THE LIFE-CYCLE
AND
MORPHOLOGY OF
TETRACERASTA BLEPTA, GEN. NOV., SP. NOV.
AND
STEGODEXAMENE CALLISTA, SP. NOV.
(TREMATODA: LEPOCREADIIDAE)
FROM THE LONG-FINNED EEL,
ANGUILLA REINHARDTII STEINDACHNER
BY
REGINALD ALAN WATSON BSc.(HONS.), MSc. (MANITOBA)
PARASITOLOGY DEPARTMENT University of Queensland, ST. LUCIA, Queensland, Australia, 4067.
A THESI S SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY WITHIN THE UNIVERSITY OF QUEENSLAND
JULY, 1982 DECLARATION
This thesis has been prepared in accordance with the rules set out in the Combined Higher Degree Handbook 1982, for the degree of Doctor of Philosophy at the University of Queensland.
The work presented is this thesis is entirely my own except where otherwise acknowledged and no part of it has been submitted for any other degree at this or any other university
R. A. Watson July 1982 ABSTRACT
Two new lepocreadiid digeneans, Tetracerasta blepta, gen. nov., sp. nov. and Stegodexamene callista, sp. nov. are described from the intestines of the long-finned freshwater eel, Anguilla reinhardtii, from the Brisbane River, Queensland, and the Australian bass, Macquaria novemaculeata, from the Richmond River, New South Wales, Australia.
Their life-cycles have been elucidated and completed in the laboratory. All developmental stages are described and illustrated.
Eggs from adults of these lepocreadiid species were obtained from eels. These hatched and miracidia from them injected a precocious sporocyst into uninfected laboratory-reared prosobranch gastropods, Posticobia brazieri. Following two generations of rediae in the digestive gland of the snail, large ophthalmotrichocercous cercariae were produced.
The cercariae of..!.:._ blepta penetrated uninfected tadpoles of Litoria lesueuri and the gudgeons, Hypseleotris compressus and ~ galii. Encystment occurred in the muscle and viscera.
The cercariae of S. callista lured fish to eat them or were inhaled. They penetrated the throat of uninfected, pond-reared rainbowfish, Nematocentris fluviatilis and uninfected perchlet, Ambassis sp. Encystment occurred in the pharyngeal muscle or liver.
Adults developed in uninfected eels, Anguilla austral is and A. reinhardtii, completing the life-cycles. The life-cycles and morphology of these species are compared to those of Stegodexamene anguillae Macfarlane, 1951 and other lepocreadiids.
i ACKNOWLEDGEMENTS
First and foremost I would like to thank Dr. J. C. Pearson for his never failing interest, his freely-given time and his valuable advice supported by years of careful observations.
I wish to thank T. Cribb, R. Hutchings, J. Field, I. Johnston, J. Waikagul, Dr. D. Blair and particularly Dr. J. Beumer and the Victorian State Fisheries for their provision of fish, specimens and field assistance.
I am very grateful for the help and support of J. Alder.
The author acknowledges the advice and assistance of Dr. C. Dobson, Dr. R. Lester and of the Queensland State Fisheries.
I would like to thank D. Scott for her technical advice and help. I am indebted to R. Manzanell for his assistance with translations and with the identification of nematodes. I am grateful to Dr. S. Edmonds for his identifications of acanthocephalans.
I acknowledge J. Hardy and the staff of the Electron Microscope Centre for their assistance.
The author most gratefully acknowledges the logistic support of the Department of Parasitol ogy; the Universi ty of Queensland, its services and particularly the provision of a University of Queensland Postgraduate Research Scholarship which helped sustain the author.
ii TABLE OF CONTENTS
ABSTRACT . i
ACKNOWLEDGEMENTS ii
LIST OF FIGURES. viii
LIST OF TABLES . v
LIST OF APPENDICES. xi
I INTRODUCTION.
II MATERIALS AND METHODS. 3
III LIFE-CYCLE
A. Tetracerasta blepta (a) First Intermediate Host . 5 (b) Second Intermediate Host. 6 (c) Definitive Host. 6
iii TABLE OF CONTENTS Continued
III LIFE-CYCLE
B. Stegodexamene callista (a) First Intermediate Host • 9 (b) Second Intermediate Host. 9 (c) Definitive Host. 9
C. Comparison 10
IV MIRACIDIUM
A. Tetracerasta blepta (a) Development . 15 (b) Hatching of the Egg 15 (c) Description . 20 (d) Swimming and Penetration of the First Intermediate Host 24
B. Stegodexamene callista (a) Development . 27 (b) Hatching of the Egg 27 (c) Description 27 (d) Swimming and Penetration of the First Intermediate Host 27
C. Comparison 28
V SPOROCYST
A. Tetracerasta blepta (a) Development 30 (b) Descri pti on . 30
iv TABLE OF CONTENTS Continued
V SPOROCYST
B. Stegodexamene callista (a) Development • 34 (b) Description 34
C. Comparison 35
VI REDIA
A. Tetracerasta blepta (a) Development . 36 (b) Description . 39
B. Stegodexamene callista (a) Development . 41 (b) Description . 43
C. Comparison 44
VII CERCARIA
A. Tetracerasta blepta (a) Development . 46 (b) Description . 49 (c) Behaviour and Swimming 50 (d) Penetration of the Second Intermediate Host 52
B. Stegodexamene callista (a) Development 55 (b) Description . 55 (c) Behaviour and Swimming 59 (d) Penetration of the Second Intermediate Host 60
v TABLE OF CONTENTS Continued
VII CERCARIA
C. Comparison 62
VIII METACERCARIA
A. Tetracerasta blepta (a) Development . 64 (b) Description 66 (c) Oral Sucker Lobes • 69 (d) Disposition in the Second Intermediate Host 71
B. Stegodexamene callista (a) Development . 72 (b) Description . 72 (c) Disposition in the Second Intermediate Host 75
C. Comparison 76
IX ADULT
A. Tetracerasta blepta (a) Development . 77 (b) Description . 77 (c) Remarks 83 (d) Generic Diagnosis . 84 (e) Specific Diagnosis. 84 ( f) Differential Diagnosis 84 (g) Disposition in the Gut of the Eel. 86
vi TABLE OF CONTENTS Continued
IX ADULT
B. Stegodexamene callista . (a) Develoµnent . 87 (b) Description . 87 (c) Remarks 93 (d) Specific Diagnosis. 94 ( e) Differential Diagnosis 94 ( f) Disposition in the Gut of the Eel. 95
X EVOLUTIONARY RELATIONSHIPS • 98
XI CONCLUSIONS • 99
APPENDICES 101
XII REFERENCES 106
KEY TO LE TTER I NG 11 3
vii LIST OF FIGURES Figure
1 A-F Development and Hatching of the Egg of Tetracerasta blepta 16 2 A-B The Vitelline Remnants of Tetracerasta blepta. 17 3 Epidermal Plates of the Miracidium of Tetracerasta blepta. 21 4 Miracidium of Tetracerasta blepta head-on • 21 5 Miracidium of Stegodexamene callista head-on . 21 6 Miracidium of Tetracerasta blepta Showing Internal Features . 22 7 A-F Penetration by the Sporocyst of Tetracerasta blepta. 22 8 Swimming Miracidium of Tetracerasta blepta 25 9 A-C Penetration by the Miracidium of Tetracerasta blepta 25 10 Sporocyst of Tetracerasta blepta 2 Weeks after Penetration 32 11 Sporocyst of Stegodexamene callista 2 Weeks after Penetration 32 12 Sporocyst of Tetracerasta blepta 2 Weeks after Penetration 33 13 Redia of Stegodexamene callista 33 14 A-B Development of the Redia of Tetracerasta blepta . 37 15 Redia of Tetracerasta blepta Showing Internal Features. 37 16 Redia of Stegodexamene callista at 4 Weeks. 42 17 Redia of Stegodexamene callista Showing Internal Features. 42 18 Developing Cercaria of Tetracerasta blepta. 47 19 Cercaria of Tetracerasta blepta 47 20 Body of the Cercaria of Tetracerasta blepta 47 21 Developing Cercaria of Tetracerasta blepta. 48 22 Live Cercaria of Tetracerasta blepta. 48 23 Anterior End of the Cercaria of Tetracerasta blepta. 48 24 Tail Setae of the Cercaria of Tetracerasta blepta 48 25 Swimming Cercaria of Tetracerasta blepta 51 26 A-D Penetration by Cercaria of Tetracerasta blepta 51 27 Developing Cercaria of Stegodexamene callista. 56 28 Body of the Cercaria of Stegodexamene callista 56 29 Live Cercaria of Stegodexamene callista. 57 30 Body of the Cercaria of Stegodexamene callista 57 31 Tail of the Cercaria of Stegodexamene callista 57 32 Ta il Setae of the Ce rcaria of Stegodexamene callista 57 33 Ve ntral Sucker of t he Cercaria of Stegodexamene calli sta 57 34 Body Spines on the Cercaria of Stegodexamene callista . 57
viii LIST OF FIGURES Continued Figure
35 Cercaria of Stegodexamene callista 58 36 Swimming Cercaria of Stegodexamene callista 58 37 Metacercaria of Tetracerasta blepta • 67 38 Oral Sucker Lobes of Tetracerasta blepta 67 39 Cyst of Tetracerasta blepta. 67 40 Metacercaria of Tetracerasta blepta . 68 41 Ventral Sucker of the Metacercaria of Tetracerasta blepta. 68 42 Oral Sucker Lobes of Metacercaria of Tetracerasta blepta 68 43 Oral Sucker Lobes Partially Protracted 68 44 Oral Sucker Lobes Protracted 68 45 Pores of Ampullae on the Oral Lobes of Tetracerasta blepta 68 46 Crystals in Caeca and Excretory Bladder in the Metacercaria of Tetracerasta blepta 68 47 Metacercaria of Stegodexamene callista . 74 48 Cyst of Stegodexamene callista. 74 49 Development of the Adult of Tetracerasta blepta • 79 50 Adult of Tetracerasta blepta 80 51 Terminal Male Genitalia of Tetracerasta blepta 80 52 Female Reproductive System of Tetracerasta blepta 80 53 Adult of Tetracerasta blepta 81 54 Anterior End of Tetracerasta blepta 81 55 Protracted Oral Lobes of Tetracerasta blepta 81 56 Ventral Sucker of Tetracerasta blepta 81 57 Terminal Male Genitalia of Tetracerasta blepta 81 58 A-D Development of the Adult of Stegodexamene callista 89 59 Adult of Stegodexamene callista 90 60 Terminal Male Genitalia of Stegodexamene callista 90 61 Female Reproductive System of Stegodexamene callista 90 62 Adult of Stegodexamene callista 91 63 Oral Sucker of Stegodexamene callista 91 64 Anterior Surface Scales of Stegodexamene callista 91 65 Ventral Sucker and Genital Opening of Stegodexamene callista. 91 66 Midbody Spines of the Adult of Stegodexamene callista 91 67 Midbody Spines of the Adult of Tetracerasta blepta . 91
ix ------LIST OF TABLES Table
I Natural and Experimental Second Intermediate Hosts. 7 II Measurements of the Miracidium 23 III Development of the Sporocyst in Experimental Infections . 31 IV Development of the Redia in Experimental Infections 38 V Measurements of the Redia . 40 VI Development of the Metacercaria of Tetracerasta blepta in Experimental Infections. 65 VII Development of the Metacercaria of Stegodexamene callista in Experimental Infections. 73 VIII Development of the Adult of Tetracerasta blepta in Experimental Infections. 78 IX Development of the Adult of Stegodexamene callista in Experimental Infections. 88
x ------LIST OF APPENDICES Appendix
I Measurements of the Cercaria • 101 II Measurements of the Metacercaria. 102 - III Measurements of the Adult . 103 IV Distribution of Adults along Gut. 105
xi I INTRODUCTION 1
Lepocreadiidae, a family of spiny digeneans of marine fishes, was beginning to take form when Stossich (1903) created the genus Lepocreadium using ~album as the type species. He added it to the Allocreadiinae which was an ill-assorted assemblage of digeneans, primarily parasites of fishes. Odhner (1905) began to organize the family Allocreadiidae by creating the subfamily Lepocreadiinae which Nicoll (1934) later elevated to the family Lepocreadiidae. Many subfamilies have been created since then, but all that maintains the unity of the family is the presence of a spiny tegument and the presence in the life-cycle of rediae which produce hairy-tailed cercariae without stylets.
Macfarlane (1951) described the morphology and life-cycle and in 1952 the bionomics of Stegodexamene anguillae from two species of New Zealand freshwater eels, Anguilla dieffenbachii Grey and A. australis schmidtii Phillipps.
The subfamily Stegodexameninae was erected by Mehra (1962) under the family Lepocreadiidae. Srivastava (1962) and Pershad (1964) both described species of Rhynchocreadium (Allocreadiidae) but although Kakaji (1969) synonymized these with Allocreadium mehra Gupta, 1956, Yamaguti (1971) included them as members of Rhynchocreadium and although these lacked a spiny tegument he wrongly made them a new subgenus of Stegodexamene in the family Lepocreadiidae.
Adults of two lepocreadiid digeneans were found in the long-finned freshwater eel, Anguilla reinhardtii Steindachner by Pearson (pers. comm.) in the Brisbane River system. He believed that one was a species of Stegodexamene and the other a new genus. His observations and experiments suggested that Posticobia brazieri (Smith) Iredale served as the first intermediate host. He had found encysted metacercariae closely resembling these two species in several small fishes and tadpoles.
The life-cycles of less than twenty lepocreadiids have been elucidated. Yamaguti (1975) provided a good summary of most of the known life-cycles. In addition, the reader is referred to descriptions of the life-cycles of Lepocreadium areolatum by Stunkard (1980a), Neopechona cablei by Stunkard (1980b) and Holorchis pycnoporus by Bartoli and Prevot (1978). I INTRODUCTION 2
The purpose of this study was to elucidate the life-cycles and to describe the morphology of these two lepocreadiid species. Laboratory-reared P. brazieri were exposed to eggs laid by these lepocreadiids taken from the "hindgut of the eel A. reinhardtii. Upon hatching their miracidia infected the snails and eventually cercariae emerged which encysted as metacercariae in cultured, parasite-free crimson-spotted rainbowfish, Nematocentris fluviatilis (Castelnau), in the case of Stegodexamene and in parasite-free tadpoles of Litoria lesueuri (Dumeril and Bibron) for the other genus. These infected fish and tadpoles were fed to uninfected eels, Anguilla australis (australis) Richardson and A. reinhardtii. Adult trematodes of these two lepocreadiid species were recovered from eel intestines, completing the life-cycles of these trematodes.
One trematode was found to be a new genus of Lepocreadiidae and named Tetracerasta (Greek, plural, neuter, meaning four horns). The type species was named Tetracerasta blepta (blepta is Greek meaning worth-seeing). The other lepocreadiid was a new species of the existing genus Stegodexamene Macfarlane, 1951 and named Stegodexamene callista (callista is Greek, singular, feminine, meaning most beautiful). II MATERIALS AND METHODS 3
Infection-free hosts were used throughout the life-cycle studies. The snails, P. brazieri, were laboratory-reared. Rainbowfish, N. fluviatilis, which were raised in pools without~ brazieri were used as the intermediate hosts of one lepocreadiid and young unparasitised tadpoles of Litoria lesueuri were used for the other.
All hosts taken from the wild were not assumed infection-free until at least 30 of a range of sizes were dissected and found free of all lepocreadiids or digeneans that might be confused with these. Such was the case for the eels, ~ australis taken from Merri Creek, Yarra River near Melbourne in Victoria and A. reinhardtii from the University of Queensland's Lake, Brisbane which were both used as the definitive hosts in the life-cycle studies.
Eels used for study of the disposition of adult lepocreadiids along the gut were dissected within 12 hours of capture and were alive until just before dissection. The stomach and pharynx were separted from the intestine and examined separately. The intestine was divided into quarters each of which was separately examined.
Snails, ~ brazieri, were exposed to miracidia by placing them in shallow dishes with containers of eggs which were screened so that the snails could not ingest the eggs. Small fishes and tadpoles were exposed to cercariae for less than a day in shallow dishes or in large jars for longer periods. Eels were kept in large filtered aquaria and were hand-fed infected fish or tadpoles. When they would not take these they were force-fed while anaesthetized with a dilute benzocaine solution.
Most observations were made on living specimens but unless otherwise stated, all morphological measurements were made on specimens killed and fixed in hot 5 to 10% formalin, stained in haemalum, dehydrated with ethanol, cleared in methylbenzoate and mounted in Canada balsam. Specimens were not flattened.
All measurements are reported in micrometers in the f orm: average (range, sample size) unless otherwise stated. II MATERIALS AND METHODS 4
The development of miracidia was followed by placing eggs in a small volume of water under a coverslip supported and sealed by petroleum jelly. The epidermal plates and pores of miracidia were demonstrated by the method of Lynch (1933). Miracidia wer e examined head-on using a modification of Anderson's (1958) method as follows: the miracidium was placed on a coverslip and the excess fluid blotted away, a small piece of glycerine jelly was placed loosely over it, the jelly was slightly and carefully heated until liquid, the miracidium was positioned head down, the jelly was allowed to harden and then the coverslip was inverted and supported by plasticine above a slide. The coverslip could be easily removed and the miracidium reorientated without further heating. Lacto-orcein in acetic acid was used to stain the nuclei of miracidia and cercariae.
Paraffin sections taken on several planes at 'ZAJm were prepared of some specimens; these sections were stained in hematoxylin and eosin or Masson's trichrome after Humason (1962). Scanning electron microscopy was used to study the surface features of some specimens.
A cine camera operating at 100 frames per second was used to record the swimming behaviour of the cercariae of Stegodexamene callista at 25° c in a coulter counting slide.
The term prevalence is defined as the percentage of hosts infected and intensity as the mean number of worms in an infected host. As there is no mother s~rocyst, the adjecti ves "mother" and "daughter" will not be used to distinguish the s~rocyst and redial generations as such use could lead to confusion. III LIFE-CYCLE 5 A. Tetracerasta blepta
(a) First Intermediate Host
The prosobranch snail Posticobia brazieri is found infected in streams and rivers where infected Anguilla reinhardtii was caught. These snails are only numerous in streams which are not brackish. No other species of snail is found naturally infected or could be experimentally infected.
Only a maximum of 0.3% of P. brazieri over 1.5 mm and none under
0.5 mm are found infected in nature with~ blepta, but all those snails over 0.5 mm tested could be infected in the laboratory.
Sporocysts from several miracidia may penetrate a single snail. These sporocysts quickly produce rediae and die. Within one month of penetration 6(2 to 20, 20) rediae are found within each snail, entangled in and feeding on the digestive gland and gonad in the posterior coils of the snails. Within six weeks the immature cercariae pass out of the rediae and develop in the surrounding body tissue.
Approximately two months after penetration of the snail by the sporocyst, the cercariae begin to emerge, usually less than 10 per snail each day, although a large, 2 to 3 mm, heavily infected snail had over 20 cercariae per day emerge from it.
Kendall (1964) believed that all ages and sizes of the preferred snail host were susceptible to infection by Fasciola hepatica but that larger snails could support a greater burden on their nutrition and thus infections of greater intensity. Cort, Ameel & Van der Woude (1954) found germinal sacs (rediae) did not develop as well within the crowded conditions of small juvenile snails as they did in larger snails.
The results of Kendall (1964) and Cort et al. support the present author's findings that larger snails produced more cercariae. It is likely that Posticobia under 0. 5 mm are incapable of providing the nutrients and space necessary for the production and development of~ blepta cercariae. Smaller snails may die as infections develop within them but I have no evidence for this. III LIFE-CYCLE 6 A. Tetracerasta blepta
(b) Second Intermediate Host
The fishes and tadpoles which were found infected in nature or which could be experimentally infected appear in Table I. Parasite-free experimental hosts were used in all life-cycle studies. Only in the cases of Gambusia affinis, Nematocentris fluviatilis and Litoria lesueuri were more than 10 individuals exposed in the laboratory to cercariae of either lepocreadiid. A minimum of five wild-caught individuals of all host species in Table I except Notesthes robusta were examined for lepocreadiid metacercariae. Only one N. robusta was examined.
Care should be taken in the interpretation of the data presented in Table I because the conditions in the laboratory were not those in nature and hosts were exposed over a short period to greater numbers of cercariae then they are likely to be exposed to in nature. A fish cable of being an intermediate host in the laboratory could be ecologically or otherwise separated from the other hosts necessary in the life-cycle.
The best experimental and natural intermediate host of _!:_ blepta was the tadpole of Litoria lesueuri. The guppy, Poecilia reticulata Schneider, and the mosquitofish, Gambusia affinis (Baird and Girard), were difficult to infect experimentally and the latter, which is a common introduced species, is very seldom infected in nature. Although the elver of Anguilla reinhardtii could be experimentally infected, it has not been found to have metacercariae in nature.
All infected fishes and tadpoles are found in streams or rivers close to infected snails and eels. There are rarely more than 10 metacercariae encysted in a wild-caught fish or tadpole.
(c) Definitive Host
The foreguts and hindguts of A. reinhardtii and the Australian bass, Macquaria novemaculeata, are found infected in the wild. The mouth almighty, Glossamia aprion, and the spangled perch, Leiopotherapon unicolor, ar e not found infected in nature and attempts to infect these fishes in the laboratory have failed. The eel, A. australis, is not found 7 TABLE I: Natural and Experimental Second Intermediate Hosts
Tetracerasta Stegodexamene Host Species
Anguilla australis (australis) 0 0
A. reinhardtii 0 + 0 + Notesthes robusta (Gunther) 0 0 Leiopotherapon unicolor (Gunther) 0 0 Glossamia aprion (Richardson) 0 0
Retropinna semoni (Weber) 0 + + * Carassius auratus Linnaeus 0 0 Tandanus tandanus Mitchell 0 0 * Gambusia affinis 0 + 0 + * Poecilia reticulata + + Nematocentris fluviatilis + + Nematocentris sp. + + Craterocephalus stercusmuscarum (Gunther) 0 + Pseudomugil signifer Kner 0 + + +
Ambassis sp. + 0 + 0 Philypnodon grandiceps (Krefft) 0 0 Gobiomorphus australis (Krefft) + 0 Gobiomorphus sp. + + 0 Mogurnda sp. 0 0 Hypseleotris compress us (Krefft) + 0 0 +
.!:!..:_ galii (Ogilby) + 0 + Myxus petardi (Castelnau) 0 0 Mugil cephalus Linnaeus 0 0
Mixophyes sp. + 0
Litoria lesueuri + + 0 + Litoria sp. + 0 * Bufo marinus Linnaeus 0 0
KEY: + positive 0 negative if blank then species not tested * introduced species Nat= wild infection Exp= experimental infection III LIFE-CYCLE 8 A. Tetracerasta blepta
infected locally or in samples from Victoria though this eel could be infected experimentally.
The smallest A. reinhardtii found infected in nature was a 14 cm specimen from the Fitzroy River, Rockhampton, Queensland, although the prevalence of~ blepta in eels under 30 cm is very low. 9 III LIFE-CYCLE B. Stegodexamene callista
(a) First Intermediate Host
As with.!..:._ blepta, the only snail which is infected in nature or which has been experimentally infected is P. brazieri. Prevalence of S. callista in wild P. brazieri is low and comparable to that of .!..:._ blepta. Often fewer cercariae emerge from snails infected with S. callista than from those with a comparable infection of.!..:._ blepta. One snail, approximately 2 mm in diameter, produced 32 cercariae in one day. The digestive gland of this snail was completely penetrated with rediae. Kendall (1964) found that when many digenean infections of snails are mature, very little is left of the snail's digestive gland.
Possibly because of the extremely low infection rates of these lepocreadiids, no snail ever had an infection of both, even though over 10,000 were examined. Even in mixed experimental infections where the prevalence of both .!..:._ blepta and S. callista was high, no snail ever produced both cercariae.
(b) Second Intermediate Host
Those fishes infected in nature or through experiment appear in Table I. Infected fishes are caught in streams or rivers in the vicinity of infected eels and snails. It was possible to infect the elver of A. reinhardtii with cercariae but only a few cysts resulted.
(c) Definitive Host
Although the eel,!:_ australis, was not found infected in nature, as are A. reinhardtii and the bass, M. novemaculeata, it was infected in the ~ ~ laboratory and several successive infections were achieved. Eggs released by adults of S. callista from experimental infections of!:_ australis were used to infect P. brazieri and normal cercariae emerged.
The smallest A. reinhardtii found infected in nature was 30 cm in l ength. 10 III LIFE-CYCLE C. Comparison
The life-cycles of less than twenty lepocreadiids have been elucidated. In addition to those summarized in Yamaguti (1975), the life-cycle of Lepocreadium areolatum has been reported by Stunkard (1980a), that of Neopechona cablei by Stunkard (1980b) and that of Holorchis pycnoporus by Bartoli and Prevot (1978). Lepocreadiids whose life-cycles are known include about five species of Lepocreadium and about eight other genera of which all but Stegodexamene anguillae have marine hosts. All use a prosobranch gastropod as their first intermediate host. It is common for species of Lepocreadium to use Nassa, Nassarius or Conus, whereas amongst the other genera of lepocreadiids, Mitrella, and Anachis are common hosts.
Cercariae usually emerge and penetrate a wide range of invertebrate hosts such as medusae, annelids, gastropods and lamellibranchs. Medusae and annelids are the most commonly used. Metacercariae often remain unencysted in these hosts. Arvy (1953) found that Lepocreadium album remained unencysted with its tail still attached in the nudibranch Phyllirhoe bucephala. Many authors suggest that the metacercariae creates little or no host response.
These invertebrate hosts are then eaten by a definitive fish host and the adult lepocreadiid becomes established in their intestine. A wide range of definitive host fishes have been reported for lepocreadiids including gobies, blennies, and flounders as well as members of the Sparidae, Scf ombridae and Labridae.
Only one lepocreadiid has been previously reported to have a life-cycle which does not fit the basic marine prosobranch - marine invertebrate - marine fish life-cycle pattern and that is Stegodexamene anguillae. It has a life-cycle using only freshwater hosts and utilising several fish species but not invertebrates as the intermediate host (Macfarlane, 1951). These fish species are mainly bottom-dwelling and sedentary.
The lepocreadiids of the present study have only freshwater hosts and use vertebrate intermediate hosts. The prosobranch snail host, Posticobia brazieri, is one of the few freshwater members of a marine family and even though the definitive eel host, Anguilla reinhardtii, lives most of its life in freshwater, it is catadromous and spawns in the sea near New III LIFE-CYCLE 11 C. Comparison
Caledonia. Eleotriids and other second division freshwater fishes can
serve as intermediate hosts. All the hosts in the life-cycles of~ blepta and S. callista except the tree frog are from marine groups or have some association with the sea. As might be supposed from the general life-cycles of lepocreadiids, there is little specificity for the second intermediate host although invertebrate hosts have not been found infected by the lepocreadiids of this study. The marine affinities of the hosts in this study and the diversity of marine lepocreadiid life-cycles suggest that Tetracerasta and Stegodexamene may be examples of the movement of trematodes into freshwater systems with their marine hosts. Cable (1974) believed that because there are so few freshwater species of lepocreadiids that this was evidence for a marine origin for the family.
Macfarlane (1951) found that Stegodexamene anguillae used Potamopyrgus
antipodum and ~ badia as its primary hosts in New Zealand. These are in the same family of snails, the Hydrobiidae, as Posticobia brazieri, the
primary host for~ blepta and S. callista. S. anguillae infected 0.7% of Potamopyrgus sampled in New Zealand (Macfarlane, 1952), this is more than twice the infection rate of Posticobia by local lepocreadiids but is still quite low.
Although~ callista can use amphibian larvae as intermediate hosts in
the laboratory, it is not common in tadpoles in nature. ~ blepta appears to be a common parasite of tadpoles. By using tadpoles in their
life-cycle, ~ blepta may become established in upland rainforest where waterfalls block the entry of all fishes but eels. In rainforest areas where Posticobia is found, tadpoles can become infected and could be eaten by eels before or after the tadpoles transform into adult frogs. The other lepocreadiid, S. callista, is found only where its fish intermediate hosts are nearby and is prevented from completing its life-cycle in the rainforest.
Macfarlane (1951) found that ~ anguillae metacercariae developed eventually into egg-producing adults, which he called "progenetic metacercariae", while encysted in Gobiomorphus cotidianus and Galaxias brevipennis but such a change may have taken two or more years (pers. comm.). III LIFE-CYCLE 12 c. Comparison
This would explain how the life-cycle of ~ anguillae is completed at Kuratau, Lake Taupo, New Zealand, where eels are largely unknown (Hine, 1978).
No encysted adults of S. callista or .'.!:..!. blepta were ever found in nature in the present study but the age of the natural infections was not known. In laboratory infections of rainbowfish, N. fluviatilis, no egg production took place within one year and very little development of the metacercariae occurred after two weeks. Maturation of metacercariae to egg-producing adults within cysts may occur in~ callista but, if so, must be quite rare. Metacercariae of.'.!:..!. blepta continue .to grow for several months in the tree frog, Litoria lesueuri, but none reached maturity after nine months.
In the laboratory the short-finned eel, A. australis, could be infected with both .'.!:..!. blepta and S. callista although only~ callista lived long enough to produce eggs. The same eel, A. australis, was not found infected with either lepocreadiid in nature.
The short-finned eel,!:._ australis, is a temperate eel and is much less common in the Brisbane River then is the tropical, long-finned eel, A. reinhardtii. As a result, less than five local!:._ australis were caught and examined but none was infected with either lepocreadiid, although the prevalence of both digeneans is high in samples of local A. reinhardtii. In samples of eels from the Franklin and Agnes Rivers, Victoria, where the temperate A. australis is common and the tropical A. reinhardtii is comparatively rare, the latter is infected with Stegodexamene, probably callista, whereas once again!:._ australis has no lepocreadiids. Why are two such di ver se fishe s as the l ong-finned eel,!:._ reinhardtii , and the Australian bass, !i..:_ novemaculeata, commonly infected in the wild, whereas the short-finned eel,!:._ australis, is uninfected? The reason may lie in their diets. Rid (1973) found that Stegodexamene anguillae was twice as prevalent in Anguilla australis schmidtii as i n !:._ dieffenbachii sampled from the Waimakariri River in New Zealand although the intensity of infecti ons was nearly equal. He believed that a difference in diet caused the difference in prevalence. A. austral is schmidtii fed more heavily on small fishe s includi ng the intermediat e hosts of ~ angui llae which ar e Gob i omorphus goboi des and Philypnodon s p. ( Cairns , 1942). III LIFE-CYCLE 13 C. Comparison
Too few of the local ~ australis were caught to allow a comparison of diet with A. reinhardtii. The high water temperatures of the Brisbane area make any stomach content analysis difficult because of the rapid decomposition of food in the stomachs.
Dietary differences may explain why~ australis is not infected with S. callista in the wild whereas when it is fed metacercariae in the laboratory, infections of gravid adults are produced, but it can not explain why laboratory infections of ~ blepta fail to become gravid and usually die within one month, regardless of the maturity of the metacercariae fed to the eel. S. callista may develop fully in both species of eels because it is less host specific then~ blepta which can only mature in the long-finned eel, ~ reinhardtii. Host specificity in
~ blepta is, however, hard to reconcile with its ability to develop in both the long-finned eel, ~ reinhardtii, and the Australian bass,
~ novemaculeata, which are fishes in different orders and would be expected to be less similar in their physiology then the congeneric A. reinhardtii and A. australis.
Macfarlane (1952) did not find eels under 35 to 40 cm in length infected with Stegodexamene anguillae; this agrees well with my findings for S. callista, but it was possible to find~ reinhardtii of half that length infected with~ blepta. This may mean that the intermediate hosts of ~ blepta can be infected at a smaller size than those of S. callista. Such hosts might be small gudgeons, Hypseleotris compressus or H. galii, or small tadpoles, which can be easily infected in the laboratory and are primarily hosts of~ blepta and not S. callista. Tadpoles less than one centimeter in length might be eaten by and i nfect smal l ( <15 cm) A. r e inhardtii .
The differences in the behaviour of eels under 30 cm and those over, as observed by Macfarlane (1952 ) and the present author, might also explain the difference i n the maxi mum size of eel infected by these t wo lepocreadiid species. I have observed that very small eels spend a large amount of time buried up to their necks in gravel or well hidden amon gst rocks on the bottom. They feed on small snails and crustaceans . They can catch only small and s l ow- mov i ng ani mals tha t frequent the bottom. Lar ger eels ar e us ually mor e exposed and gener ally feed on lar ger organisms . They III LIFE-CYCLE 14 C. Comparison
often feed on large fish but they continue to eat snails and crustaceans, though these form a smaller proportion of their diet. The behaviour of the intermediate host species, especially whether they hide near the bottom or stay in the water column, could determine if smaller eels could feed on them and acquire lepocreadiid infections from them.
Eels from about 30 to 80 cm are usually quite heavily infected and presumably eat many infected fish. Eels over 80 cm (the largest eel caught was 103 cm) are less heavily infected and stomach contents reveal that they feed on larger, uninfected fish species or birds.
Both adults and metacercariae of~ blepta and ~ callista showed no seasonal trends in prevalence. The prevalence of these lepocreadiids in snails was so low that seasonal studies were not possible. Posticobia and suitable fish intermediate hosts were common in the areas sampled but water levels fluctuated seasonally and the period of study included some unseasonably dry weather which reduced their habitat and therefore likely reduced their numbers. The movement of eels between the Brisbane River and the smaller creeks and dams which were studied may have been influenced by water levels, temperature and the availability of food, but eel numbers and movement could not be properly assessed.
Studies of marine lepocreadiids have shown seasonal cycles in prevalence. McDaniel & Coggins (1972) found that Lepocreadium setiferoides, a parasite of flounder, infected the prosobranch, Nassarius obsoletus, during all months except June, but prevalence was highest in December as the cold weather approached. Miller & Northup (1926) found that the maximum prevalence for this species in Nassa obsoleta was in December and July. They believed that it might be related to the migration of the definitive host, the flounder, the degree of the infestation of the flounder, the life-span of N. obsoletus and/or the effect of parasitism upon it. The maximum number of Nassa (Amyclina) corniculum infected with metacercariae of Lepocreadium album was in May (Palombi, 1937). IV MIRACIDIUM 15 Tetracerasta blepta
(a) Development
The zygote of the newly laid egg is a large clear cell located near the opercular end. The rest of the egg is filled with large, granular vitelline cells (Figure 1A). As the embryo develops the vitelline cells become more granular and within 2 days their opacity limits study of the developing embryo. Intact vitelline cells persist in the abopercular end of the egg for several days. Vitelline cell remnants form large refractile vacuoles at the ends of the egg. By day 5, at 20 to 25oc, most miracidia have developed some eye pigment and at day 6 most show active flame cells. Development continues rapidly and by day 7, miracidia are fully developed and active (Figure 1C). By then, the developing sporocyst within the posterior half of the miracidium shows flame-cell movement and is capable of contraction and extension independent of the miracidium.
Eggs in fresh eel faeces are not developed beyond their condition in the uterus of the adult fluke.
Development of miracidium of Bunodera sacculata reported by Cannon (1971) is similar to that of Tetracerasta blepta.
(b) Hatching of the Egg
At temperatures of 20 to 25oC, hatching begins after 7 days and at 10 to 2ooc, 14 days after the eggs are laid. Hatching occurs mostly in the first hours of daylight and light simulates the activity of the miracidium within a developed egg; very few eggs hatch in complete darkness.
The activity of a miracidium and its latitude of movement increase as hatching approaches and as a result, the vitelline remnants are pushed to a lateral position in the egg (Figure 2A). Several hours before hatching, the miracidium begins to slowly contract and stretch. By an hour before hatching it begins to press on the operculum about every 30 seconds during which vigorous ciliary activity occurs. The miracidium starts to continuously probe the operculum as though it were seeking a weak point or applying a digestive enzyme and occasionally it rapidly contracts and extends its body. Figure 1: Development and Hatching of the Egg of Tetracerasta blepta A Newly laid egg B Egg with developing miracidial embryo C Egg with fully developed, active miracidium D Miracidium pressing on egg operculum E Miracidium being ejected within vitelline membrane F Miracidium piercing vitelline membrane 1 6
/ 1
f .c.
A 8 c
I 25,Um I
D E F Figure 2: The Vitelline Remnants of Tetracerasta blepta (Arrow indicates vitelline remnant immediately before and after hatching; Scale for both 25 µn) A Miracidium immediately before hatching B Egg shell immediately after hatching (Vitelline remnants swell and burst) 17
0
..
0 IV MIRACIDIUM 18 Tetracerasta blepta
No viscous cushion is ever visible between the operculum and the miracidium. The apical end of the miracidium comes into close contact with the opercular seal when the miracidium extends itself and uses the abopercular end of the egg for purchase; later, it often uses the sides of the egg for leverage as it exerts slight pressure on the operculum by flexing it body into a C-shape (Figure 10). Flame-cell activity increases as the vitelline remnants enlarge and occasionally burst. Some miracidia rotate on their longitudinal axis immediately prior to hatching.
The miracidium continues to contact the operculum until the operculum swings open rapidly. The miracidium does not attempt to protect its apical end. As its cilia quiver, the miracidium, still enclosed in the vitelline membrane, is quickly ejected 3/4 of its length out of the shell, but is stopped when the vitelline membrane fails to pass completely through the opercular opening (Figure 1E). Within seconds, the miracidium pierces the membrane and swims away (Figure 1F). Occasionally the vitelline membrane ruptures over the opercular opening and the miracidium swims away in the anterior portion of the membrane. If the vitelline remnants are still intact they continue to swell and eventually burst (Figure 2B).
Pearson (1956) and others have found that light is an important stimulus for the hatching of digenean eggs.
When the active miracidium of Tetracerasta blepta presses on the operculum it may be applying digestive enzymes to the opercular cement or it may be damaging the vitelline membrane which allows an influx of water, or both. Rowan (1956) reported that the miracidium of Fasciola hepatica released a hatching enzyme which digested the opercular seal. Wilson (1968) believed that no such enzyme exists but that the activity of the miracidium only damages the vitelline membrane allowing water to enter. Agarwal (1959) and others believe that the miracidium simply breaks open the operculum and wriggles out.
Eggs of Tetracerasta blepta have no viscous cushion or pad under the operculum as Pearson (1956) and Rowan (1957) have observed in other trematodes. Pearson (1956) suggested that the miracidium of Alaria arisaemoides reduced or displaced this pad exposing the vitelline membrane which possibly allowed water to enter. Rowan (1957 ) observed that the pad IV MIRACIDIUM 19 Tetracerasta blepta
in eggs of Fasciola hepatica expanded as hatching neared. Wilson (1968) believed that this occurred because the miracidium damaged a membrane surrounding the pad and an influx of water caused the pad to expand.
The increase in flame-cell activity and the swelling of vitelline remnants prior to hatching of .!..:._ blepta indicates that osmosis is occurring. The resulting osmotic pressure causes the ejection of the miracidium and the subsequent rupture of the vitelline remnants. Pearson (1956, 1961) observed an increase in the flame-cell activity as hatching neared. Wilson (1968) observed that sacs within the egg expanded. Rowan (1957) measured an increase in water within the egg as hatching neared.
These authors and others have presented strong evidence that the miracidium is ejected, sometimes even backward or when dead, if the eggs are in a hypoosmotic solution. Kassim & Gilbertson (1976) and Becker (1973) have shown that the osmolarity of the medium affects hatching. Kearn (1975) reported that the hatch of the oncomiracidium of Entobdella soleae, a monogenean, does not depend on osmolarity but occurred through the application of enzymes and pressure by the oncomiracidium on the operculum.
Wilson (1968) suggested that pressure from the expansion of sacs within the egg was the main means of escape for the miracidium but in
~ blepta apparently normal hatching occurs even when the vitelline remnants have ruptured prior to hatching indicating that although the sacs contribute to the increasing osmotic pressure that ejects the miracidium, with the increased turgor and possible weakening of the opercular seal, the sacs are not essential to hatching.
Cable (1972) believed that different hatching mechanisms may occur in different species and suggested that the hatching of anoperculate eggs and of those of philophthalmids could be explained by the movements of the activated miracidium. He suggested that the elastic shells of philophthalmid eggs exerted pressure which helped the larvae rupture the shell and escape. Kassim & Gilbertson (1976) found that the osmolarity of the medium affected the rupture of the anoperculate eggs of Schistosoma mansoni. They found that eggs would hatch if the ion concentration of the medium allowed an influx of water whi ch swells and ruptures the egg, IV MIRACIDIUM 20 Tetracerasta blepta
expelling the miracidium even if it were dead.
The development and hatching of lepocreadiid eggs has seldom been reported. Stunkard (1969) found that eggs of Neopechona pyriforme hatch in 9 to 10 days at laboratory temperatures. Macfarlane (1951) found that hatching of Stegodexamene anguillae eggs occurred by contractions and extensions of the body as it pushed up the operculum rather than through the motile power of the beating cilia. He did not report the period of development.
(c) Description
Miracidium is ovoid to spheroid and its measurements appear in Table II. Cilia are arranged on 4 tiers of epidermal plates with the formula 6:9:4:2 (Figure 3). Tier I, the most anterior, has 6 triangular plates (2 subdorsal, 2 lateral, 2 subventral) with their posterior margins at the level of the eyespots; tier II had 9 rectangular plates (2 dorso-lateral, 2 lateral, 2 ventro-lateral, 2 subventral, 1 ventral); tier III has 4 trapezoidal plates (2 dorso-lateral, 2 ventro-lateral); and tier IV has 2 helmet-shaped plates (1 dorso-lateral, 1 ventro-lateral).
The asymetrical pattern formed by the apical pores appears in Figure 4. There is a lappet on the left and right sides visible only in living specimens (Figure 6).
Internally, the miracidium has two concave pigment cups which are joined medially to form an hourglass shape. Each cup has two visible rhabdomeres. Apical gland longitutionally divided, filled with granular material and lacking nucleui. The miracidium has two large flame cells which open through ducts on the right and left sides, just anterior to the most posterior row of epidermal plate s. The nuclei of these cell s are the only nuclei visible in the body of the miracidium except those of the developing sporocyst even when lacto-orcein was used to stain cell nuclei.
In the posterior half of the miracidium is the spheroidal developing sporocyst. Th is moti l e body has a po i nted anterior end and two small active flame cells which open laterally through ducts. The apical end of Figure 3: Epidermal Plates of the Miracidium of Tetracerasta blepta
Figure 4: Miracidium of Tetracerasta blepta head-on .,
Figure 5: Miracidium of Stegodexamene callista head-on 21
3
e.p.
2s_um Figure 6: Miracidium of Tetracerasta blepta Showing Internal Features
Figure 7: Penetration by the Sporocyst of Tetracerasta blepta A Miracidium soon after contacting snail ' B Sporocyst beginning to elongate c Sporocyst elongated and moving into anterior of miracidium D Sporocyst beginning to leave miracidium E Sporocyst leaving miracidium and entering snail F Sporocyst within snail tissue 22
/
/
Wll'OS
<( u 0 ·.u
?
..c.·u . c.5 WrY St --= lo... c.. '+- 23
TABLE II: Measurements of the Miracidium
Feature Tetracerasta blepta Stegodexamene callista
Body 44(41-50,18) 47(40-54,9) 33(30-36,21) 33(29-40,8)
Cilia Length 6(4-8,5) 6(4-8,16)
Epidermal Plates: Tier I 12(8-14,11) 11(11-12,12) 9<8-9' 8) 10(10-12,10) Tier II 20( 15-23, 21) 16(15-17,6) 11(9-17' 15) 9(7-13, 12) Tier III 20( 17-22, 11) 16(13-18,6) 22(16-23,5) 21( 19-23, 5) Tier IV 15(14-17,3) 13(10-15,4) 27(26-28,4) 24(20-28,4)
Pigment Cups 8(7-9. 18) 8(7-10,28) 10(8-11,16) 11(9-13,21)
Flame Cell 8 by 4 8 by 3
Sporocyst 28(25-34,20) 30(27-33,8) 22(20-25,21) 22(18-25,8)
Sporocyst Flame Cell 4 by 2 4 by 2
Number Sporocyst Cells 34(30-41,8) 41(38-44,5) Figure 8: Swimming Miracidium of Tetracerasta blepta (Scale 25 ,,um)
Figure 9: Penetration by the Miracidium of Tetracerasta blepta (Arrow indicates advancing sporocyst; Scale 25 µm) A Miracidium contacting snail B Sporocyst within miracidium elongating and moving anteriorad C Miracidium contracting toward snail 8 25
• • IV MIRACIDIUM 26 Tetracerasta blepta
Cable (1972) and others have discussed the behaviour of miracidia. They reported that some miracidia respond to the snail's presence by swimming faster and in tight cir~les because they detect some substance which diffuses from the snail into the surrounding water. Miracidia of
~ blepta often try to penetrate the glass surface over which snails have just passed as if some identifiable and attractive trace of the snail, presumably in its mucus, had rubbed off onto the glass.
Stunkard (1934) and West (1961) have observed a well developed intramiracidial redia penetrate a snail from a miracidium which had only partially penetrated the snail itself. Cable (1972) has been able to observe a sporocyst emerge from a miracidium in vitro which was he believed to be old and deteriorating. No such in vitro escape by the sporocyst from old or dying miracidia of T. blepta has been observed in the present study.
Some species of miracidia release a well developed redia soon after penetration (Bennett and Humes, 1939).
The behaviour of only one lepocreadiid miracidium has been reported, that of Stegodexamene anguillae by Macfarlane (1951). He reported that the miracidium swam rapidly toward the light, often to the surface, revolving slowly on its long axis. Although he reported an active semi-independent intramiracidial body which he believed to be a redia, he did not describe penetration of the snail which is likely to be a similar process to that of
~ blepta. IV MIRACIDIUM 27 B. Stegodexamene callista
(a) Development
The newly laid egg is very s1milar to that of ~ blepta (Figure 1A).
Development is similar to that of an egg of~ blepta. By day 7, at 20 to 25oc , and at day 10 at 15 to 2ooc, most miracidia have eyespots. Within one day of eyespot formation the miracidia show flame-cell movement. By day 8 at 20 to 25oc, the miracidium is moving within the egg.
(b) Hatching of the Egg
Eggs begin to hatch on the 9th day at 15 to 20°c (days 11 to 14 at 10 to 15°c). Light appears to stimulate hatching. The process of hatching is identical to that of~ blepta (Figure 1D-F).
(c) Description
Measurements of the miracidium appear in Table II. Its morphology is similar to that of -T. blepta (Figure 3). The pattern of apical pores appears in Figure 5 and differs somewhat from that of -T. blepta shown in Figure 4. The pores at the posterior margin of the first tier of the epidermal plates in S. callista have an asymmetrical arrangement as they do in ~ blepta. Its internal anatomy is identical to that of ~ blepta (Figure 6).
(d) Swimming and Penetration of the First Intermediate Host
Swimming and penetration of the snail host were identical to that observed for the miracidium of~ blepta (Figures 8 & 9). Miracidia can still be alive and slowly swimming 12 hours after hatching but penetration has not been observed after the first few hours and older miracidia do not seem to be stimulated by the presence of Posticobia. IV MIRACIDIUM 28 C. Comparison
The miracidia of .!.:_ blepta and S. callista are very similar in morphology and size. The pattern of apical pores though similar is not identical. Brooker (1972) reviewed the literature on the sense organs of trematode miracidia. Besides the two prominent rhabdomeres in each eyespot, a fifth rhabdomere has been found. This was in a posteriomedian extension of the left pigment cell in several species which have been sectioned. It is therefore likely that the miracidia of .!.:_ blepta and S. callista also have 5 rhabdomeres though the fifth is not visible.
The intramiracidial sporocyst of ~ callista is slightly larger and has more cells than that of.!.:_ blepta. A miracidium with a well developed redia within it has been reported by authors for several families. Stunkard (1934) observed a well developed redia in the miracidium of the cyclocoeliid, Typhlocoelum cymbium, with a pharynx, strong circular muscles and limb-like appendages with which it swam like a frog. The biggest part of the miracidium of Pseudhyptiasmus dollfusi is occupied by a large redia with masses of germ cells and a large anterior pharynx leading to a cylindrical caecum (Timon-David, 1955). Similarly well developed is the intramiracidial redia of the paramphistome, Stichorchis subtriquetrus (Bennett & Humes, 1939).
There are very few descriptions of lepocreadiid miracidia. The miracidium of Lepocreadium album is conical with two projections from the apical end formed by the ducts of two claviform gland cells. The epidermis has a few ciliated polyhedral cells. It has two flame cells, with ducts symmetrical about the midline, which open close together, just anterior to the midbody (Palombi, 1937). He did not describe eyespots. The excretory pores of.!.:_ blepta and S. callista are on opposite sides of the body but are otherwise similar to L. album. Palombi (1937) does not refer to any germinal masses, developing rediae or sporocysts. Stunkard (1969) describes the miracidium of Lepocreadium pyriforme as having ocelli with conspicuous lenses and long cilia.
Macfarlane's (1951) description of the miracidium of Stegodexamene anguillae is more complete. He illustrates two longitudinally arranged apical gland cells. Although he describes 5 rows of ciliated epidermal cells he did not count the number of cells per row. He describes two flame cells but although he described an active germinal mass which he assumed to 29 IV MIRACIDIUM C. Comparison
be a redia within the hind body of the miracidium, he did not observe any active flame cells or other structures within it. 30 V SPOROCYST A. Tetracerasta blepta
(a) Development
In the newly hatched miracidium, the sporocyst is an ovoidal mass (Figure 6) with 34(30-41,8) cells arranged so that the centre appears less dense. As the sporocyst penetrates the snail it elongates and becomes pointed anteriorly. At 2 weeks after penetration, the sporocyst is found in the anterior body of the snail near the heart. It has grown (Table III) and contains germinal bodies which will become the rediae (Figures 10 & 12). At this time a lateral birth pore is first observed. By 4 weeks, the sporocyst has further enlarged and now contains 6 or 7 rediae. The sporocyst still has only 2 flame cells as it did within the miracidium. No germinal bodies are now observed and there is no persistent germinal mass as defined by Cort et al. (1954). After 4 weeks the sporocyst is no longer observed and is assumed to have died.
(b) Description
The sporocyst is thin walled, ovoidal, 202(175-240,4) by 85(75-95,4) (Figures 10 & 12). The anterior end is usually pointed and although this end appears quite dense, no pharynx has been observed. No caecum has been observed. The excretory system consists of 2 flame cells just posterior to the midbody, one on each side of the body. Each flame cell opens on the lateral surface through a duct. There is a lateral, circular birthpore.
At 4 weeks after penetration, the sporocyst contains 6 or 7 ovoidal developing rediae, the largest of which is 38(31-46,2) by 32(31-33,2). 31
TABLE III: Development of the Sporocyst in Experimental Infections
Number Redial Size Largest Body Species Age* Length Width Bodies Length Width
Tetracerasta 0 Weeks 28(25-34,20) 22(20-25,21) blepta 2 188(175-200,2) 90(85-95,2) 65 42 4 216(192-240,2) 80(75-84,2) 6-7 38(31-46,2) 32(31-33,2)
Stegodexamene 0 30(27-33,8) 22(18-25,8) callista 2 141( 135-148,2) 74( 65-84' 2) 7 77(69-85,2) 50(45-56,2) 4 134(127-140,2) 71(69-73,2) 4 48(44-52,2) 40(36-44,2)
* Number of weeks after penetration of the snail Figure 10: Sporocyst of Tetracerasta blepta 2 Weeks after Penetration
Figure 11: Sporocyst of Stegodexamene callista 2 Weeks after Penetration 32
u• • -~
...
Wll'OS
u• • • -Cl)
... a.• Q)• • ..0 ~ wii'os \
Figure 12: Sporocyst of Tetracerasta blepta 2 Weeks after Penetration (Scale 25 ,um)
Figure 13: Redia of Stegod~xamene callista (Scale 25 ..um) 33 V SPOROCYST 34 B. Stegodexamene callista
(a) Development
The development is similar to that of .!_:_ blepta (Table III). The ovoidal sporocyst within the miracidium becomes anteriorly pointed and flattened after penetrating the snail. After 2 weeks it has enlarged and contains 7 ovoidal redial bodies, usually one much larger than the others (Figure 11). By 4 weeks after penetration the sporocyst begins to shrink and contains only a maximum of 4 redial bodies. Some sporocysts are nearly empty and contain only one redial body. No germinal cells are visible suggesting that there is no persistent germinal mass and that production of rediae has ceased. After 4 weeks the sporocyst is no longer found, suggesting that death rapidly follows senility.
(b) Description
The sporocyst of this species is similar to that of .!_:_ blepta. The thin-walled ovoidal body is 141(135-148,2) by 74(65-84,2) (Figure 11). No pharynx or caecum has been observed. Two flame cells, one on each side of the body, open on the lateral surfaces through ducts. There is a lateral birthpore. There is a maximum of 7 ovoidal, developing rediae, the largest measures 48(44-52,2) by 40(36-44,2). 35 V SPOROCYST C. Comparison
The sporocysts of ..!.:_ blepta and S. callista are very similar in morphology and development. ~ callista develops faster, has a wider size range of developing rediae within it and becomes senile faster.
Unlike a redia, the sporocysts of..!.:_ blepta and S. callista lack a definable pharynx or caecum although the anterior end is pointed and muscular. They are unlike sporocysts in that they have a lateral birthpore. In all the studies that have been made of lepocreadiids none have described the penetration of the snail and therefore it is not known whether the miracidium of other species enters the snail or injects a sporocyst or a redia. In only two studies was a sporocyst described but all studies describe a redial stage. Stunkard (1969) described a rediae-producing sporocyst for Neopechona pyriforme and Lengy & Shchory (1970) suggested that Cercaria levantina 2, likely a lepocreadiid, had a sporocyst stage in its life-cycle though they did not observe one and did not suggest why they assumed so.
Macfarlane (1951) did not find a sporocyst of Stegodexamene anguillae but assumed that if it did exist it would disappear soon after a single brood of rediae had been liberated. The germinal body he described and illustrated within the miracidium of ~ anguillae lacked any trace of a pharynx or a caecum and it is likely that this is a sporocyst which penetrates a snail as does that of S. callista. It would then quickly produce rediae and die shortly afterward.
In describing the life-cycles of lepocreadiids, it is common for authors to lump all redial stages together and to describe them as a group. Bartoli & Prevot (1978) provided one of the few descriptions of a mother redia from this family. This redia has a large pharynx, a poorly developed caecum and a 2[3+3] excretory system.
Stunkard (1964) found that the mother redia of Homalometron pallidum produced daughter rediae then later produce cercariae. There was no evidence in the present study to suggest that the sporocyst may produce cercariae. VI REDIA 36 A. Tetracerasta blepta
(a) Development
Redia are found in the digestive gland of Posticobia within 2 weeks of the penetration of the snail by the sporocyst. They are cylindrical and wider at the level of the caecum (Figure 14A). They have a well developed ovoid al pharynx and a small oval caecum which is usually empty but occasionally contains orange granular material from the snail digestive gland. Sometimes germinal bodies are visible (Table IV).
By 4 weeks, rediae are found on the surface of the snail's digestive gland. These rediae have grown, the pharynx is enlarged and is now oval (Figure 14B). The oval caecum is full of snail tissue. There are 3 to 6 ovoidal germinal bodies within the redia but no longer a posterior mass of germinal cells. At 4 weeks, much smaller rediae, believed to be the second generation of rediae are observed. These are very similar to the previous generation and can only be separated by their smaller size.
By 6 weeks after penetration of the snail, the persistence of several generations of rediae, all producing other rediae, results in a continous range of sizes of rediae so that redial generations can no longer be separated and their further development can not be followed.
The depletion of the posterior mass of germinal cells in the first generation of redia after 4 weeks suggests that they are not long lived although other generations of redia may survive longer. Rediae measured at 4, 6, 8 and 18 weeks (Table IV) showed an increase in their average body and caecal size but little growth in their pharynx between 8 and 18 weeks. An increase in the average size of redia suggests that after the first generation, rediae may be long lived and that there may be only two generations of rediae before the production of cercariae, at about 11 weeks at 15 to 20°c.
The number of generations of some species of rediae may only be limited by the available space in the snail (the.. snail's size), the snail's age and its nutritional state. Experiments by Donges (1971) suggested that the number of redial generations of the echinostomatid Isthmiophora melis may not be limited. Figure 14: Development of the Redia of Tetracerasta blepta A Redia at 2 weeks after penetration of the sporocyst B Redia at 4 weeks after penetration of the sporocyst
Figure 15: Redia of Tetracerasta blepta Showing Internal Features 3 7
. an u . .... Q) u.
I WrY!it I
I UJfl' !ft I TABLE IV: Development of the Redia in Experimental Infections
Body Pharynx Caecum # Germinal Largest Germinal Body Generation Age* Length Width Length Width Length Width Bodies Length Width
Tetracerasta I 2 133 44 27 27 23 15 blepta 4 168(115-211,9) 62(42-94,9) 36(29-42,9) 28(25-36,9) 28(19-42,6) 30(23-42,6) 3(0-6) 36(23-52,7) 28(13-38,7)
II 4 94(84-107 ,3) 54(38-63,3) 23(21-25,3) 20(17-23,3) 15 21 0(0-1) 10 8
combined 6 119(55-163,10) 36(29-46,10) 21 (15-31,9) 21(15-27,9) 28(21-44,7) 20(13-27,7) 3(0-4) 21 (19-23,6) 15(10-21,6) 8 164(92-289 ,9) 48(31-64,10) 30(20-38,10) 26(20-38,10) 35(18-51 ,8) 24(18-51,8) 3(0-6) 36(26-79,4) 35(26-44,4) 18 266(156-339,5) 64(42-79,5) 29(23-33 ,5) 31 (25-38,5) 4 7(21-77 ,5) 43(23-67,5)
Stegodexamene I 4 225(98-378,9) 57(33-94,10) 42(23-61, 10) 42(23-63'10) 50(17-92,10) 34(17-46,10) 4(0-6) 43(19-65,7) 32(19-52,7) cal/ista combined 5 321 (242-422,4) 99(63-115,4) 46(40-59,4) 49(36-63 ,4) 107(73-121,4) 96(84-104,4) 4(0-10) 64(56-73,3) 57(42-73,3) 6 189(154-246,3) 98(71-133,3) 63(49-77,3) 64(51-77,3) 67(56-77,2) 33(28-38,2) 3(0-3) 41 31 7** 230(166-340,6) 64(38-82,6) 49(26-64,6) 46(28-54,6) 45(38-51,3) 38(23-56,3) 1 (0-4) 53(23-77,3) 39(20-51,3) 11 207(105-302,12) 58(33-97,12) 42(26-64,12) 42(28-64,12) 42(26-64,10) 34(18-61,10) 3(0-4) 31 (13-46,5) 25(10-46,5) 13 222(109-303,6) 91 (52-109,6) 40(33-46,6) 43(27-52,6) 49(31-84,6) 38(25-63,6) 4(3-5) 74(19-109,6) 48(6-94,6)
wild ? 247(167-345,10) 75(42-98'10) 29(21-42,10) 31 (23-44,10) 52(25-94,9) 44(23-90,9) 2(0-6) 62(31 -94,7) 42(21-56,7) infection
* weeks following penetration of the snail by sporocyst some cercariae in snai I
V-1 co VI REDIA 39 A. Tetracerasta blepta
Immature cercariae of.!.:_ blepta first appear within the snail at 11 weeks at 15 to 20°c. Some or all of the redial generations may then be responsible for cercariae production. The stimulus for the production of cercariae is not known and may result from the crowding of rediae although snails with as few as 8 rediae have had immature cercariae.
(b) Description
Rediae are described from snails infected in nature. Each muscular sac-like redia has a well developed terminal sphincter muscle directly anterior to a muscular, ovoidal pharynx (measurements in Table V, Figure 15). The pharynx connects directly to a large, conspicuous, ovoidal caecum which is filled with a granular orange material from the snail digestive gland. The largest and most developed of the spheroidal germinal bodies (developing cercariae) is located just posterior to the caecum, lateral to the birthpore.
The redia has no trace of eye pigment. The flame-cell pattern is 2(3+3); the excretory pores are located lateral to the midline, one third of the body length from the posterior end (Figure 15). 40
TABLE V: Measurements of the Redia
Feature Tetracerasta blepta Stegodexamene callista
Body 192(143-298,20) 245(165-314,15) 67(35-91,20) 80(52-111, 15)
Pharynx 27(22-36,20) 26(17-37,15) 31(24-38,20) 26(17-37,15)
Caecum 29(14-81,20) 33(14-58,14) 42(19-81,20) 20(9-30,14)
Number of Germinal Bodies 3( 1-6' 20) 5(3-9,8)
Size of Largest Body 48(17-94,20) 53(39-70, 10) 38(17-62,20) 60(27-72, 10) VI REDIA 41 B. Stegodexamene callista
(a) Development
The development of the redia of S. callista is similar to that of T. blepta (Figure 14). Measurements of rediae were first made 4 weeks following the penetration of the snail by the sporocyst (Table IV). The redia is cylindrical to bottle-shaped with a circular pharynx and an oval caecum filled with snail material (Figure 16). There are usually 4 to 6 germinal bodies but there is no persistent mass of germinal cells as defined by Cort et al. (1954). Samples of infections of different ages showed little or no increase in the average size of the rediae with time. The average size of rediae actually decreased from infections of 5 to those of 6 weeks, although few rediae are involved. Factors like the health and size of the snail and the degree of infection may vary between experiments thus making comparisons difficult. The lack of any increase in the average size of rediae with the increasing age of the infection may be due to the continuing birth of rediae while older, larger rediae died. There may be several generations of rediae unlike the indications for rediae of ..:!:..:.. blepta.
The rediae of ~ callista are generally larger than those of ..:!:..:.. blepta at a comparable age and this corresponds to the larger size of cercariae produced by~ callista.
As in the present study, Macfarlane (1951) did not find rediae of Stegodexamene anguillae in which recognizable second generation rediae were being produced but he found forty or more mature rediae in one snail suggesting that the production of rediae must have occurred before his observations. I believe that, like the cercariae, the rediae are very immature at birth and are then only barely recognizable germinal bodies and hence they would not be recognizable within the rediae.
It is not uncommon for the cercariae produced in lepocreadiid rediae to be born while still immature. There are no reports of species of lepocreadiids in which advanced cercariae are born. Stunkard (1972) observed the birth of immature cercariae of Lepocreadium setiferoides and Macfarlane (1951) reported it for Stegodexamene anguillae. Cercariae of
!:_ blepta and ~ callista pass from rediae at so immature a stage that they are scarcely recognizable as cercariae. Figure 16: Redia of Stegodexamene catlista at 4 Weeks
Figure 17: Redia of Stegodexamene callista Showing Internal Features 42
u. CJ)
WrYOs;
. Q) d. u. u
WrYOs; VI REDIA 43 B. Stegodexamene callista
(b) Description
The redia of this species is similar to that of ..:!:...:_ blepta. The muscular pharynx is circular (measurements appear in Table V, Figure 17) and connects to an ovoid caecum filled with snail digestive gland tissue. The most anterior germinal body (developing cercariae) is the most developed and is located lateral to the birthpore, just posterior to the caecum. The redia has no eye pigment. The flame-cell pattern is 2(3+3) with the excretory pores laterally placed about half way along the body length. VI REDIA 44 C. Comparison
The redia of S. callista is generally larger than that of .!...:_ blepta. This is not surprising as the germinal bodies which the rediae of S. callista contain and the cercariae these develop into, are larger. The general morphology of these two species is very similar.
It is common for the rediae of species in the family Lepocreadiidae to be given only brief, general descriptions. Often more than one generation has been recognized but these are designated only as large and small rediae.
All lepocreadiid rediae reported in the literature are longer than the two species in the present study. That of Cercaria levantina 2 is 1,100 in length (Lengy & Schchory, 1970), Lepocreadium pegorchis is over 1000 (Bartoli, 1967), and that of Lepocreadium areolatum is up to 1600 in length (Stunkard, 1980a). These are 4 to 6 times the length of the rediae of .!...:_ blepta and S. callista and may be attributable to the comparitively small size of their snail host, Posticobia (2 to 3 mm).
Due to their much larger size, other lepocreadiid rediae contain more germ balls than those in this study. Opechona bacillaris (Kpie, 1975) and Lepocreadium setiferoides (Martin, 1938) have up to 30 immature cercariae within them.
Some lepocreadiid rediae differ in shape from those of the present study. Neopechona cablei has a distinct neck region posterior to the pharynx (Stunkard, 1980b). Lepocreadium pegorchis described by Bartoli
(1967), ~ areolatum described by Stunkard (1980a) and Neopechona pyriforme described by Stunkard (1969) all have a distinctly pointed posterior end which .!...:_ blepta and S. callista do not have. Holorchis pycnoporus has a little tail (Bartoli & Prevot, 1978). The caecum of Lepocreadium pegorchis is extremely small and peduncular (Bartoli, 1967). The excretory systems of some of these have been studied and are reported to have a 2(3+3) system as do those of the present study. Most similar to the rediae of this study is that of Homalometron pallidum which has a similar shape and internal morphology (Stunkard, 1964) but is larger than even S. callista. VI REDIA 45 C. Comparison
The mature redia of Stegodexamene anguillae is over 3 times longer than that of S. callista or .:!...:_ blepta but is much thinner and only one quarter the width. ~ anguillae contains 10 to 25 immature cercariae which is more than either.:!...:_ blepta or~ callista. The caecum of~ anguillae is also longer and thinner. Macfarlane (1951) also observed 10 flame cells per side in ~ anguillae which differs from the 6 per side of .:!...:_ blepta and S. callista. VII CERCARIA 46 A. Tetracerasta blepta
(a) Development
1he formation of the germinal bodies which become cercariae occurs at the posterior end of rediae. Few features of even the most developed cercariae within the redia can be recognized. 1he ventral sucker is sometimes observed but eyespots and tail setae are not.
1he least mature cercariae outside the rediae are found near them. 1hese cercariae have an oral sucker, a pharynx, a protruding ventral sucker, a large bladder and a stumpy ovoidal tail with no visible setae (Figures 18 & 21). 1hey are not very mobile and they are likely born passively rather than escaping from the rediae.
Mature cercariae are found near the surface of the snail digestive glands approximately 2 weeks after the birth of the first cercariae. 1hey are not usually in the immediate vicinity of the rediae which again suggests that they mature outside the rediae. As the tail grows, the setae form.
1he first cercariae emerge from the snails 70(40-84,14) days at 15 to 2o oc and 85(71-102,18) days at 10 to 20°c following the exposure of snails to miracidia.
Kendall (1964) found a direct relationship between the rate of development of the parasite and the environmental temperature of the host. Below 1o oc he found that development of Fasciola hepatica did not occur and that the rate of development increased up to 28° C when snails became unhealthy.
Stunkard (1980a) studied the development of lepocreadiid cercariae within the snail and reported that a very immature cercaria with a short non-functional tail leaves the redia and matures in the haemocoel of the snail. He reported that the cercaria of Lepocreadium areolatum matures in the haemocoel, develops tail setae shortly before emergence and emerges through the snail's gills. 1he emergence of the cercariae of~ blepta has not yet been observed but the gills may also be the site of emergence. Figure 18: Developing Cercaria of Tetracerasta blepta
Figure 19: Cercaria of Tetracerasta blepta (ventral)
Figure 20: Body of the Cercaria of Tetracerasta blepta (ventral) 4 7
ci.. . . Cl. (/). -0 . a. 0 ...... : u
0 N WrfOS
1 ' wrfos
u) en. . > ..ci 0 a.
() 0 0 0
C) 0 c 0
...co UJrfst' Figure 21: Developing Cercaria of Tetr acerasta blepta (Scale 25 ,um)
Figure 22 : Live Cercaria of Tetracerasta blepta (Scale 5 ,um)
Figure 23: Anterior End of the Cercaria of Tetracerasta blepta
Figure 24 : Tail Setae of the Cercaria of Tetracerasta blepta 48 21 • • 22 I
Cl •
•
1011 , 2~ .
10U. VII CERCARIA 49 A. Tetracerasta blepta
The species of cyclocoelids that Stunkard (1934) reported which have a well developed intramiracidial redia, differ from~ blepta and S. callista in their life-cycles, as their cercariae do not emerge but encyst in the snail.
(b) Description (Figures 19-20, 22-24)
Description is based mostly on observation of live material supplemented by measurements of 11 fixed specimens (measurements appear in Appendix I).
Cercaria distomate, pharyngeate, ophthalmotrichocercous (Figures 19 & 22). Body spinous, flattened ovoid, length 186(116-215,11), width 76(62-86,11). Oral sucker large, ovoidal, subterminal, ventral, length 37(32-44,11) and width 35(30-41,11). Ventral sucker large, circular 34(30-41,11). Tail plumose, setiferous; tail-stem cylindrical, length 212(170-252,11), widest at anterior end 18(13-22,11). Setae serrated, blade-like (Figure 24), maximum length 52(44-60,11) at midtail; 35 setae per side and 2 or 3 terminal, anterior 15 pairs bifurcate, others simple; few large dentiform scales on dorsal and ventral surface in transverse rows.
Prepharynx very short; pharynx oval, length 13(9-16,11), width 15(10-19,11); oesophagus slender, tube-like, length 39(9-16,11), bifurcation just anterior to ventral sucker; caeca thin walled, end blindly at posterior end, length 87(70-104,11) (Figure 20). Penetration glands lacrimiform, eight arranged in two groups of four, anterior and lateral to ventral sucker, four swollen ducts from each group pass forward, lateral to eyespots and open through a diamond-shaped bundle of four pores on the dorsal rim of the oral sucker. Cystogenous glands small, spherical, numerous, near dorsal surface, between oral and ventral suckers.
Rudiments of testes and ovary rarely present. Cirrus sac not visible.
Flame-cell formula 2[(3+3+3)(3+3+3)]; anterior and posterior collecting ducts join main ducts antero-lateral to ventral sucker; main ducts, with a series of flagella, join excretory bladder just posterior to VII CERCARIA 50 A. Tetracerasta blepta
ventral sucker. Bladder tubular, length 95(81-116,11), width 23(17-29,11), size varies with content, extends from lateral to ventral sucker to posterior end; wall epidermal, thin; excretory pore at tail-body junction.
Eyespots prominent, length 7, width 9, 2 lenses each.
Host: Posticobia brazieri (natural and experimental infections) Locality: Brisbane River, Queensland, Australia
(c) Behaviour and Swimming
Most cercariae emerge at dusk and show a weak but positive phototaxis. Fresh water stimulates the emergence of cercariae. The cercaria swims tail first by generating a sigmoidal curve at the tip of its tail which it sweeps toward its body (Figure 25). The anterior end of the body is bent toward the ventral sucker. The body moves in a clockwise helix through the water usually near the bottom. A newly emerged cercariae swims almost constantly, stopping briefly when it contacts the surface to extend its body and tail. Sometimes the setae are spread by pulling the tail across the rim of the ventral sucker. As a cercaria ages it spends more and more time in its "resting position". The cercaria lies dorsal side down on the bottom, body arched upward and tail outstretched, sometimes making weak swimming motions.
Cercariae swim for 6 to 8 hours after which they usually lose their tails and crawl leechlike along the bottom for several more hours. They remain infective even after they lose their tails but the chance of contact with the host is reduced. Cercariae remain active and alive less than 8 hours in pond water but one day in 0.8% saline.
Cable (1972) reported that if the second intermediate host of a trematode is a fish then the cercaria is likely to be photopositive and if the host is a bottom-dwelling invertebrate, the cercaria is likely to be photonegative. The emergence pattern of lepocreadiids varies, the cercaria of Lepocreadium setiferoides emerges day and night and is photonegative (Martin, 1938). Cercariae of Neopechona pyriforme emerge during the night Figure 25: Swimming Cercaria of Tetracerasta blepta
Figure 26: Penetration by Cercaria of Tetracerasta blepta A Swimming cercaria contacting tadpole B Cercaria parallel to host surface and penetrating c Cercarial body fully penetrated D Cercarial tail separating from body 51
0
or early morning mostly and are photonegative but become photopositive (Stunkard, 1969).
The action of pulling the tail across the edge of the ventral sucker is similar to the tail cleaning motion reported by Kbie (1975) for the cercaria of Opechona bacillaris, another lepocreadiid.
Kendall & McCullough (1951) found that when snails were shifted to fresh water then emergence was induced even in the case of i..=_ hepatica, whose cercariae they believed emerged mainly passively.
Chapman & Wilson (1973) studied the propulsion of the cercariae of Himasthla Secunda (Nicoll) and Cryptocotyle lingua in detail and of these, the swimming behaviour of the cercaria of ..!.=._ blepta is most similar to the 2-dimensional movement of H. secunda.
The cercaria of Stegodexamene anguillae crawls along the bottom but when it does swim it does so in a series of waves that cause it to move horizontally (Macfarlane, 1952). He found that it did not have a marked positive phototropism.
F.mergence of cercariae of..!.=._ blepta after dusk must be initiated by some stimulus. Since it is unlikely that even with their eyespots the cercariae can detect the lower light levels of dusk, a change in the behaviour of the snail with dusk may initiate emergence. No difference in the behaviour of the snail at dusk has been observed. When infected snails are lit artifically at dusk the emergence of ..!.=._ blepta cercariae is reduced. Cercariae can be stimulated to emerge from snails during the day by placing the infected snails in the dark. The nightly emergence pattern of cercariae is not independent of light.
It would be advantageous for ..!.=._ blepta to emerge at night because their movements near the bottom would bring them near the nocturnally less active tadpoles and fish which they infect, and make it easier for cercariae to attach to the skin as a prelude to pentrating. Adult tree frogs such as Litoria lesueuri are active at night and come from woods bordering streams to the shallow stream waters where Posticobia infected with..!.=._ blepta are found. Cercaria emerging at night could penetrate the VII CERCARIA 53 A. Tetracerasta blepta
frogs as they sit in the water although my experiments have shown the adult frog less attractive than the tadpole to the cercaria.
(d) Penetration of the Second Intermediate Host
If a swimming cercaria of..!..:._ blepta contacts an unsuitable host it may even bump into it and fail to react. The cercariae are rarely inhaled or swallowed as are the cercariae of s. callista. When the cercaria of
~ blepta contacts a tadpole it immediately elongates it anterior end (Figure 26A) and attaches to the skin. Within seconds it has brought its ventral surface into contact with the tadpole's surface (Figure 268). It appears to attach by its ventral sucker. The tail is held in a slight arc nearly parallel to the surface of the tadpole. Then, only seconds after the initial contact with the host, even the vigorous, apparently irrated movements of the host fail to dislodge it.
The anterior end continuously probes into the tadpole's skin and the cercaria soon makes its way through the skin and into the subcutaneous tissue (Figure 26C). Setae may fall off but the tail of the cercaria remains attached as the cercaria moves under the tadpole's skin until only the tail remains outside. The tail then drops off (Figure 260). Penetration usually takes less than 5 minutes. Although the tail of the cercaria has remained motionless since the initial contact with the host, it may resume swimming when it is no longer attached and may even swim away from the tadpole and remain active for one hour or more. Once the body of the cercaria has moved completely inside the tadpole it continues to burrow deeper into the muscle and viscera.
Penetration of suitable fish hosts is similar except that the cercaria may become caught fast in the mucus and unable to reach the skin. It then stretches vigorously and may weakly attempt to swim which may either free it from the mucus or allow it to contact the skin. The cercaria may worm its way through the mucus to the skin or scales. It then pushes its anterior end under the free edge of a scale and slowly pulls itself under. Penetration of a fish to this point may take 5 minutes or more. VII CERCARIA 54 A. Tetracerasta blepta
Cercariae penetrate anywhere on the surface of the tadpole, even into the thin transparent tail fins. The junction of the body and tail is the area most heavily penetrated. In fish, it is the bases of the fins which are penetrated the most.
Cercariae may occasionally be eaten by smaller fish or accidentally inhaled by fish or tadpoles and swallowed. They may then penetrate the throat region or intestine and encyst in the surrounding tissues. I have observed a cercaria inhaled into the nostril of a tadpole and as it did not reappear it most likely encysted within.
~ blepta in the present study could not detect potential hosts until contact. In his review of the literature Cable (1972) reported that cercariae in general show little indication of chemical attraction to potential hosts.
Stunkard (1969, 1980a) found that the cercaria of Neopechona pyriforme and the cercaria of Lepocreadium areolatum used their tails to initally attach to the host and later penetrated leaving behind their tails which swam by themselves for hours. Cercariae of ~ blepta were not observed to attach to their hosts by their tails but their tails did sometimes become stuck to mucous on the surface of a fish.
K~ie found that cercariae of the lepocreadiid, Opechona bacillaris, penetrate and infect medusae while the hydroid stage of the same species, which are found on the snail host, eat and digest the cercariae.
Macfarlane (1952) believed that cercariae of Stegodexamene anguillae attacked gobiids from below or from the sides while they lay in the shadows or interstices of a shingle bed. This cercaria is very similar in morphology to ~ blepta which attacks fish and tadpoles as they lie on the bottom. VII CERCARIA 55 B. Stegodexarnene callista
(a) Development
Developing cercariae within rediae occasionally have visible eyespots. The least developed cercariae free from the rediae are similar to those of
.'.!:..:_ blepta (Figure 27). As a cercaria develops, the tail and body elongate and setae are formed.
The first cercariae emerge from P. brazieri 59(57-68,6) days at 15 to 20°c and 89(71-119,64) days at 10 to 209c following exposure of the snails to miracidia.
(b) Description (Figures 28-35)
Description is based mostly on observations of live material supplemented by measurements of 12 fixed specimens (measurements appear in Appendix I).
Cercaria distomate, pharyngeate, ophthalmotrichocercous (Figures 29 & 35). Body spinous (Figure 34), cylindrical (Figure 30), length 264(254-288,12), widest at ventral sucker 75(59-99,12). Oral sucker large, ovoidal, subterminal, ventral, length 39(32-47,12), width 44(37-52,12); with eight papillae (Figure 28). Ventral sucker large, circular 43(37-52,12); with 2 concentric rings of papillae, the inner of 5 small and pointed papillae, the outer of 9 large and blunt (Figure 33). Tail very large, plumose, setiferous (Figures 29 & 35); tailstem cylindrical, length 610(464-731,9), widest at midlength 65(40-86,9). Setae maximum length 103(89-116,9) at midtail, width 1; 1140 to 2000 per side on the lateral surface in 190-250 rows; setae become transverse rows of dentiform scales on dorsal and ventral tail surface (Figures 31 & 32).
1'l- Prepharynx very short (Figure 28); pharynx length 14( l 4-19,12), width 17(11-23,12); oesophagus slender, length 80(60-100,12), bifurcation just anterior to ventral sucker; caeca thin walled, end blindly at posterior end, length 30(20-40,12). Penetration glands lacrimiform, eight arranged in two groups of four, anterior and lateral to ventral sucker, four swollen ducts from each group pass forward lateral to eyespots and open through a diamond-shaped bundle of four pores on the dorsal rim of the oral sucker. Figure 27: Developing Cercaria of Stegodexarnene callista
Figure 28: Body of the Cercaria of Stegodexarnene callista (ventral) 56
28 -. p.g.p.
pa.
27 ey.
t. ~I
ex.s. Figure 29: Live Cercaria of Stegodexamene callista (Scale 50 ,Um)
Figure 30: Body of the Cercaria of Stegodexamene callista (Scale 20 ,,um)
Figure 31 : Ta il of ·he Cercaria of Stegodexamene callista (Scale 20 ,um)
Figure 32: Tail SP. tae of t he Cercaria of Stegodexamene callista (Scale 4 ,Um)
Figure 33 : Ventral Sucker of the Cercaria of Stegodexamene callista (Arrow indicates papilla; Scale 4 tUm)
Figure 34: Body Spines on the Cercaria of Stegodexamene callista (Scale 2 ,Um)
t . . 57
29 Figure 35: Cercaria of Stegodexamene callista
Figure 36: Swimming Cercaria of Stegodexamene callista 58
35
36
E ~ 0 0 C"4 VII CERCARIA 59 B. Stegodexamene callista
Cystogenous glands small, spherical, numerous near dorsal surface between the oral and ventral suckers.
Testes two, oblique; anterior testis oval, 5 by 7, sinistral; posterior testis circular, 6. Ovary single, oval, 9 by 8, anterior to I testes, dextral. Cirrus sac poorly developed, curved, 27 by 12, sinistral to ventral sucker.
Flame-cell formula 2[(4+5+5)(5+5+5)], variable; anterior and posterior collecting ducts join main ducts at anterior border of penetration glands; main ducts, with about 10 long flagellae in lumen, join excretory bladder just posterior to ventral sucker. Bladder tubular, length 96(86-114,12), width 22(5-46,12), extends from just anterior to ventral sucker to posterior end; wall epidermal, thin; excretory pore at tail-body junction.
Eyespots two, prominent, circular, 9, 2 lenses each, with the mouth of the pigment cup directed antero-laterally.
Host: Posticobia brazieri (natural and experimental infections) Locality: Brisbane River, Queensland, Australia
(c) Behaviour and Swimming
Most cercariae of S. callista emerge at dawn when they are highly visible to feeding fish. The cercariae do not survive overnight but because they emerge at dawn this allows them the longest daylight period possible during which they can attract and be eaten by a susceptable fish host. Cable (1972) reported correlations, from his own and others work, between the time of cercarial emergence and the habits of the next host. He reported that cercariae emerge when the host is most likely to be contacted, normally when the host is most active.
Bright light and freshwater stimulate emergence of S. callista cercariae. The only other lepocreadiid tested outside the present study was Cercaria levantina 2 which was stimulated to emerge under a strong light source and rarely emerged in the dark (Lengy & Shchory, 1970). VII CERCARIA 60 B. Stegodexamene callista
To initiate swimming the tail of the cercaria generates a wave from a point near its anterior end which is transferred anteriorly to the body and posteriorly along the tail. As the swimming motion of the tail develops, the body rocks and eventually rotates in space about a nodal point on the tail which is located just anterior to where the tail wave is initiated. As the oscillation of the tail increases, another nodal point develops near the posterior end of the tail about which the end of the tail flips from side to side, passing dorsal to the rest of the tail (Figure 36). The cercaria generally swims tail first, as does the cercaria of ..!..:_ blepta. Normally if there is any directional movement it is upward in a tight helix. The cercaria of S. callista, unlike that of ..!..:_ blepta, does not usually move much laterally or vertically during its brief periods of swimming (usually less than 10 seconds) and spends longer periods in its resting position during which it hangs obliquely, body down, ventral side up and tail outstretched. The anterior end of the body is often bent upwards toward the surface as it is during swimming (Figure 36). In this position the cercariae slowly sinks or floats depending on convection currents until swimming is resumed. A complete cycle of the swimming tail takes 1.4 seconds at 25oc. Mechanical disturbances stimulate swimming. Occasionally the cercaria may coil its tail into a tight spiral.
As the cercaria ages, it spends less and less time swimming. During its prolonged resting periods it may sink until its dorsal body surface touches the bottom. Cercaria stay active for 6 to 8 hours in pond water but almost one day in 0.8% saline.
The intermittent swimming behaviour of the cercaria may allow it to survive longer by reducing its energy expenditure and thus increasing its chances of contact with the fish host.
Of the swimming behaviour described for cercariae by Chapman & Wilson (1973), S. callista most closely approximates that of Cryptocotyle lingua because both move in 3-dimensions through space unlike the cercaria of Himasthla secunda which moves in 2-dimensions using a flat sweep of its tail. VII CERCARIA 61 B. Stegodexamene callista
(d) Penetration of the Second Intermediate Host
Smaller fish eat the cercariae and larger fish inhale them. A slight mechanical shock will dislodge their large tails. Cercariae which are inhaled usually lose their tails as they strike the gill rackers and often only the tails pass back out the opercles while the bodies attach inside the gill chamber. In experiments, cercaria rapidly penetrated and encysted inside a piece of fish liver.
The cercaria may also penetrate the skin. Setae are rapidly lost from the tail or the tail lost completely as the cercaria begins to penetrate. The tail is always lost before the cercaria completely enters the fish. The cercarial tail may swim away and remain active for several hours. The cercaria may move several millimeters laterally under the skin before encysting and the process of penetration takes about 20 minutes. A cercaria on the bottom which has lost its tail may remain infective for several hours but as with..!...:_ blepta its chances of contacting the host are reduced.
Nasir & Skorza (1968) have found that the gigantic pigmented tail of the cercaria of the echinostome Stephanoprora denticulata lures small fish to eat it. VII CERCARIA 62 C. Comparison
The cercaria of~ blepta and ~ callista differ in several respects.
The body of the cercaria of ~ blepta is a flattened ovoid whereas that of S. callista is cylindrical. The tails of these cercariae are very different. The tail of S. callista is longer, wider and has several thousand more setae. The setae of T. blepta are wider, serrated and blade-like whereas those of S. callista are bristle-like. Several setae on the tail of ~ blepta are bifurcate whereas all those of S. callista are simple.
The genitalia of s. callista though immature, are more highly developed in the cercaria than in that of~ blepta. The flame-cell pattern of T. blepta is 2[(3+3+3)(3+3+3)] whereas that of S. callista is 2[(4+5+5)(5+5+5)].
The cercaria of ~ blepta is similar to that of Stegodexamene anguillae described by Macfarlane (1951). The tails though similar have a different number of setae, ~ blepta has 35 on each side and 2 or 3 terminal whereas ~ anguillae has 37 or 38 bristles on each side and one wider terminal bristle. The anterior 15 setae of~ blepta are bifurcate while the anterior 20 or 25 of~ anguillae are paired. As the cercaria of
~ anguillae is very similar to that of ~ blepta it differs from the cercaria of S. callista in the same ways that it does from~ blepta.
A characteristic of lepocreadiids is that they have cercariae with tails bearing setae. In the genus Lepocreadium, in species such as
.!::.:_ album (Stossich, 1890) Stossich, 1903, these setae are in tufts of 4 or
5 and not single as in ~ blepta or partially single and partially in pairs as in S. callista. Neopechona pyriformis (Linton, 1900) Stunkard, 1969, Opechona bacillaris (Molin, 1859) Looss, 1907, and Holorchis pycnoporus Stossich, 1901 also have tuffs of setae. Those that have single setae have a few short, thin setae, more of sensory than locomotory purpose. An example of such cercariae are those of Homalometron pallidum Stafford, 1904 which is in the Homalometronidae, a family related to Lepocreadiidae.
Of the lepocreadiids whose cercarial flame-cell formulae have been reported, none are identical to either ~ blepta or S. callista.
Neopechona cablei described by Stunkard (1980b), and~ pyriforme (Linton, 1900) Stunkard, 1969 reported by Stunkard (1969) both have VII CERCARIA 63 C. Comparison
2[(4+4+4)(4+4+4)]. Martin (1938) reported that Lepocreadium setiferoides (Miller et Northup, 1926) Martin, 1938 has 2[(3+3+3+3)(3+3+3+3)] and Stunkard (1980a) reported that L. areolatum (Linton, 1900) Stunkard, 1969 has 2[(5+5+5)(5+5+5)]. All lepocreadiids appear to have numerous flame cells.
In the cercariae of S. callista the most anterior group of flame cells joins the anterior collecting duct near its junction with the main collecting duct, just posterior to the eyespot (Figure 28), whereas in .'.!:..!. blepta the most anterior group is not the first group along the anterior collecting duct but the last (Figure 20). There are few descriptions of lepocreadiid excretory systems in sufficient detail to allow comparison with these two patterns. Those excretory systems of Opechona bacillaris described by Kfie (1975), Neopechona pyriforme described by Stunkard (1969) and Lepocreadium setiferoides described by Martin (1938) have their anterior most group of flame cells connected close to the main collecting duct as in S. callista. Descriptions of Lepocreadium areolatum by Stunkard (1980a) and Neopechona cablei by Stunkard (1980b) have the anterior group of flame cells connected at the end of the anterior collecting duct as in .'.!:..!. blepta. It appears that the excretory systems of lepocreadiids vary in the position of the anterior group of flame cells even within the genus Lepocreadium. VIII METACERCARIA 64 A. Tetracerasta blepta
(a) Development
. When the cercaria begins to encyst, it probes in all directions as though seeking to create a space large enough for its cyst. The cercaria becomes dorso-ventrally flattened and begins to move in a tight circle by following its posterior end. When the cercarial body is motionless then the cyst material is presumably secreted and when the body rotates, its anterior end probes and smooths, moulding the cyst wall. Additions to the wall are made between periods of rotation and the plane of rotation is altered so that a complete spherical wall is formed. This wall is formed within 10 minutes but the cercaria continues to rotate within the cyst for several hours although it is less active than earlier during cyst-wall formation. Cercariae have rarely encysted on the bottom of the container.
The newly formed cyst is very thin (2), elastic and transparent (Table VI). The swollen ducts from the penetration glands of the cercaria are depleted during penetration and encystment but the bodies of the penetration glands are only slightly smaller than before. Within one day the pigment granules of the eyespots begin to break apart and the surface of the parasite's cyst wall may have a granular, opaque appearance which is the start of the host capsule.
At one week after encystment the metacercaria is not active but is undergoing development and some changes of morphology. The eyespots are fu~her broken up, the bladder is rapidly filling up with granules, the oral sucker is no longer circular as in the cercaria but is developing raised areas which will become the oral sucker lobes.
After six weeks the cyst does not become larger but the metacercaria continues to develop and enlarge within the cyst (Table VI). After 10 weeks the genitalia no longer enlarges or develops possibly because the host capsule reduces the flow of nutrients to the metacercaria. Metacercariae are infective after two weeks of development and most live at least one year.
Many species of Lepocreadium have been reported to lie unencysted in their intermediate host. Stunkard (1980a) reported that the metacercaria of Lepocreadium areolatum was unencysted in medusae and ctenophores. The -dt- TABLE VI: Development of the Metacercaria of Tetracerasta blepta in experimental infections
CYST BODY MATURITY COMMENT
Host Capsule Cirrus Inf ection Diameter Thickness Length Width Testes Ovary Length Width
~..,_ 0 days 149(131 - 159, 12) as cercaria
4 days 189(151-256,20) 5 243(220- 282,12) 97(87-110,12) 8,n=6 6 28(26-30,2) 9(8-10 ,2) eyespots intact, bladder nearl y full
1 wk 222(159-264, 15) 204(161-246 ,11) 91(72-128,11) 9(8-10,4) eyespots breaking up
2 wks 202(154-282 , 14) 13 272(230-320 ,9) 96(69-123 ,8) 9(8-10 ,2) 8,n=2 38 13 bladder full, oral lobes forming
3 wks 285(264-317 ,14) 13(8-18,2) 465(370-581 , 19) 135(100-182 ,19 30(18-46 , 17) 16(13-18,15) 68(38-77, 12) 17(15-23, 13) crystal in caeca, eyespots broken up
6 wks 416(359-476,12) 25(20- 31,4) 657(475-772,11) 156(128-182 ,7) 31 (23-38,11) 22(10-33 ,9) 74(59-92 ,5) 20(15-33 ,6) oral sucker enlarged
8 wks 329(232-465 , 12) 50(31-91,11) 669(465-846, 15) 160(128-189 , 14) 33(20-51,15) 18(15-20 ,13) 65(51-77,14) 15(13-18,15)
10 wks 295(254-349 ,10) 25(15-38 ,7) 673(507-856 ,15) 135(108- 166, 15) 35(28-51, 15) 20(15-28 ,14) 71 (59-90,13) 17(13- 20,13)
12 wks 297(243-402,15) 27(18-41,15) 624(486-772 ,16) 127(110- 148, 15) 30(18-46 , 16) 16(13-18 ,14) 60(38-77,14) 15(13-20,14)
16 wks 332(264-423,12) 40(20-108,7) 688(518-793, 15) 165(143-189,15) 29(13-44 , 15) 18(13- 28, 15) 66(17-26 ,15) 17(10-26,15) some cysts dying
* at 15-20°C ** measured from live material
°'Ul Figure 37: Metacercaria of Tetracerasta blepta (only anterior concretions in excretory bladder shown, ventral view
Figure 38: Oral Sucker Lobes of Tetracerasta blepta (in various stages of protraction)
Figure 39: Cyst of Tetracerasta blepta 6 7
37 o.I. po. 38
: oe. r. I. : ,
.. : 9.d .. ' : c.s. pa.
0. E c. ~ .,,0 t.
c.w. 39 .·
E ~ 0 0 C"'4
··· ··· ...... Figure 40: Metacercaria of Tetracerasta blepta
Figure 41: Ventral Sucker of the Metacercarla of Tetracerasta blepta (arrows indicate papillae)
Figure 42: Oral Sucker Lobes of Metacercaria of Te·tracerasta blepta fully retracted (upper surface ventral)
Figure 43: Oral Sucker Lobes Partially Protracted
Figure 44: Oral Sucker Lobes Protracted
Figure 45: Pores 0f Ampullae on the Oral Lobes of Tetracerasta blepta
Figure 46: Crystals in Caeca and Excretory Bladder in the Metacercaria of Tetracerasta blepta (Scale 50 ,um)
VIII METACERCARIA 69 A. Tetracerasta blepta
Flame-cell formula 2[(3+3+3)(3+3+3)]; anterior and posterior collecting ducts join main ducts antero-lateral to ventral sucker; main ducts join excretory bladder posterior to ventral sucker. Bladder tubular, full of refractile concretions (Figure 46); length 207(94-260,10), width 19(7-32,10); extends from anterior to the ventral sucker, dorsal to the ovary and testes to the posterior end of the body.
Eyespot pigment scattered.
Cyst spherical (Figure 39), large, 320(270-381,4); cyst wall transparent, elastic, thin (4); host capsule ellipsoidal (spherical in tad_poles), length 595(577-614,4), width 422(381-456,4); capsule wall fibrous, white, translucent, thickest at capsule ends (in ellipsoidal cysts), 129(93-186,8). Both host capsule and cyst wall easily removed.
Hosts: Hypseleotris galii Gobiomorphus australis Gobiomorphus sp. Mixophyes sp. (tadpole) Litoria lesueuri (tadpole, natural and experimental infections) Location: Pharyngeal muscle, viscera and muscle at base of fins Locality: Brisbane and Fitzroy Rivers, Queensland, Australia
(c) Oral Sucker Lobes
Four knobbed, protractible lobes originate from the dorsal margin of the oral sucker (Figures 42-45). The tip of each lobe as well as the dorsal margin of the oral sucker has large polygonal ampullae which contain granular material which stains light blue with Masson's trichrome stain indicating that it contains a mucous or collagen compound. The surface of the lobes has conical projections each with an opening at the apex like a volcano (Figure 45). There is apparently a cone for each of the large ampullae underneath. Under the cap of these ampullae lies a column of muscle continuous with the musculature of the oral sucker which can retract the lobes until they are completely inside the anterior end of the worm and only slits reveal their placement (Figures 38 & 42). The lobes are retracted into a parenchymatous portion of the oral sucker at the base of VIII METACERCARIA 70 A. Tetracerasta blepta
each lobe. These regions are less dense than the surrounding tissues and contain connective tissue in addition to the gland ducts which connect the lobes with the gland cells of the anterior worm. When the lobes are protracted the musculature of the oral sucker capsule contracts, forcing the lobes out of the parenchymatous portions. Although scales cover the anterior end of the worm, they do not occur on the lobes or on the margins of the oral sucker.
From the cap of granule-containing ampullae on the oral lobes, ducts pass back in two groups laterally and connect with two large groups of nucleated cells which stain identically to the material in the ampullae. These gland cells fill most of the body between the pharynx and the cirrus sac and may be homologous to the frontal glands of other digeneans. These cells may produce the granular material, possibly a mucopolysaccaride, which the ducts carry to the oral lobe cells and possibly also to the glandular tissue on the dorsal edge of the oral sucker. These gland cells release this material which is stored in the ampullae and which may act as a digestive enzyme. The lobes may also allow better attachment of this long cylindrical worm to the eel gut which passes much coarse material such as snail shells, insect exoskeleton and fish bones.
Yamaguti (1971) reported that three genera of lepocreadiids in the subfamily Megalogoniinae have oral sucker appendages or head lobes. Megalogonia, Creptotrema, and Creptotrematina all have a pair of papilliform head lobes anterodorsally on the oral sucker.
Among the subfamilies of allocreadiids summarized by Yamaguti (1971) some have oral sucker lobes. In the subfamily Allocreadiinae, Austrocreadium has 6 papilliform appendages surmounting the oral sucker. In the Bunoderinae there is Bunodera, which has a transverse row of four or six muscular head lobes along the anterodorsal margin of the oral sucker, as well as Allobunodera and Bunoderina. In the Crepidostominae, Crepidostomum Braun, 1900 has a half-crown of six muscular papillae or lobes. It would appear that oral sucker lobes are not uncommon in these two families although none of the above possess lobes which appear very similar to those of~ blepta because they are positioned differently and glandular tissue has not been reported in association with them. VIII METACERCARIA 71 A. Tetracerasta blepta
(d) Disposition in the Second Intermediate Host
In tadpoles the cysts appear in all parts of the body. They are most common in the tail and body near the tail-body junction.
In infected fishes, cysts are commonly found near the bases of the fins. In larger (over 8 cm) fish species, like Gobiomorphus australis, the metacercarial cysts are found in the muscle just under the scales especially at the base of the dorsal fins.
In smaller infected fish, such as Hypseleotris, the pharyngeal muscle near the gills or the viscera may have cysts.
Macfarlane (1951) found that cysts of~ anguillae which formed on the fins were often ru&ed off, those remaining did not develop well due to malnutrition, but those encysting in the muscles or the gonads of gobiids developed normally. Hine & Francis (1980) found that the site of encystment of Stegodexamene anguillae may vary from one intermediate host to another. VIII METACERCARIA 72 B. Stegodexamene callista
(a) Development
Encystment is very similar to that described for.!..:_ blepta. The newly formed cyst wall is a thin (2), transparent, elastic sphere (Table VII). Metacercaria grow for about the first 2 weeks in the cyst then they stop growing. The metacercariae are infective to eels within 7 days of encystment.
(b) Description (Figures 47 & 48)
Description of the cyst, cyst wall and host capsule is based on 11 live cysts from the pharyngeal muscle of Nematocentris fluviatilis infected experimentally 4 weeks previously. Description of the metacercaria is based on 15 fixed specimens from the liver of wild Retropinna semoni supplemented by observation of live material (measurements in Appendix II).
Metacercaria flattened, ovoid (Figure 47), length 334(255-465,15), width 152(62-161,15), anterior surface covered with scales which become spines posteriorly extending to level of ovary and sparcely to posterior end (Figure 47). Suckers subequal; oral sucker subterminal, ventral, retractible, subcircular, length 42(32-49,15), width 45(37-54,15); ventral sucker circular, 40(30-52,15), with 2 concentric rings of papillae, the inner of 5 small and pointed papillae and the outer of 9 large and blunt; skin glands abundant anterior to ventral sucker.
Prepharynx very short; pharynx circular, 18(12-40,15); oesophagus short, length 75(40-110), width 8(4-12,15), bifurcation anterior to ventral sucker; caeca end blindly at posterior end, length 170(135-270,15), width 9(5-12,15).
Testes two, equal, circular, 12(7-17,15), oblique, posterior to ovary; anterior testis sinistral; cirrus sac dextral, botuliform, slightly longer than diameter of ventral sucker, length 44, width 15. Ovary circular, 7, seldom differentiated from primordium of female system. Eggs not present. Vitellaria not fully developed. TAB LE V 11: Development of the Metacercaria of Stegodexamene callista in Experimental Infections
CYST** BODY MATURITY COMMENT Infection Host Capsule Cirrus
Age~ Diameter Thickness Length Width Testes Ovary Length Width
0 days 126( 121-135,6) as cercaria
3 days 173(150-213,23) 8 271 (243-305,5) 104(85-116,5) 7(6-8,4) 5(3) eyespots breaking up, bladder empty, caeca dilating, body follicular, cirrus only a curved series of cells
1 wk 264 85 6 8
2 wks 199(158-247,35) 12(6-28,29) 365(317-433,13) 108(85-127, 13) 8(6-10,10) 8(8-9,10) 42(38-51,10) 14(13-15, 10) eyespots scattered, bladder full, caeca dilated, cirrus a curved collapsed ring of cells, skin glands numerous
4wks 204( 167-250, 12) 11(4-15,11) 328(211-486,4) 97(85-137,8) 11 (6-18,3) 13(2) 44(2) 15(2) as above
6 wks 190(167-214, 16) 12(6-13, 16) 361 (317-423,8) 110(95-137,8) 8(8-10,7) 10(1) 41 (38-44,5) 12(10-13,5) one dead cyst, brown (in colour) with thickened (23) outer wall
8 wks 201 (186-223,23) 10(4-17,23) 410(317-602,18) 98(85-116, 18) 12(9-23, 17) 9(8-10,8) 49(38-54, 11) 13(10-18,13) several dead cysts with wrinkled brown outer wall
12 wks 373(296-476,6) 106(95-116,6) 13(12-13,6) 8(8-9,4) 44(38-51,3) 14(10-13,3) unchanged
* at 15-20°C measured from live material
-...:i Vl Figure 47: Metacercaria of Stegodexamene callista (only anterior concretions of excretory bladder shown) (ventral)
Figure 48: Cyst of Stegodexamene callista 74
47
h.c. 48 VIII METACERCARIA 75 B. Stegodexamene callista
Flame-cell formula 2[(4+5+5)(5+5+5)]; anterior and posterior collecting ducts join main ducts antero-lateral to ventral sucker; main ducts join excretory bladder anterior to ovary. Bladder tubular, may fill with refractile concretions until nearly ovoidal; length 150(100-290,15), width 90(50-140,15); extends from just anterior of ventral sucker dorsal to ovary and testes, to the posterior end.
Eyespot pigment scattered in older specimens.
Cyst spheroidal (Figure 48), 204(167-250,11); cyst wall thin, transparent, elastic, 2; host capsule spheroidal, fibrous, white, translucent, thickness 11(4-15,11).
Hosts: Retropinna semoni Pseudomugil signifer Ambassis sp. Location: Pharyngeal muscle, viscera and muscle at base of fins Locality: Brisbane River, Queensland, Australia Richmond River, New South Wales, Australia
(c) Disposition in the Second Intermediate Host
The metacercaria encysts in the viscera, in the external muscle just under the scales, especially around the base of the dorsal fins and most often in the pharyngeal muscle, gill chamber lining, even on the gill arches. Most of these sites are consistent with observations which suggest that penetration usually takes place in the throat following ingestion or inhalation of the cercariae. VIII METACERCARIA 76 C. Comparison
The metacercaria of I:__ blepta develops into a larger worm than does S. callista (Appendix II). I:__ blepta has a larger oral than ventral sucker whereas the suckers are equal and smaller in S. callista. S. callista does not have the oral sucker lobes of I:__ blepta. Even as a metacercaria S. callista has numerous skin glands which are few in T. blepta.
The ovary, testes and cirrus sac of I:__ blepta are larger than those of S. callista.
When both species are found in a single infected host, the metacercariae of I:__ blepta will be found mostly in the external muscle and the bases of the fins whereas S. callista will be in the pharyngeal muscle. This difference results from the behaviour of the cercariae.
Hine & Francis (1980) observed that site segregation between Stegodexamene anguillae and Telogaster opisthorchis occurs at the metacercarial stage.
Donges (1969) classified metacercariae by the amount they developed in in their intermediate hosts. Those that developed the most needed a longer development time before they became infective and survived longer in their intermediate hosts. The metacercaria of S. callista develops less then that of T. blepta within its intermediate hosts and is infective sooner. Both are long-lived but if Donges' (1969) relationship holds true then !.=._ blepta is the longer-lived of the two.
There is considerable range of development in lepocreadiid metacercariae. Some species of lepocreadiid metacercariae such as Stegodexamene anguillae may be "progenetic" and egg producing (Macfarlane, 1951) whereas others such as Lepocreadium pegorchis are little advanced from the cercaria (Bartoli, 1967). In some, there is no observable growth or maturation but in many there is development of the reproductive system but little growth (Stunkard, 1980a and others). A comparison between the metacercariae of the present study and those of the literature would be difficult and would be less meaningful than comparison of adults because of differences in the degree of development. IX ADULT 77 A. Tetracerasta blepta
(a) Development
The least mature adults examined, 7 days after infection, were less mature than the most developed metacercariae. Experimental infections of the eels,~ australis and~ reinhardtii, maintained at 20 to 25° c yielded the results in Table VIII and Figure 49. Adults of~ blepta developed normally in~ australis for one month or less, then stopped growing and died before they became gravid. In~ reinhardtii, gravid worms developed from 19 to 47 days after infection depending on the age of the metacercariae used, older metacercariae developed into mature adults sooner. Infections did not survive longer then two months in the laboratory.
(b) Description (Figures 50-57 & 67)
Diagnosis and description are based on 15 fixed specimens, the type series, from the hindgut of Anguilla reinhardtii and supplemented by observation of live material (measurements appear in Appendix III).
Adult with the taxonomic characters of the family; cylindrical (Figure 53); length 2010(1715-2412,12), widest at ventral sucker 228(195-251,12); anterior surface covered by scales (Figure 55) which become spines posteriorly (Figure 67) extending to level of ovary and sparcely to posterior end. Oral sucker large, subterminal, ventral, retractible, oval; length 150(128-182,15), width 173(150-205,12) with glandular tissue on anterior edge; 4 lobes in a transverse row on anterior margin (Figure 54). Lobes extendible, with conical ampullae on ends (Figure 55). Ventral sucker smaller, circular 110(82-146,12), with 3 pointed papillae forming a triangle in addition to less constant papillae (Figure 56). Skin glands sparse, only anterior to ventral sucker.
Prepharynx very short (Figure 50); pharynx ovoidal, length 53(46-77,15), width 67(58-84,12); oesophagus length 206(144-397,15), width 17(8-31,12), bifurcation anterior to ventral sucker; caeca end blindly at posterior end, length 1479(1348-1776,15), width 20(10-23,12). 78
TABLE VIII: Development of the Adult of Tetracerasta blepta in Experimental Infections
Age of Metacercaria Infection Body Eel Used Age Length Width Maturity Host
4-5 weeks 7 days* 627(539-729,16) 115(92-136,15) Immature AA 4-5 30 856(814-930, 3) 131(123-141,3) Maturing AA 4-5 47 1897(1430-2177,8) 295(211-328,8) Gravid AR
6-8 28 2022 297 Gravid AR 17 19 1664(1586-1742,2) 243(n=2) Gravid AR
* at 20 to 30°c
AA= Anguilla australis AR= A. reinhardtii Figure 49: Development of the Adult of Tetracerasta blepta A 7 days of age - immature B 30 days of age - maturing c 47 days of age - gravid 79
WrY OOS •
Figure 50: Adult of Tetr~ ce rasta blepta (Holotype) (ventral, oral sucker flattened) Figure 51: Terminal Male Gcnit.3lia of Tetracerasta blepta (Holotype) (ventral
Figure 52: Female Reproductiv~ System of Tetracerasta blepta (Composite drawn from live specimens)
,, 8 0 50
Cl. a.g. 51 m.-g. g.o. pr.g. u. a.s . .
52
o.
y.r.
ex.s. Figure 53: Adult of Tetracer asta blepta
Figure 54: Anterior End of Tetracerasta blepta
Figure 55 : Protracted Oral Lobes of Tetracerasta blepta
Figure 56: Ventral Sucker of Tetracerasta blepta
Figure 57: Terminal Male Genitalia of Tetracerasta blepta (Arrows indicate cirrus spines; Scale 25 µm) 81 IX ADULT 8 2 A. Tetracerasta blepta
Testes two, ovoidal, oblique to nearly tandem, posterior to ovary; anterior testis sinistral, length 118(90-157,15), width 92(67-113,12); posterior testis dextral, length 127(102-157,15), width 98(77-123,12); sperm ducts separate, join at posterior end of cirrus sac; cirrus sac botuliform (Figure 51), extends from halfway between ovary and ventral sucker dextrally to just anterior to ventral sucker, length 242(195-335,15), width 75(63-104,11). Cirrus sac muscular enclosing bipartite seminal vesicle; posterior seminal vesicle oval, length 137(102-197,15), width 63(51-84,15), sperm-filled; anterior seminal vesicle triangular to oval, length 74(38-102,15), width 43(26-64,15), sperm-filled; pars prostatica triangular, length 74(59-102,11), width 36(26-44,15); cirrus length variable, width 43(36-59,12), 12 patches of minute spines in folds distally, minute spines in lumen just anterior to pars prostatica; genital sinus small; genital opening 27, antero-sinistral to ventral sucker; metraterm glands unicellular, 6 to 10, petal-like, in a semi-circle surrounding the end of the uterus on the anterior, dextral and posterior sides, immediately dorsal to the genital opening; accessory gland cells surrounding the metraterm glands anteriorly, externally and posteriorly.
Ovary single, oval, length 76(65-92,15) width 70(42-92,12), submedian, dextral, posterior to ventral sucker (Figures 50 & 52); seminal receptacle lacrimiform, sperm-filled, length 89(63-136,15) width 44(27-54,12); Laurer's canal 78 long, 8 wide, narrow, dextral, opposite seminal receptacle (Figure 52); yolk reservoir oval, length 41(21-56,14), width 54(42-73,12), sinistral to ovary; vitelline follicles surround the caeca dorsally, ventrally and externally, extend from anterior to testis to end of caeca, confluent posterior to testes; follicles oval, length 45(33-54,15), width 27(17-33,12); uterus entirely anterior to ovary, 3 to 5 loops, contains 16(5-45,36) eggs; eggs smooth, light brown, ovoid, length 70(62-77,16), width 48(47-52,16).
Flame-cell formula 2[(3+3+3)(3+3+3)]; anterior and posterior collecting ducts join main ducts antero-lateral to ventral sucker, at level of bifurcation of caeca; main ducts join excretory bladder anterior to ovary. Bladder long, tubular, extends from bifurcation of the caeca, dorsal to the ovary and testes, to the posterior end; length 1458(1321-1674,15), width 63(27-94,12). IX ADULT 83 A. Tetracerasta blepta
Eyespot pigment widely scattered.
Type Host: Anguilla reinhardtii (wild and experimental) Location: hindgut Type Locality: Brisbane River, Queensland, Australia Disposition of Type Specimens: Queensland Mus., Brisbane: Holotype GL 1561 2 Paratypes GL 1562-3 South Aust. Mus., Adelaide: 3 Paratypes v 3078-80 British Mus. Nat. Hi st., London: 3 Paratypes 1982. 3. 11. 4-6 U.S. Nat. Mus., Beltsville: 3 Paratypes 77029 Meguro Parasi t. Mus., Tokyo: 3 Paratypes MPM 19355 Other Hosts: Macquaria novemaculeata (wild infections) Anguilla australis (experimental infections) Other Localities: Fitzroy River, Queensland, Australia Richmond River, New South Wales, Australia Other Species Examined: Notesthes robusta (1) Leiopotherapon unicolor (15) Glossamia aprion (4) Carassius auratus (2) Tandanus tandanus (4)
(c) Remarks
The oral sucker is retractible, as has been described for the metacercaria.
The evaginated cirrus has not been observed. It is believed that the cirrus evaginates to the level of the pars prostatica. The small spines in semicircular patches at the end of the cirrus (Figure 57) would lie at the base of the evaginated cirrus and the spines in the lumen would form a ring around the distal end of the cirrus. IX ADULT 84 A. Tetracerasta blepta
(d) Generic Diagnosis
With characters of family Lepocreadiidae (Odhner, 1905) Nicoll, 1934 as summarized by Yamaguti (1971); cylindrical, scale and spine covered. Oral sucker larger than ventral sucker, with a dorsal transverse row of 4 extendible lobes with conical ampullae connected to extensive gland cells in anterior body. Prepharynx very short; esophagus long; caeca long, end blindly at posterior end. Testes two, diagonal; cirrus sac large, continues posterior to ventral sucker; seminal vesicle bipartite, no external seminal vesicle; pars prostatica well developed; cirrus spiny; uterus pre-ovarian; petal-like metraterm glands form semicircle around end of uterus; genital opening antero-sinistral to ventral sucker; eggs large and few in number; vitellaria confined posterior to base of cirrus sac. Bladder tubular extending from bifurcation of caeca to opening at posterior end. Cercaria ophthalmotrichocercous.
(e) Specific Diagnosis
As the only species is ~ blepta, then the specific diagnostic characters are those of the genus.
(f) Differential Diagnosis
Stegodexamene is morphologically the most similar genus to Tetracerasta in the family Lepocreadiidae described to date. The most similar species to ~ blepta is S. callista of the present study but there are marked differences. The body of~ blepta is quite cylindrical whereas
S. callista is flattened. ~ blepta has four oral sucker lobes which have no counterpart in S. callista. The oral sucker of~ blepta is larger than the ventral sucker whereas the suckers of S. callista are equal. The arrangement of papillae on ~ blepta is a triangle of pointed papillae while there is always at least 2 rings of papillae on the ventral sucker of
S. callista. The skin glands of ~ blepta are sparse but other glands connecting with the oral sucker lobes are extensive whereas S. callista has numerous skin glands but lacks any other complex of glands in the body anterior to the accessory glands of the genital opening. The accessory IX ADULT 85 A. Tetracerasta blepta
glands of~ blepta are less extensive than those of S. callista. The size of the pars prostatica is also less extensive in ~ blepta than in S. callista.
The spines on the end of the cirrus of ~ blepta are in 12 distinct patches but those of S. callista are more scattered and not in distinct patches.
The flame-cell pattern of~ blepta is 2[(3+3+3)(3+3+3)] whereas that of S. callista is 2[(4+5+5)(5+5+5)].
The next most similar species to ~ blepta is Stegodexamene anguillae based on Macfarlane's (1951) description and on specimens examined from Anguilla australis from Christchurch, New Zealand (deposited in the Queensland Museum, GL 1567-69). The adult of T. blepta differs from
~ anguillae in the same characters in which it differs from S. callista as discussed above; however, the cercariae are remarkably similar as discussed in section VII C.
Macfarlane (1951) did not describe cirrus spines in~ anguillae like those of ~ blepta and cirrus spines could not be observed in preserved specimens of~ anguillae in the present study. Cirrus spines have been described for other lepocreadiids including Neopechona cablei, reported by Stunkard (1980b) and Neopechona pyriforme reported by Stunkard (1969).
These spines may occur in other lepocreadiids including ~ anguillae but may not have been observed because as Stunkard (1969) found, the spines are only conspicuous in living specimens.
Besides~ anguillae which Yamaguti (1971) placed in the subfamily Stegodexameninae, the subfamily most similar to T. blepta is the Lepocreadiinae. Within this subfamily, the most similar genera are Lepocreadium Stossich, 1903 and Opechona Looss, 1907. Lepocreadium has a much shorter oesophagus and a stouter body than ~ blepta. Lepocreadium also has an external seminal vesicle which ~ blepta does not have. Various differences in the life history and life-history stages exist as discussed earlier. Although Opechona is a longer worm with a longer oesophagus than Lepocreadium, it also has an external seminal vesicle which
~ blepta lacks. In Opechona the vitellaria continue anterior to the base IX ADULT 86 A. Tetracerasta blepta
of the cirrus sac and the uterus is contained in the intercaecal field which is not true of~ blepta. As discussed in section VII A (c), several genera of Allocreadiidae and Lepocreadiidae have oral sucker lobes like
~ blepta but their anatomy otherwise is not very similar.
Allocreadiids differ from the genus Tetracerasta by at least interfamily differences; in particular, the adults of allocreadiids have no spines or scales, cercariae are produced in sporocysts not rediae and the cercariae have stylets.
(g) Disposition in the Gut of the Eel
Although~ blepta is found along the entire length of the intestine and rarely in the stomach of!.:.._ reinhardtii, distribution is not uniform (Appendix IV). Two thirds are found in the 1st quarter of the intestine and most of the remaining worms are found in the 2nd quarter of the intestine.
A similar distribution of~ blepta is found in the Australian bass, M. novemaculeata (see discussion Section IX B (f) ). IX ADULT 87 B. Stegodexamene callista
(a) Development
Experimental infections of the eel A. australis maintained at 20 to 25<{; have yielded the results in Table IX and Figure 58. The forebody, and midbody posterior to the testes grew but the region from the ventral sucker to the gonads did not. Egg production started at about 70 days after the metacercariae were ingested by the eel. The single adult remaining in the 192-day infection was thin and not active. The testes and vitelline follicles were reduced; there were very few eggs in the uterus indicating that the worm was senescent (Figure 58D). As only a single worm remained in the infection it is likely that older worms may cease to produce eggs and die.
When very young (less than 8-day-old) metacercariae were used to infect eels, the size and maturity of the adults at removal were comparable to that of adults developing from 4-week-old metacercariae, suggesting that development from young metacercariae is not a disadvantage since the metacercariae develop rapidly for only a few weeks after encystment and when the younger metacercariae excyst compensatory growth occurs.
(b) Description (Figures 59-66)
Diagnosis and description are based on 16 fixed specimens, the type series, from the hindgut of Anguilla reinhardtii and supplemented by observation of live material.
Adults with the characters of the family after Yamaguti ( 1971). Body subcylindrical, flattened slightly dorso-ventrally (Figure 62), length 1720(1450-2140,16), width 227(190-290,16), anterior body surface covered with scales (Figure 64) which become spines posteriorly and extend to the level of the ovary and sparcely to posterior end. Oral sucker subterminal, ventral, retractible, oval (Figure 63); length 73(65-94,16), width 80(71-96,16). Ventral sucker subequal, circular 82(73-104,16), with 2 concentric rings of papillae, inner with 5 small pointed, outer with 9 large blunt papillae (Figure 65). Skin glands numerous anterior to ventral sucker (Figure 59). 88
TABLE IX: Development of the Adult of Stegodexamene callista in Experimental Infections
Infection Body Age Length Width Maturity
37 days* 908(660-1135,11) 168(142-209,10) Genitalia developed, no eggs 60 1040(707-1294,7) 173(140-209,7) Genitalia developed, no eggs 71 ** 1046(761-1268,9) 179(148-211,9) Some have eggs, all with sperm 78 1716(1469-1990,10) 264(232-381,9) Egg production, healthy 192 1962 223 Egg production, unhealthy
* at 20 to 3ooc, metacercariae 4 weeks old when used in infection ** this infection only, metacercariae less than or equal to 8 days old Figure 58: Development of the Adult of Stegodexamene callista A 37 days of age - immature B 60 days of age - immature c 78 Days of age - gravid D 125 Days of age - senesant 89
WrYOOS
0 '-
u
co lft (
Figure 59 : Adult of Stegodexamene callista (Holo t ype) (ventral)
Figure 60: Terminal Genitalia of Stegodexamene callista (Holo ty,.. ~ )(ventral)
Figure 61: Female Reproductive System of Stegoctexanrene callist a (Composite drawn from live specimens)
.~ 90
59 o.s. p.
s.g.
OE:. 60 ci. a.g.
....__....~ m.g. pr.g. "'.at' ,__
a.s. c.s. p.s. f.d. o.-----"J..ll' s. r. _ _n "" t. s.d.
E ~ 0 0 an
ex ..: " -·
Figure 62: Adult of Stegodexamene callista \.
Figure 63: Oral Sucker of Stegodexamene callista
Figure 64: Anterior Surface Scales of Stegodexamene callista
Figure 65: Ventral Sucker and Genital Open ing of Stegodexamene callista (Arrow indicates one of papillae)
Figure 66: Mid body Spines of the Adult of Stegodexamene callista
Figure 67: Mi dbody Spines of t he Adult of Tetracerasta blepta 91
63 IX ADULT 92 B. Stegodexamene callista
Prepharynx very short; pharynx ovoidal, length 48(38-56,16), width 53(46-63,16); oesophagus length 183(121-242,16), width 10(6-12,16), bifurcation anterior to ventral sucker; caeca end blindly at posterior end, length 1408(1135-1776,16), width 29(21-46,16).
Testes two, ovoidal, oblique, posterior to ovary; anterior testis sinistral, length 115(102-136,16), width 90(73-104,16); posterior testis dextral, length 124(109-146,16), width 96(84-113,16); sperm ducts separate, open separately and postero-dorsally into cirrus sac; cirrus sac botuliform (Figure 60), extends from halfway between ovary and ventral sucker dextrally to just anterior to ventral sucker, length 256(214-316,16), width 75(63-104). Cirrus sac muscular enclosing bipartite seminal vesicle; posterior seminal vesicle oval, length 124(90-165,16), width 65(52-92, 16), sperm-filled; anterior seminal vesicle oval, length 51(38-63,16), width 44(33-61,16), sperm-filled; pars prostatica triangular, length 115(86-146,16), width 42(36-52,15); cirrus length variable, width 43(36-52,15), end of cirrus with small spines, small spines also in lumen; genital sinus small; genital opening 28, antero-sinistral to ventral sucker; metraterm glands 6 to 10, large, unicellular, petal-like, surrounding terminal end of uterus just dorsal to genital opening; accessory glands surround the metraterm glands anteriorly, posteriorly, and externally.
Ovary single, circular 77(65-100,16), submedian, dextral, posterior to ventral sucker; seminal receptacle lacrimiform, sperm-filled, length 85(52-107,15), width 50(36-69,15); Laurer's canal about 80 long, narrow, connects to seminal receptacle (Figure 61); yolk reservoir oval, length 45(21-75,16), width 36(17-50,16), connects to oviduct; vitelline follicles surround the caeca dorsally, ventrally and externally, extend from ovary to end of caeca, become confluent posterior to testis, ending just anterior to posterior end of body; follicles oval, length 30(21-40,16), width 23(19-29,16); uterus entirely anterior to ovary, 2 to 3 loops, contains 9(4-29,32) eggs; eggs smooth, light brown, ovoid, length 70(64-74,14), width 45(42-49,14).
Flame-cell formula 2[(4+5+5)(5+5+5)]; anterior and posterior ducts join main ducts antero-lateral to ventral sucker, anterior to bifurcation of caeca; main ducts join excretory bladder just anterior to ovary. IX ADULT 93 B. Stegodexamene callista
Bladder long, tubular, extends from bifurcation of caeca or even just posterior to pharynx, dorsal to the ovary and testes, to the posterior end; length 1411(1144-1776,16), width 46(29-75,16).
Eyespot pigment widely scattered.
Type Host: Anguilla reinhardtii (wild and experimental) Location: hindgut Type Locality: Brisbane River, Queensland, Australia Disposition of Type Specimens: Queensland Mus., Brisbane: Holotype GL 1564 2 Paratypes GL 1565-6 South Aust. Mus., Adelaide: 3 Paratypes v 3081-83 British Mus. Nat. Hist., London: 3 Paratypes 1982. 3. 11 . 1-3 U.S. Nat. Mus., Bel ts ville: 3 Para types 77030 Meguro Parasit. Mus., Tokyo: 3 Paratypes MPM 19356 Other Hosts: Macquaria novemaculeata (natural infections) Anguilla australis (experimental infections) Other Localities: Richmond River, New South Wales, Australia Franklin and Agnes Rivers, Victoria, Australia Other Species Examined: Notesthes robusta (1) Leiopotherapon unicolor (15) Glossamia aprion (4) Carassius auratus (2) Tandanus tandanus (4)
(c) Remarks
The oral sucker is retractible.
In addition to the 2 rings of papillae described for the ventral sucker there are other papillae on or near the sucker but their placement is not consistent (Figure 65).
The evaginated cirrus has not been observed and likely the cirrus evaginates to the level of the pars prostatica. The patches of spines would then lie at the base of the cirrus and those previously in the lumen 94 IX ADULT B. Stegodexamene callista
would surround its terminal end.
(d) Specific Diagnosis
With the characters of the genus Stegodexamene Macfarlane, 1951. Spines on end and in lumen of cirrus. Excretory bladder often extending to just anterior to bifurcation of caeca, flame-cell pattern 2[(4+5+5)(5+5+5)]. Cercariae large, trichocercous with up to 4000 simple setae on its large tubular tail.
(e) Differential Diagnosis
The morphology of s. callista and T. blepta the two lepocreadiid species of this study have been compared above. The adults of s. callista
and ~ anguillae are very similar and share the characteristics of the genus as given by Macfarlane (1951) and on specimens examined from Anguilla australis from Christchurch, New Zealand. S. callista is a smaller worm,
its pars prostatica is not nearly as extensive as that of~ anguillae. The excretory bladder of S. callista may extend even anterior to the
bifurcation of the caeca whereas this was not reported for~ anguillae.
The flame-cell pattern of~ anguillae illustrated by Macfarlane (1951) was 2[(3+3)(3+3+3+3+3)] or 42 in total although he believed it might be incomplete. The flame-cell pattern for S. callista is 2[(4+5+5)(5+5+5)] or 50 in total. As previously discussed, Macfarlane (1951) did not report
cirrus spines for ~ anguillae as have been observed in S. callista but they are easily overlooked.
There are small but consistent differences between the adults of S. callista and ~ anguiliae but, as discussed before, there are marked differences in their cercariae. It was first supposed that the cercaria of S. callista was that of l.:._ blepta and vise versa, in keeping with the life-history stages described by Macfarlane (1951) based on his laboratory infections of uninfected hosts. The life-cycles of T. blepta and S. callista have been completed several times in the laboratory with lab-reared or uninfected hosts and it was found that the cercaria of ~ blepta, not that of S. callista closely resembles that of ~ anguillae. IX ADULT 95 B. Stegodexamene callista
This similarity is inexplicable.
Other similar lepocreadiid genera include Lepocreadium and Opechona and these differ from S. callista in the same way as these genera differ from l..:._ blepta discussed above.
Similarly ~ callista differs from all allocreadiids in the characters of the family namely its tegument spines, having rediae which produce cercariae in the life-cycle and in having cercariae with no stylets.
Yamaguti (1971) wrongly places Rhynchocreadium, a synonymn for Allocreadium, as a subgenus of Stegodexamene. This is wrong because Rhynchocreadium lacks body spines, a characteristic of the family Lepocreadiidae. Although Rhynchocreadium has some similarities to Stegodexamene such as an elongated body, a very short prepharynx, 2 diagonally placed testes, a well developed cirrus pouch and a long excretory bladder, it has only a simple seminal vesicle, a larger, less sessile ventral sucker and its cirrus pouch does not extend posterior to the ventral sucker. Until the life history of Rhynchocreadium is known, I believe that the two species of this genus should be considered synonyms of Allocreadium mehra Gupta, 1956, an allocreadiid, following the suggestions of Kakaji (1969).
(f) Disposition in the Gut of the Eel
~ callista is more uniformly distributed along the intestine of the eel, ~ reinhardtii, than l..:._ blepta and is rarely found in the stomach (Appendix IV). More than two thirds of S. callista are found in the 2nd and 3rd quarters of the intestine. Distribution along the intestine of the
Australian bass,~ novemaculeata, is similar.
Typically A. reinhardtii has three intestinal trematodes; S. callista, T. blepta and an unidentified opecoelid. The stomachs of larger eels often have unidentified hemiurids. In a typical infection the hemiurids are found exclusively in the stomach and pharynx along with large spirurid nematodes, likely Proleptus. In the 1st quarter of the intestine there are many l..:._ blepta, and S. callista is about half as numerous. In IX ADULT 96 B. Stegodexamene callista
the 2nd quarter, the number of~ blepta is very much reduced and the number of S. callista is increased. There may be a few opecoelids. In the
3rd and 4th quarters there are very few~ blepta. In the 3rd quarter the peak of s. callista occurs and there may be a few opecoelids. In the last quarter of the intestine there is a reduced number of S. callista but numerous opecoelids.
If only S. callista or ~ blepta occurs within an eel, a rare occurrence except in experimental infections, then the worms are evenly distributed along the intestine suggesting that species interaction restricts the distribution of the worms. The only occurrence of~ blepta in the stomach was in an eel with no hemiurids which normally occupy that region. An eel without opecoelids has more numerous lepocreadiids in the last quarter where the opecoelids normally are numerous.
Macfarlane (1951), Hine (1980) and Rid (1973) found that New Zealand freshwater eels, Anguilla dieffenbachii and A. australis schmidtii normally have intestinal infections of two trematodes. Stegodexamene anguillae is found in the anterior one third of the intestine and Telogaster opisthorchis in the posterior two thirds (Macfarlane, 1945, 1951). Hine
(1980) found that if an eel had a heavy infection of~ anguillae then the midgut was more heavily infected. In the present study it was found that if T. blepta was numerous then its distribution was wider and continued into the last half of the eel intestine. An increase in population and hence the range of one lepocreadiid restricted the range of the other.
Macfarlane (1952) believed that different chemotropism, effective after ~ anguillae and ~ opisthorchis excyst, produced the site segregation. Hine (1980) believed that the species interacted and he observed an extension of the S. anguillae population posteriorly due to crowding which resulted in a restricted anterior distribution of
~ opisthorchis. This agrees with the present study.
Hine (1980) found that the host species may affect the overlap between intestinal trematodes. Overlap was 24% in the eel A. dieffenbachii but only 6% in A. australis. IX ADULT 9 7 B. Stegodexarnene callista
Hine (1980) believed that the segregated distribution of ~ anguillae and ~ opisthorchis resulted from a posterior migration of~ opisthorchis after excystment because it is unlikely that~ opisthorchis excysts more posteriorly than~ anguillae because its cyst is thinner. Differences in the thickness of the host capsule or cyst wall are not believed to contribute to the segregated distribution of S. callista and ~ blepta in the present study. X EVOLUTIONARY RELATIONSHIPS 98
There is still much debate about the evolutionary relationships within the Allocreadioidea. Lepocreadiidae is considered an advanced family in the scheme proposed by Bayssade-Dufour & Maillard (1974) but not advanced in the scheme proposed by Cable (1974).
Cort et al. (1954) considered families that lack a persistent germinal mass as primitive. The species of the present study would be considered primitive under such a scheme. With their comparatively few but large and developed cercariae, they would be considered only slightly more advanced than the amphistomes.
The cercariae of the present study have a mesostomate type of excretory system and have an epithelial bladder. They form part of the superorder Epitheliocystidia which contained the Allocreadioidea. The presence of an epithelial bladder in this scheme is not primitive but advanced. La Rue ( 1 957) acknowledged that some of the Allocreadioidea did not fit such a system and more life history work would have to be completed to sort out the superfamily.
The phylogenetic tree proposed by Cable (1974) included several changes from La Rue's (1957) scheme and depicted the Lepocreadiidae as midway between the advanced and the primitive groups. It shows the Allocreadiidae to be more advanced than the Lepocreadiidae. A scheme for the evolution of the Allocreadioidea based on the chaetotaxy of cercariae placed the lepocreadiids as the most advanced of the superfamily and the allocreadiids as more primitive (Bayssade-Dufour & Maillard, 1974). The family Lepocreadiidae was, however, represented only by Lepocreadium album in that study so the results should not be considered conclusive. The evolutionary placement of the family Lepocreadiidae awaits further life history studies. XI CONCLUSIONS 99
Two lepocreadiid digeneans found in the intestine of the long-finned eel, Anguilla reinhardtii, from the Brisbane River are new species. One is a new genus and is named Tetracerasta blepta; the other is a species related to Stegodexamene anguillae Macfarlane, 1951 and is named Stegodexamene callista.
Both lepocreadiid species use the prosobranch gastropod, Posticobia brazieri, as their primary host. After about 10 to 14 days (less time at warmer temperatures) the operculate eggs hatch as a result of light stimulating the developed miracidium to become active and damage the vitelline membrane causing an influx of water which expels the miracidium. The miracidium uses chemotaxis to locate the snail. On contact with the snail the miracidium penetrates partially and an active sporocyst within it moves into the snail aided by muscle contraction of the miracidium. The sporocyst moves to near the snail's heart, develops rapidly and produces less than a dozen rediae after which it dies. Both the sporocyst and the redia lack a persistent germinal mass. The rediae move to the digestive gland in the posterior coils of the snail and following about 2 generations of rediae, immature cercariae are born. These mature and grow outside the rediae and emerge from the snail about 2 months following penetration of the snail by the sporocyst. The cercariae are large, ophthalmotrichocercous and live less than one day if a suitable host is not penetrated.
The cercariae of I.:.. blepta emerge after dusk in small numbers and move tail-first near the bottom. They have only a weak positive phototaxis. They penetrate a variety of small fishes and are often found encysted in the pharyngeal muscle, external muscle, and viscera of fishes of the genera Gobiomorphus and Hypseleotris, and in tree frog tadpoles. The rainbowfish, Nematocentris fluviatilis, and the tadpole, Litoria lesueuri, serve as hosts in the laboratory. Penetration is rapid and after the initial contact with the host the cercaria can not be shaken off and encysts within 20 minutes.
The cercariae of ~ callista emerge in small numbers at dawn and during the day. They swim tail-first and have a strong positive phototaxis. They swim in a manner which keeps them in the water column and visible to fish. They are often eaten or are accidentally inhaled. Cysts are common in the pharyngeal muscle and viscera of several small fishes in XI CONCLUSIONS 100
a range of genera including Retropinna, Craterocephalus, Pseudomugil and Ambassis. Several fishes could be infected in the laboratory including the rainbowfish, N. fluviatilis. Penetration usually occurs in the gill chamber or oesophagus after the cercariae contact the gill rakers and their tails break off. Encystment occurs rapidly in a similar manner to l.!. blepta.
The metacercaria of ~ blepta continues to enlarge and develop for several months during which time the eyespots break up, the bladder and caeca fill with crystals, and the gonads and oral sucker lobes develop. The oral sucker lobes are protractible and have conical ampullae on their tips which connect via ducts to glandular cells in the forebody which likely produce digestive enzymes. After 2 weeks of development the metacercaria is infective.
The metacercaria of ~ callista only grows for the first 2 weeks following cyst formation and develops numerous skin glands. It is infective within 7 days of encystment. No precocious egg production occurs within the cyst within one year.
Adults of~ blepta and ~ callista are common in the intestine of Anguilla reinhardtii from the Brisbane and Fitzroy Rivers of Queensland and from the Australian bass, Macquaria novemaculeata, from the Richmond River
of New South Wales. ~ blepta is found mostly along the 1st quarter of the
intestine of!:_ reinhardtii whereas ~ callista is mostly along the 2nd and
3rd quarters. Uninfected eels, Anguilla reinhardtii and ~ australis, were
used for laboratory infections. ~ callista produced eggs about 2 months after the metacercariae were eaten, reached senility about 4 months later
and died shortly afterward. ~ blepta produced eggs in~ reinhardtii from 3 to 7 weeks after infection depending on the development of the metacercariae. Infections of the eel,~ australis, never reached maturity and died within one month.
Only further life-cycle studies will elucidate the evolutionary and taxonomic position of the Lepocreadiidae within the Allocreadioidea and these are necessary as present views are contradictory. 101
APPENDIX I: Measurement of Cercaria
Feature Tetracerasta blepta Stegodexamene callista
Body 186(116-215)* 264(254-288)** 76(62-86) 75(59-99) Oral Sucker 37(32-44) 39(32-47) 35(30-41) 44(37-52) Ventral Sucker (Circular) 34(30-41) 43(37-52) Distance Ventral Sucker 95(49-117) 143(126-158) from Anterior End
Tail Length 212(170-252) 610(464-731,9) Tail Width: Anterior 18(13-22) 26(7-37,9) Maximum 18(13-22) 65(40-86,9) Posterior 4(4-12) 13(7-27,9) Setae Length: Anterior 50(39-56) 87(54-106,9) Mid tail 52(44-60) 103(89-116,9) Posterior 43(35-50) 71(52-99,9)
Pharynx 13(9-16) 14(12-19) 15(10-19) 17( 11-23) Penetration Gland 19(13-24) 24(15-30) 13(8-21) 16(11-30) Oesophagus 39(17-64) 80(60-100) 3 4 Caeca 87(70-104) 30(20-40) 4(4-6) 5 Bladder 95(81-116) 96(86-114) 23(17-29) 22(5-46) Eyes pots 9(8-9) 9(7-12) 7(6-7) 9(7-10) Testes: Anterior 5 by 7 Posterior 6 by 6 Cirrus sac 27 by 12 Ovary 9 by 8
** n=11 for all measurements unless otherwise noted * n=12 for all measurements 102
APPENDIX II: Measurements of the Metacercaria
Feature Tetracerasta blepta Stegodexamene callista
Host Capsule 595(577-614,4) 209(170-233,11) 422(381-456,4) 209( 170-233, 11) Host Capsule Wall: Ends 129(93-186,8) 11(4-15, 11) Sides 49(37-74,8) 11(4-15, 11) Cyst Diameter 320(270-381,4) 204(167-250,12) Cyst Wall 2 2
Body 409(232-530)* 334(255-465)** 133(101-168) 152(62-161) Oral Sucker 80(54-101) 42(32-49) 80(54-101) 45(37-54) Ventral Sucker (circular) 63(44-77) 40(30-52) Distance Ventral Sucker 190(128-247) 151(104-198) from Anterior End
Pharynx 23(15-30) 18(12-40) 31(22-42) 18(12-40) Oesophagus 72(37-136) 75(40-110) 12(10-12) 8(4-12) Caeca 186(106-259) 170(135-270) 19(12-25) 9(5-12) Bladder 207(94-260) 150(100-290) 19( 7-32) 90(50-140) Testes 22(17-30) 12(7-17) Cirrus Sac 64 44 12 15 Ovary (circular) 12 7
* n=10 unless otherwise noted ** n=15 unless otherwise noted 103
APPENDIX III: Measurements of the Adult
Feature Tetracerasta blepta Stegodexamene callista
Body 2010(1715-2412,12) 1720(1450-2140)* 228(195-251,12) 227(190-290) Oral Sucker 150(128-182,15) 73(65-94) 173( 150-205, 12) 80(71-96) Ventral Sucker (circular) 110(82-146, 12) 82(73-104) Distance Ventral Sucker 535(465-763,15) 450(372-593) from Anterior End
Pharynx 53(46-77,15) 48(38-56) 67(58-84,12) 53(46-63) Oesophagus 206(144-397,15) 183(121-242) 17(8-31,12) 10(6-12) Caeca 1479(1348-1776,15) 1408(1135-1776) 20(10-23,12) 29(21-46) Testes: Anterior 118(90-157,15) 115(102-136) 92(67-113, 12) 90(73-104) Posterior 127(102-157,15) 124(109-146) 98(77-123,12) 96(84-113) Cirrus Sac 242(195-335,15) 256(214-316) 75(63-104, 11) 75(63-104) Seminal Vesicle: Anterior 74(38-102,15) 51(38-63) 43(26-64,15) 44( 33-61) Posterior 137(102-197,15) 124(90-165) 63(51-84,15) 65(52-92) Pars Prostatica 74(59-102, 11) 115(86-146) 36(26-44,15) 42(36-52,15) Cirrus Width 43(36-59, 12) 42(36-52, 15) Ovary 76(65-92,15) 77(65-100,16) 70(42-92, 12) 77(65-100, 16) Seminal Receptacle 89(63-136,15) 85(52-107, 15) 44(27-54,12) 50(36-69,15) Yolk Reservoir 4 1( 21 -5 6 ' 14 ) 45(21-75,16) 54(42-73, 12) 36( 17-50, 16) Laurer' s Canal 78 by 8 80 by 7 104
APPENDIX III: Measurements of the Adult cont.
Feature Tetracerasta blepta Stegodexamene callista
Vitelline Follicles 45(33-54,15) 30(21-40,16) 27(17-33,12) 23(19-29,16) Egg Number 16(5-45,36) 9(4-29,32) Egg Size 70(62-77,16) 70(64-74,14) 48(47-52, 16) 45(42-49,14) Bladder 1458(1321-1674,15) 1411(1144-1776,16) 63(27-94, 12) 46(29-75,16)
* n=16 unless otherwise noted 105
APPENDIX IV: Distribution of Adults along Gut*
Intestinal Quarter Species Stomach 2 3 4
Unidentified Hemiurid 27
Tetracerasta blepta 54 314 101 5
Stegodexamene callista 4 126 240 210 57
Unidentified Opecoelid 2 6 253
* accumulated results from 6 freshly killed !.:_ reinhardtii XII REFERENCES 106
AGARWAL, s. M. (1959). Studies on the morphology, systematics and life history of Clinostomum giganticum n. sp. (Trematoda: Clinostomatidae). Indian Journal of Helminthology 11, 75-115.
ANDERSON, R. C. (1958). Methode pour !'examen des nematodes en vue apicale. Annales de Parasitologie 33, 171-172.
ARVY, L. (1953). Presence de Cercaria setifera (Monticella, 1888 et 1914 nee John Muller, 1850) chez Phyllirhoe bucephala Per. et Les., a Villefranche-sur-mer. Annales de Parasitologie, Humaine et Comparee 28, 289-299.
BARTOLI, P. (1967). Etude du cycle evolutif d'un trematode peu connu: Lepocreadium pegorchis (M. Stossich 1900) (Trematoda, Digenea). Annales de Parasitologie 42, 605-619.
BARTOLI, P & PREVOT, G. (1978). Le cycle biologique de Holorchis pycnoporus M. Stossich, 1901 (Trematoda, Lepocreadiidae). Zeitschrift fur Parasitenkunde 58, 73-90.
BAYSSADE-DUFOUR, Ch. & MAILLARD, C. (1974). Chetotaxie de quatre cercaires d'Allocreadioidea. Annales de Parasitologie 49, 521-554.
BECKER, W. (1973). Aktivierung und Inaktivierung der Miracidien von Schistosoma mansoni innerhalb der Eischale. Zei tschrift fur Parasitenkunde 42, 235-242.
BENNETT, H. J. & HUMES, A. G. (1939). Studies on the pre-cercarial development of Stichorchis subtriquetrus (Trematoda: Paramphistomidae). Journal of Parasitology 25, 223-231.
BROOKER, B. E. (1972). The sense organs of trematode miracidia. In Behavioural aspects of parasite transmission. (Suppl. to Zoological Journal of the Linnean Society 51, 1972) (ed. E.U. Canning and C.A. Wright), pp.171-180. London: Academic Press. XII REFERENCES Continued 107
CABLE, R. M. (1972). Behaviour of digenetic trematodes. In Behavioural aspects of parasite transmission. (Suppl. to Zoological Journal of the Linnean Society 51, 1972) (ed. E.U. Canning and C.A. Wright), pp.1-18. London: Academic Press.
CABLE, R. M. (1974). Phylogeny and taxonomy of trematodes with reference to marine species. In Symbiosis in the Sea. The Bell W. Baruch Library in Marine Science Number 2, pp.173-193.
CAIRNS, D. (1942). Life-history of two species of fresh-water eels in New Zealand. Part II Food and inter-relationships with trout. New Zealand Journal of Science and Technology 23(4B), 132-178.
CANNON, L. R. G. (1971). The life cycles of Bunodera sacculata and B. luciopercae (Trematoda: Allocreadiidae) in Algonquin Park, Ontario. Canadian Journal of Zoology 49, 1417-1429.
CHAPMAN, H. D. & WILSON, R. A. (1973). The propulsion of the cercariae of Himasthla secunda (Nicoll) and Cryptocotyle lingua. Parasitology 64, 1-15.
CORT, W.W., AMEEL, D.J. & VANDERWOUDE, A. (1954). Germinal development in the sporocysts and rediae of the digenetic trematodes. In Parasitological Reviews, Experimental Parasitology 3, 185-225.
DONGES, J. (1968). Entwicklungs- und Lebensdauer von Metacercarien. Zeitschrift fUr Parasitenkunde 31, 340-366.
DONGES, J. (1971). The potential number of redial generations in Echinostomatids (Trematoda). International Journal of Parasitology 1, 51-59.
HINE, P. M. (1978). Distribution of some parasites of freshwater eels in New Zealand. New Zealand Journal of Marine & Freshwater Research 12, 179-187. XII REFERENCES Continued 108
HINE, P. M. (1980). Distribution of helminths in the digestive tracts of New Zealand freshwater eels. 1. Distribution of digeneans. New Zealand Journal of Marine & Freshwater Research 14, 329-338.
HINE, P. M. & FRANCIS, R. I. C. C. (1980). Distribution of helminths in the digestive tracts of New Zealand freshwater eels. 3. Interspecific associations and conclusions. New Zealand Journal of Marine & Freshwater Research 14, 349-356.
HUMASON, G. L. (1962). Animal Tissue Techniques. 3rd ed. San Francisco: Freeman. 641pp.
KAKAJI, V. L. (1969). Studies on helminth parasites of Indian fishes. Part III. On some species of the genus Allocreadium Looss, 1900. Annales de Parasitologie 44, 131-146.
KASSIM, O. & GILBERTSON, D. E. (1976). Hatching of Schistosoma mansoni eggs and observations on motility of miracidia. Journal of Parasitology 62, 715-720.
KEARN, G. C. (1975). The mode of hatching of the monogenean Entobdella soleae, a skin parasite of the common sole (Solea solea). Parasitology 71, 419-443.
KENDALL, S. B. (1964). Some factors influencing the development and behaviour of trematodes in their molluscan hosts. 3rd Symposium of the British Society for Parasitology, pp.51-73.
KENDALL, S. B. & MCCULLOUGH, F. S. (1951). The emergence of the cercariae of Fasciola hepatica from the snail Limnaea truncatula Journal of Helminthology 25, 77-92.
K~IE, M. (1975). On the morphology and life history of Opechona bacillaris (Molin, 1859) Looss, 1907 (Trematoda, Lepocreadiidae). Ophelia 13, 63-86. XII REFERENCES Continued 109
LA RUE, G. R. (1957). The classification of digenetic trematoda: a review and a new system. In Parasitological Reviews, Experimental Parasitology 6, 306-349.
LENGY, J. & SHCHORY, M. (1970). Studies on larval stages of digenetic trematodes in aquatic molluscs of Israel 1. On two cercariae encountered in the marine snails Littorina neritoides (L.) and Nassa circumcincta (Adams, 1851). Israel Journal of Zoology 19, 135-144.
LYNCH, J. E. (1933). The miracidium of Heronimus chelydrae Maccallum. Quarterly Journal of Microscopical Science 76, 13-33.
MACFARLANE, W. V. (1945). The life cycle of the heterophyoid trematode Telogaster opisthorchis n.g., n.sp. Transactions of the Royal Society of New Zealand 75, 218-230.
MACFARLANE, W. V. (1951). The life cycle of Stegodexamene anguillae N.G., N.Sp., an allocreadiid trematode from New Zealand. Parasitology 41, 1-10.
MACFARLANE, W. V. (1952). Bionomics of two trematode parasites of New Zealand eels. Journal of Parasitology 38, 391-397.
MARTIN, W. E. (1938). Studies on trematodes of Woods Hole: The life cycle of Lepocreadium setiferoides (Miller and Northup), Allocreadiidae, and the description of Cercaria cumingiae. Biological Bulletin 74, 463-474.
MCDANIEL, J. S. & COGGINS, J. R. (1972). Seasonal larval trematode infection dynamics in Nassarius obsoletus (Say). Journal of the Elisha Mitchell Scientific Society 88, 55-57.
MEHRA, H. R. (1962). Revision of Allocreadioidea Nicoll, 1934 Part-I. Families: Lepocreadiidae Nicoll, 1934, Deropristiidae, N. Fam., Homalometridae, N. Fam. and Maseniidae Gupta, 1953. Proceedings of the National Academy of Sciences of India 32(sect. Biol.), 1-22. XII REFERENCES Continued 110
MILLER, H. M. & NORTHUP, F. E. (1926). The seasonal infestation of Nassa obsoleta (Say) with larval trematodes. Biological Bulletin 50, 490-509.
NASIR, P. & SKORZA, J. V. (1968). Studies on freshwater larval trematodes. XVIII. The life cycle of Stephanoprora denticulata (Rudolphi, 1802) Odhner 1910 (Trematoda: Digenea: Echinostomatidae). Zeitschrift fur Parasitenkunde 30, 134-148.
NICOLL, W. (1934). Vermes. Zoological Record (1933) 70, 92.
ODHNER, T. (1905). Die Trematoden des arktischen Gebietes. Fauna Arctica 4, 291-372.
PALOMBI, A. (1937). Il ciclo biologico di Lepocreadium album Stossich sperimentalmente realizzato. Rivista di Parassitologia 1, 1-13.
PEARSON, J. C. (1956). Studies on the life cycles and morphology of the larval stages of Alaria arisaemoides Augustine and Uribe, 1927 and Alaria canis LaRue and Fallis, 1936 (Trematoda: Diplostomidae). Canadian Journal of Zoology 34, 295-387.
PEARSON, J. C. (1961). Observations on the morphology and life cycle of Neodiplostomum intermedium (Trematoda: Diplostomatidae). Parasitology 51, 133-172.
PERSHAD, R. S. (1964). On a new species of the genus Rhynchocreadium Srivastava, 1962, from the fresh water fish, Rhynchobdella aculeata in Hyderabad, Andrha Pradesh. Zoologischer Anzeiger 175, 415-417.
RID, L. E. (1973). Helminth parasites of the long-finned eel, Anguilla dieffenbachii and the short-finned eel, A. australis. Mauri Ora 1, 99-106.
ROWAN, W. B. (1956). The mode of hatching of the egg of Fasciola hepatica. Experimental Parasitology 5, 118-137. XII REFERENCES Continued 111
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.
SRIVASTAVA, C. B. (1962). A new allocreadiid trematode, Rhynchocreadium aculeatum gen. et. sp. nov., from the fresh water eel, Rhynchobdella aculeata Bloch. Indian Journal of Helminthology 14, 1-4.
STOSSICH, M. (1903). Note distomologiche. Bollettino della Societa Adriatica di Scienze in Trieste 21, 193-201.
STUNKARD, H. W. (1934). The life history of Typhlocoelum cymbium (Diesing, 1850) Kossack, 1911 (Trematoda, Cyclocoelidae). A contribution to the phylogeny of the monostomes. Bulletin de la Societe Zoologique de France 59, 447-465.
STUNKARD, H. W. (1964). The morphology, life-history and systematics of the digenetic trematode, Homalometron pallidum Stafford, 1904. Biological Bulletin 126, 163-173.
STUNKARD, H. W. (1969). The morphology ond life-history of Neopechona pyriforme (Linton, 1900) N. Gen., N. Comb. (Trematoda: Lepocreadiidae). Biological Bulletin 136, 96-113.
STUNKARD, H. W. (1972). Observations on the morphology and life-history of the digenetic trematode, Lepocreadium setiferoides (Miller and Northup, 1926) Martin, 1938. Biological Bulletin 142, 326-334.
STUNKARD, H. W. (1980a). The morphology, life-history and taxonomic relations of Lepocreadium areolatum (Linton, 1900) Stunkard, 1969 (Trematoda: Digenea). Biological Bulletin 158, 154-163.
STUNKARD, H. W. (1980b). Successive hosts and developmental stages in the life history of Neopechona cablei sp. N. (Trematoda: Lepocreadiidae). Journal of Parasitology 66, 636-641. 112 XII REFERENCES Continued
TIMON-DAVID, J. (1955). Cycle evolutif d'un trematode cyclocoelide: Pseudhyptiasmus dollufusi Timon-David 1950 recherches experimentales. Annales de Parasitologie 30, 43-61.
WEST, A. F. (1961). Studies on the biology of Philophthalmus gralli Mathis and Leger, 1910 (Trematoda: Digenea). The American Midland Naturalist 66, 363-383.
WILSON, R. A. (1968). The hatching mechanism of the egg of Fasciola hepatica L. Parasitology 58, 79-89.
YAMAGUTI, S. (1971). Synopsis of Digenetic Trematodes of Vertebrates, vol. 1. Tokyo: Keigaku. 1074pp.
YAMAGUTI, s. (1975). Review of Life Histories of Digenetic Trematodes of Vertebrates. Tokyo:Keigaku. 590pp. KEY TO LETTERING
a. apical gland o.s. oral sucker a.b. anterior end of bladder p. pharynx pa. papilla(e) a .g. accessory gland a.s. anterior seminal vesicle p.c. pigment cup b. excretory bladder p.g. penetration gland b.p. birth pore p.g.d. penetration gland duct c. caecum p.g.p. penetration gland pore c .e. cercarial embryo p.l. protracted sucker lobe c .g. cystogenous gland po. pores ci. cirrus p.p. pars prostatica cl. cilia p.s. posterior seminal vesicle er. crystals r.e. redial embryo c.s. cirrus sac r.f.c. redia flame cell c.w. cyst wall rh. rhabdomere e. egg(s) in uterus r.l. retracted sucker lobe e.p. excretory pore r.p. rings of papillae e.pl. epidermal plate s. sporocyst ex .s. excretory bladder sphincter s.c. sucker of cercaria ey. eye spot s.d. sperm duct(s) ey.p. eyespot pigment s.f.c. sporocyst flame cell f .c. flame cell s.g. skin gland f.d. flagella in main duct s.r. seminal receptacle g .c. gland cell ( s) s.v. seminal vesicle g.d. gland duct t. testes g .o. genital opening ta. tail h .c. host capsule u. uterus 1. lappet v. vitelline follicle(s) l .c. Laur er' s canal v.c. vitelline cell m. muscle v.m. vitelline membrane m.g. metraterm gland v.r. vitelline cell remnants o. ovary v.s. ventral sucker oe. oesophagus y.r. yolk reservoir 0 .1. oral sucker lobe z. zygote op. operculum