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Juvenile hormone (JH) plays a vital role in the growth, development, and reproduction of insects and other arthropods. Previous experiments have suggested that BmFAMeT6 could affect the duration of the silk moth’s larval stage. In this study, we established the BmFAMeT6 overexpression strain and BmFAMeT6 knockout strain using the GAL4/UAS binary hybrid system and CRISPR/Cas 9 system, respectively, and found that the larval stage of the overexpression strain was shorter, while the knockout strain was longer. Our results exhibited that both the JH titers and BmKr-h1 levels in the larvae of the third instar were reduced significantly by BmFAMeT6 overexpression, but were increased obviously by BmFAMeT6 knockout. In addition, injection of farnesoic acid induced changes in the JH I and JH II levels in the hemolymphs of larvae. This study is the first to directly reveal the role of BmFAMeT6 in the regulation of insect JH titers and the relationship between farnesoic acid and JH (JH I and JH II). This provides a new perspective on regulating the growth and development of insects such as Bombyx mori.

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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.

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