Interactions between allatostatins and allatotropin on spontaneous contractions of the foregut of larval Lacanobia oleracea

The interactions between the activity of three neuropeptides, Manduca sexta allatostatin (Manse-AS), M. sexta allatotropin (Manse-AT) and cydiastatin 4, on the spontaneous foregut contractions of the tomato moth, Lacanobia oleracea, were investigated. Bioassays revealed that application of Manse-AS to the foregut at high concentrations (10(-7)M) stopped contractions completely, and this inhibition could not be reversed by Manse-AT. Conversely, Manse-AS could inhibit a Manse-AT stimulated tissue. In contrast, Manse-AT reversed the inhibition of foregut peristalsis by cydiastatin 4 (10(-7)M), and cydiastatin 4 counteracted the stimulation by Manse-AT. These results imply that the Manse-AS inhibitory effect is dominant over the stimulatory action of Manse-AT. However, when two peptides with opposing actions were added together, the overall effect on foregut peristalsis was determined by the relative concentrations of each peptide, suggesting that in these experiments, no peptide was dominant over the other. When Manse-AS and cydiastatin 4 were applied to foregut tissues simultaneously the overall effect was not significantly different to the individual peptides, i.e. there was no additive effect. This suggests that the individual activities of Manse-AS and cydiastatin 4 are suppressed by an undetermined mechanism in the presence of the other peptide. These results question the need for two structurally different allatostatins that have the same physiological effect on foregut peristalsis in L. oleracea larvae.     

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.     

Endopeptidase activity of larval Lacanobia oleracea corpus allatum: metabolism of Manduca sexta allatostatin and allatotropin

The degradation of synthetic Manduca sexta allatostatin (Manse-AS) and allatotropin (Manse-AT) by enzymes associated with the corpus allatum (CA) of larvae of the tomato moth, Lacanobia oleracea, was investigated using reversed-phase high performance liquid chromatography and matrix-assisted laser desorption ionisation-time of flight mass spectrometry. Manduca sexta allatostatin was metabolised by CA extract to Manse-AS5-15, Manse-AS6-15, and Manse-AS7-15, which indicates enzymic cleavage at the C-terminal side of arginine residues R3 and R5 and the N-terminal side of R5, suggesting this is due to a trypsin-like enzyme. In support of this, the same degradation products were identified after Manse-AS was incubated with trypsin, and CA enzymic activity could be inhibited up to 79% by aprotinin. Degradation of Manse-AT by CA extract was also trypsin-like, cleaving at the C-terminal side of the basic residues K3 and R11 to produce Manse-AT4-13 and Manse-AT1-11. Metabolism by trypsin produced the same deletion peptides, but the major product due to this enzyme was Manse-AT4-11. Hydrolysis of Manse-AT by CA could only be partially inhibited by high doses of aprotinin (36%), and the CA extract also cleaved Manse-AT between M8 and T9 to produce Manse-AT1-8. A trypsin-like peptidase appears to be the major enzyme present in the CA of larval L. oleracea that acts to metabolise Manse-AS and Manse-AT. In addition, an unidentified enzyme that cleaves between M and T residues degraded Manse-AT.     

Allatoregulatory peptides in Lepidoptera, structures, distribution and functions

Allatoregulatory peptides either inhibit (allatostatins) or stimulate (allatotropins) juvenile hormone (JH) synthesis by the corpora allata (CA) of insects. However, these peptides are pleitropic, the regulation of JH biosynthesis is not their only function. There are currently three allatostatin families (A-, B-, and C-type allatostatins) that inhibit JH biosynthesis, and two structurally unrelated allatotropins. The C-type allatostatin, characterised by its blocked N-terminus and a disulphide bridge between its two cysteine residues, was originally isolated from Manduca sexta. This peptide exists only in a single from in Lepidoptera and is the only peptide that has been shown to inhibit JH synthesis by the CA in vitro in this group of insects. The C-type allatostatin also inhibits spontaneous contractions of the foregut. The A-type allatostatins, which exist in multiple forms in a single insect, have also been characterised from Lepidoptera. This family of peptides does not appear to have any regulatory effect on JH biosynthesis, but does inhibit foregut muscle contractions. Two structurally unrelated allatotropins stimulate JH biosynthesis in Lepidoptera. The first was identified in M. sexta (Manse-AT) and occurs in other moths. The second (Spofr AT2) has only been identified in Spodoptera frugiperda. Manduca sexta allatotropin also stimulates heart muscle contractions and gut peristalsis, and inhibits ion transport across the midgut of larval M. sexta. The C-terminal (amide) pentapeptide of Manse-AT is important for JH biosynthesis activity. The most active conformation of Manse-AS requires the disulphide bridge, although the aromatic residues also have a significant effect on biological activity. Both A- and C-type allatostatins and Manse-AT are localised in neurosecretory cells of the brain and are present in the corpora cardiaca, CA and ventral nerve cord, although variations in localisation exist in different moths and at different stages of development. The presence of Manse-AS and Manse-AT in the CA correlates with the biological activity of these peptides on JH biosynthesis. There is currently no explanation for the presence of A-type allatostatins in the CA. The three peptide types are also co-localised in neurosecretory cells of the frontal ganglion, and are present in the recurrent nerve that supplies the muscles of the gut, particularly the crop and stomodeal valve, in agreement with their role in the regulation of gut peristalsis. There is also evidence that they are expressed in the midgut and reproductive tissues.     

Identification of neuropeptides from brains of larval Manduca sexta and Lacanobia oleracea using MALDI-TOF mass spectrometry and post-source decay

The occurrence of neuropeptides in the brain of larvae of the tobacco hawkmoth, Manduca sexta, and tomato moth, Lacanobia oleracea, was investigated using matrix-assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry (MS) and post-source decay (PSD). Methanolic extracts of 100 brains separated by reversed-phase high performance liquid chromatography yielded numerous ion peaks, some of which were common to both species. In M. sexta six [M+H](+) ions were in agreement with peptides previously structurally characterised from M. sexta (FLRF-amides I, II and III, M. sexta allatostatin, CAP(2b) and myoinhibitory peptide VI), whereas a further five corresponded to other known lepidopteran peptides (cydiastatins 3 and 4, helicostatins 1 and 6 and helicokinin II). Of these the identities of FLRF-amide I, cydiastatins 3 and 4 and CAP(2b) were confirmed by PSD analysis. Fourteen [M+H](+) ions corresponding to known lepidopteran peptides (FLRF-amide I, cydiastatins 2, 3 and 4, helicostatins 1, 5, 6, 7 and 9, CCAP, CAP(2b), M. sexta allatostatin and myoinhibitory peptide VI) were measured in L. oleracea brain extracts. From this insect, cydiastatins 3 and 4, helicostatin 5 and FLRF-amide I were identified by PSD. These peptides had not previously been structurally characterised from L. oleracea.     

A comparison of the neuropeptides from the retrocerebral complex of adult male and female Manduca sexta using MALDI-TOF mass spectrometry

The occurrence of neuropeptides in the retrocerebral complexes of adult male and females of the tobacco hawkmoth, Manduca sexta, was investigated using matrix-assisted laser desorption time of flight (MALDI-TOF) mass spectrometry (MS), post source decay (PSD) and collision-induced dissociation (CID) MS/MS. From fractions of methanol extracts of corpora cardiaca (CC)/corpora allata (CA), separated by reversed-phase high performance liquid chromatography (RP-HPLC), a total of 11 mass ions were assigned to known peptides from M. sexta. These peptides were adipokinetic hormone (AKH), FLRFamides I, II and III, crustacean cardioactive peptide (CCAP), cardioactive peptide 2b (CAP(2b)), three myoinhibitory peptides, corazonin, and M. sexta allatostatin (Manse-AS). A further six masses were in agreement with Y/FXFGLamide allatostatins identified from other Lepidoptera. The sequence identities of FLRFamide I and AKH were confirmed using post source decay analysis. Fragmentation by collision-induced dissociation MS/MS identified an extended AKH peptide. The apparent differences in the peptides present in male and female retrocerebral complexes are most likely quantitative rather than sex specific.     

Degradation of Manduca sexta allatostatin and allatotropin by proteases associated with the foregut of Lacanobia oleracea larvae

The degradation of synthetic Manduca sexta allatostatin (Manse-AS) and allatotropin (Manse-AT), by enzymes of the foregut of larvae of the tomato moth, Lacanobia oleracea was investigated using reversed-phase high performance liquid chromatography (RP-HPLC) together with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and Edman sequencing. Metabolism of 1nmol Manse-AS by foregut extract (1microg protein) was rapid, t(1/2) approximately 5min, with two major products produced. Mass spectrometry of HPLC fractions identified cleavage products Manse-AS-(4-15) and Manse-AS-(6-15), which indicates enzymatic cleavage at the C-terminal side of arginine residues (R(3) and R(5)). This degradation of Manse-AS could be inhibited by up to 80% by the serine protease inhibitor aprotinin, but not PMSF, pepstatin, E64, EDTA, or 1,10-phenanthroline.M. sexta allatotropin was also rapidly degraded when incubated with foregut extract, t(1/2) approximately 8min, producing two metabolic products, one of which was identified as Manse-AT-(1-11), showing enzymatic cleavage at the C-terminal side of arginine (R(11)). The second product was identified as Manse-AT-(1-8). Hydrolysis of Manse-AT could only be partially inhibited by high doses of aprotinin (30%).     

Direct peptide profiling of lateral cell groups of the antennal lobes of Manduca sexta reveals specific composition and changes in neuropeptide expression during development

The paired antennal lobes are the first integration centers for odor information in the insect brain. In the sphinx moth Manduca sexta, like in other holometabolous insects, they are formed during metamorphosis. To further understand mechanisms involved in the formation of this particularly well investigated brain area, we performed a direct peptide profiling of a well defined cell group (the lateral cell group) of the antennal lobe throughout development by MALDI-TOF mass spectrometry. Although the majority of the about 100 obtained ion signals represent still unknown substances, this first peptidomic characterization of this cell group indicated the occurrence of 12 structurally known neuropeptides. Among these peptides are helicostatin 1, cydiastatins 2, 3, and 4, M. sexta-allatotropin (Mas-AT), M. sexta-FLRFamide (Mas-FLRFamide) I, II, and III, nonblocked Mas-FLRFamide I, and M. sexta-myoinhibitory peptides (Mas-MIPs) III, V, and VI. The identity of two of the allatostatins (cydiastatins 3 and 4) and Mas-AT were confirmed by tandem mass spectrometry (MALDI-TOF/TOF). During development of the antennal lobe, number and frequency of ion signals including those representing known peptides generally increased at the onset of glomeruli formation at pupal Stage P7/8, with cydiastatin 2, helicostatin 1, and Mas-MIP V being the exceptions. Cydiastatin 2 showed transient occurrence mainly during the period of glomerulus formation, helicostatin 1 was restricted to late pupae and adults, while Mas-MIP V occurred exclusively in adult antennal lobes. The power of the applied direct mass spectrometric profiling lies in the possibility of chemically identifying neuropeptides of a given cell population in a fast and reliable manner, at any developmental stage in single specimens. The identification of neuropeptides in the antennal lobes now allows to specifically address the function of these signaling molecules during the formation of the antennal lobe network.     

Immunocytochemical localization of an allatotropin in developmental stages of Heliothis virescens and Apis mellifera

Juvenile hormone biosynthesis by the corpora allata is regulated by stimulatory neuropeptides called allatotropins and inhibitory neuropeptides called allatostatins. This study localized Manduca sexta allatotropin-like material in developmental stages of the noctuid moth Heliothis virescens and the honeybee Apis mellifera. Immunocytochemical methods using both fluorescence-tagged antibodies and enzyme-coupled antibodies were used to stain the central nervous tissue of both species. H. virescens contains M. sexta allatotropin (Manse-AT)-like material consistently throughout larval development. The distribution patterns of Manse-AT immunoreactive cell bodies in the CNS persisted from one larval instar to the next. It will be discussed how larval Manse-AT distribution patterns differed from those in adults. The total number of AT-containing cells in brain and subesophageal ganglion gradually increased during larval development, whereas in the thoracic and abdominal ganglia, the number of AT-containing neurons remained constant. In the honeybee A. mellifera, Manse-AT immunoreactive cells were only found in a few brains from late last instar larvae (prepupae). Manse-AT-like material was present in a group of 6-8 cells in the pars intercerebralis. However, we did not find any Manse-AT-like material in brains of early last instar larvae, whose corpora allata (CA) are more sensitive to in vitro stimulation by Manse-AT than prepupal CA.     

In vivo effects of Manduca sexta allatostatin and allatotropin on larvae of the tomato moth, Lacanobia oleracea

Abstract. Injection of Manduca sexta allatotropin (Manse-AT) into fifth or sixth stadium larval Lacanobia oleracea had no significant effect on larval growth, development or food consumption, compared to control injected insects. In contrast, injection of M. sexta allatostatin (Manse-AS) into fifth stadium larvae resulted in a retardation of growth, reduction in feeding and increased mortality, compared to control injected insects, but had no effect on non-feeding (day 7) sixth instar larvae. Results suggest that Manse-AS is not acting on the corpora allata (CA) to inhibit Juvenile Hormone (JH) synthesis to produce the observed effects, but most likely by its myoinhibiting action on the foregut. Inhibition of foregut peristalsis by Manse-AS in vivo appears to suppress feeding, resulting in increased mortality. Foregut peristalsis may be inhibited by the intact peptide or a deletion peptide produced by cleavage of Manse-AS by haemolymph enzymes, because Manse-AS (5-15) also inhibits muscle contractions in the foregut in vitro .     

Neuropeptide co-localisation in the lepidopteran frontal ganglion studied by confocal laser scanning microscopy

A comparative study of the co-localisation of three different families of neuropeptides, viz. allatostatins of the Y/FXFGL-NH(2) type, Manduca sexta allatostatin (Mas-AS) and allatotropin, in the frontal ganglion of lepidopteran larvae has been carried out by means of immunocytochemistry and confocal laser scanning microscopy. The simultaneous application of three types of fluorochrome-conjugated antibodies reveals triple co-localisation in an anterodorsal pair of neurones in the frontal ganglion of the noctuids Heliothis virescens and Lacanobia oleracea. There is no evidence of differential axonal transport, since all parts of these neurones show complete co-localisation of all three peptides. Prominent axons of the ganglionic neurones project in the recurrent nerve to the foregut and stomodeal valve. Over the crop, lateral and sub-lateral branches follow the course of circular muscle fibres and terminate in varicosities. All three neuropeptides have previously been shown to be myoregulatory on the foregut; the Y/FXFGL-NH(2) allatostatins and Mas-AS are inhibitory, whereas allatotropin is excitatory. The morphological evidence of co-localisation of physiologically antagonistic peptides within the same terminals suggests that an extremely complex mechanism controls the contractile activities of the foregut. A posterodorsal pair of neurones in the frontal ganglion have prominent axons projecting via the frontal connectives to the brain and in the recurrent nerve to the stomodeal valve where extensive branching suggests control over the valve movements. Studies of another noctuid, Spodoptera frugiperda, and the sphingid, M. sexta, show interesting variations in the co-localisation phenomenon.     

Metabolism of Manduca sexta allatostatin by hemolymph of larvae of the tomato moth, Lacanobia oleracea

The degradation of synthetic Manduca sexta allatostatin (Manse-AS) by hemolymph from larvae of the tomato moth, Lacanobia oleracea was investigated using reversed phase-high performance liquid chromatography (RP-HPLC), and matrix assisted laser desorption ionisation-time of flight mass spectrometry. Metabolism of 1 nmole Manse-AS in diluted hemolymph was rapid, t(1/2) = 3.5 min, with a number of products produced. Mass spectrometry of HPLC fractions identified cleavage products, which indicated a sequential degradation of Manse-AS from the N-terminal to Manse-AS (7-15). The most abundant products identified were Manse-AS (5-15), (6-15), and (7-15). These metabolites were synthesized and assayed for biological activity on juvenile hormone (JH) biosynthesis in vitro. All three of the above deletion peptides showed allatostatin activity, but were not as potent as Manse-AS (1-15).     

Triple co-localisation of two types of allatostatin and an allatotropin in the frontal ganglion of the lepidopteran Lacanobia oleracea (Noctuidae): innervation and action on the foregut

The triple co-localisation of peptidergic material immunoreactive to antisera raised against allatostatins of the Y/FXFGL-NH2 type, Manduca sexta allatostatin (Mas-AS), and allatotropin has been demonstrated in a single pair of anterodorsal neurones in the frontal ganglion of the tomato moth, Lacanobia oleracea (Noctuidae). Another pair of posterior neurones contain only Y/FXFGL-NH2-type allatostatin immunoreactivity. The neurites of all four cells trifurcate, and axons project to the brain in the frontal connectives and to the foregut in the recurrent nerve. Axons from the anterior neurones, within the recurrent nerve, have prominent lateral branches supplying muscles of the crop, and axons from both anterior and posterior cells show profuse branching and terminal arborisations in the region of the stomodeal valve. The brain contributes Y/FXFGL-NH2-immunoreactive material, but not allatotropin or Mas-AS, to the recurrent nerve via NCC 1+2 and NCC 3. All three peptides have a reversible effect on the spontaneous (peristaltic) contractions of the foregut (crop) in vitro. Thus, both types of allatostatin are inhibitory at 10(-12) to 10(-7) M, whereas allatotropin is strongly myostimulatory at 10(-14) M. This is the first demonstration of the gut myoinhibitory effects of Mas-AS and, taken together with the effects of Y/FXFGL-NH2-type allatostatins and allatotropin, reveals a different functional aspect to that normally attributed to these three peptides, i.e. control of juvenile hormone synthesis by the corpus allatum.     

Molecular characterisation of cDNAs from the fall armyworm Spodoptera frugiperda encoding Manduca sexta allatotropin and allatostatin preprohormone peptides

Allatotropin (AT) is a 13-residue amidated neuropeptide, first isolated from pharate adult heads of the tobacco hornworm, Manduca sexta (Manse-AT), which strongly stimulates the biosynthesis of juvenile hormones (JH) in the corpora allata (CA) of adult moths. In Spodoptera frugiperda, a cDNA that encodes 134 amino acids, including an AT peptide, has been cloned. The S. frugiperda allatotropin mature peptide (Spofr-AT) [GFKNVEMMTARGFa] is identical to that isolated from M. sexta. The basic organization of the Spofr-AT precursor is similar to that of Agrius convolvuli, M. sexta, Pseudaletia unipuncta, and Bombyx mori with 83-93% amino acid sequence identity. The Spofr-AT gene is expressed in at least three mRNA isoforms with 134, 171 and 200 amino acids, differing from each other by alternative splicing.All allatostatins (AS) have an inhibitory action on the JH biosynthesis in the CA. A cDNA that encodes 125 amino acid residues including one copy of the Manse-AS peptide has been cloned from S. frugiperda (Spofr-AS; QVRFRQCYFNPISCF). The basic organization of the Spofr-AS precursor is similar to that of P. unipuncta with 85% amino acid sequence identity.Using one step RT-PCR for semi-quantification of the gene expression, we showed that the three mRNAs of the Spofr-AT gene and the Spofr-AS gene are expressed in brains of last instar larvae, prepupae, pupae, and adults of both sexes of S. frugiperda with variable intensity.     

Allatotropin-like peptide in Heliothis virescens: tissue localization and quantification

The mating-induced increase in juvenile hormone (JH) biosynthesis in Heliothis virescens females may be stimulated by production and/or release of stimulatory neuropeptides such as allatotropins (AT). Although there is evidence that H. virescens allatotropin may be structurally related to Manduca sexta allatotropin (Manse-AT), little is known of its occurrence and distribution in H. virescens. An enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody against Manse-AT was used to quantify concentrations of Manse-AT immunoreactivity in tissue extracts of H. virescens. In mated females, the highest concentrations of Manse-AT-like material occurred in the brain. The ventral nervous system and the accessory glands also contained considerable amounts of Manse-AT-like material, whereas concentrations were very low in ovaries, fat body, and flight muscle. The Manse-AT antibody was used for whole-mount immunocytochemistry to localize Manse-AT-immunoreactivity in the central nervous system. Several groups of Manse-AT-immunoreactive cells were discovered in the brain, subesophageal ganglion, and thoracic and abdominal ganglia of H. virescens females and males. Strong immunoreactivity was detected in axons going through the corpora cardiaca and branching out over the surface of the corpora allata. The presence of Manse-AT-like material in various locations in the central nervous system suggests that these peptides may have other as yet unknown functions. At the posterior margin of the terminal ganglion of males, a group of large immunoreactive cells was observed that was not present in females. Other than that, there were no obvious differences between virgin and mated females or males. The lack of differences in AT distribution in mated and virgin females suggests that mating-induced differences in female JH biosynthesis rates may be caused by changes in cellular response to AT at the level of the CA, rather than by changes in the amounts of AT acting on the CA.     

Effects of Manduca allatotropin and localization of Manduca allatotropin-immunoreactive cells in earwigs

Manduca sexta allatotropin (Manse-AT) was first isolated on the basis of its ability to stimulate production of juvenile hormone in that insect. We examined whether this neuropeptide affects corpus allatum activity and visceral muscle contraction in adult females of the earwig, Euborellia annulipes. We also assessed the presence of allatotropin-like material in tissues using immunocytochemistry. Manse-AT at 1 nM to 10 muM stimulated juvenile hormone production in vitro by glands of low activity from 2-day virgin females. In glands of high activity from 12-day mated females, 1 and 100 nM allatotropin were effective, but 10 muM was not. Similarly, hindguts of 2-day and 12-day females significantly increased in motility in vitro in response to Manse-AT. A monoclonal antibody to Manse-AT was used to demonstrate allatotropin-like material throughout the nervous system of 2-day, virgin females. Immunoreactivity was most pronounced within varicosities of the corpora cardiaca and perisympathetic organs. No immunofluorescence was observed in gut tissue. Lastly, we showed that extract of retrocerebral complexes also enhanced in vitro hindgut motility from 2-day virgin females, in a dose-dependent manner. These results indicate material similar to M. sexta allatotropin in female earwigs and that such peptides may modulate juvenile hormone biosynthesis and visceral muscle contractions. Sensitivity to the peptides may change with physiological stage.     

The role of allatostatic and allatotropic neuropeptides in the regulation of juvenile hormone biosynthesis in Lacanobia oleracea (Lepidoptera: Noctuidae)

In the sphinghid moth Manduca sexta, two allatoactive neuropeptides appear to be responsible for regulating juvenile hormone (JH) production by the corpora allata (CA). These peptides (M. sexta allatostatin, Mas-AS, and M. sexta allatotropin, Mas-AT) respectively inhibit and stimulate in vitro JH biosynthesis by CA in this insect. However, although Mas-AS inhibits CA in both larval and adult insects, Mas-AT is active only in adult M. sexta. The situation in other lepidopteran species is less clear-cut and, although both peptides have been detected (usually by immunologic and/or molecular techniques) in several other moths (including noctuids), their function as regulators of JH production remains uncertain. In the tomato moth Lacanobia oleracea (Lepidoptera: Noctuidae), we have previously demonstrated the occurrence of Mas-AS and/or Mas-AT in extracts of CA, brain and other organs, and have shown that both peptides are present in larval and adult forms. However, in L. oleracea, although Mas-AS inhibits larval and adult CA in vitro, it does so only at relatively high concentrations, and to a maximum of only approximately 70%. By contrast, Mas-AT (which is also present in larval and adult L. oleracea) stimulates larval and adult CA, but is substantially more potent ( approximately 100 fold) than the allatostatin. In this paper we present the results of paired, concurrent measurements (using ELISA) of levels of Mas-AS and Mas-AT in brains, CA and hemolymph (plasma and hemocytes) of L. oleracea at times when there are marked changes in JH titers. We also present data on the in vitro rates of JH biosynthesis by isolated CA, and on hemolymph JH esterase activity measured at the same critical developmental times, and discuss all of these data in relation to the putative allatoregulatory roles of the M. sexta allatotropic and allatostatic neuropeptides in L. oleracea.     

Isolation and functional characterization of an allatotropin receptor from Manduca sexta

Manduca sexta allatotropin (Manse-AT) is a multifunctional neuropeptide whose actions include the stimulation of juvenile hormone biosynthesis, myotropic stimulation, cardioacceleratory functions, and inhibition of active ion transport. Manse-AT is a member of a structurally related peptide family that is widely found in insects and also in other invertebrates. Its precise role depends on the insect species and developmental stage. In some lepidopteran insects including M. sexta, structurally-related AT-like (ATL) peptides can be derived from alternatively spliced mRNAs transcribed from the AT gene. We have isolated a cDNA for an AT receptor (ATR) from M. sexta by a PCR-based approach using the sequence of the ATR from Bombyx mori. The sequence of the M. sexta ATR is similar to several G protein-coupled receptors from other insect species and to the mammalian orexin receptor. We demonstrate that the M. sexta ATR expressed in vertebrate cell lines is activated in a dose-responsive manner by Manse-AT and each Manse-ATL peptide in the rank order ATL-I > ATL-II > ATL-III > AT, and functional analysis in multiple cell lines suggest that the receptor is coupled through elevated levels of Ca(2+) and cAMP. In feeding larvae, Manse-ATR mRNA is present at highest levels in the Malpighian tubules, followed by the midgut, hindgut, testes, and corpora allata, consistent with its action on multiple target tissues. In the adult corpora cardiaca--corpora allata complex, Manse-ATR mRNA is present at relatively low levels in both sexes.     

Alanine substitution and deletion analogues of Manduca sexta allatostatin: structure-activity relationship on the spontaneous contractions of the foregut of larval Lacanobia oleracea

Manduca sexta allatostatin (Manse-AS) is a 15-residue non-amidated peptide with a blocked N-terminus and a disulphide bridge between the cysteine residues at positions 7 and 14. Analogues of Manse-AS were used to examine the structural requirements of Manse-AS for inhibitory activity on spontaneous foregut contractions of larval tomato moth (Lacanobia oleracea). Breaking the disulphide bond between C(7) and C(14) by reduction reduced the potency of the peptide, suggesting that the conformation of Manse-AS is important for its biological activity. When either of the cysteine residues were replaced with alanine the Manse-AS analogue had no measurable bioactivity. Alanine substitution at Q(6) was as potent as Manse-AS, all other alanine substitution analogues (R(5), Y(8), F(9), N(10), P(11), I(12) and S(13)), were myoinhibitory but less potent than native Manse-AS to varying degrees. Analogues with alanine substitution at amino acids with aromatic side chains (Y(8) and F(9)) were the least active. Amino-terminal deletion analogues Manse-AS(6-15) and Manse-AS(7-15) were inactive whereas Manse-AS(5-15) was fully active but not as potent as Manse-AS. The results show that amino acid residues both inside and outside the disulphide ring are important for biological activity.     

Increased urinary C-type natriuretic peptide excretion may be an early marker of renal tubulointerstitial fibrosis

The cDNAs encoding allatotropin (AT) and allatotropin-like peptides (ATLPs) were isolated from the silkworm, Bombyx mori. Similar to those of the tobacco hornworm, Manduca sexta, four peptides (AT, ATLP1, ATLP2, and ATLP3) are present in three different variants generated by alternative splicing. RT-PCR analyses showed that these splice variants are expressed in the central nervous system with differing expression patterns in each ganglion. Immunohistochemistry using an anti-AT antibody confirmed that AT-expressing cells were located in these central nervous ganglia as well as in two large anterior cells of the frontal ganglia. Injection of synthetic AT and ATLP-1 into B. mori larvae increased the latency to feed, indicating that AT and ATLP might function in the regulation of feeding behavior in B. mori.