Studies On Molecular Identification And Fluorescence In Situ Hybridization Detection Of Alexandrium Spp.

Apparent increases in frequency, intensity and distribution of harmful algal blooms (HABs) have been observed on a global scale recently, which has become a significant environmental issue in coastal waters. To effectively prevent or mitigate the impacts of HABs, it is important to establish a valid HAB monitoring system. At present, the identification and detection of HAB causative species mainly depend on the microscopic observation of morphological features of the algae. However, the morphological observation method is not suitable for monitoring all HAB causative species. In some cases, toxic and non-toxic HAB causative species cannot be discriminated simply based on morphological differences. And some morphological features are easily affected by environmental conditions or growth stages. In addition, it's not easy to implement high-quantity and continuous monitoring with the morphological observation method, due to the complexity of the method and necessity for trained experts. To improve the efficiency in monitoring HABs, the molecular biological techniques and methods were employed to develop algal identification and detection methods in the current study.Species in genus Alexandrium are important HAB causative species. Some of them have very different toxin production capacities, but they are morphologically similar and very difficult to distinguish from one another. To develop the molecular identification method, we amplified and sequenced the whole-length ribosomal RNA gene (rDNA), including the small-subunit rDNA, the large-subunit rDNA, the 5.8S rDNA and the flanking internal transcribed spacers 1 and 2, of 9 Alexandrium strains isolated from Chinese coastal waters and 2 other Alexandrium strains from elsewhere maintained in the laboratory. Results of sequence analysis show that these Alexandrium strains include 5 ribotypes, which are Temperate Asian (TSC-TA) ribotype and West European (TSC-WE) ribotype of"tamarense species complex", Portugal (M-PO) ribotype and New Zealand ribotype (M-NZ) of A. minutum, and A. affine (AF) ribotype. The rDNA sequences were divided into several regions and subjected to phylogenetic analysis, together with sequences collected from GenBank database. It was found that the D1 and D2 region were more suitable for the molecular identification and phylogenetic analysis of Alexandrium. Meanwhile, a single-cell rDNA sequence analytical method was established. This method is suitable for identification of Alexandrium cells at various life stages, without the hard work in establishing clonal cultures.To detect the Alexandrium cells, we focused on the fluorescence in situ hybridization (FISH) method. The obtained sequences of rDNA were compared and analyzed, and several probes for the 5 ribotypes of Alexandrium were designed based on the ribotype-specific domains. For each ribotype, a specific probe with high labeling efficiency was selected. The probes for the M-PO and M-NZ ribotypes of A. minutum, as well as the TSC-WE ribotype of"tamarense species complex"are acquired for the first time. The probes for TSC-TA ribotype of"tamarense species complex"and A. affine (AF) ribotype are designed based on new target domains. Factors interfering the FISH detection efficiency were also analyzed and discussed. To validate the FISH method in monitoring of Alexandrium blooms, the technique was used to detect Alexandrium cells during the cruises on the waters near the Changjiang estuary in spring of 2007. High density of Alexandrium was detected, and the maximum cell density could reach 103cells/L. It was also found that the samples preserved for a long time showed a diminished or even extinguished fluorescence in FISH detection. Among several preservation methods, saline ethanol fixative or Paraformalclehyde/Methanol fixative were selected and showed satisfactory results in 3 months.In conclusion, we sequenced the whole-length ribosomal RNA gene (rDNA) of 9 Alexandrium strains isolated from China's coastal waters and other 2 Alexandrium strains for the first time. Based on these sequences, 5 specific probes were acquired for the 5 ribotypes of Alexandrium, of which 3 probes were obtained for the first time. The single-cell rDNA sequence analytical technique and the FISH method are promising methods in identification and detection of Alexandrium spp. in natural waters. The work will offer theoretical basis and methodological assistance in routine monitoring of Alexandrium species in China.