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United States Department of Agriculture

Agricultural Research Service

Research Project: EICOSANOID-MEDIATED AND MOLECULAR IMMUNE SIGNALING INHIBITORS IN PIERCING/SUCKING INSECT PESTS OF SMALL AND URBAN VEGETABLE FARMS

Location: Biological Control of Insects Research

Title: Identification of immune system gene silencing targets in a de novo assembly of the transcriptome of the squash bug, Anasa tristis (De Geer) (Heteroptera: coreidae)

Author
item Shelby, Kent

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: March 29, 2012
Publication Date: June 1, 2012
Citation: Shelby, K. 2012. Identification of immune system gene silencing targets in a de novo assembly of the transcriptome of the squash bug, Anasa tristis (De Geer) (Heteroptera: coreidae) [abstract]. Kansas State University Arthropod Genomics Symposium. p. 30.

Technical Abstract: The Squash bug is a major piercing/sucking pest of cucurbits causing extensive damage to plants and fruits, and transmitting phytopathogens. There are few effective biological control agents or cultural practices for controlling this highly destructive pest. A promising new approach for control is insect pest specific RNAi targeted to critical physiological systems via the plant phloem. Thus we have initiated studies to determine the feasibility of delivering immunosuppressive RNAi to phloem feeding insects such as the squash bug. First steps towards this goal include construction of the first de novo exome, identification of candidate gene silencing targets, and laboratory based per os delivery methods for gene silencing of immunity. RNA was extracted from insects challenged with bacterial and fungal immunoelicitors, insects fed on different cucurbit species, and insects from all life stages from egg to adult. All treatments and replicates were separately barcoded for subsequent analyses, then pooled for sequencing in a single lane using the Illumina HiSeq2000 platform. Over 211 million 100-base tags generated in this manner were trimmed, filtered, and cleaned, then assembled into a de novo reference transcriptome using the Broad Institute Trinity assembly algorithm. The assembly was annotated using NCBIx NR, BLAST2GO, KEGG and other databases. Of the >130,000 total assemblies 37,327 were annotated identifying the sequences of candidate gene silencing targets from immune, and other physiological systems. To accomplish expression profiling the 100-base tags from each treatment, controls and replicates were independently aligned to the annotated assembly of the A. tristis reference transcriptome. Immune system components upregulated by infection will be presented. From this first reference transcriptome a comprehensive RNA-seq expression profiling of squash bug immune defense has been completed, and candidate gene silencing targets have been identified to accomplish RNAi-based immunosuppression.

Last Modified: 9/21/2014
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