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.
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.
Industrial farming is an alternative mode for Pinctada maxima juvenile cultivation to avoid mass mortality caused by natural disasters. Suitable and enough food is crucial for successful industrial bivalve farming. To investigate the feasibility of live microalga instead of spray-dried microalgal powder in P. maxima juvenile industrial farming, this study replaces a positive control live microalgal diet [Isochrysis zhanjiangensis (L-iso) and Platymonas subcordiformis (L-pla)] with spray-dried I. zhanjiangensis powder (P-iso) and P. subcordiformis powder (P-pla). Continuous feeding trials (30 days) were conducted on the P. maxima juvenile (1.2008 ± 0.0009 g initial weight and 30.12 ± 0.05 mm initial shell length), under laboratory conditions. Survival, growth performance, and intestinal microbial community were studied and compared across the groups. Results showed that survival rate (SR) did not differ significantly across the groups (ranged from 84 to 86%, P > 0.05). The growth performance in spray-dried microalgal groups, including total weight (TW), shell height (SH), absolute growth rate (AGR), and relative growth rate (RGR) for SH and TW, was slightly lower than that in live microalgal groups, while the activities of pepsin (PES), amylase (AMS), and lipase (LPS) were significantly higher (P < 0.05). The best growth performance was observed in the L-iso group, followed by the L-pla group. A 16S rRNA-based sequencing revealed that Proteobacteria was the dominant phylum in P. maxima juvenile intestinal bacterial community under controlled conditions, which accounted for 62–82% across groups. The intestinal bacteria at the genus level were more sensitive to diets, whereas Burkholderia was the dominant genus in both L-iso (66.52 ± 6.43%) and L-pla groups (54.00 ± 5.66%), while Mycoplasma, Alphaproteobacteria, and Oxyphotobacteria were in both P-iso and P-pla groups. The P-pla group got higher ACE, Chao1, and Simpson and Shannon indices (P < 0.05). The above results suggested that the spray-dried P-iso and P-pla can serve as substitutes for live microalga in P. maxima juvenile industrial farming under controlled conditions. The finding in this study provides basic data to optimize industrial farming technology and healthy management for P. maxima juvenile.
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