Morphological And Phylogenetic Analysis And Detection By Fluorescentin Situ Hybridization Of Several Red Tide Causative Microalgae

So-called red tide is an ecologic phenomenon that seawater turns red, brown, or yellow, when oceanic plankton overly propagate or congregate. Red tide, especially harmful algae blooms have neagatively impacted the aquaculture, fisheries and tourism industries as well as the environment and public health. To understand red tide and further control and prevent the occurring of HABs, it is foremost and essential to ascertain the causative species, while the efficient detection methods should be developed to accelerate the identification and quantification aiming at monitoring the composition of organisms in the natural water. In this study, we performed morphological and phylogenetic analysis of several red tide caustative microalgae, and explore the application of fluorescent in situ hybridization (FISH) in the detection of causative species.Five Skeletonema costatum (Greville) Cleve-like diatoms isolated from different areas along the coast of the China Sea (SK-BH, SK-FQ, SK-HH, SK-DH and SK-XM) were studied with light microscopy (LM) and scanning electron microscopy (SEM), and internal transcribed spacers (containing 5.8S rDNA) (ITS) and partial large subunit ribosomal DNA (D1-D2) (LSU) sequences were obtained by PCR amplification, and included in a phylogenetic analysis with published sequences of other Skeletonema spp., aiming at exploring the relationships between our isolates and previously described Skeletonema species. The five isolates differed in morphology, only one (SK-XM) being identified as Skeletonema costatum sensu stricto. The other four isolates did not correspond morphologically to any described Skeletonema species. The LSU and ITS trees provided different topologies and phylogenetic inferences suggested that the five isolates belonged to at least three different species. Sequence distance analysis indicated that genetic similarity between the isolates paralleled their geographical location. Additionally, subtle morphological revolution could be detected among the five species. Together, all of results above proved the diversities of genus Skeletonema along the China Sea coast.Morphological investigation was perfomed on a Gymnodinium-like species isolated from a sample collected from where red tide occurred, and the relationships between this species and Gymnodinium, Gyrodinium, Karena, Karlodinium and Symbiodinium were also explored. This species was characterized with typical features similar to Gymnodinium in LM, while we failed to obtain well-preserved cells for SEM with integrative morphology. Phylogenetic analysis suggested this Gymnodinium-like species is a Symbiodinium species.We obtained LSU and ITS sequences from Heterosigma akashiwo (Hada) Hada, designed species specific probes against cytoplasmic rRNA and nuclear rDNA, established whole cell and nuclear FISH protocols for the detection of H. akashiwo, tested specificity of the probes and examined the change in the hybridization signal and detection efficiency in the whole cell cycle. The probes were specific against H. akashiwo, showing no cross-reactivity with four other test microalgae. The rRNA-targeted probe hybridized to cytoplasmic rRNA, showing strong green fluorescence throughout the whole cell, while cells labeled by rDNA-targeted probe exhibited exclusively fluorescent nucleus. The detection protocols were optimized and could be completed within an hour. For rRNA and rDNA targeted probes, about a corresponding 80% and 70% of targeted cells could be identified and quantified during the whole growth circle, despite the inapparent variability in the average probe reactivity. The established FISH was proved promising for specific, rapid, precise, and quantitative detection of H. akashiwo.Similarily, we obtained LSU and ITS sequences from Prorocentrium micans Ehrenberg, designed rRNA targeted probe, established whole cell hybridization detection protocol, and test the specificy of the probe. P. micans cells labled with the probe displayed intensive fluorescence. The designed probe did not react with other test microalgae, proving its specifiy against with P. micans.