Microbial diversity in four Mediterranean irciniid sponges.

This paper describes a dataset of microbial communities from four different sponge species: Irciniaoros (Schmidt, 1864), Irciniavariabilis (Schmidt, 1862), Sarcotragusspinosulus Schmidt, 1862 and Sarcotragusfasciculatus (Pallas, 1766). The examined sponges all belong to Demospongiae (Class); Keratosa (Subclass); Dictyoceratida (Order); Irciniidae (Family). Samples were collected by scuba diving at depths between 6-14 m from two sampling sites of rocky formations at the northern coast of Crete (Cretan Sea, eastern Mediterranean) and were subjected to metabarcoding for the V5-V6 region of the 16S rRNA gene.

Spotting disease disrupts the microbiome of infected purple sea urchins, Strongylocentrotus purpuratus.

BACKGROUND: Spotting disease infects a variety of sea urchin species across many different marine locations. The disease is characterized by discrete lesions on the body surface composed of discolored necrotic tissue that cause the loss of all surface appendages within the lesioned area. A similar, but separate disease of sea urchins called bald sea urchin disease (BSUD) has overlapping symptoms with spotting disease, resulting in confusions in distinguishing the two diseases. Previous studies have focus on identifying the underlying causative agent of spotting disease, which has resulted in the identification of a wide array of pathogenic bacteria that vary based on location and sea urchin species. Our aim was to investigate the spotting disease infection by characterizing the microbiomes of the animal surface and various tissues. RESULTS: We collected samples of the global body surface, the lesion surface, lesioned and non-lesioned body wall, and coelomic fluid, in addition to samples from healthy sea urchins. 16S rRNA gene was amplified and sequenced from the genomic DNA. Results show that the lesions are composed mainly of Cyclobacteriaceae, Cryomorphaceae, and a few other taxa, and that the microbial composition of lesions is the same for all infected sea urchins. Spotting disease also alters the microbial composition of the non-lesioned body wall and coelomic fluid of infected sea urchins. In our closed aquarium systems, sea urchins contracted spotting disease and BSUD separately and therefore direct comparisons could be made between the microbiomes from diseased and healthy sea urchins. CONCLUSION: Results show that spotting disease and BSUD are separate diseases with distinct symptoms and distinct microbial compositions.

Insights on Pinna nobilis population genetic structure in the Aegean and Ionian Sea.

The fan mussel Pinna nobilis Linnaeus, 1758 is an endemic species of the Mediterranean Sea, protected by international agreements. It is one of the largest bivalves in the world, playing an important role in the benthic communities; yet it has been recently characterized as Critically Endangered by the IUCN, due to mass mortality events. In this context, the assessment of the genetic variation of the remaining P. nobilis populations and the evaluation of connectivity among them are crucial elements for the conservation of the species. For this purpose, samples were collected from six regions of the Eastern Mediterranean Sea; the Islands of Karpathos, Lesvos and Crete; the Chalkidiki and Attica Peninsulas; and the Amvrakikos Gulf. Sampling was performed either by collecting tissue from the individuals or by using a non-invasive method, i.e., by scraping the inside of their shells aiming to collect their mucus and thus avoid stress induction to them. Conventional molecular techniques with the use of the COI and 16S rRNA mitochondrial markers were selected for the depiction of the intra-population genetic variability. The analyses included 104 samples from the present study and publicly available sequences of individuals across the whole Mediterranean Sea. The results of this work (a) suggest the use of eDNA as an efficient sampling method for protected bivalves and (b) shed light to the genetic structure of P. nobilis population in the Eastern Mediterranean; this latter knowledge might prove to be fundamental for the species conservation and hence the ecosystem resilience. The haplotype analyses reinforced the evidence that there is a certain degree of connectivity among the distinct regions of the Mediterranean; yet there is evidence of population distinction within the basin, namely between the Western and the Eastern basins. The combination of both genetic markers in the same analysis along with the inclusion of a large number of individuals produced more robust results, revealing a group of haplotypes being present only in the Eastern Mediterranean and providing insights for the species' most suitable conservation management.

Bald sea urchin disease shifts the surface microbiome on purple sea urchins in an aquarium.

Bald sea urchin disease (BSUD) is most likely a bacterial infection that occurs in a wide range of sea urchin species and causes the loss of surface appendages. The disease has a variety of additional symptoms, which may be the result of the many bacteria that are associated with BSUD. Previous studies have investigated causative agents of BSUD, however, there are few reports on the surface microbiome associated with the infection. Here, we report changes to the surface microbiome on purple sea urchins in a closed marine aquarium that contracted and then recovered from BSUD in addition to the microbiome of healthy sea urchins in a separate aquarium. 16S rRNA gene sequencing shows that microhabitats of different aquaria are characterized by different microbial compositions, and that diseased, recovered, and healthy sea urchins have distinct microbial compositions, which indicates that there is a correlation between microbial shifts and recovery from disease.

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.

Draft Genome Sequences of Three Bacterial Species from Aquatic Habitats near Washington, DC.

We report the draft genome sequences of three bacterial species isolated from freshwater ponds or features around monuments in Washington, DC, during a semester-long microbiology lab course at the George Washington University. Two of the isolates belong to potentially novel species but lost their viability and could not be revived.

Deciphering the community structure and the functional potential of a hypersaline marsh microbial mat community.

Microbial mats are vertically stratified communities of microorganisms characterized by pronounced physiochemical gradients allowing for high species diversity and a wide range of metabolic capabilities. High Throughput Sequencing has the potential to reveal the biodiversity and function of such ecosystems in the cycling of elements. The present study combines 16S rRNA amplicon sequencing and shotgun metagenomics on a hypersaline marsh in Tristomo bay (Karpathos, Greece). Samples were collected in July 2018 and November 2019 from microbial mats, deeper sediment, aggregates observed in the water overlying the sediment, as well as sediment samples with no apparent layering. Metagenomic samples' coassembly and binning revealed 250 bacterial and 39 archaeal metagenome-assembled genomes, with completeness estimates higher than 70% and contamination less than 5%. All MAGs had KEGG Orthology terms related to osmoadaptation, with the 'salt in' strategy ones being prominent. Halobacteria and Bacteroidetes were the most abundant taxa in the mats. Photosynthesis was most likely performed by purple sulphur and nonsulphur bacteria. All samples had the capacity for sulphate reduction, dissimilatory arsenic reduction, and conversion of pyruvate to oxaloacetate. Overall, both sequencing methodologies resulted in similar taxonomic compositions and revealed that the formation of the microbial mat in this marsh exhibits seasonal variation.

metaGOflow: a workflow for the analysis of marine Genomic Observatories shotgun metagenomics data.

BACKGROUND: Genomic Observatories (GOs) are sites of long-term scientific study that undertake regular assessments of the genomic biodiversity. The European Marine Omics Biodiversity Observation Network (EMO BON) is a network of GOs that conduct regular biological community samplings to generate environmental and metagenomic data of microbial communities from designated marine stations around Europe. The development of an effective workflow is essential for the analysis of the EMO BON metagenomic data in a timely and reproducible manner. FINDINGS: Based on the established MGnify resource, we developed metaGOflow. metaGOflow supports the fast inference of taxonomic profiles from GO-derived data based on ribosomal RNA genes and their functional annotation using the raw reads. Thanks to the Research Object Crate packaging, relevant metadata about the sample under study, and the details of the bioinformatics analysis it has been subjected to, are inherited to the data product while its modular implementation allows running the workflow partially. The analysis of 2 EMO BON samples and 1 Tara Oceans sample was performed as a use case. CONCLUSIONS: metaGOflow is an efficient and robust workflow that scales to the needs of projects producing big metagenomic data such as EMO BON. It highlights how containerization technologies along with modern workflow languages and metadata package approaches can support the needs of researchers when dealing with ever-increasing volumes of biological data. Despite being initially oriented to address the needs of EMO BON, metaGOflow is a flexible and easy-to-use workflow that can be broadly used for one-sample-at-a-time analysis of shotgun metagenomics data.

Micro-CT for Biological and Biomedical Studies: A Comparison of Imaging Techniques.

Several imaging techniques are used in biological and biomedical studies. Micro-computed tomography (micro-CT) is a non-destructive imaging technique that allows the rapid digitisation of internal and external structures of a sample in three dimensions and with great resolution. In this review, the strengths and weaknesses of some common imaging techniques applied in biological and biomedical fields, such as optical microscopy, confocal laser scanning microscopy, and scanning electron microscopy, are presented and compared with the micro-CT technique through five use cases. Finally, the ability of micro-CT to create non-destructively 3D anatomical and morphological data in sub-micron resolution and the necessity to develop complementary methods with other imaging techniques, in order to overcome limitations caused by each technique, is emphasised.

Benthic habitat mapping of Plazh Gradina - Zlatna ribka (Black Sea) and Karpathos and Saria Islands (Mediterranean Sea).

BACKGROUND: Habitat mapping is nеcessary for the efficient conservation and protection of marine ecosystems. In addition, it is a requirement for EU Member States as stated in the European Union (EU) Habitats Directive (92/43/EEC), as well as necessary for the achievement and maintenance of 'good environmental status (GES)' of benthic marine habitats in the framework of the EU Marine Strategy Framework Directive (2008/56/EC). NEW INFORMATION: This study provides baseline information on the marine benthic habitats of Sozopol Bay (Black Sea) and Karpathos and Saria Islands (Mediterranean Sea). These two Natura 2000 sites were selected as study sites of the RECONNECT project, which aimed at creating a transnational cooperative network to confront the environmental threats of ecosystems with a high natural and cultural interest, by the establishment of common practices and a joint regional strategy. The specific objective was to map the marine habitats using a defined a priori classification (EUNIS), with the ultimate purpose of supporting government marine spatial planning, management and decision-making processes through the development of a Decision Support System.

0s and 1s in marine molecular research: a regional HPC perspective.

High-performance computing (HPC) systems have become indispensable for modern marine research, providing support to an increasing number and diversity of users. Pairing with the impetus offered by high-throughput methods to key areas such as non-model organism studies, their operation continuously evolves to meet the corresponding computational challenges. Here, we present a Tier 2 (regional) HPC facility, operating for over a decade at the Institute of Marine Biology, Biotechnology, and Aquaculture of the Hellenic Centre for Marine Research in Greece. Strategic choices made in design and upgrades aimed to strike a balance between depth (the need for a few high-memory nodes) and breadth (a number of slimmer nodes), as dictated by the idiosyncrasy of the supported research. Qualitative computational requirement analysis of the latter revealed the diversity of marine fields, methods, and approaches adopted to translate data into knowledge. In addition, hardware and software architectures, usage statistics, policy, and user management aspects of the facility are presented. Drawing upon the last decade's experience from the different levels of operation of the Institute of Marine Biology, Biotechnology, and Aquaculture HPC facility, a number of lessons are presented; these have contributed to the facility's future directions in light of emerging distribution technologies (e.g., containers) and Research Infrastructure evolution. In combination with detailed knowledge of the facility usage and its upcoming upgrade, future collaborations in marine research and beyond are envisioned.

Benthic communities in three Mediterranean touristic ports: MAPMED project.

Background: Mediterranean ports are sources of significant economic activity and at the same time they act as recipients of considerable anthropogenic disturbance and pollution. Polluted and low-in-oxygen sediments can negatively impact benthic biodiversity and favour recruitment of opportunistic or invasive species. Macrobenthic communities are an important component of the port biota and can be used as environmental quality indicators. However, a baseline database for benthic biodiversity in Mediterranean ports has not yet been widely established. New information: Macrobenthic assemblages were recorded in three Mediterranean touristic ports under the framework of the ENPI CBC MED project MAPMED (MAnagement of Port Areas in the MEDiterranean Sea Basin). Samples were collected from Cagliari (Sardinia, Italy), Heraklion (Crete, Greece) and El Kantaoui (Tunisia) ports during February, May and September 2012. The sampling stations were selected according to the different sectors within each port (i.e. leisure, fishing, passenger/cargo vessels and shipyard). A total of 277 taxa belonging to 12 phyla were found, of which the 96 taxa were present in all three ports. El Kantaoui port hosted the highest number of macrobenthic taxa. Mollusca were the most abundant group (34%) in all ports. The highest percentage of opportunistic taxa per station was found before the touristic period in the shipyard of Heraklion port (89.3%).

Viral Metagenomic Content Reflects Seawater Ecological Quality in the Coastal Zone.

Viruses interfere with their host's metabolism through the expression of auxiliary metabolic genes (AMGs) that, until now, are mostly studied under large physicochemical gradients. Here, we focus on coastal marine ecosystems and we sequence the viral metagenome (virome) of samples with discrete levels of human-driven disturbances. We aim to describe the relevance of viromics with respect to ecological quality status, defined by the classic seawater trophic index (TRIX). Neither viral (family level) nor bacterial (family level, based on 16S rRNA sequencing) community structure correlated with TRIX. AMGs involved in the Calvin and tricarboxylic acid cycles were found at stations with poor ecological quality, supporting viral lysis by modifying the host's energy supply. AMGs involved in "non-traditional" energy-production pathways (3HP, sulfur oxidation) were found irrespective of ecological quality, highlighting the importance of recognizing the prevalent metabolic paths and their intermediate byproducts. Various AMGs explained the variability between stations with poor vs. good ecological quality. Our study confirms the pivotal role of the virome content in ecosystem functioning, acting as a "pool" of available functions that may be transferred to the hosts. Further, it suggests that AMGs could be used as an ultra-sensitive metric of energy-production pathways with relevance in the vulnerable coastal zone and its ecological quality.

PEMA: a flexible Pipeline for Environmental DNA Metabarcoding Analysis of the 16S/18S ribosomal RNA, ITS, and COI marker genes.

BACKGROUND: Environmental DNA and metabarcoding allow the identification of a mixture of species and launch a new era in bio- and eco-assessment. Many steps are required to obtain taxonomically assigned matrices from raw data. For most of these, a plethora of tools are available; each tool's execution parameters need to be tailored to reflect each experiment's idiosyncrasy. Adding to this complexity, the computation capacity of high-performance computing systems is frequently required for such analyses. To address the difficulties, bioinformatic pipelines need to combine state-of-the art technologies and algorithms with an easy to get-set-use framework, allowing researchers to tune each study. Software containerization technologies ease the sharing and running of software packages across operating systems; thus, they strongly facilitate pipeline development and usage. Likewise programming languages specialized for big data pipelines incorporate features like roll-back checkpoints and on-demand partial pipeline execution. FINDINGS: PEMA is a containerized assembly of key metabarcoding analysis tools that requires low effort in setting up, running, and customizing to researchers' needs. Based on third-party tools, PEMA performs read pre-processing, (molecular) operational taxonomic unit clustering, amplicon sequence variant inference, and taxonomy assignment for 16S and 18S ribosomal RNA, as well as ITS and COI marker gene data. Owing to its simplified parameterization and checkpoint support, PEMA allows users to explore alternative algorithms for specific steps of the pipeline without the need of a complete re-execution. PEMA was evaluated against both mock communities and previously published datasets and achieved results of comparable quality. CONCLUSIONS: A high-performance computing-based approach was used to develop PEMA; however, it can be used in personal computers as well. PEMA's time-efficient performance and good results will allow it to be used for accurate environmental DNA metabarcoding analysis, thus enhancing the applicability of next-generation biodiversity assessment studies.

Sediment microbial taxonomic and functional diversity in a natural salinity gradient challenge Remane's "species minimum" concept.

Several models have been developed for the description of diversity in estuaries and other brackish habitats, with the most recognized being Remane's Artenminimum ("species minimum") concept. It was developed for the Baltic Sea, one of the world's largest semi-enclosed brackish water body with a unique permanent salinity gradient, and it argues that taxonomic diversity of macrobenthic organisms is lowest within the horohalinicum (5 to 8 psu). The aim of the present study was to investigate the relationship between salinity and sediment microbial diversity at a freshwater-marine transect in Amvrakikos Gulf (Ionian Sea, Western Greece) and assess whether species composition and community function follow a generalized concept such as Remane's. DNA was extracted from sediment samples from six stations along the aforementioned transect and sequenced for the 16S rRNA gene using high-throughput sequencing. The metabolic functions of the OTUs were predicted and the most abundant metabolic pathways were extracted. Key abiotic variables, i.e., salinity, temperature, chlorophyll-a and oxygen concentration etc., were measured and their relation with diversity and functional patterns was explored. Microbial communities were found to differ in the three habitats examined (river, lagoon and sea) with certain taxonomic groups being more abundant in the freshwater and less in the marine environment, and vice versa. Salinity was the environmental factor with the highest correlation to the microbial community pattern, while oxygen concentration was highly correlated to the metabolic functional pattern. The total number of OTUs showed a negative relationship with increasing salinity, thus the sediment microbial OTUs in this study area do not follow Remane's concept.

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 and its utility in the fields of environmental source tracking, paleontology, and studies of microbial biogeography. To install seqenv, go to: https://github.com/xapple/seqenv.

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_greece.

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.

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/.