| Summary: | Cuttlefish (<i>Sepia officinalis</i>) haemocytes are potential sources of antimicrobial peptides (AMPs). To study the immune response to <i>Vibrio splendidus</i> and identify new AMPs, an original approach was developed based on a differential transcriptomic study and an in-depth in silico analysis using multiple tools. Two de novo transcriptomes were retrieved from cuttlefish haemocytes following challenge by <i>V</i>. <i>splendidus</i> or not. A first analysis of the annotated transcripts revealed the presence of Toll/NF-κB pathway members, including newly identified factors such as <i>So</i>-TLR-h, <i>So</i>-IKK-h and <i>So</i>-Rel/NF-κB-h. Out of the eight Toll/NF-κB pathway members, seven were found up-regulated following <i>V</i>. <i>splendidus</i> challenge. Besides, immune factors involved in the immune response were also identified and up-regulated. However, no AMP was identified based on annotation or conserved pattern searches. We therefore performed an in-depth in silico analysis of unannotated transcripts based on differential expression and sequence characteristics, using several tools available like PepTraq, a homemade software program. Finally, five AMP candidates were synthesized. Among them, NF19, AV19 and GK28 displayed antibacterial activity against Gram-negative bacteria. Each peptide had a different spectrum of activity, notably against <i>Vibrio</i> species. GK28—the most active peptide—was not haemolytic, whereas NF19 and AV19 were haemolytic at concentrations between 50 and 100 µM, 5 to 10 times higher than their minimum inhibitory concentration.
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