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Submitted to: Insects
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 11/13/2013 Publication Date: 11/26/2013 Citation: Shelby, K. 2013. Functional immunomics of the squash bug, Anasa tristis (De Geer) (Heteroptera: Coreidae). Insects. 4(4):712-730. Interpretive Summary: Cucurbits are major horticultural cash crops in the United States. In 2011 cash revenue for squash and pumpkin totaled $396 million, $350 million for cantaloupe and $543 million for watermelon. Although transmission of plant viruses to cucurbits by aphids and whiteflies is a major cause of economic loss, additional insect pests such as cucumber beetles and squash bugs physically damage both the plants and the fruit. Squash bugs also transmit a bacterial infection, Cucurbit yellow vine disease, between infected plants resulting in significant spoilage and loss of consumer appeal. In this study the first gene list for squash bugs was compiled providing actual 37 thousand sequences targeting the squash bug nervous, endocrine and immune systems. Disruption of immunity, feeding, physiology, reproduction, insecticide resistance, etc. are possible using these new resources. The publically shared squash bug gene database constructed by this study will be useful primarily by the horticultural industry for the design of control measures such as mating disruption technologies, new insecticides, and development of resistant cucurbit cultivars. Integrated pest management professionals will use the database for more effective pest insect population monitoring, biological control, resistance management and control of squash bug and other cucurbit pests. Technical Abstract: The Squash bug, Anasa tristis (De Geer), 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 I 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, endocrine, reproductive, cuticle, and other physiological systems. Expression profiling of the adult immune response was accomplished by aligning the 100-base tags from each biological replicate from each treatment and controls to the annotated reference assembly of the A. tristis transcriptome. 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. |
