Marine broth

LB: tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L.
MB: Marine Broth (Condalab, Madrid, Spain).
ISP2: glucose 4 g/L, yeast extract 4 g/L, malt extract 10 g/L.
SV2: glucose 15 g/L, peptone 15 g/L, glycerol 15 g/L, CaCO₃ 1 g/L.
All the media ingredients were dissolved in distilled water and autoclaved before use.

Two female cuttlefish were anesthetised with ethanol 3% before tissue collection. Haemolymph was withdrawn from the vena cava and branchial hearts using a 10 mL syringe. Vibrio splendidus strain CIP 107,715 was grown under agitation at 20 °C in Marine Broth (Conda, 40.20 g/L) medium for 24 h. The culture was centrifuged (4,000× g, 5 min, room temperature), washed twice, and resuspended in sterile sea water to an optical density at 600 nm (OD₆₀₀) of 1 (1 × 10⁹ CUF/mL).
Haemolymph was diluted in sterile sea water (v/v) and divided into two batches: 10 mL remained untreated, and 10 mL were challenged with 200µl of boiled bacteria (boiled 15 min at 100 °C). Haemolymph with and without heat-killed bacteria was then incubated at 16 °C at 100 rpm in a MaxQ6000 incubator (ThermoFisher) equipped with an orbital shaker. After 24 h, hct from the two conditions were harvested by centrifugation at 12,000 rpm and resuspended in TriReagent (Sigma-Aldrich, Saint Louis, MO, USA) to extract total RNA.

Bacterial strains used in this study are shown in Table 1. V. parahaemolyticus strains were initially cultured aerobically onto selective media Thiosulphate Citrate Bile Sucrose (TCBS) agar (Oxoid) at 37°C for 24 h to check for contamination. For enumeration of colony counts we used Marine Agar (Conda lab, India) at 30°C for 24 h and for routine subculturing and growth Luria-Bertani (LB) agar at 37°C for 18 h was used. For growth curves, wildtype T024 and T024:ΔmutT were grown in minimal media with aeration at 30°C and optical readings and colony counts were carried out on Marine Agar at 0, 2, 4, 6 and 8 h. For virulence assays the V. parahaemolyticus strains were grown in Marine Broth (Conda Lab, India) at 37°C.