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Early development and pathogenesis of Lobatostoma manteri Rohde (Trematoda: Aspidogastrea)
IntmmtionolJourdfor Parasitology.1975.Vol. 5. pp. 597-607.
Pergamon
Press.
Printed
in Grent
Britain.
EARLY DEVELOPMENT AND PATHOGENESIS LOBATOSTOMA MANTERI ROHDE (TREMATODA : ASPIDOGASTREA)”
OF
KLAUS ROHDE Heron Island Research Station, via Gladstone,
Queensland 4680, Australia
(Received 4 December 1974) K. 1975. Early development and pathogenesis of Lobatostoma manteri Rohde (Trematoda: Aspidogastrea). International Journal for Parasitology 5: 597-607. The prosobranch Planaxis sulcatus is reported as a new natural host of Lobatostoma manteri at Heron Island, Great Barrier Reef. Planaxis sulcatus and Cerithium moniliferum were experimentally infected with large numbers of eggs. The larvae hatch in the stomach and migrate immediately along the ducts of the digestive gland into the digestive follicles. The larvae feed on the secretion and probably epithelial cells of the follicles. The acetabulum is used for adhesion to the epithelium and contributes to its erosion. In heavily infected snails, the digestive follicles disappear gradually and the larvae live in cavities lined by a flattened epithelium, parts of which show secretory activity. In snails dissected 4749 and 65-66 days after infection, the cavities are fused, forming several large spaces which communicate with each other; only small parts of the epithelium are still secretory. Concentrations of amoebocytes occur in the walls of the digestive gland and in the wall between digestive gland and stomach of infected Planaxis. Some young worms were found in the stomach of Planaxis. No tissue reactions were seen around the stomach except in the wall between digestive gland and stomach. In Cerithium with 65-67 days old infection, the cavities contain much detritus and disintegrating cells, the epithelium is practically non-secretory and surrounded by loose connective tissue. In larvae with a body length of approximately 0.5-0.6 mm, the acetabulum begins to divide into alveoli and its anterior end grows forward; the anterior alveoli gradually increase in size and new alveoli are formed in the posterior undivided zone. In two specimens of approximately 1.3 mm body length, the whole adhesive disk was divided into half the number of alveoli usually found in adults. Allometric shifts during growth of the worms are analysed. Abstract-RoEmE
INDEX KEY WORDS: Trematoda; Aspidogastrea; host specificity; pathogenesis; development; life cycle; allometric growth.
INTRODUCTION THE REVIEW
by Rohde (1972) and the discussions in the papers by Rohde (1973~) and Rohde & Sandland (1973) show that very little is known about early development and pathogenesis in aspidogastreans. The only more or less complete life cycle of an aspidogastrean has been worked out for Lobatostoma manteri Rohde (see Rohde, 1973a), though even here some early developmental stages have not been described. Adults live in the intestine of the marine fish Truchinotus blochi (La&p&de); eggs with fully developed larvae are laid and eaten by snails; the larvae hatch in the stomach and migrate into the ducts of the digestive gland, where they grow up to full body size; the final hosts become infected by eating infected snails. * Publication from the Heron Island Research Station. Dedicated to Professor B. Rensch on the occasion of his 75th birthday.
feeding;
tissue reactions;
Rohde & Sandland (1973) also examined the pathology of the infection in two snail species, but nothing is known on the pathogenesis. There is still doubt about the route of infection of Aspidogaster conchicola Baer, one of the few other aspidogastrean species which are relatively well known (Rohde, 1972), in spite of the recent study by Bakker and Davids (1973). Hendrix & Short (1972) described the juvenile of Lophotaspis interiora Ward and Hopkins, but no details on development, etc. were given. With regard to feeding in aspidogastreans, Gentner (1971) claimed that Aspidoguster conchicola Baer and Cotylaspis insignis Leidy feed on the snails’ blood, but the possibility exists that cells derived from the worms’ epithelium (holocrine secretion) were mistaken for blood cells of the hosts (Rohde & Sandland, 1973).
MATERIALS AND METHODS The study was made at Heron Island, which belongs to the Capricorn group of coral islands (cays) at the 597
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FIG. 1. Cerithium moniriferum, l-day old infection, larva in digestive follicle. FIGS.2 & 3. Planaxis srdcatus, 47-49-day old infection, larva in digestive gland; parts of flattened
epithelium with secretion. FIG. 4. Pianaxis sukaius, l-day old infection, larva in stomach or large digestive duct. FIG. 5.Planaxis sukaf~rs, l-day old infection, larva in digestive follicle. FIG. 6. P/UPKLX~.S SI~~CQ~Z~S, 47-49-day old infection; larvae in digestive gland.
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FIG. 7. Planaxis
of Lobatosroma
srrlcatus, 47-49-day-old
infection, larvae in digestive gland.
FIGS. 8 & 9. Cerithium moniliferum, 47-49-day old infection. Note: digestive follicles replaced by cavities with flattened secretory epithelium and containing many larvae. FIG. 10. PIanuxis sulcatus, 47-49-day old infection, wall between digestive gland and stomach with many amoebocytes. FIGS. 11 & 12. Cerithium moniliferum,
4749-day
old infection. Note: eroding action of acetabulum.
Abbreviations
A-Amoebocytes AC-Acetabulum C-Caecum
CT-Connective DF-Digestive L-Lobatostoma
used
tissue follicle
S-Stomach SD-Sperm duct SC-Secretory granules
600
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FIGS. 13-H. Phaxis
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sulcatus, 65-66-day old infection. Note: larvae in large cavity of digestive gland, no typical digestive follicles left, but some parts of flattened epithehum with secretory grana. FIG. 16. Cerifhirrm ~ro~izjferum, 47-49-day old infection. Note: caecum of worm with digestive secretion of snail. FIG. 17. P/anuxis sukatus, 47-49&y old infection. Note: caecum of worm with digestive secretion of snail. FIG. lg. Planaxis sulcatus, 47-49-day old infection, contents in caecal lumen of worm (amoebocytes?, epithelial ceils of worm’s caecum?, epithelial cells of digestive follicle?).
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Cerithium monihferum, 65-67-day old infection, larvae in digestive gland. Note: no digestive follicles left, many necrotic cells around worms, some loose connective tissue.
FIGS. 19-22.
southe rn end of the Australian Great Barrier Reef, just north taf the Tropic of Capricorn, about 80 km from the mainla nd. Natural infections of the prosobranch Planaxis sulcatu s Born with Lobatostoma manteri were found during a quantitative ecological study of this snail species
and its parasites. The parasites were demonstratl ed by crushing the snails. Specimens of Planaxis sulcatu 8s and Cerithium (Clypeomorus) moniliferum Kiener (for taxonomic status of this prosobranch compare Rohde, I 973a) were exposed to large numbers of eggs laid by 27 vvorms
602
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dissected out of one fish of the species Trachinotus blochi La&p&de (Sub-nosed Dart) caught at Heron Island and containing a total number of 79 worms. Ten snails each (Planaxis 12-15 mm, Cerithium 8-15 mm high) from localities known to be free of the parasite, were put into cavity blocks with several hundred eggs for one to several days. After infection the snails were kept in
Route of infection, feeding and pathogenesis experimentally infected with Lobatostoma
in snails
manteri Rohde (1973a) showed experimentally that infection of snails occurs by ingestion of eggs and hatching of larvae in the stomach. Serial sections of Cerithium and Planaxis experimentally infected gave further evidence for this. Complete eggs and eggs with escaping larvae were demonstrated in the stomach. One day after infection, larvae were found in the stomach, in the large ducts of the digestive gland, and in the follicles of the digestive gland (Figs. 1, 4, 5); i.e. larvae hatch and migrate immediately into the digestive gland. Some l-day old larvae were attached to the epithelium of the follicles with their acetabula. Larvae were still in the follicles 22-24 days after infection. Forty-seven to forty-nine and 65-66 days after infection, the young
aerated aquaria with some pieces of beachrock, on which the snails live in their natural environment. The temperature ranged from 19” to 26°C. The snails were dissected at intervals. The worms were fixed in hot lOok formalin without pressing and stained with Grenacher’s carmine alum. For sectioning, snails were fixed in Bouin’s fixative and serial sections 10 ,um thick were made of the visceral mass. All sectioned snails were heavily infected. Crushed snails showed similar heavy infections with only one exception (Table I). Photomicrographs were taken with a Leitz Orthomat on an Orthoplan microscope.
TABLE ~-EXPERIMENTAL
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ROHDE
INFECTION OF SNAILS WITH Lobutostoma manferi
_~_. Age of Snail
species Pfanaxis sulcalus
Cerithium moniliferum
infection (days)
Size of No. of worms (mm) worms ..____-.
47-48
11
65-66
2
90-91
51
23
76
47.-49
55
47-54
94
65-61
57 - __
RESULTS NaturaI infection of Planaxis sulcatus with Lobatostoma manferi In July 1973, 782 Planaxis sulcatus were sampled
along transects on the southern beachrock at Heron Island and dissected for parasites. Two of the snails were found each to be infected with one Lobatostoma manteri. In one snail, the stomach was identified as the site of infection. One Lobatostoma was found in one of approximately the same number of snails dissected in April-May 1974. No Lobatostoma were found in similar numbers of Planaxis examined in October 1973, January 1974 and July-August 1974. Two worms were fixed in hot 10% formalin. They had a length of 3.9 and 3.5 mm respectively and each had 60 marginal alveoli. Comparison with other snails(Cerithium
monihyerum, Peristerniaaustraliensis
Reeve) shows that Pianaxis is comparatively rarely infected and that the worms have the same number of marginal alveoli in all the snail species (see Rohde & Sandland, 1973).
Remarks
0*[email protected]
(ave. 0.65) 0~62-0.67 also infected with (ave. 0.65) monostome xiphidiocercariae 0.39-071 (ave. O-57) 0.20-0.39 also infected with large (ave. 0.29) ocellate distome cercariae 0.13-0.56 (ave. @38) 0.32-0.76 (ave. 0.50) 0.28-0.75 (ave. 0.48) ._-...~~~- .__. -._ .--. __.~.
worms were in large cavities with flattened epithelium only parts of which showed secretory activity (Figs. 2,3,6-g). Large worms in Cerithium with 65-67 days old infection were located in large cavities lined by a flattened non-secretory epithelium and containing some necrotic cells (Figs. 19 & 20); smaller worms were surrounded by a dense mass of necrotic cells (Figs. 21 & 22). No digestive follicles were seen, the “digestive gland” being largely formed by much loose connective tissue (Figs. 19 & 22). Some worms were also found in the stomach of Planaxis. Whereas no tissue reactions were seen in the stomach walls, aggregations of large numbers of amoebocytes occurred in the walls of the digestive gland and in the wall between digestive gland and stomach of Planaxis (Fig. 10). Though amoebocytes were seen in the digestive gland of infected Cerithium, they never occurred in such large numbers. The undivided acetabulum of the young worms is still used for attachment to the epithelium (Figs. 11 & 12) and apparently contributes significantly to erosion
of the epithelium. The divided acetabulum (adhesive disk) of larger worms is located along the flattened epithelium of the cavities (Figs. 2, 6, 7, 13-15). Some worms in a Planaxis with 65-66-day old infection were rolled up in a “resting position” as found in large preadults in naturally infected Cerithium moniliferum (see Rohde & Sandland, 1973). In the caeca of many l-day old larvae and young worms, material was found which is identical with the secretion in the lumen of the digestive follicles and in the epithelial cells. It can easily be recognized by its large brown granules (Figs. 6, 12, 16 & 17). In addition, there are occasionally small cells with large nuclei (Fig. 18), which are either cells derived from the epithelium of the digestive follicles, amoebocytes of snails, or cells derived from the caecal epithelium of the worms (holocrine secretion). Of the 19 Cerithium moniliferum still alive 4749 days and 1 snail alive 49-54 days after infection, all except 2 died between 50 and 90 days after infection. Of the 2 living Cerithium, 1 was dissected and 1 sectioned 65-67 days after infection and found to harbour large numbers of worms. No control snails were kept but it seems probable that death was the result of the heavy infection. TABLE &-MEASUREMENTS
Early
0.12 0.13 0.12 0.12 0.14 0.12 0.14 0.13
growth
and
0.036 o+Mo 0.039 0.038 0.043 0.042 0.042 0.036
of
Lobatostoma
Dimensions
of larvae one day after hatching in are given in Table 2. The larva has previously been described by Rohde (1973a). It grows to about 3-4 times its original length without alveolus formation (Fig. 23). Growth is without significant changes in the body proportions and the caudal appendage disappears gradually. At a length of approximately 0.3-0.4 mm, the rudiments of the cirrus pouch (and metraterm?) and of the ovary and testis become distinct (Fig. 24). At a length of approximately 0.5 mm the testis is connected to the rudimentary cirrus pouch by means of a solid cord of cells, the rudiment of the sperm duct. In specimens 0.6 mm long, solid cords of cells represent the rudiments of testis-sperm ductcirrus pouch and ovary-uterus-metraterm. Formation of alveoli and marginal bodies begins in specimens approximately 0506 mm long and proceeds from the anterior to the posterior end. A rapid growth (forward) of the acetabulum soon after the appearance of the first alveoli leads to a complete transformation of the body proportions (Fig. 23). The marginal alveoli increase in size after they have been formed (Fig. 24) and there is apposition of more and Cerithium
moniliferum
OF ~-DAY OLD LARVAEOF Lobatostomamanteri
Length of acetabulum
allometries
manteri in snails
FIXEDINBOUIN'SFIXATIVEWITHOUTPRESSING
Length
603
Development of Lobatostoma
I.J.P. VOL. 5. 1975
Length of prepharyngeal body part 0.08 0.09 0.08 0.08 0.10 0.08 0.10 0.09
(mm,dia
=
FROM Cerithium monihferum, length + width)
2
Diameter of oral sucker
Diameter of pharynx
0.044 0.042 0.043 0.044
0.026 -
oGI3 0.052
07mm
Body length
FIG. 23. Lobatostoma manteri, growth from l-day old larva to young worm with 35 marginal alveoli.
604
KLAUS
b5mm Body Length
FIG. 24. Lobatustomu
marginal
alveoli,
manteri, growth from worm with 35 to worm with 41 marginal alveoli.
more alveoli in the posterior undivided zone of the acetabulum. The sheath delimiting the oral sucker disappears in specimens of about 1 mm length and the head lobes develop. In 2 specimens of approximately 1.3 mm length, the whole acetabulum was divided into 32 marginal alveoli (Fjg. 25). This may be explained in several ways: (1) The posterior un-
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divided zone is extremely small but does, in principle, not differ from the normal growth zone; (2) some specimens never acquire the full number of 60-62 alveoli; (3) there is an alternative, less common mode of alveolus formation, i.e. by interpolation of one additional alveolus between any two adjacent alveoli except for the most anterior and posterior ones. Indications for the last possibility may be that the number of marginal alveoli in both abnormal specimens was half the normal number and that lightly stained spots halfway between adjacent marginal alveoli could be marginal bodies in statu nascendi. However, serial sections were made of one specimen and no indication of developing alveoli or marginal bodies between those already present was seen. Growth of the acetabulum by apposition (new formation of alveoli in the posterior zone) and stretching (increase of size of alveoli formed already), leads to complicated allometric shifts in some organs (Figs. 26-29). The oral sucker shows negative allometric growth with an allometric exponent of 0.74 04
03
E O-2 E t
5-
.
I
‘a-
I
01
03
CG
Body
FIG.
FIG. 25. Lobatostomu munteri, young worm with apparently fully developed adhesive disk containing 32 marginal alveoli.
length,
04
0.5
0.7
I
mm
26. Lobatostomu manteri, relative growth of oral sucker. Allometric exponent 0.74.
until it disappears (Fig. 26). The pharynx has negative allometric growth with an allometric exponent of 0.65, which later decreases to 044 (Fig. 27). The anterior body part in front of the anterior margin of the acetabulum grows at first isometrically (allometric exponent 0.98) and subsequently, does not increase in size at all, a consequence of alveolus formation and forward growth of the acetabulum. At later stages it grows with slight negative allometry (allometric exponent 0.94), an expression of the fact that though most alveoli have been formed, the acetabulum still grows more rapidly than the rest
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Development
605
of ~obutostuma
Body length, mm FIG. 27. Lobu~ostoma munteri, relative growth of pharynx. Allometric (increasingly negative allometric growth).
exponents
065 and 0.44
.
O-I
0.2
03
o-4
05
07
I
2
3
4
Body length, mm FIG. 28. ~~&u~~~~o~u manreri, relative growth of anterior (preacetabular) body part. Allometric exponents 0.98 (approximately isometric growth), zero (no growth of anterior body part), and 0.94 (approximately isometric growth).
of the body because of apposition of some new alveoli and stretching of those formed already (Fig. 28). The acetabulum grows at first with very slight positive allometry (allometric exponent 1.08), then with very strong positive allometry (allometric exponent 2.20) because of alveolus formation and forward growth of its anterior part, and finally isometrically (allometric exponent 0.99) (Fig. 29). DISCUSSION Host specificity
Host records of aspidogastreans show that all species which are well known, i.e. which have been found repeatedly, occur in a variety of molluscs and in a number of different sites (Rohde, 1972).The data for Lobatostoma confirm this. The parasites were
found in the digestive gland and stomach of three snail species belonging to three families (Cerithium (Clypeumor~s) mon~l~ferum Kiener, Cerithiidae; Peristernia australiensis Reeve, Fasciolariidae; Planaxis sulcatus Born, Planaxidae). Cerithium and Peristernia are frequently infected, but only in one small habitat at Heron Island, though the snails are common in large areas (Rohde & Sandland, 1973). In the former species, a single specimen is usually found in a large cavity formed by enlargement of one (or several?) ducts of the digestive gland; only rarely are two specimens found or a specimen occurs in the stomach. In the latter species, several specimens occur usually in the stomach and less often in the ducts of the digestive gland. Pkwzaxis is only rarely infected and the site of infection in the only
606
K~nus
FIG. 29. Lo~atos#omama~~eri, relative growth of acetabulum. Aliometric exponents 1.08 (slightly positive allometric growth), 2.20 (strongly positive allometric growth)
and 0.99 (isometric growth). snail with a natural infection examined was the stomach. The fact that this species can easily be infected experimentally indicates that rare infection in the natural environment is probably due to a less frequent exposure to eggs. Planaxis lives higher on the beachrock than Cerithium and Per~ster~ia and it is for instance, possible that the final host, the fish Trachinutus biochi (La&p&de), does not visit that habitat regularly. Data on other mollusc and fish species examined and found to be negative are given by Rohde (1973a). Route of infection, feeding and pathogenes~s
At the time of publication of the review on Aspidogastrea by Rohde (1972), the route of infection in molluscs was known only for one species. Wootton (1966) stated in an abstract that larvae of CotyIogas~er (Cotylogastero~des) occidentalk are ingested by mussels and begin their development in the stomach region. No details were, however, given. There are different views on how larvae of Aspidogasfer conchicoja enter mollusc hosts. Adults occur usually in the pericardial cavity and kidneys of bivalves, and it seems possible that the larvae reach these sites either from the intestine or through the kidney funnel (compare Rohde, 1972). Bakker & Davids (1973) suggested on the basis of infection experiments that clams become infected by embryonated eggs and that infection occurs via the nephridiopore. According to these authors, “the
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larvae apparently remain in the renal cavities of the host until they mature, when they move into the pericardium”. Larvae may also hatch in the free environment but experiments to infect hosts with such larvae failed. Evidence for the suggested route of infection is indirect and it must be taken into consideration that Aspidogaster conchicola is also commonly found in the digestive gland of prosobranchs and that young worms were found in the intestine after experimental infection (compare discussion by Rohde, 1973a). Thus, the question of how larvae invade molluscs still remains unsettled. It seems possible that different routes are chosen in different hosts or even in the same host. The only species for which detailed observations on the infection route have been made is Lobatostoma manteri (compare Rohde, 1973a; Rohde & Sandland, 1973, this paper). There can be no doubt that ingestion of eggs by snails, hatching of larvae in the stomach and migration of larvae into the digestive gland are the only mode of infection in the snail species examined. The only observations on feeding in aspidogastreans are by Gentner (1971), who claimed that Aspidogaster conchicola and Cotyfaspis irtsignis feed on mollusc blood cells. The only evidence given is a similar size of cells extracted from the caeca of worms and of blood cells. Rohde (19736), however, has shown that in the aspidogastrean Multicotyle purvisi Dawes as well as in the monogenean Polystomo~des renschi Rohde, whole cells of the caecal epithelium are shed. Confusion of epithelial cells with blood cells is therefore possible. In this study, it was shown that larvae begin to feed soon after infection on secretion of the digestive follicles and probably on the secretory epithelium itself. A discussion of tissue reactions in molluscs infected with aspidogastreans was given by Rohde & Sandland (1973). These authors have shown that in natural infections of Peristernia australiensis with Lobatostoma, where the worms five predominantly in the stomach, a thick layer of fibrous tissue is found below the stomach wall. Such fibrous layer was not found in Planaxis even in cases where worms were present in the stomach. It is possible that it develops only in older infections. Aggregations of amoebocytes in the walls of the digestive gland of Planaxis correspond to similar aggregations in Cerithium moniliferum naturally infected with Lobatostoma. The smaller number of amoebocytes in experimentally infected Cerithium may be due to the younger age of infection or the abnormally high intensity of infection. Early growth and allometry Only in Multicotyie purvisi have early develop-
ment and allometric growth been studied in detail (Rohde, 1971). There is a surprising similarity of this species and Loba~osfoma ~~anferi, In both, the larva grows at first with only minor changes in body
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Development
proportions and without division of the posterior sucker into alveoli. Formation of alveoli occurs along an anterior-posterior gradient and leads to a rapid forward growth of the anterior part of the adhesive disk. Alveoli increase in size after they have been formed. As a result of these growth characteristics, relative growth of the pre-acetabular body part is in both species at first isometric, then zero and finally slightly negative-allometric. The only difference between both species is a relatively longer zerogrowth phase in Multicotyle. The allometric curves for the acetabulum have the same general shape in both species. Differences are larger allometric exponents for the first two growth phases in Lobatostoma and a more extensive second growth phase in Multicotyle. Relatively longer second growth phases of anterior body part and acetabulum in Multicotyle are apparently an expression of the greater body length of this species. Acknowledgements-I wish to thank Mrs. M. Bremhorst for making the sections and Mr. E. Grant for supplying the fish. The study was supported by a University of Queensland Research Grant.
REFERENCES K. E. & DAVIDS C. 1973. Notes on the life history of Aspidoguster conchicola Baer, 1826 (Trematoda; Aspidogastridae). Journal of Helminthology 47:
BAKKER
269-276.
of Lobatostoma
607
GENTNERH. W. 1971. Notes on the biology of Aspidogaster conchicola and Cotylaspis insignis. Zeitschrift j%
Parasitenkunde
35: 263-269.
HENDRIX S. S. & SHORT R. B. 1972. The juvenile of Lophotaspis interiora Ward and Hopkins 1931 (Trematoda: Aspidobothria). Journal of Parasitology 58: 63 -67.
ROHDE K. 1971. Untersuchungen an Multicotyle purvisi Dawes, 1941 (Trematoda: Aspidogastrea). II. Quantitative Analyse des Wachstums. Zoologische Johrbiicher, Abteilung fiir Anatomie 88: 188-202. ROHDE K. 1972. The Aspidogastrea, especially Multicotyle purvisi Dawes, 1941. Advances in Parasitology 10: 77-151.
ROHDE K. 1973~. Structure and development of Lobatostoma manteri sp. nov. (Trematoda: Aspidogastrea) from the Great Barrier Reef, Australia. Parasitology 66: 63-83.
ROHDE K. 1973b. Ultrastructure of the caecum of Polystomoides malayi Rohde and P. renschi Rohde (Monogenea : Polystomatidae). International Journal for Parasitology 3 : 46 I-466. ROHDEK. & SANDLANDR. 1973. Host-parasite relations in Lobatostoma manteri Rohde (Trematoda: Aspidogastrea). Zeitschrift fiir Parasitenkunde 42: 115-136. WOOTTON D.
M.
1966. The cotylocidium larva of (Nickerson, 1902) Yamaguti 1963 (Aspidocotylea-Trematoda). Proceedings of the First International Congress of Parasitology, Rome, 1964. 547-548 (abstract). Cotylogasteroides
occidentalis
Pergamon
Press.
Printed
in Grent
Britain.
EARLY DEVELOPMENT AND PATHOGENESIS LOBATOSTOMA MANTERI ROHDE (TREMATODA : ASPIDOGASTREA)”
OF
KLAUS ROHDE Heron Island Research Station, via Gladstone,
Queensland 4680, Australia
(Received 4 December 1974) K. 1975. Early development and pathogenesis of Lobatostoma manteri Rohde (Trematoda: Aspidogastrea). International Journal for Parasitology 5: 597-607. The prosobranch Planaxis sulcatus is reported as a new natural host of Lobatostoma manteri at Heron Island, Great Barrier Reef. Planaxis sulcatus and Cerithium moniliferum were experimentally infected with large numbers of eggs. The larvae hatch in the stomach and migrate immediately along the ducts of the digestive gland into the digestive follicles. The larvae feed on the secretion and probably epithelial cells of the follicles. The acetabulum is used for adhesion to the epithelium and contributes to its erosion. In heavily infected snails, the digestive follicles disappear gradually and the larvae live in cavities lined by a flattened epithelium, parts of which show secretory activity. In snails dissected 4749 and 65-66 days after infection, the cavities are fused, forming several large spaces which communicate with each other; only small parts of the epithelium are still secretory. Concentrations of amoebocytes occur in the walls of the digestive gland and in the wall between digestive gland and stomach of infected Planaxis. Some young worms were found in the stomach of Planaxis. No tissue reactions were seen around the stomach except in the wall between digestive gland and stomach. In Cerithium with 65-67 days old infection, the cavities contain much detritus and disintegrating cells, the epithelium is practically non-secretory and surrounded by loose connective tissue. In larvae with a body length of approximately 0.5-0.6 mm, the acetabulum begins to divide into alveoli and its anterior end grows forward; the anterior alveoli gradually increase in size and new alveoli are formed in the posterior undivided zone. In two specimens of approximately 1.3 mm body length, the whole adhesive disk was divided into half the number of alveoli usually found in adults. Allometric shifts during growth of the worms are analysed. Abstract-RoEmE
INDEX KEY WORDS: Trematoda; Aspidogastrea; host specificity; pathogenesis; development; life cycle; allometric growth.
INTRODUCTION THE REVIEW
by Rohde (1972) and the discussions in the papers by Rohde (1973~) and Rohde & Sandland (1973) show that very little is known about early development and pathogenesis in aspidogastreans. The only more or less complete life cycle of an aspidogastrean has been worked out for Lobatostoma manteri Rohde (see Rohde, 1973a), though even here some early developmental stages have not been described. Adults live in the intestine of the marine fish Truchinotus blochi (La&p&de); eggs with fully developed larvae are laid and eaten by snails; the larvae hatch in the stomach and migrate into the ducts of the digestive gland, where they grow up to full body size; the final hosts become infected by eating infected snails. * Publication from the Heron Island Research Station. Dedicated to Professor B. Rensch on the occasion of his 75th birthday.
feeding;
tissue reactions;
Rohde & Sandland (1973) also examined the pathology of the infection in two snail species, but nothing is known on the pathogenesis. There is still doubt about the route of infection of Aspidogaster conchicola Baer, one of the few other aspidogastrean species which are relatively well known (Rohde, 1972), in spite of the recent study by Bakker and Davids (1973). Hendrix & Short (1972) described the juvenile of Lophotaspis interiora Ward and Hopkins, but no details on development, etc. were given. With regard to feeding in aspidogastreans, Gentner (1971) claimed that Aspidoguster conchicola Baer and Cotylaspis insignis Leidy feed on the snails’ blood, but the possibility exists that cells derived from the worms’ epithelium (holocrine secretion) were mistaken for blood cells of the hosts (Rohde & Sandland, 1973).
MATERIALS AND METHODS The study was made at Heron Island, which belongs to the Capricorn group of coral islands (cays) at the 597
KLAUS
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FIG. 1. Cerithium moniriferum, l-day old infection, larva in digestive follicle. FIGS.2 & 3. Planaxis srdcatus, 47-49-day old infection, larva in digestive gland; parts of flattened
epithelium with secretion. FIG. 4. Pianaxis sukaius, l-day old infection, larva in stomach or large digestive duct. FIG. 5.Planaxis sukaf~rs, l-day old infection, larva in digestive follicle. FIG. 6. P/UPKLX~.S SI~~CQ~Z~S, 47-49-day old infection; larvae in digestive gland.
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FIG. 7. Planaxis
of Lobatosroma
srrlcatus, 47-49-day-old
infection, larvae in digestive gland.
FIGS. 8 & 9. Cerithium moniliferum, 47-49-day old infection. Note: digestive follicles replaced by cavities with flattened secretory epithelium and containing many larvae. FIG. 10. PIanuxis sulcatus, 47-49-day old infection, wall between digestive gland and stomach with many amoebocytes. FIGS. 11 & 12. Cerithium moniliferum,
4749-day
old infection. Note: eroding action of acetabulum.
Abbreviations
A-Amoebocytes AC-Acetabulum C-Caecum
CT-Connective DF-Digestive L-Lobatostoma
used
tissue follicle
S-Stomach SD-Sperm duct SC-Secretory granules
600
KLAUS
FIGS. 13-H. Phaxis
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sulcatus, 65-66-day old infection. Note: larvae in large cavity of digestive gland, no typical digestive follicles left, but some parts of flattened epithehum with secretory grana. FIG. 16. Cerifhirrm ~ro~izjferum, 47-49-day old infection. Note: caecum of worm with digestive secretion of snail. FIG. 17. P/anuxis sukatus, 47-49&y old infection. Note: caecum of worm with digestive secretion of snail. FIG. lg. Planaxis sulcatus, 47-49-day old infection, contents in caecal lumen of worm (amoebocytes?, epithelial ceils of worm’s caecum?, epithelial cells of digestive follicle?).
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601
Cerithium monihferum, 65-67-day old infection, larvae in digestive gland. Note: no digestive follicles left, many necrotic cells around worms, some loose connective tissue.
FIGS. 19-22.
southe rn end of the Australian Great Barrier Reef, just north taf the Tropic of Capricorn, about 80 km from the mainla nd. Natural infections of the prosobranch Planaxis sulcatu s Born with Lobatostoma manteri were found during a quantitative ecological study of this snail species
and its parasites. The parasites were demonstratl ed by crushing the snails. Specimens of Planaxis sulcatu 8s and Cerithium (Clypeomorus) moniliferum Kiener (for taxonomic status of this prosobranch compare Rohde, I 973a) were exposed to large numbers of eggs laid by 27 vvorms
602
KLAUS
dissected out of one fish of the species Trachinotus blochi La&p&de (Sub-nosed Dart) caught at Heron Island and containing a total number of 79 worms. Ten snails each (Planaxis 12-15 mm, Cerithium 8-15 mm high) from localities known to be free of the parasite, were put into cavity blocks with several hundred eggs for one to several days. After infection the snails were kept in
Route of infection, feeding and pathogenesis experimentally infected with Lobatostoma
in snails
manteri Rohde (1973a) showed experimentally that infection of snails occurs by ingestion of eggs and hatching of larvae in the stomach. Serial sections of Cerithium and Planaxis experimentally infected gave further evidence for this. Complete eggs and eggs with escaping larvae were demonstrated in the stomach. One day after infection, larvae were found in the stomach, in the large ducts of the digestive gland, and in the follicles of the digestive gland (Figs. 1, 4, 5); i.e. larvae hatch and migrate immediately into the digestive gland. Some l-day old larvae were attached to the epithelium of the follicles with their acetabula. Larvae were still in the follicles 22-24 days after infection. Forty-seven to forty-nine and 65-66 days after infection, the young
aerated aquaria with some pieces of beachrock, on which the snails live in their natural environment. The temperature ranged from 19” to 26°C. The snails were dissected at intervals. The worms were fixed in hot lOok formalin without pressing and stained with Grenacher’s carmine alum. For sectioning, snails were fixed in Bouin’s fixative and serial sections 10 ,um thick were made of the visceral mass. All sectioned snails were heavily infected. Crushed snails showed similar heavy infections with only one exception (Table I). Photomicrographs were taken with a Leitz Orthomat on an Orthoplan microscope.
TABLE ~-EXPERIMENTAL
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ROHDE
INFECTION OF SNAILS WITH Lobutostoma manferi
_~_. Age of Snail
species Pfanaxis sulcalus
Cerithium moniliferum
infection (days)
Size of No. of worms (mm) worms ..____-.
47-48
11
65-66
2
90-91
51
23
76
47.-49
55
47-54
94
65-61
57 - __
RESULTS NaturaI infection of Planaxis sulcatus with Lobatostoma manferi In July 1973, 782 Planaxis sulcatus were sampled
along transects on the southern beachrock at Heron Island and dissected for parasites. Two of the snails were found each to be infected with one Lobatostoma manteri. In one snail, the stomach was identified as the site of infection. One Lobatostoma was found in one of approximately the same number of snails dissected in April-May 1974. No Lobatostoma were found in similar numbers of Planaxis examined in October 1973, January 1974 and July-August 1974. Two worms were fixed in hot 10% formalin. They had a length of 3.9 and 3.5 mm respectively and each had 60 marginal alveoli. Comparison with other snails(Cerithium
monihyerum, Peristerniaaustraliensis
Reeve) shows that Pianaxis is comparatively rarely infected and that the worms have the same number of marginal alveoli in all the snail species (see Rohde & Sandland, 1973).
Remarks
0*[email protected]
(ave. 0.65) 0~62-0.67 also infected with (ave. 0.65) monostome xiphidiocercariae 0.39-071 (ave. O-57) 0.20-0.39 also infected with large (ave. 0.29) ocellate distome cercariae 0.13-0.56 (ave. @38) 0.32-0.76 (ave. 0.50) 0.28-0.75 (ave. 0.48) ._-...~~~- .__. -._ .--. __.~.
worms were in large cavities with flattened epithelium only parts of which showed secretory activity (Figs. 2,3,6-g). Large worms in Cerithium with 65-67 days old infection were located in large cavities lined by a flattened non-secretory epithelium and containing some necrotic cells (Figs. 19 & 20); smaller worms were surrounded by a dense mass of necrotic cells (Figs. 21 & 22). No digestive follicles were seen, the “digestive gland” being largely formed by much loose connective tissue (Figs. 19 & 22). Some worms were also found in the stomach of Planaxis. Whereas no tissue reactions were seen in the stomach walls, aggregations of large numbers of amoebocytes occurred in the walls of the digestive gland and in the wall between digestive gland and stomach of Planaxis (Fig. 10). Though amoebocytes were seen in the digestive gland of infected Cerithium, they never occurred in such large numbers. The undivided acetabulum of the young worms is still used for attachment to the epithelium (Figs. 11 & 12) and apparently contributes significantly to erosion
of the epithelium. The divided acetabulum (adhesive disk) of larger worms is located along the flattened epithelium of the cavities (Figs. 2, 6, 7, 13-15). Some worms in a Planaxis with 65-66-day old infection were rolled up in a “resting position” as found in large preadults in naturally infected Cerithium moniliferum (see Rohde & Sandland, 1973). In the caeca of many l-day old larvae and young worms, material was found which is identical with the secretion in the lumen of the digestive follicles and in the epithelial cells. It can easily be recognized by its large brown granules (Figs. 6, 12, 16 & 17). In addition, there are occasionally small cells with large nuclei (Fig. 18), which are either cells derived from the epithelium of the digestive follicles, amoebocytes of snails, or cells derived from the caecal epithelium of the worms (holocrine secretion). Of the 19 Cerithium moniliferum still alive 4749 days and 1 snail alive 49-54 days after infection, all except 2 died between 50 and 90 days after infection. Of the 2 living Cerithium, 1 was dissected and 1 sectioned 65-67 days after infection and found to harbour large numbers of worms. No control snails were kept but it seems probable that death was the result of the heavy infection. TABLE &-MEASUREMENTS
Early
0.12 0.13 0.12 0.12 0.14 0.12 0.14 0.13
growth
and
0.036 o+Mo 0.039 0.038 0.043 0.042 0.042 0.036
of
Lobatostoma
Dimensions
of larvae one day after hatching in are given in Table 2. The larva has previously been described by Rohde (1973a). It grows to about 3-4 times its original length without alveolus formation (Fig. 23). Growth is without significant changes in the body proportions and the caudal appendage disappears gradually. At a length of approximately 0.3-0.4 mm, the rudiments of the cirrus pouch (and metraterm?) and of the ovary and testis become distinct (Fig. 24). At a length of approximately 0.5 mm the testis is connected to the rudimentary cirrus pouch by means of a solid cord of cells, the rudiment of the sperm duct. In specimens 0.6 mm long, solid cords of cells represent the rudiments of testis-sperm ductcirrus pouch and ovary-uterus-metraterm. Formation of alveoli and marginal bodies begins in specimens approximately 0506 mm long and proceeds from the anterior to the posterior end. A rapid growth (forward) of the acetabulum soon after the appearance of the first alveoli leads to a complete transformation of the body proportions (Fig. 23). The marginal alveoli increase in size after they have been formed (Fig. 24) and there is apposition of more and Cerithium
moniliferum
OF ~-DAY OLD LARVAEOF Lobatostomamanteri
Length of acetabulum
allometries
manteri in snails
FIXEDINBOUIN'SFIXATIVEWITHOUTPRESSING
Length
603
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Length of prepharyngeal body part 0.08 0.09 0.08 0.08 0.10 0.08 0.10 0.09
(mm,dia
=
FROM Cerithium monihferum, length + width)
2
Diameter of oral sucker
Diameter of pharynx
0.044 0.042 0.043 0.044
0.026 -
oGI3 0.052
07mm
Body length
FIG. 23. Lobatostoma manteri, growth from l-day old larva to young worm with 35 marginal alveoli.
604
KLAUS
b5mm Body Length
FIG. 24. Lobatustomu
marginal
alveoli,
manteri, growth from worm with 35 to worm with 41 marginal alveoli.
more alveoli in the posterior undivided zone of the acetabulum. The sheath delimiting the oral sucker disappears in specimens of about 1 mm length and the head lobes develop. In 2 specimens of approximately 1.3 mm length, the whole acetabulum was divided into 32 marginal alveoli (Fjg. 25). This may be explained in several ways: (1) The posterior un-
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divided zone is extremely small but does, in principle, not differ from the normal growth zone; (2) some specimens never acquire the full number of 60-62 alveoli; (3) there is an alternative, less common mode of alveolus formation, i.e. by interpolation of one additional alveolus between any two adjacent alveoli except for the most anterior and posterior ones. Indications for the last possibility may be that the number of marginal alveoli in both abnormal specimens was half the normal number and that lightly stained spots halfway between adjacent marginal alveoli could be marginal bodies in statu nascendi. However, serial sections were made of one specimen and no indication of developing alveoli or marginal bodies between those already present was seen. Growth of the acetabulum by apposition (new formation of alveoli in the posterior zone) and stretching (increase of size of alveoli formed already), leads to complicated allometric shifts in some organs (Figs. 26-29). The oral sucker shows negative allometric growth with an allometric exponent of 0.74 04
03
E O-2 E t
5-
.
I
‘a-
I
01
03
CG
Body
FIG.
FIG. 25. Lobatostomu munteri, young worm with apparently fully developed adhesive disk containing 32 marginal alveoli.
length,
04
0.5
0.7
I
mm
26. Lobatostomu manteri, relative growth of oral sucker. Allometric exponent 0.74.
until it disappears (Fig. 26). The pharynx has negative allometric growth with an allometric exponent of 0.65, which later decreases to 044 (Fig. 27). The anterior body part in front of the anterior margin of the acetabulum grows at first isometrically (allometric exponent 0.98) and subsequently, does not increase in size at all, a consequence of alveolus formation and forward growth of the acetabulum. At later stages it grows with slight negative allometry (allometric exponent 0.94), an expression of the fact that though most alveoli have been formed, the acetabulum still grows more rapidly than the rest
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Development
605
of ~obutostuma
Body length, mm FIG. 27. Lobu~ostoma munteri, relative growth of pharynx. Allometric (increasingly negative allometric growth).
exponents
065 and 0.44
.
O-I
0.2
03
o-4
05
07
I
2
3
4
Body length, mm FIG. 28. ~~&u~~~~o~u manreri, relative growth of anterior (preacetabular) body part. Allometric exponents 0.98 (approximately isometric growth), zero (no growth of anterior body part), and 0.94 (approximately isometric growth).
of the body because of apposition of some new alveoli and stretching of those formed already (Fig. 28). The acetabulum grows at first with very slight positive allometry (allometric exponent 1.08), then with very strong positive allometry (allometric exponent 2.20) because of alveolus formation and forward growth of its anterior part, and finally isometrically (allometric exponent 0.99) (Fig. 29). DISCUSSION Host specificity
Host records of aspidogastreans show that all species which are well known, i.e. which have been found repeatedly, occur in a variety of molluscs and in a number of different sites (Rohde, 1972).The data for Lobatostoma confirm this. The parasites were
found in the digestive gland and stomach of three snail species belonging to three families (Cerithium (Clypeumor~s) mon~l~ferum Kiener, Cerithiidae; Peristernia australiensis Reeve, Fasciolariidae; Planaxis sulcatus Born, Planaxidae). Cerithium and Peristernia are frequently infected, but only in one small habitat at Heron Island, though the snails are common in large areas (Rohde & Sandland, 1973). In the former species, a single specimen is usually found in a large cavity formed by enlargement of one (or several?) ducts of the digestive gland; only rarely are two specimens found or a specimen occurs in the stomach. In the latter species, several specimens occur usually in the stomach and less often in the ducts of the digestive gland. Pkwzaxis is only rarely infected and the site of infection in the only
606
K~nus
FIG. 29. Lo~atos#omama~~eri, relative growth of acetabulum. Aliometric exponents 1.08 (slightly positive allometric growth), 2.20 (strongly positive allometric growth)
and 0.99 (isometric growth). snail with a natural infection examined was the stomach. The fact that this species can easily be infected experimentally indicates that rare infection in the natural environment is probably due to a less frequent exposure to eggs. Planaxis lives higher on the beachrock than Cerithium and Per~ster~ia and it is for instance, possible that the final host, the fish Trachinutus biochi (La&p&de), does not visit that habitat regularly. Data on other mollusc and fish species examined and found to be negative are given by Rohde (1973a). Route of infection, feeding and pathogenes~s
At the time of publication of the review on Aspidogastrea by Rohde (1972), the route of infection in molluscs was known only for one species. Wootton (1966) stated in an abstract that larvae of CotyIogas~er (Cotylogastero~des) occidentalk are ingested by mussels and begin their development in the stomach region. No details were, however, given. There are different views on how larvae of Aspidogasfer conchicoja enter mollusc hosts. Adults occur usually in the pericardial cavity and kidneys of bivalves, and it seems possible that the larvae reach these sites either from the intestine or through the kidney funnel (compare Rohde, 1972). Bakker & Davids (1973) suggested on the basis of infection experiments that clams become infected by embryonated eggs and that infection occurs via the nephridiopore. According to these authors, “the
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larvae apparently remain in the renal cavities of the host until they mature, when they move into the pericardium”. Larvae may also hatch in the free environment but experiments to infect hosts with such larvae failed. Evidence for the suggested route of infection is indirect and it must be taken into consideration that Aspidogaster conchicola is also commonly found in the digestive gland of prosobranchs and that young worms were found in the intestine after experimental infection (compare discussion by Rohde, 1973a). Thus, the question of how larvae invade molluscs still remains unsettled. It seems possible that different routes are chosen in different hosts or even in the same host. The only species for which detailed observations on the infection route have been made is Lobatostoma manteri (compare Rohde, 1973a; Rohde & Sandland, 1973, this paper). There can be no doubt that ingestion of eggs by snails, hatching of larvae in the stomach and migration of larvae into the digestive gland are the only mode of infection in the snail species examined. The only observations on feeding in aspidogastreans are by Gentner (1971), who claimed that Aspidogaster conchicola and Cotyfaspis irtsignis feed on mollusc blood cells. The only evidence given is a similar size of cells extracted from the caeca of worms and of blood cells. Rohde (19736), however, has shown that in the aspidogastrean Multicotyle purvisi Dawes as well as in the monogenean Polystomo~des renschi Rohde, whole cells of the caecal epithelium are shed. Confusion of epithelial cells with blood cells is therefore possible. In this study, it was shown that larvae begin to feed soon after infection on secretion of the digestive follicles and probably on the secretory epithelium itself. A discussion of tissue reactions in molluscs infected with aspidogastreans was given by Rohde & Sandland (1973). These authors have shown that in natural infections of Peristernia australiensis with Lobatostoma, where the worms five predominantly in the stomach, a thick layer of fibrous tissue is found below the stomach wall. Such fibrous layer was not found in Planaxis even in cases where worms were present in the stomach. It is possible that it develops only in older infections. Aggregations of amoebocytes in the walls of the digestive gland of Planaxis correspond to similar aggregations in Cerithium moniliferum naturally infected with Lobatostoma. The smaller number of amoebocytes in experimentally infected Cerithium may be due to the younger age of infection or the abnormally high intensity of infection. Early growth and allometry Only in Multicotyie purvisi have early develop-
ment and allometric growth been studied in detail (Rohde, 1971). There is a surprising similarity of this species and Loba~osfoma ~~anferi, In both, the larva grows at first with only minor changes in body
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Development
proportions and without division of the posterior sucker into alveoli. Formation of alveoli occurs along an anterior-posterior gradient and leads to a rapid forward growth of the anterior part of the adhesive disk. Alveoli increase in size after they have been formed. As a result of these growth characteristics, relative growth of the pre-acetabular body part is in both species at first isometric, then zero and finally slightly negative-allometric. The only difference between both species is a relatively longer zerogrowth phase in Multicotyle. The allometric curves for the acetabulum have the same general shape in both species. Differences are larger allometric exponents for the first two growth phases in Lobatostoma and a more extensive second growth phase in Multicotyle. Relatively longer second growth phases of anterior body part and acetabulum in Multicotyle are apparently an expression of the greater body length of this species. Acknowledgements-I wish to thank Mrs. M. Bremhorst for making the sections and Mr. E. Grant for supplying the fish. The study was supported by a University of Queensland Research Grant.
REFERENCES K. E. & DAVIDS C. 1973. Notes on the life history of Aspidoguster conchicola Baer, 1826 (Trematoda; Aspidogastridae). Journal of Helminthology 47:
BAKKER
269-276.
of Lobatostoma
607
GENTNERH. W. 1971. Notes on the biology of Aspidogaster conchicola and Cotylaspis insignis. Zeitschrift j%
Parasitenkunde
35: 263-269.
HENDRIX S. S. & SHORT R. B. 1972. The juvenile of Lophotaspis interiora Ward and Hopkins 1931 (Trematoda: Aspidobothria). Journal of Parasitology 58: 63 -67.
ROHDE K. 1971. Untersuchungen an Multicotyle purvisi Dawes, 1941 (Trematoda: Aspidogastrea). II. Quantitative Analyse des Wachstums. Zoologische Johrbiicher, Abteilung fiir Anatomie 88: 188-202. ROHDE K. 1972. The Aspidogastrea, especially Multicotyle purvisi Dawes, 1941. Advances in Parasitology 10: 77-151.
ROHDE K. 1973~. Structure and development of Lobatostoma manteri sp. nov. (Trematoda: Aspidogastrea) from the Great Barrier Reef, Australia. Parasitology 66: 63-83.
ROHDE K. 1973b. Ultrastructure of the caecum of Polystomoides malayi Rohde and P. renschi Rohde (Monogenea : Polystomatidae). International Journal for Parasitology 3 : 46 I-466. ROHDEK. & SANDLANDR. 1973. Host-parasite relations in Lobatostoma manteri Rohde (Trematoda: Aspidogastrea). Zeitschrift fiir Parasitenkunde 42: 115-136. WOOTTON D.
M.
1966. The cotylocidium larva of (Nickerson, 1902) Yamaguti 1963 (Aspidocotylea-Trematoda). Proceedings of the First International Congress of Parasitology, Rome, 1964. 547-548 (abstract). Cotylogasteroides
occidentalis