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Characterization of Neuropeptide F in the tobacco hornworm, Manduca sexta
AdvisorNuss, Andrew B
Cell and Molecular Biology
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Neuropeptide F (NPF) belongs to the RF-amide family of peptide hormones and is a homolog to vertebrate Neuropeptide Y (NPY). NPF binds to a seven transmembrane G-protein coupled receptor, NPFR, and typically inhibits adenylyl cyclase through the Gα/i subunit to induce downstream pleiotropic physiological traits. NPF-NPFR signaling has been shown to regulate feeding, food choice, growth, mating, circadian cycle, and stem cell proliferation in invertebrates, among other functions. Most of these functions are explored at molecular levels in Drosophila melanogaster but studies in other insects lag in similar genetic explorations. One feature of NPF is its expression in both the central nervous system (CNS) and midgut endocrine cells in insects. Several functions of NPF have been elucidated through manipulations of the CNS, but the role of midgut-produced NPF is much less clear. Lepidoptera produce NPF abundantly in the midgut, suggesting an important role for the peptide in this group. Manduca sexta, a model for lepidopteran physiology, was therefore used in this study.To examine the role of midgut peptide hormones and more broadly the physiology of lepidopteran food deprivation, a starvation proteomic profile was generated from midguts of starved M. sexta caterpillars for 24 and 48 h in comparison to larvae on normal food for 24 and 48 h, respectively. A total 3,047 proteins were identified, and 1,368 proteins significantly differed in abundance. Six neuropeptide and related signaling peptides were detected, and NPF was higher in abundance in starved larvae in the combined fed vs. starved analysis. To further investigate the role of NPF in M. sexta, NPF sequences were first cloned and sequenced along with the putative receptor, NPFR. Transcript levels for NPF1, NPF2 and NPFR were compared in head, midgut, nerve cord and carcass of 4th instar larvae. Immunoreactivity to NPF antiserum was also detected in brain, ventral nerve cord ganglia and midgut endocrine cells. To identify potential molecular functions of NPF, a transcriptome for NPF injected caterpillars was performed from head, midgut, fat body, hemolymph, and carcass samples. In the global analysis of all tested tissues, TCA cycle associated transcripts were significantly upregulated whereas protein processing in the endoplasmic reticulum was downregulated. However, expression in specific samples suggests tissue-specific functions as well.