3.5. Acetylcholinesterase Inhibition Assay

The crude extract of the sponge and the purified psammaplysene D (1) were dissolved in DMSO at 10 mg/mL, stored at −20 °C and diluted subsequently 100 times in H₂O prior testing. Determination of AChE-inhibitory activity was measured according the Ellman’s colorimetric method [41], in 96-well tissue culture plates. All reagents, buffers and salts were purchased from Sigma-Aldrich (Saint-Louis, MO, USA). Galantamine was used as a positive control for each analysis. Solvent controls received the same volume of water. After a 30 min incubation at 25 °C in the dark, absorbance was monitored at λ = 405 nm. Results were expressed as the percentage of enzyme inhibition calculated as the ratio: 100 × (OD_control − OD_treated)/(OD_control − OD_blank), where the blanks are the wells without AChE. Serial dilutions of the compound or extract were prepared (100, 50, 25, 12.5, 6.25, 3.12, 1.56, and 0.78 μg/mL) and tested according to the same procedure using 10 μL of each solution. IC₅₀ values were graphically determined by plotting the percentage of inhibition against the compound concentrations. Each assay was performed in triplicate and results are expressed as mean values ± S.D.

The inhibition mode of psammaplysene D on AChE was determined while measuring the enzyme activity in presence of an increasing concentration of ATCI (0.024, 0.049, 0.098, 0.19, 0.39, 0.78, 1.57, and 3.15 μM), in absence or presence of 2 μg/mL final concentration of purified psammaplysene D. The inhibition mode, V_max, and K_M values were determined by double-reciprocal Lineweaver and Burk plot analysis (1934) of the data obtained.

5 to 8 cm long guppies (Poecilia reticulata) were collected at night in a private fresh water pool at Papara (Tahiti, French Polynesia) and immediately acclimated at the laboratory temperature (25 °C). After acclimatization, fishes were parted in two 2 L tanks: one for the control group treated with solvent (N = 5). Another experiment was performed using a 1 h treatment with 100 μg/mL of crude extract of the sponge or purified psammaplysene D (1) to check their acute toxicity.

Manini (Acanthurus triostegus) were caught during new moon nights on the foreshore puddles at Tema’e public beach [42]. Recruits (fish larvae undergoing metamorphosis) and juveniles (larvae after the recruits stage) were collected and immediately used for the experiments. Natural lagoon sea-water was used and UV-sterilized prior use. Three conditions were tested on three groups: control, solvent, 1 μg/mL sponge extract treatment, with 4 or 5 fishes in each 3 L tank. The fishes were exposed under semi-static system for 72 h, the experimental solutions were renewed every 24 h to maintain clean experimental conditions. The experiments were performed with fish recruits and juveniles. Rubbles were disposed in each aquarium 1 h/day during 3 days. Feeding behavior experiment consists in counting the overall number of bites on those rubbles in each aquarium. Six videos sequences (N = 6) of 5 or 10 min per aquarium and per day were analyzed. Results are expressed in number of bites per fish and per hour.

All the values of AChE activity are represented in a boxplot distribution, because of non-normality of variable distribution (N = 4 or 5). The significant differences between controls and treatments (p-value p < 0.05) were analyzed with non-parametric test of Wilcoxon Mann Whitney. In feeding deterrent assay, pairs significant differences between different conditions (p < 0.05) were tested with non-parametric Kruskal Wallis test. All statistical assays were executed using R software (R-3.2.0, The R Foundation for Statistical Computing, Vienna, Austria).