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A Contribution to the Life History of Bucephalus longicornutus (Manter, 1954)

The Metacercaria

The Metacercaria

Development of the Metacercaria

Immediately following encystment all movements of the metacercaria cease and this quiescent phase persists for approximately 40 days. Initially, the cyst is ovoid and its wall is granular and diffuse. After 24 hours the cyst is approximately 200μ long by 85μ to 90μ wide. The metacercaria, which completely fills the cyst, is densely granular except for the distinct, translucent, fluid-filled intestine which occupies the central regions of the body, and the region of the cystogenous organ near the anterior end. This characteristic appearance is retained for about 26 days (Text-fig. 8, B).

Minor changes in the structure of the metacercaria take place during this interval. After five days the cuticular armature of the cercaria becomes patchy and is replaced by fine longitudinal and transverse striations. The metacercaria is completely striated after 15 days, and minute spines are developed at alternate intersections of the striations after 20 days (Text-fig. 8, E). Cysts do not alter appreciably in size during this interval but some growth of the metacercaria takes place. Twenty-day-old excysted metacercariae are 230μ long by 55μ to 60μ at their widest point (at approximately mid-body level).

After 28 to 30 days, the metacercaria is more translucent, and the excretory vesicle which now contains a few, small, spherical granules, is conspicuous. The cyst increases to approximately 210μ long by 130μ wide and an excysted metacercaria is 320μ long by 65μ wide. The granular and diffuse cyst wall of earlier stages is now more or less transparent, membranous and elastic, and a small amount of host tissue adheres to the cyst wall on removal from surrounding tissue.

Appreciable flexing and twitching movements of the metacercaria commence after approximately 40 days development. The intestine and excretory vesicle are almost completely filled with granules which give them an opaque appearance. The excretory vesicle is more extensive than in the cercaria, reaching to the anterior level of the intestine. The cyst is 235μ to 250μ long by 150μ to 185μ wide at this stage and retains this size (apart from a few exceptions mentioned below) during subsequent development. Cyst walls are thicker by 5μ in cysts recovered from muscle tissue than in those from the fin web.

After approximately 50 days, both the anterior sucker and cuticular spination are well developed. Activity of the metacercaria, which at this stage consists of page 26 rotating movements within the cyst, gradually increases and reaches a peak after 75 to 80 days.

The maximum size of the metacercaria is attained after approximately 80 days development, irrespective of the experimental host in which it develops. Expanded metacercariae range from 700μ to 715μ long by 80μ. to 90μ. wide; contracted, from 270μ to 275μ long by 125μ to 130μ wide. The reproductive system does not undergo any further development beyond that observed in specimens of this age.

A thin band of brown pigment is laid down on the inner surface of the cyst wall of many cysts recovered from Tripterygion sp. after 85 to 90 days. Metacercariae are still alive within these cysts but their activity is greatly diminished by comparison with 80-day-old specimens. After 95 to 100 days, metacercariae within these pigmented cysts are either dead or very inactive. Of the cysts that remain unpigmented, many contain granular masses resulting from the degeneration of the enclosed metacercariae, and they are smaller than normal cysts. The remainder contain normal, active metacercariae which, in addition to spherical granules, contain needle-shaped crystals, 10μ long, in the intestine.

After 110 days development, all metacercariae in either pigmented or unpigmented cysts recovered from Tripterygion sp. are either dead or degenerate. Dead metacercariae in pigmented cysts are themselves pigmented brown and their major morphological features are still recognisable.

Cysts of up to 120 days of age recovered from Acanthoclinus quadridactylus do not become pigmented but some metacercariae degenerate into a granular mass within the cyst after this time, and the activity of many others is diminished.

Cysts from Tripterygion sp. and A. quadridactylus, older than 110 days and 120 days development respectively, have not been examined.

Two, three or four metacercariae are occasionally confined within the same cyst (Text-fig. 8, J). These cysts, which are larger than normal cysts containing one metacercaria, were recovered from both experimental hosts.

Between two and eight normal cysts are frequently recovered from A. quadridactylus, and these are joined to one another in a chain-like fashion (Text-fig. 8, I).

Occasionally, groups of three or four larger cysts, embedded in an opaque, granular and fibrous matrix, presumably of host origin, are recovered from A. quadridactylus (Text-fig. 8, K). Apart from this phenomenon, tissue reactions induced by large numbers of cysts in both experimental hosts are slight.

Spontaneous excystment of 75- to 95-day-old metacercariae takes place from approximately 10% of the cysts recovered from A. quadridactylus and only occasionally in cysts recovered from Tripterygion sp.

Kniskern (1952) found that the metacercariae of Rhipidocotyle septpapillata reached maximum development after 12 days. By comparison, the time taken for maximum development in the present species is prolonged since it is not reached until after 75 to 80 days. Kniskern found that the contents of the cysts degenerated into a "cheesy mass" after 84 days in R. septpapillata. The appearance of these cysts may be comparable with those of the present species which contain degenerate metacercariae. Pigmentation of cysts has not been described for other bucephalid metacercariae.

Kniskern also reported that spontaneous excystment of all mature metacercariae of R. septpapillata took place; this is in striking contrast to the 10% noted in the present species obtained from A. quadridactylus.

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Schurmans-Stekhoven (1934) for Bucephalus polymorphus and Kniskern (1952) for Rhipidocotyle septpapillata have both noted that cysts may contain two or occasionally more metacercariae. With the same phenomenon recorded for the present species, it may be a characteristic feature of metacercariae of this family.

Woodhead (1929, 1930) has given no details of development of the metacercariae of either R. papillosum or B. elegans.

The fact that metacercariae remain alive longer in A. quadridactylus than in Tripterygion sp. suggests that the former may be a natural host or may be more akin to the natural host or hosts of the metacercariae than the latter.

The Mature Metacercaria
(Text-fig. 8, A-L.)

The natural fish host or hosts of the metacercariae were not discovered during the course of this study. In general, attempts were made to obtain and examine fish that fell within the size range of those successfully infected experimentally. Three attempts were made to obtain likely fish hosts from Balaena Bay, Wellington Harbour, where infected oysters are known to occur, but no fish were recovered from the fish traps used. Ten specimens of Tripterygion sp., collected with a dip net from the same locality, were not infected with metacercariae of the present species.

Small fish are not common in oyster "cultch" from oyster dredges working in Foveaux Strait. In one day spent dredging on the vessel Kumea, only 11 specimens of Trachelochismus sp. and four specimens of Acanthoclinus trilineatus (Forster) were recovered by the author but none of these proved to be infected. A day spent trawling over the oyster beds in Foveaux Strait yielded, in addition to the larger fish species listed elsewhere (p. 33), 30 small fish comprising 25 specimens of Trachelochismus sp., three specimens of Tripterygion sp. and two specimens of A. trilineatus. None of these specimens proved to be infected.

Experimentally infected specimens of A. quadridactylus and Tripterygion sp. provided the only source of metacercariae for study. For reasons stated elsewhere (p. 26), the following description is based on 80-day-old and older specimens.

Description of Metacercaria
(Based on 40 living and five fixed and stained excysted specimens).

Metacercariae are small, active organisms possessing a prominent, mobile, anterior sucker. Each is more or less translucent except for a prominent, opaque gut, excretory bladder and small, variously sized, refractile granules scattered throughout body (Text-fig. 8, C). Body is bluntly ovoid with a truncate anterior margin when fully contracted and elongated, rounded anteriorly and tapering to a blunt point posteriorly when expanded. Total length contracted 270μ to 275μ, greatest width, 125μ to 130μ, at approximately mid-body level. Total length expanded, 700μ to 715μ, greatest width, 80μ to 90μ, near anterior margin. Complete contractions of body not common. Between successive expansions, a semi-contracted state is assumed by contraction of anterior half of body (Text-fig. 8, L).

Body is covered by a thin cuticle, 3μ to 4μ thick. Small, diagonally arranged spines are embedded in cuticle except within concavity of anterior sucker. Spines are not as prominent posterior to level of pharynx as over anterior region of body.

A distinctive, mobile sucker is situated at anterior end of body. Its shape is variable, depending on its degree of contraction. Sucker is truncate anteriorly, rounded posteriorly, when contracted, with a deep anteroventral concavity shaped page 28
Text-fig. 8.—Bucephalus longicornutus. Morphology and anatomy of the metacercaria: Fig. A, lateral view of the caudal fin of Tripterygion sp. showing the location of cysts; Fig. B, ventral view of encysted, 24-hour-old, living specimen; Fig. C, ventral view of excysted, 85-day-old, living specimen; Fig. D, dorsal view of excysted, 91-day-old, fixed and stained specimen; Fig. E, diagrammatic representation of cuticular spines showing how diagonal arrangement is achieved; Fig. F, right lateral view of the contracted sucker of a 91-day-old, fixed and stained specimen; Fig. G, ventral view of the expanded sucker of a 91-day-old, living specimen; Fig. H, ventral view of the contracted sucker of a 91-day-old, fixed and stained specimen; Fig. I, aggregation of four, 38-day-old metacercarial cysts from experimentally infected Acanthoclinus quadridactylus; Fig. J, multiple cyst from A. quadridactylus; Fig. K, three larger cysts embedded in host tissue, recovered from A. quadridactylus; Fig. L, body movements of the metacercaria. For abbreviations see p. 9.

Text-fig. 8.—Bucephalus longicornutus. Morphology and anatomy of the metacercaria: Fig. A, lateral view of the caudal fin of Tripterygion sp. showing the location of cysts; Fig. B, ventral view of encysted, 24-hour-old, living specimen; Fig. C, ventral view of excysted, 85-day-old, living specimen; Fig. D, dorsal view of excysted, 91-day-old, fixed and stained specimen; Fig. E, diagrammatic representation of cuticular spines showing how diagonal arrangement is achieved; Fig. F, right lateral view of the contracted sucker of a 91-day-old, fixed and stained specimen; Fig. G, ventral view of the expanded sucker of a 91-day-old, living specimen; Fig. H, ventral view of the contracted sucker of a 91-day-old, fixed and stained specimen; Fig. I, aggregation of four, 38-day-old metacercarial cysts from experimentally infected Acanthoclinus quadridactylus; Fig. J, multiple cyst from A. quadridactylus; Fig. K, three larger cysts embedded in host tissue, recovered from A. quadridactylus; Fig. L, body movements of the metacercaria. For abbreviations see p. 9.

page 29 like a quarter-segment of a sphere (Text-fig. 8, C), and is 65μ to 70μ?wide at its anterior margin (which is slightly lobed laterally) and 50μ to 55μ from anterior margin to posterior limit. Musculature of sucker is essentially scoop-shaped with open and directed anteriorly. Thin flap of cuticle at anterior end of body closes off open end of musculature and marks anterior border of sucker concavity. In living specimens, this flap of cuticle obscures muscle fibres along anterodorsal margin of sucker. Band of radial muscles, approximately 10μ?wide, prominent around posterior, semi-circular margin of sucker concavity.

When sucker is expanded, musculature is more elongated. Sucker is 40μ to 45μ wide, approximately 60μ to 65μ long, and its concavity is relatively inconspicuous (Text-fig. 8, G). Anterodorsal muscle fibres of musculature are now relatively conspicuous, and consist of several oblique groups separated by more or less triangular areas devoid of muscle fibres (Text-fig. 8, G). These groups of fibres can be traced some distance posteriorly into dorsal musculature of sucker in fixed and stained specimens only (Text-fig. 8, D). Five areas devoid of muscle fibres are seen in ventral view; three are dorsal, two ventral (Text-fig. 8, H). Thin flap of cuticle which closes off open end of musculature when sucker contracted, is now projected anterior to musculature as a semi-circular lobe which gives anterior end a rounded appearance (Text-fig. 8, G). However, anterior end of body proper is distinctly demarcated, and flap of cuticle projects beyond this.

Floor of the sucker concavity is vesiculate, and in ventral view three, narrow, longitudinal ducts, one median and one on either side of it, can be observed in floor (Text-fig. 8, H). These ducts are continuous with non-muscular areas between groups of oblique muscle fibres along the anterodorsal margin of the sucker and are designated tentacular ducts.

Sucker musculature is essentially C-shaped in lateral view (Text-fig. 8, F). Anterior portion thickened, consisting of dorsal and ventral groups of oblique fibres which run towards dorsal and ventral surfaces of the musculature respectively from anterior margin of sucker. Posterior arm of C consists of radial muscles. Triangular area, devoid of muscles, between these three groups of fibres. Five non-muscular areas between groups of muscle fibres seen in ventral view, combined with each lateral area make a total of seven. Tentacles are not developed in 120-day-old metacercariae.

An undetermined number of gland cells are situated near anterior end of body, and comprise a conical-shaped mass dorsal to posterior half of sucker musculature, and a few scattered irregularly anterior to this mass (Text-fig. 8, G). A number of narrow, parallel ducts run anteriorly from gland cells and open at anterodorsal margin of body by several pores.

Mouth is posterior to mid-body level, varying between 75μ and 120μ from posterior extremity depending on degree of contraction of body. Pharynx 28μ to 30μ long by to 25μ?wide; oesophagus indistinct, approximately 35μ to 40μ long; intestine ovoidal and directed posteriorly. Intestine almost completely filled with spherical granules (approximately 3μ in diameter) in life but in mounted specimens these granules are inconspicuous and cavity of intestine has a vacuolate appearance (Text-fig. 8, D).

Reproductive system situated in hind-body posterior to pharynx (Text-fig. 8, D). Ovary and testes on right side of body and together, form three ovoid to spherical masses of cells lying slightly obliquely to longitudinal body axis. Most anterior mass, at approximately mouth level, is ovary which is 10μ to 12μ in diameter. It overlaps the anterior testis dorsally. Posterior testis overlaps the anterior testis dorsally. Testes are 15μ to 18μ in diameter.

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Cirrus pouch is on left side of body, 60μ long and 15μ wide at its anterior end, tapering to a blunt point posteriorly; extending from 12μ to 15μ behind posterior testis to level of genital pore which varies between 18μ to 25μ from posterior extremity. Mass of cells comprising cirrus pouch not differentiated into seminal vesicle and pars prostatica and genital atrium not developed.

Gap between cirrus pouch and posterior testis occupied by an irregularly-shaped mass of cells which merges with, and is similar in appearance to, cells of the cirrus pouch. Significance of these cells not clear. No evidence of gametogenesis, and vitelline follicles, uterine coils and Mehlis's gland not observed.

Excretory pore situated at posterior extremity. Excretory bladder long, running to left of intestine and terminating approximately 20μ to 30μ anterior to intestine. Small spherical granules almost completely fill bladder in life but most of these disappear after fixation. Flame cell formula as for cercaria.

Discussion of Metacercaria

Several differences between the anatomy of the metacercaria and cercaria are evident. These include for the metacercaria the possession of the sucker and gland cells in place of the cystogenous organ and four lips at the anterior end of the body; a more elongate excretory bladder; granulation of the excretory bladder and intestine; spination, and some further differentiation of the reproductive system. The relationship between the cystogenous organ and four lips at the anterior end of the body of the cercaria and the sucker and gland cells of the metacercaria was not determined. Accordingly, it is not known whether the sucker is a completely new development or develops by modifications of pre-existing structures at the anterior end of the body.

Although tentacles are not developed in the metacercariae examined, the present species can be confidently assigned to the genus Bucephalus even though the flap of cuticle at the anterior extremity is suggestive of the "hood" of Rhipidocotyle. The ducts observed in the sucker musculature and their confluence with gaps between the groups of muscle fibres along the anterior margin of the sucker correspond in position with the three most dorsal tentacular ducts of adult specimens of Bucephalus longicornutus (Manter, 1954) and other species of Bucephalus. Furthermore, the nature of the sucker musculature is typical of many species of Bucephalus (vide Ziegler, 1883; Manter, 1940). In Rhipidocotyle, however, tentacular ducts are not present, and the musculature of the sucker differs from Bucephalus species (vide Nagaty, 1937).

Two other Bucephalus metacercariae are known. The metacercaria of B. elegans, figured but not described by Woodhead (1930), is insufficiently characterised for it to be closely compared with the present species. The metacercaria of the present species shows some resemblance to the metacercaria of B. polymorphus, described by Ziegler (1883) and Schurmans-Stekhoven (1934), with regard to the shape of the sucker and position of the gland cells, shape of the cyst, and granulation of the intestine and excretory vesicle. However, the cirrus sac is not as fully differentiated in the present species as indicated by Ziegler for B. polymorphus. It is not clear from Schurmans-Stekhoven's description or figures whether the longitudinal ridges in the sucker concavity of B. polymorphus are comparable with the tentacular ducts of the present species.

Results of Feeding Experiments with Metacercariae

A total of 14 fish belonging to eight different species were fed on one or more occasions with specimens of Tripterygion sp. and Acanthoclinus quadridactylus that had been experimentally infected with metacercariae.

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Table IV.—Feeding Experiments.

Table IV.—Feeding Experiments.

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The experimental hosts were examined between five days and six weeks after feeding for the presence of juvenile or adult worms. Two gravid specimens of Bucephalus longicornutus were recovered from the intestine of one specimen of Scorpaena cardinalis Richardson, the scarpee or red rock cod. The posterior half of a bucephalid, considered to be B. longicornutus by the nature of the structure of the cirrus sac and genital atrium, was recovered from another specimen of S. cardinalis. This latter specimen was damaged during examination of the host's intestinal contents and despite a careful examination, its anterior half could not be found. Details of all experiments conducted are summarised in Table IV.

It was stated elsewhere (p. 26) that metacercariae do not reach maximum development until after approximately 80 days. Accordingly, the feeding of sub-mature metacercariae of only 25 to 75 days development in the above experiments might be questioned assuming that metacercariae are not infective until they have reached maximum development. The reason for feeding with sub-mature metacercariae was that some experiments were conducted before a satisfactory indication was obtained of when maximum development of the metacercariae was reached. Furthermore, the feeding of metacercariae of different ages to the one experimental host in some experiments might be questioned. Again, without the necessary knowledge of when metacercariae reached maximum development it was hoped to establish an infection and establish the age limits within which the metacercariae were infective. This was not successful. Only two experiments were performed, with host species 3 and 14, in which the metacercariae used were known beforehand to have reached maximum development.

It is of interest to note that successful results were obtained only with Scorpaena cardinalis and that this species (apart from Trachelochismus sp. which, however, was fed sub-mature metacercariae) was the only species used that was free from intestinal parasites other than the specimens of Bucephalus longicornutus. Whether there is any correlation between the successful experimental establishment of the bucephalid and the absence of other intestinal parasites can, as yet, only be conjectured.

It should be pointed out that S. plumieri from Florida which is related to S. cardinalis is infected with Bucephalus scorpaenae Manter, 1940. Therefore, it is possible that S. cardinalis could harbour a natural Bucephalus infection which would invalidate the experimental results. However, Manter (1954) examined nine specimens of S. cardinalis from Wellington and Portobello, and the author has, during the course of this study, examined 15 from Island Bay, Wellington, and no natural bucephalid infections have yet been found in this species. This supports the view accepted here, that the specimens of B. longicornutus recovered from the intestine of S. cardinalis were established experimentally.