ABSTRACT. Juvenile estuarine gastropods (Clithon retropictus), maintained in ultraviolet ray-irradiated recirculating artificial seawater with a salinity of 20 at 28掳C, preserved thermostable direct hemolysin (TDH)-producing strain D-3 of Vibrio parahaemolyticus at a level of 10 4 -10 5 colony forming units per gram (cfu/g) and TDH-non-producing strains N-18 and R-13 at a level of 10 1 -10 2 cfu/g in the alimentary tract for at least 21 days after ingestion. In adults, the numbers of the three strains decreased to a level of 10 0 cfu/g within 21 days under the same conditions. This evidence supports our recent observations that TDH-producing strains increased to a high level in the summer months in the presence of high levels of TDH-non-producing strains in the alimentary tract of juvenile C. retropictus at estuaries in Japan. Lymnaea stagnalis to Trichobilharzia ocellata [14]. Also, hemocytes of juvenile L. stagnalis were shown to be less differentiated in morphology and less active in a lysosomal enzyme, peroxidase, than those of adults [1]. Based on these findings, the present study was done to confirm that juvenile C. retropictus could preserve TDH-producing strains at higher levels than adults and that juveniles could preserve TDH-producing strains at higher levels than TDHnon-producing strains.
MATERIALS AND METHODSGastropod: Adult and juvenile C. retropictus, 3.0-4.8 g and 0.2-0.9 g weight, respectively, were maintained in aquaria with tap water at 28掳C for one week and confirmed non-detectable for V. parahaemolyticus before experiments.Bacteria: V. parahaemolyticus strains D-3 (TDHp r o d u c i n g ; n a l i d i x i c a c i d -r e s i s t a n t ) , N -1 8 (TDH-non-producing; nalidixic acid-resistant) and R-13 (TDH-non-producing; rifampicin-resistant) [9] were grown in Trypticase Soy Broth (Difco, U.S.A.) containing 1% NaCl at 37掳C for 8 hr with shaking. The cells were washed with 20 artificial seawater [9] and suspended at approximately 10 6 cfu/ml in the artificial seawater. Inoculation: A hundred gastropods were incubated in 100 ml of the bacterial suspension containing a single strain in 20 artificial seawater at 25掳C for 24 hr with aeration. Then they were washed with distilled water to remove the strains attached to the surface and maintained in aquaria with 30 l of UV-irradiated, recirculating, 20 artificial seawater at 28掳C for up to 21 days. Cultured diatom algae, Navicula strain F-1 [2], were fed to the gastropods throughout the study. Every 10-15 gastropods were sacrificed at appropriate time intervals to obtain homogenates of the alimentary tracts attached to mid-gut Thermostable-direct hemolysin(TDH)-producing strain of Vibrio parahaemolyticus is a potential pathogen of human gastroenteritis. The strain was detected at the highest level of 10 5 colony forming units per gram (cfu/g) from a neritid gastropod (Clithon retropictus) at a site of the Hashizu estuary in Japan where juveniles, less than 1.0 g weight, were predominant in a C. retropictus community. It was not, however, detected at sit...