Methyl-CpG binding domain (MBD)2/3 specifically recognizes and binds to the genomic mCpG site with a ß-sheet in the MBD to affect embryonic development in Bombyx mori.

Methyl-CpG (mCpG) binding domain (MBD) proteins especially bind with methylated DNA, and are involved in many important biological processes; however, the binding mechanism between insect MBD2/3 and mCpG remains unclear. In this study, we identified 2 isoforms of the MBD2/3 gene in Bombyx mori, MBD2/3-S and MBD2/3-L. Binding analysis of MBD2/3-L, MBD2/3-S, and 7 mutant MBD2/3-L proteins deficient in ß1-ß6 or α1 in the MBD showed that ß2-ß3-turns in the ß-sheet of the MBD are necessary for the formation of the MBD2/3-mCpG complex; furthermore, other secondary structures, namely, ß4-ß6 and an α-helix, play a role in stabilizing the ß-sheet structure to ensure that the MBD is able to bind mCpG. In addition, sequence alignment and binding analyses of different insect MBD2/3s indicated that insect MBD2/3s have an intact and conserved MBD that binds to the mCpG of target genes. Furthermore, MBD2/3 RNA interference results showed that MBD2/3-L plays a role in regulating B. mori embryonic development, similar to that of DNA methylation; however, MBD2/3-S without ß4-ß6 and α-helix does not alter embryonic development. These results suggest that MBD2/3-L recognizes and binds to mCpG through the intact ß-sheet structure in its MBD, thus ensuring silkworm embryonic development.

Knockdown of AMP-activated protein kinase increases the insecticidal efficiency of pymetrozine to Nilaparvata lugens.

Insecticides are the main tools used to control Nilaparvata lugens (Stål), a serious pest of rice in Asia. However, repeated application of insecticides has caused many negative effects. Reducing the amount of insecticide used, while maintaining good pest population control, would be valuable. AMP-activated protein kinase (AMPK), a sensor of cellular energy status, helps to maintain insect energy balance at the cellular and whole-body level. The role of AMPK in insect response to insecticide stimulation is unknown. We studied the functions of AMPK catalytic subunit alpha (NlAMPKα) in the development of N. lugens and in response to pymetrozine, an insecticide used to control insect pests with piercing-sucking mouthparts. A phylogenetic analysis of protein sequences from 12 species in six orders showed that insects have only the AMPKα 2 subtype. RNA interference against NlAMPKα demonstrated that blocking the AMPK pathway led to a decrease in the systemic ATP level and an increase in N. lugens mortality. NlAMPKα responded to the energy stress caused by pymetrozine treatment, which activated downstream energy metabolic pathways to compensate for the energy imbalance. However, the ATP level in pymetrozine- treated nymphs was not increased, suggesting that ATP is consumed more than synthesized. When NlAMPKα expression was reduced in pymetrozine-treated nymphs by RNAi, the ATP level was decreased and the mortality was significantly increased. At day eight post 0.5 g/3 L of pymetrozine and dsNlAMPKα treatment, nymph survival was 29.33%, which was similar to the 27.33% survival of 1 g/3 L pymetrozine-treated nymphs. Addition of dsNlAMPKα can reduce the concentration of pymetrozine used by 50% while providing comparable efficacy. These results indicate that AMPK helps maintain the energy metabolism of N. lugens in response to pymetrozine treatment. Knockdown of NlAMPKα increases the insecticidal efficiency of pymetrozine to N. lugens.