Gene expression in the toxic dinoflagellate, Alexandrium fundyense: Emphasis on cell cycle- and growth-regulatory genes

Molecular markers have become an important tool for obtaining in situ growth rates of phytoplankton, which is a key parameter in understanding phytoplankton population dynamics. No molecular markers for in situ growth rate of Alexandrium fundyense are available. In this dissertation, characterization of transcriptional profiles in the cell cycle and identification of potential cell cycle markers for growth studies were done by using a cDNA microarray. A custom cDNA microarray was developed containing 1,512 features to study gene expression at different growth stages and during cell cycle progression. About 2% of the array was differentially expressed between cultures in exponential and stationary phases. By time serial analysis of samples over a 25-h period, 7% of the array was differentially expressed during the cell cycle progression. Three genes (actin, putative toxin-related gene and clone 8) exhibited positive correlation in expression levels with the percentage of the cell population in the G2+M stage. This suggests involvement in cell cycle progression and that these genes are potentially useful as cell cycle markers for in situ growth rate studies.^ Diel transcriptional profiles during a natural bloom of A. fundyense were analyzed by microarray and spliced leader-based cDNA library technique. The diel pattern of the cell cycle progression observed resembled that in the laboratory culture. Microarray analyses showed that 10% of the array was differentially expressed during the light-dark and dark-light transitions, coinciding with the S and G2+M phases of the cell cycle, indicating that the expression of these genes is entrained by these transitions. Some of these genes (DNA damage protein, histone-like protein, and major basic nuclear protein) showed up-regulation only in the G2+M phase, indicative of potentials as cell cycle markers.^ Consistent with previous studies, results showed that a small fraction of the genome is regulated at the transcriptional level in A. fundyense. The identified genes either previously characterized or novel, are promising candidates for cell cycle markers and need to be further characterized for their applicability to in situ growth rate studies. The putative cell cycle inhibitors also need to be investigated further to gain insights into their roles in cell cycle regulation in this important species.

Gene expression in the toxic dinoflagellate, Alexandrium fundyense: Emphasis on cell cycle- and growth-regulatory genes

Molecular markers have become an important tool for obtaining in situ growth rates of phytoplankton, which is a key parameter in understanding phytoplankton population dynamics. No molecular markers for in situ growth rate of Alexandrium fundyense are available. In this dissertation, characterization of transcriptional profiles in the cell cycle and identification of potential cell cycle markers for growth studies were done by using a cDNA microarray. A custom cDNA microarray was developed containing 1,512 features to study gene expression at different growth stages and during cell cycle progression. About 2% of the array was differentially expressed between cultures in exponential and stationary phases. By time serial analysis of samples over a 25-h period, 7% of the array was differentially expressed during the cell cycle progression. Three genes (actin, putative toxin-related gene and clone 8) exhibited positive correlation in expression levels with the percentage of the cell population in the G2+M stage. This suggests involvement in cell cycle progression and that these genes are potentially useful as cell cycle markers for in situ growth rate studies.^ Diel transcriptional profiles during a natural bloom of A. fundyense were analyzed by microarray and spliced leader-based cDNA library technique. The diel pattern of the cell cycle progression observed resembled that in the laboratory culture. Microarray analyses showed that 10% of the array was differentially expressed during the light-dark and dark-light transitions, coinciding with the S and G2+M phases of the cell cycle, indicating that the expression of these genes is entrained by these transitions. Some of these genes (DNA damage protein, histone-like protein, and major basic nuclear protein) showed up-regulation only in the G2+M phase, indicative of potentials as cell cycle markers.^ Consistent with previous studies, results showed that a small fraction of the genome is regulated at the transcriptional level in A. fundyense. The identified genes either previously characterized or novel, are promising candidates for cell cycle markers and need to be further characterized for their applicability to in situ growth rate studies. The putative cell cycle inhibitors also need to be investigated further to gain insights into their roles in cell cycle regulation in this important species.

Characterising planktonic dinoflagellate diversity in Singapore using DNA metabarcoding

Dinoflagellates are traditionally identified morphologically using microscopy, which is a time-consuming and labour-intensive process. Hence, we explored DNA metabarcoding using high-throughput sequencing as a more efficient way to study planktonic dinoflagellate diversity in Singapore’s waters. From 29 minimally pre-sorted water samples collected at four locations in western Singapore, DNA was extracted, amplified and sequenced for a 313-bp fragment of the V4–V5 region in the 18S ribosomal RNA gene. Two sequencing runs generated 2,847,170 assembled paired-end reads, corresponding to 573,176 unique sequences. Sequences were clustered at 97% similarity and analysed with stringent thresholds (≥150 bp, ≥20 reads, ≥95% match to dinoflagellates), recovering 28 dinoflagellate taxa. Dinoflagellate diversity captured includes parasitic and symbiotic groups which are difficult to identify morphologically. Richness is similar between the inner and outer West Johor Strait, but variations in community structure are apparent, likely driven by environmental differences. None of the taxa detected in a recent phytoplankton bloom along the West Johor Strait have been recovered in our samples, suggesting that background communities are distinct from bloom communities. The voluminous data obtained in this study contribute baseline information for Singapore’s phytoplankton communities and prompt future research and monitoring to adopt the approach established here

Diversity and Abundance of the Bacterial Community of the Red Macroalga <em>Porphyra umbilicalis</em>: Did Bacterial Farmers Produce Macroalgae?

<div><p>Macroalgae harbor microbial communities whose bacterial biodiversity remains largely uncharacterized. The goals of this study were 1) to examine the composition of the bacterial community associated with <i>Porphyra umbilicalis</i> Kützing from Schoodic Point, ME, 2) determine whether there are seasonal trends in species diversity but a core group of bacteria that are always present, and 3) to determine how the microbial community associated with a laboratory strain (P.um.1) established in the presence of antibiotics has changed. <i>P. umbilicalis</i> blades (n = 5, fall 2010; n = 5, winter 2011; n = 2, clonal P.um.1) were analyzed by pyrosequencing over two variable regions of the 16 S rDNA (V5–V6 and V8; 147,880 total reads). The bacterial taxa present were classified at an 80% confidence threshold into eight phyla (Bacteroidetes, Proteobacteria, Planctomycetes, Chloroflexi, Actinobacteria, Deinococcus-Thermus, Firmicutes, and the candidate division TM7). The Bacteroidetes comprised the majority of bacterial sequences on both field and lab blades, but the Proteobacteria (Alphaproteobacteria, Gammaproteobacteria) were also abundant. Sphingobacteria (Bacteroidetes) and Flavobacteria (Bacteroidetes) had inverse abundances on natural versus P.um.1 blades. Bacterial communities were richer and more diverse on blades sampled in fall compared to winter. Significant differences were observed between microbial communities among all three groups of blades examined. Only two OTUs were found on all 12 blades, and only one of these, belonging to the Saprospiraceae (Bacteroidetes), was abundant. <i>Lewinella</i> (as 66 OTUs) was found on all field blades and was the most abundant genus. Bacteria from the Bacteroidetes, Proteobacteria and Planctomycetes that are known to digest the galactan sulfates of red algal cell walls were well-represented. Some of these taxa likely provide essential morphogenetic and beneficial nutritive factors to <i>P. umbilicalis</i> and may have had unexpected effects upon evolution of macroalgal form as well as function.</p> </div

Bacterial community recovered in the V8 library from <i>Porphyra umbilicalis</i> in fall (n = 5 blades), winter (n = 5 blades) and lab P.um.1 (n = 2 blades).

<p>Pie-charts for the mean proportion of A) sequences, B) OTUs (0.03 distance), and C) sequences in normalized samples that belong to particular phyla and classes.</p

Biplot of all blade samples in a nmds ordination (here showing an axis1-axis 3 plot) with blade OTUs that belong to the core microbiome described from the V8 library.

<p>Vectors pointing toward “axis 3” are associated with winter samples; vectors pointing toward the bottom “axis 1” label are associated with fall samples, and vectors pointing away from the “axis 1” label are associated with laboratory samples. The OTU's length indicates the strength of the association, and the direction indicates the direction of the effect; the relative positions of each sample (n = 12) are plotted in the biplot. Numbers for OTUs in the figure correspond to OTU reference numbers as follows and 9 OTUs are significantly associated with an axis at <i>p</i><0.05: 1 = OTU 1982; 2 = OTU 2387; 3 = OTU 2410; 4 = OTU 2427; 5 = OTU 2434; 6 = OTU 2521; 7 = OTU 2525; 8 = OTU 2493(NS); 9 = OTU 2408; 10 = OTU 1933(NS); 11 = OTU 2219(NS); 12 = OTU 2494(NS); 13 = OTU 2225; for taxonomic classification see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058269#pone-0058269-t004" target="_blank">Table 4</a>; also see Table S9 for significantly associated OTUs.</p

Dendrogram of the microbial community on each blade in the V8 library as clustered (thetayc distance matrix) to examine dissimilarity by group or by position along the intertidal transect for fall and winter blades.

<p>Dendrogram of the microbial community on each blade in the V8 library as clustered (thetayc distance matrix) to examine dissimilarity by group or by position along the intertidal transect for fall and winter blades.</p

OTUs richness and diversity.

<p>Richness (A) of V8 OTUs (0.03 distance) observed and those predicted (Chao1) for samples before normalization and diversity (B) calculated for samples before and after normalization with the inverse Simpson index.</p

Rarefaction analysis of V8 samples from each fall, winter and lab blade showing the mean (±95% C.I.) number of OTUs (0.03 distance) as a function of sequencing depth.

<p>Note the overlap of curves for F1, F3, F5 (top); W1, W3, W5 (middle); L1, L2 (bottom). Panel A contains the analysis for samples before normalization; Panel B shows rarefaction analysis for the normalized samples.</p

Taxonomic summary of named genera found in the V5V6 or V8 libraries (I) and taxa found in only one library (V5V6, II; V8, III).

*<p>Not present in laboratory blades</p