Oral activity of FMRFamide-related peptides on the pea aphid Acyrthosiphon pisum (Hemiptera: Aphididae) and degradation by enzymes from the aphid gut

Insect myosuppressins and myosuppressin analogues were tested for oral toxicity against the pea aphid Acyrthosiphon pisum (Harris) by incorporation into an artificial diet. Acyrthosiphon pisum myosuppressin (Acypi-MS) and leucomyosuppressin (LMS) had significant dose-dependent effects (0.1-0.5μg peptide/μl diet) on feeding suppression, mortality, reduced growth and fecundity compared with control insects, but Acypi-MS was more potent than LMS. One hundred percent of aphids had died after 10days of feeding on 0.5μg Acypi-MS/μl diet whereas 40% of aphids feeding on 0.5μg LMS/μl diet were still alive after 13days. Myosuppressins were degraded by aphid gut enzymes; degradation was most likely due to a carboxypeptidase-like protease, an aminopeptidase and a cathepsin L cysteine protease. The estimated half-life of Acypi-MS in a gut extract was 30min, whereas LMS was degraded more slowly (t?=54min). No toxicity was observed when the analogues δR(9) LMS and citrolline(9) Acypi-MS or FMRFamide were fed to the pea aphid. These findings not only help to better understand the biological effects of myosuppressins in aphids but also demonstrate the potential use of myosuppressins in a strategy to control aphid pests.     

Identification of leucomyosuppressin in the German cockroach, Blattella germanica, as an inhibitor of food intake

The feeding pattern of the adult female of Blattella germanica peaks in the middle of the vitellogenic cycle. Following the hypothesis that a factor inhibiting gut peristalsis also inhibits food intake and is involved in the regulation of feeding, we searched for the most powerful myoinhibitory peptide in brain extracts from B. germanica females collected after the peak within the feeding cycle. Through HPLC purification and sequence analysis, we obtained the peptide leucomyosuppressin (LMS): pQDVDHVFLRFamide. LMS elicited a powerful myoinhibitory effect on B. germanica foregut and hindgut, with ED(50) values around 10(-10) M. In addition, it inhibited food intake in vivo in a dose-dependent manner at doses between 5 and 50 microg. The study of the distribution of ingested food in the foregut, midgut and hindgut of B. germanica females treated with LMS showed that food accumulates in the foregut, which may be due to the myoinhibitory effects of the peptide. We propose that this accumulation inhibits food intake because of the persistence of the signals from gut stretch receptors.     

In vitro and in vivo effects of myo-active peptides on larvae of the tomato moth Lacanobia oleracea and the cotton leaf worm Spodoptera littoralis (Lepidoptera; Noctuidae)

Neuropeptides from five different neuropeptide families [Manduca sexta allatostatin (Manse-AS), and Manse-AS deletion analogue(5-15), M. sexta allatotropin (Manse-AT), leucomyosuppressin, perisulfakinin, and myoinhibitory peptide I (MIP I)] were assayed for their ability to affect the development and food consumption of penultimate and last larval instars of two lepidopteran species, L. oleracea and S. littoralis. Injections of Manse-AS deletion analogue(5-15), Manse-AT, perisulfakinin, and MIP I had no observable effects on development, food consumption, or mortality compared to controls. Single injections of Manse-AS significantly reduced the weight gain and increased mortality of L. oleracea and S. littoralis larvae compared to controls. By contrast, feeding Manse-AS to L. oleracea had no such effects. These differences were probably due to the degradation of the peptide by digestive enzymes in the foregut of L. oleracea. In studies in vitro, perisulfakinin, and MIP I had no effect on the spontaneous foregut contractions of L. oleracea larvae. Leucomyosuppressin, however, had myoinhibitory effects on the foregut. Single injections of leucomyosuppressin significantly reduced the weight gain and food consumption of L. oleracea and S. littoralis larvae and increased mortality. These data suggest that the deleterious effects observed in vivo were due to the myoinhibition by Manse-AS and leucomyosuppressin of the normal peristaltic movements of the gut either by the intact peptide or by its cleavage products resulting from degradation in the haemolymph.     

Characterization and baculovirus-directed expression of a myosuppressin encoding cDNA from the true armyworm, Pseudaletia unipuncta

Insect myosuppressins are a highly conserved sub-family of peptides which are primarily characterized by the ability to suppress contraction of visceral muscles in a variety of insect species. We have isolated a cDNA from the true armyworm, Pseudaletia unipuncta, that encodes a prohormone containing a peptide identical to ManducaFLRFamide. We have shown that this myosuppressin gene appears to be expressed in late larval and adult insects. In Manduca sexta, a number of extended-FLRFamide peptides have previously been purified including ManducaFLRFamide, F7D (DPSFLRFamide), F7G (GNSFLRFamide) and two larger peptides F24 and F39 that contain the shorter ManducaFLRFamide sequence at their C-terminus. Comparison with the true armyworm prepropeptide characterized here identifies F24 and F39 as partially processed products from the same precursor. Expression in the true armyworm was shown by in situ hybridization to occur in over 150 cells throughout the adult brain and nerve cord, and also to occur in both open and closed endocrine type cells of the gut. Overexpression of the P. unipuncta FLRFamide cDNA from a baculovirus vector in cabbage looper caterpillars was used to assess the potential for myosuppressin expression as a means of enhancing virus efficacy. Viral expression of the armyworm prohormone cDNA resulted in raised levels of RFamide-like products in the hemolymph of infected insects, but the products were found to be chemically distinguishable from authentic mature peptide and probably represent partially processed hormone.     

Mechanism of entomotoxicity of the plant lectin from Hippeastrum hybrid (Amaryllis) in Spodoptera littoralis larvae

Plant lectins have received a lot of attention because of their insecticidal properties. When orally administered in artificial diet or in transgenic plants, lectins provoke a wide range of detrimental effects, including alteration of the digestive enzyme machinery, fecundity drop, reduced feeding, changes in oviposition behavior, growth and development inhibition and mortality. Although many studies reported the entomotoxicity of lectins, only a few of them investigated the mode of action by which lectins exert toxicity. In the present paper we have studied for the first time the insecticidal potential of the plant lectin from Hippeastrum hybrid (Amaryllis) (HHA) bulbs against the larvae of the cotton leafworm (Spodoptera littoralis). Bioassays on neonate larvae showed that this mannose-specific lectin affected larval growth, causing a development retardation and larval weight decrease. Using primary cell cultures from S. littoralis midguts and confocal microscopy we have elucidated FITC-HHA binding and internalization mechanisms. We found that HHA did not exert a toxic effect on S. littoralis midgut cells, but HHA interaction with the brush border of midgut cells interfered with normal nutrient absorption in the S. littoralis midgut, thereby affecting normal larval growth in vivo. This study thus confirms the potential of mannose-specific lectins as pest control agents and sheds light on the mechanism underlying lectin entomotoxicity.     

Molecular identification of a myosuppressin receptor from the malaria mosquito Anopheles gambiae

The insect myosuppressins (X1DVX2HX3FLRFamide) are neuropeptides that generally block insect muscle activities. We have used the genomic sequence information from the malaria mosquito Anopheles gambiae Genome Project to clone a G protein-coupled receptor that was closely related to the two previously cloned and characterized myosuppressin receptors from Drosophila [Proc. Natl. Acad. Sci. USA 100 (2003) 9808]. The mosquito receptor cDNA was expressed in Chinese hamster ovary cells and was found to be activated by low concentrations of Anopheles myosuppressin (TDVDHVFLRFamide; EC50, 1.6 x 10(-8)M). The receptor was not activated by a library of 35 other insect neuropeptides and monoamines, including neuropeptides that resembled myosuppressin in their C-terminal moiety, such as PDRNFLRFamide (Anopheles FMRFamide-3), other Anopheles FMRFamide peptides, or neuropeptide F-like peptides, showing that the receptor was quite selective for myosuppressin. These results also showed that the myosuppressin receptor needs a much larger portion than the C-terminal FLRFamide sequence for its activation. The insect myosuppressins are often grouped together with the insect FMRFamides under the name FaRPs (FMRFamide-related peptides). However, this is not justified anymore, because the insect myosuppressin receptor/ligand couple is both functionally and evolutionarily fully unrelated to the insect FMRFamide receptor/ligand couple. To our knowledge, this is the first report on the molecular identification of a mosquito neuropeptide receptor.     

The cDNA for leucomyosuppressin in Blattella germanica and molecular evolution of insect myosuppressins

Myosuppressins are a group of 10-residues FMRFamide-related peptides reported in Dictyoptera, Orthoptera, Lepidoptera and Diptera. Myosuppressins inhibit visceral muscle contractions and, in the cockroach Blattella germanica, inhibit food intake. In B. germanica, the cDNA of leucomyosuppressin (LMS) has been cloned and sequenced. The deduced precursor is 96 amino acids long and contains a single copy of LMS. Brain mRNA levels remain constant during the first reproductive cycle of adult females, whereas those in the gut show a slight decline during the time of maximal food intake. Comparison of myosuppressin precursors of different species reveals that all have the same organization. Phylogenetic analysis suggests that the precursor experienced an accelerated evolution in Lepidoptera and Diptera with respect to Dictyoptera, whereas only Lepidoptera has radical changes in the bioactive peptide.     

Lateral ventricular ghrelin and fourth ventricular ghrelin induce similar increases in food intake and patterns of hypothalamic gene expression

The gut peptide ghrelin has been shown to stimulate food intake after both peripheral and central administration, and the hypothalamic arcuate nucleus has been proposed to be the major site for mediating this feeding stimulatory action. Ghrelin receptors are widely distributed in the brain, and hindbrain ghrelin administration has been shown to potently stimulate feeding, suggesting that there may be other sites for ghrelin action. In the present study, we have further assessed potential sites for ghrelin action by comparing the ability of lateral and fourth ventricular ghrelin administration to stimulate food intake and alter patterns of hypothalamic gene expression. Ghrelin (0.32, 1, or 3.2 nmol) in the lateral or fourth ventricle significantly increased food intake in the first 4 h after injection, with no ventricle-dependent differences in degree or time course of hyperphagia. One nanomole of ghrelin into either the lateral or fourth ventricle resulted in similar increases in arcuate nucleus neuropeptide Y mRNA expression. Expression levels of agouti-related peptide or proopiomelanocortin mRNA were not affected by ghrelin administration. These data demonstrate that ghrelin can affect food intake and hypothalamic gene expression through interactions at multiple brain sites.     

Growth and mitogenic effects of arylphorin in vivo and in vitro

In insects, developmental responses are organ- and tissue-specific. In previous studies of insect midgut cells in primary tissue cultures, growth-promoting and differentiation factors were identified from the growth media, hemolymph, and fat body. Recently, it was determined that the mitogenic effect of a Manduca sexta fat body extract on midgut stem cells of Heliothis virescens was due to the presence of monomeric alpha-arylphorin. Here we report that in primary midgut cell cultures, this same arylphorin stimulates stem cell proliferation in the lepidopterans M. sexta and Spodoptera littoralis, and in the beetle Leptinotarsa decemlineata. Studies using S. littoralis cells confirm that the mitogenic effect is due to free alpha-arylphorin subunits. In addition, feeding artificial diets containing arylphorin increased the growth rates of several insect species. When tested against continuous cell lines, including some with midgut and fat body origins, arylphorin had no effect; however, a cell line derived from Lymantria dispar fat body grew more rapidly in medium containing a chymotryptic digest of arylphorin.     

Bombyxin: An Insect Brain Peptide that Belongs to the Insulin Family

Bombyxin is a 5 kDa secretory brain peptide that belongs to the insulin family. Bombyxin of the silkmoth Bombyx mori can induce adult development when injected into brain-removed dormant pupae of the saturniid moth Samia cynthia ricini by activating the prothoracic glands to synthesize and release ecdysone. Bombyx bombyxin has been shown to lower the concentration of the major haemolymph sugar, trehalose, and to elevate the trehalase activity in the midgut and muscles in Bombyx, but the doses required to be effective are higher than the amounts in the feeding larvae. The exact physiological function of bombyxin in Bombyx itself is therefore still obscure, but its insulin-like structure suggests it has important roles. Bombyxin comprises a mixture of highly heterogeneous molecular forms whose amino acid sequences have 40% identity with human insulin. The Bombyx bombyxin gene encodes a precursor consisting of the signal peptide, B chain, C peptide, and A chain, in that order from the N terminus. So far, 32 bombyxin genes have been identified in Bombyx, and they are classified into 7 families, A to G, according to their sequence similarity. The bombyxin genes have no introns and cluster in unique distribution patterns. The gene arrangement in the cluster has been classified into three categories: gene pairs, gene triplets, and single genes. Nucleotide sequence analysis indicates that equal and unequal crossings-over and duplications may have generated these unique distribution patterns. The Bombyx bombyxin genes are expressed predominantly in the brain and at low levels in a number of other tissues. Genes of all 7 families are expressed in four pairs of the medial neurosecretory cells of the brain. Detailed examination indicated that only a limited number of genes in the A, B and C family members are expressed and that their expression shows a gene-arrangement-dependent pattern.     

RNA interference suggests sulfakinins as satiety effectors in the cricket Gryllus bimaculatus

In the Mediterranean field cricket, Gryllus bimaculatus, the action of sulfakinin (SK) gene expression on food intake, food transport in the gut and carbohydrate digestion (alpha-amylase activity) was investigated by using the RNA interference (RNAi) method. Injection of SK double-stranded (ds) RNA into the abdomen of female adults and last instar larvae led to a systemic silencing of the SK gene, as was shown by RT-PCR studies. In adults, suppression of SK gene expression was effective from the first day after injection up to at least the third day. Treatment of the adult crickets by injection or feeding of dsRNA led to a stimulation of the food intake. Assuming that the gene silencing is followed by a depletion of the SK in tissues and/or haemolymph implies an inhibitiory role of the native SK peptides on food intake. The alpha-amylase activity in vitro in the midgut tissue and in the secretions of adult females was not affected by silencing the SK gene.     

Peptide YY(3-36) inhibits gastric emptying and produces acute reductions in food intake in rhesus monkeys

Peptide YY3-36 [PYY(3-36)], a gastrointestinal peptide that is released into the circulation in response to ingesting a meal, has recently been suggested to play a role in controlling food intake. PYY(3-36) has been reported to inhibit food intake following peripheral administration in rodents and in human subjects. To more fully characterize the potential feeding actions of PYY(3-36), we examined the ability of a dose range of PYY(3-36) (0.3-3.0 nmol/kg) to affect liquid gastric emptying and daily 6-h food intake in male rhesus monkeys. Intramuscular PYY(3-36) produced a dose-related inhibition of saline gastric emptying that was maximal at a dose of 3 nmol/kg. Intramuscular PYY(3-36) administered before daily 6-h food access produced significant feeding reductions at doses of 1 and 3 nmol/kg. Analyses of the patterns of food intake across the 6-h period of food access revealed that PYY(3-36) increased the latency to the first meal and reduced average meal size without altering meal number. Although single doses of PYY(3-36) reduced intake, a suppressive effect on food intake was not sustained over multiple administrations across successive days. Together, these data suggest that PYY(3-36) has the ability to reduce food intake in acute test situations in nonhuman primates. Whether this is a physiological action of the endogenous peptide remains to be determined.     

The gut hormones PYY 3-36 and GLP-1 7-36 amide reduce food intake and modulate brain activity in appetite centers in humans

Obesity is a major public health issue worldwide. Understanding how the brain controls appetite offers promising inroads toward new therapies for obesity. Peptide YY (PYY) and glucagon-like peptide 1 (GLP-1) are coreleased postprandially and reduce appetite and inhibit food intake when administered to humans. However, the effects of GLP-1 and the ways in which PYY and GLP-1 act together to modulate brain activity in humans are unknown. Here, we have used functional MRI to determine these effects in healthy, normal-weight human subjects and compared them to those seen physiologically following a meal. We provide a demonstration that the combined administration of PYY(3-36) and GLP-1(7-36 amide) to fasted human subjects leads to similar reductions in subsequent energy intake and brain activity, as observed physiologically following feeding.     

Role of hypothalamic neuropeptide Y (NPY) in the change of feeding behavior induced by repeated treatment of amphetamine

Neuropeptide Y (NPY), an orexigenic peptide, is involved in the control of food intake. Repeated administration of amphetamine (AMPH), an anorectic agent, results in an anorectic effect on day 1 and a tolerant anorectic effect on the followings. In an attempt to know the role of hypothalamic NPY in these effects of AMPH, contents of hypothalamic NPY were determined by radioimmunoassay at first. In AMPH-treated groups, the contents of hypothalamic NPY decreased rapidly on day 1 but restored gradually to the normal level on the following days as observed in repeated AMPH. An involvement of hypothalamic NPY in the feeding change of repeated AMPH can thus be considered. Moreover, daily injection of NPY antisense oligonucleotide into brain (10 microg/10 microl/day, i.c.v.) to inhibit the gene expression of hypothalamic NPY were performed at 1 hour before daily 2 mg/kg AMPH. The reversion of food intake from the anorectic level to the normal level (tolerant anorexia) was abolished by this antisense pretreatment. It is suggested that hypothalamic NPY may play a role in the change of feeding behavior induced by repeated AMPH administration.     

Peptide S is a novel potent inhibitor of voluntary and fast-induced food intake in rats

Peptide S (NPS or PEPS) and its cognate receptor have been recently identified both in the central nervous system and in the periphery. NPS/PEPS promotes arousal and has potent anxiolytic-like effects when it is injected centrally in mice. In the present experiment, we tested by different approaches its central effects on feeding behaviour in Long-Evans rats. PEPS at doses of 1 and 10 microg injected in the lateral brain ventricle strongly inhibited by more than 50% chow intake in overnight fasted rats with effects of longer duration with the highest dose (P<0.0001). A similar decrease was observed for the spontaneous intake of a high-energy palatable diet (-48%; P<0.0001). This anorexigenic effect was comparable to that induced by corticotropin-releasing hormone in fasted rats at equimolar doses. However, peptide S did not modify food intake stimulated by neuropeptide Y (NPY) at equimolar doses. It also did not affect the fasting concentrations of important modulators of food intake like leptin, ghrelin, and insulin in circulation. This study therefore showed that peptide S is a new potent anorexigenic agent when centrally injected. Its inhibitory action appears to be independent of the NPY, ghrelin, and leptin pathways. Development of peptide S agonists could constitute a new approach for the treatment of obesity.     

In vitro analysis of the digestive enzymes amylase and alpha-glucosidase in the midguts of Locusta migratoria L. in response to the myosuppressin, SchistoFLRFamide

We have investigated the effect of the locust myosuppressin, SchistoFLRFamide, on the activity of amylase and alpha-glucosidase in the midgut of 2-week old male locusts. Total enzyme activity in the lumen contents and tissue extracts of midguts responds to SchistoFLRFamide in a dose-dependent manner that appears to vary with the feeding state of the locust and duration of exposure to the peptide. Starvation for 24h prior to assessment alters the distribution of enzyme activity between the midgut lumen contents and tissue extracts in response to SchistoFLRFamide when compared with fed locusts. Duration of exposure to SchistoFLRFamide also alters the distribution of total amylase and alpha-glucosidase activity; as duration of exposure increases, lower concentrations of SchistoFLRFamide increase total enzyme activity in the lumen contents while decreasing total enzyme activity in the tissue extracts. We suggest that the minimum amino acid sequence in SchistoFLRFamide necessary to increase both amylase and alpha-glucosidase activity is DHVFLRFamide. We have determined that two other peptides endogenous to the locust, AFIRFamide and GQERNFLRFamide, increase amylase and alpha-glucosidase activity in midgut lumen contents.     

Functional proteomics of neuropeptidome dynamics during the feeding process of Rhodnius prolixus

In hematophagous insects, blood intake triggers a prompt response mediated by neuropeptides, which regulates a variety of physiological processes. Here we report a quantitative proteomic analysis of the postfeeding response in the central nervous system of Rhodnius prolixus, a vector of Chagas disease. The concentration of neuropeptides NVP-like, ITG-like, kinin-precursor peptide, and neuropeptide-like precursor 1 (NPLP1) significantly changes in response to blood intake. We also performed a neuropeptidomic analysis of other feeding-related organs, namely salivary glands and gut. We identified NPLP1 in salivary glands and myosuppressin in midgut. This is the first report suggesting a role for NPLP1, involving the peptides processed from this precursor in the hormonal control of the production and/or release of saliva. Our results contribute to the understanding of the postprandial neuroendocrine response in hematophagous and provide important information for physiological and pharmacological studies aimed to the design of next-generation insecticides such as peptidomimetics.