Exploring the Potential of Metatranscriptomics to Describe Microbial Communities and Their Effects in Molluscs.

Metatranscriptomics has emerged as a very useful technology for the study of microbiomes from RNA-seq reads. This method provides additional information compared to the sequencing of ribosomal genes because the gene expression can also be analysed. In this work, we used the metatranscriptomic approach to study the whole microbiome of mussels, including bacteria, viruses, fungi, and protozoans, by mapping the RNA-seq reads to custom assembly databases (including the genomes of microorganisms publicly available). This strategy allowed us not only to describe the diversity of microorganisms but also to relate the host transcriptome and microbiome, finding the genes more affected by the pathogen load. Although some bacteria abundant in the metatranscriptomic analysis were undetectable by 16S rRNA sequencing, a common core of the taxa was detected by both methodologies (62% of the metatranscriptomic detections were also identified by 16S rRNA sequencing, the Oceanospirillales, Flavobacteriales and Vibrionales orders being the most relevant). However, the differences in the microbiome composition were observed among different tissues of Mytilus galloprovincialis, with the fungal kingdom being especially diverse, or among molluscan species. These results confirm the potential of a meta-analysis of transcriptome data to obtain new information on the molluscs' microbiome.

High-throughput sequencing as a tool for monitoring prokaryote communities in a wastewater treatment plant.

In this study, the DNA metabarcoding technique was used to explore the prokaryote diversity and community structure in wastewater collected in spring and winter 2020-2021 as well as the efficiency of the treatment in a wastewater treatment plant (WWTP) in Ría de Vigo (NW Spain). The samplings included raw wastewater from the inlet stream (M1), the discharge water after the disinfection treatment (M3) and mussels used as bioindicators of possible contamination of the marine environment. Significant differences were discovered in the microbiome of each type of sample (M1, M3 and mussels), with 92 %, 45 % and 44 % of exclusive OTUs found in mussel, M3 and M1 samples respectively. Seasonal differences were also detected in wastewater samples, with which abiotic parameters (temperature, pH) could be strongly involved. Bacteria present in raw wastewater (M1) were associated with the human gut microbiome, and therefore, potential pathogens that could be circulating in the population in specific periods were detected (e.g., Arcobacter sp. and Clostridium sp.). A considerable decrease in putative pathogenic organisms from the M1 to M3 wastewater fractions and the scarce presence in mussels (<0.5 % total reads) confirmed the effectiveness of pathogen removal in the wastewater treatment plant. Our results showed the potential of the DNA metabarcoding technique for monitoring studies and confirmed its application in wastewater-based epidemiology (WBE) and environmental contamination studies. Although this technique cannot determine if the infective pathogens are present, it can characterize the microbial communities and the putative pathogens that are circulating through the population (microbiome of M1) and also confirm the efficacy of depuration treatment, which can directly affect the aquaculture sector and even human and veterinary health.

Wastewater and marine bioindicators surveillance to anticipate COVID-19 prevalence and to explore SARS-CoV-2 diversity by next generation sequencing: One-year study.

This study presents the results of SARS-CoV-2 surveillance in sewage water of 11 municipalities and marine bioindicators in Galicia (NW of Spain) from May 2020 to May 2021. An integrated pipeline was developed including sampling, pre-treatment and biomarker quantification, RNA detection, SARS-CoV-2 sequencing, mechanistic mathematical modeling and forecasting. The viral load in the inlet stream to the wastewater treatment plants (WWTP) was used to detect new outbreaks of COVID-19, and the data of viral load in the wastewater in combination with data provided by the health system was used to predict the evolution of the pandemic in the municipalities under study within a time horizon of 7 days. Moreover, the study shows that the viral load was eliminated from the treated sewage water in the WWTP, mainly in the biological reactors and the disinfection system. As a result, we detected a minor impact of the virus in the marine environment through the analysis of seawater, marine sediments and, wild and aquacultured mussels in the final discharge point of the WWTP.

High-Throughput Sequencing of Environmental DNA as a Tool for Monitoring Eukaryotic Communities and Potential Pathogens in a Coastal Upwelling Ecosystem.

The marine environment includes diverse microeukaryotic organisms that play important functional roles in the ecosystem. With molecular approaches, eukaryotic taxonomy has been improved, complementing classical analysis. In this study, DNA metabarcoding was performed to describe putative pathogenic eukaryotic microorganisms in sediment and marine water fractions collected in Galicia (NW Spain) from 2016 to 2018. The composition of eukaryotic communities was distinct between sediment and water fractions. Protists were the most diverse group, with the clade TSAR (Stramenopiles, Alveolata, Rhizaria, and Telonemida) as the primary representative organisms in the environment. Harmful algae and invasive species were frequently detected. Potential pathogens, invasive pathogenic organisms as well as the causative agents of harmful phytoplanktonic blooms were identified in this marine ecosystem. Most of the identified pathogens have a crucial impact on the aquacultural sector or affect to relevant species in the marine ecosystem, such as diatoms. Moreover, pathogens with medical and veterinary importance worldwide were also found, as well as pathogens that affect diatoms. The evaluation of the health of a marine ecosystem that directly affects the aquacultural sector with a zoonotic concern was performed with the metabarcoding assay.