Behavioral flexibility is an important cornerstone for the ecological success of animals. Social Cataglyphis nodus ants with their age‐related polyethism characterized by age‐related behavioral phenotypes represent a prime example for behavioral flexibility. We propose neuropeptides as powerful candidates for the flexible modulation of age‐related behavioral transitions in individual ants. As the neuropeptidome of C. nodus was unknown, we collected a comprehensive peptidomic data set obtained by transcriptome analysis of the ants’ central nervous system combined with brain extract analysis by Q‐Exactive Orbitrap mass spectrometry (MS) and direct tissue profiling of different regions of the brain by matrix‐assisted laser desorption/ionization time‐of‐flight (MALDI‐TOF) MS. In total, we identified 71 peptides with likely bioactive function, encoded on 49 neuropeptide‐, neuropeptide‐like, and protein hormone prepropeptide genes, including a novel neuropeptide‐like gene (fliktin). We next characterized the spatial distribution of a subset of peptides encoded on 16 precursor proteins with high resolution by MALDI MS imaging (MALDI MSI) on 14 µm brain sections. The accuracy of our MSI data were confirmed by matching the immunostaining patterns for tachykinins with MSI ion images from consecutive brain sections. Our data provide a solid framework for future research into spatially resolved qualitative and quantitative peptidomic changes associated with stage‐specific behavioral transitions and the functional role of neuropeptides in Cataglyphis ants. image

We analysed peptide hormones of South African species of the recently described insect order Mantophasmatodea. Mass spectrometric techniques made it possible to analyse minute amounts of material stored in different neurohemal organs of single specimens. The methodological approach underlying this application is described and resulted in the first completely sequenced peptide hormones of the Mantophasmatodea, namely adipokinetic hormone, leucomyosuppressin, and a novel member of the periviscerokinins. The extensive peptide screening revealed a surprisingly clear differentation of species by peptide mass fingerprints. This is the first successful attempt to catalogue neuropeptides of insects for systematic/taxonomic purposes. In total, 14 of 32 peptides differed across species. Moreover, the data presented here show a hierarchy in the tendency for modifications among the different peptide hormones.

Mass spectrometry imaging (MSI) of neuropeptides has become a well-established method with the ability to combine spatially resolved information of immunohistochemistry with peptidomics information of mass spectrometric analysis. Several studies have conducted MSI of insect neural tissues, however; these studies did not detect a neuropeptide complement comparable to that obtained with conventional peptidomics. The aim of our study was to improve sample preparation so that MSI provides comprehensive and reproducible neuropeptidomics information. Using the cockroach retrocerebral complex, the presented protocol produces enhanced coverage of neuropeptides at 15 µm spatial resolution, confirmed by parallel analysis of tissue extracts using electrospray ionization MS. Altogether, more than 100 peptide signals from 15 neuropeptide precursor genes could be traced with high spatial resolution. In addition, MSI spectra confirmed differential prohormone processing and distinct neuropeptide-based compartmentalization of the retrocerebral complex. We believe that our workflow facilitates incorporation of MSI in neuroscience-related topics, including study of complex neuropeptide interactions within the CNS.