Joe W. Crim

Identification of a Drosophila brain-gut peptide related to the neuropeptide Y family ☆

A neuropeptide F (NPF) was isolated from the fruit fly, Drosophila mellanogaster, based on a radioimmunoassay for a gut peptide from the corn earworm, Helicoverpa zea. A partial sequence was obtained from the fly peptide, and a genomic sequence coding for NPF was cloned after inverse polymerase chain reaction and shown to exist as a single genomic copy. The encoded, putative prepropeptide can be processed into an amidated NPF with 36 residues that is related to invertebrate NPFs and the neuropeptide Y family of vertebrates. In situ hybridization and immunocytochemistry showed that Drosophila NPF was expressed in the brain and midgut of fly larvae and adults.

An insulin-like peptide regulates egg maturation and metabolism in the mosquito Aedes aegypti

Ingestion of vertebrate blood is essential for egg maturation and transmission of disease-causing parasites by female mosquitoes. Prior studies with the yellow fever mosquito, Aedes aegypti, indicated blood feeding stimulates egg production by triggering the release of hormones from medial neurosecretory cells in the mosquito brain. The ability of bovine insulin to stimulate a similar response further suggested this trigger is an endogenous insulin-like peptide (ILP). A. aegypti encodes eight predicted ILPs. Here, we report that synthetic ILP3 dose-dependently stimulated yolk uptake by oocytes and ecdysteroid production by the ovaries at lower concentrations than bovine insulin. ILP3 also exhibited metabolic activity by elevating carbohydrate and lipid storage. Binding studies using ovary membranes indicated that ILP3 had an IC50 value of 5.9 nM that was poorly competed by bovine insulin. Autoradiography and immunoblotting studies suggested that ILP3 binds the mosquito insulin receptor (MIR), whereas loss-of-function experiments showed that ILP3 activity requires MIR expression. Overall, our results identify ILP3 as a critical regulator of egg production by A. aegypti.

Characterization of a functional neuropeptide F receptor from Drosophila melanogaster

Potential receptors for Drosophila neuropeptide F (DmNPF) were identified in the genome database. One receptor (DmNPFR1) sequence resembled the Lymnaea NPY receptor, an invertebrate homolog of the vertebrate Y-receptor family. DmNPFR1 was cloned and tested for functionality in stably transfected mammalian CHO cells. In whole cell binding assays, DmNPF displaced 125I-NPF in a concentration-dependent manner (IC(50) = 65 nM). DmNPF inhibited forskolin-stimulated adenylyl cyclase activity similarly (IC(50) = 51 nM). Whole-mount in situ hybridization revealed that DmNPFR1 RNA is expressed in CNS and midgut of Drosophila larvae. DmNPFR1, a new invertebrate Y-receptor homolog, apparently is a functional receptor for DmNPF.

FMRFamide- and pancreatic polypeptidelike immunoreactivity of endocrine cells in the midgut of a mosquito

Immunocytochemical surveys of midguts from female mosquitoes, Aedes aegypti, reveal that half of the estimated 500 endocrine cells in a midgut contain a substance recognized by antisera to bovine pancreatic polypeptide and a molluscan peptide, FMRFamide (phenylalanine-methionine-arginine-phenylalanine-amide). With light microscopy the cells resemble an endocrine type because of their basal position in the epithelium, conical shape, and, in some instances, apical extensions to the lumen. At the ultrastructural level, the immunoreactive substance is contained specifically within the secretory granules of such cells. Immunoreactive cells are distributed exclusively in the midgut region where blood is stored, and ingestion of vertebrate blood reduces the number of such cells and the intensity of reaction in others. These two facts suggest that a blood meal stimulates release of the immunoreactive substance from the cells. Since the immunocytochemical localization is supplemented by a demonstrated secretory response, the cells are considered to be peptidergic endocrine cells.

Neuropeptidomics of the mosquito Aedes aegypti

Neuropeptidomic data were collected on the mosquito Ae. aegypti, which is considered the most tractable mosquito species for physiological and endocrine studies. The data were solely obtained by direct mass spectrometric profiling, including tandem fragmentation, of selected tissues from single specimens, which yielded a largely complete accounting of the putative bioactive neuropeptides; truncated neuropeptides with low abundance were not counted as mature peptides. Differential processing within the CNS was detected for the CAPA-precursor, and differential post-translational processing (pyroglutamate formation) was detected for AST-C and CAPA-PVK-2. For the first time in insects, we succeeded in the direct mass spectrometric profiling of midgut tissue which yielded a comprehensive and immediate overview of the peptides involved in the endocrine system of the gut. Head peptides which were earlier identified as the most abundant RFamides of Ae. aegypti, were not detected in any part of the CNS or midgut. This study provides a framework for future investigations on mosquito endocrinology and neurobiology. Given the high sequence similarity of neuropeptide precursors identified in other medically important mosquitoes, conclusions regarding the peptidome of Ae. aegypti likely are applicable to these mosquitoes.

Isolation and primary structure of neuropeptides from the mosquito, Aedes aegypti, immunoreactive to FMRFamide antiserum

Abstract Two novel neuropeptides, Aea-HP-I and II, have been isolated from a head extract of the mosquito, Aedes aegypti; they were detected by a FMRFamide radioimmunoassay. The peptides were purified by gel filtration, ion exchange chromatography, and reversed-phase high performance liquid chromatography. Amino acid composition and sequence analysis, combined with enzymatic digestion, established the primary structure of Aea-HP-I as pGlu-Arg-Pro-Hyp-Ser-Leu-Lys-Thr-Arg-Phe-NH2 and Aea-HP-II as Thr-Arg-Phe-NH2. Aea-HP-I was synthesized, and chromatographic properties of the synthetic peptide were the same as those of the native peptide, thus confirming the structural analysis. The peptide has three unusual residues: an amino-terminal pGlu, a Hyp in the fourth position, and a carboxyl-terminal amide. The Pro-Hyp sequence occurs in toxin peptides from the venoms of cone snails and wasps and in bradykinin analogues. Although the functions of Aea-HP-I and II have not been determined, the peptides have the same RFa sequence at the carboxyl-terminal as Lem-SK-I and II (leucosulfakinins) and Lem-MS (leucomyosuppressin) in cockroaches and FMRFamide-related peptides in molluscs.

Endogenous regulation of mosquito host-seeking behavior by a neuropeptide

Abstract Most species of mosquitoes must engage in host-seeking behavior to find a vertebrate host and ingest a meal of blood in order to initiate and complete each oogenic cycle. Female Aedes aegypti fed to repletion almost immediately fail to respond to odor cues that would otherwise lead them to a host for another blood meal. This response is delayed for approx. 24h in females that ingest small blood meals. In both instances, during this period of inhibition, the hemolymph titer of Ae. aegypti Head Peptide I (Aea-HP-I; pGlu-Arg-Pro-Hyp-Ser-Leu-Lys-Thr-Arg-Phe-NH 2 ) rises to a peak, as measured with an Aea-HP-I radioimmunoassay (RIA). This homologous RIA is based on an antiserum that specifically binds Aea-HP-I (5–100 fmol range, 1:100,000 final dilution) as compared to other related peptides, Aea-HP-I and at least one other immunoreactive peptide were identified in pooled hemolymph from sugar-fed females with HPLC fractionation and the RIA. To determine whether Aea-HP-I affects host-seeking behavior, synthetic Aea-HP-I and related peptides were injected into non-oogenic females, which actively seek a host. Host-seeking behavior was inhibited by Aea-HP-I within a dosage range of 16.5–825 pmol/female, whereas related peptides differing by a few amino acids had no effect. The relatively high doses of Aea-HP-I, which elicited behavioral inhibition, were rapidly degraded in vivo as determined with the RIA of hemolymph from experimental females. Other associated behaviors were not altered by the Aea-HP-I injection, even at high doses. These results together suggest that Aea-HP-I regulates the observed behavioral inhibition in this mosquito species.

Identification and characterization of a G protein-coupled receptor for the neuropeptide proctolin in Drosophila melanogaster

Proctolin is a bioactive neuropeptide that modulates interneuronal and neuromuscular synaptic transmission in a wide variety of arthropods. We present several lines of evidence to propose that the orphan G protein-coupled receptor CG6986 of Drosophila is a proctolin receptor. When expressed in mammalian cells, CG6986 confers second messenger activation after proctolin application, with an EC50 of 0.3 nM. In competition-based studies, the CG6986 receptor binds proctolin with high affinity (IC50 = 4 nM). By microarray analysis, CG6986 transcript is consistently detected in head mRNA of different genotypes, and under different environmental conditions. By blot analysis, anti-CG6986 antibodies detect a band in tissue homogenates similar to the predicted size of the protein. Proctolin receptor immunosignals are found in the hindgut, heart, and in distinct neuronal populations of the CNS; such patterns correlate with previous demonstrations of proctolin biological activity, and in several instances, with areas of proctolin peptide immunosignals. The identification of a bona fide proctolin receptor provides the basis for a mechanistic analysis of this critical synaptic modulator.

Neuropeptide F and its expression in the yellow fever mosquito, Aedes aegypti

A neuropeptide F (NPF) was isolated from an extract of adult Aedes aegypti mosquitoes based on its immunoreactivity in a radioimmunoassay for Drosophila NPF. After sequencing the peptide, cDNAs encoding the NPF were identified from head and midgut. These cDNAs encode a prepropeptide containing a 36 amino acid peptide with an amidated carboxyl terminus, and its sequence shows it to be a member of the neuropeptide F/Y superfamily. Immunocytochemistry and Northern blots confirmed that both the brain and midgut of females are likely sources of NPF, found at its highest hemolymph titer before and 24 h after a blood meal.

Characterization of neuropeptide F and its receptor from the African malaria mosquito, Anopheles gambiae

The genome of Anopheles gambiae contains sequences encoding a neuropeptide F (Ang-NPF) and NPF receptor (Ang-NPFR) related to the neuropeptide Y signaling family. cDNAs for each were cloned and sequenced. Ang-NPFR was stably expressed for radioligand binding analysis. Ang-NPF exhibited high affinity (IC50 approximately 3 nM) membrane binding; NPFs from Aedes aegypti (Aea-NPF) and Drosophila melanogaster (Drm-NPF) were less potent, with the rank order: Ang-NPF>Aea-NPF>Drm-NPF>Drm-NPF8-36. RT-PCR analysis revealed Ang-NPF and Ang-NPFR transcripts in all life stages. Ang-NPF and Ang-NPFR may be strategically positioned for signaling in relation to nutritional status in the African malaria mosquito.