DNA metabarcoding para análise da microbiota aquática em um experimento de mesocosmos

Abstract

Aquatic microbiota are very important in aquatic environments and perform essential ecosystem functions, such as nutrient cycling and waste decomposition. A group of organisms that make up this complex biota is the phytoplankton community. For the phytoplankton community, higher temperatures and high concentrations of nutrients such as phosphorus can lead to an increase in the biomass of algae, especially cyanobacteria, reducing biodiversity and impacting the entire aquatic ecosystem. The use of advanced molecular technologies and techniques, such as DNA metabarcoding, has enabled greater knowledge about the biodiversity of the aquatic microbiota. In this study, two rDNA regions were analyzed in order to investigate how temperature rise and trophic state variation affect the richness and composition of phytoplankton communities of prokaryotes and microeucariots in mesocosms. We employed various methods to examine these taxa (OTUs and ASVs) and evaluate the statistical pattern of response across three data sets: Total OTUs/ASVs, OTUs/ASVs classified in databases, and OTUs/ASVs classified and manually filtered for the phytoplankton community with the help of scientific articles. The samples were examined from a mesocosm experiment where various temperature and trophic state conditions were simulated. The DNA was extracted and quantified and the 16S V3-V4 and 18S V4 rDNA regions were amplified to access prokaryotic and eukaryotic organisms respectively. The amplified products were prepared and sequenced on the Illumina MiSeq platform using the 600-cycle Illumina MiSeq kit. In bioinformatics analyses, we follow the PIMBA pipeline for the identification and classification of OTUs and ASVs. A manual inspection was performed to filter the OTUs and ASVs belonging to the phytoplankton. We conducted an ANOVA and PERMANOVA to evaluate the richness and composition of OTUs and ASVs under different treatments. It was observed that there were differences in the results obtained for OTUs and ASVs in certain cases. However, we noticed that ASVs generate an inflation in the results of wealth and composition. This leads us to conclude that the OTUs approach is more appropriate when the objective is to approach the species level to evaluate aquatic microbiota communities. Our findings showed that the increase in nutrient load was harmful to richness, as it encourages the selection of specific organisms that are more suited to eutrophic environments, which, in turn, affects the composition and structure of the community of prokaryotes and phytoplankton eukaryotes. The richness and composition of OTUs and ASVs for cyanobacteria were not significantly altered by the temperature range between 25 °C and 28 °C, suggesting that the species present are well adapted to this temperature variability. For microeucariots, temperature had a positive influence on OTUs richness and no influence on ASVs richness.Temperature did not have any impact on the composition of OTUs between treatments, but the complex relationship between temperature and nutrients was crucial to distinguish these communities. In general, we saw that eutrophication was more harmful to phytoplankton communities than the increase in temperature at 3ºC.