Summary

Deep-sea sediments cover about 65% of the word surface and play an important part in biomass production and biogeochemical cycle on a global scale. These processes are largely mediated by benthic prokaryotes, which use organic detritus for biomass production and respiration.

IP2 will investigate the diversity, spatial distribution, and abundance of deep-sea prokaryotes and macrofaunal species in selected areas of the Mediterranean Sea and the Atlantic Ocean. This will allow describing biogeographic patterns of some dominant species across the Mediterranean Sea and the adjacent Atlantic Ocean, and analysing these in relation with geomorphological barriers (Gibraltar Strait and Sicilian Sill) and gradients of biotic (e.g. nutrient availability) and abiotic (e.g. thermohaline characteristics, sediment dynamics) variables. The input of the CNR-ISMAR to the CRP include two main objectives:

- To investigate the spatial patterns of diversity and activity of deep-sea prokaryotes (Bacteria and Archaea) in terms of biomass, C production, metabolism and degradation rates in the deep-sea Mediterranean Sea and the adjacent Atlantic Ocean. A special attention will be also posed in evaluating the role of viruses (viral shunt) in controlling/shaping relationship between benthic biodiversity and ecosystem functioning.

- To study the macrofaunal biodiversity in relation with physical/environmental gradients across the deep-Mediterranean Sea.

Work plan

Field work:

CNR-ISMAR leaded a Mediterranean cruise by R/V Urania in spring-summer 2008 in the central and eastern basin. Another cruise is planned in spring-summer of 2009 in western Mediterranean basin. Some deep-sea selected areas will be sampled in order to focus on different nutrient availability and sediment/slope conditions. The two cruises will cover a wide geographical area (>1000 km). At each site, in order to avoid pseudo-replication, 3 to 5 independent deployments (box corers and/or multicorer) will be carried out. This hierarchical sampling strategy will provide the opportunity to assess the pattern of spatial variability in prokaryote and macrofaunal abundance, biomass and diversity. The analysis of longitudinal gradients in hydrological and phsyico-chemical conditions will provide background information for testing the hypothesis of gradient/biodiversity relationships.  The same sampling strategy will also help to assess spatial variability in the main ecosystem functioning processes that will include measurements of prokaryote C production and extracellular enzymatic activity.

Analyses:

1. Prokaryotic biodiversity, community structure and biomass: will be investigated using epifluorescence microscopy, CARD-FISH (Catalysed Report Deposition-Fluorescence in situ Hybridization) and molecular fingerprinting technique (ARISA for Bacteria and T-RFLP for Archaea)

2. Deep-benthic prokaryotic metabolism: will be investigated in terms of extracellular enzymatic activities and heterotrophic C production in the sediments.

3. Benthic viral abundance, production and decay: will be investigated through epifluorescence microscopy and dilution techniques.

4. Macrofaunal biodiversity, community structure and biomass: will be investigated using a classical taxonomic approach and standard techniques.

Retrospective data available:

Historical information for microbial variables will be obtained from the TransMed cruise (1999) of the MATER project (10 deep-sea sites at 3000-4000 m depth crossing the entire Mediterranean basin) and others collected during the ADIOS project (2001; 2 deep-sea sites at 3000 m depth in covering both Mediterranean basins) and another TransMed cruise in the frame of MEDGOOS in 2006. Time series are available at 1000 and 1600 m depth in the Eastern Mediterranean sea. Moreover, others sample collected in the Portuguese Margin will be obtained during the HERMES project (2005/06;  deep sea sites at 1500-3000 m). For the benthic fauna will be available sample of the Sea of Crete (1999). Environmental factors will be compared with data collected during the cruise activities planned in 2007 (VECTOR project; 10 deep-sea sites at 3000-4000 m depth crossing the entire Mediterranean basin) and 2008 (HERMES project, 10 deep-sea sites at 3000-4000 m depth crossing the entire Mediterranean basin).

Deep Mediterranean Prokaryote Communities - Nucleic Acid and Culture Studies

Aims and objectives

1. To identify the elements of prokaryotic communities which play key roles in carbon processing in the deep benthic boundary layer

2. To determine the contribution of these key elements to the overall structure and biodiversity of individual prokaryotic communities and their distribution with depth and geographical location.

3. To determine the degree to which the prokaryotic community is perturbed by organic loading (natural or anthropogenic) and to obtain isolates of active members of the deep ocean prokaryotic community for further study of their  physiology and biotechnological potential.

Methodologies

Analysis of  prokaryotic community structure and biodiversity by methods based on nucleic acid sequences is, at present, the method of choice for microbial ecologists. It can provide a synoptic view of the community, enable inter-community comparisons, and indicate the phylogeny of individual community components. Nucleic-acid based methods have several disadvantages, however: The operational taxonomic units (OTU’s) used to describe the community may be poorly defined. At best they may identify a clone as represented by a specific DNA sequence. The phylogenic position of this sequence as compared with other reported sequences may be determined, but it provides no information about the clone, its physiology , metabolism etc. Typically we will have no idea as to whether the clone is active, inactive, an essential part of the functioning community or redundant.

Pressure-retainer sampler and Pressure vessel used for batch enrichment studies: a) connection for pressurizing pump; b) non-return valve; d) pressure vessel; e) vessel cap; f) air/pressure release valve

Alternative methods of community structure cannot solve this problem: prokaryote identification and determination of function by morphology is not feasible. Cultural methods provide growing clones for further  study, but are generally dismissed as dealing with only a small and possibly unrepresentative fraction of the total community.

Our approach combines nucleic acid based methods together with the use of enrichments and culture based methods, and is intended to identify the key prokaryotic elements involved in carbon cycling and their distribution.  Changes in community structure which occur during sequential batch or semi-continuous enrichments are followed by Degrading Gradient gel Electrophoresis; (DGGE: a synoptic DNA-based method).In this way we can identify the “fitter” clones (those that out-compete the other elements of the community) under the specific enrichment conditions. Changes in “cultivable” bacteria are also monitored by the use of the MPN (most probably number) method using multiwell plates. Unlike conventional plate counts, MPN facilitates the counting and isolation of clones under near-environmental conditions and thus deals with oligotrophs potentially relevant to deep ocean ecosystem functioning rather than unrepresentative copiotrophs. Enrichments under quasi-environmental conditions, combined with culture and DNA based analyses identify potential “keystone” members of the prokaryotic community and define them in terms of their DNA sequence. This information may then be used to determine their contribution to the overall structure and biodiversity of individual prokaryotic communities and their distribution with depth and geographical location. This is achieved by associating them with specific bands and peaks in DGGE “fingerprints” of natural communities and also by the use fluorescent sequence-based probes (FISH).