The last project meeting will take place in Reykjavik, Iceland, the day before the start of the 12th Deep-sea Biology Symposium. The exact programme, venue and travel information will be comunicated as the date nears. In the meantime, you can obtain general information on the Symposium site. For further information, please contact Dr. Eva Ramírez-Llodra (ezr@icm.csic.es).

Summary

Bathyal and abyssal ecosystems cover over 60% of the earth’s surface and are thought to be hotspots of marine biodiversity. Deep-sea benthic communities are the ultimate recipients of food produced in overlying surface waters by phytoplankton and play an integral role in the global carbon reservoir. The overall aims of IP 3&4 are (1) to elucidate linkages between organic matter processing and the benthic community (2) to investigate and model trophic linkages and (3) to unravel the links between community composition, trophic structure and biogeochemical cycling. 

The five main objectives of IP 3&4 are:

1. To identify the bathyal and abyssal biodiversity of macro- and megafauna in two contrasting deep-sea environments

2. To elucidate the flow of carbon to archaea, bacteria and macrofauna using in situ enrichments with stable isotopes

3. To investigate trophic linkages within the food-web

4. To link differences in trophic structure and biodiversity to local biogeochemical functioning by quantifying the OM mineralization pathways and reconstructing the carbon sequestration scheme

5. To develop a dynamic mass balance model to describe the flow of bacterial and phytodetritus carbon and nitrogen through the entire food web 

Work Plan

Cruises

NIOZ/NIOO will lead two cruises, one to Galicia Bank, N. Atlantic (Sept/Oct 2008) and another to the Algerian-Balearic Basin (W. Mediterranean, October 2009). The aim is to sample two habitats with differing food availability, productive vs. oligotrophic at 1200, 1900 and 3000 m. Each cruise will: 

1. Map/characterize the seafloor of each area using multibeam.

2. Investigate food availability and the physical characteristics of each area using moorings equipped with sediment traps, data loggers, current meters and instruments to detect optical backscatter and fluorescence. Moorings will be deployed for 12 months.

3. Characterize the food sources available both in the water column (CTD & SAPS) and at the seafloor (multicores).

4. Determine macro and megafaunal biodiversity (Agassiz trawl and boxcores both equipped with video).

5. Benthic landers (ALBEX and the newly developed MOVE!) will be deployed to measure solute fluxes and oxygen microprofiles and to carry out baited camera experiments and in situ pulsechase experiments (Mediterranean only).

6. Long-term deck incubations will be carried out for ex situ pulse-chase experiments.

Analyses

1. Mass fluxes will be determined from sediment trap material.

2. Biodiversity, biomass and abundances of macro and megafauna will be determined from video analyses of boxcores and Agassiz trawl transects in additions to samples obtained from these instruments.

3. Trophic studies will be carried out on collected faunal and food source (e.g. sediments and water) samples using natural abundance stable isotopes (bulk and compound specific), and biomarkers such as lipids and pigments.

4. Solute fluxes and oxygen microprofiles will be measured both in situ and from sediment cores

5. Stable isotope pulse-chase experiments will be carried out with enriched phytodetritus (POM) and dissolved organic matter (DOM). POM and DOM enriched in heavy isotopes (13C and 15 N) will be added to sediment communities. Total community respiration will be measured via 13C in dissolved inorganic carbon. Assimilation and uptake of phytodetritus by infaunal organisms will be determined through measurement of 13C and 15N tissues. Carbon and nitrogen assimilation by bacteria and archaea will be based on appearance of 13C or 15N in microbial biomarkers (13C in phosphor-lipid derived fatty acids for bacteria, 13C in archaeal ether lips and 13C and 15N in Dalanine, a prokaryotic biomarker). This approach allows a direct assessment on the role of the various organisms involved and will be the first study involving all three kingdoms (archaea, bacteria and eukaryotes). To study the trophic significance of heterotrophic bacteria and archaea in a deep-sea ecosystems 13C-glucose will be added to isotopically enrich the osmotrophs (bacteria and archaea). Incorporation of 13C-glucose into archaea and bacterial carbon will be traced with above mentioned biomarkers and subsequent transfer to benthic fauna will be followed through measuring the appearance of 13C of organism tissues.

6. Biodiversity and quanti-qualitative studies on the Protistan community:  living field-obtained cultures will be used to try to obtain PLF specific markers, in order to analyze the role and the importance of protozoa in the foodweb;  DNA  extracted from field samples, coupled with DGGE technique and metagenomics will be used to get an insight of the relative abundance of the major groups, and draw the community fingerprinting.

UNIVPM-DiSMAr

Aims and objectives

Benthic faunal diversity provides an ideal tool for exploring the relationships between biodiversity and ecosystem functioning, and among benthic faunal taxa, nematodes are ideal model for deep-sea investigations. Nematodes are indeed the most abundant metazoans on Earth, but in the deep sea they are even more important accounting for more than 90% of the total abundance of metazoa. This phylum is also characterised by: i) very high species richness (i.e., among the most diverse of marine Phyla); ii) distinct and easily recognisable feeding type and iii) life strategies that make it possible to also identify functional diversity traits.

We identified the following key tasks:

1)      Diversity distribution, community structure, abundance and biomass of metazoan meiofauna and identification of nematodes to species level.

2)      Physico-chemical characteristics of the water column and of the sediment-water interface (down to 20 cm depth).

3)      Biochemical and biogeochemical analyses, quantification of the organic matter bioavailable to consumers in deep-sea sediments

Work plan

Field work: Oceanographic cruises planned in spring-summer of 2008 and 2009. In order to make comparable these data with those collected in other previous occasions from deep-sea sites and hierarchical sampling strategy and sampling at fixed depths will be endorsed. At each sampling site, triplicate deployments of box-corer or multiple corers will be carried out. This hierarchical sampling strategy will provide the opportunity to assess the pattern of spatial variability in meiofaunal, abundance, biomass and diversity. The same sampling strategy will also help to assess spatial variability in the quantity, biochemical composition and bioavailability of meiofaunal food resources.

Analyses: Sediment subsamples will be collected from independent deployments of the box- or multiple corers will be stored at -20°C until the analysis of the quantity and biochemical composition and bioavailability of the sediment organic matter. These sediments will be analysed in terms of chloroplastic pigments (chlorophyll-a and phaeopigments) and of total and enzymatically hydrolyzable protein, carbohydrate and lipid concentrations (spectrophotometrically). The sum of total protein, carbohydrate and lipid C will be reported as the biopolymeric fraction of sedimentary organic C, whereas the sum of the enzymatically digestible fractions of protein, carbohydrate and lipid C will be reported as the bioavailable fraction of biopolymeric C, and will be utilised as an estimate of the bioavailability of organic C for meiofauna.

Additional sediment samples will be used for the meiofaunal study. Sediment cores obtained from separate corer deployments will be sliced into five horizons (0-1, 1-2, 2-3, 3-5, 5-15 cm) and analysed for meiofaunal abundance. Meiofauna will be extracted according to standard protocols and counted and identified to major taxa under stereomicroscope. Replicate sediment samples will be utilised for nematode extraction and analysis. Nematodes will be sorted and identified to genus and, when possible, to species level. A subsample of the nematodes extracted from each core and sediment layer will be utilized for diversity analysis based on molecular tools.

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).

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).

Aims and objectives

The overall aim of this study is to identify the role of meiofauna in the C flow through benthic deep-sea sediments of the Mediterranean in relation to their biodiversity. In order to unravel the link between meiofaunal biodiversity and function, it is essential to reveal the interactions in the benthic food web and the trophic position of different meiofauna taxa and functional groups at different locations with contrasting food input. Only in this way we can contribute to the understanding of deep-sea ecosystem functioning, and to the conservation of deep-sea ecosystems in a changing environment. In order to achieve this overall aim we identified five specific objectives which fit into 5 of the 6 main objectives of the BIOFUN project:

1. The description of the biodiversity of the meiofauna, especially Nematoda. Interpretation of the biodiversity in a geographical and bathymetrical context in relation to environmental factors and geological barriers. The bathyal (and abyssal) biodiversity of the meiofauna in contrasting deep-sea environments will be studied. Furthermore, the biogeography of some dominant meiobenthic taxa will be investigated.

2. To allocate the Carbon flux to meiofauna in an experimental approach

3. To investigate the trophic interactions in the meiofauna undertaking stable isotope and fatty acid analyses  

4. To make the link between biodiversity and functioning in meiobenthic communities  

5. To contribute to ecotrophic models

Analyses

1. Biodiversity and biogeography analysis

Samples for biodiversity analysis will be collected at three geographical locations (East and West Med basin and Atlantic Galicia Basin) and at minimum two water depths (between 1000 and 3000 m). Additional locations for biogeographical analysis will be sampled along a trans-Mediterranean transect. At these three sites the biodiversity of meiofauna, and in particular nematodes as dominant taxon of the metazoan meiofauna (representing > 90 %) will be analyzed up to species level. Since most of the deep-sea nematodes are new to science and since the dominant genera are characterized by a high species richness, all species within the most dominant genera present at all locations will be taxonomically identified in order to study the spatial turn-over and biogeographical patterns within these dominant genera. Samples from the same locations will be analyzed for molecular analysis by UNIVPM in order to allow a comparison of biodiversity patterns based on molecular and morphological data. Meiofauna biodiversity data will also be collected by HCMR and UNIVPM. Integration of these data in a larger dataset will extend the geographical range and available taxonomic information.

Working on board R/V Belgica, in the NE Atlantic, June 2008. © Ellen Pape 

2. Natural biomarker analysis

At each site meiofauna samples will be collected for analysis of natural biomarkers. Different meiofauna taxa will be analyzed separately. Nematodes will be analyzed in bulk but also sorted according to body size, depth in the sediment, morphology of the buccal cavity and other functional criteria. Additional samples for overall sediment biomarker composition will be processed, while biomarker composition of sediment trap material will be available too (CEFREM). For all these components of the BBL, stable isotope ratios will be investigated in addition to lipid composition (in collaboration with NIOO).

3. Enrichment experiments

During these experiments ¹³C or¹⁵N labelled benthic bacteria or epipelic diatoms (preferentially collected from sediment traps) will be added to the sediment and incubated for several days. In this way food pulse events are simulated which allows to follow up the uptake of label (food) by different meiofauna taxa (selectivity) and to quantify the carbon flow from these different nutritional sources to particular taxa or groups (Middelburg et al, 2000) Label incorporation will be measured by stable isotope analysis of selected meiofauna taxa. During the experiments changes respiration and SCOC will be monitored (in collaboration NIOO). Experiments will be performed in situ by means of ROV or lander technology. However, nematodes communities have proved to persist for longer time (several weeks) in mesocoms conditions if retrieved quickly from water depths above 2500 m (personal observations) and kept in lab conditions of atmospheric pressure, and in situ temperature and oxygen. Therefore parallel to some of the in situ experiments, lab mesocosm studies will be performed, allowing long term uptake.

Ellen Pape (Ellen.Pape@UGent.be)

The function of meiofauna and the importance of their biodiversity in contrasting Southern European deep-sea environments.

University: University of Gent

Supervisor: Dr Ann Vanreusel

Massimiliano Molari (m.molari@univpm.it)

Role of Archaea in the deep-sea sediments

University: Polytechnic University of Marche

Supervisor: Prof. Roberto Danovaro

Daniela Zeppilli (d.zeppilli@univpm.it)

Role of catastrophic events on benthic biodiversity and deep-sea ecosystem functioning

University: Polytechnic University of Marche

Supervisor: Prof. Roberto Danovaro

Marianna Mea (m.mea@univpm.it)

Impact of episodic events on biodiversity and ecosystem functioning in the deep-sea sediments

University: Polytechnic University of Marche

Supervisor: Prof. Roberto Danovaro

Donato Giovannelli (d.giovannelli@univpm.it)

Study of viral metagenomics in different deep-sea sediments

University: Polytechnic University of Marche

Supervisor: Prof. Roberto Danovaro

Elisa Baldrighi (elicasacca@yahoo.it)

Biodiversity of macrofauna and ecosystem functioning in the Mediterranean deep-sea sediments: analysis at different spatial scales

University: CNR-ISMAR and Polytechnic University of Marche

Supervisor: Dr. Elena Manini

Samuele Tecchio (tecchio@icm.csic.es)

Biodiversity of deep-sea megafauna and their role in ecosystem functioning in contrasting Southern European environments

University: ICM-CSIC and Universitat de Barcelona

Supervisor: Prof Francesc Sardà & Dr Eva Ramirez-Llodra

Ulla Fernandez de Arcaya (ulla@icm.csic.es)

Life cycle startegies of deep-water Mediterranean fishes

University: ICM-CSIC and Universitat de Barcelona

Supervisor: Dr Joan Baptista Company

Sam Koenig (koenig@icm.csic.es)

Contaminants in deep-water fauna from the Atlantic and Mediterranean

University: ICM-CSIC and Universitat de Barcelona

Supervisor: Montse Sole and Pilar Fernandez

Aileen Gill (a.gill9@nuigalway.ie)

Diversity and Distribution of Bacteria in Deep Mediteranean Waters

University: National University of Ireland, Galway

Supervisor: Dr. Ger Fleming

Lara Pozzato (L.Pozzato@nioo.knawnl)

Biodiversity and quanti-qualitative studies on the Protistan community

University: NIOO, NL

Supervisor: Dr. Leon Moodley

Second BIOFUN and EuroDEEP meetings (10 Nov. 2008). Valencia, Spain. The second BIOFUN and EuroDEEP meetings were held at the Ciudad de las Ciencias y las Artes in conjunction with the World Conference of Marine Biodiversity.

BIOFUN kick-off meeting (25-26 Sept. 2007). Barcelona, Spain. At this meeting, each partner presented the detailed work plan for their individual and associated projects. The study sites and common methodology was discussed and agreed upon. For further information, contact Dr Eva Ramirez-Llodra (ezr@icm.csic.es).

First EuroDEEP meeting (26-28 Nov. 2007). Taormina, Sicily. This was the first EuroDEEP science meeting, with participation of members (from students to principal investigators) from all Collaborative Research Projects (BIOFUN, CHEMCO, DEECON and MIDDLE). The scientific plans for each CRP were given and specific studies presented. For further information, contact Dr Inge Jonckheere (ijonckheere@esf.org).

Galicia Bank (June 2008). Cruise on board R/V Belgica for meiofauna sampling on the Galicia Bank, NE Atlantic. PI: Ann Vanreusel, UGent, BE. Cruise report

Coring sampling and experiments on board R/V Belgica. Photos: Ellen Pape.

Central Mediterranean (June 2008). Cruise on board R/V Urania to the Central Mediterranean for deep-water ecosystems studies, from viruses to megafauna. PI: Stefano Aliani, La Spezia, IT. Urania cruise report, 2008 (download).

Galicia Bank (Sept-Oct 2008). Cruise on board R/V Pelagia to the Galicia Bank to sample bathyal and abyssal fauna from microbes to megafauna. PI: Marc Lavaleye, NIOZ, NL.

Trans-Mediterranean (June 2009). Multidisciplinar cruise on board the new Spanish R/V Sarmiento de Gamboa. Sampling from viruses to megafauna took place in the southern Balearic Sea, the western Ionian Sea, and the Cretan Sea. PI: Francesc Sardà, ICM, ES.

Cruise report

BIOFUN planned cruises

ICM-CSIC, UB, UPC, CEAB, IMEDEA-CSIC

Summary

The Mediterranean Sea is a unique system characterised by homeothermia and a steep gradient of increasing oligotrophy from West to East. The aim of BIOFUN is to characterise, under an ecosystem approach, two deep-sea habitats – the mid-slope and abyssal plain – including for the first time the analysis from viruses to megafauna, to understand the linkages between biodiversity patterns and ecosystem functioning in relation to environmental conditions along a gradient of increased oligotrophy from West to East.  

IP1 will undertake a multidisciplinary study in collaboration with other partners in BIOFUN to characterise the mid-slope and abyssal plains in the Mediterranean Sea along a longitudinal trophic gradient with increasing oligotrophic conditions from West to East and compared to the more productive Atlantic site. In particular, the overall aim of IP1 is to investigate the biodiversity of the megafauna (fish and invertebrates) and macrofauna and to understand the trophic structure and life cycle of key species in relation with the physical and geochemical environmental conditions as well as with the other faunal size-classes. This will allow describing patterns of distribution and ecosystem functioning, providing essential data to evaluate the vulnerability of the populations to fluctuations in food input as well as to anthropogenic impact such as fisheries or pollution.

 Work plan

Cruise: ICM-CSIC will lead a trans-Mediterranean cruise along a longitudinal gradient in order to sample habitats of differing environmental characteristics and food availability. The cruise will take place in summer 2009 and will sample the two main BIOFUN areas: the Algerian-Balearic Basin and the Ionian Sea at 1200 and 3000 m depth, as well as the Levantine Basin and the Messina Abyssal Plain (4100 m). This multidisciplinary cruise will characterise the water column (CTD) and the sediment (coring) and will sample for microbes, meio and macrofauna (multicorer) and megafauna (Agassiz trawl, OTSB and Epibenthic sled). The cruise will be coordinated in time and space with the other BIOFUN cruises and it will include the participation of other BIOFUN partners, to ensure integration of sampling in time and space, which will be essential for the trophic structure analyses. In addition, we will coordinate efforts with CEFREM for the characterisation of the hydrodynamic and physico-geochemical patterns of the NW Mediterranean slope. In particular, several 3 arrays of automated sediment traps and current meters will be deployed for two 6-month periods within the NW Mediterranean experimental sites.

BIOFUN main study sites: ABB, Algerian-Balearic Basin; MAP, Messina Abyssal Plain; IS, Ionian Sea; LB, Levantin Basin 

Anaylses:

1. Physical and geochemical studies: Total mass and major constituents (organic and inorganic carbon, opal, lithogenic material) will be determined for all sediment trap samples to provide a quantification of the basic features of settling material. Physical and sedimentological characteristics (grain size, dry bulk density, water content), major constituents and sedimentation rates will be analysed in the sediment samples to provide a balance between sinking material and deposited material.

2. Biodiversity, biomass and abundance of mega and macrofauna will be quantified from trawl, sledge and corer samples and statistical comparisons will be made between sites.

3. Trophic studies will be conducted by analysing stomach contents and using biomarkers and stable isotope analyses in key megafauna species.

4. Life histories will be studied with histology, microscopy and image analyses will be used to determine gametogenetic cycles, while fecundity will be quantified.

5. Ecological models will be developed using data generated from field work.

6. Determination of contaminants will be conducted using gas chromatography coupled with mass spectrometry and ion selection mode.