Induction of larval metamorphosis of the coral Acropora millepora by tetrabromopyrrole isolated from a Pseudoalteromonas bacterium

Abstract

The induction of larval attachment and metamorphosis of benthic marine invertebrates is widely considered to rely on habitat specific cues. While microbial biofilms on marine hard substrates have received considerable attention as specific signals for a wide and phylogenetically diverse array of marine invertebrates, the presumed chemical settlement signals produced by the bacteria have to date not been characterized. Here we isolated and fully characterized the first chemical signal from bacteria that induced larval metamorphosis of acroporid coral larvae (Acropora millepora). The metamorphic cue was identified as tetrabromopyrrole (TBP) in four bacterial Pseudoalteromonas strains among a culture library of 225 isolates obtained from the crustose coralline algae Neogoniolithon fosliei and Hydrolithon onkodes. Coral planulae transformed into fully developed polyps within 6 h, but only a small proportion of these polyps attached to the substratum. The biofilm cell density of the four bacterial strains had no influence on the ratio of attached vs. non-attached polyps. Larval bioassays with ethanolic extracts of the bacterial isolates, as well as synthetic TBP resulted in consistent responses of coral planulae to various doses of TBP. The lowest bacterial density of one of the Pseudoalteromonas strains which induced metamorphosis was 7,000 cells mm(-2) in laboratory assays, which is on the order of 0.1-1% of the total numbers of bacteria typically found on such surfaces. These results, in which an actual cue from bacteria has been characterized for the first time, contribute significantly towards understanding the complex process of acroporid coral larval settlement mediated through epibiotic microbial biofilms on crustose coralline algae.

Figure 1. Phylogenetic tree of bacteria affiliated to the genus Pseudoalteromonas based on 16S rRNA gene sequences (5′-prime region, poisitions 10 to 509 E. coli equivalent).

Nucleotide distances are based on the maximum likelihood algorithm and the tree clustered using the Neighborjoining procedure.

Figure 2. Percentage of larvae undergoing metamorphosis in response to biofilms of Pseudoalteromonas strain J010 at a range of bacterial densities.

Each data point represents the mean percentage of metamorphosis (±SE) of 6 replicates containing 10 larvae each, and the mean bacterial density (±SE) of 6 replicates with 10 cell counts in each replicate.

Figure 3. Isolation of the bacterial metabolite of Pseudoalteromonas strains J010 that induced metamorphosis of coral larvae.

The asterisk marks 100% larval metamorphosis in bioassays after 6 h.

Figure 4. HPLC chromatogram of the inductive C18-chromatographic fraction of Pseudoalteromonas strain J010.

The asterisk marks the peak fraction that induced 100% metamorphosis of coral larvae.

Figure 5. Different types of larval response (settlement, metamorphosis, swimming) to a 10-fold dilution series of synthetic tetrabromopyrrole in absence (A, B) and presence (C, D) of chips of Hydrolithon onkodes after 6 h (A, C) and 24 h (B, D).

The control contained FSW only. The 1x concentration was lethal to the larvae. Each value (error bars) represents the mean (±SE) of 6 replicates with 10 larvae in each replicate.