Development of a novel molecular tool for the rapid assessment of the biodiversity changes of benthic nematodes assemblages. Mares Conference Marine Ecosystems Helath and conservation

The molecular profiling system was developed using directed terminal-restriction fragment length polymorphism (dT-RFLP) to characterize soil nematode assemblages by relative abundance of feeding guilds and validation by comparison to traditional morphological method. The good performance of these molecular tools applied to soil nematodes assemblages create an opportunity to develop a novel approach for rapid assessment of the biodiversity changes of benthic nematodes assemblages of marine and estuarine sediments. The main aim of this research is to combine morphological and molecular analysis of estuarine nematodes assemblages, to establish a tool for fast assessment of the biodiversity changes within habitat recovery of Zostera noltii seagrass beds; and validate the dT-RFLP as a high-throughput tool to assess the system recovery. It was also proposed to develop a database of sequences related to individuals identified at species level to develop a new taxonomic reference system. A molecular phylogenetic analysis of the estuarine nematodes has being performed. After morphological identification, barcoding of 18S rDNA are being determined for each nematode species and the results have shown a good degree of concordance between traditional morphology-based identification and DNA sequences. The digest strategy developed for soil nematodes is not suitable for marine nematodes. Then five samples were cloned and sequenced and the sequence data was used to design a new dT-RFLP strategy to adapt this tool to marine assemblages. Several solutions were presented by DRAT and tested empirically to select the solution that cuts most efficiently, separating the different clusters. The results of quantitative PCR showed differences in nematode density between two sampling stations according the abundance of the nematode density obtained by the traditional methods. These results suggest that qPCR could be a robust tool for enumeration of nematode abundance, saving time.

Fixing, storage time and DNA extraction applied to dT-RFLP for ecological and molecular studies of marine nematodes assemblages

The application of molecular methods offers an alternative faster than traditional methods based on morphology It is nearly impossible to process all the samples in short period using traditional methods, and the deterioration of marine sediments rapidly occurs The dT-RFLP (directed Terminal-Restriction Fragment Length Polymorphism) allows a rapid assessment of biodiversity changes of nematodes assemblages The use of a not suitable fixing, storage time and DNA extraction could be a limitation in molecular analysis like dT-RFLP and real time PCR.Objetives: the best fixative •the level of DNA degradation over the time •the best DNA extraction method for marine nematodes and suitable for dT-RFLP analysis

Development of molecular approaches to assess the diversity and abundance of marine nematodes assemblages for assessment of Good Environmental Status of the estuarine and marine ecosystems

In Europe, the concerns with the status of marine ecosystems have increased, and the Marine Directive has as main goal the achievement of Good Environmental Status (GES) of EU marine waters by 2020. Molecular tools are seen as promising and emerging approaches to improve ecosystem monitoring, and have led ecology into a new era, representing perhaps the most source of innovation in marine monitoring techniques. Benthic nematodes are considered ideal organisms to be used as biological indicator of natural and anthropogenic disturbances in aquatic ecosystems underpinning monitoring programmes on the ecological quality of marine ecosystems, very useful to assess the GES of the marine environment. dT-RFLP (directed Terminal-Restriction Fragment Length Polymorphism) allows to assess the diversity of nematode communities, but also allows studying the functioning of the ecosystem, and combined with relative real-time PCR (qPCR), provides a high-throughput semi-quantitative characterization of nematode communities. These characteristics make the two molecular tools good descriptors for the good environmental status assessment. The main aim of this study is to develop and optimize the dT-RFLP and qPCR in Mira estuary (SW coast, Portugal). A molecular phylogenetic analysis of marine and estuarine nematodes is being performed combining morphological and molecular analysis to evaluate the diversity of free-living marine nematodes in Mira estuary. After morphological identification, barcoding of 18S rDNA and COI genes are being determined for each nematode species morphologically identified. So far we generated 40 new sequences belonging to 32 different genus and 17 families, and the study has shown a good degree of concordance between traditional morphology-based identification and DNA sequences. These results will improve the assessment of marine nematode diversity and contribute to a more robust nematode taxonomy. The DNA sequences are being used to develop the dT-RFLP with the ability to easily process large sample numbers (hundreds and thousands), rather than typical of classical taxonomic or low throughput molecular analyses. A preliminary study showed that the digest enzymes used in dT-RFLP for terrestrial assemblages separated poorly the marine nematodes at taxonomic level for functional group analysis. A new digest combination was designed using the software tool DRAT (Directed Terminal Restriction Analysis Tool) to distinguished marine nematode taxa. Several solutions were provided by DRAT and tested empirically to select the solution that cuts most efficiently. A combination of three enzymes and a single digest showed to be the best solution to separate the different clusters. Parallel to this, another tool is being developed to estimate the population size (qPCR). An improvement in qPCR estimation of gene copy number using an artificial reference is being performed for marine nematodes communities to quantify the abundance. Once developed, it is proposed to validate both methodologies by determining the spatial and temporal variability of benthic nematodes assemblages across different environments. The application of these high-throughput molecular approaches for benthic nematodes will improve sample throughput and their implementation more efficient and faster as indicator of ecological status of marine ecosystems.

Mapping and characterisation of the inter-reefal benthic assemblages of the Torres Strait

A comprehensive survey of the benthic assemblages of the Torres Strait was conducted in order to provide critical baseline information for regional marine planning, assessing the environmental sustainability of fisheries and understanding the ecosystems of the region. Over 150 sites throughout the region were sampled with a modified prawn trawl, towed underwater video, pipe dredge and epibenthic sled. This manuscript provides a broad overview of the activities undertaken and data collected. Two thousand three hundred and seventy-two different nominal species were sampled by the trawl and sled, only 728 by both gears. The towed video was not able to provide the same level of taxonomic resolution of epibenthic taxa, but was particularly useful in areas where the seabed was too rough to be sampled. Data from the trawl, sled and video were combined to characterise the epibenthic assemblages of the region. Data from the towed video was also used to provide a characterisation of the inter-reefal benthic habitats, which was then analysed in combination with physical covariate data to examine relationships between the two. Levels of mud and gravel in the sediments, trawling effort and seabed current stress were the covariates most significantly correlated with the nature of the seabed habitats.

Fish Habitat Utilization Patterns and Evaluation of the Efficacy of Marine Protected Areas in Hawaii: Integration of NOAA Digital Benthic Habitat Mapping and Coral Reef Ecological Studies

Over the past four decades, the state of Hawaii has developed a system of eleven Marine Life Conservation Districts (MLCDs) to conserve and replenish marine resources around the state. Initially established to provide opportunities for public interaction with the marine environment, these MLCDs vary in size, habitat quality, and management regimes, providing an excellent opportunity to test hypotheses concerning marine protected area (MPA) design and function using multiple discreet sampling units. NOAA/NOS/NCCOS/Center for Coastal Monitoring and Assessment’s Biogeography Team developed digital benthic habitat maps for all MLCD and adjacent habitats. These maps were used to evaluate the efficacy of existing MLCDs for biodiversity conservation and fisheries replenishment, using a spatially explicit stratified random sampling design. Coupling the distribution of habitats and species habitat affinities using GIS technology elucidates species habitat utilization patterns at scales that are commensurate with ecosystem processes and is useful in defining essential fish habitat and biologically relevant boundaries for MPAs. Analysis of benthic cover validated the a priori classification of habitat types and provided justification for using these habitat strata to conduct stratified random sampling and analyses of fish habitat utilization patterns. Results showed that the abundance and distribution of species and assemblages exhibited strong correlations with habitat types. Fish assemblages in the colonized and uncolonized hardbottom habitats were found to be most similar among all of the habitat types. Much of the macroalgae habitat sampled was macroalgae growing on hard substrate, and as a result showed similarities with the other hardbottom assemblages. The fish assemblages in the sand habitats were highly variable but distinct from the other habitat types. Management regime also played an important role in the abundance and distribution of fish assemblages. MLCDs had higher values for most fish assemblage characteristics (e.g. biomass, size, diversity) compared with adjacent fished areas and Fisheries Management Areas (FMAs) across all habitat types. In addition, apex predators and other targeted resources species were more abundant and larger in the MLCDs, illustrating the effectiveness of these closures in conserving fish populations. Habitat complexity, quality, size and level of protection from fishing were important determinates of MLCD effectiveness with respect to their associated fish assemblages. (PDF contains 217 pages)

Influence of hard bottom morphology on fish assemblages of the continental shelf off Georgia, southeastern USA

Various reef types worldwide have inconsistent relationships among fish assemblage parameters and benthic characteristics, thus there is a need to identify factors driving assemblage structure specific to each reef type and locale. Limestone ledges are known to be key habitats for bottom fish on the continental shelf of the southeastern USA, however, the specific factors that link them to fish assemblages have not been quantified. Bottom fishes and habitat characteristics on ledges were surveyed at a study site located centrally in the southeastern USA continental shelf. Species richness, diversity, abundance, and biomass of fish were higher at ledges than on flat bottom. Species richness, abundance, and biomass of fish were well explained by ledge variables including percent cover of sessile invertebrates, total height, and height of undercut recesses. Multivariate analyses based on biomass of individual species at ledges revealed two fish assemblages associated with four ledge types. One assemblage was associated with ledges that were tall, heavily colonized with sessile invertebrates, large in area, and did or did not have undercuts. The other assemblage was associated with ledges that were short, not undercut, smaller in area, and were or were not heavily colonized by invertebrates. Seafloor classification schemes presently used in the region do not adequately capture hard bottom diversity to identify the location and extent of essential fish habitats for ecological and fisheries purposes. Given that ledges cover only ∼1% to 5% of the southeastern USA continental shelf, they merit the highest levels of consideration in regional research, conservation, and management plans.

Ecology and settlement of marine fouling in the Suez Bay, Egypt

This study deals with seasonal variations, natural correlations and similarities of fouling assemblages on exposure panels in the Suez Bay during January 1992 to January 1993. Three main sources of pollutions flow into the bay; industrial waste products, domestic drainage of Suez city and ships' oil and refuse.The fouling assemblages on the test pan els after various periods (1, 2 and 3 months) belonged mainly to the algae (Ulva rigida), polychaetes (Hydroides elegans), Cirripedes (Balanus amphitrite) and amphipods. The fouling at the lst station was relatively more dense than at the 2nd station during the summer and autumn seasons. The lowest productivity was achieved at the 3rd station which was considered less polluted being offshore water. The overall paucity of fouling in the bay is because of the silt covering the submerged surfaces, particularly at the 2nd station, leading to the prevention of the settlements or establishment of fouling organisms. The seasonal changes in the intensity of fouling assemblages on submerged surfaces in seawater seems to be closely related to seasonal variations in water temperature. The great fouling communities on the buoys and long exposure panels showed a remarkable variety of species and density rather than on short term exposures, which were more dense during warmer months.

Exploiting the bioactive potential of marine sponge microbiota

Endospore-forming bacteria are often isolated from different marine sponges, but their abundance varies, and they are frequently missed by culture-independent studies. Within endospore-formers, Bacillus are renowned for the production of antimicrobials and other compounds of medical and industrial importance. Although this group has been well studied in many different environments, very little is known about the actual diversity and properties of sporeformers associated with marine sponges. Identification of the endospore-forming bacteria associated with the marine sponges; Haliclona simulans, Amphilectus fucorum and Cliona celata, has uncovered an abundant and diverse microbial population composed of Bacillus, Paenibacillus, Solibacillus, Halobacillus and Viridibacillus species. This diversity appears to be overlooked by other non-targeted approaches where spore-formers are masked by more dominant species within the ecosystem. In addition to the identification of two antibiotic resistant plasmids, this bank of sporeformers produce a range of bioactive compounds. New antimicrobial compounds are urgently needed to combat the spread of multidrug resistant pathogens, as few new options are entering the drug discovery pipelines for clinical trials. Based on the results of this project, endospore-formers associated with marine sponges may hold the answer. The power of coupling functional based assays with genomic approaches has enabled us to identify a novel class 1 lantibiotic, subtilomycin, which is active against several clinically relevant pathogens. Subtilomycin is encoded in the genomes of all the marine sponge B. subtilis isolates analysed. They cluster together phylogenetically and form a distinct group from other sequenced B. subtilis strains. Regardless of its potential clinical relevance, subtilomycin may be providing these strains with a specific competitive advantage(s) within the stringent confines of the marine sponge environment. This work has outlined the industrial and biotechnological potential of marine sponge endospore-formers which appear to produce a cocktail of bioactive compounds. Genome sequencing of specific marine sponge isolates highlighted the importance of mining extreme environments and habitats for new lead compounds with potential therapeutic applications.

Assessing the conservation status of marine habitats: thoughts from a sandflat on the Isles of Scilly.

Statutory monitoring of the fauna of the ‘mudflats and sandflats not covered by seawater at low tide’ biotope complex on St Martin’s Flats, a part of the Isles of Scilly Complex Special Area of Conservation, was undertaken in 2000, 2004 and 2009. The targets set by Natural England for “characteristic biotopes” were that “composite species, abundance and diversity should not deviate significantly from an established baseline, subject to natural change”. The three specified biotopes could not be distinguished, and instead three assemblages were subjectively defined based on sediment surface features. There were statistically significant natural changes in diversity and species composition between years, especially in the association initially characterized by the razor-clam Ensis, and possible reasons for this are discussed. It is suggested that setting fixed local limits on natural variability is almost always impractical. Two possible approaches to distinguishing between natural and anthropogenic changes are suggested; a change in ecological condition as indicated by AMBI scores, and a significant change in average taxonomic distinctness (Δ+) compared with expectation. The determination of species biomasses as well as abundances might also open more possibilities for assessment. The practice of setting objectives for a marine SAC feature that include the range and number of biotopes cannot be supported, in view the difficulty in ascribing assemblages to recognised biotopes. A more realistic definition of species assemblages might best be gained from examination of the species that consistently make a substantial contribution to the Bray Curtis similarity among samples collected from specific sites.

Relationships between biodiversity and the stability of marine ecosystems: comparisons at a European scale using meta-analysis.

The relationship between biodiversity and stability of marine benthic assemblages was investigated using existing data sets (n = 28) covering various spatial (m-km) and temporal (1973-2006) scales in different benthic habitats (emergent rock, rock pools and sedimentary habitats) through meta-analyses. Assemblage stability was estimated by measuring temporal variances of species richness, total abundance (density or % cover) and community species composition and abundance structure (using multivariate analyses). Positive relationships between temporal variability in species number and richness were generally observed at both quadrat (<1 m2) and site (100 m2) scales, while no relationships were observed by multivariate analyses. Positive relationships were also observed at the scale of site between temporal variability in species number and variability in community structure with evenness estimates. This implies that the relationship between species richness or evenness and species richness variability is slightly positive and depends on the scale of observation, suggesting that biodiversity per se is important for the stability of ecosystems. Changes within community assemblages in terms of structure are, however, generally independent of biodiversity, suggesting no effect of diversity, but the potential impact of individual species, and/or environmental factors. Except for sedimentary and rock pool habitats, no relationship was observed between temporal variation of the aggregated variable of total abundances and diversity at either scale. Overall our results emphasise that relationships depend on scale of measurements, type of habitats and the marine systems (North Atlantic and Mediterranean) considered.

Phytobenthic assemblages of Addaia Bay (Menorca, Western Mediterranean): composition and distribution

Study on the composition and distribution of Phytobenthic Assemblages of Addaia Bay (Menorca, Western Mediterranean)

Species Assemblages of Leptocephali in the Southwestern Sargasso Sea

Catches of leptocephali of shelf and slope marine eels of the Chlopsidae, Congridae, Moringuidae, Muraenidae, and Ophichthidae collected during a survey in the southwestern Sargasso Sea in late September and early October 1984 were analyzed to learn about their reproductive ecology and larval transport. Sampling along a transect from the Florida Current (FC) out across the southwestern Sargasso Sea and in the Northwest Providence Channel (NWPC) of the Northern Bahamas enabled the evaluation of the larval distributions, abundances and size ranges, regional assemblage structure, and the apparent spawning areas of these marine eels. Distinctly different assemblages observed in the FC and NWPC included the congrid genera Heteroconger, Paraconger, Uroconger, and many ophichthid species, which were rare or absent offshore. Other taxa of congrids, chlopsids, muraenids and moringuids were present in all areas, but the smallest specimens of most taxa were only caught at the NWPC or FC stations. Multivariate analyses reflected higher richness and abundance in the FC and NWPC and also similar species compositions in offshore areas. The patterns of distribution of these leptocephali differed from those of anguillid, nettastomatid, and mesopelagic eel leptocephali collected in the same survey. These findings support the hypothesis that most taxa of marine eels spawn close to their adult habitats, and indicate that despite high biodiversity of marine eels in the Northern Bahamas, only some species of leptocephali appear to get transported far offshore by ocean currents.