Diversity And Distribution Of Marine Microbial Eukaryotes And Ecological Roles Of Picoprasinophytes In Arctic Seas

The microbial eukaryotes, as the base of microbial food webs, are crucial to the marine systems. Recent environmental studies based on molecular data have revealed a high diversity of microbial eukaryotic lineages in the marine environment, while they also play significant roles in the Arctic Oceans. We explored the distribution of microbial eukaryotes (≤50μm) in Kongsfjorden (Arctic) using PCR/DGGE (denaturant gradient gel electrophoresis). Samples analyzed by DGGE combined with environmental data has allowed us to investigate the microbial structure of the polar coastal sites and estimate the distribution of dominant taxa in the community. Meanwhile, we constructed SSU rRNA clone libraries to assess the diversity of microbial eukaryotes in the central Arctic Ocean during the Third Chinese National Arctic Research Expedition (CHINARE-2008, July 11-September 26). The clone library screening and fluorescence in situ hybridization (FISH) were combined to evaluate the contribution of picoprasinophytes, which was prevalent in the cold ecosystem, in the Central Artic Ocean. In addition, FISH (≤3μm) and FCM (≤5μm) were applied to evaluate the ecological role of picoeukaryotes, especially picoprasinophytes in the Arctic Oceans. Spatial distribution of picoprasinophytes and their contributions in the central Arctic Ocean determined by specific probes would help us identify the ecological dynamics of "pico-size" organisms in the pelagic systems. The main outcomes include:1. Applying DGGE to investigate the spatial distribution of microbial eukaryotes in Kongsfjorden, Svalbard during summer 2008 and 2009. DGGE band patterns showed remarkable diversity and spatial difference among the sampling sites. The results of CCA analysis showed that temperature and salinity are the main envrionmental factors that influence the structure of microbial eukaryotes. The analysis of samples from station K3 in 2009 showed that the dominant genetic eukaryotic taxa include Syndiniales, Chamaeleomyces granulomatis, Calamus sinicus, Chrysophyceae sp.. Atlantic water masses, inputs from large tidal glaciers and the ice cover in winter create steep environmental gradients in temperature, salinity and sedimentation along the length of Kongsfjorden fjord, which might be responsible for the significant difference in diveristy of microbial eukaryotes between the outer and inner fjord.2. Through. two SSU rRNA gene libraries, CNCⅢ05 and CNCⅢ51 were obtained in the 18S rRNA clone library of sample from the B80-20m and B85-0m in the Central Arctic Ocean. CNCⅢ05 contained a rich diversity of five taxon groups:alveolates, stramenopiles, metazoans, chlorophytes. and telonema. In addition to the first four clades listed above, picobiliphytes and Choanoflagellida were detected in the library of CNCIII51. A genotype related to Micromonas pusilla (Chlorophyta) was one of the most frequently occurring clades in both libraries. Species richness curve suggested that the species richness has been underestimated in this study.3. Standard FISH protocols using fluerochrome-labeled oligonucleotide probes have been successfully applied for in situ detection of prokaryotes and eukaryotes. However, the optimized protocols of FISH for specific eukaryotes in marine environment have not been developed yet. This study optimized the conditions of fluorescence in situ hybridization (FISH) by using two polar isolated microalgae. The modified conditions were as follows:(1) 10mg mL-1 lysozyme solution pretreatment at 37℃for 30min was helpful to increasing the permeability of cells by changing the structure of cell walls or membrane; (2) 20% formamide brought the right level for a low-background signal hybridization; (3) the satisfactory signal intensity and hybridization efficiency were obtained at 47℃for 6h. The cells of FISH were compared with FCM and DAPI to confirm its hybridized efficiency. The optimized protocol was also applied into the Arctic Ocean samples, which was predominant by Micromonas sp.. The modified protocol has shown a relatively high efficiency and can be successfully applied for the detection of specific microbial eukaryotes in environmental samples.4. The clone library screening and fluorescence in situ hybridization (FISH) were combined to evaluate the contribution of the picoprasinophytes which were prevalent in the cold ecosystem. The analysis of samples from Central Arctic Ocean by FISH revealed that the abundance of picoeukaryotes was 1.944x103 cells mL-1-2.37x104cells mL-1 (average 7.13x103cells mL-1). The dominant population of picoprasinophytes accounted for 25.45%-86.89% (average 44.37%) in the pico-sized eukaryotic communities. The result obtained by FCM showed that the abundance of picoeukaryotes was 0.955x103 cells mL-1-1.073x104cells mL-1. There was high abundance of picoeukaryotes in the station B81-B84. The FISH and FCM counts generated similar variability trends in abundance of all samples. Although, the abundance of picoeukaryotes in station B81-50 m obtained by FISH was much more than by FCM. Spatial distribution of picoprasinophytes, especially Micromonas sp. and their contributions in the central Arctic Ocean determined by specific probes reflected their ecological importance in the pelagic systems.5. Meanwhile, we investigated the picoeukaryotes in the microbial community at the upper water column (<50 m) of Kongsfjorden sites in 2008 by FISH. The result showed the abundance of picoeukaryotes and pico-prasinophytes was 0.821x103-23.38×103cells mL-1 and 0.25x103-6.64x103cells mL-1, respectively. The ratio of picoprasinophytes to picoeukaryotes was 11.63%-54.64%(average 29.11%). The results suggested that picoprasinophyes were playing important ecological roles in the changing ecosystem of Arctic Kongsfjord.