The AMPK-PP2A axis in insect fat body is activated by 20-hydroxyecdysone to antagonize insulin/IGF signaling and restrict growth rate.

In insects, 20-hydroxyecdysone (20E) limits the growth period by triggering developmental transitions; 20E also modulates the growth rate by antagonizing insulin/insulin-like growth factor signaling (IIS). Previous work has shown that 20E cross-talks with IIS, but the underlying molecular mechanisms are not fully understood. Here we found that, in both the silkworm Bombyx mori and the fruit fly Drosophila melanogaster, 20E antagonized IIS through the AMP-activated protein kinase (AMPK)-protein phosphatase 2A (PP2A) axis in the fat body and suppressed the growth rate. During Bombyx larval molt or Drosophila pupariation, high levels of 20E activate AMPK, a molecular sensor that maintains energy homeostasis in the insect fat body. In turn, AMPK activates PP2A, which further dephosphorylates insulin receptor and protein kinase B (AKT), thus inhibiting IIS. Activation of the AMPK-PP2A axis and inhibition of IIS in the Drosophila fat body reduced food consumption, resulting in the restriction of growth rate and body weight. Overall, our study revealed an important mechanism by which 20E antagonizes IIS in the insect fat body to restrict the larval growth rate, thereby expanding our understanding of the comprehensive regulatory mechanisms of final body size in animals.

Correction: Wu, W., et al. Tip60 Phosphorylation at Ser 99 Is Essential for Autophagy Induction in Bombyx mori. Int. J. Mol. Sci. 2020, 21, 6893.

The author wishes to make the following correction to this paper [...].

Tip60 Phosphorylation at Ser 99 Is Essential for Autophagy Induction in Bombyx mori.

Tip60, a key histone acetyltransferase of the MYST family and member of the nuclear multimeric protein complex (NuA4), regulates the activity and stability of proteins involved in the cell cycle, DNA damage responses, autophagy, etc. However, the function and regulatory mechanism of Tip60 homolog in Bombyx mori are not elucidated. In the present study, Bombyx Tip60 (BmTip60) was functionally identified. Developmental profiles showed that the protein levels and nuclear localization of BmTip60 peaked in fat body during the larval-pupal metamorphosis when autophagy was intensive; simultaneously, the BmTip60 protein migrated to form an upper band as detected by Western blot. Interestingly, the upper band of BmTip60 was reduced by λ-phosphatase treatment, indicating that it was a phosphorylated form of BmTip60. Results showed that BmTip60 was promoted by starvation but not 20-hydroxyecdysone treatment. Transcription factor AMP-activated protein kinase (AMPK) affected by starvation was pivotal for BmTip60 protein migration. In addition, one mammalian phosphorylation site was identified in BmTip60 at Ser99, the constitutive-activation mutation of Ser99 to Asp99 but not its inactive mutation to Ala99 significantly upregulated autophagy, showing the critical role of phosphorylation at Ser99 for BmTip60-mediated autophagy. In conclusion, the starvation-AMPK axis promotes BmTip60 in B. mori, which was requisite for autophagy induction. These results reveal a regulatory mechanism of histone acetyltransferase Tip60 homologs by phosphorylation in insects, and sheds light on further related studies of acetylation regulation.

The appearance of cytoplasmic cytochrome C precedes apoptosis during Drosophila salivary gland degradation.

Apoptosis is an important process for organism development that functions to eliminate cell damage, maintain homeostasis, and remove obsolete tissues during morphogenesis. In mammals, apoptosis is accompanied by the release of cytochrome C (Cyt-c) from mitochondria to the cytoplasm. However, whether this process is conserved in the fruit fly, Drosophila melanogaster, remains controversial. In this study, we discovered that during the degradation of Drosophila salivary gland, the transcription of mitochondria apoptosis factors (MAPFs), Cyt-c, and death-associated APAF1-related killer (Dark) encoding genes are all upregulated antecedent to initiator and effector caspases encoding genes. The proteins Cyt-c and the active caspase 3 appear gradually in the cytoplasm during salivary gland degradation. Meanwhile, the Cyt-c protein colocates with mito-GFP, the marker indicating cytoplasmic mitochondria, and the change in mitochondrial membrane potential coincides with the appearance of Cyt-c in the cytoplasm. Moreover, impeding or promoting 20E-induced transcription factor E93 suppresses or enhances the staining of Cyt-c and the active caspase 3 in the cytoplasm of salivary gland, and accordingly decreases or increases the mitochondrial membrane potential, respectively. Our research provides evidence that cytoplasmic Cyt-c appears before apoptosis during Drosophila salivary gland degradation, shedding light on partial conserved mechanism in apoptosis between insects and mammals.

Atg1 phosphorylation is activated by AMPK and indispensable for autophagy induction in insects.

Phosphorylation is a key post-translational modification in regulating autophagy in yeast and mammalians, yet it is not fully illustrated in invertebrates such as insects. ULK1/Atg1 is a functionally conserved serine/threonine protein kinase involved in autophagosome initiation. As a result of alternative splicing, Atg1 in the silkworm, Bombyx mori, is present as three mRNA isoforms, with BmAtg1c showing the highest expression levels. Here, we found that BmAtg1c mRNA expression, BmAtg1c protein expression and phosphorylation, and autophagy simultaneously peaked in the fat body during larval-pupal metamorphosis. Importantly, two BmAtg1c phosphorylation sites were identified at Ser269 and Ser270, which were activated by BmAMPK, the major energy-sensing kinase, upon stimulation with 20-hydroxyecdysone and starvation; additionally, these Atg1 phosphorylation sites are evolutionarily conserved in insects. The two BmAMPK-activated phosphorylation sites in BmAtg1c were found to be required for BmAMPK-induced autophagy. Moreover, the two corresponding DmAtg1 phosphorylation sites in the fruit fly, Drosophila melanogaster, are functionally conserved for autophagy induction. In conclusion, AMPK-activated Atg1 phosphorylation is indispensable for autophagy induction and evolutionarily conserved in insects, shedding light on how various groups of organisms differentially regulate ULK1/Atg1 phosphorylation for autophagy induction.

The genomic and functional landscapes of developmental plasticity in the American cockroach.

Many cockroach species have adapted to urban environments, and some have been serious pests of public health in the tropics and subtropics. Here, we present the 3.38-Gb genome and a consensus gene set of the American cockroach, Periplaneta americana. We report insights from both genomic and functional investigations into the underlying basis of its adaptation to urban environments and developmental plasticity. In comparison with other insects, expansions of gene families in P. americana exist for most core gene families likely associated with environmental adaptation, such as chemoreception and detoxification. Multiple pathways regulating metamorphic development are well conserved, and RNAi experiments inform on key roles of 20-hydroxyecdysone, juvenile hormone, insulin, and decapentaplegic signals in regulating plasticity. Our analyses reveal a high level of sequence identity in genes between the American cockroach and two termite species, advancing it as a valuable model to study the evolutionary relationships between cockroaches and termites.

Peroxiredoxin 5 from common cutworm (Spodoptera litura) acts as a potent antioxidant enzyme.

In this study, we describe the cloning and characterization of a Prx from the common cutworm Spodoptera litura (SlPrx5). The SlPrx5 cDNA contains an open reading frame of 477 bp encoding a predicted protein of 159 amino acid residues, 16.902 kDa, and an isoelectric point of 7.68. Furthermore, the deduced amino acid sequence of the SlPrx5 cDNA showed 86% identity to Papilio xuthus Prx5, 72% to Aedes aegypti Prx5, and 64-67% to other insect Prxs. A phylogenetic analysis further revealed that the deduced amino acid sequence of SlPrx5 groups within the atypical 2-Cys Prx cluster. Recombinant SlPrx5 (20 kDa) purified from baculovirus-infected insect cells was found to reduce H2O2 in the presence of electrons donated by dithiothreitol and protect super-coiled DNA from damage by metal-catalyzed oxidation in vitro. During S. litura development, SlPrx5 is constitutively expressed in the epidermis, fat body, and midgut, with the highest expression occurring in the sixth-instar larval stage in the fat body and midgut. Additionally, SlPrx5 mRNA expression was up-regulated after injection with H2O2, cumene hydroperoxide, indoxacarb, and metaflumizone. A disc diffusion assay indicated that recombinant SlPrx5 can play a functional role in protecting cells from oxidative stress in vivo. These results provide insight into the role of SlPrx5 during development and the oxidative stress response of S. litura.