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ILLINOIS BIOLOGICAL 
MONOGRAPHS 


Vol. XIV No.4 


PUBLISHED BY THE UNIVERSITY OF ILLINOIS 
UNDER THE AUSPICES OF THE GRADUATE SCHOOL 
UrBANA, ILLINOIS 


EDITORIAL COMMITTEE 


Joun THreopore BUCHHOLZ 
FrepD WILBUR TANNER 
CHARLES ZELENY 


UNIVERSITY 


c * OF ILLINOIS 
1000—9-36—9359-S 1h PRESS 1: 


THE LIFE HISTORY OF COTYLOPHORON 
COTYLOPHORUM, A TREMATODE 
FROM RUMINANTS 


WITH NINE PLATES 


By 
HARRY JACKSON BENNETT 


CONTRIBUTION FROM THE ZOOLOGICAL LABORATORY OF THE 
UNIveRSITY OF [LLINOTS 
No. 485 


DistrrButEp 
SEPTEMBER 29, 1936 


CONTENTS 


Introduction. Bow ee a eA Oe Be te Le 7 
Materials and Methods . . .. . . . . . . 8 
History of the Genus Cotylophoron . 9 
“Egg 12 
OMiracidium. . . . . 1... ee ee ee 
Development 14 

PL Clio ake e ane OL be eh. a, 5, la  & 23 
Mature Mirdcidium 2, << “ 29 as 4 8 we ew 25 
Mintermediate Host.“ .. - 2. - 2. 2. w sie « © « » 88 
Determination of the Host . . . . .. ... . 38 
Biology of Fossarta parva. . . 1. we 39 
SOOMOC st MGs. bP eee ie. Oe. ak tae we lee ee 44 
Development .....° . . « « «© « » «© &» +» «© » 44 
Mature Sporocyst . «*. «< $ «  « «© e026 «6 » 
Redig@ts WRT 4? ia ek a ke Oe SO ee 
Wevelopmewt. « wraiaes & © «@ & «a o) “ei ce 54 
NMattiretRedia jo 5's & is 2 «© + «~ « » «= weg ~ 9 00 
Daughter Redia. “2.0% 4 < 3 «2° « G + <@ Go e « 4. 68 
Scrcatiameen ty fa a. ae ee ee SS ee sw «6 
Development . . . . . . 2... «we « « . 64 
Niatinewercatia’ 2° « 2 4 vat eS Me ee Se Ge OD 
Discussion of Previously Described Amphistome Cercariae . 76 
Wetacercariave's; i 4 -S- Se + & Rb eela¥oew. oo we ue, 18 
Nau ny ee ok en en RI. Btn AD 
Experimental Infestation. . . . . . . . . . . 79 
Development. . . . . . ... pa! ake fe A uier OO 
Specific Description of Cotylophoron espe a th ee. BO 
Summary and Conclusions .-. = «< &% « = «4 » s 96 
Bibliosraphy- 3 « 2 « «© 4» + “8 ese e « «2 » 98 


mlatess ee eee a eee ee chr es Se Ow. & OT 


ACKNOWLEDGMENT 


This work was begun under the direction of 
Professor H. B. Ward, now Professor Emeritus 
of the University of Illinois, to whom I wish to 
express my appreciation for suggestions in con- 
nection with this problem. The work was com- 
pleted under the direction of Professor H. J. Van 
Cleave, to whom I wish to express my apprecia- 
tion for his inspiration and encouragement and 
for his many valuable suggestions. I wish to 
thank Dr. Maurice C. Hall and Dr. Wendell H. 
Krull for the loan of specimens, and Dr. Frank 
C. Baker for the identification of the intermedi- 
ate snail hosts. Thanks are due also to Dr. 
W. H. Gates, Dr. R. L. Mayhew, Dr. J. I. 
Martin, the late Dr. Harry Morris, and Jane 
Tobie Bennett for material aid in the preparation 
of this work. 

Grateful acknowledgment is also due to the 
Louisiana State University for aid in the publica- 
tion of this monograph by a grant of seventy- 
five dollars to be applied on the cost of the 
printing. 


INTRODUCTION 


The knowledge of North American trematode life histories is very 
limited, and in many instances the life histories which have been described 
lack completeness. This is particularly true of the amphistomes. Cary 
(1909) published a life history of Diplodiscus temperatus which, as Cort 
(1915:24-30) pointed out, must be considered erroneous. Krull and 
Price (1932:1-37) determined experimentally the life history of this 
same form but omitted a description of the sporocyst. Beaver (1929: 
13-22) found and described all of the developmental stages in the life 
history of Allassostoma parvum, with the exception of the sporocyst. 
However, Beaver did no experimental work except to infest the final 
host. Krull (1934:171-180) obtained eggs of Cotylophoron cotylophorum 
from Puerto Rico and determined experimentally the life history of this 
parasite, but he did not describe any of the developmental stages. 

Looss (1892:147-167) published the life history of Diplodiscus sub- 
clavatus (Syn. Amphistomum subclavatum) but he did not completely 
describe the miracidium nor experimentally infest the final host. He also 
described the miracidium of Gastrothylax gregarius (1896:170-177) ; the 
developmental stages of Gastrodiscus aegyptiacus (pp. 177-185) with the 
exception of the adult; and the developmental stages of Paramphistomum 
cervit (Syn. Amphistomum conicum) (pp. 185-191) with the exception of 
the adult. Takahashi (1928) described briefly some of the life history 
stages of P. cervt. 

There are two methods of attack in solving trematode life history 
problems. One is to attempt to prove specific identity between cercaria 
and adult by structural comparison, and the other is to find the relation- 
ship experimentally. Several authors have described amphistome cercariae 
and suggested the possible relationship existing between them and known 
species of adults, but thus far no one has conclusively demonstrated such 
a relationship. In the present work the experimental method was used 
and all of the developmental stages were studied successively. Eggs se- 
cured from adult worms were hatched and the intermediate snail host was 
determined by exposing many species of snails to the free-swimming 
miracidia. The life history stages consisting of the egg and its develop- 
ment, the mature free-living miracidium, the infestation of the inter- 
mediate host, the sporocyst, the redia, the cercaria, the metacercaria, the 
infestation of the final host, and the development of the parasite to sexual 
maturity in the final host are discussed. 

An attempt is made to evaluate the diagnostic value of certain morpho- 
logical features which have been considered of no specific value by recent 
writers in extensive revisions of the classification of the amphistomes. 


7 


8 ILLINOIS BIOLOGICAL MONOGRAPHS 


This report constitutes the first complete study of an amphistome life 
history and the first report of a representative of the genus Cotylophoron 
from the mainland of North America. 


MATERIALS AND-METHODS 


Material for the study of the various stages of the life history of 
Cotylophoron cotylophorum was obtained from the two kinds of host of 
this parasite. Mature worms were collected from the rumen and imma- 
ture ones from the duodenum and rumen of cows, Bos taurus, slaughtered 
at the city abattoir at Baton Rouge, Louisiana. The intermediate snail 
hosts, Fossaria parva and F. modicella, were collected from lakes, ponds, 
and drainage ditches in the vicinity of Baton Rouge. 

I-ggs deposited by worms after removal from the final host were 
studied alive only. Miracidia, sporocysts, rediae, cercariae, immature and 
mature worms were studied while alive, in toto mounts, and from sec- 
tioned material. 

Miracidia were studied alive unstained or stained intra vitam. The 
intra vitan stains which gave the best results were methylene blue, bril- 
liant cresyl violet, and neutral red. Fleming’s osmic acid, Bouin’s, or 
Bouin’s modified with urea and chromic acid, and sublimate-acetic solu- 
tion were the fixatives used but the first two were best for this material. 
Miracidia were stained in toto mounts with Biondi’s haematoxylin and 
Ehrlich’s acid haematoxylin. For sectioned material Ehrlich’s acid haema- 
toxylin was used most often. 

Sporocysts, rediae, and developing cercariae were dissected from snails 
for study while alive and from toto mounts. For sectioning, the entire 
snail was fixed in warmed Bouin’s fixative unmodified or modified with 
urea and chromic acid. Sublimate-acetic and modified Bouin’s were used 
in fixing specimens for toto mounts. The stains used most often in pre- 
paring toto mounts were borax carmine, alum cochineal, and Ehrlich’s 
acid haematoxylin. For sections the latter stain was used almost ex- 
clusively with alcoholic eosin as a counter stain. 

The mature cercariae were studied alive, in toto mounts and from 
sectioned material. Hot sublimate-acetic solution and Bouin’s were used 
as fixatives, but the fixed cercariae were always greatly contracted. Alum 
cochineal and borax carmine gave good results for toto mounts, and 
Ehrlich’s acid haematoxylin for sections. Metacercariae were studied 
alive only. 

Immature and mature worms are extremely resistant to external con- 
ditions and become relaxed in cold water only after several hours. The 
mature worms remain active from 6 to 8 hours, and the young specimens 


LIFE HISTORY OF COTYLOPHORON—BENNETT 9 


sometimes are active after 24 hours. When relaxed the worms were 
placed in warmed sublimate-acetic solution or Bouin’s fixative. For toto 
mounts borax carmine and alum cochineal gave good results, while 
Ehrlich’s acid haematoxylin, Delafield’s haematoxylin, and Mallory’s 
triple connective tissue stain followed by eosin gave excellent results in 
staining sectioned worms. 

The final host, Bos taurus, was infested by feeding metacercariae 
encysted on lettuce. The rate of development and the location of the para- 
sites in the body were determined by killing and examining the hosts. 


HISTORY OF THE GENUS COTYLOPHORON 


Cotylophoron cotylophorum (Vischoeder, 1901) Stiles and Goldberger, 
1910 was described by Fischoeder (1901:370) as Paramphistomum coty- 
lophorum. His brief description is as follows: 

Nur 5-8 mm lang, gedrungen, dorsoventral schwach abgeflacht. Ocsophagus 
stark musculos. Scharf abgegrenzter Genitalnapf. Hoden fast neben einander. 
He places this species in the family Paramphistomidae Fischoeder, 1901 
and in the subfamily Paramphistominae Fischoeder, 1901. Later (1903: 
546-550) he redescribes this species in much greater detail. 

Stiles and Goldberger (1901:15) raised the family Paramphistomidae 
to the rank of superfamily Paramphistomoidea, having practically the 
same characteristics as Paramphistomidae Fischoeder. The superfamily 
they divide into three families, the Gastrodiscidae Stiles and Goldberger, 
1910, the Gastrothylacidae Stiles and Goldberger, 1910, and the Param- 
phistomidae. Paramphistomum cotylo phorum Fischoeder, 1901 was desig- 
nated by Stiles and Goldberger (1910:63) as the type species of their 
new genus Cotylophoron. They distinguish the genus Cotylophoron from 
Paramphistomum by a single character, the presence of a genital sucker. 
Fukui (1929:309) in his work on Japanese Amphistomata considers this 
difference not important enough to be of generic value and wishes to 
preserve Cotylophoron as a subgenus of Paramphistomum. I agree with 
Fukui and believe that Cotylophoron should be preserved as a subgenus 
only. On the other hand, Maplestone (1923:151) and Stunkard (1925: 
141) consider Cotylophoron as a distinct genus. 

Stiles and Goldberger (1910:63) described a second species for the 
genus Cotylophoron which they designated as C. indicum. The similarity 
of C. indicum and C. cotylophorum is evident from the summation of the 
differences between the two forms by these writers. Their statement is 
as follows: 


Cotylophoron indicum comes close to C. cotylophorum, from which it differs 
chiefly in the structure of the oesophagus, which is provided with a bulbus thicken- 


eurisino7yT ‘asnoy uojeg 


ILLINOIS BIOLOGICAL MONOGRAPHS 


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wunsoygojkjoo uosoydojkjoy JO NOILAGIMLSIGQ] OIHAVADOAD) ONIMOHS—'T AIAV]E, 


LIFE HISTORY OF COTYLOPHORON—BENNETT 11 


ing in the latter species but is without it in the former. The two differ also in 
details of structure of the copulatory apparatus and in the position of the genital 
pore. In C. imdicum the genital sucker is less sharply delimited, projects less, 
has a much smaller genital atrium, and the genital pore is decidedly post- 
bimurcal .... 

Maplestone (1923:152-153) has pointed out that the course and length 
of the esophagus and the size of the esophageal thickening or bulb are 
subject to considerable variation in C. cotylophorum,; and that the position 
of the genital pore varies in relation to the intestinal bifurcation to such 
an extent that neither of these points is reliable for distinguishing be- 
tween C. indicum and C. cotylophorum. He further points out (p. 155) 
the variability in the size and appearance of the genital atrium in C. 
cotylophorum and states that the shape and number of chambers in this 
structure cannot be regarded as of any value for specific diagnosis. He 
concludes that Stiles and Goldberger were in all probability dealing with 
immature specimens of C. cotylophorum. His conclusion (p. 195) as 
to the diagnostic value of the copulatory structures in other amphistomes 
is given as follows: 

.... the presence or absence of a prominent genital papilla, or a genital atrium, 
are purely matters of chance, and are of no more diagnostic value in this instance 
(Gastrodiscoides hominis) than in any other species of the group Amphistomata. 

Stunkard (1925:138) attributes Maplestone’s viewpoint to a confusion 
between physiological variations due to degrees and states of functional 
activity and true structural differences. On the other hand, Fukui (1929: 
270) agrees with Maplestone that the shape of the atrium is highly vari- 
able according to the protrusion of the genital papilla and so cannot be 
used for diagnosis. He (p. 319) definitely considers C. indicum to be a 
synonym of C. cotylophorum. I am of the opinion that the conclusions of 
Maplestone and Fukui concerning the importance of the genital appa- 
ratus are unsound for reasons to be pointed out later in the discussion of 
growth changes of C. cotylophorum in the final host. However, Maple- 
stone’s conclusions concerning the position of the genital pore in regard 
to the intestinal bifurcation and the variability in the size of the 
esophageal bulb are correct. 

Leiper (1910:244-248) described two new species of trematodes, 
Paramphistomum minutum and P. sellsi, {rom the hippopotamus, which 
according to Maplestone (1923:158) should be placed in the genus 
Cotylophoron. Maplestone considers C. minutum and C. Sellsi to be 
identical. Stunkard (1925:139) and Fukui (1929:307) accept both of 
them as valid species of the genus because of the large genital sucker in 
these forms. Regarding the validity of these two species Stunkard states: 

According to the description of Leiper, C. sellsi is more than twice as large 
as C. minutum, the testes and ovary are about four times as large, whereas the 


12 ILLINOIS BIOLOGICAL MONOGRAPHS 


oral and ventral suckers are actually smaller than those of C. minutum. It seems 
incredible that these differences are mere variations and therefore I am in agree- 
ment with Leiper in regarding the two forms as distinct species. 

I am of the opinion that Stunkard and Fukui are correct in considering 
these two species as distinct. 

Only three accepted species, C. cotylophorum, C. minutum, and C. 
sellsi, have been described for the genus Cotylophoron. 

C. cotylophorum is widely distributed as indicated by reports of this 
parasite from Africa, India, Puerto Rico, and the United States (present 
paper). The hosts from which it has been reported, its position in the 
host, the localities from which the hosts came, the names of the authori- 
ties reporting the parasite, and the dates of the reports are given in 
Table 1. 

EGG 

Appearance and Structure.—The eggs of C. cotylophorum are re- 
markably uniform in appearance. The shape is nearly ovoid, there being 
a slight attenuation at the opercular end. However, variations occur in 
which the eggs are completely ovoid or are more distinctly attenuated, 
giving a pyriform shape to the eggs (Figs. 1-11). The only marking on 
the shell is a small projection opposite the opercular end. This marking is 
usually asymetrical in position. The operculum, which measures 22 by 3 p, 
articulates with the shell by means of numerous small tooth-like projec- 
tions which interdigitate with similar structures on the shell. 

The eggshell is whitish when seen with the unaided eye but is trans- 
parent when seen with the microscope. In optical sections the shell is 
seen to be variable in thickness, being 2 » at the operculum, 1.5 in the 
lateral areas, and 3 » at the posterior end. 

When deposited each egg contains from 40 to 50 yolk masses. Each 
mass is composed of a membranous envelope which is filled with a 
translucent liquid and numerous small granules. This material imparts 
to the egg its brownish-yellow appearance when studied under the 
microscope. The ovum, which is completely enclosed by the yolk cells, is 
located slightly anterior to the middle of the egg. Cleavage has not oc- 
curred in the majority of the eggs when deposited, but in some it will 
have advanced as far as the second cleavage stage. The outline of the 
ovum is easily seen in these eggs, although it is completely embedded in 
yolk, as is the developing embryo. 


Sige.—The size of the egg is of special interest because many authors 
attach specific significance to the size of the eggs, based on the extreme 
limits. Fischoeder (1903:550) in his description of Cotylophoron coty- 
lophorum gave the egg sizes as 125 to 135 » long by 65 to 68 p» wide. Stiles 


LIFE HISTORY OF COTYLOPHORON—BENNETT 13 


and Goldberger (1910), Maplestone (1923), and Stunkard (1929) re- 
described the worm but did not give the egg sizes. Krull (1934:178) 
found the average measurement of 12 eggs teased from a preserved 
specimen to be 126 by 61 » and the average measurement of 12 eggs 
collected from the faeces of an infested calf to be 132 by 68 up. 
Hundreds of eggs from many different specimens were measured 


TABLE 2.—EGG MEASUREMENTS FOR Cotylophoron cotylophorum 


Size of worm in mm...| 3.7x 1.3 | 4.5x2.2 | 6.0x 2.0 | 6.6x2.5 | 9.0x3.0 | 9.0x 3.5 
Size of eggs in microns: 
1 ee eee 121x58 | 116x 67 | 126x62 | 139x 63 | 134x 63 | 134 x 67 
De aes Se Poet een re osuenctelc 125x 67 | 120x 67 | 116x 67 | 118x 67 | 122x 67 | 134x 67 
9a OO one ae Ne 125x 67 | 120x 67 | 125x 67 | 126x 67 | 126x67 | 139x 67 
ANSASER VENA es A | olka 134x067 | 125x 67 | 125x71 | 130x 67 | 126x 67 | 143 x 67 
SNP PEC acinar 115x 68 | 120x 71 125x71 | 139x 67 | 130x067 | 143x 67 
(0) Rene een cee ee a eee 137x 68 | 126x711 125x 71 | 147x 67 | 134x060 | 143 x 67 
Lie ete ee tae ake es 137x 68 | 125x73 | 125x71 |) 122x71) 118x71 | 147x 67 
Oe eh aie core Gx i 125 x73) (0 129% 71 | 126K 71 W122 Al 130 x 76 
Oe pots apa tns, Sanaa 138x 71 | 125x76 | 129x711 | 130x71 | 126x 71 | 130x 76 
OMe esas nee sae 125x76.| 125x 76 | 133x711 | 126x71 | 118x711 | 134x 76 
Average size of eggs. .| 127x 68 | 122x 69 | 129x 69 | 131x 68 | 126x 68 138 x 70 


during this study and the variability was found to be much greater than 
that indicated by Fischoeder or Krull. [xtreme variations in size are 
rare, but eggs as small as 105 by 55 » and as large as 155 by 76 » were 
found. The size of 100 eggs deposited by worms over 6 mm in length 
varied from 113 to 143 » in length by 66 to 76 » in width. The size varia- 
tion in these eggs which were deposited by worms that had been mature 
for several months was 30 » in length and 10 » in width. The average 
size of these eggs was 134 by 69 pm. 

Table 2 presents data on the size of eggs produced by small, medium, 
and large individuals. The eggs produced by the two smaller worms 
averaged 124.5 by 68.5 » and were much more variable in size than the 
eggs produced by either the medium or large worms. The average size 
of the eggs produced by the medium-sized worms was 130 by 68.5 « while 
that for the largest worms was 132 by 69 pw. It is possible to conclude 
from these data that the average size of eggs produced by young worms 
is less than that of older ones and that egg sizes tend to become more 
uniform as age increases. 

Further study of Table 2 indicates that individuals tend to produce, 
on an average, either small or large eggs but not both. The smallest 
worm, shown in column 1, produced eggs slightly larger than the worm 
shown in column 5. Their bodies were 3.7 by 1.3 mm and 9.0 by 3.0 mm 


14 ILLINOIS BIOLOGICAL MONOGRAPHS 


respectively. On the other hand, the largest worm (column 6), which 
measured 9.0 by 3.5 mm, produced the largest eggs, and one of the 
smallest worms (column 2), which measured 4.5 by 2.2 mm, produced 
the smallest eggs. 

The average size of all these eggs in Table 2 is 129 by 68 p, which is 
only a little less than that of the eggs from worms which were on an 
average of much larger size. This tends to support the statement that 
individuals produce either small or large eggs but not both. 

The extreme range of variation for this group of eggs is 32 p in 
length and 18 » in width. Then by taking the extremes shown in Table 2 
the egg size for this species is found to be from 115 to 147 yw long by 
58 to 76 w wide, while the average size of the eggs is 129 by 68 p. This 
is approximately the same as the averages given by both Fischoeder and 
Krull. The slightly smaller average is possibly due to the inclusion of 
measurements made on eggs produced by very small worms. 


MIRACIDIUM 


DEVELOPMENT 


The development of the miracidium has been described for very few 
trematodes, and the descriptions which have been made vary considerably 
in their completeness. An accurate description of this stage in trematode 
development is comparatively difficult because of the minuteness and 
indefiniteness of the miracidial organs. Such a study necessitates both 
living and fixed materials which must be studied at very short intervals 
to determine the embryological sequence of organ development. Living 
material is sometimes difficult to study because of the opaque shell or 
the enclosed vitelline mass. Fixed materials are also difficult to study 
since this involves the fixation of embryos at known stages of develop- 
ment, sectioning and staining, followed by intensive study of the material 
under high magnification. Consequently only a few authors have at- 
tempted to describe this stage in the life history of trematodes. 

The most complete studies of this nature were made by Thomas 
(1883) on Fasciola hepatica; Looss (1892) on Diplodiscus subclavatus ; 
Looss (1896) on Gastrothylax gregarius, Gastrodiscus aegyptiacus, and 
Paramphistomum cervi; Ortmann (1908) on Fasciola hepatica; Johnson 
(1920) on Echinostoma revolutum; Stunkard (1923) on undetermined 
species of Spirorchis; Barlow (1925) and Ishii (1934) on Fasciolopsis 
buski; and Suzuki (1931) on Fasciola hepatica. Of these workers, 
Ortmann and Ishii used sectioned and living material while the others 
made their studies from living material only. 

The similarity of the results obtained by these authors as to the se- 


LIFE HISTORY OF COTYLOPHORON—BENNETT 15 


quence of organ development is remarkable. Quite naturally, however, 
the time of appearance of organs varies considerably because of the 
difference in time required for the miracida to develop under natural or 
experimental conditions. The slight variations found to occur in the 
sequence of organ development in these different species of trematodes 
may have several explanations: first, the difficulty with which such 
minute structures are recognized in either living or sectioned material; 
second, the almost simultaneous appearance of some organs; and, third, 
the lack of accurate, detailed observation. 

In the present work, the development of the miracidium of C. cotylo- 
phorum was studied in living material only. The eggs used in making 
these observations were secured by taking adult worms from the host and 
placing them in dishes of water where they would deposit eggs for several 
hours. The worms were removed before they died and the water was 
decanted. The eggs were then washed in several changes of water in 
order to remove as much débris as possible. It was found that if any 
animal tissues were left in the dishes bacteria would destroy a large per- 
centage of the eggs within a few days. This was demonstrated by allow- 
ing the eggs and worms to remain in the same dish until the worms had 
begun to decompose. In such instances only about ten per cent of the 
eggs would reach the hatching stage. In order to secure the highest 
percentages of hatching it was found necessary to change the water on the 
eggs at least twice each day. When the eggs were thus properly cared for 
about ninety per cent of them would hatch. 

Time Required for Hatching—Many eggs were obtained throughout 
the period from August 12, 1933, to June 22, 1934, and a record was kept 
as to the minimum time required for hatching (Table 3). The eggs se- 
cured between August 12, 1933, and January 4, 1934, were kept at 
laboratory temperatures, but the time required for the eggs to hatch in- 
creased as the winter advanced, due to the fact that laboratory tempera- 
tures fluctuated with outside temperatures, except during the day when 
the laboratory was heated. Between January 6 and February 23 the 
eggs obtained were subjected to outside temperatures at all times and 
none of these eggs hatched. The eggs obtained between March 28 and 
June 26 were again kept at laboratory temperatures, but throughout this 
period laboratory temperatures fluctuated day and night with outside 
temperatures. , 

No controls were established or temperature records kept for these 
egg-hatching experiments but the data indicate that temperature condi- 
tions are of primary importance in determining the time required for the 
eggs to hatch. The time required steadily increased from 15 to 29 days 
as the temperatures became lower from August 12, 1933, until February 


16 ILLINOIS BIOLOGICAL MONOGRAPHS 


23, 1934. During the period from January 6 to February 23 no eggs 
reached the hatching stage when exposed to outside temperatures due, it 
is believed, to the fact that freezing temperatures occurred a number of 
times. The eggs in this experiment would begin to develop, some de- 
veloping as far as the ciliated stage. None was observed which had 


TABLE 3.—RESULTS OF EGG-HATCHING EXPERIMENTS 


Showing the minimum time required for eggs of Cotylophoron cotylophorum to 
hatch at different times of the year 


‘ Days : Ay Days 
Deposited Hatched elapsed Deposited Hatched alates d 

Auge l2eyocas Aug. 27 15 Jan. 26.......| none hatched 
Aug. 17....... Aug. 30 13 Reb oalie race none hatched 
Aug. 18.2004 3% Sept. 1 14 Feb. 6....... none hatched 
AUG RZ OC eas Sept. 10 19 Keb: 132.2 none hatched 
Aug. 31... .< 006. Sept. 21 22 Feb. 20...... none hatched 
Octy23 ana s aa: Nov. 20 28 Feb. 23...... none hatched 
Octo 312s 2s 1! Nov. 22 23 Mar. 28...... Apr. 24 DY 
Nove link. fac Nov. 23 24 Apres, whee: Apr. 25 21 
NOVs 82.0808. Dec. 2 24 Apres Once. oa. Apr. 27 | 
Now [Sv a. Dec. 14 29 Apres 923 cea May 2 23 
Nov. 24....... Dec. 20 26 Apr. 17.2.3... May 9 22 
Dec. 6........ Dec. 31 25 Apr. 20...... May 11 21 
Dec. 20....... Jan. 14 28 Apra20. aya. May 16 20 
Jan. 3-....02.5 Feb. 1 29 May 29...... June 15 18 
Vanh 4G: Feb. 2 29 June 8....... June 20 12 
Jan Ors eaee none hatched June 22). 2.2... July 3 11 
Jan. 1965 2% none hatched June 26......| July 8 11 
Jan. 24....... none hatched | 

Average Time Elapsed...... 21 


developed beyond this condition, although living embryos were found as 
long as 35 days after the beginning of the experiment. The time required 
for the eggs to hatch during the period from March 28 to July 8 de- 
creased from 27 days at the beginning to 11 days at the end of the experi- 
ments. This decrease in time follows steadily the increase in temperature. 

In this series of experiments the time required for the eggs to hatch 
varied from 11 days during one of the warmest periods of the year to 
29 days during one of the coolest periods, even though the eggs were kept 
at laboratory temperatures. The average time required for all the eggs to 
hatch in this series of experiments, which is overbalanced in number in 
the cooler months, is 21 days. In those experiments in which no eggs 
reached the hatching stage the temperature dropped below freezing for 
a part of the time. Consequently, the conclusions which may be drawn 
are that temperature is of great importance in determining the time 
required for the eggs to hatch and that freezing temperatures are 
fatal to them. 


HIFE HISTORY OF COTYLOPHORON—BENNETT 17 
Developmental Rate—A study of the rate of development of C. 
cotylophorum miracidia was made on many of the eggs used in the hatch- 
ing experiments, and the results of those experiments show that the 
developmental rate is influenced directly by temperature. However, only 
the minimum time required for hatching is given in Table 3. The ma- 
jority of the eggs will hatch within a few days after the minimum time 
but there are others which do not complete their development until months 
afterwards. One egg culture in which hatching began on January 14 was 
kept in order to determine the time required for all of the eggs to hatch. 


TABLE 4.—-DEVELOPMENTAL RATE OF MIRACIDIA OF Cotylophoron cotylophorum 
Based on measurements (in microns) of different individuals at four-day intervals 


No. April 4 April 8 April 12 | April 16 | April 20 | April 25 

(La eee eee 25:% 20 55 x 38 90 x 43 154x38 | 169x 29 
1) Ae ara? SI 25°x 25 55 x 42 65x46 | 160x38 153 x 32 
Sen cee eee e = 25x25 59 x 42 69x46 | 101 x42 189 x 34 
Acca oasareeie ies: Ow 29x25 59 x 42 101x46 | 105x 42 197 x 34 
eee nee oe ce 25x25 59x 42 79x47 | 118x42 | 189x 36 
Ope nore 4 29 x 29 63 x 42 90 x 47 143 x 42 168 x 38 
Dire ene tics On 29 x 29 50 x 46 97 x47 103 x46 | 182 x 38 
Senge, ce ee E 29 x 29 59 x 46 72x49 | 113x460 | 193 x38 
ee ee ee a 29 x 29 71x 46 72x49 105x50 | 180x42 
WO recs caees. 34 x 34 62 x 50 80 x 50 55x55 | 21063 
PAIVEV OP Cenierreeieinr- steel ok 28 x 26 59 x 44 82x47 116x 44 184 x 39 


This culture was examined from time to time and after an interval of five 
months occasional developing embryos could be found. Other cultures 
were kept for varying lengths of time and this condition was observed 
in all of them. The cause of this slow development was not determined. 

The seemingly inherent variation in the rate of development and that 
induced by changing temperature makes the age of the embryo an unre- 
hable standard for determining the time of appearance of the structures 
of the fully developed miracidium. However, if the age and size of a 
sufficiently large number of embryos of any developmental series are 
combined a fairly reliable standard is obtained. This method was used in 
preference to following the development of one embryo. In this way the 
average rate of development can be determined, and at the same time 
the appearance of organs can be correlated with the age and size of any 
one individual. 

While the developmental rate of the miracidium was studied in many 
eggs, only the results of observations made on one developmental series 
will be discussed. This series represents the average minimum time of 
development required by all of the eggs obtained. In making this study 
the eggs were observed when deposited, and cleavage was followed for 


ILLINOIS BIOLOGICAL MONOGRAPHS 


18 


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LIFE HISTORY OF COTYLOPHORON—BENNETT 19 


several hours. The eggs were then studied at four-day intervals until 
hatching began. Measurements were made on ten embryos selected at 
random at the end of these intervals in order to obtain data on the rate 
of growth. In addition, at the end of each interval many embryos were 
studied in an attempt to determine at what size and age the various 
organs of the miracidium made their appearance. 

The data obtained on growth rate and the size at which organs can 
first be recognized in the embryo are presented in Tables 4 and 5. 


From Deposition To END oF FourtH Day 


Cleavage.—Eggs are usually deposited before cleavage begins, but 
occasionally they are deposited as far advanced in cleavage as the four-cell 
stage. The ovum or the embryo is usually situated slightly anterior to the 
middle of the egg, entirely surrounded by the vitelline cells, although it 
is sometimes peripheral in position. 

The early stages of cleavage in various trematodes were described as 
being unequal by Ortmann (1908) and Ishii (1934). Thomas (1883) 
and Johnson (1920) do not describe cleavage, but their figures show that 
the early cleavage stages result in cells of equal size. Suzuki (1931) 
figures cleavage as being very regular through all of the early stages. 
Looss (1896) and Barlow (1925) discuss the early cleavage stages but 
neither their discussions nor figures throw any light on the subject. 
Stunkard (1923) does not mention these early stages. 

The first cleavage of the ovum in this species results in two cells 
slightly unequal in size. As a result of the second cleavage there are one 
large and three small cells, one of which is considerably larger than the 
other two (Fig. 12). These stages occur in most of the eggs 12 hours 
after being deposited, but some have advanced to the eight-cell stage at the 
end of this time. It was impossible to determine accurately the size of 
the cells in the eight-cell stage due to the surrounding vitelline cells. 


Size of Embryo.—The increase in size of the embryo is slight during 
the first four-day period of incubation. The size of 10 embryos at the end 
of this period is given in Table 4, column 2. At this age and size the 
embryo appears as a rounded, semi-transparent ball of cells, in which 
the nuclei vary from 3 to 5 » in diameter. It is clearly delimited from 
the enclosing yolk material. 


Four To E1icut Days 


Sizge.—There is a marked increase in size at the end of the second 
four-day period, as indicated in Table 4, column 3. Some of the embryos 
have reached their maximum width but none of them have structures 


20 ILLINOIS BIOLOGICAL MONOGRAPHS 


which could be identified with certainty, other than the nuclei mentioned 
as being present in the earlier stages. 

Vitelline Cells——Up to this point in development there is very little 
change in the appearance of the vitelline cells. The original outlines of 
the cells are still distinct but there are fewer granules in them, giving 
them a more hyaline appearance. 


Eicut To Twetve Days 


Size.—Between the eighth and twelfth days a difference in rate of 
development becomes very evident. The largest embryo observed on the 
twelfth day measured 101 by 42 », while the smallest measured 65 by 46 p, 
which is somewhat smaller than the largest embryo recorded at the end 
of the eighth day. However, the average rate of increase in size is com- 
parable to that of the second four-day period. The majority of the 
embryos have reached their maximum width by the twelfth day and many 
structures have made their appearance. 

Cilia.—The cilia are the first structures developed which can be defi- 
nitely recognized, being seen on an embryo which measured 76 by 46 p 
(Table 5; Fig. 5). The size of the embryos on the twelfth day (Table 4, 
column 4) indicates that many of them possessed cilia on the ninth or 
tenth day of development while there were some which had not developed 
them by the twelfth day. 

The presence of the cilia is evidence that the epithelial cells have 
developed in an earlier stage but neither the nuclei nor cell boundaries 
could be seen at any stage in development, although the anterior and 
posterior limits of the cells could sometimes be seen in optical section 
at the lateral limits of the embryo. 

Primitive Gut.—Following the cilia in development are the so-called 
primitive gut and two flame cells which appear at approximately the 
same time. These structures were first observed in an embryo which 
measured 80 by 46 » (Fig. 9). The primitive gut at this stage consists of 
two large cells filled with granular material similar to that present in the 
primitive gut of the fully developed miracidium. The cells measure 17 by 
11 » and are located in the center of the body, 9 » from the anterior end. 
The nucleus of each cell is 6 » in diameter and contains a large chromatin 
knot which is 2 » in diameter. The flame cells, which are located laterally 
and slightly posterior to the middle of the body, measure 5 by 3 p. The 
ducts leading from them could not be seen. 

The exact size of the embryo at which the two primitive gut cells 
divide to form the four cells characteristic of it in the mature miracidium 
was not determined but the four-cell condition was found in an individual 


LIFE HISTORY OF COTYLOPHORON—BENNETT 21 


which measured 90 by 42 » (Fig. 2). In this individual the primitive gut 
had begun to elongate, appearing similar to that in the fully developed 
miracidium. On the other hand, some much larger individuals (Fig. 10) 
possessed a gut much less advanced in development. With the appearance 
of the gut or shortly afterwards the apical papilla can be distinguished 
as a small, non-ciliated projection at the anterior end. 


Muscle Tissue-—The muscle layers of the miracidium were not seen 
in these early stages but their presence is denoted by movement. In this 
series no movement was detected in any embryos under 90 by 43 uy, 
although it was seen in embryos of other series at a size of 85 by 45 yp. 
Movement in these earlier stages is very infrequent and consists of very 
slow and slight contractions of the anterior half of the body. 


Sube pithelial Tisswe-—The subepithelial layer (Fig. 9) became dis- 
tinguishable from the other tissues of the body during the period between 
the eighth and twelfth days. Not all of the nuclei of this layer can be 
seen, but some of them can be seen easily in optical section. They may 
be recognized by their characteristic ovoid shape and their position imme- 
diately beneath the ciliated epithelium. The nuclei, which are the only 
criteria by which this layer may be recognized, measure approximately 
oiby 3.p. 

Vitelline Membrane.—According to Ortmann (1908) and Ishii 
(1934), working on different species of trematodes, the vitelline mem- 
brane is formed by cells which break away from the embryo during the 
early developmental stages and migrate to the periphery of the vitelline 
mass where they flatten, unite, and eventually enclose the entire vitelline 
mass and the embryo. In the eggs of this series the vitelline membrane 
was not clearly distinguishable prior to the twelfth day. Its recognition 
depends upon its withdrawing from the eggshell at some point, and 
this point is always the opercular end of the egg (Figs. 2, 3). It is then 
seen as a very thin membrane. The space left at the opercular end of 
the egg is filled with a clear liquid at first, but it is finally occupied by a 
viscid, granular mass called a “mucoid plug” by Barlow (1925). 

Vitelline Cells—There is no marked change in the appearance of the 
vitelline cells or masses during the earlier developmental stages. There 1s, 
however, a gradual decrease in the number and an increase in the size of 
the masses. Perhaps this is due to the breaking down of the original 
masses and a subsequent coalescence of the liquids contained in them, 
There is also a gradual decrease in the number of vitelline granules. 

The most extensive changes, in the series under discussion, occurred 
between the eighth and twelfth days when the vitelline masses were 
broken down rapidly until there were left only a few relatively large 
masses. However, there is no uniformity in these changes. The condition 


22 ILLINOIS BIOLOGICAL MONOGRAPHS 


is true for some embryos while others of approximately equal age and 
size still have a large number of small vitelline masses (cf. Figs. 2 
and 11). 

The cilia do not break down the yolk masses in this species as they 
do in Fasciolopsis buski Barlow (1925). The cilia are very seldom in 
motion until late in development and could scarcely be of any service in 
this respect. However, since they do break down shortly after most of the 
miracidial structures are present, it is possible that the movements of the 
embryo and perhaps some secretion produced by the embryo hasten this 
process. 


Nerve Tissue.—The nervous system develops simultaneously with the 
primitive gut and the flame cells. [t consists of many cells located on the 
dorsal surface of the body between the gut and the flame cells (Figs. 9, 
10, 11). The nuclei are the only structures which can be seen distinctly, 
although there is a clear area ventral to them which probably represents 


the early stages of the fibrous brain. 


Germinal Tissue-—The primordial germ cells can be recognized in 
embryos as small as 80 by 46 ». They are massed together in the 
posterior third of the body, the only distinctive feature being the large 
nuclei characteristic of these cells. The nuclei measure 4 to 5 w in 


diameter and are surrounded by very small amounts of cytoplasm. 


Mucoid Plug.—The mucoid plug is not formed until after the embryo 
has acquired most of its organs. It was first seen in an embryo which 
measured 90 by 42 » (Fig. 2), where it appeared as a granular, trans- 
lucent mass at the opercular end of the egg. Barlow (1925) states that 
this mass is formed only at the opercular end of the egg and that perhaps 
it prevents embryonal secretions from loosening the operculum. This is 
not true in the present species since the plug may develop at either or 
both ends of the egg (Figs. 3, 6, 7). It becomes so viscid as develop- 
ment proceeds that only by extremely vigorous movements can the 
miracidium indent it. When present at the anterior end of the egg it 
forms an effective barrier between the miracidium and the operculum 
which has to be removed before the miracidium can hatch. I believe that 
this mucoid plug is nothing more than concentrated waste material 
excreted by the miracidium. Its positions in the egg and the fact that it 
does not appear until after the flame cells begin to function tend to 
support this belief, as does the fact that this plug increases in size and 
viscosity as the embryo develops. Furthermore, the concentration of this 
mass outside the vitelline membrane indicates that the membrane is 
selective and prevents the embryo from being enveloped in its excreta. 


LIFE HISTORY OF COTYLOPHORON—BENNETT 23 


TWELVE TO SIXTEEN Days 


After the twelfth day the only other structures to make their appear- 
ance are the four penetration glands which were first seen in an individual 
122 by 46 » (Fig. 11). Between the twelfth and sixteenth days there is 
a considerable increase in length and a slight decrease in width of the 
embryo (Table 4, column 5). Some of the embryos become longer than 
the egg during this period, and the posterior end of the body is flexed to 
provide for further increase in size (Figs. 3, 4, 6, 7). 

The vitelline masses are reduced in number until there are only two 
large bodies which partially enclose the embryo. These are kept pressed 
tightly against the embryo by the vitelline membrane but move freely 
when the embryo moves. A few scattered vitelline granules are still 
present at the end of this period. 


SIXTEEN TO TWENTY-ONE Days 


Between the sixteenth and the twenty-first days there was a remark- 
able increase in growth. The average size of 10 embryos on the sixteenth 
day was 116 by 44 p, while the average size of 10 embryos on the twenty- 
first day was 184 by 39 ». There was an average decrease of 5 » in width 
and an average increase in length of 58 ». The position of the miracidium 
in the egg immediately prior to hatching is shown in Fig. 7. 

The development of the miracidium as described here coincides in 
practically every detail with the development of the miracidia described 
by the workers mentioned earlier in this discussion. This result points 
to the conclusion that the chronological sequence of organ development in 
trematode miracidia is essentially the same. 


HATCHING 

The process of hatching in C. cotylophorum was found to be more 
complicated than has been described for most trematode eggs. Barlow 
(1925), in describing the hatching of the eggs of Fasciolopsis bushi, 
states that the glands of the miracidium are of importance in effecting its 
release. My observations on the present species fully support this view. 

No criterion was discovered which would seem to indicate exactly 
when the miracidium begins its hatching activities, but such efforts con- 
tinue for approximately 48 hours. The mucoid plug is located at the 
opercular end of the egg in almost every case and is the first obstacle 
which has to be removed. This plug may reach a thickness of 34 ,, so 
crowding the miracidium in the remaining space that its body is bent 
almost double. No effort was made to follow in detail the formation of 
the structure, but, as previously stated, it makes its appearance after the 


24 ILLINOIS BIOLOGICAL MONOGRAPHS 


flame cells begin to function, and it is probably formed from waste 
materials concentrated outside the vitelline membrane. If this is true 
then the plug would increase in size until the miracidium begins to 
destroy it. If it were possible to determine at just what moment the 
miracidium starts to do this then it would be possible to determine at 
what moment hatching efforts begin. 

The plug is removed or destroyed at an extremely slow rate. A num- 
ber of miracidia were observed for as long as four hours each and in no 
case did one make any measurable progress through it. However, it is 
easy to find all stages of destruction of the plug in an egg culture in which 
miracidia are hatching. 

The initial efforts consist of applying the tip of the apical papilla to 
the flat base of the plug, usually near its center, and then strongly con- 
tracting the circular muscles of the body. This gives the impression that 
the miracidium is attempting to push the plug out of the egg or to one 
side. It is most probable that such contraction stimulates glandular secre- 
tions and aids in forcing out the secretions. The body may contract at 
any point but usually the strongest contractions occur at the base of the 
papilla and at the junction of the epithelial plates. During such contrac- 
tions the ducts of the glands become more prominent than at any other 
time. 

The cilia apparently are of little service in hatching, although some- 
times they beat energetically as the miracidium applies its apical papilla 
to the plug. The short, stiff cilia present on the anterior half of the first 
tier of epithelial plates seem to serve as a brush. At times the miracidium 
slightly withdraws the apical papilla and twitches its body from side to 
side, which may brush off parts of the plug loosened by its secretions. 
The miracidium is not continuously active, each period of muscular 
activity being followed by a slightly longer period of quiescence. Neither 
of these periods exceeds more than two minutes. 

The plug is entirely removed after an undetermined length of time, 
no trace of it being found in eggs in which the miracidium is in contact 
with the operculum. The apparent necessity for completely removing 
this plug was demonstrated in one instance. In attempting to orient an 
egg under a cover slip by tapping gently on it with a needle the operculum 
was slightly loosened and a drop of water entered the egg. The plug 
immediately expanded until it filled approximately two-thirds of the egg 
and crushed the miracidium (Fig. 8). The fact that the operculum does 
not open as soon as the miracidium comes in contact with it is attributed 
to the possibility that it is cemented shut by some secretion of either the 
adult worm or the embryo. Many miracidia which were undergoing 
strong muscular contractions in an apparent attempt to liberate themselves 


LIFE HISTORY OF COTYLOPHORON—BENNETT 25 


were observed for varying lengths of time. Occasionally one would be 
successful. The operculum springs back allowing water to flow in. This 
seems to stimulate the miracidium to vigorous activity. However, it takes 
the miracidium only a few seconds to find the opening and it immediately 
begins to squirm through. The fact that the operculum remains closed, in 
some instances for hours, after the miracidium comes in contact with it 
points directly to the conclusion that it is eventually loosened by glandular 
secretion and not by intermittent muscular activity. 

The opercular opening is considerably less in diameter than the body 
of the miracidium and it takes miracidia from five seconds to twelve 
minutes to get out of the egg. The apical papilla, which is narrow, goes 
through the opening readily but the remainder of the body passes through 
in some instances only after a prolonged period of incessant activity. 
Some swim out immediately and some were observed swimming with 
the eggshell still attached to the posterior end. 

The miracidia do not rotate or turn around in the egg under normal 
conditions but they do so when the opercular opening becomes filled with 
débris and they are unable to penetrate it. Under such conditions they 
remain in constant activity until death ensues. In all egg cultures about 
two per cent of the embryos were found developing in a reversed position. 
The mucoid plug in all such cases observed was present at the opercular 
end of the egg, but the hatching efforts of the miracidium in each case 
were directed against the end opposite the operculum. None was ever 
observed to turn around. 


Mature MIRACIDIUM 


General Activity.—The miracidia hatch throughout the day and night 
but the numbers hatched between 8:00 and 11:00 a.m. are so much 
greater that the hatching may be considered periodic. Between 3:00 and 
5:00 p.m. large numbers are hatched also, but relatively much fewer than 
during the morning hours. They can be induced to hatch in considerable 
numbers at any time by stirring the egg culture while under a strong 
light. Their positive phototropism is evidenced by the fact that they 
always congregate on the lighted side of a container. 

After hatching the miracidia are extremely active. They usually swim 
in a straight line at top speed, but they follow a zigzag course at slower 
speeds. The body rotates in a counter clockwise direction. At other times 
it may contract in such a way that the anterior end of the body is pulled 
to one side and as a result it swims in a small circle. 

Shape and Size-——When swimming straight ahead the body is pyri- 
form, the greatest diameter being one-fifth of the body length from the 
anterior end. From this point the body tapers sharply anteriorly to 


26 ILLINOIS BIOLOGICAL MONOGRAPHS 


terminate in the small blunt apical papilla, while posteriorly the decrease 
in size is much more gradual, leaving the posterior end bluntly rounded 
(Figs 17): 

The ability of the miracidium to change its shape is quite marked. At 
slow rates of speed it swims along alternately contracting and extending 
its body, and when stopped it may contract to such an extent that it has 
the appearance of a slightly ovoid ball. When the anterior end is con- 
tracted the apical papilla projects from the bottom of a conical depres- 
sion formed by an invagination of the body. The miracidium very often 
swims along in this contracted state alternately thrusting out and with- 
drawing the papilla while the short cilia present at the anterior end are 
beating rapidly. The initial impression given is that the miracidium is 
feeding, because particles in the water are swept down into the funnel- 
shaped cavity toward the tip of the papilla, but nothing was ever ob- 
served to enter the gut. 

If no host is available to the miracidium it dies after 8 or 10 hours. 
When near death it becomes greatly distended and moves very slowly. 
The body becomes vesicular as the tissues begin to break down and the 
epithelial plates swell away from the body, but the cilia remain active for 
some time afterward. Motion finally ceases except for a very slow turning 
around in a small circle, which continues until the epithelial plates break 
away completely from the body. As the internal tissues break down, the 
nuclei float free and are concentrated in one or more groups within the 
body. 


4 


General Description—Descriptions of the morphology of fully de- 
veloped miracidia have been made for comparatively few species of 
trematodes and many of these are not complete. In view of the limited 
number which have been described in detail it is difficult to judge the 
completeness of any of the present descriptions. The miracidium of C. 
cotylo phorum possesses a ciliated epithelial covering, a primitive rhabdo- 
coel gut, penetration glands, an excretory system, reproductive tissue, and 
a nervous system. 

The description of the miracidium of C. cotylophorum presented here 
is based on a study of living specimens unstained and stained intra vitam, 
toto preparations, and sectioned material. 

Size.—The exact size of living miracidia is very hard to determine 
because of their incessant activity. However, an attempt was made to 
measure them by using the hanging drop method. To prepare a hanging 
drop, one or two miracidia were pipetted onto a cover slip and the excess 
water was removed. A few cotton fibers were added to the remaining 
water. The cilia of the miracidium become entangled in these fibers and 
hold it in a relatively stable position without distortion of the body. It 


LIFE HISTORY OF COTYLOPHORON—BENNETT 2 


continues to contract and extend itself but at intervals it becomes motion- 
less. The size of miracidia measured in this way was found to vary from 
153 to 210 » in length and from 32 to 63 » in width; the average size of 
10 was 164 by 39 ». An attempt was made to measure them first alive 
and then fixed, but this was unsuccessful. The vapors of warmed Bouin’s 
and Fleming’s fixatives were employed. The contraction of the miracidia 
fixed in this way was always abnormally great when compared with those 


TABLE 6.—MEASUREMENTS OF FIXED MIRAcIDIA (IN MICRONS) 


No. Length Width No. Length Width 
ieee eae yis doeite 143 38 1 ar eae 168 a 
71 ght Gece 151 38 UO crake ees 134 38 
See aa 155 38 1G egesce seer 160 34 
i) Se ee Fee 164 38 Ager ee eras Cerra 164 38 
Dyciicrtes reas nny aie 168 38 i: ee ee ee ee 151 42 
Oh thle ath 197 38 LOT erate tomar 164 34 
1 Re eee 143 38 DO tie a hall Seana 181 38 
Blerteageovtoutier ies 168 29 4 edo at eananeaee 176 38 
Oi ETE fe ise 139 42 DW Matai Be we 159 34 
(0 Ease meee eee 172 42 DS shat ea dBi 160 38 
(TL Seles lesa aoe oe 147 46 LL re ee 147 34 
i, Oe ree eee 151 50 DD Bs costa a ek Rican 168 34 

3 Bea. Be Sra 168 46 
Average..... 159.8 38.2 


fixed by other methods. The size of 25 miracidia fixed by flooding them 
with warmed Bouin’s fixative is given in Table 6. The range in size of 
these fixed specimens was 134 to 197 » in length by 29 to 50 p» in width, 
with an average size of 159.8 by 38.2 ». Others were fixed in warmed 
Fleming’s fixative but there was no perceptible difference in the results 
produced by the two fixatives. 


E pithelium.—With the exception of the apical papilla the outer surface 
of the body is covered by flattened, ciliated epidermal cells (Fig. 14). 
There are 20 of these cells arranged in 4 rows or bands which completely 
encircle the body. The anterior, or first, series consists of 6 cells, the 
second of 8, the third of 4, and the fourth of 2. Expressed by formula 
the arrangement is 6;8 ;4;2, in which the first number represents the most 
anterior row of cells. 

The 6 cells of the anterior group cover the first fifth of the body, 
terminating posteriorly at the widest point in the body. The shape of the 
anterior part of the body is such that each cell of this group is wide at its 
posterior border and narrows gradually toward the anterior end, which 
lies at the base of the apical papilla. These cells are slightly thicker than 
the cells of the second group and consequently project above them very 
noticeably, and in slightly contracted specimens may overlap them for a 


28 ILLINOIS BIOLOGICAL MONOGRAPHS 


short distance. The thickness of the anterior cells is approximately 3 p 
while that of the second group is 1.5 to 2 ». The cilia present on the 
anterior group vary from 2 » in length at the anterior tip of the cells to 
12 » at their posterior borders. The increase in length of the cilia is very 
gradual. Lynch (1933:15) states that the short cilia present at the an- 
terior ends of these cells are stiff and motionless in the miracidium of 
Heronimus chelydrae, but they are movable in the present miracidium. 
As pointed out in the discussion on the hatching of the miracidium of 
C. cotylophorum, they are directed anteriorly during the hatching process 
but in free-swimming specimens they were observed to beat in the same 
manner as the other cilia. 

The cells of the second group are rectangular and extend slightly past 
the middle of the body. The cells of the third group are also rectangular 
but are much broader, due to the fact that there are only 4 cells in this 
group and the body is only slightly smaller than in the region of the 
second group. The 2 cells in the posterior group cover the last fifth of the 
body and have a triangular shape because of the body form. Their broad 
ends are directed forward while the tapered ends cover the tapering 
posterior end of the body. All the cells of the 3 posterior groups are very 
uniform in their thickness, which is from 1.5 to 2 p». The cilia also are 
very uniform in length, being approximately 12 in length on all of these 
cells. Each cilium is rooted in a distinct basal body. These bodies may 
be seen in living specimens as rows of very fine dots. Similar basal bodies 
have been described for the cilia of Fasciola hepatica by Ortmann (1908: 
270; fig. 34a). The absence of cilia between the epithelial cells has been 
noted by most authors, but Talbot (1933:524; fig. 1) states that cilia 
cover the entire body of the miracidium of Lechriorchis primus. The 
spaces between the cells of the present miracidium are very narrow (Fig. 
14), being from 1 to 2 », but the spaces between groups of cells are very 
distinct and can be seen especially well in optical sections of living 
miracidia. These spaces are most evident between the cells of the first 
and second groups because of the greater thickness of the first tier of 
cells. Because of this thickness the cilia on the first tier of cells project 
further from the body, and in swimming specimens they have the appear- 
ance of a mantle or epaulets and for this reason this region of the body 
is sometimes designated as the “shoulder” of the miracidium. 

Ciliated plates or cells have been observed by other investigators on 
miracidia but the numbers of cells apparently are not the same even 
within a single species. A survey of the literature summed up in Table 
7 shows the counts for different miracidia. Looss (1892: pl. 19, fig. 17) 
figured these cells on the miracidium of Diplodiscus subclavatus. He also 
figured them as being present on the miracidium of Gastrothylax 


LIFE HISTORY OF COTYLOPHORON—BENNETT 


2) 


gregarius (1896: pl. 12, fig. 121) and Gastrodiscus aegyptiacus (1896: 
pl. 12, fig. 123) but did not give the number of cells present in these 
forms. However, in all of them he placed nuclei representing 4 tiers of 
cells, and it 1s very probable that the number and arrangement of cells 


TABLE 7.—SHOWING THE ARRANGEMENT AND NUMBER OF THE CILIATED EPIDERMAL 


CELLS IN MIRACIDIA 


Family 


Genus and species 


Paramphistomidae 


Echinostomidae. . . 


Strigeidae........ 


Schistosomatidae. . 


Fasciolidae....... 


Troglotrematidae......... 


Heronimidae...... 


Paramphistomum 
cervt 

Cotylophoron 
cotylophorum 

Diplodiscus 
temperatus 


Hypoderaeum 
conoideum 

Echinoparyphium 
recurvatum 

Echinostoma 
revolutum 


Strigea tarda 
Diplostomum 
flexicaudum 


Schistosomatium 
douthitts 


Fasciola hepatica 
Fasciola hepatica 
Fasciola hepatica 
Fasciola halla 
Fasciola califor- 
nica 
Fasctoloides 
magna 
Fasciolopsis 
buski 


Paragonimus 
westermannt 


Heronimus 
chelydrae 


Arrange- 

Author and ment and Total 

date number of cells 

cells 
Sinitsin 1931 | 6;6;3;4;2 21 
Bennett 1936 | 6;8;4;2 20 
Krull and 6;8;4;2 20 
Price 1932 
Mathias 1925 | 6;6;4;2 18 
Rasin 1933 6;6;4;2 18 
Beaver 1936 | 6;6;4;2 18 
Mathias 1925 | 6;8;4;3 21 
Van Haitsma | 6;8;4;3 21 
1931 
Price 1931 6;834;3 21 
Thomas 1883 | 4-5;5-6;3;4;2] 18-20 
Coe 1896 6;6;3;4;2 21 
Ortmann 1908} 6;6;3;4;2 21 
Sinitsin 1931 | 6;6;3;4;2 21 
Sinitsin 1931 | 6;6;3;4;2 21 
Sinitsin 1931 | 6;6;3;4;2 21 
Barlow 1925 | 6;6;6;6;6 30 
Ameel 1934 6;6-733;1 16-17 
Lynch 1933 4-6;6-10; 16-22 
3-6;1-2 


in each miracidium is 6;8;4;2. Unfortunately, he did not figure the 
nuclei of these cells in the miracidium of Paramphistomum cervi (1896: 
pl. 12, fig. 125). Sinitsin (1931) has given the epidermal cells of the 
miracidium of P. cervi as 6;6;3;4;2, which is very different from the 


number of cells described as being present in other miracidia belonging to 


30 ILLINOIS BIOLOGICAL MONOGRAPHS 


the family Paramphistomidae. It is probable that Sinitsin was mistaken 
in the number of epithelial cells in this form, doubtless being influenced 
by the number of cells present in the other miracidia which he has appar- 
ently described correctly. We may then assume that the number of epi- 
dermal cells for the family Paramphistomidae may be expressed by the 
formula 6;8 ;4;2. 

The formula for the epidermal cells of the Echinostomidae miracidia 
based on the same number and similar arrangement which has been de- 
scribed for the different species may be expressed as 6;6;4;2. On a 
similar basis the formula for the Strigeidae miracidia may be expressed 
as 6;8;4;2, and for the Schistosomatidae miracidia as 6;8;4;3. The 
Fasciolidae miracidia formula might be expressed as 6;6;3;4;2 were it 
not for the extremely aberrant number of cells described for the 
miracidium of Fasciolopsis buski. The cells of the Troglotrematidae and 
of the Heronimidae miracidia are not uniform in number for the single 
species described for each of these families. 

Price (1931:703) has pointed out the possibility that the number and 
arrangement of the ciliated epidermal cells may be of some value in 
establishing relationships between families or larger groups. As she has 
said, the number of these cells possibly indicates relationship between the 
Strigeidae and the Schistosomatidae. Lynch (1933:10) has expressed 
doubt that these cells are of any value in determining relationships of 
various groups. However, the preponderance of the evidence points to the 
conclusion that the number and arrangement of these cells may be of 
value for establishing relationships within the families, and there is some 
evidence that it may be of value in establishing relationships between 
families. A final conclusion cannot be based on the small amount of 
information now available. 

The nuclei of the epidermal cells were studied in living specimens 
stained with intra vitam stains, in stained toto mounts, and in stained 
sectioned material. The nuclei of the first group of cells are irregularly 
cylindrical in shape and are located near the posterior borders of the 
cells. The nucleus in each cell measures 7 to 10 » by 2 to 3 p» in surface 
view. The shape of the nuclei of the second group of cells is so irregular 
that it may be described as being lobed. These nuclei also are located near 
the posterior borders of the cells and measure approximately 6 by 3 py. 
The nuclei of the third group of cells are similar in appearance to those 
in the first group, although they are somewhat larger, being 9 to 12 » by 
2 to 3 » in size. They are located very near the posterior boundaries of 
the cells. The nuclei in the last group of cells are found very near the 
anterior borders of the cells. These nuclei are larger than any of the 
others, measuring 12 by 3 p», although their shape is much the same as 


TIPE HISTORY OF COTYLOPHORON—BENNETT ot 


that of the nuclei in the first and third groups. The positions and shapes 
of the nuclei of all these cells as seen in cross sections are shown in Figs. 
18, 19, 20, 21. The thickness of the nuclei as seen in cross section is 
about 1 up. 

The nuclei of the epidermal cells have been described for only a few 
miracidia. Thomas (1883: pl. 2, figs. 5,6) figures them in the miracidium 
of Fasciola hepatica as round and located in the posterior part of the 
cells; Leuckart (1886:63; fig. 37) figures them as round and centrally 
located; Ortmann (1908: table 14; fig. 38) figures them as round in 
sectioned material and located toward the posterior border of the cells; 
Coe (1896:565) was the first author to describe in detail their shape and 
position. Sinitsin (1931:426; fig. 8) describes and figures these nuclei as 
being long and located at the posterior borders of the cells in the miracidia 
of Fasciola halt, F. californica, Fascioloides magna, and Param plvisto- 
mum cervi. Lynch (1933:20) saw them in the miracidium of Heronimus 
chelydrae as circular and flat in cross section. He could not see them in 
surface views and his statement as to their shape is open to question, 
since one might easily gain the impression that they are round when 
seen in sectioned material only. Krull and Price (1932:5; fig. 6) have 
described nuclei for the miracidium of Diplodiscus temperatus which 
closely resemble those of the miracidium of C. cotylophorum. The chief 
difference is that the nuclei of the second and third groups of cells are 
more anteriorly placed in the miracidium of D. temperatus. 


Sube pithelium.—The subepithelium is a thin, transparent layer located 
immediately beneath the ciliated epidermal cells and is continued forward 
at the anterior end to form the apical papilla (Fig. 17). The cell 
boundaries of this layer could not be determined but its extent is clearly 
indicated by the nuclei. The layer varies in thickness with the state of 
contraction of the miracidia, but in well extended specimens it is approxi- 
mately 5 w thick. It is somewhat less than this in the posterior region of 
the body, which is distended by the germ mass, and in the region of 
the body distended by the primitive gut and the brain. Slightly anterior 
to the middle of the body, between the brain and germinal tissue, there is 
an inward protrusion of this layer in which the flame cells are embedded. 
The nuclei are elongate or almost round and contain a number of small 
chromatin masses (Fig. 22) which aid in distinguishing these nuclei from 
the others of the body. The size of these nuclei is 4 to 6 uw by 3 to 4 pn. 
Krull and Price (1932:6; fig. 7) described and figured the nuclei of this 
layer as round and showed that they are arranged in 3 definite rows en- 
circling the body of Diplodiscus temperatus. No other authors have de- 
scribed the nuclei of this layer as round or attempted to demonstrate that 
they are arranged in definite positions in the body. 


32 ILLINOIS BIOLOGICAL MONOGRAPHS 


In the present work a careful study of these nuclei was made and it 
was found that they vary from elongate to round in shape and that they 
are distributed in 4 principal groups (Fig. 15). However, these 4 groups 
do not contain all of the nuclei. There are many nuclei situated irregu- 
larly throughout the subepithelium, and a careful count of all the nuclei 
indicated that the number is far from constant. The first group, which 
is located beneath the anterior limits of the second group of ciliated cells, 
consists of from 10 to 20, with an average of 16. The second group is 
located in the middle of the body and the number of nuclei varies from 
12 to 19, with an average of 15. The third group is located immediately 
beneath the termination of the third group of ciliated cells and variation 
in this group was found to be from 6 to 11, with an average of 8. The 
fourth group, consisting of from 2 to 4 nuclei, with an average number 
of 3, is located in the posterior extremity of the body. Some of these 
nuclei were occasionally seen undergoing mitosis, indicating still more the 
futility of attempting to determine the number of cells in the subepithe- 
lium. The presence of dividing nuclei in this layer indicates that the 
miracidium may grow while free-living but no experiments were made 
to determine the correctness of this supposition. 


Muscle Tissue.—The rapid and strong contractions and extensions of 
the miracidium indicate a well developed musculature. The muscles ob- 
served in this miracidium were the circular and longitudinal layers which 
are between the ciliated epidermal plates and the subepithelium. The 
layer of circular muscles is located external to the longitudinal layer and 
the two are pressed closely together (Fig. 23). The circular muscles can 
be seen in living specimens and sectioned material as minute parallel 
bands closely set together. A single band or fiber measures approximately 
1 » in diameter and the distance between fibers is about 1 p. 

The longitudinal muscles are slightly more developed than the circular 
muscles in the anterior region of the body. These muscles are arranged 
in parallel bands also. Each band measures approximately 1.5 pm in 
diameter, and the distance between bands is about equal to their diameter. 
The greater development of the fibers at the anterior end of the body is 
due, perhaps, to the fact that they serve to retract and extend the apical 
papilla and the anterior region of the body. 

The muscles in the posterior regions of the body are extremely diff- 
cult to demonstrate but their presence is indicated by the ability of the 
miracidium to extend and contract this region. The circular muscles can 
be seen in living and stained toto mounts and the arrangement is the 
same as in the anterior region of the body. The longitudinal muscles were 
never seen clearly in this region of the body. 

The arrangement of muscles in miracidia has been described by several 


LIFE HISTORY OF COTYLOPHORON—BENNETT Jo 


authors and there is close agreement between their descriptions. Ortmann 
(1908) describes and figures the muscles of the miracidium of Fasciola 
hepatica as having an arrangement very similar to that of C. cotylo- 
phorum. Looss (1892, 1896) describes and figures the muscles of the 
miracidia previously mentioned as being the same as in the closely related 
miracidium described here. Reisinger (1923:12; fig. 3) describes and 
figures the muscles of the miracidium of Schistosoma haematobium. He 
describes the circular muscles as bands 0.6 by 0.1 « placed at intervals of 
0.8 to 1.1 «. The longitudinal muscles are similarly arranged but measure 
only 9.5 » in breadth and are placed at intervals of 2 to 4 u. The muscles 
of this miracidium are much smaller than those of C. cotylophorum but 
the two agree as to arrangement. The retractor muscles described by 
Reisinger as being present at the anterior end of the body could not be 
seen. Ishii (1934:30) describes large muscle cells containing nuclei in the 
miracidium of Fasciolopsis buski but similar structures were not seen in 
the present material. 

Primitive Gut.—The structure usually called the primitive gut by 
writers in their descriptions of miracidia is a flask-shaped structure of 
variable proportions, depending on the state of contraction of the miraci- 
dium. In elongated specimens it may extend slightly posterior to the 
middle of the body, while in contracted specimens it becomes broadened 
and occupies all of the available space in the anterior fourth of the body. 
In specimens of average extension it extends almost to the center of the 
body. It terminates anteriorly in a narrow duct which does not open to 
the outside. The coarsely granular contents first appear when the gut 
consists of only two cells (Fig. 9). Later the two original cells divide 
and the four resulting cells apparently become confluent, because no cell 
boundaries can be found. The nuclei remain in the posterior part at all 
times and are surrounded by cytoplasm which stains more darkly than 
any other region of the gut. The granular contents move freely and 
completely fill the gut with the exception of the anterior tip of the duct 
near its termination in the apical papilla. The nuclei are easily recognized 
in all preparations of the miracidia because of their position and size. 
They measure 5 to 7 » in diameter and contain several small masses of 
chromatin, usually grouped together near the center of the nucleus. 

The development of the primitive gut at some distance from the 
anterior end of the body, the size of the cells and their nuclei, the early 
development of the granular contents, the absence of a definite cell wall 
around each nucleus after the four-cell stage is reached, the concentra- 
tion of cytoplasm around the nuclei at the posterior end of the gut, the 
absence of a mouth and a lumen, and the complete disappearance of the 
contents immediately after penetration of the miracidium into the snail 


34 ILLINOIS BIOLOGICAL MONOGRAPHS 


host while the nuclei may still be identified—all give evidence in favor of 
interpreting this structure as being a gland rather than a gut. 

The earlier writers—Schauinsland (1883), Thomas (1883), Leuckart 
(1886), Looss (1892, 1896), Coe (1896), and Ortmann (1908) as well 
as some of the later writers—Faust and Meleney (1924), Mathias 
(1925), Barlow (1925), Sinitsin (1931), and Van Haitsma (1931)— 
considered this structure as a primitive or vestigial gut. More recently, 
Reisinger (1923), Manter (1926), Price (1931), and Lynch (1933) have 
presented evidence in favor of considering this structure as a gland. An 
analysis of the opinions of these writers leaves some doubt as to the 
nature of this organ, but I believe, for the above reasons, that it is a 
gland and that it functions during the penetration of the miracidium into 
the snail. 

Penetration Glands.—These glands have been described for many 
miracidia, but within the family Paramphistomidae they have been ob- 
served only in the miracidium of Diplodiscus temperatus by Krull and 
Price (1932:7; fig. 7). Looss (1892, 1896) does not describe them for 
the miracidia of the forms previously mentioned, all of which belong to 
this family. Krull and Price found two pairs of these glands, a pair 
being situated on each side of the gut. In C. cotylophorum there are two 
pairs of these glands which are extremely hard to detect. However, they 
may be observed late in the development of the miracidium as well as 
in the mature specimens (Figs. 11, 17). They are filled with a clear non- 
granular substance which is difficult to stain. They extend posteriorly 
for about one-fifth of the body length from their openings at the base 
of the apical papilla. The nuclei of these cells, which are located near 
their posterior ends, measure 4 to 5 » in diameter. 

Nervous System and Sense Organs.—The nervous system consists of 
a central fibrous mass, nerves, and nerve cells. The central fibrous mass 
is located dorsal to the posterior part of the primitive gut, where it may 
be seen easily in both living and stained specimens surrounded by the 
nuclei of the nerve cells (Fig. 22). In mounted specimens the brain 
appears to be lateral in position, due to the pressure of the cover slip 
(Fig. 17). It is oval or quadrangular in shape when viewed from the 
dorsal side and measures approximately 20 by 25 p. It is from 14 to 16 p 
in depth and characteristically forms an indentation in the dorsal side of 
the primitive gut, which can be seen in lateral view. 

No nerves passing out from the brain could be seen in living speci- 
mens stained imtra vitam or in stained toto mounts. It was in sectioned 
material only that large fibrous structures resembling nerves were found 
arising principally from the lateral surfaces of the brain, although 
smaller fibers were found arising at various other points. Two large 


LIFE HISTORY OF COTYLOPHORON—BENNETT 3D 


fibers were/found passing obliquely forward from the brain to the lateral 
processes located between the first and second rows of ciliated epidermal 
cells (Fig. 22). While similar processes or nerves were observed to leave 
the brain from its posterior lateral surfaces, they could not be traced to 
their terminations. Neither could the smaller nerves be traced to their 
terminations, although finer striations observed in longitudinal sections of 
the anterior part of the body may have been nerves passing to termina- 
tions in this region. 

The nerve cell boundaries were not seen at any time but their nuclei 
are stained readily by imtra vitam stains and by other stains used on 
sectioned material. These nuclei are located in largest numbers immedi- 
ately anterior to the brain mass, although a few were found scattered 
completely around it. These nuclei were found to vary from 12 to 20 in 
number while the average number was 17. No nerve connections could 
be traced to or from them. In appearance they greatly resemble subepithe- 
lial nuclei but they are round and slightly smaller, and the dense chro- 
matin causes them to stain more darkly. They vary in size from 2.6 to 
3.4 pw in diameter. 

Looss (1896: pl. 12, figs. 119, 121, 125) has figured a central fibrous 
nerve mass surrounded by nuclei for the miracidia of Gastrothylax 
gregarius, Gastrodiscus aegyptiacus, and Paramplistomum cervi which is 
very similar to that of the miracidium of C. cotylophorum. He also ob- 
served anterior and posterior nerves arising from each side of the fibrous 
nerve mass in the miracidium of Gastrothylax gregarius. Krull and Price 
(1932) described a central mass for Diplodiscus temperatus but did not 
find the anterior and posterior nerves. However, they did describe 6 
nerve cells located anterior to the brain which had fibers connecting the 
brain and the tip of the apical papilla. These cells were not observed in 
the present species. Reisinger (1923:16; fig. 3) in his description of 
the nervous system of the miracidium of Schistosoma haematobium 
found structures similar to those found in the present material, as did 
Lynch (1933:22; fig. 9) in the miracidium of Heronimus chelydrae. 

The only sense organs observed on the miracidium of C. cotylophorum 
were a pair of structures which have been variously named anterior ducts, 
anterior papillae, mucoid secretion, lateral papillae, and lateral processes 
by different authors. These organs are located laterally between the first 
and second rows of ciliated epidermal cells (Fig. 14). Each papilla 
appears as a clear structure, approximately hemispherical in shape. It 
was difficult to make out any internal structures connected with these 
papillae but at times in living miracidia there appeared to be a duct 
extending inwardly and posteriorly from each papilla which seemed to 
terminate in a small vesicle in the region of the central nerve mass. 


36 ILLINOIS BIOLOGICAL MONOGRAPHS 


This structure is probably the large nerve previously described as passing 
from the brain to the lateral papilla. 

Cort (1919:516) and Faust and Meleney (1924:28) called these 
papillae anterior or lateral ducts and stated that the extrusion of sub- 
stances from these structures was observed. Stunkard (1923:183) made 
similar observations on the miracidia of Spirorchis. The present observa- 
tions agree with those of Sewell (1922:287) who found no openings in 
these structures in the miracidia of Cercariae Indicae xv. Price (1931: 
705) made similar observations on the miracidia of Schistosomatium 
douthitti. Living miracidia of C. cotylophorum were observed for great 
lengths of time and even when subjected to pressures great enough to 
break the body walls no substance was observed to be extruded. 

Looss (1896: fig. 125) figures but does not discuss two small lateral 
papillae on each side of the miracidium of Paramphistomum cervi. \rull 
and Price (1932: fig. 7) figure one lateral papilla on each side of the 
miracidium of Diplodiscus temperatus but they show no structures in 
connection with them nor do they suggest the possible function of these 
papillae. 

That these structures are sensory in Schistosoma haematobium was 
clearly demonstrated by Reisinger (1923) who has described and figured 
nerves passing from the central nerve mass to them. Lynch (1933:31; 
fig. 9) has demonstrated in a similar way that these papillae are sensory 
in the miracidium of Heronimus chelydrae. The present observations 
agree in detail with those of these two workers and I believe that these 
structures are sensory. 

Many other structures designated as sensory in function have been 
described for other species of miracidia by Coe (1896), Ortmann (1908), 
Price (1931), Reisinger (1923), and Lynch (1931) but none of these 
were found in the present material. 

Germinal Tissue.—The germinal tissue was studied from its first 
appearance in the developing embryo and the conclusions arrived at are a 
result of detailed study on living material, stained toto mounts, and 
serial sections. In the youngest forms in which the germinal tissue could 
be distinguished it had a homogeneous translucent appearance. Nuclei 
4 to 5 » in diameter were present. As the miracidium develops some of 
the germ cells break loose into the central cavity. One large and several 
small germ balls usually are present when the miracidium is hatched, al- 
though some are hatched in which no definite germ balls have developed. 
In the latter cases there are many germ cells which seem to be free in 
the central cavity. The posterior three-fifths of the body is completely 
filled by the germinal tissue in the fully developed miracidium. 

There is a pronounced difference in the appearance of this tissue and 


LIFE HISTORY OF COTYLOPHORON—BENNETT of 


the enclosing subepithelium in stained specimens. The nuclei of the 
subepithelium of this region are ovoid and do not stain as darkly as do 
the round nuclei of the germinal tissue. No cell boundaries could be 
distinguished but the limits of the germinal tissue could be established 
by the numerous large, closely packed nuclei, the granular appearance of 
the tissue, and its much greater affinity for stains than the subepithelial 
layer. The germinal nuclei, representing the germ cells, form a thick 
layer in the posterior extremity of the body and are located also around 
a central cavity which extends forward past the middle of the body (Fig. 
16). These nuclei, which measure 4 to 5 » in diameter, have numerous 
small scattered chromatin masses and one large centrally located mass. 
The amount of cytoplasm surrounding each of these nuclei is very small. 
After being liberated into the central cavity the germ cells develop into 
germ balls by unequal cleavage (Fig. 17). Early in cleavage a thin 
membrane encloses the developing germ ball. This membrane is present 
around each of the germ balls and it seems to develop from very small 
cells located in the periphery of the germ balls. Ortmann (1908:287) 
discusses a similar structure in the miracidium of Fasciola hepatica and 
Lynch (1933:29) has done the same for the miracidium of Heronimus 
chelydrae. The observations made by Looss (1892), Price (1931), and 
Lynch (1933) that the germ balls are held in position by fiber-like at- 
tachments were not confirmed in the present material. Germ cells located 
in the posterior part of the body at the end of the central cavity were 
observed to be attached to the lateral germinal areas by extensions of 
the cells but no connections were found for the cells which were free in 
the cavity or for the germ balls. 

Excretory System.—The excretory system is very similar to that de- 
scribed in miracidia of many species. It consists of two laterally situated 
flame cells and their ducts (Fig. 15). The flame cells are located in the 
previously mentioned protrusion of the subepithelial layer, immediately 
anterior to the middle of the body. Each flame cell measures approxi- 
mately 10 by 3 ». Reisinger (1923) in his detailed study of the excretory 
system of the miracidium of Schistosoma haematobium states that a flame 
cell nucleus is lacking. There are several nuclei embedded in the tissue 
surrounding the flame cells of the present material, but since cell 
boundaries were not seen no nucleus could be definitely associated with 
the flame cell. However, a nucleus was found in close proximity to the 
basal plate of the cell (Fig. 13) which is probably directly associated 
with it. The excretory tubule for each flame cell extends posteriorly in 
loose coils almost to the excretory pore. Here it loops back to the flame 
cell where it again turns on itself and extends to the excretory pore 
which is located laterally between the third and fourth epidermal plates. 


38 ILLINOIS BIOLOGICAL MONOGRAPHS 


Immediately before reaching the pore the tube is expanded to form a 
small excretory bladder. Krull and Price (1932:8; fig. 8) describe two 
duct nuclei for each of the tubules in the miracidium of D. temperatus, 
but these nuclei could not be found in the miracidium of C. cotylophorum. 


INTERMEDIATE HOST 
DETERMINATION OF THE Host 


Two methods were used in determining what snail or snails would 
serve as the intermediate host of C. cotylophorum. The first consisted of 
searching known foci of infestation for infested snails, and the second 
consisted of exposing several species of snails to the free-swimming 
miracidia followed by dissection of the snails after an interval of 10 or 
15 days to discover whether or not there were any developmental stages 
present in them. 


Natural Infestation —The distribution of naturally infested snails 
depends entirely upon the distribution of infested carriers of the adult 
worm. Consequently, in order to determine what region of the country 
surrounding Baton Rouge, Louisiana, had the largest number of carriers, 
many of the cattle slaughtered at the city abattoir during the period from 
June 6, 1933, to June 27, 1934, were examined for these worms. When 
an infested cow was found, the range from which it came was located 
and searched for the intermediate host of C. cotylophorum. It soon be- 
came evident that the worms were more abundant and more frequently 
found in cows which came from the low, semi-swampy ranges located 
south and east of the city. Occasionally cows from the hilly ranges north 
of the city were found to be infested and upon examination of the ranges 
from which these came it was found that they had access to either a small 
pond or lake or to a stream which ran through open fields. It was not 
until May 26, 1934, that a natural infestation was found in specimens of 
Fossaria parva taken from the margin of an artificial pond southeast of 
Baton Rouge. 

Experimental Infestation.—The presence of strongly developed cilia 
combined with the swimming ability of the miracidium gave basis for a 
surmisal that the intermediate host was either an aquatic or amphibious 
snail. Several genera of snails are commonly present in or around 
streams, lakes, and ponds in this region. Prior to the finding of naturally 
infested intermediate hosts, snails were collected from an area semi- 
circular in shape with a radius of approximately 20 miles, being taken 
in every instance from a range on which the cattle were known to be 
infested with C. cotylophorum. The snails were examined first by placing 


LIFE HISTORY OF COTYLOPHORON—BENNETT 39 


them in water to determine whether or not fully developed cercariae were 
present, and then by dissection for the earlier developmental stages. 
When found not to be infested others were then exposed to free-swim- 
ming miracidia. The snails used in these experiments were Physa halei, 
Helisoma lentum, Succinea retusa, Succinea unicolor, Fossaria parva, 
Fossaria modicella, and undetermined species of Physa and Campeloma. 
Of these only Fossaria parva and F. modicella could be infested 
experimentally. 


F. modicella was found in only one locality near Baton Rouge, 
Louisiana, late in the spring of 1934, and consequently is not used in the 
following discussion on the development of larval stages of C. cotyloph- 
orum. Snails of this species collected from near Urbana, Illinois, by 
the writer and from Turkey Run State Park, Indiana, by Dr. H. J. Van 
Cleave were infested experimentally also. Krull (1934:171) secured this 
same species from Utah and was able to infest these snails with the 
miracidium of C. cotylophorum. F. parva was found frequently and 
doubtless is the species which serves most often as the intermediate 
host of this parasite in the vicinity of Baton Rouge. 


BroLocy oF Fossaria parva 


Fossaria parva is a small amphibious snail which rarely exceeds 7 mm 
in length. It is commonly found near the margins of small ponds, lakes, 
and streams where there is some decaying vegetation which it uses for 
food. The snails were rarely found in the water, and they are very help- 
less when caught by any current, being carried along until they drift 
against some object. They were never found in areas which were shaded 
at all times. The normal habitat is a well moistened area which is subject 
to direct sunlight for the greater part of the day. 

The snails were found in largest numbers along the margins of an 
artificial pond which served as a source of drinking water for cattle, and 
along the margins of a large unshaded drainage ditch. Observations 
were made at intervals on the snails present in the drainage ditch. They 
were first located in August, 1933, and were present in large numbers 
on the moist area at the edge of the water. As the water receded during 
dry periods it was followed by the snails and when the ditch became 
completely dry the snails burrowed into the mud where they remained 
until water was again present. Snails were collected in large numbers 
from this ditch during the autumn and early winter months but none 
could be found after December 26, 1933. Observations were continued 
through January and February, 1934, but no snails were seen before 
March 3. At this time a single specimen was found after an hour of 


\ = 


40 ILLINOIS BIOLOGICAL MONOGRAPHS 


searching. One week later 4 specimens were collected. These specimens 
were all large. By the latter part of March these older specimens were 
found frequently and very young snails were observed in steadily 
increasing numbers during the month of April. 

The first snails used for experimental purposes were collected during 
September, 1933, from the previously mentioned drainage ditch which 
was not accessible to carriers of the adult worms. However, many of 
the snails were examined for developmental stages but not a single 
naturally infested snail was found throughout the months in which col- 
lections were made from this locality. These snails were placed in 
aquaria which were prepared to approximate as nearly as possible the 
natural habitat of the snails. Large and small galvanized tin tubs were 
filled with dirt taken from the natural habitat and this dirt was banked 
to one side so that when water was added there was a dry area at the 
top of the bank, a moist area along the water’s edge, and then water 
which was kept at a fairly constant level. To make the habitat still more 
natural, grasses, weeds and aquatic vegetation were planted in the aquaria. 
These aquaria were placed against the southern wall of a large building 
where they were exposed to the sunlight for the greater part of each 
day. They remained uncovered except during rains. To supplement the 
food supply planted in the aquaria, lettuce, cabbage and cauliflower leaves 
were added when needed. None of the vegetation available to the snails 
was eaten until it was partially decayed. The snails kept under these 
conditions seemed to live as well as in their natural habitat. The rate 
of reproduction was rapid and a constant supply of laboratory raised 
snails was available after the first few weeks. Under these conditions 
the snails did not hibernate during the colder months, i.e., January and 
February, and there was some reproduction although less than in the 
warmer months. The eggs are usually deposited at the edge of the water 
but are sometimes found in moist depressions some distance from the 
water. 

The snails were very seldom found in the water or at the dry top 
of the bank, preferring in the aquaria the same habitat as in nature. 
Usually when found in water they are attached to some piece of decaying 
vegetation. 

Penetration Experiments.—The snails were infested by placing them 
singly in small glass dishes containing from 3 to 5 miracidia or by ex- 
posing a large number of snails to hundreds of free-swimming miracidia 
in large containers. The miracidia are attracted to the snails almost im- 
mediately, but apparently penetration takes place slowly and only after 
prolonged exposure of the snails to them. Observations were made many 
times on the reactions of the miracidia but none was ever observed to 


LIFE HISTORY OF COTYLOPHORON—BENNETT 41 


penetrate the snail. In experiments with only a few miracidia in a 
dish with a single snail they have been observed for as long as 3 hours 
and at the end of this time all of the miracidia were still present in the 
dish. Possibly the constant activity of the snail during observation under 
the microscope and the abnormal conditions under which the miracidia 
_are placed prevented them from entering the snail. The percentages of 
infestation were always low when the snails were exposed to only a few 
miracidia, usually about 10% becoming infested. The method of exposing 
snails to many miracidia resulted in 100% infestation in most cases, but 
there is danger of over-infestation. However, it was found that the best 
results could be obtained by exposing the snails in this way for a period 
of 2 to 4 hours. In earlier experiments snails were left overnight in 
dishes containing many miracidia, but the subsequent death rate among 
the snails was so great that only an occasional individual would live long 
enough to shed cercariae. 

The natural inclination of the snails to leave the water adds to the 
difficulties in securing 100% infestation. To secure the best results in 
this respect it was found necessary to place in the containers some food 
material which served to attract the snails into the water. Under these 
conditions the snails remain relatively motionless, giving the miracidia 
ample time and opportunity to penetrate them. The miracidia collect at 
the anterior ends of the snails where they can be seen attaching them- 
selves to the head, foot, and mantle, and at times to the shell. Their at- 
tachment usually is very brief, as they are shaken off by slight movements 
of the snail or they release themselves. They may then attach themselves 
again at the same point, select another, or swim away, finally becoming 
attached to another host. At times the snails seem to have no attraction 
at all for the miracidia. In such cases miracidia were observed swimming 
in close proximity to several snails but never made any attempt to pene- 
trate them. In view of some of the recent observations on immunity of 
infested snails this failure of miracidia to penetrate might be due to 
earlier infestation by this same or other trematode larval forms, but 
extended observations failed to yield any evidence of natural infestation 
in the stock of snails used in these experiments. Experiments also 
demonstrated that these snails do not become immune to infestation by 
the miracidia of C. cotylophorum when previously infested by this same 
species. Snails containing larval stages as much advanced as mature 
rediae were exposed to miracidia, and large numbers of the latter pene- 
trated and began development. Miracidia were observed collecting around 
snail faeces and débris scraped from the bodies of the snails. These sub- 
stances produced reactions in the miracidia alike in all respects to those 
produced by the snails themselves. Apparently the attraction was as 


42 ILLINOIS BIOLOGICAL MONOGRAPHS 


strong as that of the snails since the miracidia continued to collect around 
them even though there were many snails in the container. 

The penetration of the miracidia into the snail host was never ob- 
served, even though many hours were spent in attempting to do so. 
However, Krull (1934:176) observed that this miracidium penetrated the 
head and mantle of Fossaria modicella within 15 minutes. In order to 
determine the point of entrance in the present experiments a few medium- 
sized snails were exposed to large numbers of miracidia for 6 hours and 
were then fixed. Upon sectioning and staining these snails many miracidia 
were found to have penetrated all the exposed surfaces of the snail 
but principally the mantle and dorsal surface of the foot. 

The habitat of F. parva and the fact that it feeds on moist decaying 
vegetation at the edge of the water, where eggs deposited in the faeces 
of cattle would remain viable, led to experiments to determine whether 
or not ingested eggs would hatch in the intestine and the miracidia pene- 
trate the intestinal wall. Some small snails were placed in a dish con- 
taining eggs almost ready to hatch which were readily eaten by the 
snails. A few of the snails were fixed immediately after eating the eggs, 
while the rest were kept for 24 hours before being fixed. It was noticed 
that some of the eggs were passed in the faeces of the snails unhatched 
and unharmed since many of them were observed to hatch subsequently. 
The fixed snails were sectioned and stained, and a careful search was 
made for miracidia which might have penetrated the wall of the digestive 
tract at any point, but none was found. 

Numerous experiments were made during the fall and winter months 
of 1933 with very young, medium-sized, and old snails and it was found 
that the miracidia infested all ages equally well. However, in these ex- 
periments large numbers of miracidia were used and as a result most of 
the snails died before cercariae were shed. The young snails when 
severely infested usually die within 15 to 20 days while the older ones 
live until the liver is completely destroyed by developing cercariae. In 
the young snails which died only sporocysts and young rediae were found. 

Relation of Temperature to Development.—One group of snails in- 
fested on October 5, 1933, shed cercariae on November 17, after a lapse 
of 44 days. A second group infested on October 31 shed cercariae on 
December 23, after 54 days, and a third group infested on December 
20 shed cercariae on March 30, 1934, after 91 days. The time required 
for development increased as the winter months advanced. Late in the 
spring and in the early summer months of 1934 another series of experi- 
ments was performed and in this series the time required for develop- 
ment decreased as the temperature rose. Snails infested on April 25 


LIFE HISTORY OF COTYLOPHORON—BENNETT 43 


shed cercariae on June 1 (37 days); a second group infested on April 
26 shed cercariae on June 2 (37 days) ; a third group infested on May 5 
shed cercariae on June 5 (31 days); a fourth group infested on May 
11 shed cercariae on June 12 (32 days); a fifth group infested on 
May 15 shed cercariae on June 14 (30 days) ; and a sixth group infested 
on June 23 shed cercariae on July 24 (31 days). The results of these 
experiments are summarized in Table 8. 


TABLE 8.—DaATA SHOWING TIME REQUIRED FOR DEVELOPMENT OF CERCARIAE OF 
Cotylophoron cotylophorum AT DIFFERENT TIMES OF THE YEAR 


Date on which 


No. Date of infestation Pi CA aE Te Days elapsed 
Ree teen x ce eenege ce ee Oct. 5, 1933 Nov. 17, 1933 44 
DEN Ys: ain Siateconerae een Oct. 31, 1933 Dec. 23, 1933 54 
ce cichaceg tied taayat a Dec. 20, 1933 Mar. 30, 1934 91 
Cm A Lis or Ga rarer ade Apr. 25, 1934 June 1, 1934 37 
SR ies «is sine a ae cesake Apr. 26, 1934 June 2, 1934 37 
Cio oer ie yo knee May 5, 1934 June 5, 1934 32 
dhs. chastat a casei aieraecs 8a May 11, 1934 June 12, 1934 32 
Rent ae cen ae aera May 15, 1934 June 14, 1934 30 
0) eae a apne eeietea rera arerae June 23, 1934 July 24, 1934 31 


These data show that the effect of temperature on the rate of de- 
velopment of the stages in the snail host is very comparable to the effect 
on the rate of development of the miracidium. The time required for the 
development of the miracidium increased from 11 days in the warmer 
months to 29 days during the colder months of the year. In the same 
way the time required for the development of the cercariae increased 
from 30 to 91 days. Krull (1934:174) found that snails infested with the 
miracidium of C. cotylophorum on May 14 began to shed cercariae on 
June 19, after a lapse of 36 days. This result agrees very closely with 
the foregoing since snails infested on May 15 began to shed cercariae 
on June 14, representing a difference of only 6 days from Krull’s data. 
Krull performed only the one experiment so that no other comparisons 
can be made. Suzuki (1931:97) performed similar experiments on the 
development of Fasciola hepatica and obtained results comparable to 
those presented here. He found that a period of 30 days was required 
for the development of these stages during the summer months of July 
and August but that from 60 to 70 days were required during the winter 
months of January and February. 

The data secured on the time required for the development of the 
cercariae during the warmer months indicate that 30 days is approxi- 
mately the minimum time required. The average time required for de- 


44 ILLINOIS BIOLOGICAL MONOGRAPHS 


velopment of the cercariae during these warmer months was 33 days. 
The rate of development of the sporocyst, redia, and cercaria was studied 
in all the experiments shown in Table 8—by dissection of snails at in- 
tervals, and also by fixing, sectioning, and staining snails. In this way 
it was possible to determine at what time the various developmental 
stages make their appearance. The following discussion is based on the 
development of these stages in snails infested on May 5. Cercariae were 
shed by these snails on June 5, after a period of 32 days. 


SEOROCYs |, 


DEVELOPMENT 


Method of Study.—The earlier stages in development of the sporo- 
cyst were studied from sectioned material only. The minuteness of these 
stages combined with the opacity of the snail shell made accurate 
observations of living material impossible. To obtain representative 
stages some of the snails were fixed at 12 hours, and others after 1, 2, 
5, 10, 15, 20, 25, 30, and 35 days from the time of exposure to miracidia. 
In order to supplement the data acquired from sectioned material many 
snails were dissected after the fifth day of development, at the ends of the 
given periods and at intervals not indicated, in order to study the living 
torms. 


Sporocyst from Penetration of Miracidium to End of 12 Hours.— 
As has been stated previously, the miracidia were not observed to pene- 
trate the snail host, but it was possible to determine the points of en- 
trance from sectioned material. The miracidia were never observed to 
shed the ciliated epidermal cells and evidence from sectioned individuals 
within the snails clearly demonstrated that the epidermal cells are present 
after penetration. In several instances miracidia were found in the 
lymph spaces of the foot and in the body cavity, with all or a part of the 
cells still attached (Figs. 24, 29). The exact time at which they are lost 
was not determined but there is no evidence of their presence 12 hours 
after penetration. The cells are sloughed first from the anterior end of 
the body as indicated by the fact that many individuals were found on 
which only the last one or two tiers were present. The opposite condition 
was never observed. Looss (1892:156) observed that the ciliated cells 
of the miracidium of Diplodiscus subclavatus were retained for 24 hours 
and until it had reached the liver or ovo-testis. The miracidium of P. 
cervi according to Looss’ description (1896:186) does not lose its ciliated 
cells until after penetration and changes accompanying transformation 


LIFE HISTORY OF COTYLOPHORON—BENNETT 45 


into the sporocyst have begun. Takahashi (1928:278) made similar 
observations on the miracidium of P. cervi. Thomas (1883:114; Fig. 7) 
observed the same phenomenon in the miracidium of Fasciola hepatica. 
He states: 

The outer layer of ciliated cells is lost, whilst the embryo changes in form. The 
ciliated cells absorb water and appear as round or hemispherical vesicles with the 
cilia standing out perpendicularly from their surface... 

He does not state how soon after penetration these cells are lost, and it 
was not determined for the present miracidium. However, they were 
seen only on miracidia which had undergone the least change at the 
end of 12 hours after penetration. Doubtless these changes are initiated 
immediately after the miracidium gains entrance into the snail. 

Ameel (1934:289) observed the penetration of the miracidium of 
Paragonimus westermanm but could not determine whether or not these 
cells are lost although Nakagawa (1917:302) noted that the cells are lost 
during penetration. Mathias (1925:44; fig. 9a) has described and figured 
the loss of these cells during the penetration of the miracidium of Strigea 
tarda. Barlow (1925:34; text-fig. 6) made similar observations on the 
miracidium of Fasciolopsis buski as did Ishii (1934: figs. 1, 2, 3) for the 
same form. Rasin (1933:102) observed that the epidermal cells are 
shed by the miracidium of Echinoparyphium recurvatum before entrance 
into the snail host. In view of these observations it is only possible to 
conclude that the ciliated epidermal cells are lost by some miracidia 
during penetration but are carried into the snail host by others. 

The appearance of the miracidium is not greatly altered for some time 
after penetration, many of the structures being still recognizable. The 
structures most altered in appearance are the primitive gut and the brain. 
No contents can be seen in the gut even in miracidia which have just 
penetrated the snail, as indicated by their very superficial positions. 
The nuclei of the gut are recognizable in some miracidia but they dis- 
appear before the end of the first 12 hours after penetration. The brain 
degenerates rapidly, not being definitely recognizable in any miracidia 
after penetration. In a number of miracidia a small round structure was 
observed, occupying the position of the brain in the free-swimming forms, 
which might have been the brain in an advanced state of degeneration. 
This structure measured 12 p» in diameter. 

The nuclei of the penetration glands were not seen in miracidia after 
penetration, but occasionally individuals were seen in which the ducts of 
the glands were very conspicuous. In some individuals at the end of the 
first 12 hours the anterior part of the body possesses no recognizable 
structures other than a few nuclei (Fig. 29). 

The appearance of the subepithelial cell nuclei remains the same 


46 ILLINOIS BIOLOGICAL MONOGRAPHS 


_ through the first stages of transformation but there is a great reduction 
in number. The central cavity of the miracidium remains and one or 
two germ balls and many germ cells are present in it. The excretory 
system is unchanged from that typical of the miracidium. 

As transformation takes place the miracidium loses its elongate shape 
and becomes gradually more ovoid. A very thin cuticula is formed around 
the outside of the body. Accompanying the change in shape and the 
loss of miracidial organs is a decided decrease in size. The size of 10 
sporocysts which were approximately 12 hours of age is given in Table 9. 


TABLE 9.—SHOWING THE SIZE (IN MILLIMETERS) OF 12-HOUR AND 
24-HouR SPOROCYSTS 


No. 12 Hours No. 24 Hours 

Pee ns Pe 0.070 x 0.035 Lege ata ees eee 0.077 x 0.024 
a es ae eGR One 0.054 x 0.032 Ee Oe oe Te 0.069 x 0.038 
ered ease eter 0.052 x 0.037 a eg ee ere se 0.069 x 0.033 
Oe SEE eRe grea 0.052 x 0.025 Age 2G ae: Oe agen 0.069 x 0.024 
Dist Retr Aten 0.048 x 0.032 Di scott eel aa ee 0.064 x 0.030 
Cede he ate es eatin 2 0.046 x 0.031 Giiaus 3 flee 0.061 x 0.032 
Des ecis aia dere diet res aerate 0.046 x 0.024 Liege nee fe eee 0.061 x 0.030 
Sitarscscamhe veto ae aoeee 0.045 x 0.034 | a a se aac re, 0.061 x 0.026 
Desk eee eee 0.045 x 0.029 ae Rm ce Pa 0.061 x 0.023 
it Da ge Ree eee ee 0.039 x 0.032 LOn Ao Dhaene eee ces 0.060 x 0.018 

AVETO BES: Mics ties Riwcade 0.050x 0.031 FT RUT Tae a ea 0.065 x 0.028 


Barlow (1925:34) observed that the sporocysts of Fasciolopsis buski 
never became immobile and that the digestive tract became larger and, 
functionally more active as the sporocysts grew in size and that they 
could be observed feeding at all times. Before rediae were born he found 
that the sporocysts had migrated well into the body of the snail. These 
observations could not be confirmed in the present material. The positions 
in which the sporocysts develop indicate that some of the miracidia either 
swim in the body fluids or are passively carried by movements of the 
liquids in the cavities of the snail’s body. However, they become per- 
manently located soon after penetration. In some instances young sporo- 
cysts were found free in the body spaces surrounding the radula and 
esophagus, and subsequent findings indicate that sporocysts develop at- 
tached to the walls of this cavity. Other young sporocysts were observed 
in all parts of the foot, including the center of this muscle mass, which 
further demonstrates that some movement from the point of penetration 
does occur. The sporocysts apparently prefer the mantle tissues to any 
other tissues of the snail’s body since more of them develop in this region 
than elsewhere in the body. 


LIFE HISTORY OF COTYLOPHORON—BENNETT 47 


24-Hour Sporocyst.—The initial rate of development is very slow 
when the size of the sporocyst is taken as a criterion. Perhaps this is 
due to the fact that an almost complete transformation occurs and to the 
fact that the sporocyst must become established before it receives ade- 
quate nourishment to provide for rapid growth. There are two notable 
changes which occur by the end of the first 24 hours in the snail. The 
first is the initiation of growth, as shown in Table 9, and the second is 
the breaking down of the germ balls which were present in the miracidium 
(Fig. 28). The germ cells, separated from each other by the breaking 
down of the germ balls, are scattered in the central cavity, almost oc- 
cluding it. No cell boundaries could be distinguished, thus giving the 
body the appearance of a syncytium. The germ cell nuclei measure from 
4 to 6 » in diameter and have lost the appearance characteristic of these 
nuclei in the miracidium. The chromatin is no longer concentrated in 
one central body surrounded by distinct masses but is uniformly scat- 
tered throughout the nucleus. Suzuki (1931: figs. 12, 13) has figured 
a similar stage in the young sporocyst of Fasciola hepatica. Mathias’ 
(1925:44) description of this stage of development of the sporocyst of 
Strigea tarda is not complete but apparently it is very similar to that of 
the present material. Brooks (1930:302; fig. 1) has described and 
figured these scattered germ cells in the young sporocyst of Cercaria 
lintoni Miller which he designates as “antecedent germ cells.” 

Several elongate nuclei which measure 4 by 2 » were observed near 
the periphery of the body. These are probably subepithelial nuclei. The 
anterior end of the body at this age appears as a translucent, granular 
mass in which remains of miracidial structures can be seen occasionally. 

48-Hour Sporocyst.—After two days in the snail host the sporocyst 
no longer has any trace of the miracidial structures, although the an- 
terior end of the body is still filled with a translucent tissue in which 
only an occasional nucleus is located. There is a decided increase in size 
over the 24-hour stage (Table 9). The most important development is 
that of the embryonic rediae. The origin of these was not established 
but it is possible that they are derived from the germ cells liberated by 
the breaking down of the miracidial germ balls, the “antecedent germ 
cells’ of Brooks. The “germ mass” and “components” described by 
Brooks as being derived from the “antecedent germ cells” in the sporocyst 
of C. lintoni were not observed in the sporocyst of C. cotylophorum. The 
fact that the structures seen in the 48-hour sporocyst are embryonic rediae 
and not “germ masses” is clearly demonstrated by their subsequent 
development. 

The size and number of young rediae present in 48-hour sporocysts is 
shown in Table 10. At a very early age these embryos have a definite 


48 ILLINOIS BIOLOGICAL MONOGRAPHS 


TABLE 10.—SHOWING THE SIZE (IN MILLIMETERS) OF 48-HourR, AND 5-, 10-, AND 
15-Day SPpoRoOcYSTS, THE NUMBER OF REDIAE IN EACH, AND 
THE SIZE OF THE LARGEST REDIA IN EACH 


Age and No. Sporocyst Mom bee of Largest redia 
48 hours: 

Whe gina. ocala aires 0.070 x 0.038 1 0.023 x 0.016 
Dad pater deena Sateu Mean 0.070 x 0.053 1 0.015x0.015 
SS epee te clean 0.077 x 0.033 if 0.023 x 0.015 
Bose, wtietie manta smear! 0.082 x 0.043 2 0.025 x 0.018 
Dee Pier ey over 0.092 x 0.046 1 0.018 x 0.018 
Giana outa ee 0.100 x 0.046 3 0.021x0.015 
Ve iene ectuey Can eon 0.101 x 0.046 2 0.023 x 0.018 
Greet or cya teeta 0.107 x 0.053 3 0.021x 0.020 
Re Oe ne areas at 0.123 x 0.026 2 0.023 x 0.015 
DO i eae ace aee wear ae ese 0.123 x0.028 2 0.027 x 0.018 
PA DEVOGCS Scan oe nts 0.095 x 0.041 a 0.022 x0.017 

5 days 
Lei otrges as ate es 0.126x 0.084 3 0.042 x 0.042 
Det tate Sestak ve eos, 0.138x 0.061 3 0.053 x 0.043 
Oe a ec eee 0.161 x 0.053 3 0.046 x 0.046 
Cy ee a ee 0.168 x 0.097 4 0.063 x 0.050 
Be oe iecke ee Scenes 0.168x 0.105 5 0.067 x 0.054 
Oc eee cease 0.170 x 0.078 4 0.052 x 0.052 
(ee eet Ree 0.170'x0-110 5 0.069 x 0.053 
Sa. eo ere 0.200 x 0.086 4 0.058 x 0.058 
DT tare re accent aie aie 0.226x 0.084 5 0.073 x 0.047 
LOR ero ciotaeee 0.218x 0.092 5 0.080 x 0.063 
AUCH AGO er Acc eae: 0.181 x 0.085 ae 0.060 x 0.051 

10 days 
DFR ine eae ere 0.160 x 0.080 5 0.076 x 0.029 
Doig w tees snared Waa cena 0.218x 0.063 5 0.105 x 0.038 
Sit. oa agmere aan 0.260 x 0.134 5 0.105 x 0.055 
ee eer ee Pre 0.268x 0.151 5 0.139 x 0.092 
DS ac ice a eter ea anaes 0.294 x 0.105 8 0.151x 0.046 
Oins eecemen ce ae 0.302 x 0.100 5 0.134 x 0.088 
| ES ae ee he 0.336x 0.134 5 0.126x 0.084 
Ss drs dee acne eee eet a cae 0.395 x 0.189 5 0.168 x 0.050 
DP techs Syne es eee re 0.420 x 0.168 7 0.189 x 0.088 
SOA vide ohh is Sere 0.433 x 0.160 9 0.189 x 0.046 
AUERILDE airs oct ueon een 0.309 x 0.126 a 0.138 x 0.062 

15 days 
ear eee seer a 0.273 x 0.189 5 0.168 x 0.050 
Ditwimt cee ce eatt rer 0.294 x 0.160 6 0.197 x 0.055 
Ben eisende Co hea el aoe 0.315 x 0.156 5 0.216x0.061 
tate hoger en 0.315 x 0.210 6 0.193 x 0.046 
LL eee pe re 0.320x0.210 5 0.155 x 0.080 
CS orden ecten e a saea, 0.323% 0,151 ie 0.210x 0.050 
Des ovis a eae eiead Caer 0.370 x 0.181 8 0.168 x 0.063 
eee ee ee 0.376x 0.134 8 0.189 x 0.055 
D mraia aetinie ete ae ue BO 0.420 x 0.189 9 0.189 x 0.063 
LO sean se Se se ooeaer 0.470 x 0.285 8 0.225 x 0.080 
AVCLE IC Waals evchncteene ae 0.348 x 0.186 se 0.191 x 0.060 


LIFE HISTORY OF COTYLOPHORON—BENNETT 49 


shape and each is enclosed in a firm membrane which Dubois (1928:63) 
designates as a primitive epithelium (Fig. 31). In addition to the 
definitely formed rediae there are many germ cells present in the posterior 
part of the body. These cells measure 8 to 10 yw in diameter and have 
large nuclei which measure 6 to 7 » in diameter. The cytoplasm of these 
cells stains more darkly than the surrounding tissues in which cell bounda- 
ries are not distinguishable. The chromatin of the nuclei is arranged in 
one large body eccentrically placed and several small masses. Cleavage 
of these cells is unequal. 

Some of the 48-hour sporocysts show a definite central cavity but in 
others no cavity could be seen. The cuticula around the outside of the 
body is considerably thicker than in the 24-hour sporocyst. 


5-Day Sporocyst.—Between the second and fifth days the sporocyst 
and enclosed rediae increase very rapidly in size (Table 10). The central 
cavity becomes more distinct and the body walls become much thinner. 
At the two extremities the body is filled by a large number of cells, 
making the walls much thicker in these regions than they are laterally. 
In sectioned specimens the difference in thickness of these regions is not 
so evident as in living specimens. This is due to the fact that the de- 
veloping rediae are uniformly placed in an undisturbed sporocyst and 
keep it more extended than in a sporocyst dissected from a snail. When 
fixed quickly the sporocysts do not contract, but living specimens usually 
contract at both extremities, forcing the rediae to the center of the body 
(Fig. 25), and consequently the two extremities appear to be very thick- 
walled. The number of rediae in 5-day sporocysts is variable but is 
never found to exceed 5. The size of the largest redia in each of 10 
sporocysts of this age is shown in Table 10. No structures of the redia 
are recognizable at this stage in development. 


10-Day Sporocyst.—A very few of the sporocysts in this experiment 
reached their maximum size in 9 days and rediae were found free in the 
tissues of the snail. The size of 10 sporocysts and the largest redia in 
each is shown in Table 10. The time required for the sporocyst to reach 
this stage of development during the winter months was very much 
longer. In the experiment begun on December 20, 1933, no free rediae 
were found in the snails until February 2, 1934, representing a difference 
of 35 days required to reach similar stages. 

15-Day Sporocyst.—By the fifteenth day many rediae were found free 
in the snail, but as indicated in Table 10 only a small number of the 
sporocysts had reached their maximum size. 

An occasional sporocyst was found in snails as long as 35 days after 
infestation, which in this experiment was after cercariae were being shed. 


50 ILLINOIS BIOLOGICAL MONOGRAPHS 


These sporocysts were usually located in the foot where conditions were 
perhaps not as favorable for growth as in other parts of the body. Others 
were found in the anterior margin of the mantle. Many authors have 
reported extensive migrations by the sporocysts of other species of tre- 
matodes, but the sporocysts of the present species were never found 
posterior to the anterior margin of the kidney. In most instances the 
sporocysts were found completely surrounded by an unbroken layer of 
cells produced by the snail, which indicates the relative immobility of the 
sporocyst. 

The number of fully developed and developing rediae was never 
found to exceed 9 in a mature sporocyst (Fig. 35). Usually there are 
one or two ready to be liberated, while the remainder are in various 
stages of development. There is no birth pore in the sporocyst and the 
rediae can be liberated only by rupturing the body wall. The rediae most 
advanced in development are always located at the anterior end of the 
sporocyst, and it is this region of the body which is ruptured. A number 
of sporocysts were observed in which this rupture was evident. It was 
always at the extreme anterior end, and posterior to it the sporocyst was 
strongly contracted, causing the torn end to flare out. The constriction 
of the body prevents the less developed rediae from escaping. Thomas 
(1883:120) states that this constriction is maintained until the rupture 
is healed but this observation could not be confirmed. It is probable 
that the act of rupturing the body wall is initiated by the rediae, but 
observations made on sporocysts containing advanced rediae indicate 
that the sporocyst is an active participant in the process. When a redia 
is ready to emerge it moves or is forced into the anterior end by con- 
traction of the body wall. The sporocyst then contracts behind it forcing 
it strongly against the anterior end of the body, and in this way ap- 
parently takes a part in rupturing its own body wall (Fig. 36). 

Old sporocysts which have ruptured walls contain only a_ few 
rediae, some having been observed in which there were only 3 present. 
This fact, combined with the fact that they decrease rapidly in number 
in infested snails, points to the conclusion that each sporocyst will pro- 
duce a definite number of rediae. For this species the number is 
probably 9. 

Excretory System.—The excretory system of the sporocyst consists of 
the two original flame cells and their ducts present in the miracidium. 
The flame cells are readily visible at all times in the living specimens and 
it is quite easy to trace the ducts. These structures increase in size as the 
sporocyst develops, the flame cells reaching a size of 15 by 5 yw. The 
course of the ducts in the young sporocyst is exactly the same as in the 
miracidium (Fig. 25) but tends to become straighter as the sporocyst 


LIFE HISTORY OF COTYLOPHORON—BENNETT 51 


becomes larger (Fig. 26). The course followed by these ducts depends 
quite naturally on the state of contraction of the individual and in some 
contracted old sporocysts the convolutions performed by them are very 
similar to those in younger specimens. The small bladder present at the 
end of each duct in the miracidium is also present in the sporocyst. 

Shape.—The shape of the sporocyst as seen in sectioned material is 
ovoid at all stages of development. When young specimens are dissected 
out of the host into physiological salt solution they are capable of assum- 
ing a spherical shape, but the older specimens cannot contract to this 
extent, due to the presence of large rediae. They are able to contract the 
extremities strongly, and freed specimens usually have the posterior end 
more strongly contracted than the anterior end. 

Muscles and Activity.—The circular muscles are strongly developed 
throughout the body, as evidenced by their activity, but the longitudinal 
ones seem to be less well developed. Very young specimens when freed 
from the host assume the spherical shape immediately, and subsequent 
movements are so slight as to be hardly noticeable. The fully developed 
sporocysts are relatively active. Their activity consists of slow contrac- 
tions of the muscle layers which are too weak to produce any appreciable 
progression. 

Appearance.—The fully developed sporocyst is visible to the unaided 
eye but cannot be readily distinguished from rediae or from small par- 
ticles present in the water. The outside of the body is covered by a mucus 
which is evidenced by the ability of the sporocyst to cling to the bottom 
of a dish or to a slide and by the amount of débris which adheres to it. 
The cuticula on the outside of the body is thrown into fine transverse 
striations by the contraction of the longitudinal muscles, which gives the 
body a distinctly ridged appearance. The simultaneous contraction of 
the muscle layers at the anterior end of the body produces a knobbed 
appearance (Fig. 25). This condition is so characteristic of the living 
sporocyst that one is able to distinguish it from rediae and to orient it 
very quickly with the aid of the microscope. 


MaTuRE SPOROCYST 


The mature sporocyst is elongate, usually bluntly rounded at the two 
extremities, and circular in cross section. It is relatively simple in struc- 
ture, consisting of a wall of variable thickness surrounding a central 
cavity (Fig. 30). The walls are composed of a thin cuticula, a layer of 
circular and longitudinal muscle, and an epithelial layer. 

The cuticula is from 2 to 3 p» thick when measured in mature sec- 
tioned sporocysts, but when measured on living specimens of the same 


52 ILLINOIS BIOLOGICAL MONOGRAPHS 


age it appears to be from 5 to 6 yu thick, as seen in optical sections. This 
difference is due partially to the contracted state of the living specimens 
and partially to the fact that less accurate measurements can be made on 
living material. Immediately beneath this layer are the circular muscles, 
which can be seen distinctly. The longitudinal muscles were not seen, 
although they were looked for with a magnification of approximately 
1500 diameters. The thickness of the combined layers is only 3 un. 

The epithelial layer consists of large vacuolated cells and small cells 
with a granular, deeply-staining cytoplasm (Figs. 27, 30). The large 
cells are more numerous than the others and comprise most of the body 
wall. They measure from 30 to 40 » by 20 to 35 w and contain nuclei 
which measure from 8 to 10 yw. A distinct chromatin mass, usually eccen- 
tric in position, is present in all of them. There are also many granular 
masses of chromatin scattered throughout the nuclei. The small cells 
measure approximately 18 by 9 uw and the diameter of the nuclei is from 
6 to 7 w. The chromatin of these nuclei has the same arrangement as 
that in the nuclei of the larger cells but is more dense. These small cells 
are readily distinguished from the larger cells by the difference in size 
and by their darker staining reaction. The distribution of these cells is 
very irregular. They may be located in contact with the muscle layer 
or scattered among the larger cells, although more of them were found 
in the posterior tip of the body than elsewhere. 

That these small cells are probably germinal is indicated by their 
location and similarity in size and staining reaction to the cells of very 
young rediae (Fig. 30). Many workers have described similar cells in 
sporocysts of other species as germ cells. 

Thomas (1883:115) in his description of the life cycle of Fasciola 
hepatica says: 

The contents of the sporocyst are formed by a number of very clear rounded 
cells, some of which are the germinal cells of the embryo or cells derived from 
them by division, others are formed by a proliferation of the epithelium lining the 
cavity of the sporocyst. 

Looss (1896:187) states of the sporocyst of Paramphistomum cervt: 
Tandis que sur la paroi interne du sporocyst, on ne rencontre que rarement,.... 
des cellules germinatives normales, celles-ci se présentent amassées dans 1l’ex- 
trémité caudale ot elles vont former un véritable épithélium germinatif. 

Mathias (1925:50) describes two kinds of cells in the walls of the sporo- 
cyst of Strigea tarda which are similar to those present in the wall of 
the present sporocyst. The smaller of these he believes to be germ cells. 
Dubois (1928:63) describes similar cells irregularly dispersed in the body 
wall of the sporocyst of Cercaria helvetica v which he considers to be 
germ cells. Brooks (1930) in his detailed study of the germ cell cycle 
in 20 species of trematodes did not find any evidence to support the 


LIFE HISTORY OF COTYLOPHORON—BENNETT 53 


theory that any celis in the epithelial layer of the sporocyst wall were 
germ cells. Price (1931:709) believes that the larger of the two types 
of cells found in the sporocyst wall of Schistosomatium douthittr are 
germ cells. 

It is not my purpose to enter into the merits of the many conflicting 
viewpoints, but in the present material only 9 rediae are produced in each 
sporocyst, and I believe that the germ cells producing these are formed 
in the germinal tissue of the miracidium prior to its penetration into the 
snail host. If the small, deeply-staining cells present in the wall of the 
sporocyst are germ cells then the vast majority of them never produce 
rediae. 

The rediae develop entirely enclosed by sporocyst tissue which divides 
the cavity of the sporocyst into compartments and in which the rediae 
remain until late in development. The fibers seen attached to developing 
rediae by Looss (1892:159) in the sporocyst of Diplodiscus subclavatus 
and by Price (1931:709) in the sporocyst of Schistosomatium douthitti 
are probably the same structures described here. 


REDIA 


Rediae belonging to the family Paramphistomidae have been de- 
scribed by a number of authors. Looss (1892) studied the development 
of the redia of Diplodiscus subclavatus and described the developmental 
stages and the mature redia in great detail. He also (1896) described 
briefly the rediae of Paramphistomum cervi and Gastrodiscus aegyptiacus. 
Cort (1915) gave a few details concerning the structure of the rediae of 
Cercaria inhabilis and C. diastropha. Faust (1919, 1919a) did the same 
for the rediae of Cercaria frondosa and C. convoluta. Sewell (1922) 
described rather completely the rediae of Cercariae Indicae XXI, XXvI, 
XXIX, Xxxut. McCoy (1929) gave a brief description of the redia of 
Cercaria missouriensis. Beaver (1929) described the redia of Allassos- 
toma parvum. Le Roux (1930) mentioned the fact that daughter rediae 
occur in the life cycle of Cotylophoron cotylophorum but gave no morpho- 
logical details. Krull and Price (1932) described very briefly the redia 
of Diplodiscus temperatus. 

These rediae belong to the subfamilies Paramphistominae Fischoeder 
1901 and Diplodiscinae Cohn 1904. The rediae of P. cervi, C. Indicae 
XXVI, XXIX, XxxuI, and C. cotylophorum belong to the subfamily Param- 
phistominae. These rediae are readily distinguished from those of the 
Diplodiscinae by the absence of lateral appendages. In general, the rediae 
of the Paramphistominae are smaller and possess a smaller pharynx and 
gut, although these characteristics are not of diagnostic value. 


54 ILLINOIS BIOLOGICAL MONOGRAPHS 


DEVELOPMENT 


Structure of the Redia.—The redia is much more complex in struc- 
ture than the sporocyst. It possesses a well developed digestive tract con- 
sisting of a mouth, pharynx, esophagus, rhabdocoel intestine, and a large 
number of unicellular glands which are associated with it. The redia also 
possesses a more complex excretory system, a discernible central nervous 
system, and a birth pore. 

Rate of development.—Development of the redia in the sporocyst is 
very rapid and at the time of liberation all the structures of the mature 
redia are present, with the exception of the birth pore. In the experiment 
under discussion the first rediae free in the body of the snail host were 
found 9 days after infestation. 

The growth of the redia in the sporocyst was studied in an attempt 
to establish the chronological sequence of organ development and to de- 
termine the time of germ ball development. During the first 3 days the 
redia is spherical in shape and consists of numerous cells with indistinct 
boundaries which contain nuclei varying from 3 to 6 » in diameter. On 
the fourth or fifth day the redia begins to elongate and the primordia 
of the digestive system appear. The smallest embryo with the primordia 
measured 68 by 45 » (Fig. 42). 


Digestive System.—The primordia of the digestive system consist of 
a group of centrally located cells whose cytoplasm is finely granular. 
These cells measure 10 to 12 » in diameter and contain relatively large 
nuclei, 6 to 7 » in diameter. Looss (1892:160) states that these cells 
produce a secretion which forces them apart, thus producing the lumen 
of the digestive tract. The cause of the separation was not determined in 
the present material, but the lumen is produced by a delamination of the 
primordial cells. As the embryo continues to grow the digestive primordia 
increase rapidly in size, extending from near the anterior end almost to 
the posterior end in embryos measuring 80 to 50 #. In rediae of this size 
a small number of loosely organized cells which are destined to form the 
pharynx are present at the anterior end of the digestive tract. The lumen 
of the intestine becomes much more evident at this stage, being widest 
at the middle of its length. Anteriorly it is much narrower where it 
joins the pharyngeal cells. 

Following this stage it rapidly assumes the appearance characteristic 
of it in the mature redia. The pharynx becomes definite in shape, a basal 
membrane develops around it and the intestine, and muscle fibers begin to 
develop in the pharynx. The development of muscles in the pharynx is 
accompanied by a breaking down of the cell membranes of the cells 
from which it develops. However, the nuclei of these cells remain dis- 


LIFE HISTORY OF COTYLOPHORON—BENNETT 55 


tributed irregularly through it. In an embryo 106 by 55 yn, representing 
this stage in development, the pharynx measured 26 » wide and 16 
long and was located 14 » from the anterior end of the body. The 
intestine does not increase in length to accompany the increase of body 
length. In the above specimen it terminated 27 » from the posterior end. 

Early in development, when the embryos have reached a length of 
approximately 90 », the cuticula lining the mouth cavity, pharynx, and 
upper part of the esophagus begins to form. Six cells at the anterior 
end of the intestine, which are designated as pharyngeal cuticula cells, 
grow forward through the lumen of the pharynx but do not entirely 
occlude it. The cells are united at their anterior ends, forming a cap 
which closes over the lumen (Fig. 44). As growth continues the cells 
elongate and their anterior ends approach the surface of the body. At 
this stage the cells measure 31 p in length and 6 » in width at the posterior 
end. At the anterior ends of these cells and near the surface the primi- 
tive epithelium covering the body grows inward around them until it 
reaches the anterior margin of the pharynx, where it seems to fuse with 
the inner surfaces of the cells in embryos approximately 100 » in length. 
That this process is a result of growth and not of invagination of the 
anterior part of the body was demonstrated by study of serial sections 
of the embryo. The primitive epithelium was found to be intact over 
the entire surface of the body. That portion of the body lying directly 
anterior to the pharynx, represented by the nuclei in Fig. 44, is eventually 
sloughed and apparently forms a plug which fills the mouth cavity in 
slightly larger individuals (Fig. 49). The function of this plug is 
unknown. 

At the time of the fusion of the primitive epithelium with the pharyn- 
geal cuticula cells a substance is deposited in the surface cells next to the 
lumen of the pharynx. This substance, which forms the cuticular lining 
of a part of the mouth cavity, the pharynx, and a part of the esophagus, 
stains darkly in haematoxylin and is very difficult to destain. This charac- 
teristic indicates its extent in older forms, since the cuticula produced 
by the primitive epithelium does not stain so deeply. Accompanying the 
formation of the pharyngeal cuticula the nuclei of the cells producing 
it degenerate. The nuclei first become flat and elongate, the chromatin 
then forms a large central mass, and finally the nuclei disappear entirely, 
leaving a uniformly thin cuticula continuous with that of the body. The 
nuclei of the primitive epithelium degenerate also during its transforma- 
tion into the cuticular covering of the body, which is concurrent with the 
formation of the pharyngeal cuticula. 

Simultaneously with the formation of the pharyngeal cuticula, 6 other 
cells, which are designated as esophageal cells, become differentiated at 


56 ILLINOIS BIOLOGICAL MONOGRAPHS 


the anterior end of the intestine. These cells are broad at the base but 
become thin anteriorly at their junction with the pharyngeal cuticula 
cells (Fig. 43). The esophageal cells either produce a cuticula-like sub- 
stance or are covered externally by the secretions of the pharyngeal 
cuticula cells, since they stain in a similar manner during the early stages 
of their development. Later this staining reaction disappears. However, 
the fact that this is cuticula is proved by the stiffened esophagus which 
projects prominently into the lumen of the intestine in contracted rediae. 

In embryos 150 in length the formation of these structures is com- 
plete, the mouth is open but still contains the plug, and the entire embryo 
is still enclosed by sporocyst tissue (Fig. 40). The pharynx is 34 » wide 
and 22 yw long, and the intestine, which is approximately 40 by 30 p in 
embryos of this size, extends slightly past the middle of the body. The 
intestine consists of a single layer of distinct cells which measure 10 to 
15 » in diameter. Each contains a relatively large nucleus. The basal 
membrane which encloses both the pharynx and the intestine contains 
distinct ovoid nuclei early in development but they could not be found in 
the membrane in rediae of this size. Looss’ (1892:161) observation 
that muscles develop in this membrane was not confirmed for this redia. 

No rediae larger than 150 by 52 » were found enclosed in sporocyst 
tissue, nor any which contained the plug in the mouth cavity. Apparently 
they break out of their individual compartments shortly after reaching 
this size and the mouth opens. 

Looss’ (1892:160-161; figs. 6, 7) description and figures of the de- 
velopment of the digestive organs in the redia of D. subclavatus show it 
to be very similar to that of the present redia. He did not observe a 
sloughing of the primary cuticula, which is produced by the primitive 
epithelium and pharyngeal cuticula cells, in the redia of his material, but 
he did observe indications of such a process in the redia of Cercaria 
cystophora (1892:161) after being born. He believes that a similar 
process occurs in the redia of D. subclavatus and that a secondary cuticula 
is produced by the underlying layers of the body wall. According to his 
observations neither the mouth nor the birth pore are open until after 
birth. In C. cotylophorum the mouth of the redia is open at least two 
days before birth but there is no indication of a birth pore. At this stage 
of development no indications of sloughing of the primary cuticula other 
than that previously mentioned were observed in the present material at 
any stage of development. Since no subsequent sloughing was ob- 
served it is believed that the primary cuticula is not sloughed at any 
time and that it forms the cuticula of the mature redia. 


Germ Cells.—The germ cells appear simultaneously with the primordia 


LIFE HISTORY OF COTYLOPHORON—BENNETT 7 


of the digestive system, that is in individuals approximately 60 p» in 
length. At this size living rediae appear as a homogeneous mass of cells 
surrounded by the primitive epithelium. However, in sectioned material 
the germ cells are obvious because of their size, position, and staining 
reaction. They were always found in the posterior region of the body, 
posterior or lateral to the digestive primordia (Fig. 42). The cells do not 
have definite boundaries, and the cytoplasm, which is finely granular, 
takes a deeper stain than the cells of surrounding tissue. They measure 
approximately 10 by 8 and contain nuclei which are 6 » in diameter. 
There is no distinct central cavity in the young individuals but as the 
germ cells divide to form germ balls a small space becomes evident 
around each one. The smallest redia in which a definitely formed germ 
ball was found measured 85 by 39 ». In an individual measuring 152 by 
54 w 4 germ balls were present. Usually there are 10 to 12 present when 
the redia is born. Early in the formation of the germ balls an occasional 
ovoid nucleus is observed near the periphery. These are the nuclei of the 
cells which are destined to form the primitive epithelium around the germ 
ball. These nuclei were observed in germ balls as small as 15 p in di- 
ameter, but no definite retaining membrane is formed until they have 
reached approximately 30 » in diameter. The retention of the germ balls 
in individual compartments is not very evident in the young rediae be- 
cause of the thickness and irregularity of the body wall but this arrange- 
ment becomes very distinct in older specimens (Fig. 39). 


Excretory System.—The earliest stages in the development of the 
excretory system were not observed. Looss (1892:161) states that in 
Distomum ovocaudatum he observed the excretory system of the redia 
in the 2-flame-cell stage; each cell opened separately at the posterior end 
of the body. In the present material no stage as early as this was ob- 
served. However, I believe that the excretory system becomes functional 
when the redia is approximately 75 » in length. Small lateral tubules 
toward the posterior extremity were observed in embryos of this size but 
no flame cells could be distinguished. The smallest redia in which flame 
cells were observed was 105 by 84 ». In this individual there were 2 on 
each side. The anterior pair is located lateral to the anterior end of the 
intestine, and the posterior pair is located in the extreme posterior end 
(Fig. 35). The excretory ducts on each side unite to form a common 
duct which expands to form a small bladder on either side shortly before 
reaching the excretory pore. In individuals of this size the excretory 
pore is located approximately one-third of the body length from the 
posterior end. A third flame cell is developed on each side when the redia 
is about 160 » long, although specimens 135 » long which possessed the 


58 ILLINOIS BIOLOGICAL MONOGRAPHS 


third cell were found occasionally. This cell develops near the excretory 
pore and its duct unites with the duct trom the anterior flame cell 
(Fig. 46). 

Nervous System.—The nervous system of the redia develops at the 
same time as the digestive and excretory systems. The central fibrous 
nerve mass is located dorsal to the esophagus, and the nerve cells com- 
pletely surround it (Fig. 37). The region of the body in which it de- 
velops is filled by numerous cells at all stages of development and it is 
very difficult to determine when the nerve cells become differentiated. 
However, in rediae 100 » long some nuclei have the appearance which is 
characteristic of the nerve cell nuclei in older forias. The nuclei are 
round, measure 4 to 5 » in diameter, and contain numerous granular 
masses of chromatin which cause them to stain more darkly than other 
surrounding nuclei. The nuclei are scarce over the dorsal surface of the 
fibrous mass but are very numerous lateral to it. They extend to the 
ventral side of the redia and across the body ventral to the esophagus. 
The central fibrous mass is approximately 30 by 15 p in rediae 150 p in 
length. No nerves were observed to leave this central mass. 


Salivary Glands.—The salivary glands which surround the anterior 
part of the digestive tract become differentiated in rediae slightly over 
100 » in length, being first observed in an individual which measured 
104 by 52 p». In this redia 6 cells were found which were considered 
to be gland cells. These glands are characterized by finely granular, 
deeply-staining cytoplasm and a relatively large nucleus which contains a 
few granular masses of chromatin (Fig. 34). There is also a concen- 
tration of chromatin at the nuclear membrane, while the remainder of 
the nucleus remains comparatively clear. The cells are drop-shaped with 
a long slender projection extending anteriorly. The cells are approxi- 
mately 6 » in diameter at their posterior ends, and the nucleus, which is 
located here, is 7 by 4 ». The anterior extensions of the glands lengthen 
as the redia grows but do not reach the surface until the mouth cavity 
is being formed. Ina redia about 150 » in length 40 of these glands were 
found. Twelve of them were located around the pharynx, some lying 
anterior to it, while the remaining 28 were distributed around the esoph- 
agus and anterior part of the intestine. Looss (1892:161), Cort (1915: 
23, 25), Sewell (1922:71, 77, 86), and Krull and Price (1932:9) have 


described very similar glands in other amphistome rediae. 


Muscle Tissue—The development of the muscular tissues is seem- 
ingly very slow. No movement is noticeable in rediae under 125 p in 
length. At this size movement consists of very slow and weak contrac- 
tions of the circular and longitudinal muscles. The individual muscle 


LIFE HISTORY OF COTYLOPHORON—BENNETT 59 


layers could not be distinguished in rediae of this size, but the thickness 
of the combined layers is only 1 to 1.5 p. 


Body Wall.—tThe body wall in the young redia is similar to that of 
the young sporocyst. It consists of a very thin cuticula 1 p thick, the 
muscle layer, and an inner epithelial layer which is from 10 to 15 p thick 

in rediae developing in the sporocyst. The germinal tissue is located in 
the posterior extremity of the body, where it forms a mass which is ap- 
proximately 30 » thick. 


Redia Prior to Liberation.—By the end of the seventh or eighth day 
after infestation the largest rediae are approximately 150 » long and are 
similar in every way to mature rediae, except for their lack of a birth 
pore. The chronological sequence of organ development and germ ball 
formation is: (1) primitive epithelium, (2) digestive primordia, (3) germ 
cells, (4) excretory system, (5) nervous system, (6) gland cells, and 
(7) muscle development, as evidenced by movement. The sequence of 
organ development and the formation of early germ balls is very similar 
to that of the miracidium. 


Liberation of Rediae—Rediae are found freé in the central cavity of 
the sporocyst for one or two days before they break out into the body of 
the snail. During these days the only noticeable changes are an increase 
in the size and number of germ balls. Usually there are only one or two 
of these more advanced rediae present in the sporocyst at one time. The 
process of breaking out of the sporocyst was considered in the discussion 
of the sporocyst development. 

The fact that the rate of development is unequal may explain why 
rediae of variable sizes are liberated by the sporocysts. Some rediae were 
found free in the snail host at a size of 169 by 52 p, while on the other 
hand rediae as large as 225 by 58 » were observed still in the sporocyst. 
The average size of 10 at the time of liberation was 188 by 56 ». Looss 
(1892:162) found that the rediae of Diplodiscus subclavatus were freed 
when approximately 200 ,, in length. At the time of their liberation the 
rediae contain from 10 to 12 germ balls, the largest of which are ap- 
proximately 20 by 20 ». In an individual which measured 225 by 67 p 
the pharynx was 29 » wide by 22 » in length; the intestine was 71 p 
long by 54 » wide and terminated near the middle of the body length. 


Birth Pore.—The rediae begin feeding upon the host tissues immedi- 
ately after their release and migrate slowly into the liver and ovo-testis 
where they complete their development. The birth pore is the only struc- 
ture which develops after they leave the sporocyst. It was first observed 
in a living specimen which measured 0.27 by 0.1 mm. In contracted 
specimens it appears as a small projection on the ventral surface of the 


60 ILLINOIS BIOLOGICAL MONOGRAPHS 


body, approximately 160 » from the anterior end. Sewell (1922:71) 
found the birth pore of the redia of Cercariae Indicae xxvi to be situated 
to one side just behind the level of the pharynx, and in the rediae of 
C. Indicae xx1x he (p. 77) found it to be ventro-lateral one-fourth of 
the body length from the anterior end. Beaver (1929:16) found that the 
birth pore of the redia of Allassostoma parvum was dorsal to the an- 
terior part of the intestine, and that it was visible only when cercariae 
were emerging through it. The central nerve mass of the redia is con- 
sidered to be dorsal, and the flame cells, excretory ducts, and pore are 
considered to be lateral. By using the position of the excretory organs for 
orientation of the redia the birth pore is found to be ventral. In living 
specimens it can be seen only in a lateral view, and in sectioned material 
it is found ventral to the brain mass (Fig. 38). The time at which it 
opens was not determined. 


Increase in Size.—The increase in size of the redia is very rapid. In 
the present experiment the largest redia ever found was dissected from a 
snail 10 days after infestation (Fig. 48). This specimen was 1.02 by 
0.21 mm in size, the pharynx measured 55 by 55 yp, and the intestine was 
160 by 84 p. It contained 23 well-formed germ balls or cercariae, and 
others seemed to be developing at the posterior end of the body. The 
largest of the cercariae measured 90 by 65 . This individual was perhaps 
an aberrant form since the next largest specimen ever found measured 
0.84 by 0.18 mm and contained only 14 cercariae. Apparently the redia 
reach their maximum size immediately before the birth of the first cer- 
cariae and then decrease somewhat in size as the cercariae are born. The 
redia in Fig. 33 was taken from the snail mentioned above and was the 
largest of the remaining rediae. It measured 0.55 by 0.12 mm, the 
pharynx was 48 by 50 uw, and the intestine was 105 by 64 yp. It contained 
15 cercariae. The size of the 10 largest rediae found, the size of the 
pharynx, the position of the birth pore, and the number of cercariae 
developing in each is given in Table 11. Many rediae in all stages of 
development were constantly found in the snails used in this experiment, 
due to the fact that the sporocysts continued to produce rediae until the 
termination of the experiment, 35 days after infestation of the snails. 


MaTurRE ReEDIA 


There is no marked change in the appearance of the mature rediae 
from that of those just born. The pharynx and intestine increase in size 
but are much smaller in relation to body size than in young rediae. The 
pharynx of 10 rediae just liberated from the sporocyst averaged 32 by 
27 pu, while the average size of this structure in the rediae, given in 


LIFE HISTORY OF COTYLOPHORON—BENNETT 61 


Table 11, is 49 by 40 ». The pharynx was never observed to become 
elongate, its greatest diameter always being the transverse one. The 
intestine, which reaches the middle of the body in young specimens, does 
not extend posteriorly more than one-fifth of the body length in mature 
forms. 


TABLE 11.—MEASUREMENTS (IN MILLIMETERS) OF TEN MATURE REDIAE AND 
THE NUMBER OF CERCARIAE IN EACH 


Distance of 
z Number of 
No. Size of redia Size of pharynx birth pore cercariae 
from anterior | ~ 
me in each 
1364 Seer ee aan 1.01 x0.22 0.055 x 0.050 0.180 23 
Denyse tel aed 0.94x0.18 0.050 x 0.048 0.174 14 
Sere tae earn heed 0.77x0.21 0.050 x 0.042 0.165 18 
[Tt a 0.67x0.13 0.046 x 0.046 0.147 12 
RAE oe don, suet at 0.62x0.12 0.046 x 0.034 0.189 10 
(Oa Ae Re ae 0.60x 0.17 0.042 x 0.042 0.178 12 
LEIS Tet etasdeesd da 0.59x0.17 0.046 x 0.042 0.162 15 
{gs Pan Paya 0.58x 0.15 0.046 x 0.046 0.155 14 
(0) 0 as a a 0.57x0.17 0.046 x 0.046 0.170 16 
NOW ghia ese 0.55x0.12 0.048 x 0.040 0.148 15 
Average....... 0.59x0.16 0.049 x 0.040 0.168 15 


The excretory system is typical of all the amphistome rediae which 
have been described, with the exception of the redia of Paramphistomum 
cervt and Diplodiscus subclavatus. Looss (1892:161: fig. 10) found 
3 or 4 pairs of flame cells in the redia of D. subclavatus, and in the 
mature redia of P. cervi there are 5 pairs of flame cells (1896:188). The 
flame cells in the mature redia of C. cotylophorum are located as in 
the young forms. However, the excretory pore is situated very near the 
middle of the body length, because of the posterior extension of the 
body caused by the developing cercariae (Fig. 46). The flame cells are 
approximately 12 by 5 » and the ducts, which are only a little coiled, 
are 2 to 3 »w in diameter. The ducts from the anterior and posterior cells 
on each side unite slightly anterior to the middle of the body. The duct 
from the middle cell joins the duct from the anterior flame cell. The 
common duct formed by the union of the anterior and posterior ducts is 
approximately 50 by 10 p» in size. It expands to form a small bladder 
20 by 30 » which opens to the outside through a very small canal. The 
excretory pore when expanded measures 10 » in diameter. The nervous 
system remains unchanged in the mature redia. The salivary glands in- 
crease in size as the redia grows. In a redia which was 0.35 mm long the 
cells measured 14 by 10 », being very conspicuous in sectioned material 


62 ILLINOIS BIOLOGICAL MONOGRAPHS 


because of this increase in size and a darker staining reaction. In old 
specimens which have passed maturity these cells stain lighter in haema- 
toxylin stains and seem to be fewer in number. 

-The germinal tissue, which is located in the posterior extremity of 
the body, becomes exhausted in the older forms (Fig. 41). This ex- 
haustion was found to occur in some rediae in this experiment 25 days 
after infestation of the snail, although there were many developing cer- 
cariae still present in them. This indicates that the number of cercariae 
produced by a single redia is very limited. In Table 11 the number 
present in mature rediae is shown to vary from 10 to 23, with an average 
of 15. This average doubtless is less than the number produced by each 
individual which is believed to be nearer 25. If this estimate is approxi- 
mately correct, then the number of cercariae produced by each mira- 
cidium is 225, since each sporocyst produces 9 rediae. Naturally this 
estimate precludes the formation of daughter rediae. Takahashi (1928: 
278) found that the sporocyst of Paramphistomum cervi produces 9 
rediae, each of which gives rise to 20 cercariae, resulting in a total of 180 
cercariae produced by a single sporocyst. Krull (1934:174) attempted 
to correlate his findings with those of Takahashi based on a total number 
of cercariae shed by each snail. He found that the average production 
of each of 11 snails was 152 cercariae; but since he allowed uncounted 
numbers of miracidia to infest his snails, no accurate estimate could be 
made as to the number of cercariae produced by’a single miracidium. 
Consequently, he has no basis for his attempt at correlation with 
Takahashi’s observations. 

The body wall of the mature redia is very similar to that of the 
sporocyst. It consists of a thin cuticula covering the external surface of 
the body, a layer of circular muscles followed by a layer of longitudinal 
muscles, and a thin epithelial layer. The cuticula gives a white appear- 
ance to the redia and is wrinkled in low transverse ridges by contraction 
of the body. The cuticula appears to be 4 to 6 p thick in living specimens, 
but in well extended sectioned rediae it measures 1.5 to 2 mw in thickness. 
The fact that it is no thicker in the older forms than in young rediae 
indicates that there are no additions to it by underlying cells, and this 
condition supports the evidence concerning the transformation of the 
primitive epithelium into the cuticula. 

This study of the developing and mature redia demonstrates that the 
development of this stage in the life history of C. cotylophorum is very 
rapid. The chronological sequence of organ formation and the time of 
germ ball formation is very similar to the same processes in the mira- 
cidium. The birth of the redia occurs 9 to 10 days after the snail host is 


at 


LIFE HISTORY OF COTYLOPHORON—BENNETT 63 


infested. All the structures are formed except the birth pore. The 
average size of rediae at the time of liberation from the sporocyst is 
0.188 by 0.056 mm. Following liberation the redia reaches its maximum 
-size in 5 days and may contain as many as 23 cercariae, all of which are 
retained in individual compartments. The germinal epithelium is located 
in the posterior extremity of the body and probably becomes exhausted 
after 25 cercariae are produced. The digestive system consists of a 
mouth, a pharynx, an esophagus, and a rhabdocoel intestine. The ex- 
cretory system is similar to that of other amphistome rediae, consisting 
of an anterior, a median, and a posterior flame cell and their ducts on 
each side of the body. The nervous system consists of a mass of fibrous 
tissue and many associated ganglion cells located principally in the dorsal 
region of the body in the esophageal region. The body wall is composed 
of an outer cuticula, a circular and a longitudinal layer of muscles, and 
an inner epithelial lining. The average size of mature rediae is 0.59 by 
0.163 mm and the birth pore is located on the ventral surface 0.168 mm 
from the anterior end of the body. 


DAUGHTER REDIA 


Daughter rediae have been reported for only three species of amphis- 
tomes. Looss (1896:184) mentions that daughter rediae occur in the 
life cycle of Gastrodiscus aegyptiacus and that occasionally both rediae 
and cercariae were found developing in the same mother redia. Looss 
(1896:189) also found that in the life cycle of Paramphistomum cervi 
two and sometimes three generations of rediae were produced. Beaver 
(1929:17) in his work on the life cycle of Allassostoma parvum found 
that daughter rediae were produced in mother rediae in which the 
pharynx and intestine were larger in proportion to the size of the body 
than in daughter rediae. He found, too, that the mother redia possessed 
only one pair of appendages and that the body was greatly distorted. 

Only a single mother redia was found in the present material although 
many infested snails were examined over a period of nearly a year. This 
one specimen was found in a snail on August 4, 1934, one month after it 
was infested. It was not observed until too distorted by pressure to make 
accurate observations. However, it contained three well developed and 
three developing rediae. In the more developed individuals the digestive 
and excretory systems were fully formed and one or two germ balls 
were present. In this same snail many young cercariae were found in the 
liver as well as many other rediae which contained developing cercariae 
only. 


64 ILLINOIS BIOLOGICAL MONOGRAPHS 


CERCARIA 


DEVELOPMENT 


-The cercaria acquires most of its structures while still within the 
redia, although some are represented by primordia only. In the experi- 
mental series under discussion mature cercariae were shed 32 days after 
the infestation of the snail. The first cercariae were freed from the 
rediae 15 days after infestation in a very immature condition. They were 
about one-third of the size of mature cercariae. 

The first differentiation which occurs is the formation of the primi- 
tive epithelium, which does not develop until the germ ball or cercaria 
has reached a diameter of 30 ». The appearance of the primitive 
epithelium is very similar to that surrounding developing rediae but it 
loses its cellular nature much more quickly than in the redia. No nuclei 
were distinguished in this layer in cercaria more than 60 p in length. 

The next structures to appear are the cystogenous gland cells. The 
first of these were found irregularly scattered through the body of a 
cercaria which measured 50 by 46 p. These cells are easily recognized 
because of their size and characteristic appearance (Fig. 52). The cells 
are 8 to 10 » and have nuclei 6 to 7 » in diameter. A distinct deeply- 
staining chromatin body, 2 » in diameter, is located in the nucleus. The 
remainder of the nucleus is finely granular while the cytoplasm is very 
transparent. The number of these cells increases rapidly as the cercaria 
develops. 

Shortly after this stage in development the cercaria becomes dis- 
tinctly ovoid, and at a length of 65 » the primordia of the digestive and 
excretory systems appear. The excretory system consists of two lateral 
ducts which open separately at the posterior end of the body. Looss 
(1892:162) observed a similar condition in the developing cercaria of 
Diplodiscus subclavatus and was able to see flame cells at the internal 
ends of the ducts. In the present material no flame cells were seen prior 
to the birth of the cercaria. The digestive primordia consist of a group 
of cells located centrally near the anterior end of the body, in which 
lumina appear very early (Fig. 52). The most anterior lumen is that of 
the oral sucker and the most posterior that of the rhabdocoel intestine, 
which Looss (1892:163) considers as probably homologous to the intes- 
tine of the redia. The esophagus appears as a solid cord of cells connect- 
ing the oral sucker and the intestine. The number of cells entering into 
the formation of these primordia is much greater than in the redia. No 
cell boundaries could be distinguished but the number of cells present is 
indicated by the many small, closely packed nuclei. 

Shortly after the formation of these structures a basal membrane in 


LIFE HISTORY OF COTYLOPHORON—BENNETT 65 


which an occasional ovoid nucleus is located develops around them. The 
nuclei of this layer can be distinguished from those of the surrounding 
tissues by their position only. The ultimate development of this layer 
was not determined in the present material, but Looss (1892:165) states 
that these cells are derived from body parenchyma and give rise to the 
muscle layers surrounding these organs. 

A mass of deeply-staining cells located immediately posterior to the 
blind end of the intestine at this stage in development represents the 
primordia of the male and female genital systems. The cells of this mass 
can be distinguished from surrounding cells by their position and staining 
reaction only, but by following them in their subsequent dévelopment their 
function is discovered. 

In embryos of approximately 90 w in length the tail is formed as a 
broad but short part of the body at the posterior end, which becomes set 
off rapidly from the body by ventral and lateral constrictions. The cells 
entering into the formation of the tail are similar in every respect to 
those of the body. As the constrictions deepen the excretory vessels in 
the tail become confluent, but retain their separate openings, which 
become lateral in position because of the narrowing and lengthening of 
the tail (Fig. 57). The union of the excretory ducts occurs first in the 
tail but continues into the posterior part of the body for a short distance. 
As a result of this fusion the excretory system consists of a tail duct with 
its two lateral openings and a lateral duct on each side of the body. At 
the junction of the tail duct and the two lateral ducts another pore is 
formed in the median dorsal line of the body. This is the pore of the 
future excretory bladder in both the mature cercaria and the mature 
worm. This pore is formed prior to the birth of the cercaria. 

While these changes are taking place in the excretory system the 
digestive system is becoming complete. The rhabdocoel intestine bi- 
furcates and a mass of cells is formed at the end of each fork which 
grows laterally and posteriorly on each side. These cells are the primordia 
of the caeca (Fig. 55). As the caeca elongate a small lumen is formed in 
them. They develop dorsal to the excretory ducts. 

At this stage of development the cuticula-producing cells of the oral 
sucker still retain their nuclei and the mouth is not yet open. The lumen 
of the esophagus is present and the esophagus is lined by a thin cuticula 
continuous with that of the oral sucker. The cuticula lining both the 
esophagus and the oral sucker is thrown into longitudinal folds which 
are continued a short distance anterior to the oral sucker. It is possible 
that the mouth opening is formed in the cercaria as in the redia but no 
observations were made on the details of this process. However, the 
mouth opening is formed before the cercaria is born, being similar in 


66 ILLINOIS BIOLOGICAL MONOGRAPHS 


this respect to the redia, but the nuclei of the cuticular cells are retained 
until after birth. Looss (1892:164) assumes that the primary cuticula 
is sloughed by the cercaria as in the redia but he made no observations 
on this process. In the present material no sloughing was observed and 
it is believed that the primary cuticula produced by the primitive 
epithelium and the cells lining the oral sucker is retained throughout the 
life of the individual. 

Simultaneously with the formation of the tail the nervous system and 
eyes appear. The nervous system at this stage of development consists of 
a small ganglion on each side of the esophagus. These ganglia are con- 
nected by a commissure passing dorsal to the esophagus immediately 
posterior to the primordium of the oral sucker. Several small nerves can 
be traced a short distance from each of the ganglia (Fig. 57). Many 
nuclei were observed arranged in a close series which completely encircle 
the ganglia and commissure clearly delimiting them from the surround- 
ing parenchyma cells. Looss (1892:164) described a similar nervous 
system for the developing cercaria of Diplodiscus subclavatus and was 
able to trace the nerves much farther than could be done in the present 
material. At the time of the formation of the nervous system and the tail 
the cells producing the eyes become evident. The pigmented part of each 
eye is produced from a single cell located laterally above each ganglion. 
These cells could not be distinguished from the surrounding cells prior 
to the formation of the pigment. After the formation of the pigment the 
cells are conspicuous, having the appearance shown in Fig. 64. At this 
stage in development the cell measures 10 to 12 » in diameter and the 
nucleus 6 ». The nucleus contains a large central chromatin mass 3 p» in 
diameter. Looss (1892:165) observed three cells which contributed to 
the eye formation in the early developmental stages of the cercaria of 
Diplodiscus subclavatus. As he pointed out, one cell produced the pig- 
mented part and the other two the lens of the eye. These latter two cells 
were not identified in the present material prior to the birth of the 
cercaria and then were recognized in sectioned material only (Fig. 65). 
These cells lie against the cuticula, between it and the pigment cell, and 
seem to give rise to a refractive substance which fills the space in the 
pigment cone. The arrangement of these cells was determined in cercariae 
which had a body length of 130 p.w< 

Thus in the cercaria the following structures can be identified before 
it leaves the redia (in order of appearance): primitive epithelium, cysto- 
genous glands, excretory and digestive primordia, reproductive tissues, 
nervous system, pigment cells of the eyes, and the tail. The only struc- 
ture which develops after birth and of which there is no indication before 
birth is the acetabulum. 


LIFE HISTORY OF COTYLOPHORON—BENNETT 67 


Birth occurs shortly after the formation of the tail but the size at 
which the cercariae are born is not uniform. The approximate size of 
the body is 120 by 55 » and of the tail 37 by 33 uw. The cercaria is 
capable of very weak movement which is sufficient to break the enclosing 
strands of tissue but the process of birth was never observed. The 
mouth is open and the caeca which extend the length of the body are 
provided with a small lumen, and so it is possible for the cercaria to 
begin feeding at once.. Immediately after birth the eyes rapidly become 
more prominent and the other structures of the body also increase rapidly 
in size (Fig. 56); the primordia of the reproductive systems divide, 
forming two large masses which remain connected by a small cord of cells. 

The acetabulum first appears as a solid mass of cells at the ventro- 
posterior surface of the body in cercariae approximately 140 by 65 u. 
This mass projects prominently and measures about 45 » in width and 
30 » in length. A lumen becomes conspicuous in the acetabulum when 
the cercaria has reached a size of 190 by 75 yp. At this stage the 
acetabulum appears as a wide flat mass of cells with a small concavity 
near its center. It is 50 » wide and 20 p» thick. 

Shortly after birth the eyes acquire a very heavy pigmentation and 
their characteristic oval shape which is retained until the cercaria is 150 
in length. Pigment then begins to grow out from the eyes in lateral 
finger-like projections which completely encircle the body. A short time 
later projections are formed at the posterior surface also. At the same 
time the eyes become surrounded by a solid mass of pigment which 
entirely obscures their original outline (Fig. 61). As the growth of the 
pigment continues it is arranged in an irregular dendritic pattern over 
the dorsal and lateral surfaces of the body (Fig. 61). The branches 
break up into small irregularly arranged patches which remain attached 
to each other by very small strands of pigment. A short time before 
maturity is reached only a few large branches of pigment, which extend 
laterally and posteriorly from the eyes, are present (Fig. 59). The eyes 
assume their original shape but remain connected by a conspicuous band 
of pigment. It is possible that these later concentrations of pigment lie 
directly above the large nerves of the body as suggested by Sewell 
(1922:75). In the mature cercaria all of the pigment is uniformly dis- 
tributed, there being no definite concentrations into lines or large patches 
as in the developing forms. Consequently the eyes, which retain their 
pigmentation, become very conspicuous. 

The pigment is entirely superficial in position, being located imme- 
diately beneath the cuticula with the exception of small granules which 
are scattered throughout the body. The concentration of pigment on the 


68 ILLINOIS BIOLOGICAL MONOGRAPHS 


dorsal and lateral surfaces is much heavier than on the ventral surface, 
but no area was found entirely devoid of pigment. 

Simultaneously with the formation of the acetabulum the two lateral 
excretory ducts become united by a cross connection located across the 
middle of the body near the dorsal surface. The lateral excretory ducts 
pass outward and forward from their union with the caudal duct, then 
turn forward and inward and at their most inward position the cross 
connection is formed. From this point they turn outward and forward 
and pass ventral and mesial to the eyes to reach the sides of the oral 
sucker. Here they turn sharply and pass back toward the posterior end 
of the body. In cercariae between 150 and 175 yw in length a small 
median anterior diverticulum is formed on the cross connection and a 
lateral diverticulum grows out from each lateral duct immediately 
posterior to the eyes. The excretory system is shown in Fig. 50. 

As has been stated previously an excretory pore is formed dorsal to 
the union of caudal and lateral excretory ducts prior to the birth of the 
cercaria. The excretory duct of the bladder is lined for a considerable 
distance by cuticula, which makes it evident that the pore is formed by an 
invagination of the body wall which comes in contact with the excretory 
ducts at their union. As the cercaria increases in length the posterior ends 
of the lateral ducts become situated more posteriorly. This change in 
position is slight but necessitates an anterior extension from their -point 
of union to the excretory pore. At first, in cercariae of not over 0.2 mm 
in length, this anterior extension is no wider than the ducts, but as 
growth continues it assumes the appearance of the bladder or excretory 
vesicle in the mature cercaria. The lateral ducts empty at all stages of 
development into the posterior lateral margins of the bladder. The caudal 
excretory duct opens into the excretory duct from the bladder (Fig. 45). 
Consequently, the bladder is formed by first an extension and later an 
expansion of the ends of the lateral excretory ducts at their point of 
union. 

The cystogenous glands which increase in number as the cercaria 
develops begin to produce cystogenous rods or granules when the cercaria 
is approximately 140 » in length. Previous to this stage in development 
the cercaria stains very deeply in both toto mounts and sectioned material, 
but with the formation of the cystogenous granules this staining reaction 
largely disappears. This is due to the fact that the granules are very 
difficult to stain. The increase in number of these granules is so rapid 
and the area they occupy is so great that in cercaria of 200 yu in length 
there is comparatively very little tissue in the body. The body parenchyma 
is almost completely obliterated, only an occasional nucleus surrounded 
by a small amount of cytoplasm being found. The cystogenous cells are 


LIFE HISTORY OF COTYLOPHORON—BENNETT 69 


round and vary from 13 to 21 » in diameter when filled with the granules. 
The rod-shaped granules are arranged in parallel series in the cells and 
measure 12 by 4 pn. 

All of the structures of mature cercariae are present but not fully 
developed in individuals which measure approximately 0.175 by 0.09 mm. 
The structures continue to increase in size but marked changes occur 
only in the pigmentation, the size of the excretory bladder, and the size 
of the tail. The changes occurring in the first two have been discussed 
previously. 

In its early stages of development the tail consists of a dense mass of 
cells set off from the body. It is much broader than long when first 
formed and at the time of the birth of the cercaria the dimensions are 
practically equal. It is also capable of very slight movements which 
consist of slow contractions and extensions. It becomes longer than wide 
immediately after birth, and the length increases rapidly. Both the body 
and tail of the cercaria are subject to considerable variation in size in 
older individuals, but in well extended fixed specimens the tail and body 
become approximately equal in length when both are about 0.2 mm long. 
Three or four days before maturity is reached the tail is sufficiently 
strong to enable the cercaria to swim for brief intervals. Such cercaria 
will swim for a‘short time and then apparently attempt to encyst but 
cannot. In cercaria of this age the tail is approximately twice the 
length of the body. 

The number of cells comprising the tail appears to remain unchanged. 
They are very small and numerous in the young stages, as indicated by 
the nuclei, but become large in mature individuals (Fig. 54), giving the 
impression that the cells increase in size but not in number as the tail 
grows. 


MaTuRE CERCARIA 


Age at Birth.—Mature cercariae escaped from the snails in this experi- 
ment 32 days after infestation. The development of the cercaria when 
compared to that of the redia is very slow. It will be recalled that the 
first rediae were freed from the sporocyst in 9 days and at the time 
contained developing cercariae 5 days old. The cercariae continued to 
develop in the redia for another 5 days before the first of the cercariae 
were born at an age of 10 days. Following their birth these oldest 
cercariae developed for another 22-day period in the liver of the snail 
before they made their escape. 

Size—Because of their size and heavy pigmentation the free- 
swimming cercariae are readily distinguishable while swimming if they 
are placed over a white background. The cercaria is extremely variable 


70 ILLINOIS BIOLOGICAL MONOGRAPHS 


in form, and since it is constantly active exact measurements are hard 
to secure. When fully extended the body may be three times its width 
and very flat. When fully contracted the width is slightly greater than the 
length and the body may be one-half as thick as it is wide. When fixed 
in warmed Bouin’s or corrosive sublimate fixatives the body contracts 
still more. In the contracted state the anterior end of the body is pulled 


ventrally. 


TABLE 12.—MEASUREMENTS (IN MIcRONS) OF TEN CERCARIAE EXTENDED, 
CONTRACTED, AND FIXED 


Body measurements Tail measurements 
No. 
Extended |Contracted| Fixed Extended |Contracted| Fixed 

Dee sare ee 302x151 | 151x235 | 168x184 | 670x47 | 352x100 | 336x 84 
ee eee 302 x 252 | 201x201 | 252x184 | 655x60 | 302x 841! 369x 84 
Stes eee eto ee 336x 168 | 168x252 | 176x189 | 672x50 | 420x 84 | 378x71 
a ee 386x117 | 189x231 | 218x184} 588x50 | 302x 84 | 554x67 
Suhre aeeenale 403x108 | 186x240 | 218x184! 630x50 | 302x 84] 554x67 
Osa.r een 420x134 | 235x268 | 235x210 | 588x60 | 319x 84 | 420x71 
(Eee re 431x156 | 245x303 |.216x%216 | 621x45 | .312x= 89.| °353:x55 
ul Fe: dans nee 451x184 | 196x235 | 176x196 | 686x58 | 294x° 98 | 333x59 
) ae ee 460x156 | 196x274 | 187x187 | 706x54 | 392x 89 | 460x 54 
10 sence te 490 x 134 | 235x314 | 319x268 | 672x70 | 470x100 | 420x 84 
Average..... 398 x 153 | 200x252 | 236x200 | 668x55 | 346x 89 | 417x69 


The tail size varies considerably also. When fully extended it may 
be approximately twice as long as when contracted, but when fixed it is 
usually slightly longer than when contracted. 

The measurements made on 10 cercariae when extended, contracted, 
and fixed are given in Table 12. When the average length of the body 
and tail of the 10 are combined the total length of the cercaria is 1.066 
mm when fully extended and only 0.546 mm when fully contracted. The 
same measurement on fixed specimens was 0.653 mm. 

Body Wall.—In contracted specimens the surface of the body is 
covered by a thin cuticula in which is seen a series of clear longitudinal 
lines running parallel to each other. Sewell (1922:75) saw similar lines 
in Cercariae Indicae and thought that they might denote longitudinal 
muscle fibers. However, since these lines are superficial in position, large 
and distinct, and can be seen only in living contracted specimens (not 
being seen at all in toto mounts or sectioned material) it is believed that 
they are only folds in the cuticula. 


Pigment.—The pigment of the body is distributed over the entire 
body in a solid layer which is about 3 times as thick on the dorsal and 


LIFE HISTORY OF COTYLOPHORON—BENNETT ie 


lateral surface as it is ventrally. A few pigment granules are scattered 
irregularly throughout the body (Fig. 58). 


Eyes.—The eyes are located far forward, lateral to and immediately 
posterior to the oral sucker (Fig. 51). They are conical in shape with 
the base located immediately beneath the cuticula and with the apex 
directed ventro-posteriorly in the body. The base is surmounted by a 
clear refractile lens in which are two nuclei representing the cells from 
which the lens is formed. The nuclei measure approximately 7 by 5 p 
and each contains a small distinct chromatin body. The cone is 30 p 
long and 18 ,» in diameter at its base. When the cercaria is swimming 
the anterior end of the body is drawn ventrally, leaving the eyes located 
at the most anterior and dorsal point of the body. 


Acetabulum.—The acetabulum in the relaxed condition in living 
cercaria measures 65 . in diameter. In contracted, fixed, and sectioned 
material the acetabulum is very much smaller. It measures 43 p. in 
diameter under such conditions. 


Digestive System.—An accurate study of the digestive structures 
cannot be made in either living cercaria or toto mounts because of the 
heavy pigmentation and the cystogenous glands. The extent of the 
esophagus cannot be determined and the caeca are not visible in living 
specimens. In toto mounts these structures can be seen indistinctly. The 
oral sucker is terminal and usually ovoid in shape, its length slightly 
exceeding its width. Its average size in living cercariae is 47 » in length 
and 46 » in width; in fixed sectioned material the average size is 32 by 
32 u. When the oral sucker is retracted a distinct oral cavity is formed 
which is lined by smooth cuticula. 

The esophagus originates near the ventral surface of the oral sucker 
and passes dorsally and posteriorly (Fig. 58). It bifurcates posterior to 
the eyes and in the dorsal part of the body to form the intestinal caeca. 
It is lined by a cuticula continuous with that of the oral sucker. The 
walls of the esophagus are thin at its origin but increase in thickness 
posteriorly. There is no definite sphincter muscle at its termination but 
the increase in thickness gives it a bulbous appearance. Deeply-staining 
glandular cells, the esophageal glands, completely surround the esophagus 
throughout its length (Fig. 47). . 

The caeca are small, measuring 15 1 in diameter, and terminate above 
the anterior margin of the acetabulum. 

Nervous System.—No parts of the nervous system can be seen in 
living specimens or toto mounts and only the larger parts can be traced 
through sections. The central nervous system consists of two ganglia 
connected by a transverse commissure. The ganglia are located directly 


dz ILLINOIS BIOLOGICAL MONOGRAPHS 


beneath the eyes and the commissure passes across the body dorsal to the 
esophagus, immediately posterior to the dorsal surface of the oral sucker. 
The ganglia are approximately 15 » in diameter and the commissure is 
9 » in diameter in the median line of the body. Each ganglion gives rise 
to several nerves. Three were found to pass anteriorly and terminate on 
the body surface lateral to the oral sucker, two being ventral and one 
dorsal. Another nerve passes dorsally and terminates in contact with the 
inner end of the eye (Fig. 66). A posterior nerve passes from the 
ganglion to the ventral surface of the caecum on the same side where it 
disappears from view. The ganglia, the commissure, and the nerves are 
entirely enclosed by numerous small nuclei which probably represent 
nerve cells. 


Reproductive System.—The primordia of the male and female re- 
productive systems are distinguishable in the mature cercaria. These 
primordia consist of two large groups of cells connected by a single cord 
of cells (Fig. 58). The most posterior mass which represents the ovary, 
Mehlis’ gland, and testes is located immediately dorsal to the anterior 
margin of the acetabulum. It consists of numerous deeply-staining cells 
and nuclei. This mass may vary from 22 to 29 p by 16 to 20 p in con- 
tracted specimens. From it a cord of cells passes anteriorly in the center 
of the body which connects with the second large group of cells located 
immediately posterior and slightly ventral to the intestinal bifurcation. 
From its ventral side a cord of cells passes ventrally, terminating against 
the surface of the body. Its point of termination marks the position of 
the genital pore but no opening could be found in the cercaria. The cells 
of these primordia are entirely similar. 


Excretory System.—The excretory system of the mature cercaria is 
essentially the same as in the immature cercaria. The presence of the 
heavy pigmentation and the cystogenous glands makes it impossible to 
determine the flame cell pattern or the position of the smaller excretory 
tubules. Sixteen flame cells were found in developing cercariae irregularly 
distributed but principally around the acetabulum and oral sucker prior to 
the spreading of the pigment to these regions. Consequently, only the 
larger units are described here. 

The excretory bladder is located dorsal to the acetabulum and the 
posterior genital complex (Fig. 58). A short cuticula-lined duct passes 
from its anterior end dorsally to the excretory pore located in the dorsal 
median line above the anterior margin of the acetabulum. In extended 
cercariae there may be a slight change in these relationships. In such speci- 
mens the duct passes dorso-anteriorly from the bladder and the excretory 
pore may be as much as 10 or 15 p» anterior to the acetabulum. Two large 
excretory ducts join the posterior lateral margin of the bladder. From 


LIFE HISTORY OF COTYLOPHORON—BENNETT 73 


this point they pass outward and forward for a short distance and then 
turn inward. Slightly posterior to the middle of the body the cross con- 
nection previously described passes across the median line and sends off 
a small median diverticulum. The main ducts are ventral to the caeca 
but the cross connection passes dorsally and the diverticulum is located 
near the dorsal surface of the body a short distance posterior to the 
intestinal bifurcation. No tributaries were seen emptying into this 
diverticulum. Anterior to the cross connection the main ducts turn out- 
ward and then turn inward again posterior to the eyes. They pass for- 
ward ventral to the eyes and ganglia to reach the sides of the oral sucker. 
Here they turn sharply and pass back to the posterior end of the body, 
terminating near the acetabulum. Diverticula are formed on the main 
ducts also immediately posterior to the eyes. As in the median diverti- 
culum no ducts were seen opening into them. 

The main ducts from the bladder to the oral sucker, the cross con- 
nection, and the diverticula were filled with excretory granules varying 
from 2 to 10 » in diameter.. The smaller granules were located in the 
extremities of the ducts while the larger ones were located in the main 
ducts near the center of the body. 

The wide caudal excretory tube joins the oe duct just before 
it reaches the pore. It passes backward through the center of the tail 
and enlarges to form a small bladder at its posterior end a short distance 
from the end of the tail. From this enlargement a short duct passes 
posteriorly and laterally on each side to reach the surface. These ducts 
are the ends of the two original excretory ducts of the very young 
cercaria but the presence or absence of an opening for these was not 
determined for older ones. 


Free-Living Cercariae.—It was observed that these cercariae escaped 
from the infested snails during a definite period each day and at no 
other time unless artificially stimulated. The time and numbers of 
cercariae escaping from individual snails are shown in Table 13. The 
snails used in this experiment were infested on May 5, 1934, and began 
shedding cercariae on June 5, 1934. There were 35 snails in this group, 
all of which were shedding cercariae at the same time, but only 15 were 
used to illustrate the periodicity of shedding. 

Five snails were placed in individual finger bowls which contained a 
small amount of water and a piece of lettuce. The size and pigmentation 
of the cercariae makes them easy to see, especially when placed over a 
white background. It had been observed previously that the snails would 
shed cercariae at any time if they were kept out of water for as long as 
24 hours and then put back into water. The first snails used in this 
experiment were taken from an aquarium (in which water was present at 


74 ILLINOIS BIOLOGICAL MONOGRAPHS 


all times) at 8:00 a.m. on June 14 and placed in the finger bowls. The 
cercariae began escaping a short time after 11:30 a.m. and continued to 
escape until 5:30 p.m. The snails were under observation continuously 
and the cercariae were removed as they emerged. Group 1 of Table 13 
shows the results of this experiment. The snails were kept under obser- 
vation in the bowls until 2 a.m. June 15 but no more cercariae escaped 
until about noon of the following day. A similar experiment was made 
with 5 other snails on June 15 and June 19. The results indicate definitely 
that cercariae escape in largest numbers during the brightest hours of the 


TABLE 13.—SHOWING PERIODIC SHEDDING OF CERCARIAE BY THREE DIFFERENT 
Groups OF FIvE SNAILS EACH 


Group I Group II . Group III 
Time 
June 14, 1934 June 15, 1934 June 19, 1934 

10:30-11:30| 0) 0} 0).0)] 0} 0] 0|-.0} 0) (0) 0} 0) OOo 
11:30-12:30} 55 5 (104 | 0 1-5 1310 7 | 32 | 32 | 14 |126 |125 | 184)" (00/838 
12:30-1:30 4 | 88 |101 0} O}:9 1170 | 4] 16 |-23 |-39 | 45-| - O07) 2054 
1:30-2:30.. 0} 0; Of} 90} O| 21 | 46] 2 8 1 014 | 0) (O° xO 0 
2:30-3:30. 0 | 27.) 20 1 0 1/72) 0} 0)] 0] 164 O% OE. Os RRO 
3:30-4:30.. 0 1 S2 40, OO) OO” Oe: 68 1 0; 0 1 0 
4:30-5:30.. 01 °01/121 0} O} 0) 0} 20) 07 0) 25) 70) Oy Sore 
5:30-6:30.. 0; 0] Of O} 0} OF OF} O] OF O} OF Ofe-OFPxOF iO 

Total 59 |119 |249 | 95 5 1341 |225 | 38 | 56 | 38 {201 |170 | 18 1 |142 


day. June 15 was a rainy day and much darker than either June 14 and 
19, and cercariae escaped through a period of 3 hours only, while on the 
other days which were very bright this period increased to 6 hours in 
some instances. This response of the cercariae to light is demonstrated 
by free-swimming cercariae also. Immediately after escaping they collect 
in the dish where the light is most intense, and in a short time begin to 
encyst at the surface of the water. If vegetation is placed in the dish the 
majority of the cercariae encyst on the part at which the light is most 
intense. However, they select the vegetation for encystment in preference 
to the glass even though the light is less intense near it. This response 
to light is further demonstrated by the fact that cercariae make their 
escape at any time of the day if the snails are placed directly beneath a 
strong light. This response is most easily demonstrated in the following 
manner. If a number of cercariae are drawn into a pipette which is then 
held perpendicular to the edge of a lamp shade covering a lighted electric 
lamp with the pipette exposed to the light, the cercariae move back and 
forth in it. If the pipette is then drawn past the shade so that the upper 
end is darkened the cercariae move into the lighted area and never leave 


LIFE HISTORY OF COTYLOPHORON—BENNETT Za 


it for more than a few seconds. By continuing to raise the pipette the 
cercariae can be concentrated at the end of the pipette. This method of 
concentration is rapid and useful in securing large numbers of cercariae 
in a minimum amount of water. 

The cercariae are active swimmers but do not swim for long periods. 
After emerging from the snail they swim in a small circle for a short 
time and then proceed to the most illuminated part of the dish. They 
swim for a few seconds and then drop to the bottom of the dish or come 
to rest on small bits of débris or vegetation for a short time. At the sides 
of the dish they swim at the surface briefly then settle slowly to the bot- 
tom but are soon back at the surface again. This activity may continue 
for as long as an hour if there is no vegetation on which to encyst but 
none were found to remain unencysted for longer periods unless con- 
stantly disturbed. If vegetation is present some cercariae begin to encyst 
at once but others require as long as 30 minutes. They encyst readily on 
many kinds of vegetation but lettuce was used in most instances. If 
cercariae are place in the hollow of a lettuce leaf they collect in the small 
folds of the leaf but always on the side turned toward the light. 

The number of cercariae escaping from the snails (Table 13) was 
observed to vary considerably. If a snail produced a large number of 
cercariae in one day, it was 8 days before any more escaped in some 
instances. However, the time interval may be as short as one day, but 
the number emerging on the second day is always small. That this 
variation is not due to a loss of infestation was demonstrated by dissecting 
some of the snails shown in Table 13 after an interval of 4 or 5 
days during which no cercariae were shed. In every snail examined many 
developing rediae, mature rediae, and developing cercariae were found in 
large numbers. 

Cort (1922:183) observed that cercariae escaped at definite periods 
each day and that these periods remained the same for specific cercariae 
but differed for different species. He also observed that there was con- 
siderable variation from day to day in the numbers produced. Krull and 
Price (1932:20) observed that the cercariae of Diplodiscus temperatus 
escaped at any time of the day but that largest numbers escaped between 
11:00 a.m. and 5:30 p.m. They also observed that there is a very definite 
heliotropic response in these cercariae. Krull (1934:175) found that the 
cercariae of Cotylophoron cotylophorum escaped from the snail host 
between 8:30 a.m. and 1:00 p.m., the peak being about 8:30 a.m. This 
observation shows that the cercariae escaped periodically, but the time at 
which the largest numbers are shed is quite different from the findings in 
the present observations on the same species. Krull also found that these 
cercariae occasionally escaped at other hours of the day. 


76 ILLINOIS BIOLOGICAL MONOGRAPHS 


Krull and Price (1932) and Krull (1934) noted that the number of 
cercariae escaping from the snails increased the length of the period of 
escape. That this was not the case in the present experiments can be 
seen from the results shown in Table 13. Several instances are shown in 
which a larger number of cercariae escaped in less time than that required 
for the escape of smaller numbers from other snails. 

The length of time snails remain infested was not determined. How- 
ever, the time would depend on the rate of development of the various 
stages. In the present work snails infested during the coldest months of 
the year shed cercariae only after 91 days, and in the warmest months 
cercariae were shed 30 days after infestation of the snails. That a definite 
number of rediae is produced by each sporocyst and a definite number of 
cercariae is produced by each redia has been pointed out. Consequently 
the snails do become free of infestation but only after a prolonged 
period. Under optimum developmental conditions the sporocyst remains 
productive for fully 30 days, the redia for another 30 days, and the 
cercaria requires 22 days to develop after leaving the redia. Thus under 
theoretical optimum conditions snails remain infested for at least 78 days 
and probably remain infested under natural conditions for periods ranging 
from 4 to 6 months, which is doubtless equal to or exceeds the life of the 
snail. Krull (1934:175) was able to keep infested snails alive for 6 
weeks after cercariae began to escape, 36 days after infestation. The 
number of days these snails remained alive after infestation, 76 days, is 
very close to the theoretical time limit they will remain infested under 
optimum conditions. Krull’s work was done in the summer months of 
May, June, July, and August which may be considered as the optimum 
developmental period for the parasite. 


Discussion OF PREVIOUSLY DESCRIBED AMPHISTOME CERCARIAE 


Sixteen species of amphistome cercariae have been described, two of 
which have been considered doubtful. Cary (1909:604-607) described an 
amphistome cercaria which he considered to be that of Diplodiscus 
temperatus. However, Cort (1915:23-30) has pointed out that Cary was 
mistaken in considering the cercaria he studied as being that of D. 
temperatus. Ward (1916:17-19) described Cercaria gorgonocephala and 
expresses his own uncertainty as to its exact systematic position, and as 
it is not closely related to the cercaria of C. cotylophorum it needs no 
further consideration here. 

Looss (1892:162-166) described in detail the cercaria of Diplodiscus 
subclavatus. Later he (1896:185-191) described the cercariae of Param- 
phistomum cervi and Gastrodiscus aegvptiacus (1896:177-185). His 


LIFE HISTORY OF COTYLOPHORON—BENNETT 77 


conclusions in the latter case, however, rest on the structural comparison 
of the cercaria and the adult. 

Cort (1915:24) described Cercaria diastropha and C. inhabilis and 
called attention to the fact that the 5 species of amphistome cercariae 
then described belong to two subfamilies of the Paramphistomidae. He 
assigned the cercaria of Paramplistomum cervi to the subfamily Param- 
phistominae; and Cercaria diastropha and C. inhabils, the cercaria of 
Diplodiscus subclavatus, and that of Gastrodiscus aeygyptiacus, he assigned 
to the subfamily Diplodiscinae. Sewell (1922:66) accepted Cort’s classi- 
fication and added 3 new species, Cercariae Indicae XXvi, XXIx, and 
xxx to the former group; and C. frondosa Cawston (1918), C. corti 
O’Roke (1917:165-180) and a new species C. /ndicae xx1, to the latter. 
Sewell omitted C. convoluta Faust (1919:172), but Faust states that it 
probably belongs to the Diplodiscinae. 

Sewell (1922:67, 80) prepared a key for the separation ot two dis- 
tinct groups for which he proposes the names ‘“‘Pigmentata”’ and 
“Diplocotylea.” He assigned the cercaria of Paramphistomum cervi, 
Cercariae Indicae XXv1, XXIx, and xxx1r to the former group, and the 
other cercariae mentioned above he assigned to the latter group. To this 
latter group, Diplocotylea, McCoy (1929:200) added Cercaria missourt- 
ensis. Beaver (1929:20) described the cercaria of Allassostoma parvum 
and assigned it to the Diplocotylea also, and he points out that Ad. parvum 
belongs to the subfamily Schizamphistominae Looss 1912. He further 
points out the mistake made by Cort (1915:24) in assigning amphistome 
cercariae to subfamilies based on larval characteristics only, since the 
diagnostic characteristics of amphistomes are restricted to characters 
which are not present in the larval forms. His conclusion that only 
Sewell’s classification can be rightly used is considered to be entirely 
correct. 

C. cotylophorum belongs to the Paramphistominae, so that the present 
cercaria may be assigned to that subfamily. The cercaria possesses all of 
the characteristics of the “Pigmentata” and can be assigned to that group 
also. 

This cercaria can be readily distinguished from that of P. cervi by 
its much smaller size, shorter tail, smaller suckers, and particularly by 
the presence of the evaginations from the excretory vessels. These 
evaginations are lacking in the cercaria of P. cervt. 

Cercariae Indicae xxvi differs from the present cercaria in that C. 
Indicae is very much larger; the suckers are nearly twice as large; the 
esophagus is narrow without any trace of a pharynx or sphincter muscle ; 
the genital system is well developed and shows a differentiation into the 


78 ILLINOIS BIOLOGICAL MONOGRAPHS 


respective organs, and the vitelline glands are well developed. In view of 
these differences there can be no doubt that the two cercaria are entirely 
different. 


Cercariae Indicae XX1x presents fewer differences than the two above 
but can be distinguished from the present cercaria by a few characteristics 
of diagnostic value. This cercaria is slightly larger than the cercaria of 
C. cotylophorum yet possesses suckers almost twice as large; there is no 
thickening of the circular muscle layer around the esophagus, and the 
“anlage” of the vitelline glands are present. Consequently, these two 
cercariae must be considered as different species. 

The only other cercaria assigned to the ‘‘Pigmentata” group is C. 
Indicae xxx. This form is readily distinguished from the present 
cercaria also. C. /ndicae xxxi1 is slightly larger and possesses an oral 
sucker 3 times as large, and an acetabulum more than twice as large as 
these structures are in the cercaria of C. cotylophorum. It differs also in 
possessing a very long esophagus with a well marked sphincter muscle 
at its posterior end, and in that the excretory pore opens posteriorly. 

It is quite evident from a comparison of these forms that the cercaria 
of C. cotylophorum has not been described previously. Le Roux (1930: 
247) mentions finding an amphistome cercaria in a snail, Bulinus schako1, 
which he considers as practically identical with C. frondosa Cawston as 
described by Faust (1919:172), and which he thinks is the cercaria of 
C. cotylophorum. He did not describe it, however, and it is impossible to 
determine what cercaria he found. Faust describes C. frondosa as having 
pharyngeal pockets and does not describe the cross connection between 
the excretory ducts which is characteristic of the group to which the 
cercaria of C. cotylophorum belongs. The possession of pharyngeal 
pockets and the absence of the connecting duct clearly places C. frondosa 
in the “Diplocotylea” group. Consequently, if the cercaria found by Le 
Roux were C. frondosa it was not the cercaria of C. cotylophorum. 


METACERCARIA 


Cercariae remain free-swimming from 10 minutes to an hour after 
escaping from the snail, but the usual time for them to remain unencysted 
when vegetation on which to encyst is present is from 20 to 30 minutes. 
When encystment begins the cercaria actively elongates and contracts the 
body while the tail is beating violently. This movement alternates with 
one in which the body is thrown from side to side by the movements of 
the tail as in swimming, but no progress is made. At the same time the 
cystogenous material is breaking down and flowing out all over the body 
surface. The cuticula seems to loosen and the body contracts until a con- 


LIFE HISTORY OF COTYLOPHORON—BENNETT 79 


siderable space is left between it and the cuticula. The cystogenous ma- 
terial collects on this membrane, producing at first a roughened surface 
both externally and internally. The cercaria is constantly in motion during 
this process, twisting the body from side to side. These movements 
smooth the internal wall of the cyst and distribute the cystogenous 
material evenly. The tail continues to beat rapidly, and as the cyst is 
formed it becomes loosened from the body but may remain attached to 
the cyst wall for some time. When freed from the cyst it swims away 
and continues to swim for as long as 8 hours. The process of encystment 
is usually complete after 20 minutes but the metacercaria may continue to 
contract from side to side for a much longer period. 

The completed cyst is round in surface view, and dome-shaped in 
lateral view, since it is slightly flared at the base (Fig. 53). The base is 
hollow and acts as a support for the remainder of the cyst. The cyst 
proper averages 154 yw in diameter, and the base 184 ,; the total 
height averages 130 uw. The cyst wall is transparent but appears whitish 
to the unaided eye. The metacercaria retains its pigmentation, and the 
only structures even slightly visible are the oral sucker, the acetabulum, 
the eyes, the excretory pore, and the longitudinal striations in the cuticula 
described as being present in the contracted cercaria. 

Under optimum conditions the metacercaria probably lives for several 
months as was pointed out by Krull (1934:176). To determine the 
longevity of the metacercaria 50 cercariae were allowed to encyst on the 
bottom of small dishes which were set aside and covered. Encystment 
occurred on June 5, 1934. On August 5, 1934, 56% were alive, and on 
September 5, 1934, 33% were alive. This experiment was carried no 
further but it is evident that under optimum natural conditions the 
metacercariae may live as long as 5 or 6 months. Krull kept them alive 
for 5 months under experimental conditions. 


ADULT 


EXPERIMENTAL JNFESTATION 


Development of Amphistome Parasites in the Final Host.—The infor- 
mation on this subject is very meager, consisting of chance observations 
and of only one concluded experimental problem. Looss (1892:166) 
observed that Diplodiscus subclavatus develops slowly in frogs during the 
winter months but he did not determine the time required for the worms 
to become mature. Beaver (1929:13) observed that a cercaria from 
Planorbis trivolvis was found to encyst on crayfish and frog larvae, and 
when fed to bullfrogs (Rana catesbiana) and snapper turtles (Chelydra 
serpentina) developed into a known species, Allassostoma parvum 


80 ILLINOIS BIOLOGICAL MONOGRAPHS 


Stunkard 1916. However, Beaver did not determine the time required for 
this species to become mature. Krull and Price (1932:34) determined 
experimentally that in lightly infested frogs Diplodiscus temperatus 
reaches maturity in 27 days but that in most hosts 2 or 3 months is the 
usual time required. Le Roux (1930:248) states that young forms of 
C. cotylophorum probably live in the duodenum of the final host 6 to 8 
weeks before migrating into the rumen, where they become mature in 
another 6 to 8 weeks. Le Roux’s statement was not supported by experi- 
mental evidence. Krull (1934:177) determined experimentally that C. 
cotylophorum matures in the latter part of the fourth month after 


TABLE 14.—SHOWING RESULTS OF EXPERIMENTS IN WHICH CALVES WERE FED 
METACERCARIAE OF Cotylophoron cotylophorum 


Waresor Daren Number of | Number of Number. of 


Host i sfesent aera metacer- worms in worms in 
saint cis Soe cariae fed | duodenum rumen 

Galt enue Nov. 19, 1933 | Jan. 4, 1934 150 0 6 
June 4, 1934 | 300 

CalfIhi" 33.2. June 11, 1934 |} Jan. 25, 1934 300 199 4 
Tune 18, 1934 || 300 
June 4, 1934 300 

Cali Vee. June 11, 1934 300 
June 18, 1934 |{ July 25, 1934 | 399 id 34 
June 25, 1934 300 


infestation. His determination was based on the findings of eggs in the 
faeces of the host. Krull did not attempt to determine the age or size at 
which the parasite migrates from the duodenum to the rumen nor the 
size at which the parasite becomes mature after reaching the rumen. 


Experimental Infestation of the Final Host of C. cotylophorum.—On 
June 28, 1933, two 7 or 8 months old calves were received at the animal 
pathology department of Louisiana State University. These animals were 
kept in a dry, well ventilated room when being used for experimental 
purposes but were allowed to graze in a small grassy plot at other times. 
No snails were present in this area so that there was no opportunity for 
the calves to become infested by C. cotylophorum. 

These calves were obtained for experimental work in connection with 
this problem early in November, 1933. They were placed in the room 
previously mentioned on November 10, where they remained until the 
end of the experiment on January 4, 1934. The faeces of both calves 
were examined several times for the eggs of C. cotylophorum but were 
found to be negative. On November 19, 1933, they were separated by a 


LIFE HISTORY OF COTYLOPHORON—BENNETT 81 


partition placed in the room, and Calf I was fed 150 metacercariae. 
Calf Il was kept as a control (Table 14). Subsequently both animals 
were given the same type of feed and were given water from the same 
source. 


TABLE 15.—SizeE (IN MILLIMETERS) OF Cotylophoron cotylophorum IN RUMEN OF 
EXPERIMENTAL CALVES 
Note: In Calf I, infested 46 days, 6 worms were found in the rumen; in Calf III, 
infested 21 days, 4 worms; in Calf IV, infested 51 days, 34 worms (sizes of 20 are given 


here). 


No. Calf I Calf III Calf IV 
detects has ae ees eh anche Sap Aoi a eile fal 2.06x 0.63 1.95 x 0.78 
Dea Mer aics Mev tn tay os OREEDLS 2.10x 0.63 1.95x0.91 
SIS AE Mitte ote osc ens cae es 355X261 2229 S015 2.08x0.72 
A Seen a eet merch 3.60 x 2.20 2.95 x 0.84 2.08x 0.85 
Dei eaera a clske teeta tue a. SOFC 2 008 I aware: 2.13 x 1.04 
Opnara deter e Saneaanis 6 Oe OrOo es 2.45 ee | ceteerdas.ae 2.28x0.85 
[MTR ees) wesitceistascce | nae eas eben 2.31x0.91 
Sere Cree | 5 oer c rc ccce ml |) Oe wether eshte 2.34x0.80 
Oe ej ase oe NL Meter aaies ahi 2.34x0.96 
HORM) sinc atci cate) | UT Ogee eda sbictean 2.34x 1.04 

eel | sae naticarr: , UNL eri wnwhaterddien 2.47x 0.85 
(Qe eye e eer | cu ccoicnicis al Ol) @ onda beans 2.54x0.78 
Ree eRe err ett A aitnietatotea fe | aie ce vevour eh 2.60x 0.78 
Lee erence || erika ccc 6 1) = BAUS cde Vat 2.60x 0.85 
[Seer wanes Peas, \p af athe sage 2.60x0.91 
ING} Soh Gevsich Gh © © Ss are oc) Se Um ee ete | 2.60 x 0.98 
IGA sec ws ef Bec Le AD cote A OS eae a a 2.62 x 0.85 
Loe r errr sw ctiesdtos, |) 9 aad Grd eo 2.70 x 0.83 
LORE ce eer) Cetceani@scor.. 1 ame sc ain gue 2.73 x 0.88 
PAU Beiter teeect Ae Peet | estate sic, we Qi [hy > ~-chaidead ar etare 2.86x0.99 

AVCV EGER elite sess oe Ree. ony 2.32)x.0. 71 2.41x 0.84 


The faeces of both calves were examined at 2-day intervals after the 
twenty-first day of infestation. However, no eggs were found. Both 
animals were killed on January 4, 1934, aiter 46 days of infestation, in an 
attempt to establish the time of migration of the parasites. No parasites 
were found in Calf II, but 6 small mature specimens of C. cotylophorum 
were obtained from the rumen of Calf I. None was present in the 
duodenum. The parasites recovered varied in length from 3.25 to 3.85 
mm and in width from 1.71 to 2.45 mm (Table 15). Only a few eggs 
were present in the uterus and only a few were deposited by the worms, 
although they were kept alive in physiological salt solution for 4 hours. 

The presence of mature specimens in Calf I indicates that maturity 
is reached in less than 46 days, or that the worms were present as a 
result of natural infestation before the calf was confined and were 
depositing so few eggs that they were missed in the faecal examinations. 
The latter supposition is doubtless correct in view of the fact that Krull 


82 ILLINOIS BIOLOGICAL MONOGRAPHS 


(1934:177) determined in a series of experiments that this parasite 
becomes mature in approximately three and one-half months. Unfor- 
tunately, Krull did not determine the size at which this species becomes 
mature nor does he give the size of the worms recovered from the rumen 
of one of his experimental animals. However, Krull kindly loaned me one 
toto mount and one specimen serially sectioned, and a comparison of the 
present material to his is possible. Krull’s specimens were approximately 
6 months and 20 days old and the present material was 6 months and 7 
days old, if we assume that Calf I became infested a short time before 
being confined. A comparison of size of the oral sucker, the esophageal 
thickening, the acetabulum, the testes, ovary, Mehlis’ gland, and the 


TABLE 16.—SHOWING NUMBER, SIZE (IN MILLIMETERS), AND DISTRIBUTION OF 
Cotylophoron cotylophorum IN THE DUODENUM OF EXPERIMENTAL CaLF III 


Ne 1st foot 2nd foot 3rd foot Ath foot 5th foot 6th foot 
, (54 present)|(40 present)|(34 present)|(14 present)| (6 present) |(12 present) 

i ke eee 1.22x0.72 | 0.99 x 0.36 | 1.35x 1.05 | 2.08x0.78 | 1.61x0.39 | 1.56x 0.80 
Die Bae 1.50x 0.95 | 1.43x0.85 | 1.43x0.75 | 2.26x 0.99 | 1.90x 1.06 | 1.97 x 1.06 
S| ena ae 1.69x 0.93 | 1.45x 1.05 | 1.43x0.85 | 2.39x 0.80 | 2.50x0.93 | 2.26x0.85 
At Shans 1.60 x 0.80 | 1.69x 0.96 | 1.71x 1.04 | 2.60x 0.78 | 2.54x 1.04 | 2.54x0.91 
Se eae 1:90'x 0,95. 1,85x 1,10.) 2.13% 1:04 |-2:67 x ROL | 275 x DOL) 2 oUF 
ORS ete 2.00x 0.85 | 2.13x 1.04 | 2.30x 1.15 | 2.73 x 0.93 | 3.09x 1.04 | 2.75 x 0.96 
ee 2:00'x 1.05°| 2.25% 05751-2550 x1.25 | 25/5 x 1,01 | 2k 2.75 x 1.01 
toe reset 2.05x 1.10 | 2.25x0.80 | 2.70x1.05 | 2.76x1.04| ........ 2.149 x%41.14 
OAR cer 215:X.1.05) | 22550195 2575 x1 U5" 12?8050 1806) | eee 2.99 x 1.04 
LO ass 3105.x0:95 |2,60ise1510) 12580105: | 2.99101) fio ee 3.09 x 1.04 
Average..| 1.94x 0.94 | 1.95 x 0.90 } 2.11x 0.96 | 2.61x 0.94 | 2.23x 0.90 | 2.63 x 0.99 


genital sucker of Krull’s sectioned material and similar structures in the 
smallest of the worms from Calf I demonstrates that the specimens are 
approximately the same age. The finding of no immature specimens in 
either the rumen or the duodenum indicates that none of the metacercariae 
fed to the calf developed. 

This experiment was repeated in another attempt to determine the 
time of migration of parasites from the duodenum to the rumen. Two 
4 months old calves which had not had access to infestation were obtained 
and kept under conditions similar to those of the first experiment. Calf 
III was fed 300 metacercariae on June 4, 1934, 300 on June 11, and 300 
on June 18, making a total of 900 fed at 7-day intervals. Calf IV was 
fed a total of 1200 metacercariae at 7-day intervals from June 4 to 
June 25. 

Calf III was examined on June 25, 1934, 21 days after the first 
infestation, and 203 immature specimens of C. cotylophorum were 
recovered. Of these, 199 were distributed in the anterior 6 feet of the 


LIFE HISTORY OF :COTYLOPHORON—BENNETT 83 


duodenum and 4 were found in the rumen. The other parts of the 
stomach were examined but no parasites were found. The smallest 
specimen from the duodenum measured 0.99 by 0.36 mm and the largest 
was 3.09 by 1.04 mm. There was no distinct grouping according to size 
so that it was impossible to determine the exact age of any one specimen. 
However, it may be safely assumed that the smallest worms were only 7 
days old, whereas the largest were 21 days old. The large and small 
individuals were distributed irregularly in the duodenum so that it was 
again impossible to group them according to their distribution (Table 16). 
However, the largest number of small specimens was found in the upper 
3 feet. of the duodenum, indicating that the metacercariae probably excyst 
in this region. Most of the worms distributed posteriorly were much 


TABLE 17.—SHOWING NUMBER, SIZE (IN MILLIMETERS), AND DISTRIBUTION OF 
Cotylophoron cotylophorum IN THE DUODENUM OF EXPERIMENTAL CALF IV 


No. 1st foot 2nd foot 3rd foot 4th foot 5th foot 
(eee Re are 2.13x1.04 | 1.71x0.78 | 2.21x0.88 | 1.69x0.83 | 2.41x0.80 
DAG et Grates cos Deen 2.31x0.96 | 2.08x0.78 | 2.34x0.88 | 2.36x0.91 | .......... 
SR eae De sac OrOle|e22 62x. 0872. | 234 OC9L Wn uw tes ae. || ceca sete oe 
AR te eet hs DESO 1 O4 oer ereae. D3 OxO Soy ll ko cee a sesssevn Clk seseeosee wiace 
Se rere eerie. toe. Ilan siSeaeh DEAT OL OGR Serre ets ener ee 
Oe ers ns aN oe eS asdsaletes DESO OP 96ell Soak. ohare Al oars eee tr 
pee erent Se No oom hyo oe | aretha alte ys De SACO Sele es ee aeons tia tsere okt 
CP ee ie lata etry || od gh Straten 2360 6.092 |b githc huss tote. We fees a oe 
ORE siecle bl) Agliaaeenses DRT SOOO eects ildeeis eae: 

Average...... 2.28x0.99 | 1.60x0.76 | 2.44x0.91 | 0.02x0.87 | 2.41x0.80 


larger than the smallest worms, indicating that the worms either migrate 
after becoming excysted anteriorly or that some metacercariae are carried 
farther before being liberated. The average size of the 199 specimens 
was 2.28 by 0.94 mm. 

The 4 specimens found in the rumen of this host indicate that migra- 
tion begins at the end of the first 3 weeks after infestation. These 
specimens varied from 2.06 to 2.95 mm in length and from 0.63 to 0.84 
mm in width (Table 15). The average size of the 4 was 2.32 by 0.71 mm. 

Calf IV was examined on July 25, 1934, 51 days after the first 
infestation and 30 days after the last infestation. Nineteen specimens 
were recovered from the first 5 feet of the duodenum, 34 from the 
rumen, and 1 from the pylorus (Table 17). Those from the duodenum 
were much more uniform in size than those from the duodenum of Calf 
III (Table 16). The smallest specimen measured 1.95 by 0.78 mm and 
the largest 2.86 by 0.99 mm. The average size of the 19 from the 
duodenum was 2.32 by 0.75 mm, while the average size of the 34 from 


84 ILLINOIS BIOLOGICAL MONOGRAPHS 
the rumen was 2.33 by 0.83 mm. The one specimen from the pylorus 
measured 2.43 by 0.81 mm. 

The small number of parasites present in this host and their uni- 
formity in size indicates that probably only one group of metacercariae 


TABLE 18—DatTA ON SIzE (IN MILLIMETERS) OF Cotylophoron cotylophorum IN TRE 
DUODENUM AND RUMEN OF NATURALLY INFESTED Hosts 


Age of host: 2 
Age of host: 6 months Age of host: 6 months years (none pres- 
(16 present in duodenum) | (7 present in duodenum) | ent in duodenum) 
a: (7 present in rumen) (39 present in rumen) (128 present in 
No. rumen) 
Duodenum Rumen Duodenum Rumen Rumen 
(7 smallest) (20 smallest) 
Neca arte 1.04x0.41 | 2.54x1.17 | 0.96x0.83 | 2.44x1.19 1.°82'x'1,:22 
oD puss asthe 1.22x0.52 | 2.62x1.30 | 1.35x0.78 | 2.60x 1.30 1.87x 1.08 
Sieh 1.97x0.80 | 2.63x1.04 | 1.43x0.85 | 2.62x 1.56 1.92x 1.04 
Aide apt eae 2.00x0.91 | 2.70x0.98 | 1.56x0.91 | 2.71x1.56 1.92x1.17 
Sch soe 2.10x0.96 | 2.99x 1.22 | 1.87x1.06 | 2.83x1.53 2.00 x 1.06 
ORM gic: 2.15x0.85 | 3.12x1.30 | 2.08x 0.98 | 3.12 1.45 2.00x 1.17 
Nex Roraa 2.21x0.65 | 3.30x1.01 | 2.39x0.78 | 3.12 x1.58 2.02x1.17 
Oia D2 SX OLS uly va eo te tee, een ate Me oe 2.05x1.17 
Oren ae DAZ Orxa OYA Oia Ws hee teeny | en ae rere rere || pays Aen 2.08 x 1.04 
Oe Mee eyes 2520 Sa0 Gln) Casa heck at ee ee ee erenea nae 2.08x 1.09 
1 eee se DDO: S80 BhSs | Bs uses. ee ee | oe ce een | ee nee ee 2.08x 1.11 
A ee De SAO 18h Mee) ae eek | eet Sere: Gee eee 2.08x 1.17 
13 race DIS OXI" OSG Tie. 4s Seco een | eee ae eee | ioe eer ee 2.08x 1.17 
1 eae DPS 2 XAOS Bia ce ecco eae Meek eerie cet || coe 2 AS x71 
WSR acts 2EO2 50 ORO SM ime serves on mee rtetpty ee mei | eevee epee De 2 lexelet 
Glee sere Pei Oo e{ 0 Fao Kea gamers Ao | ae AG, PaeOar Poe gee 0d 2h 2.21x 1.30 
Lak Seah oh aeavirah oe: bie a. oe) Rie rice, eal Mee eae ne eee FIP nas eer oe 2.26x 1.06 
US eee saves tsvel| py as ees e og te Se al Se | DOS, Sueseeeateer em ap rere 2.34x1.17 
LO ree eats cclll Marlatt qe ettarsionesa||4 goed care ees rn | sg eee ae Ome | ete en ee Qe] XA 22 
DO on camnal we 8 chet: Galas dean! Vete Sate ASG | dan eee 2°52:x,1. 30 
Average} 2.15x0.72 | 2.86x1.15 | 1.66x0.88 | 2.78x 1.45 2.10x1.14 


fed to the calf produced the infestation. The average size of worms from 
the duodenum of Calf IV is only slightly greater than that of the worms 
from the duodenum of Calf III, which seems to indicate that it was the 
last feeding of metacercariae to Calf IV which produced the infestation. 
A comparison of the average size of the parasites from the duodenum 
and the rumen of Calf IV clearly indicates that the worms were 
migrating and that very little growth occurs during their passage through 
the other parts of the stomach. That this passage is rapid is indicated 
by the fact that only one specimen was found in other parts of the 
stomach. The variability of the size of the worms in the rumen com- 
bined with the fact that there is a decided overlapping of size with those 
in the duodenum indicates that the worms migrate singly and that the 


LIFE HISTORY OF COTYLOPHORON—BENNETT 85 


migratory period for any group of worms of the same age may extend 
over several days. Thus in Calf III, only 4 of 203 worms had migrated 
at the end of 21 days, and in Calf IV, 34 of 54 had migrated at the end 
of 30 days. 

The size at which the worms migrated from the duodenum to the 
rumen in the experimental animals can be correlated with findings in 
naturally infested hosts. Many naturally infested animals were examined 
which contained the parasites in both the duodenum and the rumen. 
Others were examined in which only the duodenum or rumen was 
infested. Three such cases are presented in Table 18. The host repre- 
sented in the first column was infested by 128 specimens in the rumen, 
15 of which were mature. The average size of 20 of the smallest speci- 
mens is 2.1 by 1.14 mm. These worms are slightly shorter but wider than 
those from the rumen of experimental animals. 

The parasites shown in column 2 of Table 18 were collected from 
the duodenum and rumen of a 6 months old calf. Sixteen were found 
in the duodenum which had an average size of 2.15 by 0.79 mm. Only 
7 were found in the rumen. These averaged 2.86 by 1.15 mm. The 
parasites shown in column 3 were collected from another 6 months old 
calf. In the duodenum of this calf there were 7 parasites which averaged 
1.66 by 0.88 mm. There were 39 specimens in the rumen, the largest of 
which measured 5.33 by 1.82 mm. The average size of 7 of the smallest 
specimens was 2.78 by 1.45 mm. 

In these three cases there is further evidence that the worms migrate 
from the duodenum into the rumen of the final host when considerably 
less than 3 mm in length. A graph made using the data on specimens 
from both experimentally and naturally infested animals demonstrates 
that the greater number of worms migrate at a size of 2.37 by 0.98 mm. 

From the above data it is possible to conclude that the metacercariae 
become excysted in the duodenum where they develop for 3 to 5 weeks. 
Following this period they migrate to the rumen’ at an average size of 


2.37 to 0.98 mm. 
DEVELOPMENT 


The metacercariae excyst in the upper part of the duodenum but some 
may be carried as far posterior as 6 feet, as has been previously pointed 
out. The distribution of the various sizes found in the duodenum of 
experimental hosts indicates that some migration may occur within the 
limits in which they were found. The young forms are very active and 
migration for considerable distances is probably a matter of a very short 
time. 

In the duodenum they are found attached to the mucosa by a power- 


86 ILLINOIS BIOLOGICAL MONOGRAPHS 


fully developed acetabulum, elongating and weaving their bodies from 
side to side. The worms are capable of moving rapidly from one position 
to another in the measuring worm manner. The color of the worm is 
reddish, which makes it very inconspicuous against the background of 
mucosa. To collect the worms, sections of the duodenum were placed in 
warm physiological salt solution. This increases their activity which 
makes them more easily seen. They were then scraped off and shaken 
free of all tissue from the duodenum. 

The shape of the young worm is very much like that of the adult, 
being attenuated at the anterior end and widest in the testicular region. 
The dorsal surface is convex and the ventral surface is slightly concave. 
In cross section the body is nearly ovoid or round. The acetabulum is 
subterminal in living specimens, but when allowed to die unfixed the 
acetabulum opens posteriorly and the body becomes much flatter. The 
young worms are more active than the adults and are able to extend the 
body three times their contracted length while the adults are not capable 
of extending the body more than one and a half times their contracted 
length. Young individuals are also much more resistant than the adults. 
Some of the young specimens from the duodenum remain alive for 24 
hours in cold physiological salt solution while older worms remain alive 
only 6 to 8 hours under similar conditions. 

The age and size at which these parasites become mature was not 
determined experimentally, but by examining naturally infested animals 
a series of developmental stages varying from 1 to 11 mm in length was 
obtained. The smallest mature specimens measure 2.86 by 1.22 mm. 
Many are mature at a length of 3 mm. Krull (1934) has shown that 
these worms reach maturity in approximately three and one-half months, 
and the correctness of his findings has been pointed out previously (page 
82). Since the worms migrate at an average size of 2.37 by 0.98 mm 
during the fourth and fifth weeks after infestation of the host and 
mature at the sizes given above it is evident that the rate of growth in 
the rumen is slow. This is shown also by the average size of the worms 
from the rumen of Calf I. Those worms which were 6 or 7 months old 
averaged only 3.55 by 2.32 mm. 

The results obtained from the examination of a bull brought in to 
the animal pathology department of Louisiana State University are also 
of value in demonstrating the slow growth rate of these forms. This bull 
was brought in for observation on August 10, 1933, and was kept, as 
were the experimental calves, with no chance of becoming infested with 
C. cotylophorum. Upon examination on June 12, 1934, 26 specimens of 
C. cotylophorum were found in the rumen. The smallest of these worms 
measured 5.2 by 2.1 mm and the largest 7.0 by 2.75 mm. The average 


LIFE HISTORY OF COTYLOPHORON—BENNETT 87 


size of the 26 was 5.6 by 2.34 mm. This bull had been confined slightly 
more than 10 months so that the smallest of the specimens must neces- 
sarily have been somewhat over 10 months of age. The largest specimens 
found from other naturally infested hosts never exceeded 11 by 3 mm. 
Specimens of this size were probably well over one year in age and 
represent the maximum size attained by the parasites in this host. No 
information other than the above was obtained concerning the longevity 
of these forms. 

The adult of this species has been fully described by Fischoeder (1901, 
1903), Stiles and Goldberger (1910), Maplestone (1923), Bennett 
(1928), and Stunkard (1929) so that no detailed descriptions of struc- 
tures will be given here. Descriptions of the development of structures 
of diagnostic importance and of structures which have not been described 
for this parasite are included. 


Digestive Tract.—In the smallest individuals the digestive tract is 
identical in appearance to that of the largest worms. The caeca are in 
the same position in both, that is, in the dorso-lateral part of the body 
and terminate dorsal to the acetabulum. The posterior ends may be 
curved ventrally anterior to the acetabulum, but such variations are of 
no importance as pointed out by Maplestone (1923) and can be explained 
on the basis of differences in the degree of contraction. 

The oral sucker changes consist of an increase in size only. Its 
growth, however, is not proportionate to that of the body. Its length 
in 1 mm worms as compared with the body length is in the ratio of about 
[25 ine toroenim worms the ratio is 126 or 1:7“ and in 4 to 5 mm 
worms the ratio is 1:6. The oral sucker attains its maximum size in 
worms of 6 to 7 mm and the ratio is about 1:9. In a well extended 
specimen measuring 6 by 2.5 mm the oral sucker is 0.74 mm long, 0.58 
mm wide and 0.46 mm dorso-ventrally. The worms may reach a size 
of 11 by 3 mm but there is no further increase in the size of the sucker. 

The esophagus is the only structure of the digestive tract which 
possesses characteristics of specific importance. These are its length 
and the gradual increase in thickness of its walls from its anterior to its 
posterior end. The length of the esophagus is subject to considerable 
variation because of contraction but it increases in length as the worms 
develop, reaching its maximum length in worms of 6 to 7 mm in length 
as does the oral sucker. In the smallest worms obtained the esophagus 
is about 0.3 mm long and it increases steadily as the worms grow, but 
it never exceeds 0.9 mm in length. It bifurcates to form the intestinal 
caeca in the region of the genital pore in worms under 7 mm long (Figs. 
81, 82, 83). However, the worms reach 11 mm in length and as a re- 
sult the genital pore becomes distinctly post-bifurcal in position. 


88 ILLINOIS BIOLOGICAL MONOGRAPHS 


Stiles and Goldberger (1910:72) state that the genital pore of C. 
indicum is decidedly post-bifurcal and designate this as one of the 
differences between C. indicum and C. cotylophorum. Maplestone 
(1923:152) has pointed out that this characteristic is too variable to be 
of diagnostic importance for these two species, and the present findings 
support his contention. 

The muscular thickening of the esophagus in C. cotylophorum is 
evident in the cercaria and becomes more evident as the worm develops 
in the final host. In the small worms the muscle wall at the proximal 
end of the esophagus is about 5 » thick and at its distal end is 15 p 
thick, while the diameter of the esophagus at the proximal end includ- 
ing the esophageal glands is 37 » and at the distal end is 85 p. The ratio 
of these measurements is found to vary from 1:2 in young worms to 
1:4 in old worms. The thickness of the muscle wall at the proximal end 
does not exceed 20 p, and it does not exceed 60 p at the distal end. In 
the largest of the worms the total diameter of the proximal end does not 
exceed 0.17 mm and does not exceed 0.32 at the distal end. All of these 
measurements are subject to considerable variation but there is a normal 
increase in the size of the esophagus concurrent with growth. However, 
growth of the esophagus stops when the worms reach a size of 6 or 
7 mm. 

The difference in the thickness of walls of the esophagus at the two 
ends is very evident at all ages (Figs. 70-73), and the appearance of 
this structure is very similar to that in C. cotylophorum as described by 
Fischoeder (1903:547) and Maplestone (1923-152). 

Maplestone (p. 152) attempts to show that the esophagus of C. 
indicum and C. cotylophorum are identical, but I cannot agree with him. 
As stated, the esophageal thickening in the present material is very 
evident at all ages and sizes of worms. Stiles’ and Goldberger’s ma- 
terial possessed no such thickening, although their figure (1910:fig. 45, 
p. 66) shows that the walls of the esophagus are thick. The apparent 
muscular thickening shown in their figure is due to an increase in the 
lumen rather than to an increase of thickness of the walls. I have been 
able to determine this point from a sectioned specimen of a worm identi- 
fied as C. indicum by these writers. The thickness of the walls of the 
esophagus in this material does not exceed 10 p at its proximal end and 
does not exceed 20 p at its distal end. The diameters of the two ends 
are 0.105 and 0.195 mm respectively. The above worm, which was not 
mature, measured 5.16 by 1.68 mm. These differences are too great to 
be the result of normal variation, as determined by a comparison with 
the present material. 


Development of Sex Organs.—The primordia of the genital organs 


PIPE FISTORY OF “COTY LOPHORON—BENNETT 89 


are present in the cercaria, as previously described. In the smallest 
worms recovered from the final host the ovary, Mehlis’ gland, and testes 
were differentiated (Fig. 77). The ovary and Mehlis’ gland are repre- 
sented by two small masses of cells located near the center of the body 
above the anterior margin of the acetabulum. From Mehlis’ gland a cord 
of cells, in which there is no lumen, passes ventrally over the anterior 
margin of the acetabulum to the ventral region of the body. It turns 
forward and continues anteriorly near the ventral surface to reach a 
mass of cells located above the position of the future genital pore. The 
testes are located laterally, one on each side of the cord immediately 
anterior to the acetabulum. Their method of formation was not de- 
termined, but their position suggests that they are set off from the 
ventral side of the mass of cells located anterior and dorsal to the aceta- 
bulum in the cercaria. The ovary and Mehlis’ gland doubtless are formed 
from the dorsal part of this mass. There is no indication of a lumen in 
the cord of cells at any place. The testes are surrounded by a thin 
membrane but no vasa efferentia were observed. The genital pore is 
not yet formed, and the only indication of a genital sucker is a slight 
thickening of the body wall and a mass of deeply-staining cells which 
are probably cells of the prostate gland. 

The degree of development described above is reached in worms of 
approximately 1.0 by 0.65 mm. In individuals of this size the ovary 
measures 0.045 by 0.03 mm, Mehlis’ gland 0.037 by 0.022 mm, the testes 
0.065 by 0.045 mm, the diameter of the cord of cells 0.045 mm, and the 
mass of cells surrounding the genital pore 0.097 by 0.032 mm. 

The vasa efferentia, the vas deferens, the uterus, and a genital pore 
were clearly distinguished in an individual 1.17 by 0.8 mm. The uterus 
follows a course from Mebhlis’ gland to the ventral side of the body 
similar to that of the cord of cells described in the above form and is 
doubtless formed from it. Here it turns dorsally and anteriorly until 
it reaches the center of the body. It passes forward for a short distance 
and then turns ventrally. Near the ventral surface it turns forward to 
the genital pore. It joins the vas deferens, and the common duct thus 
formed, the ductus hermaphroditicus, opens to the outside (Fig. 63). 

In this same specimen the testes have taken the tandem arrangement 
characteristic of the mature worm. The vasa efferentia are conspicuous 
and are easily traced. The one from the posterior testis passes anteriorly 
and dorsally until it reaches the mesial side of the right caecum where it 
turns forward. The vas efferens from the anterior testis passes forward 
mesial to the left caecum. The two unite in the center of the body a 
short distance posterior to the genital pore and immediately anterior to 
the descending uterus. The vas deferens thus formed coils ventrally and 


90 ILLINOIS BIOLOGICAL MONOGRAPHS 


anteriorly to the genital pore. It is located dorsal to the uterus and unites 
with it to form the ductus hermaphroditicus. 

The position of the vasa efferentia and vas deferens indicates that 
these structures are formed from the dorsal part of the cord of cells 
described in the smaller individuals. The ventral position of the uterus 
near the genital pore indicates that it is derived from the ventral part of 
the cord. The descent of the uterus posterior to the loop formed by the 
union of the vasa efferentia supports this conclusion also. 

The genital sucker becomes more evident in specimens of this size, 
although as yet there are no definitely differentiated muscles in it. It 
consists of a compact mass of cells surrounding the terminations of the 
vas deferens, the uterus, and the ductus hermaphroditicus. This mass 
of cells is approximately 0.12 mm long, 0.108 mm wide, and 0.072 mm 
thick (Fig. 63). 

All of the male and female genital structures, with the exception 
of the vitellaria, are formed before the worms migrate from the duo- 
denum to the rumen. 

Following the stage just described the other structures character- 
istic of the genitalia of the mature worm are rapidly developed, being 
present in worms 2.5 mm long and less than 3 weeks old (Fig. 76). 
The vas deferens is differentiated into several distinct regions: a seminal 
vesicle, a pars musculosa, the prostate gland, and the ductus ejacula- 
torius, which joins the ductus hermaphroditicus. The ductus hermaphro- 
diticus passes through the center of a minute hermaphroditic papilla 
which opens into a small genital atrium in the genital papilla. The 
copulatory structures and the terminations of the male and female ducts 
are enclosed in the genital sucker. 

Laurer’s canal is well developed in individuals of this size also. It 
passes from the oviduct dorsally and laterally to open to the exterior 
behind the excretory pore and to the left of the median line. The 
only other development in the female system is the formation of the 
metraterm. 

There is no recognizable change in the worms immediately follow- 
ing their migration into the rumen, which is a second indication that the 
time required for migration is very short. The fact that only one worm 
was found in other parts of the stomach other than the rumen has been 
considered above as an indication that the worms migrate from the 
duodenum to the rumen rather quickly. 

No worms of known age were studied from the time of migration 
but many specimens representing all sizes and stages of development 
were secured from naturally infested hosts. There is very little increase 
in size before the worms reach sexual maturity and this increase is in 


LIFE HISTORY OF COTYLOPHORON—BENNETT 91 


diameter. Thé smallest mature worm collected measured 2.86 by 1.43 
mm. The most conspicuous changes in the small mature worms as com- 
pared to the immature ones are the increase in size of the genital organs 
and the development of the vitellaria (cf. Figs. 76 and 79). In the 
smallest of the mature worms the vitellaria are very sparsely developed 
in the lateral regions of the body and extend from the esophagus to the 
acetabulum. The testes in immature worms at the time of migration are 
round and smooth and measure only 0.1 mm in diameter. In the smallest 
of the mature worms the testes are 0.36 by 0.26 mm; they extend dorso- 
ventrally for a distance of 0.48 mm and are distinctly lobed. There is 
an increase in the size of the ovary from 0.075 mm to 0.196 mm in 
diameter. The uterus in both the immature and small mature worms is 
straight but its diameter increases from 0.032 mm in the immature to 
0.081 mm in the mature worms. The vasa efferentia remain unchanged 
but the vas deferens becomes greatly coiled. The genital sucker and 
copulatory structures are also much more conspicuous in the small 
mature worms. 

The genital sucker in worms which have just migrated to the rumen 
measures 0.17 mm in diameter and the muscle mass is about 0.14 mm 
thick (Fig. 76). In small mature worms this structure measures 0.4 mm 
in diameter and is 0.22 mm thick. 

The only marked change which occurs after sexual maturity is 
attained is the rapid development of the vitellaria. In worms only 3.5 
mm in length they have reached a state of development comparable to 
that in the largest worms (Fig. 80). They extend in closely grouped 
follicular masses from the oral sucker to the acetabulum, principally in 
the extra-caecal zones but approach the median line both dorsally and 
ventrally. The uterus becomes more coiled as larger numbers of eggs 
are produced and fills all available space between the acetabulum and 
the posterior testis. It is coiled transversely dorsal to the testes, and 
more coils are formed in the ventral region of the body anterior to the 
anterior testis. 

Stunkard (1929:244) found that C. cotylophorum reached maturity 
at a much smaller size in calves than in antelopes, but he could not ex- 
plain the incongruity. He did not give the size of the mature worms 
from the calves but specimens as long as 6 mm from the antelopes were 
not mature. These findings clearly indicate that these parasites mature 
at a much smaller size in some hosts than in others. 

The genital organs are much larger in fully grown worms than in 
those just reaching maturity (cf. Figs. 79 and 81). The testes are 
located in tandem arrangement near the center of the body and occupy 
approximately two-fifths of the body length. The seminal vesicle is 


92 ILLINOIS BIOLOGICAL MONOGRAPHS 


greatly expanded and coiled; the pars musculosa is thick, muscular- 
walled, and coiled; the pars prostatica located directly above the genital 
pore is straight and measures about 0.20 by 0.22 mm. Its lumen is large 
but narrows abruptly as it becomes continuous with the ductus ejacula- 
torius. The ductus ejaculatorius is about 0.19 mm long and opens into 
the ductus hermaphroditicus. The hermaphroditic papilla varies in length 
with its state of contraction but its length is about 0.1 mm. The genital 
papilla encloses a small atrium into which the protrusible hermaphro- 
ditic papilla projects. The walls of the genital papilla are very muscular 
and are about 50 p thick. The genital papilla when protruded measures 
about 0.19 by 0.14 mm and projects into the cavity of the genital sucker 
(Fig. 74). 

The ovary and Mehlis’ gland in fully grown worms are located above 
the anterior margin of the acetabulum. The uterus is crowded with 
eggs and is coiled anterior to the acetabulum, dorsal to the testes, and in 
the ventral region of the body anterior to the anterior testis. The mus- 
cular metraterm joins the ductus hermaphroditicus. The union of the 
male and female ducts forms a distinct but small vesicle at the inner 
end of the ductus hermaphroditicus. 

The genital sucker consists of a muscular mass which encloses the 
terminal ends of the male ducts and the copulatory apparatus. This 
structure increases in size as the worms develop but does not exceed 0.7 
mm in diameter in any of the present material. Its walls are approxi- 
mately 0.3 mm thick. The cavity of the sucker is considered as a genital 
atrium by Fischoeder (1903) and Maplestone (1923). The genital 
pore is the opening of the sucker while the pore of the ductus hermaphro- 
diticus is the porus hermaphroditicus. 

When the worms are emitting eggs or sperm the genital papilla can 
be protruded for a short distance beyond the edge of the genital sucker. 
At the same time rapid contractions and relaxations of the muscles of 
the genital sucker occur. 

The genital atrium, the genital papilla, and the genital sucker are 
subject to considerable change in shape and size. The hermaphroditic 
papilla is also subject to great changes in size and appearance. However, 
in the present material after their development was completed these 
structures were evident in all ages and sizes of worms, and in all states 
of contraction (Figs. 68, 69, 74, 75). 

Maplestone (1923:153-155, figs. 8, 9) describes in detail the extreme 
variability in the appearance of the genital sucker and copulatory 
apparatus of C. cotylophorum and concludes that these structures are 
of no diagnostic value. His figures (Figs. 8, Al, A2, B) represent 
the appearance of the genital sucker and copulatory apparatus of C. 


TIFE HISTORY OF COTYLOPHORON—BENNETT 93 


cotylophorum as described by Fischoeder (1903:548, fig. 38) and of 
C. indicum as described by Stiles and Goldberger (1910:69, fig. 48). As 
a result of his observations on these structures and on the variability 
of the esophagus of C. cotylophorum he considers C. indicum as a 
synonym of C. cotylophorum. 

Fukui (1929:319) agrees with Maplestone and designates C. indicum 
as a synonym of C. cotylophorum. 

As originally described by Fischoeder the genital sucker of C. cotyl- 
ophorum is much larger and much more distinctly set off from the body 
parenchyma than in Maplestone’s or the present material (Fig. 67). 
Fischoeder does not describe a genital papilla in C. cotylophorum and the 
male and female ducts remain separate in the hermaphroditic papilla. 

The genital sucker of C. indicum as described by Stiles and Gold- 
berger is also distinctly set off from the body parenchyma and they do 
not describe a genital papilla as being present. In the present study some 
of the original material of Stiles and Goldberger was studied and no 
genital papilla was found. There is also a distinct difference in the ap- 
pearance of the genital sucker of C. indicum and that of C. cotylophorum 
as described and figured by Maplestone (1923:156, fig. 9) and as de- 
scribed in this paper. 

Stunkard (1923:138) believes that Maplestone’s conclusions as to 
the importance of these structures are erroneous. I am of the same 
opinion, and in view of the decided differences in the appearance of the 
genital sucker, the copulatory apparatus, and esophagus of C. indicum 
as compared to C. cotylophorum | cannot consider these two species as 
synonymous. 

The present specimens of C. cotylophorum agree in every detail with 
Maplestone’s description of this species with the exception of the ex- 
treme variability of the copulatory apparatus as pointed out above, with 
Stunkard’s description (1929:244-251), and with specimens loaned me 
by Krull. However, | believe that the differences between the genital 
sucker and copulatory apparatus of Fischoeder’s material and the present 
material are of diagnostic importance. Since these differences are the 
only ones which have been observed | am hesitant in considering these 
differences as being of specific value in view of Maplestone’s and Fukui’s 
findings. 

Excretory System.—The arrangement of the excretory system in 
the young and mature specimens is very similar to that of the cercaria 
(Fig. 60). The details of the system were not studied. Only living 
immature specimens under pressure were studied. It was possible in 
this way to determine the course and extent of the larger ducts and the 
position of the bladder. 


94 ILLINOIS BIOLOGICAL MONOGRAPHS 


The bladder is an elongate structure located dorsally in the posterior 
region of the body, extending from near the posterior margin of the 
acetabulum to slightly past the anterior margin. It opens to the exterior 
through a narrow short muscular duct lined with cuticula continuous 
with that of the body surface. It may pass directly to the surface from 
the middle or anterior end of the bladder or may extend forward from 
it (Figs. 77, 78). In young specimens the former condition is more 
often found, and it is only in the more fully developed and very ex- 
tended individuals that the duct opens very far in front of the bladder. 
The pore is located in the medial dorsal line, usually directly above the 
posterior margin of the posterior testis in mature specimens, but it may 
be as far forward as the middle of the anterior testis, or as far posterior 
as the anterior margin of the acetabulum. Its position relative to these 
organs depends entirely on the age of the individual and its state of con- 
traction. However, the position of the pore may be considered as pre- 
vesicular, being dorsal to the bladder only in immature or contracted 
mature specimens. Maplestone (1923:157, text-fig. 11) has described 
and shown similar conditions in specimens of C. cotylophorum of 
different ages. I did not observe the pore to be post-vesicular in any of 
my material as Maplestone has figured it in a very young specimen. 
Fukui (1929:275) has described similar variations in Paramphistomum 
explanatum, P. cervi, and P. orthocoelium. He states that the pore is 
very variable in position and cannot be used for exact diagnostic purpose, 
but that it is roughly definite for species. 

The main excretory canals are located the same as in the cercaria. 
From their union with the posterio-lateral angle of the bladder on each 
side they pass outward and forward. Immediately posterior to the 
middle of the body length these canals bend mesially and a cross con- 
nection passes across the middle line and sends off a forward diver- 
ticulum. The main canals then curve outward and forward. The diver- 
ticulum present just posterior to the eye in the cercaria is present in 
these older worms, and in them it receives a small duct which in turn 
receives branches from the esophageal region. The main canals continue 
forward from this diverticulum until they reach the posterior margin of 
the oral sucker. Here they turn abuptly on themselves and pass pos- 
teriorly in the lateral regions of the body. These posterior extensions 
could not be traced to their terminations but doubtless they extend as 
far back as the acetabulum, as they do in the cercaria. A small duct on 
each side extends forward from the turning point of the main canals 
which drains the anterior region of the body. Numerous small ducts 
which are symmetrically located empty into the larger canals throughout 
their course. 


LIFE AISTORY OF COTYLOPHORON—BENNETT 95 


The anterior diverticulum from the cross connection becomes greatly 
enlarged and it also receives small ducts from the anterior dorsal region 
of the body. 

A detailed description of the excretory system was made from pre- 
served material by Bennett (1928:22-23). 

The excretory system of this material is very similar to that of 
Gastrothylax and Paramphistomum as described by Fukui (1929:272). 
This type of system he designates as Type A and calls it H-shaped. 


SPECIFIC DESCRIPTION OF Cotylophoron cotylophorum 


The following specific description of C. cotylophorum is based en- 
tirely on the characteristics of the material used in the present study. 

Body of mature worm 3 to 11 mm long by 1.15 to 3 mm wide; conical 
in form, greatest width in testicular region; tapers to bluntly pointed 
anterior end, posterior end broadly rounded; dorsal surface convex 
longitudinally and transversely, ventral surface concave longitudinally, 
convex transversely; oval to round in cross section. Surface without 
spines or papillae. Genital pore bifurcal or slightly post-bifurcal, at 
junction of first and second body thirds, surrounded by genital sucker 
0.4 to 0.7 mm in diameter which forms a distinct projection in the 
median ventral line. Acetabulum at posterior end, distinctly subterminal 
0.75 to 1.36 mm in diameter. Mouth at blunt anterior extremity; oral 
sucker pyriform in sagittal section; 0.52 mm long, 0.45 mm wide and 
0.39 mm in dorso-ventral diameter in small mature worms, its maximum 
in fully grown individuals 0.74 mm long, 0.58 mm wide and 0.45 mm 
in dorso-ventral diameter; esophagus slightly longer than oral sucker; 
its walls increase in thickness posteriorly, ratio of thickness of anterior 
wall to posterior wall 1.3; caeca arise from dorso-lateral aspects of end 
of esophagus, terminate in acetabular zone. Excretory pore in median 
dorsal line about at junction of median and posterior body thirds; ex- 
cretory bladder extends posteriorly from pore above acetabulum; lateral 
excretory tubes extend from ventral and postero-lateral margin of bladder 
to oral sucker, turn sharply posterior to acetabulum; cross connection 
between lateral ducts in dorsal region and near middle of body length, 
median diverticulum extends forward for short distance from cross 
connection, lateral diverticulum from each lateral duct a short distance 
posterior to oral sucker. 

Testes large, lobate, about size of oral sucker in young mature worms, 
larger than acetabulum in old specimens, in median line, tandem arrange- 
ment; union of vasa efferentia slightly anterior to anterior testis; vas 
deferens coiled; its vesicula seminalis coiled, expanded; pars musculosa 
coiled, narrow; pars prostatica straight, located directly above genital 


96 ILLINOIS BIOLOGICAL MONOGRAPHS 


sucker ; ductus ejaculatorius short, unites with metraterm to form ductus 
hermaphroditicus ; hermaphroditic papilla short, protrusible, arises from 
the vertex of a conspicuous genital papilla, almost filling the cavity of 
the papilla; genital papilla in turn surrounded by the genital sucker. 

Ovary and Mehlis’ gland above anterior margin of acetabulum; 
Laurer’s canal passes over excretory bladder, opens posterior to excre- 
tory pore and left of median dorsal line; uterus coiled anterior to ace- 
tabulum, passes anteriorly dorsal to testis, descends vertically over the 
anterior margin of anterior testis, anteriorly again ventral to vas defer- 
ens, enters genital sucker; and metraterm unites with ductus ejacu- 
latorius. 


SUMMARY AND CONCLUSIONS 


Cotylophoron cotylophorum is a widely distributed parasite of rumi- 
nants but has not been previously reported from the mainland of North 
America. 

The time required for the miracidium to develop varies directly with 
temperature. In the present experiments eggs kept at room tempera- 
tures hatched in 11 to 29 days. 

The structures of the miracidium develop in sequence and are rec- 
ognizable before hatching occurs. , 

The miracidium is similar to other amphistome miracidia. A study 
of the descriptions of 18 different species of miracidia indicates that the 
number and arrangement of ciliated epidermal cells is of taxonomic 
value. 

The snails Fossaria parva and F. modicella are capable of serving 
as the intermediate hosts of C. cotylophorum. The former is the natural 
host of this parasite in Louisiana. 

The miracidium penetrates the mantle, head, and foot of the snail, 
loses its ciliated epidermal cells, and transforms into a sporocyst. 

The sporocyst develops rapidly and produces 9 rediae. 

The rediae are born at an average size of 0.188 by 0.056 mm and 
migrate into the liver and ovo-testis where their development is complete. 
Each redia produces approximately 25 cercariae. 

Mother rediae may occur in the life cycle but were observed in only 
one instance. 

Cercariae are born in an undeveloped condition and continue their 
development in the liver and ovo-testis. 

The time required for the development of the sporocyst, redia, and 
cercaria varies directly with temperature. Infested snails kept under 
natural temperature conditions shed cercariae in 30 to 91 days. 


LIFE HISTORY OF COTYLOPHORON—BENNETT 97 


The cercariae encyst on vegetation and the metacercaria lives for 
over 3 months. 

The metacercariae become excysted in the duodenum of the final 
host. Migration from the duodenum to the rumen begins in 21 days at 
an average size of 2.37 by 0.98 mm and may continue over a period of 
about 14 days. The worms do not migrate at the same age or size. 

The worms become mature after reaching the rumen at an age of 
about three and a half months, at a size of approximately 3.0 by 1.15 
mm. They reach their maximum size in about one year. 

The time required to complete the life cycle of C. cotylophorum 
varies from about 5 to 8 months. 

C. indicum is not a synonym of C. cotylophorum. 


98 ILLINOIS BIOLOGICAL MONOGRAPHS 


BIBLIOGRAPHY 
AMEEL, D. J. 


1934. Paragonimus, Its Life History and Distribution in North America and 
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Bartow, C. H. 
1925. The Life Cycle of the Human Intestinal Fluke Fasciolopsis buski 
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1929. Studies on the Development of Allassostoma parvum Stunkard. Jour. 
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BENNETT, H. J. 
1928. A new Species of Cotylophoron from Bos taurus. Collected in 
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Brooks, F. G. 
1930. Studies on the Germ Cycle of Trematodes. Amer. Jour. Hyg., 12: 
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Carvel wR: 
1909. The Life History of Diplodiscus temperatus Stafford. Zool. Jahrb., 
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Cor, W. R. 
1896. Notizen uber den Bau des Embryos von Distomum hepaticum. Zool. 
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Cort, W. W. 
1915. Some North American Larval Trematodes. Il. Biol. Monographs, 1, 
no. 4:1-86, 8 pls. 
1919. Notes on the Eggs and Miracidia of the Human Schistosomes. Univ. 
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Dusots, G. 
1928. Les Cercaires de la Région de Neuchatel. Soc. Neuchateloise des Sci. 
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Faust, E. C. 
1919. Notes on Some South African Cercariae. Jour. Parasitol., 5:164-175, 
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Faust, E. C., and MELeney, H. E. 
1924. Studies on Schistosomiasis Japonica. Amer. four, Hyg., Monographic 
Series, No. 3. 
FISCHOEDER, F. 
1901. Die Paramphistomiden der Satgethiere. Zool. Anz., 24: Zool. Anx., 
24:365-375. 
1903. Die Paramphistomiden der Saugethiere. Zool. Jahrb. f. Syst. Geog. 
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Fuxul, T. 
1929. Studies on Japanese Amphistomatous Parasites, with Revision of the 
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LIFE HISTORY OF COTYLOPHORON—BENNETT 99 


IsHu, Y. 
1934. Studies on the Development of Fasciolopsis buski, Part I, Jour. Med. 
Assoc. Formosa, 33:349-378, 1 pl., 10 tables, 1 text-fig. 
1934a. Studies on the Development of Fasciolopsis buski, Part II, Jour. Med. 
Assoc. Formosa, 33:379-390, 1 pl., 2 tables. 
1934b. Studies on the Development of Fasciolopsis buski, Part III. Jour. 
Med. Assoc. Formosa, 33:391-412, 1 pl., 2 charts. 
Jounson, J. C. 
1920. The Life Cycle of Echinostoma revolutum (Froelich). Univ. Calif. 
Publ. Zool., 19:335-388, pls. 19-25, 1 text-fig, 
Kruii, W. H. 
1932. Studies on the Life History of Cotylophoron cotylophorum (Fischoeder, 
1901) Stiles and Goldberger, 1910. Jour. Parasitol., 19: 165-166. 
1934. Life History Studies on Cotylophoron cotylophorum (Fischoeder, 1901) 
Stiles and Goldberger, 1910. Jour. Parasitol., 20:173-180, 1 text-fig., 
1 table: 
Kru.i, W. H., and Price, HELEN F. 
1932. Studies on the Life History of Diplodiscus temperatus Stafford from 
the Frog. Occasional Papers from Mus. of Zool., Univ. of Mich., 
no. 237. 
Lerper, R. T. 
1910. The Entozoa of the Hippopotamus. Proc. Zool. Soc. London, 1:233-251, 
9 text-figs. 
Le Roux, P. L. 
1930. A Preliminary Communication on the Life Cycle of Cotylophoron 
cotylophorum and Its Pathogenicity for Sheep and Cattle. 16. Rep. 
Director Vet. Serv., Dept. Agric. Union South Africa, Pretoria, pp. 
243-253, 7 figs. 
Leucxkart, R. 
1886. Die Parasiten des Menschen. Leipzig. Vol. II, p. 72. 
Looss, A. 
1892. Ueber Amphistomum subclavatum Rud. und seine Entwicklung. Fests. 
zum 70. Geburts. R. Leuckarts, Seite 147 bis 167, 2 Tafeln. Leipzig. 
1896. Recherches sur les faune parasitaire de l’Egypte, I. Mem. I’Inst. Egypt., 
3:1-252, 16 pls. 
Lyncu, J. E. 
1932. The Miracidium of Heronimus chelydrae MacCallum. Quar. Jour. 
Micro. Sci., 76:13-33, 2 pls., 2 text-figs. 
McCoy, O. R. 
1929. Notes on Cercariae from Missouri. Jour. Parasit., 15:199-208, 1 pl. 
Manter, H. W. 
1926. Some North American Fish Trematodes. Ill. Biol. Monographs, 10: 
No. 2:1-138, 6 pls., 2 charts, 1 text-fig. 
MAPLESTONE, P. A. 
1923. A Revision of the Amphistomata of Mammals. Ann. Trop. Med. and 
Parasitol., 17:114-212, 4 pls., 32 text-figs., 10 tables. 
Matuias, P. 
1925. Recherches expérimentales sur le cycle évolutif de quelques Trématodes. 
Bull. Sci. de la France et de la Belgique, 59:1-124, 4 pls., 13 text-figs. 
Naxkacawa, K. 
1917. Human Pulmonary Distomiasis caused by Paragonimus westermanit. 
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100 ILLINOIS BIOLOGICAL MONOGRAPHS 


O’Roxg, E. C. 
1917. Larval Trematodes from Kansas Fresh Water Snails. Kans. Univ. Sci. 
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OrTMANN, W. 
1908. Zur Embryonalentwicklung der Echinorhynchen. Zool. Jahrb. f. Anat., 
26:255-293, 2 pls. 
Price, HELen F. 
1931. Life History of Schistosomatium douthitti (Cort). Amer, Jour. Hyg., 
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1933. Echinoparynphium recurvatum (Linstow, 1873) und seine Entwicklung. 
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1883. Beitrag zur Kenntniss der Embryonalentwicklung der Trematoden. 
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1922. Cercariae Indicae. Ind. Jour. Med. Res., 10, Supplement. June: 1-366. 
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1931. Studien tber die Phylogenie der Trematoden. IV. Zeit. f. wiss. Zool., 
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Suzukl, 5S. 
1931. Researches into the Life-History of Fasciola hepatica and its Distribu- 
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1928. Ueber die Entwicklungsgeschichte des Paramphistomum cervi. (Ab- 
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1933. Life History Studies on Trematodes of the Subfamily Reniferinae. 
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1883. The Life History of the Liver-Fluke (Fasciola hepatica). Quar. Jour. 
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Parasitol., 3:10-20, 1 pl. 


LIFE HISTORY OF COTYLOPHORON—BENNETT 101 


EXPLANATION OF PLATES 


All figures were made with the aid of a camera lucida with the ex- 
ception of Fig. 80 which is a graphic reconstruction. 


Abbreviations Used 


ere tee acetabulum PGS ast 
Dijdas Gia c® anterior nerve rd ae 
Oya aoe apical papilla 1 tee 
DM an. basement membrane Ppae ss 
ae birth pore PSec i. 
Oe ane brain {Ls pane 
Chek eee cuticula hp...... 
Cosine caecum ere 
cap..... primordium of caecum GO Cx 
COM Fee oe central cavity LGR 
(Alene 5 ee caudal excretory bladder LO ers oar: 
CEG. ner caudal excretory duct led..... 
COD tear caudal excretory pore Im...... 
CO eee cercaria eee 
Cone eye: cerebral ganglion ME... 6s. 
CM oe sae circular muscles Mg..... 
COM «a. commissure Wiican ee 
CLG... cuticular cell NG 
CVsCee ee cystogenous cell fee ee 
CV ie cystogenous granule WU ese = acdhe 
(| een ductus ejaculatorius OP. ..... 
GN, takin ductus hermaphroditicus OS care Sevier 
CUS eine excretory bladder OU ee es 
OCs tes cstins epithelial cell Danae? 
Qe ole he excretory duct pe...... 
CNL S erune: embryo LL See 
Cigee eye nerve ped..... 
CPi ones epidermal cell, row 1 ph...... 
Cpa. ine epidermal cell, row 2 phe... 
Cp3...... epidermal cell, row 3 PUD coe 
eps. ..... epidermal cell, row 4 pm..... 
epm..... epidermal cell nucleus, row 1 DPrcne. 
epny..... epidermal cell nucleus, row 2 DNn...... 
epn;.....epidermal cell nucleus, row 3 SO eateriats 
epns..... epidermal cell nucleus, row 4 ty seen 
CS ee Keke esophagus SP ae aie 
es; ......esophageal cell Sina! ts 
Gin css ote excretory tubule Sees 
XP. ..6%. excretory pore Gaicaros) sus 
CYR re dsteks eye te 
Gees os flame cell TE ree 
DUR ert se gut Ue eee 
Ct ee oe genital atrium WO 6 ates ans 


2D verso aie. germ ball UE o eer ae 


germ cell 
gut nucleus 
genital pore 


.genital papilla 


genital sucker 
germinal tissue 
hermaphroditic papilla 
intestine 


..Laurer’s canal 
_lens cell 


lateral evagination 


.lateral excretory duct 


longitudinal muscle 
metraterm 

median evagination 
Mehlis’ gland 

nerve 

nerve cell nucleus 
nerve fiber 

nucleus 

oral plug 

oral sucker 

ovary 

plug 

primitive epithelium 
penetration gland 
penetration gland duct 
pharynx 


. pharnygeal cuticular cell 


pigment 

pars musculosa 

pars prostatica 
posterior nerve 
salivary gland 
subepithelial nucleus 
sensory papilla 
sporocyst tissue 
seminal vesicle 

tail 


.. testis 


uterus 
vitellaria 
vas deferens 
vas efferens 


102 ILLINOIS BIOLOGICAL MONOGRAPHS 


PLATE I 


Fics. 1-11—Developing miracidia. Scale 0.05 mm. 


Fic. 12.—Four-cell stage in development of the miracidium. 
Scale 0.03 mm. 


Fic. 13.—Flame cell of miracidium. Scale 0.01 mm, 


LIFE HISTORY OF COTYLOPHORON—BENNETT 103 


PLALE 


104 


ILLINOIS BIOLOGICAL MONOGRAPHS 


PLATE II 


Fics. 14-17,—Mature miracidia. Scale 0.05 mm. 


Fics. 18-21.—Cross sections of miracidia. Scale 0.02 mm. 


Fic. 22—Frontal section of anterior end of miracidium. Scale 
0.02 mm. 


Fic. 23.—Frontal section of miracidium. Scale 0.02 mm. 


105 


HiPe HISTORY OF COTYLOPHORON—BENNETT 


PLATE II 


106 ILLINOIS BIOLOGICAL MONOGRAPHS 


PLATE. II 


Fic. 24—Miracidium in lymph duct of snail. Scale 0.03 mm. 
Fic. 25.—Five-day sporocyst. Scale 0.05 mm. 
Fic. 26.—Mature sporocyst. Scale 0.1 mm. 


Fic. 27—Germinal and epithelial cells in body wall of 
sporocyst. Scale 0.01 mm. 


Fic. 28—Twenty-four-hour sporocyst. Scale 0.03 mm. 
Fic. 29—Twelve-hour sporocyst. Scale 0.02 mm. 
Fic. 30.—Longitudinal section of sporocyst. Scale 0.1 mm. 


Fic. 31—Longitudinal section of forty-eight-hour sporocyst. 
Scale 0.03 mm. 


Fic. 32.—Body wall of sporocyst. Scale 0.05 mm. 


LIFE HISTORY OF COTYLOPHORON—BENNETT 107 


PLATE III 


108 ILLINOIS BIOLOGICAL MONOGRAPHS 


PLATE IV 

Fic. 33.—Mature redia. Scale 0.3 mm. 

Fic. 34.—Frontal section of redia showing salivary glands. 
Scale 0.05 mm. 

Fro. 35.—Mature sporocyst. Scale 0.1 mm. 

Fic. 36.—Redia about to escape from sporocyst. Scale 0.05 
mm. 

Fic. 37.—Frontal section of redia showing brain. Scale 0.05 
mm. 

Fic. 38.—Sagittal section of anterior end of redia. Scale 
0.1 mm. 

Fic. 39.—Posterior end of redia showing germ cells and de- 
veloping cercariae. Scale 0.1 mm. 

Fic. 40.—Redia in pocket of sporocyst tissue. Scale 0.3 mm. 

Fic. 41.—Posterior end of redia showing exhaustion of germ 
cells. Scale 0.1 mm. 

Fic. 42—Longitudinal section of immature redia. Scale 0.03 
mm, 


LIFE HISTORY OF COTYLOPHORON—BENNETT 109 


PLATE IV 


110 


ILLINOIS BIOLOGICAL MONOGRAPHS 


PLATE V 
Fics. 43, 44—Longitudinal section of anterior end of imma- 
ture redia. Scale 0.03 mm. 


Fic. 45.—Lateral view of posterior end of cercaria. Scale 
0.05 mm. 


Fic. 46.—Mature redia. Scale 0.2 mm. 

Fic. 47.—Oral sucker and esophagus of mature cercaria, 
sagittal section. Scale 0.02 mm. 

Fic. 48.—Mature redia. Scale 0.2 mm. 


Fic. 49 —Longitudinal section of anterior end of immature 
redia. Scale 0.03 mm. 


LIFE HISTORY OF COTYLOPHORON—BENNETT 111 


PLATE V 


112 


ILLINOIS BIOLOGICAL MONOGRAPHS 


PLATE. Vi 


.50.—Immature cercaria, dorsal view. Scale 0.05 mm. 
;. 51.—Mature cercaria, ventral view. Scale 0.1 mm. 

;, 52.—Immature cercaria, dorsal view. Scale 0.05 mm. 
.53.—Metacercaria, lateral view. Scale 0.01 mm. 


.54.—Cross section of tail of mature cercaria. Scale 


0.04 mm. 


;,55.—Frontal section of immature cercaria. Scale 0.05 


mm. 


;.56.—Immature cercaria, dorsal view. Scale 0.05 mm. 
;,57.—Immature cercaria, ventral view. Scale 0.05 mm. 


5. 58.—Mature cercaria, sagittal section. Scale 0.05 mm. 


113 


BENNETT 


PIPE. DISLORY (OF COTYLOPHORON 


®) ae 


zs 


ON’ oS - ‘ 
220: 20'g'0 coes 


PLATE VI 


114 


ILLINOIS BIOLOGICAL MONOGRAPHS 


PLATES Vit 
Fic. 59.—Immature cercaria, dorsal view showing pigment. 
Scale 0.1 mm. 
Fic. 60.—Excretory system, immature specimen. Scale 0.2 mm. 


Fic. 61—Immature cercaria, lateral view showing develop- 
ment of pigment. Scale 0.1 mm. 


Fic. 62.—Section of developing eye. Scale 0.61 mm. 


Fic. 63.—Cross section through genital sucker of a worm 
1.17 x 0.84 mm. Scale 0.1 mm. 


Fics. 64,65.—Sections of developing eye. Scale 0.02 mm. 


Fic. 66.—Anterior end of mature cercaria, sagittal section. 
Scale 0.04 mm. 


Fic. 67.—Cross section through genital sucker of a worm 
3.65 x 2.45 mm. Scale 0.5 mm. 


LIFE HISTORY OF COTYLOPHORON—BENNETT 115 


PLATE VII 


116 


ILLINOIS BIOLOGICAL MONOGRAPHS 


PLATE VIII 

Fic. 68.—Sagittal section through genital complex of a speci- 
men 2.5x0.72 mm. Scale 0.2 mm. 

Fic. 69.—Cross section through genital sucker of a specimen 
2.95x1.13 mm. Scale 0.1 mm. 

Fic. 70.—Sagittal section of anterior end of a _ specimen 
1.09x 0.39 mm. Scale 0.2 mm. 

I'ic.71.—Sagittal section of anterior end of a _ specimen 
2.46 x 0.63 mm. Scale 0.5 mm. 

Fic. 72—Sagittal section of anterior end of a _ specimen 
6.0x 2.75 mm. Scale 1.0 mm. 


Fic. 73.—Sagittal section of anterior end of a _ specimen 
9.0x 2.75 mm. Scale 1.0 mm. 

Fic. 74.—Cross section through genital sucker of a specimen 
8.0x 2.75 mm. Scale 0.1 mm. 

Fic. 75.—Cross section through genital sucker of a specimen 
40x1.15 mm. Scale 0.1 mm. 


LIFE GISTORY OF COTYLOPHORON-—-BENNETT 


PLATE VIII 


117 


118 


ILLINOIS BIOLOGICAL MONOGRAPHS 


PLATE IX 
Fic. 76.—Sagittal section of a specimen of migration size, 
2.46x 0.65 mm. Scale 1.0 mm. 


Fic. 77.—Sagittal section of a very young specimen. Scale 
1.0 mm. 


Fic. 78.—Sagittal section of a mature specimen 2.8 x 1.26 mm. 
Scale 1.0 mm. 


Fic. 79.—Frontal section of a mature specimen 2.99 x 1.61 mm. 
Scale 1.0 mm. 


Fic. 80.—Graphic reconstruction of a mature specimen. Scale 
1.0 mm, 


Fic. 81.—Sagittal section of a mature specimen. Scale 1.0 mm. 


LIFE HISTORY OF COTYLOPHORON—BENNETT 119 


Pats, AGES 
ic : (28 Xooo 
MAGI FSS O52 9 
Cah ee 


lace oN rz Frere <p 
ay WRAL 
Hee aM 
af 4 \Ya Z(\s 
ie A f LEV A 


T Ss ov 
Se 
1 Sp 


PLATE IX 


dl ‘ 
a\ - i | 4) : } . 
LIBRARY 


OF THE 
UNIVERSITY OF ILLINOIS 


UNIVERSITY OF ILLINOIS BULLETIN 


oy September 29, 1936 No. 9 


ILLINOIS BIOLOGICAL MONOGRAPHS 
Vol. XIV, No. 4, Price $1.50 


The Life History of 
Cotylophoron cotylophorum 


a trematode from ruminants 


BY 


Harry JACKSON BENNETT 


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a “ILLINOIS BIOLOGICAL MONOGRAPHS 


Vol. I 


rision of the Cestode Family Proteocephalidae. With 16 plates, By G. R. 
La Rue. $2.00. 


o a the Cestode Family Anoplocephalidae. With 6 plates. By H. Douthitt. 
netss oo 
North American Larval Trematodes. With 8 plates. By W. W. Cort. $1.20. 


: Vol. II 
Jassification of Lepidopterous Larvae. With 10 plates. By S.‘B. Fracker. $1.50. 
in the Osteolesy of Some of the Loricati. With 5 plates. By J. E. Gutberlit. 5o cts. 


Bins: Vol. Til 
Stud es on ‘the Pactars Controlling the Rate of Regeneration. By C. Zeleny. $1.25. 
the Reson and Mouth-parts of Diptera. With 25 plates. By A. Peterson. 


tudies on North American Polystomidae, Aspidogastridae, and Paramphistomidae. 
wes With 11 plates. By H. W. Stunkard. $1.25. 


Color and Color-pattern Mechanism of Tiger Beetles. With 29 black and 3 colored 
A wites. By V. E. Shelford. $2.00. 


Ree ; Vol, IV 
Net aie Life History Studies on Montana Trematodes. With 9 plates. By E. C. Faust. $2.00. 


: No.2. The Goldfish (Carassius carassius) as a Test Animal in the Study of Toxicity. By 
E. B. Powers. $1.00. 


ee ee and Biology of Some Turbellaria from the Mississippi Basin. With 3 
plates. By Ruth Higley. $1.25. 


No, 4. North American Pseudophyllidean Cestodes from Fishes. With 13 plates. By A. R. 
Pitot same Cooper. $2.00. 
Vol. V 


a No, 1. The Skull of Amiurus. With 8 plates. By J. E. Kindred. $1.25. 


No. we Contributions to the Life Histories of Gordius robustus Leidy and Paragordius varius 
(Leidy). With 21 plates. By H. G. May. $1.50. 


got 3-4. Studies on Myxosporidia. A Synopsis of Genera and Species of Myxosporidia. 
With 25 plates and 2 text-figures. By R. Kudo. $3.00. 


Vol. VI 
No.1. The Nasal Organ in Amphibia. With 10 plates. By G. M. Higgins. $1.00. 


Nos. 2-3. Revision of the North American and West Indian Species of Cuscuta. With 13 
plates. By T. G. Yuncker. $2.00. 


No. 4. The Larvae of the Coccinellidae. With 6 plates. By J. H. Gage. 75 cts. 


Vol. VII 


No.1. Studies on Gregarines, II. Synopsis of the Polycystid Gregarines. With 4 plates. 
By Minnie Watson Kamm. $1.00. 

No. 2. The Molluscan Fauna of the Big Vermilion River, Illinois. With 15 plates. By F. C. 
Baker. $1.25. 

No. 3. North American Monostomes. With 9 plates. By E. C. Harrah. $1.25. 

No. 4. A Classification of the Larvae of the Tenthredinoidea. With 14 plates. By H. Yuasa. 


$2.00. 
Vol. VIII 
No.1. The Head-capsule of Coleoptera. With 26 plates. By F. S. Stickney. $2.00. 
No. 2. Comparative Studies on Certain Features of Nematodes and Their Significance. With 
4 plates. By D. C. Hetherington. $1.00. 
No. 3. oe Fungi from British Guiana and Trinidad. With 19 plates. By F. L. Stevens. 
1.25. 
No.4. The External Morphology and Postembryology of Noctuid Larvae. With 8 plates. 
By L. B. Ripley. $1.25. 


[Continued on back cover.] 


ILLINOIS BIOLOGICAL MONOGRAPHS 2 


Vol. IX 


No.1. The Calciferous Glands of Lumbricidae and Diplocardia. With 12 plates. By Frat 
Smith. $1.25. ee 


Nos. 2-3. A Biologic and Taxonomic Study of the Microsporidia. With 27 plates and 9 text 
figures. By R. Kudo. $3.00. bs 


No. 4. Aa Ecology of an Illinois Elm-maple Forest. With 7 plates. By A. O, Weese. 
1.25. 


2 Bis Yee 


Vol. X 3 


No.1. Studies on the Avian Species of the Cestode Family Hymenolepididae. With 9 plates oF 
and 2 text-figures. By R. L. Mayhew. $1.50. 


No. 2. Some North American Fish Trematodes. With 6 plates, 2 charts, and 1 text he nd 
_ By H. W. Manter. $1.50. : es 


No. 3. Comparative Studies on Furcocerous Cercariae. With 8 plates and 2 text-figures. By 2 5 
H. M. Miller. $1.25. BAS 


No. 4. A Comparison of the Animal Commitee of Concer ons and Deciduous Forests. aac 
With 16 plates. By I. H. Blake. $1.50. i 


Vol. XI 


No.1, An Seay Study of Southern Wisconsin Fishes. With 16 plates. By A. R. Cahn. * 
1.50. Deere Ne 


No. 2. Fungi from Costa Rica and Panama. With 18 plates. By F. L. Stevens. $1.25. 


No.3. The  eceeis and Development of Corallobothrium. With 5 plates. By H. E. Essex. e, 
1.00 


No. 4. see “6 ay Caryophyllaeidae of North America. With 7 plates. Ey G. W. Hunter, — 
. $1.25. 


Vol. XII _ 
No. 1. ae Studies of the Genus Cercospora. With 4 plates. By W.G. Solheim, _ 
1.00 


No.2. Morphology, Taxonomy, and Biology of Larval Scarabaeoidea. With 15 plates. By 
W. P. Hayes. $1.25. 


No. 3. Sawflies of the Sub-Family Dolerinae of America North of Mexico. With 6 plates. 
By H. H. Ross. $1.25. 


No.4. A Study of Fresh-water Plankton Communities. By Samuel Eddy. $1.00. 


Vol. XIII 


No.1. Studies on Some Protozoan Parasites of Fishes of Illinois. With 8 plates. By R. R. 
Kudo. 75 cents. 


No.2. The Papillose Allocreadiidae: A Study of Their Morphology, Life Histories, and 
Relationships. With 4 plates and 6 text-figures. By S. H. Hopkins. $1.00, 


No. 3. Evolution of Foliar Types, Dwarf Shoots, and Cone Scales of Pinus, with Remarks 
oncerning Similar Structures in Related Forms. With 32 text-figures, By 
C. C. Doak. $1.50. 


No. 4. A ar ii Rearrangement of Lophodermium. With 5 plates. By L. R. Tehon. 
2.00. 


Vol. XIV 


No. 1. ke rae of the Pectoral Limb of Necturus maculosus. With 11 plates. By H. K. 
hen. $1.00. 


No. 2. Studies on North American Cercariae. With 8 plates. By E. L. Miller. $1.50. 


No. 3. Studies on the Morphology and Life History of Nematodes in the Genus Spironoura. 
With 5 plates and 2 text-figures. By J. G. Mackin. $1.00. 


No. 4. The Life History of Cotylophoron cotoylophorum, a Trematode from Ruminants. 
With 9 plates. By H. J. Bennett. $1.50. 


Address orders: Director, University of Illinois Press, Urbana, Illinois. 


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