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Viruses are the most abundant life forms in the world's oceans and they are key drivers of biogeochemical cycles, but their impact on the microbial assemblages inhabiting hydrothermal vent ecosystems is still largely unknown. Here, we analysed the viral life strategies and virus-host interactions in the sediments of a newly discovered shallow-water hydrothermal field of the Mediterranean Sea. Our study reveals that temperate viruses, once experimentally induced to replicate, can cause large mortality of vent microbes, significantly reducing the chemoautotrophic carbon production, while enhancing the metabolism of microbial heterotrophs and the re-cycling of the organic matter. These results provide new insights on the factors controlling primary and secondary production processes in hydrothermal vents, suggesting that the inducible provirus-host interactions occurring in these systems can profoundly influence the functioning of the microbial food web and the efficiency in the energy transfer to the higher trophic levels.

Viruses profoundly influence benthic marine ecosystems by infecting and subsequently killing their prokaryotic hosts, thereby impacting the cycling of carbon and nutrients. Previously conducted studies, based on different methodologies, have provided widely differing estimates of the relevance of viruses on benthic prokaryotes. There has been no attempt so far to compare these independent approaches, including contextual comparisons among different approaches for sample manipulation (i.e., dilution or not of the sediments during incubations), between methods based on epifluorescence microscopy (EFM) or radiotracers, and between the use of different radiotracers. Therefore, it has been difficult to identify the most suitable methodologies and protocols to be used as standard approaches for the quantification of viral infections of prokaryotes. Here, we compared for the first time different methods for determining viral and prokaryotic production rates in marine sediments collected at two benthic sites, differing in depth and environmental conditions. We used a highly replicated experimental design, testing the potential biases associated to the incubation of sediments as diluted or undiluted. In parallel, we also compared EFM counts with the 3H-thymidine incubations for the determination of viral production rates, and the use of 3H-thymidine versus 3H-leucine radiotracers for the determination of prokaryotic production. We show here that, independent from sediment dilution, EFM-based values of viral production ranged from 1.4 to 4.6 x 107 viruses g-1 h-1, and were similar but overall less variable compared to those obtained by the 3H-thymidine method (0.3 to 9.0 x 107 viruses g-1h-1). In addition, the prokaryotic production rates were not affected by sediment dilution, and the use of different radiotracers provided very consistent estimates (10.3–35.1 and 9.3–34.6 ngC g-1h-1 using the 3H-thymidine or 3H-leucine method, respectively). These results indicated that viral lysis was responsible for the abatement of 55–81% of the prokaryotic heterotrophic production, corroborating previous findings of the major role of viruses in benthic deep-sea ecosystems. Moreover, our methodological comparison for the analysis of viral production in marine sediments suggests that microscopy-based approaches are simpler and more cost-effective than those based on radiotracers. These approaches also reduce time to results and overcome issues related to generation of radioactive waste.

Molecular approaches that target the total DNA pool recovered from permanently anoxic marine ecosystems have revealed an extraordinary diversity of prokaryotes and unicellular eukaryotes. However, the presence of gene sequences contained within the extracellular DNA pool is still largely neglected. We have investigated the preservation, origin and genetic imprint of extracellular DNA recovered from permanently anoxic deep-sea sediments of the Black Sea. Despite high DNase activities, huge amounts of total extracellular DNA were found in both the surface and subsurface sediment layers, suggesting reduced availability of the extracellular DNA pool to nuclease degradation. The reduced degradation of the total extracellular DNA was confirmed by its low decay rate and the high accumulation in the deeper sediment layers. The copy numbers of 16S and 18S rDNA contained within the extracellular DNA pool in both the surface and subsurface sediment layers was very high, indicating that permanently anoxic sediments of the deep Black Sea are hot spots of preserved extracellular gene sequences. The extracellular DNA recovered from these sediment layers also contained highly diversified 18S rDNA sequences. These were not only representative of the major protistan lineages, but also of new very divergent lineages, branching as independent clades at the base of the tree. Our findings indicate that the extracellular DNA pool is a major archive of present/past eukaryotic gene sequences, and they highlight the importance of integrating molecular cell-oriented approaches with molecular analyses of the extracellular DNA pool, for a better assessment of microbial diversity and temporal changes in marine benthic ecosystems. © 2010 Blackwell Publishing Ltd.

Although nematodes play an important ecological role in all major ecosystems, their diversity is still largely unknown. The gap between ‘known’ and estimated species richness may be due to the limitations of morphological identification which is difficult, laborious and requires taxonomic expertise, meaning that biodiversity of the group is typically underestimated. To better understand the relationships between biodiversity and ecosystem functioning is crucially important a full assessment of biodiversity. This is particularly important for marine species and small-sized metazoa. The advent of molecular techniques and overall the development of next generation sequencing techniques (i.e. 454 sequencing) provide new perspectives to investigate the biodiversity of nematode assemblages.

Viruses have been recognized as the most abundant and diversified component of aquatic environments. The development of metagenomic approaches, indeed, has provided an unprecedented level of access to viral genomes from different environments, making it possible to characterize their taxonomic and functional diversity. Despite this, viral diversity in benthic deep-­-sea ecosystems is still completely unknown. Multiple bioinformatic tools were leveraged to analyze viral sequences in environmental samples and evaluate performance in virome analyses. In this study we compared three metagenomic sequence annotation pipelines (MG-­-RAST, VMGAP, MetaVir) to test their efficiency in the analysis of viromes from different deep-­-sea sediment samples. MetaVir identified a higher number of viral strains compared to MG-­-RAST and VMGAP (80% vs.45-­-55%, respectively) and a different pattern of assemblage composition. Instead, contrary to MetaVir, MG-­-RAST and VMGAP allowed the identification of putative proteins and functions providing important information on the hypothetical metabolic role of the viromes analysed. This study indicates that the integration of information from different bioinformatic tools can help to shed light on the enormous genetic diversity contained in viruses inhabiting the largest ecosystem of Earth.

Metagenetics allows the study of marine biodiversity through sequencing of a target gene shared by a pool of different species of organisms. This approach is useful to study the origin of invasive and cosmopolite species and to identify unknown, cryptic and of the species for which classical taxonomic tools are limited. Nematodes, the dominant meiofaunal taxon (up to 90% of meiofauna in the deep sea), have been largely utilized as indicators of environmental changes due to their ubiquity, high abundance, high taxonomic and functional diversity. However, so far, the biodiversity of free-living deep-sea nematodes has been studied almost exclusively on the basis of their morphological features, not allowing to recognize cryptic species and, thus, effecting the actual estimates of biodiversity. In the present study we set up and optimized a molecular approach, based on DNA extraction and purification of nematodes recovered from different culture, coastal and deep-sea sediments worldwide. Pyrosequencing technology of the eukaryotic 18S rRNA gene was utilized to evaluate the estimates of the nematode biodiversity, which were compared to those obtained by using the classical taxonomic approach.

We have investigated here the structure of the pelagic microbial food web and quantified the carbon fluxes from viruses to microplankton along trophic gradients in the Mediterranean Sea. To explore the complex trophic pathways of the pelagic food web, we conducted independent and replicated experiments to measure (i) predation on prokaryotes by microzooplankton, (ii) predation on prokaryotes by heterotrophic nanoflagellates, (iii) virus-induced prokaryotic mortality, and (iv) microzooplankton grazing on nanoplankton and microphytoplankton. Our study covered more than 5000 km, from the Atlantic Ocean to the Levantine basin, and from conditions of high primary production and nutrient availability to ultraoligotrophic and phosphate-limited waters. Microphytoplankton abundance and biomass were typically scarce across the entire Mediterranean basin, with almost negligible levels in the eastern part. Also, nanoplankton biomass was typically low. Conversely, prokaryotes, and particularly the heterotrophic components, were abundant and represented the only significant food source for both nanoplankton and microplankton grazers. Viral infections were not the primary agents of prokaryotic mortality, but in some areas, such as the Ligurian Sea, they had a key role in prokaryotic dynamics. The scenario depicted in this study in summer reveals the pivotal role of microzooplankton in the pelagic food web of the Mediterranean Sea, with a key role in the potential transfer of biomass to higher trophic levels. We also show that converse to theoretical expectations, the microbial food web was relatively complex under the mesotrophic conditions (Atlantic and western Mediterranean) and was much more simplified in the ultraoligotrophic conditions of the eastern Mediterranean.