Chemical defense against microfouling by allelopathic active metabolites of Halymenia floresii (Rhodophyta)

Abstract

During the experimental cultivation of the red alga Halymenia floresii under Integrated MultiTrophic Aquaculture (IMTA), the establishment of opportunist green algae and the colonization of sessile invertebrates, which were usually disturbing the cultivation, were not observed. The culture tanks were clean and the surface of the H. floresii was remarkably free from any fouling organisms. This phenomenon could reveal that the presence of H. floresii may prevent biofilm formation by releasing allelopathic active compounds that ultimately interfere with the settlement and growth of competitors. In order to understand this phenomenon, H. floresii was cultivated under controlled environmental conditions and analyzed for its surface chemical defense metabolites. The surface-associated metabolites were extracted using the DIP extraction method, using different solvents with increasing polarity and immersion periods. Using epifluorescence microscopy, n-hexane was found to be the suitable immersion solvent for H. floresii for a period of 10 to 60 s to extract surface metabolites. The whole cell metabolites were extracted exhaustively with the same solvent for a period of 24 h. The chemical profiling of the surface compounds was performed by liquid chromatography mass spectroscopy (LC-MS), followed by a Mass Bank search and compared with those obtained from the whole-cell extracts. The mean concentration of H. floresii surface metabolites was 600 ng cm−2 (c. 60 g of a fresh sample) whereas the whole-cell metabolite concentration was around 4.5 μg mg−1 (400 mg of the lyophilized sample). The bioactivity of the H. floresii surface extracts was studied by evaluating their quorum quenching behavior on the surface-associated bacteria. The cultivable bacteria isolated from the surface of H. floresii were identified as Vibrio owensii (B3IM), Alteromonas sp. (B7CC), Pseudoalteromonas arabiensis (B4BC), Ruegeria sp. (B4CC), Tenacibaculum sp.(B9BC), Maribacter sp. (B9IM) and Aquimarina sp. (B9.1CC). All the isolated strains belonged to Alphaproteobacteria, Gammaproteobacteria and Bacteroides. The results of this bioactivity proved that the surface-associated metabolite extract (DIP) interferes with the communication signals produced by the bacteria isolates with the reporter strain employed. According to the Mass Bank compound analysis, we hypothesized that flavonoids and/or halogenated compounds might have contributed to this activity. This work provides an understanding of the influence of surface-associated metabolites on the associated bacterial community and by which H. floresii manages to control the biofouling on its surface.