Evaluation of Antifouling Potential and Ecotoxicity of Secondary Metabolites Derived from Red Algae of the Genus Laurencia

Red algae of the genus Laurencia are known to biosynthesize and secrete an immense variety of secondary metabolites possessing a spectrum of biological activities against bacteria, invertebrates and mammalian cell lines. Following a rigorous cross-species screening process, herein we report the antifouling potential of 25 secondary metabolites derived from species of the genus Laurencia, as well as the thorough evaluation of the ecotoxicity of selected metabolites against non-target marine arthropods and vertebrate cell lines. A number of these secondary metabolites exhibited potent antifouling activity and performed well in all screening tests. Our results show that perforenol (9) possesses similar antifouling activity with that already described for bromosphaerol, which is used herein as a benchmark.


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Values are expressed as the percentage of animals that did not react to light mechanical stimuli from the total number of animals placed in a well of a 24-well tissue culture plate with varying concentrations of the indicated metabolites. The cumulative results from 3-4 independent experiments with 5 replicates each is shown.
S5 Figure S3. Inhibition of metamorphosis of A. amphitrite cyprids in the presence of varying concentrations of 25 secondary metabolites isolated from Laurencia and Aplysia species. Values are expressed as the percentage of animals that neither settled, nor were dead from the total number of animals placed in a well of a 24-well tissue culture plate with varying concentrations of the indicated metabolites. The cumulative results from 3-4 independent experiments with 5 replicates each is shown.
S6 Figure S4. Results of settlement bioassays of A. amphitrite cyprids in the presence of varying concentrations of bromosphaerol. Values are expressed as the percentage of animals that settled (settlement) or did not react to light mechanical stimuli (mortality) or neither settled nor were dead (no metamorphosis) from the total number of animals placed in a well of a 24-well tissue culture plate with varying concentrations of bromosphaerol.
The cumulative results from 3 independent experiments with 5 replicates each is shown.
S7 Figure S5. Results of Artemia salina toxicity assays in the presence of 8 secondary metabolites isolated from Laurencia and Aplysia species and bromosphaerol. Values are expressed as the percentage of animals that showed no movement of the appendages from the total number of animals placed in a well of a 24well tissue culture plate with varying concentrations of the indicated metabolites. The cumulative results from 3 independent experiments with 5 replicates each is shown.
S8 Figure S6. Results of Chaetoceros gracilis toxicity assays in the presence of 6 secondary metabolites isolated from Laurencia and Aplysia species and bromosphaerol. Values are expressed as cell density of Chaetoceros gracilis diatoms in the presence of varying concentrations of the indicated metabolites for 96 h. The cumulative results from 3 independent experiments with 5 replicates each is shown. S9 Figure S7. Results of RTL-W1 cell viability assays in the presence of 3 secondary metabolites isolated from Laurencia and Aplysia species and bromosphaerol. Values are expressed as % cell viability or cytotoxicity (see Materials and Methods for details) in the presence of varying concentrations of the indicated metabolites. The cumulative results from 3 independent experiments with 5 replicates each is shown. S10 Figure S8. Results of HEK293 cell viability assays in the presence of 3 secondary metabolites isolated from Laurencia and Aplysia species and bromosphaerol. Values are expressed as % cell viability or cytotoxicity (see Materials and Methods for details) in the presence of varying concentrations of the indicated metabolites. The cumulative results from 3 independent experiments with 5 replicates each is shown. S11 Figure S9. Results of marine bacteria growth assays in the presence of 3 secondary metabolites isolated from Laurencia and Aplysia species and bromosphaerol. Marine agar plates (Zobel marine agar medium) were split in 4 sections (each corresponding to a serial dilutions (0.1, 1, 10, 100 μΜ) of each tested metabolite) and a sterile filter paper disc (4 mm) (GF/F; Whatman), loaded with a 40 μL sample of each tested metabolite, was placed on each section of the agar plates that were then seeded with a single strain of bacteria and incubated for 24 h in 25 o C (see Materials and Methods for details). Indicative plates from 3 independent experiments with 5 replicates each are shown.