More Drosophila enteroendocrine peptides: Orcokinin B and the CCHamides 1 and 2

Antisera to orcokinin B, CCHamide 1, and CCHamide 2 recognize enteroendocrine cells in the midgut of the fruitfly Drosophila melanogaster and its larvae. Although the antisera to CCHamide 1 and 2 are mutually cross-reactive, polyclonal mouse antisera raised to the C-terminals of their respective precursors allowed the identification of the two different peptides. In both larva and adult, CCHamide 2 immunoreactive endocrine cells are large and abundant in the anterior midgut and are also present in the anterior part of the posterior midgut. The CCHamide 2 immunoreactive endocrine cells in the posterior midgut are also immunoreactive with antiserum to allatostatin C. CCHamide 1 immunoreactivity is localized in endocrine cells in different regions of the midgut; those in the caudal part of the posterior midgut are identical with the allatostatin A cells. In the larva, CCHamide 1 enteroendocrine cells are also present in the endocrine junction and in the anterior part of the posterior midgut. Like in other insect species, the Drosophila orcokinin gene produces two different transcripts, A and B. Antiserum to the predicted biologically active peptide from the B-transcript recognizes enteroendocrine cells in both larva and adult. These are the same cells as those expressing β-galactosidase in transgenic flies in which the promoter of the orcokinin gene drives expression of this enzyme. In the larva, a variable number of orcokinin-expressing enteroendocrine cells are found at the end of the middle midgut, while in the adult, those cells are most abundant in the middle midgut, while smaller numbers are present in the anterior midgut. In both larva and adult, these cells also express allatostatin C. We also made a specific polyclonal antiserum to the NPF precursor in order to determine more precisely the expression of this peptide in the midgut. Using this antiserum, we find expression in the midgut to be the same as described previously using transgenic flies, while in the adult, midgut expression appears to be concentrated in the middle midgut, thus suggesting that in the anterior midgut only minor quantities of NPF are produced.