Characterization of biofilm exudates and their effects on settlement of mussel (Perna canaliculus) larvae

Settlement-inducing cues for larvae of the green-lipped mussel, Perna canaliculus, were characterized from biofilm exudates of mono-species bacteria. Biofilm exudates of Macrococcus sp. AMGM1 and Bacillus sp. AMGB1, previously known to induce settlement of P. canaliculus larvae, were chosen for this study. An innovative step-wise multi-technique approach, including physical methods (molecular weight fractionation and heat treatment) and chemical methods (crude fractionation, sub-sample fractionation, sequential fractionation, lipid fractionation and protease treatment), was used to characterize the chemical cues from these bacterial biofilm exudates. The resulting fractions obtained from these methods were individually tested for their ability to induce larval settlement. Results from the settlement assays indicate that settlement cues from Macrococcus sp. AMGM1 were between 1–3, 3–10 and 10–30 kDa. These molecules were thermo-labile to over 70 °C, and contained polar proteins and non-polar lipoprotein molecules. Protease-treated biofilm exudates did not elicit a settlement response, indicating the involvement of proteins from Macrococcus sp. AMGM1 in the larval settlement process. In addition, settlement cues from Bacillus sp. AMGB1 were found to be between 10–30 and 30–50 kDa, were stable to up to 100 °C, and contained glycolipid molecules. These results provide evidence for the presence of multiple settlement cues in the biofilm exudates from both Macrococcus sp. AMGM1 and Bacillus sp. AMGB1. This study provides a comprehensive and inexpensive set of biochemical techniques, which can be used in a step-wise manner, to deduce partial chemical identity of both inductive and non-inductive settlement cues produced by biofilm exudates. The results can be used to decipher complex bacterially mediated larval settlement processes for P. canaliculus and other marine invertebrates.

► Innovative step-wise multi-technique approach to characterize chemical cues.
► Characterization of mussel settlement cues from bacterial biofilm exudates.
► Proteins from Macrococcus sp. AMGM1 induce P. canaliculus settlement.
► Glycolipid from Bacillus sp. AMGB1 induces P. canaliculus settlement.
► Cost-effective approach to characterize chemical cues from natural products.