Quantitative real-time PCR for detecting germination of heterosigma akashiwo and chattonella subsalsa cysts from delaware's inland bays, USA

Abstract

Cyst germination of strains of the harmful algal species Heterosigma akashiwo and Chattonella subsalsa (Raphidophyceae) from Delaware's (USA) Inland Bays was studied both in the field and laboratory during the spring and early summer seasons. Quantitative real-time PCR was employed for detection and quantification of cells in natural sediments and of germinated vegetative cells in the water column. Temperature, salinity, and dissolved nutrient concentrations were examined in field mesocosm experiments to identify physicochemical factors associated with germination, while the effects of temperature and light on germination were examined in laboratory experiments. We detected and monitored a wide range of cyst abundances of H. akashiwo (from 164 to 2820 cysts cm-3 wet sediment) and C. subsalsa cysts (from 2 to 135 cysts cm-3 wet sediment) in environmental sediments. Germinated H. akashiwo cells were detected in situ after temperatures reached 15°C. However, in laboratory studies, H. akashiwo germination occurred at even lower temperatures (10°C), which was considerably lower than typical germination temperatures from similar Japanese strains. In contrast, a temperature of 20°C stimulated C. subsalsa germination in both field and laboratory studies, although germination still occurred at low temperatures (10°C). The presence or absence of light did not affect the germination of C. subsalsa. The low quantities of detected vegetative cells from cyst germination for both H. akashiwo and C. subsalsa suggest the inoculation of a small number of vegetative cells into the water column during the spring and early summer months. © Inter-Research 2009.