Application of microarrays (phylochips) for analysis of community diversity by species identification

Abstract Molecular probe-based methods (Fluorescent in-situ hybridisation or FISH, Next Generation Sequencing or NGS) have proved successful in improving both the efficiency and accuracy of the identification of microorganisms, especially those that lack distinct morphological features, such as picoplankton. However, FISH methods have the major drawback that they can only identify one or just a few species at a time because of the reduced number of available fluorochromes that can be added to the probe. Although the length of sequence that can be obtained is continually improving, NGS still requires a great deal of handling time, its analysis time is still months and with a PCR step it will always be sensitive to natural enzyme inhibitors. With the use of DNA microarrays, it is possible to identify large numbers of taxa on a single-glass slide, the so-called phylochip, which can be semi-quantitative. This review details the major steps in probe design, design and production of a phylochip and validation of the array. Finally, major microarray studies in the phytoplankton community are reviewed to demonstrate the scope of the method.

Application of microarrays (phylochips) for analysis of community diversity by species identification

Abstract Molecular probe-based methods (Fluorescent in-situ hybridisation or FISH, Next Generation Sequencing or NGS) have proved successful in improving both the efficiency and accuracy of the identification of microorganisms, especially those that lack distinct morphological features, such as picoplankton. However, FISH methods have the major drawback that they can only identify one or just a few species at a time because of the reduced number of available fluorochromes that can be added to the probe. Although the length of sequence that can be obtained is continually improving, NGS still requires a great deal of handling time, its analysis time is still months and with a PCR step it will always be sensitive to natural enzyme inhibitors. With the use of DNA microarrays, it is possible to identify large numbers of taxa on a single-glass slide, the so-called phylochip, which can be semi-quantitative. This review details the major steps in probe design, design and production of a phylochip and validation of the array. Finally, major microarray studies in the phytoplankton community are reviewed to demonstrate the scope of the method.

Increased blooms of a dinoflagellate in the NW Atlantic

Sampling by the Continuous Plankton Recorder (CPR) over the NW Atlantic from 1960 to 2000 has enabled long-term studies of the larger components of the phytoplankton community, highlighting various changes, particularly during the 1990s. Analysis of an index of phytoplankton biomass, the Phytoplankton Colour Index (PCI) has revealed an increase over the past decade, most marked during the winter (December to February) months. Examination of the structure of the community using multiple linear-regression models indicates that the winter phytoplankton community composition has changed markedly in the 1990s compared to the 1960s. One phytoplankter, the dinoflagellate Ceratium arcticum (Cleve), has undergone dramatic changes in abundance during this period, with pronounced large winter blooms and decreased autumnal levels, and its contribution to the Phytoplankton Colour index values has increased significantly. Other dominant species in the phytoplankton community, both diatoms and dinoflagellates, did not show the same variations over the examined time period. It is suggested that the response of C. arcticum is probably a result of previously reported changes in stratification in the NW Atlantic, due to dynamic hydro-climatic (freshening and cooling) events.

Reduction in photosystem II efficiency during a virus-controlled Emiliania huxleyi bloom

During viral infection of Emiliania huxleyi, laboratory studies have shown that photo-system (PS) II efficiency declines during the days post-infection and is thought to be associated with viral-induced interruption of electron transport rates between photosystems. However,measuring the impact of viral infection on PSII function in E. huxleyi populations from natural,taxonomically diverse phytoplankton communities is difficult, and whether this phenomenon occurs in nature is presently unknown. Here, chlorophyll fluorescence analysis was used to assess changes in PSII efficiency throughout an E. huxleyi bloom during a mesocosm experiment off the coast of Norway. Specifically, we aimed to determine whether a measurable suppression of the efficiency of PSII photochemistry could be observed due to viral infection of the natural E. huxleyi populations. During the major infection period prior to bloom collapse, there was a significant reduction in PSII efficiency with an average decrease in maximum PSII photochemical efficiency (Fv/Fm) of 17% and a corresponding 75% increase in maximum PSII effective absorption cross section(σPSII); this was concurrent with a significant decrease in E. huxleyi growth rates and an increase in E. huxleyi virus (EhV) production. As E. huxleyi populations dominated the phytoplankton community and potentially contributed up to 100% of the chlorophyll a pool, we believe that the variable chlorophyll fluorescence signal measured during this period was derived predominantly from E. huxleyi and, thus, reflects changes occurring within E. huxleyi cells. This is the first demonstration of suppression of PSII photochemistry occurring during viral infection of natural coccolithophore populations.

Bridging the gap between marine biogeochemical and fisheries sciences; configuring the zooplankton link

Exploring climate and anthropogenic impacts on marine ecosystems requires an understanding of how trophic components interact. However, integrative end-to-end ecosystem studies (experimental and/or modelling) are rare. Experimental investigations often concentrate on a particular group or individual species within a trophic level, while tropho-dynamic field studies typically employ either a bottom-up approach concentrating on the phytoplankton community or a top-down approach concentrating on the fish community. Likewise the emphasis within modelling studies is usually placed upon phytoplankton-dominated biogeochemistry or on aspects of fisheries regulation. In consequence the roles of zooplankton communities (protists and metazoans) linking phytoplankton and fish communities are typically under-represented if not (especially in fisheries models) ignored. Where represented in ecosystem models, zooplankton are usually incorporated in an extremely simplistic fashion, using empirical descriptions merging various interacting physiological functions governing zooplankton growth and development, and thence ignoring physiological feedback mechanisms. Here we demonstrate, within a modelled plankton food-web system, how trophic dynamics are sensitive to small changes in parameter values describing zooplankton vital rates and thus the importance of using appropriate zooplankton descriptors. Through a comprehensive review, we reveal the mismatch between empirical understanding and modelling activities identifying important issues that warrant further experimental and modelling investigation. These include: food selectivity, kinetics of prey consumption and interactions with assimilation and growth, form of voided material, mortality rates at different age-stages relative to prior nutrient history. In particular there is a need for dynamic data series in which predator and prey of known nutrient history are studied interacting under varied pH and temperature regimes.

Challenges in integrative approaches to modelling the marine ecosystems of the North Atlantic: Physics to fish and coasts to ocean

It has long been recognised that there are strong interactions and feedbacks between climate, upper ocean biogeochemistry and marine food webs, and also that food web structure and phytoplankton community distribution are important determinants of variability in carbon production and export from the euphotic zone. Numerical models provide a vital tool to explore these interactions, given their capability to investigate multiple connected components of the system and the sensitivity to multiple drivers, including potential future conditions. A major driver for ecosystem model development is the demand for quantitative tools to support ecosystem-based management initiatives. The purpose of this paper is to review approaches to the modelling of marine ecosystems with a focus on the North Atlantic Ocean and its adjacent shelf seas, and to highlight the challenges they face and suggest ways forward. We consider the state of the art in simulating oceans and shelf sea physics, planktonic and higher trophic level ecosystems, and look towards building an integrative approach with these existing tools. We note how the different approaches have evolved historically and that many of the previous obstacles to harmonisation may no longer be present. We illustrate this with examples from the on-going and planned modelling effort in the Integrative Modelling Work Package of the EURO-BASIN programme.

The assessment of a global marine ecosystem model on the basis of emergent properties and ecosystem function: a case study with ERSEM

Ecosystem models are often assessed using quantitative metrics of absolute ecosystem state, but these model-data comparisons are disproportionately vulnerable to discrepancies in the location of important circulation features. An alternative method is to demonstrate the models capacity to represent ecosystem function; the emergence of a coherent natural relationship in a simulation indicates that the model may have an appropriate representation of the ecosystem functions that lead to the emergent relationship. Furthermore, as emergent properties are large-scale properties of the system, model validation with emergent properties is possible even when there is very little or no appropriate data for the region under study, or when the hydrodynamic component of the model differs significantly from that observed in nature at the same location and time. A selection of published meta-analyses are used to establish the validity of a complex marine ecosystem model and to demonstrate the power of validation with emergent properties. These relationships include the phytoplankton community structure, the ratio of carbon to chlorophyll in phytoplankton and particulate organic matter, the ratio of particulate organic carbon to particulate organic nitrogen and the stoichiometric balance of the ecosystem. These metrics can also inform aspects of the marine ecosystem model not available from traditional quantitative and qualitative methods. For instance, these emergent properties can be used to validate the design decisions of the model, such as the range of phytoplankton functional types and their behaviour, the stoichiometric flexibility with regards to each nutrient, and the choice of fixed or variable carbon to nitrogen ratios.

The significance of nitrogen regeneration for new production within a filament of the Mauritanian upwelling system

The Lagrangian progression of a biological community was followed in a filament of the Mauritanian upwelling system, north-west Africa, during offshore advection. The inert dual tracers sulfur hexafluoride and helium-3 labelled a freshly upwelled patch of water that was mapped for 8 days. Changes in biological, physical, and chemical characteristics were measured, including phytoplankton productivity, nitrogen assimilation, and regeneration. Freshly upwelled water contained high nutrient concentrations but was depleted in N compared to Redfield stoichiometry. The highest rate of primary productivity was measured on the continental shelf, associated with high rates of nitrogen assimilation and a phytoplankton community dominated by diatoms and flagellates. Indicators of phytoplankton abundance and activity decreased as the labelled water mass transited the continental shelf slope into deeper water, possibly linked to the mixed layer depth exceeding the light penetration depth. By the end of the study, the primary productivity rate decreased and was associated with lower rates of nitrogen assimilation and lower nutrient concentrations. Nitrogen regeneration and assimilation took place simultaneously. Results highlighted the importance of regenerated NHC 4 in sustaining phytoplankton productivity and indicate that the upwelled NO3 pool contained an increasing fraction of regenerated NO3 as it advected offshore. By calculating this fraction and incorporating it into an f ratio formulation, we estimated that of the 12:38Tg C of annual regional production, 4:73Tg C was exportable.

The significance of nitrogen regeneration for new production within a filament of the Mauritanian upwelling system

The Lagrangian progression of a biological community was followed in a filament of the Mauritanian upwelling system, north-west Africa, during offshore advection. The inert dual tracers sulfur hexafluoride and helium-3 labelled a freshly upwelled patch of water that was mapped for 8 days. Changes in biological, physical, and chemical characteristics were measured, including phytoplankton productivity, nitrogen assimilation, and regeneration. Freshly upwelled water contained high nutrient concentrations but was depleted in N compared to Redfield stoichiometry. The highest rate of primary productivity was measured on the continental shelf, associated with high rates of nitrogen assimilation and a phytoplankton community dominated by diatoms and flagellates. Indicators of phytoplankton abundance and activity decreased as the labelled water mass transited the continental shelf slope into deeper water, possibly linked to the mixed layer depth exceeding the light penetration depth. By the end of the study, the primary productivity rate decreased and was associated with lower rates of nitrogen assimilation and lower nutrient concentrations. Nitrogen regeneration and assimilation took place simultaneously. Results highlighted the importance of regenerated NHC 4 in sustaining phytoplankton productivity and indicate that the upwelled NO3 pool contained an increasing fraction of regenerated NO3 as it advected offshore. By calculating this fraction and incorporating it into an f ratio formulation, we estimated that of the 12:38Tg C of annual regional production, 4:73Tg C was exportable.

Differential contribution of diatoms and dinoflagellates to phytoplankton biomass in the NE Atlantic Ocean and the North Sea

Sampling by the continuous plankton recorder (CPR) survey over the North Atlantic Ocean and the North Sea has enabled long-term studies of phytoplankton biomass. Analysis of an index of phytoplankton biomass, the phytoplankton colour index (PCI), has previously shown an increase in phytoplankton biomass in the NE Atlantic. In the current study, further investigations were conducted to determine the contribution of diatom and dinoflagellate cell counts to the PCI, their fluctuations over the last 45 yr and their geographical variations in the eastern North Atlantic and the North Sea. An increased contribution of dinoflagellates to the PCI was revealed over the south NE Atlantic and the northern North Sea. In contrast, the contribution of diatoms decreased in the north NE Atlantic and the northern North Sea. No discernible trends were found in the other regions of the North Sea. The relative contributions of diatoms and dinoflagellates to the PCI led to the identification of 3 geographically distinct dynamic regimes in the diatom/dinoflagellate dynamics in the NE Atlantic and the North Sea. Finally, it is stressed that the discrepancy observed in the patterns of PCI and diatom and dinoflagellate cell counts suggests that changes in PCI do not reflect changes in the community structure and that the exclusive use of PCI is not adequate to investigate the long-term trends in the trophic link between phytoplankton and herbivorous zooplankton.

Impacts of fisheries on plankton community structure

There has been much debate on the extent to which resource availability (bottom-up) versus predation pressure from fish (top-down) modulates the dynamics of plankton in marine systems. Physico/chemical bottom-up forcing has been considered to be the main mechanism structuring marine ecosystems, although some field observations and empirical correlations support top-down modulation. Models have indicated possible feedback loops to the plankton and other studies have interpreted a grazing impact from long-term changes in fish stocks. In freshwater systems, evidence for top-down forcing by fish and trophic cascading is well documented. First, evidence for equivalent top-down effects in the marine environment is presented, with an overview of relevant publications. In the second part, time series, averaged for the North Sea (when possible from 1948 to 1997), of fish catch, recruitment, and spawning stock biomass are related to the abundance of species or larger groupings of zooplankton and phytoplankton from the Continuous Plankton Recorder survey and selected environmental parameters. Preliminary analysis suggests that there is strong interaction between different fish species and the plankton and that the fishery, through top-down control, may at times be an important contributor to changes in the North Sea ecosystem.

Plankton Of The Fladen Ground During Flex-76 .1. Spring Development Of The Plankton Community

Results from plankton sampling in the northern North Sea with the Continuous Plankton Recorder (CPR) and the Undulating Oceanographic Recorder (UOR) during the Fladen Ground Experiment in 1976 (FLEX 76) are summarised. The first evidence of the spring outbreak of phytoplankton was on 19 April, the day after the first signs of vertical stability of the water column were observed. This was followed by spawning of the euphausiid Thysanoessa inermis and rapid increase in the numbers of Calanus finmarchicus. C. finmarchicus was the most abundant species over the FLEX period (19 March to 3 June) and, together with T. inermis, accounted for over 80% of the dry weight of the zooplankton standing stock. By early June the standing crop of phytoplankton had been depleted and nutrients levels were reduced to very low concentrations in the upper 50 m.

Ocean acidification shows negligible impacts on high-latitude bacterial community structure in coastal pelagic mesocosms

The impact of ocean acidification and carbonation on microbial community structure was assessed during a large-scale in situ costal pelagic mesocosm study, included as part of the EPOCA 2010 Arctic campaign. The mesocosm experiment included ambient conditions (fjord) and nine mesocosms with pCO(2) levels ranging from similar to 145 to similar to 1420 mu atm. Samples for the present study were collected at ten time points (t-1, t1, t5, t7, t12, t14, t18, t22, t26 to t28) in seven treatments (ambient fjord (similar to 145), 2x similar to 185, similar to 270, similar to 685, similar to 820, similar to 1050 mu atm) and were analysed for "small" and "large" size fraction microbial community composition using 16S rRNA (ribosomal ribonucleic acid) amplicon sequencing. This high-throughput sequencing analysis produced similar to 20 000 000 16S rRNA V4 reads, which comprised 7000OTUs. The main variables structuring these communities were sample origins (fjord or mesocosms) and the community size fraction (small or large size fraction). The community was significantly different between the unenclosed fjord water and enclosed mesocosms (both control and elevated CO2 treatments) after nutrients were added to the mesocosms, suggesting that the addition of nutrients is the primary driver of the change in mesocosm community structure. The relative importance of each structuring variable depended greatly on the time at which the community was sampled in relation to the phytoplankton bloom. The sampling strategy of separating the small and large size fraction was the second most important factor for community structure. When the small and large size fraction bacteria were analysed separately at different time points, the only taxon pCO(2) was found to significantly affect were the Gammaproteobacteria after nutrient addition. Finally, pCO(2) treatment was found to be significantly correlated (non-linear) with 15 rare taxa, most of which increased in abundance with higher CO2.