Multiple diptericins of the black soldier fly (Hermetia illucens) differentially respond to bacterial challenges

Due to its significant bioconversion potential, the black soldier fly (BSF), Hermetia illucens, shows great promise as a cost-effective alternative for recycling biological waste. BSF larvae (BSFL) are constantly exposed high levels of pathogenic microorganisms, including bacteria and fungi, which endows BSFL with a robust immune system. Diptericin, a type of glycine-rich antimicrobial peptide (AMP) that exhibits activity against gram-negative bacteria, contains proline-rich domains (P-domains) and glycine-rich domains (G-domains); these domains are separated by a furin cleavage site. Although the presence and expression patterns of BSFL diptericins have been documented, their basic molecular properties remain unclear. To the best of our knowledge, in the present study, we report, for the first time, the molecular characteristics of seven full-length cDNA sequences of H. illucens diptericins and their expression patterns following challenges with gram-positive or gram-negative bacteria. Seven diptericin paralogs are located in tandem on chromosome 2, spanning a total length of 38.6 kb, with an average intergenic distance of approximately 5.5 kb. Sequence analysis revealed that three diptericins (HipDptA/B/C) are pseudogenized due to premature stop codons. In contrast, the other diptericins (HiDpt1/2/3/4) possess mature-sized G-domains rich in glycine at the C-terminus, which are essential for AMP activity, along with proline-rich domain (P-domain) in the N-terminal and either two (HiDpt1/2/3) or one (HiDpt4) putative furin cleavage sites (R-X-R/K-R) between these domains. These furin cleavage sites possibly produce a glycine-rich AMP and one or two additional peptides with unknown functions. Similar to other diptericins, the expression of HiDpt1/2/3/4 mRNAs is predominantly induced by gram-negative bacteria, increasing typically by ≥ 1,000-fold (up to 5,000-fold). Additionally, HiDpt1/3/4 show significant responses to gram-positive bacteria such as Micrococcus luteus, though not as strongly as to gram-negative bacteria. These findings suggest that HiDpts function as a rapid, effective, and broad-spectrum first-line defense mechanism in the BSFL immune system.