Nezara viridula (Hemiptera: Pentatomidae) transcriptomic analysis and neuropeptidomics

Abstract

Stinkbugs (Hemiptera: Pentatomidae) are of major economic importance as pest of crops. Among the species composing the stinkbug complex, Nezara viridula is one of the most abundant in Brazil, Argentina and the Southern USA. However, this species has been poorly characterized at the genetic and physiological level. Here we sequenced and analyzed the complete transcriptome of N. viridula male and female adults. We identified neuropeptide precursor genes and G-protein coupled receptors for neuropeptides in this transcriptome. Mature neuropeptides were identified in N. viridula brain extracts by liquid chromatography-tandem mass spectrometry. We also analyzed the neuropeptide precursor complement in the genome sequence of Halyomorpha halys, another pentatomid of economic relevance. We compared the results in both pentatomids with the well-characterized neuropeptide repertoire from the kissing bug Rhodnius prolixus (Hemiptera: Reduviidae). We identified both group-specific features (which could be related to the different feeding habits) and similarities that could be characteristic of Heteroptera. This work contributes to a deeper knowledge of the genetic information of these pests, with a focus on neuroendocrine system characterization.

Figure 1

Multiple sequence alignment of neuropeptide precursors that are specific to N. viridula and H. halys. The sequences of R. prolixus were used as a reference. Predicted convertase cleavage sites, according to the rules proposed by Veenstra, are shadowed in red. Glycine residues shadowed in pink indicate predicted amidation sites. The green shadows indicate the predicted signal peptides. The peptides found by mass spectrometry and deduced from transcriptomic sequences are underlined. Black background indicates a fully conserved residue, gray background indicates a conservative substitution. Conserved cysteine residues in neuroparsin A and insulin-like peptides are shadowed in yellow. In Insulin-like peptide, B chain, C peptide and A chain are boxed.

Figure 2

Multiple sequence alignment of neuropeptide precursors that are specific to the heteropteran species analyzed here. The sequences of R. prolixus were used as a reference. Predicted convertase cleavage sites, according to the rules proposed by Veenstra, are shadowed in red. Glycine residues shadowed in pink indicate predicted amidation sites. The green shadows indicate the predicted signal peptides. The peptides found by mass spectrometry and deduced from transcriptomic sequences are underlined. Black background indicates a fully conserved residue, gray background indicates a conservative substitution.

Figure 3

Bayesian phylogenetic analysis of neuroparsin A precursors from R. prolixus, H. halys and N. viridula. N. viridula transcripts are indicated with a black circle. The scale bar represents genetic distance. The number at each node indicates the posterior probabilities.

Figure 4

Mass spectrometry spectra verifying the presence of neuropeptides encoded in the precursors described, in brain extracts from male and female adult N. viridula.

Figure 5

Bayesian phylogenetic analysis of GPCRs for neuropeptides and protein hormones from D. melanogaster (Dm), R. prolixus (Rp) and N. viridula (Nv). N. viridula transcripts are indicated with a black circle. The scale bar represents genetic distance. The accession number in FlyBase is indicated for D. melanogaster GPCRs; for R. prolixus, either GeneBank accession number, contig number or transcript number in vectorbase/VectorBase (www.vectorbase.org) are indicated (for reconstructed R. prolixus GPCRs see predicted sequences in^,,,). Family A GPCRs are indicated by red branches; family B GPCRs are indicated by violet branches. Each GPCR family is indicated by different color shadows. The names of the receptors are indicated in the base of each clade. The scale bar represents genetic distance. The number at each node indicates the posterior probabilities.