Dataset: extract allelopathy
Deployment: Fiji_2011

To assess allelochemical induction in Sargassum and Galaxaura after 8 days of competition with coral, we generated hydrophobic extracts from treatment and control thalli and tested their allelopathic effects on new Porites in the field. We previously identified two allelopathic compounds produced by G. filamentosa-both terpenoid loliolide derivatives. Although these two molecules may be involved in the induction of allelopathy in Galaxaura, monitoring changes in their concentrations pre- versus post-competition to evaluate induction would be insufficient and potentially misleading, because they represent only two of at least six compounds in an allelopathic mixture produced by Galaxaura. The other allelopathic compounds remain unidentified due to their low yield and/or instability following isolation. Given that multiple compounds within Galaxaura act together to produce its allelopathic effect and that most of these compounds cannot be identified and thus quantified, the only rigorous way to evaluate induction was to assess the allelopathic strength of the extract from seaweeds that had competed with corals versus those that had not. We assessed this for hydrophobic crude extracts because previous studies demonstrated that hydrophilic fractions produced no allelopathic effect and the effect of the whole seaweed was reproduced by the hydrophobic extract alone.

For each seaweed species, we created bulk extracts by removing a small portion of thallus from each seaweed and grouping them by treatment or control. We determined the volumetric displacement of the grouped thalli from each species, exhaustively extracted each in 100% methanol, and dried each by rotary evaporation. We then partitioned each extract between water and ethyl acetate, and retained the hydrophobic (ethyl acetate) fraction of each for testing its allelopathic effects on Porites. To test its effects on Porites, hydrophobic extracts from treatment and control thalli were re-suspended in solvent and incorporated at natural volumetric concentration into a series of approximately 1 cm2 Phytagel (Sigma-Aldrich, USA) squares (n = 10 extract^-1 species^-1) hardened on window screen backing [37,38]. We also prepared squares with solvent but no seaweed extract as controls for the effects of the Phytagel, solvent and backing (n = 10; ‘Phytagel control’). Phytagel squares with and without seaweed extracts differed in colour and clarity but because the bioactivity of Sargassum extract squares (dark) and Phytagel control squares (light) did not differ (figure 1), nor did several pigmented seaweed extract versus Phytagel control contrasts in previous assays, we assumed that colour did not affect pulse-amplitude-modulated (PAM) readings to a detectable degree and thus avoided the use of dyes (with unknown effects) to standardize colour/clarity.

Phytagel squares were wrapped and cable-tied at mid-height on individual branches of Porites (6-8 cm height, planted as above) and were interspersed on an un-caged rack in the reserve. After 24 h, we assessed the effects of seaweed extracts versus Phytagel controls on coral photophysiology by taking a single PAM fluorometry measurement (fibre-optic diameter = 5.5 mm, distance = 9-10 mm, angle = perpendicular) under the centre of each square (see paper for detailed methods).