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copyright 1966, by Victoria University of Wellington
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Modified from part of a thesis submitted in partial fulfilment for the degree of M.Sc. in Zoology at Victoria University,
Present address: Department of Zoology, Australian National University, Canberra.
The sporocyst, cercaria, metacercaria and adult of Bucephalus longicornutus (Manter, Alcicornis longicornutus Manter,
Sporocysts were recovered from the visceral mass, gills and pericardium of the New Zealand mud-oyster, Ostrea lutaria Hutton, Tripterygion sp. and Acanthoclinus quadridactylus (Forster). Adult flukes were recovered from the intestine of Scorpaena cardinalis Richardson, 35 days after feeding mature metacercariae, and from the intestine and pyloric caeca of Kathetostoma giganteum Haast, the definitive host.
All previous reports of bucephalid cercariae are listed. Reference is made to some cercariae which have been incorrectly assigned to adult genera. Possible taxonomic implications arising from variations found in adult fluke characters are discussed.
Bucephalus longicornutus (Manter, Ostrea lutaria Hutton,
Members of the family Bucephalidae, commonly referred to as bucephalids or gasterostomes, constitute an aberrant group of digenetic trematodes which, in their adult stage, infect the pyloric caeca, intestine, and more rarely the stomach and body cavity of both marine and fresh-water teleosts. The diagnostic feature of the family is that the mouth is on the mid-ventral surface and leads into a saccular intestine. Other morphological features are essentially the same as for the prosostome digenea (vide Dawes,
The presence of the ventral mouth and sac-like gut have served to link the family Bucephalidae with the Turbellaria, and the family is considered by some authors (e.g., Yamaguti,
The life history of members of the group is complex, involving three parasitic and two free-living phases, and has been determined for three fresh-water species only. A similar life history is postulated for marine species on the basis of work by Tennent (
The systematics of the family is based entirely on adult morphology. To date, some 140 adult species distributed among 12 genera are known. Hopkins (
The general uniformity of internal structure found among the adult members of the Bucephalidae has necessitated the use of minor characters to separate species. Judging by the variability of egg size and the relative positions of internal organs exhibited by some species, the validity of established species using such characters is questionable.
Yamaguti (
To date, 23 bucephalid cercariae have been described. Of these, the adult status of three fresh-water North American species has been determined experimentally and that of one fresh-water European species has also been established on the basis of a solitary recorded species in Europe. The remainder cannot be assigned their adult status under the present system of classification since adult characters are not exhibited by cercariae. Nevertheless, many authors have placed cercariae in adult genera without experimental evidence to substantiate their views.
Many bucephalid cercariae are inadequately described for comparative purposes. Bucephalopsis haimeanus, for example, has been reported from 14 different species of bivalve molluscs. Regarding this, Hopkins (
Hitherto, bucephalid sporocysts have been recovered from eight commercially exploited species of marine bivalve molluscs, and it is perhaps surprising to find that the life histories of all of these are unknown although they may at times affect seriously the host stock. It is hoped, therefore, that the present study will stimulate further life history work on this important group of trematodes.
Von Siebold (Bucephalus polymorphus von Baer, B. fimbriatus (von Siebold, Gasterostomum fimbriatum), from European fresh-water predaceous fishes, on the basis of the ventral mouth and sac-like intestine in each case.
Wagener (B. polymorphus. He pointed out that the sporocysts occurred in fresh-water bivalves; cercariae were formed in the sporocyst and when mature were liberated and became free-swimming; encysted, immature bucephalids occurred on the gills of small cyprinid fishes and B. fimbriatus occurred in fresh-water perches. Ziegler (B. fimbriatus was a synonym of B. polymorphus.
Only one species of bucephalid is recognised at present from European freshwater fishes and, therefore, there is no reason to doubt the validity of the suggested life history of B. polymorphus. In fact, Poche (B. fimbriatus a synonym of B. polymorphus.
Rudolphi (B. polymorphus.
Tennent (B. cuculus McCrady, Crassostrea virginica (Gmelin). However, Tennent disregarded McCrady's name for the cercaria by calling it B. haimeanus Lacaze-Duthiers, B. haimeanus. The close similarity between the known bucephalid cercariae on the one hand, and adults on the other, led Tennent to believe that all forms were merely "physiological varieties" of the one species. It is clear from the systematic work of many earlier and subsequent authors on the group that this opinion held by Tennent is erroneous.
Hopkins (Menidia and adults in Strongylura and assumed they were the same species as the oyster cercariae. He never obtained experimental evidence of a connection between these forms. He did prove that encysted forms in Menidia would excyst, live and grow when fed to predaceous fishes but he never completed these experiments ... Tennent did prove that some unknown gasterostome in Lepisosteus was capable of infecting oysters and developing in oysters at least to the sporocyst stage." However, Tennent did not describe the adult from Lepisosteus, assuming it to be the same species as other bucephalid adults previously described.
Hopkins (Strongylura harbours three species of bucephalids whose excretory systems exclude any possibility that they might develop from the
B. cuculus. He also described Rhipidocotyle lepisostei from Lepisosteus spatula and on the basis of the excretory system and Tennent's work, states that this species could possibly develop from B. cuculus. However, no infection experiments have been made to confirm this probable relationship.
Levinsen (B. crux from the marine mussel Modiolaria discors. This cercaria was distinct from B. haimeanus in having a posterior, sucker-like extension of the tail-stem. Odhner (Prosorhynchus squamatum Odhner, Cottus scorpio by Levinsen but identified by this author as G. armatum Molin.
Cole (B. mytili (which resembles B. crux) and, although he was apparently unaware of Levinsen's B. crux and Odhner's suggested relationship between B. crux and P. squamatum, he considered that the identity of B. mytili "with a species of Prosorhynchus might yet be established."
Chubrik (P. squamatum. He followed Odhner by assuming that a cercaria of the B. crux-type developed into a species of Prosorhynchus.
There is no experimental evidence to substantiate any of these views. Hopkins (
Carrere (Atherina sp. from the Mediterranean Sea. These developed experimentally into a new species, Dolichoenterum lamirandi, in Labrax lupes and Hyla arborea. This, along with Tennent's observation that an unidentified bucephalid in Lepisosteus sp. could infect oysters and develop to the sporocyst stage, constitutes the only published experimental proof of a connection between the various stages in the postulated life history of marine bucephalids.
Yamaguti (Prosorhynchus uniporus Ozaki,
Three life histories of North American fresh-water bucephalids have been determined experimentally. They are Rhipidocotyle papillosum (Woodhead, B. elegans Woodhead, R. septpapillata Krull,
Live oysters for experimental purposes were obtained from Foveaux Strait between June and October,
Oysters showing infection were transferred to new bowls and those showing no infection had their water changed daily. If the oysters did not liberate cercariae
The term infection experiment, used throughout the text, denotes an experiment in which cercariae were used to infect second intermediate hosts to obtain metacercariae. Infection experiments were carried out with small specimens of Tripterygion sp., Acanthoclinus quadridactylus (Forster), Helcogramma medium (Gunther), and Trachelochismus sp. as experimental hosts. These fish were collected from Island Bay, Wellington, a locality where, because there are few bivalve molluscs, the chance of prior infection of these fish with bucephalid metacercariae was thought to be unlikely. Confirming this, examination of the fin webs of the fish for encysted metacercariae gave no evidence of infection. However, the fin webs of A. quadridactylus are not transparent and consequently no easy check on prior infection could be made.
One to three fish were transferred to each finger bowl containing cercariae on the basis of one fish to every 1,000 cercariae (see p. 6 for method of estimation). The water was stirred to disperse the cercariae. Attachment of cercariae was checked and a note made of the approximate numbers attached to various parts of the fish. The finger bowls were then stacked on shelves, aerated, and left undisturbed for 18 to 24 hours. After this period the water was changed daily and the fish fed on chopped mussel (checked as free from bucephalid sporocysts).
Four invertebrate species comprising a crab, Cyclograpsus lavauxi, a bivalve, Mytilus edulis, a coelenterate, Phlyctenactis retifera, and a polychaete, Nereis sp., were collected from rock pools at Island Bay, placed in finger bowls containing cercariae and treated in a similar manner to the experimental fish hosts in attempts to obtain metacercariae.
The term feeding experiment, as used here, denotes an attempt to obtain the adults of the metacercariae established experimentally in the small fish listed above by feeding these small fish to larger fish.
The larger fish selected as experimental hosts for feeding experiments were those in which bucephalid infections had not been reported by Manter (Helcogramma medium, Acanthoclinus quadridactylus, Tripterygion sp. and Trachelochismus sp. collected with a dip net from rock pools at Island Bay; Geniagnus monopterygius (Bloch and Schneider) and Pseudolabrus celidotus (Forster) collected by otter-trawl in Wellington Harbour; Scorpaena cardinalis Richardson and Pseudolabrus coccineus (Forster) caught on a hand line at Island Bay. These fish were kept in aquaria supplied with running sea-water from the open sea at Island Bay.
Helcogramma medium, Tripterygion sp. and Trachelochismus sp. were fed chopped up pieces of fish that had been experimentally infected with metacercariae. Geniagnus monopterygius, P. celidotus and P. coccineus would not feed naturally on live or recently killed infected fish and had to be force fed. Acanthoclinus quadridactylus and S. cardinalis readily took live infected fish placed in the aquaria.
Eggs were recovered from gravid adult specimens of Bucephalus longicornutus that had been established experimentally in Scorpaena cardinalis. The eggs were divided into two samples and placed in separate Syracuse watch glasses, one of which contained sea-water and the other, sea-water diluted by two and a-half times
(a) Experiments were made to determine the reaction of cercariae to increasingly diluted sea-water. Twenty 4in diameter finger bowls were set up, each containing approximately 100 cercariae in 40cc of sea-water. Five cc of distilled water were added to the first finger bowl, 10 to the second, 15 to the third, etc.
(b) The body wall muscles of a freshly killed Tripterygion sp. were ground up with a mortar and pestle. The few cc of liquor obtained were decanted from the residue into a Syracuse watch glass. Approximately 10 cercariae were added to this liquor with care to exclude as much sea-water as possible.
Estimations of the number of cercariae liberated from infected oysters were made during
Portions of sporocysts were teased out of the visceral mass of infected oysters and mounted in sea-water. 0.5% neutral red and 0.5% methyl green in sea-water were used as vital stains but these were of little value in clarifying details of structure as stain tended to concentrate in the sporocyst wall and embryonic cercariae. Portions of sporocysts were fixed in warm formol-acetic-alcohol (FAA), and stained in acetic-acid-alum-carmine (AAAC), cleared in xylol and mounted in balsam.
Histological details of the sporocyst were obtained by sectioning portions of the gills and visceral mass of infected oysters. The best fixative was found to be Dubosq-Brasil followed by treatment with Lenoir's fluid for picric acid removal (Gray,
Free-swimming cercariae were examined alive and 0.5% neutral red was occasionally used as an intra vital stain.
Some cercariae were fixed in FAA and stained in AAAC. A tangled cluster of approximately 50 cercariae was fixed in warm FAA and sectioned at 3 to 5μ. No care was taken to orientate the cercariae before embedding since adequate numbers ensured that at least some would be suitably orientated. Sections stained in Delafield's haematoxylin and alcoholic eosin gave the best results.
Experimentally infected small fish were used as a source of metacercariae for study. After killing the fish, the fins were severed from the body, the two halves
B. longicornutus.
Cysts were freed from host tissue and mounted in diluted sea-water. The metacercariae were readily released from cysts by applying light pressure to the coverslip. Metacercariae were occasionally dissected out of their cysts before being examined, and a few mature metacercariae were found to excyst spontaneously in diluted sea-water.
Permanent mounts of metacercariae were made after fixing under slight pressure in warm FAA and staining in AAAC.
The technique suggested by Manter (Kathetostoma giganteum, from Foveaux Strait were fixed in formalin before being sent to Wellington and those from Cook Strait were delayed in transit. As a result, of the 75 adult specimens of B. longicornutus obtained from this material only 24 were suitable for making whole mounts. These were washed and flattened under slight pressure of a coverslip. Warm FAA was drawn under the coverslip and after approximately five minutes, the slide, still covered by the coverslip, was immersed in a dish of 70% iso-propyl alcohol and left for 18 to 24 hours. This usually ensured that specimens remained flat during staining. Flattened specimens were stained with AAAC using standard procedures. Three adult worms were sectioned, and stained in Delafield's haematoxylin and alcoholic eosin.
Living material examined was limited to the few adults recovered from successful feeding experiments.
A sporocyst is milky-white, elongate, rounded, hollow, branching, and swollen at irregular intervals as brood chambers which contain developmental stages of cercariae (Text-fig. 1, B, E, F, G). Brood chambers range from 120μ to 750μ. in diameter, and are up to 3.5cm long. Narrower regions separating brood chambers, 45μ. to 110μ in diameter by 20μ to 3.5cm long, may exhibit a beaded appearance.
Longest portion of a single sporocyst isolated, 6.1cm long, branched dichotomously at an average of every 2.2cm. Total length and branchings of a single sporocyst not determined, due to its fragility and complex inter-twinings with its own branches and the branches of other sporocysts. No early developmental stages of sporocysts were recovered from dissection of 44 oysters, and sectioning 8 oysters, all of which were not obviously infected with sporocysts.
A sporocyst is hollow in transverse section (Text-fig. 1, A), covered by cuticle, 2μ to 4μ, thick, which is thinner over brood chambers (Text-fig. 1, D). Nucleated layer of sporocyst is internal to cuticle and individual cell membranes in this layer are incomplete and indistinct. This layer is 15μ to 22μ wide in narrower regions and 10μ to 12μ. wide in brood chamber regions. Cytoplasm is continuous suggesting a syncytium, and contains three types of nuclei. Ovoid to spherical vesicular nuclei,
Germinal nuclei lie in clusters in syncytium after several divisions, and, after further divisions, bulge into sporocyst lumen as a germinal cyst (Text-fig. 2, B). Germinal cysts are analogous with ovaries. Mitotic figures only located in those parts of sporocyst wall where germinal nuclei occur.
Cytoplasm of syncytium essentially hyaline, faintly granular in irregular patches. Cytoplasm forming a distinct band approximately 1μ to 2μ wide around germinal nuclei, is more densely granular than granular patches of cytoplasm in other parts of syncytium (Text-fig. 2, A).
Terminal region of sporocyst is densely nucleated, containing both germinal and mesenchymal nuclei. Cell membranes are more distinct in this region.
No muscle cells found in sporocyst wall and no movements of sporocyst were observed. No indications of an excretory system, testes or specialised feeding or nutritive branches of the sporocyst were observed.
List of Abbreviations for all Figures
Published accounts of the sporocysts of bucephalids show that there are slight differences in form and structure among the different species.
The beaded appearance of narrow regions alternating with dilated regions or brood chambers has been noted for Bucephalus cuculus by Tennent (Rhipidocotyle papillosum by Woodhead (Bucephalopsis haimeanus by Lacaze-Duthiers (Bucephalus elegans by Woodhead (R. septpapillata by Kniskern (
R. papillosum by Ciordia (
Both Woodhead (R. papillosum and R. septpapillata respectively. The former observed brownish, empty tubules which he interpreted as being spent sporocysts of the previous year. Kniskern observed tubules devoid of cellular content which he termed nutritive branches. However, polymorphism of the sporocyst tubules has not been noted in the present species.
The general structure of the sporocyst wall, namely, cuticle, cellular layer and lumen, is most similar to the sporocyst of R. septpapillata as described by Kniskern (
Muscle cells were not observed in the sporocyst wall of the present species. However, Tennent (Bucephalus cuculus and R. papillosum respectively, but Ciordia's figures do not substantiate the presence of such fibres. Apart from Woodhead (R. papillosum but did not show muscle cells in his figures, there seems to be general agreement that no appreciable movement of the sporocysts takes place and accordingly, the presence of "muscle fibres" in these two species is unusual. It seems probable that either Kniskern (Rhipidocotyle.
The various interpretations of the cellular layer of bucephalid sporocysts differ markedly. Haswell (Bucephalus sp. was a "germinal epithelium" and any part of it was capable of proliferation to give rise to cercariae.
Tennent (B. cuculus. The larger of these, which he termed propagatory cells, occurred in the tip of the sporocyst and in parts of the wall nearer the tip. They resemble the germinal nuclei of the present species. These nuclei, as they appear in his
Woodhead (B. pusillus and R. papillosum. He also described the occurrence of a redial generation in the sporocyst lumen in both species. However, he did not give any details or figures of the other types of nuclei found in the sporocyst wall. Woodhead's observations were not confirmed by Ciordia (R. papillosum. No testes, true ovaries, or redial generation were found in the terminal regions of the sporocysts of the present species and, apart from Woodhead's observations, have not been found in other bucephalid species.
Kniskern (
Ciordia (R. papillosum. These were stated to be mesenchymal and germinal nuclei. The latter were found in greatest number at the tips of branches and more sparsely, in clusters or singly, among mesenchymal nuclei throughout the remainder of the sporocyst wall. The germinal nuclei were characterised by the irregular and dense disposition of chromatin giving a pyknotic appearance, thus differing from the germinal nuclei of the present species. The mesenchymal nuclei were larger than the germinal nuclei and, in disposition of chromatin, resembled the larger mesenchymal nuclei of the present species. Ciordia stated that the mesenchymal nuclei had distinct cellular outlines but her figures show these outlines to be incomplete.
Analysis of the above observations coupled with those for the present species clearly show that the cellular layer of bucephalid sporocysts is a syncytium. Incomplete cell membranes are distinguishable in the syncytium which contains at least two types of nuclei (and three in the present species). The nuclei are mesenchymal and germinal.
Germinal nuclei have been located by Tennent (O. lutaria represent growing points, since the sporocyst wall in the vicinity of this region is the only region where germinal nuclei and mitotic figures have been located. Haswell's inference, that the whole cellular layer is a germinal epithelium, is probably incorrect. Characterisation of nuclei in descriptions and figures for many species is inadequate and does not permit close comparisons to be made with the present species, and it is evident that the sporocysts of most species need critical restudy.
(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).
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 (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 (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 (Bucephalopsis can only be used for the cercaria Bucephalopsis haimeanus (Lacaze-Duthiers, Bucephalopsis modiolae Faust, C. modiolae (Faust,
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.
Data concerning the number of cercariae liberated from 11 infected oysters are given in Table III, which covers a 53-day period during
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,
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.
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 (Bucephalus elegans. By comparison with the present species, however, B. elegans requires about three minutes to recover from the shock. Dawes (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 (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
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.
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, Rhipidocotyle septpapillata, is 24 hours (Kniskern,
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 (R. septpapillata by Kniskern (
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 (R. septpapillata was achieved within a few hours, the cercariae died.
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
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.
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
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.
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
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 (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.
Schurmans-Stekhoven (Bucephalus polymorphus and Kniskern (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 (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 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.
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
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.
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.
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, Bucephalus. Furthermore, the nature of the sucker musculature is typical of many species of Bucephalus (vide Ziegler, Rhipidocotyle, however, tentacular ducts are not present, and the musculature of the sucker differs from Bucephalus species (vide Nagaty,
Two other Bucephalus metacercariae are known. The metacercaria of B. elegans, figured but not described by Woodhead (B. polymorphus, described by Ziegler (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.
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.
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, S. cardinalis could harbour a natural Bucephalus infection which would invalidate the experimental results. However, Manter (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.
Two gravid specimens and the posterior half of another gravid specimen of Bucephalus longicornutus (Manter, Scorpaena cardinalis Richardson. The definitive host was found to be Kathetostoma giganteum Haast, the mcnkfish, and examination of several specimens from different localities combined with the experimental material provided numbers of B. longicornutus for study as follows: from the intestine of Scorpaena cardinalis used as an experimental host, two complete specimens in one host, and the posterior half of a specimen in another host; from the intestine and pyloric caeca of Kathetostoma giganteum from Foveaux Strait, 74 specimens from 10 of 13 hosts; from K. giganteum from Cook Strait, one specimen from one of four hosts.
K. giganteum from Foveaux Strait showed a 77% incidence of infection, and in 38% of the infected specimens, 5 or more adult worms were recovered.
K. giganteum from Cook Strait showed a 25% incidence of infection and only one adult worm was recovered. This is a similar incidence of infection to that obtained by Manter (
A number of fish species were obtained from Foveaux Strait in order to determine whether the range of host species for B. longicornutus could be extended. These included three specimens of Physiculus bachus (Bloch and Schneider); two Trigla kumu (Lesson and Garnot); three Neophrinichthys latus (Hutton); one Genypterus blacodes (Bloch and Schneider); two Parapercias colias (Forster); and two Arnoglossus scapha (Forster). However, no bucephalid infections were found. It was not possible to obtain a large number of each host species and thus the zero infection found may not be significant.
Alcicornis longicornutus Manter, p. 482.
Host: Kathetostoma giganteum Haast, monkfish; family Uranoscopidae.
Location : Pyloric caeca and intestine.
Locality: Cook Strait (type locality); Foveaux Strait.
Description (24 gravid specimens were studied and 12 were measured; 3 specimens were sectioned; measurements in mm) : Body elongate, truncate anteriorly tapering posteriorly. Total length varies between 0.73 and 2.86 (average approximately 1.6) and greatest width varies between 0.3 and 0.66 at approximately ovary level. Cuticle spinose, apart from anterior sucker concavity, tentacles and ventral to prominent radial muscles of anterior sucker. Spines more sparse over posterior third of body. Anterior sucker 0.2 to 0.3 long by 0.23 to 0.26 wide at its anterior margin. Seven tentacles surmount anterior sucker; three dorsal, two lateral, two ventral. Each approximately 0.09 to 0.11 long by 0.02 wide, having one thorn-like process, approximately 0.03 long, near base (Text-fig. 9, B). Tentacles retracted in most specimens but a complete series from almost fully extended to fully retracted obtained. Each tentacle, with muscular core which extends into the thorn-like process, bears a slight depression at its truncate or occasionally rounded distal end. Sucker musculature essentially scoop-shaped and three, occasionally five, tentacular ducts, continuous with free ends of tentacles, prominent in dorsal musculature. Prominent gaps between groups of oblique muscles along anterior margin of sucker coincide with positions of emergence of free ends of tentacles. Ducts for ventral pair of tentacles not observed. When ventral tentacles are completely retracted their points of emergence from musculature are marked by small, but conspicuous, ventral depressions. Prominent band of radial muscles, 0.05 to 0.06 wide, U-shaped or semicircular, around posterior margin of sucker concavity. Cuticle overhangs these muscles and demarcates posterior limit of the sucker concavity. Cuticle does not completely close off open end of musculature and spines on this part of cuticle are not always seen. Musculature U-shaped in transverse section and seven gaps between muscle fibres seen corresponding to seven tentacular ducts.
Mouth generally anterior to mid-body level, varying between 0.33 and 0.56 from anterior extremity. Pharynx, ovoid to spherical, 0.07 to 0.12 long by 0.07 to 0.12 wide. Eggs obscure oesophagus in material examined. Intestine ovoid, 0.2 to 0.3 long by 0.13 to 0.19 wide, dorsal in position and directed posteriorly, often obscured by eggs.
Testes, ovoid to spherical, 0.13 to 0.2 long by 0.1 to 0.2 wide, on right side of the body, generally between intestinal and mid cirrus sac levels, diagonal in most specimens but occasionally tandem. Anterior testis usually touches posterior margin of ovary but in one specimen it overlaps ovary ventrally by approximately
Ovary generally spherical (but in five specimens ovoid), varies between 0.1 and 0.13 in diameter or 0.1 to 0.11 long and 0.12 wide, on right side of body at approximately intestinal level. Oviduct emerges from right side of ovary, opens into ootype, 0.01 to 0.02 posterior to ovary and gives off Laurer's canal approximately one-third from its proximal end. Laurer's canal runs obliquely towards dorsal midline and opens to exterior at approximately anterior testis level. Uterus emerges from posterior margin of ootype and was traced posteriorly to intertesticular level. Remaining coils indistinct.
Vitellaria arranged in two lateral groups between sucker and pharynx. Individual follicles clumped or spread and this can be related to number of eggs in uterus. Most often, 16 follicles on each side but this number can vary between 14 and 18. Each follicle more or less spherical, between 0.04 and 0.06 in diameter. A difference of 0.005 generally exists between extremes in sizes of follicles in a given specimen. Left vitelline duct runs obliquely to posterior testis level then loops and runs anteriorly on right side of body to enter vitelline reservoir immediately median to ootype. Right vitelline duct runs slightly obliquely and enters vitelline reservoir. Short duct connects vitelline reservoir with ootype (Text-fig. 9, F).
Eggs occupy most of body in most specimens. Their anterior extent is variable. In two specimens they extend just beyond pharynx level but in most they reach to within 0.1 to 0.18 of posterior limit of sucker. Eggs extend posteriorly to level of genital pore. They are ovoid, distinctly operculate, 0.021 to 0.035 long by 0.015 to 0.022 wide (Text-fig. 9, H). Two specimens contained a few abnormal eggs (Text-fig. 9, I, J).
Excretory pore terminal and saccular excretory bladder extends just anterior to pharynx. Flame cell formula not determined.
Compared with the metacercaria, adult B. longicornutus have tentacles surmounting the anterior sucker and show considerable development of the reproductive system. The development of elements of the latter in the hindbody has a considerable bearing on the position of the mouth and intestine, a feature which differs considerably between metacercaria and adult. Remaining differences are relatively minor. Spines are absent from the cuticle overlying the band of radial muscles of the adult sucker; and the flap of cuticle closing off the open end of the sucker musculature in the metacercaria, while still present in the adult, is not conspicuous. Both of these differences may possibly result from the effects of attachment to the intestinal wall of the definitive host.
Only three immature worms were recovered from the material examined. Two of these had their tentacles partially extended and all three, apart from the absence of eggs, were very similar to sexually mature adults.
Manter (Alcicornis McCallum, Kathetostoma giganteum from Cook Strait was mounted in lateral view and the sucker-like nature of the anterior end was not evident, appearing instead rhynchus-like, and this is diagnostic of the genus Alcicornis. Manter compared the species with other known species of Alcicornis. However, because of the change in generic status proposed here it is now necessary to compare Bucephalus longicornutus with related species of Bucephalus.
In the possession of one thorn-like process at the base of the tentacle, B. longicornutus resembles B. polymorphus von Baer, B. introversus Manter, B. scorpaenae Manter, B. polymorphus in that the cirrus sac is much longer in relation to the total length, the tentacles do not taper to a point and the thorn-like process on the tentacle is situated nearer the base of the tentacle; from B. introversus in that the anterior sucker does not introvert into the anterior end of the body and the structure of the genital atrium is not as complex; and from B. scorpaenae in that the excretory vesicle is shorter, the pars prostatica is not subdivided and the genital lobe is simpler in structure.
B. longicornutus does show some resemblance to B. kathetostomae (Manter, Kathetostoma albigutta Bean. However, it differs from B. kathetostomae in the absence of the muscular sphincter surrounding the genital pore, shorter genital atrium, the absence of processes on the eggs and considerably shorter excretory vesicle.
There are two main differences between Manter's description of B. longicornutus and that given above. These are the bipartite rather than ovoid seminal vesicle, and the longer tentacles. Both of these characters are considered to be subject to variation. The shape of the seminal vesicle is probably dependent on the amount of sperm present and the length of the tentacles on their degree of contraction.
The variations noted for B. longicornutus are considerable. The position of the internal organs is apt to be governed by the degree of contraction of the body on fixation and by the number of eggs in the uterus. The arrangement of the vitelline follicles, which has been mentioned above, illustrates this latter point. Egg size is extremely variable between specimens from different hosts but is generally more or less constant within a group of specimens from the same host. A similar phenomenon has been noted by Manter (B. varicus Manter, B. polymorphus of Nagaty, B. longicornutus which he examined. Egg size cannot be correlated with the length of the specimen, e.g., a specimen of 1.718 long had eggs 0.021 by 0.016 while a specimen 1.818 long had eggs 0.032 by 0.020; but can, however, be correlated with the number of eggs present, e.g., a specimen 1.110 long had few eggs and these were large, being 0.035 by 0.022 while a specimen 1.496 long had many eggs and these were relatively small, being 0.024 by 0.016.
B. longicornutus parallels B. varicus in the range of variation exhibited. Such a range has not been noted for other species of the genus. However, many of these species are either insufficiently described (vide van Beneden, vide Velasquez, 2μ to 3μ in egg size have, in some cases, been used to separate species (vide Velasquez, B. varicus and B. longicornutus, two species which have been collected from a number of different host species and the same host species respectively, these specific distinctions should be regarded as tentative and possibly subject to alteration after reference to further material.
The most constant features noted in those specimens of B. longicornutus examined were the nature of the genital atrium and genital lobe, and the floor of the anterior sucker. Unfortunately, descriptions of these features are absent from many systematic papers dealing with species of Bucephalus and they may, in future, prove more satisfactory criteria for differentiating between species than those used in the past.
The eggs of adult specimens of Bucephalus longicornutus, obtained and set up as described elsewhere (p. 5), were examined on 20 consecutive days in order to detect miracidia. None was found and no eggs showed signs of embryonation. The eggs may not have been viable but this point needs further investigation.
The major features of the life history of Bucephalus longicornutus agree with those described for fresh-water bucephalids by Woodhead (
A consideration of the life history of B. longicornutus shows there is still no way of determining the generic status of a cercaria other than by infection experiments although an alternative means has been suggested (p. 22). The latter means is hypothetical and yet to be tested.
Generic identity can be assigned to the metacercaria but its specific identity must be obtained from experimental infections.
It should be stressed again that future investigators must give more accurate descriptions of sporocysts; cercariae and metacercariae than have previously been given by many authors. Because of the close morphological similarity of bucephalid cercariae, information on flame cell formulae, behaviour and all essential details of spination and granulation will be necessary for separation of species. The variability of adult B. varicus and B. longicornutus noted on p. 36 may also apply to other species and genera of the family, and systematists are urged to avoid the tenuous criteria that have, hitherto, been used to differentiate between species.
In a study of this nature one becomes indebted to many institutions and individuals for their assistance.
The Marine Department chartered the trawler Miss Southland, for which grateful thanks are offered. Their officers at Bluff, in particular Mr G. C. Crowther, were most co-operative in forwarding live oysters and collecting fishes, and arranging for a day's dredging on the Kumea. It would have been impossible to undertake this study without their assistance.
I would also like to thank the Internal Research Fund Committee, Victoria University, for the grant (9/64) which enabled me to spend a week at Bluff during
Professor H. W. Manter, University of Nebraska (U.S.A.), and Dr Sewell Hopkins, Texas A. and M. College (U.S.A.), made many helpful suggestions during the course of this study. For this assistance I am indeed grateful.
Finally, I would like to express my gratitude to Drs P. H. J. Castle, J. A. F. Garrick and R. B. Pike for helpful criticisms of various sections of the manuscript; Mr J. M. Moreland for identifying fishes; and the staff of the Victoria University of Wellington Library for assistance with literature. Publication of this paper has been assisted by a grant from the Victoria University Publications Fund.
Not seen.