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Generation of a Transcriptome in a Model Lepidopteran Pest, Heliothis virescens, Using Multiple Sequencing Strategies for Profiling Midgut Gene Expression.

Perera OP, Shelby KS, Popham HJ, Gould F, Adang MJ, Jurat-Fuentes JL - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Southern Insect Management Research Unit, USDA, Agricultural Research Service, Stoneville, MS, 38776, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

RNA-Seq determination of transcript expression level differences between tissues.Venn diagram demonstrating expression differences between RNA-Seq reads from whole insect, midgut, and hemocyte of H. virescens. The number of transcripts with significantly different expression levels (≥2-fold, p≤0.01) are shown within parenthesis.
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pone.0128563.g004: RNA-Seq determination of transcript expression level differences between tissues.Venn diagram demonstrating expression differences between RNA-Seq reads from whole insect, midgut, and hemocyte of H. virescens. The number of transcripts with significantly different expression levels (≥2-fold, p≤0.01) are shown within parenthesis.

Mentions: In order to identify transcripts unique to the midgut tissue we compared gene expression levels using RNA-Seq in whole insect, hemocytes and midgut tissues. Of the 7,765 transcripts with significant differential expression (≥2-fold, p≤0.01) between whole insect and midgut tissue, 1,895 showed ≥2-fold higher expression in the midgut. A larger number of differentially expressed genes (11,455) were detected when comparing hemocytes and midgut tissue, of which 5,334 were expressed ≥2-fold higher in the midgut. When considering the intersection of the three comparisons between pairs of samples (whole body vs. midgut, whole body vs. hemocyte, and midgut vs. hemocytes), 1,464 transcripts were identified as common (Fig 4). There were 1104 sequence contigs highly expressed in the midgut (≥2-fold, P<0.01) compared to the other two samples (S4A Table), although only 296 transcripts had annotations. Among these transcripts with significantly higher expression (>2-fold, p<0.01) in the midgut, we detected 13 aminopeptidases, four cadherin–like proteins, 107 proteases (chymotrypsins, trypsins, and serine proteases), 35 carboxyl esterases, 37 cytochrome P450 monooxygenases, three amino acid transporters, and four each ABC class B and C transporters (S4 Table).


Generation of a Transcriptome in a Model Lepidopteran Pest, Heliothis virescens, Using Multiple Sequencing Strategies for Profiling Midgut Gene Expression.

Perera OP, Shelby KS, Popham HJ, Gould F, Adang MJ, Jurat-Fuentes JL - PLoS ONE (2015)

RNA-Seq determination of transcript expression level differences between tissues.Venn diagram demonstrating expression differences between RNA-Seq reads from whole insect, midgut, and hemocyte of H. virescens. The number of transcripts with significantly different expression levels (≥2-fold, p≤0.01) are shown within parenthesis.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4457788&req=5

pone.0128563.g004: RNA-Seq determination of transcript expression level differences between tissues.Venn diagram demonstrating expression differences between RNA-Seq reads from whole insect, midgut, and hemocyte of H. virescens. The number of transcripts with significantly different expression levels (≥2-fold, p≤0.01) are shown within parenthesis.
Mentions: In order to identify transcripts unique to the midgut tissue we compared gene expression levels using RNA-Seq in whole insect, hemocytes and midgut tissues. Of the 7,765 transcripts with significant differential expression (≥2-fold, p≤0.01) between whole insect and midgut tissue, 1,895 showed ≥2-fold higher expression in the midgut. A larger number of differentially expressed genes (11,455) were detected when comparing hemocytes and midgut tissue, of which 5,334 were expressed ≥2-fold higher in the midgut. When considering the intersection of the three comparisons between pairs of samples (whole body vs. midgut, whole body vs. hemocyte, and midgut vs. hemocytes), 1,464 transcripts were identified as common (Fig 4). There were 1104 sequence contigs highly expressed in the midgut (≥2-fold, P<0.01) compared to the other two samples (S4A Table), although only 296 transcripts had annotations. Among these transcripts with significantly higher expression (>2-fold, p<0.01) in the midgut, we detected 13 aminopeptidases, four cadherin–like proteins, 107 proteases (chymotrypsins, trypsins, and serine proteases), 35 carboxyl esterases, 37 cytochrome P450 monooxygenases, three amino acid transporters, and four each ABC class B and C transporters (S4 Table).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Southern Insect Management Research Unit, USDA, Agricultural Research Service, Stoneville, MS, 38776, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus