Publications by authors named "Subba Reddy Palli"

80 Publications

MicroRNA miR-8 promotes cell growth of corpus allatum and juvenile hormone biosynthesis independent of insulin/IGF signaling in Drosophila melanogaster.

Insect Biochem Mol Biol 2021 Jun 26;136:103611. Epub 2021 Jun 26.

Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology & School of Life Sciences, South China Normal University, Guangzhou, 510631, China. Electronic address:

The Drosophila melanogaster corpus allatum (CA) produces and releases three types of sesquiterpenoid hormones, including juvenile hormone III bisepoxide (JHB3), juvenile hormone III (JH III), and methyl farnesoate (MF). JH biosynthesis involves multiple discrete enzymatic reactions and is subjected to a comprehensive regulatory network including microRNAs (miRNAs). Using a high throughput sequencing approach, we have identified abundant miRNAs in the D. melanogaster ring gland, which consists of the CA, prothoracic gland, and corpus cardiaca. Genetic and qPCR screens were then performed in an attempt to uncover the full repertoire of CA miRNAs that are involved in regulating metamorphosis. miR-8 was identified as a potential candidate and further studied for its role in the CA. Overexpression of miR-8 in the CA increased cell size of the gland and expression of Jhamt (a gene coding for a key regulatory enzyme in JH biosynthesis), resulting in pupal lethality. By contrast, sponge-mediated reduction of miR-8 in the CA decreased cell size and Jhamt expression, but did not cause lethality. Further investigation revealed that miR-8 promotes cell growth independent of insulin/IGF signaling. Taken together, these experiments show that miR-8 is highly expressed in the CA and exerts its positive effects on cell growth and JH biosynthesis. The miRNAs data in the ring gland also provide a useful resource to study how miRNAs collaboratively regulate hormone synthesis in D. melanogaster.
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http://dx.doi.org/10.1016/j.ibmb.2021.103611DOI Listing
June 2021

Transcript level is a key factor affecting RNAi efficiency.

Pestic Biochem Physiol 2021 Jul 6;176:104872. Epub 2021 May 6.

The Key Laboratory of Monitoring and Management of Plant Diseases and Insects / Department of Entomology, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China. Electronic address:

Efficiency is the basis for the application of RNA interference (RNAi) technology. Actually, RNAi efficiency varies greatly among insect species, tissues and genes. Previous efforts have revealed the mechanisms for variation among insect species and tissues. Here, we investigated the reason for variable efficiency among the target genes in the same insect. First, we tested the genes sampled randomly from Tribolium castaneum, Locusta migratoria and Drosophila S2 cells for both their expression levels and sensitivity to RNAi. The results indicated that the genes with higher expression levels were more sensitive to RNAi. Statistical analysis showed that the correlation coefficients between transcript levels and knockdown efficiencies were 0.8036 (n = 90), 0.7255 (n = 18) and 0.9505 (n = 13), respectively in T. castaneum, L. migratoria and Drosophila S2 cells. Subsequently, ten genes with varied expression level in different tissues (midgut and carcass without midgut) of T. castaneum were tested. The results indicated that the higher knockdown efficiency was always obtained in the tissue where the target gene expressed higher. In addition, three genes were tested in different developmental stages, larvae and pupae of T. castaneum. The results found that when the expression level increased after insect pupation, these genes became more sensitive to RNAi. Thus, all the proofs support unanimously that transcript level is a key factor affecting RNAi sensitivity. This finding allows for a better understanding of the RNAi efficiency variation and lead to effective or efficient use of RNAi technology.
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http://dx.doi.org/10.1016/j.pestbp.2021.104872DOI Listing
July 2021

Identification of juvenile hormone-induced posttranslational modifications of methoprene tolerant and Krüppel homolog 1 in the yellow fever mosquito, Aedes aegypti.

J Proteomics 2021 06 4;242:104257. Epub 2021 May 4.

Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, S-225 Agricultural Science Center North Lexington, KY 40546-0091, United States. Electronic address:

Recent studies reported that JH-regulated phosphorylation status of the JH-receptor complex contributes to its transcription activity in Aedes aegypti. However, phosphorylation sites of these proteins have not yet been identified. In this study, we found that the fusion of an EGFP tag to Ae. aegypti Kr-h1 (AaKr-h1) and Met (AaMet) improved their stability in mosquito Aag-2 cells, which allowed their purification. The liquid chromatography and tandem mass spectrometry analysis of the purified AaKr-h1 showed that the phosphoserine residue at position 694, located in the evolutionarily conserved SVIQ motif, is dephosphorylated when the cells are exposed to JH. The AaKr-h1 dephosphorylation mutant (S694V) showed significantly higher activity in inducing the luciferase gene regulated by JH response elements. The phosphorylation profile of Met also changed after exposing Aag-2 cells to JH III. The Ser-77 and Ser-710 residues of Met were phosphorylated after JH III treatment. In contrast, the two phosphoserine residues at positions 73 and 747 were dephosphorylated after JH III treatment. JH exposure also induced transient and reversible phosphorylation of Thr-664 and Ser-723 residues. Overall, these data show that JH induces changes in post-translational modifications of AaMet and AaKr-h1. SIGNIFICANCE: Female Aedes aegypti mosquitoes are known to vector many disease agents, including Zika virus, dengue virus chikungunya virus, and Mayaro and yellow fever virus. In the present study, we developed an efficient method to prepare Ae. aegypti Met and Kr-h1, which are typically difficult to produce and purify, using a mosquito cell line expression system. A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approaches were utilized to map the phosphorylation profiles of the isolated proteins. We then monitored the changes induced by JH activation in the phosphorylation profiles to check if the JH modulates post-translation modification of its key transcription factors. We found that the JH induced alterations in the phosphorylation profiles of the multiple residues of AaMet. In contrast, activation of the JH signaling pathway was accompanied by dephosphorylation of AaKr-h1 at phosphoserine-694, increasing its transcriptional activity. In addition, S694 of AaKr-h1 was located in the RMSSVIQYA motif highly conserved in orthologous proteins from other insect species. These results can help us further understand how JH modulates its key transcription factors and provide a basis for the development of novel insect control strategies.
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http://dx.doi.org/10.1016/j.jprot.2021.104257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8218339PMC
June 2021

Stage-specific action of juvenile hormone analogs.

J Pestic Sci 2021 Feb;46(1):16-22

Department of Entomology, University of Kentucky, College of Agriculture, Food and Environment, Lexington, KY, USA.

The discovery of juvenile hormones (JH) and their synthetic analogs (JHA) generated excitement and hope that these compounds will replace first- and second-generation insecticides that have not so desirable environmental and human safety profiles. However, JHAs used commercially during the past four decades did not meet these expectations. The recent availability of advanced molecular and histological methods and the discovery of key players involved in JH action provided some insights into the functioning of JHA in a stage and species-specific manner. In this review, we will summarize recent findings and stage-specific action of JHA, focusing on three commercially used JHA, methoprene, hydroprene and pyriproxyfen and economically important pests, the red flour beetle, , and the tobacco budworm, , and disease vector, the yellow fever mosquito, .
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http://dx.doi.org/10.1584/jpestics.D20-084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7953018PMC
February 2021

Changes in both trans- and cis-regulatory elements mediate insecticide resistance in a lepidopteron pest, Spodoptera exigua.

PLoS Genet 2021 03 9;17(3):e1009403. Epub 2021 Mar 9.

Department of Entomology, University of Kentucky, Lexington, Kentucky, United States of America.

The evolution of insect resistance to insecticides is frequently associated with overexpression of one or more cytochrome P450 enzyme genes. Although overexpression of CYP450 genes is a well-known mechanism of insecticide resistance, the underlying regulatory mechanisms are poorly understood. Here we uncovered the mechanisms of overexpression of the P450 gene, CYP321A8 in a major pest insect, Spodoptera exigua that is resistant to multiple insecticides. CYP321A8 confers resistance to organophosphate (chlorpyrifos) and pyrethroid (cypermethrin and deltamethrin) insecticides in this insect. Constitutive upregulation of transcription factors CncC/Maf are partially responsible for upregulated expression of CYP321A8 in the resistant strain. Reporter gene assays and site-directed mutagenesis analyses demonstrated that CncC/Maf enhanced the expression of CYP321A8 by binding to specific sites in the promoter. Additional cis-regulatory elements resulting from a mutation in the CYP321A8 promoter in the resistant strain facilitates the binding of the orphan nuclear receptor, Knirps, and enhances the promoter activity. These results demonstrate that two independent mechanisms; overexpression of transcription factors and mutations in the promoter region resulting in a new cis-regulatory element that facilitates binding of the orphan nuclear receptor are involved in overexpression of CYP321A8 in insecticide-resistant S. exigua.
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http://dx.doi.org/10.1371/journal.pgen.1009403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7978377PMC
March 2021

Intragenic DNA methylation regulates insect gene expression and reproduction through the MBD/Tip60 complex.

iScience 2021 Feb 7;24(2):102040. Epub 2021 Jan 7.

Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology, School of Life Sciences, South China Normal University, Guangzhou 510631, China.

DNA methylation is an important epigenetic modification. However, the regulations and functions of insect intragenic DNA methylation remain unknown. Here, we demonstrate that a regulatory mechanism involving intragenic DNA methylation controls ovarian and embryonic developmental processes in . In DNA methylation is found near the transcription start site (TSS) of ovarian genes. By promoter activity analysis, we observed that 5' UTR methylation enhances gene expression. Moreover, methyl-DNA-binding domain protein 2/3 (MBD2/3) binds to the intragenic methyl-CpG fragment and recruits acetyltransferase Tip60 to promote histone H3K27 acetylation and gene expression. Additionally, genome-wide analyses showed that the peak of H3K27 acetylation appears near the TSS of methyl-modified genes, and DNA methylation is enriched in genes involved in protein synthesis in the ovary, with MBD2/3 knockdown resulting in decreased fecundity. These data uncover a mechanism of gene body methylation for regulating insect gene expression and reproduction.
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http://dx.doi.org/10.1016/j.isci.2021.102040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7820559PMC
February 2021

Expanding the Toolkit for Genome Editing in a Disease Vector, Transgenic Lines Expressing Cas9 and Single Guide RNA Induce Efficient Mutagenesis.

CRISPR J 2021 Jan 15. Epub 2021 Jan 15.

Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, Kentucky, USA.

CRISPR-Cas9 mediated genome editing methods are being used for the analysis of gene function. However, it is hard to identify gene knockout mutants for genes whose knockout does not cause distinct phenotypes. To overcome this issue in the disease vector, , a transgenic Cas9/single guide RNA (sgRNA) method, was used to knock out the eye marker gene, (), and the juvenile hormone receptor, (). PiggyBac transformation vectors were prepared to express sgRNAs targeting and under the control of the U6 promoter. Transgenic expressing -sgRNA or -sgRNA under the control of the U6 promoter and enhanced green fluorescent protein (eGFP) under the control of the hr5ie1 promoter were produced. The U6-sgRNA adults were mated with AAEL010097-Cas9 adults. The progeny were screened, and the insects expressing eGFP and DsRed were selected and evaluated for mutations in target genes. About 77% and 78% of the progeny that were positive for both eGFP and DsRed in -sgRNA and -sgRNA groups, respectively, showed mutations in their target genes.
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http://dx.doi.org/10.1089/crispr.2020.0052DOI Listing
January 2021

Off-target effects of RNAi correlate with the mismatch rate between dsRNA and non-target mRNA.

RNA Biol 2021 Jan 4:1-13. Epub 2021 Jan 4.

The Key Laboratory of Monitoring and Management of Plant Diseases and Insects/Department of Entomology, College of Plant Protection, Nanjing Agricultural University, Nanjing, China.

RNAi is a potent technique for the knockdown of target genes. However, its potential off-target effects limit the widespread applications in both reverse genetic analysis and genetic manipulation. Previous efforts have uncovered rules underlying specificity of siRNA-based silencing, which has broad applications in humans, but the basis for specificity of dsRNAs, which are better suited for use as insecticides, is poorly understood. Here, we investigated the rules governing dsRNA specificity. Mutational analyses showed that dsRNAs with >80% sequence identity with target genes triggered RNAi efficiently. dsRNAs with ≥16 bp segments of perfectly matched sequence or >26 bp segments of almost perfectly matched sequence with one or two mismatches scarcely distributed (single mismatches inserted between ≥5 bp matching segments or mismatched couplets inserted between ≥8 bp matching segments) also able to trigger RNAi. Using these parameters to predict off-target risk, dsRNAs can be designed to optimize specificity and efficiency, paving the way to the widespread, rational application of RNAi in pest control.
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http://dx.doi.org/10.1080/15476286.2020.1868680DOI Listing
January 2021

systemic RNA interference defective protein 1 enhances RNAi efficiency in a lepidopteran insect, the fall armyworm, in a tissue-specific manner.

RNA Biol 2020 Nov 9:1-9. Epub 2020 Nov 9.

Department of Botany, Kongunadu Arts and Science College (Autonomous), Bharathiar University, Coimbatore, India.

RNA interference (RNAi) is an important tool for gene function studies in insects, especially in non-model insects. This technology is also being developed for pest control. However, variable RNAi efficiency among insects is limiting its use in insects. Systemic RNAi in requires systemic RNA interference defective protein 1 (. The expression of Ce in insect cell lines was shown to improve RNAi. However, the mechanisms through which this double-stranded RNA (dsRNA) transporter improves RNAi efficiency in insects is not known. We stably expressed in two cell lines, Sf9 and Sf17 cells derived from ovary and midgut, respectively. Expression of enhanced RNAi efficiency in ovarian Sf9 cells, but not in midgut Sf17 cells. Reduced accumulation of dsRNA in late endosomes and successful processing dsRNA to siRNA contribute to enhanced RNAi efficiency in Sf9 cells. Transgenic expressing were produced and tested for RNAi efficiency. RNAi efficiency enhancement due to expression showed tissue specificity. Compared to RNAi efficiency in wild-type expressing transgenic showed a significant improvement of RNAi in tissues such as Verson's glands. In contrast, no improvement in RNAi was observed in tissues such as midgut. The cell-type specific and tissue-specific enhancement of RNAi efficiency by in provides valuable information for improving RNAi in insects such as those belonging to order Lepidoptera where RNAi is variable and inefficient.
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http://dx.doi.org/10.1080/15476286.2020.1842632DOI Listing
November 2020

Insulin/IGF signaling and TORC1 promote vitellogenesis via inducing juvenile hormone biosynthesis in the American cockroach.

Development 2020 10 23;147(20). Epub 2020 Oct 23.

Guangdong Provincial Key Laboratory of Insect Developmental Biology and Applied Technology, Institute of Insect Science and Technology and School of Life Sciences, South China Normal University, Guangzhou 510631, China

Vitellogenesis, including vitellogenin (Vg) production in the fat body and Vg uptake by maturing oocytes, is of great importance for the successful reproduction of adult females. The endocrinal and nutritional regulation of vitellogenesis differs distinctly in insects. Here, the complex crosstalk between juvenile hormone (JH) and the two nutrient sensors insulin/IGF signaling (IIS) and target of rapamycin complex1 (TORC1), was investigated to elucidate the molecular mechanisms of vitellogenesis regulation in the American cockroach, Our data showed that a block of JH biosynthesis or JH action arrested vitellogenesis, in part by inhibiting the expression of (), a key transcription factor gene involved in the sex determination cascade. Depletion of IIS or TORC1 blocked both JH biosynthesis and vitellogenesis. Importantly, the JH analog methoprene, but not bovine insulin (to restore IIS) and amino acids (to restore TORC1 activity), restored vitellogenesis in the neck-ligated (IIS-, TORC1- and JH-deficient) and rapamycin-treated (TORC1- and JH-deficient) cockroaches. Combining classic physiology with modern molecular techniques, we have demonstrated that IIS and TORC1 promote vitellogenesis, mainly via inducing JH biosynthesis in the American cockroach.
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http://dx.doi.org/10.1242/dev.188805DOI Listing
October 2020

Epigenetic regulation of post-embryonic development.

Curr Opin Insect Sci 2021 02 14;43:63-69. Epub 2020 Oct 14.

Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, S225 Ag. Science N, Lexington, KY 40546, United States. Electronic address:

Modifications to DNA and core histones influence chromatin organization and expression of the genome. DNA methylation plays a significant role in the regulation of multiple biological processes that regulate behavior and caste differentiation in social insects. Histone modifications play significant roles in the regulation of development and reproduction in other insects. Genes coding for acetyltransferases, deacetylases, methyltransferases, and demethylases that modify core histones have been identified in genomes of multiple insects. Studies on the function and mechanisms of action of some of these enzymes uncovered their contribution to post-embryonic development. The results from studies on epigenetic modifiers could help in the identification of inhibitors of epigenetic modifiers that could be developed to control pests and disease vectors.
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http://dx.doi.org/10.1016/j.cois.2020.09.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8044252PMC
February 2021

Identification and characterization of highly active promoters from the fall armyworm, Spodoptera frugiperda.

Insect Biochem Mol Biol 2020 11 19;126:103455. Epub 2020 Aug 19.

Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, 40546, United States. Electronic address:

The cell lines derived from the fall armyworm (FAW), Spodoptera frugiperda, have been widely used for production of recombinant proteins for applications in both basic research and applications in medicine and agriculture. Promoters from the nucleopolyhedrovirus (NPV) are commonly used in these expression systems. These promoters have some limitations, which may be overcome by using promoters of genes from S. frugiperda. However, information on these promoters is not available. We identified several highly expressed genes from the transcriptomes of S. frugiperda midgut, fat body, epidermis, ovarian cell line (Sf9), and a midgut cell line (Sf17). The activity of potential promoters of 21 highly expressed genes was evaluated in Sf9 and Sf17 cells. Two of these promoters, SfHSC70-P1780 and SfPub-P2009, showed higher activity than commonly used hr5/ie1 (baculovirus enhancer element, hr5 and immediate early gene 1, ie1) promoter. Interestingly, the activity of these two promoters increased after adding hr5 enhancer element. The hr5/SfPub-P2009 promoter performance was evaluated by expressing an exogenous P450 protein in Sf9 cells using a plasmid-based expression system. The activity of this promoter was also evaluated in the FAW by expressing green fluorescence protein using the baculovirus expression system. In both cases, the hr5/SfPub-P2009 promoter performed better than the commonly used hr5/ie1 promoter. These strong endogenous promoters will be useful for studies in S. frugiperda and other lepidopteran insects for multiple applications, including protein expression, genome editing, and transgenic insects.
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http://dx.doi.org/10.1016/j.ibmb.2020.103455DOI Listing
November 2020

Histone Deacetylase 11 Knockdown Blocks Larval Development and Metamorphosis in the Red Flour Beetle, .

Front Genet 2020 3;11:683. Epub 2020 Jul 3.

Department of Entomology, University of Kentucky, Lexington, KY, United States.

Post-translational modifications (PTM) such as methylation, acetylation, phosphorylation, and ubiquitination of histones and other proteins regulate expression of genes. The acetylation levels of these proteins are determined by the balance of expression of histone acetyltransferase (HATs) and histone deacetylases (HDACs). We recently reported that class I HDACs (HDAC1 and HDAC3) play important roles in juvenile hormone (JH) suppression of metamorphosis in the red flour beetle, . Here, we report on the function of a single class IV HDAC member, HDAC11. Injection of dsRNA targeting gene into newly molted last instar larvae induced knockdown of the target gene and arrested larval development and prevented metamorphosis into the pupal stage. Dark melanized areas were detected in larvae that showed developmental arrest and mortality. Developmental expression studies showed an increase in HDAC11 mRNA levels beginning at the end of the penultimate larval stage. These higher levels were maintained during the final instar larval and pupal stages. A JH analog, hydroprene, suppressed expression in the larvae. Sequencing of RNA isolated from control and dsHDAC11 injected larvae identified several differentially expressed genes, including those involved in JH action, ecdysone response, and melanization. The acetylation levels of core histones showed an increase in TcA cells exposed to dsHDAC11. Also, an increase in histone H3 acetylation, specifically H3K9, H3K18 and H3K27, were detected in HDAC11 knockdown larvae. These studies report the function of HDAC11 in insects other than for the first time and show that HDAC11 influences the acetylation levels of histones and expression of multiple genes involved in larval development.
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http://dx.doi.org/10.3389/fgene.2020.00683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7348043PMC
July 2020

Histone deacetylase 3 is required for development and metamorphosis in the red flour beetle, Tribolium castaneum.

BMC Genomics 2020 Jun 22;21(1):420. Epub 2020 Jun 22.

Department of Entomology, University of Kentucky, Lexington, KY, 40546, USA.

Background: Hormones are chemical communication signaling molecules released into the body fluids to stimulate target cells of multicellular organisms. We recently showed that histone deacetylase 1 (HDAC1) plays an important role in juvenile hormone (JH) suppression of metamorphosis in the red flour beetle, Tribolium castaneum. Here, we investigated the function of another class I HDAC member, HDAC3, and show that it is required for the normal development of T. castaneum.

Results: RNA interference-mediated knockdown of the HDAC3 gene affected development resulting in abnormally folded wings in pupae and adults. JH analog, hydroprene, suppressed the expression of HDAC3 in T. castaneum larvae. The knockdown of HDAC3 during the final instar larval stage resulted in an increase in the expression of genes coding for proteins involved in JH action. Sequencing of RNA isolated from larvae injected with dsRNA targeting malE (E. coli gene, control) or HDAC3 followed by differential gene expression analysis identified 148 and 741 differentially expressed genes based on the P-value < 0.01 and four-fold difference, and the P-value < 0.05 and two-fold difference, respectively. Several genes, including those coding for myosin-I heavy chain (Myosin 22), Shaven, and nuclear receptor corepressor 1 were identified as differentially expressed genes in HDAC3 knockdown larvae. An increase in histone H3 acetylation, specifically H3K9, H3K18, and H3K27, was detected in HDAC3 knockdown insects.

Conclusion: Overall, these data suggest that HDAC3 affects the acetylation levels of histones and influences the expression of genes coding for proteins involved in the regulation of growth, development, and metamorphosis.
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http://dx.doi.org/10.1186/s12864-020-06840-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7310253PMC
June 2020

Xenobiotic transcription factors CncC and maf regulate expression of CYP321A16 and CYP332A1 that mediate chlorpyrifos resistance in Spodoptera exigua.

J Hazard Mater 2020 11 26;398:122971. Epub 2020 May 26.

College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China. Electronic address:

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.
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http://dx.doi.org/10.1016/j.jhazmat.2020.122971DOI Listing
November 2020

Transport of orally delivered dsRNA in southern green stink bug, Nezara viridula.

Arch Insect Biochem Physiol 2020 Aug 22;104(4):e21692. Epub 2020 May 22.

Department of Entomology, University of Kentucky, Lexington, Kentucky.

The southern green stink bug (SGSB, Nezara viridula) is an emerging polyphagous pest in many regions of the world. RNA interference (RNAi) is a valuable method for understanding gene function and holds great potential for pest management. However, RNAi efficiency is variable among insects and the differences in transport of double-stranded RNA (dsRNA) are one of the major factors that contribute to this variability. In this study, Cy3 labeled dsRNA was used to track the transport of dsRNA in SGSB tissues. Cy3_dsRNA was detected in the hemocytes, fat body (FB), epidermis, and midgut tissues at 24-72 hr after injection. Orally delivered Cy3_dsRNA or Cypher-5E labeled dsRNA was mostly detected in the midgut and a few signals were detected in parts of the FB and epidermis. Both injected and fed Cy3_dsRNA showed stronger signals in SGSB tissues when compared to Cy3_siRNA (small interfering RNA) or Cy3_shRNA (short hairpin RNA). dsRNA targeting the gene for a vacuolar-sorting protein, SNF7, induced higher knockdown of the target gene and greater SGSB mortality compared to siRNA or shRNA targeting this gene. P-labeled dsRNA injected into SGSB was processed into siRNA, but fed P-labeled dsRNA was not efficiently processed into siRNA. These data suggest that transport of orally delivered dsRNA across the midgut epithelium is not efficient in SGSB which may contribute to variable RNAi efficiency. Targeting genes expressed in the midgut rather than other tissues and using dsRNA instead of siRNA or shRNA would be more effective for RNAi-mediated control of this pest.
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http://dx.doi.org/10.1002/arch.21692DOI Listing
August 2020

RNAi in Sf9 Cells via Nanomaterial Mediated Delivery of dsRNA: A Comparison of Poly-l-arginine Polyplexes and Poly-l-arginine-Functionalized Au Nanoparticles.

ACS Appl Mater Interfaces 2020 Jun 28;12(23):25645-25657. Epub 2020 May 28.

This work focused on the delivery of dsRNA either complexed with poly-l-arginine (PLR-polyplex) or loaded onto poly-l-arginine functionalized 20 nm Au nanoparticles (PLR/Au NPs). RNA interference (RNAi) of a gene expressed in Sf9 stable cell line () was used as a model system. The binding affinity of dsRNA with two modes of functionalization of Au NPs was investigated: the electrostatic binding of PLR on citrate stabilized Au NPs (e-PLR/Au NPs) via the layer-by-layer method or the covalent-linking reaction of the polymer with NHS groups on the Au NPs surface (c-PLR/Au NPs) with excess groups quenched with either hydroxylamine (c-PLR/Au/Hyd NPs) or bovine serum albumin (c-PLR/Au/BSA NPs). The formation of PLR-polyplex particles resulting from the complexation of PLR and dsRNA were revealed by transmission electron microscope (TEM), scanning transmission electron microscope (STEM), and elemental mapping by energy dispersive X-ray spectroscopy (EDS). gene knockdown was evaluated after exposure of Sf9 cells for 72 h to 600 ng of dsRNA. Gene knockdown (GK) was found to be more efficient by exposing Sf9 cells to nanoscaled (size <100 nm) PLR-polyplex (58% GK), in contrast to microscaled (size >1 μm) PLR-polyplex (20% GK) or covalent PLR/Au/Hyd (7% GK) particles. The replacement of hydroxylamine by bovine serum albumin as ligand has significantly enhanced the efficiency of GK (31% GK). Investigation of endosomal escape, a key physiological barrier for dsRNA delivery, with CypHer5E labeled dsRNA showed the good ability for the dsRNA conjugated to the different nanosystems to enter the cells compared to the unconjugated one. This study provides valuable information concerning the required properties of materials used for dsRNA delivery in lepidopteran cells such as nanoparticle size, stability in the cell culture media, the polymer molecular weight and binding strength to the nanoparticle, and the nature of ligands on the surface.
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http://dx.doi.org/10.1021/acsami.0c06234DOI Listing
June 2020

The effect of E93 knockdown on female reproduction in the red flour beetle, Tribolium castaneum.

Arch Insect Biochem Physiol 2020 Aug 11;104(4):e21688. Epub 2020 May 11.

Department of Entomology, University of Kentucky, Lexington, Kentucky.

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.
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http://dx.doi.org/10.1002/arch.21688DOI Listing
August 2020

Development of RNAi methods to control the harlequin bug, Murgantia histrionica.

Arch Insect Biochem Physiol 2020 Aug 11;104(4):e21690. Epub 2020 May 11.

Department of Entomology, University of Kentucky, Lexington, Kentucky.

The harlequin bug (HB), Murgantia histrionica, is a major pest of cabbage family plants throughout its range in the United States. RNA interference (RNAi) is a posttranscriptional gene silencing mechanism that is showing promise as a biopesticide due to the ability to target species-specific genes necessary for growth and/or survival with synthetic double-stranded RNA (dsRNA). In the present study, dsRNA stability assays revealed that nucleases present in the saliva of harlequin bugs did not rapidly degrade dsRNA. We tracked the movement and localization of radioactively labeled dsRNA in both mustard plant seedlings and harlequin bug nymphs that fed on treated host plants. Movement of P-labeled-dsRNA from soil to plant and plant to insect was detected. The efficacy of RNAi in inducing mortality in harlequin bug adults and nymphs injected or fed with dsRNA targeting inhibitor of apoptosis (IAP), ATPase N2B (ATPase), serine/threonine-protein phosphatase PP1-β catalytic subunit (PP1), signal recognition particle 54 kDa protein (SRP), and G protein-coupled receptor 161-like (GPCR) genes was evaluated. Injection of dsRNA targeting candidate genes into adults caused between 40% and 75% mortality and induced significant knockdown of target gene expression. Feeding dsRNA targeting the IAP gene to nymphs by plant-mediated and droplet feeding methods induced knockdown of the target gene and caused 40-55% mortality. These findings suggest that RNAi may be a viable approach for managing this pest.
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http://dx.doi.org/10.1002/arch.21690DOI Listing
August 2020

CREB-binding protein regulates metamorphosis and compound eye development in the yellow fever mosquito, Aedes aegypti.

Biochim Biophys Acta Gene Regul Mech 2020 08 7;1863(8):194576. Epub 2020 May 7.

Department of Entomology, University of Kentucky, Lexington, KY 40546, USA. Electronic address:

Juvenile hormones (JH) and ecdysone coordinately regulate metamorphosis in Aedes aegypti. We studied the function of an epigenetic regulator and multifunctional transactivator, CREB binding protein (CBP) in A. aegypti. RNAi-mediated knockdown of CBP in Ae. aegypti larvae resulted in suppression of JH primary response gene, Krüppel-homolog 1 (Kr-h1), and induction of primary ecdysone response gene, E93, resulting in multiple effects including early metamorphosis, larval-pupal intermediate formation, mortality and inhibition of compound eye development. RNA sequencing identified hundreds of genes, including JH and ecdysone response genes regulated by CBP. In the presence of JH, CBP upregulates Kr-h1 by acetylating core histones at the Kr-h1 promoter and facilitating the recruitment of JH receptor and other proteins. CBP suppresses metamorphosis regulators, EcR-A, USP-A, BR-C, and E93 through the upregulation of Kr-h1 and E75A. CBP regulates the expression of core eye specification genes including those involved in TGF-β and EGFR signaling. These studies demonstrate that CBP is an essential player in JH and 20E action and regulates metamorphosis and compound eye development in Ae. aegypti.
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http://dx.doi.org/10.1016/j.bbagrm.2020.194576DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7339023PMC
August 2020

Orally delivered dsRNA induces knockdown of target genes and mortality in the Asian long-horned beetle, Anoplophora glabripennis.

Arch Insect Biochem Physiol 2020 Aug 15;104(4):e21679. Epub 2020 Apr 15.

Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, Kentucky.

The Asian long-horned beetle (ALB) Anoplophora glabripennis is a serious invasive forest pest in several countries, including the United States. Methods available to manage or eradicate this pest are extremely limited, but RNA interference (RNAi) technology is a potentially effective method to control ALB. In this study, we used sucrose feeding bioassay for oral delivery of double-strand RNA (dsRNA) to ALB larvae. P-labeled dsRNA orally delivered to ALB larvae using the sucrose droplet feeding method was processed to small interfering RNA. Feeding neonate larvae with dsRNA targeting genes coding for the inhibitor of apoptosis (IAP), vacuolar sorting protein SNF7 (SNF7), and snakeskin (SSK) induced knockdown of target genes and mortality. Feeding 2 µg of dsRNA per day for 3 days did not induce a significant decrease in the expression of target genes or mortality. However, feeding 5 or 10 µg of dsRNA per day for 3 days induced a significant decrease in the expression of target genes and 50-90% mortality. Interestingly, feeding 2.5 µg each of dsIAP plus dsSNF7, dsIAP plus dsSSK, or dsSNF7 plus dsSSK per day for 3 days induced a significant decrease in the expression of both target genes and approximately 80% mortality. Our findings demonstrate that orally delivered dsRNA induces target gene knockdown and mortality in ALB neonate larvae and RNAi technology may have the potential for effective ALB control.
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http://dx.doi.org/10.1002/arch.21679DOI Listing
August 2020

Lipids help double-stranded RNA in endosomal escape and improve RNA interference in the fall armyworm, Spodoptera frugiperda.

Arch Insect Biochem Physiol 2020 Aug 15;104(4):e21678. Epub 2020 Apr 15.

Department of Entomology, University of Kentucky, Lexington, Kentucky, 40546, USA.

RNA interference (RNAi) is a valuable method for understanding the gene function and holds great potential for insect pest management. While RNAi is efficient and systemic in coleopteran insects, RNAi is inefficient in lepidopteran insects. In this study, we explored the possibility of improving RNAi in the fall armyworm (FAW), Spodoptera frugiperda cells by formulating dsRNA with Cellfectin II (CFII) transfection reagent. The CFII formulated dsRNA was protected from degradation by endonucleases present in Sf9 cells conditioned medium, hemolymph and midgut lumen contents collected from the FAW larvae. Lipid formulated dsRNA also showed reduced accumulation in the endosomes of Sf9 cells and FAW tissues. Exposing Sf9 cells and tissues to CFII formulated dsRNA caused a significant knockdown of endogenous genes. CFII formulated dsIAP fed to FAW larvae induced knockdown of iap gene, growth retardation and mortality. Processing of dsRNA into siRNA was detected in Sf9 cells and Spodoptera frugiperda larvae treated with CFII conjugated P-UTP labeled dsGFP. Overall, the present study concluded that delivering dsRNA formulated with CFII transfection reagent helps dsRNA escapes from the endosomal accumulation and improved RNAi efficiency in the FAW cells and tissues.
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http://dx.doi.org/10.1002/arch.21678DOI Listing
August 2020

CncC/Maf-mediated xenobiotic response pathway in insects.

Arch Insect Biochem Physiol 2020 Jun 12;104(2):e21674. Epub 2020 Apr 12.

Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, Kentucky.

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.
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http://dx.doi.org/10.1002/arch.21674DOI Listing
June 2020

Genome editing in the fall armyworm, Spodoptera frugiperda: Multiple sgRNA/Cas9 method for identification of knockouts in one generation.

Insect Biochem Mol Biol 2020 07 7;122:103373. Epub 2020 Apr 7.

Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, 40546, USA. Electronic address:

The CRISPR/Cas9 system is an efficient genome editing method that can be used in functional genomics research. The fall armyworm, Spodoptera frugiperda, is a serious agricultural pest that has spread over most of the world. However, very little information is available on functional genomics for this insect. We performed CRISPR/Cas9-mediated site-specific mutagenesis of three target genes: two marker genes [Biogenesis of lysosome-related organelles complex 1 subunit 2 (BLOS2) and tryptophan 2, 3-dioxygenase (TO)], and a developmental gene, E93 (a key ecdysone-induced transcription factor that promotes adult development). The knockouts (KO) of BLOS2, TO and E93 induced translucent mosaic integument, olive eye color, and larval-pupal intermediate phenotypes, respectively. Sequencing RNA isolated from wild-type and E93 KO insects showed that E93 promotes adult development by influencing the expression of the genes coding for transcription factor, Krüppel homolog 1, the pupal specifier, Broad-Complex, serine proteases, and heat shock proteins. Often, gene-edited insects display mosaicism in which only a fraction of the cells are edited as intended, and establishing a homozygous line is both costly and time-consuming. To overcome these limitations, a method to completely KO the target gene in S. frugiperda by injecting the Cas9 protein and multiple sgRNAs targeting one exon of the E93 gene into embryos was developed. Ten percent of the G0 larvae exhibited larval-pupal intermediates. The mutations were confirmed by T7E1 assay, and the mutation frequency was determined as >80%. Complete KO of the E93 gene was achieved in one generation using the multiple sgRNA method, demonstrating a powerful approach to improve genome editing in lepidopteran and other non-model insects.
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http://dx.doi.org/10.1016/j.ibmb.2020.103373DOI Listing
July 2020

Identification and functional analysis of promoters of heat-shock genes from the fall armyworm, Spodoptera frugiperda.

Sci Rep 2020 02 11;10(1):2363. Epub 2020 Feb 11.

Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY, 40546, United States of America.

The functional information on heat-shock proteins (Hsp) and heat-shock promoters from an important agricultural insect pest, Spodoptera frugiperda, is still lacking. We conducted a genome-wide identification of Hsp genes and identified a total of 21 genes belonging to four major insect Hsp families (small heat-shock proteins, Hsp60, Hsp70, and Hsp90) in S. frugiperda. Expression of most of S. frugiperda (SfHsp) genes could be detected in Sf9 cells, embryos and larval tissues of S. frugiperda. The heat-inducible activity of heat-shock promoters from several SfHsp genes was tested in Sf9 cells and embryos. The promoter of SfHsp70D showed the high constitutive activity in cell line and embryos, while the activity of SfHsp20.15 and SfHsp20.71 promoters was most dramatically induced in Sf9 cells and embryos. In embryos, the heat-induced activity of SfHsp20.71 and SfHsp70D promoters outperformed commercially used ie1 and ie2 promoters. The heat-induced activity of SfHsp70D and SfHsp19.07 promoters were more robust than ie2 promoter in Sf9 cells. These SfHsp promoters with high basal activity or with heat-induced activity from low basal activity, could be used in S. frugiperda or other lepidopteran insects for many applications including transgenesis and genome editing.
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http://dx.doi.org/10.1038/s41598-020-59197-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012861PMC
February 2020

Identification and characterization of multiple dsRNases from a lepidopteran insect, the tobacco cutworm, Spodoptera litura (Lepidoptera: Noctuidae).

Pestic Biochem Physiol 2020 Jan 23;162:86-95. Epub 2019 Oct 23.

The Agricultural Ministry Key Laboratory of Monitoring and Management of Plant Diseases and Insects, Department of Entomology, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China. Electronic address:

RNA interference (RNAi) efficiency varies among insects. RNAi is highly efficient and systemic in coleopteran insects but quite variable and inefficient in lepidopteran insects. Degradation of double-stranded RNA (dsRNA) by double-stranded ribonucleases (dsRNases) is thought to contribute to the variability in RNAi efficiency observed among insects. One or two dsRNases involved in dsRNA digestion have been identified in a few insects. To understand the contribution of dsRNases to reduced RNAi efficiency in lepidopteran insects, we searched the transcriptome of Spodoptera litura and identified six genes coding for DNA/RNA non-specific endonucleases. Phylogenetic analysis revealed the evolutionary expansion of dsRNase genes in insects. The mRNA levels of three midgut-specific dsRNases increased during the larval stage, and the highest dsRNA-degrading activity was detected in third-instar larvae. Proteins produced via the expression of three midgut-specific dsRNases, and the widely expressed dsRNase3, in a baculovirus system showed dsRNase activity for four out of five dsRNases tested. In addition, the increase in dsRNA-degrading activity and upregulation of dsRNase1 and 2 in larvae fed on cabbage leaves suggests that the diet of S. litura can influence dsRNase expression, dsRNA stability, and thus probably RNAi efficiency. This is the first report that multiple dsRNases function together in an RNAi-recalcitrant insect. The data included in this paper suggest that multiple dsRNases coded by the S. litura genome might contribute to the lower and variable RNAi efficiency reported in this and other lepidopteran insects.
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http://dx.doi.org/10.1016/j.pestbp.2019.09.011DOI Listing
January 2020

Mechanisms, Applications, and Challenges of Insect RNA Interference.

Annu Rev Entomol 2020 01 14;65:293-311. Epub 2019 Oct 14.

Department of Entomology, University of Kentucky, Lexington, Kentucky 40546, USA; email:

The RNA interference (RNAi) triggered by short/small interfering RNA (siRNA) was discovered in nematodes and found to function in most living organisms. RNAi has been widely used as a research tool to study gene functions and has shown great potential for the development of novel pest management strategies. RNAi is highly efficient and systemic in coleopterans but highly variable or inefficient in many other insects. Differences in double-stranded RNA (dsRNA) degradation, cellular uptake, inter- and intracellular transports, processing of dsRNA to siRNA, and RNA-induced silencing complex formation influence RNAi efficiency. The basic dsRNA delivery methods include microinjection, feeding, and soaking. To improve dsRNA delivery, various new technologies, including cationic liposome-assisted, nanoparticle-enabled, symbiont-mediated, and plant-mediated deliveries, have been developed. Major challenges to widespread use of RNAi in insect pest management include variable RNAi efficiency among insects, lack of reliable dsRNA delivery methods, off-target and nontarget effects, and potential development of resistance in insect populations.
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http://dx.doi.org/10.1146/annurev-ento-011019-025224DOI Listing
January 2020

Knockout of juvenile hormone receptor, Methoprene-tolerant, induces black larval phenotype in the yellow fever mosquito, .

Proc Natl Acad Sci U S A 2019 10 30;116(43):21501-21507. Epub 2019 Sep 30.

Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, KY 40546;

The yellow fever mosquito, , 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 () gene coding for a JH receptor. The knockout larvae exhibited a black larval phenotype during the L3 (third instar larvae) and L4 (fourth instar larvae) stages and died before pupation. However, 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 , 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 mutants. These data suggest that, during the L3 and L4 stages, 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 .
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http://dx.doi.org/10.1073/pnas.1905729116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6815201PMC
October 2019

Histone deacetylase 1 suppresses Krüppel homolog 1 gene expression and influences juvenile hormone action in .

Proc Natl Acad Sci U S A 2019 09 22;116(36):17759-17764. Epub 2019 Aug 22.

Department of Entomology, University of Kentucky, Lexington, KY 40546

Posttranslational modifications, including acetylation and deacetylation of histones and other proteins, modulate hormone action. In TcA cells, Trichostatin A, a histone deacetylase (HDAC) inhibitor, mimics juvenile hormone (JH) in inducing JH response genes (e.g., ), suggesting that HDACs may be involved in JH action. To test this hypothesis, we identified genes coding for HDACs in and studied their function. Knockdown of 12 genes showed variable phenotypes; the most severe phenotype was detected in insects injected with double-stranded RNA targeting (dsHDAC1). The dsHDAC1-injected insects showed arrested growth and development and eventually died. Application of JH analogs hydroprene to larvae and JH III to TcA cells suppressed 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 promoter region of cells exposed to JH III or dsHDAC1. Overexpression or knockdown of , , or both resulted in a decrease or increase in mRNA levels and its promoter activity, respectively. Overexpression of the JH receptor Methoprene tolerant () was unable to induce 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.
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http://dx.doi.org/10.1073/pnas.1909554116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6731676PMC
September 2019

Development of CS-TPP-dsRNA nanoparticles to enhance RNAi efficiency in the yellow fever mosquito, Aedes aegypti.

Sci Rep 2019 06 19;9(1):8775. Epub 2019 Jun 19.

Department of Entomology, University of Kentucky, Lexington, Kentucky, 40546, USA.

Mosquito-borne diseases are a major threat to human health and are responsible for millions of deaths globally each year. Vector control is one of the most important approaches used in reducing the incidence of these diseases. However, increasing mosquito resistance to chemical insecticides presents challenges to this approach. Therefore, new strategies are necessary to develop the next generation vector control methods. Because of the target specificity of dsRNA, RNAi-based control measures are an attractive alternative to current insecticides used to control disease vectors. In this study, Chitosan (CS) was cross-linked to sodium tripolyphosphate (TPP) to produce nano-sized polyelectrolyte complexes with dsRNA. CS-TPP-dsRNA nanoparticles were prepared by ionic gelation method. The encapsulation efficiency, protection of dsRNA from nucleases, cellular uptake, in vivo biodistribution, larval mortality and gene knockdown efficiency of CS-TPP-dsRNA nanoparticles were determined. The results showed that at a 5:1 weight ratio of CS-TPP to dsRNA, nanoparticles of less than 200 nm mean diameter and a positive surface charge were formed. Confocal microscopy revealed the distribution of the fed CS-TPP-dsRNA nanoparticles in midgut, fat body and epidermis of yellow fever mosquito, Aedes aegypti larvae. Bioassays showed significant mortality of larvae fed on CS-TPP-dsRNA nanoparticles. These assays also showed knockdown of a target gene in CS-TPP-dsRNA nanoparticle fed larvae. These data suggest that CS-TPP nanoparticles may be used for delivery of dsRNA to mosquito larvae.
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http://dx.doi.org/10.1038/s41598-019-45019-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6584730PMC
June 2019
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