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Insect cytochrome P450 s (P450 s) are associated with the metabolic detoxification of toxic xenobiotics and their constitutive upregulation is often associated with resistance to natural and synthetic toxicants. The P450 s CYP321A16 and CYP332A1 are constitutively overexpressed in an insecticide-resistant strain of beet armyworm, Spodoptera exigua. However, the function and upstream regulation of these two P450 s remain unknown. Here, we investigated the function of CYP321A16 and CYP332A1 in resistance to the organophosphate insecticide, chlorpyrifos and their regulation by the transcription factors CncC and Maf. Transgenic strains of Drosophila melanogaster expressing CYP321A16 or CYP332A1 showed higher levels of tolerance to chlorpyrifos than the control flies with the same genetic background. Furthermore, recombinant CYP321A16 and CYP332A1 proteins metabolized chlorpyrifos. Analysis of the putative promoter sequences of the genes coding for CYP321A16 and CYP332A1 revealed conserved CncC/Maf binding sites. Transfection of luciferase reporter plasmids containing the promoter of CYP450 gene together with CncC and Maf expression plasmids significantly enhanced the activity of the reporter. Promoter truncation identified a site in the promoter of CYP321A16 that is critical for the CncC/Maf binding. These data demonstrate that resistance to chlorpyrifos in S. exigua is conferred by the combined action of CYP321A16 and CYP332A1 and uncovered their regulation by the transcription factors CncC and Maf., Competing Interests: Declaration of Competing Interest The authors have declared that no conflict of interests exist., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The E93 transcription factor is a member of helix-turn-helix transcription factor family containing a Pip-squeak motif. This ecdysone primary response gene was identified as a regulator of cell death in Drosophila melanogaster where it is involved in ecdysone-induced autophagy and caspase activity that mediate degeneration of larval tissues during metamorphosis from larva to pupa. However, its function in adult insects is not well studied. To study E93 function in the red flour beetle, Tribolium castaneum, double-stranded RNA (dsRNA) targeting E93 (dsE93) was injected into newly emerged adults. Knockdown of E93 caused a decrease in the synthesis of vitellogenin (Vg), oocyte development, and egg-laying. Sequencing of RNA isolated from adults injected with dsE93 and control dsmalE (dsRNA targeting Escherichia coli malE gene) followed by differential gene expression analysis showed upregulation of genes involved in the metabolism of reserved nutrients. E93 knockdown induced changes in gene expression resulted in a decrease in Vg synthesis in the fat body and oocyte maturation in ovaries. Mating experiments showed that females injected with dsE93 did not lay eggs. Knockdown of E93 caused a reduction in the number and size of lipid droplets in the fat body when compared with that in control beetles injected with dsmalE. These data suggest that during the first 2-3 days after the emergence of adult females, E93 suppresses genes coding for enzymes that metabolize reserved nutrients until initiation of vitellogenesis and oogenesis., (© 2020 Wiley Periodicals LLC.)

Insects have evolved resistance to almost all insecticides developed for their control. Multiple mechanisms of resistance, including enhanced metabolism and excretion of insecticides, target-site insensitivity, reduced penetration of insecticides, and avoidance behavior, have been reported. The genes coding for proteins involved in resistance have been identified in numerous insects. The enzymes and transporters required for all three phases of insecticide metabolism and excretion including cytochrome P450 monooxygenases, glutathione S-transferases, UDP-glucuronosyltransferases, carboxylesterases, and ATP-binding cassette transmembrane transporters have been identified. Recent research in multiple insect species identified CNC-bZIP transcription factor superfamily members as regulators of genes coding for enzymes and transporters involved in insecticide metabolic resistance. The information on the pathway including reactive oxygen species, cap "n" collar isoform-C, and its heterodimer partner, muscle aponeurosis fibromatosis transcription factors involved in overexpression of enzymes and transporters involved insecticide resistance will be summarized., (© 2020 Wiley Periodicals, Inc.)

Background: Doublesex (dsx), the downstream gene in the insect sex-determination pathway, is a key regulator of sexually dimorphic development and behavior across a variety of insects. Manipulating expression of dsx could be useful in the genetic control of insects. However, information on the sex-specific function of dsx in non-model insects is lacking., Results: In this work, we isolated a dsx homolog, which is alternatively spliced into six female-specific and one male-specific isoforms, from an important agricultural pest, the black cutworm, Agrotis ipsilon. Studies on the expression of sex-specific Aidsx mRNA during embryonic development showed that the sixth hour post oviposition is the key stage for sex determination in A. ipsilon. Functional analysis of Aidsx was conducted using a CRISPR/Cas9 system targeting female- and male-specific Aidsx exons. Disruptions of sex-specific Aidsx exons resulted in sex-specific, sexually dimorphic defects in external genitals, gonads and antennae, and expression of sex-specific genes as well as production of offspring in both sexes., Conclusion: Our results not only demonstrate that dsx is a key player determining A. ipsilon sexually dimorphic traits, but also provide a potential method for the genetic control of this pest. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

The Colorado potato beetle (CPB), Leptinotarsa decemlineata developed resistance to imidacloprid after exposure to this insecticide for multiple generations. Our previous studies showed that xenobiotic transcription factor, cap 'n' collar isoform C (CncC) regulates the expression of multiple cytochrome P450 genes, which play essential roles in resistance to plant allelochemicals and insecticides. In this study, we sought to obtain a comprehensive picture of the genes regulated by CncC in imidacloprid-resistant CPB. We performed sequencing of RNA isolated from imidacloprid-resistant CPB treated with dsRNA targeting CncC or gene coding for green fluorescent protein (control). Comparative transcriptome analysis showed that CncC regulated the expression of 1798 genes, out of which 1499 genes were downregulated in CncC knockdown beetles. Interestingly, expression of 79% of imidacloprid induced P450 genes requires CncC. We performed quantitative real-time PCR to verify the reduction in the expression of 20 genes including those coding for detoxification enzymes (P450s, glutathione S-transferases, and esterases) and ABC transporters. The genes coding for ABC transporters are induced in insecticide resistant CPB and require CncC for their expression. Knockdown of genes coding for ABC transporters simultaneously or individually caused an increase in imidacloprid-induced mortality in resistant beetles confirming their contribution to insecticide resistance. These studies identified CncC as a transcription factor involved in regulation of genes responsible for imidacloprid resistance. Small molecule inhibitors of CncC or suppression of CncC by RNAi could provide effective synergists for pest control or management of insecticide resistance., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Guanine-rich and cytosine-rich DNA can form four-stranded DNA secondary structures called G-quadruplex (G4) and i-motif, respectively. These structures widely exist in genomes and play important roles in transcription, replication, translation and protection of telomeres. In this study, G4 and i-motif structures were identified in the promoter of the transcription factor gene BmPOUM2, which regulates the expression of the wing disc cuticle protein gene (BmWCP4) during metamorphosis. Disruption of the i-motif structure by base mutation, anti-sense oligonucleotides (ASOs) or inhibitory ligands resulted in significant decrease in the activity of the BmPOUM2 promoter. A novel i-motif binding protein (BmILF) was identified by pull-down experiment. BmILF specifically bound to the i-motif and activated the transcription of BmPOUM2. The promoter activity of BmPOUM2 was enhanced when BmILF was over-expressed and decreased when BmILF was knocked-down by RNA interference. This study for the first time demonstrated that BmILF and the i-motif structure participated in the regulation of gene transcription in insect metamorphosis and provides new insights into the molecular mechanism of the secondary structures in epigenetic regulation of gene transcription.

In invertebrates, a heterodimer of xenobiotic transcription factors, cap n collar C isoform (CncC) and muscle aponeurosis fibromatosis (Maf) mediate cellular defense. In insects, these proteins regulate expression of genes involved in insecticide detoxification. In the current study, we performed sequencing of cDNA copied from RNA isolated from Tribolium castaneum pyrethroid resistant strain (QTC279) beetles injected with CncC or green fluorescence protein (GFP, control) dsRNA. Differential expression analysis of sequences identified 662 genes that showed a decrease and 91 genes that showed an increase in expression (p value ≤ 0.01 and log2 fold change of ≥ 1.5) in CncC knockdown insects when compared to their expression in control insects. We selected a subset of 27 downregulated genes and verified their differential expression using qRT-PCR. This subset of 27 genes included 21 genes with a predicted function in xenobiotic detoxification. RNAi and insecticide bioassays were employed to study the function of six of these genes coding for CYP4G7, CYP4G14, GST-1 and four ABC transporters, ABCA-UB, ABCA-A1 and ABCA-A1L and ABCA-9B involved in all three phases of insecticide detoxification. These data suggest that CncC regulates genes coding for proteins involved in detoxification of insecticides., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The common bed bug is an obligate hematophagous parasite of humans. We studied the regulation of molting and metamorphosis in bed bugs with a goal to identify key players involved. qRT-PCR studies on the expression of genes known to be involved in molting and metamorphosis showed high levels of Krüppel homolog 1 [Kr-h1, a transcription factor that plays key roles in juvenile hormone (JH) action] mRNA in the penultimate nymphal stage (N4). However, low levels of Kr-h1 mRNA were detected in the fifth and last nymphal stage (N5). Knockdown of Kr-h1 in N4 resulted in a precocious development of adult structures. Kr-h1 maintains the immature stage by suppressing E93 (early ecdysone response gene) in N4. E93 expression increases during the N5 in the absence of Kr-h1 and promotes the development of adult structures. Knockdown of E93 in N5 results in the formation of supernumerary nymphs. The role of JH in the suppression of adult structures through interaction with Kr-h1 and E93 was also studied by the topical application of JH analog, methoprene, to N5. Methoprene induced Kr-h1 and suppressed E93 and induced formation of the supernumerary nymph. These data show interactions between Kr-h1, E93 and JH in the regulation of metamorphosis in the bed bugs.

Insecticide resistance is a global problem that presents an ongoing challenge to control insects that destroy crops, trees and transmit diseases. Dramatic progress has been made during the last decade on identification of insecticide resistance-associated genes. In one of the most common resistance mechanisms, insects acquire resistance by increasing the levels of their detoxification enzymes especially the cytochrome P450 monooxygenases (P450's). Previous studies in our laboratory showed that the pyrethroid resistance in QTC279 strain of Tribolium castaneum is achieved through constitutive overexpression of the P450 gene CYP6BQ9 by 200-fold higher in the resistant strain as compared to that in the susceptible strain. RNAi-aided knockdown in the expression of probable genes that regulate P450 gene expression in QTC279 identified cap 'n' collar C (CncC) and muscle aponeurosis fibromatosis (Maf) family transcription factors as the key regulator of these genes, CncC and Maf regulate expression of multiple genes in the CYP6BQ cluster. Studies on the promoters of these genes using reporter assays identified binding sites that mediate CncC and Maf regulation of CYP6BQ gene expression., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Ecdysteroids and juvenile hormones (JH) regulate a variety of developmental, physiological, behavioral, and metabolic processes. Ecdysteroids function through a heterodimeric complex of two nuclear receptors, ecdysone receptor (EcR) and ultraspiracle (USP). An 85 kDa protein identified in Drosophila melanogaster methoprene-tolerant (Met) mutant binds to JH III with high affinity, and the mutant flies are resistant to juvenile hormone analog (JHA), methoprene. Reporter assays using the yeast two-hybrid system were performed in order to study the molecular interactions between EcR, USP and Met. As expected, EcR fused to the B42 activation domain and USP fused to the LexA DNA binding domain interacted with each other and supported induction of the reporter gene in the presence of stable ecdysteroid analog, RG-102240 or steroids, muristerone A and ponasterone A. The USP:USP homodimers supported expression of the reporter gene in the absence of ligand, and there was no significant increase in the reporter activity after addition of a JHA, methoprene. Similarly, Met:Met homodimers as well as Met:EcR and Met:USP heterodimers induced reporter activity in the absence of ligand and addition of ecdysteroid or JH analogs did not increase the reporter activity regulated by either homodimers or heterodimers of Met protein. Two-hybrid assays in insect cells and in vitro pull-down assays confirmed the interaction of Met with EcR and USP. These data suggest that the proteins that are involved in signal transduction of ecdysteroids (EcR and USP) and juvenile hormones (Met) interact to mediate cross-talk between these two important hormones. Arch. Insect Biochem. Physiol. 2008. (c) 2008 Wiley-Liss, Inc.

Ecdysteroids regulate insect growth and development through a heterodimeric complex of nuclear receptors consisting of ecdysone receptor (EcR) and ultraspiracle (USP). In the red flour beetle, Tribolium castaneum, two isoforms each of EcR and USP have been identified. Quantitative real-time reverse-transcriptase PCR (qRT-PCR) analysis showed isoform-specific developmental expression of both EcR and USP in the epidermis and the midgut dissected from the final instar larvae and pupae. Injection of double-stranded RNA (dsRNA) prepared using the common or isoform-specific regions of EcR or USP as templates caused derailment of development. EcR common region (EcRC) or EcRA dsRNA caused more severe effects, and most of the treated larvae died prior to pupation. EcRB dsRNA caused less severe effects and most of the treated larvae became pupae but showed developmental defects. Only dsRNA prepared against USP common region but not against USPA or USPB isoform-specific region caused developmental defects during larval-pupal metamorphosis. Determination of mRNA levels of EcR isoforms and 20-hydroxyecdysone-response (20E) genes (broad, E75, E74, HR3 and FTZ-F1) by qRT-PCR in the larvae injected with EcRA, EcRB or EcRC dsRNA showed that EcRA initiates ecdysteroid action by regulation the expression of EcRB and 20E-response genes. These data suggest that the EcR but not USP isoforms play distinct roles during the larval-pupal metamorphosis and EcRA plays a dominant role in transduction of ecdysteroid response in T. castaneum.

The Colorado potato beetle is one of the most challenging agricultural pests to manage. It has shown a spectacular ability to adapt to a variety of solanaceaeous plants and variable climates during its global invasion, and, notably, to rapidly evolve insecticide resistance. To examine evidence of rapid evolutionary change, and to understand the genetic basis of herbivory and insecticide resistance, we tested for structural and functional genomic changes relative to other arthropod species using genome sequencing, transcriptomics, and community annotation. Two factors that might facilitate rapid evolutionary change include transposable elements, which comprise at least 17% of the genome and are rapidly evolving compared to other Coleoptera, and high levels of nucleotide diversity in rapidly growing pest populations. Adaptations to plant feeding are evident in gene expansions and differential expression of digestive enzymes in gut tissues, as well as expansions of gustatory receptors for bitter tasting. Surprisingly, the suite of genes involved in insecticide resistance is similar to other beetles. Finally, duplications in the RNAi pathway might explain why Leptinotarsa decemlineata has high sensitivity to dsRNA. The L. decemlineata genome provides opportunities to investigate a broad range of phenotypes and to develop sustainable methods to control this widely successful pest.

Colorado potato beetle (CPB), Leptinotarsa decemlineata is a notorious pest of potato. Co-evolution with Solanaceae plants containing high levels of toxins (glycoalkaloids) helped this insect to develop an efficient detoxification system and resist almost every chemical insecticide introduced for its control. Even though the cross-resistance between plant allelochemicals and insecticides is well acknowledged, the underlying molecular mechanisms are not understood. Here, we investigated the molecular mechanisms involved in detoxification of potato plant allelochemicals and imidacloprid resistance in the field-collected CPB. Our results showed that the imidacloprid-resistant beetles employ metabolic detoxification of both potato plant allelochemicals and imidacloprid by upregulation of common cytochrome P450 genes. RNAi aided knockdown identified four cytochromes P450 genes (CYP6BJ a/b , CYP6BJ1v1, CYP9Z25, and CYP9Z29) that are required for defense against both natural and synthetic chemicals. These P450 genes are regulated by the xenobiotic transcription factors Cap n Collar C, CncC and muscle aponeurosis fibromatosis, Maf. Studies on the CYP9Z25 promoter using the luciferase reporter assay identified two binding sites (i.e. GCAGAAT and GTACTGA) for CncC and Maf. Overall, these data showed that CPB employs the metabolic resistance mediated through xenobiotic transcription factors CncC and Maf to regulate multiple P450 genes and detoxify both imidacloprid and potato plant allelochemicals. [ABSTRACT FROM AUTHOR]

Following the publication of this article [1], the authors found that the primers listed for CREB-binding protein were not correct. This mistake occurred during assembly of the primer table and the authors apologize for this error. This correction does not change the data included in the paper, their interpretation nor the conclusions drawn. [ABSTRACT FROM AUTHOR]