RESEARCH ARTICLE CCHamide-2 Is an Orexigenic Brain-Gut Peptide in Drosophila

The neuroendocrine peptides CCHamide-1 and-2, encoded by the genes ccha1 and-2, are produced by endocrine cells in the midgut and by neurons in the brain of Drosophila melanogaster. Here, we used the CRISPR/Cas9 technique to disrupt the ccha1 and-2 genes and identify mutant phenotypes with a focus on ccha-2mutants. We found that both larval and adult ccha2mutants showed a significantly reduced food intake as measured in adult flies by the Capillary Feeding (CAFE) assay (up to 72 % reduced food intake compared to wild-type). Locomotion tests in adult flies showed that ccha2mutants had a significantly reduced locomotor activity especially around 8 a.m. and 8 p.m., where adult Drosophila nor-mally feeds (up to 70 % reduced locomotor activity compared to wild-type). Reduced larval feeding is normally coupled to a delayed larval development, a process that is mediated by insulin. Accordingly, we found that the ccha2mutants had a remarkably delayed develop-ment, showing pupariation 70 hours after the pupariation time point of the wild-type. In con-trast, the ccha-1mutants were not developmentally delayed. We also found that the ccha2 mutants had up to 80 % reduced mRNA concentrations coding for the Drosophila insulin-like-peptides-2 and-3, while these concentrations were unchanged for the ccha1mutants. From these experiments we conclude that CCHamide-2 is an orexigenic peptide and an important factor for controlling developmental timing in Drosophila