gbpA as a Novel qPCR Target for the Species-Specific Detection of Vibrio cholerae O1, O139, Non-O1/Non-O139 in Environmental, Stool, and Historical Continuous Plankton Recorder Samples.

The Vibrio cholerae N-acetyl glucosamine-binding protein A (GbpA) is a chitin-binding protein involved in V. cholerae attachment to environmental chitin surfaces and human intestinal cells. We previously investigated the distribution and genetic variations of gbpA in a large collection of V. cholerae strains and found that the gene is consistently present and highly conserved in this species. Primers and probe were designed from the gbpA sequence of V. cholerae and a new Taq-based qPCR protocol was developed for diagnostic detection and quantification of the bacterium in environmental and stool samples. In addition, the positions of primers targeting the gbpA gene region were selected to obtain a short amplified fragment of 206 bp and the protocol was optimized for the analysis of formalin-fixed samples, such as historical Continuous Plankton Recorder (CPR) samples. Overall, the method is sensitive (50 gene copies), highly specific for V. cholerae and failed to amplify strains of the closely-related species Vibrio mimicus. The sensitivity of the assay applied to environmental and stool samples spiked with V. cholerae ATCC 39315 was comparable to that of pure cultures and was of 102 genomic units/l for drinking and seawater samples, 101 genomic units/g for sediment and 102 genomic units/g for bivalve and stool samples. The method also performs well when tested on artificially formalin-fixed and degraded genomic samples and was able to amplify V. cholerae DNA in historical CPR samples, the earliest of which date back to August 1966. The detection of V. cholerae in CPR samples collected in cholera endemic areas such as the Benguela Current Large Marine Ecosystem (BCLME) is of particular significance and represents a proof of concept for the possible use of the CPR technology and the developed qPCR assay in cholera studies.

Sustained UK marine observations. Where have we been? Where are we now? Where are we going?

This introduction traces the earliest interaction of ancient humans with their marine environment, through marine explorations in the Middle Ages and Renaissance, to the development of early marine science in the Enlightenment. This sets the scene for how marine observations developed in the modern era and explains the status of today's marine observation networks. The paper concludes with an assessment of the future needs and constraints of sustained marine observation networks and suggests the lessons from a long history might be the key to the future.

Bacterial diversity in the bacterioneuston (sea surface microlayer): the bacterioneuston through the looking glass.

The bacterioneuston is defined as the community of bacteria present within the neuston or sea surface microlayer. Bacteria within this layer were sampled using a membrane filter technique and bacterial diversity was compared with that in the underlying pelagic coastal seawater using molecular ecological techniques. 16S rRNA gene libraries of approximately 500 clones were constructed from both bacterioneuston and the pelagic water samples and representative clones from each library were sequenced for comparison of bacterial diversity. The bacterioneuston was found to have a significantly lower bacterial diversity than the pelagic seawater, with only nine clone types (ecotaxa) as opposed to 46 ecotaxa in the pelagic seawater library. Surprisingly, the bacterioneuston clone library was dominated by 16S rRNA gene sequences affiliated to two groups of organisms, Vibrio spp. which accounted for over 68% of clones and Pseudoalteromonas spp. accounting for 21% of the library. The dominance of these two 16S rRNA gene sequence types within the bacterioneuston clone library was confirmed in a subsequent gene probing experiment. 16S rRNA gene probes specific for these groups of bacteria were designed and used to probe new libraries of 1000 clones from both the bacterioneuston and pelagic seawater DNA samples. This revealed that 57% of clones from the bacterioneuston library hybridized to a Vibrio sp.-specific 16S rRNA gene probe and 32% hybridized to a Pseudoalteromonas sp.-specific 16S rRNA gene probe. In contrast, the pelagic seawater library resulted in only 13% and 8% of 16S rRNA gene clones hybridizing to the Vibrio sp. and Pseudoalteromonas sp. probes respectively. Results from this study suggest that the bacterioneuston contains a distinct population of bacteria and warrants further detailed study at the molecular level.

The large capacity for dark nitrate-assimilation in diatoms may overcome nitrate limitation of growth.

• The ability of the diatom Thalassiosira weissflogii to assimilate inorganic N in darkness is compared with that seen in flagellates. • Experiments were conducted with T. weissflogii grown in N-replete and in N-limiting cultures and the rates and capacity for ammonium and nitrate assimilation were determined. • High daily growth rates in the diatom under high-light nitrate-replete conditions are only attainable by continuing nitrate assimilation in darkness using excess C accumulated in the light when nitrate assimilation cannot match C-fixation. The ability to use ammonium in darkness is greater than for nitrate but the ratio of dark to light assimilation for each N source is similar over a wide range of cellular N : C ratios. These capabilities are in strong contrast with those in the flagellates Heterosigma carterae and Heterocapsa illdefina, which are incapable of high nitrate use in darkness. • While the possession of large capacity for dark nitrate-assimilation in diatoms may provide a mechanism that overcomes nitrate limitation of growth, the explanation for the lower capabilities exhibited by flagellates is less clear.

Modelling suggests that optimization of dark nitrogen-assimilation need not be a critical selective feature in phytoplankton.

• Alternative strategies for the dark assimilation of ammonium and nitrate into microalgae are explored using a mechanistic model of algal physiology. • The standard diatom strategy, continuation of N assimilation at high rates in darkness as long as reserve C remains, is the most advantageous. The flagellate strategy, incorporating ammonium but not nitrate at a reasonable rate in darkness, is best suited to organisms with high metabolic costs, inhabiting waters with relatively high concentrations of ammonium. The strategy of vertically migrating diatoms - accumulation of nitrate in internal pools for assimilation after return to the photic zone - is best suited to slow-growing cells in low-ammonium environments. • Differences between the strategies become less significant with increasing N-source limitation (the situation more typically encountered by flagellates and migratory species) because transport rather than post-transport assimilatory processes become most limiting. • It is suggested that optimization of dark N-assimilation is not a critical selective feature; organisms with contrasting abilities in this regard usually inhabit different water bodies and have other more fundamental phenotypic differences (e.g. motility or silicon requirements).