Specimens of P. hoplura were collected from farmed oysters
(Crassostrea gigas) from Saldanha Bay, South Africa in November 2012.
Oysters were placed in an aquarium filled with artificial seawater
(Seachem, Georgia USA) kept at a temperature of 21 °C and a salinity
of 33. Oysters were fed a mixture of ground fish feed (Tetramin, Melle
Germany) and cultured algae (Isochrysis galbana and Nitzschia
closterium) obtained from the Department of Agriculture Forestry and
Fisheries (DAFF), South Africa. After an acclimatization period of 24 h,
some oysters were shucked and shells were broken with pliers to extract
worms from their burrows.Worms were identified using the dissecting
microscope, fitted into 1.2–1.4 mm diameter glass capillary
tubes and placed in a 60 × 15 mm petri dish (one worm/dish) where
theywere fed every other day. Specimens of B. proboscideawere collected
in February 2012 and August 2013 from sediment in the outflow
path of an abalone farm in Gansbaai, South Africa. Worms were
transported to the laboratory in plastic containers sealed with parafilm
and identified using the dissecting microscope. Worms were then
placed in a 21 °C aquarium with sediment taken from the worm's habitat.
Prior to use, the sedimentwas sieved using a 500-μmfilter,washed
with freshwater and frozen overnight to kill any fauna and to remove
any dead organic material that could produce unwanted products of decomposition.
Pilot experiments found that B. proboscidea suffered high
mortalities when cultured in 100% artificial seawater therefore filtered
seawater was obtained from the abalone farm and adjusted with
Seachem sea salts to a salinity of 33 when necessary. All other conditions
(feeding, temperature regime, etc) were similar to those outlined
above for P. hoplura.