Transcriptomics and Neuropeptidomics of the Cockroach <i>Rhyparobia maderae</i> to Characterize the Neuropeptide Landscape of the Cockroach’s Circadian Clock

The circadian clock of <i>Rhyparobia maderae</i> is the accessory medulla (AME) in the optic lobes of the brain. Controlled by compound ocular photoreceptors, the cockroach clock orchestrates circadian rhythms in physiology and behavior synchronized with environmental light-dark cycles. Approximately 240 neurons, rich in partially colocalized neuropeptides, innervate the AME. Since the identity and function of most of these neuropeptides are unknown in the cockroach, the transcriptome of the Madeira cockroach was prepared, revealing 68 neuropeptides, neuropeptide-like and protein hormone genes. To identify the neuropeptidome, a combination of analyses of brain/retrocerebral complex extracts by Q-Exactive Orbitrap mass spectrometry (MS) and direct tissue profiling of different parts of the nervous system by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS was used. The resulting data set revealed 192 mature neuropeptides, with 155 identified for the first time and 57 precursor peptides. The AME neuropeptidomic profile revealed 166 mature neuropeptides with potential bioactive circadian clock functions. The precision of our MS data was validated by immunostaining, as illustrated by an example of immunoreactive patterns for proctolin in AME neurons. This peptidergic data set serves as the foundation for future research on neuropeptide-based functional studies that regulate the timing of disparate neuronal circuits.