Around the world, marine fouling can have serious impacts on human activities and other organisms. To survive in the highly competitive arena of the marine environment, many organisms have developed unique protection mechanisms against fouling, including tolerance, avoidance, low surface energy and the secretion of natural compounds. Some species of sponge and coral are rarely epiphytized because of the production of secondary metabolites against fouling. Unlike common man-made antifouling compounds, these natural chemicals are environmentally friendly in marine ecological systems.
The majority of marine fouling organisms are algae, coelenterates, polychaetes, bivalves, bryozoans and barnacles. Of them, the acorn barnacle Balanus reticulatus is one of the most important dominant species in the fouling communities of tropical and subtropical waters, particularly in the East and South China Sea. Thus is can be considered as an appropriate test representative for antifouling bioassays.
Barnacle nauplii were obtained by dissecting freshly collected adult barnacles from the aquaculture facilities at Daya Bay, Shenzhen, China. Barnacle larvae were reared in darkness, at 30℃ and on a diet of the green algae Platymonas subcordiformis. Seawater was changed and more algae added as necessary. Cyprid larvae developed after around 5 days and were collected and stored at 4℃ for subsequent use. Sponges of the genus Callyspongia are widely distributed in the southern waters of China (e.g. coastal water of Fujian, Guangdong, Guangxi and Hainan Provinces). They are well known as sources of biologically active natural products, including polyacetylenes, peptides, alkaloids, fatty acids, polyketides, and sterols. Some of these compounds possess antimicrobial, anti-tumor, cytotoxicic, and HIV reverse transcriptase inhibition properties.
Freshly collected samples of Callyspongia were minced and extracts taken by washing with dilute ethanol three times. The aqueous ethanol extracts were concentrated under vacuum. The combined extracts were partitioned into ethyl acetate and water. The ethyl acetate soluble portion was separated using column chromatography. A silica gel column was eluted with petroleum ether / ethyl acetate and then ethyl acetate / acetone. Similar isolates were identified using thin layer chromatography (TLC). The structures of all isolates were determined using one dimensional and two dimensional nuclear resonance microscopy (NMR), mass spectroscopy (MS) analyses, and by comparison of the spectroscopic data with those reported in the literature. A total of eight compounds were isolated and purified: Cyclo-(S-Pro-Gly), Cyclo-Pro-Ala, 1-(2'-deoxy-β-D-erythro-pentofuranosyl)-1-H-1,2,4-triazole, uracil, 4-(1-phenylethyl) phenol, 6-NH₂-purine, 1-H-1,2,4,triazole and cholest-5-ene-3β,7α-diol.
For antifouling tests, each compound was dissolved in 1ml methanol (or chloroform) and transferred into Petri dishes. Petri dishes were dried on a shaker prior to the addition of filtered seawater and larvae. Control dishes, using 1 ml of solvent (pre dried as above) and filtered seawater only were also prepared. The mean values of larval survival rate were compared among samples using least significant difference tests. Six compounds had no influence on the behavior and survival rate of larvae (P>0.05): 1-(2'-deoxy-β-D-erythro-pentofuranosyl)-1-H-1,2,4-triazole, Cyclo-(S-Pro-Gly), Cyclo-Pro-Ala, uracil, 1-H-1,2,4,triazole and cholest-5-ene-3β,7α-diol. The other two compounds 6-NH₂-purine (7.1 μg/cm²) and 4-(1-phenylethyl) phenol (5.9 μg/cm²) had significant toxic effects on the cyprid larvae of the acorn barnacle Balanus reticulatus (P<0.05). The synergistic effect of these compounds may play an important role in the antifouling activities of the sponge.