Recovery of cercariae

Live oysters for experimental purposes were obtained from Foveaux Strait between June and October, 1963, and March and July, 1964. Each oyster, right valve uppermost, was kept alive in a 7in diameter finger bowl two-thirds filled with sea-water. Daily examinations of the water were made for liberated cercariae. The greatest concentration of cercariae was usually found on the bottom of the finger bowl opposite the exhalent chamber of the oyster.

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 page 5 within a week they were opened and a superficial examination was made of their visceral mass for the presence of sporocysts. If sporocysts were absent, the oysters were dissected or sectioned for any deep-seated infection.

Injection experiments

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.

Feeding experiments

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 (1954) or recorded by the author during the course of independent studies. These included 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.

Attempts to obtain miracidia

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 page 6 its volume with distilled water to make it isosmotic with fish body fluids. The latter is termed diluted sea-water in the remainder of the text. It was unknown whether the eggs might hatch in sea-water or in the rectum of the host and therefore the two possibilities were considered for this experiment. The eggs were examined daily.

Formation of the cyst wall by the cercaria

(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.

Estimation of cercariae liberated

Estimations of the number of cercariae liberated from infected oysters were made during June and July, 1964. The procedure was as follows: 11 infected oysters were set up in separate bowls each with one litre of sea-water. Bowls were examined daily. The oyster was first removed from the bowl, the water agitated vigorously to disperse the cercariae and then allowed to settle. The total number of cercariae in the field of view of a Zeiss "Opton" microscope using a ×10 eyepiece and 1.6 objective was counted and averaged over 10 counts. This gave the average number of cercariae in roughly 5cc of water. This method gave a means of repeatable, relative approximations. When very few cercariae were present they were counted individually.

Sporocysts

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, 1953). Sections were stained with Heidenhain's haemotoxylin with alcoholic eosin, or Weigert's haematoxylin with picro-ponceau S.

Cercariae

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.

Metacercariae

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 page 7 of each fin web separated, and the relative abundance of cysts on the various fins recorded. The skin was then removed from the head, and the underlying tissues, together with the eyes, pharynx and gills, were examined for cysts. Finally, the skin was removed from the remainder of the body, and the body wall muscles and viscera were examined. A similar dissection procedure was used for small fish obtained from the Foveaux Strait oyster beds and Wellington Harbour in attempts to find natural infections of the metacercaria of 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.

Adults

The technique suggested by Manter (1954) was followed in the examination of all fish for adult worms. Unfortunately, specimens of naturally infected definitive hosts, 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.