A secreted phospholipase A₂ (BmsPLA₂ ) regulates melanization of immunity through BmDDC in the silkworm Bombyx mori

Insect immune-associated phospholipase A₂ (PLA₂ ) is an important target of pathogen invasion. Melanization, an effective defense response, has significant correlations with other immune responses to coordinate immune attack against invaders. However, the effect of PLA₂ on melanization has not yet been reported in insects or other arthropods. In this work, we cloned a PLA₂ gene (BmsPLA₂ ), and its protein had characteristic features of secreted PLA₂ (sPLA₂ ). After injection of bacteria, BmsPLA₂ expression and sPLA₂ activity in hemolymph significantly increased. BmsPLA₂ fluorescence was transferred from the cytoplasm to the cell membranes of circulating hemocytes. These results indicated that BmsPLA₂ was related to hemolymph immunity in silkworms. Interestingly, reducing BmsPLA₂ by RNA interference decreased melanosis (melanistic hemocytes) levels in vivo and in vitro, while BmsPLA₂ overexpression had the opposite effect. The larval survival and melanization rate in the hemocoel both slowed depending on the PLA₂ inhibitor dosage. These results demonstrated that BmsPLA₂ plays a role in melanization during the immune process of silkworms. Surprisingly, the level of BmDDC matched the degree of melanization in various observations. BmDDC expression showed a significant increase, with the peak occurring later than that of BmsPLA₂ after injection of bacteria, implying that BmsPLA₂ was activated prior to BmDDC. Moreover, the alteration of BmsPLA₂ by RNA interference or overexpression led to altered BmDDC levels. These results suggested that BmsPLA₂ regulates the melanization response in silkworms through BmDDC. Our study proposes a new regulatory mechanism of the melanization response and new directions for understanding the complex immune networks of insects.