Two c-type lysozymes boost the innate immune system of the invasive ladybird Harmonia axyridis

The invasive ladybird beetle Harmonia axyridis has a two-layered immune system, featuring the constitutive production of the low-molecular-mass antimicrobial compound harmonine and the inducible production of a broad range of antimicrobial peptides (AMPs). Here we show that the immune system also features two c-type lysozymes, the acidic c-lys3 (pI = 5.46) and the basic c-lys4 (pI = 8.18). The injection of bacteria into H.axyridis boosted c-lys4 gene expression 8-fold in the gut, whereas the c-lys3 gene was expressed at comparable levels in both naïve and challenged beetles. Both c-lys3 and c-lys4 were expressed in Pichia pastoris and the bacteriolytic activity of the recombinant proteins was found to be calcium-dependent with pH maxima of 6.0 and 6.5, respectively. In a Bacillus subtilis growth inhibition assay, the antimicrobial activity of harmonine and two highly-inducible H.axyridis AMPs (coleoptericins) was potentiated in the presence of c-lys4 but not c-lys3, resulting in 4-fold (harmonine) and up to 16-fold (AMP) lower minimum inhibitory concentrations. Our results suggest that two structurally and functionally distinct lysozymes contribute to innate immune responses of H.axyridis and augment the harmonine and AMP components of the immune response.