SURIAYA MOGHAL M. Sc

STUDIES ON LUNG FLUKES OF RANA SPP.

A thesis submitted for the Degree of Master of Philosophy of the University of London and for the Diploma of Membership of the Imperial College.

October, 1979

Department of Zoology and Applied Entomology, Imperial College of Science and Technology, South Kensington, London, SW 7. 2

ABSTRACT

Miss Suriaya Moghal. "Studies on lung flukes of Rana spp" Studies were made on (1) the life cycle of Haematoloechus similis and H. variegatus from Rana ridibunda (2) Egg hatching in vitro of a, Haplometra cylindracea (from Rana temporaria) b, Haema- toloechus spp from Rana pipiens and R. ridibunda (3) In vitro culture of lung of Rana temporaria and R. pipiens. The development of Haematoloechus similis and H. variegatus in their intermediate hosts was investigated. Planorbis planorbis was found to serve as the first intermediate host for both these species. The second intermediate host Coenagrion puella was exposed to cercariae of Haematoloechus similis and 1 - 2 unencysted metacercariae were found free in the haemocoele on the following day. Coenagrion puella was also exposed to the cercariae of H. variegatus but no penetration was observed, again 1 - 2 metacercarial cysts were found. Out of 10 nymphs unexposed to cercariae which were used as controls, one was positive. Hence an experimental infection was not unequivocally demonstrated. The complete life cycle of Haematoloechus similis and H. variegatus in the U.K. thus remains undetermined. Attempts were made to obtain experimental infections of Haplometra cylindracea in snails of various species of laboratory bred snails such as Physa sp, Planorbis planorbis, Lymnaea peregra, L. stagnalis, L. palustris and L. truncatula. All these attempts were unsuccessful. It was, therefore, decided to attempt to hatch the eggs in vitro. The effects of different factors on the eggs of Haplometra cylindracea in vitro, such as osmotic pressure, hydrogen ion concentration, digestive enzymes, temperature, gas - 3 - phase, reducing agents, bile salts, NaOCland various other factors were examined. All attempts to hatch the eggs outside the host were unsuccessful but hatching took place in NaOC1, but only non-motile miracidia were observed since NaOClis toxic to miracidia. Eggs of Haematoloechus spp. hatched when placed in "snail conditioned water" (using Planorbis Planorbis) suggesting that some factor or factors which induce hatching, such as an enzyme, are released from the snail. Attempts were made to culture the lung tissue of Rana temporaria and R. pipiens in vitro in the hope that this might make possible culture of trematodes within cultured lung tissue. The maximum time of survival of this tissue culture was 114 days. 4

ACKNOWLEDGEMENT

It is my first duty to express my sincerest thanks to my supervisor, Professor J. D. Smyth, for introducing me into such an interesting field, for his encouragement, continuous help and patience which have been priceless throughout my work and the preparation of my thesis. My sincere thanks to Dr D. P. McManus and Dr C. J. Davies for helpful advice and encouragement throughout the course of my work. Thanks are also due to Dr Heather Paterson and Mrs Nicola Barrett for their advice and suggestions. Special thanks to Mrs G. Adams for her advice and encouragement during my stay. I would like to thank Mrs A. Meredith for her care in typing the manuscript. I am very grateful to Mr C. L. Meredith, Mr I. J. French, Mr J. Harmer, Miss S.R. Harper, Mrs A. Merry, Mr R. Krafft, Mrs I. Gadd, Mrs B. Stronach, Mr S. V. Stammers and others for help in various ways. I am grateful to all my colleagues and friends. Thanks are due to Dr Paul Sterry, Dr S. Cole, Mrs J. Yeadon and other librarian staff. The work was supported by a central training overseas scheme of the Government of Pakistan. I am grateful to the Government of Pakistan for awarding me the scholarship for this re search. Finally, my gratitude is due to my parents, M.A. Moghal and Dr Sanjida Moghal for their help and moral support. 5

CONTENTS

Page No.

Title Page ...... 1

Abstract ...... 2

Acknowledgement ...... 4

Contents ...... 5

List of Figures ...... 8

List of Plates . , . . • ...... 10

List of Tables ...... 12

General Introduction • ••• •• • ••• ••• •.. 16

Part I The life cycle of Haematoloechus similis

and H. variegatus ••• ••• ••• ••• ••• ••• ••• 18

Introduction ••• ••• ••• ••• ••• ••• ••• ••• ,.. 19

Materials and Methods ••• ••• •• • • .. ••• ••• 36 Results of the examination of Planorbis planorbis exposed to infection of Haematoloechus egg. ... . •• 63 Discussion ...... 99 Summary . .•• ••• 112 - 6

Page No.

Part II

1. In vitro hatching of the eggs of 114 Haplometra cylindracea (R. temporaria)

2. In vitro hatching of the eggs of Haematoloechus (R. pipiens)

3. In vitro hatching of the eggs of Haematoloechus (R. ridibunda)

Introduction ••• ••• ••• ••• ••• ••• ••• • •• 115

Materials and Methods ,,, ,,, 120 I In vitro hatching of the eggs of Haplometra cylindracea , , , 124

Experimental procedure and results

II In vitro hatching of the eggs of Haematoloechus (R. pipiens) 148

Experimental procedure and results

III In vitro hatching of the eggs of Haematoloechus (R. ridibunda) 152

Experimental procedure and results

Discus sion ••• ••• ••• ••• ••• ••• ••• ••• 165

Summary •• • ••• ••• ••• ••• ••• ••• ••• 176

Part III In vitro culture of lung of Rana temporaria

and R. pipiens ••• ••• ••• ••• ••• ••• ••• 177

Introduction ••• ••• ••• ••• ••• ••• ••• ••• 178

Materials and Methods ••• ••• ••• ••• 183 7

Page No.

Result and Discussion ...... 185 Summary ...... 190

Conclusions and scope for further work 191

References...... 192 - 8 -

LIST OF FIGURES

Figure Page No.

1. Mother sporocyst, 4 days old, of Haematoloechus sp. 65

2. Daughter sporocysts, 32 days old, of Haematoloechus sp. 65

3. Early cercarial stages, 32 days old, of Haematoloechus sp. 65 - 66

4. Mature daughter sporocyst containing an immature cercaria, 49 days after infection of Haematoloechus sp. 68

5. Tail of cercaria of Haematoloechus similis. 84

6. Stylet of cercaria of Haematoloechus similis. 84

7. Tail of cercaria of Haematoloechus variegatus. 88

8. Stylet of cercaria of Haematoloechus variegatus. 88

9. Metacercarial cyst removed from the nymph Coenagrion puella. 95

10. The life cycle of Haematoloechus similis. 97 9

Figure Page No.

11. (a) Cercaria of Haematoloechus similis. 106

(b) Stylet of cercaria of Haematoloechus similis.

12. Cercaria of Haematoloechus variegatus. 110

1.3. Complete anaerobic condition applied in the case of eggs of Haplometra cylindracea in vitro. 12.3

14. The digestive system of Lymnaea stagnalis . 164 - 10 -

LIST OF PLATES

Plate Page No.

1. Maintenance of uninfected and infected Snails, Planorbis planorbis. 39

2. Dorsal view of second intermediate host damsel fly nymph, Coenagrion puella. 41

3. Maintenance of second intermediate host damsel fly nymph, Coenagrion puella. 43

4. Maintenance of experimental infected second intermediate host damselfly nymph, Coenagrion puella. 45

5. Haematoloechus similis, a common parasite

in the lungs of frog Rana ridibunda. 49

6. Adult Haematoloechus variegatus, a common lung fluke of Rana ridibunda. 55

7. Experimentally infected Snail, Planorbis planorbis. 62

8. Photomicrograph of 42 days old daughter sporocyst of Haematoloechus sp. 71

9. Photomicrograph of 42 days old immature cercaria of haematoloechus sp. 73 Plate Page No.

10. Photomicrograph of 49 days old section of infected snail, Planorbis planorbis containing daughter sporocysts . 75

11. Encysted metacercarial cyst removed from the second intermediate host, the damselfly

nymph, Coenagrion puella. 93

12. Excysted metacercaria. 93

13. Eggs of lung fluke, Haplometra cylindracea

of frog Rana temporaria. 143

14. Egg hatching of the lung fluke, Haplometra

cylindracea of frog Rana temporaria. 143

15- 19. Photomicrographs showing hatching of the

egg of the lung fluke Haematoloechus sp. 155 - 158

20. Lung of Rana pipiens (not cultured). 189

21. Section of lung of Rana pipiens after 114 days in culture. Mitotic divisions. 189 - 12 -

LIST OF TABLES

Table Page No.

1. The complete life cycle of the species of the genus Haematoloechus as reported by various workers. 25 - 27

2. Lists of the species of genus Coenagrion Kirby, 1890 and their distribution (After Hammond, 1977). 35

3. Dimensions of Haematoloechus similis. 50

4. Variability of Haematoloechus similis as recorded by the various authors. (After Prokopic & Krivanec, 1974). 51 - 52

5. Dimensions of Haematoloechus variegatus. 56

6. Variability of Haematoloechus variegatus as recorded by the various authors (After Prokopic & Krivanec, 1974). 57 - 58

7. Experimental infection of snails Planorbis planorbis with Haematoloechus sp. 76 - 77

8. Comparison of sporocysts sizes of the species of the genus Haematoloechus. 78 - 13 -

Table Page No.

9. Comparative dimensions of cercariae of Skrjabinoeces similis = Haematoloechus similis and present material (After Ginetsinskaia & Dobrovol'skii, 1968). 80

10. Exposure of cercariae of Haematoloechus similis to the damselfly Coenagrion puella. 82

11. Comparison of measurements made in different conditions of cercariae of Haematoloechus variegatus. 86

12. Comparative dimensions of cercariae of Pneumonoeces variegatus and present data. (After Ginetsinskaia & Dobrovol'skii, 1968). 89

13. Exposure of cercariae of Haematoloechus variegatus to the damselfly Coenagrion puella. 91

14. Effect of various concentrations of Sodium hypochlorite and enzymes on the hatching of the eggs of Haplometra cylindracea. 144

15. Effect of 1% Sodium hypochlorite with different pHs on the eggs of Haplometra cylindracea. 145

16. The eggs of Haplometra cylindracea (17 days) placed in cavity block, dried and rewetted with the media. 146 - 14 -

Table Page No.

17. Effect of "Snail conditioned" water on the eggs of Haplometra cylindracea. 147

18. Effect of enzymes on the eggs of Haematoloechus (R. pipiens). 150

19. Effect of Sodium hypochlorite on the eggs of Haematoloechus (R. pipiens). 151

20. Effect of "Snail conditioned" water on the egg

hatching of Haematoloechus sp. (R. ridibunda). 159

1 The effect of "Snail water" (from tank) on 26 days old eggs of Haematoloechus

(Rana ridibunda). 160

22. Effect of mechanical pressure on the egg of Haematoloechus (R. ridibunda). 161

23. Effect of enzymes and bile on the eggs of Haematoloechus spp. (R. ridibunda). 162

24. Blood ionic composition of Lymnaea stagnalis (After Schoffeniles & Gilles, 1972). 169

25. Summary of methods used in lung tissue culture by various workers and the mitotic incidence in cultured lung tissue. 181 - 182 - 15 -

Table Page No.

26. Methods used in lung tissue culture and results (incubated at 20°C and medium was changed

once in a week). 186

27. The lung portion of Rana temporaria was used. Some were contaminated and discarded on day *. 187 - 16 -

GENERAL INTRODUCTION

The thesis has been divided into three parts and a detailed introduction has been provided in each part. Part I - includes the life cycle of Haematoloechus similis and Haematoloechus variegatus from the frog, Rana ridibunda imported from Yugoslavia. The development of Haematoloechus similis and H. .variegatus in their intermediate hosts was investigated. Laboratory bred snails Planorbis planorbis collected from Romney on June 14, 1977 were found to serve as the first intermediate host for both these species. No attempt was made to rear the second intermediate host damselfly Coenagrion puella. Larvae of these species were exposed to cercariae of Haematoloechus similis and 1 - 2 number of free unencysted metacercariae were found in the haemocoele. The cercariae of Haematoloechus variegatus were exposed to Coenagrion puella but no penetration was observed; 1 2 metacercarial cysts were found. Out of 10 used as a control, one was positive. The complete life cycle of Haematoloechus similis and H. variegatus in the U.K. thus remains undetermined. Part II - deals with the problem of hatching of lung flukes of Rana sp. in vitro. (1) Haplometra cylindracea, the common lung fluke of Rana temporaria. Attempts were made to hatch the eggs in vitro. Factors such as osmotic pressure, hydrogen ion concentration, enzymes, temperature, gas phase, reducing agents, bile salts and NaOCl and various others were examined. Only in NaOCl were non-motile miracidia observed, but these solutions killed the miracidia. (2) The eggs of Haematoloechus (from Rana pipiens) were tried with different enzymes and again only non-motile miracidia were released in NaOC1. (3) The eggs of Haematoloechus (from Rana ridibunda) were tried in different species of "Snail conditioned" water; hatching occurred only in the snail conditioned - 17 - water of Planorbis planorbis. Part III - deals with the problem of the in vitro culture of lung tissue of Rana temporaria and R. pipiens incubated at 20°C. This was maintained for periods up to 114 days. Although mitoses were observed in vitro cultured tissue, it was not possible to accurately assess the growth rate nor to count the mitoses or statistically analyse the data. - 18 -

PART I

The life cycle of Haematoloechus similis and H. variegatus - 19 -

INTRODUCTION

The flukes of the family Plagiorchiidae are mainly parasites of amphibia and birds. In the subfamily Haematoloechinae, they are parasitic in the lungs of frogs and toads, where they feed on the lung tissue and blood (Halton, 1967). Krull (1931) observed in parasite- free frogs that the lungs are spongy, thin and elastic while the lungs from infected frogs are less spongy, more brittle and more readily torn. In general the presence of flukes in the lung apparently does little damage, but the pulmonary epithelium becomes columnar (Erasmus, 1972; Smyth, 1976). There is very little information on pathological changes in frog lungs as a result of Haematoloechus parasitism. Cain & French (1975). described the effects of parasitism by Haematoloechus medioplexus on lung fatty acid and sterol composition in the bull frog, Rana cate sbiana. The adult flukes lay large numbers of eggs, which are carried out of the respiratory tract by ciliary action through the glottis into the mouth cavity. They are swallowed and pass through the gastrointestinal tract and are voided with the faeces. The genus Haematoloechus is one of the commonest genera. The earliest record of the genus Haematoloechus in the lungs of amphibia is provided by Zeder (1800). Since then, numerous other workers have reported the trematodes present in lungs of various species. Krull (1931) gave an historical record of the discovery of adults in frogs. Rudolphi (1809) was unable to see the ventral sucker but later (1819) described as Distoma variegatum the lung fluke of Rana esculenta. Looss (1894) described it and later (1899) created a separate genus Haematoloechus and divided it into three species, (1) Haematoloechus variegatus, (2) H. similis and (3) H. asper. Looss (1902) changed the generic name to Pneumonoeces because of the existance of Stal's hemipteron genus Haematoloecha - 20 - established in 1874. Leidy (1851) reported the first record of a lung fluke in America. Stafford (1902) described five species of the genus Haematoloechus: (1) H. longiplexus, (2) H. breviplexus, (3) H. similiplexus, (4) H. medioplexus and (5) H. varioplexus. But later (1905) he changed the name of the genus Haematoloechus to Pneumonoeces. Seeley (1906) discovered another fluke Pneumonoe- ces complexus. Cort (1915a) re-described the species of Stafford & Seeley and introduced Pneumonoeces coloradensis. Irwin (1929) described the lung fluke Pneumonoeces parviplexus. There are three main features of the genus Haematoloechus Looss, 1899; (Odening, 1960). (1.) The possession of long intestinal caeca.

(a) Extracaecal uterine loops. (3) A ventral sucker. Odening (1960) recognised three sub genera Skrjabinoeces, Haematoloechus and Anomolecithus, based on the arrangement of the vitellaria. The classification of the genus Haematoloechus of digenetic trematodes as described by Yamaguti (1958) is as follows:-

Order:- Digenea van Beneden, 1858 S. order:- Prosostomata Odhner, 1905 Family:- Plagiorchiidae Ward, 1917 S. family:- Haematoloechinae nomemend for Haematoloecinae Teixeira de Freitas et Lent, 1939

Genus:- Haematoloechus Looss, 1899 nec. Haematoloecha Stāl, 1874 syn. Pneumonoeces Looss, 1902 Ostiolum Pratt, 1903 Pneumobite s Ward, 1917 Skrjabinoeces Sudarikov, 1950 - 21 -

In the family Plagiorchiidae the chief characteristics are either the uterus passing between two testes or not very often reaching to their posterior extremity. In the sub family Haematoloechinae, the chief characteristics are the presence of extracecal loops, cirrus pouch, preace- tabular genital pore ventral to pharynx or oseophagus and vitellaria forming distinct bunches.

Key to Haematoloechinae genera:

(1) Lateral, anteriorly-directed hinder uterine loops absent.

0 stiolum

On both sides of the body lateral, anteriorly-directed hinder

uterine loop present ......

(2) Ventral sucker absent in adult

Neohaematoloechus

Ventral sucker present ...... 3

(3) Testes disposed laterally to intestinal caeca.

Metahaematoloechus Testes between intestinal caeca ...... 4

(4) Vitelline follicles extending posteriorly beyond anterior testis.

Haematoloechus

Vitelline follicles not extending posteriorly beyond testes. Skrjabinoeces - 22 -

Life cycles:- Considerable attention has been given to the life cycle of digenetic trematodes because of the larval stages and the nature of their multiplication. The life cycles of various species of the genus Haematoloechus have been studied and reported by numerous workers (Table 1). Van Thiel (1930) fed metacercariae from anopheline larvae to frogs and found mature flukes of Haematoloechus variegatus (Rud, 1819) in the lungs of Rana spp. Probably, the most comprehensive studies on the life cycle of frog lung flukes Haematoloechus medioplexus (Stafford, 1902) and. H. parviplexus (Irwin, 1929) have been reported by Krull (1931). Krull (1932) found free metacercariae of Haematoloechus longiplexus (Stafford, 1902) in the adult damselfly Lestes vigilax and these grow rapidly when fed to the bull frog, Rana catesbeiana. The snail host was unknown. Krull (1933, 1934) also reported the life cycle of Haematoloechus complexus (Seely, 1906). Ingles (1933) investigated the life history of Haematoloechus oxyorchis Ingles, 1932 from the lung of Rana aurora. Rankin (1939) found cercariae from Pseudosuccinea columella. The cercariae merchanti resembled the cercariae of lung flukes of amphibians (Van Thiel, 1930; Krull, 1931; Ingles, 1933), but differed by having small papillae at the anterior end and a longer and narrower stylet. Part of the life cycle has been deter- mined experimentally, but not completed. Smith (1959) investigated the ancylid snails, the first intermediate host. He found cercariae from ancylid snail Ferressia parallela resembled the ceracriae of Haematoloechus Grabda (1960) reported the life cycle of Haematoloechus similis. Dollfus et al. (1960) described the cercariae of Haematoloechus bombynae. Combes (1965) in his description of the life history of - 23 -

Haematoloechus pyrenaicus reported Ancylus fluviatilis as the first intermediate host, Cordulegaster annulatus; Gamphus sp; Aeschna sp; Perla sp and Nemura as the second intermediate hosts and Rana temporaria and B. bufo as the final hosts. The life cycle of Haematoloechus breviplexus (Stafford, 1902) from Rana pretiosa was described by Schell (1965). The snail host was Gyraulus similaris and Aeschna multicolor acted as the second intermediate host. Hollis (1972) found a difference in seasonal incidence of Haematoloechus medioplexus between male and female Rana pipiens. Dronen (1975) reported the life cycle of Haematoloechus coloradensis (Cort, 1915) from Rana pipiens. Physa virgata was the snail host and Tramea sp; Libellula sp; Anax sp. (dragonfly nymphs) and Enallagma sp (damselfly nymph) served as second intermediate hosts. Underwood and Dronen (1977) noticed that when eggs of H. breviplexus were fed to Gyraulus parvus, Helisoma trivolvis, Physa virgata and a species of Ferrissia (an ancylid) only the ancylids become infected after 35 days. The cercariae which they shed differed from those of H. breviplexus as described by Schell (1965). Dronen (1977) has reported the host-parasite population dynamics of Haematoloechus coloradensis, noting that the incidence of infection and number of parasites per host varied seasonally in all hosts. Bourgat & Kulo (1978) recently illustrated the life cycle of Metahaematoloechus exoterorchis (Rees, 1964), a parasite of the lung of the frog Dicroglossus occipitalis in Togoland. The sporocysts and xiphidio cercariae developed in the snail Segment- orbis kanisaensis; experiments showed that Gyraulus chudeaui and Bulinus forskalii were vectors. The larvae of dragonflies serve as the second intermediate host and adults have been shown experi- - 24 -

mentally developing in the frog Conraua derooi. Details of the life cycles of the species of the genus Haematoloechus were summarised in Table 1. TABLE 1

The complete life-Cyele of the species of the genus Haematoloechus as reported by various workers

Parasite Host First inter- Second inter- Final host Authors mediate host mediate host

Haematoloechus Rana pipiens Planorbula Sympetrum Rana pipiens Krull medioplexus (Leopardfrog) armigera obtrusum, Bufo ameri- (1930, 31) S. rubicundulum canus

Haematoloechus Rana clamitans Gyraulus parvus Sympetrurn Rana clami- Krull parviplexus rubicundulum, tans (1930) S. obtrusum

Haematoloechus Rana. clamitans Pseudo succinea Sympetrum vicinum, Rana clami- Krull complexus columella Pachydiplex longi- tans (1933, 34) pennis, Halotania incesta, Tetrago- neuria cynosura, Chromagrian conditum, Enallagma divagans Le s to s vigilax, Agria sp. Parasite Host First inter- Second inter- Final host Authors mediate host mediate host

Haematoloechus Rana aurora Planorbis parvus, Sympetrum illotum Rana aurora Ingles oxyorchis Planorbis vermic- (Adult) (1933) ularis Plathemis lydia

Haematoloechus - Lymnaea truncatula Culicid mosquito Bombina Dollfus et al. bombynae - larvae and vire :ata (1960) Theobaldia and Rana sp (quoted in Yamaguti 1975)

Haematoloechus Rana esculenta Planorbis planorbis Coenagrion Rana Grabda similis hastulatum esculenta (1960)

Haematoloechus Rana pretiosa Gyraulus similaris Aeschna Rana Schell breviplexus multicolor pretiosa (1965)

Haematoloechus Rana temporaria Ancylus fluvia Cordulegaster R. temporaria Combes pyrenaicus and Bufo bufo tilus annulatus, and B. bufo (1965) Gom•hus sp. , Aeschna sp., Perla sp. , and Nemura Parasite Host First inter- Second inter- Final host Authors • mediate host mediate host

Haematoloechus Rana pipiens Physa virgata Tramea sp., Rana pipiens Dronen coloradensis Libellula sp., Ambystoma (1975) Anax sp., tigrinum (dragonfly nymph) Enallagma sp. (damselfly nymph)

Metahaemato- Dicroglossus Gyraulus Larvae of Conrauade- Bourgat & loechus occipitalis chudeaui dragonflies rooi Kulo exoterorchis and Bulinus (1978) for skalii

- 28 -

Obiect:- The purpose of this project was to trace the life cycle of the species of the genus Haematoloechus and to study its development in the intermediate hosts. Haematoloechus similis and H. variegatus are lung flukes common in the frog Rana ridibunda. The frog host Rana ridibunda was obtained from Yugoslavia. The green frog, Rana ridibunda (Marsh or Lake frog) is a noisy, aquatic species up to 120 mm in size. It has a big pointed snout and green or olive and dark spots are present on the back. At each corner of the mouth, the male has a grey vocal sac. The hind legs are long and metatarsal tubercle is small, about ā to the length of the first hind toe (Arnold, Burton 1978). It differs from Rana temporaria by closely set eyes and vocal sacs at the corner of the mouth in the males. In the U.K. it was introduced to Romney Marsh in 1935.

Species of Haematoloechus in R. ridibunda

Prudhoe & Bray (personal communication) listed the following species of the genus Haematoloechus common in Rana ridibunda:- 1. Rana ridibunda Palla s (1) Haematoloechus asper (2) Haematoloe chu s asper lipsiensis (3) Haematoloechus odeningi (4) Haematoloechus schultzei (5) Haematoloe chu s variegatus (6) Haematoloechus variegatus dubininae (7) Haematoloechus spp. innom. (8) Skrjabinoe ce s breviansa (9) Skrjabinoe ce s donicus (10) Skrjabinoe ce s minimus - 29 -

(11) Skrjabinoeces similis (12) Skrjabinoeces sp. innom

2. Rana ridibunda pe re zi Seoane (1) Haematoloechus pyreniacus (2) Haematoloechus varie gatus (3) Skrjabinoeces similis

3. Rana ridibunda ridibunda Pallas (1) Haematoloechus variegatus (2) Haematoloechus similis - 30 -

Amphibian ecology and parasite fauna:- Amphibians are generally associated during part of their life cycle with aquatic habitats and the latter play an important role in parasitic infection. The natural diet of amphibians consists of aquatic insects. The cerceriae of trematodes often infect the aqautic insects, their intermediate host, through the exoskeleton or its thinner parts. The digenetic trematodes are all parasites of vertebrate and produce eggs which pass out in the faeces or urine. The egg liberates a miracidium outside or inside the snail, the first intermediate host. The snail releases cercariae, which may encyst on vegetation or may penetrate and encyst in a second intermediate host which is the food of the final host and develops inside the final host. The second intermediate host always has the same habitat as the final host in order to complete the cycle. The phylum Arthropoda contains numerous forms which serve as intermediate hosts. Arthropods are a large group of animals which constitute the food supply for many higher animals such as fish, amphibians, birds and some mammals. Hall (1929) has reviewed arthropods as intermediate hosts of helminths and listed the members of the trematode families having their metacercarial stage in various aquatic insects. In the e,Y4l xD p t.-ds family Plagiorchiidae, the various aquatic which serve as intermediate hosts are members of the orders Decapoda, Amphipoda, Diptera, Trichoptera, Plecoptera, Ephemerida and Coleoptera. Jones (1950) described anopheline larvae as intermediate hosts for larva trematodes. Timon (1958) described the role of insects as intermediate hosts in the life cycle of digenetic trematodes. Shevchenko & Barabashova (1960) examined the aquatic arthropods as intermediate hosts for helminths. Schevchenko (1962) reported the helminth larvae in aquatic insects. The defence - 31 -

reactions to metazoan parasites have been reviewed by Salt (1963). Chibichenko (1964) reported the aquatic invertebrates of kirgizia as intermediate hosts of helminths. The incidence and morphology of digenean metacercariae in dragonflies was described by Krasnolobova (1970). Skrjabinoeces sp and S. similis were also reported. The work done in Turkmenia on the insect intermediate hosts of helminths of wild and domestic animals was summarized by Mushkambarova (1970). Nikulin (1971) described the role of odonata in spreading trematode infections of birds. Infections were heaviest in the spring and summer. Ginetsinskaya & Dobrovol'skii (1972) reviewed the work accomplished in the study of invertebrates as intermediate hosts of helminths. Ilyushina (1973, 1975) stated the aquatic insects as second intermediate hosts of trematodes and review data from other authors. Stadnychenko (1974) studied the host-parasite relationships between larval trematodes and their hosts. - 32 -

Odonata as intermediate hosts:- The dragonflies are insects comprising the order Odonata and are divided into two suborders.

(1) Anisoptera (Dragonflies) Distinguished by the presence of fore and hind wings. When the insect is at rest it opens its wings widely. The large compound eyes touch or nearly touch each other on the top of the head.

(2) Zygoptera (Damselflies) The wings are narrow near the point of attachment and are partially or completely folded together over the back when the insect is at rest. The eyes are widely separated on the head.

The "nymphs" or "naiads", the young stages of all dragonflies are completely aquatic and carnivorous. They have various structures which are absent in adults. The eggs hatch in a month or six weeks or wait until the spring time. The nymph grows by successive moults or "ecdyses". These separate stages or "instars" vary in number from about 9 to 14; the length of time spent in the nymph stage varies with the species. If the food is plentiful the larvae complete their development more quickly. Towards the end of the final instar the tissues inside the larval cuticle change into adults; these internal changes constitute a "metamorphosis". When metamorphosis is complete, it leaves the water and undergoes its moult where ittransforms into the adult. The process is known as "emergence" and the larval skin or "exuvia" remains at the site of emergence (Corbet, 1962). In temperate regions, several zygoptera and anisoptera move to deeper and more sheltered situations during the winter months. The nymphs of the suborder Zygoptera are long and slender, abdominal pleurites infolded, abdomen terminating in three caudal - 33 - lamellae. The nymphs of the suborder anisoptera are shorter and more stoutly built, abdomen with ventral pleurites, abdomen terminating in fine spine like appendages (Gardner, 1977). The nymphs of the family Coenagriidae (suborder Zygoptera) are found on the surface of the mud or among weeds. The identification of the dragonflies used in this work were made according to Gardner (1977). Useful characteristics for identification are based on the caudal lamellae. - 34 -

Species in the U.K. :- (Table 2) Hammond (1977) listed the following species of the genus Coenagrion Kirby 1890 present in Great Britain and Ireland.

(1) C. armatum (Charpentier, 1840), Norfolk Coenagrion, Norfolk damselfly.

(2) C. hastulatum (Charpentier, 1825), Northern Coenagrion, Northern damselfly.

(3) C. mercuriale (Charpentier, 1840), Southern Coenagrion, Southern damselfly.

(4) C. puella (Linnaeus, 1758), Common Coenagrion, Azure damselfly.

(5) C. pulchellum (van der Linden, 1825), variable Coenagrion, Variable damselfly.

(6) C. scitulum (Rambur, 1842), Dainty Coenagrion, Dainty damselfly. TABLE 2

List of the species of genus Coenagrion Kirby, 1980 and their distribution (after Hammond, 1977)

Distribution

(1) C. armatum (Charpentier, 1840), Near Stalham, Norfolk The Norfolk Coenagrion, The Norfolk Damselfly

(2) C. hastulatum (Charpentier, 1825) Scotland The Northern Coenagrion The Northern Damselfly

(3) C. mercuriale (Charpentier, 1840) Southern and South-Western counties The Southern Coenagrion The Southern Damselfly

(4) C. puella (Linnaeus, 1758) England and Ireland, local in Scotland The common Coenagrion, The Azure Damselfly

(5) C. pulchellum (van der Linden, Norfolk Broads 1825) The variable Coenagrion, The variable Damselfly

(6) C. scitulum (Rambur, 1842) Essex The Dainty Coenagrion, The Dainty Damselfly - 36 -

MATERIALS AND METHODS

(1) Embryonation of eggs:- Mature specimens of Haematoloechus similis and H. variegatus were obtained from the lungs of Rana ridibunda. The latter was maintained in the laboratory and fed on insects. The lungs were examined for parasites in amphibian saline under a dissecting microscope. The worms were removed immediately and placed in distilled water and teased when necessary to release the eggs from the uterus. The eggs were washed several times with distilled water to remove any adhering tissues which tended to encourage the growth of protozoa and algae. The eggs were incubated in covered watch glasses at 25°C for two weeks to allow the development of miracidia. The water was changed every week.

(ii) Snail culture and maintenance:- Specimens of the fresh water snails, Planorbis planorbis were collected from Romney on June 14, 1977.. Identification of the snail was based on shell morphology and the identification was confirmed by the Mollusca department, British Museum. The snails were maintained in plastic aquaria which had a capacity of 13 litres. The aquaria were aerated by an electrical air-pump and porous stones were placed on the ends of air-hoses to produce a regular stream of small air-bubbles, (Plate 1). The tap water contained a considerable amount of chlorine which is toxic. In order to dechlorinate the water, it was allowed to stand in an open container for a few days. This "aged" tap water was used. Aquaria were covered with glass panels to exclude the dust and to prevent the snails from escaping. One third of the water was changed once in a week (a change of water causes an increased ovipositian) and the snail faeces were removed. The routine diet of snails was boiled and dried lettuce leaves. These were washed several times with distilled water before boiling to remove any plant fertilizer - 37 -

which might be present and which might affect the snails. Old leaves were removed before decay set in. The snails which died were replaced. During winter the temperature was approximately 6 - 14°C; the production of egg masses and growth ceased, oviposition was induced artifically by liaising the temperature of maintenance to 21°C. The snail room was lighted with fluorescent ceiling light.

(iii) Culture method of nymph:- No attempt was made to rear insects in the laboratory. The nymphs of the damselfly Coenagrion puella (Plate 2) were received from Biosery Ltd and maintained in the laboratory as described by Galtsoff et al. (1937).' The nymphs have a labium with premental setae 5 + 5, and labial palpi with 6 setae. Narrow lanceolate caudal lamellae with bluntly pointed apices are pres ent. Wing sheets are green and lamellae are brown. They breed in weedy ponds, lakes and canals; they are common in England, Ireland and Scotlands (Table 2). The nymphs are aquatic and carnivorous. For maintenance separate containers were used; each nymph was placed in a small boat. The frame of the boat was made of light expanded poly- styrene and the support of the vessel contained fine pores, in order to permit circulation of water. The boats were maintained in a large tank with a continuous flow of water (plate 3). The water was aerated with the help of electrically operated pumps (Lacy-Hulbert model 1200). The air was broken into fine bubbles by use of air- stones attached to plastic tubing. The pupae of mosquito and Daphnia were given as a food twice a week. The cultures of Daphnia were maintained in the laboratory. After exposure to cercariae the nymphs were individually isolated in small beakers to prevent cannibalism (plate 4). - 38 -

Plate 1. Maintenance of uninfected and infected Snails, Planorbis planorbis (photograph). 11 II II II II II II II II II II 11 II I_I II Ii 11 II II II II II

II II II II II ĪI II II II - 40 -

Plate 2. Dorsal view of second intermediate host damselfly nymph, Coenagrion puella (photograph). Pf

O - 42 -

Plate 3. Maintenance of second intermediate host damselfly nymph, Coenagrion puella (photograph).

Plate 4. Maintenance of experimental infected second intermediate host damselfly nymph, Coenagrion puella (photograph).

- 46 -

Handling and measuring of specimens:- For general morphology, the adult specimens were fixed in 70% alcohol under coverslip pressure overnight, stained with Gower's carmine for two hours, dehydrated, cleared in xylene and mounted in canada balsam. All measurements were given in millimeters. Identification was based on descriptions of Prokopic & Krivanec (1974) and confirmed by Dr Bray, British Museum (Natural History).

Haematoloechus similis (Looss, 1890) Syn:- Distoma simile Looss, 1899, nec Sonsino, 1890; Haematoloechus similigenus of Stiles & Hassal, 1902; Distoma variegatum of Looss, 1894; in part; Pneumonoeces similis of Klein, 1905; Liihe, 1909 and other writers; P. simili genus of Cort, 1915; Nicoll, 1926.

Description:- (Plate 5) The following description (Table 3) was based on 4 mounted specimens. Elongate shape, body covered with spines. The length of the spine is 0. 01 mm. Length of the body 2. 580 - 3. 65 mm; width 0. 860 - 1. 290 mm. The oral sucker is larger than the ventral sucker. The diameter of the oral sucker is 0. 27 - 0. 36 X 0. 27 - 0. 36 mm. The diameter of the ventral sucker is 0. 18 - 0. 27 X 0. 18 - 0. 27 mm. The ventral sucker is 1. 5 mm from the anterior end of the body. The oral sucker is followed by a pharynx 0. 18 X 0. 09 - 0. 18 mm. Behind the pharynx is the bifurcation of the gut. Caecal branches extend to the end of the body. Ovary ovoid, 0. 18 - 0. 45 X 0. 18 - 0. 36 mm in diameter. The seminal receptacle near the ovary (0. 1 8 - 0. 27 X 0. 27 mm). The diameter of the anterior testis is 0. 36 - 0.63 X 0. 27 - 0.63 mm. The diameter of the posterior testis is 0.36 - 0.63 X 0.45 - 0.72 mm. Uterus descending in moderate loops downwards from the ventral - 47 - sucker and forms two lateral extracaecal uterine branches in the posterior portion of the body. The vitelline follicles 5 - 15 (follicles per cluster) are situated in mid-body. Eggs are ovoid, length 0. 03 mm and width 0. 02 mm. The variability of Haematoloechus similis as recorded by the various authors is tabulated in Table 4. - 48 -

Plate 5. Haematoloechus similis, a common parasite in the lungs of frog Rana ridibunda (photomicrograph) X b.9.

A ,a- L144,.. _ 3. o ',11•

0 - 50 -

TABLE 3

Dimensions of Haematoloechus similis:- (in mm)

1. Body measurements Width 0. 860 - 1. 290 Length 2.580 - 3.655 Len 2. Ratio of of body 3: 2. 8 Width

3. Oral Sucker Width 0.27 - 0.36 Length 0.27 - 0.36 4. Ventral Sucker Width 0.18 - 0. 27 Length 0.18 - 0.27 0 5. Ratio of Sucker 1.: 1

6. Pharynx Width 0.18 Length 0.09 - 0.18 7. Ovary Width 0. 1.8 - 0. 45 Length 0.18 - 0.36 8. Oesophagus Width 0.09 Length 0.09 9. Seminal receptacle Width 0.18 - 0.27 Length 0.27 10. Anterior testis Width 0.36 - 0.63 Length 0.27 - 0.63 11. Posterior testis Width 0.36 - 0.63 Length 0.45 - 0.72 12. Vitelline follicles Width 0. 18 - 0. 27 Length 0.27 - 0.36 13. Egg Width 0.02 Length 0. 03 14. Length of Spine 0.01 TABLE 4

Variability of Haematoloechus similis as recorded by the various authors (after Prokopic & Krivanec, 1974)

('1'Ya,aS'u,yQwVZa..}5 it w-s1 Travassos H. minimus IL 4p Prokopic & Darriba Sevēenko Seveenko Krivanec 1974 1930 1965 1965

(1) Body measurements Width 4. 6 - 9. 4 2. 5 - 3. 3 4. 3 2. 54 - 8. 34 Length 1.0 - 1.6 0.7 - 1.0 1.0 0.65 - 1.91

Length Ratio 1) 1) (2) Width of body (4. 0 - 4. 2: (3. 0 - 3. 7: 4. 3: 1 (3. 37 - 5. 2: 1 4. 20: 1) (3) Oral Sucker Width 0. 35 - 0. 50 0. 25 - 0. 30 0. 353 0. 218 - 0.415 Length 0.40 - 0.45 0.22 - 0.27 0.321 0.210 - 0.377

(4) Ventral Sucker Width 0.28 - 0.36 0.15 - 0.23 0.107 0.143 - 0.276 Length 0.24 - 0.35 0.15 - 0.21 0.257 0.143 - 0.304 Oral (5) Ratio of sucker (1. 5: 1) 1. 6: 1 1 Ventral 1. 3 - 1. 9: 1.25 - 1.69: 1 (1. 52: 1) Travassos H. minimus H. sp. Prokopic &Krivanec

(6) Pharynx Width 0.107 - 0.139 0.170 0.118 - 0.202 Length ? 0.085 - 0.107 0.150 0.101 - 0.160

(7) Oesophagus Width 0.042 - 0.126 (0. 20) (0. 15) Length 0.042 - 0.252

(8) Ovary Width 0.35 - 0.50 0.25 - 0.35 0.278 0.193 - 0.746 Length 0. 35 - 0. 60 0. 17 - 0. 7 (?) 0. 342 0. 218 - 0. 609

Seminal Width 0. 386 - 0. 746 (9) bigger than 0.45 receptacle Length ovary ( ) 0.386 - 0.788

(10) Anterior Width 0.50 - 0.71 0.30 - 0.55 0.428 0.210 - 1.044 testis Length 0.45 - 0.85 0.38 - 0.42 0.492 0.184 - 1.160

(11) Posterior Width 0.50 - 0.78 0.34 - 0.47 0.470 0.176 - 0.899 testis Length 0.71 - 1.10 0.47 - 0.57 0.750 0.176 - 1.546

(12) ant 1: 1.05 - 2. 07 Ratio of post body (1: 1.25 - 1.9) (1: 1.48 - 1.7) 1: 1.1 (1: 1.46)

(13) Vitelline Width ? ? ? 0.092 - 0.353 follicles Length

(14) Eggs Width 0.016 - 0.026 0.017 - 0.025 0.016 - 0.029 Length 0.032 - 0.040 0.029 - 0.032 0.031 - 0.054

(15) Length of spines ? 0. 006 - 0. 010 (0. 008) 0.007 - 0.009

- 53 -

Haematoloechus variegatus (Rudolphi, 1819) Looss, 1899 Syn:- Distoma variegatus Rudolphi, 1819 Penumonoeces variegatus (Rudolphi) of Looss, 1902, nec D. variegatum Looss, 1894.

The following description was based on 3 mounted specimens (in mm) (Table 5).

Description:- (Plate 6) Body elongate, length of adult is 4. 30 - 9. 030 mm; width 1. 290 - 1. 935 mm. Cuticle smooth, the length of the spine is 0. 01 mm. Oral sucker is larger than ventral sucker; the diameter of oral sucker is 0. 36 - 0. 54 X 0. 36 - 0. 54 mm. Ventral sucker is 0. 27 0. 45 X 0. 27 0. 45 mm. From anterior end of the body at 2. 1.5 mm, the oral sucker is followed by a spherical pharynx (0. 18 - 0. 27 X 0. 18 - 0. 27 mm). Behind the pharynx the gut bifurcates. Caecal branches extend to the end of the body. Ovary is ovoid, 0. 36 - 0. 63 X 0. 63 1. 075 mm in diamter. Situated in the region of the ventral sucker. The seminal receptacle (0. 27 - 0. 36 X 0. 27 0.45 mm in diameter) is near the ovary. Testes below the ovary; the dimater of anterior testis is 0.63 - 0. 645 X 0. 81 - 1. 290 mm while the diameter of posterior testis is 0. 54 - 0. 860 X 0. 81 - 1. 290 mm. The uterus descends from the ventral sucker to the posterior end of the body, forming two lateral extracaecal branches. The ascending portion of the uterus continues upwards, mostly between the testis. Vitelline glands consist of 7 - 15 rosette shaped follicles arranged in two groups, one in the anterior and one in the posterior portion of the body. Egg is 0. 03 mm long and 0. 02 mm wide. The variability of Haematoloechus variegatus as recorded by the various authors is given in Table 6. - 54 -

Plate 6. Adult Haematoloechus variegates, a common lung fluke of Rana ridibunda (photograph).

- 56 -

TABLE 5

Dimensions (in mounted specimens were based on three specimens of Haematoloechus variegatris (in mm)

1. Body measurement Width 1.290 - 1. 935 Length 4.30 - 9.030

Ratio of L'e of body 3. 4 4. 6 2. Width

3. Oral Sucker Width 0. 36 - 0. 54 Length 0.36 - 0.54 4. Ventral Sucker Width 0.27 - 0.45 Length 0.27 0.45 O 5. Ratio of Sucker 1 : 1

6. Pharynx Width 0. 18 - 0. 27 Length 0.18 - 0.27 7. Oesophagus Width 0.09 Length 0.09 - 0.45 8. Ovary Width 0.36 - 0.63 Length 0.63 - 1.075 Seminal receptacle 9. Width 0. 27 - 0. 36 Length 0.27 - 0.45 10. Anterior testis Width 0.63 - 0.645 Length 0.81 - 1.290 11. Posterior testis Width 0.54 - 0.860 Length 0.81 - 1.290 12. Vitelline follicles Width 0. 215 - 0. 430 Length 0.270 - 0.645 anterior testis 1. 2 1. 5 13. Ratio of posterior : 14. Eggs Width 0.02 Length 0.03 15. Length of Spine 0. 01 TABLE 6

Variability of Haematoloechus variegatus as recorded by the various authors (after Prokopic & Krivanec (1974)

C I i RS~YC~~12K H. variegatus H. var. dubininae H. variegatus (Odening 1958) Odening 1958 Prokopic & Krivanec 1974

(1) Body measurements rWidth 10.0 8. 5 - 10.0 2.450 - 13. 26 (5. 66) `Length 1. 7 2.0 - 2. 2 0. 510 - 2. 560 (1.06)

Length 3.84, - 8.83: 1 (2) Ratio of body 5. 88: 1 3. 86 - 5. 0: 1 Width (5. 60: 1)

(3) Oral sucker Width 0.438 0.429 - 0. 515 0. 178 - 0. 540 (0. 354) Length 0.478 0.452 - 0.482 0. 186 - 0. 586 (0. 359)

(4) Ventral sucker Width 0. 340 0. 334 - 0.415 0. 136 - 0. 374 (0.222) Length 0. 335 0. 317 - 0. 370 0. 144 - 0. 310 (0. 208)

oral - 1.66: 1 (5) Ratio of sucker 1. 35: 1 1. 16 - 1. 48:1 1.04 ventral (1.48: 1)

(6) Pharynx Width 0. 233 0. 267 - 0. 275 0. 099 - 0. 234 (0. 168) Length 0. 227 0. 239 - 0. 274 0. 114 - 0. 258 (0. 203) H. variegates H. var. dubininae H. variegatus

(7) Oesophagus Width 0.233 _ 0.010 - 0.030 (0.17) Length 0.227 0.010 - 0.515 (0.193)

(8) Ovary Width Smaller than 0.423 - 0.455 0. 087 - 0. 374 (0. 225) Length testis 0.995 - 1.164 0.129 - 1.250 (0.625)

(9) Seminal Width Smaller than 0.441 - 0. 635 0. 076 - 0.450 (0. 222) receptacle Length testis 0.476 - 0.481 0.151 - 0.900 (0.394)

(10) Anterior Width 0.338 0.550 - 0.714 0.083 - 0.510 (0.322) testis Length 1.180 0.952 - 1.111 0.256 - 1.770 (0.709)

(11) Posterior Width 0.582 0.529 - 0.635 0.129 - 0.875 (0.324) testis Length 1.130 1.010 - 1.058 0.293 - 2.210 (0.852)

(12) ant 1: 0.85 - 2.31 Ratio of body 1: 1. 65 (1: 1. 50 - 1. 56) post (1: 1.5)

(13) Eggs Width 0.012 - 0.015 0.014 - 0.020 0.015 - 0.024 Length 0.025 - 0.028 0.027 - 0.030 0.020 - 0.035

(14) Vitelline follicles - - 0. 053 - 0. 360 - 59 -

Experimental infection of Snails:- Very young laboratory bred snails, Planorbis planorbis (length 0.645 mm - 2. 15 mm and width 0. 860 - 2. 15 mm), were allowed to feed on eggs for 45 minutes; their movements were observed through a microscope. After 45 minutes their faeces contained a large number of empty shells, after which they were removed and rinsed with water to remove excess eggs and placed in finger bowls containing water. The snails were dissected at regular intervals to detect the non-motile and immature parasitic stages. Infected tissues and parasitic stages were transferred to a slide and examined alive under coverslip pressure. Some infected and non-infected snails were fixed in Bouin's fluid. The shell fragments were separated to prevent damage to the microtome, the soft tissues were then dehydrated, embedded in paraffin wax (melting point 56 - 58°C) and serially sectioned at 7 pm and stained with haematoxylin and eosin. The snails Planorbis planorbis have transparent shells which permitted observation of developing sporocysts (plate 7). Water from cultures of exposed snails was examined daily to determine cercarial emergence. The majority of observations were made on living material; 0. 01% neutral red (aqueous) was used where appropriate. Diagrams were made with the help of a camera lucida. Photomicrograph of selected sections were taken. As temperature varies, the intra-molluscan development time was altered only in winte-r. Cercariae appear in the water in greatest number in the early morning. Emergence of cercariae was stimulated by light (Miller & Mahaffey, 1930) - a 60-watt bulb placed about 15 inches above the beaker for two hours. Forced shedding was undertaken only twice a week, as infected snails do not tolerate more frequent exposures to strong light. Cercariae were first studied alive for the excretory system (Looss, 1894). Some were stained with neutral red and chilled in a refrigerator for about an hour (Etges, 1960). This system made - 60 - the excretory system more easily observed as the excretory system of the cercariae were most difficult to make out. In this connection the best results can only be obtained from high power studies of an abundance of living material. The life cycle of Haematoloechus similis is shown in fig. 10. - 61 -

Plate 7. Experimentally infected Snail, Planorbis planorbis (photograph).

ŪZe3 ka~' o w e te1( - 0 - 63 -

RESULT OF THE EXAMINATION OF PLANORBIS PLANORBIS EXPOSED TO INFECTION OF HAEMATOLOECHUS EGG

43 experiments were conducted from June 20, 1978 to February 28, 1979; results are shown in Table 7. The egg:- The eggs containing not fully developed miracidia when deposited were kept in a shallow dish at 25°C for a period of two weeks. They remained viable but unhatched until eaten by laboratory raised specimens of Planorbis planorbis. Fully embryonated eggs of Haematoloechus are ovoid, operculate and dark brown. Twenty-two embryonated eggs were measured, the average length being 0.03 mm and the average width 0. 02 mm (plate 15). The miracidium is stimulated to emerge from the embryonated egg by the pressure of a cover glass; the mean length is 0. 04 mm and width 0. 02 mm (average of 5). The miracidium is not described here in detail. The snails Planorbis planorbis became infected when exposed to eggs. The internal anatomy of the snail is essential to an understanding of the migration of the parasite within the tissues. The relevant morphological information has been provided by Runham (1975) (Fig. 14). After 45 minutes and 24 hours the faecal material of the snails were examined and the parasite eggs present were repre- sented by the shells only, indicating that the miracidia had escaped. The egg hatched in the digestive tract of the snails, but active penetration by the miracidia was not observed in the present study. The incubation time of the egg is from 20 days onward and 88 days old eggs were still infective. Mother sporocyst At the 4th day, the mother sporocyst consisted of a round structure embedded in the intestinal wall. The mother sporocyst measured 0. 03 mm X 0. 02 mm (Fig. 1). - 64 -

Figure 1. Mother sporocyst, 4 days old, of Haematoloechus sp.

Figure 2. Daughter sporocysts, 32 days old, of Haematoloechus sp.

Figure 3. Early cercarial stages, 32 days old, of Haematoloechus sp. Fig. 1

Fig. 2

Fig. 3

germ balls 154Am

Lu',V C - 67 -

Figure 4. Mature daughter sporocyst containing an immature cercaria, 49 days after infection, of Haematoloechus sp. Fig.4

mature daughter sporocyst - 69 -

Daughter sporocyst At 21 days post infection, many milky white elongate oval structures with single cells and cell masses were found free in haemocoele. Each contained numbers of refractile granules which give the sporocyst an opaque appearance. At 32 days post infection, the daughter sporocysts measured: length 0. 175 mm - 0. 45 mm and width, 0.125 - 0. 180 mm; the organism had irregular constructions and a number of cercarial germ balls in various stages of development (Figs. 2 and 3). The sporocysts were loosely threaded in and out of the digestive gland. The outlines of the sporocysts were traced using a camera lucida and measurements were made with an eye piece graticule through the microscope and photographs were taken. 42 days post infection the snail contained tailed and immature cercariae (plate$&9) At 49 days post infection the mature sporocyst contained all stages of development from germ balls to tailed cercariae. Cercaria develop in oval sporocyst measuring: length 0. 36 mm and width 0. 18 mm (Fig. 4). In one sporocyst a cercaria with a tail length 0. 27 mm and width 0. 18 mm was found. Photographs of 49 days old (sectioned) from experimentally infected snails were taken (plate 10). Development in the snails virtually ceases during winter, but it was artifically induced to continue by raising the temperature. In one snail which died 115 days later large numbers of daughter sporocysts were found in the digestive gland. Twenty one sporocysts were measured, the length being 0. 100 mm - 0. 25 mm and the width 0. 050 mm - 0. 125 mm. Comparison of sporocysts sizes of the species of Haematoloechus with other workers are shown in Table 8. - 70 -

Plate 8. Photomicrograph of 42 days old daughter sporocyst of Haematoloechus sp. X 250.

- 72 -

Plate 9. Photomicrograph of 42 days old immature cercaria of Haematoloechus sp. X 350. I ® - 74 -

Plate 10. Photomicrograph of 49 days old, section of infected snail, Planorbis planorbis containing daughter sporocysts. Haematoxylin & eosin X 466. 4 - 76 -

TABLE 7

Experimental infection of snails Planorbis planorbis with Haematoloechus sp.

No. of Age of the Days after egg (Days) Exper- Date infected experimen- Dead iment incubation tal infection at 25°C (Cercarial shedding)

A. 1 20. 6. 78 20 56 21. 11. 78 A. 2 21.6.78 21 63 9.11.78 A. 3 22.6.78 21 - 3. 7.78 A. 4 23.6.78 23 - 8. 8.78 A. 5 27.6.78 27 56 4. 1.79 A. 6 1. 7. 78 26 - 4. 7. 78 A. 7 6.7.78 30 - 2. 8.78 A. 8 10. 7. 78 35 - 17. 7. 78 A. 9 14. 7. 78 39 - 15. 7. 78 A. 10 17. 7. 78 41 - 26. 9. 78 A. 11 27. 7. 78 42 - 1. 8. 78 *A. 12 1. 8. 78 47 76 5. 2. 79 A. 13 3.8.78 47 - 28. 9.78 *A. 14 9.8.78 55 97 13.12.78 A. 15 14. 8. 78 52 - 18. 8. 78 *A. 16 21.8.78 68 158 7. 2.79 A. 17 23.8.78 61 - 30. 8.78 *A. 18 30.8.78 77 121 5. 2.79 *A. 19 30.8.78 77 146 7. 2.79 *A. 20 31.8.78 78 137 21. 3.79 A. 21 1. 9. 78 75 - 6. 9. 78 *A. 22 4.9.78 76 133 7. 2.79 - 77 -

No. of Age of the Days after Exper- Date infected egg (Days) experimen- iment incubation tal infection Dead at 25°C (Cercarial shedding)

A. 23 5. 9.78 79 - 14. 9.78 *A. 24 8. 9.78 82 129 28. 2.79 *A. 25 12. 9. 78 86 144 12. 3.79 *A. 26 14. 9.78 88 128 19. 2. 79 *A. 27 14. 9. 78 88 - 27. 9. 78 A. 28 20. 9.78 46 - 10.10.78 *A. 29 25. 9.78 32 - 22. 1.79 A. 30 28. 9.78 56 - 20.10.78 A. 31 10. 10. 78 21 - 6. 11. 78 A. 32 18. 10. 78 55 - 6. 11. 78 A. 33 4. 1.79 82 - 11. 1.79 A. 34 9. 1.79 87 - 16. 1.79 A. 35 10. 1.79 88 - 10. 3.79 A. 36 11. 1.79 112 - 16. 1.79 A. 37 17. 1.79 95 - 10. 4.79 A. 38 22. 1.79 148 - 12. 2.79 A. 39 2. 2.79 120 - 8. 2.79 A. 40 5. 2.79 113 - 13. 2.79 A. 41 13. 2.79 237 - 21. 3.79 A. 42 20. 2.79 244 - 21. 3.79 A. 43 28. 2.79 136 - 10. 4.79

* = moved to C. T. room @ 21°C on 2. 1. 78 = Not applicable - 78 -

TABLE 8

Comparison of sporocysts sizes of the species of the genus Haematoloechus

Authors Species Sporocysts size (mm)

(1) Krull (1930, 31) H. medioplexus 0.4 X 0. 08

(2) Krull (1931) H. parviplexus 0.25 X 0. 075

(3) Krull (1933, 34) H. complexus 0. 19 - 0. 25 X 0. 08 - 0. 16 (4) Ingles (1933) H. oxyorchis 0.250 - 0. 125

(5) Grabda (1960) H. similis 0. 163 - 0. 600 X (a) 0.132 - 0.273

(b) Smirnova & 0. 32 - 0. 521 X Ibrasheva (1967) 0. 189 - 0. 28 (c) Present data 0. 36 - 0. 18

(6) Dollfus et al. H. bombynae 0. 25 - 0. 35 X (1960) 0.08 - 0.12

(7) Schell (1965) H. breviplexus 0. 18 X 0. 066

(8) Combes (1965) H. pyrenaicus 1.5 X 0. 3

(9) Dobrobol'skii H. as •er 0. 14 - 0. 21 X (1965) 0.11 - 0.15

(10) Dronen (1975) H. coloradensis 0. 34 X 0. 19 - 79 -

Behaviour of cercaria:-

The time required in order to complete development of the cercariae from penetration by miracidia to shedding of first mature cercariae from the snail host varied in the laboratory from 56 days during summer and to 128 days during winter (Table 7). Cercariae emerge abundantly during the early morning hours and much less at other times. When the cercariae are released from the snail they swim at all levels of the water. The swimming motion of the cercaria is continuous until they become exhausted. As soon as the body relaxes and the tail ceases to beat, the cercaria begins to sink to the bottom. Cercaria emerging display no phototropism. Some cercariae were attached to long delicate strands of a mucus-like substance emitted from the snail. One snail continued to produce cercariae for as long as 100 days. This indicates that an infection once acquired, persists for many months. TABLE 9

Comparative dimensions of cercariae of *Skrjabinoeces (=Haematoloechus) similis and present material (in mm) (after Ginetsinskaia & Dobrovol'ski 1968)

Dimensions Present data Ginetsinskaia & After Odening After Grabda Dobroval'skii (1968) (1962) (1960)

(1) Length of 0. 125 - 0. 166 0. 084 - 0. 148 0. 081 - 0. 109 0. 130 - 0. 164 body

(2) Width of 0. 100 0. 042 - 0. 092 0. 072 - 0. 081 body 0. 087 - 0. 104

(3) Length of 0. 125 042 - 0. 126 0. 107 - 0. 140 tail 0. 0. 102 - 0. 136

(4) Diameter of 0. 050 X 0.029 - 0.034 X 0.034 - 0.042 X 0. 026 - 0. 032 oral sucker 0. 050 0. 030 - 0. 039 0. 037 - 0. 046

(5) Diameter of 0. 025 X 0. 014 - 0. 020 X 0. 014 - 0. 022 0. 020 - 0. 022 X ventral sucker 0. 050 0. 014 - 0. 020 0. 020 - 0. 024

(6) Length of 0. 050 0. 026 0. 030 Stylet 0. 032 - 0. 034

* Grabda believed this to be Haematoloechus, at first, but had reservations; no other authors concluded it was Skrjabinoeces. - 81 -

Cercaria of Haematoloechus similis

The measurements of the cercarial body, tail, oral sucker and ventral sucker in the above description were based on 5 cercariae. The body was 0. 125 - 0. 166 mm long and 0. 100 mm broad. The oral sucker was 0. 050 mm long and 0. 050 mm broad. Inside the oral sucker was a stylet 0. 050 mm long and 0. 025 mm broad (Fig. 6). The ventral sucker was 0. 025 mm long and 0. 050 mm broad. The number of penetration glands, 5 - 6 pairs, were not fully confirmed. Each gland has large nuclei and fine granules. The excretory system vesicle was Y- shaped. The flame cells formula was unknown. The tail was 0. 125 mm long and 0. 75 mm broad. The caudal fin was absent (Fig. 5). The comparative dimensions of cercariae with those of other workers are given in Table 9.

Experimental infections of damselfly Coenagrion puella with cercaria of Haematoloechus similis:-

Coenagrion puella was exposed to 15, 30, 40, 45, 50, 63, 64 and 116 numbers of cercariae. Attraction towards the nymph was observed under the microscope. The cercariae penetrated the soft part of the thorax, anterior part of the leg and wings. The cercariae shed their tails on entering. The nymphs were dissected out at different intervals. The unencysted free metacercariae were found moving in haemocoele on the following day. Four nymphs were opened as a control and found to be negative. The results are summarised in Table 10. - 82 -

TABLE 10

Exposure of cercaria of Haematoloechus similis to the damselfly Coenagrion puella

Expt. Number of Cercariae Days after Number of No. nymphs per nymph Infection (unencysted) free metacercariae

1 1 15 1 - ve

2 1 30 1 2

3 1 40 2 -ve

4 1 45 4 - ve

5 1 50 1 1

6 1 63 • 5 - ve

7 1 64 2 -ve

8 1 116 3 - ve

Control 4 - - - ve

- ve = Negative - = Not applicable - 83 -

Figure 5. Tail of cercaria of Haematoloechus similis.

Figure 6. Stylet of cercaria of Haematoloechus similis. Fig.5

tail

Fig. 6

stylet - 85 -

Cercaria of Haematoloechus variegatus:-

The measurements of the cercarial body, tail, oral sucker and ventral sucker in the above description were based on 5 cercariae killed in hot 10% formalin. The body of cercaria was 0. 100 - 0. 150 mm long and 0. 100 mm broad. The oral sucker was 0. 050 mm long and 0. 050 mm broad. The stylet was embedded in the anterior part of the oral sucker (Fig. 8). Length of stylet was 0. 025 mm and with marked shouldering. The ventral sucker was 0. 025 mm long and 0. 025 mm broad. The penetration glands and exretory systems were not described. The tail was with caudal fin; the tail was 0. 100 - 0. 150 mm long (Fig. 7). Comparison of measurements of 5 cercariae made after different conditions of fixation are given in Table 11. The comparative dimensions of cercariae with other workers are given in Table 12. - 86 -

TABLE 11

Comparison of measurements made in different conditions of cercariae of Haematoloechus variegates (in mm)

Conditions

Hot water Neutral red 10% hot formalin

(1) Body Length 0. 150 0. 125 - 0. 200 0. 100 - 0. 150 Width 0. 075 0. 100 - 0. 125 0. 100

(2) Oral Sucker Length 0. 050 0. 050 0. 050 Width 0. 050 0. 050 - 0. 075 0.050

(3) Ventral Sucker Length 0. 025 0. 025 - 0. 100 0. 025 Width 0. 025 0. 025 - 0. 050 0. 025

(4) Stylet Length 0. 025 0. 025 0. 025

(5) Tail Length 0. 100 - 0. 125 0. 075 - 0. 125 0. 100 - 0. 150 Width 0. 025 0. 025 - 0. 125 0. 025

(6) Tail fin Dorsal 0. 05 - - Ventral 0. 04

= Not applicable - 87 -

Figure 7. Tail of cercaria of Haematoloechus variegatus.

Figure 8. Stylet of cercaria of Haematoloechus variegatus. Fig.7

4IE

E o N

Fig. 8 d

2E 1 stylet tail fin TABLE 12

Comparative dimensions of cercariae of Penumonoeces variegatus and present data (in mm) (After Ginetsinskaia & Dobrovol' skii, 1968)

Dimensions Present data Ginetsinskaia and After Von Thiel (10% formalin Hot) Dobrovol'skii, 1968 1922 (Hot formalin *) (Living)

(1) Length of body • 0. 100 - 0. 150 0. 084 - 0. 116 0. 168 - 0. 192

(2) Width of body 0. 100 0. 074 - 0. 101 0. 036 - 0. 080

(3) Length of tail 0. 100 - 0. 150 0. 066 - 0. 140 0. 140 (4) Diameter of oral 0. Sucker 050 X O. 050 • 0. 028 - 0. 038 0. 040 X O. 032

(5) Diameter of ventral 0. Sucker 025 X 0. 025 0. 026 - 0. 034 0. 036

(6) Length of Stylet 0. 025 0. 021 0. 021

the living cercariae are anaesthetised with neutral red and fixed with hot formalin. - 90 -

Experimental infection of the damselfly Coenagrion puella with cercariae of Haematoloechus variegatus:- The nymphs were exposed to 15, 15, 21, 28, 30, 36, 43, 45 and 45 cercariae of H. variegatus; but the cercarial penetration was not observed. The nymphs were dissected out at different intervals. 1 & 2 cysts respectively were found in the nymphs. In 10 nymphs unexposed to cercariae, which were used as controls, one was positive. The results are summarised in Table 13. - 91 -

TABLE 13

Exposure of cercariae of Haematoloechus variegatus to the damselfly Coenagrion puella

Expt. Number Cercariae Days after Number of No. of nymphs per nymph incubation Cysts

1 1 15 17 hours 1

2 1 15 8 1

3 1 21 29 1

4 1 28 11 0

5 1 30 8 0

6 1 36 27 1

7 1 43 17 0

8 1 45 1 0

9 1 45 14 2

Control 9 - - 0 1 - - 1

= Not applicable 0 = Not found Plate 11. Encysted metacercarial cyst removed from the second intermediate host, the damselfly nymph Coenagrion puella X 440.

Plate 12. Excysted metacercaria X 88. ED - 94 -

Figure .9. Metacercarial cyst removed from the nymph, Coenagrion puella. Fig. 9

oral sucker

ventral sucker

excretory concretions - 96 -

Figure 10. The life cycle of Haematoloechus similis. Fig. 10

adult in Frog's lung eaten by frog

eggs released metamorphosis to adult in faeces }

metacercaria (in haemocoel )

eaten by penetrates Planorbis pianorbis Coenagrion(nymph)

xiphidiocercaria hatching on ingestion emerges T mother s orocyst

daughter sporocyst - 98 -

Cysts were found usually in the haemocoel or either attached by a few strands of connective tissue or free in the body cavity. The cyst was 0. 075 mm long and body with spines (Fig. 9) (plate 11). The excysted metacercaria was obtained following treatment of 0. 5% trypsin (pH 6. 2) for 20 minutes at room temperature. The metacercaria which became active and revolved within the cyst measured 0. 18 r4m long and 0. 18 mm broad. The excretory vesicle was filled with cā.rcretions (plate 12). The metacercaria and the developing adult stage are not described. Attempts to maintain the complete life cycle stages of Haematoloechus similis and H. variegatus have not been successful. Only the cercarial stages in the life history were obtained. The life cycle of H. similis is shown in fig. 10. Cercariae were studied in the living condition using both phase contrast and bright field microscopy because certain morphological features were not easily observed in killed and fixed specimens. The excretory system of the cercariae was most difficult to make out. The best result can be obtained from high power studies of an abundance of living material. - 99 -

DISCUSSION

Haematoloechus is a common trematode in the lungs of frog. The complete life cycle of various species of this genus has been studied and numerous molluscan and insects as intermediate hosts have been reported from other workers; these data are summarised in Table 1. The eggs discharged in the lungs are carried in the fluid of the lung by ciliary activity through the glottis into the mouth cavity, then they are swallowed, pass through the gastro-intestinal tract and are voided with the faeces. The miracidia hatch only when the eggs are ingested by a proper snail. It hatches in the host's intestine and enters the wall and develops to the mother sporocyst. When the mother sporocyst is mature it bursts to release the first generation of daughter sporocysts; these, in turn, produce several more generation of daughters eventually packing the digestive gland and gonad. The daughter sporocysts produce the cercariae. When freshly collected adults were crushed in distilled water at room temperature, no miracidia emerged. It seems that an incubation period is necessary for emergence of miracidia and that in nature such a period occurs before ingestion of viable eggs by the snail host. Young snails, in general, were more susceptible to successful penetration and succumbed more readily and the size of the host snail has a considerable effect on the development of both mother and daughter sporocysts. Snails sized 0. 645 mm - 2. 15 mm long and 0.860 - 2. 15 mm broad gave the best result in the present experiment. Temperature is important in the rate of development of the inter molluscan stages of digenetic trematodes (Smyth, 1966 and Wright, 1971). Development in the snails ceases during the winter, but it was artifically induced to continue by raising the temperature to 21°C. The eggs after 20 days incubation were fed to the snail Planorbis planorbis and 88 days old eggs (stored at 25°C) were still - 100 - infective; the latter began to shed cercariae after 128 days (Table 7) Kagan (1951) observed in the life history of Neoleucochlor- idium problematicum, that the eggs were viable for 195 days after removal from the bird host. Hatching normally took place after the eggs were eaten by the intermediate host, but it may occur after incubation in saline under refrigeration. Pearson (1956) while tracing the life cycles of Alaria arisaemoides and Alaria canis, observed that the eggs of A. arisaemoides were hatched out in about two weeks at 21 - 24°C. The first fully developed miracidia were seen within the egg after six weeks; a few had hatched out after three months. When 1 year old eggs were put together with snails, Planorbula armigera, they began to shed cercariae 18 days later, demonstrating that the miracidia were still infective. Wright & Bennett (1964) studied the life cycle of Notocotylus attenuatus. The eggs of the parasite hatch only after ingestion by the snail host and are not infective to snails after about 3 days in water at room temperature. They noticed that eggs kept for 105 days in a refrigerator were still infective. Kingston (1965), studied the life cycle of Tanaisia zarudnyi, when fully developed eggs pass out of the kidney of the host with urinary excretions and the egg is ingested by the first intermediate host and it hatches in the host's intestine. The eggs were resistant to low temperature (- 15°C) for 30 - 60 days and - 49°C for 24 - 48 hours and remained unhatched and viable for periods exceeding three years at 2°C. The purpose of this investigation was to. determine the life cycle of Haematoloechus similis and H. variegatus and to study its development in the intermediate hosts. The intermediate host has a very important role in biology. The successful completion of a life cycle depends on the fact that intermediate and definitive hosts occur in the same ecosystem. Snails, insects and frogs all associated with an aquatic environment, serve as first, second and - 101 -

definitive hosts respectively. The snail host Planorbis planorbis serves as a first intermediate hosts for both species of Haematoloechus similis and H. variegatus. Grabda (1960) was the first to describe the life cycle of Haematoloechus similis. The snail first intermediate host Planorbis planorbis was exposed to eggs and after 24 hours their faeces contained a large number of empty egg shells. After 40 days, the snails were examined. The sporocysts are small bags of spherical or oval shape. Each sporocyst contains 1 - 3 mature cercariae. The measurements of the sporocysts are 0. 163 to 0. 600 mm by 0.132. to 0. 273 mm. Attempts to determine the life cycle of Haematoloechus similis were made by exposing laboratory-reared snails Planorbis planorbis to eggs. At 32 days old, the daughter sporocysts varied from 0.175 mm - 0. 45 mm long and 0.125 - 0. 18 mm wide. The sporocyst with irregular constrictions and a number of cercarial germ balls in various stages of development. Xiphidiocercariae were first observed to emerge from the snail on the 56th day during summer. Grabda (1960) exposed Coenagrion hastulatum and C. armatum to 15 to 20 cercariae and 4 to 6 mature metacercariae developed after a month. In C. pulchellum the metacercariae were found dead on the second day only. Coenagrion puella is common in England and Ireland and local in Scotland (Table 2). When 30, 50 numbers of cercariae Haematoloechus similis were allowed to attach to Coenagrion puella, tailless (unencysted) metacercariae were found moving free in the body cavity on the following day, while on 2, 3, 4 and 5 days no metacercariae were found (Table 10). Ecological specificity is an important factor and can influence the course of the whole life cycle; like miracidia, the cercariae may also be selectively attracted to

certain hosts. The cercariae of H. similis shows penetration to

- 102 -

Coenagrion puella but failed to develop further and died. The xiphidiocercariae are well known by the following important characters:-

(1) The structure of the excretory system, i.e. the excretory formula and the shape of the vesicle.

(2) The structure and degree of development of the digestive system.

(3) The number and character of arrangement of the gland cells in the body of the larva.

(4) The armament.

(5) The presence or absence of a fin-fold on the tail.

(6) The number of sensillae and the pattern on the body of the ce rcaria.

(Ginetsinskaia at Dobrovol'skii, 1968).

Dawes (1968) divided the xiphidiocercariae (stylet cercariae) into the following groups:-

1. Cercariae Microcotylae:- (1) Cercariae are very small.

(2) The ventral sucker is smaller than the oral and situated behind the mid-body.

(3) The tail is not forked, no fin-fold.

(4) The tail is not of very different length from the body.

(5) The excretory system is very simple.

2. Cercariae virgula:- (1) Presence of two pyriform sacs are fused in the median line known as "Virgula organ". Situated near the oral sucker.

(2) Ventral sucker is smaller than the oral.

- 103 -

(3) Fin-fold absent.

(4) Excretory vesicle V-shaped.

3. Cercariae ornatae :-

It is divided into two species:-

(1) Cercaria ornata:- Fin- fold -extending along the entire tail; ventral sucker larger than the oral.

(2) Cercaria prima:-

Fin-fold extending only along the distal half of the tail; ventral sucker smaller than the oral.

4. Cercariae armatae:- (1) The body and tail are of equal length.

(2) Fin-fold and virgula are absent.

(3) Oral and ventral suckers of equal size.

(4) Y- shaped excretory vesicle.

Ginetsinskaia & Dobrovol'skii (1968) in their review article gave the key for identification of stylet cercariae to the family level (does not include many families of the Plagiorchiata suborder).

1. (2) Excretory formula 2[(1 + 1) + (1 + 1] = 8 or 2 1(2 + 2) + (2 + 2)] = 16. Cercariae develop in marine Prosobranchia. Microphallidae, Travasov, 1920.

2. (1) Excretory formula 2 [(2 + 2 + 2) + (2 + 2 + 2)] = 24 or 2 [3+ 3+ 3)+(3+ 3+ 3).]= 36.

3. (10) Cercaria lacking in virgula.

4. (9) Four pairs or more of penetration glands. Ciliated cells grouped in twos or threes. Cercariae develop in molluscs of the Pulmonate order. - 104 -

5. (6) Principal collecting canals empty into lateral branches of vesicle terminally or slightly subterminally. Fin-fold on tail present or absent. Plagiorchiidae Liihe, 1901.

6. (5) Principal collecting canals empty into branches of vesicle about midway along their length. Fin-fold absent on tail.

7. (8) Branches of vesicle relatively short and in uncontracted cercaria do not go beyond the level of the posterior border of the ventral sucker. Telorchiidae. Stunkard, 1924.

8. (7) Branches of vesicle long and extend far anteriorly beyond level of posterior border of ventral sucker. Ochetosoma- tidae Leao, 1945.

9. (4) Not more than four pairs of penetration glands. Excretory formula 2 C(2 + 2 + 2) + (2 + 2 + 2)] = 24. Cercariae develop in fresh water Prosobranchia (genus Bithynia). Prosthogonimidae Nicoll, 1924. 10. (3) Cercaria have virgula. Lecithodendriidae Odhner, 1910..

One of the most important features of the xiphidiocercariae group is the presence of the stylet, inside the oral sucker, which can penetrate into the body of the second intermediate host. The stylet cercariae are larvae of trematodes of families Plagiorchiidae, Lecithodendriidae, Ochetosomatidae, Telorchiidae, Prosthogoni- midae, Ophisthogonimidae, Lissorchiidae (Ginetsinskaya & Dobrovol'skii, 1968). Molluscs, larvae of aquatic insects, tadpoles, crustaceans can serve as the second intermediate host for stylet cercariae. Cercariae penetrate molluscs, oligochaetes, hirudineans and polychaetes which serve as intermediate hosts. The cercaria of Haematoloechus similis is very small in size and the body proportions and the structure of sporocysts shows resemblance to the "prima" group of cercariae ornatae, but lack of - 105 -

Figure 11. (a) Cercaria of Haematoloechus similis.

(b) Stylet of cercaria of Haematoloechus similis (After Grabda, 1960). Fig. 11

oral sucker

ventral sucker penetration glands

excretory bladder

stylet - 107 - fin-folds on tail and excretory system resemble Cercariae armatae. According to Grabda (1960) the specific features are well developed and differently situated penetration glands and lack of cystogenous glands. (Fig. 11). - 108 -

Skrjabinoeces similis (Looss, 1899) Sudarikov,- 1950

Synonymy:- Distoma simile Looss, 1899, nec Sonsino, 1890, Haematoloechus similis Looss, 1899, Pneumonoeces similis Looss, 1899, Pneumonoeces similis Looss, 1902, Haematoloechus similigenus Stile & Hassall, 1902.

Stiles & Hassall (1902, 1920) proposed this name for Haematoloechus similis Looss, because Looss initially called the worm Distoma simile, which is a stillborn homonym of Distoma simile Sonsino, 1890 (Prudhoe & Bray, in preparation). The life cycle of Haematoloechus (= Skrjabinoces) similis is important for the taxonomy of subfamilies of the Pneumonoecinae (syn. Haematoloechina.). In 1950, V. E. Sudarikov, subdivided the genus Haematoloechus into three genera: Pneumonoeces, Ostiolum and Skrjabinoeces. Odening (1958) suggested all three genera as as subgenera of the genus Haematoloechus, but in 1960 he accepted Ostiolum as separate genus and Neohaematoloechus as another genus. He regarded the groups Skrjabinoeces and Pneumonoeces as subgenera. Species of the genus Pneumonoeces have cercariae belonging to the ornata group, whereas the cercariae of Skrjabinoeces belong to the armata group and have a completely different type of structure. In the review article of Ginetsinskaya & Dobrovol'skii (1968) they suggested that such different cercariae cannot be included in the same genus. Von Thiel, (1930), discovered that cercariae from Planorbis vortex penetrated the larvae of Anopheles maculipennis forming metacercariae different from those described by Sinicyn (1905). Grabda (1960) found metacercariae in the genus Coenagrion identical to those described by Sinicyn as H. variegatus. Ginetsinskaya & Dobrovol'skii (1968) found the shape of cercaria Pneumonoeces variegatus (Fig. 12) is very similar to that described by Von Thiel (1922) - 109 -

Figure 12. Cercaria of Haematoloechus variegatus (After Ginetsinskaia & Dobrovol'skii, 1968). Fig. 12

oral sucker

penetration glands

flame cells ventral sucker

excretory bladder

tail fin When the eggs of Haematoloechus variegatus were experimentally fed to Planorbis planorbis, during this work, the cercaria emerged. The nymphs Coenagrion puella were exposed to cercariae of H. variegatus. Cercarial penetration was not observed but cysts were found (Table 13). Out of 10 nymphs unexposed to cercariae which were opened as a control, 1 was positive; no attempt was made to rear the insect in the laboratory. The insects might be naturally infected. It is better to rear the damselflies in the laboratory; they should then be parasite free and the absence of snails should ensure that the insect would be free from infection. Unfortunately, said material was not available for the present studies . In other studies on related species, Krull (1933) observed no penetration of the cuticle by cercariae and the nymphs became infected only through the rectum. Ingles (1933) noticed the cercariae were indifferent to the nymphs and later ate the cercariae. Schell (1965) observed no penetration when young nymphs of the dragonfly Aeschna multicolor were placed for several hours with cercariae of Haematolochecus breviplexus but found encysted cercariae in the walls of the branchial basket of the rectum. - 112 - SUMMARY

Specimens of Planorbis planorbis, the fresh water snails, were collected from Romney on June 14, 1977, maintained in plastic aquaria and aerated by an electrical air-pump. One third of the water was changed once in a week. The routine diet of the snails was boiled and dried lettuce leaves. Rana ridibunda from Yugoslavia were purchased from a supplier. Haematoloechus similis and H. variegatus are common lung flukes of the frog. The eggs were teased out of the uterus of Haematoloechus spp. and incubated at 25°C for two weeks. The water was changed every week to provide aeration, reduce bacterial and fungal growth. Very young laboratory bred snails Planorbis planorbis were allowed to feed on the eggs of Haematoloechus and a number of snails were dissected at various intervals. The intermolluscan phase of development took 56 days in summer and snails started shedding cercariae from then onwards. Larval stages were recovered from the snails by crushing the shell. Infected tissues and parasitic stages were transferred to a slide and examined, alive under coverslip pressure. 43 experiments were conducted from June 20, 1978 to February 28, 1979. Development in the snails ceased during winter, but it was artifically induced to continue by raising the temperature. Planorbis planorbis was found to serve as first intermediate host for both species. Cercariae appear in the water in greatest numbers in the early morning. - No attempt was made to rear the insect, the intermediate host, in the laboratory. Coenagrion puella common in England, Ireland and Scotland were received from Biosery Ltd. andmaintained in the laboratory. These were exposed to cercariae of H. similis and the cercarial penetration was observed. Unencysted slow moving metacercariae were found on the following day but failed to develop and died. - 113 -

Coenagrion puella were exposed to cercariae of H. variegates; no cercarial penetration was observed. The nymphs were dissected out at different intervals. Cysts 1 & 2 were found in the nymphs. Out of 10 nymphs unexposed to cercariae which were opened as a control, 1 was positive. Unfortunately the quoted intermediate insect host Coenagrion hastulatum was not available for the present studies. The metacercaria and developing adult stages are, therefore, not described. - 114 -

PART II

1. In vitro hatching of the eggs of Haplometra cylindracea (R.. temporaria)

2. In vitro hatching of the eggs of Haematoloechus (R. pipiens)

3. In vitro hatching of the eggs of Haematoloechus (R. ridibunda) - 115 - INTRODUCTION

Egg hatching:-

Two types of hatching are found in trematode eggs. Type I egg:- Those that hatch in water before reaching the specific host; the eggs are large and have thin shells. These are common in Schistosomatidae, Fascioloidae and Troglotrematidae. Type II egg:- Those that hatch on ingestion by a suitable host; the eggs are generally smaller than type I eggs and have a thick shell. These are common in Opisthorchiidae, Brachylaemidae and Plagior chiidae . Very few attempts have been made to examine the hatching of the eggs in vitro of those species whose eggs hatch inside the mollusc host. Considerable work, however, has been carried out on eggs that hatch in water. The hatching of eggs Type II in vitro has been examined by Russel (1954), Ratcliffe (1968) and Mitterer (1975). As stated earlier the eggs in this group are generally small and thick shelled. The hatching occurs only after the eggs have been ingested by a suitable host snail; presumably physical and chemical factors present in the host's gut bring about hatching. A trematode egg consists of a well developed egg shell or capsule which encloses the ovum and a number of vitelline cells, an operculum is present except in the Schistosomatidae. The formation of the egg has been reviewed by Smyth (1976) and is generally well known. The release of ova from the ovary into the oviduct is apparently controlled by a spincter muscle or oocapt (= ovicapt); a number of vitelline cells are released from the vitellaria, together with spermatozoa from the receptaculum seminis. The ootype is the chamber where the eggs are formed; it is surrounded by a complex collection of gland cells called the "Mehlis gland". The function of the gland is not clear, but their - 116 -

secretion may assist the release of the shell globules released from the vitelline cells. Another view is that it forms a lipo protein coat on the eggshell. The shape of the egg appears to be dependent upon the shape of the uterus. In the majority of trematodes the egg shell is made up of Sclerotin, a tanned protein, covered by a lipo protein (part of vitelline cells). Phenolase Phenol quinone` tanned = Sclerotin protein Protein. ) Protein In the Paramphistomidae (e. g. Paramphistomum), the chief structural protein in the eggshell appears to be keratin (Madhavi 1966, 1968). The egg shell formation and histochemical methods for examining it in trematodes are given by Smyth (1954, 1966) and Clegg (1959), Burton (1963), Erasmus (1972) Ramalingam (1973) and various other workers. The fully embryonated egg is lined by a thin vitelline membrane, which also encloses a pad-like viscous cushion between the anterior end of the miracidium and the operculum. The viscous cushion is apparently not present in every egg, e. g. Neodiplostomum intermedium (Pearson, 1961), Paramphistomum hibernia (Willmott, 1952). Trematode eggs when laid may be (a) fully embryonated, e.g. Haplometra cylindracea (b) partially embryonated, e.g. Haematoloechus or (c) unembryonated, e. g. Fasciola hepatica. In the case of (b) and (c) in order to complete the embryonation, the time required varies from one week to three weeks. The chief factors controlling embryonation appears to be temperature, oxygen, moister and pH. Fully embryonated eggs clearly represent the resting infective stage. Hatching of infective eggs, may be regarded as a - 117 -

part of the process of infection. "Hatching" of the egg as used here means the separation of the fully developed miracidium from the egg shell. Factors affecting hatching: Type I egg:- Hatching of the egg in water depend upon the salinity, osmotic pressure and various other factors. Some or all of these factors stimulate the activation of miracidia, the vigorous beating of cilia and probably the action of hatching-enzyme cause the release of operculum. The hatching mechanism in several species (especially Fasciola) has been described by several workers such as Krull (1934), Rowan (1956, 1957), Wilson (1968), Azimov & Nazarov (1972), and Mitterer (1975). The hatching mechanism of Schistosome egg has been ' studied by Faust & Hoffman (1934), Ingalls et al (1949), Maldonado et al (1950), Sugiura et al (1954), Standen (1951), Lengy (1962), Newsome (1962), Abousenna & Bassaly (1964), Kusel (1970), Pitchford & Visser (1972), Becker (1973), Bair & Etges (1973), and Kassim & Gilbertson (1976). Friedl (1960) reported the hatching of Fascioloides magna egg. Iwasaki (1960), observed the egg of Gigantobilharzia sturniae. Kawashima & Miyazaki (1961), studied the egg of Paragonimus ohirai while Tongson & Semilla (1965) of Fasciola gigan.tica egg. Type II egg:- Factors which are likely to be important in the hatching of this type of egg include (1) the feeding habits of the snail (2) physico- chemical nature of the gut especially (a) pH (b) Redox potential or reducing conditions (i. e. Eh) (c) oxygen (P02) (d) digestive enzymes (e) carbondioxide PCO2 (f) temperature (g) osmotic pressure. The digestive system of Pulmonates has been well reviewed - 118 -

by Hyman (1967) and Owen (1966). A major early contribution was the work of Carriker (1946). Although this work was published thirty-three years ago, detailed information is available for Lymnaea stagnalis (Carriker, 1946). The sorting, retention time and rate of transport of food and of non-food particles in the digestive system of Lymnaea stagnalis has been studied by Veldhuijzen (1974). Lymnaea stagnalis is generally considered to be herbivorous. Veldhuijzen (1974) suggested that it is more omnivorous than herbivorous as it is feeding on dead snails, larvae and faeces. Jager's (1971) and Veldhuijzen (1974) found it to be a continuous feeder. The enzymes present in the crop juices of pulmonate s have been reported by Evans & Jones (1962 a, b) Stone & Morton (1958), Jeuniaux (1954), Myers & Northcote (1958), Boer et al (1967) and various others. Present experiments:- The eggs of Haplometra cylindracea hatch only on ingestion by the snail. Linstow (1890), Lūhe (1909), Wesenberg Lund (1934), Combes (1968), Grabda-kazubska (1970), Dobrovol'skii & Rajhel (1973) worked out the life cycle of Haplometra cylindracea. Dobrovol'skii & Rajhel (1973) found Lymnaea ovata to act as intermediate host. Grabda-kazubska (1970) found infection in L. palustris. The structure of the egg and the morphology of mature miracidium of Haplometra cylindracea has been studied by Dobrovol'skii (1965). In the present experiment attempts were made to obtain experimental infection in snails of various species of laboratory breeds of healthy snails such as Physa sp, Planorbis planorbis, Lymnaea peregra, L. stagnalis, L. palustris and L. truncatulus. Unfortunately only negative results were obtained. The snail host and asexual generation, therefore, - 119 -

remain undetermined. It was, therefore, decided to attempt to hatch the eggs in vitro attempting to relate the physico-chemical conditions employed in vitro to the physiological environment in vivo. These results also were almost entirely negative, but methods are reported here for the benefit of future workers. - 120 -

MATERIALS AND METHODS

Incubation of eggs:-

Haplometra cylindracea was obtained from Rana temporaria from commercial sources, and eggs were removed as for Haematoloechus (R. ridibunda). After washing the eggs were incubated at 25°C for two weeks. The water was changed every week. A stock solution of 0. 4% NaCl was prepared both in distilled water and triple distilled water. All test solutions used were made up in. 0.4% NaCl, Hank's solution or buffer solution. The stock buffer solutions of disodium hydrogen phosphate-sodium dihydrogen phosphate (0. 1M) were prepared according to Dowson et al (1959). The pH of solutions was adjusted by addition of 0.1N NaOH or 0. 1.NHC1.with pH meter. Egg hatching experiments were conducted (a) aerobically (i.e. without gassing)(b) anaerobically, in a gas phase of 95% Nitrogen and 5% CO2 . (The term "gas" used throughout refers to this phase). To produce completely anaerobic conditions a (30 ml) Falcon flask was sealed with rubber bung, two syringes were placed through the bung and inserted into a rubber tubing inside the flask, one being connected by tubing to a gas cylinder and the other to a compressed air outlet. After gassing or the addition of eggs or solution, the syringes were sealed (Fig. 13 ). In some experiments using watch glasses and Leighton tubes, the gas was bubbled through the open vessel but completely anaerobic conditions were not established. In determining the effect of osmotic pressure, enzymes, reducing agent, temperature, bile salts, gas and sodium hypochlorite on egg hatching, a series of vial tubes (30 ml) and Leighton tubes (Bellco) were employed. After every treatment the eggs were washed with 0. 4% NaCl. The repeated washing served to remove the traces of enzymes, reducing agent, bile salts and sodium hypochlorite, etc. Most of the experiments were conducted at - 121 - room temperature but the experiments conducted in constant temperature were carried out in incubators. Observations were made every 1 hr to 6 hrs. Sometimes the observations were made over 8 hrs. The eggs were discarded after observation. The enzymes were prepared immediately prior to use. The reducing agent solution was prepared fresh for each experiment. Distilled water was added to the eggs in the controls. Reagents used in Egghatching:- The solutions were freshly made each time they were used. Enzymes:- (1) Amylase (Sigma)

(2;) Amyloglucosidase (Sigma) (3) Cellulase (Sigma) (4) Chitinase (Sigma) (5) Diastase (Sigma) (6) Papain (Harrington Brothers Ltd. ) (7) Pancreatin (Sigma) (8) Pepsin (Sigma) (9) Trypsin (Sigma) Bile:- (1) Sodium glycocholate (Sigma) (2) Taurocholic acid (Sodium Salt) (Sigma) (3) Taurocholic acid (crude) (ICN Pharmacueticals Inc.) Reducing agents:- (1) Glutathione (crystalline) (Sigma) (2) Sodium dithionite (Sigma) (3) L-cysteine (Hydrochloride Hydrate) (Sigma) (4) L-ascorbic acid (BDH) Other reagents:- (1) Disodium hydrogen phosphate (BDH) (2) Dihydrogen sodium phosphate (BDH) (3) Sodium bicarbonate (analar) (Hopkin & Williams) (4) Hank's solution (Difco) (5) Sodium hypochlorite (BDH) (6) Sodium chloride (analar - BDH) - 122 -

Figure 13. Complete anaerobic condition applied in the case eggs of Haplometra cylindracea in vitro. Fig. 13

cling film

falcon flask syringe

tubing - 124 -

I IN VITRO HATCHING OF THE EGGS OF HAPLOMETRA CYLINDRACEA

Experimental procedure and results:-

1. Effect of osmotic pressure:- To determine the effect of osmotic pressure on the egg, the experiments were conducted with concentrations of NaC1 solution from 0. 1% to 0. 8%. Results were negative but in 0. 3% NaC1 non-motile miracidia came out of 3-4 eggs on the following day.

2. Effect of Hydrogen, ion concentration:- To determine the effect of hydrogen ion concentration on the egg, experiments were conduced in which the pH varied from 2. 5 -

11. 1..

2.1 Experiment:- The eggs were placed in Pancreatin. a., 0.05% (pH 11.1). b, 0. 1% (pH 10. 9). c, 0.. 2% (pH 9.8). d, 0.3% (pH' 9.2). e, 0.4% (pH 9.0). f, 0.5% (pH 8.6). The results were negative .

2.2 Experiment:- The eggs were exposed to gas for 2 minutes followed by 0. 5% amylase with different pH 5. 0, 6, 8, 7. 0 and 7. 2.

2.3 Experiment:- The eggs were incubated in 0. 05% pepsin (pH 2. 6), 0. 1% (pH 2. 6), 0. 5% (pH 2. 5) and 1% pepsin (pH 2. 6) for 1 hr and rinsed twice with 0.4% sodium chloride to remove the pepsin followed by 0. 02% sodium taurocholate (pH 7. 6) and 0. 02M sodium dithionite (pH 7. 2) and were then exposed to gas for 1 minute. Movement inside the egg in 0. 05%, 0. 1% and 0. 5% pepsin was observed. - 125 -

2.4 Experiment:- The eggs were incubated with 0.1M phosphate buffer in which the pH varied from 7. 2 - 8. 0 and were exposed to gas for 1 minute and a few drops of 0. 02M cysteine were added. The eggs were held anaerobically. 2.5 Experiment:- The eggs were incubated in 0. 5% pepsin (pH 2. 5) for 1 hr and washed with two changes of 0. 4% NaCl. This was followed by 0. 5% pancreatin (pH 6. 8, 7. 0, 7. 8 and 8. 2). The results were negative. 2. 6 Experiment:- The eggs were incubated in 0. 5% pancreatin (in Hank's solution .with different pH 6. 8, 7. 0, 7. 8 and 8. 2). 2..7 Experiment: 0. 4% and 0. 5% NaCT with a range of pH 5. 5 8. 5 were used. The eggs were exposed to gas. 2.8 Experiment:- The eggs were incubated in 0. 1M buffer solution in which the pH varied from 6. 2 - 8. 0 and 1 ml of 0.02M cysteine were added and exposed to gas for 1 minute. The results were negative. 2. 9 Experiment:- The eggs were placed in constant pH 2. 5 of triple distilled water, 0. 4% NaC1, Hank's solution, 0. 5% pepsin and 1% pepsin. The results were negative. 2. 10 Experiment:- The eggs were placed in 0. 5% pepsin (pH 2. 5) for 20 minutes. The eggs were washed free of pepsin with 0. 4% NaC1 twice followed by in solution of 50 ml of 0. 02% sodium taurocholate and 50 ml of 0. 5% trypsin (pH 6. 2, 6. 4, 7. 0, 7. 4 and 8. 0). They were exposed to gas. One set was without gas exposure. The results were negative. 2. 11 Experiment:- The eggs were placed in a mixture of 50 ml of 0. 02% sodium taurocholate and 50 ml of 0. 5% trypsin (pH 6. 2 - 8. 0). They were exposed to gas. The results were negative. - 126 -

2. 12 Experiment:- The eggs were placed in an equal amount of 0.1N sodium bicarbonate and 0. 03N sodium dithionite diluted into distilled water (pH 7. 1, 7. 4, 7. 7 and 8.0). Then they were exposed to gas for 1 minute. The results were negative. 2. 13 Experiment:- The eggs were placed in 0. 1M phosphate buffer at pH value from 6. 2 - 8. 0. In one set, a few drops of 0. 03M ascorbic acid and in another a few drops of 0. 4M cysteine were added. They were exposed to gas for 1 minute. The results were negative. 2. 14 Experiment:- The eggs were kept in 0. 5% amylase at a pH of 5. 0, 6. 8, 7. 0 and 7'. 2. The results were negative. 2.15 Experiment:- The eggs were placed in 0. 5% trypsin at a range of pH of 6.4, 7. 5 and 8. 0 for 1 hr 40 minutes followed by two rinses in 0.4% NaCl and incubated in 0. 4% sodium chloride. The results were negative. 3. Effect of pepsin and trypsin To determine the effect of pepsin and trypsin on the egg, experiments were conducted at concentrations 0. 025 - 0. 5% and at a range of pHs from 2. 0 - 7. 5. 3.1 Experiment:- The eggs were pre-treated with 0. 5% pepsin (pH 2. 0) for 1 hr followed by washing twice in 0. 4% NaCI and held in 0. 3% trypsin (pH 6.8). The results were negative. 3.2 Experiment:- The eggs were placed in 0. 025% pepsin (pH 2. 5 in Hank's solution) and exposed to gas for 1 minute. One set was without gas exposure. The results were negative. 3.3 Experiment:- The eggs were placed in 0. 3% trypsin (pH 7. 5)andwere exposed to gas for 1 minute followed by two rinses in 0. 4% NaC1 - 127 - then incubated in 0. 5% pepsin (pH 2. 3). The results were negative. 3.4 Experiment:- The eggs were placed in 0. 5% pepsin (pH 2. 6) for 40 minutes and followed by two rinses in 0. 4% NaCl and held in 0. 3% trypsin (pH 3. 0). The results were negative. 3.5 Experiment:- The eggs were placed in 0. 5% pepsin (pH 2. 5 and 2. 3) for 30 minutes and followed by washing twice in 0. 4% NaCl and held in 0. 3% trypsin (pH 6. 6 and 7. 5).

4. Effect of temperature:- Experiments were conducted to find out whether the temperature played an important part in the hatching of eggs. 4. 1 Experiment: The eggs were placed in 0. 5% pepsin (pH 2. 2). They were exposed to gas for 5 minutes and were held at 30°C for 22 hrs then incubated at 20t,.. 25°C and 30°C. The internal structure shrunk. 4. 2. Experiment:- The eggs were placed in 0. 5% pepsin (pH 3. 2) and 2ml of 2% ascorbic acid. Each set was held at 4°C, 20°C, 27°C and 30°C and a few drops of 6% cysteine were added after 2 hrs and 5 minutes. At 4°C and 27°C a few eggs showed movement inside the eggs after 2 hrs. 4.3 Experiment:- The eggs experiments were conducted anaerobically with 0. 3% trypsin and 2% sodium taurocholate (a) a solution of 50 ml of 0. 3% trypsin and 5 ml of 2% sodium taurocholate was used at 24t. (b) a solution of 50 ml of 0. 3% trypsin and 5 ml of 2% sodium taurocholate at 37°C was held for 1 hr and then shifted to room temperature at 24°C. The results were negative. 4.4 Experiment:- The eggs were placed in 0. 5% pepsin and incubated at various temperatures - 20°C, 24°C, 28t and 37°C. At 20°C, 24°C - 128 - and 28%. the outer-most layers of the eggs were separated; this was observed after one hour. 4.5 Experiment:- The eggs were placed in 0. 5% pepsin (pH 2. 5) plus a few drops of 6% cysteine, a few drops of 2% ascorbic acid and incubated at 4°C, 20°C, 24°C and 30°C. At 20°C and 30°C the internal structure of some eggs were shrunk. 4. 6 Experiment: The eggs were placed in 0. 5% amylase (pH 7. 2) and 3 drops of 0. 4M cysteine; they were exposed to gas for 5 minutes then incubated in a roller drum for one hour. Movement in some eggs was observed. 4. 7 Experiment:- The eggs were kept in 0. 5% amylase (pH 7. 2) and 3 drops of 0. 4M cysteine and were exposed to gas for 5 minutes and incubated at 20°C, 25°C and 37°C. The results were negative. 4.8 Experiment:- The eggs were placed in 0. 5% pepsin at 20°C, 25°C, 30°C and 37°C. The results were negative. 4.9 Experiment:- The eggs were kept in 0. 5% amylase (pH 7. 2) and 3 drops of 0.4M L-cysteine were added; they were exposed to gas for 5 minutes. One set was incubated at 4°C. The outer-most layer became separated off and movement inside the egg was observed. 4. 10 Experiment:- The eggs were placed in 0. 5% oc-amylase (pH 9. 2) and a few drops of 0. 4M L-cysteine. They were exposed to gas for 5 minutes and incubated for 1 hour on the roller drum; they were then placed at 4°C, 17. 8°C, 20 C, 24. 3°C and 30 C. The results were negative. 4. 11 Experiment:- The eggs were incubated at 0. 5% amylase (pH 9. 0) and 2m1 of 6% cysteine, and were exposed to gas for 1 minute and - 129 - incubated at 4°C, 20°C, 18.9°C, 24. 3°C and 31°C. The results were negative. 4. 12 Experiment:- The eggs were placed in a mixture of 0. 5% pepsin (pH 3. 2) and a few drops of 2% ascorbic acid and 6% cysteine. They were exposed to gas 2 minutes and incubated at 4°C, 15.6°C, 20°Q, 27°C, 30°C, and 37°C for 5 hours and 25 minutes, then they were placed at room temperature 27°C. Several miracidia were seen moving inside the eggs after 5 hrs and 15 minutes. 5. Effect of gas To determine the effect of gas on the egg, experiments were conducted in which the time of exposure of gas varied from 1 to 15 minutes. 5.1 Experiment:- The eggs were placed in 0.4% NaCl and were exposed to gas for 1 to 15 minutes. The results were negative. 5.2 Experiment:- The eggs were placed in tap water, distilled water and "snail conditioned" water. They were exposed to gas. The gas flow rates varied from 1 to 3 minutes. The results were negative. 6.. Effect of reducing agent:- To determine the effect of reducing agents on the eggs, the experiments were conducted with different concentration of cysteine, ascorbic acid and sodium dithionite. 6.1 Experiment:- Eggs were placed in 0. 5% pepsin (pH 2. 5) and equal amounts of 6% cysteine and 2% ascorbic acid. After one hour 35 minutes, there were slight movements inside the eggs. One egg showed the beating of cilia. 6.2 Experiment:- The eggs were placed in (a) 0.4% pepsin (pH 3. 2) (b) 0. 4% trypsin (pH 7. 0) and various combinations of reducing solutions (c) 0. 4% pepsin and 0. 1% ascorbic acid and (d) 0. 4% pepsin and - 130 -

0. 1% cysteine (e) 0. 4% pepsin, 0. 1% ascorbic acid and 0. 1% cysteine (f) 0. 4% trypsin (pH 7. 0) and 0. 1% ascorbic acid (g) 0. 4% trypsin (pH 7. 0), 0. 1% ascorbic acid and 0. 1% cysteine. Each set was incubated on a roller drum for one hour and placed at 27°C. Some movement was observed in 0. 4% pepsin (pH 3. 2). 6. 3. Experiment:- After the pretreatment of 0. 5% pepsin (pH 3. 2) for one hour followed by two changes of NaC1 to remove the pepsin, eggs were held at trypsin 0. 5% (pH 8. 2). The eggs were then placed in various combinations of solutions (a) a mixture of 1% ascorbic acid and 1% cysteine (b) 1% cysteine (c) 1% ascorbic acid (d) 3 drops of mixture of 5m1 of 1% cysteine and 5 ml of 1% ascorbic acid (e) 5 drops of 1% cyste-ine. There was movement in the eggs in trypsin 0.5% (pH 8.2) (c) and (d). 6.4 Experiment:- The eggs were kept in 0. 1M buffer solution in which the pH varied from 6.2 - 7.0 and 0. O1M, 0. 02M, 0. 03M, 0. 04M and 0. 05M ascorbic acid were used and the eggs were then exposed to gas for 1 minute. Negative results were obtained. 6.5 Experiment:- The gas was bubbled through the eggs for 2 minutes, 5 minutes and 10 minutes, and 0. 03M sodium dithionite was used. Negative results were obtained. 6.6 Experiment:- Each tube was exposed to the gas for 5 minutes, the eggs were added anaerobically in 0. 3% trypsin at pH 7. 0. In another set the eggs were already in the 0. 3% trypsin (pH 7. 0) and then gassed for 5 minutes. The results were negative. 6. 7 Experiment:- The eggs were placed in 0. 02M sodium dithionite and 0. 3% trypsin (diluted into buffer solution pH 7. 0) and were exposed to the gas for 1 minute. The experiment was conducted anaerobically. The results were negative. - 131 -

6. 8 Experiment:- The eggs were placed in 0. 5% pancreatin (pH 2.6) and 5 m1 of 0. 4M cysteine and exposed to the gas for 5 minutes. The results were negative. 6.9. Experiment:- The eggs were in 0. 3% trypsin (pH 7. 0) and in various combinations of solution (b) 0. 3% trypsin and 0. 02M sodium dithionite (c) 0. 02M sodium dithionite. They were exposed to gas for 1 minute. The results were negative.

6. 1.0 Experiment:- The eggs were placed in various combinations of solution

(a.) 1.0 ml of 0. 3% trypsin and. 5 m1 of 0. 02M sodium dithionite

(b) 1.0 ml of 0. 3% trypsin (pH 7. 6) and 5 ml of 0. 02M sodium dithionite (c) 10 ml of 0. 02.5% pepsin (pH 2. 1, 2. 2 and 2. 5) and 5 ml of 0. 02M sodium dithionite were used. They were exposed to gas. for 1 minute. 6. 11 Experiment:- The eggs were pretreated with 0. 025% pepsin (pH 2. 1, 2. 2: and 2. 5) followed by two changes of 0. 4% NaCl in order to remove the traces of pepsin; this was followed by 0. 02M ascorbic acid for twenty minutes. They were rinsed twice in 0. 4% Na Cl and were held in 0. 3% trypsin (pH 7. 6) and gassed for 1 minute. The results were negative. 6. 12 Experiment:- The eggs were kept in 0. 3% trypsin (pH 7. 6) and 0.02M ascorbic acid. They were exposed to gas for 1 minute. The results were negative. 6. 13 Experiment:- The eggs were kept in various combinations of solution (a) 0.4% trypsin (pH 5) and 4 drops of 1% ascorbic acid (b) 0. 4% trypsin (pH 5) and 4 drops of 1% cysteine (c) 0. 4% trypsin and 4 drops of 1% ascorbic acid and 1% cysteine. They were incubated at - 132 -

at 20°C and 27°C. The results were negative. 6. 14 Experiment:- The eggs were placed in 0.4% amylase (pH 10) with different combinations of (a) 1% cysteine (b) 1% ascorbic acid (c), a & b were incubated at room temperature and 20°C. The results were negative. 6. 15 Experiment:- The eggs were placed in (a) 0. 5% amylase (pH 9. 6) (b) 0. 5% trypsin (pH 8. 6) (c) a & b (d) 0. 5% trypsin (pH 8. 6) and 1 drop of 1% ascorbic acid (e) 0. 5% trypsin (pH 8. 6) and 1 drop of 1% cysteine (1) 0. 5% trypsin (pH 8. 6) and 1 drop of 1% ascorbic acid an.d 1% cysteine (g) 5 ml of 0.1% trypsin and 0. 5%amylase and l drop of 1% ascorbic acid (h) 5 ml of trypsin 0. 5% (pH 8. 6) and 0. 5% amylase and 1 drop of 1% cysteine were used. In (c) slow movement inside the eggs was observed. 6.16 Experiment: The eggs were kept in 0. 5% pepsin (pH 2. 6) and 1 drop of 1% glutathione and 1M cysteine. They were incubated in a roller drum for, 1 hr. The results were negative. 6.17 Experiment:- The eggs were kept in 0. 5% pepsin (pH 2. 6) with the combination of (a) 3 drops of 1M cysteine (b) 3 drops of 1% glutathione (c) a & b were used. Some movement inside the eggs in (a.), (b) and (c) was observed. 6. 18 Experiment:- The eggs were exposed to gas for 5 minutes and 0.02M sodium dithionite was added. One set was exposed to gas after 0. 02M sodium dithionite. The experiment was conducted anaerobically. The results were negative. 6. 19 Experiment:- The eggs were placed in 0. 5% pepsin (pH 3. 2), with a few drops of 1% ascorbic acid and 6% cysteine and were incubated at 27°C. There was some movement of miracidia inside the eggs after 2 hours and 53 minutes. - 133 -

7. Effect of bile salts:- To determine the effect of bile salts on the egg, the experiments were conducted with different concentrations of taurocholic acid (Sodium-salt), Sodium glycocholate. 7.1 Experiment:- The eggs were placed in 0. 5% pepsin (pH 2. 6) followed by two changes of 0. 4% NaC1, to remove the pepsin and were incubated with 2% Sodium taurocholate. The results were negative. 7.2 Experiment:- The eggs were pretreated with 0. 5% pepsin (pH 2. 0) for 1 hour followed by two washings with 0.4% NaC1, were incubated in 0.. 3% trypsin (pH 6. 8) for 1 hour, then rinsed twice with 0.4% NaC1, then they were held in 0. 01%, 0. 02% and 0.03% Sodium taurocholate (pHs 7. 2, 7. 5 and 7. 2). Negative results were obtained. 7.3 Experiment:- The eggs were placed in 0. 5% pepsin pH 2. 0 for 1 hour, followed by two rinses in 0.4% NaCl then placed in various combinations of mixtures of (a) 5 ml of 0. 01% Sodium taurocholate and 10 ml of 0. 3% trypsin (pH 7. 4) (b) 5 ml of 0. 02% Sodium taurocholate and 10 ml of 0.3% trypsin (pH 7.4) (c) 5 ml of 0.03% Sodium taurocholate and 10 ml of 0. 3% trypsin (pH 7. 0). One set was tried without the pretreatment of 0. 5 % pepsin. The results were negative. 7.4 Experiment:- The eggs were placed in 0. 5% papain (pHs 6. 1, 7.7 and 8. 0) for 1 hr. followed by two rinses in 0.4% NaCl. They were held in 0. 3% trypsin and 0. 1% Sodium taurocholate (pH 7.4). Results were negative. 7.5 Experiment:- The eggs were placed in various combinations of (a) 0. 5% pancreatin, 1. 0% Sodium bicarbonate and 0. 8 gm Sodium taurocholate (pHs 7. 5 and 8. 2). They were exposed to gas for - 134 -

1 minute. The movements in a (i), (ii) in some eggs. 7.6 Experiment:- The eggs were placed in 0. 025% pepsin (in Hank's pH 2. 5) for 15 minutes followed by two washings with Hank's solution. They were held in 0. 3 gm of trypsin and 0. 05 gm of Sodium taurocholate diluted into 100 ml of Hank's solution (pHs 6. 7, 7. 0, 7. 3 and 8. 0). They were exposed to gas for 1 minute. The results were negative. 7.7 Experiment:- The eggs were placed in a solution of 0. 3 gm of trypsin and 0.025 gm of Sodium taurocholate (crude) diluted into 100 ml of Hank's solution (pHs 6. 7, 7. 0, 7. 3 and 8.0). To each set one drop 0. 02M ascorbic acid was added. The results were negative. 7.8 Experiment:- The eggs were placed in pepsin 0. 5% in Hank's (pHs 2. 1, 2. 5, 2. 6 and 2. 8) for 3 hours. followed by two changes of Hank's to remove the traces of pepsin. The eggs were held in a mixture of 0. 3% trypsin and 0. 2% Sodium taurocholate (pHs (a) 6. 5, (b) 7. 0 (c) 7. 5). There was movement in 0. 5% pepsin (pH 2. 5) in many eggs. 7. 9 Experiment:- The eggs were pretreated with 0. 5% pepsin (pH 2. 5) for 4.0 minutes, followed by two changes of Hank's solution. They were held. in 50 ml of 0. 3% trypsin and 5 ml of 0. 1% Sodium taurocholic acid (pHs 6. 7, 7.4 and 7. 8). The results were negative. 7. 10 Experiment:- The eggs were pretreated in 0. 5% pepsin (pH 2. 5) for 40 minutes followed by two rinses of Hank's solution. They were held in a solution of 50 ml of 0. 5% trypsin and 5 ml of 0. 1% Sodium taurocholic acid with (pHs 6. 8, 7. 4 and 7. 6). In 0. 5% pepsin (pH 2. 5) many eggs showed movement inside. 7. 11 Experiment:- The eggs were placed in 0. 5% trypsin for 2 hrs and 20 - 135 -

minutes followed by two rinses of Hank's solution. They were held in a mixture of 50 ml of 0. 5% trypsin and 5 ml of 0. 1% Sodium taurocholate (pHs 6. 8, 7. 4 and 7. 6). The results were negative. 7. 12 Experiment:- The eggs were placed in 0. 1% Sodium taurocholate (in Hank's solution). The result was negative. 7.13 Experiment:- The eggs were pretreated with 0. 025% pepsin (pH 2. 0) for 15 minutes followed by two washings with 0.4% NaCl. They were placed in 0. 02M Sodium dithi.onite for 15 minutes followed by two rinses with 0. 4% NaCl. They were held in 0. 3 gm trypsin and 0. 05 gm Sodium glycocholate then exposed to gas for 1 minute. The results were negative. 7.14 Experiment:- The eggs were placed in 0. 3% trypsin and 0. 05 gm of Sodium glycocholate (pH 7. 4). They were exposed to gas. The results were negative.

7. 1. 5 Experiment:- The eggs were placed in 0. 025% pepsin (pH 2. 0) for 15 minutes followed by two rinses with NaCl. They were held in 0. 3% trypsin and 0. 025% Sodium glycocholate (pH 7. 4). The results were negative.

7. 1.6 Experiment:- The eggs were exposed to gas for 1 minute. They were held in (a) 0. 025% pepsin (pH 2. 0) (b) mixture of 0. 3 gm trypsin and 0.05 gm Sodium glycocholate (pH 7.4). The results were negative. 7. 17 Experiment:- The eggs were placed in 0. 02 % Sodium taurocholate and exposed to gas. The result was negative. 7. 18 Experiment:- The eggs were placed in 0. 5% pepsin (pH 2. 2) for 40 - 136 -

minutes followed by two washings with 0. 4% NaC1 and held in a mixture of 0. 3% trypsin and 0. 2% Sodium taurocholate (pHs 6. 6, 7. 2, 7. 5 and 8. 4). They were exposed to gas for 1 minute. The results were negative. 7. 19 Experiment:- The eggs were pretreated with 2 ml of 1% pepsin (pH 2. 0) for 30 minutes followed by two rinses of 0. 4% NaCl. They were held in a mixture of 0. 5% trypsin and 0. 5% Sodium taurocholic acid (pHs 7. 0, 7. 3, 7. 6, 8. 1 and 8. 5). They were exposed to gas for 1 minute. The result was negative. 7. 20 Experiment: The eggs were placed in (a) 0. 3% pepsin (pH 2. 3) (b) 0. 5% pepsin (pH 2. 4) for 40 minutes, followed by two changes of 0. 4% NaCl. They were held in 0. 3% trypsin (pH 4. 5) and 0. 5% trypsin (pH 4. 5) for 1 hour. They were washed twice with 0. 4% NaC1 and were held in 1% Sodium taurocholate (pH 8. 2). The results were negative. 7.21 Experiment:- The eggs were placed in (a) 0. 3% trypsin (pH 6. 8) and (b) 0. 5% trypsin (pH 6. 6). Washed twice with 0. 4% NaCl. Then they were held in 1% Sodium taurocholate (pH 8. 2). The result was negative. 7. 22 Experiment:- The eggs were placed in a combination of (a) 50 ml of 0. 3% trypsin and 5 ml of 1% Sodium taurocholate (pH 7. 5) (b) 50 ml of 0. 5% trypsin and 5 ml of 1% Sodium taurocholate (pH 7. 3). The results were negative. 7. 23 Experiment:- The eggs were placed in (a) 0. 5% pepsin (pH 2. 2) for 45 minutes (b) 0. 3% trypsin. (pH 6. 1) for 45 minutes. They were exposed to gas for 1 minute, followed by two changes of 0.4% NaCl. They were held in a solution of 50 ml of 0. 3% trypsin and 2% - 137 -

Sodium taurochalate (pH 7.4). The results were negative. 7. 24 Experiment:- The experiment was conducted anaerobically and the eggs were placed in 0. 5% pepsin (pH 2. 2) (b) 0. 3% trypsin (pH 6. 1), (c) 2% Sodium taurocholate (pH 6. 3) (d) 50 ml of trypsin 0. 3% and 5 ml. of 2% Sodium taurocholate (pH 7. 4). They were further gassed for 1 minute. The result was negative. 7. 25 Experiment:- The eggs were placed in 0. 5% pepsin (pH 2. 3) for 1 hr and 40 minutes and gassed for 1 minute. They were washed twice with 0. 4% NaC1 and then kept in 0. 02M Sodium dithionite (pH 6. 5) for 15 minutes followed by two changes of 0.4% NaCl. They were then held. in 0.05% Sodium glycocholate (pH 7.0) and exposed to gas for 1 minute. The result was negative. 7. 26 Experiment:- The eggs were placed in 0. 3% trypsin (pH 7. 5) for 1 hr and 40 minutes and exposed to gas for 1 minute followed by two changes with 0. 4% NaC1, then were placed in 0. 02M Sodium dithionite (pH 6. 5) for 15 minutes, followed by two changes of 0. 4% NaCl. They were then held in 0. 05% Sodium glycocholate (pH 7. 0) and exposed to gas for 1 minute. The results were negative. 7. 27 Experiment:- The experiment was conducted anaerobically. The eggs were placed in the tube and exposed to gas, a solution of 5 ml of 0. 5% trypsin, 0. 3% Sodium taurocholate and 0. 3% Sodium bicarbonate (pHs 7. 0, 7. 3 and 7. 5). They were exposed to gas for two minutes. The result was negative. 7. 28 Experiment:- The eggs were placed in 0. 5% Diastase (pHs 6. 4, 7. 0, 7. 5, 8. 0 and 9. 0) for 3 hrs and 27 minutes. Washed twice with 0. 4% NaCl. Then they were placed in 0. 02M Sodium dithionite (pH 7. 0) for half an hour, followed by two rinses free of Sodium dithionite and held in 50 ml of 0. 5% trypsin and 0. 5% Sodium glycocholate (pH 7. 5). They 138 - were exposed to gas for 1 minute. The result was negative. 7. 29 Experiment:- The eggs were exposed to gas for 1 minute and 10 minutes and placed in 0. 5% trypsin for one hour, followed by two changes with 0.4% Na Cl. They were placed in 5 ml of 0. 5% Sodium glycocholate (pH 7. 8) and small crystal of sodium dithionite was added. The result was negative. 7. 30 Experiment:- The eggs were placed in 0. 5% pepsin (pH 2. 5) for 45 minutes followed by two changes of 0. 4% NaCl. Then they were placed in 0. 02M Sodium dithionite for 15 minutes and followed by two washings of 0. 4% NaCl and held in 10 ml of 0. 5% trypsin (pH. 6. 6) and. 5 ml of. 1% Sodium taurocholate (pH 7.6). They were exposed to gas for two minutes. The result was negative. 7. 31 Experiment: The eggs were placed in 0. 5% amylase (pHs 5. 0, 6. 8, 7. 0 and 7.2) for• 3 hrs and 25 minutes, followed by two changes of 0. 4% NaCl.. Then they were held in a combination of different solutions (a) 5 ml. of 0. 5% trypsin, 5 ml of 2% Sodium taurocholate and 0. 5% amylase (pH 7. 4), (b) 5 ml of 0. 5% trypsin and 5 ml of 2% Sodium taurocholate (pH 7. 4), (c) 5 ml of 0. 5% trypsin and 5 ml of 0. 5% amylase (pH 7. 4), (d) 5 ml of 0. 5% amylase and 5 ml of 2% Sodium taurocholate (pH 7. 1). They were exposed to gas for two minutes. The results were negative. 7. 32 Experiment: - The eggs were placed in a combination of solutions (a) 5 ml of 0. 5% trypsin, 5 ml of 2% Sodium taurocholate and 0. 5% of Amylase (pH 7. 4), (b) 5 ml of 0. 5% trypsin and 5 ml of 2% Sodium taurocholate (pH 7.4), (c) 5 ml of 0. 5% trypsin and 5 ml of 0. 5% Amylase (pH 7. 4), (d) 5 ml of Amylase 0. 5% and 5 ml of 2% taurocholic acid (pH 7. 1). They were exposed to gas for two minutes. The results were negative. 7. 33 Experiment:- The eggs were placed in a combination of solution of 2 gms - 139 - of trypsin and 2 gms Sodium taurocholate diluted into 100 ml of 0. 4% NaCl (pH 7. 4). They were exposed to gas. The results were negative. 7. 34 Experiment:- The eggs were placed in 0. 5% pepsin (pH 2. 5) for 20 minutes, followed by washing in two changes of 0. 4% NaCl. Then placed in 0.02M Sodium dithionite for 8 minutes, followed by two rinses of 0. 4% NaCl. The eggs were held in 0. 03%, 0. 04% and 0. 05% Sodium taurocholate (pH 7. 5, 7. 3 and 7. 4). The results were ne gative . 7. 35 Experiment:- The eggs were placed in (a) 0. 025% pepsin (pH 2. 5), (b.) 0. 5% pepsin (pH 2. 5), (c) 1% pepsin (pH 2. 5) for twenty minutes followed by two changes of 0. 4% NaCl. Then placed in 0. 02M Sodium dithionite for 30 minutes and exposed to gas for 1 minute, followed by two changes of 0. 4% NaCl. Then they were held in (1) 0. 03% Sodium taurocholate (pH 7. 5), (2) 0. 04% Sodium taurocholate (pH 7. 3), (3) 0. 05% Sodium taurocholate (pH 7. 5). The results were negative. 7. 36 Experiment:- The eggs were placed in 0. 5% trypsin (pH 6. 4, 7. 5 and 8. 0) for one hour, followed by two changes with 0. 4% NaC1. They were then held in 1% Sodium taurocholate (pH 7. 8). The result was negative. 7. 37 Experiment:- The eggs were placed in the combination of solutions (a) 20 ml of 1% Sodium taurocholate and 20 ml of 0. 5% trypsin (pH 7. 6), (b) 1% Sodium taurocholate (pH 7. 8), (c) 0. 5% trypsin (pH 4. 3) for 1 hr and 25 minutes. They were washed twice with 0.4% NaCl. They were held in 1% Sodium taurocholate (pH 7. 8) 20 ml of 1% Sodium taurocholate and 20 ml of 0. 5% trypsin (pH 7. 6). The results were negative. - 140 -

7. 38 Experiment:- The eggs were placed in a mixture of 50 ml of 0. 3% trypsin and 5 ml of 2% Sodium taurocholate (in. Hank's). They were incubated at 20°C and 31°C. The results were negative. 7. 39 Experiment:- The eggs were placed in 0. 3% trypsin (pH 3. 0) and 2% Sodium taurocholate and incubated for 5 hrs at 20°C., followed by two changes of 0. 4% NaCl. They were held in 0. 5% pepsin (pH 2. 6) at 20°C. The results were negative. 7. 40 Experiment.:- The eggs were placed in 0. 3% trypsin (pH 3. 0), 2% taurocholic acid. They were incubated at 17. 8°C for 5 hrs, followed by two changes with 0. 4% NaCl. They were held in 0. 5% pepsin (pH 2. 6) and incubated at 17. 8°C. The results were negative. 8. Effect of Sodium hypochlorite (Na0C1) To determine the effect of NaOCI on the egg, the experiments were conducted with different concentrations of NaO Clat 1 - 5%. 8.1 Experiment:- The eggs were placed in 1% NaO Cl (pH 8. 5) for 1 hr. Washed several times with (a) tap water (b) distilled water (c) 0. 4% NaC1 (d) "snail- conditioned"water. The opercula opened in most eggs and hatching took place, but the miracidia were non-motile. 8.2 Experiment:- Following this position of result, different concentrations of NaO Gland enzymes were tried. The results are shown in Table 14. 8.3 Experiment:- The eggs were placed in 1% NaOC1 (pHs 7. 2, 8. 0, 8. 5, 10. 0, 11.0). The results are shown in Table 15. 9. Effect of other factors:- 9. 1 Experiment:- The eggs of Haplometra cylindracea (17 days old) were placed in a watch glass, dried and rewetted (Laws, 1968) with the - 141 -

different media. In water a few non-motile miracidia were observed; the sudden release of the pressure apprently shatters the eggs. The results are shown in Table 16. 9.2 Experiment:- The eggs were placed in"snail conditioned"water. The results are shown in Table 17. 9. 3 Experiment:- The eggs were placed in formal saline. No hatching occurred. 9. 4 Experiment:- The eggs were placed in teased snail tissue. No hatching occurred.. 9. 5 Experiment:- Attempts were made by addition of cold water to warm water. no hatching occurred. 9.6 Experiment:- Non-motile miracidia hatched artificially by mechanical pressure to cover slip. This resulted in breakage of the egg capsule at the operculum (plates 13 and 14). 9. 7 Experiment:- 8 months old eggs were placed in a watch glass containing L. palustris (from Belfast) for 4 hrs. Many non-motile miracidia were found out of the egg shell. Some miracidia had burst. 142. -

Plate 13. Eggs of lung fluke, Haplometra cylindracea of frog Rana temporaria X 216.

Plate 14. Egg hatching of the lung fluke Haplometra cylindracea of frog Rana temporaria X 216. 4 - 144 -

TABLE 14

Effect of various concentrations of Sodium hypochlorite and enzymes on the hatching of the eggs of Haplometra cylindracea

Concen- Time of Washing Washing Trypsin tration of incuba- many ' 2 x with 0. 5% & Sodium tion times Pepsin 0.4% 1% ascor- Result hypo- with dist. NaC1 bic acid & chlorite (minutes) water cysteine

1% 1 minute + 0.. 2% - - - ve pH 2.3

(a) 2% 1 minute + 0. 5% + + - ve. pH 3. 2 for 3 hrs & 10 minutes

(3) 3% 45 min- + - - - non- ute s motile miracidia

(4) 5% i minute + 0. 5% - - - ve pH 2.6

(5) 5% 15 min- + - - - Many utes non- mo tile miracidia

+ = Applied - = Not applied ve = Negative - 145 -

TABLE 15

Effect of 1% Sodium hypochlorite with diffe re nt pHs on the eggs ofHaplometra cylindracea

pHs Incubation Washed many Result time times as soon as the lid separated

(1) 7. 2 1 hour + Few non motile miracidia

it (2.) 8. 0 + Few non motile miracidia

(3) 8. 5 It + Several non-motile miracidia

(4) 10.0 " + Few non motile miracidia

(5) 11.0 " + Few non motile miracidia

+ = applied - 146 -

TABLE 16

The eggs of Haplometra cylindracea (17 days) placed in cavity block, dried and rewetted with the following media

Condition of Water Result

(1) Aged tap water 3 non-motile miracidia

(2.) Boiled tap water 5 non-motile miracidia

(3) "Snail conditionecrwater - ve of L. stagnalis

(4) 'Snail. conditioned'.'water - ve of L. palustris

(5) "Snail conditioneduwater - ve of L. peregra

- ve = Negative - 147 -

TABLE 17

Effect of "Snail conditioned"water on the egg of Haplometra cylindracea

Expt Age of. the Incubation Species of Number Result egg after of snails in Snails of incubation tap water Snails at 25°C (100 ml) (Days) (Hrs)

1 12 3 Lymnaea 13 - ve stagnalis

2 12. 3 L. palus- 11 - ve tris

3 12 3 L. •er- 6 - ve egra

- ve = Negative - 148 -

II IN VITRO HATCHING OF THE EGGS OF HAEMATOLOECHUS (RANA PIPIENS)

Incubation of eggs:-

The lung fluke Haematoloechus was obtained from Rana pipiens from commercial sources; the eggs were removed as for other lung flukes (Haematoloechus from Rana ridibunda and Haplometra cylindracea from Rana temporaria). After washing the eggs were incubated at 25°C for two weeks. The water was changed every week. Experimental procedure and results:- H 1. 1 Experiment:- 2% NaOCl was used and observed as soon as the miracidia started to come out from the egg followed by three washings with 0.4% NaCl to remove the hypochlorite solution and three drops of 0. 01% neutral red which gives the pink colour in the region of operculum separated off. After. 23 minutes, non-motile miracidia were observed. H 1.. 2 Experiment:- The. eggs were placed in 3% and 4% NaOClfor 17 minutes and 8 minutes followed by two washings and were placed in 0. 5% cellulase. H 1. 3 Experiment:- The eggs were placed in 2% NaO Cl for 45 minutes and washed twice with "snail water. Hatching took place and non-motile miracidia were observed. H 1. 4 Experiment:- The eggs were placed in "snail conditionedilwater and exposed to gas for 5 minutes and 10 minutes. The results were negative. H 1. 5 Experiment:- The eggs were placed in teased tissue of snails. The results were negative. - 149 -

H 1. 6 Experiment:- The eggs were placed in 0. 5% of pepsin, chitinase, trypsin, cellulase, papain, diastase and amylase at different temperatures. The results are shown in Table 18. H 1.7 Experiment:- The eggs were placed in 1%, 2%, 4% and 5% NaO Clat different intervals. The results were tabulated in Table 19. - 150 -

TABLE 18

Effect of enzymes on the eggs of Haematoloechus (R. pipiens)

Enzymes Gas phase Temperature Egg hatching 0.5% (N = 95% CO2= 5%) time

(1.) Pepsin - 4°C - ve - 18°C - ve - 20°C - ve 2, minutes 25°C - ve

(2) Chitinase 2 minutes 25°C - ve

(3) Trypsin 2 minutes 25°C -• ve

(4) Cellulase 2 minutes 25°C - ve

(5) Papain 2 minutes 25°C - ve

(6) Diastase 2 minutes 25°C - ve

(7) Amylase 2 minutes 25°C - ve

= Not applied - ve = Negative - 151 -

TABLE 19

Effect of Sodium hypochlorite on the egg of Haematoloechus (R. pipiens)

Expt Concentration Gas phase Time of Result No. of NaOCl (95% N, incubation 5% CO2) Time

(1.) 1% 5 minutes 2 hrs Non motile miracidia

(2.) 2% - 1. hr Non motile miracidia

(3) 4% -• 8 minutes Non motile miracidia

(4) 5% - 3 minutes Non motile miracidia

= not applied - 152 -

III IN VITRO HATCHING OF THE EGGS OF HAEMA TO LOE CHUS (RANA RIDIBUNDA )

Incubation of eggs of Haematoloechus:-

Haematoloechus was obtained from R. ridibunda from Yugoslavia. The eggs were removed as described before (Materials and Methods, Part I). The eggs were washed several times with distilled water to remove any tissue and incubated at 25°C for two weeks. The water was changed every week. Experimental procedures and results:- H. 2.- 1 Experiment:- The eggs were placed in"snail conditioned'water to determine whether some substance may leak out from the snail and induce hatching. Positive results were obtained in the case of Planorbis planorbis only and tabulated in Table 20. H 2.2. Experiment:- 26 days old eggs were placed in "snail conditioned water (from tank of Planorbis planorbis, L. stagnalis, L. peregra and L. palustris). The results are shown in Table 21. H 2. 3 Experiment:- The eggs were tried to induce hatching by mechanical pressure. The result is shown in Table 22 (plates 15 - 19). H 2. 4 Experiment:- An attempt was made to infect the snail Planorbis planorbis for 45 minutes; the"snail water"was found to have a number of motile miracidia. H 2. 5 Experiment:- 7 specimens of Planorbis planorbis were kept for 30 minutes in water. The "snail conditioned water was boiled and cooled. The eggs were placed in the boiled water but showed no signs of hatching. H 2.6 Experiment:- 7 specimens of Planorbis planorbis were kept for 30 minutes in water and dried in an incubator at 25°C and the deposit then dissolved in 0. 4% Na and eggs were added. No hatching occurred. - 153 -

H 2. 7 Experiment:- The eggs were placed in 0. 5% pepsin (pH 2. 4) followed by two washings of NaCl, then placed in 1% trypsin (pH 6. 2), then two washings of NaCl, held in 0. 02% Sodium taurocholate (pH 7. 4). The results are given in Table 23. - 154 -

Plates 15 - 19. Photomicrographs showing hatching of the egg of the lung fluke Haematoloechus sp.

Plate 1.5 X 248 16 X. 313 17 X 550 18 X. 1.00 1.9 X 250

- 159 -

TABLE 20

Effect of "Snail conditioned water" on the egg hatching of Haematoloechus sp. (R. ridibunda)

Exper- Age of Duration Species of Result iment egg time Snail No. (Days) (minutes)

1 19 55 Planorbis Many motile planor.bis miracidia

2 1.9 60 Physa One non-motile acuta miracidia

3 19 55 L. peregra One non-motile miracidia

4- 19 75 L. palustris One non-motile miracidia

5 19 90 L. stag- - ve nalis

Control 19 60 - - ve

- ve = Negative = Not applicable - 160 -

TABLE 21

The effect of "Snail water"(from tank) on 26 days old eggs of Haematoloechus. (Rana ridibunda)

Expt. Time Species Egg hatching duration of Snail (hrs)

1 4 hrs Planorbis 2 or 3 motile miracidia 15 min- •lano rbi.s ute s

2. 4 hrs Lymnaea. - ve 15 min- stagnalis ute s

3 4 hrs L. peregra - ve 15 minute s

4 4 hrs L. palustris - ve 15 minutes

5 4 hrs control - ve 15 min- tap water ute s

- ve = Negative - 161 -

TABLE 22

Effect of mechanical pressure on the egg of Haematoloechus (R. ridibunda)

Expt. Age of the egg Egg hatching No. after incubation at 25°C (Days)

(1) 4 Few, not active

(2.) 1.3 many motile

(3) 1.6 many active

(4:) 17 many active

(5) 19 many active

(6) 22 many active

* motile miracidia were seen moving outside the eggs for 6 minutes. - 162 -

TABLE 23 Effect of enzymes and bile on the eggs of Haematoloechus spp (R. ridibunda)

Expt. Pepsin Washing Trypsin Washing Na tauro- Result 0. 5% 2 X 1% 2 X cholate pH 2.4 0.4% pH= 6.2 0.4% 0.02% NaG1 NaCl' pH= 7.4

(1) + + + - + - ve

(2.) - - + + + - ve

(3) - - - - + - ve

(4) + + + + + - ve

+= Used = Not applicable ve = Negative - 163 -

Figure 14. The digestive system of Lymnaea stagnalis (from Runham, 1975). Fig. 14

buccal mass

nerve ring pro -oesophagus salivary gland post-oesophagus crop

gizzard

rectu m stomach

digestive gland - 165 -

DISCUSSION

Infection of the first intermediate hosts of digenetic trematodes is, in many species, brought about by the actual ingestion of the egg. Thus, the eggs of Haplometra cylindracea, Haematoloechus (R. pipiens) and Haematoloechus (Rana ridibunda) do not hatch until they are eaten by the snail host. Infection with miracidia involves a metamorphosis of the parasite and the host restarts a developmental mechanism which is incomplete in the "resting" infective stage (Rogers, 1962). External stimuli are generally required to initiate the hatching of the eggs, and presumably components in the alimentary tract induce hatching. Presumably the hatching of the eggs inside the gut depends to some extent upon the feeding mechanism of the host. If the snail is not the actual intermediate host, the eggs may die in the alimentary tract or be passed out. Pulmonates are one of three subclasses of Gastropoda, which has two well known superorders: the Basommatophora with one pair of contractile cephalic tentacles bearing eyes at the base, and the Stylommatophora, with two pairs of tentacles with eyes at the summit of the hinder pair. Pulmonates are mostly herbivorous but some are carnivores. Runham (1975) gave a general anatomy of the digestive system of Lymnaea stagnalis (Basommatophora) (Fig. 14). The mouth leads via a short oral tube into the buccal cavity which has the jaw in its roof; projecting from its floor is the odontophore supported by the odontophoral cartilage. On part of the surface of the odontophore is the radula. The buccal mass is a complex arrangement of muscles which are responsible for movement of the odontophore and radula. Paired salivary glands open via ducts into the buccal cavity. Food passes from the buccal cavity into the long oesophagus and then into the greatly dilated short crop (characteristic basommatophorean feature). From the crop, the food passes into the stomach. 'The presence of gizzard inside the - 166 -

stomach is a characteristic basommatophorean feature. Ducts from the stomach lead to the two lobes of the digestive gland (one of the largest organs in the body). Faeces form in the style sac (the last part of the stomach) and are passed out into the anus through intestine and rectum. On hatching from their eggs, snails and slugs have a different diet from that of the adult. In Lymnaea, the posterior oesophagus and crop appear to function as a temporary store for food before its passage into the gizzard. It has a very large, highly muscular, bilobed gizzard containing sand grains. Behind the gizzard there is a thin walled chamber called the "pylorus". Along the pylorus there are three well developed folds in the wall, two dorsal and a single ventral. The sand grains are essential for trituration (Carriker, 1946b) and present in the gizzard, crop and pylorus. In Basommatophora the part of the gut between buccal mass and stomach is divisible into pro and post oesophagus and crop. (In stylommatophora only oesophagus and crop). The crop is larger than the oesophagus in the stylommatophora and is filled with a crop juice, containing an enzyme; it is sold commercially. The function of digestive gland is absorption of food material and its digestion, secretion of enzymes, storage of reserve material and excretion. The number of enzymes present in the tissue extracts of the digestive gland have been reported by various workers (refer to introduction). Following ingestion of embryonated eggs, miracidia hatch, penetrate the gastropod's digestive tract, then elongate to form mother sporocysts on the outer surface of the intestine. Hatching of infective eggs may be regarded as part of the process of infection and as the result of some simple or complex stimulus provided by the host. In nematodes most stimulus which restart development are physico-chemical and the act of hatching becomes a combination of chemical (enzyme) and physical (pressure) (Rogers & Sommerville, 1963). The physico-chemical nature of the gut depends on pH, redox-potential, 02, digestive enzyme, CO2 and temperature. - 167 -

Rogers (1961) suggested that the host stimulus which restarts the development of the infective agent is in three steps:- (1) The host supplies a stimulus that acts on the "receptor" in the infective egg or larva. (a) As the result of the stimulus the first indication is the secretion of a "hatching fluid" or an "exsheathing fluid". (3) These fluids attack the layers of the egg shell or the sheath, and the infective agent emerges. Lackie (1975) has reviewed the activation of infective stages of endoparasites of vertebrates. The majority of the infective stages of helminths have a low resting metabolism. A stimulus is required to induce semi-dormant infective stages to continue their development. The host's digestive physiology may have a direct and/or an indirect effect on the activation of infective stages of many parasites. A direct effect is provided by the digestive activity of the enzymes in the host's alimentary tract; an indirect effect may stimulate activity of the larva. Hence there may be a relationship between the digestive physiology of the host and the responses of the parasite. Dasymetra conferta is common in the mouth and oesophagus of Water Snakes (Natrix laurenti) and it hatches after it is ingested by the snail host, Physa spp. The life-cycle of Dasymetra conferta described by Byrd & Maples (1964) suggests that the digestive tract provides the factors necessary to induce hatching and the stimulus supplied by the host could be:- Scraping action of radula. The swallowing action. The action of salivary juices. The movement of muscular stomach. The concentration of juices from the hepatic diverticulum. Action of juices from the small intestine. Physico-chemical changes such as temperature, pH, ion. exchange. - 168 -

Becker (1973) observed the following steps at the time of activation of miracidia of Schistosoma mansoni within the egg shell: (1) Flame beating in the protonephridia. (2) Contractions of whole animal. (3) Movement of the cilia at the anterior end of the miracidium. (4) Movement of the cilia on the whole surface. (5) Opening of the egg-capsule. (6) Emergence of the miracidium. Studies on the in vitro hatching of the egg are aimed at understanding the mechanism of the process. Infective eggs of many helminths, especially nematodes and cestodes, can be induced to hatch in vitro in the presence of stimuli which resemble those present in the intestine of the host. Attempts were made here to determine the conditions necessary for the eggs of Haplometra cylindracea and Haematoloechus (Rana pipiens), examining the effects of osmotic pressure, hydrogen ion concentration, proteolytic enzymes (such as pepsin, trypsin and papain) and other enzymes, temperature, gas phase, reducing agents, bile salts, NaOC1 and various other factors. In relation to the effects of osmotic pressure, general surveys of osmotic and ionic regulation have been given by Krogh (1965) and iono-regulation and osmo-regulation in Mollusca has been well reviewed by Schoffeniels & Gilles (1972). Fresh water gastropods and bivalves maintain very low concentrations of ions. Hyper osmotic regulation is effected by active uptake of ions such as Na+ and Cl+ from fresh water by excretion. Determinations of blood ionic composition of Lymnaea stagnalis have been made by Huf (1934), Florkin & Duchāteau (1950) and Burton (1968b) and are given in Table 24. Hatching of the egg in the snail may be dependent upon the presence of different factors or a change in the concentration of ions within the egg or its environment, resulting in a changed osmotic pressure within the egg. - 169 -

TABLE 24 Blood Ionic composition of Lymnaea stagnalis (After Schoffeniles & Gilles, 1972)

Expt. Ionic concentration in millimoles References per litre of blood plasma

Na K Ca Mg Cl

(1) 47.4 2.8 1.5 2.4 42.6 Huf, 1934

(2) 31.2 1. 2 7. 1 2. 0 27.2 Florkin & Duchāteau, 1950

(3) 49.1 2.0 4.0 1.5 - Burton, 1968b - 170 -

Experimentally, the osmotic pressures were adjusted by alterations in the concentration of NaCI but had almost no effect; only in 0. 3% NaCI were a few non-motile miracidia observed. Osmotic pressure may have some effect on hatching but does not seem to be an essential factor. Ratcliffe (1968) found that at 0. 8% NaC1 an inhibited hatching of Dicrocoelium lanceolatum occurred. Mitterer (1975) made observations on the hatching of miracidia of Dicrocoelium dendriticum and presented, "the evidence of an oligosaccharide liberated from 'Spaltraum' during egg opening and the determination of the osmotic pressure of the hatching process. (50% hatching in 1. 2 1.4 osmols sucrose/ 1000 ml H2O) led to the following hypothesis of hatching mechanism. After the activation of the granular gland of the miracidium an enzyme is released into the extra-embryonic 'Spaltraum'. A polysaccharide is digested to an oligosaccharide which cannot permeate the egg shell and the embryonic membrane. The rising osmotic pressure bursts off the operculum." The word "Proteolysis" means the hydrolysis of proteins into their constituent amino acids. The enzymes responsible are known as proteolytic enzymes (Proteases). The activity of enzymes is greatly influenced by acidity or alkalinity. Proteolytic enzymes such as pepsin (activated by HC1) in the gastric juice and trypsin secreted by the pancreas (intestinal secretion). Proteolytic enzymes and other enzymes with varying pH and concentration were shown to play no part in hatching of the eggs of Haplometra cylindracea and Haematoloechus (R. pipiens). Combined action of gastric and intenstinal secretion were tried and proved unsuccessful. Some stimulation of the miracidia apparently occurred in pepsin- HCI. In carnivores snails the buccal mass is extremely long, (Smith, 1970; Crampton, 1973) and the intestine is short so that - 171 -

the concentration of CO2 is comparatively low. In herbivorous snails the intestine is long. The pH of the intestine is slightly more alkaline than the stomach. The pH varies in different portions of the gut. To determine the different effect of hydrogen ion concentration on the egg, experiments were conducted in which the pH varied from 2.5 - 11. 1. These experiments proved unsuccessful. The snails were kept at temperatures from 4°C - • 37°C. Different experiments were conducted to find out whether the temperature played an important part in hatching; again they were unsuccessful. Ratcliffe (1968) found the rapid hatching of eggs of Dicrocoelium lanceolatum at 33°C. In cestodes, Voge & Turner (1956). studied development of cysticercoids of Hymenolepis diminuta at temperatures from 15 to 37°C. The body temperatures of the flour beetle changes from day to night temperature. Collings & Hutchins (1965) hypothesised that daily temperature change triggers the activity of the eggs preliminary to hatching in the intermediate host of Hymenolepis microstoma. A gas phase 95% N, 5% CO2 was used to determine the effect on egg hatching. The time of exposure to gas varied from 1 to 15 minutes. No hatching occurred. In nematodes, some trematodes (e. g. Fasciola) and protozoans, the concentration of undissociated carbonic acid or dissolved carbon dioxide plays an important part in hatching mechanism (Rogers, 1960). But Anya (1966) found carbon dioxide does not appear an important factor in the hatching of eggs of Aspiculuris tetraptera. Some experiments were conducted anaerobically (Fig 13). Friedl (1960) found mass hatching of miracidia of Fascioloides magna by exposure to nitrogen and under partial vacuum. When embryonated eggs were placed in a bell jar under reduced pressure of 5 to 15 centimeters of mercury in the light at room temperature for over 60 minutes, he found 80 per cent egg hatching. - 172 -

He also found partial anaerobiosis is a common factor in the activation of the miracidium, but clearly the exposure of nitrogen to egg hatching is unlikely to occur in nature. Reducing agents produce anaerobic conditions as well as changes in redox potential. Intestinal bacteria provide reducing conditions; the effect of this is to increase the rate of activation. The presence of cellulase and chitinase in the crop of the snail is well known and it was believed these were not produced by animals, only by bacteria from the crop juice. However, Strasdine & Whitaker (1963) confirmed that the digestive gland in

Helix poma.tia contained cellulase and chitinase and was free of bacteria. Parnas (1961) concluded that cellulase was produced by the digestive gland and bacteria. Enzymes from the crop juice must be present amongst the faecal material and bacterial action. Ste,fanski (1965) studied the bacterial flora and suggested that it constitutes one of the ecological factors affecting the establishment of parasites in the intestine of their host. "Bacteria play an important role in meeting the demands of certain vitamins. A. host organism plays with regard to its parasite the same role as the external environment plays for each free living organism. " Each segment of the alimentary tract has its own bacterial flora. The mechanism of the activity and effect of bacteria on parasites is not known. It probably forms an important source of the vitamin requirements of parasites. The reducing agents such as ascorbic acid, cysteine and glutathione when used with pepsin, trypsin and amylase produce some activity of the miracidia. Ratcliffe (1968) used ascorbic acid and cysteine over a period of 5 hours at room temperature to determine the action of reducing agents. The egg of Dicrocoelium lanceolatum hatched only after ingestion by an intermediate host and reducing conditions - 173 - increase the activation. Bile salts are well known as biological surfactants and have been shown to trigger or enhance activation of infective stages of a wide variety of intestinal parasites (Lackie, 1974). The combined action of pancreatin, sodium bicarbonate and sodium taurocholate stimulated the movement of miracidia of Haplometra cylindracea. The bile solution was ineffective in producing hatching. NaOClacts as a strong oxidizing agent and breaks the sulphide bonds of tissue (Esser, 1972). The effect of NaOC1 on the eggs was to induce a number of miracidia to hatch but it killed them. The phenolically tanned eggs bleached after the treatment with NaOC1. Hypochlorite attacks proteins readily and is less effective against polysaccharides (Roberts & Gibbons, 1966). The NaOC1 may be attacking a protein substance sealing the operculum. 8 months old eggs (after incubation) were placed in a watch glass with L. palustris (Belfast Snail). After 4 hrs, non motile miracidia were found out of the shell. Some were burst. Abnormal hatching of the egg was attempted by mounting incubated eggs on a slide and applying slight mechanical pressure to the cover slip. This resulted in breakage of the egg capsule at the operculum. All attempts to hatch Haplometra cylindracea eggs outside the digestive tract of the proper host was constantly negative. Control of the physio-chemical conditions in order to induce hatching in vitro is difficult. In vivo it may be possible that the mechanical forces, with the help of digestive enzymes and/or the presence of bacteria in the snail's alimentary canal induce hatching of the eggs. Mitterer (1975) found hatching of miracidia of Dicrocoelium dendriticum when he used the intestinal juice of the Roman Snail Helix pomatia which were dependent on the absence of 02 and the presence of bacteria. - 174 -

The eggs of Haematoloechus when placed in different species of "snail conditioned"water gave some positive results. Some motile miracidia were hatched but only in the "snail conditioned"water of Planorbis planorbis (Table 20 ). Presumably some substance(s) leaked out from the snails and induce hatching. This positive result is clearly one merit for the investigators. When boiled "snail conditioned"water of Planorbis planorbis and dried deposit of "snail conditioned"water (diluted into 0. 4% NaCl) were used, no hatching was induced. The function of the saliva is believed to be lubricatory, feeding and assisting with the removal of food from the radula, and with its passage back into the oesophagus. Analysis of the salivary gland indicates the presence of amylase together with small amounts of a trypsin-like protease (Carriker, 1946b, Beor et al, 1967 and Walker, 1970b). Observation on the life history of Haematoloechus has been made when the eggs were fed in a dish to Planorbis planorbis. Some hatching of uningested eggs took place and some miracidia were hatched in vitro. It is not known whether these miracidia can be infective for snails or not. Schell (1962) while tracing the life-cycle of Telorchis bonnerensis noticed that when snail hosts Physa gyrina and P. propinqua (the natural and experimental hosts) were used, some miracidia hatched in aquarium water. While hatching occurs after the eggs have been ingested by a suitable host snail, it is not known if the miracidia in vitro can be infective to the snail or not. Schell (1975) noticed that when the gravid specimens of Lecithaster salmonis, were examined in sea watef on a slide, the miracidia were hatched from 3 of the eggs in the terminal part of the uterus. The hatching of the miracidia were regarded as exceptional; the embryonated eggs normally hatch only in the intestine of suitable gastropod snails. - 175 -

The effect of mechanical pressure on the four day old eggs of Haematoloechus (after incubation) was examined. A few injured non-motile miracidia were obtained (Table 22). The sudden release of pressure shatters the eggs. Several motile miracidia were observed in the eggs which had been incubated for 13, 16, 17, 19 and 22 days. It means that miracidium develop within a period of 2 weeks and the constant degree of hatching occurs in the intestine of living snails. MacInnis (1976) in his review article described the release of chemicals by potential hosts and the roles which the chemicals serve in the processes of host-finding by parasites. MacInnis (1965) stated that certain amino acids, short chain fatty acids and a sialic acid attracted miracidia to stimulate attachment and attempt to penetrate the agar. The mucus of Lymnaea truncatula contains glucose, and 16 amino acids and lipid fractions which may serve to stimulate miracidia of Fasciola hepatica (Wilson, 1968; Wilson & Denison, 1970). Wright &. Ronald (1972) and MacInnis et aL (1974) stated the water conditioned by snails contained amino acids. Stibbs et al. (1976) observed that snails emit substances called "miraxone s" which alter the swimming behaviour of miracidia and help them to locate and attack the snails. Sponholtz & Short (1976) found that inorganic ions Caa+ and Mg++ affect behaviour of S. mansoni miracidia and suggested that the ratio of these ions may be important in attracting miracidia to snails. In vitro the hatching of the eggs of Haematoloechus (Rana ridibunda) indicate that activation is triggered by one or more factors in the alimentary tract of the host. It is possible that it is a digestive enzyme which leaks out from the snail which is responsible for the induction of hatching, perhaps by attacking the seal of the operculum. - 176 -

SUMMARY

Attempts were made to obtain infections of Haplometra cylindracea in various species of snails. These proved to be unsuccessful. It was decided to attempt to hatch the eggs in vitro. The effect of different factors such as osmotic pressure, hydrogen ion concentration, different enzymes, temperature, gas, reducing agent, bile salts, sodium hypochlorite, "snail conditioned" water, teased snail tissue, mechanical pressure, etc., were examined. All attempts to hatch the eggs outside the host were unsuccessful but hatching took place in. Na0Clwhen only non-motile miracidia were observed. The eggs of Haematoloechus (R. pipiens) were tried with enzymes, snail teased tissue, "snail conditioned" water and NaOC1. Hatching took place and non-motile miracidia were observed in the case of. NaOC1. The eggs of Haematoloechus (Rana ridibunda.) were tried in different species of "snail conditioned" water. The hatching occurred only in the "snail conditioned" water of Planorbis planorbis. The effect of enzymes on the egg of Haematoloechus proved unsuccessful in inducing hatching. - 177 -

PART III

In vitro culture of lung of Rana temporaria and R. pipiens - 178 -

INTRODUCTION

In vitro culture of lung of Rana temporaria and R. pipiens:- The aim of this project was to attempt to culture lung tissue in vitro in the hope that this might make possible the culture of trematodes within the same tissue. "Tissue or organ culture" is defined as the maintenance or growth of tissues, organ primordia, or the whole or parts of an organ in vitro in a way that may allow differentiation and preserva- tion of the architecture and/or function (Fedoroff, 1967). The ready availability, ease of maintenance of adult frogs and the large cell size are characteristic of all amphibians. Amphibian tissues, having cells larger than those of most homoiothermic animals, are particularly suitable for the examination of intracellular detail. Microbial contamination is also more easily controlled at the low temperatures supporting amphibian cell growth than at the warmer temperatures required by homoiothermic vertebrate cells. Ross Harrison (1907) the "father of the tissue culture technique" was the first worker to culture amphibian tissues. He removed asceptically small fragments from the wall of the frog neural crest and cultivated them in coagulated frog lymph. A comprehensive review of cell and tissue culture has been given by Paul (1975) and review of methodology in organ culture by Hodges (1976) and Ryley & Wilson (1978) discussed the cell and tissue culture method. Amphibian cell cultures have been reviewed by Freed & Mazger-Freed (1969) and by Rafferty (1976). The major factors influencing the growth behaviour of tissue of culture are well known. They are as follows:-

1. Temperature 2. Osmolality - 179 -

3. Gas phase 4. Substrates 5. Media 6. pH

In order to get the best growth the tissues have to be grown at temperatures near the "normal" temperature of the animal. The effect of temperature on amphibian tissues in organ culture has been reviewed by Monnickendam & Balls (1973). The partial pressure of carbon dioxide in amphibian blood is much lower than that of mammals (Balls et al. 1975). The great majority of media used for cold blooded vertebrate cell culture employ balanced salt solutions including NaH CO3 for buffering. Most cell culture media include a buffer system because naturally CO 2-bicarbonate systems occur in blood plasma. CO.2 is readily lost from the media causing a rise in pH., and moreover it is quite toxic for some cells. Nutritional requirements of amphibian cells in culture has been reported recently by Chinchar & Sinclair (1978. a, b).

Morphology of the Amphibian lung:- The amphibian lung is a much simpler structure than the mammalian or avian lung and it gives rise to a different type of outgrowth in culture. In the amphibians, owing to reduction of ribs, the lungs are filled by "swallowing" air - a force pump mechanism (Romer, 1949). In the adult frog there are simple paired saccular lungs opening directly into the pharynx. The vascularised epithelial lining of the lungs is smooth, in very young frogs, but as the animal grows older the lining is thrown into folds (septae) separating infundibula lined with thin squamous respiratory epithelium which thus forms blind-ended alveoli (Leake, 1975) (plate Lung rudiments are capable of continuing development to a remarkable degree in vitro. Lung cultures always give rise to an outgrowth of both mesencymal and epithelial cells (Aydelotte, 1965). - 180 - The respiratory tract in culture has been reviewed by Aydelotte (1965). The lung of the newt, Triturus viridescens, is of the simple type. In vitro the lung produced two types of epithelium, a pulmonary and a ciliated sheet. In cultures older than 7 days abnormal mitoses and fragmentation of the nuclei occurred in pulmonary cells but not in ciliated cells (Danes, 1949). The lung of Xenopus laevis laevis became much denser due to collapse of the air spaces. Mitoses were found in cells lining the air spaces and there was increased mitotic activity in vitro (Simnett & Balls, 1969). In the lungs of immature Xenopus, mature Xenopus, mature Rana pipiens and mature Pleurodeles, the mitotic incidences are higher than those in. vivo (Balls, et al. 1969). Amphiuma lungs are cylindrical organs. No increase in the proliferative activity of the epithelium has been found in any of the media used. The lung fragments can survive for long periods but there is no increase in mitotic activity in vitro (Monnickendam & Balls, 1972). A summary of methods used in lung tissue culture and the mitotic incidence in cultured lung tissue by various workers is showh) in Table 25. This study was undertaken to observe the lung of R. temporaria and R. pipiens in tissue culture with or without serum and pointing out their differences and relating activity in vitro with function in vivo. It is hoped that the observations may possibly be of value as a basis for future experimental studies on problems of pulmonary respiration as well as in parasitology. TABLE 25

Summary of methods used in lung tissue culture by various workers and the mitotic incidence in cultured lung tissue.

Method Temper- Gas Basic medium Maxi- Mitotic Species Reference ature Phase serum, etc. mum time incidence in culture in vitro (days) compared in vivo

Hanging drop 25.5 - Equal parts of 42 Abnormal Triturus Danes (1949) preparations, 26.5°C embryonic mitoses viridescens roller tubes extract from l- and carrel days chicks and flasks cockeral plasma

Fused silica 25°C Air Leibovitz L-15 6 Increased Xenopus Simnett & dish Fetal bovine laevis Balls (1969) serum laevis

Rigid 25°C Air Leibovitz L-15 7 Increased Immature Balls et al. platforms foetal calf Xenopus (1969) or grids serum Method Temp. G. phase Basic medium Max. time Mitotic Species Reference incidence

Rigid 25°C Air Leibovitz L-15 7 Increased Mature platforms foetal calf Xeno .us or grids serum

7 Increased Mature Rana

18 Increased Mature Pleurodele s

Cultured 25°C Air 70%, 30%, or 35 No change Amphiuma Monnickendam in liquid 45% L-15, 8 & Balls, (1972) medium or 0. 8% serum - 183 -

MATERIALS AND METHODS

The frogs Rana temporaria and R. pipiens were purchased from a biological supplier (Xenopus Ltd). They were maintained in the laboratory and fed on insects. Culture technique:- Sterile glassware was used throughout. All procedures were performed in a , laminar flow cabinet with a sterile air-supply. The frogs were killed by pithing. The dissection was carried out under sterile conditions. The surface of the abdomen was sterlised by swabbing with iodine in alcohol, allowed to dry and an incision was made along the length of the ventral surface of the animal, which was fixed to a dissection board. The lungs were immediately removed asceptically in a sterlised Hank's solution. The tissue was cut with fine scissors into small pieces and, because of its non-sterile lumen, several changes of Hank's solution were used: the tissue was then transferred into a disposable Falcon flask (25 cm2) containing 10 ml of medium. The lung tissues (sample as a control) were taken for determination of initial histology. The medium changes were done every week; a small amount of old medium was left before adding fresh medium. All cultures were incubated at 200C in an incubator (Gallenkamp) regulated at a cooled CO2 air atmosphere. The culture flask was laid horizontally to facilitate gaseous exchange between the medium and the atmosphere. As soon as the contamination was detected, the culture was discarded. The cultures were examined every day under an inverted microscope and micrographs of selected areas were taken with a camera attached to a microscope. Media:- Two types of media SM III and SM IV were used. Asceptic procedures were followed in the preparation of the media. Stock Solution:- Powdered NCTC 135 and 2. 2 gms NaHCO3 diluted into 1000 ml of sterile triple distilled water. The medium was sterilised - 184 -

by filteration under vacuum. It was incubated for 24 hrs at 37°C and then removed to 20°C.

(i) Medium SM III 200 ml of stock solution diluted by 50 ml of sterlised triple distilled water.

(ii) Medium SM IV 100 ml of SM III diluted with 25 ml of foetal calf serum (heat inactivated at 56°C for 30 minutes). The stock solution, media, SMIII and SMIV were gassed with a mixture of 5% CO2 and 95% Nitrogen. The media were changed when the pH fell below 7. All stock solutions, media, SM.III and SMIV were kept in temperature and humidity controlled incubator at 20°C.

Antibiotics For the prevention of microbial contamination 2 m1/100 ml penicillin streptomycin was added in the Stock Solution.

Reagents:- 1. NCTC 135 medium (Difco)

2. Foetal calf serum (Gibco)

3. Penicillin Streptomycin (Flow)

4. Sterile triple distilled water (Difco)

5. Sodium bicarbonate (BDH)

6. Hank's solution (Difco)

Histology: - Uncultivated and cultivated tissues were fixed in Bouin' s fluid for histological examination. The tissues were selected at 7 )zm and stained with haematoxylin and eosin. - 185 -

RESULT AND DISCUSSION

Observations were made on in vitro cultures of lung tissues of Rana temporaria and R. pipiens. The number of mitotic figures were counted with an oil immersion lens (plate 21). The lung rudiments are capable of continuing development to a remarkable degree in vitro. New growth was indicated by the appearance of bud on 9th day cultures and the growth was more at the periphery than in the centre. Active numbers of buds were observed at 17 days. The maximum time of culture of the lung tissue of Rana temporaria and

R. pipiens was 11.4- days.. The aim of the study was to establish an environment for cell cultivation in regard to isotonicity pH and nutritional requirements. Calf serum is the preferred growth medium supplement for the great majority of poikilothermic vertebrate cell types. Sooy & Mezger-.Freed (1970) found that a macromolecular fraction of foetal calf serum, together with added purines, supported the growth of a number of amphibian cell lines. Methods used in lung tissue are shown in Table 26. In some experiments the lung tissue of Rana temporaria was contaminated and discarded on different days as shown in Table 27. The cells in tissue culture differ in form and general appearance from the cells in uncultivated tissues. As the culture ages, an increasing proportion of cells appear with numerous small nuclei of various sizes and irregularly shaped nuclei. It is well known that growth media used for tissue culture must contain serum in order to support rapid proliferation of cells in vitro. Two media SM III and SM IV were used in order to see the rapid growth in the media. Mitoses were observed in vitro cultured tissue but were generally only found in the lung in vivo. However, it was not feasible to accurately count the mitosis or to subject the data to statistical analysis. Due to lack of time, the work was not extended further, but additional work might prove valuable. TABLE 26

Methods used in lung tissue culture and results (incubated at 20°C and medium was changed once in a week)

Method Temper- Gas Basic Serum Maximum Species ature phase medium Foetal calf time in serum culture days

Cultured in 20°C 5% CO2 NCTC - 114 R. temporaria liquid medium 95% N2 135

Cultured in 206C 5% CO2 NCTC FCS 114 R. temporaria liquid medium 95% N2 135

Cultured in 20°C 5% CO2 NCTC - 114 R. pipiens liquid medium 95% N2 135

Cultured in 20°C 5% CO2 NCTC FCS 114 R. pipiens liquid medium 95% N2 135

= Not applied - 187 -

TABLE 27

The lung portion of Rana temporaria was used. Some were contaminated and discarded on day *

Basic medium Serum FCS Days* NCTC 135

(1) + - 8

(a) + - 15

(3) + - 16

(4) + - 35

(5) + - 88

(6) + 3 8

(7) + + 14

(8) + + 22

+ = Applicable = Not applicable - 188 -

Plate 20. Lung of Rana pipiens (not cultured) photograph.

Plate 21.. Section of lung of Rana pipiens after 114 days in culture. Mitotic divisions. Haematoxylin & eosin X 189. .

- 190 -

SUMMARY

The lung tissue of adult Rana temporaria and R. pipiens incubated at 20°C was maintained in SM III and SM IV media for periods up to 114 days. Mitosis was observed in vitro cultured 4 tissue, but It was not possible to accurately count the mitoses or statistically analyse the data.

W qS - 191 -

CONCLUSIONS AND SCOPE FOR FURTHER WORK

Detailed discussions have been written in each part. Part I includes the life cycle of Haematoloechus similis and H. variegatus. Only the first intermediate host Planorbis planorbis was investigated. Grabda (1960) was the first to trace the complete life cycle of Haematoloechus similis. The second intermediate host as quoted by her is Coenagrion hastulatum but this was not available for the present study. Metacercariae and adults have not, therefore, been fully described. The present research has presented some data which may be of value to future workers who may wish to complete the life cycle. The nature of the factors present in "Snail conditioned" water of Planorbis planorbis which induced hatching of the eggs of Haematoloechus (from Rana ridibunda) offers scope for further investigation. Tissue culture of the lung of Rana temporaria and R. pipiens offers some hope that this observation may possibly be of value as a basis for future experimental studies on problems of pulmonary respiration as well as in parasitology. 192 -

REFERENCES

Abou Senna, H.O. & Bassaly, M. , 1964. [Observations on the hatching of miracidia from Schistosome eggs. Medskaya Parazit., 33 (6), 704 - 706 (In Russian). Helminth.Abstr. 36, 399.

Anya, A.O. , 1966. Experimental studies on the physiology of hatching of eggs of Aspi.culu.ristetraptera Schulz (Oxyuridea: Nematoda). Parasitology, 56, 733 - 744.

Arnold, E.. N. &. Burton, J.A. , 197'8.. A. fiend guide to the reptiles and amphibians of Britain and. Europe. London, Collins, 1978.

Aydelotte, M. B. , 1965.. Respiratory tract. In "Cells & Tissues in Culture".

(E.. N. Willrne.r, ed). Vol 2., pp 569 - 589. Academic Press, London & New York.

Balls, M. , Simnett, J. D. & Arthur, E. , 1969. Organ cultures of normal and neoplastic tissues. In "Biology of Amphibian Tumors". (M. Mizell, ed. ), pp 385 389. Springer-Verlag, New York.

Balls, M., Brown, D. & Fleming, N., 1975. "Long-Term Amphibian Organ Culture". In "Methods in Cell. Biology" (D.M. Prescott, ed. ), Vol XIII pp 213 - 238. Academic Press, New York, San Francisco London.

Bair, R.D. & Etges, F.J. , 1973. Schistosoma mansoni`Factors affecting hatching of eggs. Exp. Parasitol, 33, 155 - 167. - 193 -

Becker, W., 1973. [Activation and inactivation of the miracidia of Schistosoma mansoni within the egg shell Zeitschrift far Parasiten- kunde 42 (31 235 - 242. (De, en) Helminth. Abstr. 43 (5), No. 1717.

Boer, H.H., Bonga, S.E.W. & van Rooyen 1967 . N. Light and electron microscopical investigations on the salivary glands of Lymnaea stagnalis L. Z Zellforsch. mikrosk Anat 76, 228 - 247. quoted by Runham (1975).

B'ou.rgat; R..& Kulo, S D. , 1-978. Research on. the- life.-cycle of Metahaema.toloechus exoterorchis (Rees, 1.964.r A-tre;matode parasite from the lungs of Dic:rogl.ossu.s occipitalis (Gunther, 1858) in Togoland. Annales de Parasitolgie Humaine et Comparee 53 (2.), 195 - 200.

Burton, P. R. , 1964.. The ultrastructure of the integument of the frog lung-fluke, Haematoloechus medioplexas (Trematoda: Plagiorchiidae) Journal of Morphology, 115, 305 - 318. quoted by Smyth (1976).

Burton, R. F. , 1968b. Ionic balance in the blood of pulmonata. Comp. Biochem. Physiol., 25, 509 - 516.

Byrd, E.E. & Maples, W.P. , 1964. Developmental stages in the Digenea V. The egg, miracidium and brood mass in Dasymetra conferta Nicoll, 1911. (Trematoda: Plagiorchioidea: Ochetosomatinae) Parasitology, 54, 295 312. - 194 -

Cain, G. D. , & French, J. A. , 1975. Effects of parasitism by the lung fluke, Haematoloechus medioplexus, on lung fatty acid and sterol composition on the bull frog, Rana catesbiana. Int. J. for Parasitology, 5, 159 - 164.

Carrike r, M. R. , . 1946b Observations on the functioning of the alimentary system of the snail Lymnaea stagnalis appressa Say. Biol. Bull mar. biol. Lab Woods Hole 91, 88 - 111.

Collings, S. B. , & Hutchins, C. P. , 1965. Motility and hatching of Hymenolepis microstoma Oncospheres in Sera, Beetle extracts and Salines. Experimental Parasitology, 16, 53 - 56.

Cbmbes, C. , 1965.. Cycle Biologique De Haematoloechus pyreniacus In Biologie, ecologie des cycles et biog6ographie de digene s et monogenēs d' amphibiens dans les Pyrenees.. Memoires du Museum National d' Histoire Naturelle, 51, 50 - 55 (1968).

Cōmbe s, C., 1968. Biologie, ecologie de cycles et biogēographie de digēnes et monogēnes d' amphibians dans 1' est des Pyrēnēes. Memoires du Museum nationale d' histoire naturelle. 51, 1- 195.

Corbet, P. S. , 1962. A Biology of Dragonflies. Witherby, London.

Crampton, D. M. , 1973. "The functional anatomy of the buccal mass of one opisthobranch and three pulmonate gastropod molluscs". - 195 -

Phd. Thesis, University of Reading. In "Pulmonates"

(V. Fretter & Peake, J. Eds.) Vol I, p xxiii Academic Press. New York, London.

Chibichenko, N. T., 1964. Aquatic invertebrates of Kirgizia as intermediate hosts of helminths . [Abstracf . Mater. nauch. Konf. uses. Obshch Gelmint. , Year 1964, Part II, pp 224 - 226. (In Russian). Helminth. Abstract 38, No. 3.17'0.

Chinchar, G. D. , & Sinclair, J. H. , 1978. Amphibian cells in. culture I. Nutritional studies. J. Cell Physiol., 96, 333 342.

Chinchar, G. D. , & Sinclair, J. H. , 1978 Amphibian cells in culture II Isolation of drug resistant variants and an asparagine--independent variant. J. Cell Physiol. 96, 343 - 354.

Cort, W.W., 1915a North American frog lung flukes. Trans. Amer. Micr. Soc. 34, 203 240. quoted by Krull (1931).

Danes, B. , 1949. Pulmonary epithelium of the newt, Triturus viride scens, studied in living cultures with cinematographic apparatus and phase contrast technique. J. of expt. Zool. 112, 417 - 447.

Dawson, R.M. C., et al. , 1959 "Data for biochemical research". Oxford University Press. - 196 -

Dawes, B. , 1968. "The trematoda", Cambridge University Press.

Dobrovol'skii, A.A., 1965. Life cycle of Pneumonoeces asper Looss, 1899 (Plagiorchiidae) Materials for Science Conference, All-Sov. Union Soc. Helm. 59 - 64. quoted by Yamaguti (1975).

Dobrovol'skii, A.A., 1965. Uber die Einscheitlichkeit des Bauplanes von Mirazidien der. Uberfamilie Plagiochioidea. Angew. Parasitol. , Rd, 6, H. 3.

Dobrovol'skii, A.A., Raikhel, A. S., 1973.. [The life-cycle of Haplometra cylindracea] . Zeder 1800 (Trematoda, Plagiorchiidae). Vestnik Leningradskogo Univer.siteta, Biologiya. No. 3 (1) 5 13 [Ru, en].

Helminth. Abstr. , 43, No. 1.818

Dollfus, R. P. , Doby, J. , & Lauret, P. , 1960. Sur une xiphidiocercaraire parasitant Limnaea truncatula (O.. F. Muller) en. Haute-savoie et S'enkystant clans des larves de moustiques. Bull Soc. Zool. France 85, 331 - 347. quoted by Yamaguti (1975).

Dronen, N.O., Jr., 1975. The life cycle of Haematoloechus coloradensis Cort. 1915 (Digenea: Plagiorchiidae), with emphasis on host susceptibility to infection. J. Parasit. , 61, 657 - 660.

Dronen, N.O. , Jr., 1977. Studies on the population structures of two species of Haematoloechus Looss., 1899 (Digenea: Plagiorchiidae) in - 197 -

raniid frog in New Mexico. Proc. helminth. Soc. Wash., 44 (1), 68 - 72.

Dronen, N.O. , Jr. , 1977. Host-parasite population dynamics of Haematoloechus

coloradensis Cort, 1915. (Digenea: Plagiorchiidae). American Midland Naturalist 99 (2), 330 - 349.

Erasmus, D. A.. , 1. 972. The Biology of. Trematodes. Edward Arnold, London.

E:sse.r., P. R.. , 1972. Effect of Sodium hypochlorite concentrations on selected genera of nematodes. Proc. Helm. Soc. Wash 39, (1)

1.08 1.1.4

Etge s, F.. J. , 1960. On the life history of Prosthodendrium (Acanthatrium) anaplocami. N. sp. (Trematoda: Lecithodendriidae)

J. pa.rasit., 46, 235 - 239.

Evans, W.A. L. , &. Jones, E.G. , 1962a Carbohydrases in the alimentary tract of the slug Arion a.te.r L. Comp. Biochem. Physiol. 5, 149 - 160. In "Pulmonates" (V. Fretter & Peake, J. Ed.) Vol. I, 92. Academic Press, London, New York.

Evans, W.A. L. , & Jones, E. G. , 1962b A note on the proteinase activity in the alimentary tract of the slug Arion ater L. Comp. Biochem. Physiol. 5, 223 - 5. In "Pulmonates" (V. Fretter & Peake, J. Ed. ) Vol. I, 92. Academic Press London, New York.

Faust, E. C. & Hoffman, W.A., 1934. Studies on Schistosomiasis mansoni in Puerto Rico III. - 198 -

Biological studies. I The extra-mammalian phases of the life cycle. Puerto Rico J. publ. Hlth trop. Med. 10, 1 - 47.

Fedoroff, S., 1967. Proposed usage of animal tissue culture terms. Expl. Cell Res, 46, 642 - 648.

Freed, J.J., & Mezger-Freed, L., 1969. Culture methods for anuran cells. In "Methods in Cell Physiology" (D. M. Prescott, ed. ), Vol. IV, pp 19 - 47. Academic Press, New York.

Freed,. J..J.., &.Me:zge:r-Freed, L.., 1.970. Cixltu.re: methods for anuran cells. Meth. Cell Physiol. 19 --

Friedl, F. E. 1960 Induced, hatching of operculate eggs. J. Pa.rasit., 46, 454.

Frie.dl, F. E. , 1961 Studies on larval Fascioloides magna III. Mass hatching of miracidia by exposure to nitrogen. J. Parasit. , Vol. 47, 770 772.

Florkin, M., & Duchāteau,G. , 1950. Concentrations cellulaire et plasmatique du potassium, du calcium et du magnesium chez Serie d'animaux dulcicoles C. r. Seanc. Soc. Biol. 144, 1132 1133. quoted by Schoffeniels & Gilles (1972).

Galtsoff, P. S. , et al. , 1937. Culture methods for the damselfly, Ischnura verticalis. "In culture Methods for invertebrate animals". pp 268 - 270. Ithaca, New York, Comstock Publishing Company, Inc. - 199 -

Gardner, A. E. , 1977. A key to larvae. In "The dragonflies of Great Britian and Ireland" (C.O. Hammond, ed. ), pp 72 - 79. The Curwen Press Ltd. , London.

Ginetsinskaya, T.A. , & Dobrovol'skii, A.A. , 1968.

Larval trematodes of fresh water molluscs in the Volga delta. Trudy Astrakh Zapovednika 11, 29 - 95.

G.ine:tsinskaya, T. A.., Zdun, V. I. , Dobrovol' skii, A.A. , 1972. [Results, and prospects in the study of parasites of aquatic. invertebrates]. In. Parazity i bole zni ryb i. vodnykh bespozvonochnykh. Moscow, USSR: Izdatel'stvo "Nauka" 1.56 1.66 [Ru]. Leningrad. State Univ. , Leningrad. Helminth. Abstr.., 43; No. 3895.

G:rabda., B. 1.960. Life: cycle of Haematoloechus similis (Looss, 1899) (Trematoda -• Plagiorchiidae). Acta Parasitologica. Polonica, Vol 8, 357 367.

Grabda-kazubska, 1970. Studies on the life-cycle of Haplometra cylindracea (Z'ede.r, 1800) (Trematoda: Plagiorchiidae). Acta. Parasitologica 'Polonica, Vol. 1.8, 497 - 512.

Hall, M. C., 1929.. Arthropods as intermediate hosts. Smithson. misc. Collns, 81, 1 77.

Halton, D.W. , 1967. Observations on the nutrition of digenetic trematodes. Parasitology, 57, 639 - 660. - 200 -

Hammond, C. O. , 1977. The dragonflies of Great Britian and Ireland. The Curwen Press Ltd. , London.

Harrison, R. G. , 1907. Observations on the living developing nerve fiber. Proc. Soc. Exp. Biol. Med. 4, 140 - 143. quoted by Rafferty (1976).

Hodges, G. M. , 1976. A. review of methodology in organ culture. In "Organ Culture in. Biomedical Research", (ed. M. Balls & N.. Monnckendam), pp 15 - 59. Cambridge University Press.

Hollis, P. D. , 1972. Host sex influence on the seasonal incidence of Haematoloechus medio.plexus (Trematoda.: Piagiorchiidae) in. Rana. pipiens. J. Pa.rasit. , vol. 58, (1.) 128.

H,f, E. , 1934. Ue.ber den Einfluss der Narkose auf den Wasserund Mineralhaushalt bei Susswassertieren. Pflug. Arch. ges. Physiol. 235, 129 40. quoted by Schoffeniels & Gilles (1972).

Hyman, L. H. , (1967). "The Invertebrates 6 Mollusca I" McGraw-Hill, New York.

Ilyushina, T. L., 1973. [Aquatic insects of the Karasuk lakes as second intermediate hosts of trematodes] Trudy Gel'mintologicheskoi Laboratorii (Ekologiya i taksonomiya gel'mintov) 23, 55 - 64 [Ru] . Helminth. Abstr. 43, No. 4940. - 201 -

Ilyushina, T. L., 1975. [Role of aquatic insects in trematode life-cycles]. Trudy Biologicheskogo Instituta Sibirskogo Otdeleniya Akademii Nauk USSR (Parazity v prirodnykh Kompleksakh Sever not Kulundy) 17, 53 - 94. [Ru] . Helminth.Abstr. 45, No. 648.

Ingles, L. G. , 1933. Studies on the structure and life history of Ostiolum oxyorchis (Ingles) from the California red-legged frog, Rana aur.ora. draytoni. Univ. Calif. Publ. Zool., 39, 1.35 1.61..

Ingalls,. J. W. , et al.• , 1.949 The molluscan intermediate host and Schistosomiasis japonica. I Observations on the conditions governing hatching of the eggs of Schistosoma japonicum.

J. Paras•it-., 35, 1.47 - 151.

Irwin, M. S. , 1.929.

A new lung fluke from Rana clami.tans . Latre ille . Trans. Amer. Micr. Soc. 48. 74 - 79. quoted by Krull (1931).

Iwasaki, H., 1960. •[Studies on the miracidium of Gigantobilharzia sturniae (Tanabe, 1948) 2. Ecology of the miracidium of G. sturniae (Tanabe, 1948)]. Japanese Journal of Parasitology, 9 (5), 582 588 [In Japanese] . Helminth. Abstr., 30, No: 1990.

Jager, J. C., 1971 A quantative study of a chemores ponse to sugars in Lymnaea stagnalis. Neth. J. Zool. 21, 1 - 59. quoted by Veldhuijzen (1974). - 202 -

Jeuniaux, C., 1954. Sur la chitinase et la flora bactērienne intestinale des mollusques gastēropodes. Mēm. acad. roy Belg. Sci 28, 1 - 45. quoted by Runham (1975).

Jones, T.W. T., 1950. Anopheline larvae as intermediate hosts for larval trematodes. Parasitology, 40, 144 - 148.

Kagan, I. G. , 1951. Aspects in the life history of Neoleucochloridium

problematicum. (Magath, 192.0) n. comb. and

Leu.c:ochloridium cyanocittae McIntosh, 1.932 (Trematoda:

Bra.chylaeroidae). Tr. Am. Micr. Soc.. 70 (4), 281 318.

Kassim,O. & Gilbertson, D. E. , 1976. Hatching of Schistosoma Mansoni eggs and observations

on motility of. miracidia. J. Parasite ; 62., 715 - 72.0.

Kctwa.shima, K., Tada., I. & Miya. zaki, I., 1961. "Ecological analysis on the mechanism of the host preference of miracidia of Paragonimus ohirai Miya zaki, 1939 in natural condition". Kyushu Journal of Medical Science, 12 (4), 143 - 151. Helminth.Abstr. 31; No: 1334.

Kingston, N., 1965. On the morphology and life cycle of the trematode Tanaisia zarudnyi (Skrjabin, 1924) Byrd et Denton, 1950, from the ruffed grouse, Bonasa umbellus L. Canad. J. Zool. 43 (6)," 953 - 969.

Krasnolobova, T.A., 1970. [nfe ction of dragon-flies with metacercariae of trematodes in the Latvian SSR]. Zool. Zh. , 49 (9), 1290 - 1297 [In Russian)] Helminth.Abstr.,, 40, No. 1701. - 203 -

Krull, W . H. , 1930. Life history of two North American frog lung flukes. J. Parasit. , ,16, 207 - 212.

Krull, W.H., 1931. Life history studies on two frog lung flukes, Pneumonoeces

medioplexus and Pneumobites parviplexus. Trans. Amer. Microsc. Soc. 50, 215 - 277.

Krull, W.H. , 1932. Studies on the life history of Pneumobites longiplexus (Stafford.) Zool. Anz. 99, 231 239.

Krull, W.H., 1.933. Studies on the life history of a. frog lung fluke, Haematoloechus complexus (Seely, 1906) Krull, n. comb. Z. Parasitenkd 6, 192 - 206.

Krull, W. H. , 1934. Sōrne additional notes on the life history of a frog lung fluke, Haematoloechus complexus (Seely, 1906). Krull. Trans. Amer. Microsc. Soc. 53, 196 - 199.

Krull, W.H.., 1934. "Notes on the hatchability and infectivity of refrigerated eggs of Fasciola hepatica Linn". Proceedings of the Iowa Academy of Science 41, 309 - 311. Helminth. Abstr. , 28 (3/6), No. 460.

Krogh, A., 1965. Osmotic regulation in aquatic animals pp 53 - 64. Dover Publications Inc., New York.

Kusel, J.R. , 1970. Studies on the structure and hatching of the eggs of Schistosoma mansoni. Parasitology 60, 79 - 88. - 204 -

Lackie, A. M. , 1974. The activation of cystacanths of Polymorphus minutus (Acanthocephala) in vitro. Parasitology, 68, 135 - 146.

Lackie, A. M. , 1975. The activation of infective stages of endoparasites of Vertebrates. Biol. Rev., 50, 285 - 323.

Laws, G. F , 1968. The hatching of taeniid eggs. Expt. Parasitology 23,

1 1.0..

Leake, L. D._, 1975... Comparative histology: An introduction to the micro- scopic structure of animal pp 575 581. Academic Press, London, New York.

Leidy, J. , 1851. Contributions to helminthology. Proc. Acad. Nat. Sci.. Phila. 5, 2.05 209. quoted by Krull (1931).

Lengy, J., 1962. Studies on Schistosoma bovis in Israel. Bull. Res. Coun. Israel E. 10, 1 - 36. quoted by Mitterer (1975).

Linstow, 0. , 1890. Uber den Bau und die Entwicklung des Distomum cylindraceum Zed. Arch. mikrosk. Anat. EntwMeck. , 36, 173 - 190. quoted by Grabda Kazubska (1970).

Loos s, A. , 1894. Die Distomen unserer Fische und Frāsche Biblioth. Zgol. 16, 296 pp. quoted by Krull (1931) &Stunkard (1930). - 205 -

Looss, A., 1899. Weitere BeitrUge Zur Kenntniss der Trematoden-fauna Aegyptens. Zool. Jahrb. Syst. 12, 521 - 784. quoted by Krull (1931).

Looss, A., 1902. Ueber neue und bekannte Trematoden aus Seeschildkroten. Zool. Jahrb. Syst. 16, 411 - 891. quoted by Krull (1931).

Liihe, M. , 1909.. Parasitis che Plattwiirme r I. Trematodes. Siisswa.s se rfauna Deutsch.. , Bd. 17. quoted by Dobrovol.'skii & Raijhel. (1.973)..

MacInnis, A. J. , 1965... Responses of Schistosoma mansoni miracidia to chemical attractants. J. Parasit. , 51, 731. - 746.

Maclnnis, A. J. , 1.976.. Host parasites find hosts: some thoughts on the inception of host-•parasite integration. In "Ecological aspects of. Parasitology" (C. R. Kennedy ed.) pp 15 - 20. North Holland Publishing Company - Amsterdam/Oxford.

Maclnnis, A. J. , 1976. In "Ecological aspects of Parasitology" (C. R. Kennedy Ed. ) pp 3 - 20. North Holland Publishing Company, Amsterdam.

Maclnnis, A. J. , Bethel, W. M. , & Cornford, E. M. , 1974. Identification of chemicals of snail origin that attract Schistosoma mansoni miracidia. Nature, 248, 361 - 363. quoted by Maclnnis (1976).

Madhavi, R. , 1966. Egg-shell in Paramphistomatidae (Trematoda: Digenea). Experimentia, 22, 93 - 94. quoted by Smyth (1976). - 206 -

Madhavi, R. , 1968. Diplodiscus mehrai: chemical nature of egg-shell. Experimental Parasitology, 23, 392 - 397. quoted by Smyth (1976).

Maldonado, J. F. , Ascosta-Matienzo, J , & Velez, H. F. , 1950. Biological studies on the miracidium of Schistosoma mansoni Part 3. The role of light and temperature in hatching. Puerto Rico J. Pub. Health & Trip. Med. 26, 85 - 91. quoted by Kassim & Gilbertson (1976).

Miller, H. M. & Mahaffy, E. E. , 1930.. Reactions of c.erca.r.ia hama.ta to light and to mechanical stimuli. Biol. Bull, 59, 95 - 103.

Mitte:rer, K. E. , 1975... The hatching of the eggs of Fasciola hepatica under various Co2 concentrations]. Zeitschrift fu r Parasitenkunde 47 (1), 35 - 43 [De, eni. Helminth. Abstr., 45 (2): No. 812.

Mitte re r, K. E. , 1975... Studies on the hatching of miracidia of Dicrocoelium dendriticum Zeitschrift fltr Parasitenkunde 48(1)35-45.

Monnickendam, M. A. , & Balls, M. , 1972. The long-term organ culture of tissues from adult Amphiuma, the Congo eel. J. of Cell Science, 11, 799- 813.

Monnickendam, M. A. , & Balls, M. , 1973. Amphibian organ culture. Experientia, 29, 1 - 17. - 207 -

Mushkambarova, M. G. , 1970. [The insect intermediate hosts of helminths in Turkmenia] In: Tashliev, A. O. , Shagalina, L. M [Editors] , [Academician K. I. Skryabin and the development of helminthological Science in Turkmenia]. Ashkhabad: Izdat. "ILIM", pp 74 - 78. [In Russian]. Helminth. Abstr, 41; No. 364.

Myers, F. L. & Northcote, D. H. , 1958. A survey of the enzymes from the gastro-intestinal tract of Helix. pomatia.. J. exp. Biol., 35, 639 - 648.

Newsome., J. , 1962.. Maturation of Schistosome eggs in vitro.

Nature, London 195, (4842) 72.2 723.

Nikulin, T. G. , 1971. [Odona.ta of the lake area of Byelorussia and their role in spreading trematode infections of birds]. Sbornik Rabot po Gel'mintologii posvyaschen 90-letiyu so dnya rozhdeniya Akademika K•.L. Skryabina. Moscow: Izdatel'stvo "Kolos", pp 2.84 - 289 [Ru] . Vitebsk veterinary Inst., Byelorussia, USSR. Helmint .Abstr., 41, No. 4172.

Odening, K., 1958. Zur Systematik von Haematoloechus (Trematoda: Plagiorchiidae). Mitt Zool. Mus. Berl 34, 53 - 108. quoted by Rees (1964).

Odening, K., 1960.. Revision der unterfamilie Haematoloechinae Freitas & Lent, 1939. (Trematoda: Plagiorchiidae). Sond. Monats. dtsch. Akad. Wiss. Berl. 2 (7), 449- 54.quoted by Rees (1964). - 208 -

Owen, G. , 1966. In "Physiology of Mollusca" (K. M. Wilbur & C.M. Younge, Eds. ), Vol. 2, pp 1 - 51. Academic Press, New York and London.

Parnas, I. , 1961. The cellulolytic activity in the Snail Levantina hierosolyma Boiss. J. Cell Comp. Physiol., 58, 195 - 201.

Paul, J., 1975. "Cell and Tissue Culture", 5th edition. Edinbrugh: E. & S. Livingstone.

Pearson, J. C. , 1956. Studies on the life cycles and morphology of the larval stages of Alaria a.risae moides Augustine et Uribe, 1927 & Alaria canis La Rue et Fallis, 1936 (Trematoda: Diplo- stomidae). Canad. J. Zool., 34, 295 - 387.

Pear.•son, J. C. , 196.1.. Observations on the morphology and life cycle of Neodi.plostomum intermedium (Trematoda: Diplostomatidae) Parasitology, 51, 133 - 172. quoted by Erasmus (1972).

Pitchford, R. J. & Visser, P. S. , 1972. Some observations on the hatching pattern of Schistosoma mansoni eggs. Annals of Tropical Medicine and Parasitology, 66 (3),399 - 407.

Prokopic, J. , & Krivanec, K. , 1974. Trematodes of the genus Haematoloechus Looss, and their variability. Helminthologia Bratislava, 15 (1/4) 779 - 802.

Prudhoe, S. & Bray, R.A. (In press). Platyhelminth Parasites of Amphibia. - 209 -

Ratcliffe, L. H. , 1968. Hatching of Dicrocoelium lanceolatum eggs. Exp. Parasitology, 23, 67 - 78.

Rafferty, K.A., Jr., 1976. The physiology of amphibian cells in culture. In "Physiology of the Amphibia". (B. Lofts, ed.) Academic Press, New York, Vol. III, pp 111 - 162.

Ramalingam, K., 1973. The chemical nature of the egg-shell of helminths. I. Absence of quinone tanning in the eggshell of the liver

fluke;, Fasciola. hepatica. International Journal for Parasitology, 3;. 67 - 75. quoted by Smyth (1976)

Rankin, J. S. , 1939. Cercaria pseudo burti n. sp., a strigeid cercaria from Wes•tern Massachusetts. J. Pa.rasit. 25 (1), 87 - 91.

Rankin, S. J. , 1939. Ecological studies on larval trematodes from Western Massachusetts. J. Parasit., 25, 309 - 328.

Rees, G., 1964. Two new species of the genus Haematoloechus (Digenea: Plagiorchiidae) from Rana occipitalis (Gunher) in Ghana. Parasitology, 54, 345 - 368.

Roberts, G. P. & Gibbons, R. A. , 1966. The action of neutral hypochlorite on epithelial mucopolysaccharides. Biochemical Journal, 98, 426 - 437.

Rogers, W. P. , 1960. The physiology of infective processes of nematode parasites: the stimulus from the animal host. Proceedings of the Royal Society (London) Ser. B., 152, 367 - 386. - 210 -

Rogers, W.P., 1961. "The nature of parasitism. The relationship of some metazoan parasites to their hosts" Academic Press, New York.

Rogers, W. P. , 1966 Exsheathment and hatching mechanisms in helminths. In "biology of parasites" (Soulsby, E. J. L. , ed. ) pp 33 - 40. Academic Press, New York and London.

Rogers, W. P. & Sommerville, R.I.., 1.963. The infective stage of nematode parasites and its significance in parasitism. Advances in parasitology 1,

pp109-- 177..

Romer, A. S. , 1949.. The vertebrate body pp 333. Philadelphia W. B. Saunders Company.

Rowan, W. B. , 1956. The mode of hatching of the egg of Fasciola hepatica. Experimental Parasitology, 5, 118 - 137.

Rowan, W. B. , 1957. The mode of hatching of the egg of Fasciola hepatica II. Colloidal nature of the viscous cushion. Experimental Parasitology, 6; 131 - 142.

Rudolphi, C.A., 1809. Entozoorum sive vermium intestinalium historia naturalis 2, 386 pp. Amstelae dami. quoted by Krull (1931).

Rudolphi, C.A., 1819. Entozoorum synopsis cui accedunt mantissa duplex et indices locupletissi mi 10; 811 pp. Berolini. quoted by Krull (1931). - 211 -

Runham, N.W. , 1975. Alimentary canal. In "Pulmonates" (V. Fretter & Peake, J. Eds.) pp 53 - 104. Academic Press, London, New York, San Francisco.

Russell, C.M. , 1954. The effects of various environmental factors and the hatching of eggs of Plagitura salamandra Hall (Trematoda: Plagiorchiidae). J. Parasit. 40, 461 - 4.

Ryley, J. F.., & Wilson, R. G. , 1978. "Cell. and. Tissue Culture". In "Methods of cultivating Parasites in vitro". , (e d. A. E. R. Taylor & J. R. Baker). pp 11.1. -. 128. Academic Press.

Salt, G. , 1963. The defence reactions of insects to metazoan parasites.

Parasitology, 53, 527 642...

Schell, S.C., 1962... The life history of Telorchis bonnerensis Waitz (Renife ridae ), a parasite of the long-toed Salamander, Amblystoma macrodactylum. Baird. Tr. Am. Micr. Soc. 81 (2.), 137 - 146.

Schell, S. C. , 1965. The life history of Haematoloechus breviplexus Stafford 1902 (Trematoda: Haplometridae McMullen, 1937), With emphasis on the development of the sporocysts. J. Parasit., Vol. 51 (4), 587 - 593.

Schell, S. C. , 1975. The miracidium of Lecithaster salmonis Yamaguti, 1934. (Trematoda: Hemiuroidea). J. Parasit., 63, 562 - 563. - 212 -

Schoffeniels, E. , & Gilles, R. , 1972. Ionoregulation and osmoregulation in Mollusca. In "Chemical Zoology" (M. Florkin & Scheer, B. T. Eds. Vol VII Mollusca pp 393 - 420. Academic Press, New York and London.

Seeley, L. B. , 1906. Two distomes. Biol. Bull., 10; 249 - 254. quoted by Krull (1931).

Shevchenko, N. N. , 1962.. [Helminth larvae in aquatic insects in the North Donets and its flood. plains] . Dokladi Akademii Na.uk SSSR,

1.42: (4) 972. 975 [In Russian]. Helminth.Abstr. 31, 1777

Shevchenko, N. N., &. Barabashova, V. N. , 1960.. [Aquatic arthropods as intermediate hosts of helminths . Dopovidi Akademii Nauk Ukrainskoi. RSR, No. 11,

pp 1.555 1. 559 [in Ukrainian]. Helminth.Abstr, 34, No. 825.

Stadnychenko, A. , 1974. Studies on host-parasite relationships between larval trematodes and their hosts :Title only] . In third International Congress of Parasitology. Munich.

August 2.5 - 31. Proceedings, Vol. I. Vienna, Austria. FACTA Publication 346. Helminth.Abstr., 44, No. 2312.

Stafford, J., 1902. American representatives of Distomum variegatum. Zogl. Jahrb. Syst., 16; 895 - 912 quoted by Krull (1931).

Stafford, J. , 1905. Trematodes from Canadian vertebrates. Zool. Anz. , 28,' 681 - 694. quoted by Krull (1931). - 213 -

Simnett, J. D. , & Balls, M. , 1969. Cell proliferation in Xenopus tissues, a comparison of mitotic incidence in vivo and in organ culture. J. Morph. 127, 363 - 372.

Sinicyn, D. 0. , 1905. Materijaly po estestvennoj istorii trematod. Distomyryb iljagusek Okrest nostiej Va.rsavy. 1 - 210, Varsava. quoted by Grabda (1960).

Smirnova, V. A. , & Ibrasheva, S. I. , 1.967. Larval trematodes from freshwater molluscs of the western Kazakhstan Trudy Inst.. Zool.. Akad. Nauk Kazakhstan SSR 27, 53 57. quoted by Yamaguti (1975).

Smith, B. J. , 197'0.

Notes on. the anatomy of Victaphanta atramentaria. (Shu.ttlewor.th) and V. compacta (Cox & Hedley) and the designation of a neotype for V. atr.amentaria. J'. malac. Soc. Aust. 2, 13 21. In "Pulmonates" (V.. Fretter & Peake, J. Eds.) Vol. 1 p xxiii. Academic Press, London, New York, San Francisco.

Smith, R. J. , 19 59 . Ancylid Snails: First intermediate host to certain trematodes with notes on ancylids as a new host for Megalodiscus and Haematoloechus. Trans. Amer. Microsc. Soc., 78,<. 228 - 231.

Smyth, J. D. , 1954.. A technique for the histochemical demonstration of poly- phenol oxidase and its application to egg-shellformation in helminths and byssus formation in Mytilus . quarterly Journal of Microscopical Science, 95; 139 - 152. quoted by Smyth (1976). - 214 -

Smyth, J. D. , 1966. The Physiology of Trematodes. Oliver & Boyd, Edinburgh and London.

Smyth, J. D. , 1976. Introduction to animal parasitology 2nd edition. pp 159- 161 Hodder & Stoughton, London, Sydney, Auckland, Toronto.

Smyth, J. D. , & Clegg, J . A. , 1959. Egg-shell formation in trematodes and cestodes. Experimental Parasitology, 8, 286 - 323 quoted by Srnyth (1.976).

Sponholtz, G. M. & Short, R. B. , 1976. . "Schistosoma mansoni Miracidia: Stimulation by calcium and Magnesium". J. Parasit., 62, 155 157.

Sugiura, . S., et al., 1954. A. study of several factors influencing hatching of Schistosoma japonicum eggs. J. Parasit., 40, 381 386..

Sooy, L.E., & Mezg,er-Freed, L., 1970. A serum macromolecule-supplemented medium for frog cell lines. Expl. Cell. Res., 60, 482 - 485.

Standen, O. D. , 1951. The effect of temperature, light and salinity upon the hatching of the ova of Schistosoma mansoni. Transactions of the Royal Society of Tropical Medicine & Hygiene 45, 225 - 241.

Stefan'ski, W. , 1965.

Bacterial flora as one of ecological factors affecting the establishment of parasites in the intestine of their hosts. Acta Parasitologica Polonica 13, 1, 1 - 6. - 215 -

Stibbs, H. H. , Chernin, E. , Ward, S. , and Karnovsky, M. L. , 1976. Magnesium emitted by snails alters swimming behaviour of Schistosoma mansohi Miracidia. Nature, 260, 702 - 703.

Stiles, C.W. & Hassall, A., 1902. Haematoloechus similigenus, new name for the trematode H. similis Looss, 1899. Bull (35) Bur. Anim. Ind. U.S. Dept. Agr: 20.

Stone, B.A., & Morton, J.E., 1958. The distribution of cellulases and related enzymes in Mollusca. Proc. Malacol. Soc. London, 33, 127 141..

Strasdine, G. A. , & Whitaker, D. R. , 1963. On the origin of the cellulase and chitinase of Helix pomatia Can. J. Biochem. Physiol., 41, 1621 - 1626. quoted by Runham (1975).

Stunkard, H. W . , 1930.. An analysis of the methods used in the study of larval trematodes. Parasitology,22, 268 - 273.

Sudarikov, V. E. , 1950. On trematode fauna. of Vertebrates in Middle Volga Provinces (Russian text). Trudy Gel'mint Lab. Akad. Nauk. SSSR, pp 131 - 41. quoted by Ginetsinskaya & Dobrovol'skii (1968).

Timon-David, J. , 1958. "Rale des insectes comme hates inter-mediaires dans les cycles des trematodes digene tique s" . International Congress of Entomology (10th), Montreal, 1956. Proceedings, vol. 3, pp 657 - 662. Helminth. Abstr., 31, 1778. - 216 -

Tongson, M. S. , & Semilla, S. S. , 1965. "The development and hatchability of Fasciola gigantica ova at different temperatures and hydrogen-ion concentrations". Philipp. J. Vet. Med. , 3 (2), 161 - 170. Helminth. Abstr., 40, No. 2423.

Underwood, H. T. , & Dronen, NM., 1977. The molluscan intermediate hosts for species of Haematoloechus Looss 1899. (Digenea: Plagiorchiidae) from raniid frogs of Texas. J. Parasit., 63, (1) 122.

Va:n. Thiel, 1922.. Aantee•ke.ninge:n over Agamodistomum anophelis Tijdsch.rift voor ve:rgelijkende Genee skunde, Deel VII, AfT... 2, pp 1 -- 1.7. quoted by Ginetsinskaya & Dobr.ovol'skii. (1968).

Van. Thiel, P. H..., 1930. Die, Entwicklung von. Agamodistomum anoph.elis zum Pneumonoec.es varie:gatus Rud. Centr. Bakt. 117, 103 - 112. quoted by Rankin (1939).

Veldhuijzen, J. P. , 1974. Sorting and retention time of particles in the digestive system of Lymnaea stagnalis. Netherlands Journal of Zoology, 24 (1), 10 - 21.

Voge, M. & Turner, J.A., 1956. Effect of temperature on larval development of the cestode Hymenolepis diminuta. Experimental parasitology, 5, 580 - 586. - 217 -

Walker, G., 1970b Light and electron microscope investigations on the salivary glands of the slug, Agriolimax reticulatus (Muller). Protoplasma 71, 111 - 126. quoted by Runham (1975).

Wesenberg-Lund, C. , 1934.

Contributions to the development of the Trematoda Digenea. II The biology of freshwater cercariae in Danish freshwaters. Mem. Acad. Roy. Soc. Let de Denemark Sect. Sci. Ser 9, Vol. 5, No. 3. quoted by Dobrovol'skii & Raikhel (1973).

Wilson,.. R. A. , 1968,. The hatching mechanism of the egg of Fasciola. hepatica. L. Parasitology, 58, 79 89.

Wilson, R.A. , 1968, An investigation into mucus produced by Lymnaea truncatula, the snail host of Fasciola, hepatica. Comp. Bioche.ra. Physiol. 2.4, 629 633. quoted by Maclnnis (1976) In. "Ecological aspects of Parasitology" (Ed. E. R. Kennedy) pp 3 - 2.0.

Wilson, R.A. , & Denison, J., 1970 . Short chain fatty acids as stimulants of turning activity by the miracidium of Fasciola hepatica. Comp. Biochem. Physiol., 32, 511 - 517. quoted by Maclnnis (1976).

Willmott, S. , 1952. The development and morphology of the miracidium of Paramphistomum hiberniae Willmott, 1950. J. Helminth., 26, 123 - 32. quoted by Erasmus (1972).

Wright, C.A. , 1971 . "Flukes and Snails". George Allen & Unwin Ltd. , London. - 218 -

Wright, C. A. , & Bennet, M. S. , 1964. The life cycle of Notocotylus attenuatus. Parasitology, 54 (4),

p. 14.

W right, D. G. S. , & Ronald, K. , 1972'. Effects of amino acids and light on the behaviour of Miracidia of Schistosomatium douthitti. Can. J. Zool., 50, 855 - 860. quoted by Maclnnis (1976).

Yamaguti, S. , 1958. Systema helrninthum Vol. I. The digentic trematodes of Vertebrates. Inter. science publishers Inc. New York

Iinte:r• s cience. Publishers Ltd.. ,. London..

Y.amaguti, S., 1975. A. synoptical review of life histories of digenetic trematodes of Vertebrates. Keigaku Publishing Co.

Zeder, J. H. , 1.800.. Erster Nachtrag Zur Naturgeschichte der Eingeweidewiirmer

Leipzig 32.0 pp. as quoted by Krull (1931).