Figure 1.

Retrospective assessment of relative abundance of vibrios, Vibrio relative abundance index (VAI), in the North Sea. Methods used to calculate the relative abundance of vibrios (VAI index) from formalin-fixed plankton samples collected by the CPR survey off the Rhine and Humber estuaries, in August, from 1961 to 2005. (a) CPR samples were collected in the North Sea from 1961 to 2005 by the CPR survey. (b) Back in the laboratory, the silk containing the entrapped plankton (under 4–10% buffered formalin) is cut into blocks, each representing 10 nautical miles of tow (3 m³ of filtered seawater). (c) Five replicate 1-cm² sections are prepared from each CPR block for microbiological molecular analyses. (d) Genomic DNA is extracted from each section and purified. (e) The ratio of Vibrio spp. cells to total bacterial cells is assessed by real-time PCR of 10 ng of genomic DNA from each section using genus-specific and universal primers, respectively, producing small amplicons of similar size (113 vs 98 bp) to avoid age- and formalin-induced bias (see main text). The VAI is then calculated for each sample by average values of this ratio for the five replicate measurements.