This dissertation has been 64—6978 microfilmed exactly as received

WILLIAMS, Russell Raymond, 1926- LIFE HISTORY STUDIES ON FOUR DIGENETIC TREMATODES THAT UTILIZE LYMNAEA (STAG- NICOLA) REFLEXA (SAY) AS THEIR FIRST INTERMEDIATE HOST IN A TEMPORARY POND HABITAT. \ The Ohio State University, Ph.D., 1963 ^^m f^rsity Microfilms, Inc., Ann Arbor, Michigan LIFE HISTORY STUDIES ON POUR DIGENECIC TREMATODES THAT UTILIZE

LYMNAEA (STAGNICOLA) REFF.EXA (SAY) AS THEIR FIRST

INTERMEDIATE HOST IN A TEMPORARY POND HABITAT

DISSERTATION

Presented in Partial fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

Russell Raymond Williams, B. Sc., M. Sc.

■5H H H H H t

The Ohio State University 1963

Approved by

Adviser tent of Zoology Entomology /

ACKNOWLEDGEMENTS

I wish to extend ny sincere, appreciation to the many persons whose help has been invaluable in the completion of this study.

For the help and guidance given me during the preparation of this thesis I am especially indebted to my adviser, Dr. Joseph N. Miller.

In addition, his patience and understanding have been greatly appreciated throughout my years of graduate training. I wish to express my thanks to Dr. Allen McIntosh for the loan of specimens from the U. S. National Museum Helminthological Collection, and to

Dr. Henry van der Schalie for identifying the species of lymnaeid snail used in this research. I wish also to thank the following:

Dr. Robert Shields for sectioning and staining material for me as well as for his many helpful suggestions} Mr. James McGraw for helping me in the laboratory and on collecting trips; and Dr. John

Crites and Dr. Wilbur Tidd for their interest, encouragement, and helpful suggestions. In addition, I wish to thank Mr. Lutz May for permitting me to work and collect at Calamus Swamp. To the many other persons who have helped either directly or indirectly,

I am also greatly indebted. Not to be forgotten is ny wife, Carol, whose constant encouragement contributed to the completion of this study.

ii TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS...... ii

LEST OF TABLES...... ▼

LIST OF ILLUSTRATIONS...... vi

INTRODUCTION...... ^. . . 1

MATERIALS AND METHODS...... h

OBSERVATIONS AND RESULTS...... 10

The First Intermediate H o s t ...... 10

The Trematode Parasites ...... U The Taxonomic Positions of the Four Trematodes Encountered in this Investigation ...... 11 Diplostomum micradenum (Cort and Brackett, 1938)...... 13 Introduction...... 13 The Daughter Sporocyst...... 18 The Cercaria...... 19 Penetration Experiments...... 21 The Metacercaria...... 2k Second Intermediate Hosts...... 25 Recovery From Eye and Br a i n ...... 26 Feeding Experiments with Metacercariae...... 29 The Adult...... 32 Discussion...... 33 Protechinostoma mucronisertulatum Beaver, 19U3...... 37 Introduction...... 37 The Redia...... U5 The Cercaria...... U5> Encystment...... h i The Metacercaria...... h i Excystment...... 1*8 Feeding Experiments with Metacercariae...... 1*9 The Adult...... 51 Discussion...... 53 Notocotylus stagnicolae Herber, 19U2...... 55 Introduction...... 55 The Redia ...... 59

iii TABLE OF C OUT EM'S (CONTINUED)

Page

The Cerceria...... 60 The Metacercaria...... 62 Feeding Experiments withMetacercariae ...... 63 The Adnlt ...... 6U Discussion...... 66 Plagiorchis noblei Park, 1 9 3 6 ...... 67 Introduction...... 67 The Daughter Sporocyst...... 72 The Cercaria...... 73 Penetration of SecondIntermediate Host .... 75 The Metacercaria...... 77 Feeding Experiments with Metacercariae...... 77 The A d u l t ...... 79 The Egg and Miracidium...... 81 Discussion...... 83

SUMMARY...... 89

LITERATURE CITED...... 92

AUTOBIOGRAHIT...... 96

iv LIST OF TABLES

Table Page

1 1961 Collection and Infection Data for Iymnaea (S.) reflexa...... “ ...... I k

2 1962 Collection and Infection Data for lynmaea (S.) reflexa...... - ...... 15

3 1963 Collection and Infection Data for Lymnaea (S.) reflexa...... “ ...... 16

U Diplostomum micradenum (Cort and Brackett, 1938) Exposure”of fish and Amphibia to Cercariae...... 22

5 Diplostomum micradenum (Cort and Brackett, 1938) Recovery of Metacercariae from Rana pipiens Tadpoles 30 Days after Exposure to $0 CercariW Each...... 28

6 A Two-Way Analysis of Variance for the Metacercarial Data from 19 Rana pipiens Exposed to 50 Cercariae Bach . 29

7 Diplostomum micradenum (Cort and Brackett, 1938) % Results of1 Feeding Metacercariae from Rana pipiens Tadpoles to One-Day-Old Domestic Du c k s ...... 30

8 Diplostomum micradenum (Cort and Brackett, 1938) Results of deeding Kfetacercariae from Rana pipiens Tadpoles to One-Day-Old DomesticCh i c k e n s ...... 31

9 Protechlnostoma mucronisertulatum Beaver, 19b3 Results of Feeding Metacercariae to Various Vertebrate Animals...... 50

10 Notocotylus stagnicolae Herber, 191*2 Results of Feeding 1- to 1;-Day-Old Metacercariae to Domestic Chickens...... 63

11 Plagiorchis noblei Park, 1936 Results of Feeding Metacercariae to Vertebrate Animals...... 78

v LIST OF ILLUSTRATIONS

PIATE I ......

Life History Stages of Diplostomum micradenum. Fig. 1.— Metacercariae in Retina of Eye of Rana pipiens Tadpole.

PIATE II ......

Life History Stages of Diplostomum micradenum. Fig. 1.--Metacercariae in Vesicles of Cerebral Hemispheres of Rana pipiens Tadpole.

H A T E H I ......

Life History Stages of Dfmlostomum micradenum. fig. 1.— Cercaria. Ventral View. Fig. 2.— Cercaria Shoving Normal Resting Position, fig. 3• —Daughter Sporocysts. Fig. U>— Eleven-Day-Old Metacercaria. Ventral View. Fig. 5>*— Thirteen-Day-Old Metacercaria. Ventral View, fig. 6 . — Adult fluke. Ventral View.

PIATE IV ......

Life History Stages of Protechlnostoma mucronisertulatum. Fig. 1.— Cercaria. Dorsal View. Fig. 2.— Cercaria Shoving fin Fold. Lateral View. Jig* 3«— Mature Redia Containing Cercariae. fig. — Immature Redia. fig. 5.— Encysted Metacercaria. fig. 6 .— Excysted Metacercaria with Cyst Still Present, fig. 7.--Adult Fluke. Ventral View.

HATE V ......

Life History Stages of Notoeotylus stagnicolae. fig. 1.— Daughter Redia Containing a Single Cercaria. Fig. 2.— Cercaria. Dorsal View, fig. 3.— Encysted Metacercaria. Fig. it.— Adult fluke. Ventral View, fig. 5-— filamentous Egg. LIST OF ILLUSTRATIONS (CONTINUED)

PLATE V I ......

Life History Stages of Plagiorchis noblei. Fig. 1.— Daughter Sporocyst Containing Cercariae. Fig. 2.— Cercaria. Ventral View. Fig. 3•— Stylet. Dorsal View. Jig. It.--Stylet. Lateral View. Jig. 5 .--Oral Sucker of Cercaria Showing Position of Stylet. Lateral View. Jig. 6 .--Encysted Metacercaria. Jig. 7.— Six-Day-Old Experimental Adult Jluke. Ventral View. Fig. 8 .— Adult Fluke from Natural Infection in Red-Winged Blackbird. Ventral View.

PLATE VII ......

Life History Stages of Plagiorchis noblei. Jig. 1.— Egg from Near Metraterm of Uterus. Jig. 2.— Egg Possessing Miracidium after Jive Days of Incubation. fig. 3«~Adult Jluke. Transverse Section Through Region of Genital Pore. Fig. h-— Adult Jluke. Transverse Section Through Region of Acetabulum. Jig. 5 .--Adult Jluke. Transverse Section Through Region Posterior to Testes. INTRODUCTION

This represents a stu

Snails of this species were collected over a two-year period at

Calamus Swamp which is a natural temporary pond near Circleville in

Pickaway County, Ohio. This swamp covers approximately ten acres when full. Calamus Swamp was included in the March, 19llt, geological survey edition of the Circleville Quadrangle, and undoubtedly dates back to many years before that time. According to Stansbery (personal communication), the underlying floor of the swamp consists primarily of a clay lens which forms an impervious layer in the glacial till of the region. Water drains into the swamp only from adjoining fields.

Although considered a temporary pond, there are summers, e.g., 1961, when the water level remains high and there is standing water the entire twelve-month period. During the year 1962, however, there was no standing water in August, September, or October, and relatively little in November. The swamp is bordered by trees and forms a well protected habitat for numerous birds. This pond has been of interest to zoologists and botanists alike and is visited frequently each year by both. Although parasitologists have at times taken an interest in the helminth fauna of this area, no long-term studies have been made on any of the numerous helminths known to be present. Six trematode species were found utilizing Iymnaea (S.) reflexa as their first intermediate host in this habitat. Two of these,

Echinostoma revolutum (Froelich, 1802) and Apatemon gracilis (Rudolph!,

1819), are cosmopolitan in distribution, and have been covered thor­ oughly in the literature. Beaver (1937) did a monographic study on

E. revolutum which stands as a masterpiece to the present day. The strigeid fluke, A. gracilis, has received considerable attention by

Szidat (1931) in Europe, Yamaguti (1933) in Japan, and Stunkard, Willey,

and Rabinowitz (19U1) in the United States. Although life history work was done with these two flukes at Calamus Swamp, this work will not be covered in the present study. The present work is a study on four addi­ tional trematodes3 Diplostomum micradenum (Cort and Brackett, 1938);

Protechinostoma mucronisertulatum Beaver, 19U3j Rotocotylus stagnicolae

Herber, 19U2; and Plagiorchis noblei Park, 1936, all of which are

considerably less well known. Plagiorchis noblei has been known previ­

ously only in the adult form. The egg, miracidium, sporocyst, cercaria,

and metacercaria of this trematode are described here for the first tire.

Although life history work was done by Olivier (19U0) on Diplostomum

micradenum, his study was discredited by Dubois (1953) who placed this

species in synonymy with Hysteromorpha triloba (Rudolphi, 1819) • It is

intended that the present life history study on Diplostomum micradenum

will strengthen Olivier's previous study and clarify the taxonomic

position of this trematode. Life history studies also have been

conducted on Protechinostoma mucronisertulatum and Notocotylus stagni­

colae . Little is known about the distribution of these four species of

trematodes. Twenty-four collections, tnfca.lH.ng 1,252 specimens of Iymnaea

(S.) reflexa, were made from March 23, 1961, through May 2 k, 1963. An examination of these snails revealed that 2h per cent were positive for

Protechlnostoma mucronisertulatum, 3-3 per cent for EcMnostoma revolutum, 3*3 per cent for Plagiorchis noblei, 2.5 per cent for

Apatemon gracilis, 1.0 per cent for Diplostomum micradenum, and O.U per cent for Notocotylus stagnicolae. Since this study centers around only four of these species, no further information will be presented on

Echinostoma revolutum and Apatemon gracilis.

While other species of snails, present at Calamus Swamp, also were examined for cercariae, this was not done so extensively as it was for Iymnaea (S.) reflexa. Other snails include the aquatic pulmonates,

Helisoma trivolvis (Say), Gyraulus parvus (Say), and Physa gyrina (Say), as well as two terrestrial forms, Suecinea avara (Say), and Oxyloma retusa (Lea), which were found associated with Calamus Swamp. None of these were infected with any of the four species of trematodes treated in this stu^y. MATERIALS AND METHODS

Specimens of Iymnaea (S.) reflexa were collected by hand from

Calamus Swamp and brought back to the laboratory where they were placed individually into small, wide-mouth glass jars, three-fourths filled with tap water. Each jar was checked for cercariae each morning and afternoon for a period of three weeks. By holding a jar at eye level while looking through it toward a well-lighted window, cercariae in the water could be seen easily. It was necessary to rotate the jar gently during this observation in order to bring up into the water column any cercariae which might be present but resting on the bottom of the container. When cercariae were detected, the jar was then viewed beneath a stereoscopic microscope and a sample taken of the cercariae with a fine pipette. A wet mount, containing the living cercariae, was then made and checked beneath the 10X and

U3X objectives of a compound microscope equipped with a 10X ocular.

Living material, both stained and unstained, was examined. The vital

stain that was used was neutral red. To make observations of the

excretory system of a cercaria, it was necessary to gradually flatten

the specimen by slowly removing water from beneath the coverglass.

This was accomplished easily by using pieces of absorbent paper towel.

By careful manipulation one could control the rate of water removal.

Fixed cercariae for measurements were prepared by adding water

containing the cercariae to an equal volume of 6 per cent formalin. These cercariae received no further treatment, and measurements were made from prepared wet mounts.

Sporocysts and redial stages were obtained most frequently by crushing the snails and removing the soft parts containing these immature stages. The sporocysts and rediae were dissected out, and wet mounts were made for immediate examination of the living material. In order to make measurements of these stages they were killed and fixed by the same method that was used for the cercariae.

Various invertebrate animals were collected frequently from ponds and streams in the immediate area and checked as possible second intermediate hosts. Midge and caddisfly larvae, damselfly and dragonfly naiads, and mayfly nymphs were collected for penetration experiments with xiphidiocercariae, but they were never used for infection experi­ ments since it was not known if they had been infected previously. For infection experiments, where insects probably serve as the second intermediate hosts, larvae of Aedes aegypti were used. Eggs of this mosquito were obtained from the mosquito rearing room in the Department

of Zoology and Entomology. The mosquitoes were reared in a white opaque plastic container having a fine-mesh screen top. When needed, larvae

of known age and size were removed from this container and placed in

small glass jars with cercariae. Uninfected lymnaeid snails were

obtained from a known lymnaeid population reared in the Department

greenhouse.

Numerous vertebrate animals were also used as possible second

intermediate hosts. These consisted of both fish and amphibia. The

fish came from three different sources. The brook sticklebacks, Eacalia inconstans, were from a stream in Champaign County; the black bullheads, Ictalurus melas, and the green sunfish, Lepomis cyanellus, were from the Olentangy River in Eranklin County; and the goldfish,

Cyprinus auratus, from a local department store. These fish were tested as possible second intermediate hosts for Diplostomum micradenum. Each of 26 fish was exposed to approximately 200 cercariae in a crystallization dish for a period of two hours. It was possible that these fish could have been infected with metacercariae of strigeid trematodes, and, therefore, controls for each group also were established.

With the exception of Ambystoma opacum, all amphibians used as possible second intermediate hosts were reared from eggs, either brought to the laboratory as egg masses or laid by the animals in the labora­ tory. Larvae of Ambystoma opacum were collected from a shallow pool in

Crane Hollow in Hocking County which was free of any lymnaeid snails.

Examinations of Ambystoma larvae from such pools in Crane Hollow have

always shown these larvae to be uninfected with trematodes. In March,

1962, three pairs of Rana sylvatica frogs in amplexus were brought

to the laboratory and permitted to continue breeding, resulting in three

fertilized egg masses being produced. Other egg masses of R. sylvatica

were obtained from shallow pools in Crane Hollow, as were egg masses of

Pseudacris brachyphona, Ambystoma jeffersonianum, and a species of Bufo.

Eggs of Rana pipiens were obtained from Calamus Swamp. The frog tadpoles

and salamander larvae were reared at ambient room temperature in shallow,

clear, plastic containers, twelve by ten by four inches, the water being

kept at a depth of about two inches. Two to three hundred tadpoles were

kept in each container. Their food consisted of boiled egg yolk and concentrated yeast which was placed in the containers daily. Tap water was always used to refill the containers, being changed only when it

appeared cloudy from waste materials. A few tadpoles from each aquarium were placed in separate containers where conditions were less

crowded. These were allowed to complete metamorphosis. All of the metamorphosed frogs and toads were probably smaller than they would

have been normally under natural conditions; however, with the exception

of the toads, they could be identified to species.

Most of the animals that were used as possible experimental

definitive hosts were laboratory reared. Juvenile frogs, which had

metamorphosed from laboratory reared tadpoles, were used on occasion.

Young mice came from known laboratory stock. Chicken eggs which were

obtained from The Ohio State University Poultry Department and white

Pekin duck eggs from the Ridgeway Hatcheries in La Rue, Ohio, were

incubated in the laboratory in a hl6-egg, 187-watt incubator. In most

instances only one-day-old unfed chickens and ducks were used.

In addition, six one-day-old chicks and three four-week-old, cage-reared

chickens were obtained from The Ohio State University Poultry Department.

Only four vertebrate animals that were tested as possible definitive

hosts were collected from the field. One wood frog, Rana sylvatica,

and one green frog, R. clamitans, came from Crane Hollow in Hocking

County; one juvenile robin, Turdus migratorius, and one adult starling,

Sturnus vulgaris, came from The Ohio State University farm on Kenny

Road. Metacercariae of the various parasites were introduced orally

into these animals. Metacercariae that were free from any tissue were

introduced by means of a medicine dropper, whereas whole organisms containing metacercariae were force fed to these vertebrate animals with the aid of forceps. Following the introduction of metacercariae, alls chickens and ducks were kept in suitable pens in the laboratory and fed commercially prepared feed.

Adult trematodes were handled differently than were larval

stages. It was necessary to make some observations on living specimens, but measurements could not be made accurately on such material. Adult worms were most frequently heat killed. This entailed placing the

living specimens in a watch glass with a few milliliters of Ringers

Warm and then quickly pouring a large volume of boiling water directly

on the specimens. Following this killing process, the specimens were

transferred to Lavdowsky’s formula of AFA fixing fluid. Occasionally worms were killed and fixed by introducing AFA beneath a coverglass

under which they were being flattened. Following their fixation in

AFA, many of the worms were stained with Semichon’s carmine, dehydrated

in ethyl alcohol, cleared in xylene, and mounted in piccolyte on

standard 3" x I” microscope slides. Other flukes, not so treated, were

transferred to one dram screw cap vials containing 75 per cent ethyl

alcohol.

Several frog tadpoles and two adult flukes were sectioned for

use in this study. The tadpoles were fixed in alcoholic Bouin's

solution and the flukes in Lavdowsky’s formula of AFA. All material

was dehydrated in ethyl alcohol, cleared in xylene, and imbedded in

paraffin. This imbedded material was cut with a rotary microtome at

thicknesses of eight and ten microns. A modified Azo-Carmine-G stain was employed to stain the sectioned material prior to dehydrating, clearing, and mounting it.

Prog tadpoles were exposed to Diplostomum micradenum in

Syracuse watch glasses. By using a fine pipette, drops of water containing cercariae weresplaced separately in a watch glass. A count of the cercariae in each drop was made beneath a stereoscopic type of microscope. Following the introduction of the cercariae, two to three milliliters of water were added and all of the drops combined, leaving a single reservoir of water and cercariae. Prior to the introduction of a tadpole, one side of the watch glass was raised and permitted to rest on the edge of a second watch glass. A whole series of these was prepared at one time, each resting on one edge of another.

The tadpoles were introduced into the troughs of water, one to each watch glass. This method assured close contact between the cercariae

and the tadpole. Following a thirty-minute exposure, each tadpole was

removed and placed in an appropriate aquarium. In only a few instances

cercariae were found in a watch glass following this thirty-minute

exposure.

Drawings prepared for this study frequently were made free

hand or with the aid of either a camera lucida or a Bioscope projector.

Measurements on all trematodes were made with the aid of a micrometer

eyepiece calibrated for use with an A-0 Spencer compound monocular

microscope. "With only a few exceptions, all measurements in this

research are expressed in. millimeters, and in most instances a range,

from minimum to maximum, is given for each structure, followed by the

mean in parentheses. OBSERVATIONS AND RESULTS

The First Intermediate Host

The species of pulmonate snail at Calamus Swamp serving as the first intermediate host for the four trematodes considered in

this research was identified as Lymnaea (Stagnicola) reflexa (Say).

According to van der Schalie (personal coiranunication), this species may well he a part of the Lymnaea palustris complex. There are

relatively little anatomical differences in many of the related

groups of Iymnaeids.

Calamus Swamp appears to be an ideal habitat for Iymnaea (S.)

reflexa. This species, according to Baker (1928), is found in small

pools or ponds \hich may become more or less dry in summer. In

Wisconsin frequently he found this snail in swales in woods or fields,

but never in any of the large streams or lakes. At Calamus Swamp this

form was relatively abundant, but because of its behavior it was

sometimes difficult to find. In early March, soon after the ice has

disappeared from the pond and the water level has risen, many of these

snails were found clinging to dead cattail stems and other aquatic

vegetation near the surface on warm sunny days. If the day was cloudy,

cold, and windy, relatively few snails were found. In this respect,

this species may behave similar to Lymnaea palustris. Cheatum (193U)

observed that Iymnaea palustris migrated from the surface to deeper 11 water •when the water temperature dropped from 21° C to 10° C. Their habit of migrating in the spring from deeper water to the shore, where

they breed, often made hand collecting of snails a problem. As long

as they were at the surface some distance from the shore there was no

great difficulty involved in locating them, but along the shore they became increasingly difficult to find. The color of their shells

blended with the leaf litter over which they crawled.

At Calamus Swamp mature Iymnaea (S.) reflexa varied considerably

in size. When the summer was dry and many of the snails were forced to

aestivate, early collections made the following spring seldom contained

snails with shells greater than 2f> mm in length. However, if the pond

remained filled the entire summer, the first snails collected the

following spring were 30 to 35 mm long. Some snails of this species

attained a length of 39 to i|0 mm.

Most of the mature snails, if not infected, were kept alive

under laboratory conditions for two or three months, but at no time was

I able to keep any of the newly hatched snails alive for more than one

or two weeks.

The Trematode Parasites

The Taxonomic Positions of the Four Trematodes

Encountered in this Investigation

CLASS: Tr emat oda

SUPERORDER: Anepitheliocystidia La Rue, 1957

ORDER: Strigeatoidea La Rue, 1926 SUBORDER: Strigeata La Rue, 1926

SUPERFAMILY: Strigeoidea Railliet, 1919

FAMILY: Diplostomidae Poirier, 1886

SUBFAMILY: Diplostominae Mbnticelli, 1892

Diplostomum micradenum (Cort and

Brackett, 1938)

ORDER: Echinostomida La Rue, 1957

SUBORDER: Echinostomata Szidat, 1939

SUPERFAMILY: Echinostomatoidea Faust, 1929

FAMILY: Echinostomatidae Looss, 1902

SUBFAMILY: Echinostomatinae Faust, 1929

Protechinostoma mucronisertulatum

Beaver, 1914-3

SUBORDER: Paramphistomata Szidat, 1936

SUPERFAMILY: Notocotyloidea La Rue, 1957

FAMILY: Notocotylidae L\ihe, 1909

SUBFAMILY: Notocotylinae Kossack, 1911

Notocotylus stagnicolae Herber, 19U2

SUPERORDER: Epitheliocystidia La Rue, 1957

ORDER: Plagiorchiida La Rue, 1957

SUBORDER: Flagiorchiata La Rue, 1957

SUPERFAMILY: Plagiorchioidea Dollfus, 1930

FAMILY: Flagiorchiidae Ward, 1917 13

SUBFAMILY: Plagiorchiinae Pratt, 1902

Plagiorchis noblei Park, 1936

Diplostomum micradenum (Cort and Brackett, 1938) (Plates I, II, I H )

Introduction

The cercariae of Diplostomum micradenum made their appearance in early collections of Iymnaea (S.) reflexa, being most abundant during late April and early May (Tables 1, 2, 3). This was true for both 1961 and 1962, but in the spring of 1963, following an exceptionally dry summer this species was particularly scarce. Only two infected snails were found, and the first snail shed only a few hundred cercariae. Of the 1,2]?2 snails examined over the two-year period, only twelve, or

1 per cent, were found shedding cercariae of Diplostomum micradenum.

Cort and Brackett (1938) described Cercaria micradena from the pulmonate snail, Stagnicola palustris elodes in the Douglas Lake region of Michigan. Of 555 snails that they collected, only one was shedding this strigeid. This infected snail was turned over to Olivier who found that these cercariae penetrated tadpoles of Rana pipiens and developed to mature metacercariae in the central nervous system of this host.

Olivier (19U0) continued his life history studies on this fluke during the summers of 1938 and 1939 and published a description of Diplostomum micradenum (n. comb.) after recovering adult worms experimentally from the pigeon. Of 2,705 Stagnicola palustris elodes collected from the

Douglas Lake region during the summers of 1937, 1938, and 1939, only four snails were found harboring this trematode. With the aid of TABLE 1

1961 COLLECTION AND INFECTION DATA FOR LIMNAEA (S.) REFLEXA

Number Shedding Cercariae Date Number Diplostomum Protechinostoma Notocotylus Plagiorchis Collected Collected micradenum mucronisertulatum s'tagnicolae noblei

March 23 105 2 (l.9)a ll5 U12.9) 0 (0.0) 5 0i.8)

April 23 65 1 (1.5) 3li (52.li) 0 (0.0) 1 (1.5)

May 3 7 0 (0.0) 2 (28.6) 0 (0.0) 0 (0 .0 )

May 22 111 0 (0.0) 2i (28.6) 0 (0.0) 1 (7.2)

August 1 20 0 (0.0) 1 (5.0) 0 (0.0) 0 (0.0)

aFigures in parentheses represent percentage of infection. TABLE 2

1962 COLLECTION AND INFECTION DATA FOR LYMNAEA (S.) REFLEXA

Nranber Shedding Cercariae Date Number Diplostomum Protechinostoma Notocotylus Plagiorchis Collected Collected micradenum mucronisertulatum stagnicolae noblei

March 21 60 1 (l.7)a 28 (1*6.7) 0 (0.0) 5 (8.3)

March 28 8 0 (0.0) 3 (37.6) 0 (0.0) 0 (0.0)

March 31 6 0 (0.0) 2 (33.lt) 0 (0 .0) 0 (0.0) I April 21 10 1 (10.0) 1 (10.0) 0 (0.0) 0 (0.0)

May 1 10 1 (10.0) 2 (20.0) 0 (0.0 ) 1 (10.0)

Nay $ 32 3 (9.h) 5 (15 .6 ) 0 (0.0) 1 (3 .1)

May 27 81* 1 (1.2) 3 (3.6) 0 (0 .0 ) 0 (0.0)

June 26 173 0 (0 .0 ) 3 (1.7) 1 (0 .6 ) 8 (It.6)

Nov. 11 7 0 (0.0) 1 (Ht.3) 0 (0.0) 0 (0 .0)

Nov. 2h 9 0 (0 .0) 0 (0.0) 0 (0 .0) 0 (0.0)

aFigures in parentheses represent percentage of infection. is

TABLE 3

1963 COLLECTION AND INFECTION DATA FDR LYMNAEA (S.) REFLEXA

Number Shedding Cercariae Date Number Diplostomum Protechinostoma Notocotylus I’lagiorchis Collected Collected micradenum mucronisertulatum siagnicolae noblei PJ _* O 0 • O March 17 206 V 13 (6.3) 1 (0.5) 7 (3.10

March 23 226 1 (o.li) 9 (lt.O) 2 (0.9) 5 (2.2)

March 30 8 0 (0.0) 1 (12.5) 0 (0.0) 0 (0.0)

March 31 8 0 (0.0) 2 (25.0 ) 0 (0.0) 0 (0.0)

April 7 Sh 0 (0.0) 18 (33.lt) 0 (0.0) k (7.10

April 111 35 0 (0.0) 1 (2.9) 0 (0.0) 0 (0.0)

April 28 2li 0 (0.0) 5 (20.8) 0 (0.0) 2 (8.3 )

May 2 53 1 (1.9) 9 (17.0) 1 (1.9) 1 (1.9)

May 2k 28 0 (0.0) 10 (35.7) 0 (0.0) 0 (0.0)

aFigures in parentheses represent percentage of infection Olivier’s specimens, Dubois and Rausch (1950) redescribed the adult of

Diplostomum micradenum in their review of North American strigeids from birds. Later, Dubois (1953) in his monograph on strigeid trematodes considered the similarities of adults and discarded Olivier’s life history work, placing this fluke in synonomy with ffysteromorpha triloba (Rudolphi, 1819). Hugghins (l95k) made a study of the life history of Hysteromorpha triloba, but did not consider Diplostomum micradenum as a synonym.

In the genus Diplostomum the general life cycle patterns involve two intermediate hosts and a definitive host. The first intermediate host apparently is always a pulmonate snail; the second intermediate host is a vertebrate animal, lamprey, fish, or amphibian; and the definitive host most frequently is a fish eating bird. Miracidia, hatching from eggs in the water, penetrate the snail host where they develop to mother sporocysts. The mother sporocysts give rise to a second generation of sporocysts, and these daughter sporocysts produce fork-tailed cercariae. The cercariae emerge from the snail host, becoming free living for a short time in the water. For the cycle to continue, a cercaria must penetrate a second intermediate host, usually a fish in which it develops for a period as a metacercaria. In

Diplostomum micradenum, amphibia,rather than fish, serve as second intermediate hosts. Development of the metacercaria may take place in either the central nervous system or in the eye of the host. From the time of its formation the metacercaria does not encyst, but is an active stage referred to as the diplostomulum larva. Following the development of the metacercaria, generally around thirty days, the diplostomulum is 18 infective'to the definitive host. The definitive host acquires the infection when it ingests the second intermediate host. This releases the metacercaria which develops in the small intestine to a mature adult within six to ten days. Eggs that are produced by the fluke reach the outside aquatic environment in the feces of the definitive host.

The following pages include descriptions of the life history stages with which I worked and also the results of penetration experi­ ments with cercariae and feeding experiments with the metacercaria of

Diplostomum micradenum.

The daughter sporocyst (Plate ill, Fig. 3)

Long cylindrical body, threaded through the digestive gland of

the snail. Young daughter sporocysts producing cercariae, 1.5 to 2.5 mm long and 0.068 mm wide. Older sporocysts, 1 centimeter or more in

length. Anterior end bluntly rounded, with prominent protruding birth pore on one side close to anterior end. Birth canal long and narrow.

Numerous cercariae in various stages of development from spherical germ masses to mature forms. Sporocysts active; anterior end moving in

snake-like manner. Body wall containing dark granular material impart­

ing a dark appearance to the sporocyst. Old sporocysts with constric­

tions at various levels along their bodies.

Because of the blunt anterior end, prominent birth pore, and

long birth canal, these sporocysts fit the description given by Cort and

Brackett (1938) for the sporocysts of Cercaria micradena. 19

Hoffman and Hundley (1957) found that at 22-28 degrees centigrade it required twenty-two days for mother sporocysts of Diplostomum baeri eucaliae to begin producing daughter sporocysts. Cercariae were produced thirty days after infection.

The cercaria THatTTTTTFig. 1, 2)

Small furcocercous cercaria; both body and furcae shorter than

tail stem; booty- 0.132-0.178 mm (0.170 mm) long by 0.01+0-0.051 mm (0.01+5

mm) wide. Tail stem 0.251+-0.290 mm (0.275 mm) long by 0.029-0.039 mm

(0.031+ mm) wide with furcae, 0.236-0.268 ram (0.261 mm) long. Oral sucker

larger than acetabulum, 0.030-0.0it5 mm (0.032 mm) long by 0.030-0.038 mm

(0.032 mm) wide. Prepharynx short; pharynx, 0.018 mm long by 0.011 mm

wide, opening into a short esophagus. Bifurcation of intestine midway

between oral sucker and acetabulum. Ceca extending to near posterior end

of body. Acetabulum in anterior part of posterior half of body; smaller

than oral sucker and often protruding; 0.022-0.029 mm (0.027 mm) in

diameter. Acetabulum with a single row of short spines surrounding its

opening. Ten to twelve spines at anterior tip of oral cap; cap with

five or six irregular rows of spines extending about one-half length of

oral sucker. Twelve rows of spines extending from posterior edge of

oral sucker to level of acetabulum, not all completely encircling the

body. Scattered spines on sides of body posterior to level of acetabulum.

No spines on tail or furcae. Tail stem without caudal bodies, but with

twelve to fourteen pairs of long hair-like flagellets on each side.

Body with four penetration glands; one pair anterior, the other posterior

to the acetabulum, difficult to see in both living and preserved 20

specimens. Ducts of penetration glands expand in region of oral sucker.

Genital primordium vaguely visible just anterior to excretory bladder.

Execretory bladder composed of two chambers, the anterior being more

flat laterally. Flame cell formula 2/"(2 + 2 ) + (2 + 2 + {2 )J = 20.

Flame cells of tail stem evenly distributed in its anterior half.

The cercariae of Diplostomum micradenum were shed mainly during

the early morning hours. Jars examined at 6:00 a.m. already had heavy

concentrations of cercariae. A single snail, at room temperature in

the laboratory, shed an estimated 5*000 to 10,000 cercariae per day.

Shedding remained high for four or five days, after which time there was

a rapid decrease in the number. By the tenth day, there may be only a

few hundred shed. By the end of two weeks, only a few cercariae were

present. Snails examined at this time contained long daughter sporocysts

having only an occasional well-developed cercaria, but with numerous

undeveloped germinal masses present. These sporocysts were typical of

old sporocysts, being one centimeter or more in length and having

numerous constrictions along their bodies.

Cercariae of Diplostomum micradenum hang relatively motionless

in the water with their furcae at right angles to each other. Their

period of activity was shorter than their period of rest. Activity

brought about movement only through a very short distance, often not

greater than once or twice their own length. In the laboratory, most

of the cercariae were observed to remain up in the water column for

the greater part of a day. Many remained alive and would penetrate

tadpoles up to U8 hours. 21

Penetration experiments.— Twenty-six fish, consisting of eight goldfish, Cyprinus auratus; four black bullheads, Ictalurus melasj eight brook sticklebacks, Eucalia inconstans; and six green sunfish, Lepomis cyanellus were exposed to the cercariae of Diplostomum micradenum. Each fish was exposed to approximately 200 cercariae (Table U). Since the sunfish and bullheads were young, all of the fish were relatively small and could be exposed and observed easily. Of the many cercariae seen coming in contact with fish, none were ever seen to penetrate. An examination of the fish five to ten days after exposure to cercariae showed that cercariae had not entered in a single instance. Each fish was examined thoroughly, and each was found not to be infected with metacercariae of Diplostomum micradenum.

Frog and toad tadpoles, however, proved to be excellent hosts.

When cercariae came in contact with tadpoles of Rana plpiens, R. sylvatica, Pseudacris brachyphona, and a species of Bufo, they immediately began to penetrate. When in contact with the integument of a tadpole the body of the cercaria lies parallel to the surface of the host. The anterior end then becomes active and produces an opening in the integument. As the cercaria begins to enter this opening, there is a rapid jerking motion of the body, resulting in the tail being cast off. Complete penetration takes place within one to two minutes.

Observations on cercariae that had penetrated the clear tail region of tadpoles revealed that cercariae were capable of moving slowly through the tissues of this host. When encountering capillaries, migrating cercariae entered these blood vessels and crawled rapidly along the lumen. Cercariae were observed entering capillaries on several 22

TABLE U

DIPLOSTOMUM MICRADENUM (CORT AND BRACKETT, 1938) EXPOSURE OF FISH AND AMPHIBIA TO CERCARIAE

Classification and Number Number Number Scientific Name Exposed Examined Infected

Class: Teleostomi Family: Cyprinidae

Cyprinus auratus 8 8 0 family: Ictaluridae

Ictalurus melas h k 0 Family: Gasterosteidae

Eucalia inconstans 8 8 0

Family: Centrarchidae

Lepomis cyanellus 6 6 0

Class: Amphibia® Family: Ranidae

Rana pipiens 350+ 132 132

Rana sylvatica 150+ 76 76

Family: Hylidae

Pseudacris brachyphona 21 18 18

Family: Bufonidae

Bufo. sp. 5o+ 21 21

Family: Ambystomidae

Ambystoma opacum Hi lit 3

Ambystoma jeffersonianum 18 13 k

aOnly larval stages of Amphibia exposed to cercariae. occasions, often blocking the flow of blood in these until they crawled into larger blood vessels. Several times I observed cercariae being swept away in the blood stream as they entered veins from the smaller venules. The entering of a blood vessel simply appeared to be by chance, however, and some cercariae were observed still moving through tissues

for well over twenty minutes after penetration.

Sectioned tadpoles of Rana pipiens (2.0 cm in length) revealed

that cercariae reach their site of infection in the central nervous

system within eight hours after penetration. By twelve hours none were

found outside the central nervous system. Sectioned material also

revealed that the cercariae (now early metacercariae) actually burrow

into the nerve tissue at this early age. Previous workers have reported

fatal cerebral hemorrhages being produced in fish by the burrowing of

large numbers of cercariae of Diplostomum spathaceum. Rees (l955)

observed in the case of Diplostomum phoxini that when penetration of

the cercariae of this form was gradual over a period of time fatal

hemorrhage did not occur. I found that tadpoles, 2.5 to 3.0 centimeters

in length could be exposed to fifty cercariae at one time without fatal

results. However, when the number was increased to one hundred for the

same size tadpoles, about 50 per cent of them died.

Cercariae of Diplostomum micradenum were found to penetrate

larvae of Ambystoma opacum and A. jeffersonianum, but the metacercariae

did not develop properly in these animals. 2h

The metacercaria maiTTTT,“Bi7 U, 5)

Unlike most metacercariae, those of Diplostomum micradenum do not encyst but remain free and active in the central nervous system and eyes of the second intermediate host. From the time of its origin a metacercaria undergoes considerable morphological change. During the first three days, however, little internal change was observed. The oral sucker, acetabulum, digestive system, and excretory system remained relatively unchanged from that of the cercaria, but there was a notice­ able change in the size of the body. There was a growth in length from approximately 0.170 mm to 0.2S>0 mm. Generally on the fourth or fifth day a reserve excretory system began to develop, first as bud-like projections from the excretory bladder, then as long branching ducts extending anteriorly. As the reserve system developed there was a gradual increase in the size of the metacercaria. Seven-day-old meta­

cercariae were 0.376-0.U31 mm (0.399 mm) long by 0.103-0.130 mm (0.117 mm) wide. Also by the seventh day the reserve excretory ducts that

extend medially form a commissure in the region of the acetabulum as well as additional branches. Two such branches extend anteriorly along

the lateral margins of the body and two, one anterior and one posterior,

extend from the commissure in the median zone of the body. By the ninth

day the metacercariae were 0.U51-0.520 mm (0.i|6l mm) long by 0.137-

0.178 mm (0.159 mm) wide. In addition, the excretory system underwent

considerable change. By the eleventh day all of the major ducts of the

reserve system were formed, including another lateral commissure just

posterior to the oral sucker with a medial posterior duct joining the

posterior commissure. Also by the eleventh day a pair of muscular, sucker-like organs have begun their development, one on each side of the oral sucker. In addition, the hind-body has started to form dorsally from the posterior end of the fore-body. A muscular structure, the holdfast organ, also forms on the ventral surface of the fore-body just posterior to the acetabulum. By the thirteenth day the metacercaria appears to have reached full development. The muscular pseudo-suckers on either side of the oral sucker are well developed, the hind-body is decidedly formed, the holdfast organ has taken definite shape, and all of the many branches of the reserve excretory system are complete. The only observable change after the thirteenth day was that of the numerous concretions that form at the ends of the reserve excretory ducts. These concretions increased from 2^ on the fourteenth day to 7^ on the twentieth day.

When diplostomula were removed from the intermediate host they remained active in Ringers Cold for a long period of time. It was not uncommon for them to live in Ringers Cold for an entire day.

Under pressure of a coverglass some of the flame cells were discernible, but the pattern and number was not determined. Rees

(1955) observed flame cells in the diplostomulum of Diplostomum phoxini and established a formula for this species of {"(12) + (20)3 +

(20)7 “ 10U.

Second intermediate hosts.— In addition to Rana pipiens, R. sylvatica, Pseudacris braehyphona, and Bufo sp., that served well as second intermediate hosts in the laboratory, animals having natural infections were obtained from Calamus Swamp. Twelve out of thirteen tadpoles of Rana pipiens collected on May 3, 1961, were infected with 26

immature metacercariae of Diplostomum micradenum, the lowest infection being one and the highest twelve. Four additional tadpoles of t he same

species, collected May 22, 1961, were also infected. At this time, however, the lowest infection was twelve and the highest forty-three.

Only two adult Rana pipiens were collected, but both were infected with metacercariae. One collected March 20, 1962, was an adult male, and

probably was one of the first arrivals at the pond. This frog had 170

fully developed metacercariae in its central nervous system. It was at

least one-year old, and may have been older. Since adult frogs are

refractive to cercariae of Diplostomum micradenum it is probable that

it had obtained this heavy infection as a tadpole. One of two chorus

frogs, Pseudacris triseriata, was infected with twenty-thref meta- —

cercariae. In addition, two tadpoles and one adult Rana clc;-,ritans

were obtained, but all three were uninfected with D. micradenum.

Olivier (l9i|0) mentioned that while cercariae of D. micradenum

penetrated R. clamitans, they did not develop successfully in this frog.

He examined eight tadpoles twenty-one days after they had been exposed

to cercariae, and found only one tadpole to be infected. Moreover,

this one yielded only four poorly developed metacercariae.

In August, 1962, when there was no standing water at Calamus

Swamp, fourteen Acris crepitans were collected from the damp area near

the middle of the pond. None of these were infected with metacercariae.

Recovery from eye and brain. — Metacercariae of Diplostomum

micradenum were always found in greater numbers in the cavities of the

brain and central canal of the spinal cord of the second intermediate

host than they were in the eyes (Plate II, Fig. 1). Within the brain 27 these diplostomula invaded every cavity, and also were found occasion­ ally just beneath the meninges. The metacercariae were found more frequently around the fourth ventricle, optic lobes, and cerebral hemispheres of the brain and to a lesser degree in the central canal of the cord.

Olivier (191*0) did not mention finding diplostomula of

Diplostomum micradenum in the eyes of any of the second intermediate hosts with which he worked. However, the majority of diplostomula of

other species of the genus Diplostomum have been recorded more

frequently from the eyes of the host than from the brain. In almost

all of the life history studies in the genus Diplostomum, involving

fish as second intermediate hosts, the eyes have been reported as

common sites of infection as well as the central nervous system.

Berrie (i960) demonstrated that two distinctly different diplostomula

inhabited the eyes of the stickelback, Gasterosteus aculeatus, one

species in the lens and the other in the retina. In Rana pipiens, I

found metacercariae in the retina of the eye, but never in the lens

(Plate I, Fig. l).

An experiment was conducted in the spring of 1962 in which each

of nineteen Rana pipiens was exposed to fifty cercariae (Table 5).

These tadpoles were examined thirty days after exposure to the cercariae

and a count was made of the metacercariae recovered from the eyes and

also the central nervous system. Jive was the greatest number ever

recovered from the eyes of any individual tadpole, whereas forty-three

were recovered from the brain on one occasion. From a two-way analysis

of variance on these data it was concluded that there were no TABLE 5 28

DIPLOSTOMUM MICRADENUM (CORT AND BRACKETT, 1938)

RECOVERY OF METACERCARIAE FROM RANA PIPIENS TADPOLES 30 DAYS AFTER EXPOSURE TO ^ T S e RCARIAE EACH

V

Number In HQSt Central Nervous Number in Total Number Per Cent No. ______System______Eyes______Recovered______Recovered

1 27 0 27 51*

2 ho 0 1*0 80

3 30 0 30 60

k 31 1 32 61*

5 la 0 Ul 82

6 35 0 35 70

7 23 2 25 50

8 18 2 20 1*0

9 26 5 31 62

10 28 3 31 62

11 37 3 1*0 80

12 36 2 38 76

13 35 1 36 72

U* 1*3 2 1*5 90

19 37 h la ■ 82

16 35 1 36 72

17 37 1 38 76

18 30 2 32 61*

19 31 1 32 61* significant differences between total numbers of metacercariae removed from individual Rana pipiens tadpoles, but that differences between the numbers of metacercariae found at the two locations (eyes and central nervous system) were highly significant at the 1 per cent level (Table 6).

TABLE 6

A TWO-WAY ANALYSTS OF VARIANCE POR THE METACERCARIAL RECOVERY DATA PROM 19 RANA PIPIENS EXPOSED TO SO CERCARIAE U C H

Source of Sum of Degrees of Mean F (Test Critical Variation Squares Freedom Square Statistic) Value

Between locations 9,160.52 1 9,160.52 387.66 8.28

Between individuals 351-58 18 19.53 0.82 3.1

Error U25.18 18 23.62 — —

Total 9,937.58 37 — ——

Feeding experiments with metacercariae.— Both domestic chickens and ducks were used as experimental definitive hosts

(Table 7). Chickens served in this capacity better than did ducks.

Metacercariae of known age were removed from tadpoles and counted in

Ringers Cold. They were then fed to the chickens and ducks orally with the aid of a medicine dropper. Although three adult flukes were obtained from a duck that had been fed twenty-two-day-old meta­ cercariae, no consistent results were obtained with metacercariae 30

TABLE 7

DIPLOSTOMUM MICRADENUM (CORT AND BRACKETT, 1938)

RESULTS OF FEEDING METACERCARIAE FROM RANA PIPIENS TADPOLES TO ONE-DAY-OLD DOMESTIC DUCKS

Number of Age of Time in Host Metacercariae Metacercariae Definitive Host Worms Recovered No. Fed to Host (Days) (Days) Immature Adult

1 26 21 5 0 0

2 31 21 5 0 0

3 m 22 5 0 0

h 38 22 5 0 3

5 36 23 5 0 0

6 29 23 5 0 0

7 95 26 6 0 0

8 7U 26 6 0 2

9 82 28 5 0 0

10 96 J?8 5 0 0

11 103 29 5 0 0

12 119 29 5 0 u

13 115 30 5 0 0

lit 6k 31 5 0 0

13 U5 31 5 0 1

16 113 33 10 0 1

17 69 k l k 0 0 31 older than twenty-two days. In two instances where twenty-nine-day-old metacercariae were fed, only one of these ducks yielded flukes even though both ducks had been fed approximately the same number of meta­ cercariae. At no time was the yield of worms from ducks satisfactory.

Chickens produced much better results, with flukes recovered from all four hosts (Table 8). Since metacercariae that were younger

TABLE 8

DIPLOSTOMUM MICRADENUM (CORT AND BRACKETT, 1938)

RESULTS OF FEEDING METACERCARIAE FROM RANA PIPIENS TADPOLES TO ONE-DAY-OLD DOMESTIC CHTCKENS

Number of Age of Time in Host Metacercariae Metacercariae Definitive Host Worms Recovered No. Fed to Host (Days) (Days) Immature Adult

1 92 28 9 k 10 2 •> 33 6 k u

3 U8 3h I* 15 0

k 63 36 6 0 U

than twenty-eight days were not fed to chickens, nothing can be concluded

about the age at which metacercariae of Diplostomum micradenum first

become infective. Olivier used domestic pigeons as definitive hosts in

his work with this species, but he obtained flukes from only a single

bird which had been, fed metacercariae that were fifty-one days old. He

fed other metacercariae from fourteen to forty-three days old to pigeons but without establishing infections. The recovery of adults by me from chickens and ducks that were fed metacercariae as young as twenty-two to twenty-eight days old brings the age of infectivity within the time normally found for metacercariae of other species in the genus

DiplostoTTtum.

The adult (Plate T H , Fig. 6)

Body consisting of two regions, the anterior ("fore-bocfy«) being flattened and the posterior ("hind-body"), containing the reproductive organs, being cylindrical. Over-all length of specimens that were stained, cleared, and mounted 0.87-1.15 mm (1.02 mm). Fore-body round to oval, 0.1*7-0.57 mm (0.1*9 mm) long by 0.1*0-0.56 mm (0.1*6 mm) wide, concave ventrally. Hind-body more cylindrical, usually slightly longer than fore-body, 0.1*0-0.58 mm (0.5l mm) long by 0.23-0.35 mm

(0.28 mm) wide. Subterminal oral sucker slightly smaller than acetabulum, 0.073-0.087 mm (0.080 mm) long by 0.080-0.093 mm (0.081* mm) wide. A pair of "pseudosuckers" ventro-laterally situated at anterior end, separated by oral sucker. Pharynx posterior and dorsal to oral sucker, 0.05i*-0.080 mm (0.065 mm) long by 0.01*1-0.057 mm (0.051 mm) wide. Esophagus short, bifurcating slightly beyond pharynx j ceca dorso-lateral in fore-body, ventrolateral in hind-body, extending to near posterior end. Vitellaria extend from bifurcation of esophagus to posterior end of body, with maximum density of follicles in fore- body, larger but less dense in hind-body. Follicles predominantly ventral in hind-body. Ovary ovoid, median, in anterior end of hind- body, 0.062-0.100 mm (0.083 mm) long by 0.080-0.152 mm (0.108 mm) wide. 33

Testes posterior to ovary, differ in shape and si v - from each other ; anterior testis asymmetrical longer than wide, on left side of median zone of body, 0.118-0.167 mm (0.1it3 mm) long by 0.097-0.139 mm (0.113 mm) wide; posterior testis in median zone, wider than long; 0 .092-0 .1I4.8 ram

(0.120 mm) long by 0.175-0.256 mm (0.195 mm) wide. Uterus short, not coiled, containing lj-7 eggs. Eggs 0.081; mm long by 0.056 mm wide.

Site of infection: Small intestine.

Definitive host: Anas domestica and Gallus domesticus

(Experimental).

Type specimens: Homotype No. 59670 deposited in U. S.

National Museum Helminthological Collection.

Ten-day-old specimens obtained by Olivier (19U0) from a domestic pigeon averaged 1.15 mm in length, whereas my five and six- day-old specimens averaged 1.02 mm in length. However, the age differences might well account for this. In addition, most of the specimens with which I worked showed a more definite division between the fore-and hind-body than was described by Olivier (I9lt0) and again by Dubois and Rausch (1950) for this species. Different methods of fixation could account for this difference to some degree. In view of no major morphological differences between my adult specimens and those described by Olivier, I must consider them conspecific.

Discussion

Olivier's publication in 19U0 is the only known report of life history work on Diplostomum micradenum. The natural definitive host

is not known to the present day. Lacking additional information on 3U this fluke, Dubois (1953) concluded that Diplostomum micradenum was a synonym of Hysteromorpha triloba (Rudolphi, 1819), a strigeid of cormorants, the metacercariae of which were found encysted in the flesh of fish. Dubois based this taxonomic change on the similarities of the adults of the two forms and discarded Olivier's life histoiy work on D. micradenum, believing D. micradenum to have been worked through an aberrant second intermediate host. The following is directly from

Dubois' 1953 monograph:

Le Diplostome qu'Olivier obtint chez le Pigeon domestique par infestation experimentale au moyen de metacercaires hebergees par des tetards et provenant du developpement de Cercaria micradena Cort et Brack., 1938, nous parait identique & Hysteromorpha triloba (Rud). L 1etude morpholo- gique des adultes ne revele aucune difference ... La cercaire differe de celle des Diplostomes (s. str.) par la situation de ses quatre cellules glandulaires de penetration (deux pre- et deux postacetabulaires). Quant a la meta- cercaire, Ciurea (1930, p. 300-305) la trouve enkystee dans les muscles de Poissons de la f ami lie des Cyprinides, tandis qu'Olivier (op. cit., p. kSh) la de'couvre et l'obtieirfc experimentalement' dans le systeme nerveux central du tetard et 'de l'adulte de Rana pipiens Schr.j il 1' observe egalement chez la larve de Bufo amencanus Hoi. Les resultats plutot negatifs des infestation du Pigeon auraient pour cause, d'apres Olivier, la maturite insuffisante des metacercaires libres, agees de IL a 55 jours et dont la taille (3U0-U50/200-250fA a l'^tat de fixation) est tres inferieure £. celle des larves ehkystees, decrites par Ciurea (690-76o/i|20-550/J , Egalement fix^ees et meme contractees). Certes, on doit tenir compte du fait constate pour diverses especes de Strig^id^s, que 1'enkystement est gen^ralement tardif et que la larve ne devient infestante qu'une fois encapsulee, mais on peut aussi supposer que les metacercaires engagees dans les tetards font fausse route, ont des localisations erratiques et n'achevent leur developpement que difficilement. Le regime piscivore des Cormorans justifie cette supposition. Quoi qu'il en soit, la forme adulte a e te obtenue (longueur: 1 ,0)4. a 1,35 mm), mais le descripteur n'indique pas les mesures des oeufs representes dans la fig. U . Pour les raisons susdites, nous consid^rons Diplostomum micradenum (Cort et Brack.) comme synonyms de Hysteromorpha triloba (Rud.). 35

The life history of Hysteromorpha triloba is known, the most recent work being that by Hugghins (195U) who made a complete stingy of its cycle. In describing the cercaria, Hugghins stated that it has three rows of spines surrounding the acetabulum, that neither pair of penetration glands is posterior to the acetabulum, and that the flame cells of the tail are evenly distributed along the entire length of the tail stem. In contrast, Diplostomum micradenum has a single row of spines around the acetabulum, only one pair of penetration glands posterior to the acetabulum, and the flame cells are about evenly distributed in the anterior half of the tail stem only. As indicated by the measurements obtained by Hugghins, the cercaria of Hystero­ morpha triloba is also larger than that of Diplostomum micradenum. In addition, the resting cercaria of D. micradenum does not exhibit a hook-like shape which is characteristic of H. triloba.

The differences are even more striking when we compare the metacercariae. According to Hugghins, the metacercariae of H. triloba encyst in the flesh of fish, primarily black bullheads, and attain a length of 1.52 mm when mature. On the other hand, it was found that

the cercariae of Diplostomum micradenum did not penetrate any of the

fish used in this study, including black bullheads. These cercariae

penetrated only amphibia, and in Rana pipiens and R. sylvatica the metacercariae attained a length of never more than 0.60 mm when mature.

In addition, twelve weeks were required under laboratory conditions for

the metacercariae of Hysteromorpha triloba to reach maturity, and at

this time they were infective to double-crested cormorants,

Hialacrocorax auritus auritus, but never to chickens and ducks. Here again the two differ. Metacercariae of Diplostomum micradenum reached maturity within twenty-two to twenty-eight days, being infective at this time to both domestic chickens and ducks.

Differences in the adults were not so apparent. Dubois probably obtained his information for D. micradenum from one of

Olivier's specimens which he and Rausch examined and redescribed in

1950. Olivier's specimen was more pyriform than any specimens that I obtained. It also had a deep ventral concavity and no definite separation of fore- and hind-body. The method of fixation might possibly bring about these conditions. Hugghins believed that Lutz

(1931), in his work with flukes in herons, may actually have had the adult of Diplostomum micradenum, but unknowingly described it as

Hysteromorpha triloba.

It may also be worth noting that species of Diplostomum most frequently have a lymnaeid snail as their first intermediate host while Hysteromorpha triloba utilizes a planorbid snail, Gyraulus hirsutus.

Considering the dissimilarities between Diplostomum micradenum and Hysteromorpha triloba with respect to their life history stages, it is evident that these two are not conspecific, and, therefore, that

Diplostomum micradenum (Cort and Brackett, 1938) cannot be a synonym

of Hysteromorpha triloba (Rudolphi, 1819).

Anas domestica and Gallus domesticus are new experimental

definitive hosts for Diplostomum micradenum, and the finding of the

cercariae of this species at Calamus Swamp establishes a new

distribution record for this parasite. Prior to this research the 37 metacercaria of D. micradenum had never been reported from the eyes of

the second intermediate host.

Protechinostoma mucronisertulatum Beaver, 19U3 (Plate IV)

Introduction

Cercariae of Protechinostoma mucronisertulatum were encountered

in collections of Lymnaea (s.) reflexa more frequently than any other

cercariae. Of the 1,2J?2 specimens of L. reflexa collected and examined

from March 23, 1961, through May 2h, 1963, twenty-four per cent shed

cercariae of this trematode (Tables 1-3). The highest incidences were

in the early spring. Forty-three per cent of the first snails collected

in 1961 shed cercariae of this trematode. In 1962, forty-seven per cent

of the first snails shed cercariae of this species. Of the twenty-four

collections, only one (November Hi, 1962) failed to have snails shedding

cercariae of this trematode.

Feldman (l9Ul) was the first to publish an account of this

species; however, he associated the cercarial stage with Cercaria

reflexae Cort, 191ii, failing to recognize the weak collar of spines.

He considered this trematode to be a collarless echinostome-like form,

and accordingly placed it in the genus Psilostomujn, naming it

Psilostomum reflexae after Cort's cercaria.

Beaver (19U3), working with the cercaria of this same trematode

from Stagnicola palustris elodes, published an account of his findings

two years later. Beaver claimed that Cercaria reflexae could not be

the cercaria of Psilostomum reflexae since size and absence of crown PIATE I

Life history stages of Diplostomum micradenum.

Fig. 1.— Metacercariae in retina of eye of Rana pipiens tadpole.

38 39

PLATE I 7

PIATE U

Life history stages of Diplostomum micradenum.

Fig. 1.— Metacercariae in vesicles of cerebral hemispheres of Rana pipiens tadpole.

1*0 h i PLATE H

I PIATE III

Life history stages of Diplostomum micradenum.

Tig. 1.--Cercaria. Ventral view.

Fig. 2.— Cercaria. Showing normal resting position.

Tig. 3*— Daughter sporocysts.

Tig. U*--Eleven-day-old metacercaria. Ventral view.

Fig. 5>.— Thirteen-day-old metacercaria. Ventral view.

Tig. 6.— Adult fluke. Ventral view.

U2 in 31Vld spines in C. reflexae were not in agreement with the cercaria of

Psilostomum reflexae. The cercaria described by Beaver possessed a relatively small collar with two rows of inconspicuous short spines.

Also, these two rows of spines were apparent in the adult. On the basis of this collar of spines Beaver considered this worm to be an echinostome. Since it did not possess a conspicuous collar of

spines, as do members of the genus Echinostoma, Beaver constructed a new genus, Protechinostoma, for this trematode. In addition, he found it necessary to change the trivial name since the cercaria was shown not to be Cercaria reflexae. Accordingly, he renamed the fluke

Protechinostoma mucronisertulatum.

Although miracidia, sporocysts, and mother rediae have never been reported for Protechinostoma mucronisertulatum, it is probable that this fluke has a life cycle very much like other echinostomes.

For echinostomes in general, free-swimming miracidia hatch from eggs

in feces that is deposited in the water by infected bird or mammal

definitive hosts. The miracidia penetrate pulmonate snails where they become mother sporocysts from which develop the mother rediae. These

rediae give rise to daughter rediae which in turn produce cercariae.

The cercariae emerge from the snail host and swim about rapidly and

continuously until entry into the respiratory orifice of the snail from which they were shed or another snail in the vicinity. Once in the

respiratory cavity of the snail, they encyst and undergo a period of

development as metacercariae. Following the ingestion of infected

snails by a suitable definitive host, the metacercariae excyst and mature to adults in the small intestine. us The redia (Plate iV, Fig. 3)

Mature cercariae-producing rediae elongate, 1.36-2.19 mm

(1.67 mm) long by 0.31-0.39 mm (0.35 mm) wide; in digestive gland of snail. A distinct collar composed of four lobes near anterior end; two lobe-like locomotor appendages near posterior end. Birth pore slightly posterior to collar; oral opening not surrounded by muscular sucker. Muscular pharynx, 0.068-0.075 mm (0.070 mm) in diameter, opening into a rhabdocoel gut, extending approximately three-fourths length of body. Cecum contains considerable pigmented material. Wall of redia with distinct orange-colored granules. Younger rediae display a more noticeable anterior collar and posterior locomotor appendages.

These immature rediae lack the pigmentation of the mature forms.

When a snail is removed from its shell, the presence of a large number of these rediae imparts a dark orange color to its digestive gland. Young snails, 15-20 mm, may support 200-300 of these rediae, whereas older snails, 20-35 mm, may carry burdens well above

500. At no time were sporocysts found.

The cercaria T E U t e lV, Fig. 1, 2)

Body large, elongate, with narrowing anterior end displaying a small collar with minute spines. Body of formalin-fixed specimens,

0.U92-0.55U mm (0.517 mm) long by 0.205-0.226 mm (0.216 mm) wide.

Oral sucker subterminal, conspicuous, 0.055-0.068 mm (O.063 mm) long by 0.055-0.062 mm (0.056 mm) wide. Openings of penetration glands anterior to oral sucker. Acetabulum larger than oral sucker; 0.062- 0.082 mm (0.072 mm) long by 0.075-0.096 mm (0.079 mm) wide. Acetabulum in posterior half of body, its anterior margin 0.250-0.269 mm (0.258 mm) from anterior end. Prepharynx short; pharynx oval, 0.029-0.032 mm

(0.031 mm) long by 0.013 mm vide; joining a postpharyngeal esophagus,

0.135-0.175 ran (0.158 mm) long. Bifurcation of intestine just anterior to acetabulum. Ceca extend to near posterior end of body. Excretory

system echinostome-like; main canals extend forward as enlarged ducts to anterior end and then extend back to extreme posterior end.

Refractile bodies fill main excretory ducts. Flame cells numerous and

difficult to count; in three groups; 11-12 anterior to bifurcation of

intestine, 6-8 in acetabular region, and 22-28 posterior to acetabulum

on each side. Tail longer than body, 0.630-0.732 mm (0.703 mm) long by

0.096-0.103 mm (0.101 mm) wide at base. Long fins on both dorsal and

ventral sides. Dorsal fin low at base but widening one-third distance

from base and narrowing again at distal end. Ventral fin wide at base,

narrowing approximately one-third distance from base and then widening

again in distal half of tail.

This cercaria is a powerful swimmer and moves rapidly and

continuously from the time of its emergence from the first intermediate

host until its entrance into a second intermediate snail host or at

death. Most cercariae of Protechinostoma mucronisertulatum remained

active for eight to ten hours; however, a few were observed to live as

long as twenty-four hours. The time of emergence does not seem critical

for this specd.es. Although the largest numbers of cercariae were present

in the shedding jar from 7:30 a.m. to 9:00 a.m. other emergences took

place later in the day. Many Lymnaea (S.) reflexa that had been brought directly to the laboratory from the field shed cercariae immediately upon being placed into jars.

Encystment.— In the laboratory the cercariae of Protechinostoma mucronisertulatum were allowed to re-enter and encyst in the snails from which they had emerged. 'When a swimming cercaria comes in contact with the soft parts of the snail, it exhibits a crawling behavior, resulting in its being directed toward the respiratory opening of the snail. As the cercaria crawls through this opening, its tail is shed. Encystment follows within one to two hours in the respiratory cavity. Since the cercariae of Echinostoma revolutum are known to enter tadpoles where they encyst in the excretory system, tadpoles of Rana pipiens and R. sylvatica were exposed to cercariae of P. mucronisertulatum for three hours. All such attempts to infect these tadpoles were unsuccessful.

The metacercaria (Plate IV, Fig. 5, 6)

Metacercariae in clear, colorless, spherical cysts, 0.255-

0.272 mm (0.265 mm) in diameter; loose in respiratory cavity of snail host or may be grouped together in a cluster in wall of cavity. Cyst wall smooth and hyaline, consisting of three layers with a combined thickness of 0.021 mm; innermost layer narrower and darker than outer two. Metacercaria quite active within cyst. Refractile granules of excretory system, oral sucker, acetabulum, and cuticular spines can be

seen clearly. Excysted metacercaria, 0.75 mm long by 0.18 mm wide, with well-developed oral sucker, 0.068 mm in diameterj pharynx 0.037 mm long;

and acetabulum 0.089 mm in diameter. Intestinal ceca and branching

excretory system well developed. Metacercariae of Protechinostoma mucronisertulatum were found occurring naturally in Lymnaea (S.) reflexa, Helisoma trivolvis, Physa gyrina, and Succinea avara at Calamus Swamp. Individually isolated lymnaea (S.) reflexa, which were shedding cercariae of this species, yielded hundreds of metacercariae after a three-week period. These isolated snails, with heavy burdens of metacercariae, frequently died much sooner than snails that contained only a few (twenty to forty) metacercariae.

Excystment.— Feldman (l9lil) reported that he had no success in metacercariae of Protechinostoma mucronisertulatum to excyst by either puncturing the cyst or by using artificial digestive means. By using

1 per cent and l/lO per cent trypsin solutions and keeping the material in an egg incubator at 98° Fahrenheit, I was successful in getting approximately 30 per cent of the metacercariae to excyst between eight and ten hours. One hundred per cent excysted by twelve hours. I was not successful in keeping any of these alive, however. Amylopsin mixtures failed to give any results within the above time limits and yielded no more than 10 per cent excysted forms after twenty-four hours.

Encysted metacercariae died within two hours in Ringers Warm, but lived for as long as two days in tap water at this temperature. Empty cysts failed to be digested in trypsin solutions even when left for as long as two days. Excystment is apparently initiated by the metacercaria.

This stage literally squeezes out through a small opening in the cyst wall which apparently it has made from within. The chemical makeup of the cyst wall is not known. Feeding experiments with metacercariae.— In working with the metacercariae I ran into the same problem that was encountered by both

Feldman and Beaver. They found that their experimental animals were not reliable definitive hosts. Feldman fed metacercariae to forty-seven chickens and succeeded in infecting only eighteen of these, which yielded a total of fifty-seven worms after five days. This represented

2 per cent of the metacercariae fed. Feldman’s experiments with mice proved unsuccessful as did those of mine. Although Beaver had difficulty obtaining adult worms, he did manage to infect mammals with some degree of success. One mouse was fed one hundred metacercariae each day for four consecutive days and then examined on the fifth day. In this mouse there were seventy-two worms of various stages of maturity. Beaver was unsuccessful, however, in his attempts with chickens and pigeons.

My best results were obtained when whole snails were fed, each of which might have had a hundred or more metacercariae (Table 9). The two field hosts, a juvenile robin and a mature starling, gave the best results. The starling, in particular, yielded sixty-two immature worms from a feeding of thirteen snails. It was highly improbable that these birds were infected with P. mucronisertulatum prior to my work with them in the laboratory. One duck (not represented in the table) was given

119 encysted metacercariae, and then examined two hours later. Forty- eight encysted metacercariae were obtained from the gizzard; two encysted metacercariae were located midway in the small intestine; two empty cysts, one excysted metacercaria, and ten encysted metacercariae were found two-thirds the length of the small intestine; and one empty cyst was located near the distal end of the small intestine. Numerous 50 TABLE 9

PROTECmOSTOMA. MUCRONISERTULATUM BEAVER, 191+3

RESULTS OF FEEDING METACERCARIAE TO VARIOUS VERTEBRATE ANIMALS

Age of Number of Time in Vertebrate Animal Metacercariae Vert. Animal Worms Recovered Animal (Days) Fed to Animal (Days) Immature Adult

Rana pipiens 10 75 1 8 0

n ii 10 75 2 0 0 Rana sylvatica ? 100 2 0 0

Rana clamitans ? 75 1 2 0

Domestic duck J.T 100 1 0 0

tt n 1 112 15 0 0 ti ii 1 (It snails )a l 0 0

it ii 1 (3 snails) it 2 2

ti ii 1 (5 snails) 1+ 6 2

it it 3 150 5 0 0

it it 5 200 5 0 0 n tt 6 (8 snails) 5 0 2

it it 12 (10 snails) 5 0 0

it ii 17 (17 snails) 2-5 0 0

Domestic chicken 1 200+ 2 2 0

Turdus migratorius ? (1+5 snails) 1-5 36 1

Sturnus vulgaris (13 snails) 2 (died) 62 0

Mus musculus juvenile 200+ 5 0 0

it ti juvenile 200+ 5 0 0

^fliole snails given to host. Number of metacercariae not known. encysted metacercariae were found in the void feces, but no count was made. This indicated that excystment can take place in a relatively

short time, but, as shown by other feeding experiments, many of these

excysted metacercariae apparently were not retained in the intestine.

The first clues to the identification of Protechinostoma imicronisertulatum came from observations made on immature flukes

recovered from a juvenile Rana pipiens which had been given seventy-

five metacercariae. For being only one-day old these immature worms

furnished considerable information about the adult form. Both the

developing ovary and testes were apparent and the cirrus pouch was

forming anterior to the acetabulum. Body spines could be seen easily

and the pattern of the crown spines was obvious. These immature worms

were removed from the large intestine of this host, and, as shown by

another Rana pipiens, no worms were present by the end of two days

(Table 9). Amphibia had not been used by either Feldman or Beaver.

I successfully obtained adult worms from three ducks out of ten;

however, these produced only two adult worms each. One of the two

chickens that had been fed metacercariae was infected with two

immature flukes. The two mice were not infected five days after being

fed metacercariae.

The adult TPlate TV, Fig. 7)

Body slipper-shaped, bluntly rounded at both ends. Length,

1.16-1.27 mm (1.21 mm); body width greater anterior to acetabulum,

0.29-0.36 mm (0.33 mm) wide. Cuticular spines extend to level of

anterior testis on ventral surface, but only to level of ovary on dorsal surface. Two rows of crown spines in region of oral sucker,

similar in size but distinctly separate from body spines. Crown

spines variable in number, 1*9-53. Oral sucker subterminal, 0.067-

0.072 mm (0.071 mm) long by 0.062-0.092 mm (0.076 mm) wide. Pre­ pharynx short; pharynx oval, 0 .01*8-0.053 mm (0.050 mm) long by 0 .035-

O.OI46 mm (0.01*0 mm) wide. Postpharyngeal esophagus long and narrow,

0 .11*3-0 .231; mm (0.193 mm) long by 0 .019-0 .021* mm (0.022 mm) wide,

dividing just anterior to cirrus pouch. Ceca extend to near posterior

end of body, widening posterior to acetabulum. Acetabulum larger than

oral sucker, 0.099-0.122 mm (0.111 mm) long by 0 .111-0.126 mm (0.120

mm) wide. Ovary relatively spherical, 0.051-0.088 mm (0.073 mm) long

by 0.072-0.086 mm (0.079 mm) wide; posterior to acetabulum and

slightly on the left side. Mehlis' gland well developed; between

ovary and anterior testis. Vitelline follicles large; extending to

midacetabular level; widely separated and paralleling intestinal ceca

anterior to testes, but converging posterior to testes in median zone.

Uterus short and containing two to four eggs. Eggs large, 0.060-0.095

mm (0.082 mm) long by 0.01*5-0.065 mm (0.055 mm) wide. Testes tandem;

somewhat oval; in median line of body. Anterior testis, 0.076-0.127 mm

(0.103 mm) long by 0.138-0.178 mm (0.152 mm) wide. Posterior testis,

0.108-0.11*3 mm (0.125 mm) long by 0.130-0.175 mm (0.11*5 mm) wide.

Cirrus sac oval; dorsal; between acetabulum and bifurcation of

intestine. Seminal vesicle well developed; 0.155 mm long; cirrus

slender and coiled. Genital opening just posterior to bifurcation of

intestine. Excretory pore ventral, main tubules observable only in

living specimens. 53

Site of infection; Small intestine.

Definitive host: Anas domestics, Turdus migratorius, and

Sturnus vulgaris (Experimental).

Type specimens: Hbmotype No. 60114; deposited in the U. S.

National Museum Helminthological Collection.

Although the adult specimens reported in this research were generally smaller than those obtained by Feldman and Beaver, there was no reason to believe that they were not the same species. The number of crown spines, arrangement of cuticular spines, and position and extent of internal structures agreed favorably with specimens of

Protechinostoma mucronisertulatum described by Feldman and Beaver.

Protechinostoma mucronisertulatum is midway between the spineless echinostome-like psilostomes and the true echinostomes, and according to Yamaguti (1958) is the only species in the genus

Protechinostoma. The collar is so inconspicuous and the spines so small that these could be overlooked if extreme care was not taken.

With the size of the adult being less than two millimeters, it is conceivable that this species could be overlooked easily in survey studies where frozen or formalin preserved material is examined. In locating these worms in the intestine of a bird, their movements were indeed helpful.

Discussion

The domestic duck, Anas domestica; the robin, Turdus migra­ torius; and the starling, Sturnus vulgaris, are new experimental definitive hosts for Protechinostoma mucronisertulatum, but they appear to serve in this capacity no better than do chickens and mice.

Frogs, although not used by either Feldman or Beaver, served only as temporary hosts where excystment could take place. I believe that frogs would not have harbored mature adults. The natural definitive host at

Calamus Swamp is not known. Only five red-winged blackbirds, Agelaius phoenieeus phoeniceus, were examined from the area, but they were negative for P. mucronisertulatum. To my knowledge this species has not been reported in any helminth surveys of birds, and the discovery by Beaver of this trematode in the sora rail, Porzana Carolina, near

Black Creek, Wisconsin, stands as the only known natural record. Since cercariae were found in all snail collections except one, it seems

logical that the natural definitive host is a resident of Calamus

Swamp, and that the snail, Lymnaea (S.) reflexa is picking up infections

throughout the spring, summer, and autumn. Rails have been reported

from Calamus Swamp. Feldman's cercarial material was obtained near

Edina, Minnesota, but he did not find any natural definitive host in

that area.

The present research is the only known study on Protechinostoma mucronisertulatum since Beaver's work in 191+3. Wo outstanding differ­

ences were noted between my material and that found by Beaver, and the

finding of P. mucronisertulatum at Calamus Swamp in Pickaway County,

Ohio, stands as a new distribution record for this species.

Additional work on the excystment of the metacercariae of this

trematode might prove rewarding. Since metacercariae can be obtained

in large numbers free from any host tissue and can be kept alive in tap

water for several days, they might be useful in culturing experiments. The fact that only four or five days are required for this species to reach maturity in the intesting of its definitive host would also be of great advantage in such experimental work. '

Notocotylus stagnicolae Herber, 191*2 (Plate V)

Introduction

Only five Lymnea (S.) reflexa, from the 1,252 collected, shed cercariae of Notocotylus stagnicolae. This was the lowest incidence encountered in this research, being only 0.1* per cent. No cercariae of N. stagnicolae were observed in the 1961 collections and only one in the 1962 material. Lymnaea (S.) reflexa collected in 1963 produced cercariae in both March and May (Tables 1-3).

Herber (191*2) described and named this species after completing its life cycle. He found the cercariae of Notocotylus stagnicolae being shed by Stagnicola emerginata angulata and S. e. candensis in the Douglas Lake Region of Michigan, and was successful in getting both domestic chickens and ducks, as well as blue-winged teal,

Querquedula discors, and mergansers, Mergus merganser, to serve as experimental definitive hosts. Eleven specimens were found occurring naturally in a semi-palmated plover, Aegialitis semipalmata, by

Brackett at Douglas Lake.

Wu (1953) obtained cercariae of Notocotylus stagnicolae from

Stagnicola palustris in the Ottawa River in Canada, and obtained adults PLATE IV

Life history stages of Protechinostoma mucronisertulatum.

Pig. 1.— Cercaria. Dorsal view.

Pig. 2.— Cercaria showing fin folds. Lateral view.

Pig. 3.— Mature redia containing cercariae.

Pig. 1*.— Immature redia.

Pig. £.— Encysted metacercaria.

Pig. 6.— Excysted metacercaria with cyst still present.

Pig. 7-— Adult fluke. Ventral view.

£6 PLATE 12 experimentally in domestic chickens. Wu observed a few minor morpho­ logical differences from those of Herber, but concluded that his material was Notocotylus stagnicolae.

Although Herber recognized that the adults of Notocotylus attenuatus (Rudolphi, 1809), Notocotylus magniovatus Yamaguti, 193U, and N. imbricates (looss, 1896) were very similar to N. stagnicolae, he emphasized the importance of the larval stages in determining species in the genus Notocotylus. Herber pointed out that the larval stages of each of these differed from those of N. stagnicolae, and concluded that no species had been described with characters identical in both larval and adult stages with N. stagnicolae. Both Skrjabin

(1953) and Yamaguti (1958) recognized N. stagnicolae as a valid species

in their comprehensive volumes on the digenetic trematodes.

Members of the genus Notocotylus have comparatively simple life

cycles. Miracidia, hatching from a filamentous egg, penetrate suitable

aquatic snail hosts and give rise to mother sporocysts. The sporocysts

produce a generation of rediae which then produce cercariae. In some

species in this genus two redial generations have been observed. The

cercariae that emerge from the snail host quickly encyst on various

objects, including vegetation and the shell of the snail itself. The

metacercariae must then be ingested by suitable definitive hosts,

frequently members of the family Anatidae, where they excyst and mature

to adult flukes. The ceca are the most common sites inhabited by the

adult flukes. Eggs from the adult worms reach the outside aquatic

environment in the feces of the definitive host. Relatively little 59 definitive host specificity has been demonstrated for the various species in this group, at least among hosts belonging to the same class.

The following descriptions of the larval and adult stages of

Notocotylus stagnicolae were based on material originating from a single snail host. Although cercarial stages from all five snails were compared and determined to be Notocotylus stagnicolae, it was desirable to use metacercariae for feeding experiments from only a single host. Thus, in comparing flukes of different ages there was no doubt that they were the same species.

The redia TFlate V, Fig. 1)

Rediae elongate, without collar and posterior locomotor processes. Formalin-fixed mature rediae, 0.63-0.96 mm (0.81 mm) long by 0.19-0.27 mm (0.23 mm) wide. Pharynx well developed, 0.072-0.080 mm (0.076 mm) long by 0.056-0.076 ram (0.068 mm) wide, leading into

3hort esophagus. Intestine long, broad, and folded; extending to near posterior end and containing dark particles of various sizes. Reddish- brown pigment scattered through body proper. Germ masses concentrated

in posterior half of body. Usually one or two well-developed cercariae with eye spots per redia. In living specimens, anterior end of body

frequently constricts posterior to pharnyx. Body wall highly muscular

over entire length. One redia was found, 0.73 mm long by 0.19 mm wide,

containing a single daughter redia. Daughter redia 0 .1+X mm long with

pharynx, 0.0U3 mm in diameter.

The mature rediae that I observed were not sufficiently

different from those found by Herber to warrant any special attention. 60

No comparisons could be made with Wu's material since Wu did not describe any of the redial stages. Since these rediae had no posterior laterial processes, they could not have been rediae of Notocotylus magniovatus Yamaguti, 193U-

The cercaria (]*late V, fig. 2)

Body of formalin-fixed specimens, brown, elongate, bluntly

rounded at anterior end, with muscular oral sucker opening ventrally.

Bodies, 0.iUi-0.50 mm (0.1*6 mm) long by 0.12-0.18 mm (0.16 mm) wide.

Oral sucker 0.0i|0-0.052 mm (O.OiUl mm) in diameter. Three prominent

eye spots, median one less well formed and anterior to lateral two,

near posterior margin of oral sucker. Lateral eye spots dark, hollow,

cone-like bodies, 0.027 mm long by 0.016 mm wide. Main parts of

excretory system consisting of two ducts joined anteriorly just

behind median eye spot and posteriorly into the lateral walls of the

small excretory bladder. Ducts filled with refractile bodies, 3 p

in diameter at anterior end of ducts and 2p in diameter at posterior

end. Intestinal ceca not distinct, intestine bifurcating slightly

posterior to median eye spot and extending to near posterior end of

body, ending at level of excretory bladder. Two blunt organs of

attachment arising dorsally at the caudal end. Tail at caudal end

arising ventrally, 0.61-0.78 mm (0.72 mm) long by 0.062-0.075 mm

(0.069 mm) wide at base.

Living cercariae were extremely active and difficult to observe

for any long period of time. Cercariae that were removed for observa­

tion frequently encysted on the slide before the coverglass could be V

6l placed over the material. During encystment the cercaria rounds up and begins to rotate, first in one direction and then in another. The tail is cast off and secretions are discharged from the body, first a granular substance from the bladder which clears and forms an outer layer and then a fibrous-like material which forms a wall around the cercarial body. In the shedding jar these cercariae encysted on the sides of the container or on the snail from which they emerged.

The cercariae of Notocotylus stagnicolae were punctual, appear­ ing each day at approximately 10:30 a.m. and continuing to be shed until noon. These cercariae moved rapidly through the water very much like echinostome cercariae. They were distinguished from echinostomes, however, by their much darker bodies. Most of the cercariae encysted within fifteen minutes after emergence. The cast off tails of the cercariae remained active for as long as three to four hours.

Care had to be taken to make measurements of emerged cercariae for comparison with Herber's material, rather than that of free

cercariae obtained by crushing the snailEvidently the tail is the

last structure to complete its development. Body measurements of

formalin-fixed nonemerged cercariae were the same as those for emerged

cercariae, but tail measurements were considerably different. The

tails of emerged cercariae were longer than their bodies, but those of

non-emerged cercariae were definitely shorter. The tail of a living

emerged cercaria is capable of elongating to twice the length of the

body. 62

The metacercaria TEEate V, fig. 3)

Dark brown subspherical cyst. Formalin-fixed specimens, 0.171-

0.193 Mm (0.176 mm) in diameter. Lateral eye spots prominent; some

dark particles in excretory ducts; oral sucker inconspicuous. Wall of

cyst 0.017 mm thick, consisting of a thin, light, inner layer and

thicker outer fibrous layer. Irregular adhesive layer outside of wall

not included in measurements.

Herber obtained measurements of 0.20-0.25 mm for the diameter

of the metacercariae of Notocotylus stagnicolae. However, he did not

state whether these measurements included the outer irregular layer.

Wu obtained measurements of 0.157-0.198 mm for the metacercariae, which

were more in agreement with those that I obtained. Wu did not include

the outer layer. In ny specimens, had this outer layer been included,

the diameter measurements would probably have been over 0.20 mm.

As was mentioned previously, the metacercariae of Notocotylus

stagnicolae were found encysted on the sides and bottom of the

shedding jar as well as on the shell of the snail from which they

emerged. They were attached strongly to the surface of the glass and

a sharp probe was required to loosen them. A number were removed by

this means to use in feeding experiments. A better way of obtaining

metacercariae was discovered on the day following a heavy emergence of

cercariae. The snail had loosened numerous metacercariae from the glass

and had ingested them. Living metacercariae appeared in long strands

of fecal material from the snail, not having been killed and digested by this organism. These loose strands were removed with a pipette and fed directly to the definitive host.

Feeding experiments with metacercariae. — Four domestic chickens were used as definitive hosts for Notocotylus stagnicolae (Table 10).

TABLE 10

NOTOCOTYLUS STAGNICOLAE HERBER, W bZ

RESULTS OF FEEDING 1- TO U-DAY-OLD METACERCARIAE TO DOMESTIC CHICKENS

Age of Host Fed Number of Time in Host Metacercariae Metacercariae Definitive Host Worms Recovered No. (Days) Fed to Host (Days) Immature Adult

1 1 26 2 lit o

2 1 70 8 36 0

3 1 25 10 8 0

h 28 200+ 12 6 82

Three of these hosts were one-day old and one was twenty-eight days

old when fed metacercariae. Metacercariae from cercariae that had

emerged from a single lymnaea (S.) reflexa were used so as to be

certain of having the same species. All metacercariae were given

orally to the birds by means of a medicine dropper. With the

exception of the two-day-old flukes, all others were found only in the

intestinal ceca of the host. Eight of the two-day-old flukes were in

the intestine and six in the ceca. Notocotylus stagnicolae matured in

twelve days in the domestic chicken. Ten-day-old flukes were almost mature, but the developing eggs lacked the polar filaments, the pattern of the ventral glands was not identical to that of mature forms, and the body measurements were generally less. The fact that eighty-eight worms were recovered from a chicken that was twenty-eight days old when fed metacercariae shows that chickens that have been given food previously are not refractive to this parasite.

The adult (Plate V, Fig. 1;)

Body longer than broad; concave ventrally; highly muscular; with three ventral rows of glands, lianas often everted, having narrow slit-like openings. Twelve-day-old, heat killed, stained, cleared, mounted specimens, 3.97-U-37 ram (U.17 mm) long by 0.95-1.05 mm (1.00 mm) wide, with sixteen to seventeen glands in each lateral row and fourteen to fifteen glands in median row. Most common gland pattern, 16-15-16. Usually only one gland in median row anterior to most anterior glands in lateral rows. Posterior glands in lateral rows always posterior to most posterior gland in median row.

Anterior ventral surface covered with numerous small spines arranged in oblique rows. Conspicuous terminal oral sucker, opening ventrally,

0.150-0.161; mm (0.160 mm) long by 0.16l;-0.l81; mm (0.172 mm) wide.

Esophagus 0.171-0.205 mm (0.183 mm) long, bifurcating anterior to genital pore. Ceca long, extending to posterior median border of testes. Testes arranged bilaterally, with six to twelve lobes on lateral margins; right testis, 0.560-0.680 mm (O.616 mm) long by 0.238-

0.289 mm (0.270 mm) wide; left testis, 0.U9U—0.6U5 mm (0.578 mm) long by 0.20l;~0.272 mm (0.258 mm) wide. Posterior margin of testes, 0.187-0.255 iran (0.215 mm) from posterior end of body. Cirrus pouch,

1.27-1.1+1 mm (1.36 mm) long, containing tuberclated cirrus and conspicuous seminal vesicle. Follicular vitellaria lateral to ceca, twenty-three to thirty-six groups of follicles on each side.

Vitellaria 1.26-1.38 mm (1.33 mm) long, extending forward from anterior border of testes. Ovary smaller than testesj lobedj 0.255-

0.306 mm (0.280 mm) long by 0.20J+-0.238 mm (0.215 mm) wide. Uterus in close transverse intracecal loops, extending forward from ootype at anterior border of testes to posterior end of cirrus pouch. Five to eight loops anterior to anterior border of vitellaria. Section of uterus without transverse loops extending anteriorly, from posterior border of cirrus pouch along its left margin to genital pore. Eggs,

0.022-0.021+ mm (0.022 mm) long by 0.011-0.013 mm (0.013 mm) wide, with a filament at each end, O.U3-0.1+7 mm (0.14+ mm) long. Ventral excretory opening at caudal end of body.

Site of infection: Ceca.

Definitive host: Gallus domesticus. (Experimental)

Type specimens: Homotype No. 60159 deposited in U. S. National

Museum Helminthological Collection.

The body in general differed from that described by Herber only

in details. My specimens were larger, but differences in fixation could well account for some of this. My specimens were heat killed and placed

in A.F.A. fixing reagent, whereas Herber1 s material was killed and fixed

in Gilson's fluid. Measurements of the adults agreed more favorably with those described by Wu. Wu's specimens were twenty-one to thirty-

one days old, whereas mine were only twelve days old. Considering that the differences are of a minor nature only, there is no reason to believe that the present material is not Notocotylus stagnicolae.

Discussion

Szidat and Szidat (1933) suggested that the pattern of the ventral glands is constant in a given species, and the number of glands per row reasonably constant as well as independent of size or age of the specimen. This was not entirely true for Notocotylus stagnicolae. A comparison between mature and immature worms showed slight differences in the number and arrangement of the ventral glands. The pattern in eight- and ten-day-old worms varied from that of mature twelve-day-old flukes. Each lateral row of glands in eight- and ten-day-old specimens contained fourteen to sixteen glands (generally fifteen), whereas the median row contained thirteen to fourteen (generally fourteen) glands. The most common combination was 15-1U-15. In addition, there were generally two median glands anterior to the anterior-most glands in the lateral rows rather than one, which is characteristic of twelve-day-old worms. Twelve-day-old flukes characteristically have a 16-15-16 pattern of glands. Since considerable importance has been placed on the number and arrangement

of the ventral glands, it is very important that only adult worms be used for comparison.

The discovery of Notocotylus stagnicolae at Calamus Swamp

establishes a new distribution record for this species. Although only

red-winged blackbirds were examined from this area, and found to be uninfected for this species, it is quite probable that wood ducks or

other members of the Anatidae harbor this fluke at Calamus Swamp.

Considering the great similarity of the adult of Notocotylus

stagnicolae with those of N. attenuatus, N. nagniovatus, and N.

imbricatus, it is conceivable that these may constitute a single

species. A serious comparative study involving all of the life

history stages of each of these is needed.

Plagiorchis noblei Park, 1936 CftHeTTTrvil)

Introduction

Forty-one lymnaea (S.) reflexa, or 3.3 per cent, were infected

with cercariae of Plagiorchis noblei Park, 1936. Cercariae of this

species appeared in both early and late spring collections and were

present in twelve of the twenty-four collections made over the two-

year period (Tables 1-3).

This species was originally described by Park (1936) who dis­

covered twenty-one adult plagiorchiid worms, which he named Plagiorchis

noblei, in the small intestine of a red-winged blackbird, Agelaius

phoeniceus californicus, at Stockton, California. There is no univer­

sal agreement on the systematics of this species. Olsen (1937), in

making a taxonomic stucfcr of the subfamily Plagiorchiinae, removed P.

noblei from the genus Plagiorchis Liihe, 1899 and placed it in a new

genus, Flagiorchoides, which he established for trematodes of this type

that possess a seminal receptacle. Skrjabin and Antipin (1958) recog­

nized Plagiorchoides as being a valid genus, but Yamaguti (1958) revised

the genus Plagiorchis to include flukes in which the seminal PIATE Y

Life history stages of Notocotylus stagnicolae.

Fig. 1.— Daughter redia containing a single cercaria.

Fig. 2.— Cercaria. Dorsal view.

Fig. 3-— Encysted metacercaria.

Fig. k -— Adult fluke. Yentral view.

Fig* 5-— Filamentous egg.

68 PLATE 3E 69

0.05 70 receptacle is either absent or rudimentary, thereby returning Park’s species to the genus Plagiorchis. In P. noblei the seminal receptacle is not prominent, and is difficult to demonstrate in some specimens.

Some workers, namely Rees (1952), Skrjabin and Antipin (1958), and Dollfus (i960) recognize two subgenera, Plagiorchis and

Multiglandularis Schultz and Sworzov, 1931. These subgenera are based on the nature of the vitellaria in the adult trematodes. In members of the subgenus Plagiorchis the vitelline follicles of the region anterior to the acetabulum are confined to the lateral margins of the body, with only a few follicles ever in the median zone. In Multiglandularis the follicles extend across the body as a broad continuous band near the anterior end. Yamaguti (1958) and Najarian (1961) did not recognize these subgenera. The nature of the vitelline follicles in some species is not so clearly defined as it is in others. Dividing the genus on this basis seems undesirable. I have chosen to accept

Yamaguti*s classification, and shall refer to this species as

Plagiorchis noblei Park, 1936.

No life history study has been conducted on P. noblei prior to the present research, but life cycle studies have been conducted on

Plagiorchis proximus Barker, 1915; P* muris Tanabe, 1922; P. micracanthos Macy, 1931; P. jaenschi Johnston and Angel, 1951; and P. parorchis Macy, 1956 from mammals; P. cirratus (Rudolphi, 1802); P. maculosus (Rudolphi, 1802); P. arcuatus Strom, 1921).; P. goodmani

(Najarian, 1952); and P. megalorchis Rees,, 1952 from birds; and P. ramlianus (Looss, 1896) from amphibians and reptiles. 71 The general life cycle involves two invertebrate intermediate hosts. Eggs, containing miracidia, are ingested by the first inter­ mediate host, a snail. The miracidia hatch from operculated eggs within the snail host and develop into mother sporocysts. These in turn give rise to daughter sporocysts in which xiphidiocercariae form.

Emerging cercariae penetrate larval stages of various species of aquatic insects where they encyst as metacercariae. Only a few days are required for the metacercariae to develop fully in the second intermediate host. Following the ingestion of either the larval or adult infected insects by a suitable definitive host, the metacercariae excyst and mature to adults in the distal half of the small intestine.

Eggs reach the outside aquatic environment in the feces of the defini­ tive host. Members of this genus of flukes are principally parasites of birds and mammals; however, a few species are known from amphibians and reptiles, but none from piscine hosts.

Although field collected insect larvae and nymphs were used in penetration experiments, none were used in determining the life history of this species. Immature and adult stages of Plagiorchis noblei were worked entirely through laboratory reared hosts -which could not have been infected by any trematode prior to their use with this species.

After I had obtained the adult fluke experimentally and had identified it as Plagiorchis noblei, I collected five red-winged blackbirds,

Agelaius phoeniceus phoeniceus, at Calamus Swamp, one of which was naturally infected with three adult specimens of this fluke. 72

The daughter sporocyst matTTr;-^ TJ '

Small sausage-shaped bodies, 0.622-1.313 mm (0.852 mm) long by

0.136-0.168 mm (0.1b3 mm) wide; scattered throughout digestive gland or attached to intestine of snail host; containing both immature and mature cercariae as well as germinal masses. Usually eight to twelve well-developed cercariae per mature sporocyst. Body walls of sporo­ cysts attached to intestine had thick outer cell layers containing reddish-brown pigment. Other sporocysts, more or less free in gland, had relatively thin, clear outer membranes.

I was not successful in establishing infections in lymnaeid snails, and have no information on the early formation of sporocysts in this species. Cort and Olivier (I9b3) observed development of the larval stages of Plagiorchis muris in the snail and observed that there were two sporocyst generations. They also observed that the mother sporocysts were attached to the intestine of the snail and frequently did not rid themselves completely of their daughter sporocysts. The daughter sporocysts that remained produced cercariae as well as did other daughter sporocysts that were free in the digestive gland.

The thick-walled, heavily pigmented sporocysts that I observed in

Plagiorchis noblei may well have been mother sporocysts containing daughter sporocysts. According to Cort and Olivier, daughter sporo­ cysts of P. muris continue to produce cercariae long after the first

cercariae have matured and have left the sporocyst. Considering the number of germ cell masses and developing cercariae in the daughter 73 sporocysts of Plagiorchis noblei, I suspect the production of cercariae in this species is very much like that of P. muris.

The cercaria T^Tate Vf, Pig. 2, 3, It, 5)

Xiphiaiocercaria of the Conniae division of the Polydena group.

Body of formalin-fixed specimens pear-shaped, with posterior half being more narrow than anterior half; heavy concentration of cystogenous glands mainly along lateral margins of body from level of pharynx to posterior end of body. Body 0.212-0.2it6 mm (0.226 mm) long by 0.102-

0.136 mm (0.121 mm) wide, with entire surface covered with spines.

Oral sucker larger than acetabulum, 0.0l|3-0.052 mm (0.0it7 mm) long by

0.0i;9-0.059 mm (0.035 mm) wide, with ventral opening and dorsally placed stylet. Stylet, 0.032-0.033 mm (0.032 mm) long by 0.005 mm wide at base; without basal bulb, strong, curved dorsally, with prominent dorso-lateral shoulders one-third distance from tip, 0.008 mm across.

Tip of stylet directed forward from a narrow channel in anterior end of oral sucker. Pharynx 0.016-0.021 mm (0.018 mm) long by 0.017-0.019 mm (0.019 mm) wide. Esophagus and ceca not visible. Acetabulum 0.029-

0.038 mm (0.031; mm) long by 0.032-0.OUl mm (0.037 mm) wide. Excretory bladder thick-walled, with single posterior chamber joined by median duct to two anterior horns which extend to posterior edge of acetabulum.

Main excretory ducts arise from anterior end of excretory horns and divide into anterior and posterior lateral collecting tubes at approxi­ mately mid-acetabular level. No bubble-like refractile bodies present. Penetration glands difficult to see, eight per side in pre- and mid- acetabular region. Numerous flattened living specimens revealed a flame cell pattern of 2/"(3 + 3 + 3) + (3 + 3 + 31/ = 36.

The cercariae of Plagiorchis noblei were shed most frequently during the early morning hours in groups of several hundred each. Once shed from the snail they remained alive for as long as forty-eight hours, exhibiting two types of motion, an upward swimming action in the water and a creeping behavior on the bottom of the container. Newly emerged cercariae remained in the water column for the greater part of a day, frequently swimming by vibratory movements of both their bodies and tails. When at rest, the cercaria rounds up with its tail con­ tracted and placed in the depression formed by its ventrally folded body. Later in the day cercariae were found on the bottom of the jar, creeping on the surface of the glass with the aid of their suckers.

When disturbed, these creeping cercariae often exhibited an upward swimming action. McMullen (1937), Rees (1952), and Najarian (1952) mentioned the same type of creeping action exhibited by Plagiorchis muris, P. proximus, P. micracanthos, P. megalorchis, and P. goodmani.

Many xiphidiocercariae have been described by various writers, namely, Cort (1915), Sewell (1922), Miller (l935), Brooks (19U3), and

Fain (1953). Although the characteristics of the cercaria of

Plagiorchis noblei place it in Brook's Conniae division, and although it shows morphological relationships with cercariae of other known members of the genus Plagiorchis, it also shows relationships with

Cercaria dorotti Brooks, 19U3 and Cercaria granulata Fain, 1953. The measurements of the body, oral sucker, pharynx, acetabulum, and stylet of Plagiorchis noblei agree favorably with Brooks' formalin-fixed specimens of C. dorotti; however, Brooks did not determine the number and pattern of the flame cells, nor was he positive of the number of penetration glands. He stated that there were at least five pairs of penetration glands and that he was unable to determine the flame cell pattern because of the heavy concentration of cystogenous glands.

Both of these cercariae possess many cystogenous glands, especially along the lateral margins of the body posterior to the pharynx.

Cercaria granulata is similar to the cercaria of Plagiorchis noblei with respect to measurements of general body structures, but the former differs principally by having spines on its tail and by possessing a

longer and broader stylet. I am not aware of any cercaria that is

absolutely identical to that of Plagiorchis noblei.

Penetration of the second intermediate host.— In order to

determine the species of Plagiorchis which which I was working, it was

necessary to infect susceptible second intermediate hosts and obtain metacercariae for feeding experiments. Xiphidiocercariae of members

of the genus Plagiorchis characteristically have strong stylets,

generally dorsally curved, and possessing prominent dorso-lateral

shoulders. Cercariae having strong stylets of this nature are known

to penetrate various aquatic insect larvae, naiads, and nymphs. The

cercariae of Plagiorchis noblei were no exception. They penetrated

larval stages of various species of mayflies, caddisfLies, damselflies,

mosquitoes, and midges. I had no success with dragonfly naiads nor

with any amphipods, cladocera, or ostracods. Since all of these animals were collected in the field, I could not be certain about their not being infected prior to my work with them in the laboratory.

Therefore, larvae of the mosquito, Aedes aegypti, were reared in the laboratory and used as experimental second intermediate hosts. I

discovered, however, that second stage larvae (or older) had to be used. First stage larvae were too small and the entrance of a single

cercaria into a larva of this size caused its death.

When a cercaria comes in contact with the surface of a mosquito

larva it exhibits the crawling-type action often demonstrated by a

cercaria on the bottom of a container. The cercaria generally crawls

to an area where two body segments join before it begins any penetra­

tion activity. Normally the stylet is carried in a relatively

horizontal position, but when the cercaria begins to penetrate, the

posterior end of the stylet is pulled dorsally and the anterior end

directed downward at about a U5 degree angle. Numerous downward and

forward thrusts of the dorsally curved stylet scratches the surface of

the cuticle. The oral sucker helps hold the s tylet against the cuticle.

After an opening is produced, the body of the cercaria begins to enter

the larva. The tail may be shed at the time the cercaria begins its

scratching activity or later when its body is about half way through

the cuticle of the insect host. Once inside, the cercaria may encyst

immediately or it may migrate to another part of the body of the host

before encysting. While cercariae most often entered the abdomen, they

encysted in the head region of Aedes aegypti. 77

Although cercariae of Plagiorchis noblei were shown to pene­

trate damselfly naiads and mosquito larvae collected from Calamus Swamp,

no natural infections were found in twenty-one damselfly and forty-two mosquito larvae examined from this pond.

The metacercaria "(Plate Y l, tfig. 6)

Metacercariae in thin-walled spherical cysts, 0.127-0.11*5 mm

(0.137 mm) in diameter, in tissues of intermediate host. Wall of cyst

3pt thick. Oral sucker, stylet, acetabulum, excretory bladder, and

body spination clearly visible. Metacercaria often quite active in

cyst; stylet generally free from oral sucker; excretory bladder of

four-day-old metacercaria filled with dark refractile granules about

51u in diameter.

There was no great change in the size of the metacercarial

cyst from the time of its formation; however, pronounced changes did

take place in the excretory bladder. At the time of encystment the

excretory bladder was free of any concretions, but by the fourth day

it was completely filled with dark bubble-like bodies. The stylet

was generally free in the cyst after the first day. No metacercariae

were found encysted precosciously in the daughter sporocysts of this

species. Metacercariae are known to encyst in the daughter sporocysts

of Plagiorchis muris, P. proximus, and P. goodmani, but not in those

P. micracanthos.

Feeding experiments with metacercariae.— Entire mosquito

larvae, pupae, and adults were force fed to several vertebrate animals

(Table 11). A sample was taken from each group of insects that was to 78

TABLE 11

PIAGIORCHIS NOBLEI PARK, 1936

RESULTS OF FEEDING METACERCARIAE TO VERTEBRATE ANIMALS

Age of Time in Metacercariae Vertebrate Animal Worms Recovered Host (Days) (Days) Immature Adult

Rana pipiens $ 3 0 0

Mus musculus 3-6 5 0 0

ti n 3-6 7 0 0

n n 6-8 7 0 0

Eptesicus fuscus fuscus 6-12 10 0 0

Gallus domesticus k-6 6 0 2

ii ii U—6 6 0 lli

ii i i U-6 7 0 2

be used and was checked for the presence of metacercariae, but no total

count of the metacercariae was made. Since the cercariae and meta­

cercariae resembled closely those of a known mammalian plagiorchiid

trematode, Plagiorchis micracanthos, mice were the first experimental

animals to be fed infected mosquitoes. Young mice, however, failed to

yield any worms when fed three- to six-day-old and six- to eight-day-

old metacercariae. Another mammal, a mature, big brown bat, Eptesicus

fuscus fuscus, which had been obtained from a hall in the Botany and

Zoology building was fed metacercariae that were six to twelve days old. This animal also was found to be uninfected when examined ten days later.

A single adult amphibian, Rana pipiens, also was uninfected three days after being fed five-day-old metacercariae. Three domestic chickens were the only experimental animals that became infected with flukes.

One chicken produced fourteen adult worms six days after being fed seventy Aedes aegypti larvae. Two additional chickens received fewer larvae, pupae, and adults, but were infected with two adults each.

The adult (Plate VI, Pig. 7, 8; Plate VII, Pig. 3, it, 5)

Body elongate; bluntly rounded anteriorly; somewhat pointed at posterior end. B o d i e s Q f heat killed specimens, stained, cleared, and mounted, measured 1.89-2.66 mm (2.25 mm) long by 0.66-0.82 mm (0.77 mm) wide. Minute spines barely projecting above cuticle cover four-fifths of body anteriorly. Spines dense in anterior third of body but gradually diminish toward posterior end. Subterminal oral sucker larger than acetabulum, 0.18U-0.27U mm (0.218 mm) long by 0.l8U-0.2l*0 mm (0.209 mm) wide. Prepharynx extremely short; pharynx 0.095-0.130 mm

(O.llU mm) long by 0.095-0.137 mm (0.113 mm) wide, opening into short postpharyngeal esophagus that can be seen only in living or greatly relaxed specimens. Ceca, 1.61*-1.92 mm (1.80 ram) long, extending to near posterior end of bo<%r. Muscular acetabulum preequatorial, 0.157-

0.212 mm (0.181* mm) long by 0.16U-0.212 mm (0.187 ram) wide. Cirrus sac, 0.376-0.753 mm (0.612 mm) long by 0.089 mm wide; curving from level of ovary dorsally around right side of acetabulum. Prominent seminal vesicle divided by a constriction. Cirrus long and coiled, opening into genital atrium slightly anterior to acetabulum. Testes globular, postequatorial, obliquely situated one behind the other; anterior testis, 0.212-0.287 mm (0.236 mm) long by '0.191-0.260 mm (0.219 mm) wide; posterior testis more medially located, 0.205-0.308 mm (0.262 mm) long by 0.171-0.260 mm (0.211 mm) wide. Ovary anterior to and smaller than testes, generally on right side, 0.171-0.205 mm (0.195 mm) long by 0.150-0.191 mm (0.166 mm) wide. Ootype slightly posterior to ovary; Mehlis' gland prominent, seminal receptacle rudimentary, and

Laurer's canal present. Vitellaria principally along lateral margins of body from anterior edge of acetabulum to posterior end of body.

Follicles extend medially only in caudal region posterior to testes.

Beginning at ootype, descending tract of uterus extends between testes to extreme caudal end of body where it forms a single loop.

Ascending tract passes between testes to level of cirrus sac where

dorsal muscular metraterm curves around left side of acetabulum and

opens into genital atrium. Genital pore slightly anterior to

acetabulum. Operculated yellowish-brown eggs numerous, small, 0.035-

O.OiiO mm (0.037 mm) long by 0.019-0.020 mm (0.019 mm) wide.

Site of infection; Small intestine.

Definitive host: Gallus domesticus (experimental), and

Agelaius phoeniceus phoeniceus.

Type specimens; Homotype No. 601ii5 deposited in the U. S.

National Museum Helminthological Collection.

The nature of the excretory bladder was observed in transverse

sections of Plagiorchis noblei. Two horns of a Y-shaped bladder begin

at a level slightly anterior to the genital opening and are ventro­

lateral to the ceca in this area. On tracing the horns posteriorly, 81

I found them extending dorso-medially in the region of the anterior

1 testis, finally converging dorsally and forming a single duct which extended to the very distal end of the body'where it opens to the outside. Sectioned material also revealed the genital atrium into which both the cirrus sac and metraterm open. Prom the atrium there is a single genital pore opening to the ventral surface just anterior to the acetabulum. The vitellaria are shown to extend dorso-medially behind the posterior testis, but at no other region.

Three adult flukes obtained from a red-winged blackbird at

Calamus Swamp were slightly larger than were those obtained experi­ mentally from chickens. In addition, the uteri of these three trematodes were more fully packed with eggs. These differences simply indicated to me that the flukes from the red-winged blackbird were

older than the six- to seven-day-old flukes from chickens. No addi­

tional discernible differences, other than those just pointed out, were

observed between the two groups of specimens. Both the naturally and

experimentally obtained flukes fit well within the measurements given

for Plagiorchis noblei by Park in 1936. I believe that my material

is conspecific with that obtained by Park from the red-winged blackbird

in California.

The egg and miracidium TPlate VXE, Pig. 1, 2)

One of the seven-day-old flukes recovered from the domestic

chicken was placed in a Syracuse watch glass with tap water. This worm

was then cut in half with a fine needle so as to permit the exposure

and removal of the eggs. Several yellowish-brown operculate eggs from near the metraterm were examined beneath a compound microscope. Each egg contained a single uncleaved mass surrounded by coarse vitelline material. This opened fluke was set aside in the laboratory at room temperature and was not examined again until the following day, at which time each egg that was examined contained an early embryo in the morula stage. The eggs were once again set aside and not examined

again until the fifth day. At this time several eggs contained

actively moving miracidia and several irregular concretions of various

sizes. The miracidium was pear-shaped, with its greatest width being

near the posterior end. The body was 0.030-0.032 mm long and had a

medially located anterior papilla from which extended a granular mass

of material, probably penetration glands. Although not clearly

discernible, the miracidium appeared to have cilia.

At this time an attempt was made to infect lymnaeid snails.

Approximately one-half of the eggs were transferred to a small stender

dish with three lymnaeid snails. Since the dish was small, the chances

of the eggs being ingested were good. Two of these snails were opened

eleven days later and. also found to be uninfected.

The remaining half of the eggs were incubated at room

temperature for another two days and then examined. The seven-day-old

micracidium showed no observable change from that of the five-day-old

form. These eggs were transferred to a stender dish with four lymnaeid

snails. Two of these snails were examined eight days later and found

to be uninfected for sporocysts as were the two remaining snails which

were examined twelve days from the time that they were placed with the

embryonated eggs. From all indications the eggs were ingested by the snails, but sporocysts had not developed. The snails were from a lymnaeid population in the Botany and Zoology greenhouse and may not have been Iymnaea (S.) reflexa.

Discussion

Park (1936) stated that Plagiorchis noblei closely resembled

Plagiorchis maculosus and Plagiorchis elegans, but that it could be

distinguished easily from these by the nature of its vitellaria. The vitellaria in P. maculosus extend anterior to the level of the

esophagus, while those of P. elegans extend forward beyond the posterior limits of the pharynx. In P. noblei the vitellaria extend only slightly in advance of the anterior margin of the acetabulum.

A portion of the life history of P. maculosus was worked by N&ller

and Ullrich in 1927 and again by Strenzke (1952). The cercaria of

P. maculosus has a larger body, oral sucker, and acetabulum than does

the cercaria of P. noblei. The stylet of P. maculosus is smaller than

that of P. noblei, being 25-27JJ long.

Of the known life cycles in this group, P. noblei most closely

parallels that of Plagiorchis micracanthos, a bat fluke. Although

the large number of bubble-like concretions in the cercaria of P.

micracanthos readily distinguish it from that of P. noblei, the body

and organ measurements, as well as measurements and appearance of the

stylet, are similar. Neither species encysts precosciously in daughter

sporocysts, and both form approximately the same size cyst. This

continued similarity is carried over to the adult as well. The extent

of the vitellaria is similar in both species as is the extent and nature of the uterus. A small seminal receptacle is known for

Plagiorchis noblei, but no such structure has been reported for

Plagiorchis micracanthos. Najarian (1961) reported a seminal receptacle for Plagiorchis goodmani, but this species differs from

P. noblei principally by the nature of its vitellaria. The vitellaria of P. goodmani extend forward to the anterior level of the pharynx and also medially in this area. In addition, this species has separate

genital openings which is not characteristic of P. noblei.

To my knowledge no life history work has been conducted with

Plagiorchis noblei prior to this study. The micracidium, sporocysts,

cercaria, and metacercarial stages are, therefore, new to science.

In addition, the finding of adults of P. noblei in the red-winged

blackbird, Agelaius phoeniceus phoeniceus, at Calamus Swamp establishes

a new distribution record for this parasite. The domestic chicken,

Gallus domesticus, also becomes the only known experimental definitive

host.

Considering that the life history stages of Plagiorchis noblei

resemble closely the corresponding stages of other species in the genus

Plagiorchis, it can be concluded ohat P. noblei is closely related to

these other species. H A T E VI

Life history stages of Plagiorchis noblei.

Fig. 1.— Daughter sporocyst containing cercariae.

Pig. 2.— Cercaria. Ventral view.

Pig. 3-— Stylet. Dorsal view.

Pig. h•— Stylet. Lateral view.

Fig. 5.— Oral sucker of cercaria showing position of stylet. Lateral view.

Pig. 6.— Encysted metacercaria.

Pig. 7-— Six-day-old experimental adult fluke. Ventral view.

Pig. 8.— Adult fluke from natural infection in red-winged blackbird. Ventral view.

85 PLATE 3ZI 66 PIATE VII

Life history stages of Plagiorchis noblei.

Fig. 1.— Egg from near metraterm of uterus.

Fig. 2.— Egg possessing miracidium after five days of incubation.

Pig. 3 .— Adult fluke. Transverse section through region of genital pore.

Fig. — Adult fluke. Transverse section through region of acetabulum.

Fig. 5.— Adult fluke. Transverse section through region posterior to testes.

AC - Acetabulum

ATJ - Ascending uterus

B - Excretory bladder

C - Cirrus sac

DU - Descending uterus

E - Egg

ED - Excretory duct

GA - Genital atrium

IC - Intestinal cecum

M - Metraterm

V - Vitellaria

87 PLATE 3ZE 88 SUMMARY

1. Twenty-four collections, totalling 1,252 specimens of

Lymnaea (Stagnicola) reflexa (Say), were made over a two-year period from Calamus Swamp, a natural temporary pond in Pickaway County, Ohio.

All of these snails were examined for cercariae.

2. Six species of trematodes were found utilizing this snail as their first intermediate host. Life history information is presented for four of these, the larval stages of one not being known prior to this research.

3. One per cent of the snails were infected with develop­ mental stages of Diplostomum micradenum (Cort and Brackett, 1938)j

2k per cent with the stages of Protechinostoma mucronisertulatum

Beaver, 19U3; O.Oi* per cent with those of Notocotylus stagnicolae

Herber, 19U2; and 3.3 per cent with the larval stages of Plagiorchis noblei Park, 1936.

U. Cercariae of Diplostomum micradenum did not penetrate the fish, Cyprinus auratus, Ictalurus melas, Eucalia inconstans, and

Lepomis cyanellus, but did penetrate tadpoles of Rana pipiens, R. sylvatica, Pseudacris brachyphona, and Bufo sp., as well as larvae of

Amfoystoma opacum and A. jeffersonianum. The metacercariae did not develop in the salamander larvae.

5. Cercariae of Diplostomum micradenum were observed to enter the blood stream of tadpole intermediate hosts, and were obtained i

89 90 from their final site of infection, the central nervous system, within eight hours after penetration.

6. In addition to the central nervous system the metacercariae of D. micradenum were found developing in the retina of the eyes of

Rana pipiens and R. sylvatica tadpoles, a site not disclosed previously for this species of trematode.

7. Metacercariae of D. micradenum, as young as twenty-two to twenty-eight days, were infective to domestic chickens and ducks.

8. Anas domestica and Gallus domesticus were established as new experimental definitive hosts for D. micradenum. However, the natural definitive host of this species has never been found.

9. On the basis of information from this study and from that

of Hugghins (l95k) on Hysteromorpha triloba, it was concluded that

Diplostomum micradenum is a valid species and not a synonym of

Hysteromorpha triloba (Rudolphi, 1819).

10. The present life history study on Protechinostoma

mucronisertulatum confirmed previous work by Feldman and Beaver, and

my material is considered to be conspecific with theirs.

11. The discovery of Protechinostoma mucronisertulatum

constitutes a new distribution record for this trematode, and Anas

domestica, Turdus migratorius, and Stamus vulgaris become additional

experimental definitive hosts.

12. Life history work revealed that Notocotylus stagnicolae

matures to an adult in twelve days in the domestic chicken, and that

the ventral gland pattern in eight- and ten-day-old flukes is

different than that of twelve-day-old adults. 91

13- The present life history work on Notocotylus stagnicolae confirms previous studies by Herber (19U2) and provides an additional distribution record for this parasite.

111. Life history studies disclosed that the xiphidiocercaria of the Conniae division of the Polydena, group, being shed by Lymnaea

(S.) reflexa, was the cercaria of Plagiorchis noblei, the larval stages of which have never been described prior to this study.

15- The cercariae of Plagiorchis noblei were found to be very similar to those of other species in the genus Plagiorchis, having eight pairs of penetration glands and a flame cell pattern of

2 / b + 3 + 3) + (3 + 3 + 317 = 36.

16. The cercariae of Plagiorchis noblei were observed to penetrate the larval stages of caddisflies, midges, mosquitoes, damselflies, and mayflies. The metacercariae of this species became infective in four to six days in laboratory reared Aedes aegypti.

17 • Adults of Plagiorchis noblei were obtained in six days from the small intestine of Gallus domesticus.

18. A natural infection, consisting of three adult flukes of

Plagiorchis noblei was found in one of five red-winged blackbirds,

Agelaius phoeniceus phoeniceus, collected at Calamus Swamp in

Pickaway County, Ohio.

19. Eggs of Plagiorchis noblei produced active miracidia in

five days at room temperature, but these miracidia did not produce

infections in seven lymnaeid snails to which the embryonated eggs were fed. LITERATURE CITED

Baker, F. G. 1928. The fresh water mollusca of Wisconsin. Part 1. Gastropoda. Wisconsin Geol. and Wat. Hist. Survey Bull., 70: 1-570.

Beaver, P. C. 1937. Experimental studies on Echinostoma revolutum (Eroel.), a fluke from birds and mammals. Illinois Biol. Monogr., 15: 1-96.

______19k3- Studies on Protechinostoma mucronisertulatum, n. g., n. n. (Psilostomum reflexae Feldman, 19U1), a trematode (Echinostomidae) from the sora rail. J. Parasitol., 29: 65-70.

Berrie, A. D. I960. Two Diplostomulum larvae (Strigeida, Trematoda) in the eyes of sticklebacks (.Gasterosteus aculeatus L.). J. Helminthol., 3 k ‘- 211-216.

Brooks, F. G. 1914-3 Larval trematodes of Northwestern Iowa. I. Nine new xiphidiocercariae. J. Parasitol., 29: 330-339.

Cheatum, E. P. 193k- Limnological investigations on respiration, annual migratory cycle, and other related phenomena in fresh­ water pulmonate snails. Trans. Am. Microscop. Soc., 53: 3U8-1407.

Cort, W. W. 1915* Some North American larval trematodes. Illinois Biol. Monogr., 1: 1-87=

Cort, W. W. and S. Brackett. 1938. Two new species of strigeid cercariae in Stagnicola palustris elodes (Say) from the Douglas Lake region, Michigan. Trans. Am. Microscop. Soc., 57: 27U-281.

Cort, W. W. and L. Olivier. 19U3. The development of the larval stages of Plagiorchis muris Tanabe, 1922, in the first intermediate host. J. Parasitol., 29: 81-99.

Dollfus, R. P. I960. Recherches sur le developpement et 1'identi­ fication de Plagiorchis (Multiglandularis) cirratus (Rudolphi 1802). II. Description et identification. Ann. de Parasitol. Humaine et Comparee, 35: 282-291.

92 93

Dubois, G. 1953- Systeimatique des Strigeida. Mem. Soc. Neuchateloise Sci. Nat., 8: 1-lUl.

Dubois, G. and R. Rausch. 1950. A contribution to the study of North American strigeids (Trematoda). Am. Midland Naturalist, h 3 ’ 1-31.

Fain, A. 1953. Contribution a 1*etude des formes larvaires des trematodes au Congo Beige et specialemente de la larve de Schistosoma mansoni. Part VI. Xiphidiocercariae. Mem. Inst. ftoyal. Colon. Beige, 22: 1-312.

Feldman, S. I. 19^1. Studies on the morphology and biology of a psilostome fluke. J. Parasitol., 27: 525-533.

Herber, E. C. 19i|2. Life history studies on two trematodes of the subfamily Notocotylinae. J. Parasitol., 28: 179-196.

Hoffman, G. L. and J. B. Hundley. 1957. The life-cycle of Diplostomum baeri eucaliae n. subsp. (Trematoda: Strigeida). Jl. Parasitol., h 3 ‘- 613-627.

Hugghins, E. J. 195U. Life history of a strigeid trematode, Hysteromorpha triloba (Rudolphi, 1819) Lutz, 1931- II. Sporocyst through adult. Trans. Am. Microscop. Soc., 73: 221- 236.

Lutz, A. 1931. Contribuiqao ao conhecimento da ontogenia das Strigeidas. I. Ontogenia de Hemistomum trilobum (Rudolphi, 1819). Mem, Inst. Oswaldo Cruz, 25: 333-3L2. German translation: 3h3-353.

McMullen, D. B. 1937. The life histories of three trematodes, parasitic in birds and mammals, belonging to the genus Plagiorchis. J. Parasitol., 23: 235-21*3.

Miller, E. L. 1935. Studies on North American cercariae. Illinois Biol. Monogr., Hi: 1-125.

Najarian, H. H. 1952. A new xiphidiocercaria, C. goodmani, from Iymnaea palustris. J. Parasitol., 38: 157-160.

______1961. The life cycle of Plagiorchis goodmani n. comb. (Trematoda: Plagiorchiidae). jT Parasitol., U?: 625-631*.

Olivier, L. 191*0. Life history studies on two strigeid trematodes of the Douglas Lake region, Michigan. J. Parasitol., 26: UU7 —U77- 9U

Olsen, 0. W. 1937* A systematic stucfcr of the trematode subfamily Flagiorchiinae Pratt, 1902. Trans. Am, Microscop. Soc., 56s 311-339.

Park, J. T. 1936. New trematodes from birds, Plagiorchis noblei sp. nov. (Flagiorchildae) and Galactosomum faumpargari sp. nov. (Heterophyidae). Trans. Am. Microscop. Soc., 55* 360-365.

Rees, G. 1952. The structure of the adult and larval stages of Plagiorchis (Multiglandularis) megalorchis n. nom. from the turkey and an experimental demonstration of the life history. Parasitology, 1*2: 92-113.

1955. The adult and diplostomulum stage (Diplostomulum pfaoxini (Faust) of Diplostomum pelmatoides Dubois and an experimental demonsiration of part of the life cycle. Parasitology, U5: 295-302.

Sewell, R. B. S. 1922. Cercariae Indicae. Indian J. Mied. Res., 10: 1-370. (Suppl. no.)

Skrjabin, K. I. 1953. Family Notocotylidae Duhe, 1909. In K. I. Skrjabin: Trematodes of animals and man. U.S.S.R. Academy of Sciences Publishers, Moscow, 8: 9-211.

Skrjabin, K. I. and D. N. Antipin. 1958. Superfamily Plagiorchioidea Dollfus, 1930. In K. I. Skrjabin: Trematodes of animals and man. U.S.S.R. Academy of Sciences Publishers, Moscow, lU: 75-666.

Strenzke, K . ' 1952. Der Wirtswechsel von Plagiorchis maculosus. Zeitschr. Parasitenk., 15: 369-391.

Stunkard, H. W., C. H. Willey, and T. Rabinowitz. 19Ul. Cercaria burti Miller, 1923* a larval stage of Apatemon gracilis (Rudolphi, 1819) Szidat, 1928. Trans.Am. Microscop. Soc., 60: U85-U97.

Szidat, L. 1931. Beitrage zur Entwicklungsgeschichte der Holosto- miden. TV. Die Cercarie des Entenparasiten Apatemon (Strigea) gracilis Rud. und ihre Entwicklung im Blutgeftesystem de Zwischenwirtes (Herpobdella atomaria Gar.). Zeitschr. Parasitenk., 3s 160-172.

Szidat, L. and U. Szidat. 1933. Beitrage zur Kenntnis der Trematoden der Monostomidengattung Notocotylus Diesing. Centr. Bakt., 129: IP1-U22.

Wu, L. T. 1953. On the life history and biology of Notocotylus stagnicolae Herber, 19U2 (Family NotocotylidaeTi Canadian 7Tf55T7731: 522-527. 95 Yamaguti, S. 1933. Studies of the helminth fauna of Japan. Part I. Trematodes of birds, reptiles and mammals. Japanese J. Zool., 5: 1-134

______1958. Systema Helminthum. Vol. 1. The digenetic trematodes of vertebrates. Interscience Publishers, Inc., New York, Parts I and II, 1575 pp. AUTOBIOGRAFET

I, Russell Raymond Williams, was born at Lost Creek, West

Virginia on April 11, 1926. I received by secondary-school education in the public school in Gambier, Ohio, and completed one year of undergraduate work at Kenyon College in the same village. For five and one-half years I was a chemical laboratory technician for the

B. F. Goodrich Chemical Company in Avon Lake, Ohio. In 1950, I entered military service where I served as a medical laboratory techni­ cian in the Department of Medical Zoology of the U06th Medical General

Laboratory in Tokyo, Japan. Following my separation from the service in 1952, I entered The Ohio State University from which I received the degree Bachelor of Science in 1955 and the degree Master of Science in

1957. While at The Ohio State University, I held the following posi­ tions in the Department of Zoology and Entomology: Graduate Assistant from 1955 to 1957; Assistant Instructor from 1957 to 1961; and

Instructor from 1961 to 1963. In 1953, I married Carol Anne Garber of

Avon Lake, Ohio. We presently have three children: Russell Dean,

David Wayne, and Susan Carol.

I have accepted a position as Assistant Professor of Biology at

Waynesburg College in Waynesburg, Pennsylvania.

96