Metatranscriptome Library Preparation Influences Analyses of Viral Community Activity During a Brown Tide Bloom

There is growing interest in the use of metatranscriptomics to study virus community dynamics. We used RNA samples collected from harmful brown tides caused by the eukaryotic alga Aureococcus anophagefferens within New York (United States) estuaries and in the process observed how preprocessing of libraries by either selection for polyadenylation or reduction in ribosomal RNA (rRNA) influenced virus community analyses. As expected, more reads mapped to the A. anophagefferens genome in polyadenylation-selected libraries compared to the rRNA-reduced libraries, with reads mapped in each sample correlating to one another regardless of preprocessing of libraries. Yet, this trend was not seen for reads mapping to the Aureococcus anophagefferens Virus (AaV), where significantly more reads (approximately two orders of magnitude) were mapped to the AaV genome in the rRNA-reduced libraries. In the rRNA-reduced libraries, there was a strong and significant correlation between reads mappings to AaV and A. anophagefferens. Overall, polyadenylation-selected libraries produced fewer viral contigs, fewer reads mapped to viral contigs, and different proportions across viral realms and families, compared to their rRNA-reduced pairs. This study provides evidence that libraries generated by rRNA reduction and not selected for polyadenylation are more appropriate for quantitative characterization of viral communities in aquatic ecosystems by metatranscriptomics.

A Novel Immunofluorescence Flow Cytometry Technique Detects the Expansion of Brown Tides Caused by Aureoumbra lagunensis to the Caribbean Sea

ABSTRACTDuring the past 3 decades, brown tides caused by the pelagophytesAureococcus anophagefferensandAureoumbra lagunensishave caused ecological and economic damage to coastal ecosystems across the globe. While blooms ofA. lagunensishad previously been confined to Texas, in 2012, an expansive brown tide occurred on Florida's East Coast, causing widespread disruption within the Indian River and Mosquito Lagoons and generating renewed interest in this organism. A major impediment to detailed investigations ofA. lagunensisin an ecosystem setting has been the absence of a rapid and reliable method for cell quantification. The combination of their small size (3 to 5 μm) and nondescript extracellular features makes identification and enumeration of these cells with conventional methods a challenge. Here we report the development of an immunological-based flow cytometry method that uses a fluorescently labeled antibody developed againstA. lagunensis. This method is species specific, sensitive (detection limit of 1.5 × 103cells ml−1), precise (1% relative standard deviation of replicated samples), and accurate (108% ± 8% recovery of spiked samples) over a wide range of cell concentrations. Furthermore, this method effectively quantifiesA. lagunensisin both glutaraldehyde- and formalin-preserved samples, yields a high throughput of samples (∼35 samples h−1), and is cost-effective, making it an ideal tool for managers and scientists. This method successfully documented the recurrence of a brown tide bloom in Florida in 2013. Bloom densities were highest in June (>2.0 × 106cells ml−1) and spanned >60 km from the Ponce de Leon inlet in the northern Mosquito Lagoon south to Titusville in the Indian River Lagoon. Low levels ofA. lagunensiscells were found >250 km south of this region. This method also quickly and accurately identifiedA. lagunensisas the causative agent of a 2013 brown tide bloom in Guantanamo Bay, Cuba, and thus should prove useful for both quantifying the dynamics of ongoing blooms ofA. lagunensisas well as documenting new outbreaks of this harmful alga.