Purification and characterization of a novel antibacterial peptide from black soldier fly (Hermetia illucens) larvae

• A novel peptide (defensin-like peptide, DLP) was identified from Hermetia illucens.
• The cDNA sequences of DLPs were determined.
• DLP4 showed potent antimicrobial activity against Gram-positive bacteria including MRSA.
• The mRNA transcripts of DLP4 were mainly expressed in fat body after bacterial challenge.

In this study, we induced and purified a novel antimicrobial peptide exhibiting activity against Gram-positive bacteria from the immunized hemolymph of Hermetia illucens larvae. The immunized hemolymph was extracted, and the novel defensin-like peptide 4 (DLP4) was purified using solid-phase extraction and reverse-phase chromatography. The purified DLP4 demonstrated a molecular weight of 4267 Da, as determined using the matrix-assisted laser desorption/ionization–time-of-flight (MALDI–TOF) method. From analysis of DLP4 by N-terminal amino acid sequencing using Edman degradation, combined with MALDI–TOF and rapid amplification of cDNA ends–polymerase chain reaction (RACE–PCR), the amino acid sequence of the mature peptide was determined to be ATCDLLSPFKVGHAACAAHCIARGKRGGWCDKRAVCNCRK. In NCBI BLAST, the amino acid sequence of DPL4 was found to be 75% identical to the Phlebotomus duboscqi defensin. Analysis of the minimal inhibitory concentration (MIC) revealed that DLP4 have antibacterial effects against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA). The expression of DLP4 transcripts in several tissues after bacterial challenge was measured by quantitative real-time PCR. Expression of the DLP4 gene hardly occurred throughout the body before immunization, but was mostly evident in the fat body after immunization.