PROCEEDINGS

The Academy of Natural Sciences

OF

PHILADELPHIA

VOLI VIE l.\\ III

PH N

. s

r H I DEMY OF N

A

In i \i u>i mi op Natural. Sen mis of Philadelphia.

.1 \\i \i;> !.">, I '.117.

I hereby certify thai printed copies o! the Proceedings for L916 were mailed as follow -

33 64

li.-. s|

•■

197 31 l

315 378

••

127 171

175 538

•• 5

-•is

March	7	L916
March	i:.	t916
March	:;i	L916
May	'.'	L916
M.,y	23	1916
June	L'l	1916
Augual	11	L916
Augusl	30	L916
Novembei	_>l	1916
1 December	7	L916
January	29	1(117
EDWARD .1. MH.AN,
L'i cording Secretary.

PUBLICATION COMMITTEE!

Henry -Kiwii;. M.D. Sc.D., Witmhi; Stone, A.M., Sc.D.,

Henri A. Pil&brt, Sc.D., William . I. Fox,

V'.irx \i;i. .1. Nolan, M.D.. Sc.D. '//" /' i - mi n. Gibson Dixon, M.D., LL.D., cx-o(fwio.

II 'I Toll: Ki>\\ LRD .1. NOLAN, M.D., Sc.D.

I () N'T K X T S.

innouncemenls, Reports, etc., set General Index.

PAGE

Alexander, Charles P. New <>r little-known crane-flies

from the United States and Canada: Tipulidse, Ptychop-

teridffi, Diptera. Part 3 (Plates XXV XXXI) 180

Banks, Nathan. Revision of Cayuga Lake spiders (Plates

X, XI 68

Barringer, Daniel M. A possible partial explanation of the

visibility and brilliancy of comets. With an addendum

by Elihu Thomson 172

Berry, S. Stillman. Cephalopoda of the Kermadec Islands

Plati - VI. \ M. \ III. LX) 45

Cockerell, T. I ). A. >i>\;\<- bees from Australia, Tasmania,

and the New Hebrides 360

Colton, Harold, S. On aome varieties of Thai- lapillus in

the M "i i ni Deserl region, a stud} of individual ecology 1 1" Crawley, Howard. The sexual evolution of Sarcocystis muris

(Plates I. II. MI. I\ ::

The zoological position of Sarcosporidia 379

Dall, William II. A new species of Onchidiopsis from

!'•• • ing Sea 376

Daniels, L. E. See Henders in, Ji mi b. Fowler, Henri W. Cold-blooded vertebrates from Costa

and the < 'anal Zone 389

Notes on fishes of the order- Haplomi and \I\ crocyprini 1 15 M in, Harold. The aervous system of Crepidula adunca

and n - d>\ elopmenl a I7'.»

Hebard, Morgan. See Rehn, James \ < '>. Hend Ji six s, and L. E. I >aniels. Hunting Mollu

in Utahand [dahol Plates \ V. \\ I. \ \ 1 1. \ \ 1 1 1 315

Jacobs, Merkel II., Ph.D. Temperature and the activiti

<>i animal- v",

iv rONTEN

I'M, I

Pilsbry, Henri \ Notes on the anatomj of Oreohelix, with :i catalogue of the species (Plates XIX, \ \ . \ \ I Wll 340

IIi.hv James \ G and Morgan Hebard. Studie* in the Dermaptera and Orthoptera ol the Coastal Plain and Piedmont Section of the southeastern United States Plates XII, XIII, XI\ 87

Thomson, Elihi . See B irringer, I >. M

Viere* k. Henri I.. New species of North American bees <>!' the genus Andrena from wesl of the littitli Meridian contains! in the collections of The Academy of Natural

aces of Philadelphia 550

Wade, Bru< e. New genera and species of Gastropoda from

the Upper Cretaceous i Plates Will. X X 1 \ 155

PROCEEDINGS

OF THE

ACADEMY OF NATURAL SCIENCES

OF

PHILADELPHIA.

1916.

January lis. The President, Sami el <j. Dixon, M.l >.. LL.D., in the Chair.

Thirty-six persona presenl .

The death of Daniel Giraud Elliot, a member, December 22, 1915, was announced.

The Publication Committee announced the presentation of papers under tin- following i itles:

Revision of Cayuga Lake spiders, " by Nathan Banks, December 2, 1915.

"Studies in the Dermaptera and < >rthoptera <>i' the < loastal Plains and Piedmonl Region of the southeastern United States," by James A. < i. Rehn and Morgan Hebard, January 17.

Fossil bird by Dr. R. \Y. Shufeldt, January 18.

Prof. William P. Magie and Daniel Moreai Barrinoer made communications on the Meteor Craterof Arizona, illustrated by lantern slides. No absl ra

The following were elected members: \\ iltei Sonni '■■ William S. Huntington. Samuel I . Bodine. David Wilbur Horn, Ph.D.

The following wan ordered t<> !«• printed:

pro< EEDiNoa <>i the \< \m \n of [Jan.,

THE SEXUAL EVOLUTION OF SARCOCYSTIS MURIS. HV llo\\ URD I B WYl.KI .

A number of years ago, at the University of Pennsylvania, an investigation of the early stages of the evolution of Sarcocystis nvuris

in the intestinal cells «>t" the mouse was undertaken. This investiga- tion was interrupted for several year-. bu1 was resumed at the laboratory of the Zoological Division of the Bureau of Animal [ndustry, Washington, D. ('.. and the first definite results obtained were outlined in a preliminary note published in l'.U 1 (< Jrawley, 1914).

In this note it was shown that following ingestion of the so-called -pure- «>f tin- parasite, penetration of the intestinal cell- of the mouse was effected within aboul two hour-. Once within the cells the spores rapidly underwent profound changes and after the lapse of aboul nine hours they had separated into two categories, which were interpreted to represenl male- and females. In the case of the supposed male-, development took the form of a loss of most if not all of the cytoplasm, so that the parasite became reduced to a nucleus, which, however, wa- of considerably Larger size than that of the original spore. Later, the chromatin of this nucleus became col- lected itito a number of -mall rounded masses placed at the periphery. These masses, at fir-t granular, later became solid and eventually transformed themselves into elongated, thread-like bodies, which were interpreted to he microgametes. This evolution was completed • the end of is hour-.

Meanwhile others of the original spores went through a wholly

differenl course Of development, which wa- not at the time considered in detail. It wa- evident, however, that these elements retained their cytoplasm and eventually transformed themselves into oval cell-, with rather dense cytoplasm and a vesicular nucleus containing a large karyosome. These, which were interpreted to be females, al-o reached the end of their development within 18 hour-.

Finally, appearances suggesting fertilization were noted.

At the time when this preliminary not ice was prepared, my material for the later stages, from 12 to L8 horn-, was abundant and the conclusions a- published were based on the findings in a number of mice ( > 1 1 the other hand, for the early stages only three mice were

1916.] NATURAL SCTEN< ES OF PHILADELPHIA. 3

available, these representing, respectively, the 2 to 2] hour, the 3 to Sh hour, and the '■'> and (> hour stages, the last being a mouse given two infecting feeds between which was an interval of 3 hours. and killed 3 hour- after the second meal. It was therefore con- sidered desirable to obtain more material for these earlier stages, and the results to be set forth herein are based upon the examination of a number of mice killed at periods of from one hour onward.

Material and Methods.

A li-t of the mice used, with appropriate explanatory matter, is placed at the end of the present section. During the entire course <>!' the studies on sarcosporidiosis, every mouse obtained in whatsoever manner was given a [lumber. In many cases these were trapped gray mice, <>r else white mice which had never been inoculated. Hence those used for the microscopical study of the evolution of the parasites in the cells represent but a portion of the entire series. It ha- been considered better, however, to retain the numbers origin- ally given, since no confusion can possibly arise from such a procedure.

In the li-t appended 'he time in hour- elapsing between the infect- ing meal and the death of the mouse has been placed immediately

aftei the number of the mouse, since this is the most important datum. After this the fixing fluid is noted, and finally ;i statement with reference to the quantity of infectious material which the mouse ate is appended. The omission of these data in a number

of cases indicate- a failun to keep the record complete.

The li-t a- given include- 38 mice, and the conclusions a- se1 forth herein are thu- based on thi- number of experimental animal-. It i- desired to lay emphasis on thi- point on account of the possibility of confusion with other intestinal Protozoa, 3uch :i- < loccidia.

Erdmann (1914 endeavored to obtain mice in which the possi- bility of extraneous infection was excluded. The procedure was to raise mice from birth under a- sterile condition- a- possible. Tin-.

of course, LS 'he ideal method, but. a- ladinaiin -tale-, it i- tedious ami difficult. The other method is to u-e a large Series Of mice.

which will presumably yield results that cannot be questioned. Thus, if. after feeding, parasites are found in the cells which are evidently Sarcocystia -pore- and if. a- time passes, these intracellular element- undergo serial changes it would seem to be hypercritical to question their identity a- stages in the evolution of Si /nun's. For it would be Decessarj to assume that each experimental mouse harbored Coccidia in addition to the Sarcosporidia and thai

1 pRi ii ii i.i\. - • 'i i in if \i>i.\n of [Jan.,

in i -arli and every case the ( loccidia happened to be in precisely such a stage as to rest ruble the Sarcosporidia. The mathematical proba- bility of this taking place diminishes with the number of mice used and when this number is large becomes a vanishing quantity.

since protozoan infections naturally tend to assume the ■utie form, it' one or two mice from a given cage were found to be infected with Coccidia, the surmise would be warranted thai many or all of the others were so infected. Hut in the case of the series upon which the present studies wen' based, the mice were obtained from various sources and from various places and in many cases had never been in contact. It is therefore believed thai the results as stated are valid, so far as concern- the possibility of confusion with ( "occidia.

The mice -elected for inoculation were deprived of food for 24 hour-. This served a two-fold purpose. It rendered them more prone to ea1 the infectious material when given and it served to free the intestine of half-digested vegetable food, the presence of which interferes with sectioning. For the shorl periods, up to 18 hours or so, the mice were given a piece of infected muscle of what was regarded as an appropriate size. This was larger or smaller, according to the number of cysts it contained: and when these were abundanl the portion given had a weighl of the order of one-tenth of a mam.

When the mouse is given it- infecting meal, any one of several things may happen. Some mice positively refuse to touch the meat while other- merely play with it for a time and then abandon it. More usually, however, the mouse feed-, and the customary method is for it to hold the food in its forepaws and nibble at it until it i- all consumed. This procedure, however, may be inter- rupted by delays, hut if the entire time required to finish the meal i- -hort in comparison with thai to elapse between feeding and death, the mouse may be used. Finally, in some cases the meal was bolted in the manner in which a dog feeds.

At the end of the proper period the mouse was chloroformed, ted, and the alimentary canal removed. In nearly all cases it was placed in the killing fluid entire, being neither cut into pieces nor -lit open. The int. -tine of a mouse has such thin walls that the fluid- have no difficulty in penetrating, and this procedure does away with the rough handling necessary in slitting the intestine. It more- over retained the intestinal content-, an obvious advantage, and a comparison with -lit intestines showed thai the fixation was equally accurate. The only disadvantage was that al times the penetration

1916.] NATURAL SCIENCES OF PHILADELPHIA. 5

of the fluids from the muscularis toward the epithelium resulted in the latter being torn loose from the underlying connective tissue. At least this phenomenon was not infrequently manifest and is presumably to be credited to the direction of entrance of the fluids.

Following fixation, each intestine was cut into pieces of a con- venient length for embedding in paraffin. In general, the small int. '-tin.' was cut into 25 to 30 pieces which were numbered, as a rule, from the anterior to the posterior end. Thus, int. 1 of a given mouse indicated the piece immediately following the stomach, the highest <>r last number that piece immediately in front of the caecum. Sometimes, however, this process w- - reversed, the last piece of the intestine being designated as int. 1: the next to the last, int. 2 and so on, the negative signs serving to distinguish such cases from the more usual procedure. This, as already aoted, is applicable to the small intestine alone, the caecum and large intestine being given other designations. The procedure as outlined above was not, however, always followed.

The fixing fluids used were:

1. Hermann's fluid, stronger formula.

2. Zenker's fluid.

:;. Picro-acetic acid, made by reducing a saturated aqueous solution «»t" picric arid t<> one-half strength with water, and adding 1 per cent, glacial acetic acid.

I. An alcoholic-corrosive-acetic mixture, designated in the text a- A. ( '. A. The formula for this i- a- follow-:

Saturated aqueous solution of mercuric chloride 50 parts.

Alcohol. «.t."i per cenl 50 parts.

( ilacial acetic acid ."> parts.

these, Hermann's fluid and the picro-acetic mixture, the latter de~piie Lee's strictun 'he most delicate fixation. Zenker's

fluid ia not to he recommended, since it leaves 'lie tissues in poor

condition tor staining and i- at best a mediocre fixative.

Tie v I \. fluid, while scarcely so accurate a- Hermann's fluid, i- none the less a very good fixative. It i-. moreover, very con- venient, since the tissues can he passed directly from it into alcohol, and it leaves the material in excellent condition for staining

The material was stained both in bulk and on the slide. While there i- a prejudice against the former method for delicate cytological work. Delafield's hematoxylin counterstained on the slide with alcoholic .ip-iii or acid fuchsin dissolved in 95 per cent, alcohi

6 pro< bedinos "i mi: u vdemi of [Jan.,

results nut far short of the besl slide staining. The use of counter- stains in alcohol is :i ureal time-saving device, since the preparation has bu1 tn be passed from xylol, to absolute alcohol, to 95 per cent. alcohol, to the stain and back again to be ready for the < !anada bal- sam. M over, if acid fuchsin be used, one quick dip in the stain is sufficient.

Fur slide staining, iron hsematoxylin, Wright's and Giemsa stains and thionin were used either with or without counterstains. The blood stains used alone are unsatisfactory, since only the blue ingre- dients seem to take hold of the tissues. Wright's stain, counter- stained with alcoholic eosin, however, gave very good results. The technique was as follows: The slide holding the sections was first treated as a blood smear and allowed to lie with the mixture of -tain and water upon it tor Id to 1"> minute-. It was then washed first in water and then in 95 per cent, alcohol until all of the pre- cipitated -tain had dissolved. It was then stained with eosin dis- solved in absolute alcohol, next passed into clean absolute alcohol, and finally into xylol. Wright's stain come- out very rapidly in alcohol, hut the whole procedure a- above outlined can be completed in a very -hurt time.

Thionin preparations counterstained in either alcoholic eosin or acid fuchsin in alcohol weir largely used and »a\e in -ome respect - the besl results. Preparations so stained display beautifully sharp and clear-cut pictures, and they are very good when it is a question of bringing out the chemical qualities of different parts of the para- ►n the other hand, thionin fails to bring oul certain granules in these Sarcosporidia which are perhaps significant, and slides stained in thionin are nut permanent.

Accordingly, most of the slides were stained with iron hematoxylin. With material fixed in Hermann's fluid, a counterstain is not neces- sary, although it wa- often used. With the other fixative- a counterstain wa- necessary, and acid fuchsin wa- the one most

generally em	ployed.
The li-t of	mire used follows:
~~		l.'i hours.
		:; and 6 hours.
		'.i hours	A. C \	1 [eavy meal.
L20		\Q\ and 17 hours		1 [eavy meal.
121		117 hours		1 [eavy meal.
1 _'.".		ours	1 fermann's fluid.
1 2 i		:; lli'	Picro-acel ic.
		v', hours	1 [ermann'e fluid	1 [eavy meal.
		'.» hours	1 [ennann's fluid	1 [eavy meal.
t t.i		1 1 1 hours	1 [ermann'e fluid	Moderate mi

1916.] NATURAL SCIENCES OF PHILADELPHIA. t

L46 Hi hours Hermann's fluid Moderate meal.

117 21 hours Hermann's fluid Modi rate meal.

lis L8hours Hermann's fluid Moderate meal.

H'.i 24hours Hermann's fluid Moderate meal.

[50 24hours Hermann's fluid Moderate meal.

L52 18 hours Hermann's fluid.

l.",:; 24hours Hermann's fluid Heavymeal.

L5 \ 24 hours.

L75 Yl\ hours Hermann's fluid.

L76 13J hours Hermann's fluid.

177 i i \ hours Hermann's fluid.

17s 15| hours Hermann's fluid.

17'.» lti|. hours Hermann's fluid.

isii \~\ hours Hermann's fluid.

181 L8£ hours Hermann's fluid.

L82 18 hours Hermann's fluid.

246 1 hour A. C. A Lighl meal.

•J 17 1 hours A. C. A Heavy meal.

248 2 hours A. ('.A Moderate meal.

249 3 hours A. ('. A Moderate meal.

•_'.■)() :. hours A. ('. A Heavy meal.

251 i j hours A ( !. A Very lighl meal.

252 2J hours AC. A Very light meal.

253 6 hours \ C. A Heavy meal. 257 3 hours Hermann's fluid Moderate meal.

I hours A. C A Moderate meal.

261 I hours Hermann's fluid Heavymeal.

262 .", hum- Hermann's fluid Moderate meal.

Prior i<> taking up the description of the findings in the cells, it is desirable to call attention to a phenomenon firsl signalized by Erd- iii.-itin (1910). This is tin exfoliation of the intestinal epithelium which appears to follow ingestion of the spores of Sarcocystis. The natural inference would be thai this was due to the invasion and subsequenl destruction of the cells by the parasites, and Buch was my original idea (Crawley, 1913). No doubl :i certain amounf of exfoliation is t<» I"- accounted for in this way, but other Factors are involved. Thus Erdmann found thai the ingestion of an extrad ol Sarcocystis cysts, from which tin- spores themselves had been removed, was followed by exfoliation, and data thai I -hall now give -how that the exfoliation takes place before any extensive invasion of the

cell..

The intestines of a series of mice, all of which had been killed within <i hour- after feeding, were examined and the conditions presented by the epithelium in various part- of the intestine noted. The results of this examination are tabulated below. Where no lation was demonstrable, the condition i- indicated by the word "none." Where, however, it i- in evidence it is designated "slight," "moderate," or "severe," according to its deg

PROCEEDINGS OF THE ICADElfi OP [Jan.

Moi -l 246, l-HOl R Si

IMt i ilial i • * 1 1 -lij:lit .

Exfolial mil moderate, olial ion none. 7 Exfoliation slighl ■

Exfoliation slight 1 1 Exfolial ion moderate.

13 Exfolial ion severe.

I.", Exfolial ion Bevere.

17 olial ion Blighl

l'» Exfolial ion nunc.

2] Exfoliation severe.

Exfoliation slight. 25 Exfolial ion none.

Mm se 251, I '-Hui i; Stage.

Int. i Exfoliation none.

Exfolial i"ii none 7 Exfoliation none,

g Exfoliation Done.

Mm -t 2 18, 2-hoi i; Stage.

I,,t Exfoliation >lij:lit .

x Exfolial ion none,

Exfoliation none l.", Exfoliation slight.

_M Exfoliation slight.

Exfoliation none

27 I rfolial ion none.

28 Exfolial ion none.

Exfoliation very -liiilii . Exfoliation very Blighl .

Moi -i. 249, 3-HOi i: Stage.

Iin j Exfoliation moderate

r. Exfolial inn nunc

v Exfoliation none.

in Exfolial ion none.

1_» Exfoliation nunc

17 Exfolial inn moderate

is Exfoliation slight.

i'i Exfoliation Blighl .

Exfoliation very .-li^lit. Exfoliation none. _' \ Exfoliation nunc.

Exfoliation Blight. Exfolial ion none. :;i Exfoliation none.

Moi -' 261 . 1-noiu Stage.

-1 Exfolial ion none.

— :', iliation Blight.

Exfoliation slight.

Mot BE 2 t7. l-iioi i: Si \'i Int. 12 ! Ixfoliation none.

1 1 I Ixfoliation none.

1916.] NATURAL SCIENCES OF PHILADELPHIA. 9

Hi Exfoliation slight.

Is Exfoliation slight.

19 Exfoliation slight.

30 Exfoliation slight.

:; 1 Exfoliation very slight .

Moi si 262, 5 to 3-houb Stage.

Im. _ 1 Exfolial ion none.

—2 Exfoliation slighl .

Exfoliation slight. _ j • Exfoliation sevi re.

— ;, Exfoliation moderate.

_ti Exfoliation mod* rate.

Moi be 250, 5-HOi r Stage.

lnt . _M ' Exfoliation none.

S2 Exfolial i'»n Done.

30 Exfoliation moderate.

:;i Exfohal ion moderate.

Exfoliation slight.

Exfoliation mod< rate.

Moi -i. 253, 6-hoi R m \'.i-

Ini. 10 Exfoliation Blight.

1.", Exfoliation slighl .

Id Exfolial ion none.

pi Exfoliation Questionable.

Exfoliation slighl ,

27 ■ dial ion none

28 I folial ion none.

Exfoliation noi

As already stated, as a resull of the method of fixing the mouse intestine, the epithelial row was at times torn loose from the sub- epithelial connective tissue. It thus results thai in some cases the materia] presented a decidedly battered appearance. While, how- ever, the epithelial row itself migb.1 thus be torn loose and mor< or less broken, the individual cells uric not thereby injured and their appearance indicated an accurate fixation.

< »n the other hand, it was frequently possible to see thai the cells al the tips of the villi were abnormal, this abnormality expressing itself in :i loss of staining capacity on the part of the cytoplasm and an obvious degeneracy of the nuclei. This degeneracy, a1 firsl rting only the cells al the tips of the villi, passes into a condition in which these cells have disappeared, while those lying along the sides of the villi are affected. This condition, in it- turn, passes into one in which the villi arc represented merely by stumps oi connective tissue, the epithelium being present only in the regions between the bases of the villi. Finally a stage is reached in which the intestine is wholly denuded of epithelium. In the list* given

in pro< i i DINQ8 OF i in \' \i" M1" OF [Jan..

the iitui "slight " defines those conditions in which epithelial degen- eration i- just beginning to be manifest, and "moderate" conditions where the tips of the villi are seriousl) affected, and "severe" con- ditions where the destructive influence has gone further.

The data given in the above lists seem to establish the facl thai ation of the epithelium is correlated with ingestion of the spores of Sarcosporidia, but they are no1 consistenl amongst them- selves. Thus, mouse 248, a 2-hour stage, is ao1 so badly affected as mouse 246, killed only one hour alter feeding, whereas mouse 251, a 1 j-hour stage, -how- do exfoliation at all, although in this lasl caa rvations were confined to only a small pari of the intestine.

In the cases of Nos. 249 and 261, both 3-hour stag< s, and No. 247, 1-hour, exfoliation is qo1 extensive, while No. 262, 5- to 3-hour, -how- a considerable amounl near the posterior end of the intestine, as doe- also No. 250, 5-hour. On the other hand, mouse 253, killed 6 hour- after feeding, is bu1 slightly affected. In view of the rather contradictory nature of the data, it is impossible to attempt any explanation of the modus operandi of loss of the epithelium.

Krdmanii suggested that the destruction of the epithelium was an adaptation having for it- purpose the easier penetration of the spores into the tissues of the mouse. Presumably this destruction i- correlated with ingestion of the parasites, bu1 if it be of any value to hosl or parasite it seems more plausible to regard it a- a defensive move «.n the part "f the former. In their attack upon the mouse the parasites fir-t invade the epithelial cells and this they do within the first two or three hour-. Obviously; then, the destruction of this epithelium, either before or after penetration by the parasites can only work to their disadvantage. Hence, while it is possible to look upon this exfoliation as an adaptation on the part of the host, there seems no good reason for so doing. It is a matter of observation that exfoliation follow- the ingestion of sarcosporidian cysts, but it i- also a matter of observation that such ingestion is practically always followed by infection of the muscles. Hence,

the exfoliation i- Obviously Q01 protective.

The matter i-. however, one of minor importance and the data

are merely given for what they are worth.

EVOL1 ["ION OF THE PARASITES l\ THE CELLS.

The account of the findings in the cell- may appropriately be

. in with mouse '_M»i. killed one hour after feeding. As indicated in the table on p. 8, slides were prepared from the alternate pii

1916.] NATURAL SCIEN< ES OF PHILADELPHIA. 11

of the intestine from one end to the other. The anterior portions, Nbs. 1 to 11. were wholly negative, there being no spores either in the lumen or in the cell-. Beginning with int. 13, however, spores weir presenl in the lumen, and they weir seen in the cells in int. 19, 23, and '2'). Their absence from int. 2] i- to be credited to the severe exfoliation there present.

From im. 13, where the spores weir first seen in the lumen, then was a rapid increase in their numbers in each successive piece, and in the lumina of im. 23 and 25 they were presenl in enormous numbers. From this it i< evideni thai it requires but one hour for the ingested spores to reach the extreme posterior end of the -mall intestine, and as shown both by this and other mire, apparently the greal majority of them reach this situation very quickly. While they have also been found in the caecum in very early stages, they evidently do not - from the small intestine into the caecum a- readily a- they pass along the -mall intestine itself. This is evidenced by the facl that in the earlier stages up to <) hour- or thereabouts the la-t two or three centimeters of the -mall intestine always harbor spores free in the lumen.

Then- i- thus broughl aboul a 3tat< of affair- of some interest when the earlier stages of the evolution of the parasites is under consideration. It i- evident that the spores pass along the -mall intestine very rapidly until the posterior end i- reached. The length of time required for this stream of 3pores to pa-- a given point will obviously vary in the different mice. In those cases when-. ;,-

u!t of prolonged nibbling, the ingested meat reaches the stomach thoroughly comminuted, we may presume that it- stay in the stomach i- shortened and it- movement along the -mall intestine more rapid. On the other hand, when the meal is swallowed in large pieces, the

imption i- that it will remain in the stomach until it i- softened and disintegrated, and in consequence it- progress along the -mall intestine will he delayed. Nevertheless, digestion in mice of purely animal matter i- rapid, and in general at the end of a few hour- sp<

scarce in tin- lumen of i he upper and middle portions of the -mall intestim I is a pure guess, we maj assume that the stream oi spores requires on.- hour to pass a given point, thru the spores within the cells in any particular part of tin intestine (except tin posterior end) will all have entered the cells within an hour of one another. < >n the other hand, a- we have seen, great numbers <»i the Bpores reach tin- posterior part of the intestine within our hour, and remain there for several hour-, n- i- mown bj finding them in tin-

L2 proceedings oi phe \> \m.\i\ of [Jan.,

situation in 9-hour stages. During the whole of this time it cannot Im> questioned thai individuals are constantly penetrating the cells.

Therefore it seems reasonable to assume thai within certain limits. the intracellular spores in a given section of the upper or middle parts of the intestine will be in somewhal the same developmental On the contrary, in the posterior pari of the intestine, the intracellular spores will represenl a series in the development, covering the greater part »>t' the period of time elapsing between feeding and the death <>t' the mouse. Thus, in a 9-hour mouse, the parasites in the eells 0f this extreme posterior part of the intestine migh1 represenl form- which had been in the cells from only a few minutes up to seven or eight hours, and in Nos. 132 and 133 it was evident thai this was the case.

It further follows that in these posterior portions many more cells are parasitized than elsewhere, since there is here maintained for several hour- a targe supply of extracellular spores.

Returning to the condition- as found in mouse 246, intracellular -pop,- in small numbers were found in int. 19, 23, and 25. These, of course, represented the very earliesl stages in the development and in the main were not to be distinguished from those in the lumen. In some cases, however, development had begun, thus demonstrating the extreme rapidity with which these parasites go through with their evolution. The mounted material of this mouse, however, was prepared with a view of getting a general survey of the condition- rather than for detailed cytological study. Hence, no figures of the parasites a- found here have been made.

Moua .'■'>!. Mouse 251 was killed 11 hours after feeding. According to the observations made at the time of the infecting : it receive.! "a very light meal." bul when the stomach and intestine were prepared for study the findings suggested that whereas relatively the meal may have been very light, positively such a definition seemed scarcely appropriate. In the stomach, from which the epithelium had largely disappeared, there were abundant cysts of the parasite, many of which were more or less intact and contained the greater number of 'he spores. In int. 1, \\. 5. and 7 there were abundant spores in the lumen, and in int. <) they were nt both in the lumen and in t he cells.

Comparing the conditions found here with those in mouse 246,

it is to be note.) that in the latter the 3pores were further hack toward

the end of the intestine. In No. 246 the upper portion- of the

-tine were free ot spores, whereas in 25] these same portion-

1916. N All RAL SCIENCES OF PHILADELPI11 \. L3

contained them in considerable abundance. These differences are. as already -uuiie-ted. probably to be accredited to differences in the manner in which the two mice fed.

Taking up now the evolution of the spores within the intestine of the mouse, we may advantageously use as a point of departure the spore as it occurs free in the lumen, for it is evidenl that developmenl begins here.

Plate I, fig. 2, portrays such a -pore and may be taken as the point nt' departure, although in all the early stages the spores in the lumen are identical with many of those in the cells. This particular case i- from mouse 248, a 2-hour stage, but it is valid for any of the early stag

The characteristics of this stage are as follows: The contours of the cell are -harp and clear cut, and there is a distinct bounding line or periplast. The cytoplasm, while obviously alveolar, is dense and ordinarily stains well. Granules may or may not be present. The aucleus, which i- conspicuous, is round and gives the appearance as though in life it were turgid with nuclear sap. There is a distincl nuclear membrane and a more or less distincl nuclear ne1 is always present.

With regard t « • it - shape, t he spore in the lumen maj be ;i smooth oval, as, i'ii" example-, are tin intracellular spores shown in Plate I. figs. '■> and I. or it may have the sides more or less bulged oul in the region of the nucleus. This latter phenomenon is due to the incn in size of the nucleus, a process initiated very shortly after the spore reaches the alimentary canal of the mouse.

The appearance of the spores in the intestine, whether they be in the lumen or in the cells, offers a considerable cbntrasl to thai of spores removed from the cysts. With regard t" these latter, a ription i- herewith given, although they :w- familiar objects in the literature ami have been described and figured n numb times.

Such -pore- an' shown in Plate I. fit:. I. \s i- here indicated, one end df the -pure i- I ,ii >ad< t. the other end narrower, and the nucleus

lie- nearer the narrow end.

The internal structure is obscure. The nucleus is m clear-cut vesicle, and to all appearances is provided with ;i definitive mem brane. No internal structure can he made out. ami the staining reaction is feeble. In Giemsa preparations it stains a pah' reddish

color.

cytoplasm with < liemsa stains :i dense blue. It- structur

1 J rif"> i i DINOS "i i in \' IDEM! "i [Jan..

scarcely be determined, bu1 it may be inferred thai it is alveolar. It is densesl in the immediate vicinitj of the nucleus. The broad end of tlie spore is frequently much less dense than elsewhere and at times -how- a more or less well defined oval area. This appear- ance, however, i- probably due merely to the fad thai here the

ilasm has a lower affinity for the stain than elsewhere. The same phenomenon is shown by the spores of Sarcocystis

■ •■■ Crawley, 191 l , In the case of the rabbil parasite, a- 1 have endeavored t<> show, one end of the spore seems t<> be differen-

1 into a sorl of rostrum, the cytoplasm of which doe- not -tain

a- densely :i- doe- that of the halance of the spore. We are prohahk

dealing with the same thing in the case of Sarcocystis maris, hut in this parasite the differentiation of the rostrum is not bo sharp. It may furthermore he suggested that it i- this clearer region in the e which ha- given rise to the belief, expressed by some author-. that the sarcosporidian -pore possess) ~ a polar capsule.

• Comparing the spores taken directly from a cyst with those in the intestine, the latter are broader, more oval bodies, and, although this i- not shown by the figures, there ha- been a loss of the granules which are such a characteristic feature of the former. The most noteworthy change, however, concerns the nucleus, which has _• r. apparently much more turgid, and begins to show a nuclear net.

It i- thus evident that evolution begins :i- soon ;i- tic- -pore reaches

atestine of the mouse, and apparently the most important step

greal increase in activity of the nucleus. This evolution, however.

- not appear to he carried far mile-.- the spore £iain> a resting place

within a cell of the host.

The cells invaded are the cylinder cells. At least this is so in the

vast majority of cases. Now and then, however, parasites are to In'

I in mucous cell-, hut since the presence of a parasite in a cell

may result in mucoid degeneration, it i- difficull to diagnose such

inces. The parasite may have invaded a mucous cell or it may

have caused mucoid degeneration of a cylinder cell. This question,

appear- to he of no greal importance.

It i- also well to emphasize the point that the -pore is a naked

protoplasm and that the only differentiation displayed by

the cytoplasm i- the peripheral condensation into a periplastic layer.

: ,t- to the effect that the -pore open- and releases an amcebula

wholly without warrant, and polar filament-, either coiled up

within one end of the cell or discharged, do not exist. Claims of

1916.] NATURAL B< IKN< ES OF PHILADELPHIA. 15

this sort, which have appeared in the literature from nmc to time, seem to be due to an unfortunate desire to correlate the Sarcospo- ridia with the Myxosporidia. These two groups may of course be closely related, but as ye1 there is no conclusive evidence on hand to show T ha1 they are.

Plate I. figs. 3 and 1 -how spores in the cells of int. 9 of mouse 251 . a 1 '-'■ »ur stage. It is to I"- noted thai each of these lies in a vacuole. Pi ably they have not been in the cells for more than an hour. In both of them the cytoplasm is dense, compact, and deeply staining. In both of them, also, the nucleus has enlarged and is separated from the periplast only by very narrow strips of cytoplasm.

There is, however, a difference in the nature of the net in these

two nuclei. Iu the case of fig. •'». the meshes in the centre are solidly

filled with chromatin, whereas in that of fig. I the chromatin is divided

separate masses. In all of these early stages the chromatin has

a low affinity for -tain-, and, following the rule which seems to hold

rally true in Protozoa, takes the acid rather than the basic

-tain-. < >n this account it re-ults that these nuclei are rather difficult microscopical objects, and seen with power- of less than 1,000 diame- ters, a nucleus like that of fig. 3 look- like a vesicle containing only a central granule. But with a magnification of 1,500 to 2,000, and an intense artificial light, the -tincture a- shown in the figure is brought out. A- will l»e -hown later, fit:. 3 represents the more primitive condition, in which the chromatin i- concentrated into a single ma — . within which, however, the meshes of the nuclear net can he traced. In the ca-e nf fig. i. the chromatin occur- in separate masses and the centra] meshes of the net are largely clear. Nuclei precisely like either of these may be seen in -pore- in the lumen.

M Mouse 248 was killed two hour- after feeding. It-

intestine was cut into 32 pieces, int. 32 representing the extreme posterior portion. Observations were made on int. :;. 9, 15, 21, 27, and 32. In the upper portion- there were scattered -pore- m tin' lumen and occasional specimens in the cell.-, hut. following the ral rule, parasitization \\.i- not extensive until the more posterior

■ached.

I'lat-- I fig. 5, from int. 27, shows a parasite lying in the usual vacuole

• to th< 'lie epithelium. The cytoplasm i- dense,

while the nucleus -how- very distinctly a nuclear net supporting

; chromatin granules. The uucleus, in thi lies near

one end ol 'he cell. I _ •. from int. :;it, also represent* what i-

clearlj :i verj earl) The cytoplasm is dense and compact,

16 PBO< i BDDfOfl < i \< m»i Ml of [Jan.j

the cell contours are sharp and a periplasl can be seen. The aucleus, however, shows merely as ;i Faintlj stained vacuole containing some formed substance, the details of which could no1 be made out. It may here be remarked thai in mosl of the cell parasites the nucleus appears as a vesicle containing a nuclear net, and thai in general nuclear nel is the only pari of the nucleus thai stains. Hence tiif nit appears as though projected againsl a clear background. < >n the other hand, it is frequently to be observed in the spores free in the lumen, and much less frequently in those in the cells, thai the

nuclear Bap as well as the nuclear net has taken the stain, thus obscuring the details of the latter. This condition is illustrated by

fig. ...

In the preliminary note it was stated that at leasl a portion of the

intracellular -pun- rapidly underwent a change which expressed itself in a reduction of the cytoplasm and an increase in the size of the nucleus, the ultimate resull of which was the production of a

body which was apparently only the enlarged original nucleus, the cytoplasm having apparently all disappeared. This, it was stated, was interpreted to he the male form.

This evolution is illustrated in Plate I, figs. 7 to 1 1 . Fig. 7 shows the nuclear enlargemenl with no urea t amount of cytoplasmic reduction.

The nuclei;- is large and turgid, it causes the sides of the parasite to bulge and -how- a- distincl net . Tin- net consists of a cent ral aggre- gation from which strands run to the periphery. As already indicated in the discussion of figs. :; and I, whereas the appearance of this central aggregation differ- considerably in the different specimens, it- structure appears to he fundamentally the same throughout. It seems to he merely the close-meshed central pari of the nuclear net, with the inter-pace- sometimes empty and sometimes filled in with faintly staining chromatin. The latter is the more primitive condition.

The parasite shown in fig. 7 was from int. 15, or about the middle