Settlement of larvae of Crassostrea gigas on shell chips (SC) prepared from shells of 11 different species of mollusks was investigated. Furthermore, the settlement inducing compound in the shell of C. gigas was extracted and subjected to various treatments to characterize the chemical cue. C. gigas larvae settled on SC of all species tested except on Patinopecten yessoensis and Atrina pinnata. In SC of species that induced C. gigas larvae to settle, settlement was proportionate to the amount of SC supplied to the larvae. When compared to C. gigas SC, all species except Crassostrea nippona showed lower settlement inducing activities, suggesting that the cue may be more abundant or in a more available form to the larvae in shells of conspecific and C. nippona than in other species. The settlement inducing activity of C. gigas SC remained intact after antibiotic treatment. Extraction of C. gigas SC with diethyl ether (Et2O-ex), ethanol (EtOH-ex), and water (Aq-ex) did not induce larval settlement of C. gigas larvae. However, extraction of C. gigas SC with 2N of hydrochloric acid (HCl-ex) induced larval settlement that was at the same level as the SC. The settlement inducing compound in the HCl-ex was stable at 100°C but was destroyed or degraded after pepsin, trypsin, PNGase F and trifluoromethanesulfonic acid treatments. This chemical cue eluted between the molecular mass range of 45 and 150 kDa after gel filtration and revealed a major band at 55 kDa on the SDS-PAGE gel after staining with Stains-all. Thus, a 55 kDa glycoprotein component in the organic matrix of C. gigas shells is hypothesized to be the chemical basis of larval settlement on conspecifics.

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Larval settlement of the Pacific oyster Crassostrea gigas on microbial biofilms obtained by immersing half-size glass slides in the sea (1.0 m depth) off Taira-cho, Nagasaki, Japan for 1 to 24 days during the period between May 2009 and January 2010 was investigated. Settlement inducing activities of biofilms that were subjected to heat (80°C), formalin (FA) and antibiotic mixture (AM) treatments were also investigated. Moreover, the settlement inducing activities of 4 bacterial strains isolated from the biofilm were investigated. C. gigas larvae settled in response to microbial biofilms. The percentage of post larvae increased with immersion period in biofilms obtained during Jan to Mar and Oct to Dec. Larval settlement also increased with the bacterial density of biofilms in Jan to Jun and Oct to Dec, and with the diatom density in Apr to Jun. By contrast, larval settlement did not linearly correlate with the dry weight of the biofilms. FA treatment did not affect the activity of the biofilm but heat and AM treatments of the biofilm resulted in significantly low percentage of post larvae. Of the 4 bacterial isolates tested, Pseudoalteromonas tetraodonis and Pseudoalteromonas sp. induced the highest percentage of post larvae but their activities were reduced with formalin treatment. Thus, microbial biofilms may possess a cue that remains intact even after killing the components of the film by FA treatment, and this cue may be distinct from the water soluble metabolite produced by specific bacterial species, such as Pseudoalteromonas sp.

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Glass slips were submerged in the sea (1 m depth) off Taira-cho, Nagasaki, Japan for 1, 2, 3 and 4 weeks every month from Jul 2010 to Oct 2012, and the dry weights, chlorophyll contents and diatom community structures of the marine biofilms were investigated. Glass slips immersed in the sea acquired biofilms that consisted mainly of diatoms. Young thalli of Ulva compressa also occurred in the biofilms from Apr to Dec. Marine biofilms increased in mass with longer immersion periods, indicating that growth of marine biofilms should be looked into for up to a month. Colonization on glass slips by invertebrate macroorganisms occurred from Jul to Sep, consequently making the estimation of biofilm biomass inaccurate during this period. Dry weights of biofilms were linearly correlated with the amounts of chlorophylls-a and -c, indicating that these pigments can be used as indices to estimate biomass growth of marine biofilms, which primarily consist of periphytic microalgae. Chlorophyll-a contents and diatom densities of biofilms both showed temporal and seasonal variations, whereas chlorophyll-c contents of biofilms showed seasonal variation. Decrease in the chlorophyll (-a and -c) contents and diatom densities of biofilms in Jul to Sep may probably be due to the disturbance of biofilms caused by the attachment of invertebrate macroorganisms. Navicula almost always was the dominant diatom in established marine biofilms.Keywords: marine biofilm, biofilm growth, periphytic diatoms, chlorophyll-a, chlorophyll-c, algal biomass Surfaces submerged in the sea initially acquire a thin layer of organic materials, referred to as a conditioning film, which is then colonized by bacteria, fungi, diatoms and protozoans, the main organisms that comprise marine biofilms (Wahl, 1989). Colonization by these micro-organisms follows no particular order (Marszalek et al., 1979).In benthic ecosystems, marine biofilms are known to be an important source of chemical cues that either facilitate or inhibit settlement of algal spores (e.g. Joint et al., 2000;Silva-Aciares and Riquelme, 2008;Shin, 2008) and invertebrate larvae (see review by Qian et al., 2007;Dobretsov et al., 2013). While bacteria influence larval settlement by their production of waterborne-metabolites (e.g. Fitt et al., 1989;1990; Bao et al., 2007a), extracellular polymeric substances From an economic standpoint, biofilms are considered a nuisance to maritime industries because it is a precursor phenomenon to the biofouling of macro-organisms and it influences corrosion of metallic materials leading to economic losses (Yebra et al., 2004). On the other hand, hatcheries in Japan typically grow diatom-based biofilms on artificial substrates to induce larval settlement and feed juveniles of sea urchins (Ito, 1984), sea cucumbers (Ito and Kitamura, 1997) and abalones (Kawamura et al., 1995;Takami et al., 1996;.Success in the production of these economically valuable species will rely on how hatcheries can control production of diatom-based biofilms that will consistently ...