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 NPF's 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.     

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.     

A comparative review of short and long neuropeptide F signaling in invertebrates: Any similarities to vertebrate neuropeptide Y signaling?

Neuropeptides referred to as neuropeptide F (NPF) and short neuropeptide F (sNPF) have been identified in numerous invertebrate species. Sequence information has expanded tremendously due to recent genome sequencing and EST projects. Analysis of sequences of the peptides and prepropeptides strongly suggest that NPFs and sNPFs are not closely related. However, the NPFs are likely to be ancestrally related to the vertebrate family of neuropeptide Y (NPY) peptides. Peptide diversification may have been accomplished by different mechanisms in NPFs and sNPFs; in the former by gene duplications followed by diversification and in the sNPFs by internal duplications resulting in paracopies of peptides. We discuss the distribution and functions of NPFs and their receptors in several model invertebrates. Signaling with sNPF, however, has been investigated mainly in insects, especially in Drosophila. Both in invertebrates and in mammals NPF/NPY play roles in feeding, metabolism, reproduction and stress responses. Several other NPF functions have been studied in Drosophila that may be shared with mammals. In Drosophila sNPFs are widely distributed in numerous neurons of the CNS and some gut endocrines and their functions may be truly pleiotropic. Peptide distribution and experiments suggest roles of sNPF in feeding and growth, stress responses, modulation of locomotion and olfactory inputs, hormone release, as well as learning and memory. Available data indicate that NPF and sNPF signaling systems are distinct and not likely to play redundant roles.     

Duplication of neuropeptide Y and peptide YY in Nile tilapia Oreochromis niloticus and their roles in food intake regulation

In vertebrates, the neuropeptide Y (NPY) family peptides have been recognized as key players in food intake regulation. NPY centrally promotes feeding, while peptide YY (PYY) and pancreatic polypeptide (PP) mediate satiety. The teleost tetraploidization is well-known to generate duplicates of both NPY and PYY; however, the functional diversification between the duplicate genes, especially in the regulation of food intake, remains unknown. In this study, we identified the two duplicates of NPY and PYY in Nile tilapia (Oreochromis niloticus). Both NPYa and NPYb were primarily expressed in the central nervous system (CNS), but the mRNA levels of NPYb were markedly lower than those of NPYa. Hypothalamic mRNA expression of NPYa, but not NPYb, decreased after feeding and increased after 7-days of fasting. However, both NPYa and NPYb caused a significant increase in food intake after an intracranial injection of 50 ng/g body weight dose. PYYb, one of the duplicates of PYY, had an extremely high expression in the foregut and midgut, whereas another form of duplicate PYYa showed only moderate expression in the CNS. Both hypothalamic PYYa and foregut PYYb mRNA expression increased after feeding and decreased after 7-days of fasting. Furthermore, the intracranial injection of PYYb decreased food intake, but PYYa had no significant effect. Our results suggested that although the mature peptides of NPYa and NPYb can both stimulate food intake, NPYa is the main endogenous functional NPY for feeding regulation. A functional division has been identified in the duplicates of PYY, which deems PYYb as a gut-derived anorexigenic peptide and PYYa as a CNS-specific PYY in Nile tilapia.     

The effects of the broad-specificity lipase inhibitor, tetrahydrolipstatin, on the growth, development and survival of the larvae of Epiphyas postvittana (Walker) (Tortricidae, Lepidoptera)

The effects of the lipase inhibitor, tetrahydrolipstatin (THL), on neonate Epiphyas postvittana (Walker) (Lepidoptera, Tortricidae) larvae were investigated by feeding on control artificial diets (with and without 2% ethanol) and diets containing 2% ethanol and one of three concentrations of THL (0.011%, 0.037% and 0.11%). Small but significant reductions in growth rate, percent pupation and time to pupation were observed for larvae feeding on 2% ethanol control diet compared with standard control diet, but larger reductions in all parameters occurred with increasing THL concentration. Third instar larvae fed 0.011% THL in the diet had 40% of the midgut lipase activity in the relevant control larvae and showed up-regulation of gene expression of the gastric lipase-like family but not the pancreatic lipase-like family of midgut lipases.     

Compensatory proteolytic responses to dietary proteinase inhibitors from Albizia lebbeck seeds in the Helicoverpa armigera larvae

Plant proteinase inhibitors (PIs) have been shown to reduce the growth rates in larvae of numerous insect species. On the other hand, insects can also regulate their proteinases against plant PIs. In the present study, we report the compensatory activities of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) gut proteinases against the PIs of Albizia lebbeck seeds. Total of ten proteinase inhibitor bands were detected in the seed extract of A. lebbeck. Bioassays were conducted by feeding H. armigera larvae on diet containing partially purified PIs from A. lebbeck seeds. Results show that larval growth and survival was significantly reduced by A. lebbeck PIs. We found that higher activity H. armigera gut proteinase (HGP) isoforms observed in the midgut of control larvae were inhibited in the midgut of larvae fed on test diet. Some HGP isoforms were induced in the larvae fed on PI containing test diet; however, these isoforms showed lower activity in the larvae fed on control diet. Aminopeptidase activities were significantly increased in the midgut of larvae fed on test diet. A population of susceptible and resistant enzymes was observed in the midgut of H. armigera, when fed on diet containing PIs from A. lebbeck seeds. Our initial observations indicate that H. armigera can regulate its digestive proteinase activity against non-host plant PIs, too. It is important to study the exact biochemical and molecular mechanisms underlying this phenomenon in order to develop PI-based insect control strategies.     

A new microsporidian species, Vairimorpha ocinarae n. sp., isolated from Ocinara lida Moore (Lepidoptera: Bombycidae) in Taiwan

A new microsporidium was isolated from Ocinara lida Moore (Lepidoptera: Bombycidae), a pest of Ficus microcarpa L. f. in Taiwan. The microsporidium produces systemic infections in O. lida larvae; the midgut epithelium, Malpighian tubules, and midgut muscle tissues were the target tissues for this isolate, and atrophied fat body tissues were found in heavily infected larvae. Two types of spores were observed, diplokaroytic spores with 11-13 coils of polar tube, and monokaryotic spores with 12 coils of the polar tube that developed within a sporophorous vesicle to form octospores. Electron-dense granules were abundant in the episporontal space of the sporophorous vesicles, and were similar to those of Vairimorpha invictae isolated from Solenopsis invicta, but different from granules or inclusions of other Vairimorpha species. Based on the phylogenetic analysis of the small subunit ribosomal DNA sequence, this isolate is unique within the Vairimorpha complex. Morphological and genetic characters showed this isolate to be a new species. It is placed in the genus Vairimorpha and is described as Vairimorpha ocinarae n. sp.     

Expressing a moth abcc2 gene in transgenic Drosophila causes susceptibility to Bt Cry1Ac without requiring a cadherin-like protein receptor

Bt toxins ingested by insect pests can bind to midgut receptors and cause death, although several steps in this process remain unclear. Multiple Bt toxin receptors have been identified in Lepidoptera, including a cadherin-like protein (CaLP), which is central to several models explaining Bt toxins’ mode of action. Mutations in the Plutella xylostella ATP-dependent binding cassette transporter C2 (Px-abcc2), rather than CaLP, are genetically linked with Bt Cry1Ac resistance. Here we expressed Px-abcc2 in Drosophila and performed larval bioassays to determine whether this protein acts as an effective Bt receptor. Cry1Ac had no effect on larvae expressing Px-abcc2 in salivary glands, yet larvae expressing Px-abcc2 in the midgut were highly susceptible to both Cry1Ac protoxin and trypsin activated toxin. Furthermore, the CaLP orthologue has been lost from the Drosophila genome, making this a useful system for investigating the role of CaLP peptides from Manduca sexta (CR12-MPED), which are known to act as Bt synergists in larval feeding assays. Drosophila larvae expressing Px-ABCC2 in the midgut were fed LD₅₀ concentrations of Cry1Ac toxin or protoxin, plus purified CR12-MPED cloned from M. sexta or P. xylostella. The M. sexta CR12-MPED protein acted synergistically with Cry1Ac protoxin and activated toxin significantly more effectively than the P. xylostella peptide. This work demonstrates ABCC2 is the major functional Cry1Ac receptor for P. xylostella and the importance of CaLP proteins in Bt mode of action may vary between different lepidopteran species.     

Regulatory peptides in fruit fly midgut

Regulatory peptides were immunolocalized in the midgut of the fruit fly Drosophila melanogaster. Endocrine cells were found to produce six different peptides: allatostatins A, B and C, neuropeptide F, diuretic hormone 31, and the tachykinins. Small neuropeptide-F (sNPF) was found in neurons in the hypocerebral ganglion innervating the anterior midgut, whereas pigment-dispersing factor was found in nerves on the most posterior part of the posterior midgut. Neuropeptide-F (NPF)-producing endocrine cells were located in the anterior and middle midgut and in the very first part of the posterior midgut. All NPF endocrine cells also produced tachykinins. Endocrine cells containing diuretic hormone 31 were found in the caudal half of the posterior midgut; these cells also produced tachykinins. Other endocrine cells produced exclusively tachykinins in the anterior and posterior extemities of the midgut. Allatostatin-immunoreactive endocrine cells were present throughout the midgut. Those in the caudal half of the posterior midgut produced allatostatins A, whereas those in the anterior, middle, and first half of the posterior midgut produced allatostatin C. In the middle of the posterior midgut, some endocrine cells produced both allatostatins A and C. Allatostatin-C-immunoreactive endocrine cells were particularly prominent in the first half of the posterior midgut. Allatostatin B/MIP-immunoreactive cells were not consistently found and, when present, were only weakly immunoreactive, forming a subgroup of the allatostatin-C-immunoreactive cells in the posterior midgut. Previous work on Drosophila and other insect species suggested that (FM)RFamide-immunoreactive endocrine cells in the insect midgut could produce NPF, sNPF, myosuppressin, and/or sulfakinins. Using a combination of specific antisera to these peptides and transgenic fly models, we showed that the endocrine cells in the adult Drosophila midgut produced exclusively NPF. Although the Drosophila insulin gene Ilp3 was abundantly expressed in the midgut, Ilp3 was not expressed in endocrine cells, but in midgut muscle.     

Neuropeptide F signaling regulates parasitoid-specific germline development and egg-laying in Drosophila

Drosophila larvae and pupae are at high risk of parasitoid infection in nature. To circumvent parasitic stress, fruit flies have developed various survival strategies, including cellular and behavioral defenses. We show that adult Drosophila females exposed to the parasitic wasps, Leptopilina boulardi, decrease their total egg-lay by deploying at least two strategies: Retention of fully developed follicles reduces the number of eggs laid, while induction of caspase-mediated apoptosis eliminates the vitellogenic follicles. These reproductive defense strategies require both visual and olfactory cues, but not the MB247-positive mushroom body neuronal function, suggesting a novel mode of sensory integration mediates reduced egg-laying in the presence of a parasitoid. We further show that neuropeptide F (NPF) signaling is necessary for both retaining matured follicles and activating apoptosis in vitellogenic follicles. Whereas previous studies have found that gut-derived NPF controls germ stem cell proliferation, we show that sensory-induced changes in germ cell development specifically require brain-derived NPF signaling, which recruits a subset of NPFR-expressing cell-types that control follicle development and retention. Importantly, we found that reduced egg-lay behavior is specific to parasitic wasps that infect the developing Drosophila larvae, but not the pupae. Our findings demonstrate that female fruit flies use multimodal sensory integration and neuroendocrine signaling via NPF to engage in parasite-specific cellular and behavioral survival strategies.     

Nosema invadens sp. n. (Microsporida: Nosematidae), a pathogen causing inflammatory response in Lepidoptera

States in the life cycle of Nosema invadens sp. n. are described from the raisin moth, Cadra figulilella, and the almond moth, Cadra cautella. In laboratory tests, larvae of the following Lepidoptera were also susceptible: Ephestia elutella, Plodia interpunctella, Galleria mellonella, and Paramyelois transitella. The midgut wall and Malpighian tubules were invaded first, but subsequently most other tissues were also infected. Severe inflammatory response accompanied infection, including hemocytic encapsulation of infected areas and hemocytic infiltration of fat tissue.     

Identification of the novel bioactive peptides dRYamide-1 and dRYamide-2, ligands for a neuropeptide Y-like receptor in Drosophila

A number of bioactive peptides are involved in regulating a wide range of animal behaviors, including food consumption. Vertebrate neuropeptide Y (NPY) is a potent stimulator of appetitive behavior. Recently, Drosophila neuropeptide F (dNPF) and short NPF (sNPF), the Drosophila homologs of the vertebrate NPY, were identified to characterize the functions of NPFs in the feeding behaviors of this insect. Dm-NPFR1 and NPFR76F are the receptors for dNPF and sNPF, respectively; both receptors are G protein-coupled receptors (GPCRs). Another GPCR (CG5811; NepYR) was indentified in Drosophila as a neuropeptide Y-like receptor. Here, we identified 2 ligands of CG5811, dRYamide-1 and dRYamide-2. Both peptides are derived from the same precursor (CG40733) and have no significant structural similarities to known bioactive peptides. The C-terminal sequence RYamide of dRYamides is identical to that of NPY family peptides; on the other hand, dNPF and sNPF have C-terminal RFamide. When administered to blowflies, dRYamide-1 suppressed feeding motivation. We propose that dRYamides are related to the NPY family in vertebrates, similar to dNPF and sNPF.     

Antigenicity to neuropeptide F (NPF) in Stenostomum leucops and Microstomum lineare

The first native flatworm regulatory peptide, neuropeptide F (NPF) has recently been isolated and sequenced from the cestode Moniezia expansa (see Maule et al., 1991) and the turbellarian Artioposthia triangulata, (see Curry et al., 1992). NPF belongs to the neuropeptide Y (NPY) superfamily and the antiserum is known to show cross-reactivity to the vertebrate neuropeptides of the NPY superfamily. It terminates in RFamide, like the invertebrate neuropeptides FMRFamide and RFamide, and may cross-react with neuropeptides of the FMRFamide family. Strong immunoreactivity (IR) to FMRF- and RF-amide has been demonstrated in members of most flatworm groups. In the present study, IR to NPF (diluted 1:1000) is demonstrated in Stenostomum leucops (Catenulida) and Microstomum lineare (Macrostomida). The controls included: omitting primary antibody, using non-immune serum and liquid-phase absorption with the homologous antigen (1000 ng ml^–1). The NPF IR pattern was compared to the FMRF and RF-amide IR patterns in order to reveal differences or co-localization. In addition, the sequential appearance of NPF-positive cells in developing zooids was followed and double staining with a-5-HT made to complete the study.     

Characterization of neuropeptide F-like immunoreactivity in the blood-feeding hemipteran, Rhodnius prolixus

The invertebrate neuropeptide Y (NPY) homolog, neuropeptide F (NPF), has been characterized for a wide range of invertebrate phyla, including platyhelminthes, molluscs, and arthropods. Current hypotheses suggest that NPF may be capable of regulating responses to diverse external cues related to nutritional status and feeding. The qualitative and quantitative distribution of an NPF-like peptide in fifth instar Rhodnius prolixus was undertaken using an antiserum raised against Drosophila NPF. Immunohistochemistry reveals NPF-like immunoreactive neurons and processes in the central nervous system, stomatogastric nervous system and peripheral nervous system. The distribution of NPF-like immunoreactivity within the medial neurosecretory cells of the brain and neurohemal areas of the corpus cardiacum and dorsal vessel, suggests NPF may act as a neurohormone. Immunoreactive processes are present over the surface of the hindgut and the immunoreactivity in these processes is greatly reduced in intensity 24h post-feeding. The quantification of partially purified NPF-like material in the CNS of R. prolixus was conducted by HPLC fractionation and radioimmunoassay. The results suggest that NPF-like material is present in fifth instar R. prolixus and likely released into the hemolymph following a blood meal.