Life Sciences Commons^™

Emerald Ash Borer Specific Gene Silencing Has No Effect On Non-Target Organisms, Flavia Pampolini, Lynne K. Rieske

Entomology Faculty Publications

The sequence complementarity of the RNA interference (RNAi) pathway allows for targeted suppression of genes essential for insect survival, and enables development of pest management strategies specific to a given species while reducing the likelihood of adversely impacting non-target organisms (NTOs). The feasibility of manipulating the RNAi pathway to cause mortality in the highly invasive emerald ash borer (EAB) has been demonstrated. Here the spectrum of activity of three double stranded RNAs (dsRNAs) targeting the genes hsp, shi, and sn-rnp in EAB was evaluated in model insects representing five functional guilds including herbivore, predator, detritivore, pollinator, parasitoid; …

Oral Ingestion Of Bacterially Expressed Dsrna Can Silence Genes And Cause Mortality In A Highly Invasive, Tree-Killing Pest, The Emerald Ash Borer, Ramya Shanivarsanthe Leelesh, Lynne K. Rieske

Entomology Faculty Publications

RNA interference (RNAi) is a naturally occurring process inhibiting gene expression, and recent advances in our understanding of the mechanism have allowed its development as a tool against insect pests. A major challenge for deployment in the field is the development of convenient and efficient methods for production of double stranded RNA (dsRNA). We assessed the potential for deploying bacterially produced dsRNA as a bio-pesticide against an invasive forest pest, the emerald ash borer (EAB). EAB feeds on the cambial tissue of ash trees (Fraxinus spp.), causing rapid death. EAB has killed millions of trees in North America since …

Identification And Characterization Of Epigenetic Regulators In The Yellow Fever Mosquito, Aedes Aegypti, Shankar C. R. R. Chereddy

Theses and Dissertations--Entomology

Due to the increased interactions between the sylvatic and urban cycles of Aedes aegypti life cycle, there has been an increase in the incidence of vector-borne diseases. The risk involved through the transmission of viruses like Dengue, Zika, Yellow-fever and Chikungunya calls for an effective control method for the deadly vector. The possibility of pesticide resistance and the costs involved in producing new pesticides have pushed Sterile Insect Technique (SIT) into prominence. However, current methods used to sterize insects for SIT are not highly efficient. This study tackles current issues in mosquito control by identifying and characterizing epigenetic regulators that …

Development Of Cs-Tpp-Dsrna Nanoparticles To Enhance Rnai Efficiency In The Yellow Fever Mosquito, Aedes Aegypti, Ramesh Kumar Dhandapani, Dhandapani Gurusamy, Jeffrey L. Howell, Subba Reddy Palli

Entomology Faculty Publications

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 …

Rna Interference And Validation Of Reference Genes For Gene Expression Analyses Using Qpcr In Southern Pine Beetle, Dendroctonus Frontalis, Bethany R. Kyre, Thais B. Rodrigues, Lynne K. Rieske

Entomology Faculty Publications

RNA interference (RNAi) is a highly specific gene-silencing mechanism that can cause rapid insect mortality when essential genes are targeted. RNAi is being developed as a tool for integrated pest management of some crop pests. Here we focus on an aggressive forest pest that kills extensive tracts of pine forests, the southern pine beetle (SPB), Dendroctonus frontalis. We sought to identify reference genes for quantitative real-time PCR (qPCR) and validate RNAi responses in SPB by mortality and gene silencing analysis. Using an adult beetle feeding bioassay for oral ingestion of dsRNA, we measured the expression and demonstrated knockdown of …

Molecular Evolutionary Trends And Feeding Ecology Diversification In The Hemiptera, Anchored By The Milkweed Bug Genome, Kristen A. Panfilio, Iris M. Vargas Jentzsch, Joshua B. Benoit, Deniz Erezyilmaz, Yuichiro Suzuki, Stefano Colella, Hugh M. Robertson, Monica F. Poelchau, Robert M. Waterhouse, Panagiotis Ioannidis, Matthew T. Weirauch, Daniel S. T. Hughes, Shwetha C. Murali, John H. Werren, Chris G. C. Jacobs, Elizabeth J. Duncan, David Armisén, Barbara M. I. Vreede, Patrice Baa-Puyoulet, Chloé S. Berger, Chun-Che Chang, Hsu Chao, Mei-Ju M. Chen, Yen-Ta Chen, Christopher P. Childers, Ariel D. Chipman, Andrew G. Cridge, Antonin J. J. Crumière, Peter K. Dearden, Elise M. Didion, Subba Reddy Palli, Jayendra Nath Shukla

Entomology Faculty Publications

Background: The Hemiptera (aphids, cicadas, and true bugs) are a key insect order, with high diversity for feeding ecology and excellent experimental tractability for molecular genetics. Building upon recent sequencing of hemipteran pests such as phloem-feeding aphids and blood-feeding bed bugs, we present the genome sequence and comparative analyses centered on the milkweed bug Oncopeltus fasciatus, a seed feeder of the family Lygaeidae.

Results: The 926-Mb Oncopeltus genome is well represented by the current assembly and official gene set. We use our genomic and RNA-seq data not only to characterize the protein-coding gene repertoire and perform isoform-specific RNAi, but …

Rna Sequencing, Selection Of Reference Genes And Demonstration Of Feeding Rnai In Thrips Tabaci (Lind.) (Thysanoptera: Thripidae), Satnam Singh, Mridula Gupta, Suneet Pandher, Gurmeet Kaur, Neha Goel, Pankaj Rathore, Subba Reddy Palli

Entomology Faculty Publications

Background: Thrips tabaci is a severe pest of onion and cotton. Due to lack of information on its genome or transcriptome, not much is known about this insect at the molecular level. To initiate molecular studies in this insect, RNA was sequenced; de novo transcriptome assembly and analysis were performed. The RNAseq data was used to identify reference and RNAi pathway genes in this insect. Additionally, feeding RNAi was demonstrated in T. tabaci for the first time.

Results: From the assembled transcriptome, 27,836 coding sequence (CDS) with an average size of 1236 bp per CDS were identified. About 85.4% of …

Identification Of Target Genes For Rnai-Mediated Control Of The Twospotted Spider Mite, June-Sun Yoon, Dipak K. Sahoo, Indu B. Maiti, Subba Reddy Palli

Entomology Faculty Publications

RNA interference (RNAi) is being developed for the management of pests that destroy crops. The twospotted Spider Mite (TSSM), Tetranychus urticae is a worldwide pest due to its unique physiological and behavioral characteristics including extraordinary ability to detoxify a wide range of pesticides and feed on many host plants. In this study, we conducted experiments to identify target genes that could be used for the development of RNAi-based methods to control TSSM. Leaf disc feeding assays revealed that knockdown in the expression genes coding for proteins involved in the biosynthesis and action of juvenile hormone (JH) and action of ecdysteroids …

Identification Of Highly Effective Target Genes For Rnai-Mediated Control Of Emerald Ash Borer, Agrilus Planipennis, Thais B. Rodrigues, Jian J. Duan, Subba Reddy Palli, Lynne K. Rieske

Entomology Faculty Publications

Recent study has shown that RNA interference (RNAi) is efficient in emerald ash borer (EAB), Agrilus planipennis, and that ingestion of double-stranded RNA (dsRNA) targeting specific genes causes gene silencing and mortality in neonates. Here, we report on the identification of highly effective target genes for RNAi-mediated control of EAB. We screened 13 candidate genes in neonate larvae and selected the most effective target genes for further investigation, including their effect on EAB adults and on a non-target organism, Tribolium castaneum. The two most efficient target genes selected, hsp (heat shock 70-kDa protein cognate 3) and shi (shibire), …

Improving Rnai In The Brown Marmorated Stink Bug: Identification Of Target Genes And Reference Genes For Rt-Qpcr, Kanakachari Mogilicherla, Jeffrey L. Howell, Subba Reddy Palli

Entomology Faculty Publications

The brown marmorated stink bug (BMSB) is native to Asia and recently invaded the USA. RNA interference (RNAi) is a gene silencing mechanism in which the introduction of double-stranded RNA (dsRNA) inhibits gene function by degrading target mRNA. In dsRNA stability assays, the dsRNases present in the hemolymph and salivary gland secretions of BMSB showed lower activity than those in the hemolymph of Heliothis virescens. We evaluated six housekeeping genes (18S rRNA, EF1-α, Actin, Ubiquitin, 60S RP and β-Tubulin) across dsRNA treatments (injection and feeding) in nymphs and adults of BMSB and identified …

Multiple Functions Of Creb-Binding Protein During Postembryonic Development: Identification Of Target Genes, Amit Roy, Smitha George, Subba Reddy Palli

Entomology Faculty Publications

Background: Juvenile hormones (JH) and ecdysteroids control postembryonic development in insects. They serve as valuable targets for pest management. Hence, understanding the molecular mechanisms of their action is of crucial importance. CREB-binding protein (CBP) is a universal transcriptional co-regulator. It controls the expression of several genes including those from hormone signaling pathways through co-activation of many transcription factors. However, the role of CBP during postembryonic development in insects is not well understood. Therefore, we have studied the role of CBP in postembryonic development in Tribolium, a model coleopteran insect.

Results: CBP is ubiquitously expressed in the red flour beetle, …

Comparative Analysis Of Double-Stranded Rna Degradation And Processing In Insects, Indrakant K. Singh, Satnam Singh, Kanakachari Mogilicherla, Jayendra Nath Shukla, Subba R. Palli

Entomology Faculty Publications

RNA interference (RNAi) based methods are being developed for pest management. A few products for control of coleopteran pests are expected to be commercialized soon. However, variability in RNAi efficiency among insects is preventing the widespread use of this technology. In this study, we conducted research to identify reasons for variability in RNAi efficiency among thirty-seven (37) insects belonging to five orders. Studies on double-stranded RNA (dsRNA) degradation by dsRNases and processing of labeled dsRNA to siRNA showed that both dsRNA degradation and processing are variable among insects belonging to different orders as well as among different insect species within …

Rna Interference In The Asian Longhorned Beetle: Identification Of Key Rnai Genes And Reference Genes For Rt-Qpcr, Thais B. Rodrigues, Ramesh Kumar Dhandapani, Jian J. Duan, Subba Reddy Palli

Entomology Faculty Publications

Asian Longhorned Beetle (ALB) Anoplophora glabripennis is a serious invasive forest pest in several countries including the United States, Canada, and Europe. RNA interference (RNAi) technology is being developed as a novel method for pest management. Here, we identified the ALB core RNAi genes including those coding for Dicer, Argonaute, and double-stranded RNA-binding proteins (dsRBP) as well as for proteins involved in dsRNA transport and the systemic RNAi. We also compared expression of six potential reference genes that could be used to normalize gene expression and selected gapdh and rpl32 as the most reliable genes among different tissues and stages …

Development Of Rnai Method For Screening Candidate Genes To Control Emerald Ash Borer, Agrilus Planipennis, Thais B. Rodrigues, Lynne K. Rieske, Jian J. Duan, Kanakachari Mogilicherla, Subba Reddy Palli

Entomology Faculty Publications

The ingestion of double-strand RNAs (dsRNA) targeting essential genes in an insect could cause mortality. Based on this principle, a new generation of insect control methods using RNA interference (RNAi) are being developed. In this work, we developed a bioassay for oral delivery of dsRNA to an invasive forest and urban tree pest, the emerald ash borer (EAB, Agrilus planipennis). EAB feeds and develops beneath the bark, killing trees rapidly. This behavior, coupled with the lack of a reliable artificial diet for rearing larvae and adults, make them difficult to study. We found that dsRNA is transported and processed …

Risk Parameters And Assessment Of Dietary Dsrna Exposure In Folsomia Candida, Jeffrey Edward Noland

Theses and Dissertations--Entomology

Assessing the risk of transgenic crop products is essential when determining the safety of a crop for deregulation and commercialization. The Organization of Economic and Cooperative Development (OECD), International Standards Organization (ISO), and governmental regulatory agencies require a battery of tests to demonstrate the safety of a GM product against several surrogate species of organisms that perform various ecosystem services. Assays are performed using toxicology methods established for pesticides. These methods have been applied to testing the safety, specificity and fate of Bacillus thuringiensis (Bt) Cry protein toxins engineered into crop plants and information exists on the effects on non-target …

Selection Of Reference Genes For Rt-Qpcr Analysis In A Predatory Biological Control Agent, Coleomegilla Maculata (Coleoptera: Coccinellidae), Chunxiao Yang, Huipeng Pan, Jeffrey Edward Noland, Deyong Zhang, Zhanhong Zhang, Yong Liu, Xuguo Zhou

Entomology Faculty Publications

Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for quantifying gene expression across various biological processes, of which requires a set of suited reference genes to normalize the expression data. Coleomegilla maculata (Coleoptera: Coccinellidae), is one of the most extensively used biological control agents in the field to manage arthropod pest species. In this study, expression profiles of 16 housekeeping genes selected from C. maculata were cloned and investigated. The performance of these candidates as endogenous controls under specific experimental conditions was evaluated by dedicated algorithms, including geNorm, Normfinder, BestKeeper, and ΔCt method. In addition, …

Biosafety Research For Non-Target Organism Risk Assessment Of Rnai-Based Ge Plants, Andrew F. Roberts, Yann Devos, Godwin N. Y. Lemgo, Xuguo Zhou

Entomology Faculty Publications

RNA interference, or RNAi, refers to a set of biological processes that make use of conserved cellular machinery to silence genes. Although there are several variations in the source and mechanism, they are all triggered by double stranded RNA (dsRNA) which is processed by a protein complex into small, single stranded RNA, referred to as small interfering RNAs (siRNA) with complementarity to sequences in genes targeted for silencing. The use of the RNAi mechanism to develop new traits in plants has fueled a discussion about the environmental safety of the technology for these applications, and this was the subject of …

Mirnas Regulated Overexpression Of Ryanodine Receptor Is Involved In Chlorantraniliprole Resistance In Plutella Xylostella (L.), Xiuxia Li, Lei Guo, Xuguo Zhou, Xiwu Gao, Pei Liang

Entomology Faculty Publications

The amino acid mutations in ryanodine receptor (RyR) and elevated activity of detoxification enzymes have been associated with the diamide insecticide resistance in the diamondback moth, Plutella xylostella (L.). The up-regulation of P. xylostella RyR mRNA (PxRyR) expression has also been reported in field populations of different graphical origin. However, whether the up-regulation of PxRyR is involved in diamide resistance remains unknown. In this paper, 2.28- to 4.14-fold higher expression of PxRyR was detected in five field collected resistant populations, compared to that in a susceptible population. The expression of PxRyR was up-regulated 5.0- and 7.2-fold, respectively, after …

Nutrition Regulation Of Male Accessory Gland Growth And Maturation In Tribolium Castaneum, Jingjing Xu, Ashlee L. Anciro, Subba R. Palli

Entomology Faculty Publications

Insulin/IGF-1 signaling (IIS) pathway is known to control growth, development and reproduction. Insulin-like peptide mediated body size plasticity in Drosophila melanogaster has been reported. Here, our studies showed that IIS pathway and nutrition regulate growth and maturation of the male accessory gland (MAG) in the red flour beetle, Tribolium castaneum. The size of MAG increased from day 1 to day 5 post-adult emergence (PAE). This increase in the size of MAG is contributed by an increase in cell size, but not cell number. The growth of MAG was impaired after double-stranded RNA (dsRNA)-mediated knockdown in the expression of genes …