Performing Department
Entomology
Non Technical Summary
Neuropeptide F (NPF) is a predominant peptide hormone in the midgut endocrine system of insect pests, but nothing is known about its function. This project will determine the function of NPF in mosquitoes and corn earworm and the feasibility of developing chemical mimics that can be applied with traditional methods or engineered into plant hosts to interfere with the feeding and growth of insect pests.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The endocrine system in the midgut of insects is an important source of peptide hormones presumed to regulate digestion, metabolism, and appetite in the same ways as the gastric-enteropancreatic endocrine system in vertebrates. At first, these putative insect hormones were purified from midgut extracts based on their immunoreactivity to antisera specific for peptide hormones characterized in mammals or other invertebrates. Now that a catalog of peptide hormone genes exists for the fruit fly and a mosquito, the presence of such gene transcripts in the midgut of other species can be confirmed with PCR, microarrays, or EST libraries and bioinformatics. Yet, almost nothing is known about the functional significance of these midgut peptides in insects. Our past efforts have focused on the characterization of a particular peptide hormone, neuropeptide F (NPF), that is found in tens to thousands of endocrine cells scattered throughout the midgut of larval and adult insects. The gene encoding NPF in all insects examined to date is structurally related to the neuropeptide Y (NPY) family in vertebrates. Neuropeptide Y plays pivotal roles in the regulation of food intake, circadian rhythms, and other processes in mammals. Related peptides, peptide YY and pancreatic polypeptide (PP), localized in the vertebrate gut, regulate enzyme secretion and motility. We were the first to isolate and characterize NPF from the corn earworm, fruit fly, yellow fever mosquito, and African malaria mosquito. As determined by immunocytochemistry and other molecular techniques, NPF is expressed in relatively few cells in the nervous system and throughout the midgut endocrine system of these insects. Recent work with Brian Forschler and Andrew Nuss, a Ph.D student, in Entomology, UGA confirmed this same distribution of NPF in different castes of a termite and led to its isolation and molecular and functional characterization. For the past project, we also characterized the NPF receptor in two dipteran species. One such receptor with striking similarity to mammalian PP receptors was first identified in the Drosophila genome. It was expressed in mammalian cells and shown to bind NPF specifically and inhibit production of a key intracellular messenger, cAMP, the gold standard of NPY action in mammalian cells. We then completed a comparable study of a mosquito NPF receptor. For this proposal, our first goal is to determine whether NPF has specific effects on midgut specific processes, such as enzyme secretion, nutrient transport, and motility, in larval and adult mosquito stages and in lepidopteran larvae. Once the regulation of a particular midgut process is defined, our second goal will be to block it in two ways: 1. Target expression of NPF and its receptor by RNA interference 2. Identify molecular agents known to be agonists or antagonists of NPY/PP action in mammalian systems that are effective in the insect model study. Application of a blocking agent, as well, may have significant consequences on appetite and digestion in that insect and possibly delay development or reproduction.
Project Methods
Experimental insects: Eggs from a colony of corn earworm at a UGA Experiment Station in Tifton, GA will be hatched and reared to last instar larvae for experiments. All stages of the mosquito species, Aedes aegypti, are available in the Brown laboratory for experiments. Bioassays: Synthetic NPF for both species will be produced by Kevin Clark, Entomology, UGA. Agonists and antagonists of NPY and PP are available from Sigma. These reagents will be tested for effects on gut motility and feeding with bioassays developed for earworms and mosquitoes in the laboratories of Crim and Brown. Bioassays to test their effects on metabolite transport and digestive enzyme activity in midguts will be adapted for these insects. Results will be statistically evaluated for significance. Molecular identity of earworm NPF and its receptor (NPFR): Since genes for earworm NPF and NPFR are not characterized, partial nucleotide sequences will be amplified by PCR from larval midgut cDNA with degenerate nucleotide primers designed to the known region of the earworm NPF and to one or more conserved regions of homolog NPFRs in other insects. Once an authentic nucleotide sequence for the NPF and NPFR is known, specific primers will be used in PCR to complete the nucleotide sequence of cDNAs. Their expression will be confirmed in the earworm midgut. The mosquito NPFR has been predicted from the genome database and will be cloned with specific primers by PCR, and its tissue expression confirmed. RNA interference: Following procedures developed in the Brown laboratory and that of Michael Strand, Entomology at UGA, dsRNA will be amplified from the NPF and NPFR cDNAs and injected into the insects at times and conditions to be determined experimentally, along with the proper controls (GFP dsRNA). After one to four days, transcript levels of the genes in the midgut will be assessed with RT-PCR, and a reduction in their levels in dsRNA treated animals will confirm the efficacy of experimental conditions. Once these conditions are established, physiological effects of dsRNA treatment will be explored at the midgut level with bioassays in which NPF has a demonstrated effect. It is expected that pretreatment of midguts with NPFR dsRNA will block NPFR gene expression through the mechanism of RNA mediated interference, and when those midguts are treated with NPF in the bioassay, its effects will be negligible. Effects of dsRNA on gene transcript expression will be assessed with RT-PCR or quantitative PCR and statistically evaluated for significance. Additionally, effects of dsRNA treatments will be evaluated in live insects with different feeding and development bioassays. If NPF dsRNA treatment has an effect in such an assay, then it is expected that NPF injection would revitalize the normal condition. In contrast, injection of NPF would not restore the normal condition in aberrant insects treated with NPFR dsRNA, since expression of the NPFR is blocked. Either way, NPF signaling and its effects would be explored in tissues associated with the aberrant response to elucidate a novel or organismal NPF function.