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RNA interference (RNAi) is more efficient in coleopteran insects than other insects. StaufenC (StauC), a coleopteran-specific double-stranded RNA (dsRNA)-binding protein, is required for efficient RNAi in coleopterans. We investigated the function of StauC in the intracellular transport of dsRNA into the cytosol, where dsRNA is digested by Dicer enzymes and recruited by Argonauts to RNA-induced silencing complexes. Confocal microscopy and cellular organelle fractionation studies have shown that dsRNA is trafficked through the endoplasmic reticulum (ER) in coleopteran Colorado potato beetle (CPB) cells. StauC is localized to the ER in CPB cells, and StauC-knockdown caused the accumulation of dsRNA in the ER and a decrease in the cytosol, suggesting that StauC plays a key role in the intracellular transport of dsRNA through the ER. Using immunoprecipitation, we showed that StauC is required for dsRNA interaction with ER proteins in the ER-associated protein degradation (ERAD) pathway, and these interactions are required for RNAi in CPB cells. These results suggest that StauC works with the ERAD pathway to transport dsRNA through the ER to the cytosol. ^his information could be used to develop dsRNA delivery methods aimed at improving RNAi. [ABSTRACT FROM AUTHOR]

The yellow fever mosquito, Aedes aegypti, is distributed worldwide and transmits viruses that cause many diseases, including dengue, yellow fever, chikungunya, and zika. Epigenetic modifications such as acetylation of histones regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs) control insect development. We recently reported that the Creb-binding protein (a HAT) regulates the metamorphosis of A. aegypti. However, the function of HDACs in A. aegypti is not known. In this study, we identified 10 genes coding for HDACs in A. aegypti and determined their function in larval development using RNA interference (RNAi). Knockdown of each HDAC has a distinct effect on the growth, development, and metamorphosis of A. aegypti. Knockdown of HDAC3 severely affected the larval survival, indicating its indispensable role in larval development. HDAC3 is highly expressed during the larval stages, and its messenger RNA (mRNA) levels correlate with the juvenile hormone (JH) titers. JH induces the expression of HDAC3 through its receptor, methoprenetolerant (Met). Knockdown of HDAC3 resulted in increased expression of proapoptotic genes involved in apoptosis of larval midgut cells. This consequently decreased midgut size and led to larval death. HDAC3 deacetylates histone H4 localized at the promoters of proapoptotic genes and suppresses their expression. In addition, a corepressor, SMRTER, is required for HDAC3-mediated suppression of proapoptotic genes. Interestingly, ecdysone attenuates HDAC3-mediated repression of proapoptotic genes. These data demonstrate that JH-induced HDAC3 is a key player in JH suppression of precocious larval cell death and metamorphosis in A. aegypti. [ABSTRACT FROM AUTHOR]

It is well documented that the juvenile hormone (JH) can function as a gonadotropic hormone that stimulates vitellogenesis by activating the production and uptake of vitellogenin in insects. Here, we describe a phenotype associated with mutations in the Drosophila JH receptor genes, Met and Gce: the accumulation of mature eggs with reduced egg length in the ovary. JH signaling is mainly activated in ovarian muscle cells and induces laminin gene expression in these cells. Meanwhile, JH signaling induces collagen IV gene expression in the adult fat body, from which collagen IV is secreted and deposited onto the ovarian muscles. Laminin locally and collagen IV remotely contribute to the assembly of ovarian muscle extracellular matrix (ECM); moreover, the ECM components are indispensable for ovarian muscle contraction. Furthermore, ovarian muscle contraction externally generates a mechanical force to promote ovulation and maintain egg shape. This work reveals an important mechanism for JHregulated insect reproduction. [ABSTRACT FROM AUTHOR]

The yellow fever mosquito, Aedes aegypti, vectors human pathogens. Juvenile hormones (JH) control almost every aspect of an insect’s life, and JH analogs are currently used to control mosquito larvae. Since RNA interference does not work efficiently during the larval stages of this insect, JH regulation of larval development and mode of action of JH analogs are not well studied. To overcome this limitation, we used a multiple single guide RNA-based CRISPR/Cas9 genome-editingmethod to knockout the methoprene-tolerant (Met) gene coding for a JH receptor. The Met knockout larvae exhibited a black larval phenotype during the L3 (third instar larvae) and L4 (fourth instar larvae) stages and died before pupation. However, Met knockout did not affect embryonic development or the L1 and L2 stages. Microscopy studies revealed the precocious synthesis of a dark pupal cuticle during the L3 and L4 stages. Gene expression analysis showed that Krüppel homolog 1, a key transcription factor in JH action, was down-regulated, but genes coding for proteins involved in melanization, pupal and adult cuticle synthesis, and blood meal digestion in adults were up-regulated in L4 Met mutants. These data suggest that, during the L3 and L4 stages, Met mediates JH suppression of pupal/adult genes involved in the synthesis and melanization of the cuticle and blood meal digestion. These results help to advance our knowledge of JH regulation of larval development and the mode of action of JH analogs in Ae. aegypti. [ABSTRACT FROM AUTHOR]

Posttranslational modifications, including acetylation and deacetylation of histones and other proteins, modulate hormone action. In Tribolium castaneum TcA cells, Trichostatin A, a histone deacetylase (HDAC) inhibitor, mimics juvenile hormone (JH) in inducing JH response genes (e.g., Kr-h1), suggesting that HDACs may be involved in JH action. To test this hypothesis, we identified genes coding for HDACs in T. castaneum and studied their function. Knockdown of 12 HDAC genes showed variable phenotypes; the most severe phenotype was detected in insects injected with double-stranded RNA targeting HDAC1 (dsHDAC1). The dsHDAC1-injected insects showed arrested growth and development and eventually died. Application of JH analogs hydroprene to T. castaneum larvae and JH III to TcA cells suppressed HDAC1 expression. Sequencing of RNA isolated from control and dsHDAC1-injected larvae identified 1,720 differentially expressed genes, of which 1,664 were up-regulated in dsHDAC1-treated insects. The acetylation levels of core histones were increased in TcA cells exposed to dsHDAC1 or JH III. ChIP assays performed using histone H2BK5ac antibodies showed an increase in acetylation in the Kr-h1 promoter region of cells exposed to JH III or dsHDAC1. Overexpression or knockdown of HDAC1, SIN3, or both resulted in a decrease or increase in Kr-h1 mRNA levels and its promoter activity, respectively. Overexpression of the JH receptor Methoprene tolerant (Met) was unable to induce Kr-h1 in the presence of HDAC1 or SIN3. These data suggest that epigenetic modifications influence JH action by modulating acetylation levels of histones and by affecting the recruitment of proteins involved in the regulation of JH response genes. [ABSTRACT FROM AUTHOR]

RNA interference (RNAi) is being used to develop methods to control pests and disease vectors. RNAi is robust and systemic in coleopteran insects but is quite variable in other insects. The determinants of efficient RNAi in coleopterans, as well as its potential mechanisms of resistance, are not known. RNAi screen identified a double-stranded RNA binding protein (StaufenC) as a major player in RNAi. StaufenC homologs have been identified in only coleopteran insects. Experiments in two coleopteran insects, Leptinotarsa decemlineata and Tribolium castaneum, showed the requirement of StaufenC for RNAi, especially for processing of double-stranded RNA (dsRNA) to small interfering RNA. RNAi-resistant cells were selected by exposing L. decemlineata, Lepd-SL1 cells to the inhibitor of apoptosis 1 dsRNA for multiple generations. The resistant cells showed lower levels of StaufenC expression compared with its expression in susceptible cells. These studies showed that coleopteran-specific StaufenC is required for RNAi and is a potential target for RNAi resistance. The data included in this article will help improve RNAi in noncoleopteran insects and manage RNAi resistance in coleopteran insects. [ABSTRACT FROM AUTHOR]

The article discusses the role of the reactive oxygen species (ROS) and protein arginine N-methyltransferase 1 (PRMT1) in the modification of histones and DNA, focusing on the paper "Reactive oxygen species extend insect life span using components of the insulin-signaling pathway," by Zhang and colleagues. It mentions that Zhang and colleagues explored the regulation of pupal diapause in insect pest Helicoverpa armigera, reporting the direct involvement of ROS and PRMT1 in diapause.

It is well documented that the juvenile hormone (JH) can function as a gonadotropic hormone that stimulates vitellogenesis by activating the production and uptake of vitellogenin in insects. Here, we describe a phenotype associated with mutations in the Drosophila JH receptor genes, Met and Gce : the accumulation of mature eggs with reduced egg length in the ovary. JH signaling is mainly activated in ovarian muscle cells and induces laminin gene expression in these cells. Meanwhile, JH signaling induces collagen IV gene expression in the adult fat body, from which collagen IV is secreted and deposited onto the ovarian muscles. Laminin locally and collagen IV remotely contribute to the assembly of ovarian muscle extracellular matrix (ECM); moreover, the ECM components are indispensable for ovarian muscle contraction. Furthermore, ovarian muscle contraction externally generates a mechanical force to promote ovulation and maintain egg shape. This work reveals an important mechanism for JH-regulated insect reproduction., Competing Interests: The authors declare no competing interest.

The yellow fever mosquito, Aedes aegypti , vectors human pathogens. Juvenile hormones (JH) control almost every aspect of an insect's life, and JH analogs are currently used to control mosquito larvae. Since RNA interference does not work efficiently during the larval stages of this insect, JH regulation of larval development and mode of action of JH analogs are not well studied. To overcome this limitation, we used a multiple single guide RNA-based CRISPR/Cas9 genome-editing method to knockout the methoprene-tolerant ( Met ) gene coding for a JH receptor. The Met knockout larvae exhibited a black larval phenotype during the L3 (third instar larvae) and L4 (fourth instar larvae) stages and died before pupation. However, Met knockout did not affect embryonic development or the L1 and L2 stages. Microscopy studies revealed the precocious synthesis of a dark pupal cuticle during the L3 and L4 stages. Gene expression analysis showed that Krüppel homolog 1 , a key transcription factor in JH action, was down-regulated, but genes coding for proteins involved in melanization, pupal and adult cuticle synthesis, and blood meal digestion in adults were up-regulated in L4 Met mutants. These data suggest that, during the L3 and L4 stages, Met mediates JH suppression of pupal/adult genes involved in the synthesis and melanization of the cuticle and blood meal digestion. These results help to advance our knowledge of JH regulation of larval development and the mode of action of JH analogs in Ae. aegypti ., Competing Interests: The authors declare no competing interest., (Copyright © 2019 the Author(s). Published by PNAS.)

RNA interference (RNAi) is being used to develop methods to control pests and disease vectors. RNAi is robust and systemic in coleopteran insects but is quite variable in other insects. The determinants of efficient RNAi in coleopterans, as well as its potential mechanisms of resistance, are not known. RNAi screen identified a double-stranded RNA binding protein (StaufenC) as a major player in RNAi. StaufenC homologs have been identified in only coleopteran insects. Experiments in two coleopteran insects, Leptinotarsa decemlineata and Tribolium castaneum , showed the requirement of StaufenC for RNAi, especially for processing of double-stranded RNA (dsRNA) to small interfering RNA. RNAi-resistant cells were selected by exposing L. decemlineata , Lepd-SL1 cells to the inhibitor of apoptosis 1 dsRNA for multiple generations. The resistant cells showed lower levels of StaufenC expression compared with its expression in susceptible cells. These studies showed that coleopteran-specific StaufenC is required for RNAi and is a potential target for RNAi resistance. The data included in this article will help improve RNAi in noncoleopteran insects and manage RNAi resistance in coleopteran insects., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)