STUDY ON DETECTION OF IN SITU GROWTH RATE OF TAKAYAMA PULCHELLUM

Harmful algal blooms are getting more and more serious problems in the water ecosystem in the world than before, for its blooming areas are expanding in many places, such as freshwater and coastal water.It is important to measure the in situ growth rate to understand the dynamics of harmful algal blooms in the field, because the temporal change of algal species abundance at a fixed point can be affected by many factors such as growth (cell division), grazing mortality caused by zooplankton, sedimentation , parasite or virus infections, autolysis due to unfavourable growth conditions, etc.Moreover, cell abundance is affected by concentration or dilution due to physical processes, such as physical advection and diffusion.The abundance of a certain phytoplankton species in natural systems is a result of a large array of processes.To investigate causal relationships behind algal bloom proliferation and decline, growth rate must be measured independently of cell abundance and different loss processes.There are several methods available for estimating phytoplankton growth.The usual technique to date makes use of specific cell cycle markers such as cellular DNA content or presence of cell cycle-related proteins.The combination of growth rate measured using cell cycle analysis and the net change in cell abundancemeasured by microscopy or flow cytometry can provide both growth and loss rates for the studied species.This approach has the potential to allow the separation of population changes directly caused by factors affecting HAB growth (cell division).The PNA (Peptide nucleic acid)probe is an artificial synthetic DNA analogs, in which the sugar phosphate backbone of the DNA helix is replaced with an uncharged structurally homomorphous pseudopeptide backbone.The synthetic backbone provides PNA probes with unique hybridization characteristics such as more rapid and stronger binding to complementary targets according to the Watson and Crick basepairing rules.Takayama pulchellum was the first recorded in Japan as an ichthyotoxic dinoflagellate.It was initially described as Gymnodinium pulchellum, but recently renamed as T.pulchellum.T.pulchellum formed HABs in America and Australia, causing large numbers of fish kills, and in the recent years it blooms occurred in Xiamen Bay.The specific growth rate estimated by PNA probe in this study to measure the in situ potential growth rate of T.pulchellum is very important.Because currently available approaches for estimating harmful algal bloom species' growth rates can be difficult to apply in the field,we have been exploring the feasibility of using quantitative rRNA by peptide nucleic acid, chl a and protein concentration measurements as the basis for making such estimates.In this study, we examined the relationship between rRNA and growth rate of T.pulchellum strains (TPXM).Whole-cell hybridization with fluorescence labeled peptide nucleic acid (PNA)probe was used in conjunction with flow cytometry and the professional image analysis software to quantify rRNA on a per cell basis.The result showed that cellular rRNA levels in different growth periods were well correlated with its corresponding specific growth rates (R2=0.7293, p<0.01, n=14);cellular protein concentration, cellular rRNA level and the cell cycle also presented good correlation (R2=0.6984, p <0.01, n=14).All the results suggested that cellular rRNA level was a good indicator to analyze cell cycle and cell growth rate of T.pulchellum.