A simple and effective method for high quality co-extraction of genomic DNA and total RNA from low biomass Ectocarpus siliculosus, the model brown alga.

The brown seaweed Ectocarpus siliculosus is an emerging model species distributed worldwide in temperate coastal ecosystems. Over 1500 strains of E. siliculosus are available in culture from a broad range of geographic locations and ecological niches. To elucidate the molecular mechanisms underlying its capacity to cope with different environmental and biotic stressors, genomic and transcriptomic studies are necessary; this requires the co-isolation of genomic DNA and total RNA. In brown algae, extraction of nucleic acids is hindered by high concentrations of secondary metabolites that co-precipitate with nucleic acids. Here, we propose a reliable, rapid and cost-effective procedure for the co-isolation of high-quality nucleic acids using small quantities of biomass (25-, 50- and 100 mg) from strains of E. siliculosus (RHO12; LIA4A; EC524 and REP10-11) isolated from sites with different environmental conditions. The procedure employs a high pH extraction buffer (pH 9.5) which contains 100 mM Tris-HCl and 150 mM NaCl, with the addition of 5 mM DTT and 1% sarkosyl to ensure maximum solubility of nucleic acids, effective inhibition of nuclease activity and removal of interfering contaminants (e.g. polysaccharides, polyphenols). The use of sodium acetate together with isopropanol shortened precipitation time and enhanced the yields of DNA/RNA. A phenol:chlorophorm:isoamyl alcohol step was subsequently used to purify the nucleic acids. The present protocol produces high yields of nucleic acids from only 25 mg of fresh algal biomass (0.195 and 0.284 µg mg(-1) fresh weigh of RNA and DNA, respectively) and the high quality of the extracted nucleic acids was confirmed through spectrophotometric and electrophoretic analyses. The isolated RNA can be used directly in downstream applications such as RT-PCR and the genomic DNA was suitable for PCR, producing reliable restriction enzyme digestion patterns. Co-isolation of DNA/RNA from different strains indicates that this method is likely to have wider applications for intra- and inter-specific studies on other brown algae

Gender differences in the utilization of health-care services among the older adult population of Spain

BACKGROUND: Compared to men, women report greater morbidity and make greater use of health-care services. This study examines potential determinants of gender differences in the utilization of health-care services among the elderly. METHODS: Cross-sectional study covering 3030 subjects, representative of the non-institutionalized Spanish population aged 60 years and over. Potential determinants of gender differences in the utilization of health services were classified into predisposing factors (age and head-of-family status), need factors (lifestyles, chronic diseases, functional status, cognitive deficit and health-related quality of life (HRQL)) and enabling factors (educational level, marital status, head-of-family employment status and social network). Relative differences in the use of each service between women and men were summarized using odds ratios (OR), obtained from logistic regression. The contribution of the variables of interest to the gender differences in the use of such services was evaluated by comparing the OR before and after adjustment for such variables. RESULTS: As compared to men, a higher percentage of women visited a medical practitioner (OR: 1.24; 95% confidence limits (CL): 1.07–1.44), received home medical visits (OR: 1.67; 95% CL: 1.34–2.10) and took ≥3 medications (OR: 1.54; 95% CL: 1.34–1.79), but there were no gender differences in hospital admission or influenza vaccination. Adjustment for need or enabling factors led to a reduction in the OR of women compared to men for utilization of a number of services studied. On adjusting for the number of chronic diseases, the OR (95% CL) of women versus men for ingestion of ≥3 medications was 1.24 (1.06–1.45). After adjustment for HRQL, the OR was 1.03 (0.89–1.21) for visits to medical practitioners, 1.24 (0.98–1.58) for home medical visits, 0.71 (0.58–0.87) for hospitalization, and 1.14 (0.97–1.33) for intake of ≥3 medications. After adjustment for the number of chronic diseases and HRQL, the OR of hospitalization among women versus men was 0.68 (0.56–0.84). CONCLUSION: The factors that best explain the greater utilization of health-care services by elderly women versus men are the number of chronic diseases and HRQL. For equal need, certain inequality was observed in hospital admission, in that it proved less frequent among women

Reconciliation of the carbon budget in the ocean’s twilight zone

Photosynthesis in the surface ocean produces approximately 100 gigatonnes of organic carbon per year, of which 5 to 15 per cent is exported to the deep ocean1, 2. The rate at which the sinking carbon is converted into carbon dioxide by heterotrophic organisms at depth is important in controlling oceanic carbon storage3. It remains uncertain, however, to what extent surface ocean carbon supply meets the demand of water-column biota; the discrepancy between known carbon sources and sinks is as much as two orders of magnitude4, 5, 6, 7, 8. Here we present field measurements, respiration rate estimates and a steady-state model that allow us to balance carbon sources and sinks to within observational uncertainties at the Porcupine Abyssal Plain site in the eastern North Atlantic Ocean. We find that prokaryotes are responsible for 70 to 92 per cent of the estimated remineralization in the twilight zone (depths of 50 to 1,000 metres) despite the fact that much of the organic carbon is exported in the form of large, fast-sinking particles accessible to larger zooplankton. We suggest that this occurs because zooplankton fragment and ingest half of the fast-sinking particles, of which more than 30 per cent may be released as suspended and slowly sinking matter, stimulating the deep-ocean microbial loop. The synergy between microbes and zooplankton in the twilight zone is important to our understanding of the processes controlling the oceanic carbon sink

Seasonal dynamics of active SAR11 ecotypes in the oligotrophic Northwest Mediterranean Sea

A seven-year oceanographic time series in NW Mediterranean surface waters was combined with pyrosequencing of ribosomal RNA (16S rRNA) and ribosomal RNA gene copies (16S rDNA) to examine the environmental controls on SAR11 ecotype dynamics and potential activity. SAR11 diversity exhibited pronounced seasonal cycles remarkably similar to total bacterial diversity. The timing of diversity maxima was similar across narrow and broad phylogenetic clades and strongly associated with deep winter mixing. Diversity minima were associated with periods of stratification that were low in nutrients and phytoplankton biomass and characterised by intense phosphate limitation (turnover time80%) by SAR11 Ia. A partial least squares (PLS) regression model was developed that could reliably predict sequence abundances of SAR11 ecotypes (Q2=0.70) from measured environmental variables, of which mixed layer depth was quantitatively the most important. Comparison of clade-level SAR11 rRNA:rDNA signals with leucine incorporation enabled us to partially validate the use of these ratios as an in-situ activity measure. However, temporal trends in the activity of SAR11 ecotypes and their relationship to environmental variables were unclear. The strong and predictable temporal patterns observed in SAR11 sequence abundance was not linked to metabolic activity of different ecotypes at the phylogenetic and temporal resolution of our study

Composition Influences the Pathway but not the Outcome of the Metabolic Response of Bacterioplankton to Resource Shifts

Bacterioplankton community metabolism is central to the functioning of aquatic ecosystems, and strongly reactive to changes in the environment, yet the processes underlying this response remain unclear. Here we explore the role that community composition plays in shaping the bacterial metabolic response to resource gradients that occur along aquatic ecotones in a complex watershed in Québec. Our results show that the response is mediated by complex shifts in community structure, and structural equation analysis confirmed two main pathways, one involving adjustments in the level of activity of existing phylotypes, and the other the replacement of the dominant phylotypes. These contrasting response pathways were not determined by the type or the intensity of the gradients involved, as we had hypothesized, but rather it would appear that some compositional configurations may be intrinsically more plastic than others. Our results suggest that community composition determines this overall level of community plasticity, but that composition itself may be driven by factors independent of the environmental gradients themselves, such that the response of bacterial communities to a given type of gradient may alternate between the adjustment and replacement pathways. We conclude that community composition influences the pathways of response in these bacterial communities, but not the metabolic outcome itself, which is driven by the environment, and which can be attained through multiple alternative configurations

A metagenomic assessment of winter and summer bacterioplankton from Antarctica Peninsula coastal surface waters

© The Author(s), 2012. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in The ISME Journal 6 (2012): 1901-1915, doi:10.1038/ismej.2012.31.Antarctic surface oceans are well-studied during summer when irradiance levels are high, sea ice is melting and primary productivity is at a maximum. Coincident with this timing, the bacterioplankton respond with significant increases in secondary productivity. Little is known about bacterioplankton in winter when darkness and sea-ice cover inhibit photoautotrophic primary production. We report here an environmental genomic and small subunit ribosomal RNA (SSU rRNA) analysis of winter and summer Antarctic Peninsula coastal seawater bacterioplankton. Intense inter-seasonal differences were reflected through shifts in community composition and functional capacities encoded in winter and summer environmental genomes with significantly higher phylogenetic and functional diversity in winter. In general, inferred metabolisms of summer bacterioplankton were characterized by chemoheterotrophy, photoheterotrophy and aerobic anoxygenic photosynthesis while the winter community included the capacity for bacterial and archaeal chemolithoautotrophy. Chemolithoautotrophic pathways were dominant in winter and were similar to those recently reported in global ‘dark ocean’ mesopelagic waters. If chemolithoautotrophy is widespread in the Southern Ocean in winter, this process may be a previously unaccounted carbon sink and may help account for the unexplained anomalies in surface inorganic nitrogen content.CSR was supported by an NSF Postdoctoral Fellowship in Biological Informatics (DBI-0532893). The research was supported by National Science Foundation awards: ANT 0632389 (to AEM and JJG), and ANT 0632278 and 0217282 (to HWD), all from the Antarctic Organisms and Ecosystems Program

Coalescent Simulations Reveal Hybridization and Incomplete Lineage Sorting in Mediterranean Linaria

We examined the phylogenetic history of Linaria with special emphasis on the Mediterranean sect. Supinae (44 species). We revealed extensive highly supported incongruence among two nuclear (ITS, AGT1) and two plastid regions (rpl32-trnLUAG, trnS-trnG). Coalescent simulations, a hybrid detection test and species tree inference in *BEAST revealed that incomplete lineage sorting and hybridization may both be responsible for the incongruent pattern observed. Additionally, we present a multilabelled *BEAST species tree as an alternative approach that allows the possibility of observing multiple placements in the species tree for the same taxa. That permitted the incorporation of processes such as hybridization within the tree while not violating the assumptions of the *BEAST model. This methodology is presented as a functional tool to disclose the evolutionary history of species complexes that have experienced both hybridization and incomplete lineage sorting. The drastic climatic events that have occurred in the Mediterranean since the late Miocene, including the Quaternary-type climatic oscillations, may have made both processes highly recurrent in the Mediterranean flora