The chemical cue tetrabromopyrrole from a biofilm bacterium induces settlement of multiple Caribbean corals

Abstract

Microbial biofilms induce larval settlement for some invertebrates, including corals; however, the chemical cues involved have rarely been identified. Here, we demonstrate the role of microbial biofilms in inducing larval settlement with the Caribbean coral Porites astreoides and report the first instance of a chemical cue isolated from a marine biofilm bacterium that induces complete settlement (attachment and metamorphosis) of Caribbean coral larvae. Larvae settled in response to natural biofilms, and the response was eliminated when biofilms were treated with antibiotics. A similar settlement response was elicited by monospecific biofilms of a single bacterial strain, Pseudoalteromonas sp. PS5, isolated from the surface biofilm of a crustose coralline alga. The activity of Pseudoalteromonas sp. PS5 was attributed to the production of a single compound, tetrabromopyrrole (TBP), which has been shown previously to induce metamorphosis without attachment in Pacific acroporid corals. In addition to inducing settlement of brooded larvae (P. astreoides), TBP also induced larval settlement for two broadcast-spawning species, Orbicella (formerly Montastraea) franksi and Acropora palmata, indicating that this compound may have widespread importance among Caribbean coral species.

Keywords: Pseudoalteromonas; biofilm; chemical ecology; coral recruitment; larval settlement.

Figure 1.

Response of P. astreoides larvae to 15- and 21-day-old biofilms on limestone tiles. FSW control contained only FSW and no tile, and was not included in statistical analyses. Bars represent mean percentage metamorphosis after 48 h including attached (white) and unattached (hatched) larvae (n = 8, 40 larvae per replicate). Error bars represent ±s.e. for total metamorphosis (upper) and attached (lower). Letters above bars represent significant differences (p = 0.012) in settlement (attached) between treatments according to a t-test; p-values given for total metamorphosis.

Figure 2.

Response of P. astreoides larvae to natural 21-day-old biofilms and 21-day-old biofilms treated with antibiotics. FSW control contained only FSW and no tile, and was not included in statistical analyses. Bars represent mean percentage metamorphosis after 24 h including attached (white) and unattached (hatched) larvae (n = 8, 50 larvae per replicate). Error bars represent ±s.e. for total metamorphosis (upper) and attached (lower). Letters above bars represent significant differences (p < 0.001) in settlement (attached) between treatments according to a t-test; p-values given for total metamorphosis.

Figure 3.

Response of P. astreoides larvae to monospecific biofilms of bacterial strain PS-MBA-5 (Pseudoalteromonas sp. PS5) compared with sterile MB media controls. FSW control contained only FSW and no tile, and was not included in statistical analyses. Bars represent mean percentage metamorphosis after 24 h including attached (white) and unattached (hatched) larvae (n = 5, 20 larvae per replicate). Error bars represent ±s.e. for total metamorphosis (upper) and attached (lower). Letters above bars represent significant differences (p < 0.002) in settlement (attached) between treatments according to a t-test; p-values given for total metamorphosis.

Figure 4.

Phylogenetic relationship of Pseudoalteromonas strains that have been tested for their ability to induce coral larval settlement. Strains that have been shown to induce coral larval settlement are indicated by a plus symbol (+); Pseudoalteromonas sp. PS5 from this study is shown in bold. Tree is based on 1360 bp of the 16S rRNA gene using Minimum Evolution method (Jukes–Cantor model). Numbers beside nodes indicate the level of bootstrap support as a percentage out of 1000 replicates. Evolutionary analyses were conducted in MEGA5. Superscript numbers denote as follows: ¹Tebben et al. [16], ²Negri et al. [15], ³Tran & Hadfield [14].

Figure 5.

Response of P. astreoides larvae to crude extracts (625 ng ml⁻¹) from bacterial strain PS-MBA-5 (Pseudoalteromonas sp. PS5) compared to solvent (DMSO) and Hydrolithon boergesenii (CCA) controls. FSW controls were not included in statistical analyses. Bars represent mean percentage metamorphosis after (a) 6 h and (b) 24 h including attached (white) and unattached (hatched) larvae (n = 10, 10 larvae per replicate). Error bars represent ±s.e. for total metamorphosis (upper) and attached (lower). There was a significant effect of treatment on settlement and total metamorphosis after both 6 and 24 h according to a one-way ANOVA or ANOVA on ranks (p < 0.001 for each). Letters above bars represent significant differences (p < 0.05) in settlement (attached) among treatments according to a Tukey test.

Figure 6.

Bioassay-guided isolation of the settlement inducing compound TBP. Solvents used are shown in boxes with the percentage settlement (mean ± s.e.) of P. astreoides and extract concentrations below.

Figure 7.

Response of P. astreoides larvae to different concentrations of TBP after (a) 6 h and (b) 24 h. Hydrolithon boergesenii (CCA) controls were not included in statistical analyses. Bars represent mean percentage metamorphosis after 6 and 24 h including attached (white) and unattached (hatched) larvae (n = 5, 10 larvae per replicate). Error bars represent ±s.e. for total metamorphosis (upper) and attached (lower). There was a significant effect of treatment on settlement and total metamorphosis after both 6 and 24 h according to a one-way ANOVA or ANOVA on ranks (p < 0.001 for each). Symbols above bars represent significant differences (p < 0.05) in settlement (*) and total metamorphosis (+) from the solvent control (SC) according to Dunnett's test.

Figure 8.

Response of (a) O. franksi and (b) A. palmata larvae to different concentrations of TBP after 24 h. Hydrolithon boergesenii (CCA) controls were not included in statistical analyses. Bars represent mean percentage metamorphosis after 24 h including attached (white) and unattached (hatched) larvae (n = 5 except for 25 and 500 ng ml⁻¹, n = 6; 10 larvae per replicate). Error bars represent ±s.e. for total metamorphosis (upper) and attached (lower). There was a significant effect of treatment on settlement and total metamorphosis according to a one-way ANOVA or ANOVA on ranks (p < 0.001 for each). Symbols above bars represent significant differences (p < 0.05) in settlement (*) and total metamorphosis (+) from the solvent control (SC) according to Dunnett's test.