The role of <i>BmsPLA2‐4</i> in tissue defense against pathogens in silkworm, <i>Bombyx mori</i>

Lepidoptera, one of the largest orders within the class Insecta, accounts for approximately 70% of known pests affecting crops and forests. Therefore, immune research in the lepidopteran model Bombyx mori offers valuable insights for refining pest control strategies. Although numerous studies have investigated the role of phospholipase A2 (PLA2) in immunity, research on its role in tissues remains lacking in insects. We previously found that BmsPLA2-4 is highly expressed in the midgut, a key immune tissue in silkworms. In this study, we successfully generated BmsPLA2-4 overexpressing and knockout transgenic strains using the piggyBac system, microinjection, and fluorescence screening. The target sequence was inserted into NSCAF6497 on chromosome 15 in overexpressing individuals, and deletions or mutations of 13 or 14 bp occurred near genomic guide RNA targets in knockout individuals. Upon oral bacterial challenge, midgut bacterial load was significantly reduced in overexpressing individuals and markedly elevated in knockout individuals. Furthermore, bacterial growth was significantly promoted by hemolymph from knockout individuals. Expression levels of various immunity-related genes in the midgut were significantly altered in transgenic individuals following pathogen infection. These suggested that BmsPLA2-4 plays an important role in immune tissue resistance to pathogens in silkworm. Interestingly, BmsPLA2-4 protein expressed in vitro in eukaryotic systems inhibits bacterial growth without binding to bacteria, providing new data for studying the mechanism of BmsPLA2-4 in immunity. Our work, for the first time in insects, demonstrates PLA2's role in tissues against pathogens, offering reliable data for applications in silkworms or other insects.