Description

(Based on 50 living and 10 fixed and stained specimens. Body, tail stem and furcae dimensions are given in Table I, p. 15).

A cercaria is small, opaque, and consists of a body, tail stem and furcae (Text-fig. 3, A). Body tapered anteriorly, pear-shaped and depressed when contracted (Text-fig. 3, A; 4, D), elongate and cylindrical when expanded (Text-fig. 4, A; 4, F). Greatest width contracted is at about mid-body level. Small, diagonally arranged, blunt spines are distributed over anterior half of body, and these merge with irregularly shaped cuticular plates which form a mosaic over posterior half of body (Text-fig. 3, C). Numerous, irregularly shaped cystogenous granules, 4μ to 15μ by 2μ to 9μ,? are distributed throughout parenchyma and impart opacity to body. These granules are concentrated in posterior half of body, becoming sparser anterior to pharynx. Central region of body is almost completely devoid of granules and accordingly less opaque. Flask-shaped cystogenous organ, 32μ to 35μ long by 22μ to 25μ at its widest point, is situated near anterior extremity (Text-fig. 3, A). Its external surface is partly covered by granules of similar appearance and texture to cystogenous granules in parenchyma. Neck of cystogenous organ encloses posterior half of a narrow duct at posterior end of which, and within cystogenous organ, is a mass of gland cells. This duct of cystogenous organ opens anteriorly by a small pore which is surrounded by four lips. Neutral red stains cystogenous granules in body stain dull pink to orange indicating neutrality, and cystogenous organ bright red indicating acidity.

An H-shaped ganglionic mass lies just posterior to cystogenous organ (Text-fig. 4, A, C). Anterior and longitudinal nerve trunks arising from this were not seen.

Mouth is a small transverse slit, 5μ to 6μ wide, surrounded by cuticular plates, situated 30μ to 40μ from posterior extremity depending on state of contraction of body. Little or no prepharynx. Pharynx more or less spherical, 12μ to 16μ in diameter, followed by a short oesophagus; intestine sacculate, up to 75μ long by 20μ to 25μ wide, and lined by a cuboidal epithelium. Peristaltic contractions of intestine occur, but no food inclusions seen in intestine.

Usual number of body wall layers present (Text-fig. 3, F). A band of muscles (2μ?thick) lies immediately within cuticle but separate longitudinal and circular fibres could not be seen. Parenchyma contains a few oblique muscle fibres. Mucus cells surround oesophagus.

Rudimentary genital pore is situated 10μ?from posterior extremity. Genital anlagen consists of two lobed masses of densely staining nuclei posterior to pharynx, each about 30μ long by 10μ wide (Text-fig. 4, A and C).

Excretory pore is at posterior extremity of body. Excretory vesicle is saccular and extends to left of pharynx. Excretory tubules open into excretory vesicle approximately midway along its length. Flame cell formula is 2[(3 + 3 + 3) + (3 + 3 + 3)] = 36 (Text-fig. 4, B). Elimination of liquid from the excretory vesicle takes place as indicated in Text-fig. 5, A-C.

Tail stem is unspined. A parenchymatous layer within the cuticle is continuous with an axial strand of parenchyma and muscle fibres. A single row of cystogenous granules lies within cuticle along posterior surface of tail stem, apart from axial strand region where granules may be four or five rows deep. A few scattered granules are distributed through axial strand. Lumen of tail stem contains a few oily droplets.

Furcae are sub-circular in section, unspined, tapered distally and flattened against tail stem proximally. Highly contractile, varying between 1.3 and 18 times as long as body. Two rows of cystogenous granules, 6μ wide by 2μ long in ventral view, by 6μ tall in lateral view, lie along ventral surface of furcae (Text-fig. 3, D and E).

Text-fig. 5.—Bucephalus longicornutus. Elimination of liquid from the excretory vesicle. For abbreviations see p. 9.

Table II.—Previous Reports of Bucephalid Cercariae from Bivalve Molluscs.

Date	Author	Host	Name given to Species	Locality
1827	von Baer	Unio pictorum	Bucephalus polymorphus	Europe (F)
1827	von Baer	Anodonta anatina	B. polymorphus	Europe (F)
1827	von Baer	A. cellensis	B. polymorphus	Europe (F)page 16
1848	Siebold	A. cellensis	B. polymorphus	Europe (F)
1854	Lacaze-Duthiers	Ostrea edulis	B. haimeanus	Mediterranean (S)
1854	Lacaze-Duthiers	Cardium rusticum	B. haimeanus	Mediterranean (S)
1857	Pagenstecher	A. anatina	B. polymorphus	Europe (F)
1863	Claparede	Found free swimming cercaria	B. haimeanus	Normandy (S)
1874	McCrady	Crassostrea virginica	B. cuculus	Carolina (S)
1878	Ulicny	A. cellensis	B. intermedius 1	Europe (F)
1881	Ercolani	U. pictorum	Cercaria bucephalus 1	Europe (F)
1881	Levinsen	Modiolaria discors	B. crux	Egedesminde (S)
1882	Ercolani	A. anatina	C. bucephalus 1	Europe (F)
1883	Ziegler	—	B. polymorphus	Europe (F)
1888	Huet	Cardium edule	B. haimeanus	Normandy (S)
1890	Nelson	Crassostrea virginica	"Gregarinoid parasites"	New Jersey, U.S.A. (S)
1893	Huet	Mactra solida	B. haimeanus	Normandy (S)
1893	Nelson	Crassostrea virginica	B. cuculus	New Jersey, U.S.A. (S) Europe (S)
1894	Vaullegeard	Tapes pullastra	B. haimeanus
1894	Vaullegeard	Tapes decussatus	B. haimeanus	Europe (S)
1899	Kelly	Unionidae	B. polymorphus	Mississippi R. (F)
1903	Haswell	Mytilus latus	B. sp.	New Zealand (S)
1904, 05	Johnstone	Cardium edule	B. haimeanus	England (S)
1905, 06 09	Tennent	Crassostrea virginica	B. haimeanus 2	East Coast, U.S.A. (S)
1906	Pelseneer	Syndosmya alba	B. haimeanus	Europe (S)
1906	Pelseneer	Cardium edule	B. haimeanus	Europe (S)
1907	Pelseneer	Mactra solida	B. haimeanus	Europe (S)
1907	Pelseneer	Mactra subtruncata	B. haimeanus	Europe (S)
1907	Pelseneer	Donax trunculus	B. haimeanus	Europe (S)
1909	Sinitzin	Dreissensia polymorpha	B. polymorphus	Warsaw (F)
1909	Sinitzin	A. mutabilis	B. polymorphus	Warsaw (F)
1909	Sinitzin	Tapes rugatus	B. haimeanus	Black Sea (S)
1911	Sinitzin	Tapes rugatus	C. hydriformis 3	Black Sea (S)
1911	Lebour	Cardium edule	B. haimeanus	England (S)page 17
1911	Lebour	S. alba	B. syndosmyae	England (S)
1924	Wunder	—	B. polymorphus	Europe (F)
1925	Miller	Pinna carnea	Cercaria N	Tortugas (S)
1928	Faust	Modiola capensis	Bucephalopsis modiolae	Sth. Africa (S)
1929	Woodhead	Elliptio dilatatus	Bucephalus papillosum 4,8	Michigan (F)
1930	Woodhead	Eurynia iris	B. elegans 8	Michigan (F)
1933	Roughley	O. commercialis	B. haimeanus	N.S.W. (S)
1933	Roughley	O. angasi	B. haimeanus	N.S.W. (S)
1934	Ozaki and Ishibashi	Pinctada martensi	B. margaritae	Japan (S)
1934	Palombi	Tapes decussatus	Bucephalopsis haimeana 6	Naples (S)
1934	Palombi	Tapes aureus	B. haimeana 6	Naples (S)
1934	Wesenberg-Lund	—	Bucephalus polymorphus	Europe (F)
1935	Cole	Mytilus edulis	B. mytili	England (S)
1936	Woodhead	A. grandis	C. argi	Michigan (F)
1936	Woodhead	Lampsilis siliquoidea	C. basi 5	Michigan (F)
1936	Woodhead	Eurynia iris	C. scioti	Michigan (F)
1939	Rees	Cardium edule	B. haimeanus	England (S)
1949	Andreu	Tapes aureus	Bucephalopsis haimeana 6	Spain (S)
1952	Chubrik	Mytilus latus	Prosorhynchus squamatus 7	Arctic? (S)
1952	Kniskern	L. siliquoidea	Rhipidocotyle septpapillata 8	Michigan (F)
1954?	Ozaki	?	Bucephalus itabo?	Japan (S)
1954	Hopkins	Crassostrea virginica	B. cuculus	U.S.A. (S)
1956	Cable	Donax denticulatus	C. caribbea XLII	Puerto Rico (S)
1956	Cable	Tellina lintea	C. caribbea XLII	Puerto Rico (S)
1958	Hopkins	Donax variabilis	B. loeschi	Mustang Is. (S)
1960	Ozaki	Caecella chinensis	P. caecellae	Japan (S)
1960	Ozaki	Gryphaea gigas	P. magakii	Japan (S)
1961	Angel	Velesunionis ambiguus	C. velesunionis	Australia (F)
1961	Holliman	Mulinia lateralis	C. apalachiensis	Florida (S)
1961	Laird	Ostrea belcheri	unidentified sp.	Pakistan (S)

Key to Table II

F	Fresh water species.
S	Marine species.
1	Considered as synonyms of B. polymorphus.page 18
2	Synonymous with B. cuculus
3	≡ B. haimeanus of Sinitzin, 1909.
4	Now R. papillosum
5	Synonymous with R. septpapillata
6	≡ Bucephalopsis haimeanus
7	Adult status not determined experimentally but based on morphological resemblances.
8	Adult status determined experimentally.

Discussion of Cercaria

All previous descriptions or reports of bucephalid cercariae known to the author are listed in Table II. Of the descriptions of marine species, only those given by Levinsen (1881), Haswell (1903), Ozaki and Ishibashi (1934), Cole (1935), Chubrik (1952), Hopkins (1954), Cable (1956), Hopkins (1958) and Holliman (1961) are adequate for close comparisons with the present species. Bucephalus crux, B. mytili and the cercaria of Prosorhynchus squamatus described by Levinsen, Cole and Chubrik respectively have a posterior sucker-like extension of the tail stem and are thus quite distinct from the present species; B. margaritae Ozaki and Ishibashi differs in details of spination, the absence of an axial strand and has "dermal gland cells" rather than cystogenous granules distributed through the body; B. sp. Haswell differs in the shape of the intestine and has two slender processes at the anterior end of the body; B. cuculus McCrady, as described by Hopkins (1954), has a flame cell formula of 2 [(2 + 2 + 2) + (2 + 2 + 2)] = 24; the behaviour, spination and granulation, number of lips surrounding the opening of the cystogenous organ pore, and shape and structure of the tail stem of Cercaria caribbea XLII Cable and C. apalachiensis Holliman differ from the present species; B. loeschi Hopkins has a shorter excretory bladder and a smaller intestine.

As stated elsewhere (p. 2) and as can be seen from Table II, many authors have placed cercariae in adult genera without any experimental evidence to substantiate their views. It is therefore recommended that all bucephalid cercariae described as species of Bucephalus or Prosorhynchus without experimental proof be transferred to the group name Cercaria to agree with the usual practice regarding cercariae whose adult status is unsure. Furthermore, as shown by Hopkins (1954), Bucephalopsis can only be used for the cercaria Bucephalopsis haimeanus (Lacaze-Duthiers, 1854). Thus Bucephalopsis modiolae Faust, 1928, must now become C. modiolae (Faust, 1928).

The probable error of many authors in synonymising the cercariae they have found with Bucephalopsis haimeanus has already been referred to (p. 2). This practice is a reflection on the inadequate descriptions that have been given and it is obvious that the majority of different cercariae need re-describing to rectify this situation.

Number of Cercariae Liberated from Infected Oysters

Data concerning the number of cercariae liberated from 11 infected oysters are given in Table III, which covers a 53-day period during June and July, 1964. There was a general tendency for peak liberations of more than 1,000 cercariae to be followed by lulls with few or no cercariae liberated. The duration of peaks and lulls in liberation varied considerably in the individual oysters under observation. A peak was maintained for 11 days in oyster 4 (June 10–20), while peak liberation lasted for only 24 hours in all oysters on various occasions. Peaks were generally, however, of the order of 1.5 days in duration. A lull persisted for 13 days in oyster 8 (July 3–15) but only for 24 hours in many instances. Lulls were generally more variable in duration than peaks.

Table III.—Numbers of Gercariae Liberated from 11 Infected Oysters over a 53-day period During June and July, 1964.

The maximum number of cercariae liberated from one oyster over a 24-hour period was 10,000, and this occurred on one occasion in oysters 2 and 11, and on two occasions in oysters 4 and 10.

Towards the end of the observation period there was a marked tendency for peaks in liberation to become less frequent. This might suggest that the infection was abating but this has been discounted elsewhere (Howell, 1966, in press). More probably, only one change of water per day for each oyster is insufficient to provide the necessary food requirements to maintain both oyster and parasite and this is reflected in fewer cercariae produced and liberated. It is difficult to adjudge from the results obtained any effect of temperature (which fluctuated between 10–15°C) on the production and liberation of cercariae. However, despite these points, the net results suggest that liberation of cercariae is neither cyclic nor continuous but essentially an intermittent phenomenon.

It should be noted that it was possible to determine the state of the infection in individual oysters when they were opened after completing the above observations. As expected, the number of cercariae liberated from a given oyster could be correlated with the state of the infection. Oysters 3, 6, 7, 8 and 9 proved to be relatively lightly infected; oysters 1 and 11 moderately heavily infected; and oysters 2, 4, 5 and 10 heavily infected.

Movement and Behaviour of Cercariae

Examination of liberated cercariae in an undisturbed finger bowl shows that they are located in greatest numbers opposite the exhalent chamber. The cercariae are generally in a resting position with the furcae contracted, encircling the body, and almost meeting in front of the anterior extremities (Text-fig. 6, B).

The furcae rapidly expand and tend to coil on stimulation by light (Text-fig. 6, F). If the water is then agitated any one of the positions illustrated in Text-figs. 6, A, C or E is assumed with the body suspended vertically and the furcae streaming above. The angle between the furcae is acute when the cercaria is nearer the bottom of the bowl, but obtuse nearer the surface. If strong currents are set up in the water the furcae bend with the current (Text-fig. 6, A). The sudden flexure and coiling of one furca of a suspended cercaria results in horizontal movement in calm water (Text-fig. 6, D). A cercaria may remain suspended for at least one hour in calm water, but after this time slowly sinks to the bottom of the bowl and coils the furcae which are eventually fully contracted to revert to the resting position (Text-fig. 6, B).

Light tapping of a finger bowl containing cercariae with the furcae expanded causes immediate and complete contraction of the furcae. The furcae normally revert to their expanded state when the tapping ceases. A similar reaction to mechanical shock has been described by Woodhead (1930) for the cercaria Bucephalus elegans. By comparison with the present species, however, B. elegans requires about three minutes to recover from the shock. Dawes (1946) does not describe the reaction of B. polymorphus to mechanical shock but he does describe the position of the furcae in a resting cercaria which is identical with that of the present species. Both Woodhead and Dawes stated that B. elegans and B. polymorphus respectively, are able to swim by alternate contractions and expansions of the furcae, but this was not observed in the present species.

Hopkins (1954) and Holliman (1961) stated that true swimming movements of Cercaria cuculus and C. apalachiensis do not occur and the cercariae in each case rely on turbulence in the water to remain suspended. With mechanical shock, the furcae of the latter species violently contract to about twice body length and are temporarily orientated at about 60° to the body in a rod-like manner. The resultant appearance of this cercaria can be contrasted with that described above page 21

Text-fig. 6.—Bucephalus longicornutus. Behaviour of the cercaria: Fig. A, suspended cercaria (semi-diagrammatic) in a strong current; Fig. B, resting position or position induced by light mechanical shock; Fig. C, suspended cercaria some distance below the surface of the water; Fig. D, suspended cercaria undergoing lateral movement by flexure of one of the furcae; Fig. E, suspended cercaria near the surface of the water; Fig. F, relaxed position assumed with the furcae expanded and partly coiled. For abbreviations see p. 9.

page 22 for the present species, as well as B. polymorphus and B. elegans. It is suggested that this contrast may reflect a generic distinction between C. apalachiensis and the three Bucephalus cercariae. In fact, behaviour of the furcae may reflect the generic identity of a given cercaria.

Body movements, independent of furcae movements, take place. From a contracted state a peristaltic wave commences at the anterior extremity of the body. When this has reached about one-third body length from the anterior extremity, a similar wave commences at the posterior extremity. When these two waves meet at approximately mouth level, the body of the cercaria is fully expanded. This is followed by overall contraction of the body to revert to the initial position.

Attachment and Penetration of Cercariae

The attachment of a cercaria to a fish commences with contact of any portion of the furcae with the body surface of the fish, particularly the fins (Text-fig. 7, A). The furcae adhere at the point of contact and as the body of the cercaria drifts in the current the tail stem is eventually brought into contact with the skin (Text-fig. 7, B). The body of the cercaria then elongates, and bends into a U-shape so that its anterior extremity touches the skin (Text-fig. 7, C). The anterior half of the body revolves and thrusts and, possibly with the aid of acidic and enzymic secretions from the cystogenous organ, erodes a point of penetration. Once the anterior end is firmly attached the tail stem parts from the body, and the body straightens out to lie almost at right angles to the skin (Text-fig. 7, D). The tail stem and furcae eventually dislodge from the skin and disintegrate. After a series of revolving and thrusting movements, the body of the cercaria completely penetrates the skin, settles under the malpighian layer or in muscle tissue, and encysts (Text-fig. 7, E). Attachment, penetration and encystment are accomplished within one half to one hour and fish do not appear to be irritated by the process. By comparison, the time taken for attachment, penetration and encystment by B. elegans is a few minutes (Woodhead, 1930) and by Rhipidocotyle septpapillata, is 24 hours (Kniskern, 1952).

Cysts are most frequently found near the base of the fin web and in the extrinsic muscles of the fin of experimental hosts (Text-fig. 8, A). They are only occasionally found under scales, under the skin of the head and branchial chamber, in the conjunctiva of the eye and in the deeper muscle layers of the body wall. No cysts were found in the coelom or gut of experimental hosts.

The method of attachment and entry into fish in this species is unlike that described for B. elegans by Woodhead (1930) or R. septpapillata by Kniskern (1952). In both of these species the furcae play a particularly important part in the process since they force the body of the cercaria, by alternate expansions and contractions, into the body of the host.

Free-swimming cercariae remain alive and active for 36 to 38 hours at 13.0°C. However, the tail stem and furcae begin to degenerate after this time and this would appear to make attachment to host fish improbable. Some cercariae appear to lose the tail stem and furcae before this time. Attachment in these cases would also appear to be improbable. By comparison, Kniskern (1952) stated that unless attachment and encystment of the cercariae of R. septpapillata was achieved within a few hours, the cercariae died.

Encystment of Cercariae

Observations were made to determine the effect of successive dilutions of sea-water on cercariae. There appeared to be no visible effects until the sea-water was diluted 50% by distilled water. At this dilution, the body of the cercaria expanded slightly, and within five minutes, individual cystogenous granules were page 23

Text-fig. 7.—Bucephalus longicornutus. Attachment and method of entry of the cercaria, into the fish intermediate host (semi-diagrammatic): Fig. A, adherence of furcae; Fig. B, attachment of tail-stem; Fig. G, U-shape assumed by body as the enterior end is brought into, contact with the skin of host; Fig. D, penetration of the anterior end of the cercaria and dislodgement of the tail stem and furcae from the body; Fig. E, complete penetration and encystment. For abbreviations see p. 9.

page 24 broken down into a large number of minute, vibrating particles. These particles were compacted together in the shapes of the individual cystogenous granules from which they were derived. Approximately one minute later, the particles were exuded through the cuticle and enveloped the body of the cercaria as a mucus secretion. Simultaneously, the tail stem and furcae become detached from the body.

With still further dilutions, the same effect was observed but it occurred more rapidly. After 100cc of distilled water had been added a fluid concentration corresponding approximately to that of fish body fluids was reached. The formation of the enveloping secretion around the body of the cercaria then occurred after approximately two minutes.

Precisely the same sequence as that already described was observed in cercariae treated with the body fluids from Tripterygion sp., when encystment took place in approximately three minutes.

These experiments strongly suggest that encystment of the cercaria is a phenomenon controlled osmotically. It is assumed that the body fluids of the cercaria are more or less isotonic with sea-water whereas those of the fish are hypotonic. Hence, when a cercaria penetrates the host, osmosis occurs which results in water uptake by the body of the cercaria. This appears to initiate the breakdown of the cystogenous granules into small particles which are eventually exuded through the cuticle to restore osmotic equilibrium and form the initial cyst wall.

Results of Infection Experiments with Cercariae

Forty-three of forty-five specimens of Tripterygion sp., ranging in size from 30mm to 50mm total length, and 30 of 40 specimens of Acanthoclinus quadridactylus (Forster), ranging in size from 30mm to 60mm total length, were successfully infected with metacercariae. Those specimens of each of these species which were not infected were 20mm to 25mm greater in total length than the largest specimens successfully infected.

Attachment of cercariae to 143 specimens of Helcogramma medium (Gunther) was observed and encystment took place in 111. However, the metacercariae did not develop. None were found alive in 47 specimens 20 days after exposure to cercariae, and cysts had decreased in size and their contents had degenerated in 25 specimens after 35 days. Subsequently, cysts underwent a further decrease in size and it was assumed that they were in the process of being resorbed. The range in size of specimens from which cysts were recovered was 25mm to 50mm. Seven specimens greater than 60mm in total length, were found to be uninfected five days after exposure to cercariae. The remaining 25 specimens which were found to be uninfected fell within the size range of those in which encystment took place, but they were examined 60 days after exposure to cercariae. In these cases, complete resorption of the cysts was assumed to have taken place.

Attachment of cercariae to two specimens of Trachelochismus sp., both 40mm total length, was observed but metacercariae were not recovered from either specimen six days after exposure to cercariae.

Metacercariae were not recovered from any of the invertebrate species (see p. 5 although attachment to each took place. With these experimental hosts, examination of the soft parts was made three days after exposure to cercariae.

A total of 12 fish died at various intervals after exposure to cercariae but there was no evidence to suggest that this was due to the infection. In fact, fewer cysts were recovered from those that died than from the majority of those that remained page 25 alive. Kniskern (1952) considered that a heavy infection with the metacercariae of Rhipidocotyle septpapillata resulted in death of the host fish.

Three main conclusions can be drawn from the results of these experiments. First, the cercariae show some measure of host specificity in that encystment did not occur in Trachelochismus sp., and the metacercariae show some measure of specificity in that development did not proceed beyond 20 days in H. medium. Secondly, penetration and encystment did not take place in specimens of H. medium, A. quadridactylus and Tripterygion sp. greater than 50mm, 60mm and 50mm total length respectively. This suggests age immunity of the experimental hosts in these cases. Thirdly, invertebrate species are unlikely to be natural hosts of the metacercariae.

A. quadridactylus proved to be more susceptible to infection than Tripterygion sp. Specimens of the former always yielded more cysts than the latter after both had been exposed to similar numbers of cercariae. The maximum number of cysts recovered from a specimen of A. quadridactylus was approximately 325; from Tripterygion sp., approximately 250.