We have recently Alpelisib isolated antimicrobial compound-producing strains from oyster haemolymph, suggesting that microbiota may confer a health benefit on the host (Defer et al., 2013). In this study, we have explored the cultivable haemolymph-associated bacteria in four bivalves (oyster, clam, mussel and scallop) for their antimicrobial activity. The most potent ones were also investigated for hemocyte cytotoxicity. Results are clearly in

line with the hologenome concept. Moreover, they suggest that haemolymph-associated bacteria are a potential source of aquaculture probiotics. To limit the impact of anthropic pressure, bivalve specimens were collected by deep-sea diving in the Glenan Archipelago (47°43′N, 4°01′W, WGS84 system), a Natura 2000 TSA HDAC research buy area (FR5300023), during winter

2009 and spring 2010. Selected species were the oyster (Crassostrea gigas), the blue mussel (Mytilus edulis), the scallop (Pecten maximus), and the pink clam (Tapes rhomboides). Haemolymph of each individual was collected aseptically by inserting a 25-gauge needle attached to a 1-mL syringe directly into the adductor muscle. For C. gigas, haemolymph was collected from the pericardium. A volume ranging from 0.5 to 1 mL of haemolymph was drawn from each mollusc and placed in ice to prevent the hemocyte aggregation. Each sample was microscopically examined to check the presence of healthy hemocytes. Checked haemolymph (50 μL) was spread onto Marine agar Petri dishes using a Wasp® automated spiral plater (AES Lab). Plates were further incubated for 72 h at 18 °C. To isolate as many different bacteria as possible, 1–10 macroscopically distinguishable colonies were picked and subcultured in Marine Broth for 48 h at 18 °C with shaking (100 r.p.m.). Bacterial purity was assessed Ketotifen by streaking on Marine Agar. For long-term storage, sterile glycerol was added to 1 mL bacterial culture (25% v/v) in cryogenic vials that were stored at −80 °C. Cell-free supernatants coming from culturable haemolymph-associated bacteria were assayed for antibacterial activity against a panel of 12 aquaculture pathogens (Table 1).

After growth (72 h, 18 °C, 100 r.p.m.), the culture supernatant (1 mL) was collected by centrifugation (6000 g for 10 min at 4 °C) and filtration (0.22 μm, SFCA serum Filter Unit, Nalgene). To detect antibacterial activity, the well-diffusion method was used (Wiegand et al., 2008; Defer et al., 2013). Specific agar medium according to bacterial target was inoculated with an 8-h-old culture broth of the indicator strain to a bacterial concentration of 1.106 CFU mL−1. Wells (diameter 4 mm) were punched into the agar medium and cell-free supernatants (20 μL) or controls (Marine Broth for negative control and polymyxin B sulphate and Nisaplin® at 1 mg mL−1 as positive control against respectively Gram-negative and Gram-positive target bacteria) were created.