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

Anatomical Studies

Anatomical Studies

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.