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University Microfilms International 300 North Zeeb Road Ann Arbor. Michigan 48106 USA St. John's Road, Tyler's Green High Wycombe, Bucks, England HP10 8HR 77-29,829 COOK, Thomas Wayne, 1947- LOCALIZATION OF FIBRICOLA CRATERA (TREMATODA: DIPLOSTOMATIDAE) METACERCARIAE IN RANA PIPIENS. Iowa State University, Ph.D., 1977 Zoology

Xerox UniVOrSity Microfilms, Ann Arbor, Michigan 48106 Localization of Fibricola cratera

(Trematoda; Diplostomatidae) metacercariae

in Rana pipiens

Thomas Wayne Cook

A Dissertation Submitted to the

Graduate Faculty in Partial Fulfillment of

The Requirements for the Degree of

DOCTOR OF PHILOSOPHY

Department; Zoology Major; Zoology (Parasitology)

Approved:

Signature was redacted for privacy.

Signature was redacted for privacy. For the Major Department

Signature was redacted for privacy.

Iowa State University Ames, Iowa

1977 ii

TABLE OF CONTENTS

Page

INTRODUCTION 1

LITERATURE REVIEW 3

MATERIALS AND METHODS 27

RESULTS AND DISCUSSION 36

SUMMARY AND CONCLUSIONS 80

LITERATURE CITED 83

ACKNOWLEDGEMENTS 96

PLATES 97 1

INTRODUCTION

A well-known attribute of parasitism is that parasites localize in their inter mediate or definitive hosts. This so-called "site-finding behavior" involves entry into the proper host (i.e. host selection) and, once in the intermediate or defini tive host, localizing in a suitable microhabitat for establishment and survival.

Research into parasite localization is a relatively new area of investigation for parasitologists. Only recently have the complexities of parasitic behavior be gun to be examined experimentally. This dissertation is an invest%ation of the larval localization of the d%enetic trematode Fibricola cratera (Barker and Noll,

1915) Dubois, 1932, a common duodenal parasite of raccoons (Procyon lotor) whose metacercariae (diplostomula) occur in tadpoles and frogs.

Although Hoffman (1955) and Turner (1958) briefly demonstrated and de scribed all stages in the life-cycle of this parasite, certain questions relative to the localization of the metacercariae remain to be answered. Hoffman's (1955) description of the infection of tadpole embryos was vague; no mention in his report is made of numbers of cercariae used in experimental infections of tadpoles nor of the exact age of the tadpoles.

lue phenomenon of metacercarial migration of ^ cratera has never been studied in any detail. Although migration of metacercariae during frog metamor

phosis has been known for 35 years, no published data are available on the significance of frog metamorphosis on the life-cycle of F. cratera. Furthermore, 2 no descriptive account of the progress and route of m^rating metacercariae from the peritoneal cavity to the hind leg musculature appears in the literature. These aspects of the life-cycle have been investigated in detail and are presented in this dissertation. 3

LITERATURE REVIEW

Historical Review

The genus Fibricola was established by Dubois (1932). The type species cratera was assigned to a diplostomatid trematode described by Barker (1915) as

Hemistomum craterum (Barker and Noll, 1915) recovered from a muskrat

(Ondatra zibethicus) intestine. Since 1915,cratera has been reported in al least 15 species of hosts from 14 states and two provinces in Canada. This in formation is summarized in Table 1.

According to Dubois (1963, 1970), two species of the genus Fibricola occur in the United States namely; Fibricola cratera (Barker and Noll, 1915) Dubois,

1932, and F. lucida (La Rue and Bosma, 1927) Dubois and Rausch, 1950. Five ad ditional species of Fibricola presently accepted by Dubois (1963, 1970) include:

F. caballeroi Zerecero, 1943 from Rattus norvégiens, F. intermedins (Pearson,

1959) Sudarikov, 1960, from Rattus as similis and Hydromys chrysogaster,

F. minor Dubois, 1936 from Hydromys chrysogastér, F. sarcophilus Sandars,

1957 from Sarcophilus harrisi, and F. sudarikovi Sadovskaja, 1952 from Ondatra

zibethicus, Apodemus agrarius and Cricetulus triton nestor.

Fibricola is placed in the order Strigeatida La Rue. 1926 and within the

superfamily Strigeoidea Rallliet, 1919. Fibricola, a typical strigeoid with a dis

tinct fore- and hindbody, possesses a tribocytic (holdfast) organ in addition to

oral and ventral suckers. Table 1. Definitive hoats of Fibricola ctatera reported In the literature

Host Locality Author(s)

Blarina brevlcauda Minnesota Cuckler, 1940

Chickens^ Florida Leigh, 1954 Iowa Ulmer, 1955

Didelphis vlrginiana Georgia Byrd et al., 1942 Tennessee

Michigan Chandler and Rausch, 1946

Eudocimus albus Louisiana Lumsden, 1961

Mephitis mephitis Wyoming Dubois and Rausch, 1950

Mephitis nigra Michigan Chandler and Rausch, 1946

Mesocrlcetus auratus Florida Leigh, 1954

Mustt.la vison Ontario, Canada Law and Kennedy, 1932 Texas Read, 1948 Louisiana Lumsden and Zischke, 1961

Ondatra zlbethicus Nebraska Barker, 1915 Minnesota Cuckler, 1940 Illinois Gilford, 1954 Ohio Beckett and Galllcbhlo, 1967 Utah Grundmann and Tsal, 1967 '^Experimental host. Table 1. (Continued)

Host Locality Author(s)

Procyon lotor Quebec, Canada Miller, 1940 Iowa Morgan and Waller, 1940 Texas Chandler, 1942 Michigan Chandler and Rausch, 1946 Iowa Hoffman, 1955 Georgia Babero and Shepperson, 1958 Georgia Sawyer, 1958 Iowa IXirner, 1958 Louisiana Lumsden and Zischke, 1961 Cape Island, B.C. Harkema and Miller, 1962 North Carolina Harkema and Miller, 1964 Soutli Carolina Harkema and Miller, 1964 Florida Harkema and Miller, 1964 Iowa Present Study

Rattus norvegicus Wisconsin Schiller and Morgan, 1949 Michigan DeGlustl, 1957 Florida Leigh, 1954 Iowa® Ulmer, 1954 Iowa® Hoffman, 1955 Iowa® Turner, 1958

Sclurus hudsonicus Michigan Chandler and Rausch, 1946

Tamlasciurus hudsonicus Several North Central States Rausch and Tiner, 1948 Table 1. (Continued)

Host Locality Author(s)

Taxldea taxus Iowa Wittrock and Ulmer, 1974 a White mice Florida Leigh, 1954 lowa^ Ulmer, 1955 lowa^ Turner, 1958 7

Dubois (1953, 1963, 1970) placed Fibricola within the subfamily Alariinae

(Family Diplostomatidae) because it is parasitic in mammals and its vitelline folli cles are limited chiefly to the forebody. Members of a second subfamily, Diplo- stomatinae, are parasites of birds and have vitelline follicles limited chiefly to the hindbody. Dubois (1963, 1970) separated Fibricola from other genera in the

Alariinae because of the following characteristics: spoon-shaped forebody (much longer than hindbody from which it is distinctly separated by a transverse constric tion), asymmetrical anterior testis, the absence of pseudosuckers and auricles, presence of a circular or elliptical tribocytic organ, and a normally developed ventral sucker.

Early observations of the Fibricola cratera life cycle were reported by

Cuckler (1940 and 1949), who demonstrated that Physa sp. and several species of tadpoles and frogs serve as intermediate hosts. Further life cycle data were pre sented by Ulmer (1954, 1955) and by Hoffman (1955). Detailed observations and descriptions of larval stages were reported by Turner (1957, 1958). Extensive observations of the infection in tadpoles, migration of metacercariae in the frog host, and effects of blocked and accelerated metamorphosis to migrating larvae are presented in this study.

Localization of Trematode Metacercariae

It has been well-established that many trematode metacercariae show a pro nounced localization within their hosts. Most observations have come from life 8 history studies. In many cases, investigators have noted a particular organ spe cificity or migration of trematode metacercariae within the hosts. Experimental

work to investigate reasons of such localization is almost totally lacking.

Szidat (1969) has theorized that metacercariae evolve an "engram" or a spe

cific memory guiding them to a particular site within a host. Szidat bases his

hypothesis on experimental evidence that lower invertebrates, such as planarians,

seem to develop memories (Best, 1963; McConnell et al., 1959) from past ex

periences .

Holmes (1973) suggests that continued interactions between parasites over

long periods of time lead to their occupying a particular "niche" within the host.

Such interactions between parasites result in site segregation and, consequently,

narrow site specificity. By using Hymenolepsis diminuta as an example. Holmes

(1973) presents a strong argument that parasites respond to the regular presence

of competitors by niche specialization much the same as free living organisms.

Holmes interprets parasite faunas exhibited today as mature communities estab

lished, to an important extent, through biotic interactions.

Croll (1976) believes that localization is due to the presence of suitable habi

tats within the host which meet the needs of the parasite. According to Croll, the

term "site" when used to describe parasite localization is misused. The term

site has come to mean a "place" which may be physico-chemically described as

the location where the parasite is found. A better concept, Croll states, would be

"habitat" because location of parasites is undoubtedly due to a combination of host-

produced environmental factors. 9

The literature contains many reports of localization of trematode metacer cariae (Table 2). How well these reports fit the theories of Szidat, Holmes, or

Croll remains to be proven. Only Szidat's theory focuses on larval parasites; re search by the other two authors was on adult organisms. However, the concepts of niche specialization or habitats could easily be applied to the localization of metacercariae within the host. Conceivably, all three factors might be at work influencing the location of larval parasites. Table 2, Localization oi; trematode metacercarlae reported In the literature

Species Host(s) Observations Reference

Alarla arlsaemoides Rana piplens Mesocercarlae widely scat Pearson, 1956 Augustine and R. sylvatica tered in the tadpole, between Uribe, 1927 Pseudacris n%rata muscles ventrally in the distal part of thighs or between sternum and hyoid of adult frog.

A. canis Bufo amerleaiiUs Mesocercariae migrate from Pearson, 1956 LaRue and Fallls, Canids gut to lungs; diplostomula de 1934 velop in lungs, migrate to tra chea where they are swallowed, and return to intestine.

A. intermedia Girnlvores Mesocercariae migrate through Cuckler, 1949 (Olivier and Odlaug, intestinal wall into abdominal 1938) Odlaug, 1940 and tiioracic cavities to lungs and develop into metacercarlae. A. marcianae Mature metacercarlae migrate (LaRue, 1917) Walton, via bronchi to trachea to mouth, 1950 are swallowed to reach intestine.

A. mustelae Rat, mouse Metacercarlae migrate to lungs Bosma, 1934 Bosma, 1931 from gut.

A. arlsaemoides Chorioallantoic Metacercarlae migrate to Madsen and A. mustelae membrane (CAM) mesenchyme layer of CAM. Johnson, 1974 Table 2. (Continued)

Species Host(s) Observations Reference

Allassogonoporus Dicomoecus sp. Cercariae penetrate insect Burns, 1961 vespertilionis larvae to form motile unencysted Macy, 1940 metacercariae in body cavity. Metacercariae encyst in thoracic muscles of imago.

Alloglossidium cortt Dragonfly naiads Metacercariae encyst ordi- Crawford, 1937 (Lament, 1921) Van narily among muscle fibers of Cleave and Mueller, abdominal body wall. 1934

Amblosoma suwaense Sinoatria quadrata Metacercariae unencysted be- Shimazu, 1974 Shimazu, 1974 tween digestive gland and shell.

Apatemon annuligerum Perca fluviatilis Metacercariae migrate to eye Kozicka, 1972 (von Nordmann, 1832) by unknown route. Kozika, 1961

Apatemon sp. Cyprinid fry Metacercariae found only in Kozicka, 1972 Szidat, 1928 brain of fry, not adults.

Apophallus brevis Perca flavescens Metacercariae orient themselves Sinclair, 1972 Ransom, 1920 with long axis parallel to peri pheral blood vessels.

Ascocotyle mcintoshi Poecilia latipinna Cercariae swallowed; meta- Leigh, 1974 Price, 1936 Gambusia affinis cercariae penetrate wall of intestine and encyst in coelom. Table 2. (Continued)

Species Host(s) Observations Reference

Ascocotyle pachycystis Cyprinodon variegatus Metacercarial cysts found Schroeder and Schroeder and Leigh, under endothelium of the Leigh, 1965 1965 bulbus arteriosus.

A. sexlchigita Fundulus parvipennis Encysted metacercariae found Martin and Martin and Steele, only on stomach and intestinal Steele, 1970 1970 walls.

Brachylecithum sp. Camportis herculeanis Metacercariae localize in Carney, 1969 Shtrom, 1940 C. pennsylvanicus supraesophageal ganglion.

Brachylecithum sp. Phalangium opilo Metacercariae located in adi Gabrion and pose tissue and muscles of Ormieres, 1973 abdomen.

Bucephalus elegans Lepomis macrorchis Metacercariae encyst under Woodhead, 1929 Woodhead, 1929 skin of fins.

B. gracilescens Ciliata mustela Metacercariae migrate to Matthews, 1974 (Rudolphi, 1819) cranial nerves by unknown Pratt, 1916 route and encyst.

Bucephalopsis labiatus Leuresthes tenuis Metacercariae encyst in Olson, 1975 Manter and Van Cleave, muscle of heart ventricle. 1951

Cephalophallus obscurans Astacus trowbridgii Cysts containing metacercariae Macy and Macy and Moore, 1954 located primarily in abdominal Moore, 1954 muscles. Table 2. (Continued)

Species Host(s) Observations Reference

Cercarla X Gasterosteus aculeatus Metacercariae found only in eye, Erasmus, 1958, Baylls, 1930 migrating via connective tissue 1959 and muscle. Clinostomum complanatum Perca flavescens and Metacercariae encyst In sub Hunter and (Rudolph!, 1814) other centrarchlds cutaneous tissues and muscles. Dal ton, 1939 Braun, 1899

Cotylurus erratlcus Salmo galrdneril Metacercariae always found in Olson, 1970 (Rudolphl, 1809) Salvellnus fontlnalls the pericardial cavity. Circu Johnson, 1971 Szldat, 1928 Oncorhynchus klsutch latory system and loose connec O. nerka tive tissue serve as migratory Thymallus cutlcus routes. M Crassiphiala bulboglossa Fishes of; Metacercariae encyst in skin Hoffman, 1956 Van Haltsma, 1925 Cyprinidae causing black spot. Cyprlnodontldae Esocldae Etheostomldae Percidae Umbridae

Crepldostomum cooper 1 Hexagenia llmbrata Metacercariae encyst in Hopkins, 1934 Hopkins, 1931 H. recurrata muscles, usually those of gill Polymitarys sp. bearing filaments.

C. cooper! Hexagenia llmbrata Metacercariae infect females Esch and more than males (90% vs. 72%). Coggins, 1974 Table 2. (Continued)

Species Host(s) Observations Reference

Dicrocoelium lanceatum Formica pratensis Cercariae migrate from Anokhin, 1966 Stiles and Hassall, abdominal cavity to subeso- 1896 phageal ganglion.

Dlplostomum baeri eucaliae Eucalia inconstans Diplostomula localize in optic Hoffman and Hoffman and Hundley, lobes, optic nerves, cornea, Hundley, 1958 1957 and retina.

D. ellipticus Puntius ticto Metacercariae found free in the Chakrabarti Chakrabarti and P. stigma cranial cavity. and Baugh, 1973 Baugh, 1973 Oxygaster bacaila

D. flexicaudum Salmo irideus Metacercariae migrate to lens Ferguson, 1943 (Cort and Brooks, Plmephales promelas of eye. 1928) Van Haitsma, 1930

D. lucknowensis Mystus vittatus Metacercariae found in cranial Chakrabarti and Chakrabarti and cavity and eyes, Baugh, 1973 Baugh, 1973

D. micradenum Rana pipiens Metacercariae migrate to cen Oliver, 1940 Cort and Brackett, tral nervous system by unknown 1938 pathway; encyst in cavities of brain and spinal cord.

D. murryensis Oryzias latipes Metacercariae migrate only to Johnston and (Johnston and Cleland, lens of eye. Simpson, 1939 1938) Johnston and Simpson, 1939 Table 2. (Continued)

Species Host(s) Observations References

Diplostomum ophthalmi Puntius sp. Metacercariae located in Pandey, 1970 Pandey, 1970 Oxygaster bacaila cranial cavity and eyes. Trichogaster fasialus Heteropneustes fossilis

D. pelmatoides Phoxinus phoxinus Metacercariae occur in large Rees, 1955 Dubois, 1932 numbers in brain, spinal cord, and eyes. Most worms appear in cavities of brain, especially 4th ventricle.

D. phoxini Phoxinus tschekanowskii Metacercariae localize in brain. Agapova and (Faust, 1918) Galieva, 1972 Arvy and Buttner, 1954

D. pusillum Noemacheilus stauchi Metacercariae found in eyes. Agapova and (Dubois, 1927) Galieva, 1972 Dubois, 1932

D. scheuringi Perca flavescens Metacercariae occur mainly in Hughes, 1929 (Hughes, 1929) vitreous humor of eye, never in Bangham and Hunter, lens. 1939

D. spathaceum Phoxinus phoxinus Cercariae initially penetrate gill Ratanarat- (Rudolphi, 1819) chamber and fin peduncles; mi- Brockelman, Olsson, 1876 gration through muscles and sub- 1974 cutaneous tissues tp reach eye. Table 2. (Continued)

Species Host(s) Observations References

Diplostomum tulsipurensis Clarias batrachus Metacercariae found in body Chakrabarti and Chakrabarti and Baugh, cavity. Baugh, 1973 1973

D. (lylodelphus) xenopodis Xenopus laevis Metacercariae lie free within Tinsley and Southwell and Kirshner, pericardial sac of host. Many Sweeting, 1974 1937 localize in membranes enclos ing apex of heart.

Echinoparyphium aconiatium Rana climitans Metacercariae localize in renal Najarian, 1954 Dletz, 1909 R. pipiens tissue of host. R. sylvatica Pseudacris triseriata Hyla crucifer Lymnea palustris Physa gyrina Gyraulus parvus

Echinostoma ivaniosi Indoplanorbls exustus Metacercariae found in the peri- Mohandas, 1973 Mohandas, 1973 Idiopoma dis similis cardial sac of Indoplanorbls exustus, but also in kidney region of Idiopoma dissimilis.

Echinostoma revolutum Various tadpoles and Metacercariae creep into cloaca Johnson, 1920 (Froelich, 1802) silurid fishes and migrate via ureters into Looss, 1899 kidneys and encyst. Table 2. (Continuée^

Species Host(s) Observations Reference

Echiaostoma sp. Lymnea rubiginosa Metacercariae migrate to renal Heyneman, 1966 ( Rudolph!, 1809) Indoplanorbl» exustus organs after being implanted in host body cavity.

Echinostomum ilocanum Lymnea rubigtnoaa Metacercariae in pericardial Lie Kian Joe Vogel, 1933 Tadpoles sac of snail and in tadpole and Nasemary, kidneys. 1973

Ectostphonus rhomboidous Ventridens ILgera Metacercariae develop in sali Villella, 1953 Sinitsin, 1931 vary glands, kidney, and pedal sinus of host.

Euclinostomum Opîiiocephalus sp. Metacercariae in liver of host. Bilquees, 1972 gasterocaecum

E. heterostomum Tilapia zillii Metacercariae localize dorsal Donges, 1974 (Rudolphi, 1809) to swim bladder or in kidney. . Travassos, 1928

E. ihdicum marulii Opîiiocephalus sp. Metacercariae in kidney. Bilquees, 1972 Bhalerao, 1942

Euclinostomum Ophiocephalus sp. Metacercariae in kidney. Bilquees, 1972 nephrostomum Bilquees, 1972 Table 2. (Continued)

Species Host(s) Observations Reference

Euhaplorchis Fundulus parvipinnis Metacercariae migrate to re Martin, 1950 oalifomiensis gion of the brain by unknown Martin, 1950 route, encysting near or on the meninx or rarely within outer region of brain,

Euryhelmis sguamula Rana temporaria Cercariae penetrate and encyst Combes and (Rudolphi, 1819) under skin of young frogs, but Richard, 1974 Poche, 1926 not tadpoles.

Fibricola cratera Rana pipiens Diplostomula migrate to hindleg Cuckler, 1949 (Barker and Noll, 1915) muscles from peritoneal cavity Hoffman, 1955 Dubois, 1932 of metamorphosing frog. Turner, 1958

Galactosomum timondavidi Mugil auratus Metacercariae migrate and lo Prevot, 1973 Pearson and Prevot, Syngnathus abaster calize in optic lobes of host. 1971

Gorgodera amplicava Rana sp. Metacercariae creep over surface Goodchild, 1948 Looss, 1899 of intestine for 10-15 minutes, then burrow into wall of intestine and encyst within it.

Gymnophallus choledochus Nereis diversicolor Metacercariae of both species Bartoli, 1974 Odhner, 1900 encyst in ^ di versicolor ad jacent to the ventral and more rarely the dorsal setae. Table 2. (Continued)

Species Host(s) Observations Reference

Gynaecotyla nassicola Talorchestia longicornis Cercariae penetrate branchial Rankin, 1940 (Cable and Hunninen, lamellae of gills. Metacercariae 1938) Rankin, 1940 work their way towards dorsal pericardial cavity. Cysts found free in pericardial cavity.

Halipegus eccentricus Rana sp. Metacercariae remain in cardiac Trtiomas, 1939 Thomas, 1937 end of stomach until tadpole metamorphoses into adult; then migrate into eustachian tubes and develop to maturity.

Haplometra cylindrica Rana temporaria Metacercariae in body cavity. Grabda- (Zeder, 1800) Looss, Kazubska, 1974 1899

Lechriorchis primus Rana pipiens Metacercariae move into con Talbot, 1933 Stafford, 1905 R. clamltans nective tissue between muscle bundles and promptly encyst.

Lillatrema sobolevi Hexagrammos otakll Metacercariae encysted only Ohabayashi and Gubanov, 1953 In musculature. Arak, 1974

Lissorchis mutabile Chaetogaster sp. Cercariae eaten by host whose Wallace, 1941 Cort, 1919 Dugesia sp. gut wall they penetrate. In Chaetogaster, encystment occurs in parenchyma. Table 2. (Continued)

Species Host(s) Observations Reference

Macravestibulum eversum Goniobasls livescens Encystment of metacercarlae Hsu, 1937 Hsu, 1937 only on protected surface of operculum of snail host.

Megalodlscus temperatus I^a ^p^ Metacercarlae encyst In stratum Krull and (Stafford, 1905) cornum of darkly pigmented Price, 1932 Harwood, 1932 areas of frog.

Metoliophllus uvaticus Acroneuria pacifica Metacercarlal cysts almost en- Macy and Bell, Macy and Bell, 1968 tlrely in fat bodies of nymphs. 1968

Metorchis conjunctus Catostomus oommersoni Metacercarlae encyst In lateral Cameron, 1944 g (Cobbald, 1860) body muscles from level of Looss, 1899 dorsal fin to tall.

Microphallus bitti Larus argentatus Metacercarlae encyst in nerves Prevot, 1973 Prevot, 1972 of locomotor appendages, mainly coxopodltes.

Neodlplostomum Rana n%romaculata Metacercarlae only In liver. K If une and brachypterus Takao, 1973 Chatter jl, 1942

N. Intermedium Hyla sp. Dlplostomula encapsulate Pearson, 1961 Pearson, 1959 mostly between muscle fibers of trunk muscles around notochord. Table 2. (Continued)

Species Host(s) Observation Reference

Neogogotea kentuckiensis 10 sp. of freshwater fish Cercariae penetrate and migrate Hoffman and Cable, 1935 into musculature very quickly. Dunbar, 1963 Most have localized in muscula ture within two to three hours.

Neoleuchochloridium Succinea ovalis Broodsacs (sporocysts) migrate Kagan, 1952 problematicum S. avara to tentacles and pulsate in re (Magath, 1920) Oxyloma retusa sponse to light. Kagan, 1950

Panopistus price! Ventridens ligera Metacercariae recovered from Villella, 1954 Slnitsin, 1931 Stent rema monodon heart and pericardial cavity of Zonltoides nttidus hosts. Deroceras laeve

Paragonimus kellicoti Cambarus sp. Encystment occurs on surface Ameel, 1934 Ward, 1908 of heart. Metacercariae gen erally align in a transverse band across the dorsal side of heart between two ostia.

P. miyazakli Albino rat Metacercariae implanted in Hashiguchl Kamo, Nishlda, pleural cavity migrate to liver and Takei, 1971 Hatsushika, and or abdominal wall musculature Toraimura, 1961 6 to 15 days after implanta tion. Metacercariae then return to lungs and develop into adults. Migration to liver appears neces sary for maturation. Table 2. (Continued)

Species Host(s) Observations Reference

Petasiger neocomense Fish Metacercariae localize in Nasir, Gonzalez, Fuhrmann, 1928 musculature near gills. and Diaz, 1972

P. novemdecium Lebistes reticulatus Metacercariae found in gills Nasir, Gonzalez, Lutz, 1928 and inner layer of esophagus. and Diaz, 1972

P.hilophthalmus grallt Duck, chicken, peafowl, Metacercariae migrate from Alicata, 1962 Mathis and Leager, and goose mouth and crop to conjunctival 1910 sac through nasolacrimal duct. Migration takes one to five days.

P. hegeneri Chicken 98 percent of metacercariae Lauer and Fried, Penna and Fried, placed in eyes reach area of 19741 . 1963 nictitating membrane in three weeks.

P. megaluris Chicken and embryonic Metacercariae migrate to Danley, 1973 (Cort, 1914) Harderian glands Harderian glands in vivo and Cable and Hayes, 1963 in vitro.

Phyllodistomum solidum Ischuria venticales Metacercariae found encysted Goodchild, 1943 Rankin, 1937 Argia sp., Libelula sp., in thoracic hemocoel. Enallagina ap.

P. staffordi Sphaerium lacustre Metacercariae within sporo- Waffle, 1968 Pearse, 1924 cysts in gill lamellae and inter- lamellar gill spaces. Table 2. (Continued)

Species Host(s) Observations Reference

Plagioporus sinitsini Gonlobasis Hvescens Sporocysts with infective meta- Dobrovolny, Mueller, 1934 cercariae migrate from liver 1939 to rectum by unknown route.

Plagitura salamandra Pseudosuccinea columella Metacercariae encyst in foot of Owen, 1956 Holl, 1928 Diving beetle snail, but localize in gill lamellae and branchial chamber of diving beetle.

Pleurogenoides tener Trithemis annulata Encysted metacercariae in tissue Macy, 1964 (Looss, 1898) Crocothemis erythraea of branchial basket. N) Travassos, 1921 Anax imperator CO A. parthenope

Pneumonoeces medloplexus Sympetrum sp. Metacercariae encyst on wall Krull, 1931 (Stafford, 1902) and gills of branchial basket Stafford, 1905 only.

Podocotyle reflexa Pandalus gonlurus Metacercariae localize in Shimazu, 1973 (Creplin, 1825) muscles of posterior thorax. Odhner, 1905

Postharmostomum helicis Anguispira alternata Metacercariae migrate and Ulmer, 1951a, (Leidy, 1847) Polygyra sp. localize in pericardial chamber 1951b Robinson, 1949 Deroceras of host.

Posthodiplostomum minimum fiupomotis glbbosus Metacercariae localize in liver Hunter, 1936 (MacCallum, 1921) and kidney. Dubois, 1936 Table 2. (Continued)

Species Host(s) Observations Reference

P. minimum Metacercariae migrate towards Pettigrew and a thermal gradient in vitro - 16° Fried, 1973 to 45° C.

Procytrema marsupiformes Procyon lotor Diplostomula develop within pan Harris, Harkema and Miller, creas , migrate down pancreatic Harkema, and 1959 duct to intestine when mature. Miller, 1970

ProsorKynchus crucibulus Mentlcirrhusi americanus Metacercariae found only in Kohn and (Rudolphi, 1819) renal parenchyma. Jurburg, 1965 Odhner, 1905 If».to Proathogonlmus macrochis Leucorrhina sp. Metacercariae migrate to Macy, 1934 Macy, 1934 Tetragoneura sp. muscles of body wall, pri Epicordulia op. marily those on posterior Gomphus sp. of abdomen. Mesothermis sp.

Psilostomum sp. Rana cyanopliyctis Metacercariae encyst only in Nath, 1972 Looss, 1899 kidneys.

Ribeiroia ondatrae Perca flavescens Metacercariae encyst pri- Beaver, 1939 (Price, 1931) Ambloplites rupestrts marily on lateral line canal Price, 1942 Lepomis palfldus of fishes and in cloaca of AMeiurus tadpoles. Tadpoles Table 2. (Continued)

Species Host(s) Observations Reference

Stictodora cursitans Fundulus grandis Metacercariae in tongue mus Kinsella and Kinsella and Heard, F. confluentis culature of F. grandis, but in Heard, 1974 1974 F. similis muscles of thoracic body wall in F. confluentis and similis.

Strigea elegans Water snakes and ducks Tetracotyles locate in subcu Pearson, 1959 Chandler and Rausch, taneous tissues of host. 1947

Tetracotyle glossogobii Glossogabiu» giuris Tetracotyles in cranial cavity, Chakrabarti, Chakrabarti, 1970 most free, some attached to 1970 nervous tissue.

Uvulifer ambloplitis Micropterus dolomieu Metacercariae encyst in skin. Hoffman and (Hughes, 1927) Putz, 1965 Ehibois, 1938 26

Review of Life Cycle

Cuckler (1940, 1949), Hoffman (1955), and Turner (1958) have demonstrated all stages in the life cycle of Fibricola cratera.

Adult flukes are commonly found in the duodenum of raccoons (Procyon lotor). Dark amber, operculate eggs are voided with feces into water six to eight days after infection. Eggs hatch after nine to 33 days of incubation. Hatched miracidia penetrate the snail Physa gyrina. Within the snail, two generations of sporocysts are produced. Furcocercous cercariae emerge in 24 to 31 days from infected snails and penetrate tadpoles of several species, particularly Rana pipiens.

Metacercariae (diplostomula) develop in the peritoneal cavity of the tadpole. Upon metamorphosis of the tadpole, metacercariae migrate from the peritoneal cavity to the hind leg musculature, principally the gastrocnemius. Here, diplostomula become embedded intercellularly amoi^ the muscle fibers, and then undergo slight encapsulation with connective tissue produced by the host. In 30 to 35 days, encapsulated diplostomula are infective. When eaten by the definitive mammalian host, metacercariae rapidly develop into adult trematodes. Intrauterine eggs are observed six and one-half days post feeding. 27

MA TEEIAI5 AND METHODS

Maintenance of Hosts

Raccoons (Procyon lotor), used as definitive hosts for all experiments pre sented in this dissertation, included three females trapped at a very early age at

Lakeside Laboratory at Lake Okoboji, Iowa. Raccoons were kept in galvanized dog cages. Two raccoons served as the primary source of Fibricola cratera eggs used in the research. They were fed a ration of dogfood and table scraps; bedding below the cages was changed every 72 hours. All raccoons were given dog and cat distemper shots as a precautionary measure.

Laboratory-reared Physa gyrina, used as the first intermediate host in this study, were kept in ten or 20 gallon aerated aquaria and fed as needed with lettuce.

E3q)osed snails were maintained in aerated jars and, prior to the initial release of cercariae, each was isolated in a 4" culture dish and then observed daily for shed ding.

Laboratory-reared tadpoles (Rana pipiens) were obtained by using methods outlined by Rugh (1962) for rearing artificially fertilized eggs. Newly hatched tadr- poles were kept in flat enamel pans and were "aged" using the criteria published by Shumway (1940) and listed in Table 3. Naturally-infected frogs and tadpoles were collected from Jemmerson Sloi^ in Dickinson County, Iowa. Laboratory- reared and naturally infected tadpoles older than two weeks were maintained in

50 gallon aquaria filled with 20 liters of artificial lake water (Ulmer, 1970) 28

Table 3. Normal stages in the development of Rana pipiens embryos at 18° C (Adapted from Shumway, 1940)

Stage Number Age (Hours) Diagnostic Feature(s)

1 0 Unfertilized egg

2 1 Fertilized egg, grey crescent present

3 3.5 First cleavage

4 4.5 Second cleavage, four cells

5 5.7 Third cleavage, eight cells

6 6.5 Fourth cleavage, sixteen cells

7 7.5 Fifth cleavage, thirty-two cells

8 16 Early blastula

9 21 Late blastula

10 26 Early dorsal lip

11 34 Active gastrulation, yolk plug formed

12 42 Yolk plug disappears

13 50 Neural plate and groove

14 62 Neural folds

15 67 Neural groove and rotation

16 72 Neurala - neural tube closed

17 84 Early tail bud

18 96 Muscular response, length 4 mm

19 118 Initial heart beat, myotomes present, length 5 mm 29

Table 3. (Continued)

Stage Number Age (Hours) Diagnostic Feature(s)

20 140 Hatching, gill development, 6 mm long

21 162 Mouth open, cornea transparent, length, 7 mm

22 192 Tail fin circulation, length 8 mm

23 216 Opercular fold, teeth, length 9 mm

24 240 Operculum closed on right, length 10 mm

25 284 Operculum complete, length 11 mm 3(»

which was constantly aerated and changed every 48 hours. Tadpoles selected for exposure were kept in 4" culture dishes and one gallon aquaria filled with 200 ml and one liter of artificial lake water, respectively. Water was changed daily.

Tadpoles were fed canned spinach daily. Spinach was first rinsed with distilled

water to remove excess salt, then frozen and thawed as needed. Tadpoles under

going metamorphosis were aged (Table 4) according to stages outlined by Taylor

and Kollros (1946).

Adult frogs were housed in fish tanks of 50 or 100 gallon capacity filled with

40 liters (100 gallon tank) or 20 liters (50 gallon tank) of tap water to which 1.5 g

of sodium chloride and 0.16 g of calcium chloride per liter were added. This

dilute salt solution prevented "redleg" and other fungal or bacterial infections.

The calcium chloride prevented muscular spasms and other difficulties associated

with lab-reared frogs. Frogs were fed mealworm beatles (larvae and adults) or

pork liver dusted with "Sergeant's Vitapet Powder- (Miller-Morton Co., Richmond,

Va.) to insure all dietary needs.

Exposure and Infection of Hosts

Raccoons were exposed by feedii^ them two or three frog legs heavily in

fected with Fibricola cratera diplostomula. One week after exposure, feces were

checked for presence of trematode eggs. Sufficient egg production resulted if

hosts were fed frog legs every six to nine weeks. Table 4. Stages in the metamorphosis of Rana pipiens (Adapted from Taylor and Kollros, 1946)

Stage Number Age (Days) Length (mm) Diagnostic Features

I 5 12 Oral sucker absent, four rows of labial teeth present

n 7 15 Height of limb bud elevation equal to one-half its diameter

in 10 23 Length of limb bud equal to its diameter

rv 20 33 Length of limb bud equal to one and one-half times its diameter

V 23 40 Length of limb bud equal to twice its diameter; distal half of limb bud bent ventrad

VI 27 45 Limb bud flattened mediolaterally to form foot paddle

VII 30 50 4th and 5th toe proml nences separated by a slight indentation

Vin 35 55 Margin of foot paddle indented between toes 5-4

IX 37 57 Margin of foot paddle Indented between toes 5-4,4-3, and 3-3

X 39 59 Margin of foot paddle indented between all five toes

XI 42 61 5th toe web pointed toward tip of 2nd toe

XII 46 65 5th toe web pointed toward tip of 1st toe

Xm 52 68 5th toe web pointed toward prehallux Table 4. (Continued)

Stage Number Age (Days) Length (mm) Diagnostic Features

XIV 58 70 Pigment free patches appear at metatarsophalangeal joints

XV 62 72 Proximal toe pads appear at metatarsophalangeal joints

XVI 65 73 Middle toe pads appear

XVn 67 73 Distal toe pad appears at second interphalangeal joint on toe four

XVm 71 74 Cloacal tail piece disappears

XIX 72 72 Skin window of gill chamber clears

XX 75 70 One or both forelegs protruded

XXI 76 64 Angle of mouth midway between nostril and anterior margin of eye

XXn 79 45 Angle of mouth reaches middle of eye; tail shorter than ex tended hind limb

XXin 81 33 Angle of mouth reaches posterior margin of eye

xxrv 34 26 Stub of tail still remains

XXV 90 25 Tail completely resorbed 33

Eggs were separated from feces by standard sedimentation techniques.

Small portions of the washed fecal sample were examined under the dissecting microscope and eggs observed were transferred by pipette to a 1" culture dish.

E^s were incubated for nine days at 30° C at which time the culture dish was refrigerated at 5° C overnight. Eggs were then removed from the refrigerator, exposed to bright light for three to four hours and then checked for the presence of miracidia.

Each snail was exposed to three miracidia and checked daily for shedding

28 days after exposure.

Lab-reared, thiourea-treated, thyroxin-treated, and control tadpoles were exposed to cercariae pooled from several sheddii^ snails. Four to six tadpoles

were exposed simultaneously in one gallon aquaria filled with 250 ml of artificial

lake water containing approximately 500 cercariae. Embryonic tadpoles were ex

posed singly in 1" watch glasses filled with 1 ml of artificial lake water containing

35 cercariae.

Experiments involving tadpoles exposed only on the tail surface were conduct

ed by placing the body of the tadpole, anesthetized with tricane methanesulphonate

(Finquel Ayerst Laboratories, Inc.), in a water-filled rubber cup (Fig. 1) fashioned

from the tip of the finger of a surgical glove. The tadpole's tail, drawn through a

slit in the bottom of the cup, was then placed in a 1" culture dish containing approxi

mately 200 cercariae. After a 15 minute exposure, the tadpole was removed 34

from the culture dish, rinsed off to remove any cercariae that had not penetrated, and then examined later for evidence of metacercariae within the peritoneal cavity.

Alteration of Normal Rate of Frog Metamorphosis

Metamorphosis was blocked by immersing tadpoles previously exposed to cercariae in a solution of 0.033% thiourea in artificial lakewater. These tadpoles were maintained singly in 4" culture dishes; fresh thiourea solution was added daily.

Effects of accelerated metamorphosis were also studied. A stock solution of

thyroxin (1:100,000) was prepared by dissolving 10 ug of L-thyroxin (sodium) penta^ hydrate in 5 ml of 1% NaOH and adding this to one liter of distilled water. Subse

quent dilutions were made from this stock solution. Tadpoles selected for treat

ment were first exposed to cercariae, then immersed in artificial lake water con-

tainit^ increasing strengths of thyroxin over a period of four weeks. The initial

concentration of thyroxin was 1 ppb on day one. Every three days the concentration

was doubled so that after 27 days, the level of hormone reached 256 ppb, an amount

sufficient to complete metamorphosis.

Histological and Physiological Techniques

Embryonic tadpoles were fixed in Zenker's fixative, cleared in toluene,

stained with Harris' hemato:!qrlin and eosin or Mallory's triple connective tissue

stain, and sectioned at 12 u thickness to determine the presence of larvae. 35

Larval tadpoles (stages I-XV) were fixed in Bouin's fixative, cleared in toluene, stained with Harris' hemato^lin and eosin or Mallory's triple connective tissue stain, and sectioned at 15 u to determine the location of metacercariae.

Whole mounts of metacercariae removed from thiourea-treated tadpoles were prepared by fixing diplostomula in hot alcohol-formalin-acetic acid. Speci mens were stained with Mayer's paracarmine and fast green, then cleared with methyl salicylate.

Naturally-infected frogs and thyroxin^-treated frogs undergoing metamorpho sis were fixed in Bouin's fixative and Zenker's fixative, respectively; both were de calcified in Jenkin fluid, cleared in toluene, stained with Harris' hematojgrlin or

Mallory's triple connective tissue stain, and sectioned at 15 u to determine metacercarial location.

Standard Warburg respirometer apparatus was used to measure oxygen con

sumption of heavily infected versus non-infected gastrocnemius muscles. Each

muscle was removed from the tibio-fibula, weighed, placed in a reaction flask

with two ml of frog Ringer's solution, and incubated for one hour. 36

RESULTS AND DISCUSSION

Exposure of Embryonic Tadpoles to Fibricola cratera cercariae

Exposure of embryonic R. pipiens tadpoles to Fibricola cratera cercariae re vealed that only the late stages of embryonic development are susceptible to infec tion. Artificially fertilized pipiens embryonic tadpoles were e3q)osed to cercar iae through all 25 Shumway st^es (Table 5).

Cercariae were not stimulated to attach to nor did they make attempts to pene trate egg masses. When sii^le eggs were placed in 1" culture dishes, no response from the cercariae was noted. Furthermore, when the egg was removed from its gelatinous capsule, no response was noted. Experimental exposures of tadpoles from stages 1 throi^h 19 also proved negative.

Embryonic tadpoles hatohed at stage 20, about 132 hours after fertilization.

When each embryo was placed in a watch glass filled 1 ml of water containii^

35 cercariae from experimentally-infected, lab-reared snails, cercariae made no

response to the presence of the tadpole. Similar results occurred with stage 21,

about 150 hours after fertilization.

Cercariae appeared to show a slight attraction to the tadpole at stage 22 (180

hours). They often touched the body briefly, then swam away. One cercaria after

20 minutes of observation attached for several minutes, attempted unsuccessfully

to penetrate, and then swam off.

The embryonic integument is clearly a factor in preventing cercarial penetra

tion in tadpoles between stages 20 and 22. The integument consists of a basal layer Table 5. Exposure of Kana piplens embryos to 35 Fibrlcola cratera cercariae (embryos raised in artificial lake water at 21® C)

Average number of cercarial attachments No. Hosts Time After 'rime After Length after: Stage Exposed Fertilization Hatching (mm) 5 min. 10 min. 15 min. Total (hrs.) (hrs.)

1-19 2/stage 0-131 —— — ——— 0 0 0 0

20 3 132 0 6 0 0 0 0

21 3 150 18 7 0 0 0 0

22 3 180 47 8 0 0 0 1^

23 3 205 67 9 1.3 2 1.6 4.9

24 3 230 98 10 3 3 3.6 9.6

25 4 275 143 11 5.5 3.75 3.5 12.75

^One cercarial attachment noted after 20 minutes of observation. 38

(the true epidermis) and a ciliated peridermal layer (Witschi, 1956). Peridermal

cilia create constant water currents that not only sweep cercariae away from the

integument but also seem to induce cercariae to swim vigorously away when they

drift into the water current (Fig. 2).

At stî^e 23 (205 hours), an average of almost five cercarial attachments

were recorded in 15 minutes(Table 5 and Graph 1). This increase is apparently

related to the partial loss of cilia on the tadpole embryo body. As development

continues, cilia disappear except on the tail, the only area on which they are re

tained in stage 25 (Shumway, 1940). Although cercariae may attach and penetrate

(Figs. 4-7) at this stage, most are incapable of penetration. In such cercariae

the tail breaks off, the cercarial body moves about for several minutes on the tad

pole integument, and then falls off.

Hoffman (1955) stated that cercariae would not penetrate frog embryos

prior to hatching but readily penetrated tadpoles one day old. My experimental

data confirm the former but dispute the latter. Sections of tadpoles one day old

(i.e. 24 hours after hatching) fail to show any evidence of larvae. Two reasons

may account for the discrepancy. Firstly, Hoffman (1955) worked with three spe

cies of frogs: Rana pipiens, Pseudacris nigrita tricerata, and Rana clamitaas.

He did not state which species were used in these embryonic exposures. It is

possible that the latter two species affect cercarial penetration differently than

does Rana pipiens, and perhaps cercarial penetration occurs at an earlier time.

Secondly, embryonic development is influenced to some extent by temperature; Graph 1. Number of cercarial attachments recorded over a period of 15 minutes with three different stages of R. pipiens tadpole embryos. 15

4^ >C^' o

10 15

Stage 23 Stage 25—— 41

thus, if Hoffman's tadpoles were readed In an environment warmer than 21° C

(the temperature used in this study) development would be accelerated, accounting for the shorter time period. Because of the variability in chronological develop mental times, the use of Shumway stages is far more accurate. Shumway stages are delineated by the appearance of anatomical features that are used to index experimental exposures.

The rate of cercarial attachment rises sharply at stage 24 (230 hours). Cer- cariae have less difficulty in attaching to the tadpole and are now able to penetrate

(Table 5, Graph 1, and F%s. 8-11). By stage 25 (275 hours) cercariae appear to encounter no difficulty attaching to and penetrating the tadpole's integument (Figs.

12-14). Stage 25 marks the end of embryonic development in Rana pipiens. From this point the tadpole is considered to be a larva undergoing metamorphosis.

In conclusion, responsiveness of cercariae to the tadpole is apparently keyed to loss of the periderm (beginning at stage 23) and concurrent acquisition of the thicker larval integument. These processes may be associated with a rise in cercarial chemotactic response as the embryonic integument more and more comes to resemble the larval integument.

The transition point where typical cercarial attachment response begins is not distinct. Rather, a gradual increase in cercarial attachment and penetration occurs, beginning at stage 23 and increasing to a maximum at stage 25. After 42

stage 25, cercarial response does not differ from that described by Turner (1958) on older larval tadpoles.

Entry of Cercariae into the Body Cavity of Rana pipiens

Whole body exposures

Six lab-reared stage X tadpoles were exposed to Fibricola cratera cercariae

obtained from lab-reared, experimentally-infected snails at intervals of 15, 30,

60, 120, 240, and 260 minutes (Table 6). Sections of tadpoles revealed that larvae

usually did not move immediately to the peritoneal cavity. The majority remained

under the dermis and did not penetrate the peritoneum until after approximately

60 minutes (Fig. 15). Larvae were also noted under the pharyngeal integument

(Fig. 16). Almost all larvae were in the peritoneal cavity after 120 minutes

(Fig. 17). None are found under the pharyngeal integument after this time, ap

parently havii^ migrated into the peritoneal cavity.

The apparent time lag may be due to the amount of energy expended by larvae

penetrating the integument. Once through the int^ument, larvae must then break

through the body wall muscles and peritoneum to make their way to the body cavity.

A similar time lag, attributed to a "resting period" occurs in Schistosoma mansoni

cercariae while lying in the stratum comeum before penetration into deeper tissues

(Gordon and Griffiths, 1951). Another factor is the location of initial cercarial

penetration. Some cercariae, as previously noted, penetrate initially in the area 43

Table 6. Location of Fibricola cratera larvae in Rana pipiens larval tadpoles after exposure to cercariae

Time after Exposure (min.) 15 30 60 120 240 360

Underneath dermis of peritoneal cavity area

Underneath dermis of pharyngeal area 0 0

Within peritoneal cavity 2 10 10 10 44

of the pharynx and require some time to reach the body cavity. Penetration of cercariae in the tail (described in the following section) requires a more extensive

migration.

Tail surface exposures

Many cercariae attached to and penetrated the tail surface of Rana pipiens

tadpoles (Fig. 18). Experiments were conducted to determine if entry via the tail

resulted in subsequent development.

In a series of preliminary experiments, excised tadpole tails were placed in

water containing cercariae and then observed through a dissecting microscope.

Cercariae readily attached to and penetrated the excised tail. Their movements

through the translucent tail fin were observed and recorded at five-minute inter

vals for 20 minutes (Fig. 3). Study of these larvae terminated when they reached

the opaque area of tail muscles, which made further observations difficult. These

experiments establish that larvae do have the ability to move within the tail tissue

(Figs. 19-21).

In subsequent experiments, ten lab-reared stage X or XI tadpoles were

anesthetized and each tadpole had its posterior two-thirds of the tail surface ex

posed for 15 minutes to water containii^ a heavy concentration of cercariae ob

tained from lab-reared, experimentally-infected snails. Only three tadpoles sur

vived more than 24 hours following such exposure.

After four days, one of the surviving tadpoles was sacrificed and the con

tents of the body cavity flushed out. Four diplostomula were recovered. These 45

diplostomula were found crawling actively over the intestine. Since only the tail surface of the tadpole had been exposed, it is assumed larvae arrived there via migration through tail tissue.

One week after exposure, the two remaining tadpoles were sacrificed and sectioned. Examination of stained and sectioned material revealed the presence of metacercariae. In one tadpole, a metacercaria was found within the pericardial cavity; in the other tadpole, eight metacercariae were found between the coils of the intestine (Fig. 20) and one metacercaria within the pericardial cavity. Diplosto mula found in the pericardial cavity in both tadpoles were located between the heart muscle and the pericardial membrane.

Apparently not all larvae are successful in completing this migration from tail tissue to body cavity. An enormous amount of tissue must be traversed which undoubtedly consumes a great deal of energy. Even after m%rating this dis

tance (up to 40 mm or longer), larvae must then penetrate body wall muscles and

peritoneum. Many larvae wandered aimlessly in the tail fin fold tissue without any

migration anteriorly. These experiments indicate that some larvae are capable of

reaching the coelomic cavity by penetrating the tail, although easier access to this

site is afforded by cercarial penetration of the body proper.

Migration of Metacercariae in Metamorphosing Tadpoles

Metacercariae within tadpoles in the limb bud stages (I-V) are limited exclu

sively to the body cavity. No preference for location within the peritoneal cavily 46

could be demonstrated. In some heavily infected, lab-reared tadpoles, metacercariae were also observed within the pericardial cavity. The transverse septum separating these two cavities is incomplete in the tadpole (Shumway, 1940), thus providing access to the pericardial chamber. Results in paddle stages (VI-X) and in premetamorphic stages (XI-XVIU) were similar with reference to metacercarial location (Figs. 22-23).

Migration of diplostomula to the legs in naturally-infected tadpoles takes place during metamorphic stages (XVEI-XXV). Although the legs are well-formed at stages XX, XXI, and XXn, no metacercariae are found there. These legs are essentially fully developed with skeletal and muscular elements identical to those of postmetamorphic frogs (Witschi, 1956). The major anatomical difference is that of size. Legs of the aforementioned stages are somewhat smaller than those of postmetamorphic frogs, but could still easily support an influx of metacercariae.

The stimulatory mechanism signaling commencement of migration at stage

XXm (Table 7) is most likely the high hormonal and metabolic changes takLt^ place during metamorphosis. The level of thyroxin rises from 3 ppb to 250 ppb from

Stage XX to stage XXIV (Etkin, 1964). If metacercariae are directly influenced by

thyroxin as are frog tissues, it is interesting to speculate on mechanisms responsi

ble for metacercarial reaction to the rising level of hormone.

Two theories as to how frog tissue reacts to rising thyroxin levels have been

presented. Etkin's views (1935, 1955), which have gained wide acceptance, are Table 7. Location of Fibricola crater a metacercariae ia naturally-infected Rana tadpoles and adults. Percent^ age of observed metacercariae in parentheses

No. Hosts Total No. of Location of Metacercariae Stage Examined Metacercariae Body cavity Thigh Gastrocnemius

XX 2 41 41 (100) 0(0) 0(0)

XXI 2 39 39 (100) 0(0) 0(0) xxn 2 4(5 46 (100) 0 (0) 0(0) xxm 2 92 64 (70) 28 (30) 0(0) xxrv 2 56 30 (54) 26 (46) 0(0)

XXIV + 3 days 2 52 17 (33) 34 (66) 1(1)

XXV 3 66 6(10) 51 (77) 8(13)

XXV + 5 days 2 50 4(7.5) 33 (67) 13 (25.5)

XXV4- 10 days 5 195 11 (5) 120 (61) 64 (33)

XXV + 15 days 2 70 3(3) 34 (49) 33 (48)

XXV f 20 days 2 82 Ml) 30 (32) 51 (67)

XXV + 25 days 1 24 0(0) 3(13) 21 (87)

XXV+ 30 days 2 82 0(0) 0(0) 82 (100) 48

that hormonal concentration (i.e., a stoichiometric factor) determines the pattern of metamorphic events by controlling the speed of metamorphic incidents. Etkin

(1964) believes that each tissue is characterized by a specific total requirement of hormone to complete metamorphic change.

Kollros (1959, 1961) believes threshold levels in the tissue are affected by the increasii^ amount of thyroid hormone. Thus, increasing concentrations of thyroxin match the threshold level of the tissue resulting in metamorphosis.

Both concepts on the events of metamorphosis are based on continuously rising concentrations and the continuity of action of thyroxin. If metacercariae of

F. cratera are directly affected by thyroxin, migration becomes explainable in terms of sensitivity (threshold level) or reactivity (reaction rate) to thyroxin. Al though metacercariae are in the peritoneal cavity, not in direct contact with thy

roxin circulating in the blood, they could still be exposed to high hormone levels

likely to be present in peritoneal interstitial fluid. Water and solutes (containing

hormones) move from blood capillaries into peritoneal interstitial fluid (Deyrup,

196^ and are returned to the blood stream via blood capillaries or renal nephro-

stomes.

One must also consider that larvae may be affected only indirectly by thy

roxin. The diverse metabolic changes associated with metamorphosis may be

initiatir^ migration. Metamorphosis causes dramatic losses in body fluids and

organic matter. Body water decreases of 56 to 66% are common in metamor**

phosing anurans (Weber, 1967). Organic matter (carbohydrates), nitrogen and lipids) decreases 44 to 60% (Weber, 1967). These losses are due to intestinal . evacuation and the atrophy of larval structures such as the tail (Weber, 1967).

Such losses begin at stage XX and are at their peak during the climactic metamorphic stages XXm, XXIV, and XXV.

The phenomenon of tail resorption might also be implicated as a triggering mechanism. According to Aleschin (1926), tail tissue, upon resorption, under goes a dramatic increase in acidosis. His measurements show the pH for normal tissues is around 7.5, while that of rpressing tail tissue is 6.6. Blacher and

Liosner, (1932) reported a similar increase in proteolytic enzyme activiiy as indi cated by an 82% rise in free amino nitrogen. Metacercariae may be responding to the decreased pH or to the increased acid hydrolase activity.

Observations of freshly dissected, naturally-infected metamorphosing frogs revealed that many metacercariae were crawling in the vicinity of the tail stub.

Turner (1958) made a similar observation. Many metacercariae were also found localized in tail stub tissue (Table 8 and Figs. 30-31) and encapsulated in naturally- infected, post-metamorphic frogs. These diplostomula apparently become encap sulated earlier than their counterparts in the gastrocnemius. Di a fror at stage

XXIV + 3 days, for example, 43 (53%) of the observed diplostomula had encap sulated.

Following stage XXm, there is a consistent and steep decline (Graph 2 and

Table 7) in the number of metacercariae within the peritoneal cavity. The great bulk of these diplostomula have moved out of the body cavity by stage XXV. 50

Table 8. Fibricola cratera metacercariae found in the tail stub tissue of recently metamorphosed naturally-infected Rana pipiens. Percentage of observed metacercariae in parentheses.

Number of Metacercariae Number of Encapsulated Stage of Frog Observed Metacercariae Observed

XXrV + 3 days 81 43 (53)

XXV + 7 days 16 9 (58)

XXV + 9 days 47 32 (67)

XXV + 10 days 16 11 (68)

XXV + 12 days 44 35 (79)

XXV + 21 days 55 49 (88)

XXV + 24 days 108 102 (95) Graph 2. Metacercariae observed in the peritoneal cavity, thigh area, and area of gastrocnemius muscle of naturally-infected metamorphoriiig R. pipiens 100 / / / / V/

/ /\

i y >2 23 24 2540 25+20 ^5*30 Stage days days days In Peritoneal Cavity In Area of Thigh In Area of Gastrocnemius 53

Metacercariae are found in the area of the thigh musculature commencing at stage XXm (Fig. 24). Sectioned material of migrating diplostomula indicates that the majority travel between the fascia of the muscle and the basal layer of dermis.

Some, however, migrate between individual thigh muscle bundles (Figs. 25 and 26).

Such metacercariae may never reach the gastrocnemius but instead eventually be come encapsulated in the thigh musculature.

Migration through the thigh area reaches its peak at the beginning of stage

XXV (Graph 2 and Table 7). This coincides with the end of the largest influx of metacercariae from the peritoneal cavity. Numbers of diplostomula in the thigh region decline steadily after this peak as more and more larvae make their way to the gastrocnemius.

At stage XXIV + 3 days, a considerable number of migrating metacercariae occur in the thigh, but a few early arrivals are moving further into the gastro cnemius (Fig. 27). Stage XXV f 10 days marks the appearance of large numbers

of diplostomula in this muscle (Fig. 28). Numbers of metacercariae increase

steadily (Graph 2 and Table 7) in accordance with the decline in numbers observed

in the peritoneal cavity and thigh area. All metacercariae have localized in the

gastrocnemius by stage XXV + 30 days (Fig. 29).

It is interesting to speculate on what selective advantage miration must

have had in the evolution of this host^parasite relationship. When frogs are eaten

by raccoons, hind legs are the preferred part of the meal. This has been observed 54

in laboratory-infected hosts. The raccoon grasps the body of the frog so that the legs are eaten first. The skin is also stripped from the legs and from the tail stub region and ingested. Often the body of the frog and the viscera are discarded or only partially eaten. Evolutionary pressure, it seems, may have favored those metacercariae having migrated to the legs for this may ensure greater numbers of larvae capable of completing the life cycle.

Physiologically, the presence of metacercariae seems to have no effect on frog leg muscle. Although no experimental data are at hand, regular observation of field-collected frogs maintained in the laboratory indicated no detectable differ ence in the jumping ability of frogs later found to be heavily infected when com pared with non-infected specimens. Warburg respirometry revealed no detectable difference in oxygen consumption between similar ly sized gastrocnemius infected with heavy numbers of diplostomula and controls (Table 9) when incubated for one hour.

Effect of Blocked Metamorphosis on Localization of

Fibricola cratera Metacercariae

Fifty lab-reared pipiens larval tadpoles were e3q)0sed to cercariae ob

tained from lab-reared experimentally infected snails (P. gyrina). Tadpoles were

maintained in aquaria where water changed daily contained 0.033% thiourea to

block metamorphosis. These tadpoles, maintained from nine to 121 days,

were compared with untreated lab-reared controls over the 55

Table 9. Amount of oxygen consumed in one hour by gastrocnemius muscles from field-collected Rana pipiens. Muscles were incubated at 30° C in frog Ringer's solution. Naturally-infected muscles contained large numbers F. cratera metacercariae

Non-Infected We%ht mm^ Og/g Muscle No. or Infected (mg) hour

1 Non-infected 488 .040

2 Non-infected 450 .043

3 Non-infected 450 .061

4 Infected 468 .036

5 Lifected 470 .058 56

same period of time to ascertain any differences in subsequent localization of metacercariae.

Five control tadpoles progressed from stage I to stage XXV after a period of

96 days and had metamorphosed into adult frogs. Metacercariae in these frogs were m%rating and were found in the area of the gastrocnemius muscle (Table 10).

Only nine (7.5%) metacercariae remained in the peritoneal cavity of the frog at

stage XXV.

Thiourea-treated tadpoles, however, reached stags xn only after 96 days

(Table 10). All metacercariae recovered were found within the peritoneal cavity

(Fig. 47) crawling on the surface of the viscera. No evidence of migration nor of

metacercariae undergoing encapsulation could be discerned.

One thiourea-treated tadpole lived until metacercariae were 109 days old

and two additional hosts were maintained until their metacercariae were 114 days

old. In these tadpoles, metacercariae were frequently observed in areas other

than the peritoneal cavity (Graph 3). The pharyngeal musculature and connective

tissue and pericardial cavity of these hosts contained a far higher proportion of

diplostomula than were found in younger tadpoles (Figs. 48-51). No 109 day old

diplostcmula were encapsulated. However, many of the 114 day old metacercar

iae were encapsulated in pharyngeal musculature lying ventral to the chondo-

cranium and eye (F%. 50). Some encapsulated metacercariae were found loosely

attached to the parietal peritoneum (Fig. 49). 57

Table 10. Effect of blocked metamorphosis on the location of cratera metacercariae in H. pipiens larval tadpoles

Stage Number of Metacercariae Found in Peritoneal Host Age Thiourea Cavity (Days) Control Treated^ Control Thiourea Treated

77 XX X 52 (100)^ 46 (100)

84 xxn X 47 (100) 18 (100)

91 XXIV XI 18 ( 50) 29 (100)

96 XXV xn <î( 7.5) 35 (100)

^Treatment commenced when tadpole was seven days old, ^Per cent of total recovered is in parentheses. Graph 3. Location of observed Fibricola cratera metacercariae in e3q)erimentally- infected, thiourea^treated pipiens tadpoles. Black bar indicates tad poles that were 11-82 days old. White bar indicates tadpoles that were 116-121 days old 59

100 —

within within within encapsulated peritoneal pericardial pharyngeal cavity cavity connective and muscular tissue

"no. observed 60

Blocking metamorphosis with thiourea results in complete disruption of the normal localization behavior of Fibricola crater a metacercariae. This effect is associated with the removal of normal stimulation metacercariae need to begin their migration to the legs.

The anteriad migration of the 114 day old metacercariae was an entirely unexpected result. Reasons for such a shift in location are as yet unknown. A

rise in thyroxin level, as a stimulus, is clearly not indicated.

Growth of Metacercariae in Thiourea-Treated Tadpoles

Measurements were taken of metacercariae flushed from the peritoneal cavitv

of thiourea^treated tadpoles. These larvae range in age from two to 114 days

old (Table 11 and F%s. 32-42).

Metacercariae grow with steady increases in size until 96 days. The great

est increase of body lei^th and width occurs between two and 21 days of age

(Graph 4). The oral sucker and acetabulum show more moderate growth (Graphs

5 and 6) and seem to reach essentially maximum size in approximately 63 days.

The holdfast shows continuous growth through 96 days (Graph 7). The genital

primordium does not change significantly in size after 28 days (Table 11). One

hundred fourteen day old metacercariae showed unexplainable declines in body

length and width, oral sucker and acetabulum size, and holdfast width.

Comparative measurements of ten 96 day old metacercariae removed from

a control frog at stage XXV in the area of the gastrocnemius and ten 96 Table 11. Comparative measurements of Flbricola cratera metacercarlae removed from thiourea-treated tad poles. Measurements are basecf on ten specimens expressed as means and range in parentheses

Age Measurement 2 days 11 days 14 days

Body length 159^ (146.5-173.2) 239.5 (255-315) 328.5 (305-360) Body width 42 (40.0-44.4) 188.5 (170-205) 192.0 (150-220)

Oral sucker length 28.3 (24.4-31.1) 38.8 (33.3-42.2) 40.4 (37.7-44.4) Oral sucker width 28.9 (22.2-32.2) 36.9 (26.6-42.2) 40.5 (35.5-44.4)

Acetabulum length 22.2 (18.9-25.0) 31.5 (28.9-42.2) 35.5 (28.9-59.9) Acetabulum width 22.2 (18.9-31.1) 34.2 (31.1-40.0) 36.4 (33.3-40.0)

Holdfast length — 44.9 (35.5-55.5) 55.7 (40.0-68.8) Holdfast width 42.1 (35.5-49.9) 47.4 (37.7-55.5)

Genital primordium length 21.3 (15.4-26.6) 24.6 (22.2-31.1)

Genital primordium width — — 16.2 (13.3-26.6) 19.3 (13,3-26.6)

Distance anterior end to 84.4 (75.5-102.1) 153.3 (125-178) 176.5 (150-200) acetabulum

Distance anterior end to 47.4 (44.4-53.3) 62.4 (48-77.7) 77.1 (60-120) end of esophagus

^ Microns. Table 11. (Continued)

Age Measurement 21 days 28 days 42 days

Body length 357.5 (285-420) 365 (425-405 388 (340-440) Body width 358.5 (230-300) 248.5 (240-280) 306 (260-350)

Oral sucker length 45.9 (37.7-51.1) 47.9 (44.4-51.1) 47.1 (42.2-51.1) Oral sucker width 40.2 (33.3-48.8) 42.6 (37.7-46.6) 46.4 (44.4-50.0)

Acetabulum length 41.3 (35.5-46.6) 40.0 (37.7-44.4) 45.3 (37.7-55.5) Acetabulum width 42.9 (37.7-50.0) 44.2 (40.0-46.6) 49.5 (44.4-59.9)

Holdfast length 57.3 (51.1-66.6) 59.7 (44.4-71.0) 63.9 (57.7-68.8) Holdfast width 52.2 (44.4-60.0) 51.7 (44.4-62.2) 54.6 (48.8-59.9)

Genital primordium length 25.3 (20.0-33.3) 26.6 (20.0-33.3) 29.3 (26.6-33.3) Genital primordium width 24.5 (17.8-33.3) 26.4 (15.5-35.5) 26.6 (22.2-31.1)

Distance anterior end to 172.5 (120-240) 207.5 (170-230) 204.2 (180-230) acetabulum

Distance anterior end to 89.3 ( 50-120) 123.0 (100-150) 131.4 (117-140) end of esophagus Table 11. (Continue*^

Age Measurement 56 days 63 days 82 days

Body length 411.5 (360-480) 426 (360-490) 453 (400-490) Body width 282.5 (260-310) 328 (260-410) 327 (280-360)

Oral sucker length 50.5 (46.6-51.1) 53.9 (44.4^57.7/ 55.7 (51.0-57.0) Oral sucker width 46.7 (43.3-55.5) 47.5 (42.2-53.3) 49.5 (46.6-53.3)

Acetabulum length 44.3 (41.1-48.8) 42.6 (37.7-48.8) 49.7 (44.4^-53.3) Acetabulum width 47.9 (44.4-48.8) 48.4 (44.4-53.3) 50.4 (42.2-55.5)

Holdfast length 70.5 (55.5-77.7) 69.7 (59.9-79.9) 82.8-77.7-91.0) Holdfast width 54.8 (46.6-66.6) 62.8 (57.7-71.0) 62.7 (51.0-66.0)

Genital primordium leng;th 27.7 (24./t-33.3) 31.1 (26.6-37.7) 29.7 (26.6-37.7) Genital primordium width 30.4 (24./t-35.5) 41.3 (31.1-55.5) 37.5 (31.1-44.4)

Distance anterior end to 222.5 (170-270) 229.5 (180-290) 218 (180-250) acetabulum

Distance anterior end to 127.5 (110-150) 117.5 (100-140) 124 (105-140) end of esophagus Table 11. (Continued)

Age Measurement 96 days 114 days

Body length 457 (410-495) 421.5 (360-560) Body width 361,5 (315-400) 283.0 (250-350)

Oral sucker length 53.5 (48.8-60.0) 51.1 (48.8-55.5) Oral sucker width 48.0 (42.2-53.3) 43.2 (36.6-48.8)

Acetabulum length 48.0 (37.7-55.5) 41.2 (28.8-48.8) Acetabulum width 53.0 (42.2-62.2) 46.9 (37.7-57.7)

Holdfast length 72.3 (66.6-79.9) 71.8 (64.4-79.9) Holdfast width 60.1 (55.5-64.0) 55.2 (48.8-64.4)

Genital primordium length 28.4 (25.5-37.9) 27.4 (22.2-35.5) Genital primordium width 39.3 (26.6-48.1) 32.7 (26.6-40.0)

Distance anterior end to 223.5 (190-270) 217.0 (160-260) acetabulum

Distance anterior end to 125.0 (100-150) 131.5 (100-160) end of esophagus Graph 4. Increase in body lei^h (solid line) and width (dashed line) in metacercariae removed from experimentally-infected, thiourea-treated • pipiens tadpoles. Measurements are based on the average of ten speci mens in each age group 66

500

400

M C O 300 V à

200

100 2 11 14 21 28 42 56 63 82 114

Age in Days Graph 5. Increase in oral sucker length (solid line) and width (dashed line) in metacercariae removed from experimentally-infected, thiourea-treated R. pipiens tadpoles. Measurements are based on the average of ten specimens in each age group 68 Graph 6. Increase in acetabulum lençch (solid line) and width (dashed line) in metacercariae removed fvom experimentally-infected, thiourea-treated R. pipiens tadpoles. Measurements are based on the average of ten specimens in each age group 70

11 21 42 63 96 Age in Days Graph 7. Increase in holdfast length (solid line) and width (dashed line) from ex perimentally- infected, thiourea-treated R. pipiens tadpoles. Measure ments are based on the averse of ten specimens in each age group 72 73

day old metacercariae from a thiourea-treated tadpole at stage XI (Table 12, Fig.

42) revealed that the former is somewhat larger in some respects but not signifi cantly so. These measurements indicate that metacercariae reach the same size in thiourea^treated or naturally-metamorphosing hosts.

Effect of Accelerated Metamorphosis on Localization

of Fibricola cratera metacercariae

Five laboratory-reared stage VI tadpoles were treated with increasir^ doses

of thyroxin (0-256 ppb) to accelerate metamorphosis after exposure to Fibricola

cratera cercariae obtained from laboratory-reared experimentally infected snails.

Such tadpoles were compared with untreated controls to detect differences in local

ization of metacercariae. The time period involved in the thyroxin treatment was

38 days.

The control group of tadpoles, after 38 days, were only at stage X. Meta^

cercariae were located within the peritoneal cavity of these hosts (Table 13).

In the thyroxin-treated group, however, metacercariae started to migrate

into the thigh area after 31 days (Table 13). In one frog at stage XXII, a single

metacercaria was found in the area of the gastrocnemius (Figs. 49 and 50). At 38

days, two frogs had reached stage XXIV of metamorphosis. Only 46 (34%) of meta^

cercariae remained within the peritoneal cavity, 68 (50%) having migrated to the

thigh area, and 22 (16%) having reached the gastrocnemius in these frogs. No en

capsulated diplostomula were found. Table 12. Comparative measurements of Fibricola cratera metacercariae. Measurements are of 96 day old control metacercariae versus 96 day old metacercariae removed from a thiourea-treated tadpole. Measurements based on ten specimens and are in microns expressed as means and range in parentheses

Source of Metacercariae Measurement "Itiourea-treated tadpole Control frog

Body length 457 (410-495) 469 (380-530) Body width 361.5 (315-400) 301 (260-385)

Oral sucker length 53.5 (48.8-60.0) 40.5 (36.6-51.1) Oral sucker width 48.0 (42.2-53.3) 42.8 (35.5-51.1)

Acetabulum length 48.0 (37.7-55.5) 40.6 (26.6-46.6) Acetabulum width 53.0 (42.2-62.2) 46.3 (42.2-53.3)

Holdfast length 72.3 (66.6-79.9) 75.0 (62.2-85.0) Holdfast width 60.1 (55.5-64.0) 61.1 (51.1-75.5)

Genital primordium length 28.4 (25.5-37.9) 31.8 (24.4-40.0) Genital primordium width 39.3 (26.6-48.1) 28.2 (23.3-31.1)

Distance anterior end to 223.5 (190-270) 252.0 (200-290) acetabulum

Distance anterior end to 125.0 (100-150) 120.0 ( 90-140) end of esophagus Table 13. Effect of accelerated metamorphosis on the migration of Fibricola cratera metacercariae

Stage Location of Metacercariae

Hiyroxin Body cavity Thigh musculature Gastrocnemius Control Treated Control Treated Control Treated Control Treated

VI (31)^ XXn (31) 65 (100)^ 78 (81) 0(0) 6(19) 0(0) 0(0)

VII (34) XXn (34) 66 (100) 119 (83.2) 0(0) 23 (16) 0(0) 1 (0. 8)

Vn (33) XXin (33) 59 (100) 45 (64) 0(0) 25 (36) 0(0) 0(0)

X (38) XXIV (38) 52 (100) 24 (33.3) 0(0) 36 (50) 0(0) 12 (16.7)

X (38) XXIV (38) 43 (100) 22 (34.6) 0(0) 32 (50) 0(0) 10 (15.4)

^Figures in parentheses represent tlie age in days from hatching. Figures denote number and per cent ( ) metacercariae observed. 76

Acceleration of metamorphosis by thyroxin results in precocious metacercarial migration resulting in a change in their location from peritoneal cavity to the region of the thigh and lower leg (Figs. 53-56).

The experimentally-induced frog metamorphosis does not appear to differ physiologically or morphologically from that of normal metamorphosis; only the chronological sequence of metamorphic events is compressed into a much shorter time period.

Metacercariae are apparently responding to the same set of triggers that would normally stimulate migration at about three months, ^th accelerated meta^ morphosis, metacercariae follow the same localization pattern as older meta cercariae albeit two months earlier than normal.

Examination of sectioned 31 to 38 day old thyroxin-treated frogs revealed metacercariae in the pericardial cavity (Fig. 55) unattached to either heart muscle or parietal pericardium. The transverse septum, incomplete in the tadr- pole, closed upon metamorphosis (Shumway, 1940). Apparently these diplostomula were unable to leave the pericardial cavity to begin their migration to the gas trocnemius.

Unfortunately, none of the thyroxin-treated frogs lived long enough for meta cercariae to complete their migration. Although measurements of ten metacercar iae in situ (Table 14) indicate that they were approximately 60 per cent of con trols, data were not obtained as to their ability to encapsulate or become infective 77

Table 14. Measurements of Fibricola cratera metacercariae located within thy roxin-treated frogs 31 to 38 days old. Measurements in microns are based on ten specimens expressed as a means and range in parentheses

Measurement Mean and Range

Body length 312.5 (270-390) Body width 226 (180-260)

Oral sucker lei^h 40.2 (36.6-44.4) Oral sucker width 43.5 (37.7-48.8)

Acetabulum length 29.5 (26.6-31.1) Acetabulum width 32.2 (26.6-37.7)

Holdfast length 55.5 (45.5-66.6) Holdfast width 44.3 (38.8-48.8)

Genital primordium length 31.1 (28.8-34.4) Genital primordium width 26.6 (22.2-28.8)

Distance anterior end to 162.3 (120-200) acetabulum 78

to the definitive host. Normally, metacercariae require a 30 day period after en capsulation to become infective.

These experiments involving thiourea- and thyroxin-treated tadpoles show the profound effects exerted by metamorphosis upon localization of cratera meta cercariae. How such a migratory pattern became established may never be known, but certain speculations based on recent theories of parasite localization may be cited. Szidat's (1969) "engram theory" would explain such migration as a result of generations of metacercariae having genetically acquired a type of "memory" relative to their localization within the frog host. Usii^ Holmes' (1973) theory of niche specialization, one m%ht envision competition between cratera metacer cariae and other parasites for available sites within the host. Over long periods of time, this competition may have resulted in F. cratera's becoming limited to the peritoneal cavity of the tadpole and the gastrocnemius of the adult frog. However, there appears to be little evidence that such competition has occurred, if one ac cepts Croll's (1976) theory which interprets localization as being associated with the most suitable habitat meeting the needs of the parasite, F. cratera metacer cariae could be considered as responding to a shift from the peritoneal cavity to a more favorable habitat of voluntary muscle. Thus, the peritoneal cavity is suitable during the larval tadpole stage of the frog, but upon metamorphosis, certain host- produced environmental factors provide the stimulatory mechanism(s) resulting in metacercarial localization in the more favorable area of the gastrocnemius. Such 79

a stimulatory mechanism or mechanisms appear to be provided by the series of complex physiological changes occurring in metamorphosis. Definitive data on these intriguing problems, however, must await development of suitable culture techniques of the post-cercarial stages of F. cratera. 80

SUMMARY AND CONCLUSIONS

1. Localization of Fibricola cratera metacercariae in laboratory-reared and

naturally-infected Rana pipiens was investigated.

2. The ability of R. pipiens embryos to harbor F. cratera metacercariae (diplo-

stomuls) was examined usit^ standard Shumway stages to index embryonic age.

3. Tadpole embryos are refractive to infection from stages 1-22. Cercariae are

slightly attracted to stage 22 embryos, but no penetration occurs.

4. The embryonic ciliated periderm prevents cercarial penetration from stage 20

(hatching) until stage 23 by creating constant water currents that keep cercar

iae away from the integument.

5. Stage 23 is the earliest embryonic stage at which cercarial penetration may

occur. By stage 25 (end of embryonic development), cercariae appear to en

counter no difficulty attaching to and penetrating the integument.

6. Cercarial responsiveness to the tadpole is apparently keyed to the loss of

ciliated periderm (beginning at stage 23) and the acquisition of larval epider

mis.

7. Cercariae, when exposed to lab-reared tadpoles, do not move immediately to

the peritoneal cavity. Most remain under the dermis for approximately 60

minutes. Almost all larvae are in the peritoneal cavity after 120

minutes.

8. It is suggested that this time lag may be due to larvae restit^ after the energy

expenditure of penetrating the integument, before breaking through body wall 81

and peritoneum. Some cercariae penetrate sites other than over the peritoneal

cavity and must require some time to reach the abdominal cavity.

9. Cercariae readily penetrate the tail surface of tadpoles. Three ^ pipiens tad

poles, experimentally exposed on only the posterior two-thirds of the tail sur

face, were subsequently shown to have metacercariae in the peritoneal cavity.

10, The migratory route of metacercariae was observed ia naturally-infected

metamorphosing frogs, using Taylor and Kollros stages to correlate progress

of metacercariae with morphological events of the frog.

11, Metacercariae are limited to the peritoneal cavity until stage XXm, at which

stage they begin their migration to the gastrocnemius.

12, The stimulatory mechanism is most likely the high hormonal or metabolic

changes taking place during metamorphosis.

13, Metacercariae are frequently found encapsulated in the tail stub of naturally

infected frogs. It is suggested that the mechanism of tail resorption might

also be implicated as a stimulatory mechanism for diplostomular migration.

14, By stage XXV, the great bulk of dlplostomula have moved out of the peritoneal

cavity, mlgratir^ thence through the thigh area, between the basal layer of

dermis and muscle fascia. A few dlplostomula migrate between thi^ muscle

bundles.

15, At stage XXV + 10 days, large numbers of dlplostomula are in the gastro

cnemius. Their number increases steadily in accordance with their decline

observed in the peritoneal cavity and thigh area. 82

16. Warburg respirometry revealed no difference in oxygen oonsuraption between

infected and non-infected gastrocnemius muscles.

17. Blocking host metamorphosis with thiourea causes complete disruption of

normal localization, presumably because of the removal of normal triggers.

Metacercariae remain within the peritoneal cavity lor^er and in greater num

bers than do those in controls. Metacercariae 114 days old migrated and en

capsulated in pharyi^eal musculature and connective tissue below the chondro-

cranium and eye for reasons as yet unknown.

18. Metacercariae, removed from thiourea^treated tadpoles, grow with steady

increases in size until 96 days of age. Greatest growth occurs between two

and 21 days of age. MeLacorcariiio 114 days old showed une?qf)lainable de

clines in body length and width, oral sucker, and acetabulum size, and hold

fast width. 18. Accelerated metamorphosis causes precocious migration of metacercariae by

31 to 38 days, which is approximately 60 days earlier than normal. Some

metacercariae become entrapped in the pericardial cavity when transverse

septum closes.

20. It is concluded that although certain current theories of parasite localization

may explain the migration of F. cratera metacercariae, namely, an ancestral

memory engram, niche specialization within the host, or habitat preference

wiûûn the host, definitive data will require the development of suitable culture

techniques of post^cercarial stages. 83

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ACKNOWLEDGEMENTS

The author wishes to deeply thank Dr. Martin J. Ulmer for his unselfish help, advice, and patience given throughout the course of this investigation.

Sincere appreciation is extended to my wife Kathleen for her love and under standing which were vital to the successful completion of this dissertation.

Thanks are also due to the Graduate College and Science and Humanities Re search Institute for providing funds for this investigation. 97

PLATES

Abbreviations

A - Acetabulum

AT - Atrium

BP - Body wall musculature and parietal peritoneum

C - Cercaria

CC - Cranial cavity

CD - Culture dish

D - Disc

DC - Diencephalon

DM - Dermis

E - Eye

EP - Epidermis

ES - Esophagus

F - Femur

G - Gill

GA - Gastrocnemius muscle

GP - Genital primordium

GS - Gill sac

HB - Hindbody

HF - Holdfast

I - Intestine 98

IC - Intestinal crura

IE - Inner ear

IL - nio-fibularis muscle

IS - Ischium

IT - Integument

L - Liver

M - Metacercaria

MG - Mucous gland

N - Notochord

O - Oral sucker

P - Pharynx

PC - Peritoneal cavity

PE - Pericardial cavity

PN - Pronephros

PP - Parietal peritoneum

PR - Peroneus muscle

PT - Premetamorphic tadpole

RC - Rubber cap

S - Spinal cord

SE - Semimembranosus muscle

SF - Segmental tail fin muscle

SM - Segmental muscle ST - Stomach

TE - Temporalis muscle

TF - Triceps femoris

TFN- TaU fin

TI - Tibio-fibula

TS - Transverse septum

V - Ventricle

W - Water y - Yolk PLATE I

Fig. 1. Apparatus for exposing the tail surface of Rana pipiens tadpoles. A premetamorphic tadpole (PT) is placed in a rubber cup (RC) containing arti ficial lake water (W). The cup is supported by a plastic disc (D) resting atop a 1" culture dish (CD) containing cercariae (C). 101

î PLATE n

Fig. 2. Diagram illustrating water currents (WC) generated by peridermal cilia in embryonic tadpoles.

Fig. 3. Migratory movements of Fibricola cratera larvae after penetration of an excised tail of Rana pipiens tadpole over a 20 minute period. Black cir cles represent sites of initial penetration. Each white circle represents position of larva 5 minutes later. 103 PLATE ni

Figs. 4-7. Infections of embryonic ^ pipiens tadpoles at stage 23.

Fig. 4. Stage 23 embryo.

Fig. 5. Metacercaria (arrow) in peritoneal cavity of sagittally-sectioned stage 23 embryo.

Fig. 6. As above, but higher power.

Fig. 7. Penetration of cercarla into embryonic integument near body-taU junction. 105 PLATE IV

Figs. 8-11. Infection of embryonic R. pipiens tadpoles at stage 24.

Fig. 8. Frontal section of a stage 24 embryo showing presence of metacercaria (arrow) within peritoneal cavity.

Fig. 9. As above.

Fig. 10. As above, same scale as showii in Fig. 9.

Fig. 11. Higher magnification of Fig. 10. Note oral sucker and acetabulum. 107 PLATE V

Figs. 12 - 14. Infection of embryonic R. pipiens tadpoles at stage 25.

Fig. 12. Stage 25 embryo.

Fig. 13. Metacercaria (arrow) underneath integument.

Fig. 14. Metacercaria (arrow) within peritoneal cavity. 109 PLATE VI

Figs. 15 - 17. Infections of larval R. pipiens tadpoles involving whole body exposures.

Fig. 15. 15 minutes after exposure. Note metacercaria lying between dermis and body wall musculature.

Fig. 16. 30 minutes after exposure. Metacercaria located beneath pharyngeal integument. Frontal section.

Fig. 17. Two hours after exposure. Metacercariae (arrows) within peri toneal cavity. Frontal section. Ill PLATE Vn

Fig. 18. Penetration of Fibricola cratera cercariae into a larval st%e X Rana pipiens tadpole. Note that many cercariae penetrate the tail as well as the body proper. 0.2mm PLATE Vm

. 19-21. Infections of larval tadpoles involving exposure of tail surface to cercariae.

Fig. 19. Living metacercariae in the tail fin of Rana pipiens photographed through a dissecting micro scope, lateral view.

Fig. 20. Metacercaria within peritoneal cavity of tadpole having had only tail surface exposed, one week post-exposure.

Fig. 21. Metacercaria in the tail fin, 15 minutes post-exposure to a stage X larval tadpole. 115 PLATE DC

Figs. 22 - 23. Metacercariae in larval ^ plpiens tadpoles.

Fig. 22. Metacercariae within peritoneal cavity of a 35 day old stage vn laboratory-reared tadpole.

Fig. 23. Metacercariae within the poritoneal cavity of an 80 day old stage XXI laboratory-reared tadpole. Same scale as shown in Fig. 22.

Figs. 24 - 25. Metacercariae In naturally-infected metamorphosing frogs.

Fig. 24. Stage XXEII. Metacercariae in region of the triceps femoris muscle.

Fig. 25. Stage XXIV. Frontal section showing metacercariae (arrows) entering area of thigh musculature. Same scale as shown in Fig. 22. 117 PLATE X

Figs. 26 - 28. Migration ot metacercariae in naturally-infected metamorphosing Rana pipiens.

Fig. 26. St£^ XXIV. Metacercaria located deep within ilio-fibularis muscle.

Fig. 27. Stage XXIV + 3 days. Metacercaria (arrow) entering area of gas trocnemius. Sagittal section.

Fig. 28. Stage XXV + 10 days. Metacercaria on surface of gastrocnemius muscle. Same scale as shown in Fig. 26. 119 PLATE XI

Figs. 29 - 31. Encapsulation of F. cratera metacercariae within naturally-infect ed Rana pipiens adult frogs.

Fig. 29. Stage XXV + 30 days. Metacercariae encapsulated in gastrocnem ius muscle.

Fig. 30. Stage XXV. Metacercariae encapsulated in the tail stub tissue. Frontal section, same scale as shown in Fig. 29.

Fig. 31. As above, higher magnification. 121 PLATE Xn

Figs. 32 - 37. Growth of metacercariae (dlplostomula) removed from laboratory- reared thiourea-treated tadpoles. All figures in same scale as shown in Fig. 32.

Fig. 32. Two-day old metacercaria.

Fig. 33. 11 days old.

Fig. 34. 14 days old.

Fig. 35. 21 days old.

Fig. 36. 28 days old.

Fig. 37. 42 days old. S2I PLATE Xm

Figs. 38 - 42. Metacercariae removed from laboratory-reared, thiourea-treated tadpoles. All figures to same scale as shown in Fig. 39.

Fig. 38. 56 day old metacercaria.

Fig. 39. 63-days old.

Fig. 40. 82 days old.

Fig. 41. 96 days old.

Fig. 42. 114 days old.

Fig. 43. 96 days old. From area of gastrocnemius in untreated control under going metamorphosis. 125 PLATE XIV

Figs. 44- 47. Metacercariae within laboratory-reared, thiourea-treated tadpoles.

Fig. 44. 23-day old metacercaria witliin peritoneal cavity of a 35-day old tadpole at stage V.

Fig. 45. 63-day old metacercariae within peritoneal cavity of a 70-day old tadpole at stage IX. Same scale as shown In Fig. 44.

Fig. 46. 92-day old metacercariae within peritoneal cavity of an 89-day old tadpole at stage X.

Fig. 47. 96-day old metacercariae within peritoneal cavity of a 105-day old tadpole at stage Xn. Same scale as shown in Fig. 44.

PLATE XV

Figs. 48 - 51. Metacercariae within laboratory-reared, thiourea-treated tadpoles.

Fig. 48. 109-day old metacercariae within pericardial cavity of a 1'16-day old tadpole.

Fig. 49. 114-day old metacercaria (arrow) located beneath parietal peritoneum of a 121-day old tadpole. Same scale as shown in Fig. 48.

Fig. 50. 114-day old metacercariae encapsulated in pharyngeal area in a 121-day old tadpole. Sagittal section. Same scale as shown in Fig. 48.

Fig. 51. As above, higher magnification.

PLATE XVI

Figs. 52 - 54. Precocious migration of metacercariae in laboratory-reared, thyroxin-treated R. pipiens.

F%. 52. 31-day old frog at stage XXn photographed from life. Note small size for metamorphic stage. Normal size would be 2" or more in length.

Fig. 53. Metacercariae in peritoneal caviiy and thigh musculature of 33-day old frog at stage XXm, sagittal section.

Fig. 54. Metacercariae (arrows) in a 33-day old stage XXm frog, frontal section. Same scale as shown in Fig. 53. ImcHES PLATE XVn

Figs. 55 - 56. Precocious migration of metacercariae in laboratory-reared, thyroxin-treated R. pipiens.

Fig. 55. Metacercariae within pericardial cavity of a 38-day old frog at stage XXEV, oblique section.

Fig. 56. Metacercaria (arrow) in area of gastrocnemius, 38-day old stage XXIV frog. Sagittal section. 133