The effects of bacterial cell suspensions on mussel (Perna canaliculus) larval settlement

A technique was developed to separate and test bacterial cell surface-bound compounds and their exudates (biofilm matrix) for marine invertebrate larval settlement experiments. A broth culture (preventing biofilm formation) was used to obtain planktonic cell suspensions without exudates (PCo). Biofilm cell suspensions with their exudates (BCx) were obtained by growing the cells on marine agar (allowing biofilm formation) before suspending in seawater. A biofilm cell suspension without exudates (BCo) was obtained by washing BCx cells thoroughly before suspending in seawater. Three separate marine bacteria (Macrococcus sp. AMGM1, Bacillus sp. AMGB1 and Pseudoalteromonas sp. AMGP1) were used to test PCo, BCx, and BCo suspensions for their ability to induce settlement of mussel larvae (Perna canaliculus). BCx suspensions of Macrococcus sp. AMGM1 and Bacillus sp. AMGB1 significantly induced settlement (over 70%) compared with their corresponding PCo and BCo suspensions and the sterile control plates. Conversely, BCx suspensions of Pseudoalteromonas sp. AMGP1 did not induce mussel larval settlement, and were highly toxic to the larvae. In addition, PCo and BCo treatments of Pseudoalteromonas sp. AMGP1 had no effect on larval settlement or mortality, compared with control plates. Results show that the tested bacterial cells alone (devoid of their biofilm exudates) do not induce settlement or mortality in this mussel species, and that their exudates contain the chemical cues that affect larval development and survivability. Thus, this work highlights the importance of appropriately separating exudates from bacterial cells to test surface-bound compounds in settlement cue experiments.

► New technique separates bacterial cell surface-bound compounds from biofilm exudates.
► Biofilm cell suspensions with exudates induced mussel larval settlement.
► Biofilm cell suspensions were highly toxic to mussel larvae.
► Bacterial cells without exudates had no effect on larval settlement or mortality.
► Bacterial biofilms without exudates had no effect on larval settlement or mortality.