Microbial community functioning at hypoxic sediments revealed by targeted metagenomics and RNA stable isotope probing

Microorganisms are instrumental to the structure and functioning of marine ecosystems and to the chemistry of the ocean due to their essential part in the cycling of the elements and in the recycling of the organic matter. Two of the most critical ocean biogeochemical cycles are those of nitrogen and sulfur, since they can influence the synthesis of nucleic acids and proteins, primary productivity and microbial community structure. Oxygen concentration in marine environments is one of the environmental variables that have been largely affected by anthropogenic activities; its decline induces hypoxic events which affect benthic organisms and fisheries. Hypoxia has been traditionally defined based on the level of oxygen below which most animal life cannot be sustained. Hypoxic conditions impact microbial composition and activity since anaerobic reactions and pathways are favoured, at the expense of the aerobic ones. Naturally occurring hypoxia can be found in areas where water circulation is restricted, such as coastal lagoons, and in areas where oxygen-depleted water is driven into the continental shelf, i.e. coastal upwelling regions. Coastal lagoons are highly dynamic aquatic systems, particularly vulnerable to human activities and susceptible to changes induced by natural events. For the purpose of this PhD project, the lagoonal complex of Amvrakikos Gulf, one of the largest semi-enclosed gulfs in the Mediterranean Sea, was chosen as a study site. Coastal upwelling regions are another type of environment limited in oxygen, where also formation of oxygen minimum zones (OMZs) has been reported. Sediment in upwelling regions is rich in organic matter and bottom water is often depleted of oxygen because of intense heterotrophic respiration. For the purpose of this PhD project, the chosen coastal upwelling system was the Benguela system off Namibia, situated along the coast of south western Africa. The aim of this PhD project was to study the microbial community assemblages of hypoxic ecosystems and to identify a potential link between their identity and function, with a particular emphasis on the microorganisms involved in the nitrogen and sulfur cycles. The methodology that was applied included targeted metagenomics and RNA stable isotope probing (SIP). It has been shown that the microbial community diversity pattern can be differentiated based on habitat type, i.e. between riverine, lagoonal and marine environments. Moreover, the studied habitats were functionally distinctive. Apart from salinity, which was the abiotic variable best correlated with the microbial community pattern, oxygen concentration was highly correlated with the predicted metabolic pattern of the microbial communities. In addition, when the total number of Operational Taxonomic Units (OTUs) was taken into consideration, a negative linear relationship with salinity was identified (see Chapter 2). Microbial community diversity patterns can also be differentiated based on the lagoon under study since each lagoon hosts a different sulfate-reducing microbial (SRM) community, again highly correlated with salinity. Moreover, the majority of environmental terms that characterized the SRM communities were classified to the marine biome, but terms belonging to the freshwater or brackish biomes were also found in stations were a freshwater effect was more evident (see Chapter 3). Taxonomic groups that were expected to be thriving in the sediments of the Benguela coastal upwelling system were absent or present but in very low abundances. Epsilonproteobacteria dominated the anaerobic assimilation of acetate as confirmed by their isotopic enrichment in the SIP experiments. Enhancement of known sulfate-reducers was not achieved under sulfate addition, possibly due to competition for electron donors among nitrate-reducers and sulfate-reducers, to the inability of certain sulfate-reducing bacteria to use acetate as electron donor or to the short duration of the incubations (see Chapter 4). Future research should focus more on the community functioning of such habitats; an increased understanding of the biogeochemical cycles that characterize these hypoxic ecosystems will perhaps allow for predictions regarding the intensity and direction of the cycling of elements, especially of nitrogen and sulfur given their biological importance. Regulation of hypoxic episodes will aid the end-users of these ecosystems to possibly achieve higher productivity, in terms of fish catches, which otherwise is largely compromised by the elevated hydrogen sulfide concentrations

Macrofaunal assemblages associated with the sponge Sarcotragus foetidus Schmidt, 1862 (Porifera: Demospongiae) at the coasts of Cyprus and Greece

Background: This paper describes a dataset of macrofaunal organisms associated with the sponge Sarcotragus foetidus Schmidt, 1862, collected by scuba diving from two sampling sites: one in Greece (North Aegean Sea) and one in Cyprus (Levantine Sea). New information: This dataset includes macrofaunal taxa inhabiting the demosponge Sarcotragus foetidus and contributes to the ongoing efforts of the Ocean Biogeographic Information System (OBIS) which aims at filling the gaps in our current knowledge of the world's oceans. This is the first paper, to our knowledge, where the macrofauna associated with S. foetidus from the Levantine Basin is being recorded. In total, 90 taxa were recorded, from which 83 were identified to the species level. Eight of these species are new records for the Levantine Basin. The dataset contains 213 occurrence records, fully annotated with all required metadata. It is accessible at http://lifewww-00.her.hcmr.gr:8080/medobis/resource.do?r=organismic_assemblages_sarcotragus_foetidus_cyprus_greec

Environmental variability and heavy metal concentrations from five lagoons in the Ionian Sea (Amvrakikos Gulf, W Greece)

Background: Coastal lagoons are ecosystems of major importance as they host a number of species tolerant to disturbances and they are highly productive. Therefore, these ecosystems should be protected to ensure stability and resilience. The lagoons of Amvrakikos Gulf form one of the most important lagoonal complexes in Greece. The optimal ecological status of these lagoons is crucial for the well-being of the biodiversity and the economic prosperity of the local communities. Thus, monitoring of the area is necessary to detect possible sources of disturbance and restore stability. New information: The environmental variables and heavy metals concentrations, from five lagoons of Amvrakikos Gulf were measured from seasonal samplings and compared to the findings of previous studies in the area, in order to check for possible sources of disturbance. The analysis, showed that i) the values of the abiotic parameters vary with time (season), space (lagoon) and with space over time; ii) the variability of the environmental factors and enrichment in certain elements is naturally induced and no source of contamination is detected in the lagoons

Phytoplankton community composition in relation to environmental variability in the Urdaibai estuary (SE Bay of Biscay): Microscopy and eDNA metabarcoding

Phytoplankton monitoring is essential for the global understanding of aquatic ecosystems. The present research studies the phytoplankton community of the Urdaibai estuary, combining microscopy and eDNA metabarcoding for the first time in the area. The main aims were to describe the phytoplankton community composition in relation to the environmental conditions of the estuary, and to compare the two methods used. Diatoms Minutocellus polymorphus and Chaetoceros tenuissimus dominated the outer estuary, being replaced by Teleaulax acuta (cryptophyte), Kryptoperidinium foliaceum (dinoflagellate) and Cyclotella spp. (diatom) towards the inner area. This change was mainly prompted by salinity and nutrients. Metabarcoding revealed the presence of 223 species that were not observed by microscopy in previous studies in the estuary. However, several characteristic species (e.g., K. foliaceum) were only detected with microscopy. Additionally, microscopy covered the limitations of eDNA metabarcoding concerning quantification. Thus, to give a full insight, a combination of techniques is recommended.This research was supported in part through computational resources provided by IMBBC (Institute of Marine Biology, Biotechnology and Aquaculture) of the HCMR (Hellenic Centre of Marine Research). Funding for establishing the IMBBC HPC has been received by the MARBIGEN (EU Regpot) project, LifeWatchGreece RI and the CMBR (Center for the study and sustainable exploitation of Marine Biological Resources) RI. In addition, this study was partially supported by the project PPG17/67 funded by the University of the Basque Country (UPV/EHU) and J. Bilbao was funded by a grant from the University of the Basque Country (UPV/EHU – PIF 18/306). This paper is contribution number 1181 from AZTI (Marine Research Division)

Metagenomics : tools and insights for analyzing next-generation sequencing data derived from biodiversity studies

Advances in next-generation sequencing (NGS) have allowed significant breakthroughs in microbial ecology studies. This has led to the rapid expansion of research in the field and the establishment of “metagenomics”, often defined as the analysis of DNA from microbial communities in environmental samples without prior need for culturing. Many metagenomics statistical/computational tools and databases have been developed in order to allow the exploitation of the huge influx of data. In this review article, we provide an overview of the sequencing technologies and how they are uniquely suited to various types of metagenomic studies. We focus on the currently available bioinformatics techniques, tools, and methodologies for performing each individual step of a typical metagenomic dataset analysis. We also provide future trends in the field with respect to tools and technologies currently under development. Moreover, we discuss data management, distribution, and integration tools that are capable of performing comparative metagenomic analyses of multiple datasets using well-established databases, as well as commonly used annotation standards

Polytraits : a database on biological traits of marine polychaetes

The study of ecosystem functioning – the role which organisms play in an ecosystem – is becoming increasingly important in marine ecological research. The functional structure of a community can be represented by a set of functional traits assigned to behavioural, reproductive and morphological characteristics. The collection of these traits from the literature is however a laborious and time-consuming process, and gaps of knowledge and restricted availability of literature are a common problem. Trait data are not yet readily being shared by research communities, and even if they are, a lack of trait data repositories and standards for data formats leads to the publication of trait information in forms which cannot be processed by computers. This paper describes Polytraits (http://polytraits.lifewatchgreece.eu), a database on biological traits of marine polychaetes (bristle worms, Polychaeta: Annelida). At present, the database contains almost 20,000 records on morphological, behavioural and reproductive characteristics of more than 1,000 marine polychaete species, all referenced by literature sources. All data can be freely accessed through the project website in different ways and formats, both human-readable and machine-readable, and have been submitted to the Encyclopedia of Life for archival and integration with trait information from other sources

Optimized R functions for analysis of ecological community data using the R virtual laboratory (RvLab)

Background: Parallel data manipulation using R has previously been addressed by members of the R community, however most of these studies produce ad hoc solutions that are not readily available to the average R user. Our targeted users, ranging from the expert ecologist/microbiologists to computational biologists, often experience difficulties in finding optimal ways to exploit the full capacity of their computational resources. In addition, improving performance of commonly used R scripts becomes increasingly difficult especially with large datasets. Furthermore, the implementations described here can be of significant interest to expert bioinformaticians or R developers. Therefore, our goals can be summarized as: (i) description of a complete methodology for the analysis of large datasets by combining capabilities of diverse R packages, (ii) presentation of their application through a virtual R laboratory (RvLab) that makes execution of complex functions and visualization of results easy and readily available to the end-user. New information: In this paper, the novelty stems from implementations of parallel methodologies which rely on the processing of data on different levels of abstraction and the availability of these processes through an integrated portal. Parallel implementation R packages, such as the pbdMPI (Programming with Big Data – Interface to MPI) package, are used to implement Single Program Multiple Data (SPMD) parallelization on primitive mathematical operations, allowing for interplay with functions of the vegan package. The dplyr and RPostgreSQL R packages are further integrated offering connections to dataframe like objects (databases) as secondary storage solutions whenever memory demands exceed available RAM resources. The RvLab is running on a PC cluster, using version 3.1.2 (2014-10-31) on a x86_64-pc-linux-gnu (64-bit) platform, and offers an intuitive virtual environmet interface enabling users to perform analysis of ecological and microbial communities based on optimized vegan functions. A beta version of the RvLab is available after registration at: https://portal.lifewatchgreece.eu

ENVIRONMENTS and EOL : identification of Environment Ontology terms in text and the annotation of the Encyclopedia of Life

© The Author(s), 2015. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Bioinformatics 31 (2015): 1872-1874, doi:10.1093/bioinformatics/btv045.The association of organisms to their environments is a key issue in exploring biodiversity patterns. This knowledge has traditionally been scattered, but textual descriptions of taxa and their habitats are now being consolidated in centralized resources. However, structured annotations are needed to facilitate large-scale analyses. Therefore, we developed ENVIRONMENTS, a fast dictionary-based tagger capable of identifying Environment Ontology (ENVO) terms in text. We evaluate the accuracy of the tagger on a new manually curated corpus of 600 Encyclopedia of Life (EOL) species pages. We use the tagger to associate taxa with environments by tagging EOL text content monthly, and integrate the results into the EOL to disseminate them to a broad audience of users.The Encyclopedia Of Life Rubenstein Fellows Program [CRDF EOL-33066-13/E33066], the LifeWatchGreece Research Infrastructure [384676-94/GSRT/ NSRF(C&E)] and the Novo Nordisk Foundation Center for Protein Research [NNF14CC0001]

Seqenv : linking sequences to environments through text mining

Understanding the distribution of taxa and associated traits across different environments is one of the central questions in microbial ecology. High-throughput sequencing (HTS) studies are presently generating huge volumes of data to address this biogeographical topic. However, these studies are often focused on specific environment types or processes leading to the production of individual, unconnected datasets. The large amounts of legacy sequence data with associated metadata that exist can be harnessed to better place the genetic information found in these surveys into a wider environmental context. Here we introduce a software program, seqenv, to carry out precisely such a task. It automatically performs similarity searches of short sequences against the ‘‘nt’’ nucleotide database provided by NCBI and, out of every hit, extracts–if it is available–the textual metadata field. After collecting all the isolation sources from all the search results, we run a text mining algorithm to identify and parse words that are associated with the Environmental Ontology (EnvO) controlled vocabulary. This, in turn, enables us to determine both in which environments individual sequences or taxa have previously been observed and, by weighted summation of those results, to summarize complete samples. We present two demonstrative applications of seqenv to a survey of ammonia oxidizing archaea as well as to a plankton paleome dataset from the Black Sea. These demonstrate the ability of the tool to reveal novel patterns in HTS How to cite this article Sinclair et al. (2016), Seqenv: linking sequences to environments through text mining. PeerJ 4:e2690; DOI 10.7717/peerj.2690 and its utility in the fields of environmental source tracking, paleontology, and studies of microbial biogeography

Wide-Geographic and Long-Term Analysis of the Role of Pathogens in the Decline of Pinna nobilis to Critically Endangered Species

A mass mortality event (MME) affecting the fan mussel Pinna nobilis was first detected in Spain in autumn 2016 and spread north- and eastward through the Mediterranean Sea. Various pathogens have been blamed for contributing to the MME, with emphasis in Haplosporidium pinnae, Mycobacterium sp. and Vibrio spp. In this study, samples from 762 fan mussels (necropsies from 263 individuals, mantle biopsies from 499) of various health conditions, with wide geographic and age range, taken before and during the MME spread from various environments along Mediterranean Sea, were used to assess the role of pathogens in the MME. The number of samples processed by both histological and molecular methods was 83. The most important factor playing a main role on the onset of the mass mortality of P. nobilis throughout the Mediterranean Sea was the infection by H. pinnae. It was the only non-detected pathogen before the MME while, during MME spreading, its prevalence was higher in sick and dead individuals than in asymptomatic ones, in MME-affected areas than in non-affected sites, and it was not associated with host size, infecting both juveniles and adults. Conversely, infection with mycobacteria was independent from the period (before or during MME), from the affection of the area by MME and from the host health condition, and it was associated with host size. Gram (-) bacteria neither appeared associated with MME.En prens