Molecular and Functional Characterization of Pyrokinin-Like Peptides in the Western Tarnished Plant Bug Lygus hesperus (Hemiptera: Miridae)

SIMPLE SUMMARY: Neuropeptides regulate most insect biological functions. One such group of peptides, the pyrokinins (PKs), are distinguished by a C-terminal FXPRLamide. While widely distributed in most insects, they are poorly characterized in plant bugs. To address this limitation, we identified the PK transcript in the western tarnished plant bug (Lygus hesperus) and examined its expression. The Lygus PK transcript is predicted to yield three PK-like peptides but only two (LyghePKa and LyghePKb) have the characteristic C-terminal amide. The transcript is expressed throughout development and is most abundant in heads. A custom FXPRLamide antibody revealed immunoreactive cells throughout the Lygus central nervous system consistent with typical neuropeptide expression. To assess potential functional roles of the peptides, a fluorescence-based Ca(2+) influx assay using cultured insect cells stably expressing a moth PK receptor was performed. LyghePKa was unable to stimulate receptor activation, whereas LyghePKb triggered a robust response. The in vivo pheromonotropic activity of the two peptides was likewise assessed using three different moth species. Like the cell culture system, only the LyghePKb peptide was active. The study suggests evolutionary divergence of the PK gene in plant bugs and provides critical insights into likely biological functions in the western tarnished plant bug. ABSTRACT: The pyrokinin (PK) family of insect neuropeptides, characterized by C termini consisting of either WFGPRLamide (i.e., PK1) or FXPRLamide (i.e., PK2), are encoded on the capa and pk genes. Although implicated in diverse biological functions, characterization of PKs in hemipteran pests has been largely limited to genomic, transcriptomic, and/or peptidomic datasets. The Lygus hesperus (western tarnished plant bug) PK transcript encodes a prepropeptide predicted to yield three PK2 FXPRLamide-like peptides with C-terminal sequences characterized by FQPRSamide (LyghePKa), FAPRLamide (LyghePKb), and a non-amidated YSPRF. The transcript is expressed throughout L. hesperus development with greatest abundance in adult heads. PRXamide-like immunoreactivity, which recognizes both pk- and capa-derived peptides, is localized to cells in the cerebral ganglia, gnathal ganglia/suboesophageal ganglion, thoracic ganglia, and abdominal ganglia. Immunoreactivity in the abdominal ganglia is largely consistent with capa-derived peptide expression, whereas the atypical fourth pair of immunoreactive cells may reflect pk-based expression. In vitro activation of a PK receptor heterologously expressed in cultured insect cells was only observed in response to LyghePKb, while no effects were observed with LyghePKa. Similarly, in vivo pheromonotropic effects were only observed following LyghePKb injections. Comparison of PK2 prepropeptides from multiple hemipterans suggests mirid-specific diversification of the pk gene.