The role of bacterial biofilms on the settlement and nutrition of mussel (Perna canaliculus) larvae and juveniles

Ganesan, Annapoorna Maitrayee
Alfaro, Andrea. C.
Brooks, John. D.
Higgins, Colleen. M.
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Doctor of Health Science
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Auckland University of Technology

The green-lipped mussel, Perna canaliculus is indigenous to New Zealand, and is of significant commercial importance, contributing over NZ $200 million per annum in exports. Despite the economic importance of this mussel species, very little is known about its early life. Larvae at the pediveliger stage may remain planktonic for several weeks until individuals find a suitable settlement site. A range of filamentous macro-algae provides the primary settlement site and source of nutrition for larvae until they make the transition from primary substrates on to rocky shores. The underlying mechanism for choosing specific substrates remains largely unknown, but may be related to the epiphytic bacterial biofilms present on those surfaces. Thus, the present study is the first to investigate bacterial biofilm-mediated settlement and nutrition for P. canaliculus larvae and juveniles through laboratory-guided experiments.

To investigate a role for specific bacterial biofilms in mussel larval settlement, experimental conditions were optimised to achieve exemplar settlement assays, and to obtain efficient and reproducible results. Settlement assays analysed pediveliger larvae between 19–21 days’ old which were subjected to a range of settlement substrates and assay media. Larvae were found to settle uniformly on polystyrene plates, glass slides and noble agar media, indicating that the physicochemical properties of the settlement substrates were not important for the settlement of larvae. In addition, sterile natural seawater was preferred as a culturing environment compared with artificial seawater, which was found to be toxic for P. canaliculus larvae. The 48-hour assay period resulted in minimal variation within replicate plates, hence it was found to be most suited for conducting ideal settlement experiments.

Marine bacteria were cultured from diverse marine origins, isolated through traditional culturing techniques, and their ability to induce settlement was investigated. Out of fourteen bacteria isolated, three mono-species biofilms were exclusively chosen for further study. With the aid of phylogenetic analyses, these three bacterial strains were found to be Macrococcus sp. AMGM1, Bacillus sp. AMGB1 and Pseudoalteromonas sp. AMGP1. The biofilms of the former two species of bacteria were shown to induce greater settlement (over 60%) compared with controls. Conversely, the latter species was showed to have similar settlement levels compared with sterile controls. However, this latter bacterium produced toxic molecules for mussel larvae and induced mortalities of over 70% compared with the other species of bacteria and controls.

Petri plates, glass slides and noble agar were investigated along with biofilms for their collective ability to modulate settlement of larvae. The study demonstrated that some species of bacteria were able to interact with the settlement substrate and modify their capacity to induce mussel larval settlement. However, the three key mono-species bacteria (Macrococcus sp., Bacillus sp. and Pseudoalteromonas sp.) continued to show consistent levels of settlement across all three substrates, indicating that the settlement cue was confined to bacteria, and was not a result of combined physicochemical cues of biofilm and their substrates.

These results led to the investigation of the origin of the settlement cues present in the biofilms of mono-species marine bacteria. The biofilm cells and the chemicals from the biofilms (exudates) of Macrococcus sp. and Bacillus sp. resulted in similar patterns of settlement inducing properties. Conversely, washed cells of biofilms and cells grown in the planktonic form resulted in reduced settlement inducing properties. Unlike washed biofilm cells and planktonic bacteria, bacterial biofilms and biofilm exudates of Pseudoalteromonas sp. induced greater levels of larval mortality. These results revealed the origin of the respective settlement and toxic cues to be present in the exudates of these mono-species bacteria.

Further characterisation of the settlement cues from biofilm exudates of respective mono-species bacteria revealed their relative molecular identity. The settlement cues from exudates of Macrococcus sp. AMGM1 were between 1–3, 3–10 and 10–30 kDa, thermo-labile over 70°C, and contained polar proteins and non-polar lipoprotein molecules. 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. The bacterial biofilm toxin from Pseudoalteromonas sp. AMGP1 was identified as a heat labile (> 70°C) polar protein molecule of < 1kDa in size. In addition, a settlement inductive molecule was found from this bacterial species to be a heat labile non-polar lipoprotein of 30–50 kDa.

Settlement assays were used to determine the responses of juvenile mussels. These assays showed that mussels at this stage of development also responded to settlement cues from mono-species bacteria. Juveniles were shown to favour low wettable polystyrene substrates and settled in response to the physicochemical properties of the settlement substrates. Addition of Macrococcus sp. and Bacillus sp. biofilms enhanced settlement on glass slides and coir substrates, whereas no effect was observed on substrates with biofilms of Pseudoalteromonas sp. Moreover, retention was shown to improve during the initial 24-hour period of the assay on glass slides and ropes containing biofilms of Macrococcus sp. and Bacillus sp. These results indicated that biofilms were able to mediate settlement and early retention of juvenile mussels.

Finally, nutrition assays were conducted to identify whether mussel larvae and juveniles obtained nutrition from bacteria during the 48-hour settlement assay period. Ytterbium-labelled bacteria were fed to mussel larvae and juveniles, which were analysed for the presence of these metals after 48-hour incubation. Both mussel larvae and juveniles were shown to be bacterivorous through this assay. In addition, a sole diet containing biofilm cell suspensions of only Macrococcus sp. or Bacillus sp. and mixed diets of these bacteria together with microalgae showed similar levels of metamorphosis and survival.

This study used a multi-disciplinary approach to highlight the importance of bacterial cues for settlement of mussel larvae and juveniles and their bacterivorous nature. These specific biofilms or their chemicals have the potential for use in hatchery settings to achieve improved settlement rates and production. In addition, bacteria may a cheap alternative diet to rear mussels.

Green-lipped mussel , Bacterial biofilms , Larval settlement cue , Perna canaliculus , Biofilm exudates , Bacterivory , Larval nutrition , Spat retention
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