Peptide hormones synthesized by secretory neurons in the CNS are important regulators of physiology, behavior, and development. Like other neuropeptides, they are synthesized from larger precursor molecules by a specific set of enzymes. Using a combination of neurogenetics, immunostainings, and direct mass spectrometric profiling, we show that the presence of Drosophila prohormone convertase 2 encoded by the gene amontillado (amon) is a prerequisite for the proper processing of neuropeptide hormones from the major neurohemal organs of the CNS. A loss of amon correlates with a loss of neuropeptide hormone signals from the larval ring gland and perisympathetic organs. Neuropeptide hormone signals were still detectable in the adult corpora cardiaca of older amon-deficient flies which were amon heat-shock-rescued until eclosion. A semiquantification by direct peptide profiling using stable isotopic standards showed, however, that their neuropeptide hormone levels are strongly reduced. Targeted expression of GFP under the control of amon regulatory regions revealed a co-localization with the investigated peptide hormones in secretory neurons of the brain and ventral nerve cord. The lack of AMON activity resulted in a deficiency of L3 larva to enter the wandering phase. In conclusion, our findings provide the first direct evidence that AMON is a key enzyme in the production of neuropeptides in the fruitfly.

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.