This d isserta tio n has been m icrofilm ed exactly as received

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1 This d isserta tio n has been m icrofilm ed exactly as received WILLIAMS, R u ssell Raymond, 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 U niversity, Ph.D., 1963 ^ ^ m f^ r sity Microfilms, Inc., Ann Arbor, Michigan \

2 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

3 / 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

4 TABLE OF CONTENTS Page ACKNOWLEDGEMENTS... ii LEST OF TABLES... LIST OF ILLUSTRATIONS... vi INTRODUCTION... ^... 1 MATERIALS AND METHODS... h OBSERVATIONS AND RESULTS The First Intermediate H o s t The Trematode Parasites... U The Taxonomic Positions of the Four Trematodes Encountered in this Investigation Diplostomum micradenum (Cort and Brackett, 1938) Introduction The Daughter Sporocyst The Cercaria Penetration Experiments The Metacercaria... 2k Second Intermediate Hosts Recovery From Eye and Brain Feeding Experiments with Metacercariae The Adult Discussion Protechinostoma mucronisertulatum Beaver, 19U Introduction The Redia... U5 The Cercaria... U5> Encystment.... h i The Metacercaria... h i Excystment... 1*8 Feeding Experiments with Metacercariae... 1*9 The Adult Discussion Notocotylus stagnicolae Herber, 19U Introduction The Redia iii

5 TABLE OF C OUT EM'S (CONTINUED) Page The Cerceria The Metacercaria Feeding Experiments with Metacercariae The Adnlt... 6U Discussion Plagiorchis noblei Park, Introduction The Daughter Sporocyst The Cercaria Penetration of Second Intermediate Host The Metacercaria Feeding Experiments with Metacercariae The Adult The Egg and Miracidium Discussion SUMMARY LITERATURE CITED AUTOBIOGRAHIT iv

6 LIST OF TABLES Table Collection and Infection Data for Iymnaea (S.) reflexa Page I k Collection and Infection Data for lynmaea (S.) reflexa Collection and Infection Data for Lymnaea (S.) reflexa U Diplostomum micradenum (Cort and Brackett, 1938) Exposure of fish and Amphibia to Cercariae Diplostomum micradenum (Cort and Brackett, 1938) Recovery of Metacercariae from Rana pipiens Tadpoles 30 Days after Exposure to $0 CercariW Each A Two-Way Analysis of Variance for the Metacercarial Data from 19 Rana pipiens Exposed to 50 Cercariae Bach Diplostomum micradenum (Cort and Brackett, 1938) % Results of1feeding Metacercariae from Rana pipiens Tadpoles to One-Day-Old Domestic D u cks Diplostomum micradenum (Cort and Brackett, 1938) Results of deeding Kfetacercariae from Rana pipiens Tadpoles to One-Day-Old Domestic C h ickens Protechlnostoma mucronisertulatum Beaver, 19b3 Results of Feeding Metacercariae to Various Vertebrate Animals Notocotylus stagnicolae Herber, 191*2 Results of Feeding 1- to 1;-Day-Old Metacercariae to Domestic Chickens Plagiorchis noblei Park, 1936 Results of Feeding Metacercariae to Vertebrate Animals v

7 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. HATE 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.

8 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.

9 INTRODUCTION This represents a stu<fy of the life histories of four species of digenetic trematodes, the asexually reproducing stages of which were found infecting the pulmonate snail, lymnaea (S.) reflexa (Say). 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.

10 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 thoroughly 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 additional 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 previously 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 stagnicolae. Little is known about the distribution of these four species of trematodes.

11 Twenty-four collections, tnfca.lh.ng 1,252 specimens of Iymnaea (S.) reflexa, were made from March 23, 1961, through May 2k, 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.

12 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.

13 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 experiments 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,

14 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 laboratory. 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

15 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

16 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

17 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.

18 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

19 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

20 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, 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

21 13 SUBFAMILY: Plagiorchiinae Pratt, 1902 Plagiorchis noblei Park, 1936 Diplostomum micradenum (Cort and Brackett, 1938) (Plates I, II, IH) 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

22 TABLE COLLECTION AND INFECTION DATA FOR LIMNAEA (S.) REFLEXA Date Collected Number Collected Diplostomum micradenum Number Shedding Cercariae Protechinostoma mucronisertulatum Notocotylus s'tagnicolae Plagiorchis noblei March (l.9)a ll5 U12.9) 0 (0.0) 5 0i.8) April (1.5) 3li (52.li) 0 (0.0) 1 (1.5) May (0.0) 2 (28.6) 0 (0.0) 0 (0.0) May (0.0) 2i (28.6) 0 (0.0) 1 (7.2) August (0.0) 1 (5.0) 0 (0.0) 0 (0.0) afigures in parentheses represent percentage of infection.

23 TABLE COLLECTION AND INFECTION DATA FOR LYMNAEA (S.) REFLEXA Date Collected Number Collected Diplostomum micradenum Nranber Shedding Cercariae Protechinostoma Notocotylus mucronisertulatum stagnicolae Plagiorchis noblei March (l.7)a 28 (1*6.7) 0 (0.0) 5 (8.3) March (0.0) 3 (37.6) 0 (0.0) 0 (0.0) March (0.0) 2 (33.lt) I 0 (0.0) 0 (0.0) April (10.0) 1 (10.0) 0 (0.0) 0 (0.0) May (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 (0.0) 3 (1.7) 1 (0.6) 8 (It.6) Nov (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.

24 is TABLE COLLECTION AND INFECTION DATA FDR LYMNAEA (S.) REFLEXA Date Collected Number Collected Diplostomum micradenum Number Shedding Cercariae Protechinostoma mucronisertulatum Notocotylus siagnicolae I lagiorchis noblei March O O V _ * PJ 13 (6.3) 1 (0.5) 7 (3.10 March (o.li) 9 (lt.o) 2 (0.9) 5 (2.2) March (0.0) 1 (12.5) 0 (0.0) 0 (0.0) March (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 (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 (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

25 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

26 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 experiments 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 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 imparting a dark appearance to the sporocyst. Old sporocysts with constrictions 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.

27 19 Hoffman and Hundley (1957) found that at 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 T H att T T T T F ig. 1, 2) Small furcocercous cercaria; both body and furcae shorter than tail stem; booty mm (0.170 mm) long by mm ( mm) wide. Tail stem mm (0.275 mm) long by mm ( mm) wide with furcae, ram (0.261 mm) long. Oral sucker larger than acetabulum, it5 mm (0.032 mm) long by mm (0.032 mm) wide. Prepharynx short; pharynx, mm long by 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; 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

28 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 )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.

29 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

30 22 TABLE U DIPLOSTOMUM MICRADENUM (CORT AND BRACKETT, 1938) EXPOSURE OF FISH AND AMPHIBIA TO CERCARIAE Classification and Scientific Name Number Exposed Number Examined Number Infected Class: Teleostomi Family: Cyprinidae Cyprinus auratus family: Ictaluridae Ictalurus melas h k 0 Family: Gasterosteidae Eucalia inconstans Family: Centrarchidae Lepomis cyanellus Class: Amphibia Family: Ranidae Rana pipiens Rana sylvatica Family: Hylidae Pseudacris brachyphona Family: Bufonidae Bufo. sp. 5o Family: Ambystomidae Ambystoma opacum Hi lit 3 Ambystoma jeffersonianum k aonly larval stages of Amphibia exposed to cercariae.

31 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.

32 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 noticeable change in the size of the body. There was a growth in length from approximately 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 metacercariae were U31 mm (0.399 mm) long by 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.U mm (0.i 6l mm) long by 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,

33 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

34 immature metacercariae of Diplostomum micradenum, the lowest infection 26 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

35 27 these diplostomula invaded every cavity, and also were found occasionally 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

36 TABLE 5 28 DIPLOSTOMUM MICRADENUM (CORT AND BRACKETT, 1938) RECOVERY OF METACERCARIAE FROM RANA PIPIENS TADPOLES 30 DAYS AFTER EXPOSURE TO ^ T S ercariae EACH V HQSt No k U* Number In Central Nervous Number in Total Number Per Cent System Eyes Recovered Recovered * ho 0 1* * la 0 Ul * * *3 2 1* h la * *

37 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 Variation Sum of Squares Degrees of Freedom Mean Square F (Test Statistic) Critical Value Between locations 9, , Between individuals Error U Total 9, 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 metacercariae, no consistent results were obtained with metacercariae

38 30 TABLE 7 DIPLOSTOMUM MICRADENUM (CORT AND BRACKETT, 1938) RESULTS OF FEEDING METACERCARIAE FROM RANA PIPIENS TADPOLES TO ONE-DAY-OLD DOMESTIC DUCKS Host No. Number of Metacercariae Fed to Host Age of Metacercariae (Days) Time in Definitive Host (Days) Worms Recovered Immature Adult m h U J? u lit 6k U k l k 0 0

39 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 metacercariae. 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 Host No. Number of Metacercariae Fed to Host Age of Metacercariae (Days) Time in Definitive Host (Days) Worms Recovered Immature Adult k 10 2 > 33 6 k u 3 U8 3h I* 15 0 k 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

40 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 TH, 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 mm (1.02 mm). Fore-body round to oval, 0.1* mm (0.1*9 mm) long by 0.1* mm (0.1*6 mm) wide, concave ventrally. Hind-body more cylindrical, usually slightly longer than fore-body, 0.1* mm (0.5l mm) long by mm (0.28 mm) wide. Subterminal oral sucker slightly smaller than acetabulum, mm (0.080 mm) long by 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* mm (0.065 mm) long by 0.01* 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, mm (0.083 mm) long by mm (0.108 mm) wide.

41 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, mm (0.1it3 mm) long by mm (0.113 mm) wide; posterior testis in median zone, wider than long; I4.8 ram (0.120 mm) long by mm (0.195 mm) wide. Uterus short, not coiled, containing lj-7 eggs. Eggs 0.081; mm long by mm wide. Site of infection: Small intestine. Definitive host: Anas domestica and Gallus domesticus (Experimental). Type specimens: Homotype No 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 sixday-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

42 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 morphologique 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 metacercaire, Ciurea (1930, p ) 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/ fA a l'^tat de fixation) est tres inferieure. celle des larves ehkystees, decrites par Ciurea (690-76o/i /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 t e 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.).

43 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 Hysteromorpha 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

44 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 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

45 metacercaria of D. micradenum had never been reported from the eyes of the second intermediate host. 37 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

46 PIATE I Life history stages of Diplostomum micradenum. Fig. 1. Metacercariae in retina of eye of Rana pipiens tadpole. 38

47 PLATE I 39