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Title: Generation of a transcriptome in a model lepidopteran pest, Heliothis virescens, using multiple sequencing strategies for profiling midgut gene expression

Author
item Perera, Omaththage
item Shelby, Kent
item Popham, Holly
item GOULD, FRED - North Carolina State University
item ADANG, MICHAEL - University Of Georgia
item JURAT-FUENTES, JUAN LUIS - University Of Tennessee

Submitted to: PLoS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/13/2015
Publication Date: 6/5/2015
Publication URL: http://handle.nal.usda.gov/10113/61099
Citation: Perera, O.P., Shelby, K., Popham, H.J., Gould, F., Adang, M.A., Jurat-Fuentes, J. 2015. Generation of a transcriptome in a model lepidopteran pest, Heliothis virescens, using multiple sequencing strategies for profiling midgut gene expression. PLoS One. 10(6):e0128563. DOI:10.1371/journal.pone.0128563.

Interpretive Summary: Several species of moths of the subfamily Heliothentinae are serious pests of crops and ornamental plants in the Americas. Tobacco budworm, Heliothis virescens (F.) is one of the major pests of cotton. Efforts directed at improving tobacco budworm control measures as well as its use as a relevant biological model would be substantially accelerated by making available a variety of molecular resources for this pest species. The work described here demonstrates the utility of next-generation sequencing technologies for rapid molecular resource generation from this species for which no annotated genome is available. High-throughput sequencing technologies and standard sequencing of cloned complementary DNA (cDNA) were used to obtain a large number of genes expressed in the pest moth tobacco budworm. Pooling of RNA extracted from insects exposed to diverse environmental conditions, developmental stages, infections with entomopathogens used for biological control, and various chemicals allowed a thorough sampling of gene transcripts produced under a wide range of physiological conditions. Among these identified tobacco budworm genes were immune effectors, signal transduction pathways, olfactory receptors, hormone biosynthetic pathways, peptide hormones and their receptors, digestive enzymes, insecticide resistance enzymes, among others. We demonstrate the utility of this genetic resource to study gene expression profiling of larval midguts during intoxication with toxins from Bacillus thuringiensis (Bt). Availability of these substantial molecular resources for this destructive pest will allow the full range of biological experimentation needed to further control efforts for this and related Noctuid pest moths and the development of H. virescens as a relevant biological model for functional genomics.

Technical Abstract: Heliothine pests such as the tobacco budworm, Heliothis virescens (F.), pose a significant threat to production of a variety of crops and ornamental plants and are models for developmental and physiological studies. The efforts to develop new control measures for H. virescens, as well as its use as a relevant biological model, are hampered by a lack of molecular resources. The present work demonstrates the utility of next-generation sequencing technologies for rapid molecular resource generation from this species for which lacks a sequenced genome. In order to amass a de novo transcriptome for this moth, transcript sequences generated from Illumina, Roche 454, and Sanger sequencing platforms were merged into a single de novo transcriptome assembly. This pooling strategy allowed a thorough sampling of transcripts produced under diverse environmental conditions, developmental stages, tissues, and infections with entomopathogens used for biological control, to provide the most complete transcriptome to date for this species. Over 138 million reads from the three platforms were assembled into the final set of 63,648 contigs. Of these, 29,978 had significant BLAST scores indicating orthologous relationships to transcripts of other insect species, with the top-hit species being the monarch butterfly (Danaus plexippus) and silkworm (Bombyx mori). Among identified H. virescens orthologs were immune effectors, signal transduction pathways, olfactory receptors, hormone biosynthetic pathways, peptide hormones and their receptors, digestive enzymes, and insecticide resistance enzymes. As an example, we demonstrate the utility of this transcriptomic resource to study gene expression profiling of larval midguts and detect transcripts of putative Bacillus thuringiensis (Bt) Cry toxin receptors. The substantial molecular resources described in this study will facilitate development of H. virescens as a relevant biological model for functional genomics and for new biological experimentation needed to develop efficient control efforts for this and related Noctuid pest moths.