Increased settlement on bacterial biofilms has been demonstrated for a number of marine invertebrate larvae, but the nature of the cue(s) responsible is not well understood. We tested the hypothesis that the bay barnacle Balanus improvisus utilises the bacterial signal molecules N-acylhomoserine lactones (AHLs) as a cue for the selection of sites for permanent attachment. Single species biofilms of the AHL-producing bacteria Vibrio anguillarum, Aeromonas hydrophila and Sulfitobacter sp. BR1 were attractive to settling cypris larvae of Balanus improvisus. However, when AHL production was inactivated, either by mutation of the AHL synthetic genes or by expression of an AHL-degrading gene (aiiA), the ability of the bacteria to attract cyprids was abolished. In addition, cyprids actively explored biofilms of E. coli expressing recombinant AHL synthase genes, but not on E. coli that did not produce AHLs. Finally, concentrations of synthetic AHLs similar to those found within natural biofilms (5 µM) resulted in increased cyprid settlement. Thus, the attraction of B. improvisus larvae to biofilms appears to be mediated by AHL signalling bacteria in the laboratory. This adds to our understanding of how quorum sensing inhibition may be used as a means of biofouling control. Nonetheless, the significance of our results for larvae settling naturally in the field, and the mechanisms that underlay the observed responses to AHLs, are as yet unknown.
Tait and Havenhand Biofilm data
Biofilms data.txt: This data records the number of Balanus improvisus cyprids exploring (Day 2 data only) and the number of cyprids settled (Days 3 to 7 data) on biofilms of signal-producing and signal-deficient biofilms. Biofilms either had mutations to their AHL synthase gene or were expressing the recombinant AHL inactivation protein AiiA. The biofilms were: Vibrio anguillarum NB10, a vanM mutant of this strain, and a variant expressing aiiA; A. hydrophila and an ahylI mutant and Sulfitobacter sp. BR1 and a sulI mutant. Each experiment also contained no biofilm controls. Experiments with V. anguillarum were conducted with 3 batches of cyprids, Sulfitobacter BR1 with 2 batches of cyprids and A. hydrophila with 1 batch of cyprids. This data was recorded by Karen Tait, Plymouth Marine Laboratory (ktait@pml.ac.uk) at the Department of Biological and Environmental Sciences, Tjarno, Sweden in June 2009. Column headings: V. anguillarum Batch 1; V. anguillarum Batch 2; V. anguillarum Batch 3; A. hydrophila Batch 1; Sulfitobacter Batch 1; Sulfitobacter Batch 2. Sub-headings: Biofilm Type (WT, mutant or no biofilm); Vessel No.; Day 2 (percent number of cyprids actively exploring biofilm); Day 3 (percent number of cyprids settled); Day 4 (percent number of cyprids settled); Day 5 (percent number of cyprids settled); Day 6 (percent number of cyprids settled); Day 7 (percent number of cyprids settled). Mutant biofilm descriptions are as follows: vanM – V. anguillarum, mutation to the AHL synthase, vanM; aiiA – V. anguillarum expressing the AHL degradation protein AiiA; ahyI – A. hydrophila, mutation to the AHL synthase, ahyl; sulI – Sulfitobacter BR1, mutation to the AHL synthase, sulI.
Tait and Havenhand E. coli data
E. coli data.txt: This data records the number of Balanus improvisus cyprids exploring biofilms of E. coli expressing recombinant AHL synthases after 2 days incubation. For each AHL synthase gene, this is compared to a background control (E. coli carrying the same plasmid without the AHL synthase insertion). Data is the percent number of cyprids actively exploring (crawling) on the biofilms. This data was recorded by Karen Tait, Plymouth Marine Laboratory (ktait@pml.ac.uk) at the Department of Biological and Environmental Sciences, Tjarno, Sweden in June 2009. Column headings: Vessel number (numbered 1 – 21); no biofilm (vessel contained no biofilm); E. coli pGEM (control biofilm); E. coli pGEM sulI (produces the AHLs C4-HSL, HC6-HSL, C8-HSL and OC10-HSL); E. coli pT3T7 (control biofilm); E. coli pT3T7 luxI (produces the AHL OC6-HSL); E. coli pUCP18 (control biofilm); E. coli pUCP18 rhlI (produces the AHL C4-HSL); E. coli pET3a (control biofilm); E. coli pET3a vanI (produces the AHL OC10-HSL).
Tait and Havenhand Synthetic AHL data
Synthetic AHL data.txt: This data records the number of Balanus improvisus cyprids exploring agarose films containing 5 µM C6-HSL, C8-HSL, OC10-HSL and C12-HSL after 2 days incubation. Data is the percent number of cyprids actively exploring (crawling) on the biofilms. Also recorded is the numbers of cyprids settled within vessels containing the same synthetic AHLs dissolved in seawater to the following concentrations: 0.5, 5 and 50 µM. Data is the percent number of cyprids settled within the vessels. This data was recorded by Karen Tait, Plymouth Marine Laboratory (ktait@pml.ac.uk) at the Department of Biological and Environmental Sciences, Tjarno, Sweden in June 2009. Column headings: Synthetic AHL in agarose film; Vessel number (numbered 1 – 18); C6; C8; OC10; C12; no AHL. Synthetic AHL in seawater; AHL concentration (0, 0.5, 5 or 50 µM); Vessel number (numbered 1 – 21 for each treatment); C6; C8; OC10; C12.
