Year-round dynamics of amplicon sequence variant communities differ among eukaryotes, Imitervirales and prokaryotes in a coastal ecosystem.

Coastal microbial communities are affected by seasonal environmental change, biotic interactions and fluctuating nutrient availability. We investigated the seasonal dynamics of communities of eukaryotes, a major group of double-stranded DNA viruses that infect eukaryotes (order Imitervirales; phylum Nucleocytoviricota), and prokaryotes in the Uranouchi Inlet, Kochi, Japan. We performed metabarcoding using ribosomal RNA genes and viral polB genes as markers in 43 seawater samples collected over 20 months. Eukaryotes, prokaryotes and Imitervirales communities characterized by the compositions of amplicon sequence variants (ASVs) showed synchronic seasonal cycles. However, the community dynamics showed intriguing differences in several aspects, such as the recovery rate after a year. We also showed that the differences in community dynamics were at least partially explained by differences in recurrence/persistence levels of individual ASVs among eukaryotes, prokaryotes and Imitervirales. Prokaryotic ASVs were the most persistent, followed by eukaryotic ASVs and Imitervirales ASVs, which were the least persistent. We argue that the differences in the specificity of interactions (virus-eukaryote vs prokaryote-eukaryote) as well as the niche breadth of community members were at the origin of the distinct community dynamics among eukaryotes, their viruses and prokaryotes.

An Optimized Metabarcoding Method for Mimiviridae.

Mimiviridae is a group of viruses with large genomes and virions. Ecological relevance of Mimiviridae in marine environments has been increasingly recognized through the discoveries of novel isolates and metagenomic studies. To facilitate ecological profiling of Mimiviridae, we previously proposed a meta-barcoding approach based on 82 degenerate primer pairs (i.e., MEGAPRIMER) targeting the DNA polymerase gene of Mimiviridae. The method detected a larger number of operational taxonomic units (OTUs) in environmental samples than previous methods. However, it required large quantities of DNA and was laborious due to the use of individual primer pairs. Here, we examined coastal seawater samples using varying PCR conditions and purification protocols to streamline the MEGAPRIMER method. Mixing primer pairs in "cocktails" reduced the required amount of environmental DNA by 90%, while reproducing the results obtained by the original protocol. We compared the results obtained by the meta-barcoding approach with quantifications using qPCR for selected OTUs. This revealed possible amplification biases among different OTUs, but the frequency profiles for individual OTUs across multiple samples were similar to those obtained by qPCR. We anticipate that the newly developed MEGAPRIMER protocols will be useful for ecological investigation of Mimiviridae in a larger set of environmental samples.

Human amniotic membrane as newly identified source of amniotic fluid pulmonary surfactant.

Pulmonary surfactant (PS) reduces surface tension at the air-liquid interface in the alveolar epithelium of the lung, which is required for breathing and for the pulmonary maturity of the developing foetus. However, the origin of PS had never been thoroughly investigated, although it was assumed to be secreted from the foetal developing lung. Human amniotic membrane (hAM), particularly its epithelial cell layer, composes the amniotic sac enclosing the amniotic fluid. In this study, we therefore aimed to investigate a potential contribution of the cellular components of the hAM to pulmonary surfactant found in amniotic fluid. We identified that cells within the native membrane contain lamellar bodies and express all four surfactant proteins as well as ABCA3. Lipidomic profiling by nanoESI - MS/MS revealed the presence of the essential lipid species as found in PS. Also, the biophysical activity of conditioned cell culture supernatant obtained from hAM was tested with captive bubble surfactometry. hAM supernatant showed the ability to reduce surface tension, similar to human PS obtained from bronchoalveolar lavage. This means that hAM produces the essential PS-associated components and can therefore contribute as second potential source of PS in amniotic fluid aside from the foetal lung.