A Transcriptome Survey Spanning Life Stages and Sexes of the Harlequin Bug, Murgantia histrionica

Abstract

The harlequin bug, Murgantia histrionica (Hahn), is an agricultural pest in the continental United States, particularly in southern states. Reliable gene sequence data are especially useful to the development of species-specific, environmentally friendly molecular biopesticides and effective biolures for this insect. Here, mRNAs were sampled from whole insects at the 2nd and 4th nymphal instars, as well as sexed adults, and sequenced using Illumina RNA-Seq technology. A global assembly of these data identified 72,540 putative unique transcripts bearing high levels of similarity to transcripts identified in other taxa, with over 99% of conserved single-copy orthologs among insects being detected. Gene ontology and protein family analyses were conducted to explore the functional potential of the harlequin bug's gene repertoire, and phylogenetic analyses were conducted on gene families germane to xenobiotic detoxification, including glutathione S-transferases, carboxylesterases and cytochrome P450s. Genic content in harlequin bug was compared with that of the closely related invasive pest, the brown marmorated stink bug, Halyomorpha halys (Stål). Quantitative analyses of harlequin bug gene expression levels, experimentally validated using quantitative real-time PCR, identified genes differentially expressed between life stages and/or sexes.

Keywords: RNAi targets; agricultural pest; harlequin bug; insect pheromones; invasive insects; transcriptomics; xenobiotic detoxification.

Figure 1

Mevalonate (MVA) and Juvenile Hormone (JH) biosynthetic steps and the proposed pathway for the formation of the aggregation pheromone murgantiol. Multiple enzyme conversions are represented with dashed arrows.

Figure 2

Quantitative real-time PCR validation of select harlequin bug transcript expression patterns.

Figure 3

Maximum likelihood-based glutathione S-transferase phylogeny for H. halys and M. histrionica proteins. Four distinct clades corresponding to GST class were observed: delta (red), theta (blue), sigma (brown) and microsomal (green). Two prostaglandin E synthase isoforms (pink) were placed in the microsomal GST clade. Bootstrap support (100 replicates) is indicated on branches.

Figure 4

Segment of scaffold 261 from the Halyomorpha halys genome assembly harboring an array of eleven E4/FE4 esterase genes. The H. halys COE genes are show in the top track, in grey (note that gene model XM_014420119.1 appears to correspond to an unrelated lipase gene) a total of six unique M. histrionica proteins (bottom track, purple) could be aligned to this chromosomal region, and these associated with three distinct COE loci present in the H. halys genome sequence.

Figure 5

Cytochrome P450s from H. halys (126 sequences) and M. histrionica (86 sequences) plus two from Rhodnius prolixus and one from Riptortus pedestris were aligned using Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/) at EBI. An NJ midpoint-rooted tree was made. The tree was drawn using Figtree v1.3.1 and labeled in Adobe Illustrator CC ver17.0.0. P450 clans are colored. Sequences less than 175 amino acids were not included.