Abstract.In an attempt to clarify the influence of marine bacteria on the settling of fouling invertebrate larvae, we screened for inhibitors, produced by marine bacteria, of settling by cyprids of the barnacle, Balanus amphitrite. We found that the culture broth of Alteromonas sp. strain number KK10304, which was associated with the marine sponge, Halichondria okadai, effectively inhibited settling of the cyprids. Bioassay-guided isolation indicated ubiquinone-8 (1) as an effective inhibitor of cyprid settling. As ubiquinones are widely distributed in bacteria, several related compounds were also tested.Key words. Settlement inhibitor; barnacle; ubiquinone; sponge; Halichondria okadai; sponge associated bacteria; Alteromonas sp.Marine fouling invertebrates, such as barnacles and blue mussels, cause serious problems on ship hulls, in cooling systems of power plants, and in aquaculture systems. Organotin compounds such as TBTO [bis-(n-tributyltin)-oxide] have been used as effective antifouling agents against those organisms. However, stern warnings have been issued regarding the toxic effects of such heavymetal compounds on marine environments and wildlife, including fish and shellfish. Therefore, antifouling substances with no or reduced toxicity must be found and developed. In our screening for antifouling substances, using laboratory-reared larvae of the barnacle, Balanus amphitrite, we have reported fatty acids ~, steroids 1, a sesquiterpene hydrocarbon 2, a betaine 3, a furanoterpenoic acid 4, a pukalide derivative 4, a gramine derivative s and others as settling inhibitors in marine invertebrates. Crisp 7 reported that the larvae of fouling invertebrates were recruited to the settling surface in response to surface associated stimuli. He also revealed 7 that the stimuli played an important role not only in attracting larvae but also in inducing their metamorphosis. Mitchell and Kirchman s proposed that the stimuli were produced by bacteria growing on the solid surface. Maki et al. 9 reported that films of bacteria on solid substrate could positively or negatively influence the settling of marine invertebrate larvae. They 9 tested the effects of culture media of 18 different strains of marine bacteria on the settling of cyprids of the barnacle, B.amphitrite. They found that 8 out of 18 culture media showed inhibition of the settling and that the culture medium of Deleya (Pseudomonas) marina showed the strongest activity. These papers have suggested the existence of settling inhibitors produced by marine bacteria, although many of these compounds have remained unknown. In the present paper, we report the isolation and identification of the settling inhibitors from bacteria associated with a marine sponge.
Materials and methods
Isolation of bacteria.A common marine sponge, Ha[ichondria okadai, was collected from Numazu area in Suruga Bay of Shizuoka Prefecture, in 1990. This specimen was cut into small pieces (5 g), homogenized with 10 ml of sterilized seawater and diluted in a series of 10-fold dilutions. Each...
