liparis dispar. Biol. Centralbl. VIII.
Schleip. W.

'08 Die Richtingskorperbildung im Ei von Formica sanguinea. Zool. Jahrb.,

Anat. u. Ontog., XXVI.
Silvestri, F.

'06 Contribuzioni alia conoscenza biologica degli Imenotteri Parassiti, I. Bio-
logica del Litomastix truncatellus (Dalm.). Annali della R. Scuola Sup.
d'Argic. di Portici, Vol. VI.
'08 Contribuzioni, etc., II.-IV. Boll, del Lab. di zool. gen. e agrar. della R.

Scuola Sup. d'Agric. di Portici, Vol. III.
Stevens. N. M.

'05a A Study of the Germ-cells of Aphis rosae and Aphis CEnotherse. Jour.

Exp. Zool., II., 3.
'05ft Studies in Spermatogenesis. Publication No. 36, Carnegie Institution of

Washington.

'06u Studies on the Germ-cells of Aphids. Ibid., No. 51.
'Q6>> Studies in Spermatogenesis, II. Ibid., No. 36, part II.
'09 An Unpaired Heterochromosome in the Aphids. Jour. Exp. Zool., VI.
Stschelkanovzew, J. P.

'04 Ueber die Eireifung bei viviparen Aphiden. Biol. Centralbl., XXIV.
Tannreuther, C. W.

'07 History of the Germ Cells and Early Embryology of Certain Aphids. Zool.

Jahrb., Anat. u. Ontog., XXIV.
Wheeler. W. M.

'89 The Embryology of Blatta gemanica and Doryphora decemlineata. Jour.

Morph., III.
Wilson, E. B.

'05d Studies on Chromosomes, I. Jour. Exp. Zool., II., 3.

'05b Studies on Chromosomes, II. Ibid., II., 4.

'06 Studies on Chromosomes, III. Ibid., III., i.

'07 The case of Anasa tristis. Science, N. S., XXV., February.

'09a Recent Researches on the Determination and Heredity of Sex. Science,

N. S., XXIX., January.

'09/< Studies on Chromosomes, IV. Jour. Exp. Zool., VI., i.
'09c Studies on Chromosomes, V. Ibid., VI., 2.

'09d The Female Chromosome Groups in Syromastes and Pyrrhocoris. Biol.
Bull., XVI., 4.

124 CHARMS V. MORRILL.

EXPLANATION OF PLATE I.

Archimerus alternatus. a, formation of the first polar body — the mitotic figure
is in final anaphase, the inner daughter group showing eight chromosomes; the
cytoplasm contains a number of yolk spheres of different sizes and staining capacity.

b, formation of the second polar body — the inner group of chromosomes have been
transformed into the female pronucleus; those of the outer group are still separate.

c, the female pronucleus advancing into the egg in its cytoplasmic "island"; tin-
latter also contains two yolk spheres, d, the sperm-head advancing into the egg,
in its cytoplasmic "island"; it is preceded by an aster and surrounded by a clear
area. The magnification is 1,375 diameters.

BIOLOGICAL BULLETIN, VOL. XIX.

PLATE I.

CHARLES V. MORHILL.

126 CHARLES V. MORRILL.

EXPLANATION OF PLATE II.

a-c, Archimerus allernatus. a, a stage letter than PI. I., d — -the sperm head has
been transformed into the male pronucleus and is still advancing into the egg pre-
ceded by its aster and a small clear area, b, an early stage in the copulation of
the male and female pronuclei — each is in contact with a clear area and an amphi-
aster lies between them, c, a later stage in the copulation of the pronuclei — the
nuclear membranes are still intact; seven chromosomes appear in the lower nucleus,
the wz-chromosome being absent; a faint aster appears on the right, d, Protenor
belfragel: First cleavage prophase, polar view — the male and female groups are
still separate, one of them being incomplete; an idiochromosome appears in each
group. The magnification is 1,375 diameters.

BIOLOGICAL 6ULLFTIN, VO .

PLATE II.

* •

CMAHlES V MO«B II.

rOCRTSHIl' IX DVSDERA CROCATA.

ALEXANDER PETRUNKEVITCH. PH.D.

:nale and a male of this species of spiders were found under
' <int flat stones on a sunny hillside in Montclair on the

• uing of April 21. On the same day they were put in a ivc-
t. uvular small glass dish with a little earth at the bottom, awn-o-
lid and a partition of wire-net in the middle to separate the
spider-* from each other. They at once tried to get at each otl
>-tii king their legs through the net. After a while they de-i-ted
and -at quietly on the ground. The next morning the female
und in the corner farthest from the light and next to the
partition, in a little excavation in the ground, which \\a-
pi«iii-i ted on all side* with web. The web was destroyed and a
ll\ ua- held in a forceps before the spider. She bit at it but \\a-
• •it In i in: able or did not want to kill it. The male made no ex-
iti«m or web. When the partition was removed the male ami
all- met several times in a rather threatening way, opening
\\ide thi-ir mandibles and at times seizing each other by tin
inamlibli--. but the male would pat the female with his four
ti"iit leet on the sides and back and then they would separate
l>ly. After a short time the female dug rapidly a hole in
the Around under a little stone. The male approached several
time-. 1 1 inching her legs with his front legs and again going away.
I in >u removed the stone to observe how the female digs the hole,
^lie at once beg. in to work, using all parts of her body. The
Miiall • lumps of soil she removes by pushing them with palpi.
In >MI to i and mandibles with the fangs drawn in. Heavy clump-
"t the size of her own Ixxly she grasp* in her fangs and either
pu-lie- or pulls backward out of the excavation. The hole i-
f(.riiiied by web which she spins in a very interesting manner.
She -tand* head down with only the abdomen out of the hole ami
nio\e- the latter in a half circle about the petiolus with spinncrei-
i.m-iretched, I'a-tening the thread first at one, then at the otlu-r
end <>t the half circle. After doing this several time-, -he turns
in th- hole to spin the other half circle in the same manner.

'=7

128 ALEXAN'DER PETKUNKEV1TCH.

Meanwhile the male came quite close and was evidently watching
her, at the same time cleaning his legs and palpi by drawing them
through his mandibles. After a while he again began his courtship ;
patted her back, standing face to face and tried to bring his
front legs under her body. Later she left the hole without finish-
ing it, returning to it from time to time. They met several
times on the ground, always with mandibles wide open and
touching each other with the four front legs. At times the female
would make a threatening move toward the male upon which
he would dra\v back, but invariably a rapid trembling would
seize his four front legs, which lasted from 10-15 seconds. At
times when she was out of the hole as though in search of some-
thing, he would occuply her place in it. On returning, she gently
displaced him. Later as the female showed no intention of ac-
cepting the male, I separated them by means of the wire screen.

The next day was cold and rainy and both spiders were found
in the morning in separate excavations covered up with earth
and web. On the third day after a stormy night it became wrarm.
and the glass was placed in the sunshine. The screen w-as re-
moved and the spiders pushed out of their holes. The male at
once went to the female, meeting her face to face. He crept
under her sternum and took her firmly by the petiolus between
his mandibles with fangs drawn in. With his legs he embraced
her in such a way that his right front leg passed between the
mandibles (with fangs open) of the female and rested on her back.
His left front leg passed between her right mandible and palpus
and also rested on her back. The other legs passed similarly
between the legs of the female and touched her back. She seemed
to make no objection whatever. They tried several times to
change their position, the female creeping from place to place
while the male continued to cling to her. Finally she lay down
on her side and he applied the palpus (at 10.43 A. M.), all the
while patting her with his third leg. The coitus lasted five
minutes, after which they slowly separated and going to opposite
sides of the dish hid themselves in the ground. During the
whole time the back of the male was in contact with the sternum
of the female, a position not common to the majority of spiders.

In a recent paper on the courtship of araneids Professor Mont-

COURTSHIP IN DYSDERA CROCATA. 1 2Q

gomery objects to my conclusion that the sense of sight is the
only sense th.it guides hunting spiders in finding the females
during tin- mating period. He says that he has "frequently
noticed m. tics of even diurnal attids and lycosids first recognizing
tin- iViii.ile by touch." It seems to me however very doubtful
th.it a male should approach a female without having previously
noticed her or without having been noticed by her, so to say
imau an-- on the part of both. The fact that the female may stay
<|tii»-t U no proof that she has not noticed him. From my obser-
\ation- .md experiments on hunting spiders I am convinced that
they n-adily see moving objects. If frightened they run away:
i| cons* i"ii- of their advantage and hungry or if cornered, they
ai tack. \\ hat Montgomery looks upon as recognition by touch
ma\ l.e merely an attempt on the part of the male to find out
\\heiher the female is inclined to accept him. I do not deny
that -IK h a chance meeting is possible, but from what we know
o| the habits of spiders, I should expect the female to be either
-tartled or resentful on being touched unawares. In the case of
rocala, which may in a certain sense be regarded as a
hunting -pider, the male shows every indication of perceiving
mo\ inv; objects and of recognizing the female by sight. Several
times h, distinctly watched the end of a hatpin with which I was
bi. -.iking up the hard clumps of earth in a jar. Invariably on
m\ . ea-ing to do this, he approached the spot and scratched at it
\\ iih \\\- front leg*. A- I have already stated he also approach. -d
ih« t, male whenever she began to dig. Reversing the conclusion
: -mery that touch occupies the first place in the sen-e-

• ignition and sight the second, I repeat therefore thai
-ight i- the only sense of sex-recognition in hunting spider-.
Alter sex has been recognized, courtship begins, and touch is the
chid means by which the male excites the female and tests her
\\illiiune>s to accept him.

AN ABERRANT LASIUS^FROM] JAPAN.1

•

WILLIAM MORTON WHEELER.

In a small collection of Japanese ants recently sent me for
identification by Professor S. J. Kuwana, of the Imperial Agri-
cultural Experiment Station at Nishigahara, near Tokio, I find
a single female specimen of such unusual conformation that I at
first supposed it to represent an undescribed genus. On closer
examination, however, it proves to be a Lasins strikingly different
from the females of any of the known species, and suggests two
hypotheses for both of which provision will be made in the fol-
lowing paragraphs. The specimen may represent either a new
species or merely an aberrant female phase of some one of the
known Japanese Lasii. The latter supposition will be considered
at length in the sequel; the former calls for the following, perhaps
merely provisional, taxonomic description:

Lasius spathepus sp. nov. (Fig. i, A and B.)

Female (dealated). Length 6 mm.

Head cordate, slightly broader than long, with notched pos-
terior border and rounded, convex posterior corners and sides;
convex above; gula concave, with a median longitudinal ridge.
Mandibles small, flattened; apparently 5-toothed, with concave
external borders. Clypeus depressed, broadly rounded in front,
obscurely carinate in the middle. Frontal area obsolescent;
frontal groove distinct. Eyes rather large; ocelli small. Antennal
scapes broad and compressed, reaching well beyond the posterior
corners of the head; funiculi slender, not clavate; all the joints
distinctly longer than broad; joints 1-3 more than twice as long
as broad ; terminal twice as long as the penultimate joint. Thorax
much narrower than the head, fully twice as long as broad ; meso-
notum and scutellum flattened above; epinotum short, rounded
above, with the declivity abrupt, straight in profile and longer
than the base. Petiole with an erect scale, compressed antero-
posteriorly and with its upper margin rather sharp and distinctly
notched in the middle. Gaster very short, but little longer than

Contributions from the Entomological Laboratory of the Bussey Institution,
Harvard University. No. 22.

130

AN ABERRANT LASIUS FROM JAPAN.

I 3

AIM! i apilla prominent. Legs very long; femora, tibia?
and i) etatarsi dilated and compressed anteroposteriorly; remain-

tarsal joint- growing successively narrower.
Bod 'I appendages smooth and shining, very finely and
inconspi< uously punctate. Pleura? and especially the sides of the
• pin-. tn in more opaque and somewhat more coarsely punctate.
M.indiM. .|ue, finely and sharply striated.

yrllou ish, very short and sparse on the body, denser and
- appr, — ocl on the flat surfaces of the legs, but absent on tin-

• i A. Lasius s path f pus sp. nov.. deflated female ; B, head of same.

-harp dot-al and ventral edges of these appendages. Anterior
I 'oidn ot t l\ priis with a row of short, stout bristles. Border of
prin -lr .ind posterior edge of each gastric segment with a sinje
row oi liaits. Circlet of anal cilia long and coarse.

HI d\ drip chestnut brown; scapes, legs and articulations of
\\ini:- p. ill i ,uid more reddish; corners of clypeus and posterior
bordei -trie segments sordid yellow.

I lii- tnr.ale may be at once distinguished from any of the

kiioun ti in. ile Las ii by its peculiar heart-shaped head, short

• r .ind dilated and flattened metatarsi. The last character.

in lari, i> nut nu-t with in any other known ant, except Melisso-

tnr.^iis. \shirli l.mery regards as an aberrant Ponerine.

Tin- Mippo-ition that L. spatliepus may not be a new species,
bin merely an extraordinary female form of some one of the \\rll-
knoun Japanese Lasii, is supported by the following considera-
tion-. Main years ago Walsh1 described an aberrant female

1 'n tin ' .f Aphidac found in the United Suit'1-." I 'roc. J-'.nl. Soc. Phila..

I . N". •;. i y -1)4-311.

132 WILLIAM MORTON \YHKELER.

Lasius from Illinois as L. latipes, and in 1903 McClendon and I1
showed that this ant has two forms of females: the one described
by Walsh and characterized by extremely flat, dilated femora and
lilii.i-. >m. ill, feeble tarsi, strongly clavate antennal scapes, short
funicular joints and long, fulvous pilosity; and another of a
darker color, with less flattened legs, less clavate scapes, longer
funicular joints, longer tarsi and sparser, shorter pilosity. The
latter we designated as the a-, the former as the /3-female. We
found most colonies at the height of the breeding season to con-
tain only /3-females, but in three colonies from different localities
both forms occurred simultaneously. These observations sug-
gest that L. spathepus may be the /3-female of some Japanese
Lasius, which in its worker and male phases show's no departure
from the usual generic type of structure. Five Lasii are known
from Japan, namely, L. niger L., niger alienus Forster, myops
Forel, umbratus Nyl. and L. fuliginosus Latr. All of these are
well-known European species and, in all probability, common
also throughout temperate Asia.2 The only one of these species
of which spathepus could be a /3-female is L. fuliginosus. I possess
males and workers of this species collected by Mr. Hans Sauter in
Kanagawa, Japan, and there were three workers in the collection
sent me by Professor Kuwana, but as these bear a special number
they were probably not taken in the nest containing the spathepus.
All the Japanese workers and males of fuliginosus are indistin-
guishable from specimens in my collection from several European
countries (England, France, Germany, Switzerland, Austria and
Russia). In Europe, however, this ant is known to have only
one form of female, which is in no respect extraordinary (Fig. 2, A
and B) though it would bear to spathepus about the same relation
that the a-female of latipes bears to the cospecific /3-female. Com-
parison of the figures accompanying this article shows that the
head of spathepus in its outline is in some respects more like that

'"Dimorphic Queens in an American Ant (Lasius talipes Walsh)," BIOL. BULL.,
IV., No. 4. 1903. PP- 149-163.

-L. fuliginosus is cited by Forel from lower altitudes in the Himalaya ("Les
Fourmis de 1'Himalaya," Hull. Soc. Vaitd. Sc. Nat., 5 ser., XLII., 1906, p. 85).
Du Buysson in a paper which I have not seen ("Les fourmis fuligineuses au Japon."
Rev. Ent., 1906, pp. 101-102) gives some notes on the occurrence of this ant in
Japan.

AN ABERRANT LASIUS FROM JAPAN.

133

of the worker fnli^inosus than the latter is like that of the Euro-
pean female of the species. If spathepus is an aberrant female
fiiliziHtisuv, as M'-ms not only possible, but probable, we must
therefore ;t--ume either that this species in Japan has two females,
• iparalil.- to the a- and /3-females of tetipes, or that ii has only
''"• .Mi in ili , \\hile the a-female alone is retained in Europe

I /. ; in fit : 'tuius 1. Mr., ilr.ili female ; B. head of same; C,

I!\ i« lent K i his question can be decided only by exhaustive obser-
\ aiioii- in J.i|i.in.

Tin- tr.iinoi h\ potheses suggested by spathepus isnot terminated
.ii tlii- |)i)int. Recent investigations make us look with increasing
inti TI •-( mi .ill .tin rrant female ants, for it has been found that
oit.iin species of Formica, A phccnogaster and Bothriomyrmex-
\\ hirh h.i\ e Ifni. ilt - rit her of unusually small size, glabrous integu-
ni. f\n.i«'nliii.ti \ color or pilosity, or with unusual morpho-
if.il characters, also exhibit correlative ethological peculiaritii ->.
Sin h t« malt--, during the establishment of their formicaries, an-,
as a rulr, temporary parasites on workers of allied species whose
tVmalr- n-tain tin- t\'pical generic characters. The question
tlit-ifton- arises as to whether the aberrant female, which I have
t alK-d 1.. tfxitlh'pHs, may not be a temporary parasite on some
otht i moiv common species of Lasius. Here the siiiyo-iion that
.s/x;///<7>//.s ma> 1 >r a /J-female of fiiliginosus receives a little support

134 WILLIAM MORTON WHEELER.

from some recent European investigations. Forel1 long ago
showed that this ant is unique among the old world Lasii umd
the new world species may be included in this statement) in its
odor, the great size of its colonies, its habit of foraging in long
files in the broad day-light and in constructing carton nests in
old tree-trunks. \Yasmann2 has recently called attention to its
ability to form new colonies by sending off detachments of queens
and workers after the manner of Formica rufa. Like rufa it also
possesses another method of colony formation, namely, through
temporary parasitism. Unlike the queens of the common Lasius
niger, the queen of fuliginosus, after fecundation on her marriage
flight and on returning to the earth, is unable to start a colony
unaided, and if prevented from rejoining the maternal colony
or a detachment of workers of her own species, has to seek out a
colony of L. umbratus and have her young brought up by the
workers of this ant. The umbratus queen must be killed either
by her own workers or by the intrusive fuliginosus queen, so that
the host species is destined eventually to die off and leave a pure
and thriving fuliginosus colony. That this method of colony
formation is actually adopted by fuliginosus queens is clearly
indicated by the following observations which have been slowly
accumulating during the past few years: In 1908 de Lannoy
stated that in 1904 he found at Knoche-sur-Mer in Belgium a few
workers of L. mixtus (a subspecies of umbratus} living in a large
colony of fuliginosus, and that in 1906 he found several similar
colonies. Emery4 and Forel5 interpreted these observations to
mean that the queen fuliginosus founds her colony with the aid of
umbratus workers, in a manner analogous to that employed by the
North American and European Formica of the rnfa, exsecta and

'"Les Fourmis de la Suisse," Nouv. Mhn. Soc. Helv. Sc. Nat. Zurich. XXVI.,
1874, pp. J-447. 2 pis.

"'Ueber gemischte Kolonien von Lasius-Arten," Zoo/. Anzeig., XXXV., 1909,
pp. 129-141.

'"Notes sur le Lasius niger et le Lasius fuliginosus," Ann. Soc. Enl. Belg., LI I.,
1908. pp. 47-53-

4"Remarc|ues sur les observations de M. de Lannoy touchant 1'existcnce de
L. mixtus dans les fourmilieres de L. fuliginosus," Ann. Soc. Eiit. Bclg., LIL, 1908,
pp. 182, 183.

6"Lettre a la Societc Entomologique de Belgique," Ann. Soc. Ent. Belg., LIL,
1908. pp. 180, 181.

AN ABERRANT LASIUS FROM JAPAN. 155

niicrogyna groups when they enter nests of F. fusca and incerta.
\Ya-mann Inco citato] accepts the interpretation given by Emery
and Ford, and now recalls that he has on several occasions found
mixed colonies <»f L. nmbratus and fuliginosus. Donisthorpe1
states ili at in 1X97 he recorded the occurrence of a large colony
of fnli'jninsns in a hollow tree at Lymington, England, living with
\\li.it In In-lit -\« -d at the time to he L.flavus but has since decided
must ha\<- Ix-rn nmbratus. He also says that Crawley has re-
ccntK l«Mind nn;!>rntns workers in company with fuliginosus.

Hut even ilii- is apparently not the whole story. CrawK-y-
foimd ili.n i In- ijucen of nmbratus may be adopted by a colony of
L > nid \\.i-mann (loco citato} has shown that the former

am is, .ii I. ast occasionally, a temporary parasite on niger, for he
loi 1 1 id .1 inixid i -i i|i my of the two species which could only U
inii i ]uvii d mi this supposition. He believes, therefore, that we
m.r Ii i\«- In •!<• a case of social hyperparasitism — nmbratus found-
sith the aid of niger, and fuliginosus with the aid
ol nmhrtitns! In these observations it is, of course, the female of
tin- Knro| .1 -.in 'nliginosus which exhibits temporary social para-
Hti-m, and it ^ />nthff>ns is really the /^-female of this species, it is
al-o. in .dl pri'liability, addicted to the same form of parasitism,
IK rli.ip- MM -, ,nie other species of Lasins, although nmbratus, as
I lia\c -i.iird. i- known to occur in Japan.

rim- ii .ip|M-ars th.it in the old world the genus Lasins, like
ilic ;o'iiu- /"''iiicd, is made up of two sets of species — one
(AircincK abundant and widely distributed, with queens able
t<> r-iaMNi their colonies independently, the other rare and
>poradir in tin -ir occurrence, with (jueens that require the assi-t-
aint ni' \\orkn-~ ol other species of the genus when engaged in
founding iluir commonwealths. To the former set belong L.
I nid ii> various subspecies (alienns Forster, brnmnas
I. at r.. , »:,ir^iinilns Fabr.. lasioides Emery) and L. flavus; to the
latter /.. nnif'nitns Nyl., and its subspecies mixttts Nyl., bicornis
\ -tt-r and affinis Schenck, L. carniolicus Mayr and fuliginosus
Lair. I'l i rarity of carniolicus and the very small

'"On th<' Kiiim Him »i Nests by Ants; an 1 a few n^tes on Myrnr-c •philes,"'

l:nt. R,. XXII . No. |. IQIO, 4pp.
:. Mxtiti: \l.:. . \-}>«), p. 94.

136 WILLIAM MORTON WHEELER.

of its females point unmistakably to parasitic habits. The same-
is probably true of L. crinitus described by F. Smith1 and Mayr-
from Cashmir. Only the female of this species is known ;m<l
this has long yellow hairs like the North American Formica
ciliata Mayr and crinita Wheeler, which are, in all probability,
temporary parasites on varieties of F. schaufussi or fusca.

In North America, the genus Lasius, which embraces the sub-
genus (Acanthomyops) not represented in Eurasia, seems to pre-
sent a corresponding division of its species into those with inde-
pendent and those with parasitic queens, although the data un
which this assertion is based are at present very meager. Here,
too, the forms of L. niger, namely the varieties americanus Emery
and neoniger Emery, L. flavus var. nearcticus Wheeler and brevi-
cornis Emery establish their colonies independently. This I can
affirm from many observations in the field. The same is true of
L. (Acanthomyops} daviger Roger and probably also of L. (A.} in-
ter jectus Mayr. But I have never seen any of the females of our
itmbratus forms (mixtus var. aphidicola Walsh, subumbratus
Viereck, minutus Emery and speculiventris Emery) in the act of
founding their colonies independently, and it is quite probable
that they are temporary parasites on the extremely common /..
americanus. Equally negative have been my observations on L
(A.} latipes, which has the a- and /3-females to which I alluded
in a preceding paragraph. That this species is a temporary
parasite on L. americanus is indicated by the fact that near Cole-
brook, Conn., I found four small mixed colonies.3 L. (A.)
murphyi Forel and occidentalis Wheeler, which are closely related
to latipes and have females covered with singular fulvous hairs,
are also very probably to be regarded as parasitic in the early
stages of colony formation.

The genus Lasius, which comprises some of the commonest and
most characteristic ants of the north temperate zone, has never
attracted a large number of students, probably because the

'"Catalogue of Hymenopteroua Insects in the Collection of the British Museum,"
Pt. VI.. Formicidae. 1858, p. 13.

"Myrmecologische Studien," Verh. K. K. Zoo/, hot. Ges. Wien., XII.. 1862.
p. 700.

'Sec Wheeler, "Ants: Their Structure, Development and Behavior," 1910, p.
504, nola.

AN ABERRANT LASIUS FROM JAPAN. 137

species are in the main subterranean and are so much more
moii' )t on' Hi- in their habits than the species of Formica. Nevcr-
th< -It •->, an intensive study of the ethology of the European and
North Ann ri< .in Lasii is bound to bring to light many surprising
I'.K ts. This is sufticiently indicated in the preceding paragraphs
notwithstanding the large amount of conjecture which they con-
t.iin

SPERMATOGENESIS OF THE MYRIOPODS.

\'I. AN ANALYSIS OF THE CHROMOSOME GROUP OF

Scolopenda heros.1

M. \V. HI.. \CKMAN.

I hiring the last few years a number of attempts have been made
to analyze the chromosome complex of various animals. These
attempts have met with such apparent success in the case of
several insects, notably Orthoptera, that I have been led to
attempt a similar analysis of the chromosome group in Scolopendra
heros. Indeed, before the appearance of earlier papers upon this
species such an attempt had been made, but it had met with but
small success owing, as I now know, to deficiencies in the optical
apparatus employed. With the facilities then at my disposal, it
was impossible to secure definite clear-cut images of the chromo-
somes at a magnification greater than 1,500 diameters. As the
chromosomes in Scolopendra heros, although exceedingly clear-
cut and definite in outline, are considerably smaller than in some
insects, a greater magnification- than 1,500 diameters is necessary
if the study is to be at all convincing, either to the investigator or
to those reading his report. However, by the use of a Zeiss 2-
mm. apochromatic objective and a number 12 compensating
ocular, the source of light being a Welsbach mantle, a magnifica-
tion of 2,300 diameters was obtained with no perceptible loss of
definition in the image.

The material used in this study is the same which served as a
basis of several previous papers2 (Blackmail :oi, 103, 105), the

'From the Zoological Laboratory, Syracuse University, Syracuse, New York.

'Blackman, M. W., :oi, "The Spermatogenesis of the Myriapods — I., Notes on
the Spermatocytes and Spermatids of Scolopendra," Kans. Univ. Quart.. Vol. 10,
pp. 61-76, pi. 5-7.

Blackman, M. W., 103, "The Spermatogenesis of the Myriapods — II., On the
Chromatin in the Spermatocytes of Scolopendra heros," BIOL. BULL., Vol. 5, pp.
187-217, 22 fig.

Blackman, M. \V., 105, "The Spermatogenesis of the Myriapods — III., The
Spermatogenesis of Scolopendra heros," Bull. Mia. Comp. Zoo/., Harvard ('<•//.
Vol. 48, No. i., pp. 1-137, 9 pl-

138

SPERMATOGENESIS OF THE MYRIOPOn-. 139

majority of the -lides having been mounted nine years, but the
-tain Heidenhain'- iron-alum haematoxylin), except where a por-
tion of i In- -ection extends from under the cover-glass, is as perfect
a- \\ hen tir-t mounted.

Sutton,1 :O2, Robertson,2 :o8, and Xowlin,3 :o8, working upon
ih»- male cell- of ( )rthoptera have found that the chromosomes
during both the -permatogonial and spermatocyte generations
may be arranged in a graded series as regards size. In the sper-
tin- -eries is a double one, the two chromosomes of a
-i/r repre-enting the similar elements derived from the
t\\'> parent-. The-e -imilar chromosomes unite during synap-i-
• Snt ton. :<t2.t>f>. fit.), and give rise to the single series character -
i-tic "f tin -pcrm.itocytes. The extreme difference in the si/e
of the chionio-,,nn-s in the Orthoptera is so marked that it i-
IK -I iceable at a glance (see Sutton, :02, Fig. 6) and after stiuh inu
preparation- of thi«, material one cannot doubt the accuracy "f
their observation- ,,r deny the strength of their conclusions, that
in the-e lorin- the chromosomes at any given stage bear a certain
-i/e i el at ion to t-ach other, and that this presents strong evidence
in -uppoit IP! tlu- theory of the individuality of the chromosomes.

lint it ue i^rani the>e conclusions with regard to the form-
-tndied. dm - it necessarily follow that these conclusions should
be made nioiv general and applied to the chromosomes of all
animal-.' What shall we say as regards the application of this
te-t to tin- chromosomes of a form in which the difference in
-i/e i- not - ..... arked or in \\hich the chromosomes all appear of
nearU one size? Such is apparently the case in Scolopendra. At
ordinary magnification there is very little difference in the si/e
« it the chroiim-c .me- as seen in a metaphase of the first spermato-
( \ t. dillereiice is to be detected even at a magnification

a- lnu as i.ooo diameters, but this is so slight that if size alone
be u-ed a- a criterion it would seem impossible to distinguish be-
t \\een the chroino-omes farther than to say: "This is one of the
-mallei one-" or "one of the larger ones."

. \V S.. :o2. "On the Morphology of the Chromosome group in Bra-

'i'i," Him- BVLL., \"ol. 4, pp. 24-39, " "8-
K. i .. rtson, \\ . K. B., :o8, "The Chromosome Complex of Syrbulii ailmira^ilis."

S null.. \'ol. 1\".. pp. 275-305, 5 plates.

'Nmvlin. Na.line, :o8. "The Chromosome Complex nf Melanoplus biiittatus
- i. Intll.. \'<>1. I\"., pp. 265-271. 2 plates.

I4O M. \V. lil.ACK.MAX.

But other tests may be applied and have been applied. Baum-
gartner1 (:O4), made an attempt to distinguish the chromosomes
in Gryllns by differences in form. He reaches the conclusion
that in Gryllns certain definite shapes constantly occur and estab-
lishes the probability that there is a fixed number of each type.
Davis2 (:o8), working upon various Orthoptera reaches the con-
clusion that "In addition to the difference in volume, the bivalent
autosomes (chromosomes) show constant and characteristic dif-
ferences in form. In general several more or less distinct morpho-
logical types can be distinguished, and the members of each type
appear to bear a constant numerical relationship to each other."

Robertson, :o8 (op. cit.}, does not consider the shape of the
chromosome of first importance in establishing its identity but
considers size as the primary characteristic, while shape is second-
ary and to a certain extent dependent upon size or at least upon
the degree of lengthening. The main criticism I wish to make
regarding Robertson's conclusions on this point is that in his
study of the chromosomes, he has not drawn them from the best
view point to establish any characteristic difference in shape.
His drawings are all or nearly all of chromosomes as seen in polar
view, whereas a view at right angles to the spindle is more satis-
factory in determining both the shape of the chromosomes and
their relation to the mantle fibers.

In Scolopendra, as I have already implied, it is impossible to
establish the identity of many of the chromosomes on the basis of
size alone. Early in my work, however, after six or eight chromo-
some groups had been carefully drawn, it became evident that the
chromosomes as seen in a lateral view of the metaphase of the
first spermatocyte are of several distinct types as regards shape
and that the size relation of the chromosomes of each type are
such as to make it possible to distinguish the individual chromo-
somes with some degree of certainty. This, I think, will be
apparent from a Mudy of the figures of plates I. and II., although
it must be borne in mind that the figures are of course much less
satisfactory for this comparison than the actual chromosomes,

"Baumgartner, \V. J., 104. "Some New Evidences for the Individuality of the
Chromosomes," BIOL. BTLL , Vol. 8, pp. 1-23, 3 plates.

'Davis, H. S., :o8, "Spermatogenesis in Acridickc and Locustida?," Bull. Mm.
Comp. Zoo/. Harvard Coll., Vol. LIII., pp. 59-158, 9 plates.

-l'I .ENES1S OF THK .MM<|o: j .1

due to tin- i.i' i ill. it many of the chromosomes do not lie at right
the I iii.- of vision and must, therefore, appear fore-
-hortcned in ,m outline drawing.

l-efore di the individual characteristics of the various

chromo-ome- , n in metaphase, it might be well to gh

l.rii-l" review of thi-ir history in the prophase. The spermatocvte
< lii-om- nteen in number. Of these, sixteen are

l.i\al< in element- formed by the end to end union of univalent

imosomes during the tetophase of the last spermatogonial
di\i-ion Hl.ickman. :o^. :o5, op. cit.}. The seventeenth element ,
'In < liron;,,-<imc, is univalent in character, being de-

ii\«d dinctU from .1 si ngle specialized chromosome, the accessory
chromosome oi tl matogonium. The growth period following

i|>-i- i Duration in Stolopendra, and during this period

• ill o| i In- ( In. mi* are grouped together to form a nucleolu —

like -inn tun- to which I have given the name karyosphnv.
\\hile in tin- karyi-pluTe the chromosomes arc so closely a^.

d ih.it i luir indi\idual outlines cannot be distinguished \\iili

'I In •>, lio\\e\rr, enter the karyospherc as distinct in-

dixidnal- .uid I.i -e from it as definite chroma tin segment-.

-iinil.tr in every resj>ect except for their greater size. These

Is \\oiild -rnn to argue f(>r, rather than against, the individu-

.ilit\ oi the chromosomes during this stage.

In tin- proph.i-r the chromosomes arising from the karyosph<
.tn i\|.it,ill\ long. -l«-inler threads of granular chromatin, which
in\ .iri.tl'K -h«'\\ near their middle an interruption of the chro-
in.itin thi- n |'K -niting the point at which the chromosomes
united during -\ n.t|i-i-. The two spermatocyte divisions al\\

li in .i loiigitudin.il and a cross division of these bivalent
rlrimnt-. The longitudinal division as a rule seems to occur
lir-t. although, a- \\ e -hall see later, this is not invariable, CM n
tor the ordinary chromosomes. The cross division or reduction
di\i-ioii iv-uh- in the separation of entire spermatogonical chro-
nio-oim--, tin' di\ i-ion occurring at the point at which they united
dining synapsis. However, although the results of these di-
\ i-ion- .tic tin -ame for all of the chromosomes (with the excep-
tion- to In- iioud later), the changes through which the tetrad
pass in the projih.i-e and the shapes they assume during the pro-

142

M. \V BLACK. MAN

phase and later during the metaphase differ to some extent. A-
this difference in shape is one characteristic by which we must
hope to identify the various chromosomes, occasion may be taken
here to describe briefly the processes by which these various
forms arise.

What I shall call type A is represented in the text-figure I.
The origin and evolution of this type of tetrad is described in
sufficient detail in previous papers (Blackmail, :O3, 105, op. cit.);
so it will not be necessary to repeat the description in detail.

/ «

h i

FIG. I. Semi-diagrammatic representation shewing the formation and history
of the cross-shaped type of tetrads; a, bivalent chromatin segment as it appears
in the very early prophase; b, planes of longitudinal and transverse cleavage
established; c, d, later stage in evolution of prophase tetrad; e, f, tetrads as seen
in early metaphase; g, tetrad in act of division, showing the manner in which the
component parts glide over each other; h, early anaphase showing distortion of
halves of tetrad due to their close adhesion; ;, daughter chromosome in metaphase
of second spermatocyte.

FIG. II. Corresponding stages in evolution of the clouble-V type of tetrad.

I wish, however, to emphasize two points. First the points at
the ends of the shorter arms of the cross-like figure (Fig. I, b, c,d)
represents the point at which union occurred during synapsis.
The attachment of the mantle fibers in the metaphase is not at
this point as it is said to be in Syrbula by Robertson (:o8, op. cit.},
but is at the ends of the longer arms of the cross as shown in
Fig. I, c,f, g.

Robertson believes that the attachment of the mantle fibers

5PERMATOGENESIS OF THE MVRIOPODS. 143

coim id.-- with the point of synaptic union of the elements and
thai cadi bivalent chromosome during its division undergoes a
"change of it- long axis from a longitudinal to a trail-verse direc-
tion." Thi- i- accomplished by a rotation of the chromatids
over ea< h other in such a manner as to result in a longitudinal
division of tin tetrad in the first spermatocyte. In Scolopendra,

I have -ho\\n in previous papers (op. cit.}, no such complicated
process occurs in division. The long axis of the tetrad in mo-t
< ases remain- p.ir.illel to the line of longitudinal clea\age and in
tin- mci.ipli.i-. the two halves glide over each other during the
.HI n| di\i-inii. A- may be seen in the semi-diagrammatic
dra\\ii. I I , A) and in several of the chromosomes of this
t\pe in tin- .1- < ompanying plates, the two halves of tin- tetrad
11 to .idhere raihcr closely and there is often considerable di--
toriion. In I -ig. I. /;, drawn from my preparations direct it \\ill
be -ecu ih. n the parts of the two daughter chromosomes remain-
in^ longest in I'Hit.ict are considerably lengthened and di-iorted
appareinK due to the firm adhesion of the two parts.

I lie -e< ..ml i\pe of spcTmatocy to chromosome is the "double-
V" iiii.id de-cribed b\ me in a previous paper (op. cit.}. This
t\pe u-ii. ill\ aii-es from the bivalent chromosomes of the early
I 'i i .] ihase \v Inch are bent at a sharp angle at the point of synap-i~.
\liei tin lmiv;iiiidinal cleavage of the chromatin thread has oc-
< -lined i he doubli- thread becomes shorter and thicker, resulting
in the double \ -haped structure shown in Fig. II, r. There i-.n
.ill time- .1 very apparent interruption of the chromatin at the
.mje n| e. irli Hue, id (point of synapsis), and it is at this point
th. H i In cross di\i-ion occurs later. In the late prophase there
i- .i -till further condensation of the chromatin and shortening of
the Hue. id, re-ulting in the closer apposition of the ends of the
t lire. id- l.uthe-t limn the point of synapsis, resulting in a chronx >-
-nine of i he -h.ipe -hown in Fig. II, e,f. At the time of the forma-
tion ol the -pindle the mantle fibers come to be attached t» the
di-i.il end- .ml- farthest from point of synapsis) of the tetrad in
-nth a manner iFig. II, e, f, ij) that the chromosome i-di\idid
along it- longitudinal axis. In this type al^o, the two halves of
the chroiiio-oiiie -ec'in to adhere closely and to divide reluctantly
Fij II. . //, al-o 1'igs. 6, /, and 17, j).

144

M. W. 1U.ACKMAN.

The chromosomes of the third type arise from thread-like
structures similar to those from which type A and B arise. This
thread may be either approximately straight, or it may be curved
slightly in various ways, but is never bent at a sharp angle at the
point of synapsis. The filament undergoes a longitudinal cleav-
age just as with the other types. The two resulting threads, as a
usual thing, lie parallel to each other (Fig. Ill, ft, c) but in some

IV.

FIG. III. Evolution of the double-rod-shaped tetrads, a, bivalent chromatin
segment; b, c, d, formation of tetrad; e, f, tetrads as seen in early stages of the
spindle; g, i, ordinary tetrad in two stages of longitudinal division; h, rod-shaped
tetrad apparently in act of transverse division; j. dyad as seen in metaphase
(secondary spermatocyte).

FIG. IV. Variation of double-rod-shaped tetrad. In early prophase the double
chromatin segment is often twisted as shown in b. The shortening of chromatin
thread results in less and less twisting; so that the two parts of the metaphase
chromosome merely overlie each other at an angle or are only partially wrapped
about each other.

cases they are .twisted about each other, so as to form a rope-like
structure (Fig. IV, ft, r). In such cases the resulting chromosome
has a somewhat different shape. In this type of chromosome the
tetrad resulting appears rod-shaped or double rod-shaped de-
pending upon the angle from which the structure is viewed.

SPERMATOGENESIS OF THE MYRIOPOlv-. 145

er tlu- plain '•- of longitudinal and cross division are establi>hed.
the further change- involve merely a shortening and condensation
of tin- < MI in- -tructure. In Fig. Ill, c, d, two stages in this pro<
an- -hown, lioth cleavages being very evident. L.ner, as the
omden-ai i<m i.mtinues these planes of cleavage are obscured to
-nine extent .md usually the only evidence we have of the longi-
tudinal cleavage i- a very definite notch at each end of the chro-
mosome, \\hile the plane of cross division is usually shown merely
b\ .1 >liyhtl\ [i d diameter of the tetrad near its middle (Fig.

Ill,'/, - I me cases, however (Fig. 3, g, //), the plain- «\

longitudinal and transverse cleavage may be seen very distinctly
;i in the metaphase (Fig. 4. N, 13, ;/, 16, ;;, etc.). In nu ta-
kine-i- a longitudinal division is accomplished by a gliding apart
«.| the i\\o h.il\i - of the tetrad (Fig. Ill, g, i) in a manner

'•iitialK -imilar to the division of the cruciform tetrads.

In a number <>i the cells studied there seems to be one of the
rim mi"- HIM •- <>l this type, which presents quite a different ap-
pearance dining the act of division. A constriction appear- in
thi- rhiomo-t.nic at about the middle point (the plane of tran —
verse clea This is so pronounced that there is in many

dependent on the stage of division) a partial or even a
(i.inpleie interruption of the chromatic material at this point
I igs. ;. \, |. N, 9, N, 10, N, etc.). From a careful study of thi>
dm uin i-iinir in inaiiN1 cells, the conclusion seems inevitable that
thi- i me tetrad undergoes a transverse division, while the rest of
tin i hn mn '-miles are dividing longitudinally.

A \aiiat i-'ii i>l the rod-shaped tetrad is shown in text-fig. IV.
1 hit alter the longitudinal cleavage has been established, the t\\ o
ilmad-, in-tead «\ King side by side, are twisted about each
other in -iuh a \\a\ as to form a rope-like structure. The result -
IIH tapha-e chromosome differs somewhat in appearance fnmi
other- i.|' t hi- t\pe, although in all essentials it is identical. As
the thread- -Imrten the twisting gradually becomes less and less
pi.mniinced I i-. IV, b, c,d, e) until in the completed chromosomes
there i- onh a -li^ht twisting and usually the two part- merely
overlie • ich other at an angle. The division accomplished by
the ln-t mat nr.it ion mitosis is a longitudinal one.

He-itle- the-e three types of ordinary chromosomes all of which

146 M. \V. BLACKMAX.

are bivalent there is one element which is univalent in character,
being derived directly from a single spermatogonial chromosome.
This is the accessory chromosome and in the metaphase can
always be distinguished by its characteristic shape, and especially
by the fact that it is connected by mantle fibers to only one pole
of the spindle. There is usually no indication of the plane of
division in the accessory chromosomes at this time, although in
the prophase a longitudinal constriction is often shown.

The chromosomes of Scolopendra heros are seventeen in number,
sixteen of which are bivalent, while one is univalent. The sixteen
bivalent chromosomes undergo longitudinal and transverse divi-
sions during the prophase and at the beginning of the metaphase
are of several different types as regards shape. After studying a
large number of metaphases of both the large and small (Black-
man, 105) type of spermatocytes it has been found to be a rule
that in each cell the chromosomes of the several types bear a
definite and constant numerical relationship to each other. This
fact can best be appreciated by referring to the accompanying
plates. It will be seen at a glance that the cruciform tetrads
which have been described above as type A are in all cases six in
number. Furthermore, among the chromosomes of this typical
form a definite size relation exists which makes it possible to
arrange the cross-shaped chromosomes in a graded series on the
basis of bulk. To be sure, the difference in size is not so striking
as that existing between the largest and smallest chromosomes in
some insect material, but I believe is as great as the difference in
size between adjacent chromosomes of the graded series in insects.
It is perhaps unnecessary to explain that the actual size difference
is in many cases greater than appears in the camera lucida draw-
ings, owing to the fact that some of the chromosomes are fore-
shortened on account of the angle from which they are viewed.
For this reason, the drawings are much less convincing than the
preparations.

The shape of the chromosomes, aside from showing an apparent
modification due to the angle of vision, actually docs vary con-
siderably but each of the group of six chromosomes in question
are always reducible- to the cruciform tetrad described as type A.
The variations in shape have to do only with the degree to which

OF THE MYRIOPOP>. 147

the shorl arm f.f the cross is drawn out and to apparent differences
incident to tin- an;Je of vision. When the short arm of the cr« —
i- drawn out hut little the tetrad approaches the rod-shaped
( hromosomes of type C. Then, too, the cruciform tetrads vary
in -li.ipc in <!iti. rent stages of the metaphase. The limbs of the
re more likely to be of nearly equal size in the early spindle
ill. m uhen division has actually begun. As the chronio-
son n in i lie same equatorial plate, are not all in exact ly

tin- same staj division at the same time- this factor should

M « • i\ e ' • 'ii-ideration.

An at ii-mpt u a- made to learn whether any of the chromox imes
i on-taiitK either lag behind or precede the others in division. 1 1
\\a- found that chromosome A, the largest of the cross-shaped
tetrad-. -ho\\- .i tendency to lag somewhat behind the others of
tlii- t\pe. In many of the chromosome groups this is very evi-
dent. In -oii;«- cells it is not yet oriented in the characteristic
manner in the equatorial plate at a time when some of the others
ha\e he^un to -hou tlu- characteristic change in shape incident to
the eliding apart of their component elements. This can be seen
h\ i oiiipaniii; ( hromosome A with other chromosomes of similar
t \ pe in 1 [ i. >, 4. etc. Then, too, this chromosome often
pre-eiit- a l< — « lear-cut outline than do the others, approaching
tin granular condition characteristic of the prophase.

1 M tin- i hiomosonies of tyjx' H there are five present in the
metapha-c oi >', m'upcndra hcros. This is the type of chromosome
uhicli -h..\\- the characteristic double-Y shajx« in the propha-e.
I he dil!eu-i,.i- in -hape between the tetrads of this type and tin
h.iped elements is usually cjuite striking. E\ren more char-
acteri^tic of thi- t \ pe is the attachment of the mantle fibers and
the orientation ..f the chromosomes in the equatorial plate. As
-i .n in >ide \ ie\\ . the chromosome is more or less rectangular in
-hape. hut one end is usually wider than the other and to the
angles of tin- .-ml the mantle fibers are attached. The chromo-
-oine n-uall> lie- with this end toward the center of the spindle,
\\ hile the free end that to which the mantle fibers do not attach >
extends oiituard. This free end is often notched and this notch
indicate- the plane of longitudinal division. In end view (Figs,
i , /. <>. ./. etc. i the appearance is not so characteristic in the draw-

14$ M- W. 111.. \CK.M.\.\.

ings, although in the preparations there is no difficulty in recog-
nizing the true shape of the chromosome, the apparent difference
in shape being due to the view-point from which it is seen.

The five chromosomes of type B form a graded series as regards
size, just as with those of type A. The largest one is very per-
ceptibly greater than the smallest, and the intermediate ones
differ in size to such an extent that there is usually little difficult)"
in assigning them to their proper place in the series. No indi-
vidual of this type shows any constant precocity or tardiness in
division, although in some cells one or more of them are farther
along than the others (Figs. 6, 7, 17, J).

The rod-shaped tetrads (type C) are five in number in Scolo-
pendra heros. They show the same constancy in size relation as
do the other types, and may be readily arranged in a graded series-
Usually one or more of this type of chromosome show the com-
ponent parts overlapping each other at an angle or partially
wrapped around each other, indicating that they arise from the
twisted threads often seen in the prophase and already described.
However, these are not constant in occurrence and this condition
seems to depend largely upon chance.

A fact which has proved rather puzzling was brought out by a
careful study of the various chromosomes of this type. \Yhile
it cannot be doubted that four of the rod-shaped chromosomes
divide longitudinally in the first spermatocyte division, the fifth
tetrad of this shape apparently divides transversely. In all cases
in which this element is well advanced in the metaphase there is
a very evident constriction at its middle point, and in some cases
this amounts to a nearly complete interruption ol the chromatic
material. This is especially evident in Figs. 3, 4, 10, chromosome
N. Indeed, it seems hardly possible to escape the conclusion
that at the same time the other fifteen bivalent chromosomes are
undergoing longitudinal division, this one element is divided
reductionally.

This, however, is no less to be expected than is the behavior of
the accessory chromosome in this same division. It differs from
the other chromosomes in being univalent (i. e., it has no synaptic
mate), while the rest are bivalent. Alter the formation of the
spindle it lies among the other chromosomes and is scarcely dis-
tinguishable from the rod-shaped ones aside from the fact that

SPERM ATOGENESIS OF THE MYRIOPODS. 149

it i- connected by mantle fibers to only one pole of the spindle.
It i> not divided by the first spermatocyte division but passes to
one pole entire. Thus the result is in a sense similar to a reduc-
tion. il division, the two cells differing as regards the distribution
of tin- element. It has no synaptic mate and is, therefore, dis-
tributed to only one of the resulting cells (Figs. 19, 20).

1 1 i- exident tli.it the unequal distribution of the accessory
chromosomes produces two sorts of secondary spermatocytes—
OIK- half li,i\in;^ only the sixteen ordinary chromosomes and one
halt" ha\in^ t lie accessory chromosome in addition. By the
second -peim.itoi \ te division, the accessory chromosome is di-
vided ,nid ,i- it 01 curs in but half of the second spermatocytes it i-
distributed to only half of the spermatids, thus giving rise to a
dimorphism among the spermatids and spermatozoa. The signi-
ficance ot tin- dimorphism has been discussed by a number of
investigators McClung1 (:O2), Wilson2 (:o6), Stevens3 (:o8, :o8a,
:o<,i Uoring' i 107) and others — and, as I have nothing new in the
\\.i\ ot ol.MT\ations to offer it would appear hardly profitable to
consider the -ul>jeci in detail. I believe, however, that when the
i-hromo-ome- o| the female germ cells of Scolopemlra are studied
it \\ill be toiind that these are thirty-four in number in the
ovogonia, and that the following fertilization formula? of \\'ilson
\\ill hold good for this species:

.V / including \

• >on I = A ••' nuile).

2 2 \ accessory /

\ N ( accessory \

.-miatozoon - i I 1 = AT - i (male).

K. t I . :02. "The Accessory Chromosome — Sex Determinant?"
Hi. .I BULL., Vol. <. ]>p- 43-84.

\\ ilson, I l< -uidies on Chromosomes — III.. The Sexual Differences of

thr ( hi. .111. -.MIL- i -i. .up in Hemiptcra. with some Considerations on the Deter-
inin.Lticin .in.l lnh.-iit.ince of Sex." Journ. Exp. Zoo/., Vol. III., pp. 1-40. 6 fig.

'Si \ \I., :o8, "A Study of the Germ Cells of Certain Diptera with

Ri-i.-iciii i- t.> tin- 11- :• -.-chromosomes and the Phenomena of Synapsis," Journ.
E*i Vol. \'.. pp. 359-374. 4 plates.

xt,\, , "The Chromosomes in Diabrotica -.iltala, etc.," Journ.

\ ..1. \ .. pp. 453-469, 3 plates.

Stevens, X. M . :OQ, "Further studies on the Chromosomes of Coleoptera."
J«ur>i. l:.\r,r. /. / . \".-l. \"I., pp. 101-113. 4 plates.

'BmiiK. Ali.v M.. 107. "A Study of the Spermatogenesis of Twenty-two Species
i.t tin- NK inbi.n • 1 i , Jassidae. Cercopidse and Fulgoridse." Journ. Kxp. Zoo/., Vol.
1\ .. pp. 4'.<>-5i2, 9 plates.

I5O . M. W. BLACK MAN.

In ScoJopendra (Blackman, 105, op. cit.} there are two distinct
types of spermatocytes readily divisible on the basis of size.
Those characterized by the larger size are about twice the average
diameter of the smaller ones and vary from them in behavior in
the two maturation divisions. But this variation in behavior
concerns the achromatic structures of .the cell and seems to be
due to the much greater amount of cytoplasmic and archoplasmic
material present in the larger cells. It is extremely interesting
to note that as regards the behavior of the chromosomes these
two sizes of spermatocytes are essentially identical. Indeed, the
chromosome groups of the small cells differ in no respect from
those of the large type. The elements are no smaller than in
many of the large cells and present the same constancy in form
and the same size relations characteristic of the large type of cells.
Figs. 1 6, 17, 1 8 represent the chromosome groups of the small
type of spermatocytes. Many more were carefully studied and
drawn and all show the same characteristic shapes and size rela-
tions typical of the spermatocyte chromosomes. In fact, the
only reason more of these were not used is that they are not so
desirable for study owning to the difficulty in drawing them due
to their closer crowding in the metaphase.

The shape of the daughter chromosomes as they move apart
to the poles in the anaphase of the first spermatocyte mitosis is
quite characteristically different for the different types of chro-
mosomes (Figs. I, II, IV, //, III, i). Those resulting from the
division of cross-shaped tetrads have the three lobed appearance
shown in Fig. I, //. The daughter chromosomes resulting from
the division of the double-V-shaped tetrads have the shape shown
in Fig. II, //, and are essentially single-V-shaped chromosomes,
as is shown at a later stage. Those resulting from the division
of the double-rod tetrads, as they move toward the poles, have
the form of single rods, slightly constricted near the middle. I
have been unable to identify positively the division products of
the tetrad which undergoes its reduction division in the first
mitosis, but in several anaphases six of the daughter chromosomes
of each group are V-shaped, and it is probable that the sixth one
of this shape is the chromosome in question. This is rendered
more certain by the observation that in these cells there are but

-I I.KMATOGENESIS OF THE MYRIOI'O 151

four of the rod --haped chromosomes exclusive of the accessory,
which, of cour-e. i- present in but one of the chromosome groups.
The chromo-oine groups, as seen in the metaphase of the two
cell- derived from one primary spermatocyte are shown in Plate
II., I ;L-. MI, 20. The most striking fact to be observed is the
absence of ih- -ory chromosome in one of the cells. Kvena

MI per t"n ial examination, however, shows that in shape and relation
to the inaiitli- titters the chromosomes are of several different.
t\pe-, am! that these characteristics coincide with what would
'1 fmm a study of their earlier history. The chromo-
somes deri\ed fn-m the cross-shaped tetrads have altered their
-hape i on-.ider.ibly since last seen in the anaphase ol the ln>t
maturation <li\i-ioii. They are much shorter and thicker ami
are m>\\ l>ilo|..-d bodies — the constriction between the lobes repre-
senting the plain- of transverse cleavage. The attachment ol
ill. mantle liber-; seems to be at no particular point but may be
ai an\ pan of each Unite ned end of the dyad.

Tin- -inn itiral peculiarities of the chromosomes derived from
the double Y--haped tetrads are much more characteristic. In
-hap.- tin- d\ad- of this type resemble those just described to
Mime .Atl-iit. but, except in si/e, bear a more striking resemblance
i,, iln double- Y-shaped chromosomes from which they are de-
rived. < >n.- end. usually the one nearest the center of the equa-
torial plate, is broader than the other, and the entire structure
\ei\ , -\idenil\ correspond-, to the single-V-shaped chromosomes
ol the iir-t spermatocyte anaphase. The mantle fibers are always
attached to the broader end of the dyad, this fact being even
more characteristic than the shape. The appearance of these
chromosomes \\hilt- in the act of division might lead one to believe
that the it Milling division is a longitudinal one, but such a con-
clusion \\ould ignore entirely the previous history of this type o|
chromo-, .me- during the prophase and metaphase of the fn-t
maturation di\ i-ion.

Chromosomes 1.. M. O and P. in the second spermutocyte
(Kiu-. to. jo are the product of the longitudinal division o! the
rod--haped tetrads. They are dumbbell-shaped d\ad> with a
manile liber attached to each end. The constriction at the
mid. lie of each represents the plane of tran>\ er-e division. Chro-

152 M. \V. r.LACKMAN.

mosome N shows a considerably different shape, corresponding
to its different history. It is shorter and in one plane broader
than the other dyads derived from the double-rod-shaped tetrads
of the first spermatocyte. It has been already shown that chro-
mosome N of the first spermatocyte probably undergoes a trans-
verse division while the other tetrads are dividing longitudinally,
and we would, therefore, expect the products of this division to
present a different appearance from the other dyads. As a
matter of fact, it is of quite a different shape from the others
derived from the double-rod tetrads.

The differences in size between the various chromosomes of
the different types is, of course, only half as great in the second
spermatocyte, as it is in the first spermatocyte, and therefore
there is not such certainty in identifying the various individuals
of the different types. But the same size ratio seems to exist
and the chromosomes of the different types can readily be ar-
ranged in a graded series as regards size, just as in the first
spermatocyte.

It has been shown by this study that the chromosomes of
Scolopendra heros cannot be considered as ephemeral structures,
which have one appearance in one cell and present an entirely
different form in another cell of similar history. Any study
except a very superficial one must lead to an entirely different
conclusion. By a study of many hundreds of cells in various
stages of mitosis it has been found that the number of chromo-
somes in the primary spermatocytes is absolutely constant and
invariable. Furthermore, these chromosomes show other char-
acteristics, which speak very strongly for their individuality.
The ordinary chromosomes arc divisible into three types on the
basis of the shape they assume in the prophase and metaphase of
the first maturation division, and in their relation to the mantle
fibers of the spindle. The individuals of each type of structure
are invariably of the same number and in all favorable cases each
chromosome of a given type is distinguishable from the others of
a similar shape by a difference in size.

In addition, several of the chromosomes possess certain in-
dividual peculiarities aside from shape and size, which serve

SPERMATOGENESIS OF THE MVR1OPODS. 153

further to characterize them. One of the cross-shaped tetrads
:i'lv in ori< -ming itself in the plane of the spindle. Another
chromo-ome. o-ie of the rod-shaped ones, shows a much more
striking and fundamental peculiarity, in that it differs from all
of the rest "f the bivalent chromosomes in the plane of its divi-
-ion- in tin- fir-t and second spermatocytic mitoses. The ac-
orv ( hromo-ome shows still more striking peculiarities, differ-
in mi the others in its origin, valence, behavior in the propha-c,
n -l.nioii ID I!H- mantle fibers of the spindle and in its distribution
ID 1. 111 Dm halt of the resulting cells.

All of i he t'.n i- enumerated above offer evidence which seems
cDiiclu-i\c thai tin- chromosomes of Scolopendra heros, during the
-]>i Tinato< \ ic stages ;ii least, must be considered as di-tinci
cntiiic-. each "tie possessing certain well defined peculiarities
\\liith .in .1- characteristic for any given chromosome of the
spermato. is are the peculiarities of a species of animals. I

belie\e ih.it (Mutually in many animals it will be possible to
make tin- -tatenient still broader and to demonstrate the c<>n-
linniiN of the individual elements from cell generation to cell
generation. \V«- \\ill. then, be able to say that, while in different
cell '0 -i icr.iti.>ii -or different conditions of cell activity, the appear-
.iii.e ,ni<l bch.i\ior of any given chromosome may be quite dif-
iit. jn-t .1- i- true of many animals in different stages of their
c\i-icin . . \» t in .1 -imilar cell generation any particular chromo-
-ome \\ill | iic-.nt the same appearance and will behave in the
-.line 111. inner. I believe that the condition described above is
inn- iii Scolopendra heros but several facts conspire to make it
impo— ible D|' ph\-ical demonstration.

The-e difficulties are mechanical difficulties and have to do
\\ith the -mall -i/e of the chromosomes in the spermatogonial
stages and the close aggregation of these elements in the karyo-
sphere <>t the growth period. The difficulties due to the small
>i/e ..t the chromosomes in the spermatogonial stages appeal -
in-iiriiionniable. and the only evidences of individuality which
the\ pre-cin ha\ e to do with their absolute constancy in numb-r.
and \\ith the very characteristic behavior of the acce— ory — it
being the only element which can be identified at all Mages.
\Ye might rea-on from this that because one of the elements

154 M- xv- W.ACKMAX.

displays unmistakable individuality all of the chromosomes pos-
sess individuality. This argument has been made in other cases
and, while the continuity of the accessory chromosome does offer
valuable evidence in support of the individual continuity of the
chromosomes in general, it cannot be said to establish the truth
of the general theory.

The difficulty of establishing the individuality of the chromo-
somes during the growth period would seem fully as great as
during the spermatogonial period. During all the stages in which
the karyosphere exists the chromosomes are so densely aggregated
that it is impossible to distinguish the separate elements. But
even at this time it is possible in favorable cases to distinguish
the accessory chromosomes and to discern the outlines of some
of the other elements. Furthermore, as I have shown in previous
papers (Blackman, 105, op. cit.) the chromosomes enter the karyo-
sphere as distinct bivalent elements, and at the end of the growth
period arise from it as distinct chromatic segments of the same
number and character as in the earlier stage.

The chromatin segments entering the karyosphere are bivalent
threads formed by the union and subsequent diffusion of two
spermatogonial chromosomes. The point of union of synapsis
shows very plainly as a distinct interruption of the chromatin
granules near the middle of the segment, the interval being
bridged by linin fibers. In favorable sections of the karyosphere
(i. e., those in which the stain has been sufficiently extracted) it
is seen that this body is made up of a number of chromatin seg-
ments closely massed about the accessory. The chromosomes
on leaving the karyosphere are of the same structure as when
they entered, are of the same number and in appearance differ
from those of an earlier period in size only. In fact, the larger
spermatocyte chromosomes possess nearly as great a bulk as the
entire chromosome group of the spermatogonium, this immense
increase in size being accompanied by a growth of other parts of
the cell, which is proportionally even greater.

It would appear then, that during certain stages of the sper-
matogenesis of Scolopendra it is possible to demonstrate absolutely
that each chromosome is a distinct unit rhararteri/ed by certain
definite and constant peculiarities and that the continuity of

SPERM ATOGENESIS OF THE MVKIOPODS. 155

each clement can be traced from the early prophase of the first
spermatocyte to the anaphase of the second maturation division.
In other word-, it is evident that during this very important
period of their hi-tory the chromosomes show complete individu-
ality. In other namely, in the spermatogonia and during
the Drouth period, it cannot be claimed that the continuity of
the clironio-ome- is actually demonstrated in Scolopoidra, al-
though e\idencr -trongly supporting such a vie\v undoubtedly
exists.

SlM MARY.

The ( hroino-ome ^roup of the primary spermatocytes of Scolo-
f>i-inlni ln-r<>\ i- made up of sixteen bivalent chromosomes tetrad-
and one uni\ alcut chromosome (dyad), the accessory chnnno-
some.

The i liromo-.oin< - -how such constancy in shape in tin- pm-
pha-e and inetapha-e of the primary spermatocytes, and in their
relation i., tin- mantle fibers of the first maturation spindle, that
tln\ -eein naturally to group themselves under four di-tinct
t\l" I In -. ma\ lie designated respectively, as the cn>---
-haped t. -trad-, the double- Y-shaped tetrads, the nxl-shaped
tetrad-, and a -in Je-n Kl-shaped dyad.

Tin- cro — liaped tetrads arc six in number and may be ar-
ranged in led series as regards size, the difference in bulk
beini; -nun ientl\ ^reat to allow the individual chromosomes of
thi- t\pe to be di-tiiuui^hed. ( )ne of the chromosomes of thi->
t\pe tin lai:ce-t one can furthermore often be identified by it>
I. ml. in \ to la- behind the others during the early metaphase.

I i\e of the tetrad-* are of the double-V shape. The individual-
of thi- t\|.e al-o ma\ be distinguished by differences in bulk.

The rod--haped tetrads are present to the number of li\e.
Tlu'M- sho\\ con-taut -i/.e relations and may readily be arrant «l
in a graded -erie- as regards magnitude. One of the tetrads <>l
tin- t\pe differ- from the others in the form it assumes during
actual di\ i-ion. It -eem- to divide transversely, while the others
are di\ idin- longitudinally.

The accessory chromosome is univalent and passes to one of
the -econdary -permatocytes without divi-ion. During the

156 M. W. BLACKMAN.

metaphase it is connected by mantle fibers to only one pole of the
spindle.

As a result of the first spermatocyte mitosis fifteen of the
tetrads are divided longitudinally (equationally), while the one
remaining tetrad divides transversely (reductionally). The fail-
ure of the accessory chromosome to divide is, also, in effect a
reductional division.

During the later stages of the first maturation division and
during the metaphase of the second spermatocyte, it is possible to
distinguish the daughter chromosomes derived from the several
types of tetrads, by their shape and their relations to the mantle
fibers. The individuals of the various types show the same size
ratio as exists between the chromosomes of the first spermatocyte,
although, of course, the actual difference in bulk is but half as
great.

The above results seem to establish as a fact, or at least as a
very strong probability, that the chromosomes of Scolopendra
heros are distinct and definite individuals, which, under similar
circumstances, i. e., in the same cell generation, show a remark-
able constancy in form, relative size, and in their attachment to
the mantle fibers. This constancy of form, size and behavior,
affords a strong argument in favor of the theory of the individu-
ality of the chromosomes in this species in particular and adds
support to the evidence derived from the study of other forms,
to the general application of the theory.

LABORATORY OF ZOOLOGY, '

SYRACUSE UNIVERSITY,
April ii, 1910.

158 M. \V. IU.ACKMAN.

EXPLANATION OF PLATE I.

All drawings were made by the author with the aid of a camera lucida. The
optical equipment consisted of a Zeiss apochromatic objective of 2 mm. focus and
a number 12 compensating ocular, the source of the light being a Welsbach mantle
The original magnification was 2,300 diameters and the drawings were reduced
one fifth in reproduction, making the final magnification 1,840 diameters.

The seventeen chromosomes arranged in each horizontal row represent the
chromosomes of a single cell, as seen in a side view of the spindle in the metaphase.
They are arranged as follows: the six which show the characteristic cruciform shape
comprise the first six of each row and are lettered A, B, C, D. E and F. Those
showing the double-V shape — five in number — are lettered G, H, I. J and K.
Those corresponding to the double-rod type of structure are lettered L, M, N, O
and P. The seventeenth and last chromosome in each row is the accessory and
is distinguished by the letter Q. The individuals of each type of structure are
further arranged in a graded series as regards size, the largest first, etc.

Figs. 1-15 represent the chromosome groups in the metaphase of the large type
of first spermatocytes.

BIOLOGICAL BULLETIN, VOL. XIX. PLATE !•

A B C D E FGHIJKLMNOPQ

f \ ii f

V. A. BLACKMAN

l6o M. \V. BLACK MAN.

EXPLANATION' OF PLATE II.

Figs. 16-18 represent the chromosomes of the small type of spermatocyte in a
similar stage.

Figs. 19 and 20 represent the chromosomes of the two second spermatocytes,
derived from one primary spermatocyte. The individual chromosomes are arranged
so as to correspond in position to the parent chromosomes as seen in the other
figures. As will be seen, in one cell the accessory chromosome is not present, it
being distributed to only half of the secondary spermatocytes.

BIOLOGICAL BULLETIN, VOL. XIX. PLATE II.

A B CDEFGHIJKL M N O P Q

- W

/ 1

« »»»«* » J I it • I

/ j I / I

f ; «> » » » » I * | * .

I \

/ 7

f, > * 4*4 PP^MC tf

:. v * •«»»»!•«» i ./.

] ' 1

M. A. BLACKMAN

\\>l. XIX. August, 1 910. A~

BIOLOGICAL BULLETIN

M. \\r..\\ESE OF THE LAMELLIBRANCHS.

H. C. BRADLEY.

Dl I : "K PHYSIOUK.Y. L'SIVERSITY OF WISCONSIN. MADISON. \\ 1^

lii |N')2 ( iritliths pul)lishe(l an account of the finding of man-
gam -i- in i In- MIMM! of the lamellibranch, Pinna sqnanwsa.1
far ,i~ I .mi aware this result has never been confirmed nor until

nil\ ha- .111 examination of other molluscs led to an extension
of this isolated fact. To the student of comparative physiol
such a finding must be of considerable interest, adding one m<>iv
respiratoiA mechaiii-m to the list of five or six with which we
are familiar. At the same time it is highly improbable thai
I'inna squanwsa is the only mollusc utilizing manganese in a
respiratory compound; we should ex|>ect to find the element
in ni her forms more or less closely related to it. It is a matin
• •I i omiiion observation that the respiratory proteins of the more
hi.chK or-aiii/ed animals fall into a few general types, such as
ha-mo;Joliiii ,,r ha-mocyanin, and that while individual bloods
ma\ -ho\\ >u bile biological differences within one of these groups,
there i> ne\er an\ ilitliculty in determining chemically whether
a blood pigment is a ha-mocyanin, a haemoglobin, or some
oilier t\pical ((.niplex. The effective respiratory mechanisms
are thn- utiiie limited, so that we do not expect to find a single
member of a family possessed of a blood protein totally unlike
the other members of that family.

For thi- iva-on we have extended the investigation of thi-
point Muucuhat with a view to determining what other lamelli-
braiuhs are pn.\i<led with a respiratory mechanism similar to
that of rinmi sqnanwsa. The most notable group which we

•/. rend, de I'Acad. dfs Set., CXIV.. p. 840.
161

l62 H. C. BRADLEY

have thus far found to utilize the element manganese is the
Unionidse, the common fresh-water mussels. Since 1906 when
the element was first noticed in the specimens obtained from
the Madison Lakes of Wisconsin,1 we have examined many
hundreds of specimens from the Mississippi basin, St. Lawrence
and Atlantic coast drainage. In not a single specimen has the
element been wanting or small in amount. It is obvious that a
single individual which failed to show manganese, or contained
only a trace of it would be sufficient to cast grave doubts upon
the normality of the element and lead one to ascribe an adven-
titious character to it. But in every case manganese has been
abundant. The reactions for its identification are fortunately
brilliant and decisive and at the same time indicate very well
the relative amount of the element. The quantitative deter-
minations show that the metal occurs in quite uniform amounts
in the various specimens examined.

To summarize briefly the results: Some twenty-four analyses
were made quantitatively upon material from about Madison.
Some of these analyses were made upon single specimens of
Anadonta or Unio, more were made upon a sample taken from
the dried and pulverized tissues from a large number of speci-
mens secured at one time from a given locality. Many of these
analyses therefore represent the average of fifty or a hundred
individuals. The average of the twenty-four analyses shows
21.8 per cent, of ash in the tissues, 4.52 per cent, manganese
present in the ash and 0.95 per cent, manganese in the tissues.
Mussels from the Wisconsin River averaged about 14.5 per cent,
ash, 2.4 per cent, manganese in the ash and 0.35 per cent, in
the tissues. From the Temagami Reserve of Ontario mussels
averaged 15.4 per cent, ash, 3.1 per cent, manganese in the ash
and 0.45 per cent, in the tissues. Specimens obtained from a
great number of localities in Michigan, Illinois, Wisconsin,
Indiana and Iowa average about 17.0 per cent, ash, 3.4 per cent,
manganese in the ash and 0.60 per cent, in the dry tissue. A
number of normal, average sized specimens from Lake Mendota
were dissected into their more prominent tissues or organs.
Analyses of these fractions gave the following results:

1 Bradley, Jr. Biological Chem., III., 151, 1907.

MAM.ANESE OF THE LAMELLIBRANCHS. 163

Tissue. Per Cent. Per Cent. Per Cent.

Ash. Mn in Ash. Mn in Tissue.

Mi; 6.0 4.87 0.293

is part .... 14.5 5.73 0.831

brous part 32.0 4.66 1-492

V ins ....27.0 5.31 J-434

<•']' ---33-5 4-89 1-638

M.I: . . .48.0 5.12 2.457

..39.0 5.85 2.107

I .37.0 2.024 1-749

I1' rh.ip- the most interesting result of these analyses was the
pp •-. -in ( • , ,1 ill,- 1 1. mem in the eggs and embryos, showing clearly
that manganese is not an adventitious element picked up by
tin- ,i<ltili .UK! ln-lil in the tissues from inability to excrete it—
..nnple. iron compounds may be in the mammuli.m

. ••!! after -'Main diseases involving great destruction of red
les.

Another inl ng |>oint brought out in the above table

i- i In- i mineral content of such a tissue as tin

in. inilr. h- a-h content, of 48.0 per cent, puts it in a class with

m. mini. ili. mi is tissue, though unlike the latter the mantle

• li .ui'l pliable. This is a type of tissue resembling no verte-

n <>r tissue of which we are aware. It seems probal-1
that it- I inn ti.in as a gland secreting the shell must have some
n \\ith the high mineral content.

II ivii !>li-hed as we In-lieve the normality of the element

ni.m^.iiH-M- in the tissues of the I'nionida?, the question as to
it- . infill naturally presents itself. It is hardly to be supposed
th.it .m .iniin.il of so great complexity as a lamellibranch would
trate the element from its highly dilute solu-
tion-, in lake- .md streams. Such concentration is usually ]>• r-
tdiiiu-il l>\ lc.\\rr forms of lift- which are then obtained as fo<><l.
In tin the Tnioniche the food origin of manganese i-

nnu h iii"iv .-It-.irly apparent than is the food origin of copper
in m. m\ ••!' the h.emocyanin-bearing animals. The water- in
\\ hi. h t he mn— els are found have invariably contained the I >n >\\ n
m. i— e- .M the manganiferous crenothrix mixed \\iili di.ii«.m-,
.md ether pl.mrton forms which very probably contain m.m-.i-
IUM- .il-o. And this brown slime seems to be the normal l"o<,d
M! these mn— el- so far as our observations exti-nd. In Ontario

164 H. C. BRADLEY.

there are many lakes set in clean rocky basins, and fed by
streams which leave little or no manganese stain on the rocks,
and which appear to be free from the masses of crenothrix.
In such lakes we have been unable to find mussels. In other lakes
in the same region where seepage through glacial drift was ap-
parent, or where the tributary streams flowed through such
drift, discoloration of the stones, evidence of the presence of
crenothrix, and the presence of the mussels seemed always to
go together. For example in the Temagami Reserve, Lake
Temagami itself is characterized by its clear water, and its clean
rock basin. In certain parts occur limited areas of drift — sand
and gravel — which are of insignificant amount. But though
the bottom afforded, where the lake washed such drift areas,
looked promising no mussels were found and the sandy stretches
were apparently free from crenothrix. To the north of Lake
Temagami are several lakes which lie in basins of glacial drift.
In Sucker Gut, for example, sand and gravel beaches are abun-
dant, the tributary stream flows through .many miles of drift
and carries enough manganese and iron in solution to stain its
stones and pebbles strongly brown and black. The sand itself
is stained with iron, and the brown slimy masses of crenothrix
are abundant. In this lake and its tributary stream we found
enormous numbers of small mussels wedged in thickly between
pebbles or projecting from the sandy bottom. The many obvious
examples in this region of the simultaneous presence of manga-
nese, crenothrix and mussels, or of the absence of all three is
probably more than a mere coincidence. We believe that more
careful examination would show thai the mussels require such
manganiferous food as crenothrix and that they cannot live in
\vaters where such food does not thrive.

In growing the mussels in aquaria the specimens always carry
enough of the manganiferous organisms clinging to them so that
in a few days an abundant development of the bacteria results.
In this way several hundred grams of the dry organisms have
been obtained for analysis. Such specimens are mixtures of
a great variety of organisms and thus show large differences in
chemical content. The ash content of such plancton crops
vary from 24 to 76 per cent, of the dry weight ; the manganese

MANGANESE OF THE LAMELLII5RANCH-. 165

fn>m o.i.} i" i-*4 per cent, of the dry weight. It has thus been

• obtain, through the agency of these organisms, several

gram- »\ manganese from running water which contained about

r rent, of that element. The concentrating effi-

eiei. lower forms is therefore of a high order.

1 1, in;.; the shell, the Unionida? deposit salts of man-

-.iii' '11 as of calcium and magnesium. The nacer of

tin- -lull-, r.iivtully freed from contaminating material, al\va>s

ion for manganese; its presence in the shell
ts its presence in any of the tissues. It was
Him -lore that an examination of fossil shells <>t the
\\onld be of interest in determining whether the man-
ganese i- "t < »mparativdy recent occurrence in these animals
»r \\liether it i- a metabolic characteristic of long standing.

\\« II.IM- h.i«l the opportunity to examine but one well pr< -

il -hell. This was a specimen obtained through the

c.nii if Dr. G -e Wagner who published a description

..I it in .\<:ii!i!in. \'ol. iK. The nacer of this shell was perfectly

retaining its luster, though friable and crumbling

i ]>i.\\d«T ea-ily. The fossil nacer gave 0.085 per cent, of

ni.r , \\hili- fresh shells of the present jwriod frequently

contain as mm-h as 0.14* |HT cent. Thus it can be definitely

I that the 1'nionuhe in pre- Pliocene times were u>iii^

the < li -IIH ut manganese as we find them today. It seems pn>b-

.iblr tliat tin marine ancestors of the l*nionicl«u were thcmscKe-

inan^.initi -r.-u-.. The fact that at least one marine lamelli-

liran.h i- kii"\\n makes such an assumption the more plausible.

I'., determine \\lu-ther mlu-r marine lamellibranchs utili/e

inan^an.-M- in thi- same way, an examination of the common

I. uni- aloni: the coast of southern Massachusetts was made at

ih. \\ 1- Molt- laboratory. In several forms the elements

could u-ualK l.e detected as a trace, but in such cases no import
(an be atta. hed to its presence except as indicating that then-
i- -oine marine low form of life serving as food for lamellibranchs,
\\hirh also . arries manganese. In Pectcn the man^am •-<• was
\ariaMe. -ometimes large in amount, at others very small. It
was frequently found abundant in the stomarh contents. In
nwtliolus the element was present in every specimen

1 66 H. C. BRADLEY.

examined, and it seemed to be rather uniform in amount. It
was present in every tissue, and in the nephridial organs it was
really abundant. In most of the tissues it was not at all com-
parable to the amount present in the Unionidae, approximately
o.i per cent, or less of the dry material. It will be remembered
that the nephridial organs of Modiola modiolus are usually pig-
mented a dark brown — in all of some fifty specimens examined
by us this brown pigmentation was prominent. It is possible
that this lamellibranch deposits manganese obtained with its
food in the nephridia in an attempt to excrete the element;
that it is in this case adventitious and analogous to deposits of
iron-containing pigment in mammalian tissues as the result of
pathological conditions. It is interesting to note however that
this marine mussel which stands morphologically fairly close to
the Unionidse, should appear to utilize the element so charac-
teristic of the latter family.

It is our belief that other lamellibranchs will be found which,
like Pinna squamosa, the Unionidae and perhaps Modiola modi-
olus, utilize the element manganese in their metabolic processes.
Such a chemical relationship may be useful in suggesting the
lines of the evolutionary process which has led to the develop-
ment of the present forms. It is our expectation to continue
this line of investigation as opportunity permits.

THE SPERMATOGENESIS OF EUCHROMA

GIGAXTEA.

M. LOUISE NICHOLS.

Tin- lar^e-4 of the huprestid beetles, Eitchroma gigaiitcn. i-
n;iti\c to Central and South America and is commonly found
sunning it -elf on the trunks of trees. In such situations tin-
ties are not difficult to capture, as their movements are
rather -lir^i-h until they become thoroughly alarmed. The
-pei imen- trom which the present study was made were taken
at ( nlel.ra. Panama, in the month of August, at which time
some "I i In- l>eetles were mating, the male apparently attracting
tin ti in, ill- by a clicking sound produced by the elytra.

I pi MI M-< -i inning the testes, I was surprised to find a complete
verie- nt -t.e^es from the spermatogonia to the mature sperma-
tozoa, tin- \niinger stages not being confined to the larva,- <>r
pnp.e. .1- i- ln-i|iu-iitly the case in insects. The testes were fixi ••!
in ( lil-nn's mi nuro-acetic-nitric solution or in Fleming's strong
-olution and stained with iron-luematoxylin or with saffranin
ami malai hite green.

In i In- development of the germ cells of insects, as is well known
through tin- results of the researches of Montgomery, Wilson,
Stevens and others, there are present chromosomes which have
been lallnl heterochromosomes or idiochromosomes. Wilson
(1909 ha- slin\\n for the Hemiptera that in certain forms tin-
idiochromosomes are equally well developed in both sexes, in
oilier- the male possesses one well developed, the other rediK-'l
in si/c, \\hiU- in still others one is entirely lacking in the malf.
Mr\<-n- [906 t ound somewhat similar conditions in the Coleop-
tera. llm-. tin l^laterida? and Lampyrida- possess only ilu
in Id i-hn uni -si unes. while the families Chrysomelida?, Cocci-
nelidae, Scarabidae, Silphidae and Buprestida- show one of tin- idin-
clii-oino-i'ini-s reduced in size. In Carabida^ some nu-nil'»-r-
have an unequal pair of heterochromosomes, others an odd
din >nu !-• mie. Enchroma gigantea, as a member of the taniih
Hupu-stida-. belongs in the second of these groups (Figs. 21-22).

167

1 68 M. LOUISE NICHOLS.

Besides the idiochromosomes, Wilson discovered in the He-
miptera a pair of chromosomes equal in size but noticeably
smaller than the others, which he designated as w-chromosomes.
According to the researches of Stevens these are occasionally
present in the Coleoptera, i. e., in Trirhabda virgata and T.
canadense and in an unidentified buprestid. They likewise are
represented in Eiichroma (Figs. 19, 23, 24). In addition there
are, in the spermatocytes, eleven chromosomes of more nearly
equal size, making the total reduced number thirteen.

In most forms heretofore studied, the idiochromosomes are
evident not only at the time of mitosis but also in the resting
stage and prophases, for while the other chromosomes become
resolved into the nuclear network, the idiochromosomes remain
compact. It is in the manner of formation of the chromosomes
during the prophases of the first maturation division and in the
fact that neither at that time nor in the previous stages are the
idiochromosomes distinctly different in behavior from the other
chromosomes that the chief interest of the spermatogenesis of
this beetle lies.

The nuclear network of the last generation of spermatogonia
is of delicate texture. Chromatin masses occur at intervals,
at first few in number and without constancy of position or shape
(Fig. 5). The masses gradually become more distinct and form
elongated threads near the center of the nucleus (Figs. 6, 7).
The network breaks away from the nuclear wall and the synapsis
is inaugurated (Figs. 8, 9). During this time there is no evidence
of the idiochromosomes being isolated from the synaptic threads
or failing to take part in their formation, nor, in the resting
spermatocyte, do the idiochromosomes differ from the others.
Stevens (1906) has reported a somewhat similar condition in
the beetle Tenebrio molitor.

The nuclear network of the resting spermatocyte is more
clearly defined than that of the spermatogonia and bears chro-
matin masses distributed with a fair degree of regularity (Fig.
10). This condition, however, does not continue. Instead of
the usual spireme formation, the chromatin granules commence
to migrate towards a specialized area within the nucleus (Figs.
11-14). The final result of this process is the formation of a

-PF.KMATOGENES1S OF EUCHROMA GIOANTEA. 169

den-e ma-- - -t" chromatin in one part of the nucleus, the remainder
of the nucK-ii- being occupied by a fine network. It sometinu-
happen- thai mop- than one of these areas of aggregation de-
velop- Figs, i - i s).

The outline- of the separate chromosomes may be seen, al-
though the\ lie very close together. The nuclear network
iio\v a parallel arrangement of thread- prepara-
to the formation of the spindle (Figs. 17-18).

A- i- tli- with other members of this order, the small

lir<>m<>-"ine is separated from the larger by the hr-t divi-
-ioii Fig. 2l). In mitosis the w-chromosomes tend to divide
-..meuhai later than the others (Figs. 23-25).

. \tier tli d «li\i>ion the spindle fibers persist in the cyto-

plasm. Ill--, .radiially cca.se to run parallel to each other,

• in. in. .n- or less interlaced, and finally are arranged in

spiral for: i 26-28). Later they are converted into the

tail ..I i he -|MTP • 'm (.Fig. 32).

The (hroMiatin "I the s|K-rmatid at first condenses in lar-e
measure it ihe -ide .>t the nucleus nearest the spindle fibtr-.
but a- tlu laiter 1< --e th< ir regularity of arrangement, the chro-
maiin i- di-p<T-ed throughout the network (Figs. 26-27). It
M. -\t l.nak- into small fragments which migrate to the center
..! i he nucleus, one mass, however, (probably the heterochro-
m.i-. .m. . i. -m. lining distinct from the rest (Figs. 29-32). As
the nu. K -u- api-r. -aches maturity, it elongates and the chromatin
becomes im. 1\ -i nuilar, although fragments in chain form are
(li-iiucui-lial-1. Fig. 33).

1 Mscussinx OF RKSULTS.

\ numb» r "i \\.-rkers (see Blackman, 1903) have described

;ir.. matin nucleoli." They occur in a wide variety (jf plant

an.l animal groups and usually during a [period of growth. In

enesis and -permatogencsis they may be present in the 06-
aiiil -|>ermat.'U"iiia as well as in the later stages. Among the

up- in which they have been discovered are echinoderms,
inolln-c-. arthro|»ods, amphibians and mammalian-, al-o in
pn -t../. -a and plants. It may prove instructive to compare
some "t" the more -triking of these cases with that of

I/O M. LOUISE NICHOLS.

Sometimes the karyosphere is formed immediately after syn-
apsis. Illustrations of this are furnished by the oogenesis of
the dragonfly, PUitln-mis (McGill, 1906), and the pollen develop-
ment of Sarracenia (Nichols, 1908). In this plant the karyo-
some is formed from the synaptic threads through an absorp-
tion of the chromatin substance by a nucleolus (Plate III., Fig.
i). The achromatic substance (linin) remains as a dense mass
after the chromatin has passed by drops into the closely con-
tiguous nucleolus. There is, therefore, a rather conspicuous
separation here of chromatin and linin.1

In Plathemis the synaptic threads gather closely around the
nucleolus and form what Miss McGill calls a double nucleolus
(oxyphil + basophil). There is plainly an interchange of material
between the two parts of the nucleolus and, as the network
again expands, masses of basophil substance are elaborated
within the nucleolus and pass out on to the network.

In Euchroma the transfer of chromatic material to the kary-
osome is less direct, inasmuch as the synaptic threads are first
extended into the network of the resting spermatocyte (Fig. 10;
Plate III, Fig. 4, a). The latter possesses no large nucleolus such
as is present in the plant nucleus. Nevertheless a center of activ-
ity arises, towards which the chromatin passes and simulta-
neously also the linin, at first in the form of streamers radiating
from the karyosphere (Figs. 11-12). Later, as the chromosomes
become distinct within the karyosphere, the linin is incorporated
with them (Figs. 16-18).

Blackman (1903) interprets the karyosphere of the spermato-
cytic prophase of myriapods as a mass of fine, granular filaments
closely gathered about the accessory chromosome. As the
nucleus approaches mitosis, the threads emerge from the karo-
sphere, shorten and thicken to become the mitotic chromosomes
(Plate III, Fig. 3, a and b). There is here no separation of chro-
matic from achromatic substance, only a strong attraction for both
to a definite region of the nucleus. As compared with Euchroma
this tendency manifests itself much earlier, for the karyosphere
has already begun to resolve itself into definite chrojnosomes in

'A closely similar behavior of chromatin has been described for Spirogyra by
Berghs (1906).

SPERMATOGENES1S OF EUCHROMA GIGANTEA. 1 7 I

itpcndra at a period when in Euchroma it is condensing
(Plate- III., Figs. 3 and 4). In Scolopendra there i- likewise a
kar\o-phere present in the spermatogonia, wherea- it i- Inking
in I: in hrti ni<i.

\ -onieuhat different condition is described by Eisen (1901 >
for Hulniilniseps. A karyosphere (chromoplast) is present in
tin-, obj« t. .\i fir-t it lies free in a vacuole, but later appear-
tttrai i "leaders" which might be compared to the radiating
- of l-.uchron:.: I igs. I I -12).

Through the-e. leaders small particles of chromatin are pn>-
jei led into the karyosphere and again emerge from it. The c«m-
the Naders towards the karyosphere apparently
corresponds t" a -\napsis stage, and as they finally break apart
eat h i hroiiio-oin.il thread receives a portion of the karyo>pln n
\\hiih i- ^radnalK di-t ribiited through the length of the thread
Plate Ml I b and c).

'I lie ("iidition in echinoderm eggs treated with Mg salt-
\\il-on. [901 and in normal mouse eggs (Sobotta, 1895) is
in -nine i imil.ii t" that of Euchroma, for although then

i- ,i|ipan nil paration of liniii and chromatin, the kary>-

^]>here bre.ik- up din i ilv into the mitotic chromosomes (Plate
111, FigS. 5. </ ami b).

What l.e.iiiiu have these facts on the question of the indi-
\idnalit\ <-t the chromosomes? In the pajxT on Sarracenia 1
suggested that tin- phenomena there "might be explained on
the a--mnptioii that the morphological basis of the chromosomes
remain- in the linin \\hile that portion of their substance which
caii.-c- i hem to col,, i deeply is absorbed by the nucleolus. It
.i -imilar inti r|.retaiioit be applied to the case of Batrachoscf^,
it \\ill In- -« en that tin linin retains its individuality more clearly
than the »hromatin. \\hich may be transferred to the kary-
>pheie. In the m\riapi.ds there is apparently no separation ot
chroin.it in .UK! linin, but a tight coil of threads, consisting ol

I r work on Batrachoseps. remarks: "il imu-.

i|ii<- 1,-- ]>lastes resultaient de la soudure tios intim-- dea

in.i-,. in. i laveur de rapprochement." "II semble qu'un,

-t.in.r inti-11-.fiiifiu -i.l> n.pliilc. une sorte de nucleine, soit venue etnpau-r t,.ut
1.- poll- ,!i- l.i tii^iiii- K litre ace moment. II se pi-ut qu -,• la d'un t-xlat

i lit,. in- ix-inOiues."

1/2 M. LOUISE NICHOLS.

both, forms the karyosphere. Possibly this might be regarded
as a continuation and exaggeration of the synaptic condition,
and, if this were true, a series of consecutive stages might be con-
ceived between the typical synapsis and post-synapsis of most
animals and plants and the extreme conditions presented by
Sarracenia.

In the echinoderms, in MHS and in Euchroma a similar ten-
dency to condense reappears and in the germ cells later than
synapsis, owing, no doubt, to a chemical condition of the nu-
cleus varying from the usual type. Here there is in one case
(Euchroma) a more rapid condensation of chromatin than of linin
and a consequent partial separation, while in the other (Mus,
echinoderms) no such separation is apparent, chromatin and linin
condensing simultaneously (Plate III., Figs. 4 and 5).

There is, however, little reason to believe that the difference
in the method of formation of the spermatocytic chromosomes
of Euchroma and most others insects is fundamental. The
gradual change in coloration as resting chromatin becomes
active goes to show that there is a chemical change in progress
from less acid to more acid condition, accompanied by a conden-
sation of substance. According to the differing constitution
of different nuclei, this chemical activity might be confined to
one center or distributed through the nucleus around several
centers. If the latter were the case, the network of resting
spermatocytes would break at various points and, condensing,
form the chromosomes, but if there were but one center, the
condensation would occur within a more circumscribed area.
In Euchroma, while the latter method is more common, it may
happen that the centers of condensation are multiplied (Fig. 15).

An interesting question concerning the relation of the chemistry
of the nucleus to the individuality of the chromosomes presents
itself at this point. If it be true that the aggregation of chro-
matin is accompanied by a decomposition of nucleo-proteids
and a reduction of chromatin to nucleic acid or a simple com-
pound of that acid and also true' that the chromatin may be
separated from the linin and gathered into a karyosphere, may
it not be possible that the linin network is not homogeneous as
regards its chemical character, but that in different areas are

SPERMATOGENESIS OF EUCHROMA GIGAXTEA. 173

developed ditYerx-nt proteid substances which, when combined
with the nucleic acid of the karyosphere, become active and color-
aMc by chromatin stains. It may be that usually in the devel-
opment ot tin UITIII cells the nucleic acid becomes chemically
<li"o< 'fated. Inn not visibly separated from these proteid -ub-
Btan !i-e<|iiuuly no karyosphere is present, simply

rliroMio-oiix •- coii-iMing of a condensed linin framework sur-
rounded by nucleic acid.

PHIL \HI.I rin \ N«KMAL SCHOOL,

Jt;:

LITERATURE.

Berghs, J.

:06 I • chez Ic Spirogyra. La Cellule. XXIII.. i.

Blackman. M. B.

:03 I 1. i tl»e Myriapods. Biol. Bull.. \'., 4.

Eisen, G.

:00 S|.« n hoscps. Jour. Morph.. XVII.. i.

Jannsens, F. A.

:06 Kv«ilini'>i: vtcs males du Batrachoseps attenuatu?. l..i

McGill, C.

:06 I li«- 1 !eoli During Oogenesis with Especial Refen-n. <•

J.V

Nichols. M. I

:08 III- 1 >. •>. '..|iiin-nt of the Pollen of Sarracenia. Bot. Gaz.. 45.
Stevens, N. M.

:06 i. A ' of Hi-UTochromosomes in Certain Species of

Hemiptera and Lepidoptera, with Especial Reference to Sex
•.rinin.it: trnegie Institute. 36.

:09 .'. 1'iiitlni Mti«lii-« i. M tin- Chromosomes of Coleoptera. Jour. Exp. Zoo!.,

VI . i.
Sobotta, J.

'05 I M. I'.. iiu> lining und Furchung des Eies der Maus. A. m. A., XL\'.
Wilson, E. B.

:01 i :iu-iit.il Minlii-s in Cytology. Archiv f. Entwicklungs-ni.

:Q(i M>mes in Syromastes and Pyrrochoris with a Ci.ni-

|i.n.ui\i- K.-\ i. \\ i-t I ypes of Sex Differentiation of Chromosome

Jour K\i>. /.'..| . \ 1., i.

174 M- LOUISE NICHOLS.

EXPLANATION OF PLATE I.

The drawings were made with the camera lucida, Zeiss microscope, oc. 12, obj.
oil immersion 1/12. They have been enlarged to twice the diameter and reduced
one half.

FIGS. 1-4. Spermatogonia. Prophases.

FIGS. 5-7. Last generation of spermatogonia. Prophases.

FIGS. 8-0. Synapsis.

FIG. 10. Resting spermatocyte.

FIGS. 11-18. Spermatocytes. Prophases of the first maturation division.

FIGS. 19-20. Equatorial plates of the first maturation division. 13 chromo-
somes.

FIGS. 21. The heterochromosome x.

BIOLOGICAL BULLETIN, VOL. XIX.

:
-

-i^ \ o

. Wr

M. LOUISE MCHOL8.

176 M. LOUISE NICHOLS.

EXPLANATION OF PLATE II.

FIG. 22. The heterochromosome x.

FIG. 23. Side view of the first maturation division. Metaphase.

FIG. 24. Anaphase. Late division of the microchromosomes.

FIG. 25. Telophase. Traces of the late division of the microchromosomes.

FIGS. 26-33. Spermatids.

BIOIOGIICAL BULLETIN, VOL. XIX.

PLATE ll>

u~>
<S

1*5

M. LOUISE NICHOLS.

EXPLANATION OF PLATE III.

FIGS. 1-5. Diagrams showing the relation of the karyosphere to chromatin
and linin.

BIOLOGIC* L VOL. XIX.

PLATE III.

Sarracenia.

Batrachoseps.

Etiihroma.

!•*«

Echinodenn.

M. LOUISE MCHOIS.

• )\ I Hi; \.\ Tl KK OF THE CAXALICULAR APPARATl-

OF ANIMAL CELLS.1

R. R. BENSLEY.

I Miring i In- i \\vl\r years that have elapsed since the discovery
•In- internal reticular apparatus of the nerve cell,
mini) .itti -nti'iii h.i-, l>een devoted by investigators to the c»>n-
-ider.ttii.n "t -iinil.tr reticular structures in cells. Our knowledge
h.i- pi 1 nig several different lines, which have been

determined IIH .re . >r less by the techniques employed by dit-
nt in\ . and although all are not agreed as to the

t tin -inn tures so revealed, their consideration to-
gether in-tilk-d by the great similarity in configu-
ration .UK! 1 . \\hiih these elements jx)ssess.

It i- ii"' in this paper to review in extenso the liter-

. it iin i .11 i hi- t"i.ir. for that is well covered by the summary
vM\en by Holmgren (*O2) in Mi-rkel und Bonnet, Ergebnisse,
. in \\hirh also the difTert-nt theories of the nature of these
Mrii< tun > are \\rll brought out. The more recent contribu-
tions .lit ti.ii-idt !••<! in the article of von Bergen ('04) and that
ft I .t o n.lre (.'oS-'OQ). It will suffice here to summarize the
|.ri>^rr-«. th.it has been made along the different lines of inves-
ition, antl to considi r the- interpretations of these structures
uhith ha\t I .« t n .nlvanced by different workers.

1 lir-t described the internal reticular apparatus

in tin 1 1 II I'-irkifije of the cerebellar cortex, where he dem n-
-tr.itt (1 it by iiK.in- < .1 a miKlification of his well-known chrome
>il\ir imprr^n.itinti nu-thod. IK- describes it as a closed net
"I lint- tiU-r- LI fiiji\ing the intermediate zone of the cell pro-
ii.pl.i-m and separated by a distinct interval from the nucl« M-
. .n tin- LIU- hand .unl from the surface of the cell on the other,
that i- to -.i\. tin re was a zone of protoplasm on the periphery
..t tin (rll \\ltirh \\.i- wholly free from the fibers con-tit mini;
tin- nrt\\ork. T«.\. id the nucleus the net sent out fine fi ber-
th.it the ].t riiiinK ar space was not \\holly de\i.id ot fibers.
1 i. >in tin- Hull I ;il'. .ratory of Anatomy, t'ni . ;o.

I SO K. R. BENSLEY.

In his later studies Golgi confirmed this result for other types
of nerve cells including spinal ganglion cells, spinal cord cells
and cells of the cerebral cortex. In some of these cases he found
the network provided with freely ending branches which ter-
minated in a small swelling. In some cases, too, the fibers of
which the network was composed had varicose swellings on
them, and nodal enlargements, and, in some cells, he even found
two concentric nets which differed inter se by the amplitude of
the meshes and the size of the fibers. Golgi in all of his papers
expressed himself with great reserve as to the nature of the net-
work, but was confident that they had nothing to do with the
neurofibrils and that they were not canals which had been filled
with the silver precipitate. He was moreover certain that they
were entirely intracellular and that they had no communication
with extracellular structures.

In the meantime Golgi himself and his students had been ex-
tending the field of investigation to other than nervous tissues,
and it had developed from these investigations that the retic-
ular apparatus was not confined by any means to nerve cells
but was present in a large variety of cells from different sources.
For example, Negri ('oo) demonstrated an apparatus of this
sort in the cells of the pancreas and of the parotid gland of the
cat, and in the thyroid epithelium of the dog. In these cells
the situation of the network was quite characteristic and recalled
the observations of Golgi that in young nerve cells with excen-
tric nucleus the reticular apparatus was also excentric and lo-
cated for the most part at one pole of the nucleus. In the epi-
thelial cells, namely, it was found that the reticular apparatus
was located near the nucleus, but between the latter and the
free border on the lumen or surface, that is, it was distal to the
blood vessels. Later, similar nets were found in the cells of the
epididymus by Negri, in cartilage cells by Pensa ('oi), and in
striated muscle fibers by Veratti ('02).

The observations of Golgi were confirmed by a number of ob-
servers, using this method or one of the silver reduction methods
of Cajal. ketzius, for example, obtained good impregnations
of the apparatus in the nerve cells of the cat and rabbit, which
corresponded in their salient characters \\ith those <>l ('.<>l^i but

CANALICULAR APPARATUS OF ANIMAL CELL-. l8l

were n»t ne.irh so complete judging by the figures published.
In view of thi- it i> surprising to note that Retzius ('oo) found,
in -om«- nf the cells, the fibers of the network communicai
by .t branch with the surface of the cell, which Golgi had never
oli-er\ed in hi- more perfect preparations.

In a recent publication Golgi has adopted a new method for
the demon-iration of the reticulum \vhich is based on the -il\er
reduction method of Cajal. In view of this fact it is proper to
mention hen- that < ajal ('07) has also studied the reticular ap-
liaratu- in the nerve cell, which he accepts, contrary to Golgi.

a tubular a]i|iaratus to which he applies the name "Conduii-
de Golgi-Holmgren," thus accepting the interpretation of Holm-
gren that they are the same as the so-called juice canals describe*!
l>\ him. II' i- .ids the. appearances seen in his preparations

lue i" the pn >f canals filled with a coagulable substance

which ha- an at'finit\ lor colloidal silver. He also notes dit'ler-

es in the I eha\iot ( if the apparatus in different animals, from
\\hich he- • on. hull •- that the contents of the canals in different
tell- i'iav ha\e ililtereiit chemical pro[KTties.

I. •' • line met In. 1 1- as those employed by Cajal, Sam li< /
ilemon-i rait il an c\t vrdingly interesting system in the striated
inn-tie tibet- of mammals and insects. In the former this sys-
tt in did not communicate with structures outside of the cell
but -eni free ending branches \\hich terminated just under the
-aivi.lrmma. In tin- insects, however, he made the surprising
nb-er\ ation thai the intercellular network was in continuity
\\ ith the irai In al -ystcm.

I'lie -eciuitl line of progress in the study of the reticular ap-
paratu-. bi^an \\iih the discovery by Kopsch that it could be
-taineil b\ pn>lon^etl immersion of the tissues in a two per cent,
-olutiun of o-niic acid. In a short paper (Kopsch, '02) he de-
-> til .til hi- inethoil and contributed the results of his application
of it. Tin n -nit- obtained corresponded very closely to those
.. I i ,..l-i but \\,n- obtained \,'ith greater certainty. Like < ',ol-i
he wa- unable to find any communications between the apparatus
ami the Mirfacc ol the cell, although in addition to the osmic
acid met hot 1 he eni|)loycd the resorcin-fuch-iii method of Holm-

II, to \\hich reft re .ice will be made later.

1 82 R. R. BENSLEY.

The method of Kopsch has been exploited in particular by
Misch ('03), and von Bergen ('04). The former found that the
apparatus was not present in all cells and that in some cells it
presented itself in the form of fragments, or of rows of granules.
He found, moreover, in agreement with Golgi and Kopsch that
the network never communicated with the surface of the cell,
nor did it penetrate the nucleus. Von Bergen's studies extended
to a very large category of cells ranging from wander cells to
nerve cells. To show how general these structures are in animal
cells a list of the elements in which von Bergen obtained positive
results would have considerable interest. In addition to nerve
cells he found a reticular apparatus in the following elements:
prostate epithelium, pancreas cells, demilunes and mucous cells
of the submaxillary gland of the cat, glandular epithelium from
the trachea, chief cells of the fundus glands of the stomach,
ciliated epithelium of the trachea, epithelium of the sweat glands,
wander cells and many leucocytes, fixed connective tissue cells,
cartilage cells, endothelium, smooth muscle, interstitial cells of
the testis. The wide range of these observations taken in con-
nection with the observations of Golgi and Cajal and their pupils
indicate that the reticular apparatus is by no means a structure
confined to a single cell category but is a cell organ of almost if
not quite universal occurrence in the. protoplasm of animal cells.

Before passing to a review of the investigations that have
been made from the standpoint of the canalicular apparatus of
Holmgren and others it may be of interest to refer briefly to the
studies of Golgi on the development of the reticular apparatus.
In the nerve cells of the foetal calves of two or three months, he
found the apparatus greatly reduced, often consisting of but a
single fiber, with short branches running in various directions. In
these cells the apparatus has a distinctly excentric position at
one pole of the nucleus. In the new-born animal the net often
extended around the nucleus, but left the perinuclear zone as
well as the peripheral protoplasmic zone entirely free of such
fibers. In old animals the apparatus was sometimes broken
up into peculiar island-like fragments which however were con-
nected with one another by single fibers. These observations
suggest strongly that the apparatus constitutes a unit in its ori-
gin and developmental history.

CANALICL'LAR APPARATUS OF ANIMAL CELI.S. iS;

'I In- hi-tory of the intracellular canalicular apparatus, con-

n-d apart fn.ni the positive impregnations of Golgi and his

follourr-. I.'-- ins with the discovery by Holmgren ('99) of endo-

cellular net- of juice-canals in nerve cells which he said was

exhibited particularly well in preparations made from ral>l>it

tissui s. Almo-t at the same time Nelis ('99) described, in nerve

• ill- tixed in -ublimate or osmic acid and stained in iron-lia-ma-

uliar coil-like bands to which he gave the name

t spin " the nature of which, however, remained

to him fully ob-cure.

h ml publication (Holmgren, '99, 2) Holmgren described

in . n ater d- tail the canalicular apparatus in the spinal ganglion
cell- of ill. rabbit, fixed in picric acid-sublimate and stained with
toluidi IK- blue and erythrosin. Hi- found in these cells nn«ler-
atel\ Inn- i anal- of fairly uniform calil>er which, anastomo-ing
ly, loriiu-il a ! airly dense network. The latter extended in
' at- nmd the nucleus but often was found at one pole of the
inn Iru-. more rarely at both |w>lcs. Here and there he found
ihe-e i anal- communicating with pericellular canals, and at tl
point- In \\.(- able to make out a distinct wall staining with
er\ thro-iii. Hi ~scd the opinion that the canals were of

Kmphatic n, nun \\ithout however stating \\hether they were of
extracellular or int racellulur origin.

In i^o'i ^indnit l.a ('99) also described the canals in the pro-
topla-m of ih. ganglion cells of the trigeminus of Petromyzon

and al.-o in tin -pinal ganglion cells, in the nerve cells of the
medulla obloii-aia and the cells of Reissner of the same animal.
II. explained tl.. n of the canals as due to the union of a

t"\\ ot \aciiolt--. .;iid Kiid that while many of the canals had
-month contour-. \t t in others might easily l)e seen the constit-
uent \aciiole- from \\hich they had arisen. In a foot-note In-
remarked that IK- had not found in his objects the connection
\\iih extrai ellnlar -trncturcs descril)ed by Holmgren, although
h. admitted that the canals opened on the surface into tin peri-
i rllnlar -p.

In a series "f papers dating from 1899 Holmgren has described
the n -nits of hi.- in\e-iigations on this topic, covering a wide
range of material including not only nerve cells, but cells from

184 R- R- BENSLEY.

various epithi-lia ,ind from other sources. The existence of tin-
canals has also been confirmed by a large number of observers
including Kolster Coo), Fragnito foo), Lugaro ('oo), Donaggio
( — ), Pugnat ('oi), Sjovall ('01), Smirnow ('01), von Bergen ('04)
and others.

F"or comparison with the results of the Golgi and Kopsch
techniques an enumeration of the different types of cells in which
a canalicular apparatus has been found may be of interest.
Holmgren demonstrated the canals in the following cells: gland
cells of the pancreas and parotid, intestinal and gastric epithe-
lium, epithelium of the epididymus, biliary duct epithelium,
uterine epithelium, thyroid epithelium, liver cells, epithelium
of the suprarenal gland. Retzius ('oi) described similar canals
in the giant cells of the bone-marrow, which, like Holmgren, he
considered to be in direct connection with pericellular spaces.

It is to be noted that many of the objects studied by Holm-
gren coincide with those studied by Golgi and his pupils, and
with those investigated by von Bergen, and that where this is the
case, the canalicular structures described by Holmgren correspond
closely in tln-ir location and in their configuration with those
demonstrated by the other methods. Whatever conclusion we
may reach with regard to the relation between the canalicular
apparatus of Holmgren and the reticular apparatus in nerve
cells, few who have studied the actual preparations made ac-
cording to these different techniques in respect to epithelial
cells and cartilage cells will deny their substantial identity. Ii
is true that there are differences in the appearances obtained,
but, in the opinion of many, these are sufficiently accounted for
by the differences in the thickness of the sections studied in the
different methods, and so, in the completeness of the apparatus
which is brought to expression in a single preparation.

In his later papers dealing with these structures Holmgren
has abandoned his original opinion that the canals are lymphatic
in nature and constructed an entirely ne\v hypothesis as to their
nature. This hypothesis is based on the confirmation by him
of the interesting observations of Nansen ('86) and Rhode ('91,
'93. '95). that the nerve cells of certain Crustacea (NaiiM-n, '86),
and those of certain Gastropoda and Hirudinea i Rhode, loc.

CANALICUI.AK A IT. \KATUS OF ANIMAL CELLS. 185

cit.) were penetrated by a network derived from surrounding
capsul.tr cell-. Holmgren found the nerve cells of Helix pomatia
particularly suitable for the demonstration of these intracellular
nets of capsular origin. He found here that UK- nerve cells
were provided \vith a richer or poorer network of juice-canals,
which were formed in the interior of a network of processes
derived from other cells. He even found nucleated strands
within the bo- lie- of t lie nerve cell. In later publications \ Holm-
gren, '01, '02, et< he has developed this hypothesis on tlie ba-i-
of results obt. lined by the employment of a new method, lie
fixed hi- m.iteri.i! in trichloracetic acid, or trichlorlactic acid,
and -lained it v\ith a fre-hly prepared solution of Weigert's

• rcin-fucli-in. H\ this method the protoplasm of the nerve
cell- -taiii'-d faintly luit thai of the intracapsular cell- stained
dark \iolet. as did al-o the processes of the latter. By this
me. in- lie wa- able t., -. r processes of the darkly stained intra-
cap-ular ci 11- \\hich penetrated the nerve cells, branched within
them, and ana-h >ni"-ed with one another, in order to produce
an intracellular uet\\ork. lie applied this observation al-o to
the ner\e cell- . s, and came to the conclusion that

ihe latter were ])«-netrat«-d by processes of other cells which
branched and aiia-iomo-<-d I ret 1\'. to form in the interior <.| the
iiti\e cell- a "spongioplasma" which, however, in no wise be-
lonvied to the neixetell. but was of extraneous origin. In the
interior • t the-e mi- juice canals coukl arise, which communi-

. d directK \\ith -imilar -paces in the interior of the matrix-
tell- of thi- iKi. lo ihi- net of cxtraneou- origin Holmgren
.e tin- name " t n»pho-| ...n^ium." He regarded therefore the
tro|iho-|>..n-ia not a- tixe.l -tmctures, but as undergoing a om-
-tant change, v. hich depended upon the physico-chemical proc-
i 5S( - in ih. cell, and thought that, while at one moment the net-
work of cell pi might sacrifice itself by liquefaction to
the need- of the ner\e cell, it might later be regenerated, by
new gro\\t h ••!' the pro..--- fnun without. He ilm- abandoned
com])letel\ hi- t'ornu r view that the canals nprc-cined circu-
lator\ or l\'iupliatic -tructures. or a drainage -\-HMII, in la\or

Foi m] ; l..lingren. E., "Ni - !•• ti g< zui

ili-t /rllr," in "Merkel-Bonnet ! \'"l. ti, p.

1 86 R. R. BENSLEV.

of the view that they represented the transitory phases of the
reciprocal nutritive inter-relations of the capsular and nerve cells.

The object of the foregoing brief and incomplete resume of
the literature, has been to show that from three different lines
of investigation we have evidence of the wide occurrence in
animal cells, ranging in diversity from leucocytes to nerve cells
and muscle cells, of a reticular apparatus, which exhibits itself
in the form of a network of canals with colorless contents, or
of a stained network according to the technique employed. The
uniformity with which this apparatus has been discovered in
those types of cells in which it has been sought justifies the expec-
tation that similar methods will reveal similar structures in
cells which have thus far not been investigated with this point
in view. We are thus dealing with a cell organ of almost if not
quite universal distribution in animal cells.

The question now arises — What is the significance of this
structure?

The trophospongium theory of Holmgren, as far as I am aware,
has found no support. Even if it were admitted for the nerve
cells there are many categories of cells in which a reticular ap-
paratus, or a canalicular apparatus is to be found, to which the
theory is wholly inapplicable. For example, it is difficult to con-
ceive how the reticular canalicular apparatus of the cartilage cells
and of leucocytes, could be derived from the liquefaction of pene-
trating processes of other cells. Holmgren, it is true, has made an
attempt to adapt his hypothesis to the canalicu ar apparatus of
epithelial cells, and has described in the pancreas of the sala-
mander the continuity of the intracellular network \vitli mtra-
cellular strands which go to the periglandular connective tissue
cells or to the centro-acinous cells. However, all of Holmgren's
figures of preparations made by the trichloracetic acid, resorcin-
fuchsin method are explainable on the basis of the canals having
a precipi table content which when precipitated by the fixative,
has an elective affinity for the dye. It is not by any means
certain that the figures which Holmgren has given us of intra-
cellular nets stained by fuchsin, in continuity with processes of
capsular cells, do not really represent two different structures
brought into apparent relation with one another by a common

CANAIJCUIAR APPARATUS OF ANIMAL CEI.I -. I S,~

affinit\- for the dye. It is certain, moreover, that the networks
apparently composed of solid fibers denvm-trated in the pancrca-
epit helium l>y the resorcin-fuchsin method, are not solid, for.
in preparation- made by this method, every cell in the section
\\ill -ho\\ Mich a deeply stained network while section- of the
same pancreas fixed in Kopsch's formol-bichromate solution
and -i. lined with iron-huematoxylin, will show in every cell a

i) of c.m.il- •\itti unstiined contents.

AccordingK . u« must either reject Holmgren's hypothesi-
or .i--ume ili.n then .ire two sorts of these nets, those of cartii
c.-ll- .nid epithelial cell- and leucocytes being different from th >-c

of Del \e cell-.

The -tatemeiu of Legeiidre foS-'og) that these structure- arc
either wholl\ .tb-ein or are the result of pathological chai
i- not to be -eriou-ly considered, in view of the fact that thi>
author in .itu-aiptin^ to explain the positive observations in
tlii- regard ot so m.my experienced investigators, is compelled
io re-ort to ihe \\holl\ unwarranted assumption that their re-
sults ha\e been due to the selection of unhealthy animal-, or
to the 1 1 \.nioi i ot ii-Mie- .ttier several days of inanition, or ^e\eral
hour- .liter de.tth.

Main observers, includiiu Retxius ('oo), are inclined to
|.elie\e that the\ repiv-eiit an int racelluUir system of nutritive or
drain. t^e canals ha\ing direct relations with the lymphatic
system. I'lu- extracellular communications are, however, denied

\>\ < ml^i and hi- jmpiU. who have never seen them in their
prep.n.iti..ii- nude b\ the chrome-silver impregnation method-.
nor by the -il\er reduction method. They are equally denied
by \on Rer^en. \\ho. however, admits the existence of canal- in
no \\IM ..nine, led \vith tlu-se, which he regards as artefact-.
\\hich do open on the -urface of the cell.

\ "ii I'ti^'ii 114 who studied these structures by all three
method- but, in particular, by the osmic acid method of Kop-ch,
agrees \\ith < iol^i that the structures are for the most part
net u i 'i k- 1 1| liber- compx >-ed of a -ul stance which reduce- • i-mic
.icid. but e\|>lain> the discontinuous elements found b\- him in
main cell- studied, b\- the assumption that the>" represent dif-
lerent stages in tlu- formation or destruction of the apparatu-

I 88 R. R. BENS LEY.

which thus would have a variable structure from moment to
moment in the cell. He claims that the reticular apparatus
arises by the appearance in the cell protoplasm of granules or
droplets which arrange themselves in net-like or tortuous n>\\-
which fuse to form more continuous fibers, and further, that the
network so formed can undergo vital changes, by virtue of which
it loses its stainability and becomes dissolved, the canals so
formed finally disappearing by absorption of their contents.

In view of the almost universal occurrence of these structures
in all of the tissue cells of mammals, and in many of those of
lower vertebrates and invertebrates, it seemed probable that
they would not be wholly absent from the cells of the other great
division of living organisms, namely from the cells of plants.
Accordingly, I have studied with this end in view the structure
of certain plant cells, using for this purpose in addition to the
conventional methods of plant histology, those methods which
in my experience were best for demonstration of the canalicular
system in animal cells. It seemed probable, in view of the con-
ditions found in the animal cell, that, if a homologue of the canal
network of the animal cell were to be found in plant cells, it
would be studied with greatest ease in those plant cells in which
the vacuolar system had not yet reached its full development,
namely in meristem tissues, sporogenous tissues and their prod-
ucts, cambium and embryonic tissues. The three last, however,
did not lend themselves readily to this investigation because
of the difficulty introduced by the slow penetration of the fixing
agents, so that I have been obliged for the present to content
myself with the results obtained in the root-tips of AUiuni.
Lilinm and Iris, and in the tapetum of the lily. Whether tin-
consistent results obtained from the study of these cells are
generally applicable or not to plant cells, future investigation
will show. In the meantime, because of the fact that the result -
are at variance with the accepted views of the structure of tin
cells in question, because they furnish a new interpretation <>t
the history of the vacuole of these cells and in particular because
they seem to throw an interesting light on the question of de-
nature of the canalicular apparatus of animal cells, it seem- \\i-<-
to put these preliminary observations on record.

\AL1CULAR APPARATUS OF ANIMAL CELLS. I Sg

The de-cription- which follow have lx.-t.-n ilrawn largely from
iht- -tudy <>f preparations of the root-tip of the onion, but the
r\ation- m.i'li- on the roots of the other genera mentioned
an- in lull accord with them.

In hi- -tiidy of the vacuolar system of tin- cells of plant-.
\V« : -he young cells of the onion root-tip

follou-: "In tin- \oimgest cells of roots two or three niillinu
in diameti-r I -a\\ a -reat miml>er of ver>- small vaciiole-; the
lar^e-t had a diameter of four mikra, the smallest of one mikron."
-mall \ai iiole- he claims reproduction by di\i>ion, in
the sense of the tonoplast theory of DeYries ('85'. He al-o
deri\ e- i In \ at IK .It- i .t t he older cell from these multiple \ arm >le-.

•ice.

In preparation- made after fixation in Flemming's .-tn>ng tluid,
I In •maim'- tluid. /.< iikn'- lluid, Carnoy's Huid, etc., and >tain«-d
in iron h.i-mato\> lin. or in the three-color process of Flemm

• tiding to these were obtaineil, that i- io
tin- \oim- cell- contained a multitude of small vaciioK -
\\hich l>\ their coalescence seemed to form the large central \

Hole of tile i.Mer Cell.

( }\\ the other hand, preparations made by methods whieh I
had found to 1 -I • mo ii\e for the demonstration of the canal -

ieular apparatus in animal cells, gave results which were wholly
diltireiii. In the-e iluie \vas no trace in the youngest cells of

the rout tip <i| the multiple
\ at IK .le- de-i ril-ed l'\ \\ «-nt and

other-. l>ul in-lead. < .1. h tell »^>

^r *MI* X%
po--e--ed an imri.aie network * ^L4!' * *

o| canals the .omponeiit ele- . '

ment- ot -.\hieh in tin- \oimgcst

II- \\en- often of extreme fine-

Fir,. I. Cell of outer \ tin-

111 " !1" inals were best root tip of onion showing fin,

a in the dermato^en cells on in the cytoplasm. X 800.
the -urfai-e of the root but were

i. . o^ni/able a- -ueh, though less well preserve* 1. in the . ell> of the
plenum-. I i-. i -lio\\- a cell in which this >\-tem i- romp<
of extremelx tine .anal-. In this figure it will be -et-n that the
ranalicular -\-tem tend- in the-e cell- a- in the animaljvl1

*'* *

190

R. K. 'BENSLEV.

leave a peripheral zone of cytoplasm wholly free from the canals
which constitute it. In Fig. 2 are shown four of the large wedge-
shaped cells from the region of most active division of the der-
matogen. in which the type of the canalicular system is well
brought out. Here it will be seen that in these plant cells, as
in the animal cells, the network tends to be concentrated on one
side of the nucleus, and, as in the epithelial cells, this point of
concentration is not one of the division poles of the nucleus but

FIG. 2. Four cells of outer layer of root tip of onion, showing more advanced
condition of the cytoplasmic canals. X 800.

corresponds to one side of the equator of the future spindle.
The system constitutes a closed system of canals, lying in very
close relation to the nucleus, never, however, invading, in dividing
cells, the spindle territory. From this network run out branches
which end freely often near the cell wall in a small expansion.
Many of the canals in some preparations, and this is particularly
true of the smallest canals, such as those shown in Fig. I, show
moniliform enlargements, as if they were on the point of breaking
up into a row of vacuoles, or possibly, as if they had just been
formed by the coalescence of a row of vacuoles. Again fre-
quently, the canals show a spiral or tortuous course, as if they
were fixed while in a condition of internal tension, which re-
sembles very closely the spiral or tortuous condition found in many
nerve cells (etat spiremateux of Nelis).

Tracing this system in the older and older cells of the root tip
it is found that as the cell retreats from the growing point, the
canals become progressively larger and larger. In the inter-
mediate stages of this process the condition depicted in Fig. 3
is obtained. Here there is still a continuous system of ran.il-
but they are fewer in number and broader than in the younger

CANALICL'LAK APPARATUS uF ANIMAL CELLS. IQI

• ell-, ritimately by a continuation of this process we ha\c
ill'- f.tiuiliar picture ot' the plant cell with a large central vacuole
\\hich run -trand- of protoplasm which are the la.-t at-
tenuated remain- «.f i In- protoplasmic partitions between t lu-
ll-.

A -imilar inn hani-m is revealed by the same technique in
the ia|n-ial M-ll- "t Liliitm candid nm. In preparations made
alter fixation in I lemming- fluid, the protoplasmic tip of^the
t i-ll pre-eni- a t< >am--t ru« t urt- o\\ing to the presence in it of a

• J^

'• otiowinj; the expanded

..ile.

e nuinl'( i . >t inn- \.u ii.il.--. In .11-. however, which

ha\i- !.»< 11 madr l>\ the tr. hni(|ii<- to above, it is seen

thai in-icad <>| a mull il in !•• • >l minutr \a« u«.lr- < >r alveola-, there

-trin . r canal- t'Tiniiii; a network which

"]>t n- at intt i\al- int.. the large vacuole which ncmpies the base

..I the . ell.

Apart Irmn the « 1« ineni- \\hich i"ii-tilute the tubes or vac-

ii"l« to the UK tlmtl of preparation, the cytoplasm of

ihe-e celU -In >\\ - M, . i ndi< at i« >n whate\'er of an alveolar struc-
uire under the mil r« •-• ' >|M-. It i- composed of an optically
homogeneous ui.'iind -ub-ianci- in which are imbedded the mit<>-

• ln>ndria and ciher granular eleiiieni-. t.>r example |)Ia-tid-,
\\ hich ma\ I »e pre-i-nt.

The tun ditlereiit ti chiiique-. therefore, jjve u- t \vo entirely
dittereni • • .nceptioii- of" the hi-inry of" the \-aciinle. According
to ihe lir-t ihe \a, uole ari-e- from the 't preexi-iin;^

innunieral'le -mall \acuoK--. According !•> the new method-,
the \acuole in the-e cell- con-iitute- a unit elemenl from the
verj I'l-^innin^. beini; re|>re-<-uted in the N'oiin^er cell- l.\ a -ii
-\ -tern of ana-tomo-ini; canal-.

192 K. R. BENSLEY.

The decision as to which of these views expresses the condition
in the living cell must of necessity rest on the examination of
living cells. Before taking up this question, however, we may
discuss the significance of these observations in the interpretation
of the canalicular apparatus of the animal cell for this inter-
pretation does not of necessity imply the assumption that the
canalicular structure so demonstrated in the plant cell has a
real preexistence in that form in the living cell. We may, on
the contrary, treat the technique as an experimental method
and discuss the results comparatively on this basis.

For the demonstration of the vacuolar system of plant cells
as a network of canals I have found the following fixing fluids
best adapted:

i. FORMALINE, BICHROMATE, SUBLIMATE.

Neutral formaline (freshly distilled) 10 c.c.

Water 90 c.c.

Potassium bichromate 2.5 gr.

Mercuric chloride 5 gr.

2. KOPSCH'S FLUID.

Potassium bichromate 2.5 per cent, in water 75 c.c.

Neutral formaline 25 c.c.

With these fluids, as indicated above, the cells of the root tip
show a network of canals, whereas the same tissues fixed in
Flemming's solution show, instead of canals, multiple small
vacuoles. The same statement holds good for animal cells
similarly treated. For example, the epithelial cells of the in-
testinal glands fixed in the formaline-bichromate-sublimate
mixture, or in Kopsch's fluid, show a beautiful canalicular
system, while the same cells fixed in Flemming's fluid show at
the site of the canals merely a large number of exceedingly fine
vacuoles. Thus whether we accept the multiply vacuolated
condition, or the canalicular condition, as the preexisting one
in the living cell, the analogy between these structures in the
animal and vegetable cell holds.

On the basis of the similarity in constitution of the canalicular
apparatus of the plant cell to that of the animal cell, and of the
similarity in behavior of this system when treated by the same
methods and an account of the part these canals in the plant cell

N'AUCfLAK AH'AKATl'.- OF ANIMAL

take in the hi- .tcuole of the latter. I think we are iu-u-

•r the present, to be sure, only as a working hy-

•hat t: -.v-rk of canal- found in so many animal

(i-ll- i- the ph\ -i'.l-or and morphologic equivalent of the vacn-

ol.ir '-11.

\\'e ma- 'urn to the consii a of the question

\\heiln-r tin- e.malii tilar -\-tem represents the true structure of

the vacuolar mechanisn g plant cells or not. Thisquesti<>n

f course, b !« -nl > l-\ observations of the living cells

tin ni-i -1\< •-. and the ii it ion of these is beset byextraordi-

dittH ultie- in r cell due in particular to the

impo— ibiliu nidi in \\hirh to examine the cell-.

\\hiih i-in.t it-i-l!' injurim!-. In theet't'ort to find a suitable fluid

;hi- pin |" •-< 1 tri( inin nitrate, of sodium

rhlnndr. .im! ntrations, but found

in all i hat t he -nrl.u «• .ipid changes of tin

-true tun- <•! tin !.i-m \\ hit h i 'ill to study t In

uinaliK uninini' 5. 1 was linalU

i.l>li;^( il tu re a mounting medium

the ire-lil- of similar ti- (though even in this

the t «-ll- i-i tin rsoffree-hai underwent more

-lip\\l\ tin , In these s so mounted the

cell- -hn\\ed the iiuilti|)le \ aeuolar o >ndii i« -n ili-cribed by Went.
In tin the contrary, in set >i the onion i

tip, one emild see, \\ ith ditiu tilty to l>e sure, but still unmistakably
the canal system i entedii 1.2 and 3. As these cell-

\\atehed. however, t !n ' anals are seen to break up slowly int"
munded \aeu»li- thu- 1 about the ronditimi i:enerall\-

1 in th» -i . rll-. I n _trd, therefore, the canalicular -
ti in a- the in iition intra vitam of the vacuolar apparatus in

ilu--e .-ell -of the n" -t lip. a ml believe that the multiply vacuol.

• lition i- • !idar> origin due in most cases to injur

the (ell.

REFERENCES.

i ; \vitli •

Holmgren, E.
'02 \

-.\

194 K- K- BENSLEY.

von Bergen, F.

'04 Zur Kenntniss gewisser Strukturbilder (Xctz-apparate, "Saftkanalchen,"
"Trophospongien") im Protoplasma verschiedener Zellenarten. Arch. f.
mikr. Anat., Bonn, 1904, Vol. LXIV., pp. 498-574.
Cajal, S. R.

'07 L'appareil reticulaire de Golgi-Holmgren colore par le nitrate d'argent.
Travaux du laboratoire de recherches biologiques, del'universite de Madrid,
Madrid, 1907, Vol. V., pp. 151-155.
Golgi, C.

'08 Di un metodo per la facile e pronta dimostrazione dell' apparato reticolare
interne delle cellule nervose. Boll, della Societa medico-chirugica, Pavia,
1908, Anno XXII., della Societa, No. 2.
Legendre, R.

'08- '09 Contribution a la connaissance de la cellule nerveuse. La cellule
nerveuse d'Helix pomatia. Arch. d. anat. micr., Par., 1908-09, Vol. X.,
pp. 287-555.
Sanchez, D.

'07 I.'appareil reticulaire de Cajal-Fusari des muscles stries. Travaux du
laboratoire de recherches biologiques de 1'universite de Madrid, Madrid,
1907, Vol. V., pp. 155-69.

A STUDY OF CHROMOSOMES OF TOXOPNEUSTES

VARIEGAT1 - WHICH SHO\\ IM >IYII>r.\l.

!'i;< I LIARITIES OF FORM.

BARBARA HEFFNER.

INTRODUCTION.

The observation- descril>ed in this paper were made during
tlic \\inter ami -priii;,;. 1909-10. in the Biological Lai >• .rati »ry
o| Br\n Maur ( 'ml< . Last Novemlnr after my arrival in

Hr\n Ma\\r rp>te—m I « iinen t suggested to me a -tudy of the
• hr"iu< • tain echinoderms with -pecial tvl-

« in e to cl iliarities of form, a question e-|Hviall\

-unilic.ini since tlu- appearance of Baltxer's ('090) pa|*r mi llu-
<lii«'in ..I > ntrotus livid us and Echinus »;:

•.\hiili h.i- thrown ne\\ li^ht on tie indi\ idnalit \
"t i hn. in. hinoid-. I'revious to this author'^ lir-t

publication mi tin- -nl.;«-ii 'oHj there existed only a stigj;c-iimi

!>\ lioM-ii 'ui .iml '1.7 i hat in some echinoids there occur chro-
mosomes "I a chai tic shajx.-. Baltxer pointed out that

in l-.<i:inn \ ;;:.'- • there are two very long rotl->hapcd

chromosomes, t\\'' long hook-shaped ones, and two or thn-r
horseshoe-shaped chrmni-mnes, while in Strongylocentrotus livid us
then- are t\\o I .l--ha|>ed chromosomes, two long ho»k-

-ha|)«-«l mil •- and in a part of the eggs one smaller hook-shaped
chrmin'-i-ii.. \-> ihi- laiu-rone occurs only in a purl of tin-

in al>mn mu- half "I them — Baltxer suggests ('oga, p. 5
that it i^ ppiliai'!> an idi< x hromosome whose smaller matt- i-
1'iic of the ^horu-r P-d-~hai>ed chromosomes. The sanu- -ui;-
i"ii i- in. iili- in p-ard to one of the horseshoe-shaped chro-
nio-oiiH-- in /-., itinio. in cases \\hcre there are three of that t\pc.
\\'hile idiochromo-mm-> and other heterochromosonie- ha\e for
-mne ti.iie been kuo\\n in insects, arachnids and

I t.ikr .nU.int j>|Mittiniiiy to express in; th.uiks i^r •

:."],ir>liip ,iu • ••. M>I\\I .iii-l t<>r i-iu-nuranini; -uv;-

and Dr. Su-vriis ihirin^ ill- »i" my \vi>rk

in their lalmiat^

196 BARBARA HEFFNER.

they have only quite recently been discovered by (.ulick in tin-
nematode Heterakis (Boveri, '10), by Baltzer in the ediinoid-.
and by Guyer ('090 and b) in vertebrates.

Observations on metakinesis stages (Baltzer '090, Plate
XXXVII., Fig. 9) have shown that the hook-shape of certain
chromosomes in Strongylocentrotus is due to the fact that the
spindle fiber from each pole is attached at a point about one
third of the length of the chromosome from one end, so that a
shorter and a longer arm are formed. In the horseshoe-shaped
chromosomes in Echinus the fiber is attached about half way
between the two ends, so that the two arms are nearly of the
same length (ibid., Fig. 10). As for the origin of the hook-
shaped and horseshoe-shaped heterochromosomes, observations

Strongylocentrotus c
on cross-fertilized eggs, „ , . < - , and on multipolar

mitoses of Strongylocentrotus have shown that they come from
the female pronucleus, the corresponding pair in the male being
rod-shaped. Apparently the female has an unequal pair of
heterochromosomes, one hook- or horseshoe-shaped, the other
rod-shaped; while the male has a corresponding equal pair of
rod-shaped chromosomes. It may be mentioned that this is the
reverse of what is found in insects, but as in most insects the
female nucleus must obtain more chromatin than the male

nucleus.

MATERIAL AND METHODS.

My observations were made upon eggs of Toxopneustes and
Arbacia, collected and preserved by Professor Tennent. As
preserving fluid either picro-acetic or sublimate-acetic was used.
Sections of 5 /* thickness were stained with Heidenhain's iron
haematoxylin, except in a few cases mentioned later.

The figures are all drawn with Abbe's drawing camera, Zeiss
oil immersion 2 mm. apochr. objective, oc. 12, enlarged to twice
or four time£ the original diameter and reduced one half.

ARBACIA PUNCTULATA.

The eggs of this species are quite unfavorable for detailed
cytological studies. Not only are the chromosomes very small
but the cytoplasm of the egg is filled \\ith pigment granules so

i:S OF TOXOI'NEO I !.- VARIEGA1 5. 197

that a -harp differentiation of plasma and chromosome- i- im-
possible. 1 ollowing Dr. Stevens's suggestion, I tried to bleach
tin ii with H^i >.,, a method -ucce--fully applied in -ome

other cases, I. ut entireh useless in Arbacia. I therefore gave
up further -tudv of the chromosomes of tin- -peci. -

-OPNEUSTES VARIEi.A I l -.

'I In- o|,-rr\ at ion- were made on two series of ruu> from \\\«
different loealiti' from Beaufort, X. C., the other from the

Tortugas. The results obtained are the same for both. I U .

\\iih the -iu«l\ ol the chromosomes in the first segmentation
-pindle ami found in . neustes as Baltzer ('09'," had done

in nd l:<>::nus a considerable variation in

the length and form of the chromosomes.

As iii /•• and St ntrotns there are t\\o i-\tremel\

Ion- chroino -"in. ~ in «-.n h daughter plate. Their beha\ ioi -i,
-einM< •- that dt-iiil.td \>\ I'.alt/er in thai these long rod-bha
< •linimo-c.iiu-- lair in .splitting and moving to the p< >li s

ii|>an- Tc\t i ,ith Balt/er's '090. 1M. XXX\ II.. i

5. </ and I hr-r « In • i ncs may also be seen in my l-'iu;-. i,

_• and ;v Sonietinii- the-e long chromosomes are contra.
inoiv and then ajipear thicker and shorter (Fig. I, a, the lett pair
nt Ion- rod--haped chromosomes).

A t\; hroiiio-oinc of |>eculiar shajx-, found in all of the

in- \\hi<li i- n-nally \'-shajK'd, but sometimes more
horseshoe-shape* -. 4 and 5). Very frequently in late ana-

pha-t •- tin- t\\o arm- are |.arallel. or nearly so, and one may
p.niK o\( -rlie the other, I nit there is hardly ever any doubi
to \\hether there i- oiu .hroinosome with two arms present or
tuo -eparate rod -h.iped ones (Fig. 3, a and b). As may l'«
O|P-« r\ed from I [g. 4 tin- length cf the two arms in these chro-
iiio-oine- ma\ \ 'i^htly when the arms are lying in OIK

optical plane. I tried to determine whether this different! i-
confined onl> to certain chromosomes or to chromosome- in cer-
taii l>ut no regularity seems to exist. The probable origin

of the difference in length will be discussed la

Ihe-. V-shaped chromo- . r in all tertili/ed 1<>.\<>{>-

ncii nd tin either two or three present. .\monw ;.(

198

BARBARA HEFFNER.

one-cell stages examined with reference to this point, I found in 16
cases two, and in 18 three such chromosomes. Fig. I, a, illus-
trates a case with two V-shaped chromosomes in each daughter
plate. Their antagonistic position proves that they are division

FIGS. 1-5.

products of the same chromosome, their regular number, that
they are not merely incidental features. The other chromo-
somes are very crowded, as most of them occur in one section.
Their position has been slightly changed in cases where they were

TOXOPNEUS.TES VAKIKi.ATl -

over <>r under tin- V--haped chromosomes, and this holds f.-r
all -imilar 1'iL'iirr-. >p< « ial care was of course taken to keep tin
'i'.n «•!" the Ion- rod-shaped and the \*-shaped chromosome^
iiratcly as possible.

I- i. shows daughter plates of a late anaphase where th<

arc tlin-c V--haped (hi :iies \vith more nearly parallel arm>.

T\\o ot the-e t hronio-<,ni( - are very close together, one partly

mother in each plate. Fig. 3 also shows in a aiv

three \--haped < hn.mo-. ,me- in early anaphase. One pair of

the-e ehronio-ome- apjM-.ir- -mailer than the two others, but

in evui'imiu othei I I '1 no regularity in the apparent

the ilin-e pair-, Sometimes all three pairs vary a little,

sometime- ti tly the same si/e, sometimes

onU i lie member- ..! <>ne pair \ apparent size. This dit-

ma\ IM <hie to dii- ictimi or to an original

diltei.iK. in length of the i hroino-oiiies. As Balt/er's ('090,

urements of the hook-^hajn-d chromosomes show,

i In length of ch<»mosomes of a certain t\|H- is (jiiite variable;

it ma\ \ar\ Irom <>-^ ijo mm. rr is no n-st-inblance

ho\\i \( i to the t •omlitioii-N in :is in res|X.'Cl to the

thinl jiair <»f hook--ha|M-<l ehromo-onn-, \\hith always is coiisid-

• 1\ -mailer than t he oiht-r two.

< IIM- of the three Y-sha|>ed chromosomes is probably a hetero-
. hronii-onie. as Ralt/er assumes to account for the conditions
loimd in -ntrotus and Echinus. Studies in orogenesis

and -|>eriiiat"^eii( -i- u-.nld be necessar\- to obtain evidence
li T ' 'i a^ain-t tin -tion ma<le.

\- it i- dilfu uh to ic. tint the total number of chromosomes in
a lateial \ie\\ o| the daughter plates I counted the number in
I ic ilar \ie\\- \\here tort unateU' the X'-shajK-d chromosomes sho\\
\er\ i learly.

l-i. ! presents two succeeding sections through

t\\o anapha-e daughter j)lates; the \"-shaped chromosome- are
the t\\o double ones, finished in solid black. The number ot
fhromo-oine- in earh j'late is 36. l;i^. ~ shows a p»lar \ ieu with
thn. \ -hajted cliromo-<»mes; here also the number i- 36.

I (..tinted 14 anaphase plates from the pole, \\ith a clear
arrangement of chromosomes, and found the average number

2OO BARBARA HEFFXER.

36, always counting the two arms of the Y-shaped chromosome
as one.

Although I did not have very much material I was never-
theless able to trace the V-shaped chromosomes in the 2-, 4-,
8-, 12- and 16- to 32-cell stages.

• . . . ..•••;"

O 0 ~" O rt Q n O

°°n°*° o° ° 0 o° °°

0 0 o O o o

o o o a

6a 6b 7

FIG. 6. FIG. 7.

Fig. 8, a and &, shows a metaphase from a 2-cell stage, with
the three V-shaped chromosomes distinguishable by their char-
acteristic splitting figures, which are fully explained below.

Fig. 9, A and B, represents two adjoining cells of a 16- to
32-cell stage. In cell A, gb, we see three V-shaped chromosomes

gfll

FIG. 8.

and the two long ones. In B, gc, we see again two of the V--
shaped chromosomes; and in gd the third one. The two long
ones are distributed between c and d. The chromosomes in
this stage are so small and crowded together that an accurate
count of their total number is impossible.

The figures 10-15, enlarged 4 diameters and in publication
reduced one half, show splitting, or metakinesis, stages of the

en;

201

V--haped rhroii From these : it is evident that

the Y--haped ehronio-omes of the anaphase come from a long
r«nl- iromo- > which the spindle fibers are attached

with m<>re <>r It •— re-ul.trity in the middle of the chromosome
Fig. 12 It tin it ion of the daughter-chromosomes

i than at the other (Fig. 13) or one

.inn of tin- < hrom" «n traded more than the other, or,

a- I i^. 14 ie hindrance on one side prevents one

..
f

i -ml I'nun iiM\ii rd tin pole as rapidly as the other end,

thea tin \aiiation-, in ih' h of the arms mentioned above

Fig i ap|>«-.ir. In comparing these figures with Baltzer's '090,

I'l. \\X\1I . Fig i" v< cannot fail to find a dose resemblance.

In K.ili/i T'- |>a|H-i '«>«) n, p. <)o;, we notice a suggestion that in
tin- h.M.k--hap«-d rhp-in. •-, mu-s there may be a union of two chn>-
mosomes, i-nd tn md at tin- jx)int where spindle fibers are at-
tached. Tin -on for this suggestion are tin- extra-
ordinary len-th nt' the hook-shaped chromosomes, and the fan
that all other ehnuno-onii-- are attached to the spindle til.i-r-
1>\ one end. Thi- suggi 'ii is a very natural one, for Mich
apparently hoim .-eneou- but pllirixalenl chromosomes are

2O2 HAKI1ARA HEFFNER.

known in Ascaris, and compound chromosomes are found in
the maturation mitoses of certain insects (McClung, '05, Payin-.
'09). In some of these latter cases (Payne, '09) the plurivak'iu
chromosome has a spindle fiber attached to each unit in the early
metaphase and in many cases two spindle fibers from each pole
are attached to one of each of the four units of a tetrad in primary
maturation mitoses (Stevens, '10).

Since the Y-shaped chromosome of To \~opneustes seems to be
exactly comparable to the hook-shaped chromosome of Echinus
and StrongyJocentrotus, the question arose whether, assuming
that the V-shaped chromosomes of Toxopneustes may be biva-
lent, one might by careful observation be able to trace in the early
metaphase two spindle fibers from each pole attached to each
of them. As the spindle fibers were not especially clear in the
preparation stained with iron-ha?matoxylin alone, a few slides
were counter-stained with Rubin S. Among 42 cases I found
only two where I was inclined to count two fibers; in all other
cases I was certain that only one fiber from each pole was attached
to each V-shaped chromosome. My observations have there-
fore failed to add any facts supporting Baltzer's suggestion,
which, however, future investigation may verify.

Baltzer ('090) traced the heterochromosome in Strongylo-
centrotus and Echinus to the female pronucleus. Unfortunately
I was not able to obtain suitable material for this purpose, but
further investigation will probably reveal the same conditions
as in Echinus and Strongylocentrotus.

DISCUSSION.

Comparing the chromosomes with peculiar shape in Echinus
and Strongylocentrotus with those in Toxopneustes we find that
the hook-shaped chromosomes in Echinus and Strongylocen-
trotus have no exact equivalent in Toxopneustes. The two ex-
tremely long rod-shaped ones are found in the three spr< -i< •-.
The Y-shaped chromosomes in Toxopneustes are very similar
to the horseshoe-shaped chromosomes of Echinus in rrs|xrt to
their formation and the equal length of their two arms. They
differ from the Echinus chromosomes as already mentioiu-d in
their length and slender ness. As in Echinus we are n<>t aliK i..

CHROMOSOMES OF TOXOPNEUSTES VARIECA 1 i - 2O3

di-tin^uish a particular one of these three V-shaped chromosomes

a heterochromosome.

The discovery of individuality of form among the chromo-
~-.nx •- in <•< hinoid- is a very valuable factor in support of Boveri's
"Individualitatstheorie" of the chromosomes. One also wrl-
con ry MI- h nu-ans of distinguishing parental chroni'.-»!ii<^

in < ross-fertilized Toxopneustes for instance has been'used

for -p :li/ati<m 'Tennent, '07 and *io). These rhnnnu-

somes which show marked individuality of form will be of^special
\ahii- in - fertilized eggs, where, as shown l>y llerbst

('09 .iii-l Kilt/- .•'•), the chromosomes of one parent arv al-

most entii minau-d during the first segmentation divi-imis.

II! I KATl'RE.
Baltzer. F.

'On i Chromosomen bci Sceigcleiern. Vcrh. -1. -I.

'OOa I •>•!. liv. u. Ech. micr. Arch. f. Zcllt"i-i h..

H.l II.

•

'OOb I u-Bastardc mil bes. Bcril- uny.

'I. ' Anz., B<l. XXX\'.

Boveri. Th.

'01 IV. Jena.

'07 VI Jena.

'10 I n bei Ncmatodon. Arch. f. Zcllforsi li..

l\ .
Guycr, M. F.

Domestic Guinea (Numida meleagris doni.).
An.it. An/ IV.

"OOti lie Domestic Chicken (Callus gallus dom.). An.it.

CIV.
Herbst, C.

'00 \ VI. Arch. f. Entw. Mech.. Bel. XXVII.. Heft 2.

McClung, C. E.

"05 '1 In- > :nplex of Orthopteran Spermatocytes. Biol. Bull.,

V..1. IX,
Payne, F.

'0<J Miosomc Distribution and Their Relation to S

Bi..l. Hull., XVI.
Stevens, N. M.

'10 An l'iu-(|ii.il I Ik-ierochromosomes in Furlkula. Joiirn.

/,.,,!.. \,,1
Tennent, D. H.

'07 Tin- > i i.inoiil K.UK*. Biol. Bull., XV.

'10 Tin- I >. .mill. mi i- ••! M.iifiiuil ami »l Piiti-rnul Character-; in Ecliino'li-ini
llvl.ri.l-, Arch. i. Hutu. Mech., H.I. XXIX.

ON THE INHERITANCE OF COLOR IN THE
AMERICAN HARNESS HORSE.

A. H. STURTEVANT, JR.

In a study of the English thoroughbred horse C. C. Hurst1 has
shown that chestnut is recessive to bay and brown. He supposes
that the presence of black in the coat is the dominant character.
Now black, gray and most roan horses also have black in their
coats, but 95 per cent, of the English thoroughbreds are bay,
brown or chestnut, so that Hurst was unable to verify his supposi-
tion. The American trotting and pacing horse, however, a close
relative of the English thoroughbred, exhibits colors in proportions
much more favorable for an investigation of this kind. These
proportions are about as follows: bay, 53 per cent.; black,
13 percent.; brown, 15 percent.; chestnut, 14 per cent.; gray,
3 per cent.; roan, 2 per cent.; dun, .1 per cent.

Perhaps before going further it will be well to give a brief dis-
cussion of these colors. According to Miss F. M. Durham, as
quoted by \V. Bateson,2 there are three pigments, yellow, black
and chocolate, concerned in the color of horses, as in mice, rabbits
and other animals. Chestnuts have the yellow pigment alone.3
Bays have both yellow and black pigments, and browns are only
very dark bays, shading into the self-colored blacks on the
other extreme. Grays have black hairs mixed with white ones,
usually in a dapple pattern. Roans are of at least three types.
The most common are the bay, red or strawberry roans, which
have yellow-black hairs* intimately mixed with white ones.
The black, blue or gray roans appear to differ from grays chiefly in
that their black and white hairs are more intimately mixed. The
chestnut roans have yellow and white hairs. As will appear later
the fact that there is no black in this class introduces a possible
source of error into my calculations. However, these chr-iiiut

lProc. Royal Soc., Vol. 77. B., 1906, p. 388.
"Mendel's Principles of Heredity," p. 125.

'According to Bateson some chestnuts are really chocolates, but these an
the yellows in having no black.

204

COLOR IN THE AMERICAN HARNESS HORSE. 2O5

roans are ran . forming something less than 10 per cent of the

il mimlicr of roan- -eeii on the streets of New York, and nearly

all nl' tho-e -eeii an- heav y draught horses, so that I feel sure they

verv ran indeed among blooded trotters. The official record -

dn ii": di-iingui-h between these three types of roans, but in

the journal- it i- not ran to see a horse described as belonging

to on.- ui" tin- two i..niinoiu-r .lasses, though I have never yet

n in tin in a reien-n.-e i.. ,( chestnut roan. There are several

ty|x-- ol dun-. Inn. in the list above, all are rare.

In a lew lainilic- dun- -< . in in lx? dominant to bay, brown and.

Ma. k. and out- v. 1 \\ith gray, but l>eyond this I have

Iniind mulling aliout tin- color, l.a-tly there are a very few

>poiird trmti-r-. luit ihe-e arc all poorly bred ones, with short

and 1 have done nothing with them.

I ha\c tried to-lm\\ Inn- that Hur-t's discovery of the domi-
nance nl l»a> andMouiii t nut holds good for the American
harm — hoi -c, and th.c . and roan, or all other colors
containing Mai k, are al-o dominant to chestnut. In order to
avoid coiitu-ioii I -hall call tin- dominant factor for the presence
o| Mack Hurst's factor. Apparently all tn liters have the factor
l"i i In -unit, which I -hall represent by C. This factor is hypo-
-tati. in all the others here mentioned. 1 he factor next highest
in the -.ale i- thai for black, or Hur-i'- . //, its absence
In in;< //. \. \t higher is that for bay, B, its absence being b.
At the to|i -land the i^ray and roan factors, (/ and R. Now
nio-t horses have neither of these last two, and are therefore
\ .he-tnut will al\va\s be Cfili, but may have any com-
bination »l the other la. tors and their absences, since they pro-
duce no vi-iMe eiieci in the al>-ence of Hur-t's factor. N-ll
Mack- are ( 7///;';> or i //'.•. -ince bay is epistatic to sc'lf black.
Ha\- have .me or two (">. //'.<, and B's. Gra\'s ha\'e C, II and
(/', and loan- have i '. // and 7^. Whether these last two mu-t
have /•>' or nm i» not clear. I -hall discuss the three epi-iatic
color- more lullv and give my theory as to brown when I have
I'lVM-nu-d the evidence a- to Hurst's factor.

The chief authoritic- for the statistics and color pedign .--
given here ha\e ln-eii \\alla«e'- '^"ear liook of Trotting and
Pacing" and Walla. <•'- "American Trolling Kegi-ier." l»>ih of
which are oiticial record-.

2O6 A. H. STURTEVANT.

It is a recognized fact among the breeders of harness horses
that certain stallions never produce chestnut foals. In Wallace's
Monthly for February, 1880, there is an article by "Truth," in
which he says: "I have learned that neither of the brothers
[Volunteer and Sentinel] have ever sired a chestnut colt." W. H.
Marrett, in the September, 1890, issue of the same paper, tells
us that the two bay sires Volunteer and Electioneer never had
chestnut foals. Both were by a bay sire (Rysdyk's Hamble-
tonian, which appears in the first table below), one being from
a bay mare, the other from a brown. In a sale catalogue issued
in 1903 C. \V. Williams says of the browrn stallion Belsire: "His
get are . . . bays, browns and blacks." This horse is a son
of the Electioneer mentioned above and of a black mare whose
sire was a black and dam bay. He is a full brother to the bay
Chimes which appears in the table below, and to Bow Bells,
bay, and St. Bel, black, both also probably homozygous.

I have found a good many sires homozygous for Hurst's factor.
The small number of gray and roan sires in the table below is
to be explained by the small number of those colors existing.
It will be noticed that two of the number have one chestnut
foal each recorded. Director's was found in an advertisement
in a horse journal — obviously a poor authority, as the pedigree
might easily have been false. That by Jay Bird is Cardenas,
trotting record 2:263/4. from a chestnut mare. He is recorded
as a chestnut by the "Year Book." But the "Year Book" does
sometimes make mistakes in the matter of color. Among others
I could mention is the case of the bay stallion Charley Wilkhurst,
recorded as a gray gelding.1 In this connection it is worth noting
that Hurst found about I per cent, of exceptions recorded in his
investigation, but wras able to explain most of them by showing
them to be probably mistakes.

'See The Horse Review for December 12, 1905, p. 1424.

OR IN THE AMERICAN HARNESS HORSE.

2O;

TABLE <>F SIRES HOMOZYGOUS FOR HURST'S FACTOR.

on.

Cblmi

•

v Mciliutii .
: ••-

Lfl

Or., \\J|K, -

• -
'.'.

bay brown 40

n bay i i 5

brown brown 12

black 15

bay k i 33

k 12
^ n

or 12
roan

bay 94

gray 3

gray 49

12
19

4
7
5

.
"

•

J
pg

-s

bay

black

bay

bay bay

59 404

«5

61 i

116

3 63

a 30

...
....

Pilot M«-'liiim. . .
••I ....
Mir-1

•H.i'i "I tlii'-r : the ch- ng.

mares.
nut marea.

I ii iv< ;. 'inid only 69 cases of chestnuts bc-ing mated togeth
Inn in .ill ihrse the rf-ult was chestnut. They are from t lu-
ll ,lli '\\ • [ illi' >n->: .

N u t wood

Color
fn>m Cli-

Sire. l'.mi.

bay gray
bay bay
. bay ?
bay ?

7
6

5
4
nut 4
4

II
69

\ttci[ nrv

I >;«!.

•

Wilk
M. mil'! ino K

•1

bav chest

. black ?

uith tv ....
\\ i 1 1 . (i

' 1

Tin- timlinu « >t t'n.ils from heterozygous sires and
iii.uv- i- ,i \«T\ -l->\v and laborious task, but 1 have found
t'ri'in tlu- I'cllowin^ -millions:

208

A. H. STURTEVANT.

Foals fr

om Chestnut Mares.

Chestnuts.

Total Found of all Colors.

Alcvone

bav

-i

Axtell . . '

bay

Boreal

bav

Guy Wilkes

bay

Norval

bay

Onward .

bay

Red Wilkes

bav

T 1

Strathmore

bay

•j

Total bay sires

bav

Grattan

black

Simmons . . .

black

Total black sires

black

j j

Alcantara

brown

Allerton

brown

I 7

Total brown sires . ...

brown

Alcrvon .... ...

gray

Re-election

srav

Total gray sires

gray

Grand Total

121

Exnectation. .

6ol4

121

Here again I have been handicapped in working with gray and
roan by the small number of sires of those colors, and also by
the fact that most of the best known of those existing seem to be
homozygous. However, I have found some sires of those colors
which throw a fair percentage of chestnut foals, as shown in the
following table, which shows all known foals. Two blacks are
also included.

Stallion.

Color.

Chestnut
Foals.

Foals of All Colors.

Bellini

black

Manibrino Patchcn

black

Alcryon . .

gray

•?o

Pilot, Jr

gray

Jay Hawker1

roan

Roan Wilkes

roan

Tom Hal, Jr.2

roan

1 The one chestnut from Jay Hawker is scarcely to be doubted, as he is Coun-
try Jay 2:07^, world's champion trotter under saddle, and one of the most prom-
inent race horses of the season of 1909.

'-This one chestnut also is not a doubtful one. His name is in fact Chestnut
Hal.

COLOR IN THE AMERICAN HARNESS HOR- 2OQ

It seems to me that we have here sufficient proof that H ur-
inal -uppo-ition is right — that the dominant character i- the
Mack in the coat. However his idea that the Mark, it" pre-ent.
- '.n ili,- tVt locks, seems to me to be unjustified, as Joe
I1 t< IK n. a h- gous Mack, has white feet, though the.- white

do,-- not n-ach as far up as the fetlock on the left hind one.
('•rattan, anotlu-r heterozygous black, also has three white fet-
1"< k-. It i- not at all rare to see pictures of bays or l»n>\\n-
\\ith one foot or more white as far up as the fetlock or further.
II-! amples: bays — Capo, both hinds; Moko,

i hind: Aii" l.eyburn, left hind; Allerworthy, both left>;
Hail ('loud. riv;lit front; browns — Rcdlac, both hind-: The
II lett hind; Searchlight, both hinds. At lea-t on.

the-. Moko is : lozygous.

In th< the next factor, bay, a complication arises in

nl to l>ro\\n. A- explained lx.-fore the presence of Ui\ i-

dominant e, and the color next below it in tin -cale

i- Mai k. l'.r>.\\: T l»ctwei-n these two, shading into both

• \i ' brown is usually a heterozygous color,

I 'lit that bay also is quite often het« i
uid that l'io\\n may occasionally be either of the homo/v-

i- I\|H--.( 1 1 /l /•> or t 'I I fib. This suggests the idea that the line
Ixtuieii Mack and bay should l>e drawn somewhere near the
black limit ot l.n.un. The obvious result of this complication
i- the creation ol considerable confusion in the numerical propor-
tion- of the t\\o (uli.r-.. It is evident that, except for this com-
plication and the appearance of some chestnuts, bay will acl
as ihou-h it \\ere an ordinary dominant to black.

I'.. l..\\ i- a taMe -Imwing twelve sires homozygous for the l>a\
factor. >i\ of them are bays, and one is the only brown certainly
kiio\\M to In- homo/\ gous for this factor. Two are ihe-tnuts,
and therefore la. k Hurst's factor. The other three are a K'"-I>'
and t \\o roan-, and it \\ ill IK? noted that all three of them appeared
in the taMe of -ires homozygous for HurstV factor. They arc-
not l.a\- Ucaii-c they also bear other fa» I -hall explain
later, uf the bays two are homozygous and four are heten>/y-
gousfor H in -t'- factor. The single black from KoU-n Mc(',r.
i- l>oM.\ Good, pacer, 2:II14, out of a daughter of A-hland

Wilkes.

210

A. H. STURTEVANT.

^oals.

Stallion.

•j.

C
tn
o

c
o
c

(f.

Ashland Wilkes

bay

bav

'•>

114

Rysdyk's

bay

brown

Hambletonian

Happy Medium.

bay

brown

Onward

bay

brown

bav

24.

117

Red Wilkes

bay

brown

I2J.

Sphinx

bay

chestnut

Prodigal

brown

bav

•3

TT6

Axworthy

chestnut

bay

bav

-J-3

•32

Robert

McGregor ....

chestnut

bay

Pilot Medium . .

gray

bay

gray

116

Tav Bird .

roan

brown

roan

- ,

I c

•J

Tl6

Mar crave .

roan

brown

roan

6=;

The mating together of blacks should produce only blacks and
a few chestnuts, since chestnut is the only color hypostatic to
black. However it does produce some browns and is recorded
as producing occasional bays. The bays so far found are: Kip-
ling 2 :2i^, by Gambetta Wilkes ex Margaret W., and Gipsey Bel
2 130, by St. Bel ex Gipsey A. Now there is some reason to doubt
the color of St. Bel. His dam was the black Beautiful Bells,
but his sire was the bay Electioneer, and he is the only black
among the eleven foals by Electioneer from black mares that I
have found. Moreover, counting St. Bel, I have found only
two black foals out of a total of 52 from Electioneer. Since
Electioneer was homozygous for Hurst's factor this small pro-
portion cannot be partly explained by supposing that more of the
52 would have been black had they had that factor. Neither
is it possible to suppose that the small proportion is due to sup-
pression by the gray or roan factor, since the 52 include only
two grays and no roans. It looks very much as though Elec-
tioneer were homozygous for the bay factor. St. Bel died young,
leaving few foals, so that I have been unable to get much data
about his descendants. As to the other apparent exception,
Kipling, I shall only call attention to the fact that neither he
nor his dam are very well known. The following table shows
foals from two black parents.

The case of foals from heterozygous sires and black mares is

COLOR IN THE AMERICAN HARNESS HORSE.

21 I

Bla Brown-

Chestoi

Bellini . . .

2 0
3 0
3 o
8 o

7 i

1.1 A

o
o

Gam'
Ci rat tan

\Vilkcs i

ben \\

\ViIk
Simp.

...
1

Nin«- i.tli'-r

g i

th<- cm- ulu-p- the uncertainty about brown causes tin-

trouble. Since the presence of Hurst's factor is nece— ary before

"I" the time colors in question can ap|x\ir I have left out

tin- 1 he-unit foals in the following tal)le of foals fnun black mare-.

•] ......

•

AN an)

Nti i v

•vn
trn

•nut

t.,v».

Blacks.

Tin- numbers in tin- .ibovc table are small, and, as in the similar
e \\ith Ibir-i >r, I have supplemented it with one

all kiK'un t'l-.ils, chrstnuts being again left out.

Mnu I.

nun
Mi k;it:;. •

ii \\ ilk .\vn

: AVn

•\vn

: >WI1

bn '\vn

-tnut
M.iiiil)riiii> Kini; . . . chi -inut

'1 In- !•'.!! 1 . • ' -tnut

('iiii'i i;ray

I'ilnt. Ji. . . .

: Hiiil .

Rays.

lilack*.

urn*.

-4

o
o

2
O

o
1

o
o
8

In
O

4
0

o
o
0

Ni»\\ t.> tin ii to the gray factor. In tin- first place. I in. ike no
claims th.it .ill -ray is epistatic to the four Usual colors. Perhaps

212

A. H. STURTEVANT.

it will be best to take up the grays by families, and I will first
treat of those in which it is epistatic, and then of the one in which
it seems not to be.

Most of the high-bred grays of to-day go back to Pilot, Jr.,
through an unbroken line of grays. This horse was a gray, son
of a black sire and of a mare of untraced breeding whose color
I have been unable to find. The gray sires in the next table all
get their gray from him. This table includes all known foals
except chestnuts, these being omitted for the same reason as
in the last case. Since gray is an unpopular color it is safe to
say that nearly all these foals were from recessive (gg) mares. I
have so far found only one case of grays being mated together,
and, since the produce of that mating was never heard from after
racing, I know of no horse homozygous for the gray factor, G.

Gray Stallion.

. Sire's Color.

Dam's Color.

Foals not Gray
or Chestnut.

Gray Foals.

Bavard

tjrav

Pilot, Jr

black

Pilot Medium

bay

erav

Re-election

bay

eray

I ;

Total .

The following sires are all sons of gray members of the Pilot,
Jr., family. Several of them have gray foals in this table, but
all of these are from gray mares.

Q to 1 linn

]

roals

Bays.

lilacks.

Browns.

Grays.

Roans.

Lord Russell

bay

Peter the Great

! M '

Darknight

black

Klectricitv

brown

Kxpedit ion

brown

High wood

brown

2J.

Mambrino Russell ....
Nutwood

chestnut
chestnut

6
70

0
0

There can be little doubt that in the Pilot, Jr., family gray is
an ordinary dominant, and there are other families where it seems
to be, though there is not as much evidence. One of these goes
back to the mare Sontag Mohawk, and through her probably
to imported Messenger, the foundation of the breed of American

COLOR IN THE AMI N HARNESS HORSE. 21

harm--- IP \n< >t h< r ^oes to the m;: »haw Belle, daughter

of Y'.unw Ba-haw. ^ray. Of the three li.>r-r- K-l»\\. Conduct. >r

M»hawk, Manager is a grand x>n of Ba>haw
ml Ah r\"ii i- "lit of Lady Blanche, daughter <>t I'm au -i-r.

I>am'> Color.

• r Chestnut.

gray 14 7

! .ay gray 7

•Jiut gray ii i-

Kn>- ,iinl Walnut Hall, a brother and son, respectively, <>t

h lirowns, have no gray foals anmn- tin
I lia\«- fdiinil.

n which gray apjx?ars not to In- rpUtatir.
I In- in -t IP.I : iinily that I know of is General \\ il

\\ilkes (an ordinary brown which ap|><ai-

in t\\<> i.f tin- iaMi •- .iln-a.K given and has nine di!tn\-nt x-n-
in ilicni am! in. ire. This stallion had some gra) !«M!-,

(•I' \\lii' I have found no record. But In- had t\\<.

\\hiih ha\i- prtnluced many gray foals. 1 In \ an
hi-puir. Ma. k. .,ii.l Bubby Burns, bay. I am not sure of tin-
color "I ill- dam- «•! any of Dipute's gray foals, but in the •
l'...l.l.\ 1 : -<MIH- (jf them are from bay mares. Dispu

inau-nial «.•!,, r |xduive I do not know, but Bobby Burn- i-
fr. .MI hixii. a ba\ dau^liter of the brown Dictator appearing
in -niiif . .f iln- lir-i tables in this paper. The colors of all foaU
fiitind lie. in l>i-pute and Bobby Burns are:
Pi-puti-: bay, ,\; black. 2; gray, 4; all. 9.
l'...l,|,\ I'.uin-: Lay, 40; black, 10; brown, lo; choimit, 3;

;',- all. i

1 'hi- i- n-rtainK a dil'u-rent kind of gray from the otlur- ju-t
. ril'td. but I ha\r not enough data to try to rxplain it.1

'In 'i..n with tlie bay and gray factors I may q i..ll,.\\i:

tin- IH-II arson ("The Law of Ancestral Heredity,1 i-ika>

th»ui;h it was written about tli i-lil.n.| I:

lil.u- : in lu.rses were 'ret' \\li.-n tu» }•'.

-h..ulil rxi'«vt ciily Mack oil-spring, but black can <li i'..r a Ki-iu-ration

>r even two and then ir. Or. take a case like that of a gray h. unt,

2I4

A. H. STUKTEVANT.

The last character I have to deal with is roan. This, like gray,
is epistatic to the four usual colors in most families, but may
not be in all.

Many of the roans of to-day go back to the old roan race-mare
Lady Franklin, through her daughter Lady Frank and grandson
Jay Bird, both roans. Jay Bird sired Eagle Bird, Jay Hawker,
Allerton and Jackdaw, and Jay Hawker sired Jay McGregor.
The following table shows all foals but chestnuts.

als.

Stallion.

Color.

Bays.

Blacks.

Browns

Grays.

Roans.

All not
Roan.

Eagle Bird

roan

I c

Jay Bird .

roan

"54

I c

•2

Jay Hawker

roan

Total

•}

,S I

Jay McGregor

bav

Allerton

brown

•12

Tackdaw. .

brown

2=;

Allerton's roan foal is from a roan daughter of Jay Bird.

Another family goes back to Laura Fair, roan, through her
roan granddaughter Spanish Maiden. This last mare produced
I bay and 3 roans, including the sire Margrave. Tom Hal, Jr.,
founded another family of roans, and another goes to the roan
mare Tilla, which had 4 bay foals, I brown and 3 roans, the latter
including Fred S. Wilkes. The Brown Hal appearing in the
table below is a son of Tom Hal, Jr. Chestnut foals are omitted
as before.

Foals.

Stallion.

Color.

Not Chestnut
or Roan.

Roan.

Fred S. Wilkes

roan

I -i

Margrave

roan

•ic

•?o

Tom Hal, Jr

roan

Total

4=5

Brown Hal .

brown

where gray remained dominant for three generations only to disappear before the
chestnut of the mare Blue Stocking in the Viscount and Blue Stocking filly Miss
Johanna!" Just what that passage was intended to mean is a problem which I
have not yet solved. What do blacks produce when mated together, and what
has that to do with skipping a generation or so? And if a recessive cannot skip
what can? Certainly not a dominant. What is to prevent us from supposing
Viscount a heterozygote?

COLOR IN THE AMERICAN HARNESS HORSE. J I ;

roan ami also the two gray foals of Brown Hal probably
get their color from their dams, since in all cases the-e \\cre.
daughter- "f r. MII or gray sires. As to the kind of roan- these
I can on! that Margrave and one of his foals, and one

of tin- foal- of Tom Hal, Jr., are all red roans.

I li md .-ix roan foals which had neither pan-in roan.

( )m- was from a dun man-. One was from two bays and another
was from ut and a bay, the chestnut beinv; Robert

M' • , which almost certainly carried no gray factor. An-

other \\.i- from a bay sire and a chestnut dam. The other i\\..
hhada . , and at least one of them was a black roan.

d i iced by the thousands of cases of mat in-
tin r hor-e- ,in isa very small jx-rcentage. It i- to be
noii. eel thai are not closely related. Even tin t\\<>
• rued .11 I'ilot, Jr., and one Sontag Mohauk.
I eems to me probable that red roan at least is an ordinary
dominant . ami that all but oiu? of the above cases are mi -take- or

• ption-. h i- (|iiii' ible that all horses having the fai

A' .(I- i .n. the i\| an depending upon the color the II«IM

\\iiulil ha\e been if he had not had that factor. This, if cor-
.plain- away the dilhculty presented in the next paragraph
in -o far as r- Mil is concerned.

1 he [elation I K-t \veen gray and roan is not clear. It seem-
probable that some of the black roans may be connected \\iih
. , bin an examination of the tables given above will convince
one i hat. in 1. the two colors are quite distinct. 1 ha\e

found niil\ t\\o in-tances of the mating together of grays and
roan-, ami in both the result was gray. Of course much im-iv
e\ idem e \\oiild be m -eded in order to find out how tin \ ,ut
io\\ard each other. The relation between these two laci.>r-
aml the bay factm i-> also not quite clear. It is evident that the
presence of eitln r can conceal bay, but whether or not either
can .i|>pear iii the absence of the bay factor is not certain. I am
inclined in think that they can. If gray cannot we ha\e an
explanation of the gray foals from the black Pi-pute, but
m> help in the much harder problem concerning hi- bay half-
brother, liobby Burns. One would expect to tmd -<»ine ch.
nut- carrying gray or roan factors (if the ChhR horses are ch<

2l6 A. H. STURTEVANT.

nut roans there should be no chestnuts carrying the roan factor).
With one possible exception in the case of each color I have found
no such case. How unsatisfactory these two cases are will
appear from the color pedigrees of the horses concerned.

( Banker Rothschild, f Rothschild.

brown. { Pilot Anna, gray.

i Lady Forrester, ] Royal George, chestnut.

chestnut. \ Belle of Saratoga, brown.

f Young Clay Pilot. / Clay Pilot, bay.

North Wind, roan. bay , R Executorf brown.

I Lola M., chestnut. { Ljght ^ chestnut<

SUMMARY.

This study of the pedigrees of blooded trotters indicates that
the color of such horses is usually controlled by five factors, as
follows. First, a factor for chestnut, C, present in all the horses
studied. Second, a factor for black, Hurst's factor, H, epistatic
to the factor C, and hypostatic to the three following. Third,
a factor for bay, B. Fourth, a factor for roan, R. Fifth, a
factor for gray, G. R or G inhibits B if it is present, but whether
they depend upon its presence for their own appearance or not
is not clear.

COLUMBIA UNIVERSITY,
June, 1910.

THl \I\K-l PITM OF THE AXOI >< >\ II N.E.

In the June number of the BIOLOGICAL BULLETIN (p. ;i . G.

nl \\ . ( . Curtis have published a paper on "tin- Mar-

siipium of tin- I 'nionida'." in which they say that the lateral

• •inlar r tubes cut off from the original (primary) water

tubi-- in the in.ir-upimn of the Anodontinae, described by myself

in \...,' ru.iry, 1910, are not present. In order to show

thi-.. tin \ publish three figures of horizontal sections throiisji the

m.ir-upia of tin. ies of Anodontiiuc.

I nisunderstanding, I want to point out, that, in

t\M.i.f the figun vrcd to, these secondan- water tube- \KI

pin SENT, in. v and typically in Fiji. I. In 1 L

ti.t, :hein -able, while in Fi^. 2 they are not yet

develop : I tul>csarc not blood vessels, as might be be-

ln \t-.l .ifti-r -u|M-riiei.il ii. \i-tination.

|.,r tlie I nnot go into detail, but must refer t<> in\

funiif publie.ition illu-trated by micmphotographs) in the
.17. • :*• Museum, where additional facts \\ill be

pul ili-hnl.

A. E. OKTMANN.
: PITTSBURGH. PA.,

217

Vol. AV.V. September, 1910. No. ./

BIOLOGICAL BULLETIN

ACCESSORY CHROMOSOMES IX MAN.

MICHAEL F. GUYER.
UNIVERSITY OF CINCINNATI.

•iblir.it ion of my papers on the sperm, i isis of

tin guinea and of the rhirken respectively (Guyc-r '090, '<

in \\ liirh was recorded tin- finding of a chromosome or chromosome.

complex comparable- to the "odd," "accessory" or "X-elrmeiu,"

ile-i rilHxl so frequently of late as occurring in a \\i<le r.m-i . .f

tin- Artliropoda, particularly the Tracheata, I have rx.imiiinl

inatri-i.il from otlu-r \ertebratcs and can at present record it-;

presence iii the rat, its probable occurrence in tin pigeon

.ihhoii-li this \\ill require some further corroboration), ami ii>

piiiious occurrence in man. Inasmuch as the material

u- stu< l\ in the rat is in the hands of a student lor further

iii\e-ti^aiion. I >hall confine myself in this pa|>er to a de-rrip-

tioii ot tlu- t wo accessory chromosomes as found in man, together

\\iih other leaimv> of human sjx;rmatoR:enesi-

I i >r m\ studies on man 1 have l>een fortunate in l>eiivsr able

•ibtain CM epti. -nally PIKK! material through the courte>\ of

m\ (ollea^iii. I >r. Paul. < '• \Voolley. The subject from which

the testicular material was secured was a negro thirty years of

\\ ho h.nl ilie<l MuliK-nly from the rupture of an aortic aneuri-m

1 • no. 151,1.}; Pathological Records, the Cincinnati City

II. spital). \ - was removed within l)etween an hour and

an hour and a half after death while the body was still warm

and >li< e- \\etv placed immediately into Gil-on'v and into

-. fluid-.

The mounted sections were from ti\e to twelve microns thick.
(,f tlu-m \\ere >tained in Heidrnhain'.- iron-lurmatoxylin

22O MICHAEL F. GUVER.

and counter-stained with Congo red or acid fuchsin, although
Delafield's haematoxylin was used with some.

An abundance of cell divisions were found to have been in
progress at the time of death. In a given field of the microscope,
in a favorable region, it was not unusual to observe as high as
six or seven cells in various phases of division. As many as five
or six of such areas might exist in a single section, although it
was more usual to find only one or two. The material was very
uneven in that slides would be found in which section after sec-
tion showed division stages, while in others divisions were scarce.
These facts indicate that there were proliferating and resting
zones in the testis. The stages found in most abundance were
the metaphases and late prophases of the primary spermatocytes.
It was a comparatively simple matter to find spindles on which
the ordinary chromosomes were in metaphase with the two
accessories, closely associated, well removed toward, or at, one
pole (Figs. 6, 7, 8 and 9).

In the literature of the subject much confusion prevails re-
garding the number of chromosomes characteristic of man.
There is wide disagreement in the counts of different observers
and there seems to have been a great dearth of material showing
division stages. Most of the enumerations are based on obser-
vations of from two to eighteen cells and these often in ques-
tionable stages of preservation. The great difficulty apparently
has been to secure material which was sufficiently fresh or which
was not diseased tissue that is notoriously irregular as regards
karyokinetic phenomena.

As early as 1881 Flemming discussed mitosis in the case of
man illustrating it with some six figures (Taf. 3, Figs. 11-16)
of which Fig. 16 is from leucocytes of leucemic blood, the others,
from the corneal epithelium of two different subjects from each
of whom an eye had been removed because of affection of the
bulbus. Although at this time he made no definite record of
the number of chromosomes, his drawings show them to be
considerably in excess of sixteen, the number later announced
by Bardeleben ('92).

Writing several years later, however, in response to the 1892
paper of Bardeleben, Flemming ('97), from a reexamination ot

ACCESSORY CHROMOSOMES IN MAN. 22 \

old in. iii-rial. - the number of chrumnsom« \\vmy-

foiir. He < ites tin- papers of Hansemann ('91, '93 as the earlie-t
attempi- kn«.\vn to him to make a count of the fhn>m<»«>nu-- of
man. Bui sin< e 1 l.msemann records eighteen in one case, twenty-
four in another, and forty in a third, the latter apparently

:nattd Irom the spireme stage, and inasmuch as he him-elt
admit- ihat his count was very uncertain, concluding with the

•• -nient that, "die Zahl sicher holier als 24 sei," we may fairly
di -regard it. I think, in the light of modern technique. In this

ml papi r 1 lemming ('97) states that his count i- ba-ed <>n
iinl\ ti.ur cell-divisions in which the chromosomes had ju-t
split preparatory to separation. His exact statement of hi>
ol,-«-r\ ations is as follows: "Es gelang das zwar bei keinen
>i< her. alx-r bei zweien der vier darin enthaltenen Mito-< n
ann.iheriid ; es scheinen in beiden I'iillen 24 Doppelchromosomen
zu sein. Bei beiden sind es jedenfalls mehr als 22 und. \\ ie ich

• •n /u konnen glaul>e, weniger als 28; an einigcr Stellen di ( ken
sie sich so, dasx eine exacte Zahlung mir unmoglich \\ird."
I !• -mining's material had IK.VII fixed in one sixth per cent, chromic

I and stained \\ith safranin.

Kirdelelx-n has published thn-e paj>ers ('92, '97, '^s ' mi

the spermatogenesis of mammals including man in which he

comes l«> the conclusion that the number of chromosomes in

the spermatogonia and siK-rmatocytes of man are sixteen and

•ectively, and in his later pajKTs he sets down four

as i lu nnmlH-r that ultimately reaches the spermatids. That is,

there i- in successive divisions a reduction in numbers from six-

ijn and then from eight to four. This is much the

• lit ion thai I have found prevailing in birds (Guyer, '02, '<
\\ilc.'\ ulied sections from a testis which had been re-
moved 1 1 oin a man lit ty-four years old, in an operation for hernia.
Alihon'ji -,T,, i al swelling had existed for a year previous to tin-
operation the te-tis seemed to be normal in size and appearan.v.
He repon-, that, "In my material the number seemed to be

iteen, the difterent counts resulting in ti ran-in^ troin

titteeii to nineteen." lie remarks howc\'er u|«»n the Mrikiii^
absence, of kar\okinetic stages, so that his ob-ervation-, were
ba-ed upon a \er\ limited number ot di\-i>ion>. liec.m-e of

222 MICHAEL F. GUVER.

this lack of favorable stages he says: "The whole organ was,
therefore, sectioned and in the great number of sections thus ob-
tained, not more than twenty cells were found in mitotic con-
dition."

Wilcox continues: 'The few cases of mitosis observed were
in spermatocytes of the first order. One could easily distinguish
spermatogonia, spermatocytes of the first and second order,
spermatids, and numerous nearly mature spermatozoa. The
number of the latter to be seen was very large and precludes the
assumption that the testis was functionally impaired by age or
by hernia. In the opinion of the writer, this condition merely
indicates that all the various processes in the spermatogenetic
series are not necessarily to be observed as taking place at the
same time. I can see no reason why there might not become
established in the testis periods of cellular activity alternating
with periods of cellular rest."

Unfortunately Wilcox gives no drawings with his paper nor
does he state definitely whether he regards the eighteen chro-
mosomes seen in the spermatocytes of the first order as the
reduced number or not. He does remark, however, that, "in
many cases they were plainly arranged in the tetrad or ring for-
mation which has been observed in a pretty general variety of
investigated species," consequently the inference would be that
a synapsis had occurred and that one might expect to find in
the neighborhood of thirty-six as the somatic number.

The latest investigation on the number of chromosomes in
man with which I am acquainted is that of Duesberg ('06).
He reviews the work of Hansemann, von Bardeleben, and Flem-
ming and on the strength of his own observations concludes
that Flemming's count of twenty-four is correct. The excessive
number found by Hansemann he would account for on the basis
of the abnormal increase in the number of chromosomes which
is likely to occur in pathological tissues. In the case of Bar-
deleben he is inclined to believe that very thin sections (three
microns) are responsible for the smallness of the count since he
regards it as probable that part of the cell had been cut away.

The tissue upon which Duesberg worked had been fixed in
Flemming's or in Hermann's fluid and stained by the iron-hat-

V CHROMOSOMES IN MAN. 223

matoxylin method. However, the number, twenty-four, which
he records for in. in was not 'determined by direct count but was
intVrn-d i'r«>m tl that he found two or three clear cases of

Ive « hromo-imit •- in the primary spermatocytes. That his
find in-.: "t t \\i-l\r in the primary spermatocytes was comvt is
l...rm- MUI by in rvations but he is not justified, in con-r-

qm-iiri-. in -i.ttin- i hat there must be twenty-four in SJKTIU.I-

•nial <>r -om.ni. nil-divisions. My material shows that
two "t tin- lui-Kr chromosomes are the univalent accessor
and a cl< int of favorable spermatogonial chromosomes

n \i-.d- .>nly t\\« -nty-two. This means in all probability that
• •I' iln i \\d\i- c hromosomes of the primary spermatocyte,
.in lii\.iltni .Mul two accessories. Although Duesberg examined
tin- spermatogonial chromosomes he states that he was unaliK-
to ' "inn tin-in « \.niK U-yond determining that, contrary to
the «i|iiniuii of \'on Hardrlt-U-n. there were clearly more than six-
teen. II' : I- - lunhi i p. 477) that, "Je n'ai pas pu les compter
rx.u i. UK nt. taut ;\ i-aii-r de la petitcsse dcs cellules qnr du
iioml.iv assez -1<\. dr- chromosomes, mais dans quelqucs cas
l.i\ o|-.iMi - .,11 li-ur iniii u a pu el re tMitreprise, j'ai obtenu

dr- n-uli.ii^ trrs M>i-iu> dc 24, jamais sup6rieurs ;\ ce nombre
d.in- lr- n llules normales." And in conclusion he says: "II r£-
Miltrdr l.i i |iu lr nombre des chromosomes est certainement a mon
a\ i-. di- u dan-, les sj)ermatocytes et par cons6(juent de 24 dans

;permatogoni< U-- i»llules somatiques. C'est la confir-

matiiin dc r«i|iininn dr I Innniin^."

In i In 'o-iu-r.il •>« h< nir of the s|K-rmatogcnesis of man there ap-
|n-ar> IK In- in >i hin- unique. One can readily recognize the usual
fniir m 'in -r. uio n- n i-i-IU; viz., spermatogonia, primary sper-

in. r i -|K rniai'n ytcs of the first order), secondary sper-

matocytes "i ~\» mi.ii,>. \ tes of the second order), and la>il\
spermatids which transform directly into the spermatozoa. An
abundant' • i-il\ identified Sertoli or nurse cells are in evi-
dence. < '< > a-i«mal centrosomes were observed in suitably stained
pri'i>aratii'ns Inn I have not pictured any in my drawings U--
cause the preparations from which the latter were made were all

'K-nJx d<-( i ilori/rd that the stain had evidently o>m])K-ii-l\-
di-.ip|u .IK d I'mni an\ centTOSOmes which mi^hi ha\v Urn |>iv-nn .

224 MICHAEL F. GUYER.

The matter of counting the spermatogonial chromosomes, it
must be admitted, is one of great difficulty. In the late prophase
or equatorial plate stage, the only time at which a count is possible,
they lie for the most part in an irregular band around a central
clearer area. In the vast majority of cases only a deeply stained
mass of small contiguous or overlapping chromosomes is visible
in this band and an accurate count is out of the question although
one can frequently determine that there are over twenty. In sev-
eral instances, however, in which the positions of the chromo-
somes and the degree of the staining were favorable, twenty-two

distinct chromosomes, never more, were visible.

•

There is considerable range in size among the individual chro-
mosomes of the spermatogonia as well as observable differences of
form. Most of them were rod-like or oval in shape although some
were more nearly spherical. In several though by no means all
instances two chromosomes, closely associated, were seen lying
at some distance away from the main band, out in the cytoplasm.
Taking into account this isolation, the rounded shape of these
chromosomes and their relative sizes, it seems very probable that
they are the two accessory chromosomes which do not manifest
their presence for a certainty until the next division. It will be
observed that one is somewhat smaller than the other. This con-
dition obtains also between the twro chromatin nucleoli of subse-
quent stages as well as between the accessory chromosomes where-
ever they can be identified, and one is led in consequence to
strongly suspect that they are all one and the same thing. This
inference is all the more justifiable when the relation between the
chromatin nucleoli and the accessory chromosomes in some of the
lower forms is recalled.

Fig. 2 represents a nucleus of the primary spcrmatocyte in the
spireme stage which shows the two chromatin nucleoli in question.
In deeply stained specimens these nucleoli, especially the smaller
one, are not always evident but in preparations stained by the iron-
hsematoxylin method and then almost entirely decolorized, even
as regards the ordinary chromatin of the spireme they are usually
conspicuously visible. It should be mentioned that occasionally
other small nucleolus-like granules were observable but since there
was no constancy in their presence, size or relationship, I h;i\r

ACCESSORY CHROMOSOMES IN MAN. 225

ft It iu-titied in ignoring them in the prc-ent di-cn— inn. FL
repre-em- th* me in the contraction pha-e which is not very

pronounced in m. in. It will be observred that the two character-
i-tic ( hroinatin nudeoli still persist.

I • primar\ -i«-nnatocytes when ready for divi-ion. a- has

alre.i<l\ }>< • < ' 1, reveal twelve chromosomes in late prophase

irly metaphase (Figs. 4, 5). In Fig. 4 the two a< are

seen at and the remaining chromosomes, jud^in- from

their in< n-a-ed -i/e and changed form, are bivalent, ivpn. -cnt-

iin-il imivalent chromosomes of the spermatogonium.

Thai i ^inal twenty-two chromosomes t \\rnty ha\e

jiain-d io ii.nn the ten bivalent* of the primary s|x-nnai md

i\\o h.i\e r< inained unpaired as the accessory chromosomes. In

\ ii i- no! i \idcntjust which two are the accessories although

i \\el\e i In - are present.

li i- ob\ ion- iroin tin- the figures (Figs. 4-9) that there is con-
-id< r. (Mr dil!- in the si/e of the various chromosomes ot tin-

|irim.i! Although the attempt was made ii \\a>

not found |>o--il»lr t. . .il\\ i\ -s idrntif\' the individual chroino-'u;
Tin :•• i|n\\ n in -i/r from some three or fcmr large ones to i \\. .

or ilin . -mall oiu-^ luit the fluctuations in sixe, prohabh dm- lor tin-
in. -i pan to diitrtvnces in the effects of fixation to-ctlicr \\ith
dill, n in . of extraction of the stain, were too great to render

idniiilii aiion -nrc. In \vry strongly tlecolori/t-d -ret ion-,

ill\ \\ln n < oiinterstained with Congo red, one large chromo-
some in p.inirular fretjuently exhibited a tetrad-like formati -n.
\\hilc ilu- oth.-r l.ii^e ones at times showed in. in or less drliniu-
indication- oi l«.|.in-. 1 n some cases this was siillicu-ntly niarki-d
to interfere with accurate counting. In a very fi-w in>i.u:
i« \\ I think as io U« practically negligible, tlu-n- app«-arcrl t» In-
foiirir-'ii in-tr.ul of the customary twelve chroino-omes, but tin-
r\tra < •lironio-miu •- always took the form of a tiny pair which I am
inclined to think had Ixxrome split off from one of the ordinary
tetrad- or \\hich had through some chance never entered into tin-
proper tetrad formation. They were always united by linin-like
-trand- to one or two of the larger chromo-om

I i--. (.. ~. ^ .md 9 show the two accessories in characteristic
p. .-it ion-, ^ide \<\ side. the\ alwa\ s pass entire, considerably in

ail\ ance of the di\ ided ordinarx chromox .me-. io\\ard one pole.

226 MICHAEL F. GUYER.

Of the ordinary daughter chromosomes of this first spermato-
cytic division, a pair of small elongated ones not infrequently
are the first to emerge from the general equatorial mass as shown
in Fig. 7. One is led to suspect that they may possibly be compar-
able to the small pair of chromosomes found so constantly in cer-
tain of the Tracheata although the evidence is not sufficiently
decisive to make this an established fact.

It is inferred that the division of the primary spermatocyte
is the reducing division, not simply because such a division ordi-
narily occurs at this stage, but from the fact that the chromo-
somes after divergence (Figs. 10, n) when compared with corre-
sponding divisions of the secondary spermatocytes are seen to
resume more the elongate, rod-like appearance that characterizes
the univalent spermatogonial chromosomes, and also because
the accessory chromosomes pass over entire to one pole here
while they are halved in the next division.

It is evident from the foregoing that as regards chromatin con-
tent the result of the division of the primary spermatocyte is the
production of two dissimilar cells, one of which receives ten, the
other, twelve chromosomes. Fig. 10 is a drawing of one end of a
late anaphase of such a division showing twelve chromosomes (10
plus 2 accessory). Fig. n, in which only ten chromosomes are
visible, was drawn from what is probably the reverse end of a some-
what later anaphase than that shown in Fig. 10. It is just pos-
sible that it is a prophase of division in a secondary spermatocyte
where univalent chromosomes come to the equator, but if so it is
the exception rather than the rule, as the secondary spermatocytes
ordinarily divide according to a different scheme. In any event
the drawing serves to illustrate the fact that some daughter cells
of the primary spermatocytes have twelve chromosomes and some
only ten.

In places both primary and secondary spermatocytes were found
dividing in the same field and one is led to conclude that either
there was no intervening period of rest between the two divisions
or that it was a very brief one. In other instances, however, un-
doubted resting stages of secondary spermatocyte nuclei were seen
in abundance. Approximately half of them showed, under
proper decolorization, two chromatin nucleoli of which one
was somewhat smaller than the other.

sQRY CHROMOSOMES IN MAN. 22~

VYhile at the < -inclusion of the divisions of the primary sper-
mat'K M and twelve chromosomes respectively were delivered

\'< the pair- of daughter cells, nevertheless, when the latter a-
•ndary Bpermatocytes become ready for division, half of them
-how h\e. mil i In- remainder seven chromosomes. A -.-cond pair-
inc "t tin- ordinary chromosomes has evidently occurred, so that
tln-n- an- li\<- l.ivalent chromosomes in each type of cell and the
additional i v. ssories in the one type. 1 i_ u i- a dra\\ in-

ns secondary spermatocytes; th< -ho\\> five

bivalent < hroino-oim-s in late prophase, the other more than ti\v
chr"iiio~.,iiir^ in nu-taphase. These two cells art- undoubtedly
tin i\\o daii^hii r n Us of the same primary spermat'>i > tr. i
i ; • "Hi- d.iiii;htiT cell containing a group ol i rhn>ino-

:iidil)' a late ana phase of division which shows at one

i-nd five chromosomes. The number of chromosomes at tlu-oppo-
site i»'li "I the second cell should of course be five although l>r-
cause "1 ilu- dm-*- ma--ini; it could not he |M)>iti\d\ drti-rinincd.

i} represents a late anaphase of division in a aecondar) -pn-

ma' \\hiih niaiiitt -ily had had seven chromosome*- in nu-ia-

pli. .

Both acccssoi nosomes divide in this semiid -pi-niia1

•!i\i-iini period so that each resulting spennaiid receives

n chroinMsunu-. (Fig. 16). Fig. 15 reprr-ml> an aiia|)lia-r

o| ili\ixi,,n in a secondary spermatocyte showing -till at tin- f|iia-

i"i c.t iln- ~;iindle a laKgmis chnnnatic mass. Smli a rendition

\\as toiind in -«-\«-ral instances and while I l»«-lif\«- ii to In- tin- two

hromosomes which happened merely to !•«• unta\oralil\

pla.iillor I.I.M r\ alion, I rould not positi\'rl\ idrniilv it a^ -nch.

I r. -in tin relative positions of the chromosomes as seen in 11-. i'«

onr \\onld inli-r that the two sets of acce--oric- \\ • -n- the la-i to

have passed from the equator to the poles of the spindle M<>r»
over, -ni-h a ^ of the accessory in this divi-ion was ob-

served in 1-oth the guinea and the chicken (Guyer, 'oo .

It .should l.t- mentioned that occasional di\-isinn stages \\>
\isjMc which, judging from the smalhu-ss ol tin- cell and the -\/c
and sli,i|«e M|" the chromosomes, looked a- it" they miijit he secon-
dary spermatocytes preparing to divide with the univalent t\pe
iten or t\\rl\i- of chromosome. It i- po— ihK-, for instance, that

228 MICHAEL F. GUVER.

Fig. 1 1 represents a prophasc of the secondary rather than an ana-
phase of the primary division although I am inclined to think it is
the latter. If such simple divisions do take place, however, they
are certainly scarce in the material which I have examined so far.

From the foregoing evidence it is manifest that there are in all
two distinct groups of spermatids equal in number; namely,
those which have received five and those which have received
seven chromosomes. These chromosomes soon lose their visible
identity and the spermatids are apparently all alike except for the
significant fact that approximately half of them, in such prepara-
tions as have been stained by the iron-haematoxylin method and
then all but entirely decolorized show two chromatin nucleoli.
It would seem probable that these nucleoli stand in direct genetic
continuity with the two eccentric chromosomes seen in the sper-
matogonia and the two chromatin nucleoli and the accessory
chromosomes of the spermatocytes. Fig. 18 represents two con-
tiguous spermatids, one of which shows no nucleoli, the other, two.
Comparison with Fig. 19 shows the relative conditions of size be-
tween the nucleoli of the spermatid and those of a primary sper-
matocyte.

As to the meaning of the second conjugation there seems to be
at present no clew. I have commented on it briefly in a former
paper ('090, p. 509). It is not peculiar to man for I have observed
it also in the pigeon ('02, '03), the guinea ('oga) and the rooster
('096). Undoubtedly Bardeleben ('97, '98) still earlier saw the
same phenomenon in man, for although my results do not agree
numerically with his count of sixteen, eight and four respectively,
evidently, from the relative proportions in his counts, he had come
upon this second curious numerical reduction.

Assuming that the respective ehromosomes are more or less
qualitatively differentiated, such a numerical reduction, however,
by no means necessarily implies that there has also been a second
qualitative reduction. Aside from the improbability of such a
reduction, the general appearance of the divided chromosomes
would not warrant this interpretation; for instead of the elongated
univalent type as seen in the spermatogonia or in anaphases <>t tin;
divisions of spermatocytes of the first order, the daughter chromo-
somes here retain the rounded appearance and increased size that

ACCESSORY CHROMOSOMES IN MAN.

liivalent types (compare Figs. i. i'>.m<l 11.
[3, 14. [5, P. and i; Thus while half of the spermatids receive

. .iml hall" seven chromosomes, in terms of uni\ alence- the num-
ber- \\ould in all probability be ten and twelve r< ively.

Ina-mn< h .1- tin- -(lermatids transform directly inio -|HTinato-
ii !<>1|(,\\ - th.it there must be be two classes of the latter diltt r-
uith r- to whether they have or do not have tin t\\.

chpimo-oinrs. Thus the conditions in man appear t" be
much the -aim- that Wilson ('09) describes for .Vvrc
<>n< <|iia-li-biu lamily (Coreulze).

\tiuii P. u- ;•!»•> of such dimorphism of tin- -|H-rmai'

ha\e I 'led iii various invertebrates, particularly in in-

irachni apods, and it has Ix'en clearK dtni«>n-

-n.iiid Hi by spermatozoa which J>O-M -- thi-

< hromosome group (there may lu-om , t\\<>,
tin. chromatic bodies, depending upon the

1«.|> ii >, those fertilized by spnin.n

\\hirhd<i!i<>i [M)t>sess it, lop into males. Hence th< •>!>

me to id. il by some of our most careful and expe-

workei in actual sex determinant. In any event it i-
o|i\inii-l> : \\ith tin- determination of sex either a- t.ui-.

i In tin li^lit of numerous recent researches both «.n
|ilani- and animal- tin- idea has rapidly gained ground that
ari-r- n. 'i .1- \\as lojig l>elieved, as a response of the developing
:IHHH t.i ^timuli from without, but that under normal condi-
tion- at least, il is automatically determined by some- internal
ph\ ii .il mechanism.

Ina-inn. Ii a- [\\\> intricate matter has been repeatedly and ex-
haustive!) di-.n-M.l pro and con during the past ten year.- it i-
unncn---ar\ I"! in. to enter into a review of the subject anew.
Forth general reader who may not have kept in touch with the
current literature «>f the subject, two excellent critique- are now
available in the recent papers of Wilson ('090) and Morgan '10
In tlu-e |«aper- oii«- will also find thorougliK"iiVv di-cu— ion- . .1" the
subtle problem a.- to whether, assuming that th. sories are

sex determinant-, the matter of sex determination i- to !„• re^ardi-d
a- a qualitative pi effected by soi ne inherent peculiaritN' of the

accesson i-hn>ino-. mu-. or whetlu-r the relation o| -uch .1 thro-

230 MICHAEL F. GUYER.

mosome or group of chromosomes to sex is merely a quanti-
tative one, the female type resulting when a greater amount of
active chromatin is present. Extensive bibliographies will be
found in the recent papers of Wilson ('05, '06, '09), Payne ('09),
Morse ('09) and Morgan ('10).

In conclusion I wish merely to point out that as regards ac-
cessory chromosomes, conditions prevail among vertebrates
(guinea, chicken, rat, man, etc.) similar to those found among
numerous Tracheata (and probably certain other invertebrates)
where the accessories are undoubtedly associated in some way
with the phenomena of sexuality. In Syromastes (Wilson, '096),
which seems to parallel most nearly the condition found in man,
half of the spermatids were found to possess two more chro-
mosomes than the remainder. It was predicted by Wilson
that in consequence the somatic cells of the female of this species
would show two more chromosomes than the somatic cells of
the male. Later the facts were found to be in exact accord
with his prediction, the somatic cells of the female containing
twenty-four, of the male twenty-two chromosomes. Similar
verifications have been made in other tracheate forms.

In the light of these facts we should expect the somatic cells
of man to contain twenty-two, and of woman, twenty-four
chromosomes. The tissues of the female have not yet been
studied with this in mind. Flemming ('97) records the somatic
number of chromosomes, determined from corneal cells, as twenty-
four but unfortunately he does not record the sex of the sub-
jects from which the material was obtained. If it were a female
his count would bear out the interpretation given above.

SUMMARY.

1. Twenty-two chromosomes differing considerably in size
occur in all spermatogonia in which a definite count could be
made. In a few instances two, apparently the two accessory
chromosomes, were seen considerably to one side of the main
mass of chromosomes, surrounded by a small clear court of
cytoplasm.

2. Twelve chromosomes appear for division in the primary
spermatocyte, of which ten are evidently bivalent and two
accessories.

V CHROMOSOME IN MAN. 2U

;v The t\\o a< •« -t-sory chromosomes pass undivided to one
• •t" the -pindle considerably in advance of the other chro-
\vith tin- re-ult that half of the daughter cells in thi>
division re< «-i\e twelve, and half, only ten univalent chronic-. >im-
'1 hi- i- evidently the reduction division.

4. The ten univalent chromosomes \vhich passed t<> the one

• ndary -perm e unite again in pairs, at least in the ina-

j«>ri • form five bivalent chromosomes which appear

at the e(|u.it'»r of the spindle when the cell is ready for di\ i-ion.

Tin di\i-ion lii-re is presumably an equation and not a >ec«»nd

reduction di\i-i"M. judging from the size, shape and general

a|i|K -aram , the resulting daughter chromosom Thus

while . • the- s|XTin.uiiU formed as a result of this division

live chonv -.the latter are bivalent and equiv-

.il< lit to ten of the somatic or spermatogonial chromo-'-nu ~.

'Ih, -me -li^ln i that the secondary sjxTnuito,

in.r. ion.ilK di\i«le \\ith these chromosomes in their origin. il

:diiion ol uni\.ilei

5. !• 11 oi' tin- (\\il\.- i hronioMnnes which passed to the othrr
pol.- of tin- spindle in the primary s|x«rmatocyte behave in
i iselj the s.uue way as descril)ed in the last paragraph. The
t\\, ies come to the equator of the spindle

in i h« d.irv s|H-rin.itoc\ te with the five bivalents thus

making in .ill seven, l.uh accessory now divides so that the

h receive seven chromosomes; that

i-, ii\i liixalent |ihis t\\o accessory, or the equivalent of twel\e
uni\ alent i hroni"-' -ines.

o. In reality, then, ol' the total number of spermatids, lull
h.i\e in .ill |noli.il.ilit\ recei\-ed ten. and half, twelve do plus 2}
uni\ alent chronio-omes. Inasmuch as the spermatids transform
direct l\ into s^-nnato/oa, there must lx- two classes of the latter
difleriiu \\ith i. to whether they have or do not have the

t\\. ;iromosonies.

7. It is a si-niiiiant fact that approximately half tin resting

s|HTinatid- \\lun >trongly decolorized after iron-haematoxylin

staining. -ho\\ two chromatin nucleoli and halt do not. h

nis probable that these nucleoli may corn-pond to the ac-

son diromos, uncs and are to lx? identilied \\ith the two

232 MICHAEL F. GUYER.

nucleoli of the primary sperm atocyte and the two eccentric
chromosomes seen in the spermatogonia.

8. It is probable that in man and certain other vertebrates,
as in the insects, myriapods and arachnids, the accessory chro-
mosomes are in some way associated with the determination

of sex.

LITERATURE.
Bardeleben, K. von

'92 Ueber Spermatogenese bei Saugetieren. Verhandl. Anat. Gesellsch. Wien.
'97 Beitrage zur Histologie des Hodens und zur Spermatogenese beim Menschen.

Arch. f. Anat. und Physiol., Anat. Abth., Supplement.
'98 Weitcre Beitrage zur Spermatogenese beim Menschen. Jenaische Zeitschr.

f. Naturw., XXXI.
Duesberg, J.

'06 Sur Ic nombre des chromosomes chez 1'homme. Anat. Anz., XXVIII.
Flemming, Von W.

'81 Beitrage zur Kenntnis der Zelle, III. Arch. f. mikr. Anat., XX.
'97 Ueber die Chromosomenzahl beim Menschen. Anat. Anz., XIV.
Guyer, M. F.

'02 Hybridism and the Germ-Cell. Univ. of Cincinnati Bull., Scr. II., Vol.

II., No. 21.
'03 Spermatogenesis of Normal and of Hybrid Pigeons. Reprint of an earlier

('oo) thesis. Univ. of Cincinnati Bui., Ser. II., Vol. III., No. 22.
'09a The Spermatogenesis of the Guinea. Anat. Anz., XXXIV., Nos. 20-21.
'09b The Spermatogenesis of the Domestic Chicken. Anat. Anz., XXXIV.,

Nos. 22-24.
Hansemann

'91 Ueber pathologische Mitosen. Virchovv's Archiv, CXXIII.

'93 Studien iiber die Specificitat, der Altruismus und die Anaplasie der Zelle,

etc., p. 64.
Morgan, T. H.

'10 A Biological and Cytological Study of Sex Determination in Phylloxerans

and Aphids. Jour. Exp. Zool., VII., 2.
Morse, Max

'09 The Nuclear Components of the Sex Cells of Four Species of Cockroaches.

Arch. f. Zellforsch., III., 3.
Payne, Fernandus

'09 Some New Types of Chromosome Distribution and their Relation to Sex.

Biol. Bull., XVI.
Wilcox, E. V.

'00 Human Spermatogenesis. Anat. Anz., XVII.
Wilson, E. B.

'05 Studies on Chromosomes, I., II. Jour. Exp. Zool., II.

'06 Studies, etc., III. Ibid., III.

'09a Recent Researches on the Determination and Heredity of Sex. Science,

XXIX., Jan. 8.
'09b Studies on Chromosomes, IV., V. Jour. Exp. Zool., VI.

ACCESSORY CHROMOSOMES IN MAN.

•LAXATIOX OF PLATI I

• VI re made with the aid of a camera luoi-hi; their nuit;ni-

1,550 diameters. While :

Me, no attempt has been ma the

•> beyond general appearances and relations. Ir
'>mes were not in the same local plane they h..

My in the most favorable plane and then corr --iMi-

i run ae drawings of such individual <

:ng.

1 ate p;- phase of spcrmatogonial division showing tui-nty-tu
Chromosomes lying to one side of the in
lie two accessories.

us of primary spermatocyte showing -
>li.

.. 3. Nucleus of primary spermatocyte showing the
• • nuclear contents, also two persisting nucleoli.
Flu. 4. I. ate prophasc of division in a primary spermato-
tiiosomes. The two lying to one side of the main group

• . 5- I. air of division in a primary spermatocyte sho\\inu t\\

• the two accessories are not readily identified.

I-'ius. 6. 7. 8. 9. Mclnphases of divisions in primary
•\vo accessories in characteristic positions passing ea:
AS also two precociously diverging daughter chromosomes.

.. 10. One end of a late ana phase of division in .1
twelve chromosomes (10 plus a accessory).

.. n. I one end of a late anaphase of divi < ptim.u

tig ten chromosomes, the accessory ch;
•. a prophasc of division in a
in which tl have remained univalent.

I- 1 i«»us secondary spermatocytes ol \\ !

i late prophasc, the other more than live chn>!
two accessory) in met.i: 1 lu-se two •

niK tli. :.>n of a primary spri te in which t«-n •

and ten plus the two accc> "tlu-r.

li i-condary spermalocytes. Oiu-, ! :i\i. !••.!.

-ln'u at one pole; the chromoson

unting although there shnul-1 !»• li\'
il.ii' \-en chromosomes in late pn>ph..

I- h ise of division in a \\hidi

,i.| tin- t\\" iiromosomes, showing seven clirnnin-iini-- in all.

l-'h.. i :i in a sf'"iiilai •. -hi >\\ini; still at

Hiati'i ,i l.iuuin.c cliii'in.itic mass which i- pi"li.il.l\- the t\\- ry chm-

.iltlii>ui;h it cuiilil nut he pn>iti\'ely ident1 :ich.

234 MICHAEL F. GUYER.

FIG. 16. Late anaphase of division in a secondary spermatocyte which has
received the two accessory chromosomes. Each of the latter divides as an inde-
pendent chromosome at this time.

FIG. 17. One end of a late anaphase of a division in a secondary spermatocyte
which had not received the accessory chromosomes.

FIG. 1 8. Two contiguous spermatids, one without chromatin nucleoli, the
other with two. The spermatids in general are about equally divided into these
two classes.

FIG. 19. Nucleus of a primary spermatocyte showing two chromatin nucleoli.

BIOLOGICAL BUIIETIN, VOL. xix.

PLATE I

-•

-_;-

'• •>;••

MICHAEL F. GUYER.

ON I HI. RIGHTING MOVEMENTS Ol TNI. STARFISH .'

A. R. MOORE.

In an iirtirlc entitled "The Behavior of the Starti-h, Aster ins
For re r i de Lariol,"2 Jennings discusses the movements of that
aniinal in righting itself. He assumes that tin- -tarfi-h makes
it- IIK.M mi nts in order to adapt itself to it- environment and
that t re these movements are purposeful. 1 "mm thi- as-

sumption he concludes that "when the starfish is turned over on
ii- dor-al surface locomotion is impossible, the finding and capture
«-f fo.-d must stop; the delicate gills are pressed a^ain-t tin- l.<.i-
loin. injuring them and impeding respiration; and di-plat t un -m-
oi tin- inicrnal organs must occur that may be harmful io their
per functioning. We find, as might lie anticipated, that tin it-
is a regulation of these bad effects by movement: tin -tarii-h
turn- again on it- \« ntral -urface.'

A much simpler explanation of the righting mo\ement- h.i-

IM-III given by I.oeb.' He points out that the ttil-c ire

iti\ely stereotropic. Therefore the arms twist and turn until

all of tin tul't- feet are in a position to be in contact \\ith a

Mirfai 6.

I have ma< le • >l>servations on about thirty specimen^ < >\ A stcritm
minnita and a like number of Asterias ochra«<i. \\ith a \'ie\\ to
determining the nature of the righting movement-.

lit n- look tir-i at the cau-es, which Jenniiu- ha- ^i\eii u~,
tor the -larli-h riv;litinv: ii-elf.

In regard to the tir-t, \i/.. that locomoti<'ii i- one of the ends
which a -tarti-h ha- in \ iew in righting it-elf, I ha\f found that
\ei\ tret|iientl\- the starfish crawls up the -ide of the- aquarium
and, upon reaching the -urfare of the \\ater, thru-t- out three or
four arm- dorsal side downward, their tube Icet clin^ini; to the
surface film of the \\ater. In >udi a po-itioii the> n-mained

l i. 'in tin- I rchLaboi ' .il.

. Vul. 4, pp. .vi 1 85.

I (X !|. rative l'h>>ii>ln>;y nf tin- Hrain," ('luipti-r 3.

236 A. K. MOORE.

sometimes for more than an hour, although further locomotion
was impossible, and no attempt at righting was made. In fact,
the starfish often retained such a slight attachment to the wall
of the aquarium that the surface film of the water could no
longer support the weight of the animal, with the consequence
that the latter fell to the bottom of the tank. In such cases the
tube feet cling to the surface film of the water because the film
acts as a solid surface; it cannot, however, bear the animal's
weight. Romanes1 speaks of these movements as follows: "On
reaching the surface, the animal does not wish (!) to leave its
native element . . . and neither does it wish ( !) again to descend
into the levels from which it has just ascended. It therefore
begins to feel about for rocks or sea weeds at the surface, by
crawling along the side of the tank and every now and then
throwing back its uppermost ray or rays along the surface of
the water to feel for any solid support that may be within reach."
Romanes evidently was not familiar with surface tension. Had
he known that the surface film of a liquid acts like a solid surface
he would have been prevented from attributing intelligence to
the starfish.

In order to see whether pressure on the gills might, as Jennings
states, cause the starfish to turn over, I supported a glass plate
in the aquarium, at a height just sufficient to press lightly on the
dorsal side of a starfish moving over the floor of the tank. This
was placed in the path of an approaching starfish. The latter
did not change its direction when the plate was touched, but
pursued its course, although the gills were pressed down. Fur-
thermore, if a starfish is allowed to attach itself to a glass plate
and is then suspended dorsal side downward so that it touches
the bottom, its movements continue normally, although it could
easily right itself if that were necessary. Clearly, then, pressure
on the gills is not one of the factors which causes a starfish to
right itself.

The displacements of the internal organs which, we are assured,
"must occur" when the dorsal side is down, can only be due to
gravity. I have frequently observed large numbers of st.tNi^h
clinging, dorsal side downward, to overhanging ledges, feeding

'Romanes, "Jellyfish. Starfish and Sea Urchins," p. 268.

KIGHT: MENTS OF THE STARFISH. 257

on barnacle- .ni'l mollu-k-. Surely, "the displacements of the
internal or^an- which must occur" when the dorsal -ide is down-
ward, do not interfere in the lea-t with the in^c-ti. >n and di^c-tion
of f. M id. Such "di-placcments" can, therefore, hardly be con-
-id- rion-b. 'or the riijitin^ nio\ ement- taking

pl.f

\Ye arc forced Delude, from the oli~cr\ ation^ dr-i-ril'cd,

thai, a- Loeb h ted, the starfish cca-c-. it- effort- to ri-ht

If the inoincni all the tube feet can be brought into contact

with a solid surface. ( -ra\ it\- plays no part in the riijitin;.; nio\ c-

Illcllt-.

I 'he i«lc,i ha- I'ccn ad\aiiced by Loeb1 that the mechani-m of
the i i^hiin^ movements i- the result of coordinating and inhil>it-
i in pn l-c-, \\ hit h arc I ran -in itt eel to the \ ariou- arm- b\ the
\ cut ral nei \ c n

^i\ ili-timt method- ot the rii-htin.n reaction ha\c been de-

-« ribed b\ Jciinin^-. but he has made no anal\ -i- of them on the

is of inhibiting and ci ninlinat inv; impul-e-. M\ ob-ciA at ion-

A i' !i Loeb's assumption and give a rather simper < \plana-

t ion i il t he I >ch.i\ i< M ( it t he -lai li-h.

\- a rule, \\heii a -tarti-h is placed upon it- dor-al -idc, the
arm \\ho-e tube feet lir-t touch bottom deti-rmine- the coiir-e
of the ri.uhtiniL;. This arm be^in- at it- di-tal end to t\\i-t the

•np.irativi- I'hy-i ; ,:i." ("li.ipti i

238 A. R. MOORE.

dorsal side upward, and as rapidly as the twisting is accomplished
the tube feet secure a hold on the bottom. Next, if not simul-
taneously, the arm adjacent to the ventral side of the arm which
is obtaining a hold, twists, so that the ventral surfaces of the
two arms face each other, and secures itself in the same manner
as the first. If A and B have in this way attached themselves
to the bottom, inhibiting impulses are sent to C and D. The
latter release themselves if they have already seized the bottom,
rise ventrally, dragging E which either remains passive or bends
dorsally, even catching the bottom with its tube feet in some
cases. The righting is completed by C, D and E passing over
A and B and attaching.

This simple and useful method of righting may be modified
by (i) inequalities in the length of the arms, (2) injuries to certain
of the arms, (3) any initial twist an arm may have due to its
position before the animal was laid upon its dorsal side.

As to (i) short arms are more sluggish than ones of normal
length, (2) injury to an arm inhibits the active twisting and
seizing of the surface with the tube feet of this arm, (3) if an
arm is partially twisted its tube feet reach the bottom more quickly
than they otherwise would. As a result we have the following
modifications of the normal method of righting.

1. If four arms are injured, their activity is inhibited and the
righting is accomplished by the one uninjured arm. It may
force an adjacent arm to coordinate weakly.

2. If A and C (Fig. i) twist so as to face each other with their
ventral surfaces, B receives two impulses, from opposite direc-
tions, to coordinate, and therefore does not twist either way but
bends under dorsally, allowing A and C to accomplish what A
and B did in the normal case. The same result may be brought
about by injuring B, D and E.

3. Sometimes inhibitions are weak and A, B, C and D may all
remain attached, C and. B facing ventrally toward A and D.
E alone is inhibited and the righting is accomplished by A and B
walking backward under C and D.

I found, as Jennings noted, that in a few cases a starfish per-
sistently refused to use a certain arm for initiating the righting
movements. In most cases this was clearly due to an injury or

<»N THE RIGHTINi, MOVEMENTS OF THE STARFISH. 239

malformation -if the inactive arm. According to the author cited,
such a -tarri-h could be "taught" to use the idle arm by giving
the animal a lar^e number of "lessons" ( 180 in one case) in which
arm- ordinarily acti\ <• were pn A en led from taking hold by "stim-
ulatiiu their tube iV.-i with a ;Ja-- r- >d" \\ • IK-IK- \ er they attempted
to at tach thrm-el\ •

I wa- able to compel -tarli-h of thi-^ -ort to use the idle
arm l>y injuring tin- active "in-- in the following ways: (l)
Irritating tin- \i-ntral . of the arm by rubbing it \\iih a

ida-- r«>d, j t; the tip- < >f the arm with a fe\\ drop- <-t

w/io arid. r\v«. or thn-e aj»plicati«>n- a few minute- apart u-ually
-ut'tn ed to render the arm inacti\e. In tin- \\a> I \\a-~ able to
"teach" the -tarti-h in one "lesson," -|>ontaiH-ou-l\ i.i u-e an
arm pre\i.m-l\ inactive. I he length of time the !<•—.. n \\ as
"remembered" depended upon tlie<: t the injur\ . It -eem>

exident fn.m tin- that Jenni "lessons" c«.n-i-ted men-l\ in

inhibitii'ii- due to the injur\ cau-i-d b\ hi- irritat iii;< the mbe
,,! i he active arm-. lint an inhibition i by a -inde

or per-i-teiit -limnl.ition i- n..t identical \\ith tin- |>heiiomena • >\
A manili--ted in the \<:

5l MM \K\ .

i . The righting movements of a -tarti-h whicli ha- been placed

on it- d"i-al -ide are due only to tin- po-iti\e -ten-ot mpism of
the tube I.

2. An injury t" an arm inhibit- it- beiii;^ u-ed t->r the initiation
of i iijitiiii; m«i\ einent

3. A -tarli-h cann«-t be taught t" n-e an arm which i- ordinarily
passive, but b\ injuring the other I'mir arm- the-e can be pre-
\entrd from initiating ri^htin^ mo\enu-nt- and tin- filth arm
then initiate- the-e m«'\ eiiu-nt-.

I wi-h to express my -incere thank- t^ Professor Loeb I'-r his

helpful >u;^e-tious and critici-m.

A SIMPLE COOLER FOR USE WITH THE MICROTOME.

CASWELL GRAVE AND OTTO C. GLASER.

The microtomist's ability to prepare thin paraffin sections,
depends, among other things, on the hardness of his imbedding
medium, and this, in turn, on the temperature of the laboratory.
Usually this circumstance offers no insurmountable difficulties,
but there are times and places when this is not true. To meet
such conditions several devices have been suggested and used
by various investigators, but we know of none so simple, or as
little likely to make difficulties, as the one about to be described.

The apparatus, which is shown, set up for action, in Fig. i, is
essentially a hollow truncated pyramid, open at both ends, and
suspended in an inverted position from a standard, so adjusted
that the lower end of the shoot is at a convenient distance above
the knife. At [the upper end of the inverted pyramid, and
surrounded by it, is a tray whose dimensions are less than those
of the base of the shoot. This tray is filled with crushed ice,
and from one corner of it a drain leads the water to the escape
from the lower end of the air-channel. At that point a rubber
tube connects the pipe with a suitable receptacle.

The cooler is easily set up, interferes in nowise with the opera-
tor, and is thoroughly effective. When the air of the room
strikes the melting ice in the tray, it is chilled and immediately
falls between the tray and the walls of the pyramid. In this
way a constant stream of cold air pours from the lower end of the
shoot, and as this may be placed directly above the paraffin-block
and knife-edge, both of these are cooled, and make it possible
to cut sections very much thinner than the unmodified temper-
ature of the room would allow.

The extent to which it is desirable to cool the paratim and
knife varies with each specific case, but the cooler is adjustable
in at least two ways. In the first place the distance of the block
from the end of the shoot can be changed within comparatively

wide limits; in the second place the temperature of the air de-

240

A SIMI'I.K CnoI.l.K 1 OK USE WITH 11IK MICKOTOM; -^4 I

li\cred in.i> 1/c fiirtlu-r lowered by the addition of N.iCI to the
ice. Uther -alt- can be used should a greater (K']in---i(in of the
K-rnpi-ral urv l.r ; ;ry.

Tin- lollou in- ia M<- i- the record of a test made at a room tem-
perature • I Tin- material in thi> particular case could

Fie. i. PI

in. : tru 'in M •

•.ii'l.inl.

imi IK- iinlicildcd in parai'tin with hi^h melting-point ami satis-
factory sections, c\cii as thick a- u niicra, could not In- cut.
With the aid of the (.idler ho\\r\er. a perfect -eric-, ^ niicra in
thicknc--. was ca-il\ |ucparcd fnnil the -ailic Nock of 45 .

paraffin.

242 CASWELL GRAVE AND OTTO C. GLASER.

TEST OF COOLER.
Room temperature 3i°C. . . .87.8°F

Contents of Tray. Distance below

Mouth of Shoot.

Crushed ice 6cm. 24.5 76.1

Crushed ice +NaCI 6 ' 23 73.4

Crushed ice 3' 18 64.4

Crushed ice+NaCl 3 " 17 62.6

Several coolers, varying somewhat in size, but all modeled af-
ter our original one at Johns Hopkins University, are now in use in
different laboratories. The measurements given in connection
with Fig. i, are those of the cooler at the University of Michigan.
This particular one does not have the advantage of a removable
ice-pan. In general, size is of little consequence unless it in-
volves too great a reduction in the capacity of the ice-tray, or is
conducive to too much absorption of heat by the sides of the
pyramid. This latter difficulty is easily overcome by lining the
shoot with asbestos paper.

JOHNS HOPKINS UNIVERSITY,
UNIVERSITY OF MICHIGAN.

July 15, 1910.

THE ni ESTION OF REVERSAL OF ASYMMETRY IN
THE REGENERATING CHEIwE OF CRUSTACEA.

CHARLES R. STOCKARD.

I >urin- the summer of 1909 while at the Tortuga- Laboratory
• •I ill.- Carnegie Institution I undertook a further analysis of
the re\.T-al phenomenon in regenerating specimen- < >\ tin- p-iu-ra
Alf>hfn\ and .^ynul Aliens. These small Crustacea commonly
call. -«1 -napping shrimp, on account of their habit of snapping
tin- lar^<- fin -l.i- \\ ith such force as to produce a -urpri-ingly loud
noi-.-, an abundantly found in the "logger-he. id" sponge and
in the holes of disintegrating coral rock on the Toruiga- reels.
There .ne a number of species five of \\hieh, Alpheus formosus
and tirniillatns and Synalpheus minus and two other unideiit itied
^pecies of Synalphens, were employed in these experiment-.

The several species differ in si/e and body color but are .
sentially similar in general structure. The lir-t pair ..I" appen-
es is decidedly asymmetrical in both sexes. < >m member <>t
the pair, either the right or the left, is extremely laixe. in some
cases being more than half the size of the bod\ it -ell". The
general t\'pe of this chela in the five specie- follou- IIK.K ..r
< l.p-ely the description given by Wilson,1 for the vivai ehela
"I" AlplifHs lictcrochelis. It is greatly rounded or s\\olK-n \\iili
tran-\er-e grooves on either side of the proiiodu-. \ar\in.u in
de|)th with tin- -jwcies, and presents characteri-tie color patterns
beini; tip|)ed with a li\'cly rose color in Synalphcus minus while
in the other species it is bluish, dark or brown. On tin- e"iiea\e
sidt- of the dart\ln- is a swollen knob forming tin- "hami
which lit- intii a corresponding socket on the outer side of the
propodu- claw. M\- extending the dactylu- and then suddenly
-na|»pini; the tlaus together the "hammer" is forced into tin-
socket with the surprisingly loud sound.

XYil-i'ii'- de-cription further applies in that the large chela
ha- essentially the same structure in both sexes, while the small

!•". H., "Not.-- mi tin- Ki-vrr.-ul <>t A.-ynniK-try in the Regeneration • -i
tlu- I'lu-l.i- in Alphciis hftcrochdis." BlOL. BrtL., IV'., pp. iv7 -'i-i. 1903.

=43

244 CHAkLES R. STOCKARD.

chela shows characteristic sexual differences, we shall be mainly
concerned, however, with the fact that the small chela is always
typically different from the large chela in shape as well as in size.

Przibram1 discovered that in several species of Alpheus after
the removal of the large chela a chela of the small type regener-
ated from its base while the small chela of the opposite side meta-
morphosed or developed into a great chela of typical form at the
following moult. In other words, the asymmetry was reversed.
Further, when both first chelae are removed they regenerate in
their original conditions, no reversal following.

Zeleny2 found an exactly similar phenomenon to occur after
removal of the functional operculum in the worm, Hydroides.
In this case the rudimentary operculum of the opposite side de-
velops into a functional operculum while a rudimentary organ
regenerated from the base of the former functional one. The
principle involved in this reversal phenomenon is doubtless the
same as that in the Crustacea.

Przibram3 later found a similar reversal to occur in other
species of Crustacea, while in others the removal of either chela
is followed by the regeneration of one of the simpler or smaller
type without a regulatory change taking place in the uninjured
chela of the other side. In still other cases, as for example the
lobster, Homorus, a chela similar to the one removed invariably
regenerates whether the original chela was a large crusher claw
or the slender nipping claw.

The Crustacea thus present a series from those forms which
regenerate appendages of the type of the ones removed, others
which regenerate appendages of the simpler type without a com-
pensatory change taking place in the uninjured chela, and finally
such forms as Alpheus in which (lie simpler type of chela is re-
generated after the removal of the more specialized chela while
the uninjured small chela develops into the more modified type,

'Przibram, H., "Experimentelle Studien uber Regeneration," Arch, fur Entw.-
Mech.. XI., 1901.

2Zeleny, C., "A Case of Compensatory Regeneration in the Regeneration of
Hydroides dianthus," Arch. fur. Entw.-Mech., XIII.. 4, 1902.

"Przibram, H., "Experimentelle Studien uber Regeneration, II.," Arch, fur
Entw.-Mech., XIII., 1901-1902; "Equilibrium of Animal Form," Jour. Exp. Zoo/.,
V., p. 259, 1907-1908.

REYER.-AL OF ASYMMETRY IN CRUSTACEA. 245

.iii'l tint- by a -uri <it compensatory regulation the animal'- asym-
metrical • ondition i- quickly reestablished.

\Vil-on repeated Przibram's experiment- on Alphcns Itctcro-
chflis with -imilar results, hut carried tin- experiment- further,
hoping to analyze the factors concerned in the reversal pro., --.
After removing the great chela the nerve trunk leading to the
-in. ill i lii-la <»t the opposite side was Hipped in order io u-t
\\hetln-r tin-re- was a nervous control determining the growth of
i In- -mall chela into a large one. After Mich an operation the
-mall chda wa- generally thrown off and only \\\« -]•«•» -inn n- are
-,iid io I -I- l"-\ ond (|uestion, >'et "one of the-e did in it moult quite
noniialK and the other not at all.". The evident e, ihen. doc- not
warrant conclusions as to the CailSC of reversal of ,i-\mmeir\ ill
i he cln-I.e. Wilson finally helievcs that the initial factor that sets
iii moiiiui the complex process of different iat imi of \\hich either
-id< 'lahle, is primarily only ;i dilh n me in the amount of

material < m the two sides. "Removal ot t he lari^i chela < <\ >\ i< .u-l\
d the asymmetry in respect to the amount of material and
nm-t. l«-mporaril\', at least. K-ad to a functional IUT\OU- dillci-
eip Such a suggestion may easily he submitted to ( \pi ri-

mrntal test, for example, after removal of the l.ir^c « hcl.i fr..m
one -ide of the body if several posterior appendage- be ivmo\ed
lioni the other side the greater amount of material may -till
remain on the original large chela side. I u. I, .1, illust rate-
tin- operation. I'nder the-e conditions \\ill a large chela re-
generate I nun the -tump ot tin old one, in-tead of ari-in^ I
gi«i\\ th o| the -mall In -t chela of the oppo-ile -ide.'

X^ain, tin- propn-iiion ma>- he tested b\ removing hoth the

a and -mall chela- o| t he lir-t pair and in addition amputating

several legs "ii the -ide of iln- large chela; the operation i- illu--

tratedb\ Fig. i, J fh( greater amount of material is no\N on

the original -mall chela -ide; \\ill thi- extra amoiini caU5(

it chela to regenerate from the small stump instead of from the

-tump of the great chela which i> on the -ide \\ith le— material'
Lastly, \\hen onl\ a |iortion of the great chela i- amputated
doe- it regenerate in the original condition Of become a Miiall
chela, while a large chela appear- on the opposite -ide through
a metamorphosis o| the -mall tir-t chela.'

FIG. i. Diagrams illustrating the manner of operation in the several experi-
ments. A, removal of the great chela and the posterior appendages of the opposite
side, causing the larger amount of material to still remain on the great chela side;
B, the opposite operation as a control; C, the heavy lines show the places at which
portions of the great chela may be cut away without reversal; D and E, removal of
both first chelae and posterior appendages from either the small, D, or great, E,
chela side, to determine the influence of the lateral amount of material on the regen-
eration of the first chelae; L. large chela stump; S, small chela stump.

REVERSAL OF ASVMMKTKV IN CRUSTACEA. 247

Aiming towards an answer for these que-tion- a number of
experiment - \vep- performed the re-ult- of which may now be
i onsidered.

Fifty healthy -pecimens representing the five different >JKM
\\en- selected, and tested as to their tendency t<> reverse the
:nniftry of tin- first pair of chela1 during tin- regeneration
following tin- removal of the great chela. Without exception
all ot" the specimens responded as Przibram had found, a -mall
chela regenerated Irom the stump of the original great one and
the small ( hela of the opposite side metamorphosed into a ^reat
chela. Forty of the specimens favorably survived the experi-
ments.

FIRST SERIES.

Nineteen individuals had the great chela removed and in addi-
tion a numU-r of posterior appendages were amputated from
the opposite or small chela side, so as to allow tin- greater ma —
oi material to remain on the original great (hela -ide. The
opt 'rat ion i- illustrated by Fig. I, A, and the opposite or < "in ml
operation by Fig. I, B, or specimens lo, 12 and ,}i in the table.
\\< '• rrii . to the table the results of such an experiment ma\ be
ertained.

The left side of the table gives the date of the oprratitni. the
number of the specimen, and the appendages rrmo\ed are indi-
cated b\ .v, (/and 5 in the first appendage column indicate the
it and -mall chela. The right side of the table i;i\e- the
time ot moulting and the manner of regeneration, the r signifies
a new or : :ated Kv. (/and .V in the tir-t appendage column

m indicate the great and small chela. The horizontal lines
ot the table are BO arranged that the appendages on tin- right
side of an indi\idual ate gi\en immediately above those on the
left of the same animal, c. g., specimen I had the great chela and
the -ei ond leg removed from the right side and the fourth leg
from the left side in the first instance. Kach -pecinieli, as tin-
table sh,.\\s, \\as operated upon a second time during the experi-
ments.

Of the nineteen cases having the great chela removed from one
side and other appendage- than the lifst chela trotn the opposite
-ide i he -mall chela. e\ en though it wa> uii the -ide of less mate-

248 CHARLES R. STOCKARD.

rial, retained the power to grow into a large chela of typical form
in seventeen cases. One of these cases, specimen 23, is remark-
able, since at the first operation the small chela which was on
the left side and the other four left appendages were all removed
and regenerated at the next moult. After this moult the large
chela of the right side was amputated along with the four pos-
terior legs of the left side, thus leaving only the regenerated
small chela on the left side while the four posterior legs remained
on the right side. Nevertheless, the left small chela grew into
a great chela and the four more posterior left appendages re-
generated for the second time. The case seems an extreme test
of the power of one side to regenerate all of its posterior legs for a
second time and in addition to change the first chela from the
small to the large size and type. Specimen 25 further indicates
this remarkable power of the side of the individual with less
material to replace all lost parts and at the same time increase
the size and type of its first chela.

The remaining two of the nineteen cases, specimens 9 and 18,
present the first chela? equal in size after the moult following
the second operation. Specimen 9 had in the first case the great
chela removed from the right side and the third, fourth and
fifth appendages from the left side. The greater amount of
material was, therefore, still on the right side, yet the small
first chela of the left side became larger after the moult. The
second operation removed the great left chela and the second,
third, fourth and fifth legs of the right side. After the moult
all of the amputated legs were regenerated but the regenerated
left first was small, and the right first appendage had not in-
creased in size. Thus the first pair were symmetrical in respect
to size yet the right first or old chela had slightly approached
the large chela type. The specimen 18 responded in a closely
similar fashion.

The type of the chela is equally, if not more, important than
the size since Przibram found the chelae to be of almost equal
size in some cases but of reversed type, and the great type in-
variably increases in size at the following moult.

The experimental evidence in the first case, then, does not
support the idea that the side with most appendage material

"K ASVMMETKY IN EA. 249

moM powt-r n, produce a great chela of typical size ami form,
'lie t<-ii<li-n. . produce a chela of tin- ;>;rcat -i/e an<l t\pe

tn.m ilu- uninjured first chela, even though this In.- tin- original
-mall . lu-l.i and i- located on the body side which has >uft<
the I"— "t -til -.ihcr walking appendages. There seem- to \>c no
e\idt -in •«• In -in the-e experiments to suggest a bilateral distribu-
ii"ji .,i ^n.uth energy accompanying distribution of appendage
material.

SKCUXD SKRIKS.

Tin- '|nr-tiun of a bilateral distribution of i;ro\\th rmn:\ re-
lated to, «»r accompanying, the amount of appendage man-rial
"ii n side was furtlu-r tested in the follu\\ini; nianiui.

I specimens were operated upon so as to remove both chelae

• •I ill. tii^t pair. It was known that when only tin •-«• i\\<. ( IK la
moved that they regenerated in tln-ir original cniidiiii.ii.
a lai<r chela from the base of the original large cln-la and ,t
-mall i licla from the opposite side. If now in addition to du-
• I of the first pair of appendages a number of mon pos-
terior appnidav;es also be removed from tin- large chela -idr.
HiU sidi- will have less appendage material remaining and i-.
therefore, called upon to regenerate a greater amount of man-rial
to n place the posterior legs. Will this side of the body at tin
-aim- time br more capable of producing a first chela of larger
and -prfi.ilixed type than the opposite side which is calli-d
upon in it-place only the first chela? Such an operation is illus-
iratrd |.\ I j-. i, /{, and Fig. I, D, forms a control experiment in
\\hirh tin- additional appendages are removed from the small

chela -ide.

Hie iiMial idea of regulation would require the side with only
ilie in -i N - removed to regenerate a large chela while the other
^ide n plat i d tl • ral posterior legs and produced a small

lir-i chela. It i- found on examining the table that alter an
operation to remo\e l>oth first chelae and one or more posterior

on 1-itln-r tli' it or small chela sidr that M'\ of (if;

specimens regenerated the chela- of tin- lii>i pair etjual in >i/e.

noi out- individual re\er-etl the t>|>e of the hr-t clu-la-. and
eiijit re-i-m-raied the lir-i ehela- iii their original condiiion a>
thou-h no .11 Mi i ion a I appendage- had In t n reino\ ed. < >ne speci-
men died liffoic the experiment \\a- completed.

350 CHARLES K. STOCKAKD.

Considering the six specimens that regenerated the chela- oi
the first pair equal in size it is important to find that tour o!
these cases, specimens 16, 20, 21 and 39, had the larger number
of posterior appendages removed from the side of the original
small chela and not from that of the large chela, so that the
greater amount of material remained on the large chela side.
Such operations were intended as a control for the results fol-
lowing the removal of posterior appendages from the large chela
side. Although in these four specimens there was more material
on the original large chela side and this side was called upon lo
regenerate fewer appendages it failed to produce a great chela
from the stump of the original one.

In specimen 16 the first chelse remained equal in size and were
both of the small chela type after a second moult. The left
chela was then removed and after the next moult the right de-
veloped into a great chela and the left again regenerated small.
Both first chelae in specimens 20 and 21 were also of the small
chela type, while in specimen 39 the small chela failed to re-
generate at the first moult after the operation though a chela
of the small type regenerated from the base of the great chela
and remained small while the right small chela regenerated at
the next moult .

Specimens 31 and 32 had both first chela- and a number of
appendages, four and three, on the great chela side removed.
After the moult following the operation the first chela? were equal
in size. Yet again specimens 27, 35 and 38 were operated upon in
an identical fashion and after the moult they were able to produce-
a great chela from the original great chela stump even though
this side was called upon to regenerate three other appendages.

Of the fifteen cases tried, therefore, eight regenerate their first
chela; in the original condition of asymmetry while six regenerate
the chehe of the first pair equal in sixc- and usually similar in
type whether additional appendages are amputated from the
great chela side or from the small chela side. Such a fact would
seem to indicate that the amount of appendage material present
on either side is an unimportant factor in determining the typr
of the first chela on a given side, and it seems to show lurtln-r
that there is no clearly evident bilateral distribution of growth
energy in these regenerating specimens.

ERSAL OF A-VMMKTKV IN CIU'STACKA.

25]

TABLE I.

I HI: EFFECT OF KU-I.-VKRATIOS ON THE ASYMMETRIC xi. CONDITION OF THE

FIRM PAIR • >i- < IIKL.E is FIVE SPECIES OF Synalphftis AND Alphetis*

.III. .11.

'.adages.

14 \ i

Appeii

Jin:

Jill:

linn- '. ( .
I

May 24
Jum

Ma;

.linn- 6

i i .\

Jllllr f. '• ,
Mas ,, 7 ,

..x

June 4

June 16

June i

June 15

Sr
Gr

/"•

3°

'1 i hrl.i i

it< term
t<> t\|>< !.i.

;liinl ami tittli an- >>nly

May 29
Juno 13

June 4

x JUnC I3

Im.K. nut ml!
erat<-'l.

Only the <lartvl rnn..\i-.| an. I

r I I Also liiniillril oilr <i.i\ altrl

the operation luit hail
ited

•• i

Sr
G
G

Sr I

, Sr r i ure of third and fourth

G r O O r legs to regennat. inu'lu
account for first dn-la
coming large.

June 12 |Sr

June 3

Moiiltrd few hours after op-
eration on May 24 ami
right first chela began to

JIIIK- '•
Ma

\< ' .x

x x x

r ' r

I ,X

May 30

Juni

June 14

v»

i »

Jim-

.,;<

June 14

S
Sr

»;

> .X

r r

Also moulted two days afti-r
operation but no ri
tion.

Moult followed so so. .n uin-r
operation that left lir-t
chela had not ati.iim-d lull
size, but di<l - lew

days.

. inolllti alli-l np-

erati'Hi. Ri.uht fir-t -.iiin-
wliat -imilai (•• hela

luit --niall.

252

rilAKI.ES R. STOCKAKH.

TABLE I. — Continued.

"=!

rt t

-£•

Specimen
Number.

Appendages.

i.£

Appendages
Regenerated.

Remarks.

June 7

10 R

x i

' Sr
June 14 ~

Operated immediately after a
moult on June 7.

May 24

May 30 |

June 7

•.

June 13

Sr
G

May 24

12 L

June 2

Sr
G

June 7

12 J

June 15

May 24

13 J

X
X

May 29

Sr
G

r
r

, R

Died after operation.

June 7

•' 1. <",x

May 24

I4L

—

June i

Only the dactylus and index
cut from great chela, no re-

versal.

June 7

14 L

June 14

Second operation and result
the same as first.

May 24

June 3

June 7

15*

C,x

June 15

Sr
G

May 24

16 L

Gx
Sx

X
X

May 31

Sr
Sr

r
r

After a second moult on June
7 the first chelae were still

of equal size.

June 7

I6L

June 13

The left first chela was re-
moved and right became

large and Irlt ir.ui-iu-ialrd

small.

May 25

18 I

June I

June 7

I8L

June 14

Sr
S

I'incer of left first seems

r slightly ncaic-r tin- gi<-ai

chela type.

May 25

IQ L

June 3

June 8

19 J

Sx
Gx

June 15

Si
Gr

May 25

20 L

June 6

June 8

20 I

Gx
Sx

< X

June 19

Both first chelae of the small
type and equal in size.

May 25

-I

i x

May 30

June 8

21 L

Gx
Sx

X
X

June 13

Sr
Si

r
r

Both first chelae of equal size
and small type.

( ', r

May 25

••

x June 4

r r

June 7

23 L

••.

•.

x •l""" tS

Sr
G

Second regeneration of all pos-
terior left legs, yet left first

became large chela.

May 25

2SjL X X

Juno i

REVERSAL <>F A^YMMF.TKV IN CRUSTA

253

TABLE I. — Continued.

• —

Appendages.

e j.

Append...

Jum

June 17

Gr
Sr

r
r

ration on

i. in- -Mr y t ih-t chela of

thi^ -i'li- In - • .n..

-•'. ^

Gx x June 6

Juni

I>iil not moult tin- «ivonil
time.

June 4 Sr

r
r

Juni

X .

June 17 Sr

1 x 1 , Sr ' 1 r
June 2 c

Junr s

X
X

i G r
June 14 S| ,

Second pair i-t 1. i
VI.:' : ti-i than ll-tlal.

M.I-. .•
I

June 6 J

Molllti-,1 d.iy IM-I..II- tin- npi-I-

ation.

Jinn- 8

29 L

June 16

*•*

May 29

G-
o

Moult followed so so"ti .iitn
operation that little in-

cre;t-i- in liylit chi-!

generation of lei'

second moult Jun

Innc 10

Gx xxx

• 1 1 1 l< '1 1 \ ' •! 1 I j 111 I »

1 June 16 without inoult-

- L,

\ I . i \ .•

; G

_ In ne 7 —

X X

Jtin.

C June i

First ch> il in size and
of small ty|H'.

xxx, G

31 Sr

JllMi- [0

^ June 15

Sr
Sr

Both first chela- smaller than
n .iin.il hut of equal

i* l

Ma:

; • r

May 30

J 1 1 1 1 •

i .\ \ \ \

June 1 6

r o

are of 3 and 4 to n-.o-m-r-
rate may account for In t

\ tli.

• ,x

Jim-

i . \ \ \ x

Sr
Gr

first chela great tlmnvjli
rcKeiii-iatiiii; thn-i- ntlu-r

. < on sann- -ii|<-.

Ma> a ; 56

x x June 3

Sr
G

motilti-d J -lay- atli-r

operation.

Jnn.

June 15

Ma\ .' 17 ,

x May 30 Sr

Mniilt'-'l av;ain Juin- <>

Juni

57 J

X June 15

254

CHARLES R. STOCKAKO.

TABLE I. — Continued.

•-.!

S u
B%

'o e

Appendages.

Appendages
u "= Regenerated.

Remarks.

J5 ^
— —

— f-

v.^

May 25

38?

May 30

June 8

Gx
Sx

June 15

Also moulted June o day after
operation.

May 25

39 L

Sx
Gx

June 6

o
Sr

»s

June 14

Sr
Sr

First chelae finally regener-
ated equal in size and re-

mained so after next moult

May 25

4°L

Sx
Gx

May 30

Sr
Gr

Moulted again June 6.

June 8

K Sx
40 L Gx

June 15

Sr
Gr

Each specimen was operated upon twice as indicated. R and L following
the specimen number signifies right and left sides of the animal; G indicates the
great and S the small first chela; x indicates the appendages removed and r the
appendages regenerated.

THIRD SERIES.

Finally, an attempt was made to determine how large a portion
of the great first chela might be removed without causing it to
regenerate small; or to cause the small chela of the opposite
side to grow into the great type. When a large portion of the
chela was quickly clipped off with sharp scissors or a knife the
remaining portion was soon thrown off at the breaking joint.
The only successful operations consisted in the removal of the
dactylus or most distal segment which forms part ol the claw,
and in the removal of the entire pinccr or dactylus and distal
end of the propodus, as is indicated by the lines drawn across
the chela in Fig. i, C. In the last case a stump-like appendage
without a pincer remains.

Following either of these operations the great chela was fully
reformed or renewed at the next moult, no reversal taking place.

A small portion of the great chela may then be regenerated in
its original form. \Yhrn tin entire1 chela is removed the small
chela of the opposite side- invariably grows into a great chela
and a small chela regenerates from the stump of the orii'.inal
great one. This reversal of asymmetry may be shifted back and
forth for a number of times and occurs in a manner as decidedly
pronounced after several operations as it does after the first.

REVERSAL <)T ASVMMETKV IN CRUSTACEA. 255

CONCLUSIONS.

Tin- power in reverse the asymmetry of the tir-t chela- when

in-rating .1 ure.it claw in Alphens dor- not -eem t<> l>c clo-dy

a--ot i.itrd with .1 difference in the amount of material on the

tuo -ide- of the liorly nor with a bilateral di-trihution of growth

or regenerative < n« •

Although in certain cases there seems to lie a tendency to
aerate tin- clu-hi' of the first pair equal in -i/e and >imilar
in i\|ie. -mh a tendency is manifest under condition- -o \aried
in re-pi-d io the bilateral clistriltution of ajipi'iida^e material
and < all upon the powers of regenerative eneiu\ that the pre-cnt
com Ill-ion j- warranted. The amount of material on a ;J\»n
-ide of the animal, or the amount of t< ^ -m -ration requited of
ihi- -id«- .in- negative factors in determining tin- al>ilit\ of the
-ide to pid<ltK e a great chela instead of a HIM 1 1 one.

N \i'i i .-. July 5. 1910.

Vol. XIX. October, 1910. No. 5

BIOLOGICAL BULLETIN

EXPERIMENTS OX COLOR-VISK >N or THE

HONEY BEE.

C. H. TURNER.

INTRODUCTION.

Whether insects can or cannot di-tini;ui-h color- i- a mailer
of much theoretical importance, for the correct interpret. ui<>n
ol tin- n-l.iiion of insects to flowers depends upon thi- an-\\er.
Mo-t -indents of natural selection believed, at one time, that the
form- and colors of flowers were adaptations to insect \i-itoi>.
Lately i line lias been a reaction based on the general cmi-ensus
of opinion, among morphological entomo|o-i-i-, concerning the
poorness of insect vision. Kellogg1 writes: "The fixed -hort focal
<li-iaiu-e, the incompleteness and lack of detail incident to a
mo-aic image, and the lack of accommodation 'onl\ partly pro-
vided f.,r l)\ the shifting of the peripheral pigment) to var\in^
liijit intensity, which are admitted conditions of insect vision,
make it -eem difficult to account for the intricacy in pattern
common to man\ tlo\\ers on a basis of adaptation to animal
\ i-itor^ of -uch | r -eein- capacity as insects.

"Experimental e\ idence touching this criticism is singularly
meaner \\heii one coii-ider- the importance of the subject. If
in-eci- can accnrateK di-i in^ni-h i«'lors, and at some di>ta:
and can pen ei\ e the tine deiail> of color-pattern at a \vr\ -hort
di-iance. then the explanation of floral structure and pattern as
adaptation to in-ect \i-itors has -olid foundation for even the
ama/in.uK- lar-e and \aried results which il attempt- to explain:
if not, it i> hard to understand how the explanation i- \alid (at

\ 1 . ".\im-ii.\m In-tvts." Ilriiry II"It ^S: Co., second LMlitimi. revised.

-57

258 C. H. TURNER.

least in any such all-sufficient degree as commonly held), despite
its logical character (in light of our knowledge of the nearly
limitless capacity for modification of natural selection) and the
abundant confirmatory evidence.

"Most of the experimental evidence so far offered is that in-
cluded in Darwin's account ('On the Fertilization of Flowers by
Insects'); in Lubbock's account of his experiments on honey-
bees, familiar because of its presentation in his readable book,
'Ants, Bees and Wasps'; and in Plateau's account of his more
recent but less familiarly known experiments with various insects
including bees. Both Lubbock and Plateau are investigators
ingenious in device, keen in deduction, and of unquestioned
scientific honesty. Yet their conclusions are a direct contradic-
tion. Lubbock believes that bees recognize colors at a consider-
able distance, that they 'prefer one color to another, and that
blue is distinctly their favorite.' Plateau finds that neither the
form nor the brilliant colors of flowers seem to have any important
attractive role, 'as insects visit flowers whose colors and forms
are masked by green leaves, as well as to continue to visit flowers
which have been almost totally denuded of colored parts' ; that
insects show no preference or antipathy for different colors which
flowers of different varieties of the same or of allied species
may show; that flowers concealed by foliage are readily dis-
covered and visited; that insects ordinarily pay no attention
to flowers artificially made of colored paper or of cloth whether
these artifacts are provided or not with honey, while, on the
contrary, flowers artificially made of living green leaves and pro-
vided with honey are visited (from the attraction of the 'natural
vegetable odor'). From these observations Plateau concludes
that 'insects are guided with certainty to flowers with pollen or
nectar by a sense other than that of vision and which can only be
that of smell,' and finds particular proof of this in the facts, ac-
cording to his observations, (i) that insects tend, without hesita-
tion, towards flowers usually neglected by reason of the absence
or poverty of nectar, from the moment that one supplies these
flowers with artificial nectar, represented by honey; (2) that
insects cease their visits when one cuts out the nectary without
injuring the colored parts, and re-begin their visit if one replaces

EXPERIMENTS ON COLOR-VISION OF THE HONEY DEE. 259

the destroyed nectary by honey; (3) that it suffices to attract
numerous insects if one puts honey on or in normally anemophil-
ou- flowers, simply green or brown in color, which an- normally
pr,« -ti< -.illy invisible and almost never visited by in-ivts; and
1 thai the visiting of flower- artificially made- of fresh green
leaves and containing honey demonstrate-, plainly the role of
i In sense of smell.

"It must be said that, despite main ju-t critici-m^ that may
be madf on the character of hi> experiment-, Plateau has made
necessary more experimentation for the relief of the general
theory that floral adaptation of (dor i- due t.. c<.lor prefi fences
of in-eei \ i-itors."

Forel nd von Buttel-Reepen1 are opposed to Plateau1- \ ie\\ -,
but Bet he' is in accord with Plateau.

I ' • ;• -t his conclusions, Forel repeated, in the follov. in- manner,
Plateau's dahlia experiment. ( I j Paper dahlias were di-tribi
amoii- some dahlias from \\hich a large number of bees \\eie
e <\\ct tin^ honey. The bees paid no attention to these artit.n ts.
Honey was placed on these artifacts, and. }<\ -killtul manipu-
lation, brought to the attention of one of the bees. Immediately
that bee neglected the real dahlias for these artificial <•;

(iradually all of the bees neglected the dahlias for those
an it. n t- \\ it h their inexhaustible supply of honey- inexhaustible
luse ii was constantly replenished by Forel. 141 The artifacts
\\eii remo\ed. After a lapse of several days, similar artifacts,
bin i I'liiaininv; no honey, were scattered among those dahlias.
Immediate!) tin- bee- n ejected the dahlias for the artifacts,
\\hich the\ -e. ii dud for hone\-. I'orel thinks this experiment
>ln>\\- that bees ha\e -pace, form and color perception.

\ on Hut iel l\. . ] M n ba-e> hi> opposition to Plateau's \ie\\-
larueK upon information furni-hed him by Herr Roth, leader of
the Haden bee-keepei- -• liool. and a teacher named Staeliclin.

•ii-l. Ain;.. "l>ir p-\i In-, ln-ii l-'.i -i del Ai:i' Uen und i-ini.o-r am!-

Iii-i-lxii-n." Miii-iu-lii-ii. I'j'ii. "Ants and - tlirir In-iiiu-i-." .\/. •»;:-.'. \-nl. i }.

. )•

-Miii! ii, II. von, ">md dit- Mii-in-ii ReSex-maschinen?, I-!\i"-iinu-iit;il

Mi-iti.iv;,- /ui Minli>i;ii- <kt 1 1. mi.nlii.'iii-." Biul. Ctnlralhl., Bd. -'". n;uo. "An- i
Rctl. \ \l.i. liiiif- '" iran-l.itril \>y Maiy 11. (ivi-li-r. Medina. <>.. iv

*Bftlu'. A.. "I>ic Hi-iinlalii^kcit drr . \nn-i-i-n und Hii-nni /inn Thril naoh ni-ucn
ii-hrn." /: • '•. , I'.'l. 22, IQO2.

26O C. H. TURNER.

Von Buttel-Reepen states: "We have seen above that the flight
[of bees] becomes very unsafe in the dusk; therefore it is evident
that gloomy weather influences considerably the ability to orient.
'One of my former neighbors,' Roth says in his communication,
'painted the gable of his house over the apiary with a sky-blue
(luftblau) color. The same bees which always flew over the
gable, on the next dark day, bumped against it with their heads,
trying to fly through it.' A teacher, Staehelin, made the following
observations: A weak after-swarm, mostly of young bees from
a hive painted blue, dispersed among the masses of humming bees
which were just taking their flight of orientation out of the other
hives (which, as is usually the case in Germany, Switzerland, and
Austria, were standing close together), and settled here and there
in clumps. After a short time they flew back to the bee-house;
but only a few found the right hive; the rest flew to other
colonies, and to which? Only to those where a blue door invited
them did they attempt an entrance, but nowhere else. Unfor-
tunately they were so hostilely received that the ground in front
of all of the blue hives was covered with bees."

Bethe had a swarm of bees lodged in a brown hive which rested
on a table. He painted the outside of the hive blue and covered
the table with green branches. Instead of the backgrond of
trees, he substituted one of white and yellow flowered cloth.
No change was produced in the home-coming of the bees. This
Bethe considers conclusive proof that bees are not guided home
by memory picture contributed by the eyes.

So far as my knowledge goes, M. Gaston Bonnier1 is the only
recent investigator who furnishes any experimental evidence that
supports Bethe's view. He found that bees, the eyes of which
had been rendered opaque with pigmented collodion, would pass
direct to the hive from any distance less than three kilometers.
This observation, which is not in harmony with Forel's experi-
ence,2 supports Bethe's contention, but it has no direct bearing
upon color vision.

The purpose of this paper is not to discuss the homing of the

'Bonnier, M. Gaston, "Le sens de la direction chcz les abeilles," C. R. Acad.
Sci., Paris, T. CXLVIII., 1909, pp. 1019-1022.

2Forel always found that bees, the eyes of which had been rendered opaque,
could not find their way home.

EXPERIMENTS ON COLOR-VISION OF THE HONEY BEE, 26l

honey bee; but, by means of simple experiments, to throw some
light upon the question "Can bees distinguish color

DESCRIPTION OF Tin; Kxri KIMKNTS.

The following experiments were performed in a large held just
\\<-i "i ( »'!•". illon Park, St. Louis, Mo. The \\hite sweet elover
Mclilntiis alba Lam.), with its long raivme- uf white papiliona-
ceoiH llower-,, was abundant in den^-e pan -In--; luit there were a
t«-\\ vacant places in the field. Forajn- bee- \\ere \i-itin- this
white meliloi in large numbers.

Series I. (July 12, 2 P.M. .

The discs used in this scries o!" experiment- \\en- cut I'nnn
colored cardboard, and each was six centimeters in diameter.

I \ri imiKNT I. — / placed six discs of red cardboard on the top

that had been erected in the midst of a patch of wlii.''
clover. The rods were so adjusted that the top of each was about
<»i n /err/ with the tops of the weeds. Six similar discs were attached,
nt ii : heights, to the branches of the weeds. Honey was />/..

mi nil nl ///or i/iscs.

Mure 1 1). in an hour passed by and no response was made to

the-e di-es by the bees; but both Hies and wasps visited them.

Hie \\eeils were lull of bees that were continuous!) living to

and !i<> in tin- immediate vicinity of these artilaets with their

eopimi- >iippl\ <>f honey. Were the odor of honey alone sufficient

MI r. iet l>ee- reilexlv, ilu-e bees should have been attracted

e.nl\. Alter \\.iitiiu h.ilt an hour, I decided to force the bees

to .ttteiitl to m\ artil.i'

EXPERIMENT 2. .1 . :!>tured in a wide-month bottle and

tin- bottle, with the cork >ted over one of the red <li*i

experiment i, until th, <->pped upon the disc; the bottle

then rennn-eii. This ;.•<;. v tried with six ditjerent bees.

Ill e.ich Case tile bee ,il\\ ,i\ - ,i~< flldt d to the top (»t the but I le

.mil .utempteil to escape. After sc\-eral futile effort ^ it \\ould
drop, either l>\ .u « idem or from exhaii-tion, upon the di-e. At
that moment, 1 al\\.i\- reiimx ed tin bottk-. Immediately the
liee \\oiild lea\ e ne\ er to return. Some of the I M i S I ell into the
hone\ ; but, e\en ill that case, the\ did not re-turn.

262 C. H. TURNER.

EXPERIMENT 3. — A branch containing blossoms on which a bee
was foraging was gently removed from the plant and so manipulated
that the bee was less than two centimeters from the honey of one of
the discs of experiment i. This was tried with six bees.

In no case did the bee pay any attention either to the honey
or to my discs. The bee always left immediately and went to
one of the blossoms of the melilotus.

EXPERIMENT 4. — Whenever a bee alighted on a blossom near one
of the discs of experiment i, I gently moved the sprig until the bee
was brought to within less than two centimeters of the honey. This
was tried with a dozen bees.

No response was made to the honey.

In a cluster of weeds about a yard from the one in which most
of my discs were located, I had placed, at the beginning of this
series of experiments, a red disc so copiously supplied with honey
that it overflowed upon the weed. This disc was so situated
that by simply raising my eyes I could see it. Although the
melilotus was swarming with bees, that disc remained in that
place for nearly two hours before receiving its first visit from a
bee. At that time, however, a bee hovered at the edge of the
disc and began to sip the honey. It then alighted on the edge
of the disc and continued to sip the honey. Almost immediately
another bee flew up to this one. They both circled about for a
moment and then alighted on the disc; one on the edge and the
other near the center of the upper surface. From this time on,
all of my attention was focused upon this plant.

EXPERIMENT 5. — Near this disc was a blossom which I had wet
with honey. While the two bees mentioned in the above experiment
were foraging on disc one, a bee alighted on this blossom. I gently
moved the sprig until the bee was within about a centimeter of the
two bees just mentioned.

It left the blossom and, alighting on the disc, began to forage.

EXPERIMENT 6. — While these bees were imbibing honey, I at-
tached two other red discs, each supplied with honey, to other
branches of the weed. (For descriptive purposes, starting wiih
the disc upon which the bees were feeding, we will designate
them disc one, disc two, disc three.)

One by one, the three bees on disc one departed for the hi\i .

EXPERIMENTS ON COLOR-VISION OF THE HONEY BEE. 263

On leaving, each hovered a moment above the disc, circled around
it. made two or more short circles about the weed, then, ascend-
in^, depart. •<!. In about five minutes, two of the bee- had re-
turned to the weed. They did not visit any ot" tin- blossoms;
hut, alter circling in the vicinity of the disc for a moment,
alighted on ihe disc and began to imbibe the honey. They
arri\ed I.--- ih.m half a minute apart. Alter >eciiring a supply
of lione\ , they <leparted in the same manner that they did before.
In It •-- than ti\e minutes, the two had returned. < >ne . .f th.
alter arming at disc one, left it and went to di-c \\\» and ob-
tained honey. These three bees were watched carefully for half
an hour. In that time fourteen visits were made to the red <li
ele\en io <li-c one and three to disc two. No vi-it- \\ere made
i" disc three. On two occasions three and on two other occa-
sions t\\o bees were on disc one at the -ame time. On each of
the-.- occasions, before departing for the hive, the bee- al\\a\-
explored the neighborhood of the disc.

I or the half hour or more that I was conducting this experi-
ment, I was too much occupied to pay anj attention to the di
on the other weed. I now watched them continuous!) f<>r fifteen
minute-. Although the weed was alive with U es, no bee vi-iie.|
the red <li-c-. Had any of the bees discovered tho-e di-c- and
had tin \ begun to collect honey from them, some of them should
ha\e made a return trip while I was watching. It i- reasonable
to conclude that no bees hail visited them.

Thu-. although over a dozen discs, well supplied with lumey,
\\ere e\po-ed. on weeds that were alive with bee-, for fully three
hour-, \et onl\ tliree bees visited those disc- and their vint-
\\ere confined to two of them!1

I \i'i KiMi-\i 7. — On leaving for home, all of the discs were rc-
n:, >:;-(l from the field except the three from which (lie l>ces were collect-
ing honey, (hi those discs I placed all of the honey that they icould
hold. Ihi- \\a- done hoping that, before dark, other bee-, by
imitation, would learn to collect honey from those di-<

tric<l i\\\< .-aim- t>rpe of experiments at oth^r tim.--. Altli»iiv;li
«-i.ilil<- time aluay- rl.i|»r.l l.i-i.ire the di- nded to 1'V tin- l»-t-~. yt-t the

timr \va- -rlili'in as Imij; a- thi>.

264 C. H. TURNER.

Series II. (July 13, 8 A.M.)

On my arrival at the field this morning, I noticed that all of
the honey had been removed from the discs; and, in i\\o cases,
that much of the color had been removed in spots. It looked
as though the bees had attempted to carry off even the paper
that had been saturated with honey.

EXPERIMENT 8. — Among the branches of the same plants of
melilotus from which, yesterday, a few bees learned to collect hoin-y,
I placed six red and six blue discs. Two of the red discs (i, 4)
and one of the blue were attached to the tops of rods five feet high
(the height of the weeds}, the others were pinned, at different levels ,
to the branches of the weed. In the center of each red disc, honey
was placed. The red discs were numbered from i to 6, the blue
from 7 to 12. There was no honey on the blue discs.

Almost immediately a bee alighted on disc one. These discs
were watched continuously for a little less than a half hour.
During that time no bees visited the blue discs, but they made
thirty-nine visits1 to the red discs. These visits were distributed
as follows: disc one, seven; disc two, two; disc three, nine; disc
four, seven; disc five, six; disc six, eight. The bees visited the
discs on the rods (i, 4) just as readily as they did those attached
to the weeds; they visited those high up (i, 2, 4, 6) just as
frequently as they did those low down (3, 5). \Yhenever a bee
was ready to depart for the hive, it always made, in the manner
already described, a careful orienting flight. From now on the
bees began to visit the red discs in such large numbers that it
was impossible to keep an accurate record of the number of visits
Sometimes as many as ten bees would visit the' same di^c at
the same time.2 The significance of this marked change in the1
behavior of the bees will be discussed later.

EXPERIMENT 9. — / selected a red disc from which four bees had
been collecting honey and, while the bees were away, placed it al out
six inches lower on the plant. In its place I placed a blue dis< .
The blue disc did not have any honey on it.

'In all of these experiments, whenever a bee alighted on one of my artifacts, it
was counted a visit, whether it was the arrival of a new bee or a return visit of a
former visitor.

!Whcn these experiments were first planned, it was my intention to mark each
bee that participated; but, at this stage of the work, I rcali/i-d tluit such a. pro-
cedure would be impracticable and my paint and brush were put auay.

EXPERIMENTS ON COLOR-VISION OF THE HONEY BEE. 265

The bees that arrived at the weed in the vicinity <>t" the blue
disc dropped at once to the red disc, without even pausing before

th<- MIII-.

Exi'KKiMMNT 10. — I placed three more rods in the midst of the
san: :'; on two of them I placed blue discs without honey, and

on the othrr »n>- ,i red disc with honey.

At the end of fifteen minutes, all <>f tin- red di-r- were bring
vi-iied by numerous bees; none of the blur di-r- \\ere bi-ini;
visited.

EXI-I KIMI \ i i i. — Two rods were pla< ir each other that

a spaec of >i<>t more than tu'O centimeters separated the discs, (hie
diM 7.(/v ri'd and the other blue. The red di>> ^upplied with

honey, I In- him- was not. After they had been in one position for fifteen
-minutes, the red disc was placed where the blue had :nd the

hi ite placed in //' - from which the red had been taken.

I >urin;< ilit- tc\\ minutes that the-e di-r-. \\cn- under observa-
tion, 1 1 I.LIU bees \ i-ited the red di-r: >ne occasion, three 1

\\ere i. lit. lining h»ne\ in.m it at the -.tine time. ( )nly one bee
\i-ilt •« I the blue, and, rvideniK, -he \\.i- not fura-in- |..r hoiie\ .
She -| ten i .it le.i-i ten minute- on the di-c and mo-t of that time
was spent in one place. A part of the time -lie \\a- rubbing her
. .iin-t the edge of the di-r, the remainder -he -eiiind to
be -impK re-ting.

I \i-i KIMI.NI u. FiVi om a red disc contain;

honey. I ; ntainin^ honey.

I Hi rin- the ten minute- that the-e di-( -, \\cre \\atched. nvd\ e
Visits were m.nK- to the red di-r and only one t.t the blue. < >n
three dillerriit occasions, tlu le \\eie three bee- on t hf rrt bee \ i-itcd the blue di-c.

Just as soon as t'i:> d the honey and left the blue

disc, the a replaced icith a blue disc that i.as nut supplied

icith honey.

\ Hiring the next ti\ \- minute-, ti\ e \i-it- \\ere made to the red
and none to the blue. < >ne bee hovered momen t ai il\ above the
blue and t hen \\ent to the red.

K MM ixi\ii \ i i.v /;/ a different place from that ichere experiment
•med, I arranged, close together on rods, one blue

266 C. H. TURNER.

and tico red discs. On the blue disc and on one of the red discs
honey was placed.

During the time these discs were under continuous observation,
fifteen visits were made to the red disc that was supplied with
honey, one bee alighted on the red disc that did not bear honey,
and three bees alighted on the blue disc. These three bees visited
the blue disc at the same time; one bee alighted on the disc,
and then almost immediately the other two followed.

While the three bees just mentioned were on the blue disc, the rod
supporting that disc was gently removed to a portion of the melilotus
patch that did not contain any of my experimental discs.

One by one, the bees made a careful orienting flight and then
flew away. These discs were no longer kept under continuous
observation; but, at regular intervals, they were visited and the
honey replenished. On those occasions I would watch each disc
for about five minutes. On each trip I found three bees visiting
the blue disc. The disc might be free from bees when I arrived ;
in a short time, however, three bees would arrive. They did
not arrive simultaneously; but, before the first arrival had left
two more would be there. I therefore concluded that the same
three bees that discovered the honey on the blue disc had con-
tinued to visit it, and that no other bees had grasped the signifi-
cance of that blue disc. (Unfortunately, for the reason mentioned
above, these bees were not marked and one cannot be absolutely
certain of their identity; but, from a knowledge of the habits of
bees when foraging and of the time required to make a trip to
the hive, I feel certain that they were the same bees.)

EXPERIMENT 14. — While several bees were collecting honey from
one of the red discs that capped one of my rods, the rod was gently
carried fifteen feet in the direction of the hive and erected in another
patch of melilotus.

One by one, the bees made a careful orienting flight and then
flew away. In a short time they had returned. Often eight
or ten bees would be on the disc at the same time. While I was
taking my notes, some of the bees hovered within a short distance
of the small pad (13X8 cm.) on which I was writing, as though
they were examining it. From now on this behavior was common.

EXPERIMENT 15. — While ten bees were foraging on the red disc

EXPERIMENT- ON COLOR-VISION OF THE HoM V BEE. 267

used in experiment fourteen, the rod was gently carried fifteen feet
nearer the hire and erected in a place that was free from tall weeds;

•: the grass had been cropped short by a horse that had been

:/;/;' //;•

The bees, on leaving, hovered about tin- di-c a Ion- time, even
mining the cork to which the di-c \\a- piniu-d. 1 hen, alter
dea riliinu .1 -hallow spiral, they flew a\\a\ . In a -lion iinu- -i\
h.id reiumed to this disc. On her return, each bee ile\\ to the
i <.:k tir-t and then to the top of the di-c. I was ii"\\ t'orcrd to
leave the experiment for half an hour. On my return, the di-c
\\.i- l.iiirc \i-iied liy numerous bees. 1 hiring the !i\e minntc-
th.tt I \\.itched it, twenty-six visits were made to it. There \\< n
.il\\a\ - from six to ten bees on the disc.

I \ri KIM! vi 16. — From the melilotus weed in which the first
r.v/ !s of this series were perform •<:. I •'..'.. ' a rod

whii h . •! with a red disc upon ichich ten '

tim: en feet further away from the hire, in another patch

>rer.

The bees on leaving made a careful orienting Ili^ht. I left
the di^c for about twenty minutes. On m\ return. 1 found
seven bee- re-ting on the disc and imbibing hoi,.

I \v\ -NIMKNT 17. At that end of the field most distant

from the hire, and at about fifty yards from th< ^ in which the

first experiments of this series were performed, there was a l<.
patch of the same plants, from the racemes of : numerous

.'/<•( //;;;• honey. In the midst of these weeds, hut well exposed,
I placed a red .// poured some honey.

I inn, lined b\ thi- i »d for tuenty minutes, but no bee a|)-
pn. ached it. At intei\al> of ten minutes, I made six vi-it- to
thi> di-c. l-'.ach time I leinained h\'e minutes. At no time did

1 III id an\ bee- \ i-itili^ the di-c.

l;..\ri KIMI -NT 18.— In a space / •»! tall weeds, about thirty

vtirds nearer the hire than the icccd in which the first experiments of
this scries were performed, I erected < my free foot rods and on

its top placed a red disc well supplied with honey.

At interxals of ten minute-, 1 made four vi-it- to thi- di-<
Tin- tn-t time 1 I'mmd one bee on thedi>c; the second lime, three;
the third time, t\\o, and the fourth, four.

268 C. H. TURNER.

At this stage, I removed all of the discs from the field except the
one used in this experiment.

At intervals of ten minutes, I made two additional visits to
this disc. On the first trip I found ten bees on the disc; on the
second trip, I found eight.

On leaving for home all of the discs were removed from the field.

Series III. (July 14, 7:30 A.M.)

Apparatus. — The cornucopias used in this series of experiments
were made in the following manner: A piece of cardboard,
colored on both sides, was cut the shape and dimensions shown
in Fig. i. It was folded along the dotted lines and the flaps

FIG. i.

fastened where they lapped. About one centimeter of the apex
of the cone was bent over and fastened. When finished, each
cornucopia was nine centimeters long, with an elliptical lip
six centimeters wide and three centimeters high. The lip was
used for attaching the cornucopia to some support. Incidentally
it furnished a platform for the bees. Some of the cornucopias
were red and some were green.

EXPERIMENT 19. — In the weeds that were the seat of the experi-
ments of yesterday, several cornucopias were arranged at dijlcroit

EXPERIMENTS ON COLOR-VISION OF THE HONEY BEE. 269

levels. Half of these were red and half were green and they were
arranged in pairs, one red and one green constituting a pair. The
openings of both members of a pair faced in the same direction. All
of these cornucopias were attached to parts of the weed. The red
ones were supplied with honey, but the green were not.

Immediately .ifi<-r I had pinned the tir-t red cornucopia to
tin- l>u-h, .1 her entered it and began t<> collect tin- honey. Two of
thr-r ]),iir- \\ere kept under observation for nearly an hour.
During that time numerous bees entered the red cornucopias,
luit not .1 bee eniercd the green ones.

1 ,\IM KIMI \ i Jo. — Five red corn nco pi >nt<iining honey,

wrrc arranged on rods in the following manner: A, in a patch of
melilotns a/iout ntty yards further from the hire than th<
that were the site of experiment nineteen; H, ('. />. arranged in a
lin> <>n the patch that was the site of experiment nint

inls the hire, H twenty feet from the patch, (' thirt
and /) fifty feet; I .' yards from th< patch mentioned

a/>ore in a line which, at the ex/ <'/, made an an^i*

45° with the line containing B, C, and D. 1 these cornucopias

ited once nery twenty minutes, at which tin: .matched

closely for three minutes. The number oj n the cornucopia

when I arrived were counted, the number to arri:< I did ;

noted anil the sum of the two numbers recorded.

The results of the above experiment are ivionK-d in the |dl-
t.il 'le :

< i\\

N.I ,-ia.

N n n 1 1 n i I on trip i

Nuuilii : I "ii trip 2

Niiinl" s I nil trip 3

Niiniln • 1 mi trip 4

! "ii trip 5

Niirnl"-: ! "ii trip 6

of bees obsiT\i-.l ..n tri|i 7-

2 8

U ii

12 Hi

X X

Bel ;•• I<M\ \\\\i tin- IMT in.i -itim; tli^lit. Sin- i-xaiiiiru-il the <

i.i "ii all >ulr- -inic-. n-i-iui'ii-'l it thro- tim.-s. .in. I. -iiibing

ic\v i-iir vi-. tlr\\ ,i\\.iy t" tin- hi\

1 I:* re \\.i- .in inti-r\.il "! an Imur lu-turcn trip -ix an'l trip -rvni.
>T\vii ntlii-r IM-I-- h"\i •[•••! in ai. luit ili.l n.it i-mrr. Tin- "nc that t-iui-rt-d l.-u
iinincili.iti-ly. Tin- i-i>riuio>pia \v.i~ ~\var riling with ant-.

X nii-.ui- that tin- I»T< wen- so numerous that it wa< iinp"--il>li- tn make an ac-
curate i-niint.

2/O C. H. TURNER.

EXPERIMENT 21. — Side by side, on one of the branches of the
melilotus weed upon which most of these experiments -were conducted,
I arranged a red and a green cornucopia, and placed honey in each.

During the first five minutes that these were under observation
twenty-five bees entered the red cornucopia and three the green.

EXPERIMENT 22. On rods erected in the open space between the
experiment weed and the hive, and about three feet from disc C of
experiment 20, a red and a green cornucopia were arranged side by
side. Each contained honey.

During the first five minutes that these cornucopias were
watched, sixteen bees entered the red cornucopia and four the
green.

EXPERIMENT 23. — The green cornucopia of experiment 22 was
replaced by a red cornucopia which did not contain, and never had
contained, honey. This placed two red cornucopias side by side,
one containing honey and the other empty.

During the five minutes that these cornucopias were under
observation, so many bees entered the red cornucopia which
contained honey that it was impossible to count them; five en-
tered the empty red cornucopia.

The empty red cornucopia was now placed where the one containing
honey had been and the one containing honey placed in its stead.

During the first five minutes that they were observed, so many
bees entered the cornucopia which contained honey that it was
'impossible to count them; many bees hovered around the empty
cornucopia, but none entered.

The cornucopia that contained the honey was removed, the bees
shaken out, and the cornucopia put out of sight. This left an empty
red cornucopia in a part of the field which, for more than half an
hour, had contained at least one red cornucopia which was well
supplied with honey.

At first the bees circled around the cornucopia, presently one
entered and then left immediately. Within ten minutes twenty-
five bees had entered the cornucopia. (This does not mean twenty-
five different bees, for the same bee entered more than once
and was counted each time.) At first each IKV left as soon as
she had reached the inner depths of the cornucopia. Soon, how-
ever, the bees began to enter so rapidly and in such large numbers

.PEKIMENTS ON COLOR-VISION OF THE HONEY BEE. 271

that it \\a- impo— ible for those that had reached the inner depths
to leave without a struggle; and, in less than tliirty minute-,
tin- cornucopia \\ a- packed almost full of strutting bees, and
numerou- others were hovering around the mouth. -ecking a
place to enter.

Ivxi'i KIMI.NT 24. — Ever since the beginning of this of ex-

periments, the red cornucopias on the melilotus had been kept well
supplied :cith honey. At this time the w> >:>ained ei'J;t red

• -.nenpins and an equal number of empt • • . Into the

upper pnrlmn tif this weed, I placed an empty red eornn>

1'iiiii,^ i!ic five minutes that this cornucopia \\a- uatched,
t \\ el\ c I »ees i -n tercel, one at a time, tarried a m< micnt ami i lu-n left .

I \ri KIMI NT 25. — In the open, three feet from //';•

•:n<t>pid of experiment 23, I placed, on a rod, a>:
i uniin npiii. In this place, earlier in the morning, there had
d cornucopia well supplied with honey.

1 Miring the ten minutes that this cornucopia was ol)MT\r<l,
man\ I •»•(•- liovered around it ; one alighted on the front platform,
but MOIH- entered.

All of the cornucopias were removed from . '</ except the

empty red cornucopia of experiment 23 and :• '>ty ^reen

•his experiment. This left only two corns in the field;

red and one green, neither of which had ever contained honey.

I hi i^reen cornucopia was watched continuous!) for tc-n min-
utt-. I'miiii; that time many bees ho\'ered around the green
conmcopi.i ; t \\ o aliijiied on the front platform, and one entered.
\i i lir close "t i In- icn minutes, I walked over to the empty red
cornucopia and ton ml it almost full of strui^liiiK bees, and ntinier-
ou- other I iee- \\en- ho\ering around the entrance seeking admit -
tai;.

Series IV. (July 15, 3 P.M.)

This >erie> < •!" e\periinciil s was I'ontliu'ted with sj>ecial (.ud-

boanllH.v h consisting of a rectangular outer case (8 > 5.2

X 2.5 cm.) with a porch-like cxtcn-ioii in front and open end-,
into \\hich then' \\a- -ho\.il. from the rear, .1 canll'oanl t ra\-

5-5 Xvs X 2.4 cm. \< ir one side of the front end of this tray an

i-iiirance \\ a- m.ule. In most cases this entrance was a reciaii-u-
lar optMiinu 2 X 1. 2 cm.; in a >pecial ca-e it wa> circular and 1.5

2/2

C. H. TURNER.

cm. in diameter. The tray was shoved in from the rear until
its rear end was just inside of the rear edge of the outer case.

In constructing the outer case, a piece of cardboard was cut
the shape and dimensions of figure two, folded along the dotted
lines and glued where the sides overlap. In constructing the
inner tray, a piece of cardboard was cut the shape and dimensions
of figure three, folded along the dotted lines and glued, by the
flaps, to the inner portions of the adjacent sides.

EXPERIMENT 26. — In the same weed that has been the site of most
of these experiments, I placed one green and two red boxes. The
green box was placed near one of the red boxes. The red boxes con-
tained honey, the green was empty.

It had been raining all morning and it was still quite cloudy,
and only a few bees were afield. About five minutes after the
beginning of the experiment a bee noticed one of the^red boxes.
She examined it carefully from all sides, found the entrance

FIG. 2.

and entered. Soon after she entered, a heavy shower of rain
began to fall and I took shelter under a tree. On my return
(about fifteen minutes later) I found a large digger wasp in box
number one and bees visiting red box number two. As long as
that wasp remained in box number one, no bee would enter it.
During the time that these boxes were under continuous observa-
tion, fifteen bees visited red box number one and twenty-ri^ht
visited red box number two. Towards the close of this cxpcri-

EXPERIMENTS ON COLOR-VISION OF THE HONEY BEE. 2~ \

no alien-

. 1 •

ment, some of the bees would fly into the tray without first
alighting on the portico. Not once was the green box visited
by a 1 •

Kxn.kiMi .\ i 27. — On my return after the rain mentioned in
experiment j 'itch-glass, seven centimeters in diameter, con-

tain in cd on the ground within \ of the weed in

which the boxes mentioned in experiment 26 were located. It was
:tt, on such a cloudy afternoon, it was not cons pic u-

I >iin'ii^ ilx- time that I was watching expi-rinu-nt
tion w.i- 1 1. 1 id to the watch-
gla--. At the close of that
• liinciit the watch-glass
was "b-rrvi'd continuously
t i-ii minutes. During
t In! i nut- not a single bee vis-
i i i-i| the watch-glass. Since
i IK trip to the hive required
ill. in live- minutes, any
that had succeeded in
li nd inv; this watch-glass full
"i honey would have made
.it Ir.ist one visit whilr I
\\ .1- \\ .1 irli in- ; hrnce it is log-
it .il in i < iiu hide that no bee had visited it.

At this point rain caused a recess until eight A. M., July 16.
On /cc. the boxes were removed from the weeds

and the watci: >: the ground.

I AIM KIM i \ i 28. >/</« by side, in the same weed that has been
the s,'<it of the majority of these experiments, I placed tu'o red experi-
ment />o\, .iained honey and the other was empty.

\~ -"mi ,1- I .ippr.uvd on the scene, the bees began to IIDMT
aliDiit IIH-. .ind. brl'dri- I could pin the red box with it- -upply <>f
li(MH'\ to tlu- \\rrd, .1 1 >oe had entered its tray. In a feu minutes
so many bees \\< r<- \ i -it ing the ml lm\ that cmitaiiu-d the honey
that it \\a- imp. >— il >]<• to count tlu-m. l;m|iirntly bees would

the i-mpty ml b«.\, but tln-\ \v«.uld not tarry long.
Exn.RiMi.N r 2'). — /;; ///(• open spa. ,-n the experiment weed

2/4 C. H. TURNER.

and the hive, I arranged, on poles, about ten centimeters apart, three
red boxes containing honey, one empty red box and one empty green
box. The arrangement of the boxes in the group was altered once in
ten minutes.

These boxes were under continuous observation for about an
hour. Immediately the bees began to visit the boxes that con-
tained honey in such large numbers that it was impossible to
count them. Occasionally a bee would enter the empty red box
and frequently the}' would hover in front of the entrance to its
tray. No bee entered the green box, although occasionally a
bee would alight on its top and pause long enough to clean
its legs on an edge of it, and frequently one would pause a
moment before some portion of the box. Whenever the honey
was exhausted from one of the trays, the number of visitors would
drop off. Seldom would a bee pass through the entrance; fre-
quently one wrould hover momentarily before the door and then
pass on. As soon as I had replenished the honey, the bees would
begin to revisit it.

EXPERIMENT 30. — After the above experiment had been under
way for an hour, all of the boxes were removed from the field, except
the empty red and the empty green box of experiment 29.

Immediately the bees began to enter the red box more frequently
than they had hitherto; as soon as one got well inside, it would
leave. After a lapse of a few minutes, the bees began to rush
for the entrance in such large numbers that those that had en-
tered and wanted to leave could not do so without a struggle.
As a result the tray and the portico were crowded with struggling
bees, and numerous others were hovering about the entrance,
seeking admittance.

At first no bees entered the green box, although many circled
about it. After a lapse of ten minutes a few began to enter.
During the period of observation, ten were noticed to enter it
and leave immediately, and about twice that number were noticed
to alight in the portico.

EXPERIMENT 31. — Standing about three feet from the above boxes,
I held, in my hand, a red box containing honey.

Immediately a few bees approached and entered the box. I
held the box in my hand for about five minutes. Throughout

KXl'EKIMEXTS OX COLOR-VI-K >X OF THE HOXEY BEE. 275

that period there were from one to four bees inside of the box
all the time.

I .\ri.ki\n \T 32. — Standing in the same place mentioned in ex-
periment 3i,I held an empty red box in my hand. This box had
'nitnincd honey.

Several bee-, approached and hovered around the box, and t\\<-
entered.

IN I 1 KI'RETATION OF THE Ex PI-KIM I NTS.

Vision or smell or some combination of the t\\o are instrumental
in '^iiidiii'4 in-ects to flowers. Lately some observers have

•••d ilia! \i-ion plays no part in thi- belia\ior; indeed, ii is
' la in ii -d ilia i ii is not even olfactory perception-, but odors a< i ini;
n-ilexK that lead insects to flowers. No one \\lin ha- made
ol.ser\ alii -i i- lor himself would think of claiming thai smell pla\ s
in- role in in-ect behavior; but, the experiment- described al-o\e
-ho\\ i -i. in lu-i\e|y that odors, acting rcllexh . do not lead I
lo llouer-. In I • -i a lilies where bees are m-i accu- turned to obi. i in
hone\ from anything but flowers, hone) ma\ be placed on small

di-i I l, 17 , or ill open ve— > 1- I \. Jo .1 and /•>'. J7 and
left «-\|'i>~ei| l<-r a li-ni; time without bein^ re-pi. nded t<. by tin-
I'.- e-, captured in In. tile- and turned Im.-e upon <IUc- that
are \\ell xii|.|ilicd with In -ne\ , will usually di-part \\ itlmut pa\ ini;
an\ atteniion to the honey (Ex. 2). If a llo\\er upon which
Mich a In -ni; is so manipulated as t<- bring the bee in

clo~e pn-ximii \ to the honey of one of those di-c-. -he \\ ill n-uall\
depart \\ithoiii re-ponding to cither the hi-ne\ or the disc (Ex.
3, 4 '. It i- incredible that an od<.r of hone\ loo \\eak to at tract
3 a feu mil!imeter> oft i- able to entice them from several
meters iii the air. Then, too, each »\ my di-o contained more than
a thou-and times as much honey as any one of those !lo\\ t i -
did nectar, and the cornucopias thai 1 u-ed contained more than

tile di-c-; \ct the bee- pa— ed 1 >\ the tea-t of hmieN prepared

for them to sip tin- meaner -ii|i|il> of nectar stored in the neighbor-
ing; -mall llower-. ^uch behaxior \\ould be impossible if their
mo\emcnl- \\ere controlled b\ the hone\-oilor actiiiL; retlexK .

To claim that the nectar of the tl«. \\i-r h reater attractive

power than the IIOIHA \\--uld be illo-ical for: 1st, hone\ i- con-
centrated nectar: and 2nd, these same bee-, after they ha\e

2/6 C. II. TURNER.

learned to collect honey from objects other than flowers, will
visit such objects as soon as they are attached to the support
(Ex. 8, 19), at times they will even enter them while they are
being attached to the support (Ex. 28), or they may even enter
such an object while held in the hand (Ex. 31, 32).

If a set of bees has become accustomed to collect honey
from artifacts, and paper discs, arranged in pairs of one red
and one blue (or discs of any other two colors) are scattered on
weeds or placed on weed-high rods, and honey is placed on the
discs of one color and none placed on those of the other color,
the bees will make regular visits to the color that bears the honey,
but will not so respond to the other color (Ex. 8, 9, 10, u). If
these experiments are repeated, using cornucopias (Ex. 19) or
boxes with small openings (Ex. 29) in place of the discs, the
results will be the same. If bees have become accustomed to col-
lect honey from an artifact of a certain color, and empty artifacts
of the same kind and color are placed along side of those that
contain honey, many of the bees will enter those artifacts that
never have contained honey; empty artifacts of a different color
are not responded to in that manner (Ex. 23, 24, 28, 29). After
bees have, for a long time, been collecting honey from artifacts
of a certain color, if all the artifacts be removed from the field
except two that never have contained honey, but one of which
is the color of the artifacts from which the bees have been col-
lecting honey, numerous bees will flock into the empty artifact
of the same color as those from which the bees have been foraging;
but none, or nearly none, will visit the other artifact (Ex. 25, 30).
When bees have become accustomed to collecting honey from other
sources than flowers, if receptacles of two different colors are
placed on a bush, all of one color containing honey and all of
the other color being empty, and if, after the bees have been busy
for a long time, honey is placed in one of the artifacts of the color
that has been empty all along, it will remain on the bush some
time before it will be visited by any bees (Ex. 12, 13, 21, 22).
All of the facts recorded in this paragraph indicate that the be-
havior of foraging bees is influenced by colors.

That these bees not only respond to colors, but that they are
capable of recognizing them at a distance is evidenced by the

EXPERIMENTS ON COLOR-VISION OF THE HONEY KEE. 2"

following facts : (i) If, in an open space between their hive and
a number of artifacts from which the bees are collecting honey,
you place an artifact of the same kind and color and supply it
with hoiH-y. it will be vi-ited by bees almost immediate!} l.\. [8,
29). (2 > When bees are collecting honey from red artifacts situ-
ated in two different Stations, one of which is much nearer the
hive than the other and beneath the line of flight of tin i
collet tin- from the more distant station, if all of the artifact^
are removed from the more distant -tation. immediately the
number of visitors to the other station i- much increa-ed (Ex.
'I hi- was the case even when the artifact in the nearer
station ditl not contain honey (Ex. 30). Kmpty artifact- nl
another color than the honey-bearing i-"l--r \\<-re n-'t re-ponded
to iii tin- in. inner (Kx. 30.).

< »u lea\iir^ one of these artifacts the bee u-uall\ made an
orient in;.; flight. < )n the first few vi-it- tin- \\a- thorough l\ done.
I ing tin artifact and keeping about one centimeter tn m it-
surface, -he \\«nld -idle, in a xig/ag line, around the -tincture
two or more times and occasional!) neuter it one or more tin
I In n -he \\oiild de-i libe one or more spiral-, pau-ing at certain
plai e- in the environment as though examining landmark-. In
-nine of the cases I was, apparently, one of the objects tlm.-
-t mi in i/e<l. In harmony with her other beha\ ior. it -eems plan--
l-ile to interpret this as an act by which menior\ |iicturesof the
en\ iidiinieiit ate formed.

llo\\ minute are the details that bees obser\ e 1 am not pre-
pared to -a\; but that the\ do observe details is indicated by the
following observations. i In the bo\e- u-cd in the experiments
• •!' •>« i i« - I \ . t he t ra\ 5 of t he 1 -t >\e> used were entered, frt un t he
portico, by mean- of an e< > « ntric opeiiin^. In most of the 1"
thi-dooi\\a\ \\a- i< ular, in others it was circular, \\lnn

a bee ln-t appioached one of the-i- 1 .o\. 5, -he had to search tor
the entrance. After a te\\ trip- -he \\oiild land on the portico
directK in front of the entrance, and. in -ome . asi 5, she \\oiild
ll\ into tin- tra\ \\ithoiit e\eti pau-inu . .n the jmrtico. u I or
about an hour bee!- had been collecting hone\ from -ome red
artitai t-. It -eeined that llearK all of the bee- that \i-iled that
of the field \\ere collet tin- from t lm-e artifact-. Alonu-ide

2/8 C. H. TURNER.

one of the red boxes I placed a red box on the sides and top of
which I had pasted bits of white paper. This gave a box with
red front and spotted sides. Into this box I placed some honey.
The bees that approached this box from the front always entered
immediately; the majority of those that approached the sides
paused a moment, then went to the nearest red box.

Whether this is a true color vision or simply a greyness dis-
crimination is no easy question to answer; indeed, from our view-
point, it does not seem an important one. If what to us is red
and green appears to the bees as two distinct sensations, as a
factor for controlling behavior, it will have the same value to
the bee whether it is a red-green discrimination or a grey-grey
discrimination. However, the following line of reasoning has
led me to believe this a case of true color-vision. Bees that had
learned to respond to red boxes in the shadow of the weeds would
respond, without hesitation, to similar boxes placed in the sun-
shine. They responded to the boxes when the sun was shining
brightly just as readily as they did when a dark cloud hid the
face of the sun. The brightness content of a body in the bright
sunlight is quite unlike the brightness content of the same body
when in the shadow of weeds; the brightness content of a body
in the sunshine is quite unlike the brightness content of the
same body beneath a cloudy sky. The only factor common to
all of these cases is redness; hence I feel that, with the bees, it
is a case of true color vision.

Although odor as a incitive to reflex actions does not play any
part in leading bees to flowers, yet odor as a sensation does. If
a large number of bees are collecting honey from a cluster of
boxes that are all of the same color and you allow the honey of
some of those boxes to become practically exhausted while that
in the others is constantly replenished, when the workers ap-
proach the boxes that are practically exhausted, they, as a rule,
do not pass inside; but, pausing momentarily before the box,
pass on to one of those with an abundant store of honey. Re-
plenish the empty tray with honey, and the next bee that ap-
proaches that box will enter (Ex. 29). This, to my mind, shows
that when a bee approaches a box and finds the honey-odor weak
she immediately departs for a box where the honey-odor is
stronger.

EXPKKIMKN COLOR-VISION OF THE HONKV HI I . 279

These experiments prove that, to tin- bee, my colored di-cs,
my colored < -onim opias, and my colored boxc- were something
more i ban mere -en-ations; it seems t<> me that they were true

• I it-. T<. the bees those things had acquired a mean:
tlio-.- -trance n-d things had come to mean "honey-bean
and th' :tr^e green things and strange blue tiling- had come

to in. -an "not-honey-bearers." Hence, whenever the bees -aw
the n-d tiling-, they made the appropriate movement- tor -ecurinji
the lioiie\ , and when they saw the blue thin-- . .r the ureeti thi
the\ pa — i-d on. This explains why, in the experiments oi series
One, di-« - -i\ centimeters in diameter and \\ell -upplied with
hone\ i ould remain in the presence of hundred- of bee- \\ ith.mt
b.-in^ r. -ponded to by them; and yet, tho-e -aim- bee-, a te\\
da\- lai«-r, when those things had acquired a meaning, \\oiild
em. i m\ red boxes even before I had had an opportunit\ to
.ittai h them to their supports. In their past experience tl
tiling had never accjuired a meaning, while the -mall blo--.>in-
ot the melilotus had come to mean "honey-bear. -i-": lum. the>
tened l»y the feast that had been prepared for them and ru-hed
i he meager supply of nectar in the blo--«un- of the \\hite
-\\iet clover.

Although Plateau's conclusions are diametrically oppo-ed io
the re-ults of this series of investigations, yet the facts related
b\ him are in accord \\ith them.

While proving that bees have color-vision, these experiments
thio\\ no liyjn upon the color preferences of in-ects. That has
not been the purpose of these researches. The aim has been to
an-\\er the i|iie-tion. C 'an bees distinguish colors? The experi-
ment- seem to demon-! rate that foraging bees have percepts and
that t\\.. i.i, tors \\hich enter into those percepts are color sen-a-
tion- and olfactor\ -eii-at ions.

Mo.,

July is. i..

BIOLOGICAL STUDIES ON CORYMORPHA. IV.1

BUDDING AND FISSION IN HETEROMORPHIC PIECES AND THE

CONTROL OF POLARITY.

HARRY BEAL TORREY.

The large solitary hydroid Corymorpha exhibits the phenome-
non of heteromorphosis in forms even more striking than those
under which it appears in the related Tubularia. At the same
time, its normal polarity is in several respects more obviously
marked. As against a stem; in Tubularia, that presents little
or no indication of axial differentiation, the column of Corymorpha
is divided into several regions sharply characterized by differences
in form, structure and function. Its diameter varies, being
greatest near the base, which is enveloped, for about one third
the total length, in a thin layer of perisarc. Beyond the edge
of the latter, the naked ectoderm is thicker, its cells are more
narrowly columnar, and there is a marked increase in the number
of nematocysts. Within the perisarc is the zone of frustules, or
rootlets, that form the holdfast and have been homologized with
the stolonal processes of Tubularia, although they are far more
specialized structures. The proximal extremity, conical in form,
is furnished with an amoeboid ectoderm, by means of which the
polyp creeps about.

Not only in structure does one find evidence of regional dif-
ferentiation, but in capacity for regeneration as well. A hy-
dranth is replaced after section of the column, with a velocity that
decreases wdth the distance from the distal end of the intact
hydroid. The differences in velocity are so slight as to be ap-
preciated with difficulty in the distal half of the column, but are
easily recognizable in a comparison of rates of regeneration in
distal and proximal thirds. Furthermore, heteromorphosis,

'Contribution 32 from the Laboratory of the Marine Biological Association of
San Diego. Preceding numbers of the Biological Studies on Corymorpha have
appeared as follows: I., C. palma and Environment, J. E. Z., i (1904), p. 395;
II., The Development of C. palma from the Egg, Univ. Calif. Publ. Zool., 3 (1907),
p. 253; III., Regeneration of Hydranth and Holdfast, ibid., 6 (1910), p. 205.

280

BIOLOGICAL STUDIES ON7 CORVMORPHA.

28l

•

though it may occur after section of the column even below the
fru-tular /one, in the extreme basal region, is most frequent in
piece- (lit from the column in its distal half.

At the very 1>< -ginning of my observations on the regeneration
of C'i>r\nii>r{fhii, I was Struck with two facts : (I) that a -egment
from tin- di-tal half of the column and including the hydraiuh,
do.-- IK it d.-\elo|. ,i h \dranth at the proximal end until the original
hydrant h i- removed; (2) that when the original hydranth ha-
lii-eu reino\ . -d. the proximal hydranth dexelop-, umler normal
condition-, in. ire slowly than the distal— another indication, it
ma\ Li- mentioned, in passing, of the initial polarization ol the
( < .luinii.

Tin-, facts suggested the possibility that, 1>\ dela\ing the
development of the distal hydranth on a regenerating piece until
the pioxiiiial h\dranth should
h.i\c n.ii lud an advanced stage
oi di \clopiueiit, the initial polar-
i/atii-ii mi-Jit lie completely re-
ed. A. . ' .idingly, in the
Mimim-i . .1 i'M>2, I performed the
I'ollou ing experiment.1 A seg-
nieiit \\a-cut I loin the distal hall
o| .111 a\erage polyp (Fig. I, A).
h \\a- linn inserted, and the
di-lal cut Mirface held against
tin- gla--- IK. it on i . .f th.- aquarium F

|.\ the uei^lit "I a -trrl iici-dle
through the distal region I
1. />'). The |.n. \imal end was
and the Mem \ertical. At
tin' end of three da\ -. a ludianth lloiiri>hed at p, though tin re
\\a- -ign neithi-r "t tentacle- nor fru-tule- at <l. The needli- was

removed. I"\M> <la\ - later a -mail hydranth had appeared al <1

(Fij [, I .\\ith three di-tal and f. -ur pn.vim.d tentacli'>, sonu-
\\hat iriegularK arranged, probal >1\ < .\\ing to the \\oiind let't l'\
the needle and t.. other adverse conditions to \\hich that end
max ha\e l>eeii -ul.jected \\heii |>re— ed against the -til stratum.

nnti'.l in tin ixurred under starvation conditions.

FIG. i.

282 HARRY BEAL TORREY.

It will be noted that while a distal hydranth failed to develop
in contact with the substratum, it soon appeared when freed
from this contact, in spite of the presence of the large promixal
hydranth. On the supposition, however, that the result may
not have fully indicated the real state of affairs in the hetero-
morphic segment, the latter was sectioned at the level x. In two
days frustules were appearing at p' ; the original polarity of this
portion of the column was preserved. But frustules were also
appearing at d' , and two days later, were unmistakably defined.
In this latter region, therefore, the original polarity was reversed;
on a segment of a given polyp, not only had hydranth appeared in the
customary position of holdfast, but holdfast had appeared in the cus-
tomary position of hydranth.

The outcome of this experiment recalls the reversal of polarity
which Loeb later obtained in Tubularia crocea when, after ac-
celerating the development of the proximal hydranth by inhibiting
the development of the distal, he cut a segment just distal to the
proximal hydranth and found that a proximal was now produced
more rapidly than a distal hydranth.1 Morgan and Stevens
obtained a similar result on T. marina although the polarity of
the stem was reversed for but a very short distance from the
proximal end in this species.2

II.

The suggestion, coming from the above experiment with Cory-
morpha, that section of the column between the hydranths merely
disclosed a reversed polarity that already existed but was not,
under the conditions, expressed in structural differentiation, led
to a number of similar experiments which showed that the original
result was in no sense exceptional. I will consider three series
of these experiments.

In the first, nine heteromorphic pieces were sectioned at dif-
ferent levels to determine the extent to which each hydranth
might control the intermediate region in regeneration. In no. I,
the distal hydranth was removed by a cut immediately below it

uger's Arch., 102 (1904), p. 152; trans, in Univ. Calif. Publ. Physiol., I
(1904), p. 151.

*J. E. Z., i (1904). P- 559-

BIOLOGICAL STUDIES OX CORYMORPHA.

283

(Fig. 2, a); it was replaced by another hydrunth that at the end
of ten day- \va- in tin- condition shown in Fit;. 2, b. No. 2 wa-
a similar < ase. In neither of these case- wa- re\er-al inhibited.
No. ;> died. Mo. 4 is represented in i the proximal hydranth

bcin^ in in -1 1 li ---d» -\ doped than the distal, and the plain- of -cction
g near but not immediately distal to it. In four days both

FIG. 3.

nentB p"--e--ed frustulcs; the proximal set; men t had, accord-
ingly, r No. 6 wa> a
-iinil.ii No 7. i ut \\ln-n in the c-«.ndition shown in 1 i.

appeared, five days later, as -ho\\n in l-'i^. 7. No. 8, a similar

case, exhibited -imil.irl\ a complete re\ er-.il in li\ e days. No. M
aibled I-'I'L;. 2. ,1 ; but it was the smaller, proximal hydranth
that was removed, by a CUl immediately di-tal to it ; in -e\ en d.i\'s
a lieu ludranth \\a> e-tabli-hed in it- pla.

\< . • >rdint; to the-i- iv-ult-. heteroinorphic piece- pnuluce hold-
fa -ts .11 the \\ound when sectioned approximately midway bet \\een

the h\draiuh>, but i)ro<luce ludranth- at the wound on tin- longer
piei ••.-- iii tho- 9 in \\hich either ludranth has been removed

by a cut immediately bclo\\ it.

284

HARRY DEAL TORREV.

The second series of experiments involved 14 heteromorphic
pieces whose proportions and stage of development are represented
in the accompanying diagrams (Fig. 8). These pieces were cut
as indicated in the diagrams. Four days later, both parts of a, c,

FIG. 3.

FIG. 4.

d, e,f, h, i, I, were attached and possessed frustules; the polarity
of one of the pieces in each of these cases, accordingly, was com-
pletely reversed. At the same time, both portions of b were

FIG. 5,

regenerating as single polyps, one being attached and furnished
with frustules, the other being unattached and lacking frustules.
Under g, four heteromorphic pieces were grouped. Four days

BIOL'K.IiJAI. VU-IUKS ON COKVMORPHA.

at't.-r cutting, all were regenerating as -in-le polyps, six being
.cht-d .uid j^i,- g frustules, two being unattached and lack-

ing" fru-tulr-. Moth pieces of k were attached, three days after

d.K

JV-

FIG. 8.

iiii;, but i'\\iivvr !<> acrideiital nei^U'Ct, d i-i nt < -grated lie-lore
forming frustule-.

286

HARRY DEAL TORREY.

It should be noticed especially that all the heteromorphic
pieces used in this series were short, and none were above medium
diameter. In not a single case, tinder these conditions, did the
polarity fail to reverse in one of the portions into which the hetero-
morphic pieces were divided.

The third series shows the importance of this consideration of
length. Three heteromorphic pieces, absolutely and relatively
much longer than those of the second series, one of them con-
siderably larger than the other two, were sec-
tioned as shown in Fig. 9. Five days after
section, all six pieces wrere heteromorphic which
indicated that in none of them, whether distal
or proximal, was development at the wound
dominated by the conditions, existing at the
other end of the piece.

The same fact is brought into clear relief
by a comparison of the following figures. Of
14 segments representing the distal half of the
column of 14 polyps of moderate size, 12 were
heteromorphic in 3 days. Of 81 very short
segments from several small polyps, only 6,
j/ or 7.4 per cent., became heteromorphic.

Further, of 13 segments of approximately
the same length and diameter as the pieces
obtained by cutting the heteromorphic pieces
in series 2, 8 became heteromorphic. Of 15 similar segments,
10 became heteromorphic.

Besides these figures, there is a mass of evidence, obtained by
repeated experiments on large numbers of individuals, demon-
strating that the presence of the original hyd ninth on a segment
of the column inhibits the development of a proximal hydranth.
It is clear, then, in the light of the facts cited in this section,
(i) that reversals of polarity profound enough to effect entire seg-
ments of the column as units are readily produced in Corymorpha;
and (2) that the stage of differentiation at one end of a piece will
under certain conditions control differentiation at the other end. That
reversals of polarity, in cases of heteromorphosis, are often shown,
by form changes, to affect considerable areas of the column,
without aid from the knife, will appear in the following section.

FIG. 9.

BIOLOGICAL STL I 'IKS ON CORVMORPHA.

III.

287

Aa tin- fir-t evidence in this direct inn I may refer at once t<>
certain I'--haped figure- formed from hcteromorphic segment- 1 >y
tin- attaclnncnt ••!" tin- latter to the bottom of tin- a«(iiariuni di-h
by mean- of adhc-i\e ectoderm developed mi one -ide of the
column. Sin h a case is shown in Fig. lo, \\hich rcpre-ent- the
condition of a M _iin nt sixteen days after it^ remo\al fnnn the
di-ial hall" "I a i «ilunin. A constriction define- the al»>ral limits
1. 1 the n-^eneratinv; polyps. Frustules are pre-ent in two

10.

ri'iii:hl\- |ir<«|mrti"iial in numbers to the >f the |>«.l\|)s to

which they belong, and situated in the p"-iti'»n nnrmall\ nccupieil
l'\ I'rii-tule- ill the adult. The smaller p"I> p appn>ache- nmre
rl»-el\ tn the proportions of the larva. < >n the lnll«i\\ in^ <la> ,
thi- process <>t li— imi had been n'mpleted. pn>l.ably 1>\ rupture,
althi'ti.uh the pn-ximal end- uf the t\\«p p»lvp- were then n>uudrd
and -month \\ithuut trace- nl -uch a p'

Throiisji m\ failure, after repeated attempt-, to obtain many
Cases "f li — ii.n nf thi- -<>rt, 1 found that a neces-ary ek-ment in
the (iroce— was the adheii-nce of the hctcromorphic piece to the

288

HARRY BEAL TORREY.

substratum. I pointed out some years ago1 that Corymorpha
is negatively geotropic, even small fractions of the column re-
acting with great definiteness. This tendency to bend away
from the center of the earth can be effective, however, only when
the reacting piece is properly anchored. Pieces free from the

FIG. ii.

substratum never exhibit the reaction. As soon as they are at-
tached, it appears; and U- and Y-shaped figures are formed when
pieces are heteromorphic and the point of attachment is between
the developing ends.

FIG. 12.

Each Y-shaped figure is formed from a straight piece by the
development of a protuberance that corresponds to the stem of
the Y, and is terminally adhesive (Figs, n, 12, in both of which
the limbs of the Y have become much attenuated during the

1J. E. Z., i (1904), p. 395; Univ. Calif. Publ. Zool., 2 (1905). P- 335-

BIOLOGICAL STUDIES ON CORYMORPHA. jSo,

development). Such protuberances and Y-shaped figures are
found in nature only in the most exceptional cases, where they
probably ari-e in re-ponse to the same conditions that bring
tin-in forth in the laboratory. To present these conditions with
-iithcient ftillne-s, I must refer briefly to otlu-r abnormal forms
that lia\t- <,|i. -n appeared in neglected aquaria.

It \\a- i-it.-n difficult, in warm weather, in tin- absence of
running \vater, to prevent the growth of bacteria in the di>hes
in which fre-hly collected polyps were placed, ("nder the in-
lluein • Mientative changes induced by the-e condition-, the

hyiranth- \\ould cast their large proximal tentacle- and medu-i-
u- peduncles, the distal tentacles would be ab-orbcd. and the
(oluinn uoiild come rapidly to be surmounted by a in less

rounded ina-s of (i»ue that might show two or three knobby

.nl.iiities. The columns were alle. i. d in a much !•
it .it all. In fact it is quite easy to avoid these ditlicultie- alto-
gether by removing the hydranths as soon as the polyps are <<.!-

• d. This fact is doubtless due to the relatively lar^e ma
pioi.,p|.i-ni in, ami the greater differentiation of the ti--iir- of the
hydranths.

I pon the reduction of the hydranths to the knobby ma-
ju-i meiiiioned, and the remo\'al of the delui- composed of di--
inie^raiing tentacles ami medusa' with their peduncles, the
< iia \\oultl disappear, fermentative processes would lead to
t he Mib-t it ut ion, for the original hydranths, of \ at i> >u* monstn -11-
fonn- double ludrantlis. hydranths with double or triple
proboscea in \ai\in^ dt ^rees of independence, combinations of
hydranths with \ai\iiu numbers of probosces, etc. These phe-
nomena indicated i he breaking up of the original single ph\-io-
al -\Mem into several, the first sign of this multiplication
appearing in the irregular form of the terminal mass of tissue.
I ^ularit\ \\a> the center of a budding proce--. And

\\ ithoiit la\ in 14 an\ empha-i- mi the manner of it- initiation. «-ach
budding process ma\ In- i'om|ian-il direi'tK \\ilh the prore— b\
\\hich the >tem i> |iroduced in the Y->haped ti.^nre- \\ e ha\e been

considering.

In one imporianl respect lhe-e budding processes n-emble each
other; the\ -land, n.inieh . for tl eertain t !i-or^ani/.ation I how

290

HARRY BEAL TORREY.

produced may, for the time, not detain us) in the original physio-
logical system. In another respect they differ, in that they lead,
in the one case, to a hydranth, or part of a hydranth, in the other
to a holdfast. This difference is essentially an expression of the
different conditions controlling their development, of which the
influence of adjacent parts is the chief. The intimacy of this
coordination is obviously a function of the physiological isolation

of the parts concerned. It is a
conspicuous fact that Y figures
are formed almost invariably
from short heteromorphic seg-
ments; shrunken, starving, slow
developing (Fig. 13, which shows
eth beginning of a bud) pieces give an especially large proportion
of them. Frequently that portion of a segment of the column
which lies against the floor of the aquarium puts forth tentacles
more slowly than the upper surface. This retardation in develop-

FIG. 13.

FIG. 14.

ment, due probably to diminished supply of oxygen next the sub-
stratum, is accompanied by a bending of the segment by means of
a contraction of the affected side. It is on the opposite, convex,
aspect of the column that the bud develops (Fig. 14, in which
tentacles and gonads are only partly drawn), not, therefore, in
direct contact with the substratum. And it is significant that,
with the rarest exceptions that are referable to exceptional con-

BIOLOGICAL STUDIES ON CORYMORPHA. 2Q I

ditions, such buds arising on heteromorphic pieces become
holdfast

The-e fact- indicate that while the origin of the bud depends
upon a degree of disorganization in the original physiological
system, its fate depends upon a secondary physiological coordi-
nation with the hydranths between which it develops. The bud
.11 quires 'In- distinctive character of a holdfast, namely, its adhe-
siveni ss, indi -pendent of any influence of tin- -ul-tratum. It has

;i pointed out already that a hydranth at one end of a pi

i profound influence upon the differentiation that may
occur al the other end, depending on its own -ta^e ot dc\ elopim-nt
and it- di-tance from that end. In the V ligim--, then, there i- a
de\ eloping region between the two hy-
dr.mths whose differentiation is con-
trolU-d to some extent by them. An
intere-ting case of the coordination of
parts in a -hort piece developing as a
Y figure, i- represented in Fig. 15. A
m -u axis ai ri-Jit angles to the original Fjc

a\i- ha- IMI n established. And the

orientation of gonads and tentacles — especially the latter i-
dearly a resultant of the redistribution of forces correlated \\ith
that ( liange.

l-'i--ion may be considered as a special case of budding, depend-
ing upon the length of the heteromorphic piece in\ol\ ed. A- the
piece It 1 1- 1 hen- the tendency to bud vanishes, until the < ha rat ter-
i-tic of a«lhe-i\eiie-- alone remains. This indicates that the
region bei \\een the t\\o li\drantlis is still controlled by them,
while the constriction and the frustules on either side of i' 1 ig.
10) mark the incn-a-ing elYecti\ eness of their independence —or
of the systems ot" \\hich they form important parts.

It i- onl\ on heteromorphic pie. es of moderate length, ho\\e\er.
that li— ion of thi- ty|>e ha- been ob-er\ed. Ju-t a- il ua- from
|-elaii\el\ -hort heteromorphic piece- only that the re\er-al-
de-cribcd in the pre\ ion- -cctioii were obiained, the proximal
elements (>f tlu- longer jiiece- failing to produce holdfa-'- at the
\\oiind, so it ha- been only in the -horter heteromorphic pieces
thai llie column between the hydranth- ha- -ho\\ n any sign
budding, con-triciinn or fni-iule form.ition.

2Q2 HARRY BEAL TORREY.

The longer pieces failed to differentiate in these directions,
although attempts were made to encourage such developments, as
follows :

1. Nine long heteromorphic segments were held against the
aquarium bottom by weighted glass needles laid across their
middle. One was cut in two in two days; neither portion had
developed frustules twenty-four hours later. One was cut in
two in three days; both portions had aboral processes, but no
frustules developed in the following twenty-four hours. Four
escaped from their needles on the third day, and showed no change
at the end of another day. One was almost cut through by the
needle, and three processes were formed at the wound, but no
frustules or adhesiveness at the end of another day. One re-
mained under the needle unchanged for four days, when the exper-
iment terminated with my departure from the laboratory.

2. Discontinuity was produced by ligature on 16 long hetero-
morphic pieces two days after regeneration had begun. In two
days, 3 had broken into two parts. Two days later, 6 more had
done so; the next day, 7 more. Seven days after the ligatures
were applied, 2 had frustules at the ligatures. Both were con-
tracted and opaque — signs of structural degradation; 3 possessed
no frustules at the ligature; on the contrary, the proximal seg-
ment of one possessed a hydranth there. All the other ligatured
pieces had separated into two portions, 18 in all, of which but 4
were attached aborally and possessed frustules.

The first of these experiments serves to emphasize the feeble-
ness of contact as a formative influence, while the second adds
to the evidence that differentiation in the region between hetero-
morphic hydranths depends in an important degree upon the
distance between them, which other things equal, is an index of
their control.

That the longer pieces do not show signs of fission, then, is to
be attributed, I believe, to the freedom of the intermediate region
in each case from the effective control of the physiological systems
on either side, that may be conceived as extending over it from
opposite directions. Where these systems are near enough to-
gether, a new compound system is created, of which a bud may
form a part. \Yhen farther apart, their disharmony may appear
in the phenomnea of fiisson.

BIOLOGICAL STUDIES ON CORYMORPHA. 293

These statement- are <>b\ -ioiisly very general. Since all short
heteromorphic pit-re- <\« not either hud or divide by process of
h--i(ni. ilicn- mu-t IK- a factor still undefined that determines the
la< k <-f uniformity. There is no doubt thai external a^encie-
i an fai ilitaii- either budding or fission. Small \\ound- in the
-ide ut' tin- column may lead to a variety of rc-ult-. including
sporadic tentacles, and buds furni-hcd \\ iih ti-ntai le- • >v tru-tule-,
or neither. In this connection two heteromophic piece-, cut in
the middle region, hall" way through the column, ga\e the tol-
louing results. On the longer piece, a narrow bud de\ eloped
in four da>- at the wound, with neither fru-tule- nor tentacle-.
( )n the -horter piece, frustules developed around a blunter bud
at the \\oinnl, before attachment took place. In thi- the

\\ound ua- -iilticicnt to break up the original -\-tem exi-tmg
at that point and initiate a ne\\ de\ elojnnent . The tact thai
fru-iule- appeared shows the control of both h\dranlh- on that

de\ elopinellt .

I '. lidding OC< in -, ho\\ e\ er. \\ hen t here i- no -i^n . .| local injury
I loin \\ ithout . And it i- ditfn ult to aC( oimt on thi- ^louiid alone
for the fact that the large niajoritx of luid- aii-e appn >\imatel\-
nii.luax b«-t \\eeii the hydranths. To m\ mind, far more -signifi-
cant i-s the fatt that buds develop so of ten on pieces obviously in
i ph\ -iol. ,J. al condition generally. It i-s then that theph\-io-
. al continuity of the piece through the tran-itional middle
on miuht be • -\ peeled to be especially afl'e. ted b\ «li-rupti\e
leiideii. ies -i>iin:^in:< from the antagonism of proximal and di-tal
-\-lein- -o iib\iou-I\ antagonistic in the fi>-ioii -ho\\ n in 1 ig.
lo. In that case, the canals are complett l> obliterated at the
coii-trit -tion and the ti--ue is opaque and apparently impo\er-
i-hed. Thi- con-tricted region is under ten-ion, uhich probabh
accounts in part for its form. The ten-ion i- produced by the
acti\e migration of the l\\<> pol\ p- a\\a\ from each other, in the
manner of the oppo-ite hal\e- of an anemone in process of ti--i<.n
b\ rupture.1 The initial di-coiitimiit\ i- thu- a< < entuated by the
acti\ itie- of the po|\ p- them-el\

Porrey, J 1'ubl. Zoo/., i ;«. Ji i.

294 HARRY DEAL TORREY.

IV.

Discontinuity can be established experimentally not only by
the knife, which entails a wound, but, as already indicated, by
ligature, by the use of which a wound can be avoided and condi-
tions obtained that more nearly approximate those described
in the last section.

This method has been used on Tubularia by Driesch, Morgan,
Loeb, and Morgan and Stevens, and results obtained which are
of interest in the present connection. By ligating segments of
the stem, not only is the production of aboral (proximal) hy-
dranths assured, but accelerated; and only exceptionally, after
much longer periods, is there any development at the ligature
itself. Loeb succeeded in showing that the acceleration of the
development of the aboral hydranth is an indication of reversed
polarity that exhibits a certain stability in regeneration. This is
in accord with what I had already observed in Corymorpha,
where reversals of polarity accomplished without the aid of the
ligature are even more marked.

The experiments with ligatures have been repeated so many
times on Tubularia, that it is hardly necessary for me to refer at
present to similar experiments of my own farther than to say
that the ligature accelerated the development of the aboral but
not of the oral hydranth, and in no case was there any develop-
ment at the ligature, on either side of it.

In Corymorpha, as in Tubularia, ligatures accelerate the velocity
of development at the proximal ends of segments of the column. The
fact does not stand out with such dramatic clearness, however,
partly because there is greater individual variation in rate of
regeneration, partly because the time intervening between the
appearance of distal and proximal hydranths is much shorter.
That such an acceleration occurs can be shown by an experiment
like the following: Segments about 2 cm. long wrere cut from the
distal half of 20 polyps, a ligature being passed tightly around
each near its distal end. Segments of similar length were cut
from the distal halves of 21 polyps of similar size; these were not
ligatured. All were placed together in the same dish. In 28
hours, there were signs of proximal hvdranths on 13 ligatured
segments, and 14 on non-ligatured segments. The condition of

BIOLOGICAL STUDIES OX CORYMORPHA. 295

affairs at the end of 56 hours is shown in the following table, in
which the serial numbers represent stages in the development
arbitrarily sel for purposes of classification. I'mler each

of tin -«• appear- tin- number (l) of proximal hvdranths in that
1'ipment on the ligatured segments, (2) of proximal
h\ 'Ira nth- on non-ligatured segments, and (3) <>f di-tal h\ drain hs
on non-ligatured -e^ments.

TAHLE I.

I '•••.. -tages I 234567 I'-t.iU.

i.l o 5 3 5 5 i i

4 4 5 <> i i o

ist.) 3 2841.'! Ji

In -pile of i he individual variation represented by thr-e tunr
tin1. ncy in the proximal etui '< hydrant/:

rapidly on the ligatured segments, and alii: >af>idly

on tin- latter as the distal hydranths on non-li^atur

Although < 'orymorpha responds proximally like 'I'n'mlaria \\hen
nenta of tin- column are ligatured as al>o\e, there i- an im-
1 1- M taut diltereiH e iii its response at
tin- li^at me, namely, in the rapidity

\\itli \\hich h \dranths are formed
immediaieU below it. In Tubit-
Itiria, a hydranth very rarely ap-
peals immediate!) brlow the liga-
ture, and then oiil\ alter the lapse

.'t inaii\ days. In i>rf>lia,

on the ei.ntraiA, hydranth» form
ieadil\ and fre<ineiitl\ in thi- A,
position.

That a lixdranth >hotild arise

immediately below a ligature in

yinorpha mii;ht \n- antii'ipated
n the occasional occurrence of
Mich monsters as that sh(,\\n in \^ /

l;iu. 16, \\hich represents a re-

FIG. 16.

generating Moment ol the column.

The proximal ludranth ^belo\\ the'an^le) is not so far along as

296 HARRY DEAL TORREY.

the distal hydranth, and has apparently developed later, below
an interruption in the physiological continuity of the column
comparable with what a ligature might produce. In fact, just
such cases have been produced experimentally several times.
A typical experiment may be recorded, showing incidentally the
difficulties that made the number of positive cases so small.

The column of Corymorpha is very mobile, capable of con-
siderable changes in length and bulk, and its tissues are very
delicate and easily ruptured. So it has been difficult to make
ligatures tight enough to interrupt the currents in the canals,
as well as possible diffusions through the axial cells, without so
weakening the column as to lead to complete rupture in two or
three days. This has been accomplished, however, in a number
of cases sufficient for the present purpose.

EXPERIMENT i.

April 29, 1910, 3.30 P. M. Sectioned 20 polyps of similar size,
about midway of the column, and ligated each just below
wound, leaving a small segment of tissue above the ligature.

May i, 9.30 A. M. Four stumps removed, ligature having come
away with terminal button of tissue.

May 2, 5 P. M. Thirteen more removed for similar reason.
There are hydranths on these stumps that seem to be too far
along, under the conditions, for 31^/2 hours (i. e., assuming
the separation to have occurred immediately after the previous
survey of them, which is not probable).

Of the remaining 3, 2 show nothing below the ligature,
while the third appears as in the semi-diagrammatic Fig. 17,
which bears a striking resemblance to Fig. 16.

May 3, 9.45 A. M. The two hydranths of Fig. 17 have fallen
apart.

May 4, 10.00 A. M. Of the 2 stumps showing no development
below the ligature of May 2, one (a) has now budded a set of
tentacles just below the ligature; the other (b) as before.

May 6. The tentacles of (a) are larger.

May 9. Still no change in (b). Exp. abandoned.

In a second experiment, 6 columns were tied as indicated in

the diagram (Fig. 18, a), after removal of hydrant li.

BIOLOGICAL STUDIES ON CORYMORPHA.

297

EXPERIMENT 2.

April 7 . vOO P. M. Hydranths removed and column- tied.

April 8, i."" 1'. M. (a) Distal ligature and tip fragment (i)
liroken away in 2 cases. (6) The three segments have sepa-
rated in i • -• > cases in original condition.

April <». j.oo I'. M . . First case. Distal hydranth appearing

on SCg. J. also On ; l>Ut ii"t SO far alon^. Second «

l)i-tal h\diantli a|ipcarin.; • -n -< g. 2; nol .i|ipan-nl on >».•-. 3,
(b) • — . (c) l-'ir-t case. 1 lie Moment •> ha\ e >i-parati-d \\hile
under i»b-er\ alion. There are signs ot" ,1 very >li^ht rii|iture.
and indication- ol tentacle- on seg. l di-tall\-. Second case.

fhe segments separated at [o.oo A. M. Se-. j -ho\\ jof

lentai-le- at l>oth end-. In seg. .;. tentacle- an- heinv; >hado\\. d
di-tally. Third case. The segments have separated,

298 HARRY BEAL TORREY.

seg. 2 showing faint signs of tentacles distally; seg. 3 in poor

shape, removed.
April 10, 9.00 A. M. (a) First case. See Fig. 18, b. Note

tentacles appearing just below ligature. Second case. Similar

to first case; tentacles on seg. 3 not quite so far advanced.
April ii, 2.15 P. M. (a) Segments in both cases broken apart;

exp. abandoned.

Of the six cases considered in this experiment, three show
development of hydranths immediately below the ligature.

In a third experiment, the hydranth was removed from a polyp
and the column ligatured near the cut and near the base. In
three days, tentacles had budded just proximal to the distal
ligature, as well as on the small distal segment. The next day,
the latter was loosely joined to the segment proximal to it; both
segments possessed hydranths with both sets of tentacles.

These results show that hydranths form readily immediately
below the ligature in experiments like the foregoing.

VI.

The failure of Tubularia in such experiments to form a hy-
dranth immediately below the ligature with the facility exhibited
by Corymorpha is correlated with an important structural dif-
ference. The stem of Tubularia is encased in a stiff, chitinous
layer of perisarc, that offers a certain barrier to the diffusion of
gases between coenosarc and the surrounding medium. The
column of Corymorpha, furnished with a thin, rudimentary peri-
sarc about its base, is naked for more than half its distal length;
in this naked distal region, the ligatures wrere in all cases located.

When a ligature is passed tightly around a stem of Tubularia,
the coenosarc is not only ruptured, but the perisarc, itself intact
in most cases, is drawn closely about each end of the coenosarc
thus produced. The result is that, while discontinuity has been
established, the ccenosarc remains, as before, separated by the
perisarc from the surrounding medium. When a ligature is
passed tightly around a column of Corymorpha^ discontinuity is
established without in any way interfering in a comparable de-
gree, if at all, with the diffusion of gases between ccenosarc and
sea water. Now, when it is remembered that a discontinuity

BIOLOGICAL STUDIES ON CORYMi >KI'HA. 299

brought about by a knife- in Tnbularia leads promptly to regenera-
tion at the- wound, tin- f, n t suggests that Tnbularia develops \\ ith
•.ter difticulty than Cormyorpha at the ligature because the
coenosarc recei\e- a -mailer supply of oxygen in that region.

Tin- \ie\\ vi I by the following experiment, in \\hich

the factor .if contact, which tends to inhibit development orally
in L'orytnnrpha, wa- eliminated.

Fifteen .-h about 2 cm. lonu were cut from fifteen

Tnbnlnria -tem- of -imilar size and condition, ju-t below the
hxdranth. The di-tal end of each wa- in-erted in a capillary
Vila-- tubi- . l.,-ed at one end by a para Hi in- pln^' into \\hich it
fitted easily, without terminal contact, nnill quan-

tity : the proximal end \\a- free. A- a ntrol, \<>

-imilar pie< es were cut, both end- remaining i\>

1 "rt\-h\c hoiir> later, the piece- in the tube- had de\elopeil
nine h\dranth- (tn the outer (proximal' end-, nothing «\\ the
inner end-. In the control, though titteen ..f the -ixtecn pi.

ed di-tal h\drainhs, no proximal Indranth- \\ere \i-ible.

Twenty-five hours later still, all the pii-i e- in the tube- j»i--

• •d pr"\imal hydranths; nothing had de\ eloped on the inner

idi-tali end- In tin- control, only eiijit proximal h\dranth-

\\eie present.

l\«iii«'\ed now from t he tubi--, all the ]iie» e- rapidl> prudmed
ii"i mal h\<lranths distally.

Tin- experiment -eems to e>tabli.-h (l) that the failure >i<-

lariii si : ninths, 'chcn ligatured, immediately /'.

the ligature, is due to lack of oxygen; (2) that >i» tin

distal end in a 'Jass cap leads to the sa>> deration of

'.of»ncnt <»/ ///<• proximal hydra nth as does the f)re »f a

ligature; and « ;vi ///(;/ accordingly, sn<ii iicceleration is due to the
inhibit; the distal hydranth in the absence of

an adequate supply of oxygen, rather than to an interruption of or
other change in the the circulation in the canals. The

( in illation i- merely a transportation -v-tein, carrying -nbstances
fa\i'iiii- de\elopment that are rcinoxed from the -tie. mi by those
tissues e-pecially that have I: 6SS to ox\-gen, that i-, b\' the

tissues al the open ends of the peri-arcal t ube. The po— ibility
that oxygen mi-ht |ila\ -nch a role in thi- pn.ce— of selection

3OO HARRY DEAL TORREY.

was suggested by Loeb,1 though in a somewhat different form in
connection with a discussion of the function of the red pigment
granules of the circulation.

The acceleration of proximal development in ligated pieces of
Corymorpha is connected, not so much with any effect the ligature
may have upon the supply of oxygen to the neighboring tissues,
as with the inhibitory factors of contact and necrotic change that
it introduces. The same factors may play a certain part in the
inhibitory effect of the ligature in Tubularia also.

It will be remembered that if a piece of Tubularia or Cory-
morpha is ligated in the middle, development at the distal wound
does not exhibit the acceleration characteristic of development at
the proximal wound. This difference is to be explained, I believe,
in the following way. Inhibition of development at the ligature
on its proximal side hinders the utilization of a certain quantity
of substances that would be used up proximally were the ligature
not present. That the availability of this material for the distal
end does not obviously accelerate the development distally is
due, probably, to the initial acceleration of the distal over the
proximal development in the absence of the ligature, that is,
under conditions of active competition with the proximal end.

VII.

The considerations in the foregoing sections lead to the con-
clusion that the polarity of Corymorpha, of which the initial ac-
celeration just mentioned is one expression, is a product of con.
ditions under which the organism develops, changing as they
change; that it is essentially but an inclusive designation for
certain phenomena that depend upon both internal and external
conditions, all of which can be experimentally controlled. The
internal conditions appear in the effect of the continuity of tissue
in an intact stem, and the presence of the original hydranth on a
segment of stem, both inhibiting the development of a hydranth.
The external conditions are represented by oxygen, contact, and
necrotic changes such as are produced by ligatures. The first is
necessary to all development while the others inhibit the develop-
ment of the hydranth.

'Loc. cit.

BIOLOGICAL STUDIES ON CORYMORPHA. 30 1

With the exception of the third, these are conditions that
govern tin- development . whether cnil i \ nuiic or regenerutixv and
they are ju-t tin- . •< .millions by whose manipulation the original
pol.irity of the column can be reversed. Since such rever-al
depend- upon .in a< « <-leration of proximal relative to di-tal de\el-
Opment, it WOUld seem reasonable to -u-pect that a local accelera-
tion in the development of the embryo might be the efficient
cause of ihc initial polarity in the individual.

S :• h a local growth area appear- in the embr\ o at the point
which hr-t lea\e- iln- e^g case and thereupon define- the oral
cxtremitN.1 Thi- eMremity may emerge at any point not ad-
herent to the -iil.-iratum; \\hich qualiti.at ion indicate- that c..n-
• limit- to -.-i<lerablt • the an-a in which the oral

pole inu-t appear. Since i\]< rtain barrier to the

diffusion of oxygen into the egg if i- po--ible that \ariation in it-
thii kne-- ina\ be an important lactor in determining the po-iiion
o| i he oi.il p. .|e in ihi- ai-

i. \i I \in.k \ i-

!•:.:•: IV.

\\>l. XIX. November, igio. No. 6.

BIOLOGICAL BULLETIN

BIOLOGY OF THI-: SHAWNEE CAVE SPIDERS.

N< >KM.\N E. M. IN! II

N'ERAL IN I K« 'I'l i I I< iN.

I rom v< pteinber 7, KjoX, to "September ~. [QOO,, ilu- author
held lln- Sp,-|,.,,l,,-ie.il I i M..U -hip in Indian. i I niver-itv with
it tin- I niver-ity'- <".i\«- I. inn three mile- ea-t of
Miti hell, Ind Tin- present paper einbodie- the re-ult- of the
ob-er\.ition^ during tin- linn- on tin- h.ilni- ••!' -|udt-r- in
and in tin- laboratory.

Tin- \\ork ha- lu-i-n larrinl mi under tin- dirn-tioii ot I >r.
< Ml igenmann, prot'i---or nt ;n Indiana I 'ni\ i r-ii \ .

to \\hoin I am indebted l'or - ions and the lo.m ot hi- cave

literature. I \\i-h to expre^ in\ thank- to Dr. Cliarle- /el. n\ ,
associate pr"!. ->or of /o<.|, . : main- heljiful Mi^e-t ions.

I .mi indebted t«> I >r. \le\.mder 1'et rnnke\ ii. h (Or the ideinili-
ealion ot the -|ie( inien- ; to I >i . \. M. M.mia. for the lo.m <•! his
entire eolleetioii o| ,.i\e spider-, and to Mr. Will Scott, tor

I MI i oi the map of this s. He surveyed and mapped

the cave t'roin "i" to "37" or the I pi>er halton (see map, i

.UM in the aiitnnin o| iv"7: the ant h<>r a--i-ted b\- Mr. l-'rank
'•n snr\e\ed and mapped the I "|>|»r 1 >altoii t'roin "37" t11

"1.4" in < October, 19

I'KI vrioi - \\'< IKK.

1'nbli-hed ob-er\ation- on the habit- of cave -|>ider- are
limited to .i ir\\ -(.it it-red paper- \\hirh ^i\e li-t- of species and
l.ualities.1 Tlu- be.-t studies [iiibli-hed are tho-e of I'a< kard-
and Bain

I'.- al 06) ami Hl.it. hi. •> ('96) v;iv li>t> ot Arai'hniil- I'MUII Indiana Caves.
W, ..:. A :• I . I\'.. 1888.

"l-'.iuna i«l Maylii-ld " IQO?.

303

304 NORMAN E. M'INDOO.

THE SPECIES STUDIED.

Two species of spiders permanently live in the Mitchell caves.
One, Troglohyphantes (Willibalda) cavernicola Keyserling, a
linyphid1 is a true cave form; the other, Meta menardi Latreille,
is an epeiriclicl and also lives outside of caves. According to
Emerton (1902, 190-) the latter arachnid "lives in caves and simi-
lar cool and shady places in various parts of this country and
also in Europe."

Banta (1907, 65) reports Erigone infer nalis Key. from the
Twin Cave at Mitchell, Ind. I have been unable to find it
here, but have taken it in Mayfield's Cave at Bloomington, Ind.

Troglohyphantes has been observed in detail in order to get
as far as possible the life history of a typical cave spider. The
distribution, food and results of the experiments of Meta are
given in order to show how an outside form is able to adapt
itself to a subterranean life. All notes unless otherwise stated
refer to Troglohyphantes. The numbers in quotation marks
refer to localities in the caves (see maps, pages 321 and 323).

In Troglohyphantes there are all degrees of differences in
coloration, and in the degeneration of the eyes. The abdomen
varies in color from black, dark brown, light to white. The
cephalothorax varies from dark, pinkish, light to a white color.
The most common combination of colors is a light brown ab-
domen with a pinkish cephalothorax. The adult females range
in length (cephalothorax and abdomen) from 2.4 mm. to 3.7 mm.,
while the adult males vary from 2.2 mm. to 3 mm.

In the adults the eyes range from eight in number, each with
a maximum size of 0.036 mm. in diameter to no external signs
of eyes. I have not seen Keyserling's description, but from his
figure, which Packard2 has copied, the eyes are small, and the
front middle ones extremely minute.

PHYSICAL ENVIRONMENT.

These spiders are found only in total darkness, where the
atmosphere is saturated, and in places suitable for the construc-
tion of snares. They are never found where the walls are per-
pendicular with water covering the entire floor; nor are they

'Banks, '06, classification.
2i888, Plate XV., Fig. 32.

BIOLOG -!IA\VNKE CAVE SHIM 305

found where the walls and floor are dry. Distance from the
entrance does not necessarily limit the distribution it" the three
necessary condition- an- present, nor does -carcity of food limit
their distribution to a very great decree.

A- . . lV\v were collected last fall but none ha- been found
since. Thi- i- 200 feet from the entrance in total darkm
At "o" a feu more than at "2" have been found; at "lo" a y,reat
many ha\e been taken and "13" wa> my best collecting ground.
Tlii- pla< c from the mouth. Un several occa-ions

1 h. i In-red two or three do/en individuals hen- and -uch

whole-ale < olle< tiim at one plaee -eemcd at the time to exhaust
the -upply, but a month alter -in h a collection had been made,
I ha\e been able to duplicate tin- record. Main havWbecn
• oil,, ted al "14" and at "l<» " The latter localit> i- tin- "B

Room," 1.7001. : from the entran Three w< n at "23"

and oi, |\ a leu \\ere .ib-er\ed in a branch at "31." Thi- latter
plai <• i- in total darkne-- 200 feet from the mouth of the l.ouer

T \\ill Cave at \f>t on,- ha- berll ob-cr\ed bet\'.

;" and A IV\\ have b.-en , ..11< . ted in a branch at "

in t lie 1 pp< r I >allon. Thi- loi at ion i^ in total dark lie-- I 50
from the entrain e. A number \\eiv taken from brain he- at
"46" uhnh i- 1,024 feet from the entrain. 1 hi- l<>«alit\ \\a-
m\ collecting ground, partii ularly for tin ons.

\ ', \\ u. i. taken at "50." i >ne was caught in the "P.u koom"
at "57 " and * \<ral -nar- re seen. At a mile

from the eiitrani-e of I'pp.-r l>ali->n. three \\en- observed and
-< \<-ral \\eb- \\en among tin i o( k^ at a "i a\e-in."

'/'' al-o .|iiitc abundant in llanu-r'- Ca\e

oin- halt mile west "f tln-e «a\es. None ua- loiiinl nearer than
Vo |\.,-i ti-otn the entrar

Thi- an hnid -hare- a- \\ide a distribution as the blind bet tie,
AnophthalntUS tains Horn (Hlatchle\. "96). It far outnumber^
the latter in iin li\ i.lu.il-. but i- le-- e\.-nl\- distributed. Hy
attual D unt there are t \\iie a- man\ teinale- as male- in all
the rollet'tion- made from the \arioii- cavi

Mt-!<: i- found both in t\\ilii;ht and total darkne--. In the
Shaunee ('a\e the\ an- <|uite abundant from the entrance at
" i" to "4." The lat ler place i- o\ t r 2o<> feet from the entrance

306 NORMAN E. M'lNDOO.

in total darkness. Several were seen at "6," 100 feet from the
mouth. An adult male was observed on the roof at "14"; nine
days later, it was seen under a rock on the floor and had con-
structed a snare. One immature specimen was seen in a branch
at "23." At "30," 200 feet from the entrance of Lower Twin,
a few live, and at "32" and "33" they are quite numerous.

May 17, I collected eighteen Metas and placed them on the
north wall by a large pile of rocks at "19" in the "Big Room."
August 3, I saw six of them. They had scattered along the wall
about 50 feet and among the rock pile. Each had built an orb-
web and remains of small diptera were seen in the snares. August
17, after a brief search I saw only three of the eighteen. This
absence does not mean that they had died, but that it was im-
possible to find them.

LOCOMOTION.

This arachnid is very agile and is a good runner. Its long
legs and slender body enable it to move from place to place,
and to avoid an obstacle with much ease. When not irritated
it moves along slowly and gently. When stimulated mechan-
ically, it gives a quick jump, runs and dodges whatever obstacle
may be in its way. However, it runs only a few inches and if
stimulated a second or third time often drops in an instant and
hangs to its web. Sometimes it climbs the web to the place
from which it dropped, at other times it lets itself down to the
floor and then runs off.

Several were placed on the roof among beads of water and other
small obstacles. A pencil was used to stimulate and guide them
so that they were obliged to run up against the obstacles. They
use the first pair of legs as feelers. These are kept well in advance
of the other legs and head so that they can detect an object in
front of them the length of the body. While walking or running
slowly, they are able to dodge an object every time. If caused
to run swiftly, they run against the object, or if the object be
a low bead of water, they run over it and pass on.

WEBS AND SNARE.

This spider usually spins a web wherever it goes. It is im-
possible to see a single thread in the cave with a carbide bicycle

IMOLOi.V OF THE SHAWXEE CAVE SPIDERS. 307

lamp link--- the- thread is coated with water. Unite often <>n
the root in a -lit U-tweeii two strata of rock-, nr in crevices in
th<- u.ill- .ire found ...lie. tion- < >i \\el>- which .erally coaled

with water. The-e wel .- do not seem to In- of any -er\ ice ID
tin- -pider after oner -pun. They, ho\\e\ er. show h<>\\ it \\anders
from pl.i< e to pi. ire. It makes a Hat sheet-like snare under
which it li\e~. I ht Miare is slightly curved downward and may
lie in -u<h a jio-inon as to form any amjr l»etwe\-n o' and 90°
with the iloor. The t li reads are tine and the >naiv SO transparent
that ii i- dil'tieult to see unless it i- ted \\ith water. The
IIM -he- are -o minute that the snan- turn- \\ater. The Miare i>
supported hy many fine thr> nun the ^iiles, the length of

the-e \,u to the surroundiiu-. \\heii in-e(t> ll\-

into the xn.tre the\ .ire taken through it \>\ the spider \\hich is
on the under -ide.

siiai« • iiio~t .ilumdant in the older part- of the < .1

u here I he pa 1 -lo. k< d up \\ i t h i la \ . At Mich places the

( la\ haul. :i< ular and the \\aler has

\\a-lled ollt lllall\ Mil. ill CT< ^"Illetime-. lari^e M<uk

clay tumble down into a heap. la\ bl< « k-

and in MI. h • the .1 r\ numerous.

In a crevice ti\e in. In--. \\ i«le I once saw tin Imilt • •

al"'\e the other not more tli. in three inche-. .ip.irt. All t:
\\. re |Mialli 1 \\ith the floor.

h. il pl.t jiiite ( oil-pit noils in the an

I tet \\eeii the : :ul \\all. In ML ili-'ii- the spider- ha\e

litttei route- i.-r travel from on.- snai mother than in the

t la\ li.ink-.

The third pl.n e \\ here -nar. :• umd i- anioir^ the ro. k- at

"cave-ins." The rough cornered appear to form good pla

lor attachment and quite frequently dripping \\at.-r i- present
\\ hich this >] : ppan in l\ iii'' ••

1 • KDD.

\\hile in capti\'it\ I have fed them small iin.it-, -mall li;
and the spiderlings "t Mi-la.

In the caves the following; oli-er\alion- \\ere made: At "13"
while \\atchiiiL; a nuiK1 \\lio \\a- tr\in;4 to (.-otirt a female 1 -aw

308 NORMAN E. M'lNDOO.

a white-winged dipteron fly into the snare. In an instant the
female seized the insect, then ran back to the male. The latter
then ran to the other side of the snare. In a few minutes another
white-winged dipteron flew into the snare near the male, and he
lost no time in seizing it. Several other times these diptera
flew against the snare but failed to be entangled in its meshes.
In each case the spiders made a dash for the would-be victim
while holding one insect in their mandibles. At an earlier date
a spider at this same place was seen eating a white-winged dip-
teron. On various dates at "14" one was caught eating a
myriopod; one, a small gnat; and several were caught under an
old turtle's shell with thysanurans.1 At "19" two or three were
observed in a mouse trap with some cheese. At "40" one was
caught under an old piece of meat with a small white thysanuran
in its mandibles. At "43" one was eating a white-winged
dipteron, this spider was running on the wall. At "50" one
was seen in its snare eating a small myriopod.

This spider is usually very peaceful. Neither in the caves
nor in captivity have I ever seen them to show the least signs
of pugnacity when they came in contact with each other. Never-
theless it appears certain that they at times eat each other. A
few times their remains have been found in the snares. A few
remains were observed in the glass cases in which they were
sometimes kept in the laboratory. Quite a number of times
remains were found in the collecting vials the next morning when
three or four were left in the same vial over night.

When bits of dirt were thrown into the snares, the spiders
ran away quickly. Blind beetles were caught and tossed into
the snares. At the instant when the beetles struck the snare,
the spiders ran and with a jump seized the victim. Neither the
web, nor spider, nor both together were strong enough to hold
the beetle. Large flies were also thrown into the snare \\iih
the same results. The spiders always seized their prey and held
on tenaciously until the last second. Small flies and mosquitoes
were likewise tossed into the snares. In each case the spider
made a quirk run and with a jump seized the victims and held

'Here as elsewhere used in broad sense to include both Tliysannra and Collem-
bola.

BIOLOGY OF THE SHAWNEE CAVE SPIDER- . 309

mi so firmly that both prey and spider were torn loose from the
\vd> by pirkiii^ the prey up with forceps.

\"i one -;><•< imeii lid- even been seen drinking water. Since
they alwa\ - live in a -atura'ed atmosphere all the water required
i- probably ab-orbed through the ^kin.

In «-a|)tivity I have fed Mfln Hies, mosquitoes, i^nats and
variou- her arachnids -mailer than thcmsel

\\'hen two <>r more of them are placed in the sam- he lai\

invariabl Mir- all the others in a short time.

In tl.' ;s have been ob-er\vd at xarimi- Incali:

eatiiu juitoes, flies and . ricket-. ( )ne u as

11 eatil 'iall moth and another an old dried m\ riop.nl.

I •. • "

\- i ir as m> "b-tTVaiii>n- L;O, \\](^- -pidcr> ha\e im eiieii
bi--ide- iht-ii: Ii i- \ id»m thai one can find the

remain mien in tin- webs and a- mentioned above no <

ol li-hiiii^ h • \\iine--etl. Ai a mile

troin the entrance the onl\ other in^ Mini ua- the blind

tie. l>"Ml.'l i"nll\ i ervations at this locality

\\ill |iro\e that other in-eM- are al-> present.

I Ml'l K \ I I i
I >lll 11:. the la-t tlir. • "{->\>{

ha\e been taken at "!<)." or in tin Room." I'm; the

tein]>eratiire ; 11.5 C. for January, February, March,

Ajiril and Ma\; June 11.7 : |u' . ; Aii^ti-t and September

u.; October t2.2°; Noveml ; 11.9°; ami I ber 11.7°.

Since |o,,.l i- in- -re abundant and the three necessar) ph\>iial
condition , \identl\ suitable at "J." "M" and ".Vs," \\ e tan

pri'bal.K attribute the -mail number of -pecimeii-. to the une\eii
tempi-rature. Vgain, -in* i i- e\tremel\ scarce a halt mile

from the entrance \\hile the number of -peeimeii- i- few, [x.-rhap>

the -mall number of -piders i- due to the scarcity of f 1. Mid-

\\a\ betueeii the-e t u <• loralilie- fo« >< 1 is eoini >arat i\ el \ ]ilentiful,
the temperature i- practically e\eii throughout the \ear. and
thi> combination i- probably re-poii-ibk- for the i^n at number ol
indi\ idllal-.

'09.

NORMAN E. M'lNDOO.

Specimens have been kept in captivity in the laboratory
throughout the year. In cold weather they are less active than
in warm and are very fretful when the vials become too warm,
and often die.

COURTSHIP.1

November 16 two were seen copulating. In order to see
them I got too close and my breath irritated the web. This
caused them to separate. After an absence of five minutes I
returned and found them together again. A second time they
were disturbed. Returning after an absence of fifteen minutes,
they were found close together. While copulating, they were
lying one under the other with anterior and posterior ends re-
versed and with the ventral parts of their cephalothorax in
contact. November 19, a pair was seen pairing. December 17,
two wrere observed copulating, these were both under the snare,
and the anterior and posterior ends of the cephalothorax were
reversed. The dorsal surface of the cephalothorax of the male
was pressed against the corresponding ventral part of the female.
The male placed his palp on the epigynum once, this lasted only
a few seconds, then they parted. December 14 and 22, two
couples were seen copulating. Those on the latter date were first
seen at 2:25 P.M. and then disturbed by breath. At 2:27 they
were together again after the male had circled once around the
female. This pair was on top of the horizontal snare all the time.
The male used his palps alternatively three times in three minutes,
each time lasted only a few seconds. They were disturbed at
2:30. January 4, two were observed copulating June 7, a
male was seen courting a female. Both were under the snare
within one and one-half inches of each other. They were first
seen at 3:45 P.M. When the male tried to advance toward the
female, she caused him to keep his distance, the result of which
caused the male to circle completely around her clockwise in
six minutes. Most of the time she kept the posterior end of
her abdomen toward him, while he had his head facing her all
the time. At 4:00 I left them and a half hour later upon my
return they were still in the same position. They were placed
in a vial, taken to the house and were put in a case. In this

'McCook, '93, describes only courtship of outside form

BIOLOGY OF THE SHANYNEE CAVE SPIDERS. 3! I

case they lived two weeks where they died. This pair like all
others when in captivity had no inclination to mate.

Coco- >NS AND Er,<,-

One cocoon wa- made in a -l.t-- case < Vt<>U-r 4. It contained
six eggs. Another was made in a case I >« • <-ml>cr 1 1. January
21 , MIII- with ! • c.m-tructed in a vial. ( Hhrr cocoons

wen- made in \i.d- on the following dates: April J>> : May 5. 8,
jj. 27 ,uid ,}i; June 3, J.s and 28; Jul\ > and 27; August 6, 19
and J I .

< Mi tin- follouiiu dates cocoons with eggs \\ere collected in
tin- cavi Octobei - one with two t. ne with ei-h

Janu.in. 2O; |anuar\ Jo, one containing; ei-hl ne\\ly hatched
spider- .ind one \\ith -< M-II eggs; another \\itli -e\t : M.in h

2\ M.in li i<>, i •. -is, one with egii» and tin- other \\itli

ncul\ h.iiiln-d; M.i\ j}. one with four i-i;y; July -. OIK- \\ith
loin J"ly '•'. at various localities collected -<-\(-n cocoons,

OIK o| \\hiili i iiiitainrd fijn eggs, and t \\ o other- IM. h hcl<l
tour yum-, .tt \.irioii- 1... ,ilitii-> in 1'pprr Malion Au^n-i jj
(-..lleitrd seven ns. S<jme f)f lh> 'itaim-d yoiinv; ju-t

hatched, and others young r«.iil\ to l< ,i\(- tli. ns.

Tin- i -ii- in (!:• ^ are u-uall\ c- >n>i nu tf<l in ^ccludnl

pi. i' • - and .in- ditlu nit to find unless i.nr examine- r\t-r\ little

rnce and looks under the li k-\<r\ ran-iull\.

timr- tin \ are lound .ittaihnl !«• the undi r-idr o| rocks

on tin- !lo.,r. din mi.tv »llrn undi r litllr Ird^r^ •>! fm-k- and in

tlir amtr aii'^lr- ( .(' -mall i fr\ i.

In i nlof 1 1 1, -\ .in- -now \\ hiir and an- di-< -like in -h.r Thr

tlat part o| tlir di-i i- I'.i-tnird lirmK to tin- P., ' '!'!..

-i/r i- (. mm. in diameter l.y ;, mm. in depth, although -i.metime-
a i moon i i. in, lining the minimum numl-er ol i- .1- large as

one i-oiiiaiiiiiiii the maximum number. I ha\e ne\ rr l.reii
lortunaie eiion-h to \\itnr-- .1 lemale making her CQCOOn, luit
on examination, .1 , , ,, „ . -n i- compn-ed ..f .1 mofe or less linn ami
rlo-el\ woven circular base. The - re pil< d into a heap in

the middle ol this IMM- and then the convex part i- -pun over
them in Mich a crude and iniMilistantial \\.ty that one can gener-
ally count the eggs through this covering. In detachini; the
M.'nm<'iiu-ry. '06, describes the cocoons and eggs of an allied out-ide form.

312 NORMAN E. M INDOO.

cocoon from the rock one must use precaution for fear the eggs
fall through the covering.

In number the eggs vary from two to eight with five for an
average cocoon. They are transparent whitish in color and
are perfect spheres with an average diameter of 0.6 mm. During
the embryological stages, they soon take on a yellowish color,
become oblong in shape, and the outline of the embryos is dis-
cernible through the covering. Some of these embryos assume
the shape of the profile of a man's head.

YOUNG.

When hatched they remain for an indefinite period inside the
cocoon and when strong enough emerge through a small circular
hole.

March 22, three of the seven eggs in a cocoon collected March 2,
hatched; April 3, two of these spiderlings were dead, they with
the remaining eggs were covered with mold. June 4, two of the
four eggs in a cocoon collected May 24, were hatched, one spider-
ling was dead and the other alive on this date. Neither one had
any eyes. July 29, a cocoon collected July 19, was examined and
contained three young. Each one was examined both alive
and dead. All eyes, except the anterior middle ones, were dis-
cernible. Female no. 139 made a cocoon and laid four eggs
May 5. On May 23 all four eggs were hatched, but the young
were still inside the cocoon. Each spiderling had all eight eyes
except the anterior middle ones. The eyes had a uniform diam-
eter of 0.018 mm. While alive under the microscope their little
eyes shone like small electric lights. Their mother had no signs
of external eyes. Many other newly hatched spiderlings were
observed both alive and dead. The anterior middle eyes are
never discernible. In some, the other six eyes are present and
in others no eyes can be seen. All the other eggs laid in the
laboratory failed to hatch. Perhaps this was due to uneven
temperature.

The young are much thicker-set than the old. The legs are
thick and stubby. The cephalothorax and legs are transparent
whitish while the abdomen is light cream in color. The latter
has a few longitudinal rows of hairs. The length varies from
0.6 to 0.8 mm.

I;: THE ?H, \\VNEE CAVE SPIDERS. 313

While in captivity -even individuals moulted, three of which
iund dead shortly • r the skins were cast off . The deaths
wen- probably partial! to an - of water in tin- vial-

for inm,< diatelv aft< r the old -kii • -hed the spiders lay

lifele-- in tin- water. The -kins v. • aded by thread-

the ujipiT The moults -how that the -kin >plits

on i • -f i!i-- cephalothorax at the <l<>r-.il -i-le of \\here the

!• hed. Ili-ni «-. ilu- moulted -kin of ml mouth

ntrul hall" of tin- moult, and the ronieal
belonf 'lie dorsal half. All the old hair- an

with the -kin, new OIK-S take the plare of the old \\hith

•iiMy brighter. The moulted >kins

lie al-doinrii \\.-re ciih.-r mi- rolleil up into littK- \\ad-

•liat one i oiild not tell prei-Udy how they \s •

M' IB rALITY.

In tl, tmd- dr. id Bpecimi US In eaptix it\'

mortalitN i- not I h • mos! important n-i|iiirement i- to

pi. n • i hem in a sat urati-d atm< >Sph< Jit. l'\\o

third- of ih. and dead in I he i . .ll.-i t in^ \ial-

tlu- follouin- morning \\hen l<-tt in tin- vial- without a dro:
v\ati r over night. l; i- imp"~-il'l<- t" kre]i tin-in Ion- in anv -
tiling not .lir-ti^hl, hov lul on< p them -upplii d

with water. "l'h«- !»«•-! device i- -mall vial- with air-ti'Jit cork
-topper-. In -urh vial- they may \»- kept for month- without
food. < >m- rau-Jn 5 i her I o was placed in a small vial con-

taining two dr-' < >n Jann.i: more water wa-

added whirh alnio-t drowned t imeii. ( »n Januarv Jo it

dii-d. Dunn:; all thi- time it had had nothii it. Another

indiv idual wa- placed in a -mall v ial January ~, and died April \<t,
dm- to l.iek of nioi-t utv or lood.

In ca|)tivit\- tlu-v i|iiite often di<- soon after moulting. In
vial- -unshine kill- them in a tew minute-. The heat from a
Mudent'.- lamp i- al-o fatal.

l.Ii.ll 1 I-'.M'l klMl N I-.

In tin- caves one may throw the li^ht from a carlude lii< vrl«-
lamp on Tro'Johyplnintt-* for a half hour or more without pro-

314 NORMAN E. M'INDOO.

ducing any effect. Such is not true with Meta. Just as soon
as the light strikes their eyes, they run into the dark. If the
light is repeatedly thrown on their eyes, they may be turned in
any direction and often can be driven into places where the light
cannot reach them.

The following apparatus was used in the laboratory: For the
adult Metas, slender lo-inch bottles; for the medium sized Metas,
6-inch test-tubes; for the spiderlings of Meta and for Troglo-
hyphantes, small 5-inch vials. The closed end and the lower
half of each vessel were covered with black carbon paper. The
open ends were securely closed with air-tight cork stoppers.
One specimen with one or two drops of water were placed in
each vessel. In a very short time the water forms a thin film of
moisture all over the inner surface of the vessel. This saturated
the air in both ends equally. The vessels were then placed
on an inclined rack by a south window in order to give each
an equal amount of light. Occasionally they were rotated so
that the light always fell directly upon the spiders' eyes. At
various times the carbon paper was transferred to the cork end,
thus throwing the specimen into the light or dark as the case
may be. Those that were strongly negatively phototropic
never lost much time in finding the dark end, regardless of the
number of times the carbon paper was changed. Such individuals
often pass into the dark in three minutes. The few that were
strongly positively phototropic always changed from the dark
end to the light end whenever they were thrown into the dark.
Some were thus experimented with for thirty days, but experi-
ence taught that their actions were reliable for only the first
four or five days. Darkness and cloudy weather had much to
do with the final results. Time was counted from the period
when first placed in the vessel, and each morning when first
observed until 6:00 P.M. each day for four consecutive days.
When first placed in the vessels and for a short time after the
carbon paper was transferred, they were noticed every few
minutes, after that irregularly five times every day.

The adult Metas were always in the dark end during clear
and cloudy weather, and always in the light end when it was
dark. The medium sized Metas were ahva\N found in the dark

BIOLOGY OF THE SHAWNEE CAVE SPIDERS. 315

end in clear weather, one half the time in the dark end during
cloudy weather, and usually in the lii;ht end when dark. The
-piderl: remained in the dark end one half the time

in clear \\eather. one third the time in the dark end during cloudy
her and mo-t of the time in the li^ht end when dark.

All ei^ht eyes in the .1/VM\ \\ere present and presumabK well
de\elo])ed. The anterior middle OIH-- v nerallx a little

^mailer than the other- an 1 ••« , a-nmally an eye \\a- found anioii-
tin- other- which wa- about one half -i/e. In all the-e experiments
forty .!/<.',." \\ere n-ed.

The following table gives the re-ult- of the liijit experiments
tor / >!yf}hnn!'^. <>uthe left i- entered the number of the

specimen, the localil .'. he t her mature or immature

and the four \MI. -land- for anterior middl*

I'M I tor |io-terior middl' \H for anterior -ide • itnl

I'^l for po-ti rior -ide < • All mea-uremeiit - \\etv made \\ith

a mil lometer -lide in-ide the i wo inch ocular \\ith i u o third- or

lo\\ nbjet li\e. \\hellthee\e-\v • 1 \ 1 1 i -< elllible t he olie-

iin li o, ul.ir \\as -iib-lituied for the i \\o-inch. A- a unit of meas-
urement tor the • in- lifty-tiflh of a millimeter O.OlB mm

I. Phe f 1 nal |.at 'mil are mil> approxi-

mate. The — '- are U-ed uhell tile e\"- are joined together, if
sej.arate einplo\ed. 1* stand- tor pigment Speck. The

under i- -eli-e\planat

The thirl\ example- included in tin- table \\ i : I, not

on a photoiropic ba-i-, but • nt the \ariou- localilie-.

the • iteration o| "the • :n 1 • --lorat ion. It another

table \\ere made Iroiu th.- -]>eciinen- not included in thi- one, t he
re-uli- \\oiild be -imil.ir If .' c. .rn-ciii .11 could be made for
rloudx \\eatln r and for the ti: ii)tied in ^oin^ inlo the dark,

the total i .t. of .).) for tho-e in the- dark column would be

ion-id -rabl\ larger than the total per lent, ol 51 lor tho-e in the

li.uht column. In all these experiments 225 specimens have been

1 and I am po-iii\elv (crtain that the result- ..- .i\en in the
tabl • are correct.

Summari/in- the following table and other data not included
therein \\>- have the t'ollou iiv.; statements:

T\\ent\--ix per cent of all the indixidual- examined had no

3i6

NORMAN E. M INDOO.

LIGHT EXPERIMENTS FOR TROGLOHYPHANTES CAVERNICOLA KEY.

•6

e •

»d

•

•J.

-^.

«§

•i-

. C

— ^ . '

—

_c
"o

j= —

at =

1=1

~~ i

£ s

397

Im.

2.2

•S+-S

-7— -7

• 7— -7

light

pink

II.4O

24-45

712

Mat.

2.8

• 5— -5

i— -5

i— i

i-5— 1-5

brown

< i

15-20

21.05

322

Im.

1.0

o — o

0 — 0

o — o

light

IO.OO

25-15

324

4 4

2-5

-5+-5

i — i

0 0

4 4

pink

15.15

20.00

327

T n

Mat.
Tm

2.6

2^7

o — o

c -1- c

o — o

0 0

brown

4 t

4 1

1 i o-li f

13.00

8 or

22.15

28.00

O 1

*7 *7

325

1 \J

i 111 .

Mat.

•7
2.8

• 5 T^-5
I — I

2 2

1.5—1.5

I— I

I I

llgfll

pink

•25
30.15

5.00

77
15

326

4 I

2-5

I + I

2 — 2

I— I

light

18.00

17-15

399

T A

4 4

2.8

2*7

-5+-S

74-7

• 5— -7

I— I

4 4

1 4

« 4

I 4

17-55

O1 T /~l

18.30

T "J T C

o *7

747

*-f

Im.

• /
2.O

• 7 i-7
0 — 0

2 2

4 4

Jj.lU

2-45

I3-I5
33-35

37
92

729

Mat.

2.8

•7— -7

I I

I— I

i-5— 1-5

4 4

4 I

22.05

I4.I5

741

3-0

o — o

0 I

7T
1

0 — I

4 4

15-50

20.30

709

' *

3-0

o — o

0 0

o — o

4 4

16.05

20.20

73 *

4 4

3-0

• 7—1

I O

I— I

1-5— 1-5

brown

4 4

30-30

5-50

735

4 4

3.1

i — i

I I

o — i

0—1.5

dark

30.50

2.30

730

3-0

• 7—1

I I

o — i

i.S— 2

light

4 4

36.20

o.oo

IOO

359

1 4

2.8

• 5— -5

PT
i

0 0

o — i

brown

v 14

13-05

21.40

374

4 4

2.9

• 5— -5

• 7— -7

.7— o

• 7— -7

1 1

4 4

29-30

9.00

356

• 5— -5

o— .7

o — o

28.05

6.40

355

Mat.

2.8

• 5—1

• 5— -5

I— I

I— 0

brown

15.10

19-35

37i

4 4

3-0

0 0

0 — O

0 0

light

2.25

32.20

375

Im.

3-0

o — o

0 0

o — o

dark

4 4

16.30

22.00

753

Mat.

3-2

•5 — !

I — I

.7— -7

0 0

1 1

33-40

O.OO

IOO

755

* *

2.4

i — i

I — I

0 0

I— I

light

25-45

7-55

725

< i

3-0

-7— -7

I — I

.7— o

I— I

brown

pink

12.40

23.00

726

2-9

0 — 0

I — I

I— O

I— I

black

i i

10.25

25.I5

721

4 4

3-2

-7— -7

I — I

I— I

dark

4 4

14-55

21. 2O

722

Im.

2.3

o — o

0 0

o — o

light

12.05

24.IO

33i

* *

2.O

0—0

0— I

0 0

o — o

black

pink

17-45

I7-I5

1')

rotal

549-25

522.30

[ ? Abdomen was lost. ]

external eyes. Sometimes the eyes are not in their natural
position. Often black pigment specks are found where the eyes
are absent. The largest eyes are two fifty-fifths millimeter
(0.036 mm.) in diameter, being twice as large as those of the newly
hatched, but such individuals are comparatively rare. Hence
as a rule, the eyes do not grow larger after birth, while the speci-
mens more than thribble themselves in size. Neither locality
nor size of the specimen determines the degree of degeneration
in the eyes, or the shade of coloration. Generally, the lighter
colored the individual, the more degenerated the eyes, and vice
versa. Specimens totally devoid of eyes always stay in the dark
more than fifty per cent, of the time; those with one or more

l:l»I-<",Y nl- IMF. SHAWNEE CAVE SPIDERS. $1*

either in the IK; In or dark more than fifty per cent.
of the time, the per cent. depending mi the amount of degen-
eration.

HUMIDITY.

Apparatus. < ".la-s tube- with one-half-inch l>ore ami twelve
iii( lie- lon.y were u-e 1. The opening and three inches of one
end w« I with Mack carlioii paper. The other end \\as

< lo-ed \\itli .1 <-otion cloth. A -pider and two drop- of water
wen pla< <-d in the Ikht end of ea< h tnl.e ami the tube- were placed
in th<- li.yht the -aim- as in the liijn experiment-. When lirM
pla< ed in the lube- the -|>ecimen- wandered from one end to
the oil), r. In iu.-t a h w moment- th« -ed their wandering

and rein. lined within reach of the drop- of water. A- it \\.i-
ini|>o--iM«- to \\.it< h i xjKTiments .ill the time, (|tiite fre-

quently tin drop- ot \\.itir c\ .iporati d 1-efore m-u ones could
:ddid. ^oiiu-tiim-- when the ttil-e- liti.nne dr\ . the -pidcrs
were tound in th< d.irk end, other linn - in the li-ht end. 1 nder
MU h loiidition- -opie u«n- ,iM«- to li\e only one or t \\ o d.i\-,
BOme tour or ti\e da\- while other- -ur\i\ed ten days. < Mil of
two do/en indi\ idn.il- n-<-i| not one .it .ui\ time w.i~ ever found
in the dark end when the li^ht end w.i- wet. Mach -|>ei imeii w.i-

evimined. • had eyes and others were devoid of < Jud^-

llom the |.leied: inilit- oil lii^llt the -peiimeiis

i|e\oid ot ' Mould ha\- • foiiml in the dark end at time-,

pi"\idiiK there wa- no other tatior -u«'ii^er than ne-ati\e
photOtTOpism. Nm <• t he-e -pi i iim -i- remained n.-ar the drop- of
water all the time in-tead 0 - into the dark, \\ e i • •ncludc

that hiimidilx i- a -ti tor than nei;ati\e plmtoi ropi-in.

The -ami- e\]u rinieiit- were iii<l with the -piderlii

and medium -i/i d -]K •« inn n- ot .\ !,:>:. At time- the-e were found
in the dark end when the li.uht end \\a- wet, tliereli^re pn>l>aMy
humidit\- with them i- not greater than ne;4ati\e photot ropi-m.

( 'll ANi.l "I Hi MII'I l\.

•

Afiparatns. The -aim- tul>c- a- u-ed in the |irecediii^ e\[ieri-
inent- for humidity, al-o a lu^rometer wa- eni]»loy«-d. A -pei i-
men wa- |>laced in i-ach lul'e and wa- <.l-er\ed -e\cral linn-
each day. The follow in- re-ult- -how the relatixe humidity and
the number of hour- and minutes various individuals lived.

318 NORMAN E. M'INDOO.

No. of Specimens. Relative Humidity. Hrs. Min.

4 38—36 3 15

70—66—59 5 30

I 38—36—44 8 15

4 63—93 8 15

i 95—82—84—88— 92—78 23 35

I 81—60—76—73—100—75—89 31 45

.1 ioo — 65 — 55 — 60 — 72 — 66 — 60 33 45

On various dates at the entrance and at the different localities
in the caves the relative humidity was recorded. At the entrance
it varied considerably on different days, but in the caves, the
hygrometer always stood at ioo (saturation point).

These arachnids always live in a saturated atmosphere and it is
impossible for them to survive long outside the caves where the
variation in the degree of humidity is great. As a general rule
the higher the relative humidity (with but a gradual and small
amount of variability), the longer they live. Since the above
experiments were prosecuted from May 18 to June 8, when the
change in temperature was not such as to materially affect these
spiders, we must attribute their deaths to the hygrometric
conditions.

SUMMARY.

1. Troglohyphantes cavernicola Keys, is found everywhere in
these caves, where the three following necessary conditions
exist — total darkness, a saturated atmosphere, and a suitable
place for the construction of snares.

2. The first pair of legs are used as tactile organs.

3. All small winged insects, thysanurans and small myriopods
serve as food. Scarcity of food does not entirely limit their
distribution.

4. They have no known enemies other than themselves.

5. While temperature outside the caves does not materially
affect the adult spiders themselves, it is probable that to the
even temperature at localities between 600 and 1,700 feet from
the entrance is due the great number of specimens found at this
place.

6. Courtship is similar to that of some outside forms.

7. Cocooning is rudimentary. The eggs are few and com-
paratively large.

1 THE SHAWXEE CAVE SFII'l 319

-. I white and arc thicker set than tin- adults,

had lied with eyes, while other- are entirely blind.

9. Moultin. 'ively rare and is often fatal. There

are all -hade- from white in Mack in coloration.

i". !• try from a -mall pigment -peck 100.036

millimeter in diameter. A- a rule, after birth the • <• to

.yrow while :!ie -pe.-imeii- more than thribblc them-el\e- in
size. T\\i it. of all the individual- are entirely

de\oid ,,!

l I . 'I !:< tion iu t! - and the -hade of

(ol., ration ai .ined by eitlur loralii of the

imeii.

12. I !H lighter • "lored the specimen the in* generated

th«

. I he m"-- eel the eye- tin gative

phototropi-m. am! .1.

I}. lliimidit\ ; router factor than m . photropi-m

!ll detei iiiiliilU the |o. atioil of S|H-ciiiiell- in the (\perilllellLal

till

15 ( h.c :elaii\e humidi(\- i- fatal in a few h«.iir-

LITERA1

Banks. Nathan

'06 A 1': : :: : ::!i K

Banta A. M
'07

Blatchley. W S.

'96 1 1, .'U.

EiRenmann C H

'09 I be U!in.| \". Publii .it ;• •!! v-

: :i~Ututi"ll.
Emerton J 11
'02 I in- < '.imiiMii Ginn and Co.. B ton.

Emerton. J. H.

'75 N"tt^ "ii tin- S iroin Ki-ntui-ky. \'ir.v;ini:i. and Indi.in.i. I'.i. k.ud's

\ nth \in.ii.. i. M.-in. X.it. A. .id. Sci., I\ . iS88, 57-58.

McCook. H. C.

'93 Aiuciicaii S|.; : tlu-ir Spiunii:^ \\ . .1 k. I'hil.Aoad. i vols.

Montgomery. Thorn. H . Jr.

'06 I In- < )vi|)n-itii>ii. ( '.K-iH.iiinu .nid Hat, hin.; ,,i" an Aiam-.id. T In-rid ium T«-pi-
d. Hi.. rum • k.sh. Mi-.l. Hull.. \'..l. XI. I ... N". I. Dec.. 1906.

32O NORMAN E. M INDOO.

EXPLANATION OF MAP.

Shawnee Cave (the outlet). Sec. I., No. i.

Closed chamber caused by collapse of roof at Sec. I., Nos. 2-3.

Cascade. Sec. I., No. 6.

Double passage. Sec. I., Nos. 7-8.

Old cross cave. Sec. I., Nos. 9-10.

New passages. Sec. I., Nos. 1-8 and 11-13.

Opening in roof leading to upper older levels of cave. Sec. I., No. 14.

"Big Room." Sec. I., Nos. 15, 16, 17, 18, 19, 20, 21, 22.

"Fallen Rock." Sec. I., No. 31.

Lower Twin Cave. Sec. I., No. 32.

Upper Twin Cave. Sec. I., No. 33.

Roof too low for passage of boat. Sec. I., No. 34.

Deepest water in cave, 10 feet 4 inches. Sec. I., No. 35.

Lower Dalton Cave. Sec. I., No. 36.

Upper Dalton Cave. Sec. I., No. 37.

• - : \ V. Ml CAVE -111': -

321

1 I., i \I.ip <>t Hi.r.< MVtion I. I'ri'in >h.i\MH-.- t.i I nun

th |. is i l<-vt. - to ill'' IIH h.

322 NORMAN E. M'lXDOO.

EXPLANATION OF MAP.

Upper Dalton Cave. Sec. II., No. 37.

"Cross bedding" in limestone. Sec. II., Nos. 46-47.

"Old passages." Sec. II., Nos. 56-57.

Obstruction past which boat cannot be taken. Sec. II., No. 63.

End of exploration. Sec. II., No. 64.

Y "I 'lllK SHAWNEE CAVK .-TIDERS.

3^3

I-'n;. J. Map ct sliawn- -mn _•, iii.m !.<>\wr I >aiton to unexplored

A \K\V SPECIES OF PAR. \MECIUM (P. MULTIMICRO-
XUCLEATA) EXPERIMENTALLY DETERMINED.1

J H. POWERS AND CLAUDE MITCHELL.

On September 27, 1909, I received from Dr. Powers two sample
cultures of Paramecia with the request that I investigate them
as to type and purity of culture. To this end I first killed, fixed,
mounted and examined 1,000 individuals. They proved to be
neither typical Paramecium cdiitJatnni nor Paramecium anrclhi,
although most of their characters differed but little from these
well-known types. Their length ranged between 144 and 288 /z.
Their anterior end was a little blunter and the posterior end a
little more pointed than even in P. caudatitm. The cytoplasm
was more dense and more opaque. Their chief difference,
however, from hitherto described types of Paramecia lay in the
matter of the micronucleus, for, instead of the single micronucleus
of P. caudatum or the two micronuclei of its variety P. a arc/ in,
there is a number of very small bodies, evidently micronuclei,
ranging in diameter from about .7 to 1.15 // (Fig. 3). The char-
acteristic position of micronuclei is fully retained, these bodies
lying either in slight grooves or in shallow impocketings of the
macronucleus. Like the micromiclei of other types, these deli-
cate bodies are always surrounded by a nuclear membrane.

Of the I ,OOO individuals examined 875 distinctly showed
Iroin two to six of these small micronuclei, 1 24 showed apparently
no micronuclei whatever, while one appeared at first to possess
a micronucleus of the type found in P. cditdatitni. This single
instance, however, turned out upon careful study to be a ca>e
in which a detached fragment of the macronucleii> chanced to
simulate in size and appearance the regular micronucleus. As
to the 124 which appeared \\iihout micromiclei, entire degenera-
tion of these bodies may h.i\ e been possible, but it is more prob-
able that a slight oxerstain obscured I hem, especially when
lying behind the macronucleus; the same explanation is doubt-
studies from the Zoological I.al.oi .itoiy, the I 'nivrrsity n\ \VI,i .i^ka, No. 101.

324

A NK\V SPEI [ES OF PA RAM EC I I'M. 525

true <>f the individuals in which luit two or tlmv micronuclei
u.-re found, other- e\i-iin- l»ut in a les- vi-ible location.

Finding ihu- that tin- cultures in hand contained nothing but
thi- -aim- type of Paramecinni, I next pron-edi-d to u-t the per-
manence of the type. On October o. I i-ol.ued live indi\ idual-,
pl.t, i h in a rle.m watch glass c< mtainini; a definite pro-

portion -.f -terile and bacterially infected water. Thc-e indi-
vidual-, however, lived but .1 day. I then a^ain -elected live
more, \ar\inu tin- proportion- of the- fluid media. Of the-e. the
l u . i which were pl.u ed in u ater c« miaininv; the hi'Jie-t perceir
of ba<teria lived, uhile the other- did not. Three more were
Marled in the -ame manner ,1- the t u •• successful ones and all
the' e lixinu cultures. ( >\ the li\e li\iirc culture- thu-

obi, lined tuo pp.\ed much -iron-er than the other-, de-pile the
lh. it the condition- \\ere kept as con-t.mt .1- po--ible in all
two iii' I rapidly in number-, uhile the other-

but litlle and lill.tlK died olll allel li\e \\eek-.
the-e tuo strong culture-, |ott\ in. li\ idual- were kill..!,
-t. lined .ind mounted on \o\einber JJ and about lill\ more OH
I ). . ember 17. All of ihe-e pi . '\ ed identical in l\p«- uith tin-
ori-inal uild stock. The minute micronuclei \\.re pre-eiit .1-
before, and ,uain -eeiiie.l (o \ar\ from ihr.e to -e\eii in number,
uhich ditleieiice .|e|u-ii<led. in |iart at least, upon the -lain and
the iraii-pareiic\ of ihe indi\ idual.

Unfortunately during <'hri-tma- \\eek extreme ...Id uealhcr
and |),ulial failure of heating (ilaiit c.m-ed the death of .ill i-..l,i-
tion culture-, the ..luinal cullure. houe\ mainin. 1 rom

thi- lailer. -in^le individual- Wl iiu i-olated and neu cul-

ture- -I art ed on January 1\ , the culture media I •• tried OS in

|)n-\ ion- in-i.ince-. Thi- re-ulted auain in culture- of \ar\ini;
degrees of -irengih. One of the best, which I will de-i-n.ite as
culture X. u.i- chosen and le-ted \>\ mounting a number of
individual- all of uhich a^ain i>ro\ed [« be of the mull imicro-
nucleaie t\pc. Thi- culture X was now accordingly taken as a

basis for all furllu'r uoik. l;roin it -ix culture- were -larted,
the medium bi'invi modified in (hi- case b\ ihe u-e of different
pro|)ortion- of agar agar infected uith the customary bacteri.i.
Of the-e six culture- one, culture V, \\a- \\orihy of e>pecial

326 J. H. POWERS AND CLAUDE MITCHELL.

note in that it produced a few conjugants. Early in April this
culture became infected with a minute unicellular alga and, pos-
sibly as the result of this, the paramecia became more active
and increased more rapidly in number. They also ingested the
algse until they became greenish in color. On April 15 six pairs
of conjugants appeared. Three of these were killed in about the
three-hour stage of conjugation, another in about the seventeenth
hour of conjugation, while the other two pairs were isolated,
allowed to complete the act of conjugation, and the ex-conju-
gants used to start new cultures. It was hoped that stronger
cultures would thereby be obtained, but this did not follow.
They lived and divided slowly for about three weeks only.

The pairs of conjugants which had been killed were stained
and mounted in toto, and are of interest as showing, not only
that this type of Paramecium is capable of conjugation, but
something of the nuclear phenomena undergone during the proc-
ess. In all cases the micronuclei, or at least a part of them,
could be made out. In those killed at the three-hour stage
(Fig. i) all were in pairs, indicating no doubt the customary
divisions preceding nuclear exchange. In one case three of these
pairs were really single nuclei in advanced division. With dif-
ficulty the nuclear membrane could be made out, extending, as
in the case of the larger dividing micronuclei of P. caudalum,
between the separating portions of the dividing nucleus. The
micronuclei forming the pairs in these three-hour conjugants were
smaller than those in non-conjugants. The macronucleus in this
stage is still unchanged except that its surface is more or less
furrowed.

In the pair of conjugants killed at the seventeen-hour stage
the micronuclei are also present, some again in pairs or in division,
some single. The macronucleus on the other hand has now
broken up into bands and curved segments, simulating a reticu-
lum. This breaking up of the macronucleus at an early stage
does not occur with P. caudatum, and, in case further study
shows it to be habitual with the present type, this will constitute
further proof of its independence.

The limited number of conjugants at our disposal and the
consequent inability to procure all the stages ha\r prevented

A NEW SPEC IKS OF I 'A KAM KCIL'M.

our demonstration of the actual nuclear exchange during c.m-
n. Ian -uch exchano- i- naturally \» In- inferred from the
ry divi-ion ()f the micronuclei ami from -ul>-e<iuein
loun of ill.- niai roniit leu-. K\ery feature of the fc\v
•.amined indicated normal pi • - ami con-

dition

Although tin- i ultiire- -tarted t'mni th, , \ , onjugants I'an-d SO
•il\ it : h noting th.it -h«Ttl\- .ilt.-r tlu- lattrr \\.icdi —

d in Culture Y this culture nnd.-r\u-iu a rapid at i ( Icrati.'ii
i..\\ ih .nid di\ i-i-.i! I is n . lia\»- 1-ri-n dm- to limit

• urrini; in the cull u: M i In- < -t lu-r hand

it ina\ ha-. ..I -tiniulu-. u liich it-i-lt"

I r-iin thi- rapidk ^r<p\\in^ culture

tin ih.-i -I- •• r i have been mounted, and, as bel

all |M"\r 1. 1 l.»- «.t" tin- multimacronucli - dona 1

also been und. and d uith iron h.i-inaln\\lin I "in

dittrn-nl i;r.iup- imiiintrd during lli. llu-N ha\e I'ully

IH.MI,- ..til lln- it -uli- ••! l In- in. m- IIUIIHT..U- ti-l«» nitnint-.

All t.ild the i lllllll.-- lia\e ! 'iitlllt ted. tin- ti: up tfl

ihiff. and lli* • ni.iiith-. llif\ ha\r imt ln-m as

str« • "U It I In- \\ i-ln d. I UK tin- In -i i ulturr- t'l.tainrd -h<>\\rd

at lra-t n tO 'lit- "ill ami a( thr cl-.-r "t the U"ik

ciilinit- Y \\a> multiplying im>rr r.i|iidl> than at an\ |ut\i"ii-
liin. II nl "in trinpi-iaiuri- d -mlit i< m«> ln-i-n imuv unilHrin ami
!a\"ial.lr, \\t- -hoiild pM.|iaM\ ha\«- lu-di al.l. !i imirc-

t "pi. ni- « nh in. 3. Iht- t-ni in- nnil"iiiiit\ nl" tin- t Y|'r I hn m;< limit

thr-i- culture- -eelll i e\it|t : il- peniiailelHe ami the

ppilialiility lhat it deserv< ilic rank.

LUD1 \\'M. Mi n in i i..

I tan I ill I \ \ i >uch I't-r the meth"d- ami resultswhich ni\ -tin lei it ,
Mr. Mitchell, h »rde«l in ihe lir>t part "I" thi- pap. r. I

ina\ -|'e.ik a tfu \\"i.l- lunlu-r a- in nu • >\\ n experience \\ith
/'. muUimicronucleata. It i> not, in the \\riter'- \icinit\-. a rare

or attidental t\|>e. Tin • Mi^hoiit a lUlinlit-r ol years of \\ork in
eastern .V-lira-ka it ha- lu-en a frc-<|iienl and t roulile-oiiie in-
truder in mv I\ir<i»ii'(inni culture-. The ;u«.-t ]>er-i-teiit elT"rl>

J. H. POWERS AND CLAUDE M1TCHELF..

have often failed to procure, from wild slock, pure cultures of
P. caudatum. A portion, usually the bulk, and frequently the
whole, of any culture obtained from pond or river water would
turn out to be of this multimicronucleate type.

I did not at first recognize the minute micronuclei. I regarded
the individuals, which careful and elaborate technique showed
to be lacking in the typical micronuclei of P. caudatum and P.
ditrelia, as degenerates in the sense of Maupas' contention. As
however the hypothesis of the degeneration of the micronucleus
became more and more discredited, I reexamined mounted slides
of these Paramecia under high magnifications, with the result
that the minute bodies in question were visible in every case.
That this type of Paramecium was not related to degeneration
was further shown by the fact that many pure cultures, unlike
those with which Mr. Mitchell has labored so assiduously, have
been vigorous and strong growrers. I may further mention the
fact that in several very large aquaria supplied with running
water and a small amount of fresh meat added occasionally,
this type of Paramecium apparently maintained itself continu-
ously for several years. As often as the organic matter was
supplied the animals would multiply and appear in vast swarms
in the corners and protected portions of their space; whenever
examined they proved of this type and of this type only.

The existence of an undescribed species of Paramecium seems
improbable. The protozoa are considered of universal distribu-
tion, and Paramecium is the most-studied genus in existence.
Nevertheless much of the study of mircoorganisms is superficial;
many have failed to develop a suitable technique, easy as this is,
for the certain demonstration of micronuclei ; and as to the
hypothesis of universal distribution, it is certainly assumed much
further than it is proven. Thus, for the last six years, I have
made careful search among cultures derived from very numerous
wild stocks, for Paramecium of the aitrdni lype, i. e., with thr
well-known two micronuclei. But, aside from a very few isolated
individuals derived experimentally from P. caudal nm, not a single
example has been found.

All in all, it seems that, in the light of Mr. Mitchell's results,
the type in question deserves specific rank, although this rank

A NT IES OF I'AKAMKCIt'M.

- upon nuclear difference- only. Tin- external character-
mentioned l.s Mr. Mitchell, although holding good for the- ma-
terial in\. iiim and for many other lot- a- \\ell, are
not univer-al, ii«.r have [l ted • rnal character that i-.
When /'. multiti AH in the -a mo culturo \\ith

i\\\ -:tni, the t\\o can u-nally l>e readily

•ed |.\ ..in- or i ;ial characui- 1 requeiitly the

/'. m nltim: :e uniformK than the accompanying

• iiolntnn: hut die\ do n ;]]e kno\\n dirndl-ion- ..f
the commoner lorin. and. in -ome culture- the\ are uniformly
-mallei - \\ith length form of end-, opaquoi'

The iin. -I unit". tin (ll.r 1 lia\e -eell i- tll.lt the

neu I \ pe i~ a liirh n -m. luil^ini: le-- at tho

point . iriit /' appioa; he- this

I'M III ill -la! \ ed CllltllV

I he pro; :|»on IHK lear < harai ter- «n\y

depend upon their :i< y. ( "alkin- ha- -ho\\ n

dial id \\ilh ind doul.le microiim lei

i\el\ an- not \\ h«i||\ i <.n-i -ion.il iran-ili-'n- taking

pla< e in l.oth dii- II.. then ; -rom .1111. ed die t \ pe-

\ aiieiii But the conclusion h tiled in question,

the illtlei|llel ill-ilio|| leadilU Iv'l.'id to re-illlel pl'el

die phen. uneiia a- in-lain e- o| nnitalion.

I ha\ e m\ -tit 'ii-iiletaMe part of

the pie-, in year, pieliminai\ experiment- on /'. mmlntnii:, -nl»-
ieciin- them to dill.t, nt (.-ndiiioii- uilh a \ie\\ to a-certainin^
iheir |>o--il)ilitir- <.| \arialioii. The oiil\ -irik lilt- ha\e

Occurred as the c,.n-e(|Ueiiee- ol in leedinii

habit.

/'. i.utdntitni i- alm«-t e\clu-i\el\ a kicteria U-t-der. Hut a-
Mr Mill hell ha- re. orded tli<-\ occ.i-ionalK de\iale l" '-llier
minute \e^elal>le Organisms. Thi- Near I ha\e -ucceeded ill
inducing a cerlain percentage of the individual- irom a pun-
culture of \ ei\ lat^e and -trolly -ro\\in- /'. mndatnm to h-,(| on
minor animal or-aiii-in-. lir-i on Magellan- ( 'hilonwini?- • and
then, lo a con-ider.ible extent. ii])oii -mailer ciliate-. Th«--e \ et \
:-trikinu i han-e- in fo,.d habit produced \ er\ -irikinj; \ariatioiis
in die Parameria. 1-oth nuclear and c\'to|ila-mic. I will not

J. H. POWERS AND CLAUDE MITCHELL.

describe these at the present time save in so far as they relate
to the present discussion. Many of the nuclear changes were
erratic and possibly pathological: Macronuclei greatly enlarged,
micronuclei unchanged, or sometimes apparently absent, or again
enlarged, even more in proportion than the macronucleus, and
sometimes divided.

Among the large mass of such material, stained, mounted and
examined, I discovered a very few instances of individuals with
two typical micronuclei. In fission these micronuclei divided
simultaneously and normally. The number of these individuals
was very few, probably not exceeding one to several thousand,
but they confirm, to some extent, Calkins's observation that P.
aurelia may arise from P. caitdatum.

Among the different types of variants I sought assiduously
for examples of P. multimicronucleata. But none of the exact
type were found. Evidently this type is farther separated from
P. caudatum than is P. aurelia. A considerable number of indi-
viduals were found however which showed an approach to P.
multimicronucleata, in that the micronucleus was divided, usually,
again, into but two bodies, perfectly normal in appearance, but
much smaller than the typical micronuclei of the genus, though a
little larger than those of the new type. This variant was one of
the most constant and frequent results of the changed diet. In
other characters, however, it did resemble closely P. multimicro-
nucleata or, for that matter, any recognized type of the genus.
I regard it merely as an instance of the well-known law that a
powerful stimulus to variation applied to any species brings out,,
not only new characters, but characters of existing allied species
as well. The phenomena, to the writer, serve to confirm, rather
than to refute, the specific independence of the new type. But
they are of interest in themselves as showing possible lines of
experiment leading to nuclear variation. In the present instance
it seemed especially worth while to record them, and indeed this
is the chief reason for the entire studs', in thai Paramecium is-
more and more being made the subject of extensive experimental
research. So far, little of this study has had regard to other
than external characters, but this admitted limitation must soon
be remedied, and to this end it is e»ential that we kn<>\\ the typec

A NE\V SPECIES « »F I'AKAM ECICM. 33!

"' nuclear structun ent in the ditkTvnt r varieties

"f tllr ?enus :l as the lin^ of variation to which UK-V arc

subject.

J. II. I1' >\\ i

Julj- 30.

332 J. H. POWERS AND CLAUDE MITCHELL.

EXPLANATION OF PLATE I.

Parameciiim multiinicronucleata.

FIG. i. Conjugation near the three-hour stage, showing micronuclei in pairs
or in division.

FIG. 2. Conjugation at about seventeen hours. Macronucleus already broken
into band-like portions. Micronuclei visible in part, in pairs or single.

FIG. 3. Section of typical P. miiltimicronucleata, an unusual number of the
micronuclei chancing to lie in one plane.

BIOIC

PLATE t.

>>O*mS AND MITCMELl..

III! CENTRAL NERVOUS SVSTKM AS A FACTOR
IN I Hi: REGENERATION <>F POLYCLAD

I! KhKI.I.AKIA.

M. CHILD.

Someyeai I V. Morgan1 described a series of experiments

• • ninij tl f removal of the cephalic ganglia upon the

COUl :.iti'«n, particularly of tin- anterior re-inn, in the

( 'aliforniaii poK.l.nl. >nilis. IK-r conclu-ion i-

lh.it mull Million- . |i.irtirul.irly in tin- piv-rnce

n|' M--UI- .iiiti-ii'ii- in t ; :i<T.nion incur- .1- rr.ulily

and as completely in thea!.- . .mjia a- \\ hrn thr\ an-

At .iliiuii tin- -aim- lime tin- rc-nlt- of ni\ o\\ n i-xprri-
incnt - • in /.. ptoplc I I' i 'Mini that \\ hen

h.ili i.t less th. in h.ilt i»t tin- -nil- ti--m- \\.i- rcin< >\ nl, re-

generation inii;lit In 1 1| 'It -If .UK I a- r. i piil .1- \\ hen the i;.

Were iininjiin-il, Imt th.il uhcii niMir th.in hall <>f the

!Vllln\ ril. -ii.ll \\ ' '>lll\- -s|n\\rr lull l(

than uhni lh«-\ \\ . i , iiiiiiijn:

I aUn li'timl, h' its n«t int'ivi|iiriiily

appcaiiil in . :i;^lia 1 lu-in-rh r- \\ i i • . SO I'.r

rmilil I"- lU-lt i ininnl. i <>ni|ilt ld\- rrnn >\ cil, and thai tin- aiimunt
nl antni'T lu-u ii--in- in all -in i ih. in in tl

- uhi-M- r\c -p..t- ili,l n, ,t .ippcar, tlimi^h if^ciirratii ui \\as

never complete. M-. general conclusion from tl .;UT|IIH-IHS

was that the Cfinr.il in-r\i.u- -\-tt-in in thr-r lOn: :

funciioii.il -timiiln- t" the ^r«i\\ili (>|" iu-\v ii--nc, incn-a»in^ lioth
tlu- r.ipiiliu ami the amount ,,f growth.

The al'o\i- nii-ntionfil «-\pi-riin»-in - of I.. V. Mm-. m seem to

1 V. Morgan. "Incomplel •. imi in Hi«- A .1 "tin;

in / '•' ii. I \ "5.

"V. -Tin- Rfl.uion !i''t\vi-<-ii '

itinii," Jiuirn. I

on !<• \ I. -Tin' Kt-laiiciii. etc.: Anterior

ami I. atria! l< .nun," ./ , I., 4. i

i liilil. "Stin In- on Ki i^ulati.in. \'L." I i,;. ]>. $22, l-'i-^-. \\ i ;, p. 526.

3 > ^

334 c- M- CHILD.

indicate that under certain conditions this is not the case. In
view of the apparent disagreement between her results and my
own further experiment seemed desirable and during the autumn
of 1907 and the summer of 1910 I took the opportunity to
examine several species of Leptoplanida* which occur at La Jollar
Calif., with reference to this point. In 1905 I had worked with
L. littoralis at Pacific Grove and obtained results similar to those
described for L. tremellaris.

My conclusions from this later work are essentially the same
as those reached in my earlier paper. Removal of the ganglia
with as little of the surrounding tissue as possible always results
in decreased rapidity and completeness of regeneration, wrhatever
the method of operation employed. In many cases, however,
groups of eye spots appear in the new tissue, even when the
ganglia are wholly absent, and in such cases the regeneration is
always more rapid and more nearly complete than when the
eye spots do not appear.

When the ganglia are removed by a cut from one side of the
head, as in some of . Morgan's experiments, more new tissue is
often formed, or it forms more rapidly, in the deep cleft made by
the cuts than on a nearly flat terminal surface. This, however,
is not due to any specific effect of the anterior tissue, but is merely
a very general characteristic of wound-healing, not only in Tur-
beUaria, but in many other forms, and is doubtless due to the
fact that nutritive and other conditions for growth are better in
such a cleft, where the growing parts are in contact on both sides
with other tissue, than on surfaces where such contact exists
only on one side.

I believe that the important point in connection with the
problem of the influence of the central nervous system on re-
generation in these forms lies in the question as to what con-
stitutes the central nervous system. As Morgan states, thecepha-
lic ganglia in the polyclads are enclosed in a definite sheath, but
a further point of great importance which she does not consider
at all is that the nerve roots contain numerous ganglion cells
for a considerable diMauee from their point of origin in the ganglia.
Reference to Lang's monograph of the polyclads1 is sufficient to

'Lang. "Die Poly I;I.|I-M >\<-~- Golfes \<>n Nr.iprl." Fnumi nn<l Flora des Colfes
von Neapel, XL, Leipzig, 1884.

GENERATION OF POLYCLAD TCKHEM.AKI A. 335

e-i.ibli-h tin- [M)int. There i- thru every reason in believe that
the central ner\ou- a comprises, not the yanijia alone, but

tin- u .niylia phi- the nerve roots for a certain greater or le>s
di-tan.e I nun their origin. Kveii when the ^anijia arc com-
plt-ii-l;. removed, tin- capacitie- of tlu- central nervou- -v-u-m for
in-raiioii and -timulation are not wholly lo-t , if sufficient
portion- of the ner\ e roots near the u.nulia remain. In Mich
CS the amount of n-. ii.-ration i- ^rcaier than \\lu-n the IHT\ e
root- are al-o remo\ed. .iinl Croups of eye -pm^ ma\ appear.
In !a< t. in one < as I ob-erved th- eratioii of a ^mall luit

di-iiiH . lionic nia-- .ifter the apparently complete remov.il

of ili. ganglia. h seems not improbable that if our techni(|in-
ueie -iitth ienilv nermit removal of the ;.;.in;.;lia \\ithoiit

iujinv io the iiei\i • |it at their origin, the ri'-eiierat ion

even oi i; them-el\ well as of other parts, might

In- a lino- 1 . ,i quit • <uii)ili -te a- v, hen the -.m^lia remain.

I,. \ . Morgan's I if !'•-' "I ihe regenerated anterior end of the
nervous system alti-r n ni"\al of tl. _ lia -ho\\ - mils tilirillar

>l i in lure and -he states thai miK fibril la- are pre-ent in I he mass.
Hut \\hen \\e lec.ill the l.ii i- as to the h i -I ol"^ii a I -iriictnre of
the iiei\e ioot^ it -eem- extreiiieK improbable that iMiiiJion
i i II- art tot.ill\ ab-eiit I'mm -in h i e-i net ai ec 1 masses. In all
• - of the kind, \\hich I ha\e ob-ei\ed. -oine (cll- a> \\ell as
t he 1 1 bl ilia- h.t\ e alu a\ - been ple-eiit ill the knot of ti--lle formed
b\ the union o| tin- nei \ i

The de\elopment ol -pot- iii many of the cases \\iihoiit

^.in-lia de-i ribi-d b\ Nb'i-aii i- lindonbledK due to re-eiiei al i\ 'e
pio, iii the remainin. . lionic ner\e roots. In m\ o\\ n

experimeiil^ I ha\e found that in all cases, \\hate\er the method
of operation, \\heie the ^an^lia phi- a -ulln ieiit jiortion ol the
nei\e roots are ivmo\ed the re-ein-raiioii i- al\\a\- -li^ht, e\ e
>|>oi- do not de\elop and the animal ne\er -ho\\ - aii\ recovery
from il- -hi^^i-h nnn>poii-i\ e eondiiion, ;. c., it behaxe- in all
respects like a he. idle— animal. <>n the other hand, where the
roots are laixelx intact . i e-eiierat i<ui i- more rapid and pio< eeds
lai I her, eye Spots often appear and the re. o\ erv of motor acti\ ity
and a|)pan-nt >poiilaneit\ 1 rei [iieiit 1\ OCCUrs to a \'er\ marked

extent.

'« hild, 'Studi* },eti . \'I . Fig i;. p. 526.

336 C. M. CHILD.

Since my later experiments confirm in all respects my earlier
work, it seems unnecessary to describe them in detail and to
figure all the various methods of operation and the results. I
can only conclude that the apparent absence of effect of gan-
glionic removal upon regeneration in certain cases is due to one
of two things, viz., failure to remove the ganglia completely, or
the presence of the intact nerve roots. Morgan's experiments
do not in any way prove that the central nervous system does
not exert an influence upon the rapidity and amount, and so
far as the sense organs are concerned, upon the character of
regeneration in the polyclads.

As regards one point, however, Morgan's results as stated in
her paper disagree so completely with my own that some further
consideration is necessary. In the concluding paragraph of her
paper the statement appears that "regeneration of the anterior
tip of the worm, that is when the worm has been cut off anterior
to the ganglia, occurs in the absence of the ganglia as well as
when they are present." Individuals with the ganglia removed
and the end cut off anterior to the ganglionic region regenerated
as rapidly and completely as controls with uninjured ganglia
and the anterior end cut oft" at the same level. In these experi-
ments the ganglia were removed by using the cut end of a straw
as a punch and after the wound thus made had healed the anterior
region of the head was cut off.

I have performed this experiment a large number of times and
on various species of Leptoplanidae and with essentially uniform
results, viz., that in all cases where the ganglia were actually
completely removed, regeneration \\.is less rapid and less complete
than in control experiments with uninjured ganglia. Moreover
the larger the portion of the nerve roots removed in addition to
the ganglia themselves, the less rapid and less complete the re-
generation. The operation is by no means easy to perform suc-
cessfully and in many cases larger or smaller portions of the
ganglia remain: such cases show all gradations from complete
regeneration to a condition essentially like the pieces from which
the ganglia are totally absent, but they must of course be regarded
as unsuccessful experiments for our present purpose.

In my experiments the ganglia \\ere removed with a straw in

ENERATION <iF 1'oI.YCI.A I > TURBELLARIA.

33,

the manner de-eribed above: two week- later, after the wound

had completely healed and the ganglionie region was tilled in with

new ti--ue. the aiitt-ri«>r end «»f the liead region A\a- removed by

Li .1- indii ated in Fig. I. At the same time the anterior ends

6 removed .it the -aim- level from am it her < >t~ indi\ idual-

with uniniiin . lia.

•

The condition df the .mimal- \\ithou; lia .1 \\ « « k after the

• ud operation i- indieated in I • ami ,>, \\hile I ig. .} -hi'U->

the i.-iidition . ,f the c.mtr«'U. Tin- dilli-reiiee i- marke«l and
reqniit- iin (''inmetit. After two \M •« k- n .-ration i^

alim»t or quite coini >lcit- in the eontroU. \\hile tlie animal- \\ith-
OUt i;.iiiv:lia remain e--eiiti.ill\ a . eiieration ne\ er

jjrocee.U further in them.

In the-e e\peiiment- ^ivat care was u-ed to lie certain that the
uan^lia were entirely relinked. In \ariou- -|it.-cie~, tin- ^an-lia
Can be seen quite dearlj from the \ ciitral -in face and examination
in. m thi- -id. alter the ojieration \\ill u-u.ill\ -how e\eii rather
Miiall p of the uan-lia if the\ n-inain. M-ir^an doe- not

^tate ho\\ the total al>-cnce o| the ^aii^lia was determined in
her experiment- and it -eem- probable that in i ises u here tin-
anterior end of the head regenerated a- rapidl> and a- completeK"
in animal- -uppo-edl\ \\ithoiit i;an;;lia a- in 'Im-e \\ith uninjured

338 C. M. CHILD.

ganglia some portions of the ganglia remained. It is possible
that in some cases where the nerve roots were largely intact
regeneration might be almost as rapid and complete as when the
ganglia are present, but it is certainly impossible to make an ex-
tensive series of operations which are uniform in this respect.
Morgan's experiments of this kind included however, only
"several" worms.

The only conclusion possible seems to be then that the central
nervous system, i. e., the nerve roots near their origin from the
ganglia, as well as the ganglia themselves, do affect in marked
degree the rapidity and amount of regeneration of the anterior
regions and, at least as regards the sense organs, its character as
well. More-over, where the ganglia, or the ganglia together with
the nerve roots, are removed the method of operation makes no
essential difference in the result. As most experiments, not only
on the turbellaria but on other forms, indicate, it is probable
that the early stages of the formation of new tissue are largely
or wholly independent of the nervous system, but it is difficult
to understand how the nervous system of an adult animal could
fail to affect the amount and rapidity of growth in a regenerating
part composed largely of muscles and sense organs. Absence
of such an affect would be in direct opposition to the well estab-
lished fact of the functional influence of the nervous s\>iem on
various parts of the organism. The rate of metabolism and
consequently the rate of growth — i. e., provided nutritive material
is present — in such parts must be in greater or less degree de-
pendent upon nerve stimuli. Such an influence of the nervous
-\-tem upon growth must, however, be- sharply distinguished
from ilie determination of differentiation of parts: the effect of
the functional stimulus in the stricter sense is primarily quauliia-
tive rather than qualitative, so far as structure i- concerned.
The-e point-, \\ere emplia-i/ed in my earlier paper-.

/< .c.| I M.|! \\ |. \110K \ [( .KY,
l'M\ KKXI IV oh ( UK \(.O,
Octnlicl, KJIO.

THE FORMATION OF GERM LAYERS IN
.VI IMA BERMI DENSIS \ l.RR.

I.HWIS R. CARV.
•-IVI,K-UY.

While .it the Bermuda BioloJ, .il st.uion <luriii^ ilu- -ninmer

<if i ',0. i I attempted to secure ma it -rial for a -tndy of the de\ elop-

menl 1. 1' Aitinin bermuc \'.-rrill. a \i\iparoii- artinian that

i- almmlant 1 ii tide mark- in the -haded lime-tone caverns

aloni; tin- shon •!" the -mailer inland-. Sim e tin- material

.<•(! in l.c lacking in tin- t-arlic- in tin- «K-\ (•!• ipim-nt ,

•hat tin- i iiiii]ili-ii- finlir\«ili >^\ i"iil«l nut In- \\nrki-il <>ut. I

lia\c tlimi^ht it ail\i-aM<- tu puMi>h tin- fi >\\< >\\ ii. . ant as

it covers one of 1 • !.-il p. iim- in tin- cU-\r|(.|nnrnt 1.1'

anl Im/naii-.

Vccordii McMurrich 1891 , the onl) \\cll aiitlu-nn< att-d

1 < nirrata in uhidi tin- «-m|mlrnn aii-c^ h\-

li«ni are lii ami /' \»>\\i Sr\ ph< >nic-

liuinlaii '111111 ill" the I'lirmatiiin »\ the nnlo-

in . '. lii- -.11 tin- lia-i- i •!" hi- i.u n

observations "ii .\' . l\"\\alr\-

>ki-' J (187 •! the ; \\ hirli

in tin- "li-inal \\.i- i; :Mc alike I' MiMnrrich

and m\-ell". lie ili-ini— e- in ihe -aim- in. inner -a\in^ that il \\a-
pn 'I'.iliK an ei n .r in inter|irelal i- .n.

Appellot I iidoderill |c ifllial i< Ml ill \\\i>

Species "t ai-tinian-: / and .\<tini<i
(K/Huni. His observations on the latter species confirm the » pin-

it ni <>l Mi M nrrirh, iianieK , that there \\a^ im true invagination.

In tin MII the other hand, he de-erilie- ami

li^nre- a true imagination, \\hich from hi- figure- nuild \\<>\ In-
con-ideied a> an error of interpretation, -inn he \\orked \\ith

-erial sections.

Kanroi [907 ha- de-rrilied a tnu- although rather imu-iial
t\[>e of imagination in the drvelopmrnt of .^(i^irlia parasitica
and Ailtinisia jxilliatd.

339

340

LEWIS R. GARY.

In Metridium, according to McMurrich, the result of segmenta-
tion is the formation of a hollow blastula with a considerable
cavity. Later the inner ends of the cells are constricted off-
by the appearance of vacuoles in the line where the separation
is to occur — to form the endoderm. At the time when this proc-
ess is finished there appears at one pole of the blastula a slight de-

FIG. i. Pseudogastrulation in Metri- FIG. 2. Later stage in the pseudogastrula-
diiim. After McMurrich. tion of Metridium. After McMurrich.

pression which gives the embryo, when it is seen in optical section,
an appearance similar to the early stages of a true invagination
(Fig. i).

When the mouth has broken through, the resemblance to an
invaginate gastrula is even more complete, so that until such
embryos had been seen in sections it would be almost certain to
mislead any observer. In reality, however, the two layered
condition had been readied before the- mouth was formed. This
so called "Pseudo-gastrula" McMurrich held to be the true con-
dition in those forms in which invagination had been reported
to occur.

All the material of A. bermudensis was obtained by slitting
the adult individuals longitudinally, and then \\.i--l ling the em-
bryos into a dish of sea water with the stream from a pipette.
All stages including the young in which the second series of
tentacles was complete, and which wen- ready to be liberated
from the body of the parent were capable of swimming about
actively by means of their cilia.

The earliest segmentation stages were never found among the

FORMATION OF liERM LAVEKS IN ACTINIA IIKRMUI >KN-1S. 54!

material obtained by washing out the adults, nor did -Actions of

th.- mr-ri the adult show any segmenting eggs, so the

[>r<> M-adin.y up to the formation of the Ma-tula cannot be

:. In the -eetioii represented in 1 u. .>. the Ma-tula

• • 1).

.nipleteK formed. l'< cells are very numerous and of pi

tie.ilK tin- -ame height over the mi in- circumference <>t the
lila-tula. llir inti tin- Ma-tllla i- tO a great extent tilled

with a i ..mparati\. ly thin. li^litK -tainiiiv;. i>la-ma-like matnial,
in uhich tlnir are man\ yolk y;ranulr-.

Tin- maniirr in \\liiih tl.' -ati-'ii "I tin- \olk -|)h«-n-- to.ik

pi. i. r iltninv; tl,' :cntatioii i.m In- o m jiTtmv.l onl> . l-ut,

from tin- appi-.uaiit •• of' the u-l ilr-eril >«-d it would -n-in

pii.l.al.N- that, ju-t as in Urticino, tln-n- i- ix-vtT an extensive

Ma-tula IM\ it \ . I n-trad t In- > • -Ik matt-rial i- pn.liaM\ separated
li»m tin- e\ to|>la-mic portion nf the ft-11- in an carl\ -ta^e of

^mentation.

In a lain s | •' Ma-tula ha- ln-i-ume moi, i-l..n-

i;ati-d. I'lu- i-t-11- ha\t- become relati\-ely thinner, and higher,
\\hile at one pole tlu-n- i- the tir-t in<lii\iti< m of the depression
that mark.- the lie-inning; of the infolding of one portion ..f the
Ma-tula \\all to form the endoderm.

\\ithin the interior uf the Ma-tula, there has been a marked
increase in the relative amount of space unoccupied by nutritixe

342

LEWIS R. GARY.

materials. The plasma-like substance has disappeared for a con-
siderable proportion and the yolk spheres have undergone an
apparent disintegration. Their outlines are no longer distinct,
as in the earlier stages, and in many instances, they can be ob-

FIG. 4. Blastula of A. bermudensis just at the beginning of the invagination.

served breaking up into rather coarse granules which are being
spread out through the surrounding plasma-like material.

In the stage shown in Fig. 5 the process of invagination has
gone on until the section shows a w'ell-markecl early gastrula,
and puts beyond any question the type of endoderm formation
in this species. The character of the cells in the invaginating

^^' V-;-" ' Ipp^

I' ii.. 5. Invagination of A. bermudensis.

area has not as yet undergone any change to distinguish them
structurally from those making up the ectoderm.

The nutritive material within tlu- original blastoccel shows
practically the same conditions as nok-d for the stage last de-

IN OF GERM LAYERS IN ACTINIA lU-lKMl'IiFN-l -. 343

<•< rilied. \\iihin the ga>trula cavity there have appeared some
lev, masses that an- apparently composed of the rather o>ar-e

granule'- thai come fn.in tin- lux-akin- up of the yolk -phere-.

In the older v;a-trula. almo-i all of the nutritive material

lit- within tl. rocoel, only a comparatively few

ol tin- in .ranule-, re-ultiiu from tlu- di-inu-^ration of

the >olk ^p! '•niainiiii; in the original 1. la-tula ca\it\ .

Apprllo! 1 itioll of the

Illltlime inal« lial dmi: nation I |, ralsGB

the iiiii--iion . hether the yolk i in the ^a-in*

alter the in\ !>.ii i- e..nij ill i hat 54 • n in

the Ma-t'M oil- 0 h"\\ the 1 rail-fer

< on Id take pi.:' . Of 1 — il'ililie- in the \\a\ of 3 Uall-fer

nieiilioii-: In-l that the yolk ini^ht I .e al.-orlied by

the fell- < -I the ill\ axilla ! i i: ^ !a\ • '. all<l then i tO Colled

iii the . .il and second 1) that the yolk elements pass "-i»-h

dian^eii odd -n the celU of the in-

inatini: la\er. • hi- interpretation, his material

Mlpport- the la-t mentioned \ie\\ as many of the t ells appear
shrunken in diameter \\hile \"lk -pheie- ma\- he >i-en lu-t\\
MU h cell- -eparatiii'c their lateral \\all- one Innn another. He
mention- be-ide- that the \\all- at the inner ends of the cell-
are I'ften very indi-tinct durini; the time- when this proces- i-

344

LEWIS R. GARY.

going on, although over the remainder of the gastrula wall there
is no appreciable change in the characteristics of the cells.

Appellof makes no direct mention, although his Fig. 13, PI. 2,
shows, that while the transfer of the nutritive material is taking
place the yolk spheres are breaking up rapidly, so that to infer
that the spheres pass through the invagination layer in their
original condition is unnecessary. In the older gastrulse of A.
bermudensis a complete yolk sphere was scarcely ever found in
the gastrocoel. While many of the masses of nutritive material
still retained their identity and practically their original volume
it could be observed in every instance, that the sharp outline
was no longer apparent, and usually the granules were separated
from one another. It is also noticeable in the section shown in
Fig. 6, that nearly all of the yolk material present in the blastoccel
is massed about the invaginating cell layer and that the inner
ends of these cells are much less sharply defined than they were
in the younger stages, Fig. 5, or than they are more laterally in
the invaginating area. The central part of the invaginating area
is more densely filled with granules than are those parts farther
to the sides. The granules in this denser area are also markedly
larger than the granules in the cytoplasm of the more laterally
placed cells.

It seems, then, beyond question that in A. bermudensis, just
as in Urticina crassicornis, there is an actual passing of the yolk
material, in a practically unaltered state, through the layer of
invaginating cells to the forming gastroccel. As the cells of the
invaginating layer approach those of the outer gastrula wall all
of the yolk passes through so that the two layers come into con-
tact and the supporting layer is secreted between them.

In an older embryo, Fig. 7, in which the formation of the
stomodeum has begun, the gastroccel — gastro-vascular cavity-
still contains a considerable amount of the yolk material which
now appears as distinct granules. In nearly all instances, how-
ever, the granules are arranged in groups which show clearly their
origin from an originally more circumscribed mass.

In this last-mentioned stage the cells making up the endoderm
have undergone a considerable change so that tlu-ir histological
characteristics are very different from those of the ectoderm cells

I"KM.\TION OF GERM LAYKRS IN ACTINIA r.EK.ML'HENSIS. 345

ha .ined very nearly the appearance of the original

lila-tul.i < clU. The endoderm cells have in»\v become much
bro.ider in proportion to their height, tlu-ir cytoplasm is much
[ess d.-n-e than formerly, and take- the -tain- [ess readily, \vhile
the IHH l<-i have bi-come markedly le-- con-picuous. In -ome
ons, uherc the body of the embr\o i- mo-t contracted, espe-
cially about thi n of the forming -tomodeiim. the oiitline-

o! the i-ndoderm cell- are \«-r\ iudi-tinct , and indeed iinpo--ihle
lo m.iki- ..in al the proximal end- of the cell-.

I eitl 7 .in.l ;.

Iii d tO the int. :i.-n ,,t the optic. d na -ho\\ n

in McMuiiiih'- I ig. [Q, I'l. XIII.. it i- int.-ir-t ii: .
m\ I ig. o. \\ith the ' of tin- mil': ' •:/«/

v;i\«-n bj \ppellof. In the la-t iiu-nt i< nied form, \\heiv th<- eixlo-
<lenn arises \>\ di-lamination. thei LfS .it the time of the

I'leaUiiu through o| the m«iUth o; ideil thinniiu

I'nih i;ei-m la\er- at the point \\here the mouth « >\ pciiiii'< \\ill
appear. When the mouth ha- l»eeii formed tlu- ti--ue- are \er\-
thin all around it, and in a longitudinal -ectioii there ap|iear-
, iln .lit the mouth thi- thin area in-tea<l of the more thickened
ana \\hich is found about the ^a-tro]iore <>i an iiua^in.:
trula. So that in some in-tance- at least, McMurrii h'- critici-m
of the interpretation of optie.il -ecti»n-. of \\hole mount- \\ould
not appl\ .

346 LEWIS R. GARY.

REFERENCES.
Appellbf, A.

'oo Studien uber Actinien-EnUvicklung. Bergens Museum Aarbog for 1900.

Faurot, L.

'07 Nouvelles Recherches sur le Developpement du Pharynx et des Cloissons
chez les Hexactinies. Arch. d. Zool. Exper. et Gen., Ser. 4, T. 6, 1907.

Jourdan, E.

'78 Recherches Zool. et Histolog. sur les Zoanthiares du Golfe de Marseille.
Ann. Sci. Nat., Ser. 6, T. 10, 1878.

McMurrich, J. P.

'91 Contributions to the Morphology of the Actinozoa, II. On the Develop-
ment of the Hexactinse. Jour, of Alorphology, Vol. 4, 1891.

MHI. WHOI I

UH 17JQ G

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