Filling in the gaps: A reevaluation of the Lygus hesperus peptidome using an expanded de novo assembled transcriptome and molecular cloning

Abstract

Peptides are the largest and most diverse class of molecules modulating physiology and behavior. Previously, we predicted a peptidome for the western tarnished plant bug, Lygus hesperus, using transcriptomic data produced from whole individuals. A potential limitation of that analysis was the masking of underrepresented genes, in particular tissue-specific transcripts. Here, we reassessed the L. hesperus peptidome using a more comprehensive dataset comprised of the previous transcriptomic data as well as tissue-specific reads produced from heads and accessory glands. This augmented assembly significantly improves coverage depth providing confirmatory transcripts for essentially all of the previously identified families and new transcripts encoding a number of new peptide precursors corresponding to 14 peptide families. Several families not targeted in our initial study were identified in the expanded assembly, including agatoxin-like peptide, CNMamide, neuropeptide-like precursor 1, and periviscerokinin. To increase confidence in the in silico data, open reading frames of a subset of the newly identified transcripts were amplified using RT-PCR and sequence validated. Further PCR-based profiling of the putative L. hesperus agatoxin-like peptide precursor revealed evidence of alternative splicing with near ubiquitous expression across L. hesperus development, suggesting the peptide serves functional roles beyond that of a toxin. The peptides predicted here, in combination with those identified in our earlier study, expand the L. hesperus peptidome to 42 family members and provide an improved platform for initiating molecular and physiological investigations into peptidergic functionality in this non-model agricultural pest.